BBL433

Experiment 3:
ASSAY OF β-GLUCOSIDASE

DATE OF EXPT : Monday, 5/02/2024 (Grp.4)

UTKARSH ANAND 2021BB10322

MEENAKSHI MINA 2021BB10012
Background
β-Glucosidase is an enzyme which acts upon the β 1,4 linkages between two glucose or glucose-substituted
molecules. It catalyzes the hydrolysis of terminal non-reducing residues in β-D- glucosides with the release of
glucose. β – glucosidase activity can be measured by monitoring the formation of a chromogenic product p-
nitrophenol, which is formed by hydrolysis of p-nitrophenol – β- D – glucopyranoside (PNPG). The amount of
PNP formed can be measured by determining the absorbance at 405nm. The amount of PNP produced is
proportional to the amount of β- glucosidase at the time of the reaction. The reaction is quenched by adding
Na2CO3 which shifts the pH of the reaction mixture to alkaline (pH = 11.0). At this pH most of the PNP is
converted to the yellow-colored anionic form and βglucosidase is inactivated.
Materials And Equipment Required :-
 1. Spectrophotometer
 2. Water Bath ( at 50 degree C and 30 degree C )
 3. Vortex Mixer
 4. Cuvettes
 5. Pipettes and Tips
 6. Eppendorfs
 7. Test tubes / Falcons

Reagents Required:-
 1. Citrate Buffer (50mM) (pH: 4.8) (7ml)
 2. PNPG solution (1mM) in Citrate buffer (4ml) 3. PNP
solution (1mM) in Citrate buffer (1ml)
 4. Sodium Carbonate solution (1N) in distilled water
(10ml)
 5. Enzyme (300 ul)
Procedure
1. I. For standard Curve:- Serially diluted PNP solution (1mM) to prepare dilutions of 20µM,
40µM, 60µM, 80µM, 100µM with citrate buffer to the final volume of 1mL (Note: Citrate
buffer was used for dilution of PNP; not water)
2. 0.5mL of 1N Na2CO3 was added to each tube and OD was measured at 405nm against the
reagent blank (Blank= 1 mL Citrate buffer + 0.5 mL 1N Na2CO3)

3. II. For Enzyme Assay:- 1. In two tubes each containing 100µL of the enzyme, 0.9 mL of
PNPG solution was added
4. The solution in one tube was incubated in a water bath at 50 degree C while solution in
another tube was incubated in a water bath at 30 degree C for exactly 10 minutes
5. After incubation 0.5 mL of 1N Na2CO3 was added to each tube to stop the reaction
6. Absorbance of both the solutions was measured at 405nm [Blank= 0.1mL of Citrate buffer
+ 0.9mL PNPG + 0.5 mL of 1N Na2CO3]
Observations

Absorbance of samples at 405nm after 10 minutes of incubation at:

(i) Room Temperature: 0.411
(ii) 30ºC : 0.620
(iii) 50ºC : 0.845

Equation of standard curve from graph: y =0.0106x + 0.0753

Calculations
• Absorbance of samples at 405nm after 10 minutes of incubation at:
(i) Room Temperature: 0.411
(ii) 30ºC : 0.620
(iii) 50ºC : 0.845
• Equation of standard curve from graph: y =0.0106x + 0.0753
• Plugging in the values of A(405nm) at different temperatures into the obtained
equation, we get the following concentrations of PNP:
(i) Room Temp: 31.67 μM
(ii) 30ºC : 51.39 μM
(iii) 50ºC : 72.61 μM
•For 1.5 mL of sample each, amount of PNP formed:
(i) Room Temp: (31.67 μM)*1.5 mL= 47.5 nanomoles
(ii) 30ºC : ( 51.39 μM )*1.5 mL= 77.1 nanomoles
(iii) 50ºC : ( 72.61 μM )*1.5 mL= 108.94 nanomoles
Calculations
Calculation of enzyme activity:-

Enzyme dilution = 1; Incubation time= 10 minutes; Vol. of enzyme = 100 μL = 0.1mL

 For Room temp. , Enzyme activity= 47.5*10^(-3)*1/ 10*0.1 = 0.048 IU/mL

 For 30 degree C, Enzyme activity= 77.1*10^(-3)*1/ 10*0.1 = 0.077 IU/mL
 For 50 degree C, Enzyme activity = 108.94*10^(-3)*1 / 10*0.1 = 0.109 IU/mL
 Results & Conclusion

From the above experiment we can conclude that as the temperature increases
beta-glucosidase enzyme activity also increases.

This is because higher temperature generally causes more collisions among the
molecules and therefore increases the rate of a reaction. More collisions increase
the likelihood that substrate will collide with the active site of the enzyme, thus
increasing the rate of an enzyme-catalyzed reaction.
Precautions
 The dilutions should be carefully made from the stock solution in citrate buffer.
 Make sure the reagents are not contaminated.
 Cuvettes should be clean.
 Use the pre-incubated PNPG at 50 °C.
 The incubation time should be exactly 10 minutes

Thank You!