Hindawi Publishing Corporation
Enzyme Research
Volume 2013, Article ID 438645, 7 pages
http://dx.doi.org/10.1155/2013/438645

Research Article
Purification and Properties of Polygalacturonase Produced by
Thermophilic Fungus Thermoascus aurantiacus CBMAI-756 on
Solid-State Fermentation

Eduardo da Silva Martins,1 Rodrigo Simões Ribeiro Leite,2

Roberto da Silva,3 and Eleni Gomes3

1 Laboratório de Microbiologia, Universidade do Estado de Minas Gerais (UEMG), Avenida Prof. Mario Palmerio 1000,
38200-000 Frutal, MG, Brazil

2 Faculdade de Ciências Biológicas e Ambientais (FCBA), Universidade Federal da Grande Dourados (UFGD),
Rodovia Dourados-Itahum, Km 12, 79804-970 Dourados, MS, Brazil

3 Laboratório de Bioquı́mica e Microbiologia Aplicada, Instituto de Biociências, Universidade Estadual Paulista (UNESP),
Rua Cristovão Colombo 2265, Jd. Nazareth, 15054-000 São José do Rio Preto, SP, Brazil

Correspondence should be addressed to Eduardo da Silva Martins; edusmartins@yahoo.com.br

Received 25 February 2013; Revised 9 August 2013; Accepted 11 August 2013

Academic Editor: Toshihisa Ohshima

Copyright © 2013 Eduardo da Silva Martins 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.

Polygalacturonases are enzymes involved in the degradation of pectic substances, being extensively used in food industries,
textile processing, degumming of plant rough fibres, and treatment of pectic wastewaters. Polygalacturonase (PG) production by
thermophilic fungusThermoascus aurantiacus on solid-state fermentation was carried out in culture media containing sugar cane
bagasse and orange bagasse in proportions of 30% and 70% (w/w) at 45∘C for 4 days. PG obtained was purified by gel filtration and
ion-exchange chromatography. The highest activity was found between pH 4.5 and 5.5, and the enzyme preserved more than 80%
of its activity at pH values between 5.0 and 6.5. At pH values between 3.0 and 4.5, PG retained about 73% of the original activity,
whereas at pH 10.0 it remained around 44%. The optimum temperature was 60–65∘C. The enzyme was completely stable when
incubated for 1 hour at 50∘C. At 55∘C and 60∘C, the activity decreased 55% and 90%, respectively. The apparent molecular weight
was 29.3 kDa, 𝐾

𝑚

of 1.58mg/mL and 𝑉max of 1553.1 𝜇mol/min/mg. The presence of Zn+2, Mn+2, and Hg+2 inhibited 59%, 77%, and
100% of enzyme activity, respectively.The hydrolysis product suggests that polygalacturonase was shown to be an endo/exoenzyme.

1. Introduction

Pectinases are a heterogeneous group of enzymes that hydro-
lyze the pectic substances present in plantmaterial.The classi-
fication of pectinases is based on their mode of attack on the
galacturan backbone of the pectin molecule, on specificity by
substrate, or according to region of molecule where it acts [1].

The polygalacturonases catalyze the hydrolysis of gly-
cosidic 𝛼-1-4 linkages in pectic acid and are of two types:
endo-polygalacturonases (endo PG, EC 3.2.1.15), which act
by hydrolysis of internal glycosidic bonds 𝛼-1-4 of polygalac-
turonic acid at random form, resulting in molecule depoly-
merization with release of oligogalacturonic acids, and exo-
polygalacturonases (exo PG, EC 3.2.1.67) which hydrolyse

alternate 𝛼-1-4 glycosidic linkages of polygalacturonic acid
from the nonreducing end, releasing unsaturated mono- or
digalacturonic acids [2, 3].

This group of enzymes has been widely used in the food
industry process such as clarification and viscosity reduction
of fruit juices, preliminary treatment of grape juice for wine
industries, tomato pulp extraction, oil extraction, and tea
fermentation and in the textile industry in fibers degumming
[4, 5].

In the literature, it has been reported that the type
of fermentation influences the enzymes properties, such
as thermostability and tolerance to pH variations [6, 7].
According to Acuña-Argüelles et al. [8], pectinases obtained



2 Enzyme Research

by Aspergillus oryzae cultivation in solid-state fermentation
(SSF) were more resistant to pH and temperature changes
compared to those obtained by submerged fermentation
(SmF).Moreover,Martin et al. [9] reported that polygalactur-
onase obtained by Thermomucor indicae-seudaticae showed
higher thermostability in SmF than that in SSF. Thus, the
present study aimed to purify polygalacturonase produced by
thermophilic fungus Thermoascus aurantiacus in solid-state
fermentation and compare its properties with those of the
purified polygalacturonase produced by the same fungus in
SmF, in a work reported by Martins et al. [10].

