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Modelling the enzymatic activity of two lipases


isoenzymes commonly used in the food industry
Modelado de la actividad enzimática de dos
isoenzimas lipasas comúnmente utilizadas en la
industria alimentaria
a a a a
Nelson P. Guerra , María Pernas , Lorenzo Pastrana , Ana Torrado , Martín Míguez
a a a a a
, Clara Fuciños , Natalia Estévez , Cristina Sobrosa , Roberto González , Pablo
a a
Fuciños & María Luisa Rúa
a
Departamento de Química Analítica y Alimentaria, Facultade de Ciencias de Ourense,
Universidade de Vigo, As Lagoas s/n, 32004, Ourense, Spain

Available online: 04 Nov 2011

To cite this article: Nelson P. Guerra, María Pernas, Lorenzo Pastrana, Ana Torrado, Martín Míguez, Clara Fuciños, Natalia
Estévez, Cristina Sobrosa, Roberto González, Pablo Fuciños & María Luisa Rúa (2011): Modelling the enzymatic activity of
two lipases isoenzymes commonly used in the food industry Modelado de la actividad enzimática de dos isoenzimas lipasas
comúnmente utilizadas en la industria alimentaria, CyTA - Journal of Food, 9:4, 307-313

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CyTA – Journal of Food
Vol. 9, No. 4, December 2011, 307–313

Modelling the enzymatic activity of two lipases isoenzymes commonly used in the food industry
Modelado de la actividad enzimática de dos isoenzimas lipasas comúnmente utilizadas en la
industria alimentaria
Nelson P. Guerra*, Marı́a Pernas, Lorenzo Pastrana, Ana Torrado, Martı́n Mı́guez, Clara Fuciños,
Natalia Estévez, Cristina Sobrosa, Roberto González, Pablo Fuciños and Marı́a Luisa Rúa
Departamento de Quı´mica Analı´tica y Alimentaria, Facultade de Ciencias de Ourense, Universidade de Vigo, As Lagoas s/n, 32004,
Ourense, Spain
(Received 4 May 2011; final version received 27 June 2011)

An in-depth analysis of the kinetics of two lipases isoenzymes (Lip1 and Lip2) in triacetin hydrolysis in absence and
in presence of hexane was carried out. The addition of hexane led to an increase in enzymatic activities of both
enzymes for all triacetin concentrations, and the kinetic data described a hyperbola which was consistent with the
classical Michaelis–Menten model. Without hexane, the time-course of the triacetin hydrolysis by Lip1 and Lip2 did
not follow a Michaelian behaviour. In this case, a first phase of low enzymatic activity (at triacetin concentrations
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from 0 to 250 mM) was followed by a rapid increase in velocity at triacetin concentrations 250 mM. The
Michaelis–Menten model was unable to describe the first phase due to the linear (nonhyperbolic) relationship
between the velocity and the triacetin concentration, meanwhile the logistic model provided a satisfactory description
of the experimental data corresponding to the second phase of activity.
Keywords: lipases; food; modelling; triacetin; hexane; Michaelis–Menten model; logistic model

En este trabajo se llevó a cabo un profundo análisis de la cinética de dos isoenzimas lipasas (Lip1 y Lip2) en la
hidrólisis de triacetina, en ausencia y en presencia de hexano. La adición de hexano a la mezcla de reacción
incrementó las actividades enzimáticas de ambas enzimas para todas las concentraciones de triacetina, obteniéndose
una relación hiperbólica compatible con el modelo clásico de Michaelis-Menten. En ausencia de hexano, la actividad
de Lip1 y Lip2 no mostró un comportamiento Michaeliano, observándose una fase inicial de baja velocidad a
concentraciones de triacetina entre 0–250 mM, seguida de un rápido incremento en la actividad enzimática
([triacetina]  250 mM). El modelo de Michaelis-Menten no pudo ser utilizado para describir la primera fase debido
al incremento lineal (no hiperbólico) de la velocidad con la concentración de triacetina, mientras el modelo logı́stico
describió adecuadamente la cinética de hidrólisis en la segunda fase.
Palabras clave: lipasas; alimentos; modelado; triacetina; hexano; modelo de Michaelis–Menten; modelo logı́stico

