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.
Soluble alcohol oxidase (AO) activity was detected in the supernatant fraction of a high-speed centrifugation procedure after ballistic cellular homo-genization to break the mycelium from a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing different carbon sources, including alcohols and hydrocarbons but not in glucose. In previous work, zymogram analysis conducted with crude extracts from aerobic mycelium of YR-1 strain indicated the existence of two AO enzymes originally named AO-1 and AO-2. In the present study, we were able to separate the AO-1 band into two bands depending on culture conditions, carbon source, and polyacrylamide gel electrophoresis (PAGE) separation conditions; the enzyme activity pattern in zymograms from cell-free extracts exhibited three different bands after native PAGE. New nomenclature was used for upper bands AO-1 and AO-2 and lower band AO-3, respectively. The expression of AO activity was studied in the absence of glucose in the culture media and in the presence of hydrocarbons or petroleum as sole carbon source, suggesting that AO expression could be subjected to two regulatory possibilities: carbon catabolite regulation by glucose and induction by hydrocarbons. The possibility of catabolic inhibition of AO by glucose in the active enzyme was also tested, and the results confirm that this kind of regulatory mechanism is not present in AO activity.
A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde. Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2). Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20,38, and 46 kDa that could be part of the native enzyme. In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha. The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol. Km values obtained for methanol and hexa-decanol indicated a higher affinity for the latter. Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.
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