International Journal of Pharmacy Research and Technology

2013, Volume 3, Issue 2, 20-25

ISSN 2250 – 0944 (Online)
ISSN 2250 – 1150 (Print)
Research Article

Immobilization and Characterization of Porcine pancreas lipase on “Gellasan”

Solanki S N1*, Pokharkar V B2, Solanki N R1

1
Department of Biotechnology, Vidyabharati Trust College of Pharmacy, Umrakh,Ta: Bardoli, Dist: Surat,
b
Department of Biotechnology, Poona College of Pharmacy, BharatiVidyapeethUniversity, Erandwane, Pune, Maharashtra.
* Corresponding AuthorE-mail: sonalpawar_life@yahoo.comMob No: +919016454096
Received: 20/05/2013, Revised: 25/06/2013 Accepted: 30/06/2013
ABSTRACT
Porcine pancreas lipase was immobilized by entrapment in ‘Gellasan’ beads, a polyionic hydrogel obtained by
complexation between gellan gum and chitosan. The activity of free and immobilized lipase was assayed in aqueous and
organic solvents using p-NPP as the substrate. The comparison of activities of free and immobilized lipase in aqueous and
organic media resulted in higher activity in the aqueous media. Amongst the organic solvents, non-polar solvents like
cyclohexane, carbon tetrachloride and n-heptane gave higher activity than polar solvents. Similarly, the amount of added
water upto 100 µl in the organic medium of cyclohexane increased the reaction rate of the immobilized than free. The
immobilized enzyme exhibited a shift towards alkaline pH for its optimal activity. The apparent optimum temperature for the
immobilized enzyme was 8-100C higher than that for the free enzyme, stabilizing the immobilized enzyme against heat
treatment and thereby improving its thermal stability. The optimum loading obtained was 500 mg lipase/g of the support.
The immobilizate showed a decent operational stability retaining almost 50% activity in aqueous medium and 40% activity
in organic medium after four reuses. The storage stability was remarkable too since, the enzyme retained 25% activity even
after three months at room temperature.

Keywords: Gellasan; Lipase Assay; non-conventional media; protein loading; Enzyme stability

INTRODUCTION disadvantageous[12,13]. Thus, hydrogels are the ideal

Numerous advances have been made in the techniques
of enzyme immobilization since 1960’s in various areas
like food industry, industrial chemistry, medicine and
modern diagnostics [1,2]. Enzymes display a great specificity
and are not modified permanently in the reaction mixtures
which makes them cost-effective and to be used more than
once. However, if they are in the solution with reactants
and products it is much difficult to separate them. Hence,
immobilization not only permits the easy biocatalyst-
product separation but also improves the operational
performance of the enzyme thus accruing economical
advantage [3].
Gel entrapment is the most widely used technique
since, it does not alter the enzyme structure and thereby
retaining its activity and stability. It involves encapsulation
of the enzyme within a gel matrix. This immobilization
technique consists of enclosing the enzyme in an aqueous
solution inside a semipermeable membrane capsule[4-7].
Mainly, there are two advantages of this method of
immobilization. Firstly, the particle structure allows contact
between the substrate and enzyme. Secondly it is possible
to immobilize several enzymes at the same time[8,9]. But,
most entrapment techniques have 2 major limitations viz.
(1) they entrap the protein with low entrapment efficiency
(2) they frequently require the use of volatile organic
solvents which may alter the structure and function of
proteins. Several researchers have also reported low
enzyme activity and low enzyme loading[10,11]. Inspite of
these limitations, researchers favour this technique of
immobilization due to the fact that hydrogels provide
maximum stabilization to the active structure and are easily
wetted allowing the free access to the substrate. Generally
hydrophillic supports enhance the enzyme stability while
immobilization on hydrophobic matrices appears to be
supports for immobilization thus providing less expensive,
more stable and reusable alternatives to the free enzymes.
Lipases (triacylglycerol ester hydrolase, EC 3.1.1.3 )
are very significant enzymes from industrial, therapeutic,
and biotechnological point of view. The vast potential of
these biocatalysts for industrial applications has been
recognized till date. Till now, lipases are the only enzymes
which have attracted considerable attention, showing
significant activity in organic media, since they are widely
used to catalyze the esterification andtransesterification
reactions.
A promising property of lipases is their activation in
presence of hydrophobic interfaces which was first reported
by Sarada and Desnulle[14].
Natural polysaccharides and their derivatives are the
polymers widely used in pharmaceutical formulations.
These polymers also play a fundamental role in determining
the release rate from the dosage forms[15]. With this regard,
chitosan has been utilized as its biochemical properties
have proved its suitability as a sustained release matrix
[16,17]
. Chitosan [poly (β-(1→4)-2amino-2dedoxy-d-
glucose)] is a natural cationic polysaccharide derived from
chitin which is a copolymer of glucosamine and N-acetyl
glucosamine units combined together.
In our previous studies we have already found that
gellan and chitosan are excellent matrices for the
immobilization of lipase. We report here the studies carried
out on ‘Gellasan’ which is a polyionic hydrogel, a solid
porous matrix which was used to immobilize the enzyme
lipase in our lab. Gellasan has been formed by the ionic
bondings as well as the Vanderwaal’s interactions between
the two polymers gellan and chitosan[18]. Gellan is an
extracellular polysaccharide produced by Pseudomonas
elodea and is composed of tetrasaccharide repeat units: D-

