food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596

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Food and Bioproducts Processing

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Review

Enzymatic production of pectic oligosaccharides from

polygalacturonic acid with commercial pectinase
preparations

Agnan Marie Michel Combo ∗ , Mario Aguedo, Dorothée Goffin, Bernard Wathelet,
Michel Paquot
University of Liège, Gembloux Agro-Bio Tech, Department of Industrial Biological Chemistry, Passage des Déportés, 2, 5030 Gembloux,
Belgium

a b s t r a c t

The present study investigates the individual efficiency of six commercial pectinase preparations (Endopolygalac-
turonase M2, Pectinase, Viscozyme L, Pectinex Ultra SP-L, Pectinase 62L and Macer8 FJ) in catalyzing the liberation
of pectic oligosaccharides (POS) from polygalacturonic acid. On the basis of high-performance anion-exchange chro-
matography with pulsed amperometric detection (HPAEC-PAD) analysis of the enzymatic hydrolysates, products
release kinetics revealed a random cleavage pattern and an exo mode of cleavage for all the enzymes except for
Endopolygalacturonase M2.
All six enzymes generated oligoGalA with different degree of polymerization (DP); the quantitative composition
of oligoGalA depended on the enzyme specificity and the time of enzymatic reaction. Endopolygalacturonase M2
was the best enzyme preparation for production of oligoGalA, with 18% (wt) of digalacturonic acid and 58% (wt) of
trigalacturonic acid after 2 h of reaction. Concerning galacturonic acid production, Pectinase 62L was superior to the
other enzyme preparations with 47% (wt) after 1 h of reaction.
© 2011 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Polygalacturonic acid; Pectic oligosaccharides; Pectolytic enzymes; Degree of polymerization; HPAEC-PAD

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
2.1. Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
2.2. Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
2.3. Enzyme activity measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
2.4. Protein determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
2.5. SDS-polyacrylamide gel electrophoresis profiling of commercial pectinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
2.6. Enzymatic hydrolysis of polygalacturonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
2.7. Analysis of oligogalacturonic acids released from polygalacturonic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
3.1. Enzymes characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
3.2. HPAEC-PAD analysis of hydrolysis products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594
3.3. Characterization of oligoGalA profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 594

∗
Corresponding author. Tel.: +32 081 62 22 32; fax: +32 081 62 22 31.
E-mail addresses: ammcombo@student.ulg.ac.be, comboagnan@yahoo.fr (A.M.M. Combo).
Received 17 January 2011; Received in revised form 10 August 2011; Accepted 29 September 2011
0960-3085/$ – see front matter © 2011 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.fbp.2011.09.003
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596 589

4. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595

1. Introduction and specificity of the different commercial enzymes, and

thus to establish an activity pattern for each prepara-
Biomass from plant material can be employed as a source tion.
of polymeric and oligomeric carbohydrates. In recent years,
oligosaccharides have found applications in various fields, 2. Materials and methods
notably because of their specific biological activities. The
potential of plant cell wall polysaccharides as sources of novel 2.1. Chemicals
high value-added oligosaccharides has also received special
attention. Pectic substances can be employed as functional Polygalacturonic acid (PGA), citrus peel pectin (galacturonic
foods. The conversion of pectic substances by microorganisms acid content approx. 74% and methoxy content approx. 6.7%),
or enzymes (Zykwinska et al., 2008; Martínez et al., 2009) con- d-galacturonic acid monohydrate (AGA), digalacturonic acid
stitutes promising biological processes because they allow the (DiAGA) and trigalacturonic acid (TriAGA) were from Sigma
production of specific oligosaccharides without any formation Chemical Co., (St. Louis, MO, USA). A standard of saturated
of undesirable by-products (Leitão et al., 1995; Cabrera and Van oligogalacturonic acids was provided by Prof. P. Van Cutsem
Cutsem, 2005). Most food-grade pectic oligosaccharides (POS) (Notre Dame de le Paix University, Namur, Belgium).
studied to date have been produced by pectinases. Commer- All chemicals and reagents were of analytical or HPLC
cial pectinase is a term that generally refers to mixtures of grade.
three different enzymatic activities: polygalacturonase (PG),
pectin lyase (PL) and pectin esterase (PE). All three activities 2.2. Enzymes
contribute to the breakdown and modifications of pectic sub-
stances from a wide variety of plant materials. Pectinases can All enzyme preparations or pectinases were commercially
be isolated from plants or from microorganisms such as bac- available. A total of six food grade pectinases from differ-
teria and fungi (Kaur et al., 2004; Jayani et al., 2005). Most ent companies were studied. According to the suppliers, the
commercial pectinases are produced by Aspergillus spp. and enzymes were generally produced by Aspergillus varieties.
are mixtures of pectinolytic enzymes. In spite of the exten- Sources, suppliers and experimental conditions for the com-
sive application of pectinases in food industry, commercial mercial pectinases used in this work are given in Table 1.
pectinase preparations have been investigated. The enzymatic
degradation of pectic substances gives galacturonic acid and 2.3. Enzyme activity measurements
oligomers (POS).
Galacturonic acid and its derivates can be used in food Polygalacturonase (PG) activity was assayed for 10 min with
industry as acidic agents, in chemical industry as washing a 0.2% solution of polygalacturonic acid. The number of
powder agents and as non-ionic or anionic biodegradable sur- reducing groups, expressed as galacturonic acid released by
factants and in pharmaceutic industry in the production of enzymatic action was quantified by the 2-cyano-acetamide
vitamin C (Molnár et al., 2009; Burana-Osot et al., 2010). POS reagent assay and monitored by the absorbance of resulting
have also various applications, since they are important signal colored mixture at 276 nm (Verlent et al., 2005). One unit (U)
molecules in plant defences and play roles in plant growth and of PG activity was defined as the amount of enzyme releasing
development processes (Marfà et al., 1991; Ridley et al., 2001; 1 ␮mol of galacturonic acid per min under assay conditions.
Baldan et al., 2003) and in food industry as potential ingredi- Experiment was carried out in duplicate.
ents (Willats et al., 2006). Moreover, the prebiotic potential of Preliminary experiments were conducted to determine
POS has been reported because they selectively increased the optimum pH and temperature of pectinase from Aspergillus
populations of beneficial bacteria in human gastrointestinal niger because no information was given by the supplier. The
tract such as bifidobacteria and Eubacterium rectale (Manderson
et al., 2005). Furthermore, additional functionalities of POS
120 EPG-M2
were reported including the repression of lipid accumulation
Pectinase
Decrease in viscosity (%)

in rats liver, an anti-bacterial activity (Iwasaki et al., 1998), the

100
protection of colonocytes against Escherichia coli verocytotox- Viscozyme L

ins (Olano-Martin et al., 2003) and the stimulation of apoptosis Pectinex Ultra SP-L
80
of colon cancer cells (Chauhan et al., 2005). Pectinase 62L
The aim of the present work was to characterize a series Macer8 FJ
60
of commercial pectinases in order to produce galacturonic
acid and pectic oligomers. The enzyme content of six dif-
40
ferent food-grade commercial pectinase preparations was
profiled. Then, the enzymatic hydrolysis of polygalactur-
20
onic acid was carried out using the commercial pectinase
0 10 20 30
preparations; a comparison of the production yields by the
Time (min)
different pectinase preparations was assessed. The released
monomer and oligomers were analyzed by high-performance Fig. 1 – Decrease of viscosity catalyzed by commercial
anion-exchange chromatography with pulsed amperometric pectinase preparations. The results are expressed as
detection (HPAEC-PAD) in order to compare the efficiency means of two tests.
590 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596

Table 1 – Enzymes, supplier, sources and working conditions used.

Enzyme Supplier Source Working conditions

Temperature (◦ C) pH

EPG-M2 Megazyme A. aculeatus 40 5.5

Pectinase Sigma A. niger 20–60 (40) 3–5.0 (3.8)
Viscozyme L Sigma A. aculeatus 25–55 (35) 3.3–5.5 (4.0)
Pectinex Ultra SP-L Novozymes A. aculeatus Below 45 (35) 4.5–6 (4.5)
Pectinase 62L Biocatalysts A. sp 10–55 (37) 3.0–5.0 (5.0)
Macer8 FJ Biocatalysts A. sp 40–60 (40) 3.0–5.0 (5.0)

(): Experimental conditions chosen in the present work.

