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Abstract The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application.

The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application. [ABSTRACT FROM AUTHOR]

Abstract Objective New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. Results In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

SUMMARY: Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP‐sugar‐dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl‐glucose‐dependent glucosyltransferase activity modifying cyanidin 3‐xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3‐type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells. Significance Statement: Glucosylation of the purple carrot anthocyanin cyanidin 3‐O‐(2″‐O‐xylosyl)‐(6″‐O‐sinapoylglucosyl)‐galactoside is mediated by a glycoside hydrolase family 3 vacuolar enzyme that catalyzes a reaction that uses acyl‐glucose as the glucosyl donor. [ABSTRACT FROM AUTHOR]

Objective: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase. Results: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists. [ABSTRACT FROM AUTHOR]

The sesaminol triglucoside (STG)-hydrolyzing β-glucosidase from Paenibacillus sp. (PSTG1), which belongs to glycoside hydrolase family 3 (GH3), is a promising catalyst for the industrial production of sesaminol. We determined the X-ray crystal structure of PSTG1 with bound glycerol molecule in the putative active site. PSTG1 monomer contained typical three domains of GH3 with the active site in domain 1 (TIM barrel). In addition, PSTG1 contained an additional domain (domain 4) at the C-terminus that interacts with the active site of the other protomer as a lid in the dimer unit. Interestingly, the interface of domain 4 and the active site forms a hydrophobic cavity probably for recognizing the hydrophobic aglycone moiety of substrate. The short flexible loop region of TIM barrel was found to be approaching the interface of domain 4 and the active site. We found that n -heptyl-β-D-thioglucopyranoside detergent acts as an inhibitor for PSTG1. Thus, we propose that the recognition of hydrophobic aglycone moiety is important for PSTG1-catalyzed reactions. Domain 4 might be a potential target for elucidating the aglycone recognition mechanism of PSTG1 as well as for engineering PSTG1 to create a further excellent enzyme to degrade STG more efficiently to produce sesaminol. [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 3 (GH3) β-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its β-glucosidase has not been identified. In this study, a GH3 β-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p -nitrophenyl β-glycosides, MnBG3A showed activity on d -glucoside (pNP-Glc) and slight activity on d -xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d -glucose caused competitive inhibition (K i = 0.5 m m). MnBG3A acted on β-glucobioses with β1-3, -6, -4, and -2 linkages, in descending order of kcat / Km. In contrast, the regioselectivity for newly formed products was limited to β1-6 linkage. MnBG3A has similar features to those of β-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects. [ABSTRACT FROM AUTHOR]

β-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 β-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction. The structure of the E88A mutant of AnBX, determined at 2.5-Å resolution, contains two molecules in the asymmetric unit, each of which is composed of three domains, namely an N-terminal (β/α)8 TIM-barrel-like domain, an (α/β)6 sandwich domain, and a C-terminal fibronectin type III domain. Asp288 and Glu500 of AnBX were experimentally confirmed to act as the catalytic nucleophile and acid/base catalyst, respectively. The crystal structure revealed that Trp86, Glu88 and Cys289, which formed a disulfide bond with Cys321, were located at subsite -1. Although the E88D and C289W mutations reduced catalytic efficiency toward all four substrates tested, the substitution of Trp86 with Ala, Asp and Ser increased the substrate preference for glucoside relative to xyloside substrates, indicating that Trp86 is responsible for the xyloside specificity of AnBX. The structural and biochemical information of AnBX obtained in this study provides invaluable insight into modulating the enzymatic properties for the hydrolysis of lignocellulosic biomass. Key points: • Asp288 and Glu500 of AnBX are the nucleophile and acid/base catalyst, respectively • Glu88 and the Cys289-Cys321 disulfide bond are crucial for the catalytic activity of AnBX • The W86A and W86S mutations in AnBX increased the preference for glucoside substrates [ABSTRACT FROM AUTHOR]

