Arabinosidase from Aspergillus niger Induced by Utilizing Soybean Pulp as Carbon Source

Jurnal ILMU DASAR, Vol. 3 No. 2, 2002: 80-85 80
Arabinosidase from Aspergillus niger
Induced by Utilizing Soybean Pulp as Carbon Source
Kahar Muzakhar
Department of Biology
Faculty of Mathematics and Natural Sciences, The University of Jember
ABSTRACT
The production of Arabinosidase from Aspergillus niger was studied by introducing the different simple and
complex carbon sources. Soybean pulp was found potential to be an inducer of arabinan degrading enzyme.
The enzyme was purified after growing in medium employed with fresh soybean pulp. The optimum for
Arabinosidase production was obtained after 6 days cultivation at 37oC. The enzyme was purified and
characterized to have molecular weight of 97,000 Da, stable in pH 4 – 8 and temperature below 50oC, and
optimum activity at pH 5 and temperature 50oC.
Keywords : arabinase, carbon source
INTRODUCTION
The suspension was neutralized with acetic
Arabinosyl residues are widely distributed in
acid and the pellet was collected by adding
the form of hemicellulose (Manin et al., 1994)
ethanol 1:1 (v/v) followed by centrifugation at
such as arabinan, arabinoxylan gum arabic, and
4000 rpm. Then the pellet was again 3 times
arabinogalactan. Arabinosyl also found in the
washed in 50% ethanol and re-centrifugation at
major component of the hemicellulose fraction
same rpm for removing oligosaccharides.
of the soybean pulp (Muzakhar et.al., 1998). It
constitute after galactose the most abundant
Optimization of Arabinosidase Production
hydrolyzate during solid state fermentation of
The Arabinosidase production was optimized
soybean pulp by introducing A. niger
by cultivation of A. niger in 20 flasks of
(Muzakhar et al., 1998). For industrial
Erlenmeyer 100 ml. Each flask contains 10g of
application, this simple sugar can be used as
sterilized soybean pulp as medium. The culture
carbon source for fermentation in alcohol
was harvested everyday and the crude enzymes
production (Blanco et al., 1999).
was collected using NaCl extraction as
Some potential applications for arabinan
described by Muzakhar et al., 1998. This crude
enzymes in alcohol industry would require the
enzyme was stored at 4oC till used for
use of enzymes worked in wide range of pH.
Arabinosidase purification.
The enzymes were mostly found in
The production of Arabinosidase was also
microorganisms especially in fungus (Blanco et
observed by employing various carbon source
al., 1999; Gessesse et al., 1998). The fungus
such as L-arabinose, D-glucose, D-galactose,
has therefore attracted considerable attention as
pectin (citrus), bran extract, arabinoxylan, and
sources of Arabinosidase degrading enzyme for
arabinan (beet).
arabinan hydrolysis.
Up to now, there have been many studies of
Enzyme assay
Arabinosidase but only a few studies of the
The Arabinosidase activity was determined by
production by utilizing raw material of
measuring of releasing reducing sugar using
agriculture wastes. The production of
the method of Nelson (1944) as modified by
Arabinosidase by utilizing soybean pulp is
Somogy Nelson (1952). For this measurement,
expected and will enable us to utilize it
the arabinan was used as substrate.
effectively of hemicellulose and in minimizing
environmental problem of wastes.
Analysis Degree of Hydrolysis of
Arabinosidase
EXPERIMENTAL
The degree of hydrolysis of Arabinosidase is
Preparation of Soybean Pulp Alkali Extract
determined by employing 1% arabinan
(SPAE) Substrate
substrate in 0.1 M pH 5 acetate buffer and
Five hundred of soybean pulp was hydrolyzed
incubated at 37oC. The releasing sugar was
over night (12 hour) using 0.5 M of NaOH.
analyzed every 1 hour. The degree of

