Content uploaded by Dr-Arafat Tanash
Author content
All content in this area was uploaded by Dr-Arafat Tanash
Content may be subject to copyright.
ORIGINAL PAPER
Properties of Aspergillus subolivaceus free and immobilized
dextranase
A. B. El-Tanash •E. El-Baz •A. A. Sherief
Received: 9 July 2011 / Revised: 10 August 2011 / Accepted: 22 August 2011 / Published online: 4 September 2011
ÓSpringer-Verlag 2011
Abstract Aspergillus subolivaceus dextranase is immo-
bilized on several carriers by entrapment and covalent
binding with cross-linking. Dextranase immobilized on
BSA with a cross-linking agent shows the highest activity
and considerable immobilization yield (66.7%). The opti-
mum pH of the immobilized enzyme is shifted to pH 6.0 as
compared with the free enzyme (pH 5.5). The optimum
temperature of the reaction is resulted at 60 °C for both
free and immobilized enzyme. Thermal and pH stability
are significantly improved by the immobilization process.
The calculated K
m
of the immobilized dextranase
(14.24 mg mL
-1
) is higher than that of the free dextranase
(11.47 mg mL
-1
), while V
max
of the immobilized enzyme
(2.80 U lg protein
-1
) is lower than that of the free dex-
tranase (11.75 U lg protein
-1
). The immobilized enzyme
was able to retain 76% of the initial catalytic activity after
5.0 cycles.
Keywords Dextranase Dextran degradation
Enzyme immobilization Aspergillus subolivaceus
Introduction
Dextran is a homoglycan of a-D-1,6-glucopyranose
molecules with a-1, 6 linkages with side with side-chains
of a-1,2, a-1,3, and a-1,4 to the main chains [1]. Leu-
conostoc,Lactobacillus, and Streptococcus catalyze a
primary reaction for enzymatic synthesis of dextran from
sucrose, maltose, and isomaltose producing insoluble
glucans [2]. Beside the important of dextran, it has
harmful effects on sugar cane and sugar beets containing
sucrose in food and sugar industries [3,4]. Dextrans
represent also a structural component of dental plaque
that causes the development of dental caries [1].
Simultaneous use of dextran hydrolyzing enzymes
could be advantageous to overcome these problems
[5,6].
Dextranase is commonly used to release D-glucose and
shorter oligosaccharides causing decrease the de-branching
degree and increase the solubility of higher molecular
weights glucans removing the undesirable sliming in sugar
industry [7] or as possible mouthwash ingredients [8].
Dextranolytic enzymes are also being used in the synthesis
of potentially valuable prebiotic oligosaccharides [9].
Dextranase can also be used as universal targeting method
for therapeutic agents to activate the cancer antibodies and
delayed the efficiency of penicillin and temafloxacin to be
active for a long time [1]. Furthermore, hydrolysis of
dextran by microbial dextranase is of significant interest in
drug formulation, vaccines, cosmetics, and other food
industries [1,10].
Dextranases are produced by various microorganisms,
including bacteria [11,12], yeast [13] and filamentous
fungi [1,5,14]. Fungal dextranase has attracted much
attention due to higher enzyme activity and due to the
synthesis of isomaltooligosaccharides (IMOs) [15].
Due to the potential applications of dextranase, the
objective of this paper is aimed to immobilize dextranase
to improve its thermal, pH and operational stabilities as
compared with free enzyme.
A. B. El-Tanash (&)E. El-Baz A. A. Sherief (&)
Botany Department, Faculty of Science, Mansoura University,
Mansoura, Egypt
e-mail: arafattanash@yahoo.com
A. A. Sherief
e-mail: dshrif2004@yahoo.com
123
Eur Food Res Technol (2011) 233:735–742
DOI 10.1007/s00217-011-1570-1
Materials and methods
Microorganism and culture maintenance
The fungal strain used in the present study was locally
isolated from soil sample on dextran containing medium
and identified as Aspergillus subolivaceus by Regional
Center for Mycology and Biotechnology (RCMB),
Al-Azhar University, EGYPT. The strain was sub-cultured
on modified agar media containing 1.0% dextran as a sole
carbon source at 30 °C for 7.0 days and maintained at
4°C. Induced slant of A. subolivaceus is mixed with
10 mL basal medium for preparing spore suspension. The
spore count in the suspension was 2.0 910
7
spore mL
-1
.
