omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.
Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-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-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-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-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.
Yun
Hyungdon
H
Institute for Molecular Biology and Genetics and School of Chemical Engineering, Seoul National University, Seoul 151-742, Korea.
Lim
Seongyop
S
Cho
Byung-Kwan
BK
Kim
Byung-Gee
BG
D042441:Achromobacter denitrificans / Q000201:enzymology* / Q000235:genetics; D000421:Alcaligenes / Q000201:enzymology* / Q000235:genetics; D000588:Amines / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000302:isolation & purification; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D051278:beta-Alanine-Pyruvate Transaminase
D000588:Amines; D000596:Amino Acids; D017931:DNA Primers; D004269:DNA, Bacterial; D000637:Transaminases; D051278:beta-Alanine-Pyruvate Transaminase