Carbohydrate base co-polymers as an efficient immobilization matrix to enhance lipase activity for potential biocatalytic applications

Article abstract of DOI:10.1016/j.carbpol.2015.08.036

In the present study, we have synthesized biocompatible hybrid blend of cellulosic polymers of hydroxypropyl-methyl-cellulose (HPMC) and chitosan (CHY) for the immobilization of Candida rugosa lipase (CRL). The immobilized biocatalyst HPMC:CHY:CRL was subjected for characterization such as SEM, TGA, water content analysis, lipase activity, specific activity and protein content analysis. The kinetic parameter study (R_max/K_m) demonstrated improved biocatalytic activity of lipase after immobilization on carbohydrate co-polymers of HPMC:CHY. This biocatalyst was then employed to study practical biocatalytic applications for kinetic resolution which provided 50% conversion and >94% enantiomeric excess of substrate/product (ees/eep). The protocol demonstrated excellent recyclability upto five cycles. Finally, we studied influence of immobilization on cellulosic polymers for substrate, structure and reactivity for kinetic resolution. Hence, we investigated R₀ (initial reaction rate), E-value (enantioselectivity) and E_a (activation energy). This study confirms that, lipase immobilized on carbohydrate polymers had 3-4 folds higher biocatalytic activity as compared to crude CRL.

Full text of DOI:10.1016/j.carbpol.2015.08.036

Accepted Manuscript
Title: Carbohydrate base co-polymers as an efficient
immobilization matrix to enhance lipase activity for potential
biocatalytic applications
Author: Kirtikumar Chandulal Badgujar Bhalchandra
Mahadeo Bhanage
PII:
S0144-8617(15)00777-8
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.carbpol.2015.08.036
CARP 10239
To appear in:
Received date:
Revised date:
Accepted date:
21-5-2015
12-8-2015
13-8-2015
Please cite this article as: Badgujar, K. C., and Bhanage, B. M.,Carbohydrate
base co-polymers as an efficient immobilization matrix to enhance lipase
activity for potential biocatalytic applications, Carbohydrate Polymers (2015),
http://dx.doi.org/10.1016/j.carbpol.2015.08.036
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Carbohydrate base co-polymers as an efficient immobilization matrix to
enhance lipase activity for potential biocatalytic applications
Kirtikumar Chandulal Badgujar and Bhalchandra Mahadeo Bhanage∗
Department of Chemistry, Institute of Chemical Technology, Matunga,
Mumbai 400 019, India
Graphical abstract
Enzyme anchored
on Carbohydrate
Polymers blend
Free Enzyme
Physical , biochemical
characterization studies and
Biocatalytic applications
Lower (3 × 10-4)
Lower (24 - 90 %)
Lower
Initial reaction rate
Enantiomeric excess
% Conversion
Higher (10 × 10-4)
Higher (94 – 99 %)
Higher (3 times)
Higher (> 200)
5 times
Lower (< 48 )
No recyclability
Enantioselectivity (E)
Recyclability
∗Corresponding author (B. M. Bhanage): Tel.: +91-22-3361-2601/2222; Fax: +91-22-
2414-5614; Email:bm.bhanage@gmail.com
Highlights
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 Lipase was successfully immobilized on the blend of carbohydrate co-polymers
 Immobilized HPMC:CHY lipase displayed 3-4 folds activity compared to free
lipase
 Immobilized lipase fully characterized by the physical and biochemical techniques
 Immobilize lipase showed feasible Ea, R0, ees/p and E-value compared to free
lipase
 Immobilized lipase used for kinetic resolution with excellent recyclability
Abstract:
In the present study, we have synthesized biocompatible hybrid blend of cellulosic
polymers of hydroxypropyl-methyl-cellulose (HPMC) and chitosan (CHY) for the
immobilization of Candida rugosa lipase (CRL). The immobilized biocatalyst
HPMC:CHY:CRL was subjected for characterization such as SEM, TGA, water content
analysis, lipase activity, specific activity and protein content analysis. The kinetic
parameter study (Rmax/km) demonstrated improved biocatalytic activity of lipase after
immobilization on carbohydrate co-polymers of HPMC:CHY. This biocatalyst was then
employed to study practical biocatalytic applications for kinetic resolution which
provided 50% conversion and >94% enantiomeric excess of substrate/product (ees/eep).
The protocol demonstrated excellent recyclability upto five cycles. Finally, we studied
2
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influence of immobilization on cellulosic polymers for substrate, structure and reactivity
for kinetic resolution. Hence, we investigated R0 (initial reaction rate), E-value
(enantioselectivity) and Ea (activation energy). This study confirms that, lipase
immobilized on carbohydrate polymers had 3-4 folds higher biocatalytic activity as
compared to crude CRL.
Key words: Co-polymer; Immobilization; Kinetic resolution; Substrate structure
reactivity; Enhanced enzyme activity
1.
Introduction
Synthesis of enantiomerically pure compounds and kinetic resolution of racemic
organic moieties is the area of wide interest in the pharmaceuticals, agrochemicals,
flavours, aroma chemicals, fragrances and synthetic chemistry (Farina et al., 2006; FDA
statement). Specifically, optically active alcohols and acetates are important chiral
intermediates/ auxiliaries which have been synthesized by various conventional chemical
synthetic methodologies (Solano et al., 2012; Margolin, 1993). Synthesis of chiral
intermediates involve asymmetric hydrogenation of pro-chiral ketones by oxidation of
metal complexes, kinetic resolution of racemic alcohols, acylation of secondary alcohols
and selective hydrolysis of epoxides (Ni and Xu, 2012).
