Lipase immobilization on hyroxypropyl methyl cellulose support and its applications for chemo-selective synthesis of β-amino ester compounds

Article abstract of DOI:10.1016/j.procbio.2016.07.008

The present study carried out the synthesis of β-amino ester compounds using lipase immobilized on hyroxypropyl methyl cellulose (HMC) support. Initially various lipases (biocatalysts) from different origin were immobilized and subsequently screened to obtain the robust biocatalyst. The lipase Pseudomonas fluorescence (PFL) immobilized on HMC was displayed highest lipase activity, protein content and retention of activity. The physical and biochemical characterization verified immobilization of lipase PFL on the HMC support. This immobilized biocatalyst HMC:PFL (3.5:1) was successfully applied for the practical biocatalytic applications to synthesize variety of β-amino esters. Various eight reaction parameters were optimized in details to achieve the maximum yield and chemo-selectively. The developed biocatalytic protocol was successfully applied to synthesize different industrially important β-amino esters compounds (21 substrates) with an excellent yield (>90%) and remarkable chemo selectivity (>94%). Interestingly, the immobilized HMC:PFL lipase showed 2.1–2.5 folds higher bio-catalytic activity and five times recyclability as compared to the free PFL. The plausible mechanism for lipase catalyzed synthesis of β-amino ester compounds was also proposed.

Full text of DOI:10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
Process Biochemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Lipase immobilization on hyroxypropyl methyl cellulose support and
its applications for chemo-selective synthesis of ␤-amino ester
compounds
Kirtikumar C. Badgujar, Bhalchandra M. Bhanage∗
Department of Chemistry, Institute of Chemical Technology, Matunga, Mumbai 400 019, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The present study carried out the synthesis of ␤-amino ester compounds using lipase immobilized on
hyroxypropyl methyl cellulose (HMC) support. Initially various lipases (biocatalysts) from different origin
were immobilized and subsequently screened to obtain the robust biocatalyst. The lipase Pseudomonas
fluorescence (PFL) immobilized on HMC was displayed highest lipase activity, protein content and reten-
tion of activity. The physical and biochemical characterization verified immobilization of lipase PFL on
the HMC support. This immobilized biocatalyst HMC:PFL (3.5:1) was successfully applied for the practi-
cal biocatalytic applications to synthesize variety of ␤-amino esters. Various eight reaction parameters
were optimized in details to achieve the maximum yield and chemo-selectively. The developed biocat-
alytic protocol was successfully applied to synthesize different industrially important ␤-amino esters
compounds (21 substrates) with an excellent yield (>90%) and remarkable chemo selectivity (>94%).
Interestingly, the immobilized HMC:PFL lipase showed 2.1–2.5 folds higher bio-catalytic activity and five
times recyclability as compared to the free PFL. The plausible mechanism for lipase catalyzed synthesis
of ␤-amino ester compounds was also proposed.
Received 27 January 2016
Received in revised form 30 June 2016
Accepted 9 July 2016
Available online xxx
Keywords:
Lipase
Immobilization
Ester compounds
Biocatalysis
Improved activity
© 2016 Elsevier Ltd. All rights reserved.
1. Introduction
follow any green chemistry principles [3–14,17,18,19–21]. More
recently, the American Chemical Society Green Chemistry Insti-
The formation of C N bond is of great importance in the nat-
ural as well as synthetic bio- chemistry which is a backbone of
several life beginning bio-organic molecules like DNA, RNA, micro-
cycles, glycopeptides, amino-acids, proteins, amino-glycosides and
alkaloids [1–3]. Moreover, the C N bond formation is most widely
used versatile step in the synthesis of active pharmaceutical drug
intermediates (API) [2]. The conjugate addition reactions such as
aza-Michael addition of nucleophiles with ␣, ␤ unsaturated com-
pounds is considered as a powerful tool for the carbon-nitrogen
bond formation [3–14]. This aza-Michael addition reaction has
great importance in total synthesis of various important medicinal
molecules [2–16].
tute of Pharmaceutical Roundtable (ACS-GCIPR) was set up to
promote pharma/drug industries for synthesis of active pharma-
ceutical intermediates using the Green chemistry and technology
[1,19,22].
In view of this, biocatalysis is the best way for synthesis of
pharma/drug intermediates since; it offers several benefits like
regio/stereo/chemo- selectivity, mild reaction condition, optimum
temperature, low activation energy, safe use and no dispos-
able/issues [19–24]. In biocatalysis, particularly triacyl glycerol
hydrolases (lipase: E.C. 3.1.1.3) from various microbial sources
attracted the significant attention because of the broad substrate
array and capability to carry out various promiscuous reactions
[21–24]. Despite of these benefits of lipases in biocatalysis, there
are still some unavoidable issues regarding to thermal and sol-
vent stability of free lipases in non-aqueous media [20–24]. The
advance immobilization of lipase may triumph over above said
technical difficulties, as immobilization may leads to improve the
activity, stability, conversion, recycle and solvent tolerance capac-
ity [21–25].
In conventional chemical synthesis/catalysis various chemical
reagents/catalysts are reported to synthesize these ␤-amino ester
compounds such as Lewis acids of transition metals, lanthanide
halides, triflates, silica supported perchloric acid etc which hardly
∗
Corresponding author.
E-mail addresses: bm.bhanage@gmail.com, bm.bhanage@ictmumbai.edu.in
In recent time, various natural polymers have received great
importance for immobilization which having excellent proper-
(B.M. Bhanage).
http://dx.doi.org/10.1016/j.procbio.2016.07.008
1359-5113/© 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
2
ties like biodegradability, eco-friendliness and biocompatibility
for biomolecules [25–30]. Herein, we make an effort to immobi-
lize lipase on the hydroxypropyl methyl cellulose (HMC) which
finds great applications in bio-medical and bio-engineering field
for the drug delivery and tissue engineering [31]. HMC is natu-
ral polymer consist of the several monomeric linear units of ␤-1,4
glucose [31]. It possesses excellent properties like adhesiveness,
film-forming ability, biocompatibility and flexibility. Moreover,
presence of free −OH group in HMC afford enhanced immobiliza-
tion effect and binding efficiency [31]. The present immobilization
involves simple entrapment method which offering the benefits
like (i) simple methodology, (ii) no use of cross-linking chemical
agent and (iii) use of water as a greener medium. Also, immobiliza-
tion via entrapment can shield the enzyme from direct exposure to
outer environment which ultimately displayed improved activity
[1,24–26].
According to survey of reactions of pharmaceutical companies,
␤-amino ester synthesis via 1,4 addition counted almost 14–16%
of overall synthesis steps [15]. Till time few researchers have syn-
thesized these ␤-amino esters using commercially available Cal-B
enzyme [32–36]. However in present study, we used lab made
immobilized lipase for the synthesis of ␤-amino ester compounds
with chemo selective aspect and detail characterization. Thus, the
present works deal with the lipase immobilization and synthesis of
␤-amino ester compounds, process optimization, substrate appli-
cability, reusability, leaching, inhibition and mechanistic approach.
2.3.2. TGA
The thermo gravimetric analysis (TGA) was performed to inves-
tigate the thermal stability of immobilized lipase and free lipase by
using Q-series 600 analyzer. For this purpose nearly 10 mg of sam-
ples was placed in an alumina crucible and analysis was performed
from 30 to 400 ◦C along with 10 ◦C/min rise, at pure N2 atmosphere
with flow rate of 80 mL/min.
2.3.3. Film appearance and thickness determination
The thickness of immobilized HMC:PFL matrix and control HMC
matrix was measured by manual micrometre. The final thickness
was the average of 10 various random places of HMC.
2.3.4. Lipase activity
Various free lipases and immobilized HMC lipases were
subjected to study the hydrolytic lipase activity spectrophotomet-
rically at 405 nm with the slight change of reported procedure by
Pencreac’h and Baratti [36]. In a hydrolytic lipase activity assay,
we had studied hydrolysis of p-nitro phenyl butyrate (p-NPB) sub-
strate. In a reference assay consist of 2 mg of free lipase PFL (or
comparable quantity of the immobilized HMC:PFL lipase) in 1 mL
of n-hexane solvent. The reaction was begun with addition of 1 mL
of 18 mM, p-NPB substrate dissolved in 2-propanol solvent and
incubated at 35 ◦C and at 150 rpm for 10 min. Later on, 250 ␮L of
hydrolytic assay reaction mass was taken out and added by 450 ␮L
of distilled water to extract p-nitro phenol (p-NP) in an aqueous
phase. At last, 1300 ␮L of potassium phosphate buffer of pH ∼8.0
was added which provided a pale yellow colour to the extracted
p-NP. This sample (pale yellow colour) was immediately utilized
to find out the absorbance at 405 nm. The lipase activity (U) was
defined as a micro-moles (␮ mol) of p-NP released by per milligram
(mg) of lipase per minute (min) under the given standard assay
condition.
2. Material and methods
2.1. Chemical and enzymes
Lipase MJL (Mucor javanicus, activity ≥10 U/mg) and lipase
AYS (Candida rugosa, lipase, activity ≥30 U/mg) was gifted by
Amano Enzymes Pvt. Ltd. (Japan). Lipase HPL (Hog pancreas lipase,
HPL, activity ≥30 U/mg) was purchased from Fluka India Pvt. Ltd.
