Synthesis of lipase nano-bio-conjugates as an efficient biocatalyst: Characterization and activity-stability studies with potential biocatalytic applications

Article abstract of DOI:10.1039/c5ra10032a

In the present study, we have synthesized lipase-nano-bio-conjugates via immobilization of various lipases on multiwall carbon nano-tubes (MCNT), in order to construct an efficient and recyclable biocatalytic system. In a screening study lipase Pseudomonas fluorescens (PFL) acted as an efficient biocatalyst (lipase-nano-bio-conjugates) which showed higher retention of lipase activity and protein loading. Consequently the immobilization support : lipase (MCNT : PFL) composition was screened in which MCNT : PFL (2 : 1) was calculated as a robust biocatalyst composition which showed higher activity retention and protein loading. This nano-bio-conjugate was then characterized in detail with physical and biochemical techniques using SEM, TEM, FTIR, K_m, V_max, catalytic efficiency and (%) water content analysis. This developed biocatalyst was further used for practical biocatalytic applications such as O-acylation reactions. Various reaction parameters were optimized in detail like reactant molar ratio (2 : 3.5), solvent, MCNT : PFL biocatalyst amount (36 mg), temperature (50°C) etc. The developed biocatalytic protocol was then extended to synthesize several (twenty-two) industrially important acylated moieties with an excellent yield, these products are well characterized by ¹HNMR, ¹³CNMR and GCMS analysis. Moreover in the present study, we have reviewed the potential industrial applications of various synthesized compounds. Also, we have studied the thermodynamic aspect which demonstrated more feasibility of use of immobilized MCNT : PFL lipase over free lipase. Interestingly, immobilized MCNT : PFL lipase showed 2.3 fold higher catalytic activity than free PFL. Besides this, the biocatalyst was efficiently recycled for up to five cycles. Thus the present protocol demonstrated, (i) synthesis of nano-bio-conjugates as a bio-catalyst, (ii) detailed physical-biochemical characterization of nano-bio-conjugates, (iii) optimization of the biocatalytic protocol (iv) practical biocatalytic applications along with a mechanistic study (v) a thermodynamic feasibility study and (vi) recyclability study. 2015

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ARTICLE TYPE
Synthesis of lipase nano-bio-conjugates as an efficient biocatalyst: Characterization and
activity-stability studies with potential biocatalytic applications
Kirtikumar Chandulal Badgujar,*a Takehiko Sasaki,b Bhalchandra Mahadeo Bhanage*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
In present study, we have synthesized lipaseꢀnanoꢀbioꢀconjugates via immobilization of various lipases on multiwall carbon nanoꢀtube
(MCNT), in order to construct an efficient and recyclable biocatalytic system. In screening study lipase Pseudomonas fluorescens (PFL)
acted as an efficient biocatalyst (lipaseꢀnanoꢀbioꢀconjugates) which showed higher retention of lipase activity and protein loading.
Consequently immobilization support:lipase (MCNT:PFL) composition was screened in which MCNT:PFL (2:1) was worked out as
10 robust biocatalyst composition which showed higher activity retention and protein loading. This nanoꢀbioꢀconjugate was then
characterized in details with physical and biochemical techniques using SEM, TEM, FTIR, Km, Vmax, catalytic efficiency and (%)
water content analysis. This developed biocatalyst was further used for practical biocatalytic applications such as Oꢀacylation reactions.
Various reaction parameters were optimized in details like reactant molar ratio (2:3.5), solvent, MCNT:PFL biocatalyst amount (36 mg),
temperature (50 oC) etc. The developed biocatalytic protocol was then extended to synthesize several (twentyꢀtwo) industrially important
15 acylated moieties with an excellent yield, these products are well characterized by 1HNMR, 13CNMR and GCMS analysis. Moreover in
present study, we have reviewed potential industrial applications of various synthesized compounds. Also, we have studied
thermodynamic aspect which demonstrated more feasibility of use of immobilized MCNT:PFL lipase over free lipase. Interestingly,
immobilized MCNT:PFL lipase showed 2.3 folds higher catalytic activity than free PFL. Besides this, biocatalyst was efficiently
recycled upto five cycles. Thus the present protocol demonstrated, (i) synthesis of nanoꢀbioꢀconjugates as a bioꢀcatalyst, (ii) detail
20 physicalꢀbiochemical characterization of nanoꢀbioꢀconjugates, (iii) optimization of biocatalytic protocol (iv) practical biocatalytic
applications along with mechanistic study (v) thermodynamic feasibility study and (vi) recyclability study.
biocatalytic system has various greenꢀchemistry advantages such
as higher activity, recyclability, higher chemoꢀ, regioꢀ, enantioꢀ
Introduction:
selectivity, mild reaction condition, sustainable E factor, easy
Enzymes are the widely studied bioꢀmolecules in various
handling, safe synthetic protocol and no environmental
50 hazards/issues.2ꢀ12 Till time immobilization of lipases are reported
fields such as bioꢀchemical engineering, bioꢀmedical sciences,
25 bioꢀinformatics, microbiology, biotechnology, bioꢀchemistry and
on various types of carriers such as polymeric matrix, polymer
Chemistry.1,2 In particular, enzyme lipase {triacyl glycerol
beads, gels, solꢀgels, fibres, mesoꢀ, microꢀporous materials etc.
via physical adsorption, entrapment, and crossꢀlinking method.3ꢀ12
hydrolases, (E.C. 3.1.1.3)} from the hydrolases class has been
attracted significant consideration in food, flavour, dairy, leather
However, an ideal immobilisation of biocatalyst on carrier should
and pharma sector as a versatile biocatalyst to carry out organic
55 retain or improve its catalytic activity, stability and
30 synthesis in different reaction media at mild reaction condition.2ꢀ4
However, potential biocatalytic applications of these free lipase
biomolecules are greatly hampered by sensitive proteomic nature
which reflect poor solubility, activity and stability in various
organic media.3,4,5 Moreover, enzymes are active in aqueous
35 media where, organic compounds did not show any solubility.3ꢀ7
The advance skilful immobilization technique is the only
way to triumph over above proposed difficulties.2ꢀ8 Hence,
various researchers are engaged to improve activity, stability and
reuse of enzymes in nonꢀaqueous media for effective biocatalytic
40 applications.5ꢀ10 Immobilization of enzyme is the most
fundamental aspect to improve the sufficient activity and stability
of enzymes, moreover immobilization build up a heterogeneous
reaction system which can shield the sensitive enzymes from the
surrounding reaction media and also overcome the economic
45 reusability issue.5ꢀ12 Furthermore, the use of immobilized
recyclability.2ꢀ8
Nanoꢀmaterials for immobilization and their practical
synthetic applications (bioꢀnonmaterial and nanoꢀbiocatalysis) is
a fast growing research area which involves the enzyme
60 immobilization on nanoꢀmaterial and their biocatalytic
applications.10ꢀ19 Different nanoꢀstructured materials like nanoꢀ
particles, nanoꢀfibres, nanoꢀwires, and nanoꢀcomposites have
been engaged as
a
novel immobilisation support for
enzymes.10,12,14,16,18,19 These nanoꢀmaterials serves as an excellent
65 immobilization material to synthesize various nanoꢀbioꢀ
conjugates as compared to bulk solid materials. This is because of
key properties such as higher surface area, low mass transfer
resistance, effective enzyme loading, nanoꢀscale dispersion and
ease of surface functionalization/ modification.10ꢀ19
70
Thus, discovery of nanoꢀscale materials proved countless
applications and scope based on their extraordinary properties
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owing to size of nanoꢀmaterials.10ꢀ19 Among various said nanoꢀ
materials, a special mention goes to carbon based nanoꢀmaterials
such as carbon nanoꢀtubes (CNTs) which have attracted the
significant attention in biomedical science and technology.9ꢀ19
Terminalꢀend and sideꢀwalls of CNTs can be modified by various
chemical, physical and biological methods according to choice of
application.11,12 Moreover, CNTs possess wide applications
because of excellent properties such as structural, mechanical,
greener synthesis of various twentyꢀtwo food flavour and
fragrance compounds along with their commercial importance.
