Keywords:

Schéma 8.Représentation proportionnelle de l'i droite) sur de MNPs fonctionnalisées avec le ligand azoture En conclusion, ce travail nous a permis de la fonctionnalisation linéaire ou dendritique de nanoparticules, l'efficacité d'immobilisation de laccases (amino, aldéhyde et azoture) effets d'orientation induits par une immobilisation de deux types de laccases (Lac3 et Unik157) présentant des sites d'accrochesproche du site ou à 180° du site de cuivre

Dans le domaine de la biocatalyse, l'immobilisation des enzymes sur des supports a pour principal objectif d'améliorer à la fois les performances opérationnelles et économiques des procédés biocatalytiques.

Produire un biocatalyseur recyclable avec de bonnes propriétés catalytiques et une bonne stabilité reste un enjeu majeur pour aller vers des processus durables plus efficaces et plus rentables [1]. Au cours de la dernière décennie, les méthodes et les supports d'immobilisation d'enzymes ont largement été étudiés [2], et contrairement à d'autres supports, les nanomatériaux sont apparus comme des supports attrayants car ils possèdent de nombreux avantages, tels que des surfaces spécifiques élevées.Parmi ces nanomatériaux, les MNPs (nanoparticules superparamagnétiques) [3] Dans le domaine de la biocatalyse supportée un des enjeux est le contrôle de l'immobilisation de l'enzyme et l'étude des effets d'orientation à la surface des supports solides sur les propriétés des biocatalyseurs. Le contrôle d'un microenvironnement autour du site catalytique aura sans nul doute un impact sur la sélectivité et la réactivité enzymatiques des biocatalyseurs ainsi produits. En effet, dans la très grande majorité des cas, les immobilisations enzymatiques se font de manière non-orientées, ce qui est inhérent aux méthodes de greffage (adsorption physique, encapsulation, cross-linking et greffage covalent). Ces méthodes se traduisent par une faible densité et une répartition aléatoire des enzymes en surface, conduisant généralement à une faible activité des biocatalyseurs hétérogènes.

Dans cette étude, nous nous sommes concentrés sur l'optimisation des paramètres influençants l'activité spécifique des biocatalyseurs hybrides (MNP-Laccase), l'orientation des enzymes à la surface des particules et la densité enzymatique des biocatalyseurs hybrides (Schéma 2). Nous avons proposé une nouvelle stratégie pour une immobilisation enzymatique orientée, plus accessible et plus sélective. Notre schéma de greffage orienté et sélectif est basé sur la modification de surface de nanoparticules coeur-écorce d'oxyde de fer (300 nm, -Fe 2 O 3 /SiO 2 ) avec des agents de couplage linéaires ou dendritiques. La structure dendritique des agents de couplage conduit à une grande flexibilité chimique permettant un plus grand nombre de sites potentiels d'immobilisation. Nous avons ainsi modulé la nature du microenvironnement proche du site catalytique en faisant varier la taille, la forme, la flexibilité/rigidité, la polarité, l'hydrophilie, les charges et l'aromaticité des agents de couplage. Nous avons enfin étudié l'activité enzymatique des biocatalyseurs produits. . MNPs fonctionnalisées par des groupements amino, aldéhyde et azoture obtenues.

L'efficacité du couplage covalent a été contrôlé par spectroscopie IR de l'apparition des bandes de vibrations caractéristiques nouvellement introduits sur la surface des MNPs. La microscopie été utilisée pour caractériser les suspensions de particules greffées. Aussi, nous avons de surface par des mesures de potentiels zêta. En effet, un rôle fondamental dans les phénomènes d'adsorption et facteur déterminant dans le contrôle de la stabilisation électrostatique. Enfin, la caractérisation des MNPs greffées a été étudiée par microscopie électronique ( pour confirmer l'intégrité des nanoparticules et de la couche de silice (mesure des lors des procédures de greffage.

ifférentes stratégies ont été employées pour la quantification des groupes fonctionnels disponibles à la surface par titrage. La densité des groupes amino a été Les caractérisations des MNPs fonctionnalisées a été une étape cruciale car on peut raisonnablement penser que in fine la charge enzymatique du biocatalyseur sera proportionnelle, dans une certaine mesure, à la quantité de groupes fonctionnels disponibles à la surface des MNPs.

La laccase (EC 1.10.3.2,d'origine végétale, fongique ou bactérienne) est un catalyseur robuste largement étudié pour ses utilisations potentielles dans les procédés industriels, dans les applications de synthèse organique et comme catalyseur cathodique ayant la propriété de réduire le dioxygène en eau dans les biopiles [4]. Il contient un ensemble unique d'ions de cuivre constitués d'au moins un de chacun des trois types de sites de cuivre actifs en catalyse (mononucléaire de type 1 (T1) et de type 2 (T2) et un binucléaire de type 3 (T3)) dont la présence permet une caractérisation spectroscopique aisée de l'enzyme (UV/VIS et ESR). Le groupe T.Tron [5] possède une expertise dans la manipulation des laccases et dans la production à grande échelle de métallo-enzymes recombinantes. Ils se sont également récemment concentrés sur la construction de systèmes catalytiques dans lesquels l'enzyme jouerait le rôle de moteur redox. Ils ont également développé une stratégie originale de fonctionnalisation de la surface de l'enzyme basée sur la production de variants contenant un seul résidu lysine disponible pour modification de surface. C'est-à-dire que les deux seuls groupes lysine (K40 et K71) naturellement présents dans l'enzyme Lac3 ont été remplacés par mutagenèse dirigée par des acides aminés innocents; le variant sans lysine résultant (NoK) est alors une plate-forme pour la substitution de Les biocatalyseurs hybrides ainsi produits nous ont permis d'étudier la cinétique et l'activité enzymatique de la transformation d'un substrat standard (ABTS). En effet, l'ABTS est transformé en radical cation ABTS .+ par une réaction d'oxydation catalysée par la laccase (Schéma 5). Le produit radical cation (ABTS .+ ) est détecté rapidement (en 2 minutes), par spectroscopie UV-vis à 420 nm (ε= 36000 mol -1 .cm -1 , facteur de calcul : 27.8) à température ambiante dans le tampon de stockage (pH= 5.5). Green chemistry is one of the most important subsections in the modern society.

Efficient, durable, economical catalysts are essential in regard of industrial value and environment, especially to avoid the use of organic solvents, toxic metal, harsh operation conditions. The development of nano-science offers a powerful tool to promote green chemistry. Among solid apparatus for green chemistry, MNPs exhibited many advantages, compared to other inorganic matrix, they offer an easy recovery and separation from the reaction mixture. In contrast to conventional protocols of biotechnology (such as centrifugation, precipitation, etc) [1], magnetic bioseparation is highly efficient, fast, simple and low cost. Under an external magnetic field, MNPs suspensions can be magnetized and enriched, and when the magnetic field is lost, they can be re-dispersed in the solution again. At present, this method has been applied separation of material samples (DNA, RNA, cell, protein) [2], and MNPs have been widely used in many fields, such as biotechnology, biomedicine, catalysis, etc [3].

The surface modification of MNPs promotes the development of new strategies and is demanding for new materials with various geometries, better physical/chemical properties, more durable stability, higher loading. Their functionalization allows the possibility to modify the surface and change the properties of MNPs to target various biomolecules. Therefore, MNPs are promising materials due to their specific physical/chemical properties [4].

In the field of biocatalysis, the main objectives of the immobilization of enzymes on supports are to enhance both the operational and economic performances of biocatalytic processes, besides, immobilization of enzymes results in more efficient and cost-effective sustainable processes. Laccase (EC 1.10.3.2, from plant, fungi or bacteria) is a robust catalyst largely studied for its potential uses in industrial processes, in organic synthesis applications and as cathodic catalyst reducing dioxygen into water in biofuel cells [5]. T.Tron group [6] production, applications, advantages and disadvantages of various enzyme and immobilization technologies and the popular materials needed for enzyme immobilization, and reported their excellent results. In parallel, we also described the preparation, properties and modification of magnetic nanoparticles, as well as their wide range of applications. In addition, the properties and development of laccase as a widely-loved biocatalyst were also studied, including their immobilization and application in various fields.

In chapter II, first of all, the synthesis of three families of coupling agents is described, -NH 2 , -CHO, -N 3 , and all coupling agents are composed by, (i) a hydrophilic backbone to increase the solubility in aqueous media, (ii) an anchoring site (silane group) to covalently bind the silica shell, (iii) a terminal functional group (NH 2 , CHO, N 3 ) to immobilize the target enzyme. Various synthesis routes were investigated and several dendritic structures were proposed (by varying size and nature) in order to tune the nature of the surface of MNPs and thus the local environment at the enzyme catalytic site. The linear coupling agents, two branched coupling agents, and four branched coupling agents are proposed.

In chapter III, the surface modification of MNPs was achieved via covalent attachment of the silica shell and silane groups of linear or dendritic coupling agents. Optimization of surface modification of MNPs was presented. The grafting process was studied by varying parameters, such as temperature, solubility of coupling agents, media, suspension of MNPs, ratio of amount of MNPs/coupling agents. Finally the quantifications of functional groups (NH 2 , CHO, N 3 ) of grafted MNPs were performed. The use of the dendritic structure of coupling agents has been favored with the aim of improving chemical flexibility and a larger number of potential sites of immobilization. We envisaged to modulate the nature of the size, the shape, the flexibility/rigidity, the polarity, the hydrophilicity, the charges and aromaticity of the coupling agents.

In chapter IV, the enzyme immobilization was carried out. Parameters influencing the enzyme grafting were investigated such as catalyst, pH, buffer, quantities of enzymes and functional MNPs. Various immobilization routes were explored. For amino particles, heterobifunctional linker was employed to bridge amino particles and laccase followed by covalent binding through a reductive amination in mild conditions. For aldehyde particles, a direct coupling was achieved via a reductive amination in mild conditions. For azide particles, enzyme immobilization was performed via a click reaction. The immobilization strategies were adjusted depending of the activity of hybrid biocatalysts towards ABTS. Then enzyme loading was evaluated by Elisa test. Finally the specific activity of hybrid biocatalysts was calculated. At last, discussions about the influence of the specific environment close to the T1 catalytic site were presented as well as orientation effect of the enzyme on the catalytic properties of the hybrid biocatalysts. Enzymes are produced by living organism or chemical synthesis to overcome the impediments of the biocatalytic application in the industry, such as microorganism, plants, and animals. Nowadays increasingly rewarding, lots especially for industrial production. However, it is not always possible to find a proper enzyme from the commercial library, which mainly contains: 1)

Chapter I Bibliographical study

for new enzymes from nature activity with new conditions genetic engineering.

Biocatalysis

Since ancient times, humans have known that microbe fermentation, for example, beer, breed and cheese has been known

Chapter I Bibliographical study

Menten equation (Fig. Nowadays, the discovery of new enzymes is becoming increasingly rewarding, lots of enzymes are from the commercial library available, especially for industrial production. However, it is not always possible to find a the commercial library, which mainly contains: 1)

for new enzymes from nature; 2) Synthesis of new enzymes; 3) Novel enzyme new conditions, such as metal ions; 4) Altering enzymes, such as Since ancient times, humans have known that microbes have been used for for example, using microbial cells as biocatalysts in wine making, has been known for thousands of years. Modern , the discovery of new enzymes is becoming of enzymes are from the commercial library available, especially for industrial production. However, it is not always possible to find a the commercial library, which mainly contains: 1) Screening

; 3) Novel enzyme enzymes, such as have been used for in wine making, Modern concept of "enzyme" is associated with fermentation and digestion, and the term "enzyme" was coined to express the catalytic activity. Biocatalysis refers to the activities where organisms produce their own metabolism and maintain their organisms.

Nowadays, biocatalysis or biological transformation is defined as the use of enzymes or whole living cells containing enzymes to convert a specific substrate into a product. and re-use. In general, industrial production runs in the fluidized bed, and the substrate and biocatalyst should be pre-dissolved in bulk solution. Therefore, an enzyme system will interact with the environment surrounding the biocatalyst.

Besides mass transfer limitation is influenced by the substrate diffusion though porous materials. Moreover, both enzymes and products are always used in solvents, resulting in difficult and expensive separation, but these drawbacks can be overcome by immobilizing enzymes on supports, which will lead to an easy handling of enzymes in a solid form. In addition, immobilized biocatalysts present higher activity, higher stability, and greater resistance to external changes (such as pH, solvents, temperature) which ultimately reduces the overall cost of a biocatalysis reaction. In addition, the immobilization of enzymes on a support can offer a different micro-environment from that of free enzymes which can affect the activity of an enzyme in terms of reactivity and selectivity [1] [2].

Those excellent advantages greatly boost biocatalysis and lead to more and more emphasizes on research of biocatalysis. Biotechnology has been a key cornerstone in many fields, and an environmentally friendly process in chemical manufacturing affords imaginative economic potential. Some enzymes have been used in pharmaceutical industry, such as proteinases, asparaginase, urokinase, glucocerebrosidase, one famous application of biocatalysis is the production of penicillin, which is a widely used antibiotic and a well-known example facilitating the new semi-synthesis of antibiotics, such as, penicillin G acylase (PGA), which are able of hydrolyzing amide bond of the side chain of the aim molecule to produce an amine acid, 6-aminopenicillanic acid (6-APA), which is converted to produce a lot of new derivatives to overcome the problem of resistance. Compared to the traditional methods which need hazardous chemicals such as pyridine and are unfriendly to environment, leading to the hydrolysis of the cyclic amides, a biotransformation of breakdown of amide bond catalyzed by PGA is cheaper and also offers a mild condition and neutral pH to avoid decomposition of 6-aminopenicillanic acid. Besides, for industrial applications, cost and recovery of enzymes are essential, which means immobilization of PGA has been very important to recover enzymes and keep its activity and stability [3][4][5][6][7][8].

2 Immobilization of enzymes 1.2.1 Methods of immobilization

As early as 1910, the immobilization of enzymes was described, but it was not until the 1960s that the technology of enzymes immobilization was really developed [9,[START_REF] Nidhi | Nature Inspired Multienzyme Immobilization: Strategies and Concepts[END_REF], for example, Grubhofer and Schleith used polyaminostyrene resin as support, then after activation of the support by diazotization, they were able to immobilize carboxypeptidase, amylase, pepsin and ribonucleic acid. In general free enzymes are usually delicate and extremely sensitive to the change of external environment, the immobilization improved the stability or catalytic performance of enzymes and allowed their re-use thus reducing production costs. But retention of geometric configuration is a significant factor, and immobilization on a support brings another risk that enzymes lose the original geometry or its specific functions in some cases. However, numerous studies [START_REF] Mahboobi | 2-Aroylindoles and 2-Aroylbenzofurans with N-Hydroxyacrylamide Substructures as a Novel Series of Rationally Designed Histone Deacetylase Inhibitors[END_REF] have shown a positive impact of immobilization on supports due to the chemical or physical properties induced by the support, such as shape, size or porosity.

The immobilization of enzymes on organic or inorganic solid materials is based on the principle of incorporating or binding enzymes to the surface or in the micropores of materials, while retaining the catalytic activity of enzymes. In addition, immobilization of whole cells has also been reported by scientists [START_REF] Ebrahim | Preparation and characterization of monosubstituted porphyrins immobilized on nanosilica[END_REF]. In this latter case, these "living" biocatalysts convert nutriments into products by their own metabolism. Enzymes immobilization makes it possible to conserve or improve their properties, such as the stability and catalytic activity, also to increase the re-use abilities and reduce the cost of overall processes. In general, the procedure of immobilization can be divided into several categories (Fig. 1.2):

reversible binding (physical adsorption), entrapment (encapsulation), cross-linking, covalent bonding (usually irreversible), or carriers-free immobilized enzymes, i.e.

cross-linked enzyme crystals (CLECs) [START_REF] Roy | Strategies in making cross-linked enzyme crystals[END_REF], and cross-linked enzyme aggregates (CLEAs) [14].

Fig. 1.2 Various methods of enzyme immobilization [START_REF] Ahmad | Enzyme Immobilization: An Overview on Nanoparticles as Immobilization Matrix[END_REF] Adsorption at the surface of a material is the oldest and the most widely enzymatic immobilization method. This procedure is generally reversible due to the weak interaction forces involved such as van der Waals interactions, hydrogen bonding, hydrophobic interactions, electronic affinity or electrostatic binding. It can allow recycling supporting materials when enzymatic activity decreases. Solid supports commonly used include activated carbon, alumina, diatomaceous earth, porous ceramics, porous glass, silica gel, hydroxyapatite, etc. This method has industrial potential because solid supports are abundant and inexpensive, re-used easily, and do not denature enzymes. However, due to the physical adsorption, the binding of enzymes and supports is weak, and the enzyme is not firmly bonded to the carrier [15] [16] [17].

An enzyme or an enzyme-containing cell is buried in various porous carriers to immobilize an enzyme, which is called embedding, encapsulation or entrapment.

The porous carrier used mainly includes agar, agarose, sodium alginate, carrageenan, gelatin, polyacrylamide, resin, polyamide, collodion, etc, [START_REF] Bilal | Naturally-derived biopolymers: Potential platforms for enzyme immobilization[END_REF].

Depending on materials and methods, it is divided into two types: sol gel-embedding and microencapsulation. For the sol gel-embedding, enzyme molecules are encapsulated in a high polymer lattice, or directly embedded in a bead polymer. The mechanical strength of the immobilized enzymes increases and prevents the shedding of enzymes. In fact, microencapsulation is the encapsulation of an enzyme solution or suspension in a membrane that allows enzymes to be entrapped in a cell-like environment while preventing an enzyme from escaping or directly shield the microencapsulated environment, and finally products can diffuse through the membrane. Therefore it has great possibilities in the industry, like in medicine, and it is getting more and more attention from scientists. Besides that, this process does not involve the conformation and chemical changes of an enzyme due to a mild synthesis condition. However, this technology still exhibits some defects, such as enzymes leakage, diffusion limitation, the mass transfer resistance, and it is not suitable to a macromolecular substrate.

Immobilization also can be achieved in virtue of chemical bond, namely chemical covalent binding by covalent bond or an ionic bond (fig. 1.3). Formation of covalent bonds comes from functional groups on an enzyme molecule and reactive groups on the surface of solid supports, and common used carriers include natural polymers and synthetic polymers such as cellulose, agarose, starch, glucose gel, collagen, and nylon, polyamino acids, ethylene-maleic anhydride copolymers, etc. and inorganic supports (porous glass, metal oxide, etc.) [19]. Owing to an enzyme firmly linked to a carrier, the enzyme immobilized by covalent binding is not easy to fall off, exhibiting good stability and reusability, and has become one of the most widespread immobilizations. Dessouki [20] reported the immobilized pullulanase by covalent binding processes a stable activity and a longer durity, just with only a slight loss (about 20 %) after 10 times recycle, compared to the cross-linking technique whose catalytic activity declined due to a heavy leakage. For chemical attachment, an active group is essential pre-requisite, such as amine, thiol, carboxylate, etc. However, irreversible attachment also leads to violent loss of the activity of enzymes and unrecyclable carriers.

A bifunctional or multifunctional chemical cross-linker, such as glutaraldehyde, adipaldehyde, maleic anhydride, bisazobenzene is used to react with the active group of an enzyme to form an insoluble network-structure to immobilize enzymes, which is called cross-linking. In addition, for enzymes with better thermal stability, by heat-treating the enzyme-containing cells at a certain temperature to fix an enzyme in cells is also used in combination with cross-linking or other immobilization methods. However, due to intense reaction conditions, this method will result in a low activity, difficulties in handling, etc. In general, non-specific conjugation is more widely employed because these reactions are easily performed under mild conditions, and the crossing-linkers are commercial, or easily synthesized. In non-specific conjugation, coupling agents including maleimides, glutaraldehyde, carbodiimide, epoxides, etc are introduced to the supporting material afterwards to corresponding side groups of the biomolecule, such as, -NH 2 , -OH, -COOH, -SH, etc. However, enzymes attached at a supporting surface may not control the orientation, in that case, enzymes are immobilized onto the surface via non-specific conjugation due to a large number of functional groups. In addition, the active group of enzymes, such as amino acid, which contribute to the high activity, would be depleted by crossing-linking reagents. Therefore, generally, for specific bioconjugation, proteins or materials always have a particular binding site to optimize the orientation of enzymes. In that case, if the active site of an enzyme is not available to a substrate, the efficiency of catalysis would be dramatically cut down.

The principle of developing a novel enzyme immobilization is to achieve

Supporting materials for enzymes immobilization

A wide switch was made to the techniques that surface chemistry boosts greatly the supporting material for enzyme immobilization, generally supporting materials are modified instead of enzymes, because the properties of materials can influence the loading capacity, activity, conformation, stability, recycle of immobilized enzymes, besides, which is also critical to reduce cost in industrial production.

Thereby, an ideal supporting material should be suitable, re-useable, stable, bio-compatible, and should also allow an easy separation of complex with the reactor. Indeed, the nature of supporting materials, such as size, surface charge, topography, hydrophobicity or hydrophilicity, has a significant effect on an enzymatic immobilization and its activity due to the interaction (proximity) of enzymes with the support [START_REF] Sharifi | Enzyme immobilization onto the nanomaterials: Application in enzyme stability and prodrug-activated cancer therapy[END_REF]. Also, it influences the amount of immobilized enzymes (the enzyme carrying capacity), and to induce a high enzymatic charge, it is important to be able to control the specific surface of a support, for example increasing the porosity of porous materials or reducing the particles' size. Many types of support matrix have been reported by scientists and are available such as gels, resin, mesoporous silica particles, composite material, capsules [1] [24]. The development of new carrier materials for immobilization, or surface modification of supporting materials remains a major issue for immobilization of enzymes or proteins. Nowadays, many supporting materials have been used in the industrial production.

A-Inorganic porous carriers

According to definition from the International Pure and Applied Chemistry (IUPAC) materials with pore sizes include: microporous materials (less than 2 nm), mesoporous materials (2-50 nm), macroporous materials (50-200 nm), and gigaporous materials (more than 200 nm). Generally a protein molecule would be attached onto the surface, or buried inside the porous materials, and chelated in the ports. Porous materials processing specific properties, such as high surface areas, hydrophobic/hydrophilic surface offer an extensive potential for enzymes immobilization. For example, Bellino [25] produced a robust biocatalyst prepared by immobilization of α-amylase on a mesoporous thin film as a supporting matrix, leading to an easy separation and reuse-ability. The experiment of starch degradation catalyzed by the catalyst exhibited a wonderful activity, and an enhanced stability at different pH and temperature, compared to free enzyme. In addition, nanotechnology and surface chemistry emerged and promoted the material modification, particularly for porous materials, or carrier with nanoscale structure and size. A variety of inorganic matrix has been used for enzyme immobilization, such as silica, glass beads, zeolites, mesoporous silica, etc, [26], among them, silica has been favored by researchers due to its rich-availability, low-cost, and nano-scale silica or mesoporous silica has higher surface energy and biocompatibility, and has promising potentials for enzyme immobilization. In general, a mesoporous material which has a slightly larger pore diameter than the diameter of an enzyme is selected as a carrier, and after an enzyme is absorbed into the pore, the orifice is greatly reduced by a silanization to prevent enzymes leakage. In addition, mesoporous molecular sieves are also suitable for enzymes immobilization due to high specific surface area, nano-sized pores, and large pore volume. Therefore, immobilized enzymes by physical adsorption with molecular sieves as carriers exhibit high catalytic activity, mainly electrostatic or hydrophilic/hydrophobic interaction.

MCM-41 is a representative, which contains a narrow pore size distribution, usually possessing a large internal surface area. MCM-41 displayed a large potential as supports with specific pore diameters, high surface area, strong stability to immobilize enzymes, besides, many functionalizations of their surface were proposed with silane precursor, functional groups, such as -NH 2 , -COOH for the enzyme immobilization, which could modulate the microenvironment of immobilized enzymes, improve the affinity between enzymes molecule and carriers to increase the immobilized enzyme activity. P-or Ca-modified MCM-41 materials with suitable pore diameters and large pore volumes was produced by Yamashita Hiromi [START_REF] Yasutaka | Enhancement in Adsorption and Catalytic Activity of Enzymes Immobilized on Phosphorus-and Calcium-Modified MCM-41[END_REF] to immobilize enzymes, leading to increased enzyme absorption. The experiment exhibited the modification of silica surface with P-or Ca-could offer great opportunities of enzyme adsorption due to the strong negative charge of the surface or the specific interatomic interaction between these atoms and the enzyme's functional groups, in addition it could increase the adsorption efficiency, high stability in a wide pH range and storage stability, low-leaching. In many real industrial applications, the spatial distribution of enzymes on a supporting matrix will also influence the final catalytic activity of the 'complex' in the reaction medium, for those spherical solid matrix, it exhibit fair significance [START_REF] Mei | Imaging the Distribution and Secondary Structure of Immobilized Enzymes Using Infrared Microspectroscopy[END_REF].

In addition, the hydrophobicity or hydrophilicity of a solid matrix is another factor. Let's take lipases adsorption for example to explain how hydrophobicity or hydrophilicity of a supporting material could influence the catalytic activity of immobilized enzymes (see fig. 1.6). A large number of literatures [29] disclosed when the lipase was immobilized onto a hydrophobic surface of carriers, the immobilized enzyme exhibits a highly hyper activated catalytic activity. In fact, lipase is a group of enzymes, a famous star in recent years to produce the biodiesel, which can catalyze hydrolysis of ester bonds to obtain organic acids and alcohols in industry, according to the literature [START_REF] Mateo | Improvement of enzyme activity, stability and selectivity via immobilization techniques[END_REF], lipase generally exhibits two conformations, an active form, where the active site is exposed to external medium, and an inactive form, where the active site is surrounded by the polypeptide chain. When it is attached on a hydrophobic carrier in a mild condition, the special preference in open form would allow lipase a high activity. In other words, if a native lipase exists in a medium, it may work in two patterns, 'lid', or 'flap', an equilibration could occur between these two forms, in lid form which the lipase' catalytic site is blocked and can't work in a medium, whereas in another transition state 'flap', the 'lid' of lipase disappear, and the catalytic site is surrounded by the medium and can catalyze a reaction effectively, which is considered as an "active" form. Therefore, the activity of immobilized enzymes may be modulated by using different hydrophobic supporting materials, when lipase is immobilized on hydrophobic supports, a controlled structural shift would occur and lead to shift of the equilibration. And in a case of lipase used in chiral organic synthesis, Zhang [START_REF] Zhang | An efficient resolution of racemic secondary alcohols on magnetically separable biocatalyst[END_REF] prepared hydrophobic magnetic siliceous mesocellular foam functionalized with octyl groups as a support for lipase adsorption. They found immobilized lipase had a better efficient resolution of secondary alcohols, which is about four times higher activity than that of a free lipase, they believed these results are probably due to the ''interfacial adsorption'' and ''hyper-activation'' of lipases on the hydrophobic surface of the magnetic siliceous mesocellular foam. Jose M.Guisana [START_REF] Cabrera | Novozym 435 displays very different selectivity compared to lipase from Candida antarctica B adsorbed on other hydrophobic supports[END_REF] compared Novozym 435 and lipases immobilized on Lewatit VP OC 1600, with CAL-B immobilized in other hydrophobic supports (agarose coated with butyl or octyl groups and sepabeads coated with octadecyl groups), which showed the immobilized enzyme may involve different interactions to produce the catalytic changes.

B-Organic carriers

Compared to inorganic carriers, organic supports usually possess larger pore size, offering a high loading, or enhanced activity and high stability. Moreover, the large pore size and high surface areas probably reduce diffusion. Nowadays, a great number of natural or synthetic organic polymers are available as carriers for enzyme immobilization in industrial productions. Various natural organic materials have been widely used for enzyme immobilization, such as agar, agarose, chitosan, cellulose, dextran, alginate, gelatin, collagen, etc [START_REF] Eş | Principles, techniques, and applications of biocatalyst immobilization for industrial application[END_REF]. Agar is commonly used for cell encapsulation due to a proportional pore size, but it is restricted with weak mechanical properties. Chitosan is abundant in many resources, for example, fungi and crustacean shells, and have been used for biotechnology, pharmaceutical, food.

Gelatin is also inexpensive, abundant, and bio-safe material in addition to its high hydrophilicity and strong swelling power. Collagen is a potential support for immobilization due to its biocompatibility, abundant sources, and strong attachment to enzymes, whereas, the challenging job is its high cost [START_REF] Eş | Principles, techniques, and applications of biocatalyst immobilization for industrial application[END_REF]. In order to achieve miehei and Humicola sp. The result is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinity for the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted. Mitsuo Hirata [34h] immobilized urease onto HEMA-and HEA grafted ePTFE films, which investigated the activity of urease covalently immobilized onto films. The relative activity of immobilized urease maintained almost constant in the range of the immobilized amounts between 10 and 30 mg/g for both grafted ePTFE films, and decreased at higher immobilized amounts because of the crowding of immobilized urease molecules in the grafted layers. They speculated the further increase in the grafted amount resulted in decrease in the relative activity, because an increase in the immobilized amount led to the crowding of urease molecules in the PHEMA layers, resulting in spatial restrictions, blocking of active sites, and denaturation of enzymes.

C-Nanomaterials

In recent years, nanotechnology developed rapidly and special properties of nano-materials have attracted a great interest from scientists as carriers for enzymes immobilization. Nanomaterials can exhibit good biocompatibility, large specific surface area, small particle diameter, small diffusion limit, high enzyme loading and high stability in solution, generally including nanoparticles (nanospheres, magnetic nanoparticles, nanocapsules), nanofibers (nanotubes, nanowires), nanofilms, nanocomposites, nanorods, nanoblocks and nano-size sol-gel materials, etc.

