The document characterizes phenol oxidase activity in Ataulfo mango. It finds that the enzyme has the highest activity with catechol as a substrate at pH 5.6. Inhibition studies show reducing agents and metal chelators can inhibit the enzyme's activity. Kinetic analyses determine the Michaelis constants for different substrates, with acetate buffer yielding larger constants than other buffers. The total phenol content in Ataulfo mango is also quantified.

1. Characterization of phenol oxidase activity in Ataulfo mangoCharacterization of phenol oxidase activity in Ataulfo mango
Summervir Cheema
Department of Chemistry & Biochemistry, CSU East Bay
References & AcknowledgementReferences & Acknowledgement
[1] Manthey, J. A., & Perkins-Veazie, P. (2009). Influences of harvest date and location on the levels of β-carotene, ascorbic acid, total phenols, the in vitro antioxidant capacity, and
phenolic profiles of five commercial varieties of mango (Mangifera indica L.). Journal of agricultural and food chemistry, 57(22), 10825-10830.
[2] Mayer, A. M. (2006). Polyphenol oxidases in plants and fungi: Going places? A review. Phytochemistry, 67(21), 2318-2331.
[3] Wang, J., Jiang, W., Wang, B., Liu, S., Gong, Z., & Luo, Y. (2007). Partial properties of polyphenol oxidase in mango (Mangifera indica L. CV.“Tainong”) pulp. Journal of food biochemistry,
31(1), 45-55.
[4] Constabel, C. P., Bergey, D. R., & Ryan, C. A. (1995). Systemin activates synthesis of wound-inducible tomato leaf polyphenol oxidase via the octadecanoid defense signaling pathway.
Proceedings of the National Academy of Sciences, 92(2), 407-411.
[5] Munoz, J. L., Garcia-Molina, F., Varon, R., Rodriguez-Lopez, J. N., Garcia-Canovas, F., & Tudela, J. (2006). Calculating molar absorptivities for quinones: application to the measurement
of tyrosinase activity. Analytical Biochemistry, 351(1), 128-138.
[6] Ainsworth, E. A., & Gillespie, K. M. (2007). Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin–Ciocalteu reagent. Nature Protocols, 2(4), 875-
877.
This project was funded by a Faculty Support Grant from CSUEB.
MethodsMethods
A crude extract was prepared by blending 49 g Ataulfo in 245 mL 0.1M sodium
phosphate buffer, pH 6.8 with 1 % w/v polyvinylpyrrolidone. The sample was
centrifuged for 30 minutes at 37,750 g. The phenol oxidase activity was determined by
monitoring the formation of colored quinone products from various diphenol substrates
via time-dependent absorbance measurements using a Synergy H1 plate reader.
Inhibition studyInhibition study
Polyphenol oxidase activity with the
substrate catechol was monitored in
the presence of various inhibitors.
These inhibitors include reducing
agents and metal chelators.
Polyphenol oxidase activity assays were
conducted with 20µL mango extract, in a
final assay volume of 300 µL with 60 mM
sodium phosphate buffer, pH 5.8, 30 mM
catechol, and different concentrations of
the listed inhibitors at 25°C. All
measurements were performed in
duplicate.
Enzyme kineticsEnzyme kinetics
PPO activity for all four diphenolic substrates was determined in dependence of substrate
concentration and pH. Two example graphs for catechol are shown below. We used the
Michaelis-Menten model to fit our data. Notably, data recorded with the buffer acetate
exhibits larger Michaelis constants than data obtained with any other buffer.
IntroductionIntroduction
Ataulfo mango is an excellent source of
antioxidants, notably phenolic compounds [1].
This fruit is already very popular in Mexico and
is gaining consumers’ interest in the United
States. It is grown in California and Florida.
Ataulfo is easily bruised upon harvesting and
transportation. The bruising results in browning
and limits the visual appeal and palpability of the
fruit.
The browning is initiated by the enzyme catalyzed oxidation of phenolic compounds into
highly reactive quinones which polymerize into dark colored melanin [2]. The
characterization of phenol oxidase activity in mango and other fruit is of interest for the food
and agricultural industry [3]. Phenol oxidases are involved in wound healing, pathogen
defense, and several other cellular processes, such as control of oxygen levels in chloroplasts
[2, 4]. The goal of our study was to characterize phenol oxidase activity in Ataulfo with a
focus on substrate specificity and inhibitor effectiveness.
A typical assay mixture contained 20 µL mango extract in a total reaction volume of 300 µL.
The pH was controlled by using various buffers (acetate, MES, phosphate, Tris) at 60 mM
strength. Substrate concentrations were varied between 0.2 mM and 60 mM. Two controls,
without mango extract or without substrate, were subtracted from the main assay. One Unit
(abbreviated IU) of phenol oxidase activity corresponds to the formation of one µmole
quinone per minute.
Substrate
pH
optimum
Monitored
wavelength and
molar absorptivity of
quinone product
Activity in IU
at pH optimum
Michaelis
Menten
constant (Km)
at pH optimum
Catechol 5.6
420 nm
ε = 1.25 mM-1 cm-1
2.3 IU per 20 µL
mango extract
5.9 +/- 6.6 mM
3-Methyl-
catechol
5.4
400 nm
ε = 1.22 mM-1 cm-1
3.3 IU per 20 µL
mango extract
2.19 +/-
1.25mM
Pyrogallol ~ 5.8*
320 nm
ε = 2.83 mM-1 cm-1
2.1 IU per 5 µL
mango extract
0.89 +/-
0.09mM
Gallic acid 8.8
380 nm
ε = 1.85 mM-1 cm-1
1.7 IU per 5 µL
mango extract
4.0 +/- 0.85mM
* If substrate controls were not subtracted, pH optimum would appear to be in the
alkaline region as pyrogallol auto-oxidation dominates above pH 7.0.
Phenolic contentPhenolic content
The total phenol content in Ataulfo mango was determined via a Folin-Ciocalteu assay
using gallic acid as a standard [6]. One gram of fresh mango pulp contained 0.273 mg
gallic acid equivalents.
A standard curve was prepared with a dilution series of
gallic acid in 95 % v/v methanol. The Folin-Ciocalteu
assay mixtures contained 100 µL standard or sample,
200 µL of 1:10 diluted commercially available Folin
Ciocalteu reagent (Fisher Scientific), and 800 µL 0.70 M
Na2CO3. After 2 hours of incubation, the samples were
centrifuged and 200 µL supernatant were pipetted into a
micro-plate for absorbance measurements at 765 nm,
A small piece of mango pulp (0.63 g) was frozen in
liquid nitrogen and homogenized on ice with 95% v/v
methanol. The sample was stored for 48 hours in the
dark, before starting the Folin Ciocalteu assay.
The molar absorptivity values
for the quinone products were
determined by oxidizing the
diphenolic substrates with a 20-
fold excess of sodium periodate
[5]. The slopes of these
calibration curves yield the
molar absorptivity values ε. To
calculate the PPO activity in IU
the slope (absorbance/min) is
divided by ε and multiplied by
the assay volume.
Example reaction with the
substrate catechol
Other substrates
Gallic acid
(3,4,5-trihydroxy-benzoic acid)
Pyrogallol
(1,2,3-trihydroxybenzene)
3-methyl-catechol
(1,2-dihydroxy-3-methylbenzene)