65
5
2.
Material and Methods
2.1 Enzymes and Chemicals
Lipase PFL (Pseudomonas fluorescens, activity ≥ 20,000
U/g), lipase HPL (Horse pancreatic lipase, ≥ 10, 000 U/g) pꢀnitro
thermal and biocompatibility.13,15 Due to these reasons, both 70 phenyl butyrate (pꢀPNB), MCNTs were purchased from Sigma
10 singleꢀwalled carbon nanoꢀtube (SCNTs) and multiꢀwalled
carbon nanoꢀtubes (MCNTs) find great scope in biomedical,
biosensor and biotechnological applications.11,12,13 MCNTs
consist of several graphite layers surrounding a central tubule,
while a SCNTs have a central tubule without a graphitic
15 layer.13,15,16 MCNTs are 1ꢀDꢀnanoꢀparticles having numerous
features such as higher surface area, more dispersibility, more
physicoꢀchemical stability, lower cost and lesser cytoꢀtoxicity
compared to SCNTs.11,12,13,15,16 Till date, very few attempts have
Aldrich Pvt. Ltd. India. Lipase MJL (Mucor javanicus, activity ≥
10,000 U/g) was kindly gifted by Amano Enzymes (Japan). All
other solvents/chemicals were bought from the Sigma Aldrich,
Alfa Aesar and Hiꢀmdia Pvt. Ltd. India.
75
2.2
Immobilization of the Lipase
200 mg of MCNTs were dispersed by sonication almost
around 30ꢀ40 minutes in 50 mL of deionised water. Later on,
been made by various researcher to form enzyme nanoꢀbioꢀ 80 lipase PFL solution (100 mg dissolved in 6ꢀ8 mL) was added into
20 conjugates with carbonꢀbased nanoꢀmaterials, however there
potential biocatalytic applications are still unexplored, which
invoke researchers to apply bioꢀnanoꢀconjugates as a biocatalyst
for potential biocatalytic applications to synthesize commercially
important organic moieties.13,15,17
above dispersed MCNT solution. The resultant solution was also
sonicated with 50 % duty cycle (5 minutes ON/OFF mode, with
frequency 30 kHz and power 100 W) for 20 minutes. Finally this
solution was kept in an orbital shaker for 4ꢀ6 hours. Afterwards,
85 the solution mixtures were vacuumꢀfiltrated to get immobilized
enzyme and the supernatant was removed. The immobilized
enzymes (nanoꢀbioꢀconjugates denoted as MCNT:PFL) were then
washed two times with 50 mL of deionised water.
25
In present study, we have used these developed enzyme
bioꢀnanoꢀconjugates for synthesis of various acylated products
(commercially important esters) which are extensively used in
various fields.20ꢀ24 The survey of chemical reactions used in
preparation of drug candidate molecule showed that 12 %
90
30 reaction involves the acylation steps.22 Thus acylation is one of
the most important transformations, but chemical way of
acylation is atom inefficient processes which have several
disadvantages such as biꢀproduct formation, use of higher
2.3
Characterization of MCNT:PFL lipase
2.3.1 Lipase hydrolytic activity assay and protein content
The lipase activity of free and MCNT immobilized lipase
was studied in triplicate spectrophotometrically at 410 nm with
temperature, lower yieldꢀhigher waste, higher activation energy 95 minor modifications in reported procedure by Pencreac’h and
35 and use of hazardous acids or bases.22 Hence, development of
greener and waste minimizing biocatalytic methods of acylation
will considerably improve the environmental performance for
sustainability.22 Recently, the American Chemical Society Green
Baratti.27 The brief process of the lipase hydrolytic activity assay
is as follows: In hydrolytic assay, we had performed hydrolysis of
pꢀnitro phenyl butyrate (pꢀNPB) substrate. In a standard assay
condition involve reaction mixture consists of 2 mg of free lipase
Chemistry Institute (ACSꢀGCIPR) was established to promote 100 (and equivalent quantity of the immobilized MCNT:lipase) in 1
40 innovations for synthesis of valuable organic moieties using
greener chemistry and sustainable technology.23 Acylated
moieties are the essential components of the flora, fruity, grasses,
essential oils and vegetations etc which having pleasant fruity
mL of nꢀhexane. The reaction was initiated by addition of 1 mL
of 12 mM, pꢀNPB substrate dissolved in isoꢀpropanol solution
and incubated at 37 °C for 10 minutes. Afterwards, 200 ꢁL of
reaction mixture was withdrawn and added to 600 ꢁL of
smell and taste.24 Moreover, these compounds are widely used in 105 deionized water to extract pꢀnitro phenol (pꢀNP) in the aqueous
45 various pharmaceuticals, decorative cosmetics, balm, body
lotions, ointment, face cream, fragrance, flavours, shampoos,
soaps, showerꢀshaving gels and other toiletries.24 Most of these
acetates are produced in metric of tons per year; furthermore
phase. Finally, 1200 ꢁL of potassium phosphate buffer solution of
the pH 7.9ꢀ8.0 was added to above mixture in order to give a pale
yellow colour for extracted pꢀNP. This pale yellow coloured
sample was instantly used to determine the absorbance at 410 nm.
these acetate esters are listed as grantedꢀA substances by the 110 The lipase activity was defined as microꢀmoles of pꢀNP released
50 Council of Europe for their safe use in foodꢀstuffs.25 According to
BCC research survey, the worldwide market for Fragrance
compounds was estimated to US $ 11.2 bn in 2011 and projected
to raise US $ 15.7 bn upto 2017; while global market for Flavour
by per milligram of the lipase per minute under the given
standard hydrolytic assay condition.
The protein content or protein binding yield or adsorption
efficiency for the MCNT immobilized lipase was determined by
was accounted to US $11.3 bn in 2012 and projected almost US $ 115 Bradford methodology at 595 nm.28 The amount of adsorbed
55 14.5 bn up to 2017.26
Thus, considering such a wide range of applications and
scope; industries are looking to produce these flavour fragrance
compounds by an ecoꢀfriendly way so that, they could labelled
them as “Safe and Green” products.24,26 Hence in present study, 120
60 we make an attempt to develop nanoꢀbioꢀconjugates as an
efficient immobilized biocatalyst which was employed for the
protein (lipase) is the difference between amount of protein
introduced for immobilization and the amount of protein found in
the filtrate/decant after immobilization on the MCNT.
