Immobilization of α-galactosidase inside hybrid silica nanocomposites containing polysaccharides

Article abstract of DOI:10.1134/S1070427206030260

α-Galactosidase was encapsulated into sol-gel derived hybrid polysaccharide-silica nanocomposites. The main properties of the immobilized enzyme were studied. Pleiades Publishing, Inc., 2006.

Full text of DOI:10.1134/S1070427206030260

ISSN 1070-4272. Russian Journal of Applied Chemistry, 2006, Vol. 79, No. 5, pp. 827 832. Pleiades Publishing, Inc., 2006.
Original Russian Text I.Yu. Bakunina, O.I. Nedashkovskaya, T.N. Zvyagintseva, Yu.A. Shchipunov, 2006, published in Zhurnal Prikladnoi Khimii,
2006, Vol. 79, No. 5, pp. 839 844.
MACROMOLECULAR CHEMISTRY
AND POLYMERIC MATERIALS
Immobilization of -Galactosidase inside Hybrid Silica
Nanocomposites Containing Polysaccharides
I. Yu. Bakunina, O. I. Nedashkovskaya, T. N. Zvyagintseva, and Yu. A. Shchipunov
Pacific Ocean Institute of Bioorganic Chemistry, Vladivostok, Russia
Institute of Chemistry, Far-East Division, Russian Academy of Sciences, Vladivostok, Russia
Received July 12, 2005
Abstract
-Galactosidase was encapsulated into sol gel derived hybrid polysaccharide silica nanocom-
posites. The main properties of the immobilized enzyme were studied.
DOI: 10.1134/S1070427206030260
Immobilization has long been successfully used for
storage and prolongation of action of enzymes, as well
as for development of artificial biocatalysts [1, 2].
Encapsulation into a matrix or immobilization on a
support allows repeated use of enzymes and facilitates
isolation of products of enzymatic reaction, which is
essential for development of biotechnological proc-
esses. An important advantage of immobilization is
the stabilization effect, i.e., significant prolongation
of the lifetime and enhancement of the stability of en-
zymes. Therefore, new efficient supports and matrices
for encapsulation of enzymes are constantly sought
for.
ing can adversely affect the enzyme through disturb-
ing the conformation of the protein macromolecule.
In some cases this is the main reason for partial or
complete denaturation of not very stable enzymes
[8, 9]. In the case of sol gel technology, immobiliza-
tion involves no cross-linking agents. The protein
macromolecules are encapsulated into the inorganic
matrix formed in situ by polycondensation reactions.
The macromolecules undergo minimal structural
changes, and the enzymes can preserve their activity
[1 3]. -Galactosidase from Pseudoalteromonas sp.
KMM 701 sea bacterium can eliminate -1,3-bound
galactose residues from the nonreducing end of gluco-
conjugates, blood group substances, and antigene de-
terminants for erythrocytes in B(III) blood group via
suppressing their serologic activity [10]. -Galactosi-
dase is a thermally labile enzyme. In concentrated
solutions at 20 C -galactosidase remained active for
1 week, and at 30 C lost 50% activity within 10 min
[10]. Dilution of concentrated solutions of the enzyme
accelerated its inactivation. A low thermal stability
and short lifetime of -galactosidase in solution
hinder its biotechnological application.
-Galactosidase ( -D-galactoside galactohydrolase,
EC 3.2.1.22) is a promising biochemical tool for
studying the antigene structure of various biological
objects, in particular, blood cells. Immobilization of
-galactosidase was repeatedly attempted. For exam-
ple, enzyme from coffee beans was covalently bound
to dextran activated with cyanogen bromide [3].
-Galactosidase from Pycnoporus cinnabarinus
was immobilized on chitin dispersions with glutaric
dialdehyde [4]. Enzyme from Aspergillus oryzae was
immobilized on Eupergit C containing epoxy groups,
which provided covalent bonding of the protein by
amino, SH, and carboxy groups [5]. -Galactosidase
from the same source was placed into calcium alginate
gel, in which it was additionally immobilized with
glutaric dialdehyde [6]. Also, this enzyme was im-
mobilized with polyacrylamide gel [7].
Our preliminary studies showed that encapsulation
of -galactosidase into polysaccharide silica matrices
obtained by the method we suggested previously [11,
12] significantly prolonged the lifetime of the enzyme.