Introduction substrate that enters the binding pocket (Rubin, 1994;


Lipases are defined as triacylglycerol acylhydrolases Verger, 1997).
(E.C.3.1.1.3) that catalyse the hydrolysis of fats and Lipolytic enzymes have attracted an enormous
oils to mono- and di-acylglycerols, glycerol and free interest since more than 20 years ago and currently
fatty acids. In contrast to carboxyl esterases (EC constitute the most important group of biocatalysts for
3.1.1.1), catalysis occurs at the lipid–water interface, biotechnological applications (Hasan, Shah, & Ha-
and most lipases showed a phenomenon known as meed, 2006; Schmid & Verger, 1998). That is mainly
interfacial activation (Saktaweewong et al., 2011). It because although the naturally occurring triacylglycer-
describes an increase in the catalytic activity of the ides are the preferred substrates, lipases also catalyse
enzyme in presence of a hydrophobic interface as that the enantio- and regioselective hydrolysis of a wide
formed by the tryacylglyceride. The activation has been range of natural and synthetic esters and also
associated to a conformational change of a lid or flap, esterification, interesterification, and transesterification
consisting of an oligopeptide segment of the primary reactions in nonaqueous media (Schmid & Verger,
structure that covers the active site. In the inactive 1998) Therefore, lipases have been selected for a
closed conformation, the flap covers the active site but number of potential applications in the food, deter-
in the presence of the interface the flap opens (active gent, pharmaceutical, leather, cosmetic, textile and
conformation) making the active site accessible to the paper industry (Bornscheuer, 2005; Hasan et al., 2006).

*Corresponding author. Email: nelsonpg@uvigo.es

ISSN 1947-6337 print/ISSN 1947-6345 online


Ó 2011 Taylor & Francis
http://dx.doi.org/10.1080/19476337.2011.601818
http://www.tandfonline.com
308 N.P. Guerra et al.

