World J Microbiol Biotechnol (2017) 33:170
DOI 10.1007/s11274-017-2322-6
REVIEW
Ultrasound assisted intensification of enzyme activity and its
properties: a mini-review
Shamraja S. Nadar1 · Virendra K. Rathod1   
Received: 14 March 2017 / Accepted: 15 July 2017
© Springer Science+Business Media B.V. 2017
Abstract  Over the last decade, ultrasound technique has
emerged as the potential technology which shows large
applications in food and biotechnology processes. Earlier,
ultrasound has been employed as a method of enzyme inac-
tivation but recently, it has been found that ultrasound does
not inactivate all enzymes, particularly, under mild condi-
tions. It has been shown that the use of ultrasonic treat-
ment at appropriate frequencies and intensity levels can
lead to enhanced enzyme activity due to favourable con-
formational changes in protein molecules without altering
its structural integrity. The present review article gives an
overview of influence of ultrasound irradiation parameters
(intensity, duty cycle and frequency) and enzyme related
factors (enzyme concentration, temperature and pH) on the
catalytic activity of enzyme during ultrasound treatment.
Also, it includes the effect of ultrasound on thermal kinetic
parameters and Michaelis–Menten kinetic parameters (­ km
and ­Vmax) of enzymes. Further, in this review, the physical
and chemical effects of ultrasound on enzyme have been
correlated with thermodynamic parameters (enthalpy and
entropy). Various techniques used for investigating the con-
formation changes in enzyme after sonication have been
highlighted. At the end, different techniques of immobiliza-
tion for ultrasound treated enzyme have been summarized.
Keywords  Ultrasound · Enzyme · Enhanced activity ·
Thermodynamics · Structural conformation · Catalytic
performance
* Virendra K. Rathod
vk.rathod@ictmumbai.edu.in
1
Department of Chemical Engineering, Institute of Chemical
Technology, Matunga (E), Mumbai 400019, India
Introduction
The rapid progress in the field of biotechnology constantly
attracts new methods and solutions for further development
of bioprocess performances. Amongst many techniques,
ultrasound has received remarkable attention over the last
decade as one of the emerging technologies in various fields,
especially in food, biotechnology and biopharmaceutical
industries (Rokhina et al. 2009). Based on the principles of
green chemistry and engineering, it provides entirely novel
opportunities for sustainable production of existing and new
products and services. Ultrasound refers to sound waves that
exceed the hearing frequency limit of the human ear ranging
from 10 to 60 kHz. In chemical reactions, the effect of ultra-
sound is principally based on cavitation phenomenon (i.e.
the formation, growth and implosive collapse of cavities in
liquids that release large amounts of highly localized energy)
which results into induced chemical or physical changes
along with the formation of local turbulences and liquid
micro-circulations (acoustic streaming) that promote the
transport processes and eliminate mass transfer resistance
in heterogeneous systems (Gogate and Kabadi 2009; Gon-
calves et al. 2015; Rao and Rathod 2015). In the last decade,
ultrasound has been extensively used in enzyme catalysed
biotransformation, aiming to intensify the reaction processes
with higher yield of product in short time. Also, it improves
the mass transfer rates leading to better catalyst effective-
ness. Moreover, the use of ultrasound assisted synthesis
shows significant benefits such as increased selectivity, use
of less hazardous solvents, lower energy consumption, better
utilization of the raw materials and the catalyst, which make
it a sustainable process (Gogate and Kabadi 2009; Subhedar
and Gogate 2013; Delgado-Povedano and Luque de Castro
2015; Sancheti and Gogate 2017).
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Earlier, food technologists have used high frequency and
high power ultrasound (mostly above 30 kHz) for inacti-
vation of many enzymes to prevent fruits and vegetables
from various undesirable effects such as browning, loss of
nutritive value and off-flavour. An enzyme inactivation by
ultrasound is mainly due to collapse of bubbles produc-
ing extreme local increase in pressure and temperature
(O’Donnell et  al. 2010; Islam et  al. 2014). In addition,
ultrasound makes stable cavitating bubbles vibrate, creating
shock waves that trigger strong shear and micro-streaming in
the adjacent liquid. Under these extreme conditions, it could
breakdown active conformation of enzymes by rupturing
hydrogen bonding and van der Waals’ interactions in the
polypeptide chains (Huang et al. 2017). Moreover, sonoly-
sis of water leads to generation of high energy and reactive
intermediates such as hydroxyl and hydrogen-free radicals
which could damage protein usually resulting into loss of
biological activity of the enzyme (Ercan and Soysal 2011;
Baltacıoğlu et al. 2017).
Recently, various researchers have demonstrated the
potential of low-frequency ultrasound to modify physico-
chemical properties of protein without affecting its struc-
tural integrity. The periodic pressure fluctuation caused by
ultrasound wave can alter 3D structure of enzyme by per-
turbing loop and domain regions, and consequently affect
its activity (Fig. 1) (Duan et al. 2011). A number of reports
are available on ultrasound assisted enzyme catalysed bio-
transformation processes carried out in textiles, drug and
pharma, oleo-chemicals and detergents, food and perfum-
ery, cosmetics industries and also in organic enzyme cata-
lysed synthesis (Khan and Rathod 2015; Banerjee 2017;
Bansode and Rathod 2017; Nadar et al. 2017). However,
during these processes, effectiveness of the ultrasound on
overall synthesis has been considered, without focussing
on the catalytic behaviour of enzymes under the influence
of ultrasound. The changes in enzyme activity under ultra-
sonication is mainly dependent on the sonication system
parameters as well as the characteristics of the enzyme
(Kentish and Ashokkumar 2011; Goncalves et al. 2015).
