- Phosphorylation by alkaline phosphatase: immobilization and synthetic potential
- International Journal of Chemistry
- Volume | Issue number
- 5 | 3
- Pages (from-to)
- Document type
- Faculty of Science (FNWI)
- Van 't Hoff Institute for Molecular Sciences (HIMS)
Phosphatases (AP, E.C. 22.214.171.124) are hydrolytic enzymes that naturally hydrolyse phosphomonoesters but in a so-called transphosphorylation reaction these enzymes are also able to transfer a phosphate group from phosphorylated compounds to alcoholic functions. This transphosphorylation catalysed by acid phosphatases using pyrophosphate as a phosphate donor has been studied in some detail. However, the acidic pH optimum of these enzymes limits some of their applications. The catalytic features of alkaline phosphatase are similar to the acid phosphatases and its alkaline pH optimum suggests a possible application of this enzyme in phosphorylation reactions which need to be carried out at higher pH. Here we explore the synthetic potential of bovine intestine alkaline phosphatase (AP) in the phosphorylation of dihydroxyacetone (DHA) and glycerol using pyrophosphate (PPi) as phosphate donor. The phosphorylated compounds are intermediates in two multi-enzymatic cascade reactions for the synthesis of carbohydrates. The yields of dihydroxyacetone phosphate (DHAP) and glycerol-1-phosphate at pH 8 (2.6 mM and 2.2 mM, respectively) were comparable to the results obtained with the acid phosphatases at pH 4. Nevertheless, when the cascade reactions were carried out at pH 8, very low conversions were measured due to inactivation of the alkaline phosphatase by the product phosphate. To circumvent this inhibition, the alkaline phosphatase was immobilized on aldehyde-activated beads (Sepabeads EC-HA). The immobilization greatly diminished the inhibition by phosphate, and the immobilized alkaline phosphatase at pH 8 gave the same conversions in the cascade reaction starting from DHA as obtained with the acid phosphatase at pH 6. However, the immobilized enzyme was active for only one catalytic cycle and the beads could not be reused.
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