Lipase PFL (Pseudomonas fluorescens lipase, activity ≥ 20 U/mg) was
purchased from Sigma Aldrich Pvt. Ltd. India. All other chemi-
cals/solvents were purchased from reputed firms and used as such.
2.3.5. Protein content
The protein content of free PFL and protein loading of immobi-
lized HMC:PFL lipase was determined by using Bradford method
at 595 nm [37]. At first, initial protein content was determined
which was employed for immobilization into matrix. After immo-
bilization, the immobilized matrix was completely removed from
the Teflon petri dish; this dish was then rinsed and subjected to
determine the un-immobilized amount of protein. Thus, quantity
of protein immobilized on HMC matrix was the difference between
the initial/crude amount of proteins used to immobilize and un-
immobilized quantity of protein found after washing of Petri dishes.
2.2. Biocatalyst preparation
The preparation of immobilization matrix film of HMC was per-
formed in deionised-water as a greener immobilization support
and medium respectively. The HMC (0.35 g) was dissolved in 30 mL
of deionised-water in a separate beaker. The resulting solution was
vigorously stirred for 4–5 h at 1200 rpm. Afterwards, the native
lipase (0.1 g) was dissolved in 4–5 mL of deionised-water and added
to above HMC matrix solution. Resulting mixture was then mod-
erately stirred at 160–180 rpm for 50–60 min. At last, the HMC:PFL
immobilized matrix was cautiously poured in a Teflon petri dish
and allowed to dry out for almost 30–35 h in an oven at 40 ◦C. A pale
yellow colour matrix of HMC:PFL immobilized lipase was formed,
which was then cut into small pieces of 3–4 mm2 size and stored
in a deep freeze at 4–5 ◦C until use.
2.3.6. Water content
The% water contents of free PFL, HMC:PFL immobilized lipases
and control HMC support were investigated by using the Karl Fis-
cher method (784 KFP Titrino)
2.3.7. Km and Vm determination
The apparent reaction velocity (Vm) and Michaelis-Menten
constant (Km) of free PFL and immobilized HMC:PFL lipase was
investigated by the Lineweaver-Burk plot by means of hydrolytic
lipase activity assay of p-NPB in the range of 2–24 mM substrate
concentration. The method used for km and Vm determination is
similar as specified in above Section 2.3.4.
2.3. Characterization of biocatalyst
2.3.8. Determination of solvent and thermal stability
2.3.1. SEM
The effect of different organic media on activity of HMC:PFL
immobilized lipase was determined. The immobilized lipase
HMC:PFL (3.5:1) was incubated in various polar and non-polar
organic media. After incubation, the solvent was separated by fil-
tration and lipase activity was determined for immobilized lipase
PFL as indicated in the above procedure (Section 2.3.4). Similarly,
The scanning electron microscopy (SEM) images were cap-
tured by the FEI-Quanta 200 to investigate surface morphology of
HMC:PFL and control HMC support. For this purpose, sample was
placed on carbon stub and images were captured at 15 kV using LFD
detector.
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
3
the effect of various temperature on activity of HMC:PFL immobi-
lized lipase was determined in the temperature range from 40 to
60 ◦C.
which demonstrating the higher decay rate for the free lipase PFL
compared to that of immobilized HMC:PFL lipase. Thus, above
experimental observations point out enhanced thermal character-
istic of immobilized HMC:PFL lipase in contrast to free PFL.
2.3.9. Determination of lipase leaching
The immobilized lipase HMC:PFL (3.5:1) was incubated for the
4h–25 h in n-hexane solvents to investigate the possible leaching of
the enzyme from the support matrix. After incubation, the solvent
was separated by filtration and lipase activity was determined for
the filtered solvent, as indicated in the above procedure (Section
2.3.4)
3.1.3. Film appearance
The HMC:PFL immobilized matrix was (i) opaque pale-yellow
colour (ii) flexible-uniform in nature and (iii) could be easily
handled/used. However, the control support HMC matrix was
presenting very slight pale yellow colouration with sufficient trans-
parency along with the uniform and flexible nature. The control
HMC film presented thickness values in between 42 and 48 ␮m
while HMC:PFL immobilized matrix showed thickness 60–65 ␮m
because of the lipase immobilization.
2.3.10. Determination of substrate inhibition
The immobilized lipase was incubated in presence of model
amine substrate (benzyl amine) at various concentrations
(0–1.5 mmol) to investigate the possible inhibition of immobilized
lipase. After incubation, the solvent was separated by filtration and
lipase activity was determined for immobilized lipase PFL as pro-
cedure given in the Section 2.3.4.
3.1.4. Determination of lipase activity
The lipase hydrolytic activity assay was performed for the
screening purpose, it was found that various free and immobilized
HMC:lipase followed the sequence as: lipase MJL < HPL < AYS < PFL
(Table 1). All four HMC immobilized lipases demonstrated
improved catalytic activity than free lipases which might be
attributed to the interfacial activation phenomenon after immo-
bilization of lipase on the hydrophobic HMC support matrix
[39–41]. The hydrophobic support matrix may form strong inter-
action with the hydrophobic active sites of lipase molecules
after immobilization and endorsed ‘open state conformation’ of
lipases [21,36,29,39]. Thus, various HMC:PFL immobilized biocat-
alyst showed improved lipase activity as compared to free lipase
(Table 1, column 1,4), while HMC:PFL lipase showed highest% lipase
activity.
2.4. Reaction procedure and analysis
Various ␤-amino ester moieties were synthesized in a 10 mL
glass reaction vessel of 1.2 cm i.d. with a glass lid. The desired
Michael acceptor was taken into a reaction vessel and diluted
by solvent. Afterwards, Michael donor was added into the reac-
tion vessel and reaction was initiated by addition of immobilized
HMC:PFL lipase very soon at a specified temperature. The sam-
ple of reaction mixture was analyzed by using gas chromatograph
(Perkin Elmer Clarus: 400) having flame ionizing detector (FID) and
capillary column. The detector and injector temperature were kept
280 and 50 ◦C, respectively. The oven temperature of GC was kept
at 50 ◦C for 3 min constant and after that raised with 10 ◦C/min
upto the 280 ◦C. Moreover, reaction products were also verified
by GCMS (Gas-Chromatography-Mass Spectroscopy Shimadzu QP-
2010) analysis.
3.1.5. Determination of protein content
The immobilized amount of protein loading is the difference
of free (initial) amount of protein loading and un-immobilized
amount of protein present in Teflon petri dishes after removal of
support matrix [42]. It was observed that, the% protein content
for all screened lipase species was found to be greater than 91%
(Table 1, column 9). This concluded that (i) protein was immo-
bilized on the HMC support and (ii) support was capable for
the immobilization of bio-molecules like enzymes and proteins
[21,36,39–41]. It is obvious to find such higher amount of protein
loading because of the entrapment methodology of immobiliza-
tion which is generally free from the washing and decant process
(the physical adsorption losses somewhat higher amount of pro-
teins) [21]. The order of% protein immobilization was found to be
as AYS < MJL < HPL < PFL. In all after immobilization, the higher or
improved lipase activity and% protein binding showed that, HMC
polymer support was one of the best suitable choice for lipase
immobilization. The specific activity cumulatively notifies the rela-
tionship between the lipase activity and protein content (Table 1,
column 4 and 7) [42]. Moreover, the retention activity was the ratio
of specific activity of immobilized lipase to free lipase. Thus study
demonstrated that, lipase PFL showed highest% activity retention
(Table 1, column 10), while interfacial activation was the basic
cause for increase in activity of various lipases after immobilization
on HMC matrix.
3. Result and discussion
3.1. Biocatalyst characterization
3.1.1. SEM
The SEM study noticeably showed difference in the plane
support and immobilized HMC:PFL lipase since, immobilized
HMC:PFL lipase showed well scattered globules into the support
(Fig. 1) which indicating the change in texture morphology after
immobilization. However, the control support of HMC (without
immobilization) was showed planer surface.
3.1.2. TGA
The primary investigation of the thermal characteristic of free
PFL and immobilized HMC:PFL lipase was performed by TGA anal-
ysis [38]. The native lipase PFL and immobilized HMC:PFL lipase
showed initially slight weight loss at nearly 100–110 ◦C owing
to the removal of physically adsorbed bound water (TGA graph
not shown) [21,25,29]. The weight loss at the 200–220 ◦C was
owing to removal of the tightly bound water in the vicinity of
the lipases [21,25,38]. The removal of this essential bound water
(220–230 ◦C), causes sharp degradation which was attributed to
destruction of enzyme conformation [38]. Compared to HMC-PFL
lipase, the free PFL showed faster decay which was owing to dena-
ture of the proteomic structure of lipase [38]. The TGA curve of free
PFL showed almost 10% weight loss at nearly 240–250 ◦C whereas,
immobilized HMC:PFL showed 10% weight loss almost at 300 ◦C.
Besides this at 400 ◦C, the free lipase PFL showed 60% weight loss
while immobilized HMC:PFL lipase showed nearly 48% weight loss,
3.1.6. Determination of % water content
It is well known fact that, bio-catalytic activity of lipases is well
controlled by the degree of hydration which maintain the stabil-
ity of enzyme tertiary structure and conformation [20,21]. This
degree of hydration is guarded by the presence of trace amount of
bound water which preserve the optimal conformation of lipases
and promotes opening and closing movement of lipase lids for
the efficient mass transfer of substrates to active sites [21,29,39].
