Molecular insights into the hydrolysis and transglycosylation of a deep-sea Planctomycetota-derived GH16 family laminarinase.

The biochemical and structural characteristics of PtLam, a laminarinase from deep-sea Planctomycetota, have been extensively elucidated, unveiling the fundamental molecular mechanisms governing substrate recognition and enzymatic catalysis. PtLam functions as an exo-laminarinase with the ability to sequentially hydrolyze laminarin, cleaving glucose units individually. Notably, PtLam exhibits proficient transglycosylation capabilities, utilizing various sugar alcohols as acceptors, with lyxose, in particular, yielding exclusively transglycosylated products. Structural analysis of both apo-PtLam and its laminarin oligosaccharide-bound complex revealed significant conformational alterations in active residues upon substrate binding. Moreover, pivotal residues involved in substrate recognition were identified, with subsequent mutation assays indicating the contribution of positive subsites in modulating exo-hydrolysis and transglycosidic activities. These results enhance our comprehension of laminarin cycling mechanisms by marine Planctomycetota, while also providing essential enzyme components for laminarin hetero-oligosaccharide synthesis.IMPORTANCEThe ubiquitous Planctomycetota, with distinctive physiological traits, exert a significant influence on global carbon and nitrogen fluxes. Their intimate association with algae suggests a propensity for efficient polysaccharide degradation; however, research on glycoside hydrolases derived from Planctomycetota remains scarce. Herein, we unveil the GH16 family laminarinase PtLam from deep-sea Planctomycetota, shedding light on its catalytic mechanisms underlying hydrolysis and transglycosylation. Our findings elucidate the enzymatic pathways governing the marine laminarin cycle orchestrated by Planctomycetota, thereby fostering the exploration of novel polysaccharide hydrolases with promising practical implications.

Insights into the dual cleavage activity of the GH16 laminarinase enzyme class on ß-1,3 and ß-1,4 glycosidic bonds.

Glycoside hydrolases (GHs) are involved in the degradation of a wide diversity of carbohydrates and present several biotechnological applications. Many GH families are composed of enzymes with a single well-defined specificity. In contrast, enzymes from the GH16 family can act on a range of different polysaccharides, including ß-glucans and galactans. SCLam, a GH16 member derived from a soil metagenome, an endo-ß-1,3(4)-glucanase (EC 3.2.1.6), can cleave both ß-1,3 and ß-1,4 glycosidic bonds in glucans, such as laminarin, barley ß-glucan, and cello-oligosaccharides. A similar cleavage pattern was previously reported for other GH16 family members. However, the molecular mechanisms for this dual cleavage activity on (1,3)- and (1,4)-ß-D-glycosidic bonds by laminarinases have not been elucidated. In this sense, we determined the X-ray structure of a presumably inactive form of SCLam cocrystallized with different oligosaccharides. The solved structures revealed general bound products that are formed owing to residual activities of hydrolysis and transglycosylation. Biochemical and biophysical analyses and molecular dynamics simulations help to rationalize differences in activity toward different substrates. Our results depicted a bulky aromatic residue near the catalytic site critical to select the preferable configuration of glycosidic bonds in the binding cleft. Altogether, these data contribute to understanding the structural basis of recognition and hydrolysis of ß-1,3 and ß-1,4 glycosidic linkages of the laminarinase enzyme class, which is valuable for future studies on the GH16 family members and applications related to biomass conversion into feedstocks and bioproducts.

Characterization of the GH16 and GH17 laminarinases from Vibrio breoganii 1C10.

Laminarin is an abundant glucose polymer used as an energy reserve by micro- and macroalgae. Bacteria digest and consume laminarin with laminarinases. Their genomes frequently contain multiple homologs; however, the biological role for this replication remains unclear. We investigated the four laminarinases of glycoside hydrolase families GH16 and GH17 from the marine bacterium Vibrio breoganii 1C10, which can use laminarin as its sole carbon source. All four laminarinases employ an endolytic mechanism and specifically cleave the ß-1,3-glycosidic bond. Two primarily produce low-molecular weight laminarin oligomers (DP 3-4) whereas the others primarily produce high-molecular weight oligomers (DP > 8), which suggests that these enzymes sequentially degrade laminarin. The results from this work provide an overview of the laminarinases from a single marine bacterium and also provide insights regarding how multiple laminarinases are used to degrade laminarin.

Enzymatic properties of a multi-specific ß-(1,3)-glucanase from Corallococcus sp. EGB and its potential antifungal applications.

