Ester ammoniolysis in an enzymatic membrane reactor

Article abstract of DOI:10.1016/j.molcatb.2014.05.020

A form of generating amides is by reaction of esters with ammonia (ammoniolysis) or with amines (aminolysis). This reaction can be catalyzed by free or immobilized lipases. The aim of this work is to develop a gas-liquid enzymatic membrane reactor in which the ammoniolysis of ethyl octanoate, catalyzed by Candida antarctica lipase B (EC 3.1.1.3), is improved through immobilization of the biocatalyst onto a membrane surface which also serves as gas-liquid interphase. After the selection of a suitable membrane for the process, data show that the higher the initial substrate concentration, the lower the reaction rate. A Michaelis-Menten type model with competitive product inhibition correlated well with the kinetic data. In addition, the temperature dependence of the reaction rate goes through a maximum. The yield of the amide was always above 92% in the range 25-55 °C, with a maximum of 95% at 40°C. At 40°C, and after 24 h of reaction, values comparable to those reported in the literature on the free enzyme-catalyzed ammoniolysis were achieved, but with much lower enzyme/substrate ratio. Moreover, the immobilized enzyme kept its activity for a long period of time (three weeks).

Full text of DOI:10.1016/j.molcatb.2014.05.020

Accepted Manuscript
Title: Ester Ammoniolysis In An Enzymatic Membrane
Reactor
´
Author: Susana Luque Jos e´ R. Alvarez F. Petrus Cuperus
PII:
S1381-1177(14)00170-2
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2014.05.020
MOLCAB 2968
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
10-12-2013
25-5-2014
27-5-2014
´
Please cite this article as: S. Luque, J.R. Alvarez1, F.P. Cuperus, Ester Ammoniolysis
In An Enzymatic Membrane Reactor, Journal of Molecular Catalysis B: Enzymatic
(2014), http://dx.doi.org/10.1016/j.molcatb.2014.05.020
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ESTER AMMONIOLYSIS IN AN ENZYMATIC
MEMBRANE REACTOR
1
,*
1
2
Susana Luque , José R. Álvarez and F. Petrus Cuperus
1
Department of Chemical and Environmental Engineering. University of Oviedo, 33071
Oviedo, Spain.
2
SolSep BV. St Eustatius 65, NL-7335JE Apeldoorn, The Netherlands
*
Corresponding author: +Tel: +34 98 510 2998; Fax: +34 98 510 3434
e-mail: sluque@uniovi.es
Highlights:
A gas-liquid enzymatic membrane reactor was implemented for direct ammoniolysis.
Ethyl octanoate was converted to octanamide by immobilized C.antarctica lipase B.
High enzyme stability and selectivity towards the amide (95% at 40 ºC) was achieved.
1
Page 1 of 30

ABSTRACT
A form of generating amides is by reaction of esters with ammonia (ammoniolysis) or with
amines (aminolysis). This reaction can be catalyzed by free or immobilized lipases. The aim
of this work is to develop a gas-liquid enzymatic membrane reactor in which the
ammoniolysis of ethyl octanoate, catalyzed by Candida antarctica lipase B (EC 3.1.1.3), is
improved through immobilization of the biocatalyst onto a membrane surface which also
serves as gas-liquid intephase. After the selection of a suitable membrane for the process,
data show that the higher the initial substrate concentration, the lower the reaction rate. A
Michaelis-Menten type model with competitive product inhibition correlated well with the
kinetic data. In addition, the temperature dependence of the reaction rate goes through a
maximum. The yield of the amide was always above 92 % in the range 25-55 ºC, with a
maximum of 95 % at 40 ºC. At 40ºC, and after 24 h of reaction, values comparable to those
reported in the literature on the free enzyme-catalyzed ammoniolysis were achieved, but
with much lower enzyme/substrate ratio. Moreover, the immobilized enzyme kept its
activity for a long period of time (three weeks).
KEYWORDS: Candida antarctica; ester ammoniolysis; lipase; membrane reactor
2
Page 2 of 30

