Overexpression and Catalytic Function of α-L-arabinofuranosidase from Trichoderma koningii G-39

• Main Authors: Cheng-Ta Chen, 陳成達
• Other Authors: Yaw-Kuen Li
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/44855076682610026835

碩士 === 國立交通大學 === 應用化學系 === 91 === The gene from Trichoderma koingii G-39 encoding a bifunctional enzyme with both α-L-arabinofuranosidase (ABF) and β-xylosidase activities was successfully expressed in Pichia pastoris. This enzyme was further recognized as a α-L-arabinofuranosidase (EC 3.2.1.55) and belongs to the family 54 glycohydrolase. The recombination enzyme can be efficiently purified to >95 % homogeneity by SP (cationic) column. The physical and chemical property of the recombination enzyme is similar to that of the native enzyme. Based on the reversible inhibition study, we conclude that the enzyme possesses two different catalytic domains toward p-nitrophenyl-α-L- arabinofuranoside (PNPAF) and 2,4-dinitrophenyl-β-D-xylopyranoside (2,4-DNXP) with the kcat / Km values of 75512, 5370 S-1M-1, respectively. The transglycosylation activity and the pH-dependent hydrolysis (a bell-shaped curve) on both PNPAF and 2,4-DNXP reveal that the enzyme may catalyze the hydrolysis of PNPAF & 2,4-DNXP with the retention of anomeric configuration. Since no significant activity decreases on E271Q, D302N, E305Q, and E389Q. E271, D302, E305 and E389 are unlikely to be the catalytic essential group for both enzymatic functions.