Engineered dehydrogenase biocatalysts for non-natural amino acids: efficient isolation of the D-enantiomer from racemic mixtures.
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 micromole scale in 20 ml ethanolamine HCl buffer, pH 9.5, with 25 microg N145A enzyme and 100 microg 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.
Paradisi
Francesca
F
UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland. francesca.paradisi@ucd.ie
Conway
Philip A
PA
Maguire
Anita R
AR
Engel
Paul C
PC
D000596:Amino Acids / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D055162:Biocatalysis; D003067:Coenzymes / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D010649:Phenylalanine / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D000596:Amino Acids; D003067:Coenzymes; D010649:Phenylalanine; D010088:Oxidoreductases