Purification and Some Properties of Pectinesterase from Potato (Solanum tuberosum L.) Alpha Cultivar

Purification and Some Properties of Pectinesterase from
Potato (Solanum tuberosum L.) Alpha Cultivar
Julio Montañez Sáenz1, Alejandro Téllez2,3, Heliodoro de la Garza1, María de la Luz
Reyes1, Juan Carlos Contreras-Esquivel1,4 and Cristóbal Noé Aguilar1,3*
1Food Research Dept. Faculty of Chemical Sciences. Universidad Autónoma de Coahuila, Saltillo, Coah P.O.Box
252, 25000 Mexico.2Centro de Investigaciones Biologicas del CSIC, Spain. 3Biotechnology Dept. CBS. Universidad
Autónoma Metropolitana, Iztapalapa, Mexico. 4CINDEFI, Faculty of Exact Sciences, Universidad Nacional de La
Plata, La Plata, Argentina
ABSTRACT
Pectinesterase was extracted from potato alpha cultivar, purified and partially characterized The used protocol
resulted in a 58.8-fold purification (51 850.2 units/mg protein) with 15.5% recovery of pectinesterase activity. The
purified enzyme had a molecular weight of 27 kDa and its isoelectric point was around 4.5 with pH and temperature
optima of 8.0 and 60°C, respectively. The purified enzyme had a single symmetric peak of specific activity after
chromatographic steps. The homogeneity of the purified pectinesterase was confirmed by gel filtration and
polyacrylamide electrophoresis gel.
Key words: Extraction, purification, properties, pectinesterase, potato
INTRODUCTION
thermostability of the PE forms (Giovane et al.,
1990).
Pectinestarase (PE, pectin methylesterase, pectin
Control of PE activity in situ is very important in
pectylhydrolase, EC, 3.1.1.11) removes methoxyl
the food industry because of its influence on the
groups from methylated galacturonic residues of
final product quality; particularly to produce low
pectic substances (Sáez et al., 1983). This enzyme
methoxyl pectins in citrus peels (Taylor, 1982) to
is widely distributed in higher plants and can be
obtain cloudy citrus juice (Nath & Ranganna,
found in different plant tissues; mainly those
1977) and high viscosity tomato juice and puree
contained in fruits (Baron & Thibault, 1985).
(Nath et al., 1983) to improve texture and firmness
PE has been purified and characterized from
in some processed fruits and vegetables (Pilnik &
several fruit sources, including tomato (Lee &
Voragen, 1991; Stanley et al., 1995) and color,
Macmillan, 1968), orange (Versteeg et al., 1978),
limpness and other physicochemical parameters of
papaya (Lourenco & Catutani, 1984; Fayyaz et al.,
fried potatoes (Chávez et al., 1998; Aguilera-
1994), apple (Castaldo et al., 1989), kiwi (Giovane
Carbó et al., 1999). In the last case, the control of
et al., 1990), grapefruit pulp (Seymour et al.,
PE activity is a critic factor due the potato tubers
1990) and mandarin orange fruit (Rillo et al.,
of low and medium specific gravity (Alpha
1992). From other plant sources, PE has been
cultivar, 1.065-1.085) can be used to obtain fried
extracted and partially purified from potato (Puri
products in México (Aguilar, 1995; Aguilar et al.,
et al., 1982) and from seeds of Ficus awkeotsang
1997). For this reason, the extraction,
(Komae & Misaki, 1989; Komae et al.,1990).
quantification, purification and characterization is
Some reports established that plants contain
needed if one wishes to measure the effect of
multiple forms of PE (Hultin & Levine, 1963;
temperature on activation/inactivation processes
Evans & McHale, 1978) differing in molecular
during the Low Temperature-Long Time (LT-LT)
weight, charge and glycosylation degree which
blanching and to design the better blanching
affects the affinity for pectin and the
conditions (Aguilera-Carbó et al., 1996a).

