International Journal of Biological Macromolecules 271 (2024) 132538

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Enhancing efficiency of ultrasound-assisted biodiesel production catalyzed

by Eversa® Transform 2.0 at low lipase concentration: Enzyme
characterization and process optimization
Min Ying Liow , Eng-Seng Chan *, Wei Zhe Ng , Cher Pin Song *
Department of Chemical Engineering, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500 Subang Jaya, Selangor,
Malaysia
Monash-Industry Plant Oils Research Laboratory (MIPO), Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500 Subang Jaya, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Keywords: This study focused on the ultrasound-assisted transesterification of simulated low-quality feedstocks using a low-
Biodiesel cost liquid lipase Eversa® Transform 2.0 (ET2). Enzyme characterization was also performed to investigate the
Ultrasonication effect of ultrasound parameters on enzyme structure. The optimal ultrasound parameters, 40 % amplitude, and
Enzyme characterization
5 % duty cycle effectively enhanced the reaction rate compared to the conventional stirring method while
Lipase
Methanol inhibition
retaining 95 % of the enzyme activity. Analysis of circular dichroism (CD) spectra revealed the preservation of
the secondary structure of ET2 under the optimal ultrasound intensities, while fluorescence spectra indicated a
slight change in its tertiary structure. The implementation of a two-stage methanol dosing strategy in the
ultrasound-assisted reaction effectively mitigated lipase inhibition, yielding a remarkable fatty acid methyl ester
(FAME) content of 92.2 % achieved within a 12-h reaction time. Notable, this high FAME content was achieved
with only a 4:1 methanol-to-oil molar ratio and a 0.5 wt% enzyme concentration. Under these optimized con­
ditions, the ultrasound-assisted reaction also demonstrated a 15 % improvement in the final FAME content
compared to the conventional stirring method. These promising results hold significant potential for advancing
the field of biodiesel production via ultrasound technology, contributing substantively to sustainable energy
sources.

1. Introduction production processes face limitations when handling feedstocks with

high free fatty acid (FFA) content or impurities. These processes pri­
Research and development in biofuels have become a top priority in marily rely on chemical catalysts, such as sodium hydroxide or potas­
the present day. As indicated in a report published by the International sium hydroxide, for the transesterification of triglycerides present in
Energy Agency (IEA), the global demand for fossil fuels is anticipated to vegetable oils or animal fats. The interaction between the base catalysts
increase significantly in the coming decades, resulting in the depletion and the FFA present in the feedstock can lead to saponification, which
of non-renewable energy [1]. The consequential environmental impact, negatively impacts biodiesel quality [4,5].
such as the greenhouse gas emissions contributing to climate change, is The enzymatic transesterification process for producing biodiesel has
of paramount concern. The IEA report highlights that the transport gained significant attention as a promising strategy for overcoming the
sector, particularly diesel engines, accounted for 37 % of global green­ challenges associated with using low-quality feedstock in biodiesel
house gas emissions in 2021 [2]. Therefore, renewable and clean energy production. This is because enzymes are insensitive to the impurities
sources as alternative fuels, such as biodiesel (also known as fatty acid present in the feedstock, allowing for higher-quality biodiesel to be
methyl ester or FAME), have gained global attention due to their produced [6]. Moreover, enzymatic transesterification offers several
numerous benefits, such as reducing the emission of greenhouse gases advantages over traditional chemical catalysts, including lower energy
and increasing energy security [3]. Nevertheless, traditional biodiesel consumption, higher purity of the final product, and reduced

* Corresponding authors at: Department of Chemical Engineering, School of Engineering, Monash University Malaysia, Jalan Lagoon Selatan, 47500 Bandar
Sunway, Selangor, Malaysia.
E-mail addresses: [email protected] (E.-S. Chan), [email protected] (C.P. Song).

https://doi.org/10.1016/j.ijbiomac.2024.132538
Received 8 February 2024; Received in revised form 29 April 2024; Accepted 19 May 2024
Available online 21 May 2024
0141-8130/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

