Performance characteristics of cholesterol oxidase for kinetic determination of total cholesterol

✍ Scribed by Pornpen Srisawasdi; Patcharee Jearanaikoon; Martin H. Kroll; Porntip H. Lolekha

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 145 KB
• Volume: 19
• Category: Article
• ISSN: 0887-8013
• DOI: 10.1002/jcla.20086

✦ Synopsis

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 Â 10 À4 M), followed by Streptomyces (2.17 Â 10 À4 M), Cellulomonas (0.84 Â 10 À4 M), and Pseudomonas (0.61 Â 10 À4 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 Â 10 À4 to 24.89 Â 10 À4 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.