CHARACTERIZATION OF POLYPHENOL OXIDASE IN ATAULFO

Transcription

C H A R A C T E R I Z AT I O N O F P O L Y P H E N O L O X I D A S E
I N AT A U L F O M A N G O
A University Thesis Presented to the Faculty
of
California State University, East Bay
In Partial Fullfillment
of the Requirements for the Degree
Master of Science in Biochemistry
By
Summervir Cheema
December, 2015
Abstract
Ataulfo mango is prone to bruising due to its soft skin. The enzyme polyphenol
oxidase (PPO) contributes to the browning of bruised fruit which lowers their appeal
and nutritional value. PPO activity in crude extracts prepared from Ataulfo mango
was characterized in this study. Crude extracts of Ataulfo exhibited PPO activity with
the substrates pyrogallol, 3-methylcatechol, catechol, gallic acid, and protocatechuic
acid. The substrate dependent pH optima ranged from pH 5.4 to 6.4 with MichaelisMenten constants between 0.84 ± 0.09 mM and 4.6 ± 0.7 mM in 2-(N-morpholino)
ethanesulfonic acid and phosphate buffers. The use of acetate buffers resulted in
larger Michaelis-Menten constants up to 14.62 ± 2.03 mM. Sodium ascorbate,
glutathione, and kojic acid were promising inhibitors to prevent enzymatic browning
in Ataulfo. PPO remains active at temperatures up to 70°C. PPO activity increased
with ripeness and was always higher in the skin compared to the pulp. Sodium
dodecyl sulfate (SDS) enhanced PPO activity with pulp showing stronger activation
than skin. SDS-PAGE gels stained for catecholase activity showed multiple bands,
but the most prominent and ubiquitous band was located at an apparent molecular
weight of 53 kDa. Bands with higher apparent molecular weights of 112 kDa and 144
kDa were detected in skin samples or pulp samples of overripe Ataulfo. Protein
content in pulp ranged from 2.8 ± 0.7 mg/g to 21.4 ± 1.8 mg/g for different ripeness
stages of Ataulfo mango. For skin samples the protein content ranged from 3.1 ± 0.3
mg/g to 13.2 ±0.3 mg/g. PPO activity in Ataulfo mango pulp varied from 8.99 ± 0.82
IU/g to 20.74 ± 5.98 IU/g, and 5.25 ± 0.49 IU/g to 67.90 ± 14.77 IU/g in pulp and
skin of differnt ripeness stages of Ataulfo mango. Overall, this study includes
ii
important information for researchers who seek to improve mango quality as PPO
plays a major role in the browning of bruised or cut mango.
iii
Acknowledgements
This project was done with the aid and help of Dr. Sommerhalter and the
Biochemistry Department of CSU East Bay. I would like to dedicate all my work to
Dr. Sommerhalter, my professors, my lab friends Tuan, Robin, Eric, Karishma,
Stephen and the whole Department of Chemistry & Biochemistry of CSU East Bay. I
especially want to thank Khanh for helping with the Folin-Ciocalteu assay. Without
all your effort this thesis would not have been possible.
“Research is to see what everybody else has seen and to think what nobody
else has thought”
-
v
Albert Szent Gyorgyi
Table of Contents
Abstract………………………………………………………………………………..ii
Acknowledgements……………………………………………………………………v
List of Figures……………………………………………………………………….viii
List of Tables………………………………………………………………………...xii
1 Introduction
1
1.1
Mango cultivars and their economic importance…………………………...1
1.2
Bioactive compounds in mango…………………………………………….5
1.3
Importance of phenolic content in Ataulfo mango………………………....6
1.4
Effect of ripening on mango fruit…………………………………………..7
1.5
Effect of storage conditions on mango fruit………………………………..8
1.6
Polyphenol oxidase………………………………………………………....8
1.7
Scope of thesis……………………………………………………………...9
2 Methods and Materials
11
2.1
Crude extract preparation………………………………………………….11
2.2
Determination of protein content………………………………………….14
2.3
PPO activity measurements……………………………………………….16
2.4
Molar extinction coefficient determination of quinones ………………….18
2.5
Protein electrophoresis…………………………………………………….19
2.6
Inhibition of PPO activity in crude extracts of Ataulfo mango……….......22
3 Results & Discussions
23
3.1
pH optima…………………………………………………………………23
3.2
Effect of substituents present in substrates on PPO activity and pH
optima in Ataulfo mango extract……………………………………………..30
3.3
Kinetic parameters of substrate concentrations variations on PPO activity….38
vi
3.4
Thermostability of PPO…………………………………………………...43
3.5
Inhibition of PPO activity in crude extracts of Ataulfo mango…………...44
3.6 Phenolic Content determination…………………………………………...56
3.7 Dependence of PPO activity on the maturity in the skin versus pulp of
Ataulfo mango..….………………………………………..……………..…...58
3.8 Apparent molecular weight of bands with PPO actvity in partially
denaturing SDS-PAGE ………………………………………..…………...…65
Conclusions
69
References
71
vii
List of Figures
Figure 1.1.1: Ataulfo mango used for research study................................................. 1
Figure 1.1.2: Other fruits & flowering plants of Anacardiacae family ....................... 1
Figure 1.1.3: Versatile mango products ..................................................................... 2
Figure 1.1.4: World ranking production of mango .................................................... 3
Figure 1.1.5: Comparison between the gross production of mangoes, mangosteen,
and guavas in India, Pakistan, and Mexico ........................................... 4
Figure 1.1.6: Popular cultivars of mango in United States of America....................... 5
Figure 2.1.1: Ripeness stages of Ataulfo mango ...................................................... 12
Figure 2.1.2: Seperation of Ataulfo mango skin and pulp at ripeness stage three ..... 12
Figure 2.1.3: Sample preparation and extraction steps ............................................. 13
Figure 2.2.1: Bradford assay showing the increasing amout of protein
concentration after binding of protein to Commassie Reagent ............ 14
Figure 2.2.2: Bradford standard curve showing linear relationship in the
concentration range of 0.1–1.0 mg/mL total protein .......................... 15
Figure 2.3.1: PPO activity assay plate showing color formation after adding
extract, & substrates catechol, pyrogallol, gallic acid, & 3- methyl
catechol………………………………………………………………..17
Figure 2.3.2: Measurement of typical PPO activity assay in a total volume of 300
µl with 30 mM catechol substrate, 20 µl of mango extract…………...17
Figure 2.5.1: Reactiom mechanism for the formation of the blue adduct between
Orthobenzoquinone and ADA ........................................................... 21
Figure 3.1.1: pH optima profile for catechol as substrate......................................... 24
viii
Figure 3.1.2: pH optima profile for pyrogallol as substrate...................................... 25
Figure 3.1.3: pH optima profile for 3- methylcatechol as substrate .......................... 26
Figure 3.1.4: pH optima profile for gallic acid as substrate...................................... 27
Figure 3.1.5: pH optima profile for 3, 4-dihydroxybenzoic acid as substrate ........... 28
Figure 3.1.6: pH optima profile for L-DOPA as substrate ................ …....................29
Figure 3.2.1: Slope yielding the molar absorptivity values for the oxidation
product of catechol.………………………………...………...……….32
product of pyrogallol………………………………………....……….33
product of 3- methylcatechol.………………………………….…..….34
product of protocatechuic acid.………………………………...……..35
product of gallic acid………..……………………………...………....36
product of L-DOPA………..……………………………...……….....37
Figure 3.3.1: Dependence of PPO activity on the concentration of the substrate
Catechol for various buffers…………………………………………..40
Pyrogallol for various buffers ............................................................ 41
3- methyl catechol for various buffers................................................ 42
Figure 3.4.1: PPO activity of crude Ataulfo extract assayed at different
ix
temperature (30˚- 90˚C) using catechol as substrate………. ........ …..43
Figure 3.5.1: Effect of different concentrations of sodium azide inhibitor on PPO
activity….……………………………………………………………..46
Figure 3.5.2: Effect of different concentrations of EDTA inhibitor on PPO
activity….……………………………………………………………..47
Figure 3.5.3: Effect of different concentrations of sodium chloride inhibitor on
PPO activity…………………………………………………………...48
Figure 3.5.4: Effect of different concentrations of glutathione inhibitor on PPO
activity .............................................................................................. 49
Figure 3.5.5: Effect of different concentrations of benzoic acid on PPO activity ..... 50
Figure 3.5.6: Effect of different concentrations of kojic acid on PPO activity.......... 51
Figure 3.5.7: Effect of different concentrations of citric acid PPO activity .............. 52
Figure 3.5.8: Effect of different concentrations of sodium ascorbate on PPO activity ................................................................................................... 53
Figure 3.5.9: Effect of different concentrations of sodium meta-bisulfite on PPO
activity .............................................................................................. 54
Figure 3.5.10: Effect of different concentrations of beta-mercapto-ethanol on PPO
activity .............................................................................................. 54
Figure 3.6.1: Gallic acid standard curve .................................................................. 56
Figure 3.7.1: Mango pulp extracts at different ripeness stages ................................. 61
Figure 3.7.2: Mango skin extracts at different ripeness stages .................................. 61
Figure 3.7.3: PPO activity of all ripeness stages of Ataulfo mango pulp .................. 62
Figure 3.7.4: PPO activity of all ripeness stages of Ataulfo mango skin .................. 62
Figure 3.7.5: Effect of SDS (1% w/v) on PPO activity of Ataulfo mango pulp
x
and skin…………. ............................................................................. 63
Figure 3.7.6: Protein content in Ataulfo mango pulp and skin ................................. 64
Figure 3.8.1: Partially denaturing 4-20% Tris-glycine SDS-PAGE gel stained for
loaded with Ataulfo pulp samples ....................................................... 67
Figure 3.8.2: Partially denaturing 4-20% Tris-glycine SDS-PAGE gel stained for
PPO activity loaded with Ataulfo skin samples ................................... 68
xi
List of Tables
Table 1: Apparent molecular weights of proteins in the See Blue pre-stained
standard on a 4-20 % Tris-glycine gel……………………………………..20
Table 2: PPO activity for diphenolic and triphenolic substrates with information
on molar absorptivity of quinone-product and monitored wavelength ....... 31
Table 3: Enzyme kinetic parameters obtained by fitting substrate variation curves .. 39
Table 4: Effect of various inhibitors on PPO activity in crude extracts of Ataulfo ... 45
Table 5: PPO activity and protein content in dependence of ripeness stage of pulp
and skin Ataulfo samples........................................................................... 60
xii
1
1 Introduction
1.1 Mango cultivars and their economic importance
Mango (Mangifera indica) belongs to
the flowering plant family commonly known
as Anacardiaceae [1, 2, 3]. Other members of
this family include cashew or poison ivy,
poison oak, marula, pistachio, jamaica plum,
yellow mombin, sumac, smoke tree, and
cuachalalate [4, 5, 6].