Aspergillus oryzae isoprimeverose‐producing oligoxyloglucan hydrolase (IpeA) releases isoprimeverose units (α‐d‐xylopyranosyl‐(1→6)‐d‐glucose) from the non‐reducing end of xyloglucan oligosaccharides and belongs to glycoside hydrolase family 3. In this paper, we report the X‐ray crystal structure of the IpeA complexed with a xyloglucan oligosaccharide, (XXXG: Glc4Xyl3). Trp515 of IpeA plays a critical role in XXXG recognition at positive subsites. In addition, docking simulation of IpeA‐XXXG suggested that two Tyr residues (Tyr268 and Tyr445) are involved in the catalytic reaction mechanism of IpeA. Tyr268 plays an important role in product turnover, whereas Tyr445 stabilizes the acid/base Glu524 residue, which serves as a proton donor. Our findings indicate that the substrate recognition machinery of IpeA is specifically adapted to xyloglucan oligosaccharides. [ABSTRACT FROM AUTHOR]

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Background β-Glucosidases have attracted considerable attention due to their important roles in various biotechnological processes such as cellulose degradation to make energy and hydrolysis of isoflavone. Microbulbifer thermotolerans (M. thermotolerans) is isolated from deep-sea sediment and has not been researched much yet. As a potential candidate for a variety of biotechnological industries, β-glucosidases from the novel bacterial species should be researched extensively. Methods β-Glucosidase, MtBgl85, from M. thermotolerans DAU221 was purified by His-tag affinity chromatography and confirmed by SDS-PAGE and zymogram. Its biochemical and physiological properties, such as effects of temperature, pH, metal ions, and organic solvents, substrate specificity, and isoflavone hydrolysis, were investigated. Results M. thermotolerans DAU221 showed β-glucosidase activity in a marine broth plate containing 0.1% esculin and 0.25% ammonium iron (III) citrate. The β-glucosidase gene, mtbgl85, was isolated from the whole genome sequence of M. thermotolerans DAU221. The β-glucosidase gene was 2,319 bp and encoded 772 amino acids. The deduced amino acid sequence had a 43% identity with OaBGL84 from Olleya aquimaris and 35% and 32% identity with to CfBgl3A and CfBgl3C from Cellulomonas fimi among bacterial glycosyl hydrolase family 3, respectively. The optimal temperature of MtBgl85 was 50 °C and the optimum pH was 7.0. MtBgl85 activity was strongly reduced in the presence of Hg2+ and Cu2+ ions. As a result of measuring the activity at various concentrations of NaCl, it was confirmed that the activity was maintained up to the concentration of 1 M, but gradually decreased with increasing concentration. MtBgl85 showed higher enzyme stability at non-polar solvents (high Log Pow) than polar solvents (low Log Pow). The hydrolyzed products of isoflavone glycosides and arbutin were analyzed by HPLC.

Chitin, a β-1,4-linked homopolysaccharide of N -acetyl- d -glucosamine (GlcNAc), is one of the most abundant biopolymers on Earth. Paenibacillus sp. str. FPU-7 produces several different chitinases and converts chitin into N , N′ -diacetylchitobiose ((GlcNAc)2) in the culture medium. However, the mechanism by which the Paenibacillus species imports (GlcNAc)2 into the cytoplasm and divides it into the monomer GlcNAc remains unclear. The gene encoding Paenibacillus β- N -acetyl- d -glucosaminidase (PsNagA) was identified in the Paenibacillus sp. str. FPU-7 genome using an expression cloning system. The deduced amino acid sequence of PsNagA suggests that the enzyme is a part of the glycoside hydrolase family 3 (GH3). Recombinant PsNagA was successfully overexpressed in Escherichia coli and purified to homogeneity. As assessed by gel permeation chromatography, the enzyme exists as a 57-kDa monomer. PsNagA specifically hydrolyses chitin oligosaccharides, (GlcNAc)2–4, 4-nitrophenyl N -acetyl β- d -glucosamine (pNP-GlcNAc) and pNP-(GlcNAc)2–6, but has no detectable activity against 4-nitrophenyl β- d -glucose, 4-nitrophenyl β- d -galactosamine and colloidal chitin. In this study, we present a 1.9 Å crystal structure of PsNagA bound to GlcNAc. The crystal structure reveals structural features related to substrate recognition and the catalytic mechanism of PsNagA. This is the first study on the structural and functional characterization of a GH3 β- N -acetyl- d -glucosaminidase from Paenibacillus sp. [ABSTRACT FROM AUTHOR]

