Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitri lotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA mono oxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediamin ediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified a nd characterized to date. In this report, an LDA oxidase was purified to ap parent homogeneity from strain BNC1 by using a combination of eight purific ation steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reve aled a single protein band of 40 kDa, and by using size exclusion chromatog raphy, we estimated the native enzyme to be a homodimer. Flavin adenine din ucleotide was determined as its prosthetic group. The purified enzyme oxidi zed IDA to glycine and glyoxylate with the consumption of O-2. The temperat ure and pH optima for IDA oxidation were 35 degreesC and 8, respectively. T he apparent K-m for IDA was 4.0 mM with a k(cat) of 5.3 s(-1). When the N-t erminal amino acid sequence was determined, it matched exactly with that en coded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fu sion with a His tag was purified by a one-step nickel affinity chromatograp hy. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA t o ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second en zyme in both NTA and EDTA degradation pathways in strain BNC1.