On the Regulation of the Activity of Immobilized Enzymes

Glucose oxidase, hexokinase, trypsin and urease were entrapped, either separately or together, within polyacrylamide particles, using a bead polymerization technique. The pH optima, for the immobilized enzymes in 5 mM buffer, were displaced compared to those of the enzymes free in solution. Thus the pH optimum for trypsin activity towards benzoyl‐l‐arginine ethyl ester shifted 1.3 pH unit (to pH 9.6) and the pH optimum for glucose oxidase towards glucose shifted 0.3 pH units to the alkaline side (to pH 6.9). Reversely, urease activity towards urea leading to consumption of protons shifted its optimum 0.4 unit to the acidic side (to pH 5.8). The effect on glucose oxidase activity of pH changes caused by activity of coentrapped trypsin or urease was studied. It was found that glucose oxidase activity in the alkaline region was stimulated during simultaneous trypsin activity, whereas at pH values below that of the optimum for glucose oxidase inhibition occurred. A reversed effect was measured during urease activity. At pH 8.6, for example, glucose oxidase activity was increased by a factor of three to 75% of its activity found at pH optimum due to trypsin activity. On the other hand, at pH 6.0, i.e. below the optimum, it increased by a factor of two to about 80% due to urease activity. Both simultaneous trypsin or urease activity gave distorted “two‐peak” pH‐activity profiles of glucose oxidase. The effect of protons produced by trypsin on the simultaneous activities of glucose oxidase and hexokinase, both competing for the same substrate, glucose, was studied with a gel containing all three enzymes. It was found that the amount of glucose converted per minute by each enzyme reaction was effected. In one case, at pH 8.5 in the absence of trypsin activity, about one seventh of the glucose consumption was due to hexokinase activity. On addition of benzoyl‐l‐arginine ethyl ester, hexokinase activity decreased almost to zero, while glucose oxidase activity was stimulated, resulting in practically all conversion being due to the latter.