2. Materials and Methods

2.1. Microorganism. Thermophilic fungus Thermoascus
aurantiacus CBMAI756 was used. The strain is deposited
in the Coleção Brasileira de Microrganismos de Indústria e
Meio Ambiente-CBMAI, UNICAMP, Campinas, SP.

2.2. Media, Cultivation of Microorganism, and PG Production.
SSFwas carried out using a 250mLErlenmeyer flask contain-
ing 5 g of sterilized mixture of sugar cane bagasse and orange
bagasse in proportions of 30 and 70% (w/w). The material
was inoculated with 5mL of micelial suspension (14.5mg dry
micelial mass/g dry substrate) which was obtained from a 4-
day agar slant culture suspended in sterile distilled water.

After inoculation, 10mL of nutrient solution composed of
0.1% NH

4
NO
3
, 0.1% NH

4
H
2
PO
4
, 0.1% MgSO

4
⋅7H
2
O at pH

5.0 was added to each of the several flasks. The final moisture
content of the medium was approximately 70%. Cultivation
was carried out at 45∘C for 4 days. At 24 h intervals, the
material corresponding to one Erlenmeyer flask was mixed
with 40mL distilled water, stirred for 40min, filtered under
vacuum, and centrifuged.The supernatant was used as crude
enzyme solution.

2.3. Enzyme Activity Measurements. Exo-polygalacturonase
(exo-PG) activity was assayed in amixture containing 0.4mL
of 1% of citrus pectin solution (26% esterified—Sigma) in
0.2M sodium acetate buffer (pH 5.5) and 0.1% of crude
enzyme solution at 60∘C for 10min. The number of reducing
groups, expressed as galacturonic acid released by enzymatic
action, was quantified by the DNS method [11]. One unit of
enzyme activity (U) was defined as the amount of enzyme
releasing 1 𝜇mol of galacturonic acid per minute under the
assay conditions.

Endo-PG activity was measured viscosimetrically by
adding 2mL of crude enzyme to 6mL of 0.2M acetate-
NaOH buffer (pH 5.5) containing 3% of low-esterified citrus
pectin (Sigma). The reaction mixture was incubated at 60∘C
for 15min, and its viscosity was determined with a basic
viscosimeter (Fungilab). One unit of enzyme activity was
defined as the amount of enzyme that reduced the initial
viscosity by 50% per minute.

2.4. Enzyme Purification. 150mL of crude enzyme extract
was dialyzed against 10mM acetate buffer, pH 4.0, overnight.

After dialysis, it was lyophilized and resuspended in 20mL of
10mM acetate buffer, pH 4.0.

Gel filtration chromatography with Sephadex G-75 col-
umn (90.0 cm × 2.5 cm—Pharmacia) was used, and the
elution occurred with 20mM acetate buffer pH 4.0 at a flow
rate of 16.8mL/h. The PG activity (DNS assay method) and
protein content of each tube were determined. The fractions
containing the peak of enzyme activitywere joined to the next
step purification process in ion-exchange column.

For ion-exchange chromatography, SP Sepharose column
(20.0 cm × 2.5 cm—Aldrich) was used and the elution was
made with same acetate buffer and flow at salt gradient from
0 to 1.1MNaCl.The solution containing PG peak activity was
desalted by dialysis against 10mM acetate buffer, pH 4.0, at
4∘C, overnight.

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 in polyacrylamide gel, consisting of a 4%
(w/v) stacking gel and 10% (w/v) resolving gel in Tris/glycine
buffer (pH 8.3), by the method of Laemmli [12]. The molecu-
lar weight marker (SigmaM6539, 6.5–180 kDa) was used.The
protein band was visualized by silver staining.

Analytical isoelectric focusing was performed in an Ettan
IPGphor II Isoelectric Focusing system (Amersham) by
electrophoresis in a 7.5% polyacrylamide gel (14 × 15 cm)
containing 5% Pharmalyte (pH 3.0–10.0) (purchased from
Amersham Bioscience). The gel was silver-stained to reveal
protein.

