The combine use of ultrasound and lipase immobilized on co-polymer matrix for efficient biocatalytic application studies

Article abstract of DOI:10.1016/j.molcatb.2015.09.012

In this work, we have investigated the combine use of ultrasound and lipase (Pseudomonas cepacia: PCL) immobilized on co-polymer of polyvinyl alcohol (PVA) and chitosan (CHI) for biocatalytic applications. Initially, we have screened different free and immobilized biocatalysts to find-out the robust biocatalyst. The immobilized biocatalyst PVA:CHI:PCL (5:5:2.5) worked as a robust biocatalyst to provide superior conversion (99%) for the synthesis of model ultrasound assisted reaction. Subsequently, various reaction parameters were optimized in details to obtain the higher yield. Besides this, developed biocatalytic protocol was used to synthesize various industrially important butyrate compounds which provided excellent conversion of 99% under ultrasonic conditions. The developed biocatalyst showed excellent recyclability upto studied five cycles under ultrasonic condition. The immobilized PVA:CHI:PCL biocatalyst displayed 2.4 folds higher activity as compared to free lipases in ultrasonic condition. Moreover, PVA:CHI:PCL biocatalyst in ultrasound media showed 4.5 folds higher activity as compared to free lipases in conventional media. The energy assessment was performed which demonstrated feasibility of combine use of immobilization and ultrasonication to carry out efficient biocatalytic process.

Full text of DOI:10.1016/j.molcatb.2015.09.012

Accepted Manuscript
Title: The combine use of ultrasound and lipase immobilized
on co-polymer matrix for efficient biocatalytic application
studies
Author: Kirtikumar Chandulal Badgujar Bhalchandra
Mahadeo Bhanage
PII:
S1381-1177(15)30073-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.09.012
MOLCAB 3244
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
3-5-2015
14-9-2015
21-9-2015
Please cite this article as: Kirtikumar Chandulal Badgujar, Bhalchandra Mahadeo
Bhanage, The combine use of ultrasound and lipase immobilized on co-polymer
matrix for efficient biocatalytic application studies, Journal of Molecular Catalysis B:
Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.09.012
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The combine use of ultrasound and lipase immobilized on co-
polymer matrix for efficient biocatalytic application studies
Kirtikumar Chandulal Badgujar and Bhalchandra Mahadeo Bhanage∗
Department of Chemistry, Institute of Chemical Technology, Matunga,
Mumbai 400 019, India
∗Corresponding author (B. M. Bhanage): Tel.: +91-22-3361-2601/2222; Fax: +91-22-2414-
5614; Email address: bm.bhanage@ictmumbai.edu.in
bm.bhanage@gmail.com
bhalchandra_bhanage@yahoo.com
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Graphical abstract
2

Research Highlights
 Lab made immobilized PVA:CHI:PCL lipase was used as a biocatalyst in ultrasound
 Comparative optimization of 14 reaction factors under sonication/conventional mode
 Sonication & immobilization improve biocatalytic activity by 4.5 folds
 Biocatalyst showed excellent substrate array & recyclability under sonication
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Abstract:
In this work, we have investigated the combine use of ultrasound and lipase (Pseudomonas
cepacia: PCL) immobilized on co-polymer of polyvinyl alcohol (PVA) and chitosan (CHI)
for biocatalytic applications. Initially, we have screened different free and immobilized
biocatalysts to find-out the robust biocatalyst. The immobilized biocatalyst PVA:CHI:PCL
(5:5:2.5) worked as a robust biocatalyst to provide superior conversion (99 %) for the
synthesis of model ultrasound assisted reaction. Subsequently, various reaction parameters
were optimized in details to obtain the higher yield. Besides this, developed biocatalytic
protocol was used to synthesize various industrially important butyrate compounds which
provided excellent conversion of 99 % under ultrasonic conditions. The developed biocatalyst
showed excellent recyclability upto studied five cycles under ultrasonic condition. The
immobilized PVA:CHI:PCL biocatalyst displayed 2.4 folds higher activity as compared to
free lipases in ultrasonic condition. Moreover, PVA:CHI:PCL biocatalyst in ultrasound media
showed 4.5 folds higher activity as compared to free lipases in conventional media. The
energy assessment was performed which demonstrated feasibility of combine use of
immobilization and ultrasonication to carry out efficient biocatalytic process.
KeyWords: Biocatalysis; Immobilized lipase; Ultrasound; Improved activity
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1.
Introduction
The wide industrial demand and sustainable technology always encourage to
researchers to develop new eco-friendly synthetic pathways for production of the important
organic moieties which are widely used in pharmaceutical, flavour, fragrance, cosmetic,
toiletries, foods, beverage and various home-care products [1]. In view of this, biocatalysis
received special attention, since biocatalysis engaged natural enzymes for synthesis [1,2]. In
particular, enzyme lipases in biocatalysis attracted significant attention as a potent biocatalyst
due to its broad substrate array and ability to succeed various promiscuous organic
transformations in various reaction media [1-3]. However, sensitive proteneous nature of
enzymes always hampers their direct applications as a potential biocatalyst in organic media
[4]. Moreover, application of the crude (or free) enzyme has a number of disadvantages such
as no recyclability, lower activity-stability to temperature and organic solvent [5-9]. Hence,
massive efforts have been taken by number of researchers to constrain above said difficulties
and to improve/enhance the activity-stability of enzymes by the use of various skilful
immobilization techniques [1-10].
In this work, we have fabricated a binary hybrid blend of chitosan (CHI) and poly
vinyl alcohol (PVA) polymer for lipase immobilization which is further used for biocatalytic
applications. Chitosan is a polysaccharide material having straight chain β-1,4-linkage and
possessing a number of useful features for immobilization such as high mechanical strength,
adhesivity, biodegradability, lack of toxicity and most important is the inertness to chemical
reactivity/organic solvents [10]. The amino groups of CHI can perform the covalent
immobilization, which may avoids enzyme leakage. PVA is a synthetic polymeric material,
widely used in the biomedical and biochemical engineering appliances which endowed
excellent film forming and blending properties like biocompatibility, high tensile strength,
high flexibility, high interfacial adhesion, non-toxicity and inertness to organic solvent/
chemicals [4-7]. The inertness means, the support should not be react with the substrate or
product of the reaction, which is unique property of an ideal support [11].
Herein, we have immobilized bacterial lipase Pseudomonas cepacia (PCL) on a
hybrid blend of PVA:CHI, which will contribute individual advantages/ properties of CHI
and PVA into a single hybrid material. Moreover, the protocol of immobilization is simple
and the presence of free hydroxyl and amino functionality recommend a higher extent of
immobilization ability, which formulate a PVA:CHI carrier more attractive and perfect for
the lipase PCL immobilization [12]. The lipase PCL was immobilized into PVA:CHI support
5

