Analysis of Polyphenol Oxidase enzyme isolated from different sources

1. Analysis of Polyphenol Oxidase enzyme
isolated from different sources
Presented By -
Manojit Mandal
M.Sc. Biotechnology 4th semester
Roll- PG/VUOGP57/BIT-IVS No. 042
Reg. No. – 02177of 2020-2021

2. Contents :-
 Introduction
 Aims and objectives
 Methodology
 Result
 Conclusion

3. Introduction :-
 Polyphenol oxidase(PPO) are a copper containing group of enzymes.
 The enzyme works by catalyzing the ortho-hydroxylation to ortho-
diphenols to further ortho- quinones.
 Mainly responsible for enzymatic browning.
 Used in the removal of phenolic compounds from industrial effluents.
 Used in treatment of several diseases like Parkinson’s disease.

4. ● Determination of the polyphenol oxidase enzyme activity
● Determination of the inhibition effect
● Determination of the protein estimation by Lowry’s method
● Determination of the anti-oxidant activity by DPPH
Aims and Objectives :-

5. ● Fresh samples of mushroom, pomegranate and banana were bought.
● Samples were cut into small pieces.
● Citrate buffer was prepared.
● 50g of each samples were poured into the citrate buffer.
● Crushed well in a mortar pestle.
● Kept for centrifugation at 12,000 rpm for 20 minutes at 40C.
● Supernatant was collected and stored.
Methodology :-
1. Extraction of polyphenol oxidase enzyme :-

6. ● Pyrocatechol was used as substrate.
● Substrate was prepared for 3 different concentrations (0.2mM, 0.3mM
and 0.4mM).
● Sodium phosphate buffer was made.
● A reaction mixture was prepared by adding sodium phosphate buffer,
substrate and enzyme extracts for each samples.
● Then the absorbance was measured in the colorimeter in 600nm
wavelength for all the samples.
2. Polyphenol oxidase assay :-

7. ● Ascorbic acid and citric acid was used as inhibitor.
● Each inhibitor stock was prepared.
● Then reaction mixture was prepared by adding sodium phosphate
buffer, substrate, enzyme extracts and inhibitor for each sample
individually.
● At last, absorbance was measured in colorimeter in 600nm
wavelength.
3. Anti-browning activity with inhibitors :-

8. ● At first, 50ml 0.1N NaOH was prepared.
● 0.1g sodium potassium tartrate was dissolved in 10ml distilled water.
● 0.05g copper sulphate was dissolved in 10ml distilled water.
● 48ml NaOH, 1ml of sodium potassium tartrate and 1ml copper sulphate was
taken in a flask and mixed well.
● 10ml Folin-ciocalteu reagent was made.
● BSA solution was prepared for 1mg/ml concentration.
● BSA standards were made for 200 µg/ml, 400 µg/ml, 600 µg/ml, 800 µg/ml,
1000 µg/ml.
● At last, optical density was measured in colorimeter in 600nm wavelength.
4. Estimation of protein by Lowry’s method :-

9. ● At first, 100ml of 0.1mM DPPH was prepared.
● Then 2.4ml of DPPH solution and 1.6ml of enzyme sample was
added in a test tube for each sample individually.
● A control was made by adding 1.6ml methanol and 2.4ml of DPPH
solution.
● A blank was also prepared.
● Then all the test tubes were vortex and left for incubation in dark for
30 minutes.
● At last absorbance was measured in colorimeter in 520nmm
wavelength.
5. Anti-oxidant activity by DPPH :-

10. Result :-
1. Polyphenol oxidase assay :-
Time Concentration OD at 600nm
0 Sec
0.2 mM 0.25
0.3 mM 0.30
0.4mM 0.32
30 Sec
0.2 mM 0.23
0.3 mM 0.28
0.4mM 0.31
60 Sec
0.2 mM 0.24
0.3 mM 0.24
0.4mM 0.28
90 Sec
0.2 mM 0.20
0.3 mM 0.22
0.4mM 0.25
120 Sec
0.2 mM 0.15
0.3 mM 0.19
0.4mM 0.22
Time Concentration OD at 600nm
0 Sec
0.2 mM 0.28
0.3 mM 0.30
0.4mM 0.54
30 Sec
0.2 mM 0.25
0.3 mM 0.28
0.4mM 0.51
60 Sec
0.2 mM 0.24
0.3 mM 0.26
0.4mM 0.48
90 Sec
0.2 mM 0.23
0.3 mM 0.24
0.4mM 0.45
120 Sec
0.2 mM 0.21
0.3 mM 0.21
0.4mM 0.41
(i) For mushroom :- (ii) For pomegranate :- (iii)For banana :-
Time Concentration OD at 600nm
0 Sec
0.2 mM 0.45
0.3 mM 0.50
0.4mM 0.65
30 Sec
0.2 mM 0.43
0.3 mM 0.49
0.4mM 0.63
60 Sec
0.2 mM 0.42
0.3 mM 0.47
0.4mM 0.60
90 Sec
0.2 mM 0.39
0.3 mM 0.45
0.4mM 0.57
120 Sec
0.2 mM 0.37
0.3 mM 0.43
0.4mM 0.55

11. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0 30 60 90 120
Absorbance
Time ( In seconds)
0.2mM Conc.
0.3mM Conc.
0.4mM Conc.
0
0.1
0.2
0.3
0.4
0.5
0.6
0 30 60 90 120
Absorbance
Time (In seconds)
0.2mM Conc.
0.3mM Conc.
0.4mM Conc.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 30 60 90 120
Absorbance
Time (In seconds)
0.2mM Conc.
0.3mM Conc.
0.4mM Conc.
Figure 1: PPO activity in mushroom with different substrate
concentration.
Figure 2: PPO activity in pomegranate with different
substrate concentration.
Figure 3: PPO activity in Banana with different substrate concentration.

