Unravelling the role of ultrasonic energy in the enhancement of enzymatic kinetics

Article abstract of DOI:10.1016/j.molcatb.2011.08.001

The enzymatic hydrolysis of anabolic androgenic steroids excreted to urine as glucuronide conjugates has been recently reported to be improved by applying ultrasonic energy to the reaction medium. The hydrolysis time using β-glucuronidase from Escherichia coli K12 was reduced by a factor of six when ultrasonic energy was employed to enhance the enzymatic kinetic. In this study, the effect of ultrasonic energy on the enzymatic hydrolysis kinetic parameters, as well as on the enzymatic activity of β-glucuronidase from E. coli K12 was assessed. The study was conducted using the compounds 4-nitrophenyl-β-d-glucuronide and 4-nitrophenol. Experimental data suggested that the reaction follows the Michaelis-Menten kinetics type. In addition it was found that the ultrasonic energy affects the initial velocity of reaction, which is higher when ultrasound waves are employed when compared to the classical method of incubation at 55 °C. Moreover the values of V _max and k_cat are higher for the ultrasonic essay (V _max(US) = 17.1 ± 0.8 μM min^-1; k _cat(US) = 340,523 min^-1; V_{max(55 °C)} = 14.8 ± 0.7 μM min^-1; k_{cat(55 °C)} = 295,187 min^-1) whilst the Michaelis-Menten constant obtained for the two methodologies showed similar values (K_M(US) = 94.7 ± 7.2 μM; K_{M(55 °C)} = 92.5 ± 6.6 μM). Deactivation of the enzyme was also observed under ultrasonic energy, which was found particularly evident for experimental conditions with excess of substrate; enzymatic activity half-life time increased with the increase of the E/S ratio. This research work seems to support the idea that the use of ultrasonic energy affects the transition state of the enzymatic reaction and that mass transfer processes are also most likely enhanced. .

Full text of DOI:10.1016/j.molcatb.2011.08.001

Journal of Molecular Catalysis B: Enzymatic 74 (2012) 9–15
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Unravelling the role of ultrasonic energy in the enhancement of enzymatic
kinetics
M. Galesioa,∗, J. Lourenc¸ oa, D. Madeiraa, M. Diniza, J.L. Capeloa,b
a REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Monte de Caparica, Portugal
b BIOSCOPE Group, Physical Chemistry Department, Science Faculty – University of Vigo, Ourense Campus E-32004, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
The enzymatic hydrolysis of anabolic androgenic steroids excreted to urine as glucuronide conjugates
has been recently reported to be improved by applying ultrasonic energy to the reaction medium. The
hydrolysis time using ␤-glucuronidase from Escherichia coli K12 was reduced by a factor of six when
ultrasonic energy was employed to enhance the enzymatic kinetic. In this study, the effect of ultra-
sonic energy on the enzymatic hydrolysis kinetic parameters, as well as on the enzymatic activity
of ␤-glucuronidase from E. coli K12 was assessed. The study was conducted using the compounds 4-
nitrophenyl-␤-d-glucuronide and 4-nitrophenol. Experimental data suggested that the reaction follows
the Michaelis–Menten kinetics type. In addition it was found that the ultrasonic energy affects the ini-
tial velocity of reaction, which is higher when ultrasound waves are employed when compared to the
classical method of incubation at 55 ◦C. Moreover the values of Vmax and kcat are higher for the ultra-
Received 5 January 2011
Received in revised form 19 June 2011
Accepted 2 August 2011
Available online 9 August 2011
Keywords:
Enzymatic kinetic study
Ultrasound irradiation
␤-Glucuronidase
Enzymatic hydrolysis
Anti-doping
sonic essay (Vmax(US) = 17.1 0.8 ␮M min−1
;
kcat(US) = 340,523 min−1
Vmax(55 C) = 14.8 0.7 ␮M min−1
◦
; ;
kcat(55 C) = 295,187 min−1) whilst the Michaelis–Menten constant obtained for the two methodologies
◦
◦
showed similar values (KM(US) = 94.7 7.2 ␮M; KM(55 C) = 92.5 6.6 ␮M). Deactivation of the enzyme was
also observed under ultrasonic energy, which was found particularly evident for experimental conditions
with excess of substrate; enzymatic activity half-life time increased with the increase of the E/S ratio.
