The deracemization of racemic amines to yield enantioenriched amines using S-stereoselective amine oxidases (AOx) has recently been attracting attention. However, Rstereoselective AOx that are suitable for deracemization have not yet been identified. An R-stereoselective AOx was now evolved from porcine kidney d-amino acid oxidase (pkDAO) and subsequently use for the deracemization of racemic amines. The engineered pkDAO, which was obtained by directed evolution, displayed a markedly changed substrate specificity towards R amines. The mutant enzyme exhibited a high preference towards the substrate a-methylbenzylamine and was used to synthesize the S amine through deracemization. The findings of this study indicate that further investigations on the structure-activity relationship of AOx are warranted and also provide a new method for biotransformations in organic synthesis.

Mutant α‐amino‐ε‐caprolactam (ACL) racemase (L19V/L78T) from Achromobacter obae with improved substrate specificity toward phenylalaninamide was obtained by directed evolution. The mutant ACL racemase and thermostable mutant D‐amino acid amidase (DaaA) from Ochrobactrum anthropi SV3 co‐expressed in Escherichia coli (pACLmut/pDBFB40) were utilized for synthesis of (R)‐phenylalanine and non‐natural (R)‐phenylalanine derivatives (4‐OH, 4‐F, 3‐F, and 2‐F‐Phe) by dynamic kinetic resolution (DKR). Recombinant E. coli with DaaA and mutant ACL racemase genes catalyzed the synthesis of (R)‐phenylalanine with 84% yield and 99% ee from (RS)‐phenylalaninamide (400 mM) in 22 h. (R)‐Tyrosine and 4‐fluoro‐(R)‐phenylalanine were also efficiently synthesized from the corresponding amide compounds. We also co‐expresed two genes encoding mutant ACL racemase and L‐amino acid amidase from Brevundimonas diminuta in E. coli and performed the efficient production of various (S)‐phenylalanine derivatives. Moreover, 2‐aminophenylpropionitrile was converted to (R)‐phenylalanine by DKR using a combination of the non‐stereoselective nitrile hydratase from recombinamt E. coli and mutant ACL racemase and DaaA from E. coli encoding mutant ACL racemase and DaaA genes.

Oxidation of amino groups in amines or amino acids activates the sp3 Cα–H bond to form imines, making the alpha carbon atom a preferable target for nucleophilic reagents such as cyanide.

Abstract:We have succeeded in the enzymatic synthesis of (R)-a-aminobutyric acid from racemic aaminobutyronitrile. This has been demonstrated by the use of non-stereoselective nitrile hydratase (NHase) from Rhodococcus opacus 71D, d-aminopeptidase from Ochrobactrum anthropi C1-38 and a-amino-e-caprolactam (ACL) racemase from Achromobacter obae. Racemic a-aminobutyronitrile was completely converted in 6 h at 30 8C to (R)-aaminobutyric acid whose optical purity was more than 99%. (S)-a-Aminobutyric acid was also synthesized from a-aminobutyronitrile by NHase, ACL racemase and l-amino acid amidase from Brevundimonas diminuta TPU 5720. In a similar manner, other (R)-or (S)-a-amino acids with more than 97.5% ee could be synthesized from the corresponding a-aminonitriles. This is the first report on the dynamic kinetic resolution (DKR) of a-aminonitriles to form chiral a-amino acids. The key enzyme in this DKR is non-stereoselective NHase, which had been newly screened from soil samples, and its gene cloned.