Académique Documents
Professionnel Documents
Culture Documents
Departamento de Biotecnologa, Universidad Autnoma Metropolitana, P.A. 55-535, 09340 Iztapalapa, Mxico D.F., Mexico
Grupo de Procesos de Transporte y Reaccin en Sistemas Multifsicos, Departamento de Ingeniera de Procesos e Hidrulica, Universidad Autnoma Metropolitana, Mxico
D.F., Mexico
c
Department of Biochemical Engineering, University College London, London WC1E 7JE, United Kingdom
d
Universidad Politcnica de Tlaxcala, San Pedro Xalcaltzinco Tepeyanco, Tlaxcala, Mexico
b
h i g h l i g h t s
A non-reported pseudo intrinsic kinetic model was developed on basic reactions.
The reaction mechanism proposed followed the formalism of LangmuirHinshelwood.
Complexes formation of substrate inhibition and oxygen inactivation are accounted.
Afnity and inhibition constants were obtained from the kinetic model.
This is the rst report for oxygen inactivation constant (22.3 lM).
a r t i c l e
i n f o
Article history:
Received 20 September 2013
Received in revised form 18 November 2013
Accepted 23 November 2013
Available online 1 December 2013
Keywords:
Bioconversion
Cyclohexanone monooxygenase
Modelling
Kinetic parameters
Oxygen inactivation
Escherichia coli
a b s t r a c t
The aim of the current work was to develop a pseudo intrinsic kinetic model, based on elementary reactions, to describe the behavior of the bioconversion of ketones using Escherichia coli TOP10 pQR239. Since
there are no reports of the oxygen inactivation constant in the literature, this study gave new insights to
nd optimal conditions of a suitable oxygen supply during the bioconversion. In this model the reaction
mechanism proposed followed the formalism of LangmuirHinshelwood and considered both substrate
inhibition and oxygen inactivation by the formation of intermediary complexes. Therefore, approximations of the pseudo equilibrium of reaction rates or steady state intermediary species were not considered, which allowed for identifying the role of each reaction step involved in the bioconversion. This
kinetic model adequately described the observations with and without substrate inhibition and/or oxygen inactivation. And the regression and the estimated parameters were statistically signicant, making
these analyses reliable regarding the kinetic behavior of CHMO. Then, substrate and oxygen afnity and
inhibition constants were obtained from the kinetic parameters of the model. It was observed that oxygen and substrate presented similar afnity constant values. The substrate inhibition (KIS) and oxygen
inactivation (KIO2) constants were determined to be 9.98 lM and 22.3 lM, respectively, showing that
the CHMO enzyme was twice more sensitive to inhibition by an excess of substrate than oxygen.
2013 Elsevier B.V. All rights reserved.
1. Introduction
Lactones have wide applications in avorings, as precursors of
anticancer and antihypertension drugs and in the pharmaceutical
industry [1]. Lactones have been obtained by BaeyerVilliger reactions using catalytic process [2] or whole cell bioconversion with a
cyclohexanone monooxygenase (CHMO) expressed in Escherichia
Corresponding author. Tel.: +52 58044999; fax: +52 5558044712.
E-mail
addresses:
coca@xanum.uam.mx
(O.
Castillo-Araiza),
danieltm99@hotmail.com (D. Torres-Martnez), g.lye@ucl.ac.uk (G.J. Lye),
sho@xanum.uam.mx (S. Huerta-Ochoa).
1385-8947/$ - see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.cej.2013.11.047
coli TOP10 pQR239 [3]. The use of whole cells allows enzyme cofactor regeneration for the production of enantiomerically pure compounds [4]. However, it has been reported [5,6] that ketone
bioconversion using CHMO is inhibited by the substrate and
product at concentrations above 0.4 and 4 g L1, respectively. Additionally, Bennett [7] reported that enzyme inactivation may occur
due to residue oxidation of two serines close to the active site. A
number of strategies have been proposed to avoid these types of
inhibition, such as substrate feeding and in situ product removal
using Lewatit resin [8], biocatalyzer encapsulation to prevent
CHMO oxidation [9], the use of ionic liquids as an immiscible phase
substrate reservoir and in situ product removal and maintaining
Nomenclature
CHMO
NT
hE
hEO2
hEO2 S
hEO2 SS
hO2 EO2
[P]
cyclohexanone monooxygenase
enzyme total concentration (lg g1 of biomass)
free enzyme fraction
enzymeoxygen complex fraction
enzymeoxygen-substrate complex fraction
substrate inhibition complex fraction
oxygen inactivation complex fraction
product concentration (g L1)
[O2]
[S]
rn
kj
kLa
K
YP
K P ekL at kL a eK P t
K P kL a
YP
C P C P
C P C P0
coefcient, and t is the time. Fitting was performed using a non-linear regression LevenbergMarquardt algorithm in Polymath.
