ART. CELLS, BLOOD SUBS., AND IMMOB. BIOTECH.

, 27(5&6), 393-398 (1999)

Studies on the Kinetics of Isopropyl Palmitate Synthesis in Packed Bed Bioreactor

Using Immobilized Lipase
Chua Ye0 Gek Kee, Masitah Hassan and K. B. Ramachandran
Chemical Engineering Deparfment, fJniversi@of Malaya
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ABSTRACT

The objective of this research was to study the kinetics of synthesis of a commercially important ester - lsopropyl Palmitate
(IPP) using immobilized lipase (Lipozyme IM).It was studied in a packed bed differential reactor. In order to establish the
kinetics of the reaction, parameters such as linear velocity of the fluid through the reactor, particle size, substrate
concentration, substrate molar ratio, temperature and water activity were studied. Operational and storage stability of the
enzyme were also assessed. The reaction followed Michaelis-Menton kinetics as observed from the relationship of initial
rate of the reaction as a function of substrate concentration. It was found that the optimum substrate concentration was
0.15M palmitic acid and isopropyl alcohol in 1: I stoichiometric ratio. Inhibition by excess of isopropyl alcohol has been
identified. The optimum temperature for the esterification reaction was found to be around 50T. The activation energy of
this process was determined to be 43.67 kllmol. The optimum water content was 0.50%. The reaction rates were measured
in the absence of any significant external diffusional limitations. Since internal diffusional limitations could not he
eliminated. the kinetics observed is only apparent.

Keywords
Kinetics, Immobilized Lipase. Esterification, Packed Bed Reactor, Lipozyme
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INTRODUCTlON facilitates easy separation of products. Therefore,

Conventional chemical methods for producing esters
involve direct esterification at high temperature ( 1 00-
300°C) and pressure in the presence of a mineral acid as a
catalyst. These involve high energy cost, undesirable
product formation and difficulty in separating and
recovering the product (Al-Saadi and Jefieys, 1981). As a
result, triacylglycerol lipase (EC 3.1.1,3), a biocatalyst, has
been used to replace the conventional chemical
esterification process. Reactions catalyzed by lipase occur
under mild operating conditions, offering environmental
advantages and low energy costs. Also, because of their
specificity and selectivity, the fraction of unwanted
intermediates and by-products is reduced, giving products
of high purity and improved quality.
For lipase-catalyzed esterification reactions, one requires a
nonpolar environment with a low water content to shift the
thermodynamic equilibrium in favor of esterification over
hydrolysis. Therefore, most of the studies dealing with
lipase-catalyzed ester synthesis are carried out in organic
solvents (Klibanov ef al., 1986 & 1989). It also offers the
advantages of high solubility of substrate and product. and
controlling microbial contamination (Halling, 1990 &
Khmelnitsky ef a / ,1988).
In industries, utilizing soluble enzymes in large scale may
not be cost effective. Therefore, immobilization of
enzymes on appropriate support matrices is a possible
solution to the problems caused by the high cost of enzyme
isolation and the poor stability of soluble enzyme. It
provides reusability and hence reduces operational cost.
Besides, it also reduces enzyme contamination and
continuous operation is possible. An immobilized lipase
reactor is a more cost-effective alternative than a soluble
lipase reactor (Victor ef a/.,1996). Packed-bed reactor
(PER) is a configuration that is most suitable for use in
ester synthesis because they continuously remove water
and product, which is important in bringing the reaction to
completion. '
The aim of the present work is to develop environmentally
friendly processes that are based on lipase biocatalysis for
the production of lsopropyl Palmitate. Isopropyl Palmitate
is used in topical pharmaceutical and cosmetic
preparations as a non-oleaginous emollient with very good
spreading characteristic. The reaction was carried out in a
packed bed reactor with isopropyl alcohol and palmitic
acid as the substrate. Effect of various parameters on the
synthesis of isopropyl palmitate was studied. Operational
and storage stability of the enzyme were also evaluated.
MATERlALS 81METHODS
MBteI'iBlS
Lipozyme IM (LUX 0106) was supplied by Novo Nordisk
N S , Denmark. It has been derived from a selected strain
of Mucor miehei. Lipozyme IM belongs to the class of
triacylglycerol hydrolases (EC 3. I . I .3), Palmitic acid 98%
(A9816) was obtained from Palm Oleo Sdn Bhd. Kuala
Lumpur. Hexane with 99% purity was purchased from
Merck. Germany and lsopropyl alcohol from Riedel-de
Haen, Germany.

