See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/234022975

Immobilization of Lipase by Entrapment in Ca-alginate

Beads

Article  in  Journal of Bioactive and Compatible Polymers · November 2008

DOI: 10.1177/0883911508097866

CITATIONS READS

52 366

4 authors:

Indu Bhushan Rajinder Parshad

Kurukshetra University Indian Institute of Integrative Medicine
19 PUBLICATIONS   151 CITATIONS    71 PUBLICATIONS   774 CITATIONS   

SEE PROFILE SEE PROFILE

Ghulam Nabi Qazi Vijay Kumar Gupta

Jamia Hamdard University Kurukshetra University
359 PUBLICATIONS   7,119 CITATIONS    87 PUBLICATIONS   1,445 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

invitro cell culture and anticancer View project

Development of Herbal Based Bioprotective Agents Targeting Selected Antibiotic Resistant Microorganisms View project

All content following this page was uploaded by Vijay Kumar Gupta on 05 February 2016.

The user has requested enhancement of the downloaded file.

 Journal of Bioactive and
Compatible Polymers
http://jbc.sagepub.com

Immobilization of Lipase by Entrapment in Ca-alginate Beads

Indu Bhushan, Rajinder Parshad, Ghulam Nabi Qazi and Vijay Kumar Gupta
Journal of Bioactive and Compatible Polymers 2008; 23; 552
DOI: 10.1177/0883911508097866

The online version of this article can be found at:

http://jbc.sagepub.com/cgi/content/abstract/23/6/552

Published by:

http://www.sagepublications.com

Additional services and information for Journal of Bioactive and Compatible Polymers can be found
at:

Email Alerts: http://jbc.sagepub.com/cgi/alerts

Subscriptions: http://jbc.sagepub.com/subscriptions

Reprints: http://www.sagepub.com/journalsReprints.nav

Permissions: http://www.sagepub.co.uk/journalsPermissions.nav

Citations http://jbc.sagepub.com/cgi/content/refs/23/6/552

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 Immobilization of Lipase by
Entrapment in Ca-alginate Beads

INDU BHUSHAN, RAJINDER PARSHAD* AND GHULAM NABI QAZI

Indian Institute of Integrative Medicine (CSIR), Canal Road
Jammu-Tawi, (J&K) India 180001

VIJAY KUMAR GUPTA

Kurukshetra University, Kurukshetra, India 136119

ABSTRACT: The lipase-producing strain, Arthrobacter sp. (ABL), isolated was

immobilized in Ca-alginate beads by entrapment. The alginate beads were
prepared as an aqueous mixture of sodium alginate, the cells and CaCl2 to
increase its reusability, and overall enzyme stability. Various parameters like
alginate and CaCl2 concentration, lipase units loading and bead size were
evaluated for optimum immobilization yield. It was observed that with the
increase in alginate concentration, the yield of immobilized enzyme also
increased up to a limit. A similar pattern was observed with CaCl2 addition;
the optimum concentrations of alginate and CaCl2 observed were 1.5% (w/v) and
2%, respectively. The concentration of enzyme entrapped in the beads with
an activity of 5 units per gram of wet beads was obtained by the addition of
100 units in 10 mL of slurry; beyond this amount a very little increase in activity
was observed. The maximum immobilization yield was observed with a 1.2 mm
bead size; increased bead sizes decreased the yield of immobilization. After
optimization of all the parameters, a 40% yield of lipase (ABL) activity was
observed in the Ca-alginate beads. These lipase beads were used for 10 cycles for
the hydrolysis of triglycerides without any loss in activity. The entrapped lipase
was more stable over a wide range of temperatures, pH, and storage time as
compared to free enzyme.

KEY WORDS: lipase, triglyceride hydrolysis, Arthrobacter sp., enzyme

immobilization, enzyme entrapment, calcium/alginate beads, enzyme tempera-
ture stability, enzyme pH stability.