2.4. Aerated power consumption (Pg) measurement
Aerated power consumption (Pg) was measured using the methodology recently discussed by Ascanio et al. [19]. Basically, the
method is based on electrical measurements performed directly
in the bioreactor stirrer shaft motor by watt meters and ammeters.
To take into account losses occurring in the agitation system, a
blank of the measurements was rst performed with an empty bioreactor. All measurements were performed in triplicate.
2.5. Bioconversion experiments
Bioconversion experiments were carried out using 3.0 g of biomass L1. A buffer solution consisting of 50 mM phosphate pH 7.0
supplemented with 10 g glycerol L1 was used as the aqueous
phase for the bioconversion media. A full face-centered central
composite experimental design with three factors and eleven
experiments was used. Three levels of each factor were studied:
agitation rate (750, 1350 and 1950 rpm), aeration rate (0.75, 1
and 1.4 vvm) and substrate concentration (0.35, 0.80 and
1.0 g L1). During bioconversion experiments, samples of 500 lL
were taken every 3 min during the rst 15 min and then every
hour for three hours. Samples were quickly frozen to stop the reaction. For substrate and product analysis, the samples were centrifuged at 5000 rpm for 10 min to separate the biomass and the
supernatant was analyzed. The substrate and product were quantied by gas chromatography.
2.6. Analysis
Gas chromatography (GC) was used to quantify the concentrations of bicycle[3.2.0]hept-2-en-6-one and its corresponding
regioisomeric lactones. Samples (5 lL) were injected into an XL
gas chromatograph (Perkin Elmer, Norwalk, CT) tted with a
CYCLOSILB 113-6632 capillary column (30 m 530 lm) (J&W
Scientic), and concentrations were determined using an
external calibration curve. The GC injector temperature was set
at 250 C. The GC temperature program used was as follows: the
initial oven temperature was 100 C, held for 1 min and followed
by a temperature increase at 10 C min1 up to 150 C, which
was then held for 3 min. Retention times were 3.7 min and
3.95 min for the substrate (mixture of ketone isomers) and
8.5 min for the product.
3. Mathematical model
3.1. Kinetic model
r1 k1 NT hE O2 k1 NT hEO2
k1
where h represents the free or intermediary complex enzyme fractions in the reaction mechanism. EO2 interacts with the substrate
(S) to form the complex enzyme-oxygen-substrate (EO2S) at a reaction rate r3 and a stoichiometric coefcient of 2
k3
EO2 S 0 EO2 S;
k3
r3 k3 NT hEO2 S k3 NT hEO2 S
EO2 S ! E P;
r 5 k5 NT hEO2 S
However, this mechanism does not consider any type of inactivation by oxygen excess or substrate inhibition, and only proposes
the oxidationreduction dynamic of the active site of the CHMO for
product formation. The reaction mechanism proposed in this paper
(Fig. 1) takes into account the possible formation of two inactive
complexes, one involving the over-oxidation of the active site
(O2EO2) at a reaction rate r2 and a stoichiometric coefcient of 1,
k2
To develop a kinetic model for ketone bioconversion, the reaction mechanism suggested by Sheng et al. [12] was modied. The
reaction mechanism proposed in this paper follows the LangmuirHinshelwoodHougenWatson formalism [20], considering
the following assumptions:
E O2 0 EO2 ;
EO2 O2 0 O2 EO2 ;
k2
and another that represents substrate inhibition (EO2SS) at a reaction rate r4 and a stoichiometric coefcient of 1
k4
k4
NT
dhE
r5 r1
dt
NT
dhEO2
r1 r2 r3
dt
dhO2 EO2
r2
dt
10
dhEO2 SS
NT
r4
dt
11
dhEO2 S
NT
r3 r4 r5
dt
12
NT
13
dS
r3 r4
dt
14
dP
r5
dt
15
dO2
kL aO2 O2 r 1 r 2
dt
16
RSSb
nexp X
nresp
X
b ;b ;...;bp
^ij yil y
^il 1 2! min
W jl yij y
j
17
[P] g/L
0.4590
0.3790
0.2980
0.2180
0.1380
0.0571
Fig. 3. Ketone bioconversion under various operating conditions of agitation and aeration rates, and substrate concentrations: (a) 0.4 g L1; (b) 0.7 g L1; (c) 1.0 g L1.