This research work seems to support the idea that the use of ultrasonic energy affects the transition state
of the enzymatic reaction and that mass transfer processes are also most likely enhanced.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
can subsequently occur [1,3,4]. The phase II metabolism can be
characterized as the conjugation reactions that change the physic-
The administration of anabolic steroids has been the most fre-
quent offence to fair competition inside sports. Their use was first
introduced as agents supporting the athlete recuperation after
extreme stress and fatigue, but rapidly became the main group
in doping abuse [1]. In the World Anti-Doping Agency (WADA)
statistic report of 2009, the anabolic agents, in which is included
the group of androgenic anabolic steroids (AAS), represented 64.9%
of all adverse analytical findings reported by WADAs’ accredited
laboratories [2].
Due to their non-polar character, most AAS are modified in the
human body by phase-I and phase-II metabolic reactions, prior to
their excretion in urine [3]. Phase-I metabolism consists in enzy-
matic reactions (e.g., oxidation, reduction, or hydroxylation) that
inactivate the drug and increase its polarity by adding or exposing
functional sites into the AAS structure, where phase II metabolism
ochemical properties of the compounds in order to promote its
elimination from the body [1,5]. The main phase-II reactions are
the conjugation with glucuronic acid or sulfate moiety [1,3]. How-
ever, for the AAS, the formation of glucuronate conjugates is the
major metabolic pathway of conjugation and inactivation of these
compounds [1,3,6,7]. The result of both phase-I and phase-II reac-
tions is the synthesis of hydrophilic compounds that are more easily
excreted.
Doping control of AAS is based on the detection of such com-
pounds and its metabolites in urine samples from athletes [8,9].
Currently, the routine procedure for the detection of these com-
pounds, comprising both screening and confirmatory analysis, is
typically carried out by gas chromatography–mass spectrometry
(GC–MS) methodologies [10–13]. It is important to stress out that
to be analysed by GC–MS techniques, conjugated steroids must be
hydrolysed and derivatised to fit GC analysis pre-requisites and
improve its mass spectrometric characteristics.
Of crucial importance, is the step of the enzymatic hydrolysis
of the steroid glucuronides that is carried out directly on the urine
sample with the enzyme ␤-glucuronidase (GUS, EC 3.2.1.31) from
∗
Corresponding author. Tel.: +351 21 294 83 00.
E-mail address: marcogalesio@dq.fct.unl.pt (M. Galesio).
URL: http://www.bioscopegroup.org (M. Galesio).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.08.001

10
M. Galesio et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 9–15
Escherichia coli, which is a hydrolase highly specific to ␤-linked-d-
glucuronides [14]. Within anti-doping laboratories, the cleavage of
the glucuronide moiety to produce the free compound is normally
performed by conventional warming at 55 ◦C for 1 h, which was
found to be the optimum temperature to achieve maximum yield
[15–19].
Over the past years, the increasing complexity of doping control,
mainly due to the continuously introduction in anti-doping regu-
lations of new banned substances and methods and also due to the
increasing workloads inside anti-doping laboratories, has led scien-
tists to develop new strategies for fast and high throughput sample
treatments and analysis.
Germany). Sodium hydrogen phosphate and sodium phosphate
dibasic were purchased from Sigma–Aldrich. A commercial solu-
tion of ␤-glucuronidase from E. coli K12 with a specific activity of
185 U/mg at 37 ◦C and pH 7 with nitrophenyl-␤-d-glucuronidase
as substrate (1 mL contained 1.47 mg of protein) was purchased
from Roche Diagnostic (Mannheim, Germany). Methanol (MeOH)
was purchased from Sigma–Aldrich and trifluoroacetic acid (TFA,
99%) was from Riedel-de Haën.
Individual stock standard solutions of each compound with
0.05 M were prepared in 10 mL volumetric flasks with methanol.
These standard solutions were stored in the dark at −20 ◦C. Working
standard solutions were prepared by dilution of the stock standard
solutions in the appropriate volume of methanol.
Modern approaches to sample preparation show a growing
interest in ultrasonic energy as an alternative to conventional
methods. The ability of ultrasonic irradiation methods to acceler-
ate and, occasionally, to increase the chemical reactions yield have
attracted the scientific community for its use over the last decades.
Despite the widespread use of ultrasonic energy in various research
disciplines, only recently the synergetic use of ultrasonication and
enzymes to enhance reactions has been described [20–23].