Mode of fermentation and dextranase preparation
According to Shereif et al. [16], extracellular dextranase
from A. subolivaceus was obtained through submerged
fermentation medium (pH 5.5) containing 1.0% dextran;
0.3% NH
4
H
2
PO
4
; 0.05% KCl; 0.1% KH
2
PO
4
; and 0.05%
MgSO
4
–7H
2
O. In 250 mL Erlenmeyer flask, 49 mL of
medium was transferred and autoclaved at 121 °C at 15 lbs
for 20 min. The medium after cooling is inoculated by
1.0 mL spore suspension (2.0 910
7
spores) and incubated
at 30 °C for 4.0 days in incubating shaker (150 rpm). For
obtaining extracellular dextranase, mycelial pellets were
filtrated through Gough No. 1 under water pump. The
filtrate was collected and stored at deepfreeze (-20 °C) as
crude enzyme preparation till used for enzyme assay.
Assay of dextranase
Dextranase activity was determined by detecting the
amount of liberated reducing sugar from the hydrolysis of
dextran (MW 260 kDa; BDH) according to Nelson [17]
and Somogyi [18] method using Spectro UV–VIS RS
spectrophotometer. Unless specified otherwise, the assay
mixture consisted of 0.5 mL dextran (2.0% w/v) in acetate
buffer (0.1 M, pH 5.5) and 0.5 mL of enzyme solution or
weighed amount of the immobilized enzyme. The reaction
was incubated at 60 °C for 20 min. One unit of enzyme
activity (U) is defined as the amount of the enzyme that
releases 1 lmol of reducing sugars (D-glucose) per minute
under assay conditions.
Protein determination
Soluble protein was determined according to Bradford
method [19], by measuring optical density of developed
color at 595 nm. Optical density was measured against
blank. The lg of protein was estimated using standard
curve of bovine serum albumin (BSA).
Immobilization methods of dextranase
Carriers for enzyme immobilization
Chitins, gelatin and BSA were from Sigma, and Na-algi-
nate was from BDH. All other chemicals were of analytical
grade.
Covalent binding with cross-linking
Chitin (1.0 g) was shaken with 10 mL of 2.5% glutaral-
dehyde. Chitin was then collected by filtration using a
sintered glass funnel and washed with distilled water to
remove the excess glutaraldehyde. The wet chitin was
mixed with the enzyme solution (80 U; A. subolivaceus
dextranase) for 1.0 h at room temperature (25 °C). The
unbound enzymes were removed by washing with 0.1 M
acetate buffer pH 5.5 [20].
One gram of gelatin or BSA was mixed with 80 units of
A. subolivaceus dextranase (50 °C for melting gelatin).
Then, 0.7 mL (50% v/v) glutaraldehyde was added, the net
concentration of prepared gel was 10% (w/v). The mixture
was incubated overnight at 4.0 °C. The resulting gel was
washed as described above and then cut into small 0.2 cm
3
cubes [21].
Entrapment in Ca-alginate
Eighty units of A. subolivaceus dextranase were mixed
with different concentrations of sodium alginate (Pharma-
cia chemicals) with a final concentration of 3.0, 5.0, and
7.0%. The entrapment was carried out by dropping each
alginate solution in 0.1 M CaCl
2
solution. The resulting
beads were collected, washed with acetate buffer (0.1 M;
pH 5.5), and kept in the same acetate buffer at room
temperature for 2.0 h to remove unbounded enzymes [20].
Properties of the free and immobilized dextranase
Optimum pH
The optimum pH for free and immobilized dextranase was
estimated by incubating enzyme at different pH values
(0.1 M acetate buffer was used at a range of pH 4.0–5.5
and 0.1 M phosphate buffer for a range of pH 6.0–8.0 at
45 °C for 20 min using 2.0% dextran as substrate with
different controls). Dextranase activity was then assayed as
described above.
Optimum temperature
The effect of temperature was studied by incubating both
soluble and immobilized dextranase in their respective
736 Eur Food Res Technol (2011) 233:735–742
123
optimum pH at different temperatures (ranging from 30 to
80 °C) with different controls for 20 min using 2.0%
dextran as substrate.
