Gene synthesis, expression in E. coli, and in vitro refolding of Pseudomonas sp. KWI 56 and Chromobacterium viscosum lipases and their chaperones.
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.
Traub
P C
PC
Institut für Technische Biochemie, Universität Stuttgart, Germany.
Schmidt-Dannert
C
C
Schmitt
J
J
Schmid
R D
RD
D016956:Burkholderia cepacia / Q000235:genetics; D002861:Chromobacterium / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D008049:Lipase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D018832:Molecular Chaperones / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D017510:Protein Folding; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry
D004274:DNA, Recombinant; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D008049:Lipase