GH Family Research Articles

Competitive inhibition of family GH11 Aspergillus fumigatus endo-xylanase A by Oryza sativa xylanase inhibitor protein: Investigating key interface residues and non-covalent interactions

Glycoside hydrolase (GH) family 11 Aspergillus fumigatus endo-xylanase A (AfXylA11) was recombinantly expressed in Pichia pastoris X33 and secreted into culture medium. Hydrolysis of beechwood xylan by recombinant AfXylA11 (reAfXylA11) predominantly generated xylooligosaccharides (XOs), chiefly comprising xylotriose (X2) and xylotetraose (X3). These hydrolysates demonstrated significant antioxidant capabilities. Competitive inhibition of reAfXylA11 was observed in the presence of the Oryza sativa xylanase inhibitor protein (OsXIP), with an inhibition constant (Ki) of 120.98 nM. Molecular dynamics (MD) simulations and conformational analyses of the AfXylA11-OsXIP complex revealed that the extended loop (Lα4β5, K150-V158) of OsXIP penetrated the active groove of AfXylA11, thereby obstructing xylan from interacting with catalytic sites. The R153 at the bottom of the U-shaped loop, was found to form durable hydrogen bonds with E197 (acid/base catalyst) and Y97 of AfXylA11. Additionally, OsXIP established stable interactions with the “cord” and “thumb” regions of AfXylA11 during MD simulations. The binding free energy of the AfXylA11-OsXIP complex was determined to be -74.3 ± 7.6 kcal/mol, with electrostatic interactions and van der Waals forces being the predominant contributors to enzyme-inhibitor interaction. Further MD simulations demonstrated that the mutation of Y116A or S120A in the “cord” region of AfXylA11 significantly reduced its binding affinity to OsXIP by weakening interface residue interactions. Delving into the conformational dynamics of the AfXylA11-OsXIP complex yields insights into the molecular mechanisms dictating their competitive inhibition. This investigation offers useful basis for engineering mutant xylanases that resist to the inhibitory protein yet retain high catalytic efficacy.

An integrated microbiome- and metabolome-genome-wide association study reveals the role of heritable ruminal microbial carbohydrate metabolism in lactation performance in Holstein dairy cows.

Despite the growing number of studies investigating the connection between host genetics and the rumen microbiota, there remains a dearth of systematic research exploring the composition, function, and metabolic traits of highly heritable rumen microbiota influenced by host genetics. Furthermore, the impact of these highly heritable subsets on lactation performance in cows remains unknown. To address this gap, we collected and analyzed whole-genome resequencing data, rumen metagenomes, rumen metabolomes and short-chain fatty acids (SCFAs) content, and lactation performance phenotypes from a cohort of 304 dairy cows. The results indicated that the proportions of highly heritable subsets (h2 ≥ 0.2) of the rumen microbial composition (55%), function (39% KEGG and 28% CAZy), and metabolites (18%) decreased sequentially. Moreover, the highly heritable microbes can increase energy-corrected milk (ECM) production by reducing the rumen acetate/propionate ratio, according to the structural equation model (SEM) analysis (CFI = 0.898). Furthermore, the highly heritable enzymes involved in the SCFA synthesis metabolic pathway can promote the synthesis of propionate and inhibit the acetate synthesis. Next, the same significant SNP variants were used to integrate information from genome-wide association studies (GWASs), microbiome-GWASs, metabolome-GWASs, and microbiome-wide association studies (mWASs). The identified single nucleotide polymorphisms (SNPs) of rs43470227 and rs43472732 on SLC30A9 (Zn2+ transport) (P < 0.05/nSNPs) can affect the abundance of rumen microbes such as Prevotella_sp., Prevotella_sp._E15-22, Prevotella_sp._E13-27, which have the oligosaccharide-degradation enzymes genes, including the GH10, GH13, GH43, GH95, and GH115 families. The identified SNPs of chr25:11,177 on 5s_rRNA (small ribosomal RNA) (P < 0.05/nSNPs) were linked to ECM, the abundance alteration of Pseudobutyrivibrio_sp. (a genus that was also showed to be linked to the ECM production via the mWASs analysis), GH24 (lysozyme), and 9,10,13-TriHOME (linoleic acid metabolism). Moreover, ECM, and the abundances of Pseudobutyrivibrio sp., GH24, and 9,10,13-TRIHOME were significantly greater in the GG genotype than in the AG genotype at chr25:11,177 (P < 0.05). By further the SEM analysis, GH24 was positively correlated with Pseudobutyrivibrio sp., which was positively correlated with 9,10,13-triHOME and subsequently positively correlated with ECM (CFI = 0.942). Our comprehensive study revealed the distinct heritability patterns of rumen microbial composition, function, and metabolism. Additionally, we shed light on the influence of host SNP variants on the rumen microbes with carbohydrate metabolism and their subsequent effects on lactation performance. Collectively, these findings offer compelling evidence for the host-microbe interactions, wherein cows actively modulate their rumen microbiota through SNP variants to regulate their own lactation performance. Video Abstract.

