Monitoring the activity of immobilized lipase with quinizarin diester fluoro-chromogenic probe

Article abstract of DOI:10.3390/molecules22122136

Ouinizarin diester is used as a fluoro-chromogenic substrate of the activity of lipase supported in poly(methylmetacrylate) beads (CALB, Novozym^fi 435) dispersed in organic solvents. The monoester and diester of quinizarin are both non-fluorescent species contrasting with the enzymatic product quinizarin that shows optical absorption in the visible region and strong fluorescence signal. The enzymatic conversion is accomplished by spectroscopic measurements and it follows a sigmoid curve from which the mean reaction time of the enzymatic process can be determined. This parameter indicates the enzyme activity of the immobilized lipase. Its dependency with the amount of lipase allowed the determination of the ratio of the catalytic rate and the Michaelis constant (k_c/K_m) and the experimental value found was (1.0 ± 0.1) × 10^?2 mg^?1/min in the case of quinizarin diacetate.

Full text of DOI:10.3390/molecules22122136

molecules
Article
Monitoring the Activity of Immobilized Lipase with
Quinizarin Diester Fluoro-Chromogenic Probe
Carolina Aparecida Sabatini, Denis Massucatto dos Santos, Sabrina Matos de Oliveira da Silva
and Marcelo Henrique Gehlen *
Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos 13566-590, SP, Brazil;
carolinasabatini@gmail.com (C.A.S.); denis.massucatto@gmail.com (D.M.d.S.);
sabrinacarbureto@gmail.com (S.M.d.O.d.S.)
* Correspondence: marcelog@iqsc.usp.br; Tel.: +55-16-3373-9928
Received: 1 November 2017; Accepted: 1 December 2017; Published: 4 December 2017
Abstract: Quinizarin diester is used as a fluoro-chromogenic substrate of the activity of lipase
supported in poly(methylmetacrylate) beads (CALB, Novozym® 435) dispersed in organic solvents.
The monoester and diester of quinizarin are both non-fluorescent species contrasting with the
enzymatic product quinizarin that shows optical absorption in the visible region and strong
fluorescence signal. The enzymatic conversion is accomplished by spectroscopic measurements
and it follows a sigmoid curve from which the mean reaction time of the enzymatic process can be
determined. This parameter indicates the enzyme activity of the immobilized lipase. Its dependency
with the amount of lipase allowed the determination of the ratio of the catalytic rate and the Michaelis
constant (kc/Km) and the experimental value found was (1.0
quinizarin diacetate.
±
0.1)
×
10−2 mg−1/min in the case of
Keywords: fluorochromic substrate; quinizarin diester; lipase assay; enzyme kinetics
1. Introduction
The study of lipase catalyzed reactions of hydrolysis or formation of ester is an important issue
in organic biosynthesis [
and production of enantiomeric compounds using enzymes are applications with great industrial and
pharmaceutical interest [ 11]. Recent reports in the literature have shown that enzyme immobilization
1–5]. Moreover, biofuel production from trans-esterification of natural oils
6–
strategies may control the efficiency of the biocatalyst toward improved properties when compared
with the behavior of non-immobilized enzyme [12–16].
The enzyme activity may be determined from diverse analytical methods. Among those
experimental approaches to monitor enzyme activity, the use of fluorochromic compounds is one
of the most practical methods [17
and opened important frontiers in fluorescence spectroscopy and microscopy of enzymes [22
–
21]. Fluorochromic compounds have established new tools
24].
–
The enzyme screening from microorganism is highly effective with the application of fluorescence
measurements [25–27].
On the other hand, when an immobilized enzyme is reused in a bioreactor, it is important to
determine the degree of activity loss in order to decide the replacement of the biocatalyst. Thus, critical
and direct methods of enzyme activity evaluation using fluorochromic substrates have become
more appealing.
In this contribution, the supported lipase in a catalyzed hydrolysis of aromatic diester is
investigated by absorption and emission spectroscopy. We will demonstrate that quinizarin diester can
be quantitatively converted to free fluorescent quinizarin (Qz) in the presence of immobilized lipase.
This fluorochromic probe allows easy absorption and emission readout in the visible spectral region
Molecules 2017, 22, 2136; doi:10.3390/molecules22122136
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where scattering and bio-sample damage are less effective contrasting with coumarin based substrates
that requires UV excitation.
2. Experimental
The preparation of the substrates quinizarin diacetate (QDA) and quinizarin dibutyrate (QDB)
was described previously [28]. The purity of the diesters was checked by the absence of the 1H-RMN
signal at 12.9 ppm of the –OH group (data reported in the Supplementary Materials, SI) of quinizarin
and monoester. Also, the UV-visible absorption and fluorescence emission measurements help in the
characterization of the compounds.
The supported lipase in poly(methylmetacrylate) beads (CALB, Novozym® 435) was gently
provided by Novozymes Co. and it was used as received. The percentage of enzyme in the solid
support was calculated from the amount of N found in CHN standard analysis. The experimental
samples in fluorescence measurements were prepared by adding 3 mL of the quinizarin diester solution
(2.0
×
10−5 mol L−1 in hexane, cyclohexane, or in decaline) to a quartz cuvette containing a small
·
amount of immobilized lipase (typically 20 mg of the solid support containing immobilized lipase).
Absorption measurements were performed on a Cary 5G—Varian spectrophotometer, and the
corrected steady-state fluorescence spectra were recorded on a CD-900 Edinburgh spectrofluorimeter
with excitation at 480 nm. Samples were conditioned in a 1
×
1 cm quartz cuvette and thermostated at
298 K by circulating water through a jacketed cuvette-holder and were keep under constant magnetic
stirring. During spectroscopic measurement, the solid beads remained in the bottom of the cuvette and
the optical light beam passed though the clear solution region. The emission intensities were calculated
from integration of the full emission spectra. Quinizarin diester in solution without addition of the
lipase did not undergo detectable hydrolysis during several hours. Fluorescence decays of quinizarin
in solution were measured by single-photon timing technique using the picosecond spectrometer
described elsewhere [29].
3. Results and Discussion
The absorption spectrum of the quinizarin diester (QDA or QDB) solutions in the presence of
immobilized lipase in poly(methylmetacrylate) beads (CALB, Novozym® 435) changes dramatically with
reaction time as shown in Figure 1 for QDA. The fade of the UV band with maximum at about 330 nm,
assigned to the diester species, and the concomitant rise of a strong visible absorption band with maximum
about 485 nm of the free quinizarin, points out clearly the lipase activity. The diester and monoester
substrates are non-fluorescent species, and only quinizarin is fluorescent. The emission spectrum of
the samples with QDA or QDB increases with reaction time generating structured emission band with
maximum at 570 nm and a shoulder at about 610 nm as shown in the 3D plot in Figures 2 and 3.
Although the kinetics could be follow by the change in optical density with time, the fluorescence
signal is more sensible to the initial conversion and it has no effect of the presence of the intermediate
monoester that could introduce some spectral overlap in absorption measurements.
The results in absorption and emission obtained here by enzymatic hydrolysis of quinizarin diesters
are similar to the acid hydrolysis used by Ahn et al. to form quinizarin from tert-butyloxycarbonyloxy
protected dye [30,31].
Moreover, the lifetime of the solutions under reaction is about 2.3 ns (monoexponential decay
as illustrated in Figure 4) confirming the absence of emission from quinizarin diester or from its
intermediate monoester. This lifetime value agrees with the lifetime of pure quinizarin of 2.4 ns in
cyclohexane reported in literature [32,33].

