Immobilization Of Burkholderia Cepacia Lipase And Its Application In Biodiesel Preparation

Burkholderia cepacia lipase is one of the ideal catalysts for biodiesel preparation. In order to broaden its application in non-aqueous medium, a series of properties as stability, catalytic activity in organic phase and reusability are need to be improved. Immobilized lipase can match the above need. In this study, B. cepacia lipase was immobilized on macroporous ion exchange resin by adsorption. Optimization of immobilization and preliminary application in biodiesel production was investigated. The main results were listed as follows:(1) Through primarily screening on seven ion exchange resins conventionally used in industrialization, D311, D380, D699K, D418, D401, D152 and D113, D699K achieved the best immobilization effect of lipase.(2) Then, B. cepacia lipase was immobilized on anion-exchange resin D699K by ion exchange adsorption. Factors as pH of immobilized medium, loading amount of the enzyme, concentration of enzyme, temperature, and immobilized time were investigated in detail. After single factorial experiments, the optimum levels for the factors were: pH 9.0, enzyme supply 0.6 g powder per gram carrier, concentration of enzyme 0.1 g/mL, temperature 35℃, adsorption for 3 h. Under these conditions, immobilization efficiency could reach 90.02%, and the specific activity of the immobilized protein on the carrier could reach 131554.2 U/g.(3) Based on single factorial experiments, two important factors ( pH of immobilized medium and temperature ) were selected from the five factors related to the specific activity of the immobilized protein on the carrier by Plaekett-Burman method, and response surface analysis was used to optimize these two factors. Ascent procedures were applied to define the optimal response region for the two factors, and then, the optimal values for the two factors were determined using response surface methodology. Eventually, the optimal parameters of the immobilization were: pH of immobilized medium 8.6, enzyme supply 0.6 g lipase powder per gram carrier, concentration of enzyme 0.1 g/mL, temperature 32.7℃, and adsorption for 3 h. Under the optimal conditions, the measured immobilization efficiency could reach 91.68%, and the specific activity of the immobilized protein on the carrier could reach 138806.9 U/g, whereas the specific activity of free lipase was only 8348.0 U/g, enhanced 16.6 folds. (4) The properties of immobilized B. cepacia lipase were assayed. The results revealed that the optimal temperature of the immobilized lipase and the free lipase were the same, but the immobilized enzyme had a much beter thermal stability than the free lipase. The immobilized lipase also exhibited tolerance to polar alkanes such as methanol and ethanol. Moreover, acetone and other non-polar alkanes such as heptane, iso-octane could activate the immobilized lipase.(5) The immobilized B. cepacia lipase was used to catalyze transesterification reaction of soybean oil and methanol to produce biodiesel. The conditions were optimized: in solvent-free system, enzyme supply 4% and amount of water 8% of oil weight, methanol/oil molar ratio 4:1, two-plus adding methanol for 2 h interval, temperature 40℃, and under which conditions, biodiesel yield of 99.63% was obtained for 12 h. After 10 recycles of usage, the catalytic efficiency of the immobilized B. cepacia lipase only decreased 11.89% of its initial catalytic efficiency, which indicated that the immobilized lipase had a nice operational stability in the transesterification reaction.