Cloning and expression of gene, and activation of an organic solvent-stable lipase from Pseudomonas aeruginosa LST-03

Abstract

Organic solvent-tolerant Pseudomonas aeruginosa LST-03 secretes an organic solvent-stable lipase, LST-03 lipase. The gene of the LST-03 lipase (Lip9) and the gene of the lipase-specific foldase (Lif9) were cloned and expressed in Escherichia coli. In the cloned 2.6 kbps DNA fragment, two open reading frames, Lip9 consisting of 933 nucleotides which encoded 311 amino acids and Lif9 consisting of 1,020 nucleotides which encoded 340 amino acids, were found. The overexpression of the lipase gene (lip9) was achieved when T7 promoter was used and the signal peptide of the lipase was deleted. The expressed amount of the lipase was greatly increased and overexpressed lipase formed inclusion body in E. coli cell. The collected inclusion body of the lipase from the cell was easily solubilized by urea and activated by using lipase-specific foldase of which 52 or 58 amino acids of N-terminal were deleted. Especially, the N-terminal methionine of the lipase of which the signal peptide was deleted was released in E. coli and the amino acid sequence was in agreement with that of the originally-produced lipase by P. aeruginosa LST-03. Furthermore, the overexpressed and solubilized lipase of which the signal peptide was deleted was more effectively activated by lipase-specific foldase.

Co-Expression of an Organic Solvent-Tolerant Lipase and its Cognate Foldase of Pseudomonas aeruginosa CS-2 and the Application of the Immobilized Recombinant Lipase

“…To prevent the formation of inclusion bodies, the recombinant cells were cultured at a low temperature (20°C) after the addition of IPTG, for a higher temperature (e.g., 37°C) gave rise to easy formation of inclusion bodies [13]. Upon induction with IPTG, both lipase and foldase were expressed in the recombinant E. coli BL21 (DE3) as shown in SDS-PAGE gel (Fig.…”

“…Furthermore, owing to the pathogenicity of strains and/or the requirement of higher lipase productivity, some lipase genes of these strains were expressed in heterogeneous host [11][12][13][14][15]. However, most of these lipases were overexpressed in the bacterial cytoplasm as inactive inclusion bodies.…”

An Organic Solvent-Tolerant Alkaline Lipase from Cold-Adapted <i>Pseudomonas mandelii</i>: Cloning, Expression, and Characterization

“…The lipS gene was cloned using primers based on the non-coding region sequences surrounding the P. fluorescens Pf0-1 lipase gene (UniProt ID Q3KIU1). LipS showed high sequence similarity to organic solvent-tolerant lipase rPFL from Pseudomonas fluorescens JCM5963 13) (80% sequence identity) and to Lip9 from Pseudomonas aeruginosa LST-03 14,15) (44% sequence identity). We found that LipS is an alkaline lipase that demonstrates more than 80% of its activity at pH 8-10.5, is tolerant of organic solvents, and has relatively high thermal stability.…”

Optimization of an organic solvents tolerance lipase expressed in E.coli

“…Figure 6 indicated that 25 °C was the best induction temperature for the expression of functional lipase. Generally speaking, the recombinant cells were cultured at a low temperature after the addition of IPTG to prevent the formation of inclusion bodies [14]. The study by Madan et al indicated 25 °C is beneficial for lipase expression as compare with 30 °C and 37 °C [15].…”