Comparison of the coupling recoveries of immobilized aspartate aminotransferase. Specific activity, enzyme-bound coenzyme, and transaminationable active centers.

Aspartate aminotransferase (AspAT, EC 2.6.1.1) was bound on CNBr-activated Sepharose and the effects of immobilization on the maximum velocity, biologically active pyridoxal-5'-phosphate (PLP), and transaminationable active centers were studied. By comparing these parameters of soluble and immobilized enzyme the factors decreasing the observed reaction rate upon immobilization were evaluated. Ninety percent of the soluble protein in the coupling mixture was bound to the support. The amount of enzyme-bound PLP of immobilized preparation was 83% of that of the soluble one. The coupling recovery of specific activity was 46%, which was 10%-units lower than that of the transaminationable active centers. This difference depends on the fact that a part of the active centers of immobilized enzyme had lower catalytic rate, due to the enzyme-matrix interactions or internal mass transfer limitations, than the others. The immobilized catalytically active AspAT had 80% of the turnover efficiency of the soluble enzyme. The affinity of the enzyme to its substrates did not significantly change upon immobilization, neither did the pH profile.