| Lipase is an important enzyme preparation applied widely in many fields.The lipases from Aspergillus oryzae are interested for good safety and catalytic properties.The studies of enzymatic properties and dynamics of lipase from Aspergillus oryzae F0S0179 showed that the optimum temperature of the lipase was 400C,the optimum pH was 7.5,the enzyme has a good thermal stability at 35? and lipase has a better pH stability in the condition of pH7.0-pH8.0.The half-life(ti/2)of the lipase was 1.14 hours(40 0C).The enzyme activity was highest in the buffer solution with ions concentration of 0.25mol/L.When using peanut oil,olive oil,camellia oil and soybean oil as the substrate,the enzyme activity of peanut oil as the substrate had the highest relatively activity;when using p-nitrophenyl myristate(pNPM),lauric acid p-nitrophenyl ester(pNPL),decanoic acid p-nitrophenyl ester(pNPD)and octanoic acid p-nitrophenyl ester(pNPO)as the substrate,the results was that pNPD as the substrate had the highest enzyme activity.The Michaelis constant Km was 0.1g/mL when olive oil was catalyzed by lipase,and the Vm was 250000?mol/mL.min,the activation energy was 44.045 KJ/mol.The ions of Cu2+?Fe2+?Mn2+and K+inhibited the activity of the lipase,and the ions of Mg2+,Ba2+,Ca2+had an active effect on the enzyme activity;Na+and Co2+ had no obvious effect on the enzyme activity.The inhibitory effect of HgC12 on enzyme was reversible,and the inhibition constants Ki was 0.35g/mL,IC50(the inhibitor concentration of enzyme activity decreased by 50%)was 0.68mmol/L.The volume fractions of methanol,ethanol and isopropanol were 15.56%,14.69%and 10.80%respectively,which caused the 50%loss of enzyme activity.The effects of specific protein modification agent on side chain functional groups of lipase were studied,the result showed that the influence of N-bromosuccinimide on enzyme activity was very obvious,and the preliminary inference was got that the tryptophan indole was an essential group to maintain the function of lipase.Aspergillus oryzae FS0179 lipase was immobilized with sodium alginate,polyvinyl alcohol and gelatin,the results showed that the recovery rate of immobilized enzyme was 66.25%,59.81%,61.11%and 71.13%respectively,with sodium alginate gelatin,polyvinyl alcohol,sodium alginate and polyvinyl alcohol sodium alginate immobilized lipase.So the PVA alginate immobilized lipase was selected.The optimum conditions of PVA alginate immobilized lipase were 8%polyvinyl alcohol,2%sodium alginate,2%CaC12,and 1%boric acid.The ratio of enzyme to carrier was 1000U/mL,and the maximum recovery activity of immobilized enzyme was 71.13%.Studies on the enzymatic properties of the immobilized enzyme showed that the optimum temperature is 45oC.When the temperature was lower than 45oC,showed good thermal stability.The half-life time of immobilized enzyme was 18.6 hours(45?).Compared with the free enzyme,the thermal stability of the immobilized enzyme was improved obviously.The optimum pH is 7.5,the stability of pH(pH 6.5?8.5)compared with the free enzyme has a wider scope.The Km of the immobilized enzyme was 0.13g/mL,Vm was 166250?mol/mL.min.The storage stability of the immobilized enzyme was better than that of the free enzyme,and after used 8 times recyclely,the residual relatively activity still was 50.7%.Immobilized Aspergillus oryzae F0179 lipase was immobilized by magnetic nano Fe3O4 particles,diatomite and modified diatomite.The results showed that the recovery rate of immobilized enzyme activity was 51.27%,68.8%,69.19%and 86.13%,respectively,by using magnetic nano Fe304,diatomite,hydroxyl diatomite and silane diatomite.So the immobilization of lipase was carried out by using silylation of diatomite.The optimum conditions for immobilization of lipase with silylation of diatomite were 0.3%silane reagent,and the adsorption temperature was about 35oC,the adsorption time was 5h,the ratio of enzyme to carrier was 1600U/g,and the maximum recovery of enzyme activity of immobilized enzyme reached 86.13%.The studies on the enzymatic properties of immobilized enzyme showed that the optimum temperature was 45?,When the temperature was lower than 45?,it has good thermal stability,The half-life time of immobilized enzyme was 19.5 hours(45oC).Compared with the free enzyme,the immobilized enzyme thermal stability was improved obviously.The optimum pH of immobilized enzyme was 7.2,and the stability of pH(pH 6.5-8)was wider than that of free enzyme.The Kni of the immobilized enzyme was 0.12g/mL,Vm was 200000?mol/mL.min.The storage stability of the immobilized enzyme was better than that of the free enzyme,and after used 8 times recyclely,the residual relatively activity still was 65.3%.Compared with free enzyme,the anti degeneration ability of polyvinyl alcohol and sodium alginate immobilized lipase and silylated diatonite immobilized lipase was improved in organic solvents(methanol,ethanol,isopropanol,acetone,propylene glycol and n-butanol)and denaturant urea solution.Metal ions had no significant effect on the two kinds of immobilized lipase.Studies on the application of polyvinyl alcohol and sodium alginate immobilized lipase uesed for olive oil hydrolysis showed that the optimum of the hydrolysis pH was 7.8,the temperature was 35?,and the ratio of oil to water was 3:2.In the reaction system glycerol concentration showed little effect on olive oil hydrolysis catelyzed by immobilized enzyme.After the immobilized enzyme was used 9 times repeatedly for olive oil,the residual relatively activity still was 45%.Silylated diatomite immobilized lipase applied in the biodiesel synthesis was studied.The results showed that the optimum reaction conditions were the amount of immobilized enzyme 1.5g,the molar ratio of methanol to oil 1.8:1,the amount of n-hexane 1 lmL,the reaction time 30h,the temerature 45?,the final conversion rate reached 85.71%.After the immobilized enzyme was used 5 times repeatedly,the residual enzyme activity was more than 80%.After the immobilized enzyme was used 10 times repeatedly,the residual relatively enzyme activity was 55.1%. |
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