Low Enzyme ProcBioc

Kinetics of the enzymatic hydrolysis of palm oil by lipase

Sulaiman Al-Zuhair, Masitah Hasan, K.B. Ramachandran *

Department of Chemical Engineering, University of Malaya, 50603 Kuala Lumpur, Malaysia

Received 28 March 2002; received in revised form 23 August 2002; accepted 18 September 2002

Abstract

The kinetics of the enzymic hydrolysis of palm oil using lipase in a batch reactor has been investigated. The lipase enzyme used

was not ester bond position selective and its activity at the interface was higher compared to that in the bulk. A mathematical model

taking into account the mechanism of the hydrolysis reaction and the effect of interfacial area between the oil phase and the aqueous

phase containing the enzyme was developed. A correlation between the interfacial area and the operating conditions including

agitation speed and oil volume fraction was established experimentally. The kinetic parameters were estimated by fitting the data to

the model and comparing with previously reported values. The kinetic model represented the experimental data accurately.

# 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Lipase hydrolysis; Palm oil; Kinetic model; Interfacial area

1. Introduction

Hydrolysis of oil and fat is an important industrial

operation. The products, fatty acids and glycerol are

basic raw materials for a wide range of applications.

Fatty acids are used as a feedstock for the production of

oleochemicals such as fatty alcohols, fatty amines and

fatty esters. These oleochemicals are used as lubricant

greases, anti-block agents, plastisizers, and emulsifiers

and as ingredients in the manufacture of soaps, deter-

gents, and animal feed.

The present method of hydrolysis of crude palm oil to

fatty acids and glycerol involves high temperature and

pressure operation for about 2 h to achieve the desired

96/99% conversion [1]. When these extreme conditionsare employed, polymerisation of fat and by-product

formation takes place resulting in dark fatty acids and

discoloured aqueous glycerol solution. To remove the

colour and the by-products, further purification by

distillation is required. Both hydrolysis and subsequent

distillation of fatty acids are energy intensive processes

[1]. Hence, it would be advantageous to develop a low-

energy process that produces a colourless product.

Recently, enzymic splitting of fats has gained increas-

ing attention, as lipase (triacylglycerol acylhydrolase) is

now available at reasonable cost. The industrial use of

lipase for splitting lipids as an energy-saving process has

been addressed in the literature, especially for producing

high value-added products or heat sensitive fatty acids

[1]. However, a reliable kinetic model to predict the

hydrolysis rate is still lacking.

Lipase catalysed reactions take place at the interface

between the aqueous phase containing the enzyme and

the oil phase [2,3]. Hence, the interfacial area, which is

affected by mixing and substrate concentration, influ-

ences the rate of reaction. All previous studies to

establish a rate equation for the enzymic hydrolysis of

lipids in batch reactors have assumed that the total

interfacial area between oil phase and the aqueous phase

remains constant, even when the agitation speed or

substrate concentrations are varied. This assumption is

valid only if the substrate (oil) is dissolved in an organic

solvent (such as hexane), its concentration is changed

within that organic phase and the volume fractions of

the organic phase containing the substrate and the

aqueous phase containing the enzyme are kept constant.

Although the method of dissolving the oil in the organic

phase and its subsequent hydrolysis using lipase enzyme

* Corresponding author. Tel.: /60-3-7967-5293; fax: /60-3-7967-5319.

E-mail address: [email protected] (K.B. Ramachandran).

Process Biochemistry 38 (2003) 1155/1163

www.elsevier.com/locate/procbio

0032-9592/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0032-9592(02)00279-0

mailto:[email protected]

has several advantages, it requires the addition of an

organic solvent in the reaction mixture, which needs to

be separated later for reuse.The preferable method for the enzymic hydrolysis of

the oil is to bring directly in contact the aqueous phase

containing the enzyme and the oil phase. For this

system, it is not correct to assume that the total

interfacial area is constant, irrespective of the intensity

of agitation and the volume fraction of oil and water.