Activation energy (E
a
)
The activation Energy was determined from the slope of a
linear plot of the log of the enzyme activity (v) versus 1/T,
according to the Arrhenius law:
log vðÞ¼Cte Ea=RTðÞ ð1Þ
The enzyme activity (v) was expressed in
Ulg protein
-1
, the temperature (T) in Kelvin (K), the
gas constant (R=1.987), and the activation energy (E
a
)in
Kcal mol
-1
.
pH stability
The pH stability of the free and immobilized enzyme was
examined after pre-incubating enzyme samples at 25 °C
for 30 min at different pH values, followed by adjusting the
pH to the value of the standard assay system. The residual
activity was assayed under the standard conditions.
Thermal stability
The enzyme samples were incubated in 0.1 M acetate
buffer at designated temperatures of 60, 70, and 80 °C for
times ranging from 5.0 to 90 min. The residual activity was
assayed under the standard conditions for free and immo-
bilized dextranase.
Determination of the half-life (t
1/2
)
The half-life of the enzyme activity (t
1/2
), which corre-
sponds to the time necessary for the residual enzyme
activity to decrease to 50% of its initial value, can be
calculated from the equation:
t1=2¼0:693=Kdð2Þ
Determination of the deactivation energy (E
d
)
The deactivation energies of free and immobilized dex-
tranase were determined by plotting the activity data [log
of the ratio of Ar (residual activity)/A
0
(initial activity)] as
a function of time to obtain the deactivation rate constant
(K
d
) at each temperature. From Arrhenius equation:
Kd¼Kdoexp Ed=RTðÞ ð3Þ
plotting the log of K
d
as a function of the inverse of the
absolute temperature, the energy of deactivation (E
d
)is
obtained as the product of the slope of the resultant straight
line times R, the universal gas constant.
Kinetic values (K
m
and V
max
)
Different concentrations of pure dextran (BDH)
(1.0–50 mg mL
-1
) were respectively prepared for dex-
tranase assay. The enzyme activity was determined after
20 min incubation at 60 °C for free and immobilized
enzyme. The kinetic values of enzyme (K
m
and V
max
) were
investigated through Lineweaver–Burk Plot by plotting the
relation between different substrate concentrations against
the corresponding rate reciprocals using Graph-Pad Prism 5
software.
Operational stability of immobilized dextranase
BSA-immobilized dextranase (1.5 g, wet) was incubated
with 5.0 mL 2.0% (w/v) dextran in acetate buffer (0.1 M,
pH 6.0) at 60 °C for 20 min. At the end of the reaction, the
immobilized enzyme was collected by filtration, washed
with distilled water, and re-suspended in 5.0 mL freshly
prepared substrate to start a new run. The supernatant fluid
was assayed for D-glucose.
Reproducibility
All the experiments were repeated at least four times, and
the results were reproducible. The data points represent the
mean values within ±5.0% of the individual values.
Results and discussion
Dextranase from A. subolivaceus was immobilized by two
methods including: (1) covalent binding with cross-linker
on chitin, bovine serum albumin (BSA) or gelatin; and (2)
entrapment in Ca-alginate (Table 1). The immobilized
enzyme prepared by covalent binding with cross-linker to
bovine serum albumin had the highest specific activity
(1.56 U lg proteins
-1
) compared to other carriers with
immobilization yield about 66.7%; therefore, it was used in
the succeeding part of this work. In this connection,
immobilization by covalent binding using a cross-linking
agent (glutaraldehyde) probably increases the local surface
area, which contributes to minimizing the steric effect
around the active site of the immobilized enzyme [22]. In
addition, these results are similar to those reported during
immobilization of P. funiculosum dextranase [23] and
A. aculeatus tannase [24].
The immobilization yields of the immobilized enzyme
by entrapment on 3.0, 5.0, and 7.0% Ca-alginate were 55,
73.3, and 83.3%, respectively, while the specific activities
were lower as compared to the other used carriers, reaching
to 0.4, 0.59, and 0.62 U lg proteins
-1
. The lower values of
dextranase activity with entrapment may be due to enzyme
Eur Food Res Technol (2011) 233:735–742 737
123
leakage [22]. Similar observations were also reported
[23,25].