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81 Arabinosidase from .......................... ( Kahar Muzakhar )
hydrolysis is also tested with SPAE, pectin
the mixtures (containing 1% substrate in 990 ul
(citrus), bran extract, arabinoxylan, and
in 0.1 M buffer + 10 ul enzyme) after 10
arabinan (beet) as substrates.
minutes exposure at various pH and
temperatures. While the effect of pH and
Gas Chromatograh Analysis of Arabinose
temperature on stability was conducted by pre-
Production
incubating of enzyme in various pH and
The quantity of arabinose is analyzed by using
temperatures for 30 minutes, and then the
Gas Chromatograph (GC) as alditol acetates
remaining activity was measured in optimum
(Arai and Murao 1978; Hondmann et al.,
pH and at 37oC. In the pH ranges of 2 to 6
1994) with a few modification. The sample was
acetate buffer, 6 to 7 potassium phosphate
hydrolyzed with 2N HCL for 6 hours, reduced
buffer, and 8 to 10 Tris – HCl were used.
with equal amount of NaBH4 at room
temperature overnight and then a batch of
RESULT AND DISCUSSION
Dowex resin H type was added to the mixture
Optimization of Arabinosidase Production
followed by filtration. The filtrate was
Aspergillus niger can grow well on various
evaporated to dryness and residual boric acid
carbon source although the production of
was removed by repeated evaporation with
arabinosidase was different. The enzyme was
methanol. The sugar alcohol was acetylated in
produced specifically when polysaccharides
2 ml of acetic acid anhydride:pyridine (1:1) at
containing arabinose such as been arabinan,
100oC for 10 min. The mixture was diluted
arabinoxylan, bran extract, and pectin was
with chloroform:water (1:4), shaken well and
employed as carbon source (Table 1).
the supernatant removed by centrifugation at
2000 rpm for 10 minutes. Remaining pyridine
Table 1.
Arabinosidase activity produced
was removed by washing with water by
during cultivation on various carbon
removal of aqueous phase after centrifugation.
sources
The alditol acetates of sugar was dried and
Arabinosidase
Carbon Source
dissolved in chloroform to an appropriate
activity *
volume. The GC analysis was performed on
L-Arabinose
0
stainless column, 2mm I. D. × 1.83 m, packed
D-Glucose
0
with 3% (w/w) ECNSS-M on Gas Chrom Q,
100-120 Mesh. The initial column temperature
D-Galactose
0
was 190oC for 5 minutes and then using
Pectin (citrus)
0.7
gradient up to 210 oC at 1oC/min.
Bran extract
0.4
Arabinoxylan
1.9
Purification of Arabinosidase
Arabinan (beet)
4.6
The purification of Arabinosidase was
conducted at room temperature using various
Soybean Pulp
4.4
column. All step in purification 20 mM of
* : Units per milliliter of crude enzyme
acetate buffer pH 5 was used. Detail of
purification was reported in “Result and
However, no enzyme activity was detected
Discussion”.
when monosaccharide (arabinose, glucose and
galactose) was employed. High activity of
Estimation of Molecular Weight
enzyme is also found when soybean pulp
Molecular weight of purified enzyme was
hemicellulose was used as medium. As
estimated with Sodium Dodecyl Sulfate
reported in previous research (Muzakhar et al.,
Polyacrylamide Gel Electrophoresis (SDS
1998) that arbinose is also major component of
PAGE) using Phosphorylase (97,000),
soybean pulp. So that, in order to supply the
Albumin (66,300), Aldolase (42,400), Carbonic
carbon source, A. niger also secrets
Anhydrase (30,000), and Trypsin inhibitor
arabinosidase that is functioned as
(20,100) as standard proteins.
arabinosidase degrading enzyme which
hydrolyze arabinosyl residue in soybean pulp
Effect of pH and Temperature on Optimum
medium. The optimum for arabinosidase
Activity and Stability of Arabinosidase
production was found after 6 days cultivation
The effect of pH and temperature on optimum
(Figure 1).
activity were measured of releasing sugar of

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Jurnal ILMU DASAR, Vol. 3 No. 2, 2002: 80-85 82
0.6
0.5
0.4
i
t
)
n
(
U
0.3
i
t
y
t
i
v
c
A 0.2
0.1
0
1
2
3
4
5
6
7
8
Cultivation (Day)
Figure 1. Cultivation of A. niger on soybean pulp medium. The optimum cultivation period was
determined by daily monitoring of arabinosidase activities
Purification of Arabinosidase
dialyzed against 20 mM acetate buffer for
The supernatant containing arabinosidase was
overnight and reloaded again in Butyl
obtained from 6 days cultivation by NaCl
Toyopearl in same buffer using a gradient 1 M
extraction followed by centrifugation. It was
to 0 NaCl. The purification procedure is
concentrated with 70% saturated ammonium
summarized on the table 2 resulted in 18.3%
sulfate and centrifugated at 12000 rpm for 20
yield and 735 folds purification of
minutes. The pellet were dissolved in buffer.
arabinosidase.
Remaining ammonium sulfate was removed by
overnight dialysis against the same buffer at
Molecular Weight of the Purified Enzymes
4oC. The concentrated enzyme was then
The molecular weight of arabinosidase
applied to a DEAE Toyopearl 650 M column
determined by SDS-PAGE was 97,000 Da,
equilibrated with 20 mM acetate buffer (pH 5),
respectively (Figure 2). This enzymes is
the column was washed with the same buffer,
different with other arabinosidase as reported
and then the proteins were eluted with a
in previous research that has molecular weight
gradient of 0 to 0.5 M NaCl using same buffer.
64,000 Da (Muzakhar et al., 199x).
The last step, active fraction was pooled,
Table 2. Purification Summary of Arabinosidase
Purification
Total ABS-
Total Activity
Spec. Activity
Yield (%)
Fold
Step
280
(Unit)
(Unit/ABS-280)
Crude Enzyme
92,400
1,950
0.02
100
1.0
Ammonium
Sulfate
23,200
1,300
0.06
66.7
3
Precipitation
DEAE
908
467
0.51
23.9
25.5
Toyopearl
Butyl
14.2
356
14.7
18.53
735
Toyopearl

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83 Arabinosidase from .......................... ( Kahar Muzakhar )
Figure 2.
SDS-PAGE of the Purified Arabinosidase. Lane 1 is protein standards and lane 2 is
arabinosidase.

