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Rapeseed (Brassica napus L.), accounts for nearly 16% of vegetable oil, is the world's second produced oilseed. However, pod shattering has caused significant yield loses in rapeseed production, particularly during mechanical harvesting. The GH28 genes can promote pod shattering by changing the structure of the pod cell wall in Arabidopsis. However, the role of the GH28 gene family in rapeseed was largely unknown. Therefore, a genome-wide comprehensive analysis was conducted to classify the role of GH28 gene family on rapeseed pod shattering. A total of 37 BnaGH28 genes in the rapeseed genome were identified. These BnaGH28s can be divided into five groups (Group A-E), based on phylogenetic and synteny analysis. Protein property, gene structure, conserved motif, cis-acting element, and gene expression profile of BnaGH28 genes in the same group were similar. Specially, the expression level of genes in group A-D was gradually decreased, but increased in group E with the development of silique. Among eleven higher expressed genes in group E, two BnaGH28 genes (BnaA07T0199500ZS and BnaC06T0206500ZS) were significantly regulated by IAA or GA treatment. And the significant effects of BnaA07T0199500ZS variation on pod shattering resistance were also demonstrated in present study. These results could open a new window for insight into the role of BnaGH28 genes on pod shattering resistance in rapeseed.

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KEY MESSAGE: The genomic location and stage-specific expression pattern of GH9 genes reveal their critical roles during fruit abscission zone formation in Vaccinium ashei. Glycosyl hydrolase family 9 (GH9) cellulases play a crucial role in both cellulose synthesis and hydrolysis during plant growth and development. Despite this importance, there is currently no study on the involvement of GH9-encoding genes, specifically VaGH9s, in abscission zone formation of rabbiteye blueberries (Vaccinium ashei). In this study, we identified a total of 61 VaGH9s in the genome, which can be classified into 3 subclasses based on conserved motifs and domains, gene structures, and phylogenetic analyses. Our synteny analysis revealed that VaGH9s are more closely related to the GH9s of Populus L. than to those of Arabidopsis, Vitis vinifera, and Citrus sinensis. In silico structural analysis predicted that most of VaGH9s are hydrophilic, and localized in cell membrane and/or cell wall, and the variable sets of cis-acting regulatory elements and functional diversity with four categories of stress response, hormone regulation, growth and development, and transcription factor-related elements are present in the promoter sequence of VaGH9s genes. Transcriptomic analysis showed that there were 22 differentially expressed VaGH9s in fruit abscission zone tissue at the veraison stage, and the expression of VaGH9B2 and VaGH9C10 was continuously increased during fruit maturation, which were in parallel with the increasing levels of cellulase activity and oxidative stress indicators, suggesting that they are involved in the separation stage of fruit abscission in Vaccinium ashei. Our work identified 22 VaGH9s potentially involved in different stages of fruit abscission and would aid further investigation into the molecular regulation of abscission in rabbiteye blueberries fruit.

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BACKGROUND: The Tunicata or Urochordata is the only animal group with the ability to synthesize cellulose directly and cellulose is a component of the tunic that covers the entire tunicate body. The genome of Ciona intestinalis type A contains a cellulose synthase gene, CesA, that it acquired via an ancient, horizontal gene transfer. CesA is expressed in embryonic epidermal cells and functions in cellulose production. Ciona CesA is composed of both a glycosyltransferase domain, GT2, and a glycosyl hydrolase domain, GH6, which shows a mutation at a key position and seems functionless. Interestingly, the Ciona genome contains a glycosyl hydrolase gene, GH6-1, in which the GH6 domain seems intact. This suggests expression and possible functions of GH6-1 during Ciona embryogenesis. Is GH6-1 expressed during embryogenesis? If so, in what tissues is the gene expressed? Does GH6-1 serve a function? If so, what is it? Answers to these questions may advance our understanding of evolution of this unique animal group. RESULTS: Quantitative reverse transcription PCR and in situ hybridization revealed that GH6-1 is expressed in epidermis of tailbud embryos and in early swimming larvae, a pattern similar to that of CesA. Expression is downregulated at later stages and becomes undetectable in metamorphosed juveniles. The GH6-1 expression level is higher in the anterior-trunk region and caudal-tip regions of late embryos. Single-cell RNA sequencing analysis of the late tailbud stage showed that cells of three clusters with epidermal identity express GH6-1, and that some of them co-express CesA. TALEN-mediated genome editing was used to generate GH6-1 knockout Ciona larvae. Around half of TALEN-electroporated larvae showed abnormal development of adhesive papillae and altered distribution of surface cellulose. In addition, three-fourths of TALEN-electroporated animals failed to complete larval metamorphosis. CONCLUSIONS: This study showed that tunicate GH6-1, a gene that originated by horizontal gene transfer of a prokaryote gene, is recruited into the ascidian genome, and that it is expressed and functions in epidermal cells of ascidian embryos. Although further research is required, this observation demonstrates that both CesA and GH6-1 are involved in tunicate cellulose metabolism, impacting tunicate morphology and ecology.

