Self-driving autonomous machines to engineer novel enzymes.

Can one design and automate a computational and experimental platform such that each platform iteratively guides and drives the other to achieve a pre-determined goal? Rapp and colleagues (2024) describe just this possibility in a paper that details a prototype of a self-driven laboratory that can navigate autonomously to yield an engineered enzyme with a desired attribute. This laboratory, rather, the automated protocol, is referred to by an acronym - SAMPLE. This refers to Self-driving Autonomous Machines for Protein Landscape Exploration. The paper describes a prototype involving the engineering of a glycoside hydrolase for enhanced thermostability. The 'brain', the computational component behind this automated system, was designed to learn protein sequence- function relationships from a curated dataset. These designer proteins were then evaluated by a fully automated robotic system that could synthesize and experimentally characterize the designed protein and provide feedback to the agent, i.e., the computational component, to fine-tune its understanding of the system. The SAMPLE agents were thus designed to continually refine their understanding of the protein landscape by actively acquiring information in the search process. As this intelligent agent learns protein sequence-function relationships from a curated, diverse dataset, this feedback is crucial to refine landscape exploration and the design of new proteins based on the updated hypothesis. In this prototype, four SAMPLE agents were tasked with this goal. The goal of each of these agents was to navigate the glycoside hydrolase landscape and identify enzymes with enhanced thermal tolerance. Differences in the search behavior of individual agents primarily arise from experimental measurement noise. However, despite differences in their search behavior, all four agents could converge on a thermostable glycoside hydrolase - a remarkable feat as it apparently did not need any human intervention.

The α-linkage in funoran and agarose could be hydrolyzed by a GH96 family enzyme: Discovery of the α-funoranase.

Agarans represent a group of galactans extracted from red algae. Funoran and agarose are the two major types and commercially applied polysaccharides of agaran. Although the glycoside hydrolases targeting ß-glycosidic bonds of agaran have been widely investigated, those capable of degrading α-glycosidic bonds of agarose were limited, and the enzyme degrading α-linkages of funoran has not been reported till now. In this study, a GH96 family enzyme BiAF96A_Aq from a marine bacterium Aquimarina sp. AD1 was heterologously expressed in Escherichia coli. BiAF96A_Aq exhibited dual activities towards the characteristic structure of funoran and agarose, underscoring the multifunctionality of GH96 family members. Glycomics and NMR analysis revealed that BiAF96A_Aq hydrolyzed the α-1,3 glycosidic bonds between 3,6-anhydro-α-l-galactopyranose (LA) and ß-d-galactopyranose-6-sulfate (G6S) of funoran, as well as LA and ß-d-galactopyranose (G) of agarose, through an endo-acting manner. The end products of BiAF96A_Aq were majorly composed of disaccharides and tetrasaccharides. The identification of the activity of BiAF96A_Aq on funoran indicated the first discovery of the funoran hydrolase for α-1,3 linkage. Considering the novel catalytic reaction, we proposed to name this activity as "α-funoranase" and recommended the assignment of a dedicated EC number for its classification.

Genetic and functional diversity of ß-N-acetylgalactosamine-targeting glycosidases expanded by deep-sea metagenome analysis.

ß-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. ß-N-Acetylgalactosaminidases hydrolyze ß-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-ß-N-acetylgalactosaminidases that specifically act on ß-N-acetylgalactosamine residues, has been documented to date. In this study, we identify four groups distributed among all three domains of life and characterize eight ß-N-acetylgalactosaminidases and ß-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and have diversified their substrate specificities (oligosaccharide-releasing, oligosaccharide/monosaccharide-releasing, and monosaccharide-releasing) through the accumulation of mutations and insertional amino acid sequences. The diverse ß-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present evolutionary pathways for expanding their substrate specificity.

Discovery of Potent Glycosidases Enables Quantification of Smoke-Derived Phenolic Glycosides through Enzymatic Hydrolysis.

