Cloning and characterization of oah, the gene encoding oxaloacetate hydrolase in Aspergillus niger

The enzyme oxaloacetate hydrolase (EC 3.7.1.1). which is involved in oxalat e formation, was purified from Aspergillus niger. The native enzyme has a m olecular mass of 360-440 kDa, and the denatured enzyme has a molecular mass of 39 kDa, as determined by gel electrophoresis. Enzyme activity is maxima l at pH 7.0 and 45 degrees C. The Fraction containing the enzyme activity c ontained at least five proteins. The N-terminal amino acid sequences of fou r of these proteins were determined. The amino acid sequences were aligned with EST sequences from A. niger, and an EST sequence that showed 100% iden tity to all four sequences was identified. Using this EST sequence the gene encoding oxaloacetate hydrolase (oah) was cloned by inverse PCR. It consis ts of an ORF of 1227 bp with two introns of 92 and 112 bp, respectively. Th e gene encodes a protein of 341 amino acids with a molecular mass of 37 kDa . Under the growth conditions tested, the highest oah expression was found for growth on acetate as carbon source. The gene was expressed only at pH v alues higher than 4.0.