3
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Although these conventional chemical pathways are used widely for resolution
purpose, but these methodologies are severely affected by several drawbacks such as (i)
use of harsh chemicals (ii) lower enantiomeric excess (ee) (iii) lower yield (iv) bi-product
formation (v) disposable issue (vi) use of toxic chemicals (vii) higher activation energy
and (viii) long reaction time (Liu et al., 2013; Zhou et al., 2011; Badgujar and Bhanage
2014a; Badgujar and Bhanage 2014b). These above discussed limitations can be
effectively overcome by the use of enzyme catalysis, since enzymes displayed
outstanding catalytic properties (Adlercreutz, 2013; Kapoor and Gupta 2012). In enzyme
catalysis, basically lipases from the hydrolases class are extensively used because of their
ability of wide substrate array. However, use of these enzymes in free form possesses
several disadvantages which can be circumvented by skilful immobilization techniques.
Moreover, immobilization offers advantages like (i) improved catalytic activity, (ii)
stability and recyclability (Mateo 2007; Badgujar and Bhanage 2014c). Therefore, in the
present study, we first developed biocatalyst by immobilization on the cellulose base
support and then this biocatalyst was employed for the kinetic resolution study.
Various types of polymers have been widely used for the enzyme immobilization
(Badgujar et al., 2013; Badgujar and Bhanage 2015a). In recent years, natural cellulose
polymeric materials have received wide attention because of their outstanding functions
in membrane and bioreactor coating (May 1997). Kosaka et al. (2007) proposed that,
immobilization of lipase into the polysaccharide support favours the lipase activation and
stabilization. These polysaccharide materials are hydrophobic in nature and can serves as
a better medium for lipase interfacial activation (Vaidya and Singhal, 2008). Various
amino-acids such as Leucin, Alanine and Valine are primarily involved for
immobilization of lipase on the hydrophobic supports and lead to retain the ‘open state
conformation’ of lipase which displayed improved catalytic activity (Kosaka et al., 2007).
4
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In the present study, we used two different types of the support matrix namely,
HPMC and CHY as a medium for the lipase immobilization. CHY has excellent
immobilization characteristic like non-toxicity, natural safe polymer, environmental
friendliness, better mechanical stability, inertness to solvent and chemicals (Mendes et al.,
2012; Elnashar et al., 2013; Chang and Zhang 2011). Moreover, the CHY possessing free
amino (-NH2) and hydroxyl (-OH) groups which impart in covalent and H-bonding
respectively (Orrego et al., 2010). The HPMC possess more number of hydroxyl (-OH)
and propyl groups which provided higher extent of lipase immobilization via H-bonding
and hydrophobic (van der Waals) interactions (Mezdour et al., 2008; Sundari and
Balasubramanian, 1997; Kumar et al., 2006; Möller et al., 2004). Hence in the present
study, we immobilized lipase on both support individually as well as in various binary
blends. It was observed that, proper composition of the binary mode of immobilization
offer subsequent benefits for the protein immobilization and corresponding lipase activity
as compared to individual mode.
Thus, this study deals with the lipase immobilization on various supports made
from CHY and HPMC. Moreover, this immobilized lipase was well characterized by the
available physical characterization tools such as SEM, TGA, lipase activity, protein
content and kinetic parameters determination. Finally the immobilized lipase was
subsequently used for biocatalytic applications to study kinetic resolution of secondary
alcohols. All reaction parameters were well optimized to obtain higher % yield, % ee and
protocol was extended to resolve various racemic alcohol moieties. Also, kinetic
parameters/ initial reaction rate were also tested for various substrates. Finally
recyclability was checked, thus present protocol demonstrated robust applications of
cellulosic support for lipase immobilization and for potential biocatalysis.
5
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2.
Material and Methods
2.1
Enzyme and chemicals
Candida rugosa lipase (lipase CRL, ≥ 2000 U/g), Rhizopus niveus (lipase RNL,
≥1500 U/g), CHY (Brookfield viscosity > 200 cps) and p-NPA were purchased from
Sigma-Aldrich Ltd., India. The Bovine serum albumin (BSA) and Bradford reagent were
bought from Hi Media Ltd., India. Aspergillus niger lipase (lipase ANL, ≥ 2000 U/g) was
kindly gifted by the Advance enzymes. Other all chemicals/solvents/ reagents were
acquired from Hi media, Fluka India and S. D. Fine Chemicals Ltd.
2.2
Immobilization of the lipase
Immobilization of lipase on HPMC-CHY matrix was performed via entrapment
methodology in water as a greener immobilization medium at normal room temperature.
At first, HPMC (300 mg) was solubilised in deionized water (25 mL) and CHY (100 mg)
was solubilised in 1 to 1.5 % w/w CH3COOH solution (15-20 mL) in a separate beaker.
Both these solutions were stirred separately at 1200 rpm until complete solubilisation.
