Issue 19, 2008
From the journal:

Organic & Biomolecular Chemistry

Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the d-enantiomer from racemic mixtures

Francesca Paradisi,*a   Philip A. Conway,ac   Anita R. Maguireb  and  Paul C. Engel*c  

* Corresponding authors

a UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
E-mail: francesca.paradisi@ucd.ie
Fax: +353 1 716 2501
Tel: +353 1 716 2967

b Analytical and Biological Chemistry Research Facility, Department of Chemistry and School of Pharmacy, University College, Cork, Ireland

c UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
E-mail: paul.engel@ucd.ie

Abstract

With a view to their use in the kinetic resolution of racemic non-natural amino acids, five variants of the enzyme L-phenylalanine dehydrogenase, the wild-type enzyme from Bacillus sphaericus and four active-site mutants, have been tested with a range of amino acids. In each case, the rates of reaction with 0.2 mM L-amino acid and with the racemic mixture at 0.4 mM were compared, so that the starting concentration of the active substrate was kept constant. Although the D-amino acids are not substrates, they were inhibitory in all cases. The extent of inhibition, however, varied greatly from compound to compound and among the mutants. With the N145L mutant and DL 4-O-Me-Phe, the equimolar D-enantiomer gave 83.2% inhibition, and with the wild-type enzyme there was 86.7% inhibition with racemic norleucine. By contrast, with these same substrates the N145V mutant showed less than 9% and 24% inhibition respectively. The N145A mutant was selected for use with DL-4-Cl-Phe. The pH was decreased from the enzyme's optimum of 10.4 to 9.5 to minimise breakdown of the coenzyme NAD+, and the coenzyme was recycled by molecular oxygen with the assistance of a commercial diaphorase. Reaction on a 200 μmole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 μg N145A enzyme and 100 μg diaphorase, was monitored by chiral HPLC. The L-isomer was removed to an extent of >99% after 40 h, with the D-isomer peak undiminished. The pure D-isomer was isolated from the reaction mixture in 85% overall yield after ion-exchange chromatography.

Graphical abstract: Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the d-enantiomer from racemic mixtures

About
Cited by
Related
Download options Please wait...

Article information

DOI
https://doi.org/10.1039/B809147A
Article type
Paper
Submitted
29 May 2008
Accepted
07 Jul 2008
First published
05 Aug 2008

Org. Biomol. Chem., 2008,6, 3611-3615

Permissions

Request permissions

Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the D-enantiomer from racemic mixtures

F. Paradisi, P. A. Conway, A. R. Maguire and P. C. Engel, Org. Biomol. Chem., 2008, 6, 3611 DOI: 10.1039/B809147A

To request permission to reproduce material from this article, please go to the Copyright Clearance Center request page.

If you are an author contributing to an RSC publication, you do not need to request permission provided correct acknowledgement is given.

If you are the author of this article, you do not need to request permission to reproduce figures and diagrams provided correct acknowledgement is given. If you want to reproduce the whole article in a third-party publication (excluding your thesis/dissertation for which permission is not required) please go to the Copyright Clearance Center request page.

Read more about how to correctly acknowledge RSC content.

Social activity

Spotlight

Advertisements