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Insects have evolved with effective strategies to utilize cellulose as an energy source by possessing cellulolytic enzymes which can be used as an optimal resource in the bioenergy sector. The study was aimed at evaluating the cellulolytic enzyme in the larval gut of the banana pseudostem weevil, Odoiporus longicollis Olivier (Coleoptera: Curculionidae). Primarily, cellulase activity was localized along the gut, in which the midgut showed the highest activity (2858 U/mg). The thermo-tolerance of cellulase activity was found to be up to 80°C (highest at 60°C), and the enzyme was stable at a pH between 5 and 6. Various concentrations of divalent cations (CaCl2 , MgCl2 , and CuCl2 ) have differential enhancing and inhibitory effects on cellulase activity. The cellulase (OlCel) was purified using anion exchange chromatography. The molecular weight of the cellulase was determined to be 47 kDa. The physicochemical parameters of the purified enzyme were similar to that of enzyme activity of whole gut extract. Mass spectrometry results identified sequence similarities of purified cellulase to the glycosyl hydrolase family 5 (GHF5) family. The gut microbial cellulase activity as exogenous source showed no competence compared with the endogenous activity.

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Rhodotorula dairenensis ß-fructofuranosidase is a highly glycosylated enzyme with broad substrate specificity that catalyzes the synthesis of 6-kestose and a mixture of the three series of fructooligosaccharides (FOS), fructosylating a variety of carbohydrates and other molecules as alditols. We report here its three-dimensional structure, showing the expected bimodular arrangement and also a unique long elongation at its N-terminus containing extensive O-glycosylation sites that form a peculiar arrangement with a protruding loop within the dimer. This region is not required for activity but could provide a molecular tool to target the dimeric protein to its receptor cellular compartment in the yeast. A truncated inactivated form was used to obtain complexes with fructose, sucrose and raffinose, and a Bis-Tris molecule was trapped, mimicking a putative acceptor substrate. The crystal structure of the complexes reveals the major traits of the active site, with Asn387 controlling the substrate binding mode. Relevant residues were selected for mutagenesis, the variants being biochemically characterized through their hydrolytic and transfructosylating activity. All changes decrease the hydrolytic efficiency against sucrose, proving their key role in the activity. Moreover, some of the generated variants exhibit redesigned transfructosylating specificity, which may be used for biotechnological purposes to produce novel fructosyl-derivatives.

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A xylanase gene xynZT-1 from Alteromonas macleodii HY35 was cloned and expressed in Escherichia coli (E. coli). The sequencing results showed that the ORF of xynZT-1 was 831 bp. The xylanase DNA sequence encoded a 29 amino acids (aa) signal peptide and a 247-aa mature peptide. The XynZT-1 has been a calculated molecular weight (MW) of 27.93 kDa, isoelectric point (pI) of 5.11 and the formula C1266H1829N327O384S5. The amino acid sequence of the xynZT-1 had a high similarity with that of glycosyl hydrolase family 11 (GHF11) reported from other microorganisms. The DNA sequence encoding mature peptide was subcloned into pET-28a(+) expression vector. The resulted plasmid pET-28a-xynZT-1 was transformed into E. coli BL21(DE3), and the recombinant strain BL21(DE3)/xynZT-1 was obtained. The optimum temperature and pH of the recombinant XynZT-1 were 45 ℃ and 5.0, respectively.

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Transglycosylating glycoside hydrolases (GHs) offer great potential for the enzymatic synthesis of oligosaccharides. Although knowledge is progressing, there is no unique strategy to improve the transglycosylation yield. Obtaining efficient enzymatic tools for glycan synthesis with GHs remains dependent on an improved understanding of the molecular factors governing the balance between hydrolysis and transglycosylation. This enzymatic and structural study of RBcel1, a transglycosylase from the GH5_5 subfamily isolated from an uncultured bacterium, aims to unravel such factors. The size of the acceptor and donor sugars was found to be critical since transglycosylation is efficient with oligosaccharides at least the size of cellotetraose as the donor and cellotriose as the acceptor. The reaction pH is important in driving the balance between hydrolysis and transglycosylation: hydrolysis is favored at pH values below 8, while transglycosylation becomes the major reaction at basic pH. Solving the structures of two RBcel1 variants, RBcel1_E135Q and RBcel1_Y201F, in complex with ligands has brought to light some of the molecular factors behind transglycosylation. The structure of RBcel1_E135Q in complex with cellotriose allowed a +3 subsite to be defined, in accordance with the requirement for cellotriose as a transglycosylation acceptor. The structure of RBcel1_Y201F has been obtained with several transglycosylation intermediates, providing crystallographic evidence of transglycosylation. The catalytic cleft is filled with (i) donors ranging from cellotriose to cellohexaose in the negative subsites and (ii) cellobiose and cellotriose in the positive subsites. Such a structure is particularly relevant since it is the first structure of a GH5 enzyme in complex with transglycosylation products that has been obtained with neither of the catalytic glutamate residues modified.

