G Model
PRBI-10125; No. of Pages10
ARTICLE IN PRESS
2
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2014) xxx–xxx
In the present study lipase PCL was immobilized on biodegrad-
able ternary blend of CH, PVA, PLA. CH is attractively used for
immobilization because of its characteristic feature such as the
lack of toxicity, inertness to chemical reactivity and ecofriendly
nature [16]. PVA endows outstanding properties for immobiliza-
tion on film such as high flexibility, biocompatibility, non-toxicity,
adhesiveness and better resistivity to organic solvent [1,2]. PLA is a
biodegradable and ecofriendly polymer obtained from the natural
resources such as starch which maintains the biocompatibility in
ternary blend [16]. Preparing ternary blend of CH, PVA and PLA is
attractive as it offers all these individual advantages in single film
2.3. Characterization of the immobilized lipase
2
.3.1. Scanning electron microscopy analysis (SEM)
Scanning electron microscope (SEM) analysis was performed to observe the
change in surface morphology for control PLA:PVA:CH (1:6:1) and immobilized
PLA:PVA:CH:PCL (1:6:1:2) lipase polymer film by the FEI-Quanta 200, instrument.
The film sample was kept on carbon stub and images were recorded at 20 kV using
LFD detector under low vacuum.
2
.3.2. FTIR analysis
FTIR analysis was studied to confirm the presence of amide functionality after
immobilization of lipase PCL on the ternary blend polymer film. The FTIR analysis
was carried out by Perkin-Elmer, Spectrum 100.
[
1,2,16]. This ternary blend immobilized lipase (biocatalyst) was
2
.3.3. Water content analysis for various crude and immobilized lipase
The % water content of various crude, immobilized lipases (PCL, ROL, CRL) and
then applied to synthesis of geranyl acetate which is recognized as
a safe compound by U.S. Food and Drug Administration.
control support was measured using the Karl Fischer titration method (784 KFP
Titrino).
Geranyl acetate is a colourless liquid having pleasant floral-
fruity smell; it is important component of 60 natural essential oils
such as Alpinia galangal, Ocimum basilicum, Citrus sanguinello, Cym-
bopogon nardus etc. Furthermore it can be used as a flavouring agent
for lavender, lime grass, peach, berry, fig, rose and citrus etc. Syn-
thesis of food-flavour and fragrance compounds by the enzymatic
route labelled them as a “green-natural” products [1,2]. In 2010,
sales of immobilized enzymes for biocatalytic purpose were val-
ued almost around $160 million [18]; and nearly 42% of the overall
world enzyme market is covered for the food-beverage, pharma
and home-care products [18]. Thus, considering the wide scope
and importance of terpinyl esters, we make an attempt to explore
kinetic modelling study for geranyl acetate synthesis, which has
not been yet reported using biodegradable immobilized PCL as a
biocatalyst and hence finds a great scope.
2.3.4. Physical appearance and film thickness determination
The film thickness was determined by using a manual micrometre. The final
thickness was determined by ten random various places of films.
2
.3.5. Determination of lipase activity assay
Lipase activity of crude and ternary blend immobilized lipase was studied spec-
trophotometrically at 410 nm in triplicate by hydrolysis of p-nitrophenyl palmitate
ester (p-NPP) with the minor modification in reported procedure by Yea et al. [19]. In
standard condition, reaction mixture consists of the 1.5 mg of crude lipase (or equiv-
alent quantity of the ternary blend immobilized lipase) was taken into the 1.5 mL
of the cyclohexane. The reaction was started by addition of 0.5 mL of 25 mM, p-NPP
substrate at 37 ◦C. After 3 min the clear supernatant was withdrawn and 1.5 mL of
2 mM, NaOH was added in it so that p-nitro phenol (p-NP) was extracted in the
aqueous alkaline phase to give pale yellow colour [19]. It was then quickly used to
measure the optical density at 410 nm. Lipase activity was defined as mM of p-NP
released per min by per mg of lipase under given standard assay condition [19].
