Glycoside Hydrolase Family Research Articles

Identification and expression analysis of genomic regions associated with the traits contributing to lodging tolerance in wheat (Triticum aestivum L.)

Crop lodging, either stem breaking/stem bending or root lodging, is a major concern for farmers worldwide. In the present study, meta-QTLs, ortho-MQTLs and candidate genes related to lodging and its associated traits were identified using 836 of the 1269 known QTLs retrieved from 107 QTL mapping studies published during 1998–2022. The study resulted in the identification of 86 MQTLs with an average confidence interval (CI) of 1.66 cM (4.26-fold reduction) as against a CI of > 7.08 cM in the initial QTLs. Out of 86 MQTLs, a subset of 12 MQTLs demonstrated stability and robustness, earning them the title of breeders’ MQTLs. These selections were made based on criteria such as the number of initial QTLs involved, small CI, high LOD scores, and PVE values. Furthermore, leveraging synteny and collinearity facilitated the identification of 10 ortho-MQTLs shared between wheat and maize. Further, in silico expression analysis allowed the detection of 53 significantly expressed candidate genes encoding different proteins/products including the following: F-box protein, GDSL lipase/esterases, SANT/Myb domain transcription factor, pectin esterase inhibitor domain, glycoside hydrolase family, protein kinase domain, zinc finger domain, cytochrome P450, UDP-glucuronosyl/UDP-glucosyltransferase, glycosyltransferase 61 etc. In addition, 26 lodging tolerance genes from Arabidopsis, rice, and maize were also utilized to identify 34 wheat homologues in wheat MQTL regions. Among these, three genes TraesCS2D02G256600 (TaEREB114-2D), TraesCS7B02G340100 (TaIRX14-7B), and TraesCS1B02G347600 (TaGA2ox4-1B) were prioritized for qRT-PCR-based expression analysis in one lodging-tolerant (i.e., WH-1105) and one lodging-susceptible (i.e., WH-1184) variety. When comparing these two varieties, we found higher relative expression levels of these three genes in the second basal internode at the milky endosperm stage of the lodging-tolerant variety. The MQTLs, ortho-MQTLs, and candidate genes (CGs) identified in this study can be utilized for marker-assisted selection to improve lodging tolerance and enhance our understanding of the molecular basis of lodging tolerance in wheat.

Comparative genomics and fermentation flavor characterization of five selected lactic acid bacteria provide predictions for flavor biosynthesis metabolic pathways in fermented muskmelon puree

AbstractFive species of plant‐derived lactic acid bacteria (LAB), including Pediococcus pentosaceus, Lactococcus garvieae, Lactiplantibacillus paraplantarum, Weissella paramesenteroides, and Lactococcus lactis, were used for muskmelon fermentation to improve flavor characteristics. Comparative genomic analysis showed that a high proportion of annotated genes were related to carbohydrate metabolism and amino acid metabolism in all five strains. L. lactis P‐1 (Ll1) contained the highest proportion (2.02%) of genes from the glycoside hydrolase family associated with carbohydrate metabolism. Gas chromatography–mass spectrometry results showed that 89 volatile compounds in muskmelon purees were identified; the largest number of volatile compounds was detected in fermented muskmelon puree (FMP) inoculated with W. paramesenteroides FL3, followed by L. paraplantarum FL‐8. A total of 25 key volatile compounds were screened in muskmelon puree, and some unique key volatile compounds (e.g., β‐damascenone, phenylacetaldehyde, and 3‐penten‐2‐one) and taste compounds (e.g., mannitol) were produced by different LAB strains. L. garvieae Pa‐2 and Ll1 were considered to have greater potential due to their ability to produce a greater category of pleasant key aromas. Based on the correlations of potential pathways analyzed using comparative genomics and detected flavor profiles, flavor synthesis pathways in FMP were predicted. Some carbohydrate, amino acid, and fatty acid metabolism pathways, including tricarboxylic acid cycle, glycolysis, and cysteine and methionine metabolism pathways, are common to all five LAB strains. Furthermore, differences in the predicted flavor biosynthesis metabolic pathways in monoterpenoid biosynthesis, phenylalanine metabolism, fatty acid biosynthesis, and fructose and mannose metabolism pathways explained the differences in volatile profiles produced by different strains.

Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9

Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

Hydrolysis of ionic liquid–treated substrate with an Iocasia fonsfrigidae strain SP3-1 endoglucanase

Recently, we reported the discovery of a novel endoglucanase of the glycoside hydrolase family 12 (GH12), designated IfCelS12A, from the haloalkaliphilic anaerobic bacterium Iocasia fonsfrigidae strain SP3-1, which was isolated from a hypersaline pond in the Samut Sakhon province of Thailand (ca. 2017). IfCelS12A exhibits high substrate specificity on carboxymethyl cellulose and amorphous cellulose but low substrate specificity on b-1,3;1,4-glucan. Unlike some endoglucanases of the GH12 family, IfCelS12A does not exhibit hydrolytic activity on crystalline cellulose (i.e., Avicel™). High-Pressure Liquid Chromatography (HPLC) and Thin Layer Chromatography (TLC) analyses of products resulting from IfCelS12-mediated hydrolysis indicate mode of action for this enzyme. Notably, IfCelS12A preferentially hydrolyzes cellotetraoses, cellopentaoses, and cellohexaoses with negligible activity on cellobiose or cellotriose. Kinetic analysis with cellopentaose and barely b-d-glucan as cellulosic substrates were conducted. On cellopentaose, IfCelS12A demonstrates a 16-fold increase in activity (KM = 0.27 mM; kcat = 0.36 s−1; kcat/KM = 1.34 mM−1 s−1) compared to the enzymatic hydrolysis of barley b-d-glucan (KM: 0.04 mM, kcat: 0.51 s−1, kcat/KM = 0.08 mM−1 s−1). Moreover, IfCelS12A enzymatic efficacy is stable in hypersaline sodium chlorids (NaCl) solutions (up to 10% NaCl). Specifically, IfCel12A retains notable activity after 24 h at 2M NaCl (10% saline solution). IfCelS12A used as a cocktail component with other cellulolytic enzymes and in conjunction with mobile sequestration platform technology offers additional options for deconstruction of ionic liquid–pretreated cellulosic feedstock.Key points• IfCelS12A from an anaerobic alkaliphile Iocasia fronsfrigidae shows salt tolerance• IfCelS12A in cocktails with other enzymes efficiently degrades cellulosic biomass• IfCelS12A used with mobile enzyme sequestration platforms enhances hydrolysis

Enzymatic synthesis of phenolic acid glucosyl esters to test activities on cholangiocarcinoma cells.

While glycoside hydrolase family 1 (GH1) enzymes mostly catalyze hydrolysis reactions, rice Os9BGlu31 preferentially catalyzes transglycosylation to transfer a glucosyl moiety to another aglycone moiety to form a new glycosylated compound through a retaining mechanism. In this study, Os9BGlu31 was used to synthesize eight phenolic acid glucosyl esters, which were evaluated for activities in cholangiocarcinoma cells. The transglycosylation products of Os9BGlu31 wild type and its mutant variants were detected, produced on a milligram scale, and purified, and their structures were characterized by NMR spectroscopy. The transglycosylation products were evaluated by antioxidant and anti-proliferative assays, followed by an anti-migration assay for the selected phenolic acid glucosyl ester. Os9BGlu31 mutants produced higher yield and activity than wild-type enzymes on phenolic acids to produce phenolic acid glucosyl esters. Among these, gallic acid glucosyl ester (β-glucogallin) had the highest antioxidant activity and anti-proliferative activity in cholangiocarcinoma cells. It also inhibited the migration of cholangiocarcinoma cells. Our study demonstrated that rice Os9BGlu31 transglucosidase is a promising enzyme for glycosylation of bioactive compounds in one-step reactions and provides evidence that β-glucogallin inhibits cell proliferation and migration of cholangiocarcinoma cells. KEY POINTS: • Os9BGlu31 transglucosidases produced phenolic acid glucosyl esters for bioactivity testing. • Phenolic acid glucosyl esters were tested for cytotoxicity in cholangiocarcinoma cells. • β-Glucogallin displayed the highest inhibition of cholangiocarcinoma cell growth.

