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Retaining β-mannanases are glycoside hydrolases (GHs) that can potentially be applied for synthesis of glycosides by catalysis of transglycosylation reactions. A novel active-site double mutant (R171 K/E205D) of the catalytic module (CM) of the family GH5 Trichoderma reesei β-mannanase (TrMan5A) was expressed in Pichia pastoris and purified. TrMan5A, CM and CM-variants R171 K and R171 K/E205D had pH optima between pH 4.0-5.3 and showed >80% remaining activity after incubation at 40 °C for 48 h. The enzymes were screened for transglycosylation capacity toward oligomeric and polymeric donor substrates and alcohol acceptors using mass-spectrometry. Hydrolysis and transglycosylation products were analysed by a novel HPLC procedure using an NH2 column. R171 K/E205D was superior in reactions with mannotetraose and the acceptor allyl alcohol, it had twice as high propensity for transglycosylation as wild-type TrMan5A. Wild-type TrMan5A produced the highest amounts of allyl β-mannosides (with 1-3 mannosyls) from locust bean galactomannan. Applying enzyme synergy, adding the GH27 guar α-galactosidase to the reaction (to cleave off galactomannan side-groups), gave a 2.1-fold increase of allyl mannosides and simultaneously a significant production of allyl galactopyranoside, increasing overall yield of allyl glycosides 4.4-fold, from 2.2% to 9.8%. The enzymatic synthesis of reactive allyl glycosides opens up for production of novel biomaterials and glycopolymers.

Graphical abstract, There is a knowledge gap regarding the factors that impede the ruminal digestion of plant cell walls or if rumen microbiota possess the functional activities to overcome these constraints. Innovative experimental methods were adopted to provide a high-resolution understanding of plant cell wall chemistries, identify higher-order structures that resist microbial digestion, and determine how they interact with the functional activities of the rumen microbiota. We characterized the total tract indigestible residue (TTIR) from cattle fed a low-quality straw diet using two comparative glycomic approaches: ELISA-based glycome profiling and total cell wall glycosidic linkage analysis. We successfully detected numerous and diverse cell wall glycan epitopes in barley straw (BS) and TTIR and determined their relative abundance pre- and post-total tract digestion. Of these, xyloglucans and heteroxylans were of higher abundance in TTIR. To determine if the rumen microbiota can further saccharify the residual plant polysaccharides within TTIR, rumen microbiota from cattle fed a diet containing BS were incubated with BS and TTIR ex vivo in batch cultures. Transcripts coding for carbohydrate-active enzymes (CAZymes) were identified and characterized for their contribution to cell wall digestion based on glycomic analyses, comparative gene expression profiles, and associated CAZyme families. High-resolution phylogenetic fingerprinting of these sequences encoded CAZymes with activities predicted to cleave the primary linkages within heteroxylan and arabinan. This experimental platform provides unprecedented precision in the understanding of forage structure and digestibility, which can be extended to other feed-host systems and inform next-generation solutions to improve the performance of ruminants fed low-quality forages.

Endoglucanases are carbohydrate-degrading enzymes widely used for bioethanol production as part of the enzymatic cocktail. However, family 5 subfamily 5 (GH5_5) endoglucanases are still poorly explored in depth. The Trichoderma reesei representative is the most studied enzyme, presenting catalytic activity in acidic media and mild temperature conditions. Though biochemically similar, its modular structure and synergy with other components vary greatly compared to other GH5_5 members and there is still a lack of specific studies regarding their interaction with other cellulases and application on novel and better mixtures. In this regard, the threedimensional structure elucidation is a highly valuable tool to both uncover basic catalytic mechanisms and implement engineering techniques, proved by the high success rate GH5_5 endoglucanases show. GH5_5 enzymes must be carefully evaluated to fully uncover their potential in biomass-degrading cocktails: the optimal industrial conditions, synergy with other cellulases, structural studies, and enzyme engineering approaches. We aimed to provide the current understanding of these main topics, collecting all available information about characterized GH5_5 endoglucanases function, structure, and bench experiments, in order to suggest future directions to a better application of these enzymes in the industry.

1,2-β-Mannobiose phosphorylases (1,2-β-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-β-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-β-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-β-MBPs and should serve as an important guidance for enzyme engineering for further applications.

Exoglucanase (CBH) is the rate limiting enzyme in the process of cellulose degradation. The carbohydrate binding module (CBM) can improve the accessibility of cellulase to substrate, thereby promoting the enzymatic hydrolysis of cellulase. In this study, the influence of CBM on the properties of GH6 exoglucanase from Chaetomium thermophilum (CtCBH) is systematically explored from three perspectives: the fusion of exogenous CBM, the exogenous CBM replacement of its own CBM, and the deletion of its own CBM. The parental and reconstructed CtCBH presented the same optimum pH (6.0) and temperature (60 °C) for maximum activity. Fusion of exogenous CBM increased the binding capacity of CtCBH to Avicel by 8% and 9%, respectively, but it had no significant effect on its catalytic activity. The exogenous CBM replacement of its own CBM resulted in a 12% reduction in the binding ability of CtCBH to Avicel, and a 26% reduction in the catalytic activity of Avicel. The deletion of its own CBM significantly reduced the binding ability of CtCBH to Avicel by approximately 53%, but its catalytic activity was not obviously reduced. These observations suggest that binding ability of CBM is not necessary for the catalysis of CtCBH.

Traditionally, filamentous fungi and actinomycetes are well-known cellulolytic microorganisms that have been utilized in the commercial production of cellulase enzyme cocktails for industrial-scale degradation of plant biomass. Noticeably, the Ktedonobacteria lineage (phylum Chloroflexi) with actinomycetes-like morphology was identified and exhibited diverse carbohydrate utilization or degradation abilities. In this study, we performed genome-wide profiling of carbohydrate-active enzymes (CAZymes) in the filamentous Ktedonobacteria lineage. Numerous CAZymes (153–290 CAZymes, representing 63–131 glycoside hydrolases (GHs) per genome), including complex mixtures of endo- and exo-cellulases, were predicted in 15 available Ktedonobacteria genomes. Of note, 4–28 CAZymes were predicted to be extracellular enzymes, whereas 3–29 CAZymes were appended with carbohydrate-binding modules (CBMs) that may promote their binding to insoluble carbohydrate substrates. This number far exceeded other Chloroflexi lineages and were comparable to the cellulolytic actinomycetes. Six multi-modular extracellular GHs were cloned from the thermophilic Thermosporothrix hazakensis SK20-1T strain and heterologously expressed. The putative endo-glucanases of ThazG5-1, ThazG9, and ThazG12 exhibited strong cellulolytic activity, whereas the putative exo-glucanases ThazG6 and ThazG48 formed weak but observable halos on carboxymethyl cellulose plates, indicating their potential biotechnological application. The purified recombinant ThazG12 had near-neutral pH (optimal 6.0), high thermostability (60°C), and broad specificity against soluble and insoluble polysaccharide substrates. It also represented described a novel thermostable bacterial β-1,4-glucanase in the GH12 family. Together, this research revealed the underestimated cellulolytic potential of the Ktedonobacteria lineage and highlighted its potential biotechnological utility as a promising microbial resource for the discovery of industrially useful cellulases.

