GH Family Research Articles

Unravelling yeast cellulase potential: A computational approach to structural study, cellulolytic activity, and docking

The discovery of highly efficient catalysts capable of enhancing the biomass conversion represents a significant challenge within the biofuel production sector. Cellulolytic enzymes originating from microorganisms are promising catalysts for bioethanol conversion. In this study, we conducted a comparative analysis of cellulase, especially Endo β-1,4 glucanase enzymes, from five different non-conventional yeast species. These enzymes were evaluated in the presence of microcrystalline cellulose as the substrate, and their structural, physicochemical properties and molecular docking were studied using in silico screening techniques. During multiple sequence alignment, we identified conserved catalytic amino residues in yeast cellulases, highlighting the pivotal role of glutamic acid in cellulose hydrolysis. Using InterProScan, we determined that the obtained sequence belonged to the GH5 and GH45 families and domains. The key amino acid residues involved in the interaction were glutamic acid, serine, alanine, arginine, asparagine, and histidine. Notably, the docking score for Candida fabianii was significantly lower (-12.95kcal/mol) than that of the other enzymes, indicating a strong affinity for cellulose. In conclusion, our results underscore the importance of a key amino acid residue in Endo β-1,4 glucanase from Candida fabianii, which may enhance the selectivity of endoglucanase toward cellulose.

β-l-Arabinofurano-cyclitol Aziridines Are Covalent Broad-Spectrum Inhibitors and Activity-Based Probes for Retaining β-l-Arabinofuranosidases.

GH127 and GH146 microorganismal retaining β-l-arabinofuranosidases, expressed by human gut microbiomes, feature an atypical catalytic domain and an unusual mechanism of action. We recently reported that both Bacteroides thetaiotaomicron BtGH146 and Bifidobacterium longum HypBA1 are inhibited by β-l-arabinofuranosyl cyclophellitol epoxide, supporting the action of a zinc-coordinated cysteine as a catalytic nucleophile, where in most retaining GH families, an aspartate or glutamate is employed. This work presents a panel of β-l-arabinofuranosyl cyclophellitol epoxides and aziridines as mechanism-based BtGH146/HypBA1 inhibitors and activity-based probes. The β-l-arabinofuranosyl cyclophellitol aziridines both inhibit and label β-l-arabinofuranosidase efficiently (however with different activities), whereas the epoxide-derived probes favor BtGH146 over HypBA1. These findings are accompanied by X-ray structural analysis of the unmodified β-l-arabinofuranosyl cyclophellitol aziridine in complex with both isozymes, which were shown to react by nucleophilic opening of the aziridine, at the pseudoanomeric carbon, by the active site cysteine nucleophile to form a stable thioether bond. Altogether, our activity-based probes may serve as chemical tools for the detection and identification of low-abundance β-l-arabinofuranosidases in complex biological samples.

Screening, enzyme activity and genomic analysis of Paenibacillus silvae CH2

Mangrove soil is a reliable source for screening cellulose-degrading bacteria due to the high diversity of microbes. To effectively utilize crop straw resources, a cellulolytic bacterium, Paenibacillus silvae strain CH2 was isolated from mangrove soil. We determined the carboxymethyl cellulose (CMC) and filter paper assay (FPA) activities of CH2 at different incubation times, NaCl concentrations, pH and temperatures, estimated the degradation efficiencies of rice and maize straw by CH2, sequenced and analyzed the whole genome of CH2. The results showed that along with the increases of incubation time, NaCl concentration, pH and temperature, the CMC and FPA activities increased first and then decreased . The highest CMC and FPA activities were observed at incubation time of 72-84 h, NaCl concentration of 6.0 g·L-1, pH of 7 and temperature of 36 ℃. Degradation of straw assays revealed that CH2 could effectively degrade rice and maize straw. At 0 g·L-1 NaCl (the control), the 10-day degradation rates of rice and maize straw were 30.4% and 47.0%, respectively. In the presence of 15 g·L-1 NaCl, the degradation rates were not significantly different from the control, indicating that CH2 had a high tolerance to salts. The whole genome of P. silvae CH2 was 6797325 bp, containing 6312 coding genes. P. silvae CH2 contained multiple genes encoding cellulose and hemicellulose degrading enzymes. These enzymes mainly belonged to the GH family, including endo-1,4-β-xylanase, Xylan 1,4-β-xylosidase, β-glucosidase, and endoglucanase. The results indicated that the bacterium had the potential to be used in crop straw degradation.

