Enhancing the activity of cellulase enzyme using ultrasonic irradiations

Article abstract of DOI:10.1016/j.molcatb.2014.01.002

The present work investigates the effect of low intensity ultrasonic irradiation on the cellulase activity. The effect on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model, Arrhenius equation, Eyring transition state theory, Michaelis-Menten equation, fluorescence spectroscopy and circular dichroism (CD) spectroscopy. It has been established that ultrasound had a positive effect on the activity of cellulase enzyme, though the selection of operating conditions played a crucial role in deciding the intensification. The maximum cellulase activity was observed at 17.33 W/cm² intensity and ultrasonic treatment time of 30 min, under which the enzyme activity was increased by about 25% over the untreated enzyme. After the ultrasonic treatment, thermodynamic parameters E_a, ΔH, ΔS and ΔG were reduced by 64.7%, 68%, 37.3% and 1.3%, respectively. In addition, fluorescence and CD spectra revealed that the ultrasonic treatment had increased the number of tryptophan on cellulase surface, and changed the molecular structure of cellulase enzyme favourably to provide more access to the active sites.

Full text of DOI:10.1016/j.molcatb.2014.01.002

Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enhancing the activity of cellulase enzyme using ultrasonic
irradiations
Preeti B. Subhedar, Parag R. Gogate∗
Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai 400019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The present work investigates the effect of low intensity ultrasonic irradiation on the cellulase activity.
The effect on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase
enzyme was evaluated with the help of the chemical reaction kinetics model, Arrhenius equation, Eyring
transition state theory, Michaelis–Menten equation, fluorescence spectroscopy and circular dichroism
(CD) spectroscopy. It has been established that ultrasound had a positive effect on the activity of cellulase
enzyme, though the selection of operating conditions played a crucial role in deciding the intensification.
The maximum cellulase activity was observed at 17.33 W/cm2 intensity and ultrasonic treatment time of
30 min, under which the enzyme activity was increased by about 25% over the untreated enzyme. After the
ultrasonic treatment, thermodynamic parameters Ea, ꢀH, ꢀS and ꢀG were reduced by 64.7%, 68%, 37.3%
and 1.3%, respectively. In addition, fluorescence and CD spectra revealed that the ultrasonic treatment
had increased the number of tryptophan on cellulase surface, and changed the molecular structure of
cellulase enzyme favourably to provide more access to the active sites.
Received 25 October 2013
Received in revised form 8 December 2013
Accepted 4 January 2014
Available online 10 January 2014
Keywords:
Ultrasound
Cellulase
Enzyme activity
Kinetics
Thermodynamics
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
treatment a potential option for the conversion of lignocellulosic
biomass under mild conditions using cellulase for the production
In recent times power ultrasound has been shown to have sig-
nificant application in food and biotechnology processes [1]. For
several years, ultrasound has been used as a method for enzyme
inactivation but recently it has been reported that ultrasound does
not inactivate all enzymes especially under mild conditions [2].
The ultrasound generated by periodic mechanical motion of an
ultrasonic probe transfers energy into the solution and causes
alterations in pressure leading to the creation of small rapidly
growing bubbles [3]. These bubbles are enlarged during the nega-
tive pressure cycle and after undergoing oscillations in size during
multiple acoustic cycles, finally collapse violently which gener-
ates high pressures, temperatures and shear forces. Even though
high ultrasonic intensity or extended sonication time can denature
enzymes, it has been shown that use of ultrasonic treatment at
appropriate frequencies and intensity levels can lead to enhanced
enzyme activity [4]. Ultrasound also results in favourable con-
formational changes in protein molecules without altering the
structural integrity of the enzymes [5].
of bioethanol. Cellulase (endo-1,4-␤-d-glucanase) refers to a group
of enzymes produced chiefly by fungi, bacteria, and protozoans
that catalyze the process of hydrolysis of cellulose. Cellulases are
widely used in various fields such as pulp and paper industry, tex-
tile industry, bioethanol industry, wine and brewery industry, food
processing industry, animal feed industry, agricultural industries,
etc. [6]. Cellulase breaks down cellulose into smaller polysaccha-
rides or completely into ␤-glucose units which can be further
fermented to bioethanol. Considering the commercial importance
of cellulase, it is essential to find an effective method to improve
the activity of cellulase.
