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Immo5i(ization 'lecfm;ques amfHyarofytic )fctil-'itirs ofq·'rte ana Immo5i{LZea Lipases Cfiapter 3
Chapter 3
Immo6ilization Techniques andJ{ydrofytic
Jlctivities ofPree and Immo6ilized Lipases
(PfiJD. rrTiesis 49
lmmobifization Techniques ana1{ytim()'tic jIctiT-'ities of ~ree and ImmobltlZea Lipases Chapter 3
This chapter includes different lipase immobilization methods onto asymmetric
polymer matrices. The immobilized and free lipase is taken for the hydrolytic
experiments for different oils and their hydrolytic activities are compared. The
influencing factors of the hydrolytic experiments are detennined. The fabrications of
different reactors are also done.
3.1 Lipase Immobilizatioll techlliques
There arc many techniques to immobilize enzymes on polymer support. They are
mainly of three categories: 1) Physical adsorption 2) Chemical/Covalent coupling 3)
Entrapment. The techniques are detailed below:
3.1.1 Physical adsorptioll
In this approach enzyme immobilization refers to binding of enzymes through weak
attractive forces. The weak linkages are established between enzyme and polymer
support mainly by van der Waals, hydrogen bonds and hydrophobic/hydrophilic or
ionic interactions. The adsorption is schematically prcsented in Fig 3.1.
In general, physical adsorption of an enzyme is achieved by simply contacting the
enzyme solution with support, which may require minimal pretreatment and post
treatment. The immobilization depends upon the nature of polymeric materials and as
well as their surfaces (Masoom et aI., 1989; Tramper, 1983; Rouxhet, 1990).
The features of enzyme immobilization by physical adsorption are as follows:
• Simple to perfonn
• Substrate specificity usually remains unchanged
• Permits regeneration of the support with fresh enzyme
• Low cost method
rr/i. iJ). '[iiesis 50
lmmovi{rzatiot/ 7ccfiniijufs atu{Hyamfjtic )fcti'f'ities oj'Free alia lmmovifzzfa Lipases cnapter 3
Lipase -c:::1r"
Sbooplr Physkal adsorptia. MetJuod
Figure 3.1: Lipase immobilization on asymmetric membrane by physical adsorption
and glutaraldehyde method
3. I. I. I Imlllobilizatioll of lipase 011 flat sheet membralles
Asymmetric Polysulfone (I'S) membranes of area 6 cm 2 without any pnor
modification are impregnated in lipase solution (3ml) (2mg/ml) of 0.1 M of phosphate
buffer (I'll 7.0). The membranes are kept under shaking for l2h at lODe. Then they
are removed from lipase solution and washed with water to remove un-immobilized
lipase from the membrane surface. The lipase immobilized membranes are stored in
buffer solution for further activities. The immobilization experiments are done in
1'7 -55L13 triplicates.
In addition to that sometimes cross linker glutaraldehyde is applied to stable the lipase
on the I'olysulfone/ Polyether sulfone surface chemical. Glutaraldehyde considered as
one of the most effective protein crosslinking reagents. The chemistry of
glutaraldehyde structure in aqueous solution is not limited to its simplest form I.e.
monomeric dialdehyde form, but also as a dimer, trimer, and polymer (Migneault e/
a/., 2004). Scheme 3.1 shows the mechanism of lipase and glutaraldehyde
crosslinking by oligomer fonnation.
pn.(/). 'IfieslS 51
Immo6iGzatwn rtccliniqufs aniJ{yirofytic )lcthities of iFru ani Immobifi";:.ei Lipases Cliapter J
At first, membranes of area 6 cm2 are submerged in aqueous solution of
glutaraldehyde (2.5%) for 4h. Then the samples are removed from glutaraldehyde
solution and immersed in lipase solution (3ml) (2mg/ml) prepared in (UM of
phosphate buffer of pH 7.0 and kept under shaking for 12h at 10°C.
Membranes are then removed from lipase solution and washed with reverse osmosis
treated water to remove loosely adhered lipase from the membrane surface. The
experiments are done in triplicates and the results taken are average values. The lipase
immobilized biocatalytic membranes are stored in buffer solution.
CHO
I (CH,h ------I CIlO
Glutaraldehyde
CIlO
I
glutaraldehyde -linkage
ClIO
I - CIl = C-(ClI,),- CII = C-(CIl2h-CIl =
CHO
I
lliPase
Cll= N-lipase
I -CII - C-(ClI,h-CIl= C-(CH,l2- CIl =
I Nil-lipase
010
I C- (012),-
CHO
I C- (CII')2-
Scheme 3.1: The mechanism of lipase and glutaraldehyde crosslinking by oligomer
fomlation.
The reaction between enzyme and glutaraldehyde involves the conjugate addition of
protein amino groups to cthylenic double bonds (Michael-type addition) of the a,punsaturated oligomers found in the commercial aqueous solutions of glutaraldehyde
that are usually used (Scheme 3.2, reactiou ii) (Richards and Knowles, \968).
However, Monsan et al. (Monsan el al.. 1975) proposed a slightly different
mechanism in which an addition reaction occurred on the aldehydic part of the a, punsaturated polymers (and poly-glutaraldehyde) to give a Schiff base (imine)
stabilized by conjugation (Scheme 3.2, reaction i).
Pli. rD. 71zesis 52
Immo6ifizatioll "Yecfl1liques ana'J(wfrofytic )fcti1-itles of fFree and I mmobifiad Lipases Cliapter 3
~IIO ~'IIO
/'-v-~~ OIIC CliO
~~+ 2 R·\II, olle CliO
Scheme 3.2: SchIff base (I) and MIchael type (ii) reactions of glutaraldehyde with
proteins
Influencillg factors for immobilization
Nature of polymer materials (PSIPES)
It is aimed to study whether nature of polymers (hydrophobicity/hydrophilicity) has a
role on the immobilization or not. In this regard Polysulfone (PS) (C27Hn 0 4S) and
Polyether sulfone mcmbranes (PES) (C24HI606S2) arc taken. It is known PS
thermoplastic polymer as hydrophobic where as its derivative PES is relatively
hydrophilic one. It is found that hydrophobic moiety PS favors lipase immobilization
over PES.
Nature of membrane
The effect of nature of membrane i.e. porous or dense for lipase immobilization is
studied. For that wet phase and dry phase inversion techniques are employed. The
immobilization is done on porous or dense PS membranes (15 % w/w). It is seen that
asymmetric porous PS membranes provides more anchoring feasibility for lipase
compared to dense one.