Efficient Biotransformation of Icariin to Baohuoside I Using Two Novel GH1 β-Glucosidases

Epimedium Folium (EF) is a traditional Chinese herbal medicine, and its primary bioactive ingredients, such as icariin, are flavonoid glycosides. A rare EF flavonoid, baohuoside I, exhibits superior bioactivities and enhanced bioavailability compared to its metabolic precursor icariin. The biotransformation of icariin to baohuoside I can be effectively and specifically achieved by β-glucosidases. In this study, 33 candidate full-length β-glucosidase genes were screened from a previously built carbohydrate active enzyme (CAZyme) gene dataset derived from cow fecal microbiota. Thirteen of them exhibited β-glucosidase activity, with DCF-bgl-26 and DCF-bgl-27 showing relatively high expression levels and β-glucosidase activity. The maximum β-glucosidase activity of DCF-bgl-26 and DCF-bgl-27 was achieved at 45 °C and pH 6.0, with DCF-bgl-26 demonstrating better thermostability and pH tolerance compared to DCF-bgl-27. The activities of DCF-bgl-26 and DCF-bgl-27 were 123.2 U/mg protein and 157.9 U/mg protein, respectively, both of which are higher than those of many bacterial β-glucosidases. Structure analysis suggested that both β-glucosidases possess canonical (β/α)8-TIM barrel fold structure of GH1 family β-glucosidases. Thin-layer chromatography results showed that both enzymes could efficiently convert icariin to baohuoside I in 30 min, indicating they have potential application in the production of high value rare baohuoside I.

Enzymatic Production of Chitooligosaccharide Using a GH Family 46 Chitosanase from Paenibacillus elgii and Its Antioxidant Activity

Chitooligosaccharide (COS), a natural antioxidant, is a hydrolysis product of chitosan created using enzymatic or chemical methods. COS has received considerable attention recently, making its efficient bioproduction of great value. This study investigated the optimal conditions for the enzymatic method using a GH family 46 chitosanase from Paenibacillus elgii TKU051 to prepare COS based on the response surface methodology (RSM). The results showed optimal values for chitosan hydrolysis, such as a pH of 5.5, an incubation temperature of 58.3 °C, an [E]/[S] ratio of 118.494 (U/g), and an incubation time of 6.821 h. Under the optimal conditions, the highest reducing sugar level (per substrate, w/w) of the chitosan hydrolysis process that could be reached was 690.587 mg/g. The composition of the obtained COS was analyzed using the thin-layer chromatography (TLC) method, yielding (GlcN)2 and (GlcN)3 as the products. The ascorbic acid equivalent antioxidant capacity (AEAC) of the obtained COS was found to be 1246 mg/100 g (via a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging assay) and 3673 mg/100 g (via an ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical-scavenging assay). This green and efficient bioproduction method may possess excellent potential for application in bioactive COS preparation.

Crystal structures of Aspergillus oryzae exo-β-(1,3)-glucanase reveal insights into oligosaccharide binding, recognition, and hydrolysis.