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glucan, D-glucoronic acid, D-glucose and L-rhamnose in
the molar ratio of 2:1:1. Gellan gum is safe for human use
and is widely being used in the food industries [19].
The fact that lipases are activated in presence of
aqueous/hydrophobic interfaces has envisaged us to find
out the potential of lipase immobilization on hydrogels. In
our previous work, we immobilized lipase successfully on
Gellasan: a hybrid matrix of gellan and chitosan and studied
their characterization, entrapment, swelling and release
behaviour by analyzing their activity in aqueous medium.
The aim of the present study is to investigate the
enzyme loading capacity, physical, thermal and operational
stability of the immobilizate, comparison of activities in
aqueous and organic solvents and effect of water content on
lipase activity in gellasan.
MATERIALS AND METHODS
Materials
Porcine Pancreas lipase (PPL, Type II, crude, activity
on olive oil 186 IU/g) (EC 3.1.1.3) and para-
nitrophenylpalmitate (p-NPP) & BSA were purchased from
Sigma Chemical Co. (St. Louis, MO). Gellan Gum
(Kelcogel F- Low acyl) was kindly donated by C.P Kelco
Pvt. Ltd, Mumbai, India. Chitosan sample (95%
deacetylated) was a gift from A.E. Connock Pvt. Ltd,
England. Tris base was purchased from Himedia, Mumbai.
Folin Reagent, Acetic acid, sodium potassium tartarate,
cyclohexane, n-heptane, carbon tetrachloride, acetone,
ethanol were all products of Merck, Mumbai.
Preparation of Gellasan beads
The hydrogel beads were prepared as follows: To the
hydrated and preheated aqueous solution of 1.5%w/v gellan
gum which was cooled to 450C, a dispersion of 0.5g lipase
powder in 1ml 0.05M TrisHCl buffer, pH 7.4 was added.
This mixture was stirred and extruded dropwise using a
10ml plastic syringe of bore size 18G into the chitosan
solution cooled at 50C, kept on a controlled speed stirrer
(Eurostar Power control-visc, IKA, Labortechnic, Staufen,
Germany). The beads were cured for 30 mins in the same
solution after which they were separated by filtration.
Washing was completed twice using DI (deionized) water
and the beads were air-dried. The filtrate and the washings
were collected for the lipase loss measurements.
Lipase Assay
In Aqueous media
The hydrolytic activity of lipase was measured using
para-nitrophenylpalmitate (p-NPP) as substrate by
Pencreac’h and Baratti method 28. In brief, 20 mg beads
were added to a reaction mixture containing 3ml p-NPP
solution (0.04 M in 2-propanol), 1ml 0.05M TrisHCl buffer
(pH 7.4 at 370C) and 3ml DI water. A negative control
(placebo beads) and a positive control (10 mg free lipase)
were also studied simultaneously and the reaction mixture
was incubated for 30 mins in an incubator shaker
(Brunswick, Germany) at 50 rpm. The activity was
calculated at 410 nms. One enzyme unit was defined as the
amount of protein liberating 1µmol of para-nitrophenol (p-
NP) per minute in these conditions. The entrapment, release
and activity studies were carried out in triplicate.
In Organic media
The reaction rate of lipase preparation was determined
according to Pencreac’h and Baratti with only slight