Moreover, the effect of PS concentration (13, 15 and 18%) on the lipase
immobilization amount is also investigated. All three membranes are prepared by wet
phase inversion method. The porosity of the membranes differs because of its
concentration, The immobilization amount varies with porosity of the membranes. It
is seen that PS membrane (of 18'}lo w/w) shows 101Y immobilization ability because of
its relative low porosity.
<Pli,![). %esis 53
Immobifization -Techniques and'Jfydro[ytic)1ctit'ities of Pree ana Immo6i[izea Lipases Cliapter 3
Source of lipases
Lipase is produced from different sources viz. plants, animals and microbes. As plant
lipases are not used commercially, the animal and microbial Iipases are used
extensively. But somc disadvantages are associated with animallipases. Thcy cannot
be used in the processing of vegetarian food and have undesirable effect. They are
also likely to contain residual animal viruses, hOl1llOnes, etc. So, microbes are major
source oflipascs. Yeast has eamed acceptability since long and is considered natural.
Yeasts are also considered to be easy to handle and grow, in comparison to bacteria
(Kademi el al., 2003). The Iipases produced by Candida species is fast becoming one
of the most industrially used cnzymcs. This is because of their usc in a variety of
processes due to high activity, both in hydrolysis as well as synthesis (Redondo et al.
1995).
Different sources of lipase viz: Wheat genn, Pseudomonas fluorescens, Aspergillus
O/yzae. Mucor javanicus. Candida cylindracea. Rhizol11l1cor miehei are used to study
in this aspect.
Lipase from Candida cy/indracea is more effective in hydrolysis due to its ability to
liberate all types of acyl chains, regardless of their position in the triacylglycerols.
COllcen/ra/ion of cross-linker (i.e. Glu/araldehyde)
Glutaraldehyde is used as a crosslinker to immobilize Iipases on membranes.
Different concentrations (1, 2.5 and 5%) aqueous solutions of glutaraldehyde are used
to study the concentration effect of the crosslinker. It is observed that the
immobilization amount is greatly dependent on concentration of glutaraldehyde and
2.5 % glutaraldehyde shows the maximum and saturation occurs beyond it.
3. I. 1.2 Immobilization of lipase oil/in asymmetric PS globules
The Polysulfone solution (10%, 15 and 20%, w/w) is taken in injection syringe and
poured dropwise into lipase containing phosphate buffer (pH 7.0). The aerial path
length of the clrops is adjusted to .:I-Scm. After preparation, the lipase-loaded globules
arc treated with 2.5% glutaraldehyde (crosslinker) to stable lipase on them.
Pli.l]). 'J1ieszs 54
Immobi£izatioll ifechniques aniHyarofytic)lctivities of !free alii lmmobi£izea Lipases Chapter 3
The details of the compositions are presented in Table 3.l. The globules are then
washed with water and stored in the phosphate butfer. The globule preparation is
carried out at room temperature. The schematic presentation is displayed in Fig 3.2. In
Fig 3.2 A globules are prcpared with syringe without using needle (15 and 20% w/w).
PS) and in Fig 3.2 B with needle (10% w/w PS).
A
B
-·~~·~B· ps,,",,,,,
l.!pI.Sf SOUtDn n Ii..o!plutl: l:llrttr
Figure 3.2: Preparation of globules
Pos!. treatment
Post treatment
Table 3.1: Different technical conditions of PS globules formation
Sample no Droplet formation by Non-solvent
A (15 and 20%) Syringe diameter 1.2mm
B (10%) Needle diameter O.4mm
IlIjluellcillg factors for immobilization
COllcelltratioll of Polysulfolle
(volume: 50 ml)
Phosphate buffer (pH 7) with 5mllipase
Phosphate buffer (pH7) with 5mllipase
Post-treatment
Glutaraldehyde (2.5%)
Glutaraldehyde (2.5%)
Globules are prepared from differcnt concentrations of PS viz: 10, 15 and 20% w/w
solution. The porosity of the globules also depends upon the concentration. The
porosity bears inverse relationship with the concentrations taken. 'BET' surface area
and average pore diameter are also connected with PS concentrations.
P/i.c]). 'Ines;s ss
Immo6ifization rfecfiniques Qm[J{yarofytic)1.ctivities vf !Free alld Immo6ifi::.ed Liposes Cliapter 3
BET surface area increased and average pore diameter decreased with PS
concentrations. All the above mentioned parameters are studied for the lipase
immobilization amount on globules. It is seen that 20% w/w PS globules exhibit
maximum lipase loading. The cxperiments are done in triplicates and the results arc
average values.
3.1.2 Covalent/Chemical coupling
The covalent attachment is favored to increase the stability of enzymes, retained a
significant amount of activity with minimal leaching problem and preventing
reversible unfolding. A covalent bond is fonned between the active site of the support
and the groups of amino acid residues present in the enzyme backbone.
The functional groups may be 'in-built' or by incorporated by some functionality. The
'surface modification' is one of the unique approaches to incorporate functionalities
without disturbing the bulk propcl1ies of the polymer substrate. In this particular
phase two systems are dealt with for the lipase immobilized membranes. The two
systems are as follows.
:.. Polyvinyl alcohol (PVA) modified surface
:.. A zo-modified surface
3.1.2.1. PolYI'inyf alcohol (PVA) mOdified surface
The approach is basically to improve the surface property of the membranes. By
incorporation of PV A the biocompatibility of the PS membrane increases. In addition,
its chemical structure can cause protein stabilization by attachment to the polymer
chain or resemblance to, e.g., polysaccharides known as stabilizing agents for proteins
due to their impact onto the water structure (Arakawa and Timasheff, 1982). The
objective is to create specific microenvironment for the enzymes so that the activity
should not be disturbed. For this purpose photo-irradiation technique is employed to
modify the asymmetric PS membranc surface with PV A layer. The PV A layer plays a
key role in controlling the lipase loading and preventing lipase from being dissolved
into the aqueous phase so that leach ing can be avoided. PV A concentrations on
membranes are measurcd by formation of pVA-eu (Il) complex (section 2.4.2).