When grapes are exposed to wildfire smoke, certain smoke-related volatile phenols (VPs) can be absorbed into the fruit, where they can be then converted into volatile-phenol (VP) glycosides through glycosylation. These volatile-phenol glycosides can be particularly problematic from a winemaking standpoint as they can be hydrolyzed, releasing volatile phenols, which can contribute to smoke-related off-flavors. Current methods for quantitating these volatile-phenol glycosides present several challenges, including the requirement of expensive capital equipment, limited accuracy due to the molecular complexity of the glycosides, and the utilization of harsh reagents. To address these challenges, we proposed an enzymatic hydrolysis method enabled by a tailored enzyme cocktail of novel glycosidases discovered through genome mining, and the generated VPs from VP glycosides can be quantitated by gas chromatography-mass spectrometry (GC-MS). The enzyme cocktails displayed high activities and a broad substrate scope when using commercially available VP glycosides as the substrates for testing. When evaluated in an industrially relevant matrix of Cabernet Sauvignon wine and grapes, this enzymatic cocktail consistently achieved a comparable efficacy of acid hydrolysis. The proposed method offers a simple, safe, and affordable option for smoke taint analysis.

Valuation of the significant hypoglycemic activity of black currant anthocyanin extract by both starch structure transformation and glycosidase activity inhibition.

The objective of this study was to investigate the impact of anthocyanin-rich black currant extract (BCE) on the structural properties of starch and the inhibition of glycosidases, gathering data and research evidence to support the use of low glycemic index (GI) foods. The BCE induced a change in the starch crystal structure from A-type to V-type, resulting in a drop in digestibility from 81.41 % to 65.57 %. Furthermore, the inhibitory effects of BCE on glycosidases activity (α-glucosidase: IC50 = 0.13 ± 0.05 mg/mL and α-amylase: IC50 = 2.67 ± 0.16 mg/mL) by inducing a change in spatial conformation were confirmed through in vitro analysis. The presence of a 5'-OH group facilitated the interaction between anthocyanins and receptors of amylose, α-amylase, and α-glucosidase. The glycosyl moiety enhanced the affinity for amylose yet lowered the inhibitory effect on α-amylase. The in vivo analysis demonstrated that BCE resulted in a reduction of 3.96 mM·h in blood glucose levels (Area Under Curve). The significant hypoglycemic activity, particularly the decrease in postprandial blood glucose levels, highlights the potential of utilizing BCE in functional foods for preventing diabetes.

Kinetics and molecular modeling studies on the inhibition mechanism of GH13 α-glycosidases by small molecule ligands.

Alpha-glucosidase inhibitors play an important role in Diabetes Mellitus (DM) treatment since they prevent postprandial hyperglycemia. The Glycoside Hydrolase family 13 (GH13) is the major family of enzymes acting on substrates containing α-glucoside linkages, such as maltose and amylose/amylopectin chains in starch. Previously, our group identified glycoconjugate 1H-1,2,3-triazoles (GCTs) inhibiting two GH13 α-glycosidases: yeast maltase (MAL12) and porcine pancreatic amylase (PPA). Here, we combined kinetic studies and computational methods on nine GCTs to characterize their inhibitory mechanism. They all behaved as reversible inhibitors, and kinetic models encompassed noncompetitive and various mechanisms of mixed-type inhibition for both enzymes. Most potent inhibitors displayed Ki values of 30 µM for MAL12 (GPESB16) and 37 µM for PPA (GPESB15). Molecular dynamics and docking simulations indicated that on MAL12, GPESB15 and GPESB16 bind in a cavity adjacent to the active site, while on the PPA, GPESB15 was predicted to bind at the entrance of the catalytic site. Notably, despite its putative location within the active site, the binding of GPESB15 does not obstruct the substrate's access to the cleavage site. Our study contributes to paving the way for developing novel therapeutic strategies for managing DM-2 through GH13 α-glycosidases inhibition.

