Novel grafted agar disks for the covalent immobilization of β-D-galactosidase

Article abstract of DOI:10.1002/bip.22693

Novel grafted agar disks were prepared for the covalent immobilization of β-D-galactosidase (β-gal). The agar disks were activated through reacting with ethylenediamine or different molecular weights of Polyethyleneimine (PEI), followed by glutaraldehyde (GA). The modification of the agar gel and the binding of the enzyme were verified by Fourier Transform Infrared (FTIR) and elemental analysis. Moreover, the agar's activation process was optimized, and the amount of immobilized enzyme increased 3.44 folds, from 38.1 to 131.2 U/g gel, during the course of the optimization process. The immobilization of β-gal onto the activated agar disks caused its optimum temperature to increase from 45°C to 45-55°C. The optimum pH of the enzyme was also shifted towards the acidic side (3.6-4.6) after its immobilization. Additionally, the Michaelis-Menten constant (K_m) increased for the immobilized β-gal as compared to its free counterpart whereas the maximum reaction rate (V_max) decreased. The immobilized enzyme was also shown to retain 92.99% of its initial activity after being used for 15 consecutive times.

Full text of DOI:10.1002/bip.22693

Novel Grafted Agar Disks for the Covalent Immobilization
of b-D-Galactosidase
1,2
1,2
Marwa I. Wahba, Mohamed E. Hassan
1
Department of Chemistry of Natural and Microbial Products, National Research Center, El-Behooth St., Dokki, Giza, Egypt
2
Centre of Scientific Excellence-Group of Encapsulation and Nanobiotechnology, Cairo, Egypt
Received 2 March 2015; revised 31 May 2015; accepted 1 June 2015
Published online 5 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22693
This article was originally published online as an accepted
preprint. The “Published Online” date corresponds to the pre-
print version. You can request a copy of any preprints from the
past two calendar years by emailing the Biopolymers editorial
office at biopolymers@wiley.com.
ABSTRACT:
Novel grafted agar disks were prepared for the covalent
immobilization of b-D-galactosidase (b-gal). The agar
disks were activated through reacting with ethylenedia-
mine or different molecular weights of Polyethyleneimine
(
PEI), followed by glutaraldehyde (GA). The modifica-
INTRODUCTION
tion of the agar gel and the binding of the enzyme were
verified by Fourier Transform Infrared (FTIR) and ele-
mental analysis. Moreover, the agar’s activation process
was optimized, and the amount of immobilized enzyme
increased 3.44 folds, from 38.1 to 131.2 U/g gel, during
the course of the optimization process. The immobiliza-
tion of b-gal onto the activated agar disks caused its opti-
mum temperature to increase from 458C to 45–558C. The
optimum pH of the enzyme was also shifted towards the
acidic side (3.6–4.6) after its immobilization. Addition-
he enzyme b-D-galactosidase (E.C. 3.2.1.23) catalyzes
the hydrolysis of b-D-(1–3) and b-D-(1–4) glycosidic
bonds in oligo- and disaccharides. Lactose (b-D-
galactopyranosyl-(1!4)-D-glucose) is the main sub-
strate of b-D-galactosidase (b-gal). The enzymatic
1,2
T
hydrolysis of lactose by b-gal has two main biotechnological
applications; the production of low lactose milk and dairy
products for consumption by lactose intolerant persons, and
the utilization of whey, as its hydrolysates (glucose and galac-
3
tose) have higher fermentation potential.
Besides their hydrolytic activity on lactose, b-gal enzymes
may also possess transgalactosylation activity. This transferase
activity enables the synthesis of galacto-oligosaccharides
4
GOS), when lactose acts the acceptor of galactose. GOS are
ally, the Michaelis-Menten constant (K ) increased for
m
(
the immobilized b-gal as compared to its free counterpart
whereas the maximum reaction rate (Vmax) decreased.
The immobilized enzyme was also shown to retain
considered as prebiotics because they selectively stimulate the
growth of beneficial bacteria, such as bifidobacteria and lacto-
1
bacilli, in the colon. In addition to the prebiotic activity, GOS
9
2.99% of its initial activity after being used for 15 con-
have also been reported to contribute to (i) reduction of serum
cholesterol and lipid level; (ii) synthesis of B-complex vitamins;
secutive times. V
C
2015 Wiley Periodicals, Inc. Biopoly-
(iii) enhancing the absorption of dietary calcium; (iv) protec-
mers 103: 675–684, 2015.
tion from infection and decreasing of pathogenic bacteria; (v)
5
stimulating the absorption of some minerals. Moreover, b-gal
can also be used to attach galactose to other chemicals and
Keywords: agar; covalent immobilization; b-D-galacto-
sidase; polyethyleneimine; glutaraldehyde
consequently have imminent applications in the production of
6
biological active compounds.
Being an important industrial enzyme, b-gal was selected as
a model enzyme for our immobilization study. Immobilization
enhances the stability of enzymes. Furthermore, it facilitates
Correspondence to: Marwa I. Wahba; e-mail: drmarwawahba@yahoo.com
V
C
2015 Wiley Periodicals, Inc.
Biopolymers Volume 103 / Number 12
675

6
76
Wahba and Hassan
ꢀ
ꢀ
Step A which comprised the addition of different molec-
ular weights of polyethylenimine (PEI) or ethylene dia-
mine (EDA) to the agar disks. This polyamine
compound contributed both protonated and free amino
groups to the activation process. The protonated amino
groups formed an ionic complex (network) with the
sulfate and pyruvate anions of the agar gel.