Figure 1.1.1 : Ataulfo Mango used
for research study
Origin: Mexico
Season: March-July
Family:Anacardiaceae
Genus: Mangifera
Figure 1.1.2: Other fruits & flowering plant of Anacardiaceae family [6].
Mango is a dicot plant, enriched in high amounts of acids and sugars
contributing to pleasant or luscious flavor and high nutritional value [7]. Organic
2
acids and sugar predominantly impact the flavor properties of mango. The sucrose
content of the mango, resulting from starch hydrolysis is the paramount cause of
chemical change and extent sweetening in the fruit [1].
Mango varieties are used to prepare traditional products such as amchur,
amsath, lassi, chutney, sharbat, vinager, pickle, and murabba (Jam) [8, 9, 10]. Mango
fruit is beneficial for acne, anemia, and hypertension problems, and also contains
glutamine which improves the memory [9, 11]. Ripened mango is further used for
medicinal purposes as diuretic and laxative. However, arcid juice present in the stalk
of mango fruit is due to myrcene and ocimene, can give rise to allergenic constituents
such as urushiol, or 5- heptadecenylresorcinol [12].
Murraba
Pickle
Chutney
Amchur
Ampapar
Lassi
Mango
kalakand
Figure 1.1.3: Versatile mango products [12].
Mango fruit can be found in tropical countries (Indo-Malaysian region) but is
available all over the world including India, China, Burma, Indonesia, Central
3
America, Florida (USA), West Indies, Africa/Arabian Peninsula, and South America
[2, 3]. Notably, Mexico is the leading export country for mango after India and China
with 1.5 million tons production as reported by the Food and Agriculture
Organization in 2002 [13].
1st India
2nd China
3rd Thailand
4th Indonesia
5th Pakistan
6th Mexico
Figure 1.1.4: World ranking production of mango. This graph was generated using
data from reference [2].
India has the highest production of mango (13.6 millions of tons) followed by
China (4.2 millions of tons), Thailand (2.5 millions of tons), Indonesia, Philippines,
Pakistan, and Mexico [2, 14, 15]. Mango fruit is available and exported (912,853
metric tonnes in 2005) throughout the year and is gaining importance as a commodity
fruit in the Western Hemisphere [1]. A total of 912,853 metric tons were exported to
the US in 2005 with a market value of $ 543.10 million [1].
4
Figure 1.1.5: Comparison between the gross production of mangoes, mangosteen, and
guavas in India, Pakistan, and Mexico FAOSTAT 2014. Reference [10].
The following cultivars are popular in the Western Hemisphere: Tommy
Atkins, Haden, Kent, Keitt (all four developed in Florida), Francis from Haiti, and
Ataulfo discovered in Mexico [16]. The mango variant Ataufo has an attractive
vibrant yellow color, flattened oval shape, sweet taste, and string-less flesh [16].
Ataulfo is one of the mango varieties which are found in the tropical rain forest, and
are produced in warm climate with moist conditions and harvested in many parts of
Mexico, California, and Florida [2].
5
Figure 1.1.6: Popular cultivars of mango in United States of America. Reference [16]
1.2 Bioactive Compounds in Mango
Mango is an excellent source of antioxidants, vitamin A, B6, B complex, C, D,
& E, phytochemicals such as phenolic compounds, carotenoids, ascorbic acid,
flavonoids, beta-carotene, niacin, pectin, and minerals, such as calcium, iron,
potassium, and magnesium [7, 14, 17]. Ataulfo mango peel is also a great source of
amino acids such as leucine, valine, and lysine [2]. Phenolic compounds are the most
significant bioactive compounds found in peels, flesh and seeds of mango fruits, and
can act as antimicrobial or antioxidant agents [18]. Citric acid is the major organic
acid among oxalic, malic, succinic, pyruvic, adipic, galacturonic, and glucuronic acid
present in mango fruit [7, 14]. Mango fruit has antimutagens and antineoplastics
which lower the risk of carcinogenesis. It was shown that mango extracts can inhibit
6
cell proliferation in the leukemic cell line HL-60 [19]. Mango phenols such as
quercetin, isoquircitrin, astragalin, fisetin, and gallic acid might have the ability to
lower the risk of carcinogenesis [11].
Ataulfo mango is great source of dietary polyphenols [20]. Ataulfo mango
flesh (or pulp) contains gallic acid, gallotannins, mangiferin, quercetin, kaempferol, phydroxy-benzoic acid, m-coumaric acid, p-coumaric acid, and ferulic acid [14].
Mangiferin, isomangiferin, xanthone C-glucosides are found in Ataulfo mango
extracts and synthesized through cyclization of benzophenones [20]. The main
phenolic compounds found in Ataulfo mango pulp were determined to be pentagalloyl
glucose, and gallic acid by HPLC [21, 22, 23].
1.3 Importance of Phenolic Content in Ataulfo Mango
Phenolic content determination in food is crucial, due to chronic diseases
arising from free radicals [24]. In the human body, free radicals modify the structures
of lipids and DNA [25]. Therefore, antioxidant rich fruit and vegetables prevent cell
damage and lower the risk of chronic diseases by inhibiting the oxidation process
generating free radicals [24]. Mango (Mangifera indica) fruits are rich in antioxidant,
and consumed easily in diet [21, 24]. Ataulfo mango pulp accommodates the highest
phenolic content (109.3 ± 14.8 mg gallic acid equivalents per 100 g puree), among the
other varieties of mango [22]. Gallic acid was determined to have the highest Radical
Scavenging Activity (RSA = 30.47 ± 1.8%) via 2,2-diphenyl-1-picrylhydrazyl
(DPPH) quenching assay in comparison with other phenols such as protocatechuic
acid, chlorogenic acid, and vanillic acid [21]. It was suggested that the meta-position
of the third hydroxyl group in gallic acid caused high antioxidant capacity [21].