A GH19 lysozyme and peptidase from Myoviridae cyanophages lacking the typical holin-endolysin system exhibit lytic activity

Most of the dsDNA cyanophages employ holin-endolysin lysis systems to damage the host cells. This study aimed to elucidate the lytic activity of ORF91 and ORF117 in the cyanophage MaMV-DH01, which lacked a conventional cholinesterase system. These two proteins contained Lyz-like superfamily domains and were annotated as a member of GH family 19 (named DHGH19) and peptidase (named DHpeptidase), respectively. Overexpression of DHGH19 in E. coli over a 5 h course demonstrated potent bactericidal activity, evident from significant growth inhibition, membrane damage, and leakage of intracellular enzymes of E. coli cells. However, the lytic activity of DHpeptidase was relatively weaker, exhibiting a bacteriostatic effect. It was important to highlight that the specific mutation of enzyme-catalyzed residues in DHGH19 (E122 and E131) showed that these were the essential amino acids for DHGH19 to exert its bactericidal activity. Furthermore, the lytic function of DHGH19 and DHpeptidase on cyanobacteria cells was confirmed by their overexpression in the cyanobacterium Synechocystis sp. PCC6803. Overall, this study provides novel insights into the lytic mechanism of Myoviridae cyanophage, offering potential alternatives for the development of GH19 and peptidase as new antibacterial agents in the future.

Discovery of novel alkaline-tolerant xylanases from fecal microbiota of dairy cows

Xylo-oligosaccharides (XOS) are considered as a promising type of prebiotics that can be used in foods, feeds, and healthcare products. Xylanases play a key role in the production of XOS from xylan. In this study, we conducted a metagenomic analysis of the fecal microbiota from dairy cows fed with different types of fodders. Despite the diversity in their diets, the main phyla observed in all fecal microbiota were Firmicutes and Bacteroidetes. At the genus level, one group of dairy cows that were fed probiotic fermented herbal mixture-containing fodders displayed decreased abundance of Methanobrevibacter and increased growth of beneficial Akkermansia bacteria. Additionally, this group exhibited a high microbial richness and diversity. Through our analysis, we obtained a comprehensive dataset comprising over 280,000 carbohydrate-active enzyme genes. Among these, we identified a total of 163 potential xylanase genes and subsequently expressed 34 of them in Escherichia coli. Out of the 34 expressed genes, two alkaline xylanases with excellent temperature stability and pH tolerance were obtained. Notably, CDW-xyl-8 exhibited xylanase activity of 96.1 ± 7.5 U/mg protein, with an optimal working temperature of 55 ℃ and optimal pH of 8.0. CDW-xyl-16 displayed an activity of 427.3 ± 9.1 U/mg protein with an optimal pH of 8.5 and an optimal temperature at 40 ℃. Bioinformatic analyses and structural modeling suggest that CDW-xyl-8 belongs to GH10 family xylanase, and CDW-xyl-16 is a GH11 family xylanase. Both enzymes have the ability to hydrolyze beechwood xylan and produce XOS. In conclusion, this metagenomic study provides valuable insights into the fecal microbiota composition of dairy cows fed different fodder types, revealing main microbial groups and demonstrating the abundance of xylanases. Furthermore, the characterization of two novel xylanases highlights their potential application in XOS production.

