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Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis

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Abstract

The calcium-binding residues, Tyr302 and His235, and the sodium-binding residue, Asp194, on the activity of Bacillus licheniformis α-amylase were investigated using site-directed mutagenesis. Tyr302 and His235 were replaced by Asn and Asp, respectively, to produce the mutants Y302N and H235D; Asp194 was replaced by Ala to produce D194A. The mutant amylases were purified to homogeneity; each was ~53 kDa. The specific activity of the D194A was 236 U mg−1, lower than the specific activity of the wild-type enzyme by 55%. No significant changes of thermostability, optimum temperature, and optimum pH level were observed in D194A. Mutant amylases with H235D and Y302N significantly improved their specific activity by 43% (754 U mg−1) and 7% (563 U mg−1), respectively, compared with the wild-type enzyme. H235D substitution decreased its optimum pH by approx. 0.5–1 pH unit.

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Acknowledgments

We thank the staff of Jilin University and the Key Laboratory of Zoonosis Research, Ministry of Education, for the use of their facilities.

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Authors and Affiliations

  1. Laboratory of Nutrition and Functional Foods, Jilin University, 5333 Xi’an Road, Changchun, 130062, People’s Republic of China

    YaNan Qin, Zhen Fang, FengGuang Pan, YaYa Zhao, HongShan Li & Han Wu

  2. Jilin Business and Technology College, 1606 Hao’yue Road, Changchun, 130062, People’s Republic of China

    Xianmei Meng

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  1. YaNan Qin
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  2. Zhen Fang
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  3. FengGuang Pan
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  4. YaYa Zhao
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  6. Han Wu
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  7. Xianmei Meng
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Corresponding author

Correspondence to FengGuang Pan.

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Yanan Qin and Zhen Fang equally contributed to this research as co-first authors.

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Supplementary Table 1 Primers (DOC 130 kb)

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Qin, Y., Fang, Z., Pan, F. et al. Significance of Tyr302, His235 and Asp194 in the α-amylase from Bacillus licheniformis . Biotechnol Lett 34, 895–899 (2012). https://doi.org/10.1007/s10529-011-0843-x

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  • DOI: https://doi.org/10.1007/s10529-011-0843-x

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