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KMID : 0545120100200121696
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Journal of Microbiology and Biotechnology
2010 Volume.20 No. 12 p.1696 ~ p.1701
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Repeated Random Mutagenesis of α-Amylase from Bacillus licheniformis for Improved pH Performance
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Ramachandran Priyadharshini
Shankar Manoharan
Devaraj Hemalatha
Paramasamy Gunasekaran
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Abstract
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The α-amylases activity was improved by random mutagenesis and screening. A region comprising residues from the position 34-281 was randomly mutated in B. licheniformis α-amylase (AmyL), and the library with mutations ranging from low, medium, and high frequencies was generated. The library was screened using an effective liquid-phase screening method to isolate mutants with an altered pH profile. The sequencing of improved variants indicated 2-5 amino acid changes. Among them, mutant TP8H5 showed an altered pH profile as compared with that of wild type. The sequencing of variant TP8H5 indicated 2 amino acid changes, Ile157Ser and Trp193Arg, which were located in the solvent accessible flexible loop region in domain B.
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KEYWORD
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Directed evolution, High-throughput screening, Error-prone PCR, Bacillus α- amylase, random mutagenesis
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