Extraction of the Lipase from Pseudomonas Cepacia S31 and Its Applications in Food
|School||Nanjing Agricultural College|
|Course||Of Food Science|
|Keywords||Pseudomonas cepacia lipase purification L-ascorbyl fatty acid esters immobilization|
Lipases(EC 184.108.40.206), a series of enzymes that catalyze the hydrolysis of various fatty esters were widely distributed in animals, plants and microorganisms. The lipase from Pseudomonas cepacia S31 was non-selectivity of hydrolysis position, and its could improve efficiency of hydrolysis and transesterification. The lipase had been widely used in many fields, including biodiesel production, oil industry and medicine. The lipase from Pseudomonas cepacia S31 was one of the important industrial biocatalysts, can catalyze lipolysis, transesterification, ester synthesis, etc. In this study, we optimized purification conditions for lipase and its application in food. The main results were as follows:1 The purification of the lipase was studied. The lipase of Pseudomonas cepacia S31 was extracted and purified by membrane separation, ammonium sulfate fractionation, freeze drying.The lipase specific activity was from 153.2U.mL/mg to 1203.9 U.mL/mg. The purification factor and lipase yield were at 7.858,43.92%, respectively.2 The application of pseudomonas cepacia S31 lipase in wheat flour. The lipase catalyzed the TAG to MAG and oleic acid. Results indicate that adding level of lipase was 0.5U/g, the carotenoid content was 0.49μg/g. it had decresased 108.97%; the optimal adding level for the -SH and -S-S- contents of water soluble proteins and SDS soluble proteins were 0.3 U/g and 0.4U/g. Meanwhile, the lipase can promote the physical or chemical crosslinking in the water soluble protein and SDS soluble protein and rise to the highest extraction amount of globulin、gliadin、glutenin in the wheat flour by ana-Lox about 0.3 U/g、0.4U/g、0.2 U/g in respectively. The reslts were 15.48μg/g、13.32μg/g、31.24μg/g. The hardness and cohesiveness of dough achieved the highest level by lipase adding level was 0.3U/g.3 Lipase-catalyzed transesterification of lard to produce L-ascorbyl fatty acid esters was studied. The results showed that the optimum conditions for the conversion of L-ascorbic acid was as follows:The reaction medium was tert-butyl alcohol, enzyme dosage 300U,1:3 of mole ratio of L-ascorbic to lard, at 55℃for 24h. Under these reaction conditions, the conversion of L-ascorbic acid up to 31.24%. Based on the results of one-factor- one- time experiments, response surface method was used to optimize the reaction conditions (enzyme concentration, substrate ratio, reaction temperature and reaction time). By analyzing the response surface contour plots generated from the quadratic regression equation and solving the model equation using software of Design Expert. The result showed:enzyme dosage 327U, substrate molar ratio 3.14(L-ascorbic acid to lard), reaction temperature 49.3℃and reaction time 29.7h. Under these reaction conditions, the best conversion of the L-ascorbic acid was 40.69%, compared with the single factor experiments increased by nearly 10 percentage points.4 Condition optimization and stability for immobilization of lipase by macroporous absorbent resin was studied. The experimental results indicated that the optimal conditions of lipase immobilization were:Carrier absorption was DM130 macroporous absorbent resin, the appropriate temperature was 25℃, pH value was 7.5, adsorbent time was 8h, quantity of lipase was 493U per 0.5 gram carrier, crosslinking time 2h and the concentration of crosslinking agent(glutaraldehyde) was 0.05 vol%.