CHARACTERIZATION OF POLYPHENOLOXIDASE IN ATAULFO MANGO

Lowers the risk of chronic diseasesEasily Consumable AntioxidantRich

By: Summervir Cheema

REACTION MECHANISM OF TYROSINASE

REACTION MECHANISM OF CATECHOL OXIDASE

REACTION MECHANISM OF LACCASE

+ ½ O2

p-diphenol p-quinone

ACTIVE SITE OF PPO

METHODS AND MATERIALS

RS1 RS2 RS3 RS4 RS5 RS6

pulp skin

PPO ACTIVITY MEASUREMENTS

Substituents R

and R’

Wavelength

(nm)

Molar

absorptivity

(M-1 cm-1)

pH-

optimum

PPO activity

(IU/mL)a

Catechol R = R’ = H 420 1110b or 1225c 5.4-5.6 0.21 +/- 0.02

3-Methyl-

catechol

R = H,

R’ = CH3

400 1160b or

1420c

5.4-5.6 0.22 +/- 0.01

Pyrogallol R = H,

R’ = OH

320 3060b,c 5.8 0.50 +/- 0.05

Protocatechuic

acid

R = COOH,

R’ = H

420 1100b 6.2-6.4 0.059 +/-0.001

Gallic acid R = COOH,

R’ = OH

380 1610b 6.2-6.4 0.16 +/-0.01

aThe mango extract had a total protein content of 2.42 ± 0.30 mg/mL and an extraction ratio of

0.2 g mango per one mL of extraction buffer. The PPO activity was determined with 30 mM

substrate concentration. The value at the pH optimum is reported.

b,c The molar absorptivity value was determined in phosphate (b) or acetate (c) buffer, respectively.

Table 1: PPO activity for diphenolic and triphenolic substrates with information on molar

absorptivity of quinone-product and monitored wavelength.

MOLAR ABSORPTIVITY VALUES SLOPES

PH PROFILES OF SUBSTRATES

Fig. 1. Dependence of PPO activity on pH-value for the substrates catechol (white squares), 3-methylcatechol (grey triangles), pyrogallol (black circles), protocatechuic acid (grey diamonds), and gallic acid (white circles). For each substrate the relative activity values were normalized with the PPO activity values given in Table 1

Catechol 3-methylcatechol

Pyrogallol protocatechuic acid

Gallic acid

ENZYME KINETIC PARAMETERS

Figure B: Dependence of PPO activity on the concentration of the substrate catechol for various buffers: sodium acetate pH 5.4 and 5.6 (grey diamonds and circles), MES pH 5.6, 5.8, and 6.0 (white circles, triangles, and squares), sodium phosphate pH 5.8, 6.0, and 6.2 (black triangles, squares, and stars)

Figure C: Dependence of PPO activity on the concentration of the substrate pyrogalloll for various buffers: sodium acetate pH 5.4 and 5.6 (circles and plus symbols), MES pH 5.8 (triangles), sodium phosphate pH 5.8, 6.0, (black squares, stars and diamonds).

Dependence of PPO activity on the concentration of the substrate 3-methylcatechol for various buffers: sodium acetate pH 5.4 (sqaures symbols), MES pH 5.4 and 5.6 (diamonds and triangles).

ENZYME KINETIC PARAMETERSSubstrate Buffer KM (mM) vmax (IU/mL)