A Broad-Spectrum α-Glucosidase of Glycoside Hydrolase Family 13 from Marinovum sp., a Member of the Roseobacter Clade.

Glycoside hydrolases (GHs) are a diverse group of enzymes that catalyze the hydrolysis of glycosidic bonds. The Carbohydrate-Active enZymes (CAZy) classification organizes GHs into families based on sequence data and function, with fewer than 1% of the predicted proteins characterized biochemically. Consideration of genomic context can provide clues to infer possible enzyme activities for proteins of unknown function. We used the MultiGeneBLAST tool to discover a gene cluster in Marinovum sp., a member of the marine Roseobacter clade, that encodes homologues of enzymes belonging to the sulfoquinovose monooxygenase pathway for sulfosugar catabolism. This cluster lacks a gene encoding a classical family GH31 sulfoquinovosidase candidate, but which instead includes an uncharacterized family GH13 protein (MsGH13) that we hypothesized could be a non-classical sulfoquinovosidase. Surprisingly, recombinant MsGH13 lacks sulfoquinovosidase activity and is a broad-spectrum α-glucosidase that is active on a diverse array of α-linked disaccharides, including maltose, sucrose, nigerose, trehalose, isomaltose, and kojibiose. Using AlphaFold, a 3D model for the MsGH13 enzyme was constructed that predicted its active site shared close similarity with an α-glucosidase from Halomonas sp. H11 of the same GH13 subfamily that shows narrower substrate specificity.

The Complex GH32 Enzyme Orchestra from Priestia megaterium Holds the Key to Better Discriminate Sucrose-6-phosphate Hydrolases from Other β-Fructofuranosidases in Bacteria.

Inulin is widely used as a prebiotic and emerging as a priming compound to counteract plant diseases. We isolated inulin-degrading strains from the lettuce phyllosphere, identified as Bacillus subtilis and Priestia megaterium, species hosting well-known biocontrol organisms. To better understand their varying inulin degradation strategies, three intracellular β-fructofuranosidases from P. megaterium NBRC15308 were characterized after expression in Escherichia coli: a predicted sucrose-6-phosphate (Suc6P) hydrolase (SacAP1, supported by molecular docking), an exofructanase (SacAP2), and an invertase (SacAP3). Based on protein multiple sequence and structure alignments of bacterial glycoside hydrolase family 32 enzymes, we identified conserved residues predicted to be involved in binding phosphorylated (Suc6P hydrolases) or nonphosphorylated substrates (invertases and fructanases). Suc6P hydrolases feature positively charged residues near the structural catalytic pocket (histidine, arginine, or lysine), whereas other β-fructofuranosidases contain tryptophans. This correlates with our phylogenetic tree, grouping all predicted Suc6P hydrolases in a clan associated with genomic regions coding for transporters involved in substrate phosphorylation. These results will help to discriminate between Suc6P hydrolases and other β-fructofuranosidases in future studies and to better understand the interaction of B. subtilis and P. megaterium endophytes with sucrose and/or fructans, sugars naturally present in plants or exogenously applied in the context of defense priming.

In vitro evaluation of the application of an optimized xylanase cocktail for improved monogastric feed digestibility.