Hence, to verify the kinetics, a model taking into

account the change in interfacial area with agitation

speed and substrate concentration is needed. Such a

model will be useful in optimal design of a batch or a

continuous hydrolysis process.

The enzymic lipolytic reaction is an important exam-

ple of an heterogeneous catalytic reaction. The water-

soluble enzymes act at the interfaces of the insoluble

lipid substrate. The X-ray crystallography technique has

been used to show the existence of a preferable config-

uration change of the enzyme at the water/lipid inter-face [4]. This interfacial activation phenomenon is

thought to be due to the unfolding of an amphiphilic

peptidic loop, covering the active sites of the enzyme

when the enzyme is attached to the lipid at the interface.

When contact occurs with a lipid/water interface, theenzyme undergoes a conformational rearrangement,

rending the active site accessible to the substrate. It issuggested in this study that determining the activity of

the enzyme at the interface, and comparing it to that in

the bulk, would help to strengthen this understanding.

Determination of the fatty acids produced from the

enzymic hydrolysis of oil as a function of time is widely

addressed in the literature, being the direct way to

determine the reaction rate. This can be done by two

methods, namely: (1) titration of the products afterextraction using a auto-titrator and (2) gas chromato-

graphic determination of the fatty acids produced.

Unlike the titration method that gives the overall fatty

acids concentration, the gas chromatographic method

Nomenclature

a Specific free interfacial area (m1)at Specific total interfacial area (m

1)Am Enzyme molar area (m

2 mol1)C Proportionality constant defined by Eq. (8)C* Constant defined by Eq. (13)Dmean Surface mean diameter (mm)Do Overall mean diameter (mm)E Free enzyme (mole/total reactor volume) (mol m3)E* Penetrated enzyme (mole/total interfacial area) (mol m2)E*S Enzyme/substrate complex (mole/total interfacial area) (mol m2)Et Total active enzyme (mol m

3)(Et)m Total enzyme mass concentration (g m

3)k Constant defined by Eq. (19)kcat Catalytic rate constant (min

1)kd Desorption rate constant (min

1)kp Adsorption rate constant (m

2 min1)k Reaction rate constants (m3 mol1 min1)k1 Reaction rate constant (min

1)Ke Equilibrium constant of E*S (mol m

3)

Km? Apparent Michaelis constant (mol m3)

LU Lipase unitm Constant defined by Eq. (19)n Constant defined by Eq. (19)P* Interface product concentration (mole/total interfacial area) (mol m2)P Bulk product concentration (mole/total reactor volume) (mol m3)S Bulk substrate concentration (mole/total reactor volume) (mol m3)T Temperature (K)Greek letters

a Constant defined by Eq. (19)f Volume fraction of oil in the reaction mixtureu Area fractionv Agitation Speed (rpm)n Reaction rate (mol m3 min1)

S. Al-Zuhair et al. / Process Biochemistry 38 (2003) 1155/11631156

provides data on the progressive production of each

fatty acid. The results of the gas chromatography would

help in determining the selectivity of the enzyme with

respect to the ester-bonds in the glycerol chain of thepalm oil, to hydrolyse. It is known that some lipases

favour the terminal ester bonds of the oil glycerol chain,

and are called 1,3 lipases [5]. In most oils, the saturated

fatty acids are naturally situated on the terminal

positions of glycerol, while the unsaturated ones are in

the centre. Examining the progressive production of

different fatty acids, saturated such as palmitic acid, and

unsaturated such as oleic acid, would help to determinewhether or not the lipase used prefers to attack the

terminal-ester bonds.