environmental impact [7]. However, despite these advantages, the re­ sonochemical effects on the activity and conformation of ET2, aiming to
action rate of enzymatic biodiesel production is typically slower than elucidate the influence of these factors on the catalytic performance of
that of the conventional alkaline-catalyzed process, resulting in a longer ET2. Through evaluating the correlation between enzyme stability and
processing time. This lower reaction rate is due to the high specificity of the efficiency of ultrasound-assisted transesterification, the study aimed
the enzyme’s active site and mass transfer limitations between immis­ to provide insights that could lead to further improvements in reaction
cible reactants, ultimately limiting the productivity of biodiesel pro­ efficiency and process optimization.
duction [8]. To overcome these limitations, ultrasound technology has
emerged as a promising technique to intensify and enhance enzymatic 2. Materials and methods
biodiesel production.
Ultrasound technology utilizes low-frequency sound waves 2.1. Materials
(10–100 kHz) to generate pressure waves that create cavitation bubbles
in the reaction mixture. The subsequent collapse of these bubbles near Refined cooking oil (Alif Pure Vegetable Cooking Oil) was purchased
the phase boundary of two immiscible fluids results in various physical from a local supermarket in Malaysia. Free liquid lipase obtained from
effects that enhance mass transfer, diffusion, and mixing, ultimately Thermomyces lanuginosus microorganism and expressed in the Aspergillus
accelerating chemical reactions [9]. The use of ultrasound technology in oryzae microorganism (ET2) was supplied by Novozymes (Malaysia).
enzymatic transesterification reactions has been extensively studied in Methanol of 95 % purity was purchased from Classic Chemical
recent years, with several researchers reporting significant improve­ (Malaysia). HPLC-grade isopropanol and toluene of 99.9 % purity were
ments in reaction rate and product yield [10–12]. However, despite the purchased from Merck (Malaysia). Meanwhile, methyl nonadecanoate
outstanding achievement of ultrasound technology in intensifying was purchased from Larodan AB (Sweden). Lastly, lab grade sodium
enzymatic biodiesel production, it was reported that the typical enzyme hydroxide of 97 % purity was purchased from Sigma Aldrich (Malaysia).
consumption in the ultrasound-assisted reaction is relatively high [8]. All reagents and solvents used in this study were of analytical grade, and
For example, Gharat and Rathod [13] reported that an 86.6 % biodiesel no purification was required before usage.
yield was obtained in 4 h with 10 wt% of Novozym 435 lipase used in an
ultrasonic bath system. In a study conducted by Yasvanthrajan et al. 2.2. Methods
[14], a 98.7 % biodiesel yield was obtained in the ultrasounds-assisted
reaction with 5 wt% of lipase. 2.2.1. Enzymatic transesterification
Furthermore, it has been reported that prolonged and intense soni­ The conventional enzymatic transesterification was carried out in a
cation could lead to a negative impact on catalytic activity [15]. En­ modified 100 mL Duran® laboratory bottle on a hotplate stirrer (IKA C-
zymes are proteins that catalyze biological reactions, and their function MAG HS 7 control) connected to an external temperature sensor to
depends on the proper folding of the protein into a specific 3D structure. maintain a desired temperature at 40 ◦ C. The reaction mixture
The structure of an enzyme provides specific binding sites, known as comprised refined oil containing 40 % FFA, an enzyme solution con­
active sites, which play a crucial role in catalyzing reactions with the sisting of lipases at 0.2 wt% and water at 2 wt% based on the weight of
substrates. The accessibility of these active sites is essential for the feedstock. The mixture was kept at a constant temperature of 40 ◦ C and
catalytic activity of the enzyme. At the molecular level, ultrasound stirred continuously at a rate of 800 rpm for 10 min before the methanol
irradiation can induce changes in secondary and tertiary structures, as was added. Subsequently, methanol was added to the reaction mixture
well as the alteration in the active sites, potentially impacting enzyme at a constant rate within the initial 2 h using a peristaltic dosing pump
activity [16]. Consequently, this can affect the performance of enzy­ (Chonry BT100M). The predetermined reaction time was set at 4 h,
matic biodiesel production. However, there are limited studies on the during which samples were collected at specified intervals. To deacti­
structural characterization of lipase after exposure to ultrasound. Moj­ vate the enzymes, the collected samples were submerged in a water bath
tabavi et al. [17] reported a decrease in the stability of Porcine pancreas set at 90 ◦ C for a duration of 1 h. Then, the samples were centrifuged at
lipase and observed changes in its tertiary structure during the 4500 rpm for 10 min to facilitate phase separation. The upper layer of
ultrasound-assisted hydrolysis of coconut oil. On the other hand, Nadar the sample was then extracted for the analysis of FAME. The same
and Rathod [18] and Jadhav and Gogate [16] reported that the exposure experimental procedures were repeated with different methanol-to-oil
of Thermomyces lanuginosus lipase and Candida antarctica lipase to ul­ molar ratios, ranging from 1:1 to 5:1. All of the experiments were car­
trasound for a specific duration resulted in structural modifications and ried out in duplicates.
reduced activity compared to that of the unsonicated lipase. However,
these studies primarily focused on the enzyme activation mechanism of 2.2.2. Ultrasound-assisted enzymatic transesterification
lipases under ultrasound treatment for a short period [16,18]. A Ultrasound-assisted enzymatic transesterification was carried out in
comprehensive investigation has yet to be conducted on the structural a modified 100 mL Duran® laboratory bottle on a hotplate stirrer (IKA
characteristics of inactivated lipases and the direct impact of sonicated C-MAG HS 7 control) connected to an external temperature sensor in
lipase on transesterification performance. order to maintain a desired temperature at 40 ◦ C (± 3 ◦ C). A probe-type
Recently, there has been a significant interest in using liquid lipase ultrasonic device with a 20 kHz operating frequency and maximum
Eversa® Transform 2.0 (ET2), which is derived from Thermomyces output power of 250 W (Branson SFX250 Sonifier with ½” horn) was
lanuginosus and expressed in an Aspergillus oryzae strain, for biodiesel coupled on the top of the bottle. The reaction mixture comprised refined
production. ET2 has garnered attention for its cost-effectiveness, oil containing 40 % FFA, an enzyme solution consisting of lipases at
exhibiting a remarkable 100 times cheaper compared to immobilized 0.2 wt% and water at 2 wt% based on the weight of feedstock. The
lipases such as Novozyme 435 while maintaining high enzyme activity mixture was kept at a constant temperature of 40 ◦ C and stirred
even under mild operating conditions [19,20]. Moreover, its effective­ continuously at a rate of 800 rpm for 10 min before the methanol was
ness in catalyzing the transesterification of low-quality feedstock has added. Subsequently, the magnetic stirrer bar was removed, and the
garnered widespread recognition, underscoring its potential significance ultrasonicator was turned on. The ultrasound amplitude was varied from
in biodiesel production [6,21,22]. Despite these notable advantages, a 20 to 100 % and duty cycle from 2.5 (1 s on and 39 s off) to 30 % (3 s on
critical research gap exists concerning the utilization of ET2 in and 7 s off). The methanol-to-oil molar ratio of 2:1 was dosed uniformly
ultrasound-assisted biodiesel production to enhance reaction efficiency. using a peristaltic pump (Signal Fluid BS100-1A) into the reaction
Hence, this study aimed to address this research gap by focusing on the mixture in the first 2 h. The predetermined reaction time was set at 4 h,
use of ET2 at a low concentration to produce biodiesel from low-quality during which samples were collected at specified intervals. To deacti­
feedstock containing 40 % FFA. Additionally, this study investigated the vate the enzymes, the collected samples were submerged in a water bath