Currently, fat and oil modifications are one of the Therefore, in these cases, the use of the standard
prime areas in food processing industry that demands Michaelis–Menten model to fit the experimental
novel economic and green technologies (Hasan et al., velocity data could be very disadvantageous, because,
2006). For oils and fats, it is well-known that the the vmax and KM values could be underestimated or
functional (e.g. melting points) and nutritional proper- overestimated (Houston & Kenworthy, 2000). This
ties of triacylglycerols (e.g. digestion and absorption) poor estimation of both variables can difficult the
depend not only on their fatty acid profiles, but also optimal operation and control policies when the
on fatty acid distributions in the glycerol backbone enzymatic reaction is carried out in an industrial
(Xu, 2000). Structured lipids are thus defined as bioreactor (Bastin & Van Impe, 1995). For this reason,
triacylglycerol, which are modified to change the fatty it is more adequate that the adoption of more suitable
acid composition and/or positional distribution in the models resulting from a new proposed reaction
glycerol backbone (Iwasaki & Yamane, 2000). Chemi- mechanism or heuristic models that must provide the
cal interesterification is a mature technology in best fit of the experimental data as well as the optimum
industry but only randomised products can usually values for the model parameters (Taylor, 2002).
be produced. However, application of lipases in lipid However, only a few studies deal with the selection of
modification has found an enormous field of applica- models to describe the atypical rate-substrate concen-
tion as they show three main types of selectivity: tration curves (Houston & Kenworthy, 2000).
(i) sn-1,3-regioselectivity, (ii) fatty acid selectivity and Therefore, in the present study, the kinetic data
(iii) triacylglycerol selectivity (Kourist, Brundiek, & from triacetin hydrolysis by Lip1 and Lip2 in presence
Bornscheuer, 2010). This explains why many nutri- and in absence of hexane, obtained in previous works
tional and functional structured lipids have been (Mancheño et al., 2003; Pernas et al., 2001, 2009), were
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produced enzymatically since more than 20 years ago firstly modelled with the Michaelis–Menten model.
(Xu, 2000). Since Lip1 and Lip2 showed nonhyperbolic kinetics in
The paradigmatic example is cocoa butter, an absence of hexane, the enzymatic activity of these two
important major constituent of the chocolate, which lipases were then modelled by using a model developed
melts between 32–358C and re-crystallises during in this work. Finally, sensitivity and robustness of both
processing to a stable crystal mode. The uncertainty models were discussed in relation to the values of the
in the cocoa butter supply and the volatility in prices mean relative percentage deviation modulus and the
require manufacturers to seek alternatives to it. determination coefficient.
Cocoa butter equivalents are close to cocoa butter in
composition and physical properties but are produced
Materials and methods
by enzymatic interesterification reactions from low
valuable vegetable oils, mainly palm oil (Xu, 2000). Materials
Candida rugosa lipases (CRLs) are amongst the Lipase type VII from Candida rugosa was purchased
most frequently used enzymes in biotransformations. from Sigma Chemicals Co. (St Louis, USA). Triacetin
In previous papers, we have purified and characterised was from Fluka (Deisenhofen, Germany). DEAE-
three CRL isoenzymes sharing a high degree of Sephacel, Phenyl-Sepharose CL-4B, Sephacryl HR
homology at, namely, Lip1, Lip2 and Lip3 (Pernas, 100, PD-10 and Sephacryl S200 columns were from
López, Pastrana, & Rúa, 2000; Rúa, Dı́az-Mauriño, Pharmacia (Uppsala, Sweden). All chromatographic
Fernandez, Otero, & Ballesteros, 1993), for which the steps were run on a Pharmacia FPLC. All other
crystal structures have been solved (Ghosh et al., 1995; chemicals were of analytical grade.
Grochulski et al., 1993; Grochulski, Li, Schrag, &
Cygler, 1994; Mancheño et al., 2003). Contrary to
what is observed with ‘‘classical’’ esterases acting on Lipase purification
soluble substrates, the lipase kinetic properties didn’t Lip1 was purified from commercial crude preparations
describe a Michaelian kinetic (Verger & de Haas, according to Rúa and Ballesteros (1994), whereas Lip2
1976). In this way, many lipases, including CRL was obtained from postincubates of the yeast as
isoenzymes, can be activated by hydrophobic inter- described in Pernas et al. (2000) and Sánchez et al.
faces provided by the substrate (triacylglycerides) or (1999).
non-catalytic interfaces such as those formed by
detergents or organic solvents (hexane). Their activity
on soluble triglyceride molecules is nearly marginal Kinetic measurements
(Mancheño et al., 2003; Pernas, López, Rúa, & The interfacial activation of CRL isoenzymes was
Hermoso, 2001; Pernas, Pastrana, Fuciños, & Rúa, studied with triacetin as substrate using a titrimetrical
2009). That means that the activity increases signifi- pH-stat technique (Methrom, Switzerland) as de-
cantly when the substrate forms a lipid–water interface scribed in Pernas et al. (2001). Activation occurs
(interfacial activation) (Verger & de Haas, 1976; when triacetin forms emulsions above the solubility
Verger, 1997). limit of this triacylglyceride in water. Briefly, assays
CyTA – Journal of Food 309