2. The kinetic model

The mechanistic model proposed to describe the

action of lipase on palm oil hydrolysis is similar to

that proposed by Tsai and Chang [2]. The first step is thereversible adsorption of a water-soluble enzyme at the

interface to produce a penetrated enzyme, E*. In order

to develop the model equations, the absorption rate is

assumed to be proportional to the free enzyme concen-

tration, E and the specific free interfacial area, a . The

substrate, S , then binds to the adsorbed enzyme giving

an interfacial enzyme/substrate complex, E*S [2,6].This complex then generates the product, P* at theinterface, while regenerating the enzyme in the form of

E*. The product, P*, then desorbs from the interface

into the organic phase to give rise to product P . The

steps up to the production of product P* are illustrated

in Eqs. (1)/(3)

Ea?kp

kd

E (1)

ES ?k1

k1

ES (2)

ES 0kcat

EP (3)

The concentration of the enzyme/substrate complexand the adsorbed enzyme are both assumed constant

(quasi-steady state) [2,7], and the interfacial product

concentration, P*, is assumed to be proportional to the

free product concentration, P [2]. It is also assumed thatthe interfacial product concentration, P*, is low and

hence it occupies negligible fraction of the total inter-

facial area. With the above mechanism and assump-

tions, the model equations can be written as:

kpE a(kdk1S)E(k1kcat)ES0 (4)k1E S(k1kcat)ES0 (5)ataAm(EES)at (6)EtEat(EES) (7)

Since,

PCP=at (8)

The rate of product formation can be expressed as:

ydP

dt

at

C

dP

dt

at

CkcatES (9)

Solving Eqs. (4)/(9) simultaneously, the final equa-tion for the rate of reaction can be expressed as:

y

kcatat(G1 G2)

CAmS

kcat k1k1

S(10)

where

G1

kcat k1

k1at

kd

at at AmEt

at AmEt

at

S

kcat k1

k1

S

(11)

G2

G214AmEt

at

(12)

and

C2C (13)

Eqs. (10)/(13) are the corrected version of theequations derived by Tsai and Chang [2] for the

hydrolysis of lipids by lipase enzyme. This equation is

applicable for predicting the hydrolysis rate for any

enzyme concentration. Hydrolysis reactions are usuallycarried out at low enzyme concentration and it is useful

to get a simplified rate expression, applicable for such a

condition. At low enzyme concentrations, it has been

shown experimentally that the area occupied by the

enzyme that has penetrated the interface is negligible in

comparison to the total interfacial area [2,7]. In this

case, the free specific interfacial area, a , will be equal to

the total specific interfacial area, at. Replacing a with atin Eqs. (4)/(10), a simplified form of rate equation atlow enzyme concentration can be derived as given

below:

y

kcatEt

CS

(kcat k1)(kd kpa

2t )

k1kpa2t

S

(14)

The above rate equation can be further simplified to:

yk+catEtS

Ke

kd

kpa2t

1 S

(15)

where Ke(kcatk1)=k1 and k+catkcat=C/

S. Al-Zuhair et al. / Process Biochemistry 38 (2003) 1155/1163 1157

The above rate expressions agree in the basic form

with the previous models [2,6,7]. However, the rate

constants in the previous models were based on pro-

portionality constants that had no link to the reactionmechanism and this led to lumped rate constants.

3. Materials and methods

3.1. Materials

Lipase (Type-VII) from Candida rugosa was obtained

from Sigma Chemical Co., Japan. Refined palm oil usedin this study was obtained from Lam Soon (M) Berhad,

Malaysia. Analytical grade isopropanol was obtained

from Scharlau Chemicals Co., Spain. Gum Arabic, oleic

acid methyl ester, linoleic acid methyl ester, stearic acid

methyl ester and palmitic acid methyl ester were

obtained from Sigma Chemicals Co., Germany. All

other chemicals used were of analytical grade.

3.2. Hydrolysis reactor

The reactor consisted of a glass flask with a capacity

of 600 ml and an inside diameter of 9 cm. A four-bladed

paddle impeller (5 cm in diameter) immersed in the

solution at one-third-depth level was used for agitation.

No baffle plates were provided. The total volume of the

reaction mixture was 400 ml at the beginning of eachrun, consisting of palm oil and distilled water. The

reactor was placed in a temperature controlled water

bath and a cover was used to prevent evaporation of

water during the progress of the experiment. After the

desired conditions were reached in the bioreactor, the

required amount of enzyme solution was added to

initiate the reaction. Samples were withdrawn from the

reactor at regular intervals for analyses.