Improvement of thermostability by increasing rigidity in the finger regions and flexibility in the catalytic pocket area of Pseudoalteromonas porphyrae κ-carrageenase.

Poor thermostability reduces the industrial application value of κ-carrageenase. In this study, the PoPMuSiC algorithm combined with site-directed mutagenesis was applied to improve the thermostability of the alkaline κ-carrageenase from Pseudoalteromonas porphyrae. The mutant E154A with improved thermal stability was successfully obtained using this strategy after screening seven rationally designed mutants. Compared with the wild-type κ-carrageenase (WT), E154A improved the activity by 29.4% and the residual activity by 51.6% after treatment at 50 °C for 30 min. The melting temperature (Tm) values determined by circular dichroism were 66.4 °C and 64.6 °C for E154A and WT, respectively. Molecular dynamics simulation analysis of κ-carrageenase showed that the flexibility decreased within the finger regions (including F1, F2, F3, F5 and F6) and the flexibility improved in the catalytic pocket area of the mutant E154A. The catalytic tunnel dynamic simulation analysis revealed that E154A led to enlarged catalytic tunnel volume and increased rigidity of the enzyme-substrate complex. The increasing rigidity within the finger regions and more flexible catalytic pocket of P. porphyrae κ-carrageenase might be a significant factor for improvement of the thermostability of the mutant κ-carrageenase E154A. The proposed rational design strategy could be applied to improve the enzyme kinetic stability of other industrial enzymes. Moreover, the hydrolysates of κ-carrageenan digested by the mutant E154A demonstrated increased scavenging activities against hydroxyl (OH) radicals and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) radicals compared with the undigested κ-carrageenan.

Effect of enzymatic modification on the structure and rheological properties of diluted alkali-soluble pectin fraction rich in RG-I.

This study focuses on pectin covalently linked in cell walls from two sources, apples and carrots, that was extracted using diluted alkali, and it describes changes in the rheological properties of diluted alkali-soluble pectin (DASP) due to enzymatic treatment. Given DASP's richness of rhamnogalacturonan I (RG-I), RG-I acetyl esterase (RGAE), rhamnogalacturonan endolyase (RGL), and arabinofuranosidase (ABF) were employed in various combinations for targeted degradation of RG-I pectin chains. Enzymatic degradations were followed by structural studies of pectin molecules using atomic force microscopy (AFM) as well as measurements of rheological and spectral properties. AFM imaging revealed a significant increase in the length of branched molecules after incubation with ABF, suggesting that arabinose side chains limit RG-I aggregation. Structural modifications were confirmed by changes in the intensity of bands in the pectin fingerprint and anomeric region on Fourier transform infrared spectra. ABF treatment led to a decrease in the stability of pectic gels, while the simultaneous use of ABF, RGAE, and RGL enzymes did not increase the degree of aggregation compared to the control sample. These findings suggest that the association of pectin chains within the DASP fraction may rely significantly on intermolecular interactions. Two mechanisms are proposed, which involve side chains as short-range attachment points or an extended linear homogalacturonan conformation favoring inter-chain interactions over self-association.

Mutations in Glycosyltransferases and Glycosidases: Implications for Associated Diseases.

Glycosylation, a crucial and the most common post-translational modification, coordinates a multitude of biological functions through the attachment of glycans to proteins and lipids. This process, predominantly governed by glycosyltransferases (GTs) and glycoside hydrolases (GHs), decides not only biomolecular functionality but also protein stability and solubility. Mutations in these enzymes have been implicated in a spectrum of diseases, prompting critical research into the structural and functional consequences of such genetic variations. This study compiles an extensive dataset from ClinVar and UniProt, providing a nuanced analysis of 2603 variants within 343 GT and GH genes. We conduct thorough MTR score analyses for the proteins with the most documented variants using MTR3D-AF2 via AlphaFold2 (AlphaFold v2.2.4) predicted protein structure, with the analyses indicating that pathogenic mutations frequently correlate with Beta Bridge secondary structures. Further, the calculation of the solvent accessibility score and variant visualisation show that pathogenic mutations exhibit reduced solvent accessibility, suggesting the mutated residues are likely buried and their localisation is within protein cores. We also find that pathogenic variants are often found proximal to active and binding sites, which may interfere with substrate interactions. We also incorporate computational predictions to assess the impact of these mutations on protein function, utilising tools such as mCSM to predict the destabilisation effect of variants. By identifying these critical regions that are prone to disease-associated mutations, our study opens avenues for designing small molecules or biologics that can modulate enzyme function or compensate for the loss of stability due to these mutations.