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M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

set at 90 ◦ C for a duration of 1 h. Then, the samples were centrifuged at scanning rate of 100 nm/min.
4500 rpm for 10 min to facilitate phase separation. The upper layer of The intrinsic fluorescence intensity of the native and sonicated ET2
the sample was then extracted for the analysis of FAME. The same was measured by a fluorescence spectrometer (LS-55 Perkin Elmer, UK).
experimental procedures were repeated using the optimum ultrasound The fluorescence spectra of the lipase (0.02 wt%) were measured in a
parameters and different methanol-to-oil molar ratios. The methanol-to- semi-micro cuvette with a 5 mm path length at 260 nm excitation
oil molar ratio was varied from 1:1 to 5:1. All of the experiments were wavelengths, 280 nm emission onset wavelength and 480 nm emission
carried out in duplicates. termination wavelengths. The bandwidth excitation and emission slit
widths were 10 nm, and a 500 nm/min scan speed was used.
2.2.3. Determination of fatty acid methyl ester content
The quantification of FAME in samples was conducted according to 3. Results and discussion
the EN14103 method, employing an Agilent GC-7890A gas chroma­
tography equipped with a flame ionization detector (GC-FID) and an HP- 3.1. Effect of ultrasound parameters on the enzymatic reaction
INNOWax capillary column with a length of 30 m, inner diameter of
0.25 mm and film thickness of 0.25 μm. To outline the procedure, the 3.1.1. Power intensity
injector and detector temperature was set at 250 ◦ C, while nitrogen The cavitation intensity refers to the power density of cavitation,
served as the carrier gas with a flow rate of 2 mL/min. The split flow was which is a critical parameter that affects the mass transfer efficiency
maintained at 100 mL/min. The column temperature was programmed during ultrasound-assisted transesterification [24]. This cavitation in­
in the following sequence: initiating at 60 ◦ C and holding for 2 min, then tensity is directly proportional to the amplitude of the ultrasonic waves,
gradually increasing to 200 ◦ C at 10 ◦ C/min. Subsequently, it was which implies that changing the amplitude allows for controlling the
further heated from 200 ◦ C to 240 ◦ C at 5 ◦ C/min with a holding time of cavitation intensity.
7 min. For analysis, 1 μL of the sample was injected into the column. Fig. 1 depicts that the FAME content increased from 42.9 wt% to
To prepare the samples for GC-FID analysis, an approximate weight 44.6 wt% as the amplitude increased from 20 % to 40 %. This finding
of 0.01 g was precisely measured from each sample extracted from the indicated that the intensity of the pressure fluctuations induced in the
upper layer of the reaction mixture after phase separation through liquid at low ultrasonic power was insufficient to produce and sustain a
centrifugation. The measured samples were then placed into individual significant number of cavitation bubbles [25]. As a result, the level of
1.5 mL micro-centrifuge tubes. Subsequently, a 1 mL quantity of an mass transfer and mixing enhancement achieved by cavitation is limited
internal standard stock solution consisting of methyl nonadecanoate at low ultrasonic power levels. At the optimal ultrasonic intensity,
(C19:0) dissolved in toluene (0.01 g/mL) was added to the sample. The acoustic microstreaming generated by bubble cavitation can overcome
resulting mixture was thoroughly homogenized with a vortex mixer, the mass transfer limitation and thus increase the rate of reaction [8].
transferred into a 2 mL glass vial, and ready for GC-FID analysis. The Yasvanthrajan et al. [14] observed a similar increasing trend in the
FAME content was determined based on Eq. (1): FAME content with an increase in the ultrasound amplitude from 20 %
to 40 % for the production of biodiesel using immobilized lipase from
ΣA − Astd mc19:0
FAME content (wt%) = × × 100% (1) Rhizopus oryzae as the biocatalyst.
AC19:0 ms
However, for a further increase in the amplitude from 40 % to 100 %,
where ΣA denotes the total peak areas of methyl esters (C12:0 to C24:0), the FAME content decreased gradually to 40.7 wt%. It was speculated
Astd denotes the peak area of C19:0, mC19:0 denotes the mass of C19:0 (in that an increase in the ultrasonic intensity beyond the optimum level
g), and mS denotes the mass of sample (in g). could lead to changes in the enzyme conformation. The vigorous cir­
culatory motion of bubbles in the fluid generated strong eddy currents,
2.2.4. Enzyme characterization leading to high shear forces that could potentially cause damage to the
The enzyme solution (containing 0.2 wt% lipase based on the weight enzyme structure [26]. Additionally, the structural changes in the pro­
of water) was prepared in the modified 100 mL Duran® laboratory tein may also occur due to the high-energy shock waves resulting from
bottle, which was immersed in an ice bath and placed on the hotplate the collapse of cavitation bubbles [27].
stirrer (C-MAG HS 7 control) connected to an external temperature Furthermore, it was speculated that the reduction in product yield
sensor in order to maintain a desired temperature at 40 ◦ C (± 3 ◦ C). The
enzyme solution was sonicated for 1 h at different ultrasonic amplitude
(20–100 %) and duty cycles (2.5–30 %). Subsequently, the sonicated
lipase solution was subjected to enzyme characterization.
The enzyme activity of the liquid lipase was assayed immediately
using the hydrolytic activity [6,23]. 1 mL of the sonicated enzyme so­
lutions and 1 mL of refined cooking oil were added into a 1.5 mL
microcentrifuge tube. The mixture was stirred for 5 min using a vortex
mixer (IKA VORTEX Genius 3). The amount of fatty acid produced via
hydrolysis of refined oil was measured using an Auto-titrator (Met­
rohm’s Tritino Plus 484), where 1 IU represents the amount of enzyme
that produces 1 μmol of titratable fatty acid per minute. The residual
activity for the enzyme was calculated based on Eq. (2):
A
Residual activity = (2)
A0

where A is the activity of the sonicated ET2, and A0 is the activity of the
unsonicated ET2.
Circular dichroism (CD) spectra of the native and sonicated ET2 were
determined by using a circular dichroism spectrometer (J-815 Jasco,
Fig. 1. Effect of ultrasonic amplitude on FAME content. The reaction condi­
Japan). The far ultraviolet range was set to 200–260 nm at a 0.1 cm path
tions were 20 % ultrasound duty cycle, 2:1 methanol-to-oil molar ratio dosed in
length. A bandwidth of 1 nm and a response of 1 s were used, with a
2 h, 40 ◦ C, 0.2 wt% of free lipase, 2 wt% of water and 4 h of reaction.