Expression and characterization of mesophilic GH1 β-glucosidase CdBglA from acidophilic Cuniculiplasma divulgatum

β-glucosidase has important applications in food, pharmaceutics, biomass conversion and other fields, exploring β-glucosidase with strong adaptability and excellent properties thus has received extensive interest. In this study, a novel glucosidase from the GH1 family derived from Cuniculiplasma divulgatum was cloned, expressed, and characterized, aiming to find a better β-glucosidase. The amino acid sequences of GH1 family glucosidase derived from C. divulgatum were obtained from the NCBI database, and a recombinant plasmid pET-30a(+)-CdBglA was constructed. The recombinant protein was induced to express in Escherichia coli BL21(DE3). The enzymatic properties of the purified CdBglA were studied. The molecular weight of the recombinant CdBglA was 56.0 kDa. The optimum pH and temperature were 5.5 and 55 ℃, respectively. The enzyme showed good pH stability, 92.33% of the initial activity could be retained when treated under pH 5.5-11.0 for 1 h. When pNPG was used as a substrate, the kinetic parameters Km, Vmax and Kcat/Km were 0.81 mmol, 291.99 μmol/(mg·min), and 387.50 s-1 mmol-1, respectively. 90.33% of the initial enzyme activity could be retained when CdBglA was placed with various heavy metal ions at a final concentration of 5 mmol/L. The enzyme activity was increased by 28.67% under 15% ethanol solution, remained unchanged under 20% ethanol, and 43.68% of the enzyme activity could still be retained under 30% ethanol. The enzyme has an obvious activation effect at 0-1.5 mol/L NaCl and can tolerate 0.8 mol/L glucose. In conclusion, CdBglA is an acidic and mesophilic enzyme with broad pH stability and strong tolerance to most metal ions, organic solvents, NaCl and glucose. These characteristics may facilitate future theoretical research and industrial production.

Carbohydrate-active enzyme profiles of Lactiplantibacillus plantarum strain 84-3 contribute to flavor formation in fermented dairy and vegetable products

Microbes are critical for flavor formation in fermented foods; however, their mechanisms of action are not fully understood. The microbial composition of 51 dairy and 47 vegetable products was functionally annotated and the carbohydrate-active enzyme (CAZyme) profiles of Lactiplantibacillus plantarum 84-3 (Lp84-3), isolated from dairy samples, can promote resistant starch (RS) degradation, were analyzed. Lactobacillus, Streptococcus, and Lactococcus were the predominant genera in dairy products, whereas the major genera in vegetables were Lactobacillus, Weissella, and Carnimonas. Phages from Siphoviridae, Myoviridae, and Herelleviridae were also present in dairy products. Additionally, the glycosyl hydrolase (GHs) family members GH1 and GH13 and the glycosyltransferase (GTs) family members GT2 and GT4 were abundant in Lp84-3. Moreover, Lp84-3 was enriched in butanoate metabolism enzymes and butanoate metabolite compounds. Therefore, fermented food microbes, especially Lp84-3, have an abundant repertoire of enzymes that promote flavor production, as starter improving the flavor of fermented dairy and vegetable products.

Expression of β-xylosidase An-xyl from Aspergillus niger and characterization of its xylose tolerance

The hydrolysis of xylo-oligosaccharides catalyzed by β-xylosidase plays an important role in the degradation of lignocellulose. However, the enzyme is easily inhibited by its catalytic product xylose, which severely limits its application. Based on molecular docking, this paper studied the xylose affinity of Aspergillus niger β-xylosidase An-xyl, which was significantly differentially expressed in the fermentation medium of tea stalks, through cloning, expression and characterization. The synergistic degradation effect of this enzyme and cellulase on lignocellulose in tea stems was investigated. Molecular docking showed that the affinity of An-xyl to xylose was lower than that of Aspergillus oryzae β-xylosidase with poor xylose tolerance. The Ki value of xylose inhibition constant of recombinant-expressed An-xyl was 433.2 mmol/L, higher than that of most β-xylosidases of the GH3 family. The Km and Vmax towards pNPX were 3.6 mmol/L and 10 000 μmol/(min·mL), respectively. The optimum temperature of An-xyl was 65 ℃, the optimum pH was 4.0, 61% of the An-xyl activity could be retained upon treatment at 65 ℃ for 300 min, and 80% of the An-xyl activity could be retained upon treatment at pH 2.0-8.0 for 24 h. The hydrolysis of tea stem by An-xyl and cellulase produced 19.3% and 38.6% higher reducing sugar content at 2 h and 4 h, respectively, than that of using cellulase alone. This study showed that the An-xyl mined from differential expression exhibited high xylose tolerance and higher catalytic activity and stability, and could hydrolyze tea stem lignocellulose synergistically, which enriched the resource of β-xylosidase with high xylose tolerance, thus may facilitate the advanced experimental research and its application.