1. INTRODUCTION
The industrial use of enzymes is often limited by their cost because of the difficulty and
expense of the recovery step when they are used in the freely soluble form. The use of
enzymes is typically more cost-effective when they are immobilized. Immobilization of the
enzyme on a support may have several advantages: the possibility of reusing the enzyme, a
better control of the reaction (that allows a more feasible operation), the development of
continuous large scale commercial processes and less pollution of the product by the
residual biocatalyst. Moreover, it may also lead to a reduction of the inhibition and to the
improvement of properties such as stability, activity, specificity and selectivity [1].
Lipases (Triacylglycerol acylhydrolases, EC 3.1.1.3) are widely distributed in nature, and
are able to perform acyl-transfer reactions with a number of nucleophiles (i.e., water,
alcohols, peroxides, amines, hydrazines and oximes) via an acyl-enzyme intermediate [2, 3].
Since water always competes with the desired nucleophile in a kinetically controlled
process, water activity in the reaction mixture affects the selectivity of the reaction.
Enzyme use in the fat and oil industry has become important since they can catalyze
reactions under mild conditions (i.e., the industrial hydrolysis of fats and oils or the
manufacture of fatty acid amides) that otherwise would need high pressure and temperature,
and consequently, expensive equipment. In addition, enzymatic methods have been
developed in order to produce heat-sensitive amides, such as oleamide, without degradation.
This degradation often results in the need on additional distillation steps to meet purity
3
Page 3 of 30

specifications. Enzymes also allow achieving high specificities and, therefore, they can be
used to obtain high-value chemicals for food and industrial uses at competitive production
costs [4].
Amides are interesting chemicals since they can be employed in the peptide and lactam
synthesis, and they have many applications in the textile, paper, wood, metal, rubber, plastic
and coating industries. Fatty acid amides are produced in thousands of metric tons per year
and have good lubricating and surfactant properties, with applications, i.e., in the plastic
industry [5].
Lipase-catalyzed ammoniolysis reactions are those where ammonia serves as the nuclophile
[6,7], and have been successfully accomplished with high conversions under mild conditions
with, i.e., esters [3, 8 – 13], 3-substituted glutarates [14, 15] and amino acid esters [16, 17]
and in the preparation of carboxylic amides from carboxylic acids [18, 19]. One-pot lipase-
catalyzed procedures for the conversion of carboxylic acids into substituted amides via in-
situ formation of the ethyl ester and subsequent ammoniolysis have also been reported [20,
2
2
1]. Other sources for the NH group have been employed, such as urea [22], ammonium
bicarbonate [23], ammonium carbamate and several ammonium salts [24, 25]. If amines are
used, the reaction that takes place is an aminolysis instead of ammoniolysis, and secondary
amides can be prepared [6, 7, 10, 14, 15, 21, 26, 27]. Enantioselective biocatalyzed
ammoniolysis has also received significant attention in the last decade [28 – 31].
However, all of these aforementioned references use either free enzymes or enzymes
immobilized on particulate matter, mostly the commercial ones, prepared on porous support
made of polypropylene. The aim of the present paper is to develop an enzymatic membrane
reactor in which the enzyme-catalyzed ammoniolysis of a carboxylic acid ester can be
improved by means of biocatalyst immobilization onto a membrane surface.
4
Page 4 of 30

Ethyl octanoate was selected as substrate. The free enzyme-catalyzed ammoniolysis of this
substrate has been reported [3, 8], and the best results were obtained when Candida
antarctica lipase B (CALB) was used with an enzyme/substrate ratio of 20 mg/mL, bubbling
ammonia gas to a mixture of ethyl octanoate (0.5 mL) in dry tert-butanol (5 mL). The
selectivity of this reaction set was a function of the excess ammonia used. After 24 hours at
40°C, a 1:1 ratio (1.0 equivalent of ammonia per equivalent of octanoate) resulted in 43% of
the substrate to converted in to octanamide, whereas with an excess of ammonia (5:1
equivalents), 95 % of ethyl octanoate became amide. Fig. 1 shows a scheme of the reactions
involved.
2. MATERIALS AND METHODS
2.1. Materials
Two different dense membranes (designed for gas separation) in hollow-fiber arrangement
2
were used, one made of polyetheretherketone (PEEK), with an area of 0.004 m and the
other made of a 0.1 μm silicon rubber (SIR) layer supported on polyetherimide, with an
2
overall area of 0.04 m . The PEEK membrane module was produced at the Institute of
Membrane Technology- ITM-CNR in Calabria (Italy), and had the characteristics previously
reported by Giorno et al. [32] and the PEI membrane module was fabricated at the
University of Twente, following the protocol described by Roesink et al. [33].
Freeze-dried CALB and a frozen solution containing 28 g/L CALB were kind gifts of
NovoNordisk (Amersfoort, NL). The former showed less activity (120 μmol tributyrin/mg
enzyme⋅min) than the latter (275 μmol tributyrin/mg enzyme⋅min), as measured by the
5
Page 5 of 30