Ph/D. 'Iliesis 56
lmmo6i(tzatiofl '1ecfmiques aruf )fyarofytic.ftcthlitics of (Free aTia lmm06ifL:fa Lipases Cfiapur 3
For immobilization, first, PS membranes (modified with PYA and unmodified one of
area 9.1 em') are impregnated in aqueous solution of glutaraldehyde for 4h. Then the
samples are removed from glutaraldehyde. After it, membranes are dipped into lipase
solution (10 ml) (2mg/ml; in 0.1 M of phosphate buffer at pll 7). It is kept under
shaking condition for 12h at I DoC. Membranes are taken out from lipase solution and
washed with water to remove loosely adhered lipase from the membrane surface. The
experiments arc done in triplicates. In this method PYA and glutaraldehyde (glu)
coupling is used, the scheme of lipase immobilization through PYA-glu is as follows
Scheme 3.3.
Dipping in PYA
H+ ~ Glutaraldehyde
~ Lipase
PS '.<mbran.
Scheme 3.3: Mechanism of lipase immobilization through PYA and
glutaraldehyde
57
immo6ifizatioTl '1ccfztliqufs arufJfyarofytic )lctivities of t}'ree awi immo6i{l.Zea Lipases Cfiapter 3
The immobilization of lipase is also done without using glutaraldehyde with only
PYA modified PS membranes by the same manner. The immobilization is done on the
asymmetric or modified surface of the membranes. The four different possibilities
(adsorption and covalent approach) arc presented in Figure 3.3.
I II ill IV
Figure 3.3: four different possibilities, I is lipase immobilization on virgin PS without
any modification, II is on PS modified with PYA without any reagent, III
is on PS with glutaraldehyde reagent and IV is on PS modified with PYA
and using glutaraldehyde reagent
influencing factors for immobilization
Concentration of PVA during modification
The effect of concentration of PYA i.e. 0.5 and 2% on the amount of immobilization
is studied. The PYA contcnt on the membranes are incrcased with its concentration in
dipping. It is found that amount of lipase immobilization is increased with the PYA
content on the membranes and modification with 2% PYA has maximum lipase
immobilization.
Dipping time in PVA
Besides, concentration of PYA, dipping time of PS membranes in PYA solution also
has impact on lipase immobilization. For this purpose different dipping times 10, 30
and 60 min in PYA are applied to modify the membranes. It is observed that after 30
min there is no variation in the content of PY A on the membrane surface.
rpfzJD. 'Incsis 58
!mmo[nlization 'Techniques anaJfyarofytic )1ctivities of tFree ana !mfll06ifized Lipases Cliapter J
Concentration of cross-linker (i.e. glutaraldehyde)
Glutaraldehyde is used as a crosslinker to immobilize Iipases. Experiments are calTied
out by taking I, 2.5 and 5% aqucous solution to determine the effect of
Glutaraldehyde concentrations. It is seen that the immobilization amount is greatly
dependent on concentration of glutaraldehyde and 2.5 % glutaraldehyde shows the
maximum and after that steady level is reached.
3.1.2.2. llydrazille-lIloc/ijiec/ surface
PS membrane is modified by azo functionality to improve the immobilization and its
activity compared to the previous one. PS membranes are modified with acrylic acid
to activate the surface so that the surface is modified by azo functionality to
immobilize the Iipases. The consecutive processcs are described below. First, acrylic
acids of having different concentrations (I, 5 and 10 % v/v) are taken to activate the
PS membranes. PS-g (AA) membranes are taken for covalent binding of lipases. PS
g(AA) membranes, previously acid methylated are activated by acyl azide fOlmation.
For this, 50 ml of 60% (w/v) hydrazine hydrate solution are added on the activated
surface for 10 min. The reaction is kept at room temperature. The hydrazine solution
is then decanted from the PS surface and the membrane is dried at room temperamre.
Two routes are exploited for lipase immobilization on hydrazine treated PS
membranes (Fig. 3.4).
• Coulet Method
The Coulet method is basically for lipase immobilization on azo modified surface
(Coulet et aI., 1974). The hydrazinc modified PS membranes are treated with HCI and
sodium nitrite (NaN02) (50ml) in 1:5 molar ratio for 10 min. The temperature is kept
at 0° C. The hydrazine treated surface is converted to azo surface (Scheme 3.4).
After removal of excess reagents by repeated washing, the coupling of lipase is
perfomled by immersing the membranes into the lipase solution (50ml containing
2mg/ml in a.IM sodium phosphate buffer pH 7) at standard conditions of aoc for 4 h.
The modified azo functionality forms the covalent attachment with the lipascs through
its tyrosine residue part. Excess lipase solution is decanted and membranes are
thoroughly washed with sodium phosphate buffer (O.IM, pH 7) and stored in it for
Ph. 'D. 1Jiesis 59
immo6ifizatioll 7ecfmiques alU[Jfyarn(ytic j1ctivit ies of {Free ana immo61H::.ea L1l'ases Cnapter 3
further studies. The experiments are done in triplicates and the results arc average
values.
• Crosslinking through Glutaraldehyde on Hydrazine modified surface
Apart from the above approach the acyl azide modified membranes arc treated with
glutraldehyde solution (2.5 % aqueous solution v/v) for 4h (Scheme 3.4). Then the
membranes are submerged in solution of sodium phosphate buffer (0.1 M, pH 7)
containing 2mg/ml lipase (50ml). Lipase immobilization is occurred at 0° C for 12h.
Lipase is immobilized on PS membranes by involving Schiffs base formation
reaction. Lipase solution is then decanted and the membranes are thoroughly washed
with sodium phosphate buffer (0.1 M, pH 7) and stored in it for further studies. The
experiments are done in triplicates.
Immobilization methods
Ilydrazine-Cilutaraidehyde method
Hydrazine modified PS membrane
Coulet l\·lethod
A:z.o modified PSmembrane
Figure 3.4: Two rotes of immobilization on hydrazine modified membrane
!Pfi.{[). 'llicsis 60
Immo6i{jzatioll 'Tedmiques and'JfyrfrvCytic Activities of fFree alIa Immo6ifi::.ea Lipases Chapter 3
,,\CT)' lit "citl \ I 0 min) ..
; (Sminl
15 % PS mcmbr.tnc
o II C-OH
o
II C-OH
o II C-OH
Graft copolymer
o II C -Nfl-Lipase
o II C-OH
o II C -NH-Llpa,e
C olilel melhod
o II C -NH-N-Cfl-CH,-CH=N-Upase
o II C-OH
o II
•
Lipa>c-NIl~
Hill
C -NH-N=CH-CH,-CH=N-Upase
H yu razinc-Gllliaraldch ydc method
0
II S"'" H,~04 C-OM.