modifications. In standard conditions, the reaction mixture
composed of 2ml of cyclohexane containing 0.4mM p-NPP
after screening other organic solvents like n-heptane,
carbon tetrachloride, acetone and cyclohexane for which
maximum activity was obtained. The reaction was started
by addition of 10 mg free lipase and 50 mg immobilized
lipase. The mixture was incubated at 37oC for 30 mins in an
incubator shaker with 50 rpm. Then, the agitation was
stopped, and the lipase powder/ beads were allowed to
settle for 30secs and 50µl of the clear supernatant was
withdrawn and immediately mixed with 1ml of 0.1M
NaOH, directly in a 1ml cuvette of the spectrophotometer.
The pNP liberated was extracted by the aqueous alkaline
phase. It displayed yellow colour because of alkaline pH.
The absorbance was read at 410nm against a blank treated
in parallel. The molar extinction coefficient of pNP was
determined at 18.5 × 103/M/cm by using standard solutions
of pNP in cyclohexane as described previously.
Effect of loading on rate of hydrolysis
The aim of this study was to obtain an immobilized
biocatalyst with the highest activity and to minimize the
mass transfer limitations. Hence, varying concentrations of
lipase ranging from 10mg to 1g (1.109U/mg) were
immobilized on the beads to obtain a maximum loading.
The effect of loading on the rate of p-NPP hydrolysis by the
immobilized lipase was observed. Higher loading was seen
to visualize the effects on the mass transfer limitations.
Effect of pH on lipase activity and stability
To check the activity and stability of the immobilized
enzyme, 10 mg of free lipase and 20 mg of the beads were
suspended in buffer solutions of respective pH from pH 2 to
9 at 37oC for 30 mins on an incubator shaker at 50 rpm. The
activity of free and immobilized lipase was assayed by
using p-NPP as substrate.
Effect of temperature on lipase activity and stability
The free and immobilized placed in the buffer
solutions of optimum pH and incubated at various
temperatures ranging from 20 to 80oC for half an hour.
Effect of aqueous and organic medium on lipase activity
The hydrolysis of p-NPP, a well-known lipase
substrate was selected as a model reaction in the present
work. This choice allowed a direct comparison of the lipase
activity in aqueous and organic medium.Assay in the
organic medium was carried out in cyclohexane as
described previously. A range of organic solvents from
non-polar to polar were screened to check the influence of
organic solvents on the activity of free and immobilized
lipases. For this, 10 mg free lipase and 20 mg of
immobilized lipase were subjected to the same assay using
respective solvents viz. n-heptane, carbon tetrachloride,
acetone and methanol in the same manner. The caliberation
curve was plotted previously in all the solvents and the
molar extinction coefficients determined.
Effect of added water on lipase activity in organic
medium
The effect of water on lipase catalyzed reactions in
organic solvents has been studied extensively. Different
amounts of water ranging from 10µl-500 µl was added into
the reaction mixture with 10 mg free and 20 mg
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3 20 -25

