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Polymer International 44 (1997) 530È536
Comparison of b-Galactosidase
Immobilization by Entrapment in and
Adsorption on
Poly(2-hydroxyethylmethacrylate)
Membranes
TuŽ rker Baran,1,2 M. Yakup Arfca,1 Adil Denizli3 & Vasff Hasfrcf2,*
1 Department of Biology, K•r•kkale University, 71450 K•r•kkale, Turkey
2 Department of Biological Sciences, Biotechnology Research Unit, Middle East Technical University, 06531 Ankara, Turkey
3 Department of Chemistry, Biochemistry Division, Hacettepe University, 06532 Beytepe, Ankara, Turkey
(Received 27 May 1997 ; accepted 16 July 1997)
Abstract : b-Galactosidase was immobilized in/on poly(2-hydroxyethyl
methacrylate) (pHEMA) membranes by two di†erent methods : adsorption on
Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum b-galactosidase
adsorption on pHEMA-CB membranes was obtained as 95É6 kg cm~2 in
2É0 mg cm~3 enzyme solution. The adsorption phenomena appeared to follow a
typical Langmuir isotherm. In the entrapment, an increase in b-galactosidase
loading resulted in a consistent increase in membrane activity from 3É3 ] 10~2
to 17É8 ] 10~2 U cm~2 pHEMA membranes. The K values for both immobilized b-galactosidase (adsorbed 0É32 mM and entrappedm 0É81 mM) were higher than
that of the free enzyme (0É26 mM). The optimum reaction temperature of the
adsorbed enzyme was 5¡C higher than that of both the free and the entrapped
enzyme. The optimum reaction pH was 7É5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the
entrapped enzymes was 80% and 95%, respectively. The storage stability of the
enzyme was found to increase upon immobilization.
Polym. Int. 44, 530È536 (1997)
No. of Figures : 8. No. of Tables : 3. No. of References : 24
Key words : b-galactosidase, entrapment, adsorption, affinity membrane, enzyme
immobilization, pHEMA
meability to substrates and products, high chemical and
biological stability, high mechanical strength and the
presence of chemical groups which permit derivatization.5h7
Poly(2-hydroxyethyl methacrylate) (pHEMA) may be
prepared in di†erent shapes and forms e.g. membrane,
microcapsules, microspheres ; it possesses a hydrophilic
pendant group (wOH) and can form hydrogels. It is
non-toxic, biocompatible, very stable against microbial
INTRODUCTION
Membranes play an important role in the immobilization of enzymes, especially in the construction of
enzyme reactors and enzyme electrodes.1h4 The desirable properties of a membrane designed for enzyme
immobilization are high water content and high per* To whom all correspondence should be addressed.
530
( 1997 SCI. Polymer International 0959-8103/97/$17.50
Printed in Great Britain
b-Galactosidase immobilization by pHEMA membranes
contamination, and resistant to attack by a large
number of chemicals.8,9 It has previously been used in
immobilization of enzymes and cells, after polymerization by various techniques (bulk, suspension and
solution) using photochemical, thermal and cirradiation as initiators.10h12 The hydroxyethyl group
o†ers a site for derivatization. All these properties indicate that pHEMA is a suitable support for enzyme and
cell immobilization for various biotechnological and
biomedical applications.
b-Galactosidase Ðnds a great deal of use in the dairy
industry, especially in the production of lactose-free
milk products. Hydrolysis of lactose improves product
sweetness, makes milk consumption by people who
su†er from lactose intolerance possible, and increases
product quality and process efficiency in the dairy
industry. This hydrolysis reaction could also be applied
to the up-grading of cheese whey, a product of cheese
processing, the disposal of which constitutes a
problem.13,14
Many researchers have studied the enzymatic hydrolysis of lactose in order to develop an immobilized bgalactosidase product that could be used in the food
industry. Inorganic and organic materials such as glass,
ceramics, polyvinyl alcohol, collagen, gelatin and chitosan have all been tested as support materials for the
immobilization of b-galactosidase.15h20
This study was initiated to compare the immobilization capability of a very novel spacer, Cibacron Blue
F3GA, with the typical entrapment approach. Thus bgalactosidase was chosen more for the purpose of comparison because of the numerous immobilization trials
using it.
EXPERIMENTAL
Materials
b-Galactosidase (b-D-galactoside galactohydrolase ; EC
3.2.1.23 ; Grade IV from E. coli (300 units mg~1), was
purchased from Sigma Chem. Co. (St. Louis, USA) and
was used without further puriÐcation.
o-Nitrophenyl
b-D-galactopyroniside
(ONGP),
bovine serum albumin (BSA), FolinÈCiocalteu reagent,
and lactose were obtained from Sigma and were used as
received.