Exo-β-(1,3)-glucanases are promising enzymes for use in the biofuel industry as they hydrolyse sugars such as laminarin, a major constituent of the algal cell wall. This study reports structural and biochemical characterizations of Aspergillus oryzae exo-β-(1,3)-glucanase (AoBgl) belonging to the GH5 family. Purified AoBgl hydrolyses β-(1,3)-glycosidic linkages of the oligosaccharide laminaritriose and the polysaccharide laminarin effectively. We have determined three high-resolution structures of AoBgl: (a) the apo form at 1.75 Å, (b) the complexed form with bound cellobiose at 1.73 Å and (c) the glucose-bound form at 1.20 Å. The crystal structures, molecular dynamics simulation studies and site-directed mutagenesis reveal the mode of substrate binding and interactions at the active site. The results also indicate that AoBgl effectively hydrolyses trisaccharides and higher oligosaccharides. The findings from our structural and biochemical studies would aid in rational engineering efforts to generate superior AoBgl variants and similar GH5 enzymes for their industrial use.

The Genome-Wide Identification, Characterization, and Expression Patterns of the Auxin-Responsive PbGH3 Gene Family Reveal Its Crucial Role in Organ Development

The regulation of vital plant activities by hormones is governed by a family of macromolecular peptides referred to as GH3 genes. This work analyzed the expression patterns of GH3 family genes in pear tissues using transcriptome data and bioinformatics analysis. In the Bai Li pear genome, a total of 18 PbGH3 genes were identified. Comparative evolutionary studies have shown a strong association between PbGH3 and AtGH3 class I and class II proteins. The role of PbGH3 genes in growth activities and hormone regulation was revealed using gene ontology (GO) and promoter region analysis. In addition, although certain PbGH3 genes exhibited tissue-specific expression in sepals, the majority had a ubiquitous expression across all tissues. Bioinformatics and expression studies suggest that the GH3 gene family in pears may have a role in controlling the abscission of the fruit’s sepals. This work sheds light on the pear fruit sepal shedding process and may inspire further research.

Selective transformation of crocin-1 to crocetin-glucosyl esters by β-glucosidase (Lf18920) from Leifsonia sp. ZF2019: Insights from molecular docking and point mutations

Crocetin di/mono-glucosyl esters (crocin-4 and crocin-5) are rarely distributed in nature, limiting their potential applications in the food and pharmaceutical industries. In the present study, a novel GH3 family β-glucosidase Lf18920 was identified from Leifsonia sp. ZF2019, which selectively hydrolyzed crocin-1 (crocetin di-gentiobiosyl ester) to crocin-5 and crocin-4, but not to its aglycone, crocetin. Under the optimal condition of 40 °C and pH 6.0 for 120 min, Lf18920 almost completely hydrolyzed crocin-1, yielding 73.50±5.66 % crocin-4 and 16.19±1.38 % crocin-5. Molecular docking and point mutation studies revealed that Lf18920 formed a narrow binding channel that facilitated crocin-1 binding. Five single amino acid variants (D50A, D53A, W274A, G420A, and Q421A) were constructed, all of which showed reduced hydrolytic activity. Mutations at D50 and D53, located distal to the active site, increased binding energy and decreased hydrolytic activity, while mutations at W274, G420, and Q421, proximal to the active site, disrupted hydrolytic function. These findings suggest that the narrow binding channel and specific enzyme-substrate interactions are crucial for Lf18920’s selective hydrolytic activity. Overall, this study is the first to report a β-glucosidase capable of selectively transforming crocin-1 to crocetin di/mono-glucosyl esters, offering potential for synthesizing crocin-4 and crocin-5.

Probing the function of C-terminal region of recombinant α-amylase BmaN1 from Bacillus megaterium NL3.