International audience; The use of retaining glycoside hydrolases as synthetic tools for glycochemistry is highly topical and the focus of considerable research. However, due to the incomplete identification of the molecular determinants of the transglycosylation/hydrolysis partition (t/h), rational engineering of retaining glycoside hydrolases to create transglycosylases remains challenging. Therefore, to understand better the factors that underpin transglycosylation in a GH51 retaining α-l-arabinofuranosidase from Thermobacillus xylanilyticus, the investigation of this enzyme’s active site was pursued. Specifically, the properties of two mutants, F26L and L352M, located in the vicinity of the active site are described, using kinetic and 3D structural analyses and molecular dynamics simulations. The results reveal that the presence of L352M in the context of a triple mutant (also containing R69H and N216W) generates changes both in the donor and acceptor subsites, the latter being the result of a domino-like effect. Overall, the mutant R69H-N216W-L352M displays excellent transglycosylation activity (70 % yield, 78 % transfer rate and reduced secondary hydrolysis of the product). In the course of this study, the central role played by the conserved R69 residue was also reaffirmed. The mutation R69H affects both the catalytic nucleophile and the acid/base, including their flexibility, and has a determinant effect on the t/h partition. Finally, the results reveal that increased loop flexibility in the acceptor subsites creates new interactions with the acceptor, in particular with a hydrophobic binding platform composed of N216W, W248 and W302.

Carbohydrate processing enzymes are of biocatalytic interest. Glycoside hydrolases and the recently discovered lytic polysaccharide monooxygenase for their use in biomass degradation to obtain biofuels or valued chemical entities. Glycosyltransferases or engineered glycosidases and phosphorylases for the synthesis of carbohydrates and glycosylated products. Quantum mechanics-molecular mechanics (QM/MM) methods are highly contributing to establish their different chemical reaction mechanisms. Other computational methods are also used to study enzyme conformational changes, ligand pathways, and processivity, e.g. for processive glycosidases like cellobiohydrolases. There is still a long road to travel to fully understand the role of conformational dynamics in enzyme activity and also to disclose the variety of reaction mechanisms these enzymes employ. Additionally, computational tools for enzyme engineering are beginning to be applied to evaluate substrate specificity or aid in the design of new biocatalysts with increased thermostability or tailored activity, a growing field where molecular modeling is finding its way.

Lactobacillus plantarum is a useful microorganism that metabolizes galactose-containing polysaccharides. Genome analysis has shown that L. plantarum contains four β-galactosidase-related genes. Here, we cloned the β-galactosidase gene that encodes the glycoside hydrolase family 2 (GH2) enzyme LpGal2. Recombinant LpGal2 (rLpGal2, 72 kDa) is a homodimer with maximal enzymatic activity at pH 7.0 and 50 °C. Under these conditions, rLpGal2 hydrolyzes p-nitrophenyl-β-D-galactopyranoside (pNPβGal) with a specific activity of 2.16 × 10−3 U/mg and substrate specificity for β-1,6-galactobiose to produce D-Galactose. In addition, rLpGal2 can also hydrolyze β-1,6-galactan to D-Galactose, whereas other galactose-containing oligosaccharides and polysaccharides tested could not be hydrolyzed. This finding demonstrates that LpGal2 functions as an exo-β-1,6-galactosidase with narrow substrate specificity. To our knowledge, this is the first report of a β-galactosidase derived from L. plantarum with exo-β-1,6-galactosidase activity that has potential application for structure analysis of polysaccharides.

International audience; $Dictyoglomus\ thermophilum$ β-d-xylosidase DtXyl is attractive as a potential thermostable biocatalyst able to produce biologically active ginsenosides intermediates from β-(1,2)-D-xylosylated compounds, including Notoginsenoside-R1. DtXyl was expressed as an active N-terminal His-tagged protein, and its crystal structure was solved in presence or absence of d-xylose product. Modelling of notoginsenoside R1 in DtXyl active site led to the identification of several hydrophobic residues interacting in close contact to the substrate hydrophobic core. Unlike other residues involved in substrate binding, these residues are not conserved among GH39 xylosidase family, and their physico-chemical properties can be correlated to the efficient binding and subsequent hydrolysis of Notoginsenoside R1.

This review discusses the complex nature of the primary nonstarch polysaccharide (NSP) in corn with respect to the merit of debranching enzymes. Celluloses, hemicelluloses, and pectins comprise the 3 major categories of NSP that make up nearly 90% of plant cell walls. Across cereals, the hemicellulose arabinoxylan exists as the primary NSP, followed by cellulose, glucans, and others. Differences in arabinoxylan structure among cereals and cereal fractions are facilitated by cereal type, degree and pattern of substitution along the xylan backbone, phenol content, and cross-linkages. In particular, arabinoxylan (also called glucuronoarabinoxylan) in corn is heavily fortified with substituents, being more populated than in wheat and other cereal grains. Feed-grade xylanases – almost solely of the glycoside hydrolase (GH) 10 and GH 11 families – require at least 2 or 3 contiguous xylose units to be free of attachments to effectively attack the xylan chain. This canopy of attachments, along with a high phenol content and the insoluble nature of corn glucuronoarabinoxylan, confers a significant resistance to xylanase attack. Both in vitro and in vivo studies demonstrate that debranching enzymes appreciably increase xylanase access and fiber degradability by removing these attachments and breaking phenolic linkages. The enzymatic degradation of the highly branched arabinoxylan can facilitate disassembly of other fibers by increasing exposure to pertinent carbohydrases. For cereals, the arabinofuranosidases, α-glucuronidases, and esterases are some of the more germane debranching enzymes. Enzyme composites beyond the simple core mixes of xylanases, cellulases, and glucanases can exploit synergistic benefits generated by this class of enzymes. A broad scope of enzymatic activity in customized mixes can more effectively target the resilient NSP construct of cereal grains in commercial poultry diets, particularly those in corn-based feeds.

Background: Manno-oligosaccharides (MOS) is known as a kind of prebiotics. Mannanase plays a key role for the degradation of mannan to produce MOS. In this study, the mannanases of glycoside hydrolase (GH) families 5 Man5HJ14 and GH26 ManAJB13 were employed to prepare MOS from locust bean gum (LBG) and palm kernel cake (PKC). The prebiotic activity and utilization of MOS were assessed in vitro using the probiotic Lactobacillus plantarum strain. Results: Galactomannan from LBG was converted to MOS ranging in size from mannose up to mannoheptose by Man5HJ14 and ManAJB13. Mannoheptose was got from the hydrolysates produced by Man5HJ14, which mannohexaose was obtained from LBG hydrolyzed by ManAJB13. However, the same components of MOS ranging in size from mannose up to mannotetrose were observed between PKC hydrolyzed by the mannanases mentioned above. MOS stability was not affected by high-temperature and high-pressure condition at their natural pH. Based on in vitro growth study, all MOS from LBG and PKC was effective in promoting the growth of L. plantarum CICC 24202, with the strain preferring to use mannose to mannotriose, rather than above mannotetrose. Conclusions: The effect of mannanases and mannan difference on MOS composition was studied. All of MOS hydrolysates showed the stability in adversity condition and prebiotic activity of L. plantarum , which would have potential application in the biotechnological applications. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri",sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