*Author to whom correspondence should be addressed.

E-mail: rparshad@iiim.res.in, Rajan_basohli@Yahoo.co.in
Figures 1–4 appear in color online: http://jbc.sagepub.com

Journal of BIOACTIVE AND COMPATIBLE POLYMERS, Vol. 23—November 2008 552

0883-9115/08/06 0552–11 $10.00/0 DOI: 10.1177/0883911508097866
ß SAGE Publications 2008
Los Angeles, London, New Delhi and Singapore

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 Immobilization of Lipase by Entrapment in Ca-alginate Beads 553

INTRODUCTION

L ipase (triacylglycerol acylhydrolase, E.C. 3.1.1.3) is a ubiqui-

tous enzyme of considerable physiological significance and
industrial potential. Lipases are widely used in the processing of fats
and oils, detergents and degreasing formulations, food processing,
synthesis of fine chemicals and pharmaceuticals, paper manufacturing,
and in the production of cosmetics [1–4]. Lipases have emerged
as important biocatalysts in stereoselective reactions, having great
value in pharmaceutical industries, especially in enzymatic resolution of
racemic drugs [5–7].
Despite widespread research efforts in academics and industry, the
number and diversity of biocatalysts applications remain rather modest.
This may be attributed to several perceived limitations of biocatalysts,
such as, their high cost, instability, availability in small amount, and
operational stability. The operational stability of enzymes has been
improved steadily over the years through the use of genetic engineering,
immobilization, or process alterations. Enzyme immobilization is an
important strategy to impart the desirable features of conventional
heterogeneous catalysts on to biological catalysts [8–14]. Besides
enhancing stability, enzymes can acquire additional advantageous
properties via immobilization: (a) immobilized enzymes can be used
repeatedly or continuously in a variety of reactors and (b) can be
easily separated from soluble reaction products and unreacted sub-
strate, thus simplifying work-up and preventing protein contamination
in final products.
Immobilization often stabilizes an enzyme, thereby, allowing their
applications under harsher environmental conditions, such as pH,
temperature, and organic solvents. The basic concept for enzyme
immobilization is either to covalently attach or entrap the protein in
support materials; this prevents the enzyme from leaving while allowing
substrates, products, and co-factors to permeate to the enzyme [15].
Entrapment has been more extensively used for the immobilization
of cells than for enzymes. Commonly used matrices for entrapment
are polyacrylamide, calcium alignate, sol–gel powder, and cellulose
acetate [16–19]. Lipase from Candida rugosa was also immobilized in
the polymer of poly(vinyl alcohol), alginate, and boric acid. This
immobilized enzyme was reused for 10 cycles without any loss in
activity [17]. Candida rugosa lipase was also immobilized in Ca-alginate
beads and used in the hydrolysis of oil and grease originating from pet
food industrial wastewater [20]. Different matrices, such as agar,
alginate, and polyacrylamide, were employed to immobilize whole

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

554 I. BHUSHAN ET AL.

Ralstonia pickettii cells that had an optimal production and lipase

activity in 4% alginate beads [21].
In the present study, Arthrobacter sp. (RRLJ-1/95, MTCC No.5125)
cells were immobilized by entrapment in Ca-alginate beads. The lipase
produced by this strain has wide substrate specificity and high
enantioselectivity in comparison to commercially available lipases like
CCL, PSL, and PPL [5] and has been immobilized on various matrices
and used for kinetic resolution of many chiral drugs intermediates
[7,22–24]. In this investigation, whole cells (ABL) were immobilized
and reused for many reaction cycles for the hydrolysis of triglyceride
using tributyrin as the substrate. These immobilized beads have
shown enhancement in pH, thermal and storage stability compared it
the free enzyme.