Later, HPMC solution was slowly poured into the CHY solution and stirred robustly for
6-8 h at 1500 rpm until formation of complete homogenous mixture. Finally, crude CRL
was solubilised in 2 mL deionized water and added to the above HPMC-CHY blend,
which was stirred gently at almost 200-230 rpm for 60-70 minutes. The HPMC-CHY
immobilized solution was then carefully transferred into a Teflon dish and kept at 40-
45°C for drying. After 48 h, polymer of immobilized matrix (HPMC:CHY:lipase) was
removed from the Teflon Petri dish and cut off into the small pieces of 2-3 mm2 size and
stored at 6-8°C until their use. Thus, theoretically binary support of HPMC (300 mg) and
CHY (100 mg) was used to immobilize 120 mg of crude CRL which is denoted as
HPMC:CHY:CRL (300:100:120) i.e. HPMC:CHY:CRL (3:1:1.2).
6
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2.3
2.3.1. SEM analysis
The SEM analysis of immobilized lipase HPMC:CHY:CRL (300:100:120) matrix
Characterization
and HPMC:CHY (300:100) control support was examined by the scanning electron
microscopy (SEM)-FEI, Quanta 200. The sample matrix was kept on a carbon stub while
SEM images were captured at 10-20 kV using a LFD detector at lower vacuum.
2.3.2. TGA
The thermo gravimetric analysis (TGA) was performed to investigate thermo-
stability of HPMC:CHY:CRL immobilized lipase and crude CRL by using Q-series 600
TGA analyzer. For this purpose 8-10 mg of immobilized matrix and control sample was
o
kept in an alumina crucible and analysis was carried out from 30-600 C along with 10
oC/ min rise in temperature, at 99.9% pure N2 atmosphere with flow rate of 80 mL/min.
2.3.3. % water content
The water contents of crude CRL, HPMC:CHY support and HPMC:CHY:CRL
immobilized lipase matrix was determined using Karl Fischer method (784 KFP Titrino).
2.3.4. Lipase activity
The lipase hydrolytic activity was investigated for crude lipases and immobilized
HPMC:CHY:lipases spectrophotometrically at 410 nm with slight change in Pencreac’h
and Baratti (2001) method. In this assay, we have studied hydrolysis of p-nitro phenyl
acetate (p-NPA) substrate. The standard assay mixture consists of 2 mg of crude lipase
CRL (or equivalent quantity of immobilized HPMC:CHY:lipase matrix) in 1 mL of n-
hexane solvent. Hydrolytic assay was performed by adding 1 mL of 14 mM of p-NPA as
a substrate dissolved in 2-propanol as a solvent and incubated at 37 °C at 150 rpm for 10
7
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minutes. After that, 400 μL of hydrolytic assay reaction mass was withdrawn and added
by 400 μL of distilled water to extract p-nitro phenol (p-NP) in aqueous layer. At the end,
1200 μL of potassium phosphate buffer having pH nearly 8.0-8.1 was added and shaked
rapidly for a second to extract p-NP which produces pale yellow colour. This pale yellow
colour aqueous layer sample was instantly used to determine absorbance at 410 nm. The
lipase activity (U) was defined as µ mol of p-NP released by per milligram of lipase per
minute at standard assay condition.
2.3.5. Protein content
The protein content of crude CRL and % protein binding of various immobilized
HPMC:CHY:lipase was investigated by using Bradford method at 595 nm (Bradford,
1976). Initially, crude protein content used to immobilize lipase CRL was determined.
Later, immobilized matrix was completely removed from the Teflon petri dish and this
Petri dish was then rinsed properly and subjected to determine the un-immobilized
quantity of protein. Thus, quantity of protein immobilized (protein bound) on
HPMC:CHY matrix was the difference between the initial (crude) amount of proteins
utilized to immobilize and non-immobilized amount of protein found after washing of
Petri dishes.
2.4 Kinetic parameter determination
The maximum reaction rate (Rmax) and Michaelis-Menten constant (Km) of
crude CRL and immobilized CRL was determined by using Lineweaver-Burk plot via p-
NPA lipase activity assay in the range of 4-24 mM substrate concentrations. The method
used to determine Km and Rmax for various substrate concentrations is similar as
prescribed in above section 2.3.4 (Lipase activity assay). The catalytic efficiency (Rmax/
Km) was also determined for crude CRL and immobilized HPMC:CHY:CRL.
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2.5 Experimental set-up and analysis
The kinetic resolution and various desired organic moieties were synthesized in a
12 mL glass reaction vessel having 2 cm i.d. At first, (±) 1-phenyl ethanol (racemic
alcohol: 1 mmol) was taken in a reaction vessel and diluted by the given solvent (n-
hexane). Then given acyl donor (2.5 mmol) was added into the reaction vessel and
reaction was initiated by addition of 70 mg immobilized HPMC:CHY:CRL biocatalyst.
Later on, the reaction vessel was placed carefully on a rotatory shaker at mentioned
o
temperature (48 C). Samples of reaction mixture (5-10 μL) was withdrawn periodically
to analyze % conversion and ee by using the gas chromatograph (PerkinElmer Clarus-400
instrument) having flame ionizing detector (FID) and chiral capillary column CP-
Chirasil- Dex CB Column (Varian capillary column, 25m×0.25mm×0.25μm). The
detector and injector temperature were 200 and 60 oC, respectively. The oven temperature
o
of GC was kept at 60 °C for 5 min and then incremental increase of 10 C/ min upto 260
°C. The final reaction mass product was analyzed and confirmed by the GC-MS (Gas-
Chromatography-Mass Spectroscopy) instrument Shimadzu QP-2010.
