Improvement of cellulase activity using error-prone rolling circle amplification and site-directed mutagenesis
Journal of Microbiology and Biotechnology
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
... ated 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.