Chitinases play crucial roles in enzymatic conversion of chitin and biocontrol of phytopathogenic fungi. Herein, a chitinase of glycoside hydrolase (GH) family 19, SaChiB, was cloned from Streptomyces alfalfae ACCC 40021 and expressed in Escherichia coli BL21(DE3). The purified SaChiB displayed maximal activities at 45 °C and pH 8.0, and showed good stability up to 55 °C and in the range of pH 4.0–11.0, respectively. It exhibited substrate specificity towards chitin and chitooligosaccharides (degree of polymerization 3–6) with the endo-cleavage manner. In combination with the N-acetyl hexosaminidase SaHEX, it yielded a conversion rate of 95.2% from chitin powder to N-acetyl-D-glucosamine in 8 h and a product purity of >98.5%. Furthermore, the enzyme strongly inhibited the growth of tested pathogenic fungi. These results indicated that SaChiB has the great potential for applications in the conversion of raw chitinous waste in industries as well as the biocontrol of fungal diseases in agriculture.

A novel thermostable xylanase gene from Chaetomium sp. CQ31 was cloned and codon–optimized (CsXynBop). The deduced protein sequence of the gene shared the highest similarity of 75% with the glycoside hydrolase (GH) family 10 xylanase from Achaetomium sp. Xz–8. CsXynBop was over–expressed in Pichia pastoris GS115 by high–cell density fermentation, with the highest xylanase yield of 10,017 U/mL. The recombinant xylanase (CsXynBop) was purified to homogeneity and biochemically characterized. CsXynBop was optimally active at pH 6.5 and 85 °C, respectively, and stable over a broad pH range of 5.0–9.5 and up to 60 °C. The enzyme exhibited strict substrate specificity towards oat–spelt xylan (2, 489 U/mg), beechwood xylan (1522 U/mg), birchwood xylan (1067 U/mg), and showed relatively high activity towards arabinoxylan (1208 U/mg), but exhibited no activity on other tested polysaccharides. CsXynBop hydrolyzed different xylans to yield mainly xylooligosaccharides (XOSs) with degree of polymerization (DP) 2–5. The application of CsXynBop (200 U/g malt) in malt mashing substantially decreased the filtration time and viscosity of malt by 42.3% and 8.6%, respectively. These excellent characteristics of CsXynBop may make it a good candidate in beer industry.

Graphical abstract, Highlights • Obtained high accuracy AI-modeling structures of 22 GH families for the first time. • Glycoside hydrolases are structurally derived from 27 superfamilies and 16 folds. • The distribution of GHs is highly biased in only a few superfamilies and folds. • Most GH superfamilies are multifunctional, and a few are extremely versatile. • Most GH superfamilies exhibit narrow environmental microbiome distributions., Glycoside hydrolases (GHs) are greatly diverse in sequences and functions, but systematic studies of GH relationships based on structural information are lacking. Here, we report that GHs have multiple evolutionary origins and are structurally derived from 27 homologous superfamilies and 16 folds, but GHs are highly biased to distribute in a few superfamilies and folds. Six of these superfamilies are widely encoded by archaea, bacteria, and eukaryotes, indicating that they may be the most ancient in origin. Most superfamilies vary in enzyme function, and some, such as the superfamilies of (β/α)8-barrel and (α/α)6-barrel structures, exhibit extreme functional diversity; this is highly positively correlated with sequence diversity. More than one-third of glycosidase activities show a phenomenon of convergent evolution, especially the degradation functions of GHs on polysaccharides. The GHs of most superfamilies have relatively narrow environmental distributions, normally with the highest abundance in host-associated environments and a distribution preference for moderate low-temperature and acidic environments. Overall, our expanded analysis facilitates an understanding of complex GH sequence–structure–function relationships and may guide our screening and engineering of GHs.

Cold-adapted endo-β-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-β-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal β-1,4-linkages of cellulose and mixed-linkage β-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10–50 °C), which are distinguishing features from conventional thermophilic β-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-β-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.

This study was undertaken to explore alternative applications of the widely known entomopathogenic/endophytic fungus, Beauveria bassiana, besides its sole use as a biocontrol agent. B. bassiana SAN01, was investigated for the production of two glycoside hydrolases, xylanase and endoglucanase under submerged conditions. Among the different biomass tested, wheat bran provided the best results for both xylanase and endoglucanase, and their production levels were further enhanced using response surface methodology. Under optimised conditions, heightened yields of 1061 U/ml and 23.03 U/ml were observed for xylanase and endoglucanase, respectively, which were 3.44 and 1.35 folds higher than their initial yields. These are the highest ever production levels reported for xylanase and endoglucanase from any B. bassiana strain or any known entomopathogenic fungi. Furthermore, the efficacy of xylanase/endoglucanase cocktail in the saccharification of sugarcane bagasse was evaluated. The highest amount of reducing sugar released from the pretreated biomass by the action of the crude Beauveria enzyme cocktail was recorded at 30°C after 8 h incubation. The significant activities of the hydrolytic enzymes recorded with B. bassiana in this study thus present promising avenues for the use of the entomopathogen as a new source of industrial enzymes and by extension, other biotechnological applications.

Genomic analysis of the marine bacterium Wenyingzhuangia fucanilytica CZ1127T revealed the presence of four fucoidanase genes fwf1, fwf2, fwf3, fwf4 that belonged to the glycoside hydrolase family 107 (GH107, CAZy), which is located in one gene cluster putatively involved in fucoidan catabolism. Genes encoding two fucoidanases fwf1 and fwf2 were cloned, and the proteins FWf1 and FWf2 were produced in Escherichia coli cells. The recombinant fucoidanases were purified and the biochemical properties of these enzymes were studied. The amino acid sequences of FWf1 and FWf2 showed 41 and 51% identity respectively with a fucoidanase FcnA from the marine bacterium Mariniflexile fucanivorans, with the established 3D structure. Structures of the oligosaccharides produced during enzymatic hydrolysis of fucoidan by FWf1 and FWf2 have been determined by NMR spectroscopy. Detailed substrate specificities of FWf1 and FWf2 were studied using fucoidans and sulfated fucooligosaccharides with different structures. Both fucoidanases catalyzed hydrolysis of 1→4-glycosidic bonds between sulfated α-l-fucose residues but had different specificities regarding sulfation patterns of the fucose residues in fucoidan molecules. Specific cleavage sites recognizable by the fucoidanases in fucoidan molecules were determined. The obtained results provide new knowledge about differences between specificities of the fucoidanases belonging to the GH107 family.

