Synthesis of 4-Hydroxyisoleucine by the Aldolase–Transaminase Coupling Reaction and Basic Characterization of the Aldolase from Arthrobacter simplex AKU 626

Jun OGAWA; Hiroyuki YAMANAKA; Junichi MANO; Yuko DOI; Nobuyuki HORINOUCHI; Tomohiro KODERA; Noriki NIO; Sergey V. SMIRNOV; Natalya N. SAMSONOVA; Yury I. KOZLOV; Sakayu SHIMIZU

doi:10.1271/bbb.60655

Microbiology & Fermentation Technology Regular Papers

Synthesis of 4-Hydroxyisoleucine by the Aldolase–Transaminase Coupling Reaction and Basic Characterization of the Aldolase from Arthrobacter simplex AKU 626

Jun OGAWA, Hiroyuki YAMANAKA, Junichi MANO, Yuko DOI, Nobuyuki HORINOUCHI, Tomohiro KODERA, Noriki NIO, Sergey V. SMIRNOV, Natalya N. SAMSONOVA, Yury I. KOZLOV, Sakayu SHIMIZU

Author information

Jun OGAWA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Hiroyuki YAMANAKA
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Junichi MANO
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Yuko DOI
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Nobuyuki HORINOUCHI
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University
Tomohiro KODERA
Institute of Life Sciences, Ajinomoto Co., Inc.
Noriki NIO
Institute of Life Sciences, Ajinomoto Co., Inc.
Sergey V. SMIRNOV
Ajinomoto-Genetika Research Institute
Natalya N. SAMSONOVA
Ajinomoto-Genetika Research Institute
Yury I. KOZLOV
Ajinomoto-Genetika Research Institute
Sakayu SHIMIZU
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University

Keywords: 4-hydroxyisoleucine, aldolase, transaminase, Arthrobacter simplex, antidiabetics

JOURNAL FREE ACCESS

2007 Volume 71 Issue 7 Pages 1607-1615

DOI https://doi.org/10.1271/bbb.60655

View "Advance Publication" version (July 7, 2007).

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Abstract

Arthrobacter simplex AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG1655. By using resting cells of A. simplex AKU 626 and E. coli BL21(DE3)/pET-15b-ilvE, 3.2 mM 4-hydroxyisoleucine was produced from 250 mM acetaldehyde, 75 mM α-ketobutyrate, and 100 mM L-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mM Tris–HCl buffer (pH 7.5) containing 2 mM MnCl₂·4H₂O at 28 °C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplex AKU 626. Mn²⁺ and pyridoxal 5′-monophosphate were effective in stabilizing the enzyme. The native and subunit molecular masses of the purified aldolase were about 180 and 32 kDa respectively. The N-terminal amino acid sequence of the purified enzyme showed no significant homology to known aldolases.

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