were performed at 308C in 5 mM Tris/HCl buffer (Aguerre, Suarez, & Viollaz, 1989; Lomauro et al.,
(pH 7.0) containing 0.1 M CaCl2 and varying amounts 1985; Pérez-Guerra, Torrado-Agrasar, López-Macı́as,
of triacetin to cover a concentration range from Fajardo-Bernárdez, & Pastrana-Castro, 2007).
35 mM to 1 M. The pH was kept constant at pH 7.
Kinetic assays were also carried out in the presence of
Results and discussion
25% (v/v) hexane in the reaction media (Pernas et al.,
2009). In any case, one activity unit was defined as the Modelling the enzymatic activity of the two lipases
amount of enzyme, which released 1 mmol of fatty acids The activity profiles of the two Candida rugosa lipases
per min under assay conditions. (Lip1 and Lip2) using triacetin without and with
hexane are shown in Supplementary Figure 1. From
the detailed observation of the results, it can be noted
Protein concentration that in absence of hexane (upper part of Supplemen-
It was determined by the Lowry method (Lowry, tary Figure 1), both enzymes displayed two activity
Rosebrough, Farr, & Randall, 1951) using BSA as pulses: being the first one at substrate concentrations
standard. from 0 to 250 mM and the second one at substrate
concentrations from 250 to 1061 mM. In fact, the
activity of both Lip1 and Lip2 enzymes showed a clear
Statistical analyses jump at triacetin concentrations of about 250 mM, at
Individual experiments were performed in triplicate which, the first large-size droplets start to form as
and all data points are represented by the mean. Data indicated by the increase in the turbidity of the solution
sets were statistically analysed by using the software (upper part of Supplementary Figure 1).
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package SPSS Statistics 17.0 for Windows (Release From the comparison between the activity of both
17.0.1; SPSS Inc., Chicago, IL, 2008). enzymes, it can be observed that, in absence of hexane,
from all the substrate concentrations, the increase in
activity of Lip1 was smoother than for monomeric
Model parameters determination and model evaluation Lip2 (upper part of Supplementary Figure 1) but, in
The model parameters were obtained by using the presence of hexane, Lip1 was considerably more active
nonlinear curve-fitting software of SigmaPlot (version than Lip2 over the whole substrate concentration range
9.0, Systat Software, Inc., 2004), which minimised the (lower part of Supplementary Figure 1).
deviations between model predictions and experimen- On the other hand, the addition of hexane to the
tal data according to the sum of squares of errors reaction mixture led to an increase in enzymatic
(SSE) of the model fit: activities of both enzymes for all triacetin concentra-
tions, particularly below the solubility limit (0.27 M)
X
n X
m
SSE ¼ D2i; j for this substrate (upper and lower parts of Supple-
i¼1 j¼1 mentary Figure 1).
From a kinetic point of view, it can be noted that
where Di, j represents the difference between the model both enzymes showed a Michaelian behaviour only
and the experimental value, n and m represent the when triacetin was used as substrate in presence of
number of experimental data points and the number of hexane (lower part of Supplementary Figure 1), as
variables, respectively. it was demonstrated with the Michaelis–Menten
The coefficients of the models with P values lower model (1):
than 0.05 were considered statistically significant.
Parameters were removed from the models when their vmax  ½S
v¼ ð1Þ
asymptotic interval of confidence included zero. KM þ ½S
The criteria used to evaluate the goodness-of-fit of
each model were the determination coefficient (R2) where [S] was the initial triacetin concentration (M),
and the mean relative percentage deviation modulus vmax was the maximum rate of substrate conversion
(RPDM) (Lomauro, Bakshi, & Labuza, 1985): (U/mg) and KM was the Michaelis constant (M)
defined as the substrate concentration at which the
N 
  rate is 50% vmax.
100 X Xi  Xpi 
RPDM ¼ Thus, significant values for the parameters vmax and
N i¼1 Xi KM with high R2 values and low RPDM values (lower
than 10) were obtained (lower part of Supplementary
where Xi is the experimental value, Xpi is the calculated Figure 1). In contrast, non-significant values were
value, and N is the number of experimental data. The obtained for the parameters vmax and KM for the
RPDM parameter is widely used to determine the kinetics in absence of hexane (upper part of Supple-
quality of the fit, being a value of RPDM below mentary Figure 1), thus corroborating that in this
10% indicative of a good fit for practical purposes case, the activity of Lip1 and Lip2 did not show a
310 N.P. Guerra et al.

