Protein and enzyme immobilization on non‐porous microspheres of polystyrene

Abstract

The diffusion constraints with respect to substrate and product can be eliminated when an enzyme is immobilized on non‐porous carriers. In an affinity chromatographic system, non‐porous beads have the advantage of very rapid analytical and micropreparative chromatography of bioproducts. In the present study a procedure of protein immobilization on non‐porous, micro‐sized beads of polystyrene (PS) was developed. Results from Fourier transform infrared and elemental analysis indicated that both nitration and successive hydrogenation of PS were successful. Two model proteins, β‐lactamase and concanavalin A (Con A), were then immobilized by covalent binding on the resultant amino‐containing PS. In comparison with the behaviour on porous supports, the immobilization of enzyme on non‐porous PS resulted in only a small increase in the Michaelis constant. The microspheres of immobilized β‐lactamase exhibited a fast response (less than 30 s) in the substrate solution, indicating that they were suitable for packing into a precolumn placed before an enzyme thermistor for the clinical detection of penicillin G. The columns packed with immobilized Con A were found to be effective for the chromatographic analysis of p‐nitrophenyl‐α‐D‐mannopyranoside, a Con A‐specific sugar derivative.