Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterol

The enzymatic method for cholesterol determination can use either an endpoint or a kinetic method. Not much is known concerning the properties (K-m and V-max) of the commercial enzyme for the kinetic method. We measured the K-m and V-max of Brevibacterium, Streptomyces, Pseudomonas fluorescens, and Cellulomonas cholesterol oxidase. Brevibacterium gave the highest K-m value (230.3 x 10(-4) M), followed by Streptomyces (2.17 x 10(-4) M), Cellulomonas (0.84 x 10(-4) M), and Pseudomonas (0.61 X 10(-4) M). The K-m values and the linearity obtained from Streptomyces (2.6 mmol/L), Pseudomonas (2.1 mmol/L), or Cellulomonas (2.1 mmol/L) were too low. Dichlorophenol isomers, acting as inhibitors, increased the enzyme's K-m. The addition of 3,4-dichlorophenol raised the K-m of Streptomyces from 2.17 x 10(-4) to 24.89 x 10(-4) M. The linearity was increased from 2.6 to 13.0 mmol/L. The high K, of Brevibacterium resulted in an insensitive reaction and low cholesterol linearity (7.8 mmol/L). An increase in the sample-to-reagent ratio from 1:100 to 1:10 enhanced the reaction rate and the linearity from 7.8 to 20.7 mmol/L. We suggest that Brevibacterium and Streptomyces cholesterol oxidase (with the addition of 3,4 dichlorophenol) are good sources for serum cholesterol determination by the kinetic method.