3.3. Determination of enzyme activity

The method is based on the hydrolysis of tributyrin by

the enzyme and titrating the butyric acids produced with

0.05 NaOH in distilled water [8]. The alkali consump-

tion is registered as a function of time under standard

conditions of 30.0 8C and pH 7.0, using an auto-titrator(Metrohm 702 SM titrino). From the amount of alkali

consumed, the equivalent amount of butyric acid in the

samples was calculated and the enzyme activity deter-

mined. The enzyme activity is expressed in lipase unit

(LU), where the 1 LU is defined as the amount of

enzyme which liberates 1 mmol titrable butyric acid permin at 30 8C.

In addition to determining the activity of the enzymeused for the kinetic studies, additional analyses were

carried out to compare the activity of the enzyme in the

bulk to that at the interface. The interface was created

using the method proposed by Rooney and Weatherley

[9]. One gram of enzyme was dissolved in 100 ml of

distilled water and coated with a thin layer of palm oil.

The mixture was kept at room temperature for about 2 hin a 100-ml beaker. The temperature was then reduced

to /20 8C by placing the beaker in a freezer and leftovernight. The solid upper oil layer was then peeled off

and the solid ice surface wiped clear of any remaining

oil. A sharp knife was used to scrap off small samples

from the interface and its activity was then determined

as described in the previous paragraph.

3.4. Determination of fatty acids concentration

A gas chromatograph was used to determine the fatty

acid composition of the hydrolysed samples. In order to

determine the fatty acids using a gas chromatograph,

esterification of the fatty acids to their respective methyl

esters was carried out first, following the AOCS Official

Method Ce 1/62 [10]. After esterification, a sample of 1ml of the fatty acids methyl ester was withdrawn into a10-ml syringe. The sample was then injected into the gaschromatograph (Chemito GC 8610), equipped with a

flame ionisation detector, and area of each peak was

determined. The column was conditioned prior to use

and the operating conditions used for the analysis were,

oven temperature 200 8C, injector and detector tem-peratures 240 8C. The peaks areas obtained withsamples were compared with the peaks areas obtained

with three different dilutions of the standards, prepared

by dissolving different weights of oleic, linoleic, stearic

and palmitic acid methyl esters in heptane. These are the

main fatty acids produced by the hydrolysis of palm oil.

A straight-line method was used to determine the

amount of fatty acids in the reactor sample.

3.5. Determination of oil drop size

The method proposed by Mukataka et al. [7] was used

to determine the specific interfacial area at various

operating conditions. Five minutes after mixing the oil

and the aqueous phase, a sample was withdrawn and

placed on a slide for observation under microscope. A

microscope, model Leica DMLS, linked to a computer

software image analyser, VIDEO TEST / MASTER 4.0, wasused to determine the droplets size of palm oil in water.

Around 200/400 photographs of drops, which wereenlarged 4/20 times were taken. The drops were dividedinto five categories: drops of diameter smaller than 100

mm, 100/200 mm, 200/300 mm, 300/400 mm, and largerthan 400 mm. The mean diameter of each category wascalculated using the following equation:

Dmean; j X

d3j=X

d2j (16)


The overall surface mean diameter, which takes into

account the respective portions of each size category in

the oil/water mixture, was calculated using the equation:

Do1=X

(uj=Dmean;j) (17)

where the subscript j stands for each of the five different

size categories.

From the values of the overall mean diameter,interfacial area per unit volume of the oil/water mixture

in the bi-phasic system was calculated using the follow-

ing Eq. (18).

at6f=Do (18)

where f is the oil volume fraction.