On the other hand, the free amino groups were
employed during step B. This step involved the addition
of the di-aldehyde spacer arm, glutaraldehyde (GA).
The aldehyde groups present at one end of GA mole-
cules reacted with the free primary amine moieties of
PEI forming Schiff’s base. Meanwhile, the unreacted GA
side created free aldehyde groups for covalent immobili-
zation of the enzyme.
FIGURE 1 Schematic representation of agarobiose (RA2; RA4; RA6;
, Pyruvic
–
–
3
and RB2: H) and agaropectin (RA2: H, SO
acid (cyclic ketal with O ); RA6: H, CH , SO
ketal with O ), RB2: H, CH
3
; RA4: H, SO
–
6
3
3
; Pyruvic acid (cyclic
–
4
3 3
, SO ).
the separation of the enzyme from the product, thereby mini-
mizing the chances of product contamination and allowing the
easy recovery of the enzyme. This enzyme can then be reused
in a continuous industrial operation.
In this study, b-gal, as a model enzyme, was covalently
immobilized onto the novel activated agar disks. The activation
process was optimized in order to attain the upmost amount
of immobilized enzyme. Moreover, the alteration of the agar
structure was elucidated by elemental analysis and Fourier
Transform Infrared (FTIR). Finally the reusability of the
immobilized b-gal was investigated.
However, efficient commercial carriers suitable for the
7
immobilization of enzymes are fairly expensive. Available car-
riers; such as, Eupergit CVR or AgaroseVR are sold for e 6000
8
and e 3,250/kg, respectively. Hence, there is a persistent need
to prepare new enzyme carriers that are cheap. Agar (more cor-
rectly agar-agar) is a hydro gel that is available at a reasonable
9
EXPERIMENTAL
cost. Moreover, it is permitted for use in food industries.
Thus, it is an ideal candidate for enzyme immobilization.
Agar (Figure 1) is a polysaccharide consisting of a mixture
of agarose and agaropectin. Agarose, the predominant compo-
nent of agar, is responsible for the high strength gelling proper-
ties of agar. It is a linear neutral polymer, made up of the
repeating monomeric units of agarobiose. Agarobiose is a
disaccharide consisting of D-galactose and 3,6-anhydro-L-gal-
actose. The other component of agar, agaropectin, is a charged
polymer that has the same repeating units as agarose but with
Materials
b-D-galactosidase (EC 3.2.1.23) from Aspergillus oryzae, PEI
M 800, and PEI M 2000 were obtained from Sigma-Aldrich
w
w
(Germany). PEI Mw 750,000 was bought from Fluka (Switzer-
land). GA solution was purchased Bio Basic (Canada). Agar
was acquired from SD Fine chemicals (Mumbai). All other fine
chemicals were of Analar or equivalent quality.
acid groups such as sulfate and pyruvate. Agaropectin is also Methods
9
methylated.
Preparation of Agar Gel Disks. An agar solution of 5% (w/v)
There are many techniques to immobilize enzymes; such as, was prepared in distilled water by heating. Then, a glass parallel
1
4
adsorption, covalent, encapsulation, entrapment, and cross- plate equipment, with 10-mm gaps, was immersed into the
linking. Nevertheless, the covalent technique has the advantage hot agar solution, and the agar was left to solidify in the fridge.
of keeping the enzyme well bound to the carrier, avoiding The produced 10-mm thick homogeneous gel sheets were cut
10
enzyme leakage. Thus, enabling the reuse of the immobilized into disks using a cork borer. Typically, 4-mm diameter gel
enzymes tens of times, reducing their cost as well as their prod- disks of average weight 180 mg were produced.
ucts’ cost. This is why the covalent immobilization is widely
preferred on the industrial scale. Agar was activated, for the Activation of Agar Gel Disks. The agar disks were first soaked
covalent immobilization of enzymes, via its derivatization into in an amine solution (PEI or EDA) for a specified period of
11–13
glyoxyl agar (Agar-----O-CH -CHO).
In the present study, time. The excess amine was then removed by thoroughly wash-
2
we attempted a new activation method which, to our knowl- ing the disks with distilled water. Afterwards, the disks were
edge, has never been applied to agar before. This activation soaked in a GA solution, washed with distilled water, and
method consisted of two steps (Figure 2) as follows:
directly used to immobilize b-gal. It is worth mentioning that,
Biopolymers

Novel Grafted Agar Disks for the Covalent Immobilization of b-D-galactosidase
677
FIGURE 2 Schematic representation of agar gel activation and enzyme immobilization (X: sulfate
or pyruvate).
the color of the agar disks changed from translucent to orange in a thermo-stated shaking water bath at 378C. The agar disk
brown during the activation process. This orange brown color was then removed, and the absorbance of the supernatant was
was due to the formation of Schiff’s base (–N5CH–) between measured at 405 nm.