The initial specific activity exhibited by free dextranase
is 8.13 U lg protein
-1
, while the specific activity of
immobilized dextranase on BSA (1.56 U lg protein
-1
)
was retained about 19.43% of specific activity exhibited
by free dextranase. This drop in the specific activity after
immobilization may be due to diffusion limitation
(i.e., resistance to diffusion of the substrate into the
immobilization matrix and resistance to diffusion out of the
products), as reflected by the lower apparent activation
energy for immobilized dextranase (1.27 kcal mol
-1
vs.
2.31 kcal mol
-1
). Lower activation energy for the immo-
bilized enzyme has been reported to be an indication of
diffusional limitations [26]. On the other hand, the immo-
bilization of the enzyme by covalent binding could lead to
a decrease in the flexibility of the enzyme molecule, which
is commonly reflected by a decrease in catalytic activity
[27]. A decrease in specific activity after dextranase
immobilization is previously reported [23].
The optimum pH of immobilized dextranase was
increased at 5–7 range of pH, reaching its maximal activity
at pH 6.0 as compared to pH 5.5 as the optimum for free
dextranase (Fig. 1). This result may be attributed to an
ionic change around the enzyme active site as result of the
immobilization process [28]. The shift of pH optima has
been previously reported for other immobilized dextranase
[29] and for other immobilized enzymes [25]. Furthermore,
both immobilized and free dextranase of Brevibacterium
fuscum have the same optimum pH [30].
The profile of pH stability of immobilized and free
dextranase (Fig. 2) shows that the activity of immobilized
dextranase is shifted to alkaline range and significantly
stable at wide range of pH (5.5–8.0) than free enzyme (pH;
5.5–6.0). This result means that immobilized dextranase
would be more resistant to pH changes and could be used
industrially. This effect may have been caused by the
micro-environmental pH of the BSA matrix [22]. On the
other hand, immobilized dextranase from P. lilacinum is
Table 1 Immobilization of A. subolivaceus dextranase on different carriers
Immobilization method Carriers Enzyme units (U/g carrier) Immobilization
yield I/(A-B)%
Specific activity
U(lg protein
-1
)
a
Add (A) Unbound (B) Immobilized (I)
Entrapment Alginate (3%) 80 40 22 55.0 0.62
Alginate (5%) 80 35 33 73.3 0.59
Alginate (7%) 80 20 50 83.3 0.40
Covalent binding Chitin 80 37 24 55.8 0.91
Gelatin 80 30 33 66.0 1.45
BSA 80 20 40 66.7 1.56
a
Specific activity [U (lg protein
-1
)] is defined as the amount of enzyme that releases 1 lmole of reducing sugars per minute per amount of
bonded enzyme (lg protein) in the used weight of immobilized enzyme under assay conditions
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5
0
20
40
60
80
100
120
Free
Immobilized
pH level
Recovered activity (%)
Fig. 1 Effect of different pH levels on free and immobilized
A. subolivaceus dextranase
3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0
0
20
40
60
80
100
120
Free
Immobilized
pH level
Residual activity (%)
Fig. 2 pH stability of free and immobilized A. subolivaceus
dextranase
738 Eur Food Res Technol (2011) 233:735–742
123
more stable at low pH (3.5–4.0) and higher pH (6.0–7.5)
ranges [31].
The effect of temperature on the activity of the immo-
bilized and free dextranase (Fig. 3) shows that the optimum
temperatures are similar namely, 60 °C for free and
immobilized dextranase. Similarly, the optimum tempera-
ture of P. aculeatum dextranase was 50 °C for both free
and immobilized enzyme [32]. On contrary, optimum
temperature was shifted from 60 °C for free P. funiculosum
dextranase to 80 °C for immobilized enzyme [23].
The temperature data are replotted in the form of
Arrhenius plots (Fig. 4). The plots of both free and
immobilized dextranase were linear, and the activation
energy of immobilized dextranase was lower (1.27 kcal
mol
-1
) than the activation energy of free enzyme
(2.31 kcal mol
-1
). These results are more or less similar to
those reported for immobilized and free P. funiculosum
dextranase [23]. This decrease in the activation energy may
be due to the diffusion limitations of immobilized enzyme
[25].