In spite of the fact that ultrasound shows a potential to modify
the enzyme activity, very less publications have targeted to study
the effect of power ultrasound on the performance of enzymes.
Actually, majority of the papers published in this area evaluated the
effectiveness of the ultrasound-aided processes, without focussing
on the intensification of catalytic activity using ultrasound. Souza
et al. [7] carried out a comprehensive study and examined the activ-
ity of a commercial amylase enzyme after ultrasonic treatment in
an ultrasonic bath at 40 kHz. It has been reported that ultrasonic
treatment can promote enzyme activity. Glucose oxidase enzyme
activity under ultrasonic irradiation was evaluated by Guiseppi-
Elie et al. [8]. The results proved that the sonicated enzyme at
23 kHz showed a altered composition with reduced ␣-helix and
␤-sheet fractions upon extended sonication compared with the
The ability of ultrasound to increase the activity of enzymes and
to reduce the mass transfer resistances in the process makes this
∗
Corresponding author. Tel.: +91 22 33612024; fax: +91 22 33611020.
E-mail addresses: pr.gogate@ictmumbai.edu.in, paraggogate@yahoo.co.in
(P.R. Gogate).
1381-1177/$ – see front matter © 2014 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2014.01.002

P.B. Subhedar, P.R. Gogate / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
109
unsonicated enzyme. Along with the changes in the secondary
structure, small subsequent decrease in the enzymatic activity
was observed indicating that time and intensity are the important
parameters [8]. Effect of ultrasound on the activity of dextranase
has been investigated by Bashari et al. [9]. The highest activity of
dextranase was observed with ultrasound treatment at 25 kHz and
40 W for 15 min, under which the enzyme activity increased by
13.4% as compared to the untreated enzyme.
Enzymes are typically used at their optimal conditions, where
they demonstrate highest activity, thus achieving maximum reac-
tion rate. Therefore it is necessary to understand the influence
of the ultrasound on the effectiveness of the enzyme function-
ing under ideal conditions. Thus there is a necessity of more
research in this area to better understand the relationship of the
‘sonication–enzyme action’. This would facilitate the development
of effective processes in the field of sono-biotechnology. Nguyen
and Le [10] have indicated that ultrasound intensity of 12 W/ml
had a positive effect on the cellulase with 18% increase in its activ-
ity but, the mechanism of ultrasound action on cellulase has not
been reported. Therefore, the major focus of this work was to
investigate the effect of ultrasonic treatment on the activity of cel-
lulase enzyme. In order to explore the change in enzyme activity,
the effects of ultrasound on activity, thermodynamics as well as
molecular structure of cellulase were investigated with the help
of the Eyring transition state theory, Arrhenius equation, circular
dichroism (CD) spectroscopy and fluorescence spectroscopy. A sim-
ple kinetic model, based on Michaelis–Menten equation, has also
been introduced in order to investigate the variations in the kinetic
parameters.
Fig. 1. Experimental setup for ultrasonic treatment of cellulase enzyme.
540 nm with Chemito Spectroscan UV 2700 Double beam UV–Vis
spectrophotometer, which was used to calculate the concentration
of glucose released from CMC. 1 CMCU is the amount of enzyme,
which under standard conditions degrades CMC to reducing carbo-
hydrates with a reduction power corresponding to 1 ␮mol glucose
per minute.
2.3.2. Soluble protein estimation
The total soluble protein content was determined by Bradford
method, using bovine serum albumin as a standard [12]. To 0.25 ml
of protein sample, 2.5 ml of Bradford reagent was added and the
absorbance was immediately measured at 595 nm. For the calibra-
tion purpose, bovine serum albumin (BSA) was used.