Briefly, the vibrational motion transmitted to the liquid medium
induced by ultrasound waves causes alternately expansion and
compression of the medium [24]. At sufficiently high power, the
strength of the acoustic field may exceed the attractive forces of
the particles in the expansion or rarefaction cycle and create cavi-
ties in the liquid medium called cavitation bubbles, which can grow,
oscillate, split and implode [25–28]. It is this phenomenon, of cav-
itational bubble implosion, that produces the powerful shearing,
causing the molecules in the liquid to become intensely agitated,
as extreme temperatures and pressures are generated, acting like
micro-reactors inside the liquid media [25,26]. Moreover, the for-
mation of highly reactive chemical radicals may also take place
inside the liquid media [25,26].
Although the improvement of chemical reactions is easily
explained by the cavitation phenomena, the understanding of the
synergetic effect between ultrasounds and enzymes is still far from
being completely understood. Nevertheless, some authors have
pointed out that those phenomena created by ultrasonic energy,
increases the contact area between phases, which allows a reduc-
tion of mass transfer limitations in the enzyme–substrate system
[29].
In recent years, our research group has focused on the devel-
opment of ultrasonic-assisted enzymatic reactions as a tool to
enhance laborious and tedious reactions. In this sense and in col-
laboration with the WADA accredited anti-doping laboratory of
Rome, in Italy, it has been recently demonstrated that high intensity
ultrasonic energy enhances the enzymatic hydrolysis of AAS with
␤-glucuronidase [30]. Although this is a remarkable finding, there
is still a lack of fundamental research about the effects of ultrasonic
energy on the enzyme ␤-glucuronidase catalytic activity.
In the present work the effects of ultrasonic energy on the
kinetics of ␤-glucuronidase enzyme were evaluated using the com-
pound p-nitrophenyl–␤-glucuronide, which is commonly used for
␤-glucuronidase enzymatic essays. A simple theoretical kinetic
model, base on Michaelis–Menten equation, is introduced in order
to investigate the variations in the equation kinetic parameters. The
results of the ultrasonic based enzymatic studies were compared
with those obtained by the routine warming incubation at 55 ◦C.
2.2. Apparatus
A cup horn sonoreactor (SR), model from Dr. Hielscher (Tel-
tow, Germany), was used to accelerate the enzymatic hydrolysis
procedure. A minicentrifuge, model Spectrafuge-mini, from Lab-
net (Madrid, Spain), and a minicentrifuge-vortex, model Sky Line,
from ELMI (Riga, Latvia) were used throughout the sample treat-
ment. A Simplicity 185 from Millipore (Milan, Italy) was used to
obtain Milli-Q water. The enzymatic hydrolysis of 4-nitrophenyl-
␤-d-glucuronide was performed in 2 mL microtube flat cap from
Delta Lab (Barcelona, Spain).
2.3. Chromatographic system and conditions
To analyse the hydrolysis of the 4-nitrophenyl-␤-d-glucuronide,
measurements were carried out by HPLC, using a Waters 600E
gradient metering pump model equipped with a Waters dual chan-
nel UV–visible detector. Samples were injected through a 20 ␮L
loop injector valve (Rheodyne Model 7120) and the chromato-
graphic separation was performed on reversed phase 5 ␮m particle
size, L × I.D. 15 cm × 4.6 mm Discovery® C18 column from Supelco
(Bellefonte, Pennsylvania). The aqueous mobile phase (A) consisted
of HPLC-grade water with 0.01% TFA; the organic phase (B) was
MeOH. The HPLC elution was carried out under isocratic condi-
tions, with 55% of solution A and 45% of solution B. The flow
rate was 0.9 mL/min with a 10 min run time and the UV detec-
tor was set at the wavelength of 305 nm. Calibration curves were
constructed by plotting peak areas against concentrations of PNP.
Throughout the experimental work, data was collected and inte-
grated using Chromeleon Software. For quantification, the peak
areas were determined, and the concentrations calculated with
external calibration curves. The calibration curves for PNP and PNP-
G were run at the beginning of each week. The calibration curves
were calculated by the least square method. Linearity was assessed
by determining the coefficient of correlation (r2) of the points of
the curves, which was always higher than 0.9998. Both PNP and
PNP-G showed linear response in the target concentration range of
0.4–500 ␮M for PNP and 5–600 ␮M for PNP-G.
