Hydrolysis of triglyceride by lipases in two phase system

Abstract

A simple and rapid colorimetric method was developed to determine the lipase activity. Free fatty acids dissolved in isooctane were determined by observing the color developed(absorbancy) using cupric acetate-pyridine as a color developing reagent. The present method eliminates laborous solvent evaporation and centrifugation steps. It takes only a few minutes to determine the free fatty acids by this method, whereas it takes over 20 min by the conventional method. The sensitivity and reproducibility of the method were good for caproic, caprylic, capric, lauric, myristic, palmitic, stearic and oleic acids. The effect of organic solvents on the stability and catalytic activity of the microbial lipase from Candida rugosa and Rhizopus arrhizus for fat splitting and interesterification in two phase system was studied, respectively. The solvents examined were 5 hydrocarbons (n-hexane, n-heptane, n-octane, isooctane, and cyclohexane). The results revealed that isooctane is superior to the other solvents examined for both lipases in two phase system and that cyclohexane and diisopropylether for C. rugosa and R. arrhizus, respectively, are also recommendable. The protective effects of olive oil on the lipase stability against the denaturation or deactivation of protein in organic solvents were investigated. When lipases were incubated in organic solvents with olive oil, the lipases from C. rugosa in isooctane and from R. arrhizus in diisopropylether were substantially stabilized. The lipase stability increased in proportion with the concentration of olive oil in the organic solvents. Characteristics of lipases from C. rugosa and R. arrhizus during the hydrolysis of olive oil in two phase system were investigated. Isooctane was used as an organic solvent for both lipases. An agitation speed at 800 rpm was fixed for sufficient reaction. The proportion of solvent phase and aqueous phase in the reaction mixture was set at the ratio of 8:2 for the study. The lipid ...