Molecular characterization of the 56-kDa CYP153 from Acinetobacter sp. EB104.

CYP153 a cytochrome P450 from Acinetobacter sp. EB104 catalyzes the hydroxylation of unsubstituted n-alkanes. We have decided to use the CYP153 system as a model for mechanistic studies on regioselective n-alkane oxidation and the interaction of hydrophobic substrates with soluble enzymes.

Cytochromes P450 of the sophorose lipid-producing yeast Candida apicola: heterogeneity and polymerase chain reaction-mediated cloning of two genes.

Candida apicola belongs to a group of yeasts producing high amounts of surface-active extracellular glycolipids consisting of sophorose and long-chain-omega- and (omega-1)-hydroxy fatty acids. The involvement of cytochrome P450 in the synthesis of sophorose lipid by the hydroxylation of long-chain fatty acids was suggested from a simultaneous increase of cellular P450 content. Hydroxylation studies indicated the existence of multiple P450 forms capable of hydroxylating not only long-chain fatty acids, but also n-alkanes.

Purification of cytochrome P-450 from n-hexadecane-grown Acinetobacter calcoaceticus.

A convenient procedure for the purification of cytochrome P-450 from an n-alkane-assimilating strain of the bacterial species Acinetobacter calcoaceticus has been elaborated. The cytochrome P-450 of n-hexadecane-grown cells was found to be distributed in cell-free extracts among particulate and soluble subcellular fractions. For isolation, cytochrome P-450 was extracted from particulate fractions by addition of Triton X-100 to the buffer.

Alcohol oxidation by Acinetobacter calcoaceticus EB 104--a n-alkane-utilizing and cytochrome P-450-producing strain.

A soluble, NADP+-dependent alcohol dehydrogenase (ADH) with a pH optimum at 8.7 was found in A. calcoaceticus EB 104 after growth on different carbon sources.

Effect of oxygen limitation on the content of n-hexadecane-inducible cytochrome P-450 in Acinetobacter calcoaceticus strain EB 104.

The content of cytochrome P-450 as a function of oxygen supply was studied during growth of Acinetobacter on n-hexadecane in batch cultures at constant pH and agitation. The rate of growth and the content of cytochrome P-450 were not affected as long as the dissolved oxygen tension ranged above 3 to 5% of saturation. The amount of cytochrome P-450 increased when the oxygen tension declined to zero.

Influence of growth phase and carbon source on the content of rubredoxin in Acinetobacter calcoaceticus.

The rubredoxin content of Acinetobacter calcoaceticus in dependence on the carbon source (acetate, n-alkanes, succinate, L-malate) and on the growth phase was studied by means of a radioimmunoassay. The method used was specific for rubredoxin and Acinetobacter calcoaceticus. The formation of rubredoxin increased with time up to the end of the logarithmic phase when n-alkanes were the sole carbon source.

[Cytochrome composition of Acinetobacter calcoaceticus].

The qualitative and quantitative composition of cytochromes in intact cells of Acinetobacter calcoaceticus and in particle fractions obtained from cells following ultrasonic treatment by differential centrifugation were studied using spectrophotometric methods. Acinetobacter calcoaceticus contains cytochrome b, cytochrome o and low amounts of cytochrome d. Both the absolute content of cytochrome b and o and the relative composition do not essentially vary with the carbon source used (hexadecane, acetate, succinate, malate, yeast extract).

[Isolation and characterization of rubredoxin from Acinetobacter calcoaceticus].

Acinetobacter calcoaceticus growing on long-chain n-alkanes contains a soluble iron-sulfur protein, which corresponds in its properties to a rubredoxin. It was prepared from the 50000 X g supernatant of ultrasonically treated cells using ion exchange chromatography on DEAE cellulose and gel filtration on Sephadex G-75. The isolated protein is pure electrophoretically, but yields two bands corresponding to molecular weights of 6000 and 12000 respectively.