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