文献类型：期刊文献

英文题名：In vivo functional expression of a screened P. aeruginosa chaperone-dependent lipase in E-coli

作者：Wu, Xiangping[1];You, Pengyong[1];Su, Erzheng[1];Xu, Jingjing[1];Gao, Bei[1];Wei, Dongzhi[1]

机构：[1]E China Univ Sci & Technol, State Key Lab Bioreactor Engn, New World Inst Biotechnol, Shanghai 200237, Peoples R China

年份：2012

卷号：12

外文期刊名：BMC BIOTECHNOLOGY

收录：;WOS:【SCI-EXPANDED(收录号:WOS:000311053000001)】；

基金：This work was supported by the Open Funding Project of the State Key Laboratory of Bioreactor Engineering, the Fundamental Research Funds for the Central Universities and the Specialized Research Fund for the Doctoral Program (New Teachers) of Higher Education (20090074120015).

语种：英文

外文关键词：Pseudomonas aeruginosa; Lipase; Chaperone; Dual expression plasmid; Esterification

摘要：Background: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. It is a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor for functional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. As previously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and its chaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressed in E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA without in vitro refolding process. Results: In this work, we screened a lipase-produced strain named AB through the screening procedure, which was identified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate the gene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E. coli BL21/pET28a-lipAB and two dual expression plasmid systems E. coli BL21/pETDuet-lipA-lipB and E. coli BL21/pACYCDuet-lipA-lipB were successfully constructed. The lipase activities of the three expression systems were compared to choose the optimal expression method. Under the same cultured condition, the activities of the lipases expressed by E. coli BL21/pET28a-lipAB and E. coli BL21/pETDuet-lipA-lipB were 1300 U/L and 3200 U/L, respectively, while the activity of the lipase expressed by E. coli BL21/pACYCDuet-lipA-lipB was up to 8500 U/L. The lipase LipA had an optimal temperature of 30 degrees C and an optimal pH of 9 with a strong pH tolerance. The active LipA could catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meant that LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates for esterification were octylic acid and hexanol, respectively. Conclusions: The effect of different plasmid system on the active LipA expression was significantly different. pACYCDuet-lipA-lipB was more suitable for the expression of active LipA than pET28a-lipAB and pETDuet-lipA-lipB. The LipA showed obvious esterification activity and thus had potential biocatalytic applications. The expression method reported here can give reference for the expression of those enzymes that require chaperones.