Hindawi Publishing Corporation
International Journal of Microbiology
Volume 2009, Article ID 631942, 8 pages
doi:10.1155/2009/631942

Research Article

Purification of an Exopolygalacturonase from Penicillium
viridicatum RFC3 Produced in Submerged Fermentation

Eleni Gomes, Rodrigo Simões Ribeiro Leite, Roberto da Silva, and Dênis Silva

Laboratory of Biochemistry and Applied Microbiology, Ibilce, Universidade Estadual Paulista (Unesp),
Rua Cristovao Colombo, 2265, Jd. Nazareth, São José do Rio Preto, SP, CEP 15054-000, Brazil

Correspondence should be addressed to Eleni Gomes, eleni@ibilce.unesp.br

Received 11 August 2009; Revised 1 October 2009; Accepted 3 November 2009

Recommended by Michael A. Cotta

An exo-PG obtained from Penicillium viridicatum in submerged fermentation was purified to homogeneity. The apparent
molecular weight of the enzyme was 92 kDa, optimum pH and temperature for activity were pH 5 and 50–55◦C. The exo-PG
showed a profile of an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of pectin with a high degree of esterification
(D.E.). Ions Ca2+ enhanced the stability of enzyme and its activity by 30%. The Km was 1.30 in absence of Ca2+ and 1.16 mg mL−1

in presence of this ion. In relation to the Vmax the presence of this ion increased from 1.76 to 2.07 μmol min−1mg−1.

Copyright © 2009 Eleni Gomes et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Pectinases are enzyme group that degrade pectic substances
and are classified according to their mechanism of action in
methylesterases (EC.3.1.11.1) that remove methoxyl groups
from highly or partially esterified galacturonan; polygalac-
turonases catalyse the hydrolysis of the glycosidic bonds
in a random fashion (endopolygalacturonase-EC.3.2.1.15)
or from nonreducing end of homogalacturonan releasing
galacturonic or digalacturunic acid residues (exopolyglactur-
onases EC.3.2.1.67 and EC 3.2.1.82) and lyases (PL) which
cleave the glycosidic bonds by trans-elimination (pectato
lyase-EC.4.2.2.2 and exopectato lyase-EC. 4.2.2.9) [1].

Production of pectinase by fungi generally is influenced
by the composition of the medium, in particular the carbon
and nitrogen sources and physicochemical conditions such as
pH, temperature, and aeration, besides the fermentative sys-
tem employed. Solid state (SSF) or submerged fermentations
(SmF) have been proposed for enzyme production under
laboratory conditions using agricultural and agroindustrial
wastes, and several forms of PG have been purified from both
fermentative processes [2, 3].

The purification of PG is an important tool for com-
prehension of its properties and reveals its structure and
functional mechanism which are important to knowledge

of the action of these enzymes in the plant infections
process, in industrial application, and the importance of
them in the biomass degradation. Fungal PGs generally are
monomeric proteins with a carbohydrate content of 5–81%
and molecular masses in a range from 13 to 82 kDa [4–8].

In a previous paper [9], we described the purification
of an exo-PG produced by Penicillium viridicatum RFC3 in
SSF, that enzyme exhibited a molecular weight of 24 kDa
and was strongly stimulated by Ba2+. In this paper we report
the production of polygalacturonase by the same fungus in
SmF, with orange bagasse and wheat bran as carbon sources,
and the purification of another exo-PG with different
physicochemical properties from the earlier enzyme.

2. Material and Methods

2.1. Microorganism. The microorganism used was Penicil-
lium viridicatum RFC3, isolated from decaying vegetables in
São José do Rio Preto, SP, Brazil and maintained as stock
culture on Sabouraud dextrose agar (Oxoid) containing
0.3% citrus pectin at 7◦C.

2.2. Media, Cultivation of Microorganism, and Enzyme
Production. Polygalacturonase was produced by submerged
fermentation (SmF) in a 500 mL Erlenmeyer flask containing



2 International Journal of Microbiology

100 mL of sterilized (120◦C/30 minutes) liquid medium
containing 3% carbon source (1.5% ground orange bagasse,
provided by Bascitrus (Mirassol, SP) and 1.5% wheat bran)
and 1 g L−1 of (NH4)2SO4 and MgSO4.7H2O. The medium
was sterilized at 120◦C for 30 minutes, inoculated with a
volume of conidial suspension in 0.1% Tween 80 equivalent
to 107 spores per mL in the final medium and cultured at
28◦C for 4 days. Every day, one flask was removed and the
fermented material was filtered and centrifuged at 10,000 g
for 15 minutes at 4◦C. The supernatant was used as crude
enzyme solution.

