Accepted Manuscript

Characterization of polyphenol oxidase from Cape gooseberry (Physalis pe-

ruviana L.) fruit

Karent Bravo, Edison Osorio

PII: S0308-8146(15)30128-X
DOI: http://dx.doi.org/10.1016/j.foodchem.2015.10.126
Reference: FOCH 18321

To appear in: Food Chemistry

Received Date: 31 July 2015

Revised Date: 21 October 2015
Accepted Date: 25 October 2015

Please cite this article as: Bravo, K., Osorio, E., Characterization of polyphenol oxidase from Cape gooseberry
(Physalis peruviana L.) fruit, Food Chemistry (2015), doi: http://dx.doi.org/10.1016/j.foodchem.2015.10.126

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Characterization of polyphenol oxidase from Cape gooseberry (Physalis peruviana

L.) fruit

Running title: Polyphenol oxidase from Physalis peruviana.

Karent Bravo a, Edison Osorio a,*

a
Grupo de Investigación en Sustancias Bioactivas, Facultad de Ciencias Farmacéuticas

y Alimentarias, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín,

Colombia.

*
Corresponding author. Tel.: +574 2196590; fax: +574 2196590.

E-mail address: edison.osorio@udea.edu.co (E. Osorio).

1
Abstract

Cape gooseberry (Physalis peruviana) is an exotic fruit highly valued, however it is a

very rich source of polyphenol oxidase (PPO). In this study, Cape gooseberry PPO was

isolated and biochemically characterized. The enzyme was extracted and purified using

acetone and aqueous two-phase systems. The data indicated that PPO had the highest

substrate affinity for chlorogenic acid, 4-methylcatechol and catechol. Chlorogenic acid

was the most suitable substrate (Km = 0.56 ± 0.07 mM and Vmax = 53.15 ± 2.03

UPPO.mL-1.min-1). The optimal pH values were 5.5 for catechol and 4-methylcatechol

and 5.0 for chlorogenic acid. Optimal temperatures were 40ºC for catechol, 25ºC for 4-

methylcatechol and 20ºC for chlorogenic acid. In inhibition tests, the most potent

inhibitor was found to be ascorbic acid followed by L-cysteine and quercetin. This

study shows possible treatments that can be implemented during the processing of Cape

gooseberry fruits to prevent browning.

Keywords: Polyphenol oxidase; Cape gooseberry; Purification; Characterization;

Inhibition.

2
1. Introduction

Polyphenol oxidase (PPO) is a common natural copper-containing oxidoreductase and

plays important roles in many plant metabolic processes. In the presence of molecular

oxygen and various phenolic substrates, PPO catalyzes two different reactions, the o-

hydroxylation of monophenols to o-diphenols (i.e., monophenolase or cresolase

activity) and the subsequent oxidation of the o-diphenols to o-quinones (i.e.,

diphenolase or catecholase activity) (Falguera et al., 2012). These quinones are highly

reactive electrophilic molecules that can polymerize and/or react with endogenous

amino acids and proteins to form complex brown pigments, which produce highly

undesirable enzymatic browning during post-harvest physiology, storage and processing

of fruits and vegetables (Jiang, Duan, Joyce, Zhang & Li, 2004). Enzymatic browning is

one of the major challenges for the fruit and vegetable industries and is generally

considered detrimental to food quality from both sensory and nutritional points of view

(Falguera et al., 2012). Due to the importance of this reaction, PPO has been

investigated and characterized in several plants. However, few studies have reported on

the isolation and characterization of Cape gooseberry PPO (Bravo, Muñoz, Calderón &

Osorio, 2011).

Cape gooseberry (Physalis peruviana L.) is an herbaceous, semi-shrub, upright

perennial plant in subtropical zones and belongs to the Solanaceae family. Also known

as goldenberry, Cape gooseberry is an exotic fruit that attracts great interest because of

its nutritional and functional properties. It has been widely used in traditional medicine

for treating various diseases (Arun & Asha, 2007), and its diuretic, anti-infectious and

anti-parasitic properties have been attributed to its fruit (Mayorga, Knapp, Winterhalter

3
& Duque, 2001). The fruits, which are contained in a calyx, are characterized by

abundant amounts of vitamin C, vitamin A, vitamin B-complexes, minerals, tocopherols

and carotenoids (Ramadan, 2011). Recent studies have also determined their phenolic

content, ascorbic acid content and antioxidant properties, which are affected by cultivar,

harvest time and maturity state (Bravo, Sepulveda-Ortega, Lara-Guzman, Navas-

Arboleda & Osorio, 2015). As noted in the literature, the goldenberry could become a

fruit of particular interest to the food industry (Ramadan, 2011).

