Chiral stationary phases based on intact and fragmented cellobiohydrolase I immobilized on silica

Marle, Ingrid; Jónsson, S.; Isaksson, Roland; Pettersson, Curt; Pettersson, Göran

doi:10.1016/0021-9673(93)80415-5

Cited by 52 publications

(26 citation statements)

References 18 publications

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“…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].…”

Section: Phsupporting

confidence: 57%

“…a* = m app first enantiomer /m app last enantiomer (2) Another way of defining the separation between the enantiomers is the relative mobility difference:…”

Section: Calculationsmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2002

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The capillary electrophoretic enantioseparation of rac-propranolol using cellobiohydrolase Tr Cel7A as selector was optimized by an unbiased statistical experimental design. A set of pre-experiments was performed in order to identify critical experimental factors. In the definitive chemometric design pH, ranging from 5 to 7, ionic strength ranging between 0.01 and 0.02 and organic solvent additive in concentration from 1 to 19% v/v were studied. The response surface plot revealed a separation optimum in the pH interval studied. When all parameters were taken into account, a background electrolyte consisting of 0.016 M bistris-acetate buffer with pH 6.5 and 17% v/v acetonitrile gave the optimum separation. The significance of the statistical design was confirmed by the generally good agreement obtained between predicted response and actual experimental data.

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Section: Phsupporting

confidence: 57%

“…a* = m app first enantiomer /m app last enantiomer (2) Another way of defining the separation between the enantiomers is the relative mobility difference:…”

Section: Calculationsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2002

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“…One approach involves direct resolution where a chiral substance (selector) is immobilized on a silica support as a chiral stationary phase (CSP) [ 1,3,4]. A number of proteins have been used for this purpose, e. g., cq-acid glycoprotein [5], human serum albumin [6], e-chymotrypsin [7], ovomucoid [8] and cellulase [9].…”

Section: Introductionmentioning

confidence: 99%

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

et al. 1999

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Key WordsColumn liquid chromatography Glucoamylase Enantioselective retention and resolution Effect of operating parameters Ionic strength SummarySeveral mobile phase parameters were investigated for controlling enantioselective retention and resolution on a chiral stationary phase made in-house. The chiral selector was the enzyme amyloglucosidase, which was immobilized onto a silica support via reductive amination. The influences of the mobile phase pH, concentration and type of uncharged organic modifier, ionic strength and column temperature on enantioselectivity were studied. The analysis time for resolving enantiomers could be adjusted with only a minor decrease in enantiosetectivity by using a high ionic strength mobile phase buffer. This indicated a retention mechanism involving ion-exchange interactions. It was further confirmed by the decreasing enantioselectivity of amines when using a mobile phase pH below the isoelectric point of the native protein. Interesting effects were observed when the organic modifier concentration was increased and also when the column temperature was raised. Both retention and enantioselectivity increased with increasing concentration of 2-propanol in the mobile phase. Examples are given where both enantioselectivity and retention increased with increasing column temperature. Thermodynamic studies were performed to calculate the entropy and enthalpy constants. The results showed that, depending on mobile phase composition, the enantioselective retention may be caused by differences in entropy or enthalpy.

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“…It can be coupled to a solid support (silica or continuous bed) [l(L12] to form a chiral stationary phase (CSP) or used in a carrier-free solution in capillary electrophoresis [13]. A rather broad spectrum of racemic solutes can be resolved, but CBH 1 functions best with amino alcohols such as the drugs used to block [3-adrenergic receptors, so called [3-blockers. In an attempt to localize the chiral site of CBH 1, we found that both the binding and catalytic domains contained enantioselective sites for propranolol, a [3-blocker, but that the major enantioselectivity for propranolol and other solutes could be ascribed to the catalytic domain [14].…”

Section: Introductionmentioning

confidence: 99%

The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

et al. 1996

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The cellulases ceHobiohydrolase 1 (CBH 1) and endoglucanase 1 (EG 1) from the fungus Trichoderma reesei are closely related with 40% sequence identity and very similar in structure. In CBH 1 the active site is enclosed by long loops and some antiparallel ~strands forming a 40 A long tunnel, whereas in EG 1 part of those loops are missing so that the enzyme has a more common active site groove. Both enzymes were immobilized on silica and these materials were used as chiral stationary phases for chromatographic separation of the enantiomers of two chiral drugs, propranolol and alprenolol. The CBH 1 phase showed much better resolution than did the EG 1 phase, suggesting that the tunnel structure of the protein may play an important role in the chiral separation. The chiral compounds were found to be competitive inhibitors of both enzymes when p-nitrophenyl lactoside (pNPL) was used as substrate. (S)-enantiomers showed stronger inhibitory effects and also longer retention time on the stationary phases than the (R)-enantiomers. The consistency between kinetic data and retention on the stationary phases clearly shows that the enzymatically active sites of CBH 1 and EG 1 are involved in chiral recognition.

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Chiral stationary phases based on intact and fragmented cellobiohydrolase I immobilized on silica

Cited by 52 publications

References 18 publications

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

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

Use of immobilized amyloglucosidase as chiral selector in chromatography. Control of enantioselective retention and resolution in liquid chromatography

The active sites of cellulases are involved in chiral recognition: a comparison of cellobiohydrolase 1 and endoglucanase 1

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