Artificial Cells, Blood Substitutes, and Biotechnology

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Kinetics Study of Palm Oil Hydrolysis Using

Immobilized Lipase Candida Rugosa in Packed Bed
Reactor

Choo Seng Min, Subhash Bhatia & Azlina Harun Kamaruddin

To cite this article: Choo Seng Min, Subhash Bhatia & Azlina Harun Kamaruddin (1999)
Kinetics Study of Palm Oil Hydrolysis Using Immobilized Lipase Candida�Rugosa in Packed
Bed Reactor, Artificial Cells, Blood Substitutes, and Biotechnology, 27:5-6, 417-421, DOI:
10.3109/10731199909117713

To link to this article: https://doi.org/10.3109/10731199909117713

Published online: 11 Jul 2009.

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ART. CELLS, BLOOD SUBS., AND IMh4OB. BIOTECH., 27(5&6), 417421 (1999)

Kinetics Study of Palm Oil Hydrolysis Using Immobilized Lipase

Candida Rugosa in Packed Bed Reactor
Choo Seng Mia. Subhaah Bhrtir and Azlins Harun Kamrruddin

School of Chemical Engineering, Universifi Sains Malaysia

Perak Branch Campus, 31 750 Tronoh, Perak Malaysia

ABSTRACT

Continuous hydrolysis of palm oil triglyceride in organic solvent using immobilized Condida rugma on the Amberlite
ME-I as a source of immobilized lipase was studied in packed bed reactor. The enzymatic kinetics of hydrolysis
reaction was studied by changing the substrate concentration, reaction temperature and residence time(7) in the reactor.
At 55 'C. the optimum water concentration was found to be 15 %weight per volume of solution (%w/v). The
Michaelis-Menten kinetic model was used to obtain the reaction parameters, K,(app) and V,(app). The activation
energies were found to be quite low indicating that the lipase-catalyzed process is controlled by diffusion of substrates.
The Michaelis-Menten kinetic model was found to be suitable at low water concentration 10 - IS %w/v of solution. At
higher water concentration, substrate inhibition model was used for data analysis. Reactor operation was found to play
an important role in the palm oil hydrolysis kinetic.

Keyuordr
Palm Oil Hydrolysis, Kinetics Model, Amberlite ME-I, Packed Bed Reactor, Lipase

INTRODUCTION The Colgate Emery continuous oil or fat splitting

Palm oil is used mainly for edible purposes and have
become important raw materials for fatty acid
manufacture during past decade. Increased interest in the
chemistry and biotechnology of palm oil has emerged in
the recent years, which is mainly attributed to the fact
that oleochemicals are derived from renewable sources.
By virme of their economic and ecological advantages,
oleochemicals compete successfully with long chain
acids, alcohol and their derivatives of petrochemical
origin. With recent advances in bioreactor technology
and genetic engineering, many new and interesting ideas
for employing biotechnology to produce oleochemicals
from oil have been investigated.
The properties of oils depend overwhelming on their
glyceride composition and to a lesser extent on their
minor components. But glycerides composition is
usually very complex and physical properties can often
be explained with sufficient accuracy by fatty acid
composition.
Hydrolysis is a reaction that cleaves the triacylglycerol
and the main objective is for the production of free fatty
acid and glycerol. Under control conditions using
specific lipases, cleavage can occur at specific positions
for the production of fatty acid of interest, mainly
essential fatty acids (EFA) or the polyunsaturated fatty
acid. The reaction is showing below:
Triglycerides + )(Water) o Glycerol + Fatty acids
R'- (COOR), t 3H20o R'- (OH), t 3RCOOH
process is currently the most widely employed industrial
process for fat andoil hydrolysis (Fang and Yi, 1995)
Enzymatic hydrolysis of oil is an alternative process
since the reaction is carried out at ambient temperature
and pressure. The main reason for considering enzyme
application in lipid processing is because of its mildness,
both in the equipment set-up and reaction condition,
which result in the saving of energy and the
minimization of thermal degradation, hence reducing the
financial constraints encountered in the process
construction (Ibrahim Che Omar, 1997). The increasing
use of immobilized enzymes in enzymatic hydrolysis is
of particular interest. Lipase is generally immobilized on
hydrophobic microporous supports by physical
adsorption; in addition, leakage of lipase may be made
negligible (Malcata, el a/., 1990)
In previous works, continuous hydrolysis of palm kernel
olein with immobilized lipase Rhizopus arrhinus in
packed bed reactor had been done (Fang and Yi, 1995).
Optimum hydrolysis degree of palm oil by the lipase
Aspergillus niger at different operating conditions was
reported'. The effectiveness of hydrolysis olive oil with
lipase Candida rugosa by using different material
support had also been reported (Ibrahim Che Omar,
1992).
The main objective of the present work is to study the
kinetics of the immobilized lipase Candida rugosa for
the palm oil hydrolysis process in the packed bed
reactor. The kinetics is studied by determining the
enzyme reaction at different conditions. The enzyme
kinetics parameters are found by analyzing the
laboratory data that are obtained from packed bed
reactor.

