A different method of measuring and detecting mono-and dioxygenase activities |
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Authors: | Roberto Zazueta-Sandoval Vanesa Zazueta Novoa Hortencia Silva Jiménez Roberto Cabrera Ortiz |
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Institution: | (1) Instituto de Investigación en Biología Experimental, Facultad de Química, Universidad de Guanajuato, Noria Alta s/n, Apartado Postal 187, 36000 Guanajuato, Gto., México |
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Abstract: | 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 immuno-detection experiments with antibodies directed against oxygenase from
soybean. The partially purified enzyme oxidized different substrates, although higher activity was displayed with benzene.
K
m
values obtained for benzene and decane indicated a higher affinity for the latter |
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Keywords: | Oxygenases filamentous fungi hydrocarbon biodegradation petroleum contamination |
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