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
4
Fig. 1. (A) SEM image of HMC control support (2000X); (B) SEM image of HMC:PFL immobilized lipase (2000X) (C) SEM image of HMC:PFL immobilized lipase (10000 X).
Table 1
Determination of lipase activity and protein content.
Sample Free lipase
Immobilized HMC: lipase
%
%
%
Lipase activity Protein content Specific activity Lipase activity Protein content Specific activity Lipase activity Protein content Activity retention
1.HPL
2.MJL
3.PFL
4.AYS
17.49
14.69
51.32
21.37
91.23
57.89
53.79
45.79
0.00
0.19
0.25
0.95
0.47
0.00
18.07
16.58
60.56
21.81
0.00
85.85
53.77
51.81
41.90
0.00
0.21
0.30
1.17
0.53
0.00
103.30
112.92
118.01
102.06
0.00
94.10
92.88
96.32
91.50
0.00
109.78
121.58
122.52
111.53
0.00
5.HMC 0.00
aLipase activity assay and protein content determination.
Unit of lipase activity: U/mg; protein content: mg/␮g; specific activity: U/␮g.
Values given are average values, experimental error <5%.
However, higher water content leads to (i) make a reversible bio-
transformation via back hydrolysis of desired product or (ii) reduces
product selectivity [1,20–24,38]. Hence it is essential to maintain
or determine the water content for developed immobilized biocat-
alyst since, we have utilized water as an immobilization medium.
In present study, the free lipase possessing the 2.02% water content
while the HMC:PFL provided 8.03% water content (Table 2). Many
researchers have demonstrated slightly higher requirement of the
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
5
Table 2
Determination of Km, Vm and % water content.
Sample type
% Water content (w/w)
Apparent Vm value
Apparent Km value
Catalytic efficiency
Efficiency factor (␩)
% Catalytic efficiency
Free lipase PFL
Immobilized HMC:PFL
Plane HMC support
2.02
8.03
6.96
81.30
99.01
–
15.8
14.1
–
5.14
7.0
–
1.00
1.36
–
100
136
Efficiency factor (ꢀ) = (catalytic efficiency of immobilized lipase/catalytic efficiency of free lipase); Unit for Vm (␮mol/mg/min); Unit for Km (mM); Experimental error <5%.
Table 3
Effect of organic solvents on enzyme activity and determination of t(1/2) and Ki.
Entry
Solvent
Temperature (◦C)
% residual activity
t(1/2) (h)
Ki × 10−3 (h−1
)
Solvent stability study
1
2
3
4
5
6
HMC:PFL
HMC:PFL
HMC:PFL
HMC:PFL
HMC:PFL
HMC:PFL
n-hexane
Toluene
DIPE
DEE
THF
50
50
50
50
50
50
83.62
81.29
78.50
76.02
73.26
71.53
557
482
412
365
322
298
1.24
1.43
1.66
1.89
2.13
2.30
1,4 dioxane
Thermal stability study
7
8
9
10
11
12
HMC:PFL
HMC:PFL
HMC:PFL
HMC:PFL
HMC:PFL
Free PFL
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
n-hexane
40
45
50
55
60
50
84.10
83.82
83.62
80.12
78.02
35.20
576
565
557
451
402
96
1.20
1.23
1.24
1.26
1.27
7.26
DIPE: Di-isopropyl ether, DEE: Di-ethyl ether, THF: Tetra hydro furan.
Values given are average values, experimental error <5%.
water content for bacterial lipases, since more protein residues are
present in the lid region which requires higher degree of hydra-
tion for easy movements of lids [20,21,24,29]. Mendes et al. [29].
also proposed that bacterial lipase involves large interior flapping
movement of the lids to get ‘open conformation’. Thus, it could
be stated that, slightly higher water content of immobilized matrix
could assist to sustain three dimensional conformational structures
for efficient activity of lipase PFL [1,20,21,24,29,38,39].
The catalytic efficiency is a cumulative factor which expressed
the ratio of Vm to Km. The immobilized HMC:PFL lipases (7.0)
showed the higher catalytic efficacy (Vm/Km) as compared to free
lipase (5.14), while efficiency factor (␩) indicated higher catalytic
efficiency of immobilized HMC:PFL lipases (Table 2). Thus, the con-
formational changes after immobilization of lipase into HMC causes
an open state conformation which improved the substrate affin-
ity and catalytic efficiency [21,40–44]. Thus, preset kinetic study
encourages us to use the HMC:PFL immobilized lipase for efficient
organic synthesis instead of free lipase PFL.
3.1.7. Determination of Michaelis-Menten constant (Km and Vm)
The aim to study the apparent values of Km and Vm was to
determine the catalytic efficacy of immobilized lipase (HMC:PFL)
compare to that of free lipase PFL. Since many times it was observed
that, immobilization causes not only decrease in the enzyme activ-
ity but may also causes denaturation of enzymes [20,21,40]. The
apparent Vm value indicated the maximum reaction velocity, Km
value indicated the substrate affinity and enzyme conformational
changes, while catalytic efficiency (Vm/Km) indicated the overall
cumulative effect of Vm and Km on enzyme activity [43,44].
Interestingly in present study (Table 2), we found increase in
the Vm value of HMC:PFL immobilized species (99.01 U) as com-
pared to free PFL (81.3 U). This increase of Vm value could be
explained by several assumptions such as (i) lipase PFL dispersed
into the HMC immobilization matrix and make easy contact of
the substrate molecules to active sites which boost up the cat-
alytic activity rate [20,21,36,41] (ii) after immobilization of lipase
on hydrophobic surfaces cause easy opening of lids of lipases which
afforded easy access of reacting molecules to active sites of enzyme
[20,21,40,41]. It is also noticeable that, Km value of immobilized
HMC:PFL lipases (14.1 mM) was found to be lower than that of
free lipase (15.8 mM). This lower Km value confirmed higher affin-
ity of immobilized HMC:PFL lipase species towards the substrate
[41–44]. As mentioned earlier, the immobilization facilitates the
scattering of lipases into the support matrix, so that higher sub-
strate concentration was being experienced around immobilized
lipase (HMC:PFL) [40–43]. As a result of this, the HMC:PFL lipase
affinity towards the substrate was positively enhanced for corre-
sponding transformation [20,21,40–44].
3.1.8. Determination of solvent stability in organic media
Organic solvent stability of developed immobilized biocatalyst
is the important issue which needs to be address at various temper-
atures in various organic solvents. Table 3 depicted the% residual
activity, t(1/2) and Ki values. The highest residual activity was
observed in n-hexane (non-polar) solvent while lowest enzyme
activity was found in the THF and 1,4 dioxane (polar) solvents.
The increasing order of half-life time of lipase activity in various
solvent was found as: 1, 4 dioxane < THF < DEE < DIPE < toluene < n-
hexane (Table 3). Whereas, the order of inactivation rate constant
of lipase activity in various organic solvents was exactly reverse
to the half-life time. Thus, the higher t(1/2) and lower Ki value
indicated better activity and stability of the enzyme in various spec-
ified (polar/non-polar) organic solvents. Moreover it was observed
that, developed biocatalyst is sufficiently stable in various polar
and non-polar solvents. The t(1/2) value reduced while Ki value
rose with increase in the hydrophilicity of organic solvent, which
might be due to distortion of the hydrating (micro-aqueous) bound
layer present around the enzyme [1,45]. This enzyme hydrating
layer strip out in polar solvents because of more hydrophilicity of
polar solvents causes distortion the enzyme conformational sta-
bility which causes reduction of parent enzyme activity. Recently,
Dors et al. [45] reported various t(1/2) values from 76 to 1155 h
depending on the practical parameters for PFL immobilization.
3.1.9. Determination of thermal stability in organic media
The thermal stability determination is a necessary aspect for
the practical biocatalytic applications, hence thermal stability was
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
6
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
Table 4
Effect of amine on aza-Michael product formation.
No.
Effect of amine (mmol)
% Conversion obtained by
% Chemo selectivity for Aza-Michael product
Immobilized HMC:PFL
Free PFL
Con-trol
1
2
3
4
5
6
0.5
0.75
0.9
1
1.1
1.2
64
76
90
94
92
91
29
34
42
44
40
39
10
11
12
14
16
17
92.19
94.74
96.67
97.87
94.57
91.21
Effect of amine: Reaction condition: methyl acrylate (1.3 mmol), benzyl amine (0.5-1.2 mmol), solvent-toluene, biocatalyst (HMC:PFL- 350:100) (50 mg), orbital rotation
speed (160 rpm), temperature 50 ◦C, time (200 min).
Values given are average values; experimental error for aza-Michael product is <5%.
Table 5
Effect of methyl acrylate on aza-Michael product formation.