2-Hydroxyethyl methacrylate (HEMA) was obtained
from Sigma, distilled under reduced pressure in the
presence of hydroquinone and stored at 4¡C until use.
a-a@-Azobisisobutyronitrile (AIBN) was purchased from
Fluka AG (Buchs, Switzerland) and used as received.
Cibacron Blue F3GA (CB) was obtained from Polysciences (Warrington, USA). All other chemicals were of
reagent grade and purchased from Merck AG
(Darmstadt, Germany).
POLYMER INTERNATIONAL VOL. 44, NO. 4, 1997
531
Entrapment of b-galactosidase in pHEMA membrane
Entrapment of enzyme within the bulk of the pHEMA
membrane was carried out according to the procedure
described previously.12
A 5 ml mixture consisting of 2-hydroxyethyl methacrylate (2 ml monomer), AIBN (5 mg initiator), and
phosphate bu†er (3 ml, 0É1 M, pH 7É0) containing 0É125È
3É000 mg of b-galactosidase was stirred and poured into
a round glass mould (diameter 4É5 cm) and exposed to
UV irradiation (12 W lamp, P. W. Allen and Co.) for
30 min while a nitrogen atmosphere was maintained in
the mould. After completion of the polymerization, the
membrane was washed with phosphate bu†er (0É1 M, pH
7É0) and small disks were cut (diameter 1É0 cm) and
stored at 4¡C until use.
Preparation of Cibacron Blue F3GA derivatized
pHEMA membrane
pHEMA membrane was prepared as above, except that
it did not contain any enzyme and 0É1 M SnCl (3 ml)
4
was used instead of phosphate bu†er.
Cibacron Blue F3GA was covalently coupled to the
pHEMA membrane via a nucleophilic reaction between
the chloride of its triazine ring and the hydroxyl group
of the HEMA molecule under alkaline conditions. The
coupling procedure employed was previously
described.9 Cibacron Blue F3GA (100È300 mg) was dissolved in distilled water (10 ml), transferred to distilled
water (90 ml) in which pHEMA disks (diameter 1É0 cm,
thickness about 0É06 cm) were immersed. NaOH (4É0 g)
was added to the medium and the mixture was heated
for 4 h at 80¡C in a sealed reactor, while stirring. Under
these conditions HCl is eliminated, resulting in the
coupling of Cibacron Blue F3GA to the pHEMA disk
(Fig. 1). The dye solution was then cooled to room temperature, and disks were washed exhaustively, Ðrst with
distilled water and then with methanol, until all the
unbound dye was removed.
Immobilization of b-galactosidase on pHEMA
membrane with Cibacron Blue F3GA spacer
The e†ect of pH on the efficiency of b-galactosidase
binding on pHEMA-CB-membranes loaded with di†erent amounts of Cibacron F3GA (3É1, 6É3 and
10É7 mmol m~2) was studied (using an initial enzyme
concentration of 0É3 mg ml~1) at various pH values, in
acetate (5É0 ml, 50 mM, pH 4É0È5É0) and in phosphate
bu†ers (5É0 ml, 50 mM, pH 6É0È8É0). The assays were
carried out by incubating pHEMA-CB membranes (10
disks, 16 cm2) loaded with di†erent amounts of Cibacron F3GA in bu†er solutions for 2 h at 25¡C, while continuously stirring the medium.
In order to determine the e†ect of enzyme concentration in the medium on the adsorption capacity of
T . Baran et al.
532
In the determination of the activity of the free
enzyme, the reaction medium (1É5 ml) consisted of phosphate bu†er (0É1 ml, 0É15 M, pH 7É5) with 3 mM MgCl ,
2
ONPG (0É5 ml, 14 mM) and distilled water (1É4 ml). The
assay was started by the addition of the enzyme solution (0É1 ml, 6É25 kg ml~1) to the assay medium and the
absorbance of the medium was recorded at 405 nm.
The same assay medium was used for the determination of the activity of the immobilized enzyme. The
enzymatic reaction was started by the introduction of
Ðve disks into the assay medium and was carried out at
25¡C with shaking in a water bath for 10 min.
In order to determine enzyme activity, absorbances at
405 nm because of o-nitrophenol resulting from the
hydrolysis of the substrate ONPG were measured in a
Shimadzu (Model 1201, Japan) spectrophotometer. One
unit of b-galactosidase was deÐned as the amount of
enzyme which hydrolyses 1 kmol of artiÐcial substrate
ONPG per minute at 25¡C in phosphate bu†er (0É1 M,
pH 7É5).
