Studies on the enantioselective retention mechanisms of cellobiohydrolase I (CBH I) by covalent modification of the intact and fragmented protein

Abstract

In order to elucidate the chiral recognition mechanisms of the enzyme cellobiohydrolase I (CBH I), its carboxylic groups were covalently modified. The synthetic modification was carried out either in the presence or absence of cellobiose, which has proven to inhibit the enzymatic activity and if present in the mobile phase impairs enantioselectivity 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 differences between the CSPs were also in line with the chromatographic results. The selectivity factors of propranolol are almost unchanged during the reaction periods in the presence of cellobiose, while they decreased rapidly without the inhibitor. In one case, even a slight improvement in enantioselectivity was obtained, indicating that non‐stereospecific carboxylic groups were ruled out. Chirality 10:760–769, 1998. © 1998 Wiley‐Liss, Inc.

Separation of drug enantiomers by liquid chromatography and capillary electrophoresis, using immobilized proteins as chiral selectors

“…Similarly, as shown in Fig. 8, a great reduction in enantioselectivity was observed by Hedeland et al [91] after covalent modification of carboxylic groups of CBH I, thus supporting earlier findings. Although T. ree- sei endoglucanase Cel 7B (EG 1) has a very similar active site, it is worth noting that EG-1 CSPs displayed very poor enantioselectivity compared to that of CBH I CSPs [134].…”

“…In this method, CBH I was bound at pH = 7.0 to amino-derivatized silica gels using the sodium salt of N-hydroxysulfosuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC). Surprisingly, the resulting CBH I-CSPs showed better enantioselectivity and lower retention for propranolol than CBH I-aldehyde stationary phases although carboxylic groups of the catalytically active site have been found to participate in the chiral discrimination mechanism [90,91].…”

“…Enantiomeric separation of rac-propranolol on CBH I core-CSP before and after amidation. Conditions: sodium phosphate buffer, pH = 7.0, I = 0.1; UV detection (reproduced from [91] with permission, no further details on detection available).…”

A statistical experimental design to study factors affecting enantioseparation of propranolol by capillary electrophoresis with cellobiohydrolase (Cel7A) as chiral selector

“…Previous studies using LC with Cel7A as a chiral selector have shown that resolution, selectivity and retention times increase with increasing pH [1,2,6,[30][31][32][33][34][35][36] due to stronger electrostatic binding between the analyte and Cel7A. Similar pH dependence for the interaction between lipophilic amino alcohols and cellulases has been observed in CE [19].…”

“…It can be clearly seen that the effect of the pH is not linear and that an optimum can be found around pH 6. This effect has not been observed in HPLC, where a higher level of pH increased the selectivity [1,6,[30][31][32][33][34][35]. Regardless of the response used, both ionic strength and acetonitrile decrease the separation between the enantiomers, demonstrating that both these factors contribute to the interaction with the selector.…”