For each pre-specified operating conditions, the

above procedure was repeated three times; after 5, 10

and 15 min from the beginning of each experimental runand a statistical average of the three runs was deter-

mined. It has been found that the results were repro-

ducible and the standard deviation of the different runs

under the same operating conditions was in the range of

2.1/57.8 mm, which means the errors have not exceeded10%.

Many models have been previously proposed to

correlate the specific interfacial area as a function ofagitator speed and volume fraction at constant tem-

perature [11/13]. Calderbank [13] also included in hiscorrelation the effect of temperature on the average

drop diameter. After considering the equipment para-

meters and the physical properties of the fluids in the

experiment, the most suitable correlation for these

studies is that of Calderbank [13], given below:

at6f

DoavmfTk=( 1nf) (19)

The above equation was used to correlate the total

specific interfacial area to various operating conditions.

3.6. Determination of hydrolysis rate

The enzyme mixture was prepared by dissolving

different weights of solid lipase powder in 100 ml of

distilled water and 10 ml of this solution was added tothe reaction mixture. Samples of 10 ml volume were

withdrawn from the reaction mixture at desired time

intervals. The water in the samples was evaporated

under a vacuum of 300 mbar using Buchi Rota Vapor

(R-144) and the remaining fatty acids were extracted in

50 ml of isopropanol. The extracted fatty acids were

titrated with 0.05 N NaOH solution in isopropanol to

determine the concentration of fatty acids, using anauto-titrator. A plot of fatty acid concentration

(mol m3) against sample time was made and the slope

of the plot at the origin gave the initial rate of reaction.

4. Results and discussions

4.1. Interfacial enzyme concentration

To determine the lipase activity, at the interface, five

repetitive experiments were carried out and a statistical

average was determined. The results showed that the

activity of the enzyme at the interface was 15.7% higher

than that in the bulk. This observation agrees with the

result reported by Rooney and Weatherley [9] who

found that the activity at the interface was higher than

that in the bulk, however, they did not determine the

percentage increase. The increase in the activity at the

interface was explained using X-ray studies of the 3D

structure of lipase [4] and the conformation change of

the enzyme at the interface. This encourages the enzyme

to migrate to the interface from the bulk and expose the

active sites by folding the protein chains in a preferable

manner.

4.2. Fatty acids profile

The percentages of the main saturated fatty acid,

palmitic acid, and the main unsaturated one, oleic acid

produced with time is shown in Fig. 1. The figure shows

that both percentages remained almost constant with

time and equalled to that found in the palm oil itself,

i.e., 45.5% palmitic acid and 39.8% oleic acid [5]. This

indicates that the lipase used was not a position selective

enzyme, otherwise, the production of palmitic acid

would be much faster than the production of oleic

acid. In Fig. 2, the results obtained by the gas

chromatograph are compared to the results found by

the auto-titrator method. The average difference in

readings between these two methods is about 10%,

which is relatively low. Since, the auto-titrator method is

less time consuming, it was used to determine the

reaction rate in subsequent experiments.

Fig. 1. Percentages of palmitic acid and oleic acid produced with time.


4.3. Determination of interfacial area

The effect of different stirrer speeds and different oil

volume fractions on the total interfacial area was

measured at 45 8C and the results are shown in Fig. 3.It can be clearly seen that increasing the agitation speed

and/or the volume fraction resulted in increased total

interfacial area. This observed effect of agitation speed

is due to increase in shear rate on the oil droplets with

increasing agitation speed that caused the breakage of

the larger oil droplets into smaller ones. These results

agree with the earlier reported results [11,12] and the

basic empirical model proposed by Calderbank [13].

However, at high volume fraction, the increase in

interfacial area tended to decrease with increasing

agitation speed as suggested by the model of Tavlarides

and Bepat [12].

The effect of temperature on the total interfacial area

was not reported either by Albasi et al. [11] or by

Tavlarides and Bepat [12], but was reported by Tsai and

Chang [2]. The effect of temperature on the droplet size

was determined and is shown in Fig. 4. It shows that the

mean drop diameter decreases as the temperature

increases. This is due to the reduction in viscosity of

the oil and the surface tension at the interface between

water and oil, with increase in temperature [13].

