Glycoside Hydrolase Family Research Articles

A bacterial sulfoglycosidase highlights mucin O-glycan breakdown in the gut ecosystem.

Mucinolytic bacteria modulate host-microbiota symbiosis and dysbiosis through their ability to degrade mucin O-glycans. However, how and to what extent bacterial enzymes are involved in the breakdown process remains poorly understood. Here we focus on a glycoside hydrolase family 20 sulfoglycosidase (BbhII) from Bifidobacterium bifidum, which releases N-acetylglucosamine-6-sulfate from sulfated mucins. Glycomic analysis showed that, in addition to sulfatases, sulfoglycosidases are involved in mucin O-glycan breakdown in vivo and that the released N-acetylglucosamine-6-sulfate potentially affects gut microbial metabolism, both of which were also supported by a metagenomic data mining analysis. Enzymatic and structural analysis of BbhII reveals the architecture underlying its specificity and the presence of a GlcNAc-6S-specific carbohydrate-binding module (CBM) 32 with a distinct sugar recognition mode that B. bifidum takes advantage of to degrade mucin O-glycans. Comparative analysis of the genomes of prominent mucinolytic bacteria also highlights a CBM-dependent O-glycan breakdown strategy used by B. bifidum.

The Glycoside Hydrolase Family 35 β-galactosidase from Trichoderma reesei debranches xyloglucan oligosaccharides from tamarind and jatobá

Trichoderma reesei (anamorph Hypocrea jecorina) produces an extracellular beta-galactosidase from Glycoside Hydrolase Family 35 (TrBga1). Hydrolysis of xyloglucan oligosaccharides (XGOs) by TrBga1 has been studied by hydrolysis profile analysis of both tamarind (Tamarindus indica) and jatobá (Hymenaea courbaril) seed storage xyloglucans using PACE and MALDI-ToF-MS for separation, quantification and identification of the hydrolysis products. The TrBga1 substrate preference for galactosylated oligosaccharides from both the XXXG- and XXXXG-series of jatobá xyloglucan showed that the doubly galactosylated oligosaccharides were the first to be hydrolyzed. Furthermore, the TrBga1 showed more efficient hydrolysis against non-reducing end dexylosylated oligosaccharides (GLXG/GXLG and GLLG). This preference may play a key role in xyloglucan degradation, since galactosyl removal alleviates steric hindrance for other enzymes in the xyloglucanolytic complex resulting in complete xyloglucan mobilization. Indeed, mixtures of TrBga1 with the α-xylosidase from Escherichia coli (YicI), which shows a preference towards non-galactosylated xyloglucan oligosaccharides, reveals efficient depolymerization when either enzyme is applied first. This understanding of the synergistic depolymerization contributes to the knowledge of plant cell wall structure, and reveals possible evolutionary mechanisms directing the preferences of debranching enzymes acting on xyloglucan oligosaccharides.

Glycoside Hydrolase Family 48 Cellulase: A Key Player in Cellulolytic Bacteria for Lignocellulose Biorefinery

Cellulases from glycoside hydrolase family 48 (GH48) are critical components of natural lignocellulose-degrading systems. GH48 cellulases are broadly distributed in cellulolytic microorganisms. With the development of genomics and metatranscriptomics, diverse GH48 genes have been identified, especially in the highly efficient cellulose-degrading ruminal system. GH48 cellulases utilize an inverting mechanism to hydrolyze cellulose in a processive mode. Although GH48 cellulases are indispensable for cellulolytic bacteria, they exhibit intrinsically low cellulolytic activity. Great efforts have been made to improve their performance. Besides, GH48 cellulases greatly synergize with the complementary endoglucanases in free cellulase systems or cellulosome systems. In this review, we summarized the studies on the diversity of GH48 cellulases, the crystal structures, the catalytic mechanism, the synergy between GH48 cellulases and endocellulases, and the strategies and progress of GH48 engineering. According to the summarized bottlenecks in GH48 research and applications, we suggest that future studies should be focused on mining and characterizing new GH48 enzymes, thoroughly understanding the progressive activity and product inhibition, engineering GH48 enzymes to improve stability, activity, and stress resistance, and designing and developing new biocatalytic system employing the synergies between GH48 and other enzymes.

