title: The characteristics and applications of recombinant cholesterol dehydrogenase.
abstract: Mass production of an r-CDH derived from Nocardia species was made possible by gene technology. (Horinouchi et al., Applied and Environmental Microbiology, 57, 1386-1393 (1991)). However, the characteristics of the r-CDH have not been studied in detail and have not been improved enough for industrial use. We accordingly characterized both the native-CDH and the r-CDH prepared from Streptomyces lividans. Both CDHs were monomers with molecular masses of 37 kDa. The Km of r-CDH was 2.50 x 10(-3) M for cholesterol and 2.33 x 10(-4) M for NAD. The activators of CDHs were TritonX-100 and cholate. TritonX-405, Ag+, and Zn2+ inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37 degrees C, 60 min), while the r-CDH showed a residual activity of 87% (37 degrees C, 60 min). The r-CDH is an enzyme with high substrate specificity for cholesterol as well as native CDH and higher thermal stability than native CDH. We have developed a novel serum cholesterol assay using the r-CDH, which permits the direct measurement of cholesterol by measuring NADH reaction products. We conclude that this r-CDH enzyme is useful and can be used to measure cholesterol in a clinical chemistry setting.
authors.affiliation: International Reagents Corp., Kobe, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Kishi
authors.forename: Y
authors.initials: Y
authors.lastname: Watazu
authors.forename: Y
authors.initials: Y
authors.lastname: Katayama
authors.forename: H
authors.initials: H
authors.lastname: Okabe
mesh_terms: D002784:Cholesterol / Q000097:blood; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008970:Molecular Weight; D017830:Octoxynol; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D017830:Octoxynol; D002784:Cholesterol; D010088:Oxidoreductases; C019884:cholesterol dehydrogenase