tributyrin test [34]. Glycerol (≥99%), ethyl octanoate (> 98 %), tert-butanol (> 99 %) and
octanoic acid (>98%) were purchased from Merck, Netherlands. Bovine serum albumin
(
lyophilized powder, ≥98%), hexane (for HPLC, ≥97%) and 1-hexadecanol (≥99%) were
supplied by Sigma Aldrich. The ammonia was essentially water free (Hock Lios, T404 E,
99.95 %).
>
2.2. Enzyme immobilization
The enzyme was immobilized according to the procedure of Macrae et al. [35]. This
immobilization is based on physical adsorption on the outside (spongy layer) of the fibers,
which is an easy procedure with small associated costs. It basically consisted of a
preliminary wetting of the membrane by pumping an aqueous solution containing 5 %
glycerol to the shell side of the module for 24 h at negligible transmembrane pressure. This
was followed by a thorough rinsing with deionized water to remove the excess glycerol.
Alcohol wetting led to membrane swelling. Then, a 1000 ppm bovine serum albumin
solution was fed to the outside of the fibers at a transmembrane pressure of 2 kPa and room
temperature for 8 h, allowing the development of a first adsorbed layer of protein on the
membrane surface, which favored enzyme immobilization. This adsorption is enhanced by
the previous alcohol swelling step. The membrane was rinsed again with deionized water
and the enzyme solution was placed in the feed tank and recycled in the system (through the
shell side as well) with a retentate pressure around 4 kPa at room temperature. There was no
observable permeate flux during the whole process. Additionally, immobilization took place
only on the spongy layer of the fibers because the membranes were dense (i.e. non porous)
and therefore, they did not allow the passage of proteins nor enzymes.
6
Page 6 of 30

The amount of active lipase adsorbed with time was calculated by mass balance of the initial
activity in the feed enzyme solution and the residual activity in periodically withdrawn
samples of this solution. [36 – 38] Once the residual activity became constant, the enzyme
solution was removed, and the system was finally rinsed with deionized water and dried
with nitrogen overnight. No permeate was obtained during the immobilization because of
the low pressure applied.
This procedure usually ensures unchanged substrate specificity, and regeneration is possible
after treatments with strong denaturants. The performance has been found to be better when
a hydrophobic support was used, as in the present case, in terms of loses of enzyme and
activity enhancement [1, 39]
2.3. Enzyme-catalyzed reaction
The reaction mixture included ethyl octanoate dissolved in tert-butanol. Tert-butanol cannot
participate in the reaction, because of steric hindrance [3]. For the SIR membrane, the
mixture volume was kept constant at 250 mL, but the proportions of each compound varied
from 12.5 mL ester in 237.5 mL alcohol (0.25 mol/L of ethyl octanoate) to 125 mL ester in
125 mL alcohol (2.5 mol/L of ethyl octanoate). For the PEEK membrane, a volume of 100
mL (20 mL ester in 80 mL alcohol) was employed. During the reaction, each mixture was
pumped to the shell side of the hollow fiber module, whilst ammonia gas was fed to the
inside of the fibers in a dead-end mode.
The system experimental setup (Fig. 2) consists of an Ismatec variable speed gear pump, and
stainless steel pressure gauges located before and after the module in the fiber side. A
security pressure-relief valve is located at the outlet of the module in the ammonia side.
7
Page 7 of 30

