Enhancement of immobilized protease catalyzed dipeptide synthesis by the presence of insoluble protonated nucleophile
Barros, Raúl J. LU ; Wehtje, Ernst LU and Adlercreutz, Patrick LU
(1999)
In Enzyme and Microbial Technology
24(8-9).
p.480-488
- Abstract
α-Chymotrypsin immobilized on celite catalyzing a kinetically controlled dipeptide synthesis reaction in acetonitrile medium showed an odd behavior in response to additions of triethylamine to the reaction mixture. This base is used to deprotonate the nucleophilic reagent, l-alaninamide hydrochloride, in order to increase its nucleophilicity and solubility. However, the enzyme performance is apparently enhanced by additions of triethylamine below one equivalent (in the range 15-20 mm) while the used concentration of nucleophilic reagent is 30 mm. Under these conditions, the initial rate is up to 2.5 times higher and the nucleophile specificity is approximately 30% better than when one equivalent is added. The activating effect on... (More)
α-Chymotrypsin immobilized on celite catalyzing a kinetically controlled dipeptide synthesis reaction in acetonitrile medium showed an odd behavior in response to additions of triethylamine to the reaction mixture. This base is used to deprotonate the nucleophilic reagent, l-alaninamide hydrochloride, in order to increase its nucleophilicity and solubility. However, the enzyme performance is apparently enhanced by additions of triethylamine below one equivalent (in the range 15-20 mm) while the used concentration of nucleophilic reagent is 30 mm. Under these conditions, the initial rate is up to 2.5 times higher and the nucleophile specificity is approximately 30% better than when one equivalent is added. The activating effect on initial rates of dipeptide synthesis was not observed when polyamide was used as support. Unlike polyamide, celite is a material with quite low porosity. Improvement of nucleophile specificity was observed using both supports. It is shown that this activation arises due to the presence of a separate dense liquid phase of insoluble l-alaninamide hydrochloride that intimately contacts with the enzyme preparation, and does not depend on the addition of triethylamine itself. Additions of l-alaninamide hydrochloride improved initial rates of synthesis more than 2.5-fold, and nucleophile specificity more than threefold. The initial rate activation was also observed when using non-porous glass beads to immobilize the enzyme at a loading of 5 mg enzyme g-1 glass but not at 1 mg enzyme g-1 glass when no mass transfer limitations in the immobilized enzyme layer are expected to occur. The results suggest that the presence of the separate phase helps to relieve mass transfer limitations on the system caused by overloading at the supports. One possible mechanism for the initial rate activation might be that the enzyme is partially desorbed from the support particles into the separate phase of nucleophile, and the better nucleophile specificity observed is due to increased local concentrations of the nucleophile within this phase. Copyright (C) 1999 Elsevier Science Inc. All rights reserved.
(Less)
- author
- Barros, Raúl J.
LU
; Wehtje, Ernst
LU
and Adlercreutz, Patrick
LU
- organization
- publishing date
- 1999-06-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Immobilized enzyme, Kinetically controlled dipeptide synthesis, Nucleophile deprotonation, Nucleophile specificity
- in
- Enzyme and Microbial Technology
- volume
- 24
- issue
- 8-9
- pages
- 9 pages
- publisher
- Elsevier
- external identifiers
-
- scopus:0032917548
- ISSN
- 0141-0229
- DOI
- 10.1016/S0141-0229(98)00138-0
- language
- English
- LU publication?
- yes
- id
- dcdb28d2-69d9-4e42-9f34-c16adc937918
- date added to LUP
- 2019-06-20 16:02:46
- date last changed
- 2022-01-31 22:13:25
@article{dcdb28d2-69d9-4e42-9f34-c16adc937918,
abstract = {{<p>α-Chymotrypsin immobilized on celite catalyzing a kinetically controlled dipeptide synthesis reaction in acetonitrile medium showed an odd behavior in response to additions of triethylamine to the reaction mixture. This base is used to deprotonate the nucleophilic reagent, l-alaninamide hydrochloride, in order to increase its nucleophilicity and solubility. However, the enzyme performance is apparently enhanced by additions of triethylamine below one equivalent (in the range 15-20 mm) while the used concentration of nucleophilic reagent is 30 mm. Under these conditions, the initial rate is up to 2.5 times higher and the nucleophile specificity is approximately 30% better than when one equivalent is added. The activating effect on initial rates of dipeptide synthesis was not observed when polyamide was used as support. Unlike polyamide, celite is a material with quite low porosity. Improvement of nucleophile specificity was observed using both supports. It is shown that this activation arises due to the presence of a separate dense liquid phase of insoluble l-alaninamide hydrochloride that intimately contacts with the enzyme preparation, and does not depend on the addition of triethylamine itself. Additions of l-alaninamide hydrochloride improved initial rates of synthesis more than 2.5-fold, and nucleophile specificity more than threefold. The initial rate activation was also observed when using non-porous glass beads to immobilize the enzyme at a loading of 5 mg enzyme g<sup>-1</sup> glass but not at 1 mg enzyme g<sup>-1</sup> glass when no mass transfer limitations in the immobilized enzyme layer are expected to occur. The results suggest that the presence of the separate phase helps to relieve mass transfer limitations on the system caused by overloading at the supports. One possible mechanism for the initial rate activation might be that the enzyme is partially desorbed from the support particles into the separate phase of nucleophile, and the better nucleophile specificity observed is due to increased local concentrations of the nucleophile within this phase. Copyright (C) 1999 Elsevier Science Inc. All rights reserved.</p>}},
author = {{Barros, Raúl J. and Wehtje, Ernst and Adlercreutz, Patrick}},
issn = {{0141-0229}},
keywords = {{Immobilized enzyme; Kinetically controlled dipeptide synthesis; Nucleophile deprotonation; Nucleophile specificity}},
language = {{eng}},
month = {{06}},
number = {{8-9}},
pages = {{480--488}},
publisher = {{Elsevier}},
series = {{Enzyme and Microbial Technology}},
title = {{Enhancement of immobilized protease catalyzed dipeptide synthesis by the presence of insoluble protonated nucleophile}},
url = {{http://dx.doi.org/10.1016/S0141-0229(98)00138-0}},
doi = {{10.1016/S0141-0229(98)00138-0}},
volume = {{24}},
year = {{1999}},
}