Highlights • Isoprimeverose-producing enzyme (IPase) is vital for xyloglucan degradation. • IPase releases a disaccharide, isoprimeverose, from xyloglucan oligosaccharides. • The structure of IPase is solved for the first time. • Aspergillus oryzae IPase (IpeA) has subsites specific for isoprimeverose recognition. • The substrate recognition mechanisms of IPases are clarified by this study. Abstract Isoprimeverose-producing enzymes (IPases) release isoprimeverose (α- d -xylopyranosyl-(1 → 6)- d -glucopyranose) from the non-reducing end of xyloglucan oligosaccharides. Aspergillus oryzae IPase (IpeA) is classified as a member of the glycoside hydrolase family 3 (GH3); however, it has unusual substrate specificity compared with other GH3 enzymes. Xylopyranosyl branching at the non-reducing ends of xyloglucan oligosaccharides is vital for IpeA activity. We solved the crystal structure of IpeA with isoprimeverose at 2.4 Å resolution, showing that the structure of IpeA formed a dimer and was composed of three domains: an N-terminal (β/α) 8 TIM-barrel domain, α/β/α sandwich fold domain, and a C-terminal fibronectin-like domain. The catalytic TIM-barrel domain possessed a catalytic nucleophile (Asp300) and acid/base (Glu524) residues. Interestingly, we found that the cavity of the active site of IpeA was larger than that of other GH3 enzymes, and subsite −1′ played an important role in its activity. The glucopyranosyl and xylopyranosyl residues of isoprimeverose were located at subsites −1 and −1′, respectively. Gln58 and Tyr89 contributed to the interaction with the xylopyranosyl residue of isoprimeverose through hydrogen bonding and stacking effects, respectively. Our findings provide new insights into the substrate recognition of GH3 enzymes. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 3 (GH3) β‐glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β‐Glucosidases hydrolyse the β‐1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N‐glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β‐glucosidase from Rasamsonia emersonii. NcCel3A is a three‐domain protein with a number of extended loops that deepen the active‐site cleft of the enzyme. These structures characterize this subfamily of GH3 β‐glucosidases and account for the high cellobiose specificity of this subfamily. The glycoside hydrolase family 3 β‐glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N‐glycosylation. [ABSTRACT FROM AUTHOR]

A gene (PSTG2) coding for a novel β-glucosidase belonging to glycoside hydrolase family 3 was identified in the vicinity of the previously identified β-glucosidase gene [sesaminol triglucoside (STG)-hydrolyzing β-glucosidase, PSTG1] in the genome of Paenibacillus sp. strain KB0549. Compared with PSTG1, recombinant PSTG2 more specifically acted on the β-1,2-glucosidic linkage of the STG molecule to transiently accumulate a larger amount of 6-O-(β-D-glucopyranosyl)-β-D-glucopyranosylsesaminol. [ABSTRACT FROM AUTHOR]

A multifunctional β-xylosidase/α- l -arabinofuranosidase/β-glucosidase gene ( ccxyl3a ) belonging to glycoside hydrolase family 3 (GH3) was cloned from Cellulosimicrobium cellulans sp. 21 and expressed in Escherichia coli BL21 (DE3). The molecular mass of recombinant CcXyl3A was estimated to be approximately 95 kDa. With p -nitrophenyl-β- d -xyloside ( p NPβXyl) as a substrate, the purified protein presented an optimal pH of 8.5 and an optimal temperature of 45 °C. Moreover, CcXyl3A was activated in the presence of the metals K + and Na + . Purified CcXyl3A demonstrated multifunctional activities on p NPβXyl, p -nitrophenyl-β- d -glucoside ( p NPβGlc), and p -nitrophenyl-α- l -arabinofuranoside ( p NPαAraf). The greatest catalytic activity were found on p NPβXyl followed by p NPαAra f and p NPβGlc, respectively. Using xylooligosaccharides as substrate, CcXyl3A completely hydrolyzed xylobiose, xylotriose, xylotetraose and xylohexaose, xylose was the sole product. In addition, CcXyl3A synergistically acted with Thermomyces lanuginosus xylanase in the degradation of beechwood xylan, released xyloses from intermediate xylooligosaccharides produced by T. lanuginosus xylanase. To date, this is the first report to demonstrate the cloning and characterization of a multifunctional GH3 enzyme in C. cellulans that may have applications in hemicellulose degradation. [ABSTRACT FROM AUTHOR]