No
Effect of acrylate (mmol)
% Conversion obtained by
Immobilized HMC:PFL
% Chemo-selectivity for Aza-Michael product
Free PFL
Control
1
2
3
4
5
6
7
8
0.5
0.75
1
1.1
1.15
1.2
1.4
1.5
48
71
83
87
92
94
96
98
21
31
34
39
42
44
47
48
7
9
91.67
94.37
97.59
97.70
98.91
97.87
90.63
87.76
11
13
14
14
16
18
Effect of methyl acrylate: Reaction condition: methyl acrylate (0–1.5 mmol), benzyl amine (1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:100) (50 mg), orbital rotation
speed (160 rpm), temperature 50 ◦C, time (200 min).
Values given are average values, experimental error for aza-Michael product is <5%.
studied in the range of 40–60 ◦C (Table 3) [1,21,41]. It was found
that, t(1/2) slightly decreases with increases in the temperature
from 40 to 60 ◦C, which is an obvious observation attributed due to
sensitive nature of proteins to the thermal denaturation. Interest-
ingly HMC:PFL lipase displayed excellent thermal stability in term
of the t(1/2) and Ki as compared crude (free) lipase. This extra ther-
mal stability of the HMC:PFL lipase was attributed due to shielding
of lipase biomolecules inside the polymer matrix [1,41].
along with increase in the lipase loading from 0 to 100 mg. Fur-
ther, increase in the lipase loading from the 100–120 mg did not
show any more influence on the bio-catalytic conversion. This may
be due to use of excess lipase loading and availability of excess
catalytic sites which did not take part in the reaction [46,47]. Some-
times excess lipase loading on the support may causes stacking
of the useful catalytic sites of lipase and prevents active co-
operation in biocatalytic transformation [46,47]. Thus, in present
study 350:100 mg (HMC:PFL) is the optimized biocatalyst loading
used further for remaining all experiments.
3.2. Biocatalytic application of HMC:PFL for synthesis of ˇ-amino
ester compound
3.2.1. Screening of the various bio-catalysts
3.2.3. Effect of amine quantity
Initially we have screened lipases (free and HMC immo-
bilized) from various sources to obtain the robust biocatalyst
(Fig. 2A) (Scheme 1). The following order was observed for vari-
ous free or HMC immobilized biocatalysts: free HPL < free MJL < free
AYS < immobilized HPL < immobilized MJL = free PFL< immobilized
AYS < immobilized PFL. The screening study showed that, (i) various
HMC immobilized lipases offered higher% conversion compared to
respective free lipases (ii) various HMC immobilized lipase showed
1.65–2.2 folds increase in bio-catalytic activity as compared to
respective free lipase and (iii) immobilized HMC:PFL provided high-
est% conversion among screened catalysts which was used for
further study.
Influence of the substrate concentration has great impact on
reaction as (i) substrate may cause catalyst deactivation/inhibition
which results into reduction in reaction rate and (ii) use of random
quantity of substrate may greatly affect selectivity of reaction. In
present reaction system, substrate (methyl acrylate) has two active
susceptible reaction centres and hence it was necessary to check the
influence of both substrates. Talking about amines, which are highly
nucleophilic and basic in nature can be attack on the C C to give
aza-Michael product or C O to give aminolysis product [48–50].
Hence it is essential to control the concentration of amine substrate.
It was observed that, increase of amine from 0.5 to 1 mmol leads
to increase in the% conversion which was attributed to the higher
nucleophilicity of the amine. Further increase of amine from 1 to
1.2 mmol did not show any significant influence on the conversion.
Talking about chemo-selectivity (Table 4), lower concentration
of amine provided slightly lower selectivity for the aza-Michael
product (0.5–0.75). The main reason behind this was excess amount
of the methyl acrylate (1.3 mmol) as compared to the amine quan-
tity (0.5–0.75) [48,49]. The selectivity of aza-Michael product was
found to be higher at 1 mmol of amine concentration. Excess quan-
tity of the amine is often used to maximize the product yield,
however higher amine concentration (1.2 mmol) leads to favour
the aminolysis reaction due to higher nucleophilic nature of amines
3.2.2. Effect of enzyme loading on the support
After screening of biocatalysts (HMC:PFL), we have performed
screening of lipase PFL loading to fix amount of HMC support
composition (Fig. 2B). Since, it was obligatory to screen the immobi-
lization composition of the HMC:PFL lipase to sort out the suitable
robust bio-catalyst. Therefore, we have varied amount of lipase PFL
quantity (0–120) for the immobilization on HMC support (350 mg).
It was observed that, % conversion increased as the lipase PFL
loading increased (from 0 to 100 mg) on the 350 mg of HMC sup-
port, which may be owing to accessibility of more catalytic sites
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
7
Fig. 2. (A) Screening of the biocatalyst: Reaction condition: methyl acrylate (1.3 mmol), benzyl amine (1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:120) (50 mg),
orbital rotation speed (160 rpm), temperature 50 ◦C, time (200 min). (B) Screening of the biocatalyst composition (HMC:PFL): Reaction condition: methyl acrylate (1.3 mmol),
benzyl amine (1 mmol), solvent-toluene (2 mL), biocatalyst (HMC:PFL- 350:0 to 350:120) (50 mg), orbital rotation speed (160 rpm), temperature 50 ◦C, time (200 min).
Scheme 1. HMC:PFL lipase catalyzed Chemo-selective synthesis of aza-Michael product.
which again causes reduction in chemo-selectivity of aza-Michael
product [23].
Michael reaction is favoured in the polar solvents, wherein enzyme
showed less compatibility [1,20,21,24,25]. Hence in present study,
we have screened various (polar and non-polar, log ␳ 3.5 to −2) sol-
vents and results showed that% conversion was slightly decreased
with increase of solvent polarity. The decrease in the% conversion
was attributed to the lesser enzyme compatibility in polar solvents
[20,21]. In contrast to this, the control reaction without catalyst
showed increment in the% conversion with increase of solvent
polarity.
In the present study, the n-hexane and toluene are solvents
of good choice which offered better yield along with biocata-
lyst compatibility. Considering the issue of chemo-selectivity, it
was observed that chemo-selectivity for the aza-michael prod-
uct was greatly hampered by the use of polar solvents, whereas
chemo-selectivity was increased with non-polar solvent (Table 6).
It is proposed that, the solvents of low polarity may induces the
proper interaction between the oxyanion hole and the carbonyl
oxygen in the catalytic intermediate complex and thus favours
the aza-Michael product [52,53]. Finally, in competition with
the aminolysis and aza-Michael reaction, the non-polar solvents
offered better conversion and chemo-selectivity for aza-Michael
reaction. This fact encourages us to use non-polar solvent like n-
hexane and toluene for the corresponding system. Similar kinds of
results were observed to Sternberg et al. [23] and Escalante et al.
[51] also. Thus in the present study, we choose toluene as a reaction
solvent for further study which provided better conversion, bet-
ter chemo-selectivity and better dissolution capability with wide
range of substrate. In context to toluene, n-hexane is more compat-
ible for bio-catalysis; however n-hexane showed poor dissolution
of highly polar amine substrates [50].
3.2.4. Effect of acrylate
Similar to the effect of amine, there is need to investigate the
influence of the Michael acceptor (methyl acrylate), hence we var-
ied amount of the methyl acrylate (0.5–1.5) at constant amine
(1 mmol) quantity. In present study, % conversion increased from
48 to 98% with increase in the concentration of the acrylate from
0.5 to 1.5 mmol respectively. However considering the chemo-
selectivity aspect, 1.2 mmol of methyl acrylate was sufficient which
offered better conversion (94%) and selectivity (97.87%) for aza-
Michael product (Table 5). It was found that, % conversion increases
while selectivity was decreased for aza-Michael product at 1.4
and 1.5 mmol of acrylate. Since, higher amount of methyl acrylate
favours the double aza-Michael product and losses the chemo-
selectivity [49–51]. Hence, 1.2 mmol was the optimized quantity
of methyl acrylate to achieve higher% conversion and chemo-
selectivity at use of 1 mmol of amine. Thus it can be concluded that,
enzymatic aza-Michael addition is probably favoured in slightly
higher amount of the Michael acceptor, while large excess amount
of the Michael acceptor causes decrease in chemo-selectivity. Also
it is noted that, use of only primary amines (not secondary amines)
endorse the double aza-Michael product formation at excess con-
centration of Michael acceptor (methyl acrylate).
3.2.5. Effect of solvent
Use of the solvent is an important issue for the reaction system
since, solvent plays an important role in reaction which promotes
the dissolution of the substrates, make ease of substrate diffusion
process at active sites and controls the reaction chemo-selectivity
[23,51]. Lipase showed versatile catalytic activity in non-polar sol-
vents, as highly polar solvents may disturb the enzyme flexibility
by removal of the closely associated tightly bound water molecules
[23,50,51]. Hence, it was mandatory to screen the suitable solvent
which should offered higher% conversion and chemo-selectivity
without disturbing the enzyme activity. On the other hand, aza-
3.2.6. Effect of biocatalyst loading
To enhance the reaction rate there is need to use the bio-
catalyst which can provided better conversion in lesser reaction
time and provided easy biocatalyst recovery. On the other hand,
it must be noted that aza-Michael reaction is in competition with
the aminolysis reaction which may provided aminolysis product
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
8
Table 6
Effect of solvent media on aza-Michael product formation.
No.
