Tomohiko Matsuzawa

Screening, identification, and characterization of a GH43 family β-xylosidase/α-arabinofuranosidase from a compost microbial metagenome

A putative glycoside hydrolase family 43 β-xylosidase/α-arabinofuranosidase (CoXyl43) that promotes plant biomass saccharification was isolated via functional screening of a compost microbial metagenomic library and characterized. CoXyl43 promoted the saccharification of plant biomasses, including xylans (xylan and arabinoxylan), rice straw, and Erianthus, by degrading xylooligosaccharide residues to monosaccharide residues. The recombinant CoXyl43 protein exhibited both β-xylosidase and α-arabinofuranosidase activities for chromogenic substrates, with optimal activity at pH 7.5 and 55xa0°C. Both of these activities were inactivated by ethanol, dimethylsulfoxide, and zinc and copper ions but were activated by manganese ions. Only the β-xylosidase activity of recombinant CoXyl43 was enhanced in the presence of calcium ions. These results indicate that CoXyl43 exhibits unique enzymatic properties useful for biomass saccharification.

Crystal structure and identification of a key amino acid for glucose tolerance, substrate specificity, and transglycosylation activity of metagenomic β‐glucosidase Td2F2

β‐Glucosidase Td2F2 isolated from a compost metagenome has high glucose tolerance and transglycosylation activity. In this study, we determined the high‐resolution crystal structure of Td2F2. It has a unique structure at the −1 subsite that is important for substrate specificity but not for glucose tolerance. To elucidate the mechanism(s) of glucose tolerance, we isolated a glucose‐sensitive Td2F2 mutant using random mutagenesis. In this mutant, Asn223 residue located between subsites +1 and +2 was mutated. The Asn223 mutation resulted in reduced glucose tolerance and transglycosylation activity, and drastically changed substrate specificity. These results indicate that the structure between subsites +1 and +2 is critical for the glucose tolerance and substrate specificity of Td2F2. Our findings shed light on the glucose tolerance and transglycosylation activity mechanisms of glycoside hydrolase family 1 β‐glucosidases.

Key amino acid residues for the endo-processive activity of GH74 xyloglucanase.

Unlike endo‐dissociative‐xyloglucanases, Paenibacillus XEG74 is an endo‐processive xyloglucanase that contains four unique tryptophan residues in the negative subsites (W61 and W64) and the positive subsites (W318 and W319), as indicated by three‐dimensional homology modelling. Selective replacement of the positive subsite residues with alanine mutations reduced the degree of processive activity and resulted in the more endo‐dissociative‐activity. The results showed that W318 and W319, which are found in the positive subsites, are essential for processive degradation and are responsible for maintaining binding interactions with xyloglucan polysaccharide through a stacking effect.

Screening, identification, and characterization of a novel saccharide-stimulated β-glycosidase from a soil metagenomic library

MeBglD2, a β-glycosidase that is highly activated in the presence of various monosaccharides and disaccharides, was isolated from a soil metagenomic library. MeBglD2 had not only β-glucosidase activity but also β-galactosidase and β-fucosidase activities. MeBglD2 β-glucosidase activity increased in a cellobiose concentration-dependent manner and was not inhibited by a high concentration of d-glucose or cellobiose. MeBglD2 β-glucosidase and β-fucosidase activities were activated by various monosaccharides and disaccharides including d-glucose, d-xylose, d-galactose, maltose, and cellobiose. The saccharification yield of rice straw using Trichoderma reesei cellulase was improved by the addition of MeBglD2. These results show that MeBglD2 can be used to improve plant biomass saccharification, because both substrates and products can activate its enzymatic activity.

Improvement of thermostability and activity of Trichoderma reesei endo-xylanase Xyn III on insoluble substrates

Trichoderma reesei Xyn III, an endo-β-1,4-xylanase belonging to glycoside hydrolase family 10 (GH10), is vital for the saccharification of xylans in plant biomass. However, its enzymatic thermostability and hydrolytic activity on insoluble substrates are low. To overcome these difficulties, the thermostability of Xyn III was improved using random mutagenesis and directed evolution, and its hydrolytic activity on insoluble substrates was improved by creating a chimeric protein. In the screening of thermostable Xyn III mutants from a random mutagenesis library, we identified two amino acid residues, Gln286 and Asn340, which are important for the thermostability of Xyn III. The Xyn III Gln286Ala/Asn340Tyr mutant showed xylanase activity even after heat treatment at 60xa0°C for 30xa0min or 50xa0°C for 96xa0h, indicating a dramatic enhancement in thermostability. In addition, we found that the addition of a xylan-binding domain (XBD) to the C-terminal of Xyn III improved its hydrolytic activity on insoluble xylan.