via entrapment methodology since, entrapment of enzymes into a carrier matrix was carried
out at the time of support PVA-CHI matrix formation [2-5]. Some lipases like cepacia can
forms dimers in solution which are usually displayed lower catalytic activity than standard
enzyme since; active centres of enzymes are partially blocked [24]. Hence immobilization
becomes a necessary job to improve the catalytic activity [13-24]. Immobilization can
improve the catalytic activity of lipase via interfacial activation [13-24]. Also, lipases are
well scattered after immobilization and agglomeration of lipase was no longer possible [13-
24]. Immobilization can shield enzyme molecules from the direct exposure to outer reaction
environment which tends to enhance stability of biocatalyst in reaction media [13-24].
Immobilization can improve the operational stability (reusability) also [13-24]. However
sometimes, immobilization of enzyme may hamper the mass transfer diffusion of substrate to
the active sites and lowers the reaction rate which is still remains an unresolved concern [2-
5,25]. Hence, there is need of technology to accelerate the rate of enzyme catalysed reaction.
Ultrasound or sonochemical assisted synthesis is a newly emergent technology for the
process intensification [12,26]. The frequency of ultrasound travel through a liquid medium
leads to create bubbles during multiple cycles of compression and rarefaction [25-27]. These
bubbles undergo the sudden expansion and distraction to produce tremendous amount of local
heat and pressure which is responsible for higher mass transfer diffusion and acceleration of
reaction rate [25-28]. Thus, ultrasonication is considered as a best technique to overcome the
mass transfer limitation and to improve the reaction rate [25-29]. Moreover, the ultrasonic
irradiation is considered as a greener technology due to its superior efficiency, gentle reaction
condition and safer use [25-29]. Thus, ultrasound can be the best option to enhance reaction
rate (intensification) for organic synthesis [25-31]. In literature, very few reports are present
for the ultrasound assisted lipase catalyzed synthesis for production of commercially valuable
esters [28-34]. Most of reports focused on use of commercially available Novozyme 435
catalyzed ultrasound assisted synthesis [28-34]. More recently, Rodrigues et al. [35] used
combination of immobilized lipase (lecitase-ultra immobilized on styrene-divinylbenzene
beads) and ultrasound for esterification reaction. In present study, we immobilized lipase into
PVA/CHI carrier and this developed biocatalyst applied for synthesis of valuable butyrate
ester using ultrasound sonication as a greener technology.
Benzyl butyrate is a pale yellow liquid with a fruity, odour and widely used as/in
decorative cosmetics, creams, fragrances, shampoos, toiletries, detergents and various house-
hold cleaners [36,37]. Council of Europe permitted safe use of the benzyl butyrate in
foodstuffs and food ingredients [37]. It is used as a flavour and fragrance ingredient for the
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grapes, hog plum pear, peach, papaya, pineapple, raspberry, orchid etc., whereas it is
naturally occurs in various plantations like Spondias mombins, Osmanthus fragrans,
Vasconcellea pubescens, Grape cherimoya, Michelia champaca etc [36-38]. The estimated
worldwide use of the benzyl butyrate was almost ten to hundred metric tons per year [36-38].
Thus, in view of the extensive scope and commercial importance of benzyl butyrate, we make
an effort to investigate the ultrasound assisted synthesis of benzyl butyrate catalyzed by
PVA:CHI:PCL biocatalyst. Thus, present study deals with (i) synthesis of polymer supported
biocatalyst for benzyl butyrate using ultrasonic irradiation (ii) detail optimization of various
sonication/reaction factors and (iii) recyclability study.
2.
Material and methods
2.1
Chemicals and Enzymes
Lipases PCL (Pseudomonas cepacia, activity ≥ 23000 U/g, aggregated powder form),
lipases CCL (Candida cylindracea, activity ≥ 2000 U/g, aggregated powder form), CHI
(Brookfield viscosity >200), PVA (MW 9000-10000), benzyl alcohol, ethyl-, propyl-,
isoamyl alcohol and all other chemicals were purchased from Sigma Aldrich Pvt. Ltd. India.
2.2
Method of immobilization
Lipase was immobilized into PVA:CHI biocompatible matrix very simply in water at
room temperature. CHI (0.5 g) was dissolved in distilled water (1.5 % w/w glacial acetic acid
solution) while PVA (0.5 g) was dissolved in distilled water in a separate beaker and stirred
at 1400-1500 rpm for almost 2 h. Later, PVA solution was slowly added into the CHI
solution and vigorously stirred for 4-5 h at 1800-2000 rpm. The parent lipase (0.25 g)
dissolved in distilled water (5-6 mL) was added to the PVA:CHI blend and stirred moderately
at 180-200 rpm for 1 h. The immobilized polymer matrix was then carefully decanted into a
Teflon petri-plate and allowed to dry at 44-45 °C for 44-48 h. A plane matrix of biocatalyst
PVA:CHI:lipase was formed; which was then cut-off into small pieces of 4-6 mm2 size and
o
stored at 10-12 C in deep freeze. Thus, by calculation 1.25 g of immobilized matrix consist
of 0.25 g of free lipase, while this composition was denoted as CHI:PVA:PCL (5:5:2.5).
2.3
Reaction process/ procedure in ultrasonication technique
Initially, 30 mL glass reactor equipped with a mechanical stirrer was clamped at the
centre of ultrasonic bath, nearly 4 cm below the surface of water present in ultrasonic bath. In
this reaction, benzyl alcohol (10 mmol) was added, which is followed by addition of the
7

