Cytochrome P-450 enzymes
AssigneeSankyo Company, Limited
InventorsTatsuji Matsuoka, Shunichi Miyakoshi
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Abstract
Novel cytochrome P-450.sub.sca-1, P-450.sub.sca-2 and P-450.sub.sca-3 enzymes are produced by cultivation of Streptomyces carbophilus SANK 62585, and are suitable for use in hydroxylation processes.
Claims17
A soluble hydroxylation enzyme selected from the group consisting of cytochrome P-450.sub.sca-1, cytochrome P-450.sub.sca-2 and cytochrome P-450.sub.sca-3, wherein
- (a) the enzymes have a molecular weight of 46,000.+-.1,000 daltons as determined by SCS- polyacrylamide gel electrophoresis,
- (b) by hydroxyapatite chromatography, cytochrome P-450.sub.sca-1 is eluted with about 0.06M phosphate buffer, cytochrome P-450.sub.sca-2 is eluted with about 0.08M phosphate buffer, and cytochrome P-450.sub.sca-3 is eluted with about 0.10M phosphate buffer,
- (c) the enzymes are capable of hydroxylating ML-236B and milbemycin as substrates, said ML-236B being a lactone of the formula ##STR9## or in a ring-open form as a carboxylic acid of the formula ##STR10## and (d) the enzymes are capable of being induced by ML-236B as an induction agent.
A method for producing a hydroxylation enzyme selected from the group consisting of cytochrome P-450.sub.sca-1, cytochrome P-450.sub.sca-2 and cytochrome P-450.sub.sca-3, which method comprises culturing Streptomyces carbophilus SANK 62585 in a cultivation medium, adding to said cultivation medium an induction agent capable of inducing production of said enzyme, further culturing said Streptomyces carbophilus SANK 62585, and isolating said hydroxylation enzyme by hydroxyapatite chromatography, wherein cytochrome P-450.sub.sca-1 is eluted with about 0.06M phosphate buffer, cytochrome P-450.sub.sca-2 is eluted with about 0.08M phosphate buffer, and cytochrome P-450.sub.sca-3 is eluted with about 0.10M phosphate buffer.
In a process for the hydroxylation of a substrate using an enzyme, the improvement which comprises using as said enzyme a cytochrome P-450 enzyme produced by and isolated from Streptomyces carbophilus SANK 62585,
- wherein said hydroxylation enzyme is selected from the group consisting of cytochrome P-450.sub.sca-1, cytochrome P-450.sub.sca-2 and cytochrome P-450.sub.sca-3.
A hydroxylation process which comprises providing at least one hydroxylation enzyme selected from the group consisting of cytochrome P-450.sub.sca-1, cytochrome P-450.sub.sca-2 and cytochrome P-450.sub.sca-3 ;
- providing a substrate for said enzyme;
- hydroxylating said substrate using said hydroxylation enzyme;
- and obtaining a hydroxylated substrate.
Dependencies are inferred from the claim text.