| Lipase was an important hydrolase, which could hydrolyze triglycercide to fatty acid, glycerol and the intermediate products monoglyceride and diglyceride in the oil-water level, so it had good prospects for industrialization if applied in synthetizing biodiesel. Biodiesel was fatty acid methyl ester produced by transesterification of animal fat and vegetable oil with lower alcohol, and it was a clean biofuel and a nice substitute for petroleum diesel. It had many defects for the traditional chemical approach to synthetize biodiesel, such as trenchant requirements of substrates, complicated post treatment process, biggish environmental pollution and so on, but it could remedy these defects taking lipase as catalyst. At the moment, production level of lipase nethermore, enzyme activity lower, stability of enzyme activity inferior, these defects restricted its application in industries, so had positive and realistic significance to expand correlative research to increase lipase activity and enhance its stability.The erforscht geotrichum candidum PP1315 was gotten by complex mutagenesis in our lab, and batch fermentation kinetics, lipase isolation and purification, lipase immobilization and its catalyzing esterification were studied in this paper. The results were as follows:The fermentation process of the geotrichum candidum PP1315 was magnified in a 5 L fermenter, and on the optimum conditions of measure, lipase activity of fermentation liquor could reach 240.15 U/mL. Meanwhile, based on the data gotten from its batch fermentation in the 5 L fermenter, and combined mathematical reasoning, kinetic models of cell growth, synthesis of lipase and substrate consumption were established. These were as follows:(1) The kinetic model of cell growth(2) The kinetic model of synthesis of lipase (3) The kinetic model of substrate consumptionThese established models could describe well the dynamic process of synthesis of lipase by the batch fermentation of PP1315, and it could give some help on the further scale-upThe isolation and purification of lipase was studied by hyperfiltration, ammonium sulfate salting-out, Sephadex G-75 gel filtration chromatography, and the result of purification of every time was analyzed and identified. The results showed that lipase activity reached 1448.7 U/mg,9.61-fold purified lipase was obtained; recovery of lipase activity was 35.2% after purification. The purified lipase showed two clear bands by SDS-PAGE gel electrophoresis, which indicated that the lipase molecule was composed by a subunit, and their molecular weight was 35 kDa.The immobilization of lipase on indigenous LX-1000HA resin was studied. These main immobilization conditions of lipase on it were optimized by Single-factor experiments and experimental design of Box-Behnken. The results showed the optimal immobilization conditions:pH of lipase 7.486, temperature of immobilization 26.295℃, amount of carrier 2.469 g, time of immobilization 24 h, and under these conditions the lipase activity reached 409.32 U/g; recovery of lipase activity was 47.79%; fixed rate of lipase was 90.43%, which illustrated that it was feasible for LX-1000HA resin to immobilize lipase.The better conditions of preparative immobilized lipase-catalyzed transester-ification were studied by orthogonal experiments, and the results showed the best conditions:concentration of n-hexane 10%, water volume 7.5%, reaction temperature 45℃, methyl alcohol being divided 3 times to feed to reaction system and the reaction time of every addition 8h and the mole number of methyl alcohol being equal to grease.Under these conditions the best results could obtained and conversion rate fatty acid methyl ester also reached 42.3% after consecutive reaction of 10 times, and its half life was closed to 240 h, which indicated that the conversion result was better; the stability of lipase activity was higher; it had a prospect for application.
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