RESEARCH ARTICLE Open AccessIn vivo functional expression of a screenedP. aeruginosa chaperone-dependent lipase in E. coliXiangping Wu † , Pengyong You † , Erzheng Su * , Jingjing Xu, Bei Gao and Dongzhi Wei *AbstractBackground: Microbial lipases particularly Pseudomonas lipases are widely used for biotechnological applications. Itis a meaningful work to design experiments to obtain high-level active lipase. There is a limiting factor forfunctional overexpression of the Pseudomonas lipase that a chaperone is necessary for effective folding. Aspreviously reported, several methods had been used to resolve the problem. In this work, the lipase (LipA) and itschaperone (LipB) from a screened strain named AB which belongs to Pseudomonas aeruginosa were overexpressedin E. coli with two dual expression plasmid systems to enhance the production of the active lipase LipA withoutin vitro refolding process.Results: In this work, we screened a lipase-produced strain named AB through the screening procedure, which wasidentified as P. aeruginosa on the basis of 16S rDNA. Genomic DNA obtained from the strain was used to isolate thegene lipA (936 bp) and lipase specific foldase gene lipB (1023 bp). One single expression plasmid system E. coliBL21/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 werecompared to choose the optimal expression method. Under the same cultured condition, the activities of thelipases 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. Thelipase LipA had an optimal temperature of 30°C and an optimal pH of 9 with a strong pH tolerance. The active LipAcould catalyze the reaction between fatty alcohols and fatty acids to generate fatty acid alkyl esters, which meantthat LipA was able to catalyze esterification reaction. The most suitable fatty acid and alcohol substrates foresterification 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 expressionmethod reported here can give reference for the expression of those enzymes that require chaperones.Keywords: Pseudomonas aeruginosa, Lipase, Chaperone, Dual expression plasmid, EsterificationBackgroundLipases (triacylglycerol acylhydrolases, EC 3.1.1.3) catalyzethe hydrolysis and synthesis of a variety of acylglycerols atthe interface of lipid and water [1]. Owing to the proper-ties like wide substrate specificity, high enantio- andregioselectivity, lipases have a wide range of potentialapplications in industry such as organic synthesis,detergent formulation, food and pharmaceutical industries[2,3]. Pseudomonas lipases display special biochemicalcharacteristics which are not common among thelipases produced by other microorganism. For example,it has exhibited amide hydrolyzing activity [4] and highenantioselectivity towards hydrolysis of racemic esters[5,6].In spite of various potential applications of Pseudomonaslipases, their functional overexpression requires alipase-specific chaperone to fold into an active con-formation [7,8]. Although the lipases can be producedby their homologous expression in Pseudomonas hosts,* Correspondence: ezhsu@ecust.edu.cn; dzhwei@ecust.edu.cn† Equal contributorsState Key Laboratory of Bioreactor Engineering, New World Institute ofBiotechnology, East China University of Science and Technology, Shanghai200237, China© 2012 Wu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly cited.Wu et al. BMC Biotechnology 2012, 12:58http://www.biomedcentral.com/1472-6750/12/58