Michaelian behaviour (Verger, 1997; Verger & de where vM A


max ap and vmax ap are, respectively, the apparent
Haas, 1976). maximum rates (U/mg) for the Michaelian and
Conformational changes in lipases are an impor- autocatalytic processes, KMap is the apparent Michaelis
tant part of the catalytic process, because these constant (M), m is the specific rate for the autocatalytic
enzymes exist in two conformations, the inactive with process (M71) and b is a dimensionless constant of the
the flap closed and the active or flap-open conforma- equation. Other term ([S]) is as previously described.
tion. The transition between closed and open con- Although model (2) has not an underlying enzy-
formation, phenomenon know as interfacial activation, matic mechanism, its mathematical form can be easily
is triggered by the formation of the substrate lipidic related with the lipase mode of action by reparameter-
interface. Several authors have reported that the ising the logistic equation to make the substrate
addition of water insoluble organic solvents (such us concentration explicit at v ¼ vA max ap =2, which is defined
hexane) increased the enzymatic activity of lipase as KAap . Thus, b ¼ emKAap , and:
acting on isotropic solutions of triacylglycerides giving
rise to Michaelis–Menten type kinetics. These results vM vA
max ap  ½S max ap
correlated well with a conformational transition v¼ þ ð3Þ
between the open and closed conformation of the
KMap þ ½S 1 þ e½mðKAap ½SÞ
lipase promoted by the highly hydrophobic hexane
interface (Pernas et al., 2009). In this way, the parameters KMap and KAap could
The low activity over soluble substrates is then have pseudo-physical meaning (because a true reaction
explained by the small fraction of active lipase mechanism is not considered) with an operational and
molecules in the absence of the interface. Nevertheless, practical utility because they could be considered as
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for those activated lipase molecules (by the substrate the apparent affinities for substrate in each phase of the
interface or in the presence of the hexane interface), a catalytic process. As the actual amount of activated
Michaelian dependence between hydrolysis rate and lipases in the aqueous solution and that bound to the
substrate concentration should be expected. On the interface are unknown, KMap and KAap simply indicated
other hand, when a lipidic interface is created, the the amount of substrate which permitted that each
equilibrium will shift towards the active open con- semimaximum velocity was reached. The sum of vM max ap
former and a jump in the hydrolysis rate is observed and vA max ap is the potential maximum rate for the
which frequently follows the characteristic allosteric reaction. As the conversion of a lipase into its active
shape as the substrate concentration increases. The conformation and its penetration into the interface is
existence of non-linear relationships between interfa- a critical step in the catalytic process, m parameter
cial substrate and enzyme concentrations for several should include all factors related to the productive
lipases and substrates (Brockman, Momsen, & Tsujita, interaction lipase/interface. Thus, a high value of m
1988; Martinelle, Holmquist, & Hult, 1995) and/or should indicate a high interfacial activation, which is
cooperative effects in the adsorption of lipases to probably a function of the intrinsic structure of each
lipidic interfaces (Marangoni, 1994) might account for individual lipase and interface.
the observed sigmoidal activity/substrate concentration Nevertheless, in Equation (3) it is not possible to
profiles. obtain, as expected in an enzymatic reaction, a value of
Although for several lipases, the activity over water v ¼ 0 for [S] ¼ 0. To avoid this problem, it is necessary
soluble substrates is only marginal (Verger, 1997), it is to subtract the intercept with the ordinate in the
reasonable to assume that the overall hydrolysis rate at logistic part of model (3), as indicated as follows:
any substrate concentration will be the result of the
!
catalysis of both soluble and insoluble substrate (the vM
maxap  ½S 1 1
algebraic sum of rates if no interactions exist) by v¼ þ vA  
KMap þ ½S maxap 1 þ e½mðKAap ½SÞ 1 þ e½mKAap 
activated lipase molecules, the effective independent
variable in lipolysis (Verger & de Haas, 1976). In other ð4Þ
words, at any substrate concentration, a possible
heuristic model to describe the lipolysis rate should This model satisfies the essential initial and final
involve the sum of Michaelian velocity (predominant conditions for the enzymatic reactions with both
at low substrate concentrations) and allosteric or lipases. Thus, as occurred with the Michaelis–Menten
autocatalytic velocity (predominant when the substrate model (1), when [S] ¼ 0, v ¼ 0, and when ½S ! 1;
interface is created in the system). Thus, using v ! vM A R R
maxap þ vmaxap ! vmaxap , being vmaxap the overall
empirical logistic equations for the description of maximum velocity of the reaction.
autocatalytic processes, the new model can be written The results obtained with the use of model (4) are
as follows: shown in Supplementary Figure 2 (upper part) and in
vM vA Supplementary Table 1. Although the curves drawn
max ap  ½S max ap
v¼ þ ð2Þ through the experimental velocity seemed to indicate
KMap þ ½S 1 þ bm½S that this model describes adequately the trend observed
CyTA – Journal of Food 311