Preparation, purification, and biochemical of fat-degrading bacterial enzymes from pig carcass compost and its application

BackgroundA lot of kitchen waste oil is produced every day worldwide, leading to serious environmental pollution. As one of the environmental protection methods, microorganisms are widely used treating of various wastes. Lipase, as one of the cleaning agents can effectively degrade kitchen waste oil. The composting process of pig carcasses produces many lipase producing microorganisms, rendering compost products an excellent source for isolating lipase producing microorganisms. To our knowledge, there are no reports isolating of lipase producing strains from the high temperature phase of pig carcass compost.MethodologyLipase producing strains were isolated using a triglyceride medium and identified by 16S rRNA gene sequencing. The optimal fermentation conditions for maximum lipase yield were gradually optimized by single-factor tests. The extracellular lipase was purified by ammonium sulfate precipitation and Sephadex G-75 gel isolation chromatography. Amino acid sequence analysis, structure prediction, and molecular docking of the purified protein were performed. The pure lipase's enzymatic properties and application potential were evaluated by characterizing its biochemical properties.ResultsIn this study, a lipase producing strain of Bacillus sp. ZF2 was isolated from pig carcass compost products, the optimal fermentation conditions of lipase: sucrose 3 g/L, ammonium sulfate 7 g/L, Mn2+ 1.0 mmol/L, initial pH 6, inoculum 5%, temperature 25 ℃, and fermentation time 48 h. After purification, the specific activity of the purified lipase reached 317.59 U/mg, a 9.78-fold improvement. Lipase had the highest similarity to the GH family 46 chitosanase and molecular docking showed that lipase binds to fat via two hydrogen bonds at Gln146 (A) and Glu203 (A). Under different conditions (temperature, metal ions, organic solvents, and surfactants), lipase can maintain enzymatic activity. Under different types of kitchen oils, lipase has low activity only for ‘chicken oil’, in treating other substrates, the enzyme activity can exceed 50%.ConclusionsThis study reveals the potential of lipase for waste oil removal, and future research will be devoted to the application of lipase.

Glycoside hydrolase family 32 enzymes from Bombella spp. catalyze the formation of high-molecular weight fructans from sucrose

Abstract Aims Acetic acid bacteria of the genus Bombella have not been reported to produce exopolysaccharides (EPS). In this study, the formation of fructans by B. apis TMW 2.1884 and B. mellum TMW 2.1889 was investigated. Methods and Results Out of eight strains from four different Bombella species, only B. apis TMW 2.1884 and B. mellum TMW 2.1889 showed EPS formation with 50 g l−1 sucrose as substrate. Both EPS were identified as high-molecular weight (HMW) polymers (106–107 Da) by asymmetric flow field-flow fractionation coupled to multi angle laser light scattering and UV detecors (AF4-MALLS/UV) and high performance size exclusion chromatography coupled to MALLS and refractive index detectors (HPSEC-MALLS/RI) analyses. Monosaccharide analysis via trifluoroacetic acid hydrolysis showed that both EPS are fructans. Determination of glycosidic linkages by methylation analysis revealed mainly 2,6-linked fructofuranose (Fruf) units with additional 2,1-linked Fruf units (10%) and 2,1,6-Fruf branched units (7%). No glycoside hydrolase (GH) 68 family genes that are typically associated with the formation of HMW fructans in bacteria could be identified in the genomes. Through heterologous expression in Escherichia coli Top10, an enzyme of the GH32 family could be assigned to the catalysis of fructan formation. The identified fructosyltransferases could be clearly differentiated phylogenetically and structurally from other previously described bacterial fructosyltransferases. Conclusions The formation of HMW fructans by individual strains of the genus Bombella is catalyzed by enzymes of the GH32 family. Analysis of the fructans revealed an atypical structure consisting of 2,6-linked Fruf units as well as 2,1-linked Fruf units and 2,1,6-Fruf units.