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2.3.2 FT-IR
The FTꢀIR (ATR) analysis was performed by Shimadzu
FTIR to investigate the presence of amide functionality of lipase
after immobilization on MCNT. For each sample 16 scans were
ran to record the FTꢀIR spectra of MCNT, MCNT:lipase and
native lipase.
3.
3.1
Result and discussion
Immobilization of lipase on MCNT from various
microbial sources and their screening
60
Various immobilized lipases with the support composition
5
MCNT:lipase (200 mg:120 mg) was screened to determine %
lipase activity, % protein content, % activity retention and %
yield for the model cinnamyl acetate compound formation (Table
65 1). The % lipase activity is defined as the observed lipase activity
ratio of MCNT:lipase to free lipase activity,29 while trend for the
% lipase activity was found to be PFL (65.62 %) > MJL (48.06
%) > HPL (33.74 %). Thus, the hydrolysis is prime function of
the lipase which is found to be higher for the lipase PFL for given
70 activity assay condition. Moreover, the % protein adsorption is
defined as the amount of protein adsorbed to total amount of
protein used for immobilization.29 It was observed that % protein
adsorption efficiency was found to be higher for lipase PFL
(73.57 %) compared to the MJL (60.97 %) and HPL (53.71 %)
75 lipase. The protein loading was found to be moderate because of
the physical immobilization of lipase on MCNT support.30,31 The
activity retention is the ratio of specific activity of immobilized
lipase to free lipase.29 The lipase PFL showed highest % activity
retention (89.10 %) as compared to the MJL (78.71 %) and HPL
80 (62.81 %) lipase. Thus, the lipase activity assay and protein
content study indicated successful immobilization of various
studied lipases (PFL, MJL and HPL) on MCNTs (Table 1).
Among all three screened lipases, the lipase PFL showed the best
results for immobilization on MCNT which was used for further
85 characterization and biocatalytic application study.
2.3.3 % water content:
10
The moisture content was measured by the Karl Fischer
(784 KFP Titrino) analysis for support MCNT, immobilized
MCNT:lipase and free lipase PFL.
15 2.3.4 SEM and TEM
The morphology of the samples was examined using field
emission gunꢀscanning electron microscopy (FEGꢀSEM) analysis
(Tescan MIRA 3 model) while transmission electron microscopy
(TEM) images were recorded with a Phillips model CM 200 to
20 study the morphology and size of support MCNT and
immobilized MCNT:lipase sample.
2.3.5 Determination of Kinetic Parameters (Km and Vmax)
25
The Vmax (maximum reaction velocity) and Km
(MichaelisꢀMenten constant) for free and immobilized
MCNT:PFL lipase were determined by the LineweaverꢀBurk plot
using hydrolytic lipase activity assay of pꢀNPB in 3ꢀ24 mM
substrate concentration. The procedure used for km and Vmax
30 determination is same as indicated in section 2.3.1.
3.2
Influence of the PFL lipase loading on MCNT
Influence of PFL lipase loading (immobilized lipase PFL
2.3.6 Experimental set up
90 compositions MCNT:PFL) was tested to achieve higher protein
adsorption and higher lipase activity. The % lipase activity, %
protein adsorption and specific activity of immobilized lipase was
increased with increase in lipase loading from 50 to 100 mg per
200 mg of MCNT. This increase in lipase activity might be
95 attributed to availability of higher catalytic sites with higher
lipase loading which leads to extend its fullest activity.16,31
However, further increase in lipase loading from 100 to 120 mg
per 200 mg of MCNT showed decrease in the % lipase activity,
% protein adsorption and specific activity. Since, higher lipase
100 loading may causes increase in stacking of lipase bioꢀmolecules
on the support MCNT which restricts the possible mass transfer
diffusion of substrates towards the active sites of lipase.30,31
Moreover, heavy loading of lipases resulted into junk adsorption
of enzyme molecules on top of those previously immobilized
105 lipases which tends to causes jamming of active sites.13,29 Thus,
the optimized quantity of the lipase PFL loading was 100 mg per
200 mg of the MCNT, wherein we obtained highest lipase
activity, protein adsorption and specific activity. Similar type of
lower lipase activity at higher lipase loading was obtained to
110 Pujari et al.29 for lipase immobilization on polypropylene
biphasic membrane.
Cinnamyl acetate and various organic moieties were
35 synthesized in a 15 mL glass reaction vessel of 2 cm i.d. with a
glass lid. Initially desired alcohol was taken in a reaction vessel
and diluted by the solvent nꢀheptane. Then vinyl acetate was
added into the reaction vessel and reaction was initiated by
addition of immobilized MCNT:lipase. Soon after, the reaction
40 vessel was placed in an orbital shaker at a specified temperature
and rotation speed.
2.3.7 Analysis
45
The sample of reaction mixture (10 ꢁL) was taken out
periodically and analyzed by gas chromatograph (PerkinElmer
Clarusꢀ400 instrument) having flame ionizing detector (FID) and
capillary column. The detector and injector temperature were 280
and 60 oC, respectively. The oven temperature of GC was kept at
50 60 °C for 4 min constant and then raised with 10 oC/min upto 280
°C. The reaction mass product was also confirmed by the
Shimadzu QPꢀ2010 GasꢀChromatographyꢀMass Spectroscopy
analysis.
55
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Table 1: Immobilization of lipase from various microbial sources on MCNT and their screening study
Lipase MCNT:
lipase
Hydrolytic Lipase
activity
Lipase Total amount of Protein
Protein
Adsorbed
(%)
Sp. activity Activity
MCNT: retention
% yield
Activity
(%)
content
lipase
1.125
0.153
0.045
(%)
Free MCNT: lipase
Nonꢀadsorbed Adsorbed
Free MCNT:lipase
PFL 200:120 59.23
MJL 200:120 11.34
HPL 200:120 7.23
38.37
5.45
2.44
65.62
48.06
33.74
12.39
22.81
45.97
34.50
35.63
53.36
73.57
60.97
53.71
89.10
78.71
62.81
56
11
13
99
20
19
Lipase activity: U/mg; protein content: µg/mg; Specific activity: U/µg; aCinnamyl alcohol:vinyl acetate (2:3.5 mmol);; immobilized
biocatalyst MCNT:lipase (42 mg); temperature (50 oC); % Lipase activity = immobilized MCNT:PFL lipase activity/ free PFL lipase
activity; % Protein adsorbed = adsorbed amount of protein/ (nonꢀadsorbed + adsorbed amount of protein); Specific activity = MCNT:PFL
lipase activity/ protein adsorbed; % activity retention = specific activity of immobilized lipase/ Specific activity of free lipase
Table 2: PFL lipase loading on MCNT and their influence on lipase activity assay and protein content
No
MNCT:PFL
(mg)
Lipase activity
MCNT:PFL
0
% Lipase
activity protein adsorbed
Amount of
% Protein
adsorbed
0
67.96
74.35
76.85
76.66
73.57
69.96
Specific activity
MCNT:PFL
0
% Activity
retention
0
38.37
70.66
92.23
89.96
89.10
88.55
1
2
3
4
5
6
7
200:0
200:50
200:80
200:100
200:110
200:120
200:140
0
0
15.45
31.12
41.99
40.89
38.87
36.75
26.08
52.54
70.89
69.03
65.62
62.04
13.22
23.14
29.89
32.69
34.50
37.56
1.168
1.343
1.409
1.250
1.125
0.978
3.3
Influence of the immobilization time on % protein
content, % lipase activity and specific activity
An immobilized lipase preparation is a crucial parameter to
attributed to, CꢀC, CꢀN stretching and NꢀH bending
vibrations.16,20,31 Similar types of bands are attributed in present
study for the native/ free PFL lipase and immobilized MCNTꢀ
obtain an efficient nanaꢀbioꢀconjugates (biocatalyst) with the best 40 PFL lipase. However, MCNT solely made up of carbon and did
5
bioꢀcatalytic activity.13,16 Hence, there must be proper interaction
between the immobilization support and the lipase which can be
achieved by the maintaining the sufficient incubation time. Thus,
in present study, we have incubated the lipase from 0 to 5 hours
not provide any significant information except the CꢀC bond
which is parent bond for the organic functionality compounds.
