Maud Lemoine

Pi Nyvall Collén

William Helbert
email: helbert@sb-roscoff.fr

Physical state of -carrageenan modulates the mode of action ofcarrageenase from Pseudoalteromonas carrageenovora

Keywords: Carrageenan, -carrageenase, processivity, gel, mode of action 1 liquid chromatography-Multi angle laser light scattering TEM: transmission electron microscopy trisulfonic acid DMSO: dimethyl sulfoxyde FACE: fluorescence-assisted carbohydrate electrophoresis

Pseudoalteromonas carageenovora -carrageenase is a glycoside hydrolase involved in the bioconversion of carrageenans. Carrageenans are sulphated galactans that are densely packed in red algal cell walls. Previous crystallographic investigations revealed that the active site of -carrageenase has a tunnel-shaped topology, suggesting a processive mode of action for this enzyme. To biochemically characterise the enzymatic depolymerisation ofcarrageenan, soluble and solid substrates (in both gel and powder forms) were incubated with P. carageenovora -carrageenase. The average molecular weight of soluble carrageenan decreased rapidly and all possible degradation products were observed, suggesting random degradation of -carrageenan. In contrast, as expected for a processive-type carrageenase, the average molecular weight of solid carrageenan decreased very slowly and tetrasaccharide production was high. Interestingly, experimentally determined processivity was very similar for gel and powder, suggesting that, in addition to an adapted catalytic site, the substrate must be in the solid state for -carrageenase processivity to operate, whatever the level of carrageenan ordering.

Introduction

Agars and carrageenans are sulphated galactans widely used for industrial applications due to their unique physico-chemical properties [START_REF] Therkelsen | Carrageenan. In Industrial gums[END_REF][START_REF] Bixler | Recent development in manufacturing and marketing carrageenan[END_REF][START_REF] Mchugh | Carrageenan in a guide to the seaweed industry[END_REF]. They are the most abundant components of the red algal (Rhodophyta) cell wall and can represent up to 50% of algal dry weight. Agar and carrageenan are densely packed in the cell wall in a three-dimensional solid network of pseudo-crystalline fibres which assemble during the deposition of cell wall macromolecules [START_REF] Kloareg | Structure of the cell walls of marine algae and ecophysiological functions of the matrix polysaccharides[END_REF][START_REF] Craigie | Cell walls[END_REF]. These polysaccharides are made up of linear chains of galactose with alternating -(1 3) and -(1 4) linkages. In these galactans, the -linked galactose units are in the D configuration (G unit). However, while the -linked galactose units are in the L configuration in agars (L unit), they are in the D configuration in carrageenans (D unit).

Carrageenans are further classified according to the number and position of sulphate esters (S) and by the occurrence of 3,6-anhydro-bridges in the -linked residues (DA unit) found in gelling carrageenans [START_REF] Rees | Structure, conformation, and mechanism in the formation of polysaccharide gels and networks[END_REF][START_REF] Knutsen | A modified system of nomenclature for red algal galactans[END_REF]. For example, kappa-( , DA-G4S), iota-( , DA2S-G4S), and lambda-( , D2S6S-G2S) carrageenans are distinguished by the presence of one, two, or three ester-sulphate groups per repeating disaccharide unit, respectively.

Agarose, -and -carrageenans form thermo-reversible gels in aqueous solution, and their stiffness decreases with the degree of sulphation. Agarose and -carrageenan produce rigid and brittle gels, while -carrageenan gels are elastic. -Carrageenan, which lacks anhydro-bridges, gives highly viscous solutions. The gelling properties of carrageenan depend on the ionic strength and the type of salts occurring in the medium. It is well-known that potassium chloride and calcium chloride induce gelation of -and -carrageenans, respectively [START_REF] Rees | Shape and interaction of carbohydrate chains[END_REF][START_REF] Morris | Cation-specific aggregation of carrageenan helices: Domain model of polymer gel structure[END_REF]. At the molecular level, gelation is preceded by a disorder-order (helix) transition of polysaccharide conformation, followed by aggregation of helices which gives rise to semi-crystalline fibres [START_REF] Anderson | X-ray diffraction studies of polysaccharide sulphates: Double helix models for -andcarrageenans[END_REF][START_REF] Millane | The molecular structure of kappa-carrageenan and comparison with iota-carrageenan[END_REF]. X-ray crystallography analyses of oriented fibres ofcarrageenan suggest that double helices-probably parallel-stranded duplexes-are packed together in junction zones (i.e. the fibres) [START_REF] Anderson | X-ray diffraction studies of polysaccharide sulphates: Double helix models for -andcarrageenans[END_REF][START_REF] Janaswamy | Three-dimensional structure of the sodium salt of iota-carrageenan[END_REF]. In the case of -carrageenan, the conformational state of the macromolecules in gel form is still a matter of debate.

