ENZYMATIC HYDROLYSIS OF VEGETABLES OILS BY LIPASE F-AP15 Rhizopus

oryzae IN CONTINUOUS STIRRED TANK REACTOR (CSTR)

Adilene Lares-Molina
*
, Esther Carrillo-Prez, Manuel Prez-Tello y Juan Antonio Noriega-Rodrguez
Engineering Sciences: Chemical Engineering, Chemical Engineering and Metallurgy department, Universidad de
Sonora, Blvd. Luis Encinas y Rosales S/N, Col. Centro, Hermosillo, Sonora, 83000, Mxico.
*
adilenemar.laresmol@correoa.uson.mx, janoriega@guayacan.uson.mx.

Keywords: enzymatic, hydrolysis, oil, lipase

Abstract
In order to determinate the feed and output flows in a continuous stirred tank reactor (CSTR) in a
pilot plant scale (50 L), were determined hydrolysis kinetic parameters of analytic grade olive oil
and commercial oil (canola, soy, sunflower and safflower mix) on an automatic titration
equipment Titralab 856. Were studied the effect of temperature (25-40C) and the concentrations
of enzyme (0.25-0.75% w/w of oil). Hydrolysis greater than 25 C was observed and that the
lipase F-AP15 Rhizopus oryzae has not specificity on the type of fatty acids in oils.

Introduction
Hydrolysis of oils consists of a reaction between a triacylglycerol molecule with three water
molecules, where the triacylglycerol ester bond breaks to form three fatty acids and a glyceride.
In industries is used to obtain fatty acids to produce drugs, modified fats in food and cosmetics
[1]. The used of enzymes, as catalysts, in this type of processes has been increased in the last
years, because they offered greater advantages compared with other type of catalysts and
reagents, although their obtaining from various sources is costly [2]. Among the advantages of
used enzymes are: moderated reaction conditions of temperature and pressure, higher specificity
to substrates, reused, ease of recovery, if used immobilized way.

CSTR reactors, used for the type of reactions, have some particular advantages on fixed bed
reactors, inasmuch as it has lower construction costs and efficient agitation removed the presence
of concentration gradients and/or temperature, such, components concentrations are equal on
outputs and mass tanks fluid.

Objective of this work was to determine kinetics parameters of enzymatic hydrolysis of
vegetable oils needed to design industrial reactors.

Materials and Methods
Lipase F-AP15 Rhizopus oryzae (Amano Enzyme Inc.) was evaluated in different enzyme
concentrations (0.25-0.75% w/w oil) and temperature (25 and 40C) for oil hydrolysis of olive
oil analytic grade (Sigma-Aldrich) and commercial oil (canola, soy, sunflower and safflower
mix).

Experimental design. All experiments were made in 100 mL glass jacket reactors (Celstir,
Wheaton) maintain temperature with a recirculated water bath (Figure 1). Reaction mixture was
prepared homogenizing 40% v/v of phosphate buffer (0.1 M, pH 7.0). Initiating reaction,
previously dissolved lipase in 3 mL of buffer was added to the reaction mixture, according to the
experimental design (Table 1). 24 hours reaction is conducted where was added KOH 0.1 needed
to neutralized free fatty acids in mixture and maintain a pH 7.0 using an automatic titration
station Titralab 856 (radiometer Analytical) according to a agitation rate of 850 rpm.

For the experimentation was used the pre-determinate method OQ Method 2 PID, this pH-Stat
program has a PID controller.

Table 1. Experimental design for vegetable oil hydrolysis by lipase F-AP15.


Figure 1. Continuous stirred tank reactor experimental mounting analyzed by pH-Stat


Results Analysis. Free fatty acids (FFA) obtained by reaction were calculated AOCS method 5a-
40 [3]. Were determinate kinetic parameters k
0
, k
cat
, K
M
, V
max
and catalytic efficiency using a
pseudo-first deactivation order model (eq. 1) [1] and Michaelis-Menten integrated equation (eq.
2) [4].