The above activity assays were also carried out over
the pH range 4É0È9É5 and the temperature range 20È
65¡C to determine the pH and temperature proÐles for
the free and immobilized enzyme. The e†ect of substrate
concentration was tested in the range 0É17È4É67 mM
ONPG. The results for pH and temperature are presented in a normalized form with the highest value of
each set being assigned the value of 100% activity.
Storage stability
Fig. 1. Coupling of Cibacron Blue F3GA to the pHEMA
membrane.
Cibacron Blue F3GA loaded (10É7 mmol m~2) pHEMA
membrane, the concentration of b-galactosidase in the
medium (5É0 ml, 50 mM, pH 5É0) was varied between 0É3
and 2É0 mg ml. After binding of the enzyme, membranes
were washed several times with bu†er and stored in
fresh bu†er at 4¡C until use. The amounts of attached
Cibacron Blue F3GA on the pHEMA membranes were
determined by elemental analysis (CHNS-932, Leco,
USA).
Retention of the activity of free and immobilized bgalactosidase during storage in phosphate bu†er solution (50 mM, ph 7É5) at 4¡C was determined in a batch
operation mode under the experimental conditions
given above.
Repeated use of immobilized b-galactosidase
The retention of the immobilized enzyme activity was
tested as described in the “Enzyme activity assaysÏ
section. After each reaction run, the disk was washed
with distilled water and reintroduced into fresh medium
15 times successively.
Determination of immobilization efficiency
The amounts of protein in the enzyme solution and in
wash solutions were determined by the Lowry
method.21 A calibration curve based on bovine serum
albumin (BSA) (0É02È0É2 mg ml~1) was used to quantify
the enzyme content.
Enzyme activity assays
Activities of the free and immobilized b-galactosidase
were determined spectrophotometrically using the procedure reported by Craven et al.22
RESULTS AND DISCUSSION
Immobilization through entrapment
In this study, pHEMA was selected as the support
matrix because of its high mechanical strength, high stability and non-toxicity, and to take advantage of its
high water content to provide the enzymes with a
microenvironment similar to that in vivo.
The e†ect of loading on the activity of b-galactosidase
entrapped in pHEMA membrane was determined by
POLYMER INTERNATIONAL VOL. 44, NO. 4, 1997
b-Galactosidase immobilization by pHEMA membranes
533
Adsorption and retention of activity
Fig. 2. E†ect of enzyme loading on the entrapped enzyme
activity retention and enzyme-membrane activity.
varying the enzyme contents on the pHEMA membrane
(Table 1). As shown in Fig. 2 the highest retention of
enzyme activity (9É5%) was obtained with the lowest
enzyme content (1É1 kg cm~2). As the enzyme content
increased (from 1É1 to 23É5 kg cm~2), retention of activity decreased, dropping to a minimum of 2É5%.
A high enzyme load in the support generally leads to
a low retained activity. This is brought about by oversaturation of the pore space of the matrix with the
enzyme, as a result of which substrate di†usion is
restricted. However, even the low yields are comparable
(2É0% and 5É2% in alginate and K-carrageenan gels)
with those in the literature.19
With an increase in b-galactosidase content in the
membrane (1É1È23É5 kg cm~2), activity also increased
(3É3 ] 10~2
to
17É8 ] 10~2 U cm~2
pHEMA
membrane), but this was not at the same rate, because
of the loss in retained activity with increased load
(Fig. 2).
Cibacron Blue F3GA leakage studies from the dyeattached pHEMA-CB membranes showed that there
was no leakage in any of the adsorption and activity
assay media, which conÐrmed that the washing procedure was quite satisfactory for removal of physically
adsorbed Cibacron Blue F3GA molecules from the
pHEMA membrane.
The e†ects of the dye content of the membrane on the
extent of b-galactosidase adsorption is presented in
Table 2. As the concentration of the dye on the membrane was increased by about threefold (from 3É1 to
10É8 mmol m~2 pHEMA membrane), the amount of
enzyme adsorbed on the membrane was also increased
by about the same factor (from 4É7 to 13É5 kg enzyme
cm~2 pHEMA membrane). This showed that the
enzyme concentration was high enough to yield linear
adsorption.
The retention of activity was about 20-fold higher
than entrapment under similar levels of enzyme loading
(Table 1). This could only be explained by the di†usional restriction on the substrate and the product. Because
it is possible to load much higher amounts of bgalactosidase with this method, it is obviously a substantially better approach to immobilization.