7
1.4 Effects of Ripening on Mango Fruit
The ripening process has a huge impact on phenolic content, vitamin C content
and antioxidant capacity [24, 26, 27]. The main characteristic of Ataulfo mango is the
ripeness stage and the quality which highly affect the total phenolic content and
antioxidant activity of the fruit [28, 29]. The textural characteristics of mango fruit
including color change, and firmness are cultivator specific, and considerably altered
by ripeness, and the harvesting period [30]. Ataulfo mango phenolic content (174 mg
gallic acid equivilants/100 g fresh weight) and antioxidant capacity determined to be
highest at its ripeness stage with yellow surface area, followed by phenolic content of
green ripened flesh extract (prepared with 80% ethanol, or 80% acetone) of
Mangifera Indica cv. Siku Raja (70.52 ± 0.05 μg gallic acid equivilants/g sample) and
Mangifera Petandra cv. Pauh (142.57 ± 0.38 μg gallic acid equivilants/g sample) [27,
29, 31]. The unriped stage of mango has starch in great extent, which owes much of
its functionality to two major carbohydrate components known as amylose,
amylopectin, and also used in confectionary industry [3]. Starch is degraded by
amylases in the ripening process of mango, and converted into fructose, glucose, and
sucrose [32, 33]. Fructose is the predominant sugar in the mango with high sugar
content in excess of 90% of the total soluble solid content [32]. Also, acidity (pH)
plays a significant role in flavor enhancement of fruit juices and beverages [7]. During
the ripening period, the mature fruit undergoes deterioration in quality [32]. Notably,
the acidity and activity in the citrate acid cycle decrease during the maturation stage
of mango [7]. Acidity, gallic acid, gallotannins were declined due to the ripening
process with loss of astringency [14].
8
1.5 Effects of Storage Conditions on Mango Fruit
Storage conditions such as temperature and hydrostatic pressure contribute to
the quality and color change by varying total phenolic content and antioxidant
capacity [24, 26, 31]. Ataulfo mango can maintain high vitamin C levels and visual
quality for 9 days stored at 5 °C [26]. Fresh cut mango (Mangifera indica L. cv.
Ataulfo) slices stored at 5 °C, had total phenolic content under dark (9.0 ± 0.3 mg 100
g-¹ of fresh weight) and light (9.5 ± 0.2 mg 100 g-¹ of fresh weight) conditions [26,
28]. Ascorbic acid synthesis is reduced, and the initial concentration of chlorogenic
acid decreases to 38% with high hydrostatic pressure [31].
1.6 Polyphenol Oxidase
Polyphenol oxidase (PPO) is the major factor for initiating browning or
discoloration in fruit and vegetables. Browning results in a loss of nutritional value
and limits the visual appeal and palpability of the fruit [7]. PPO catalyzes the aerobic
oxidation of phenolic compounds into highly reactive quinones which polymerize into
dark colored melanin by auto-oxidation, turning the skin of the mango brown and
sometimes blackish brown [34, 35]. In some plants, PPOs also catalyze the
hydroxylation of monophenols to ortho-diphenols (monophenolase or cresolase
activity; EC 1.14.18.1) followed by the more common oxidation of ortho-diphenols to
ortho-quinones (diphenolase or catecholase activity, EC 1.10.3.1). Melanin is
produced by melanogenesis in which PPO oxidizes tyrosine into dopa-quinone which
9
further polymerize into 5,6-dihydroxyindole resulting in the formation of insoluble
polymers referred to as melanins [36]. Cross linking reactions and interactions of
quinones with the side chains of amino acids (containing - SH, -NH2) cause lack of
nutritional and functional value in food proteins [35]. PPO enzymes are ubiquitous in
seed plants (angiosperms), and are present as multiple isozymes in many plants [37].
PPO has alternative names such as tyrosinase, or catechol oxidase. PPO enzymes are
mostly localized in plastids. Their substrate specificities, and degree of inhibition vary
greatly among different plant tissues [35, 38]. The active site of PPO contains two
copper ions coordinated with three nitrogen atoms from adjacent histidine residues
[36]. PPOs are also involved in pathogen defense, and several other cellular
processes, such as control of oxygen levels in chloroplasts [34, 39]. PPO contributes
to wound healing of plants. PPO is mainly found in mango peel along with other vital
enzymes such as pectinase, protease, and xylanase [40].
1.7 Scope of thesis
The fruit has ‘majestic appeal’ to consumers all over the world but Ataulfo and
other mango varieties are easily bruised upon harvesting and transportation. When
produced in large quantities the amount of wastage is high. It is therefore valuable for
the fruit industry to identify reasons for wastage and to limit it. Studies on the enzyme
PPO extracted from mango pulp of the variety Tainong, sap and skin of the Australian
variety Kensington, and mango kernels from an African mango variety have already
been performed [41-43]. This research characterizes PPO in Ataulfo mango which is
of great interest for the food and agricultural industry. To improve handling and
processing of mango, different ways to inhibit the enzyme that catalyzes enzymatic
browning are explored. The main attention of our research was to characterize PPO
10
activity in the skin and pulp of the variety Ataulfo mango with a focus on ripeness
stage of Ataulfo, pH dependence and substrate specificity of PPO and inhibitor
effectiveness. The results of this study will help to increase the appeal of this healthy
fruit.
11
2 Methods & Materials
2.1 Crude Extract Preparation
Ataulfo mangoes were obtained from a local supermarket (Safeway in Castro
Valley, California). The fruits were stored at room temperature in a dry place in dark.
Crude extracts were prepared at six different stages of ripeness as determined by
visual and sensorial inspection. Based on the description of color and firmness,
ripeness stage R1 was very green, immature, and hard to the touch. Stage R2 was
green with yellow patches, but still hard and immature, whereas stages R3 and R4
were mature and slightly soft to the touch with increasing yellow coloration. Stages
R5 and R6 were over-ripe with increasing brown coloration. Stage R6 was very soft
to the touch. The skin and pulp of each fruit were separated from each other with a
razor blade and further cut into smaller pieces before blending three times in 30
second intervals with an extraction buffer composed of 0.1M sodium phosphate
buffer, pH 6.8 with 1% v/v Triton X-100, and 1 % w/v polyvinylpyrrolidone. The
ratio of skin or pulp weight in grams to the volume of extraction buffer in milliliters
was 1:5 with the exception of the sample R6-skin, for which a 1:10 ratio was used as
this batch resulted in a particularly dense and thick extract. To screen for best assay
conditions, another crude extract was prepared with an additional yellow, mature
Ataulfo mango (stage R3) including its skin and pulp. All extracts were clarified by
centrifugation for 30 minutes at 37,750 g and 4 ˚C. All samples were kept on ice or
stored in small aliquots at -80 °C until further use. The extract samples were tested in
the laboratory for pH optima, protein content, and the PPO activity of enzyme. The
action of polyphenol oxidase in fruits depends on the temperature, the pH value, and
the substrate which will be discussed further in this thesis.
12
Figure 2.1.1: Ripeness stages of Ataulfo mango – The visual appearance of Ataulfo
mango stored at room temperature for up to 14 days. These mangoes were bought
from safeway local grocery maket in castro valley california.
Figure 2.1.2: Seperation of Ataulfo mango skin (left), and pulp (right) at ripeness
stage three (RS3) during research study.
13
Mango
Cutting
Homogenized
Buffer
Grinding
Extract filled in
tube
Balancing
Centrifugation
Supernatent
removed
Running Assay
Micro-plate Reader
Figure 2.1.3: Sample preparation and extraction steps. These instruments and lab
equipments were used in Biochem lab, room # North 441, in C.S.U East Bay.
14
2.2 Determination of Protein Content
Total protein content was determined using the Bradford assay with a
Coomassie blue protein assay kit and bovine gamma globulin standards. The
standards and assay reagents were purchased from Thermo Scientific, and were used
in 0.125, 0.250, 0.500, 0.750, 1.000, and 1.500 mg/ml concentrations. Absorption
measurements were performed with a Synergy H1 plate reader from Biotek. The
assay was performed by mixing 250 µl of Commassie Reagent with 5 µl of sample
followed by ten minute incubation before measuring the absorbance at 595 nm.
StdCurve
1.000
0.900
0.800
595
0.700
0.600
0.500
0.400
0.300
0.000
200.000
400.000
600.000
800.000
1000.000
1200.000
1400.000
1600.000
<Concentrations/Dilutions>
Figure 2.2.1: Bradford assay showing the increasing amout of protein concentration
after binding of protein to Commassie Reagent (top). The calibration curve of the
Bradford assay (bottom) with positive slope indicates that as protein concentration
increases, absorption at 595 nm also increases. This data was observed during the
Bradford assay study.