Experimental data provide support for a dimeric structure of polysaccharides in an ordered state, and interpretations lead either to duplexes of single helices [START_REF] Smidsrød | Some physical properties of carrageenan in solution and gel state[END_REF][START_REF] Rochas | Mechanism of gel formation in -carrageenan[END_REF][START_REF] Bongaerts | Equilibrium and nonequilibrium association processes of kappa-carrageenan in aqueous salt solutions[END_REF] or, as forcarrageenan, double helices (intertwined strands) [START_REF] Rees | Shape and interaction of carbohydrate chains[END_REF][START_REF] Anderson | X-ray diffraction studies of polysaccharide sulphates: Double helix models for -andcarrageenans[END_REF][START_REF] Viebke | On the mechanism of gelation of helix forming biopolymers[END_REF]. The aggregation of agarose molecules in gels also occurs by association of single or double helices [START_REF] Arnott | Iota-carrageenan: Molecular structure and packing of polysaccharide double helices in oriented fibres of divalent cation salts[END_REF][START_REF] Ford | New x-ray diffraction results from agarose: Extended single helix structures and implications for gelation mechanism[END_REF]. Agar-and carrageenan-degrading enzymes, namely agarases and carrageenases, have so far only been found in marine bacteria belonging mainly to the Gammaproteobacteria, Bacteroides or Sphingobacteria classes [START_REF] Michel | Bioconversion of red seaweed galactans: A focus on bacterial agarases and carrageenases[END_REF]. Known -carrageenases (E.C. 3.2.1.83) belong to the GH16 glycoside hydrolase family, a polyspecific family which encompasses at least eight different enzymatic activities, including, notably, -agarases ( http://afmb.cnrsmrs.fr/CAZY/) [START_REF] Coutinho | Carbohydrate-active enzymes: An integrated database approach[END_REF]. Phylogenetic analysis and crystallographic investigations have demonstrated that the GH16 family of enzymes evolved from a common ancestor and thatcarrageenases most likely emerged from the -agarase branch [START_REF] Barbeyron | The kappacarrageenase of the marine bacterium Cytophaga drobachiensis. Structural and phylogenetic relationships within family-16 glycoside hydrolases[END_REF]. -Agarases andcarrageenases adopt a jelly-roll fold and hydrolyse -(1 4) glycosidic linkages with retention of the anomeric configuration [START_REF] Potin | Processing and hydrolytic mechanism of the cgka-encoded -carrageenase of Alteromonas carrageenovora[END_REF][START_REF] Jam | The endo-beta-agarases agaa and agab from the marine bacterium Zobellia galactanivorans: Two paralogue enzymes with different molecular organizations and catalytic behaviours[END_REF]. Analyses of the mode of action of -agarases suggest that agarose molecules undergo random depolymerisation. In the case of agarase A, in addition to the active catalytic site, a second strong binding site was observed on the catalytic module. It has been proposed that the co-occurrence of two binding sites facilitates heterogeneous phase degradation of agarose by unwinding or dissociating helices occurring in the gel state [START_REF] Allouch | Parallel substrate binding sites in a -agarase suggest a novel mode of action on double-helical agarose[END_REF]. The mode of action of -carrageenase has not been investigated biochemically. However, considering the tunnel-shaped topology of the active site, it has been suggested that this enzyme proceeds via a non-random, processive mechanism [START_REF] Michel | The kappa-carrageenase of Pseudoalteromonas.carrageenovora features a tunnel-shaped active site: A novel insight in the evolution of clan-b glycoside hydrolases[END_REF].

Processivity, also referred to as the multiple-attack mechanism, was first described in -amylases [START_REF] Robyt | Multiple attach hypothesis of alpha-amylase action: Action of porcine pancreatic, human salivary, and Aspergillus oryzae alpha-amylases[END_REF][START_REF] Robyt | Multiple attack and polarity of action of porcine pancreatic alpha-amylase[END_REF]. In this mode of action, enzymes form a complex with the target polysaccharide and remain attached to one end of the polymeric chain, thus cleaving several glycosidic linkages before dissociating. This mechanism implies that the enzyme slides along the polysaccharide chain. In the case of neutral polysaccharides, this involves intermediate enzyme-substrate complexes stabilised by hydrophobic interactions between aromatic amino acid and sugar residues [START_REF] Rouvinen | Threedimensional structure of cellobiohydrolase II from Trichoderma reesei[END_REF][START_REF] Davies | Structures and mechanisms of glycosyl hydrolases[END_REF]. Although the sliding mechanism of processive enzymes on polyanionic molecules is not well understood, several examples of processive enzymes acting on anionic polysaccharides have been reported [START_REF] Linhardt | Mode of action of heparin lyase on heparin[END_REF][START_REF] Pritchard | Characterization of the group b streptococcal hyaluronate lyase[END_REF][START_REF] Michel | The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae[END_REF]. Interestingly, -carrageenases, which form a monospecific family of glycoside hydrolases (GH82) unrelated to -carrageenases, degrade carrageenan gels according to a processive mechanism [START_REF] Michel | The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae[END_REF]. This was demonstrated both by observation of thinning carrageenan fibres using transmission electron microscopy and by detection of a particular groove in the active site using X-ray crystallography. This groove closes in the presence of substrate as a result of a shift in a protein domain and leads to a tunnel-shaped topology. This conformation allows the enzyme to progress unidirectionally without dissociating from the substrate.

The enzymatic digestion of -carrageenan, whose chemical structure is intermediate between agarose and -carrageenan, has not yet been investigated. -Agarases share a common ancestor with -carrageenase, but the physico-chemical properties as well as the anionic nature of -carrageenan chains closely resemble those of -carrageenan. The tunnel topology found in both -and -carrageenase reinforces the hypothesis of convergent evolution of these enzymes toward a mode of action adapted to substrates that share common chemical and physical properties. In this context, we sought to characterise the mode of action of the P. carrageenovora -carrageenase in order to consider the potential correlation between the tunnel-shaped topology of the active site with the enzyme's mode of action. We demonstrate that the -carrageenase is a processive enzyme when acting on solid substrates.

Nevertheless, -carrageenase has a random mode of action on soluble substrates. These observations suggest that the conditions for processivity not only require that the active site possess the adapted topology, but also that the substrate be in an appropriate physical and conformational state.