solvent iso-octane. Afterwards, specified butyrate donor (27 mmol) was added in it and
reaction was initiated by addition of the immobilized PVA:CHI:lipase biocatalyst. The
mechanical stirrer speed, duty cycle, power and frequency were kept maintained as
mentioned, along with specified temperature of the bath.
2.4
2.5
Reaction procedure in conventional technique
Reaction procedure is same as above without ultrasonication.
Analysis tool and methodology
To check the reaction progress, reaction mass sample (10 μL) was withdrawn
periodically from the reactor and further analyzed by use of the Perkin-Elmer, Clarus: 400
gas chromatograph equipped with a flame ionizing detector (FID) and Elite capillary column
(1.30 m × 0.32 mm × 0.25µm). The temperature of injector and detector were kept 50 and
250 °C, respectively. The butyrate products were confirmed by gas chromatography-mass
spectroscopy (GC-MS, Shimadzu QP- 2010) analysis.
2.6
Determination of influence of sonication on the lipase activity assay
Enzyme activity assay was performed by slight modification in the Poojari and
Clarson method. [39] The enzyme activity was determined by the esterification of 1 mmol of
1-octanol and vinyl laurate (1:1 mole ratio) catalyzed by 15 mg of free lipase PCL (or
equivalent quantity of immobilized lipase) in 1 ml of iso-octane at 50 oC for 1 h. The amount
of the product octyl laurate formed was analyzed by GC (Gas chromatography) analysis. The
one unit (U) enzyme activity was defined as, the amount of octyl laurate produced per hour
by per mg of enzyme at given specified conditions. The residual enzyme activity was defined
as the ratio of unit activity of ultrasound treated lipase to non-ultrasound treated lipase.
3. Result and Discussion
3.1
Screening of various immobilized and free lipases to achieve robust biocatalyst
Initially various lipases were screened to get robust biocatalyst for the corresponding
transformation of benzyl butyrate synthesis (Scheme 1; table 1). In screening we found two
observations as: (i) for reaction processing technique, ultrasound assisted system provided
better conversion compared to the conventional technique and (ii) immobilized biocatalyst
provided higher conversion compared to free lipase. Thus it was observed that, in each case
the conversion obtained was higher when we used (i) ultrasound irradiation and (ii)
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immobilized biocatalyst combinely. Herein present study, we have selected the immobilized
PVA:CHI:PCL as a screened robust biocatalyst which provided highest % conversion (98 %)
at the prescribed reaction conditions and used further for all remaining reactions (Table 1).
Please insert table 1 here
Please insert scheme 1 here
3.2
Effect of biocatalyst composition
Different quantities of the lipase PCL was loaded on a fixed amount of PVA:CHI
(5:5) support to get the robust biocatalyst composition with highest catalytic activity (Figure
1A). Hence, various PVA:CHI:PCL immobilized biocatalyst compositions (5:5:0 -to- 5:5:3.5)
were studied for benzyl butyrate synthesis under similar reaction parameters. It was observed
that, as amount of lipase loading on PVA:CHI (5:5) support increased then, corresponding
initial reaction rate (Ro) and % conversion for benzyl butyrate synthesis also increased. These
results were attributed to presence of more catalytic sites with higher lipase loading [40]. The
PVA:CHI:PCL (5:5:2.5) provided 99 % conversion of the desired product via ultrasound
assisted synthesis. Further increase in amount of lipase loading (5:5:2.5 and 5:5:3.5) showed
marginal increase in Ro and almost similar % conversion; which may be attributed due to (i)
presence of excess catalytic sites at higher lipase loading and/or (ii) hinder mass transfer
limitations at higher lipase loading in the heterogeneous biocatalytic system [40].
In contrast to this conventional heating showed increase in % conversion with
increase in lipase loading on PVA:CH (5:5) support. The PVA:CHI:PCL (5:5:2.5) provided
57 % conversion of the desired product via conventional technique in 3 h. The higher activity
after ultrasonication was due to effective mass transfer because of the ultrasonic cavitation,
while conventional technique has lower mass transfer effect and hence provided lower
reaction rate and conversion [41]. Similar type of the results was found to Zheng et al. for
synthesis of phytosterol compound synthesis [42].
Please insert figure 1 here
3.3
Effect of acyl donor
In chemical catalysis, acid was used more commonly as an acyl donor for synthesis of
the acylated compounds [42]. Nevertheless, acid worked as a powerful inhibitor of the
enzyme activity and hence provided lower conversion (Figure 1B). To evade acid inhibition,
various alkyl butyrate compounds are usually preferred for butyrate compounds synthesis
[43]. However, use of alkyl butyrate (methyl or ethyl butyrate) liberated respective alcohol as
9