Cape gooseberry has many cultivars from different regions and countries and is

differentiated by size, color, taste, flower shape, plant height and plant size. Three

cultivar types of particular interest originate from and are currently grown in Colombia,

Kenya and South Africa. Cape gooseberries are an important export product in

Colombia; in fact, the country is the largest producer of gooseberries, followed by South

Africa (Puente, Pinto-Muñoz, Castro & Cortés, 2011). However, fruit cracking has

dramatically affected crop production rates. It is estimated that 10 to 15% of total field

production is affected by cracking (Fischer, Piedrahita, Miranda & Romero, 2005) and

has resulted in significant economic losses for farmers. The preparation of processed

products from fresh Cape gooseberries, such as juice and pulp, will enable the

utilization of cracked fruits if they maintain their nutritional value and organoleptic

properties. Therefore, it is necessary to characterize the Cape gooseberry PPO content,

to develop effective methods for controlling oxidation and browning during the

processing of Cape gooseberries. In this study, a detailed biochemical analysis of

polyphenol oxidase from Cape gooseberry fruit was conducted to determine its kinetic

parameters, optimum reaction conditions, and thermal stability. Additionally, the effects

of various natural and synthetic inhibitors were investigated.

4
2. Materials and methods

2.1. Materials

The Colombian ecotype of Cape gooseberry was used. Fruits at commercial maturity

were obtained from a local farmer and exporter. The material was washed with

sufficient deionized water and stored at 4ºC in a refrigerator for approximately 24 h

until use in the enzyme extraction. The voucher specimen was deposited in Universidad

de Antioquia Herbarium. Catechol, 4-methylcatechol, chlorogenic acid,

polyvinylpyrrolidone (PVP), and Triton X-100 were obtained from Sigma Chemical Co.

Acetone and quercetin were purchased from Merck. Other reagents were analytical

grade, and the natural products were from the lab’s library of compounds.

2.2. Enzyme extraction

The extraction and purification of PPO Cape gooseberry were performed as previously

described (Bravo et al., 2011). One hundred grams of fruits was homogenized using a

Waring blender for 1 min in 200 mL of 0.2 M phosphate buffer (pH 6.0). The

homogenate was filtered and centrifuged at 1250 g for 20 min at 4 ºC. The supernatant

was supplemented with 0.5% (w/v) PVP and 1.5% (v/v) Triton X-100, then sonicated

for 45 min on ice and centrifuged again at 3200 g for 60 min at 4 ºC. The obtained

supernatant was used as the crude extract and was subjected to protein precipitation

with cold acetone (1:1 v/v) at -20 ºC. The protein fraction was separated by

centrifugation at 3200 g for 25 min at 4 ºC, and the supernatant was removed. The

5
precipitate was dissolved in 5 mL of 0.2 M phosphate buffer (pH 6.0) and stored at -20

ºC. This dissolved precipitate was the PPO extract and was later used for

characterization. Additional purification of the PPO extract was achieved by using

aqueous two-phase systems (ATPSs) composed of 5% polyethylene glycol 8000 and

14% phosphate, following the methodology previously described (Bravo et al., 2011).

Protein content was determined by a dye-binding method (Bradford, 1976) with

albumin protein as the standard.

2.3. PPO activity assay

PPO activity was determined by measuring the initial rate of quinone formation, as

indicated by an increased absorbance at 400 nm, using a Power Wave XS Biotek UV–

Vis spectrophotometer (BioTek Instruments, Inc., USA). The increased absorbance was

linearly correlated with time for the first 90 s. The sample well contained 240 µL of the

substrate solution in 0.2 M phosphate buffer (pH = 6.0) and 12 µL of the enzyme

solution. A blank sample contained 240 µL of the substrate solution and 12 µL of

phosphate buffer. Experiments were repeated in triplicate, and the results are expressed

as units of PPO activity. One unit of PPO (U) was defined as the amount of PPO that

produces an increase of 0.001 in the absorbance value per minute under the assay

conditions (Ünal & Şener, 2016). The PPO activity was determined using 4-

methylcatechol, catechol and chlorogenic acid as substrates. In addition, the PPO

activity was measured during the maturation process of the Cape gooseberry fruit.

These were classified in seven states according to the surface color indicated in the NTC

4580 (ICONTEC, 1999) that range from dark green (State 0) to intense orange (State 6).

6
2.4. Effects of pH and temperature on the PPO activity and stability

The effects of pH and temperature on PPO activity were examined using 100 mM

catechol, 20 mM 4-methylcatechol and 5 mM chlorogenic acid as substrates. The PPO

activity pH profiles were determined at room temperature and different pH conditions

between 3.5 and 6.5 with buffers at concentrations of 0.2 M. The buffer systems used

were acetate buffer for pH 3.5 to 5.5 and phosphate buffer for pH 6.0 to 6.5. Then, the

PPO activity was measured at pH levels that elicited the maximum activity for each

substrate at temperatures between 20 and 60 ºC.