2.6. Protein Determination. Protein concentration was deter-
mined in the concentration of 10–100 𝜇g/mL by the microas-
say method of Bradford [13], using bovine serum albumin
(BSA) as the standard.

2.7. Enzyme Characterization. For characterizing the PG
activity, the DNS assay method was used. Optimal activity
of purified PG was assayed as a function of pH, in 200mM
acetate buffer (pH 3.0–5.5), citrate-phosphate (pH 6.0–7.0),
Tris-HCl (pH 7.5–8.5), and glycine-NaOH (pH 9.0–11.0),
at 60∘C with 2% low-esterified pectin (2%) as substrate.
The pH stability of PG was evaluated by incubation of
enzymatic solution in 0.1M buffer solutions acetate (pH 3.0
to 5.5), citrate-phosphate (pH 6.0–7.0), Tris-HCl (pH 7.5–
8, 5), and glycine-NaOH (pH 9.0–11.0), in the absence of
pectin, for 24 hours. After this period, an aliquot was taken
tomeasure residual activity under conditions of optimumpH
and temperature.

The effect of temperature on enzymatic activity was
evaluated by incubation of reaction mixture at temperatures
from 40∘C to 80∘C for 10 minutes, at optimum pH. The
thermal stability was determined by measuring the residual
activity of the enzyme after 1 h of incubation, in absence of
substrate, at temperatures between 10 and 90∘C. After this
period, samples were taken to assay enzyme activity under
conditions of optimum pH and temperature.



Enzyme Research 3

Table 1: Purification of the PG produced byThermoascus aurantiacus in SSF.

Step Volume (mL) Total activity (U) Total protein (mg) Specific activity
(U/mg protein) Purification fold Yield (%)

Lyophilized extract 8.00 1800.00 30.00 60.00 1.00 100.00
Sephadex G-75 54.00 1058.40 3.19 331.60 5.53 58.80
SP Sepharose 12.60 255.80 0.0478 5351.50 89.19 14.20

To determine substrate specificity, solution of polygalac-
turonic acid, citrus pectin with 26% and 92% degree of ester-
ification, and apple pectin (Sigma) were used as substrates at
2.0% in 0.2M acetate buffer. The reaction was conducted in
optimal conditions of enzyme activity.

The influence of metallic ions on PG activity was eval-
uated by incubation of enzyme in the presence of different
ionic solutions at 2mM (Fe+3, Ag+, Ca+2, Mg+2, Mn+2,
Zn+2, K+, and Hg+2) and EDTA at final concentration in
the reaction medium of 2mM. After 10min. incubation at
4∘C, the residual activity was measured under conditions of
optimum enzyme activity.

The Michaelis constant (𝐾
𝑚
) and 𝑉max values were

determined from Lineweaver-Burk plots of enzyme activity
measured with citrus pectin with 26% degree of esterification
(Sigma) as substrates, at concentrations between 0.25 and
1.25% at optimum pH and temperature. The results were
plotted with the program Grafit 5.0.

The hydrolysis products of 26% esterified citrus pectin
and trigalacturonic acid were analyzed by chromatography
on Whatman no. 1 paper, using as solvent a mixture of n-
butanol, acetic acid, andwater at a ratio of 5 : 3 : 2, respectively,
and as developing solvent acetone and silver nitrate (to
saturation), washedwith alcoholic hydroxide silver for visual-
ization of the bands.Themono-, di-, and trigalacturonic acids
(Sigma) were used as standards.

3. Results and Discussion

3.1. Purification of PG. The crude enzyme solution obtained
by fungus culture on solid-state fermentation applied on
Sephadex G-75 gel column showed only one peak of enzyme
activity, which was detected between 160.0mL and 256.2mL
(Figure 1(a)).This step resulted in an increasing in the specific
activity from 60.0U/mg to 331.6U/mg protein, in 5.2-fold
enzyme purification and 58.8% yield (Table 1).

In the second step, 50mL of enzymatic extract was
applied on ion-exchange chromatography, using 20mM
acetate-NaOH buffer, at pH 4.0. Two protein peaks were
observed from the elution volumes of 42.0mL and 88.2mL
before the start of the salt gradient and three between 0.15M
and 0.7M NaCl. Polygalacturonase was eluted at 0.9M salt
concentration (Figure 1(b)). The specific activity increased
from 331.6U/mg to 5351.5U/mg protein, with 89.2-fold
enzyme purification and 14.2% yield (Table 1).