Experimental

Purification of -carrageenan

-Carrageenan extracted from Kappaphycus alvarezii (Eucheuma cottonii) was supplied by CP Kelco ( -carrageenan, cottonii X-6913 and low-gelling --carrageenan, cottonii X-6042). Before use, carrageenans were purified by isopropanol precipitation as follows. Carrageenan powder (5 g) was suspended in 500 mL cold ultra-pure water (Millipore Water Purifier). The suspension was heated at 70°C under slow stirring until the carrageenan was completely dissolved. The polysaccharides in solution were precipitated by drop-wise addition of 1 L of isopropanol under vigorous stirring. After centrifugation at 10,500 × g, the precipitate was collected and dissolved again in 500 mL ultra-pure water. This precipitation protocol was repeated twice. Carrageenans were then dissolved in 500 mL water prior to adding 29.22 g NaCl. This quantity of NaCl corresponds to a salt concentration 50 times greater than that of the ester sulphate groups in carrageenan. After overnight stirring, the solution was dialysed (6-8000 Da MWCO Spectra/Por®) against ultra-pure water in order to remove excess salt and non-polymeric materials in the presence of 5 mM NaOH to prevent acidification of the sample. Finally, the Na-carrageenan solution was filtered through a 0.45 µm filter (Pall Life Sciences, Acrodisc Syringe Filter, HT Tuffryn Membrane) and freeze-dried in the presence of 20 mM ammonium carbonate.

Recombinant -carrageenase

Pseudoalteromonas carrageenovora -carrageenase was recombinantly expressed in E. coli as described by [START_REF] Michel | Expression, purification, crystallization and preliminary x-ray analysis of thecarrageenase from Pseudoalteromonas carragenovora[END_REF] [START_REF] Michel | Expression, purification, crystallization and preliminary x-ray analysis of thecarrageenase from Pseudoalteromonas carragenovora[END_REF]. The over-expressed His-tagged fusion protein was purified by metal affinity chromatography on a chemical Sepharose Fast Flow column (GE Healthcare) loaded with NiSO 4 , followed by gel permeation chromatography on a MonoS column (HR 5/5, GE Healthcare). A stock solution containing 60 µg mL -1 ofcarrageenase with an activity of 0.06 mM eq. glucose min -1 µg -1 (0.125% -carrageenan in 0.2 M Tris) was stored at 4°C.

Enzymatic degradation

A stock solution of 0.4% (w/v) carrageenan in 10 mM Tris, pH 8.0 was prepared by mixing at room temperature under gentle stirring overnight. To ensure complete dissolution, the solution was heated at least for 1 h at 70°C. It was stored at 4°C for 2 weeks at most. Salt solutions containing 200 mM LiNO 3 , 2 mM citric acid (used as internal standard for chromatography experiments) and various concentrations of KCl ranging from 0 to 80 mM were prepared just prior to use.

For enzymatic degradation, the 0.4% carrageenan solution and the salt solutions were heated separately at 70°C for 10 min before being mixed at equal volume resulting in final concentrations of 0.2% carrageenan in 100 mM LiNO 3 , 1 mM citric acid and 0-40 mM KCl.

The mixture was kept at 70°C for at least 30 min before being transferred to room temperature (22°C). Gels were stabilised for at least 12 h at room temperature prior to incubation with enzyme. Given the range of salt concentrations used (from 10 to 40 mM KCl), gels were vigorously stirred and pipette tip ends were cut so that gel suspensions could be pipetted.

Enzymatic digestions of -carrageenan were conducted at 30°C. At first, the tubes were shaken vigorously and initial undigested samples were collected. -Carrageenase was then added to the incubation medium: 0.12 µg mL -1 for carrageenan solutions and 0.60 µg mL -1 for carrageenan gels. NaOH was added to a final concentration of 100 mM in A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

order to stop the enzymatic reaction. -Carrageenase is inactivated in alkaline solutions [START_REF] Potin | Purification and characterization of a new kappa-carrageenase from a marine cytophaga-like bacterium[END_REF] without any alteration of the chemical structure of -carrageenan.

Reducing-sugar assay

The amount of reducing sugars produced during the enzymatic digestion ofcarrageenan was determined using a method adapted from Kidby and Davidson [START_REF] Kidby | A convenient ferricyanide estimation of reducing sugars in the nanomole range[END_REF]. Aliquots (100 µl) of the reaction medium diluted five times were mixed with 1 mL ferricyanide solution ( 300 mg potassium hexocyanoferrate III, 29 g Na 2 CO 3 , 1 mL 5 M NaOH, completed to 1 L with water). The mixture was boiled for 10 min, cooled to room temperature and its absorbance was read at 420 nm. We defined 100% degradation as the amount of reducing sugars obtained when soluble -carrageenan was completely converted to DP4 and DP2 and DP6 (neo--hexacarrabiose) could no longer be detected by HPAEC.

Liquid chromatography -multi-angle laser light scattering (LC-MALLS)

Prior to LC-MALLS experiments, samples were extensively dialysed against ultrapure water containing 20 mM ammonium carbonate (to prevent depolymerisation which may occur during freeze-drying) at 4°C using a 100 Da membrane (Spectra/Por® 100, cellulose ester) to eliminate KCl salts that promote gelation of carrageenan. Dialysed specimens were lyophilised. Samples were then dissolved in 100 mM LiNO 3 to a final concentration of about 0.3%. Soluble carrageenan (without KCl) and carrageenan gels (with KCl) were prepared in a similar fashion.

After filtration (0.22 m, Millipore), samples (200 L) were injected on a Pharmacia Superdex 200 column (300×10 mm i.d., GE Healthcare) followed by a Superdex peptide 10/300 column (Pharmacia GE Healthcare), mounted in series. Elution was performed with 0.1 M LiNO 3 filtered at 0.1 µm (refractive index = 1.327) at a flow rate of 0.3 mL min -1 (Waters 626 pump) at 25°C. Detection was monitored by a Waters 2414 refractive index detector, used as a mass sensitive detector, set at 890 nm at 35°C. MALLS measurements were performed at 690 nm with a DAWN EOS system (Wyatt Technology, Santa Barbara, CA, USA) equipped with a 30 mW Ga-As linearly polarized laser. The intensity of scattered light was measured at 12 different angles, from 35° to 143°. Chromatographic data were collected and processed by Astra software (Wyatt Technology, Santa Barbara, CA, USA).

