Experimental demonstration of pH control for a sequential two-step enzymatic reaction

pH control for multistep enzymatic synthesis reactions is important due to its potential industrial applications; however, it becomes difficult when the optimal pH for the individual steps of the reaction pathway differs by several units. The usual approach of using a compromised pH is neither satisfactory nor always feasible. An optimal pH control technique has been developed which allows a two-step reaction to occur simultaneously in proximity to each other and at their respective optimal pH in a single reactor. This technique separates the microenvironments of the two steps of a reaction pathway by coating an immobilized enzyme pellet with a thin layer of urease. Ammonia generated by the hydrolysis of urea consumes the hydrogen ions diffusing from the acidic bulk solution into the immobilized enzyme pellet. In this paper, the pH control technique is demonstrated for a two-step reaction where salicin is first converted by β-glucosidase to saligenin and glucose, followed by the isomerization of glucose catalyzed by glucose isomerase. The technique allows this two-step reaction to take place simultaneously at their respective optimal pH values of 5.0 for β-glucosidase and 8.0 for glucose isomerase.