The α-amylase BmaN1 from Bacillus megaterium NL3 is a member of GH13_45 subfamily that has a conserved C-terminal region of approximately 30 residues. This region features a motif of five aromatic amino acids predicted to play a role in starch binding. This study aimed to unravel the role of the C-terminal region in starch hydrolysis. The full-length and C-terminally truncated forms of BmaN1 (BmaN1∆C) were expressed in Escherichia coli ArcticExpress (DE3), resulting in proteins with molecular weights of 56 kDa and 49 kDa, respectively. They exhibited comparable enzymatic activity in the hydrolysis of soluble starch, displaying versatility across a wide range of pH values, temperatures, and NaCl concentrations. BmaN1 and BmaN1∆C activities were inhibited by acarbose and were reduced by SDS and EDTA. In terms of binding and degrading the starch granules, BmaN1∆C showed lower affinity and activity in comparison to BmaN1. Our study indicates that the C-terminal region of BmaN1 significantly enhances its binding affinity and degrading the raw starches.IMPORTANCEα-Amylase (EC 3.2.1.1) stands as an endo-acting enzyme, essential for catalyzing the hydrolysis of α-1,4 glycosidic bonds within starch molecules. The relevance of α-amylases in biotechnological applications is substantial, constituting approximately 30% of the global enzyme market. Among these enzymes, BmaN1 was the first α-amylase identified to possess distinct catalytic residues within the GH13 family. BmaN1 from B. megaterium NL3 belongs to the GH13_45 subfamily. This subfamily is characterized by a conserved C-terminal region consisting of approximately 30 residues that contains a motif of five aromatic residues predicted to be involved in starch binding. Our study shows that the C-terminal effectively contributes to binding and degrading the raw starch granules. This pioneering research on BmaN1 expands our understanding of α-amylases and holds promise for innovative biotechnological advancements.

Structural studies of β-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus

β-Glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus (Bgl1) has been denoted as having an attractive catalytic profile for various industrial applications. Bgl1 catalyses the final step of in the decomposition of cellulose, an unbranched glucose polymer that has attracted the attention of researchers in recent years as it is the most abundant renewable source of reduced carbon in the biosphere. With the aim of enhancing the thermostability of Bgl1 for a broad spectrum of biotechnological processes, it has been subjected to structural studies. Crystal structures of Bgl1 and its complex with glucose were determined at 1.47 and 1.95 Å resolution, respectively. Bgl1 is a member of glycosyl hydrolase family 1 (GH1 superfamily, EC 3.2.1.21) and the results showed that the 3D structure of Bgl1 follows the overall architecture of the GH1 family, with a classical (β/α)8 TIM-barrel fold. Comparisons of Bgl1 with sequence or structural homologues of β-glucosidase reveal quite similar structures but also unique structural features in Bgl1 with plausible functional roles.

Comparative Genomics of Carbohydrate Utilization in Bacteria of the Family Sphaerochaetaceae: Evolutionary Origin of the Genes Encoding Galacturonidase and Unsaturated Rhamnogalacturonyl Hydrolase

Abstract A comparative analysis of carbohydrate degradation proteins encoded in the currently available genomic sequences of bacteria of the family Sphaerochaetaceae, namely Sphaerochaeta associata GLS2T, S. globosa BuddyT, S. pleomorpha GrapesT, S. halotolerans 4-11T, S. halotolerans 585, Sphaerochaeta sp. S2, Sphaerochaeta sp. PS, and Parasphaerochaetacoccoides SPN1T was carried out. The genomes of Sphaerochaeta spp. encode a medium-sized and diverse set of proteins potentially involved in the degradation of different classes of carbohydrates, mainly oligosaccharides. All studied genomes encode glycoside hydrolases of the GH1, GH2, GH3, GH4, GH13, GH20, GH28, GH36, GH43, GH57, GH63, GH77 and GH105 families, as well as carbohydrate esterases of the CE8 and CE9 families. All studied bacteria, with the exception of P. coccoides SPN1T, have many genes of the GH31 family proteins. The studied members of Sphaerochaetaceae do not have genes coding for endo-β-acetylmuramidase (lysozyme) of the GH23 family, which participates in peptidoglycan turnover. However, the genomes of S. associata, S. globosa, Sphaerochaeta sp. PS, and S. pleomorpha contain exo-β-acetylmuramidase genes (GH171 family). A significant part of the genes encoding carbohydrate degradation enzymes have the closest homologues among representatives of the phyla Bacillota, Bacteroidota, and Pseudomonadota. The genomes of the studied bacteria encode proteins that could potentially be involved in the degradation of pectin. The ability of the Sphaerochaetaceae members to use pectin for growth, as well as the evolutionary origin of the genes encoding potential α-galacturonidase (GH4 family) and unsaturated glucuronyl/rhamnogalacturonyl hydrolase (GH105 family), which participate in the degradation of pectin components, were studied.