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To efficiently decompose biomass, fungi have developed various enzymatic and non-enzymatic strategies and are a source of versatile biocatalysts. The endoglucanases in glycosyl hydrolase CAZy family 45 (GH45) are known for their small size, a high thermostability and a broad substrate specificity that has been employed in textile and detergent industries. Here we report the heterologous expression and characterisation of an GH45 endoglucanase from the brown rot Fomitopsis pinicola and its direct comparison to an already characterised GH45 from the white rot Phanerochaete chrysosporium. Both enzymes were recombinantly expressed in Pichia pastoris and purified by two chromatographic steps. The biochemical characterisation highlighted the acidophilic character, with an optimal pH of 4, and a preference for amorphous substrates as carboxymethyl cellulose (CMC) and substrates containing ß-1,4-glucans rather than the previously reported ß-1,3/1,4-glucans lichenan and ß-glucan. The dominating products from ß-1,4-glucans were C3-C6 oligosaccharides, whereas from ß-1,3/1,4-glucans glucose was the main reaction product. From the characterisation no differences between the brown rot and the white rot GH45 was evident.

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Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-ß-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.

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The termite Reticulitermes flavipes causes extensive damage due to the high efficiency and broad specificity of the ligno- and hemicellulolytic enzyme systems produced by its symbionts. Thus, the R. flavipes gut microbiome is expected to constitute an excellent source of enzymes that can be used for the degradation and valorization of plant biomass. The symbiont Opitutaceae bacterium strain TAV5 belongs to the phylum Verrucomicrobia and thrives in the hindgut of R. flavipes. The sequence of the gene with the locus tag opit5_10225 in the Opitutaceae bacterium strain TAV5 genome has been classified as a member of glycoside hydrolase family 5 (GH5), and provisionally annotated as an endo-ß-mannanase. We characterized biochemically and structurally the opit5_10225 gene product, and show that the enzyme, Op5Man5, is an exo-ß-1,4-mannosidase [EC 3.2.1.25] that is highly specific for ß-1,4-mannosidic bonds in mannooligosaccharides and ivory nut mannan. The structure of Op5Man5 was phased using electron cryo-microscopy and further determined and refined at 2.2 Šresolution using X-ray crystallography. Op5Man5 features a 200-kDa large homotrimer composed of three modular monomers. Despite insignificant sequence similarity, the structure of the monomer, and homotrimeric assembly are similar to that of the GH42-family ß-galactosidases and the GH164-family exo-ß-1,4-mannosidase Bs164 from Bacteroides salyersiae. To the best of our knowledge Op5Man5 is the first structure of a glycoside hydrolase from a bacterial symbiont isolated from the R. flavipes digestive tract, as well as the first example of a GH5 glycoside hydrolase with a GH42 ß-galactosidase-type homotrimeric structure.

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Chitinases are of great importance in chitin degradation and remodeling in insects. However, the genome-wide distribution of chitinase-like gene family in Bemsia tabaci, a destructive pest worldwide, is still elusive. With the help of bioinformatics, we annotated 14 genes that encode putative chitinase-like proteins, including ten chitinases (Cht), three imaginal disk growth factors (IDGF), and one endo-ß-N-acetylglucosaminidase (ENGase) in the genome of the whitefly, B. tabaci. These genes were phylogenetically grouped into eight clades, among which 13 genes were classified in the glycoside hydrolase family 18 groups and one in the ENGase group. Afterwards, developmental expression analysis suggested that BtCht10, BtCht5, and BtCht7 were highly expressed in nymphal stages and exhibit similar expression patterns, implying their underlying role in nymph ecdysis. Notably, nymphs exhibited a lower rate of survival when challenged by dsRNA targeting these three genes via a nanomaterial-promoted RNAi method. In addition, silencing of BtCht10 significantly resulted in a longer duration of development compared to control nymphs. These results indicate a key role of BtCht10, BtCht5, and BtCht7 in B. tabaci nymph molting. Our research depicts the differences of chitinase-like family genes in structure and function and identified potential targets for RNAi-based whitefly management.