2. Materials and methods
2.1. Materials
2.4. Effect of ultrasound treatment on cellulase kinetics and
thermodynamics
Cellulase enzyme was obtained as a gift sample from Advanced
Biotechnologies, Mumbai, India. The enzyme activity was 205,000
carboxymethyl cellulose unit per gram (CMCU/g). Carboxymethyl
cellulose, citric acid, 3,5-dinitrosalicylic acid (DNSA), phenol, NaOH,
and potassium sodium tartrate were procured from S.D. Fine Chem-
icals and all were of analytical grade. Bovine serum albumin was
obtained from Sigma Aldrich.
2.4.1. Ultrasonic treatment
For the determination of rate constants, 200 ml cellulase solu-
tion (1.0 g/l) was treated with ultrasound probe (20 kHz) at
17.33 W/cm2 under different temperature (20, 30, 40, 50 ◦C) for
30 min. After that, 1.0 ml of ultrasound treated enzyme solution
was added into the 3 ml of 0.5 M CMC solution. All the experiments
were carried out at pH 4.8 in citrate buffer. For the determination
of thermodynamic parameters, cellulase enzyme was treated with
ultrasound probe (20 kHz) at 17.33 W/cm2 for 30 min, and hydrol-
ysis experiments were conducted at temperature of 20, 30, 40 and
50 ◦C, respectively.
2.2. Ultrasonic treatment of cellulase
The device used for ultrasonic treatment of enzyme was a probe
sonicator obtained from Dakshin Ultrasonics, Mumbai. The ultra-
sonic irradiation at a frequency of 20 kHz was transferred through
a cylindrical horn. The experimental setup is shown in Fig. 1. Cellu-
lase powder was dissolved in citrate buffer of pH 4.8 and made up
to the final concentration of 1.0 g/l. 200 ml cellulase sample solu-
tion was put into the 250 ml beaker, and the beaker was placed in
a water bath maintained at temperature of 50 ◦C as it is the opti-
mum temperature for cellulase enzyme. The effect of ultrasound
at different ultrasonic time was investigated over the range 5 to
70 min, whereas the effect of ultrasound intensity was evaluated
over the range 2.88 to 23.10 W/cm2. The cellulase activities were
also investigated at different temperatures over the range of 20 to
50 ◦C, under ultrasonic treatment time of 30 min and 17.33 W/cm2
intensity.
2.4.2. Determination of kinetic parameters
The chemical kinetic model for cellulase enzyme used in the
current work was based on the first-order kinetics as depicted in
the following equation [13]:
C
ln
= −kt
(1)
C0
where C is the concentration of CMC at time t = t (␮g/ml), C0 is the
initial concentration of CMC, t is time, k is the total reaction rate
constant involving the rate constants of ultrasonic action kus and
intrinsic activity kc of cellulase (Eq. (3)). As it is difficult to measure
the decrease in the CMC concentration, the reaction rate can be
reflected by the increase in the amount of glucose released by CMC
as follows [14]
2.3. Analysis
ln V − V = −kt + ln V
(2)
where Vt is the concentration of glucose at time t = t (␮g/ml), V
(
)
∞
t
∞
2.3.1. Assay of cellulase activity
The activity of cellulase enzyme was determined using DNSA
method described by Miller [11]. 0.5 M CMC was used as the sub-
strate for hydrolysis by cellulase. The absorbance was measured at
∞
is the ultimate concentration of glucose (␮g/ml), which is obtained
from the hydrolysis experiment conducted under pH 4.8 at 50 ◦C

110
P.B. Subhedar, P.R. Gogate / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
for 10 h. The k value can be determined experimentally from the
slope by plotting ln V − V against t.
a)
(
)
t
∞
300
250
200
150
100
50
Compared with conventional enzymatic hydrolysis, the ultra-
sonic treated enzymatic hydrolysis reaction can be influenced by
the ultrasound effect in addition to the thermal effect. Therefore,
the k in ultrasonic treated enzymatic hydrolysis was expressed as:
k = kc + kus
(3)
where kc is the reaction rate constant induced by thermal effect in
conventional enzymatic hydrolysis, and kus is the reaction rate con-
stant induced by ultrasound effect in ultrasonic treated enzymatic
hydrolysis.