Xylanases from glycoside hydrolase (GH) families 10 and 11 are common feed additives for broiler chicken diets due to their catalytic activity on the nonstarch polysaccharide xylan. This study investigated the potential of an optimized binary GH10 and GH11 xylanase cocktail to mitigate the antinutritional effects of xylan on the digestibility of locally sourced chicken feed. Immunofluorescence visualization of the activity of the xylanase cocktail on xylan in the yellow corn of the feed showed a substantial collapse in the morphology of cell walls. Secondly, the reduction in the viscosity of the digesta of the feed by the cocktail showed an effective degradation of the soluble fraction of xylan. Analysis of the xylan degradation products from broiler feeds by the xylanase cocktail showed that xylotriose and xylopentaose were the major xylooligosaccharides (XOS) produced. In vitro evaluation of the prebiotic potential of these XOS showed that they improved the growth of the beneficial bacteria Streptococcus thermophilus and Lactobacillus bulgaricus. The antibacterial activity of broths from XOS-supplemented probiotic cultures showed a suppressive effect on the growth of the extraintestinal infectious bacterium Klebsiella pneumoniae. Supplementing the xylanase cocktail in cereal animal feeds attenuated xylan's antinutritional effects by reducing digesta viscosity and releasing entrapped nutrients. Furthermore, the production of prebiotic XOS promoted the growth of beneficial bacteria while inhibiting the growth of pathogens. Based on these effects of the xylanase cocktail on the feed, improved growth performance and better feed conversion can potentially be achieved during poultry rearing.

Clostridium butyricum and carbohydrate active enzymes contribute to the reduced fat deposition in pigs.

Pig gastrointestinal tracts harbor a heterogeneous and dynamic ecosystem populated with trillions of microbes, enhancing the ability of the host to harvest energy from dietary carbohydrates and contributing to host adipogenesis and fatness. However, the microbial community structure and related mechanisms responsible for the differences between the fatty phenotypes and the lean phenotypes of the pigs remained to be comprehensively elucidated. Herein, we first found significant differences in microbial composition and potential functional capacity among different gut locations in Jinhua pigs with distinct fatness phenotypes. Second, we identified that Jinhua pigs with lower fatness exhibited higher levels of short-chain fatty acids in the colon, highlighting their enhanced carbohydrate fermentation capacity. Third, we explored the differences in expressed carbohydrate-active enzyme (CAZyme) in pigs, indicating their involvement in modulating fat storage. Notably, Clostridium butyricummight be a representative bacterial species from Jinhua pigs with lower fatness, and a significantly higher percentage of its genome was dedicated to CAZyme glycoside hydrolase family 13 (GH13). Finally, a subsequent mouse intervention study substantiated the beneficial effects of C. butyricum isolated from experimental pigs, suggesting that it may possess characteristics that promote the utilization of carbohydrates and hinder fat accumulation. Remarkably, when Jinhua pigs were administered C. butyricum, similar alterations in the gut microbiome and host fatness traits were observed, further supporting the potential role of C. butyricum in modulating fatness. Taken together, our findings reveal previously overlooked links between C. butyricum and CAZyme function, providing insight into the basic mechanisms that connect gut microbiome functions to host fatness.

Genome-wide identification and expression analysis of the glycosyl hydrolase family 1 genes in Medicago sativa revealed their potential roles in response to multiple abiotic stresses

Glycoside hydrolase family 1 (GH1) β-glucosidases (BGLUs), are encoded by a large number of genes, which participate in the development and stress response of plants, particularly under biotic and abiotic stresses through the activation of phytohormones. However, there are few studies systematically analyzing stress or hormone-responsive BGLU genes in alfalfa. In this study, a total of 179 BGLU genes of the glycoside hydrolase family 1 were identified in the genome of alfalfa, and then were classified into five distinct clusters. Sequence alignments revealed several conserved and unique motifs among these MsBGLU proteins. Many cis-acting elements related to abiotic stresses and phytohormones were identified in the promoter of some MsBGLUs. Moreover, RNA-seq and RT-qPCR analyses showed that these MsBGLU genes exhibited distinct expression patterns in response to different abiotic stress and hormonal treatments. In summary, this study suggests that MsBGLU genes play crucial roles in response to various abiotic stresses and hormonal responses, and provides candidate genes for stress tolerance breeding in alfalfa.

Identification and analysis of a unique group of glycoside hydrolase family 188 sequences with an altered sulfonate binding residue.

Sulfoquinovosyldiacylglycerol (SQDG) is a plant sulfolipid that plays a major role in the global sulfur cycle. Bacteria contain sulfoglycolytic pathways that are responsible for metabolizing SQDG which requires initial delipidation by a sulfolipase and sulfoquinovosidase (SQase). Recently, a new group of SQases was discovered and have been categorized in a separate glycoside hydrolase family (GH188). Here we have identified a subset of GH188s with an altered sulfonate binding residue. We found that these GH188s have a distinct dimer interface and are found in unique gene clusters that may represent new sulfoglycolytic pathways. Further investigation into these enzymes could broaden our understanding of this new glycoside hydrolase family and uncover diverse sulfoglycolytic pathways.