2.3. Enzyme Activity Measurements. Exopolygalacturonase
(exo-PG) activity was assayed in a reaction mixture contain-
ing 1% citrus pectin solution with a degree of esterification
(D.E.) of 92% (Sigma) in 0.2 M sodium acetate buffer (pH
4.5) at 45◦C for 10 minutes. The number of reducing groups,
released by enzymatic action, was measured by the DNS
method [10] and expressed as galacturonic acid. One unit
of enzyme activity (U) was defined as the amount of enzyme
releasing one μmol of galacturonic acid per minute under the
assay conditions [3].

Endo-polygalacturonase (endo-PG) was measured vis-
cosimetrically by adding 2.0 mL of crude enzyme to 6.0 mL
of 0.2 M citrate-NaOH buffer (pH 5.5) containing 3% citrus
pectin with 92% D.E. (Sigma). The reaction mixture was
incubated at 45◦C for 10 minutes, after which its viscosity
was determined with a Basic viscosimeter (Fungilab). The
blank contained thermally inactivated crude enzyme. One
unit of enzyme activity (U) was defined as the amount of
enzyme that reduced the initial viscosity by 50% per minute
under assay conditions.

2.4. Enzyme Purification Procedure. A 1300 mL volume of
crude enzyme solution obtained after 96 hours of fermen-
tation was dialyzed overnight against 20 mM acetate buffer
pH 4.5 in acetate cellulose membrane (Pharmacia) and
concentrated by ultrafiltration with Quixstand Benchtop of
GE Healthcare with a 10 kDa cut-off. The retentate was
directly loaded on a Sephadex G-75 (Pharmacia) column
(2.6 × 90 cm) equilibrated with 40 mM acetate buffer (pH
5.0) and eluted with the same buffer at a flow rate of
0.3 mL min−1. Fractions of 4 mL were collected and assayed
for PG activity. The objective of this procedure was to
estimate the number of isoforms of PG present in the crude
enzyme solution.

In another assay, the same volume of crude enzyme was
concentrated by ultrafiltration with Quixstand Benchtop of
GE Healthcare with a 10 kDa cut-off and then filtered with a
50 kDa cut-off membrane and loaded directly on a Sephadex
G-75 column following the procedure described above.

Protein fractions collected from the Sephadex column,
corresponding to the PG activity peak, were pooled and
loaded on a Q Sepharose column (Aldrich 30 × 1 cm) equi-
librated with 40 mM acetate buffer, pH 4.5. The adsorbed
material was eluted with a linear gradient (0.0 to 1.0 M) of
NaCl, in the same buffer, at a flow rate of 0.6 mL min−1. The
protein fraction with PG activity was desalted overnight by
dialysis against 20 mM acetate buffer, pH 4.5, at 4◦C.

2.5. Analytical Electrophoresis. The molecular weight of the
purified enzyme was determined by SDS-PAGE in a Mini
Protean II apparatus (10 × 8 cm) (Biorad). Electrophoresis
was carried out on a 4% (w/v) polyacrylamide stacking gel
and 10% (w/v) resolving gel in Tris/glycine buffer (pH 8.3)
[11] with the Sigma molecular weight marker M6539 (6.5–
180 kDa) in a parallel lane. The protein band was visualized
by silver staining.

Analytical isoelectric focusing PAGE was performed in
Ettan IPGphor II Isoelectric focusing system (Amersham)
by electrophoresis in a 12.5% polyacrylamide gel (14 ×
15 cm) containing 5% Pharmalyte (GE Healthcare), which
established a pH gradient from pH 3.0–10.0, in accordance
with the instructions of the supplier. The gel was silver-
stained for protein determination.

2.6. Protein Estimation. Protein concentration was deter-
mined in the concentration ranges of 1–10 and 10–
100 μg mL−1 by the Bradford microassay [12], with bovine
serum albumin (BSA) as the standard.

