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Effect of mass-transfer limitations on the selectivity of immobilized α-chymotrypsin biocatalysts prepared for use in organic medium

Barros, Raúl J. LU ; Wehtje, Ernst LU and Adlercreutz, Patrick LU (2000) In Biotechnology and Bioengineering 67(3). p.319-326
Abstract

The selectivity of preparations of α-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass- transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to... (More)

The selectivity of preparations of α-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass- transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH2) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH2) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH2) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.

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author
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organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Acyl donor selectivity, Immobilized α-chymotrypsin, Internal diffusion, Kinetically controlled peptide synthesis, Nucleophile selectivity, Stereoselectivity
in
Biotechnology and Bioengineering
volume
67
issue
3
pages
8 pages
publisher
John Wiley and Sons
external identifiers
  • pmid:10620262
  • scopus:0034606988
ISSN
0006-3592
DOI
10.1002/(SICI)1097-0290(20000205)67:3<319::AID-BIT8>3.0.CO;2-S
language
English
LU publication?
yes
id
a2c2acad-398e-4b0c-880d-d8bb0ade5da7
date added to LUP
2019-06-20 15:52:13
date last changed
2021-02-17 07:19:38
@article{a2c2acad-398e-4b0c-880d-d8bb0ade5da7,
  abstract     = {<p>The selectivity of preparations of α-chymotrypsin immobilized on Celite or polyamide and carrying out syntheses of di- and tripeptides in acetonitrile medium were studied. The study concerns the effect of mass- transfer limitations on three different kinds of selectivity: acyl donor, stereo- and nucleophile selectivities, defined respectively as the ratio of initial rates with different acyl donors; the enantioselectivity factor (E); and the ratio of initial rates of peptide synthesis and hydrolysis of the acyl donor. Strong mass-transfer limitations caused by increased enzyme loading had a very strong effect on acyl donor selectivity, with reductions of up to 79%, and on stereoselectivity, with reductions of up to 77% in relation to optimum values, both on Celite. Nucleophile selectivity was not affected as strongly by mass-transfer limitations. Using a small molecule (AlaNH<sub>2</sub>) as nucleophile, the onset of these limitations caused only minor reductions in selectivity, while when using a larger nucleophilic species (AlaPheNH<sub>2</sub>) it was reduced by up to 60% when increasing enzyme loading on Celite from 2 to 100 mg/g. The different way these kinds of selectivity are affected by the onset of mass-transfer limitations can be explained by a combination of different aspects: the kinetic behavior of the enzyme toward nucleophile and acyl donor concentrations, the relative concentrations of reagents used in the reaction media, and their relative diffusion coefficients. In short, higher concentrations of nucleophile than acyl donor are generally used, and the nucleophile most often used in the experiments hereby described (AlaNH<sub>2</sub>) diffuses faster than the acyl donors employed. These factors combined are expected to give rise to concentration gradients inside porous biocatalyst particles higher for acyl donor than for nucleophile under conditions of mass-transfer limitations. This explains why acyl donor selectivity and stereoselectivity are much more influenced by mass transfer limitations than nucleophile selectivity.</p>},
  author       = {Barros, Raúl J. and Wehtje, Ernst and Adlercreutz, Patrick},
  issn         = {0006-3592},
  language     = {eng},
  month        = {02},
  number       = {3},
  pages        = {319--326},
  publisher    = {John Wiley and Sons},
  series       = {Biotechnology and Bioengineering},
  title        = {Effect of mass-transfer limitations on the selectivity of immobilized α-chymotrypsin biocatalysts prepared for use in organic medium},
  url          = {http://dx.doi.org/10.1002/(SICI)1097-0290(20000205)67:3<319::AID-BIT8>3.0.CO;2-S},
  doi          = {10.1002/(SICI)1097-0290(20000205)67:3<319::AID-BIT8>3.0.CO;2-S},
  volume       = {67},
  year         = {2000},
}