The Michaelis-Menten kinetics of the hydrolytic activity of both free and immobi­lized lipase were determined by measuring the initial velocity of the reaction by varying the concentration of the substrate at a constant enzyme concentration. The apparent kinetic constants of the immobilized lipase were determined with 524 mg of immobilized beads which is equivalent to 50 |rl of free enzyme taken under the same reaction conditions. The free and immobilized lipase in pH 7.0 was incubated with different concentrations of p-NPP substrate (100, 150, 200, and 250 |rg) for 1 min at 25°C.

Michaelis constant K and the maximum reaction velocity V are evaluated from the Lineweaver-Burk plot of 1/V versus 1/S. The Km values of free and immobilized lipase as calculated from Fig. 12.6 are 0.39 and 0.45 |iM, respectively, and corre­sponding V values are 10 and 9.09 amol/min. An increase in K after immobiliza­tion indicates that immobilized enzyme has an apparent lower affinity for the substrate which may be caused by the following reasons: (1) steric hindrance of active site by the support, (2) loss of enzyme flexibility necessary for substrate binding, and (3) dif — fusional resistance to solute transport near the particles of the support.

Reduced enzyme leakage, higher thermal stability, and better storage stability were the salient features achieved by this method of enzyme immobilization. By this work, an improved entrapment approach of lipase cross-linking followed by entrapment onto a hybrid matrix of alginate and к-carrageenan was studied. This enhanced cross-linked matrix is a step closer in design of a better immobilized lipase for the biodiesel industry.