Yun Hyungdon, Lim Seongyop, Cho Byung-Kwan, Kim Byung-Gee
Institute for Molecular Biology and Genetics and School of Chemical Engineering, Seoul National University, Seoul 151-742, Korea.
Appl Environ Microbiol. 2004 Apr;70(4):2529-34. doi: 10.1128/AEM.70.4.2529-2534.2004.
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.
通过从土壤样品中进行选择性富集然后筛选，获得了反硝化产碱菌Y2k - 2，该菌株表现出ω-氨基酸：丙酮酸转氨酶活性，可对脂肪族β-氨基酸进行动力学拆分，并克隆了相应的结构基因（aptA）。使用异丙基-β-D-硫代半乳糖苷（IPTG）诱导的pET表达系统，该基因在大肠杆菌BL21中实现了功能性表达（9.6 U/mg），纯化后的重组AptA对L-β-氨基丁酸（L-β-ABA）的比活性为77.2 U/mg。该酶以丙酮酸作为胺受体，将各种β-氨基酸和胺转化为相应的β-酮酸和酮。在存在10 mM丙酮酸的情况下，L-β-ABA的表观K(m)和V(max)分别为56 mM和500 U/mg。在存在10 mM L-β-ABA的情况下，丙酮酸的表观K(m)和V(max)分别为11 mM和370 U/mg。该酶在50 mM D,L-β-ABA的动力学拆分中表现出高立体选择性（E > 80），以53%的转化率产生光学纯的D-β-ABA（对映体过量99%）。