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O
O
OH
OH
tf = 360 min
1.5
1.0
0.5
0.0
O
O
O
O
O
CH3
CH3
ti = 0
O
300
350
400
450
500
550
λ (nm)
Figure 1. Absorption spectra of quinizarin diacetate (QDA 1.5
quinizarin (Qz) in water saturated cyclohexane containing 20 mg of Novozym 435, T = 298 K.
×
10−4 mol −1) conversion to
L
·
−5
−1
in hexane as
Figure 2. Emission spectra obtained from lipase hydrolysis of QDA 2.0
×
10 mol
·
L
a function of reaction time (λ = 484 nm, 20 mg of Novozym 435, T = 298 K).
exc
−5
−1
in hexane as
Figure 3. Emission spectra obtained from lipase hydrolysis of QDB 2.0
×
10 mol
·
L
a function of reaction time (λ = 484 nm, 20 mg of Novozym 435, T = 298 K).
exc

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−1
L
Figure 4. Fluorescence decay obtained from sample under lipase hydrolysis of QDA 2.0
×
10−5 mol
·
in hexane (λexc = 400 nm, λem = 550 nm, 20 mg of Novozym 435, T = 298 K). Fluorescence lifetime of 2.3 ns
and fitting chi-square χ2 = 1.041.
Thus, the quinizarin diester substrate in apolar solvent (water saturated solvent) upon addition
of lipase beads undergoes a hydrolysis reaction indicated by both absorption and emission changes of
the samples. The solution of diester substrate is colorless, but it becomes yellow and even light orange
when the conversion to quinizarin occurs by lipase action. Such clear change can also be eye detected
by evaporating the solvent after reaction producing orange polymer beads as illustrated by Figure 5.
(a)
(b)
Figure 5. Supported lipase in poly(methylmetacrylate) beads (CALB, Novozym® 435) before (
after (b) quinizarin diester (QDA) addition, reaction and solvent evaporation.
a) and
When spectral emission of the data in Figures 2 and 3 are integrated and plotted against time,
the curve shapes of the relative intensity are not that of a saturation-type growth function as would
be expected, but a sigmoid behavior is observed. Typical change of the integrated fluorescence
intensity with reaction time is given in Figure 6 in the case of hydrolysis of QDA in hexane, and in
Figure 7 in the case of hydrolysis of QDB. In addition, the same sigmoid shape is also observed in
the plot of absorbance at 485 nm as a function of time using data in Figure 1 (see Supplementary
Materials Figure S3). Thus, the conversion of the diester to the fluorescent quinizarin goes though the
intermediate monoester. The conversion of QDB to quinizarin (Qz) is slower when compared with the
hydrolysis rate of QDA under similar conditions.