3.
Result and Discussion
3.1. Characterization of the immobilization support
3.1.1. Lipase screening, lipase activity assay and protein content study
Initially, we have studied three lipases namely, RNL, ANL and CRL. In all three
cases, carbohydrate base immobilized lipase demonstrated better lipase activity as
compared to crude lipase (Table 1). Moreover, the specific activity was also found to be
higher in immobilized lipase. Lipases from ANL and CRL were displayed slightly lower
activity, however lipase CRL demonstrated better % protein content and % lipase activity.
Hence lipase CRL was chosen as a robust bio-catalyst to study kinetic resolution as a
9
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biocatalytic application. It was found that lipase CRL providing best compatibility and
subsequent higher activity. The improved bio-catalytic activity of various immobilized
biocatalyst was observed due to immobilization on the carbohydrate polymer which
causes the interfacial activation of lipase biomolecules (Reetz, 1997; Jomes et al., 2011;
Badgujar and Bhanage 2014b).
Table 1: Lipase activity study of crude and HPMC:CHY immobilized lipase
No
Immobilized or
crude lipase
lipase protein Specific % activity % lipase % protein
activity content activity retention
activity
--
content
--
1
2
3
4
5
6
Crude CRL
27.37
31.70
5.97
16.22
14.96
11.27
10.23
26.12
24.19
1.68
2.11
0.53
0.67
0.47
0.55
--
125
--
HPMC:CHY:CRL
Crude RNL
115.8
--
92.23
--
HPMC:CHY:RNL
Crude ANL
6.89
127
--
115.4
--
90.77
--
12.3
HPMC:CHY:ANL 13.32
117
108
92.61
Lipase activity: U/mg; protein content: µg/mg; specific activity: U/µg; % activity
retention is ratio of specific activity of immobilized lipase to free lipase.
3.1.2. Cellulose blend support composition
Support composition is also an important aspect which displayed important
observations that (i) higher amount of CHY in immobilization leads to improve the
protein content which might be attributed due to presence of the amino functionality in
CHY (Table 2). This amino functionality produces enhanced immobilization effect.
However, after 100 mg of CHY in HPMC:CHY blend leads to increase only the protein
binding yield but not lipase activity. This important observation concluded that, higher
CHY loading may deactivates the active catalytic sites via formation of Schiff’s reaction.
Moreover, we immobilized lipase on both supports individually as well as in various
binary blends. It was observed that, proper composition of the binary mode of
10
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immobilization offered subsequent benefits of protein immobilization and resultant lipase
activity as compared to individual mode. Hence, optimized support composition was
HPMC:CHY:CRL (300:100:120) which is used for further study. Earlier, Kosaka et al.
(2007) proposed that, anchoring of lipase biomolecules into polysaccharide support
favours the lipase activation and stabilization. Similar kind of higher activity was
observed in the present study after use of cellulose base support.
Table 2: Activity and protein content study for HPMC:CHY support composition
No
1
A
B
C
D
E
F
G
400:000:120
375:025:120
350:050:120
325:075:120
300:100:120
275:125:120
250:150:120
200:200:120
000:400:120
24.81
25.60
26.01
26.64
31.70
30.67
29.12
28.04
24.12
90.66
93.55
95.04
97.33
115.82
112.05
106.39
102.44
88.12
13.95
14.28
14.43
14.62
14.96
15.12
15.38
15.45
15.97
86.00
88.03
88.96
90.13
92.23
93.21
94.82
95.25
98.45
1.77
1.79
1.80
1.82
2.11
2.02
1.89
1.81
1.50
105.41
106.26
107.13
108.58
125.57
120.20
112.20
107.55
89.31
2
3
4
5
6
7
8
9
Lipase activity, protein content and specific activity of free CRL is 27.37, 16.22 and 1.68
A: Immobilized lipase support composition HPMC:CHY:CRL; B: Immobilized lipase
activity; C: % lipase activity (B/27.37×100); D: Immobilized lipase protein content; E: %
Protein content (D/16.22×100); F: Specific activity of immobilized lipase; G: % activity
retention (F/1.68×100); Units; Lipase activity: U/mg; protein content: µg/mg; specific
activity: U/µg.
3.1.3. Physical texture via microscopic analysis
The lipase immobilized species (HPMC:CHY:CRL) showed uniform dispersion
of lipase biomolecules (small globules) into the surface of HPMC:CHY matrix, while
11
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some lipase biomolecules are aggregated to from big cluster (Figure 1, B). However,
HPMC:CHY control support showed the plane surface (Figure 1, A). The present change
in surface morphological study clearly confirmed immobilization of lipase CRL on the
HPMC:CHY support as compared to the control support. Similar kind of the change in
surface morphology was reported by the Erdemir and Yilmaz (2009).