In this study we examined in more detail the condi-
tions for -galactosidase immobilization inside new
hybrid nanocomposites and the properties of immobi-
lized -galactosidase in order to select candidate nano-
composites for development of biocatalysts suitable
for biotechnological and biosensing applications.
It should be noted that, in the cited studies, the en-
zyme was encapsulated via cross-linking. Immobiliza-
tion inside a matrix or on a support by covalent bond-
827

828
BAKUNINA et al.
from additions of the precursor and polysaccharide
EXPERIMENTAL
used for immobilization.
Tetrakis(2-hydroxyethyl) orthosilicate (THEOS)
was synthesized from tetraethoxysilane (ABCR, Ger-
many) as described in [13]. Polysaccharides xanthan
and galactomannan (locust bean gum, from here on,
LBG) (Fluka) was used without additional purifica-
tion. Laminaran ( -1,3-glucan) was isolated from
Laminaria cichorioides brown alga [14], and -galac-
tosidase, from biomass of Pseudoalteromonas sp.
KMM 701 sea bacterium, cultivated at the Laboratory
of Microbiology, Pacific Ocean Institute of Bioorgan-
ic Chemistry, Far-East Division, Russian Academy of
Sciences, by the procedure from [10].
-Galactosidase from Pseudoalteromonas sp.
KMM 701 sea bacterium was immobilized inside
hybrid polysaccharide silica nanocomposites syn-
thesized using a new precursor, THEOS. It is distin-
guished by complete solubility in water and compati-
bility with biopolymers. Polysaccharides were used
because they catalyze sol gel processes [15, 16]. In
their presence gelation proceeds at any pH, as well
as at low temperatures, which is essential for ther-
molabile enzymes. In particular, to minimize denatura-
tion of -galactosidase exhibiting a low thermal
stability during encapsulation into silica matrix, im-
mobilization was carried out at a temperature close to
0 C and pH 7.2. Commonly used precursors, such as
tetramethoxy- and tetraethoxysilanes, do not form gels
under such conditions [8, 9, 17, 18].
-Galoactosidase was encapsulated into hybrid
polysaccharide silica nanocomposites by the proce-
dure described in our previous paper [12]. First, 1.9 ml
of solution containing 10 or 30 wt % precursor, dif-
ferent concentrations (0.25, 0.35, 0.75, and 1.5 wt %)
of one of the three polysaccharides, and 0.1 M sodium
phosphate buffer solution (to adjust pH) was prepared.
It was kept for some time, determined in previous ex-
periments and required for its conversion into a pregel
state by partial polymerization of silicic acids formed
from hydrolysis of THEOS. Then, 0.1 ml of the en-
To elucidate how the structure of the polysacchar-
ide and its charge affect the catalytic properties of
-galactosidase, we tested negatively charged xanthan
and neutral LBG and laminaran. Previously we
studied the possibility of immobilization of endo-1
3- -D-glucanase L from Spisula sacchalinensis and
IV
-galactosidase in hybrid polysaccharide silica nano-
composites [12]. The immobilized proteins remained
functionally active for 165 days. The activity of the
proteins varied with the formulation of the nanocom-
posite and structure of the polysaccharide, but these
aspects were not studied in detail.
1
zyme solution with an activity of 10 units ml was
added. All the solutions were precooled to 3 5 C in a
refrigerator. After mixing and thorough stirring, they
once again were placed into the refrigerator in order
to minimize denaturation of the enzyme during im-
mobilization.
Table 1 lists the activities of -galactosidase im-
mobilized inside hybrid polysaccharide silica nano-
composites with various formulations, synthesized in
this study. It was tested for 16 months after matrix
immobilization of the enzyme. It is seen that the
enzyme remained active by the end of the test in
selected cases only. To elucidate how the catalytic
properties of -galactosidase are influenced by the
structure and concentration of the polysaccharide, as
well as by the silica concentration in the matrix, we
will consider the activities determined just after gel
formation.