Effect of Solvent
% Conversion obtained by
Immobilized HMC:PFL
% Chemo-selectivity for Aza-Michael product
Free PFL
Con-trol
1
2
3
4
5
6
n-hexane
Toluene
DIPE
DEE
THF
94
94
92
86
84
82
44
44
43
36
36
32
14
15
17
20
22
24
97.87
97.87
93.48
93.02
92.86
87.80
1,4 dioxane
Effect of solvent: Reaction condition: methyl acrylate (1.2 mmol), benzyl amine (1 mmol), solvent (polar or non-polar) (2 mL), biocatalyst (HMC:PFL-350:100) (50 mg), orbital
rotation speed (160 rpm), temperature 50 ◦C, time (200 min).
Values given are average values, experimental error for aza-Michael product is <5%.
Table 7
Effect of biocatalyst loading on% conversion and% chemo-selectivity for immobilized HMC:PFL catalyzed aza-Michael reaction.
No.
Catalyst loading (mg)
% Conversion obtained by
Immobilized HMC:PFL
% Chemo-selectivity for Aza-Michael product
Free PFL
Con-trol
1
2
3
4
5
6
7
8
0
–
–
13
–
–
–
–
–
–
–
100.00
100.00
98.04
97.37
97.75
97.87
94.79
91.75
12
24
36
44
48
54
60
32
51
76
89
94
96
97
19
23
33
41
44
48
52
Effect of biocatalyst amount: Reaction condition: methyl acrylate (1.2 mmol), benzyl amine (1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:100) (0–60 mg), orbital
rotation speed (160 rpm), temperature 50 ◦C, time (200 min).
Values given are average values, experimental error for aza-Michael product is <5%.
in presence of excess biocatalyst and decreases the selectivity of
reaction (Table 7). Thus, biocatalyst loading is a crucial aspect to
optimize which enhances the reaction rate dramatically and sub-
sequently may causes decrease in the% selectivity also [47–50,54].
In present study, we have used biocatalyst amount ranging from 0
to 60 mg at given similar reaction conditions. It was observed that,
% conversion increased with increase in biocatalyst amount from 0
to 48 mg, further increase in biocatalyst amount from 48 to 60 mg
cause marginal increment in the reaction conversion. The improved
reaction rate was attributed to the presence of active bio-catalytic
sites of lipases which may fully engage for catalysis. However, selec-
tivity was reduced at large excess biocatalyst loading (above 48 mg)
due to presence of excess catalytic sites which may be engaged
into the aminolysis reaction [51]. Thus, 48 mg of biocatalyst load-
ing was the optimized quantity to achieve the better conversion
and chemo-selectivity.
present system 50 ◦C was the optimized temperature, which pro-
viding 94% conversion and 97.87% chemo-selectivity.
3.2.8. Effect of time
It is significant to consider the issue of economical feasibil-
ity that, reaction should provide higher conversion and selectivity
at lesser reaction time. Free enzyme catalyzed reactions achieved
equilibrium slowly and offered lesser conversion compared to
immobilized lipase at specified time. It is well known fact that,
long-hour reactions promoted impurity formation (side products)
and reduces selectivity of desired products which may discourage
the corresponding synthesis [3,23,49,50]. Moreover in case of aza-
Michael synthesis, long-hour reaction favours the accumulation
of competitor amidation product or double aza-Michael product
[23,50,51]. In view of this, we have studied reaction progress with
time study (Fig. 3A). It was observed that, non-enzymatic and free
enzyme provided 25 and 60% conversion after 5 h. Contrary to this,
immobilized lipase provided 94% conversion in 3 h only. Moreover
for immobilized lipase catalyzed reactions, it was observed that, %
conversion increases while chemo-selectivity decreases after 3 h.
This may be due to amidation reaction which achieved equilibrium
with time and causes amide product accumulation along with the
long reaction time [49–51,54]. Thus, 3 h was the optimal time to
achieve better conversion and selectivity for aza-Michel product.
3.2.7. Effect of temperature
Temperature plays a significant functions such as improvement
of catalytic interactions of substrate with catalyst and decrease
of viscosity of reaction medium [1,3,20–22,50,54]. Therefore, it
was obligatory to ensure the most favourable temperature for
enzymatic reactions (Table 8). Moreover it was observed that,
HMC:PFL did not show any decrease in the% conversion upto stud-
ied 65 ◦C while% conversion slowly decreased in case of free lipase
with increase in temperature from 55 to 65 ◦C, which indicating
higher stability of immobilized lipase. Torre et al. [50] found that
aminolysis reaction needs high temperature while higher tem-
perature may cause decrease in the selectivity. Similar type of
results was also observed in present study. Increase in temperature
(from 30 to 50 ◦C) causes increase in reaction rate and corre-
sponding% conversion. Further raise in temperature (from 50 to
65 ◦C) did not influence conversion, whereas chemo-selectivity was
severely affected for aza-Michael product formation beyond tem-
perature 50 ◦C. Since, higher temperature may favour the double
aza-Michael and amide product formation (aminolysis). Thus in
3.2.9. Comparison of free to immobilized lipase
It is highly recommended to look for a comparative account of
free and immobilized lipase activity for desired aza-Michael prod-
uct synthesis. Similarly, it is also essential to investigate the possible
role of support (HMC) in the reaction or not. Hence various reac-
tions were carried out as depicted in Table 9. It was observed that,
HMC:PFL immobilized lipase showed two folds higher bio-catalytic
activities compared to free lipase PFL. This was due to various rea-
sons such as (i) lipase PFL bio-molecules are well dispersed on HMC
support which make possible better substrate-biocatalyst interac-
tion and promote the reaction rate [20,36,41] (ii) contrary to this
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
9
Table 8
Effect of temperature on aza-Michael product formation.
No.
Temperature (◦C)
% Conversion obtained by
Immobilized HMC:PFL
% Chemo-selectivity for Aza-Michael product
Free PFL
Con-trol
1
2
3
4
5
6
7
8
30
35
40
45
50
55
60
65
29
49
64
81
94
95
95
96
13
22
30
37
44
44
42
38
4
6
100.00
97.96
98.44
97.53
97.87
94.74
90.53
88.54
10
12
14
17
20
25
Effect of temperature: Reaction conditions: methyl acrylate (1.2 mmol), benzyl amine (1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:100) (48 mg), orbital rotation
speed (160 rpm), temperature 30–65 ◦C, time (200 min).
Values given are average values, experimental error for aza-Michael product is <5%.
Fig. 3. (A) Effect of times: Reaction condition: methyl acrylate:benzyl amine (1.2:1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:100) (48 mg), rotation speed (160 rpm),
temperature 50 ◦C, time (0–5 h). (B) Recyclability: Reaction condition: methyl acrylate:benzyl amine (1.2:1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:100) (48 mg),
rotation speed (160 rpm), temperature 50 ◦C, time (3 h).
Table 9
insignificant conversion of 17 and 19% respectively as similar to
control experiment. Thus above facts confirmed that, active sites
of the HMC:PFL immobilized lipases are solely involved for aza-
Michael product formations [3,23].
Effect of support, BSA protein and denatured lipase on aza-Michael product
formation.
No.
Catalyst
% Conversion
1
2
3
4
5
6
Immobilized HMC:PFL
Free PFL
Plane support HMC matrix
Control (Blank)
BSA protein
Denatured lipase
94
44
13
12
17
19
3.3. Scope and applicability of the present protocol
To expand the scope and application of present protocol, we
have synthesized various ␤-amino ester compounds by using
the HMC:PFL immobilized lipase, while to compare the feasibil-
ity of the present immobilized protocol we also demonstrated
the% conversion achieved by the free lipases for each substrate.
In each case it was found that, % conversion was almost 2 times
higher by HMC:PFL immobilized lipase than that of free lipase
PFL. Various basic primary amines such as butyl amine, cyclo-
propyl amine, neo-pentyl amine, cyclopentyl amine, cyclohexyl
amine and cyclohexane methyl amine provided 95, 94, 92, 90, 88,
and 92% conversion respectively with the chemo-selectivity > 95%
(Table 10, entries 1–6). Various benzyl amine derivatives are nucle-
ophilic in nature which provided good to excellent yield of the
corresponding products with selectivity > 96% (Table 10, entries
7–12). Herein, electron donating substituent provided better con-
version in 3 h, because of higher nucleophilicity (Table 10, entries
10.11). However, electron withdrawing substituent provided bet-
ter% conversion in higher reaction time (3.5 h) due to pulling
of nucleophilicity of amine because of the electron withdrawing
nature of −Cl, −F and −O substituted derivatives (Table 10, entries
8,9,12). Interestingly heterocyclic compound such as 5-methyl fur-
furyl amine provided excellent conversion and selectivity (Table 10,
entries 13). It was observed that, aromatic amines such as aniline
and o-toluidine provided sluggish yield because of the lesser nucle-
ophilicity, since nucleophilic lone pair of amine is in conjugation
Reaction conditions: methyl acrylate:benzyl amine (1.2:1 mmol), solvent-toluene,
biocatalyst (HMC:PFL 3.5:1) (48 mg), orbital rotation speed (160 rpm), temperature
50 ◦C, time (3 h).