3.1.4. TGA
The thermal degradation stability was observed by TGA analysis which
demonstrated better tolerance of immobilized lipase to thermal degradation as compared
to the crude CRL (Figure S1: please refer supplementary information). Free CRL
o
displayed almost 40 % degradation at 300 C, while HPMC:CHY:CRL displayed 40 %
degradation at 360 oC. The better resistance of immobilized lipase to thermal degradation
was attributed to successful immobilization of lipase biomolecules into cellulosic
polymer. This immobilization protected lipase bio-molecules from thermal denaturation
(outside environment), while crude lipases are directly exposed to thermal denaturation
(Dave and Madamwar, 2006). The early (initial) weight loss at 100-120 oC was attributed
due to the removal of the physically adsorbed water molecules while; second weight loss
o
almost 220-240 C was attributed to removal of closely associated micro-aqueous
molecules (Turner and Vilfsun, 2000).
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(A)
(B)
0.09
0.06
0.03
0
40
30
20
10
0
R² = 0.9941
R² = 0.989
0
3
6
9
12
15
18
21
24
27
-0.1
0
0.1
1/[S]
0.2
0.3
S (mM)
(C)
(D)
Crude CRL
HPMC:CHY:CRL
Crude CRL
HPMC:CHY:CRL
Figure 1: (A) HPMC:CHY Control support (B) Immobilized HPMC:CHY:CRL lipase
(C) Substrate concentration effect on lipase activity (D) Lineweaver-Burk plot
3.1.5. % Water content
In the current study, free lipase CRL having almost 0.94 % of water content
whereas, immobilized HPMC:CHY:CRL lipase had 6.18 % water content (Table 3). The
HPMC:CHY control support contained 4.29 % of water content (Table 3). The higher
water content of immobilized HPMC:CHY:CRL species was attributed to use of the
water media for the immobilization. This small amount of bound water is highly essential
to maintain catalytic activity and to preserve conformational flexibility of enzymes. This
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bound water forms a hydrating layer around the enzyme and maintain the intramolecular
stabilizing forces of lipase biomolecules (Wehtje and Adlercreutz, 1997).
Table 3: Determination of kinetic parameters and water content
Reaction Catalytic % Catalytic
R2
rate (%) efficiency Efficiency square content
% Water
Sample
Km Rmax
Crude CRL
11.28 46.73
100
109
--
4.14
5.01
--
100
120
--
0.9941
0.989
--
0.94
6.18
4.29
HPMC:CHY:CRL 10.09 50.51
Control support -- --
3.1.6. Determination of enzyme kinetic parameters
The basic kinetic features are demonstrated by Rmax and Km which indicating the
maximum reaction speed and enzyme-substrate binding affinities at the enzyme catalytic
sites (Figure 1; Table 3). The higher Km value indicated lower substrate affinity of
enzyme and vice-a-versa, whereas higher Rmax value depicted higher reaction speed at
the given substrate concentration. The alteration in Km and Rmax values of immobilized
lipases is depended/ related to (i) enzyme conformational orientation/flexibility on the
support matrix (ii) formation and/or breakdown of enzyme-substrate complex (iii) change
of substrate binding efficiency at dynamic active site and (iv) mass diffusion restriction
(Ye et al., 2005). In the present study, Rmax value and substrate affinity was improved
after immobilization of lipase on HPMC:CHY as compared to crude lipase. This fact was
indicating fine dispersion of lipase bio-molecules on support, which experience larger
substrate concentration and promote mass transfer phenomenon at the catalytic sites of
immobilized enzymes. Cumulatively results of the Rmax and Km displayed higher
catalytic efficiency of the immobilized lipase (120 %) as compared to crude lipase CRL.
3.2. Biocatalytic applications studies
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3.2.1 Screening of various lipases for kinetic resolution of (±) 1-phenyl ethanol
Biocatalysis for the resolution of secondary alcohol is an important application for
the synthetic organic chemist which follows the major green chemistry advantages like (i)
better yield, (ii) mild reaction condition (iii) safer methodology (iv) use of non-toxic/ non-
hazardous chemicals (iv) higher enantiomeric excess (ees or eep) (v) easy recovery of
product and bio-catalyst (vi) efficient recyclability of immobilised lipase (vii) no-side
reactions (viii) green tag to enantio-rich isomers (ix) easy dispose of biocatalyst (x)
sustainable E-factor etc. (Kapoor and Gupta 2012; Badgujar et al., 2014c). Initially, we
have screened various HPMC:CHY immobilized and crude lipases for given model
reaction scheme 1 as indicated in figure 2. It was found that, each immobilized lipase
providing better biocatalytic activity as compared to respective crude lipase. The
immobilized lipase HPMC:CHY:CRL displayed the best biocatalytic activity as
compared to various tested biocatalyst and utilized for further study (Figure 2).
Scheme 1: Kinetic resolution of (±) 1-phenyl ethanol
15
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60
50
40
30
20
10
0
100
80
60
40
20
0
CRL
RNL
ANL
CRL
RNL
ANL
Immobilized HPMC:CHY:Lipase (blue shades)
and crude lipase (pink shades)
% Conversion
ees
eep
Figure 2: Effect of various lipases (crude/ HPMC:CHY immobilized lipase) (Reaction
conditions: 1-phenyl ethanol, 1 mmol; isopropenyl acetate, 3 mmol; solvent toluene upto
2 mL; HPMC:CHY:lipase (300:100:150) immobilized biocatalyst 78 mg/ equivalent
quantity of crude lipase; temperature 48oC; time, 13h).