Ilmin)
H'
~kOll J,5min)
0
.. II C-OH
0
II C-OMe
1) IICI! l\aNO, (10min)
2) Lipasc-l\H, (4h)
o II
1\112-1\11, 60% (5min)
o II C-NH-NH,
o II C-OH
o II C-NH-NH,
C-NH-N=CH-CH,-CHO
o II C-OH
o II C-NH-N=CH-CH,.CHO
Scheme 3.4: Reaction mechanism of immobilization
(['h.'D. 'Iliesis 61
Immo6i{ization 'Techniques alli[J(ylm(yticActivities of CFree and I",moGifued Lipases Cnapter J
Influencillg factors for immobilization
Cross-linker (i.e. Glutaraldehyde)
The surface of PS membrane is modified with hydrazine to get acyl azide
functionality. Two routes are followcd for lipase immobilization on these modified
mcmbranes i.e. with/without using crosslinkcr glutaraldehyde. In onc system coulet
method is exploited using NaNO,/HCI without glutaraldehyde. And in second system
crosslinker glutaraldchyde is uscd for lipase coupling on (amino groups containing)
modified PS membranes. It is found that with cross linker glutaraldehyde lipase
immobilization is more compared to other one.
COllcelltratioll of Acrylic acid monomer durillg modificatioll
The effcct of concentration of acrylic acid on lipase immobilization on PS membranes
is studied. Acrylic acid concentration is varied as I, 5 and 10% aqueous solution. It is
found that acrylic acid content on the membranes is increased with its concentration.
It is observed that PS membranes modified with acrylic acid 5 % (v/v) are capablc of
attaching maximum amount of lipase. It reaches steady level after that.
3.1.3 ElltrapmeJ/t
The technique is actually pore dependent one where enzymes are incorporated into the
membrane pores. The beauty of asymmetric membrane is of its inbuilt structure. The
hetcrogeneity of the layers has the advantages to entrap lipascs in it. Pressure is
applied to feasible the technique. The schematic diagram of the entrapment of lipases
is presented in Fig 3.5.
Glutaraldehyde treatment is also done to stabilize the immobilization. As the
molecular weights of lipases are of higher range, they can be entrapped in to the pores
of ultrafiltration or microfiltration range of membranes. In this particular experiment
PS membranes made from 13, 15 and 18 % w/w concentration (in DMF) is taken for
lipasc immobilization.
Ph.lD. '["esis 62
Immo5i[ization (Techniques and'J{ydrofyticYfctifJities of(Fm: alia Immo6i(t::.ea Lipases Cfiapter 3
UpaSf
Figure 3.5 Lipase entrapped within pores
The technique of using pressure to immobilizc lipase is exploited in the dead end
filtration set up. The dead end filtration setup is already shown in Fig 2.10. The set up
consists of dead end filtration cell of volume 75 ml fitted with a magnetic spin bar
which can rotate at desired speed with the help of a magnetic stirrer. A flat circular
shape PS membrane of effective area 12.57cm' is fixed at the bottom of the cell. The
ccll is connectcd to a solution reservoir. Varied pressure is applied in the test cell with
the help of nitrogen gas. Lipase solutions in sodium phosphatc buffcr (0.1 M, pH 7.0)
are used as fced solutions for immobilization. The lipase is entrapped in the pores of
the membranes. The entrapped membranes are treated with glutraldehyde solution
(2.5 % aqueous solution v/v) for 4h. Membranes are thoroughly washed with sodium
phosphate buffer (0.1 M, pI I 7) and stored in it for further studies. The experiments are
done in triplicates.
I/lflue/lcing factoTs jilT immobilization
CO/lcell/ratio/l l!f PS
The effect of PS concentration (13, 15 and 18% wlw in DMF) on the lipase
immobilization amount is investigated. The porosities of the membranes differ with
the PS concentration. The immobilization amount differs with porosity of the
membranes. It is seen that PS membrane (of 18% w/w) shows lowest immobilization
ability because of its relative low porosity.
po.r[). 'Iliesis 63
Immo6iuzation 'Techniques andJfydro{ytic jIaivitifs 0/ tFree alld Immo6ifiua Lipases Cfiapter J
Concentratioll of lipase
The effect of lipase concentration (2, 2.5 and 3mglml) on the immobilization amount
is studied. It is seen that at 2.5 mglml concentration it reaches maximum and above it
the immobilization amollnt is decreased.
Applied Pressllres
The pressure is one of the important physical controlling parameters for the
entrapment process. The effect of pressure on lipase immobilization is detemlined by
applying different pressures (0.034, 0.069, 0.10 and 0.14 MPa). It is found that at 0.10
MPa pressure, lipase immobilization is maximum and above this it becomes steady.
Tim e period
Different time periods (3,6,9, 12 and 15 h) are taken for the study. With time the
entrapment of lipases in membrane pores is increased. It follows the increasing trend
up to 12 h and then it becomcs steady above this duration.
3.2 Estimatioll of lipase immobilized contellt 011 Polymeric matrix by Lowry method
The amount of lipase immobilized on polymeric matrix is estimated by using Lowry
method. It is explained in section 2.4.2. The amount of lipase immobilized is
deteffi1ined by BSA standard curve. The amount of lipase immobilization is calculated
from the difference of lipase concentration in the solution before and after
immobilization.
It is mathematically presented as follows for membrancs
v w=(C,-C,).
A (I)
Where \V is the total immobilized amount (mglcm\ C 1 and C2 are the initial
concentration of fi'ee lipase and decant after immobilization, respectively (mg/ ml). V
is the reaction volume (ml) and A is the area of the PS membranes (cm').
Ph.!D. '[{"sis 64
immobi{fzatioll tfecfiniques anaJfydro6'tic jlctit,ities of tfree and immo6ifiud Liposes CliaptCY 3
To determine the amount of immobilized lipase on the asymmetric PS globules (made
from 10, 15 and 20% w/w) the following expression is taken.
The mathematical representation is as follows
V IV=(C, -C,).w
(2)
Where w is the total immobilized amount (mg g-l), C 1 and C2 are the concentrations
of the initial frce lipase and final in decant after immobilization, respectively
(mg mrl), V the reaction volume (ml), W the weight oflhe PS globules (g).