Pcal_0768 gene encoding an amylomaltase, a 4-α-glucanatransferase belonging to family 77 of glycosyl hydrolases, from Pyrobaculum calidifontis was cloned and expressed in Escherichia coli. The recombinant protein was produced in E. coli in soluble and active form. However, the expression level was not very high. Analysis of the mRNA of initial seven codons at the 5′-end of the gene revealed the presence of a hair pin like secondary structure. This secondary structure was removed by site directed mutagenesis, without altering the amino acids, which resulted in enhanced expression of the cloned gene. Recombinant Pcal_0768 exhibited optimal amylomaltase activity at 80 °C and pH 6.9. Under these conditions, the specific activity was 690 U/ mg. Recombinant Pcal_0768 was highly thermostable with a half-life of 6 h at 100 °C. It exhibited the highest kcat value among the characterized glucanotransferases. No metal ions were required for activity or stability of the enzyme. Recombinant Pcal_0768 was successfully employed in the synthesis of modified starch for producing thermoreversible gel. To the best of our knowledge, till now this is the most thermostable enzyme among the characterized amylomaltases. High thermostability and starch modification potential make it a novel and distinct amylomaltase.

Recently discovered Lytic Polysaccharide Mono-Oxygenase (LPMO) enhances the enzymatic deconstruction of complex polysaccharide by oxidation. The present study demonstrates the agricultural waste hydrolyzing capabilities of Paenibacillus dendritiformis CRN18, which exhibits the enzyme activity of exo-glucanase, β-glucosidase, β-glucuronidase, endo-1, 4 β-xylanases, arabinosidase, and α–galactosidase as 0.1U/ml, 0.3U/ml, 0.09U/ml, 0.1U/ml, 0.05U/ml, and 0.41U/ml, respectively. The genome analysis of strain reveals the presence of four LPMO genes, along with lignocellulolytic genes. The gene structure of LPMO and its phylogenetic analysis shows the evolutionary relatedness with the Bacillus LPMO gene. Gene position of LPMOs in the genome of strains shows the close association of two LPMOs with chitin active enzyme GH18, and the other two are associated with hemicellulases (GH39, GH23). Protein-protein interaction and gene networking of LPMO sheds light on the co-occurrence, neighborhood, and interaction of LPMOs with chitinase and xylanase enzymes. Structural prediction of LPMOs unravels the information of the LPMO's binding site. Although the LPMO has been explored for its oxidative mechanism, a little light has been shed on its gene structure. This study provides insights into the LPMO gene structure in P. dendritiformis CRN18 and its potential in lignocellulose hydrolysis.

α-1,3-Glucan is a homopolymer composed of D-glucose (Glc) and it is an extracellular polysaccharide found in dental plaque due to Streptococcus species. α-1,3-Glucanase from Streptomyces thermodiastaticus strain HF3-3 (Agl-ST) has been identified as a thermostable α-1,3-glucanase, which is classified into glycoside hydrolase family 87 (GH87) and specifically hydrolyzes α-1,3-glucan with an endo-action. The enzyme has a potential to inhibit the production of dental plaque and to be used for biotechnological applications. Here we show the structure of the catalytic unit of Agl-ST determined at 1.16 A resolution using X-ray crystallography. The catalytic unit is composed of two modules, a β-sandwich fold module, and a right-handed β-helix fold module, which resembles other structural characterized GH87 enzymes from Bacillus circulans str. KA-304 and Paenibacillus glycanilyticus str. FH11, with moderate sequence identities between each other (approximately 27% between the catalytic units). However, Agl-ST is smaller in size and more thermally stable than the others. A disulfide bond that anchors the C-terminal coil of the β-helix fold, which is expected to contribute to thermal stability only exists in the catalytic unit of Agl-ST.

The gene encoding Pcal_0842, annotated as a cellulase (accession no. ABO08268), was cloned and expressed in Escherichia coli. The gene product was produced in insoluble form in E. coli in high amounts even without addition of the inducer isopropyl β-D-1-thiogalactopyranoside. The recombinant protein was solubilized in 8 M urea and refolded by gradual removal of urea. The refolded protein exhibited both α-1,4- and β-1,4-glycosidic cleavage activities. The enzyme activity increased with the increase in temperature till 120 °C. Apart from very high optimal temperature, recombinant Pcal_0842 was extremely thermostable. There was no significant loss in activity even after heating for 100 h at 100 °C. The half-lives of Pcal_0842 were 6 and 2.5 h at 110 and 120 °C, respectively. To the best of our knowledge, Pcal_0842 is the most thermostable glycosidase characterized to date and this is the first report on cloning and characterization of an enzyme from archaea that displays both α-1,4- and β-1,4-glycosidic cleavage activities.

Increased demand for biofuels promotes the search for new biomass-degrading fungi. Acremonium strictum is an environmentally widespread filamentous fungi found on plant debris; that secretes lignocellulose-degrading enzymes. A recently isolated A. strictum strain, AAJ6; native to the Brazilian Cerrado biome was evaluated for its capacity to degrade lignocellulosic substrates. In this study, whole-genome sequencing of AAJ6 was performed and 775 CAZy domains were identified which correlated to those of A. strictum strain DS1bioAY4a and other lignocellulolytic fungi; suggesting AAJ6 is a high CAZyme producer. We expressed the glycoside hydrolase families GH74 and GH3 from plasmid or genome-integrated to evaluate the ethanol production from cellulosic substrates in Brazilian industrial Saccharomyces cerevisiae strains (PE-2 and SA-1) evolved for thermotolerance (AMY12 and AMY35). Those expressing the genome-integrated enzymes showed the highest β-glucosidase activity and growth in medium with cellobiose at 40°C. The strain AGY005 (integrated cassettes) showed 19, 23 and 46% higher ethanol production in SHF, pSSF (partial hydrolysis SSF) and SSF processes, respectively, using Avicel, and ∼50% more ethanol using pre-treated sugarcane bagasse, compared to the strain with a plasmid-based expression. These results indicate the improved performance of thermotolerant industrial strains with genome-integrated CAZymes in the SSF process for 2G ethanol.

A novel chitosanase gene, csn4, was identified through function-based screening of a marine mud metagenomic library. The encoded protein, named CSN4, which belonged to glycoside hydrolase family 46, showed its maximum identity (79%) with Methylobacter tundripaludum peptidoglycan-binding protein. CSN4 was expressed in Escherichia coli and purified. It displayed maximal activity at 30 °C and pH 7. A weakly-alkaline solution strongly inhibited the activity. The enzymatic activity was enhanced by addition of Mn2+ or Co2+. CSN4 exhibited strict substrate specificity for chitosan, and the activity was enhanced by increasing the degree of deacetylation. Thin-layer chromatography and electrospray ionization-mass spectrometry showed that CSN4 displayed an endo-type cleavage pattern, hydrolyzing chitosan mainly into (GlcN)2, (GlcN)3 and (GlcN)4. The novel characteristics of the chitosanase CSN4 make it a potential candidate to produce chitooligosaccharides from chitosan in industry.

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and β-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50–80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and β-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55–60 °C, 5.0–5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and β-galactosidase activities, but also exhibited β-xylosidase and β-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable β-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.