Site-directed mutagenesis of family GH10 Aspergillus fumigatus xylanase A and the interaction with Oryza sativa xylanase inhibitor protein

In this study three alanine mutants (V99A, D145A and V99A/D145A) of family GH10 Aspergillus fumigatus xylanase A (AfXylA10) were engineered and characterized. These variants exhibited molecular mass of approximately 52.0 kDa and optimum temperature at 53 °C. The optimum pH of V99A/D145A was pH 4.0, and that of V99A and D145A was 5.0. Three variants were competitively inhibited by Oryza sativa xylanase inhibitor protein, rePOsXIP in a typical dose-dependent manner, with Ki of 214.8, 170.3 and 126.2 nM, respectively. Mutant V99A showed lower sensitivity to rePOsXIP and lower activity compared to reAfXylA10. However, D145A obtained higher activity and similar sensitivity to rePOsXIP. The underlying interaction mechanism was delved by molecular dynamics (MD) simulations and computational analysis. Binding free energy of V99A-OsXIP, D145A-OsXIP and V99A/D145A-OsXIP systems, calculated using the MM/PBSA method, were −19.7 ± 9.4, −26.9 ± 6.0, and −45.4 ± 9.8 kcal/mol, respectively. Representational snapshot of these systems from MD simulations showed that the α7-helix of OsXIP entered the active tunnel of mutant xylanases, which would hinder xylan from entering the catalytic groove. Particularly, residue K239 in OsXIP's α7-helix formed stable hydrogen bonding with the acid-base catalytic site (E140) of these mutants. Laplacian of electron density values of E140xylanase- K239OsXIP in V99A-OsXIP, D145A-OsXIP, and V99A/D145A-OsXIP systems were 0.07545, 0.09155 and 0.09764 au., respectively. The study provided preliminary insight into how V99AfXylA10 and D145AfXylA10 residue influence enzymatic properties and the interaction with OsXIP. These findings might inform design of mutant xylanase with enhanced catalytic activity and reduced sensitivity to xylanase inhibitory proteins (XIs).

Flexibility of active center affects thermostability and activity of Penicillium canescens xylanase E

Xylanases are used in several industrial applications, such as feed additives, the bleaching of pulp and paper, and the production of bread, food, and drinks. Xylanases are required to remain active after heat treatment at 80–90 °C for 30 s to several minutes due to the conditions of feed pelleting. Also, xylanases need to be active at 60–70 °C for several hours while bleaching of pulp and paper or manufacturing of bread, food, and drinks is performed. Xylanases of the glycoside hydrolase family GH10 are good candidates for application in such processes because of their high thermostability and, in particular, as feed additives because of their insensitivity to protein inhibitors in cereal feeds. In the study, the thermostability of GH10 xylanase E from Penicillium canescens was improved to reach a half-inactivation period of 2 min at 80 °C compared to 21 s for the wild-type enzyme (WT). Enzymatic activity was increased by 22–48 % at 40–70 °C, which improved the action of the enzyme as a feed additive in the gastric system of animals and during bleaching of pulp and paper. Molecular dynamics simulations demonstrated lower flexibility of the tertiary structure of the engineered enzyme at elevated temperatures compared to WT. The residues W113, Q116, W313, and W321 in the (−1) and (−2) subsites for the substrate binding were less flexible. In the simulations, the engineered enzyme had a comparable content of α-helixes, 310-helixes, β-sheets, and β-bridges as WT, but a lower content of coils and a higher content of β-turns.