The optimal pH value for the adsorption of bgalactosidase on a pHEMA-CB membrane with a dye
loading of 10É8 mmol m~2 was investigated in the pH
range 4É0È8É0. As observed in Fig. 3, the maximum
enzyme adsorption of 15É2 kg cm~2 was obtained at pH
8É0 with an enzyme concentration of 0É3 mg ml~1.
Enzymes do not have a net charge at their isoelectric
point, and therefore the maximum adsorption from
aqueous solution is usually observed at their isoelectric
point (pI). The isoelectric point of b-galactosidase (from
E. coli K12, four identical subunits, molecular weight of
enzyme 329 000) is 5É1.23 In the present study, an
TABLE 1. Properties of the entrapped and adsorbed b-galactosidase
Type of immobilization
Enzyme loading
(mg cmÉ2)
Entrapped enzyme
1·1
2·1
3·4
7·6
14·0
23·5
Adsorbed enzyme
13·5 À 1·2
33·0 À 1·1
60·0 À 1·9
91·1 À 1·7
95·6 À 2·1
Activity retention
(%)
9·5
7·4
6·2
4·7
3·0
2·5
56
45
38
32
29
POLYMER INTERNATIONAL VOL. 44, NO. 4, 1997
Activity
(U (mg enzyme)É1)
(U (cm2 membrane)É1)
30·0
19·6
16·5
12·5
7·9
6·7
3·3 Ã 10É2
5·5 Ã 10É2
9·2 Ã 10É2
11·5 Ã 10É2
13·3 Ã 10É2
17·8 Ã 10É2
149
119
100
85
77
3·457
6·581
8·334
11·710
14·373
T . Baran et al.
534
TABLE 2. The effect of dye content of b-galactosidase
adsorption and activity
Cibacron F3GA
input (mg (100 ml)É1)
Dye content of the
pHEMA membrane
(mmol mÉ2)
Enzyme loading
(mg cmÉ2)
100
200
300
3·1
6·3
10·8
4·7 À 0·8
8·5 À 0·7
13·5 À 1·2
adsorption peak was obtained at pH 5É0, which
dropped to a minimum at pH 6É0. After this pH, a consistent increase in adsorption was obtained with
increasing medium pH. These speciÐc interactions may
result from the cooperative e†ect of di†erent mechanisms, such as hydrophobic interactions and/or ionic
e†ects, caused by the several aromatic rings and sulphonic acid groups on the Cibacron Blue F3GA and
the amino acid side-chains of the enzyme molecules.9
An adsorption isotherm was constructed to characterize the interaction of the enzyme (b-galactosidase)
with the adsorbent (Cibacron Blue F3GA attached
pHEMA membrane). This provides a relationship
between the concentration of enzyme in the solution
and the amount of enzyme adsorbed on the solid phase,
when the two phases are at equilibrium. The results of
the adsorption tests are presented in Table 1, and Fig. 4.
An increase in b-galactosidase concentration (from 0É3
to 2É0 mg ml~1) led to an increase in adsorption efficiency but this levelled o† at an enzyme concentration
of 1É2 mg ml~1. A maximum enzyme adsorption of
95É6 kg cm~2 was obtained at 2É0 mg ml~1 bgalactosidase solution. This trend is exactly the same as
that obtained with the entrapped enzyme case. In this
case, however, the retention of the activity is substantially higher, possibly because of the enzyme being in a
much less restricted environment.
Kinetic constants
Kinetic parameters, the Michaelis constant K and
m
V
, for free and immobilized b-galactosidase were
max
determined using ONPG as substrate. The properties of
free and immobilized enzymes are presented in Table 3.
For the free enzyme the K was found to be 0É26 mM,
m
and the V was calculated to be 266 U (mg enzyme)~1.
max
Kinetic constants for the immobilized b-galactosidase
were also determined using a batch-wise test. K values
m
were found to be 0É32 mM for the adsorbed and 0É81 mM
for the entrapped enzyme. Thus the apparent K for the
m
entrapped enzyme was increased by about three-fold
with respect to that of the free enzyme. This indicates an
alteration in the affinity of the enzyme towards the substrate upon entrapment within the membrane. Adsorption, however, led to an insigniÐcant change in the
Fig. 4. E†ect of enzyme content on the adsorbed enzyme
activity retention and enzyme-membrane activity.
TABLE 3. Kinetic properties of the free and immobilized b-galactosidase
Form of enzyme
Fig. 3. E†ect of pH on the adsorption of enzyme on pHEMACB membrane.
