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Applied Microbiology and Biotechnology

, Volume 80, Issue 5, pp 795–803 | Cite as

Acid stabilization of Bacillus licheniformis alpha amylase through introduction of mutations

  • Yi-han Liu
  • Fu-ping LuEmail author
  • Yu Li
  • Jian-ling Wang
  • Chen Gao
Biotechnologically Relevant Enzymes and Proteins

Abstract

This paper provided further understanding of the relationships between acid resistance and structural features of different mutants in Bacillus licheniformis alpha amylase (BLA) due to the changes of two crucial positions Leu134 and Ser320. In order to investigate effect of the two positions on the acid stability, we described the detailed characterization of wild-type and the single mutants L134R and S320A as well as the double mutant L134R/S320A. The highest kcat/Km with pH 4.5, approximately 14 times that of wild type, was observed in L134R/S320A. The kcat/Km corresponding to L134R and S320A were at an intermediate values between those for wild type and L134R/S320A. In addition, compared with wild type, which had a rapid decline of the activity, L134R/S320A could maintain its activity strongly in low pH. Meanwhile, lower tolerance of L134R and S320A in acidic conditions than that of L134R/S320A was determined. Surprisingly, the acid-resistant capability of L134R/S320A was significantly enhanced by directed evolution. These results, combined with three-dimensional structure analysis, show that the electrostatic effects play a significant role in determining the stability of BLA at two crucial positions, 134 and 320.

Keywords

Bacillus licheniformis alpha amylase Acid stability Kinetics Electrostatic field Protein structure 

Notes

Acknowledgments

The authors would like to thank Dr. Sui-Lam Wong for the supply of plasmid pWB980. Dr. Sui-Lam Wong is a senior medical scholar from University of Calgary. This work was supported by Key Program of Tianjin Science and Technology Development Plan (06YFGPSH03500).

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Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Yi-han Liu
    • 1
  • Fu-ping Lu
    • 1
    Email author
  • Yu Li
    • 1
  • Jian-ling Wang
    • 1
  • Chen Gao
    • 2
  1. 1.Tianjin Key Laboratory of Industrial Microbiology, College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China
  2. 2.College of Life ScienceNankai UniversityTianjinPeople’s Republic of China

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