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In order to improve the degradation activity of β-glucosidase (CpBgl) from Coniophora puteana , the structural modification was conducted. The enzyme activity of mutants CpBgl-Q20C and CpBgl-A240S was increased by 65.75% and 58.58%, respectively. These mutants exhibited maximum activity under the same conditions as wild-type CpBgl (65 ℃ and pH 5.0), slightly improved stabilities compared that of the wild-type, and remarkably enhanced activities in the presence of Mn2+ or Fe2+. The V max of CpBgl-Q20C and CpBgl-A240S was increased to 138.18 and 125.14 μmol/mg/min, respectively, from 81.34 μmol/mg/min of the wild-type, and the catalysis efficiency (k cat / K m) of CpBgl-Q20C (335.79 min−1/mM) and CpBgl-A240S (281.51 min−1/mM) was significantly improved compared with that of the wild-type (149.12 min−1/mM). When the mutant CpBgl-Q20C were used in the practical degradation of different biomasses, the glucose yields of filter paper, corncob residue, and fungi mycelia residue were increased by 17.68%, 25.10%, and 20.37%, respectively. The spatial locations of the mutation residues in the architecture of CpBgl and their unique roles in the enzyme-substrate binding and catalytic efficiency were probed in this work. These results laid a foundation for evolution of other glycoside hydrolases and the industrial bio-degradation of cellulosic biomass in nature. [Display omitted] • Improved β-glucosidase mutants were obtained via site-directed mutagenesis. • The activity of CpBgl-Q20C and CpBgl-A240S was increased by 65.75% and 58.58%. • The molecular mechanism of improved activity was preliminarily elucidated. • CpBgl-Q20C and CpBgl-A240S effectively promoted biomass cellulose degradation. [ABSTRACT FROM AUTHOR]

In order to improve the catalytic efficiency of cellulase for more effective utilization of lignocellulose, a novel endoglucanase (CgEndo) from Colletotrichum graminicola was expressed by Pichia pastoris X33 and modified by site-directed mutagenesis. Two mutants, Y63S and N20D/S113T, with 62.31% and 57.14% increased enzyme activities were obtained, respectively. On this basis, their biochemical properties, kinetic parameters, structural information as well as the application in biomass degradation were investigated and compared with the wild-type CgEngo. The results indicated that the mutation Y63S and N20D/S113T resulted in an improvement of proximity between enzyme and substrate through conformational changes of the catalytic region, which might contribute to the higher enzyme activities and catalysis efficiency (K cat / K m) of Y63S and N20D/S113T. These findings laid important foundation for the further engineering of this endoglucanase and practical application in efficient degradation of cellulosic biomass in nature. [Display omitted] • Improved endoglucanase CgEndo mutants were obtained via site-directed mutagenesis. • The activities of Y63S and N20D/S113T were 62.31% and 57.14% increased, respectively. • Y63S and N20D/S113T could effectively promote natural cellulose degradation. [ABSTRACT FROM AUTHOR]