The thermal stability of Cape gooseberry PPO was determined by measuring PPO

activity at different temperatures from 30 – 80ºC over 75 min at 10 min intervals using

15 mM 4-methylcatechol and 1.5 mM chlorogenic acid. A 2 mL enzyme solution was

incubated in a test tube at the required temperature for fixed time intervals. Enzyme

aliquots of 200 µL were withdrawn, rapidly cooled in an ice bath and brought to room

temperature. Then, the residual PPO activity was determined as described above, at the

optimal pH for each substrate and at room temperature. The first-orderrate constant (k)

for inactivation was calculated from the slope of the time course of denaturation, using

the equation: ln(A/Ao) = k, where Ao is the initial enzyme activity, and A is the activity

measured at time t. The percent residual PPO activity was calculated by comparison

with unheated enzyme. The half-life of the enzyme was calculated as t1/2 = 0.693/k. The

decimal reduction time (D-value) was estimated as D = 2.303/k. The z value, which is

the temperature increase required for a log10 reduction (i.e., a 90% decrease) of the D-

value, was determined from a plot of log10 D against temperature. The activation energy

7
of denaturation (Ea) was calculated by multiplying the slope of the Arrhenius plot (ln (k)

vs. 1/T) by the universal gas constant, R (kJ.mol-1 K-1).

2.5. Enzyme kinetics and substrate specificity

To determine the Michaelis-Menten constant (Km) and maximum velocity (Vmax), the

PPO activity was measured using catechol, 4-methylcatechol and chlorogenic acid as

substrates at concentrations of 25 – 200 mM, 1.25 – 20 mM and 0.2 – 4.0 mM,

respectively, under optimal pH and temperatures for each substrate and 12 µL of the

enzyme solution. Km and Vmax values of the enzyme were calculated by the

mathematical model Michaelis-Menten using the STATGRAPHICS Centurion XVI

version 16.0.07 software. Measurements were carried out in triplicate.

2.6. Effect of inhibitors on enzyme activity

The Cape gooseberry PPO was incubated in the presence of the following compounds:

ascorbic acid, butyl hydroxytoluene (BHT), calcium chloride, citric acid,

ethylenediaminetetraacetic acid (EDTA), hydroquinone, L-cysteine, sodium chloride,

sodium sulfite, tannic acid, tartaric acid, and the polyphenols quercetin, amentoflavone,

morelloflavone and fukugiside, at 0.1, 1.0 and 10 mM. Additionally, 15 mM 4-

methylcatechol and 1.5 mM chlorogenic acid substrates (final concentration) were

included at optimal conditions. The reaction mixture contained 160 µL substrate, 80 µL

inhibitor solution and 12 µL enzyme. The percent inhibition was calculated using the

following equation: Inhibition (%) = (1-(Ai / A0)) x 100, where A0 is the initial PPO

activity (i.e., without inhibitor), and Ai is the PPO activity with inhibitor. The inhibitory

8
concentration that reduced the enzyme activity by 50% (IC50) was determined for the

inhibitors with a lower percentage of PPO residual activity at concentration of 0.1 mM

using six different concentrations of inhibitor. Finally, using five different

concentrations of the substrates, PPO activities were measured at constant inhibitor

concentrations (IC50). The inhibitors having greater activity were ascorbic acid, L-

cysteine, quercetin and sodium sulfite. The Lineweaver–Burk graphs were used to

determine the type of inhibition.

2.7. Statistical analysis

All statistical analyses were done using GraphPad Prism 5 (GraphPad Software Inc.,

San Diego, CA, EUA). To evaluate significant (P <0.05) differences between samples,

analysis of variance (ANOVA) was performed.

3. Results and Discussion

3.1. Parameters for extraction of PPO and molecular weight

Although PPO has been purified from many plant sources, there are few reports

describing the purification of PPO from Cape gooseberry fruits (Bravo et al., 2011). By

applying previously reported methods for the extraction of Cape gooseberry PPO via

extraction with phosphate buffer, Triton X-100 and PVP, a homogenized PPO extract

was obtained by precipitation with acetone, and a purified extract was recovered after

purification through ATPSs. The purification parameters are shown in Table 1.

