Mechanism of the dimerization of enzymes upon adsorption on silicate adsorbents using the example of lysozyme and β-galactosidase

Article abstract of DOI:10.1134/S0036024410110282

It is shown by the kinetic analysis of lysozyme and β-galactosidase adsorption on silochrome, silica gel, and mesoporous silica that the adsorption follows a two-stage scheme, including reversible pre-adsorption and the irreversible binding of dimmers. The corresponding rate constants of adsorption, desorption, and dimerization were calculated. It was found that β-galoctosidase adsorbed on silica gel and mesoporous silica retained 20% of its activity, while β-galoctosidase adsorbed on silochrome retained more than 30% of its activity.

Full text of DOI:10.1134/S0036024410110282

ISSN 0036ꢀ0244, Russian Journal of Physical Chemistry A, 2010, Vol. 84, No. 11, pp. 1986–1992. © Pleiades Publishing, Ltd., 2010.
Original Russian Text © E.S. Chukhrai, O.S. Pilipenko, R.A. Ovsyannikov, L.F. Atyaksheva, E.E. Knyazeva, I.I. Ivanova, 2010, published in Zhurnal Fizicheskoi Khimii, 2010,
Vol. 84, No. 11, pp. 2175–2181.
BIOPHYSICAL
CHEMISTRY
Mechanism of the Dimerization of Enzymes
upon Adsorption on Silicate Adsorbents Using the Example
of Lysozyme and ꢀGalactosidase
β
E. S. Chukhrai, O. S. Pilipenko, R. A. Ovsyannikov, L. F. Atyaksheva, E. E. Knyazeva, and I. I. Ivanova
Department of Chemistry, Moscow State University, Moscow, 119991 Russia
eꢀmail: Chukhrai@phys.chem.msu.ru
Received December 22, 2009
Abstract—It is shown by the kinetic analysis of lysozyme and
chrome, silica gel, and mesoporous silica that the adsorption follows a twoꢀstage scheme, including
reversible preꢀadsorption and the irreversible binding of dimmers. The corresponding rate constants
β
ꢀgalactosidase adsorption on siloꢀ
of adsorption, desorption, and dimerization were calculated. It was found that
adsorbed on silica gel and mesoporous silica retained ~20% of its activity, while
adsorbed on silochrome retained more than 30% of its activity.
β
ꢀgaloctosidase
ꢀgaloctosidase
β
DOI: 10.1134/S0036024410110282
INTRODUCTION
rier, the specific strong contact (
E
Ωst). The occurrence
of this contact is preceded by reversible adsorption,
E + Ω ↔ Ωw
Ωw is a nonspecific weak contact that arises as
Adsorption as a method of immobilizing enzymes
allows us to attain the irreversible binding of a protein
with a solid surface without significant loss of its cataꢀ
lytic properties. The irreversibility of adsorption, howꢀ
ever, introduces fundamental difficulties into the theꢀ
oretical interpretation of the observed effects. Modern
physicochemical theories are applicable only to
extreme cases related to either the chaotic distribution
of adsorbate described by a random law, or to thermoꢀ
dynamic equilibrium layers, which is not the case for
adsorption layers. Kinetic investigations of the proꢀ
cesses of irreversible protein sorption have therefore
come to the fore.
E
,
where
E
a result of an incident collision of the enzyme moleꢀ
cule with the carrier surface. The energy of an adsorꢀ
bate’s bond with a surface is relatively low, and we can
therefore expect the reversibility of adsorption. The
following scheme corresponds to the model approxiꢀ
mation:
k
k
2
→
1
⇐⇒
E +
Ω
EΩw
EΩst
,
(1)
k
–1
where k1, k–1, and k2 are kinetic constants of the rate
of adsorption, desorption, and strong binding, correꢀ
spondingly.
The features of a protein as an adsorbate are conꢀ
sidered in detail in [1], in which the kinetic analysis of
a series of possible adsorption mechanisms and the
wellꢀgrounded selection of one of them are suggested.