The data shown in Figs. 3 and 4 were fitted to Eq. (19)

and the model parameters in the equation were esti-

mated by applying a stepwise numerical method usingEXCEL. The resultant correlation for the total specific

interfacial area is as follows:

at0:024v0:6T1:7f=(13:0f) (20)

4.4. Hydrolysis reaction

Experiments were run at different oil volume frac-

tions, temperatures, enzyme-concentrations and stirrer

speeds to determine their effect on the initial rate of

palm oil hydrolysis. Fig. 5 shows the effect of oil volume

fraction, which reflects the substrate concentration, on

the initial rate of reaction. It can be noted that, as the

volume fraction of oil increased the initial rate of

reaction also increased. The increase in reaction ratewith volume fraction at a given agitation speed is due to

an increase in substrate concentration and the increased

interfacial area, as reported in the previous section.

However, above 30/40% oil (v/v), a slight decrease in

Fig. 2. Comparison between the gas chromatograph and the auto-

titrator results (f/0.2, T/45 8C, v/1300 rpm, and (Et)m/25g m3).

Fig. 3. Effect of stirrer speed and oil volume fractions on the specific

interfacial area (T/45 8C).

Fig. 4. Effect of temperature and agitation speed on the mean drop

diameter (f/0.5).

Fig. 5. Effect of oil volume fraction on the initial rate of reaction at

various stirrer speeds (T/45 8C and (Et)m/25 g m3).


the initial rate of reaction was observed. This result

agrees with the results previously presented in the

literature [7,14], which showed that the initial rate of

reaction decreased above an oil volume fraction of 43%,

when the unfavourable phase inversion begins to take

place (i.e. the aqueous phase becomes the dispersed

one). When phase inversion occurs, dispersion of the

enzyme at the interface will be restricted, as it would be

trapped in the water droplets, where the agitation effect

is limited. It can also be observed from Fig. 5 that at a

given volume fraction of oil, the initial rate of reaction

increased as the agitation speed increased. This again is

due to the increase in mean interfacial area with increase

in agitation speed as reported in the previous section.

These results also agree with previous literature reports

[11,13,14].

The effect of increasing enzyme concentration on the

initial rate of reaction is shown in Fig. 6. The initial rate

of reaction increased linearly with enzyme concentration

at low enzyme concentrations. At high enzyme concen-

trations, this increase tends to fall. This result agrees

with that found by Albasi et al. [11] for the hydrolysis of

sunflower oil. This phenomenon is explained by hy-

pothesising that at high concentrations of enzyme the

interfacial area is totally saturated with enzyme mole-

cules. Hence, any further increase in enzyme concentra-

tion in the bulk would not enhance the reaction rate. It

can also be seen, from Fig. 6 that the point where the

effect of enzyme concentration tended to fade, shifts to

the right (higher enzyme concentration) as the agitation

speed increased. This is due to increase in interfacial

area available for the enzyme to occupy at higher

agitation speeds and hence, a higher enzyme concentra-

tion is needed to saturate the available interfacial area.

A multiple regression method, using a MATLAB

computer package was applied to the data shown in

Figs. 5 and 6, to determine the parameters of the model

(Eq. (15)). Under these conditions, the enzyme concen-

tration used was low enough to assume that the area of

coverage of the enzyme is negligible. The model

equation with the estimated rate constants can be

represented as given below:

y1:8 103(Et)mS

5:65

7:7 107

a2t 1

S

90:018 (21)

4.5. Effect of temperature

Fig. 7 shows the effect of temperature on the initial

rate of reaction. It shows, initially as the temperatureincreased, the reaction rate increased. This is mainly due

to increase in rate constant with temperature and partly

due to increase in interfacial area with temperature as

discussed in the previous section. However, the initial

rate decreased sharply after 50 8C, which was due to thedeactivation of the enzyme. It is known that most

proteins tend to decompose at temperatures above

50 8C [15]. In addition to the deactivation of theenzyme, the presence of the inactive enzyme at the

interface would block the active enzyme from penetrat-

ing the interface, which would further decrease the

reaction rate.