Regarding the protein content, 74% of the protein was removed in the fraction during

ATPSs, and the PPO activity increased by approximately 9-fold. The lower protein

9
content in the ATPSs fraction of Cape gooseberry fruit led to an increased specific

activity, indicating that the aqueous two-phase systems were effective in the partial

purification of PPO. In fact, a number of studies have focused on the liquid–liquid

extraction separation techniques of ATPSs. The systems also provide other beneficial

features, such as cost-efficiency, ease of scale-up, process integration capabilities, and

biocompatibility (Asenjo & Andrews 2012). In the separation of proteins by ATPSs, an

understanding of how a few factors affect the separation efficiency is necessary (Asenjo

et al., 2012). Reports have found that PPO partitioning is significantly affected by PEG

molecular weight and the pH of the system (Vaidya, Suthar, Kasture & Nene, 2006). In

this study, 5% polyethylene glycol 8000 and 14% phosphate provided for the selective

partitioning of PPO. The molecular weight of PPO of the Cape gooseberry fruit was

determined by SDS–PAGE (data not shown). The SDS–PAGE profile showed a single

band of approximately 31 kDa weight. There are reports in the literature that PPO from

vegetable sources have molecular weights varying from 29 kDa to 220 kDa (de Fátima

Pereira Goulart, Donizeti Alves, Murad Magalhães & Evangelista Meyer, 2003; Pinto,

Siqueira, Oliveira & Fernandes, 2008; Liu, Zhao, Wen & Ni, 2015). However, similar

PPO molecular weights have been reported, for example, for vanilla bean (34 kDa)

(Waliszewski, Márquez & Pardio, 2009) and coffee (29 kDa) (de Fátima Pereira Goulart

et al., 2003) plants, among others.

3.2. PPO activity during Cape gooseberry ripening

The results show that PPO activity was dependent on the state of ripeness of the fruit.

The enzymatic activity significantly varied (p < 0.05) throughout maturation and was

highest in the early stages of maturation and, as the fruit developed, gradually decreased

10
by approximately 63% between the immature stage (S0) and the overripe stage (S6)

(Fig. 1). It has also been reported that the total phenol content decreased during the

maturation cycle (Bravo et al., 2015). Because pre-harvested Cape gooseberry fruits are

able to maintain a stable separation between PPO and its substrates, PPO cannot directly

oxidize phenolic compounds. Thus, the total phenol content level is primarily dependent

on its synthesis. As the primary metabolism in ripe fruit is reduced, there is a resultant

lack of substrates necessary for the biosynthesis of phenols (Kobayashi, Wang &

Pomper, 2008). However, the observed PPO activity could be at the appropriate level

for ripe fruits susceptible to enzymatic reactions during storage and processing.

3.3. Effects of pH and temperature on the PPO activity and stability

The pH and temperature are two key factors that greatly influence the catalytic activity

of enzymes. The enzymatic profile of the Cape gooseberry PPO at different pH values is

shown in Fig. 2a. The pH optimum was found to be 5.5 for catechol and 4-

methylcatechol and 5.0 for chlorogenic acid (Fig. 2a). The PPO activity varied with pH

and was primarily dependent on the substrate and the sample origin, the extraction

method, the purity of the enzyme, and the localization of the enzyme in the cell

(Montero, Ávalos & Pérez-Mateos, 2001). For the substrates tested, the enzymatic

activities were significantly decreased below pH 4.5 and above pH 6.0. These results

indicated that the Cape gooseberry PPO has a narrow optimal pH range. These results

are similar to the optimum pH range between 5.4 and 6.4 reported for Ataulfo mango

PPO (Cheema & Sommerhalter, 2015), which were evaluated with different substrates.

The low activity of Cape gooseberry PPO at acidic pH could be exploited, i.e.,

11
enzymatic browning during storage and processing could be controlled with acidic

solutions.

The temperature profiles for the oxidation of catechol, 4-methylcatechol and

chlorogenic acid by the Cape gooseberry PPO are shown in Fig. 2b. The optimal

temperature was found to be 40ºC for catechol, 25ºC for 4-methylcatechol and 20ºC for

chlorogenic acid, according to the conditions established in this experiment. At 50ºC,

the enzymatic activity of PPO decreased by 70, 44 and 54% from their maximums for

catechol, 4-methylcatechol and chlorogenic acid, respectively. These results suggested

that the Cape gooseberry PPO is sensitive to heat and that its activity decreased with

increasing temperature. Similar reports for the optimal temperature have been presented

by other authors for artichoke and basil (Dogan, Turan, Dogan, Arslan & Alkan, 2005;

Doǧan, Turan, Ertürk & Arslan, 2005) using catechol as a substrate and for Ferula sp.

using 4-methylcatechol and chlorogenic acid as substrates (Erat, Sakiroglu &

Kufrevioglu, 2006).

The thermal inactivation parameters for the Cape gooseberry PPO are shown in Table 2.

When the enzyme was exposed to 70ºC for 30 min, only 30 and 20% of its original

activity was retained with 4-methylcatechol and chlorogenic acid, respectively. The

incubation at 80ºC reduced 90% of the PPO activity within 10 min of exposure.

Between 30 and 40 ºC, sharp decreases were observed in the half-life (t1/2) and the

decimal reduction time (D-value), which is defined as the time necessary for the activity

to reduce by 90% of the initial activity. This result suggested an increased sensitivity in

this temperature range. At high temperatures (60 to 80 ºC), the Cape gooseberry PPO

was more stable than the PPO from Anamur bananas (Ünal, 2007) and was less stable

12
than the PPO from vanilla bean and wolf apple plants (Batista, Batista, Alves &

Fernandes, 2014; Waliszewski et al., 2009).

