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- Title
Mutations in the substrate entrance region of β-glucosidase from Trichoderma reesei improve enzyme activity and thermostability.
- Authors
Lee, Hsiao-Lin; Chang, Chih-Kang; Jeng, Wen-Yih; Wang, Andrew H.-J.; Liang, Po-Huang
- Abstract
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.
- Subjects
GENETIC mutation; GLUCOSIDASES; PROTEIN engineering; SITE-specific mutagenesis; TRICHODERMA reesei; ENZYME kinetics; CELLOBIOSE; THIN layer chromatography
- Publication
PEDS: Protein Engineering, Design & Selection, 2012, Vol 25, Issue 11, p733
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzs073