Evaluation of phenylalanine ammonia lyase activity within cellulose nitrate membrane microcapsules

Microencapsulation of phenylalanine ammonia lyase (PAL) within cellulose nitrate membrane microcapsules causes a marked reduction in activity compared to the activity of unencapsulated enzyme. The purpose of this investigation was to identify the factors responsible for the reduction in activity of encapsulated PAL and to evaluate the contribution of each factor to the overall loss of activity of the enzyme within microcapsules. Using radiolabeled hemoglobin, the encapsulation efficiency of PAL was determined to be 70%, which suggested that incomplete entrapment might partially account for the poor activity of encapsulated PAL. The internal environment of microcapsules is composed of 10% hemoglobin solution which could affect the activity of PAL. The effect of internal environment of the microcapsules on the enzyme activity was evaluated by comparing KM and Vmax values of PAL in 10% hemoglobin solution and pH 8.5 Tris buffer. Similarity of these parameters in the two media suggested that the internal environment of the microcapsules did not contribute to the reduction in activity of the encapsulated enzyme. The cellulose nitrate membrane of microcapsules might exert a diffusional restriction for phenylalanine transport and thereby reduce the apparent activity of encapsulated PAL. The existence of this diffusional barrier was evaluated by breaking microcapsule membrane and measuring the enzyme activity in intact and broken microcapsules. PAL activity in broken microcapsules was similar to the activity of the enzyme in intact microcapsules suggesting that the membrane of microcapsules does not exert a diffusional barrier to the transport of substrate molecules. The effect of microencapsulation process on PAL activity was evaluated by comparing kinetic parameters of free and encapsulated enzyme. The similarity of K M values in these two media suggested that conformation of active PAL does not change during microencapsulation. However, the encapsulated enzyme had a markedly lower Vmax compared to the free enzyme which indicated that the encapsulation process probably denatures and deactivates a high proportion of PAL.