A novel glycoside hydrolase (GH) family 36 α-galactosidase gene (designated PtGal36A) from Paecilomyces thermophila was cloned and expressed in Escherichia coli. The deduced sequence of the gene shared the highest identity of 87% with the characterized α-galactosidase from Aspergillus nidulans FGSC A4. The recombinant enzyme (PtGal36A) was purified to homogeneity with a purification fold of 11.0 and a recovery yield of 55.2%. PtGal36A was most active at pH 5.0 and 60 °C and was stable within the pH range of 4.5-11.5 and up to 50 °C. PtGal36A displayed strict specific activity towards substrates with α-galactosyl linkages in the nonreducing ends, with the highest activity on stachyose (58.5 U/mg), followed by melibiose (39.2 U/mg) and raffinose (31.4 U/mg). The enzyme efficiently hydrolyzed raffinose family oligosaccharides in soybean meal by more than 95%. Moreover, PtGal36A showed excellent resistance (residual activities >90%) against α-chymotrypsin, proteinase K, subtilisin A, trypsin and papain. Therefore, PtGal36A should be a good candidate for the food and feed industries.

Infections by many bacterial pathogens rely on their ability to degrade host glycans by producing glycoside hydrolases (GHs). Here, we discovered a conserved multifunctional GH, SsGalNagA, containing a unique combination of two family 32 carbohydrate-binding modules (CBM), a GH16 domain and a GH20 domain, in the zoonotic pathogen Streptococcus suis 05ZYH33. Enzymatic assays revealed that the SsCBM-GH16 domain displays endo-(β1,4)-galactosidase activity specifically toward the host-derived αGal epitope Gal(α1,3)Gal(β1,4)Glc(NAc)-R, whereas the SsGH20 domain has a wide spectrum of exo-β-N-acetylhexosaminidase activities, including exo-(β1,3)-N-acetylglucosaminidase activity, and employs this activity to act in tandem with SsCBM-GH16 on the αGal-epitope glycan. Further, we found that the CBM32 domain adjacent to the SsGH16 domain is indispensable for SsGH16 catalytic activity. Surface plasmon resonance experiments uncovered that both CBM32 domains specifically bind to αGal-epitope glycan, and together they had a KD of 3.5 mm toward a pentasaccharide αGal-epitope glycan. Cell-binding and αGal epitope removal assays revealed that SsGalNagA efficiently binds to both swine erythrocytes and tracheal epithelial cells and removes the αGal epitope from these cells, suggesting that SsGalNagA functions in nutrient acquisition or alters host signaling in S. suis. Both binding and removal activities were blocked by an αGal-epitope glycan. SsGalNagA is the first enzyme reported to sequentially act on a glycan containing the αGal epitope. These findings shed detailed light on the evolution of GHs and an important host-pathogen interaction.

Epimedin C, a major flavonoid extracted from Herba Epimedii, is a precursor of minor flavonoid icaritin that is a desired drug candidate with remarkable anti-cancer activities. For enhancing the biotransformation efficiency of icaritin, a novel α- l -rhamnosidase gene was cloned from hyperthermophiles Thermotoga petrophila DSM 13995. TpeRha displayed optimal activity at a pH of 4.5 and a temperature of 90 °C. The Km and Kcat of TpeRha for p-nitrophenyl-α- l -rhamnopyranoside were 2.99 mM and 651.73 s−1, respectively. It displayed broad catalytic ability in cleavage of the outer and inner rhamnopyranosyl moieties on the C-3 carbon of epimedin C. Further, this enzyme was utilized to improve the efficiency of the co-conversion system in transforming epimedin C into icaritin, in combined with a thermostable β-glucosidase Tpebgl1. In addition, a transformation pathway (epimedin C -icariin - icariside I - icaritin) with a high efficiency for icaritin production was screened. After a two-stage transformation under optimized conditions (90 °C, pH 4.5, 80 U/mL of TpeRha and 1.2 U/mL of Tpebgl1), 1 g/L of epimedin C was transformed into 0.4337 g/L of icaritin within 150 min, with a corresponding molar conversion rate of 96.9 %. This is the first report of enzymatic transformation on preparing icaritin from epimedin C by using thermostable glycosidase.

Bacteroides thetaiotaomicron (B. thetaiotaomicron), which resides in the human intestinal tract, has a number of carbohydrate enzymes, including glycoside hydrolase (GH) family 97. Only a few GH 97 enzymes have been characterized to date. In this study, a novel α-galactosidase (Bt_3294) was cloned from B. thetaiotaomicron, expressed in Escherichia coli, and purified using affinity chromatography. This novel enzyme showed optimal activity at 60 °C and pH 7.0. Enzyme activity was reduced by 94.4% and 95.7% in the presence of 5 mM Ca2+ and Fe2+, respectively. It is interesting that Bt_3294 specifically hydrolyzed shorter α-galactosyl oligosaccharides, such as melibiose and raffinose. The D-values of Bt_3294 at 40 °C and 50 °C were about 107 and 6 min, respectively. After immobilization of Bt_3294, the D-values at 40 °C and 50 °C were about 37.6 and 29.7 times higher than those of the free enzyme, respectively. As a practical application, the immobilized Bt_3294 was used to hydrolyze raffinose family oligosaccharides (RFOs) in soy milk, decreasing the RFOs by 98.9%.

Mannosidases are a diverse group of enzymes that are important in the biological processing of mannose-containing polysaccharides and complex glycoconjugates. They are found in 12 of the >160 sequence-based glycosidase families. We discuss evidence that nature has evolved a small set of common mechanisms that unite almost all of these mannosidase families. Broadly, mannosidases (and the closely related rhamnosidases) perform catalysis through just two conformations of the oxocarbenium ion-like transition state: a B2,5 (or enantiomeric 2,5B) boat and a 3H4 half-chair. This extends to a new family (GT108) of GDPMan-dependent β-1,2-mannosyltransferases/phosphorylases that perform mannosyl transfer through a boat conformation as well as some mannosidases that are metalloenzymes and require divalent cations for catalysis. Yet, among this commonality lies diversity. New evidence shows that one unique family (GH99) of mannosidases use an unusual mechanism involving anchimeric assistance via a 1,2-anhydro sugar (epoxide) intermediate.

Bacillus amyloliquefaciens SS35 was subjected to ultraviolet irradiation to improve the enzymatic hydrolysis of lignocellulosic biomass. The resulting mutant, UV2, produced endoglucanase, carboxymethyl cellulase, CMCase-UV2 with 1.6–4.1-fold higher activity against cellulosic substrates than the wild type, CMCase-WT. CMCase-UV2 exhibited wider pH stability in the acidic range than CMCase-WT. The TLC analysis showed that the hydrolysis of CMC-Na and β-glucan by CMCase-UV2 produced glucose along with cello-oligosaccharides and cellobiose in 45 min, whereas, CMCase-WT produced, only cello-oligosaccharides and cellobiose in 120 min by endolytic mode of action. The hydrolysis of pretreated Pennisetum purpureum by CMCase-UV2 gave total reducing sugar yield 154.2 mg/g pretreated biomass in 48 h, which was 1.8-fold higher than CMCase-WT. CMCase-UV2 was the promising endoglucanase which improves the saccharification of lignocellulosic biomass therefore, it will improve the efficiency of the process, lignocellulose-based biorefineries for bioethanol production. The gene encoding cellulase was amplified from wild-type and UV2 strains using degenerate primers designed from phylogenetically related spp. Bacillus amyloliquefaciens KHG19 for family 5 glycoside hydrolase. Sequences analysis of genes from wild-type and UV2 strains showed the mutation, D233G. These results will provide information for protein engineering in designing mutant of endoglucanase for improved catalytic efficiency and pH stability.