417

Copyr~ghtQ 1999 by Marcel Dekker, Inc www.dekker.com

418 MIN,BHATIA, A N D KAMARUDDM

MATERIAL AND METHOD

Raw Material and CbemicaLc I nrrn

Palm olein oil was supplied by VESAWIT cooking oil,
which is 100% refined palm olein oil. Lipase
(EC3.1 . I .3) Type VII from Candida rugosa was supplied
by Sigma Chemicals. Lipase contains lactose as an
extender, substantially bee of a-amylase and protease.
One unit of lipase will hydrolyze 1.0 microequivalent of Solutio
fatty acid !?om olive oil in I hour at pH 7.2 at 37OC (30-
min incubation). n-heptane was used as an organic
solvent in the hydrolysis process. Support material,
which is used, is Amberlite MB-1 €rom Organo.

Preparing Immobilized Lipase Hot Plate pump

Support material Amberlite MB-l (6 g) were stirred at

2OOrpm for 2 hour at room temperature in 100 ml of
phosphate buffer solution @H 3) containing about 200 U
of enzyme activity6. Then, the immobilized enzyme
preparation was washed thoroughly with distilled water
1 D T I - Digital Temperature Indicator
RCWB - Re-circulating Water Bath
~~

and rinsed with n-heptane. Figure I:Flow Diagram for the Reactor System

Experimental Set Up
RESULTS
The system was set up as a down flow system. A
thermocouple and Digital Temperature Indicator were Initial appraisal of the main factors influencing the
used for measuring the temperature of the enzymatic hydrolysis of palm olein oil was made using packed bed
reaction. The re-circulating water bath was used for reactor (PBR). Three oil concentrations (10% wlv,
getting and maintaining the desired temperature of the 15%w/v and 2O%w/v), four temperature (45 'C, 50 OC,
enzymatic reaction. A magnetic stirrer was needed to stir 55 OC and 60 OC), and six substrate flow rate (0.5,0.75,
and mix the palm oil and water, together. A peristaltic 1.0, 2.0, 3.0, 4.0 mumin) were examined in a
pump was used to pump the feed solution to the reactor experiment. The PBR was packed with 0.2 g of
system. A by-pass valve was used to reduce the high immobilized lipase. The enzyme was changed after
pressure existing in the tygon Nbe after the pump. A completing the nine experimental runs. The conditions
rotameter controlled the flow rate of the feed solution were carefully selected in order that the reactor was
fed to the reactor system. The immobilized enzyme was operating differentially.
placed between glass filter (glass wool). Figure I shows
the flow diagram for the whole reactor system. Because of the physicochemical nature of these systems,
the meaning normally attributed to K,,, and V,, in
Experimental Procedure enzyme kinetic studies does not apply and the term
apparent K, Kdapp)l and apparent V, [Vda~p)l
The reactor was loaded with 0.2 gram of the are used here.
immobilized lipase particles. The reactor was set to the
temperature of 45'C. The substrate solution, which was The system kinetic parameters K,(app) and V,,(app)
40% w/v of oil and 15% w/v of water in n-heptane, was were estimated using the Michaelis-Menten equation.
stirred by using magnetic stirrer. Then, flow rate 0.5
mVmin was adjusted. The product was collected every 5 vm CA dCA
minutes and was analyzed by titration method using -rA= -- ______-----
(1)
NaOH'. The reaction was carried out until the reaction K, + CA dt
reached steady state condition. The above procedure was
repeated for different condition of temperature (5OoC, where rA = reaction rate of substrate palm oil
5SoC, 60°C), flow rates (0.75 mVmin, 1.0 ml/min. 2.0 V,, = the maximum reaction rate
ml/min, 3.0 mllmin, 4.0 ml/min) and substrate CA = palm oil concentration
concentrations (10%. 20% w/v of water with the same % K, = Michaelis constant
w/v of oil). t = time for experiment run
 PALM OIL HYDROLYSIS STUDY 419

