| 论文题目: | Autodisplay of an archaeal gamma-lactamase on the cell surface of Escherichia coli using Xcc_Est as an anchoring scaffold and its application for preparation of the enantiopure antiviral drug intermediate (-) vince lactam |
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| 作者: | Wang Jianjun*, Zhao Guogang, Zhang Zhiwei, Liang Qiulin, Min Cong, Wu Sheng* |
| 联系作者: | Wang Jianjun*, Wu Sheng* |
| 刊物名称: | Appl Microbiol Biotechnol |
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| 年份: | 2014 |
| 影响因子: | 3.762 |
| 论文下载: | |
| 摘要: | At present, autotransporter protein mediated surface display has opened a new dimension in the development of whole-cell biocatalysts. Here, we report the identification of a novel autotransporter Xcc_Est from Xanthomonas campestris pv campestris 8004 by bioinformatic analysis and application of Xcc_Est as an anchoring motif for surface display of gamma-lactamase (Gla) from thermophilic archaeon Sulfolobus solfataricus P2 in Escherichia coli. The localization of gamma-lactamase in the cell envelope was monitored by Western blot, activity assay and flow cytometry analysis. Either the full-length or truncated Xcc_Est could efficiently transport gamma-lactamase to the cell surface. Compared with the free enzyme, the displayed gamma-lactamase exhibited optimum temperature of 30 degrees C other than 90 degrees C, with a substantial decrease of 60 degrees C. Under the preparation system, the engineered E. coli with autodisplayed gamma-lactamase converted 100 g racemic vince lactam to produce 49.2 g (-) vince lactam at 30 degrees C within 4 h. By using chiral HPLC, the ee value of the produced (-) vince lactam was determined to be 99.5 %. The whole-cell biocatalyst exhibited excellent stability under the operational conditions. Our results indicate that the E. coli with surface displayed gamma-lactamase is an efficient and economical whole cell biocatalyst for preparing the antiviral drug intermediate (-) vince lactam at mild temperature, eliminating expensive energy cost performed at high temperature. |
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