immobilized lipase beads and the lipase assay using p-NPP

p become a problem. Hence, the further experiments were
in cyclohexane as substrate was carried out as described carried out using 0.5g protein/g of support.
previously.

Operational stability
bility of the immobilized beads
The operational stability of the immobilized enzyme is very
important economically
onomically and an increased stability could
make an immobilized enzyme more advantageous than its
free counterparts. To investigate the operational stability of
lipase on the immobilized system, 50 mg the beads of plain
gellan and gellan-chitosan beads weree used repeatedly in
batch reactions for hydrolysis of p-NPP NPP in successsive
cycles. At the end of each cycle, the immobilized beads
were removed from the reaction mixture and washed with
deionized water to remove any product or substrate
adhering to the beads.
ads. The beads were again introduced in
the fresh medium and reassayed. Activities were estimated Fig. 1Efficiency
Efficiency plot showing the effect of loading on the
at the end of each cycle and expressed as µmol/min/mg rate of hydrolysis.
catalyst. Similar process was carried out in the organic
medium. Effect of pH on n lipase activity and stability
pH is one of the vital factors affecting the enzymatic
Storage stability of the immobilized beads activities by altering the enzyme conformation
conform in the
The stability of an enzyme is of significant importance for aqueous solutions. Relativeve activity as a function of pH is
scheduling its application for a particular reaction. The depicted in Fig. 2A. The effect of pH on the activity of both
immobilized enzyme and the free enzyme were stored in the free and immobilized lipase is compared. It was found
the dry state at different temperature conditions viz. 40C, that the optimum pH value for the free lipase is about 7
Room Temperature and 40oC. Similarly at different time whereas the pH of the immobilized lipase shiftedsh to the
intervals, the free and the immobilized enzymes were stored alkaline side at about pH 7.5 to 8. But the stability of the
for 8 days upto 3 months and the activity was assayed at immobilized enzyme (Fig 2B) is found to be in the pH
RT. range of 5.5-7.5.
7.5. Similar it is noticeable that immobilized
enzyme shows 90-95% 95% activity, while the free enzyme
Table 1Effect of enzyme concentration
centration on lipase activities retained only 70% activity. This was in accordance with
Enzyme offered studies carried out by Bagi et al (1997).
Specific
(mg protein/g of Activity (U/mg)
activity
support)
10 0.9619 0.120
50 1.3 0.162
100 1.46 0.183
200 1.60 0.201
500 2.2 0.275
1000 1.85 0.232

RESULTS AND DISCUSSION

Effect off loading on rate of hydrolysis
The effect of loading on the rate of hydrolysis on the
immobilized lipase is illustrated in Fig1. The reaction rate
increased linearly upto 500 mg of the loaded lipase on the
support and thenafter, it decreased. At 500mg protein/g of Fig. 2A Effect of pH on lipase activity
support, the loading iss high enough to achieve a maximum
activity. But higher loadings led to lower activities. This
might be due to the internal diffusional limitations.
Similarly, when converted into efficiency plot
(activity/loading) (Fig. 1), lower efficiencies were obtained
obtaine
at higher loadings. This may be due to the uneven
distribution of the enzyme in the gel, possibly blocking the
access of enzyme to the active sites [21]. Another interesting
observation (Table 1) is that, there is a constant increase of
the specific activity
vity of the immobilized lipase since a
constant or decrease in specific activity has been reported
by other investigators [22,23]. Similarly, it is more obvious to
look at the specific activity at optimal loading. At low
protein loading, the enzyme may not gain an access to the
active sites on the substrates resulting into a low activity
and with higher protein loadings mass transfer limitations Fig. 2B Effect of pH stability on lipase activity

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Pharmacy Research & Technology 2013 3(2)
3 20 -25

The pH stability of the immobilized enzyme showed
increase in stability because of the partition effects. The
internal diffusion effects in the microenvironment of the
enzyme as well as the conformational effects may be
responsible for increasing the stability on the alkaline
side[24].
altering the conformational stability of the enzyme
molecule and thus resulting into enhanced activity. Our
results were in agreement with Magnin et al.(2003) who
showed that activity of free lipase was more in aqueous
medium[25].