2.7. Properties of Purified Enzyme. All enzyme catalytic
properties were assayed with 1% citrus pectin (D.E 92%,
Sigma) as substrate using the procedure for enzyme activity
determination described above and carried out with three
replicates. PG activity was assayed as a function of pH, in
McIlvaine buffer (pH 4.0–8.0), at 55◦C, and temperature,
in McIlvaine at the pH optimum, incubated at temperatures
between 35◦C and 70◦C.

The thermal stability was investigated by remeasuring the
activity of the purified enzyme solution after it had been
kept for 1 hour, in the absence of substrate, at temperatures
between 5 and 80◦C. The half-life was determined by
incubating the enzyme solution at 60◦C for 1 hour. In these
tests, the initial and final PG activities were determined at
optimum pH and temperature.

pH stability of the purified enzyme was evaluated by
dispersing (1 : 1, v/v) enzyme solution in 0.1 M McIlvaine
buffer (pH 3.0–8.0) and 0.1 M glycine-NaOH buffer (pH 8.0–
10.5) and maintaining these solutions at 25◦C for 24 hours.
An aliquot was taken to determine the remaining activity at
the optimum pH and temperature.

The Michaelis constant (Km) and Vmax values were
determined from Lineweaver-Burk plots of enzyme activity
measured with 92% D.E. citrus pectin (Sigma) as substrate,
at concentrations between 1.0 and 10.0 mg mL−1 at optimum
pH and temperature. The results were plotted with the
program Grafit 5.0.

The effects of metal ions (Ag+, K+, Na+, Cu2+, Ca2+, Ba2+,
Co2+, Hg2+, Ni2+, Mg2+, Mn2+, Zn2+, Cr3+, Al3+, Fe3+) and
EDTA on enzyme activities were assayed at concentrations of
2.0, 5.0, and 10.0 mM in the reaction mixture.

The substrate specificity was assessed by testing poly-
galacturonic acid, citrus pectin (Sigma) of 26% and 92%
D.E., and apple pectin (87% D.E.-Sigma) as substrates under
optimum conditions for enzyme activity.

The products of the hydrolysis of citrus pectin (92% D.E.)
by PG were analyzed by paper chromatography on Whatman



International Journal of Microbiology 3

0

5

10

15

20

25
B

io
m

as
s;

re
du

ci
n

g
su

ga
r

(m
g.

m
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1
)

0 24 48 72 96 120

Time (h)

0

1

2

3

4

P
G

ac
ti

vi
ty

(U
.m

L
−1

)

PG activity
Reducing sugars
Biomass

Figure 1: Production of PG by P. viridicatum RFC3 in submerged
fermentation with orange bagasse (1.5%) and wheat bran (1.5%) as
carbon sources.

no. 1 paper, with ethyl acetate/isopropanol/water (6 : 3 : 1,
by volume) as the mobile phase.

3. Results and Discussion

3.1. Production of PG by SmF. During the submerged
fermentation in medium containing orange bagasse and
wheat bran as carbon sources (Figure 1), the PG activity
increased continuously between 24 and 96 hours of batch
culture varying from 3.0 to 4.1 U mL−1 by three repetitions.
The enzyme activity was lower than that obtained in SSF with
the same fungus (18 U mL−1) [9]. Orange bagasse (pressed
peel, pulp, rag, and seeds) has an average pectin content of
223 g Kg−1 (dry weight) [13, 14] and is a good inducer of
pectinases [15, 16] while wheat bran is a good nutrient source
[17]. In spite of this, the PG activity obtained was lower than
that reported for enzyme production in SmF on other carbon
sources. Patil and Dayanand [18] obtained 30.3 U mL−1 of
exo-PG from the Aspergillus niger DMF 27 when they used
deseeded sunflower and glucose as carbon sources. The
same fungi produced 14.5 U mL−1 of pectinase in medium
containing citrus pectin [19]. The related A. sojae produced
15.5 U mL−1 of pectinases in medium supplemented with
maltrin [20].

PG production increased substantially when the reducing
sugar content dropped and after the log phase of fungal
growth. The consumption of reducing sugars promotes fun-
gal growth and ensures success in colonization of medium
[21]. On the other hand, a high level of reducing sugars
in the medium is an important factor limiting of enzyme
production, owing to catabolic repression [2].

