Electrochemical analysis of immobilised chemical and genetic biotinylated alkaline phosphatase

Abstract

Many approaches to enzyme immobilisation have been described but almost all are non-specific and result in a loss of biological activity or loss of enzyme due to weak or poor binding. In order to overcome these problems, we have investigated the fusion of biotin acceptor peptide sequences to the C-terminus of alkaline phosphatase (ALP) as a method to immobilise this enzyme through the avidin–biotin linkage. ALP from E coli. was expressed with the following sequences fused to its C-terminus: LEAIFEAQ KIEWR or LGGIFEAM KMEWR. These sequences have been reported to be biotinylated on the ε-amino group of the lysine residue (shown above in bold). Wild-type ALP was also biotinylated with a NHS derivative of a long chain biotin. Both genetically (GB) and chemically (CB) biotinylated enzymes were purified and their immobilisation to an avidin-coated electrode was investigated. The immobilised ALP was assayed electrochemically by following the conversion of p-aminophenylphosphate to p-aminophenol with amperometric determination of this product. The results showed that both biotinylated enzymes exhibited significantly higher binding to the electrode than the wild-type enzyme, which yields currents that are comparable to the background.