The samples application on gel electrophoresis indicated
that the enzyme was purified to homogeneity and hadmolec-
ular weight of 29.3 kDa (Figure 2), similar to PG produced
in submerged fermentation presented by Martins et al. [10].

0

20

40

60

80

100

120

140

160

0

5

10

15

20

25

30

4
.2

2
5
.2

4
6
.2

6
7
.2

8
8
.2

1
0
9
.2

1
3
0
.2

1
5
1
.2

1
7
2
.2

1
9
3
.2

2
1
4
.2

2
3
5
.2

2
5
6
.2

2
7
7
.2

2
9
8
.2

3
1
9
.2

3
4
0
.2

3
6
1
.2

3
8
2
.2

4
0
3
.2

4
2
4
.2

4
4
5
.2

To
ta

l p
ro

te
in

 (𝜇
g/

m
L)

PG
 ac

tiv
ity

 (U
/m

L)

Elution volume (mL)

(a)

0

0.2

0.4

0.6

0.8

1

1.2

0

5

10

15

20

25

30

4
.2 2
1

3
7
.8

5
4
.6

7
1
.4

8
8
.2

1
0
5

1
2
1
.8

1
3
8
.6

1
5
5
.4

1
7
2
.2

1
8
9

2
0
5
.8

2
2
2
.6

2
3
9
.4

2
5
6
.2

2
7
3

2
8
9
.8

3
0
6
.6

3
2
3
.4

3
4
0
.2

3
5
7

3
7
3
.8

3
9
0
.6

4
0
7
.4

4
2
4
.2

4
4
1

4
5
7
.8

N
aC

l (
M

)

PG
 ac

tiv
ity

 (U
/m

L)
To

ta
l p

ro
te

in
 (𝜇

g/
m

L)

Elution volume (mL)

(b)

Figure 1: Elution of PG activity from chromatography columns
previously equilibrated with 20mM acetate buffer, pH 4.0: (a)
Sephadex G-75 column (3.0 × 80 cm) and (b) SP Sepharose (2.5 ×
20 cm) eluted with a NaCl gradient (0–1.1M). -◼- PG activity; -◻-
protein; — NaCl gradient.

PGs with very similar molecular weight were also described
by Saito et al. [14] (29.7 kDa) studying the fungus Rhizopus
oryzae and Niture and Pant [15] (30.6 kDa) studying the
fungus Fusarium moniliforme in solid-state fermentation.

3.2. Enzyme Properties. The highest activity was found
between pH 4.5 and 5.5 (Figure 3(a)) and when maintained
for 24 h in different pH values, in the absence of substrate, the
enzyme preserved more than 80% of its activity at pH values
between 5.0 and 6.5. In more acidic pH values (3.0 to 4.5), the
enzyme retained about 73% of the original activity, whereas at
pH 10.0 it remained around 44% (Figure 3(a)).

The response to the effects of pH presented by the enzyme
produced in SSFwas quite different from that observed for the
same enzyme produced in SmF presented by Martins et al.
[10], which showed maximum activity between pH 5.5 and



4 Enzyme Research

50

30

20

10

Figure 2: SDS-PAGE of purified PG from gel filtration and ion-
exchange chromatography. The numbers of the left indicate the
positions of molecular weight markers in kDa.

6.0 and was stable in a very narrow pH range (between 5.0
and 7.5).However, it is similar to the data reported by Siddiqui
et al. [16], with the PG produced by Rhizomucor pusillus in
solid-state fermentation, which showed optimum pH of 5.0
and a wide range of pH stability.

Regarding the influence of temperature on the enzyme
activity, it was observed that the PG was most active between
60–65∘C, with a reduction of about 75% activity at 75∘C.
When incubated for 1 hour at different temperatures, in
the absence of substrate, a pure PG maintained 100% of
the original activity at 50∘C. At 55∘C and 60∘C, the activity
decreased 55% and 90%, respectively, whereas at 70∘C the
enzyme was denatured (Figure 3(b)).

This result is similar to the thermostability of PG pro-
duced in solid-state fermentation by Rhizomucor pusillus,
which showed 100% stability at 50∘C for 1 hour, but at 60∘C its
stability decreased [16]. Comparison of these data with those
for enzyme produced in submerged fermentation related by
Martins et al. [10] indicates that the enzyme obtained from
SSF was less thermostable, since the enzyme SmF retained
25% and 10% at 60∘C and 70∘C, respectively.

The half-life of PG at 60∘C was approximately 5 minutes
(Figure 4), even lower than that found for the PG obtained in
SmF [10], in which half -life was approximately 10 minutes.

