Sweet Potato Acid Phosphatase Immobilized on Glutaraldehyde-Activated Aminopropyl Controlled-Pore Glass: Activation, Repeated Use and Enzyme Fatigue

Abstract

Sweet potato acid phosphatase was covalently coupled with glutaraldehyde to aminopropyl controlled-pore glass, and used as a pre-column enzyme reactor. The immobilized enzyme reactor (IMER) was continuously operated using an automated chromatographic detection system we developed. Functional evaluation of the IMER was carried out by injecting ten samples on the same day at an injection amount of 1.25 nmol (62.5 nmol per ml) using riboflavin sodium phosphate (FMNs) as a substrate, and by prolonged use for ten months. The IMER exhibited decreased activity after repeated use for a total of 3000 samples, but about 75% of its original activity remained. The conversion rate of FMNs to riboflavin by IMER was increased from 89 to 97% by adding citrate, ethylenediaminetetraacetic acid disodium salt, etc., but especially by adding citrate. The increased conversion of FMNs to riboflavin due to the addition of citrate was probably not due to the chelation of heavy metal ions by citrate. We also investigated complex formation of acid phosphatase with the substrate FMNs using surface plasmon resonance to determine the effect of citrate on the processes of association and/or dissociation between the enzyme and substrate. Enzyme fatigue was also observed during the course of prolonged and repeated use.