ω-Amino Acid:Pyruvate Transaminase from Alcaligenes denitrificans Y2k-2: a New Catalyst for Kinetic Resolution of β-Amino Acids and Amines

Abstract

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed -amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic ␤-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-␤-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-␤-amino-n-butyric acid (L-␤-ABA). The enzyme converts various ␤-amino acids and amines to the corresponding ␤-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K m and V max for L-␤-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-␤-ABA, the apparent K m and V max for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-␤-ABA, producing optically pure D-␤-ABA (99% enantiomeric excess) with 53% conversion.

Cloning and Characterization of a Novel β-Transaminase fromMesorhizobiumsp. Strain LUK: a New Biocatalyst for the Synthesis of Enantiomerically Pure β-Amino Acids

“…In the case of the amino acceptor, 2-ketoglutarate, pyruvate ethyl ester, pyruvate, and pyruvate methyl ester showed similar reactivities, while oxaloacetate showed slightly lower reactivity. It is quite interesting that this enzyme has no striking differences in the specificities toward different amino acceptors, because transaminases generally show a clear preference either for pyruvate or for 2-ketoglutarate/oxaloacetate (33,47,48,52,56). However, considering the fact that this enzyme shows a high sequence similarity with the GSA which does not need any second substrate and catalyzes the intramolecular isomerization, it is possible that the unique amino acceptor specificity could be due to different structural reasons with other transaminases.…”

“…Moreover, transaminases allow asymmetric synthesis from prochiral ketone compounds depending upon the properties of target chemical compounds (1,4,10,11,17,49,50). Though transaminases are not widespread, we have reported an -transaminase of Alcaligenes denitrificans which can catalyze mainly the transamination between aliphatic ␤-amino acids and pyruvate (56). Other examples were recently introduced for the transamination of aliphatic and aromatic ␤-amino acids by Alcaligenes eutrophus and Variovorax paradoxus (6).…”

“…We have tried to clone the gene of this enzyme by PCR using the degenerative primers of consensus transaminase sequences (56). However, due to the large population of the homologous transaminases in the screened organism, we found a few transaminases without any activities for ␤-amino acids, even though we used genuine N-terminal amino acid sequence to make one of the degenerative primers.…”

Microbial Production of Optically Active β-Phenylalanine through Stereoselective Degradation of Racemic β-Phenylalanine

“…Concerning the microbial metabolism of -amino acids, such as -alanine, -aminoisobutyrate, -aminobutyrate and taurine, metabolism through transamination catalyzed by !-amino acid transaminases has been reported. [12][13][14] Until now, there has been no report on BPA metabolism. In this study, it was found that one type of BPA metabolism involves transamination that is the same as some other -amino acid metabolisms.…”

Computational Selection, Identification and Structural Analysis of ω-Aminotransferases with Various Substrate Specificities from the Genome Sequence ofMesorhizobium lotiMAFF303099

“…35) Mll3663 gene product, predicted as an amine:pyruvate AT, showed high activity towards secondary aromatic amines like (S)--methylbenzylamine (A1) and 1-phenylpropylamine (A2), but no or very low activity towards -amino acids (C1, C2, and D1). This enzyme showed rather narrow substrate specificity.…”