| Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution |
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| 作者姓名： | ZHANG Xiu-yan RUAN Hui MU Lin HE Guo-qing TANG Xing-jun CHEN Qi-he |
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| 作者单位： | Department of Food Science and Nutrition，Zhejiang University，Hangzhou 310029，China |
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| 基金项目： | Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of ZhejiangProvince (No. Z304076), China |
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| 摘 要： | INTRODUCTION Enzymes are efficient and specific biocatalysts widely used in food industries, but their application in industrial process often involves special properties not found in natural source enzymes. In order to ob- tain desirable enzymes, in the past ten years, scientists have developed rational (Kurth et al., 1998; Mouratou et al., 1999; DeSantis et al., 1999) and irrational de- sign methods (Babbitt and Gerlt, 1997; O’Brien and Herschlag, 1999) to improve the enzyme propert…
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| 关 键 词： | Directed evolution Error-prone PCR DNA shuffling β-1 3-1 4-glucanase Thermostability |
| 收稿时间： | 2005-10-27 |
| 修稿时间： | 2006-03-14 |
Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution |
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| ZHANG Xiu-yan RUAN Hui MU Lin HE Guo-qing TANG Xing-jun CHEN Qi-he.Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution[J].Journal of Zhejiang University Science,2006,7(11):1948-1955. |
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| Authors: | Xiu-yan Zhang Hui Ruan Lin Mu Guo-qing He Xing-jun Tang Qi-he Chen |
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| Institution: | (1) 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 T
m=62.5 °C for the wild-type enzyme to T
m=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.
Project supported by the National Natural Science Foundation of China (No. 20276064) and Natural Science Foundation of Zhejiang
Province (No. Z304076), China |
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| Keywords: | Directed evolution Error-prone PCR DNA shuffling β -1 3-1 4-glucanase Thermostability |
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