Lipase (EC 3.1.1.3) from the $\underline{Candida}$ $\underline{cylindracea}$ was immobilized on the polyacryl amid gel (Bio-Gel P-10) by covalent binding. The substrate with which lipase reacts, has long-chained fatty acids. Therefore, substrates are insoluble in water.
The kinetics of the enzymatic hydrolysis by soluble and immobilized lipase of tributyrin and triolein as the substrates, has been investigated in a batch reactor.
The optimum conditions for lipase immobilization, such as amino ethylation conditions, amount of enzyme to be attached to the gel and coupling time of protein were investigated as a preliminary step.
Emulsion of substrates were prepared to react with lipase. The emulsions containing gum arabic were prepared by homogenization followed by sonication. The surface area of emulsion particle is very important factor in the interfacial reaction of lipase. Hence, the size of emulsion particle was determined, which was 1㎛ and 2 ㎛ in diameter, for tributyrin and triolein, respectively.
Soluble lipases are inhibited severely by substrate at high concentration of substrate emulsion, but immobilization of lipase minimized the substrate inhibition. This is true especially for the triolein emulsion.
Even though apparent Km of immobilized lipase (Km) was greater than that of soluble lipase (Km), the stability of lipase was increased considerably when lipase was immobilized.
The effects of pH on the lipase were studied. Optimum pH of immobilized lipase was shifted to pH of 0.5 from 6-7, compared to soluble lipase.
As a result of immobilization, the value of activation energies were increased. The activation energy is about 8.5 Kcal/mol and 13.0 Kcal/mol, for soluble lipase and immobilized lipase for both emulsions, respectively.
The maximal specific activity was 2,000 μmol/hr/g-Gel and 1,400 μmol/hr/g-Gel for tributyrine and triolein, respectively.
