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

Arthrobacter simplexAKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, α-ketobutyrate, and L-glutamate in the presence of Escherichia coliharboring the branched chain amino acid transaminase gene (ilvE) from E. coliK12 substrain MG1655. By using resting cells of A. simplexAKU 626 and E. coliBL21(DE3)/pET-15b-ilvE, 3.2 mM4-hydroxyisoleucine was produced from 250 mMacetaldehyde, 75 mMα-ketobutyrate, and 100 mML-glutamate with a molar yield to α-ketobutyrate of 4.3% in 50 mMTris–HCl buffer (pH 7.5) containing 2 mMMnCl2·4H2O at 28 °C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and α-ketobutyrate was purified from A. simplexAKU 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.