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Engineering of a Bacillus α-Amylase with Improved Thermostability and Calcium Independency

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Abstract

Successful industrial use of amylases requires that they are sufficiently stable and active at application conditions, e.g., at high temperature in starch-liquefaction process. In the present study, site-directed mutagenesis was used to enhance the thermal stability and calcium independency of a mesophilic α-amylase from Bacillus megaterium WHO. Mutations (A53S and H58I) were designed at the calcium-binding site based on the sequence alignment. Kinetic and thermostability parameters of the mutants were analyzed and compared with that of the wild type. In the presence of calcium, the affinity of the enzymes (wild type and mutants) toward starch was increased. In comparison to the wild type, calcium ion had more effect on the catalytic efficiency, k cat/K m, and half-life (at 60 °C) of A53S mutant. In A53S, the dependence of half-life on calcium concentration showed that the enhanced calcium binding is likely to be responsible for the increased stability. In contrast, calcium-independent mutant (H58I) possessed high thermostability. In addition, thermodynamic parameters of amylolytic reaction exhibited an increase in the activation energy and the entropy of the system. Kinetics of irreversible thermal inactivation suggests that the activation energy increased by 1.4-fold in the most stable variant.

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Acknowledgement

The authors express their gratitude to the research council of Tarbiat Modares University for the financial support during the course of this project.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Faculty of Biological Science, Tarbiat Modares University, P.O Box: 14115-175, Tehran, Iran

    Marzieh Ghollasi, Khosro Khajeh & Hossein Naderi-Manesh

  2. Institute of Biochemistry and Biophysics, University of Tehran, P.O. Box: 13145-1384, Tehran, Iran

    Atiyeh Ghasemi

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  1. Marzieh Ghollasi
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  2. Khosro Khajeh
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  3. Hossein Naderi-Manesh
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  4. Atiyeh Ghasemi
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Correspondence to Khosro Khajeh.

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Ghollasi, M., Khajeh, K., Naderi-Manesh, H. et al. Engineering of a Bacillus α-Amylase with Improved Thermostability and Calcium Independency. Appl Biochem Biotechnol 162, 444–459 (2010). https://doi.org/10.1007/s12010-009-8879-2

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