The results in Fig. 5a, b and Table 2indicate that the
immobilization process improved the thermal stability of
dextranase relative to free enzyme. For example, the free
dextranase was completely inhibited after 10 min incuba-
tion at 80 °C, while the immobilized form retained 74.8%
of its original activity after the same treatment. The cal-
culated half-lives of free dextranase at 60, 70, and 80 °C
are 1.3, 1.43, and 7.2 times faster than those of immobi-
lized enzyme, respectively. Similarly, increase in half-lives
times of P. funiculosum immobilized dextranase compared
25 30 35 40 45 50 55 60 65 70 75 80 85
0
20
40
60
80
100
120
Free
Immobilized
Temperature (°C)
Recovered activity (%)
Fig. 3 Effect of different temperatures on free and immobilized
A. subolivaceus dextranase
2.9 3.0 3.1 3.2 3.3 3.4
0.0
0.2
0.4
0.6
0.8
1.0
Free
Immobilized
Slope
Free -0.5045 ± 0.02191
Slope
Immobilized -0.2766 ± 0.01729
1/K x 1000
Log of enzyme activity
Fig. 4 Arrhenius plots for the activation energy of free and
immobilized A. subolivaceus dextranase. aImmobilized dextranase,
bFree dextranase
0 10 20 30 40 50 60 70 80 90
0.0
0.5
1.0
1.5
2.0 60°C 70 °C 80 °C
1/slope
60°
C
-863.5
70°
C
-140.3
80°C
-60.85
Time (min)
Log of relative activity
a - Immobilized dextranase
0.0
0.5
1.0
1.5
2.0 70°C80°C
60°C
1/slope
60°C
-662.3
70°
C
-98.43
80°C
-8.416
Log of relative activity
b- Free dextranase
0 102030405060708090
Time (min)
Fig. 5 Thermal stability of free and immobilized A. subolivaceus
dextranase
Eur Food Res Technol (2011) 233:735–742 739
123
with free enzyme is reported [23]. Results also showed that
the immobilized dextranase had the highest thermal sta-
bility translated as the longest half-life and the lowest
deactivation rate constant at 60 °C. Furthermore, the
Arrhenius plot (Fig. 6) for the deactivation energies (E
d
)of
free and immobilized enzymes raveled that the value of E
d
of immobilized dextranase (69.8 kcal mol
-1
) is lower
as compared with the value of free enzyme (113.1 kcal
mol
-1
). This decrease in the deactivation energy of
immobilized dextranase may be attributed to diffusion
limitation at different treated temperatures. In contrast, the
deactivation energy of other immobilized enzyme was
increased compared with free form [23,24].
K
m
(Michaelis constant) and V
max
(maximum reaction
velocity) of free and immobilized dextranase were esti-
mated under optimal pH and temperature by incubating
each enzyme at different concentrations of pure dextran
(BDH) ranged from 1.0 to 40 mg mL
-1
. Linweaver Burk
plots (Fig. 7) showing that K
m
of the immobilized dex-
tranase (14.24 mg mL
-1
) is higher than the value of free
dextranase (11.47 mg mL
-1
), while V
max
(1/slope) of the
immobilized enzyme (2.80 U lg protein
-1
) is lower than
that of free dextranase (11.75 U lg protein
-1
). This
increase in the K
m
value after the immobilization may be
partially due to mass transfer resistance to diffusion into
the immobilization matrices and/or to low substrate
accessibility to the enzyme active site. On the other hand,
Table 2 Comparison between the thermal stabilities of free and A.
subolivaceus dextranase
Free
dextranase
Immobilized
dextranase
Specific activity (U lg protein
-1
) 8.13 1.56
Optimum pH 5.5 6.0
Optimum temperature 60 °C60°C
Activation energy (E
a
; kcal mol
-1
) 2.31 1.27
Half-life time (min)
60 °C 331.15 431.8
70 °C 49.22 70.15
80 °C 4.21 30.43
Deactivation rate constant K
d
(min
-1
)
60 °C 0.21 910
-2
0.16 910
-2
70 °C 1.41 910
-2
0.99 910
-2
80 °C 16.46 910
-2
2.28 910
-2
Deactivation energy (dE
a
;
kcal mol
-1
)
113.1 69.8
K
m
(mg mL
-1
) 11.47 14.24
V
max
11.75 2.80
2.80 2.85 2.90 2.95 3.00 3.05
-8
-7
-6
-5
-4
-3
-2
Free
Immobilized
Slope
Free
-24.68 ± 0.3168
Immobilized
-15.24 ± 0.07392
1/K x 1000
ln of Kd
Fig. 6 Arrhenius plots for the deactivation energy of free and
immobilized A. subolivaceus dextranase
-0.14 -0.07 0.00 0.07 0.14 0.21 0.28 0.35
0.3
0.6
0.9
1.2
1.5
1.8
Free
Immobilized
1/[S]
1/V
Fig. 7 Lineweaver-Burk plots of immobilized and free A. suboli-
vaceus dextranase acting on pure dextarn (BDH)
Table 3 Effect of different metal ions on the activity of immobilized
and free dextranase
Different mineral Relative activity (%)
Free Immobilized
Control 100.0 ±0.10 100.0 ±0.19
K
?
