Biochemical characterization and cleavage specificities analyses of three endo-1,3-fucanases within glycoside hydrolase family 174.

Sulfated fucans have garnered extensive research interest in recent decades due to their varied bioactivity. Fucanases are important tools for investigating sulfated fucans. This study reported the bioinformatic analysis and biochemical properties of three GH174 family endo-1,3-fucanases. Wherein, Fun174Rm and Fun174Sb showed the highest optimal reaction temperature among the reported fucanases, and Fun174Sb possessed favorable thermostability and catalysis efficiency. Fun174Rm displayed a random endo-acting manner, while Fun174Ri and Fun174Sb hydrolyzed sulfated fucan in processive manners. UPLC-MS and NMR analyses confirmed that the three enzymes catalyze cleavage of the α(1 â†’ 3)-bonds between Fucp2S and Fucp2S in the sulfated fucan from Isostichopus badionotus. These enzymes demonstrated novel cleavage specificities, which could accept α-Fucp2S residues at subsites -1 and + 1. The acquiring of these biotechnological tools would be beneficial to the in-depth research of sulfated fucans.

Remote loop evolution reveals a complex biological function for chitinase enzymes beyond the active site.

Loops are small secondary structural elements that play a crucial role in the emergence of new enzyme functions. However, the evolutionary molecular mechanisms how proteins acquire these loop elements and obtain new function is poorly understood. To address this question, we study glycoside hydrolase family 19 (GH19) chitinase-an essential enzyme family for pathogen degradation in plants. By revealing the evolutionary history and loops appearance of GH19 chitinase, we discover that one loop which is remote from the catalytic site, is necessary to acquire the new antifungal activity. We demonstrate that this remote loop directly accesses the fungal cell wall, and surprisingly, it needs to adopt a defined structure supported by long-range intramolecular interactions to perform its function. Our findings prove that nature applies this strategy at the molecular level to achieve a complex biological function while maintaining the original activity in the catalytic pocket, suggesting an alternative way to design new enzyme function.

Targeting PARG induces tumor cell growth inhibition and antitumor immune response by reducing phosphorylated STAT3 in ovarian cancer.

BACKGROUND: Ovarian cancer is the most lethal gynecological malignancy, with limited treatment options after failure of standard therapies. Despite the potential of poly(ADP-ribose) polymerase inhibitors in treating DNA damage response (DDR)-deficient ovarian cancer, the development of resistance and immunosuppression limit their efficacy, necessitating alternative therapeutic strategies. Inhibitors of poly(ADP-ribose) glycohydrolase (PARG) represent a novel class of inhibitors that are currently being assessed in preclinical and clinical studies for cancer treatment. METHODS: By using a PARG small-molecule inhibitor, COH34, and a cell-penetrating antibody targeting the PARG's catalytic domain, we investigated the effects of PARG inhibition on signal transducer and activator of transcription 3 (STAT3) in OVCAR8, PEO1, and Brca1-null ID8 ovarian cancer cell lines, as well as in immune cells. We examined PARG inhibition-induced effects on STAT3 phosphorylation, nuclear localization, target gene expression, and antitumor immune responses in vitro, in patient-derived tumor organoids, and in an immunocompetent Brca1-null ID8 ovarian mouse tumor model that mirrors DDR-deficient human high-grade serous ovarian cancer. We also tested the effects of overexpressing a constitutively activated STAT3 mutant on COH34-induced tumor cell growth inhibition. RESULTS: Our findings show that PARG inhibition downregulates STAT3 activity through dephosphorylation in ovarian cancer cells. Importantly, overexpression of a constitutively activated STAT3 mutant in tumor cells attenuates PARG inhibitor-induced growth inhibition. Additionally, PARG inhibition reduces STAT3 phosphorylation in immune cells, leading to the activation of antitumor immune responses, shown in immune cells cocultured with ovarian cancer patient tumor-derived organoids and in immune-competent mice-bearing mouse ovarian tumors. CONCLUSIONS: We have identified a novel antitumor mechanism underlying PARG inhibition beyond its primary antitumor effects through blocking DDR in ovarian cancer. Furthermore, targeting PARG activates antitumor immune responses, thereby potentially increasing response rates to immunotherapy in patients with ovarian cancer.

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.

Novel glycosidase from Paenibacillus lactis 154 hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins.

