Preparation and characterization of immobilized acid phosphatase used for an enzyme reactor: evaluation in flow-injection analysis and pre-column liquid chromatography Original Research Article

Acid phosphatase from various sources (plant and animals) was immobilized by attachment with glutaraldehyde to aminoalkylsilyl glass. An amino function of the enzyme was covalently coupled to the aldehyde derivative in the presence of phosphate as an enzyme inhibitor. The highest retention of enzyme activity was obtained by the immobilization of acid phosphatase from sweet potato. The double bond of Schiff base was reduced with sodium tetrahydroborate. The immobilized sweet potato acid phosphatase was active toward three substrates, p-nitrophenyl phosphate, β-glycerophosphate and riboflavin phosphate. The immobilized acid phosphatase retained almost complete activity over 4 months in a refrigerator. The immobilized acid phosphatase was packed into a stainless-steel column (10 × 4 mm i.d.) and used on-line as an immobilized enzyme reactor (IMER). The stability, reusability and utility of the IMER were verified by a flow-injection system and a liquid chromatographic pre-column reaction system. The IMER was extremely stable and reusable at room temperature, and was fully active even in 50% methanol solution.