3.2. Purification of PG. The crude enzyme solution obtained
after 96 hours of fermentation, concentrated by ultrafiltra-
tion with a 10 kDa cut-off and separated on a Sephadex G-75
column, afforded 5 peaks of PG activity suggesting a number
of isoforms (Figure 2(a)). In work published previously,

0

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25

P
ro

te
in

(m
g.

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0 100 200 300 400 500 600

Elution volume (mL)

0

2

4

6

8

10

12

P
G

ac
ti

vi
ty

(U
.m

L
−1

)

Protein
PG activity

(a)

0

1

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4

5

P
ro

te
in

(m
g.

m
L−

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)

0 200 400 600 800

Elution volume (mL)

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4

5

6

P
G

ac
ti

vi
ty

(U
.m

L
−1

)

280 nm
Protein
PG activity

(b)

Figure 2: Gel filtration chromatography of PG on Sephadex G-75
(2.5 × 90 cm) equilibrated with 40 mM acetate buffer, pH 4. (a)
Crude extract concentrated by ultrafiltration with 10 kDa cut-off,
(b) crude extract after ultrafiltration with 10 and 50 kDa cut-off.

we obtained 5 PG fractions by solid-state fermentation of
P. viridicatum RFC3 on a 1 : 1 mixture (w/w) of wheat
bran and orange bagasse at 67% moisture [17], while and
recently in another experiment, 6 PG fractions were obtained
in the same medium, but at 80% of moisture [9]. We
supposed that the moisture difference between the two media
could have influenced the expression of PG isoforms, but
now, in liquid medium, we found the same number of
isoforms as in the medium with lower moisture content. The
influence of water potential (aw) variation on the isoform
expression of extracellular enzymes has been attributed to
changes in the permeability of fungal membrane, limitation
of sugar transport, and presence or absence of inducer
[22]. We have also reported changes in the expression of
extracellular enzymes with variation of culture conditions
and fermentative techniques [17].

The sequential productions of pectinases have been
reported by other authors in various microorganisms [15].
Pectinases produced by the same microorganism exhibit
differing molecular weights, degrees of glycosylation and



4 International Journal of Microbiology

0

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60

A
bs

or
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Elution volume (mL)

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1.2

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1.8

2

P
G

ac
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vi
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(U
.m

L
−1

)

0280 nm
NaCl (0–1 M)
PG

Figure 3: Ion-exchange chromatography of PG on Q Sepharose (1
× 30 cm) equilibrated with 40 mM acetate buffer, at pH 4.0 and
eluted with NaCl (0–1 M).

specificities, due either to posttranslational modification
of a protein from a single gene or to the expression of
different genes, and such variants are important in balancing
specific modes of action (endo or exo) and substrate affinity
[5, 23].

When the crude enzyme solution was concentrated by
ultrafiltration at 10 kDa and shortly after filtered through
a 50 kDa cut-off membrane, and the concentrated material
loaded on a Sephadex G-75 column, only one polygalac-
turonase peak (PG III) was eluted, indicating that the
majority of the PG fractions from crude enzyme were of
low molecular weight and only one (PG III) was retained
by the 50 kDa membrane (Figure 2(b)). The PG peak,
corresponding to elution volume 215 to 315 mL, was applied
to a Q Sepharose column at pH 4 and, after elution with NaCl
solution (around 0.5 M), only one peak of PG was detected
(Figure 3). SDS/PAGE revealed that this PG peak was homo-
geneous (Figure 4). Three stages were necessary to reach
a 37.7-fold PG III purification, with a final yield of 3.4%
(Table 1).

3.3. Characterization of PG. Molar mass of PGIII was
estimated to be 92.2 kDa (Figure 4). Homogeneity was
confirmed by isoelectric focusing, where a single band
with isoelectric point (pI) of 5.4 was observed (figure not
shown).

Purified PG III exhibited higher activity on highly
esterified pectin (Figure 5(a)) suggesting that PG is a poly-
methylgalacturonase. However, the activity on citrus pectin
(5.1 U mL−1) was appreciably higher than that on apple
pectin, although both have high D.E. (92% and 82%, resp.).

The mode of action of the polygalacturonase on citrus
pectin (92% D.E.) was also assessed by measuring viscosity
in the reaction mixture, to estimate endo activity. Figure 5(b)
shows that the enzyme had a poor capacity to decrease

92.24

6.5

14.2
18.4
29

48.5

66

97.4

116

180

Figure 4: SDS-PAGE of purified PG.

