Mutations in the substrate entrance region of β-glucosidase from Trichoderma reesei improve enzyme activity and thermostability

Hsiao Lin Lee, Chih Kang Chang, Wen Yih Jeng, Andrew H.J. Wang, Po Huang Liang

Research output: Contribution to journalArticlepeer-review

67 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)733-740
Number of pages8
JournalProtein Engineering, Design and Selection
Volume25
Issue number11
DOIs
Publication statusPublished - Nov 2012
Externally publishedYes

Keywords

  • β-glucosidase
  • biofuel
  • protein engineering
  • site-directed mutagenesis

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Biochemistry
  • Molecular Biology

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