PEI amino groups and the GA’s aldehyde groups. Noteworthy,
Regarding the free b-gal, it was assayed by mixing 0.25 ml
the presence of the aldehyde group required for b-gal immobi- of the enzyme solution with 3.75 ml of 0.1 M acetate buffer
lization was proven through the Fourier Transform Infrared (pH 4.6). Afterwards, 1 ml of a 10 mM ONPG solution was
technique.
added to the reaction mixture. The reaction was left to proceed
for 15 min in a thermos-stated water bath at 378C. Then, the
Covalent Immobilization of b-Gal onto the Activated Agar absorbance of the solution was measured at 405 nm. One unit
Disks. About 1 g of the activated agar disks was mixed with of b-gal activity (U) was defined as the amount of enzyme that
5
ml of 0.3 M citrate-phosphate buffer (pH 4.5) containing liberates 1.0 nmol of o-nitrophenol from the ONPG per min
ꢁ200 U/ml b-gal. This mixture was agitated, using a roller stir- under standard assay conditions.
rer, at room temperature for 18 h. Afterwards, the gel disks
were filtered off, washed thoroughly with distilled water, and Structure Elucidation. Elemental Analysis. The elemental
directly assayed for b-gal activity.
composition of four samples (agar, agar/PEI, agar/PEI/GA,
and agar/PEI/GA/Enz) was determined by the elemental ana-
Determination of b-Gal Activity. The b-gal was assayed by lyzer vario El (Elementar, Germany).
using a modification of the procedure employed by Haider Fourier Transform Infrared. The infrared spectra of all formula-
15
and Husain. One b-gal loaded agar disk was soaked in 4 ml tions were recorded with Fourier Transform Infrared Spectros-
of 0.1 M acetate buffer (pH 4.6), followed by the addition of copy (FTIR-4100, Jasco, USA). FTIR spectra were taken in the
2
1
1
ml of a 10 mM o-nitrophenyl b-D-galactopyranoside wavelength region from 4000 to 400 cm at ambient temper-
ONPG) solution. The reaction was left to proceed for 15 min ature. Formula 1 (agar) formula 2 (agar/PEI), formula 3 (agar/
(
Biopolymers

6
78
Wahba and Hassan
PEI/GA), and formula 4 (agar/PEI/GA/b-gal) were examined
for the presence of the new functionalities using FTIR.
Assessment of the Covalent Bond Formed Between b-Gal
and the Activated Agar Disks. The b-gal loaded agar disks
were incubated with 1M NaCl at room temperature for 1 h.
16
After that, enzyme activity was analyzed in the supernatant.
Evaluation of the Catalytic Activity of b-Gal. Effect of
pH. Both the free and the immobilized b-gals were assayed in
buffers of various pH values (pH 2.6–8) as described in the
section on determination of b-gal activity. The buffers used
were the citrate-phosphate buffer (pH 2.6–5.6) and the sodium
phosphate buffer (pH 6–8). These buffers were employed at a
final concentration of 100 mM. The activity recorded at the
optimum pH was taken as the 100% activity, and the activities
offered at the other pH values were expressed as a percentage
of this optimum 100% activity.
FIGURE 3 Effect of the type of the polyamine compound on the
immobilization of b-gal (means 6 S.E.).
RESULTS AND DISCUSSION
Optimization of the Activation Process
Optimization of Step A. Step A, which comprised the reac-
tion of the agar disks with PEI (Figure 2), was optimized while
keeping the reaction conditions for step B constant. In all the
experiments, the agar disks were soaked in a 2% (v/v) GA solu-
tion for 1 h after their reaction with PEI.
Effect of Temperature. The enzymatic reactions of both the free
and the immobilized b-gals were carried out at different tem-
peratures (378C–658C) as described in the section on determi-
nation of b-gal activity. The activity offered at the optimum
temperature was taken as the 100% activity, and the activities
recorded at the other temperatures were expressed as a per-
centage of this 100% activity.
Selection of the Polyamine Compound. EDA together with three
different molecular weight PEI compounds (800, 2000, and
750,000 Da) were selected to test their efficiency in activating
the agar gel. EDA was previously used during the activation of
17
Determination of the Kinetic Parameters. The Michaelis-
epoxy monolithic polyacrylamide cryogels and poly(vinyl
18
Menten constant (K ) and the maximum reaction rate (Vmax)
m
chloride) microspheres for the covalent immobilization of
19
enzymes. PEI was also used to activate carrageenan disks,
for the free b-gal and its immobilized counterpart were deter-
mined using the Hanes-Woolf plot. In order to do so, the enzy-
matic reaction was carried out in the presence of varying final
concentrations of ONPG (0.5–5 mM) in 100 mM acetate
buffer of pH 4.6.
20
alginate beads, and alginate-carrageenan beads.
21
During the course of this experiment, the agar disks were
soaked for 1 h in a 1% (w/v) solution (pH 8.5) of each of these
four amine compounds. The pH 8.5 was selected as it is some-
22
what lower than the isoelectric point of EDA (PI 9.4). More-
over, it was reported that about 18% of PEI would be
Operational Stability (Reusability). The reusability of b-gal
covalently immobilized onto activated agar disks was tested.
The gel disks were assayed as in the section on determination
of b-gal activity. Afterwards, the same gel disks were washed
thoroughly with distilled water and re-assayed. This procedure
was repeated 15 times, and the initial activity was considered
as 100%. The relative activity was expressed as a percentage of
the starting operational activity.
23
protonated at pH 8.5. Hence, all the tested amine com-
pounds were partially protonated at pH 8.5. This partial proto-
nation enabled the amine compounds to interact ionically
with the negatively charged agar disks.
The results (Figure 3) showed that EDA offered the lowest
immobilization yield (38.10 U/g gel). This could be due to the
fact that each EDA molecule could only immobilize one
enzyme molecule (Figure 2). EDA has only two amino groups;
one to interact with the agar gel and the other to interact with
Statistical Analysis. Data were analyzed with the Microsoft one GA molecule and consequently one enzyme molecule.
Excel 2007. Results were expressed as mean 6 standard error.
It was noticed (Figure 3) that the amount of immobilized
The mean of the control was compared with the means of dif- b-gal increased with the increase in the PEI’s molecular
ferent treatments using the one way ANOVA. The P values less weight till reaching a maximum of 84.44 U/g gel upon
than 0.05 were considered statistically significant.
employing PEI 750,000. This could be attributed to the fact
Biopolymers

Novel Grafted Agar Disks for the Covalent Immobilization of b-D-galactosidase
679
methacrylate) microchannels for the covalent immobiliza-
25
tion of glucose oxidase.
Optimization of PEI 750,000 pH, Concentration, and Soaking
Time. In order to mark out the optimum PEI pH, 7 different
pH values (between 7 and 10) were examined. It could be seen
(Figure 4A) that the maximum amount of immobilized b-gal
was attainable at the highest inspected pH values. Where, a
whole of 93.13 U/g gel were immobilized at either pH 9.5 or
10. These high pH values were favored for the enzyme immo-
bilization, since they provided the most suitable ratio between
the PEI’s protonated and free amino groups. At such high pH
values, the majority of the PEI’s amino groups were in the free
form and only few were protonated. Actually, it was previously
2
6
reported that only ꢂ4% of PEI was protonated at pH 10.
This low level of protonation was enough for the PEI to inter-
act ionically with the low amounts of negatively charged pyru-
vate and sulfate groups located on agar. Where, agar was
27
previously reported to contain only 0.12–0.65% pyruvate
9
and about 2% sulfate. The low sulfate content was also veri-
fied for the type of agar used in the study, as its elemental anal-
ysis showed it to exhibit only 0.596% sulfur. On the other
hand, the high content of PEI’s free amino groups, that was
brought about by such high pH values, enabled the PEI treated
agar disks to react with lots of GA molecules and accordingly
to immobilize lots of enzyme molecules. It is worth mention-
ing that, a PEI 750,000 solution of pH 9.5 was also used during
the activation of the waveguide sensor surface for the immobi-
28
lization of antibodies.
The PEI concentration and soaking time were optimized
while utilizing a PEI 750,000 solution of pH 9.5. The results
(Figure 4B) displayed a gradual increase in the amount of
immobilized b-gal with the increase in the PEI’s concentration
till reaching a maximum of 106.29 U/g gel upon employing a
3% (w/v) PEI solution. It was also shown (Figure 4C) that the
optimum soaking time of PEI with the agar disks was 2.5 h.
These findings were close to those previously reported in litera-
29
30
ture where both alginate and carrageenan gel beads were
soaked in a 4% PEI solution for 3 h during their activation for
the covalent immobilization of penicillin G acylase. Moreover,
FIGURE 4 Effect of (A) PEI pH, (B) PEI concentration, and (C)
PEI soaking time on the immobilization of b-gal onto the activated
agar disks (means 6 S.E.).
19
Elnashar et al. stated that it would be optimum to employ a
concentration range of 3–5% PEI during the activation of car-
rageenan disks.
that the higher the molecular weight of PEI, the more
branched it becomes (Figure 2), and the more primary Optimization of Step B. Step B of the activation process
amino groups it attains. Those primary amino groups are involved the reaction of GA with the PEI treated agar disks.
2
4
the ones responsible for the reaction with GA. Thus, their This reaction created free aldehyde groups capable of cova-
abundance in the high molecular weight PEI would ensure lently immobilizing b-gal (Figure 2). The optimization of step
the reaction of this PEI treated agar disks with plenty of GA B was fulfilled through the investigation of the optimum GA
and in turn plenty of enzyme molecules. Noteworthy, PEI concentration and soaking time. Noteworthy, the optimum
750,000 was also employed in the activation of poly(methyl settings that were reached during the optimization of step A
Biopolymers

680
Wahba and Hassan
cantly affect the amount of immobilized b-gal. In order to ver-
ify such a postulate, we conducted a one way ANOVA test on
the results of this experiment. The P value turned out to be
0.792, indicating that there was no significant difference
between the amounts of immobilized b-gal obtained at any of
the inspected GA soaking times. A similar situation was
2
1
noticed by Elnashar et al., who reported that changing the
GA soaking time from 0.5 to 3 h did not significantly affect the
amount of penicillin G acylase covalently immobilized onto
alginate-carrageenan gel beads. They debated that the reason
behind this observation was the fact that GA crosslinks the
PEI’s free amino groups only. Most of these free amino groups
would suffer from protonation upon incubation with a GA
solution of low pH (4.52). Hence, after 30 min of incubation
with the GA solution, the PEI treated agar disks would virtually
have no more free PEI amino groups to offer for the reaction
with GA. Thus, further increasing the soaking time would not
significantly increase the amount of reacted GA and conse-
quently would not significantly increase the amount of immo-
bilized enzyme. For future work we recommend preparing the
GA solution in a buffer of pH in the range of 7–9 to limit the
protonation of the PEI’s free amino groups.