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The ability of retaining glycoside hydrolases (GHs) to transglycosylate is inherent to the double-displacement mechanism. Studying reaction intermediates, such as the glycosyl-enzyme intermediate (GEI) and the Michaelis complex, could provide valuable information to better understand the molecular factors governing the catalytic mechanism. Here, the GEI structure of RBcel1, an endo-1,4-ß-glucanase of the GH5 family endowed with transglycosylase activity, is reported. It is the first structure of a GH5 enzyme covalently bound to a natural oligosaccharide with the two catalytic glutamate residues present. The structure of the variant RBcel1_E135A in complex with cellotriose is also reported, allowing a description of the entire binding cleft of RBcel1. Taken together, the structures deliver different snapshots of the double-displacement mechanism. The structural analysis revealed a significant movement of the nucleophilic glutamate residue during the reaction. Enzymatic assays indicated that, as expected, the acid/base glutamate residue is crucial for the glycosylation step and partly contributes to deglycosylation. Moreover, a conserved tyrosine residue in the -1 subsite, Tyr201, plays a determinant role in both the glycosylation and deglycosylation steps, since the GEI was trapped in the RBcel1_Y201F variant. The approach used to obtain the GEI presented here could easily be transposed to other retaining GHs in clan GH-A.

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Stevia rebaudiana Bertoni is an important economic crop that is well known for its secondary metabolites, steviol glycosides (SGs), found in leaves. Because the enzymes of deglycosylation (glycoside hydrolases) play important roles in SGs biosynthetic processes, our study is focused on the functions of ß-glucosidases in SGs catabolism in stevia. We cloned and characterized 19 stevia GH1 genes based on transcriptomic sequences. The 19 genes were divided into five putative subfamilies in Arabidopsis. Conserved motifs in the SrGH1 proteins were analysed using the online motif-based sequence analysis tool, MEME. Most of the identified proteins contain the conserved 'TFNEP' motif (contains the catalytic acid/base) and 'ITENG' motif (contains the catalytic nucleophile). Furthermore, the steviol glycoside content and expression of these 19 genes were characterized under constant darkness. The dark treatment lowered the steviol glycoside content significantly, while SrBGLU16 responded to darkness and was markedly upregulated. This study is the first transcriptome-wide analysis of the GH1 family in Stevia rebaudiana. The sequences of 19 SrGH1 members and their expression when grown in darkness were characterized. Among the 19 genes, SrBGLU16 was markedly upregulated by darkness. Thus, we identified SrBGLU16 for further investigation as a possible steviol glycoside beta-glucosidase.

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Horizontal gene transfer (HGT) is the movement of genetic material between different species. Although HGT is less frequent in eukaryotes than in bacteria, several instances of HGT have apparently shaped animal evolution. One well-known example is the tunicate cellulose synthase gene, CesA, in which a gene, probably transferred from bacteria, greatly impacted tunicate evolution. A Glycosyl Hydrolase Family 6 (GH6) hydrolase-like domain exists at the C-terminus of tunicate CesA, but not in cellulose synthases of other organisms. The recent discovery of another GH6 hydrolase-like gene (GH6-1) in tunicate genomes further raises the question of how tunicates acquired GH6. To examine the probable origin of these genes, we analyzed the phylogenetic relationship of GH6 proteins in tunicates and other organisms. Our analyses show that tunicate GH6s, the GH6-1 gene, and the GH6 part of the CesA gene, form two independent, monophyletic gene groups. We also compared their sequence signatures and exon splice sites. All tunicate species examined have shared splice sites in GH6-containing genes, implying ancient intron acquisitions. It is likely that the tunicate CesA and GH6-1 genes existed in the common ancestor of all extant tunicates.

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Microbes, especially the uncultured microbes, have been considered as an important resource for discovery of novel cellulases. In this study, a novel bifunctional cellulase/hemicellulase (ZFYN184) was identified by functional screening of a soil metagenomic library. Sequence analysis indicated that ZFYN184 shared at best 39% identity with glycoside hydrolase family 44 (GH44) proteins and contained a glutamic acid residue at 235 acting as the catalytic proton donor in hydrolysis of polysaccharides. The recombinant ZFYN184 was expressed in Escherichia coli BL21 (DE3), and the biochemical profiles of the enzyme, including optimum pH and temperature, pH and thermal stabilities, tolerance to various additives, and substrate specificity, were determined. ZFYN184 possessed strong endo-ß-1,4-glucanase and endo-1,4-ß-mannanase activities, as well as weak xylanase activity, while all these hydrolytic activities were derived from a single catalytic domain in this GH44 enzyme. KEY POINTS: • Discovery a novel bifunctional glycosyl hydrolase from uncultured microorganism. • ZFYN184 contains a single catalytic domain belonged to GH44.

