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.
"Peng et al. reported that no lipase activity was detected in the culture supernatant of the recombinant E. coli BL21(DE3) co-expressing the lipase and its chaperone from Pseudomonas aeruginosa CS-2 . The lipase activity was also not detected in the culture supernatant by the recombinant E. coli JM109 harboring a plasmid containing both lipase and chaperone gene from P. aeruginosa LST-03 . The secretion of subfamily I.1 and I.2 lipases did not proceed properly in heterologous hosts . "
[Show abstract][Hide abstract] ABSTRACT: The lipase subfamilies I.1 and I.2 show more than 33% homology in the amino acid sequences and most members share another common property that their genes are clustered with the secondary genes whose protein products are required for folding the lipase into an active conformation and secretion into the culture medium. In previous studies, the lipase (LipA) and its chaperone (LipB) from Ralstonia sp. M1 were overexpressed in E. coli and the lipase was successfully refolded in vitro. The purpose of this study was to enhance the production of the active lipase LipA from Ralstonia sp. M1 in the heterologous host E. coli without in vitro refolding process, using two-plasmid co-expression systems and dual expression cassette plasmid systems.
To produce more active lipase from Ralstonia sp. M1 in E. coli without in vitro refolding process but with the help of overexpression of the chaperone (LipB1 and LipB3 corresponding to 56-aa truncated and 26-aa truncated chaperone LipB), six different expression systems including 2 two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) and 4 dual expression cassette plasmid systems (BL21/pELipAB-LipB1a, BL21/pELipAB-LipB3a, BL21/pELipA-LipB1a, and BL21/pELipA-LipB3a) were constructed. The two-plasmid co-expression systems (E. coli BL21/pELipABa + pELipB1k and BL21/pELipABa + pELipB3k) produced the active lipase at a level of 4 times as high as the single expression cassette plasmid system E. coli BL21/pELipABa did. For the first time, the dual expression cassette plasmid systems BL21/pELipAB-LipB1a and BL21/pELipAB-LipB3a yielded 29- and 19-fold production of the active lipase in comparison with the single expression cassette plasmid system E. coli BL21/pELipABa, respectively. Although the lipase amount was equally expressed in all these expression systems (40% of total cellular protein) and only a small fraction of the overexpressed lipase was folded in vivo into the functional lipase in soluble form whereas the main fraction was still inactive in the form of inclusion bodies. Another controversial finding was that the dual expression cassette plasmid systems E. coli BL21/pELipAB-LipB1a and E. coli/pELipAB-LipB3a secreted the active lipase into the culture medium of 51 and 29 times as high as the single expression cassette plasmid system E. coli pELipABa did, respectively, which has never been reported before. Another interesting finding was that the lipase form LipA6xHis (mature lipase fused with 6× histidine tag) expressed in the dual expression cassette plasmid systems (BL21/pELipA-LipB1a and BL21/pELipA-LipB3a) showed no lipase activity although the expression level of the lipase and two chaperone forms LipB1 and LipB3 in these systems remained as high as that in E. coli BL21/pELipABa + pELipB1k, BL21/pELipABa + pELipB3k, BL21/pELipAB-LipB1a, and BL21/pELipAB-LipB3a. The addition of Neptune oil or detergents into the LB medium increased the lipase production and secretion by up to 94%.
Our findings demonstrated that a dual expression cassette plasmid system E. coli could overproduce and secrete the active chaperone-dependent lipase (subfamilies I.1 and I.2) in vivo and an improved dual expression cassette plasmid system E. coli could be potentially applied for industrial-scale production of subfamily I.1 and I.2 lipases.
"The cloning and expression of an organic solvent-tolerant lipase, in the presence of the activation gene, was also reported by Ogino et al.  ; however, no stability test of the lipase in organic solvents was conducted. "
[Show abstract][Hide abstract] ABSTRACT: The gene coding for the intracellular organic solvent-tolerant lipase of Pseudomonas aeruginosa strain S5 was isolated from a genomic DNA library and cloned into pRSET. The cloned sequence included two open reading frames (ORF) of 1575 bp for the first ORF (ORF1), and 582 bp for the second ORF (ORF2). The ORF2, known as chaperone, plays an important role in the expression of the S5 gene. The ORF2 is located downstream of lipase gene, and functions as the act gene for ORF1. The conserved pentapeptide, Gly-X-Ser-X-Gly, is located in the ORF1. A sequence coding for a catalytic triad that resembles that of a serine protease, consisting of serine, histidine, and aspartic acid or glutamic acid residues, was present in the lipase gene. Expression of the S5 lipase gene in E. coli resulted in a 100-fold increase in enzyme activity 9 h after induction with 0.75 mM IPTG. The recombinant protein revealed a size of 60 kDa on SDS-PAGE. The Lip S5 gene was stable in the presence of 25% (v/v) n-dodecane and n-tetradecane after 2 h incubation at 37 °C.
[Show abstract][Hide abstract] ABSTRACT: Overexpression of the organic solvent-stable LST-03 lipase from organic solvent-tolerant Pseudomonas aeruginosa LST-03 was attempted using a recombinant Escherichia coli system. It was achieved by deleting the signal peptide of the lipase and using T7 promoter. Although, the expressed lipase formed an inclusion body in E. coli, it was completely solubilized with 4 M urea and 0.05 mM dithiothreitol. The denaturants used for solubilization of the lipase inclusion body were easily removed by dilution or dialysis. The solubilized lipase was effectively activated by incubation with a lipase-specific foldase. The purity of the lipase recovered from the inclusion body was high. Use of a recombinant E. coli system improved the productivity of the highly pure lipase by about 807-fold, compared to the original system using P. aeruginosa LST-03.
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