According to this concept, irreversible protein adsorpꢀ
tion on a homogeneous surface does not occur as a
oneꢀway kinetically irreversible reaction,
The aim of this work is to consider within scheme (1)
the kinetic laws of the adsorption of glycosidic linkage
hydrolysis enzymes, egg lysozyme and ꢀgalactosidase
β
from Penecillium canescens (LAC) on silicate adsorꢀ
bents with different parameters of the porous strucꢀ
ture, to determine the mechanism of adsorption in
each case, and to obtain the quantitative characterisꢀ
tics of the process.
Proteins differing by function, molecular weight,
size, and oligomeric composition were chosen as
adsorbates. Lysozyme (KF 3.2.1.17) and LAC
E + Ω →
EΩ
,
where E is an enzyme molecule, and
Ω
is a free space
on an adsorbent surface, equivalent to the molecular
area of adsorbing enzyme, Eаds
.
As a rule, the adsorption of an enzyme having a (KF 3.2.1.23) hydrolyze Oꢀglycosidic linkages in
contact area of 15 to 150 nm2 or larger occurs as a mucopolysaccharides (bacterial walls) and polysacꢀ
result of the multipoint binding of a protein with a carꢀ charides, respectively. Like all proteins, lysozyme and
rier. A contact enzyme carrier (
E ) thus arises with a LAC have very complicated topography of the moleꢀ
Ω
certain structural correspondence between strongly cule surface. For example, data from Xꢀray diffraction
interacting regions of an enzyme surface and the carꢀ studies [2] indicate that the lysozyme molecule is
1986

MECHANISM OF THE DIMERIZATION OF ENZYMES
1987
Table 1. Parameters of the porous structure of adsorbents
compact, quite stable, and has the topography of a surꢀ
face with alternating polar and hydrophobic areas,
making it promising for use as an adsorbate [3, 4]. The
same property presents a problem in ophthalmology,
since ocular liquid contains lysozyme, which is quite
actively adsorbed on a contact lens. Lysozyme moleꢀ
cule has one polypeptide chain consisting of 129 amiꢀ
noacid residuals with four disulfide linkages, Mr =
14300. This is an extended spheroid with two separate
areas and an active center. The size of the molecule’s
Adsorbent
Silica gel
S
bet, m2/g
V
pore, cm3/g
23
120
670
–
Silochrome
1.0
0.6
Mesoporous silica gel
EXPERIMENTAL
Enzymes
side is approximately
terminal part is approximately
3
×
4.5 nm, while the size of its
nm [5].
3 × 3
The adsorption of lysozyme from ovalbumin
(NPO Biokhimreaktiv) and LAC from Pen. canescens
(Fꢀ178 strain) (VNII Biotekhnika) was investigated.
The protein content in each preparation was deterꢀ
mined by the Bradford method and was found to be
15% in LAC and 52% in lysozyme.
LAC from Pen. canescens (monomer Mr = 120000)
has properties common to all known fungal enzymes;
however, the authors of [6] noted several features disꢀ
tinguishing LAC from Pen. canescens. This enzyme is
active not only in acidic media, but in neutral media as
well, setting it apart from other fungal lactases and
expanding the prospects for its practical application.
In addition to its activity in hydrolysis reactions, lacꢀ
tases participate in reactions of transglycosylation durꢀ
ing the synthesis of oligoꢀ and polysaccharides [7].
Adsorbents
Two industrial adsorbents were used, hydrothermal
wideꢀporous silica gel (silica gel) and silochrome
Sꢀ120 (silochrome). The third adsorbent, mesoporous
silicate (MS), was synthesized via the micellar temꢀ
plate method. Polyethylene glycol was used as a temꢀ
plate. The characteristics of adsorbent porous strucꢀ
ture shown in the Table 1 were determined from an
isotherm of lowꢀtemperature nitrogen adsorption,
obtained using an ASAP 2010N porosimeter
(Micromeritics). In Fig. 1, the distribution curves of
pores by sizes for silochrome and MS are shown.
The
βꢀGalactosydase from Pen. canescens is a tetꢀ
ramer, the size of a molecule being 12
×
12 nm [8].
The thermal stability and high activity of LAC Pen.
canescens make it worthwhile to immobilize this
enzyme on different solid carriers and to seek ways of
increasing its stability and activity. Tosylated cotton
cloth, chitosane, sepharoseꢀ4B, foam polyurethane,
and several other adsorbents have been used as carriers
[9, 10]. The most enzyme is sorbed on a chitosane surꢀ
face, where it exhibits high stability and activity in the
reaction of lactose hydrolysis in lactoserum.
