Screening Of High Lipase Producting Microorganisms ,Production Of Lipase Fermentation & Purification Of Lipase By ATPS

Two hundred and eight oil-stained soil samples were collected from Xiangfan city,Shiyan city and Wuhan city. By primary plate screening with bromothymol blue as indicator and second flask assay, six strains with high ability of lipases production were obtained, and the catalytic activity of the lipases were more than 5.0U/mL, among which the one from Aspergillus sp.F044 ranked the first and reached 11.18U/mL. Lipase production condition of Aspergillus sp. F044 was fleetly optimized using seriatim-factorial experiment,Plackett-Burrman design, response surface methodology (RSM) and Monofactorial experiment. The optimum carbon source and the optimum nitrogen source for Aspergillus sp.F044 screened through seriatim-factorial experiment were maltose and bovine extract, respectively. By Plackett-Burrman design eight process factors related to lipase production were evaluated, among which three factors, concentrations of olive, bovine extract and bitter salt were found to have prominent effect on lipase production. Then the first-order model about the factors was conducted to direct steepset ascent experiment. Under the optimum condition central-composite design and response surface analysis were exercised to estimate optimum culture medium composition. Optimum fermentation temperature and optimum agitation speed of rocking incubator were determined by Monofactorial experiment. The composition of the optimum culture medium was 1.5%(w/v) maltose , 7‰(w/v) ammonium sulfate,1‰(w/v) K2HPO4, 1.25%(w/v) bovine extract, 2.11‰(w/v) bitter salt, 1.41%(v/v) emulsified-olive, nature pH, and the optimum fermentation temperature and the optimum agitation speed were 30℃and 250r/min, respectively. After 72h incubation with the optimum medium under the condition of 250r/min and 30℃, a maximum lipase yield 32.15U/mL was obtained, which was 2.88-fold higher than that under beginning culture mediums. The partition behaviors of the lipase from Aspergillus sp.F044 were also studied in Aqueous Two Phase System(ATPS). The results showed that relative molecular weight of polyethylene glycol, concentration of polyethylene glycol, concentration of phosphate salt, pH value and concentration of NaCl greatly influenced on the partition equilibrium of the lipase in ATPS. The optimized purification condition of Aspergillus sp.F044 lipase was: 17.5% PEG600, 12.5% phosphate salt, 0.5% NaCl and pH7.0. Under the optimal condition partition coefficient of the lipase was 7.1, purification fold was 4.7, and lipase yield was 0.91. Microbial lipases possess great economical potential in industrial applications, and it is just on the first phase for lipase applied in industry in China at present. The main purpose of this subject is to research the problems in lipases industrial production and biodiesel-synthesis, so this study is of great significance.