in the experimental data obtained for each CRL the first seven experimental data for both enzymes.
isoenzyme (upper part of Supplementary Figure 2), This observation is clearer for Lip2 than for Lip1 (see
the values obtained for vmax and KM for Lip1 and the circle in the right lower part of Supplementary
Lip2 were not statistically significant at P 5 0.05 Figure 2). In fact, the first seven data points produced,
(Supplementary Table 1). for both enzymes, the higher contributions (15.72 and
Thus, it is necessary to discuss the incapacity of 18.63%) to the overall RPDM values. This indicates
model (4), comprised of the sum of two single models, that effectively, the experimental data belonging to the
for describing accurately the kinetic of enzymatic second activity pulse have a more weight in the fitting
reactions exhibiting two serial enzymatic activity of the model than those belonging to the first activity
pulses. In this type of kinetics, the inefficacy of double pulse.
models to fit the data, was found to be due to the However, the non-significant values obtained for
difference in the amount and values of the experimental vmax and KM when the double model (4) was used
data belonging to the first and second pulse (Pérez- (Supplementary Table 1) also suggest that the failure of
Guerra et al., 2010). this model to fit the experimental data could be also
The different amount of experimental data in each due to the incapacity of the Michaelis–Menten model
activity pulse does not seem to be a cause to explain the (1) for describing adequately the experimental data set
non significant values obtained for vmax and KM, corresponding to the first activity pulse. In an attempt
because the first activity pulse had seven points for for clarifying this hypothesis, model (1) was fitted to
both enzymes, while the second activity pulse had eight these latter experimental data. As can be seen in the
(in case of Lip1) and nine (in case of Lip2) data points. upper part of Supplementary Figure 3, the use of the
However, the values of the experimental activities Michaelis–Menten model did not produce satisfactory
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corresponding to the second activity pulse (from 1.3 to results, because only the value of vmax for Lip1 was
26.6 U/mg for Lip1 and from 8.0 to 28.2 U/mg for found to be significant. In addition, from the detailed
Lip2) were considerably higher than those of the first observation of the plotted raw data, it can be observed
activity pulse (from 0 to 0.8 U/mg for Lip1 and from 0 that the velocity of catalysis show a linear (in case of
to 5.7 U/mg for Lip2). In these conditions, the least- Lip2) or almost linear (in case of Lip1) response, rather
squares technique used for fitting the model to the than a rectangular hyperbolic response, to increasing
experimental data, that assumes constant error var- triacetin concentrations (lower part of Supplementary
iance (Meyer, 1994), weighs the data corresponding to Figure 3). This means that the velocities of the reaction
the second activity pulse, more heavily than those were directly proportional to the substrate concentra-
corresponding to the first activity pulse (Pérez-Guerra tion in this region and consequently, the enzymatic
et al., 2010; Stanescu & Chen-Charpentier, 2009). For reaction with respect to substrate concentration is of
this reason, not statistically significant values for the first order in the first activity pulse. In fact, although
parameters vmax and KM could be obtained and the values for the KM parameter (0.30 M for Lip1
consequently, model (4) should take the form of a and 6.39 M Lip2) calculated with model (1) were
simple Logistic model, whose expression is as follows: not statistically significant at P 5 0.05 (upper part of
! Supplementary Figure 3), they were higher than the
A 1 1 maximum TA concentration (0.25 M) corresponding
v ¼ vmax ap   ð5Þ
1 þ e½mðKAap ½SÞ 1 þ e½mKAap  to the first activity pulse for each enzyme.
In this case, when [S] << KM, the equation of
Although from a practical point of view, a simple Michaelis–Menten can be transformed in a linear
logistic model should not be used to describe a set of Equation (6) as follows:
experimental data showing two pulses (Pérez-Guerra
et al., 2007, 2010), model (5) was used to fit the kinetic vmax  ½S vmax  ½S
v¼ !v¼ ð6Þ
data, in order to corroborate the above mentioned KM þ ½S KM
hypothesis on the inadequacy of model (4) to describe
the formation of the two activity pulses observed in the Since the quotient vmax/KM is constant, it can be
enzymatic reactions of Lip1 and Lip2. substituted into Equation (6) by a new constant, which
The results obtained (lower part of Supplementary could be named k, therefore:
Figure 2 and Supplementary Table 2) clearly showed
that model (5) fits acceptably the experimental activity v ¼ k  ½S ð7Þ
data, because significant values for the parameters
2
vAmax ap , KMap and m and in addition, R values higher Substituting the Michaelis–Menten expression by
than 0.98 were obtained (Supplementary Table 2). Equation (7) into model (4) gives:
!
However, the values of RPDM were, in both cases, 1 1
considerably higher than 10%. From the detailed v ¼ k  ½S þ vA
maxap  
observation of lower part of Supplementary Figure 2, 1 þ e½mðKAap ½SÞ 1 þ e½mKAap 
it can be noted that model (5) did not fit satisfactorily ð8Þ
312 N.P. Guerra et al.