0
10
20
30
40
50
60
70
Time (min)
2.4.3. Determination of thermodynamic parameters
The activation energy can be calculated from Arrhenius equation
which is expressed as
b)
2
1.8
1.6
1.4
1.2
/RT
k = Ae−E
(4)
a
where A is pre-exponential factor, Ea is the activation energy (J/mol)
and R is the universal gas constant (8.314 J/mol K).
In the present study, Eyring transition state theory was used to
obtain the thermodynamic parameters.
ꢀ
ꢁ
ꢀ
ꢁ
kBT
h
−ꢁG
kBT
h
−ꢁH
ꢀS
R
k =
exp
=
exp
+
(5)
RT
RT
where T is the absolute temperature in K, kB is Boltzman constant
(1.38 × 10−23 J/K), h is Planck constant (6.6256 × 10−34 J/s). ꢀG, ꢀH
and ꢀS are the parameters of changes in free energy, enthalpy, and
entropy for the process.
0
10
20
30
40
50
60
Time (min)
Fig. 2. (a) Effect of ultrasonic treatment on cellulase activity. (b) Effect of ultrasonic
treatment on protein concentration.
2.4.4. Determination of the kinetic parameters of the
Michaelis–Menten equation
3. Results and discussion
Michaelis–Menten constant K and maximum rate of reaction
(
)
m
V
values of untreated and ultrasound treated cellulases were
)
(
max
3.1. Effect of ultrasonic irradiation on activity of cellulase
determined by measuring the enzyme activities at various concen-
trations of CMC. Km and Vmax values were calculated according to
Lineweaver–Burk plots.
The effect of ultrasonic treatment on cellulase activity has been
shown in Fig. 2(a). The graph shows that an increase in ultra-
sonic treatment time significantly increased the cellulase activity
till 30 min of irradiation time. When the treatment period was fur-
ther increased to 50 min, cellulase activity was lower than that of
control i.e. untreated enzyme. By using ultrasound, 23.4% increase
in enzyme activity of cellulase was obtained at optimum treatment
time of 30 min. Fig. 2(b) shows the effect of ultrasonic treatment
on the protein content of cellulase enzyme. From Fig. 2(b), it can
be clearly seen that there was no significant difference on the pro-
tein content after ultrasonic treatment. This result confirms that the
change in enzyme activity was not due to the change of protein con-
tent in the preparation, including both enzyme and non-enzyme
proteins.
Application of ultrasound generates the conditions of fluctua-
tions in local velocity and pressure in adjacent fluid resulting in
varied turbulence [16,17]. It can be stated that when ultrasonic
treatment was conducted for an optimum time, most of enzyme
molecules in the solution undergoes conformational change; which
results in the enhancement in the cellulase activity [18]. Also
the mass transfer resistances will be eliminated giving favourable
results for the activity. It is important to note here that further
increase in treatment time caused harmful effects, as continuous
exposure to cavitating conditions for prolonged time led to degra-
dation of the amino acid residues which contributes to the substrate
binding domain or catalytic domain of the enzyme molecules
resulting in decrease in enzyme stability [19]. Ertugay et al. [20]
also reported that increased ultrasonic treatment time caused an
2.5. Effect of ultrasonic treatment on the structure of cellulase
enzyme
2.5.1. Intrinsic fluorescence analysis
Two samples were prepared to measure the intrinsic fluo-
rescence. First sample was cellulase enzyme without ultrasonic
treatment whereas the second sample was cellulase enzyme
treated under optimized conditions of ultrasound intensity of
17.33 W/cm2 for 30 min of irradiation time. Intrinsic fluorescence
spectra of untreated (control) and ultrasound-treated sample in
water were measured at room temperature (25 1 ◦C) with fluo-
rescence spectrophotometer (Jasco FP-6500 Spectrofluorometer) at
280 nm excitation wavelength (slit = 5 nm), 300–500 nm emission
wavelength (slit = 5 nm) and 1200 nm/s of scanning speed.