Fig. 1  The schematic represen-
tation of effect of ultrasound on
enzyme structure
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World J Microbiol Biotechnol (2017) 33:170
This review article is mainly aimed to provide adequate
information on the ultrasound–enzyme working conditions
under lower intensity. Also, it briefly summarizes the mul-
tiple parameters associated with ultrasound and properties
of enzyme which influence the overall enzyme catalytic
performance (Fig. 2). Further, the physical and chemical
effects of ultrasound on enzyme have been correlated with
thermodynamic parameters (enthalpy and entropy). At the
end, several immobilization strategies will be discussed in
order to stabilize ultrasound treated enzyme.
Parameters affecting enzyme activity
Enzymes are sensitive to ultrasound irradiation being
activated or inactivated (Fig. 1). An enzyme’s tolerance
towards ultrasound might depend on the physiological
properties of enzyme as well as the operational param-
eters of ultrasound, such as power/intensity and ultrasound
frequency, which directly affect enzyme catalytic activity.
So, it is necessary to optimize those parameters related to
sonicated system for each of different enzymes so as to get
highly active conformation of enzyme (Delgado-Povedano
and Luque de Castro 2015; Huang et al. 2017). Figure 2
shows systematic flow for the ultrasonic assisted activation
of enzyme and characterization. There are two modes of
transmitting ultrasound wave: direct mode (i.e. ultrasound
horn) and indirect mode (i.e. ultrasound bath). However, in
the available literature, there are no reports which present
a comparative investigation of effect of the different types
of ultrasonic devices (horn and bath) on enzyme (Bansode
and Rathod 2017). Mostly, ultrasound has been employed
in terms of horn to treat the enzymes. Various enzymes
treated under ultrasound to intensify their activity are sum-
marized in Table 1 and the effect of different ultrasonic
parameters on enzyme activity is briefly discussed in fol-
lowing sections.
World J Microbiol Biotechnol (2017) 33:170 Page 3 of 12  170
Fig. 2  Parameters involved in studies of ultrasound effect on enzymes
Ultrasound frequency
The cavitational effect of ultrasound depends on the fre-
quency of irradiation. When enzyme is irradiated by opti-
mum ultrasound frequency, enzyme undergoes favourable
conformational change; which results in the enhancement
of enzyme activity (Huang et al. 2017). As the ultrasonic
power is entirely dependent on the operating frequency, it
becomes necessary to optimise the working power input for
getting positive effect on enzyme catalysed reaction. The
effect of ultrasound frequency on enzyme activity was stud-
ied by Wang et al. (2011). The catalytic activity of cellulase
increased by 6.56, 14.79, 17.85 and 10.45% under the dif-
ferent frequencies of 18, 20, 24 and 26 kHz, respectively.
The positive effect was due to the ability of ultrasound to
increase the surface area of enzyme molecules. The ultra-
sound energy absorbed by enzyme molecules varied with the
different ultrasound wavelengths, and also, it affects stability
of enzyme which ultimately resulted in change of the cata-
lytic activity. However, at higher ultrasound frequency i.e. at
29 kHz, the cellulase activity was decreased by 1.02% than
that of untreated cellulase due to generation of excessive
heat caused by violent collapse of large number of bubbles in
reaction medium. This has been resulted in the inhibition of
the catalytic functions and denaturation the enzyme. (Wang
et al. 2012). The ultrasound effect also depends on substrate
as well the biochemical reactions. Some substrates such as
cellulose are not water soluble and can exist in a crystalline
structure that is relatively unavailable to cellulase enzymes.
Sulaiman et al. (2013) carried out ultrasound mediated enzy-
matic hydrolysis of cellulose and carboxymethyl cellulose
(CMC). They found that at optimized sonication conditions,
improved rate of hydrolysis of CMC was achieved relative
to the control. Further, the alteration of substrate has been
reported to make it more accessible to enzyme during ultra-
sound treatment. The improved catalytic ability of enzyme
is also attributed to multiple effects such as increased mass
transfer of the substrate (Sulaiman et al. 2013).