The humidity intake in the 250/ 100 mL of mixture substrate-solvent was limited by placing
it in a closed jacketed feed tank instead of in an open one. In order to measure the ammonia
gas consumption, a known-volume stainless steel container was installed between the
ammonia pressurized cylinder and the module. By measuring the pressure in the reservoir it
is possible to determine the usage of the ammonia gas. A flask containing a weak acid
solution was placed at the end of the ammonia gas line for collecting the gas when the
experiments finished (which avoids the atmospheric release). Stainless steel and
polypropylene tubing were used for the ammonia gas line and viton tubing for the reaction
mixture line.
After the reaction, the system was rinsed with small volumes of tert-butanol (to eliminate
both the products and the remaining reactants) and dried by flowing nitrogen.
It has to be pointed out that the ammonia gas consumption could be control only to a certain
extent since some overpressure in the ammonia side of the membrane was needed to ensure
ammonia gas flow thought it and to prevent the reaction mixture flowing though the
membrane. In this study, the pressure was set to 0.05 bar in the liquid side and 0.1 bar in the
ammonia side, which led to an usage of 1 equivalent of ammonia gas every 4 h,
approximately, and kept the organic phase saturated. However, it could be possible to
further optimize this parameter if an automated control of the transmembrane pressure
would be set, keeping a transmembrane pressure as low as 0.01-0.02 and, therefore,
reducing significantly the ammonia gas usage.
2.4. Analysis
Samples of the reaction mixture were analyzed on a HRGC 5300 Megaseries Carlo Erba
equipped with a FID detector, an A200S autosampler and a CP Sil 5B column (Chrompack,
NL). Hexane (at which few drops of ethyl alcohol were added in order to dissolve the
8
Page 8 of 30

octanamide) was used as solvent and 1-hexadecanol was employed as internal standard. The
temperature program was set as follows: 1 min at 60°C (base line) before sampling, from 60
to 100°C at 4°C/min, from 100 to 300°C at 30 °C/min, stay at 300°C for 3 min and then the
temperature was decreased to baseline again. Temperature in both injector and detector was
set to 320°C. In order to improve the detection of the octanoic acid, samples were
methylated with diazomethane prior to analysis. Commercial octanoic acid and octanamide
isolated from a bulk reaction were used to determine the retention time of the reaction
products.
2.5. Mathematical modeling of the reaction
A modification
A Michaelis-Menten equation, extended for simple cases of competitive and uncompetitive
reversible inhibitions of the enzyme, has been adapted from the work of Staniszewki [40].
The scheme is represented in Fig. 3, with the typically employed nomenclature: E for
enzyme, S for substrate and P for products (being P
1
the octanamide and P
2
the octanoic
and ESP
acid). ES is the active enzyme-substrate complex, whereas EP
1
, EP , ESS, ESP
2
1
2
are the inactive complexes. By using the quasi-stationary approximation, the following
expression for the rate of substrate consumption was obtained:
(
1)
*
*
*
where KIS ,KIP1 and KIP2 are the uncompetitive inhibition constants for substrate S and
products P
1
and P
2
, respectively; whereas KIP1 and KIP2 are the competitive inhibition
9
Page 9 of 30

constants for the products. K
substrate conversion as x
intermediates ES, EP , EP , ESS, ESP
41, 42], Eq.1 becomes:
M
= (k-α +kP1 +kP2)/k
α
and v
], and assuming that the concentrations of the
and ESP are much lower than those of the products
m 0
= (kP1 +kP2)[E ]. By defining the
S
=1−[S]/[S
0
1
2
1
2
[
(
(
(
(
(
2)
3)
4)
5)
6)
The mathematical development has been compiled in Appendix A. The integration of Eq.2
gives
(
7)
3
3
. RESULTS AND DISCUSSION
.1. Enzyme immobilization and preliminary screening
The amount of immobilized enzyme varied for each type of membrane used. This was
estimated by measuring the remaining activity in the feed solution at the end of the
1
0
Page 10 of 30

