The Characteristics and Applications of Recombinant Cholesterol Dehydrogenase

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Mass production of an r-CDH derived form 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 K_m of r-CDH was 2.50×10^<-3>M for cholesterol and 2.33×10^<-4>M for NAD. The activators of CDHs were TritonX-100 and cholate. TritonX-405,Ag^+, and Zn^<2+> inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37℃, 60 min), while the r-CDH showed a residual activity of 87% (37℃, 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.
社団法人日本農芸化学会の論文
2000-07-23