15
To validate the method for crude extracts from Ataulfo, serial dilutions were
prepared with samples R4-pulp, R3-skin, and R4-skin. The Bradford assay provided a
linear response in the concentration range of 0.1–1.0 mg/mL total protein with
correlation coefficients of 0.95 or higher. All samples were diluted with deionized
water so that their protein content fell within this linear range.
1.4
R3 skin
R4 pulp
R4 skin
y = 0.0002x + 0.4198 y = 0.0003x + 0.4217 y = 0.0003x + 0.4051
R² = 0.9248
R² = 0.9444
R² = 0.9694
Absorbance at 595 nm
1.2
1
0.8
0.6
0.4
0.2
0
0
500
1000
Protein content (μg/mL)
1500
2000
Figure 2.2.2: Bradford standard curve showing linear relationship in the concentration
range of 0.1-1.0 mg/mL total protein.
16
2.3 PPO Activity Measurements
The phenol oxidase activity was determined by monitoring the formation of
colored quinone products from various di- and tri-phenol substrates via timedependent absorbance measurements using a Synergy H1 plate reader from Biotek.
The temperature was set to 25 ˚C. A typical assay mixture contained 5-20 µL mango
extract in a total reaction volume of 300 µL. The pH was controlled by using various
buffers at 60-68 mM strength in the assay. Sodium acetate was used to cover the pH
range 3.8 - 5.6. Sodium phosphate and 2- (N-morpholino) ethanesulfonic acid (MES)
were employed for the pH ranges 5.6 - 7.8 and 5.2 - 6.6, respectively. Tris-HCl was
used to cover the pH range 7.2 - 9.0. Substrate concentrations were varied between
0.2 mM and 30 mM to determine the enzyme kinetic parameter Km. Substrate
dependent PPO activity curves were fit to the Michaelis-Menten equation using the
program Enzfitter from Biosoft. In experiments to determine pH optima and substrate
specificity or inhibitor effectiveness, the final substrate concentration in the assay
mixture was 30 mM. To determine the effectiveness of various inhibitors the PPO
activity, assays were conducted in 60 mM sodium phosphate buffer, pH 5.8 with 30
mM catechol and different concentrations of inhibitors in the range 20 mM to 0.02
mM. Two controls, without mango extract or without substrate, were subtracted from
all main assays. These two control reactions were based on 30 min time dependent
absorbance measurements measured at 1 min time intervals.
17
Figure 2.3.1: PPO activity assay plate showing color formation after adding extract,
and substrates catechol (yellow color, first row), pyrogallol (dark yellow color,
second row), gallic acid (green color, third row), and 3- methyl catechol (pink color,
fourth row). Reference - Table 2.
Assay
Mango extract control
Catechol control
0.4
0.3
0.2
0.1
0
0
5
10
Time (minutes)
15
20
25
Figure 2.3.2: Measurement of typical PPO activity assay in a total volume of 300 uL
with 30 mM catechol substrate, 20 uL of mango extract. Two controls are also shown,
one without mango extract and the other without catechol. Reference -Table 2.
18
2.4 Molar Extinction Coefficient Determination of Quinones
Quinones are formed from phenols by an oxidation reaction catalyzed by PPO.
Molar absorptivity is a measure of the amount of light absorbed per unit
concentration, and per pathlength. A compound with a high molar absorptivity is very
effective at absorbing light (of the appropriate wavelength), and hence low
concentrations of a compound with high molar absorptivity can be easily detected.
The molar absorptivity values (ε) for the quinone products were determined by
oxidizing the diphenolic substrates with a 20-fold excess of sodium periodate as
described in reference [44]. The experiment was conducted by making dilution series
of 10 mM stock solution of diphenolic substrates catechol in different buffers such as
0.1 M of sodium phosphate, sodium acetate, MES, and TRIS-HCl with in pH range of
3.8-8.8. Sodium periodate stock solution at a cncentration of 500 mM was prepared
by adding 2.673 g of sodium periodate in 25 mL of deionized water. The typical assay
had subtrate, buffer, and sodium periodate in a total volume of 1000 µL of the assay.
Absorbance readings with different substrate concentrations at 1 mM, 0.75 mM, 0.5
mM ,0.25 mM, 0.1 mM were performed and recorded at wavelength of 400, 420, 320,
and 380 nm. Under these conditions, the reaction was instantaneous and the molar
absorptivity values were determined reliably. The data was fitted to Beer-Lambert
law: 𝑨 = 𝜺 ∗ 𝒄 ∗ 𝒍
Where A is the absorbance at a given wavelength, ε is the molar absorptivity
(M-1 cm-1), c is the concentration in (M), and l is the pathlength of light through the
cuvette (1 cm). To calculate the PPO activity in IU the slope (absorbance/min) is
divided by the molar absorptivity, ε, and a path length correction factor for the plate
19
reader and multiplied by the assay volume. One Unit (abbreviated IU) of PPO activity
corresponds to the formation of one µmole quinone per minute.
2.5 Protein Electrophoresis
Protein electrophoresis is a method for qualitative analysis of the proteins in a
fluid or an extract. It is a technique to find information about the molecular weight
and charges of proteins. Skin and pulp Ataulfo samples were prepared under partially
denaturing conditions by mixing them with an equal volume of loading buffer
composed of 1% w/v SDS, 20% v/v glycerol, and 100 mM TRIS-HCl, pH 6.8. The
samples were not heated or reduced. Tris-glycine gels with a 4 - 20% acrylamide
gradient and the SeeBlue pre-stained protein standard (Table 1) were procured from
Lifescience Technology. The gels were run in a cold room at 4 ˚C and 125 mV
constant voltage for approximately 1.5 hours in a running buffer composed of 3.02 g
TRIS-base, 18.8 g glycine, and 1 g SDS per liter distilled water. The gels were stained
for PPO activity according to the procedure listed in reference [45] with the exception
of replacing the tertiary butyl-catechol reagent for catechol. The method was fast and
specific and was based on a coupling reaction between o-benzoquinone and the
aromatic amine, 4-amino N, N-diethylaniline (ADA). Catecholase activity of
polyphenol oxidase appears as blue stained bands on a colourless background.
Pictures of gels were recorded with a Chemi Doc MP Imaging System from BioRad.
The determination of apparent molecular weights was performed with the software
Image Lab from BioRad.
20
Table 1: Apparent molecular weight of See Blue pre-stained standard on a 4-20%
Tris-Glysine gel. Bands color and molecular weights were obtained from the protein
kit of see - blue pre stained standard.
BANDS
PROTEIN
MOLECULAR Weight
( K da)
Myosin
200
Phosphorylase
148
BSA
98
Glutamic dehydrogenase
64
Alcohol dehydrogenase
50
Carbonic anhydrase
36
Myoglobin
30
Lysozyme
16
Aprotinin
6
Insulin B-chain
4
21
Figure 2.5.1: Reaction mechanism for the formation of blue adduct between orthobenzoquinone and ADA. (A) Oxidation of catechol to o-benzoquinone catalysed by
PPO. (B) The condensation reaction leading to the Schiff-base adduct. Reference - [4]
22
2.6 Inhibition of PPO Activity in Crude Extracts of Ataulfo Mango
The characterization of PPO inhibition is a major goal of this study as
inhibitors are essential to control food browning. In previous studies, preservatives
such as potassium metabisulfite (PMS), sodium benzoate, and citric acid were used
for the reduction of browning casued by PPO [7]. In our study, PPO activity with the
substrate catechol was monitored in the presence of various inhibitors including
reducing agents, acidulants and chelators. Polyphenol oxidase activity assays were
conducted with 20 µL 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. Listed
below are all types of inhibitors used in this study.
Sodium metabisulfite, Glutathione,
REDUCING AGENTS
Ascorbic acid, Beta-mercaptoethanol,
Sodium ascorbate
Sodium azide, Benzoic acid, EDTA,
CHELATORS
Kojic acid
ACIDULANTS
Citric acid, Ascorbic acid
ELECTROLYTES
Sodium chloride
23
3 Results & Discussion
3.1 pH Optima
PPO activity is dependent on the type of substrates, buffers and their pH
conditions used in the assay. The pH optimum for PPO activity with catechol as a
substrate in the crude extract of Ataulfo mango was determined to be 5.6 (Fig. 3.1.1),
which is close to the pH optimum of 6.5 observed for PPO activity in cashew apple
[5]. PPO activity is maximum at neutral pH (7) for marula fruit and for mango pulp in
another study [4, 41]. A buffer functions to resist changes in hydrogen ion
concentration, and may provide essential cofactors for enzymatically driven reactions.