The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant

BackgroundThe filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.ResultsTo analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.ConclusionWe conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

Screening, identification, and characterization of α-xylosidase from a soil metagenome.

A novel α-xylosidase, MeXyl31, was isolated and characterized from a soil metagenomic library. The amino acid sequence of MeXyl31 showed a slight homology with other characterized α-xylosidases. The optimal pH and temperature of recombinant MeXyl31 were pH 5.5 and 45°C, respectively. Recombinant MeXyl31 had a higher α-xylosidase activity toward pNP α-d-xylopyranoside than pNP α-d-glucopyranoside, isoprimeverose, and other xyloglucan oligosaccharides. The kcat/Km value toward pNP α-d-xylopyranoside was about 750-fold higher than that of isoprimeverose. MeXyl31 activity was strongly inactivated in the presence of zinc and copper ions. MeXyl31 is the first α-xylosidase isolated from the metagenome and, relative to other xyloglucan oligosaccharides, shows higher activity toward pNP α-d-xylopyranoside.

Draft Genome Sequence of the Yeast Starmerella bombicola NBRC10243, a Producer of Sophorolipids, Glycolipid Biosurfactants

ABSTRACT The yeast Starmerella bombicola NBRC10243 is an excellent producer of sophorolipids (SLs) from various feedstocks. Here, we report the draft genome sequence of S. bombicola NBRC10243. Analysis of the sequence may provide insight into the properties of this yeast that make it superior for use in the production of functional glycolipids and biomolecules, leading to the further development of S. bombicola NBRC10243 for industrial applications.

Identification of the gene encoding isoprimeverose-producing oligoxyloglucan hydrolase in Aspergillus oryzae

Aspergillus oryzae produces a unique β-glucosidase, isoprimeverose-producing oligoxyloglucan hydrolase (IPase), that recognizes and releases isoprimeverose (α-d-xylopyranose-(1→6)-d-glucopyranose) units from the non-reducing ends of oligoxyloglucans. A gene encoding A. oryzae IPase, termed ipeA, was identified and expressed in Pichia pastoris. With the exception of cellobiose, IpeA hydrolyzes a variety of oligoxyloglucans and is a member of the glycoside hydrolase family 3. Xylopyranosyl branching at the non-reducing ends was vital for IPase activity, and galactosylation at a α-1,6-linked xylopyranosyl side chain completely abolished IpeA activity. Hepta-oligoxyloglucan saccharide (Xyl3Glc4) substrate was preferred over tri- (Xyl1Glc2) and tetra- (Xyl2Glc2) oligoxyloglucan saccharides substrates. IpeA transferred isoprimeverose units to other saccharides, indicating transglycosylation activity. The ipeA gene was expressed in xylose and xyloglucan media and was strongly induced in the presence of xyloglucan endo-xyloglucanase-hydrolyzed products. This is the first study to report the identification of a gene encoding IPase in eukaryotes.

Improved thermostability of a metagenomic glucose-tolerant β-glycosidase based on its X-ray crystal structure

MeBglD2, a metagenomic β-glycosidase, is stimulated by various saccharides, including d-glucose, d-xylose, and maltose, and it promotes the enzymatic saccharification of plant biomass. To improve the thermostability of MeBglD2, its X-ray crystal structure was analyzed, and the amino acid residues responsible for its thermostability were identified using the structural information. Mutations in His8, Asn59, and Gly295 improved the thermostability of MeBglD2, and the combination of these mutations resulted in the highest thermostability. Compared with wild-type MeBglD2, thermostable MeBglD2 mutants promoted plant biomass saccharification using Trichoderma reesei cellulase. In addition to thermostability, the thermostable mutants exhibited higher tolerance to ethanol, dimethyl sulfoxide, and copper ions, indicating that the MeBglD2 mutants generated in this study were improved in their tolerance to not only high temperature but also to organic solvents and metal ions.