a by-product and inhibits the rate of desired benzyl butyrate synthesis [9,43]. The above said
complications can be overcome by use of activated vinyl butyrate as a butyrate donor [9,43].
Vinyl butyrate liberating vinyl alcohol as a by-product and this vinyl alcohol tautomerized
into acetaldehyde which did not further take part in the reaction. Herein also, ultrasonication
had more influence on the % conversion compared to conventional technique for various
butanoyl donor studies. Thus, vinyl butyrate provided the maximum conversion (99 %) for
benzyl butyrate synthesis and was continued for further study.
3.4
Effect of solvent
It is significant to note-down that, enzyme catalytic activity was strongly affected by
the nature (polar/ non-polar) of organic solvents [5,9,25]. In the hydrophilic solvent, enzymes
might lose their structural and conformational flexibility because of lacking or stripping of
essential bound water layer [3,7,9,25]. Moreover, the low-vapour pressure of solvent has
superior sonication effect than high-vapour solvent [44]. Therefore it is a critical task to
choose the best solvent under sonication effect, since combination of the polarity and vapour
pressure of solvent may combinely affect the reaction rate [3,5,7,9,25,44]. Hence, in the
present work, influence of several non-aqueous solvents was tested for given model reaction
in ultrasonic and conventional mode (Figure 2A). In present study it was observed that, non-
polar solvent have good compatibility with enzyme activity while, non-polar low vapour
pressure solvent (iso-octane and toluene) provided higher conversion (≈ 99 %) of the desired
product as compared to n-hexane. These non-polar low vapour pressure solvent (iso-octane
and toluene) reduces the implosion energy of bubbles which is mainly associated with the
vapours pressure prior to breaking up [44]. Moreover, essential water coating around the
enzyme remains intact and maintain the enzyme flexibility as well as catalytic activity
[3,7,9,25]. Thus the iso-octane was the solvent of choice which was used for further study.
Please insert figure 2 here
3.5
Effect of biocatalyst amount
To study the effect of biocatalyst amount, various batches were put up
simultaneously. Practically, no conversion was observed when no biocatalyst was used. From
the above observation it was cleared that, cavitation alone is not sufficient to perform the
reaction (Figure 2B). The biocatalyst was solely responsible for the reaction, while ultrasonic
waves assist to accelerate the reaction rate [41-43]. The continuous bubble formation and its
collapse eliminated the mass transfer resistance and increases subsequent events of collisions
10

[28-31]. The % conversion increases as the biocatalyst amount increases upto the 300 mg.
The higher biocatalyst amount assisted rapid enzyme-substrate complex formation which
resulted into the higher conversion [1-5]. However, excess use of the biocatalyst amount
above 300 mg showed marginal increase in the Ro, while it did not have significant effect on
the % conversion, which may be attributed due to presence of the excess catalytic sites and
hindered mass limitation at higher biocatalyst loading [31,40]. Moreover in contrast to
conventional technique (58 %), the ultrasound (99 %) assisted system provided almost two-
fold higher % conversion at the use of 300 mg of biocatalyst amount.
3.6
Effect of agitation speed
The mechanical stirring assists to improve mass transfer of reacting molecules to the
active catalytic sites and subsequent reaction rate [31-33]. The present study clearly showed
that, ultrasound alone (without stirring, at 0 rpm) offered better % conversion (41 %) because
of the improved mass-transfer which indicated a positive role of ultrasound (Figure 3A).
Thus, physical turbulence created due to only cavitation (without stirring, at 0 rpm) is not
enough to produce efficient conversion [31-34]. In contest to this, the conventional technique
alone provided sluggish conversion (6 %) of desired product at 0 rpm; while moderate
conversion (56 %) was obtained at 800 rpm by conventional mode only.
Thus, mechanical agitation along with ultrasonication is necessarily required to create
a proficient mass transfer and subsequent improvement in reaction rates [31-34]. In the
present study, mechanical stirring under ultrasonication increases % conversion (0-800 rpm).
Thus, the speed of agitation with ultrasonication has profound effect on the % conversion.
The increase in the stirring speed from 800 to 1000 rpm had insignificant effect on the Ro
and % conversion. Further, increase of stirring speed from 1000 to 1500 rpm showed slight
decrease in the Ro and % conversion. The biocatalyst particles are thrown outside of the
reaction mass and did not incorporate in reaction at higher stirring speed which results into
the lowering of conversion rate. The individual conventional technique provided almost 56 %
conversion at 800 rpm and required 1000 rpm speed to get efficient conversion.
Thus, ultrasound can provided better mass transfer at sufficient stirring speed of 800
rpm to achieve better conversion. Thus, 800 rpm was the optimized stirring speed which
provided highest % conversion and utilized for remaining all experiments. Similar type of
improved activity was observed to Waghmare et al. after use of mechanical stirring [33].
Please insert figure 3 here
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3.7
Effect of mole quantity of alcohol
Study of the mole quantity of reactants is an important factor to determine the
influence of both reactants on enzyme activity and subsequent reaction rate. Hence
investigation of this factor was checked by varying mole quantity of alcohol (benzyl alcohol)
from 7 to 12 mmol by keeping other reaction parameter constant. Figure 3B showing that,
conversion increased as mmol of benzyl alcohol increases from 7 to 10 mmol; while further
increase in benzyl alcohol quantity (from 10 to 12 mmol) decreases the % conversion. This
observation concluded that, higher amount of benzyl alcohol causes inhibition of enzyme
activity and reduces the rate of reaction. Moreover it was seen that, cavitation effect offered
almost two-fold higher conversion (99 %) as compared to conventional mode (55 %). This
higher conversion may be attributed to cavitation and mass transfer effect towards the
reaction mixture which is helpful for uniform mixing of both reactants in solvent without
disturbing the micro-aqueous environment of enzyme. Such type of superior cavitation and
mass transfer effect may not possible in conventional methodology which showed inhibition
effect on enzyme activity from 9 mmol, while ultrasonic technique showed inhibition effect
above 10 mmol quantity. The alcoholic inhibition was generally attributed to stripping of the
essential trace amount of water because of polar alcoholic nature. Thus, 10 mmol is the
optimized mole quantity of benzyl alcohol which was used for further experiments.
3.8
Effect of mole quantity of acyl donor
Similar to alcohol influence, we have investigated the influence of butyrate donor by
varying mmol quantity of butyrate donor from 10 to 35 mmol under similar reaction
conditions. Figure 4A showing that, conversion increased as mmol of vinyl butyrate increases
from 10 to 27 mmol; while further increase in quantity of vinyl butyrate from 27 to 35 mmol
did not have effect on % conversion under ultrasonic condition. Moreover, conversion
continuously increases in conventional technique as quantity increases from 10 to 35 mmol.
This fact indicated the easy and efficient mass transfer of substrate at 27 mmol quantity of
vinyl butyrate under ultrasonic irradiation to form butanoyl-enzyme complex. However in
case of the conventional mode, efficient mass transfer was achieved at 30 mmol quantity of
vinyl butyrate. The acyl-enzyme (butanoyl-enzyme) complex formation is the key step for the
successive attack of the nucleophile (benzyl alcohol) to achieve better conversion of the
desired product. This butanoyl-enzyme complex formation process is well endorsed under the
influence of sonication. Moreover, vinyl butyrate is an activated species of the butanoyl
donor, which liberated vinyl alcohol as a bi-product. This vinyl alcohol tautomerizes into the
12

