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KMID : 0545120120220050607
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Journal of Microbiology and Biotechnology
2012 Volume.22 No. 5 p.607 ~ p.613
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Improvement of Cellulase Activity Using Error-Prone Rolling Circle Amplification and Site-Directed Mutagenesis
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Van Hanh Vu
Kim Keun
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Abstract
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Improvement of endoglucanase activity was accomplished by utilizing error-prone rolling circle amplification, supplemented with 1.7 mM MnCl2. This procedure generated random mutations in the Bacillus amyloliquefaciens endoglucanase gene with a frequency of 10 mutations per kilobase. Six mutated endoglucanase genes, recovered from six colonies, possessed endoglucanase activity between 2.50- and 3.12-folds higher than wild type. We sequenced these mutants, and the different mutated sites of nucleotides were identified. The mutated endoglucanase sequences had five mutated amino acids: A15T, P24A, P26Q, G27A, and E289V. Among these five substitutions, E289V was determined to be responsible for the improved enzyme activity. This observation was confirmed with site-directed mutagenesis; the introduction of only one mutation (E289V) in the wild-type endoglucanase gene resulted in a 7.93-fold (5.55 U/mg protein) increase in its enzymatic activity compared with that (0.7 U/mg protein) of wild type.
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KEYWORD
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Endoglucanase gene, error-prone rolling circle amplification, site-directed mutagenesis, Bacillus amyloliquefaciens
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