TY - JOUR
T1 - Mutations in the substrate entrance region of β-glucosidase from Trichoderma reesei improve enzyme activity and thermostability
AU - Lee, Hsiao Lin
AU - Chang, Chih Kang
AU - Jeng, Wen Yih
AU - Wang, Andrew H.J.
AU - Liang, Po Huang
N1 - Funding Information:
This work was supported by the National Science Council, Taiwan, Republic of China (grants 99-3113-B-001-001 and 101-3113-P-008-001).
PY - 2012/11
Y1 - 2012/11
N2 - β-Glucosidase (EC 3.2.1.21) plays an essential role in biofuel production since it can cleave β-1,4-glycosidic bond to convert cellobiose into fermentable glucose. Based on the structure of Trichoderma reesei β-glucosidase 2 (TrBgl2) we solved, the amino acids in the outer channel of active site were mutated in this study. Mutants P172L and P172L/F250A showed the most enhanced kcat/Km and kcat values by 5.3- and 6.9-fold, respectively, compared to the wild type (WT) toward 4-nitrophenyl-β-d-glucopyranoside (p-NPG) substrate at 40°C. L167W and P172L/F250A mutations resulted in shift of optimal temperature to 50°C, at which WT was almost inactive. However, thin-layer chromatography analysis revealed that mutant L167W had the best synergism with T. reesei cellulases on degrading cellulosic substrates into glucose. This study enhances our understanding on the roles of amino acids in the substrate entrance region away from the active site and provides engineered T. reesei β-glucosidases with better activity and/or thermostability to hydrolyze cellobiose.
AB - β-Glucosidase (EC 3.2.1.21) plays an essential role in biofuel production since it can cleave β-1,4-glycosidic bond to convert cellobiose into fermentable glucose. Based on the structure of Trichoderma reesei β-glucosidase 2 (TrBgl2) we solved, the amino acids in the outer channel of active site were mutated in this study. Mutants P172L and P172L/F250A showed the most enhanced kcat/Km and kcat values by 5.3- and 6.9-fold, respectively, compared to the wild type (WT) toward 4-nitrophenyl-β-d-glucopyranoside (p-NPG) substrate at 40°C. L167W and P172L/F250A mutations resulted in shift of optimal temperature to 50°C, at which WT was almost inactive. However, thin-layer chromatography analysis revealed that mutant L167W had the best synergism with T. reesei cellulases on degrading cellulosic substrates into glucose. This study enhances our understanding on the roles of amino acids in the substrate entrance region away from the active site and provides engineered T. reesei β-glucosidases with better activity and/or thermostability to hydrolyze cellobiose.
UR - https://www.scopus.com/pages/publications/84868600048
UR - https://www.scopus.com/pages/publications/84868600048#tab=citedBy
U2 - 10.1093/protein/gzs073
DO - 10.1093/protein/gzs073
M3 - Article
C2 - 23077275
AN - SCOPUS:84868600048
SN - 1741-0126
VL - 25
SP - 733
EP - 740
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 11
ER -