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

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

The family GH126 – its relatedness to and differentiation from GH8 and GH48 including the intermediary sequences

The family GH126, best represented by the amylolytic enzyme CPF_2247 from Clostridium perfringens, exclusively includes proteins of bacterial origin, covering predominantly the phylum Bacillota. Although all the members should adopt the catalytic (α/α)6-barrel domain, neither the catalytic machinery nor the reaction mechanism has been determined as yet. The limited biochemical characterization, especially some uncertainty concerning the endo-vs exo-mode of action and retaining vs inverting mechanism, combined with the sequence-structural resemblance of GH126 members to inverting β-glucanases from families GH8 and GH48 (the clan GH-M), may lead to misclassification of putative proteins. The present study was therefore designed in an effort to identify unique sequence-structural features that would definitively differentiate family GH126 from both GH8 and GH48. To achieve this, a sequence logo, representing the seven GH126 conserved sequence regions established previously, was created using 1665 GH126 sequences. The logo was compared with GH8 and GH48 logos based on, respectively, 86 and 63 selected enzymes. An invariant tyrosine residue in CSR-6 was identified as a reliable marker for the family GH126. In addition, protein BLAST searches identified 87 putative proteins that taxonomically extend the family GH126 not only outside Bacillota, but also outside Bacteria to include representatives of archaeons and eukaryotes (fungi). Evolutionary analysis of the 434 sequences representing all the three families GH126 with GH8 and GH48, including the BLAST hits, revealed an intermediate group. In the future, it may either define a new GH family closely related to GH126, or at least constitute a future GH126 subfamily.

Developing endophytic Penicillium oxalicum as a source of lignocellulolytic enzymes for enhanced hydrolysis of biorefinery relevant pretreated rice straw.

Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1M NaCl). The comparative QTOF-LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), β-glucosidase (β-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies.

Genes of Streptomyces globisporus 1912-4Crt Encoding Chitin Catabolism Enzymes

Polysaccharide chitin is one of the most common biopolymers in nature. Chitin (when used as the sole source of energy, carbon, and nitrogen) has been shown to be a substrate sufficient to enable the growth and synthesis of secondary metabolites by the S. griseus NCIMB 8136 strain and some other streptomycetes. The species Streptomyces globisporus and S. griseus belong to the same lower hierarchical taxon (S. griseus clade). The aim was to find in the S. globisporus 1912-4Crt genome genes encoding proteins that are necessary for chitin fermentation and transmembrane transport of resulting products. Methods. The object of the study was a sequence of the S. globisporus 1912-4Crt genome (reference NZ_QWFA01000000.1, GenBank) on the server of NCBI (The National Center for Biotechnology Information). Streptomycete S. globisporus 1912 and its variants are producers of antibiotic landomycin E and carotenoids. Search for and analysis of nucleotide and amino acid sequences were performed using programs BLAST (Basic Local Alignment Search Tool) on the aforementioned server. Results. A set of genes that determine enzymes of chitin catabolism and Ngc-transporter proteins were identified in the S. globisporus 1912-4Crt genome. Genes encoding 10 endo-acting chitinases (from the GH18 and GH19 families) and 2 exo-acting hydrolases (GH20) were identified in the S. globisporus 1912-4Crt sequence. Several genes determining deacetylases that deacetylate both chitin and oligosaccharides were found in the genome of the strain. One gene that determines endo-acting chitosanase from the GH75 family was discovered there. The ability of the wild-type strain S. globisporus 1912 and a number of its variants (including S. globisporus 1912-4Crt) to ferment chitin was proven in vitro. Conclusions. A set of genes sufficient for chitin assimilation was established in the S. globisporus 1912-4Crt genome sequence. Streptomycetes from different clades (for example, strains of S. coelicolor (S. albidoflavus group) and S. griseus, S. globisporus (S. griseus group)) containing different complexes of chitinolytic enzymes could be suggested.