In another study, the effect of ultrasound frequency on
alliinase activity (derived from fresh garlic) was explored
13
 Table 1  Activation of enzymes by ultrasound
170  

Sr. No. Enzyme Source Ultrasound parameters Remark Reference

13
Fre- Power/intensity Duty cycle
quency
(kHz)
Page 4 of 12

1 α-Amylase – 40 132 W – The activation energy of both enzymes in the presence of ultrasound was (Leaes et al. 2013)
2 Amyloglucosidase – considerably reduced and improved the enzyme activity

3 α-Amylase Bacillus licheniformis 40 278.8 W – Ultrasound irradiation inhibited the activity of α-amylase and papain, while (Yu et al. 2014)
4 Papain Porcine gastric mucosa the activity of pepsin was enhanced which was analysed by CD, FT-IR
and fluorescence spectrometer
5 Pepsin –
6 Alcalase – 20 80 W 50% Enzyme activity was increased by 5.8% over the control. After the treat- (Ma et al. 2011)
(Total cycle 4s) ment, thermodynamics parameters ­Ea, ΔH, ΔS and ΔG were reduced by
70.0, 75.8, 34.0 and 1.3%, respectively
7 α-Amylase T. reesei 40 132 W – The effect of temperature was less pronounced in the presence of ultra- (Souza et al. 2013)
sound, resulting in a decreasing of about 80% in the activation energy in
comparison with in the absence of ultrasound
8 Cellulase – 24 15 W – The activity was increased by 18.17% over the control. Fluorescence and (Wang et al. 2012)
CD spectra revealed that number of tryptophan on cellulase surface
slightly increased and number of α-helix and random coil in cellulase
increased after ultrasound treatment
9 Cellulase – 20 17.33 W/cm2 – After the ultrasonic treatment, the enzyme activity was increased by about (Subhedar and Gogate 2014)
25% over the untreated enzyme while thermodynamic parameters ­Ea,
ΔH, ΔS and ΔG were reduced
10 Dextranase Chaetomiumerraticum 25 100 W – The enzyme activity increased by 13.43% compared the routine thermal (Bashari et al. 2013a)
incubation at 50 °C. The V
­ max and ­Km values of dextranase increased
with ultrasound-treated compared native
11 CyclodextrinGlu- Bacillus sp. SK13.002 25 30 W – The activity of CGTase was significantly increased by 22% compared to (Eibaid et al. 2014)
canotransferase the conventional
(CGTase)
12 Glucose oxidase A. niger 23 – – The enhanced activity was studied by UV/vis and CD spectroscopy, TGA (Guiseppi-Elie et al. 2009)
(GOx) and compared with untreated GOx. The CD spectra of ultrasonicated
GOx showed a different composition with reduced α-helix and β-sheet
fractions upon extended sonication
13 Lipase – 20 12.22 W/cm2 80% The maximum increase in lipase activity was two-fold which was opti- (Jadhav and Gogate 2014)
(Total cycle 25s) mized by the response surface methodology approach
14 Lipase Thermomyces 22 11.38 W/cm2 50% The low-intensity ultrasound treatment has a positive effect on both lipase (Nadar and Rathod 2016a)
lanuginosus (Total cycle 12s) activity. Nearly 39% enhanced activity of T. lanuginosus (TL) lipase
15 Candida antarctica 15.48 W/cm2 66.67% whereas 62% enhancement in CALB lipase was observed after ultrasonic
(Total cycle 12s) treatment

16 Polygalacturonase Aspergillusniger 22 4.5 W/mL – Ultrasound treatment could promote the stability of polygalacturonase at (Ma et al. 2015)
the tested temperature range of 20–60 °C. Also, The half-life value and
D-value were higher than that of the untreated enzyme at 40 and 50 °C
17 Tyrosinase Mushroom 40 100 W – The activity of tyrosinase was enhanced under ultrasound treatment. (Yu et al. 2013)
World J Microbiol Biotechnol (2017) 33:170

Atomic force microscopy image showed that the large molecular groups
of tyrosinase were broken into small after ultrasound irradiation
18 Alliinase Fresh garlic 40 0.5 W/cm2 – The low frequency ultrasound increased alliinase activity about 47.1%. (Wang et al. 2011)
Under ultrasound, the alliinase activity was inhibited by ­K+ ion and
enhanced by ­Fe2+ ion
World J Microbiol Biotechnol (2017) 33:170 Page 5 of 12  170
by exposing enzyme to different frequencies ranging from
28 to 100 kHz at power 0.5W/cm2, which displayed different
positive effects on the alliinase activity. Under the optimal
ultrasonic conditions, ultrasound irradiation accelerated the
enzyme activity by 47.1% in comparison with the control
(Wang et al. 2011). So, ultrasound irradiation with desir-
able frequency is one of the vital parameters while treating
enzyme to bring positive changes.
Ultrasonic intensity/power
Ultrasonic intensity is one of the important parameters for
amending the catalytic activity of enzyme. The ultrasound
intensity depends on the maximum power input of the device
and operating frequency (Bansode and Rathod 2017). Mild
intensity and low frequency ultrasound irradiation in liquids
causes the stable cavitation. These stable cavitation bubbles
induce oscillatory forces which lead to the change in 3D
conformation of enzyme and thus, change in the activity
of enzyme. Wang et al. (2012) investigated an influence of
ultrasound power on cellulase activity and found that the
activity was enhanced with increasing ultrasonic power up
to 15 W. The augmented activity was due to the rupture
of weak linkages like hydrogen bonds or van der Waals’
interactions by ultrasound which bring the enzyme in active
conformation. However, further increase in ultrasonic power
did not improve the activity of cellulase (Wang et al. 2012).
The high intensity ultrasonic irradiation shows deleteri-
ous effect on the enzyme activity. When ultrasonic intensity
exceeded optimum intensity, the enzyme activity decreased
gradually with an increase in the ultrasonic intensity (Tian
et al. 2004). The researchers explained that high intensity
ultrasound enhanced the cavitation effects that caused sig-
nificant shear in the liquid medium. Such extreme conditions
could cause great damage to polypeptide chains, leading to
inactivation of the enzyme (Şener et al. 2006). Addition-
ally, extreme increase in localized pressure and temperature
at higher intensity leads to the generation of free hydroxyl
and hydrogen radicals. It can react with protein backbone
ultimately resulting into modification of secondary active
conformation (Basto et al. 2007). Szabo and Csiszar (2013)
observed 25% loss in the cellulase activity at ultrasound
intensity of 43.4 W, whereas the activity increased upto
23.5% at low ultrasound intensity of 17.33 W (Szabó and
Csiszár 2013). On the contrary, Ma et al. (2011) found a pos-
itive effect on the alcalase activity under higher ultrasound
power (0–400  W). The maximum alcalase activity was
achieved at 80 W ultrasonic power, with 14.9% enhanced
activity. The authors have attributed the improved enzyme
activity to the breakdown of molecular aggregates which
make the enzyme active site more readily accessible for
reaction (Ma et al. 2011).
