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Site-directed mutagenesis of the calcium-binding site of α-amylase of Bacillus licheniformis

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Abstract

Amylases that are active under acidic conditions (pH <6), at higher temperatures (>70°C) and have less reliance on Ca2+ are required for starch hydrolysis. The α-amylase gene of Bacillus licheniformis MTCC 6598 was cloned and expressed in Escherichia coli BL21. The calcium-binding site spanning amino acid residues from 104 to 200 in the loop regions of domain B and D430 in domain C of amylase were changed by site-directed mutagenesis and the resultant mutant amylases were analyzed. Calcium-binding residues, N104, D161, D183, D200 and D430, were replaced with D104 and N161, N183, N200 and N430, respectively. Mutant amylase with N104D had a slightly decreased activity at 30°C but a significantly improved specific activity at pH 5 and 70°C, which is desirable character for a food enzyme. The amylase mutants with D183N or D200N lost all activity while the mutant amylase with D161N retained its activity at 30°C but had significantly less activity at 70°C. On the other hand, the activity of the mutant amylase with D430N was not changed at 30°C but had an improved activity at 70°C.

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Acknowledgements

The authors thank Department of Biotechnology, India for the financial assistant through project No BT/PR 4549/PID/20/176/2003 and Center for Excellence in Genomics, Madurai Kamaraj University for providing the infrastructure facilities.

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  1. Department of Genetics, Centre for Excellence in Genomic Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai, 625 021, India

    Ramachandran Priyadharshini & Paramasamy Gunasekaran

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  1. Ramachandran Priyadharshini
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  2. Paramasamy Gunasekaran
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Correspondence to Paramasamy Gunasekaran.

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Priyadharshini, R., Gunasekaran, P. Site-directed mutagenesis of the calcium-binding site of α-amylase of Bacillus licheniformis . Biotechnol Lett 29, 1493–1499 (2007). https://doi.org/10.1007/s10529-007-9428-0

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  • DOI: https://doi.org/10.1007/s10529-007-9428-0

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