The z-value and activation energy (Ea) were also calculated. In general, low z-values

typically indicate high sensitivity, and high Ea values typically indicate a sensitivity to

temperature changes. The z-value and Ea were respectively 27.1ºC and 75.9 kJ.mol-1 for

4-methylcatechol and 29.4ºC and 70.1 kJ.mol-1 for chlorogenic acid. However, a

variance analysis (ANOVA) indicated no significant differences between the two

substrates. The thermostability of PPOs varied considerably from plant to plant.

However, several researchers have indicated that plant PPOs were stable between 20

and 40ºC (Ünal, 2007; Waliszewski et al., 2009; Batista et al., 2014). The thermal

stability of the Cape gooseberry PPO correlated well with their temperature optimums

for enzymatic activity. This result implied that the storage of harvested fruits at low

temperature, for example, below 10 ºC, would be beneficial toward prolonging its shelf-

life, whereas a rapid blanching at high temperatures during its processing may not be

sufficient to inhibit enzymatic browning.

3.4. Enzyme kinetics and substrate specificity

The activity of the Cape gooseberry PPO was examined in the presence of monophenol

(L-tyrosine), diphenols (catechin, chlorogenic acid, catechol and 4-methylcatechol) and

triphenol (gallic acid) compounds as substrates. PPO was more active with the o-

diphenols (data not shown), which indicated a catecholase activity. However, PPO

showed no activity toward L-tyrosine. These results suggested that the Cape gooseberry

PPO lacked monophenolase (cresolase) activity. The PPO activity assays exhibited a

13
typical Michaelis-Menten profile in the evaluated concentration range at the optimal pH

values and temperatures for each substrate. The kinetic parameters Km of the Cape

gooseberry PPO were 203.80 ± 30.13 mM, 3.88 ± 0.39 mM and 0.56 ± 0.07 mM, and

the kinetic parameters Vmax were 12.90 ± 1.11 UPPO.ml-1.min-1, 38.51 ± 1.09 UPPO.ml-
1
.min-1 and 53.15 ± 2.03 UPPO.ml-1.min-1 for catechol, 4-methylcatechol and chlorogenic

acid, respectively. The data indicated that PPO had the highest affinity for chlorogenic

acid followed by 4-methylcatechol and catechol. The specificity of PPO toward

chlorogenic acid was approximately 7 times greater than that for 4-methylcatechol and

370 times greater than that for catechol. The binding properties and catalytic power of

the Cape gooseberry PPO increased as the side chain size increased, likely due to

stronger and more frequent functional interactions with amino acids at catalytic sites. A

higher affinity toward chlorogenic acid was also reported for the PPO of giant fennel

plants (Zucca et al., 2013), and a higher affinity toward 4-methylcatechol than catechol

was reported for the PPO from the wolf apple fruit (Batista et al., 2014).

3.5. Effect of inhibitors on enzyme activity

The effects of inhibitors on the Cape gooseberry PPO activity were studied at various

concentrations using 4-methylcatechol and chlorogenic acid as substrates at optimal

conditions. The results are provided as percentages of PPO inhibition in Table 3. In

addition, the IC50 value is presented for compounds that had an IC50 value greater than

50% at 1.0 mM. The most potent inhibitors were ascorbic acid, L-cysteine, quercetin

and sodium sulfite. Ascorbic acid is a reducing agent that acts as an antioxidant by

reducing quinones to form stable and colorless products (Ünal & Şener, 2006). L-

cysteine, a thiol compound, strongly inhibited the enzyme; 75% or more inhibition

14
occurred at 0.1 mM. Inhibition by thiol compounds is attributed to either the stable

colorless products formed by addition reactions with o-quinones or by binding to the

active center of PPO (Waliszewski et al., 2009). Quercetin was a strong inhibitor even

at low concentrations, while phenolic analogues, such as fukugiside, were only

moderately inhibitive. Typically, phenolic compounds inhibit PPO activity by

interacting with the active site of the enzyme (Kubo & Kinst-Hori, 1998). Thus, the

molecular size seems to be as important as the presence of glycosidic residues.

Additionally, phenolic derivatives act as chelating agents for Cu 2+, a metal necessary for

the activity of PPO, by bonding the hydroxyl group to the active center of the enzyme or

by forming a Schiff base through its aldehyde group; the former mechanism is

predominantly observed (Kubo et al., 1998; Basiri, Shekarforoush, Aminlari & Akbari,

2015). Among the tested sulfites and halides, sodium sulfite clearly had significantly

greater inhibitive effects than calcium chloride and sodium chloride. Sulfites are known

to inhibit PPO by reacting with intermediate quinones, resulting in the formation of

sulfoquinones, which irreversibly inhibit PPO, resulting in its complete inactivation

(Mishra, Gautam & Sharma, 2012). Thus, the inhibitor reaction mechanism is

dependent on the type of inhibitor agent.