Model (8) satisfies the essential initial condition for achieved for both enzymes (26.65 U/mg for Lip1
any enzymatic reaction, because for [S] ¼ 0, v ¼ 0, but and 28.24 U/mg for Lip2) and the minimum velocity
when [S] ! ?, it can be observed that v ! ?, and (1.0 U/mg for Lip1 and 6.1 U/mg for Lip2) in the
consequently, the final condition that implies that when second activity pulses in the hydrolysis of triacetin
[S] ! ?, v ! vmax is not satisfied in this case. In fact, without hexane.
mainly for Lip2, a clear lack of fit was observed at higher
substrate concentrations (Supplementary Figure 4).
Therefore, the models (4) and (8), which are based Conclusion
on the sum of the velocities in each activity pulse, are From the results obtained in this article, it can be
not applicable to describe the specific activities of Lip1 concluded that the enzymatic activity of Lip1 and Lip2,
and Lip2 on triacetin without hexane. that showed two activity pulses cannot be modelled by
For this reason, a new analysis of the experimental using heuristic double models (the sum of two simple
data was carried out by taking into account the models), because non-significant values were obtained
following considerations. On the one hand, the linear for all the parameters. In this case, it is necessary to
increase in the specific activities, at a triacetin split the time-series data in two and each set must be
concentration of 0.27 M for both enzymes (Supple- modelled separately with the corresponding simple
mentary Figure 3) led to maximum specific activities of model. This approach allows to determine the reasons
0.98 U/mg for Lip1 and 6.11 U/mg for Lip2. On the by which a simple model did not fit adequately a
other hand, at triacetin concentrations higher than corresponding data set, and consequently to modify
0.27 M, both the soluble and insoluble substrates are a model (if necessary) in order to obtain further
present in the reaction mixture. Therefore, the specific improvements in its predictions, by reducing the
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activities in the second activity pulse could be RPDM values and increasing the R2 values.
considered to be as the sum of the enzymatic activities
on both the soluble and insoluble triacetin. Supplementary material
Since the kinetic behaviour of the two lipases is The supplementary material for this paper is available online
affected by the formation of micelles, in this case, the at http://dx.doi.org/10.1080/19476337.2011.601818
substrate-series data must be split in two and each set
must be separately modelled by using the correspond-
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