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