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

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.