Numerous studies were reported about the enzyme immobilization on nanomaterials or nano-scale materials [35], and a great interest has been attracted by the considerable advantages nanomaterials have for its huge specific surface area, high loading capacity, controlled spatial distribution, many unique physical and chemical properties and biological characteristics, which are widely used in many fields of biology, such as drug carrier. Among them nanoparticles (NPs) are one of the most common nano-carriers used for enzyme immobilization, which generally refer to particles with a spherical or capsular structure with a size ranging from 1-1000 nm. Nanoparticles used as carriers could also bring about high activity and high mobility of complex catalysts in medium due to a smaller particle size, compared to the conventional carriers or other supporting matrix with big scale. For magnetic nanoparticles (MNPs), we will discuss in detail later, for nonmagnetic nanoparticles, such as Au, Ag, TiO 2 , ZrO 2 , etc, they exhibit ideal supporting matrix for enzyme immobilization due to specific surface areas, mass transfer resistance, and effective enzyme loading, etc. Zhang et al [36a] successfully immobilized deaminase on gold nano-particles (Au NPs) and confirmed the linkage between them by labeling, which shown the complex of deaminase immobilized on Au NPs still has good stability and catalytic activity. Hong et al [36b] found that α-chymotrypsin bound to the modified surface of nano-gold particles exhibits a strong affinity for substrates with positive charges under the combined action of steric hindrance and static electricity, and has a high catalytic activity and exhibits low affinity and catalytic activity only for substrates with negative charges, and has a good affinity and catalytic ability for neutral substrates. By contrast to porous materials with large surface area, minimizing the size of supporting matrix is preferred for some applications, nano-complex catalysts are well dispersed in the medium exhibiting brownian movement, to some degree, the complex catalyst is like a 'native' enzyme, exhibiting a high activity [11] [15]. In a case of lysozyme immobilization reported by Vertegel et al [36c], when the lysozyme was immobilized onto a smaller particle, the less structural change occurred, which demonstrated enzymes immobilization onto the smaller particles may lead to retention of activity. To increase the possibility of collisions of enzymes and substrates, scientists [37a, b] disclosed that the size of NP affects the catalytic activity, and smaller NPs increase their kinetic affinity for a substrate to promote the catalytic efficiency through the effects of particle size, shape, and properties of nanoparticles on kinetics of the immobilized enzyme. Compared to a direct enzyme-surface immobilization, to increase the frequency of collision of a catalyst and a substrate, a suitable ligand like amino acid, glutaraldehyde, etc. can be introduced by functionalizing carriers or modifying an enzyme, which could increase the chemical flexibility of immobilized enzymes, adjust the surface charge, expose the active site for substrates, and modulate the polarity and hydrophilicity.

Dordick [37c] immobilized perhydrolase S54V (AcT) onto multiwalled carbon nanotubes to produce bioactive composites, which was coated with amphiphilic poly (ethylene glycol) (PEG) as an effective linker, which is known to reduce nonspecific interactions, will not decrease the solubility of carbon nano-tubes and can enhance enzyme activity due to improved surface hydrophilicity. They found the PEG spacer was effective in increasing the specific activity of the conjugates from 7 % to 24 % and exposed the active center against hydrophobic surface.

Sardar [START_REF] Ahmad | Peroxidase-TiO 2 nanobioconjugates for the removal of phenols and dyes from aqueoussolutions[END_REF] prepared a bionanoconjugate through covalent binding peroxidase on TiO 2 nanoparticles (< 25 nm), which was activated with aminopropyl triethoxy silane. The biocatalyst could retain 83 % activity even after several times compared to a free peroxidase. Besides, the experiment showed the conjugate exhibited excellent catalytic properties, a high efficiency in removal of phenols and dyes from aqueous solutions, and showed an enhanced thermal stability without loss in the activity even at 60℃. For other forms of nanomaterials, like carbon nanotubes, seamless nanotubes made of graphite sheets. They can be combined with biomolecules, such as proteins, enzymes, and DNA on the surface or port of carbon nanotubes, which can improve their solubility in aqueous solution. Secondly, the inner surface of carbon nanotubes has strong interaction with biomolecules, which can maintain its configuration stability and retain considerable catalytic activity, and also can maintain thermal stability and tolerance to pH [START_REF] Cao | Immobilised enzymes carrier-bound or carrier-free[END_REF].

D-'Smart' materials

With the advent of new materials, more and more 'smart' materials are being applied to enzyme immobilization, 'smart' materials, as the name suggests, whose properties undergo change due to the external environment collectively in response to temperature, pH, magnetic field, ionic strength, etc. A series of smart polymer materials are preferred as carriers for enzymes immobilization, compared with traditional carriers, in terms of maintaining enzyme activity, increasing enzyme loading, and improving operational stability. Temperature-sensitive gel carriers:

temperature-responsive polymer hydro-gels are smart materials that respond to changes in the external temperature by changing their thermal properties. At present, temperature-responsive hydro-gels in immobilized enzymes mainly focus on high-temperature gels that undergo shrinkage and volume-phase transition with temperature changes, take poly-NIPAM for example, it is a thermo-responsive and biocompatible polymer, when below 32℃, the polymer can dissolve in water, and it can catalyze a reaction, whereas above 32℃, it will be insoluble due to dehydration from the polymer. Thereby, increasing the temperature above the critical point will lead to the recovery and reuse of immobilized enzymes [40], for example, a temperature-sensitive hydrogel carrier, which can achieve homogeneous and heterogeneous separation when immobilized by thermolysin through copolymerization of N-isopropylacrylamide and its derivatives. Yu et al [START_REF] Yu | Stability Properties of Thermoresponsive Poly(N-Isopropylacrylamide)-Trypsin Conjugates[END_REF],

covalently coupled trypsin to a thermo-sensitive polymer N-isopropyl acrylamide (PNIPAM) via mixing NAS into the polymer and enzymes, which retains higher activity than the enzyme coupled to PNIPAM-NAS (conjugated II) prepared by other methods. Cicek et al [START_REF] Cicek | Immobilization of α -chymotrypsin in thermally reversible isopropylacrylamide-hydroxyethylmethacrylate copolymer gel[END_REF] disclosed an embedding of α-trypsin by a thermo-sensitive copolymer carrier, isoacrylamide/hydroxyethyl isobutyric acid, when the thermo-sensitive gel exhibits linear change responding to temperature, the activity of immobilized enzymes was high, after the enzyme was used repeatedly after 30 times, the catalytic activity did not decrease significantly; pH-sensitive carrier, which can be soluble in medium, such as water, compared with a conventional carrier, pH-sensitive hydro-gels usually have acidic groups, such as carboxylic acids, sulfonic acid group, or a alkaline group, such as an amino group, when the environmental pH varies, proton transfer and volume-phase change occurs in the hydro-gel under interaction of electrostatic repulsion, hydrophobic force and hydrogen bonding. Totally, the pH-sensitive phase-separated polymer has a critical pH value, which can be reversibly dissolved-precipitate by adjusting the pH value of a solution. Multiple responsive carriers, multiple smart functional materials, are capable of responding to two or more two external stimuli simultaneously, such as light-pH response, magnet-temperature response, pH-temperature response.

E-Plasma

Plasma polymerization and plasma surface treatment were used to prepare carriers for enzyme immobilization [43a]. In general plasma polymerization is utilizing radio or microwave to convert organic monomers into plasma states to produce various active species (free radical), then a reaction occurs between the active species or between the active species and the monomer, and a coating is deposited on the surface of a material; Plasma surface treatment is to expose a material to a plasma of a non-polymerizable gas (such as N 2 or an inert gas, etc.) to produce specific functional groups on the surface (such as -OH, NH 2 , etc.). The two reactions above can modify the surface and structure of a material used for immobilization. For a carrier after the plasma polymerization or surface treatment, enzyme immobilization is achieved via physically adsorbing, encapsulate or cross linking, covalently immobilizing.

F-Ionic solution

Compared with other traditional methods, ionic liquid technology opens up new ways in enzyme chemistry as an important tool to promote enzyme reaction and separation and recovery process. Ionic solution is a new green solvent, which has many advantages in biocatalytic reactions, such as good selectivity, reactivity and stability of enzymes in ionic solutions. An ionic solution is usually an organic molten salt with a low melting point composed of organic positive ions, such as an alkylpyridinium ion, a quaternary ammonium salt ion, an alkylimidazolium ion and different negative ions. In general, it would exhibit a liquid state at room temperature or under low temperature. Usually an ionic solution, water, and conventional organic solvents are mutually soluble, but can't be insoluble with most ether, alkanes. Therefore, the product can be separated by extraction, and the enzyme and ionic solution can be reused. The polarity and hydrophilicity can be adjusted by changing the length of the alkyl carbon or the positive and negative ions.

Ionic liquids, or their combined use with other enzyme immobilization technologies, may provide new alternatives for enzyme-catalyzed synthesis in an environmentally friendly way [43b, c, d].

Enzymes immobilization on magnetic nano-particles (MNPs)

The boom of nanotechnology and nanomaterials facilitates enzyme immobilization, but the nano-enzyme biocatalyst would lead to defects, such as tiny size, difficult manipulation, potential risks to environment, which could suspense in the air or water and be hard to clean up. Compared with other nanomaterials, magnetic microspheres can overcome many disadvantages, which generally contain a magnetic metal or a metal oxide (such as iron, nickel, cobalt and an oxide or an alloy) as a core, which exhibit magnetic responsiveness, and a shell which is generally composed of organic or inorganic matrixes by embedding or polymerization, such as polymer, silica or carbon (fig.1.7). They are new promising functional materials in the past two decades, which have been widely employed in biology and medicine by scientists due to a large number of advantages, such as, 1) easy separation and recovery in a reaction system to reduce the cost; 2) when the functional magnetic carrier immobilized with targeted molecules, such as antibodies, proteins, dyes is placed in fluidized bed reactor, a external magnetic field can facilitate a simple operation in a continuous reaction system; 3) The external magnetic field is used to control the movement and direction of an enzyme, which improves the catalytic efficiency; 4) Numerous various functional groups are available to be combined with various functional molecules on the surface of magnetic nanoparticles through surface modification (such as -OH, -COOH, -CHO, -NH 2 and -SH, etc), which make it easy to attach covalently proper enzymes onto the surface. In addition, the magnetic microspheres have high large specific surface area, a high density of functional groups, and stable physicochemical properties, good mechanical strength, etc. Hence they have been applied in the field of immobilization of enzymes.

In and whose shell was considered as a platform to be functionalized to bind enzymes.

Compared to traditional carriers, magnetic nanoparticles exhibit high performance, supermagnetism properties, high mass transfer, large surface areas, etc. Moreover, larger specific surface areas can effectively increase loading of enzymes; particle with smaller diameter and small diffusion restriction can exist stably in solution. In addition, the external magnetic field can provide an efficient recovery, thereby improving purity of products and avoiding contamination of the final product.

Numerous studies have been reported about the preparation and functionalization of magnetic particles [45] [46]. To avoid agglomeration, organic polymer are usually employed to form one layer or two layers by coating chemically or physical adsorption, not only to keep native metallic particles in a stable state, but also the polymer processing functional groups allow to bind targeted molecules and avoid denaturation of biomolecules. Chu [START_REF] Pierre | Preparation and Properties of Magnetite and Polymer Magnetite Nanoparticles[END_REF] reported a synthesis of polymer-magnetic particles with core-shell structure in a single inverse microemulsion. The particles with a narrow size distribution and superparamagnetism were produced by the two-stage microemulsion process and the seed precipitation polymerization, and the shell came from a copolymerization of methacrylic acid, hydroxyethyl methacrylate, and cross-linker. In spite of enhancing stability and biocompatibility covered with polymer organic matrix, particles with organic polymer are not well stable in the air or in acidic solution or at high temperature, and the polymer is not strong enough to protect the super-active metal species. In contrast, inorganic matrix can avoid these problems, which can protect the superparamagnetic nanoparticles core against oxidation and aggregation. Silica is to be preferred, which ensures the presence of easily functionalized groups-silanol on the surface of carriers and offers a high stability on suspensions of particles. Pedro Tartaj [START_REF] Valde´s-Solı´s | Preparation, Characterization, and Enzyme Immobilization Capacities of Superparamagnetic Silica/Iron Oxide Nanocomposites with Mesostructured Porosity[END_REF] prepared a magnetic nanocomposites coated with a silica shell, processing a high surface area and high pore volume values, displaying good magnetic properties and lower size dispersion.

Moreover, these nanocomposites are able to immobilize large amounts of lysozyme.

The loading of lysozyme varies significantly with the pH, exhibiting a maximum at pH 10. The FT-IR spectrum obtained for the composite loaded with lysozyme

shows that the structure of this biomolecule is preserved after its immobilization. Li

Qi et al. [START_REF] Ma | Poly(2-Vinyl-4,4-dimethylazlactone)-Functionalized Magnetic Nanoparticles as Carriers for Enzyme Immobilization and Its Application[END_REF] also investigated immobilization of L-asparaginase on magnetic particles of Fe 3 O 4 functionalized with Poly(2-Vinyl-4,4-dimethylazlactone) as an enzyme immobilization carrier. The surface of Fe 3 O 4 is coated with a layer of silica to protect the surface of particles, prevent particles from collapsing, and also improve the surface modification of particles and reduce potential toxic effects.

Apart from the shell of magnetic nanoparticles, the particle size directly affects the interaction of enzymes and nanoparticles. The increase in particle size can facilitate filtration and stability but the large size will result in diffusion limitation.

In contrast, small particles (10-100 nm) are preferred for industrial production which generally operated in fluidized bed, where a substrate passes through a solid catalyst. Natalia et al. [50] investigated the thermal stability of α-galactosidase through an effective immobilization onto Fe Sachin Talekar et al [START_REF] Talekar | Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase[END_REF] reported that when α-amylase was immobilized with novel magnetic cross-linked CLEAs particles, its affinity to substrates was enhanced, and the thermal stability of the enzyme was also improved, even after storage for 42 days, it could maintain nearly 100 % activity, even after 6 cycles of reuse.

Gardimalla et al. [START_REF] Gardimalla | Superparamagnetic nanoparticle-supported enzymatic resolution of racemic carboxylates[END_REF] investigated the stability of lipase immobilized on Fe 2 O 3 magnetic nanoparticles, and immobilization on Fe 2 O 3 magnetic nanoparticles could achieve better reusability than free enzymes. Zhi Li et al [START_REF] Ngo | Efficient transformation of grease to biodiesel using highly active and easily recyclable magnetic nanobiocatalyst aggregates[END_REF] reported a highly active and easily recyclable magnetic nanobiocatalyst aggregates (MNA) through covalent attachment with a lipase (TLL or CALB) on MNPs, which exhibited a high yield (98 %) with high enzyme loading, high catalytic activity, and fast and easy separation under magnetic field. The general mechanism is (fig. 11) : when a substrate is oxidized, first Type However, substrates that are too large or have a high Eo can't be directly catalyzed by laccase, thence redox mediator are required to help achieve a catalytic reaction, for example, lignin is a natural aromatic polymer composed of polymerized aromatic alcohols, which is renewable resource that exists in nature but is difficult to degrade. It is also a main reason for blackening the color of wastewater from paper mills and textile mills and is one of the important sources of pollution. Laccase can't break the non-phenolic bonds of lignin, it is not considered as an important member of the lignin degradation system until 1990 that Bourbonnais reported [START_REF] Bourbonnais | Oxidation of non-phenolic substrates An expanded role for laccase in lignin biodegradation[END_REF] that the oxidation of non-phenolic substances was achieved by the successful laccase mediator system by ABTS as a mediator in the degradation of residual lignin in unbleached pulp. The laccase mediator system ("LMS") (fig. 1.12) is composed of laccase and a mediator in a mechanism that it is continuously oxidized by laccase to produce free radical species, then subsequently reduced by a substrate acting as "electron shuttle" during a catalytic process. Until now, about 100 molecules can act as mediators, such as ABTS, TEMPO, HBT, but mediators follow different catalytic mechanism depended on the (Eo) of substrates and their structure [58]. Though the mediators could accelerate the oxidation process, increase the types of substrates, and expand the application range, such as dye or lignin degradation, the restricted applications of LMS arises from the high cost and unsafety, like the active radical species may inactivate laccase in some cases. Therefore, more safe and eco-friendly mediators are in need for laccase applications.

Laccases and its immobilization 1.3.1 Introduce of laccases

Nowadays, laccase has also been found in fungi, plants, prokaryotes, etc, especially in large fungi such as basidiomycetes, ascomycetes and deuteroms. In addition, it has also been found in some animals, but the main sources of laccase are fungi and bacteria, and bacteria laccase has a high yield with a low Eo, but fungi laccase offer a low yield with a high Eo. At present, heterologous gene expression of laccase is an effective method, and a variety of proteins have been put into industrial production through heterologous gene expression, such as penicillin, invertase, etc. To increase laccase production, funga laccases have been successfully

heterologously expressed in various organisms, such as saccharomyces cerevisiae, trichoderma reesei, etc [59].

Laccases immobilization

However, its low stability and poor reusability hamper the further industrial applications of laccase. Thereby, immobilization technology offers an effective alternate to improve the stability of laccase and increase the potential industrial application, a lot of studies of laccase immobilizations have been reported by scientists, including a considerable amount of nanomaterials-carbon nanofibers, nanotubes, nanoparticles, carbon membrane, carbon capules, resin membrane, organic frameworks, polymers, etc [59]. As we expect, the immobilization technology could offer laccase a good operational performance, high stability and activity, high resistance to external factors, several examples are summarized as below. Radmila Tomovska [START_REF] Ormategui | Design of Stable and Powerful Nanobiocatalysts, Based on Enzyme Laccase Immobilized on Self-Assembled 3 D Graphene/Polymer Composite Hydrogels[END_REF] reported efficient nanobiocatalysts via covalent immobilization of laccase on graphene oxide/polymer due to its peculiar properties and multifunctionality, which open a completely new area of investigation as a support matrix to immobilize various enzymes. The biocatalysts showed strong dye discoloration ability and high stability in four successive batches of dye degradation in the oxidative degradation of the recalcitrant synthetic dye Remazol Brilliant Blue R in aqueous solutions. Enzymatic degradation showed laccase is a potential green solution for removal of pharmaceutical compounds in waste water. Surampalli [START_REF] Taheran | Covalent Immobilization of Laccase onto Nanofibrous Membrane for Degradation of Pharmaceutical Residues in Water[END_REF] investigated biocatalysts through laccase was immobilization onto composite nanofibrous membrane through covalent bonding in the removal of chlortetracycline (CTC), carbamazepine (CBZ), and diclofenac (DCF), which showed that the immobilized laccase has improved storage, temperature, and pH stability, compared to free laccase and it maintained almost 20 % of its initial activity after 10 cycles of ABTS oxidation, compared to conventional physical or chemical processes. Zhu [START_REF] Zhang | Encapsulation of Laccase in Silica Colloidosomes for Catalysis in Organic Media[END_REF] reported the encapsulation of laccase solution in silica colloidosomes of different shell structures and the catalytic performance of the encapsulated enzyme in toluene. The encapsulated laccase exhibits catalytic activity and reusability that are controlled by varying the weight ratio of silica particles to water. With the increase of Rs/w (the weight ratio of silica particles to water), the reusability of the enzyme improves, meanwhile, its activity declines. This method allows fabricating enzyme microcapsules with tailored activity and controlled releasing properties. Jing Gao [START_REF] Zheng | Biomimetic Synthesis of Magnetic Composite Particles for Laccase Immobilization[END_REF] reported an immobilized biocatalyst prepared by Fe 3 O 4 nanoparticles entrapped in 3-aminopropyltriethoxysilane (APTES). The composite particles were used to immobilize a kind of valuable bulky industrial enzyme (laccase). The results showed that the highest specific activity of immobilized laccase reached to 224 U and the activity recovery was 83.9 %. The good catalytic activity of the immobilized laccase was also demonstrated by the degradation of two phenolic substances. Sahu [START_REF] Samui | One-pot synthesis of microporous nanoscale metal organic frameworks conjugated with laccase as a promising biocatalyst[END_REF] reported they have developed amine-functionalized nanoscale metal organic framework (NMOF) NH 2 -MIL-53

(Al) as a support for laccase immobilization, which possess highest stability and activity at pH 4-6 by co-precipitation. It is observed that 625 mg laccase is immobilized per 1 g of NMOF, and the immobilized enzyme showed good reusability, retaining 63 % of its initial activity after being reused 10 times, which highlights the development of NMOFs that contain more laccase and retain stability and activity. Guo [START_REF] Hu | Immobilization of laccase on magnetic silica nanoparticles and its application in the oxidation of guaiacol, a phenolic lignin model compound[END_REF] developed a novel magnetically separable laccase immobilized system, which was constructed by immobilizing Aspergillus laccase onto amino-functionalized magnetic silica nanoparticles (AF-MSNPs) with core-shell structure via a chemical cross-linking. A large immobilization capacity, activity recovery and broader pH and temperature have been demonstrated by the immobilized laccase, compared to free laccase, besides thermal stability, storage stability and operational stability were increased. The high catalytic efficiency of the immobilized system in catalyzing the guaiacol as a phenolic lignin model compound was investigated, suggesting the immobilized laccase to catalyze lignin is a very promising technology for further applications. Amin Rostami [START_REF] Rouhani | Preparation and characterization of laccases immobilized on magnetic nanoparticles and their application as a recyclable nanobiocatalyst for the aerobic oxidation of alcohols in the presence of TEMPO[END_REF] disclosed that laccase from Trametes versicolor was immobilized on modified magnetic nanoparticles through a covalent attachment, which retained the activity and exhibited a higher resistance to pH and temperature changes. MNPs-Laccase were used as a magnetically recoverable nanobiocatalyst for a mild, environmentally friendly and selective aerobic oxidation of benzylic and allylic alcohols to corresponding aldehydes in the presence of TEMPO as a redox mediator at room temperature. The magnetic nanobiocatalyst was easily and rapidly recovered by applying an external magnet device and reused up to 6 times without considerable loss of reactivity. Lee [START_REF] Sanjay | Large-scale aerosol-assisted synthesis of biofriendly Fe 2 O 3 yolk-shell particles: a promising support for enzyme immobilization[END_REF] developed multiple-shelled Fe 2 O 3 yolk-shell particles and intended as a suitable support for the immobilization of commercial enzymes, such as glucose oxidase (GOx), horseradish peroxidase (HRP), and laccase as model enzymes. After cross-linking of immobilized laccase by glutaraldehyde, immobilization efficiency was improved from 83.5 % to 90.2 %.

The thermal stability of the immobilized enzyme was enhanced up to 18-fold upon cross-linking, and the enzyme retained 93.1 % of residual activity after ten cycles of reuse.

To optimize the immobilization, the orientation of enzymes bound to the surface of carriers need to be documented. It is known that the distribution of enzymes is able to boost the efficiency of biocatalysts in applications, if an enzyme was immobilized onto the surface in a way that the active site could approach substrates.

In addition, substrate approachability can always be of importance to increase the efficiency or is influenced by particle movement, distribution of enzymes, particle size, surface charge groups, polarity, hydrophilicity, etc. Yu [START_REF] Shi | Removal of sulfonamide antibiotics by oriented immobilized laccase on Fe 3 O 4 nanoparticles with natural mediators[END_REF] reported a novel strategy in the oriented immobilization of laccase from Echinodontium taxodii on concanavalin A-activated Fe 3 O 4 nanoparticles based on laccase surface analysis.

These nanoparticles showed higher enzyme loading and activity recovery, compared to conventional covalent binding. Along with the improvement in thermal and operational stabilities, the oriented immobilized laccase exhibited higher substrate affinity than free laccase, and were applied in the removal of sulfonamide antibiotics (SAs), in which GAMNs-Con A-L showed a higher removal rate of SAs, compared with the free counterpart in the presence of S-type compounds present in lignin structure.

Applications of laccases

In recent years, in food, soil and water bioremediation, organic synthesis, pharmaceuticals, biosensors and bio-fuel cells, sewage treatment, biobleaching of pulp, environmental protection and biodegradation of toxic compounds, such as chlorophenols, laccase has attracted much interest due to an urgent need to develop effective methods of detection of phenols, through some available tools for measuring phenols have been employed, such as liquid chromatography, gas chromatography, etc. Compared to conventional testing instruments, a biosensor composed of laccases is simple, fast, facile to miniaturization in analytical equipment, which meets requirements for application to multicomponent solutions in situ. Since the only by-product of a laccase catalytic process is water, laccase has a very significant function on reducing greenhouse gas emissions and reducing energy consumption, and is increasingly favored by researchers. In 1996, the first commercially available laccase DeniLite® I was introduced to the market by Novozymes, followed by several laccase products. In 2007, Genencor launched the Prima Green® EcoFad LT100 and IndiStarTM ColorAdjust, ECOSTONE® LCC10 was introduced AB Enzymes and Zylite was introduced from Zytex for textiles.

Sigma also has several laccase products. Increasing production and lowering costs are problems that need to be solved in the industrial production of laccases.

A-Organic synthesis

The laccase-catalyzed organic synthesis is more and more promising for organic chemists as a substitute for conventional chemical synthesis due to its simple operation, mild reaction conditions and less toxic by-products to form C-C bond, C-N bond, C-O bond, C-S bond, etc, however, only few studies were reported about the laccase-catalyzed organic synthesis, such as synthesis of aromatic aldehydes, substituted imidazoles, enzymatic oxidation of alkenes, etc [70]. Rutin is an antihypertensive drug, which could be produced [START_REF] Kurisawa | Laccase-catalyzed Synthesis and Antioxidant Property of Poly(catechin)[END_REF] by using laccase, which greatly improved the ability to scavenge superoxide radicals and inhibit the oxidation of human low-density lipoproteins. It can also catalyze the oxidation of phenolic or aromatic amine to synthesize new compounds, such as various drugs, amino acids, new antibiotics, peptides and other aspects. As an ultraviolet absorbent, benzotriazole (Tinuvin) is a very important light stabilizer, which has a very important application in prolonging the life of polymers and pigments, G.M.

Guebitz disclosed the production of tinuvin through coupling agent of methyl 3-(3-tert-butyl-4-hydroxybenzene) propionate and excessive 1H-phenylpropionate by laccase via C-N coupling [START_REF] Schroeder | Enzymatic synthesis of Tinuvin[END_REF]. In addition, laccase is used to catalyze an amination of hydroquinone, which is of great importance in the synthesis of new antibiotics and amino acid derivatives. For example, laccase was used to catalyze the amination of catechol and amino-β lactams to synthesize new cephalosporins, penicillins. Niedermeyer et al. [START_REF] Niedermeyer | Nuclear Amination Catalyzed by Fungal Laccases: Reaction Products of p-Hydroquinones and Primary Aromatic Amines[END_REF] used Trametes Sp and M. thermophil laccase to catalyze the reaction of hydroquinone and aromatic amines to synthesize diamine products at room temperature. In addition, the laccase-mediator system can also oxidize some non-phenolic functional groups, such as hydrocarbyl, ester, amine, etc, for active alcohols, such as benzyl alcohol, aromatic alcohols, propargyl alcohols and fatty alcohols, which are oxidized to aldehydes or ketones by laccases.

B-Industrial treatment

Compared to conventional chemical bleaching, biotechnology is used to delignification to efficiently increase the degradation of residual lignin. A large amount of toxic and harmful substances is generated in wastewater, leading to serious pollution and the residual lignin in the pulp deteriorates the paper quality.

The wastewater is treated by immobilized laccase, which can degrade pigments, lignin and other toxic substances, such as tannins and phenolic compounds [START_REF] Kirktk | enzymatic "combustion": the microbial degradation of lignin[END_REF],

which contributes to the clean production in the paper industry. Many industrial contaminants, such as chemical pesticides, contain a lot of toxic phenols or aromatic amines, laccase can be effective for degradation of catechol, methyl-catechol, hydroxyurea and other pollutants. In addition, chlorophenols are important industrial materials for the production of dyes, preservatives, pesticides and other chemical products. Laccase can oxidize anthracene and carcinogen benzopyrene. When degrading chlorophenol and its derivatives with relatively high molecular weight, the use of mediator ABTS can significantly enhance this effect [START_REF] Collins | Oxidation of Anthracene and Benzo [a] pyrene by Laccases from Trametes versicolor[END_REF].

C-Biosensor

Laccase as a biosensor characterized by qualitative and quantitative measurements is preferred and has attracted interests for researchers by its specificity, high speed, continuous operation, simplicity. In the aspect of biological detection, laccase immobilized on cellulose can be used to detect the phenol during tea processing [START_REF] Rawal | Polyphenol biosensor based on laccase immobilized onto silver nanoparticles/multiwalled carbon nanotube/polyaniline gold electrode[END_REF]. The enzyme device can be used more than 500 times and stored at room temperature for 2 months, such as in selective detection, morphine can be quickly detected and identified in a short time by laccase device. Biosensors can also be used to detect lignin, phenols and other substances from industrial wastewater, coal, oil, natural gas [START_REF] Rodríguez-Delgado | Laccase-based biosensors for detection of phenolic compounds[END_REF].

D-Food industry

Laccase can be used to improve wine stability, which is one of the main applications in the food industry. Wine is a complex mixture of ethanol, salts, organic acids and phenols, and the presence of phenols is an important factor in wine flavor and color. In order to remove polyphenols, D. Tanrioven,et al [78] used laccase to remove polyphenols from wines. Because phenols can affect the unique flavor and color of juice, laccase can oxidize and polymerize polyphenols, then precipitate and remove them by filtration. The interaction between polyphenols and proteins induces the formation of sediments in juice and the quality of juice. Apart from those mentioned above, as the environment become more and more polluted, the food industry needs more advanced wastewater treatment technologies, phenolic compounds (such as phenols and aromatic amines) are a major healthy and environmental hazard in water, laccase can degrade phenolic compounds well. After treatment with the laccase immobilized with an organic gel, the phenols in the water can be well removed easily.

Objectives of the research work

In this work, the first part is assigned to a general views on the recent development of the enzymes and enzyme immobilization including methods and materials, moreover, the laccase and applications were presented. The objective of this work is the development of the high active hybrid biocatalysts via oriented enzyme catalytic sites on the surface of MNPs. In the field of biocatalysis, the main objectives of the immobilization of enzymes on supports are to enhance both the operational and economic performances of biocatalytic processes. As a matter of fact, immobilization of enzymes results in more efficient and cost-effective sustainable processes. The underlying reasons for enzymes immobilization on supports as mesoporous solids, nanoparticles are: (i) a true recyclability of biocatalysts, i.e. a capacity to recover and re-use them for a prolonged period of time based on an easy separation of the catalyst from reaction products and, at the same time, a lower risk of product contamination with enzyme molecules, (ii) improved properties, e.g. higher tolerance to organic solvents, to pH and temperature variations, (iii) improved stability, e.g.

preventing dissociation-related inactivation by increasing the structural rigidity of the protein. Thus, our efforts are needed for a rational design of nanoparticles to optimize fundamental parameters of conjugation with enzyme, especially focusing on the orientation and the density of enzymes on the nanoparticles surface. The choice of the functionalization strategy is of great importance because it affects the orientation, the accessibility and in fine the bioactivity of the target enzyme.

Eventually, beyond the role of carrier that is the intimate relationship between the material and the catalyst that needs to be deeply investigated.

Therefore, major objectives of this research work are:

1-to study the influence of the dendritic functional coupling agents (dendritic effect) on the surface functionalization of MNPs for the immobilization of enzymes (Laccase).

2-to control enzymes orientation at functional surfaces in order to optimize the access to enzyme catalytic sites.

3-to study the relation material/enzyme on the biocatalytic activity and selectivity. A modulation of the micro-environment at the particle/enzyme interface will be achieved by varying the size, shape, flexibility/rigidity, polarity, hydrophilicity, charges and aromaticity of the dendritic coupling agents and the location of the grafting point at the enzyme surface.