For PBR, Effect of Temperature

X
Space time, T = CAo (I/-r) dX ------ (2) The experiments were carried out further on the effect of
the temperature with the same concentration of substrate
0 40%w/v palm oil, the same amount of immobilized
dx v, (1 - X) enzyme (0.2 g), and the same flow rate 0.5 mllmin or
Fromeq(l), - =
space time (T = Wff). The results are shown in Ftgure 3.
dt Km + C A . ( ~- X I

Space time for each run of experiment or residence time,

C o n v e r s i o n X ( % ) o f P a l m O i l vs
Km I CAoX S p a c e Time T
T=- In -+ - ------- (3) a t different Temperature
v, I -x v,, 18
W g (immobilized enzyme) 16
T= _= = (min)
14
F g/min (substrate flowate)

I
-In
1
-=
v,
- - - -__---(4)
CA0X -c 12
10
x 8
T I-X K, K,T 6

The CAo is the initial concentration of palm oil. The

system kinetic parameters K,(app) and V,(app) for
each temperature were estimated from the slope and
intercept ofthe plot - I/T In ( I - X) vs WT. 0 0.05 0.1 0.15 0.2

T (mh)
Effecf of Flow Rates ~~ ~ ~~ ~ ~~~ ~~~

Figure 3: Effect of temperatures on the Conversion. X (%) of

The experiments were carried out to study the effect of Palm oil in PBR (oil concentration 40% w/v in feed, water
concentration 15 %w/v and 0.2 g of immobilized lipase)
flowrate with the same concentration of substrate (40%
w/v) palm oil, the same amount of immobilized enzyme
(0.2 g), and the same temperature (45 'C). The results As the temperature increased, the product FFA ( p o l )
are shown in Figure 2. The different flow rates used and the conversion of palm oil (X) increased. From
were 0.5 ml/min, 0.75 mUmin, 1.0 mUmin, 2.0 ml/min, Table I , it was shown that as the operating temperature
3.0 mumin and 4.0 m h i n . increased, the K,(app) and V,,(app) will increase until
temperature 60 'C with V,(app) = 2.23 mol/(L min)
From Figure 2, the results showed that as the flow rate and K,(app) = I .59 M at 60 'C.
of the substrates increased, the conversion of palm oil
(X) decreased. From Table I , the kinetic parameters are used to
determine the activation energy of the enzymatic
reaction. The activation energy was estimated as 0.04
Conversion X (%) of Palm Oil vs Space kJ/mol and 0.06 kJ/mol calculated from K, and Vm,x
respectively as shown in Table 2.
Time T ,
at temperature 45 C
I
Table I : Apparent Kinetic Constants For Different
15 Tem erature Constant Substrate Concentration
Temperature V,(aPP)
mom min
0.89 1.18
55 1.20 I.62
I 60 1.59 2.23
0 0.05 0.1 0.15 0.2
I
T (min) I
I I Table 2: Activation Energies (E) For lmmobilizcd Lipase
Figure 2: Effect of flow rate (space time) on the Conversion Candida Rugosa
( O h ) of Palm oil in PBR (oil concentration 40 %w/v in feed.
I Activation Energy, E (kllmol)
water concentration I5 %w/v. 45'C and 0.2 g of immobilized K, I 0.04
Iipase) I V, I 0.06
420
Eflect of Water Concenlralion
The experunents were carried out to study the effect of
water concentration with the same concentration of
substrate 40%w/v palm oil, the same amount of
immobilized enzyme (0 2 g) and the same temperature
55 'C The results were presented m Ftgure 4 which
shows that for feed flowrate 0 5 mVmin, as the water
concentration mcreased from 10 % w/v to 15 O h wlv, the
conversion of palm oil mto bee fatty acids FFA was
lncreased €rom 14% to 15% As the water concentration