Oleanane-type ginsenosides are a class of compounds with remarkable pharmacological activities. However, the lack of effective preparation methods for specific rare ginsenosides has hindered the exploration of their pharmacological properties. In this study, a novel glycoside hydrolase PlGH3 was cloned from Paenibacillus lactis 154 and heterologous expressed in Escherichia coli. Sequence analysis revealed that PlGH3 consists of 749 amino acids with a molecular weight of 89.5 kDa, exhibiting the characteristic features of the glycoside hydrolase 3 family. The enzymatic characterization results of PlGH3 showed that the optimal reaction pH and temperature was 8 and 50 °C by using p-nitrophenyl-ß-D-glucopyranoside as a substrate, respectively. The Km and kcat values towards ginsenoside Ro were 79.59 ± 3.42 µM and 18.52 s-1, respectively. PlGH3 exhibits a highly specific activity on hydrolyzing the 28-O-ß-D-glucopyranosyl ester bond of oleanane-type saponins. The mechanism of hydrolysis specificity was then presumably elucidated through molecular docking. Eventually, four kinds of rare oleanane-type ginsenosides (calenduloside E, pseudoginsenoside RP1, zingibroside R1, and tarasaponin VI) were successfully prepared by biotransforming total saponins extracted from Panax japonicus. This study contributes to understanding the mechanism of enzymatic hydrolysis of the GH3 family and provides a practical route for the preparation of rare oleanane-type ginsenosides through biotransformation. KEY POINTS: • The glucose at C-28 in oleanane-type saponins can be directionally hydrolyzed. • Mechanisms to interpret PlGH3 substrate specificity by molecular docking. • Case of preparation of low-sugar alternative saponins by directed hydrolysis.

Conversion of ß-1,6-Glucans to Gentiobiose using an endo-ß-1,6-Glucanase PsGly30A from Paenibacillus sp. GKG.

A plethora of di- and oligosaccharides isolated from the natural sources are used in food and pharmaceutical industry. An enzymatic hydrolysis of fungal cell wall ß-glucans is a good alternative to produce the desired oligosaccharides with different functionalities, such as the flavour enhancer gentiobiose. We have previously identified PsGly30A as a potential yeast cell wall degrading ß-1,6-glycosidase. The aim of this study is to characterise the PsGly30A enzyme, a member of the GH30 family, and to evaluate its suitability for the production of gentiobiose from ß-1,6-glucans. An endo-ß-1,6-glucanase PsGly30A encoding gene from Paenibacillus sp. GKG has been cloned and overexpressed in Escherichia coli. The recombinant enzyme has been active towards pustulan and yeast ß-glucan, but not on laminarin from the Laminaria digitata, confirming the endo-ß-1,6-glucanase mode of action. The PsGly30A shows the highest activity at pH 5.5 and 50 °C. The specific activity of PsGly30A on pustulan (1262±82 U/mg) is among the highest reported for GH30 ß-1,6-glycosidases. Moreover, gentiobiose is the major reaction product when pustulan, yeast ß-glucan or yeast cell walls have been used as a substrate. Therefore, PsGly30A is a promising catalyst for valorisation of the yeast-related by-products.

Site-Specific Conjugation of Native Antibody: Transglutaminase-Mediated Modification of a Conserved Glutamine While Maintaining the Primary Sequence and Core Fc Glycan via Trimming with an Endoglycosidase.

A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.

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.

Functional Characterization of Endo- and Exo-Hydrolase Genes in Arabinan Degradation Gene Cluster of Bifidobacterium longum subsp. suis.

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics.

Genome-Wide Identification, Characterization, and Expression Analysis of Four Subgroup Members of the GH13 Family in Wheat (Triticum aestivum L.).

The glycoside hydrolase 13 (GH13) family is crucial for catalyzing α-glucoside linkages, and plays a key role in plant growth, development, and stress responses. Despite its significance, its role in plants remains understudied. This study targeted four GH13 subgroups in wheat, identifying 66 GH13 members from the latest wheat database (IWGSC RefSeq v2.1), including 36 α-amylase (AMY) members, 18 1,4-α-glucan-branching enzyme (SBE) members, 9 isoamylase (ISA) members, and 3 pullulanase (PU) members. Chromosomal distribution reveals a concentration of wheat group 7 chromosomes. Phylogenetic analysis underscores significant evolutionary distance variations among the subgroups, with distinct molecular structures. Replication events shaped subgroup evolution, particularly in regard to AMY members. Subcellular localization indicates AMY member predominance in extracellular and chloroplast regions, while others localize solely in chloroplasts, confirmed by the heterologous expression of TaSEB16 and TaAMY1 in tobacco. Moreover, 3D structural analysis shows the consistency of GH13 across species. Promoter cis-acting elements are suggested to be involved in growth, stress tolerance, and starch metabolism signaling. The RNA-seq data revealed TaGH13 expression changes under drought and submergence stress, and significant expression variation was observed between strong and weak gluten varieties during seed germination using quantitative real-time PCR (qRT-PCR), correlating with seed starch content. These findings demonstrate the pivotal role of GH13 family gene expression in wheat germination, concerning variety preference and environmental stress. Overall, this study advances the understanding of wheat GH13 subgroups, laying the groundwork for further functional studies.