The activity of immobilized -galactosidase was
determined with p-nitrophenyl- -D-galactopyranoside
(Sigma) as substrate. The reaction mixture was pre-
3
pared by mixing 0.35 ml of a 3.3 10 M solution of
1
substrate in the buffer solution (10 M sodium phos-
phate, pH 7.2) and 0.05 ml of solution of free -galac-
tosidase, or 0.05 g of the gel with the enzyme im-
mobilized, and incubated at 20 C. The reaction
was terminated by adding 0.6 ml of an aqueous
solution of 1 M sodium carbonate, whereupon the gel
was separated by centrifugation. The amount of
p-nitrophenol yielded by the enzymatic reaction was
Table 1 shows that the highest activity was ex-
hibited by sample no. 5 prepared by mixing 0.25 wt %
solution of xanthan and 10 wt% solution of THEOS.
It was nearly two times that of the free enzyme. Also,
it exceeded the activity of galactosidase in a matrix
with identical formulation that we examined previous-
ly [12], but in that study the content of the enzyme
was by an order of magnitude lower.
determined spectrophotometrically at 400 nm (
=
1
1
18300 l mol cm ). The enzyme activity unit U was
taken equal to the amount of the enzyme in 1 ml of
solution (free enzyme) or 1 g of gel (immobilized
6
enzyme) with which 10 mol of p-nitrophenol was
formed per minute. The control was a solution of the
enzyme (free enzyme) prepared by 20-fold dilution of
the initial solution of -galactosidase with 0.1 M
sodium phosphate buffer solution (pH 7.2). It was free
The enzymatic activity of sample nos. 2 and 3,
containing 0.25 and 0.75 wt % LBG, respectively,
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 79 No. 5 2006

IMMOBILIZATION OF -GALACTOSIDASE INSIDE HYBRID SILICA
829
Table 1. Activity of -galactosidase in relation to the immobilization conditions and storage time
1
U
103, g , at indicated storage time, days
Sample
no.
SiO2,
wt %
Polysaccharide,
wt %
0
6
19
30
43
165
417
1
2
3
4
5
6
7
0
10
10
10
10
30
10
Free enzyme*
LBG: 0.25
0.75
33.6
34.6
32.8
9.2
63.9
14.1
20.0
21.8
50.9
56.7
10.9
N.d.
N.d.
N.d.
16.5
76.8
81.8
12.0
N.d.
N.d.
N.d.
N.d.**
N.d.
N.d.
N.d.
21.7
0.1
18.7
54.1
63.5
8.4
N.d.
N.d.
N.d.
0
0
N.d.
N.d.
N.d.
N.d.
N.d.
N.d.
28.9
N.d.
9.6
N.d.
N.d.
N.d.
1.5
Xanthan: 0.25
0.25
Laminaran, 0.35
0
1
* U, ml
** Not determined.
just after immobilization was comparable to that of
the enzyme in solution. With increasing amount of
LBG in the silica matrix to 1.5 wt % (sample no. 4),
the activity decreased almost threefold. This suggests
that the activity depends on the concentration of the
polysaccharide in the hybrid nanocomposite. As
shown in [15, 16], this can be due to the fact that
the network structure of the nanocomponent becomes
thicker, which can hinder diffusion of the substrate
and the enzymatic reaction products.
containing (no. 7) samples showed that the activity of
the immobilized enzyme decreased with time. Also,
the lifetime of -galactosidase in matrices containing
these polysaccharides was shorter than that in the
previously discussed samples with LBG.
The retention of the immobilized enzymes in(on)
the support is of practical importance. To estimate the
strength of immobilization of -galactosidase in the
polysaccharide silica matrices, we tested sample
nos. 2 4 in which the enzyme was best preserved. To
1
0.1 g of gels, 0.4 ml of 10 M buffer sodium phos-
Most probably, a similar factor also operates when
the content of silica in the nanocomposite is increased.
Comparison of the data for sample nos. 4 and 5
(Table 1), containing identical amounts of xanthan
and different amounts of THEOS (10 and 30 wt %,
respectively), shows that the activities of the enzyme
immobilized differed for them by a factor of nearly 5.
Among the polysaccharides that we studied only lami-
naran in the gel did not exert a stabilizing effect on
the immobilized -galactosidase. This follows from
the fact that the enzymatic activity of control sample
no. 1 was 1.5 times that of sample no. 7 (see Table 1).
phate solution (pH 7.2) was added. After 2-day keep-
ing in a refrigerator at 4 C with intermittent stirring,
the samples were centrifuged at 10000 rpm. The ac-
tivity was determined in both supernatants and gels by
the above-described procedure.