3. 3 Hydrolysis of different oils by lipase immobilized matrices
Oils and fats are part of a group of organic compounds as esters or triglycerides, and
thcir hydrolysis essentially involves reactions with water to produce valuable free
fatty acids and glycerol Scheme 3.5. The hydrolytic activity of free and immobilized
lipases is studied for olive oil as an oil model system and for other oils namely (palm
oil and castor oil). Compositions of oils arc presented in Table 3.2. Hydrolytic
activities in immobilized lipase systems for PS and PES, PS -PVA-glu, PS-Hz-glu
and PS globules are detennined.
• ~Oll
free Fatty Acid
Scheme 3.5: Hydrolysis of triglyceride
Ph. (D. 'Iliesis
~IIZ01l
+ <;:11011
ClIzOIl
Glycerol
65
Immo6ifization 'Techniques am{J{yJrofytic jIctivities of !free a/U{ I IIIl1106ifize£Lipases Cfiapter J
Table 3.2: Composition of oils
Oils
Olive Oil
Palm Oil
Castor oil
Chemical
Compositions
Oleic acid: 55-83%,
Palmitic acid: 7.5-20%,
Linoleic acid 3.5-21 %,
Stearic acid : 0.5-5%,
Linolinic acid :> 1.5%
Palmitic acid: 44.3%,
Oleic acid 38.7%,
Stearic acid 4.6%,
myristic acid 1%,
Linoleic acid: 10.5%
Ricinoleic acid: 95%,
Oleic acid: 2%, Linoleic
acid: 1%, Linolinic
acid :0.5%, Stearic acid:
0.5%, Palmitic acid:
0.5%, Dihydroxy stearic
acid: 0.3%)
Structure
OH o
C,sHJ.02 cis-9-0ctadecenoic acid, (Oleic
acid)
o OH
C'6H)202 hexadecanoic acid (Palmitic
acid)
OH OH
o
C "H).O) cis-12-hydroxyoctadeca- 9-
enoic acid, (Ricinoleic acid)
3.3.1 Hydrolysis of oils with free ami immobilized Iipases
The hyclrolyic activities of the free and immobilized lipase are determined by
measuring the free fatty acid content in the medium by titrimetric method. The
hydrolytic activity of lipase is assayed with olive oil emulsion method (Soares et al.,
1999). Hydrolysis of other oils (Palm oil, Castor oil) is also done. The reaction
mixture is prepared by adding 5ml of oils emulsion (oil + gum acacia + sodium
Pli. <D. 'Inesis 66
lmmoliifizatiort 'Tecfiniques andJ{yaro{ytic Activities of rt"ree and lmmobifi::ea Lipases Cfiapter J
benzoate) and 5ml of 0.1 M sodium phosphate buffer (pH 8.0). The hydrolytic reaction
is initiated by adding tree lipase (I ml, 2mg/ml) or lipase immobilized membranes of
6cm2 or immobilized PS globules (I gill) in the reaction medium. The hydrolysis
reaction is carried out in a shaker (150 rpm) at 3rC and pH 8 for 30 min.
The catalytic reaction is stopped by adding acetone-ethanol solution (10 ml) in the
ratio of 1:1. The liberated fatty acids are titrated against 0.01 N NaOH solution with
phenolphthalein as an indicator. All the experiments are done in triplicates and the
results are average values.
3.3.2 Quantification offreefatty acid releasedfromhydrolysis
The produced frce fatty acid is the key analytical paramcter throughout the whole
study. The activity is defined according to the fatty acid production. Thus it IS
essential to use accurate and reliable analytical techniques in order to reduce errors.
By titration method
Titrimetry method also named pl-I- stat assay, is used for determination of free fatty
acids because of its simplicity of mcasurcmcnt and low chemical consumption. [t is a
convenient and sensitive method that is applicable to a wide range of enzyme
catalyzed reactions. The amount of free fatty acid released during hydrolysis is
estimated by titration with a titre i.e. dilute NaOH solution using phenolphthalein as
an indicator.
Steps:
I. Aliquots of the reaction mixture are withdrawn from lipase catalyzed reaction
mixture (hydrolyzed oils).
2. Sample is dissolved in 10 ml of a neutralized mixture of ethanol and diethyl
ether (1: 1 v/v) and titrated with 0.0 I N NaOH solution (standardized by oxalic
acid) using phenolphthalein as an indicator.
3. The fatty acid released is calculated against blank sample by subtracting the
initial fatty acids present in blank. sample (unhyclrolyzecl).
rFli.rJJ. 'Iliesis 67
Immo6ifizatioTl Techniques a1U[J{yarv(yticficti1/ities ofPree and Immo6i{izea Lipases C[wpter J
Released fatty acid jJ-om GC-I/lUSS lilli/lysis from their esters
The parameters related to hydrolysis (free fatty acid (FFA) and Acid value (A V) are
detcnnined from the following expression. The FFA is defined as the percentage by
weight of free acid groups in the oil where as A V is termed as the weight in mg of
alkali required neutralizing the hee acid groups in oil (Cuppett et aI., 2001). The
parameters are expressed by the following expression AOCS Official method Ce2-66
(1993).
FFA(%)(as . . I .v.'-ol:::lI::.l/l:.::e....:o'-f....:N,::.a:::O....:H:..:.c.( n:::''''/):::X..:....::S:::fI....:·e....:" g~t:::h....:(....:iI::."'--1O'--r __ n-"lQ"-"-"'ty...:.)-=X.:....:2:.::8=. 2 oleIC ael( ) = -weight of sample (gms)
(3)
(Eq. wt. of Oleic acid is 28.2)
Acid Value (AV) = %FFA (oleic acid) X 1.99
(4)
Apart from free fatty acids (produced from hydrolysis) analysis by titration, they are
also qualitatively shown by GC-mass analysis. For that, the free fatty acid samples are
converted into methyl esters using the AOCS Official method Ce 2-66. At first, the
hydrolytic mixture (Sml) is treated with 4ml NaOH (O.SN) in ethanol solution. The
mixture forms immiscible two layers viz. aqueous and organic phase. It contains free
fatty acid (mainly oleic acid) in aqueous phase and tri, di and mono glycerides in the
organic phase. The aqueous phase is then evaporated to its mass and methyl esters are
fomled by employing AOCS Official method Ce 2-66. 200mg sample is taken in
SOml reaction flask. Then 4 ml of O.SN NaOH is added, attached a condenser and
boiled for 1 min. The sample is cooled and 4 ml of BF}-methanol reagent is added
through the condenser and boiled for 1 min longer. Then, it is cooled to 40-S0°C and
Sml isooctane is added. After boiling for I min, it is removed from heat and 15ml of
saturated sodium chloride solution is added. The flask is stopperd and shaked
vigorously for 15 seconds while the solution is still tepid. Sufficient amount of
saturated sodium chloride solution is added to float the methyl esters dissolved in
isooctane into the neck of the flask. Then the isooetane layer is separated using
separating funnel. Then, Iml of isooetane solution containing the methyl esters is
trnnsferred into a test tube and a small amount of anhydrous sodium sulfate is added.