To ensure the analytical quality of the calibration curve made
at the beginning of each working week, a set of standard samples
with different PNP-G and PNP concentrations were prepared and
measured on each day at routine intervals.
2.4. Sample treatment
Spiked sample solutions of 4-nitrophenyl-␤-d-glucuronide
were prepared in sodium phosphate buffer (0.1 M; pH 6.8). The
commercial solution of ␤-glucuronidase from E. coli K12 was
diluted in the same buffer (1:13). To 2 mL of the spiked samples,
2 ␮L of the prepared enzyme solution, corresponding to 0.22 ␮g of
enzyme, was added and the enzymatic assay was carried out using
two different systems as follows: (a) using conventional warming
2. Experimental
2.1. Standards and reagents
The standards of 4-nitrophenyl-␤-d-glucuronide and 4-
nitrophenol were purchased from Sigma–Aldrich (Steinheim,

M. Galesio et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 9–15
11
NO2
NO2
O
O
O
O
OH
OH
O
HO
HO
β- glucuronidase
+
HO
OH
HO
OH
OH
OH
PNP-G
Glucuronic acid
PNP
Fig. 1. Hydrolytic conversion of substrate PNP-G to the products PNP and d-glucuronic acid through the action of ␤-glucuronidase.
at 55 ◦C and (b) using ultrasonic energy provided by a cup horn
sonoreactor (SR) operating at 60% of sonication amplitude. The
ultrasonic amplitude was chosen based on our previous study with
AAS [30]. After the hydrolysis, the reaction was quenched by the
addition of 10 ␮L of TFA.
The calibration standards were prepared in 10 mL volumetric
flasks. In order to have the same matrix than the samples, calibra-
tion solutions were made in sodium phosphate buffer (0.1 M; pH
6.8) to which was added 50 ␮L of TFA.
Recent studies focusing on the influence of ultrasonic energy in
the enzyme performance have reported both enhancement and
inactivation of enzyme activity [31–34]. Based on our expertise
concerning the application of ultrasonic energy to enhance a variety
of reactions and, particularly, enzymatic reactions, we have found
that, in fact, activation and inactivation occur and are closely linked
to the ultrasonic operating conditions [23,30,35–40]. Parameters
such as ultrasonic frequency and amplitude, vial shape, time of
reaction, as well as enzyme amount and temperature of the reac-
tion bulk, deeply affect the performance of the reaction. Therefore,
a consistent knowledge on the ultrasonic energy performance and
ultrasonic devices’ characteristics is vital to carry out a successful
experiment. For instance, when ultrasonication is applied to a liq-
uid, a slow but constant increase in the bulk temperature occurs
and, depending on the enzyme, denaturation may occur, not due to
ultrasonic waves itself but to the warming generated.
To evaluate the effect of ultrasonic irradiation in both enzymatic
activity and stability of ␤-glucuronidase, the appearance of PNP as
a function of time under ultrasonic irradiation was compared with
the conventional methodology of incubation at 55 ◦C. Experimental
data was achieved using large excess of substrate; 450 ␮M of PNP-
G and 0.22 ␮g of ␤-glucuronidase. Fig. 3 presents the appearance of
PNP as a function of time and the correspondent relative enzyme
activity, for both essays. The unit of enzymatic activity was con-
sidered as the enzyme activity that increases the rate of release of
1 ␮mol of PNP per minute. As it may be seen in Fig. 3b, the activity
of the enzyme under ultrasonic energy is, in the first minutes of the
enzymatic reaction, higher than the one obtained under thermal
incubation at 55 ◦C. Yet, the enzyme activity quickly decreases after
3. Results and discussion
The aim of this work was to compare the enzymatic activity of
␤-glucuronidase and the hydrolysis kinetic parameters under ultra-
sonic irradiation with the conventional reaction at 55 ◦C. For this
purpose PNP-G was used as substrate. Fig. 1 shows the hydrolytic
conversion of PNP-G to PNP. It is important to stress that for all
conditions assayed, three replicates were performed and that the
relative standard deviation, RSD, associated was always below 9%.