Advancements in the Heterologous Expression of Sucrose Phosphorylase and Its Molecular Modification for the Synthesis of Glycosylated Products.

Sucrose phosphorylase (SPase), a member of the glycoside hydrolase GH13 family, possesses the ability to catalyze the hydrolysis of sucrose to generate α-glucose-1-phosphate and can also glycosylate diverse substrates, showcasing a wide substrate specificity. This enzyme has found extensive utility in the fields of food, medicine, and cosmetics, and has garnered significant attention as a focal point of research in transglycosylation enzymes. Nevertheless, SPase encounters numerous obstacles in industrial settings, including low enzyme yield, inadequate thermal stability, mixed regioselectivity, and limited transglycosylation activity. In-depth exploration of efficient expression strategies and molecular modifications based on the crystal structure and functional information of SPase is now a critical research priority. This paper systematically reviews the source microorganisms, crystal structure, and catalytic mechanism of SPase, summarizes diverse heterologous expression systems based on expression hosts and vectors, and examines the application and molecular modification progress of SPase in synthesizing typical glycosylated products. Additionally, it anticipates the broad application prospects of SPase in industrial production and related research fields, laying the groundwork for its engineering modification and industrial application.

Expression in Pichia pastoris of Thermostable Endo-1,4-β-xylanase from the Actinobacterium Nocardiopsis halotolerans: Properties and Use for Saccharification of Xylan-Containing Products.

A gene encoding a polysaccharide-degrading enzyme was cloned from the genome of the bacterium Nocardiopsis halotolerans. Analysis of the amino acid sequence of the protein showed the presence of the catalytic domain of the endo-1,4-β-xylanases of the GH11 family. The gene was amplified by PCR and ligated into the pPic9m vector. A recombinant producer based on Pichia pastoria was obtained. The production of the enzyme, which we called NhX1, was carried out in a 10 L fermenter. Enzyme production was 10.4 g/L with an activity of 927 U/mL. Purification of NhX1 was carried out using Ni-NTA affinity chromatography. The purified enzyme catalyzed the hydrolysis of xylan but not other polysaccharides. Endo-1,4-β-xylanase NhX1 showed maximum activity and stability at pH 6.0-7.0. The enzyme showed high thermal stability, remaining active at 90 °C for 20 min. With beechwood xylan, the enzyme showed Km 2.16 mg/mL and Vmax 96.3 U/mg. The products of xylan hydrolysis under the action of NhX1 were xylobiose, xylotriose, xylopentaose, and xylohexaose. Endo-1,4-β-xylanase NhX1 effectively saccharified xylan-containing products used for the production of animal feed. The xylanase described herein is a thermostable enzyme with biotechnological potential produced in large quantities by P. pastoria.

Function Analysis of a Maize Endo-1,4-β-xylanase Gene ZmHSL in Response to High-Temperature Stress.

Rising temperature is a major threat to the normal growth and development of maize, resulting in low yield production and quality. The mechanism of maize in response to heat stress remains uncertain. In this study, a maize mutant Zmhsl-1 (heat sensitive leaves) with wilting and curling leaves under high temperatures was identified from maize Zheng 58 (Z58) mutant lines generated by ethyl methanesulfonate (EMS) mutagenesis. The Zmhsl-1 plants were more sensitive to increased temperature than Z58 in the field during growth season. The Zmhsl-1 plants had lower plant height, lower yield, and lower content of photosynthetic pigments. A bulked segregant analysis coupled with whole-genome sequencing (BSA-seq) enabled the identification of the corresponding gene, named ZmHSL, which encodes an endo-β-1,4-xylanase from the GH10 family. The loss-of-function of ZmHSL resulted in reduced lignin content in Zmhsl-1 plants, leading to defects in water transport and more severe leaf wilting with the increase in temperature. RNA-seq analysis revealed that the differentially expressed genes identified between Z58 and Zmhsl-1 plants are mainly related to heat stress-responsive genes and unfolded protein response genes. All these data indicated that ZmHSL plays a key role in lignin synthesis, and its defective mutation causes changes in the cell wall structure and gene expression patterns, which impedes water transport and confers higher sensitivity to high-temperature stress.

