Effects of Temperature and pH on Enzyme Stability

To evaluate the thermostability of the DCases, a cell-free extract (100 uL) was incubated at various temperatures (55-85 °C) for 10 min, the denatured protein was removed by centrifugation, and then the residual activity was measured by the standard HPLC method. The heat denatur-ation profiles of the four mutant enzymes from KNK402M, KNK404M, KNK416M, and KNK455M and the native enzyme were investigated. The relative activities in comparison with the activities of the non-heat-treated enzymes were plotted against temperature (Fig. 5). The thermosta-bilities of the mutant enzymes increased about 5-20 °C under these conditions compared to that of the native enzyme.

To examine the effect of pH on DCase stability, a cellfree extract (200 uL) was mixed with 800 uL of a pH-adjusted buffer (pH 6-10, 0.1 M each), followed by incu-

Table 4. Thermostability Analysis of Mutant DCases
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