Table 2 – Polygalacturonase and endopolygalacturonase activities in the commercial pectinase preparations (CP) per ml
and the corresponding total activities (enzyme units) used in the reaction media with the different enzymes.
CP EPG-M2 Pectinase Viscozyme L Pectinex Ultra SP-L Pectinase 62L Macer8 FJ

CP Proteins (mg/ml) 0.21 ± 0.02 15.5 ± 0.5 24.3 ± 1.8 11.8 ± 0.8 17.4 ± 0.2 12.8 ± 1.1
CP PG activity (U/ml) 295.6 ± 7.6 264.7 ± 4.6 415.4 ± 7.3 523.8 ± 6.6 561.5 ± 4.1 459.4 ± 3.1
CP EPG activity (U/ml) 47,774 ± 7900 3160 ± 756.6 11,725 ± 500 7160.8 ± 1702 15,537.5 ± 442 24,125 ± 0.0
Total PG activity (U) 4.9 4.4 6.9 8.7 9.4 7.7
Total EPG activity (U) 796.2 52.7 195.4 119.3 258.9 402.1

Values are means ± standard deviations of two replicates.

effects of pH and temperature on the enzymatic activity of
the pectinase preparation were investigated in the same way
as described earlier for polygalacturonase activity under assay
conditions within a pH range between pH 3.0 and pH 5.0 using
sodium acetate buffer (0.1 M) and a temperature range from
20 ◦ C to 60 ◦ C (20, 30, 35, 40, 45, 50, and 60 ◦ C). Commercial
pectinase solutions were also assayed for the desired enzyme
activities according to endopolygalacturonase (EPG) activity.
EPG activity was determined viscosimetrically using citrus
pectin as substrate. This method is based on the decrease
in the viscosity of a pectin solution caused by enzymatic
hydrolysis of the “endo” form of the enzyme. 1 ml of diluted
enzyme solution was added to 250 ml of 3% (w/v) citrus pectin.
The viscosity reduction of the pectin solution was determined
with a Brookfield viscometer (Model LVT, Brookfield Engineer-
ing Laboratories, MA, USA) at a shear rate of 30 rpm using
spindle no. 2 at different time intervals for up to 30 min. One
unit (U) of endopolygalacturonase activity was defined as the
amount of enzyme required to reduce the initial viscosity of
pectin solution by 50% per min under the conditions described
above. The measurement was made in duplicate. The EPG
activity was calculated from the slope of the linear part of the
viscosity reduction versus times curves. In this calculation,
the dilution factor during viscosimetric assay (reaction mix-
ture volume/diluted enzyme volume) and the enzyme dilution
prior to the assay were considered.

2.4. Protein determination

Protein content in enzyme preparation was determined by the

method of Bradford (1976) using the Bio-Rad protein assay dye
reagent. Bovine serum albumin (0.125–1 mg/ml) was used to
construct the standard curve (r2 = 0.985). Samples were tested
in triplicate.

2.5. SDS-polyacrylamide gel electrophoresis profiling

of commercial pectinases

SDS-PAGE was performed according to the method of Laemmli

(1970) using 10% resolving and 5% stacking gel. Protein
bands were visualized using coomassie brilliant blue R250
(Fluka Chemika, Buchs, Switzerland) and destained with
methanol–water containing 8% acetic acid. The wide range
SigmaMarker (S8445) containing twelve proteins from 6.5 to
200 kDa was used as molecular mass marker.

2.6. Enzymatic hydrolysis of polygalacturonic acid

Fig. 2 – SDS-PAGE analysis of commercial pectinase
preparations. Lanes: 1 molecular mass standard proteins, 2 For all pectinases, hydrolysis was performed as follow: 50 ml of
Viscozyme L, 3 Pectinase, 4 Pectinex Ultra SP-L, 5 Pectinase 0.2% polygalacturonic acid in 0.1 M acetate buffer (see Table 1
62L, 6 Macer8 FJ, 7 Endopolygalacturonase M2. for the pH used with each enzyme) were added 50 ml of (1:3000)
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596 591

