Applied Microbiology and Biotechnology

, Volume 76, Issue 5, pp 1027–1033

Enhancement of substrate recognition ability by combinatorial mutation of β-glucosidase displayed on the yeast cell surface

  • Takeshi Fukuda
  • Michiko Kato-Murai
  • Tetsuya Kadonosono
  • Hiroshi Sahara
  • Yoji Hata
  • Shin-ichiro Suye
  • Mitsuyoshi Ueda
Biotechnologically Relevant Enzymes and Proteins

Abstract

Recently, in family 3 β-glucosidase (BGL), the catalytically important Asp nucleophile has been identified in the SDW segment of the SDWG sequence by site-directed mutagenesis. However, the details about the roles of each amino acid residue of the SDWG sequence have not been investigated. W293 of the SDW segment, which is the residue next to the nucleophile (D292) in family 3 BGL, is very important for hydrolytic reaction as a binder to a substrate. G294 of the SDWG sequence might play an important role in catalysis. In this study, to obtain a functional BGL1 mutant by the substitution of G294 using a genetic engineering method, the library of mutant BGL1 from Aspergillus oryzae was rapidly constructed by yeast cell surface engineering, and the hydrolytic activities of mutants were comprehensively detected. Consequently, G294F, G294W, and G294Y, in which G was substituted with aromatic amino acids, showed higher activities for substrate recognition than the parent strain (1.5-, 1.5-, and 1.6-fold, respectively). These results suggest the presence of some interaction between the sugar rings and aromatic ring of W293 at the entrance of the catalytic pocket, which enhances the substrate recognition of β-glucosidase.

Keywords

Yeast cell surface engineering SDW segment Family 3 β-glucosidase Substrate recognition Comprehensive library of mutants 

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Takeshi Fukuda
    • 1
  • Michiko Kato-Murai
    • 2
  • Tetsuya Kadonosono
    • 2
  • Hiroshi Sahara
    • 3
  • Yoji Hata
    • 3
  • Shin-ichiro Suye
    • 1
  • Mitsuyoshi Ueda
    • 2
  1. 1.Department of Applied Chemistry and Biotechnology, Graduate School of EngineeringUniversity of FukuiFukuiJapan
  2. 2.Laboratory of Biomacromolecular Chemistry, Division of Applied Life Sciences, Department of Applied Biochemistry, Graduate School of AgricultureKyoto UniversityKyotoJapan
  3. 3.Research InstituteGekkeikan Sake Co. Ltd. ShimotobaKyotoJapan

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