A novel β-1,3-1,4-glucanase gene (AaBglu12A) from Aspergillus awamori was extracellularly expressed in Pichia pastoris. AaBglu12A showed amino acid identity of 96 % with a glycoside hydrolase family 12 cellulase from A. kawachii and 48 % with a β-1,3-1,4-glucanase from Magnaporthe oryzae. The highest β-1,3-1,4-glucanase activity of 159,500 ± 500 U/mL with protein concentration of 31.7 ± 0.3 g/L was achieved in a 5-L fermentor. AaBglu12A was purified until homogeneous with recovery yield of 92 %. Its maximal activity was found at 55 °C and pH 5.0. The enzyme was stable up to 60 °C and within the pH range of 2.0-9.0. It also demonstrated strict substrate specificity towards oat- and barley-glucans as well as lichenan. The Km values for oat-, barley-glucans, and lichenan were 2.82, 3.51, and 2.53 mg/mL, respectively. The Vmax values for oat-, barley-glucans, and lichenan were 12,068, 10,790, and 7236 μmol/min·mg, respectively. AaBglu12A hydrolyzed oat- and barley-β-glucans to produce tetra- and tri-saccharides. However, lichenan was hydrolyzed to yield trisaccharides as the main end product. The addition of AaBglu12A to the mashing process substantially decreased filtration time by 34.5 % and viscosity by 9.6 %. Therefore, the high-level production of AaBglu12A might be a promising strategy for the brewing industry owing to its favorable properties.

Chitooligosaccharides (COS) are hydrolytic products of chitosan that are essential in functional food, medicine, and other fields due to their biological activities. Commercial COS are often prepared by the hydrolysis of chitosan by chitosanase. In this study, a glycoside hydrolase family 46 cluster B chitosanase from Bacillus amyloliquefaciens (BaCsn46B) was efficiently expressed in Pichia pastoris. The recombinant enzyme was secreted into the culture medium that reached a total extracellular protein concentration of 4.5 g/L with an activity of 8907.2 U/mL in a high cell density fermenter (5 L). The molecular mass of deglycosylated BaCsn46B was 29.0 kDa. Purified BaCsn46B exhibited excellent enzymatic properties, which had high specific activity (2380.5 U/mg) under optimal reaction conditions (55 °C and pH 6.5). BaCsn46B hydrolyzed chitosan yielded a series of COS with different degrees of polymerization by endo-type cleavage. The end hydrolytic products of BaCsn46B were chitobiose and chitotriose, while no monosaccharide yield was evident in the hydrolytic reaction. The excellent secreted expression level and hydrolytic performance make the enzyme a desirable biocatalyst for the industrial preparation of COS.

Exploitation of enzymes in biocatalytic processes provides scope both in the synthesis and degradation of molecules. Enzymes have power not only in their catalytic efficiency, but their chemoselectivity, regioselectivity, and stereoselectivity means the reactions they catalyze are precise and reproducible. Focusing on carbohydrate processing enzymes, this review covers advances in biocatalysis involving carbohydrates over the last 2-3 years. Given the notorious difficulties in the chemical synthesis of carbohydrates, the use of enzymes for synthesis has potential for significant impact in the future. The use of catabolic enzymes in the degradation of biomass, which can be exploited in the production of biofuels to provide a sustainable and greener source of energy, and the synthesis of molecules that have a range of applications including in the pharmaceutical and food industries will be explored.

β-1,3-d-Glucan is a ubiquitous glucose polymer produced by plants, bacteria, and most fungi. It has been used as a diagnostic tool in patients with invasive mycoses via a highly-sensitive reagent consisting of the blood coagulation system of horseshoe crab. However, no method is currently available for measuring β-1,6-glucan, another primary β-glucan structure of fungal polysaccharides. Herein, we describe the development of an economical and highly-sensitive and specific assay for β-1,6-glucan using a modified recombinant endo-β-1,6-glucanase having diminished glucan hydrolase activity. The purified β-1,6-glucanase derivative bound to the β-1,6-glucan pustulan with a KD of 16.4 nm. We validated the specificity of this β-1,6-glucan probe by demonstrating its ability to detect cell wall β-1,6-glucan from both yeast and hyphal forms of the opportunistic fungal pathogen Candida albicans, without any detectable binding to glucan lacking the long β-1,6-glucan branch. We developed a sandwich ELISA-like assay with a low limit of quantification for pustulan (1.5 pg/ml), and we successfully employed this assay in the quantification of extracellular β-1,6-glucan released by >250 patient-derived strains of different Candida species (including Candida auris) in culture supernatant in vitro. We also used this assay to measure β-1,6-glucan in vivo in the serum and in several organs in a mouse model of systemic candidiasis. Our work describes a reliable method for β-1,6-glucan detection, which may prove useful for the diagnosis of invasive fungal infections.

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.

Chitosanases play an important role in chitosan degradation, and the enzymatic degradation products of chitosan show various biological activities. Here, a novel glycoside hydrolase family 46 chitosanase (named Csn-BAC) from Bacillus sp. MD-5 was heterologously expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was purified by Ni-NTA affinity chromatography, and its molecular weight was estimated to be 35 kDa by SDS-PAGE. Csn-BAC showed maximal activity toward colloidal chitosan at pH 7 and 40 °C. The enzymatic activity of Csn-BAC was enhanced by Mn2+, Cu2+ and Co2+ at 1 mM, and by Mn2+ at 5 mM. Thin-layer chromatography and electrospray ionization-mass spectrometry results demonstrated that Csn-BAC exhibited an endo-type cleavage pattern and hydrolyzed chitosan to yield, mainly, (GlcN)2 and (GlcN)3. The enzymatic properties of this chitosanase may make it a good candidate for use in oligosaccharide production-based industries.

Graphical abstract, Highlights • Genome-wide characterization of GH families is conducted for Mollusca. • GH9, GH10, GH18 and GH20 families are remarkably expanded in molluscs. • The wide adoption of CBMs likely facilitates the hydrolysis of polysaccharides. • Hepatopancreas is the main organ for the prominent expression of GH families. • Functional divergence of GH families possibly contributes to their adaptive roles., The hydrolysis of sugar-containing compounds by glycoside hydrolases (GHs) plays essential roles in many major biological processes, but to date our systematic understanding of the functional diversity and evolution of GH families remains largely limited to a few well-studied terrestrial animals. Molluscs represent the largest marine phylum in the animal kingdom, and many of them are herbivorous that utilize algae as a main nutritional source, making them good subjects for studying the functional diversity and adaptive evolution of GH families. In the present study, we conducted genome-wide identification and functional and evolutionary analysis of all GH families across major molluscan lineages. We revealed that the remarkable expansion of the GH9, GH10, GH18 and GH20 families and the wide adoption of carbohydrate-binding modules in molluscan expanded GH families likely contributed to the efficient hydrolysis of marine algal polysaccharides and were involved in the consolidation of molluscan algae-feeding habits. Gene expression and network analysis revealed the hepatopancreas as the main organ for the prominent expression of approximately half of the GH families (well corresponding to the digestive roles of the hepatopancreas) and key or hub GHs in the coexpression gene network with potentially diverse functionalities. We also revealed the evolutionary signs of differential expansion and functional divergence of the GH family, which possibly contributed to lineage-specific adaptation. Systematic analysis of GH families at both genomic and transcriptomic levels provides important clues for understanding the functional divergence and evolution of GH gene families in molluscs in relation to their algae-feeding biology.

