Kinetic and enantioselective behaviour of isoenzymes A and B from Candida rugosa lipase in the hydrolysis of lipids and esters

The isoenzymes A and B from C. rugosa lipase have been kinetically characterized by non-linear regression methods measuring the hydrolysis of tributyrin (lipase activity), p-nitrophenyl acetate (esterase activity) and methyl 2-chloropropionate (enantioselectivity). With tributyrin as substrate, the specificity constant (k(cat)(app)/K(m)(app)) for lipase A is about 2-fold that of the lipase B. This confirms the higher specificity of lipase A for substrates containing butyric acid moieties. However, the kinetic behaviour of both isoenzymes for the hydrolysis of p-nitrophenyl acetate is quite similar, with lipase A showing a slight preference for this substrate. Kinetic analysis with the substrates methyl (R)-(+) and methyl (S)-(-) 2-chloropropionates showed that both isoenzymes exhibit stereospecificity for the enantiomer R and display non-Michaelian kinetics of a sigmoidal type. Since this behaviour precludes the usual treatment on the basis of the enantiomeric ratio [(V(max)/K(m))(fast)/(V(max)/K(m))(slow)], enantioselectivity was quantified using the areas under the v-[S] curves. Thus, the ratio between the areas under the curves for the two enantiomers assayed increases from 1.27 for isoenzyme A to 1.68 for isoenzyme B. This result clearly shows that isolation and purification of isoenzymes may enhance the enantioselective properties of an enzymatic system. | The isoenzymes A and B from C. rugosa lipase have been kinetically characterized by non-linear regression methods measuring the hydrolysis of tributyrin (lipase activity), p-nitrophenyl acetate (esterase activity) and methyl 2-chloropropionate (enantioselectivity). With tributyrin as substrate, the specificity constant (kcat app/Km app) for lipase A is about 2-fold that of the lipase B. This confirms the higher specificity of lipase A for substrates containing butyric acid moieties. However, the kinetic behaviour of both isoenzymes for the hydrolysis of p-nitrophenyl acetate is quite similar, with lipase A showing a slight preference for this substrate. Kinetic analysis with the substrates methyl (R)-(+) and methyl (S)-(-) 2-chloropropionates showed that both isoenzymes exhibit stereospecificity for the enantiomer R and display non-Michaelian kinetics of a sigmoidal type. Since this behaviour precludes the usual treatment on the basis of the enantiomeric ratio [(Vmax/Km)fast/(Vmax/Km)slow], enantioselectivity was quantified using the areas under the ν-[S] curves. Thus, the ratio between the areas under the curves for the two enantiomers assayed increases from 1.27 for isoenzyme A to 1.68 for isoenzyme B. This result clearly shows that isolation and purification of isoenzymes may enhance the enantioselective properties of an enzymatic system.

Peer reviewed