|本期目录/Table of Contents|

[1]付玮,李友国,李一星.基于黄单胞菌冰核蛋白N端的表面展示体系建立[J].应用与环境生物学报,2014,20(03):351-356.[doi:10.3724/SP.J.1145.2014.10001]
 FU Wei,LI Youguo,LI Yixing.Establishment of cell surface display system based on N-domain of ice nucleation protein of Xanthomonas[J].Chinese Journal of Applied & Environmental Biology,2014,20(03):351-356.[doi:10.3724/SP.J.1145.2014.10001]
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基于黄单胞菌冰核蛋白N端的表面展示体系建立()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
20卷
期数:
2014年03期
页码:
351-356
栏目:
研究论文
出版日期:
2014-06-25

文章信息/Info

Title:
Establishment of cell surface display system based on N-domain of ice nucleation protein of Xanthomonas
作者:
付玮李友国李一星
华中农业大学农业微生物学国家重点实验室 武汉 430070
Author(s):
FU Wei LI Youguo LI Yixing
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
关键词:
野油菜黄单胞菌冰核蛋白细菌表面展示脂肪酶大肠杆菌
Keywords:
Xanthomonas campestris ice nucleation protein bacterial surface display lipase Escherichia coli
分类号:
Q786: Q939.96
DOI:
10.3724/SP.J.1145.2014.10001
文献标志码:
A
摘要:
为了建立一个自主、高效的细菌表面展示体系,利用野油菜黄单胞菌冰核蛋白N端结构域,将短小芽孢杆菌脂肪酶展示于大肠杆菌Rosetta 2 (DE3) 表面. 采用细胞分级、脂肪酶活测定和His-tag In-gel stain分析等检测了重组蛋白的表达情况,并进一步检测了不同底物、温度、pH和有机溶剂对酶活稳定性的影响. 结果显示融合蛋白在大肠杆菌细胞外膜成功展示,重组菌脂肪酶活为(74.6 ± 4.5) U/g冻干细胞,展示水平为每个细胞表面19 267个脂肪酶分子;展示酶对中长链脂肪酸酯表现出较高的水解活性,最适pH为11.0,最适温度为40 ℃,且在40 ℃保温一周仍保持80%以上的酶活. 本研究表明基于野油菜黄单胞菌冰核蛋白N端的展示体系是一种高效、稳定的细菌表面展示体系
Abstract:
This study aimed to establish an autonomous and efficient bacterial cell surface display system. The N-domain of ice nucleation protein from Xanthomonas campestris was used as the anchoring protein for displaying Bacillus pumilus YZ02 lipase on the cell surface of Escherichia coli Rosetta 2 (DE3). The localization of displayed protein was confirmed by cell fractionation, lipase activity assay and Invision His-tag In-gel stain analysis. Then the effects of different substrates, temperatures, pH and organic solvents on enzyme stability were analyzed. The results showed that the fusion protein was successfully displayed on cell surface of Escherichia coli. The activity of displayed lipase was 74.6 ± 4.5 U/g lyophilized cell and the number of lipase molecules displayed on each cell was estimated up to 19 267. The displayed lipase showed high hydrolytic activity toward medium and long-chain fatty acids. The optimum temperature and pH were 40 oC and 11.0, respectively. Importantly, the displayed lipase still retained over 80% of full activity after incubation at 40 oC in Tris-HCl buffer for a week. The results suggested that the display system in this study is an efficient and stable bacterial cell surface display system.

参考文献/References:

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备注/Memo

备注/Memo:
国家“863”计划项目(2013AA102802-04和2006AA06Z350)资助 Supported by the National “863” Program of China (2013AA102802-04 and 2006AA06Z350)
更新日期/Last Update: 2014-07-01