STUDIES ON THE ENANTIOSELECTIVE RETENTION MECHANISMS OF CELLOBIOHYDROLASE I (CBH-I) BY COVALENT MODIFICATION OF THE INTACT AND FRAGMENTED PROTEIN

In order to elucidate the chiral recognition mechanisms of the enzyme cellobiohydrolase I (CBH I), its carboxylic groups were covalently mod ified. The synthetic modification was carried out either in the presen ce or absence of cellobiose, which has proven to inhibit; the enzymati c activity and if present in the mobile phase impairs enantioselectivi ty of amino alcohols. Compared to the reference CSP (unmodified CBH-I silica and CBH-I core silica), the synthetically modified phases show differences both in enantioselectivity and retention. The enzymatic di fferences between the CSPs were also in line with the chromatographic results. The selectivity factors of propranolol are almost unchanged d uring the reaction periods in the presence of cellobiose, while they d ecreased rapidly without the inhibitor, In one case, even a slight imp rovement in enantioselectivity was obtained, indicating that non-stere ospecific carboxylic groups were ruled out. (C) 1998 Wiley-Liss, Inc.