Article

World Journal of Microbiology and Biotechnology

, Volume 21, Issue 4, pp 411-416

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

  • Huei-Fen LoAffiliated withDepartment of Food and Nutrition, Hungkuang University
  • , Ya-Hui ChenAffiliated withDepartment of Food and Nutrition, Hungkuang University
  • , Nai-Wan HsiaoAffiliated withGraduate Institute of Bioinformatics, Taichung Healthcare and Management University
  • , Hsiang-Ling ChenAffiliated withDepartment of Food and Nutrition, Hungkuang University
  • , Hui-Yu HuAffiliated withDepartment of Food and Nutrition, Hungkuang University
  • , Wen-Hwei HsuAffiliated withInstitute of Molecular Biology, National Chung Hsing University
  • , Long-Liu LinAffiliated withDepartment of Applied Chemistry, National Chiayi University Email author 

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Summary

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 K m value, more than 66% increase in the value of catalytic efficiency (k cat /K m ) was observed in H436D, H436K, and H436Y. At 70 °C, H436D exhibited an increased half-life with respect to the wild-type enzyme.

Keywords

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