Alteration of substrate affinity of Streptomyces cholesterol oxidase for application to the rate assay of cholesterol in serum

作者：Nishiya Y^*; Hirayama N

来源：Clinica Chimica Acta, 1999, 287(1-2): 111-122.

DOI：10.1016/S0009-8981(99)00130-8

• cholesterol oxidase
• protein engineering
• alteration of substrate affinity
• mutational effects
• rate assay of cholesterol

The Streptomyces cholesterol oxidase (ChoA) can not be adopted for the rate assay, because the K-m 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 K-m 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.

• 出版日期1999-9