Effect of the ionic liquid [bmim]Cl and high pressure on the activity of cellulase

Article abstract of DOI:10.1039/b918879g

The effect of the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl) and of high pressure on the activity of cellulase from Aspergillus niger were studied separately and in combination. The enzyme activity decreased with increasing concentration

Full text of DOI:10.1039/b918879g

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Effect of the ionic liquid [bmim]Cl and high pressure on the activity
of cellulase
ˆ
Angelo C. Salvador,† Mickael da C. Santos† and Jorge A. Saraiva*
Received 11th September 2009, Accepted 24th December 2009
First published as an Advance Article on the web 8th February 2010
DOI: 10.1039/b918879g
The effect of the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl) and of high
pressure on the activity of cellulase from Aspergillus niger were studied separately and in
combination. The enzyme activity decreased with increasing concentrations of [bmim]Cl, reaching
5
[
0% the value in aqueous buffer with 20% [bmim]Cl. However, when the enzyme is held in 10%
bmim]Cl and is then assayed in 1% [bmim]Cl, it showed only 8% reduction of activity. These
results can be explained by the fact that the activity of the enzyme in [bmim]Cl is linearly
correlated with the decrease of the thermodynamic water activity (a ). Under pressure the enzyme
w
activity varied from less 60% (at 200MPa) to equal (at 400 MPa), compared to atmospheric
pressure. In 10% [bmim]Cl under pressure, cellulase activity is improved compared to atmospheric
pressure, varying from equal (at 600 MPa) to 1.7-fold higher (at 100 MPa). This opens the
possibility to improve cellulase activity in ionic liquids, and possibly of other enzymes, by carrying
out the reaction under pressure.
3
,13
Introduction
aqueous buffer/ILs. However, in the latter case, activity of
cellulase has been shown to be lower than in aqueous solutions
for several ILs.
The limited reserves of fossil fuels have increased attention to
the use of renewable biomaterials for energy and production
of chemicals. However, production of biofuels and chemicals
from edible agricultural feedstocks raises problematic questions,
since it competes for agricultural land needed for food and feed
The Earth is predominantly a high-pressure environment,
with 62% of the total biosphere being characterized by pressures
greater than 100 bar (10 MPa). However, information on the
14
15
effects of pressure on biomolecules is still relatively scarce,
1
production. In the last few years, research has focused on the
being a field of increasing interest. Firstly used in the fields of
chemistry and physics, the use of the so called high (hydrostatic)
pressure (HP) is now an established method for cold pasteuriza-
tion of foods, using pressures of about 500 MPa (~5000 atm).
HP is also considered a green technology, since it uses water
as a compression media and is energetically efficient. In the
area of enzymology HP has the potential to change enzymes’
activity and selectivity.
can be carried out under HP in different solvent media as for
possible feasible use of cellulose, the most abundant renewable
bioresource, for the production of fermentable sugars that can
be converted to fuels such as ethanol or chemicals such as
16
1-3
lactic acid. For this possibility to become viable, a limiting
and crucial step is the hydrolysis of cellulose to fermentable
4
sugars, due to the protection of the sugar molecules against
chemical and enzymatic hydrolysis, by the tight packing of
cellulose chains in microfibrils and cellulose’s poor solubility
in aqueous solutions.
In the last few years ionic liquids (ILs) have emerged as a new
class of solvents for biocatalysis, either as single or co-solvents
17-19
Also, enzymatic catalytic reactions
20
instance in organic solvents. The effect of HP on an enzymatic
reaction rate is governed by the activation volume (Va) of the
reaction, defined as the overall molar volume difference between
the transition state and ground state of the enzyme–substrate
5
,6
and have attracted increasing attention for research. More
recently it was found that some ILs can dissolve cellulose,
2,4,7-10
21
complex. If Va is positive (negative), the reaction rate is de-
opening up interesting possibilities for the use of these solvents
to render cellulose more accessible to enzymatic hydrolysis.
The IL 1-butyl-3-methylimidazolium chloride, [bmim]Cl, is
indicated in the literature as one of the ILs with a better
capacity to dissolve cellulose. This has led to several recent
publications concerning enzymatic hydrolysis of cellulose after
creased (increased) by pressure, while if it is zero there is no effect.
The present work was initiated with the objective to study
the effect of the ionic liquid, [bmim]Cl, and of high hydrostatic
pressure up to 675 MPa, used separately and in combination, on
activity of cellulase, using carboxymethylcellulose as substrate.
Although there are already some publications on the effect of
pressure on enzymes’ activity in IL, using supercritical carbon
4,10
11,12
pretreatment with ILs
or directly (in situ) in mixtures of
22-24
dioxide,
the pressure level used in these cases was around
1
0 MPa, much below those used in this work (50–675 MPa).
Departamento de Qu ´ı mica, Universidade de Aveiro, Campus
Universit a´ rio de Santiago, 3810-193, Aveiro, Portugal.
As the authors are aware, this is the first study carried out
concerning the effect of HP on the activity of an enzyme in the
presence of an ionic liquid at these pressure levels. This approach
E-mail: jorgesaraiva@ua.pt; Fax: +351234370084; Tel: +351234401513
†
These authors contributed equally to the work.
6
32 | Green Chem., 2010, 12, 632–635
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can open interesting possibilities by conjugating the peculiar
solvent properties of ILs and the effect of HP on enzymatic
reactions.
Results and discussion
Effect of [bmim]Cl
In Fig. 1 it can be seen that in 10% [bmim]Cl the activity of
cellulase and yield of reducing sugars is lower compared to buffer
and the difference increases with the reaction time, reaching less
5
5% of the reducing sugars formed at 50 min in buffer. Turner
3
et al., also found that activity of cellulase was lower in 0.5 to 1%
[
bmim]Cl using cellulose azure as substrate. These results may
be due to the IL causing enzyme inactivation.
Fig. 2 Relative cellulase activity measured in 1% [bmim]Cl, as a
function of the previous residence time of the enzyme in a 10% of
[
bmim]Cl solution.
Switzerland). The results showed a linear dependence of a
w
with
2
[
bmim]Cl concentration (y = -0.0936x + 95.2, R = 0.958).
Cellulase activity shows a linear correlation with a
w
(Fig. 4,
2
y = 0.67x - 0.61; R = 0.960). a-Chymotripsin activity in
imidazolium-based ionic liquids was shown to be influenced by
27