Thus, the present FTꢀIR spectroscopy confirmed presence of the
amide functionality and subsequent immobilization of the lipase
with MCNT for immobilization (Figure 1). The % lipase activity 45 on the MCNT.31 Similar type of amide bond functionality after
10 and % protein content was decreased from the supernatants along
with the time span, this fact indicating the successful
immobilization of lipase on MCNT. Thus, highest % lipase
activity and protein adsorption was found to be nearly at 4 hour
incubation. In a similar way, the specific activity was found to be
15 higher at 4 h incubation. Further increase of incubation time span
(from 4 h to 6 h) did not have significant effect on the activity
and protein adsorption. Higher incubation time promotes the
proper hydrophobic interaction of the lipase molecules with the
support MCNT for physical adsorption.13,29,30 Moreover it was
20 postulated that, piꢀpi stacking interaction between the sidewalls
of MCNTs and the aromatic rings of amino acids residues could
also contributes for the physical adsorption of the lipase
molecules.31 Thus, 4h is the optimized incubation time for the
immobilization of the lipase PFL.
lipase immobilization was confirmed by Gupta et al,16 and
Pavlidis et al.31.
25
3.4
FT-IR analysis study
The lipase immobilization on the wall of MCNT was
Fig. 1: Influence of the immobilization time on % protein
verified by FTꢀIR spectroscopy (Please find supporting
information for figure). The most intensive and broadened
30 absorption band at 3500 cmꢀ1 corresponds to vibrations of NꢀH
groups associated with the amide bond. The parent amide
functionality of enzyme can be assigned by amides I, II, III
bands.16,20 In nanoꢀbioꢀconjugate (MCNT:PFL), amide I band at
1600ꢀ1700 cmꢀ1 is attributed to C=O stretching vibrations, amide
35 II band at 1450ꢀ1600 cmꢀ1attribute to NꢀH bending and CꢀN
stretching vibrations, while amide III band at 1300ꢀ1450 cmꢀ1 is
50 content, % lipase activity and specific activity
3.5
SEM and TEM analysis study
The SEM analysis clearly showed distinction in the MCNT
and immobilized MCNT since, immobilization of the lipase was
clearly observed on the wall of MCNT as shown in Figure 2.
55 SEM analysis (Figure 2A, 2B) showed the surface texture which
indicating the change in the surface morphology of MCNT after
adsorption of lipase PFL. Moreover, TEM analysis of support
4
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MCNT showed presence of clear picture (Figure 2C) while
immobilized sample showed presence of black colour globular
spots (Figure 2D) which indicated adsorption of lipase on MCNT.
Similar type of change in textural study was observed to various
researchers15,16,19,20 after immobilization of lipase.
3.7
Kinetic analysis study (km, Vmax, and catalytic
efficiency)
The LineweaverꢀBurk plot for kinetic study of hydrolytic
35
5
activity assay was used to deduce Km, Vmax and catalytic
efficiency (Table 4).1 Interestingly in present study, the Vmax
value for free lipase PFL showed slightly higher velocity (67.11)
40 as compared to MCNT immobilized lipase (58.14). In context to
this Km value is significantly decreased after the immobilization,
this may be attributed due to the immobilization of the lipase
which may facilitate the easy enzymeꢀsubstrate interaction.21
Thus, lower Km value indicated higher substrate affinity of the
45 immobilized lipase PFL.1.21 In enzyme biochemistry, the presence
of uncompetitive inhibition results in decrease of Km and Vmax
value after immobilization.1 In uncompetitive inhibition substrate
affinity increased while maximum velocity decreased due to
pseudoꢀinteraction of enzymeꢀsubstrate.1 However overall,
50 catalytic efficiency (Vmax/Km) was found to be higher for the
immobilized lipase as compared to the free lipase. Thus the %
catalytic efficiency showed 12 % higher hydrolytic activity of the
MCNT immobilized lipase.1,21,34 The higher catalytic efficiency
was attributed due to immobilization of lipase PFL on the
55 hydrophobic surface of MCNT. Thus, lipase immobilization on
the hydrophobic surfaces can provided activation of lipases
(interfacial activation) which leads to improve catalytic activity
of nanoꢀbioꢀconjugates after lipase immobilization as compared
to free lipase PFL.7,9,16,34
60
3.8
Screening of various immobilized lipase support
Fig. 2: (A) SEM analysis of control MCNT (B) SEM analysis of
composition for biocatalytic applications
immobilized MCNT:PFL (C) TEM analysis of control MCNT (D)
Initially we have screened various lipase from various
65 microbial sources as biocatalysts (free and MCNT immobilized
lipase) for the given reaction system (Scheme 1) (Table 1, last
columns). For biocatalytic applications, it was observed that
lipase PFL is the best suitable biocatalyst as compared to the
lipase MJL and HPL, thus immobilized MCNT:PFL lipase was
70 selected for further study. However, it is necessary to screen
immobilization composition of MCNT:PFL lipase to find out the
suitable robust biocatalyst composition.13,16,31 Hence, we have
varied lipase PFL immobilization quantity by keeping constant
amount of the immobilization support (200 mg). It was observed
75 that, as the loading of lipase PFL increases (from 50 to 100) then
% conversion also increased which may be attributed due to
availability of extra active sites for catalytic transformations
(Table 5, entries 1ꢀ4). Moreover, further increase in the lipase
PFL loading from (100ꢀ120 mg) did not have significant effect on
80 the catalytic transformation (Table 5, entries 4ꢀ6). Whereas,
increase in the lipase loading from 120 to 150 mg causes slight
decrease in the bioꢀcatalytic activity because of the crowding or
stacking of lipase bioꢀmolecules which may causes blocking of
the active catalytic sites of lipase PFL (Table 5, entries 6ꢀ8).13,16,31
85 Similar type of lower enzyme activity was noted by Pavlidis et
al.,31 for higher enzyme loading on functionalized carbonꢀbased
nonmaterial. Thus, enzyme to nanoꢀmaterial composition is an
essential parameter that could affect the catalytic behaviour of
immobilized enzymes.