Although magnetic nano particles offer many advantages and many efforts were devoted to the design of MNPs, it is still interesting to develop new strategy to achieve the surface modification of MNPs. Therefore, in chapter II the organic synthesis of linear and dendritic coupling agents was presented in detail, including different synthetic routes of coupling agents, the various attempts, different questions we encountered. Then in the chapter III, we were devoted to the surface modification of MNPs with comical or custom made coupling agents and their characterizations, different parameters such as solvent were investigated. At last, in chapter IV detailed the preparation of biocatalysts via enzyme (Lac3 and Unik157) immobilization onto the functionalized MNPs, moreover the enzyme activity and enzyme loading of novel hybrid biocatalysts were also studied. In order to modulate the activity and selectivity, we proposed to alter the microenvironment of the catalytic sites and the orientation of the enzyme catalytic sites on the surface of supporting materials. The spacer/linker would increase the distance and flexibility between MNPs and biomolecules after their immobilization. It can be also used to modulate properties of hybrid biocatalysts, like the colloidal stability in solutions. The spacer' nature, such as hydrophilic linker also influence the behavior of functionalized MNPs in aqueous medium, in order to achieve a well dispersion in media, hydrophilic moieties such as polyethylene glycol was introduced in the backbones.

These three functional groups -NH 2 , -CHO, -N 3 were chosen as terminal groups due to the high specificity towards the targeted biomolecules we used in our project.

For functional groups -NH 2 , the terephthalaldehyde was employed as a bifunctional heterolinker to bridge between the functional linker and the enzyme. For the functional groups -CHO, the immobilization of enzyme was achieved via a reductive alkylation catalyzed by an iridium complex. In the case of the functional group -N 3 , enzymes immobilizations were performed via a click reaction between linkers and functionalized enzymes.

In our project, the design of coupling agents had to fulfill all requirements of covalent enzyme immobilization, and a wise selection of coupling agents or functional terminal groups should increase successful biomolecule immobilization.

In this project, we introduced coupling agents with linear or dendritic structures [1][2][3][4][5]. The dendritic structure not only increases the number of branches of coupling agents, but also enhances the number of terminal groups offering large number of potential sites for immobilization. In this work, three types of linear or dendritic coupling agents were chosen with terminal functional groups -NH 2 , -CHO, -N 3 , able to bind enzymes specifically onto MNPs.

Synthesis of NH 2 coupling agents 2.2.1 Synthesis of linear NH 2 coupling agents

Based on our lab's experience and bibliographic studies, we proposed an appropriate linear coupling agent bearing functional groups-NH 2 CA1 (scheme 2.1).

Scheme 2.1 Synthesis route of linear coupling agents with protected-NH 2

Therefore, firstly it is necessary to protect one amino group of 1,2-bis(2-aminoethoxy)ethane. Numerous protecting groups of amino are available, such as di-Boc, Cbz, Fmoc, teoc, etc. We first envisaged the synthesis of the monoprotected compound 1 bearing phthalic anhydride as a protecting group. This reaction was performed in toluene by refluxing reagents at 80℃ for 3 hrs. This first protecting group was chosen due to its easy removal in low and mid alkaline conditions, but after removal of solvents, finally only black oil was observed and no desired compound was gained due to the harsh condition [6].

Secondly, di-boc was chosen as a protecting amino group, which could be cleaved by acidic hydrolysis and did not lead to further deprotection of phthalimide protecting groups. So, the protection of 1,2-bis(2-aminoethoxy)ethane with di-boc was done in DCM at room temperature and a mono-boc-protected carbamate 2 was obtained in 56 % yield as colorless oil (scheme 2.1). Next, the addition of boc-protected amine compound 2 to phthalic anhydride was performed in toluene by refluxing for 3 hrs [7], after removal of toluene, compound 3 was obtained in 53 % yield after purification by column chromatography on silica gel.

Then, TFA was used to remove the Boc functional group, solvents and TFA were evaporated to obtain compound 4 as yellow oil in 77 % yield. With compound 4 in our hands, silane group was introduced by its addition to triethoxy (3-isocyanatopropyl)silane in presence of TEA in DCM. After evaporation to remove solvents, a white solid was obtained and purified by column chromatography on silica gel to give CA1 in 55 % yield. It is worthy to mention that the chromatography must be performed rapidly due to possible hydrolysis of the ethoxysilane group. We must note that this reaction must run in an anhydrous condition, thence it is necessary to distill reagents and solvents, and it is better be synthesized just before grafting onto MNPs due to instability of the product.

Synthesis of dendritic NH 2 coupling agents

Synthesis of two branched NH 2 coupling agents

Attempts were made to synthesize the silylated coupling agent with two branched dendritic structure. The strategy relied on a Michael addition as shown in scheme Then, compound 6 was produced as a white solid via a substitution reaction between potassium phthalimide and intermediate 5 in DMF by heating overnight at 75℃, which was obtained and purified by column chromatography on silica gel in 47 % yield [8].

Then, a Michael addition reaction of compound 2 and compound 6 in methanol at 75℃ for 2 weeks was performed, but no coupling of these two substrates was achieved based on the TLC monitoring of the reaction. Then another attempt in DMF was investigated, this route also failed finally as no new compound was observed in TLC. It was speculated compound 2 may be in the form of a salt (NH 3 + ,Cl -), unable to donate an electron pair to attack the C=C bond of olefin, hereby alkali TEA was employed to activate compound 2 in DMF system, however still no target compound was observed in this route. Then, the reaction was carried out in methanol and activation of compound 2 was achieved via addition of alkali TEA to produce a mixture of one-branched and two-branched products 7.

Compound 7 was obtained and purified by column chromatography on silica gel in 27 % yield. Therefore, strong alkali sodium hydroxide solution (aq.) was proposed to treat compound 2 before Michael addition. Then compound 2 could react with compound 6 in methanol by heating at 75℃, after one week, appearance of new compounds was monitored by checking with TLC, and after evaporation to remove methanol, the target 2-branched intermediate with boc-group compound 7 was obtained and purified by column chromatography on silica gel in 43 % yield. In addition, it was not possible to increase the yield of the reaction by prolonging reaction time. Then, the de-protection of amino groups was carried out through the cleavage of boc-protecting group in presence of TFA in DCM, and the product compound 8 was obtained as a salt and purified by column chromatography on silica gel with 86 % yield.

Silylated two branched coupling agent CA2 was obtained by coupling between two branched amino compound 8 and triethoxy(3-isocyanatopropyl)silane at room temperature under argon and at RT in presence of TEA in DCM. First, all required compound need to be distilled in order to achieve anhydrous reaction conditions.

Then after evaporation of TEA, DCM, the silylated two branched coupling agent CA2 was obtained as yellow oil after purification by column chromatography on silica gel with 43 % yield. Then, a Michael type reaction of compound 6 and compound 10 in methanol was investigated at 75℃, but after 2 weeks, only starting materials were observed by TLC and no new product was observed. Then the amount of compound 6 was increased from 5 eq. to 8 eq., but still no target compound was obtained. Then, we speculated that compound 10 probably was in its protonated ammonium salt form, thereby we treated the compound 10 with a basic solution (NaOH) before addition into the mixture system. But no coupling occurred and no target compound was obtained.

Synthesis of four-branched NH 2 coupling agents

So, we proposed a new synthesis strategy (scheme 2.4).

Scheme 2.4 Synthesis of four branched NH 2 coupling agents

We proposed a nucleophilic substitution reaction in an alkaline system.

Inorganic base K 2 CO 3 was employed to activate amine compound 10 in CH 3 CN [9],

and

Synthesis of CHO coupling agents

Synthesis of linear CHO coupling agents

To design molecular structure of coupling agents with aldehyde groups, at the beginning we proposed a synthesis route (scheme 2.5) for the linear CHO coupling agent, with a similar backbone (ethyleneoxy) as for the amino coupling agent (CA1 scheme 2.1).

Scheme 2.5 Synthesis of linear coupling agents with CHO

First, 4-formylbenzoic acid was activated with NHS with help of DCC, then an active NHS-ester compound 14 was produced in 80 % yield [START_REF] Nidhi | Nature Inspired Multienzyme Immobilization: Strategies and Concepts[END_REF]. Afterwards NHS groups were displaced by the primary amine compound 2 in DCM at room temperature to produce an intermediate compound 15 as yellow oil in 62 % yield.

Then the de-protection was performed via cleavage of boc-protecting group of compound 15 with TFA in DCM, however it failed because the aldehyde group was very fragile and sensitive to external environments, like pH, temperature, etc.

Therefore aldehyde groups must be protected with functional protecting groups, such as ethane-1,2-diol, and methanol. First, ethane-1,2-diol was used to protect the aldehyde of compound 15 in toluene by refluxing for 24 hrs since the hydroxyl group of ethane-1,2-diol has relatively strong nucleophilicity and attack the carbonyl group of aldehyde with the help of lewis acid Al 2 O 3 , no target compound could be obtained [START_REF] Mahboobi | 2-Aroylindoles and 2-Aroylbenzofurans with N-Hydroxyacrylamide Substructures as a Novel Series of Rationally Designed Histone Deacetylase Inhibitors[END_REF].

Then, we decided to synthesize a more simple aldehyde functionalized linear coupling agent CA4 from compound 14, by direct coupling with aminopropyltriethoxysilane (APTES) (scheme 2.6) [START_REF] Ebrahim | Preparation and characterization of monosubstituted porphyrins immobilized on nanosilica[END_REF].

Synthesis of dendritic CHO coupling agents

Synthesis of two branched CHO coupling agents

In accordance with the design of NH 2 coupling agents backbones, we also attempted synthesis of dendritic coupling agents with CHO shown in scheme 2.7.

Compound 10 was reacted with 4-formylbenzoic acid in DCM with the help of HOBt and EDC, finally no coupling was achieved and no target compound was observed by monitoring the reaction by TLC [START_REF] Roy | Strategies in making cross-linked enzyme crystals[END_REF]. In the following process (scheme 2.9), the activation of aromatic carboxylic acid with thionyl chloride was carried out in toluene by refluxing overnight to produce compound 19 in 53 % yield. Then a nucleophilic substitution reaction of acyl chloride by compound 10 was investigated in different conditions as shown in scheme 2.9. First we tried an alkali TEA in DCM for 24 hrs at RT, but no desired compound was observed by TLC. Then pyridine was also used as a base and reaction time was also prolonged for 3 days and heated at 60℃, finally we did not find the target product. After that, DMAP as a super high-efficient nucleophilic acylation catalyst was employed, in which resonance between the electron-donating dimethylamino group and the pyridine ring can strongly activate the nitrogen atom on the ring for nucleophilic substitution and could remarkably catalyze the acylation, in the mean time we also prolonged the reaction time, however, still no target product was obtained finally [13c].

Scheme 2.9 Synthesis route of two branched coupling agents with CHO Besides, we investigated the protection of aldehyde group with excess of trimethoxymethane in methanol with drops of H 2 SO 4 (scheme 2.10). After 2 days, a protected desired compound 20 could be obtained after evaporation of excess of trimethoxymethane and methanol in a quantitative yield [14]. was obtained and isolated by column chromatography on silica gel as yellow oil in 35 % yield. However, the use of DDQ or TFA to remove the protecting group of amine did not lead to the target product since only black jelly was obtained [START_REF] Ahmad | Enzyme Immobilization: An Overview on Nanoparticles as Immobilization Matrix[END_REF].

Then another strategy was proposed with Tosyl chloride as protection of amino groups as shown in scheme 2.12. Unfortunately, the removal of Ts group was not possible by phenol and hydrogen bromide which seemed lead to the decomposition of aldehyde groups and backbones.

Owing to failure of protecting aldehyde groups and its subsequent deprotection, we had to propose a new synthesis strategy (scheme 2.13) based on requirement of the target aldehyde. First, a nucleophilic substitution of compound 2 to phenylpropyl bromide was investigated under alkaline conditions (K 2 CO 3 ) in acetonitrile, compound 33 was obtained and isolated by column chromatography on silica gel as yellow oil in 62 % yield. Then, boc protecting group was removed by TFA in DCM and finally a 2-branched amine compound 34 was obtained as yellow oil in 100 % yield. However, the last step of introduction of aldehyde group to benzene ring by titanium tetrachloride in DCM was not possible. was used to remove the protecting group benzyl chloroformate from amine, after removal of the solid by filtration, compound 39 was obtained and isolated by column chromatography on silica gel as yellow oil in 89 % yield. In general the acetal is easily hydrolyzed into aldehyde under acidic conditions, however hydrolysis of compound 39 was carried out in a hydrochloric acid-methanol system, but we did not find the signal of aldehyde in NMR spectrum and the ethoxy protecting group of aldehydes disappeared. Reaction conditions seemed to be too strong and the backbone of the product was affected.

Alternatively, ethyl carbonochloridate was chosen as a protecting group of amino due to its easy removal under alkaline conditions and should not cause damage to aldehyde groups (scheme 2.16). was obtained and isolated by column chromatography on silica gel as light yellow oil in 75 % yield. Finally, we attempted a click reaction [START_REF] Caterina | Superparamagnetic nanoparticles as versatile carriers and supporting materials for enzymes[END_REF] of these two compounds in a mixture of TEA/THF, [CuBr(PPh 3 ) 3 ] as catalysts under microwave to produce the target compound, but in 1 H MNR spectrum even if the backbone of the target compound was detected, the number of aldehyde groups didn't match the backbone and it seems that only one aldehyde group was produced .

Therefore, although many strategies were explored, no successful two branched coupling agent with aldehyde groups was obtained.

Synthesis of four branched CHO coupling agents

We investigated same backbone types for the synthesis of four branched CHO coupling agents. First a strategy was described in scheme 2.19.

Synthesis of N 3 coupling agents 2.4.1 Synthesis of linear N 3 coupling agents

According to the literature [17], a linear azide coupling agent was synthesized (scheme 2.21). The first step of the synthesis was carried out at 80℃ by the addition of sodium azide to 3-azidopropan-1-amine in water, then excess of sodium azide was removed by filtration and water was removed by evaporation, finally compound 57 was obtained as colorless oil but it is worthy to mention that this compound is volatile, a special attention must be payed to evaporation [START_REF] Bilal | Naturally-derived biopolymers: Potential platforms for enzyme immobilization[END_REF]. Then compound 58 was obtained and purified by column chromatography on silica gel as yellow oil in 15 % yield by addition of 3-azidopropan-1-amine to the acryloyl chloride in DCM in presence of TEA. Afterwards, a Michael addition reaction of compound 2 and compound 58 was investigated in methanol at 75℃ for 2 weeks, however, we did not find the desired product and we speculate instability and decomposition of compound 58 led to failure.

Then another synthesis route of two branched N 3 coupling agents was proposed, which was shown in scheme 2.23.

Scheme 2.23 Synthesis route of two branched coupling agents with N 3

In this strategy, we proposed to synthesize an acrylate, compound 60, in order to build the dendritic structure with Michael addition by using this compound. So, first compound 60 was obtained and purified by column chromatography on silica gel in 46 % yield as colorless oil via amidation of acryloyl chloride to 3-aminopropan-1-ol in DCM (TEA as a base). Then, a Michael addition of compound 60 with compound 2 was performed in methanol by heating at 75℃ for 2 weeks, after evaporation, a two branched di-ol 66 was obtained and purified by column chromatography on silica gel in 74 % yield as colorless oil. Then two methods were proposed to introduce azide groups. In first protocol, halogenations of di-ol compound 66 was proposed by adding PBr 3 in chloroform at rt for 2 hrs to further replace the bromide by sodium azide, but unfortunately no target compound was obtained. Then, we proposed to sulfonate compound 66 with methanesulfonyl chloride in presence of TEA and catalytic amount of DMAP in DCM, the mixture was stirred overnight at room temperature and solvents and TEA were removed by evaporation, however, we could not obtain the desired compound.

Another synthesis route was proposed (scheme 2.24). A substitution reaction of 3-bromopropan-1-amine salt with acryloyl chloride was carried out in DCM with the help of TEA, after filtration, to the filtrates NaOH solution was added, then solvents were removed by evaporation, and a desired product compound 63 was obtained and purified by column chromatography on silica gel in 46 % yield as yellow oil. An attempt of Michael addition of compound 2 and compound 63 in excess in methanol by heating at 75℃ for one week, however, no product was detected.

Scheme 2.24 Synthesis route of two branched coupling agents with N 3

Therefore, another new strategy was proposed based on amide coupling between a two branched amine precursor and a bromo-carboxylic acid compound as showed in scheme 2.25. NHS was further displaced with the amino groups of compound 10 in alkaline reaction conditions. Finally the substitution reaction did not occur and no coupling compound was obtained [19].

Thereby, a new proposal to produce two branched coupling agents with azide groups was made (scheme 2.26).

Scheme 2.26 Synthesis route of two branched coupling agents with N 3

Diazotization of 2-(4-aminophenyl) ethanol was carried out by using NaNO 2 solution in HCl (6N). After removal of volatiles under reduced pressure, the residue compound 72 was obtained in 100 % yield as yellow oil. In the next step, PBr 3 was added to the crude products above in chloroform at room temperature, then 30 ml of NaHCO 3 (aq.) was added to quench the reaction. After removal of volatiles under reduced pressure, the residue was purified by column chromatography and the desired compound 73 was obtained as yellow oil in 28 % yield [20].

Then a nucleophilic substitution reaction of compound 73 and compound 2 was carried out in acetonitrile, and potassium carbonate was used as a base, then after 7 days, solid alkali was removed by filtration and after removal of volatiles under reduced pressure, compound 74 was obtained in 83 % yield as yellow oil after the residue was purified by column chromatography. In the next step, TFA was employed to remove the boc group in DCM. After removal of volatiles under reduced pressure the residue was purified by column chromatography and the desired compound 75 was obtained in 80 % yield as yellow oil.

In the last step, the silylated two branched coupling agents CA6 was synthesized via formation of urea bond between amino groups of compound 75 and triethoxy (3-isocyanatopropyl)silane in distilled solvent DCM. After evaporation of TEA and DCM, the silylated two branched coupling agent CA6 was obtained as yellow oil after purification by column chromatography on silica gel in 87 % yield.

Synthesis of four branched N 3 coupling agents

The first strategy we investigated was reported in scheme 2.27.

Scheme 2.27 Synthesis route of four branched coupling agents with N 3

A substitution reaction of sodium azide and halide in DMSO was performed at rt, and 1-azido-3-chloropropane compound 77 was obtained and isolated by column chromatography on silica gel in 78 % yield after filtration of salts and evaporation of solvents. In parallel, another compound 78 1-azido-3-bromopropane was produced as yellow oil after purification by column chromatography on silica gel in 81 % yield via a substitution reaction of azide salts and halide in DMF at 50℃ for 16 hrs after the filtration of salts and evaporation. Then a nucleophilic substitution of compound 10 and compound 77 was performed in acetonitrile in presence of potassium carbonate as a base. The reaction was refluxed for 7 days and compound 79 were obtained as yellow oil after purification by column chromatography on silica gel in 8 % yield. Afterwards stronger alkaline cesium carbonate was employed in DMF at 85℃ for 24 hrs, however a nucleophilic substitution did not occur and no desired compound was obtained, so we abandoned this strategy because of its low yields. Then same reaction conditions were used in a nucleophilic substitution of compound 10 and compound 78, however, compound 79 were obtained as yellow oil after purification by column chromatography on silica gel in 5 % yield and we also abandoned this strategy because this route with low yield could not produce enough coupling agents to use it for surface modification of MNPs in chapter III. Then we tried another route with bromine derivative, which is more active than chloride. Thus, a substitution reaction between compound 73 and compound 10 was explored in acetonitrile with the help of potassium carbonate refluxed for 10 days, or refluxed in toluene in presence of DIPEA, yet no nucleophilic substitution was achieved and no target compound was obtained.

Next, a Michael addition reaction (scheme 2.28) of two branched amine compound 10 and compound 63 was explored in anhydrous DMF at 40℃, and stirred for 2 weeks to synthesize four branched N 3 coupling agents, but finally no target compound was obtained.

Scheme 2.28 Synthesis route of four branched coupling agents with N 3

Another new synthesis route was proposed (scheme 2.29).

Scheme 2.29 Synthesis route of four branched coupling agents with N 3

The reaction of acryloyl chloride and compound 72 in DCM in presence of TEA as a base was carried out. After evaporation of solvents and TEA, compound 80 was obtained as yellow oil after purification by column chromatography on silica gel in 85 % yield. In the second step, a Michael addition was performed between 2-banched amine compound 10 and compound 80 in anhydrous DMF at 40℃. The reaction was stirred for 2 weeks, but finally no target compound was obtained. Even if the amine was treated with sodium hydroxide (aq.) before its addition in Michael addition and even by heating at 75℃ for 2 weeks, no target compound can be obtained.

Conclusion

In this chapter the organic synthesis of linear and dendritic coupling agents with various targeted terminal functional groups was developed. Different synthesis strategies were investigated, including the protection and deprotection of amino and aldehyde groups. Finally we were able produce 6 coupling agents shown in scheme 2.30. First for the linear coupling agent with terminal group-NH 2 CA1, at the beginning we used the strategy based on the protection of amine groups with phthalimide via cycloaddition of phthalic anhydride, followed by deprotection of boc groups at the terminal site with TFA. In the case of the two branched coupling agent with NH 2 group, CA2, we used a strategy based on the Michael addition, to growth the dendritic structure. Nevertheless this strategy did not work for the four branched coupling agents bearing NH 2 , taking into account the difficulty of constructing four branches and steric hindrance is also an important reason for its failure, therefore we had to produce an alternative structure CA3 via a substitution reaction of amine and bromide. Then in order to produce silylated coupling agents, we performed a coupling with silylated isocyanate via formation of a urea link in strict anhydrous conditions. Then for the coupling agents bearing aldehyde functions, we tried to synthesize the coupling agent with CHO with similar backbones, but these routes were not successful, so we had to explore other possibilities. Thus we produced the linear coupling agent CA4 which has a more simple chemical structure. Although we tried many strategies towards synthesis of dendritic coupling agents with CHO, however, we were not able to solve problems we encountered. We had to manage with the sensitivity of aldehydes and the difficulty to build two or four branched backbones. At last for the coupling agents bearing azide functions, linear CA5 and two branched CA6 coupling agents with N 3 have been produced, yet four branched coupling agents with N 3 were not obtained even many attempts were made. Finally 6 coupling agents were produced and will be grafted onto the silica shell of MNPs as described in chapter III.

Chapter

Introduction

In past decades, nanoparticles have appeared to be attractive materials in bionanochemistry, among them, MNPs (superparamagnetic nanoparticles) as smart supports have grown exponentially due to their ease of use. Magnetic particles are considered as one of the most common solid phases for immobilization and detection of biomolecules because MNPs can achieve a much higher enzyme loading capacity and a significantly enhanced mass transfer efficiency as compared with enzymes immobilized on micrometric supports. Additionally, these nano-biocatalysts can be recycled very simply applying an external magnetic field as compared to more tedious conventional techniques (such as filtration) often used to separate other types of heterogeneous catalysts [1]. In this chapter the functionalization of 300 nm core-shell γ-Fe The ferrofluid consists of nano-sized particles with less than 10 nm diameter, which are stabilized in a surfactant solution. These particles exhibit superparamagnetic characters, the "superparamagnetism", that is, no residual magnetization is obtained on removal of the magnetic field. Due to the high magnetic content (70 %) of the MNPs, these magnetic particles have a fast and high answer when placed on an external magnetic field (Magnetization at saturation: approx. 40 cm 3 /g at 300 K), leading to their easy manipulation and a fast and cheap separation from the reaction medium. The uniform nonporous silica shell with 20 nm thickness is chemically and thermally stable and has significant roles: 1) it protects the core-maghemite ferrofluid (γ-Fe 2 O 3 ) against degradation and isolates it from media; 2) it prevents aggregation and increases dispersion abilities; 3) it offers adequate surface areas for subsequent functionalization with surface functional Si-OH groups for binding coupling agents [2].

Many reports of highly functionalized particles in bio-functional materials have been found in recent years [1] [2]. In this context, we are dedicated to make novel nano-particles by covalent binding of different linear or dendritic coupling agents bearing various targeted functional groups, such as NH 2 , CHO, N 3 on core-shell γ-

Grafting of dendritic coupling agents on MNPs

Principles and hydrolysis condensation reaction

In our study, an efficient surface modification was achieved via hydrolysis condensation of silanol groups (Si-OH) from the silica shell of MNPs and the alkoxysilanes groups from the silylated coupling agents to form Si-O-Si linkages.

Triethoxy organosilanes (R-Si(OEt) 3 ) were chosen, generally they are very reactive, inert for most of organic compounds and form a strong siloxane bond (Si-O-Si). In chapter II, various coupling agents with triethoxy organosilanes (R-Si(OEt) 3 )

anchoring groups were synthesized. These organosilanes generally exhibit relatively high stability [3]. A successful hydrolysis condensation involves many parameters, like the choice of solvents, reaction time, temperature, etc. For example, if speed of putting coupling agents into solutions is too fast, the MNPs could have tendency to aggregate together. Apart from those parameters, another main risk is the instability or insolubility of the targeted dendritic functional agent in grafting reaction medium, the solubility of coupling agents in the grafting medium is crucial and challenging since silanization is generally performed in aqueous solutions. In our project, to overcome this eventuality, all these silylated dendritic coupling agents have a low solubility in water and it was necessary to dissolve them in DMSO to introduce them in the reaction medium and limit aggregation during the reaction.

Yet the general process in aqueous solutions has been established [4] (Fig 3 .2):

first the silanetriols were generated through hydrolysis of the silanes from coupling agents with the help of water which could hydrolyze the ethoxy anchoring groups of coupling agents to produce Si-OH, which are available in the solution and on the surface. In the second step, these silanetriols can combine each other or with the siloxane or silanol groups from the silica shell of MNPs via physical adsorption or hydrogen bonding on the surface. Finally the silanol groups react with the free hydroxyl groups on the surface via condensation. A competing reaction can occur simultaneously in solutions during silanization via cross-linking of unreacted silanol groups to form a silane film (oligomerization) with a Si-O-Si three-dimensional structure network through polycondensation between Si-OH groups, and if this reaction continues, the aggregation will occur and produce large aggregates and a poor MNPs surface modification. Apart from this, in order to facilitate the hydrolysis during silanization on the surface in our grafting protocol, ammonia was employed as a catalyst in surface modification [5], experiments exhibited amine with exchangeable hydrogen ions, like ammonia, can catalyze silanization more effectively than amines without hydrogen ions. A probable catalytic mechanism was proposed (Fig 3 .3), the example of methoxysilane is given, but the mechanism is the same in the case of exthoxysilane. First interactions between amine and the silanol group enhance nucleophilicity of that site and organosilane was adsorbed to a nucleophilic silanol site. Then oxygen from the silica shell would attack nucleophilically the silicon of the silylated coupling agent. The next step is a transition state (a pentacoordinate intermediate). Then the leaving group absorbed protons from ammonia. At last ammonia is regenerated, and a silane-modified surface site emerged.

Grafting of NH 2 coupling agents on nano-particles and characterization

In general, this surface modification (also called grafting, or silanization) was performed in a hydro-alcoholic medium. MNPs are suspended in ethanol / Tx solution (1:1) and ammonia at 20 % by mass was employed as catalysts in the presence of a surfactant (Triton X-405 solution, 0.2 % by mass). These conditions prevent from aggregation of MNPs and lead to better dispersion of MNPs, resulting in a homogeneous grafting. The coupling agent was dissolved in DMSO prior to its addition, then it was added gradually to MNPs dispersion by a slow drop by drop, over an addition period of 2.5 hours at 25℃. At the end of the grafting, supernatant was removed by a magnet, and then particles were washed with ethanol, Tx solution and water. The latter was analyzed by infrared spectroscopy (in DRIFT mode), by TEM microscopy and by zetametry.

One can notice the importance of solvents effect on the grafting process. Indeed, in a first attempt, coupling agents were dissolved in absolute ethanol (Fig 3 .4), but they didn't dissolve enough and we observed a cloudy gel. More detailed information about grafting protocol of silylated coupling agents can be found in the experimental appendix. Based on these conditions, commercial or coupling agents synthesized in chapter II were grafted on γ-Fe 2 O 3 /SiO 2 MNPs (scheme 3.1)

The deprotection of phthalimide groups to produce amine grafted on MNPs was carried out in solutions with 40 % methylamine as shown in scheme 3.2. After the reaction, the dispersion of MNPs was sonicated for several seconds to disperse particles well, then after 5 mins, the solution was removed by a magnet, finally particles were washed with water, water/DMF, and ethanol.

FT-IR (DRiFT) study of amino MNPs

The FT-IR spectra of grafted MNPs with various coupling agents exhibited the characteristic vibration bands of MNP core and shell and also the characteristic vibration bands of the coupling agents, suggesting a direct evidence of successful functionalization of MNPs. In all cases, the main characteristic vibration bands of native MNPs core and shell are δCH 2 at 1400 cm -1 (due to the ferrofluid solvent), ν as Si-O-Si at 1100 cm -1 and νFe-O at 700-500 cm -1 . These vibration bands can be used as a benchmark for functionalized particles.

In the case of APTES grafting, the characteristic vibration bands observed in MNP@APTES, the symmetric, anti-symmetric stretching vibration bands of N-H

were also observed at 3299 cm -1 . Stretching vibration bands of CH 2 appeared also at from 2976 cm -1 . (See full spectrum in appendix).

In the case of N-(3-(triethoxysilyl)propyl)ethane-1,2-diamine as coupling agents, the vibration bands of N-H in MNPs@APTES-C2-NH 2 were observed at 3299 cm -1 , and stretching vibration bands of -CH 2 -at 2975 cm -1 , 1457 cm -1 . In this sample, we observed a high adsorption of water which largely affected the all spectrum by covering up other characteristic vibration bands, a low intensity probably exhibited the surface modification is not particularly efficient (see full spectrum in appendix).

In Fig 3.5, spectra of grafted MNPs with CA1, CA2, and CA3 coupling agents are reported in the range of 1000-2000 cm -1 . Indeed, this region is mainly affected by the grafting of the coupling agent sinceν C=C ,ν C=O can be observed.

For MNPs@CA1 as showed in figure 3.5a, a direct proof of a successful surface modification is the presence of characteristic absorption peaks of CA1 (see full spectrum in appendix) as C=O, whose out-of phrase and in phase stretching band appeared at 1714 cm -1 and 1772 cm -1 . Beyond that, another good demonstration arises from the characteristic absorption bands of amide, whose stretching vibration peaks were observed at 1641 cm -1 , 1565 cm -1 . Another peak observed from 1300 cm -1 to 1500 cm -1 was caused by the C=C of the aromatic rings of the phthalimide groups. MNP@CA2 and MNP@2-B-NH 2 ; (c) MNP@CA3 and MNP@4-B-NH 2

The figure 3.5a also highlighted deprotection of amino particles (mechanism as described in fig 3.6) and was followed by IR spectroscopy. In the case of MNP@lin-NH 2 , the vibration bands of phthalimide groups C=O (1772 cm -1 and 1714 cm -1 ) of grafted MNPs completely disappeared after deprotection, suggesting high efficient removal of the phthalimide groups. The successful grafting of MNP@CA2 was monitored by the appearance of the characteristic vibration bands of CA2 (see full spectrum in appendix). Vibration bands of C=O were observed (fig.3.5b). But the bad resolution of spectra did not allow us to discuss about the successful grafting and deprotection of MNP@2-B-NH 2 .