METHODS

Materials

Lipase (ABL) was isolated from Arthrobacter sp. (RRLJ-1/95, MTCC

No.5125) at the Indian Institute of Integrative Medicine (formerly
RRL), Jammu, India. Sodium alginate was procured from SD Fine
Chemicals Limited. Tributyrin, gum acacia, sodium hydroxide, peptone,
sodium chloride, beef extract, tris buffer, sodium dihydrogen phosphate,
disodium hydrogen phosphate, sodium acetate, acetic acid, and hydro-
chloric acid were obtained from Himedia, India. All other chemicals
and reagents were obtained from Merck, Inc.

PREPARATION OF ARTHROBACTER SP. (RRLJ-1/95)

CELL BIOMASS

The microbial strain Arthrobacter sp. was grown in a 20 L fermentor

(New Brunswicck scientific Co. USA), working volume15 L, for the cell
biomass preparation using medium comprising 1% peptone, 0.5%
sodium chloride, and 0.5% beef extract and pH adjusted at 7.0. The
fermentor was inoculated with seed culture 1% (v/v) 12-h-old, prepared
in the fermentation medium given above. The lipase producing
Arthrobacter sp. was grown in a fermentor by keeping the following
conditions; 500 rpm, air flow rate of 0.5 vvm, and temperature 308C for
21 h. After fermentation, the broth was centrifuged at 8000  g for
10 min at 48C (Beckman Coulter, Avanti J-201, USA); the cell pellet
obtained was removed and preserved at 208C till further use.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 Immobilization of Lipase by Entrapment in Ca-alginate Beads 555

ENTRAPMENT OF LIPASE (ABL) CELLS IN

CA-ALGINATE BEADS

Arthrobacter sp. cells were entrapped in Ca-alginate beads by mixing

50 mg lipase cell biomass (100 units) with 10 mL of sodium alginate
solution (1.5% w/v). The mixture was stirred and dripped through a
syringe into 10 mL of 2% CaCl2 solution so that Ca-alginate beads were
formed. The bead size was changed by using syringes with various
needle diameters. After 30 min of hardening in the same solution, the
beads were separated from the CaCl2 solution by vacuum filtration.
They were washed on a filter with 1% CaCl2 and then with 0.05 M Tris
buffer pH 7.5, so as to remove the loosely bounded enzyme. The filtered
CaCl2 solution and washings were collected and tested for lipase activity
to determine the binding efficiency. The immobilized beads were
checked for lipase activity using tributyrin as the substrate.

LIPASE ACTIVITY ASSAY (TITRIMETRIC METHOD)

Lipase activity was measured in a mechanically stirred emulsion of

tributyrin using pH stat equipment, which neutralized the fatty acids
released, by adding NaOH in order to maintain a constant pH end
point value. The titrable solution contained 13.3 mL of 1% tributyrin
in 1% gum acacia solution and 350 mL of 2% CaCl2. The reaction
mixture was incubated at 378C with constant agitation on a magnetic
stirrer and titrated against 10 mM NaOH. A unit of lipase was defined as
the lipase required to release 1 mM of fatty acid per minute from
triacylglycerols.

Operational Stability

The reusability of the entrapped lipase was analyzed using tributyrin

as the substrate, as given in ‘Lipase Activity Assay (Titrimetric
Method)’ section. Each cycle of the immobilized lipase was run for 1 h.
The residual activity was expressed as the percentage of the activity at
various cycles, in comparison to the first cycle.

Beads Size Measurement

The size of the air-dried beads was measured by using an optical

microscope equipped with a digital image analyzer. The diameter of each
bead was measured at three different angles and averaged.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

556 I. BHUSHAN ET AL.

Effect of Temperature

The thermal stability of both the free and immobilized lipase was
determined by measuring the residual enzymatic activity by pre-
incubating them at different temperatures ranging from 208C to
1008C for 1 h and assaying the residual enzyme activity at 378C by
titrimetric method using tributyrin as the substrate.