3.1. Chromatographic assay to measure the activity of
ˇ-glucuronidase
In order to assess the effect of ultrasonication on the kinetic of
the enzymatic hydrolysis, both product formation and substrate
disappearing rate were measured. Separation of the reaction mix-
ture and detection of its components were carried out by HPLC,
using UV detection at ꢀ = 305 nm, which was found to be the opti-
mal wavelength for detection of PNP-G and PNP. A representative
chromatogram showing PNP-G and PNP peaks, obtained after 4 min
of incubation with ␤-glucuronidase, is presented in Fig. 2. As it
may be seen, only two well resolved peaks corresponding to PNP-G
and PNP are observed, making the analysis of the reaction mixture
simple to achieve.
To correctly estimate the activity of ␤-glucuronidase under an
ultrasonic field, before the enzymatic assay, the chemical stability
of both reaction components was evaluated. Experiments were car-
ried out and the concentration of both components was measured
before and after ultrasonication (10 min at 60% of amplitude). For
this study five replicates were used. The results obtained showed
that both compounds remain stable after application of ultrasonic
energy to the sample’s medium, exhibiting the same concentration
before and after ultrasonication.
3.2. ˇ-Glucuronidase activity/stability under ultrasonic
irradiation
Fig. 2. Representative chromatogram obtained after 4 min of incubation of the sub-
strate PNP-G with the enzyme ␤-glucuronidase. The first peak elutes at 3.51 min and
corresponds to PNP-G, whereas the second peak elutes at 6.67 min and corresponds
to PNP.
The use of ultrasonic energy to enhance enzymatic reactions
is still a matter of great debate within the scientific community.

12
M. Galesio et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 9–15
Table 1
Enzymatic deactivation parameters for the ultrasonic irradiation procedure at different ␤-glucuronidase amounts.
␤-Glucuronidase amount (␮g)
Time until deactivationa (min)
kd (min−1
)
t1/2 (min)
R2
0.04
0.08
0.16
0.22
3.34
4.00
5.03
5.50
0.32 0.03
0.29 0.02
0.33 0.02
0.28 0.02
5.5 0.2
6.4 0.2
7.1 0.1
8.0 0.2
0.985
0.993
0.993
0.998
a
Initial reaction time with constant enzymatic activity (showing no deactivation).
120
100
80
60
40
20
0
0
2
4
6
8
10
12
Time (min)
Fig. 4. Relative enzyme activity for the release of PNP from PNP-G, applying ultra-
sonic irradiation. The concentration of PNP-G was fixed at 450 ␮M and the amount
of ␤-glucuronidase varied from 0.04, 0.08, 0.16, 0.22, 0.33 and 0.44 ␮g. Data identi-
fication: : 0.04 ␮g; ᭹: 0.08 ␮g; : 0.16 ␮g; : 0.22 ␮g; : 0.33 ␮g; : 0.44 ␮g.
To get further insight into the relation between the amount of
enzyme and the enzymatic deactivation by ultrasonic irradiation, a
set of experiments was devised in which the amount of enzyme var-
ied whilst the concentration of substrate was maintained constant
at 450 ␮M. The determination of the deactivation kinetic parame-
ters was performed using six distinct amounts of enzyme: 0.04 ␮g,
0.08 ␮g, 0.16 ␮g, 0.22 ␮g, 0.33 ␮g and 0.44 ␮g. Fig. 4 shows the rel-
ative enzyme activity for the different enzyme concentrations. As it
may be seen, the results present a strong decrease in the enzymatic
activity when lower amounts of enzyme were used; for higher
amounts of enzyme, the enzymatic activity is kept constant for
longer reaction times. With 0.33 ␮g of enzyme, the rate of PNP
appearance increased until 8 min of reaction and then decreased
abruptly. Likewise, by using 0.44 ␮g of enzyme, the rate of PNP still
reaches its maximum at 8 min, but at 10 min, the enzymatic activity
drop is not so significant and it corresponds to 86% of the maximum
activity.
Fig. 3. (a) Appearance of PNP as a function of time for both incubation at 55 ◦C and
ultrasonic irradiation essays. The experimental results were obtained using 450 ␮M
of PNP-G. (b) Correspondent enzyme activity for both experiments. The unit of enzy-
matic activity was considered as the enzyme activity that increases the rate of release
of 1 ␮mol of PNP per minute. Data identification: : 55 ◦C; : ultrasonic irradiation.