acetaldehyde and hence no reversible reaction is possible, thus equilibrium shifted towards
the forward direction. Thus, 27 mmol of the vinyl butyrate was used further as an optimal
quantity for the given model reaction under the ultrasonic mode.
Please insert figure 4 here
3.9
Effect of duty cycle
Enzymes are highly sensitive, hence it is suggested to use particular duty cycle to
reduce the possible denaturation of the enzyme due to continuous exposure of ultrasonic
waves [31,45]. Moreover, extensive cavitation can causes change in the protein conformation
which may have negative influence on the catalytic activity of enzyme [33,45]. In the present
work, various % duty cycles can be achieved by changing the ON/OFF time of ultrasound
irradiation (Figure 4B). Various ranges of duty cycles from 33.3 to 71.4 % were studied to
assess the effect of ultrasonic irradiation on enzymatic reaction. It was observed that, %
conversion and Ro increases as duty cycle increases from the 33.3 to 60 %. Further increase
in duty cycle from 60 to 66.7 % did not have any significant effect on the conversion,
whereas marginal decrease in % conversion and Ro was observed when duty cycle increased
to 71.4 %. Thus, above observations were indicating the sufficient stability of immobilized
biocatalyst upto 66 % duty cycle. The lower conversion and Ro at higher duty cycle may be
obtained due to denaturation of enzyme by increase in the exposure time under ultrasonic
irradiation [31,33]. Avhad et al. [45] reported that, higher duty cycle (extensive cavitation)
affects the proteins structure and cause subsequent denaturation. Thus, the optimized duty
cycle was 60 %, which was used for other all remaining experiments.
3.10 Effect of frequency
For ultrasound assisted reaction system, ultrasonic frequency (kHz) is an important
influencing factor [26-28]. Obliviously, high frequency ultrasound has higher energy and
mass transfer effect compared to low frequency ultrasound [27-29]. However, there is risk of
the enzyme denaturation and subsequent deactivation of catalytic activity at higher frequency
[28-31]. Moreover theoretically, physical parameters like shear force and cavitation decreases
with increase in ultrasound frequency [26-29]. In present study, the influence of ultrasonic
frequency for synthesis of benzyl butyrate was investigated in the range of 22-42 kHz at
constant ultrasonic power of 100 W (Figure 5A). It was observed that, conversion increases
as frequency increases from 22-33 kHz. However, further increase in frequency lead to slight
decrease in conversion of benzyl butyrate synthesis. This decrease in % conversion was
13

attributed to reduction in shear forces and enzyme activity with higher frequency [26-32].
Thus, 33 kHz was the optimized ultrasound frequency used for further all experiments.
Please insert figure 5 here
3.11 Effect of power
Not only ultrasonic frequency but also irradiation power plays an important role for
ultrasound assisted reaction system [26,27]. The higher ultrasonic power enhances reaction
rate to yield better conversion of desired product [28,31]. On the other hand, higher ultrasonic
power may leads to denature the proteomic structure of enzymes [32,33,45]. Hence in present
system, ultrasonic power was varied from 70 to 170 W at constant 33 kHz frequency to check
the effect of ultrasonic power on the reaction conversion (Figure 5B). It was observed that %
conversion increases as power increases from the 70 to 100 W. Further increase in the power
from 100 to 170 W leads to decrease in the % conversion which may be attributed to enzyme
deactivation or change in enzyme conformation at higher ultrasonic power [25-41]. Moreover
it could be said that, catalyst is sufficiently active upto power 100 W at specified reaction
conditions. Thus, ultrasonic power of 100 W was considered as an optimal and used further.
3.12 Effect of temperature
Temperature increases due to ultrasound irradiation while ultrasound effect reduces
with increase in temperature [26-28]. Indirectly at high temperature, ultrasonic mass transfer
effect decreases, since bubbles are collapsed with lesser intensity at higher temperature [28-
31]. Hence, it is obligatory to verify the influence of temperature for enzymatic ultrasound
assisted reactions, since enzymes are extremely sensitive to elevated temperatures [25-30].
Figure 6A showed following order of conversion at given range of temperature: ultrasound
(with immobilized lipase) > conventional (with immobilized lipase) > ultrasound (with free
lipase) > conventional (with free lipase). In all four cases, % conversion increases as the
o
temperature increases from 28 to 52 C. Interestingly, the developed immobilized biocatalyst
indicated remarkable catalytic activity-stability of immobilized lipase in ultrasonic irradiation
o
at power: 100 W; frequency: 33 kHz and temperature 52 C. The present immobilized
biocatalyst (PVA:CHI:PCL) indicated enhanced activity and sustainability as compared to
free lipase in ultrasound system. Thus the present system has benefit of use of lower
temperature, while recently Waghmare et al. [33] proposed use of 60 oC temperatures for the
Novozyme catalyzed ultrasound assisted carbonate compound synthesis. Thus, 52 oC was the
optimal temperature for the corresponding system and used for further experiments.
14