immobilization step and assuming that the difference (with the initial one) was the actual
2
immobilized amount. In the PEEK fibers, with an area of 0.004 m , the immobilized enzyme
leaded to an activity of 1920 μmol/min (equivalent to 16 mg of the freeze dried powder).
2
However, in the SIR module (0.04 m ), the estimated activity was around 15400 μmol/min
(corresponding of 56 mg of lipase coming from the frozen solution). The immobilized
2
activity of the PEEK membrane showed a value of 480,000 μmol/m min, whilst the SIR
2
membrane had a specific activity of 385,000 μmol/m min, which indicates that a square
meter of PEEK is more adsorptive than the same area of SIR. In both cases, the residual
activity in the feed solution became constant after 20 hours of recycle.
For the preliminary screening, solutions containing 1.0 mol/L of ethyl octanoate were fed to
the shell side of the hollow-fiber arrangements at 25ºC. Since the amount of enzyme
adsorbed in each membrane was different, it was decided to maintain an enzyme/ substrate
ratio around 1.0 (expressed as mg enzyme/ mL substrate) in order to allow a better
comparison between the results of both membranes. Hence, 50 mL of ethyl octanoate were
utilized (mixed with 200 mL of tert-butanol to achieve the concentration of 1.0 mol/L) when
dealing with the PEEK membrane (because there were 56 mg CALB on it), and 20 mL of
ethyl octanoate (mixed with 80 mL of tert-butanol) for the SIR membrane (where there were
16 mg CALB). Fig.4a displays the time-dependence of the overall ester conversion (i.e.
molar ratio of products at a given time divided between the initial ester concentration) and
Fig.4b shows the yield of the reactions. As expected, the highest reaction rate (and therefore,
the fastest conversion) was seen in the SIR membrane, because a gram of the enzymatic
preparation retained on this membrane is twice more active than a gram of the enzymatic
preparation absorbed on the PEEK membrane. Nevertheless, the yield difference was
surprising: the PEEK membrane reached a 36.4% after 150 hours and the SIR membrane
1
1
Page 11 of 30

gave a value of 93% after 24 hours. These behaviors cannot be explained by the reaction rate
alone, because in the PEEK membrane the amide yield tended to a plateau. Although further
work would be necessary, we hypothesize that the hydrophobicity of the material could be a
key parameter with respect to enzyme stability and the extent of a hydrolysis reaction. The
contact angles found in the literature lied between 110º and 101º for the silicon rubber [43,
44] whilst the values of 91º and 88º were reported for the other polymer [45, 46]. A more
hydrophilic surface, such that of the PEEK membrane, could favor the hydrolysis, as a
higher water activity could be expected in the proximity of the catalytic sites immobilized
onto the membrane surface
In view of these results, the module with the SIR membrane was selected for further
experimentation.
3.2. Kinetic study
Reactions with initial substrate concentrations of 2.5, 0.5 and 0.25 mol/L were performed,
and the corresponding ester conversions are displayed in Fig. 5a. Results obtained in the
previous section for 1.0 mol/L have also been included. As can be seen, the amount of ethyl
octanoate at zero-time had an important effect on the kinetics of the ammoniolysis, being
faster when less substrate was used. In Fig. 5b, it can be seen that the influence of the initial
concentration in the amide yield is only considerable in the first hours of reaction, and that
the plateau obtained beyond 20 hours is independent of the ester amount at the beginning of
the reaction.
Experimental times were fitted to Eq. 7. The values of the seven kinetic parameters were
determined by the least squares regression method, minimizing the following objective
function (OF) with the help of the mathematical routine UMPOL, implemented in the
software Fortran Power Station 4.0:
1
2
Page 12 of 30

