A different method of measuring and detecting monoand dioxygenase activities: key enzymes in hydrocarbon biodegradation.
A spectrophotometric method of measuring oxygenase activity in cell extracts or in zymograms was developed. It is an easy and cheap method that allows spectrophotometric measurement of activity by a colored reaction and reveals activity bands in a polyacrylamide gel electrophoresis (PAGE) gel as brown bands. To prove its usefulness, we report on a study with the oxygenase present in strain YR-1, isolated from petroleum-contaminated soils, that uses hydrocarbons as its sole carbon source. Soluble oxygenase activity was detected (under our conditions of cellular homogenization) in the mycelium of a filamentous fungus strain named YR-1. Oxygenase activity from aerobically grown mycelium was detected in growth medium containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited similar optimum pH for the hydroxylation of different aliphatic or aromatic substrates (decane, hexadecane, benzene, and naphthalene) to the corresponding alcohols. Zymogram analysis conducted with partially purified fractions from cell extracts from the aerobic mycelium of the YR-1 strain indicated the existence of only one oxygenase enzyme. Partially purified samples of enzyme, analyzed by sodium dodecyl sulfate PAGE, indicated the presence of one major protein band with a mol wt of 56 kDa that can be a constituent of the native enzyme. In samples of the enzyme, the 56-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against oxygenase from soybean. The partially purified enzyme oxidized different substrates, although higher activity was displayed with benzene. Km values obtained for benzene and decane indicated a higher affinity for the latter.
Zazueta-Sandoval
Roberto
R
Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Noria Alta s/n, Apartado Postal 187, Guanajuato, Gto. 36000, México.
Novoa
Vanesa Zazueta
VZ
Jiménez
Hortencia Silva
HS
Ortiz
Roberto Cabrera
RC
D001673:Biodegradation, Environmental; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D005658:Fungi / Q000201:enzymology*; D006838:Hydrocarbons / Q000493:pharmacokinetics*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity
D006838:Hydrocarbons; D006899:Mixed Function Oxygenases