Assay method for lipid component, assay composition, and process for production of enzyme used therefor

Abstract

An enzyme having enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity and 3-ketoacyl-CoA thiolase activity, all in the same enzyme, is produced by culturing the microorganism strain Pseudomonas fragi B-0771 FERM-P No. 5701, and isolating the enzyme thus produced from the culture medium. Such an enzyme is useful in an assay method for a fatty acid component in a sample, which fatty acid is originally present in the sample or is liberated from a fatty acid ester in the sample, comprising: (a) converting the fatty acid to acyl-CoA; (b) converting the thus-produced acyl-CoA to dehydroacyl-CoA; (c) converting the thus-produced dehydroacyl-CoA to hydroxyacyl-CoA; (d) converting the thus-produced hydroxyacyl-CoA to ketoacyl-CoA; (e) converting the thus-produced ketoacyl-CoA to acyl-CoA; and measuring the detectable changes in the reaction mixture. A composition suitable for such lipid assay comprises ATP or GTP, CoASH, NAD, acyl-CoA synthetase activity, acyl-CoA oxidase activity, enoyl-CoA hydratase activity, 3-hydroxyacyl-CoA dehydrogenase activity, and 3-ketoacyl-CoA thiolase activity, wherein the last three activities are supplied by the new multi-active enzyme.

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