Expression and biochemical characterization of a novel thermostable alkaline ß-1,3-1,4-glucanase (lichenase) from an alkaliphilic Bacillus lehensis G1.

New thermostable ß-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca2+. Blg29 showed high substrate activity towards lichenan where Vm, Km, Kcat, and kcat/Km values were 2040.82 µmolmin‾1mg‾1, 4.69 mg/mL, and 986.39 s‾1 and 210.32 mLs‾1mg‾1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications.

N-terminal domain truncation yielded a unique dimer of polysaccharide hydrolase with enhanced enzymatic activity, stability and calcium ion independence.

Domain engineering, including domain truncation, fusion, or swapping, has become a common strategy to improve properties of enzymes, especially glycosyl hydrolases. However, there are few reports explaining the mechanism of increased activity from a protein structure perspective. Amy703 is an alkaline amylase with a unique N-terminal domain. Prior studies have shown that N-Amy, a mutant without an N-terminal domain, exhibits improved activity, stability, and calcium ion independence. In this study, we have used X-ray crystallography to determine the crystal structure of N-Amy and used AlphaFold2 to model the Amy703 structure, respectively. We further used size exclusion chromatography to show that Amy703 existed as a monomer, whereas N-Amy formed a unique dimer. It was found that the N-terminus of one monomer of N-Amy was inserted into the catalytic domain of its symmetrical subunit, resulting in the expansion of the catalytic pocket. This also significantly increased the pKa of the hydrogen donor Glu350, thereby enhancing substrate binding affinity and contributing to increased N-Amy activity. Meanwhile, two calcium ions were found to bind to N-Amy at different binding sites, which also contributed to the stability of protein. Therefore, this study provided new structural insights into the mechanisms of various glycosyl hydrolases.

Characterization of a multi-domain exo-ß-1,3-galactanase from Paenibacillus xylanexedens.

ß-1,3-Galactanases selectively degrade ß-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo-ß-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens, consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using ß-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only ß-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo-acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 ß-1,3-galactanase for the degradation of arabinogalactan.

Bioinformatics-based identification of GH12 endoxyloglucanases in citrus-pathogenic Penicillium spp.

Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6-5.3 and 35-37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite -4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.

Chitosanase-immobilized magnetite-agar gel particles as a highly stable and reusable biocatalyst for enhanced production of physiologically active chitosan oligosaccharides.

A novel immobilized chitosanase was developed and utilized to produce chitosan oligosaccharides (COSs) via chitosan hydrolysis. Magnetite-agar gel particles (average particle diameter: 338 µm) were prepared by emulsifying an aqueous agar solution dispersing 200-nm magnetite particles with isooctane containing an emulsifier at 80 °C, followed by cooling the emulsified mixture. The chitosanase from Bacillus pumilus was immobilized on the magnetite-agar gel particles chemically activated by introducing glyoxyl groups with high immobilization yields (>80%), and the observed specific activity of the immobilized chitosanase was 16% of that of the free enzyme. This immobilized chitosanase could be rapidly recovered from aqueous solutions by applying magnetic force. The thermal stability of the immobilized chitosanase improved remarkably compared with that of free chitosanase: the deactivation rate constants at 35 °C of the free and immobilized enzymes were 8.1 × 10-5 and 3.9 × 10-8 s-1, respectively. This immobilized chitosanase could be reused for chitosan hydrolysis at 75 °C and pH 5.6, and 80% of its initial activity was maintained even after 10 cycles of use. COSs with a degree of polymerization (DP) of 2-7 were obtained using this immobilized chitosanase, and the product content of physiologically active COSs (DP ≥ 5) reached approximately 50%.