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M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

might be attributed to the increased number of cavitation bubbles due to to environmental changes caused by ultrasound. In another study,
high power density. These bubbles could potentially absorb and scatter Yasvanthrajan et al. [14] observed that a duty cycle of 50 % (15 s on and
the sound waves, thereby significantly weakening the acoustic field 15 s off) in the ultrasound-assisted enzymatic biodiesel production
strength [25,28]. Consequently, the abundance of bubble clouds near resulted in a 98.7 % yield. However, when the pulse time increased to
the transducer may alter the acoustic impedance and diminish the effi­ 30 s on and 30 s off, the yield significantly decreased to 74.2 % [14].
ciency of energy transfer from the ultrasound source to the reaction
medium, ultimately leading to a reduction in product yield [28]. Similar 3.2. Enzyme characterization
findings were reported by Bhangu et al. [29] for biodiesel production
from Candida rugosa lipase, where an increase in the ultrasonic irradi­ 3.2.1. Enzyme activity
ation power from 50 W to 200 W resulted in a decrease in the FAME The effect of ultrasonication on the activity of ET2, expressed as
yield from 35 % to 15 %. residual activity, is shown in Fig. 3. As depicted in Fig. 3(a), a decreasing
trend in the residual activity was observed with increasing ultrasound
3.1.2. Duty cycle amplitude. This finding aligned with existing literature, substantiating
The duty cycle for an ultrasonic system is defined as the ratio of the the hypothesis that higher ultrasound amplitudes lead to lower FAME
on-time to the total pulse cycle time, expressed as a percentage [14]. The content due to ultrasound-induced enzyme inhibition. However, despite
duty cycle directly affects the amount of energy dissipated in the reac­ achieving the highest FAME content at 40 % ultrasound amplitude (see
tion system, affecting the mixing efficiency. The performance of the Fig. 1), the residual activity of ET2 after sonication at 40 % amplitude
enzymatic reaction at the optimum ultrasound amplitude (i.e., 40 %) was lower than both unsonicated enzymes and those exposed to 20 %
was evaluated by varying the duty cycle from 2.5 % to 30 %, as depicted amplitude. This may be attributed to the higher mixing efficiency
in Fig. 2. At a duty cycle of 2.5 %, the observed low FAME content could resulting from strong shear forces at 40 % amplitude, potentially off­
be due to an inadequate supply of energy to the reaction system. This setting the effects of ultrasound-induced enzyme inhibition. This, in
may lead to insufficient ultrasound waves to generate necessary cavi­ turn, results in a higher FAME content at 40 % amplitude, despite the
tation, subsequently compromising the shear forces required for effec­ lower enzyme activity compared to 20 % amplitude.
tive mixing in the reaction. Interestingly, the highest FAME content of
46.6 wt% was observed at a considerably low duty cycle of 5 % (1 s on,
19 s off). At a 10 % ultrasonic duty cycle (1 s on, 9 s off), a comparable
FAME content of 46.5 wt% was obtained.
However, a further increase in the duty cycle to 30 % resulted in a
significant drop in FAME content to 41.6 wt%. The decrease in FAME
content could be attributed to continuous exposure of the enzyme to
cavitation conditions over extended periods, ultimately resulting in a
loss of enzyme activity. At a higher duty cycle where an enormous
temperature was generated at a localized level, hydroxyl and hydrogen
radicals were formed as a consequence of chemical change [26]. The
free radicals produced may react with amino acid residues of lipase that
participate in substrate binding, enzyme stability, or catalytic functions
and hence affect the enzyme activity [26,30]. According to a study
conducted by Subhedar and Gogate [25], the FAME yield was observed
to increase when the duty cycle was increased from 40 % (4 s on and 6 s
off) to 60 % (6 s on and 4 s off), followed by a constant yield as the duty
cycle was further increased to 70 %. Interestingly, there was no decrease
in the FAME yield at a high duty cycle [25]. This could be attributed to
the use of an immobilized enzyme in their study, which is more resistant

Fig. 2. Effect of ultrasonic duty cycle on FAME content. The reaction condi­ Fig. 3. Residual activity of ET2 with respect to unsonicated ET2 under (a)
tions were 40 % ultrasound amplitude, 2:1 methanol-to-oil molar ratio dosed in different ultrasound amplitudes and a constant 20 % duty cycle and (b)
2 h, 40 ◦ C, 0.2 wt% of free lipase, 2 wt% of water and 4 h of reaction. different ultrasound duty cycles and a constant 40 % amplitudes.

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M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

On the other hand, insignificant changes in the enzyme activity of spectrum of ET2 presented a positive peak at 193 nm and two broad
ET2 were observed when the ultrasound duty cycle was increased from negative peaks at 208 and 220 nm in the far-UV region, representing the
2.5 % to 10 % [see Fig. 3(b)]. However, a noticeable reduction in re­ structures of β-sheet and α-helix structures in ET2, respectively [19,31].
sidual activity was observed with further increments in the duty cycle to According to Fig. 4(a), the CD spectra of ET2 exposed to ultrasound with
20 % and 30 %, resulting in only 82 % and 73 % residual activity, amplitudes of 20 % and 40 % were nearly identical to that of the native
respectively. This observation demonstrated that the enzyme exhibited a ET2. However, at an amplitude of 60 %, the CD spectrum of ET2
relatively stable behavior within the lower range of duty cycles. How­ exhibited an upward shift in the peaks at 208 and 220 nm, suggesting a
ever, the prolonged exposure of lipase to the cavitation conditions loss of α-helix structures in ET2 and a transition to a random coil
showed a detrimental effect on the enzyme functionality. These findings conformation [32]. This decrease in α-helix content results in a less rigid
align with the FAME content results shown in Fig. 2 (Section 3.1.2), structure of the lipase due to the loss of hydrogen bonds within the
where the highest FAME content was achieved at 5 % and 10 % duty α-helix [17]. At an amplitude of 80 %, significant deviations were
cycles, indicating that ET2 demonstrated relatively high enzymatic ac­ observed in the peaks at 193 and 220 nm compared to the native lipase,
tivity during these duty cycles. accompanied by the loss of the peak at 200 nm. Furthermore, at a 100 %
ultrasound amplitude, the peaks at 195 and 208 nm disappeared, indi­
3.2.2. Circular dichroism cating a substantial alteration in the secondary structure of ET2 [32]. On
Circular dichroism (CD) spectroscopy was employed to quantita­ the contrary, as depicted in Fig. 4(b), the CD spectra of ET2 at different
tively evaluate the secondary structural changes of ET2 during soni­ duty cycles exhibited no significant changes in the peaks compared to
cation at different ultrasound intensities. As shown in Fig. 4, the CD native ET2. Hence, despite observing lower enzyme activity at higher
duty cycles, it can be concluded that the increase in ultrasound duty
cycles did not significantly impact the secondary structure of the lipase.