Further, Bashari et al. (2013a) stated that both activation
and inactivation of enzyme under low ultrasonic energy is
closely linked to the ultrasonic irradiation intensities. The
change in enzyme behaviour was attributed mainly to two
factors: (a) mild intensity ultrasound treatment causes stable
cavitation induced by the oscillation, which alters the con-
figuration of the enzyme and thereby, improving the activ-
ity of enzyme. (b) a more homogeneous reaction mixture is
obtained due to the irradiating surface and wide variation
in the energy dissipation rates resulting in strong acoustic
streaming and thereby, reducing the mass-transfer charac-
teristics (Bashari et al. 2013a).
Duty cycle
Along with power intensity of sonication, the duty cycle of
irradiation is one of the critical operating parameters. Duty
cycle helps to control the enzyme exposure time to sonica-
tion. It was seen that at lower duty cycle, the enzyme activity
reached to maximum after long time which is attributed to
ineffective micro-streaming caused due to acoustic cavitation.
On the other hand, continuous and prolonged irradiation at
higher duty cycle leads to generation of more heat around
the enzyme solution, which can lead to reduction in activity.
In one of the studies, CALB (C. Antarctica lipase from
Novozymes) was tested under varying duty cycles. The high-
est activity was found at 66.67% duty cycle (10-s on and 5-s
off time). The authors claimed that rise in activity might be
due to appropriate application of impulsive forces on the
enzyme (Jadhav and Gogate 2014). Further, Nadar et al.
(2016a) studied effect of duty cycle on lipase obtained from
two different sources viz. T. lanuginosus (TL lipase) and
CALB lipase. At 66.7% (8-s on and 4-s off time, 11.38 W/
cm2 power) duty cycle, maximum activity (162% activity
recovery) of CALB lipase was observed at 20 min of soni-
cation treatment, whereas for TL lipase, maximum activity
(139% activity recovery) was observed at 50% (4-s on and
4-s off time, 15.48 W/cm2 power) duty cycle with treatment
time of 25 min. The authors have explained that difference
in lipase molecular structure (due to variation in lid size, TL
lipase has large lid, whereas CALB lipase possesses small
lid) resulted in requirement of different exposure time to
induce the conformational changes to get improved activity.
Further increase in percentage of duty cycle led to produce
more heat around the enzyme molecules, which resulted into
unfolding and denaturation of enzymes (Nadar and Rathod
2016a). Thus, it can be inferred that optimization of enzyme
exposure to sonication system is required to achieve benefi-
cial effects on enzyme activity.
These findings claim that overall effect of ultrasound on
enzyme is mainly dependent on the energy input and irra-
diation period, as extreme sonication treatment may lead
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 170   Page 6 of 12
to enzyme aggregation, thereby, obstructing the active site
and decreasing enzyme stability. Thus, while application of
ultrasound in enzymatic reaction, appropriate duty cycles,
acoustic power, and exposure time are important in order to
retain the stability as well as activity of the enzyme.
Enzyme concentration and medium
Enzyme concentration plays an important role in determin-
ing the effects of sonication. Jadhav and Gogate (2014) stud-
ied the effect of enzyme concentration on its activity under
ultrasonic condition. At very low enzyme concentration, the
enzyme molecules are randomly distributed with very low
density so that they cannot effectively interact with micro-
streams generated due to acoustic cavitation continuously.
On the other hand, beyond the optimum concentration of
enzyme, excess enzyme molecules hinder the energy-trans-
fer process, thereby decreasing the available energy for cavi-
tational events. Also, authors have indicated the possibility
of enzyme aggregates as a result of cavitation, which can
further lead to a lower degree of intensification. The similar
effect exhibited by enzyme during kinetic studies suggested
the presence of competition for substrate at higher concen-
trations, which decreases the rate of reaction (Talekar et al.
2013b; Jadhav and Gogate 2014).
Another aspect to be noticed is that the nature of enzyme
medium must be considered while treating with ultrasound
which significantly affects magnitude on catalytic activities.
Shah and Gupta (2008) identified the effect of ultrasonic
treatment of lipase catalytic activity in different media such
as aqueous (phosphate buffer) and non-aqueous (acetoni-
trile, octane and DMF) media. The authors have reported
that after 4 h ultrasonication, lipase showed higher activity
than that of original activity except in DMF. However, lipase
in aqueous buffer showed five-fold enhanced activity after
sonication treatment as compared to lipase in organic media
(Shah and Gupta 2008).
pH and temperature
The enzyme activity is dependent on the micro-environment
provided around the enzyme molecules. In such a case, pH
and temperature play an important role to stabilize enzyme
to retain its activity (Özbek and Ülgen 2000; Talekar et al.
2013a). The ultrasound alters the behaviour of enzymes, since
they responded differently to alterations in pH and tempera-
ture. Souza et al. (2013) presented the effects of temperature
and pH on the alpha amylase activity in the presence and
absence of ultrasound irradiation by central composite design.
The effect of temperature was less pronounced in the pres-
ence of ultrasound, resulting in 80% decrease in the activation
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World J Microbiol Biotechnol (2017) 33:170
energy in comparison to absence of ultrasound irradiation. The
authors have reported about three folds higher activity of amyl-
ase for temperatures up to 40 °C in the presence of ultrasound.
