Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones

Applied Microbiology and Biotechnology
P C TraubR D Schmid

Abstract

Pseudomonas lipases are industrially used as detergent additives, in the food industry, and in organic synthesis. Currently, these lipases are either isolated from wild-type strains or overexpressed in recombinant Pseudomonas host strains which may be subject to special safety regulations and thus be unsuitable for enzyme engineering via directed evolution. Here we describe the heterologous expression of two Pseudomonas lipases in Escherichia coli. The lipase genes of Pseudomonas sp. KWI 56 (recently reclassified as Burkholderia cepacia) and Chromobacterium viscosum and the genes of their specific chaperones, which are required for correct folding, were synthesized with an optimized nucleotide sequence and overexpressed (up to 50%) in E. coli. However, both lipases were inactively expressed inside inclusion bodies. Quantitative in vitro refolding of the lipases in the presence of their specific chaperones yielded 310,000 U/g (Pseudomonas sp. KWI 56) and 190,000 U/g (C. viscosum) wet cells. In addition, these lipases could be demonstrated to refold efficiently in the presence of chaperones of related lipases.

Citations

Jul 25, 2009·Applied Microbiology and Biotechnology·Bhawna Madan, Prashant Mishra
May 15, 2010·Applied Microbiology and Biotechnology·Susan BilligWolfgang Zimmermann
May 22, 2010·Journal of Industrial Microbiology & Biotechnology·Yaping LuZhaoxin Lu
Aug 20, 2010·Environmental Technology·Mariane SchmidtPeter Stougaard
Feb 3, 1999·Annals of the New York Academy of Sciences·C Schmidt-DannertR D Schmid
Dec 17, 2014·Microbial Cell Factories·Viviane Paula MartiniNadia Krieger
Apr 10, 2008·Protein Expression and Purification·Kris Pauwels, Patrick Van Gelder
Jun 5, 2008·International Journal of Systematic and Evolutionary Microbiology·Xiang Y HanJonathan Segal
Jun 9, 2009·Microbial Cell Factories·Riccardo VillaPietro Gatti-Lafranconi
Dec 19, 2006·Chembiochem : a European Journal of Chemical Biology·Susanne WilhelmKarl-Erich Jaeger
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Nov 23, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·Janaina Marques AlmeidaNadia Krieger

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