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Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23

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Abstract

The functional and structural significance of glutamic acid 219 of a N- and C-terminally truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was explored by the approach of site-directed saturation mutagenesis. The expressed wild-type and mutant enzymes have been purified by nickel-chelate chromatography and their molecular mass was determined to be approximately 54 kDa by SDS/PAGE. Except E219F, E219P, and E219W, all other mutant enzymes exhibited a lower shift in their optimum temperatures with respect to the wild-type enzyme. A decreased thermostability was also found in all of the mutant enzymes when compared with the wild-type form of BACΔNC. Except E219F, E219P, and E219W mutant enzymes, greater than 2-fold decrease in k cat and a similar substrate affinity relative to the wild-type BACΔNC were observed for the rest mutant enzymes. Based on these observations, it is suggested that Glu-219 apparently plays an important role in the thermostability of BACΔNC.

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Acknowledgments

We are grateful to Dr. Julio Polaina for the assistance on the molecular model of BACΔNC and the valuable suggestions. This work was supported by a research grant (NSC95-2313-B-241-003) from National Science Council of Taiwan, Republic of China.

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Authors and Affiliations

  1. Department of Applied Chemistry, National Chiayi University, 300 University Road, Chiayi, 60083, Taiwan

    Long-Liu Lin

  2. Institute of Molecular and Cellular Biology, Department of Life Science, National Tsing Hua Univerisity, Hsinchu, 300, Taiwan

    Jai-Shin Liu & Wen-Ching Wang

  3. Department of Food and Nutrition, Hungkuang University, 34 Chungchie Road, Taichung, 433, Taiwan

    Szu-Han Chen, Chih-Chen Huang & Huei-Fen Lo

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  1. Long-Liu Lin
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Correspondence to Huei-Fen Lo.

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Lin, LL., Liu, JS., Wang, WC. et al. Glutamic acid 219 is critical for the thermostability of a truncated α-amylase from alkaliphilic and thermophilic Bacillus sp. strain TS-23. World J Microbiol Biotechnol 24, 619–626 (2008). https://doi.org/10.1007/s11274-007-9518-0

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