A first analysis of the deactivation kinetics was performed using
the simple exponential model by fitting the experimental data to a
first order kinetics, in which the native form is irreversibly trans-
formed into the denaturated form. The first order equation for the
enzymatic deactivation reaction is given by,
6 min of reaction, unlike the reaction under thermal incubation that
stays almost constant along the time studied.
Slow inactivation of the enzyme under high ultrasonic irradia-
tion was expected; nevertheless, when compared with our previous
work, in which the enhancement of the AAS glucuronates hydroly-
sis by ultrasonic irradiation was studied, the enzymatic inactivation
of ␤-glucuronidase occurred faster. The major difference between
the two enzymatic assay conditions, concerns the fact that the
experimental procedure developed in this study was carried out
with low amount of enzyme, at large excess of substrate. Concern-
ing our previous study on the hydrolysis of AAS, the amount of
enzyme was in large excess; almost 250 fold higher than the value
used in the present study [30].
(E)t
= exp(−kt)
(E)0
where (E)t represents the enzymatic activity at time t, (E)0 the initial
enzymatic activity, k the deactivation rate constant and t the time.
Fig. 5 presents the semi-logarithmic plot of (E)t/(E)0 against
time. It clearly shows that the stability of the enzyme is sensitive to
the amount of enzyme. By increasing the enzyme amount, the enzy-
Table 2
Catalytic parameters of ␤-glucuronidase from E. coli K12 using PNP-G as substrate.
KM (␮M)
Vm (␮M min−1
)
kcat (min−1
)
kcat/KM (␮M−1 min−1
)
Incubation at 55 ◦
Ultrasonic irradiation
C
92.5 6.6
94.8 7.2
14.8 0.7
17.1 0.8
295,187
340,523
3192
3593

M. Galesio et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 9–15
10 12
13
0
2
4
6
8
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
Time (min)
Fig. 5. Semi-logarithmic plot of (E)t/(E)0 against time. Data identification:
0.04 ␮g; ᭹: 0.08 ␮g; : 0.16 ␮g; : 0.22 ␮g; : 0.33 ␮g; : 0.44 ␮g.
:
matic activity is kept constant for longer times until deactivation
starts. After this holding period of constant activity the enzymatic
deactivation shows a linear behaviour. The values of the deactiva-
tion constant, as well as the half-life time, were determinate for the
experimental data corresponding to 0.04 ␮g, 0.08 ␮g, 0.16 ␮g and
0.22 ␮g (see Table 1). For enzyme amounts of 0.33 ␮g and 0.44 ␮g,
the activity of the enzyme is kept constant for most of the exper-
imental time and therefore, the deactivation parameters were not
calculated. The deactivation rate constant values found for the dif-
ferent amounts of enzyme, under ultrasonic irradiation, are similar,
which means that the amount of enzyme has no significant effect
on the deactivation rate constant. Unlike the constant rate, the
initial time at which the enzymatic activity is kept constant is sensi-
tive to the amount of enzyme. Higher amounts of ␤-glucuronidase
resulted in longer times at constant initial enzymatic activity, and
therefore higher half-life times.
These results demonstrate that the amount of enzyme is deter-
minant under ultrasonic irradiation. In fact, at this substrate
concentration value and until 0.22 ␮g of ␤-glucuronidase, the
enzyme already reached its substrate saturation point (see Section
3.3), meaning that most active sites of the enzyme are occupied.
Therefore, the inactivation of the enzyme by ultrasonic waves has
a deep effect on the product appearance rate. When the amount of
enzyme is increased, the slow inactivation that occurs during ultra-
sonication is suppressed by the positive effect of ultrasonication on
the enzyme activity.
Concerning our previous study on the hydrolysis of AAS, the
amount of enzyme was in large excess; as a result, in the first
10 min of the AAS hydrolysis reaction, the slow inactivation of the
enzyme was counterbalanced by the positive ultrasonic effects on
the enzyme activity. It is important to stress that, in our previous
study, the hydrolysis reaction time of AAS glucuronates was greatly
improved from 60 min to 10 min by using ultrasonic irradiation
together with ␤-glucuronidase [30].
These results clearly suggest that special attention must be
taken regarding the amount of enzyme, when ultrasonic waves
are employed to enhance the enzymatic reaction. To diminish the
effects of enzyme denaturation on the reaction rate, a slight excess
of enzyme is required.