Values given are average values, experimental error for aza-Michael product is <5%.
free enzyme may be agglomerated in organic solvents and tends to
block active catalytic sites [21,36] (iii) developed HMC:PFL immo-
bilized lipase could benefit the “open state” conformation of lipase
and offered easy mass transfer of substrate towards active cat-
alytic sites of enzyme to promote reaction rate of corresponding
transformation of desired products [29,36,40,41].
3.2.10. Influence of immobilization matrix, protein and
denatured lipase
Moreover, to investigate the role of HMC support, we have used
control HMC matrix (non-immobilized) as a catalyst for the given
reaction. Results signified that, reaction did not progress by use of
control HMC matrix as a catalyst and providing only 13% conver-
sion (similar to blank reaction) (Table 9). Also to investigate the
role of lipase proteins, we have used bovine serum albumin (BSA)
and denatured lipase as a catalyst. BSA is a protein which lacks cat-
alytically active sites but has a similar type of protein structure like
lipase [51]. It was observed that BSA or denatured enzyme provided
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
10
Table 10
Synthesis of various ␤-amino esters using HMC:PFL lipase bio-catalyst.
No Reactant
Product
Time (h) % Conversion obtained by
Blank/ Control Free PFL Immobilize HMC:PFL
% Chemo-selectivity for aza-Michael product
1
2
3
16
13
48
45
95
94
95.79
95.74
3.5
3
4
3
13
12
40
42
92
90
98.91
97.78
3.5
5
6
3.5
3
13
13
41
43
88
92
98.86
96.74
7
3
14
44
94
97.87
8
9
3.5
3.5
13
15
40
45
91
91
97.80
97.80
10
3
16
47
92
97.83
11
12
3
17
13
49
40
94
88
96.81
97.73
3.5
13
14
3
3
15
5
42
13
93
40
96.77
95.00
15
16
3
3
4
10
46
33
94
93.94
96.81
15
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
11
Table 10 (Continued)
No Reactant
Product
Time (h) % Conversion obtained by
% Chemo-selectivity for aza-Michael product
Blank/ Control Free PFL Immobilize HMC:PFL
17
18
19
20
3
16
14
12
13
48
45
35
40
93
94
88
90
95.70
95.74
98.86
96.67
3
3.5
3
21
3
10
30
72
97.22
Optimized parameters: Reaction condition: methyl acrylate (1.3 mmol), benzyl amine (1 mmol), solvent-toluene, biocatalyst (HMC:PFL-350:100) (48 mg), orbital rotation
speed (160 rpm), temperature (50 ◦C).
Values given are average values, experimental error for aza-Michael product is < 5%.
Table 11
with the benzene ring and unavailable for the further nucleophilic
attack on the Michael acceptor species (Table 10, entries 14,15).
Thermodynamic parameters for aza-Michael reaction.
Although o-toluidine having electron donating methyl group but
still it was providing lesser% conversion as compare to that of
aniline because of the slightly bulkier nature. Since aza-Michael
reaction is an addition of two molecules in which hybridization
changes from the SP2 to SP3 and hence steric crowding greatly
affect the% conversion. Similarly, the secondary amine also pro-
vided better conversion and selectivity of the aza-Michael products
(Table 10, entries 16–21). It is also noted that, secondary amines did
not provide any bis-Michael product due to unavailability of the
second replaceable hydrogen after formation of basic aza-Michael
product. Thus, the present protocol is well extended to synthesize
various industrially important moieties.
Thermodynamic parameters
HMC:PFL
Free PFL
Ea (KCal/mol)
13.14
12.40
18.60
17.88
ꢁH# (KCal/mol)
ꢁS# (Cal/mol K)
ꢁG# (KCal/mol)
−36.81
−22.69
24140
25009
late) to forms a concerted transition state (E). Next, His 224 was
getting protonated (F). The nucleophilic attack is facilitated by the
oxyanion hole, since H-bonding stabilizes the various partial neg-
ative charges developed on the carbonyl oxygen in transition state
(G). At the same time, resonance causes development of negative
charge on the ␣-carbon adjacent to carbonyl group which abstracts
the proton from protonated His 224 (H). With abstraction of this His
224 proton, final product (I) was released from the enzyme active
centre.
3.4. Mechanisms
In the reaction mechanism, the oxyanion hole of enzyme (A)
plays a significant role via H-bond formation (Scheme 2) [51,54].
The plausible reaction mechanism involves H-binding of the methyl
acrylate (methyl acceptor) carbonyl bond to the backbone amino
groups of threonine 40 (Thr 40) and glutamine 106 (Gln 106) of the
oxyanion hole (D) [52–55]. It was proposed that, the Gln 106 and
Thr 40 could better stabilize the formation of an enolate by three
hydrogen bonds to the carbonyl oxygen of the methyl acrylate (D).
At the same time the nitrogen atom of the histidine amino- acid (His
224) acts as a base to abstract a proton of amine (Michael donor) (C),
and thus increases the nucleophilicity of the Michael donor for fur-
ther attack on the ␤-carbon atom (activate the nucleophile for the
further attack). These two substrates (Michael acceptor and donor)
are then close enough to react with each other and forms complex
in the enzyme active site. Thus starting from the near attack com-
plex (D) with amine hydrogen bonded to His 224, the next step is
a proton transfer to His 224 concerted with a nucleophilic attack
of an amine on the ␤-carbon of unsaturated ester (methyl acry-
3.5. Thermodynamic feasibility study
Thermodynamic outlook (Table 11) can provide the sig-
nificant information about the reaction feasibility of various
bio-catalyzed (free of immobilized) processes [56–59]. The lower
activations energy requirement for HMC:PFL immobilized biocata-
lyst (13.14 Kcal/mol) demonstrated higher reaction rate and higher
catalytic efficiency as compared to free PFL lipase (18.60 KCal)
[1,20,21,59]. The lower ꢁH# of activation for HMC:PFL immobi-
lized lipase was owing to easier transition sate formation. Since,
lipases are well scattered on the immobilization support and facil-
itated easy substrate-biocatalyst interaction which might promote
transition state formation [56–58]. The entropy of activation ꢁS#
signifies the measurement of the local disorder of the reaction
system, according to the second law of thermodynamics; lower
entropy value indicated ease of reaction [56–59]. The entropy
change showed lesser disorder of HMC:PFL immobilized lipase
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
12
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
Scheme 2. HMC:PFL immobilized lipase catalyzed mechanism.
catalyzed reaction (−36.81 Cal/mol K), which demonstrated that
immobilized lipase processed reaction in a better way compare to
the free lipase PFL (−22.69 Cal/mol K) [57,58]. The feasibility and
ease of reaction is well expressed in terms of free energy change of
activation (ꢁG# value), lower the free energy change, more is fea-
sibility [56–59]. Thus, HMC:PFL lipase catalyzed reaction system
accounted for the lower ꢁG# value, which designated more feasi-
bility of reaction activation process as compared to free lipase PFL
catalyzed reaction.
3.7. Determination of lipase leaching and substrate inhibition
To investigate the reduction in conversion during recyclability,
we have performed leaching and substrate inhibition experiment.
Leaching was found to be nearly 2% in 20 h for the present work,
since entrapment technique was used for immobilization rather
than physical adsorption, wherein lipase biomolecules fix up into
the support matrix at the time polymer matrix formation (Fig. 4A)
[1,60]. In another experiment it was found that; amine substrate
causes the marginal inhibition (1.8% in 3 h) to the lipase activity,
when 1 mmol of amine was used in prescribed condition (Fig. 4B).
This fact indicated that, lipase was inhibited by substrate amine due
to its basic nature which may cause the Schiff’s binding with the free
carbonyl catalytic sites of lipases [66,67]. Furthermore, continuous
exposure of lipase to amine leads to reduces enzyme activity due
to its polar nature [1,20,21,60–64].
3.6. Effect of recyclability
One of the major benefit of use of immobilized lipase is their
easy isolation from the reaction mass and subsequent recovery of
bio-catalyst by the simple filtration [1,20,21,22,59–65]. In order to
investigate the recyclability (Fig. 3B), we have performed several
recycle runs at given optimized reaction conditions. Interestingly,
the present protocol demonstrated five recycles (excluding fresh) of
immobilized biocatalyst. In each cycle there was marginal decrease
in the % conversion while chemo-selectivity was remained almost
unaffected. The decrease in% conversion might be attributed to
deactivation/substrate inhibition and leaching of lipases from the
support. It is noteworthy to mention that, free lipase PFL did not
show any recyclability, which indicated the versatility of developed
biocatalyst (HMC:PFL).
Thus from above study, it can be concluded that, inhibition and
leaching was found to be marginal. However, cumulative effect of
the both causes reduction in the immobilized enzyme activity and
subsequent conversion at the time of recyclability. These lipase
biomolecules not only supported into the polymer matrix, but also
forms additional multiple interactions with the polymer matrix via
hydrogen, ionic or hydrophobic interaction. Thus, lipase stabiliza-
tion via immobilization through multipoint attachment to polymer
matrix may resulted into the marginal substrate inhibition as well
as lipase leaching [1,60–67].
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
13
1
0.8
0.6
0.4
0.2
0
2.8
2.4
2
4.5
3.6
2.7
1.8
0.9
0
100
96
1.6
1.2
0.8
0.4
0
92
88
0
0.3
0.6
Substrate conc. (mmmol)
% Residual activity
0.9
1.2
1.5
1.8
0
10
20
Time (h)
% Lipase leaching
30
(A) Activity found in supernatant
(B)
% Inhibition by substrate
Fig. 4. (A) Determination of lipase leaching (B) Determination of substrate inhibition for lipase activity.