The experimental results shown in Fig. 7 were used to

determine the effect of temperature on the rate constant,

kcat .

k+cat9:6103exp(1:2=RT) (22)

The activation energy of 1.2 kcal gmole1 is lower

than the reported activation energies for most enzyme

reactions [15]. Kim and Chung [16] reported a value of

7.0 kcal gmole1 for the hydrolysis of palm kernel oil in

reversed micelle systems. Desnulle [17] reported in an

aqueous emulsion system, the activation energy was 5.3

kcal gmole1. Our result is lower, but still comparableto those reported in the literature.

Fig. 6. Effect of enzyme concentration on the initial rate of reaction at

different stirrer speeds (f/0.20 and T/45 8C).Fig. 7. Effect of temparature on the initial rate of reaction at various

oil volume fraction (v/800 rpm and (Et)m/25 g m3).


4.6. Model verification

Figs. 8 and 9 show the comparison between the

experimental results and the proposed model curve

based on Eq. (21), for two agitation speeds. It can be

noted that the model predicts fairly well the initial rate

of reaction at various substrate concentrations for both

agitation speeds. It can be seen from Fig. 10 that the

experimental results are well represented by the model at

low enzyme concentrations. However, they tend to

diverge at high enzyme concentrations, as the model is

limited to low enzyme concentrations only. As discussed

earlier, at high concentrations of enzyme, the interfacial

area is mostly saturated with enzyme molecules and

hence the assumption that the area of occupation by the

enzyme is only a fraction of the total interfacial area in

deriving Eq. (21) breaks down. It is also seen from Fig.

10 that the enzyme concentration at which the experi-

mental results are well represented by the model, is

wider at higher agitation speeds. This is due to large

interfacial area available for the enzyme to penetrate the

interface at higher agitation speeds, and hence, even at

higher enzyme concentration the interface is not satu-

rated with the enzyme. Under these conditions a low

interfacial area of occupation by the enzyme is valid for

a wider range of enzyme concentration.

The experimentally determined values of apparent

Michaelis/Menten constant, Km? by Mukataka et al. [7]at different agitation speeds are compared in Fig. 11,

with those determined by the present model, under the

same operating conditions. It can be noted that the

apparent Km? in the present study are lower than thatreported by Mukataka et al. [7]. In the experiments of

Mukataka et al. [7], the organic phase consisted of 10%

beef tallow dissolved in isooctane, whereas the organic

phase in this study consisted of palm oil only. The

difference could be due to the different substrate used in

both studies. Fig. 11 also shows that the specific

interfacial area determined in the present study, using

palm oil, was lower than that obtained by Mukataka et

al. [7] for beef tallow dissolved in isooctane, under the

same operating conditions. This is due to the high

viscosity of palm oil in comparison with isooctane/tallow mixture.

Fig. 8. Comparison between experimental results and the proposed

model curve showing the effect of substrate concentration on the initial

rate of reaction (v/800 rpm, T/45 8C and (Et)m/25 g m3).


model curve showing the effect of substrate concentration on the initial

rate of reaction (v/1000 rpm, T/45 8C and (Et)m/25 g m3).


model curve showing the effect of enzyme concentration on the initial

rate of reaction (v/1000 and 1300 rpm, T/45 8C and S/660.7mole m3).

Fig. 11. Comparison between the present model results and the results

of Mukataka et al. [7].


5. Conclusion

A kinetic model based on the mechanism of the

reaction of the lipase-catalysed hydrolysis of palm oil inbi-phasic oil/aqueous system has been proposed, takinginto account the variation of interfacial area with

agitation speed and substrate volume fraction. This

was verified with experimental results at low enzyme

concentrations. There was a good agreement between

the model prediction and the experimental results. The

rate constants in the mathematical model were deter-

mined numerically from the experimental results. Thismodel can be used to predict the rate of hydrolysis in a

batch reactor and to determine optimal conditions. It

has been shown experimentally that the lipase enzyme

used was not ester bond position selective. In addition,

the activity of the enzyme was shown to increase at the

interface compared to that at the bulk, as previous

studies have also indicated [9].