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ß-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for ß-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding ß-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 ß-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site's narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-ß-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 ß-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 ß-glucosidases following relevant in-silico analyses.

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α-L-Rhamnosidases (α-L-Rha-ases, EC 3.2.1.40) are glycosyl hydrolases (GHs) that hydrolyze a terminal α-linked L-rhamnose residue from a wide spectrum of substrates such as heteropolysaccharides, glycosylated proteins, and natural flavonoids. As a result, they are considered catalysts of interest for various biotechnological applications. α-L-rhamnose (6-deoxy-L-mannose) is structurally similar to the rare sugar α-L-mannose. Here we have examined whether microbial α-L-Rha-ases possess α-L-mannosidase activity by synthesizing the substrate 4-nitrophenyl α-L-mannopyranoside. Four α-L-Rha-ases from GH78 and GH106 families were expressed and purified from Escherichia coli cells. All four enzymes exhibited both α-L-rhamnosyl-hydrolyzing activity and weak α-L-mannosyl-hydrolyzing activity. SpRhaM, a GH106 family α-L-Rha-ase from Sphingomonas paucimobilis FP2001, was found to have relatively higher α-L-mannosidase activity as compared with three GH78 α-L-Rha-ases. The α-L-mannosidase activity of SpRhaM showed pH dependence, with highest activity observed at pH 7.0. In summary, we have shown that α-L-Rha-ases also have α-L-mannosidase activity. Our findings will be useful in the identification and structural determination of α-L-mannose-containing polysaccharides from natural sources for use in the pharmaceutical and food industries.

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Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

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Halophilic bacteria are good bioresources for halotolerant alkaline enzymes. A multi-domain high-molecular-weight endo-ß-1,4-xylanase gene, xylM18, was cloned from a halophilic marine bacterium Marinimicrobium sp. LS-A18. XylM18 is different from any of the functionally reported xylanases. It has a glycosyl hydrolase (GH) 43 domain, a GH10 domain, and two serine-rich linkers, representing a novel family. The gene, encoding 1022 residues, was cloned and heterologously expressed in Escherichia coli BL21(DE3) cells. Purified XylM18 was proved to be a xylanase. It showed diminished activity without salt and showed activity with a broad NaCl range from 0.2 to 25% (w/v). NaCl can increase the optimal temperature from 30 °C (0% NaCl) to 50 °C (10% NaCl). The purified XylM18 was active between pH 6.0 and 10.0 and was optimally active at pH 7.0. The xylanase activities were basically unchanged at a NaCl concentration range from 10 to 20% or pH from 7 to 10 after 24 h incubation. The apparent Km and Vmax values of XylM18 for xylan were 2.76 mg/mL and 60.0 U/mg, respectively. The GH10 domain of this enzyme, XylM18-GH10, was expressed and characterized. XylM18-GH10 also showed xylanase activity and maintained halo-stable property. The apparent Km and Vmax values of XylM18-GH10 for xylan were 1.60 mg/mL and 130.1 U/mg, respectively. Other domains of XylM18 showed no xylanase activity. In summary, XylM18 is a halo-tolerant and alkali-stable endoxylanase which is a suitable candidate for xylan biodegradation in high-salt and alkali conditions. To our knowledge, this is the first report of a multidomain high-molecular-weight xylanase.

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Glycosyl hydrolases belonging to the family 4 (GH4) use a unique redox-based NAD+-dependent reaction mechanism involving anionic intermediates and requires a divalent metal ion and reducing conditions for catalytic activity. These enzymes display wide specificity and selectivity for their substrates. However, the structural basis of substrate binding, recognition and specificity remains poorly studied. Here, we report the crystal structure of Thermotoga maritima TmAgu4B, a GH4 α-glucuronidase, in complex with Co2+ and citrate. Analysis of GH4 structures show that the metal ion is present in a conserved octahedral coordination with conserved side chain atoms, the ligand atoms and an invariant water molecule. The data provides the first structural evidence for a metal-activated hydroxide ion that acts as the general base to deprotonate the C3-hydroxyl group of the glycone, a rate-limiting step in the mechanism. Furthermore, the citrate binding mode in the active site is analogous to a bound glucuronide substrate and provides insights into the mode of substrate interaction with the metal ion, the active site residues and, the structural basis of substrate recognition in a GH4 α-glucuronidase.

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