| 论文题目: | Directed evolution and structural analysis of alkaline pectate lyase from alkaliphilic Bacillus sp. N16-5 for improvement of thermostability for efficient ramie degumming |
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| 作者: | Zhou Cheng, Ye Jintong, Xue Yanfen, and Ma Yanhe*. |
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| 刊物名称: | Appl Environ Microbiol |
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| 年份: | 2015 |
| 影响因子: | 4.359 |
| 论文下载: | 下载地址 |
| 摘要: | Thermostable alkaline pectate lyases have potential applications in the textile industry as an alternative to the chemical-based ramie degumming processes. In particular, the alkaline pectate lyase from Bacillus sp. N16-5 (BspPelA) has potential for enzymatic ramie degumming because of its high specific activity under extremely alkaline conditions without the requirement for additional Ca2+. However, BspPelA displays poor thermostability and is inactive after incubation at 50 degrees C for only 30 min. Here, directed evolution was used to improve the thermostability of BspPelA for efficient and stable degumming. After two rounds of error-prone polymerase chain reaction and screening of more than 12,000 mutants, 10 mutants with improved thermostability were obtained. Sequence analysis and site-directed mutagenesis revealed that the single substitutions E124I, T178A, and S271G were responsible for improving thermostability. Structural and molecular dynamic simulation analysis indicated that formation of a hydrophobic cluster and new H-bond networks were the key factors contributing to the improvement in thermostability with these three substitutions. The most thermostable combined mutant EAET exhibited a 140-fold increase in t50 value at 50 degrees C, accompanied by an 84.3% decrease in activity compared with wild-type BspPelA, while the most advantageous combined mutant EA exhibited a 24-fold increase in t50 value at 50 degrees C, with a 23.3% increase in activity. Ramie degumming with EA mutant was more efficient than that with wild-type BspPelA. Collectively, our results suggested that the EA mutant, exhibiting remarkable improvements in thermostability and activity, had the potential for applications in ramie degumming in the textile industry. |
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