[]
[]

)] ( )


[]

(
[]
[]

[]
)

( )

Where: k
0,
is a constant for hydrolysis initial rate (mol
FFA
/mol
oil
s); k
D,
is deactivation constant
(s
-1
); V
max
, reaction rate when lipase is saturated with substrate (mol/s); K
M
, Michaelis-Menten
constant (mol/L); [S
0
] and [FFA] were initial substrate and free fatty acids concentration
respectively (mol/L).

To determine constants using previously models, Excel Solver package 2007 was used, using
mathematical methods that permitted a correlation of R2=1.0.

Exp. 1 Exp. 2 Exp. 3 Exp. 4 Exp. 5
Substrate Olive Olive Olive Commercial Commercial
Enzyme concentration (%w/w oil) 0.25 0.75 0.75 0.25 0.75
Vol. Substrate (mL) 24 24 12 24 12
Vol. Buffer (mL) 36 36 18 36 18
Temperature (C) 25 25 40 25 40
In practice, a continuous reactor is operated in stable state conditions, in other words, without
reagent accumulation or products in reactor, therefore, the expression used for this kind of
reactor is the following, which assume the simple Michaelis-Menten kinetic.

) [

] ( )

Where: D is the dilution rate (s
-1
) which is equal to the relation of caudal and reaction volume
mixture; X, is substrate to product conversion.

For reactor design has established 60 mL of reaction mixture as initial volume to scalar to a 50
liters pilot plant, which is going to be in a continuous adding of reagents necessary. Flows were
determinate based in mass balance for a continuous reactor (eq. 3), and experimental results were
by establishing input reagents flows and output products flows [5].

Results
Figure 2 shows hydrolysis progress for both oils in function of time. Pseudo-first order model
applied fit correctly (continuous lines) experimental data (symbols) for all experiments
(R
2
0.98).


Figure 2. Kinetics of enzymatic hydrolysis of olive oil (a) and commercial oil (b) by lipase F-AP15.

Was observed an increase in conversion and reaction rate when was increase enzyme
concentration (Exp. 1 & 2) 0.25% to 0.75% w/w oil, but was no proportional to amount of
enzyme aggregate. Increasing temperature (25 to 40C) activity and conversion were decreasing
(Exp. 1 & 3;4 & 5), as reported by Ben Salah (1994), this happen due microorganism nature
where was the enzyme origin [6].

In Figure 3 shows experimental data linearization (eq. 2) to obtain kinetic parameters, where
slope is K
M
and ordinate in origin is V
max
. There was not observed significant variations on slope
values, meaning that enzyme doesnt show specificity for mono and polyunsaturated fatty acids.
Was observed a good adjustment to experimental data (R2>0.99).

0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 5 10 15 20

H
y
d
r
o
l
y
s
i
s


(
[
F
F
A
]
/
[
S
0
]
)

Time (h)
A). Substrate: Olive oil
Exp. 1
Exp. 2
Exp. 3
Exp 1. [E]
0
= 0.25%p/p; 25C
Exp 2. [E]
0
= 0.75%p/p; 25C
Exp 3. [E]
0
= 0.25%p/p; 40C
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 5 10 15 20

H
y
d
r
o
l
y
s
i
s

(
F
F
A
]
/
[
S
0
]
)

Time (h)
B). Substrate: Commercial oil
Exp. 4
Exp. 5
Exp 4. [E]
0
= 0.25%p/p; 25C
Exp 5. [E]
0
= 0.75%p/p; 40C
There was not observed significant differences on conversion and reaction rate, although fatty
acids composition in oils differs between substrates, where oleic acid concentration
predominates in olive oil and linoleic acid on commercial oil.

Figure 3. Michaelis-Menten integrated equation to determine kinetic parameters.

In Table 2 resumes determinate kinetic parameters to fit mathematical models. With CSTR mass
balance (eq. 3) calculated input and output flows, considering maximum conversion obtained by
experiments and calculated kinetic constants. Last column shows input and output flows for this
reaction in 50 liters CSTR.