For 4-methylcatechol, only quercetin is a competitive inhibitor; ascorbic acid, L-

cysteine and sodium sulfite are not competitive inhibitors. For chlorogenic acid,

ascorbic acid, L-cysteine and sodium sulfite showed competitive inhibition, while

quercetin and fukugiside were not competitive inhibitors (data not shown). These

different properties for the same inhibitor against different substrates may be due to the

presence of several possible mechanisms of action. Ascorbic acid, L-cysteine and

sulfites have been shown to be very good browning inhibitors against PPOs from the

15
wolf apple (Batista et al., 2014) and eggplant (Mishra et al., 2012), among many other

fruits. The inhibition of PPO is important in the food industry, due its role in browning.

Therefore, there are substantial interests in using ascorbic acid to inhibit enzymatic

browning. However, ascorbic acid is a very reactive compound and is rapidly oxidized

to dehydroascorbic acid, which can react with other compounds and produce changes in

fruit quality. Sulfites, despite their wide effectiveness, have restrictions as a food

additive because of their adverse effects on human health. L-cysteine can negatively

affect the taste of the products. In light of these drawbacks, phenolic compounds

consequently have the highest potential to be agents for inhibiting enzymatic browning.

4. Conclusions

This work reported the purification through acetone and ATPSs and the characterization

of PPO from the Cape gooseberry fruit for the first time. The PPO activity decreased as

the fruit matured and ripened. The enzyme was more active and specific toward

chlorogenic acid substrates at optimal pH and temperature values of 5.0 and 20 ºC,

respectively. The PPO activity was strongly inactivated by quercetin, ascorbic acid and

L-cysteine. This study shows possible treatments that can be implemented during the

processing of Cape gooseberry fruits to prevent the browning effect and to allow for the

use of cracked fruits. However, further research is needed to determine enzyme

characteristics and propose more effective treatments applicable at the industrial scale.

Acknowledgements

16
This work was supported by the Committee for the Development of Research (CODI)

of the University of Antioquia (SIU-341373).

References

Arun, M., & Asha, V. V. (2007). Preliminary studies on antihepatotoxic effect of

Physalis peruviana Linn. (Solanaceae) against carbon tetrachloride induced

acute liver injury in rats. Journal of Ethnopharmacology, 111, 110-114.

Asenjo, J. A., & Andrews, B. A. (2012). Aqueous two-phase systems for protein

separation: phase separation and applications. Journal of Chromatography A,

1238, 1–10.

Basiri, S., Shekarforoush, S.S., Aminlari, M., & Akbari, S. (2015). The effect of

pomegranate peel extract (PPE) on the polyphenol oxidase (PPO) and quality of

Pacific white shrimp (Litopenaeus vannamei) during refrigerated storage. LWT -

Food Science and Technology, 60, 1025–1033.

Batista, K. A., Batista, G. L., Alves, G. L., & Fernandes, K. F. (2014). Extraction,

partial purification and characterization of polyphenol oxidase from Solanum

lycocarpum fruits. Journal of Molecular Catalysis B: Enzymatic, 102, 211-217.

Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding. Analytical

Biochemistry, 72, 248–254.

Bravo, K., Sepulveda-Ortega, S., Lara-Guzman, O., Navas-Arboleda, A. A., & Osorio,

E. (2015). Influence of cultivar and ripening time on bioactive compounds and

antioxidant properties in Cape gooseberry (Physalis peruviana L.). Journal of

the Science of Food and Agriculture, 95, 1562-1569.

17
Bravo, K., Muñoz, K., Calderón, J., & Osorio, E. (2011). Desarrollo de un método para

la extracción de polifenol oxidasa de uchuva (Physalis peruviana L.) y

aislamiento por sistemas bifásicos acuosos. Vitae, 18, 124-132.

Cheema, S., & Sommerhalter, M. (2015). Characterization of polyphenol oxidase

activity in Ataulfo mango. Food Chemistry, 171, 382-387.

de Fátima Pereira Goulart, P., Donizeti Alves, J., Murad Magalhães, M., & Evangelista

Meyer, L. (2003). Purification of polyphenoloxidase from coffee fruits. Food

Chemistry, 83, 7-11.

Dogan, S., Turan, P., Dogan, M., Arslan, O., & Alkan, M. (2005). Purification and

characterization of Ocimum basilicum L. polyphenol oxidase. Journal of

Agricultural and Food Chemistry, 53, 10224-10230.

