The kinetic behavior of dehydrogenase enzymes in solution and immobilized onto nanostructured carbon platforms

Article abstract of DOI:10.1016/j.procbio.2011.09.019

This paper describes the kinetic behavior of alcohol (ADH) and aldehyde (AldDH) dehydrogenases in solution and immobilized onto carbon platform via polyamidoamine (PAMAM) dendrimers. All the kinetic constants achieved for soluble ADH and AldDH are in agreement with literature data. The influence of pH and temperature was evaluated. Results showed that physiological conditions and ambient temperature can satisfactorily be applied to systems containing dehydrogenase enzymes, so as to ensure an environment where both ADH and AldDH display good activity. It is noteworthy that the affinity between both ADH and AldDH and their substrates and coenzyme is retained after the immobilization process. Investigation of the influence of the storage time demonstrated that there was no appreciable reduction in enzymatic activity for 50 days. Results showed that the PAMAM dendrimers provide a good environment for immobilization of dehydrogenase enzymes and that the affinity observed between the enzymes and their substrates and coenzymes seems to be retained, despite the considerable loss of enzymatic activity after immobilization. Furthermore, the anchoring methodology employed herein, namely layer-by-layer (LbL), required very low catalyst consumption.

Full text of DOI:10.1016/j.procbio.2011.09.019

Process Biochemistry 46 (2011) 2347–2352
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
The kinetic behavior of dehydrogenase enzymes in solution and immobilized
onto nanostructured carbon platforms
a
a
b
a
a,∗
Sidney Aquino Neto , Juliane C. Forti , Valtencir Zucolotto , Pietro Ciancaglini , Adalgisa R. De Andrade
a
Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo (FFCLRP-USP), 14040-901, Ribeirão Preto, SP, Brazil
Nanomedicine and Nanotoxicology Laboratory, IFSC, University of São Paulo, CP 369, 13566-590, São Carlos, SP, Brazil
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 April 2011
Received in revised form 31 August 2011
Accepted 25 September 2011
Available online 1 October 2011
This paper describes the kinetic behavior of alcohol (ADH) and aldehyde (AldDH) dehydrogenases in solu-
tion and immobilized onto carbon platform via polyamidoamine (PAMAM) dendrimers. All the kinetic
constants achieved for soluble ADH and AldDH are in agreement with literature data. The influence of pH
and temperature was evaluated. Results showed that physiological conditions and ambient temperature
can satisfactorily be applied to systems containing dehydrogenase enzymes, so as to ensure an environ-
ment where both ADH and AldDH display good activity. It is noteworthy that the affinity between both
ADH and AldDH and their substrates and coenzyme is retained after the immobilization process. Inves-
tigation of the influence of the storage time demonstrated that there was no appreciable reduction in
enzymatic activity for 50 days. Results showed that the PAMAM dendrimers provide a good environment
for immobilization of dehydrogenase enzymes and that the affinity observed between the enzymes and
their substrates and coenzymes seems to be retained, despite the considerable loss of enzymatic activity
after immobilization. Furthermore, the anchoring methodology employed herein, namely layer-by-layer
Keywords:
Alcohol dehydrogenase
Aldehyde dehydrogenase
PAMAM
Enzyme immobilization
(LbL), required very low catalyst consumption.
©
2011 Published by Elsevier Ltd.
1
. Introduction
The enzyme responsible for the second step of ethanol oxida-
tion, aldehyde dehydrogenase (AldDH), is part of a set of three
isoenzymes known as AldDH A, B, and C, also obtained from baker’s
yeast. AldDH has two identical subunits with a molecular weight of
approximately 200 kDa, and it is classified as 1.2.1.5 [7]. AldDH is
responsible for catalyzing the oxidation of aldehydes to carboxylic
acids, and it displays activity for a wide variety of aliphatic, aro-
matic, and heterocyclic aldehydes [8]. The mechanism proposed
for aldehyde oxidation involves a sequence of two substrates/two
products, with formation of binary and ternary complexes, similar
to the kinetic mechanism described for ADH.
Recently, there has been growing biotechnological interest
in the use of immobilized enzymes, such as dehydrogenases, for
many kinds of purposes; e.g., bioremediation, sensors, and biofuel
cells. The presence of several functional groups on the protein
structure allows one to employ different procedures for enzyme
anchoring onto solid supports. Enzyme immobilization is generally
accomplished by chemical or physical means. In the former case,
covalent linkage and also the cross-linking process are utilized
for binding the enzyme molecules. Sometimes, covalent binding
is not necessary, so enzyme immobilization can be achieved
by using membranes, adsorption processes, or entrapment into
polymer gels and microcapsules. In most cases, the simplicity of
the physical methodologies makes such processes advantageous
More than two hundred enzymes are known to catalyze reac-
+
tions in which nicotinamide adenine dinucleotide hydrate (NAD )
receives the hydride ion from a reduced substrate. The general
nomenclature used for this kind of enzyme is oxidoreductase
or dehydrogenase. Alcohol dehydrogenase (ADH) obtained from
baker’s yeast Saccharomyces cerevisiae was one of the first enzymes
to be isolated and purified. It is classified as 1.1.1.1 and is part of
a large family of dehydrogenase enzymes containing the element
zinc in their structure. The primary structure of ADH consists of a
tetramer formed by four identical subunits with a molecular weight
of 36 KDa each [1]. ADH specificity is restricted to primary alcohols
with linear aliphatic chains, and ethanol is by far the best substrate
for this enzyme [2]. Several studies on the ADH steady-state kinetic
mechanism have definitely shown that this enzyme follows a ran-
dom mechanism and have also indicated that the crucial stage of
the reaction is the dissociation of the NADH species from the formed
complex [3–6].
∗ Corresponding author. Tel.: +55 16 3602 3725; fax: +55 16 3633 8151.
E-mail address: ardandra@ffclrp.usp.br (A.R. De Andrade).
1
359-5113/$ – see front matter © 2011 Published by Elsevier Ltd.
doi:10.1016/j.procbio.2011.09.019