Table 3 – AGA/DiAGA ratios obtained from polygalacturonic acid by treatment with the different commercial pectinase
preparations.
CP AGA/DiAGA for the different hydrolysis times

5 min 15 min 30 min 1h 2h

EPG-M2 0.23 0.25 0.31 0.44 0.71

Pectinase 1.81 2.96 4.80 15.50 197.90
Viscozyme L 1.50 1.92 2.83 6.34 43.01
Pectinex Ultra SP-L 3.85 4.50 4.60 5.10 8.74
Pectinase 62L 4.13 60.30 147.80 117.80 100.70
Macer8 FJ 1.80 2.20 3.50 12.30 222.50

commercial pectinase solution. Aliquots were taken at differ-
ent time intervals up to 2 h and heated to 100 ◦ C for 10 min
to inactivate the enzymes. After cooling, the inactivated sam-
ples were filtered through a 0.45 ␮m nylon syringe filters and
the released galacturonic acid and oligogalacturonates (OGAs)
were analyzed with HPAEC-PAD as described below, in order
to determine the degree of polymerization (DP) of the OGAs.
and trigalacturonic acid were used as external standards for
quantification. Standard solutions were prepared in the range
of 12.5–750 mg/l and used for the calibration curves. Good lin-
earity was obtained for OGAs standards (r2 = 0.99, r2 = 0.98 and
r2 = 0.99 respectively). Data were collected and analyzed with
Dionex Chromeleon 6.80 SP3 Build 2345 software.

3. Results and discussion

2.7. Analysis of oligogalacturonic acids released from
polygalacturonic acid 3.1. Enzymes characterization

The polygalacturonic acid digests obtained after enzy-
matic treatment were characterized by high-performance
anion-exchange chromatography with pulsed amperometric
detection (HPAEC-PAD). The chromatographic system was a
Dionex ICS-3000 model (Sunnyvale, CA, USA) equipped with
an ED-3000 electrochemical detector, and a SP gradient pump.
The column was a Dionex CarboPac PA-100 (250 mm × 4 mm
i.d.) coupled to a CarboPac guard column (40 mm × 4 mm i.d.).
The mobile phase consisted of 100 mM sodium hydroxide
(eluent A), 600 mM sodium acetate in 100 mM sodium hydrox-
ide (eluent B) and 500 mM sodium hydroxide (eluent C). Elution
conditions were as follows: 95% A and 5% B over 0–5 min,
50% A and 50% B at 10 min, 20% A and 80% B over 15–35 min,
50% B and 50% C over 36–43 min and 95% A and 5% B over
44–50 min. The flow-rate was 1 ml/min and the injection vol-
ume was 25 ␮l. d-Galacturonic monohydrate, digalacturonic
In Table 1 are summarized the optimum pectinase reaction
conditions given by the suppliers or determined by us in the
case of Pectinase from Aspergillus niger.
PG and EPG enzymatic activities were determined for all
the six commercial enzyme preparations. PG activity was
detected in all the enzyme preparations, although with dif-
ferences among them (Table 2). Preparations with higher
levels of activity were Pectinase 62L (561.5 U/ml), Pectinex
Ultra SP-L (523.8 U/ml), Macer8 FJ (459.4 U/ml) and Viscozyme
L (415.4 U/ml), while EPG-M2 and Pectinase had the lowest PG
activities: 295.6 U/ml and 264.7 U/ml respectively. In the case of
EPG activity, the six preparations were also assessed by mea-
suring the reduction of the viscosity to estimate endo activity.
As shown in Fig. 1, all enzyme preparations cause a decrease of
the viscosity of pectin solution, indicating a depolymerization
of the pectin sample. Pectinase showed relatively low

Fig. 3 – HPAEC-PAD profiles of standard oligogalacturonides.