Improved catalytic efficiency of chimeric xylanase 10B from Thermotoga petrophila RKU1 and its synergy with cellulases

TpXyl10B is a glycoside hydrolase family 10 xylanase of hyperthermophile Thermotoga petrophila RKU-1. This enzyme is of considerable importance due to its thermostability. However, in its native state, this enzyme does not possess any carbohydrate-binding module (CBM) for efficient binding to plant biomass. In this study CBM6 from Clostridium thermocellum was attached to the N- and C-termini of TpXyl10B, thereby producing the variants TpXyl10B-B6C and TpXyl10B-CB6, respectively. TpXyl10B-B6C showed 5–7 folds increased activity on Beechwood xylan and the different types of plant biomass as compared to that from the catalytic domain only. However, the activity of TpXyl10B-CB6 decreased 0.6–0.8 folds on Beechwood xylan and plant biomass compared to the catalytic domain. We explained these results through molecular modeling, which showed that binding residues of CBM6's cleft B, which were previously reported to show no contribution towards binding due to steric hindrance from a loop region, were exposed in a favorable position in TpXyl10B-B6C such that they efficiently bound the substrate. In contrast, these binding residues of CBM6 in TpXyl10B-CB6 were exposed opposite to the catalytic residues; thus, binding to the substrate resulted in decreased exposure of catalytic residues to the substrate. CD spectroscopy and thermostability assays showed that TpXyl10B-B6C was highly thermostable, having a melting point > 90 °C, which is relatively higher than that of the other variant, TpXyl10B-CB6. In addition, this xylanase variant showed synergism with cellulases for the hydrolysis of plant biomass. Therefore, TpXyl10B-B6C, an engineered xylanase in this study, can be a valuable candidate for industrial applications.

Three Molecular Modification Strategies to Improve the Thermostability of Xylanase XynA from Streptomyces rameus L2001

Glycoside hydrolase family 11 (GH11) xylanases are the preferred candidates for the production of functional oligosaccharides. However, the low thermostability of natural GH11 xylanases limits their industrial applications. In this study, we investigated the following three strategies to modify the thermostability of xylanase XynA from Streptomyces rameus L2001 mutation to reduce surface entropy, intramolecular disulfide bond construction, and molecular cyclization. Changes in the thermostability of XynA mutants were analyzed using molecular simulations. All mutants showed improved thermostability and catalytic efficiency compared with XynA, except for molecular cyclization. The residual activities of high-entropy amino acid-replacement mutants Q24A and K104A increased from 18.70% to more than 41.23% when kept at 65 °C for 30 min. The catalytic efficiencies of Q24A and K143A increased to 129.99 and 92.26 mL/s/mg, respectively, compared with XynA (62.97 mL/s/mg) when using beechwood xylan as the substrate. The mutant enzyme with disulfide bonds formed between Val3 and Thr30 increased the t1/260 °C by 13.33-fold and the catalytic efficiency by 1.80-fold compared with the wild-type XynA. The high thermostabilities and hydrolytic activities of XynA mutants will be useful for enzymatic production of functional xylo-oligosaccharides.

A subfamily classification to choreograph the diverse activities within glycoside hydrolase family 31

The Carbohydrate-Active Enzyme classification groups enzymes that breakdown, assemble, or decorate glycans into protein families based on sequence similarity. The glycoside hydrolases (GH) are arranged into over 170 enzyme families, with some being very large and exhibiting distinct activities/specificities towards diverse substrates. Family GH31 is a large family that contains more than 20,000 sequences with a wide taxonomic diversity. Less than 1% of GH31 members are biochemically characterized and exhibit many different activities that include glycosidases, lyases, and transglycosidases. This diversity of activities limits our ability to predict the activities and roles of GH31 family members in their host organism and our ability to exploit these enzymes for practical purposes. Here, we established a subfamily classification using sequence similarity networks that was further validated by a structural analysis. While sequence similarity networks provide a sequence-based separation, we obtained good segregation between activities among the subfamilies. Our subclassification consists of 20 subfamilies with sixteen subfamilies containing at least one characterized member and eleven subfamilies that are monofunctional based on the available data. We also report the biochemical characterization of a member of the large subfamily 2 (GH31_2) that lacked any characterized members: RaGH31 from Rhodoferax aquaticus is an α-glucosidase with activity on a range of disaccharides including sucrose, trehalose, maltose, and nigerose. Our subclassification provides improved predictive power for the vast majority of uncharacterized proteins in family GH31 and highlights the remaining sequence space that remains to be functionally explored.