Optimization of α-L-arabinofuranosidase CcABF on clarification and beneficial active substances in fermented ginkgo kernel juice by artificial neural network and genetic algorithm.

This study aimed at using α-L-arabinofuranosidase CcABF to improve the clarity and active substances in fermented ginkgo kernel juice by artificial neural network (ANN) modeling and genetic algorithm (GA) optimization. A credible three-layer feedforward ANN model was established to predict the optimal parameters for CcABF clarification. The experiments proved the highest transmittance of 89.40% for fermented ginkgo kernel juice with this understanding, which exhibited a 25.56% increase over the unclarified group. With the clarification of CcABF, the antioxidant capacity in juice was enhanced with the increase of total phenolic and flavone contents, and the maximum DPPH and hydroxyl radical scavenging rates were increased by 89.71% and 26.65%, respectively. The contents of toxic ginkgolic acids declined markedly, while the active ingredients of ginkgetin and ginkgolide B showed a modest increase. Moreover, changes in free amino acids and volatile compounds improved the nutritive value and flavor of clarified fermented ginkgo kernel juice.

Multifaceted roles of plant glycosyl hydrolases during pathogen infections: more to discover.

MAIN CONCLUSION: Carbohydrates are hydrolyzed by a family of carbohydrate-active enzymes (CAZymes) called glycosidases or glycosyl hydrolases. Here, we have summarized the roles of various plant defense glycosidases that possess different substrate specificities. We have also highlighted the open questions in this research field. Glycosidases or glycosyl hydrolases (GHs) are a family of carbohydrate-active enzymes (CAZymes) that hydrolyze glycosidic bonds in carbohydrates and glycoconjugates. Compared to those of all other sequenced organisms, plant genomes contain a remarkable diversity of glycosidases. Plant glycosidases exhibit activities on various substrates and have been shown to play important roles during pathogen infections. Plant glycosidases from different GH families have been shown to act upon pathogen components, host cell walls, host apoplastic sugars, host secondary metabolites, and host N-glycans to mediate immunity against invading pathogens. We could classify the activities of these plant defense GHs under eleven different mechanisms through which they operate during pathogen infections. Here, we have provided comprehensive information on the catalytic activities, GH family classification, subcellular localization, domain structure, functional roles, and microbial strategies to regulate the activities of defense-related plant GHs. We have also emphasized the research gaps and potential investigations needed to advance this topic of research.

Two-step modification of pullulanase and transglucosidase: A novel way to improve the gel strength and reduce the digestibility of rice starch.

The multiscale structure, gel strength and digestibility of rice starch modified by the two-step modification of pullulanase (PUL) pretreatment and transglucosidase (TG) treatment for 6, 12, 18 and 24 h were investigated. The debranching hydrolysis of PUL produced some linear chains, which rearranged to form stable crystalline structures, reducing the digestible starch content, but weakening the gel strength. TG treatment connected some short chains to longer linear chains via α-1,6-glycosidic bonds, generating the structures of linear chain with fewer branches. The short branches promoted the interaction between starch molecules to form a more compact three-dimensional gel network structure, showing higher hardness and springiness. Moreover, these chains could form more stable crystals, reducing the digestible starch content, and the increase of branching degree inhibited digestive enzyme hydrolysis, reducing the digestion rate. The multiscale structure of starch tended to stabilize after TG treatment for 18 h, which could form a gel with stronger strength and lower digestibility than native starch gel. Therefore, the two-step modification of PUL and TG was an effective way to change the structure of rice starch to improve the gel strength and reduce the digestibility.

Analysis of molecular structure and topological properties of chitosan isolated from crab shell and mushroom.