Effect of pH

The pH stability of both the free and the immobilized enzymes was
determined by pre-incubating the free and immobilized enzymes cell
samples ranging from 5–9 pH buffers (0.01 M sodium citrate, 0.01 M
sodium phosphate and 0.1 M Tris-HCl, respectively, at 258C) at various
time intervals from 12 h till 96 h. The residual lipase enzyme activity
was estimated by comparing the activity that of the first cycle.

Storage Stability

Immobilized lipase beads were stored at 4–208C for 1 month and

the residual lipase enzyme activity was analyzed after every 5 days by
titrimetry using tributyrin as the substrate.

RESULTS AND DISCUSSION

The production of Arthrobacter sp. (RRLJ-1/95) cells were done in a

15 L (working volume) fermentor using medium containing 1% peptone,
0.5% sodium chloride, and 0.5% beef extract and pH adjusted at 7.0.
The maximum cell biomass obtained after 21 h fermentation was 13 g/L.
This cell biomass was used for further study.

Effect of Alginate, CaCl2 Concentration and Enzyme Units

Loading on the Entrapment of Lipase in Ca-Alginate Beads

The cross-linking between sodium alginate and calcium ions leads to

gelation and the gel entrapment of lipase depended on the concentra-
tions of both these constituents. Therefore, the effect of alginate, CaCl2
concentrations, and enzyme-loading efficiency on the immobilization
yield was studied. Alginate concentration was optimized by preparing
beads using 0.5–2.5% (w/v) alginate, 2% CaCl2, and 100 units of lipase
(ABL) in 10 mL slurry to obtain 8.0 g of beads. The activity of entrapped
lipase was maximum at 1.5% alginate concentration and decreased

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 Immobilization of Lipase by Entrapment in Ca-alginate Beads 557
6
5
Activity U/g beads

4
3
2
1
0
0 50 100 150 200 250 300 350
ABL unit added

Figure 1. Effect of various lipase units (ABL, cells) loading (after 250 units, no further
increase in entrapment occured), in 2% CaCl2 and 1.5% alginate beads.

with increases in the amount of alginate used. CaCl2 concentration

standardization was done with 1–3% CaCl2 in the presence of 1.5%
alginate and 100 units of lipase to provide a 10 mL slurry; 2% CaCl2 gave
optimal lipase entrapment. The optimum loading, in the presence of
1.5% alginate and 2% CaCl2, was 100 enzyme units (Figure 1).
Earlier, Hemachander et al. [21] immobilized whole cells of
R. pickettii in different matrices, such as agar, alginate, and poly-
acrylamide for lipase production and found that 4% alginate beads gave
an optimal lipase activity of 14 U, 15% polyacrylamide retained 25 U,
compared to free enzyme of 40 U/mL. Won et al. [18] reported the effects
of immobilization using different ratios, by weight, of enzyme to alginate
and beads size for C. rugosa lipase entrapment in Ca-alginate gel beads
and reused them to check their stability.

Effect of Beads Size

The alginate bead size affected the enzyme immobilization yield:

it was reported that enzymes in smaller beads would have higher
catalytic activity due to increase in surface area [18]. Two different sized
beads were generated by changing the needle size through which the
mixture of lipase and alginate was deposited into CaCl2 solution. The
average bead size, measured by an optical microscope, was 1.2 and
2.1 mm, which had 40 and 30% active enzyme binding, respectively.

Operational Stability of Entrapped ABL Cells

ABL cells were immobilized by entrapment in Ca-alginate beads

and were recycled for the hydrolysis of triglyceride using tributyrin

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

558 I. BHUSHAN ET AL.

as the substrate. Each run lasted for 1 h, after which the beads were
separated and washed with buffer (pH 7.0). The activity of freshly
prepared beads in the first run was considered as 100%. The
immobilized beads were found to be stable for 10 cycles for the
hydrolysis of triglycerides. Dave and Madamwar [17] observed similar
results by immobilizing C. rugosa lipase in poly(vinyl alcohol), alginate,
and boric acid. Won et al. [18] reported entrapment of lipase in
Ca-alginate gel beads but on reuse, activity loss was observed. On the
other hand, Desai et al. [25] observed 50% loss of activity after 50 cycles
by entrapped porcine pancreas lipase in K-carrageenan beads.
Yadav et al. [19] also found highly reusable biocatalysts with no
leaching even after fourth cycle while Jaganathan et al. [20] reported
the immobilization of C. rugosa lipase in Ca-alginate beads for the
hydrolysis of high oils and grease originating from pet food industrial
wastewater.