2.5.2. Circular dichroism (CD)
CD spectra of untreated and ultrasound treated cellulase were
recorded with a spectropolarimeter (Model MOS-450), using a
quartz cuvette of 1 mm optical path length at room temperature
(25 1 ◦C). CD spectra were scanned in the far UV range (190 to
250 nm) with three replicates at 30 nm/min with 0.1 nm as band-
width. The CD data were expressed in terms of mean residue
ellipticity, [ꢂ], in deg cm2/dmol. The secondary structures of cellu-
lase enzyme in the presence and absence of ultrasound irradiation
were analyzed using an online server DICHROWEB [15].

P.B. Subhedar, P.R. Gogate / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
111
300
250
200
150
100
50
a)
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
0
5
10
15
20
25
Time (min)
30 ºC
Ultrasonic intensity (W/cm2)
20 ºC
40 ºC
50 ºC
b)
Fig. 3. Effect of ultrasonic intensity on cellulase activity.
3
2.5
2
inactivation effect, and resulted in a decrease in catalytic activity
of lactoperoxidase in milk.
3.2. Effect of ultrasonic intensity on cellulase activity
1.5
1
Ultrasonic intensity is one of the important parameter which
affects the catalytic activity of cellulase. The cellulase activity
with varying ultrasonic intensity for optimum treatment time as
30 min has been shown in Fig. 3. The results demonstrated that,
maximum activity of cellulase was observed for ultrasonic inten-
sity of 17.33 W/cm2. Cellulase activity increased by 23.4% over
the untreated enzyme at 17.33 W/cm2 intensity. When ultrasonic
intensity exceeded 17.33 W/cm2, the activity of cellulase decreased
gradually with an increase in the ultrasonic intensity. Cavitation
phenomenon occurring during ultrasonication was responsible for
the observed behaviour of change in cellulase activity with varying
intensity. Ultrasound can rupture the weak linkages like hydro-
gen bonds or Van der Waals interactions and bring conformational
changes in protein structure [21]. Mild intensity and low fre-
quency ultrasound irradiation in liquids causes the stable cavitation
[22]. The forces induced by oscillation of stable cavitation bubbles
changes the spatial conformation of enzyme, and thus enhances the
activity of enzyme [23]. Szabo and Csiszar [24] observed 25% loss
in the activity of cellulase at ultrasound intensity of 43.4 W/cm2
whereas in the present study, cellulase activity was increased by
23.5% at ultrasound intensity of 17.33 W/cm2. Thus it can be said
that optimisation of the operating intensity is necessary so that
beneficial effects are obtained for each of the enzymes used in
sonicated system.
On the other hand, when ultrasound intensity exceeded
17.33 W/cm2, decrease in cellulase activity was observed. The prob-
able explanation for this is, high intensity ultrasound enhanced
cavitation effects that caused significant shear in the liquid
medium. Ultrasound irradiation, under these extreme conditions,
could cause great damage to polypeptide chains, leading to inacti-
vation of enzyme [25]. Additionally, extreme increase in localized
pressure and temperature at higher intensity also leads to the gen-
eration of free hydroxyl and hydrogen radicals. These free radicals
react with the enzyme causing its inactivation [26].
0.5
0
0
5
10
15
20
25
30
35
Time (min)
20 ºC 30 ºC 40 ºC 50 ºC
Fig. 4. (a) Relationship between ln V − V and reaction time for untreated cellu-
(
)
∞
t
lase. (b) Relationship between ln V − V and reaction time for ultrasound treated
(
)
∞
t
cellulase.
of hydrolysis of CMC to produce glucose. So in order to understand
the effect of ultrasound irradiation on the rate of hydrolysis of CMC,
experimental data have been fitted in the kinetic model. The ulti-
mate hydrolysis extent in the absence of ultrasound was observed
to be 31.8% of CMC.
