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 (230.3×10 −4  M), followed by Streptomyces (2.17×10 −4  M), Cellulomonas (0.84×10 −4  M), and Pseudomonas (0.61×10 −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 2.17×10 −4 to 24.89×10 −4  M. The linearity was increased from 2.6 to 13.0 mmol/L. The high K m 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. J. Clin. Lab. Anal. 19:247–252, 2005. © 2005 Wiley‐Liss, Inc.