Improving the catalytic activity of ß-glucosidase from Coniophora puteana via semi-rational design for efficient biomass cellulose degradation.

In order to improve the degradation activity of ß-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 â„ƒ 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 Vmax 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 (kcat/Km) 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.

Improvement of catalytic performance of endoglucanase CgEndo from Colletotrichum graminicola by site-directed mutagenesis.

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 (Kcat/Km) 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.

Heterologous expression and biochemical characterization of a thermostable endo-ß-1,4-glucanase from Colletotrichum orchidophilum.

To develop new cellulases for efficient utilization of the lignocellulose, an endoglucanase (CoCel5A) gene from Colletotrichum orchidophilum was synthesized and a recombinant Pichia pastoris GS115/pPIC9K/cocel5A was constructed for secretory expression of CoCel5A. After purification, the protein CoCel5A was biochemically characterized. The endoglucanase CoCel5A exhibited the optimal activity at 55-75 °C and high thermostability (about 85% residual activity) at the temperature of 55 °C after incubation for 3 h. The highest activity of CoCel5A was detected when 100 mM citric acid buffer (pH 4.0-5.0) was used and excellent pH stability (up to 95% residual activity) was observed after incubation in 100 mM citric acid buffer (pH 3.0-6.0) at 4 °C for 24 h. Carboxymethyl cellulose sodium salt (n = approx. 500) (CMC) and ß-D-glucan were the best substrates for CoCel5A among the tested substrates. The kinetic parameters Vmax, Km, and Kcat/Km values against CMC were 290.70 U/mg, 2.65 mg/mL, and 75.67 mL/mg/s, respectively; and 228.31 U/mg, 2.06 mg/mL, and 76.45 mL/mg/s against ß-D-glucan, respectively, suggesting that CoCel5A has high affinity and catalytic efficiency. These properties supported the potential application of CoCel5A in biotechnological and environmental fields.