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

1. Effect of pH 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:
100ul of enzyme (stock sol) added to 0.5ml of buffer of diff pH (1 ,5, 7, 9)
Solution were mix for 5 mins
10ul of mix was added to a cuvette and placed in colorimeter.
1ml of ONPG (lactose) solution was into cuvette at RT 22C.
Change of abs over time (disappearance of yellow) – rate of hydrolysis.
-ve control – ONPG + pH without any enzyme.
+ve control – ONPG with enzyme
Reagents needed

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 pH on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.

3. Change of abs over time were plotted.
Rate of hydrolysis – decrease in Abs over time
Slope/gradient taken over 10s
pH Rate
Abss-1
1 0.000039
5 0.003290
7 0.00750
9 0.06603
-ve
(control)
0.0003290
Data collected.
Rate increases, as pH increases, seems like enzyme
works best at slightly alkaline pH (pH 9)
Effect of pH on the rate of hydrolysis of lactose by enzyme (lactase), measured using visible spectrophotometer.
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0 2 4 6 8 10
Rate
of
hydrolysis
pH
pH vs rate of hydrolysis
-ve control - Very little breakdown of ONPG w/o any enzyme