Thermal Stability of Immobilized Lipase (ABL)

The thermal stability of the entrapped ABL cells in Ca-alginate was

studied by pre-incubating both the free and immobilized enzyme at
various temperatures ranging from 208C to 1008C for 1 h. The thermal
stability was markedly increased after immobilization. The free enzyme
was completely deactivated beyond 408C whereas the immobilized
enzyme did not exhibit any loss in activity upon incubation at 20–408C,
however, it retained 85, 60, 40, and 20% activity at 50, 60, 70, and 808C,
respectively (Figure 2). Increased thermostability by lipase entrapment
was also reported by other researchers [7,17,23–26].

120
Immobilized enzyme
100
Residual activity (%)

Free enzyme
80

60

40

20

0
0 20 40 60 80 100 120
Temperature in degree celsius
Figure 2. Thermal stability of free and immobilized lipase (ABL) in Ca-alginate beads.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 Immobilization of Lipase by Entrapment in Ca-alginate Beads 559
120

100 pH 5
pH 6
Residual activity (%)

80 pH 7
pH 8
60
pH 9

40

20

0
0 20 40 60 80 100 120
Time (h)
Figure 3. pH Stability of ABL immobilized in Ca-alginate beads.

pH Stability of Free and Immobilized Lipase (ABL)

The alginate beads have an inherent problem of dissolving in

phosphate buffer. Therefore, their pH stability was tested in
10–100 mM phosphate buffer and 10 mM phosphate buffer was found
to be suitable for incubation. The best stability for these beads was
in 100 mM Tris-HCl buffer (pH 7.0). The pH stability of lipase
immobilized on Ca-alginate beads was determined by incubating
the beads in 100 mM Tris-HCl at pH 7–9 and in 10 mM phosphate
buffer at pH 5–6. It was observed that no loss in enzyme activity was
noticed at pH 7; however, at pH 6, 8, 9, and 5, the residual activity
obtained after 96 h of incubation was 85, 85, 40, and 20%, respectively
(Figure 3). The free enzyme, however, became completely inactive
within 24 h when incubated at pH 4 and 5. The free enzyme also lost
its activity on incubation at pH 9 after 36 h (Figure 4). Similarly,
comparative loss in activity was observed at pH 6, 7, and 8. Therefore,
immobilization enzyme had greater pH stability compared to the
free lipase.

Storage Stability

Samples of Arthrobacter sp. cells entrapped in Ca-alginate beads were

kept in distilled water at 4 and 208C, for 1 month and their activity was
analyzed after every 5 days. No loss in activity was observed during this
duration. These beads were then stored in water for further use.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

560 I. BHUSHAN ET AL.

120
pH 4
pH 5
100
pH 6
Residual activity (%)

80 pH 7
pH 8
60 pH 9

40

20

0
0 20 40 60 80 100 120
Time (h)

Figure 4. pH Stability of free lipase (ABL).

CONCLUSION

Lipase cell biomass 13 g/L was produced from Arthrobacter sp. This
cell biomass was entrapped in Ca-alginate beads after optimizing the
concentration of alginate (1.5% w/v), CaCl2 (2%), lipase units loading
(100 unit in 10 mL slurry), and bead size (1.2 mm). After optimization of
all the parameters, the entrapped lipase cells yielded 40% of lipase
activity. These immobilized beads can be used for 10 cycles without any
loss in activity for the hydrolysis of triglycerides using tributyrin as the
substrate. The entrapped Arthrobacter sp. cells showed an increase in
thermal, pH, and storage stability compared to the free enzyme. These
beads could be used for biotransformation reactions and kinetic
resolution of chiral drugs or drug intermediates.

