SPECIFICITY TO LEAVING GROUP IN TRANSESTERIFICATION OF SUBSTITUTED PHENYL-ESTERS IN ORGANIC-SOLVENTS CATALYZED BY SUBTILISIN MICROGEL SOLS

Subtilisin has been conjugated to microgels possessing carboxyl functions. The effect of changing the leaving group on transesterification reactions of substituted phenyl acetate and hippurate esters catalysed by a subtilisin-microgel conjugate at pH 9.9 is shown to be governed by the following Bronsted equations (the pH is that of the stock enzyme-conjugate in water, added at a rate of 4% v/v to the organic solvent): log k(cat)/K(m) = -0.54 +/- 0.06 pK(a) + 6.92 +/- 0.53 (acetates, methanol) (i) log k(cat)/k(m) = -0.50 +/- 0.08 pK(a) + 7.23 +/- 0.65 (hippurates, methanol) (ii) log k(cat)/K(m) = -0.83 +/- 0.13 pK(a) + 9.95 +/- 1.1 (hippurates, butanol/acetonitrile at 16.7% v/v) (iii) The more negative slope (beta1g) for the butanol/acetonitrile composition is most likely due to a ground state effect on the effective charge on the esters. alpha-Chymotrypsin has been conjugated with microgels possessing primary amino comonomer components or sulphonic acid with carboxyl functions. The resultant enzyme-polymer conjugates exhibit good solubility properties in organic media over a wide range of pH for the stock aqueous solution of the conjugate. There is no diminution in activity of a solution of the subtilisin-microgel conjugate after 35 days compared with a drop of some 90% for the native enzyme over the same period under the same conditions.