3.3. Optimization of various reaction protocols
Various reaction parameters were optimized in detail to find out the best suitable
conditions for the given model reaction. Initially, we have investigated the support
compositions for the carbohydrate blend of HPMC and CHY. Hence, lipase amount from
60 to 150 mg were loaded on the HPMC:CHY (3:1) support. It was seen that, 120 mg of
lipase CRL loading on the HPMC:CHY support provided best results. The higher amount
of lipase loading over 120 mg provided almost similar % conversion and ee of substrate/
product (Figure 3A). Next to this, we have studied mole ratio of reactants. As shown in
figure 3B, as acyl donor quantity increased then % conversion and ee of substrate/
product also increased which may be attributed to easy substrate-enzyme complex
formation. Herein, the optimized mole ratio 1:2.5 was used for further study.
16
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60
50
40
30
20
10
0
100
80
60
40
20
0
60
50
40
30
20
10
0
100
85
70
55
40
60
CRL loading on HPMC:CHY (3:1) support composition
% Conversion ees eep
80
110
120
130
140
150
1:1.5
1:2
1:2.5
Alcohol:acyl donor
% Conversion ees
1:3
1:3.5
1:4
eep
(A)
(B)
60
50
40
30
20
10
0
100
95
90
85
80
75
70
60
50
40
30
20
10
0
100
80
60
40
20
0
Aacetic
acid
Methyl
acetate
Ethyl
Acetic Isopropenyl Vinyl
acetate
acetate unhyddride acetate
n-hexane
Toluene
Acetone 1.4 dioxane Chloroform
Acyl donor
% Conversion
Solvent
(C)
(D)
ees
eep
% Conversion
ees
eep
Figure 3: (A) Effect of CRL lipase loading on HPMC:CHY support composition
(Reaction conditions: 1-phenyl ethanol:isopropenyl acetate- 1:3 mmol; solvent toluene
upto 2mL; HPMC:CHY:CRL immobilized biocatalyst 78 mg; temperature 48oC; time
13h); (B) Effect of the reactant mole ratio study (Reaction conditions: 1-phenyl ethanol:
isopropenyl acetate-1:1.15 to 1:4 mmol; solvent toluene upto 2mL; HPMC:CHY:CRL
(300:100:120) immobilized biocatalyst 78 mg; temperature 48oC; time 13h); (C) Effect of
acyl donors (Reaction conditions: 1-phenyl ethanol:acyl donor- 1:2.5 mmol; solvent
toluene upto 2 mL; HPMC:CHY:CRL (300:100:120) immobilized biocatalyst 78 mg;
temperature 48oC; time 13h); (D) Effect of solvent (Reaction conditions: 1-phenyl
ethanol:vinyl acetate- 1:2.5 mmol; solvent
(polar or non-polar) upto 2 mL;
HPMC:CHY:CRL (300:100:120) immobilized biocatalyst 78 mg; temperature 48oC; time
13h).
17
Page 17 of 34

Later on, we have checked influence of various acyl donors such as acids, esters
and un-hydrides (Figure 3C). It was found that, isopropenyl acetate and vinyl acetate
provide better results for the maximum conversion and ee. In context to vinyl acetate,
isopropenyl acetate provided slightly lower % conversion which was attributed due to
bulky nature of the isopropenyl acetate (Badgujar et al., 2013). Hence, we used vinyl
acetate as a best suitable reagent for the kinetic resolution of 1-phenyl ethanol. Reaction
media played an important role in deciding the % conversion and ee of the respective
product (Figure 3D). It is well noted that, lipases demonstrated best results in non-polar
solvent (Adlercreutz, 2013); while in present study it was found that, HPMC:CHY
immobilized lipase was sufficiently stable to provide better % conversion in all tested
solvents. Enantiomeric excess of substrate (ees) was slightly affected by the nature of
solvents while enantiomeric excess of product (eep) provided better results in all tested
solvents. The suitable solvent for the present study was toluene and n-hexane, while n-
hexane was selected as solvents of choice for the further study.
The biocatalyst quantity is an important aspect for the industrial feasibility, which
showed improvement in conversion and ees (Figure 4A). Higher conversion at higher
biocatalyst amount was attributed due to reason of availability of more catalytic sites for
the corresponding transformations (Shinde and Yadav, 2015). Thus, 70 mg was optimized
quantity to obtain the higher conversion and ee. Above this quantity (70 mg), there is
availability of extra catalytic sites which did not influence reaction conversion
significantly (Shinde and Yadav, 2015). The effect of temperature is crucial parameter to
avoid the possible denaturation and deactivation of catalytic activity of lipase (Figure
4B). It was seen that, as temperature increases then % conversion also increases;
however, at higher temperature (55oC) eep decreases dramatically. These results
demonstrated that, mild temperature improve selectivity of resolution while higher
18
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temperature provided higher activation energy and promotes acylation reaction with loss
of selectivity (Li et al., 2013; Kuo et al., 2014). Thus, 48oC was optimized temperature
used for further study which provided higher conversion as well as higher enantiomeric
excess. Finally, the reaction time of the protocol was studied (Figure 4C). It was observed
that, 12 to 14 hours was the sufficient time for given kinetic resolution, while 12 to 13
hours is the optimized reaction time to achieve higher % conversion, ees, and eep.