104, сm3/(g
Å)
Lysozyme proved to be a good adsorbate upon its
adsorption on mesoporous molecular sieves of the
MSMꢀ41 and SBAꢀ15 types [3, 4, 11]. The maximal
lysozyme adsorption on AlSBAꢀ15 (Ssp = 604 m2/g
(
12
dV
/dD
)
×
and
Dpor ~ 12 nm) was found to be 580 mg/g (pH 9.6).
The lysozyme sorption curves at
T
= const in the pH
8
range of 6.5–10.5 correspond to the model of a Langꢀ
muir isotherm (type L), while the sorption curves
obtained at pH 12 correspond to the model of an
Sꢀshaped isotherm (type S) [3]. In [4, 11], the data on
the adsorption of lysozyme on molecular mesoporous
sieves of the SBAꢀ15 type are given, and it is shown
that maximum adsorption depends on pH and rises
from 250 to 500 mg/g with pH rising from 5 to 8. The
value of adsorption is approximately equal on samples
with pore sizes of 8 and 24 nm, and is considerably
lower on samples with a pore size of 4 nm. The authors
of [4, 11] assumed that lysozyme sorption occurs only
on the outer surface of the carrier on SBAꢀ15 mesopoꢀ
rous silicate with a pore diameter of 4 nm.
1
4
2
0
500
1000
Dpore
,
Å
Fig. 1. Distribution of pore volume by diameter (
silochrome and ( ) for mesoporous silica.
1) for
2
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1988
CHUKHRAI et al.
Adsorptive Measurements
where k2 is the rate constant of dimerization through
the solution.
According to scheme (2), the irreversible stage is
associated with the surface appearance of irreversibly
bound dimmer E2, the stability of which is maintained
by interprotein bonds and multipoint interaction with
the surface.
Lysozyme adsorption was performed using a phosꢀ
phate buffer solution, pH 6.0. The kinetic of enzymes
adsorption was traced by the change in protein conꢀ
centration in the contact solutions. Lysozyme concenꢀ
tration was determined spectrophotometrically at
280 nm. Adsorption measurements were performed at
The following equation can be used to determine
the kinetic constants in both cases:
an initial lysozyme concentration of 25–70
adsorption time varied from 0.5 to 7 h.
μm, and
LAC adsorption was performed at the optimum pH
of enzyme activity (pH 4.5) using a phosphate–citrate
buffer. LAC concentration in the contact solutions
were considered proportional to the enzyme activity.
The enzyme activity was determined according to the
1
t
1 – aθ
1 – θ
⎛
⎞
ꢀ ln ꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀꢀ = η1 + η2t,
(3)
⎝
⎠
where
θ
=
Eаds
/
Emax is the degree of filling of a carrier
with an enzyme; Eаds and Emax are the amount of an
enzyme in the adsorptive layer and upon monolayer
filling, respectively; and a = Emax/E0V, where V is the
volume of solution. The values of η1 and η2 are correꢀ
initial rate of hydrolysis of synthetic substrate
phenylꢀ ꢀDꢀgalactopyranoside (Sigma). The
o
ꢀnitroꢀ
β
оꢀNitroꢀ
phenol content in the reaction products of hydrolysis
were analyzed spectrophotometrically at 420 nm.
spondingly equal to
Since the coloration of оꢀnitrophenol appears only in
η1 = (1 – a)k1E0,
alkali media, and the hydrolysis reaction was perꢀ
formed at the optimum activity of the enzyme at
pH 4.5, test portions with a volume of 0.5 ml were colꢀ
lected from the reaction media at certain intervals and
placed into a vial with 1 M Na2CO3 solution. The iniꢀ
tial concentration of the enzyme was varied over an
η2 = (a – 1)/2xk1E0(k–1 – k2E0) [1].
In the figures plotted by the experimental data in the
coordinates of linear equation (3), where VE0
(i.e., a ≠ 1), the intercept on the ordinate is equal to
≠
Emax
interval of 0.07–7 M, and adsorption measurements
μ
Rорд = k1(1 – a)E0.