Expression, purification, characterization and glycoside production potential of rice β-d-glucan glucohydrolase I (OsExoI)

β-d-Glucan glucohydrolases from glycoside hydrolase family 3 (GH3) act to break-down β-glucans, which is critical to growth and cell wall remodeling in plants. To investigate the function and application of the rice GH3 β-d-glucan glucohydrolases OsExoI, we produced the protein from a codon-optimized cDNA in the heterogenous expression host Pichia pastoris. After purification from the medium, OsExoI was found to be heterogeneously glycosylated and could be separated into pools with different glycosylation levels and specific activities by size exclusion chromatography (SEC). The total glycosylated protein pool had specific activity similar to that of protein from which N-linked carbohydrate had been removed with endoglycosidase H. Purified enzyme hydrolyzed β-1,3- and 1,4- linked oligosaccharides, polysaccharides, and synthetic p-nitrophenyl-β-d-glucopyranoside. The deglycosylated OsExoI had catalytic efficiency (kcat/Km) slightly higher than pooled glycosylated OsExoI, although their overall biochemical properties were similar. OsExoI also exhibits transglycosylation activity using pNP-β-d-glucopyranoside, oligosaccharides, or polysaccharides as glucosyl donor to glycosylate a variety of alcohols. These results suggest that the OsExoI can be applied to alkyl glycoside production using natural polysaccharides or oligosaccharides released during biomass degradation as a source of glucosyl moieties.

Comparison of the gut microbiome and resistome in captive African and Asian elephants on the same diet.

Elephants are endangered species and threatened with extinction. They are monogastric herbivorous, hindgut fermenters and their digestive strategy requires them to consume large amounts of low quality forage. The gut microbiome is important to their metabolism, immune regulation, and ecological adaptation. Our study investigated the structure and function of the gut microbiota as well as the antibiotic resistance genes (ARGs) in captive African and Asian elephants on the same diet. Results showed that captive African and Asian elephants had distinct gut bacterial composition. MetaStats analysis showed that the relative abundance of Spirochaetes (FDR = 0.00) and Verrucomicrobia (FDR = 0.01) at the phylum level as well as Spirochaetaceae (FDR = 0.01) and Akkermansiaceae (FDR = 0.02) at the family level varied between captive African and Asian elephants. Among the top ten functional subcategories at level 2 (57 seed pathway) of Kyoto Encyclopedia of Genes and Genomes (KEGG) database, the relative gene abundance of cellular community-prokaryotes, membrane transport, and carbohydrate metabolism in African elephants were significantly lower than those in Asian elephants (0.98 vs. 1.03%, FDR = 0.04; 1.25 vs. 1.43%, FDR = 0.03; 3.39 vs. 3.63%; FDR = 0.02). Among the top ten functional subcategories at level 2 (CAZy family) of CAZy database, MetaStats analysis showed that African elephants had higher relative gene abundance of Glycoside Hydrolases family 28 (GH 28) compared to Asian elephants (0.10 vs. 0.08%, FDR = 0.03). Regarding the antibiotic resistance genes carried by gut microbes, MetaStats analysis showed that African elephants had significantly higher relative abundance of vanO (FDR = 0.00), tetQ (FDR = 0.04), and efrA (FDR = 0.04) than Asian elephants encoding resistance for glycopeptide, tetracycline, and macrolide/rifamycin/fluoroquinolone antibiotic, respectively. In conclusion, captive African and Asian elephants on the same diet have distinct gut microbial communities. Our findings established the ground work for future research on improving gut health of captive elephants.