REFERENCES

1. Vulfson, E.N. (1994). Industrial Applications of Lipases, In: Woolley, P. and

Peterson, S.B. (eds), Lipases: Structure, Biochemistry and Applications,
pp. 271–88, Cambridge University Press, Cambridge.
2. Gupta, R., Gupta, N. and Rathi, P. (2004). Bacterial Lipases: An Overview of
Production, Purification and Biochemical Properties, Appl. Microbiol.
Biotechnol., 64: 763–781.
3. Desai, P.D., Dave, A.M. and Devi, S. (2006). Alcoholysis of Salicornia Oil
Using Free and Covalently Bound Lipase Onto Chitosan Beads, Food Chem.,
95: 193–199.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 Immobilization of Lipase by Entrapment in Ca-alginate Beads 561

4. Fernandez-Lorente, G., Palomo, J.M., Cabrera, Z., Guisan, J.M. and

Fernandez-Lafuente, R. (2007). Specificity Enhancement towards
Hydrophobic Substrates by Immobilization of Lipases by Interfacial
Activation on Hydrophobic Supports, Enzyme Microb. Technol., 41: 565–569.
5. Koul, S., Taneja, S.C., Parshad, R. and Qazi, G.N. (1998). Enantioselective
Hydrolysis of Alkyl Ester of Substituted 1-phenyl Ethanols using Newly
Isolated Strain of Arthrobacter sp. Abl Comparative Study with Known
Commercial Lipase, Tetrahedron: Asymmetry, 9: 3395–3399.
6. Anand, N., Kapoor, M., Koul, S., Taneja, S.C., Sharma, R.L. and Qazi, G.N.
(2004). Chemoenzymatic Approach to Optically Active Phenylglycidates:
Resolution of Bromo and Iodohydrin, Tetrahedron: Asymmetry, 15:
3131–3138.
7. Chaubey, A., Parshad, R., Koul, S., Taneja, S.C. and Qazi, G.N. (2006).
Arthrobacter sp. Lipase Immobilization for Improvement in Stability and
Enantioselectivity, Appl. Microbiol. Biotechnol., 73: 598–606.
8. Dulik, D. and Fenselau, C. (1998). Use of Immobilized Enzymes in Drugs
Metabolism Studies, FASEB, 2: 2235–2240.
9. Nagayama, K., Yamasaki, N. and Imai, M. (2002). Fatty Acid Esterification
Catalyzed by Candida rugosa Lipase in Lecithin Microemulsion-based
Organogels, Biochem. Eng. J., 12: 231–236.
10. Ceynowa, J. and Rauchfleisz, M. (2003). High Enantioselective Resolution of
Racemic 2-arylpropionic Acids in an Enzyme Membrane Reactor, J. Mol.
Catal B: Enzym., 23: 43–51.
11. Deng, H.T., Xu, Z.K., Liu, Z.M., Wu, J. and Ye, P. (2004). Adsorption
Immobilization of Candida rugosa Lipases on Polypropylene Hollow
Fiber Microfiltration Membranes Modified by Hydrophobic Polypeptides,
Enzyme Microb. Technol., 35: 437–443.
12. Chiou, S.H. and Wu, W.T. (2004). Immobilization of Candida rugosa
Lipase on Chitosan with Activation of the Hydroxyl Groups, Biomaterials,
25: 197–204.
13. Gallego, F.L., Betancor, L., Hidalgo, A., Ortiz, G.D., Mateo, C., Lafuente,
R.F. et al. (2007). Stabilization of Different Alcohol Oxidases via
Immobilization and Post Immobilization Techniques, Enzyme Microb.
Technol., 40: 278–284.
14. Ye, P., Xu, Z.K., Wu, J., Innocent, C. and Seta, P. (2006). Nanofibrous
Poly (acrylonitrile-co-maleic acid) Membranes Functionalized with Gelatin
and Chitosan for Lipase Immobilization, Biomaterials, 27: 4169–4176.
15. Martinek, K. and Mozhaev, V. (1985). Immobilization of Enzymes: An
Approach to Fundamental Studies in Biochemistry, Advances in
Enzymology, Vol. 57, pp. 179–247, John Wiley and Sons, New York.
16. Chen, J.-P. and Lin, W.S. (2003). Sol-Gel Powders and Supported Sol Gel
Polymers for Immobilization of Lipase in Ester Synthesis, Enzyme Microb.
Technol., 32: 801–811.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