The lamC gene encoding a novel ß-(1,3)-glucanase was cloned from Corallococcus sp. EGB and successfully expressed in the industrial yeast Pichia pastoris. The mature protein without the initial 26 residues of signal peptide, designated LamC27, was found to be composed of fascin-like module and laminarinase-like catalytic module. The purified recombinant enzyme (rLamC27) with a calculated molecular mass of 45.3 kDa displays activities toward a broad range of ß-linked polysaccharides, including laminarin, curdlan, pachyman, lichenan, and CMC. Enzymological characterization showed that rLamC27 performes its optimal activity under the condition of 45 °C and pH 7.0, respectively, and preferentially catalyzes the hydrolysis of glucans with a ß-1,3-linkage, which is similar to the LamC previously expressed in E. coli. TherLamC27 enzyme was activated by Mn2+ and Ba2+, while it was inhibited by Cu2+, Zn2+, and Co2+. Moreover, rLamC27 was strongly inhibited by 10 mM EDTA with 7.5% of its original activity remiaining, and weakly by SDS and Triton X-100. In antifungal assay, rLamC27 was conformed to possess lytic and antifungal activity against rice blast fungus. Specifically, a significant decrease germ tube and appressorium formation ratios from 94% to 59% and 97%-51%, respectively, were observed following exposure to rLamC27. H2DCFDA and CFW staining further demonstrated that the fungistasis capability of rLamC27 could be contributed by its ability to hydrolyze components of the cell wall. All these favorable properties indicate a promising potential for using rLamC27 as a biological antifungal agent in areas such as plant protection and food preservation.

Clostridium fungisolvens sp. nov., a new ß-1,3-glucan-decomposing bacterium isolated from anoxic soil subjected to biological soil disinfestation.

Biological soil disinfestation (BSD) or reductive soil disinfestation (RSD) is a bioremediation method used to suppress or eliminate soil-borne plant pathogens by stimulating activities of indigenous anaerobic bacteria of the soil. An anaerobic bacterial strain (TW1T) was isolated from an anoxic soil sample subjected to the BSD treatment and comprehensively characterized. Cells of the strain were Gram-stain-positive, slightly curved and motile rods producing terminal spores. The strain was aerotolerant. Strain TW1T was saccharolytic and produced acetate, butyrate, H2 and CO2 as fermentation end products. Strain TW1T decomposed ß-1,3-glucan (curdlan and laminarin) and degraded mycelial cells of an ascomycete Fusarium plant pathogen. Major cellular fatty acids of strain TW1T were C14 : 0, C14 : 0 dimethylacetal (DMA), C16 : 0 aldehyde and C16 : 0 DMA. Strain TW1T made a group on the phylogenetic tree constructed based on 16S rRNA gene sequences with species such as Clostridium fallax (96.3 %) and Clostridium polyendosporum (96.0 %). Whole genome analysis of strain TW1T showed that the total length of the genome was 5.28 Mb with the DNA G+C content of 31.3 mol%. The average nucleotide identity (ANIb) between strain TW1T and C. fallax was 71.2 %. Presence of the genes encoding laminarinase or GH16 ß-glucosidase was confirmed from the genome analysis of strain TW1T. Based on the genomic, phylogenetic and phenotypic properties obtained, we propose strain TW1T should be assigned in the genus Clostridium in the family Clostridiaceae as Clostridium fungisolvens sp. nov. The type strain TW1T (=NBRC 112097T=DSM 110791T).

Accurate Quantification of Laminarin in Marine Organic Matter with Enzymes from Marine Microbes.

