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  • omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines.

Applied and environmental microbiology (2004-04-07)
Hyungdon Yun, Seongyop Lim, Byung-Kwan Cho, Byung-Gee Kim
ABSTRACT

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.

MATERIALS
Product Number
Brand
Product Description

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