Nevertheless, under ultrasound irradiation, antagonistic effects
on enzyme activity have been observed due to the pH of media
(Souza et al. 2013).
Generally, cavitation is better attained at lower temperature
when the ultrasonic power of the generator is constant. An
appropriate temperature range is required to disrupt strong
solute-matrix interaction such as Van der Waals forces, hydro-
gen bonding and dipole attractions between the solute mol-
ecules and active sites on the matrix besides the diffusion rate
of the material. Hence, a compromise between temperature
and cavitation must be achieved for better performance of the
ultrasonic process. It is obvious that the applied sonication has
a significant effect on the temperature of the liquid in the reac-
tion chamber. Depending on the amplitude, the temperature
of the liquid medium is increased from room temperature to
50, 70 and 85 °C at 40, 60 and 80% amplitudes, respectively,
within 10 min of continuous ultrasound exposure (Szabó and
Csiszár 2013). Bashari et al. (2013a) reported the effect of
temperature (in the range of 25–70 °C) on the dextranase activ-
ity in the absence and presence of ultrasound. The authors
observed that under ultrasound irradiation, enzyme activity
was higher (by multi-folds) than control without affecting the
enzyme’s optimal temperature (Bashari et al. 2013a). Similar
results have been shown in the case of cyclodextrin glucano-
transferase under ultrasound irradiation (Eibaid et al. 2014).
The behaviour concerning enzyme under different tempera-
tures was explained by Wang et al. (2012). They stated that
the sonication of a liquid causes two primary effects, namely
cavitation and heating. Although, the heating effect of ultra-
sound irradiation was eliminated by temperature controlled
water bath, the local shear stress caused by the collapse of
bubbles can promote a slight heating that is not measured due
to the position of sensor, that can result in a local increase
of temperature, leading to the less pronounced effect of tem-
perature in the enzyme activity in the presence of ultrasound
(Wang et al. 2012). Concerning the effect of pH on the activity,
it is very well known that ultrasound breaks weak interactions
and induces conformational changes in protein structures,
which may have the difference in the magnitude of effects on
the activity among the experiments (Özbek and Ülgen 2000;
Batistella et al. 2012).
Effect of ultrasonic treatment on enzyme
properties
Kinetic parameters
The kinetic parameters ­(Vmax and ­Km) were calculated
from non-linear regression fitting of the initial reaction
World J Microbiol Biotechnol (2017) 33:170 Page 7 of 12  170
rates corresponding to different substrate concentrations by
Lineweaver–Burk or Hill plot. The V ­ max reflects the limit-
ing rate of the enzymatic reaction at substrate saturation,
whereas, ­km value indicates the affinity of enzyme towards
substrate. The ultrasonic irradiation might change enzyme
structure, and active centre exposure results into consider-
able change in movement of the substrate towards active
site of enzyme.
Guiseppi-Elie et al. (2009) explored the effect of ultra-
sound on kinetic parameters for glucose oxidase (GOx) with
respect to time. The authors noticed that both k­ cat (turnover
number of the enzyme, V ­ max/enzyme concentration) and
­Vmax values decreased with increasing irradiation time upto
60 min. Meanwhile, the ­Km values decreased stepwise to
approximately 50% as compared to native GOx. Further,
it has been indicated that ultrasound irradiation brings the
physicochemical changes in the enzyme, resulting into
improvement in ­Vmax. However, subtle changes in its struc-
ture following an extended ultrasonication time may reduce
the activity significantly (Guiseppi-Elie et al. 2009). Simi-
larly, Sulaiman et al. (2013) examined the enhancement in
­Vmax value and reduction in K ­ m value for cellulase after
ultrasound treatment at 11.8 W/cm2 power intensity which
displayed better catalytic activity with improved affinity
towards the substrate as compared to control. Souza et al.
(2013) determined ­Vmax and ­Km values for amylase at dif-
ferent temperatures (35 and 65 °C). It has been reported that
­km value remained constant, whereas ­Vmax value increased
slightly with increase in temperature under the influence of
ultrasound. On the other hand, in the absence of ultrasound,
­km value decreased by 65% at higher temperature, whereas
­Vmax increased up to 190% with the temperature (Souza et al.
2013). Changes in the enzymatic kinetic parameters can be
attributed to intense pressure, shear force and temperature
which were seen as a result of the ultrasound cavitation. It
demonstrated that the product formation was more rapid and
efficient after ultrasound irradiation, with higher affinity for
substrate (Ma et al. 2016).
Thermodynamic parameters
Thermodynamic stability of the enzyme is important for its
catalytic activity. Several biological and physical parameters
have been identified as the factors affecting protein stabili-
zation. Changes in the spatial configuration and the activity
of enzyme can be influenced by physical parameters (tem-
perature, pH, chemical agents, autolysis or ionic strength)
and physico–chemical parameters (ultrasonic parameters).
Activation energy ­(Ea) is the minimum amount of energy
required to boost the initial materials to the transition state
to form the product. E ­ a was determined by linear fit of the
Arrhenius plot. Generally, the ­Ea value is in the range of
40–400 kJ/mol for most of the reactions. If the value is lower
than 40 kJ/mol, the reaction completes very rapidly (Ma
et al. 2011). Mostly, the reduced ­Ea value after ultrasound
treatment indicates that the reaction catalysed by enzyme
could occur very easily due to remarkable reduction in the
energy barrier to catalyse the reaction. Leaes et al. (2013)
found that the use of ultrasound was favourable for both
alpha amylase and amyloglucosidase, since ­Ea was reduced
by around 60 and 40% respectively after sonication. The
decrease in ΔEa value has been attributed to the exposure of
active sites by ultrasound assisted favourable conformational
changes occurring in enzyme, which help to accelerate the
reaction rate (Leaes et al. 2013).