Engineered Glycosidase for Significantly Improved Production of Naturally Rare Vina-Ginsenoside R7.

Ginsenosides are the main bioactive ingredients in plants of the genus Panax. Vina-ginsenoside R7 (VG-R7) is one of the rare high-value ginsenosides with health benefits. The only reported method for preparing VG-R7 involves inefficient and low-yield isolation from highly valuable natural resources. Notoginsenoside Fc (NG-Fc) isolated in the leaves and stems of Panax notoginseng is a suitable substrate for the preparation of VG-R7 via specific hydrolysis of the outside xylose at the C-20 position. Here, we first screened putative enzymes belonging to the glycoside hydrolase (GH) families 1, 3, and 43 and found that KfGH01 can specifically hydrolyze the β-d-xylopyranosyl-(1 → 6)-β-d-glucopyranoside linkage of NG-Fc to form VG-R7. The I248F/Y410R variant of KfGH01 obtained by protein engineering displayed a kcat/KM value (305.3 min-1 mM-1) for the reaction enhanced by approximately 270-fold compared with wild-type KfGH01. A change in the shape of the substrate binding pockets in the mutant allows the substrate to sit closer to the catalytic residues which may explain the enhanced catalytic efficiency of the engineered enzyme. This study identifies the first glycosidase for bioconversion of a ginsenoside with more than four sugar units, and it will inspire efforts to investigate other promising enzymes to obtain valuable natural products.

Comparative Genomic Analyses of Cellulolytic Machinery Reveal Two Nutritional Strategies of Marine Labyrinthulomycetes Protists.

Labyrinthulomycetes are a group of ubiquitous and diverse unicellular Stramenopiles and have long been known for their vital role in ocean carbon cycling. However, their ecological function from the perspective of organic matter degradation remains poorly understood. This study reports high-quality genomes of two newly isolated Labyrinthulomycetes strains, namely, Botryochytrium sp. strain S-28 and Oblongichytrium sp. strain S-429, and provides molecular analysis of their ecological functions using comparative genomics and a biochemical assay. Our results suggest that Labyrinthulomycetes may occupy multiple ecological niches in marine ecosystems because of the significant differences in gene function among different genera. Certain strains could degrade wheat bran independently by secreting cellulase. The key glycoside hydrolase families (GH1, GH5, and GH9) related to cellulase and the functional domains of carbohydrate-active enzymes (CAZymes) were more enriched in their genomes. This group can actively participate in marine biochemical cycles as decomposers. In contrast, other strains that could not produce cellulase may thrive as "leftover scavengers" and act as a source of nutrients to the higher-trophic-level plankton. In addition, our findings emphasize the dual roles of endoglucanase, acting as both exo- and endoglucanases, in the process of cellulose degradation. Using genomic, biochemical, and phylogenetic analyses, our study provides a broader insight into the nutritional patterns and ecological functions of Labyrinthulomycetes. IMPORTANCE Unicellular heterotrophic eukaryotes are an important component of marine ecosystems. However, their ecological functions and modes of nutrition remain largely unknown. Our current understanding of marine microbial ecology is incomplete without integrating these heterotrophic microeukaryotes into the food web models. This study focuses on the unicellular fungus-like protists Labyrinthulomycetes and provides two high-quality genomes of cellulase-producing Labyrinthulomycetes. Our study uncovers the basis of their cellulase production by deciphering the results of genomic, biochemical, and phylogenetic analyses. This study instigates a further investigation of the molecular mechanism of organic matter utilization by Labyrinthulomycetes in the world's oceans.

