Kinetic and enantioselective behavior of the lipase from Candida cylindracea: A comparative study between the soluble enzyme and the enzyme immobilized on agarose and silica gels

Candida cylindracea lipase has previously been covalently immobilized on agarose by the tosylation method and on silica by the trichlorotiazine method. We describe a simultaneous study of the soluble enzyme and its counterpart immobilized derivatives and quantify the differences between the kinetic behavior of the three forms. The dependence of lipase activity on Na(I) and Ca(II) concentrations has been examined for the three forms of the enzyme, and results interpreted. Kinetic studies with p-nitrophenyl acetate (esterase activity) and tributyrine (lipase activity) as substrates revealed that the immobilized derivatives have lower kcatapp and Kmapp values than the soluble enzyme. With both substrates, the parallel decrease in kcatapp and Kmapp produces the favorable effect of maintaining the kcatappKmapp ratios of the supported enzymes at values comparable to those of the soluble enzyme. Kinetic studies with methyl (R)-(+) and methyl (S)-(−)-2-chloro-propionates (enantioselective activity) point to certain deviations in the Michaelis-Menten behavior. Accordingly, the v versus [S] curves were fitted by the cubic splines method and interpreted using the areas under the curves. A new type of average enantiomeric excess calculated from these areas varied from 6.4% for the soluble enzyme to 24 and 42% when immobilized on agarose and silica, respectively. This is interpreted in terms of a rigidification of the enzyme produced by the covalent immobilization.