increased from 15 %w/v to 20 % w/v, the conversion of
palm oil lnto 6ee fatty acids FFA was decreased to
12 86%
Commion X(%) w Water COrtEBhation( O h w/v)
~
I "1
Lk'5
T-- -----I
I0 15 20
water concerntion (%M/v)
FigurF4 Effect of w a t e r e x - ( % w / v ) o n t h e
Conversion, X (%) of Palm oil in PBR at oil concentration 40
%w/v in feed, temperature 55 'C, 0.5 mumin and 0.2 g of
immobilized lipase
Table 3 shows that the kinetic parameters for different
concentration of water. For IO%w/v and 15%w/v water
concentration, the kinetic parameters were nearly
constant with KJapp] = 1.2 M and V,[app] = 1.62
moVL-min. But for high water concentration 20% w/v,
the kinetic parameters were different.
Table 3: Apparent Kinetic ConstantsCalculated For Different
Water
K.(~PP) "-(VP)
Concentration (MI (mom min)
(% w/v)
10 I .30 1.53
I5 I .20 I .62
20 2.03 2.04
DISCUSSION
Residence time or space time (F W/F) for hydrolysis
reaction of palm oil and water in the reactor decreased as
the flow rate of the substrate increased. As the flowrate
of the feed substrates increased, the hydraulic forces
produced by the flow will be higher than the interaction
forces between substrates and the active site of the
enzyme. As a result, substrates will directly flow through
the enzyme without interacting with the active site of the
enzyme. Besides that, some of the interaction bond
between the substrates and active site will be broken by
MIN, BHATIA, AND KAMARUDDIN
the high hydraulic flow of the substrates. Chances for the
palm oil and water to contact with the active site of the
enzyme will decrease as the flow rate of the substrates
increased. lntraparticle or internal diffusional resistance
influenced the kinetics of immobilized enzyme reaction.
The enzyme is usually distributed within a porous carrier
or support matrix, and substrate molecules have to
diffise through the carrier or support to reach the
reaction site.
By using the immobilized enzyme, the enzyme can be
used at higher temperature operation in hydrolysis of
palm oil. It is due to the thermal stability of the pure
enzyme Candida rugosa was highly increased by the
immobilization operation with the carrier or support
Amberlite ME-I. The optimum temperature for free
lipase Condido rugosa is 37 OC (Malcata, er al., 1992)
I
The activation energies, which are 0.04 and 0.06 Wlmol
are very low and may indicate that the lipase-catalyzed
process is controlled by diffusion of substrates.
The optimum water concentration to get the highest
conversion of palm oil to free fatty acids FFA (the
highest hydrolysis) was at 15 %w/v with the oil
25 concentration 40%w/v. The conversion of palm oil at
water concentration 20%w/v was lesser than at water
concentration 10% w/v. It is due to water concentration
having an effect on the hydrolysis rate and excess water
(bulk water) can also act as subsbate inhibitor for the
enzyme. High water concentration or excess water will
deactivate the lipase rapidly when the lipase comes in
contact with bulk water. As the water concentration was
high, the water molecule will compete with the
triglyceride molecule for attaching the active site of the
enzyme. This will lead to lower conversion of palm oil.
The kinetic model "Michaelis-Menten mechanism" was
applied to describe lipase-catalyzed reaction in the