10 TEM analysis of immobilized MCNT:PFL
3.6
% Water content analysis study
The catalytic and synthetic activity of lipases is well
15 dependent on the degree of hydration which is controlled by trace
amount of tightly bound water.1,32,33 Since, minimum amount of
bound water is essentially needed to uphold the optimal
conformation of the lipase which involves opening and closing
movement of lipase lids.3,7,9 Moreover, the bacterial lipase needed
20 higher amount of water content since bacterial lipases such as
Pseudomonas fluorescens (PFL) involves large internal
movement of lids because of more protein residues.33 Thus to
carry out an efficient bioꢀcatalysis there is need of the essential
trace amount of the water layer around enzyme. In present study,
25 we have used water as a medium for the immobilization and
hence water content for MCNT immobilized lipase was found to
be higher (7.8 %) as compared to that of the free PFL (1.23 %)
(Table 3). Thus, it is expected that higher % water content could
assist to (i) maintain the conformational changes of three
30 dimensional structures of the enzymes as well as (ii) to improve
the catalytic activity and conversion of the desired product.33
Table 3: Determination of the % Water content
No
Sample
Water content
1
MCNT only
2.2 %
90
2
3
Free PFL (Lipase)
1.23 %
7.8 %
Immobilized lipase MCNT:PFL (2:1)
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Table 4: Determination of hydrolytic catalytic efficiency and various kinetic parameters (Vmax and Km)
No
Sample
% water
Vmax
Km
Catalytic
efficiency
% catalytic
efficiency
R2
content (µmol/mg/min) (mM)
1
2
Free lipase PFL
Immobilized lipase MCNT:PFL (2:1)
1.23
7.8
67.11
58.14
8.83
6.89
7.59
8.501
100
111.94
0.978
0.992
3
CNT only
2.2
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀ
ꢀꢀꢀꢀꢀ
ꢀꢀꢀꢀ
5
Scheme 1: Immobilized MCNT:PFL lipase synthesis of cinnamyl acetate
Table 5: Screening of various immobilized support compositions for biocatalytic applicationsa
% yield
Immobilized CNT:PFL
Improved Catalytic
activity in folds
Immobilized
MNCT:PFL (mg)
200:0
Immobilized composition
No
MNCT:PFL
0
Free lipase PFL
1
2
3
4
5
6
7
8
0
0
0
200:50
200:80
200:100
200:110
200:120
200:140
200:150
2:0.5
2:0.8
2:1
2:1.1
2:1.2
2:1.4
2:1.5
48
79
99
99
99
97
96
22
38
52
54
56
58
61
2.18
2.07
1.92
1.83
1.76
1.67
1.58
aCinnamyl alcohol (2 mmol); vinyl acetate (3.5 mmol); Orbital rotation speed (200 rpm); immobilized biocatalyst MCNT:PFL (42
mg); temperature (50 oC)
which avoids distortion of lipase conformations and hence
offered best catalytic efficiency.1ꢀ3 Since, polar solvent causes
distortion of enzyme confirmation and subsequent catalytic
activity due to stripping of essential bound water.23,32,33
3.9
Optimization of developed biocatalytic protocol
To achieve maximum conversion of desired product, molar
40
Influence of the mass transfer is an important factor to
achieve proper substrate and catalyst interaction.8,23 Hence mass
transfer effect was studied in the range of the 0 to 200 rpm (Table
6, entries 16ꢀ21). It was seen that, % conversion was increased as
the rotation speed increases from 0 to 175 rpm. Thus the mass
10 ratio of cinnamyl alcohol to vinyl acetate was varied from 2:0.5
to 2:4 (Table 6, entries 1ꢀ8). Experimentally, it was observed that
increase of moles of vinyl acetate from 0.5 to 3.5 led to increase
in the % conversion of the desired product when reaction
catalyzed by immobilized lipase. However, further increase of
15 moles of vinyl acetate from 3.5 to 4 did not offer any significant
increment in the conversion of desired product. In context to this,
% conversion was increased with increase in moles of vinyl
acetate from 0.5 to 4 mmol when reaction is catalyzed by free
lipase PFL (yield showed in parentheses, table 6, last column).
20 When vinyl acetate used as an acyl donor then vinyl alcohol was
formed as a byꢀproduct which is an unstable species, tautomerizes
into acetaldehyde and did not take part in subsequent reaction.4
Thus, 2:3.5 was optimized mole ratio quantity providing 98 %
conversion of desired product and was chosen to carry out
25 remaining all experiments.
To find out suitable reaction media, we have tested various
polar and nonꢀpolar organic solvents having Log ρ value in the
range of 0.4 to 4.0 for the given model reaction. In present study,
the polar solvents such as 1,4 dioxane, diethyl ether, acetone and
30 chlorinated solvent such as chloroform gave moderate to good
conversion of corresponding product while, nonꢀpolar solvents
like toluene and nꢀheptane were found to be promising solvent to
offer excellent conversion of the desired product (Table 6, entries
9ꢀ15). This was anticipated due to better hold up of essential
35 bound water from enzyme coat in presence of nonꢀpolar solvent
45 transfer barrier was achieved at the 175 rpm which providing
highest % conversion. Higher rotation (> 230 rpm) speed leads to
decrease in the % conversion, as immobilized MCNT:PFL
biocatalyst was thrown outside of reaction media and did not take
part in further reaction. Hence, 175 rpm was the optimized
50 rotation speed for the given reaction system.
In order to maintain an economic feasibility, we studied
effect of the biocatalyst amount to find out optimum
concentration of biocatalyst.16,18 The immobilized lipases were
loaded ranging from 0ꢀ40 mg while free/ crude lipase were also
55 loaded in w/w equivalent amount (Table 6, entries 22ꢀ27). It was
observed that biocatalyst loading (free as well as immobilized
lipase) showed positive relationship with % conversion which
was attributed to active participation of the available catalytic
sites to carry out reaction. The maximum yield (99 %) of
60 cinnamyl acetate was obtained when reaction was catalyzed by 36
mg of immobilized MCNT:PFL lipase. In contrast to this
equivalent quantity of free lipase PFL provided almost 2 fold
lesser conversion. Further, increase in the biocatalyst amount to
40 mg did not afford significant improvement in the %
65 conversion.
6
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It is well known fact that, enzymes are usually active at
optimum temperatures whereas their catalytic activityꢀstability 10 displayed similar % conversion at given reaction condition. The
be optimum temperature for immobilized lipase, since 55 oC
goes on decreasing beyond the optimum temperature due to
confirmation destabilization.3,6,9,18 Hence, in current study, the
MCNT immobilized lipase was subjected for acetate synthesis at
higher energy provided activation energy to the reacting
molecules and speed up the reaction rate. These results were
signifying that, 50 C was chosen as optimized temperature and
o
5
o
temperature ranging from 25ꢀ55 C (Table 6, entries 28ꢀ34). The
used for further all experiments. Thus the optimized reaction
increase of temperature from 25ꢀ50 oC led to increase in % 15 parameters are: reactant molar ratio (2:3.5), biocatalyst amount
conversion of the desired product and thus 50 oC was observed to
(36 mg), rotation speed (175 rpm) and temperature (50 oC) etc.