Fig. 4(a) represents the plots of ln V − V versus time for the
(
)
∞
t
untreated cellulase whereas Fig. 4(b) shows the plots of ln V − V
(
)
∞
t
versus time for the ultrasound treated cellulase at 17.33 W/cm2 for
30 min. It can be seen from Fig. 4(a) and (b) that, hydrolysis of CMC
by untreated as well as ultrasound treated cellulase obeyed the first
order kinetics. The rate constants k, kc and kus at different temper-
atures have been summarized in Table 1. It can be seen that rate
constant k increased with increase in temperature from 20 to 50 ◦C.
This can be attributed to the increase in collision frequency between
the CMC i.e. substrate and cellulase enzyme at higher temperature
[27].
3.4. Effect of ultrasound on the thermodynamic parameters
Activation energy is the minimum amount of energy required
to convert a normal stable molecule into a reactive molecule and
Table 1
3.3. Effect of ultrasound on kinetic rate constants
Reaction rate constants of untreated and ultrasound treated cellulase.
Temperature (K)
kc (min−1
)
kus (min−1
)
k (min−1
)
In chemical kinetics, value of rate constant is an important
parameter which is independent of the substrate concentration,
and is mainly dependent on the temperature of reaction, medium
and catalyst. The change in the cellulase activity occurred due to
ultrasonic treatment will result in the change in the rate constants
293
303
313
323
0.0112
0.0212
0.0453
0.083
0.0432
0.0563
0.0687
0.0897
0.0544
0.0775
0.114
0.1727

112
P.B. Subhedar, P.R. Gogate / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
-1.5
-2
-2.5
-3
-3.5
-4
-4.5
-5
3.4
3.5
3.3
3
3.1
3.2
1/T (K-1)
Untreated
Ultrasound treated
Fig. 7. Plots of the V0 values obtained as
a
function of the
S
[ ]
using
Fig. 5. Relationship between lnk and 1/T.
Lineweaver–linearization.
-7
to alteration in the protein structure. ꢀS represent the variation
in the extent of local disordering between transition state and the
ground state. The 37.3% decrease in the ꢀS can be attributed to
the oxidative modification of amino acid residues and initiation
of cross-linking and aggregation which leads to the increase in
enzyme activity [14]. Qu et al. [29] have also reported the sim-
ilar results for change in thermodynamic parameters of alcalase
enzyme after ultrasonic irradiation, where ꢀH, ꢀS and ꢀG were
reduced by 74.1%, 34.3% and 1.4%, respectively. These values are in
well agreement with the values obtained in the present work.
-8
-9
-10
-11
-12
3.05
3.10
3.15
3.20
3.25
1/T (K -1
3.30
3.35
3.40
3.45
3.5. Effect of ultrasonic treatment on the kinetic parameters
)
By using a linear transformation of the Michaelis–Menten equa-
tion, the Lineweaver–Burk plot was made where the reciprocal of
Untreated
Ultrasound treated
ꢂ
ꢃ
the initial reaction rate
V was plotted against the reciprocal of
)
0
(
Fig. 6. Relationship between ln(k/T) and 1/T
.
the substrate concentration
S as shown in Fig. 7. From Fig. 7,
[ ]
maximum rate of reaction, when the enzyme was saturated with
substrate and the Michaelis constant were obtained.
V
max
K
)
m
(
)
(
it reflects the rapidity of chemical reactions [28]. Arrhenius plots
of ln k against reciprocal of absolute temperature (K−1) have been
shown in Fig. 5. Activation energy was determined from the slope
of the Arrhenius plot. For the untreated and the ultrasound treated
cellulase, the activation energy values were 53.3 and 18.8 kJ/mol,
respectively, which shows that ultrasound decreases the energy
barrier necessary for reaction remarkably. Ultrasonic treatment
of cellulase reduced the activation energy by 65% than that of
untreated cellulase. The greatly reduced Ea value by ultrasonic
treatment indicates that the hydrolysis reaction catalyzed by cel-
lulase could occur very easily.