All buffers have an optimal pH range over which they are able to moderate changes in
hydrogen ion concentration. The optimum pH which was examined with the usage of
buffer 0.1 M sodium acetate (pH 3.8 to 5.6), 0.1 M MES (pH 5.2-6.6), 0.1 M sodium
phosphate (pH 5.8-7.8), and 0.1 M Tris (pH 7-9) at 25 ˚C. Control assays without
mango extract and substrate were performed and subtracted from the presented data.
All measurements were performed in triplicate and duplicate. Each value represents
the mean of triplicate/duplicate, and error bars represented as standard deviations.
Other diphenolic or triphenolic substrates such as 3-methyl-catechol, gallic acid,
pyrogallol, L-DOPA, and protocatechuic acid were tested for pH optima, and their
profiles are shown below.
24
9
8
7
Sodium acetate
buffer
mOD/min at 420 nm
6
MES buffer
5
Sodium phosphate
buffer
4
3
2
1
0
3
-1
5
7
9
pH value
Figure 3.1.1: pH optima profile for catechol as substrate. The typical assay had 30 µL
of mango extract, buffers at 60 mM, and substrate with 30 mM final concentration in
a total reaction volume of 300 µL. Reference - Table 2.
25
25
20
mOD/min at 320 nm
15
Sodium acetate buffer
10
MES buffer
Sodium phosphate
buffer
5
0
3
-5
4
5
6
7
8
pH value
Figure 3.1.2: pH optima profile for pyrogallol as substrate. The typical assay had 5 µL
26
16
14
mOD/min at 400 nm
12
10
8
Sodium phosphate buffer
6
MES buffer
4
2
0
3
4
5
6
7
8
pH values
Figure 3.1.3: pH optima profile for 3-methylcatechol as substrate. The typical assay
had 20 µL of mango extract, buffers at 63 mM, and substrate with 30 mM final
concentration in a total reaction volume of 300 µL. Reference - Table 2.
27
4
3.5
3
mOD/min at 380 nm
2.5
Sodium phosphate
buffer
2
TRIS buffer
1.5
1
0.5
0
3
4
5
6
7
8
-0.5
pH values
Figure 3.1.4: pH optima profile for gallic acid as substrate. The typical assay had 5 µL
28
4.5
4
3.5
mOD/min at 420 nm
3
2.5
2
MES buffer
1.5
TRIS buffer
1
0.5
0
3
-0.5
5
7
9
pH values
Figure 3.1.5: pH optima profile for 3, 4-dihydroxybenzoic acid as substrate. The
typical assay had 20 µL of mango extract, buffers at 63 mM, and substrate with 30
mM final concentration in a total reaction volume of 300 µL. Reference - Table 2.
29
3
2.5
MES buffer
mOD /min at 475 nm
2
Sodium phosphate
buffer
TRIS buffer
1.5
1
0.5
0
2
3
4
5
pH values
6
7
8
Figure 3.1.6: pH optima profile for L-DOPA as substrate. The typical assay had 20 µL
30
3.2 Effect of substituents present in substrates on PPO activity and pH optima in
Ataulfo mango extract
The most commonly used substrates to assess PPO activity in fruit or
vegetables are phenolic compounds that derive from catechol. As summarized in
Table 2 we employed catechol, 3-methylcatechol, pyrogallol, protocatechuic acid, and
gallic acid in this study. The tri-phenolic compound pyrogallol exhibited the highest
PPO activity, whereas substrates carrying a carboxylic acid group displayed lower
PPO activity than catechol. Catechol and 3-methylcatechol showed similar reactivity
and the same pH optimum at 5.4 - 5.6. The presence of an additional hydroxyl group
or a carboxylic acid group raised the pH optimum of the PPO catalyzed reaction. At
pH values above 7.0, control assays with the triphenolic compounds pyrogallol and
gallic acid showed a high auto-oxidation rate in the absence of mango extract,
approaching and almost exceeding the rate of the enzyme catalyzed reaction. For all
other conditions control assays showed rates that were two orders of magnitude
smaller than the main catalyzed reaction. Plots of slopes yielding the molar
absorptivity values of each sbstrate are also shown below.
31
Table 2: PPO activity for diphenolic and triphenolic substrates with information on
molar absorptivity of quinone-product and monitored wavelength.
Substituents R
Wavelength
Molar
pH-
PPO activity
and R’
(nm)
absorptivity
optimum
(IU/mL)a
(M-1 cm-1)
Catechol
R = R’ = H
420
1110b or 1225c
5.4-5.6
0.21 +/- 0.02
3-Methyl-
R = H,
400
1160b or 1420c
5.4-5.6
0.22 +/- 0.01
320
3060b,c
5.8
0.50 +/- 0.05
420
1100b
6.2-6.4
0.059 +/-0.001
380
1610b
6.2-6.4
0.16 +/-0.01
catechol
Pyrogallol
R’ = CH3
R = H,
R’ = OH
Protocatechuic
acid
Gallic acid
R = COOH,
R’ = H
R = COOH,
R’ = OH
a
The 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.
32
1.6
y = 1.3562x
R² = 0.9209
y = 1.2503x
R² = 0.9852
1.4
y = 0.8666x
R² = 0.9745
1.2
Sodium acetate
buffer pH 5.4
1
Sodium acetate
buffer pH 5.6
0.8
0.6
Sodium phosphate
buffer pH 5.8
0.4
0.2
0
0
0.2
0.4
0.6
0.8
catechol (mM)
1
1.2
Figure 3.2.1: Slope yielding the molar absorbptivity values for the oxidation product
of catechol. The typical assay had 0-1.5 mM concentration of catechol, 0-20 mM of
sodium periodate, 86-100 mM buffer strength in a total volume of 1000 µL.
Reference - Table 2.
33
3.5
y = 3.015x
R² = 0.8947
3
y = 3.0867x
R² = 0.8947
y = 2.8383x
R² = 0.9743
2.5
2
MES buffer pH 5.8
1.5
pH 6
1
Sodium acetate buffer pH
5.6
0.5
0
0
0.2
0.4
0.6
0.8
pyrogallol (mM)
1
1.2
of pyrogallol. The typical assay had 0-1.5 mM concentration of pyrogallol, 0-20 mM
of sodium periodate, 86-100 mM buffer strength in a total volume of 1000 µL.
34
1.6
y = 1.4367x
R² = 0.9213
1.4
y = 1.2022x
R² = 0.9748
y = 1.4175x
R² = 0.9896
1.2
1
0.8
Sodium acetate pH 5.4
0.6
Sodium phosphate pH 5.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
3 methyl catechol (mM)
1
1.2
of 3-methylcatechol. The typical assay had 0-1.5 mM concentration of 3 methyl
catechol, 0-20 mM of sodium periodate, 86-100 mM buffer strength in a total volume
of 1000 µL. Reference - Table 2.
35
5
4.5
y = 10.494x
R² = 0.9986
y = 8.9228x
R² = 0.9806
y = 8.8393x
R² = 0.9943
y = 0.9786x
R² = 0.939
4
3.5
Sodium phosphate pH
5.8
3
2.5
2
1.5
1
0.5
0
0
0.2
0.4
0.6
0.8
protocatechuic acid (mM)
1
1.2
of protocatechuic acid. The typical assay had 0-1.5 mM concentration of
protocatechuic acid, 0-20 mM of sodium periodate, 86-100 mM buffer strength in a
total volume of 1000 µL. Reference - Table 2.
36
4
y = 3.5893x
R² = 0.9439
y = 3.2958x
R² = 0.9649
y = 0.9318x
R² = 0.988
3.5
3
2.5
2
1.5
1
0.5
0
0
0.5
1
1.5
gallic acid (mM)
of gallic acid. The typical assay had 0-1.5 mM concentration of gallic acid, 0-20 mM
of sodium periodate, 86-100 mM buffer strength in a total volume of 1000 µL.
37
4.5
y = 3.5428x
R² = 0.9229
y = 3.9002x
R² = 0.9564
4
3.5
3
2.5
2
1.5
1
0.5
0
0
0.5
1
L-DOPA (mM)
1.5
of L-DOPA. The typical assay had 0-1.5 mM concentration of L-DOPA, 0-20 mM of
sodium periodate, 86-100 mM buffer strength in a total volume of 1000 µL.