Here, the value of k1
k1 = RордV/(VE0 – Emax).
(4)
were performed at time intervals of 1–300 h.
The adsorption value was calculated by the differꢀ
ence between the initial and current enzyme concenꢀ
trations.
(5)
The intercept Rabs on the abscissa axis is different
for the different kinetic schemes [1].
(i) If the straight line in the coordinates of Eq. (3)
is parallel to the abcissa axis, the adsorption proceeds
irreversibly in one stage.
(ii) If the value Rabs does not depend on E0, kinetic
scheme (1), i.e., the reversible adsorption of an
enzyme followed by its strong binding, is valid.
RESULTS AND DISCUSSION
The first data on lysozyme structure obtained in
1960s [2] noted the discontinuity of the protein surface
topography and the presence of unequal contact areas
responsible both for interprotein binding with the forꢀ
mation of associates of the dimmer type, and the posꢀ
sibility of multipoint protein binding with an adsorꢀ
bent. One complication of the actual scheme of proꢀ
tein sorption is that even with the irreversibility of the
binding, a reversible “preliminary adsorption” stage
can be determined experimentally [1, 12, 13]. That the
elementary kinetic scheme of enzyme adsorption on a
solid carrier allows the presence of a reversible stage
was suggested in [1] and can be written as (1).
Rаbs = 2/k–1.
(6)
(iii) If Rаbs depends on the E0 value, scheme (2) is
realized. In this case, the focus is the association of
adsorbate via the addition of molecules from the soluꢀ
tion volume. The graph plotted in the coordinates of
the equation 2/Rаbs
(E0)
according to (7) allows us to
calculate k–1 and k2
2/Rаbs = k–1 – k2E0.
(7)
The detailed kinetic analysis of this scheme is preꢀ
sented in [1], and the methods of calculating the
kinetic constants are also given in [12–14] using the
example of LAC and peroxidase and hemoglobin.
In Fig. 2a, the kinetic curves of lysozyme adsorpꢀ
tion on silochrome at three different protein concenꢀ
trations are presented. The curves in Fig. 2b are preꢀ
sented in double reverse coordinates for calculating
As was shown in [13] for LAC, if the strong bonds
are not monomers but E2 dimmers, the kinetic
scheme is
the ultimate adsorption (Eult) at a given initial protein
concentration, and for further calculating maximum
adsorption (Emax) upon monolayer filling. To deterꢀ
mine the mechanism of adsorption, the kinetic curves
in Fig. 2c are presented in the coordinates of Eq. (3).
In Fig. 3, similar dependences for the adsorption of
k
1
E + Ω ⇐⇒ EΩw,
k
–1
(2)
k
2
→
E + Ω + EΩсл
E2Ω2,
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MECHANISM OF THE DIMERIZATION OF ENZYMES
1989
3
40
30
20
10
12
2
1
2
8
1
3
4
(a)
(b)
0
2
2
4
6
0
2
4
1
t
, h
1/t
, h−
t
, h
8
t, h
8
0
4
6
0
2
4
6
4
0.2
0.4
3
0.04
0.08
0.12
0.16
3
2
1
2
1
(c)
(d)
Fig. 2. Kinetics of lysozyme adsorption on (a) silochrome at initial concentrations of the protein of 33 (
The test portion of the adsorbent was 50 mg; = 3 ml; phosphate buffer, pH 6.0; (b) determination of limiting adsorption (
for the kinetic curves of Fig. 1a in double reversible coordinates; (c) determination of the kinetic constants of reversible lysozyme
sorption in the coordinates of Eq. (3); = (1 – )/(1 – ; (d) kinetics of lysozyme adsorption on mesoporous silica in the coorꢀ
dinates of Eq. (3) at initial protein concentrations 28.6 (
20 mg; = 2 ml; phosphate buffer, pH 6.0.
1
), 46 (
2
), and 60 (
3
)
E
μ
m.
