Most of the restriction enzymes have an optimum temperature of 37°C and an optimum pH of pH 7.2 or pH 8.5. These physical environment requirements must be met to ensure that the enzyme reaches its maximum activity. Incubation temperatures affect the activity of restriction enzymes greatly. The enzyme can denature outside the desirable physical condition range. Enzymes work very specifically; therefore, they will not work properly if their structure deteriorates. As each restriction enzyme is isolated from different bacteria and have different optimum reaction conditions, they require specific buffers for each restriction reaction.
Table 1: Enzyme activities. The number for each buffer signifies the activity in the respective enzyme. If there is a * the enzymes may exhibit star activity in this buffer.