This investigation aimed to scrutinize the chemical and structural analogies between chitosan extracted from crab exoskeleton (High Molecular Weight Chitosan, HMWC) and chitosan obtained from mushrooms (Mushroom-derived Chitosan, MRC), and to assess their biological functionalities. The resulting hydrolysates from the hydrolysis of HMWC by chitosanase were categorized as chitosan oligosaccharides (csCOS), while those from MRC were denoted as mrCOS. The molecular weights (MW) of csCOS and mrCOS were determined using Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. Furthermore, structural resemblances of csCOS and mrCOS were assessed utilizing X-ray powder diffraction (XRD) and Fourier transform infrared (FT-IR) spectroscopy. Intriguingly, no apparent structural disparity between csCOS and mrCOS was noted in terms of the glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) composition ratios. Consequently, the enzymatic activities of chitosanase for HMWC and MRC exhibited remarkable similarity. A topological examination was performed between the enzyme and the substrate to deduce the alteration in MW of COSs following enzymatic hydrolysis. Moreover, the evaluation of antioxidant activity for each COS revealed insignificance in the structural disparity between HMWC and MRC. In summary, grounded on the chemical structural similarity of HMWC and MRC, we propose the potential substitution of HMWC with MRC, incorporating diverse biological functionalities.

The CBM91 module enhances the activity of ß-xylosidase/α-L-arabinofuranosidase PphXyl43B from Paenibacillus physcomitrellae XB by adopting a unique loop conformation at the top of the active pocket.

Carbohydrate-binding module (CBM) family 91 is a novel module primarily associated with glycoside hydrolase (GH) family 43 enzymes. However, our current understanding of its function remains limited. PphXyl43B is a ß-xylosidase/α-L-arabinofuranosidase bifunctional enzyme from physcomitrellae patens XB belonging to the GH43_11 subfamily and containing CBM91 at its C terminus. To fully elucidate the contributions of the CBM91 module, the truncated proteins consisting only the GH43_11 catalytic module (rPphXyl43B-dCBM91) and only the CBM91 module (rCBM91) of PphXyl43B were constructed, respectively. The result showed that rPphXyl43B-dCBM91 completely lost hydrolysis activity against both p-nitrophenyl-ß-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside; it also exhibited significantly reduced activity towards xylobiose, xylotriose, oat spelt xylan and corncob xylan compared to the control. Thus, the CBM91 module is crucial for the ß-xylosidase/α-L-arabinofuranosidase activities in PphXyl43B. However, rCBM91 did not exhibit any binding capability towards corncob xylan. Structural analysis indicated that CBM91 of PphXyl43B might adopt a loop conformation (residues 496-511: ILSDDYVVQSYGGFFT) to actively contribute to the catalytic pocket formation rather than substrate binding capability. This study provides important insights into understanding the function of CBM91 and can be used as a reference for analyzing the action mechanism of GH43_11 enzymes and their application in biomass energy conversion.

Trends in the development and current perspective of thermostable bacterial hemicellulases with their industrial endeavors: A review.

Hemicellulases are enzymes that hydrolyze hemicelluloses, common polysaccharides in nature. Thermophilic hemicellulases, derived from microbial strains, are extensively studied as natural biofuel sources due to the complex structure of hemicelluloses. Recent research aims to elucidate the catalytic principles, mechanisms and specificity of hemicellulases through investigations into their high-temperature stability and structural features, which have applications in biotechnology and industry. This review article targets to serve as a comprehensive resource, highlighting the significant progress in the field and emphasizing the vital role of thermophilic hemicellulases in eco-friendly catalysis. The primary goal is to improve the reliability of hemicellulase enzymes obtained from thermophilic bacterial strains. Additionally, with their ability to break down lignocellulosic materials, hemicellulases hold immense potential for biofuel production. Despite their potential, the commercial viability is hindered by their high enzyme costs, necessitating the development of efficient bioprocesses involving waste pretreatment with microbial consortia to overcome this challenge.