* Author for correspondence

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Recently, we published the procedures to develop
Protein Assay. The protein concentration was
and optimize an enzyme extraction, which were
determined according to the microassay of
used for further studies on in situ PE activation
Bradford (BIO-RAD®) using a calibration curve
which improved the quality of fried potato
made with bovine serum albumin.
products (Aguilera-Carbó et al., 1996b and 1999;
Contreras-Esquivel et al., 1999). A report was
Pretreatment of the PE extract crude. PE crude
published on partial purification of PE from potato
extract obtained from potato suspension was
(Puri et al., 1982). It was considered important to
dialyzed in cellulose membrane against a
develop a protoccol of total purification for the PE
phosphate buffer (pH 7.0) during overnight for 12
from potato, which could give a complete
h at 4°C. Then the enzyme was microfiltrated
information of such enzyme. In the present paper,
using Nalgene filter of nylon membrane of 0.2
we describe the PE purification presenting some
mM.
characteristics of this enzyme extracted from
potato tubers (Solanum tuberosum L.) alpha
Pre-purification of PE. Dialyzed and micro-
cultivar, which could be of interest for optimize
filtrated PE extract was used to carry out a
the LT-LT blanching of potato strips and chips and
chromatography as preliminary fractionation on a
improves the quality of these products.
precolumn EconoPac® (Bio-Rad®) conected to
BioLogic LP System® (Bio-Rad®).
MATERIAL AND METHODS
Anion exchange chromatography. Two fractions
obtained as active fractions in the preliminary
Potato tubers (Solanum tuberosum L) alpha
chromatography were used to performed an anion
cultivar were purchased in a local market of
exchange chromatography using a column Mono
Saltillo, Coahuila, México with a specific gravity
Q (Bio-Rad®) in the same BioLogic LP System.
of 1.087. Citrus pectin (P-9125) with an
The concentrated samples were applied to a
esterification degree of 68%, sodium chloride used
column of anion exchange, equilibrated with 0.02
for enzyme extraction and some other analytical
M sodium phosphate buffer (pH 7.5) containing
grade chemicals were obtained from Sigma. BIO-
0.3 M NaCl and 0.02% sodium azide.
RAD® products supplied all chemicals needed for
purification.
Gel filtration chromatography. The molecular
weight of the native PE was determined by gel
Enzyme extraction. PE from potato tubers were
filtration chromatography using a FPLC system
extracted according to the methods developed by
(Bio-Rad®) with a column of Sephadex G-100.
Aguilera-Carbó et al., (1996b) and Contreras-
The column was equilibrated using the following
Esquivel et al., (1999).
proteins: ovoalbumin (43 kDa), carbonic
anhydrase (30 kDa), chymotripsinogen A (25
Determination of PE activity. PE activity was
kDa), ribonuclease (14 kDa) and aprotinin (6.5
assayed by the titration method proposed by
kDa). The column was eluted at flow rate of 0.5
Kertesz (1955). This method involves the
mL/min with a phosphate buffer (0.02 M, pH 7.5)
measurement of the releasing rate of carboxyl
and 300 mM NaCl.
groups in a pectin solution (1% w/v), at 30°C and
pH of 7.0. The substrate was prepared and stored
Gel electrophoresis analysis. Gel electrophoresis
according to the procedure described by Rouse &
in denaturing conditions was performed in sodium
Atkins (1955). The initial rate or reaction was
dodecyl sulfate (SDS-PAGE) according to the
obtained when the free carboxyl groups were titred
method of Laemmli (1970). The gels contained a
with 20 mM NaOH, considering that the
final acrylamide concentration of 10%. Staining of
equivalent amount of NaOH solution used is
slab gels was done with Coommassie Brilliant
proportional to the PE activity. One PE activity
Blue and Silver Nitrate. The molecular weight
was defined as the amount of the enzyme able to
markers were ovoalbumin (43 kDa) carbonic
 
 
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anhydrase (30 kDa) trypsinogen (24 kDa),
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under the above mentioned reaction conditions.
lysosyme (14.3 kDa) and aprotinin (6.5 kDa).

