The catalytic properties and stability of β-galactosidases from fungi

Article abstract of DOI:10.1134/S0036024408130165

The catalytic activity of β-galactosidases from fungi Penicillium canescens and Aspergillus oryzae is maximum in a weakly acidic medium and does not depend on the presence of magnesium cations in the reaction medium. The enzyme from Aspergillus oryzae fungi is more active, and that from Penicillium canescens is stabler. One of stability indications is the presence of an induction period in the kinetic curves of thermal inactivation. This period disappears at 54°C for the enzyme from Aspergillus oryzae and at 59°C for the enzyme from Penicillium canescens. The temperature dependences of the effective rate constants for the inactivation of the tetrameric enzyme from Penicillium canescens show that the main reason for enzyme inactivation is the dissociation of oligomeric forms below 66°C (E _act = 85 kJ/mol) and enzyme denaturation at higher temperatures (E _act = 480 kJ/mol). The dissociation stage is absent for monomeric β-galactosidase from Aspergillus oryzae fungi, and the activation energy of inactivation is 450 kJ/mol over the whole temperature range studied (53-60°C). Pleiades Publishing, Ltd., 2008.

Full text of DOI:10.1134/S0036024408130165

ISSN 0036-0244, Russian Journal of Physical Chemistry A, 2008, Vol. 82, No. 13, pp. 2250–2254. © Pleiades Publishing, Ltd., 2008.
CHEMICAL KINETICS
AND CATALYSIS
The Catalytic Properties and Stability
of β-Galactosidases from Fungi
O. S. Pilipenko, L. F. Atyaksheva, O. M. Poltorak, and E. S. Chukhrai
Faculty of Chemistry, Moscow State University, Moscow, 119992 Russia
e-mail: poltorak@phys.chem.msu.ru
Received March 27, 2008
Abstract—The catalytic activity of β-galactosidases from fungi Penicillium canescens and Aspergillus oryzae
is maximum in a weakly acidic medium and does not depend on the presence of magnesium cations in the reac-
tion medium. The enzyme from Aspergillus oryzae fungi is more active, and that from Penicillium canescens
is stabler. One of stability indications is the presence of an induction period in the kinetic curves of thermal
inactivation. This period disappears at 54°C for the enzyme from Aspergillus oryzae and at 59°C for the enzyme
from Penicillium canescens. The temperature dependences of the effective rate constants for the inactivation of
the tetrameric enzyme from Penicillium canescens show that the main reason for enzyme inactivation is the dis-
sociation of oligomeric forms below 66°C (Eact = 85 kJ/mol) and enzyme denaturation at higher temperatures
(
Eact = 480 kJ/mol). The dissociation stage is absent for monomeric β-galactosidase from Aspergillus oryzae
fungi, and the activation energy of inactivation is 450 kJ/mol over the whole temperature range studied (53–
0°C).
6
DOI: 10.1134/S0036024408130165
INTRODUCTION
are planar tetramers according to the electron micro-
scopic data [9]. It is natural to expect that the difference
in the oligomeric composition of β-galactosidases
should influence their catalytic activity and stability.
β-Galactosidases from Aspergillus and Penicillium
fungi along with β-galactosidases from yeast Kluyvero-
myces are most often used in the production of dietetic
milk products with a decreased lactose content. In
recent years, the possibility of using the transglycosi-
dase activity of these β-galactosidases for the prepara-
tion of galactooligosaccharides [1–5], for instance,
lactulose prebiotic (4-o-β-D-galactopyranosyl-D-fruc-
tose), has been discussed.
EXPERIMENTAL
We studied lyophilized preparations of β-galactosi-
dases from Penicillium canescens and Aspergillus
oryzae fungi. Their physicochemical properties are
listed in Table 1.
The purpose of this work was a comparative study
of the catalytic properties and thermal stability of two
β-galactosidases from Penicillium canescens and
The content of protein in the samples was deter-
Aspergillus oryzae fungi. Both enzymes are proteogly- mined according to Bradford. Catalytic activity was
canes, belong to the family of 35 glycosylhydrolases, measured over the (protein) concentration range 0.35–
and have close molecular weights (~110 kD). The 35 mg/l at room temperature from the initial rate of the
amino acid sequence of β-galactosidase from Penicil- hydrolysis of 2-nitrophenyl-β-D-galactopyranoside in
lium canescens includes 1011 residues [6], and that of 0.1 M phosphate–citrate buffer solutions. The hydroly-
β-galactosidase from Aspergillus oryzae, 1005 residues sis product, o-nitrophenol, was determined colorimetri-
[
7]. β-Galactosidase from Aspergillus oryzae, however, cally at λ = 400 nm. Preliminarily, a reaction mixture
exists in the form of active monomers [8], whereas mol- sample was introduced into a 1 M solution of Na CO
2
3
ecules of β-galactosidase from Penicillium canescens to stop the reaction and reveal nitrophenol coloring.
Table 1. Physicochemical properties of β-galactosidases
Vmax
,
Source of enzyme
m, %
Optimum pH
K , mM
M
µM/(min mg protein)
Penicillium canescens fungi (Biotekhnika)
Aspergillus oryzae fungi (Sigma)
Note: m is the content of protein.
15
8
4.2–4.5
4.2–4.5
1.0
1.6
19.5
62.5
2
250