Co-culture of red swamp crayfish Procambarus clarkia influenced glycoside hydrolase families and fungal communities in the rice-paddy soils

Integrated rice–crayfish farming has expanded rapidly, and an in-depth understanding of this model is urgently required. In the present study, metagenomics and real-time polymerase chain reaction were carried out to determine whether and how the introduction of the red swamp crayfish Procambarus clarkia affected glycoside hydrolase (GH) families and the abundance and community composition of fungi in soils. Investigations comprised the ecosystems of rice–crayfish co-culture fields, crayfish culture ponds, and rice paddy fields. Further, we sought to investigate the influence of the fungal community on bacterial chitinase gene abundance. The results showed that although rice cultivation induced variations in GH families and fungal communities in rice paddy soils, the introduction of crayfish was the main factor responsible for these variations. Additionally, the proliferation of bacteria with chitinase genes was not induced by the crayfish exoskeleton (in the form of living animals) but by the abundance of fungi in the soils. Therefore, bacterial chitinase in soils may not affect the exoskeleton of living crayfish but is likely the main regulator of the abundance of soil fungi. The impact of crayfish on fungal diversity was likely due to direct biological effects, while its inhibitory effect on fungal abundance was mainly attributed to the low nitrogen content in soils.

Diversity of lignocellulolytic functional genes and heterogeneity of thermophilic microbes during different wastes composting

The goal of this study was to investigate the heterogeneity of thermophilic microorganisms and their lignocellulose-degrading gene diversity during composting. In this study, bagasse pith/dairy manure (BAG) and sawdust/dairy manure (SAW) were used as experimental subjects. The pour plate method indicated that thermophilic bacteria and thermophilic actinobacteria were more culturable than thermophilic fungi. Metagenomics analysis showed that the Actinobacteria, Firmicutes and Proteobacteria were the dominant phyla during composting. In addition, auxiliary activity and glycoside hydrolase families were critical for lignocellulosic degradation, which were found to be more abundant in BAG. As a result, the degradation rates of cellulose, hemicellulose and lignin in BAG (7.36%, 13.99% and 5.68%) were observably higher than those in SAW (6.13%, 12.09% and 2.62%). These findings contribute to understanding how thermophilic microbial communities play a role in the deconstruction of different lignocelluloses and provide a potential strategy to comprehensively utilize the resources of lignocellulosic biomass.

Populus endo-glucanase 16 localizes to the cell walls of developing tissues.

The hemicelluloses comprise a group of matrix glycans that interact with cellulose microfibrils in plant cell walls and play important roles in establishing wall architecture. The structures of hemicelluloses are determined by carbohydrate-active enzymes (CAZymes) that synthesize, integrate, and break down these polymers. Specifically, endo-glucanase 16 (EG16) enzymes, which are related to the well-known xyloglucan endotransglycosylase/hydrolase (XTH) gene products in Glycoside Hydrolase Family 16 (GH16), have been implicated in the degradation of the β(1,4)-linked backbone of mixed-linkage β(1,3);β(1,4)-glucans (MLG) and xyloglucans. EG16 members are single-copy genes found in most plant clades but are absent from many eudicots, including the model plant Arabidopsis thaliana. Until recently, EG16 members had only been characterized in vitro, establishing their substrate specificity, protein structure, and phylogenetic history, but their biological function was unknown. Here we used a hybrid polar, Populus alba× Populus grandidentata (P39), as a model to examine EG16 expression, subcellular localization, and pheno- and chemotypes of EG16-downregulated P39 plants. Populus EG16 expression is strong in young tissues, but RNAi-mediated downregulation did not impact plant growth nor the fine structure of the hemicellulose xyloglucan, suggesting a restricted or currently unknown role in angiosperm physiology.

Structural and functional insights of the catalytic GH5 and Calx-β domains from the metagenome-derived endoglucanase CelE2