101.0 ±0.07 101.1 ±0.57
Na
?
15.40 ±0.21 40.80 ±0.20
Fe
2?
58.50 ±0.07 82.60 ±0.65
Cu
2?
07.10 ±0.51 43.50 ±0.04
Hg
2?
00.00 ±0.00 00.00 ±0.00
Mg
2?
102.1 ±0.09 105.4 ±0.43
Mn
2?
77.40 ±0.09 93.80 ±0.58
Zn
2?
78.60 ±0.04 92.10 ±0.36
Ca
2?
101.0 ±0.41 101.0 ±0.14
740 Eur Food Res Technol (2011) 233:735–742
123
fixation of the enzyme on the immobilization matrix could
lead to a decrease in the flexibility of the enzyme molecule,
which is commonly reflected by a decrease in the catalytic
activity [22]. Consequently, the maximum rate of the
reaction catalyzed by the immobilized enzymes was lower
than that of the free enzyme. Several researchers reported
an increase in K
m
and decrease in V
max
for dextranase due
to immobilization [23,33]. On the other hand, little
increases in K
m
value after immobilization of dextranase of
Chaetomium erraticum is recorded [34].
In this experiment, free and immobilized enzymes were
incubated with different metal ions in solution at room
temperature for 30 min. Then the residual activity was
measured at optimum conditions. The results in Table 3
show that Mg
2?
,K
?
, and Ca
2?
ions are slightly activate
both free and immobilized dextranase. While Hg
2?
was
completely inhibited the activities of both free and
immobilized dextranase. BSA as a carrier appears to
protect dextranase against the inhibitory effect of other
metal ions; hence, it was generally observed that the
inhibitory effects of the ions were less pronounced in
immobilized dextranase compared with the free enzyme.
This protection may be due to the following: (1) structural
changes in the enzyme molecule introduced by the
immobilization procedure, lower the accessibility of
inhibiting ions to the active site of the enzyme and (2) the
chelating effect of BSA, which is known to be a very
powerful chelating agent, especially when cross-linked
with glutaraldehyde forming glutaraldehyde-cross-linked
BSA particles [35].
Enzyme inactivation by heavy metals, including mer-
cury (Hg
2?
), proceeds by the reduction in the thiol group in
cysteine residues, with the formation of mercaptides, or the
reduction in disulfide bridges, leading to S–Hg–S bonds
[36]. These results are in agreement with those obtained for
other immobilized dextranase [23]. In this connection,
more stability of immobilized Streptomyces anulatus
dextranase to Pb
?2
,Cu
?2
,Al
?3
ions than free enzyme is
reported [37].