the hydration level, in a way similar to that in organic solvents.
The effect of a on cellulase activity explains the results obtained
w
after transference of the enzyme from 10% to 1% [bmim]Cl.
Recovery of activity is due to the fact that the enzyme activity
in 1% [bmim]Cl is assayed in a higher a condition.
w
Fig. 1 Reducing sugars production by cellulase in buffer (ꢀ) and in
1
0% [bmim]Cl (ꢀ).
To test for possible inactivation of cellulase in [bmim]Cl,
experiments were run in which the enzyme was held in 10%
bmim]Cl for different periods of time, and afterwards an aliquot
[
was taken and added to a solution of 1% CMC in buffer to
quantify the activity. This procedure resulted in dilution of
[bmim]Cl to 1% in the reaction media.
Fig. 2 shows that there is a linear correlation between cellulase
activity and the previous residence time of the enzyme in 10%
[
[
bmim]Cl. For 20 min of residence of the enzyme in 10%
bmim]Cl, when activity is measured in 1% [bmim]Cl, there is
only 8% reduction of cellulase activity. This value is much lower
than the difference verified in Fig. 1, indicating that the decrease
of activity verified in 10% of [bmim]Cl is mostly reversible.
Similar results were obtained when horseradish peroxidase was
transferred from several ILs at different concentrations to buffer
Fig. 3 Relative cellulase activity vs. concentration of [bmim]Cl.
25
with 0.5-2% of the ILs.
Effect of high pressure
Cellulase activity decreases with increasing concentrations of
bmim]Cl, showing about 50% of the activity in buffer in 20%
bmim]Cl for 20 min reaction time (Fig. 3). The reduction of
[
[
To verify whether possible effects of high pressure on cellulase
activity were due to changes on CMC and not on the hydrolysis
reaction, CMC was subjected to pressure at 300 and 500 MPa
the thermodynamic water activity (a
w
) has been shown to be
◦
the determinant key parameter for the activity of enzymes in
at 30 C for 15 min. Cellulase activity was quantified at
26
organic solvents, by controlling the hydration level of enzymes.
Since [bmim]Cl is a salt and salts decrease the water activity, the
value of the different [bmim]Cl solutions was determined
atmospheric pressure (during 5, 10, 20 and 40 min) using
this high pressure processed CMC as substrate and no effects
were observed (results not shown). Also, to verify whether the
pressure treatments used could inactivate cellulase, the enzyme
a
w
using a Rotronic Hygrosckop DV-2 hygrometer (Bassersdorf,
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Table 1 Activation volumes (Va) obtained for activity of cellulase
under pressure in buffer and in 10% [bmim]Cl
3
-1
Pressure range (MPa)
Activation volume/cm mol
Buffer
2
0
2
5
1
2
.1–200
00–400
00–675
0% [bmim]Cl
00–600
6.17 (R = 0.990)
2
-6.33 (R = 0.991)
2
1.68 (R = 0.992)
2
1.58 (R = 0.915)
while the opposite is verified between 200–400 MPa (Va =
6.33 cm mol ). Between 500 and 675, the reaction is slightly
3
-1
-
3
-1
inhibited by pressure (Va = 1.68 cm mol ).
Cellulase activity under pressure in the presence of 10%
[
bmim]Cl (Fig. 5) was not affected at 600 MPa and was higher at
Fig. 4 Cellulase activity vs. a
w
values of the [bmim]Cl solutions used.
the other pressures studied (respectively, 1.7-, 1.3-, and 1.2-fold
at 100, 200, and 400 MPa). At 100 MPa with 10% [bmim]Cl
the cellulase activity reaches 85% the value in aqueous buffer at
atmospheric pressure.
◦
was processed at 400 and 600 MPa at 30 C for 15 min and no
change of activity was verified at atmospheric pressure (results
not shown).
Fig. 5 shows that cellulase activity under pressure first
decreases (from 50 to 200 MPa), then increases (from 200 to
The activation volume determined for cellulase activity with
3
-1
bmim[Cl], from 200 to 600 MPa was 1.58 cm mol , meaning
that is this pressure range the reaction is slightly inhibited by
pressure.
4
4
00 MPa), and then decreases again (from 400 to 675 MPa). At
00 MPa cellulase activity is not affected by pressure, while
for the other pressures it varies from 60–80% the value at
atmospheric pressure. These results show that cellulase is active
under pressure and so has the potential to be used in these
Overall, while in aqueous buffer the activity under pressure
varies from equal to lower the value at atmospheric pressure,
in 10% [bmim]Cl it varies from equal (at 600 MPa) to higher
(1.7-fold at 100 MPa). This opens good possibilities to improve
cellulase activity in ILs using high pressure, as well as other
enzymes. The reasons for such an effect of HP in cellulase activity
in presence of [bmim]Cl are not known and should be studied
in further work.
28
conditions. Murao et al. reported that while activity of several
◦
cellulases at 37 C was enhanced by pressure (in the range of
2
00 to 400 MPa), some other cellulases showed lower activity
under pressure. For cellulase from Aspergillus niger (the same
used in the present work) these authors only indicated that
the best result was obtained at 400 MPa (115% the activity
at atmospheric pressure). However, no activation volumes were
reported to mathematically describe the effect of pressure on
cellulase activity.
Conclusions
Cellulase activity in 5–20% [bmim]Cl solutions is lower (30
to 50%) compared to buffer and correlates linearly with the
decrease of the thermodynamic water activity (a
bmim]Cl. When cellulase is held in 10% [bmim]Cl and then
assayed in 1% [bmim]Cl, activity recovery occurs, the activity
being higher, this fact being explained by the higher a of 1%
bmim]Cl solution. Under pressure, cellulase activity in buffer
w
) caused by
[
w
[
varies from 60% to 100% (at 400 MPa) the value at atmospheric
pressure. In 10% [bmim]Cl cellulase activity under pressure
varies from equal to 1.7-fold higher (at 100 MPa) the value
at atmospheric pressure. These results open the possibility to
improve cellulase activity, and possibly of other enzymes in ILs,
by carrying out the reaction under pressure.
Materials and methods
Lyophilized cellulase (E.C. # 3.2.1.4) from Aspergillus niger was
purchased from Fluka (St. Louis, MO) and used dissolved in
Fig. 5 Effect of high pressure on cellulase activity in buffer (ꢀ) and in
at 400 and 600 MPa with IL, were repeated in a second run and these
results are the average of four replicates).
1
0% [bmim]Cl (ꢀ) (the experiments at 300 and 500 MPa without IL and
0
.1 M acetate buffer, pH 5.0. Sodium carboxymethylcellulose
(CMC) and [bmim]Cl were purchased from Sigma (St. Louis,
MO) and Fluka (St. Louis, MO), respectively. Sodium acetate
and acetic acid were purchased from Fluka (St. Louis, MO) and
used as 0.1 M acetate buffer, pH 5.0. All the other reagents were
of analytical grade.
The activation volumes obtained in this work are presented
in Table 1, showing that between 0.1 to 200 MPa the activity
3
-1
of the enzyme is inhibited by pressure (Va = 6.17 cm mol ),
6
34 | Green Chem., 2010, 12, 632–635
This journal is © The Royal Society of Chemistry 2010