Free
Entrapped
Adsorbed
K
m
(mM)
0·26
0·81
0·32
V
max
(U (mg enzyme)É1)
266
30
149
POLYMER INTERNATIONAL VOL. 44, NO. 4, 1997
b-Galactosidase immobilization by pHEMA membranes
affinity. This could be interpreted as entrapment
causing more physical deformation and restriction of
the enzyme than a simple adsorption. Similar signiÐcant
changes in the K
value, twofold in Nm
isopropylacrylamide18 and threefold in alginate,19 were
also reported earlier.15,17h19
The V
values of immobilized enzyme for entrapped
max
and for adsorbed preparations were calculated at 30 U
(mg entrapped enzyme)~1 and 149 U (mg adsorbed
enzyme)~1, respectively. Thus, it appears that V
max
values decreased about two- and ninefold for adsorbed
and entrapped enzyme, with respect to free enzyme. It
appears that immobilization with both methods
changed the V
of the enzyme but as in the case of K
max
m
a signiÐcantly better result was observed with adsorption.
Effect of temperature
The temperature dependence of the activities of the free
and immobilized b-galactosidase were studied in phosphate bu†er (0É1 M, pH 7É5) in the temperature range
25È65¡C (Fig. 5). The data revealed bell-shaped curves
with optimum activity at 50¡C for both the free and
entrapped enzyme, but with an optimum at 55¡C for the
adsorbed enzyme. The temperature proÐle of the
entrapped enzyme is slightly broader than that of free
enzyme. This was quite expected because in the literature a shift has been reported upon entrapment of
various enzymes in a polymeric matrix. The reason for
shift towards higher temperatures, with adsorbed
enzyme could be multipoint attachment, which consequently leads to an increase in the activation energy for
the enzyme to reorganize to the proper conformation
for binding to substrate.
Fig. 5. Temperature proÐles of free and immobilized bgalactosidase.
POLYMER INTERNATIONAL VOL. 44, NO. 4, 1997
535
Effect of pH
The optimal pH values for ONPG hydrolysis by bgalactosidase in free and immobilized form were investigated in the pH range 4É0È9É5 at 25¡C. The reactions
were carried out in acetate, phosphate and tris-acetate
bu†ers and the results are presented in Fig. 6. Optimum
hydrolysis was obtained at about pH 8É0 for native and
entrapped preparations. A plateau region is seen for
both native and adsorbed enzyme at pH values of 7É5È
8É2 and 6É8È7É5, respectively. Similar changes in pH
proÐle upon immobilization have been reported by
several researchers15,17,24 and are probably caused in
the case of adsorbed b-galactosidase, by interactions
between high concentrations of free amino groups in the
enzyme structure and various ionic groups on the
Cibacron Blue F3-GA attached pHEMA membrane.
The reason for change in pH proÐle upon entrapment,
is because of di†usional limitations of the entrapped
enzyme molecules in the substrate and product.
Stability in repeated use
The stability of immobilized enzyme systems is very
important economically, and an increased stability
could make the immobilized enzyme more advantageous than its free counterparts. The e†ect of repeated
use on the residual activity of the entrapped and
adsorbed b-galactosidase is presented in Fig. 7. The
activity of entrapped enzyme in the pHEMA membrane
is retained without signiÐcant loss after the batch reaction was repeated 15 times. At the end of the last use
the retained activity is still 95%. With the adsorbed
enzyme, the retained activity was about 80% after 15
successive uses. Both these results are signiÐcantly
better than those reported in the literature.15,20,24 The
entrapped enzyme is probably slightly more stable
because of the protection provided by the hydrogel
environment.
Fig. 6. pH proÐles of free and immobilized b-galactosidase.
T . Baran et al.
536
mining the activities once a week (Fig. 8). All the
samples showed a rapid decrease in activity in this condition. The free enzyme activity was decreased to one
half during the Ðrst week of storage, while 90% and
80% residual activity was observed for entrapped and
adsorbed enzymes, respectively. The free enzyme lost all
its activity at the end of 28 days. Both the entrapped
and the adsorbed preparations lost about 90% of their
activities after 56 days. This decrease in activity was
explained as a time-dependent natural loss in enzyme
activity, which was prevented to a signiÐcant degree
upon immobilization.
REFERENCES
Fig. 7. E†ect of repeated use on the stability of immobilized
b-galactosidase.
Storage stability
A shelf-life or storage stability test was carried out by
incubating the free and immobilized preparations in
phosphate bu†er (0É1 M, pH 7É5) at 4¡C and by deter-
Fig. 8. Storage stability of free and immobilized enzymes.
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POLYMER INTERNATIONAL VOL. 44, NO. 4, 1997
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