Recombinant Expression in Bacillus megaterium and Biochemical Characterization of Exo-Mannered Glycosyl Hydrolase Family 43 α-L-Arabinofuranosidase from the Korean Black Goat Rumen Metagenome

There is no doubt that ruminants have the capability to digest lignocellulosic compounds and to utilize them as an absorbable form of energy by tapping into enzymes produced by the microbial population in their rumens. Among the rumens of various ruminants, this study focused on Korean goat rumens because of their unique digestibility of lignocellulosic biomasses. Therefore, a novel Gene12 gene was screened and unmasked from the constructed rumen metagenomic library of a Korean black goat and expressed in a Bacillus megaterium system. The recombinant protein was distinguished as a novel α-L-arabinofuranosidase enzyme from glycosyl hydrolase family 43 (GH43) for its capability to hydrolyze the non-reducing end of α-1,5-L-arabinofuranose linkages in α-L-arabinofuranosyl groups. The enzyme can also break apart α-L-arabinofuranosidic linkages and act synergistically with other hemicellulolytic enzymes to release α-1,2- and α-1,3-L-arabinofuranosyl groups from L-arabinose-comprising polysaccharides. In silico, phylogenetic, and computational analyses proclaimed that the Gene12 gene encodes a novel carbohydrate-active enzyme possessing a V-shaped indentation of the GH43 catalytic and functional domain (carbohydrate-binding module 6). The recombinant Gene12 protein has shared 81% sequence homology with other members of the GH43 family. Enzymic synopses (optimal pH, temperatures, and stability studies) of the recombinant Gene12 enzyme and its substrate specificity (synthetic and natural substrates) profiling were considered. The recombinant Gene12 α-L-arabinofuranosidase works best at pH 6.0 and 40 °C, and it is stable at pH 4.0 to 7.0 at temperatures of 20 to 50 °C. Additionally, 5-blended β-sheets were identified through a tertiary (3D) structure analysis along with the high substrate specificity against p-nitrophenyl-D-arabinofuranoside (pNPA). The highest substrate specificity of pNPA for Gene12 α-L-arabinofuranosidase indicated its confirmation as an exo-type arabinofuronidase. The results thus propose using the Gene12 protein as an exo-mannered GH43 α-L-arabinofuranosidase (EC 3.2.1.55) enzyme.

Deterministic assembly processes shaping habitat‐specific glycoside hydrolase composition

AbstractAimGlycoside hydrolases (GHs) encoded by prokaryotes play a crucial role in the degradation of carbohydrates, which is a key step in the carbon cycle. However, their global distribution remains poorly understood. Here, we conducted a comprehensive investigation of the global biogeographic patterns of prokaryotic‐encoded GHs.LocationGlobal.Time periodPresent.Major taxa studiedProkaryotes.MethodsWe conducted a global survey of GH distribution based on 10,000 samples from the Earth Microbiome Project (EMP) and 17,923 completely sequenced prokaryotic genomes. The samples were retrieved from seven continents, covering all 17 EMP Ontology environments. We evaluated the assembly process and influencing factors of GH functional genes within prokaryotic communities, and analysed the rank abundance distributions (RADs), occupancy frequency distributions (OFDs), and taxonomic sources of GH families across various environments.ResultsThe median number of GH genes per thousand genes encoded by global prokaryotic communities was 9.11 (7.16–11.58), with a higher GH abundance in host‐associated communities than in free‐living communities. The median number of GH families in prokaryotic communities was 105 (86–121), with fewer families but tighter functional connections between families in host‐associated communities compared to free‐living communities. In any environment, the RAD of GH families followed a lognormal model, and their OFD was bimodal, with a strong positive correlation between the two. In addition, most GH families had complex and diverse taxonomic sources across different environments.Main conclusionsThis study provides a panoramic view of the functional potentials for carbohydrate degradation across prokaryotic communities under diverse environmental conditions. The composition of GHs encoded by prokaryotic communities exhibits clear habitat specificity, which is driven by a combination of biotic and abiotic factors and generated through highly deterministic assembly processes.