HvBGlu3, a GH1 β-glucosidase enzyme gene, negatively influences β-glucan content in barley grains.

HvBGlu3, a β-glucosidase enzyme gene, negatively influences β-glucan content in barley grains by mediating starch and sucrose metabolism in developing grains. Barley grains are rich in β-glucan, an important factor affecting end-use quality. Previously, we identified several stable marker-trait associations (MTAs) and novel candidate genes associated with β-glucan content in barley grains using GWAS (Genome Wide Association Study) analysis. The gene HORVU3Hr1G096910, encoding β-glucosidase 3, named HvBGlu3, is found to be associated with β-glucan content in barley grains. In this study, conserved domain analysis suggested that HvBGlu3 belongs to glycoside hydrolase family 1 (GH1). Gene knockout assay revealed that HvBGlu3 negatively influenced β-glucan content in barley grains. Transcriptome analysis of developing grains of hvbglu3 mutant and the wild type indicated that the knockout of the gene led to the increased expression level of genes involved in starch and sucrose metabolism. Glucose metabolism analysis showed that the contents of many sugars in developing grains were significantly changed in hvbglu3 mutants. In conclusion, HvBGlu3 modulates β-glucan content in barley grains by mediating starch and sucrose metabolism in developing grains. The obtained results may be useful for breeders to breed elite barley cultivars for food use by screening barley lines with loss of function of HvBGlu3 in barley breeding.

Purification and Characterization of α-Mannosidase from Onion, Allium cepa.

α-Mannosidase (ALMAN) extracted from onion (Allium cepa) was purified by column chromatography such as hydrophobic and gel filtration. ALMAN is an acidic α-mannosidase that exhibits maximum activity against pNP-α-Man at pH 4.0-5.0 at 50°C. Amino acid sequence analysis of ALMAN was consistent with α-mannosidase deduced from Allium cepa transcriptome analysis. The gene alman was amplified by PCR using mRNA extracted from onions, and a full-length gene of 3,054 bp encoding a protein of 1,018 amino acid residues was revealed. ALMAN is classified as Glycoside Hydrolase Family (GH) 38 and showed homology with other plant-derived α-mannosidases such as tomato and hot pepper.

Characterization of a novel cold-adapted GH1 β-glucosidase from Psychrobacillus glaciei and its application in the hydrolysis of soybean isoflavone glycosides

The novel β-glucosidase gene (pgbgl1) of glycoside hydrolase (GH) family 1 from the psychrotrophic bacterium Psychrobacillus glaciei sp. PB01 was successfully expressed in Escherichia coli BL21 (DE3). The deduced PgBgl1 contained 447 amino acid residues with a calculated molecular mass of 51.4 kDa. PgBgl1 showed its maximum activity at pH 7.0 and 40 °C, and still retained over 10% activity at 0 °C, suggesting that the recombinant PgBgl1 is a cold-adapted enzyme. The substrate specificity, Km, Vmax, and Kcat/Km for the p-Nitrophenyl-β-D-glucopyranoside (pNPG) as the substrate were 1063.89 U/mg, 0.36 mM, 1208.31 U/mg and 3871.92/s, respectively. Furthermore, PgBgl1 demonstrated remarkable stimulation of monosaccharides such as glucose, xylose, and galactose, as well as NaCl. PgBgl1 also demonstrated a high capacity to convert the primary soybean isoflavone glycosides (daidzin, genistin, and glycitin) into their respective aglycones. Overall, PgBgl1 exhibited high catalytic activity towards aryl glycosides, suggesting promising application prospects in the food, animal feed, and pharmaceutical industries.

In vitro fermentation reveals an interplay relationship between oat β-glucan and human gut Bacteroides and their potential role in regulating gut cytokines.