was used, while the F test was used to obtain the regression
signicance. In this sense, statistical condence in the development of the kinetic model will allow us to relate the kinetic
-1
Estimated value
1
k1
0
k1
k2
0
k2
k3
0
k3
k4
0
k4
1653.33 (Lg s
3.32 (s1)
1
1
1
1.11 10 (Lg s
7.85 105 (s1)
1
k5 (kcat)
F
Ftab = 3.98 for 1 a = 0.95
2200
0.7
0.6
-1
Kinetic parameters
Table 1
Estimated kinetic parameters.
0.5
Substrate
Product
Oxygen
0.4
5
4
0.3
3
0.2
0.1
0.0
0
10
20
30
40
50
60
70
80
0
90
KMO2 (lM)
10
1530
62.72
KMS (lM)
0.5
6
6.8
80
1.4
70.47
KIO2 (lM)
22.30
20.5
9.98
0.9
KIS (lM)
[EO2]
[EO2S]
0.8
Complex fraction
References
1.0
[E]
O2
0.7
E
0.6
0.5
k1
k
k
1
EO2
0.4
k
3
0.3
EO2S
0.2
0.1
0.0
0
10
20
30
40
50
60
70
80
90
aeration rates, which relate the oxygen mass transfer from the
gas phase to the aqueous phase. The decrease in the percentage
of bioconversion when the substrate concentration was increased
from 0.4 to 1 g L1 was mainly due to substrate inhibition. For
the same biocatalyzer, Doig et al. [22], working under various
substrate concentrations (0.26 g L1), observed that greater
ketone bioconversion was achieved in the range of 0.20.4 g L1
in a 1.0 L ask with a volume of 20 mL at 250 rpm. Modication
and control of the kLa value (180290 h1) could increase oxygen
transfer from the gas phase to the aqueous phase by maintaining
a constant concentration of dissolved oxygen; however, this
mechanism causes oxidation in the biocatalyzer and a decrease
in bioconversion. Bennett [7] reported that oxygen excess in the
reaction medium caused oxidation of two peripheral serine residues to form sulfonic acid, causing a change and permanent inactivation of CHMO. These results were also observed by Opperman
and Reetz [11] who designed a CHMO by identifying the surface
amino acids susceptible to oxidation and folding back them inside
the enzyme. The mutated enzyme retained 40% activity in H2O2
(0.2 M), while the wild-type enzyme lost all activity in 5 mM H2O2.