Mechanistic Insights into the Halophilic Xylosidase Xylo-1 and Its Role in Xylose Production.

The Xylo-1 xylosidase, which belongs to the GH43 family, exhibits a high salt tolerance. The present study demonstrated that the catalytic activity of Xylo-1 increased by 195% in the presence of 5 M NaCl. Additionally, the half-life of Xylo-1 increased 25.9-fold in the presence of 1 M NaCl. Through comprehensive analysis including circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations, we elucidated that the presence of Na+ ions increased the contact frequency between the surface acidic amino acids and the surrounding water molecules. This resulted in the stabilization of the surrounding hydration layer of Xylo-1. Additionally, Na+ ions also stabilized the substrate-binding conformation and the fluctuation of water molecules within the active site, which enhanced the catalytic activity of Xylo-1 by increasing the nucleophilic attack by the water molecules. Ultimately, the optimal reaction conditions for the production of xylose by synergistic catalysis with Xylo-1 and xylanase were determined. The results demonstrated that the conversion yield of the method was high for various sources of xylan, indicating the method could have potential industrial applications. This study explored the structure-activity relationship of catalysis in Xylo-1 under high-salt conditions, provides novel insights into the mechanism of halophilic enzymes, and serves as a reference for the industrial application of Xylo-1.

Conformational Switch of the 250s Loop Enables the Efficient Transglycosylation in GH Family 77.

Amylomaltases (AMs) play important roles in glycogen and maltose metabolism. However, the molecular mechanism is elusive. Here, we investigated the conformational dynamics of the 250s loop and catalytic mechanism of Thermus aquaticus TaAM using path-metadynamics and QM/MM MD simulations. The results demonstrate that the transition of the 250s loop from an open to closed conformation promotes polysaccharide sliding, leading to the ideal positioning of the acid/base. Furthermore, the conformational dynamics can also modulate the selectivity of hydrolysis and transglycosylation. The closed conformation of the 250s loop enables the tight packing of the active site for transglycosylation, reducing the energy penalty and efficiently preventing the penetration of water into the active site. Conversely, the partially closed conformation for hydrolysis results in a loosely packed active site, destabilizing the transition state. These computational findings guide mutation experiments and enable the identification of mutants with an improved disproportionation/hydrolysis ratio. The present mechanism is in line with experimental data, highlighting the critical role of conformational dynamics in regulating the catalytic reactivity of GHs.

RETRACTED: Biochemical characterization of a cold-adapted κ-carrageenase from Colwellia echini: A biotechnological tool for efficient preparation of carrageenan oligosaccharides

κ-Carrageenase (EC 3.2.1.83) is a glycoside hydrolase that efficiently breaks down κ-carrageenan into κ-carrageenan oligosaccharides (κ-COS). In this study, a novel GH16 family κ-carrageenase called CeCgkB was cloned from the genome of Colwellia echini. Escherichia coli BL21 (DE3) was used to express CeCgkB. CeCgkB has a complete length of 987 bp, encoding 329 amino acid residues, and a molecular weight of 37.44 kDa. Biochemical characterization indicated that CeCgkB exhibits cold-adapted properties and was able to reach a maximum activity of 1530 U/mg at pH 8.0 and 35 °C. Kinetic parameters showed that CeCgkB has a low Km value (2 mg/mL), indicating high substrate affinity. As an endo-type κ-carrageenase, CeCgkB can degrade κ-carrageenan into products of varying DP, with a preference for DP4, DP6, and DP10. ESI-MS analysis revealed that the major products of CeCgkB consist of κ-carratetraose, κ-carrahexaose, and κ-carradecaose. As a result of CeCgkB's high activity, cold-adapted, and principal products, it is a promising biotechnological tool for the efficient preparation of κ-carrageenan oligosaccharides.