In the case of CA3, the characteristic vibration bands of C=O from amide I at 1673 cm -1 , and the vibration of the amide II (C-N and C-N-H) was observed at 1558 cm -1 . The band at 1714 cm -1 , and 1774 cm -1 corresponded to the appearance of out of phase and in phase vibration bands of C=O. The characteristic vibration bands of CH 2 were observed at 2934 cm -1 , 2880 cm -1 . The characteristic vibration bands from 1300 cm -1 to 1500 cm -1 was caused by the C=C of the aromatic rings of the phthalimide groups (see full spectrum in appendix).

In the case of MNP@CA3 (fig. 3.5c), the several characteristic absorption peaks of CA3 were observed and confirmed the successful surface modification. Strong out of phase vibration bands of C=O appeared at 1714 cm -1 , and another in phrase absorption peak appeared at 1767 cm -1 were observed. In addition to that, the characteristic stretching bands of amide were observed at 1660 cm -1 , 1552 cm -1 .

Then, stretching peak of N-H was observed at 3300 cm -1 , and symmetrical, anti-symmetrical stretching vibration bands of -CH 2 -appeared at 2941 cm -1 , 2867 cm -1 , and its in-plane bending vibration appeared at 1471 cm -1 . Another peak observed from 1300 cm -1 to 1500 cm -1 was caused by the C=C of the aromatic rings of the phthalimide groups.

In fig 3.5c, vibration bands of phthalimide groups C=O (1774 cm -1 and 1714 cm -1 ) of grafted MNPs were not observed after deprotection in MNP@4-B-NH 2 . This confirms the successful deprotection of functionalized MNPs.

In fig 3.7, TEM images of grafted MNPs were presented, from these pictures, we can calculate that average value of diameter of grafted particles and its thickness. Measurement results are gathered in table 3.1. For the native paticles, the average diameter is about 278±62 nm, and its silica shell thickness is about 18.9±1.3.

MNP@APTES has 258±47 nm diameters, and its silica thickness is about 18.9±

2.1 nm. Then for MNP@lin-NH 2 , its diameter is 278±39 nm, and its silica thickness is about 18.1±1.1 nm. As for MNP@2-B-NH 2 , its diameter is about 458

±134 nm, and its silica thickness is about 29.2±2.5 nm. For the MNP@4-B-NH 2 , its diameter is 260±45 nm, and its silica thickness is about 18.45±1.3 nm. In almost all cases, no significant increase of silica shell thickness is observed when the grafting of coupling agents is performed on MNPs. Also, the stability of silica shell when performing the deprotection step is demonstrated for all the grafted particles. However, for the 2 branched coupling agents, a significant increase of the silica shell is observed (from 19 nm for native to 29 nm). It may arise from a non dense multilayer formation. (ζ-potential), that is, Zeta potential is the potential between the continuous phase and the fluid stable layer attached to the dispersed particles. Generally, the Zeta potential can be measured directly, and the thicker the electrical double layer, the higher the Zeta potential. Zeta potential is generally measured by electrophoresis.

The charged particles move under an electric field, and the movement speed (called the electrophoresis speed) is proportional to the Zeta potential and can be calculated theoretically.

Typically the Zeta potential value may vary from +100 mV to -100 mV, and within +30 mV to -30 mV, colloidal system was considered to be unstable, therefore particles with Zeta potential more than -30 mV _represent stable suspensions.

Generally the most important factor which affect the zeta potential is the pH, thereby the effect of pH on the surface charges of functionalized and native MNPs were investigated by measuring zeta potentials in aqueous media, which was performed at room temperature by varying pH from 3.5 to 9.0. In this experiment, Zeta potential measurements of aqueous suspensions of grafted MNPs and native

MNPs were performed at 25℃ in water.

The figures 3.8 showed that the Zeta potential of native MNPs and MNP@CA1, MNP@CA2, MNP@CA3, MNP@lin-NH 2 , MNP@2-B-NH 2 , MNP@4-B-NH 2 at various pH. MNP@CA3 4.9

MNP@4-B-NH 2 5.9 We observed that the IEP of phthalimide functionalized MNPs (MNP@CA1, MNP@CA2, MNP@CA3) is about 4.5 while the IEP of amine functionalized MNPs (MNP@lin-NH 2 , MNP@4-B-NH 2 ) is about 6. In the case of MNP@2-B-NH 2 , it seems that the value of IEP remained unchanged before and after the phthalimde deprotection. This result has to be confirmed. In the other cases, this shift of IEP also confirmed the successful deprotection of phthalimide groups.

Besides we could note that the dispersion of MNPs is more stable, when the pH is higher than from 5 to 6.6. At these pH, the grafted MNPs suspensions are stabilized by electrostatic repulsion of negative charges of MNPs' surface and the presence of hydroxide ion (OH -) in the solution. When the pH gets smaller than 5, the hydrogen ions (H + ) in the solution could neutralize the negative charge on the surface of the ions to reduce the electrostatic repulsion between the ions and lead to aggregation of particles. Therefore it can be concluded that particles are relatively stable in neutral or alkaline solutions, but not stable in acidic conditions. for the size of those aggregates, it varied from 600 nm to 2000 nm for the MNP@APTES, and for another grafted particles MNP@APTES-C2-NH 2 , its aggregates were situated at about 500 nm to 5000 nm. For MNP@lin-NH 2 , aggregates process the size at 600 nm to 1500 nm, and for MNP@2-B-NH 2 , the aggregates varied from 600 nm to 1100 nm, finally for MNP@4-B-NH 2 , the size of aggregates changed from 600 nm to 1200 nm. These analyses are usually performed at the end of the grafting procedure in order to control the colloidal state of the grafted particles. Especially these analyses help us to know if some sonication or additional washing are necessary to well disperse the particles before storage and further use. The amino groups dosing of functionalized MNPs was performed with a back titration method as described in scheme 3.3 [6]. The first step consists in the reaction of NH 2 functions of MNPs with an excess of 4-nitrobenzaldehyde. After completion of the reaction, the product is recovered. Then, in the second step, hydrolysis of the product and reagents nitrobenzaldehyde was carried out.

Scheme 3.3 Dosing of amino functionalized MNPs by back titration

Finally the reaction mixture and the washings are recovered in order to quantify the amount of 4-nitrobenzaldehyde in solution by UV-Visible spectroscopy.

According to a calibration curve at 264 nm, the maximum absorbance of 4nitrobenzaldehyde, its concentration was determined in the final reaction mixture using the Beer-Lambert law. Thus indirectly the concentration of NH 2 functions per gram of amino MNPs was calculated. More detailed information are shown in appendix.

Results are gathered in table 3.3 demonstrated the hydro-alcoholic grafting method allowed an efficient homogeneous surface modification of MNPs, but it can be seen that the number of NH 2 /g MNPs after functionalization varied for each sample. The low values observed, comparing to the usual values obtained in our group (usually 20-400 μmol NH 2 /g MNPs) can be assigned to the non reproducibility of the grafting process. Indeed, various parameters can occur during silanization, such as hydrolysis, adsorption, hydrogen bond, chemical bond, condensation, polarity, solvation, affinity, etc, and it is reported that the hydrolysis-condensation on the surface of MNPs is complex and difficult to control [7]. In addition, the density of coupling agents grafted on the MNPs was similar for commercial compounds-APTES and N- (3-(triethoxysilyl) orientation is impossible on the surface. Therefore an uncontrolled condensation on the surface led to a various density of accessible amino groups. Besides the hydrogen bonding between amino groups, hydroxyls and hydrogen bounded water prevented the polymerization and promoted the adsorption of amino groups on the surface, promoting the disordered hydrolysis-condensation. Then, by contrast to the results of commercial compounds, the number of NH 2 /g MNPs of synthetic coupling agents after surface modification decreased. Finally, it seemed that the MNPs grafted with synthesized linear or dendritic coupling agents didn't improve the amount of amino groups on the surface.

Surface modification of core-shell γ-Fe 2 O 3 /SiO 2 magnetic nano-particles with CHO coupling agents and characterizations

As previously described for coupling agents bearing NH 2 on MNPs, the grafting of linear CHO coupling agents CA4 onto MNPs was performed in similar conditions. This silanization was carried out in a hydro-alcoholic medium (scheme o, we can conclude to the efficient grafting of , a good suspension in aggregates still could be observed, and the size of those aggregates varied from 500 nm to 6500 nm. In figure 3.16, it can be seen that Zeta potential vs pH of MNP@CA4 is different from the one of native MNPs. The zeta potential is higher than -30 mV when the particles were suspended in aqueous medium with the pH more than 5.4, exhibiting stable suspensions of MNPs and colloidal system was considered to be stable. Then, the isoelectric point of these grafted particles (IEP= 4.2) has shifted compared to that of native particles (IEP= 3.0). In figure 3.17, MNP@CA4 has 286±51 nm diameters, and its silica thickness is about 25.1±1.6 nm. The TEM demonstrated the graftig procedure did not affect the size and shape of MNPs. An increase of silica shell thickness is observed when the grafting of coupling agents is performed on MNPs. Also, the stability of silica shell when performing the deprotection step is demonstrated for all the grafted particles.

Then, the quantification of CHO functional groups of MNP@CA4 was performed by a dosing with dansyl-hydrazine as shown in scheme 3.5 [8].

Dansyl-hydrazine reacted with the CHO groups on the surface on MNPs in DMF/acetic acid mixture solutions. Then, the mixture was shaked at room temperature for 30 mins. MNPs were magnetized. Then, the fluorescence emission of the unreacted dansyl-hydrazine in the supernatant was analyzed using dilutions to fit with suitable detection, then finally the value of 1913 μmol CHO/g MNPs was calculated (see full experiment details in the appendix). The grafting of MNP@CA5, shown in figure 3.18a, the characteristic vibration peak of ν N3 was observed at 2117 cm -1 , and the stretching vibration bands of ν CH2 appeared from 2800 cm -1 to 3100 cm -1 , which certified an efficient surface modification.

Chapter III Functionalization of MNPs with linear and dendritic coupling agents 123

Then the figure 3.18b exhibited the grafting of MNP@CA6, in which the vibration bands ofν N3 also was observed at 2117 cm -1 . In addition, the symmetrical, anti-symmetrical stretching vibration bands of -CH 2 -appeared at 2860 cm -1 and 2957 cm -1 . Then characteristic absorption of the amide I was observed at 1640 cm -1 , and the characteristic absorption of the amide II appeared at 1536 cm -1 . The characteristic absorption from 1400 cm -1 to 1600 cm -1 corresponded to the aromatic ring. In figure 3.19, it can be seen that Zeta potential vs pH of MNP@CA5 and MNP@CA6 is different from the one of native MNPs, and the zeta potential is higher than -30 mV when the particles were suspended in aqueous medium with the pH more than 5.2, exhibiting stable suspensions of MNPs and colloidal system was considered to be stable, and the isoelectric point of these grafted particles (IEP of mnp@CA5= 4.2, IEP of mnp@CA5= 4.4) has shifted compared to that of native particles (IEP= 3.0). , we can note MNP@CA5 remained intact (size and shape).

be easily observed they are composed of irons core and silica shell with thickness at about 20 nm. In previous work, it was already occurred after surface modification. MNP@CA5 has 242±54 nm diameters, and its silica thickness is about 18.8±1.8 nm. MNP@CA6 has 245±43 nm diameters, and its silica thickness is about 18.4±1.3 nm. The TEM demonstrated the size and shape of MNPs remained constant after the graftig procedure. An increase of silica shell thickness is observed when the grafting of coupling agents is performed on MNPs.

Finally, the dosing of azide groups on the particles was performed (scheme 3.7)

by click reaction with a cleavable fluorescent compound coumarin derivative [9].

An excess of C≡C-CMRN was added to a solution of MNPs in presence of CuSO 4 •5H 2 O and sodium ascorbate to produce a complex MNPs@C≡C-CMRN, after 24 hrs supernatant were removed by a magnet, then particles were washed with methanol and water 5 times. After click reaction, in the second step a cleavage was performed via sodium hydroxide (aq.) to generate the fluorescent compound, which was quantified by fluorescence measurements. Finally, we can calculate the amount of azide groups available on the surface: for MNPs@CA5: 800 mol N 3 /g MNPs was obtained; for MNPs@CA6: 317 mol N 3 /g MNPs was obtained (see the experiment details in the appendix). An efficient surface modification was achieved for both particles. One can notice that the number of N 3 per g MNPs for two branched azide particles is lower than that for the linear one. We believe that aromatic and dendritic structure of two branched coupling agents results in a lack of reactivity of terminal azide groups towards coumarin mainly because of steric hindrance between the two azide groups. For these reasons, we think that this latter value is underestimated.

Scheme 3.7 Dosing of azide groups of MNP@CA5, MNP@CA6 by back titration of coumarin

Conclusion

In this chapter the functionalization of core-shell γ-Fe 2 O 3 /SiO 2 MNPs was investigated, and the surface modification was achieved via the covalent attachment of three families of coupling agents, amino, aldehyde, azide, and with linear and dendritic structures.

The surface modifications were monitored by the IR spectroscopy, displaying the main characteristic vibration of grafted coupling agents. It can be seen obviously that the grafting process depends on many parameters, such as the solubility of coupling agents in medium, temperature, solvents, the hydrophilic/ hydrophobic properties, etc, and the grafting procedure has to be optimized for each coupling agent.

The Zeta potential measurement revealed the surface charge of grafted MNPs and thus successful surface modification of γ-Fe 2 O 3 /SiO 2 MNPs. Optical microscopy studies exhibited good suspension of MNPs in aqueous solutions and good colloidal state of grafted MNPs. Also, the integrity of MNPs after functionalization was confirmed by TEM microscopy. Besides in regards of stability of grafted particles, these particles can be stored in triton x-405 solution, in water, in fridge at least for few months.

Finally the quantification of grafted MNPs by indirect titration was performed to monitor the number of functional groups available on the surface. For the amino particles, p-nitrobenzaldehyde was used to quantify the functional groups at the surface of MNPs. For the aldehyde particles the dansyl-hydrazine was used in the dosing procedure, and allowed a quantification of functional groups on surface of MNPs. One can notice that this amount of CHO functions (1913 μmol CHO/g MNPs) has been compared to commercial aldehyde particles [START_REF] Nidhi | Nature Inspired Multienzyme Immobilization: Strategies and Concepts[END_REF] and was found to be quite comparable and even higher. Then for the azide particles, clickable and cleavable fluorescent compound coumarin derivatives were employed in a click reaction for azide detection of dosing.

ChapterⅣ Oriented immobilization of laccase onto functionalized MNPs

Introduction

In the bibliographic chapter, we have discussed about laccase, including its physicochemical properties, immobilization and its applications. In this chapter we focus on the enzyme immobilization, which could enhance both the operational and economic performances of biocatalytic processes, improve the stability and reusability of the bio-catalyst, resulting in a more efficient and cost-effective sustainable process. Moreover, an easy separation of the catalyst from reaction products and reuse of the bio-catalyst can be achieved. Besides, in order to study the relationship of material/enzyme on the biocatalytic activity, a modulation of micro-environment at the particle/enzyme interface was achieved by varying the size, shape, flexibility/rigidity, polarity, hydrophilicity, charges and aromaticity of the dendritic coupling agents and the location of the grafting point at the enzyme surface. In addition, according to literature [1], enzymes immobilization on magnetic particles, such as hydrolase, glucose oxidase, and alcohol dehydrogenase exhibited high specificity and high efficiency with a possible recovery in some cases. Laccase immobilization onto different materials have been also reported, displaying good stability, thermal resistance, improved activities, such as inorganic silica, metal oxides, like titania, iron oxides, organic polymer, etc [2]. Nevertheless, achievement of controlling enzymes orientation at functional surfaces in order to optimize the access to enzyme catalytic sites is still to be properly documented, since random orientations are usually reported. The production of large amounts of recombinant enzymes (LAC3, and their variants) is a pre-requisite (site directed mutagenesis and fermentation) for the production of recoverable biocatalysts. The well established methodology of heterologous expression in the filamentous fungus Aspergillus niger will allow the production of recombinant enzymes variants at the g.L -1 scale in T.TRON group [3a]. The downstream processing is involving a couple of chromatographic steps at semi-prep scale and will be followed by a complete characterization of the purified enzymes variants. T.Tron group [3] has developed an original strategy enzymes mutagenesis based on variants containing a single lysine residue available for modification. In brief, the only two lysine groups (K40 and K71) naturally present in LAC3 enzyme have been replaced by innocent aminoacids by side directed mutagenesis; the resulting lysine free variant (NoK) is then a platform for a substitution of any surface aminoacid by a lysine group. As opposed to functionalization methods that lead to a random orientation of the catalyst at the surface of a given material, this strategy offers a perspective to orient an enzyme at the surface of materials. Once immobilized, enzymes are sophisticated nano-reactors performing chemical transformations even under harsh conditions. Thereby in this chapter laccases immobilization on functionalized MNPs were done to obtain a homogenous distribution of oriented enzyme molecules at the surface of the supporting material and to investigate whether orientation effect will be obtained by immobilizing laccase and its variant-unik157 onto functionalized magnetic nano-particles. The study of the effects of oriented immobilization of enzyme on the enzyme activity via changing the position of active site of laccase on surface of MNPs and recycle ability of biocatalysts will be examined. Then laccase catalyzed reactions will be performed to study the enzyme kinetics.

In order to introduce an orientation effect to this biocatalyst, two variants of laccase were employed, shown in fig 4 .1. The enzyme LAC3 from the fungus Trametes sp. C30 possesses only two lysines, offering reactive free NH 2 groups: K40 and K71 located diametrically opposed to the T1 copper site. Simultaneously Prof. T.Tron group created a laccase variant which processes single lysine groups near T1 copper site by site mutagenesis. When immobilizing LAC3 onto MNPs, T1 site will be exposed to reaction media, that is more accessible to substrates theoretically. Conversely exposition of T1 site to the surface of MNPs was achieved by immobilizing Unik157 onto MNPs, in that case substrates should be confronted to a steric hindrance to approach T1 copper site. To study this phenomenon, covalent binding of functionalized MNPs with different terminal functional groups (NH 2 , CHO, N 3 )

and laccases was investigated. Different immobilization strategies were developed to explore the best immobilization reaction conditions by varying temperature, concentration, catalysts, buffers, etc. Then to study the kinetics of immobilized enzymes, ABTS was used as a substrate (Fig 4 .2). Finally the characterizations of this hybrid biocatalyst were also discussed and the recovery and stability of the biocatalyst were also investigated.

In the regards of kinetics study, enzyme activity measurement was performed on a spectrophotometer (JASCO V700 spectrophotometer). Activity of free laccases and grafted laccases on MNPs towards ABTS was measured at RT in the storage buffer (pH= 5.5). Transformation of ABTS was catalyzed by laccase to produce a detectable ABTS cation free radicals (ABTS .+ ) in 2 mins, which was detected by UV-vis spectroscopy at 420 nm (ε= 36000 mol -1 .cm -1 , calculation factor : 27.8). For free laccase, in 1 mL cuvette, 890 μL of storage buffer was added, then 100 μL of 50 mM ABTS substrate was introduced to the cuvette, and 10 μL of the appropriately diluted enzyme was added to the mixture, then the reaction was followed on spectrophotometer for 2 mins. In the case of grafted laccase, 20 μL (from a solution of 0.5 mg/250 μL) of grafted laccase was introduced to a mixture of 880 μL of storage buffer and 100 μL of 50 mM ABTS substrate in 1 mL cuvette, then activity was followed on spectrophotometer for 2 mins. In each experiment, three reactions were performed independently (triplicate) and every sample was tested three times. Finally, an average value of nine measurements was calculated.

In order to study the specific activity of hybrid biocatalysts, conventional methods of enzyme loading, like UV-vis spectroscopy detection (i.e. bradford test), in which the enzyme concentration measurement is based on the detection of amount of proteins in solutions before and after reaction, exhibits some limitations as low sensitivity. Therefore, in our project a higher sensitivity technique Enzyme-Linked ImmunoSorbent Assay (ELISA) was employed. Enzyme-Linked ImmunoSorbent Assay (ELISA) exhibits many advantages, like it just need a low level of antigen (low-mid picogram), and it is also more sensitive and more accurate due to the combination of antigens and antibodies. ELISA has been widely used in many industrial applications such as medical research, food, diagnosis of a variety of bacteria and viruses, animal quarantine, etc, which generally is an enzyme labeled solid-phase assay based on the immunological binding adsorption between antigen and antibody.

In ELISA assay [4], the immobilization of antigen or antibody does not affect its immunological activity during the labeling process. Throughout the assay, the binding of antigen and antibody is carried out on a solid support (microplate), and the detection is based on the color or fluorescence or luminescence after the reaction of the enzyme and its substrate. The intensity of the color or fluorescence or luminescence is proportional or inversely proportional to the concentration of the tested substrate. The general principle of ELISA is described below:

1) Coating: Antigens or antibodies can be directly adsorbed on the surface of the solid carrier (microplate), which depends on adsorption between the protein and the surface of microplates, and this procedure can maintain immune activity of antigen or antibody;

2) Blocking: a blocking protein is added to cover all unsaturated binding sites of a microplate well.

3) Marking: Antigen or antibody can be linked to enzymes by covalent binding to form enzyme conjugate which can still preserve its activity and catalytic efficiency;

4) Signal measurement: After the enzyme substrate is added, an enzymatic reaction can occur to generate a detectable product which is proportional to the amount of antigen in the sample.

In our study, we have used a sandwich Elisa technique. It is often used for the detection of antigens and proteins because the target is sandwiched between the captured antibody and the detection antibody. The detection steps are: first coating of the purified corresponding antibody on the solid phase carrier, addition of the sample, which will bind to the antibody, then washing off the impurities, and then addition of the enzyme-labeled detection antibody. Finally the excess enzyme-labeled antibody is washed away, and the substrate is added.

In our case, grafted enzymes or free laccases were considered to be antigens, which could be bonded to the surface of microplate wells (fig 4 .3).

Chapter IV Oriented immobilization of laccase onto functionalized MNPs

The known free laccases to be a standard enzyme Chapter IV Oriented immobilization of laccase onto functionalized MNPs

Immobilization of laccases onto amino MNPs

Optimization of reaction conditions

In some literatures, amino groups of enzymes are used to immobilize them and this strategy often exhibit good performances in terms of grafting and catalytic activity of the immobilized enzymes. T.TRON group has used this strategy and has reported a reductive alkylation of proteins in mild conditions [3]. Laccases bearing amino groups from the lysines residues on its surface can be grafted on the amino functionalized magnetic nanoparticles with the use of a bis-aldehyde heterolinker in reductive alkylation conditions, in that case an iridium catalyst was used to reduce In this subsection, amino grafted particles described in chapter III were employed to study immobilization reaction conditions. We studied the influence of the buffers, the effect of Ir catalyst, the enzyme concentration, the ratio of enzymes/MNPs, and then we investigated the orientation of laccases immobilization on MNPs in terms of activity of the hybrid biocatalyst. Finally, the experimental results of biocatalytic activities and characterization of hybrid biocatalysts discussed. The general procedure of enzyme immobilization on amino particles was presented in Fig 4 .5. In the first step, the amino particles were coupled with terephthalaldehyde to produce aldehyde-functionalized MNPs.

Terephthalaldehyde was chosen as heterolinker instead of the very common glutaraldehyde according to the work of Lu REN (PhD, Univ. Aix-Marseille, 2019).

She observed that terephthalaldehyde can more easily react than aliphatic aldehydes.

Then, in the second step two types of laccases lac3 and unik157 were immobilized onto amino particles in a reductive alkylation condition. Generally, 2 mg particles were divided by 4 (triplicate for the reactions, and 1 for blank). And the total volume of enzyme immobilization reaction in each tube was 250 l (0.5 mg/250 l).

More detailed informations are given in appendix.

A-Immobilization of enzymes on MNP@APTES

In the case of MNP@APTES, reaction conditions of our first attempt were First of all, the particles must be dispersed well in solutions to avoid aggregation phenomenon which cause both loss of surface areas and decrease functional groups accessibility in the enzyme immobilization reaction. Thereby different methods were used to prevent the aggregation of particles in solutions. First the vortexing was used to disperse particles in solutions for 1-3 mins, particles can be dispersed well in aqueous solutions even though some aggregates can be observed. Besides bath sonication was investigated, where mild ultrasonic exposure (from 2 mins to 10 mins) did not break the large aggregates into small ones. Then more powerful probe sonication (60 w, 10-30 sec) was also employed to decrease aggregation and break big aggregates, sonication for several mins can be performed. Generally, MNPs can be dispersed well in reaction media.

In absolute ethanol, particles could be dispersed well but easily aggregate at the bottom of tubes when it is placed still, thereby reactions which run in ethanol must be stirred relatively strongly. By contrast particles exhibited good suspension in other buffers. Given the solubility of terephthalaldehyde, ethanol was preferred to be a medium before and after reaction, as particles were washed with ethanol to remove excess terephthalaldehyde after the reaction completion.

The reaction medium is an essential parameter in an enzyme immobilization reaction. Various reaction media gathered in table 4.1 were investigated in the both steps of the immobilization reactions of MNP@APTES with Lac3 in presence of Ir catalysts, such as absolute ethanol (commercial absolute ethanol stored on 4 Å molecular sieve), RA (Reductive Amination) Buffer, 0. At the beginning, the immobilization of lac3 was carried out in RA buffer (table 4.2, entry 1), however after immobilization, the hybrid biocatalyst exhibited no activity, suggesting RA buffer may be not appropriate. The bad solubility of terephthalaldehyde in RA buffer may explain why the activity was not observed. In order to test enzyme leaching, syringaldazine was used to check the last washing.

Syringaldazine (SGZ) is one of the most common substrates for laccase and can be oxidized to produce corresponding quinine, which can be seen visually by observing the appearance of a pink color in the reaction medium. At the end of enzyme immobilization reaction, washings were performed with corresponding buffer 5 times, and the last washing was tested with syringaldazine, in the case of table 4.2 entry1, a pink color was not observed, indicating no laccase in the solution.

So, two possibilities, either no enzyme was grafted or enzyme was grafted, but no leaching was observed.

Then an enzyme immobilization reaction was investigated in 0.1 M MES pH= 6.0 buffer (table 4.2, entry 2), and MNP@APTES@lac3 system produced a high activity (10.7 U/L, 7.1 U/L, 8.1 U/L), simultaneously a good enzyme loading (30.7 μg/mg, 37.7 μg/mg, 36.4 μg/mg) was obtained by Elisa test. Subsequently a good specific activity (8.7 U/mg, 4.7 U/mg, 5.5 U/mg) can be calculated. Ir cat. amount were from 0.1 eq. to [E] to 10 eq. to [E]. The amount of catalyst can be as low as 0.1 eq. to enzymes, and no enzyme leaching was observed. Then, last washing supernatant was also checked with a syringaldazine solution, no pink color appeared finally even after vortexing and socination, suggesting no enzyme leakage was observed after washing and sonication.

Then taking into account the solubility of terephthalaldehyde in first step, 0.1 M MES buffer was replaced by ethanol, but ethanol must be removed in the second step to avoid inactivation of laccases, and lac3 was immobilized onto particles in 0.1 M MES pH= 6.0 buffer in the second step (table 4.2, entry 3). A relatively higher enzyme activity (14.2 U/L, 9.9 U/L, 11.9 U/L) was observed, and a good enzyme loading (33.2 g/mg, 37.9 g/mg, 45.7 g/mg) was obtained in Elisa test, then a good specific activity (7.5 U/mg, 4.7 U/mg, 7.0 U/mg) was calculated. We concluded that no significant difference of results was observed compared to the result of entry 2. Also, at the end of enzyme immobilization reaction, washings were performed with corresponding buffer 5 times, and the last washing was tested with syringaldazine, a pink color was not observed, indicating no laccase in the solution.

Thereby another experiment was carried out in 0.1 M MES + 0.1 M HCOONa pH= 6.0 buffer (table 4.2, entry 4). Indeed, HCOONa is mandatory in the reductive alkylation step. However, this MNP@APTES@lac3 system exhibited no enzyme activity, but a medium enzyme loading (34.4 μg/mg, 28.0 μg/mg, 21.1 μg/mg) was obtained in Elisa test, an explanation is that laccase is sensitive to the HCOONa, after immobilization washing with acetate buffer is not enough or not efficient to remove HCOONa.

Finally to conclude on the grafting and activity of MNP@APTES@Lac3, the best reaction conditions for the grafting are 0.1 M MES buffer with concentration of 6 M in enzymes and in presence of Ir catlyst.

Parallelly, unik157 immobilization onto amino particles was performed in presence of Ir catalysts as shown in table 4.3. First, this MNP@APTES@Unik157 system was carried out in 0.1 M MES pH= 6.0 buffer in both steps (table 4.3, entry 1), a good enzyme activity (average activity of 3 samples, 4.6 U/L, 2.6 U/L, 5.5 U/L) was observed, and a medium enzyme loading (29.9 μg/mg, 17.1 μg/mg, 16.5 μg/mg) was observed in Elisa test, indicating the 0.1 M MES pH= 6.0 buffer was considered to be a suitable medium for this system. Then a good specific activity can be obtained.

Then, ethanol was used in the first step in order to increase the solubility of terephthalaldehyde, and in the second step 0.1 M MES pH= 6.0 buffer was employed (table 4.3, entry 2). A higher enzyme activity (8.9 U/L, 8.1 U/L, 17.4 U/L) was also observed, and a high enzyme loading (92.5 μg/mg, 77.0 μg/mg, 128.9 μg/mg) was obtained in Elisa test. It can be seen that in first step of this protocol ethanol may be more suitable than 0.1 M MES pH= 6.0 buffer due to a good solubility of terephthalaldehyde. Thus, an increased enzyme loading was observed but led to a reduced specific activity because of a comparable enzyme activity. specific activity was obtained. Also, same conclusions can be drawn for the MNP@APTES@Unik157 as same as MNP@APTES@Lac3. Also, syringaldazine test was performed after washings, no pink indicating no leaching was observed.

B-Immobilization of enzymes on MNP@APTES-C2-NH 2

Then enzyme immobilization reactions were also performed with amino functionalized particles with long arm, MNP@APTES-C2-NH 2 , in different media as showed in table 4.4 (for lac3) and table 4.5 (for unik157).

MNP@APTES-C2-NH 2 @Lac3 As for the high enzyme activity and enzyme loading, one hypothesis is that enzymes may be absorbed physically on the particles' surface via non covalent bonding. So we decided to use another strategy for the immobilization of enzymes at the surface of amino functionalized particles. This strategy is described in the next paragraph.