FIGURE 5 Effect of (A) GA concentration and (B) GA soaking Structure Elucidation
time on the immobilization of b-gal onto the activated agar disks
The incorporation of new functionalities into the agar gel struc-
ture, during the activation and the immobilization processes,
was proven via the elemental analysis and the FTIR of agar,
agar/PEI, agar/PEI/GA, and agar/PEI/GA/Enz formulations.
(
means 6 S.E.).
were employed here; i.e., the agar disks were soaked for 2.5 h
in a 3% (w/v) PEI 750,000 solution of pH 9.5 prior to treating
them with GA.
Elemental Analysis. The elemental analysis data for the agar
Optimization of GA Concentration. Figure 5A revealed that
increasing the GA concentration from 1% to 3% (v/v) caused
the amount of immobilized b-gal to increase from 86.56 to
disks was 36.29% (C), 7.126% (H), 0% (N), 0.596% (S), and
5
5.988% (O). Meanwhile, the elemental composition of the
agar/PEI disks was 40.17% (C), 8.792% (H), 8.59% (N),
.37% (S), and 44.078% (O). The absence of nitrogen in the
agar disks and its presence in the PEI treated disks proved the
126.23 U/g gel. This 1.46-fold increase in the immobilization
0
yield reflected the increase in the amount of the free aldehyde
groups capable of covalently immobilizing b-gal.
19
Further increasing the GA concentration to 3.5% (v/v) incorporation of PEI in the agar gel. Elnashar et al. also
caused the amount of immobilized b-gal to drop slightly proved the incorporation of PEI into carrageenan gel by using
reaching 122.52 U/g gel. Thus, the optimum GA concentration the elemental analysis. Where, nitrogen was absent in case of
was considered to be 3% (v/v). This value was close to the one carrageenan gel, but it was present at a concentration of
31
reported by Kishore et al., who stated that employing a 15.059% in the PEI treated carrageenan gel.
3
.34% (v/v) GA solution was optimal for the covalent immobi-
The elemental analysis of the agar/PEI/GA disks was
47.32% (C), 9.002% (H), 4.47% (N), 0.4% (S), and 38.808%
lization of b-gal onto functionalized graphene nano-sheets.
Optimization of GA Soaking Time. In this experiment, six dif- (O). The rise in the carbon percent from 40.17%, in case of
ferent GA soaking times, ranging from 0.5 to 3 h, were investi- the agar/PEI disks, to 47.32%, in case of the Agar/PEI/GA
gated. The amount of immobilized b-gal varied slightly, from disks, reflected the incorporation of GA (C
26.26 to 131.20 U/g gel, along the entire tested time range structure. Noteworthy, the incorporation of GA into alginate-
Figure 5B). This slight variation (3.8%) could indicate that carrageenan/PEI gel beads was also previously proven by the
H O ) in the gel
5
8 2
1
(
changing the GA soaking time from 0.5 to 3 h did not signifi- rise in the carbon percent from 30.64%, for the alginate-
Biopolymers

Novel Grafted Agar Disks for the Covalent Immobilization of b-D-galactosidase
681
2
590–1650 cm . Both bands were overlapped with the broad-
1
1
21
band of the –OH and the –COOH groups at 3300–3600 cm
21
and 1630–1670 cm , respectively. It is worth mentioning
that, the sulfate band of the agar was perturbed from
2
1
21
088 cm to 1075 cm after the formation of the gel com-
1
plex with PEI. This observation could be due to the strong
polyelectrolyte interaction between the positively charged –
1
NH3 of the PEI and the negatively charged –OSO of agar.
–
3
21
A similar shift was also observed by Elnashar et al. where, the
21
sulfate band of carrageenan was perturbed from 1050 cm to
21
1
030 cm after the formation of a gel-complex with PEI.
As for the (agar/PEI/GA) formulation, it was postulated
that the aldehyde groups present at one end of the GA mole-
cules reacted with the free amine moieties of PEI forming
Schiff’s base. This Schiff’s base (–C5N–) appeared as a
–1
shoulder at 1616 cm . Moreover, it was also assumed that the
unreacted GA side created free aldehyde groups for covalent
immobilization of b-gal. These aldehyde groups appeared as a
–1
shoulder at 2730 cm . This shoulder smoothed out in the
agar/PEI/GA/b-gal) FTIR indicating that the free GA aldehyde
(
groups were employed to covalently immobilize b-gal through
the formation of Schiff’s base with the b-gal amino groups.
FIGURE 6 The FTIR bands of the (agar), (agar/PEI), (agar/PEI/ The Schiff’s base peak of the (agar/PEI/GA/b-gal) formulation
GA), and (agar/PEI/GA/b-gal).
overlapped with a band, that was previously located at
–1
1
557 cm in the (agar/PEI/GA) FTIR, forming a single band
carrageenan/PEI beads, to 40.45%, for the alginate-carra-
–1
from 1560–1616 cm . Noteworthy, the Schiff’s base formed
21
geenan/PEI/GA beads.
between the aldehyde groups of GA and the amino groups of
32
the immobilized enzymes was also noticed by Bahman et al.
