Enzyme Kinetics (activity)

Effect of Temperature on Enzyme Activity

      1. Add 2.5 ml of buffer to a tube (this is the BLANK)
      2. Add 2.5 ml of starch (substrate) to each tube
        • one on ice (0°C), one on the bench (25°C) and one in a 40°C water bath
      3. Add 2 drops of iodine to each tube and mix: Blank, 0°C, 25°C, 40°C
      4. Read the Blank in the spectrophotometer and calibrate it to 100% transmittance at 560nm
      5. Read each tube in the spectrophotometer. This is time 0 min.
      6. Add 35 drops of amylase solution to each tube simultaneously, mix and ensure incubation is occurring at the correct temperature.
      7. At 2 minute intervals, quickly read ALL tubes in the spectrophotometer and immediately return the tubes to the proper temperature.
      8. Continue reading the samples every 2 minutes until you reach 22 minutes on the table below.

Time (min)

0ºC

% Trans

25ºC

% Trans

40ºC

% Trans

0

2

4

6

8

10

12

14

16

18

20

22