In the present study, we have characterized the ternary
blend film (by SEM, FTIR, % water content, protein content,
lipase activity) and then studied kinetics of geranyl acetate
synthesis which involves study of the influence of various reac-
tion parameters, energy of activation (Ea), half life time (t1/2),
2.3.6. Determination of protein content
The amount of immobilized enzyme on the ternary blend was determined by
following Folin-Ciocalteu method at 660 nm [2]. The initial amount of protein used
to load on support was determined. Finally, after removal of ternary blend immo-
bilized lipase polymer; the Teflon petri dish was rinsed and subjected to determine
un-immobilized amount of protein. Thus, amount of protein immobilized is the
difference between the initial amounts of protein used to load on support and un-
immobilized amount of protein found after washing petri dishes. BSA was used as
an internal standard to construct the calibration curve. % Protein loading, specific
activity and activity yield was determined by following equations:
deactivation rate constant (K ) for various solvents and kinetic
d
parameters (Vmax, Ki(A), Km(A), Km(B)). Furthermore alkyl ester
and alcohol chain length effect was studied to understand the
influence of the chain length on immobilized lipase catalytic
activity.
%
%
protein loading = amount of protein immobilized ÷ initial amount of protein loaded
activity yield = immobilized lipase activity ÷ crude lipase activity
2. Materials and methods
2.1. Enzymes and chemicals
specific activity = lipase activity ÷ lipase protein content
Candida rugosa (CRL, Lipase AYS, ≥30,000 U/g) and Pseudomonas cepacia lipase
(
PCL, lipase PS, ≥23,000 U/g) was gifted by Amano Enzymes (Nagoya, Japan) while
Rhizopus oryzae (ROL, lipase ROL, ≥30,000 U/g), PVA (Mw – 9000–10,000), CH
2.3.7. Effect of the organic solvent on activity and stability of immobilized lipase
The effect of various five organic solvents on the activity and stability of
CH:PVA:PLA:PCL (1:6:1:2) immobilized lipase was determined. The immobilized
lipase was placed in various five organic solvents at 140 rpm for incubation. After
incubation, the solvent was removed by filtration and lipase activity was determined
for immobilized lipase PCL as above indicated procedure.
(
Brookfield viscosity > 200.0 cps), p-NPP were purchased from Sigma–Aldrich Pvt.
Ltd., India. Bovine serum albumin (BSA) and Folin-Ciocalteu reagent to measure
protein content was purchased from Hi Media Pvt. Ltd., India. Other all chem-
icals were purchased from S.D. Fine Chemicals Ltd. with their highest purity
available.
2
.3.8. Determination of the half-life time (t1/2) and deactivation rate constant (Kd)
2.2. Immobilization of lipase
In another set of experiments, half-life time (t1/2) and deactivation rate constant
Kd) was determined for immobilized lipase in different organic solvents using lipase
(
Preparation of support matrix ternary blend film of PVA, CH, and PLA was carried
activity assay.
out with a marginal modification in the method described by the Grande et al. [16]
and subsequently lipase PCL was immobilized. The PVA (300 mg) was dissolved in
distilled water (2%, w/w solution) while CH (50 mg) was dissolved in distilled water
2.4. Experimental setup and analysis
(
1%, w/w acetic acid solution) and PLA (50 mg) was dissolved in chloroform (2%, w/w
Geranyl acetate synthesis involves the addition of geraniol (1 mmol) and vinyl
acetate (4 mmol) in a 10 mL glass reaction vessel of 1.6 cm i.d. with a glass lid. The
reaction mixture was diluted by toluene to make volume of 3 mL. Later on, 50 mg of
solution) separately. Each solution was stirred for the 40 min at 1000–1200 rpm.
Initially PVA and CH were mixed together vigorously for 30 min at speed of 2000 rpm,
later on PLA solution was added in it and stirred vigorously for 2 h. On completion
of 2 h, native lipase (100 mg dissolved in 1–2 mL of deionised water) was added to
the ternary blend and moderately stirred at 120 rpm for 50 min. The mixture was
carefully poured in a Teflon dish and allowed to dry at 40 C for 40 h. A thin film of
immobilized lipase PCL was formed which was then cut into small pieces of 2–4 mm
1
◦
immobilized lipase PCL was added to initiate the reaction and was placed at 55 C in
orbital shaker with an agitation speed of 140 rpm (Scheme 1). Reaction mass sample
10 l was withdrawn periodically and analyzed by using the Perkin Elmer, Clarus-
400 gas chromatograph equipped with flame ionizing detector and capillary column.
◦
2
◦
◦
−1
The oven temperature was kept at 80 C for 3 min with a rise of 10 C min up to
◦
240 C for 30 min. The temperature of the detector and injector was maintained 260
◦
00 mg of crude lipase.
and 60 C respectively.
Please cite this article in press as: Badgujar KC, Bhanage BM. 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 (2014),