There are few reports in the literature on the influence
of temperature on the pectinases activity from thermophilic
fungi. Kaur et al. [17] reported the partial purification
and characterization of a polygalacturonase produced by
thermophilic fungus Sporotrichum thermophile in submerged
fermentation, which showed optimum temperature of 55∘C.
The purified enzyme by the fungusAcrophialophora nainiana
showed greater activity at 60∘C [18].

Kumar and Palanivelu [19] reported that purified PG of
the thermophilic fungus Thermomyces lanuginosus retained
only 4% of activity at 60∘C and was completely inhibited
when exposed for 1 hour at 70∘C.

0

10

20

30

40

50

60

70

80

90

100

0

5

10

15

20

25

2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5

Re
la

tiv
e a

ct
iv

ity
 (%

)

PG
 ac

tiv
ity

 (U
/m

L)

pH

(a)

0

10

20

30

40

50

60

70

80

90

100

110

0

5

10

15

20

25

30

35

0 10 20 30 35 40 45 50 55 60 65 70 75 80 85 90

Re
la

tiv
e a

ct
iv

ity
 (%

)

PG
 ac

tiv
ity

 (U
/m

L)

Temperature (∘C)

(b)

Figure 3: Effect of pH (a) and temperature (b) on the PGactivity and
stability. -◼- activity in the presence of substrate expressed in U/mL;
-◻- stability in absence of substrate expressed in % of the original
activity.

Pectinases obtained by Aspergillus oryzae cultivation in
solid-state fermentation (SSF) were more resistant to pH
and temperature changes compared to those obtained by
submerged fermentation (SmF) [8]. Moreover, Martin et al.
[9] reported that polygalacturonase obtained by Thermomu-
cor indicae-seudaticae showed higher thermostability in SmF
than that in SSF.

In relation to substrate preference, PG showed the highest
activity with 26% esterified citrus pectin (Table 2) similar
to that observed for the enzyme obtained from SmF by
Martins et al. [10], indicating that this fungal strain has a
polygalacturonase with a preference for hydrolyzing low-
esterified pectin.

To evaluate the influence of ions on PG activity, it was
observed that ion Ag+ caused 18% decrease in PG activity
(Table 3), while the same enzyme produced in SmF by
Martins et al. [10] had a 10% increase in its activity in their
presence.

The ions Mg+2, Zn+2 Mn+2, and EDTA also partially
inhibited the enzyme activity, with decrease of 24%, 59%,
77%, and 27%, respectively (Table 3), similar to the results
obtained for PG of SmF [10].



Enzyme Research 5

0

10

20

30

40

50

60

70

80

90

100

110

0 5 10 15 20 25 30 35 40 45 50 55 60 65

Re
la

tiv
e a

ct
iv

ity
 (%

)

Time (min)

Figure 4: Stability of PG at 60∘C in the absence of substrate
expressed in % of the original activity.

Table 2: Substrate specificity of PG from T. aurantiacus produced
on SSF.

Substrate PG activity (U/mL)
Citrus pectin 26% DE methoxylation 22.5
Citrus pectin 92% DE 9.6
Apple pectin 3.6
Polygalacturonic acid 2.4

The ion Mg+2 (2mM) also inhibited about 50% enzyme
activity of polygalacturonase produced by Fusarium oxyspo-
rum in SmF [20].On the other hand, the PGactivity produced
by Sporotrichum thermophile was inhibited by 78% with this
ion at 1mM [17]. Regarding Zn+2, similar results were found
for other fungal PGs, which also suffered inhibition when
exposed to this ion. The polygalacturonase produced by
Thermomyces lanuginosus was inhibited by 53% [19], while
that produced by Sporotrichum thermophile was inhibited by
around 50% with this ion at 1mM [17].

The PG activity was completely inhibited in the presence
of Hg+2 (Table 3), similar to that observed for PG obtained
by SmF [10]. Inhibition by thiol group blocking agents such
as Hg+2 suggests a possible involvement of this group in the
enzyme active site. Three polygalacturonases purified from
Aspergillus carbonarius were also inhibited by this ion, even
at very low concentration (0.02mM) [21]. The effect of ions
in oxidative enzyme stability can be attributed to cysteine
oxidation, which causes the formation of intramolecular and
intermolecular disulfide bridges or rearranging these links,
leading to the formation of sulfuric acid, resulting in enzyme
structural change [22].

