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

Abstract

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 λPRL 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 inE. 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.

The cold‐active lipase of Pseudomonas fragi

“…Several Pseudomonas lipases have been reported to require a chaperone (or helper) protein for efficient secretion and folding of the active lipase [9]. Therefore, coexpression of the lipase-and helper-encoding genes has been successfully exploited as a tool to obtain high levels of recombinant active lipase in heterologous bacterial hosts [29]. However, coexpression of rPFL with the foldase of P. aeruginosa did not produce significant improvements in the fraction of soluble lipase.…”

Monoseptic growth of fungal lipase producers under minimized sterile conditions: Cultivation of Phialemonium curvatum in 350 L scale

“…Decades of development of recombinant strains revealed high titers and high productivity. E. coli strains co-expressing a modified chaperone were found to produce 314 000 U/g wet biomass in short time [28]. Fungi, e.g.…”

Cloning and heterologous expression of two cold-active lipases from the Antarctic bacterium Psychrobacter sp. G

“…However, the expression in an active form of lipase belonging to subfamilies I.1 and I.2 depends on a chaperone protein named lipase‐specific foldase, the gene of which is located downstream of the lipase gene for the efficient secretion and folding of active lipase (Arpigny et al. 1999; Quyen et al. 1999), whereas the folding of subfamily I.3 lipases does not require the assistance of any molecular chaperone (Angkawidjaja & Kanaya 2006).…”

“…1999), whereas the folding of subfamily I.3 lipases does not require the assistance of any molecular chaperone (Angkawidjaja & Kanaya 2006). For example, a lipase belonging to subfamily I.1 or I.2 was heterologously expressed in E. coli , and the chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U mg −1 , whereas in a simple and rapid in vitro refolding procedure with its modified and truncated chaperone, functionally active lipase was obtained with a specific activity of up to 4850 U mg −1 and a yield of 31 400 U g −1 of E. coli wet cells (Quyen et al. 1999).…”

“…A dozen genes encoding cold‐active lipolytic enzymes have been cloned from different species and environmental metagenomes (Choo et al. 1998; Quyen et al. 1999; Alquati et al.…”