38
3.3 Kinetic Parameters of Substrate Concentrations Variations on PPO Activity
PPO activity assays with varied substrate concentrations exhibited a typical
Michaelis-Menten profile in the concentration range of 0.2 to 30 mM. Higher
substrate concentrations often resulted in the formation of opaque solutions or
precipitation. This might be due to subsequent reactions involving the highly reactive
quinone products formed in the activity assay. The Michaelis-Menten parameters are
1.25 ± 0.14 mM, 1.31 ± 0.18 mM, 0.84 ± 0.09 mM, 4.6 ± 0.7 mM, for the substrates
catechol, 3-methylcatechol, pyrogallol, and gallic acid, determined at their respective
optimum pH-value using MES or sodium phosphate buffers. For the substrates
catechol, 3-methylcatechol, and pyrogallol we recorded Michaelis-Menten profiles at
different pH values in acetate, MES, and sodium phosphate buffers. Related graphic
parameters are shown in Figure 3.3.1 for the substrate catechol, Figure 3.3.2 for
pyrogallol, Figure 3.3.3 for 3-methylcatechol. All determined enzyme kinetic
parameters are summarized in Table 3. We noticed that all data recorded with acetate
buffers exhibited a pronounced shift to larger Km values.
39
Table 3: Enzyme kinetic parameters obtained by fitting substrate variation curves.
An example for catechol is shown in Figure 3.3.1.
Substrate
Buffer
KM (mM)
vmax (IU/mL)
Catechol
Sodium acetate, pH 5.4
14.62 ± 2.03a
0.28 ± 0.02b
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
0.63 ± 0.20
0.19 ± 0.01
0.41 ± 0.09
0.17 ± 0.01
3.69 ± 0.51
0.24 ± 0.02
MES, pH 5.6
1.31 ± 0.18
0.26 ± 0.02
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
0.83 ± 0.09
0.49 ± 0.01
3-Methylcatechol
Pyrogallol
a
Data is presented as value of the fit ± error of the fit.
b
The unit conversion for vmax into IU/mL was carried out as described in the
experimental section.
40
8
7
mOD/min at 420 nm
6
5
4
5.4
5.6
MES buffer pH 5.6
3
MES buffer pH 5.8
2
MES buffer pH 6
Sodium phosphate buffer pH
5.8
1
0
0
5
10
15
catechol (mM)
20
25
30
catechol for various buffers: sodium acetate pH 5.4 and 5.6, MES pH 5.6, 5.8, and
6.0, sodium phosphate pH 5.8, 6.0, and 6.2. The fitting parameters are summarized in
Table 3. Each data set contains double measurements with controls subtracted.
Parameters include 300 µL total assay volume, with 20 µL of mango extract,
catechol concentration ranging from 3 to 30 mM. Reference - Table 3.
41
90
80
70
mOD/min at 320 nm
60
50
Sodium phosphate buffer pH 5.8
40
Sodium phosphate buffer pH 5.8
30
Sodium acetate buffer pH 5.6
20
Sodium phosphate buffer pH 6
10
MES buffer pH 5.8
0
0
10
20
pyrogallol (mM)
30
40
pyrogalloll for various buffers: sodium acetate pH 5.4 and 5.6, MES pH 5.8, sodium
phosphate pH 5.8, 6.0. The fitting parameters are summarized in Table 3. Each data
set contains double measurements with controls subtracted. Parameters include 300
µL total assay volume, with 20 µL of mango extract, catechol concentration ranging
from 0.2 to 50 mM. Reference - Table 3.
42
20
18
16
mOD/min at 400 nm
14
12
10
8
6
4
MES buffer pH 5.6
2
MES buffer pH 5.4
0
0
20
40
60
3 methyl catechol (mM)
80
100
Figure 3.3.3: Dependence of PPO activity on the concentration of the substrate 3methylcatechol for various buffers: sodium acetate pH 5.4, MES pH 5.4 and 5.6. The
fitting parameters are summarized in Table 3. Each data set contains double
measurements with controls subtracted. Parameters include 300 µL total assay
volume, with 20 µL of mango extract, catechol concentration ranging from 0.1 to 100
mM. Reference - Table 3.
43
3.4 Thermostability of PPO
To investigate the temperature stability of PPO, crude Ataulfo extracts were
placed for a duration of 10, 20, 30, 40, 50 and 60 minutes into a water bath set to
either 30 °C, 50 °C, or 70 °C. The crude extracts were cooled in an ice water bath and
let recover to room temperature before resuming with PPO activity measurements.
More than 30 minutes at 70 °C were necessary to reduce the PPO activity by 50%.
PPO activity in crude extracts prepared from the skin of the mango variety
Kensington also revealed high thermostability [46]. Heating is therefore not a valid
treatment to prevent the browning of Ataulfo and Kensington mango cultivars. The
other alternative ways used to control enzymatic browning is the use of physical
treatments such as freezing, refrigeration, dehydration and high pressure [35].
35
mOD/min at 420nm
30
25
20
30 °C
15
50 °C
10
70 °C
5
90 °C
0
-5 0
10
20
30
40
50
Incubation time (min)
60
70
Figure 3.4.1: PPO activity of crude Ataulfo extract assayed at different temperatures
(30-90 °C) using catechol as substrate. Reference - [46] .
44
3.5 Inhibition of PPO activity in crude extracts of Ataulfo mango
Inhibition can act in three ways, i.e., directly on the enzyme, by removing the
substrate like oxygen or phenolic compounds, or by changing the product
composition. Reducing agents are known for the reduction of disulfide bonds within
proteins and peptides. Their mode of action can also inactivate the enzyme activity by
removing oxygen present at the active site or by reducing the metal ion cofactors in
the active site. Chelating agents are known for removal of metal ions, and acidulants
for reducing pH.
Compounds with carboxylic acid groups, such as citric acid and malic acid
have been shown to inhibit PPO in other fruit [35]. Our screen for PPO inhibitors (see
Table 4) also showed that citric acid and to a much lesser extent, benzoic acid are
potential agents to prevent enzymatic browning of Ataulfo. This inhibitory effect
might simply be due to the lowering of the pH value (acidulating agents) or a metalchelating effect for inhibitors with multiple carboxylic acid groups.
According to the data shown in Table 4, the thiol-containing compound betamercaptoethanol and the copper binding ligand azide are highly effective, but also
very toxic [47]. It has been shown in previous studies that SH or thiol containing
compound are strong inhibitors of enzyme PPO in fruits and vegetables [48]. Sodium
metabisulfite is also very effective, but this compound was banned as a food additive
by the FDA in 1995 [49]. Commercially available anti-browning mixtures often
contain ascorbic and citric acid [50]. Sodium ascorbate was a more effective inhibitor
compared to citric acid in their individual application. Synergistic effects were not
investigated in this study. Glutathione (another thiol-containing compound) and kojic
acid show potential as useful inhibitors. Inhibition of PPO activity was also observed
45
for EDTA, sodium chloride, and benzoic acid albeit at higher concentrations
compared to any of the other compound. Effects of various inhibitors on PPO activity
are shown below in bar graphs and summarized in Table 4.
Table 4: Effect of various inhibitors on PPO activity in crude extracts of Ataulfo
Inhibitor
20 mM
2 mM
0.2 mM
0.02 mM
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%
a
100% 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%.
46
4
sodium azide 20 mM
3.5
catechol oxidase activity mOD @ 420 nm/min
sodium azide 2 mM
3
2.5
2
sodium azide 0.2 mM
sodium azide 0.02 mM
1.5
1
0.5
0
-0.5
sodium azide concentrations
Figure 3.5.1: Effect of different concentrations of sodium azide on (PPO) activity.
Polyphenol oxidase activity assays were conducted with 20 µL 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 inhibitor at 25 °C. All measurements
were performed in triplicate or duplicate. Error bars represent standard deviations.
47
7
EDTA 20 mM
6
EDTA 2 mM
5
EDTA 0.2 mM
4
EDTA 0.02 mM
3
2
1
0
-1
EDTA concentrations
Figure 3.5.2: Effect of different concentration of EDTA on (PPO) activity. Polyphenol
oxidase activity assays were conducted with 20 µL 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 inhibitor at 25 °C. All measurements were
performed in triplicate or duplicate. Error bars represent standard deviations.
