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

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          Abstract

          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°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.

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          Most cited references 25

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          Cleavage of structural proteins during the assembly of the head of bacteriophage T4.

           U K Laemmli (1970)
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            Bacterial lipolytic enzymes: classification and properties.

            Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
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              Lipases for biotechnology.

              Lipases constitute the most important group of biocatalysts for biotechnological applications. The high-level production of microbial lipases requires not only the efficient overexpression of the corresponding genes but also a detailed understanding of the molecular mechanisms governing their folding and secretion. The optimisation of industrially relevant lipase properties can be achieved by directed evolution. Furthermore, novel biotechnological applications have been successfully established using lipases for the synthesis of biopolymers and biodiesel, the production of enantiopure pharmaceuticals, agrochemicals, and flavour compounds.
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                Author and article information

                Journal
                BMC Biotechnol
                BMC Biotechnol
                BMC Biotechnology
                BioMed Central
                1472-6750
                2012
                6 September 2012
                : 12
                : 58
                Affiliations
                [1 ]State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai, 200237, China
                1472-6750-12-58
                10.1186/1472-6750-12-58
                3497882
                22950599
                Copyright ©2012 Wu et al.; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Research Article

                Biotechnology

                chaperone, pseudomonas aeruginosa, lipase, dual expression plasmid, esterification

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