Immobilization of Trypsin at the Microcapsule Surface and Enzymatic Activity of the Immobilized Trypsin

Abstract

In an attempt to make a new type of artificial cell that has enzymatic activity on the cell surface, polyamide microcapsules having many carboxyl groups in their membrane were prepared using an interfacial polymerization technique, and trypsin was covalently bound directly or via a spacer to their surface. The enzymatic activity of the immobilized trypsin was measured and compared with that of intact trypsin in solution.
When N^α-benzoyl-DL-argininep-p-nitroanilide, a low-molecular-weight substrate, was used, specific activity was appreciably higher for trypsin in the immobilized state than in solution. On the other hand, with casein, a high-molecular-weight substrate, trypsin immobilized without a spacer showed specific activity only a half as high as trypsin bound to the microcapsule surface via a spacer probably because of steric hindrance. The activity of both f orms of the immobilized trypsin was much lower than that of free trypsin. Immobilization caused a slight shift of optimum pH for enzymatic activity of trypsin to the alkaline side because the hydrogen ion concentration was higher on the microcapsule surface than in the bulk due to negative charges of the dissociated surface carboxyl groups (Figs.1 and 2). Both Michaelis constant, K_m, and maximum reaction velocity, Vmax, were found considerably higher for free trypsin in solution than for the immobilized enzyme (Table 2). Thermal stability of the enzyme was remarkably increased by immobilization to the microcapsule surface (Fig.5).