Cellulose is the most abundant natural polymer on Earth, representing an attractive feedstock for bioproducts and biofuel production. Cellulases promote the depolymerization of cellulose, generating short oligosaccharides and glucose, which are useful in biotechnological applications. Among the classical cellulases, those from glycoside hydrolase family 5 (GH5) are one of the most abundant in Nature, displaying several modular architectures with other accessory domains attached to its catalytic core, such as carbohydrate-binding modules (CBMs), Ig-like, FN3-like, and Calx-β domains, which can influence the enzyme activity. The metagenome-derived endoglucanase CelE2 has in its modular architecture an N-terminal domain belonging to the GH5 family and a C-terminal domain with a high identity to the Calx-β domain. In this study, the GH5 and the Calx-β domains were subcloned and heterologously expressed in E. coli, to evaluate the structural and functional properties of the individualized domains of CelE2. Thermostability analysis by circular dichroism (CD) revealed a decrease in the denaturation temperature values around 4.6 °C for the catalytic domain (CelE21–381) compared to CelE2 full-length. The CD analyses revealed that the Calx-β domain (CelE2382–477) was unfolded, suggesting that this domain requires to be attached to the catalytic core to become structurally stable. The three-dimensional structure of the catalytic domain CelE21–381 was determined at 2.1 Å resolution, showing a typical (α/β)8-barrel fold and a narrow active site compared to other cellulases from the same family. The biochemical characterization showed that the deletion of the Calx-β domain increased more than 3-fold the activity of the catalytic domain CelE21–381 towards the insoluble substrate Avicel. The main functional properties of CelE2, such as substrate specificity, optimal pH and temperature, thermal stability, and activation by CaCl2, were not altered after the deletion of the accessory domain. Furthermore, the Small Angle X-ray Scattering (SAXS) analyses showed that the addition of CaCl2 was beneficial CelE21–381 protein solvency. This work contributed to fundamental concepts about the structure and function of cellulases, which are useful in applications involving lignocellulosic materials degradation into food and feedstuffs and biofuel production.

Cassava pullulanase and its synergistic debranching action with isoamylase 3 in starch catabolism.

Pullulanase (EC 3.2.1.41, PUL), a debranching enzyme belonging to glycoside hydrolase family 13 subfamily 13, catalyses the cleavage of α-1,6 linkages of pullulan and β-limit dextrin. The present work studied PUL from cassava Manihot esculenta Crantz (MePUL) tubers, an important economic crop. The Mepul gene was successfully cloned and expressed in E. coli and rMePUL was biochemically characterised. MePUL was present as monomer and homodimer, as judged by apparent mass of ~ 84 - 197 kDa by gel permeation chromatography analysis. Optimal pH and temperature were at pH 6.0 and 50 °C, and enzyme activity was enhanced by the addition of Ca2+ ions. Pullulan is the most favourable substrate for rMePUL, followed by β-limit dextrin. Additionally, maltooligosaccharides were potential allosteric modulators of rMePUL. Interestingly, short-chain maltooligosaccharides (DP 2 - 4) were significantly revealed at a higher level when rMePUL was mixed with cassava isoamylase 3 (rMeISA3), compared to that of each single enzyme reaction. This suggests that MePUL and MeISA3 debranch β-limit dextrin in a synergistic manner, which represents a major starch catabolising process in dicots. Additionally, subcellular localisation suggested the involvement of MePUL in starch catabolism, which normally takes place in plastids.

Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis.

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C.pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C.pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C.pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.

Galactomannan utilization by Cellvibrio japonicus relies on a single essential α-galactosidase encoded by the aga27A gene.

Plant mannans are a component of lignocellulose that can have diverse compositions in terms of its backbone and side-chain substitutions. Consequently, the degradation of mannan substrates requires a cadre of enzymes for complete reduction to substituent monosaccharides that can include mannose, galactose, and/or glucose. One bacterium that possesses this suite of enzymes is the Gram-negative saprophyte Cellvibrio japonicus, which has 10 predicted mannanases from the Glycoside Hydrolase (GH) families 5, 26, and 27. Here we describe a systems biology approach to identify and characterize the essential mannan-degrading components in this bacterium. The transcriptomic analysis uncovered significant changes in gene expression for most mannanases, as well as many genes that encode carbohydrate active enzymes (CAZymes) when mannan was actively being degraded. A comprehensive mutational analysis characterized 54 CAZyme-encoding genes in the context of mannan utilization. Growth analysis of the mutant strains found that the man26C, aga27A, and man5D genes, which encode a mannobiohydrolase, α-galactosidase, and mannosidase, respectively, were important for the deconstruction of galactomannan, with Aga27A being essential. Our updated model of mannan degradation in C. japonicus proposes that the removal of galactose sidechains from substituted mannans constitutes a crucial step for the complete degradation of this hemicellulose.