In this study, among the 10 genes that encode putative β-glucosidases in the glycoside hydrolase family 3 (GH3) with a signal peptide in the Aspergillus oryzae genome, we found a novel gene (AO090038000425) encoding β-1,6-glucosidase with a substrate specificity for gentiobiose. The transformant harboring AO090038000425, which we named bglH , was overexpressed under the control of the improved glaA gene promoter to form a small clear zone around the colony in a plate assay using 4-methylumbelliferyl β- d -glucopyranoside as the fluorogenic substrate for β-glucosidase. We purified BglH to homogeneity and enzymatically characterize this enzyme. The thermal and pH stabilities of BglH were higher than those of other previously studied A. oryzae β-glucosidases, and BglH was stable over a wide temperature range (4°C–60°C). BglH was inhibited by Hg 2+ , Zn 2+ , glucono-δ-lactone, glucose, dimethyl sulfoxide, and ethanol, but not by ethylenediaminetetraacetic acid. Interestingly, BglH preferentially hydrolyzed gentiobiose rather than other oligosaccharides and aryl β-glucosides, thereby demonstrating that this enzyme is a β-1,6-glucosidase. To the best of our knowledge, this is the first report of the purification and characterization of β-1,6-glucosidase from Aspergillus fungi or from other eukaryotes. This study suggests that it may be possible to find a more suitable β-glucosidase such as BglH for reducing the bitter taste of gentiobiose, and thus for controlling the sweetness of starch hydrolysates in the food industry via genome mining. [ABSTRACT FROM AUTHOR]

β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag. The expression level was increased to 5.7 g/l in a fermenter system as a result of optimization of only five codons near the 5′ end of the α-MF sequence. The recombinant AnBX was purified to homogeneity through a single-step Phenyl Sepharose chromatography. The enzyme exhibited an optimal activity at 70 °C and at pH 4.0–4.5, and a very high kinetic efficiency toward a xyloside substrate. AnBX demonstrated an exo-type activity with retention of the β-configuration, and a synergistic action with xylanase in hydrolysis of beechwood xylan. This study provides comprehensive data on characterization of a glycoside hydrolase family 3 β-xylosidase that have not been determined in any prior investigations. Our results suggested that AnBX may be useful for degradation of lignocellulosic biomass in bioethanol production, pulp bleaching process and beverage industry. [ABSTRACT FROM AUTHOR]

The β-glucosidase gene bgl3a from Myceliophthora thermophila, member of the fungal glycosyl hydrolase (GH) family 3, was cloned and expressed in Pichia pastoris. The mature β-glucosidase gene, which results after the excision of one intron and the secreting signal peptide, was placed under the control of the strong alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The recombinant enzyme (90 kDa) was purified and characterized in order to evaluate its biotechnological potential. Recombinant P. pastoris efficiently secreted β-glucosidase into the medium and produced high level of enzymatic activity (41 U/ml) after 192 h of growth, under methanol induction. MtBgl3a was able to hydrolyze low molecular weight substrates and polysaccharides containing β-glucosidic residues. The Km was found to be 0.39 mM on p-β-NPG and 2.64 mM on cellobiose. Optimal pH and temperature for the p-β-NPG hydrolysis were 5.0 and 70 °C. The β-glucosidase exhibits a half life of 143 min at 60 °C. Kinetic parameters of inhibition were determined for D-glucose, D-xylose and D-gluconic acid, indicating tolerance of the enzyme for these sugars and oxidized products. The recombinant enzyme was stimulated by short chain alcohols and has been shown to efficiently synthesize methyl-D-glucoside in the presence of methanol due to its transglycosylation activity. The stability of MtBgl3a in ethanol was prominent, and it retained most of its original activity after we exposed it to 50% ethanol for 6 h. The high catalytic performance, good thermal stability and tolerance to elevated concentrations of ethanol, D-xylose and D-glucose qualify this enzyme for use in the hydrolysis of lignocellulosic biomass for biofuel production, as part of an efficient complete multi-enzyme cocktail.

