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| Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution |
| ZHANG Xiu-yan, RUAN Hui, MU Lin, HE Guo-qing, TANG Xing-jun, CHEN Qi-he |
| Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China |
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Abstract In order to improve the thermostability of β-1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGs1 and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from Tm=62.5 °C for the wild-type enzyme to Tm=65.5 °C for the mutant EGs1 and 67.5 °C for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher (28%) for the former or much lower (21.6%) for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.
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Received: 27 October 2005
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