Please insert figure 6 here
3.13 Determination of the activation energy
For the present system, energy of activation (Ea) was estimated by the Arrhenius plot
method. The Ea at ultrasound or conventional technique for free or immobilized lipase was
found in order as: ultrasound (with immobilized lipase) < conventional (with immobilized
lipase) < ultrasound (with free lipase) < conventional (with free lipase) (Table 2). The lower
Ea was found for developed immobilized biocatalyst in ultrasound technique (45.75 KJ/mol)
which indicated (i) higher catalytic efficiency, (ii) lower energy requirement and (iii) greater
reaction rate for the given reaction system as compared to other catalyzed reaction system
(free lipase and conventional technique) (Table 2, entries 1-4) [5,6,8,46]. Moreover, the Ea
study indicated kinetically controlled synthesis of benzyl butyrate, as activation energy of
present system was found be higher than 15 KJ/mol.
The biocatalyst loading study showed linear relationship with % conversion, which
also supports the kinetically controlled synthesis [47]. Moreover, the kinetically controlled
synthesis involved activated acyl donor which readily reacts with the catalytic triad (serine
residue) of the lipase to form a butanoyl-enzyme intermediate [47]. Herein there are two
possibilities; first possibility involve nucleophilic attack of alcohol on the butanoyl-enzyme
intermediate which is a kinetically controlled synthesis and leads to provide good yield of
desired product swiftly. In second possibility involves hydrolysis of butanoyl-enzyme
intermediate which is thermodynamically controlled process and leads to form back
hydrolysis product. However in this work, we used non-aqueous reaction media and hence
possibility of back hydrolysis reaction is ruled out. Thus, present work is lipase (PVA:CHI:
PCL) catalyzed kinetically controlled synthesis of the benzyl butyrate in ultrasonic media
Please insert table 2 here
3.14 Effect of time
The progress of conventional and ultrasound assisted immobilized lipase (PVA/CHI
immobilized) catalysed reaction was evaluated to study the influence of reaction time (up to 4
h). At prescribed reaction conditions (Figure 6B), ultrasound assisted reaction showed higher
% conversion as compared to conventional mode. The slower rate of reaction after initial
hour (2 h) was attributed to increase of product concentration and subsequent decrease of
reactants concentration with respect to reaction time-span [1,5,8,31,44]. Moreover it was
found that, % conversion of the desired product continuously increases with respect to time
15

upto 3 h which signifying that (i) reaction was carried out in forward direction and (ii) final
product formed was stable one [31,44]. Thus, ultrasound assisted synthesis provided 99 %
conversion in 3h, while conventional mode provided almost 57 % conversions in 3h.
3.15 Effect of reusability
Several reuses of the immobilised biocatalyst (PVA:CHI:PCL) were performed for
the synthesis of benzyl butyrate (Figure 7A). Experimentally it was found that, biocatalytic
activity of immobilized PVA:CHI:PCL biocatalyst steadily decreased from first to fifth run.
The % conversion for the first and fifth run was found to be 99 % and 66 % respectively. The
reduction in % conversion was attributed due to loss of enzyme activity during each cycle
because of the continuous exposure of biocatalyst to ultrasonic irradiation [9,28,29,48]. There
are several reports in literatures which reported the physical breakage of immobilization
support during the reaction due to continuous exposure of biocatalyst to ultrasonic irradiation
[27,31-33,43]. Moreover, some studies reported change in the enzyme conformation due to
continuous exposure of biocatalyst to ultrasonic irradiation [28,31-33,43.48]. In addition to
this, some studies reported alcohol inhibition causes reduction in the enzyme activity
[2,9,43,49]. However in present study, we observed insignificant breakage or fractures of the
immobilized support biocatalyst after three cycles. It can be said that, alcohol inhibition was
main cause of the decrease in enzyme activity; whereas change in the enzyme conformation
was also a possible cause of decrease in enzyme activity [2,31]. Thus recyclability study
indicated that, biocatalyst was sufficiently stable and recycled.
Please insert figure 7 here
3.16 Effect of ultrasound vs. conventional technique for immobilized vs. free lipase
Each optimized experiment showed comparison for conversion of ultrasound assisted
and conventional stirring mode. It was observed that, % conversion obtained with ultrasound
assisted synthesis is more efficient as compared to the conventional mode in each optimized
experiment (Figure 1-7). Furthermore compared to free lipase, developed immobilized lipase
(PVA:CHI:lipase) showed excellent catalytic activity and stability in ultrasound irradiation
(Figure 7B). Free lipase provided almost three fold lesser conversion as compared to
immobilized lipase in ultrasound irradiation. The higher conversion by immobilized lipase
(compared to free lipase) in ultrasound irradiation was attributed to reasons like (i) lipase
scattered into co-polymer matrix could easily contact with reacting substrates molecules,
which enhances the catalytic interaction [2,3,4] (ii) immobilization via entrapment protect
16