3.2.3. Intrinsic fluorescence

Fluorescence spectroscopy can be used to investigate the change in
the tertiary structure of protein due to the surface tryptophan emission,
which is highly sensitive to the polarity of the microenvironment along
the transition [27,33]. The fluorescence spectrum shifts to a shorter
wavelength, and the fluorescence intensity increases when the polarity
of the environment surrounding the tryptophan residue decreases
[34,35]. According to Fig. 5(a), a slight increase in fluorescence in­
tensity was observed for lower ultrasound amplitudes (e.g., 20 % and
40 %). However, upon sonication of the lipase at 60 % amplitude, a
significant increase in fluorescence intensity was observed, accompa­
nied by a shift in the optimal fluorescence emission wavelength towards
a shorter wavelength by 5 nm, compared to the unsonicated lipase. On
the contrary, the impact of the duty cycle on the change in fluorescence
intensity was relatively less pronounced compared to the amplitude,
with only a slight increase observed, as depicted in Fig. 5(b). The in­
crease in fluorescence intensity and the shift in wavelength may be
attributed to the partial unfolding of the native structure of lipase
induced by ultrasound. This process leads to the exposure of hydro­
phobic amino acids that are typically buried within the interior of pro­
teins to the protein surface, resulting in a less polar environment
surrounding the surface tryptophan [36,37].
Exposure of ET2 to excessive ultrasound (e.g., 80 and 100 %
amplitude) resulted in a decrease in the fluorescence intensity of the
protein, as shown in Fig. 5(a). This reduction at higher ultrasonic in­
tensities may be attributed to the quenching of tryptophan fluorescence
by H2O2, which was generated during the sonolysis of water molecules
under localized high temperature and violent pressure conditions [26].
As an oxidizing agent, H2O2 oxidizes tryptophan to produce kynurenine,
consequently leading to a decrease in fluorescence intensity [38]. This
observation aligns with the enzyme activity analysis in Fig. 3 (Section
3.2.1), where the decrease in enzyme activity, attributed to the lipase
structural modification, was reflected by the change in fluorescence
intensity. Consequently, this finding further supports the observed
reduction in FAME yield at high ultrasound amplitudes and duty cycles.
A similar trend was observed in the fluorescence intensity of α-amylase,
as reported by Yu et al. [36]. The fluorescence peak intensity exhibited
an increase as enzyme activity decreased, followed by a decrease to an
intensity lower than that of the native amylase with continuous soni­
cation [36]. Therefore, based on the above findings, the optimal ultra­
sound parameters, 40 % amplitude and 5 % duty cycle, proven to
enhance the reaction rate with minimal impact on enzyme activity, will
Fig. 4. Circular dichroism spectra of native ET2 and sonicated ET2 under (a) be employed in the subsequent studies.
different amplitudes with a constant 20 % duty cycle and (b) different duty
cycles with a constant 40 % amplitudes.

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3.3. Effect of methanol-to-oil molar ratio

Methanol, a short-chain alcohol, has been demonstrated to signifi­

cantly influence enzyme catalytic performance in the transesterification
reaction [6]. This study investigated the impact of the methanol-to-oil
molar ratio on FAME content in ultrasonic-assisted reactions. The con­
ventional stirring method served as a control for a comparative analysis
of ultrasound effects on methanol inhibition of lipase in enzymatic
biodiesel production. The predetermined methanol molar ratio was fully
dosed at a constant rate within the initial 2 h of the 4-h reaction period.
Fig. 6(a) and (b) depict the changes in FAME content during a 4-h
reaction under conventional stirring and ultrasonic irradiation, respec­
tively. The results showed that for both mixing methods, the initial rate
of reaction increased as the methanol-to-oil molar ratio increased from
1:1 to 5:1. This could be attributed to the presence of an excess
methanol-to-oil molar ratio at the beginning of the reaction, shifting the
equilibrium of the reversible transesterification reaction towards prod­
ucts formation and facilitating FAME synthesis [39]. According to Fig. 7,
an increase in the methanol molar ratio from 1:1 to 3:1 resulted in a
significant increase in the final FAME content (i.e., 16.3 wt% to 43.6 wt
% for conventional stirring and 26.7 wt% to 50.1 wt% for ultrasound
irradiation). However, beyond the 3:1 methanol molar ratio, it was
observed that the reaction kinetics of all methods exhibited a decline,
ultimately reaching a plateau, leading to a reduction in FAME content.
This may be due to the inhibitory effect of higher methanol concentra­
tion on the catalytic activity of the liquid lipase [40].
By comparing the FAME content between ultrasound-assisted and
conventional stirring reactions, it can be observed that the intensifica­
tion effect of ultrasound diminished with an increase in the methanol-to-
oil molar ratio (see Fig. 7). The ultrasound-assisted reaction showed a
decrease in the percentage improvement in final FAME content from
63.8 % to 14.9 % when the methanol molar ratio increased from 1:1 to
3:1. Interestingly, beyond a 3:1 methanol molar ratio, the conventional
stirring method outperformed the ultrasound irradiation, achieving
7.9 wt% and 10.8 wt% higher FAME content at 4:1 and 5:1 methanol-to-
oil molar ratios, respectively. Hence, it was speculated that ultrasound
not only promoted mixing between the reactants but also intensified the
methanol inhibition on lipase.