Effect of temperature on n lipase activity and stability

The knowledge of thermal stability of the immobilized
enzyme is useful in exploring the potential applications of
the enzyme. Relative Activity as a function of time is
illustrated in fig 3A. It is observed that the optimum
temperature of the free lipase is 26-28 26 0C while
immobilization shifts the optimum temperature of lipase
from about 30 to 400C. Similarly, it is seen (Fig. 3B) that
the activity of free lipase is more at 300C than the
immobilized but the stability of the immobilized
counterpart seems to be far more than the free. It may be
explained as: incase of temperature profile, immobilized
enzyme is degraded faster due to the exposure of the
Fig. 4 Effect of aqueous and organic medium on lipase
enzyme on the surface of the matrix to high temperatures.
activity
But after 1 hour, the enzyme inn the core of the matrix which
is naturally less exposed shows more stability than the free.
Similarly, amongst the organic solvents used, the non-
non
Thus, immobilized lipase is less prone to denaturation due
polar solvents viz. cyclohexane, carbon tetrachloride and n-
n
to temperature making the gellasanimmobilizate an
heptane gave a relatively higher activity than polar solvents
attractive matrix for enzyme entrapment.
like acetone and ethanol. This can be explained as polar
solvents were reported to sustain poor enzyme activity
because they disrupt the essential water layer
l around the
enzyme[26].
The better performance of the immobilized enzymes
over the free may be ascribed to the more favourable
diffusion and repartition of substrates and products to the
enzyme.

Effect of added water on lipase pase activity in organic

medium
The rate of p-NPP
NPP hydrolysis is shown in the Fig. 5 at
different amounts of water (10-- 500µl) added into the
Fig. 3A Effect
ect of temperature on lipase activity reaction mixtures of both free and immobilized systems. It
was observed that with no addition of water, the rate of
hydrolysis was significant. Thus, the amount of water in the
system was enough for the high enzyme activity. The
increment of the amount of water from 10-500
10 µl added led
to an increase in the activity of both free and the
immobilized lipase. This change in lipase activity
activi could be
attributed to the change in the lipase conformation to a
more flexible one, and easy to access the substrate for the
reaction. Also, water might affect the reaction rate by its
direct participation as a reactant in the hydrolysis
reaction[27].

Fig. 3BEffect
Effect of temperature stability on lipase activity

Effect of aqueous and organic

ganic medium on lipase activity
The effect of organic solvents on enzyme has been the
focus of numerous studies. Similarly, non-polar
non solvents
are believed to be well suited for biocatalysis. It was found
in Fig.4 that the activity in the aqueous phase is
significantly more as compared too any of the organic
solvents used. This may be attributed to the fact that water Fig. 5:: Effect of added water on Lipase activity in organic
plays a major role of molecular lubricant and thereby medium.

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3 20 -25

It was also seen that the increase in the activity was not at the immobilized system offers good storage stability at
the same rate for the free and the immobilized lipase room temperature up to 3 months.
preparations. The relative increase in activity was higher for
the immobilized
mobilized lipase than for its free counterpart. This Table 2Storage
Storage stability of free and immobilized at
could be attributed as the support being hydrogels absorb different temperatures.
more water hence the immobilized system shows better Temp Initial Activity Activity (U/mg) after
activity than its free counterparts[35]. (0C) (U/mg) 8days 1month 3 months
Free 159.52 - - -
Operational Stability Studies 4
Immobilized 284 - - -
Immobilized system offers an advantage of reusability. Free 709 225 NA NA
Fig. 6A and 6B show the effect of repeated use on the RT
Immobilized 683 540 403 118.5
activities of immobilized enzymes on both plain gellan and Free 141.6 - - -
Gellasan beads both in aqueous and organic media. It was 40
Immobilized 157.6 - - -
found that Gellasanbeads retain nearly 50% activity
activit viz.
47% in aqueous and 41% in organic medium after 4 cycles.
While plain gellan beads after 4 cycles show almost a
decline in activity with 28.22% in aqueous and 15% in
organic medium. The results pointed out to the
inactivation of the enzyme and leakage
age of protein from the
support on subsequent reuse. It can also be seen that the
gellasan beads are more efficient than the plain gellan beads
this may be due to the better crosslinking which retains
more enzyme on the support thereby giving more activity.
activity
Thus, reusability was more efficient in aqueous medium.

