Electrochemical analysis of immobilised chemical and genetic biotinylated alkaline phosphatase

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 en zyme due to weak or poor binding. In order to overcome these problems, we h ave investigated the fusion of biotin acceptor peptide sequences to the C-t erminus 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: LEAIFEAQKIEWR or LGGIFEAMKMEW R. These sequences have been reported to be biotinylated on the epsilon-ami no group of the lysine residue (shown above in bold). Wild-type ALP was als o biotinylated with a NHS derivative of a long chain biotin. Both genetical ly (GB) and chemically (CB) biotinylated enzymes were purified and their im mobilisation to an avidin-coated electrode was investigated. The immobilise d ALP was assayed electrochemically by following the conversion of p-aminop henylphosphate top-aminophenol with amperometric determination of this prod uct. The results showed that both biotinylated enzymes exhibited significan tly higher binding to the electrode than the wild-type enzyme, which yields currents that are comparable to the background. (C) 2000 Elsevier Science B.V. All rights reserved.