'1'ti/D. 'lJiesis 68
fmmo6ifization rrtcfiniques andJ{yararytic)tctivities of ifree and lmmobifized Lipases Cliapter J
The dry isooctane is injected directly 111 GC -Mass. The esterification process IS
presented in Scheme 3.6.
Esterification of free fatty acids (For quantification of [TA)
o o II BF, - Methanol II
R-C-OII R-C- OCH3
frec fatty aeid Fatty acid methyl ester
Scheme 3.6: Esterification of free fatty acids
EIl:yme Activity Ullit alld Specific Activity
Enzyme activity unit (e. \I.) is the most commonly used standard unit to describe an
enzymatic activity sometimes referred to as the International Unit (lU). One unit of
enzymc activity can be defined as the amount of enzymc that catalyzes the
consumption of I ).lmol of substrate, or the liberation of [ ).lmol of product per minute
under the specified conditions (temperature, pH, buffer strength etc).
J.l. mol 1 e.u.= ---
rom (5)
An enzyme activity unit (e. u.) can quantify the activity of a certain amount of
enzyme. However, the activity unit is not enough to determine the quantity of the
cnzyme. Thus, an enzyme's activity is described by its specific activity. Specific
activity is defined as the number of enzyme activity units per unit mass of enzyme. [n
this particular enzyme, lipase, fatty aeids released by lipase is used as a function to
express the lipase activity. Thercfore, onc unit of lipase activity is defined as I [Imol
fatty acids released per minute of reaction time.
Activirj Specific activity = ------
Protein (mg)
(6)
Q'Il.<D. 'Inesis 69
I mmo6ifizatioll fJ'er:Imiques Qm{'}{yarofytic )leti'!lities of rrree alia Immobifizei Lipases Chapter J
Comparison with free enzyme activity can be done by retention of specific activity
(Pujari e/ ai., 2006).
Sp_ activiLY of immobilized biocatalytic membrane
Retention of specific <lctivity (%) = X 100
Sp_ activity or free lipase (7)
3.3.3 Influencing factors in hydrolytic reactions
The hydrolytic reaction is carried out at various pH's, temperatures, incubation times
and substrate concentrations and their effect on hydrolytic activity of free as well as
immobilized lipase is determined.
pH of the reaction medium
The effect of pH in the range of (5.0-9.0) on the activity of the free and immobilized
lipase is investigated at 30 and 37°C temperature respectively. As pH affects the
stability, structure, and function of globular proteins due to its ability to influence the
electrostatic interactions, the extent of hydrolysis is different at different pH. It is
observed that at pH 8.0 the hydrolytic activities of both free as well as immobilized
lipases are found maximum.
Temperature of the reactioll medium
For temperature dependence study the activity of free and immobilized lipase is
measured at temperature ranging from 20 to 50°C. As the temperature increases, more
molecules have enough kinetic energy to undergo the reaction. But, each enzyme has
an optimum temperature at which it works best. It is found that lipase activity is
maximum at 30°C for free lipase and 37°C for immobilized one for olive oil
hydrolysis.
Incubotioll time
The effect of incubation time in the range of (10-50 min) is detcnnined. Hydrolytic
activity of lipase greatly depends upon incubation time. It is seen that at 30 minutes
incubation time the hydrolytic activity of lipases is maximum and beyond it, activity
bccomcs stcady.
!J'fz.r]). 'Inesis 10
Immobifization rr'edmiqueJ <waJ{yarof),tic Jlctit:ities of rJ"ree and Imm06ifj~e' LipaseJ Chapter J
Substrate cOllcelltratioll
The effect of substrate concentration is studied by preparing oil emulsions having
concentrations in the range of (30-270111M). It is seen that at 150mM of olive oil
concentration hydrolytic activities of immobilized lipascs are maximum.
3.4 Killetics of Hydrolytic reactiolls
The kinetic paramctcrs (Km and Vmax in Michaelis-Menton cquation) are dctcnnined
for hydrolytic reaction of oils by examining initial reaction rates (Jericevic and
Kuster, 2005). The hydrolytic reaction is initiated by adding free lipase (lml, 2mglml)
or lipase immobilized membranes of 6cm2 or immobilized PS globules (lgm) in the
reaction medium. The reaction is carried out in a shaker (ISO rpm) with an emulsion
containing varying concentrations (30-270mM) of oils at pH 8 for 30 min. The assays
are perfom1cd at 30 0 e with free lipase and at 37°e tempcratures for immobilized
lipases.
The evaluation of kinetic parameters is carried out experimentally. By Michaelis
Menten equation kinetic parameters (Vrnax and Km) for both free and immobilized
lipase are calculated by fitting into two linear fom1s (Lineweaver-Burk and Hanes).
Linewcaver-Burk equation (Lineweaver and Burk, 1934)
1 Kill 1 1 -=----+--V V =, [S] V"" (8)
Hanes equation (Hanes, 1932)
S S Km -=--+--V Vrnax Vma.~ (9)
Where V is the initial reaction velocity, Km is the Michelis-Menten constant, Vmox is
the maximum reaction velocity and [S] is the substrate concentration
Km is independent of the enzyme and substrate concentrations and indicates the extent
of binding and atTinity between the cnzyme and its substrate for a given substrate
concentration, a lower Krn indicates a greater extent of binding/atTinity. Vrna, is defined
as maximum reaction velocity and depends on the enzyme concentration. K", and v,,,,,,.
Pri.rD. 'TIlesis 71
Immo6ifization rrecnniques amfJ(yarl)[ytic jlcth'ities of Cfree ana Immo6ifizea Lipases Cnapter J
both may be influenced by the cllarge and conformation of the enzyme and substrate,
which are detennined by the solution pH, temperature, ionic strength and other
factors.
