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

Hongbin Henriksson, Jerry Ståhlberg, Anu Koivula, Göran Pettersson, Christina Divne, Ludmila Valtcheva, Roland Isaksson (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

In order to investigate the basis for chiral separation in cellobiohydrolase 1 (CBH 1) and the closely related enzyme endoglucanase 1 (EG 1) from Trichoderma reesei, the wildtype proteins of CBH 1 and EG 1, as well as three catalytically deficient mutants of CBH 1 (E212Q, D214N and E217Q) were immobilised to silica and used as chiral stationary phases (CSPs) in HPLC. A large group of enantiomers could be completely resolved on the wildtype CBH 1-silica CSP while the corresponding EG 1-silica CSP only gave a partial separation of the same set of compounds. Of the CBH 1-mutant CSPs, only the D214N-CSP retained enantioselectivity whereas the selectivity was completely lost for the E212Q and E217Q-CSPs. The loss of enantioselectivity follows the same pattern as the loss of catalytic activity for the mutants which was determined from kinetic experiments using oligosaccharides as substrates. Mexiletine, a basic drug which could not be separated on the wildtype CBH 1-CSP, was successfully separated on one of the mutant phases. This demonstrates how protein engineering can be used to tailor new chiral selectors.

Original languageEnglish
Pages (from-to)115 - 125
Number of pages11
JournalJournal of Biotechnology
Volume57
Issue number1 - 3
DOIs
Publication statusPublished - 1997
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Amino acids
Catalytic Domain
Enantioselectivity
Silica
Amino Acids
Silicon Dioxide
Proteins
Oligosaccharides
Enantiomers
Catalyst activity
Enzymes
Mexiletine
Protein Engineering
Trichoderma
Kinetics
Substrates
Experiments
High Pressure Liquid Chromatography
Pharmaceutical Preparations

Cite this

Henriksson, Hongbin ; Ståhlberg, Jerry ; Koivula, Anu ; Pettersson, Göran ; Divne, Christina ; Valtcheva, Ludmila ; Isaksson, Roland. / The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition. In: Journal of Biotechnology. 1997 ; Vol. 57, No. 1 - 3. pp. 115 - 125.
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title = "The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition",
abstract = "In order to investigate the basis for chiral separation in cellobiohydrolase 1 (CBH 1) and the closely related enzyme endoglucanase 1 (EG 1) from Trichoderma reesei, the wildtype proteins of CBH 1 and EG 1, as well as three catalytically deficient mutants of CBH 1 (E212Q, D214N and E217Q) were immobilised to silica and used as chiral stationary phases (CSPs) in HPLC. A large group of enantiomers could be completely resolved on the wildtype CBH 1-silica CSP while the corresponding EG 1-silica CSP only gave a partial separation of the same set of compounds. Of the CBH 1-mutant CSPs, only the D214N-CSP retained enantioselectivity whereas the selectivity was completely lost for the E212Q and E217Q-CSPs. The loss of enantioselectivity follows the same pattern as the loss of catalytic activity for the mutants which was determined from kinetic experiments using oligosaccharides as substrates. Mexiletine, a basic drug which could not be separated on the wildtype CBH 1-CSP, was successfully separated on one of the mutant phases. This demonstrates how protein engineering can be used to tailor new chiral selectors.",
author = "Hongbin Henriksson and Jerry St{\aa}hlberg and Anu Koivula and G{\"o}ran Pettersson and Christina Divne and Ludmila Valtcheva and Roland Isaksson",
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Henriksson, H, Ståhlberg, J, Koivula, A, Pettersson, G, Divne, C, Valtcheva, L & Isaksson, R 1997, 'The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition', Journal of Biotechnology, vol. 57, no. 1 - 3, pp. 115 - 125. https://doi.org/10.1016/S0168-1656(97)00094-1

The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition. / Henriksson, Hongbin; Ståhlberg, Jerry; Koivula, Anu; Pettersson, Göran; Divne, Christina; Valtcheva, Ludmila; Isaksson, Roland (Corresponding Author).

In: Journal of Biotechnology, Vol. 57, No. 1 - 3, 1997, p. 115 - 125.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - The catalytic amino-acid residues in the active site of cellobiohydrolase 1 are involved in chiral recognition

AU - Henriksson, Hongbin

AU - Ståhlberg, Jerry

AU - Koivula, Anu

AU - Pettersson, Göran

AU - Divne, Christina

AU - Valtcheva, Ludmila

AU - Isaksson, Roland

PY - 1997

Y1 - 1997

N2 - In order to investigate the basis for chiral separation in cellobiohydrolase 1 (CBH 1) and the closely related enzyme endoglucanase 1 (EG 1) from Trichoderma reesei, the wildtype proteins of CBH 1 and EG 1, as well as three catalytically deficient mutants of CBH 1 (E212Q, D214N and E217Q) were immobilised to silica and used as chiral stationary phases (CSPs) in HPLC. A large group of enantiomers could be completely resolved on the wildtype CBH 1-silica CSP while the corresponding EG 1-silica CSP only gave a partial separation of the same set of compounds. Of the CBH 1-mutant CSPs, only the D214N-CSP retained enantioselectivity whereas the selectivity was completely lost for the E212Q and E217Q-CSPs. The loss of enantioselectivity follows the same pattern as the loss of catalytic activity for the mutants which was determined from kinetic experiments using oligosaccharides as substrates. Mexiletine, a basic drug which could not be separated on the wildtype CBH 1-CSP, was successfully separated on one of the mutant phases. This demonstrates how protein engineering can be used to tailor new chiral selectors.

AB - In order to investigate the basis for chiral separation in cellobiohydrolase 1 (CBH 1) and the closely related enzyme endoglucanase 1 (EG 1) from Trichoderma reesei, the wildtype proteins of CBH 1 and EG 1, as well as three catalytically deficient mutants of CBH 1 (E212Q, D214N and E217Q) were immobilised to silica and used as chiral stationary phases (CSPs) in HPLC. A large group of enantiomers could be completely resolved on the wildtype CBH 1-silica CSP while the corresponding EG 1-silica CSP only gave a partial separation of the same set of compounds. Of the CBH 1-mutant CSPs, only the D214N-CSP retained enantioselectivity whereas the selectivity was completely lost for the E212Q and E217Q-CSPs. The loss of enantioselectivity follows the same pattern as the loss of catalytic activity for the mutants which was determined from kinetic experiments using oligosaccharides as substrates. Mexiletine, a basic drug which could not be separated on the wildtype CBH 1-CSP, was successfully separated on one of the mutant phases. This demonstrates how protein engineering can be used to tailor new chiral selectors.

U2 - 10.1016/S0168-1656(97)00094-1

DO - 10.1016/S0168-1656(97)00094-1

M3 - Article

VL - 57

SP - 115

EP - 125

JO - Journal of Biotechnology

JF - Journal of Biotechnology

SN - 0168-1656

IS - 1 - 3

ER -