The Protein Journal

, 25:232

Identification of Glutamate Residues Important for Catalytic Activity or Thermostability of a Truncated Bacillus sp. Strain TS-23 α-amylase by Site-directed Mutagenesis

Authors

  • Long-Liu Lin
    • Department of Applied ChemistryNational Chiayi University
  • Pei-Jing Chen
    • Department of Food and NutritionHungkuang University
  • Jai-Shin Liu
    • Institute of Molecular and Cellular Biology, and Department of Life ScienceNational Tsing Hua University
  • Wen-Ching Wang
    • Institute of Molecular and Cellular Biology, and Department of Life ScienceNational Tsing Hua University
    • Department of Food and NutritionHungkuang University
Article

DOI: 10.1007/s10930-006-9006-7

Cite this article as:
Lin, L., Chen, P., Liu, J. et al. Protein J (2006) 25: 232. doi:10.1007/s10930-006-9006-7

Abstract

The importance of 17 glutamate residues of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was investigated by site-directed mutagenesis. The Ala- and Asp-substituted variants were overexpressed in the recombinant E. coli cells and the 54-kDa proteins were purified to nearly homologous by nickel-chelate chromatography. Glu-295, which locates in the conserved region III of amylolytic enzymes, mutations resulted in a complete loss of enzyme activity. The specific activity for E151A was decreased by more than 30%, while other variants showed activity comparable to that of BACΔNC. A decreased half-life at 70°C was observed for Glu-219 variants with respective to the wild-type enzyme, suggesting that replacement of Glu-219 by either Ala or Asp might have a significant destabilizing effect on the protein structure.

Keywords

α-AmylaseBacillus sp. strain TS-23catalytic reactionsite-directed mutagenesisthermostability

Abbreviations

BACΔNC

the N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase

SDS

sodium dodecyl sulfate

SDS-PAGE

SDS-polyacrylamide gel electrophoresis

TEMED

N,N,N′,N′-tetramethylethylenediamine

IPTG

isopropyl-β-d-thiogalactopyranoside

Ni2+-NTA

nickel nitrilotriacetate

BLA

Bacillus licheniformis α-amylase

Copyright information

© Springer Science+Business Media, Inc. 2006