Purification and characterization of iron-containing urethanase from Bacillus licheniformis.
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 Km 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.
Zhao
C J
CJ
Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Japan.
Kobashi
K
K
D000581:Amidohydrolases / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification*; D001407:Bacillus / Q000201:enzymology*; D002614:Chelating Agents / Q000494:pharmacology; D005591:Chemical Fractionation; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003470:Culture Media; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005290:Ferric Compounds / Q000378:metabolism*; D006868:Hydrolysis; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D013379:Substrate Specificity
D002614:Chelating Agents; D003470:Culture Media; D005290:Ferric Compounds; D010618:Phenanthrolines; D000581:Amidohydrolases; C074526:urethanase