Marine algae produce a variety of glycans, which fulfill diverse biological functions and fuel the carbon and energy demands of heterotrophic microbes. A common approach to analysis of marine organic matter uses acid to hydrolyze the glycans into measurable monosaccharides. The monosaccharides may be derived from different glycans that are built with the same monosaccharides, however, and this approach does not distinguish between glycans in natural samples. Here we use enzymes to digest selectively and thereby quantify laminarin in particulate organic matter. Environmental metaproteome data revealed carbohydrate-active enzymes from marine flavobacteria as tools for selective hydrolysis of the algal ß-glucan laminarin. The enzymes digested laminarin into glucose and oligosaccharides, which we measured with standard methods to establish the amounts of laminarin in the samples. We cloned, expressed, purified, and characterized three new glycoside hydrolases (GHs) of Formosa bacteria: two are endo-ß-1,3-glucanases, of the GH16 and GH17 families, and the other is a GH30 exo-ß-1,6-glucanase. Formosa sp. nov strain Hel1_33_131 GH30 (FbGH30) removed the ß-1,6-glucose side chains, and Formosa agariphila GH17A (FaGH17A) and FaGH16A hydrolyzed the ß-1,3-glucose backbone of laminarin. Specificity profiling with a library of glucan oligosaccharides and polysaccharides revealed that FaGH17A and FbGH30 were highly specific enzymes, while FaGH16A also hydrolyzed mixed-linked glucans with ß-1,4-glucose. Therefore, we chose the more specific FaGH17A and FbGH30 to quantify laminarin in two cultured diatoms, namely, Thalassiosira weissflogii and Thalassiosira pseudonana, and in seawater samples from the North Sea and the Arctic Ocean. Combined, these results demonstrate the potential of enzymes for faster, stereospecific, and sequence-specific analysis of select glycans in marine organic matter.IMPORTANCE Marine algae synthesize substantial amounts of the glucose polymer laminarin for energy and carbon storage. Its concentrations, rates of production by autotrophic organisms, and rates of digestion by heterotrophic organisms remain unknown. Here we present a method based on enzymes that hydrolyze laminarin and enable its quantification even in crude substrate mixtures, without purification. Compared to the commonly used acid hydrolysis, the enzymatic method presented here is faster and stereospecific and selectively cleaves laminarin in mixtures of glycans, releasing only glucose and oligosaccharides, which can be easily quantified with reducing sugar assays.

A new recombinant endo-1,3-ß-D-glucanase from the marine bacterium Formosa algae KMM 3553: enzyme characteristics and transglycosylation products analysis.

A specific endo-1,3-ß-D-glucanase (GFA) gene was found in genome of marine bacterium Formosa algae KMM 3553. For today this is the only characterized endo-1,3-ß-D-glucanase (EC 3.2.1.39) in Formosa genus and the only bacterial EC 3.2.1.39 GH16 endo-1,3-ß-D-glucanase with described transglycosylation activity. It was expressed in E. coli and isolated in homogeneous state. Investigating the products of polysaccharides digestion with GFA allowed to establish it's substrate specificity and classify this enzyme as glucan endo-1,3-ß-D-glucosidase (EC 3.2.1.39). The amino-acid sequence of GFA consists of 556 residues and shows sequence similarity of 45-85% to ß-1,3-glucanases of bacteria belonging to the CAZy 16th structural family of glycoside hydrolases GH16. Enzyme has molecular weight 61 kDa, exhibits maximum of catalytic activity at 45 °C, pH 5.5. Half-life period at 45 °Ð¡ is 20 min, complete inactivation happens at 55 °C within 10 min. Km for hydrolysis of laminarin is 0.388 mM. GFA glucanase from marine bacteria F. algae is one of rare enzymes capable to catalyze reactions of transglycosylation. It catalyzed transfer of glyconic part of substrate molecule on methyl-ß-D-xylopyranoside, glycerol and methyl-α-D-glucopyranoside. The enzyme can be used in structure determination of ß-1,3-glucans (or mixed 1,3;1,4- and 1,3;1,6-ß-D-glucans) and enzymatic synthesis of new carbohydrate-containing compounds.

Biochemical and genetic characterization of ß-1,3 glucanase from a deep subseafloor Laceyella putida.

A ß-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

The ß-glucanase ZgLamA from Zobellia galactanivorans evolved a bent active site adapted for efficient degradation of algal laminarin.

Laminarinase is commonly used to describe ß-1,3-glucanases widespread throughout Archaea, bacteria, and several eukaryotic lineages. Some ß-1,3-glucanases have already been structurally and biochemically characterized, but very few from organisms that are in contact with genuine laminarin, the storage polysaccharide of brown algae. Here we report the heterologous expression and subsequent biochemical and structural characterization of ZgLamAGH16 from Zobellia galactanivorans, the first GH16 laminarinase from a marine bacterium associated with seaweeds. ZgLamAGH16 contains a unique additional loop, compared with other GH16 laminarinases, which is composed of 17 amino acids and gives a bent shape to the active site cleft of the enzyme. This particular topology is perfectly adapted to the U-shaped conformation of laminarin chains in solution and thus explains the predominant specificity of ZgLamAGH16 for this substrate. The three-dimensional structure of the enzyme and two enzyme-substrate complexes, one with laminaritetraose and the other with a trisaccharide of 1,3-1,4-ß-d-glucan, have been determined at 1.5, 1.35, and 1.13 Å resolution, respectively. The structural comparison of substrate recognition pattern between these complexes allows the proposition that ZgLamAGH16 likely diverged from an ancestral broad specificity GH16 ß-glucanase and evolved toward a bent active site topology adapted to efficient degradation of algal laminarin.