(
11)
where NDP is the number of data points and superscripts “calc” and “exp” mean
*
“
calculated” and “experimental”, respectively. The obtained values of v
, K
m M
, KIS, KIP1, KIP2
,
*
*
K
IP1 and KIP2 were introduced Eq. 2, which was solved numerically by the finite differences
approach, and the goodness of the fitting was evaluated by means of the mean absolute
2
percentage error (MAPE) and the coefficient of determination (R ) with regards to x
S
.
(
(
12)
13)
being
the average experimental conversion. The best fitting parameters are listed in
*
*
*
the first column of Table 1.The high values of the equilibrium constants KIS, KIP1 and KIP2
indicate that the uncompetitive inhibition of the enzyme is negligible and that the dominant
mechanism is the competitive inhibition by the two products because the low value of KIP1
m M
and KIP2. Therefore, the model was refitted with only four kinetic parameters (v , K , KIP1,
IP2
K )
(
14)
1
3
Page 13 of 30

and the values obtained are those reported in the second column of Table 1. The
2
corresponding MAPE and R are almost the same than those of the Eqs. 2 – 5. Moroever,
since KIP2 was higher than KIP1, we have further simplified the model by assuming that only
the octanamide causes inhibition
(
15)
As can be seen in the third column of Table 1, the quality of the fitting is as good as in the
two previous cases. Additional experiments are needed in order to know with security if
both products inhibit the reaction or only the major one does. Calculated conversions for the
three-parameter model are also shown in Fig. 5a.
The amide yield was between 93.4 and 89.9%, depending on the initial ester concentration.
3
.3. Effect of Temperature
As shown in Fig.6, temperature had an effect of the initial reaction rate of the mixture of
2.5 mL ethyl octanoate in 237.5 mL tert-butanol, but its influence on the final conversion
1
or the selectivity was marginal The rate increased as temperature rose from 25 to 40 °C, but
a further increase up to 55 °C did not show the same trend. Amide yields after 10 hours also
increased from 91.65% at 25ºC to 95% at 40ºC, but decreased again to 92% at 55ºC. The
experimentally determined value of 95% at 40ºC is identical to that reported by Zoete et al.
[
3, 8] for the same reaction time (24 hours), but in this work it is obtained with a much
lower enzyme/substrate ratio (4.48 mg/mL), although the ester conversion is not complete
92.4%).
(
1
4
Page 14 of 30

The poorer performance of the system at the highest temperature could be the result of the
enzyme being partially deactivated.
3.4. Enzyme stability
Since the enzyme was immobilize only once in the SIR membrane and the whole set of
experiments were then run, the first reaction studied was repeated after every three
experiments, to check the enzyme stability and eventually, enzyme deactivation. During the
whole set of experiments (on a period over 3 weeks) the enzyme was shown to remain active
as it was at the beginning. Only after the last experiment at the highest temperature (55 °C),
some deactivation occurred. This great stability of the immobilized enzyme, which allowed
its reusability for a long period of time, is of capital importance in industrial applications
due to the resulting savings in operating costs.
5
. CONCLUSIONS
The immobilized enzyme remains active for a long period of time (over 3 weeks), being
affected only by exposure to high temperature. During that period, high conversions were
achieved, with a high yield of the desired product, without the need of purifying the reagents
or the solvent. The enzyme/substrate ratio is nearly 4.5 times lower than that reported
previously in the literature, and taking into account the reuse of the enzyme, that ratio could
be further reduced. The ammonia gas dosage through the membrane directly reached the
catalyst and therefore was more effective than that in the multiphasic system with free
enzyme, which could lead to a decrease of the ammonia gas consumption if an automated
control of the transmembrane pressure would be set.
1
5
Page 15 of 30

The reaction was influenced by the purity of the reagents (traces of water limit the
selectivity of the ammoniolysis) and membrane material. The immobilized enzymes were
stable up to 40ºC. At this temperature, initial rates of reaction were the highest and the final
conversion and selectivity were the same as those obtained in the free enzyme system.
ACKNOWLEDGMENTS
The University of Oviedo is a gratefully acknowledged for the grant that allowed the stay of
Susana Luque in Wageningen. Authors also want to thank Gerard Boswinkel and Gerard
F.H. Kramer for their technical assistance.
APPENDIX A
The mass balance to the substrate gave
[S
0
] = [S] + [ES] + [EP
1
] + [EP
2
] + [ESS] + [ESP
1
] + [ESP
2
] + [P
1
] + [P
2
]
(A1)
By assuming that the concentrations of the intermediates are low when compared with those
of the products, the balance can be written as
[
S
0
] ≈ [S] + [P
1
] + [P
2
]
(A2)
(A3)
Introducing the conversion
[S ] ≈ [P ] + [P
x
S
0
1
2
]
1
6
Page 16 of 30

and taking into account that the instantaneous selectivity can be defined as the ratio between
the kinetic constants
(
(
A4)
A5)
Finally, substitution into Eq. 1:
(
A6)
A slight manipulation of Eq. A6 gave Eqs. 2 – 5.
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2
0
Page 20 of 30