From the Lineweaver–Burk plot, the value of Vmax can be obtained
from the intercept and the value of Km/Vmax from the slope.
Results of the kinetic experiments have been summarized in
Table 3. Studies of the ultrasonic effects on cellulase kinetics were
carried out under the optimal ultrasonic conditions and compared
to the control without the use of ultrasound. As can be seen in
Table 3, the result showed that, Vmax of cellulase increased whereas
Km decreased for the ultrasound-treated enzyme compared to the
untreated enzyme. Vmax reflect the limiting rate of the enzymatic
reaction at substrate saturation, whereas Km value indicates the
affinity between the enzyme and the substrate. An increase in
Vmax indicates that a considerable movement of reactants to the
active site of the enzyme and the reaction products to the medium
were achieved under an ultrasonic field [23]. On the other hand,
decrease in Km under ultrasonic irradiation might be attributed to
intense pressure, shear force and temperature which were gen-
erated as a result of the ultrasound cavitation. The exposure of
active centre would make cellulase combine with the substrate
easily and display better reaction ability [30]. Sulaiman et al. [31]
studied the ultrasound assisted enzymatic hydrolysis of CMC. The
ꢂ
ꢃ
Eyring plots of ln k/T versus reciprocal absolute temperature,
1/T has been shown in Fig. 6. The plot resulted in good linear fits
and ꢀH was estimated from slopes of the curves and ꢀS calculated
from the intercepts. The values of ꢀH, ꢀS, and ꢀG obtained from
the data have been given in Table 2. It can be seen that ꢀG decreased
by 1.3% after ultrasonic treatment, which represents the increase
in cellulase activity. After ultrasonic treatment, ꢀH decreased by
68%, attributed possibly to the ultrasonically induced rupture of
hydrogen bonds stabilizing the enzyme at ground state and the
distraction of the internal hydrophobic core, both of which leads
Table 2
Table 3
Thermodynamic parameters of untreated and ultrasound treated cellulase.
Kinetics parameters of untreated and ultrasound treated cellulase.
Treatment
Ea (kJ/mol)
ꢀH (kJ/mol)
ꢀS (kJ/mol)
ꢀG (kJ/mol)
Treatment
Km (␮M)
Vmax (␮M/s)
Untreated
Ultrasound treated
53.23
18.8
50.67
16.24
−109.37
−142.72
84.90
83.67
Untreated
Ultrasound treated
9.73
8.31
3.62
4.76

P.B. Subhedar, P.R. Gogate / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
113
Table 4
Contents of secondary structure of untreated and ultrasound treated cellulase.
Treatment
␣-Helix (%)
␤-Sheet (%)
␤-Turn (%)
Random coil (%)
Enzyme activity (U/ml)
Untreated
Ultrasound treated
25.93
22.72
26.31
24.58
20.94
22.86
24.97
32.36
205.07
252.87
optimum ultrasonic intensity for enhancing the hydrolysis of CMC
was found to be 11.8 W/cm2. Sonication at the power intensity of
11.8 W/cm2 enhanced the Vmax by 84% compared to untreated sam-
ple and reduced the value of Km by 41% compared to control sample.
These results are in well agreement with the present study where
17.33 W/cm2 was found to be the optimum power intensity for the
hydrolysis of CMC with an increase in Vmax by 34% and decrease in
Km by 17% as compared to that of the untreated sample.
a)
1000
900
800
700
600
500
400
300
200
100
0
3.6. Effect of ultrasonic treatment on the structure of cellulase
In general, enzymes are monomeric globular proteins whose
catalytic activity depends on the native configuration of their active
sites. The irradiation with ultrasound might cause the changes in
the secondary structure leading to better exposure of active sites of
enzyme. Thus in order to get a better understanding about the effect
of ultrasound irradiation on the activity of cellulase and the sec-
ondary structure and tertiary structure, the conformation changes
in cellulase were evaluated by measuring the intrinsic fluorescence
and CD spectra in the presence and absence of ultrasound.