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Figure 6. Relative fluorescence intensity (RFI) from integrated emission spectra obtained from lipase
−5
−1
hydrolysis of QDA 2.0
×
10 mol
·
L
in hexane as a function of reaction time (
λ
exc
= 484 nm, 20 mg
of Novozym 435). T = 298 K. The line is the fitting with the model given by Equation (1) (fitting quality
evaluated by R2 > 0.98).
Figure 7. Relative fluorescence intensity (RFI) from integrated emission spectra obtained from lipase
−5
−1
hydrolysis of QDB 2.0
×
10 mol
·
L
in hexane as a function of reaction time (
λ
= 484 nm, 20 mg
exc
of Novozym 435). T = 298 K. The line is the fitting with the model given by Equation (1) (fitting quality
evaluated by R2 > 0.98).
The structures of species in solution during hydrolysis, the diester (S2), monoester (S1) and
quinizarin (P0), are given in scheme of Figure 8.
The S2 and S1 substrates are non-fluorescent species because their excited state of low energy
have a strong n
singlet state due to the increase the aromatic resonance of the dye provided by the two intramolecular
H-bonds formed (see P0 structure in Figure 8) [32 33]. Thus, the fluorescence intensity can grow from
a sequential hydrolysis of the quinizarin diester and monoester intermediate, a fact that may explain the
π* character, while quinizarin has emission in apolar solvents from a low energy ππ*
,

Molecules 2017, 22, 2136
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S shape observed. A kinetic mechanism where two sequential Michaelis-Menten kinetics are arranged
to described the double hydrolysis of the diester is represented in the reaction Scheme 1 below.
O
O
H
O
O
R
O
O
R
O
O
O
O
O
O
H
R
O
O
H
O
S2
S1
P0
Figure 8. Molecular structures of the diester (S2) monoester (S1) and quinizarin product (P0).
k2
kc2
(ES2)
(ES1)
S2
S1
S1
P0
E
E
E
E
+
+
+
+
k-2
k1
kc1
k-1
Scheme 1. Enzymatic reaction mechanism with two sequential Michaelis-Menten steps.
In the proposed model, E is the lipase, and ES2 and ES1 are the enzyme substrate complexes
with diester and monoester, respectively. k1,2 and k
represent the association and dissociation
−1,−2
rate constant of the complexes ES1,2. The catalytic rate constants of first and second hydrolysis
step are represented by kc1,2. For symmetrical diester as in the present case, the rate constants of
association, dissociation, and enzymatic catalysis of the double hydrolysis mechanism may be similar
in the first and in the second steps. Thus under such assumption, the kinetics becomes a function
of a single parameter k = kc[E]/Km, where kc is the general catalytic rate constant, [E] is the enzyme
concentration and Km is the Michaelis-Menten parameter. In this situation, an approximate solution of
the fluorescence intensity may be written as,
I(t) = I∞(1 − (1 + k∗t) exp[−k∗t])
(1)
where I∞ is the fluorescence intensity in the saturation (long time limit) or the maximum intensity
observed, and k* is a first order rate constant that scales with k. In the present model, the hydrolysis of
the diester in the experimental condition used is considered non reversible due to the stabilization of
quinizarin by the intramolecular hydrogen bonding in apolar solvent (see Figure 8).
The experimental results of hydrolysis of quinizarin diacetate and dibutyrate in hexane solution
are illustrated in Figures 6 and 7 with data fitting using Equation (1), respectively. We observe
a good performance of the proposed model to describe the kinetics of the lipase hydrolysis of the
diester. The global rate k* of these two systems in hexane is calculated as 2.8
×
10−2 min−1 and
−3
4.7
×
10 min−1, for QDA and QDB, respectively. Considering that the average reaction time may be
defined as 2/k*, (see Supplementary Materials) the corresponding values obtained experimentally are
about 71 min and 426 min for QDA and QDB substrates in the presence of 20 mg of Novozym 435
supported lipase, respectively.
A simple analysis of Equation (1) reveals an interesting behavior for the initial rate of the enzymatic
process. The expansion of the exponential term of Equation (1) results in a quadratic dependence the

