For repeated batch reactions with the immobilized DCase, a substrate solution (3%) of c-D-HPG (pH 7.0) was prepared, with nitrogen gas bubbling at 40 °C. The DCase immobilized on Duolite A-568 and the prepared substrate
Figure 7. Construction profile of pNT4553.
solution were stirred at 40 °C under a stream of nitrogen gas while the pH was controlled at pH 7.0 with 2 M HCl. Samples were taken for activity measurement at 10 and 60 min, the reaction being continued for 23.5 h in total. The reaction mixture was removed by suction, after which a fresh substrate solution was introduced and allowed to react in the same manner as already described. The residual activity was measured as the relative activity compared with the initial activity and plotted against the reaction times (Fig. 8). The stability of the mutant en zyme increased in comparison with that of the native enzyme.
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