Moderately thermostable GH1 β-glucosidases from hyperacidophilic archaeon Cuniculiplasma divulgatum S5.

Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus, and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5-6.0 (CIB12) and 4.5-5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma.

Regional variation and adaptive evolution in Bifidobacterium pseudocatenulatum: Insights into genomic and functional diversity in human gut

Bifidobacterium pseudocatenulatum is a prevalent gut microbe in humans of all ages and plays a crucial role in host health. However, its adaptive evolutionary characteristics remain poorly understood. This study analyzed the genome of 247 B. pseudocatenulatum isolates from Chinese, Vietnamese, Japanese and other region populations using population genomics and functional genomics. Our findings revealed high genetic heterogeneity and regional clustering within B. pseudocatenulatum isolates. Significant differences were observed in genome characteristics, phylogeny, and functional genes. Specifically, Chinese and Vietnamese isolates exhibited a higher abundance of genes involved in the metabolism of plant-derived carbohydrates (GH13, GH43, and GH5 enzyme families), aligning with the predominantly vegetable-, wheat- and fruit-based diets of these populations. Additionally, we found widespread transmission of antibiotic resistance genes (tetO and tetW) through mobile genetic elements, such as genomic islands (GIs), resulting in substantial intra-regional differences. Our findings highlight distinct adaptive evolution in B. pseudocatenulatum driven by gene specialization, possibly in response to regional variations in diet and lifestyle. This study sheds light on bifidobacteria colonization mechanisms in the host gut. ImportanceGut microbiota, as a key link in the gut-brain axis, helps to maintain the health of the organism, among which, Bifidobacterium pseudocatenulatum (B. pseudocatenulatum) is an important constituent member of the gut microbiota, which plays an important role in maintaining the balance of gut microbiota. The probiotic properties of B. pseudocatenulatum have been widely elaborated, and in order to excavate its evolutionary features at the genomic level, here we focused on the genetic background and evolutionary mechanism of the B. pseudocatenulatum genomes isolated from the intestinal tracts of different populations. Ultimately, based on the phylogenetic tree, we found that B. pseudocatenulatum has high genetic diversity and regional clustering phenomenon, in which plant-derived carbohydrate metabolism genes (GH13, GH43, GH5) showed significant regional differences, and this genetic differentiation drove the adaptive evolution, which likely shaped by diet and lifestyle.

Fibrobacter sp. HC4, a newly isolated strain, demonstrates a high cellulolytic activity as revealed by enzymatic measurements and in vitro assay.

Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% of the detected glycoside hydrolases (GHs) were involved in cellulolysis. These cellulases belong to the GH5, GH8, GH9, GH44, GH45, and GH51 families. Both carboxymethyl cellulase and xylanase activities of Fibrobacter sp. HC4 were detected using the Congo red method and were higher than those of F. succinogenes S85, the type strain. The in vitro addition of Fibrobacter sp. HC4 to a fecal microbial ecosystem of horses with large intestinal acidosis significantly enhanced fibrolytic activity as measured by the increase in gas and volatile fatty acids production during the first 48 h. According to this, the pH decreased and the disappearance of dry matter increased at a faster rate with Fibrobacter sp. HC4. Our data suggest a high specialization of the new strain in cellulose degradation. Such a strain could be of interest for future exploitation of its probiotic potential, which needs to be further determined by in vivo studies.IMPORTANCECellulose is the most abundant of plant cell wall fiber and can only be degraded by the large intestine microbiota, resulting in the production of volatile fatty acids that are essential for the host nutrition and health. Consequently, cellulolytic bacteria are of major importance to herbivores. However, these bacteria are challenged by various factors, such as high starch diets, which acidify the ecosystem and reduce their numbers and activity. This can lead to an imbalance in the gut microbiota and digestive problems such as colic, a major cause of mortality in horses. In this work, we characterized a newly isolated cellulolytic strain, Fibrobacter sp. HC4, from the equine intestinal microbiota. Due to its high cellulolytic capacity, reintroduction of this strain into an equine fecal ecosystem stimulates hay fermentation in vitro. Isolating and describing cellulolytic bacteria is a prerequisite for using them as probiotics to restore intestinal balance.