THE CATALYTIC PROPERTIES AND STABILITY OF β-GALACTOSIDASES FROM FUNGI
2251
–
1
a/amax
1/V, [µmol/min mg (protein)]
2
2
1
1
0
.5
.0
.5
.0
.5
1
0
0
.0
.6
.2
1
2
1
2
3
3
4
5
6
7
pH
–1
0
1
2
3
–
3
–1
1
0 /S, M
Fig. 1. pH dependence of the relative activity of β-galactosi-
dases from (1) Penicillium canescens and (2) Aspergillus
oryzae.
Fig. 2. Dependences of the activity of β-galactosidase from
Penicillium canescens on the concentration of 2-nitrophe-
nyl-β-D-galactopyranoside in the Lainuiver–Berk coordi-
nates in the presence of (1) 0.1 M Mg and (2) 0.1 M Mn
and (3) in their absence.
2
+
2+
The thermal inactivation of the enzymes was per-
formed in a phosphate–citrate buffer solution (pH 4.5)
in the absence of a substrate over the temperature
ranges 50–60°C for the enzyme from Aspergillus
oryzae and 50–70°C for the enzyme from Penicillium
canescens. No kinetic measurements were made at
higher temperatures, because the enzymes lost more
this work remains constant when their concentration in
solution changes by 100 times, whereas the catalytic
activity of other β-galactosidases depends on dilution [11]
because of the association and dissociation of oligo-
than 90% catalytic activity during the first 15 min. The meric forms. However, constant specific activity values
lower temperature limit (50°C) was selected because, at over a wide concentration range are not unambiguous
this temperature, the catalytic activity of β-galactosi- evidence of the absence of association and dissociation
dase from Aspergillus oryzae remained constant for 6 h,
and that of β-galactosidase from Penicillium cane-
scens, for more than 10 h.
The protein concentration in thermal inactivation
experiments was varied from 15 to 300 mg/l. During
in solution, because oligomers of different composi-
tions can possess identical catalytic properties.
A comparison of the catalytic properties of the
β-galactosidases studied shows that their activities have
thermal treatment, enzyme samples were periodically similar dependences on pH (Fig. 1). At the same time,
taken, and their activity was determined by the standard β-galactosidase from Aspergillus oryzae is three times
procedure at pH 4.5 using saturated substrate solutions. more active than the enzyme from Penicillium cane-
The v/v ratio was used as a measure of enzyme inac- scens (Table 1). The Michaelis constants differ approx-
t
0
tivation; here, v is the rate of the reaction before ther-
0
imately by a factor of 1.5 and are 1.6 and 1.0 mM for
β-galactosidases from Aspergillus oryzae and Penicil-
lium canescens, respectively. The dependence of the
activity of β-galactosidase from Penicillium canescens
on substrate concentration is shown in Fig. 2 in the
Lainuiver–Berk coordinates in the presence of 0.1 M
mal treatment and v is the rate of the reaction after the
t
thermal treatment of the enzyme during time t.
RESULTS AND DISCUSSION
The catalytic properties of β-galactosidases from
fungi differ from the properties of β-galactosidases
from other sources. Their activity is maximum at
pH 4.0–4.5 (Fig. 1), whereas β-galactosidases from
2
+
2+
Mg cations or 0.1 M Mn cations and in their
2
+
absence. These data show that the presence of Mg or
2
+
Mn in the reaction medium does not influence the cat-
other sources show the highest catalytic activity at alytic activity of the enzyme. Similar dependences
pH 7.0–7.5. The catalytic activity of the β-galactosi- were obtained for β-galactosidase from Aspergillus
dases from fungi under consideration does not depend oryzae.
on the presence of magnesium cations in the reaction
Studies of the thermal inactivation of β-galactosi-
dases from fungi gave dependences corresponding to
mixture (Fig. 2), whereas the activity and stability of
some other β-galactosidases increases upon the addi-
tion of magnesium cations [10]. The specific catalytic two kinetic mechanisms of thermal inactivation [12],
activity of the β-galactosidases from fungi studied in inactivation according to a scheme of sequential trans-
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 82 No. 13 2008