Structural and biochemical characterization of the laminarinase ZgLamCGH16 from Zobellia galactanivorans suggests preferred recognition of branched laminarin.

Laminarin is a ß-1,3-D-glucan displaying occasional ß-1,6 branches. This storage polysaccharide of brown algae constitutes an abundant source of carbon for marine bacteria such as Zobellia galactanivorans. This marine member of the Bacteroidetes possesses five putative ß-1,3-glucanases [four belonging to glycosyl hydrolase family 16 (GH16) and one to GH64] with various modular architectures. Here, the characterization of the ß-glucanase ZgLamC is reported. The catalytic GH16 module (ZgLamCGH16) was produced in Escherichia coli and purified. This recombinant enzyme has a preferential specificity for laminarin but also a significant activity on mixed-linked glucan (MLG). The structure of an inactive mutant of ZgLamCGH16 in complex with a thio-ß-1,3-hexaglucan substrate unravelled a straight active-site cleft with three additional pockets flanking subsites -1, -2 and -3. These lateral pockets are occupied by a glycerol, an acetate ion and a chloride ion, respectively. The presence of these molecules in the vicinity of the O6 hydroxyl group of each glucose moiety suggests that ZgLamCGH16 accommodates branched laminarins as substrates. Altogether, ZgLamC is a secreted laminarinase that is likely to be involved in the initial step of degradation of branched laminarin, while the previously characterized ZgLamA efficiently degrades unbranched laminarin and oligo-laminarins.

Genes for laminarin degradation are dispersed in the genomes of particle-associated Maribacter species.

Laminarin is a cytosolic storage polysaccharide of phytoplankton and macroalgae and accounts for over 10% of the world's annually fixed carbon dioxide. Algal disruption, for example, by viral lysis releases laminarin. The soluble sugar is rapidly utilized by free-living planktonic bacteria, in which sugar transporters and the degrading enzymes are frequently encoded in polysaccharide utilization loci. The annotation of flavobacterial genomes failed to identify canonical laminarin utilization loci in several particle-associated bacteria, in particular in strains of Maribacter. In this study, we report in vivo utilization of laminarin by Maribacter forsetii accompanied by additional cell growth and proliferation. Laminarin utilization coincided with the induction of an extracellular endo-laminarinase, SusC/D outer membrane oligosaccharide transporters, and a periplasmic glycosyl hydrolase family 3 protein. An ABC transport system and sugar kinases were expressed. Endo-laminarinase activity was also observed in Maribacter sp. MAR_2009_72, Maribacter sp. Hel_I_7, and Maribacter dokdonensis MAR_2009_60. Maribacter dokdonensis MAR_2009_71 lacked the large endo-laminarinase gene in the genome and had no endo-laminarinase activity. In all genomes, genes of induced proteins were scattered across the genome rather than clustered in a laminarin utilization locus. These observations revealed that the Maribacter strains investigated in this study participate in laminarin utilization, but in contrast to many free-living bacteria, there is no co-localization of genes encoding the enzymatic machinery for laminarin utilization.

Microbacterium oxydans, a novel alginate- and laminarin-degrading bacterium for the reutilization of brown-seaweed waste.

There is a growing demand for the efficient treatment of seaweed waste. We identified six bacterial strains from the marine environment for the reutilization of brown-seaweed waste, and the most potentially useful strain, Microbacterium oxydans, was chosen and further investigated. Plate assays indicated that this bacterial isolate possessed both alginate lyase and laminarinase activities. The optimal inoculum size, pH, temperature and substrate concentration for the degradation of brown-seaweed polysaccharides by the isolate were as follows: 20% (v v(-1)), pH 6.0, 37 °C, and 5 g L(-1) for alginate and 20% (v v(-1)), pH 6.0, 30 °C, and 10 g L(-1) for laminarin, respectively. During 6 d in culture under the optimal conditions, the isolate produced 0.17 g L(-1) of reducing sugars from alginate with 11.0 U mL(-1) of maximal alginate lyase activity, and 5.11 and 2.88 g L(-1) of reducing sugars and glucose from laminarin, respectively. In particular, a fair amount of laminarin was degraded to glucose (28.8%) due to the isolate's exolytic laminarinase activity. As a result, the reutilization of brown-seaweed waste by this isolate appears to be possible for the production of reducing sugars as a valuable resource. This is the first study to directly demonstrate the ability of M. oxydans to degrade both alginate and laminarin.