Doǧan, S., Turan, Y., Ertürk, H., & Arslan, O. (2005). Characterization and purification

of polyphenol oxidase from Artichoke (Cynara scolymus L.). Journal of

Agricultural and Food Chemistry, 53, 776-785.

Erat, M., Sakiroglu, H., & Kufrevioglu, O. I. (2006). Purification and characterization

of polyphenol oxidase from Ferula sp. Food Chemistry, 95, 503-508.

Falguera, V., Sánchez-Riaño, A., Quintero-Cerón, J., Rivera-Barrero, C., Méndez-

Arteaga, J., & Ibarz, A. (2012). Characterization of polyphenol oxidase activity

in juices from 12 underutilized tropical fruits with high agroindustrial potential.

Food and Bioprocess Technology, 5, 2921–2927.

Fischer, G., Piedrahita, W., Miranda, D., & Romero, J. (2005). Avances en cultivo,

poscosecha y exportación dephysalis peruviana en Colombia. Bogotá::

Universidad Nacional de Colombia.

18
ICONTEC. Norma Técnica Colombiana 4580. Frutas frescas, uchuva, especificaciones.

Bogotá-Colombia, 1999. URL http://tienda.icontec.org/brief/NTC4580.pdf.

Accessed 01/07/2015.

Jiang, Y., Duan, X., Joyce, D., Zhang, Z., & Li, J. (2004). Advances in understanding of

enzymatic browning in harvested litchi fruit. Food Chemistry, 88, 443–446.

Kobayashi, H., Wang, C., & Pomper, K. W. (2008). Phenolic content and antioxidant

capacity of Pawpaw fruit (Asimina triloba L.) at different ripening stages.

HortScience, 43, 268-270.

Kubo, I., & Kinst-Hori, I. (1998). Tyrosinase Inhibitors from Cumin. Journal of

Agricultural and Food Chemistry, 46, 5338–5341.

Liu, F., Zhao, J.-H., Wen, X., & Ni, Y.-Y. (2015). Purification and structural analysis of

membrane-bound polyphenol oxidase from Fuji apple. Food Chemistry, 183,

72–77.

Mayorga, H., Knapp, H., Winterhalter, P., & Duque, C. (2001). Glycosidically bound

flavor compounds of cape gooseberry (Physalis peruviana L.). Journal of

Agricultural and Food Chemistry, 49, 1904-1908.

Mishra, B. B., Gautam, S. & Sharma, A. (2012). Purification and characterisation of

polyphenol oxidase (PPO) from eggplant (Solanum melongena). Food

Chemistry, 134, 1855–1861.

Montero, P., Ávalos, A., & Pérez-Mateos, M. (2001). Characterization of

polyphenoloxidase of prawns (Penaeus japonicus). Alternatives to inhibition:

additives and high-pressure treatment. Food Chemistry, 75, 317-324.

Pinto, M. S., Siqueira, F. P., Oliveira, A. E., & Fernandes, K. V. (2008). A wounding-

induced PPO from cowpea (Vigna unguiculata) seedlings. Phytochemistry, 69,

2297-2302.

19
Puente, L. A., Pinto-Muñoz, C. A., Castro, E. S., & Cortés, M. (2011). Physalis

peruviana Linnaeus, the multiple properties of a highly functional fruit: A

review. Food Research International, 44, 1733–1740.

Ramadan, M. F. (2011). Bioactive phytochemicals, nutritional value, and functional

properties of cape gooseberry (Physalis peruviana): An overview. Food

Research International, 44, 1830-1836.

Solomon, E. I., Sundaram, U. M., & Machonkin, T. E. (1996). Multicopper oxidases

and oxygenases. Chemical Reviews, 96, 2563–2605.

Ünal, M. Ü., & Şener, A. (2006). Determination of some biochemical properties of

polyphenol oxidase from Emir grape (Vitis vinifera L. cv. Emir). Journal of the

Science of Food and Agriculture, 86, 2374–2379.

Ünal, M. Ü. (2007). Properties of polyphenol oxidase from Anamur banana (Musa

cavendishii). Food Chemistry, 100, 909-913.

Ünal, M. Ü., & Şener, A. (2016). Two-year comparison of the biochemical properties of

polyphenol oxidase from Turkish Alyanak apricot (Prunus armenica L.). Food

Chemistry, 190, 741-747.

Vaidya, B. K., Suthar, H. K., Kasture, S., & Nene, S. (2006). Purification of potato

polyphenol oxidase (PPO) by partitioning in aqueous two-phase system.

Biochemical Engineering Journal, 28, 161–166.

Waliszewski, K. N., Márquez, O., & Pardio, V. T. (2009). Quantification and

characterisation of polyphenol oxidase from vanilla bean. Food Chemistry, 117,

196-203.

Zucca, P., Sanjust, E., Loi, M., Sollai, F., Ballero, M., Pintus, M., & Rescigno, A.