References

[1] Arbidge MV, Pitcher WH. Industrial enzymology: a look towards

the future. Trends Biotech 1989;7:330/5.[2] Tsai SW, Chang CS. Kinetics of lipase-catalysed hydrolysis of

lipids in biphasic organic-aqueous systems. J Chem Tech Biotech

1993;57:147/54.[3] Knezevic ZD, Siler-Marinkovic SS, Mojovic LV. Kinetics of

lipase-catalysed hydrolysis of palm oil in lecithin/isooctane

reversed micelles. Appl Microbiol Biotechnol 1998;49:267/71.[4] Panalotov I, Verger R. Physical chemistry of biological interfaces.

New York: Marcel Dekker Inc, 2000.

[5] George S, Arumughan C. Positional distribution of fatty acids in

the triacylglycerols of developing oil palm fruit. J Am Oil Chem

Soc 1993;70(12):1255/8.[6] Verger R, Maria CEM, Gerard HD. Action of phospholipase A

at interfaces. J Biological Chem 1972;218(11):4028/34.[7] Mukataka S, Tetsuo K, Joji T. Kinetics of enzymatic hydrolysis

of lipids in biphasic organic-aqueous systems. J Ferment Technol

1985;63(5):461/6.[8] NOVO Industrials. Analytical methods handout, Enzyme Process

Division, NOVO industrials, Denmark; 1995.

[9] Rooney D, Weatherley LR. The effect of reaction con-

ditions upon lipase catalysed hydrolysis of high oleate sunflower

oil in a stirred liquid/liquid reactor. Process Biochem2001;36:947/53.

[10] A.O.C.S. Official Method Ce 1/62. Fatty acid composition by gaschromatography. Sampling and analysis of commercial fats and

oils; 1990. pp. 1/4.[11] Albasi C, Bertrand N, Riba JP. Enzymatic hydrolysis of sun-

flower oil in a standardised agitated tank reactor. Bioprocess Eng

1999;20:77/81.[12] Tavlarides LL, Bepat PM. Models for scale-up of dispersed phase

liquid/liquid reactors. Proceedings of ISEC83, AIChE Symp1983;80(238):12/46.

[13] Calderbank PH. Physical rate processes in industrial fermenta-

tion, Part I: the interfacial area in gas/liquid contacting withmechanical agitation. Tans Inst Chem Eng 1958;36:443.

[14] Tsai SW, Wu GH, Chiang CL. Kinetics of enzymatic hydrolysis

of olive oil in biphasic organic-aqueous systems. Biotech Bioeng

1991;38(7):762/6.[15] Baily JE, Ollis DF. Biochemical engineering fundamentals, second

ed.. New York: McGraw Hill Book Company, 1986.

[16] Kim T, Chung K. Some characteristics of palm oil kernel olein

hydrolysis by Rhizopus arrhizus lipase in reversed micelle of AOT

in iso-octane and additive effects. Enzyme Microbial Technol

1989;11:528/31.[17] Desnulle D. Pancreatic lipase. Adv Enzymology 1961;23:129/61.


Kinetics of the enzymatic hydrolysis of palm oil by lipaseIntroductionThe kinetic modelMaterials and methodsMaterialsHydrolysis reactorDetermination of enzyme activityDetermination of fatty acids concentrationDetermination of oil drop sizeDetermination of hydrolysis rate

Results and discussionsInterfacial enzyme concentrationFatty acids profileDetermination of interfacial areaHydrolysis reactionEffect of temperatureModel verification

ConclusionReferences

Low Enzyme ProcBioc

Documents

Transcript of Low Enzyme ProcBioc