Immobilization Of Lipase From Pseudomonas Cepacia And Its Application In The Production Of Biodiesel

Lipase immobilization technology is widely used because it can improve lipase stability, maintain its activity, which renders lipase be reused in biochemical processing for several hundred times thus reduce production cost. In this paper lipase from Pseudomonas cepacia Burkholder was entrapped in hybrid organic-inorganic sol-gel powder prepared by base-catalyzed polymerization of tetramethoxysilane (TMOS) and methyltreimethoxysila (MTMS). When TMOS 2 mmol and MTMS 10 mmol, optimum preparation conditions were water/silane molar ratio 10, enzyme supply 200 mg, and PEG400 120μL. The percentage of protein immobilization was 93.7% and the resulting lipase specific activity of hydrolyzing pNPP was 1.54 times higher than that of the free lipase.The characteristics of immobilized lipase were studied. The results showed that immobilized lipase remained over 80% of its activity when temperature was below 80℃while the residual activity of free one was decreased rapidly when temperature was over 50℃, and only 10% was remained when temperature reached 80℃. After incubating 48 h in 50% methanol solvent, the residual activities of immobilized lipase and free one were 85% and 69% respectively, and after incubating 48 h in 90% ethanol solvent, the residual activities of immobilized lipase and free one were 31% and 0 respectively. The kinetic parameters of Vmax and Km obtained using Lineweaver-Burk plot method were 12.3mmol/min·mg and 4.04mmol/L for immobilized lipase, 8.0mmol/min·mg and 13.64mmol/L for free lipase, respectively.Seven different free lipases from AMANO ENZYME INC.(JAPAN) were tested in transesterification experiments. Transesterification capacity of Lipase PS from P. cepacia ranked the highest and the lipase was selected as the experimental enzyme to be immobilized in sol-gel matrix, and then the immobilized lipase was used to catalyze the transesterification reactions of soybean oil with methanol to produce biodiesel. The factors such as effects of water content, methanol/oil molar ratio, enzyme loading, temperature, organic solvents and time course in the reaction were studied. The results showed that the optimal conditions for converting 4.5g soybean oil were: temperature 40℃, 4:1methanol/oil molar ratio, 6% water content and 646mg immobilized lipase, under which conditions, methyl esters formation of 96.33% was obtained after 72 h, which was 16.3% higher than that of the free one. After continuously used for 10 times, 60% residual activity of immobilized lipase was remained. These results indicated the stability and activity of the lipase when immobilized were highly improved.