Dietary oat β-glucan regulates the gut microbial composition and structure; however, the interplay relationship between oat β-glucan and the gut microbiota is unclear. In this study, we aim to investigate the interaction between oat β-glucan and human gut Bacteroides, a versatile carbohydrate utilizer, and explore the effect of their interaction on gut immunity homeostasis. The results of in vitro fermentation showed that oat β-glucan significantly increased the abundance of gut Bacteroides at the genus level. Then, Bacteroides strains were isolated from human gut microbiota and 9 strains of Bacteroides could grow on oat β-glucan and degrade oat β-glucan to reducing sugars. Notably, strains Bacteroides xylanisolvens Bac02 and Bacteroides koreensis Bac08 possessed the strongest degradation capacity towards oat β-glucan. Genome analysis and functional annotations suggested that B. xylanisolvens Bac02 and B. koreensis Bac08 contained abundant genes encoding glycoside hydrolases family 3 (GH3) and GH16, which might be responsible for β-glucan degradation. Moreover, cell experiments revealed that the metabolites from oat β-glucan fermentation by these 9 strains of Bacteroides could regulate the polarization of macrophages and maintain gut immunity homeostasis. Our study provides a novel insight into research on the interplay between dietary compounds and the gut microbiota.

Gossypium hirsutum gene of unknown function Gohir.A02G131900 encodes a potential plant-specific, dual-domain exo-1,3-β-glucosidase.

A gene of unknown function, Gohir.A02G131900.1, identified in Gossypium hirsutum was studied using computational sequence and structure bioinformatic tools. The encoded protein GhGH5BG-A0A1U8NW40 (UniProt A0A1U8NW40) is predicted to be secreted and localized to the cell wall. Homology and conserved residues indicate it belongs to a plant-specific subgroup of the glycoside hydrolase family 5 and likely has exo-1,3-β-glucosidase activity. This subgroup is unique in containing a fascin-like domain which may have evolved a unique glucan binding site of interest for further research.

Reassigning the role of a mesophilic xylan hydrolysing family GH43 β-xylosidase from Bacteroides ovatus, BoExXyl43A as exo-β-1,4-xylosidase

The recombinant 40 kDa BoExXyl43A glycoside hydrolase family 43 (GH43) from bacterium Bacteroides ovatus exhibited highest specific activity (U/mg) against corn cob xylan (136.8), followed by Beechwood xylan (81.1), Carbosynth xylan (69.3), 4-O-D-methylglucuronoxylan (61.4) and Birchwood xylan (59.9). BoExXyl43A demonstrated optimal performance at 37 °C and pH 7.6 with Vmax and Km of 141.8 U/mg and 4.0 mg/mL as well as 64.1 U/mg and 6.0 mg/mL against corn cob and Birchwood xylan, respectively. The activity of BoExXyl43A increased by 48 % by addition of 10 mM Ca2+ ions, while 1 mM EDTA or 1 mM EGTA decreased its activity by 100 % or 42.5 %, respectively, highlighting its calcium-ion dependence. Thin-layer chromatography (TLC) analysis of BoExXyl43A hydrolysates of Birchwood and Beechwood xylan as well as that of various xylooligosaccharides (DP2-DP9) from corn cob xylan showed the release of D-xylose, identifying it as an exo-β-1,4-xylosidase/exo-β-1,4-xylanase (EC 3.2.1.-/3.2.1.37). Moreover, the time-dependent TLC analysis of xylobiose hydrolysis showed release of D-xylose units, confirming its β-xylosidase activity. BoExXyl43A also exhibited exo-1,4-β-xylosidase activity on Larchwood and Carbosynth xylans. Notably, it released D-xylose from α-L-Araf2-xylotriose demonstrating its activity against decorated xylooligosaccharides. BoExXyl43A's exo-1,4-β-xylosidase and residual β-xylosidase activity on xylan and xylobiose, respectively, could potentially enhance xylan saccharification efficiency in bioethanol-based refineries. The molecular modeling showed that BoExXyl43A has 5-bladed β-propeller structure with a very shallow active-site having −1, +1 and + 2 subsites, which could accommodate three D-xylose units of longer xylan like xylododecaose thus supporting its exoxylosidase activity.

