Horseradish peroxidase (HRP), which catalyzes oxidation reduction reactions of large number of substrates, was entrapped in K-carrageenan beads using polyethyleneimine as hardening agent. The heat and storage stability was found to be better for entrapped horseradish peroxidase than free enzyme. The entrapped enzyme showed 50% retention of its activity after 4 cycles. Effective diffusion coefficient for diffusion of hydroquinone into Kcarrageenan beads was found to be 0.27 ϫ 10 Ϫ10 m 2 /s during enzyme-catalyzed oxidation of hydroquinone. Kinetic parameters calculated from Lineweaver-Burke plots were observed to be K m ϭ 8 ϫ 10Ϫ5 and V max ϭ 1.53 for free enzyme, and K m ϭ 8.3 ϫ 10 Ϫ5 and V max ϭ 2.18 for entrapped enzyme when enzyme concentration was kept constant and K m ϭ 4 ϫ 10 Ϫ11 and V max ϭ 0.45 for free enzyme and K m ϭ 4.5 ϫ 10 Ϫ11 and V max ϭ 0.58 for entrapped enzyme when substrate concentration was kept constant. This indicates that there is no conformational change during entrapment.

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Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2-hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K m ) and maximum reaction velocity (V max ) were 1.1 ϫ 10 Ϫ6 and 0.87 for the free enzyme and 1.2 ϫ 10 Ϫ6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K m and V max were 6.73 ϫ 10 Ϫ11 and 0.47 for the free enzyme and 7.59 ϫ 10 Ϫ11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration-dependent. The hydrolysis of casein was carried out with a fixed-bed reactor (17 cm ϫ 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm 3 /min flow rate at 35°C with a 1 mM casein solution.

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