Optimization Of Culture Conditions Of Lipase From Pseudomonas Cepacia G-63 And Purification By ATPS

The lipase from Pseudomonas cepacia G-63, an important industrial biocatalysts, can catalyze lipolysis, transesterification, ester synthesis, etc. The lipase has been widely used in many fields, including biodiesel production, oil industry and medicine. However, there is no merchandise production made in China, which blocks further R & D concerning its application. In this study, we optimized formation conditions for lipase production and the technics for its separation and purification. The main results were as follows:Lipase production conditions of Pseudomonas cepacia G-63 were fleetly optimized using Monofactorial experiment, Plackett-Burrman design and response surface methodology (RSM). According to single factorial experiments, among the various examined materials, maltose and soybean cake meal were the most suitable carbon and nitrogen sources. Based on the result, screening design methodology Plackett-Burman was used to evaluate the eleven factors related to lipase production, and three statistically significant factors (soybean cake meal, olive oil, and initial pH) were selected. Then, the first-order model about the factors was conducted to direct steepset ascent experiment. Steepest ascent procedures were applied to define the optimal response region for these three factors, and the optimal concentrations of the three factors were determined using RSM. They were soybean cake meal 4.34%, olive oil 1.96% and initial pH 8.4. Under the optimal conditions, the enzyme activity was improved from 13.45 U/mL to 44.39 U/mL, enhanced more than 3 folds. Based on the optimal conditions, different oils were used as inducer to study the effect on the lipase production. The result showed that bean oil or pig fat could alternate olive oil to induce lipase production, which can decrease the cost of lipase production.Pseudomonas cepacia G-63 was fermented under the optimal conditions, and the lipase was extracted and purified by PEG/phosphate double aqueous phase system. The effects of the relative molecular mass, concentration of PEG, phosphate, NaCl and pH on the distribution coefficient, yield, purification factor and phase separation time were investigated. The results showed that lipase is recovered with the highest yield when concentrations with mass fraction of PEG 2000, phosphate and NaCl in the system were at 10%, 15%, 1%, and pH at 8.0. Under the above conditions, the distribution coefficient, purification factor and lipase yield were at 4.36, 3.98, 87.25%, respectively.