Table 6: Optimization of the various reaction parameters for developed biocatalytic protocol
No
Solvent
Alcohol: acyl donor
(mmol)
Rotation
(rpm)
Biocatalyst
(mg)
Temperature
(oC)
Conversion a
(%)
Effect of mole ratio
1
2
3
4
5
6
7
8
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
Toluene
2:0.5
2:1
2:1.5
2:2
2:2.5
2:3
2:3.5
2:4
200
200
200
200
200
200
200
200
36
36
36
36
36
36
36
36
50
50
50
50
50
50
50
50
9 (0)
24 (5)
40 (10)
53 (18)
72 (25)
85 (36)
98 (45)
98 (55)
Effect of solvent
nꢀheptane
10 Toluene
11 1,4 dioxane
12 Diethyl ether
13 Acetone
9
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
200
200
200
200
200
200
200
36
36
36
36
36
36
36
50
50
50
50
50
50
50
99 (46)
98 (45)
82 (26)
78 (22)
76 (20)
28 (4)
14 Acetonitrile
15 Chloroform
Effect of rotation
16 nꢀheptane
17 nꢀheptane
18 nꢀheptane
19 nꢀheptane
20 nꢀheptane
21 nꢀheptane
Effect of biocatalyst
22 nꢀheptane
23 nꢀheptane
24 nꢀheptane
25 nꢀheptane
26 nꢀheptane
27 nꢀheptane
Effect of temperature
28 nꢀheptane
29 nꢀheptane
30 nꢀheptane
31 nꢀheptane
32 nꢀheptane
33 nꢀheptane
34 nꢀheptane
64 (7)
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
0
50
100
150
175
200
36
36
36
36
36
36
50
50
50
50
50
50
21 (06)
48 (20)
68 (30)
93 (41)
99 (48)
99 (46)
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
175
175
175
175
175
175
0
50
50
50
50
50
50
0 (0)
10
20
30
36
40
40 (14)
68 (27)
90 (45)
99 (48)
99 (50)
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
2:3.5
175
175
175
175
175
175
175
36
36
36
36
36
36
36
25
30
35
40
45
50
55
21 (5)
38 (11)
54 (22)
68 (35)
85 (47)
99 (48)
99 (48)
aConversion indicated in parenthesis is obtained by free lipase PFL, while % conversion indicated outside of parenthesis is obtained
by immobilized MCNT:PFL lipase (nanoꢀbioꢀconjugates)
25 the great importance and application as a food, flavour/ fragrance
compounds in various commercial fields.24,25,35ꢀ39 Most of these
3.10
Application of MCNT:PFL nano-bio-conjugates for
compounds have natural occurrence and can be obtained by
tedious costly natural extraction process. In context to this,
enzymatic synthesis is more efficient which can labelled these
20 syntheses of acylated moieties and their potential applications
The potential biocatalytic applications of lipase nanoꢀbioꢀ
conjugates were tested for synthesis of various important organic 30 products as “Green and Natural” for safe use in day to day life.
moieties by using optimized reaction parameters. Enzymatically
synthesized compounds are listed in Table 7, which possessing
The allylic alcohols such as cinnamyl alcohol and prenol
(possessing floralꢀrose soapy smell) (Table 7, entries 1,2) gave 99
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% yield of corresponding product in 4 h. Acetates of the 20 20) which can be synthesized enzymatically and are recognized
saturated, linear and branched primary alcohols (having sweet
fruity olfaction) also offered excellent yield up to 99 % of
respective desired product (Table 7, entries 3ꢀ10) in 4ꢀ6 h.
Aromatic sideꢀchain alcohols as like benzyl alcohol, 2ꢀphenyl
as Safe compounds by Council of Europe25 and Flavour
Manufacturers.35 In addition to this, these synthesized compounds
are approved by the Food Drug Administration (FDA)36 and the
Joint Expert Committee on Food Additives (JECFA)37 for safe
5
ethanol and 3ꢀphenyl propanol (possessing fruityꢀflavour and 25 use as an additives in foods. Thus, many of the above synthesized
fragrance) gave excellent yield (99 %) of the desired aromatic
acetate product in 4 h (Table 7, entries 11ꢀ13). Various acetates
of substituted benzyl alcohols (Table 7, entries 14ꢀ17) were
compounds were also used essentially as an ingredient for the
various perfumery and fragrances compounds (Table 7, entries 1ꢀ
20).36,37 The universal production and use of each flavour or
fragrance compound is in the range of 1 to 1000 metric tons per
10 synthesized efficiently with 99 % yield within 4ꢀ5 h. αꢀ Methyl
benzyl acetate (Table 7, entry 18) gave 46 % yield of desired 30 annum.38 The Environmental legislation, competitiveness and
product in 12 h while alicyclic secondary acetates of menthol as
well as cyclohexanol (Table 7, entry 19,20) were synthesized
with 44 and 57 % yield in 12 h respectively. Phenol and pꢀcresol
15 (Table 7, entry 21,22) were reacted slowly because of acidic
social responsibility always directing several industries towards
development of more ecofriendly, safer and greener industrial
commercial processes for the sustainable future.39 Thus looking
to the above importance, aspects and wide substrate array, the
nature to provide moderate yield (63 and 69 % respectively) of 35 present protocol demonstrated
a promising alternative to
desired acetates in 10 h.
synthesize commercially important organic moieties with greener
outlook for sustainable future.
Many of these above organic moieties are of great
importance in day to day life compounds24,25 (Table 7, entries 1ꢀ
Table 7: Application of nanoꢀbioꢀconjugate MCNT:PFL immobilized lipase for syntheses of various organic compounds and their potential
applicationsa
No
Flavour-fragrance
compound
Time
(h)
% Yield
Application in food
flavour-fragrance
Natural occurrence
in plants and fruits
Immobilized MCNT:
PFL
Free
PFL
Psidium guajava
Laurus nobilis,
Cinnamomum verum
Cinnamon, oriental, rose,
apricot, guava
1
4
99
48
Pear, Banana, Lavender,
Melon, pineapple, peach
2
3
4
99
99
42
36
Cananga odorata
Strawberry, tomato,
cherry, melon, plum,
Averrhoa carambola
Theobroma cacao
4.5
Apple, banana, pear,
mulberry, papaya,
mango, plum
Citrus bergamia
Michelia champaca
Fragaria vesca
4
5
5
5
99
99
38
37
Tea green peach, apple,
gardenia,
Boswellia carterii
Lavandula angustifolia
Quince, orchid, fig,
gooseberry, jackfruit,
melon
Jasminum flexile,
Vaccinium myrtillus,
matricaria sp.