The agar/PEI/GA/Enz disks exhibited a somewhat different
elemental composition from that of the agar/PEI/GA disks. The
carbon, hydrogen, nitrogen, and sulfur contents of the enzyme
loaded disks decreased reaching 41.21%, 8.19%, 4.24%, and
33
and Ren et al. at 1648 cm and 1639 cm , respectively.
21
–1
Assessment of the Covalent Bond Formed Between
b-Gal and the Activated Agar Disks
0
.22%, respectively. Meanwhile, its oxygen content increased to
the level of 46.14%. This alteration in the elemental composi-
tion of the agar/PEI/GA/Enz disks could be attributed to the
covalent immobilization of b-gal onto the agar/PEI/GA disks.
In order to ascertain that b-gal was covalently immobilized
onto the activated agar disks and not just adsorbed onto the
agar surface, the enzyme loaded disks were incubated with 1M
NaCl. NaCl would cause the adsorbed enzyme to be eluted
from the agar surface into the supernatant liquid, where it
could be detected. The results of this experiment revealed that
the supernatant was devoid of any b-gal activity. Hence, it
could be concluded that all the immobilized b-gal was cova-
21 lently attached to the agar disks and not adsorbed. Noteworthy,
Fourier Transform Infrared. The FTIR bands of the (agar),
(agar/PEI), (agar/PEI/GA), and (agar/PEI/GA/b-gal) were
shown in Figure 6. Regarding the agar formulation, it could be
–
3
noticed that the agar’s sulfate groups (–OSO ) appeared as a
21
band at 1088 cm . Moreover, the pyruvate’s carboxylate
–
groups (–COO ) were represented as a band at 1633 cm
21
b-gal was shown to be covalently immobilized onto GA acti-
and a broad band at 1370–1457 cm . This broad band
resulted from the overlapping of the carboxylate band with the
bands of the agar’s primary and secondary hydroxyl groups
that were supposed to appear at 1400–1460 and 1345–
34
vated carrageenan-chitosan disk, magnetic polysiloxane par-
3
5
ticles coated with polyaniline, magnetic Fe O -chitosan
4
3
36
37
nanoparticles, and multiwalled carbon nanotubes.
21
1
475 cm , respectively. Considering the (agar/PEI) formula-
tion, the PEI was expected to reveal two characteristic bands Evaluation of the Catalytic Activity of b-Gal
for the amine groups. These two bands were: a stretching Effect of pH. The free b-gal was shown to exhibit a broad pH
2
1
vibration at 3300–3400 cm
and a bending vibration at optimum (4.6–6.5) (Figure 7A). This optimum pH range was
Biopolymers

6
82
Wahba and Hassan
Table I Kinetic parameters for b-gal catalyzed ONPG hydrolysis
a
Kinetic parameters
Sample
K
m
(mM)
2.01
V
max (mmol/min/mg enzyme)
Free b-gal
Immobilized b-gal
1.275
0.968
10.37
a
m
Values of K and Vmax were calculated from the Hanes-Woolf plot
(
Figure 8).
ular cage around the enzyme. This molecular cage protected
34
the enzyme’s molecule from the bulk temperature.
Determination of the Kinetic Parameters. The results (Table
I) showed that the Km for the free b-gal amounted to
2
.01 mM. This value was close to the one reported by Vera
43
et al. who proved that the free Aspergillus oryzae b-gal exhib-
ited a K of 2.27 mM. Immobilizing b-gal onto the activated
m
agar disks caused the Km to increase reaching 10.37 mM
whereas the Vmax decreased. Mass transfer resistance might be
the reason behind the increase in Km after immobilization.
Mass transfer resistance was shown to be significant for macro-
molecular substrates such as ONPG because the substrate
should contact the immobilized enzyme on its carrier. Addi-
tionally, the immobilization of b-gal might have restricted the
FIGURE 7 Effect of (A) pH and (B) temperature on the activities
of the free and the immobilized b-gals (means 6 S.E.).
somewhat broader than the one reported by Elnashar and Yas- enzyme’s ability to undergo conformational changes intrinsic
3
4
sin who stated that the Aspergillus oryzae b-gal exhibited an to the enzyme-substrate interactions. This restriction would
42
optimum pH range of 4.5–5. The immobilization of b-gal cause the Vmax of the immobilized enzyme to decrease. The
onto the activated agar disks caused its pH optimum to shift increase in the K and the decrease in the V
for the immo-
m
max
towards the acidic side (3.6–4.6). The shifting of pH optima to bilized b-gal were reported before for the Kluyveromyces lactis
37
more acidic values after immobilization was previously b-gal immobilized on modified carbon nanotubes and the
reported for other enzymes, such as, the silica immobilized Aspergillus oryzae b-gal immobilized onto cellulose acetate-
3
8
39
42
invertase and the gelatin immobilized tannase. The change polymethylmethacrylate membrane.
in the pH optima of immobilized enzymes could be attributed
to the ionic changes that occurred around the enzyme’s active
40
site as a result of the immobilization process.