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Isoelectric point determination. The evaluation
other hand, this value was approximately equal to
of isoelectric point (pI) was performed on a Phast
the values obtained for tomato PE (Korner et al.,
System apparatus (Pharmacia) in the pH range 3-9
1980) and mandarin orange PE (96%) reported by
using a calibration kit containing proteins with pI
Rillo et al., (1992).
ranging over 8.7-3.5.
Preliminary chromatography (third step) gave a
42.12% yield, but it did not show a good
pH optimum and thermal stability of PE. The
selectivity for PE. By this step, a purification
pH dependence of potato PE was evaluated in a
factor of 2.2 was obtained. However, this step was
pH range 3.0-9.0 at 25°C, using the titrimetric
useful to eliminate many proteins presents in the
method of Kertesz (1955). Each sample of PE (1.0
dialyzed extract, giving two active fractions. These
mL) was preheated for 30 min at each temperature
results were similar to those reported to PE from
tested and immediately the PE activity was
mandarin orange fruit by Rillo et al., (1992) who
assayed.
reported a yield of 48.6 during the affinity
chromatography.
During the fourth step, the possibility of two
RESULTS AND DISCUSSION
enzymatic forms was rejected due to that one
active fraction was obtained. In this step, the
A summary of the steps utilized to purify the PE
enzyme was found to be eluted as a single peak.
from potato tissue is given in Table 1. The process
After this step, a 36.11% yield was reached and
of PE purification was achieved with a protocol
the PE was purified 4.3-folds.
consisting of five steps.
For further purification of the potato PE, a gel
The protocol carried out was repeated several
filtration chromatography was used. The purified
times and was found to be highly reproducible.
PE gave a 15.55% yield and 58.8-fold purification.
The second step gave a 100% yield. This value
This step was the major feature of the protocol
was higher to the values obtained for apple PE
proposed due it showed a good selectivity for PE,
(Castaldo et al., 1989) and for Ficus awkeotsang
in fact, by this step the purification factor was
PE (Lin et al., 1989), which were 75%. On the
higher.
Table 1. Purification of pectinesterase from potato Alpha cultivar
Purification steps
PE activity
Total activity
Specific activity
Purification
Yield
(U/mL)
(Units)
(Units/mg protein)
(fold)
(%)
Crude extract
16.2
810.0
881.2
100
-
Dialyzed extract
18.2
910.0
1121.3
100
-
Preliminary chromatography
112.5
341.2
1934.3
42.12
2.2
Anion exchange chromatography
115.3
292.5
3826.4
36.11
4.3
Gel filtration chromatography
97.4
126.4
5185.2
15.55
58.8
The specific activity (5185.2 units/mg protein) of
single polypeptide chain. A molecular weight of
the purified pectinesterase obtained in this
27 kDa was estimated.
procedure was five times higher to the specific
According to several reports of the same enzyme
activities of tomato PE and kiwi PE (1159.93 and
purified from other sources, the molecular weight
974 units/mg protein) reported by Lee &
obtained in this work was in the range proposed by
Macmillan (1968) and Giovane et al., (1990)
Delincee & Radola (1970). Puri et al., (1982)
respectively.
reported an apparent molecular weight of 25 kDa
The gel obtained from SDS-PAGE from the active
for potato Russet Burbank Cultivar, while in our
fraction corresponding to PE enzyme is presented
case, the PE from potato Alpha cultivar showed a
in Figure 1. One band of protein was observed.
molecular wieght of 27 kDa, which demostrated
SDS-PAGE showed that the enzyme consists of a
that tissues of potato of different cultivars have
PE´s with similar molecular characteristics.

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A comparison of molecular weights reported for
The thermal stability of PE (Figure 2) was calculated
PEs from several sources is presented in Table 2.
by incubing the enzyme for 30 min at increasing
Isoelectrofocusing determination showed a pI
temperature. The activity was substantially increased
value of 4.5, which was different of that reported
up to 60 °C and then it decreased to about 30% at 70
by Rillo et al., (1992). They observed a pI value
°C and 70% at 90 °C. The PE from potato alpha
higher than 8.65, however this value was
uncertainly determined due to the PE focalization
cultivar appeared thermostable. The thermal stability
at the end of the gel. Our findings were
of this enzyme was considerably higher than that
comparable to that reported by Komae et al.,
found for other PEs, which generally was up to 60°C.
(1990) for PE from Ficus awkeotsang. It is
These results are not similar to those reported by Puri
important to state that the pI values of PE from
et al., (1982), who obtained an optimum temperature
fruit sources are different (higher than 8.0) with
of 55°C and a Q10 of 1.33 in the temperature range
respect to those obtained from vegetable sources
of 15 to 45°C. It is important to note, that the PE
(lower than 6.0) (Versteeg et al., 1978; Rillo et al.,
from potato Alpha Cultivar was highly stable in a
1992).
wide temperature range (30 up to 90°C), which could
be very attractive for the thermal processing of this
cultivar. Last point is very important because, LT-LT
blanching process is generally carried out at
temperature range between 50 – 70 °C with the
objective of an in situ activation of native PE. This
study demonstrates that the maximum activity of
potato PE is around 60 °C, and this temperature is
the optimum for reach better textures in potato tissue
by LT-LT blanching.
The effect of pH on PE activity is shown in Figure 3.
The PE enzyme showed a maximum activity at pH 8
and was underdetectable below pH 5.0. The pH
optimum found for potato PE was similar to that
found for PEs from fruit sources, which generally
Figure 1. SDS-PAGE of final purification step of PE
was in the range 7-9.
from potato. Lane 1 are Molecular weight markers (see
Materials and Methods) and Lane 2 is potato PE.
120
) 100
(
%
i
t
y
Table 2. Molecular weights of pectinesterases from
80
t
i
v
several sources.
ac
60
l
Source
Molecular weight
Reference
a
40
(KDa)
i
du
s
Tomato
24.0 – 28.0
Delincee & Radola (1970)
e
20
R
Tomato
23.7 – 35.5
Pressey & Avants (1972)
0
Orange
35.5 – 37.3
Versteeg (1979)
0
20
40
60
80
100
Papaya
53.0
Lourenco & Catutani (1984)
Var. solo
Temperature (° C)
Papaya
21.0
Lim & Chung (1989)
Mandariine
37.0
Rillo et al., (1992
Figure 2. Thermal stability determination of potato PE.
Orange fruit
Papaya
32.0
Fayyaz et al., (1994)
Var.exotica
Potato
25.0
Puri et al., (1982)
Potato
27.0
This study
Var. alpha

