Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction and basic characterization of the aldolase from Arthrobacter simplex AKU 626

Arthrobacter simpler AKU 626 was found to synthesize 4-hydroxyisoleucine from acetaldehyde, alpha-ketobutyrate, and L-glutamate in the presence of Escherichia coli harboring the branched chain amino acid transaminase gene (ilvE) from E. coli K12 substrain MG-1655. 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 a-ketobutyrate, and 100 mm L-glutamate with a molar yield to a-ketobutyrate of 4.3% in 50mM Tris-HCI buffer (pH 7.5) containing 2 mm MnCl2 center dot 4H(2)O at 28 degrees C for 2 h. An aldolase that catalyzes the aldol condensation of acetaldehyde and alpha-ketobutyrate was purified from A. simplex AKU 626. Mn2+ and pyridoxal 5'-mono-phosphate 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.