3.4. Effect of excess methanol

Fig. 5. Fluorescence spectra of native ET2 and sonicated ET2 under (a) The effect of ultrasound irradiation and conventional stirring on the
different amplitudes with a constant 20 % duty cycle and (b) different duty
methanol tolerance of the enzyme was investigated by evaluating the
cycles with a constant 40 % amplitudes.
instantaneous rate of reaction under each mixing method in the presence
of excess methanol. Excess methanol, referred to as unreacted methanol
throughout the reaction, was quantified by calculating its consumption

Fig. 6. Changes of FAME content at different methanol-to-oil molar ratios under (a) conventional stirring and (b) ultrasound irradiation at 40 % amplitude and 5 %
duty cycle. The reaction conditions were 40 ◦ C, 0.2 wt% of free lipase, 2 wt% of water and 4 h of reaction.

6
M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

Fig. 7. Effect of methanol-to-oil molar ratio on FAME content under conventional stirring and ultrasound irradiation (40 % amplitude and 5 % duty cycle). The
reaction conditions were 40 ◦ C, 0.2 wt% of free lipase, 2 wt% of water and 4 h of reaction.

via stoichiometric back-calculation at specific time intervals. This assisted reaction with a 3:1 methanol molar ratio (i.e., 50.1 wt%), a
calculation was based on the molar concentration of FAME produced, 2:1 M ratio resulted in a more significant improvement (i.e., from 35.9 to
with the assumptions of negligible loss of methanol through evaporation 47.2 wt% FAME content). Hence, a 2:1 methanol-to-oil molar ratio was
and constant reaction conditions. As depicted in Fig. 8, both conven­ determined to be the optimal ratio as it provided a sufficient excess of
tional stirring and ultrasound-assisted reactions demonstrated a similar methanol to facilitate FAME production while effectively minimizing
trend, where the reaction rate increased with increasing total excess enzyme inhibition. To introduce the remaining required methanol for
methanol. It can be observed that ultrasound-assisted reaction exhibited driving the reversible reaction towards the product formation, a two-
a higher maximum instantaneous rate of reaction and a lower optimal stage methanol dosing strategy was implemented.
excess of methanol compared to conventional stirring. However, beyond
its optimal range (i.e., 0.06 to 0.08 mol), the instantaneous rate of re­ 3.5. Two-stage methanol dosing strategy
action decreased and eventually became even lower than that of the
conventional stirring method. As a result, it was hypothesized that ul­ Based on the previous studies, the implementation of a two-stage
trasound significantly enhanced the reaction rate by improving the methanol dosing strategy in enzymatic biodiesel production was found
mixing and mass transfer between the reactants, but only at low meth­ to enhance conversion efficiency by optimizing reaction kinetics
anol concentrations within the lipase tolerance limit. At high methanol [42,43]. This strategy also effectively minimizes enzyme deactivation by
concentrations, the inhibitory effect of methanol on the enzyme was controlling methanol addition. According to Section 3.4, when the total
more significant in ultrasound-assisted enzymatic reaction compared to excess methanol exceeded the optimal range, the instantaneous rate of
conventional stirring. This may be due to the potential of ultrasound in reaction decreased, indicating that a higher FAME content cannot be
enhancing the methanol diffusion into the enzyme active sites, pre­ achieved by a further increase in methanol concentration in a single
venting the effective binding of the enzyme with reactants and reducing dose, due to the methanol inhibition effect surpassing the ultrasound-
the reaction rates [41]. As a result, this inhibition effect outweighed the induced rate acceleration. Therefore, a two-stage methanol dosing
rate acceleration effect by the ultrasound, leading to lower FAME con­ strategy was implemented, and the ultrasound-assisted reaction was
tent compared to conventional stirring. prolonged to 12 h. A total methanol-to-oil molar ratio of 4:1 was used in
Although the highest FAME content was achieved in an ultrasound- order to promote the reaction while minimizing enzyme inhibition. In
Stage 1, methanol was dosed uniformly at the optimal 2:1 M ratio for the
initial 2 h. In Stage 2, the remaining 2:1 M ratio was dosed at different
flow rates and completely dosed within the subsequent 2, 4 and 6 h. The
total dosing time for the 4:1 methanol molar ratio was 4, 6, and 8 h in
this study.
Based on the findings depicted in Fig. 9(b), it was shown that
reducing the methanol dosing flow rate in the second stage led to an
increase in the final FAME content, from 75.6 to 79.9 wt%, when the
total dosing time was increased from 4 to 6 h. This result suggested that
the risk of enzyme inhibition by methanol was reduced when the
methanol concentration was distributed over a longer duration [44].
However, a further decrease in the methanol flow rate by extending the
total dosing time to 8 h did not significantly improve the final FAME
content, with 80.9 wt% of FAME content obtained. Furthermore, it can
be seen that the FAME content during the 3 to 7-h interval was slightly
higher when the total dosing time was 6 h compared to 8 h [see Fig. 9
(a)]. This could potentially be attributed to the higher amount of excess
methanol at the interval when methanol was supplied over a shorter
Fig. 8. Changes in the instantaneous reaction rate under different excess duration of 6 h. The synergetic effect between ultrasound and excess
methanol-to-oil molar ratios during conventional stirring and ultrasound irra­ methanol could facilitate the equilibrium shift and react further with all
diation (40 % amplitude and 5 % duty cycle). The reaction conditions were the available triglycerides [45]. In comparison, the slower dosing rate of
40 ◦ C, 0.2 wt% of free lipase, 2 wt% of water and 4 h of reaction. methanol over 6 h in the second stage (i.e., total dosing time of 8 h)

7
M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

3.6. Effect of enzyme concentration

In the ultrasound-assisted enzymatic biodiesel production process,

the concentration of enzymes has a significant impact on the achievable
FAME content, as it directly affects the number of active sites available
for the reaction and thereby influences the reaction rate. Fig. 10(a)
shows that the initial rate of reaction increased as the enzyme concen­
tration increased from 0.2 to 2.0 wt%, with a significant enhancement
explicitly observed in the range of 0.2 to 0.5 wt%. Additionally, the final
FAME content increased substantially from 79.9 to 96.6 wt%, with an
increase in the enzyme concentration from 0.2 to 1.0 wt%. Nevertheless,
higher concentrations up to 2.0 wt% yielded negligible changes in the
final FAME content [see Fig. 10(b)]. When the enzyme concentration is
low (i.e., 0.2 wt%), the reaction rate in ultrasound-assisted biodiesel
production may be limited by the availability of active enzyme sites,
resulting in slower reaction rates and lower FAME yields. Increasing the
enzyme concentration could overcome this limitation by providing more
enzyme active sites, which may increase the reaction rate and the
product yield. Additionally, increasing the enzyme concentration could
also help to maintain a high reaction rate throughout the course of the
reaction. The prolonged exposure to ultrasound may result in enzyme