Second strategy for laccase immobilization onto amino particles

As shown in Fig 4.7, the first step consists in a functionalization of laccases with terephthalaldehyde. First laccase-aldehyde complex was produced via condensation of amino groups from enzyme and terephthalaldehyde in presence of Ir catalyst in RA buffer, then immobilizations were performed in the same reaction conditions as in the previous paragraphs, however, MNP@APTES-C2-NH 2 @lac3 exhibited no activity or a very low activity. In the case of immobilization of unik157, a better activity but still low (3 samples: 2.1 U/L, 1.8 U/L, 2.1 U/L) and a good enzyme loading (90.8 μg/mg, 70.8 μg/mg, 71.8 μg/mg) were observed. We concluded that this strategy did not work efficiently for amino particles, especially because of the use of HCOONa reagent which seems to be hard to eliminate by washing after the reaction.

Conclusion of laccase immobilization onto amino particles

From these experiments, a lot of informations can be obtained: 1) the best immobilization condition is: 0.1 M MES buffer with enzyme concentration of 6 M in presence of Ir catalyst, and 0.1 M MES buffer was considered to be the best medium for the amino particles; 2) Ir catalyst must be used to reduce the imine into amine in the first step, and the HCOONa reduce the activity due to the sensitivity of lac3 and unik157 in reducing reaction media; 3) compared to the free enzyme activity (about 100 U/L, both for lac3 and unik157), after immobilization the activity and specific activity dramatically decreased which may arise from the mass transfer limitation, or the changes in the spatial structure of enzymes, also the interface of enzyme/MNPs and may have affected the enzyme activity; 4) the covalent bonding between enzymes and particles still need to be documented; 5) the new strategy which consisted in the modification of enzymes by aldehyde function did not work efficiently.

Laccases immobilization onto aldehyde MNPs

Immobilization of laccases reaction

Based on the study realized in paragraph 4.2, we used the best reaction conditions for the grafting of enzymes (Lac3 and Unik157) onto aldehyde MNPs In optimized reaction conditions, 2 mg MNP@CA4 were weighted and transferred to a centrifuge tube. Resuspend the beads by adding 1 ml RA buffer and mix the beads by vigorously vortexing for 1-2 minutes. If some beads aggregated after suspendeding, sonicated mildly for 10-30 seconds. Place the tube on the magnetic separator for 1-3 minutes. Remove the supernatant while the tube remains on the magnet. And resuspend the beads with According to the experimental results, for MNP@CA4@Lac3 (entry1), the activity towards ABTS is similar to the one for MNP@CA4@Unik157 (entry2), but a significant difference of enzyme loading can be observed (70.4 μg/mg for MNP@CA4@Lac3 and 139.5 μg/mg for MNP@CA4@Unik157). Thus, the ratio of specific activity of grafted lac3/unik157 is 1.4, and this result highlight an orientation effect on the enzyme activity was obtained after immobilization of enzymes. Once again, in these immobilizations all activities of grafted enzymes decreased significantly compared to the initial free enzymes, for the same reasons we have already evoked in the case of amino functionalized MNPs in paragraph 4.2.3. The orientation effect after immobilization can be explained as the T1 copper site in MNP@CHO@Lac3 is oriented towards the reaction media and substrates are easily to get access to the active site, in contrary in MNP@CHO@Unik157, the T1 site is close to the surface of MNPs, leading to a steric hindrance to substrates.

In addition, the reusability and the recovery of these hybrid biocatalysts were investigated. At the beginning, we attempted starting with about 1 mg of grafted MNPs at similar concentration (0.07 M) of substrates ABTS as described in the case of activity measurement of amino particles and aldehyde particles, but even if significant values of activities were obtained, no particles remained in the tube after several recycles due to the loss of particles during magnetization. Then, in another attempt, we increased the amount of grafted MNPs from 1 mg to 5 mg, yet no UV spectra was obtained because the particles or high intensity of ABTS solution saturated the UV detection of ABTS cation free radicals (ABTS It can be seen that the activity decreased with increased recovery cycles, which may come from the loss of MNPs during magnetization since particles are sticking on the walls of the vial. Indeed, UV of the last recovery solution showed that only 0.1955 mg of MNPs remained in suspension (90 % loss) (fig 4.9). We postulated on a linear mass loss during the recovery cycles. Considering the decrease of activity with a constant loss of MNPs, we concluded that the activity of the biocatalysts remain constant over 10 recovery cycles. aggregation (1-10 µm). That confirmed that they would become more and more difficult to magnetize.

Conclusion for laccase immobilization onto aldehyde MNPs

From these studies, we can observe that the enzyme activity and specific activity both decreased after immobilization; A good enzyme loading was obtained for lac3 and unik157; Enzyme loading was more efficient for unik157 than for lac3; A high enzyme loading was obtained for unik157, corresponding to the saturation of the MNPs surface [7], considering the size of particles and the size of enzymes, the theoretical maximal enzyme loading is 120 μg/mg of MNPs. Then, the ratio of specific activity (lac3/unik157) before immobilization this ratio increased from 1 to 1.4. This trend highlights a general effect exerted on the oxidation site of the enzyme by the proximity of the surface of the particles. At the pH of the reactions, MNPs are negatively charged particles (see zeta potential measurements). Moreover, it is reasonable to think that native electrostatic contribution of the enzyme to the overall charge of the particles can only strengthen this negatively charged surface.

This likely provides a negative repulsive environment for a negatively charged ABTS substrate. On the other hand, laccase substrate oxidation is an outer sphere mechanism (i.e. there is no coordination of the substrate to the metal) and there is not a truly defined substrate binding site. Therefore, it is tempting to think that the weight of factors like the limitation of diffusion at the liquid/solid interface and steric hindrance may here prevail on that of a deformation of the "active site". At last, the recovery test demonstrated a good stability of hybrid biocatalysts with no loss of activity after 10 recovery cycles. 11 Mechanism of copper-catalyzed azide-alkyne cycloaddition [7] Then in order to improve the coupling of enzymes and particles, more catalysts (CuSO 4 .5H 2 O: 100 eq. to enzyme) were employed to boost the click reaction.

Laccases immobilization onto azide MNPs

Immobilization reactions were performed under that condition as shown in table Finally, we concluded that a click reaction was achieved, and after immobilization, the activity and specific activity decreased drastically compared to the free enzyme. The ratio of SA (unik157/lac3) after reaction is 3.6, this value is significant. The orientation effect of enzymes on the activity towards ABTS is on the opposite of the one we found in the case of MNP@CHO particles. The unik157 immobilized enzyme is more active towards ABTS than lac3 immobilized enzyme.

MNP@CA6 (two branched azide)

Laccase immobilization onto MNP@CA6 was performed. specific activity (0.3 U/mg, 0.6 U/mg), which may be explained by an over loading of enzymes at the surface of the particles, a too crowded microenvironment on the surface or strong interactions between enzymes on the surface resulted in the change of the spatial structure of laccases, leading to a low catalytic efficiency.

In regarding of MNP@CA6@unik157 (table 4.14, entry 2), good enzyme activities (12.1 U/L, 6.3 U/L) were observed, and good enzyme loading (79.9 μg/mg, 55.5 μg/mg) were obtained in Elisa test, and specific activities of 3.8 U/mg, 2.9 U/mg were calculated. The ratio of SA (unik157/lac3) is 7.1, which means an orientation effect was observed for enzymes immobilized on MNP@CA6. We think that the dendritic effect introduced by dendritic coupling agents may modify the micro-environment (hydrophilicity, charges and aromaticity) at the particle/enzyme interface more than a linear ligand. The flexible dendritic structure led to an enlarged passageway for substrates.

Conclusion for laccases immobilization onto

MNP@CA5@Lac3 MNP@CA5@Unik157

Loading: 115 µg/mg Loading: SA: 2.9 U/mg SA: for MNP@CA6@Unik157 resulting in a decrease of the enzymatic activity.

Conclusion

In this chapter, laccase including the amino, aldehyde, azide.

Conclusion for laccases immobilization onto azide

MNP@CA5@Unik157 MNP@CA6@Lac3 MNP@CA Loading: 19 µg/mg Loading: 197 µg/mg Loading:

SA: 10 U/mg SA: 0.5 U/mg rtional explanation of enzyme immobilization on azide MNPs in fig 4 .12, regarding MNP@CA5, laccases almost have same activity towards ABTS, and compared to that of free laccases, the enzyme activity Moreover, enzyme loading is higher for Lac3 than for maybe because Lac3 has 2 free positions for grafting, in contrary Unik157 has only one free position available for grafting. Moreover, a high loading for MNP@CA6@Unik157 induces a crowded environment close to active site resulting in a decrease of the enzymatic activity.

this chapter, laccase immobilization was explored with several particles, including the amino, aldehyde, azide. The evaluation of enzyme activities was investigated with the substrate ABTS, also the enzyme loading was detected by pecific activity and orientation effect were also studied.

the activity of free enzymes and immobilized ones, it can be seen all enzyme activities and the specific activity decreased dra immobilization, which can be explained by the changes in the spatial structure of First we tried to optimize the enzyme immobilization onto MNP@NH 2 particles by using the heterolinker (terephtaraldehyde) to bridge particles and enzymes.

However IR study displayed no characteristic adsorption of C=O corresponding to the aldehyde newly formed were observed in IR spectra, suggesting no coupling by covalent bond was achieved and the enzymes are fixed to the particles in a specific way.

Then for the MNP@CHO particles, a reductive alkylation of enzymes in mild conditions facilitated by iridium catalyst was used for enzyme immobilization. A good enzyme activity and enzyme loading were obtained and also the orientation effect was observed since the ratio of SA of lac3/unik157 = 1.4, indicating that the orientation of the active sites T1 to the reaction medium could increase the catalytic efficiency of biocatalysts. By contrast when the active sites T1 was close to the surface of the particles, the activity decreased due to the trouble of substrate to access close to the enzyme catalytic site At last, the laccase immobilization was performed on MNP@N 3 by a click reaction. A good activity and enzyme loading were observed for both azide particles, MNP@CA5 and MNP@CA6. Compared to that of free laccases, the enzyme activity and the specific activity decreased drastically. Because Lac3 has 2 free positions for grafting and Unik157 has only one free position available, enzyme loading is higher for Lac3 than for Unik157. However the specific activity of hybrid biocatalyst with lac3 decreased more dramatically than with unik157 for the same reasons evoked above.

In summary, some hypothesis to explain these catalytic activities differences could rely on (i) the distances between the enzyme and the surface of the particles (linker effect) and (ii) the nature of the environment close to the catalytic sites.

Indeed, these parameters are varying from aldehyde to azide particles. It is generally considered that short linker convey more rigidity to the system whereas a longer linker (especially for the dendritic linkers) reduces the steric hindrance. Also, a positively charged and aromatic environment will be more accessible to the negatively charged and aromatic ABTS substrate. Comparing SA ratios of enzymes grafted on MNP@CHO and MNP@azide, these constructions seem to agree with these considerations.

Indeed, in the case of the enzymes immobilized on MNP@CHO particles, we observed a higher SA towards ABTS (voluminous, aromatic and negatively charged substrate) in an unconstrained environment (lac3 immobilization) than in a constrained environment (unik157 immobilization). Also, the negatively charged character of the substrate is an additional issue unfavoring its approach close to the particles. In this case, the medium length, aromatic and slightly positively charged linker of MNP@CHO is favoring the activity of the biocatalyst, compared to other linkers [6a].

In contrary, in the case of enzymes immobilized on MNP@azide particles, we observed a higher SA towards ABTS (voluminous, aromatic and negatively charged substrate) in the case of the unik157 immobilizations (Fig 4.12). In that case, the environment close to the catalytic site is more positively charged (induced by the numerous amine groups of the linker), and the linker is longer than is the case of enzymes immobilized on MNP@CHO particles (table 4.5). So, ABTS substrate can easily access to the catalytic sites in the case of MNP@N 3 particles. Thus, the increase of the activity (for unik157 immobilization) may be due to a concentration effect of the substrate close to the catalytic site due to the steric hindrance of this environment. This can explain why ABTS is more easily oxidized by MNP@azide@unik157 biocatalyst.

Then regarding perspectives, we have to keep in mind that amino particles can be good enzyme carriers due to the high non-covalent loading. For the aldehyde particles, they seemed to be a good platform in order to get stable enzyme immobilization by covalent bonding. For the azide particles, the dendritic coupling agents lead to a high enzyme loading compared to the linear one, however, a high enzyme loading does not mean a high activity, "over" loading of enzyme will result in non-specific immobilization, and thus changes in spatial structure of enzymes, and interactions between enzymes.

In the field of biocatalysis, the main objectives of the immobilization of enzymes on supports are to enhance both the operational and economic performances of biocatalytic processes. As a matter of fact, immobilization of enzymes results in more efficient and cost-effective sustainable processes. In this work, nanoparticles (superparamagnetic nanoparticles) were used because they are attractive supports that can achieve high specific surface areas in contrast with other supports. Also, these nano-biocatalysts can be recycled very easily applying an external magnetic field as compared to more tedious conventional techniques (such as filtration). In Finally, enzyme immobilization was carried out via covalent binding of enzymes on functional MNPs. Catalytic activity measurements of these hybrid biocatalysts were performed via oxidation of the common substrate ABTS. Then the enzyme loading was evaluated by Elisa tests. Afterwards the specific activity of this hybrid catalyst could be calculated. In the case of amino MNPs, a reductive alkylation in mild conditions was used and the hybrid biocatalysts displayed good activity and high enzyme loadings. However, the IR spectroscopy showed no covalent binding of the enzymes was achieved, it was speculated laccase has adsorbed onto MNPs in a non-specific way. Then the aldehyde particles appeared as an ideal platform for laccase immobilization in a reductive alkylation in mild conditions. Orientation effect due to the steric hindrance close to the T1 copper site may limit the accessibility to substrates in the case of unik157 immobilization. On the contrary, when the immobilization site is located diametrically opposed in the case of lac3 immobilization, the T1 copper site of laccase could be exposed to reaction medium and substrates, promoting a higher activity of the hybrid biocatalysts. Then for azide particles, a click reaction was used to couple alkyne modified laccase and MNPs. Also, good activity and high enzyme loading were observed, orientation effect was observed and MNP@azide@Unik157 appeared to be more active biocatalysts compared to MNP@azide@Lac3. Finally, the recovery over 10 times with no loss of activity demonstrated the good stability of the hybrid biocatalyst.

Finally, as we expected, dendritic coupling agents improved immobilization of enzymes on grafted MNPs. Also, we demonstrated that a modulation of the microenvironment of hybrid biocatalyst close to the T1 site of the enzyme was achieved (by varying the size, shape, flexibility/rigidity, polarity, hydrophilicity, charges and aromaticity of the coupling agents) and led to an orientation effect on the enzymes activity towards ABTS substrate.

In terms of perspective and further the development of the project, we will prepare other high performance recoverable nano-biocatalysts for biocatalysis through the functionalization of MNPs with dendritic structures enabling a dense grafting of the particles surface with properly oriented robust enzymes. Also, new dendritic coupling agents should be synthesized with other terminal functional groups. Then, the functionalization strategy can also be extended to other nanomaterials or other enzymes. And the potential influence on both mass transfers and enzyme cooperativity will be studied. A high density of biocatalyst grafted per surface unit is intrinsically a potential modulator of the grafted biocatalysts performances.

was extracted with DCM (3*50 ml). The organic phases were washed with water (3*150 ml), and the organic phases were dried over MgSO 4 . Concentrated under reduced pressure and obtained as colorless oil in 56.4 % yield; 1 H NMR (400 MHz, CDCl 3 ) δ: 3.65-3.62 (m, 4 H), 3.59-3.54 (m, 4 H), 3.35-3.34 (m, 2 H), 2.93-2.90 (t, J = 12 Hz, 2 H), 1.47 (s, 9 H); 13 C NMR (101 MHz, CDCl 3 ) δ: 156. 48, 79.64, 73.73, 70.68, 42.14, 40.81, 28.87

Compound3

Phthalic anhydride (1.13 eq., 0.67 g) was dissolved in 20 ml toluene, compound 2

(1 eq., 1.0 g) was dissolved in 60 ml toluene, and was added to the mixture. Then refluxed at 120℃ for 3 hrs, then the mixture was evaporated, and the residue was purified by a silica gel chromatography and the pure compound was obtained as a solid in 53 % yield; 20, 155.94, 133.92, 132.07, 123.19, 70.22, 69.88, 67.88, 40.33, 37.13, 28.39

Compound4

TFA (18 ml) was added into a solution of compound 3 (0.8 g, 1 eq.) in 10 ml CH 2 Cl 2, then stirred at RT for 1 h. Then the mixture was evaporated under reduced in 50 ml DCM, and added dropwise to the reaction under agitation over 1 h, then stirred for 3.5 hrs at room temperature. Then filtered, to the filtrate, 8 g NaOH in 30

ml water was added, the aqueous layer was separated with CH 2 Cl 2 (2 x 100 mL).

The combined organic extracts were washed with brine (100 ml), dried over (MgSO 4 ) and the solvent was evaporated and obtained as a yellow oil in 70 % yield.

Then, potassium phthalimide (1 eq., 18.13 g) was dissolved in 25 ml DMF, N-(3-chloropropyl)acrylamide (1 eq., 14.4 g) was dissolved in 25 ml DMF, and quickly added to the last solution. Then the reaction was stirred for 15 hrs at 75℃.

Then filtered, and the residue was rinsed with DMF, and the filtrate was evaporated and obtained as a white solid in 47 % yield; 1 H NMR (400 MHz, CDCl 3 ) δ:

7.86-7.84 [START_REF] Ksoy | Stability of α-amylase immobilized on poly(methyl methacrylate-acrylic acid) microspheres[END_REF]2 H),2 H),2 H),1 H),J = 12 Hz,2 H),2 H),2 H); 13 C NMR (101 MHz, CDCl 3 ) δ: 168.97, 165.76, 134.36, 132.01, 131.16, 126.43, 123.53, 36.04, 34.98, 28.30 Compound 2 (1 eq., 0.12 g) in 20 ml CH 2 Cl 2 was treated with 20 ml saturated NaOH (aq.), then separated and dried over MgSO 4 . Then concentrated under reduced pressure and redissolved in 10 ml methanol. Compound 6 (5 eq., 0.12 g) was dissolved in 15 ml methanol, and was heated at 75 ℃ until the solid disappeared. Then compound 2 solution was added to the mixture, and the mixture was stirred for 7 days at 75℃. Then evaporated under reduced pressure, the residue was purified by a silica gel chromatography and the pure compound was obtained for 24 hrs, then evaporated to afford yellow oil (compound 21) in 94 % yield without further purification for next step. The residue (compound 21) (1eq., 1 g) and TEA (1.2 eq., 0.56 g) were mixed in 20 ml DCM, acryloyl chloride (1 eq., 0.38 g) was added dropwise, then stirred overnight at RT, then evaporated and the residue was purified by a silica gel chromatography to afford a white solid in 84 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.81-7.78 (d,J = 9 Hz,2 H),J = 9 Hz,2 H),2 H), (dd, J = 3 Hz, 1 H), 5.37 (s, 1 H), 3.55-3.50 (m, 4 H), 3.28 (s, 6 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 167.92, 166.84, 141.17, 133.67, 130.40, 126.73, 126.57, 126.21, 102.08, 52.36, 40.50, 39.38

Compound23

Compound 2 (0.6 g, 1 eq.) was treated with 20 ml NaOH solutions, then separated, dried, and dissolved in 20 ml methanol, compound 22 (3 g, 5 eq.) was dissolved in 20 ml methanol, and was added to compound 2. The mixture was stirred at 75℃ for 20 days. Then solvents were evaporated, and the residue was purified by a silica gel chromatography and the pure compound was obtained as a white solid in 83 % yield without further purification; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.81-7.79 (d,J = 6 Hz,2 H),J = 9 Hz,2 H),5.29 (s,2 H),16 H),3.21 (s,12 H),2 H),4 H),2 H),4 H),1.32(s,9 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 173.47, 167.66, 156.09, 141.27, 134.07, 127.11, 126.69, 102.35, 78.93, 70.00, 68.50, 52.62, 50.49, 46.00, 40.53, 39.01, 33.42, 28.30 3.63-3.59 (m, 6 H), 3.50-3.47 (m, 2 H), 2.86-2.78 (m, 4 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 158. 57, 132.45, 129.32, 113.73, 73.47, 70.63, 70.31, 55.24, 53.31, 48.62, 41.79 269.1820.

Compound26

1-bromo-3-phenylpropane (10 g, 1.15 eq.) was dissolved in 100 ml dry dichloromethane and the mixture was cooled to 0℃ in an ice bath. A gas-trap was connected to the setup, TiCl 4 (15 g, 1.8 eq.) was added carefully but quickly.

Dichloromethylmethyl ether (5 g, 1 eq.) was added dropwise in approximately 20 minutes to the cold mixture, while the temperature was kept at 0℃. The mixture was stirred for 5 minutes, slowly heated to room temperature and subsequently stirred at 35℃ for 15 minutes. After this, the reaction mixture was slowly poured in a beaker filled with ice and subsequently transferred to a separation funnel and extracted with dichloromethane. The organic layer was collected and the aqueous phase was extracted two more times with dichloromethane. The combined organic layers were washed with a saturated NaHCO 3 solution, and dichloromethane was evaporated in vacuum. The crude product was purified by column chromatography.

The pure product was obtained as yellow oil in 91 % yield; 191.56, 147.62, 134.44, 129.73, 128.91, 33.81, 33.26, 32.36

Compound27

Ethylene glycol (10 g, 5 eq.) and Al 2 O 3 (8.8 g) were added to solution of compound 26 (9.5 g, 1 eq.) in 100 ml toluene. Then, the mixture was refluxed for 4 days, then filtered, evaporated in vacuum. The crude product was purified by column chromatography and the pure product was obtained as yellow oil in 73 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.34-7.31 (d, J = 9 Hz, 2 H), 7.14-7.12 (d, J = 6 Hz, 2 H), 5.70 (s, 1 H), 4.04-3.93 (m, 4 H), 3.31-3.26 141.69, 135.84, 128.61, 126.69, 103.69, 65.34, 34.08, 33.75, 33.03

Compound28

Compound 27 (2 g, 5 eq.) was added to the solution of compound 25 (0.4 g, 1 eq.) in 100 ml CH 3 CN, then K 2 CO 3 (1 g, 5 eq.) was added the mixture. Afterwards the mixture was stirred at 75 for 14 days. Then, filtered, concentrated under reduced ℃ pressure and the residue was purified by a silica gel chromatography and the pure compound was obtained as a white solid in 31 % yield without further purification;

1 H NMR (300 MHz, CDCl 3 ) δ: 7. 42-7.18 (m, 12 H), 5.77 (s, 2 H), 4.09-3.97 (m, 8 H), 3.76 (s, 3 H), 3.58-3.56 (m, 8 H), 2.71-2.52 (m, 12 H), 1.92-1.80 (m, 4 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 158. 61, 143.35, 135.46, 130.07, 129.91, 128.40, 126.55, 113.58, 103.73, 70.49, 70.41, 69.82, 65.25, 58.31, 55.08, 53.98, 53.46, 52.96, 33.30, 28.62 Compound 22 (5.4 g, 5 eq.) in 20 ml methanol was added to the solution of compound 25 (1 g, 1 eq.) in 10 ml methanol, then the mixture was stirred at 75℃ for 14 days. Then, concentrated under reduced pressure and the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 35 % yield without further purification; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.82-7.78 (q, J = 12 Hz, 4 H), 7.43-7.40 (q, J = 9 Hz, 4 H), 7.09-7.06 (d,J = 9 Hz,2 H),J = 9 Hz,2 H),5.33 (s,2 H),3.70 (s,2 H),18 H),3.25 (s,12 H),8 H),4 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 174.44, 173.79, 167.68, 158.86, 141.37, 134.14, 130.20, 127.10, 126.83, 113.78, 102.39, 70.20, 70.05, 68.66, 58.05, 55.18, 52.65, 52.39, 50.63, 49.97, 41.05, 38.97, 33.89 Compound 36 (4.5 g, 3 eq.) was mixed with compound 41 (1.5 g, 1 eq.) in 40 ml methanol. Then the mixture was stirred at 75 ℃ for 2 weeks. Afterwards evaporated in vacuum, and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 18 % yield; 1 H NMR (400 MHz, CDCl 3 ) δ: 4.45-4.42 (t, J = 9 Hz, 2 H), 4.09-4.05 (q, J = 12 Hz, 2 H), 16 H),2 H),4 H),6 H),J = 9 Hz,4 H),8 H),15 H); 13 C NMR ( 76MHz, CD 3 OD) δ: 172.45, 163.46, 102.68, 70.24, 70.20, 69.05, 61.38, 53.21, 50.53, 40.66, 39.04, 33.80, 31.14, 30.57, 24.70, 15.29, 14.63 Compound 42 (0.8 g, 1 eq.) was dissolved in 15 ml methanol, then 5 M NaOH (3 ml) was added. Then the mixture was refluxed for 18 hrs, afterwards adjusted to pH= 3.0, then concentrated under reduced pressure and the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 42 % yield without further purification; 13 C NMR (76 MHz, CD 3 OD) δ: 173.30, 102.76, 70.00, 68.96, 67.13, 61.23, 52.60, 50.18, 39.49, 38.79, 33.16, 30.96, 24.42, 14.53

Compound45

dropwise over 1 h. The mixture was stirred overnight at ambient temperature and then washed with an aqueous hydrochloric acid solution pH= 2.0 (30 ml) and washed with water (2*30 ml). The combined aqueous phases were extracted with dichloromethane (3* 50 ml and then 3*100 ml). The combined organic extracts were washed with brine (100 mL), dried over (MgSO 4 ) and the solvent was evaporated to afford yellow oil in a quantitative yield without further purification.

Compound55

To a solution of compound 54 (39 mmol of mesyl function) in absolute ethanol (100 ml) was added sodium azide (3.55 g, 54.6 mmol, 1.4 eq.). The mixture was heated at 80℃ under stirring for 48 hours, then the mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by chromatography over silica gel as colorless oil in 70 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 3.69-3.54 (m, 14 H), 3.36-3.33 (m, 2 H); 13 C NMR (126 MHz, CDCl 3 ) δ: 72.47, 70.69, 70.65, 70.58, 70.34, 70.04, 61.73, 50.65

Compound56

To a solution of compound 55 (1.06 g, 4.84 mmol ) in distilled tetrahydrofuran (20 ml) under inert atomosphere was added sodium hydride (60 % dispersed in mineral oil, 251 mg, 6.29 mmol, 1.3 eq.) in portions. The mixture was stirred for 10 minutes and allyl bromide (0.543 ml, 6.29 mmol, 1.3 eq.) was added. Stirring was continued overnight at ambient temperature before the mixture was quenched with water (4 ml), the mixture was concentrated under reduced pressure to half the volume and brine (100 ml) was added. The mixture was extracted with dichloromethane (2* 80 ml and then 2*50 ml). The combined organic extracts were washed with brine (100 mL), dried over (MgSO 4 ), filtered and the solvent was evaporated to afford slightly yellow oil in 84 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 5.95-5.85 (m, 1 H), 5.29-5.14 (m, 2 H), 5.14-3.99 (m, 2 H), 3.68-3.57 (m, 14 H), 3.39-3.35 (m, 2 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 134.76, 117.06, 72.22, 70.70, 70.67, 70.64, 70.02, 69.42, 50.69

Compound CA5

Under argon, compound 56 (1 eq., 0.6 g) was dissolved in HSi(OEt) 3 (10 eq., 4.14 ml), then Karstedt'cat (0.002 eq., 100 µl) was added. The mixture was heated at 60℃ overnight. Then the mixture was evaporated and the residue was purified by a silica gel chromatography and the pure compound was obtained as slightly yellow oil in 42 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 3.85-3.78 (m, 6 H), 3.78-3.57 (m, 14 H), 3.5-3.37 (m, 4 H), 1.75 -1.65 (m, 2 H), 1.25-1.20 (m, 9 H), 1.20-0.60 (m, 2 H); 13 C NMR (101 MHz, CDCl 3 ) δ: 74.09, 71.03, 70.46, 70.41, 58.78, 51.09, 23.30, 18.73, 6.82

Compound57

Sodium azide (4 g, 4 eq.) was added to the solution of 3-azidopropan-1-amine ( 2g, 1 eq.) in 30 ml H 2 O. Then the mixture was stirred at 75℃ for 20 hrs. afterwards NaOH pellet was added, extracted with ether (3*20 ml), and evaporated to obtain colorless oil in 76 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 3.34-3.29 (t, J = 15 Hz, 2 H), 2.77-2.72 (t, J = 15 Hz, 2 H), 1.72-1.63 (m, 2 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 49.13, 39.26, 32.28. 3-bromopropan-1-amine salt (10 g, 1 eq.) in 50 ml DCM, then TEA (10 g, 2.15 eq.) was added to the solution above slow over 1 h. Then the mixture was stirred at rt for 4 hrs. Then evaporated in vacuum and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 46 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 6. 19-6.15 (m, 2 H), 5.59-5.55 (m, 1 H), 3.43-3.37 (m, 4 H), 2.09-1.96 (m, 2 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 166.02, 134. 63, 130.86, 126.34, 123.61, 38.07, 32.21, 31.

Compound66

Compound 2 (0.8 g, 1 eq.) was treated with NaOH (aq.), then dissolved in 10 ml methanol. Then compound 60 (2.4 g, 5 eq.) was added to the solution of compound 2. Then the mixture was stirred at 75℃ for 2 weeks. Then evaporated in vacuum and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 74 % yield; 1 H NMR (300 MHz, CD 3 OD) δ: 3.56-3.44 (m, 12 H), 3.24-3.15 (m, 6 H) 65, 156.99, 78.67, 70.01, 69.65, 68.84, 59.01, 52.60, 50.13, 39.88, 35.96, 33.12, 31.92, 27.46

Compound69

At 0℃, H 2 SO 4 (15.7 ml) was added to 48 % HBr aqueous solution (67 ml), then mg of biocatalyst were mixed, then adjust the total volume to 10 ml, and sonicated for several seconds. Then, 1 ml mixture solution above was added to in 1 ml cuvette and absorbance was measured immediately by UV to determine activity of biocatalysts towards ABTS (see the general method in paragraph 2.6.1). Then after measurement, 1 ml was put back to the initial solution, then placed on a magnet for 30 mins to separate solution and recover biocatalysts, afterwards particles were washed with storage buffer 3-6 times for next use. Then this protocol was repeated for each recovery cycle. The average of absorbance can be obtained based on the original data after removal of blank reactions. According to the formula of calibration, the concentration of laccases grafted on MNPs can be calculated, afterwards the amount of laccases can be obtained. In each well, various amounts of particles were placed (0.002 mg/well, 0.001 mg/well, 0.0004 mg/well, 0.0008 mg/well), then we can calculate the ratio of laccase/MNPs, finally the average mass of enzymes on MNPs (47.3275 μg/mg) can be calculated. Then the specific activity was calculated as below: for activity measurement, we take 20 μL of particles, which has 40 μg of particles (20 μL*2 mg/ml), in 1 ml cuvette, 40 μg particles*47.3275 μg/mg particles= 1.8931 μg (Lac3), then for 1 L, we have 1.8931 mg of enzymes. The enzyme activity of sample 2 was obtained as: 14.1633 U/L, therefore, the specific activity is: 14.1633 U/1.8931 mg= 7.4815 U/mg (Lac3).