Effect of Temperature. Figure 7B revealed that the free Asper-
gillus oryzae b-gal offered its highest activity at 458C. A compa-
4
1
rable result was reported by Tanaka et al. who stated that
the Aspergillus oryzae b-gal exhibited a temperature optimum
of 468C. The temperature optimum for the agar immobilized
b-gal was broadened from 45 to 558C (Figure 7B). The
immobilization of b-gal onto the cellulose acetate-
polymethylmethacrylate membrane also caused its temperature
42
optimum to broaden from 50 to 608C. The shift of an
enzyme’s optimum temperature towards higher values after its
FIGURE 8 The Hanes–Woolf plot used to determine the kinetic
immobilization could be regarded to the formation of a molec- parameters for the free and the immobilized b-gals.
Biopolymers

Novel Grafted Agar Disks for the Covalent Immobilization of b-D-galactosidase
683
mally activated agar disks managed to immobilize a whole of
126.26 U b-gal/g gel, and the immobilized b-gal retained
92.99% of its initial activity after being used for 15 consecutive
times. Hence, this immobilized b-gal could be regarded as a
good candidate for application in industry. These results also
indicate the superiority of these novel grafted agar disks as
enzyme carriers and would encourage using them to covalently
immobilize other enzymes.
REFERENCES
1
. Maksimainen, M. M.; Lampio, A.; Mertanen, M.; Turunen, O.;
Rouvinen, J. Int J Biol Macromol 2013, 60, 109–115.
FIGURE 9 Reusability of immobilized b-gal (means 6 S.E.).
2
. Deressa, T.; Stoecklinger, A.; Wallner, M.; Himly, M.; Kofler, S.;
Hainz, K.; Brandstetter, H.; Thalhamer, J.; Hammer, P. PLoS
One 2014, 9, e102280, doi: 10.1371/journal.pone.0102280.
Reusability
3. Dwevedi, A.; Kayastha, A. M. Bioresour Technol 2009, 100,
2667–2675.
The reusability of b-gal covalently immobilized onto the acti-
vated agar disks was evaluated (Figure 9). The data revealed an
initial increase in the enzyme’s relative activity upon reusing it.
This initial increase persisted up to the sixth reusability cycle
4
5
6
. Iliev, I.; Vasileva, T. J Biosci Bioeng 2013, 1, 149–153.
. Regil, R.; Sandoval, G. Biomolecules 2013, 3, 812–847.
. Panesar, P. S.; Panesar, R.; Singh, R. S.; Kennedy, J. F.; Kumar, H.
J Chem Technol Biotechnol 2006, 81, 530–543.
(103.06%). The initial increase in the relative activity of immo-
7
. Bickerstaff, G. F. Genet Eng Biotechnol 1995, 15, 13–30.
bilized b-gal was previously reported for both the covalently
8. Elnashar, M. M. In Biopolymers; Elnashar, M. M., Ed.; Sciyo:
Croatia, 2010; Chapter 1.
34
44
immobilized and the entrapped b-gal. It was hypothesized
that the reason behind such an initial increase in the relative
activity of covalently immobilized enzymes was the enzymes’
need for a relaxation time in contact with their substrate in
9
. Delattre, C.; Fenoradosoa, T. A.; Michaud, P. Braz Arch Biol
Technol 2011, 54, 1075–1092.
1
1
0. Tanriseven, A.; Do ꢀg an, S. Process Biochem 2001, 36, 1081–1083.
1. Ichikawa, S.; Takano, K.; Kuroiwa, T.; Hiruta, O.; Sato, S.;
Mukataka, S. J Biosci Bioeng 2002, 93, 201–206.
19
order to reach their maximum efficiency.
The results also showed (Figure 7) that the immobilized b- 12. Kuroiwa, T.; Shoda, H.; Ichikawa, S.; Sato, S.; Mukataka, S.
Process Biochem 2005, 40, 2637–2642.
gal retained a whole of 92.99% of its initial activity after 15
1
1
3. Li, T.; Li, S.; Wang, N.; Tain, L. Food Chem 2008, 109, 703–708.
4. Elnashar, M. M.; Millner, P.; Johnson, A.; Gibson, T. Biotechnol
Lett 2005, 27, 737–739.
cycles. That is to say, the immobilized b-gal lost only 7.01% of
its original activity after 3.75 working hours. This result was
31
close to the one reported by Kishore et al. who stated that b- 15. Haider, T.; Husain, Q. Int J Biol Macromol 2007, 41, 72–80.
gal covalently immobilized onto functionalized graphene nano- 16. L oꢁ pez-Gallego, F.; Betancor, L.; Mateo, C.; Hidalgo, A.; Alonso-
Morales, N.; Dellamora-Ortiz, G.; Guis ꢁa n, J. M.; Fern ꢁa ndez-
sheets retained more than 92% of its enzymatic activity after 10
Lafuente, R. J Biotechnol 2005, 119, 70–75.
cycles of repeated use. On the other hand, the lactose pretreated
1
1
7. Johnson, W.; Makame, Y. M. M. Tanz J Sci 2012, 38, 54–64.
8. Mohy Eldin, M. S.; Elaassar, M. R.; Elzatahry, A. A.; Al-Sabah,
M. M.; Hassan, E. A. J Appl Polym Sci 2012, 125, 1724–1735.
b-gal covalently immobilized onto silica gel retained only
45
63.9% of its original activity being retained after 10 reuses.