393

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 394 KEE, HASSAN, AND RAMACHANDRAN

Reactor
The reactor (Chromatlex OR Column) consisted of a
tubular glass column of 10 mm ID and 30 cm long. It was
also provided with a water jacket for temperature control.
The immobilized enzyme packed into the reactor was
retained in place by means of a fine wire mesh with cotton
wool. The substrates were fed from the bottom by a
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rate employed was 1.0 ml/min. The duration for the
experiment was 24 hrs for 40°C and 6 hrs each for 50°C &
60°C. Figure I shows that the enzyme is stable when used
continuously for 24 hrs at 4 0 T . The activity of the
enzyme was unchanged when operated at higher
temperature continuously for 6 hrs.
Besides, operational stability in the short term, stability of

plunger type HPLC pump (Waters 510) and the products the enzyme in the long term was also studied. The
were withdrawn from the top. Water from a constant experiments were carried out at two concentrations, 0.05M
temperature bath was circulated through the jacket by a and 0.15M,at 45°C at a flow rate of 0.5 ml/min. The
peristaltic pump (B. Braun FE41 I ) at high speed (EOrpm) duration of the experiment was about 4 days. Figure 2
for temperature control. shows the drop in % initial activity of the enzyme with
respect to time. The half-life of the enzymes is 44 hours
Experimental Methods for low concentration and 32 hours for high concentration
respectively. Since the enzyme was stable for 6 hrs at all
Enzyme was packed into the column by settling it in the temperatures, for kinetic studies, one batch of enzyme was
substrate solution. Esterification reactions were then not used for more than 6 hrs. After each 6 hrs of operation
carried out by passing the substrate through the column. fresh enzyme was loaded into the column.
The temperature was maintained at the desired value by
passing water through the column jacket. Samples were
withdrawn for analysis after passing four-bed volume of
substrate through the column at each flow rate. For the
study of external mass transfer resistance. substrate in a
conical flask immersed in a temperature control bath was
circulated through the column at different flow rates for 90
For personal use only.

minutes. At each time interval of 15 min, 1 ml of sample

was withdrawn for analysis. Under all experiments,
conversion across the reactor was kept small so that the
reactor can be assumed to operate as a differential reactor.

Analysis

The samples withdrawn were first dissolved in 20 ml

lsopropyl alcohol and assayed by titration for the residual
acid content using 0.01 M sodium hydroxide with Figure I : Operational stability of the enzyme at different
phenolphthalein as an indicator. The residual acid temperatures
concentration was obtained from a standard graph. Since
the column was operated as a differential reactor, average
concentration of substrate can be calculated from the 120
following equation.
CAvr = % (c#n tout)
and the corresponding initial reaction rate was calculated
from
r = F (C," - C,,,) / Wt
where r = initial rate, g/hr.g immobilized enzyme
F = flow rate, Iihr
Wt = weight of immobilized enzyme
C,", Cau,= concentration at inlet and outlet

RESULTS AND DISCUSSIONS

Operational Stability Figure 2: Operational stability of enzyme at different

substrate concentration
This experiment was carried out at three different
temperatures, 40°C. 50°C and 60°C using 0.01M
equimolar palmitic acid and isopropyl alcohol. The flow
 KINETICS OF ISOPROPYL PALMITATE SYNTHESIS 395