lipase from direct exposure to ultrasound energy wave, while free lipase exposes directly to
high energy ultrasound wave and may be susceptible to denature [2,3,5,16].
The overall enhancement of % conversion in ultrasonic irradiation (as compared to
conventional) was owing to (i) improved mass transfer due to cavitation [27,28] and (ii) use
of developed immobilized biocatalyst which provided better shielding and activity-stability
under specified ultrasonic irradiation condition [2,28]. From the power calculations [50], it
was found that, ultrasonic irradiation provided double efficiency with 26 % additional power
than conventional technique. Thus, ultrasonication was a feasible technique by considering
the view of energy (Table 3). In case of scale-up of the process, uniform distribution of
irradiation power is necessary which can be achieve by using multiple transducers [51].
Gogate et al. [51] reported the various parameters for the scale-up sonochemical reactor
operation. For successful implementation of the scale-up ultrasonic processing there is need
of joint efforts of chemists, physicists, biochemical and chemical engineers.
Please insert table 3 here
Please insert scheme 2 here
3.17 Effect of various substrate
The practical feasibility of developed PVA/CHI immobilization protocol was verified
by synthesis of various industrially important butyrate compounds in ultrasound and
conventional mode (Scheme 2). Various valuable compounds were synthesized to provide 99
% yield in 3 h, whereas conventional technique provided much lesser yield as compared to
ultrasound technique (Table 4; entries 1-6). The small chain aliphatic alcohol provided
excellent conversion of 99 % in 3.5 h, herein reaction rate was slightly reduced because of
decrease in enzyme activity due to polar substrate (Table 4; entries 7,9) [2,34,43,52-57].
Various branched alcohol provided excellent conversion of the 99 % in 3 h (Table 4; entries
11,13). Cyclopentyl alcohol is a secondary alcohol that furnished lethargic conversion of 64
% of the corresponding butyrate in 5 h, due to presence of the steric crowding at the adjacent
α-carbon atom (Table 4; entry 15) [2,3,34]. In all studied substrates, (i) ultrasound assisted
immobilized lipase catalyzed synthesis provided excellent conversion of desired product
(Table 4; entries 1,3,5,7,9,11,13) (ii) whereas conventional technique provided much lesser
(almost 2 fold lesser) conversion (Table 4 entries 2,4,6,8,10,12,14,16) [28,29,52-58]. Thus,
present protocol demonstrated (i) better activity-stability of PVA:CHI immobilized lipase
under ultrasound condition and (ii) potential biocatalytic applications to synthesize a variety
of industrially important butyrate compounds via Greener Enzymatic-Ultrasound Technique.
17

All these synthesized compounds are widely used for the commercial purpose as/ in food,
fragrance, bakery, toiletries, flavour, pharma, cosmetic sector and other home-care products.
Please insert table 4 here
3.18 Influence of the ultrasonic radiation on the free and immobilized lipase activity
Finally one of the important part of this work is the investigation of influence of
ultrasonication on the lipase activity of free and PVA:CHI:PCL immobilized lipase. Since it
is necessary that, biocatalyst must be stable under the ultrasonic/ reaction condition to carry
out efficient biocatalysis [1-5,59]. It was observed that, PVA:CHI immobilized lipase
demonstrated better catalytic activity as compared to that of free lipase PCL. It was observed
that, free lipase demonstrated 73 % residual activity, while immobilized lipase demonstrated
94 % residual activity at given incubation condition (Table 5).
Thus, higher stability and activity was attributed to entrapment of lipase within the
polymer matrix. This immobilized support provided a more rigid external backbone for the
lipase molecules which protect lipase molecules from the effect of sonication and avoids the
breaking of various stabilizing forces. However, case is different for the free lipase which
directly exposed to outer sonication environment and causes destabilization or breaking of the
important stabilizing forces. Thus, immobilization protects lipase from direct exposure to
ultrasound energy wave, while free lipase exposes directly to high energy ultrasound wave
and causes the denaturation.
Please insert table 5 here
Conclusion:
In conclusion, we have successfully employed ultrasound energy and immobilized lipase
PVA:CHI:PCL for the potential biocatalytic synthesis of various commercially valuable
butyrate esters. Screening study showed that, lipase PVA:CHI:PCL (5:5:2.5) worked as a
robust biocatalyst to provide superior conversion of 99 %. Various optimized reaction
parameters of developed protocol were used to synthesize several industrially important
butyrate esters which furnished excellent conversion of 99 % via developed biocatalyst in
ultrasonic irradiation. The PVA:CHI:PCL (5:5:2.5) biocatalyst showed excellent recyclability
upto studied five cycles. Furthermore, immobilized lipase PVA:CHI:PCL (5:5:2.5)
demonstrated almost 4.5 folds higher activity under ultrasound condition as compared to free
lipases in conventional media. The lipase activity assay study also demonstrated better
activity-stability of immobilized lipase in ultrasonic medium. Finally, energy assessment was
18

performed to indicate feasibility of developed protocol. Thus, present protocol demonstrated
superior biocatalytic activity by combine use of ultrasound and immobilized PCL biocatalyst.
Acknowledgement:
Author Kirtikumar C. Badgujar is grateful to the Department of Biotechnology-DBT, India
(BT/PR4192/PID/6/623/2011) and the Council of Scientific & Industrial Research-CSIR,
India (09/991(0015)/2011-EMR-I) for providing research funds and fellowship. Authors are
greatly thankful to Bhagawat Patil and Miachhindra Bhalerao for their kind help.
19