592 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596

Fig. 4 – HPAEC-PAD profiles of oligogalacturonides released by the action of commercial pectinases from polygalacturonic
acid for different incubation times: (1) 5 min; (2) 15 min; (3) 30 min; (4) 60 min; (5) 120 min. (a) Endopolygalacturonase M2; (b)
Viscozyme L; (c) Pectinase; (d) Pectinex Ultra SP-L; (e) Pectinase 62L; (f) Macer8 FJ. The inset is a zoom of the indicated region
of the chromatogram.
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596 593

Fig. 4 – (continued ).

diminution of viscosity as compared to the others. EPG-M2
cause pronounced reduction in viscosity of pectin solution
much more rapidly than the other enzymes. A sharp decrease
in viscosity was observed after a 2 min incubation and a 50%
decrease in viscosity was achieved within 10 min. EPG activity
of all enzyme preparations was calculated. Results are shown
in Table 2. As can be observed, values of EPG activity obtained
for the enzyme samples showed great difference. Among
the enzyme samples used in this study, EPG-M2 has a high
EPG activity followed by Macer8 FJ, Pectinase 62L, Viscozyme
L, Pectinex Ultra SP-L and finally Pectinase. According to
SDS-PAGE analysis of the commercial enzyme preparations,
594 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596
Table 4 – (TriAGA + DiAGA)/AGA ratios obtained from polygalacturonic acid by treatment with the different commercial
pectinase preparations.
CP (TriAGA + DiAGA)/AGA for the different hydrolysis times
5 min 15 min
EPG-M2 17.15 15.15
Pectinase 1.09 0.73
Viscozyme L 1.90 1.13
Pectinex Ultra SP-L 1.00 0.95
Pectinase 62L 0.52 0.02
Macer8 FJ 1.40 1.09
all had several bands visualized when stained with Coomassie
Brilliant Blue, except EPG-M2 that was pure (Fig. 2). So, all, but
EPG-M2, were composed of heterogeneous protein mixtures.
The band corresponding to the EPG in EPG-M2 was also present
in three other commercial preparations (Viscozyme L, Pectinex
Ultra SP-L and Pectinase 62L). This does not mean that the two
other preparations (Pectinase and Macer8 FJ) do not contain it,
since EPG mass below or beyond 41 kDa have been reported
in literature (De Vries and Visser, 2001; Contrevas Esquivel
and Voget, 2004). The heterogeneity of the tested commercial
enzyme preparations makes the detection of polygalactur-
onase or EPG activity difficult because of synergism interaction
effects. The different activities in the commercial enzyme
preparations may indeed interfere on the determination of
endo and exoPG activities: EPG may also release galacturonic
acid, depending for example on the degree of methylation of
the pectin chain.
3.2. HPAEC-PAD analysis of hydrolysis products
In order to identify fragments released in the reaction mix-
ture, the composition of the hydrolysates was determined
by HPAEC-PAD. Separation was performed at alkali pH in
order to selectively separate galacturonic acid and oligomers.
The products of the hydrolysis of polygalacturonic acid were
identified by comparison of their retention times with a
preparation of oligoGalA (DP 1–9). Figs. 3 and 4 give elution
profiles respectively of the standard compounds and of the
degradation products of polygalacturonic acid by commer-
cial pectinases. After only 5 min of digestion, commercial
pectinases produced a mixture of oligoGalA from polygalac-
turonic acid showing the feasibility of the enzymatically
catalyzed production of POS. The pattern of oligoGalA pro-
Fig. 5 – Yields of oligogalacturonic acids (DP 1–3) obtained from polygalacturonic acid.
30 min 1h 2h
11.33 8.00 5.92
0.37 0.09 0.01
0.56 0.18 0.03
0.93 0.88 0.51
0.01 0.04 0.02
0.55 0.10 0.01
duced at the beginning of the degradation of polygalacturonic
acid with the enzymes indicated that these enzymes act
by a mechanism of random cleavage of polygalacturonic
acid, which confirms the endo nature of the enzymes. It
is generally accepted that the EPG acts randomly on poly-
galacturonic acid and releases long chain oligoGalA, which
are subsequently hydrolyzed to short chain oligomers (Benen
et al., 1999). Also, the release of DP1 to DP3 as main prod-
ucts by EPG-M2 confirms its EPG activity. These results are
in good agreement with those of Contrevas Esquivel and
Voget (2004) when they used Endo-polygalacturonase from
Aspergillus kawachii on polygalacturonate. During extended
