Characteristics of lipase from candida rugosa and hydrolysis of triglycerides by the enzyme in AOT-isooctane reversed micellar system

Abstract

For enzymatic production of faty acids, the potential of reversed micelles as reaction system for the hydrolysis of triglycerides is investigated. Lipase from $\mbox{\underline{Candida}}$ $\mbox{\underline{rugosa}}$ can be solubilized in reversed micelles formed by bis (2-ethylhexy) soidum sulfosuccinate and water in isooctane and catalyze the hydrolysis of triglycerides, so that kinetic analysis of the lipase-catalyzed reaction is possible in this system. The enzyme obeys Michaelis-Menten kinetics in the investigated concentration range with Km, app value which is not substantially different from the finding in an aqueous emulsion system and neither are temperature effect and pH profile. Initial velocity of lipase in reversed micelles is 6 times faster than that in emulsion system, which may be due to the enormous interfacial area provided by the reversed micelles. Among various chemical compounds, $Cu^{2+}$, $Hg^{2+}$, and $Fe^{3+}$ inhibit the lipase severely, but its activity is restorable partially by adding histidine or glycine with the metals. The enzyme activity and stability are dependent on the molar ratio of water to surfactant (abbreviated as R) and R values which maximize initial velocity and stability are 10.5. and 5.5, respectively. Addition of glycerol to reversed micelles affects the activity in different fashion according to R. Batch hydrolysis of olive oil by lipase in reversed micelles has also been carried out. Olive oil (5%, v/v or 0.1557 M as ester bond concentration) can be almost completely hydrolyzed at R value of 10 and at 100 mM AOT, since lipase from $\mbox{\underline{Candida}}$ $\mbox{\underline{rugosa}}$ has no positional specificity. Initial water concentration and R are found to be the very important factors that determine equilibrium fractional conversion and the hydrolyzing rate. The lipolysis rate and the stability of the enzyme are affected by stirring and addition of histidine or glycerol. To interpret further the results ...