Protein and enzyme immobilization on non-porous microspheres of polystyrene

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 nonporous, 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, beta-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 beta-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-alpha-D-mannopyranoside, a Con A-specific sugar derivative.