300
320
340
360
380
400
Wavelength (nm)
Untreated
Ultrasound treated
b)
0
Fluorescence spectroscopy is a useful method to analyze the
structure alteration in proteins since the intrinsic fluorescence
of aromatic amino acid residues is susceptible to the polarity of
microenvironments along the transition [32]. The intrinsic fluores-
cence of enzyme is due to their amino acid groups; and mainly
attributed to Trp, Tyr and Phe residues, particularly to Trp residue
[33]. In the present work, changes in the enzyme conformation
were investigated by Trp fluorescence spectrum (the maximum
fluorescence emission wavelength is 348 nm). It can be seen from
Fig. 8(a) that, the fluorescence intensity of the ultrasound treated
cellulase decreased compared to that of untreated cellulase, which
clearly confirmed that, the ultrasonic treatment increased the
number of tryptophan on cellulase surface [34]. Additionally, the
optimum fluorescence emission wavelength did not show a red or
blue shift. Therefore, results confirmed that, ultrasound irradiation
induced molecular unfolding of protein, destroyed hydrophobic
interactions of protein molecules, caused more groups and regions
inside the molecules to expose and therefore, decreased the fluo-
rescence of cellulase [35].
-10
-20
-30
-40
190
200
210
220
230
240
250
Wavelength (nm)
Untreated
Ultrasound treated
Fig. 8. (a) Intrinsic fluorescence spectra of untreated and ultrasound treated cellu-
lase. (b) CD spectra of untreated and ultrasound treated cellulase.
CD is being increasingly being considered as a helpful tech-
nique for investigating the structure of proteins in the solution.
This technique has been established to be easy and consistent for
rapid evaluation of protein structure and monitoring conforma-
tional changes [36]. The results of the analysis of the CD spectra of
untreated and ultrasound treated cellulase are shown in Fig. 8(b)
and are summarized in Table 4. The content of ␣-helix, ␤-sheet
and random coil of cellulase was calculated to understand the
connection between enzyme activity and secondary structure. As
can be seen in Table 4, the fraction of ␣-helix increased due to
the ultrasound irradiation. Such an increase in the ␣-helical frac-
tions in cellulase as a result of the ultrasonic treatment could be
attributed to the pressure alterations and turbulence [37] and the
induced structural transformations, which may perhaps affect the
active site of the enzyme. Under optimum ultrasound condition, the
contents of ␣-helix decreased by 12.4%, and random coil increased
by 29.6%, respectively compared with that of untreated enzyme.
These changes made cellulase show more uniformity and flexibil-
ity, which are useful for the enhancement of their activity and hence
in the catalytic efficiency. Wang et al. [38] have also reported sim-
ilar results for change in composition in cellulase structure with
8.85% decrease in ␣-helix content and 29.5% increase in random
coil. These results are in well agreement with the results obtained
in the present work.
4. Conclusions
The present work has clearly established that, low intensity
ultrasound has a positive effect on cellulase activity. The maxi-
mum activity of cellulase was observed when the enzyme was
treated with ultrasound at 17.33 W/cm2 intensity for 30 min,
under which the enzyme activity increased by ∼25%compared
to the untreated sample. Significant reduction in thermodynam-
ics parameters was observed after ultrasonic irradiation. Ea, ꢀH,
ꢀS and ꢀG were reduced by 64.7%, 68%, 37.3% and 1.3%, respec-
tively. Results showed that, hydrolysis reaction under ultrasound
irradiation obeyed Michaelis–Menten kinetics. Under the optimal

114
P.B. Subhedar, P.R. Gogate / Journal of Molecular Catalysis B: Enzymatic 101 (2014) 108–114
ultrasound conditions, the values of Vmax and Km were higher com-
pared to untreated enzyme. Fluorescence spectra reflected that the
ultrasonic treatment had increased the number of tryptophan on
cellulase surface. The molecular structure of cellulase as established
using CD spectra showed favourable changes in terms of composi-
tion of ␣-helix, ␤-sheets and random coil.
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