Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium

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An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEgl5A showed a molecular mass of approximately 40 kDa as determined by SDS–PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley β-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade β-linkage polysaccharides.

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Strain and culture conditions

T. matsutake strain NBRC30605 was obtained from the NITE Bioresource Research Center (Chiba, Japan). T. matsutake mycelia were grown in SY medium (2% soluble starch from corn, 0.3% yeast extract and 2% agar, w/v, pH 5.1) for 40 days at 24°C with constant conditions. Standard solid-state medium contained 30 g of rolled barley and 15 g of vermiculite with a 60% moisture content in 100 mL Erlenmeyer flask. Liquid contents of the media were prepared with 0.3% yeast extract, although the water

Purification and identification of the endoglucanase from T. matsutake grown in solid-state culture

To determine the productivity of the endoglucanase activity in T. matsutake, we examined cultures in the presence of several compositions of barley based solid-state medium (40 days incubation) and GYL medium (30 and 60 days incubation), as shown in Fig. 1. Of the media tested, rolled barley gave the fastest vegetative mycelial growth, followed by a mixed weight ratio of 2:1 of rolled barley and vermiculite (Fig. 1A). Moreover, this condition had the highest activities with barley β-glucan and

Discussion

Complete hydrolysis of cellulose requires the cooperative actions of three types of cellulases: endoglucanase (EC 3.2.1.4) that randomly cleaves the internal β-1,4-glycosidic bonds; two types of cellobiohydrolases, CBHI and CBHII (EC 3.2.1.176 and EC 3.2.1.91) that processivity act on the chain termini to release cellobiose; and β-glucosidase (EC 3.2.1.21) that hydrolyzes cellobiose to glucose (28). Endoglucanase also hydrolyzes the β-1,3-1,4-branched β-glucans converting into oligosaccharides.

Acknowledgments

This study was supported in part by a grant by the Strategic Research Foundation Grant-aided Project for Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), 2015-2017 (S1512004).

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