2
252
PILIPENKO et al.
V/V0
thermal inactivation (Fig. 3). During this period, the
catalytic activity of the enzyme remains constant. The
presence of an induction period can be explained on the
assumption of the existence of several “stable” and
1
0
0
0
0
.0
.9
.8
.7
.6
“
unstable” (labile) intermediate enzyme forms with
approximately equal catalytic activities. With mono-
meric β-galactosidase from Aspergillus oryzae, confor-
mational changes that occur in the globule during the
induction period do not affect the catalytic domain, and
the activity of the enzyme remains constant. With tet-
rameric β-galactosidase from Penicillium canescens,
conformational changes in the oligomer or oligomer
dissociation to active subunits,
1
2
3
0
50
100
150
200
250
τ, min
K
E4
1
4E ,
1
K
2
K
E4
2E2
4E1,
Fig. 3. Kinetic curves of the thermal inactivation of β-galac-
tosidases from (1) Aspergillus oryzae and (2, 3) Penicillium
canescens at (1) 53, (2) 57, and (3) 58°C; pH 4.5.
can occur during the induction period.
The initial inactivation stages not accompanied by
changes in the catalytic activity of the enzymes but
influencing their stability can also occur during enzyme
storage. The kinetic dependences of thermal inactiva-
tion of β-galactosidase from Penicillium canescens at
formations with an active intermediate form (Fig. 3)
and irreversible first-order inactivation (Fig. 4).
Inactivation involving sequential first-order trans-
formations can in a general form be represented as
6
0°C in a freshly prepared solution and after solution
storage in a refrigerator for two weeks are shown in
Fig. 5. The activity of the enzyme did not change after
storage, but the rate constant for inactivation increased
kn
(
n)
(n – 1)
E
E
…
k–n
k1
kd
(1)
…
E
Ed,
–4
k–1
by almost an order of magnitude (from 1.7 × 10 to
–
3 –1
1
.2 × 10 s ).
(
n)
where E is the active and stable enzyme form,
The kinetic dependences with an induction period
(n − 1)
(1)
E
…E are the active labile forms, and E is the
d
were obtained over different temperature ranges for the
denaturated enzyme.
β-galactosidases studied. The induction period disap-
If transformations occur according to this scheme, peared at 54°C from the kinetic curves for the thermal
an induction period appears in the kinetic curves of inactivation of β-galactosidase from Aspergillus oryzae
τ, min
250
τ, min
120
0
50
100
150
200
0
20
40
60
80
100
0
1
.5
.0
1
2
1
1
4
2
1.5
2
–
.0
2
3
3
–
ln(V/V0)
ln(V/V0)
Fig. 5. Kinetic dependences of the thermal inactivation of
β-galactosidase from Penicillium canescens at 60°C and
pH 4.5 (1) in a freshly prepared solution and (2) in the same
solution stored in a refrigerator for two weeks. The activity
of the enzyme did not change after storage.
Fig. 4. Kinetic curves of the thermal inactivation of β-galac-
tosidases from (1–3) Aspergillus oryzae and (4) Penicillium
canescens in the coordinates of the first-order equation at
(1) 55, (2) 57, (3) 59, and (4) 60°C; pH 4.5.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 82 No. 13 2008

