Enhancement of substrate recognition ability by combinatorial mutation of beta-glucosidase displayed on the yeast cell surface.
Recently, in family 3 beta-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 beta-glucosidase.
Fukuda
Takeshi
T
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1, Bunkyo, Fukui 910-8507, Japan.
Kato-Murai
Michiko
M
Kadonosono
Tetsuya
T
Sahara
Hiroshi
H
Hata
Yoji
Y
Suye
Shin-ichiro
S
Ueda
Mitsuyoshi
M
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods; D007700:Kinetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D001617:beta-Glucosidase