Effect o f Mass Transfer Resistance
250 ml of 0. I M of equimolar solution o f palmitic acid and
isopropyl alcohol was circulated through the column at
45°C. Esteritication was carried out at different circulation
rates: 1.0 ml/min, 3.0 ml/min, 6.0 mllmin and 7.0 mllmin,
which correspond to linear velocity o f 0.02 cmis. 0.06
cmis, 0.13 cmis and 0.15 cmis. Results are shown in
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substrates for reaction, so the initial rate increased.
However, at higher concentration, the increases o f initial
rate were not as high as at lower concentration. This might
be due to the saturation o f the available active sites For
the enzyme that has been ground into powder form, the
initial rates dropped. This might be due to the deactivation
o f enzyme caused by the grinding process, which
destroyed the three dimensional structure o f the enzyme

Figure 3. Hansen and Eigtved (1986) have also re orted the

diffusional resistance in the porous Lipozyme I' particles
When all the experiments were run under the same in their interesteritication activity. Since their internal
conditions but recycled at different linear velocity, the mass transfer resistance is significant. the observed
conversion o f palmitic acid to product was found 10 be kinetics are only apparent
almost the same at the same intervals o f time for all the
linear velocity from 0.06 c d s and above. I f external mass
transfer resistance was present, increase in linear velocity
will increase the conversion o f palmitic acid since the
substrate will be brought to the reaction sites at a faster ndm
rate. However, there are no increase in conversion when
linear velocity higher than 0.06 cmls was employed. As a
result. it can be concluded that there are no external mass
transfer limitations when operating at linear velocity of
0.06 cm/s or at an equivalent flow rate o f 3.0 m l h i n . To
*\\
eliminate the effect of external diffusional limitation on the
observed kinetics, all further experiments were conducted
above this flow rate.
For personal use only.

MI am nm nts nm .as nm as
- B l l m 4 Y

Figure 4: Effect o f particle siLe on initial rate

Effect o f Substrate Concentration

This experiment was carried out at seven different

substrate concentrations in equimolar: 0.0 IM. 0.05M.

-!
a0
o
~~,
o
,
n
.
o
~~-~
-~-.
a a m a m
1

m
i
( t ~ ~ ~ p ~ ~ & 6 ; ~ a 1 5 &

m
O.IOM, 0.15M, 0.2OM. 0.25M and 0.30M. The reaction
temperature was maintained at 45°C
-nk