12. 2. Anti-browning activity with inhibitors :-
SL
No.
Concentration Inhibitor OD
1 0.2 mM
Ascorbic acid 0.82
Citric acid 0.80
2 0.3 mM
Ascorbic acid 0.83
Citric acid 0.82
3 0.4 mM
Ascorbic acid 0.85
Citric acid 0.84
0.77
0.78
0.79
0.8
0.81
0.82
0.83
0.84
0.85
0.86
Ascorbic
acid
Ascorbic
acid
Ascorbic
acid
Citric
acid
Citric
acid
Citric
acid
0.2 0.3 0.4 0.2 0.3 0.4
Absorbance
Inhibitors and concentrations
OD
SL
No.
Concentration Inhibitor OD
1 0.2 mM
Ascorbic acid 0.59
Citric acid 0.71
2 0.3 mM
Ascorbic acid 0.41
Citric acid 0.70
3 0.4 mM
Ascorbic acid 0.40
Citric acid 0.65
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Ascorbic
acid
Ascorbic
acid
Ascorbic
acid
Citric acid Citric acid Citric acid
0.2 0.3 0.4 0.2 0.3 0.4
OD
Figure 4: Inhibition of PPO in Mushroom by ascorbic acid and citric acid.
Figure 5: Inhibition of PPO in pomegranate by ascorbic acid and citric acid.
(i) For mushroom :-
(ii) For pomegranate :-

13. SL No. Concentration Inhibitor Optical
density
1 0.2 mM
Ascorbic acid 0.68
Citric acid 0.77
2 0.3 mM
Ascorbic acid 0.65
Citric acid 0.71
3 0.4 mM
Ascorbic acid 0.63
Citric acid 0.69
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Ascorbic
acid
Ascorbic
acid
Ascorbic
acid
Citric acid Citric acid Citric acid
0.2 0.3 0.4 0.2 0.3 0.4
Absorbance
Inhibitors and concentrations
OD
Figure 6: Inhibition of PPO in Banana by ascorbic acid and citric acid.
(iii) For banana:-
Fig: Inhibition of the samples by ascorbic acid
and citric acid.

14. 3. Estimation of protein by Lowry’s method :-
Sl No
Concentration of
BSA(µg/ml)
Volume of
BSA added
(ml)
Volume of
DH2O
added
(ml)
Volume of
Reagent I
(ml)
Incubation
at
room
temperature
for
10
mins.
Volume of
Reagent II
(ml)
Incubation
at
dark
room
temperature
for
30
mins
OD at 600
nm
1 Blank 0 1
4 0.5
0
2 200 0.2 0.8 0.25
3 400 0.4 0.6 0.40
4 600 0.6 0.4 0.64
5 800 0.8 0.2 0.81
6 1000 1 0 0.92
7 Unknown (Mushroom) 0.5 0.5 0.82
8 Unknown (Banana) 0.5 0.5 0.85
9
Unknown
(Pomegranate)
0.5 0.5 0.84

15. 0.25
0.4
0.64
0.81
0.92
0.82 0.85 0.84
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Absorbance
Concentration
Protein Estimation
Fig: Protein estimation by Lowry’s method.

16. 4. Anti-oxidant activity by DPPH :-
Sl.
No
Test
samples
Volume of
enzyme
extracts
added (ml)
Volume of
DPPH
added (ml)
Optical density at
520nm
wavelength
1 Mushroom 1.6 2.4 0.18
2 Pomegranate 1.6 2.4 0.20
3 Banana 1.6 2.4 019
4 Ascorbic acid 1.6 2.4 0.12
Control
Methanol
added
DPPH
added
OD
1.6 ml 2.4ml 0.42
absorbance of control – absorbance of sample
absorbance of control
×100%
Anti-oxidant activity =
Sample Anti oxidant %
Mushroom 57.14%
Pomegranate 52.38%
Banana 54.76 %
Standard (ascorbic acid) 71.42%

17. Fig: Anti-oxidant activity using DPPH.
Figure 8: Anti-oxidant% of all the test samples.
57.14%
52.38%
54.76%
71.42%
Mushroom
Pomegranate
Banana
Ascorbic acid
Anti-oxidant %
Samples
Anti-oxidant%
Anti-oxidant%

18.  The comparative analysis of the enzyme polyphenol oxidase isolated from
different sources was studied.
 At first, PPO activity was measured and it was observed that the initial optical
density was minimal at 0.2mM concentration and maximum at 0.4mM
concentration and the optical density was seen decreasing after interval of
time.
 Adding of inhibitors (ascorbic acid and citric acid), it was observed that the
optical density(OD) remained almost constant and it showed no decreasing of
OD.
 It showed a higher protein content in Lowry’s method for all the test samples.
 Highest anti-oxidant percentage was seen in mushroom sample and the
lowest anti-oxidant percentage was observed in pomegranate sample.
Conclusion:-
Future scopes:-
 Protein identification and purification by advanced molecular techniques.
 It’s anti microbial activity.
 It's use as bio fertilizer.
 Role of the isolated proteins as therapeutic tool.

19. THANK YOU