Mechanism of selective reactions used in chapter II

  présentent un énorme potentiel en biocatalyse en raison de leur simplicité d'utilisation, d'une capacité d'immobilisation enzymatique élevée, d'un transfert de masse efficace et d'une recyclabilité par un simple un champ magnétique externe. En effet, cette dernière propriété est un avantage majeur par rapport aux techniques conventionnelles plus fastidieuses (telles que comme la filtration, l'ultracentrifugation, etc).

Schéma 1 .

 1 Recyclage des MNPs par aimantation magnétique.

Schéma 2 .Schéma 3 .Tableau 1 .

 231 Représentation du biocatalyseur hybride MNPs-Laccase Dans un premier temps, nous avons donc proposé un choix d'agents de couplage permettant des conjugaisons spécifiques avec les biomolécules cibles et également permettant d'augmenter trois familles d'agents de couplage avec différents groupes fonctionnels terminaux (NH 2 , CHO, N 3 ). Les squelettes poly(éthylèneglycol) (PEG) poly(amidoamine) qui colloïdale en milieu polaire et en particulier Nous avons alors réalisé (Schéma 3). Ces MNP milieux aqueux. Ce paramètre est considéré lors de l'immobilisation enzymatique lors hybride. Principe de la f boules violettes L'enveloppe de silice permet réactionnel en créant une réactions enzymatiques fonctionnalisation par un greffage covalent des agents de d'ancrage (silane). Le greffage est basé sur une réaction d'hydrolyse entre la silice (SiO 2 ) hydro-alcoolique basique élevée des particules. Ce greffage efficace. Les conditions de greffage (solvant, tensioactifs…) ont été optimisées en tenant compte de la solubilité des agents de couplage Résumé augmenter le potentiel de charge enzymatique. Nous avons synthétisé trois familles d'agents de couplage avec différents groupes fonctionnels terminaux Les squelettes de ces agents de couplage sont composés de chaînes poly(éthylèneglycol) (PEG) et éventuellement d'une partie dendritique confèrent aux MNPs fonctionnalisées une grande stabilité polaire et en particulier aqueux. Nous avons alors réalisé la modification de surface des MNP es MNPs fonctionnalisés ont une bonne stabilité colloïdale milieux aqueux. Ce paramètre est d'une grande importance et sera également considéré lors de l'immobilisation enzymatique lors de la formation du Principe de la fonctionnalisation covalente des surfaces de MNPs ( boules violettes représentent les groupes fonctionnels (NH 2 , CHO, N enveloppe de silice permet d'isoler efficacement le noyau magnétique réactionnel en créant une barrière empêchant les oxydes de fer d'interférer réactions enzymatiques. Également, cette couche de silice permet par un greffage covalent des agents de couplages par leur fonction e greffage est basé sur une réaction d'hydrolyse et les fonctions silane des agents de couplage, alcoolique basique. Cette couche de silice permet aussi une stabilité colloïdale Ce bon état colloïdal des suspensions est nécessaire pour un greffage efficace. Les conditions de greffage (solvant, tensioactifs…) ont été optimisées en tenant compte de la solubilité des agents de couplage 4 le potentiel de charge enzymatique. Nous avons synthétisé trois familles d'agents de couplage avec différents groupes fonctionnels terminaux sont composés de chaînes d'une partie dendritique une grande stabilité modification de surface des MNPs (-Fe 2 O 3 /SiO 2 ) colloïdale dans les et sera également de la formation du biocatalyseur s (-Fe 2 O 3 /SiO 2 ). Les , CHO, N 3 ). noyau magnétique du milieu barrière empêchant les oxydes de fer d'interférer dans les . Également, cette couche de silice permet une couplages par leur fonction e greffage est basé sur une réaction d'hydrolyse-condensation fonctions silane des agents de couplage, en milieu une stabilité colloïdale est nécessaire pour un greffage efficace. Les conditions de greffage (solvant, tensioactifs…) ont été optimisées en tenant compte de la solubilité des agents de couplage dans le milieu réactionnel de greffage représentées dans le tableau 1. MNPs fonctionnalisées par des groupements amino, aldéhyde et azoture obtenues. L'efficacité du couplage covalent a été analyse de l'apparition couplage nouvellement introduit été utilisée pour caractériser l évalué les charges de surface par de surface jouent un rôle fondamental dans le facteur déterminant dans le contrôle de la stabilisation électrostatique. Enfin, la caractérisation des MNP pour confirmer l'intégrité des nanoparticules et de l épaisseurs) lors des procédures de greffag Différentes stratégies ont été employées pour la quantification des groupes fonctionnels disponibles à la surface par titrage. La densité des groupes amino a été Résumé réactionnel de greffage. Les différentes MNPs fonctionnalisées obtenues sont représentées dans le tableau 1.

5

  Les différentes MNPs fonctionnalisées obtenues sont . MNPs fonctionnalisées par des groupements amino, aldéhyde et azoture obtenues. par spectroscopie IR-DRIFT, par caractéristiques des agents de microscopie optique a de particules greffées. Aussi, nous avons . En effet, les charges d'adsorption et sont un facteur déterminant dans le contrôle de la stabilisation électrostatique. Enfin, la microscopie électronique (TEM) silice (mesure des ifférentes stratégies ont été employées pour la quantification des groupes fonctionnels disponibles à la surface par titrage. La densité des groupes amino a été déterminée indirectement par un couplage avec du 4-nitrobenzaldéhyde. Les concentrations de ce chromophore dans les milieux réactionnels ont été mesurées par spectroscopie UV-Vis. La quantification des fonctions aldéhydes a été réalisée par un dosage avec du dansyl-hydrazine, et les concentrations de ce fluorophore dans les milieux réactionnels ont été déterminées par émission de fluorescence. Enfin, un dérivé cliquable et clivable de coumarine a été utilisé pour quantifier la densité des groupes azoture par mesures des émissions de fluorescence des milieux réactionnels.

  de surface par un nouveau groupe lysine. Dans notre étude, le groupe arginine 157 de l'enzyme Lac3 est remplacé par une lysine permettant l'obtention du variant UniK157. Les méthodes d'immobilisation classiques des enzymes à la surface d'un matériau donné conduisent naturellement à une orientation aléatoire du catalyseur. La stratégie que nous proposons et qui consiste à immobiliser les enzymes Lac3 et UniK157 offre la perspective d'orienter l'enzyme dans un sens ou dans le sens opposé, à la surface des MNPs (Schéma 4).

Schéma 4 .

 4 Représentation de l'orientation de l'enzyme à la surface des MNPs.

Schéma 5 . 7 .

 57 Transformation de l'ABTS en radical cation ABTS .+ en présence d'oxygène et catalysée par la laccase. Afin d'étudier l'activité spécifique des biocatalyseurs hybrides, un test ELISA surface des MNPs après immobilisation des enzymes cibles 6). Dans ce type de test son activité immunologique pendant le processus de marquage. Tout au long du dosage, la liaison de l'antigène et de l'anticorps est réalisée sur un support solide (microplaque), et la détection est basée sur la couleur ou la fluorescence ou la luminescence après la réaction de l'enzyme et de son substrat. L'intensité de la couleur ou de la fluorescence ou de la luminescence est proportionnelle ou inversement proportionnelle à la concent Schéma 6. Test Elisa utilisé dans ce sur la microplaque, capture l'antigène LAC3 (greffé sur les MNPs). L'anticorps secondaire, le même anticorps polyclonalbiotinylé anti rendue possible grâce à l'enzymeconjugué AIP phosphate disodiumhexahydrate) est hydrolysé en un produit Nous avons donc réalisé les différents types de MNP été immobilisée sur les MNP douces via un bis-hétérolinker pontant, Résumé ImmunoSorbent Assay) a été utilisé pour déterminer la à la surface des MNPs après immobilisation des enzymes cibles de test, l'immobilisation de l'antigène ou de l'anticorps n'affecte pas son activité immunologique pendant le processus de marquage. Tout au long du dosage, la liaison de l'antigène et de l'anticorps est réalisée sur un support solide détection est basée sur la couleur ou la fluorescence ou la luminescence après la réaction de l'enzyme et de son substrat. L'intensité de la couleur ou de la fluorescence ou de la luminescence est proportionnelle ou inversement proportionnelle à la concentration du substrat testé. Elisa utilisé dans ce travail. [6] L'anticorps polyclonal anti-LAC3 (Ab capture l'antigène LAC3 (greffé sur les MNPs). L'anticorps secondaire, le même anticorps polyclonalbiotinylé anti-LAC3 (Ab-LAC3-biotin) sert pour la détection. La détection est à l'enzymeconjugué AIP-Streptavidin. Le substrat pNPP (4 phosphate disodiumhexahydrate) est hydrolysé en un produit détectable par spectroscopie UV nm. réalisé l'immobilisation enzymatique de Lac3 et MNPs greffées. Dans le cas des particules amino, la laccase a été immobilisée sur les MNPs par une alkylation réductrice dans des conditions hétérolinker pontant, le téréphtalaldéhyde. Les conditions 8 pour déterminer la charge à la surface des MNPs après immobilisation des enzymes cibles (Schéma , l'immobilisation de l'antigène ou de l'anticorps n'affecte pas son activité immunologique pendant le processus de marquage. Tout au long du dosage, la liaison de l'antigène et de l'anticorps est réalisée sur un support solide détection est basée sur la couleur ou la fluorescence ou la luminescence après la réaction de l'enzyme et de son substrat. L'intensité de la couleur ou de la fluorescence ou de la luminescence est proportionnelle ou inversement LAC3 (Ab-LAC3) déposé capture l'antigène LAC3 (greffé sur les MNPs). L'anticorps secondaire, le même biotin) sert pour la détection. La détection est Streptavidin. Le substrat pNPP (4-nitrophenyl par spectroscopie UV-Vis à 405 Lac3 et de UniK157 sur particules amino, la laccase a par une alkylation réductrice dans des conditions le téréphtalaldéhyde. Les conditions d'immobilisation (tampon, catalyseur, dispersion…) ont été optimisées afin de définir les meilleures conditions, et différentes stratégies d'immobilisation ont été proposées pour obtenir des catalyseurs efficaces ayant une grande stabilité après l'étape de greffage. Une bonne activité enzymatique vis-à-vis de l'ABTS a été observée, ainsi qu'une charge enzymatique élevée. Enfin, une bonne activité spécifique de ce biocatalyseur hybride a été déterminée. Cependant, il est à noter que l'activité enzymatique après réaction d'immobilisation a considérablement diminué par rapport à celle de l'enzyme libre, cela peut être la conséquence d'une dénaturation de l'enzyme (dépliement de la structure tridimensionnelle globulaire native), ou d'une limitation du transfert de masse provenant de l'immobilisation de l'enzyme sur le support. Néanmoins, ces résultats d'activité enzymatique sont à relativiser car lors de l'étape de couplage avec le bis-hétérolinker (téréphtalaldéhyde) avec les MNP-amino dans une condition d'alkylation réductrice, aucune liaison covalente n'a pu être observée par spectroscopie IR. Nous n'avons donc pas pu démontrer un lien covalent entre les billes MNPs-amino et les laccases. Dans le cas des MNPs fonctionnalisées avec des groupements aldéhyde, les laccases ont été immobilisées par condensation de groupes amino libres de la laccase dans des conditions douces d'alkylation réductrice. Malgré une forte diminution de l'activité enzymatique identique à celle des particules amino, une bonne activité enzymatique a été observée, une charge enzymatique élevée a également été obtenue dans le test Elisa. Par conséquent une bonne activité spécifique de ce biocatalyseur a été calculée. Les MNPs fonctionnalisées avec des groupements aldéhyde se sont révélés être une plate-forme appropriée pour immobilisation enzymatique. De plus, un effet d'orientation de l'enzyme a été observé sur l'activité en biocatalyse en faveur de l'enzyme Lac3. Enfin, une bonne stabilité de ce biocatalyseur hybride a été démontrée par une étude de recyclage par aimantation magnétique, en effet, l'activité catalytique du biocatalyseur est restée constante après 10 cycles de recyclage. Dans le cas des MNPs fonctionnalisées avec des groupements azoture, l'immobilisation de l'enzyme a été obtenue via une réaction clic entre la laccase modifiée par un groupement conditions d'immobilisation ont été optimisées observée pour les deux types de élevée a été mesurée et par conséquent Nous avons observé un effet d'orientation su ligand azoture linéaire. En effet, au d'immobilisation (100 µg/mg pour Lac3 contre 20 le rapport des activités spécifiques est de 1,2 en fav Schéma Représentation proportionnelle de l'i droite) sur de MNPs fonctionnalisées avec le ligand azoture linéaire. Par contre dans le cas du ligand azoture dendritique, une charge supérieure à la charge maximale théorique calculée a été observée dans le cas de Lac3 (200 µg/mg) et pour UniK157, a été observée (70 µg/mg) (Schéma peut-être démontré du fait de la gauche) dans les quelles l'orientation des enzymes ne peut être assurée. noter une meilleure immobilisation des lac fonctionnalisées avec des agents de couplages dendritiques par des agents de couplages linéaires. Ainsi, pour Lac3, l'immobilisation à 200 µg/mg et pour UniK157, l'immobilisation passe de 20 à 70 respectivement pour les particules possédant un agent de couplage linéaire etdendritique. Résumé un groupement alcyne et des groupes azoture à la surface des conditions d'immobilisation ont été optimisées et une bonne activité enzymatique a été types de particules azoture, également une charge enzymatique et par conséquent une bonne activité spécifique a Nous avons observé un effet d'orientation sur les particules fonctionnalisées linéaire. En effet, au-delà d'une différence notable d'efficacité µg/mg pour Lac3 contre 20 µg/mg pour Unik157) (Schéma 7) le rapport des activités spécifiques est de 1,2 en faveur de Lac3. Représentation proportionnelle de l'immobilisation de Lac3 (à gauche) et de Uni droite) sur de MNPs fonctionnalisées avec le ligand azoture linéaire. Par contre dans le cas du ligand azoture dendritique, une charge supérieure à la charge maximale théorique calculée a été observée dans le cas de Lac3 pour UniK157, une immobilisation supérieure à son analogue linéaire µg/mg) (Schéma 8). Par contre, aucun effet d'orientation du fait de la saturation enzymatique sur les MNPs quelles l'orientation des enzymes ne peut être assurée. noter une meilleure immobilisation des laccases (Lac3 et UniK157) par les MNPs fonctionnalisées avec des agents de couplages dendritiques que celles fonctionnalisées des agents de couplages linéaires. Ainsi, pour Lac3, l'immobilisation à 200 µg/mg et pour UniK157, l'immobilisation passe de 20 à 70 les particules possédant un agent de couplage linéaire 10 à la surface des MNPs. Les une bonne activité enzymatique a été particules azoture, également une charge enzymatique une bonne activité spécifique a pu être calculée. fonctionnalisées avec le delà d'une différence notable d'efficacité µg/mg pour Unik157) (Schéma 7) gauche) et de UniK157 (à droite) sur de MNPs fonctionnalisées avec le ligand azoture linéaire. Par contre dans le cas du ligand azoture dendritique, une charge enzymatique supérieure à la charge maximale théorique calculée a été observée dans le cas de Lac3 une immobilisation supérieure à son analogue linéaire 8). Par contre, aucun effet d'orientation ne MNPs-Lac3 (Schéma 8, quelles l'orientation des enzymes ne peut être assurée. Enfin, on peut ases (Lac3 et UniK157) par les MNPs que celles fonctionnalisées des agents de couplages linéaires. Ainsi, pour Lac3, l'immobilisation passe de 100 à 200 µg/mg et pour UniK157, l'immobilisation passe de 20 à 70 µg/mg les particules possédant un agent de couplage linéaire

Fig1

  Fig1 Surface models of the laccase LAC3 (K residues in lavender) and its variant UniK (K

Fig 2

 2 Fig 2 Orientation effect of laccase for the hybrid biocatalyst

Fig. 1

 1 Fig.1.1 Michaelis-Menten equation

  by living organism or chemical synthesis to overcome the impediments of the biocatalytic application in the industry, such as microorganism,

Fig. 1 . 3

 13 Fig.1.3 Enzyme immobilization by covalent binding[19]

Fig. 1 . 4 5 Fig. 1 . 5

 14515 Fig.1.4 Carrier-free immobilized enzymes [21] [22] ( (a) crystallization; (b) aggregation; (c) spray-drying; (d) direct cross-linking. AGG, aggregates; CRY, crystals; SDE, spray-dried

Fig. 1 . 6

 16 Fig.1.6 Lipase in aqueous media (a), and immobilization of lipase on hydrophobic carriers (b), hyperactivated-lipase [29]

Fig. 1 . 7

 17 Fig.1.7 Magnetic nanoparticles

  recent years, magnetic materials are widely used in biomedical (clinical diagnosis, targeted drugs and enzyme labels, protein purification), cytology (cell labeling, cell separation, medical imaging), bioengineering (enzyme immobilization), and separation engineering [44]. The specific structure of MNPs (core-shell), mainly based on Fe 3 O 4 and Fe 2 O 3 cores are more suitable for enzyme immobilization, whose core allows to operate easily by the external magnetic field

  3 O 4 magnetic nano-aggregates polymerized with polyethylene glycol, which was affected by the physicochemical properties of nanoparticles and the diameter of nanoparticles. Besides, the surface could allow MNPs to reach a high loading, to increase the catalytic efficiency, etc. Wang et al[START_REF] Wang | Immobilization of Lipases on Alkyl Silane Modified Magnetic Nanoparticles: Effect of Alkyl Chain Length on Enzyme Activity[END_REF] investigated the correlation between the effect of surface hydrophobicity, i.e. the alkyl chain length of the modifying molecules, and catalytic activity through immobilization of lipase onto functionalized Fe 3 O 4 nanoparticles by conjugation with various alkyl silane with different chain lengths by hydrophobic interaction. The experiment showed the activity and stability of immobilized enzymes are related to the length of the alkyl chain, besides, the reusability of enzymes increased after immobilization on the modified Fe 3 O 4 particles. All as we expect the immobilization on MNPs could offer good results, such as strong binding capacity, high stability, high activity, etc.

Fig. 1 . 8

 18 Fig.1.8 Three-dimensional representation of laccase [56]

Fig. 1 . 9

 19 Fig.1.9 Structure and types of copper center of laccases including relevant ligands [56] [70a]

Fig. 1 .

 1 Fig.1.10 Representative substrates of laccase [70a]

Fig. 1 .

 1 Fig.1.11 A proposed mechanism for the reduction of O 2 to H 2 O at the active site of laccases [60] [70]

Fig. 1 .

 1 Fig.1.12 Schematic representation of laccase-catalyzed redox cycles

Fig 2 . 1

 21 Fig 2.1 Schematic representation of coupling agents: a) linear coupling agents; b) 2-branched

Fig 2 . 2

 22 Fig 2.2 Schematic representation of surface modification (FG= functional groups)

Scheme 2 . 2

 22 Scheme 2.2 Synthesis route of two branched coupling agents bearing NH 2

  bromopropyl)isoindoline-1,3-dione. The reaction was performed at 75℃ for 4 days, the four branched intermediate compound 12 was obtained and purified by column chromatography on silica gel in 42 % yield. The deprotection of amino groups was carried out through the cleavage of boc-protecting group of the intermediate 12 in presence of TFA in DCM. A four branched coupling agent compound 13 was obtained and purified by column chromatography on silica gel as yellow oil with 91 % yield. In order to obtain a silylated four branched coupling agent, four branched compound 13 was reacted with triethoxy(3-isocyanatopropyl)silane. Triethoxy(3-isocyanatopropyl)silane was introduced to a mixture system of four branched intermediate compound 13 and TEA in DCM at room temperature. Then silylated four branched coupling agent yield as yellow oil after evaporation of DCM and TEA.

Scheme 2 . 6

 26 Scheme 2.6 Synthesis route of linear coupling agents with CHO

10 Scheme 2 . 7 Scheme 2 . 8

 102728 Scheme 2.7 Synthesis route of two branched coupling agents with CHO

Scheme 2 . 10 Scheme 2 . 11

 210211 Scheme 2.10 Synthesis route of two branched coupling agents with CHO

Scheme 2 . 12

 212 Scheme 2.12 Synthesis route of two branched coupling agents with CHO

Scheme 2 . 13

 213 Scheme 2.13 Synthesis route of two branched coupling agents with CHO

Scheme 2 .

 2 Scheme 2.21 Synthesis route of linear coupling agents with N 3

Scheme 2 .

 2 Scheme 2.25 Synthesis route of two branched coupling agents with N 3

CA6Scheme 2 . 30

 230 Scheme 2.30 Coupling agents for functionalization of MNPs

  ........................................................................................................... 3.2 Grafting of dendritic coupling agents on MNPs ................................................. 3.2.1 Principles and hydrolysis condensation reaction .......................................... 3.2.2 Grafting of NH 2 coupling agents on nano-particles and characterization .... 3.2.2.1 FT-IR (DRiFT) study of amino MNPs .............................................. 3.2.2.2 Zeta potential measurements of phhtalimide and amino functionalized γ-Fe 2 O 3 /SiO 2 MNPs ....................................................................................... 3.2.2.3 Transmitted light microscopy study of phthalimide functionalized γ-Fe 2 O 3 /SiO 2 MNPs ....................................................................................... 3.2.2.4 Quantification Analysis of amino functionalized γ-Fe 2 O 3 /SiO 2 MNPs ....................................................................................................................... 3.2.3 Surface modification of core-shell γ-Fe 2 O 3 /SiO 2 magnetic nano-particles with CHO coupling agents and characterizations ......................................................... 3.2.4 Surface modification of core-shell γ-Fe 2 O 3 /SiO 2 magnetic nano-particles with N 3 coupling agents and characterizations .............................................................. 3.3 Conclusion ..........................................................................................................

2 O 3 /Fig 3 . 1

 2331 Fig 3.1 Schematic representation of core-shell γ-Fe 2 O 3 /SiO 2 magnetic nano-particles

Fe 2 O 3 /

 23 SiO 2 MNPs to produce new functionalized nanoparticles for application in biochemistry and nano-materials. Characterization and quantification of functionalized MNPs have been performed. However, beyond a very well established proof of concept, a generalization of the use of enzyme/MNP hybrids may be hampered by strategic choices: the use of functional coupling agents and non-specific grafting procedures for enzymes. These procedures can result in low density of randomly distributed enzymes on the MNP surface and a low specific activity of enzyme/MNP hybrids. Thus, many efforts are needed for a rational design of coupling agents and functionalization of nanoparticles to optimize fundamental parameters of conjugation with enzymes, especially focusing on the orientation on the surface of nanoparticles, the density of enzymes and microenvironment close to the catalytic site of hybrid biocatalysts. The choice of the functionalization strategy is of great importance because it affects the number of functional groups available on the surface, the colloidal properties, the subsequent accessibility and the bioactivity and the orientation of the target enzyme.At the beginning, we will present the detailed informations about surface modification strategies of MNPs with different target coupling agents. Next, the detailed grafting procedures will be illustrated, including the study of various parameters which have impacted the surface modification, such as temperature, reaction time, solvents, etc. Fourier Transformed Infrared Spectroscopy (FT-IR) characterizations of functional groups of the functionalized MNPs will be presented, besides, the colloidal states of functionalized MNPs were analyzed by Transmitted Light Microscopy in water and Zeta Potential (ζ) was used to characterize the surface charge of MNPs and the stability of the colloidal systems.

Fig 3 . 2

 32 Fig 3.2 General mechanism of silanization formation

Fig 3 . 3

 33 Fig 3.3 General mechanism of silanization formation: (a) amine catalysis of silylation reaction;

Fig 3 . 4 Scheme 3 . 1

 3431 Fig 3.4 Linear coupling agents with ethoxy anchoring groups dissolved in ethanol

Scheme 3 . 2

 32 Scheme 3.2 Deprotection of NH 2 coupling agents on MNPs

Fig 3 . 5

 35 Fig 3.5DRiFT FT-IR spectra of grafted MNPs: (a) MNP@CA1 and MNP@lin-NH 2 ;(b)

Fig 3 . 6

 36 Fig 3.6 Mechanism of deprotection of NH 2 coupling agents in presence of methylamine

Fig 3 . 7

 37 Fig 3.7 TEM images of grafted MNPs

Fig 3 . 8

 38 Fig 3.8Zeta potential of (a) native MNPs, MNP@CA1, MNP@CA2, MNP@CA3;(b)

Figures 3 .

 3 Figures 3.9 to 3.13 exhibited the colloidal state of functionalized MNPs in Mili-Q water pH= 5.5. According to these images recorded with the magnification (400×), the particles are well dispersed in water. Only few aggregations are observed. And

Fig 3 . 9 Fig 3 . 10 Fig 3 . 11 2 Fig 3 . 12 2 Fig 3 . 13 3

 3931031123122313 Fig 3.9 Representative Transmitted Light Microscopy image of MNP@APTES

3. 4 )Scheme 3 . 4 Fig

 434 Scheme 3.4 Grafting of silylated linear

Fig 3 .

 3 Fig 3.14 DRIFT-IR spectrum of MNP@CA4

Fig 3 . 15

 315 Fig 3.15 Representative Transmitted Light Microscopy image of MNP@CA4

Fig 3 . 16 Fig 3 . 17

 316317 Fig 3.16 Zeta potential of MNP@CA4

Scheme 3 . 5 Scheme 3 . 6

 3536 Scheme 3.5 Dosing of aldehyde MNPs (MNP@CA4) by back titration

Fig 3 . 18

 318 Fig 3.18 DRIFT IR spectra of grafted MNPs: (a) MNP@CA5; (b) MNP@CA6

Fig 3 . 19 Fig 3 . 20 Fig 3 . 21 Scheme 3 . 6

 31932032136 Fig 3.19 Zeta potential

Fig 3 .

 3 Fig 3.22 Scanning/ Transmission electron microscopy (S/TEM) images of MNP@CA5:

Fig 4 . 1

 41 Fig 4.1 Illustration of LAC3 bearing two lysine groups opposed to T1 site and Unik157

Fig 4 . 2

 42 Fig 4.2 General principle of kinetics study of grafted biocatalyst towards ABTS

  . Then a blocking protein used to cover remaining unsaturated binding sites of a microplate after microplate wells being coated with free or grafted laccases. A biotinylated anti detection antibody was afterwards an alkaline phosphatase enzyme (A employed to combine with the transformation of the substrate p yellow water-soluble reaction product to produce the absorbance, which is p laccases (see experimental details in appendix).

Fig 4 . 3

 43 Fig 4.3 Scheme of ELISA detection[5]

Fig 4 . 4

 44 Fig 4.4 The transformation of the substrate p-Nitrophenyl Phosphate (PNPP) to form a yellow

Fig 4 . 5

 45 Fig 4.5 Illustration of enzyme immobilization onto amino MNPs in a reductive alkylation

Fig 4 . 6

 46 Fig 4.6 Iridium complex used in a reductive alkylation (2b used in our study)

Fig 4 . 7

 47 Fig 4.7 Illustration of new strategy for enzyme immobilization onto amino MNPs

(

  see fig4.8). Aldehyde particles (MNP@CA4) were grafted with enzymes via condensation of free amino groups from the laccase under reductive alkylation in a mild condition and in the presence of iridium catalysts. Due to the tendency to aggregate in solutions and to avoid the loss of specific area surface, particles still need to be treated by vortexing or sonication for several mins to keep them dispersed well in corresponding buffers. This immobilization reaction was first performed by Lu Ren (PhD, Univ. Aix-Marseille, 2019) for the lac3 and unik161[6a].

Fig 4 . 8

 48 Fig 4.8 The reductive alkylation used for the enzyme immobilization onto aldehyde MNPs

Fig 4 . 9

 49 Fig 4.9 Activity of grafted MNPs during recovery cycles

Fig 4 . 10

 410 Fig 4.10 Optical microscopy image of grafted MNPs after 10 times recovery cycles (X400)

4. 4 . 1

 41 Optimization of the click reaction conditions of laccases immobilizationExperimental conditions, such as preparation of particles, amounts of particles, reaction temperature, enzyme loading measured by Elisa test, etc, have been established for the amino particles and aldehyde particles. In this subsection, we would focus on the optimization of click reaction conditions for the immobilization of alkyne modified laccases and azide MNPs (MNP@CA5 and MNP@CA6) by varying catalysts amount, buffers, reaction time and temperature in regard of activity and specific activity obtained(Fig 4.11).

Fig 4 . 11

 411 Fig 4.11 Route for laccases immobilization on azide particles

Fig 4 . 12

 412 Fig 4.12 Illustration of laccases immobilization onto azide particles

Fig 4 .

 4 Fig 4.11Mechanism of copper-catalyzed azide-alkyne cycloaddition[7]

Fig 4. 12

 12 Fig 4.12 Proportion

  investigated with the substrate ABTS, Elisa test, finally the specific activity and orientation By comparing the activity of free enzymes and immobilized ones, it can be seen all enzyme activities and the specific activity decreased dra immobilization, which can be explained by enzymes, the microenvironment at the interface of enzyme/MNPs immobilization and the mass Chapter IV Oriented immobilization of laccase onto functionalized MNPs

,

  the microenvironment at the interface of enzyme/MNPs the mass transfer limitation, and a crowed environment close to Chapter IV Oriented immobilization of laccase onto functionalized MNPs @CA5, laccases almost have same activity towards ABTS, and compared to that of free laccases, the enzyme activity gher for Lac3 than for grafting, in contrary Unik157 high loading observed induces a crowded environment close to active sites explored with several particles, he evaluation of enzyme activities was also the enzyme loading was detected by were also studied. the activity of free enzymes and immobilized ones, it can be seen all enzyme activities and the specific activity decreased drastically after hanges in the spatial structure of , the microenvironment at the interface of enzyme/MNPs after limitation, and a crowed environment close to the catalytic site.

  this study, different functional coupling agents were synthesized (amino, aldehyde, azide). Then, the surface modification of MNPs was achieved through hydrolysis condensation reaction and we investigated the influence of the functional coupling agents on the surface modification of MNPs as well as a dendritic effect in the aim of immobilization of laccase. Finally, we immobilized laccases onto grafted MNPs by oriented covalent binding and we proposed to control the micro-environment at the particle/enzyme interface to study and optimize the access of the substrate to enzyme catalytic sites. The biocatalytic activities towards ABTS were explored in acetate buffer by UV spectrophotometry. Then, the enzyme loadings were measured by Elisa tests through an antigen-antibody reaction.First of all, we started with the design and synthesis of coupling agents bearing linear or dendritic structures. Three families of coupling agents were prepared successfully, as -NH 2 , -CHO, -N 3 . The inner backbones of the coupling agents (linear or dendritic) are composed of poly(ethylene glycol) (PEG) chains as well as a poly(amidoamine) (PAMAM) dendritic parts that confers a high colloidal stability in aqueous medium to the functionalized MNPs. Most of reactions for biotechnology are performed in aqueous medium, then PEG chains or/and PAMAM chains are important blocks in the design of hydrophilic coupling agents.In the second part of this project, we focused on the surface modification of -Fe 2 O 3 /silica shell, 300 nm. The functionalization was achieved via hydrolysis condensation reaction of silane groups of coupling agents onto the silica shell of MNPs. Then, the characterizations by FT-IR spectroscopy and Zeta potential confirmed the surface modification. Optical microscopy was used to control the suspension of grafted MNPs in water, and S/TEM displayed size characterization of MNPs and particularly the modification of the thickness of silica shells. Finally different strategies were investigated for the quantification of functional groups available on the surface of grafted MNPs. For the amino particles, nitrobenzaldehyde was employed to quantify the amount of amino groups at the surface by a back titration method. For the aldehyde particles, the quantification of functional groups was performed by a dosing with dansyl-hydrazine by a back titration method. At last, for the azide particles, a clickable and cleavable fluorescent compound coumarin derivative was used for quantification by a click reaction.