(2013). Isolation and characterization of polyphenol oxidase from Sardinian

20
poisonous and non-poisonous chemotypes of Ferula communis (L.).

Phytochemistry, 90, 16-24.

21
Figure captions

Figure 1. PPO activity during Cape gooseberry fruit ripening using 20 mM 4-

methylcatechol as substrate. The values of a column with the same letter are not

significantly different at the 0.05 level according to ANOVA (P < 0.05). The results

represent the means ± SEM of three experiments performed in duplicate.

Figure 2. Effect to pH and temperature on activity of PPO from Cape gooseberry. a)

Effect to pH. Activity evaluated to room temperature. b) Effect to temperature. Activity

evaluated to optimal pH. Circles represent 100 mM Catechol; square represent 20 mM

4-methylcatechol and triangles represent 5 mM chlorogenic acid.

22
Table 1. Purification parameter to PPO from Cape gooseberry fruit.

Purification steps Total activity Protein content Specific activity Purification Recovery

(UPPO) (mg mL-1) (UPPO mg-1) factor (%)

Crude extract 25.38 1.40 1.21 100.0

Acetone precipitation 15.13 0.39 38.79 32.1 59.6

ATPSs precipitation 3.52 0.36 204.69 169.4 13.9

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Table 2. Kinetic parameters for the thermal inactivation of Cape gooseberry PPO at different temperatures.

Temperature 20 mM 4-methylcatechol 5 mM chlorogenic acid

(ºC) k (10 -2) t1/2 D value k (10-2) t1/2 D value

(min-1) (min) (min) (min-1) (min) (min)

30 0.0976 794.0 2638.2 0.0873 711.5 2364.1

40 0.5297 89.2 296.3 0.7770 130.8 434.7

50 0.9366 55.5 184.5 1.2480 74.0 245.8

60 1.2260 49.8 165.4 1.3920 56.5 187.8

70 3.9900 19.0 63.2 3.6420 17.3 57.6

80 6.6400 5.4 17.8 12.94 10.4 34.7

24
 Table 3. Effect of inhibitors on PPO activity from Cape gooseberry.

20 mM 4-methylcatechol 5 mM chlorogenic acid

Inhibidor 0.1 mM 1.0 mM 10 mM IC50 (mM) 0.1 mM 1.0 mM 10 mM IC50 (mM)

Amentoflavone 0 --- --- --- 0 --- --- ---

Ascorbic acid 89.9 % 93.0 % 96.4 % 0.045 82.9 % 95.6 % 97.9 % 0.060
BHT 0 --- --- --- 0 --- --- ---
Calcium chloride 0a 7.1 %b 34.5 %c 30.96 10.5 %a 61.0 %b 92.5 %c 3.560
Citric acid 0 0 9.3 % --- 0 0 12.7 % ---
EDTA 11.2 % 12.8 % 22.6 % --- 1.8 % 11.4 % 14.7 % ---
Fukugiside 11.7 % 86.6 % --- 0.632 34.9 % 66.2 % --- 0.536
Hidroquinone 27.5 % 67.0 % 92.3 % 0.563 70.5 % 94.9 % 97.0 % 0.044
L-Cysteine 83.6 % 91.0 % 98.0 % 0.052 75.9 % 88.0 % 91.4 % 0.044
Morelloflavone 0 --- --- --- 24.0 % --- --- ---
Quercetine 71.7 % --- --- 0.049 100.0 % --- --- 0.002
Sodium sulphite 41.1 % 95.9 % 98.1 % 0.150 26.2 % 94.3 % 99.8 % 0.157
Sodium chloride 31.8 %a 45.5 %b 79.3 %c 39.54 17.2 %a 66.2 %b 95.3 %c 5.010
Tannic acid 17.2 % 52.7 % 63.5 % 0.372 70.7 % 82.5 % 80.8 % 0.062
Tartaric acid 20.2 % 32.6 % 42.0 % --- 0 0 3.4 % ---

0: No inhibition; ---: No evaluated.

a
: Evaluated to 1.5 mM; b: Evaluated to 15 mM; c: Evaluated to 150 mM.

25
 125 a
Relative activity (%)

100

75
b
bc
50 b b
b
c
25

0
0 1 2 3 4 5 6

Maturation Stage

Figure 1.

26
a) b)
125 125

100 100
Relative activity (%)

Relative activity (%)

75 75

50 50

25 25

0 0
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 15 20 25 30 35 40 45 50 55

pH Temperature (°C)

Figure 2.

27
Highlights

• Biochemical properties of PPO from Cape gooseberry were analyzed.

• The optimal pH and temperature for three substrates were determined.

• The Cape gooseberry PPO activity decreased over time with fruit ripening.

• Different substrates were analyzed to determine their affinities with PPO.

• PPO activity was strongly inactivated by quercetin and ascorbic acid.

28