Figure 3: Effect o f linear velocity on the conversion o f

016
.
Palmitic Acid

In order to check whether there are internal mass transfer

resistance. the immobilized enzyme was reduced in size
using mortar and pestle. This is to expose the active sites
available on the pores inside the enzyme. Two batches o f
enzymes were used for this study. One hatch was coarsely
ground, while the other batch was ground nearly powder a35
form. Both batches were then used to run a series o f
experiments using different concentration o f palmitic acid
and isopropyl alcohol in stoichiometric ratio. The Figure 5: Initial Rate o f reaction at various substrate
concentrations employed were 0.01M, 0.05M. 0. IOM. concentrations
0.15M. 0.20M, 0.25M and 0.30M, at a temperature of
45°C.The results are shown in Figure 4. Figure 5 shows the relationship o f the initial rate o f
esterification o f palmitic acid and isopropyl alcohol in
From Figure 4. it can be concluded that there is an internal hexane as a function o f substrate concentration. As the
mass transfer limitation on the system. When the enzyme substrate concentration increased, the initial rate o f the
was coarsely ground, more active sites were exposed to the reaction increased. It reached a maximum at 0.15 M.
 396
Further increase of substrate concentration to 0.30 M
decreased the initial rate. This may be attributed to the
kinetic limitations, such as active site limitation, substrate
or product inhibition etc. that became significant at higher
substrate concentrations. When double-reciprocal plot of
the initial rate of esterification at varying palmitic acid
concentrations were plotted according to Lineweaver-Burk
equation, the trend o f the curve showed a typical plot for
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substrate inhibition (Figure 6)
0. -~ ~ -
D B I n m w l m
TIC.
Figure 6 Lineweaver-Burk Plot
Effect of Substrate Molar Ratio
For personal use only.
Figure 7 shows the effect of isopropyl alcohol
concentration on the initial rate of the reaction for fixed
palmitic acid concentration of 0.15M. For a fixed
concentration of palmitic acid, the initial rate of the
reaction increased with an increase in the concentration of
isopropyl alcohol. It reached the maximum at the
stoichiometric ratio of 1: I . Beyond this point, further
increase of isopropyl alcohol concentration resulted in a
drop in the initial rate. It is known that hydrophilic
solvents can strip water essential for catalytic activity of
enzymes. Therefore, it is possible that isopropyl alcohol is
a substrate inhibitor for the lipase-catalyzed esterification.
PIEr
~
h encounter hydrophilic interactions. Therefore, substrate
Figure 7: Effect of isopropyl alcohol concentration on the
initial rate of esterification
KEE, HASSAN, AND RAMACHANDRAN
The upward curvature that obtained from the Linveweaver-
Burk confirmed that high substrate inhibition by isopropyl
alcohol, competitive with palmitic acid occurred
Figure 8: Effect of palmitic acid concentration on the
initial rate of esterification reaction
From Figure 8, we can see that for a fixed isopropyl
alcohol concentration of 0.15M, the initial rate increases
with an increase in palmitic acid concentration. Further
increase the concentration of palmitic acid beyond 0. I5 M
did not increase the initial rate of the reaction. Ramamurthi
and McCurdy (1994) also encountered this effect in their
esterification of oleic acid with methanol. They suggested
the presence of a critical concentration value of methanol,
above that the reaction rate decreased. Ramamurthi and
McCurdy (1994) together with other researchers (Manjon
ef a/., 1991 & Marlot el a/,, 1985) reported that at high
concentrations, alcohol became a substrate inhibitor for
lipase-catalyzed esterification reactions. This was
attributed to methanol as a short chain alcohol with its high
polarity. It undergoes hydrophilic interaction with the
aqueous boundary layer on the lipase surface causing
disruption of the protein ternary structure and hence
enzyme inactivation. The substrate inhibition phenomenon
was indicated by a decline in the initial rate versus
concentration curve after a certain concentration value.
Also, the slope o f the Lineweaver-Burk plot was typically
positive. Compared with methanol, isopropyl alcohol or
propan-2-01 is also a short chain alcohol, hence would also
inhibition would also be encountered in this system.
Effect of Temperature
In the present system, the effect of temperature on the
progress of the esterification reaction was investigated in
the temperature range of 30°C to 6 0 T . As before, the
reaction mixture consisted of 0.15M equimolar palmitic
acid and isopropyl alcohol. Figure 9 shows that when the
temperature increased, the rate of the esterification
reaction also increased. It reached optimum at around
50°C. Then, the reaction rate started to decline when the
temperature was further increased to 60T. At
temperatures above this point the enzyme rapidly
 KINETICS OF ISOPROPYL PALMITATE SYNTHESIS
deactivates (not shown). However, there is evidence that
Ergan er a/. (1990) could carry out triolein synthesis at
temperatures as high as 80°C without any deactivation.
