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Biotechnology and Applied Biochemistry (1998) 27, (225–230) (Printed in Great Britain)

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Protein and enzyme immobilization on non-porous microspheres of polystyrene

Cheng-We Wu*, Jin-Gang Lee*, Wen-Chien Lee*

*Department of Chemical Engineering, National Chung Cheng University, Chiayi, 621, Taiwan

Abbreviations: Con A, concanavalin A; DABCO, 1,4-diazo-(2,2,2)-bicyclo-octane; PNPM, p-nitrophenyl a-d-mannopyranoside; PS, polystyrene; PVP, polyvinylpyrrolidone.

Correspondence: Wen-Chien Lee.

To whom correspondence should be addressed.

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, b-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 b-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-a-d-mannopyranoside, a Con A-specific sugar derivative.

(Received 8 September 1997/2 January 1998; accepted 6 January 1998)

The Biochemical Society and the Medical Research Society © 1998