60
50
40
30
20
10
0
100
75
60
50
40
30
20
10
0
100
85
70
55
40
25
50
25
25 mg 36 mg 50 mg 60 mg 70 mg 80 mg
Biocatalyst amount
26
32
40
48
55
60
Temperature (oC)
(A)
(B)
% Conversion
ees
eep
% Conversion
ees
eep
70
56
42
28
14
0
100
80
60
40
20
0
60
50
40
30
20
10
0
100
95
90
85
80
75
70
0
120
240
360
Time (minutes)
ees
480
600
720
eep
840
Fresh
I
II
Recycle run
% Conversion
III
ees
IV
(C)
(D)
% Conversion
eep
Figure 4: (A) Effect of biocatalyst amount (Reaction conditions: 1-phenyl ethanol:vinyl
acetate 1:2.5 mmol; n-hexane solvent upto 2 mL; HPMC:CHY:CRL (300:100:120)
immobilized biocatalyst 25-80 mg; temperature 48oC; time 13h); (B) Effect of
temperature (Reaction conditions: 1-phenyl ethanol:vinyl acetate 1:2.5 mmol; solvent n-
hexane upto 2 mL; HPMC:CHY:CRL (300:100:120) immobilized biocatalyst 70 mg;
temperature 26-60oC; time 13h); (C) Effect of reaction time (Reaction conditions: 1-
19
Page 19 of 34

phenyl ethanol:vinyl acetate 1:2.5 mmol; solvent n-hexane upto 2 mL; immobilized
biocatalyst HPMC:CHY:CRL (300:100:120) 70 mg; temperature 48oC; time 0-14h); (D)
Effect of recyclability (Reaction conditions: 1-phenyl ethanol:vinyl acetate 1:2.5 mmol;
solvent n-hexane upto 2 mL; HPMC:CHY:CRL (300:100:120) immobilized biocatalyst
70 mg; temperature 48oC; time 12-13 h).
3.4. Recyclability study:
In order to investigate the feasibility of the present biocatalytic protocol, we
studied recyclability of biocatalyst HPMC:CHY:CRL for the kinetic resolution. It was
found that, immobilized biocatalyst was sufficiently reused upto studied four cycles
(Figure 4D). There was marginal (1-2%) decrease of enantiomeric excess of product (eep)
in studied all cycles. In context to this, substrate alcohol displayed moderate decrease in
the enantiomeric excess (ees) value for studied four recycles. The decrease in ees and
subsequent conversion was attributed to deactivation of lipase due to continuous exposure
of alcoholic substrate (Kuo et al., 2014; Badgujar and Bhanage 2015b).
3.5. Influence of substrate structure and reactivity on enzyme activity for kinetic
resolution
In order to investigate the broad applicability of HPMC:CHY:CRL immobilized
lipase, we have studied the influence of substrate structure and reactivity on enzyme
activity for kinetic resolution. It was observed that, cellulose based immobilized lipase
displayed almost three folds higher catalytic activity as compared to crude lipase CRL
(Table 4, entries 1,2). This was attributed to the successful immobilization on cellulose
base supports (Jomes et al., 2011). The higher E-value indicated improvement in
enantioselectivity, when reaction was catalyzed by immobilized lipase.
20
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An electron donating substituent improved the conversion and ees however, it was
slightly decreases the eep because of the higher reactivity (Table 4, entries 2,4). At the
same time, change in acylation donor from acyl to butanoyl causes decrease in reaction
rate, subsequent conversion and ee (Table 4, entries 2,6). This was attributed to the higher
mass transfer restriction because of the hydrophobic long chain of butanoyl (Mateo et al.,
2007; Badgujar and Bhanage 2014a, Badgujar and Bhanage 2015b). This longer chain
causes slower rate of diffusion and reduces the reaction rate with increase in the reaction
time span. Substitution of more nucleophilic atom -NH2 instead of -OH causes increment
in the nucleophilicity of reactant (Table 4, entries 2,8). This highly nucleophilic species
provided better % conversion, ees and decrement of reaction time span. Moreover, the R0
value displayed initial reaction rate which is also found to be higher for electron rich and
highly nucleophilic reactant species. The activation energy was found to be lesser for
electron rich and highly nucleophilic reaction, the lesser value demonstrated higher
reaction rate for given substrate (Zhou et al., 2011; Badgujar and Bhanage 2014a;
Badgujar and Bhanage 2014c; Badgujar et al., 2015c; Badgujar and Bhanage 2015d ).
The crude lipase demonstrated higher Ea value which indicated lower catalytic
efficiency and sluggish reaction rate (R0) which was attributed due to aggregation of
crude lipase CRL in the organic media (Adlercreutz et al., 2013). Finally, talking about an
important aspect of kinetic resolution it was observed that, the crude lipase demonstrated
lower enantioselectivity (E-value) and selectivity of resolution value (ees/ eep) as
compared to HPMC:CHY immobilized lipase. Thus, cellulose based immobilized
HPMC:CHY:CRL biocatalyst showed best bio-catalytic activity to resolve various
racemic molecules with an excellent enantioselectivity and ees/eep value.