6
7
5
99
99
41
41
Peach, lemon,
blackberry, Papaya,
lavender
Citrus bergamia,
Elettaria cardamomum
5.5
Pineapple, current,
Butter, raspberry
Theobroma cacao,
Fragaria vesca
8
9
4.5
4
99
99
38
39
Vanilla, plum, peach,
date
Theobroma cacao
Musa sapientum
8
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Pea, bean, fungus
mushroom, oriental
10
11
5
4
99
99
38
48
-----
Jasmine, green apple,
strawberry, banana,
orchid,
Acacia farnesiana
Hyacinthus sp. Jasminum
Llaurus nobilis
Narcissus spp.
Psidium guajava
Blackberry, cinnamon,
lily, cranberry
12
13
4
4
99
99
46
47
Apricot, Peach Vanilla,
Tuttiꢀfrutti
freesia magnolia
Hyacinth reseda
Cyclamen persicum
Cyclamen purpurescens
14
15
5
99
99
30
39
ꢀꢀꢀꢀꢀ
Juniper berry, raspberry,
vanilla, fig, coconut
cherry
Cananga odorata
Clavija euerganea
Jacquinia keyensis
4.5
Bayberry, fig, oriental,
vanilla
16
5.5
99
29
----
Apricot, peach,
17
18
4.5
12
99
46
39
17
raspberry, woody, cherry Cichorium endivia
strawberry
Barosma betulina
Sweet pea, pear, apple,
Eucalyptus globules
grape, plum, berry
Mentha piperita
Peppermint, mint,
blueberry, citrus,
lavender, peach
Barosma betulina
Eucalyptus globules
Mentha piperita
19
20
12
12
44
57
15
20
Apple, pear, currant, fig,
jackfruit
Sauerkraut (Sour cabbage)
Silene italic
Silene vulgaris
21
22
12
12
63
69
23
29
ꢀꢀꢀꢀꢀ
Apple blossom, cherry
blossom
Prangos uechtritzii
Cananga odorata
aAlcohol (2 mmol); vinyl acetate (3.5 mmol); Orbital rotation speed (175 rpm); immobilized biocatalyst MCNT:PFL (36 mg);
temperature (50 oC)
3.11 Time versus conversion plot and comparison of bio-
The figure 3 depicted the profile of the product formation
by free and immobilized CNT:PFL lipase with respect to reaction
time progress. The profile of desired product formation with
10 respect to time progress demonstrated improved biocatalytic
5
catalytic activity between immobilized MCNT:PFL
and free PFL
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activity of the immobilized MCNT:PFL lipase compared to the
corresponding free lipase PFL. The highest conversion of
cinnamyl acetate 99 % was achieved in 240 minutes (4 h) when
3.13 Thermo-kinetic investigation of the present
biocatalytic protocol
Arrhenius equation is useful to deduce the kinetic
reaction was catalyzed by immobilized MCNT:PFL lipase, 50 parameters such as energy of activation while, Eyring equation is
5
whereas free PFL provided almost 2 fold lesser conversion of the
desired product. The continuous increase in the % conversion
representing that, reaction was progressed towards forward
direction only to offer desired stable product. The improved
useful to deduce the various thermodynamic parameters such as
ꢂG*, ꢂS*, ꢂH*. Herein equation 1, 2 and 3 involves; k, A, Ea, T,
kB, h, R, ꢂG*, ꢂS*, and ꢂH* are reaction rate constant,
Arrhenius collision factor, energy of activation, absolute
conversion of the immobilized MCNT:PFL lipase was attributed 55 temperature, Boltzmann constant, Planck’s constant real gas
10 to (i) interfacial activation of lipase PFL after immobilization on
hydrophobic MCNT7.9 (ii) more catalytic sites are easily
accessible after immobilization of lipase PFL on MCNT, which
facilities the easy substrate binding to liftꢀup reaction rate31 and
(iii) lipases PFL are well scatters after immobilization on MCNT
15 which favours easy mass transfer of the reactant/ product.7,9,31
constant, Gibbs free energy, entropy and enthalpy of activation
respectively.
k = Aexp(Ea / RT )................................(1)
ꢀG* / T = (ꢀH* / T ) − ꢀS .........................(2)
kh = Tk
exp(ꢀS* / R)exp(−ꢀH* / RT).......(3)
B
60
Various thermo kinetic parameters were calculated in the
range of range of 303 to 323 K (Table 8). The Ea for free PFL
and immobilized MCNT:PFL was found to be 74,149 and 58,671
J/mol respectively. The lower Ea value for the MCNT
3.12 Mechanism of MCNT:PFL lipase catalyzed cinnamyl
acetate synthesis
o
The active centre present in enzyme lipase is known as the
20 catalytic triad which is mainly consist of the serine, aspartic acid
and histidine residue, these three residues plays an important role
to perform the bioꢀcatalytic activity.40 (i) Initially nanoꢀbioꢀ
conjugate MCNT:PFL forms an enzyme substrate complex along
with the acyl donor vinyl acetate (Scheme 2). (ii) Afterwards, the
25 serine group attacks on the electron deficient carbonyl part of the
vinyl acetate and tend to form the acylꢀlipase intermediate. This
acyl enzyme intermediate causes elimination of the biꢀproduct
vinyl alcohol which tautomerized into a low boiling acetaldehyde
compounds (21 oC). (iii) Soon after, cinnamyl alcohol
30 incorporates in the reaction and attacks on the electron deficient
carbonyl group and again forms a tetravalent intermediate with
lipase. (iv) This tetravalent intermediate reorganized itself and
breaks down into the corresponding desired acetate product and
leads to free up lipase molecules (Scheme 2). During overall
35 biocatalytic process, activation of the histidine residue was
carried out by the aspartic acid which imparts in the abstraction
of the proton. Whereas negative charge developed on the each
tetravalent intermediate is significant for the formation of
respective product and biꢀproduct. Moreover, various aminoꢀacid
40 residues are responsible for the formation of Hꢀbonding which
65 immobilized lipase PFL indicated the (i) higher reaction rate (ii)
higher catalytic efficacy and (iii) lesser energy requirement as
compared to free PFL.41 The lesser enthalpy of activation (ꢂH*
value) value for MCNT immobilized lipase PFL was attributed
due to easy formation of acylꢀenzyme complex; while ꢂH* was
70 found to be higher for free PFL lipase catalyzed reaction which
point to complexity to tetrahedral acylꢀenzyme complex
formation.42,43 Since, free lipases PFL were agglomerated in
organic solvents and created diffusion restrictions for substrate
towards active catalytic sites.42ꢀ44 The ꢂS* value stands for the
75 difference in the extent of local disorder or randomness between
ground state and transition state.42ꢀ45 The free lipase PFL showed
higher disorder (ꢀ106.83 J/MolꢃK), while immobilized
MCNT:PFL lipase demonstrated lesser disorder (ꢀ146.65 J/Molꢃ
K) of reaction. Thus, immobilized MCNT:PFL favoured the
80 reaction with lesser extent of disorder compared to crude lipase
PFL. The feasibility of reaction system is best accounted by ꢂG*
value, lesser is the Gibb’s free energy change; higher is the
feasibility of reaction and viceꢀaꢀversa. Thus immobilized
MCNT:PFL offered smaller ꢂG* value (101.97 J/mol), which
85 demonstrating the higher feasibility of reaction as compared to
free PFL (105.18 J/mol).42ꢀ46 Thus from above all thermodynamic
parameters, the immobilized MCNT:PFL offered ease of reaction
system compared to that of free PFL. These results indicating the
lower thermodynamic energy assessment and improved catalytic
90 activity of hydrophobic MCNT supported immobilized
MCNT:PFL lipase (nanoꢀbioꢀconjugate).