The Zimm fit method was applied for molecular weight determinations Molecular weight was then measured by high performance steric chromatography using dextran sulphate as a standard (1, 5, 8, 100, 250 kDa). The chromatographic system and the elution conditions used were the same as described above.

Liquid chromatography of oligosaccharides labelled with 8-aminonaphthalene-1,3,6trisulfonic acid (ANTS)

Prior to labelling, the enzymatic reaction was stopped by adding 500 µL of ethanol to an equivalent volume of sample. The mixture was maintained at 80°C for about 10 min to ensure that the enzyme was denatured. The mixture was then was dried under vacuum. Then, 40 µL of 0.15 M the ANTS solution (ANTS disodium salt in 15% acetic acid) was added to the dried sample and kept at 37°C for 30 min. Then, 75 µL of 1 M NaBH 4 CN in DMSO was added and the sample was kept at 37°C for 16 h (i.e. overnight). The sample was dried under vacuum and dissolved in 2.5 mL of water, which corresponds to a 5X dilution of the initial sample concentration. For solid substrates (gel and powder), the final concentration of KCl was 5 mM, a concentration at which carrageenan does not gel. At this stage, ANTS-labelled oligocarrageenans were visualised after migration in a 27% (w/v) carbohydrate polyacrylamide gel (fluorescence-assisted carbohydrate electrophoresis, FACE) according to [START_REF] Guibet | Degradation of lambda-carrageenan by Pseudoalteromonas carrageenovora lambda-carrageenase: A new family of glycoside hydrolases unrelated to kappa-and iota-carrageenases[END_REF].

The sample was filtered (0.22 m, Millipore) and 100 µL was injected on a Superdex peptide 10/300 column (Pharmacia GE Healthcare) using the Dionex Ultimate 3000 chromatography system. Elution was performed with 50 mM ammonium carbonate at a flow rate of 0.3 mL min -1 . The ANTS-labelled oligosaccharides were detected using a UV-visible detector (VWD-3400RS, Dionex) operating at 365 nm. Acquisition data was achieved using the Chromeleon software (Dionex) and the surface areas of the peaks were integrated using PeakFit 4.12 software (Seasolve software Inc.). A standard curve using ANTS-labelled maltose was used to determine oligo-carrageenan concentrations.

High performance anion-exchange chromatography (HPAEC)

Degradation products were analysed using a Dionex chromatograph DX 500 equipped with a 20 µl injection loop, an AS 100XR automated injection system (Thermo Separation Products) and an AS11 anion exchange column (4 mm × 250 mm, Dionex IonPac®) with a AG11 pre-column (4 mm × 50 mm, Dionex IonPac®). The system was operated in conductivity mode using a ED40 detector (Dionex) and a Dionex ASRS ultra-4 mm suppressor with a current of 300 mA. Mobile phases were ultra-purified in water and 290 mM NaOH. Elution was conducted at a flow rate of 0.5 mL min -1 with GP40 Gradient Pump (Dionex). A linear gradient starting from 0% to 100% (290 mM NaOH) was applied for 33 min. Between each injection, the column was equilibrated with the mobile phase for 10 min. The Chromeleon-Peak Net software (Dionex) program was used for data acquisition and treatment. The area of the oligosaccharide peaks was integrated and normalised using the peak of 1 mM citric acid which was systematically included in the carrageenan preparations and used as an internal standard.

Polarimetry

Optical rotation measurements were performed using a Perkin-Elmer 341 polarimeter operating at a wavelength of 365 nm produced by Hg lamp. Samples were kept at 25°C in a thermostated jacketed cell having an optical path length of 10 cm.

Transmission electron microscopy (TEM)

One drop of each -carrageenan sample was deposited on carbon-coated copper grids.

After 30 s, the excess solution was removed using filter paper and the sample was allowed to dry. Samples were negatively stained using 1% uranyl acetate solution. One drop of stain was deposited on the grid and contact with the sample was maintained for 20 s. Excess stain was removed with filter paper and the remaining thin film of uranyl acetate solution was allowed to dry. Sample preparation was performed at room temperature and protected from direct light. Electron micrographs were recorded with a JEOL 1200X transmission electron microscope operating at 80 kV.

Results

In order to obtain soluble and gelled substrates, carrageenans were dissolved by heating in solutions containing increasing amounts of KCl from 0 to 40 mM. In the absence of KCl, the carrageenan solution remained in liquid form at room temperature, corresponding to complete dissolution. Gelation was observed macroscopically at room temperature when the occurrence of aggregation were confirmed by optical rotation (Figure 2B) and by light scattering (not shown), respectively. At KCl concentrations of 10 mM, a fragile, elastic, easily-broken gel was obtained. At KCl concentrations greater than 10 mM, gels increased in strength but could still be broken by shaking. The transmission electron micrograph of negatively stained -carrageenan gel prepared in 20 mM KCl showed that it was composed of interconnected elongated fibres of about 10-20 nm wide (Figure 1A). The fibres were made up of a thin substructure ascribed to helix duplexes or aggregated helices [START_REF] Hermansson | Rheological and microstructural evidence for transient state during gelation of kappa-carrageenan in presence of potassium[END_REF][START_REF] Borgström | On the structure of aggregated kappa-carrageenan helices. A study by cryo-tem, optical rotation and viscometry[END_REF].

