Effects of pH, temperature and reaction products on the performance of an immobilized creatininasecreatinasesarcosine oxidase enzyme system for creatinine determination

Abstract

The effects of pH, temperature ( t) and reaction products on the performance of enzyme reactors containing immobilized creatininase (CA), creatinase (CI) and sarcosine oxidase (SO) for the determination of creatinine were studied by flow-injection analysis with amperometric detection of the resulting hydrogen peroxide. The optimum performance of the coupled enzyme system was found at pH 7.7 and 25°C. Some of the CI and SO activity was lost irreversibly at t ⩾ 30°C. In contrast, the activity of CA increased with t up to at least 40°C. The effects of the reaction products on the enzyme activities were examined. Glycine caused the CA activity to increase and the SO activity to decrease, whereas the CI activity was unaffected by this compound. Sarcosine caused a decrease in the CI activity. The activities of all three enzymes were insensitive towards the presence of formaldehyde and urea and so was the activity of SO in the presence of creatine and hydrogen peroxide. The fraction, α, of the injected creatinine (or creatine) equilibrated by the CA reactor is introduced as a quantitative measure of the CA activity, and was between 10 and 72% depending on the enzyme loading. The unused immobilized enzymes were found to maintain their activity for at least 6 months. When in heavy daily use, CA and SO lost ca. 25% of the activity over a period of 20–30 days, whereas the activity of CI was found to be essentially unchanged.