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The Protein Journal

, 25:232 | Cite as

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

  • Long-Liu Lin
  • Pei-Jing Chen
  • Jai-Shin Liu
  • Wen-Ching Wang
  • Huei-Fen Lo
Article

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-Amylase Bacillus sp. strain TS-23 catalytic reaction site-directed mutagenesis thermostability 

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

Notes

Acknowledgments

This work was supported by Grants NSC 93-2313-B-241-005 and NSC 94-2313-B-241-005 from National Science Council of Taiwan, Republic of China.

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Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • Long-Liu Lin
    • 1
  • Pei-Jing Chen
    • 2
  • Jai-Shin Liu
    • 3
  • Wen-Ching Wang
    • 3
  • Huei-Fen Lo
    • 2
  1. 1.Department of Applied ChemistryNational Chiayi UniversityChiayiTaiwan
  2. 2.Department of Food and NutritionHungkuang UniversityTaichungTaiwan
  3. 3.Institute of Molecular and Cellular Biology, and Department of Life ScienceNational Tsing Hua UniversityHsinchuTaiwan

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