)
V
lim
F
aθ
θ
)
1), 40.7 (
2
), 56.3 (
3
), and 68.3 (
4
) m. The test portion of adsorbent was
μ
V
Eаds, mg/g
t, h
0
50
100
(a)
30
20
10
5
4
4
1
2
3
3
3
2
1
2
1
(b)
0
100
200
300
1
t
, h
−
100/(
tlnF
), h−
Fig. 3. Kinetics of LAC sorption on (a) mesoporous silica at initial protein concentrations of 0.07 (
and 0.67 ( m. The test portion of adsorbent was 50 mg; = 5 ml; 0.1 M phosphate–citrate buffer, pH 4.5; (b) determination
of the kinetic constants of LAC adsorption in the coordinates of Eq. (3) at initial protein concentrations of 0.13 ( ), 0.27 ( ),
0.53 ( ), and 0.67 ( m; = (1 – )/(1 –
1), 0.13 (2), 0.27 (3), 0.53 (4),
5)
μ
V
1
2
3
4)
μ
F
aθ
θ
).
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1990
CHUKHRAI et al.
Table 2. Calculating the kinetic parameters of scheme (2)
for lysozyme adsorption on silochrome ( ) and mesoporous
silica gel (MS)
A comparison of the values of the bimolecular conꢀ
stants of adsorption rate k1 and dimerization rate k2 for
each enzyme shows that they increase with an increase
in the pore sizes of the adsorbent. The minimal
adsorption rates on MS are apparently due to the
existing steric limitations of its nonuniform porous
structure.
S
E0
,
μ
m
–R
ord, h–1
Rads, h
a
k
1, М–1 s–1
S,
Emax = 100 mg/g
,
k–1 = 8
×
10–5 s–1
,
k
2 = 0.9 М–1 s–1
33.0
46.0
60.0
0.46
0.38
0.30
10.7
17.3
3.6
2.6
1.5
1.4
1.4
If we compare the rate constants of the monomoꢀ
lecular desorption process, we can see that the k–1
value is minimal (0.43 s–1) upon the adsorption of a
large LAC molecule on an adsorbent with smaller pore
size. The highest value of this constant is 8 s–1 and corꢀ
responds to lysozyme adsorption on a sylochrome surꢀ
face.
20
2
МS,
Emax = 110 mg/g
,
k–1 = 6.4
×
10–5 s–1,
k
2 = 0.4 М–1 s–1
28.6
40.7
56.3
68.3
0.16
0.16
0.07
0.04
12.0
2.75
0.9
1.2
0.9
0.9
11.8
1.9
1.4
1.2
16.8
We should note the values of the molecular areas of
lysozyme and LAC on the adsorbent surfaces. Comꢀ
pared to the theoretical area (14 nm2) for lysozyme, it
is 29 nm2 on a silochrome surface and 145 nm2 on
mesoporous silica. This means that lysozyme occuꢀ
pied ~50% of the silochrome surface and only ~10% of
the silicate MS surface. Allowing for the nonuniform
distribution of pores by size in the case of this adsorꢀ
bent (see Fig. 1), we may consider that for lysozyme
adsorption, pores with sizes greater than 7 nm are
accessible, and partial adsorption occurs at the pore
mouth and on the outer surface. The molecular areas
of LAC on the surface of the same adsorbents are conꢀ
siderably greater than for lysozyme, since a molecule
of this enzyme (a tetramer) is substantially larger then
a lysozyme molecule. For LAC adsorption, the outer
surface of a pore and pores appreciably larger than
those of lysozyme could be accessible.
14.6
LAC on MS at different protein concentrations are
presented. It is seen from Figs. 2c and 3c that the Rabs
value depends on the E0 value and scheme (2) is conꢀ
sequently observed. This means that
E molecules on
the first stage of adsorption are reversibly bound to the
surface. Protein molecules from the contact solution
are additionally sorbed onto the sorbed molecules, and
irreversibly sorbed associates arise on the surface. This
mechanism is also observed upon the adsorption of
LAC on silica gel [13]. Despite the differences in the
porous structure parameters of the employed adsorꢀ
bents (see Fig. 1 and Table 1) and the sizes of the proꢀ
tein molecules, the same kinetic mechanism is thus
observed upon their adsorption.
In Table 4, the kinetic parameters of the hydrolysis
Data from calculating the kinetic parameters of
lysozyme adsorption on silochrome and mesoporous
silica according to scheme (2) are presented in Table 2.