Noteworthy, the decline in the enzyme activity by the fifteenth 19. Elnashar, M. M. M.; Yassin, M. A.; Kahil, T. J Appl Polym Sci
008, 109, 4105–4111.
2
run could be attributed to the distortion in the active site of the
46
20. Danial, E. N.; Elnashar, M. M. M.; Awad, G. E. Ind Eng Chem
Res 2010, 49, 3120–3125.
enzyme due to its frequent encountering with the substrate.
2
1. Elnashar, M. M. M.; Wahba, M. I.; Amin, M. A.; Eldiwany, A. I.
J Appl Polym Sci 2014, 131, doi: 10.1002/app.40295.
2. Michelin, M. A.; Crott, L. S.; Assis-Pandochi, A. I.; Coimbra, T.
M.; Teixeira, J. E.; Barbosa, J. E. Int J Clin Exp Pathol 2002, 83,
105–110.
CONCLUSION
2
It could be concluded from the results of this study that the
agar disks would be optimally activated through soaking them
in a 3% (w/v) PEI solution (750,000 Da) of pH 9.5 for 2.5 h,
followed by immersing them in a 3% (v/v) GA solution. As
regards to the GA soaking time, a thirty minutes period would
be enough, as further increasing the GA soaking time would
not significantly increase the immobilization yield. These opti-
2
2
2
3. Nagaya, J.; Homma, M.; Tanioka, A.; Minakata, A. Biophys
Chem 1996, 60, 45–51.
4. Carey, F. A. Organic Chemistry; McGraw-Hill Companies: New
York, 2000; Chapter 17.
5. Cerqueira, M. R. F.; Grasseschi, D.; Matos, R. C.; Angnes, L.
Talanta 2014, 126, 20–26.
Biopolymers

684
Wahba and Hassan
2
2
2
6. Gutierrez, E.; Wu, S. R.; Racherla, U. 1999, U.S. Patent No. 36. Pan, C.; Hu, B.; Li, W.; Sun, Y.; Ye, H.; Zeng, X. J Mol Catal B:
,904,735.
Enzym2009, 61, 208–215.
7. Freile-Pelegr ꢁı n, Y.; Robledo, D.; Armis ꢁe n, R.; Garc ꢁı a-Reina, G. 37. Ansari, S. A.; Satar, R.; Chibber, S.; Khan, M. J. J Mol Catal B:
J Appl Phycol 1996, 8, 239–246.
Enzym2013, 97, 258–263.
8. Kurunczi, S.; Hainard, A.; Juhasz, K.; Patko, D.; Orgovan, N.; 38. Bergamasco, R.; Bassetti, F. J.; Moraes, F. F.; Zanin, G. M. Braz J
5
Chem Eng 2000, 17, 873–880.
9. EL-Tanash, A. B.; Sherief, A. A.; Nour, A. Braz J Chem Eng
Turck, N.; Sanchez, J. C.; Horvath, R. Sens Actuators 2013, B
81, 71–76.
3
4
4
4
4
4
4
4
1
2
011, 28, 381–391.
2
3
3
3
9. Elnashar, M. M. M.; Ali, A. O.; Abdallah, M. S.; Hamed, R. R.
J Colloid Sci Biotechnol 2012, 1, 1–7.
0. Krajewska, B.; Leszko, M.; Zaborska, W. J Chem Technol
Biotechnol 1990, 48, 337–350.
1. Tanaka, Y.; Kagamiishi, A.; Kiuchi, A.; Horiuchi, T. J Biochem
0. Elnashar, M. M. M.; Hassan, M. E.; Awad, G. E. A. J Colloid Sci
Biotechnol 2013, 2, 1–7.
1
975, 77, 241–247.
1. Kishore, D.; Talat, M.; Srivastava, O. N.; Kayastha, A. M. PLoS
One 2012, 7, 1–13.
2. Ansari, S. A.; Satar, R.; Zaidi, S. K.; Ahmad, A. Int Scholarly Res
Not 2014, 2014, doi: 10.1155/2014/163987.
3. Vera, C.; Guerrero, C.; Illanes, A. Carbohydr Res 2011, 346,
2. Bahman, M. S.; Mohammad, A. S.; Akbarzadeh, A.; Mona, S.;
Gigloo, S. H.; Ali, F.; Dariush, N. Br Biotechnol J 2013, 3, 367–
7
45–752.
376.
4. Shen, Q.; Yang, R.; Hua, X.; Ye, F.; Zhang, W.; Zhao, W. Process
Biochem 2011, 46, 1565–1571.
5. Song, Y. S.; Lee, J. H.; Kang, S. W.; Kim, S. W. Food Chem 2010,
3
3
3
3. Ren, L.; He, J.; Zhang, S.; Evans, D. G.; Duan, X. J Mol Catal B:
Enzym 2002, 18, 3–11.
4. Elnashar, M. M. M.; Yassin, M. A. Appl Biochem Biotechnol
123, 1–5.
6. Kishore, D.; Kayastha, A. M. Food Chem 2012, 134, 1650–1657.
2009, 159, 426–437.
5. Neri, D. F. M.; Balc ~a o, V. M.; Dourado, F. O. Q.; Oliveira, J. M.
B.; Carvalho, L. B., Jr.; Teixeira, J. A. React Funct Polym 2009,
69, 246–251.
Reviewing Editor: C. Allen Bush
Biopolymers