Screening Of High-Yield Phospholipase A1 Strain

Phospholipase A1 (PLA1) have a specific capacity to hydrolyze the carboxylic ester bond at the sn-1 positions of natural phospholipids to produce L-β1ysophospholipids,mainly obtained from snake venom or animal pancreas, however, its total amount is far from requirement of the industrial production. It has currently been found that a variety of microorganism could produce phospholipase A1 abroad, but which seldom reported in home. Therefore the enzyme which the oil enterprises use totally purchased from the Danish Novozymes Company at a high price. The phospholipase may be commercially used in the refinery process (oil degum) to remove phospholipids. Hence, it has its realistic significance in screening the phospholipase Al effectively and utilizing in the vegetable oil degumming.The strain named PL-06 which has higher phospholipase A1 about 3.96U/ml is obtained from the rich oil soil by using lecithin as the only carbon source and adding bromocresol purple as a type of color development reagent. Through a series of physiological and biochemical identification and 16SrDNA identification, we can determine the strain belong to Serratia profundus strain.The selection of the optimum parent strain producing PLA1 is conducted with an efficient and convenient biological screening method. The obtained strain was then mutagenized by UV light and sodium nitrite in the shake flask culture.A primary optimization for medium composition in shake flask showes that the best nitrogen source was NH4Cl and the best complex carbon source was the combination of malt sugar and lecithin, the density respectively is 2.0% and 0.5%, and the best salt is FeSO4. In such condition, the activity of phospholipase Al of PL-06 can reach 6.41U/ml which is 0.62 times higher than that of the non-optimilized condition(3.96U/ml) after 48 hours fermentation.The results show that:No. PL-06 strains for the original strains of phospholipase A1 activity to 6.41U/ml. Through UV after sodium nitrite compound mutation, obtained the enzyme activity to enhance 94.38% No.PL-06-Fl strain, its enzyme activity is 12.46U/ml. In addition,the optimal conditions for UV mutagenesis is 30W conditions, irradiation distance 30cm, irradiation 1min; And 120s,0.5mol/L as a mutation of the sodium nitrite dose is the most appropriate conditionsBesides, this passage through the effects on fermentation time, fermentation temperature, pH, rotatory velocity and inoculums volume to improve the production of enzyme strain.The optimum conditions for the PLA1 production were 35℃,180 rpm,7.0 of initial pH,10% of inoculum and fermentation which will last 48hours.In order to improve the capacity of enzyme production of the screening of high-yield strains PL-06-F1, in PLA1 fermentation process, we should take the medium into account which is on the growth of production strains of phospholipase Al in PLA1 fermentation process. Based on the results of the previous experiment, three factors influence the select production of phospholipase Al high-yield strains PL-06-F1, which including the lecithin:maltose, ammonium chloride, ferrous sulfate. Then the response surface methodology (RSM) is used to optimize the three factors. By solving the quadratic regression model equation, the optimal conditions of these variables were determined as:proportion of Lecithin and maltose 4.8:1, ammonium chloride 1.52g/L, ferrous sulfate 0.78 uM/L. After culturing under the optimized condition for 48h, phospholipase Al activity reach 18.44U/ml, increased by 47.52% over the previous one.