KINETIC-ANALYSIS OF ENZYMATIC CHIRAL RESOLUTION BY PROGRESS CURVE EVALUATION

The present study deals with kinetic modeling of enzyme-catalyzed reac tions by integral progress curve analysis, and shows how to apply this technique to kinetic resolution of enantiomers. It is shown that kine tic parameters for both enantiomers and the enantioselectivity of the enzyme may be obtained from the progress curve measurement of a racema te only. A parameter estimation procedure has been established and it is shown that the covariance matrix of the obtained parameters is a us eful statistical tool in the selection and verification of the model s tructure. Standard deviations calculated from this matrix have shown t hat progress curve analysis yields parameter values with high accuraci es. Potential sources of systematic errors in (multiple) progress curv e analysis are addressed in this article. Amongst these, the following needed to be dealt with: (1) the true initial substrate concentration s were obtained from the final amount of product experimentally measur ed (mass balancing); (2)systematic errors in the initial enzyme concen tration were corrected by incorporating this variable in the fitting p rocedure as an extra parameter per curve; and (3) enzyme inactivation is included in the model and a first-order inactivation constant is de termined. Experimental verification was carried out by continuous moni toring of the hydrolysis of ethyl 2-chloropropionate by carboxylestera se NP and the alpha-chymotrypsin-catalyzed hydrolysis of benzoylalanin e methyl ester in a pH-stat system. Kinetic parameter values were obta ined with high accuracies and model predictions were in good agreement with independent measurements of enantiomeric excess values or litera ture data. (C) 1994 John Wiley and Sons, Inc.