Characterization of a GH12 Endoglucanase from Volvariella volvacea Exhibiting Broad Substrate Specificity and Potential Synergy with Crude Cellulase

Zhen Wang, Yimei Hu, Liangkun Long, Shaojun Ding

Abstract


Fungal glycoside hydrolase family GH12 has a single catalytic domain, exhibiting a great diversity of properties and application potentials in biomass biorefinery, feed, and textile industries. To discover new GH12 enzymes from white- and brown-rot basidiomycetes for application in the saccharification of lignocelluloses, two putative genes, VvGH12A and VvGH12B, were identified from the Volvariella volvacea genome and classified into basidiomycetous subfamily GH12-1 and GH12-2, respectively. One enzyme VvGH12A was successfully expressed in Pichia pastoris, and characterized. VvGH12A was the most active on CMC but with broad substrate specificities on polysaccharides with -1,4 linked and -1,3-1,4-mixed glucans. Furthermore, VvGH12A was also active on xylan and mannan. Unlike other fungal GH12 endoglucanases, VvGH12A showed a weak processivity independent of the carbohydrate-binding module (CBM) due to both “endo” and “exo” types of enzyme activity. The pH-optimum was significantly affected by the acidity and basicity of amino acid at site 98. The enzyme optimum pH was engineered to a higher neutral or alkaline pH (from pH 6.5 to pH 7.0-8.0) when Asp98 was replaced with nonpolar or neutral or amide residue. VvGH12A exhibited synergistic action with crude cellulase from Trichoderma reesei D-86271 (Rut C-30) in saccharification of delignified wheat straw, suggesting that VvGH12A plays a functional role in efficiently hydrolyzing plant cell wall polysaccharides.

Keywords


Volvariella volvacea; Glycoside hydrolase 12; Synergistic action; Processivity; pH-Profile engineering

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