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Aguilar, C.N., Anzaldúa-Morales, A., Talamás, R. &
Gastelum, G. (1997), Low Temperature blanch
improves textural quality in french fries. J Food Sci.
62: 568-571.
Aguilera-Carbó, A., Aguilar, C.N., Contreras-Esquivel,
J.C., De la Garza, H. & Vidal, A. (1996a), Effect of
in situ activation of pectinesterase on color and
texture of french fried potatoes. Iberoam Appl
Biotechnol CIIDIR. 1: 7-14
Aguilera-Carbó, A., Montañez, J.C., Correa, C.,
Contreras-Esquivel, J.C. & Aguilar, C.N. (1996b),
Extraction of pectin methyl esterase from potato
tissue (Alpha cultivar). Conference presented at 3rd
Figure 3. pH optimum determination of potato PE.
International Congress of chemical-biological
Sciences, 10-12 March, UDLA, México
Aguilera-Carbó, A., Montañez, J.C., Anzaldúa-Morales,
AKNOWLEDGEMENTS
A., Reyes, M.L., Contreras-Esquivel, J.C. & Aguilar,
C.N. (1999), Improvement of color and limpness of
Authors wish to thank National Council for
fried potatoes by in situ pectinesterase activation. Eur
Science & Technology (CONACYT, México
Food Res Technol. 210: 49-52
Baron, A. & Thibault, J.F. (1985), Les enzymes
83076/124550). This research was partially
pectolytiques. In: Hydrolases et Dépolimérases, ed.
supported by the CGEPI-UAdeC and forms part of
A. Mouranche & C.Costes. Gauthier-Villars, Paris,
the thesis work of Julio Cesar Montañez. Authors
pp. 143-164
wish to thank researchers of PP-4 of
Castaldo, D., Quagliuolo, L., Servillo, L., Balestrieri, C.
Biotechnology Dept, in the Universidad Autonoma
& Giovane, A. (1989), Isolation and characterization
Metropolitana Iztapalapa for facilities and critical
of pectinmethylesterase from apple fruit. J Food Sci.
opinions offered.
54: 653-655, 673
Chávez, S.N., De la Garza-Toledo, H., Aguilera-Carbó,
A., Montañez, J.C., Contreras-Esquivel, J.C. &
RESUMO
Aguilar, C.N. (1998), Effect of no ordinary
blanchings on physico-chemical and microbiological
quality of fried potato strips. Ind Alim. 20: 19-22
A pectinesterase foi extraída da batata (cultivar do
Contreras-Esquivel, J.C., Correa-Robles, C., Aguilar,
alfa), purificada e parcialmente caracterizada. O
C.N., Rodríguez, J., Romero, J. & Hours, R.A.
protocolo usado levou a uma proteína purificada
(1999), Pectinesterase extraction from mexican lime
58,8 vezes (51 850,2 units/mg da proteína) com
(Citrus aurantifolia Swingle) and prickly pear
uma recuperação de 15,5 % da atividade da
(Opuntia ficus indica L.) peels. Food Chem. 65: 153-
proteína. A enzima purificada apresentou um peso
156
molecular de 27 kDa e seu ponto isoelétrico foi ao
Delincee, H. & Radola, B.J. (1970), Some size and
redor 4,5. A pectinesterase exibiu pH e
charge properties of tomato pectin methylesterase.
temperatura ótimos de respectivamente 8,0 e 60°C.
Biochem Biophys Acta. 47: 183-185
A enzima purificada apresentou um único pico
Evans, R. & McHale, D. (1978), Multiple forms of
pectinesterase in limes and oranges. Phytochemistry.
simétrico de atividade específica após as etapas de
17: 1073-1075
cromatografia. A homogeneidade da pectinesterase
Fayyaz, A., Asbi, B.A., Ghazali, H.M., Che Man, Y.B.
purificada foi confirmada por filtração em gel e
& Jinap, S. (1994), Purification and molecular
por eletroforese em gel de poliacrilamida.
properties of papaya pectinesterase. Food Chem. 49:
373-378
Giovane, A., Quagliulo, L., Castaldo, D., Servillo, L. &
REFERENCES
Balestrieri, C. (1990), Pectin methylesterase from
Actinidia chinensis fruits. Phytochemistry. 29: 2821-
Aguilar, C.N. (1995), Application of no ordinary
2823
blanching to improves the quality of processed fruits
Hultin, H. & Levine, A. (1963), On the occurrence of
and vegetables. In: Research and technological
multiple molecular forms of pectinesterase. Arch
development in Coahuila, ed. COECYT-Coahuila,
Biochem Biophys. 101: 396-402
Saltillo, México. 11-16