Fig. 6. Observed time courses for the hydrolytic conversion of PNP-G into PNP (a)
at 55 ◦C and (b) under continuous ultrasonic irradiation, with increased PNP-G con-
centration. Data identification: ‡: 7.7 ␮M; : 15.4 ␮M; ꢁ: 32.1 ␮M; ×: 62.9 ␮M;
:
127.3 ␮M; : 190.1 ␮M; : 257.0 ␮M; ♦: 352.4 ␮M; : 448.3 ␮M; ꢀ: 539.3 ␮M.
(c) Plots of the initial velocity values obtained as a function of the correspondent
PNP-G concentration values and respective Lineweaver–Burk linearization. Enzy-
matic activity was assayed with 0.22 ␮g of enzyme at pH 6.5. For Lineweaver–Burk
representation, the x and y axes indicate the reciprocals of the initial concentra-
tion of PNP-G and initial velocity, respectively. The value of Vmax is given from the
intercept and the value of KM/Vmax from the slope. Data identification: line: incuba-
tion at 55 ◦C essay; y = (6.3 0.2)x + (0.067 0.003), (r2 = 0.9992); line: ultrasonic
irradiation essay; y = (5.5 0.1)x + (0.059 0.003), (r2 = 0.9979).
3.3. ˇ-Glucuronidase kinetic parameters
Through a brief literature search, in which we found sev-
eral reports highlighting the use of ultrasound for numerous
applications, it became evident that few attempts were made to

14
M. Galesio et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 9–15
understand the effect of ultrasonic irradiation on the kinetic param-
eters of the mentioned enzymatic reactions.
energy is mainly due to the stabilisation of the transition state
and the enhancement of mass transfer processes. It has been
also proved that, at substrate excess, the positive effects of high
intensity ultrasonication are quickly surpassed by the inactivation
of ␤-glucuronidase enzyme. Hence, when ultrasonic energy is
used to enhance enzymatic reactions, a concentration of enzyme
superior to the one regularly used is advised.
The results obtained in this study represent an important step
to understand and identify the effects of ultrasonication in the
enzyme–substrate complex interaction. When compared with our
previous study conducted with AAS, the effects of ultrasonication
to enhance ␤-glucuronidase activity, using PNP-G as substrate are
less accentuated. We attribute this result to the nature of substrate;
the harder is the hydrolysis reaction achieved by conventional ther-
mal incubation, more evident and advantageous is the use of high
intensity ultrasonication.
In this work, to assess the effect of ultrasonic irradiation on the
reaction kinetic parameters, the enzymatic study was performed
using initial rate experiments, by varying the concentration of sub-
strate in the presence of a fixed concentration of enzyme. The initial
velocity for a given concentration of PNP-G was determined by plot-
ting the enzyme reaction time as a function of PNP concentration.
Ten PNP-G concentration values were used in this study, ranging
from 7.75 to 540 ␮M. Although the reaction time ranged from 20 s
to 10 min, the initial velocities were calculated using the first 2 min
of the reaction. Fig. 6 presents the time courses for the appearance
of PNP and the initial velocity values obtained as a function of the
correspondent PNP-G concentration values, for both ultrasonic and
incubation at 55 ◦C essays. As it was expected, the reaction velocity
increased with the increase in PNP-G concentration until it reaches
the substrate saturation for 0.22 ␮g of enzyme. The enzyme sub-
strate saturation for the ultrasonic irradiation essay occurred at a
higher concentration value than for the thermal incubation essay.
The kinetic parameters were determined through curve fitting,
using a linear transformation of the Michaelis–Menten equation,
the Lineweaver–Burk plot, in which the reciprocal of the initial
rate, 1/V0, was considered against the reciprocal of the substrate
concentration 1/[PNP-G] (see Fig. 6). The data obtained was used
to display the four fundamental kinetic constants for one substrate
reactions: the maximal velocity of the reaction (Vmax), the catalytic
constant (kcat), the Michaelis constant (KM), and the specificity con-
stant (kcat/KM). With the equation line of the Lineweaver–Burk
plot, the value of Vmax can be obtained from the intercept and
the value of KM/Vmax from the slope. The catalytic constant is
obtained from the total concentration of enzyme in the reaction
medium, 5.01E−05 ␮M, and the value of the maximal velocity of
the reaction. Results are shown in Table 2. The KM values found for
both incubation at 55 ◦C and ultrasonic irradiation are very similar,
meaning that the affinity between the enzyme and the substrate is
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