Molecular cloning, expression, purification, and characterization of Bacillus subtilis hydrolyzed ginsenoside Rc of α-L-arabinofuranosidase in Escherichia coli.

The α-L-arabinofuranosidase enzyme plays a crucial role in the degradation of ginsenosides. In this study, we successfully cloned and expressed a novel α-L-arabinofuranosidase bsafs gene (1503 bp, 501 amino acids, 55 kDa, and pI = 5.4) belonging to glycosyl hydrolase (GH) family 51 from Bacillus subtilis genome in Escherichia coli BL21 cells. The recombinant protein Bsafs was purified using Ni2+ sepharose fastflow affinity chromatography and exhibited a specific activity of 2.91 U/mg. Bsafs effectively hydrolyzed the α-L-arabinofuranoside at C20 site of ginsenoside Rc to produce Rd as the product. The Km values for hydrolysis of pNP-α-L-arabinofuranoside (pNPαAraf) and ginsenoside Rc were determined as 0.74 and 4.59 mmol/L, respectively; while the Vmax values for these substrates were found to be 24 and 164 µmol/min/mg, respectively; furthermore, the Kcat values for these enzymes were calculated as 22.3 and 1.58 S-1 correspondingly.

Correlation between seminal plasma biochemical markers and semen parameters in idiopathic oligoasthenoteratospermia: identification of biomarkers for L-carnitine therapy.

Background: L-carnitine therapy for idiopathic sperm abnormalities exhibits variable effectiveness, and currently, there are no established criteria to predict patient response. This study investigated correlations between seminal plasma markers and semen parameters to identify biomarkers that can guide indications for L-carnitine therapy indications in patients with idiopathic sperm abnormalities. Methods: A retrospective review was conducted on 223 male patients with idiopathic oligoasthenoteratospermia, who sought medical attention at our clinic between January 2020 and October 2022. These patients underwent a pretreatment seminal plasma biochemical analysis, followed by a three-month continuous L-carnitine treatment. The correlation between seminal plasma biochemical parameters and pretreatment semen parameters was analyzed. Semen quality was compared between cases with normal and abnormal seminal plasma biochemical parameters, both pretreatment and posttreatment. The correlation between the changes in semen parameters after treatment and seminal plasma biochemical parameters were investigated. Results: Correlation analyses revealed significant associations between all pretreatment semen parameters and seminal plasma biochemical markers, except for liquefying time and the ratio of normal morphology. Subgroup analysis, stratified by seminal fructose, zinc, citric acid, and neutral glycosidase levels, demonstrated that abnormal groups exhibited significantly different levels of semen parameters compared with the normal groups. The changing difference and changing ratio in the ratio of forward motile sperm showed a negative correlation with seminal fructose levels (r=-0.165 and -0.144). The changing difference in semen volume was negatively correlated with the level of seminal neutral glycosidase (r=-0.158). The changing ratio in semen volume, sperm concentration, total sperm count, and count of forward motile sperm all exhibited negative correlations with the levels of seminal neutral glycosidase (range from -0.178 to -0.224). Conclusion: Seminal plasma biochemical markers, particularly fructose and neutral glycosidase, may serve as valuable indicators for determining the eligibility of patients with idiopathic sperm abnormalities for L-carnitine therapy.

Heat-induced pressed gels from canola press cakes: Exploring the impact of starting materials, stirring conditions, and carbohydrase pretreatment.