Fig. 2 . 1 Fig. 2 . 2 Fig. 2 . 3 Fig. 2 . 4 Fig. 2 . 5 Fig. 2 . 6

 212223242526 Fig. 2.1 Mechanism of the protection of a primary amine with di-tert-butyl dicarbonate

Fig. 2 . 7 Fig. 2 . 8 Fig 3 . 5 Fig 3 . 6 Fig 3 . 13 Fig 3 . 15 Fig 3 . 26 Fig 3 . 27 Fig 3 . 28

 27283536313315326327328 Fig. 2.7 Mechanism of activated ester produced with DCC and NHS and mechanism of

Fig 3 .Fig 3 . 30 Fig 3 . 31 Fig 3 . 32

 3330331332 Fig 3.29 Zeta potential measurement of MNP@CA3 in water at various pH

Fig 2 . 1

 21 Fig 2.1 (a) The scheme of microplate used in ELISA test; (b) Image of microplate

1 Enzymes and Biocatalysis 1.1.1 Nature of the enzymes

  Bibliographical study .................................................................................................... 1.1 Enzymes and Biocatalysis ....................................................................................... 1.1.1 Nature of the enzymes ...................................................................................... 1.1.2 Biocatalysis .................................................................................................... 1. 2 Immobilization of enzymes .................................................................................. 1.2.1 Methods of immobilization ............................................................................ 1.2.2 Supporting materials for enzymes immobilization......................................... 1.2.3 Enzymes immobilization on magnetic nano-particles (MNPs) ......................

	Chapter I Bibliographical study
	Contents:
	Chapter I Our knowledge of enzyme chemistry derived from yeast fermentation, an
	enzyme is a unique and effective substance which catalyzes chemical reactions.
	Enzymes are proteins with an unfolding shape, a proper size, or displaying highly
	specific function with respect to substrates and co-factors. As dynamic molecules,
	enzymes have capacity of distinguishing its targeted substrates and binding to a

1.3 Laccases and its immobilization ........................................................................... 1.3.1 Introduce of laccases ...................................................................................... 1.3.2 Laccases immobilization ................................................................................ 1.3.3 Applications of laccases ................................................................................. 1.4 Objectives of the research work ............................................................................

1.

specific substrate depending on their interaction with other molecules. A general spatial structure of enzymes or proteins is constructed by forming an 'active center' and an 'essential group', or cofactor, which has a three-dimensional structure of an enzyme molecule, to bind a potential substrate and catalyze a conversion of a substrate into a product. However, the active center of enzymes is only a small part of an enzyme molecule and the specificity of an enzyme-catalyzed reaction is actually determined by the binding group, catalytic group and its spatial structure of the enzyme. Functional groups other than the enzyme active center are also necessary to form and maintain a spatial conformation of an enzyme, they are called essential groups.

The transition state and mechanism of enzymatic catalytic reactions are crucial to examine what happen to enzymes and substrates and to understand how enzymes work, such as an early model proposed by Emil Fisher in 1892. He proposed a 'key-lock' scheme, a substrate acting as a key, whereas the active group of an enzyme is a lock, responding to a specific substrate. In 1902, another theory was proposed by Henri that in an enzymatic reaction, an enzyme (E) first combines with a substrate (S) to form an unstable intermediate called central complex (ES), which finally produces a product (P) and releases an enzyme. The formula is: S + E = ES →E + P.

In 1913, a fundamental equation (equilibrium-state model), called

  1.1) was developed by Michaelis and

	Menten equation (V max = Maximum enzyme-catalyzed reaction speed
	Substrate concentration, K m = Michaelis constant)
	me disadvantages of the key-lock model, 'induced
	Koshland. D. E [1a] as: 1) The active center of

structurally malleable and flexible; 2) When a substrate is close to le enzyme configuration changes; 3) An enzyme and a substrate conform to form an intermediate complex to catalyze a reaction.

In 1961, French scientist [1b]

disclosed "Genetic regulation mechanism in protein synthesis", proposing the theory of 'operons' and initiating research on gene s are produced by living organism or chemical synthesis to overcome the impediments of the biocatalytic application in the industry, such as microorganism,

  To be exact, fermentation differs from modern biocatalysis, in a fermentation process a microorganism is supplied with sugar and other nutriments, then products are obtained after metabolism in a low yield, by contrast, bio-transformation is to use particular and specific enzymes to convert specific

reactants to desired products in a high yield. Enzymes can act outside biological organisms to catalyze many synthetic reactions, which are carried out by conventional chemistry.

Unlike conventional chemistry, biological conversions are catalyzed by enzymes, which generally work under mild conditions, moderate temperature, medium pH range, generally no heavy metals required, need less energy, and produce less contaminants. Thanks to new technologies booming in the industry, it is now possible to produce a great variety of enzymes at acceptable prices. In the past decades, developments in the biotechnology and material-engineering have made the enzyme-catalyzed reaction more efficient, more eco-friendly and more cost-effective as a widespread and key technology in green and sustainable synthesis of fine chemicals, pharmaceuticals, vitamins, bioengineering, clinical diagnosis, chemical analysis, environmental protection, energy industry, food industry, and others. However, biocatalysis in industrial applications still are challenging and hampered by vulnerable operational stability, ability for recycling

1

  Cu 2+ and type 2 Cu 2+ respectively receive an electron from a substrate and Cu2+ is Cu + and type 3 Cu 2 2+ to generate a photoactive oxygen intermediate (Cu 2 O) 3+ , and release one water, type 1 Cu + and type 3 Cu 2 2+ is oxidized to Cu 2+ . Oxygen intermediate is connected with type 3 Cu 2 2+ , and leave from type 3 Cu 2 4+ until the fourth electron is reduced to water, and laccase is in oxidation state. The redox potential of T1 site directly determines whether a reaction can occur or not and the catalytic rate, in general, laccase could catalyze those compounds whose potential is lower than the redox potential (E

	reduced to Cu 1+	, then type 1 Cu 2+ and type 2 Cu 2+ transfer electrons to type 3 Cu 2 4+
	through intramolecular electron transfer. Afterwards type 3 Cu 2	4+ is reduced to Cu 1+ .

When type 2 Cu 2+ and type 2 Cu 2+ received one electron from a substrate again, all four copper ions and the laccase was in a reduced state. In the presence of O 2 , O 2 quickly accepts three electrons from type 1 o ) of laccase T1 site, and fungal laccases have much higher redox potential than plants or bacteria laccases, therefore, and industrial production of laccases with high Eo still need to be documented.

  One of the aims of our work is to immobilize enzymes onto magnetic particles (MNPs). Functionalization of MNPs without changing nature of MNPs is an essential step to make suitable and to satisfy requirements of our objective. Thence, the surface modification of MNPs can be achieved through grafting of elaborate coupling agents, which offer not only various functional terminal groups, but allow to tune properties of functionalized MNPs. Indeed, surface charge, hydrophilic/hydrophobic ability, even colloidal behavior, etc, can be modulated.

	Fernández-Fernández, et al. "Recent developments and applications of immobilized 2.1 Introduction
	laccase." Biotechnology Advances 2013 31 (8): 1808-1825; (c) A. Arca-Ramos, E.
	M. Ammann, C. A. Gasser, et al. Assessing the use of nanoimmobilized laccases to
	remove	micropollutants	from	wastewater. Environ	Sci	Pollut	Res
	2016 23, 3217-3228; (d) S. Ba, et al. "Laccase immobilization and insolubilization:
	from fundamentals to applications for the elimination of emerging contaminants in
	wastewater treatment." Critical Reviews in Biotechnology 2013 33 (4): 404-418; (e)
	M. Taheran, et al. "Covalent Immobilization of Laccase onto Nanofibrous
	Membrane for Degradation of Pharmaceutical Residues in Water." ACS Sustainable
	Chemistry & Engineering 2017, 5 (11): 10430-10438.
		ChapterⅡ Synthesis of linear and
		dendritic coupling agents

Finally this functionalization of MNPs will affect properties of the grafted enzyme by modulating micro-environment of the catalytic site. The design or the choice of coupling agents will provide stable covalent bond between the enzyme and the carrier, a specificity to couple the target molecule and a possibility to modulate the surface of functionalized MNPs. We propose to synthesize coupling agents with silane anchoring groups, three types of functional groups (amine, aldehyde, azide) and various hydrophilic linear or dendritic linkers (fig.

2

.1).

  The reaction was run in anhydrous methanol at 50℃ for 3 days. Excess of methyl acrylate and solvents were evaporated and light yellow oil compound 9 was obtained in 87 % yield without further purification. After that, a two branched

First intermediate compound 9 was synthesized (scheme 2.3) through a Michael addition between the primary amine, compound 2 and methyl acrylate added in excess.

intermediate 10 could be synthesized by amidation, which was carried out in anhydrous methanol in presence of a large amount of ethane-1,2-diamine at room temperature for 5 days, then the mixture was evaporated, and intermediate 10 was obtained in 100 % yield as light yellow oil without further purification. Scheme 2.3 Synthesis of four branched coupling agents bearing NH 2

  chromatography on silica gel as yellow oil in 53 % yield. Hereafter an intermediate compound 42 was obtained and isolated by column chromatography on silica gel as yellow oil in 18 % yield via a Michael addition between compound 41 and compound 36 in methanol by heating at 75℃ for 2 weeks. Sodium hydroxide solution was employed to remove the ethyl formyl and release amines in methanol by refluxing. Then, an amine intermediate compound 43 was obtained and isolated by column chromatography on silica gel as light yellow oil in 42 % yield, afterwards a hydrolysis of compound 43 was investigated under various acidic conditions (hydrochloric acid from 6 N to 0.1 N; or 1 % hydrobromic acid; or 10 eq. Synthesis route of two branched coupling agents with CHO At last we tried the introduction of aldehyde part via click chemistry as described in scheme 2.18.

	Scheme 2.18 Synthesis route of two branched coupling agents with CHO
	Scheme 2.16 Synthesis route of two branched coupling agents with CHO First, compound 48 was synthesized by substitution of sodium azide to
	compound 26 and isolated by column chromatography on silica gel as yellow oil in
	1,2-bis (2-aminoethoxy)ethane was reacted with ethyl carbonochloridate in DCM 96 % yield. In parallel, a substitution of APTES to propargyl bromide in presence of

and TEA to produce a mixture of mono-protecting amine and di-protecting amine in a good yield, then mono-protecting amine compound 41 was obtained and isolated by column acetic acid), but we did not find the signal of aldehyde in NMR spectrum of the final product and it was speculated the aldehyde group was decomposed. Then another synthesis through a Michael addition was explored and an intermediate compound 45 was obtained and isolated by column chromatography on silica gel as yellow oil in 44 % yield. However, the removal of ethyl carbonochloridate with sodium hydroxide solution was not possible, finally the backbone was decomposed and no signal of aldehyde was detected.

Then another Michael addition was investigated (scheme 2.17) in methanol by heating at 75℃ for two weeks, after evaporation to remove solvents, compound 46 was obtained and isolated by column chromatography on silica gel as light yellow oil in 20 % yield, then the removal of the protecting group Benzyl chloroformate from amine was explored in presence of 10 % Pd/C and formamide, but benzyl chloroformate was still detected in 1 H MNR, indicating the incomplete deprotection of the amine. Scheme 2.17 calcium hydride as catalysts was carried out, 2-branched backbone compound 49

  Scheme 2.19 Synthesis route of four branched coupling agents with CHO We started with the reaction of compound 10 and an excess of methyl acrylate in methanol by heating at 50℃ to build a four branched backbone. Compound 51 was obtained as light yellow oil in a quantitative yield without further purification. Then methanol was carried out at room temperature for 5 days, finally a four branched intermediate compound 52 was obtained as black oil in 75 % yield after evaporation of excess of ethane-1,2-diamine plus methanol without further purification. Then, we attempted introducing CHO groups by reaction of this four-branched intermediate with 4-formylbenzoyl chloride in presence of DMAP, yet no coupling was achieved via this strategy.A second strategy was investigated as shown in scheme 2.20. A nucleophilic substitution reaction of compound 10 and the intermediate 27 in alkaline conditions was explored by heating at 75℃ for 2 weeks. Then, after filtration and evaporation, the target compound 53 was obtained and isolated by column chromatography on silica gel as light yellow oil in 28 % yield. Finally, we tried the deprotection of compound 53 with TFA in DCM at room temperature, the excess of TFA and solvents were evaporated, and a four branched compound was obtained but CHO groups were missing in 1 H spectrum, meaning that TFA may have damaged the CHO functions.

	Scheme 2.20 Synthesis route of four branched coupling agents with CHO
	Until now, no four branched coupling agents with aldehyde were obtained though
	the various synthesis routes investigated.

Table 3 . 1

 31 Data of diameters and silica thickness of grafted MNPs

3.2.2.

2 Zeta potential measurements of phhtalimide and amino functionalized γ-Fe 2 O 3 /SiO 2 MNPs

According to the theory of Stern electrical double layer, composed of namely the Stern layer and the diffusion layer, Zeta potential is defined as potential measured at the sliding plane. The potential at a certain point in the fluid is called Zeta potential

Table 3 . 2

 32 Isoelectric points for grafted particles

Table 3 . 3

 33 Concentration of NH 2 functions per gram of amino MNPs

Table 4 . 1

 41 gathered in table 4.1. Preliminary immobilization reaction conditions

	MNPs	Enzyme	buffer		Ir cat.
	2 mg	6 M	RA	or	10 eq./0.1
			MES	or	eq. to
			ethanol		enzymes

  1 M MES pH= 6.0 buffer, 0.1 M MES buffer + 0.1 M HCOONa pH= 6.0, and activities of hybrid biocatalysts were investigated. All these experiments are gathered in table 4.2. In all cases, Ir catalyst was used(Fig 4.6) in order to prevent the enzyme leakage and make sure terephthalaldehyde can bridge laccases and MNPs covalently. The Ir catalyst is of importance in an enzyme immobilization reaction and was used to reduce imine to produce a stable secondary amine in table 4.2. According to the literature [4b], sodium formate (present in RA buffer) as a stoichiometric hydride source was mandatory in a reductive alkylation catalyzed by an iridium complex.

	MNP@	1 st step	2 st step	Enzyme	Enzyme	Specific
	APTES@			activity	loading	Activity
	Lac3			(U/L)	(μg/mg)	(U/mg)
	1	RA buffer	RA buffer	no

Table 4 . 2

 42 Enzyme immobilization reaction conditions and catalytic activities of

	MNP@APTES@Lac3

Table 4 .

 4

	3 Enzyme immobilization reaction conditions and catalytic activities of
	MNP@APTES@Unik157

Table 4 .

 4

	4 Enzyme immobilization reaction conditions and catalytic activities of
	MNP@APTES-C2-NH 2 @Lac3
	MNP@APTES-C2-NH 2 @Lac3 system exhibited various activities under

Table 4 . 5

 45 Enzyme immobilization reaction conditions and catalytic activities of MNP@APTES-C2-NH 2 @Unik157 Then we performed IR spectroscopy in order to detect the vibration bands of carboxyl groupsν C=O that should be present after the first step of immobilization. Indeed, the first step corresponded to the coupling of terephthalaldehyde with the amino groups present at the surface of the particles. But in all cases reported in table 4.6, no characteristic vibration band of C=O were observed, suggesting no reaction between terephthalaldehyde and amino groups occurred in the first step. This suggests that no covalent binding of enzymes on the surface was achieved.

		RA	0.1	M	Ethanol 0.1 M MES +	25	mM
		buffer	MES			25	mM	HCOONa
			buffer			HCOONa pH=	in
						6.0 buffer	ethanol/H 2 O
	MNP@APTES	no			no	no	no
	MNP@		no
	APTES-C2-NH 2

Table 4 . 6

 46 Detection of theνC=O vibration band in amino particles functionalized with terephthalaldehyde by IR spectroscopy

3.2 Activity towards ABTS and specific activity of free and immobilized laccase on aldehyde MNPs and enzyme loading measured by ELISA test

  1 ml RA buffer by pipetting, ready for use. 2 mg MNPs were separated by 4, every tube has 0.50 mg/250 μl, 3 for triplicate reactions, one for blank. The reaction system was mixed well by vortexing, and incubated on the thermomixer at 30 ℃ for 24 hrs. After reactions, the supernatant was removed. After that, particles were washed with storage buffer (each time 0.5 ml) 5 times to remove excess of laccase. Finally, particles were kept in 250 μl of storage buffer for next test.

	The activity measurement and calculations were performed, and the activities of
	grafted laccase on MNPs are presented in table 4.7.
	Entry MNP@	Enzyme	Enzyme loading	Specific	Ratio of SA
	CHO	activity	(μg/mg)	Activity	lac3/unik157
		(U/L)		(U/mg)
	1 Lac3	7.8	65.3	3.0
		7.8	75.6	2.6
	2 Unik157	6.2	129.2	1.2	1.4
		7.1	151.2	1.2
		8.9	138.2	1.6

4.

Table 4 .

 4

	7 Activity, enzyme loading and specific activity of MNP@CA4@Lac3 and

Table 4 . 8

 48 Recovery of hybrid biocatalysts

4.4.2 Activity towards ABTS and specific activity of immobilized laccase on azide particles and enzyme loading measured by ELISA test 4.4.2.1-MNP@CA5 (linear azide)

	Enzyme immobilization onto MNP@CA5 was performed: 2 mg were divided by
	4, triplicate for immobilization and one for blank control, then particles were mixed
	with laccase-alkyne in click buffer with total volume 250 μl, and the CuSO 4 .5H 2 O
	at concentration of about 30 equivalents to enzymes and sodium ascorbate (ratio 4:1
	to CuSO 4 .5H 2 O) were added to the mixture, stirred at thermomixer at 950 rpm for
	24 hours at room temperature. Then, MNPs were washed with storage buffer 5
	times, and supernatants were removed and tested with SGZ to guarantee laccases
	did not leach from the hybrid biocatalysts. Then, the activity of this hybrid
	biocatalyst was tested with ABTS as a substrate in storage buffer and enzyme
	loading was measured by Elisa test.
	First, enzyme immobilization reactions were performed with laccase
	concentration at 10 μM and in presence of CuSO 4 .5H 2 O (about 30 eq. to enzymes),
	results of activity, enzyme loading and specific activity are displayed in table 4.9.
	MNP@CA5 Enzyme	activity	Enzyme	loading	Specific	activity
		(U/L)		(μg/mg)		(U/mg)
	Lac3	0.1		3.1		1.7
		0.1
		0.1
	Unik157	0.06		2.3		1.3
		0.1
		0.04

Table 4 .

 4

	9 Activity, enzyme loading and specific activity of MNP@CA5@Lac3 and

Table 4 .10 Optimization

 4

	of immobilization reactions on MNP@CA5@Lac3
	We observed that prolonging reaction times (table 4.10, entry 1) and increasing
	reaction temperature (table 4.10, entry 2) can't lead to a better immobilization.
	Taking into account the existence of the active catalytical species Cu I in presence
	of sodium ascorbate (fig 4.11), a repetition of an immobilization reaction was
	carried out in glove box in order to avoid oxidation of the active catalytical
	intermediate in the air (table 4.10, entry 3), but still no improvement of activity
	towards ABTS was achieved.

Table 4 .

 4

	Entry Laccases	Reaction	CuSO 4 .5H 2 O	Sodium		Enzyme	Enzyme	Specific
		time	(eq, to enzyme)	ascrobate	activity	loading	activity
				(eq,	to	(average	(μg/mg)	(U/mg)
				enzyme)	U/L)
	1 Unik157	24 hrs	100	400		3.6	74.5	0.6
						3.6	47.4	1
						3.8	30.9	1.5
	2 Lac3	24 hrs	100	400		0.5	-	-
	3 Lac3	24 hrs	200	800		0.4	-	-
		11 Optimization of immobilization reactions on MNP@CA5@Lac3 and
		MNP@CA5@Unik157 by increasing catalyst amount
	Increased Enzyme activities were observed (3.6 U/L, 3.6 U/L, 3.8 U/L) for
	Uink157 (

4.11.

table 4

 4 Thiswas confirmed by increase of the enzyme loading(74.5 μg/mg, 47.4 μg/mg, 30.9 μg/mg) in Elisa test. Moreover, even after powerful probe sonication, no enzyme leakage was observed checked with SGZ, displaying a robust immobilization of laccases onto particles.

	In the case of lac3 immobilization onto MNP@CA5 at concentration of 100
	equivalents (table 4.11, entry 2), immobilization reaction produced a low enzyme
	activity (0.5 U/L). Another experiment also generated a low enzyme activity
	(average: 0.4 U/L) even though adding more copper (200 eq.) and sodium ascorbate
	(800 eq.) (

.11, entry 1)

, which demonstrated a click reaction of enzymes and azide particles was improved by adding more catalysts (from 33 eq to 100 eq).

table 4 .

 4 11, entry 3). It can be seen that the click reaction of Lac3 and azide particles would not improve just by increasing catalyst amount.Based on these results of Lac3 (table4.11), only increasing the amount of catalyst can not improve the coupling of Lac3 and azide particles, therefore we attempted increasing the amount of enzymes to enhance the immobilization of Lac3 onto particles, thus enzyme immobilizations were performed with the increased concentration of laccases from 10 μM to 50 μM as showed in table 4.12.

	Entry	laccase	Enzyme	CuSO 4 .5H 2 O	Sodium		Enzyme	Enzyme	Specific	Ratio
			concentration	(eq.	to	Ascorbate	activity	loading	activity	of SA
				enzymes)		(eq.	to	(U/L)	(μg/mg)	(U/mg)	(Unik
						enzyme)				157
										/Lac3)
	1	Lac3	50 μM	100		400	11.2	132.9	2.1
								14.2	97.6	3.6
	2	Lac3	50 μM	200		800	2.9
								0.8
								3.2		3.6
	3 Unik	50 μM	100		400	5.7	15.9	9.0
		157						10.	22.0	11.4

Table 4 .

 4

	12 Optimization of immobilization reactions on MNP@CA5@Lac3 and
	MNP@CA5@Unik157

U/mg were obtained. Moreover, a powerful sonication was used to verify a good coupling of laccases and particles. These experiments showed increasing the concentration of enzymes can improve the click reaction between laccase and MNPs.

  2 mg particles were divided by 4 (triplicate for immobilization and one for blank control), then MNPs were mixed with laccase-alkyne at 10 μM in click buffer with total volume 250 μl, and the CuSO 4 .5H 2 O at concentration of 30 equivalents to enzymes and sodium ascorbate (ratio 4:1) were added to the mixture, stirred at thermomixer at 950 rpm for 24 hours at room temperature, and afterwards supernatants were removed and tested with SGZ to make sure laccase-alkyne still remain catalytically active. Then, particles were washed with pH 5.7 storage buffer 5 times, last washing was tested with SGZ to ensure enzymes do not leak from particles. Then the activity of this hybrid catalyst was tested with ABTS as a substrate in storage buffer at 420 nm with UV and the enzyme loading was measured by Elisa test.

	MNP@CA6	Enzyme	Enzyme	Specific
		activity	loading	activity
		(U/L)	(μg/mg)	(U/mg)
	Lac3	0.06	7.8	0.4
	Unik157	0.06	5.9	0.5

Table 4 .

 4 [START_REF] Roy | Strategies in making cross-linked enzyme crystals[END_REF] Laccases immobilization onto MNP@CA6 From the table 4.13, we used non optimized reaction condition(10 μM in

	enzyme), and no enzyme activity were observed for both lac3 and unik157. The low
	loading demonstrated few laccases were immobilized onto particles by covalent
	bonding, which can explain the low catalytic efficiency.
	Afterwards, in order to improve the click reaction between enzymes and azide
	particles, we increased the amount of laccase to 50 μM and catalyst amount as
	shown in table 4.14.

Table 4 .14 Optimization

 4

of immobilization reactions on MNP@CA6 It can be seen that in the case of lac3 immobilization onto MNP@CA6 (table

4

.14, entry 1), good enzyme activities (2.6 U/L, 4.8 U/L) were observed, and good enzyme loading (204.3 μg/mg, 189.1 μg/mg) were obtained in Elisa test. But a low

  ; MS (EI): calcd for C 11 H 24 N 2 O 4 : 248.3193, found [C 11 H 24 N 2 O 4 H]: 249.1806.

  ; MS (EI): calcd for C 19 H 26 N 2 O 6 :378.4195, found [C 19 H 26 N 2 O 6 Na]: 401.1683.

  ; MS (EI): calcd for C 14 H 14 N 2 O 3 : 258.2726, found [C 14 H 14 N 2 O 3 Na]: 281.0892.

  ; MS (ESI): calcd for C 15 H 20 N 2 O 4 : 292.3303, found [C 15 H 20 N 2 O 4 Na]: 315.1309.

  ; MS (ESI): calcd for C 41 H 64 N 6 O 12 : 832.9799, found [C 41 H 64 N 6 O 12 H]: 833.4600.-methoxybenzene (1 g, 1 eq.), and DIPEA (1.6 g, 2 eq.) were mixed in 80 ml toluene, and the mixture was refluxed for 7 hrs, then evaporated to dryness to obtain yellow oil in a quantitative yield; 1 H NMR (300 MHz, CDCl 3 ) δ: J = 9 Hz, 2 H), J = 9 Hz, 2 H), 3.78 (s, 3 H), 3.74 (s, 2 H),

	Compound25
				O
				H	Chemical formula: C 14 H 24 N 2 O 3
	H 2 N	O	O	N	M.W: 268.3520 g/mol
	2,2'-(ethane-1,2-diylbis(oxy))diethanamine	(9.5	g,	10	eq.),
	1-(chloromethyl)-4

  ; MS (ESI): calcd for C 14 H 24 N 2 O 3 : 268.3520, found [C 14 H 24 N 2 O 3 H]:

  1 H NMR (300 MHz, CDCl 3 ) δ: 9.96 (s, 1 H), J = 6 Hz, 2 H), J = 9 Hz, 2 H), 3.40-3.36 (t, J = 6 Hz, 2 H), 2.88-2.83 (t, J = 6 Hz, 2 H), 2.23-2.13 (m, 2 H);13 C NMR (76 MHz, CDCl 3 ) δ:

  ; MS (ESI): calcd for C 10 H 11 BrO: 227.0977, found [C 10 H 11 BrOH]: 227.0066.

  (t, J = 15 Hz, 2 H), 2.72-2.67 (t, J = 15 Hz, 2 H), 2.08-2.04 (m, 2 H);13 C NMR (76 MHz, CDCl 3 ) δ:

  ; MS (ESI): calcd for C 12 H 15 BrO 2 : 271.1503, found [C 12 H 15 BrO 2 H]: 272.0235.

  ; MS (ESI): calcd for C 38 H 52 N 2 O 7 : 648.8287, found [C 38 H 52 N 2 O 7 Na]: 671.3667.

  ; MS (ESI): calcd for C 44 H 64 N 6 O 11 : 853.0126, found [C 44 H 64 N 6 O 11 H]:

  ; MS (ESI): calcd for C 31 H 62 N 4 O 10 : 650.8448, found [C 31 H 62 N 4 O 10 H]: 651.4514.

	Compound43
					O
					O
				O	NH	Chemical formula: C 28 H 58 N 4 O 8
						M.W: 578.7821 g/mol
					H	O
	H 2 N	O	O	N	N	O
					O

  1 H NMR (300 MHz, CD 3 OD) δ: 4.54-4.51 (t, J = 9 Hz, 2 H), 3.75-3.49 (m, 16 H), 3.24-3.08 (m, 6 H), 2.87-2.82 (m, 4 H), 2.76-2.59 (m, 2 H), 2.41-2.37 (m, 4 H), 1.66-1.54 (m, 8 H), 1.22-1.17 (t, J = 9 Hz, 12 H);

  ; MS (ESI): calcd for C 28 H 58 N 4 O 8 : 578.7821, found [C 28 H 58 N 4 O 8 H]: 577.4170.

  ; MS (EI): calcd for C 8 H 17 N 3 O 4 : 219.2383, found [C 8 H 17 N 3 O 4 H]: 220.1306.

  ; MS (EI): calcd for C 11 H 21 N 3 O 4 : 259.3021, found [C 11 H 21 N 3 O 4 Na]: 282.1500.

  ; MS (EI): calcd for C 17 H 37 N 3 O 7 Si: 423.5771, found [C 17 H 37 N 3 O 7 SiNa]: 446.2298.

  03; MS (ESI): calcd for C 6 H 10 BrNO: 192.0537, found [C 6 H 10 BrNOH]: 129.9925.

  , 2.79-2.74(t, J = 15 Hz, 4 H), (t, J = 12 Hz, 2 H), 2.33-2.29(t, J = 12 Hz, 4 H), 4 H), 1.39 (s, 9 H);

13

C NMR (76 MHz, CD 3 OD) δ: 173.

  ; MS (ESI): calcd for C 23 H 46 N 4 O 8 : 506.6333, found [C 23 H 46 N 4 O 8 H]: 507.3349.