21
Page 21 of 34

1
Table 4: Influence of substrate, structure and reactivity on enzyme activity for kinetic resolution
No
Reactant
Acyl
donor
Product
(ester)
Product
(alcohol)
Biocatalyst
R0
Time
(h)
%
ees
eep
E-
Ea
(10-4)
Conversion
value (KJ/mol)
Crude CRL
3.1±0.17
13
16±2.51
24±1.73
94±1.0
94±0.57
98±1.0
41
60.9±3.94
46.1±2.35
1
2
>200
(354)
HPMC:CHY:CRL
10.4±0.56
13
49±0.57
Crude CRL
HPMC:CHY:CRL
Crude CRL
4.1±0.26
11.1±0.20
2.7±0.47
13
13
14
18±1.52
50±1.0
38±2.51
98±0.57
22±1.15
95±1.15
96±1.0
95±2.0
57
58.2±3.36
43.6±1.08
67.7±3.57
3
4
>200
(226)
13±1.52
48
114
43
5
6
HPMC: CHY:CRL
Crude CRL
8.4±0.36
4.6±0.45
12.1±0.52
14
12
12
45±0.57
20±2.0
88±1.0
44±1.52
98±0.57
95±0.57
93±1.52
95±1.73
50.4±2.44
50.3±3.15
41.9±1.3
7
8
HPMC:CHY:CRL
50±1.15
180
2
3
4
Effect of substrate study (Reaction conditions: racemic alcohol, 1 mmol; vinyl acetate, 2.5 mmol; solvent n-hexane upto 2 mL; immobilized
HPMC:CHY:CRL (300:100:120)- 70 mg (or equivalent to crude lipase CRL); temperature 48oC; R0 indicated initial reaction rate; E-value
(enantioselectivity), Ea (activation energy). For calculation of the E-value: average values of conversion, ees and eep are used.
22
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Conclusion:
In the present study, we have successfully synthesized a biocompatible hybrid blend of
cellulosic support made up of HPMC and CHY for lipase CRL immobilization. The
immobilized biocatalyst HPMC:CHY:CRL was characterized successfully by SEM,
TGA, % water content, lipase activity and protein content analysis. The kinetic
parameters Rmax and km demonstrated improved catalytic activity of HPMC:CHY:CRL.
This HPMC:CHY:CRL biocatalyst was then applied for kinetic resolution of secondary
alcohols which provided almost 50 % conversion and > 94 % enantiomeric excess of
substrate as well as product. The developed HPMC:CHY:CRL biocatalytic protocol was
sufficiently recycled upto the five successive cycles. We have studied the influence of
substrate, structure and reactivity of substrate on the immobilized enzyme activity for
kinetic resolution; hence various reaction aspects such as R0, E-value, Ea were
determined. On the basic of these three aspects, we have discussed the effect of various
substrates for the catalytic activity of developed biocatalyst. Moreover, carbohydrate
polymer based immobilized lipase demonstrated three folds higher biocatalytic activity
compared to crude lipase.
Acknowledgement:
Kirtikumar is grateful to Department of Biotechnology-DBT, India (BT/PR4192/PID/6/
623/2011), and Council of Scientific & Industrial Research-CSIR, India (09/991(0015)/
2011-EMR-I), for providing research funds.
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Captions to Figures:
(Captions already included in the manuscript below respective figure)
Figure 1: (A) HPMC:CHY Control support (B) Immobilized HPMC:CHY:CRL lipase
(C) Substrate concentration effect on lipase activity (D) Lineweaver-Burk plot
Figure 2: Effect of various lipases (crude/ HPMC:CHY immobilized lipase) (Reaction
conditions: 1-phenyl ethanol, 1 mmol; isopropenyl acetate, 3 mmol; solvent toluene upto
2 mL; HPMC:CHY:lipase (300:100:150) immobilized biocatalyst 78 mg/ equivalent
quantity of crude lipase; temperature 48oC; time, 13h).
Figure 3: (A) Effect of CRL lipase loading on HPMC:CHY support composition
(Reaction conditions: 1-phenyl ethanol:isopropenyl acetate- 1:3 mmol; solvent toluene
upto 2mL; HPMC:CHY:CRL immobilized biocatalyst 78 mg; temperature 48oC; time
13h); (B) Effect of the reactant mole ratio study (Reaction conditions: 1-phenyl ethanol:
isopropenyl acetate-1:1.15 to 1:4 mmol; solvent toluene upto 2mL; HPMC:CHY:CRL
(300:100:120) immobilized biocatalyst 78 mg; temperature 48oC; time 13h); (C) Effect of
acyl donors (Reaction conditions: 1-phenyl ethanol:acyl donor- 1:2.5 mmol; solvent
toluene upto 2 mL; HPMC:CHY:CRL (300:100:120) immobilized biocatalyst 78 mg;
temperature 48oC; time 13h); (D) Effect of solvent (Reaction conditions: 1-phenyl
ethanol:vinyl acetate- 1:2.5 mmol; solvent
(polar or non-polar) upto 2 mL;
HPMC:CHY:CRL (300:100:120) immobilized biocatalyst 78 mg; temperature 48oC; time
13h).
29
Page 29 of 34