The solutions in which the biocatalysts were kept
and separated by centrifugation virtually lack en-
zymatic activity (Table. 2). This suggests a fairly
strong immobilization of the enzyme macromolecules
in the gel matrix, when only insignificant number, if
any, of them can liberate. However, the activity in gel
nos. 2 and 3 proper proved to be significantly lower
than the initial activity (Table 2). Probably, centri-
fugation was responsible for compaction of gels,
which do not exhibit high mechanical strength, and
A 16-month testing of the samples showed that, by
the end of this time, -galactosidase lost its enzymatic
activity in most cases (Table 1). The situation with the
free enzyme was similar. The activity was preserved
by sample nos. 2 4 only: By the end of the test period
it was virtually identical to that in the beginning of
the experiment. However, the activity of the biocata-
lysts varied during the tests. For sample nos. 2 and 3
it increased more than twofold within the first 19 days
and then tended to decrease. The activity of sample
no. 4 significantly varied in the beginning of the ex-
periment, whereupon it remained virtually unchanged
throughout the test period.
Table 2. Retention of -galactosidase in the silica nano-
composite (10 wt %) containing different amounts of LBG
Sam-
ple
no.
U 103
LBG, U 103,
1
wt %
g
in 1 ml of supernatant in 1 g of gel
2
3
4
0.25
0.75
1.50
34.6
32.8
9.2
0.076
0.018
0.009
3.7
12.5
12.6
Tests of xanthan- (nos. 5 and 6) and laminaran-
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 79 No. 5 2006

830
U/U0, %
BAKUNINA et al.
the ratio, %, of the activity U at various pH values to
that of the enzyme at optimal pH values, U . Figure 1
0
presents the pH dependences of the residual activity
of the enzyme for free (curve 1) and immobilized
-galactosidase in gels with LBG (sample no. 2) and
xanthan (sample no. 3) (curves 2 and 3, respectively).
It is seen that encapsulation into a silica matrix caused
the pH range to broaden to a certain extent, but left
the pH corresponding to the optimal activity of the
enzyme virtually unaffected.
As shown by analysis of published data, immobili-
zation by cross-linking noticeably shifted the pH cor-
responding to the highest activity of -galactosidases.
For example, for enzyme isolated from A. oryzae, im-
mobilized inside an alginate matrix with glutaric dial-
dehyde, this pH shifted to the acid region by 0.3
pH unit [6], and for -galactosidases from Gibberella
fujikuroi in polyacrylamide gel and from green coffee
beans in dextran, by 0.4 pH unit [3, 7]. In the case
of sepharose-immobilized enzyme isolated from fig,
the pH corresponding to the best activity shifted by
1 pH unit [19]. These published data suggest that
the properties of -galactosidase changed more sub-
stantially in the case of immobilization by cross-link-
ing than in the case of the sol gel technology that
we applied here.
Fig. 1. pH dependence of the residual activity U of (1) free
and (2, 3) immobilized -galactosidase in gel nos. 4 (2) and
(3) 5. 0.1 M sodium phosphate buffer solution.
U/U0, %
Another problem addressed by this study is the
thermal stability of -galactosidase. Gel (0.05 g) with
the enzyme immobilized was placed into a thermostat,
kept at the required temperature for 10 or 30 min, and
cooled to room temperature, whereupon the residual
activity was estimated as described above. The control
sample was subjected to analogous manipulations.
T, C
Figure 2 shows the temperature dependences of the
residual activity for free (curves 1 and 2) and im-
mobilized -galactosidase in gels with LBG (sample
no. 3) and xanthan (sample no. 5) (curves 3 and 4,
respectively). The keeping time was 10 min (curves 2
and 4) and 30 min (curves 1 and 3). Analysis of these
dependences showed that, after 10-min keeping, free
-galactosidase lost its activity at 37 C (curve 2), and
after 30-min keeping, at 32 C (curve 1). Under ident-
ical conditions, the immobilized enzyme preserved
from 50 to 70% of its activity (curves 3 and 4). Major
shift of curves 3 and 4 to higher temperatures relative
to curves 1 and 2 suggests that encapsulation into
the polysaccharide silica gel causes the thermal stabil-
ity of -galactosidase to increase. This can be due to
a stabilizing effect of the inorganic matrix, hindering
temperature-induced conformational transitions of
the protein molecule. This effect was observed, for
example, in the case of proteins immobilized inside
Fig. 2. Temperature dependence of the residual activity U
of (1, 2) free and (3, 4) immobilized -galactosidase in
sample nos. (3) 4 and (4) 5. 0.1 M sodium phosphate buffer
solution, pH 7.3. Time of keeping at the required tempera-
ture, min: (1, 3) 30 and (2, 4) 10.
this either hindered the accessibility of the enzyme for
the substrate, or inactivated the enzyme immobilized.