(A)
(B)
100
100
)
)
(
%
(
%
80
80
i
t
y
i
t
y
t
i
v
60
t
i
v
60
c
c
A
A
e
40
e
40
t
i
v
t
i
v
l
a
l
a
20
20
e
e
R
R
0
0
20
30
40
50
60
70
80
2
3
4
5
6
7
8
9
10
Temperature (0C)
pH
Figure 3. Effect of Temperature (A) and pH (B) on Activity and Stability. The optimum
 
temperature ( ), temperature stability (
), optimum pH ( ), and pH stability
¡
¢
(
)were assayed in 1 ml total volume of buffer containing 1% arabinan substrate at
¡
37oC.
Effect of pH and Temperature on Optimum
enzyme activity was not affected by EDTA
Activity and Stability of Arabinosidase
(10mM), dithiothreitol (10mM) or p-
The effect of temperature and pH on the
chloromercuribenzoic acid (0.2mM). The
optimum activity and stability were examined.
remaining relative activity in percent was
The optimum activity for this enzyme was
presented in table 3
found at temperature 50oC and stability below
50oC. The optimum pH for activity for this
enzyme was found at pH 5. The enzyme
Table 3. Degree of Hydrolysis Toward
showed 95% activity in the pH range from 4 to
Various Substrates
8.
Degree of
Substrate
Hydrolysis (%)
Effect of Metal ions and Reagents
Pectin (citrus)
5
The influence of certain inhibitors or activators
SPAE
26
on Arabinosidase activity was also studied. The
Arabinoxylan
12
enzyme did not work if Ca2+, Mg2+, Mn2+ (each
Arabinan (beet)
87
at 5mM), or Co2+ (0.5 mM) present. However,

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Jurnal ILMU DASAR, Vol. 3 No. 2, 2002: 80-85 84
Table 4. Effect of Metal ions and Reagents
Degree of Hydrolysis of Arabinosidase and
Substrate Specifity
Metal ions and
Remaining Relative
The degree of hydrolysis of purified enzyme
Reagents
Activity (%)
was observed toward various substrates after
12 hour incubation at 37oC. The arabinosidase
Ca2+
5
hydrolyzed arabinan efficiently (87%) while it
Mg2+,
21
lesser efficiencies (less than 30%) when the
Mn2+
17
substrates pectin, arabinoxylan or SPAE were
Co2+
15
used (Table 3). Furthermore, quality analysis of
EDTA
98
the hydrolyzates using GC showed that the
Dithiothreitol
97
hydrolysis of arabinan resulted in a single peak
p-
100
and it suggested only monosaccharide form
Chloromercuribenzoic
(arabinose) was produced, respectively (Figure
acid
4).
Figure 4. Gas Chromatograph Analysis of Hydrolyzate. The hydrolyzate (HYD) of arabinan after
enzymatic hydrolysis was determined by GC and the standard arabinose (ARA) was
used.
CONCLUSION
REFERENCES
Inducing of arabinosidase from A. niger was
Arai, M., and S. Murao. 1978. Characterization
done successfully by employing soybean pulp
of Oligosaccharides from an
to the cultivation. The enzyme was purified and
Enzymatic Hydrolyzate of red yeast
resulted in 18.3% yield and 735 folds
cell walls by lytic enzyme. Agric. Biol.
purification. The molecular weight of enzyme
Chem. 42:1651-1659.
is 97,000 Da, and it has optimum working at
Blanco, P., Sieiro, C., and Villa, T. G., 1999.
pH 5 and 50oC while the stability between pH 4
Production of pectic enzyme in yeast.
to 8 and below 50oC. GC analysis showed that
FEMS Microbiol. Letters, 175:1-9.
this enzyme hydrolyzed arabinan and release
Gessesse, A., 1998. Purification and Properties
only monosaccharide arabinose, suggested that
of Two Thermostable Alkaline
mode of action of this enzyme is exohydrolase.
Xylanase from an Alkaliphilic
Bacillus sp. App. Envi. Microbiol., 64:
3533-3535.

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85 Arabinosidase from .......................... ( Kahar Muzakhar )
Hondmann, Dirk H. A. and J. Visser. 1994.
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Manin, C., Shareek, F., Morosoli, R., and
Cellulose Degradation, K. Ohmiya et
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lividans 66 and DNA sequence of the
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determination of glucose. J. Biol.
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