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Cloning, sequencing, and characterization of lipase genes from a polyhydroxyalkanoate (PHA)-synthesizing Pseudomonas resinovorans

Author:
Lee, Jeung Hee, Ashby, Richard D., Needleman, David S., Lee, Ki-Teak, Solaiman, Daniel K. Y.
Source:
Applied microbiology and biotechnology 2012 v.96 no.4 pp. 993
ISSN:
0175-7598
Subject:
Pseudomonas resinovorans, active sites, amino acids, carbon, clones, culture media, fluorescence, free fatty acids, gene expression, genes, mutants, nucleotide sequences, olive oil, plasmids, sequence analysis, tallow, triacylglycerol lipase, tributyrin
Abstract:
Lipase (lip) and lipase-specific foldase (lif) genes of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans NRRL B-2649 were cloned using primers based on consensus sequences, followed by PCR-based genome walking. Sequence analyses showed a putative Lip gene-product (314 amino acids, a.a.) with its catalytic active-site (Ser111, Asp258 and His280) identified. The foldase lif gene that is located 55 bps downstream of lip, codes for a putative Lif (345 a.a.). To verify the biological function of the cloned lip gene for lipase expression in P. resinovorans, we constructed a lip knock-out mutant (lip::Tn5<KAN-2>) by transposon-insertion. Complementation of the lip knock-out P. resinovorans mutant with a lipase expression plasmid (pBS29-P2-lip) was constructed, and lipase expression was investigated. The wild-type P. resinovorans and the lip::Tn5<KAN-2>[pBS29-P2-lip] recombinant (but not the lip::Tn5<KAN-2> mutant), showed fluorescence on rhodamine B plates indicative of lipase activity. The wild-type exhibited extracellular lipase activity when grown on medium containing triacylglycerol substrates (tallow, olive oil and tributyrin) as sole carbon sources, but the lip::Tn5<KAN-2> mutant did not show such activity. Lipase activity of various strains was also confirmed by TLC analysis of the composition of acylglycerols and free fatty acid in the extracts of the spent culture medium. We further found that tributyrin was more effective than olive oil in inducing lipase expression in P. resinovorans.
Agid:
60253
Handle:
10113/60253