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Twnpatum, 'C
Figure 9: Reaction Rate of esterification reaction at various
temperatures
In general, not only is every rate constant in the catalytic
reaction likely to change with temperature, but also the
three-dimensional structure of the enzyme can be affected.
Nearly all enzymes suffer a loss of structure known as
thermal denaturation at temperature above SOT. As a
result, the activity of an enzyme passes through a
maximum then declines as denaturation effect becomes
dominant. In order to obtain accurate thermal analysis, it is
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better to work in the temperature range where thermal
denaturation is insignificant. This is between 30°C to 45°C
for this immobilized enzyme.
A plot on a semi-log scale known as an Arrhenius plot,
should be a straight line of slope - E n . Figure 10 shows
the Arrhenius plot for the present system. Calculation
shows that the apparent activation energy for the system is
around 43.67kJlmol. a value typical for enzymatic
reactions. The result obtained agreed with lipase-catalyzed
lauric acid esterification carried out by Ahmed Zaidi er a/
(1995). They obtained an activation energy of 1 I k c a h o l
(45.98 kJ/mol). However, it is a bit higher if compared to
24.7 kJ/mol reported by Yee Peng Yong & Bushra AI-Duri
(1996) who used Lipozyme 10 000 for ester synthesis of
oleic acid and octanol in a solvent-free system.
3 -~
1K
Figure 10. Arrhenius Plot
397
Effect of Water Content
Water content in the isopropyl alcohol (IPA) was varied
from 0.1% to the maximum of 1.0%. The temperature and
substrate concentrations were 4 5 T and 0.15M
respectively. From Figure 11, it can be seen that the rate
increases when the water content in IPA increases. It
reaches the maximum at 0.50% water content. The rate
decreases when water content was further increased.
In esterification reactions, lipase sometimes shows an
optimum initial activity at a certain content of water in the
reaction medium (Yamane ef a/., 1989). The initial water
content in the Lipozyme IM is 2-3%. which is however
may be not sufficient for the enzyme to achieve its
optimum catalytic activity. Therefore, adding water in the
substrate increased the initial rate of esterification. This
activating effect of water dominates only at water contents
below the optimum. At higher water contents, net
esterification rates decreases. This might be due to the
thermodynamic equilibrium of the reaction that was
adversely affected, and the reverse reaction, hydrolysis
occurred (Yamane ef a/., 1989).
Figure I I : Effect of water content on the initial rate
CONCLUSIONS
From the results obtained, it is shown that an immobilized
lipase can be used to catalyze the synthesis of isopropyl
palmitate in nearly anhydrous hexane. Thus, the following
can be concluded :
I. The immobilized lipase appears to be quite stable and
can be reused repeatedly.
2. There are no external mass transfer limitations in the
system above a linear velocity of 0.06 c d s .
3. Internal mass transfer limitation exists for this system.
4. The optimum substrate concentration for this system
is 0.15M equimolar palmitic acid and isopropyl
alcohol.
5. lsopropyl alcohol is a substrate inhibitor for this
lipase-catalyzed reaction.
 398
6. The optimum temperature for the lipase-catalyzed
esterification reaction is 5 0 T , whereas. the activation
energy for the present system is around 43.67 kJ/mol.
7. The half-life of the enzyme at 0.0SM is 44 hours and
32hoursfor0.15M.
8. The optimum water content in isopropyl alcohol for
this system is 0.50%.
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Fatty Acid Esterification Using Nylon-Immobilized
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Ergan, F., Trani, M., Andre, G., (1990), Production of
glycerides from glycerol and fatty acid by immobilized
lipases in non-aqueous media, Biotechnoloa
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Halling P. 1.. Biofechnol. Bioeng., (1990) 35: 691
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lipase for interesterification and ester synthesis,
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Khmelnitsky Y., A. Levashov, N. Klyachko & K.
Martinek, (1988). Enrq’mr Microb. Techno/. 1 0 710.
KEE, HASSAN, AND RAMACHANDRAN
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Klibanov, A. M., (1989) Enzymatic catalysis in anhydrous
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Manjon, A,. Iborra. J. L. & Arocas. A,, (1991), Short-chain
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Marlot, C. G., Langrand, G., Triantaphylides, C. & Baratti.
J., (1985), Ester synthesis in organic solvent catalyzed by
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Ramamurthi, S. & McCurdy A. R., (1994). Lipase-
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hexane A kinetic study, JAOCS, 71(9) : 927-30.
~
Victor M. Balcao, Ana L. Paiva & F. Xavier Malcata,
(1996), Bioreactors with immobilized lipases: State of the
art, Enzyme Microb. Tehcnol., 18: 392-4 16,
Yamane, T., Kojima, Y., Ichiryu, T., Nagata, M. &
Shimizu, S., (1989), Intramolecular esterification by lipase
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