Open AccessPerformance characteristics of cholesterol oxidase for kinetic determination of total cholesterol
Author(s) -
Srisawasdi Pornpen,
Jearanaikoon Patcharee,
Kroll Martin H.,
Lolekha Porntip H.
Publication year - 2005
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.20086
Subject(s) - brevibacterium , cholesterol oxidase , chemistry , pseudomonas fluorescens , streptomyces , reagent , enzyme , nuclear chemistry , chromatography , bacteria , biochemistry , biology , organic chemistry , microorganism , genetics
Abstract 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.