Fig. 9. Effect of methanol dosing strategies on (a) FAME content over time and
(b) final FAME content. The reaction conditions were 40 % ultrasound ampli­
tude, 5 % ultrasound duty cycle, 2:1 methanol-to-oil molar ratio dosed in 2 h
(Stage 1), 40 ◦ C, 0.2 wt% of free lipase, 2 wt% of water and 12 h of reaction.

might limit the availability of excess methanol, resulting in a slower rate

of reaction. Nevertheless, as the reaction time progressed and the
methanol was dosed entirely by the 8th h, both strategies eventually
resulted in a similar amount of excess methanol, resulting in a compa­
rable FAME content.
Additionally, it is noteworthy that when a total molar ratio of 4:1 was
used under the implementation of the two-stage dosing strategy in the
ultrasound-assisted reaction, a substantial enhancement in the reaction
kinetics was observed, as demonstrated by comparing Fig. 6(b) and
Fig. 9(a). In the single-stage dosing reaction, the reaction kinetics started
to plateau at 2 h with only 27 wt% of FAME content obtained [see Fig. 6
(b)]. However, in the two-stage dosing reaction, with a 2:1 methanol-to-
oil molar ratio dosed over the first 2 h and another 2:1 M ratio dosed
over 4 h, the reaction kinetics continued to increase and achieved 80 wt
% of final FAME content at the 12th h. This difference in performance
could be attributed to the distribution of methanol concentration across
two stages in the two-stage dosing reaction [21]. Therefore, imple­
menting a two-stage dosing strategy in ultrasound-assisted biodiesel
production reduced the lipase’s overall exposure to methanol, lowering
the risk of enzyme inactivation and leading to a more efficient oil con­ Fig. 10. Effect of enzyme concentration on (a) FAME content over time and (b)
version to FAME. final FAME content. The reaction conditions were 40 % ultrasound amplitude,
5 % ultrasound duty cycle, 2:1 methanol-to-oil molar ratio dosed in 2 h (Stage
1) followed by additional 2:1 methanol-to-oil molar ratio dosed in 4 h (Stage 2),
40 ◦ C, 2 wt% of water and 12 h of reaction.

8
M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

deactivation over a period of time, leading to a decrease in both reaction due to the formation and collapse of bubbles during cavitation, which
rate and product yield [46]. Therefore, increasing the enzyme concen­ produced strong localized liquid jets and high-speed liquid micro
tration could counteract this effect by ensuring the availability of streams [9,48]. These fluid movements created a highly efficient mixing
adequate active sites to catalyze the reaction continuously. environment, promoting the dispersion of reactants and facilitating
However, it was speculated that substrate limitation occurred at high contact between the reactants [49]. Consequently, the interfacial sur­
enzyme concentrations due to an excess of enzymes compared to the face area between the reactants increased, allowing for more frequent
available substrate molecules. Consequently, the enzyme’s active sites and efficient collisions between molecules. This enhanced mass transfer
became saturated with substrate molecules [47]. As a result, further could facilitate faster reaction kinetics and higher reaction rates
increasing the enzyme concentration to 2.0 wt% had minimal effect on compared to conventional stirring, where mixing is generally less
the reaction rate and the product yield. Sivaramakrishnan and Inchar­ efficient.
oensakdi [46] reported a similar result in which the FAME yield Additionally, ultrasound irradiation significantly shortened the re­
increased when the enzyme concentration was increased from 5 wt% to action time compared to conventional stirring in the enzymatic biodiesel
10 wt%, and insignificant improvement in FAME yield was observed production process. With a FAME content of 90 %, conventional stirring
above his level of enzyme dosage [46]. Although there was a negligible required 24 h of reaction time, whereas ultrasound irradiation reduced
effect on the final FAME yield, it is worth noting that the utilization of a this to only 8 h. This substantial reduction underscores the enhanced
higher concentration, 2.0 wt%, exhibited an effective reduction in the productivity facilitated by ultrasound irradiation. Despite the additional
reaction time to 8 h while achieving a product yield of 97.1 % for the operating cost associated with the electricity-powered ultrasound sys­
ultrasound-assisted reaction. Despite the demonstrated advantages of tem potentially leading to a reduction in profit per run, the increased
using an enzyme concentration of 2.0 wt%, which reduced the reaction productivity enabled the synthesis of a greater amount of FAME within
time to reach equilibrium and achieve the highest yield, it is important the same timeframe. This enhanced productivity is expected to result in
to consider the potential challenges associated with high enzyme con­ higher overall profits in the long run.
centration in downstream processing steps, such as enzyme separation Gholami et al. [50] conducted a comprehensive techno-economic
and recovery [21]. These challenges may result in additional efforts and evaluation of biodiesel production from canola oil using ultrasound
costs involved in the purification process, ultimately reducing the eco­ technology. Their findings highlighted the cost-effectiveness and oper­
nomic feasibility of the overall process of biodiesel production. ational advantages of the ultrasonic process over the conventional stir­
ring method. The ultrasonic process demonstrated a 20.8 % lower total
investment, a 23.5 % reduction in equipment cost, a 5.2 % decrease in
3.7. Comparison of conventional and ultrasound-assisted biodiesel
production cost, and a 6.9 % reduction in energy consumption compared
production catalyzed by Eversa® Transform 2.0
to the mechanical stirring process [50]. Despite its cost-effectiveness,
scaling up biodiesel production using ultrasound technology encoun­
A comparison was made between the results obtained from the
ters several limitations. These include difficulties in designing efficient
conventional stirring and ultrasound-assisted biodiesel production in
ultrasonic reactors due to complex flow patterns and non-uniform
this study. Based on Fig. 11, it can be observed that the ultrasound-
acoustic fields [8,51]. Moreover, changes in the reaction mixture’s
assisted reaction exhibited a significantly higher rate of reaction
rheological properties may influence bubble dynamics, consequently
compared to the conventional stirring method. Incorporating ultrasound
affecting the reaction [51]. Thus, further research is necessary to address
technology into the biodiesel production process, catalyzed ET2, resul­
the limitations of ultrasonic systems for large-scale enzymatic biodiesel
ted in a significant improvement of 15 % in the final FAME content, as
production.
demonstrated by an increase in the FAME content from 80.2 to 92.2 wt
%. This result agreed well with the literature, as Gharat and Rathod [13]
4. Conclusion
reported a similar observation where a FAME yield of 57.68 % was
obtained at the 4th h reaction time under ultrasound irradiation,
The present work reported the transesterification of simulated low-
whereas the conventional stirring method only yielded 38.7 %. This was
quality feedstocks under ultrasound irradiation, using a low-cost
liquid lipase Eversa® Transform 2.0 (ET2). The study determined that
100 92.2 the optimal ultrasound amplitude and duty cycle were 40 % and 5 %,
respectively, showcasing their potential to enhance the reaction rate
80.2 while exerting minimal impact on enzyme activity. Under these opti­
80 mized conditions, 95 % of the enzyme activity of ET2 was retained,
FAME content (wt%)

