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 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 Zn²⁺ inhibited both enzymes. The residual activity of native CDH after heat treatment was 32% (37°C, 60 min), while the r-CDH showed a residual activity of 87% (37°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.