-1
0.5
0.4
Substrate
Product
Oxygen
0.3
5
4
0.2
3
0.1
0.0
0
10
20
30
40
50
60
70
80
90
0.6
0.5
0.4
Substrate
Product
Oxygen
0.3
4
3
0.1
0.0
0
10
20
50
60
70
80
[EO2]
90
O2
0.6
E
k
k1
O2
kk
1
0.4
EO2
EO2S
20
30
40
50
60
O2EO2
k
2
k
3
0.2
70
E
k5
Complex fraction
[E]
10
O2
0.8
[EO2S]
Complex fraction
40
[EO2O2]
30
1.0
0.8
0.0
0.2
-1
0.6
0.7
-1
0.7
-1
0.6
EO2
k3
EO2S
[EO2]
0.4
k1
k1
k
[EO2SS]
kk4
[EO2S]
k3 S
k4
EO2SS
[E]
0.2
80
0.0
90
10
20
30
40
50
60
70
80
90
Table 2 shows the afnity and inhibition constants of the substrate and oxygen obtained from the kinetic parameters of the
model (Table 1), expressed in lM for comparison with those
reported experimentally by other authors for monooxygenases enzymes. Afnity and inhibition constants (Eq. (18)) were dened as
the inverse of the equilibrium constants of the individual
reactions:
0
kj
kj
18
0
0.6
0.5
6
0.4
Substrate
Product
Oxygen
0.3
5
4
0.2
3
0.1
0.0
-1
0.7
-1
and Woodley [6] observed a specic rate of 0.65 g lactone g biomass1 h1 at 2.0 g biomass L1 and 0.5 g ketone L1. The concentration proles of the estimated intermediates present inside the
cell during the bioconversion versus bioconversion time are shown
in Fig. 4b. CHMO oxidation occurs in the rst few minutes, generating the [EO2] complex that is essential in the formation of the
[EO2S] complex which generates the product [P]. Analyzing the
afnity and kinetic parameters estimated for this case study, in
0
the rst reversible reaction, it was observed that k1 was smaller
than k1, favoring the reaction equilibrium toward the formation
of the [EO2] complex. With these kinetic parameters, substrate
afnity constant (KMO2) value of 62.72 lM was obtained (Table 2),
which is on the same order of magnitude as the values reported by
Shannon et al. [25] and Torres-Pazmio et al. [16], i.e. 1530 and
0
10 lM, respectively. In the second reversible reaction, k3 was smaller than k3, favoring the reaction equilibrium toward the formation
of the [EO2S] complex. With these kinetic parameters, an substrate
afnity constant (KMS) value of 70.47 lM was obtained (Table 2),
which is reasonably similar to that reported by Torres-Pazmio
et al. [16] (Table 2). In the last reaction, the k5 parameter was
56.66 s1 (Table 1), which is the reaction rate constant (kcat) for
product [P] formation. Kamerbeek et al. [26] reported kcat values
of 14 and 3.7 s1 for a wild type CHMO (E. coli TOP10 pQR230)
and its mutant, respectively.
In the case study where oxygen inactivation was present since
oxygen mass transfer in the reaction medium was increased, the
operation conditions were 1950 rpm and 1.4 vvm (kLa = 290 h1),
and 0.35 g substrate L1. The comparison between the experimental and calculated data versus bioconversion time is shown in
Fig. 5a. It was observed that the model was able to predict the decrease in lactone production due to CHMO oxidation. The relevant
0
kinetic parameters estimated for this case study were k2 and k2 . It
0
was observed that k2 ok2 by four orders of magnitude; this high k2
value indicates that the CHMO oxidation reaction was virtually
irreversible and coincides with that reported by Bennett [7], who
mentioned that oxidation causes permanent enzyme inactivation.
With these kinetic parameters, an oxygen inactivation constant
(KIO2) value of 22.3 lM was obtained (Table 2). There are no reports
of the oxygen inactivation constant in the literature. Under these
operational conditions, the percentage of experimental bioconversion was 42%. The concentration proles of the estimated intermediates present inside the cell during the bioconversion versus
bioconversion time are presented in Fig. 5b. It was observed that
the formation of the rst [EO2] complex was decreased, rapidly
forming the [O2EO2] and [EO2S] complexes, oxygen inactivation
complex and a complex essential for the formation of the product
[P], respectively. The linear trend in [O2EO2] complex formation in
relation to CHMO oxidation shows that exposure to these conditions will cause total enzyme inactivation.