When the LBG concentration in the gel (sample no. 4)
was increased, the mechanical exposure affected the
enzyme activity to a significantly lesser extent.
We determined the pH corresponding to the op-
timal activity of immobilized -galactosidase. To
0.05 ml of free enzyme preparation, or 0.05 g of gel,
0.25 ml of 0.1 M sodium phosphate buffer solution
2
with pH under study and 0.1 ml of 10 M aqueous
solution of the substrate were added. The activity of
the enzyme was determined by the above-described
procedure. The residual activity was calculated as
RUSSIAN JOURNAL OF APPLIED CHEMISTRY Vol. 79 No. 5 2006

IMMOBILIZATION OF -GALACTOSIDASE INSIDE HYBRID SILICA
831
tially affect the catalytic properties of the enzyme.
1
v
1 [ mol min ]
A minor and a twofold increase in K , respectively,
m
3
for the enzyme in gel nos. 2 (0.42 10 M) and 5
3
(0.72 10 M) compared to that for free -galactosi-
3
dase (0.36 10 ) can be due to hindered diffusion
of the substrate and reaction products in the silica
matrix pores. A similar increase in K was observed,
m
e.g., for - and -galactosidases immobilized in poly-
acrylamide and alginate gels using cross-linking
agents [5, 6, 15].
CONCLUSIONS
(1) -Galactosidase was immobilized inside sol
gel derived hybrid polysaccharide silica nanocom-
posites with different formulations.
S
1 [mM]
1
Fig. 3. Reciprocal rate of accumulation of p-nitrophenol v
vs. the reciprocal concentration of p-nitrophenyl galactoside
for -galactosidase (1) in solution and (2, 3) in im-
mobilized state in sample nos. (2) 2 and (3) 5.
1
S
(2) The catalytic activity of -galactosidase varies
with the formulation of the silica polysaccharide
matrix. The enzymatic activity tends to decrease with
increasing content of the inorganic component. The
activity and stability of the enzyme are also influenced
by the structure of the polysaccharide and its concen-
tration in the matrix.
silica hydrogel [20 23]. Immobilization typically
increases the thermal stability of enzymes. In the
majority of studies with -galactosidase it increased
by several degrees [3, 5 7, 19]. At the same time,
Mitsutomi [4] reported that the thermal stability of
-galactosidase from P. cinnabarinus, immobilized
in chitin, did not change relative to free -galactosi-
dase [4].
(3) -Galactosidase immobilized inside a tetrakis-
(2-hydroxyethyl) orthosilicate (10 wt %) matrix con-
taining galactomannan (0.25 and 0.75%) preserved the
catalytic activity for a long time; its thermal stability
increased by 10 C. The immobilized -galactosidase
exhibited the maximal activity at the same pH values
To compare the catalytic properties of free and
gel-immobilized -galactosidase, we determined the
corresponding Michaelis constants K . We tested gels
m
as the free enzyme; the Michaelis constant K did not
m
containing 0.75 wt % LBG (sample no. 2) and
0.25 wt % xanthan (sample no. 5) at 20 C in 0.1 M
sodium phosphate buffer solution, pH 7.7, at a time
when the free and immobilized gels still exhibited
the maximal activity. The concentration of the sub-
strate, p-nitrophenyl galactopyranoside, was varied
from 0.12 10 to 2.49 10 M. To 0.05 ml of
dilute enzyme, or to 0.05 g of gel, 0.350 ml of the
buffer solution of the substrate was added. The reac-
tion was terminated by introducing 0.6 ml of aqueous
solution of 1 M sodium carbonate. The samples with
the gels immobilized were centrifuged at 10000 rpm,
whereupon the concentration of p-nitrophenol in the
supernatant was determined by the above-described
substantially change.
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