Carrageenan powder, obtained by freeze-drying Na-carrageenan solubilised in hot water, was suspended in 20 mM KCl without heating to avoid the formation of extended helices that induce gelation. As expected, TEM revealed that the specimen was mainly composed of poorly organised or amorphous carrageenan aggregates. However, as illustrated in Figure 1B, very thin fibre-like structures 2-5 nm wide were observed. The thin fibres were probably already present in the carrageenan powder but may also result from the rearrangement of carrageenan molecules when the lyophilised powder was re-hydrated in the KCl solution. In what follows, we considered gel and powder forms of carrageenan as organised and poorly organised solid substrates, respectively.

Hydrolysis was quantified by measuring the amount of reducing sugars produced during the incubation of the -carrageenan with the recombinant P. carrageenovoracarrageenase. The degradation kinetics of -carrageenan allowed to gel in various concentrations of KCl is shown in Figure 2A. In the absence of KCl, the degradation appeared to follow an exponential increase in cleavage which reached a maximum after 360 min. At this stage, no more reducing ends could be obtained after addition of more enzyme. For increasing amounts of KCl, trends were similar with rapid increases at very short incubation times that levelled off later in the experiment with only marginal increases in digestion. For low KCl concentrations (0-10 mM), degradation was rapid and 100% degradation was attained in 360 min. At concentrations greater than 15 mM, i.e. conditions at which gelation was observed in the test tube and by microscopy, the kinetics and yield of hydrolysis were greatly reduced. For example, only 35% degradation was observed after 360 min for 20 mM KCl gel.

Hydrolysis velocities recorded at the beginning of incubation (< 15 min) are given in concentrations reached 30 mM. Kinetic experiments were also conducted with -carrageenan containing about 15% -carrabiose units, which is a carrageenan well known for its low gelling capacity. The overall kinetic curves were very similar to that of pure -carrageenan and 100% degradation was also attained in 360 min (data not shown). However, in contrast to pure -carrageenan, the amount KCl in the medium had less effect on the apparent activity of the enzyme. As illustrated in Figure 2B, the initial velocity decreased slowly and linearly as a function of KCl concentrations and about 20% loss of activity was observed in presence of 30 mM KCl.

The depolymerisation of -carrageenan induced by -carrageenase activity was followed by gel permeation chromatography coupled with a MALLS detector. In order to analyse all components occurring in the incubation medium, including gelled and soluble fractions of carrageenan, the samples were dialysed against water until buffer and KCl salts were removed. This procedure led to the complete dissolution of all carrageenan molecules and released any carrageenan oligosaccharides still entrapped in gel networks. As a consequence, the chromatography experiments presented hereafter are pictures of the overall molecular distribution of the incubation medium. In Figures 3A and3B, the time courses of depolymerisation of soluble (0 mM KCl) and gelled -carrageenan (20 mM KCl), respectively, are presented. The chromatogram corresponding to undigested samples showed a single peak eluting in the exclusion volume (at 16 mL retention volume) attributed to a 750 kDa molecular weight polysaccharide (Figure 3). As the degradation of soluble substrate proceeded (Figure 3A), the signal corresponding to the polymer fraction shifted toward higher elution volumes indicating a decrease in molecular weight. This was correlated to the MALLS measurements shown in Figure 4A which shows the drop in the molecular weight to 40 kDa after 10% hydrolysis. In addition, the shape of the peak became broader indicating an increase in polydispersity. Similar observations were done with low gelling -carrageenan incubated in 0 and 20 mM KCl. Permeation gel chromatograms as well as variation in molecular weight quantified by MALLS followed the same patterns (data not shown) whatever the KCl concentration. The decrease in molecular weight of low-gelling carrageenan in KCl shown in Figure 4 clearly demonstrates that depolymerisation followed a pattern similar to that of soluble carrageenan incubated without KCl. Recorded after 15% hydrolysis, the gel permeation chromatogram (Figure 3A) shows well-resolved signals that were ascribed to neooligocarrabioses from DP2 to DP12 (DP, degree of polymerisation, where DP2 indicates a disaccharide). The rapid depolymerisation, the increase of polydispersity and the production of all possible oligosaccharides are features of an endo mode of action.

In contrast to soluble substrates, the degradation pattern of the 20 mM KCl carrageenan gel showed a bimodal distribution of high and low molecular weight degradation products (Figure 3B). The high molecular weight fraction eluting at 16 mL remained in the exclusion volume, with signal intensity decreasing with the extent of digestion. MALLS analyses of this fraction indicated that the molecular weight decreased very slowly (Figure 4) and that polydispersity was constant (data not shown). In the low molecular weight fraction, only neo-tetracarrabiose (DP4) and neo-carrabiose (DP2) were observed at 33 and 36 mL elution volumes, and not other oligosaccharides such as neo-hexacarrabiose (DP6). Finally, no carrageenan fragments of intermediate molecular weight eluting between 18 and 32 mL were detected. Similar chromatograms were recorded for -carrageenan powder suspensions in 20 mM KCl suggesting that the enzyme adopts a similar mode of action.

The time course of oligosaccharide production was determined after labelling the reducing ends with the ANTS fluorophore. Using this approach, it was possible to directly measure the signal intensity of DP2 which eluted too close to salts (Figure 3) to directly determine its abundance by size-exclusion chromatography. Although HPAEC made it possible to visualise all the other oligosaccharides produced, again DP2 co-eluted with salts (not shown) hindering direct estimation of its production. The ANTS-labelling method offers the additional advantage of making it possible to quantitatively determine the amount of all the oligosaccharides produced. As expected, at low percentages of degradation of soluble carrageenan, a wide range of fluorescent oligosaccharides could be visualised by electrophoresis (Figure 5A). For example, after 15% degradation, it was possible to distinguish oligosaccharides from DP2 to DP26 which were converted to only DP4 and DP2 at the end of the kinetics experiment. Precise determination of oligosaccharide concentration from DP2 to DP8 was achieved by gel permeation chromatography coupled to a UV detector allowing the measurement of signal intensity corresponding to labelled oligosaccharides (Figure 6). The time-course production of the intermediate products DP6 and DP8 had a bell shape, while the amount of end-products DP2 and DP4 increased as the degradation proceeded. After complete degradation, the DP4:DP2 ratio was calculated to be, on average, abundances comparable to those observed with pure soluble -carrageenan, but hybrid -/carrageenan oligosaccharides were also present in the digest (results not shown).

