Generation of a pH gradient in an immobilized enzyme system

Abstract

Several examples of two-step sequential reactions exist where, because of the poor equilibrium conversion by the first reaction, it is desirable to conduct the two reactions simultaneously. In such a scheme, the product of the first reaction is continuously removed by the second reaction, thus not allowing the first reaction to approach chemical equilibrium. Therefore, the first reaction is allowed to proceed in the desired direction at an appreciable rate. However, in many biochemical applications where enzyme catalysts are involved, the enzyme's activities are strong functions of pH. Where the pH optima of the first and second reaction differ by three to four units, the above reaction scheme would be difficult to implement. In these cases, the two reactions can be separated by a thin permeable membrane across which the desired pH gradient is maintained. In this article, it was shown, both by theory and experiment, that a thin, flat membrane of immobilized urease can accomplish this goal when one face of the membrane is exposed to the acidic bulk solution (pH(b) = 4.5) containing a small quantity of urea (0.01 M). In this particular case, the ammonia that was produced in the membrane consumed the incoming hydrogen ions and thus maintained the desired pH gradient. Experimental results indicate that with sufficient urease loading, the face of the membrane opposite to the bulk solution could be maintained at a pH that would allow many enzymes to realize their maximum activities ( approximately 7.5). It was also found that this pH gradient could be maintained even in the presence of a buffer, which greatly enhances the transport of protons into the membrane.

Demonstration of pH control in a commercial immobilized glucose isomerase

“…As expected, the initial rate for this case is higher than the second run that includes urea. The equilibrium xylulose concentration was reached in only 6 h. This is due to the fact that for the second run a pH gradient develops near the surface of the enzyme pellet (Byers et al, 1993). In this region, where the glucose isomerase and urease are coimmobilized, the pH is less than the optimal value of glucose isomerase, which results in a very thin shell region of reduced isomerase activity.…”

“…Prior to entering the reactor, the bulk liquid was preheated to the desired reaction temperature by passing it through a long thin metal tube in good thermal contact with the constant temperature bath. The pH of the bulk solution was maintained at the desired value using the Radiometer pH stat system as described previously (Byers et al, 1993).…”

“…This requires that the reactions in the synthesis pathway occur simultaneously and in proximity to one another, and at their respective optimal pH values. In preceding studies (Byers et al, 1993(Byers et al, , 1994, the feasibility has been demonstrated by both theory and experiment of maintaining pH differences of three or more units in the same reaction system. Conceptually, the technique involves the separation of the microenvironments of the two steps of a reaction pathway by a thin, permeable, immobilized-* To whom all correspondence should be addressed.…”

“…Telephone: 419-530-8035; fax: 419-530-8076; e-mail: rfoumi@uoft02.utoledo.edu enzyme layer that catalyzes an acid-or base-forming reaction. We have shown (Byers et al, 1993) experimentally that a very thin layer of immobilized urease separating an acidic (pH -4.5) from a basic (pH -7.5) environment was able to maintain this huge pH difference-even in the presence of buffers-when only a small concentration of urea (-0.01 M) was maintained in the acidic environment. Conversion of urea by the immobilized urease generates a base which consumes the hydrogen ions diffusing into the layer of immobilized enzymes from the acidic bulk solution.…”

“…The proposed pH control system works well for the commercial immobilized glucose isomerase; that is, this enzyme exhibits much of the same activity it displays at a pH of 7.5, even though the pH of the bulk liquid is maintained at a value of 4.6. For the case of xylose isomerization to xylulose, this would allow for the simultaneous fermentation of the xylulose to ethanol in the bulk solution at its optimal acidic pH (Byers et al, 1992(Byers et al, , 1993.…”