The 𝐾
𝑚

of the PG was 1.58mg/mL and 𝑉max was
1553.1 𝜇mol/min/mgprotein.The enzyme obtained fromSmF
showed𝐾

𝑚
of 0.62mg/mL, and𝑉max of 2433.2 𝜇mol/min/mg

[10]. These values indicate that the enzyme from solid-state
fermentation (SSF) showed lower affinity for the substrate
compared with that of SmF, since the value of𝐾

𝑚
was high.

According to Mohamed et al. [23], the 𝐾
𝑚

of fungal
polygalacturonases generally ranges from 0.12 to 6.7mg/mL.
These authors purified two polygalacturonases from Tricho-
derma reesei which had 𝐾

𝑚
values of 0.15mg/mL (PG I)

Table 3: Influence of ions on the PG activity.

Ion Residual activity (%)
Control 100
Fe+3 94
Ag+ 82
Ca+2 94
Mg+2 76
Mn+2 23
Zn+2 41
K+ 100
Hg+2 0
EDTA 73

and 0.93mg/mL (PG II). Kaur et al. [17] purified the
PG of the fungus Sporotrichum thermophile, with a 𝐾

𝑚

of 0.416mg/mL. The PG purified from the thermophilic
fungus Acrophialophora nainiana showed a 𝐾

𝑚
quite high

(4.22mg/mL), indicating low affinity for citrus pectin [18].
The isoelectric point of the PG obtained from SSFwas 6.6,

a value different from that observed for enzyme produced in
SmF which was 7.8 [10]. These values indicate the presence of
higher amount of negatively charged residues in the amino
acids of PG obtained by SSF, which showed lower PI.

The pI values of the two PGs obtained by T. aurantiacus
are similar to those of other fungal strains described in the
literature. GarćıaMaceira et al. [20] purified a PGof Fusarium
oxysporum, which showed pI 7.0. Cabanne and Donèche
[24] reported the purification of two pectinases, an endo-
PG with pI 7.8 and an exo-PG with pI 8.0. Niture and Pant
[15] described the purification of a polygalacturonase with
pI 8.6. The pI is a characteristic that varies widely between
pectinases obtained by different microorganisms and even
between different strains of the same fungal species. This fact
can be illustrated by the results found by Pashkoulov et al.
[25], who reported the purification and characterization of
PGs isolated fromfive different strains ofBotrytis cinerea.The
authors observed that the enzyme from each strain showed
different isoelectric points, ranging between 5.0 and 9.0.

After incubation for 5min at 65∘C in 1% citrus pectin 26%
DE methoxylation, the polygalacturonase released a mixture
of mono-, di-, tri-, and oligogalacturonic acids (Figure 5(a))
and was not able to hydrolyze the trigalacturonic acid
(Figure 5(b)).

The PG activity measured by viscosimetric assay method
(specific activity of 3542.8U/mg) reduced afforded 56% of
viscosity of 1% citrus pectin in 10min.These results indicated
an endo-PG activity. On the other hand, di and mono-
galacturonates were released at the initial stages of the
incubation period, suggesting that PG degraded the substrate
by multiple attacks.

This profile was very similar to that observed for the
SmF enzyme by Martins et al. [10], which was considered
an enzyme with endo/exoactivity. Similar results were also
found by Contreras Esquivel and Voget [26], who observed
this same attack mode with the polygalacturonase produced



6 Enzyme Research

(a) (b)

Figure 5: Paper chromatographic analysis of hydrolysis products of PG fromThermoascus aurantiacus. (a) Acting on 1% (w/v) citrus pectin
at 65∘C, for 10min. (b) Acting on 1% (w/v) trigalacturonic acid at 65∘C, for 10min. 3G: trigalacturonic acid; 2G: digalacturonic acid; 1G:
galacturonic acid; H: enzyme hydrolyzed pectin.

by Aspergillus kawachii in SmF. This PG was also unable to
degrade digalacturonic and trigalacturonic acids but released
unsaturated mono-, di-, tri-, and oligogalacturonic acids,
indicating that it presents action in multiple attacks.

4. Conclusions

The polygalacturonase obtained by thermophilic fungus
Thermoascus aurantiacus CBMAI756 in solid-state fermen-
tation showed higher tolerance to variations of pH compared
with PG produced in SmF by the same fungus, though that
was less thermostable than SmF enzyme. Other enzymes
features, such as isoelectric point,𝐾

𝑚
, and influence of some

ions in the activity, were also different in relation to the
fermentation process employed.

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 the financial support.

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