The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition

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.…”

“…The protein has either been immobilized to supporting silica particles [1,2], continuous beds [3,4] or used as a chiral mobile phase additive [5]. Extensive studies of the liquid chromatographic enantiomer separations show that the enantioseparation is strongly dependent on pH, ionic strength, the presence of charged or uncharged modifier in the mobile phase [1,[6][7][8] and temperature [9][10][11]. The threedimensional crystal structure of the Cel7A catalytic domain [12][13][14] reveals a 50 Å long cellulose-binding tunnel [12,13].…”

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

“…The effects of synthetic amidation of the protein could be compared to the site-directed mutagenesis. 16 The amino alcohols show similar trends in the two kinds of modifications of the carboxylates. The acidic substances, however, experience lower retention times at pH 7 after the mutagenesis, while their retentions rise after the synthetic amidation.…”

“…The site-directed mutagenesis showed that two of the three carboxylates seem to be crucial for the chiral recognition. 16 Probably, the three acid residues have different reactivity in the amidation process and a number of different sites might occur after a nonquantitative synthesis. However, there was no significant change in peak asymmetry after the amidation, which might have been expected if the heterogenity of the stationary phase had increased.…”