Table 2. Kinetic parameters and necessary flows for enzymatic hydrolysis for vegetables oils by lipase F-AP15 in
continuous reactor (CSTR).


Conclusion
The mathematical models used to determinate kinetic parameters fit appropriately to
experimental data (R
2
>0.98). Models are appropriate for this kind of reactions due kinetic
behavior analysis from the beginning to the reaction end.

When was greater the enzyme concentration, although, the rate and conversion percent was
increased, there wasnt proportional to the amount of released enzyme. Therefore is
recommendable to increase enzyme amount where is a long time reaction.

Was observed temperature effect on reaction, concluding that optimal temperature was at 25C.
Lipase didnt show specificity to mono and polyunsaturated fatty acids on oils used in
experiments. With kinetics parameters was proposed a 50 liters pilot plant continuous reactor
design.


Exp 1. y = 128.67x - 2.2546
R = 0.9927
Exp 3. y = 144.94-.02595
R = 0.998
Exp 2. y = 128.51x - 0.4174
R = 0.9988
0
0.05
0.1
0.15
0.2
0.25
0 0.05 0.1 0.15 0.2 0.25
[
F
F
A
]
/
t

(Ln(([FFA]/[So]-[FFA])+1))/t
Exp 5. y = 144.44x - 0.1076
R = 0.9997
Exp 4. y = 128.85x + 0.9216
R = 0.992
0
2
4
6
8
10
12
14
16
18
20
0 0.05 0.1 0.15
[
F
F
A
]
/
t

(Ln(([FFA]/[S
0
]-[FFA])+1))/t
Exp. Conversion
k
0
x 10
5

(mol/mol s)
k
cat
x 10
8

(s
-1
)
V
max
x
10
8
(mol/s)
K
M
x 10
3
(mol/L)
K
M
/k
cat

(mol/L s)
Q x 10
3
(60 mL)
(m
3
/s)
Q (50 L)
(m
3
/s)
1 0.6 2.46 1.624 2.98 0.129 0.357 3.2 0.27
2 0.7 5.38 2.113 11.6 0.129 11.62 13.8 1.15
3 0.2 1.72 0.125 0.69 0.145 1318 8.5 0.71
4 0.6 2.83 1.388 2.97 0.129 0.788 3.1 0.25
5 0.12 1.16 0.538 0.10 0.145 483.3 6.3 0.52
Nomenclature

CSTR continuous stirred tank reactor (1)
D Dilution rate (s
-1
)
FFA Free Fatty Acids (1)
[FFA] Free Fatty Acids concentration (mol/L)
k
cat
Catalytic constant (s
-1
)
k
D
deactivation constant (s
-1
)
K
M
Michaelis-Menten constant (mol/L)
k
0
constant for hydrolysis initial rate (mol
FFA
/mol
oil
s)
KOH Potassium hydroxide (1)
PID proportional-integral-derivative (1)
rpm revolutions per minute (1)
S
0
initial substrate (1)
[S
0
] initial substrate concentration (mol/L)
V
max
reaction rate when lipase is saturated with substrate (mol/s)
X substrate to product conversion (1)


References

Noriega, J A. Purificacin de una Lipasa de las Vsceras de la Sardina (Sardinops sagax caerulea) y Evaluacin de
su Actividad Lipoltica Sobre los cidos Grasos Poliinsaturados Tesis de Doctorado, Instituto Tecnolgico de
Veracruz, 2010.
1. Ramachandra, F. H. Hydrolysis of Oils by Using Immobilized Lipase Enzyme: A Review Biotechnol.
Bioprocess Eng. 7:57-66, 2002.
2. AOCS, Official Methods and Recommended Practices of the AOCS, 6th Edition. (2000).
3. Bisswager, H. Enzyme Kinetics: Principles and Methods, Weinheim: Wiley-VCH, 2002.
4. Gacesa, P. Tecnologia de las Enzimas. Editorial Acribia S.A, 1990.
5. Ben Salah, A. Revue franaaise des corps gras, 41:133-137, 1994.