whereas higher ultrasound intensities resulted in a significant reduction

in enzyme activity (i.e., a reduction ranging from 13 to 40 %). The
60
evaluation of lipase conformation through circular dichroism (CD) and
fluorescence spectroscopy revealed the preservation of the secondary
structure and slight modifications in the tertiary structure at the opti­
40
mized condition. By employing the optimal ultrasound parameters,
biodiesel with a fatty acid methyl ester (FAME) content of 92.2 wt% was
20
Ultrasound irradiation achieved within 12 h. This was achieved with a low methanol-to-oil
molar ratio of 4:1 and a low enzyme concentration of 0.5 wt%. The
Conventional stirring
ultrasound-assisted reaction demonstrated a significant enhancement in
0 the initial rate of reaction compared to the conventional stirring method,
0 2 4 6 8 10 12 resulting in a 15 % improvement in the final FAME content. Therefore,
despite observing a lower enzyme activity (i.e., 95 % residual activity) at
Time (h) the optimal ultrasound intensity, the enhanced mixing effect of ultra­
Fig. 11. Comparison of FAME content under ultrasound irradiation (40 % sound during transesterification outweighed the inhibitory impact on
amplitude and 5 % duty cycle) and conventional stirring method. The reaction ET2, resulting in a higher FAME content. In conclusion, this study
conditions were 2:1 methanol-to-oil molar ratio dosed in 2 h (Stage 1) followed highlighted the potential of ultrasound technology in enhancing the
by an additional 2:1 methanol-to-oil molar ratio dosed in 4 h (Stage 2), 40 ◦ C, efficiency of biodiesel production while providing crucial insights into
0.5 wt% of free lipase, 2 wt% of water and 12 h of reaction. structural changes within ET2 during ultrasound treatment. Future

9
M.Y. Liow et al. International Journal of Biological Macromolecules 271 (2024) 132538

research could focus on further enhancing the biodiesel yield by [10] P. Adewale, M.-J. Dumont, M. Ngadi, Enzyme-catalyzed synthesis and kinetics of
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CRediT authorship contribution statement [13] N. Gharat, V.K. Rathod, Ultrasound assisted enzyme catalyzed transesterification of
waste cooking oil with dimethyl carbonate, Ultrason. Sonochem. 20 (3) (2013)
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Min Ying Liow: Writing – original draft, Validation, Investigation, [14] N. Yasvanthrajan, P. Sivakumar, K. Muthukumar, T. Murugesan, A. Arunagiri,
Formal analysis, Data curation. Eng-Seng Chan: Writing – review & Production of biodiesel from waste bio-oil through ultrasound assisted
editing, Supervision, Methodology, Formal analysis, Conceptualization. transesterification using immobilized lipase, Environ. Technol. Innov. 21 (2021)
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Declaration of competing interest ultrasonic irradiation and stability studies, Ind. Eng. Chem. Res. 53 (4) (2014)
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[17] S. Mojtabavi, M. Hervé, H. Forootanfar, H. Jafari-Nodoushan, G. Sharifian,
The authors declare that they have no known competing financial N. Samadi, A. Ameri, M.A. Faramarzi, A survey on the stabilizing effect of
interests or personal relationships that could have appeared to influence osmolytes on the ultrasound-irradiated lipase for efficient enzymatic hydrolysis of
coconut oil, Colloids Surf. B: Biointerfaces 220 (2022) 112910, https://doi.org/
the work reported in this paper. 10.1016/j.colsurfb.2022.112910.
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Data availability commercial lipases, Appl. Biochem. Biotechnol. 181 (4) (2017) 1435–1453,
https://doi.org/10.1007/s12010-016-2294-2.
[19] F.C. Fraga, A. Valério, V.A. de Oliveira, M. Di Luccio, D. de Oliveira, Effect of
Data will be made available on request. magnetic field on the Eversa® Transform 2.0 enzyme: enzymatic activity and
structural conformation, Int. J. Biol. Macromol. 122 (2019) 653–658, https://doi.
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Acknowledgments [20] S. Sun, J. Guo, X. Chen, Biodiesel preparation from Semen Abutili (Abutilon
theophrasti Medic.) seed oil using low-cost liquid lipase Eversa® transform 2.0 as a
The authors would like to thank the Ministry of Higher Education catalyst, Ind. Crop. Prod. 169 (2021) 113643, https://doi.org/10.1016/j.
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(MOHE) Malaysia for providing funding through Fundamental Research [21] J.M. Loh, Amelia, W. Gourich, C.L. Chew, C.P. Song, E.-S. Chan, Improved
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Ying Liow would also like to thank Monash University Malaysia for under low-input process conditions, Renew. Energy 177 (2021) 348–358, https://
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