Purification and Characterization of Iron-Containing Urethanase from Bacillus licheniformis

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Urethane is potentially carcinogenic and teratogenic to human and has been reported to be a contaminant of various kinds of alcoholic beverages. Enzymatic removal of urethane is one possible approach to remove this cancer-causing chemical from alcoholic beverages. Among Bacillus licheniformis strains, IFO 12107 showed the highest urethane hydrolyzing activity when cultivated in a urethane-containing white medium. The enzyme was purified about 300-fold by means of several chromatographic steps to homogeneity. The enzyme activity was strongly inhibited by batho- and o-phenanthroline. The complete loss of enzyme activity following treatment with bathophenanthroline was fully restored by the addition of Fe³⁺ at a ratio of 4 iron atoms to 1 mol apoenzyme. This result was obtained by the incorporation of ⁵⁹Fe³⁺ into apourethanase. ESR spectroscopy showed that the enzyme contained a typical high-spin Fe³⁺. The urethanase hydrolyzed carbamyl ester derivatives more rapidly than amide derivatives. The molecular weight of the native enzyme was about 160 kDa (gel-filtration), and that of the subunit was 42 kDa (sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE). This shows the enzyme to be a homotetramer. The pI and K_m values were 5.5 and 0.17 mM, respectively. The enzyme was considerably resistant to high concentrations of ethanol, which is a great advantage for the industrial removal of urethane from alcoholic beverages.