Immobilization of alpha-chymotrypsin on poly(urethane-graft-acrylic acid)

A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.