The constants of the desorption rate (k–1) and dimerꢀ
ization through the solution (k2) were calculated using
Eq. (7). Equation (5) was used to calculate the adsorpꢀ
reaction of
oꢀnitrophenylꢀ ꢀDꢀgalactopyranoside
β
catalyzed by LAC in solution and in the adsorption
layers on surfaces of MS, silochrome, and silica gel are
presented. The results of our research show that an
enzyme adsorbed on MS, silochrome, and silica gel is
active. The Michaelis constant for catalytic system
LAC/silica gel does not change, in contrast to the
homogeneous system, indicating the continuance of
the kinetic mechanism of the reaction in the adsorpꢀ
tion layer and the lack of diffusion limitations. As
expected, the greatest diffusion limitations correspond
tion rate constant, k1
.
The values of lysozyme and LAC adsorption on difꢀ
ferent adsorbents and the values of kinetic constants of
the adsorption process, as calculated from the experiꢀ
mental data, are presented in Table 3.
Table 3. Adsorption value (
A, mg/g), kinetic constants, and areas occupied by a protein molecule on an adsorbent surface
(S
M, nm2)
Sample
А
А
SM
k
1, M–1 s–1
k–1
×
105, s–1
k
2, m–1 s–1
Lysozyme/MS
Lysozyme/silochrome
LAC/MC
110
100
60
0.16
0.83
0.09
0.14
0.40
145
29
0.9
1.4
9.0
37
6.4
0.4
0.9
3.7
–
8.0
0.43
1.6
8900
5700
LAC/silochrome
LAC/silica gel
16.7
9.2
2000
2
60
1.7
5.5
2
Note: The theoretical molecular areas are 14 nm for lysozyme and 144 nm for the tetramer of LAC. The interval of the studied concentraꢀ
tions is 30–70 μM for lysozyme and 0.07–0.7 μM for LAC.
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MECHANISM OF THE DIMERIZATION OF ENZYMES
1991
ALAC
,
μ
m/min mg
(a)
1/ALAC
,
min mg/μm
0.6
(b)
8
0.4
0.2
4
0
0.4
0.8
x
0
0.4
0.8
x
Fig. 4. (a) Dependence of the activity of sorbed LAC on the degree of filling (
x
) of a silica gel surface; (b) determination of the
m/min mg.
specific activity of isolated LAC globules in the coordinates of Eq. (3); = 5.2
A
μ
0
to the LAC/MS sample. The Michaelis constant in ity of isolated molecules of the adsorbed enzyme.
this case is 3.6 (i.e., a threefold increase over the
enzyme in solution). Here, the catalytic activity of
both heterogeneous catalysts is roughly the same. The
highest catalytic activity among the heterogeneous
samples has a LAC/silochrome catalyst. In this case, it
is possible to preserve more than 30% of the enzyme
activity in the solution.
Here, the associates of any composition are assumed
to be inactive. In Fig. 4b, we can see that the experiꢀ
mental dependence of LAC activity on the degree of
filling is straight in the coordinates of Eq. (8), enabling
us to determine the activity of isolated globules,
a.
This was found to be 20.8 mol/(min mg), or approxꢀ
μ
imately oneꢀthird that of the activity of the enzyme in
the solution. Such a reduction in activity is the result
of conformational changes in the LAC active center
under the effect of the carrier. The further reduction in
enzyme activity in the adsorptive layer with an
In Fig. 4, it is shown that the activity of LAC
adsorbed on silica gel depends on the degree of filling, .
θ
The following linear equation corresponds to the
monotone decrease of specific catalytic activity
(Fig. 4a):
increase in
θ
is the result of interprotein interactions.