GH3 (glycoside hydrolase family 3) BGLs (ß-glucosidases) from filamentous fungi have been widely and commercially used for the supplementation of cellulases. AaBGL1 (Aspergillus aculeatus BGL1) belongs to the GH3 and shows high activity towards cellooligosaccharides up to high degree of polymerization. In the present study we determined the crystal structure of AaBGL1. In addition to the substrate-free structure, the structures of complexes with glucose and various inhibitors were determined. The structure of AaBGL1 is highly glycosylated with 88 monosaccharides (18 N-glycan chains) in the dimer. The largest N-glycan chain comprises ten monosaccharides and is one of the largest glycans ever observed in protein crystal structures. A prominent insertion region exists in a fibronectin type III domain, and this region extends to cover a wide surface area of the enzyme. The subsite +1 of AaBGL1 is highly hydrophobic. Three aromatic residues are present at subsite +1 and are located in short loop regions that are uniquely present in this enzyme. There is a long cleft extending from subsite +1, which appears to be suitable for binding long cellooligosaccharides. The crystal structures of AaBGL1 from the present study provide an important structural basis for the technical improvement of enzymatic cellulosic biomass conversion. [ABSTRACT FROM AUTHOR]

Abstract: Background: The commercially important glycoside hydrolase family 3 (GH3) β-glucosidases from Aspergillus niger are anomeric-configuration-retaining enzymes that operate through the canonical double-displacement glycosidase mechanism. Whereas the catalytic nucleophile is readily identified across all GH3 members by sequence alignments, the acid/base catalyst in this family is phylogenetically variable and less readily divined. Methods: In this report, we employed three-dimensional structure homology modeling and detailed kinetic analysis of site-directed mutants to identify the catalytic acid/base of a GH3 β-glucosidase from A. niger ASKU28. Results: In comparison to the wild-type enzyme and other mutants, the E490A variant exhibited greatly reduced kcat and kcat /Km values toward the natural substrate cellobiose (67,000- and 61,000-fold, respectively). Correspondingly smaller kinetic effects were observed for artificial chromogenic substrates p-nitrophenyl β-d-glucoside and 2,4-dinitrophenyl β-d-glucoside, the aglycone leaving groups of which are less dependent on acid catalysis, although changes in the rate-determining catalytic step were revealed for both. pH-rate profile analyses also implicated E490 as the general acid/base catalyst. Addition of azide as an exogenous nucleophile partially rescued the activity of the E490A variant with the aryl β-glucosides and yielded β-glucosyl azide as a product. Conclusions and general significance: These results strongly support the assignment of E490 as the acid/base catalyst in a β-glucosidase from A. niger ASKU28, and provide crucial experimental support for the bioinformatic identification of the homologous residue in a range of related GH3 subfamily members. [Copyright &y& Elsevier]

Abstract: Two β-xylosidases of glycoside hydrolase family 3 (GH 3) from Aspergillus nidulans FGSC A4, BxlA and BxlB were produced recombinantly in Pichia pastoris and secreted to the culture supernatants in yields of 16 and 118mg/L, respectively. BxlA showed about sixfold higher catalytic efficiency (k cat/K m) than BxlB towards para-nitrophenyl β-d-xylopyranoside (pNPX) and β-1,4-xylo-oligosaccharides (degree of polymerisation 2–6). For both enzymes k cat/K m decreased with increasing β-1,4-xylo-oligosaccharide chain length. Using pNPX as donor with 9 monosaccharides, 7 disaccharides and two sugar alcohols as acceptors 18 different β-xylosyl-oligosaccharides were synthesised in 2–36% (BxlA) and 6–66% (BxlB) yields by transxylosylation. BxlA utilised the monosaccharides d-mannose, d-lyxose, d-talose, d-xylose, d-arabinose, l-fucose, d-glucose, d-galactose and d-fructose as acceptors, whereas BxlB used the same except for d-lyxose, d-arabinose and l-fucose. BxlB transxylosylated the disaccharides xylobiose, lactulose, sucrose, lactose and turanose in upto 35% yield, while BxlA gave inferior yields on these acceptors. The regioselectivity was acceptor dependent and primarily involved β-1,4 or 1,6 product linkage formation although minor products with different linkages were also obtained. Five of the 18 transxylosylation products obtained from d-lyxose, d-galactose, turanose and sucrose (two products) as acceptors were novel xylosyl-oligosaccharides, β-d-Xylp-(1→4)-d-Lyxp, β-d-Xylp-(1→6)-d-Galp, β-d-Xylp-(1→4)-α-d-Glcp-(1→3)-β-d-Fruf, β-d-Xylp-(1→4)-α-d-Glcp-(1→2)-β-d-Fruf, and β-d-Xylp-(1→6)-β-d-Fruf-(2→1)-α-d-Glcp, as structure-determined by 2D NMR, indicating that GH3 β-xylosidases are able to transxylosylate a larger variety of carbohydrate acceptors than earlier reported. Furthermore, transxylosylation of certain acceptors resulted in mixtures. Some of these products are also novel, but the structures of the individual products could not be determined. [Copyright &y& Elsevier]