48
6
sodium chloride 20 mM
5
sodium chloride 2 mM
4
sodium chloride 0.2 mM
sodium chloride 0.02 mM
3
2
1
0
sodium chloride concentrations
Figure 3.5.3: Effect of different concentrations of sodium chloride on (PPO) activity.
49
6
glutathione 20 mM
5
glutathione 2 mM
4
glutathione 0.2 mM
3
glutathione 0.02 mM
2
1
0
glutathione concentrations
Figure 3.5.4: Effect of different concentrations of glutathione on (PPO) activity.
50
4
benzoic acid 0.2 mM
3.5
benzoic acid 2 mM
3
benzoic acid 0.2 mM
2.5
benzoic acid 0.02 mM
2
1.5
1
0.5
0
benzoic acid concentrations
Figure 3.5.5: Effect of differnt concentrations of benzoic acid on (PPO) activity.
51
6
kojic acid 20 mM
5
kojic acid 2 mM
kojic acid 0.2 mM
4
kojic acid 0.02 mM
3
2
1
0
kojic acid concentrations
Figure 3.5.6: Effect of different concentrations of kojic acid on (PPO) activity.
52
6
citric acid 20 mM
5
citric acid 2 mM
citric acid 0.2 mM
4
citric acid 0.02 mM
3
2
1
0
-1
citric acid concentrations
Figure 3.5.7: Effect of differnt concentration of citric acid on (PPO) activity.
53
7.5
sodium ascorbate 20 mM
6.5
sodium ascorbate 2 mM
5.5
sodium ascorbate 0.2 mM
4.5
sodium ascorbate 0.02 mM
3.5
2.5
1.5
0.5
-0.5
sodium ascorbate concentrations
Figure 3.5.8: Effect of different concentrations of sodium ascorbate on (PPO) activity.
54
3
sodium metabisulfite 20 mM
2.5
sodium metabisulfite 2 mM
2
sodium metabisulfite 0.2 mM
1.5
sodium metabisulfite 0.02 mM
1
0.5
0
-0.5
-1
sodium metabisulfite concentrations
Figure 3.5.9: Effect of differnet concentrations of sodium metabisulfite on (PPO)
activity. Polyphenol oxidase activity assays were conducted with 20 µL 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 inhibitor at 25 °C. All
measurements were performed in triplicate or duplicate. Error bars represent standard
deviations. Reference - Table 4.
55
2
beta-mercaptoethanol 20 mM
1.5
beta-mercaptoethanol 2 mM
beta-mercaptoethanol 0.2 mM
1
beta-mercaptoethanol 0.02 mM
0.5
0
-0.5
-1
beta-mercaptoethanol concentrations
Figure 3.5.10: Effect of different concentrations of beta-mercaptoethanol on (PPO)
activity. Polyphenol oxidase activity assays were conducted with 20 µL 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 inhibitor at 25 °C. All
measurements were performed in triplicate or duplicate. Error bars represent standard
deviations. Reference - Table 4.
56
3.6 Phenolic Content determination
The total phenol content in Ataulfo mango was determined via a FolinCiocalteu assay using gallic acid as a standard [17]. One gram of fresh mango pulp
contained 0.273 mg gallic acid equivalents. A small piece of mango pulp (0.63 g) was
frozen in liquid nitrogen and homogenized on ice with 95% v/v aqueous methanol.
The sample was stored for 48 hours in the dark, before starting the Folin Ciocalteu
assay.
0.8
0.7
y = 0.7592x
R² = 0.9936
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.25
0.5
gallic acid (mM)
0.75
1
Figure 3.6.1: Gallic Acid Standard Curve. Reference - [17].
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 sodium carbonate. After 2 hours of incubation, the
57
samples were centrifuged and 200 µL supernatant were pipetted into a micro-plate for
absorbance measurements at 765 nm.
Gallic acid has been listed as the main phenolic compound in mango fruit [51].
According to recent studies on mango fruit, it has been demonstrated that chlorogenic
acid levels can exceed gallic acid levels as the fruit ripens [52]. For ripeness stage
RS4 chlorogenic acid content was 301 mg, gallic acid 98.7 mg, vanillic acid 24.4 mg,
and protocatechuic acid 1.1 mg per 100g dry weight of Ataulfo [2, 52]. Mango pulp is
known to be acidic with pH values at or below pH 4 [53, 54]. The major organic acids
present in the pulp of mangoes are citric acid and malic acid [55]. At such low pH
values, gallic acid and protocatechuic acid are very poor substrates for PPO. Also, as
long as the cell organelles are still intact, most of the phenolic compounds will be
enclosed in a vacuole separate from the sub-cellular location of PPO [56]. PPO
initiated browning with internal substrates such as gallic acid will only start to matter
as the integrity of cellular compartments is destroyed, oxygen is available, and the
local pH is not too acidic.
58
3.7 Dependence of PPO activity on the maturity in the skin versus the pulp of
Ataulfo mango
We separated skin and pulp of our mango samples from the Ataulfo variety as
shown in Figure 3.7.1 & 3.7.2. All samples (see Table 5) showed higher PPO activity
per gram skin than per gram pulp. These differences ranged from 5-fold to 25-fold.
Sample R1 was an exception with only a 2-fold difference between the PPO activities
of pulp and skin. This sample from the most green and unripe Ataulfo also showed the
lowest protein content for pulp and skin Figure 3.7.6 and the sample R1-pulp
exhibited a surprisingly high activity of 0.60 ± 0.05 IU/g. All other samples tend to
show an increase in activity with maturation as shown in Figure 3.7.3 and 3.7.4. The
most overripe Ataulfo (R6) clearly displayed the highest PPO activities in pulp and
skin. In agreement with our observations, studies on Kensington variety of mango
also obseved a significant difference in PPO activity for skin and pulp samples of a
ripe mango with 25-fold higher activity for the skin compared to the pulp sample [42].
No PPO activity was detected in the pulp of unripe Kensington. The skin of unripe
Kensington displayed 2.5-fold reduced activity compared to the ripe Kensington skin
sample [35]. As fruit ripen and soften, several biochemical processes change [57]. It is
not uncommon for fruit to show higher activity for specific enzymes in more mature
stages. For example the activity of the enzyme β-galactosidase increases during the
ripening of mango [58]. Antioxidant capacity, quantity of phenolic compounds, and
respiration were also shown to increase during the first three stages of Ataulfo
maturation followed by a slight decrease in the later stages of ripeness [29].
Many researchers observed that PPO activity can be enhanced by adding the
anionic detergent SDS. The degree of SDS activation varies greatly with plant
59
material and experimental conditions. In a study review, a range of 4-fold to 119-fold
increase in PPO activity has been shown upon SDS addition [35]. It is conceivable
that PPO is present in latent form with a regulatory domain blocking the catalytic site
of PPO, which becomes more accessible after SDS addition, acidification, or
proteolytic treatment [35]. Notably, our data in Table 5 showed much higher SDS
activation for pulp compared to skin samples. In fact, with an addition of 1% w/v
SDS, most pulp samples approach or exceed the activity values of skin samples at the
same ripeness stage as shown in (Figure 3.7.5). We tentatively suggest a difference in
latency for PPO in the pulp versus the skin of Ataulfo.
60
Table 5: PPO activity and protein content in dependence of ripeness stage of pulp and
skin Ataulfo samples
Sample Ripeness
Stage
Protein
PPO activity
PPO activity SDS
content
(IU/g)a
with 1%
(mg/g)a
Pulp
Skin
enhancement
SDS (IU/g)a
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
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 ±
5
14.77
a
Protein content and PPO activity are reported per gram mango (skin or pulp). All
measurements were performed in triplicate. Data is presented as mean ± standard
deviation.
61
Figure 3.7.1: Mango pulp extracts at different ripeness stages
Figure 3.7.2: Mango skin extracts at different ripeness stages
PPO activity in IU/g
62
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
0.00
green pulp 1
green pulp 2
yellow pulp1
yellow pulp2
brown pulp1
brown pulp 2
Ripeness stage of mango pulp
Figure 3.7.3: PPO activity of all ripeness stages of Ataulfo mango pulp. PPO activity
is reported per gram mango pulp. All measurements were performed in triplicate at 25
°C. In a total vol of 300 µL of activity assay, 190 µL of 0.1 M sodium phosphate
buffer pH 6.8, 30 mM catechol, 20 µL of each type of extract were added. Data is
presented as mean ± standard deviation. Reference - Table 5.
