ARS | Publication request: Cloning, sequencing and characterization of lipase from a polyhydroxyalkanoate- (PHA-) synthesizing Pseudomonas resinovorans

Submitted to: Annual Meeting and Expo of the American Oil Chemists' Society
Publication Type: Abstract Only
Publication Acceptance Date: January 31, 2012
Publication Date: N/A

Technical Abstract: Lipase gene (lip) of a biodegradable polyhydroxyalkanoate- (PHA-) synthesizing bacterium P. resinovorans NRRL B-2649 was cloned, sequenced and characterized by using consensus primers and PCR-based genome walking method. The ORF of the putative Lip (314 amino acids) and its active site (Ser111, Asp258 and His280) forming a catalytic triad were identified. The biological function of the cloned lip gene in the originating P. resinovorans was verified via the construction of a lip-knockout mutant by a transposon-insertion method. Both the wild type and the lip-knockout mutant were then grown for 48 h in a medium E containing triacylglycerol substrates (tallow, olive oil and tributyrin) as only carbon sources. The results showed that only the wild type produced an extracellular lipase activity as determined by detection of fluorescence emission of cell culture and cell-free culture medium in a rhodamine B plate assay, and by TLC analysis of the composition of acylglycerols and free fatty acid in the extracts of the spent culture medium, thus establishing that the cloned lip gene is responsible for the lipase activity of P. resinovorans. Our study further showed that the substrate tributryin is a more effective inducer for lipase production of P. resinovorans than olive oil. The outcome of this study contributes to the basic knowledge of the fatty acid metabolism of PHA-producing P. resinovorans that could use agricultural fats and oils to make bioplastics. The cloned gene is also useful for producing lipase catalyst needed in various food and non-food applications.