As previously observed with unlabelled degradation products of carrageenan gels and powder, electrophoresis profiles revealed that only DP2 and DP4 were produced during incubation with -carrageenase (Figures 5A and5B). As illustrated in Figure 6B, the amount of DP2 and DP4 increased linearly with the extent of reaction for both gel and powder forms.

The DP4:DP2 ratio calculated for gel and powder forms had similar values: 0.72 ± 0.04 and 0.69 ± 0.03, respectively.

Discussion

The yields as well as the velocities of degradation of -carrageenan by P. carrageenovora -carrageenase decreased with increasing amounts of KCl in the incubation medium (Figure 2). This apparent decrease in activity was correlated with the aggregation of carrageenan molecules promoted by KCl ions. This aggregation led to the formation of a three-dimensional network of fibres visualised by TEM (Figure 1). In the solid state, fewer glycosidic linkages are exposed to the enzyme and, moreover, the network of fibres reduces the ability of the enzyme to diffuse. In low-gelling / -carrageenan, the velocity of degradation decreased very little with increasing KCl concentrations (Figure 2B). -Carrabiose units are known to hinder gelation of -carrageenan because of the irregularities or kinks in the chain that they induce [START_REF] Rees | Structure, conformation, and mechanism in the formation of polysaccharide gels and networks[END_REF][START_REF] Van De Velde | Coil-helix transition of -carrageenan as a function of chain regularity[END_REF]. Nevertheless, addition of KCl stiffened thecarrabiose segments occurring in the hybrid chains, and as a consequence, increased the viscosity of the medium thereby reducing enzyme and substrate diffusion. When KCl concentrations were greater than 200 mM, concentrations at which -/ -carrageenan starts to form a gel, we observed a decrease in the apparent activity of -carrageenase, equivalent to that observed for pure -carrageenan in 30 mM (data not shown). In addition, in both soluble carrageenan and low-gelling carrageenan incubated in 0 and 20 mM KCl, an endo mode of action was revealed, given the rapid decrease in molecular weight and slow production of diand tetrasaccharides as a function of catalytic events. Consequently, it is likely that KCl ions themselves do not play a direct role in the mechanism of action of -carrageenase but rather strongly modulate the physico-chemical properties of -carrageenan as observed by optical rotation and light-scattering experiments. The apparent decrease in enzymatic activity on pure -carrageenan should be considered as a decrease in substrate accessibility and reduced diffusion of the enzyme. A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Analysis of the mode of action of glycoside hydrolases incubated with substrates in different conformational or physical states can bring the different characteristics of an enzyme to light. Soluble or poorly organised solid substrates are known to facilitate the detection of an endo mode of action of an enzyme. For example, endo-acting cellulases are more efficient on carboxymethylcellulose or phosphoric acid swollen cellulose (PASC) than on crystalline cellulose (e.g. ref. [START_REF] Von Ossowski | Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, cel7a. A comparison with Phanerochaete chrysosporium cel7d[END_REF][START_REF] Zhou | Kinetic studies of Thermobifida fusca cel9a active site mutant enzymes[END_REF][START_REF] Stalbrand | Analysis of molecular size distributions of cellulose molecules during hydrolysis of cellulose by recombinant Cellulomonas fimi beta-1,4-glucanases[END_REF]). Similarly, chitosan (partly deacetylated chitin) is preferred over solid and unmodified chitin for identifying endo-acting chitinases [START_REF] Sikorski | Development and application of a model for chitosan hydrolysis by a family 18 chitinase[END_REF][START_REF] Sikorski | Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan[END_REF]. Although processivity can be observed on soluble substrates [START_REF] Robyt | Multiple attach hypothesis of alpha-amylase action: Action of porcine pancreatic, human salivary, and Aspergillus oryzae alpha-amylases[END_REF][START_REF] Sikorski | Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan[END_REF], it is more straightforward to demonstrate in well-organised or crystalline material observed by electron microscopy [START_REF] Chanzy | Electron microscopy investigation of the enzymatic hydrolysis of valonia cellulose[END_REF][START_REF] Helbert | Electron microscopic investigation of the diffusion of Bacillus licheniformis alpha-amylase into corn starch granules[END_REF][START_REF] Boisset | Imaging the enzymatic digestion of bacterial cellulose ribbons reveals the endo character of the cellobiohydrolase cel6a from Humicola insolens and its mode of synergy with cellobiohydrolase cel7a[END_REF][START_REF] Uchiyama | Roles of the exposed aromatic residues in crystalline chitin hydrolysis by chitinase a from Serratia marcescens 2170[END_REF] or by chromatography methods [START_REF] Stalbrand | Analysis of molecular size distributions of cellulose molecules during hydrolysis of cellulose by recombinant Cellulomonas fimi beta-1,4-glucanases[END_REF]. By adopting a similar strategy, we prepared soluble carrageenan as well as poorly (powder) and well-organised (gel) carrageenan in the solid state. In solution, -carrageenan adopted a flexible rod conformation and enzymatic depolymerisation led to the production of all likely neo-oligo-carrageenans (Figure 3A and5A). This wide range of products was accompanied by a rapid decrease in molecular weight and an increase of polydispersity (Figure 4). Consequently, in these conditions, P. carrageenovora -carrageenase can be defined as an endo-acting hydrolase. The same conclusions were drawn from analyses of enzymatic degradation products of the low-gelling -/ -carrageenan (results not shown).