0

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5

6

P
G

ac
ti

vi
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(U
.m

L
−1

)

1 2 3 4 5

Substrates

(a)

0

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2

3

4

5

6

P
G

ac
ti

vi
ty

(U
.m

L
−1

)

Endo Exo

(b)

Figure 5: Properties of exo-PG III. (a) Substrate specificity: 1:
polygalacturonic acid; 2: citrus pectin (26% D.E.); 3: citrus pectin
(92% D.E.); 4: apple pectin (82 D.E.); 5: commercial citrus pectin
(87% D.E.); (b) mode of action of PG (endo- and exo-activity).



International Journal of Microbiology 5

Table 1: Summary of purification of exo-PG III from Penicillium viridicatum RFC3.

Step
Volume PG Protein

U total
S.A.∗ Yield Fold

(mL) (U mL−1) (mg mL−1) (U mg−1) (%) purification

Crude enzyme 1300 3.8 0.08 4966 49.0 100 —

Ultrafiltration

45 63.9 0.72 2875.5.4 88.8 57.9 9.6
in 10 and 50

kDa cut-off

membranes

Fraction I from
198 5.1 0.03 1003.9 195.0 20.2 21.2Sephadex G75

column

Fraction I from
35 4.9 0.014 169.8 346.4 3.4 37.7Q Sepharose

column.
∗S.A. (Specific Activity).

3G 2G 1G H

Figure 6: Products from hydrolysis of citrus pectin (92% D.E.) by
exo-PG III—3G: trigalacturonic acid, 2G: digalacturonic acid, 1G:
galacturonic acid, H: enzyme hydrolyzate.

the viscosity of the pectin solution, indicating mainly exo-
activity. Analysis by paper chromatography revealed that
galacturonic acid was the sole product of enzyme hydrolysis
after 5 minutes of incubation (Figure 6), reinforcing the
evidence for an exopolygalacturonase (exo-PG).

The effect of ions on exo-PG III activity was tested at
concentrations of 2.0, 5.0, and 10.0 mM. The ions Hg2+,
Zn2+, Mg2+, Fe3+ and Cu2+ strongly inhibited the exo-PG
activity while, Cr3+, and K+ only partially inhibited it. On
the other hand, ions such as Na+, Mn2+, and Ca2+, at 5 and
2 mM, enhanced the enzymatic activity by 10% (and Ca2+

by 30% at 5 mM). EDTA inhibited the enzyme activity by 7–
17%, depending on the concentration (Table 2).

Na+ has been described as an inducer of PG activity
by Chun and Huber [24]. Divalent ions such as Ca2+ act
directly on the pectin molecule, stabilizing the negatively
charged carboxyl groups and indirectly stimulating the
polygacturonase activity [25, 26].

The concentration of the ions Cr3+, Al3+, Ag+, K+, and
Ni2+ in the reaction mixture was related anomalously to
their inhibitory effects, which were highest at the lowest
concentration.

Exo-PG III showed maximum activity at pH 5.0 and
50% of its maximum activity at pH 8.0 (Figure 7(a)). It was
stable in the pH range 4–5, but the presence of Ca2+ ions in
the reaction mixture resulted in an increase in the enzyme
stability, with 90–100% of the full activity in a broader pH
range of 4.0–9.0 (Figure 7(b)).

With respect to temperature, optimal PG activity was
observed at 50–55◦C (Figure 8(a)). In the absence of sub-
strate for 1 hour, exo-PG III showed 85–100% of the original
activity at 5–35◦C, while at 70◦C, the enzyme lost 55% of its
initial activity. However, in the presence of Ca2+ (10 mM),
the PG maintained 80–100% of the initial activity at 5–55◦C
and around 70% when maintained at 70◦C (Figure 8(b)).
In another experiment, exo-PG III was maintained at 60◦C
for 1 hour (samples taken every 5 minutes) in the presence
and absence of Ca2+. Without this ion, the enzyme preserved
50% of its initial activity for 25 minutes, while in its
presence, the enzyme half-life increased to 37 minutes
(Figure 8(c)). According to Hernández et al. [27], Ca2+

protects enzymes against thermal denaturation and plays a
vital part in maintaining their active configuration at high
temperatures.

