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Applied and Environmental Microbiology, February 1999, p. 787-794, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

High-Level Formation of Active Pseudomonas cepacia Lipase after Heterologous Expression of the Encoding Gene and Its Modified Chaperone in Escherichia coli and Rapid In Vitro Refolding

Dinh Thi Quyen, Claudia Schmidt-Dannert, and Rolf D. Schmid*

Institut für Technische Biochemie, Universität Stuttgart, Stuttgart, Germany

Received 11 September 1998/Accepted 23 November 1998

The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible lambda PRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5' region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5' region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed in E. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

* Corresponding author. Mailing address: Institut für Technische Biochemie, Universität Stuttgart, Allmandring 31, D-70569 Stuttgart, Germany. Phone: 49-711-685-3192. Fax: 49-711-685-4569. E-mail: rolf.d.schmid{at}rus.uni-stuttgart.de.

Applied and Environmental Microbiology, February 1999, p. 787-794, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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