Fig. 7 Storage stability of free and immobilized at room

temperature

Fig. 6AOperational
Operational stability of gellan-chitosan
gellan and plain
gellan beads in aqueous medium.
Fig. 6B Operational stability of gellan-chitosan
gellan and plain
gellan beads in organic medium.
Storage Stability
The stability of an enzyme is of prime importance in
all biotechnological processes. It is found that at 40C and
400C, the free and the immobilized enzyme enzyme retains
only one-fifth
fifth of the total enzyme activity than at room
temperature. Similarly
arly 80% activity is retained after 8 days
for immobilized enzyme. Also, one-fourthfourth of activity is
retained in the immobilized system while no activity is
obtained for free enzyme after 3 months. This shows that,
CONCLUSION
Porcine pancreas lipase entrapped in Gellasan beads
was found to be superior to the free enzyme under all
conditions tested for enzyme stability and efficacy. The
hydrolysis reaction of p-NPP
NPP in aqueous and organic media
by the immobilized enzyme were assayed
assa and compared
with those of the free enzyme. In aqueous medium,
maximum enzyme activity was obtained since, water plays
a major role of molecular lubricant thereby maintaining the
conformational stability of the enzyme molecule and thus
resulting into enhanced
nhanced activity. Similarly, amongst organic
solvents, hydrophobic solvents showed higher activity than
the polar solvents as they seem to disrupt the essential
water layer around the enzyme. Significant rate of
hydrolysis was achieved in aqueous medium with wi no
addition of water suggesting that the amount of water in the
system was high enough for enzyme activity. On the other
hand, in organic medium water added upto 100µl led to an
increase of lipase activity due to the change in lipase
conformation. It was as found that Gellasanbeads retain
nearly 50% activity viz. 47% in aqueous and 41% in
organic medium after 4 reuses. Thus, immobilization
improved the operational stability of Porcine pancreas
lipase which justifies the use of this immobilizate in
industrial
al bioreactors instead of the soluble lipase.
Immobilized system showed no significant loss of enzyme
activity, when stored at Room Temperature for three
months; with maximum enzyme activity (118.5 IU), as
compared to 4ºC and 40ºC.
Although several types of natural and synthetic gels
are used for protein immobilization, one still looks for
matrices that are cheaper, biocompatible and do a
reasonably good job. In our estimates, a matrix with a good
activity in aqueous as well as organic media, a decent
release,
ase, and optimum storage and operational stability will

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 Solanki et al / International Journal of Pharmacy Research & Technology 2013 3(2) 20 -25
always have an edge over other matrices. The hybrid
polymer is further explored for kinetic studies.
ACKNOWLEDGEMENTS
We thank A.E. Connock Pvt. Ltd, England and C.P
Kelco Pvt. Ltd, Mumbai, India for providing gift samples of
Chitosan and Gellan Gum respectively. We also gratefully
acknowledge Dr. (Mrs.) AA. Prabhune, NCL (National
Chemical Laboratory, Pune) for necessary suggestions. We
extend a deep gratitude to Mr. RajdeepDongre (Agharkar
Research Institute, Pune) and Mrs. Kulkarni (Pune
University, Pune) for Scanning electron microscopy.
Financial assistance for this work was provided by AICTE
(All India Council for Technical Education, India).
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