Optimization Of Culture Conditions Of Lipase From Pseudomonas Cepacia G63 And Homologous Expression Of The Lipase And Its Chaperone Gene

90 oil-stained soil samples were collected from Xiangfan city,Changsha city and Wuhan city. By primary plate screening with Rhodamine B as indicator and second flask assay, four strains with their lipase's catalytic activity more than 5.0U/mL were obtained, among which the one from Pseudomonas cepacia G63 ranked the first and reached 15.5U/mL.Lipase production condition of Pseudomonas cepacia G63 was fleetly optimized using Monofactorial experiment and Orthogonal test. The optimum carbon source and nitrogen source for Pseudomonas cepacia G63 screened through Monofactorial experiment were Glucose and Yeast extract, respectively. Three factors(A: Glucose, B: Yeast extract, C: olive) were investigated by Orthogonal test in order to obtain optimal medium. The effect of pH and temperature on the production of lipase was also examined with the optimized medium by Monofactorial experiment. The culture optimal conditions was : Yeast extract 2%, Glucose 0.5%, K2HPO4 0.2%, MgSO4 0.05%, Olive oil 1%, initial pH 7.5, and inoculating at 28℃for 60 h. The lipase activity was enhanced 2.66 fold and reached 45.23 U/ml.The enzyme showed maximum activity at pH 9.0 and at 70℃and was fairly stable between pH 5.0 and 9.5 and at temperatures below 70℃. The enzyme showed high stability in four tested short-chain alcohol(Methanol; Ethanol; Propyl alcohol; Isopropyl alcohol). All these mean that the enzyme has good potential for the transesterification of vegetable oils and animal fat, which are industrially and economically important for the production of biodiesel.The rapid cloning of the lipase and its Chaperone gene was done by two primers. The Open Reading Frame (ORF) of the lipase gene is 1092bps and encodes 364 amino acids. Digested with KpnI and HindIII, the lipase gene was cloned into the broad host range plasmid pBBR1Tp, and transformed into Escherichia coli DH5a. And then, the recombinant plasmids were transferred into Pseudomonas cepacia G63 strain by a triparental mating, using the pRK2013 plasmid as a helper. The recombinant Pseudomonas cepacia G63 preduced 3.6 fold higher of active lipase than the starting one did.