Kinetics of olive oil hydrolysis by free and immobilized Candida rugosa lipase

ABSTRACT KINETICS OF OLIVE OIL HYDROLYSIS BY FREE AND IMMOBILIZED CANDIDA RUGOSA LIPASE FADILO?LU, Sibel Ph.D. in Food Engineering Supervisor: Assoc. Prof. Dr. Zerrin SÖYLEMEZ September 1996, 93 pages Lipase catalyzed hydrolysis of olive oil has been studied in oil- in-water emulsion. The fraction of pure lipase in the commercial enzyme preparation was 1.1 %. The molecular weight of monomeric enzyme was determined as 56 000 Daltons. The amount of fatty acids produced was linearly proportional to an enzyme concentration up to 11 ug enzyme/ml. The specific enzyme activity was 2037 units/mg enzyme, at 37°C in 25 mM phosphate buffer, pH 7.0 at 25 % (v/v) olive oil concentration. The hydrolytic reaction obeys Michaelis-Menten kinetics with Vm3vann and Kmanri of 3472 units/mg enzyme and 16.7 %, v/v (0.457 M), respectively. Maximum activity was obtained at pH 7.0, at low buffer ionic strength, 25 mM and 40°C. The enzyme was found to be inhibited by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and o- phthaldialdehyde in a biphasic manner. Candida rugosa lipase was immobilized by two techniques. Entrapment in polyacrylamide gel and adsorption onto celite changed the optimum pH and optimum temperature in the same manner being 6.5 and 45°C, respectively. Entrapment in polyacrylamide gel resulted in large losses of lipase activity. Immobilized lipase exhibited higher productivity toward tributyrin compared to olive oil whereas free enzyme gave higher rate when olive oil was the substrate under the same experimental conditions. mThe enzyme loading used for adsorption on celite was high, being 20 mg protein/gr celite. Adsorption of lipase in a side-on fashion is calculated to give a monolayer at approximately 0.7 my gr celite which is less than the surface area of celite. The specific activity of celite immobilized lipase preparation was lower than that of free enzyme (i\ = 0.90). Under optimum experimental conditions 90 % hydrolysis was reached at 12 hours reaction time. The activity retained was found to be low during repetitive use. Lipase adsorbed on celite gave higher activity compared to polyacrylamide gel entrapped enzyme. Keywords: olive oil, Candida rugosa lipase, hydrolysis, immobilization w