Chemical modification of polysaccharides can introduce a bias in the interpretation

and comparison of the mode of action of enzymes in homogenous and heterogeneous phases.

Consequently, carrageenan gels and powder were prepared with polysaccharides having the same chemical structure and same macromolecular parameters (molecular weight, polydispersity) as those studied in the soluble state. Since these carrageenans were not subjected to any chemical modification, our observations were directly correlated to the conformational and physical states of the substrate. When carrageenan was incubated in a solid state (gel and powder), we only observed the end products DP4 and DP2. We assume that any solubilised intermediate oligosaccharides were quickly degraded into DP4 and DP2. Furthermore, these intermediate oligosaccharides were probably cleaved faster than more recalcitrant carrageenan chains located in the solid polysaccharide network. As a consequence, although degradation of carrageenan powder was faster than that of the gel, the production of DP4 and DP2 increased linearly in both cases. The production of DP4 was greater when the substrate was solid (gel or powder) than when the substrate was soluble. Moreover, when carrageenan was incubated in the solid state, the molecular weight ofcarrageenan and its polydispersity did not change much between 0 and 30% degradation (Figure 4).

We interpreted these features as evidence for a processive mode of action ofcarrageenase. Due to the alternation of -(1 4) and -(1 3) linkages in carrageenan, two successive -(1 4) linkages are in opposite orientations, pointing up or down. Consequently, as depicted in Figure 7, processive -carrageenase, as it slides along the polysaccharide chain, would encounter a -(1 4) linkage in the correct orientation for cleavage only every two disaccharide units. Therefore, in order to processively digest long-chain polysaccharides, the -carrageenase must dissociate from the substrate molecule thereby releasing DP4 or oligocarrageenan multiples of DP4 [START_REF] Michel | The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae[END_REF]. In this context, the DP4 to DP2 ratio made it possible to depict variation in the processivity of -carrageenase since DP4 is produced by random and processive modes of action while DP2 is exclusively produced by random depolymerisation.

A comparison of DP4 to DP2 ratios revealed that the processivity of -carrageenase was nearly identical when incubated with carrageenan gel or powder (0.72 ± 0.04 and 0.69 ± 0.03 respectively), and higher than when incubated with soluble carrageenan (0.576 ± 0.005). We have thus demonstrated that -carrageeenase acts in an endo manner in soluble substrates (with and without KCl) but in a processive manner in solid substrates. Consequently, it appears that P. carrageenovora -carrageenase is an endo-processive enzyme.

We observed that purified DP4 can be slowly converted to DP2 (results not shown), but the time and enzyme concentration required were greater than those used in the experimental conditions for the complete degradation of soluble substrate. In the case of solid substrates, enzyme concentrations used for degradation kinetics were twice as great as those used for the soluble substrate and degradation only reached 65% and 70% for gel and powder forms, respectively. After increasing enzyme concentrations four-fold, we were able to almost completely degrade carrageenan powder. At this concentration, the DP4:DP2 ratio was 0.65 ± 0.02, a value that is slightly lower than when enzyme concentration was only doubled. With the four-fold enzyme concentration, we obtained about 75% degradation of the gel with a DP4:DP2 ratio of 0.64 ± 0.02. In both cases, the ratio of DP4 to DP2 in gel and powder forms was higher than with soluble substrate, which provides additional support in favour of the processive character of -carrageenase. The lower ratio of DP4 to DP2 at a higher enzyme underestimation the processive character of -carrageenase. Therefore, all our investigations on solid carrageenan may have underestimated the processive character of -carrageenase.

The processive mode of action of P. carrageenovora -carrageenase in heterogeneous phases is in accordance with the prediction of [START_REF] Michel | The kappa-carrageenase of Pseudoalteromonas.carrageenovora features a tunnel-shaped active site: A novel insight in the evolution of clan-b glycoside hydrolases[END_REF] [START_REF] Michel | The kappa-carrageenase of Pseudoalteromonas.carrageenovora features a tunnel-shaped active site: A novel insight in the evolution of clan-b glycoside hydrolases[END_REF] who suggested this type of mode of action after observing the tunnel or closed-groove topology of the active site.

In homogenous phases, we demonstrated that the -carrageenan chain is internally cleaved, implying that the tunnel opens and, as a consequence, that the binding loop is flexible enough to allow for repeated binding and dissociation with carrageenan chains. This is consistent with crystallography data which revealed that amino acids constituting the loop presented higher temperature factors than the core of the enzyme [START_REF] Michel | The kappa-carrageenase of Pseudoalteromonas.carrageenovora features a tunnel-shaped active site: A novel insight in the evolution of clan-b glycoside hydrolases[END_REF]. Opening of a tunnel has already been observed in other endo-processive enzymes such as cellulases [START_REF] Armand | A bifunctionalized fluorogenic tetrasaccharide as a substrate to study cellulases[END_REF][START_REF] Varrot | Structural basis for ligand binding and processivity in cellobiohydrolase cel6a from Humicola insolens[END_REF], chitinases [START_REF] Sikorski | Serratia marcescens chitinases with tunnel-shaped substrate-binding grooves show endo activity and different degrees of processivity during enzymatic hydrolysis of chitosan[END_REF] andcarrageenase [START_REF] Michel | The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae[END_REF]. The acquisition of the tunnel-shaped topology of the active site may be attributed to convergent evolution of processive glycoside hydrolases acting on neutral and anionic polysaccharides. Although the exact role of the binding loop is not well-understood, it is supposed that it facilitates the extraction of polysaccharides from solid material [START_REF] Von Ossowski | Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, cel7a. A comparison with Phanerochaete chrysosporium cel7d[END_REF][START_REF] Koivula | Tryptophan 272: An essential determinant of crystalline cellulose degradation by Trichoderma reesei cellobiohydrolase cel6a[END_REF]. In addition, and as for -carrageenase, the tunnel topology, and more specifically the loop, ofcarrageenase may be involved in unwinding the -carrageenan double helix [START_REF] Michel | The structural bases of the processive degradation of iota-carrageenan, a main cell wall polysaccharide of red algae[END_REF].