Improving the thermostability of GH49 dextranase AoDex by site-directed mutagenesis

As an indispensable enzyme for the hydrolysis of dextran, dextranase has been widely used in the fields of food and medicine. It should be noted that the weak thermostability of dextranase has become a restricted factor for industrial applications. This study aims to improve the thermostability of dextranase AoDex in glycoside hydrolase (GH) family 49 that derived from Arthrobacter oxydans KQ11. Some mutants were predicted and constructed based on B-factor analysis, PoPMuSiC and HotMuSiC algorithms, and four mutants exhibited higher heat resistance. Compared with the wild-type, mutant S357P showed the best improved thermostability with a 5.4-fold increase of half-life at 60 °C, and a 2.1-fold increase of half-life at 65 °C. Furthermore, S357V displayed the most obvious increase in enzymatic activity and thermostability simultaneously. Structural modeling analysis indicated that the improved thermostability of mutants might be attributed to the introduction of proline and hydrophobic effects, which generated the rigid optimization of the structural conformation. These results illustrated that it was effective to improve the thermostability of dextranase AoDex by rational design and site-directed mutagenesis. The thermostable mutant of dextranase AoDex has potential application value, and it can also provide references for engineering other thermostable dextranases of the GH49 family.

The In Silico Characterization of Monocotyledonous α-l-Arabinofuranosidases on the Example of Maize

Plant α-l-arabinofuranosidases remove terminal arabinose from arabinose-containing substrates such as plant cell wall polysaccharides, including arabinoxylans, arabinogalactans, and arabinans. In plants, de-arabinosylation of cell wall polysaccharides accompanies different physiological processes such as fruit ripening and elongation growth. In this report, we address the diversity of plant α-l-arabinofuranosidases of the glycoside hydrolase (GH) family 51 through their phylogenetic analysis as well as their structural features. The CBM4-like domain at N-terminus was found to exist only in GH51 family proteins and was detected in almost 90% of plant sequences. This domain is similar to bacterial CBM4, but due to substitutions of key amino acid residues, it does not appear to be able to bind carbohydrates. Despite isoenzymes of GH51 being abundant, in particular in cereals, almost half of the GH51 proteins in Poales have a mutation of the acid/base residue in the catalytic site, making them potentially inactive. Open-source data on the transcription and translation of GH51 isoforms in maize were analyzed to discuss possible functions of individual isoenzymes. The results of homology modeling and molecular docking showed that the substrate binding site can accurately accommodate terminal arabinofuranose and that arabinoxylan is a more favorable ligand for all maize GH51 enzymes than arabinan.

Characterization of a novel GH26 β-mannanase from Paenibacillus polymyxa and its application in the production of mannooligosaccharides

A novel glycoside hydrolase family 26 β-mannanase gene ppman26a was cloned from Paenibacillus polymyxa KF-1. The full-length enzyme PpMan26A and its truncated products CBM35pp (aa 35–328) and PpMan26A-Δ205 (aa 206–656) were overexpressed in Escherichia coli. PpMan26A hydrolyzed locust bean gum, guar gum, konjac gum and ivory nut mannan, with the highest specific activity toward konjac gum. The Km and kcat values for konjac gum were 2.13 mg/mL and 416.66 s–1, respectively. The oligosaccharides fraction obtained from the hydrolysis of konjac gum by PpMan26A was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometer (MALDI-TOF-MS). The degradation products were mainly mannooligosaccharides with a degree of polymerization of 3–8. CBM35pp exerted strong binding activity toward mannans but without β-mannanase activity. PpMan26A-Δ205, with the deletion of the N-terminal CBM domain, showed lower substrate binding capacity, resulting in reduced enzymatic activity and thermostability. This study complements our understanding of GH26 β-mannanases and expands the potential industrial application of PpMan26A.