A technique based exclusively on chiral reversed-phase liquid chromatography has been shown to greatly facilitate studies of enantioselectivity in lipase-catalyzed hydrolysis of chiral organic esters. Only two sets of experimental data are needed to calculate the enantioselectivity (E) of a kinetically controlled enantiomer-differentiating reaction of this kind, viz. the enantiomeric excess of the product (ee,) or substrate (ee,), and the degree of substrate conversion (c). The product enantiomers are well separated on a BSA-based column, giving eep directly. In addition, separation of the (unresolved) ester substrate from the enantiomeric products gives c by integration. Via an optimization of the mobile phase used in the chiral chromatographic system, both these parameters can often be determined in a single run. Highly precise and detailed kinetic studies of the enzymatic reaction can thus be performed. In this way, E-values have been determined for a series of 2-chloroethyl 2-arylpropanoates hydrolyzed in the presence of a Candah cykndracea lipase at pH 6.0 and 7.1. Effects on the E-values from a partial purification and further processing of the lipase have also been studied.
A method for the determination of lipase activity in terms of rate and enantioselectivity of hydrolysis of a chiral ester substrate has been developed. When this method was applied to fractions, isolated from preparative, column chromatographic separations (anion-exchange, molecular sieve) of the lipase, significant differences in enantioselectivity (E) was found between the fractions. The highest enantioselectivity was found in the first main peak obtained on DEAE-Sepharose chromatography, meaning that the enzyme with the highest isoelectric point shows the highest esterolytic enantioselectivity .The experimental results are discussed in the light of some earlier reported results and with respect to the possible existence of subunit aggregates and isoenzymes.
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