Abstract: A cell-associated enzyme exhibiting β-xylosidase activity was purified from the cell extract of the dimorphic fungus, Aureobasidium pullulans strain ATCC 20524, grown on oat spelt xylan. The purified enzyme was homogeneous as judged by SDS–PAGE, which showed an apparent M r of 88.5 kDa. β-Xylosidase activity was optimal at pH 3.5 and 70 °C. The enzyme exhibited apparent K m and V max values of 3.5 mM and 263 μmol/mg/min, respectively, for p-nitrophenyl-β-d-xylopyranoside. The enzyme also showed some α-l-arabinofuranosidase activity. Southern blot analysis indicated that the β-xylosidase gene (xylI) was present as a single copy in the genome. The genomic DNA and cDNA encoding this protein were cloned and sequenced. The open reading frame of 2415 bp was interrupted by two introns of 54 and 52 bp, and it encoded a presumed signal peptide of 20 residues and a mature protein of 785 residues with a calculated M r of 85,045 Da and a deduced isoelectric point of 4.57. The protein was N-glycosylated and possessed 16 potential N-glycosylation sites. Two distinct transcription start points of the xylI gene were present at nt −32 (A) and −26 (A) from the start codon. The xylI cDNA was functionally expressed in the yeast Pichia pastoris. The deduced amino acid sequence of the xylI gene product was 49% identical to the Talaromyces emersonii β-xylosidase Bxl1, which belongs to the glycoside hydrolase family 3. [Copyright &y& Elsevier]

Abstract: A high β-glucosidase (BGL)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer rDNA gene sequence. Under the optimal culture conditions, a maximum BGL specific activity of 3.2Uml−1 (83Umg-protein−1), one of the highest levels among BGL-producing microorganisms was obtained. An extracellular BGL was purified to homogeneity by sequential chromatography of P. pinophilum culture supernatants on a DEAE-Sepharose column, a gel filtration column, and then on a Mono Q column. The relative molecular weight of P. pinophilum BGL was determined to be 120kDa by SDS-PAGE and size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 3.5 and a temperature optimum of 32°C. P. pinophilum BGL showed a higher activity (V max =1120Umg-protein−1) than most BGLs purified from other sources. The internal amino acid sequences of P. pinophilum BGL showed a significant homology with hydrolases from glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, P. pinophilum BGL is distinguished from other BGLs by its high activity. [Copyright &y& Elsevier]

An efficient ß-1,4-glucosidase (BGL) producing strain, Fomitopsis pinicola KMJ812, was isolated and identified based on morphological features and sequence analysis of internal transcribed spacer rDNA. An extracellular BGL was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The relative molecular weight of F. pinicola BGL was determined to be 105 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, or 110 kDa by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the BGL had a pH optimum of 4.5 and a temperature optimum of 50°C. The enzyme showed high substrate specificity and high catalytic efficiency ( kcat = 2,990 s−1, Km = 1.76 mM, kcat/ Km = 1,700 mM−1 s−1) for p-nitrophenyl-β- d-glucopyranoside. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 3, indicating that the F. pinicola BGL is a member of glycoside hydrolase family 3. Although BGLs have been purified and characterized from several other sources, F. pinicola BGL is distinguished from other BGLs by its high catalytic efficiency and strict substrate specificity. [ABSTRACT FROM AUTHOR]

An extracellular protein exhibiting β-xylosidase activity was purified from the culture filtrate of a filamentous fungus, Aspergillus japonicus strain MU-2, grown on oat spelt xylan. The purified enzyme was a monomeric glycoprotein with an apparent M r of 113.2 kDa as estimated by SDS–PAGE. β-Xylosidase activity was optimal at pH 4.0 and 70°C. The enzyme also showed β-glucosidase and α-l-arabinofuranosidase activities. The genomic DNA and cDNA encoding this protein were cloned and sequenced. Southern blot analysis indicated that the β-xylosidase gene (xylA) was present as a single copy in the genome. An open reading frame, consisting of 2412 bp, was not interrupted by introns, and it encoded a presumed signal peptide of 17 amino acids and a mature protein of 787 amino acids. The deduced amino acid sequence of the xylA gene product showed a high degree of identity (69%) to the primary structure of the Aspergillus niger β-xylosidase XlnD that belongs to the glycoside hydrolase family 3. Moreover, the xylA gene was functionally expressed in the yeast Pichia pastoris. [Copyright &y& Elsevier]

