Rigid Support Materials for the Immobilization of Enzymes

The purpose of the work presented here was to prepare a support material for enzymes and “affinity ligands” with the following characteristics: low cost, durability, rigidity, and high capacity. Our study encompassed conjugates of porous and nonporous silicas with organic polymers and macroporous ion-exchange resins. Poly-ethyleneimine (PEI), polyacrylic acid (PAA), poly (methyl vinyl ether/maleic anhydride) were attached to porous glass and silica in various combinations. The composite of silica beads with PEI and PAA is a good support for the enzyme trypsin as judged by the activity against N-α-benzoyl-L-arginine ethyl ester.

Amberlyst (macroporous, sulfonated polystyrene) was activated by treatment with thionyl chloride; the resulting resin was either used directly or reacted with a diamine. The diamine derivative was used for enzyme coupling or transformed further to the succinyl or p-aminobenzoyl derivative. None of these derivatives were particularly good as supports for the enzyme trypsin. Duolite converted to a PAA, succinyl, or succinimide derivative was a good support. The enzyme-resin adduct has good activity and stability.

The resin is quite durable and of low cost. The Duolite-trypsin has good activity against protein. In addition, this derivative was active in 7 M urea. The proteolytic activity was nearly doubled by urea, presumably as a result of substrate (casein) denaturation. The michaelis constants and pH dependences are compared for trypsin conjugates with Duolite A-7, Silica-PEI-PAA, agarose, and porous glass. A cost comparison reveals that the Duolite and silica derivatives are much less expensive than agarose and glass.