THE CATALYTIC PROPERTIES AND STABILITY OF β-GALACTOSIDASES FROM FUNGI
Table 2. Stability parameters of β-galactosidases from fungi
2253
4
–1
Source of enzyme
τind, h
Tind, °C
keff × 10 , s
E, kJ/mol
∆T, °C
Penicillium canescens
>10
59
1.7
85
60–65
66–70
53–60
4
80
Aspergillus oryzae
6
54
12.0
450
Note: τind is the duration of induction period at 50°C, T is the temperature of induction period disappearance, k is the effective rate
ind
eff
constant of inactivation at 60°C, E is the activation energy, and ∆T is the temperature interval over which the activation energy was
determined.
and at 59°C for β-galactosidase from Penicillium cane- lower temperatures, the activation energy of the reac-
scens. At 53°C, the duration of the induction period was tion decreases to 85 kJ/mol. Since β-galactosidase from
4
0 min for β-galactosidase from Aspergillus oryzae and Penicillium canescens has a quaternary structure [9],
more than 6 h for β-galactosidase from Penicillium the conclusion can be drawn that, below 66°C, the inac-
canescens, which is evidence of a much higher stability tivation of the enzyme occurs as a result of the dissoci-
of the latter.
The kinetic curves of thermal inactivation without
an induction period can be linearized in the coordinates
of a first-order equation (Fig. 4). The rate constants
determined this way are effective values and character-
ize the half-transformation periods of active enzymes.
With β-galactosidase from Aspergillus oryzae, the
ation of the oligomer to inactive protomers. The tem-
perature dependence of the effective rate constant for
inactivation allowed us to determine the limits of the
temperature intervals in which different stages of the
dissociative thermal inactivation of the oligomeric
enzyme predominated.
Some kinetic parameters of inactivation are listed in
effective rate constants likely correspond to the irre- Table 2. These parameters are evidence of a higher sta-
versible denaturation of the monomeric enzyme. The bility of β-galactosidase from Penicillium canescens
temperature dependence of the rate constants for inac- compared with β-galactosidase from Aspergillus
tivation can be used to determine the activation energy oryzae.
of denaturation (Fig. 6). It is 450 kJ/mol and corre-
Our study showed that there is a fairly narrow tem-
sponds to the activation energies of the denaturation of
perature interval over which the rate of the thermal
protein molecules determined by differential scanning
inactivation of the enzymes sharply increases. This is
calorimetry [13]. Similar activation energy values were
6
5–70°C for β-galactosidase from Penicillium cane-
obtained in [14] for the thermal inactivation of this
scens and 54–60°C for β-galactosidase from Aspergil-
lus oryzae. The existence of a narrow temperature inter-
val of irreversible enzyme denaturation is caused by the
cooperative all-or-nothing character of the denaturation
β-galactosidase at pH 7.4 over the temperature range
4
5–55°C.
The scheme of dissociative thermal inactivation of
the tetrameric enzyme from Penicillium canescens
fungi can be written as [12]
3
–1
k1
k2
kd
10 /T, K
3.05
E4
2E2
4E1
4Ed.
2.90
6
2.95
3.00
k–1
k–2
Here, E and E are the active tetramers and dimers, E
1
4
2
is the inactive (or active) monomer capable of revers-
ible association, and E is the denaturated enzyme.
d
1
7
8
9
Under the conditions of our experiments, separate
stages of the process were kinetically indistinguishable,
and we were therefore unable to determine kinetic
parameters for each stage. We could only find the effec-
tive rate constants by the linearization of the experi-
mental data in the coordinates of a first-order equation
2
(
Fig. 4). Their values are determined by the temperature
and independent of the concentrations of the enzymes
15–300 mg/l). The temperature dependence of the
(
10
lnk
effective rate constant for the inactivation of
β-galactosidase from Penicillium canescens in the
coordinates of the Arrhenius equation (Fig. 6) has a
kink corresponding to 66°C. The activation energy of
the reaction at 66–70°C is 480 kJ/mol, which corre-
sponds to the denaturation of the protein molecule. At
–
Fig. 6. Temperature dependences of the effective rate con-
stants for the inactivation of β-galactosidases from (1) Pen-
icillium canescens and (2) Aspergillus oryzae in the coordi-
nates of the Arrhenius equation.
RUSSIAN JOURNAL OF PHYSICAL CHEMISTRY A Vol. 82 No. 13 2008

2
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PILIPENKO et al.
of proteins. For such transitions, only the initial (active)
and final (denaturated) states are observed in noticeable
amounts. Transitions of the all-or-nothing type are a
microscopic analogue of the first-order phase transition
in macroscopic systems. As distinct from the true phase
transition, an enzyme loses its catalytic activity over a
narrow temperature interval. For this reason, the ther-
mal denaturation of proteins is characterized by high
activation energies and is accompanied by strong heat
effects. The enthalpy of denaturation is higher for the
proteins whose thermal denaturation occurs at higher
temperatures [15]. The reason for a higher stability of
β-galactosidase from Penicillium canescens is in our
view the stabilization of the tetramer by the conforma-
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