CANDy: Automated analysis of domain architectures in carbohydrate-active enzymes.

Carbohydrate-active enzymes (CAZymes) can be found in all domains of life and play a crucial role in metabolic and physiological processes. CAZymes often possess a modular structure, comprising not only catalytic domains but also associated domains such as carbohydrate-binding modules (CBMs) and linker domains. By exploring the modular diversity of CAZy families, catalysts with novel properties can be discovered and further insight in their biological functions and evolutionary relationships can be obtained. Here we present the carbohydrate-active enzyme domain analysis tool (CANDy), an assembly of several novel scripts, tools and databases that allows users to analyze the domain architecture of all protein sequences in a given CAZy family. CANDy's usability is shown on glycoside hydrolase family 48, a small yet underexplored family containing multi-domain enzymes. Our analysis reveals the existence of 35 distinct domain assemblies, including eight known architectures, with the remaining assemblies awaiting characterization. Moreover, we substantiate the occurrence of horizontal gene transfer from prokaryotes to insect orthologs and provide evidence for the subsequent removal of auxiliary domains, likely through a gene fission event. CANDy is available at https://github.com/PyEED/CANDy.

Novel β-galactosidase activity and first crystal structure of Glycoside Hydrolase family 154

Polysaccharide Utilization Loci (PULs) are physically linked gene clusters conserved in the Gram-negative phylum of Bacteroidota and are valuable sources for Carbohydrate Active enZyme (CAZyme) discovery. This study focuses on BD-β-Gal, an enzyme encoded in a metagenomic PUL and member of the Glycoside Hydrolase family 154 (GH154). BD-β-Gal showed exo-β-galactosidase activity with regiopreference for hydrolyzing β-d-(1,6) glycosidic linkages. Notably, it exhibited a preference for d-glucopyranosyl (d-Glcp) over d-galactopyranosyl (d-Galp) and d-fructofuranosyl (d-Fruf) at the reducing end of the investigated disaccharides. In addition, we determined the high resolution crystal structure of BD-β-Gal, thus providing the first structural characterization of a GH154 enzyme. Surprisingly, this revealed an (α/α)6 topology, which has not been observed before for β-galactosidases. BD-β-Gal displayed low structural homology with characterized CAZymes, but conservation analysis suggested that the active site was located in a central cavity, with conserved E73, R252, and D253 as putative catalytic residues. Interestingly, BD-β-Gal has a tetrameric structure and a flexible loop from a neighboring protomer may contribute to its reaction specificity. Finally, we showed that the founding member of GH154, BT3677 from Bacteroides thetaiotaomicron, described as β-glucuronidase, displayed exo-β-galactosidase activity like BD-β-Gal but lacked a tetrameric structure.

Transformation of corncob into high-value xylooligosaccharides using glycoside hydrolase families 10 and 11 xylanases from Trichoderma asperellum ND-1

Effective production of xylooligosaccharides (XOS) with lower proportion of xylose entails unique and robust xylanases. In this study, two novel xylanases from Trichoderma asperellum ND-1 belonging to glycoside hydrolase families 10 (XynTR10) and 11 (XynTR11) were over-expressed in Komagataella phaffii X-33 and characterized to be robust enzymes with high halotolerance and ethanol tolerant. Both enzymes displayed strict substrate specificity towards beechwood xylan and wheat arabinoxylan. (Glu153/Glu258) and (Glu161/Glu252) were key catalytic sites for XynTR10 and XynTR11. Notably, XynTR11 could rapidly degrade xylan/XOS into xylobiose without xylose via transglycosylation. Direct degradation of corncob using XynTR10 and XynTR111 displayed that while XynTR10 yielded 77% xylobiose and 25% xylose, XynTR11 yielded much less xylose (11%) and comparable amounts of xylobiose (63%). XynTR10 or XynTR111 has great potential as a catalyst for bioconversion of xylan-containing agricultural waste into high-value products (biofuel or XOS), which is of significant benefit for the economy and environment.