experiment. It was suitable to use for low water
concentrations of 10 % w/v to 15 %wlv. It was not
suitable for describing the lipase-catalyzed reactions in
20 %wlv water concentration. Water in high
concentration will act as a substrate inhibitor for the
enzyme. They will compete with the triglyceride
molecule for the active site in the enzyme. The new
kinetic model (Substrate Inhibitor Model) for high water
concentration was used to describe the lipase-catalyzed
reaction for the system at high water concentration and
is shown as follows:
E +S F) ES + E + P
+
ESI
where E : immobilized enzyme
S : substrate (palm oil or tryglyceride)
ESI : enzyme-substrate-inhibitorcomplex
P : product
I : substrate inhibitor (water)
PALM OIL HYDROLYSIS STUDY
The rate of triglyceride disappeared per unit volume of
reacting fluid, rA, will be represented in term of this
mechanism as:
v- CA dcA
- rA = -
- - _________
K, + C,, + CA'I Ksl dt
where Ks, : Substrate inhibitor constant
This new kinetic model (Substrate Inhibitor Model) will
be used to testify soon whether it is suitable for
describing the lipasetatalyzed reaction at high water
concentration.
CONCLUSIONS
As operating temperature increased from 45 OC to 60 'C.
the yield of the free fatty acids or the conversion of palm
oil into free fatty acids (FFA) was increased. The
enzyme did not denaturated at high temperature because
it was stabilized by immobilization.
As the flow rate increased, the yield of free fatty acids
FFA decreased because there was not enough time for
the substrate to interact with the enzyme. When the flow
rate was higher, space time for the substrate staying in
the enzyme area in the reactor was lesser. The optimum
water concentration for highest yield of free fatty acids
was 15 %w/v with 40% wlv of palm oil.
The activation energies of the enzymatic reaction were
0.04 kJ/mol and 0.06 kJ/mol. The activation energies
were quite low indicating that the lipase-catalyzed
process is controlled by diffusion of substrates.
ACKNOWLEDGEMENT
The authors acknowledge the research grant provided by
Univeniti Sains Malaysia, Penang that has resulted in
this article.
42 1
REFERENCES
Fang C. H. and H.S. Yi (1995), Continuous Hydrolysis
of Palm Kemal Olein with Immobilized Rhizopus
arrhizus Lipase in a Packed Bed Reactor, Journal of The
Chin. I. Ch. E., Vol. 26, no. 6.
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lbrahim Che Omar, (1997), Biotechnology of Lipolytic
Enzymes for the Oleochemical Industries: A Research
Perpective", Journal ofBioscience, Vol. 8, Issue I.
lbrahim Che Omar. Naor lzani Noor Jamil, (1992).
Hydrolytic behaviour of Palm Oil by the Lipase of
Aspergillus Niger", Proc. 2" Symp Ma1 Soc Appl Biol.,
96-103.
lbrahim Che Omar,R. Suguna, (1993), Characteristics of
Immobilized Lipase by Adsorption Method on
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Malcata, F.X., H.R.Reyes, H.S.Garcia, C.G.HiII,
C.H.Amundson, "Immobilized Lipase Reactors for
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the American Oil Chemisr's Society, Vol. 67, no. 12,
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Malcata, F.X., H.R.Reyes, H.S.Garcia, C.G.HiII,
C.H.Amundson, June (1992). Kinetics and Mechanisms
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