Molecules 2017, 22, 2136
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relative fluorescence intensity with initial reaction time and enzyme concentration [E] (assuming that
k* is proportional to k).
I(t)
≈ (kc[E]t/Km)2
(2)
I∞
Experiments of hydrolysis of QDA performed with different amount of supported enzyme in
decalin are illustrated in Figure 9. All of these results follow quadratic dependence of initial relative
fluorescence intensity (RFI) with reaction time, and they are well fitted with the proposed model of
Equation (1) (see curve fitting in Figure 9).
−5
−1
L
Figure 9. Relative fluorescence intensity obtained from lipase hydrolysis of QDA 2.0
×
10 mol
·
in 3 mL of decaline as a function of reaction time and amount of supported lipase added converted
to enzyme mass (
ꢀ
) 0.7 mg, (
#
) 1.5 mg, (
∆
) 2.2 mg. The line curves are the fitting of the data with
Equation (1). λ = 484 nm (fitting quality evaluated by R2 was always >0.98).
exc
The change of k* with enzyme amount for QDA hydrolysis is plotted in Figure 10 for different
solvent used. The empirical rate constant k* increases with the amount of added enzyme, with a similar
behavior in the three nonpolar solvents. The lipase amount in the supported matrix was determined as
7.2% in weight. Such result was calculated by the CHN analysis of 1.2% of N in the solid samples and
using the amino-acid composition of lipase with molecular weight of 33 kDa previously reported [34].
Using this information, it was possible to estimate the amount of enzyme in mg used in the experiments
with different mass of supported lipase (Novozym® 435).
Considering that substrates and product are in low concentration and are highly soluble in
the organic phase, there will be no saturation of the enzyme capacity in the condition used in our
experiments, and the concentration of enzyme non-complexed with the substrate will be slightly small
but proportional to total enzyme concentration. It follows that the slope of the plot of Figure 8 is an
estimation of kc/Km and the value found is (1.0
±
0.1)
×
10−2 mg−1/min. This rate is one hundred
times slower than the hydrolysis rate of p-nitrophenyl butyrate by CALB Novozym® 435, a standard
UV chromogenic substrate commonly used in the activity test of lipase [35]. It means that lipase
turnover in a double hydrolysis of a larger aromatic diester as in the case of quinizarin is a slow
process when compared with hydrolysis rate of p-nitrophenyl butyrate. On the other hand, lipase
immobilization may result in less enzyme activity in ester hydrolysis but on the other hand, it usually
gives more enzyme stability and possibility of reuse of the biocatalyst [9]. Further investigation of our
system by fluorescence optical microscopy of in situ lipase beads could provide a much faster and

Molecules 2017, 22, 2136
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reliable method of direct enzyme activity determination in the visible region using quinizarin diester
as fluorochromogenic probe.
Figure 10. First order rate constant k* of the enzymatic hydrolysis of QDA by lipase as a function of the
amount of supported lipase added (Novozym 435) converted to enzyme mass in three apolar solvents.
cyclohexane (#), decalin (∆) and hexane (ꢀ). Solution volume of 3 mL, T = 298 K.
4. Conclusions
The enzymatic hydrolysis of quinizarin diester by supported lipase beads (CALB Novozym®
435) producing the highly fluorescent quinizarin was studied by measuring the relative fluorescence
intensity with reaction time in the cases of methyl and butyl derivatives. We showed that quinizarin
diesters are potential fluorochromic probes to investigate the activity of immobilized lipase or to check
whether the enzyme beads are still active to be reused in organic biosynthesis. The fitting of the
fluorescence intensity using a functional with a single parameter k* gave satisfactory representation of
the time evolution of quinizarin formation and allowed the estimation of the average reaction time of
the immobilized lipase in the biocatalytic process.
Supplementary Materials: Supplementary materials are available online. Supplementary data concerning
1H-NMR spectra of the compounds, and the numerical solution of the set of reaction rates of the enzymatic
mechanism expressed in Scheme 1 and comparison with Equation (1) are available free of charge as PDF file.
Acknowledgments: The authors thank FAPESP, CNPq and CAPES for financial support of this work and
Novozymes Latin America for a sample of Novozym® 435. This work is also within the scope of INCT-Catálise
Research Center in Brazil.
Author Contributions: C.A.S. and D.M.d.S. performed the experiments. S.M.d.O.d.S. contributed to the analysis
of the data and M.H.G. conceived the idea, implemented the simulations and wrote the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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