K2θ
ꢀꢀꢀꢀꢀ = ꢀꢀꢀꢀꢀ + ꢀꢀꢀꢀꢀꢀꢀ ,
Asp aC aC
1
1
(8)
CONCLUSIONS
where
a
is the activity of the isolated molecules of an
Biocatalyst LAC/silochrome with an activity of
>30% than that of enzyme in solution was thus
obtained in this work. Two other biocatalysts
(LAC/MS and LAC/silica gel) retained ~20% of their
enzyme activity in solution. For the biocatalyst
LAC/silica gel, it was also shown that only isolated
molecules possess any activity, while associates of any
composition are inactive. Since LAC is a glycoproteid,
we can use, e.g., the combined adsorption of LAC with
polysaccharides amylose and heparin to obtain an
increase in the activity of isolated globules upon
adsorption [16]. Here, the activity of adsorbed isolated
LAC globules rises to the activity of the enzyme in
solution. The second way of increasing the activity of
heterogeneous biocatalysts based on adsorbed LAC is
to find new adsorbents that obstruct unproductive
association in the adsorption layers of an enzyme. Of
the three adsorbents we studied, the best was siloꢀ
chrome with a maximum of ~50 nm on the curve of
pore distribution sizes.
adsorbed enzyme, and is a parameter proportional
C
to the product of the catalyst surface and the number
of enzyme molecules on a unit of monolayer surface.
K2 is the equilibrium constant of protein association in
the adsorption layer [15]. Use of this equation enables
us to separate the effects of surface influence and
interprotein interactions, and to determine the activꢀ
Table 4. Kinetic parameters of the hydrolysis reaction of
o
ꢀnitrophenylꢀ ꢀDꢀgalactopyranoside at pH 4.5 in a 0.1 M
β
citrate–phosphate buffer, catalyzed by LAC in solution and
in adsorptive layers
Catalyst
KM, mM
V,
μmol/(min mg)
Aspec, %
LAC in solution
LAC/silica gel
LAC silchrome
LAC/MS
1.2
1.2
1.5
3.6
66.4
11.6
21
100
17
32
10.5
16
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
Vol. 84
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1992
CHUKHRAI et al.
9. W. Bednarski and A. Kulikowska, Chem. Papers 61
364 (2006).
10. S. Budriene, N. Gorochovceva, T. Romaskevic, et al.,
Centr. Eur. J. Chem. , 95 (2005).
,
REFERENCES
1. O. M. Poltorak, A. N. Pryakhin, and E. S. Chukhrai,
Vestn. Mosk. Univ., Ser. Khim. 23, 527 (1982).
2. C. C. F. Blake, D. F. Koenig, G. A. Vair, et al., Nature
206, 757 (1965).
3. A. Vinu, V. Murugesan, and M. Hartman, J. Phyz.
Chem. B 108, 7323 (2004).
4. A. Katiyar, L. Li, P. Smirniotis, and N. G. Pinto,
3
11. A. Katiyar, S. Yadav, P. Smirniotis, and N. G. Pinto,
J. Chromatogr. A 1122, 13 (2006).
12. L. F. Atyaksheva, E. E. Knyazeva, O. M. Poltorak,
et al., Zh. Fiz. Khim. 78, 2088 (2004) [Russ. J. Phys.
Chem. A 78, 1849 (2004)].
13. E. S. Chukhrai, L. F. Atyaksheva, O. M. Poltorak, and
O. S. Pilipenko, Zh. Fiz. Khim. 76, 2251 (2002) [Russ.
J. Phys. Chem. A 76, 2047 (2002)].
J. Chromatogr. A 1069, 119 (2005).
5. D. P. Kharakoz and A. P. Sarvazzyan, Biopolymers 33
,
11 (1993).
6. I. V. Nikolaev, O. M. Khodova, E. A. Timokhina, et al.,
14. L. F. Atyaksheva, O. M. Poltorak, E. S. Chukhrai, et al.,
Zh. Fiz. Khim. (in press).
Biokhimiya 54, 1294 (1989).
7. W. Bednarski and A. Kulikowska, Biotechnologia,
No. 2, 137 (2007).
15. O. M. Poltorak, Zh. Fiz. Khim. 41, 2544 (1967).
16. L. F. Atyaksheva, O. S. Pilipenko, and O. M. Poltorak,
8. A. A. Shishkin, O. M. Poltorak, E. S. Chukhrai, et al.,
Zh. Fiz. Khim. 75, 2256 (2001) [Russ. J. Phys. Chem.
A 75, 2076 (2001)].
Zh. Fiz. Khim. 64, 2169 (1990).
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A
Vol. 84
No. 11
2010