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22

Figure Captions
Figure 1:
(A) Effect of support:lipase (PVA:CHI:PCL) composition: Reaction condition: solvent:
iso-octane; biocatalyst: 320 mg; stirrer speed: 900 rpm; alcohol: 10 mmol; vinyl butyrate: 30
o
mmol; duty cycle: 63 %; frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 C.;
conventional technique did not involve sonication. Initial reaction rate (Ro) in
mmol/mL/hour.
(B) Effect of the butanoyl donor: Reaction condition: solvent: iso-octane; biocatalyst: 320
mg; stirrer speed: 900 rpm; alcohol: 10 mmol; butyrate donor: 30 mmol; duty cycle: 63 %;
o
frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 C; conventional technique did
not involve sonication.
Figure 2:
(A) Effect of solvent: Reaction condition: solvent: polar/non-polar; biocatalyst: 320 mg;
stirrer speed: 900 rpm; alcohol: 10 mmol; vinyl butyrate: 30 mmol; duty cycle: 63 %;
frequency: 33 kHz; power: 100 W; time: 3 h; temperature: 52 oC; conventional technique did
not involve sonication. Initial reaction rate (Ro) in mmol/mL/hour.
(B) Effect of biocatalyst amount: Reaction condition: solvent: iso-octane; biocatalyst: 0-350
mg; stirrer speed: 900 rpm; alcohol: 10 mmol; vinyl butyrate: 30 mmol; duty cycle: 63 %;
o
frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 C; conventional technique did
not involve sonication. Initial reaction rate (Ro) in mmol/mL/hour.
Figure 3:
(A) Effect of rotation speed: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg;
stirrer speed: 0-1200 rpm; alcohol: 10 mmol; vinyl butyrate: 30 mmol; duty cycle: 63 %;
frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 oC.; conventional technique did
not involve sonication. Initial reaction rate (Ro) in mmol/mL/hour.
(B) Effect of alcohol: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg; stirrer
speed: 800 rpm; alcohol: 5-12 mmol; vinyl butyrate: 30 mmol; duty cycle: 63 %; frequency:
33 kHz; power: 100 W; time: 3h; temperature: 52 oC; conventional technique did not involve
sonication. Initial reaction rate (Ro) in mmol/mL/hour.
23

Figure 4:
(A) Effect of butyrate donor: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg;
stirrer speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 10-35 mmol; duty cycle: 63 %;
frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 oC.; conventional technique did
not involve sonication. Initial reaction rate (Ro) in mmol/mL/hour.
(B) Effect of duty cycle: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg; stirrer
speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 27 mmol; duty cycle: 38.5 to 71.4 %;
o
frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 C. Initial reaction rate (Ro) in
mmol/mL/hour.
Figure 5:
(A) Effect of frequency: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg; stirrer
speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 27 mmol; duty cycle: 60 %; frequency:
o
22-42 kHz; power: 100 W; time: 3h; temperature: 52 C. Initial reaction rate (Ro) in
mmol/mL/hour.
(B) Effect of power: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg; stirrer
speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 27 mmol; duty cycle: 60 %; frequency: 33
o
kHz; power: 70-170 W; time: 3h; temperature: 52 C. Initial reaction rate (Ro) in
mmol/mL/hour.
Figure 6:
(A) Effect of temperature: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg;
stirrer speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 27 mmol; duty cycle: 60 %;
frequency: 33 kHz; power: 100 W; time: 3 h; temperature: 28-52 oC.; conventional technique
did not involve sonication
(B) Effect of time: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg; stirrer
speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 27 mmol; duty cycle: 60 %; frequency: 33
kHz; power: 100 W; time: 1-4 h; temperature: 52 oC.; conventional technique did not involve
sonication
Figure 7:
24

(A) Effect of recyclability: Reaction condition: solvent: iso-octane; biocatalyst: 300 mg;
stirrer speed: 800 rpm; alcohol: 10 mmol; vinyl butyrate: 27 mmol; duty cycle: 60 %;
frequency: 33 kHz; power: 100 W; time: 3h; temperature: 52 oC.
(B) Comparison of ultrasound versus conventional technique: Reaction condition:
solvent: iso-octane; biocatalyst: 300 mg; stirrer speed: 800 rpm; alcohol: 10 mmol; vinyl
butyrate: 27 mmol; duty cycle: 60 %; frequency: 33 kHz; power: 100 W; time: 3h;
temperature: 52 oC.; conventional technique did not involve sonication.
25

Figures
Figure 1:
(Colour image for web version)
100
80
60
40
20
0
2.5
2
100
80
60
40
20
0
1.5
1
0.5
0
Butyric
acid
Ethyl
Methyl
Vinyl
(A)
(B)
butyrate butyrate butyrate
Butyrate donor
Biocatalyst composition ratio
% Conv. by ultrasound
Initial rate by ultrasound
% Conv. by conventional
% Ultrasound
% Conventional
(Grey scale image for print version)
26

Figure 2:
(Colour image for web version)
100
2.5
2
100
80
60
40
20
2.5
2
80
1.5
1
60
1.5
1
40
0.5
0
20
0.5
0
Iso- n- hexane Toluene THF
octane
1,4
dioxane
0
50 100 150 180 220 250 270 300 325 350
Biocalyst loading (mg)
(A)
(B)
Solvent
% Conv. by ultrasound
Initial rate by ultrasound
% Conv. by conventional
% Conv. by ultrasound
Initial rate by ultrasound
% Conv. by conventional
(Grey scale image for print version)
27

Figure 3:
(Colour image for web version)
100
80
2.4
2
100
80
60
40
20
0
2.5
2
60
1.5
1
1.6
1.2
40
20
0.5
7
9
9.5 10 10.5 11
12
0
300 600 700 800 900 1000 1200 1500
Stirring speed (rpm)
(A)
(B)
mmol of benzyl lacohol
% Conv. by ultrasound
Initial rate by ultrasound
% Conv. by conventional
% Conv. by ultrasound
Initial rate by ultrasound
% Conv. by conventional
(Grey scale image for print version)
28

Figure 4:
(Colour image for web version)
100
80
2.5
2
100
80
60
40
20
0
2.5
2
60
1.5
1
1.5
1
40
0.5
0
20
0.5
0
0
10
15
20
25
27
30
35
33.3 50.0 55.6 60.0 62.5 66.7 71.4
Duty cycle (%)
(A)
mmol of vinyl butyrate
(B)
% Conv. by ultrasound
% Conv. by conventional
% Conv. by ultrasound
Initial rate by ultrasound
Initial rate by ultrasound
(Grey scale image for print version)
29