 562 I. BHUSHAN ET AL.

17. Dave, R. and Madamwar, D. (2005). Esterification in Organic Solvents by

Lipase Immobilized in Polymer of PAV-Alginate-Boric Acid, Process
Biochem., 41: 951–955.
18. Won, K., Kim, S., Kim, K.J., Park, H.W. and Moon, S.J. (2005). Optimization
of Lipase Entrapment in Ca-alginate Gel Beads, Process Biochem., 40:
2149–2154.
19. Yadav, G.D. and Jadhav, S.R. (2005). Synthesis of Reusable Lipases by
Immobilization on Hexagonal Mesoporous Silica and Encapsulation in
Calcium Alignate: Transesterification in Non Aqueous Medium,
Microporous and Mesoporous Mater., 86: 215–222.
20. Jaganathan, J., Bassi, A. and Nakhla, G. (2006). Pre-treatment of High Oil
and Grease Pet Food Industrial Wastewaters using Immobilized Lipase
Hydrolyzation, J. Hazard. Materials., B 137: 121–128.
21. Hemachander, C., Bose, N. and Puvanakrishnan, R. (2001). Whole Cells
Immobilization of Ralstonia pickettii for Lipase Production, Process
Biochem., 36: 629–633.
22. Johri, S., Verma, V., Parshad, R., Koul, S., Taneja, S.C. and Qazi, G.N.
(2001). Purification and Characterization of an Ester Hydrolase from a
Strain of Arthrobacter sp. Its Application in Asymmetrisation of 2-benzyl-1,
3-propanediol Acylates, Bioorg. Med. Chem., 9: 269–273.
23. Bhushan, I., Parshad, R., Qazi, G.N., Ingavle, G., Jamalpure, T.M., Rajan,
C.R. et al. (2007). Macroporous Beads for Lipase Immobilization: Kinetic
Resolution of a Racemic Drug Intermediate, J. Bioact. Compat. Polym.,
22: 174–194.
24. Bhushan, I., Parshad, R., Qazi, G.N., Ingavle, G., Rajan, C.R., Ponrathnam,
S. et al. (2008). Lipase Enzyme Immobilization on Synthetic Beaded
Macroporous Co-Polymers for Kinetic Resolution of Chiral Drugs
Intermediates, Process Biochem., 43: 321–330.
25. Desai, P.D., Dave, A.M. and Devi, S. (2004). Entrapment of Lipase into
K-carrageenan Beads and its Use in Hydrolysis of Olive oil in Biphasic
System, J. Mol. Catal B: Enzym., 31: 143–150.
26. Rassy, H.E.I., Perrard, A. and Pierrr, A.C. (2004). Application of Lipase
Encapsulated in Silica Aerogels to a Transesterification Reaction in
Hydrophobic and Hydrophilic Solvents: Bi-Bi Ping-Pong Kinetics, J. Mol.
Catal B: Enzym., 30: 137–150.

Downloaded from http://jbc.sagepub.com at REG RSRCH LAB RRL-JAMMU on November 5, 2008

View publication stats