The operational stability of immobilized dextranase is
the most important factor affecting dextran biodegradation
in many industrial applications such as food, beverage, and
sugar cane industries and other undesirable effects of
dextran [32]. The operational stability of immobilized
dextranase is evaluated in repeated batch processes. After
each run, the immobilized dextranase was washed and
reused at optimum conditions for another reaction. The
percent of residual dextranase activity was determined for
6 cycles. The immobilized enzyme was able to maintain a
good yield of reducing sugars, while drop in activity was
possible after many cycles due to the release of bound
enzyme from the carrier. The results in Fig. 8indicate that
immobilized dextranase retains 76% of its original activity
after 5 cycles. The result also indicated that the loss rate of
immobilized dextranase activity was 4.6% cycle
-1
. These
results are more or less similar with that obtained for
P. funiculosum immobilized on chitosan [23].
Conclusions
In this study, A. subolivaceus dextranase produced by
submerged fermentation on dextran was efficiently immo-
bilized by two methods of entrapment and covalent bond-
ing with cross-linking. Cross-linking within BSA in the
presence of glutaraldehyde gave immobilization yield
(66.7%) and highest specific activity (1.56 U lg protein
-1
)
compared with the other carriers used. The immobilized
dextranase remained stable for longer periods of time and
also at higher temperatures as compared to the free
enzyme. In addition, pH studies indicated that the enzyme
remained significantly active over a broader pH range
(5.5–8.0) compared to the free enzyme in solution. The
kinetic properties of dextranase revealed a lower affinity
of the immobilized enzyme with a higher K
m
(14.24 mg mL
-1
) compared to free dextranase. In addition,
after five operational cycles, it was observed that the
immobilized dextranase retained 76% of its original
activity. The properties of the immobilized dextranase
described here suggest its value for industrial applications
that would not be feasible with the free enzyme system.
Further studies will be subject in future works are needed
to use immobilized and thermal-stable dextranase in the
continuous removal of undesirable dextran molecules in
food, juice, sugar cane industries, and oligosaccharide
synthesis.
0 1 2 3 4 5 6 7
0
20
40
60
80
100
Cycle number
Residual activity (%)
Fig. 8 Operational stability of immobilized A. subolivaceus
dextranase
Eur Food Res Technol (2011) 233:735–742 741
123
References
1. Khalikova E, Susi P, Korpela T (2005) Microbial dextran-
hydrolyzing enzymes: fundamentals and applications. Microbiol
Mol Biol Rev 69(2):306–324
2. Patel S, Goyal A (2011) Functional oligosaccharides: production,
properties and applications. World J Microbiol Biotechnol
27:1119–1128
3. Eggleston G, Monge A, Montes B, Stewart D (2007) Factory
trials to optimize the industrial application of dextranase in raw
sugar manufacture part II. Intern Sugar J 109:757–764
4. Milintawisamai N, Niamsanit S, Ngasan C, Pliansinchai U,
Weerathaworn P (2009) Dextran producing microorganisms
from Mitr Phuveing sugar factory, Thailand. Sugar Tech
11(2):196–199
5. Jime
´nez ER (2009) Dextranase in sugar industry: a review. Sugar
Tech 11(2):124–134
6. Eggleston G, Monge A, Montes B, Stewart D (2009) Application
of dextranases in sugarcane factory: overcoming practical prob-
lems. Sugar Tech 11(2):135–141
7. Lee JH, Kim SH, Cho DL, Day DF, Kim D (2006) Treatment of a
glucanhydrolase from Lipomyces starkeyi for the removal of
soluble polysaccharides in sugar processing. J Microbiol Bio-
technol 16:983–987
8. Kim D, Ryu SJ, Son EJ, Chung HJ, Kim SH, Kim DW, Day DF
(2002) Glucanhydrolase from Lipomyces starkeyi KSM 22 as
potential mouthwash ingredient. J Microbiol Biotechnol
12:993–997
9. Chen L, Zhou X, Fan W, Zhang Y (2007) Expression, purification
and characterization of a recombinant Lipomyces starkey dex-
tranase in Pichia pastoris. Protein Expres Purif 58:87–93
10. Goulas AK, Cooper JM, Grandison AS, Rastall RA (2004)
Synthesis of isomaltooligosaccharides and oligodextrans in a
recycle membrane reactor by the combined use of dextransucrase
and dextranase. Biotechnol Bioeng 88:778–787
11. Kim Y, Ko E, Kang H, Kim D (2009) Construction, expression
and characterization of fusion enzyme from Arthrobacter oxy-
dans dextranase and Klebsiella pneumoniae amylase. Biotechnol
Lett 31:1019–1024
12. Lee JH, Nam SH, Park HJ, Kim YM, Kim N, Kim G, Seo ES,
Kang SS, Kim D (2010) Biochemical characterization of dex-
tranase from Arthrobacter oxydans and its cloning and expression
in Escherichia coli. Food Sci Biotechnol 19(3):757–762
13. Millson SH, Evans IH (2007) Multiple dextranases from the yeast
Lipomyces starkeyi. Antonie van Leeuwenhoek 92:399–404
14. Erhardt FA, Stammen S, Jordening H (2008) Production, char-
acterization and (co-) immobilization of dextranase from Peni-
cillium aculeatum. Biotechnol Lett 30:1069–1073
15. Thitaram SN, Chung CH, Day DF, Hinton A, Bailey JS, Siragusa
GR (2005) Isomaltooligosaccharide increases cecal Bifidobacte-
rium population in young broiler chickens 1. Poult Sci 84(7):998–
1003
16. Sherief AA, El-Tanash AB, El-Baz E (2010) Production and
characterization of dextranase from some local isolated fungi.
