Alteration of substrate affinity of Streptomyces cholesterol oxidase for application to the rate assay of cholesterol in serum.
The Streptomyces cholesterol oxidase (ChoA) can not be adopted for the rate assay, because the Km value of the enzyme for cholesterol is very small. The choA gene was subjected to random mutagenesis in vivo, and a mutant ChoA (designated E-ChoA) that showed altered substrate affinity was obtained by screening. The Km value of E-ChoA was approximately ten times larger than that of the wild type. Unexpectedly, the thermal stability was also improved. The amino acid substitutions of E-ChoA were identified to be the valine to glutamate at position 145, which has been previously identified as one of the thermostable mutations, and the glycine to serine at position 405. The mutational effects on the structure of E-ChoA are discussed on the basis of a three-dimensional model. E-ChoA has been successfully applied to the rate assay of cholesterol in serum.
Nishiya
Y
Y
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., Toyo-cho, Tsuruga, Fukui, Japan. yoshiaki_nishiya@bio.toyobo.co.jp
Hirayama
N
N
D002784:Cholesterol / Q000097:blood*; D002789:Cholesterol Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D010957:Plasmids; D011487:Protein Conformation; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity
D002784:Cholesterol; D002789:Cholesterol Oxidase