18.00
PPO activity in IU/g
16.00
14.00
green skin 1
12.00
grren skin 2
10.00
yellow skin 1
8.00
yellow skin 2
6.00
brown skin 1
4.00
brown skin 2
2.00
0.00
Ripeness stage of mango skin
Figure 3.7.4: PPO activity of all ripeness stages of Ataulfo mango skin. PPO activity
is reported per gram mango skin. All measurements were performed in triplicate at 25
°C. In a total vol of 300 µL of activity assay, 190 µL of 0.1 M sodium phosphate
buffer pH 6.8, 30 mM catechol, 20 µL of each type of extract were added. Data is
presented as mean ± standard deviation. Reference - Table 5.
63
PPO activity with 1% SDS
(IU/g)
30.00
25.00
green pulp 1
20.00
green pulp 2
yellow pulp 1
15.00
yellow pulp 2
10.00
brown pulp 1
brown pulp 2
5.00
0.00
Ripeness stage of mango pulp
PPO activity with 1 % SDS
(IU/g)
90.00
80.00
70.00
green skin 1
60.00
green skin 2
50.00
yellow skin 1
40.00
yellow skin 2
30.00
brown skin 1
20.00
brown skin 2
10.00
0.00
Figure 3.7.5: 1% SDS (activator) effect on PPO activity of Ataulfo mango pulp (top)
and skin (bottom). 1% w/v SDS corresponds to adding 30 µL of 10 % w/v SDS into
an assay of 300 µL total volume. In addition to 1% SDS, 175 µL of 0.1 M sodium
phosphate buffer pH 6.8, 30 mM catechol, and 5 µL of extract were added in the 300
uL total volume assay. Reference - Table 5.
64
Protein content of mango (mg/g)
30.00
25.00
green pulp 1
20.00
green pulp 2
yellow pulp 1
15.00
yellow pulp 2
10.00
brown pulp 1
brown pulp 2
5.00
0.00
Ripeness state of mango pulp
Protein content of mango (mg/g)
16.00
14.00
12.00
green skin 1
10.00
green skin 2
8.00
yellow skin 1
6.00
yellow skin 2
brown skin 1
4.00
brown skin 2
2.00
0.00
Figure 3.7.6: Protein content (mg/g) in mango pulp (top) and skin (bottom). PPO
protein content are reported per gram mango (pulp or skin). In a total vol of 300 µL of
activity assay, 190 µL of 0.1 M sodium phosphate buffer pH 6.8, 30 mM catechol, 20
µL of each type of extract were added. All measurements were performed in
triplicate. Data is presented as mean ± standard deviation. Reference - Table 5.
65
3.8 Apparent molecular weight of bands with PPO activity in partially
denaturing SDS-PAGE
SDS-PAGE gels stained for PPO activity with the substrate catechol are
presented in Figure 3.8.1 and 3.8.2. With the exception of R1, all pulp and skin
samples show a band with an apparent molecular weight of approximately 53 kDa.
Sample R1-pulp which exhibited an unusually high PPO activity value for a pulp
sample of an unripe, green mango, displayed a band at approximately 144 kDa that is
also found in all skin samples. The two brown pulp samples (R5 and R6) also show a
band located at 112 kDa. Less prominently stained bands are located at 23 kDa (R2pulp) and 72 kDa (R5-pulp). Multiple forms of PPO differing in their electrophoretic
mobility have been observed in a large variety of plants with molecular weights
ranging from 32 kDa to over 200 kDa, with most molecular weights between 35 kDa
and 70 kDa [35]. Possible reasons for this multiplicity are the attachment of phenolic
oxidation products or carbohydrates, proteolysis, conformational changes,
oligomerization, and finally the presence of distinctly different genes. Information on
PPO genes from mango is limited to the deposition of one partial mRNA coding
sequence from Mangifera indica, Linn (GenBank: GU266283.1). Mango belongs to
the subclass Rosidae (malvids). Within this subclass four full-length mRNA coding
sequences are available yielding calculated molecular weights of 68.5 kDa for the
Canarium album cultivar Huiyuan (GenBank: JQ319005.1), 67.2 kDa for Gossypium
hirsutum (GenBank: JQ345705.1), 69.5 kDa for Gossypium hirsutum clone ZS1
(GenBank: JX966316.1), and 66.2 kDa for Citrus clementine (NCBI Reference
Sequence: XM_006449228.1). Since the samples applied to the SDS-PAGE gels in
Figure 3.8.1 and 3.8.2 are only partially denatured the band positions indicate only
66
apparent molecular weights. It is conceivable that the bands located at 53 kDa or 72
kDa represent the main PPO isoform either with or without a proteolytic cut. Research
has demonstrated that PPO from coffee and broad bean leaves can undergo
proteolysis without loss of catalytic capacity as the apparent molecular weight is
reduced from 67 kDa to 45 kDa or 60 kDa to 42 kDa, respectively [46, 59]. The bands
located at higher molecular weights of 112 kDa and 144 kDa might represent dimeric
forms, but the presence of artifacts due to attachment of polyphenol oxidation
products cannot be ruled out. The higher molecular weight bands were only apparent
in samples with PPO activity values above 0.60 ± 0.05 IU per gram mango. The
higher the intrinsic activity is, the more likely the attachment of polyphenol oxidation
products. Further work on purified PPO from Ataulfo and a full nucleotide sequence
determination will be necessary to clarify the origin of the multiple PPO forms.
67
Figure 3.8.1: Partially denaturing 4-20% Tris-glycine SDS-PAGE gels stained for
PPO activity. Ataulfo pulp samples with increasing ripeness (stages R1-R6) were
loaded into the lanes 2 to 7. Lanes 1 and 8 contain the pre-stained protein standard
SeeBlue from Life Technologies. Reference - Table 1.
68
Figure 3.8.2: Partially denaturing 4-20% Tris-glycine SDS-PAGE gels stained for
PPO activity. Ataulfo skin samples with increasing ripeness (stages R1-R6) were
loaded into the lanes 2 to 7. Lanes 1 and 8 contain the pre-stained protein standard
SeeBlue from Life Technologies. Reference - Table 1.
69
Conclusions
PPO activity in crude extracts of Ataulfo mango was observed for di- and triphenolic substrates with pH optima between pH 5.4 and pH 6.4. The enzyme was
fairly thermo-stable, but can be inhibited effectively with sodium ascorbate in
millimolar concentrations. Ascorbic acid is listed as a GRAS compound by the FDA.
GRAS stands for “Generally Recognized As Safe”. To supress browning of mango,
ascorbic acid or sodium ascorbate could be suitable inhibitors. These chemical agents
are administrated by chemical treatments for example coating of fruits [60]. These
treatments are needed to allow the diffusion of chemical agents for the prevention of
enzymatic browning. Ascorbic acid is also used as a chemical preservative in foods
and as a nutrient and/or dietary supplement [61]. Coating formulations containing
carnauba wax, shellac, zein, and cellulose derivatives were also identified to increase
the lifespan of mango [65].
Many foods consumed today are either genetically modified (GM), or derived
from gene modification technology. This technology has the potential to extend the
lifespan of mango, papaya, and bananas as well as tomato [62]. Enhancement of
tomato shelf life is possible by suppressing N-glycan processing enzymes, similarly
enhancement of mango shelf life is possible by suppressing polyphenol oxidase,
galactosidase and hexosaminidase enzymes [63, 64]. Despite the benefits of genetic
engineering of foods, the technology is surrounded by controversy including fears of
alteration in nutritional quality of foods, potential toxicity, allergenicity and
carcinogenicity [62].
PPO activity as well as PPO isoform distribution depended on ripeness stage
and part of the fruit. The highest PPO activity was found in skin samples of very over-
70
ripe Ataulfo. Samples with high PPO activity displayed at least two PPO isoforms (53
kDa and 112 kDa or 144 kDa), whereas samples with low PPO activity showed only
one major band at 53 kDa in partially denaturing SDS-PAGE gels stained for
catecholase activity. The anionic detergent SDS was an activator of PPO. SDS
enhancement was notably stronger for pulp compared to skin samples. The ripeness
stage and the part of the plant that was used for extract preparation should therefore
always be considered in the evaluation or comparison of enzymatic browning
reactions. Ripeness also significantly influences characteristics such as color, taste,
and aroma. The determination and characterization of phenolic content, and enzymes
such as PPO in fruit and vegetables can contribute to the monitoring of food quality.
71
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CHARACTERIZATION OF POLYPHENOL OXIDASE IN ATAULFO

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