IA on effect of temperature on the rate of hydrolysis of lactose (ONPG) by enzyme (lactase), measured using visible spectrophotometer.

1. Effect of temp on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.
Lactase enzyme from baby drops used. 10ul enzyme added to 1ml water as stock sol.
Lactose solution (ONPG) 5mM prepared, by dissolving 0.15g in 100ml water.
Yellow solution produce when enzyme breaks down ONPG (lactose)
Rate of hydrolysis is measure by change in abs over time
ONPG is lactose analogue which break down by enzyme lactase to produce
yellow substance which can be measure with colorimeter
Procedure:
ONPG solution were incubated at various temp (28, 37, 50, 55)
20ul enzyme were also incubated separately at various temp.
Both solution were added/mix into a cuvette and placed in colorimeter.
Change of abs over time (disappearance of yellow) – rate of hydrolysis.
Reagents needed
Mixing is crucial to ensure, expt work, it is advisable to add enzyme to the
bottom of cuvette and then pipette in lactose sol to mix them well

2. Go to expt – press calibrate Insert cuvette (yellow sol), press collect
Insert a blank containing water
Press stop, and click on rainbow icon. Select abs vs time. λ max at 416nm will
be automatically chosen
Abs vs time
Effect of temp on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.

3. Rate increases, as temp increases, seems like enzyme
works best at around 40C and starts to break down at temp > 40C
-0.01
-0.005
0
0.005
0.01
0.015
0.02
0.025
0.03
0.035
0.04
0 10 20 30 40 50 60
rate
of
hydrolysis
temp/C
temp/C vs rate of hydrolysis
Change of abs over time were plotted.
Rate of hydrolysis – decrease in Abs over time
Slope/gradient taken over 5s
Temp/C Rate
Abss-1
28 0.01304
37 0.03430
50 0.005294
55 0.001276
Data collected.
Effect of temp on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.