Stabilization of a truncated Bacillus sp. strain TS-23 α-amylase by replacing histidine-436 with aspartate

  • Huei-Fen Lo
  • Ya-Hui Chen
  • Nai-Wan Hsiao
  • Hsiang-Ling Chen
  • Hui-Yu Hu
  • Wen-Hwei Hsu
  • Long-Liu Lin


Histidine-436 of a truncated Bacillus sp. strain TS-23 α-amylase (His6-tagged ΔNC) has been known to be responsible for thermostability of the enzyme. To understand further the structural role of this residue, site-directed mutagenesis was conducted to replace His-436 of His6-tagged ΔNC with aspartate, lysine, tyrosine or threonine. Starch-plate assay showed that all Escherichia coli M15 transformants conferring the mutated amylase genes retained the amylolytic activity. The over-expressed proteins have been purified to near homogeneity by nickel-chelate chromatography and the molecular mass of the purified enzymes was approximately 54 kDa. The specific activity for H436T was decreased by more than 56%, while H436D, H436K, and H436Y showed a higher activity to that of the wild-type enzyme. Although the mutations did not lead to a significant change in the Km value, more than 66% increase in the value of catalytic efficiency (kcat/Km) was observed in H436D, H436K, and H436Y. At 70 °C, H436D exhibited an increased half-life with respect to the wild-type enzyme.


Bacillus sp. strain TS-23 α-amylase site-directed mutagenesis histidine thermostability 


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Copyright information

© Springer 2005

Authors and Affiliations

  • Huei-Fen Lo
    • 1
  • Ya-Hui Chen
    • 1
  • Nai-Wan Hsiao
    • 2
  • Hsiang-Ling Chen
    • 1
  • Hui-Yu Hu
    • 1
  • Wen-Hwei Hsu
    • 3
  • Long-Liu Lin
    • 4
  1. 1.Department of Food and NutritionHungkuang UniversityTaiwan
  2. 2.Graduate Institute of BioinformaticsTaichung Healthcare and Management UniversityTaiwan
  3. 3.Institute of Molecular BiologyNational Chung Hsing UniversityTaiwan
  4. 4.Department of Applied ChemistryNational Chiayi UniversityChiayiTaiwan

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