The post-translational modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) dynamically programmes cellular physiology to maintain homoeostasis and tailor biochemical pathways to meet context-dependent cellular needs. Despite diverse roles of played by O-GlcNAc, only two enzymes act antagonistically to govern its cycling; O-GlcNAc transferase installs the monosaccharide on target proteins, and O-GlcNAc hydrolase removes it. The recent literature has exposed a network of mechanisms regulating these two enzymes to choreograph global, and target-specific, O-GlcNAc cycling in response to cellular stress and nutrient availability. Herein, we amalgamate these emerging mechanisms from a structural and molecular perspective to explore how the cell exerts fine control to regulate O-GlcNAcylation of diverse proteins in a selective fashion.

As the scope of modern genomics technologies increases, so does the need for informative chemical tools to study functional biology. Activity-based probes (ABPs) provide a powerful suite of reagents to probe the biochemistry of living organisms. These probes, featuring a specificity motif, a reactive chemical group and a reporter tag, are opening-up large swathes of protein chemistry to investigation in vitro, as well as in cellular extracts, cells and living organisms in vivo. Glycoside hydrolases, by virtue of their prominent biological and applied roles, provide a broad canvas on which ABPs may illustrate their functions. Here we provide an overview of glycosidase ABP mechanisms, and review recent ABP work in the glycoside hydrolase field, encompassing their use in medical diagnosis, their application for generating chemical genetic disease models, their fine-tuning through conformational and reactivity insight, their use for high-throughput inhibitor discovery, and their deployment for enzyme discovery and dynamic characterization.

A truncated β-agarase gene without the Carbohydrate-binding Modules (Aga16A-△CBM) from a marine bacterium Microbulbifer sp. BH-1 was cloned and successfully expressed in Escherichia coli. It encoded 279 amino acid (aa) with a predicted molecular mass of 42.1 kDa. The recombinant β-agarase (Aga16A-△CBM) showed the highest sequence identity of 49% with the glycoside hydrolase (GH) family 16 β-agarases AgaA and AgaB from Zobellia galactanivorans. Aga16A-△CBM showed optimal activity at 55°C and pH 8.0, respectively. It was stable within the pH range of 5.0–9.5 and up to 50°C. Km and Vmax values of Aga16A-△CBM for agarose were 1.32 mg ml−1 and 860.9 μmol min−1mg−1, respectively. In additional, agarose was hydrolyzed by Aga16A-△CBM to produce 18.4 mg mL−1 neoagarotetraose with the high yield of 93.2% (w/w). These desirable properties of Aga16A-△CBM may make it attractive in neoagarotetraose production.

GH42 enzymes are potential candidates for bifunctional β-galactosidase/α-L-arabinopyranosidase. A novel GH42 enzyme (BaBgal42A) from Bacillus was identified, the recombinant BaBgal42A hydrolyzed not only β-D-galactopyranosidic bonds in pNP-β-D-galactopyranoside, oNP-β-D-galactopyranoside, lactose, galactan, and arabinan but also α-L-arabinopyranosidic linkages in pNP-α-L-arabinopyranoside, wheat arabinoxylan and galactan. The Km values of BaBgal42A for pNP-β-D-galactopyranoside and pNP-α-L-arabinopyranoside were 2.76 and 16.23 mM, respectively. Investigation of cooperative activities of BaBgal42A with cognate enzymes revealed that BaBgal42A showed obvious synergy with an endo-β-1,4-galactanase (BaGal53A) in the decomposition of galactan, supplementing BaBgal42A resulted in a 0.56-fold increase in the release of reducing sugars; BaBgal42A also exhibited a little synergy with its cognate endoxylanase (BaXynA)/α-L-arabinofuranosidase (BaAraA) in hydrolyzing wheat arabinoxylan/arabinan, addition of BaBgal42A released 12.7%/7.8% more reducing sugars than that produced by BaXynA/BaAraA alone. Moreover, BaBgal42A is a cold-adapted enzyme, exhibiting 28–46% of the maximal activity at the range of 5–20 °C and its activity was slightly stimulated by addition of Na+, K+, or Ca2+ at low concentrations. This study not only expands the diversity within GH42 family, but also provides new insights into the role of microbial GH42 enzymes, which would contribute to its potential application in polysaccharides degradation and milk lactose hydrolysis.

N-Linked glycans play important roles in various cellular and immunological events. Endo-β-N-acetylglucosaminidase (ENGase) can release or transglycosylate N-glycans and is a promising tool for the chemoenzymatic synthesis of glycoproteins with homogeneously modified glycans. The ability of ENGases to act on core-fucosylated glycans is a key factor determining their therapeutic utility because mammalian N-glycans are frequently α-1,6-fucosylated. Although the biochemistries and structures of various ENGases have been studied extensively, the structural basis for the recognition of the core fucose and the asparagine-linked GlcNAc is unclear. Herein, we determined the crystal structures of a core fucose-specific ENGase from the caterpillar fungus Cordyceps militaris (Endo-CoM), which belongs to glycoside hydrolase family 18. Structures complexed with fucose-containing ligands were determined at 1.75-2.35 A resolutions. The fucose moiety linked to GlcNAc is extensively recognized by protein residues in a round-shaped pocket, whereas the asparagine moiety linked to the GlcNAc is exposed to the solvent. The N-glycan-binding cleft of Endo-CoM is Y-shaped, and several lysine and arginine residues are present at its terminal regions. These structural features were consistent with the activity of Endo-CoM on fucose-containing glycans on rituximab (IgG) and its preference for a sialobiantennary substrate. Comparisons with other ENGases provided structural insights into their core fucose tolerance and specificity. In particular, Endo-F3, a known core fucose-specific ENGase, has a similar fucose-binding pocket, but the surrounding residues are not shared with Endo-CoM. Our study provides a foothold for protein engineering to develop enzymatic tools for the preparation of more effective therapeutic antibodies.