Alternative plant protein sources offer excellent solutions for tackling the current challenge of food insecurity and sustainability. Inspired by soy tofu, pressed gels represent a robust and versatile way to create protein-enriched plant products. Here, production of heat-induced pressed gels from canola cold-pressed cakes (CPC) and hot-pressed cakes (HPC) was investigated under varied stirring conditions. Pressed gels prepared from CPC resulted in a greater yield and protein recovery than that of HPC. While using carbohydrases as a pretreatment was ineffective in improving yield and protein recovery, applying a stirring condition during heating increased the protein recovery up to 38.3%. Also, stirring condition was proved to be able to modulate the textural properties by controlling the compactness and the size of aggregates. It is revealed that pressed gels are stabilized through a combination of hydrogen bonds, hydrophobic interactions, and disulfide bonds. In comparison to canola press cake, the pressed gels contained less glucosinolates and phenolic compounds, but more phytic acid. A mechanism of formation has been hypothesized based on the nucleation-growth mechanism, and a shift was proposed from diffusion-limited processes in non-stirred pressed gels to reaction-limited process in stirred pressed gels. In conclusion, the potential of canola heat-induced pressed gels was demonstrated both as a stand-alone product and a micro-structured protein extract.

A novel glycoside hydrolase 43-like enzyme from Clostridium boliviensis is an endo-xylanase and a candidate for xylooligosaccharide production from different xylan substrates.

An uncharacterized gene encoding a glycoside hydrolase family 43-like enzyme from Clostridium boliviensis strain E-1 was identified from genomic sequence data, and the encoded enzyme, CbE1Xyn43-l, was produced in Escherichia coli. CbE1Xyn43-l (52.9 kDa) is a two-domain endo-ß-xylanase consisting of a C-terminal CBM6 and a GH43-like catalytic domain. The positions of the catalytic dyad conserved in GH43, the catalytic base (Asp74), and proton donor (Glu240) were identified in alignments including GH43-enzymes of known 3D-structure from different subfamilies. CbE1Xyn43-l is active at pH 7.0-9.0, with optimum temperature at 65°C, and a more than 7 days' half-life in irreversible deactivation studies at this temperature. The enzyme hydrolyzed birchwood xylan, quinoa stalks glucuronoarabinoxylan, and wheat arabinoxylan with xylotriose and xylotetraose as major hydrolysis products. CbE1Xyn43-l also released xylobiose from pNPX2 with low turnover (kcat of 0.044 s-1) but was inactive on pNPX, showing that a degree of polymerization of three (DP3) was the smallest hydrolyzable substrate. Divalent ions affected the specific activity on xylan substrates, which dependent on the ion could be increased or decreased. In conclusion, CbE1Xyn43-l from C. boliviensis strain E-1 is the first characterized member of a large group of homologous hypothetical proteins annotated as GH43-like and is a thermostable endo-xylanase, producing xylooligosaccharides of high DP (xylotriose and xylotetraose) producer. IMPORTANCE: The genome of Clostridium boliviensis strain E-1 encodes a number of hypothetical enzymes, annotated as glycoside hydrolase-like but not classified in the Carbohydrate Active Enzyme Database (CAZy). A novel thermostable GH43-like enzyme is here characterized as an endo-ß-xylanase of interest in the production of prebiotic xylooligosaccharides (XOs) from different xylan sources. CbE1Xyn43-l is a two-domain enzyme composed of a catalytic GH43-l domain and a CBM6 domain, producing xylotriose as main XO product. The enzyme has homologs in many related Clostridium strains which may indicate a similar function and be a previously unknown type of endo-xylanase in this evolutionary lineage of microorganisms.

A ß-Primeverosidase-like Enzyme in Soybean [Glycine max (L.) Merr] Hypocotyls: Specificity toward 1-Octen-3-yl and 3-Octanyl ß-Primeverosides.

A novel ß-primeverosidase-like enzyme, originating from the hypocotyl of soybeans, was isolated and characterized. This enzyme, with an estimated molecular weight of 44 kDa, was identified as a monomer and exhibited peak activity at 55 °C and pH 5.5. It demonstrated a specific and efficient hydrolysis of 1-octen-3-yl ß-primeveroside (1-octen-3-yl prim) and 3-octanyl ß-primeveroside (3-octanyl prim) but did not act on glucopyranosides. Mn2+ significantly enhanced its activity, while Zn2+, Cu2+, and Hg2+ exerted inhibitory effects. Kinetic analysis revealed a higher hydrolytic capacity toward 1-octen-3-yl prim. Partial amino acid sequences were determined and the N-terminal amino acid sequence was determined to be AIVAYAL ALSKRAIAAQ. The binding energy and binding free energy between the ß-primeverosidase enzyme and its substrates were observed to be higher than that of ß-glucosidase, thus validating its superior hydrolysis efficiency. Hydrogen bonds and hydrophobic interactions were the main types of interactions between ß-primeverosidase enzyme and 1-octen-3-yl prim and 3-octanyl prim, involving amino acid residues such as GLU-470, TRP-463, GLU-416, TRP-471, GLN-53, and GLN-477 (hydrogen bonds) and PHE-389, TYR-345, LEU-216, and TYR-275 (hydrophobic interactions). This study contributes to the application of a ß-primeverosidase-like enzyme in improving the release efficiency of glycosidically conjugated flavor substances.