Catechol Sodium acetate, pH 5.4 14.62 ± 2.03a 0.28 ± 0.02b

Sodium acetate, pH 5.6 10.59 ± 0.78 0.29 ± 0.01

MES, pH 5.6 1.25 ± 0.14 0.24 ± 0.01

MES, pH 5.8 1.07 ± 0.11 0.22 ± 0.01

MES, pH 6.0 0.68 ± 0.16 0.19 ± 0.01

Sodium phosphate, pH 5.8 0.87 ± 0.15 0.20 ± 0.01

Sodium phosphate, pH 6.0 0.63 ± 0.20 0.19 ± 0.01

Sodium phosphate, pH 6.2 0.41 ± 0.09 0.17 ± 0.01

3-Methylcatechol Sodium acetate, pH 5.4 3.69 ± 0.51 0.24 ± 0.02

MES, pH 5.6 1.31 ± 0.18 0.26 ± 0.02

Pyrogallol Sodium acetate, pH 5.6 7.31 ± 0.77 0.53 ± 0.02

MES, pH 5.4 1.26 ± 0.32 0.48 ± 0.03

MES, pH 5.8 0.84 ± 0.09 0.46 ± 0.01

MES, pH 6.0 0.79 ± 0.09 0.43 ± 0.01

Sodium phosphate, pH 5.8 0.83 ± 0.09 0.49 ± 0.01

aData is presented as value of the fit ± error of the fit.

bThe unit conversion for vmax into IU/mL was carried out as described in the experimental section.

INHIBITOR EFFECTS ON PPO ACTIVITY

INHIBITOR EFFECTS ON PPO ACTIVITYInhibitor 20mM 2mM 0.2mM 0.02mM

β-Mercaptoethanol 0%a 0% 0% 0%

Sodium metabisulfite 0% 0% 0% 24%

Sodium azide 0% 0% 1% 47%

Sodium ascorbate 0% 0% 6% 93%

Glutathione 0% 0% 6% 102%

Kojic acid 0% 4% 40% 96%

Citric acid 0% 53% 88% 101%

EDTA 23% 27% 69% 100%

Sodium chloride 21% 46% 81% 102%

Benzoic acid 44% 101% 103% 97%

Table 3: Effect of various inhibitors on PPO activity in crude extracts of Ataulfo

a100% relative PPO activity corresponds to reaction rates that are identical to a

reference condition without inhibitor. Measurements were performed in duplicate or

triplicate with standard deviations ranging from 2% to 16%.

PPO ACTIVITY IN PULP VS. SKIN

PTOTEIN CONTENT IN PULP & SKIN

PULP SKIN

Sample Ripeness

Stage

Protein

content

(mg/g)a

PPO activity

(IU/g)a

PPO activity

with 1% SDS

(IU/g)a

SDS

enhancement

Pulp R1 2.8 ± 0.7 0.60 ± 0.05 8.99 ± 0.82 15

R2 10.6 ± 0.4 0.22 ± 0.06 8.00 ± 2.34 36

R3 20.6 ± 4.5 0.19 ± 0.05 11.06 ± 1.03 58

R4 21.4 ± 1.8 0.56 ± 0.13 13.01 ± 2.66 23

R5 10.7 ± 1.3 0.85 ± 0.20 20.74 ± 5.98 24

R6 16.7 ± 2.6 2.70 ± 1.71 20.65 ± 4.27 8

Skin R1 3.1 ± 0.3 1.12 ± 0.16 5.25 ± 0.49 5

R2 9.2 ± 0.9 3.49 ± 0.96 12.62 ± 3.64 4

R3 9.4 ± 0.7 4.71 ± 0.53 10.69 ± 0.70 2

R4 13.2 ± 0.3 8.26 ± 1.91 12.63 ± 1.76 2

R5 11.5 ± 0.6 7.23 ± 0.97 20.45 ± 2.05 3

R6 7.7 ± 0.9 14.78 ± 1.24 67.90 ± 14.77 5

a Protein content and PPO activity are reported per gram mango (skin or pulp).

Table 4: PPO activity and protein content in dependence of ripeness stage of pulp and

skin Ataulfo samples

GEL ELECTROPHORESIS

PULP SKIN

CONCLUSION Substrate dependent ph optima ranged

from 5.4-6.4. Acetate buffer –Larger Km values…... Sodium ascorbate, Glutathione, Kojic

acid-promising inhibitors PPO ACTIVITY-HIGHER in SKIN Prominent Band- 53 K Dalton…

REFERENCES S. Monika; C. Summervir. Characterization of Polyphenol oxidase activity in ataulfo mango. J. Food Chem. 2015, 171, 382-387.