In order to understand microscopic effect of ultrasound
irradiation on enzyme, the thermodynamic parameters such
as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) are
analysed on the basis of experimental data. Further, these
changes in thermodynamic parameters have been correlated
with the folding/unfolding of the enzymes to determine
physico–chemical effects of cavitation/ultrasound (Jadhav
and Gogate 2014).
Generally, it has been observed that ΔG decreases after
ultrasonic treatment which indicates that the enhancement
in enzyme activity might be due to the favourable change
in enzyme conformation (Sojitra et al. 2016a; Talekar et al.
2017). Also, after ultrasonic treatment, ΔH value decreases,
which can be attributed to the breakdown of non-covalent
bonds (hydrogen bonds, internal hydrophobic interaction
and van der Waals’ force) resulting in the irreversible con-
formational changes in enzyme which eased to stabilize the
enzyme at ground state in open active conformation (Subh-
edar and Gogate 2014). The ΔS value signifies the alteration
in the extent of local disordering between transition state
and the ground state. The relatively large negative ΔS value
than that of untreated enzyme was observed after ultrasonic
treatment. This can be attributed to the sonolysis of water
generating OH΄ radicals which brought about conforma-
tional changes in enzyme (Malani et al. 2014). Further, Ma
et al. (2015) explained that oxidative modification of amino
acid residues and initiation of cross-linking and aggrega-
tion improves the enzyme activity. Also, chemical effects of
ultrasound irradiation, the micro-turbulence and acoustic (or
shock) waves caused by sonication (either by bubble oscilla-
tion or implosion) resulted into elevation of static pressure
and hydrodynamic stresses that can contribute to breakage
of weak linkages like hydrogen, electrostatic and van der
Waals’ bonds. The spontaneity of the reaction also increased
due to the enhanced activity of enzyme, as indicated by the
higher (−ΔS) values for sono-enzymatic treatment at atmos-
pheric pressure at optimized conditions (Ma et al. 2015).
Similar trends of ΔH, ΔS and ΔG have been reported for
lipase, cellulase and alcalase under ultrasonic treatment (Ma
et al. 2011; Subhedar and Gogate 2014; Nadar and Rathod
2016a).
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Effect on conformational structure
Enzyme configuration plays a crucial role in the catalytic
efficiency, stability and selectivity. Several reports validated
that ultrasound irradiation brings the changes in second-
ary–tertiary structure and alteration in active sites which
results into the change in enzyme activity (Hoshino et al.
2006). In most of the studies, the conformation changes in
enzyme after ultrasound treatment have been investigated
by spectroscopic techniques such as intrinsic fluorescence,
circular dichroism (CD) spectroscopy and Fourier trans-
form infrared spectroscopy (FT–IR) spectra data analysis
tools (Secundo 2013; Talekar et al. 2014; Nadar and Rathod
2017).
An intrinsic fluorescence measurement has been per-
ceived as an effective indicator of tertiary structure transition
and conformation changes in proteins. Fluorescence behav-
iour of protein is mainly attributed to its aromatic amino acid
residues, especially tryptophan residue which is sensitive to
the polarity of microenvironments during the transition. The
fluorescence spectra of enzyme exist typically at 280 nm.
Yu et al. (2014) studied the effect of ultrasound on confor-
mation of three different enzymes (α-amylase, papain and
pepsin) on the basis of tryptophan fluorescence measure-
ment. They found that papain showed gradual decrease in
fluorescence intensity with increase of ultrasonic irradiation
time. On the other hand, native amylase and pepsin showed
slightly lower fluorescence intensity than that of the ultra-
sonic-treated enzyme at optimized conditions. These results
suggested that sonication considerably altered enzyme struc-
ture by the means of tryptophan position. However, continu-
ous ultrasound irradiation resulted into apparent decrease
in fluorescence intensity, which indicated that ultrasound
could induce molecular unfolding and denaturation of pro-
tein caused due to strong shear and micro-streaming arising
from collapsing bubbles as well as by destruction of hydro-
phobic interactions between protein molecules, causing dif-
ferent movements of hydrophobic groups (Yu et al. 2014).
Also, it has been reported that chemical effect of ultrasound
can lead to the thermo-sonolysis of water molecules to form
intermediates such as hydroxyl and hydrogen radicals, which
can further react with tryptophan and oxidize into kynure-
nine (Chowdhury et al. 1995). Hence, the phenomenon of
decreased fluorescence intensity takes place with increasing
ultrasonic treatment.
Circular dichroism (CD) spectroscopy is well-studied
and is the most frequently used method for evaluation of
protein secondary structure, folding and binding proper-
ties of macromolecules. The method has been proven to be
adequately simple and reliable for rapid determination of
protein structure or monitoring conformational changes. In
CD spectra, enzyme (protein) is exposed to far-UV wave-
length (ranging from 190 to 250 nm) leading to electronic
13
World J Microbiol Biotechnol (2017) 33:170
transitions between molecular orbitals in ground and excited
states and hence seeming as very useful measurement for
protein conformational change. The changes in character-
istic CD spectra are assigned to ɳ⟶𝜋* and 𝜋0⟶𝜋* tran-
sitions of polypeptide chains of enzyme. Yu et al. (2014)
observed that ultrasonic treatment significantly changed
the secondary structural component, especially β-sheet.