hydrolysis, a change in the profiles of oligoGalA was observed;
higher DPs were hydrolyzed into smaller DPs. In contrast to
EPG-M2, other commercial preparations promoted the accu-
mulation of DP1 as the main product after 2 h of reaction.
This suggests that these enzymes have more affinity for the
oligoGalA or another hypothesis would be the presence of
exopolygalacturonase (exoPG) activity within these commer-
cial preparations. Sakamoto et al. (2002) have shown that
the presence of exoPG could be evaluated by calculating
the concentration ratio of galacturonate to digalacturonate
in polygalacturonic acid hydrolysis products: enzymes with
high exoPG activity have higher ratios. According to such
calculations (Table 3), in the present work, all the enzymes
displayed exoPG activity except EPG-M2. Therefore, PG activity
depends on the synergistic action of these enzymes (exo and
EPG).
3.3. Characterization of oligoGalA profiles
The influence of commercial enzymes on the concentra-
tion of oligoGalA against time is shown in Fig. 5. Due to
 food and bioproducts processing 9 0 ( 2 0 1 2 ) 588–596
unavailability of commercial standards, the oligoGalA up to
DP3 were quantified. The concentration of oligoGalA was cal-
culated from the HPAECs and reported as a percentage of the
total. EPG-M2 yielded by far the highest concentration of oli-
goGalA. The oligoGalA produced were DP3 > DP2 > DP1 after 2 h
of reaction and the yields were 58%, 18% and 13% respectively.
For the other enzymes, generally low amounts of DiAGA and
TriAGA were obtained with regard to galacturonic acid. With
Viscozyme L, pectinex Ultra SP-L and Pectinase 62L, maximum
yields for DiAGA and TriAGA were observed in the first 5 min
and were respectively 9.5% and 17.5%; 4.3% and 12.2%, 7.6%
and 8.7%. Maximum yields for Pectinase and Macer8 FJ were
obtained after 15 min of hydrolysis (9.1% and 10.7%, 8.8% and
12% respectively).
The yield of galacturonic acid increased, whereas lower
yields were obtained for oligogalacturonic acids (Fig. 5). This
tendency indicated that depolymerization of oligosaccharides
went on with increasing reaction time. On the other hand,
the treatment with Pectinase 62L released mainly galactur-
onic acid, amounting to 47% (wt) after 1 h of reaction time. The
resulting high galacturonic acid concentration also inhibits
the formation of POS (Bélafi-Bako et al., 2007). As observed
from the results, these enzymes (Pectinase 62L, Viscozyme
L, Pectinase, Macer8 FJ and Pectinex Ultra SP-L) can be used
to produce galacturonic acid. The efficiency of each enzyme
preparation for the preparation of POS could be evaluated by
the ratio of TriAGA and DiAGA to AGA: the lowest the value, the
highest the content of the monomeric acid (Table 4). The ratios
clearly showed the much higher efficiency of EPG-M2 when
compared to the other tested enzymes. The poorest ratios
were obtained with Pectinase 62L, while all the other enzymes
showed comparable values. In summary, significant oligoGalA
concentrations can be achieved in the hydrolysates of the
tested commercial pectinases. The maximum POS concentra-
tions depend on the enzyme preparation and the hydrolysis
time. The long hydrolysis times were not favourable here since
the released POS are then progressively hydrolyzed. Using
EPG like EPG-M2, it was possible to produce a mixture rich
in DP3.
4. Conclusion
The commercial pectinases of the present study contained
different activities (exoPG, EPG and other) except EPG-M2.
Hydrolysis of polygalacturonic acid with these enzymes
allowed the release of the monomer and of several oligomers,
depending on the hydrolysis conditions. The enzymes showed
a high activity on polygalacturonic acid and acted in endo
and exo modes. A mixture of oligoGalA was rapidly obtained
which was progressively converted into monomeric acid. Con-
trary to the other tested enzymes, EPG-M2 exhibited a low
conversion rate of oligoGalA. These results are useful in the
scope of POS production: reaction times should be controlled
if avoiding the production of large amounts of monomers is
the objective. Therefore, the present work brings information
on the choice of enzymes to be used in order to improve POS
production.
Acknowledgements
The authors are grateful to Biocatalysts and Novozymes for
the kind donation of enzyme preparations.
595
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