Cloning, expression, and molecular modification of glycoside hydrolase family 5 genes from Thermoascus aurantiacus.

In this paper, a novel bifunctional cellulase gene cel1 was cloned from Thermoascus aurantiacus by PCR and heterologously expressed in Pichia pastoris GS115. Bioinformatics and other related tools were used to compare the nucleotide homology of target genes, and analyze the signal peptide, transmembrane domain, hydrophilicity, secondary and tertiary structure of proteins. It was concluded that cel1 has similar endoglucanase nucleotide sequences and falls under the GH5 family. It was also found that cel1 has nucleotide sequences similar to glucosidase, which can infer that cel1 may have the properties of glucosidase, indicating that cel1 is multifunctional. At the same time, a part of the nucleotide sequence of the gene was removed to obtain a new gene cel2, and after highly efficient heterologous expression, its specific activity was found to be 2.1 times higher. Its enhancement is related to the exposure of the protein's hollow three-dimensional structure. This paper provides good material for exploring the relationship between the structure of bifunctional enzymes and their functions, which lays a solid foundation for further research and applications, and provides useful insight for gene mining of other novel enzymes.

Isolation of a newly Trichoderma asperellum LYS1 with abundant cellulase-hemicellulase enzyme cocktail for lignocellulosic biomass degradation

As the most abundant and renewable natural resource in the world, lignocellulose is a promising alternative to fossil energy to relieve environmental concerns and resource depletion. However, due to its recalcitrant structure, strains with efficient degradation capability still need exploring. In this study, a fungus was successfully isolated from decayed wood and named as Trichoderma asperellum LYS1 by phylogenetic and draft genomic analysis. The further investigations showed that strain LYS1 had an outstanding performance on lignocellulose degradation, especially for hemicellulose-rich biomass. After the analysis of encoded CAZymes, mainly on GH family, a large amount of genes coding β-glucosidase and xylanase may contribute to the high degradation of cellulose and hemicellulose. Collectively, the results generated in this study demonstrated that T. asperellum LYS1 is a potential cell factory for lignocellulose biorefinery.

Profiling of the β-glucosidases identified in the genome of Penicillium funiculosum: insights from genomics, transcriptomics, proteomics, and homology-modeling studies.

The enzymatic conversion of lignocellulosic biomass to bioethanol depends on efficient enzyme systems with β-glucosidase as one of the key components. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus Penicillium funiculosum using genomics, transcriptomics, proteomics, and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in the P. funiculosum genome, three were predicted to be extracellular based on signal peptide prediction and abundance in the secretome. Among the three secreted β-glucosidases, two belonged to the GH3 family and one belonged to the GH1 family. Homology models of these proteins predicted a deep and narrow active site for the GH3 β-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 β-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum-secreted proteins showed high β-glucosidase activities with prominent bands on the 4-methylumbelliferyl β-D-glucopyranoside zymogram. To understand the contributory effects of each of the three secreted β-glucosidases (PfBgls), the corresponding gene was deleted separately, and the effect of the deletion on the β-glucosidase activity of the secretome was examined. Although not the most abundant, PfBgl3A was found to be one of the most important β-glucosidases, as evidenced by a 42% reduction in β-glucosidase activity in the ΔPfBgl3A strain. Our results advance the understanding of the genetic and biochemical nature of all β-glucosidases produced by P. funiculosum and pave the way to design a superior biocatalyst for the hydrolysis of lignocellulosic biomass. IMPORTANCE Commercially available cellulases are primarily produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous β-glucosidase is often required to achieve the desired optimal saccharification of cellulosic feedstocks. This challenge has led to the exploration of other cellulase-producing strains. The nonmodel hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases mainly due to its ability to produce a balanced concoction of cellulolytic enzymes, including β-glucosidases. Various genetic interventions targeted at strain improvement for cellulase production have been performed; however, the β-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all eight β-glucosidases of P. funiculosum via molecular and computational approaches. The results of this study provide useful insights that will establish the background for future engineering strategies to transform this fungus into an industrial workhorse.