Studies On Sereening High-yield Phospholipase A1Strain And Its Oil Degumming Performance

Phospholipase A1(Phospholipase A1, PLA1, EC3.1.1.32) can catalytic and hydrolysis the first ester acyl bond of phospholipids to generate lysophospholipids and fatty acids.Enzymatic degumming is a relatively efficient, economical, environmentally friendly method of removal phospholipids in the vegetable oil production, and phospholipase A1is the key of the enzymatic degumming. Combined with the actual production problem, this thesis mainly makes the following researches:(1) Determining the activity assay method of Phospholipase A1. Three common methods of phospholipase A1activity were alkali titration, double-plate method and spectrophotometry. The standard of phospholipase A1and laboratory strains that produced phospholipase A1activity were measured to compare these three methods in time, accuracy and precision differences. The results obtained that spectrophotometry possesses short time-consuming, less substrate, good stability and accuracy.(2) Screening and preliminary identification of the strain producing phospholipase A1. By increasing the concentration of lecithin in the medium, combined with the screening plate colony hydrolysis circle method, strains that grow well、resistant to high concentrations of lecithin and produce turbid ring were obtained. The strain named Dx208with high phospholipase Al activity was singled out by screening repeatedly and its initial activity can reach4.52U/mL. Dx208strain was identified as Streptomyces griseus based on morphological physiological characters and16S rDNA gene sequence. The sequence JN673819was applicated in Genbank.(3) Optimizating the conditions of fermentation of strain Dx208. Studied the impact of fermentation conditions of the strain Dx208by shake flasks. The results showed that Dx208’s activity of phospholipase A1could reach7.15U/mL under the conditions of initial pH7.0, temperature30℃, fermentation time5d and shaking speed200r/min. The activity was1.58times before optimization.(4) Mutation breeding of phospholipase A1-producing strain of Dx208. Screened phospholipase A1-producing strain by UV, nitrosoguanidine mutagenesis, the results showed that:the optimal dose of UV irradiation of90s, NTG concentration of1500μg/mL, and the processing time is90min. Through single and combined mutagenesis after UV and nitrosoguanidine (NTG), screened the strain Dx208-FH12which phospholipase Al fermentation activity was11.68U/mL, and its activity was the original strain Dx208(7.15U/mL)1.63times.(5) Optimizating of fermentation medium components. Studied the impact of fermentation medium composition to the strain of phospholipase Al-producing, including different carbon sources, nitrogen sources, inorganic salts. The results showed that the best medium components of the strain Dx208-FH12enzyme production were as follows:carbon source maltose, nitrogen source (NH)4SO4+NH4Cl, inorganic salts CaCl2. By the optimization of medium components, the enzyme activity of the strain was13.57U/mL, and its activity was the original strain Dx208(7.15U/mL)1.90times.(6) Studying oil degumming performance of phospholipase Al produced by Dx208-FH12. When phospholipase A1produced by the strain Dx208-FH12used for degumming, screened by Plackett-Burman (PB) experimental design, four factors were impact on degumming greater.there were reaction time, reaction temperature, pH and the amount of enzyme. Followed by Box-Behnken central composite and response surface analysis, for the initial phosphorus content is254.30mg/kg of rapeseed oil, a better prediction of model regression equation of phospholipase A1degumming effect was got, and the phospholipase A1produced by the strains Dx208-FH12, and the optimal value was determined as follows:the reaction time3.0h, reaction temperature52℃, pH value of5.0and the amount of enzyme1.8mL/50g oil, and in that conditions degummed oil phosphorus content can be reduced to18.57mg/kg.