-Carrageenan forms physical gels in vitro that mimic the conformational and physical properties of algal cell walls [START_REF] Rochas | Solid state 13 C-nmr spectroscopy of red seaweeds, agars and carrageenans[END_REF][START_REF] Gordon-Mills | Use of solid and gel state 13 C nmr spectroscopy for differenciation between agarophytes and carrageenophytes[END_REF]. By definition, gels are not as well organised as highly crystalline materials such as cellulose, chitin or starch. In addition, the chemical structure of carrageenans in vivo are not regular, but usually hybrid or co-polymeric structures [START_REF] Bixler | Recent development in manufacturing and marketing carrageenan[END_REF][START_REF] Craigie | Cell walls[END_REF]. This chemical heterogeneity, assumed to play a role in regulating functional properties in the cell wall as well as allowing for dense packing of macromolecules, creates defects which hinder the formation of a highly organised structure [START_REF] Craigie | Cell walls[END_REF]. Consequently, in this context, the flexibility of the loop that opens the catalytic site, enabling it to accommodate one carrageenan chain, may allow it to better extract polysaccharide chains from poorly organised regions which are probably more abundant in cell walls than in gels prepared in vitro. Nevertheless, in the algal cell wall, carrageenan is found in the solid state. Sliding along carrageenan chains would increase enzyme efficiency by reducing its three-dimensional diffusion in the medium to find glycosidic bonds. This may explain why carrageenan gels and powders were degraded in a very similar processivity mechanism despite differences in molecular ordering of the carrageenan macromolecule. Therefore, conditions required for the -carrageenase processivity depend not only on the adapted configuration of the active site (amino acids, loop), but also on the physical state of the substrate. Based on our results, we cannot rule out the possibility that -carrageenase also processively degrades soluble substrates. However determining the processivity index of -carrageenase-or other depolymerising enzymesusing soluble substrates probably greatly underestimates their true processivity on solid substrate and, as a consequence, in vivo. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

7

  Biochemical Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619

  . The calculated dn/dc 8 Biochemical Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  was 0.115 ml g -1 . Bovine serum albumin monomer (Sigma, St.Louis, MO) was used to normalize the signals recorded at various angles of detection, with the signal measured at 90°. Below 100 kDa, scattering of carrageenan was too low to determine molecular weight.

  mixture contained at least 10 mM KCl. The formation of carrageenan helices and the 10 Biochemical Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 2B .

 2B Figure 2B. As observed for the degradation kinetics (Figure2A), velocity decreased with increasing amounts of KCl, with the minimum velocity being measured when KCl

  0.576 ± 0.005 (n = 6) Similarly, chromatograms of low-gelling -/ -carrageenan incubated without KCl and with 20 mM KCl showed a wide distribution of oligosaccharides with 13 Biochemical Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619

  concentration can be explained by a low rate of conversion of DP4 to DP2. Unlike DP2, DP4 can be produced processively; thus the conversion of DP4 to DP2 would result in a 16 Biochemical Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 05 Feb 2009 as manuscript BJ20080619 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20080619A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 :

 1 Figure 1: Transmission electron micrograph of negatively stained -carrageenan gels in 20 mM KCl (A) and -carrageenan powder suspended in 20 mM KCl (B).

Figure 2 :

 2 Figure 2: (A) Degradation kinetics of 0.2% carrageenan by P. carrageenovora carrageenase subjected to gelation in increasing concentrations of KCl. (B) Initial velocity of degradation of -carrageenan ( ) and low-gelling -carrageenan (containing 15% -carrabiose units) ( ) with respect to KCl concentration. Optical rotation recorded on -carrageenan preparation as a function of KCl concentration ( ).

Figure 3 :Figure 4 :

 34 Figure 3: Degradation kinetics of soluble (A) and 20 mM KCl gelled -carrageenan (B) monitored by size-exclusion chromatography as a function of the percentage of degradation as defined in the Experimental section. DP: degree of polymerisation (i.e. DP4 indicates tetrasaccharides). RF: Resistant fraction. * hybrid oligosaccharides

Figure 5 :

 5 Figure 5: FACE of oligosaccharides released during the digestion of soluble (A), gelled (B) and powder (C) forms of carrageenan at various percentages of degradation. Oligosaccharides were labelled with fluorescent ANTS. * Hybrid -/ -oligosaccharides [53].

Figure 6 :

 6 Figure 6: Amount of oligosaccharides produced during incubation of soluble ( -carrageenan in 0 mM KCl) and solid (20 mM KCl) forms of carrageenan. Oligosaccharide concentrations were determined by ANTS labelling and integration of the signal from gel permeation chromatography.

Figure 7 :

 7 Figure 7: A) Schematic representation of the endolytic mode of action of P. carrageenovora -carrageenase which produces all possible oligosaccharides. B) The processive degradation of -carrageenase which slides from the reducing (R) to the non-reducing end (NR) and leads to the production of DP4. The enzyme has six sub-sites and arrowheads indicate position of catalytic amino acids (25).

3 Biochemical

 3 FIGURE 2

Biochemical

  FIGURE 4

  FIGURE 6

  FIGURE 7

© 2009 The Authors Journal compilation © 2009 Portland Press Limited

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2009 The Authors Journal compilation © 2009 Portland Press Limited

Acknowledgements

This work was supported by the French National Center for Scientific Research (CNRS ATIPE program) and Pierre and Marie Curie University (Paris VI). The authors acknowledge Muriel Jam for her help in producing recombinant enzymes.