| Pseudomonas cepacia lipase, with better resistance to heat, various organic solvents (alcohol, etc.), oxidants, surfactants, detergents, protease, etc, now is widely used in organic synthesis, detergent additives, biodiesel production. In this paper, with lipase-producing strain Pseudomonas cepacia PCL-3, optimizations of fermentation conditions of lipase production in flask and 10L fermentor were conducted, and the initial separation and purification of the lipase were also examined. The main results were as follows:(1) According to single factorial experiments, dextrin was the most suitable carbon source and peptone and urea were the suitable compound nitrogen sources, respectively, among the various tested materials. Three important factors (urea,inoculum and initial pH) were selected from the eight factors related to lipase production by Plaekett-Burman method, and response surface analysis was used to optimize these three factors. Steepest ascent procedures were applied to define the optimal response region for the three factors, and then, the optimal concentrations for the three factors were determined as urea 0.15%, inoculum 3.05% and initial pH 8.38 using response surface methodology. Under the optimal conditions, the enzyme activity improved from 25.37U/ml to 48.88U/ml (pNPP assay), enhanced 1.93 folds. Based on the optimal conditions, the impacts of different surfactants, inducers and defoamers on the lipase production were investigated. The results showed that loading a certain amount of surfactant to the medium could significantly increase the production of liapse. Tung oil, tea oil, rapeseed oil and soybean oil could alternate olive oil to induce lipase production properly, which could decrease the cost of lipase production. The concentration of organic silicon defoamer lower than 0.05% in the medium was beneficial to the production of lipase.(2) The highest lipase activity of flask fermentation, batch fermentation and fed-batch fermentation were 19.5U/ml, 18U/ml, 22.75U/ml, respectively. The largest dry cell weights were 11.96g/l, 7.39g/l, 10.019g/l, respectively. The highest lipase activity of batch fermentation was a little lower than that of the flask fermentation. Compared with the batch fermentation, fed-batch operations increased cell density and lipase activity, the highest activity increased 16.67% more than that of the flask fermentation.The optimal operatiion conditions of batch fermentation were temperature 28℃, shake speed 300 rpm, ventilation volume 0.694vvm, initial pH 8.2, dissolved oxygen control more than 10%. Based on the optimal batch fermentation parameters, the optimal operation conditions of fed-batch were 7h from the start of fermentation,adding soybean oil at 4.1 ml/h of continuous-feeding rate till the oil final concentration reached 1%.(3) The Pseudomonas cepacia PCL-3 lipase was purified by using ammonium sulfate precipitation and ethanol precipitation. Its specific activity was improved in saturation range from 20%to 40%, enhanced 1.87 folds. After further dialysis to precipitation, the specific activity was enhanced up to 2.65 folds ,and lipase yield was 72.8%. In the process of ethanol precipitation, adjustmenting the medium pH to 5.0, and adding alginate to a final concentration of 2 mg/ml were beneficial to the deposition of the lipase. The results showed that adding 1 fold volume of ethanol to fermentation supernatant could improve the specific activity to 1.81 folds, and lipase yield was 76.7%. It was improved to some extent with different batches of the fermentation supernatant after the freeze-drying.The maximal improvement enhanced 1.32 folds. Surfactant, such as SDS, Triton X-100, and Tween 80 had certain activation to the lipase, while Triton X-100 had the best effect among those studied surfactants, and the specific activity was enhanced 1.7 folds after 2h interaction with crude enzyme solution. The Ca2+ concentration at between 1mM and 3mM in crude enzyme solution could improve lipase stability.
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