TABLES
Table 1.
Parameters in the kinetic models
2
1
Page 21 of 30

FIGURE CAPTIONS
Figure 1. CALB – catalyzed reaction of ethyl octanoate (I) with ammonia to produce
octanamide (II) and with water to generate octanoic acid (III).
Figure 2. Scheme of the enzymatic membrane reactor
Figure 3. Schematic illustration of the kinetic model with competitive and uncompetitive
reversible inhibition.
Figure 4. Influence of the membrane on the ammoniolysis of ethyl octanoate catalyzed by
immobilized CALB at 25ºC and 1 mol/L of substrate. (a) Ester conversion; (b) yield of the
amide.
Figure 5. Influence of initial substrate concentration on the ammoniolysis of ethyl
octanoate catalyzed by CALB immobilized on a SIR membrane at 25ºC. (a) Substrate
conversion; (b) yield of the amide. Solid lines are the values calculated with Eq.8.
Figure 6. Influence of temperature on the conversion of 0.25 mol/L of ethyl octanoate
catalyzed by CALB immobilized on a SIR membrane.
2
2
Page 22 of 30

Table 1.
Parameters in the kinetic models
Eqs. 2 – 5
Eqs. 2 and 14 Eqs. 2 and 15
v
m
(mol/L/h)
0.09139
0.08936
393618
0.01850
0.05828
1821.26
1184.42
9.363
0.09152
0.08969
0.09149
0.08937
K
K
M
(mol/L)
*
IS (mol/L)
IP1 (mol/L)
K
0.01912
0.02849
0.01795
K
K
K
IP2 (mol/L)
*
IP1 (mol/L)
*
IP2 (mol/L)
MAPE (%)
9.361
0.988
9.363
0.988
2
R
0.988
2
3
Page 23 of 30

GRAPHICAL ABSTRACT
Stainless steel
container
Hollow fiber reactor
Pressure relief
valve
Gear pump
Ethyl octanoate
mixed with tert-butanol)
Flask with acid
(
2
4
Page 24 of 30

OEt
OEt
NH3
NH2
+
EtOH
EtOH
O
CALB
O
(
I)
(II)
H2O
OH
+
O
CALB
O
(
I)
(III)
Figure 1. CALB – catalyzed reaction of ethyl octanoate (I) with ammonia to produce
octanamide (II) and with water to generate octanoic acid (III).
2
5
Page 25 of 30

Stainless steel
container
Hollow fiber reactor
Pressure relief
valve
Gear pump
Ethyl octanoate
mixed with tert-butanol)
Flask with acid
(
Figure 2. Scheme of the enzymatic membrane reactor
2
6
Page 26 of 30

EP2
ESS
*
KIP2
K
IS
P2
+
E + S
+
S
k
P1
+ E + P
ES
+ + E + P
1
kα
k-
α
k
P2
2
P1
P1 P2
*
IP1
*
IP2
KIP1
K
K
EP1
ESP ESP2
1
Figure 3. Schematic illustration of the kinetic model with competitive and uncompetitive
reversible inhibition.
2
7
Page 27 of 30

(
a)
(
b)
Figure 4. Influence of the membrane on the ammoniolysis of ethyl octanoate catalyzed by
immobilized CALB at 25ºC and 1 mol/L of substrate. (a) Ester conversion; (b) yield of the
amide.
2
8
Page 28 of 30

(
a)
(
b)
Figure 5. Influence of initial substrate concentration on the ammoniolysis of ethyl
octanoate catalyzed by CALB immobilized on a SIR membrane at 25ºC. (a) Substrate
conversion; (b) yield of the amide. Solid lines are the values calculated with Eq.8
2
9
Page 29 of 30