Mutation of conserved residues in the laminarinase Lam1092 increases the antioxidant activity of the laminarin product hydrolysates.

Laminarinases from the glycoside hydrolase 16 (GH16) family are hydrolases that break ß-1,3-glycosidic bonds in laminarin, which is the major storage polysaccharide present in brown algae or microalgae. We explored a laminarinase from the marine Flavobacteriaceae species Tamlana sp. PT2-4 at the structural and functional levels. Based on a homology model of Lam1092-substrate interactions, the large active groove crossing Lam1092 was deemed a reasonable pathway for the bent substrates for hydrolysis. Eight residues (Gly361, Asn364, Arg400, His466, Asp449, Glu452, Ser477 and Thr538) were selected for mutagenesis based on the interactions of Lam1092 in complex with Lam4/Lam6. Ultimately, we generated eight mutants of Lam1092, and the antioxidant activities of the hydrolysates of two mutants (G361A and H466A) showed significant improvement. These results show that the antioxidant activity of laminarin can be improved by laminarinase mutation, which will be beneficial for developing efficient approaches to engineer the substrate specificity of laminarinases and improve the application of bioactive laminarioligosaccharides.

Safety evaluation of the food enzyme endo-1,3(4)-ß-glucanase from the non-genetically modified Rasamsonia composticola strain 427-FS.

The food enzyme endo-1,3(4)-ß-glucanase (3-(1-3,1-4)-ß-d-glucan 3(4)-glucanohydrolase; EC 3.2.1.6) is produced with the non-genetically modified Rasamsonia composticola 427-FS strain by Kerry Ingredients & Flavours Ltd. The food enzyme is free from viable cells of the production organism. The food enzyme is intended to be used in six manufacturing processes, i.e. baking processes, other cereal-based processes, brewing processes, grain treatment for the production of starch and gluten fractions, distilled alcohol production and yeast processing. Since residual amounts of total organic solids (TOS) are removed by distillation and during grain processing, dietary exposure was calculated only for the remaining four processes. It was estimated to be up to 0.809 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 866 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1,070. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Simple Methods for the Preparation of Colloidal Chitin, Cell Free Supernatant and Estimation of Laminarinase.

Colloidal chitin (CC) is a common substrate used in research work involving chitin-active enzymes (chitinases). Cell free supernatant (CFS) is prepared from fermented broth. Preparation of CC and CFS usually involve large amounts of liquid, which must be separated from the solids. This necessitates the use of a large volume centrifugation facility, which may not be accessible to everyone. Filtration is a viable alternative to centrifugation, and several filter elements are described in the literature. Each of those elements has its own set of disadvantages like non-availability, high cost, fragility, and non-reusability. Here we describe the use of lab coat clothing material (LCCM) for the preparation of CC and CFS. For filtration purposes, the LCCM was found to be functional, rugged, reusable, and cost-effective. Also described here is a new method for the estimation of laminarinase using a laminarin infused agarose gel plate. An easily available optical fabric brightener (OFB) was used as a stain for the agarose plate. The laminarin infused agarose plate assay is simple, inexpensive, and was found to be impervious to high amounts of ammonium sulfate (AS) in enzyme precipitates.

Enzymatic properties and the gene structure of a cold-adapted laminarinase from Pseudoalteromonas species LA.

We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15-50°C and pH 5.0-9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.

The ß-glucosidase secreted by Talaromyces amestolkiae under carbon starvation: a versatile catalyst for biofuel production from plant and algal biomass.