The increase or reduction of β-sheet structures caused by
ultrasound might be due to the structural re-arrangement
and conversion of different domains to β-turn and random
coil (Yu et al. 2014). In another study, Wang et al. (2012)
investigated the effect of different ultrasound frequencies on
secondary structure of cellulase and it was correlated with
cellulase activity. It was found that prolonged ultrasound
exposure with high frequency (29 kHz, 50 W for 30 min)
led to decrease in cellulase activity as a result of decrease
in random coil and increase in α-helix content (Wang et al.
2012).
Another method of determination of structural changes
is based on FT–IR spectroscopy. Molecular geometry and
hydrogen bonding pattern of each enzyme is unique (Sojitra
et al. 2016b). Among complete FT–IR spectra, 1600–1700/
cm band (representing amid I) is most sensitive spectral
region for structural component of protein. However, due
to the extensive overlapping of the broad underlying com-
ponent bands, which lie in close proximity to one another,
the secondary FT–IR derivative of amide I band was used
to identify the structural components which was calculated
by considering the multicomponent peak areas (Nadar et al.
2016a, b). Nadar et al. (2016a) identified the structural
changes in TL lipase and CALB lipase before and after
sonication treatment. In both the cases, decrease in α-helix,
β-sheets and β-turns fraction was observed. The structural
changes have been attributed to the cavitation effect lead-
ing to hydrodynamic stresses that can contribute to break-
age of weak bonds and forces (Nadar and Rathod 2016a).
Similarly, Wang et al. (2012) have also reported similar
structural composition for cellulase after treatment under
ultrasound.
Thermogravimetric analysis (TGA) is also employed
to provide critical information regarding conformational
changes on the basis of difference in the amount of water
associated with the enzyme before and after sonication.
Guiseppi-Elie et al. (2009) performed TGA for ultrason-
icated and native glucose oxidase (GOx) enzyme. The
higher mass loss was found for sonicated GOx during
first transition between 25 and 100 °C which was associ-
ated with the broad endothermic effect of the protein. The
difference in bonded water to enzyme surface may be due
to alteration in overall surface topology and conforma-
tional structure caused by the combined ultrasonication
and local lyophilization (Guiseppi-Elie et al. 2009).
World J Microbiol Biotechnol (2017) 33:170 Page 9 of 12  170
Effect on particle size distribution
Besides the conformation changes in enzyme, the particle
size of enzyme plays very important role during the cata-
lytic performance. The behaviour concerning the size of
enzyme was explained in the work of Nadar et al. (2016a).
They revealed that untreated lipase contained mixture of
smaller amount (about 30%) of 8 nm enzyme molecules
and higher amount (about 70%) of 40 nm enzyme aggre-
gates, therefore, it showed less activity of lipase. How-
ever, after treating lipase at optimum ultrasound condi-
tions, effective dismantling of molecular agglomerates by
disruption of the van der Waals’s forces and hydrophobic
interaction has been reported. This decreases the parti-
cle size, narrows their distribution and increases surface
area as well as accessibility, thereby, enhancing the lipase
activity (Nadar and Rathod 2016a). However, continuous
ultrasound exposure had contributed to denaturation and
fragmentation which again led to the formation of aggre-
gates (Gülseren et al. 2007; Jambrak et al. 2014). Fur-
ther, Yu et al. (2013) studied interactions among amino-
acid residues of native and ultrasonic treated tyrosinase
by observing molecular structure under atomic force
microscopy. They reported that the cavitation effect of
ultrasound can lead to the strong shear, micro-streaming
and the extreme localized pressure and temperature. It
further leads to water lysis which results into intermedi-
ates (such as hydroxyl and hydrogen free radicals) forma-
tion. These free radicals might react with some amino
acid residues that participate in enzyme stability, which
contribute to the formation of large aggregates (Yu et al.
2013). Furthermore, the morphological changes and size
distribution after ultrasound treatment were studied using
scanning electron microscopy (SEM) by Shah and Gupta
(2008). SEM images showed that untreated lipase was
more or less monolithic in nature, however, after ultra-
sonic treatment, fine powder and small spheres were seen,
which helped to increase the surface area of the catalyst,
and hence, exhibited enhanced activity (Shah and Gupta
2008).
Therefore, the enhanced activity of enzymes is a com-
bined result of conformational changes in 3D structure
and narrow scaled size distribution with increased avail-
ability of molecular groups, which might be attributed to
the physical and chemical effect of ultrasound.
Immobilization
There are several reports suggesting a gradual decrease in the
activity of the sonicated enzyme when kept at room tempera-
ture for some time (mostly 40–50 min). This was observed
due to low structural stability which mainly attributed to
the reconfiguration of the enzyme toward its original state
and a decrease in the surface area of the substrate because
of de-emulsification. As the stability of enzyme after soni-
cation is very low, the modified enzyme conformation can
be retained by using different immobilization techniques
(Nadar and Rathod 2016b). Immobilization of enzyme not
only stabilizes the enzyme in active conformation but also
improves the operational parameters (temperature and pH),
thermal stability, reusability and storage stability (Cao et al.
