The physiology, biochemistry and molecular cloning of a Pseudomonas lipase.
thesisposted on 19.11.2015, 09:08 by Elizabeth Jane. Gilbert
The physiological regulation of lipase production by a newly-isolated strain of Pseudomonas aeruginosa (strain EF2) was investigated during growth in batch, fed-batch and continuous cultures. Lipase activity (measured as the rate of olive oil hydrolysis) was strongly induced by long- chain fatty acyl esters (especially Tween 80), weakly induced by carbon and/or energy limitation, and repressed by long-chain fatty acids, including oleic acid. Lipase production by Ps. aeruginosa EF2 was optimised with respect to temperature and pH in a Tween 80-limited continuous culture using statistical response surface analysis at a fixed dilution rate of 0.05 h-1 (optimum values 35.5 C, pH 6.5). Maximum activities were obtained under these conditions at a dilution rate of 0.04 h-1 (39 LU [mg cells]-1; where 1 LU equalled 1 ?mol titratable fatty acid released min-1). These were over eight- times greater than the maximum activities detected following growth on Tween 80 (8.3 LU mg cells-1), and over forty-times greater than growth on glucose (1 LU mg cells-1) in batch cultures. Esterase activities (measured as both the rate of hydrolysis of both p-nitrophenyl acetate and Tween 80) varied approximately in parallel with lipase activities under nearly all growth conditions, suggesting that a single enzyme catalysed both activities. Lipase was isolated from a high activity continuous culture of Ps. aeruginosa EF2. The enzyme was purified to homogeneity (99.5 % pure from SDS-PAGE analysis) by ultrafiltration of the culture supernatant, followed by anion-exchange and gel-filtration FPLC. The lipase was composed of a single subunit protein which appeared to aggregate variably under non-dissociating conditions. The enzyme was a true lipase that exhibited some esterase activity towards p-nitrophenyl acetate and Tween 80. The enzyme preferentially hydrolysed the 1,3-oleyl residues of radiolabelled triolein, was relatively stable at moderate temperatures and very stable to freezing and thawing. The enzyme was only weakly inhibited by the serine- active reagent 3,4-dichloroisocoumarin, and not inhibited by the chelating agent EDTA. The N-terminal amino acid sequence of the Ps. aeruginosa EF2 lipase showed a significant homology with those of other Pseudomonas lipases. The lipase gene of Ps. aeruginosa EF2 was identified by Southern analysis of restricted chromosomal DNA using a synthetic oligonucleotide probe based on the N-terminal amino acid sequence of the purified lipase. The hybridizing EcoR I fragments were ligated into the plasmid vectors pKT230 (pJG1) and pUC18 (pJG5 and pJG31). These constructs were used to transform the lipase-negative E. coli JM109 and the resultant clones (JG1, JG5 and JG31) were able to grow on Tween 80. pJG5 was further analysed by restriction with various endonucleases and Southern blotting. Sub-clones of pJG5 were constructed (containing either a 4.3 kbp BamlH I, 2.5 kbp Sal I or 3.2 kbp Sph I fragment), but these failed to confer the lipase-positive phenotype to E. coli JM109. Esterase activity (measured as p-nitrophenyl acetate hydrolysis) of E. coli JG5, unlike that of Ps. aeruginosa EF2, was not subject to substrate-induction/end-product repression and was very low.