For the case study where substrate inhibition was present due
to a greater substrate concentration in the reaction medium, the
operation conditions were 1350 rpm and 1.0 vvm (kLa = 180 h1),
and 0.70 g of substrate L1. The comparison between the experimental and calculated data obtained from the t of the model versus bioconversion time is shown in Fig. 6a. Under these operational
conditions, the experimental bioconversion was 20%. It was observed that the model was also able to predict the decrease in lactone production due to substrate inhibition. The relevant kinetic
0
parameters estimated for this case study were k4 and k4 . With
these kinetic parameters, a substrate inhibition constant (KIS) value
of 9.98 lM was obtained (Table 2), which is half of that reported by
Bucko et al. [9] (Table 2). It was found that k4 was three orders of
0
magnitude greater than k4 ; this means that for substrate concen1
trations above 0.4 g L , the global reaction will tend to form more
0
of the substrate inhibitory [EO2SS] complex than the product [P]. k4
is the kinetic dissociation constant of the reversible reaction of
2
0
10
20
30
40
50
60
70
80
90
O2
E
k
0.8
Complex fraction
k1
O2
kk
EO2
P
EO2S
k
4
O2EO2
k
2
k
3
0.6
k
4
3
S
EO2SS
0.4
[EO2]
[EO2O2]
[EO2SS]
0.2
[EO2S]
[E]
0.0
0
10
20
30
40
50
60
70
80
90
5. Conclusions
In this study, we developed a non-reported pseudo intrinsic kinetic model based on elementary reactions accounting for substrate inhibition and oxygen inactivation for bicyclic ketone
bioconversion. The kinetic model was coupled to the reactor model
taking into account interfacial oxygen mass transport, adequately
tted observations and was validated in predictive mode describing observations that were not used in the parameter estimation.
The regression and these estimated parameters were statistically
signicant, making their analysis reliable regarding the kinetic
behavior of CHMO, particularly allowing identify/understand the
kinetic role of each substrate, intermediate and reaction product
on the CHMO enzyme. In further studies, this kinetic model will
be essential to model and scale-up a partitioning bioreactor using
this CHMO enzyme expressed in whole cells of E. coli TOP10
pQR239.
References
[1] M.J. Fink, F. Rudroff, M.D. Mihovilovic, BaeyerVilliger monooxygenases in
aroma compound synthesis, Bioorg. Med. Chem. Lett. 21 (2011) 61356138.
[2] Z. Kang, X. Zhang, H. Liu, J. Qiu, K. Lun Yeung, A rapid synthesis route for Snbeta zeolites by steam-assisted conversion and their catalytic performance in
BaeyerVilliger oxidation, Chem. Eng. J. 218 (2013) 425432.
[3] S.D. Doig, P.J. Avenell, P.A. Bird, P. Gallati, K.S. Lander, G.J. Lye, R. Wohlgemuth,
M.J. Woodley, Reactor operation and scale-up of whole cell BaeyerVilliger
catalyzed lactone synthesis, Biotechnol. Prog. 18 (2002) 10391046.
[4] H. Pfruender, R. Jones, V. Weuster-Botz, Water immiscible ionic liquids as
solvents for whole cell biocatalysis, J. Biotechnol. 124 (2006) 182190.
[5] S.D. Doig, L.M. OSullivan, S. Patel, J.M. Ward, J.M. Woodley, Large scale
production of cyclohexanone monooxygenase from Escherichia coli TOP10
pQR239, Enzyme Microb. Technol. 28 (2001) 265274.
[6] C. Baldwin, M.J. Woodley, On oxygen limitation in a whole cell biocatalytic
BaeyerVilliger oxidation process, Biotechnol. Bioeng. 95 (2006) 362369.
[7] A. Bennett, Mechanism of oxidative inactivation of acinetobacter sp. NCIMB
9871. Cyclohexanone monooxygenase, J. Undergrad. Res. 6(1) (2004) pp. 19.
[8] K. Geitner, J. Rehdorf, R. Snajdrova, U.T. Bornscheuer, Scale-up of Baeyer
Villiger monooxygenase-catalyzed synthesis of enantiopure compounds, Appl.
Microbiol. Biotechnol. 88 (5) (2010) 10871093.
[9] M. Bucko, A. Schenkmayerov, P. Gemeinera, A. Vikartovsk, M. Mihovilovibc, I.
Lackc, Continuous testing system for BaeyerVilliger biooxidation using
recombinant Escherichia coli expressing cyclohexanone monooxygenase
encapsulated in polyelectrolyte complex capsules, Enzyme Microb. Technol.
49 (2011) 284288.