2003; Tran and Balkus 2011; Talekar et al. 2012; Hanefeld
et al. 2013). There are three ways of employing ultrasound
during immobilization: (i) ultrasound treatment before
immobilization of enzyme. (ii) ultrasound treatment during
immobilization process and (iii) ultrasound treatment after
immobilization.
Jadhav and Gogate (2014) pre-treated lipase under ultra-
sonic irradiation and successively immobilized them by
physical adsorption on HP20 and calcium alginate entrap-
ment. The maximum lipase activity was retained in the case
of physical adsorption than the entrapment by calcium algi-
nate. This was because of absence of interference with the
folding pattern of the enzyme during the immobilization
via adsorption. Further, the immobilized sonicated enzyme
showed superior thermal stability (in the range of 50–70 °C)
as compared to free sonicated enzyme (Jadhav and Gogate
2014).
Bashari et al. (2013b) carried out ultrasound-assisted
dextranase (immobilization of the enzyme was carried out
in one-step ultrasound process) entrapment into calcium-
alginate gel beads. The immobilized dextranase was pre-
pared with an ultrasonic irradiation (25  kHz, 40  W for
15 min) and found that ultrasound-assisted immobilization
showed higher enzyme activity along with increased load-
ing efficiency (by 27.21%) and increased the immobilization
yield (by 18.77%) as compared to immobilization without
ultrasonication irradiation. Also, the immobilized enzymes
prepared with ultrasound irradiation exhibited enhanced rate
of reaction (­ Vmax) as well as excellent recyclability (Bashari
et al. 2013b).
Recently, Ma et al. (2017) immobilized pectinase in the
presence of ultrasound within sodium alginate which fur-
ther cross-linked with glutaraldehyde. After ultrasound treat-
ment, pectinase activity increased by 30.05% with higher
immobilization yield of 92.28% as compared to control.
Also, kinetic parameters ­(Vmax and ­Km) were improved,
which indicated the increased catalytic efficiency and
enhanced affinity towards substrate. Furthermore, ultra-
sound-treated pectinase exhibited higher thermal stability
in the range of 40–60 °C (the residual activity for free pec-
tinase preserved for 60 min at 40, 50 and 60 °C were 31.98,
19.90 and 6.57%, respectively, while for immobilized pec-
tinase showed 67.42, 35.04 and 19.71% residual activity at
respective temperature) and reaction stability (up to 60 min),
13
 170   Page 10 of 12
as compared to free untreated form of pectinase (Ma et al.
2017).
More recently, Ladole et al. (2017) carried out hyper-acti-
vation of cellulase immobilized onto magnetic nanoparticles
(MNPs) under the influence of ultrasound. The cellulase was
immobilized onto MNPs (cellulase@MNPs). After immobi-
lization process, cellulase@MNPs were exposed to optimum
ultrasonic condition (i.e. at 6.3 W exposed for 6 min). It
was found that the catalytic activity of enzyme MNPs was
enhanced almost by 3.4 folds over the control. The authors
claimed that the increase in the catalytic activity can be
attributed to more accessibility of enzyme and that of the
substrate leading to more interaction of enzyme-substrate
complex which forms more products. Further, the fraction
of secondary structure in free and cellulase@MNPs, before
and after sonication was estimated and it was observed that
the β-sheet and random coil increased by 3.54 and 0.71% in
cellulase@MNPs before sonication and 6.23 and 0.69% in
cellulase@MNPs after sonication over the control; whereas
α-helix and β-turn content reduced by 1.5 and 1.91% in cel-
lulase@MNPs before sonication and 1.8 and 2.11% in cel-
lulase@MNPs after sonication over the control. Hence, the
immobilization of enzyme after ultrasonic treatment helps to
maintain highly active conformation of the enzyme (Ladole
et al. 2017; Talekar et al. 2017).
Conclusion and future scope
In summary, low intensity ultrasound irradiation is a prom-
ising technology to be used in the activation of enzyme
by altering their structural conformation, which is totally
dependent on the ultrasound parameters and enzyme prop-
erties. Ultrasound treatment can improve enzyme stability,
thermal kinetic parameters as well as Michaelis–Menten
kinetic parameters. The ultrasonic effect on enzyme can
be explored by investigating structural and conformational
changes by spectroscopic techniques such as the intrinsic
fluorescence, circular dichroism (CD) spectroscopy and
FT–IR spectra analysis data tools. Further, the physical
and chemical effects of ultrasound on enzyme have been
correlated with thermodynamic parameters (enthalpy and
entropy). However, there is lack of information on the US
enzyme–working conditions. Hence, it seems necessary
to study how and to what extent ultrasonic effects vary
under different experimental conditions in order to develop
mathematical model that can quantify the contribution of
variable parameters to the overall effect of ultrasound and
consequently predict changes in the efficiency of the pro-
cess. The maintaining uniform distribution of cavitational
activity is one of the most important design aspects in the
sono-chemical reactors which need to be optimized by mak-
ing a proper choice of the operating conditions and using
13
World J Microbiol Biotechnol (2017) 33:170
larger irradiation surface. Also, after sonication, enzyme
stability is a major hurdle to maintain its improved structure
properties and enhanced activity. So, novel immobilization
techniques need to be developed so as to use highly active
enzyme for biotransformation processes. Also, combined
enzyme–ultrasound systems have a synergistic behaviour
which provide benefits such as reduce the operational cost,
develop entirely new functional products with concomitant
quality enhancement and the environmentally sustainable
processing.
Acknowledgements  The authors would like to acknowledge Uni-
versity Grants Commission (UGC) of India for their financial support
in our research work.
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