The half-life time at 60◦C of exo-PG III from P. viridi-
catum was higher than that described by Shanley et al. [28]
for the three polygalacturonases purified from P. pinophilum.
Those half-lifes were 10.6 minutes for PGI, 16.5 minutes
for PGII, and 9.5 minutes for PGIII. However, the PG from
Acrophialophora nainiana purified by Celestino et al. [29]
had a half-life of 20 minutes at 60◦C and 3 minutes at 70◦C.

The exo-PG III affinity for citrus pectin (92% D.E.) was
determined by the Lineweaver-Burk plot. In the presence
of Ca2+, the substrate affinity increased, with Km falling
from 1.30 (±0.04) to 1.16 (±0.05) mg mL−1. Similarly, an
improvement was observed in Vmax in the in presence



6 International Journal of Microbiology

0

0.5

1

1.5

2

2.5

3

3.5

4

P
G

ac
ti

vi
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(U
.m

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−1

)

3 4 5 6 7 8 9

pH

(a)

0

20

40

60

80

100

R
el

at
iv

e
ac

ti
vi

ty
(%

)

4 5 6 7 8 9 10

pH

(b)

Figure 7: Properties of exo-PG III. (a) Effect of pH on enzyme
activity; (b) effect of pH on enzyme stability. : PG alone ©: PG
plus Ca2+.

of this ion, with values rising from 1.76 (±0.06) to 2.07
(±0.03) μmol min−1 mg−1.

Other purified exo-PGs have shown widely differing
values in their kinetic parameters, which range from
0.11 mg mL−1 to 4.47 mg mL−1 for Km and 1.68 μmol
min−1 mg−1 to 1100 μmol min−1 mg−1 for Vmax [30–33].

The properties of exo-PG III from P. viridicatum
described in this work revealed quite different properties
from those of another PG (exo-PG II) from the same fungus
[9] such as molecular weight (92 and 24 kDa, resp.) and
optimum pH of activity (5.0 and 6.0, resp.). Besides, the PG
III, purified in this work, proved to be an exo-PG inhibited
by to Ba2+ at 10 mM which enhanced the stability of exo-
PG II, on the other hand, its stability against pH variation,
thermostability, and substrate affinity were improved in
presence of Ca2+.

0

1

2

3

4

P
G

ac
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(U
.m

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−1

)

30 40 50 60 70

Temperature (◦C)

(a)

0

20

40

60

80

100

120

R
el

at
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e
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ti
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ty
(%

)

0 10 20 30 40 50 60 70 80

Temperature (◦C)

(b)

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50

60

70

80

90

100

110

R
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ty
(%

)

0 5 10 15 20 25 30 35 40 45 50 55 60

Time (min)

(c)

Figure 8: Properties of exo-PGIII. (a) Effect of temperature on
enzyme activity; (b) effect of temperature on enzyme stability. (c)
Enzyme stability at 60◦C (half-life). �: PG alone; �: PG plus Ca2+.

The effect of Ca2+ and Ba2+ on stability and activity
of exo-PGs brings out one step closer for improvement of
pectinase efficiency when used in bioprocess application as
juice extraction.



International Journal of Microbiology 7

Table 2: Effect of metal ions on the exo-PG III activity.

Ions Residual activity (%)

Ion concentration (mM)

2 S 5 s 10

Control 100 100 100

Al3+ 67 ±3.0 90 ±10 99 ±4

Fe3+ 43 ±5 15 ±2 ND

Cr3+ 60 ±5 83 ±4 93 ±5

Zn2+ 3 0 0

Hg2+ 0 0 0

Mn2+ 110 ±2 93 ±3 90 ±4

Mg2+ 27 ±2 ND ND

Cu2+ 33 ±4 ND ND

Ca2+ 110 ±2 130 ±3 107 ±1

Co2+ 107 ±3 100 ±5 93 ±2

Ag+ 87 ±3 90 ±1 93 ±1

Na+ 123 ±3 107 ±2 98 ±2

K+ 63 ±4 77 ±5 84 ±3

Ba2+ 90 ±1 90 ±2 20 ±1

Ni2+ 63 ±3 78 ±2 81 ±1

EDTA 93 ±2 83 ±3 83 ±3

ND: not determined.
Data are mean of three repetitions.

Acknowledgments

The authors wish to thank the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP) and Con-
selho Nacional de Pesquisa e Desenvolvimento Tecnológico
(CNPq) for financial support.

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