3.5 Reusability featllre alld variation of hydrolytic activity of lipase with treatmellt
(pH amI temperatllre)
Though the immobilization of the lipase facilitates the reuse of the enzyme retaining
its activity, the repeated use of the immobilized enzyme might lead to a little
deactivation of the enzyme. So, the experiments are carried out to find out the
durability of lipase immobilized membranes/globules towards their reuse. The
reaction is done for hydrolysis of olive oil at optimum conditions (pH 8, temp 37°C
and reaction time 30 min). At the end of each batch, lipase immobilized membranes
are separated and used for the next hydrolysis cycle. Five such cycles of hydrolysis
reactions are conducted and the residual activity is compared with the first run
(activity defined as 100%).
Enzymes have their activity in only an optimum pH and temperature range. So, the
pH and thennal stability studies of enzymes are very important parameters. The pH
stabilities of the free and immobilized lipascs arc assayed by immersing them in
buffer solutions in the pH range of 3-13 for different time periods (0.5-4h) at 37°C.
After immersion they are used to detennine their hydrolytic activities. The relative
activities are calculated as the ratio of the activity of immobilized lipase/free lipase
after incubation in different pH and the activity at the optimum pH.
The thennal stabilities of the free and immobilized lipases are also assayed by
immersing them in buffer solution (O.IM, pH 8.0) at the temperature range of 20-
70°C for different time periods (20-80 min). Then the membranes with immobilized
lipases and free lipases are used to study their activities. The relative activities are
calculated as mentioned above.
lPn/D. 'Iliesls 72
Immo6ifization 'Tecliniques anaJfyarofyticfictivities of (Free ana 1 mmo6iftzea Lipases Cliapter J
Exploitatioll Of immobilized ellzyme ill large scale applicatiolls
The catalytic PS mcmbranes and globules prepared by above immobilization methods
are used in immobilized enzyme reactors. These reactors are applied for large scale
applications in hydrolytic reactions (section 1.5.2). The important paramcters of these
reactors are type of reaction, solvent (if any), substrate(s), reactor configuration,
support matrix, and immobilization method. An overview of the various reactor
configurations cmploying immobilized lipases is presented below.
3.6 Fabricatioll of immobilized ellzyme reactors
Thrce types of immobilized enzyme rcactors are configurcd at laboratory scalc viz.
Biphasic membrane, Packed bed and Hollow fiber reactor. These reactors are applied
for different oil hydrolysis.
The degree of hydrolysis, X is calculated as below:
X% (m! NaOH used) (molanty ofNaOH) (average molecular weight offatty acid)
IO(weight of sample)
3.6.1 Bip/wsic ellzyme membralle reactor
(10)
It is of II- shaped. The two anns are tilled with organic and aqueous phases. It is made
up of glass and consisted of two identical flat channels with arrangemcnt for holding
membrane (membrane area: 9.1 cm2, membrane shape: circular disc) between two
compartments. The immobilized membranes are fixed at the junction of the two arms.
As the two arms are filled with two phases it is called biphasic one. The two phases
are separated by a biocatalytic membrane on which lipase is immobilized. Fig.3.6
shows the experimental set-up of the biphasic enzyme membrane reactor. The set up
is placed on magnetic stirrers. Lipasc immobilizcd PS mcmbranc (15% w/w) is
prepared according to the procedure described in section 3.1.1.1.
The reaction and the simultaneous separation at the membrane surface take place,
where the membrane is used both as catalyst support to provide better/more selective
contact with the reactant and for selcctive removal of product(s). The hydrolytic
experiment is controlled by diffusivc way. The reactor offers high specific surface
area, simultaneous reaction, reuse of the enzyme and continuous operation of the
process.
Ph.'D. 'ITtesis 73
imfTIobifizatioll tTecfi.niques ana:Hyaroljtic jfctivities of (j"ree ana imlllo6ifizea Lipases C!iapter 3
In order to perform an enzymatic reaction measurement, 50ml of olive oil in isooctane
is filled in one atm facing the immobilized PS membrane side, while 50ml of O.IM
phosphate buffer solution at pH 8.0 is filled in another ann. All experiments are
can·ied out at 30°C. A series of oil concentrations in the range of (0.05- 0.30 M) in
isooctane solvent are used. Sample (0.51111) is taken from the organic phase at ditTerent
time intervals and free fatty acid concentration is detennined. Various parameters are
studied for hydrolysis of different oils (olive oil, palm oil, castor oil).
Diameter 4.5 <In i-+--.;oi
AI]1Jl"I1US
phase
Lipase immobilized PS membrane
Organic phaSE!
Figure 3.6: The experimental set-up of the biphasic enzyme membrane reactor
Influencing factors for hydrolysis
pH of aqueolls phase
The effect of pH in aqueous phase on hydrolysis is studied in the range of 5.0 to 9.0. It
is avoided to carry out the experiment in drastic pH conditions (strongly
acidic/alkaline) as lipases are unstable in that condition. Moreover, the partition of
fatty acid into aqueous and organic phase is affected unfavorably at lower pH. It is
observed that hydrolysis is maximnm at pH 8.0 for all the three oils.
Different solvents
As the catalytic activity of lipases strongly depends on the solvents (Carrea el al.,
2000) the experiments arc carried out for hexane, heptane and iso-octane solvents.
The effect of these three different solvents is evaluated for the hydrolysis of oils in
enzyme membrane reactor. It is seen that in iso-octane solvent hydrolysis is maximum
for all the three oils.
Ph/D. 'Iliesis 74
Immo6iCizatioIL rrecfiniques amf'){yarvfytic )tcti1;ities of tFree alia Immo6ifized Lipases Cliapter J
Reaction tillle
The effect of time period is detennined for the perf01111ance of enzyme membrane
reactor. The reactor is operated at different time intervals (2, 4, 8, 12,24,28 h). It is
seen that up to 24 h, the degrce of hydrolysis is in incrcasing trend and it bccomes
steady above this duration.
Substrate concentration
The effect of substrate conccntration is examined for the hydrolysis reaction. Enzyme
membrane reactor is operated at different concentrations (0.05, 0.10, 0.15 and 0.30M)
of oils (olive oil, palm oil, castor oil) dissolvcd in iso-octanc. The experiments
showed that hydrolysis is maximum at 0.05 M for the three oils.
Nature of oils
The hydrolytic experiments are carried out for different oils (olive oil, palm oil, castor
oil). The reaction is done at 30 0e for 24 h using O.IM of phosphate buffer of pH 8.0
m aqueous phase and 0.05M oils in orgamc phase with lipase immobilized PS
membrane.
