A different method of measuring and detecting monoand dioxygenase activities: key enzymes in hydrocarbon biodegradation.

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 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.

authors

Zazueta-Sandoval R,Novoa VZ,Jiménez HS,Ortiz RC

doi

10.1385/abab:108:1-3:725

subject

Has Abstract

pub_date

2003-04-01 00:00:00

pages

725-36

eissn

0273-2289

issn

1559-0291

pii

ABAB-108-1-3-725

journal_volume

105 -108

pub_type

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