Amphiphilic α-Peptoid-deoxynojirimycin Conjugate-based Multivalent Glycosidase Inhibitor for Hypoglycemic Effect and Fluorescence Imaging.

Multivalent glycosidase inhibitors based on 1-deoxynojirimycin derivatives against α-glucosidases have been rapidly developed. Nonetheless, the mechanism based on self-assembled multivalent glucosidase inhibitors in living systems needs to be further studied. It remains to be determined whether the self-assembly possesses sufficient stability to endure transit through the small intestine and subsequently bind to the glycosidases located therein. In this paper, two amphiphilic compounds, 1-deoxynojirimycin and α-peptoid conjugates (LP-4DNJ-3C and LP-4DNJ-6C), were designed. Their self-assembling behaviors, multivalent α-glucosidase inhibition effect, and fluorescence imaging on living organs were studied. LP-4DNJ-6C exhibited better multivalent α-glucosidase inhibition activities in vitro. Moreover, the self-assembly of LP-4DNJ-6C could effectively form a complex with Nile red. The complex showed fluorescence quenching effect upon binding with α-glucosidases and exhibited potent fluorescence imaging in the small intestine. This result suggests that a multivalent hypoglycemic effect achieved through self-assembly in the intestine is a viable approach, enabling the rational design of multivalent hypoglycemic drugs.

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.

Chemical stability of carboxylpyranocyanidin-3-O-glucoside under ß-glucosidase treatment and description of their interaction.

Anthocyanins are susceptible to degradation by ß-glycosidase, resulting in color loss. This study analyzed the impact of ß-glycosidase on carboxylpyranocyanidin-3-O-glucoside (Carboxyl-pycy-3-gluc) and its precursor cyanidin-3-O-glucoside (Cy-3-gluc). Carboxyl-pycy-3-gluc exhibited enhanced stability upon treatment with ß-glucosidase. Ultraviolet-visible and circular dichroism spectroscopy revealed slight changes in the microenvironment and secondary structure of ß-glycosidase when carboxyl-pycy-3-gluc was present. The fluorescence experiment indicated that anthocyanins quench the fluorescence of ß-glycosidase through static quenching via hydrophobic interactions. Molecular docking of six types of carboxylpyranoanthocyanins and their precursors with ß-glycosidase revealed that carboxylpyranoanthocyanins exhibited lower binding affinity than their precursors, consistent with the enzyme kinetic experiment results. The incorporation carboxyl-pycy-3-gluc into Sanhua Plum Juice and Wine endowed them with vivid and stable coloration. The study illustrated that carboxyl-pycy-3-gluc exhibits low binding affinity with ß-glycosidase, thereby maintaining stability and confirming its potential as a colorant.

Pullulanase pretreatment of highly concentrated maltodextrin solution improves maltose yield during ß-amylase-catalyzed saccharification.

Increasing the substrate concentration can effectively reduce energy consumption and result in more economic benefits in the industrial production of maltose, but this process remarkably increases the viscosity, which has a negative effect on saccharification. To improve saccharification efficiency, pullulanase is usually employed. In the conventional process of maltose production, pullulanase is added at the same time with ß-amylase or later, but this process seems inefficient when the substrate concentration is high. Herein, a novel method was introduced to enhance the maltose yield under high substrate concentration. The results indicated that the pullulanase pretreatment of highly concentrated maltodextrin solution for 2 h greatly affects the final conversion rate of ß-amylase-catalyzed saccharification. The maltose yield reached 80.95 %, which is 11.8 % above the control value. Further examination confirmed that pullulanase pretreatment decreased the number of branch points of maltodextrin and resulted in a high content of oligosaccharides. These linear chains were suitable for ß-amylase-catalyzed saccharification to produce maltose. This research offers a new effective and green strategy for starch sugar production.

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.