3.6.2. Packed bed En:;;yme reactor
The packed bed reactor is one of the most commonly employed for catalysis. In this
study, the packed bed reactor system is employed for lipase-catalyzed hydrolysis of
palm oil. Thc Packed bed reactor column with dimensions of 1.2 cm (i.d) x 56 cm
length is used. The lipase loaded PS globules (already described) are used as packing
material in this colunm. These globules are prepared by wet phase inversion technique
using syringc. The globulcs arc porous and have asymmetric structure with lipase
entrapped on surface and in pores. The attractive feature of these globules is 'lipase
immobilization' that occurs at the time of globule preparation. Fig. 3.7 shows the
expcrimental set-up of the Packed bed reactor. It allows reuse of the cnzymc without
need of a prior separation and permits to handle substrates of low solubility by using
largc volumcs containing low concentrations of substratc. It lcads to morc consistcnt
product quality and improved enzyme stability due to the ease of automation and
control. It is suitable for long-tcml and industrial scale production compared to
stirred-tank reactor whcrc cnzymc particles are susceptiblc to breaking because of thc
CPh.!fJ. 'Tflesis 75
Immo6ifization rTl!cfiniques amf'){),aro(yticfictiy;ities of 'Free ana Imm06iazea Lipases Cfiapter 3
mechanical shear stress. It is more cost effective than the batch operation (Xu et aI.,
1998; Laudania ct aI., 2007).
Palm oil cmulsion is used as substrate for reaction mixturc. The reaction is donc at
Jooe using 0.05 M palm oil emulsion in O.IM of phosphate buffcr (pH 8.0). The bcd
height and flow rate is varied. The substrate mixture is fed upwards through the
column. The present work is also paying attention on the reaction parameters that
affected lipase-catalyzed hydrolysis of palm oil to better understand the relationships
between the reaction variables (packed bed height, flow rate, pH, time and substrate
concentration).
Lipase loaded
PS globules
Figure 3.7: The experimental set-up of the Packed bed reactor
Ph. rD. 'Iliesis 16
L I' (Ji
I mmoMizatio" rechniques ani 1fyiro(ytic .WcW,ii~~ bY1'm Ii\,llir"j~jj,uO.2'lipases Chapter 3
lllflllellcillg factars for hydrolysis:
pH ofreactiollllledilllll
IIydrolysis reaction is dependent on pH of reaction medium. The effect of pH is
dctcl111ined in the range of (5.0, 6.0, 7.0, 8.0 and 9.0) for palm oil hydrolysis. 1t is
observed, that at pH 8.0 degree of hydrolysis is maximum in reactor.
SlIbstrate cOllcelltratioll
The elTect of substrate concentration is examined for palm oil in the range of (0.05,
0.10, 0.15 and 0.30 M). The reaction is operated at 30°C using palm oil emulsion in
O.IM of phosphate buffer (pH 8.0). It is observed, that at 0.05 M of palm oil the
degree of hydrolysis is maximum.
Reactioll time
The effect of reaction time on the hydrolytic activity of lipase is investigated by
operating the reactor at different time intervals (1- Sh). It is seen that up to 6 h degree
of hydrolysis is in increasing trend and it becomes steady above this duration.
Physical parameters (bell height, flow rette)
The effect of bed height is evaluated for (16, 36, 56 cm). The reactor is opcrated at
different bed heights for 6h and at 200 mllmin flow rate. It is found, that at 56 cm bed
height the hydrolysis is maximum for the reactor.
The effect of flow rate is also studied in the range of (100,200, and 400 ml/min) on
the perfol111ance of reactor. Results show that at 200 mllmin flow rate, degree of
hydrolysis is maximal for the reactor.
3.6.3 Hollow jiber Ellzyme reactor
In this reactor hollow fiber membranes are used as a carricr to immobilize lipase for
hydrolysis of oil. Hollow fibers are cylindrical in shape and hollow in nature. The
operation throngh reactor is simple and easy. Compared to flat sheet it is of high
specitic surface area. The high specific area is of advantageous in terms of
simultaneous reaction and continuous operation. As there is the feasibility in lipase
immobilization because of its high surface area the extent of hydrolysis is more in the
pli. (D. 'Thesis 77
Immobifization 'Tediniques alJ([1{yaroryticActivities of 'Free alia Immobi(iua Lipases Cliapter J
hollow fiber EMR. In the present study commercial hollow-fiber reactor module
having 80 fibers and area 213.52 cm2 is used. The fibers are made up of 20 % PS,
inner diameter 0.5 mm, outer diameter 0.6 mm and length 17cm. The set up of hollow
fiber reactor is presented in Fig. 3.8. 150 ml lipase solution (containing 2.5 mg/ml in
Phosphate buffer, pH 7.0) is circulatcd up to 15 h at flow rate of 185 mi!min and
O.IMPa pressure at room temperature to immobilize lipase on hollow fibers. The
immobilization depends on the duration (I-ISh) is studied. Finally, 100 ml of the
same phosphate buffer solution is pumped through the reactor to remove any soluble
enzyme that had not adsorbed during the inmlObilization procedure. Palm oil
hydrolysis is carried out in this hollow fiber EMR. The hydrolytic reaction is carried
out using 0.05 M palm oil emulsion (200ml) in 0.1 M of phosphate buffer of pH 8.0.
The emulsion is circulated in the lumen of the hollow fibers for 6 h at 0.069 MPa
pressure at 30oe. The free fatty acid analysis is done in 30 min interval using
titremetric method.
Oil reservoir
Pressure
Hollow fiber module
with lipase immobili2ed
Pump
Figure 3.8: The set up of hollow liber reactor
Ph.i]). 'Iliesls 78
Imm06ifization Techniques antfJ0,'tfrofjtic)lctivities of (Free alia lmmobifizea Lipases Chapter J
Illfluencing factors for hydrolysis
Applied pressure
The effect of applied pressure is studied up to 0.1 MPa for hydrolysis of palm oil. The
flow rate dcpends upon pressure and degree of hydrolysis is on flow rates. It is
observed that upto 0.069 l'v1Pa pressure the extent of hydrolysis is increased.
Reaction tillle
The effect of reaction time IS studied by operating the reactor at different time
intervals (1/2- 8 h) for hydrolysis of palm oil. It is seen that up to 6 h the degree of
hydrolysis is in increasing order and it becomes steady above this duration.
The chapter is dealt with different immobilization methods and hydrolytic activities
are also experimented. The results and discussion of all the above experiments are
detailed in following chapter.
rpfz.<D. 'Iliesis 79