4. Conclusion
(+)-sedamine, (+)-allosedamine, and (+)-coniine, Org. Lett. 9 (2007)
5283–5286.
[8] J.B. Samarat, T.B. Kraïem, H. Ayed Amri, An efficient synthetic route to
functionalized (-lactams), Tetrahedron 64 (2008) 9540–9543.
[9] S. Chandrasekhar, R.V.N.S. Murali, C.R. Reddy, Enantioselective synthesis of
(−)-lasubine II, Tetrahedron Lett. 50 (2009) 5686–5688.
The present study concluded successful immobilization of var-
ious lipases on the HMC support. The biocatalysts HMC:PFL (3.5:1)
demonstrated highest lipase activity, protein content and retention
of activity. The physical and biochemical characterization veri-
fied the immobilization of lipase PFL on HMC. This immobilized
biocatalyst HMC:PFL (3.5:1) was successfully applied for practi-
cal biocatalytic applications to synthesize 21 different industrially
important ␤-amino ester compounds with excellent conversion
and chemo-selectivity. Moreover, the thermodynamic analysis was
indicated more feasibility of immobilized HMC:PFL biocatalyst over
the free lipase. Interestingly, the immobilized lipase showed 2.1
folds higher bio-catalytic activity and five times reusability as com-
pared to free PFL. Thus, present protocol is a robust, efficient and
reusable protocol which carries fabrication of robust bio-catalyst,
and their potential practical bio-catalytic applications in detail.
[10] S. Comesse, M. Sanselme, A. Daïch, New and expeditious tandem sequence
aza-Michael/intramolecular nucleophilic substitution route to substituted
clactams: synthesis of the tricyclic core of ( )-martinellines, J. Org Chem. 73
(2008) 5566–5569.
[11] S. Sternativo, F. Marini, F.D. Verme, A. Calandriello, L. Testaferri, M. Tiecco,
One pot synthesis of aziridines from vinyl selenones and variously
functionalized primary amines, Tetrahedron 66 (2010) 6851–6857.
[12] E. Matveeva, P. Petrovskii, L. Odinets, Efficient synthesis of racemic
baminophosphonates via aza-Michael reaction in water, Tetrahedron Lett. 49
(2008) 6129–6133.
[13] P.R. Krishna, A. Sreeshailam, A stereoselective formal total synthesis of
(+)-hyperaspine via a tandem aza-Michael reaction, Tetrahedron Lett. 48
(2007) 6924–6927.
[14] M. Görmen, R.L. Goff, A.M. Lawson, A. Daïch, S. Comesse, Tandem
aza-Michael/spiro-ring closure sequence: access to a versatile scaffold and
total synthesis of ( )-coerulescine, Tetrahedron Lett. 54 (2013) 2174–2176.
[15] R.W. Dugger, J.A. Ragan, D.H.B. Ripin, Survey of GMP bulk reactions run in a
research facility between 1985 and 2002, Org. Process Res. Dev. 9 (2005)
253–258.
Acknowledgements
[16] C.A. Olsen, Peptoid–peptide hybrid backbone architectures, Chembiochem 11
(2010) 152.
[17] V. Polshettiwar, R.S. Varma, Tandem bis-aza-Michael addition reaction of
amines in aqueous medium promoted by polystyrenesulfonic acid,
Tetrahedron Lett. 48 (2007) 8735–8738.
[18] S. Azad, A. Kobayashi, K. Nakano, Y. Ichikawa, H. Kotsuki, Efficient brønsted
acid-catalyzed aza-Michael reaction of amides and ureas with ␣-bunsaturated
enones under high-pressure conditions, Tetrahedron Lett. 50 (2009) 48–50.
[19] U.T. Bornscheuer, R.J. Kazlauskas, Enzymatic catalytic promiscuity and the
design of new enzyme catalyzed reactions, in: Enzymes in Organic Synthesis,
Wiley-VCH, 2012, pp. 1695.
The author Kirtikumar Badgujar is grateful to the Depart-
ment 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 fund and fel-
lowship respectively.
Appendix A. Supplementary data
[20] K.C. Badgujar, B.M. Bhanage, Application of lipase immobilized on the
biocompatible ternary blend polymer matrix for synthesis of citronellyl
acetate in non-aqueous media: kinetic modelling study, Enzyme Microb.
Technol. 57 (2014) 16–25.
[21] P. Adlercreutz, Immobilisation and application of lipases in organic media,
Chem. Soc. Rev. 42 (2013) 6406–6436.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.procbio.2016.07.
008.
[22] David J.C. Constable, et al., Key green chemistry research areas—a perspective
from the pharmaceutical manufacturers, Green Chem. 9 (2007) 411–420.
[23] P. Steunenberg, M. Zuilhof, J. Sanders, E. Scott, M. Franssen, Lipase-catalyzed
aza-Michael reaction on acrylate derivatives, J. Org. Chem. 78 (2013)
3802–3813.
References
[1] K.C. Badgujar, K.P. Dhake, B.M. Bhanage, Immobilization of Candida
cylindracea lipase on poly lactic acid, polyvinyl alcohol and chitosan based
ternary blend film: characterization, activity, stability and its application for
N-acylation reactions, Process Biochem. 48 (2013) 1335–1347.
[2] J.S. Carey, D. Laffan, C. Thomson, M.T. Williams, Analysis of the reactions used
for the preparation of drug candidate molecules, Org. Biomol. Chem. 4 (2006)
2337–2347.
[3] M.N. Monsalve, F. Gillanders, A. Baldessari, Promiscuous behavior of
Rhizomucor miehei lipase in the synthesis of N-substituted ␤-amino esters,
Eur. J. Org. Chem. (2012) 2012.
[4] Q. Lin, D. Meloni, Y. Pan, M. Xia, J. Rodgers, Enantioselective synthesis of janus
kinase inhibitor INCB018424 via an organocatalytic aza-Michael reaction,
Org. Lett. 11 (2009) 1999–2002.
[5] S. Fustero, S. Monteagudo, M. Sánchez-Roselló, S. Flores, P. Barrio, C. del Pozo,
N-sulfinyl amines as a nitrogen source in the asymmetric intramolecular
aza-Michael reaction: total synthesis of (−)-pinidinol, Chem. Eur. J. 16 (2010)
9835–9845.
[24] M. Kapoor, M.N. Gupta, Lipase promiscuity and its biochemical applications,
Process Biochem. 47 (2012) 555–569.
[25] C. Matero, J.M. Palomo, G. Fernandez-Lorente, R. Fernandez-Lafuente, J.M.
Guisan, Improvement of enzyme activity, stability and selectivity via
immobilization technique, Enzyme Microb. Technol. 40 (2007) 1451–1463.
[26] W. Pronk, G. Boswinkel, K.V. Riet, Parameters influencing hydrolysis kinetics
of lipase in a hydrophilic membrane bioreactor, Enzyme Microb. Technol. 14
(1992) 214–220.
[27] D.J. Macquarrie, J.J.E. Hardy, Applications of functionalized chitosan in
catalysis, Ind. Eng. Chem. Res. 44 (2005) 8499–8520.
[28] M.M.M. Elnashar, H. Mostafa, N.A. Morsy, G.E.A. Awad, Biocatalysts: isolation,
identification, and immobilization of thermally stable lipase onto three novel
biopolymeric supports, Ind. Eng. Chem. Res. 52 (2013) 14760–14767.
[29] A.A. Mendes, P.C. Oliveira, A.M. Veı´lez, R.C. Giordano, R.L.C. Giordano, H.F. de
Castro, Evaluation of immobilized lipases on poly-hydroxybutyrate beads to
catalyze biodiesel synthesis, Int. J. Biol. Macromol. 50 (2012) 503–511.
[30] L. Ma, M. Persson, P. Adlercreutz, Water activity dependence of lipase
catalysis in organic media, Enzyme Microb. Technol. 31 (2002) 1024–1029.
[6] S.W. Liu, H.S. Hsu, C. Chang, H.G. Tsai, D.R. Hou, Asymmetric synthesis of
(−)-lentiginosine by double aza-Michael reaction, Eur J. Org. Chem. (2010)
4771–4773.
[7] F. Fustero, D. Jiménez, J. Moscardó, C. Catalán, C. Pozo, Enantioselective
organocatalytic intramolecular aza-Michael reaction: a concise synthesis of
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008

G Model
PRBI-10739; No. of Pages14
ARTICLE IN PRESS
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2016) xxx–xxx
14
[31] H. Akinosho, S. Hawkins, L. Wicker, Hydroxypropyl methylcellulose
substituent analysis and rheological properties, Carbohydr. Polym. 98 (2013)
276–281.
[32] K.P. Dhake, P.J. Tambade, R.S. Singhal, B.M. Bhanage, Promiscuous: Candida
antarctica lipase B-catalyzed synthesis of ␤-amino esters via aza-. Michael
addition of amines to acrylates, Tetrahedron Lett. 51 (2010) 4455–4458.
