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.

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.

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.

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.

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.

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.

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.

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.

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.

Improvement of the catalytic performance of chitosanase Csn-PD from Paenibacillus dendritiformis by semi-rational design.

Chitooligosaccharides (COS) possess versatile functional properties that have found extensive applications across various fields. Chitosanase can specifically hydrolyze ß-1,4 glycosidic bonds in chitosan to produce COS. In this study, Csn-PD, a glycoside hydrolase family 46 chitosanase from Paenibacillus dendritiformis, which produces (GlcN)2 as its main product, was rationally redesigned aiming to improve its catalytic performance. Based on the results of molecular docking analysis and multiple sequence alignment, four amino acid residues in Csn-PD (I101, T120, T220, and Y259) were pinpointed for targeted mutations. Beneficial mutations in terms of enhanced catalytic activity were then combined by site-directed mutagenesis. Notably, the most promising variant, Csn-PDT6 (Csn-PD I101M/T120E/T220G), exhibited an impressive eight-fold surge in activity compared to the wild-type Csn-PD. This heightened enzymatic activity was complemented by an enhanced pH stability profile. A compelling feature of Csn-PDT6 is its preservation of the hydrolytic product profile observed in Csn-PD. This characteristic further accentuates its candidacy for the targeted production of (GlcN)2. The success of our strategic approach is vividly illustrated by the significant improvements achieved in the catalytic performance of the chitosanase, encompassing both its activity and stability. These developments offer a valuable model that may have implications for the semi-rational design of other enzymes.

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.

A trapped covalent intermediate as a key catalytic element in the hydrolysis of a GH3 ß-glucosidase: An X-ray crystallographic and biochemical study.

Glycoside hydrolases (GHs) are industrially important enzymes that hydrolyze glycosidic bonds in glycoconjugates. In this study, we found a GH3 ß-glucosidase (CcBgl3B) from Cellulosimicrobium cellulans sp. 21 was able to selectively hydrolyze the ß-1,6-glucosidic bond linked glucose of ginsenosides. X-ray crystallographic studies of the ligand complex ginsenoside-specific ß-glucosidase provided a novel finding that support the catalytic mechanism of GH3. The substrate was clearly identified within the catalytic center of wild-type CcBgl3B, revealing that the C1 atom of the glucose was covalently bound to the Oδ1 group of the conserved catalytic nucleophile Asp264 as an enzyme-glycosyl intermediate. The glycosylated Asp264 could be identified by mass spectrometry. Through site-directed mutagenesis studies with Asp264, it was found that the covalent intermediate state formed by Asp264 and the substrate was critical for catalysis. In addition, Glu525 variants (E525A, E525Q and E525D) showed no or marginal activity against pNPßGlc; thus, this residue could supply a proton for the reaction. Overall, our study provides an insight into the catalytic mechanism of the GH3 enzyme CcBgl3B.

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.

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.

Expression and characterization of α-1,3-glucanase from Paenibacillus alginolyticus NBRC15375, which is classified into subgroup 2 (minor group) of GH family 87.

Bacterial α-1,3-glucanase, classified as glycoside hydrolase (GH) family 87, has been divided into 3 subgroups based on differences in gene sequences in the catalytic domain. The enzymatic properties of subgroups 1 and 3 of several bacteria have been previously investigated and reported; however, the chemical characterization of subgroup 2 enzymes has not been previously conducted. The α-1,3-glucanase gene from Paenibacillus alginolyticus NBRC15375 (PaAgl) belonging to subgroup 2 of GH family 87 was cloned and expressed in Escherichia coli. PgAgl-N1 (subgroup 3) and PgAgl-N2 (subgroup 1) from P. glycanilyticus NBRC16188 were expressed in E. coli, and their enzymatic characteristics were compared. The amino acid sequence of PaAgl demonstrated that the homology was significantly lower in other subgroups when only the catalytic domain was compared. The oligosaccharide products of the mutan-degrading reaction seemed to have different characteristics among subgroups 1, 2, and 3 in GH family 87.

Structural and functional analysis of SpGlu64A: a novel glycoside hydrolase family 64 laminaripentaose-producing ß-1,3-glucanase from Streptomyces pratensis.

Laminaripentaose (L5)-producing ß-1,3-glucanases can preferentially cleave the triple-helix curdlan into ß-1,3-glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, ß-1,3-glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a ß-1,3-glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·mg-1). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (α/ß) domain, which formed an unusually wide groove with a crescent-like structure. In the two complex structures (SpGlu64A-L3 and SpGlu64A-L4), two oligosaccharide chains were captured and the triple-helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple-helical ß-1,3-glucan. A catalytic framework (ß6-ß9-ß10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype-like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A-CBM) and a mutant (Y161F; by site-directed mutation) were obtained, with 1.2- and 1.7-fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A-CBM and -Y161F improved the enzyme activity by 2.63-fold. The study will not only be helpful in understanding the reaction mechanism of ß-1,3-glucanases but will also provide a basis for further enzyme engineering.

Efficient biosynthesis of prunin in methanol cosolvent system by an organic solvent-tolerant α-L-rhamnosidase from Spirochaeta thermophila.

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The Km of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while kcat/Km value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin.

QM/MM study of the catalytic reaction of aphid myrosinase.

Brevicoryne brassicae, an aphid species, exclusively consumes plants from the Brassicaceae family and employs a sophisticated defense mechanism involving a myrosinase enzyme that breaks down glucosinolates obtained from its host plants. In this work, we employed combined quantum mechanical and molecular mechanical (QM/MM) calculations and molecular dynamics (MD) simulations to study the catalytic reaction of aphid myrosinase. A proper QM region to study the myrosinase reaction should contain the whole substrate, models of Gln-19, His-122, Asp-124, Asn-166, Glu-167, Lys-173, Tyr-180, Val-228, Tyr-309, Tyr-346, Ile-347, Glu-374, Glu-423, Trp-424, and a water molecule. The calculations show that Asp-124 and Glu-423 must be charged, His-122 must be protonated on NE2, and Glu-167 must be protonated on OE2. Our model reproduces the anomeric retaining characteristic of myrosinase and indicates that the deglycosylation reaction is the rate-determining step of the reaction. Based on the calculations, we propose a reaction mechanism for aphid myrosinase-mediated hydrolysis of glucosinolates with an overall barrier of 15.2 kcal/mol. According to the results, removing a proton from Arg-312 or altering it to valine or methionine increases glycosylation barriers but decreases the deglycosylation barrier.