Thesis (M.Sc.)--Chulalongkorn University, 2015

Lipases (Triacylglycerol acylhydrolases EC 3.1.1.3) are enzymes which catalyze the hydrolysis of triglycerides at the oil water interphase and generally applicable by many industries such as detergent, baking, oil and fats, hard surface cleaning, organic synthesis, leather, paper and in particular, the production of biodiesel, a renewable green energy. Although lipases can be naturally found in various animals and plants, the microbial lipases have played more important roles for industrial applications due to their rapid growth and high production. Nevertheless, the cost remains the obstacle by which the immobilization can alleviate the problem with higher stability and reusability of the enzymes. In this work, four types of microorganisms from natural sources and the recombinants were comparatively investigated for the potential source of lipase production. Consequently, the recombinant lipase from Aureobasidium pullulans (rAPL) expressed in methylotrophic yeast, Pichia pastoris was selected for the immobilization. In order to obtain the high production of lipase, the induction of expression was optimized by the addition of 2% pure methanol in the culture every 24 hours for the period of 120 hours. The obtained activity of the rAPL was 58.14 μmole/min/mg protein. Then, the optimal conditions for the immobilization of rAPL on the hydrophobic polymeric support, Amberlite XAD7HP by physical adsorption were obtained. 15 mg of rAPL in 50 mM acetate buffer at pH 5 were immobilized on 1 g of Amberlite XAD7HP and continuously stirred at 50°C for 2 hours. The activity of the immobilized rAPL was 5.67 μmole/min/g support. When free and immobilized rAPL were used to catalyze the transesterification for the production of biodiesel using palm oil as the substrate at 40°C for 12 hours, the percentages of the obtained product from the analysis by high performance liquid chromatography were found to be approximately 56 and 61%, respectively. The immobilized rAPL on Amberlite XAD7HP was stable at 40°C and still retained 86% of relative transesterification activity after second cycle use. Overall results indicated that biodiesel production could be accomplished by transesterification catalyzed by the immobilized rAPL on Amberlite XAD7HP.