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Kertesz, Z. (1955), Pectic enzymes. In: Methods in
Puri, A., Solomos, T. & Kramer, A. (1982), Partial
enzymology (Vol. 1), ed.S.P. Colowik & N.O.
purification and characterization of potato
Kaplan. Academic press, New York, USA. 158
pectinesterase. Food Chem. 8: 203-213
Komae, K., and Misaki, A. (1989), Isolation and
Rillo, L., Castaldo, D., Giovane, A., Servillo, L.,
characterization of gel-forming polygalacturonoide
Balestrieri, C. & Quagliuolo, L. (1992), Purification
from seeds of Ficus awkeotsang. Agric Biol Chem.
and properties ofpectin methylesterase from mandarin
53: 1237-1245
orange fruit, J Agric Food Chem. 40: 591-593
Komae, K., Sone, Y., Kakuta, M. & Misaki, A. (1990),
Rouse, A. & Atkins, C. (1955), Pectinesterase and
Purification and characterization of pectinesterase
pectin in commercial citrus juices as determined by
from Ficus awkeotsang. Agric Biol Chem. 54: 1469-
methods used at the Citrus Experimental Station. Fla
1476
Agr Expt Sta Bull. 570
Korner, B., Zimmermann, G. & Berk, Z. (1980),
Sáez, S., Villarroel, P., Parraguirre, V. & Pennachiotti,
Orange pectinesterase: purification, properties and
I. (1983), Determinación de actividad pectinesterásica
effect on cloud stability. J Food Sci. 45: 1203-1206
y extracción de pectina a partir de sub-productos
Laemmli, U. K. (1970), Cleavage of structural proteins
cítricos. Alimentos (Chile). 8: 38-39
during the assembly of the head of bacterophage, T4.
Seymour, T., Preston, J., Wicker, L., Lindsay, J.A. &
Nature. 227: 680
Marshall, M.R. (1990), Purification and properties of
Lee, M. & Macmillan, J.D. (1968), Mode of action of
pectinesterase of marsh white grapefruit pulp, J Agric
pectic enzymes. I. Purification and certain properties
Food Chem. 39: 1080-1085
of tomato pectinesterase. Biochemistry. 7: 4005-4010
Stanley, D.W., Bourne, M., Stone, A. & Wismer, W.
Lin, T., Liu, C., Chen, S. & Wang, W. (1989),
(1995), Low temperature blanching effects on
Purification and characterization of
chemistry, firmness and structure of canned green
pectinmehtylesterase from Ficus aweotsang Makino
beans and carrots. J Food Sci. 60: 327-333
Achenes. Plant Physio. 91: 1445-1453
Taylor, A. (1982), Intramolecular distribution of
Lourenco, E. J. & Catutani, A.T. (1984), Purification
carboxyl groups in low methoxyl pectins-a review.
and properties of pectinesterase from papaya. J Sci
Carbohydr Polym. 2: 9-17
Food Agric. 35: 1120-1127
Versteeg, C., Rombouts, F. & Pilnik, W. (1978),
Nath, N. & Ranganna, S. (1977), Time/temperature
Purification and some characteristics of two
relationship for thermal inactivation of pectinesterase
pectinesterase insoenzymes from orange. Lebensm
in mandarin orange (Citrus reticulata Blanco) juice. J
Wiss Technol. 11: 267-274
Food Technol. 12: 411-419
Nath, N., Rao, A. & Gupta, R. (1983), Thermal
resistance of pectin methyl esterase in juice of pusa-
Received: December 16, 1999;
ruby tomatoes. Ind Food Packer. 37: 30-38
Revised: June 12, 2000;
Accepted: July 10, 2000.
Pilnik, W. & Voragen, A. (1991), The significance of
endogenous and exogenous pectic enzymes in fruit
and vegetable processing. In: Food Enzymology, Vol.
1, ed. P.F. Fox. Elsevier Applied Science, London.
304-336

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