[33] X. Fan, W. Qiongsi, C. Xiaoyang, L. Xianfu, W. Qi, A single lipase-catalysed
one-pot protocol combining aminolysis resolution and aza-Michael addition:
an easy and efficient way to synthesise ␤-Amino acid esters, Eur. J. Org. Chem.
24 (2015) 5393–5401.
[34] H. Christian, C.S. Mihaiela, D. Anders, K. Anzar, Aza-Michael addition reaction:
post-polymerization modification and preparation of PEI/PEG-based polyester
hydrogels from enzymatically synthesize, React. Polym. 53 (2015) 745–749.
[35] D. José, R. Rivera, E. Jaime, L. Agustín, M. Alain, C. Edmundo,
Thermodynamically controlled chemoselectivity in lipase-catalyzed
aza-Michael additions, J. Mol. Catal. B: Enzym. 112 (2015) 76–82.
[36] G. Pencreacha, J.C. Barattia, Comparison of hydrolytic activity in water and
heptane for thirty-two commercial lipase preparations, Enzyme Microb.
Technol. 28 (2001) 473–479.
[37] M.M.A. Bradford, Rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding,
Analyt. Biochem. 72 (1976) 248–254.
[38] N.A. Turner, E.N. Vulfson, At what temperature can enzymes maintain their
catalytic activity, Enzyme Microb. Technol. 27 (2000) 108–113.
[39] K.C. Badgujar, B.M. Bhanage, Synthesis of geranyl acetate in non-aqueous
media using immobilized Pseudomonas cepacia lipase on biodegradable
polymer film: kinetic modelling and chain length effect study, Process
Biochem. 49 (2014) 1304–1313.
[40] O. Barbosa, M. Ruiz, C. Ortiz, M. Fernández, R. Torres, R. Fernández-Lafuente,
Modulation of the properties of immobilized CALB by chemical modification
with 2,3,4-trinitrobenzenesulfonate or ethylendiamine. Advantages of using
adsorbed lipases on hydrophobic supports, Process Biochem. 47 (2012)
867–876.
[41] G. Fernandez-Lorente, Z. Cabrera, C. Godoy, R. Fernandez-Lafuente, J.M.
Palomo, J.M. Guisan, Interfacially activated lipases against hydrophobic
supports: effect of the support nature on the biocatalytic properties, Process
Biochem. 43 (2008) 1061–1067.
[50] O. Torre, V. Gotor-Fernandez, I. Alfonso, F.L. Garca-Alles, V. Gotor, Study of the
chemoselectivity in the aminolysis reaction of methyl acrylate catalysed by
lipase B from candida antarctica, Adv. Synth. Catal. 347 (2005) 1007–1014.
[51] J. Priego, C. Ortız-Nava, M. Carrillo-Morales, A. Lopez-Munguıa, J. Escalante, E.
Castillo, Solvent engineering: an effective tool to direct chemoselectivity in a
lipase-catalyzed Michael addition, Tetrahedron 65 (2009) 536–539.
[52] K. Hult, P. Berglund, Enzyme promiscuity: mechanism and applications, Cell
25 (2007) 231.
[53] P. Carlqvist, M. Svedendahl, C. Branneby, K. Hult, T. Brinck, P. Berglund,
Exploring the active-site of a rationally redesigned lipase for catalysis of
michael-type additions, Chembiochem 6 (2005) 331–336.
[54] R. Jerome, P. Teyssie, Chemo-, regio-, stereo- and chrono-selectivity control in
polymerization and organic processes by catalytic coordination aggregates: a
topochemistry in solution? Curr. Org. Chem. 6 (2002) 109–119.
[55] M. Svedendahl, K. Hult, P. Berglund, Fast carbon-carbon bond formation by a
Promiscuous lipase, J. Am. Chem. Soc. 127 (2005) 17988–17989.
[56] D. Menezes-Blackburna, M. Jorquer, M. Gianfreda, R. Rao, E. Greiner, M.L.
Garrido, Activity stabilization of Aspergillus niger and Escherichia coli
phytases immobilized on allophanic synthetic compounds and
montmorillonite nanoclays, Bioresour. Technol. 102 (2011) 9360.
[57] S. Jonsson, E. Wehtje, P. Adlercreutz, B. Mattiasson, Thermodynamic and
kinetic aspects on water vs: organic solvent as reaction media in the
enzyme-catalysed reduction of ketones, Biochim. Biophys. Acta 1430 (1999)
313–322.
[58] H.N. Bhatti, M.H. Rashid, R. Nawaz, A.M. Khalid, M. Asgher, A. Jabbar, Effect of
aniline coupling on kinetic and thermodynamic properties of Fusarium solani
glucoamylase, Appl. Microbiol. Biotechnol. 73 (2007) 1290–1298.
[59] K.C. Badgujar, B.M. Bhanage, Thermo-chemical energy assessment for
production of energy-rich fuel additive compounds by using levulinic acid
and immobilized lipase, Fuel Process. Technol. 138 (2015) 139–146.
[60] K.C. Badgujar, T. Sasaki, B.M. Bhanage, Synthesis of lipase nano-bio-conjugates
as an efficient biocatalyst: characterization and activity-stability studies with
potential biocatalytic applications, RSC Adv. 5 (2015) 55238–55251.
[61] K.C. Badgujar, B.M. Bhanage, Carbohydrate base co-polymers as an efficient
immobilization matrix to enhance lipase activity for potential biocatalytic
applications, Carbohydr. Polym. 134 (2015) 709–717.
[62] K.C. Badgujar, B.M. Bhanage, Immobilization of lipase on biocompatible
co-polymer of polyvinyl alcohol and chitosan for synthesis of laurate
compounds in supercritical carbon dioxide using response surface
methodology, Process Biochem. 50 (2015) 1224–1236.
[42] S. Chang, J. Shaw, K. Yang, S. Chang, C. Shieh, Studies of optimum conditions
for covalent immobilization of Candida rugosa lipase on poly(c-glutamic acid)
by RSM, Bioresour. Technol. 99 (2008) 2800–2805.
[43] G. Ren, H. Yu, Oriented adsorptive immobilization of esterase BioH based on
protein structure analysis, Biochem. Eng. J. 53 (2011) 286–291.
[44] K.C. Badgujar, B.M. Bhanage, The solvent stability study with thermodynamic
analysis and superior biocatalytic activity of Burkholderia cepacia lipase
immobilized on biocompatible hybrid matrix of polyvinyl alcohol and
hypromellose, J. Phys. Chem. B 118 (2014) 14808–14819.
[63] K.C. Badgujar, B.M. Bhanage, The combine use of ultrasound and lipase
immobilized on co-polymer matrix for efficient biocatalytic application
studies, J. Mol. Catal. B: Enzym. 122 (2015) 255–264.
[64] K.C. Badgujar, B.M. Bhanage, The green metric evaluation and synthesis of
diesel-blend compounds from biomass derived levulinic acid in supercritical
carbon dioxide, Biomass Bioenergy 84 (2016) 12–21.
[45] G. Dors, L. Freitas, A.A. Mendes, A. Furigo, H.F. Castro, Transesterification of
palm oil catalyzed by pseudomonas fluorescens lipase in a packed-bed
reactor, Energy Fuels 26 (2012) 5977–5982.
[65] K.C. Badgujar, B.M. Bhanage, Enhanced biocatalytic activity of immobilized
Pseudomonas cepacia lipase under sonicated condition, Bioprocess Biosyst.
Eng. 39 (2016) 211–221.
[46] A. Salis, I. Svensson, M. Monduzzi, V. Solinas, P. Adlercreutz, The atypical
lipase B from Candida antarctica is better adapted for organic media than the
typical lipase from Thermomyces lanuginose, Biochim. Biophys. Acta 1646
(2003) 145–151.
[47] K.C. Badgujar, B.M. Bhanage, Enhanced biocatalytic activity of lipase
immobilized on biodegradable copolymer of chitosan and polyvinyl alcohol
support for synthesis of propionate ester: kinetic approach, Ind. Eng. Chem.
Res. 53 (2014) 18806–18815.
[66] P. Worsztynowicz, M. Napierała, W. Białas, W. Grajek, M. Olkowicz, Pancreatic
␣-amylase and lipase inhibitory activity of polyphenolic compounds present
in the extract of black chokeberry, Process Biochem. 49 (2014) 1457–1463.
[67] A. Rahim, Y. Takahashi, K. Yamaki, Mode of pancreatic lipase inhibition
activity in vitro by some flavonoids and non-flavonoid polyphenols, Food Res.
Int. 75 (2015) 289–294.
[48] G. Bosica, J. Spiteri, C. Borg, Aza-Michael reaction: selective mono- versus
bis-addition under environmentally-friendly conditions, Tetrahedron 70
(2014) 2449–2454.
[49] B.D. Mather, K.P. Viswanathan, K.P. Miller, T.E. Long, Michael addition
reactions in macromolecular design for emerging technologies, Prog. Polym.
Sci. 31 (2006) 487–531.
Please cite this article in press as: K.C. Badgujar, B.M. Bhanage, Lipase immobilization on hyroxypropyl methyl cellu-
lose support and its applications for chemo-selective synthesis of ␤-amino ester compounds, Process Biochem (2016),
http://dx.doi.org/10.1016/j.procbio.2016.07.008