Inhibition the activity of glycosidase is an effective method for the treatment and prevention of diabetes. In this study, enzymatic kinetics, fluorescence spectrum experiment, starch granule digestion, molecular docking studies and animal's studies were used to investigate the interaction mechanism of carnosic acid against two glycosidase (α-amylase and α-glucosidase). Enzymatic kinetics showed that carnosic acid inhibited α-amylase activity in a competitive manner and α-glucosidase activity in a non-competitive manner. The half inhibitory concentrations (IC50) of carnosic acid to α-amylase and α- glucosidase were (1.12 ± 0.31) and (0.08 ± 0.17), respectively. The fluorescence quenching experiments showed that the intrinsic fluorescence of α-amylase or α-glucosidase was quenched by forming a complex with carnosic acid, and there was only one binding site between carnosic acid and glycosidase. The starch granules were no longer hydrolyzed by α-amylase after the addition of carnosic acid, which indicated that carnosic acid inhibited the activity of α-amylase. Molecular docking study showed that carnosic acid binds to the amino acid residues of glycosidase through hydrogen bond and van der Waals force, which leads to the change of the molecular conformation of glycosidase and thus reduces the activity of glycosidase. The experiment on mice showed that carnosic acid could effectively reduce postprandial blood glucose in mice.

Glycoside hydrolase (GH) family 45 is one of the smallest and poorly studied endoglucanase family with a broad biotechnological application ranging from treatment of textiles to conversion of complex cell wall polysaccharides into simple oligo- and monosaccharides. In a present study, GH45 cellulase from Neurospora crassa OR74A (NcCel45A) was characterized both biochemically and structurally. HPLC analysis of the hydrolytic products confirmed the endo-β(1,4) mode of action of the enzyme. Moreover, such pattern revealed that NcCel45A cannot hydrolyze efficiently oligosaccharides with a degree of polymerization smaller than six. The crystal structure of NcCel45A catalytic domain in the apo-form was determined at 1.9 A resolution and the structure of the enzyme bound to cellobiose was solved and refined to 1.8 A resolution. Comparative structural analyses and molecular dynamics simulations show that the enzyme dynamics is affected by substrate binding. Taken together, MD simulations and statistical coupling analysis revealed previously unknown correlation of a loop 6 with the breakdown of cellulose substrates by GH45.

A novel gene aga3027 from the genome of Flammeovirga sp. OC4, isolated from the deep sea, was screened and expressed in Escherichia coli BL21. This gene encoded the genetic information of a potential agarase that consists of 851 amino acids and belongs to 16 β-agarase family of glycoside hydrolase. Purified recombinant Aga3027 demonstrated the maximum activity of agarase at 40 °C and pH 9.0, displaying excellent thermostability and pH-stability. The agarase retained more than 80% of its maximum activity after incubation at 30–40 °C for 48 h, or after incubation at pH 6.0–9.0 for 60 min, which indicated that this agarase was suitable for industrial applications. Silica gel chromatography was used to purify the hydrolysates of agar treated by agarase from the recombinant Aga3027. The hydrolysates were identified as neoagarotetraose and neoagarohexaose by thin layer chromatography and further confirmed by ion chromatography.

Sulfolobus solfataricus β-glycosidase (SS-βGly) belongs to Glycosyl Hydrolase family1 (GH1) with broad substrate specificity. SS-βGly catalyzes both hydrolysis and transglycosylation reactions. SS-βGly is commonly used to synthesize variety of galacto-oligosaccharides. A comparison of SS-βGly with bacterial and eukaryotic homologs, using DALI search, revealed unique inserts. Free enzyme molecular dynamics (MD) simulation was performed under two different pH conditions (pH 6.5 and 2.5) at a constant temperature of 65 °C using GROMACS. A probable active-site loop (residues 331–364) in SS-βGly was identified. Dynamics of substrate binding cavity revealed that it was buried and inaccessible during most timeframes at pH 6.5 whereas open and accessible at pH 2.5. New cavities identified during both simulations may act as probable water channel or product egress path. Analyses of docked complexes of 3D structures obtained at every 1ns interval with compounds, involved in hydrolysis and tranglycosylation reactions, demonstrated that conformational states sampled by SS-βGly during free enzyme dynamics mimic the stages in enzyme catalysis thereby providing a mechanistic perspective. Current study revealed that conformational changes were conducive for hydrolysis at pH 6.5 and multiple cycles of transglycosylation at pH 2.5. Probable role of salt-bridge interactions in determining the type of reaction mechanism was also explored.

As a new food source, Moringa oleifera Lam. leaves containing large amounts of proteins attract increasing attention. This study detected and characterized hydrolytic and milk-clotting enzymes in these leaves. The proteins were examined by proteomics. A total of 3378 proteins were identified, mostly comprising enzymes of carbohydrate and protein metabolism, including 676 hydrolases, 548 oxidoreductases, glycoside hydrolases and proteinases. A serine/threonine-endopeptidase with a molecular mass of 56.146 kDa and an isoelectric point of 5.27 with milk-clotting activity was obtained from M. oleifera leaves by multistage ultrafiltration and anion exchange chromatography, and characterized by ESI mass spectrometry. The milk-clotting activity was inhibited over 95% by chymostatin, which confirmed its chymotrypsin-like serine protease nature. The protease had a milk-clotting activity/proteolytic activity (MCA/PA) activity ratio of 126.76, an optimal pH of 8.0, and an optimal temperature of 65 °C, indicating its potential use for cheese production and in food industry.

The β-galactosidase from Aspergillus oryzae produces galactooligosaccharides with beneficial prebiotic effects through the transglycosylation of lactose. However, its low galactooligosaccharide yield greatly limits its use in industrial applications. Rational design and site-directed mutagenesis were used to produce three mutants of A. oryzae β-galactosidase: N140C, W806F, and N140C/W806F. The galactooligosaccharide yields of N140C (50.7%), W806F (49.3%), and N140C/W806F (59.8%) represent substantial improvements over that of the wild-type (35.7%). The galactooligosaccharide yield of N140C/W806F is the highest reported to date. Our rational design approach suggests novel strategies for further study of the β-galactosidase reaction mechanism and has produced mutants may be more useful in industrial applications than wild-type A. oryzae β-galactosidase.

A novel β-galactosidase gene (Bgal) was cloned from the marine bacterium Alteromonas sp. ML117. Bgal from Alteromonas sp. ML117 was heterologously expressed in Escherichia coli BL21 (DE3) cells to study the enzymatic properties of the recombinant β-galactosidase. Using gel-filtration chromatography and SDS-PAGE, the recombinant enzyme was identified to be a tetrameric enzyme that belonged to the glycoside hydrolase family GH2. The purified recombinant Bgal was able to hydrolyze lactose and o-nitrophenyl-β- d -galactopyranoside (ONPG), and showed maximum activity at pH 8.0 and 30 °C to hydrolyze ONPG and at 35 °C to hydrolyze lactose. The enzyme’s activity level quickly diminished when incubated at 35 °C, suggesting that it was a cold-adapted variant. At 10 °C, the purified enzyme had Km values of 1.6 mM for ONPG and 3.8 mM for lactose. The enzyme tolerated NaCl, and retained 80% of its activity in a 30% ethanol reaction system. In addition, the recombinant Bgal hydrolyzed 86% of the lactose from milk over 24 h at 10 °C. Taken together, this study identified a recombinant Alteromonas sp. ML117 β-galactosidase that may be used for industrial applications.