Mansoura J Biol 36(2):33–49
17. Nelson N (1944) A photometric adaptation of Somogyi method
for determination of glucose. J Bio Chem 153:375–380
18. Somogyi M (1952) Notes on sugar determination. J Bio Chem
195:19–23
19. Bradford M (1976) A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 72:248–254
20. Abdel-Naby MA (1993) Immobilization of Aspergillus niger
NRC 107 xylanase and b-xylosidase and properties of the
immobilized enzymes. Appl Biochem Biotechnol 38:69–81
21. Ghosh M, Nanda G (1993) Thermostability of b-xylosidase from
Aspergillus sydowii MG49. Federation of European Biochemical
Societies (FEBS) 330(3):275–278
22. Cao L (2005) Carrier-bound immobilized enzymes: principles,
application and design. WILEY-VCH Verlag GmbH and Co.
KGaA, Weinheim. ISBN:3-527-31232-3
23. Abdel-Naby MA, Ismail S, Abdel-Fattah AM, Abdel-Fattah AF
(1999) Preparation and some properties of immobilized Penicil-
lium funiculosum. Process Biochem 34:391–398
24. El-Tanash AB, Sherief AA, Nour A (2011) Catalytic properties of
immobilized tannase produced from Aspergillus aculeatus com-
pared with the free enzyme. Braz. J Chem Eng 28(3):381–391
25. Yu X, Li Y, Wang C, Wang D (2004) Immobilization of
Aspergillus niger tannase by micro encapsulation and its kinetic
characterization. Biotechnol Appl Bioc 40:151–155
26. Kitano H, Nakamura K, Ise N (1982) Kinetic studies of enzyme
immobilized on anionic polymer lattices. J Appl Biochem
4:34–40
27. Gottschalk N, Jaenicke R (1991) Authenticity and reconstitution
of immobilized enzymes: characterization and denaturation/
renaturation of glucoamylase 11. Biotechno Appl Bioc 14:324–
335
28. Krajewska B, Leszko M, Zaborska W (1990) Urease immobilized
on chitosan membrane: preparation and properties. J Chem
Technol Biot 48:337–350
29. Ramesh V, Singh C (1980) Bacterial dextranase immobilized on
zirconia coated alkylamine glass using glutaraldehyde. Biochem
Biophys Res Commun 97:779–786
30. Sugiura M, Ito A (1975) Studies on dextranase. VIII. Some
enzymatic properties of immobilized dextranase from Brevibac-
terium fuscum var. dextranlyticum. Chem Pharm Bull 23(12):
3223–3227
31. Aslan Y, Tanriseven A (2007) Immobilization of Penicillium li-
lacinum dextranase to produce isomaltooligosaccharides from
dextran. Biochem Eng J 34:8–12
32. Madhu GLS, Prabhu KA (1985) Studies on dextranasa from
Penicillium aculeatum. Enzyme Microb Technol 6:217–220
33. Rogalski J, Szczodrak M, Pleszczyhka J, Fiedurek J (1997)
Immobilisation and kinetics of Penicillium notatum dextranase on
controlled porous glass. J Mol Catal B-Enzym 3:271–283
34. Erhardt FA, Jo
¨rdening HJ (2007) Immobilization of dextranase
from Chaetomium erraticum. J Biotechnol 131(4):440–447
35. Kennedy JF, Kalogerakis B (1980) Immobilization of glucoam-
ylase on gelatin by transition-metal chelation. Biochimie 62(8–9):
549–561
36. Vallee BL, Ulmer DD (1972) Biochemical effects of mercury,
cadmium, and lead. Ann Rev Biochem 41:91–128
37. Mahmoud D, Helmy W (2009) Application of cold-active dex-
tranase in dextran degradation and isomaltotriose synthesis
by micro-reaction technology. Aust J Basic Appl Sci 3(4):3808–
3817
742 Eur Food Res Technol (2011) 233:735–742
123
