G Model
PRBI-10411; No. of Pages13
K.C. Badgujar, B.M. Bhanage / Process Biochemistry xxx (2015) xxx–xxx
2
industrial process economics because of poor solvent-, thermal-
and operational stability [10–12]. To conquer above said limita-
tions, various advanced immobilization methods were developed
of various enzymes [11–14]. Among above immobilization pro-
tocols, the use of natural polymer matrices for immobilization is
preparation of such type of active matrices can be achieved by dif-
ferent immobilization techniques such as entrapment, adsorption,
ionic-binding, covalent-linkage, etc. which leads to form a highly
such as cellulose, carrageenan, alginate, -glucan, poly (hydroxy-
butyrate), hydroxypropyl methyl cellulose, polylactic acid etc. were
employed in the form of spherical beads or films for immobilization
of different enzymes [11–16].
response surface methodology (RSM) is a mathematical and multi-
variate statistical technique to optimize the process, which is aimed
to reduce the cost of expensive analysis methods [24]. To the best
of our knowledge, such type of membrane or polymer base biocat-
alyst (PVA:CHI:lipase) for their potential biocatalytic applications
in SC-CO2 was not explored in details, which inspire us to explore
the influence of various reaction parameters on the enzyme activ-
ity in SC-CO2 and corresponding synthetic applications of designed
biocatalyst. In present study, we (i) synthesized biocatalyst, (ii)
characterized it, (iii) used for citronellyl laurate synthesis using
RSM, (iv) explored various substrate scope as well as recyclabil-
ity in SC-CO2, and (iv) finally studied effect of SC-CO2 parameters
on the immobilized biocatalyst activity.
2. Materials and methods
used as an attractive immobilization support. PVA was extensively
used in bio-medical applications as a potential bio-polymer with
excellent properties such as biocompatibility, high adhesivity, high
flexibility, high tensile strength, non-toxicity, better resistivity to
chemicals and organic solvent [17]. CHI is a linear -1,4-linked
polysaccharide matrix which possess various useful features such
as high mechanical strength, inertness to chemical reactivity, good
adhesion, lack of toxicity and biodegradability which can hold
up enzyme immobilization ability [18]. Both of these polymers
possessing the hydroxyl group while, free-amino groups of each
unit of CHI offer a higher extent of immobilization [17,18]. This
PVA:CHI:BCL biocatalyst was then applied to synthesize citronellyl
laurate as a biocatalytic application. The Commission of European
community has restricted the use of volatile organic solvents for
synthesis of the food ingredients to maintain its purity, which
appeals to researchers to look for alternative greener solvent for
synthesis of valuable food ingredient additives/commodities [19].
Use of these volatile organic solvent is restricted as, these sol-
vents are the major source of volatile organic compounds (VOCs)
which severely affect the environment and human health [20]. The
utilization of the volatile organic solvent can cause inhibition of
enzyme catalytic activity, difficulty in use because of flammable
nature, expensive down-streaming process and increment of E-
factor (E-factor is defined as the ratio of mass of waste generated per
unit of desired product; higher the E factor more is the waste and
subsequently, having negative environmental impact) [17,20,21].
Hence, use of supercritical carbon dioxide (SC-CO2) as a solvent
is the best alternative for synthesis of drug and food-additives
over, employment of the SC-CO2 has been accepted as a ‘Clean
and Green’ solvent with noteworthy potential for commercial pur-
reduces work-up procedure and provide final product by simple
depressurization [20–22]. Additionally, this SC-CO2 is available in
large quantity in environment and SC-CO2 is a low-viscous sol-
vent which may endorse easy mass-transfer phenomenon between
reaction mass and active sites of catalyst [17–22]. Thus, use of
enzyme catalysis in SC-CO2 is extremely attractive system and can
be considered as an “Eco-friendly and Safe” technique [17–22].
Citronellyl laurate is a colourless liquid having pleasant fruity
Citus (lemon type) aroma which is widely used in pharma, cosmet-
ics, emulsification, perfumery and food-flavour ingredient. In 2009,
the overall international estimated market for the essential food,
flavour and fragrance was nearly 20 thousands million USD, which
increases upto 24 thousands million USD by 2013 [23]. Hence,
in view of the present extensive scope and importance of these
valuable fatty-acids esters, we make an attempt to investigate the
synthesis of citronellyl laurate using PVA:CHI:lipase as an immobi-
lized biocatalyst in SC-CO2 by response surface methodology. The
2.1. Enzymes and chemicals
The lipase BCL (Burkholderia cepacia lipase, BCL), CHI (Brook-
field viscosity >200), PVA (Mw 9000–10,000), vinyl laurate (VL), and
p-nitro phenyl butyrate (p-PNB) citronellyl alcohol, bovine serum
albumin (BSA) and all other solvents or chemicals were purchased
from Sigma–Aldrich Pvt. Ltd.
2.2. Immobilization of lipase
The lipase was immobilized onto the PVA/CHI biocompatible
matrix simply in water as a greener solvent at room tempera-
ture (∼30 ◦C). The PVA (600 mg) was dissolved in distilled water
(40–50 mL) while CHI (400 mg) was dissolved in distilled water
(1.5%, w/w, acetic acid solution) in a separate beaker and stirred
at 1200–1400 rpm for 60 min. Both these solutions were then fil-
tered to remove the undissolved particles. Finally, both solutions
were mixed and stirred vigorously for 4–5 h at 1500–1600 rpm.
After that, parent lipase BCL (250 mg) was dissolved in deionised
water (6–8 mL) which was added to the PVA/CHI blend and stirred
it gently at 160–180 rpm for 60 min. The PVA/CHI/BCL immobi-
lized lipase blend was then carefully poured into a Teflon-dish and
allowed it to dry at 40–46 ◦C for 45–48 h. A uniform plane thin film
of PVA:CHI:BCL was formed, which was afterwards cut off into the
small pieces of 2–3 mm2 size and stored at 8–12 ◦C in freeze. Thus,
the theoretically 1000 mg (1 g) support was loaded by 250 mg of
native lipase, and this composition was denoted as PVA:CHI:BCL
(6:4:2.5) means PVA:CHI:BCL (600:400:250).
2.3. Characterization of immobilized lipase
2.3.1. Surface texture analysis
Scanning electron microscope (SEM) analysis was performed to
observe the change in surface texture of the control PVA:CHI and
immobilized PVA:CHI:lipase by the FEI-Quanta 200, instrument.
The film sample was placed on a carbon stub and images were
captured at 15–20 kV using LFD detector under the lower vacuum.
The film thickness of control support and immobilized lipase was
determined by using a manual micrometre at 8–10 random places
of films.
2.3.2. TGA analysis
The thermo gravimetric analysis (TGA) was performed using Q-
series 600 analyzer; for this 7–8 mg of sample was kept in ceramic
crucible and the analysis was examined from 30 to 600 ◦C with
10 ◦C/min rise in temperature, under the 99.99% pure nitrogen
atmosphere with flow of 100 mL/min. The reference control run
was performed with an empty sample crucible pan.
Please cite this article in press as: Badgujar KC, Bhanage BM. 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