Table 2 . 1

 21 ). Finally, grafted MNPs without laccases were prepared as a blank control in fig.2.1 (E10/H12). Afterwards various values of absorbance of grafted MNPs were obtained. Then, we calculate the corresponding relationship of the absorbance and the amount of laccases grafted on MNPs. We must note the bare grafted MNPs blank also have small absorbance at 405 nm, which must be deducted. Example of calculation is shown in table 2.1. Absorbance and calculation of the example of sample 2 in MNP@APTES@lac3

	Absorbance			Average	Remove blank	Concentration	Mass=C*V	V	of	Particle	E/MNP
						(μg/ml)	(μg)	particles		mass (mg)	(μg/mg)
	0.5380	0.6252	0.5940	0.587	0.4589	0.8403	0.08402	100 μl		0.002	42.0123
	0.3455	0.4096	0.2765	0.3439	0.2505	0.4586	0.04586	50 μl		0.001	45.8604
	0.2222	0.2649	0.2362	0.2411	0.1460	0.26733	0.02673	20 μl		0.0004	66.8315
	0.1447	0.1529	0.1391	0.1456	0.0378	0.06921	0.00692	10 μl		0.0002	34.6059

nm in the ultraviolet-visible spectrum, but it has diamagnet. Type 2 Cu 2+ and

3.2.2.3 Transmitted light microscopy study of phthalimide functionalized γ-Fe 2 O 3 /SiO 2 MNPs

MNP@2-B-NH 2

MNP@4-B-NH 2

General conclusion

Experimental part

were recovered with a magnetic magnetization (2 mins); (2) Then, in 15 ml vial, the γ-Fe 2 O 3 @SiO 2 MNPs were dispersed in 0.75 ml 0.21 % Tx-405, 0.75 ml ethanol,

Content

Appendix of chapter II 1. Materials and instruments

All manipulations were carried out under argon using standard Schlenk techniques.

All glassware was oven dried prior to use. All solvents were purified and dried according to standard methods prior to use, unless stated otherwise. All reagents were obtained from commercial sources and used without further purification. Thin-layer chromatography (TLC) was performed using 60 mesh silica gel plates visualized with short-wavelength UV light (254 nm). Silica gel 60 (200-300 mesh) was used for column chromatography. 1 H NMR spectra were obtained at 300 MHz and recorded relative to the tetramethylsilane signal (0 ppm) or residual proton-solvent. 13 C NMR spectra were obtained at 100 MHz, and chemical shifts were recorded relative to the solvent resonance (CDCl 3 ,77.16 ppm). Data for 1 H NMR are recorded as follows: chemical shift (δ, ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet or unresolved, br = broad singlet, coupling constant. (s) in Hz, integration. IR spectra were recorded on a Nicolet FT-IRspectrometer and only major peaks are reported in cm -1 . UV-Vis spectra were recorded on a JASCO V-770.

Mass spectra were performed by the Centre d'Etude Structurale et d'Analyse des Molecules Organiques, ISM, CNRS-University of Bordeaux.

Synthesis of coupling agents Compound2

Di-boc (1 eq., 10 g) was dissolved in 100 ml DCM, then added dropwise to 1,2-bis(2-aminoethoxy)ethane (10 eq., 74 g), under strong stirring at 0 for 8 hrs, ℃ then at room temperature for 16 hrs, then 300 ml water was added and the product pressure and obtained as a white solid in 77 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ:

7.85-7.67 (m, 2 H), 7.51-7.37 (m, 2 H), 3.65-3.47 (m, 10 H), 3.03-3.01 (m, 2 H); [START_REF] Roy | Strategies in making cross-linked enzyme crystals[END_REF] C NMR (76 MHz,CDCl 3 ) δ: 168.81,134.27,131.31,123.28,69.86,69.49,68.70,65.90,39.87,37.45

Compound CA1

Under argon, compound 4 (1 eq., 1.2 g) was dissolved in 10 ml distilled CH 2 Cl 2 , then distilled TEA (3.66 g) was added to the mixture. After 10 mins, distilled isocyanatopropyltriethoxysilane (1.2 eq., 1.1 g) was added and stirred at RT overnight. Then the mixture was evaporated, the residue was purified by a silica gel chromatography and the pure compound was obtained as a white solid in 55 % yield;

1 H NMR (300 MHz, CDCl 3 ) δ: 7.82-7.79 (m, 2 H), 7.71-7.68 (m, 2 H), 3.90-3.86 (t, J = 12 Hz, 2 H), 3.80-3.64 (m, 8 H), 3.57-3.53 (m, 2 H), 3.50-3.47 (m, 2 H), 3.44-3.40 (m, 2 H), 3.29-3.28 (m, 2 H), 3.21-3.09 (m, 2 H), 1.62-1.57 (m, 2 H), 1.57-1.13 (m, 9 H), 0.64-0.59 (m, 2 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 168. 57, 158.53, 134.11, 131.92, 123.28, 70.55, 70.02, 69.87, 68.00, 58.30, 42.91, 39.81, 37.61, 23.77, 18.25, 7.67

Compound6

Amino-3-chloropropane hydrochloride (1 eq., 11 g) was dissolved in 50 ml DCM, at 0℃, acryloyl chloride (1.2 eq., 9.87 ml) was dissolved in 50 ml DCM and quickly added to the solution under argon. Then TEA (2.2 eq., 30.5 ml) was diluted as yellow oil in 43 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.84-7.80 (m, 4 H), 7.75-7.69 (m, 4 H), 3.78-3.72 (m, 4 H), 3.60-3.52 (m, 8 H), 3.31-3.23 (m, 6 H), 2.89-2.79 (m, 6 H), 2.47 (s, 4 H), 1.91-1.85 (m, 4 H), 1.45 (s, 9 H); 13 C NMR ( 76MHz, CDCl 3 ) δ: 172. 28, 168.47, 156.00, 134.03, 131.97, 123.23, 70.22, 68.89, 53.07, 50.53, 36.31, 35.42, 33.84, 29.68, 28.58, 28.42

Compound8

TFA (10 eq., 1.7 g) was added into a solution of compound 7 (1 eq., 1 g) in 20 ml CH 2 Cl 2 at RT and stirred overnight. Then Concentrated under reduced pressure, the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 86 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.79-7.76 (m, 4 H), 7.69-7.66 (m, 4 H), 3.84 (s, 2 H), 3.70-3.53 (m, 14 H), 3.40 (s, 2 H), 3.25-3.19 (m, 6 H), 2.84-2.80 (m, 4 H), 1.90-1.81 (m, 4 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 170. 75, 168.59, 134.16, 131.97, 123.29, 70.17, 66.75, 64.92, 53.21, 50.57, 39.67, 37.00, 35.50, 29.72, 28.

Compound CA2

Under argon, compound 8 (1 eq., 0.25 g) was dissolved in 10 ml distilled CH 2 Cl 2 , then distilled TEA (8.4 eq., 0.76 g) was added to the mixture. After 10 mins, distilled isocyanatopropyltriethoxysilane (1.2 eq., 0.23 g) was added and stirred at RT overnight. Then the mixture was evaporated, the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 43 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 1 H NMR (300 MHz, CDCl 3 ) δ: 7.79-7.74 (m, 4 H), 7.68-7.65(m, 4 H), 3.79-3.67(m, 10 H), 3.55-3.43 (m, 8 H), 3.31-3.19(m, 6 H), 3.12-3.10(m, 2 H) Hz, 2 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 72.72, 168.44, 158.82, 134.05, 131.89, 123.19, 71.02, 70.44, 70.27, 69.26, 58.30, 53.05, 50.65, 42.83, 40.04, 36.37, 35.40, 33.88, 29.62, 28.47, 23.75, 18.27, 7.65

Compound9

Compound 2 (1 eq., 4.0 g) was dissolved in 60 ml methanol and methylacrylate (10 eq., 13.87 g) was added and stirred at 50℃ for 3 days. Then solvents and excess of methylacrylate were evaporated and obtained in 87. 172.99, 156.03, 79.14, 70.44, 70.29, 69.75, 53.29, 51.57, 49.98, 40.41, 32.64, 28.47 13 C NMR (76 MHz, CDCl 3 ) δ: 172. 71, 156.09, 78.91, 70.14, 69.09, 53.46, 50.74, 44.63, 41.99, 41.32, 40.21, 34.05, 28.36

Compound12

Compound 10 (1 eq., 0.2 g) was mixed with K 2 CO 3 (5 eq., 0.23 g) in 20 ml acetonitrile, refluxed for 2 hrs. Then N-(3-bromopropyl) phthalimide (5 eq., 0.58 g) were added into the mixture, and stirred for 4 days. After that, solid was filtered, and the solvent was concentrated under reduced pressure, the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 42 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.79-7.76 (m, 8 H), 7.67-7.64 (m, 8 H), 3.83-3.45 (m, 18 H), 3.28-3.24 (m, 6 H), 2.90-2.76 (m, 8 H), 2.53-2.45 (m, 14 H), 1.81-1.71 (m, 8 H), 1.39 (s, 9 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 172.59, 168.41, 156.13, 133.97, 132.09, 123.23, 79.10, 70.24, 69.06, 59.12, 52.90, 52.78, 51.21, 50.29, 40.37, 37.12, 36.13, 33.32, 28.47, 26. (m, 16 H), 7.85-7.82 (m, 2 H), 7.72-7.66 (m, 14 H), 7.56-7.43 (m, 10 H), 7.31-7.26 (m, 13 H), 7.14-7.11 (m, 2 H), 6.81-6.77 (m, 4 H), 6.11-6.06 (m, 8 H); 13 C NMR (76 MHz, CD 3 OD) δ: 172. 10, 168.42, 134.06, 131.75, 122.82, 69.98, 66.50, 64.30, 52.08, 50.97, 50.09, 39.24, 34.61, 34.31, 33.62, 28.42, 23.12

Compound CA3

Under argon, compound 13 (1 eq., 0.55 g) was dissolved in 10 ml distilled CH 2 Cl 2 , then distilled TEA (8.4 eq., 0.42 g) was added to the mixture. After 10 mins, distilled isocyanatopropyltriethoxysilane (1.2 eq., 0.15 g) was added and stirred at RT overnight. Then the mixture was evaporated. The residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 44.1 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 7.81-7.75 (m, 8 H), 7.72-7.64 (m, 8 H), 3.83-3.68 (m, 16 H), 3.56-3.48 (m, 10 H), 3.39-3.30 (m, 6 H), 3.20-3.13 (m, 2 H), 2.89-2.39 (m, 18 H), 1.87-1.78 (m, 8 H), 1.62-1.57 (m, 2 H), 1.23-1.18 (m, 9 H), 0.66-0.60 (m, 2 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 168.34, 158.67, 133.88, 132.07, 132.02, 123.16, 123.10, 77.46, 77.04, 76.62, 71.09, 70.51, 70.33, 58.34, 53.10, 52.91, 52.18, 51.14, 50.90, 42.92, 40.14, 37.06, 36.09, 25.99, 23.82, 18.31, 7.68 (m, 4 H), 3.57-3.39 (m, 10 H), 3.17-3.12 (m, 2 H), 1.28 (s, 9 H); 13 C NMR (76 MHz, CDCl 3 ) δ: 191.61, 166.45, 155.96, 139.63, 137.91, 129.54, 127.78, 79.08, 70.01, 69.44, 40.17, 39.88, 28.28

Compound CA4

Compound 14 (1 eq., 1 g) was dissolved in 20 ml THF, then APTES (1.2 eq., 1.1 g), TEA (1.2 eq., 0.5 g) were added under Ar, and stirred for 18 hrs at RT. Then filtered and evaporated under reduced pressure, the residue was purified by a silica gel chromatography and the pure compound was obtained as colorless oil in 34 % yield; 139.78, 137.58, 129.30, 127.30, 58.12, 42.05, 22.36, 17.87, 7.50 SOCl 2 (15.9 g, 2 eq.) was added to the solution of 4-carboxybenzaldehyde (10 g, 1 eq.) in 100 ml toluene. Then, the mixture was refluxed overnight. Afterwards the solvent and an excess of SOCl 2 were evaporated, cyclohexane (100 ml) was added to the residues at 0℃, after 4 hrs, the white crystals were filtered and obtained in 166.60, 142.87, 130.06, 129.36, 126.69, 102.13, 52.40, 51.91.

Compound22

Compound 20 (1eq., 14 g) was added to 100 ml ethylene diamine, then refluxed 854.4687

Compound30

Toluenesulfonyl Chloride (5 g, 1 eq.) was added to the mixture of 2,2'-(ethane-1,2-diylbis(oxy))diethanamine (7.76 g, 2 eq.) and Et 3 N (3.9 g, 1.5 eq.) in 100 ml THF over 30 mins at 0℃, then up to RT. Afterwards the mixture was stirred at rt for 24 hrs, then concentrated under reduced pressure and the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 58 % yield without further purification; 3031334.

Compound31

Compound 30 (1 g, 1 eq.) was mixed with compound 27 (2.68 g, 3 eq.) in 100 ml CH 3 CN, then K 2 CO 3 (2.3 g, 5 eq.) was added to the mixture. Then the mixture was refluxed for 2 weeks. Then, filtered, concentrated under reduced pressure and the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 44 % yield without further purification; 1 H NMR ( 400MHz, CDCl 3 ) δ: 7.42-7.17 (m, 12 H), 5.78 (s, 2 H), 4.12-3.98 (m, 8 H), 3.62-3.52 (m, 8 H), 3.30-3.19 (m, 4 H) 139.89, 128.41, 128.35, 126.31, 69.97, 69.91, 66.45, 63.93, 58.29, 39.12, 31.41, 23.

Compound36

At 0℃, 4,4-diethoxybutan-1-amine (6 g, 1 eq.) was mixed with TEA (6 g, 1.6 eq.) in 100 ml DCM. After 15 mins, acryloyl chloride (4 g, 1.2 eq.) was added to the mixture dropwise. Then the mixture was stirred at rt overnight, afterwards evaporated in vacuum. The crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 75 % yield;

Compound37

At 0℃, 2,2'-(ethane-1,2-diylbis(oxy))diethanamine (33 g, 10 eq.) was mixed with TEA (2.84 g, 1.2 eq.) in 100 ml DCM, then benzyl chloroformate (4 g, 1 eq.) was added dropwise. Then the mixture was stirred at rt overnight. Afterwards evaporated in vacuum, and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 45 % yield; 1 H NMR (400 MHz, CDCl 3 ) δ: 7.32-7.30 (m, 5 H), 3.80 (s, 2 H), 3.62-3.59 (m, 6 H),

The crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 89 % yield; 1 H NMR (300 MHz, CD 3 OD) δ:

4.54-4.51 (t, J = 9 Hz, 2 H), 3.75-3.49 (m, 16 H), 3.24-3.08 (m, 6 H), 2.87-2.82 (m, 4 H) ,2 H),4 H),8 H),.17 (t, J = 15 Hz, 12 H); 13 C NMR (76 MHz, CD 3 OD) δ: 173. 30, 102.76, 70.00, 68.96, 67.13, 61.23, 52.60, 50.18, 39.49, 38.79, 33.16, 30.96, 24.42, 14.53 11g, 10 eq.) was mixed TEA (16 g, 1.9 eq.) in 100 ml DCM. After 15 mins, ethyl carbonochloridate (6 g, 1 eq.) was added dropwise. Then the mixture was stirred for 2 hrs, afterwards 40 ml water was added and separated, the combined aqueous phases were extracted with dichloromethane (3* 50 ml and then 3*100 ml). The combined organic extracts were washed with brine (100 mL), dried over (MgSO 4 ) and the solvent was evaporated. The crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 53 % yield; 1 H NMR (300 MHz, CDCl 3 ) δ: 4.07-4.00 (q, J = 9 Hz, 2 H), 3.57-3.44 (m, 8 H), 3.34-3.29 (m, 2 H) Hz, 3 H); 13 C NMR (101 MHz, CDCl 3 ) δ: 173.70, 167.80, 156.91, 141.42, 134.14, 127.12, 126.81, 102.40, 70.12, 69.97, 68.85, 60.61, 53.16, 52.66, 50.69, 40.81, 40.59, 39.04, 33.77, 14.61

Compound46

Compound 37 (0.5 g, 1 eq.) was mixed with compound 22 (1.5 g, 3 eq.) in 30 ml methanol. Then the mixture was stirred at 75℃ for 2 weeks. Afterwards, concentrated under reduced pressure and the residue was purified by a silica gel chromatography and the pure compound was obtained as yellow oil in 20 % yield without further purification;

Compound58

At 0℃, compound 57 (7 g, 1 eq.) was mixed with TEA (4.2 g, 0.6 eq.) in 150 ml DCM, then acrylyl chloride (7.6 g, 1.2 eq.) was added to the solution dropwise. The mixture was stirred at rt overnight. Afterwards evaporated in vacuum and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 15 % yield;

Compound60

At 0℃, under argon acryloyl chloride (10 g, 1 eq.) was added to the solution of 3-aminopropan-1-ol (16.3 g, 2 eq.) in 100 ml DCM. Then the mixture was stirred at rt overnight. Then evaporated in vacuum and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 46 % yield;

Compound63

At 0℃, under argon acryloyl chloride (5 g, 1.2 eq.) was added to the solution of γ-butyrolactone (10 g, 1 eq.) was added to the acidic solution. Then the mixture was stirred at rt for 2 hrs, afterwards refluxed for 12 hrs. Then extracted with diethyl ether, and evaporated in vacuum and the crude product was purified by a silica gel column chromatography and the pure product was obtained as yellow oil in 41 % yield; 158. 53, 137.67, 137.07, 130.02, 118.86, 70.84, 70.32, 69.64, 58.30, 56.36, 53.25, 42.88, 40.14, 32.85, 23.65, 18.24, 7.62

Compound77

NaN 3 (6.5 g, 1 eq.) was added to the solution of 1-bromo-3-chloropropane (9.9 ml, 1 eq.) in 100 ml DMSO. Then the mixture was stirred at rt for 12 hrs.

Afterwards 30 ml H 2 O was added, and extracted with pentane (3* 20 ml), then the combined organic extracts were washed with brine (100 mL), dried over (MgSO 4 ), filtered and the solvent was evaporated to afford slightly yellow oil in 78 % yield;

Appendix of chapter III

1. Materials and Instruments

Zetametry measurements

Zetametry measurements for the surface charge state of grafted particles were performed on a Nano-Partica SZ-100 apparatus (at ISM, IPM group).

FT-IR

IR-DRiFT spectroscopy has been performed on FTIR Nicolet iS50 spectrometer (at ISM, GSM group).

General protocol of grafting of particles

The colloidal state of grafted particles and hybrid biocatalysts dispersed in corresponding medium was observed under Transmitted Light Microscope equipped with an Axiocam digital camera. The Zeta potential measurements were performed on Horiba Scientific nanoparticles analyzer SZ-100 at ISM, and the measurements were carried out at RT for diluted aqueous suspension of particles. MNPs were washed with 0.21 % Tx-405 (1 ml*5), ethanol (1 ml*5), water (1 ml*5), then were dispersed in 3 ml of water.

Grafting of APTES coupling agents

Grafting of linear phthalimide coupling agents CA1

(1) Washing: 10 mg of γ-Fe 2 O 3 @SiO 2 MNPs were washed with water (1 ml*1) and 0.21 % Tx-405 (1 ml*1), and was sonicated at 60 W for 10 seconds. Then MNPs was recovered with a magnetic magnetization (2 mins); (2) Then, in 15 ml vial, the γ-Fe 2 O 3 @SiO 2 MNPs were dispersed in 0.75 ml 0.21 % Tx-405, 0.75 ml ethanol, dissolved into 0.5 ml dry DMSO and was sonicated at 60 W for 10 seconds and was kept under N 2 ; (4) The MNPs suspension was stirred in the thermomixer (750 rpm) at 25℃. Then, during 2.5 hrs, the coupling agent solution was added into the MNPs suspension (8 µl, every 2 mins). During grafting, the mixture was sonicated several times; (5) Then the mixture was finally stirred for another 1 h at 40℃; (6) Then, the mixture was separated by magnetic decantation and MNPs were washed with 0.21 % Tx-405 (1 ml*1), ethanol (1 ml*1), and water (1 ml*1), and then were dispersed in 3 ml of water.

Grafting of two branched phthalimide coupling agents CA2

(1) Washing: 10 mg of γ-Fe 2 O 3 @SiO 2 MNPs were washed with water (1 ml*1) and 0.21 % Tx-405 (1 ml*1), and were sonicated at 60 W for 10 seconds. And MNPs were recovered with a magnetic magnetization (2 mins); ( 2) Then in 15 ml vial, the γ-Fe 2 O 3 @SiO 2 MNPs were dispersed in 0.75 ml 0.21 % Tx-405, 0.75 ml ethanol, 87 µl 30 % NH 4 OH; (3) Then, 0.1114 g (0.111 mmol) of coupling agent CA2 was dissolved into 0.5 ml dry DMSO and were sonicated at 60 W for 10 seconds; (3)

The MNPs suspension was in the thermomixer (700 rpm) at 25℃. And during 2.5 hrs, the coupling agent solution was added into the MNPs suspension (13 µl, every 2 mins) under N 2 ; (4) The mixture was finally stirred for another 2.5 hrs at 40℃; (5) The mixture was separated by magnetic decantation and MNPs were washed with 0.21 % Tx-405 (1 ml*1), ethanol (1 ml*1), water (1 ml*1), then were dispersed in 3 ml of water.

Grafting of four branched phthalimide coupling agents CA3

(1) Washing: 10 mg of γ-Fe 2 O 3 @SiO 2 MNPs were washed with water (1 ml*1) and 0.21 % Tx-405 (1 ml*1), and were sonicated at 60 W for 10 seconds. And MNPs dissolved into 0.5 ml dry DMSO and were sonicated at 60 W for 10 seconds; (4)

The MNPs suspension was in the thermomixer (300 rpm) at 25℃. And during 2.5 hrs, the coupling agent solution was added into the MNPs suspension (8 µl, every 2 mins) under N 2 ; (5) Then, the mixture was finally stirred for another 1 h at 40℃; (6) Then, the mixture was separated by magnetic decantation, and MNPs were washed with 0.21 % Tx-405 (1 ml*1), ethanol (1 ml*1), water (1 ml*1), then were dispersed in 3 ml of water.

General protocol of phthalimide group deprotection for

MNP@CA1, MNP@CA2, MNP@CA3 30 mg grafted particles were sonicated and water was removed by magnetic decantation. Then 10 ml 40 % MeNH 2 in water solution was added. The mixture was sonicated for 10 seconds, and was kept for 5 mins. Then, the MNPs were separated by magnetic decantation and solution was removed. Particles were washed with 10 ml of mili-Q water, 10 ml of 10 % DMF in water, then 10 ml of ethanol, then 5 ml*3 of water, and finally redispersed in 3 ml of water.

Grafting of linear CHO coupling agents CA4

(1) Washing: 10 mg of γ-Fe 2 O 3 @SiO 2 MNPs were washed with water (1 ml*1) and 0.21 % Tx-405 (1 ml*1), and were sonicated at 60 W for 10 seconds, and MNPs were recovered with a magnetic magnetization (2 mins); (2) Then, in 15 ml vial, the γ-Fe 2 O 3 @SiO 2 MNPs were dispersed in 0.75 ml 0.21 % Tx-405, 0.75 ml ethanol, 87 µl 30 % NH 4 OH; (3) Then, 0.044 g (0.125 mmol) of coupling agent CA4 was dissolved into 0.5 ml dry DMSO, and were sonicated at 60 W for 10 seconds. The MNPs suspension was in the thermomixer (300 rpm) at 25℃. And during 2.5 hrs, the coupling agent solution was added into the MNPs suspension (8 µl, every 2 mins) under N 2 ; (4) Then, the mixture was stirred for another 1 h at 40℃; (5) Then, the mixture was separated by magnetic decantation, and MNPs were washed with 0.21 % Tx-405 (1 ml*1), ethanol (1 ml*1), water (1 ml*1), then were dispersed in 3 ml of water.

Grafting of linear N 3 coupling agents CA5

(1) Washing: 10 mg γ-Fe 2 O 3 @SiO 2 MNPs were washed with water (1 ml*1) and 0.21 % Tx-405 (1 ml*1), and were sonicated at 60 W for 10 seconds and MNPs were recovered with a magnetic magnetization (2 mins); ( 2) Then, in 15 ml vial, the γ-Fe 2 O 3 @SiO 2 MNPs were dispersed in 0.75 ml 0.21 % Tx-405, 0.75 ml ethanol, 87 µl 30 % NH 4 OH; (3) Then, 0.42 g (0.99 mmol) of coupling agent CA5 was dissolved into 0.5 ml dry DMSO, and were sonicated at 60 W for 10 seconds. The MNPs suspension was in the thermomixer (300 rpm) at 25℃. And during 2.5 hrs, the coupling agent solution was added into the MNPs suspension (8 µl, every 2 mins) under N 2 ; (4) The mixture was stirred for another 2 hrs at 25℃, then at 40℃ overnight; (5) The mixture was separated by magnetic decantation, and MNPs were washed with 0.21 % Tx-405 (1 ml*1), ethanol (1 ml*1), water (1 ml*1), then were dispersed in 3 ml of water.

Grafting of two branched N 3 coupling agents CA6

(1) Washing: 10 mg γ-Fe 2 O 3 @SiO 2 MNPs were washed with water (1 ml*1) and 0.21 % Tx-405 (1 ml*1), and were sonicated at 60 W for 10 seconds and MNPs were recovered with a magnetic magnetization (2 mins); (2) Then, in 15 ml vial, the 10 mg γ-Fe 2 O 3 @SiO 2 MNPs were dispersed in 0.75 ml 0.21 % Tx-405, 0.75 ml ethanol, 87 µl 30 % NH 4 OH; (3) Then, 0.68 g (1 mmol) of coupling agent CA6 was dissolved into 0.5 ml dry DMSO, and were sonicated at 60 W for 10 seconds. The MNPs suspension was in the thermomixer (700 rpm) at 25℃. And during 2.5 hrs, the coupling agent solution was added into the MNPs suspension (8 µl, every 2 mins) under N 2 ; (4) Then, the mixture was stirred at 40℃ for 1 h; (5) Then, the mixture was separated by magnetic decantation, and MNPs were washed with 0.21 % Tx-405 (1 ml*1), ethanol (1 ml*1), water (1 ml*1), then were dispersed in 3 ml of water.

Quantitative analysis

General procedure for quantification of amino groups on MNPs by UV-Vis spectroscopy

In the dosing of amino MNPs, p-nitrobenzaldehyde was used to quantify the functional MNPs (fig 3.1). Generally in first step, excess p-nitrobenzaldehyde reacted with functionalized MNPs in solution. Then when the functional groups-NH 2 was consumed, remaining p-nitrobenzaldehyde was removed by washing MNPs with ethanol. Afterwards in the second step, a hydrolysis of grafted MNPs@ p-nitrobenzaldehyde complex was performed in water with help of acetic acid at 30℃ for 1 h. Finally the reaction mixture and the washings are recovered and collected in order to quantify the amount of 4-nitrobenzaldehyde in solution by UV-Visible spectroscopy. According to the maximum absorbance at 264 nm of 4nitrobenzaldehyde, its concentration was determined using the Beer-Lambert law.

Thus indirectly the concentration of NH 2 functions per gram of amino MNPs was calculated. functions per gram of MNP@SiO 2 @NH 2 was obtained.

1.0x10 -5 1.5x10 -5 2.0x10 -5 2.5x10 -5 3.0x10 -5 3.5x10 -5 4.0x10 -5 4.5x10 -5 5.0x10

Concentration measurement of laccases

The laccase concentration was measured by UV-visible spectroscopy (JASCO V-770) at ε 610 nm = 5600 M -1 .cm -1 for the active copper site T1.

Microplate reader

Elisa test measurements were performed at Bordeaux Science Agro on Thermo Scientific™ Multiskan™ FC 51119100 Microplate Photometer, Software SkanIt 5.0.

Buffers

(1) RA (Reductive Amination) Buffer: 50 mM aqueous phosphate with 0.

Enzyme Immobilization

Preparing enzymes

Changing buffer of laccases with Vivispin 2 (30 KDa) in RA buffer, then concentrated with the centrifuge apparatus (3000 r/min, 25 mins), and measured concentration and activity of free enzymes with UV adsorption according to the calibration curve.

Enzyme immobilization on amino particles

(1) Weight and transfer 2 mg Magnetic beads to a centrifuge tube. Washing particles with 0.1 M MES buffer pH= 6.0. Then resuspend beads by adding 1 ml 0.1 M MES buffer pH= 6.0 and mix beads by vigorously vortexing for 1-2 minutes, if some beads aggregated after suspendeding, sonicated mildly for 10-30 seconds, 60 w; (2) Remove the supernatant while the tube remains on the separator, and repeated again;

(3) 2 mg/mL of MNPs were separated by 4, every tube has 0.

Enzyme immobilization on aldehyde particles

(1) Weight and transfer 2 mg Magnetic beads to a centrifuge tube. Wash particles with 1 ml RA buffer. Then resuspend beads in 1 ml RA buffer and mix beads by

MQ-water each;

Step7: Weight and transfer 2 mg of MNPs to a centrifuge tube. Wash particles with 1 ml click buffer (50 mM aqueous phosphate, pH= 7.5). Then resuspend beads in 1 ml click buffer and mix beads by vigorously vortexing for 1-2 minutes, 60 w, if some beads aggregated after suspendeding, sonicated mildly for 10-30 seconds, and remove the supernatant while the tube remains on the magnetic separator, and repeated again;

Step8: 2 mg/mL of MNPs were separated by 4, every tube has 0.50 mg/250 μl, 3 for triplicate reactions, one for blank, then remove the tube containing particles from the magnetic separator;

Step9: Calculate amount of reactants: Add the catalysts, buffer, enzymes to the 3 tubes containing azide functionalized MNPs, and another tube for blank control as calculated;

Step10: The mixture was degassed and shaked on a thermomixer (850 rpm/min) at room temperature for 24 hrs;

Step11: After reaction, MNPs were magnetically separated and the supernatant was kept, then wash beads 5 times with 0.5 mL storage buffer at each time, and the last washing solution was tested with SGZ. Particles were kept in 250 μL storage buffer for next use.

Recovery of hybrid biocatalysts

In a 15 ml tube, ABTS (1.3 ml, from 50 mM solution), 8 ml of storage buffer, 2

Then calculation about the activity can be calculated; (1) Prepare a series of standard samples: dilute the standard enzymes to an appropriate concentration in coating buffer, and then added at the plate in triplicate, allowing sufficient volume of 100 μL in each well.

(2) Cover the plate, incubate at room temperature for 1 hour in order to immobilize enzymes at the bottom of each well. Remove the supernatant and washing 3 times with 200 μl washing buffer.

(3) And take 10 μl of each sample dilute by 100 with coating buffer or washing buffer, for A, add 100 μl, for B, add 50 μl, for C, add 20 μl, for D, add 10 μl. Then waiting for several mins, then put them on the magnetic magnet to keep particles, and remove the supernatant, then washing these particles 3 times with 200 μl washing buffer.

(4) Then add 200 μl blocking buffer to each well, cover the plate with para film and put it on the thermomixer at 400 rpm for 1 h. Then remove the blocking buffer with magnetic supporter.