induces the sufficient polarisation in
a molecule so that
electrophilic species can easily attacked by nucleophilic species.40
120
100
80
60
40
20
0
105
90
75
60
45
30
15
0
Table 8: Thermoꢀkinetic investigation of the present
biocatalytic protocol
No. Thermo-kinetic factor
MCNT-PFL
Free PFL
1
2
3
4
Ea (J/mol)
58671.80
56072.11
ꢀ146.65
101.97
74149.24
71549.45
ꢀ106.83
105.18
ꢂH* (J/mol)
ꢂS* (J/Molꢃ K)
ꢂG* (J/mol)
0
45
90
135
180
225
270
315
Time (minutes)
95
Reactant consumption (MCNT:PFL)
Product formation (MCNT:PFL)
Reactant consumption (free PFL)
Product formation (freePFL)
45
Fig. 3: Product formation profile and comparison of immobilized
MCNT:PFL with free PFL for biocatalytic application
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Scheme 2: Mechanism of the MCNT:PFL catalyzed reaction for synthesis of cinnamyl acetate
3.14 Recyclability and leaching study
% conversion is also a cause of handling loss and inhibition by
inhibition complex and reduces the enzyme activity.18,4,6
5
The recyclability of immobilised lipase is a key aspect for 25 alcoholic substrate (cinnamyl alcohol) which forms a deadꢀend
potential industrial applications and economical feasibility; hence
reusability was studied for given model reaction under identical
optimised reaction conditions. After each cycle immobilized
lipase was washed/ rinsed 2 times by nꢀheptane to remove any
10
8
100
80
10 stick of products or reactants. It was then dried for 10ꢀ12 hours
and stored at 0ꢀ5 oC until used for next recycle study. It was
observed that, conversion decreases as number of recycles
increases, while 63 % conversion was achieved at the end of the
fifth cycle (including fresh) (Figure 4A). The decrease in %
15 conversion may attributed by leakage of enzyme from MCNT
surface, as lipases are physically adsorbed on the surface of
MCNT.18,23,47,48 For the leaching study, we have performed an
experiment, wherein lipase was placed in reaction medium nꢀ
heptane (without reactants) for 1ꢀ10 hours at 175 rpm (Figure
20 4B). It was observed that lipase was slowly leached from the
surface of MCNT which may be attributed to physical adsorption
type of immobilization. Thus the leaching was found to be 6.5 %
and 9 % at interval of 6 and 10 hours. Moreover, the decrease in
60
6
40
4
20
2
0
0
I
II
III
IV
V
2
4
6
8
10
12
(A)
(B) 0
Recycle run number
Time (h)
Fig. 4: Recyclability and leaching study
30
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Journal Name, [year], [vol], 00–00 | 11

RSC Advances
Page 12 of 13
View Article Online
DOI: 10.1039/C5RA10032A
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H. Liu, Ind. Eng. Chem. Res. 2012, 51, 13173ꢀ13181
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Sci. Technol., 2015, 5, 1840ꢀ1846
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8. K. C. Badgujar, B. M. Bhanage, Ind. Eng. Chem. Res., 2014, 53,
18806ꢀ18815
Conclusions
In present study, we have successfully immobilized lipase PFL on
the MCNT, this nanoꢀbioꢀconjugate worked as robust biocatalyst.
The composition MCNT:PFL (2:1) showed highest % lipase
activity, % protein binding yield and % activity retention. The
immobilization of the lipase PFL on the MCNT was confirmed
by physical and biochemical characterization such as SEM, TEM,
FTIR, Km, Vmax, catalytic efficiency and (%) water content
analysis. This immobilized biocatalyst MCNT:PFL (2:1) was
5
9. G. FernandezꢀLorente, Z. Cabrera, C. Godoy, R. Fernandezꢀ
65
Lafuente, J. M. Palomo, J. M. Guisan, Process Biochem, 2008,
43, 1061ꢀ1067
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Process Biochem, 2006, 41, 1193ꢀ1197
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Gournis, H. Stamatis, Process Biochem. 2013, 48, 1010ꢀ1017
13. S. S. Karajanagi, A. A. Vertegel, R. S. Kane, J. S. Dordick,
10 successfully applied for Oꢀacylation reactions as a practical
biocatalytic applications. For Oꢀacylation reaction, various five
reaction parameters were optimized in details. Furthermore these
developed biocatalytic protocol was then successfully extended to
synthesize several (twentyꢀtwo) industrially important acylated
15 moieties with excellent yields. These products are well
characterized by the available tools such as H NMR, 13C NMR
1
and GCꢀMS analysis. Besides this, we have reviewed the
potential commercial applications of the synthesized moieties.
Moreover, the thermodynamic study indicating more feasibility
20 of immobilized MCNT:PFL lipase (nanoꢀbioꢀconjugate) over free
lipase PFL. Also, immobilized MCNT:PFL lipase showed 2.2
folds higher catalytic activity and five times recyclability
compared to free PFL. Thus present protocol is a robust, efficient
and recyclable which carry out the (i) synthesis of nanoꢀbioꢀ
25 conjugate as a bioꢀcatalyst, (ii) detail physicalꢀbiochemical
characterization of biocatalyst, (iii) optimization of biocatalytic
protocol (iv) practical biocatalytic applications along with
mechanistic study (v) thermodynamic feasibility study and (vi)
recyclability study.
75
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Mathur, Process Biochem. 2013, 48, 124ꢀ132
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Dordick, Biotechnol. Bioeng. 2006, 95, 804ꢀ811.
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2014, 61-62, 28ꢀ34
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42, 332ꢀ339
20. K. C. Badgujar, B.M. Bhanage. Enzyme Microbial Technol.
2014, 57, 16-25,
90 21. O. Barbosa, C. Ortiz, Á. BerenguerꢀMurcia, R. Torres, R.C.
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1600
The author Kirtikumar Badgujar is grateful to the Department of
BiotechnologyꢀDBT, India (BT/PR4192/PID/6/623/2011), and
the Council of Scientific & Industrial ResearchꢀCSIR, India
(09/991(0015)/ 2011ꢀEMRꢀI), for providing research funds. Also
35 author is greatly thankful to Ms. Poorna Pai and Mr. Rajesh Patil
for their kind help in characterization/ analysis.
22. J. S. Carey, D. Laffan, C. Thomson, M. T. Williams. Org.
Biomol. Chem. 2006, 4, 2337ꢀ2347.
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bDepartment of Complexity Science and Engineering, Graduate
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