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Quantification of cellulase activity
Where P is the pressure (MPa); v is the activity of the
-
1
-1
enzyme (mmol min mL ); Va is the activation volume
◦
Cellulase activity was determined at 30 C by the spectrophoto-
metric quantification of the reducing sugars produced, using the
3
-1
(
(
cm mol ); T is the absolute T/K, A is a constant, and R
3
-1
-1
-1
8.314 cm MPa mol K ) is the universal gas constant. The
29
3
,5-dinitrosalicylic acid (DNS) method, as glucose equivalents,
activation volumes were calculate by linear regression analysis.
2
by means of a standard curve of glucose (R = 0.996). Previous
experiments confirmed a linear relation between activity and
enzyme concentration and reaction time. A solution of 1% CMC
was used as a substrate, which was dissolved under continuous
stirring, in the acetate buffer solution at room temperature
during 45 min. To 450 mL of substrate solution, 35 mL of
buffer were added and the reaction started by the addition
of 15 mL of enzyme solution (2.5 mg mL ). After 20 min the
reaction was stopped by the addition of 1.0 mL DNS, followed
by boiling during 5 min, cooling, addition of 1.0 mL water
and measurement of absorbency at 540 nm using a Shimadzu,
UV/VIS 1240 spectrophotometer (Kyoto, Japan). In the case of
activity of cellulase in [bmim]Cl, the substrate solution consisted
of 1% cellulose dissolved in mixtures of buffer and 5–20%
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PVa
ln
(
V
)
= ln
(
A
)
−
(1)
RT
This journal is © The Royal Society of Chemistry 2010
Green Chem., 2010, 12, 632–635 | 635