β-Glucosidases belong to families 1, 3 and 9 of the glycoside hydrolases and act on cello-oligosaccharides. Family 1 and 3 enzymes are retaining and are reported to have transglycosylation activity, which can be used to produce oligosaccharides and glycoconjugates. Family 3 enzymes are less well characterized than their family 1 homologues and to date only two crystal structures have been solved. Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. Crystals of selenomethionine-substituted protein have also been grown. The crystals belong to space group C2221, with unit-cell parameters a = 74.9, b = 127.0, c = 175.2 Å. Native data have been collected to 2.4 Å resolution and the structure has been solved to 2.7 Å using the selenomethionine MAD method. Model building and refinement of the structure are under way. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases are often members of a multigene family, suggesting individual roles for each isoenzyme. Various extracellular glycoside hydrolases have an important but poorly understood function in remodelling the cell wall during plant growth. Here, MsXyl1, a concanavalin A-binding protein from alfalfa (Medicago sativa L.) belonging to the glycoside hydrolase family 3 (β-D-xylosidase branch) is characterized. Transcripts of MsXyl1 were detected in roots (particularly root tips), root nodules, and flowers. MsXyl1 under the control of the CaMV 35S promoter was expressed in the model legume Medicago truncatula (Gaertner). Concanavalin A-binding proteins from the transgenic plants exhibited 5–8-fold increased activities towards three p-nitrophenyl (PNP) glycosides, namely PNP-β-D-xyloside, PNP-α-L-arabinofuranoside, and PNP-α-L-arabinopyranoside. An antiserum raised against a synthetic peptide recognized MsXyl1, which was processed to a 65 kDa form. To characterize the substrate specificity of MsXyl1, the recombinant protein was purified from transgenic M. truncatula leaves by concanavalin A and anion chromatography. MsXyl1cleaved β-1,4-linked D-xylo-oligosaccharides and α-1,5-linked L-arabino-oligosaccharides. Arabinoxylan (from wheat) and arabinan (from sugar beet) were substrates for MsXyl1, whereas xylan (from oat spelts) was resistant to degradation. Furthermore, MsXyl1 released xylose and arabinose from cell wall polysaccharides isolated from alfalfa roots. These data suggest that MsXyl1 is a multifunctional β-xylosidase/α-L-arabinofuranosidase/α-L-arabinopyranosidase implicated in cell wall turnover of arabinose and xylose, particularly in rapidly growing root tips. Moreover, the findings of this study demonstrate that stable transgenic M. truncatula plants serve as an excellent expression system for purification and characterization of proteins. [ABSTRACT FROM PUBLISHER]

Abstract: Alkyl glycosides are surfactants with good biodegradability and low toxicity, attractive to produce by an enzymatic method to get a well-defined product. In this paper, we report a novel thermostable variant of a family 3 β-glucosidase to be an efficient catalyst in alkyl-glucoside forming reactions using transglycosylation with hexanol or octanol as the acceptor molecule. The enzyme has an apparent optimum for hydrolysis at 90°C, which coincides with its unfolding temperature. The total activity is lower at lower temperature (60°C), but the ratio of alcoholysis/hydrolysis is slightly more favourable. This ratio is however more heavily influenced by the water content and the pH. Optimal reaction conditions for hexyl glucoside synthesis from p-nitrophenyl-β-glucopyranoside were a water/hexanol two-phase system containing 16% (v/v) water, pH 5.8, and a temperature of 60°C. Under these conditions, the total initial reaction rate was 153μmolmin−1 mg−1 and the alcoholysis/hydrolysis ratio was 5.1. Comparing with alcoholysis/hydrolysis ratios of other β-glycosidases, TnBgl3B can be considered to be a very promising catalyst for alkyl glucoside production. [Copyright &y& Elsevier]