BACKGROUND: In the last years, the most outstanding trend for obtaining high added-value components and second-generation (2G) biofuels consisted on exploitation of plant biomass. But recently, 3G biofuels, based in algae biomass, have emerged as a great alternative for production of energy. RESULTS: In this work, a versatile ß-glucosidase from the ascomycete fungus Talaromyces amestolkiae has been purified, characterized, and heterologously expressed. The synthesis of this ß-glucosidase (BGL-3) was not induced by cellulose, and the presence of a specific carbon source is not required for its production, which is uncommon for ß-glucosidases. BGL-3, which was obtained from a basal medium with glucose as carbon source, was profusely secreted under carbon starvation conditions, which was corroborated by qRT-PCR assays. BGL-3 was purified from T. amestolkiae cultures in one step, and biochemically characterized. The enzyme showed high thermal stability, and very high efficiency on pNPG (Km of 0.14 mM and Vmax of 381.1 U/mg), cellobiose (Km of 0.48 mM and Vmax of 447.1 U/mg), and other cello-oligosaccharides. Surprisingly, it also showed remarkable ability to hydrolyze laminarin, a ß-1,3-glucan present in algae. The recombinant enzyme, obtained in the yeast Pichia pastoris, exhibited kinetic and physicochemical properties similar to those found for the native protein. Enzyme efficiency was examined in wheat straw saccharification processes, in which BGL-3 worked better supplementing Celluclast 1.5L than the commercial cellulase cocktail N-50010. Besides, BGL-3 hydrolyzed laminarin more efficiently than a commercial laminarinase. CONCLUSIONS: A very efficient 1,4-ß-glucosidase, which also showed activity over 1,3-ß-glucose bonds, has been produced, purified, and characterized. This is the first report of such versatility in a 1,4-ß-glucosidase. The application of this enzyme for saccharification of wheat straw and laminarin and its comparison with commercial enzymes suggest that it could be an interesting tool for the production of 2G and 3G biofuels.

Laminarin Quantification in Microalgae with Enzymes from Marine Microbes.

The marine beta-glucan laminarin is an abundant storage polysaccharide in microalgae. High production rates and rapid digestion by heterotrophic bacteria turn laminarin into an ideal carbon and energy source, and it is therefore a key player in the marine carbon cycle. As a main storage glucan laminarin also plays a central role in the energy metabolism of the microalgae (Percival and Ross, 1951; Myklestad, 1974; Painter, 1983). We take advantage of enzymes that digest laminarin selectively and can thereby quantify only this polysaccharide in environmental samples. These enzymes hydrolyze laminarin into glucose and oligosaccharides, which are measured with a standard reducing sugar assay to obtain the laminarin concentration. Prior to this assay, the three enzymes need to be produced via heterologous expression and purification. The assay can be used to monitor laminarin concentrations in environmental microalgae, which were concentrated from seawater by filtering, or in samples derived from algal lab cultures.

Disturbed processing of the carbohydrate-binding module of family 54 significantly impairs its binding to polysaccharides.

Carbohydrate-binding modules of the family 54 (CBM54) are characterized by spontaneous rupture of the peptide bond Asn266-Ser267 (numbering corresponds to that of laminarinase Lic16A of Ruminiclostridium thermocellum). As a result of processing, two parts are formed noncovalently connected to each other. Here, to gain insights into the functional significance of the internal cleavage, we made modifications of the family-conserved processing site in CBM54 of Lic16A. We demonstrate that the introduced mutations of residues G264 or S267 to alanine block the hydrolysis. Unprocessed, modified proteins bind insoluble polysaccharides pustulan, chitin, xylan, Avicel, phosphoric acid-swollen cellulose, and ß-d-glucan of the yeast cell wall 2-20 times worse than the wild-type module. The data obtained are the first to demonstrate that processing is important for the functioning of CBM54s.

Unraveling the multivalent binding of a marine family 6 carbohydrate-binding module with its native laminarin ligand.

UNLABELLED: Laminarin is an abundant brown algal storage polysaccharide. Marine microorganisms, such as Zobellia galactanivorans, produce laminarinases for its degradation, which are important for the processing of this organic matter in the ocean carbon cycle. These laminarinases are often modular, as is the case with ZgLamC which has an N-terminal GH16 module, a central family 6 carbohydrate-binding module (CBM) and a C-terminal PorSS module. To date, no studies have characterized a true marine laminarin-binding CBM6 with its natural carbohydrate ligand. The crystal structure of ZgLamCCBM6 indicates that this CBM has two clefts for binding sugar (variable loop site, VLS; and concave face site, CFS). The ZgLamCCBM6 VLS binds in an exo-manner and the CFS interacts in an endo-manner with laminarin. Isothermal titration calorimetry (ITC) experiments on native and mutant ZgLamCCBM6 confirm that these binding sites have different modes of recognition for laminarin, in agreement with the 'regional model' postulated for CBM6-binding modules. Based on ITC data and structural data, we propose a model of ZgLamCCBM6 interacting with different chains of laminarin in a multivalent manner, forming a complex cross-linked protein-polysaccharide network. DATABASE: PDB code 5FUI.