2
348
S. Aquino Neto et al. / Process Biochemistry 46 (2011) 2347–2352
over chemical methods [9]. One of the most important points to
consider for enzyme anchoring onto solid platforms is enzyme
stability, so it is very important to ensure that the enzyme is placed
in a friendly environment, so that it can resist sudden changes in
temperature, pH, and solution composition, which could inactivate
the anchored enzymes. In this context, the choice of a suitable
immobilization process for enzyme anchoring onto solid platforms
is of great importance, because it directly affects the lifetime of the
immobilized enzyme.
The PAMAM dendrimer represents a class of branched and
monodisperse polymers. Unlike classical polymers, dendrimers
exhibit larger uniformity, narrow molecular weight distribution,
and highly functionalized terminal surface. Due to their organized
structure and adsorption characteristics, dendrimers have been
extensively exploited for production of film layers that can be used
as sensors for detection of many different compounds [10]. Recent
papers have described the viability of anchoring enzymes onto
PAMAM dendrimers using the layer-by-layer technique. Perinotto
et al. [11] have shown that ADH can be anchored with PAMAM onto
Au electrodes, and that the resulting electrodes can be applied for
ethanol detection with a detection limit of 1 ppm.
In this context, the study of the kinetic behavior of enzymes
is very helpful for comparison of the effectiveness of the different
methodologies employed for enzyme immobilization. Our group
has evaluated two immobilization processes, namely LbL and pas-
sive adsorption technique, and has observed that the methodology
employed for enzyme immobilization directly influences the enzy-
matic activity [12,13]. In fact, our previous work on enzymatic
biofuel cells has demonstrated that the combination of the LbL
technique with PAMAM dendrimers seems to be a better and more
feasible way of anchoring enzymes onto carbon platforms, since
good control of enzyme disposition onto the surface of the bioanode
is obtained with very low enzyme consumption [13].
Despite the several literature studies on the structure and
kinetic mechanism of dehydrogenase enzymes, there are few com-
parative studies of the kinetic behavior of immobilized enzymes.
In this paper, the kinetic behavior of both ADH and AldDH dehy-
drogenase enzymes in solution and of the corresponding enzymes
anchored onto carbon platforms using PAMAM dendrimers is
investigated.
Fig. 1. Mechanism for the two-step oxidation of ethanol to acetate catalyzed by
ADH and AldDH, with concomitant NAD+ consumption.
in order to keep the same architecture of the generally employed bioanodes, a sta-
ble methylene green film was electropolymerized at the carbon support before the
immobilization step [18].
After formation of poly(methylene green), enzyme immobilization was per-
formed by anchoring the dehydrogenase enzymes onto both sides of a 1 cm2 Toray®
paper (pretreated with nitric acid, in order to enhance the hydrophilicity of the sur-
face) using the LbL technique [13]. Briefly, sample preparation was carried out by
−
1
immersing the substrate into the PAMAM solution (2 mg mL ) for 5 min, followed
−
1
by immersion onto the enzyme solutions (1 mg mL ) for 15 min. The substrates
were rinsed with the buffer solution after each deposition, followed by drying [13].
2
.3. Determination of enzymatic activity by the continuous method
The two-step oxidation of ethanol to acetate catalyzed by ADH and AldDH occurs
+
with concomitant NAD consumption (Fig. 1). So, the substrate hydrolysis activity
◦
of dehydrogenase enzymes was investigated at 25 C, by folowing the reduction of
+
−1
−1
NAD to NADH at 340 nm (ε340 nm, pH 7.5 = 6.220 L mol cm ) in a UV/vis spectropho-
tometer Ultrospec 5300 pro from Amersham Biosciences, using thermostatic quartz
cells of 1 cm path length. The assays were accomplished in phosphate buffer, pH
7.2, to a final volume of 1 mL. The reaction was initiated by addition of the soluble
enzymes or the substrate containing the immobilized proteins, depending on the
study that was being performed. Enzymatic activity was determined by quantifica-
tion of NADH formation, as measured by the increase in absorbance at 340 nm. The
absorbances were recorded for 5 min (with an interval of 2 s between each mea-
surement), and the initial velocity was calculated by linear regression during the
first 2–3 min of reaction. Assays were conducted in triplicate, and controls without
added enzyme were included in each experiment, to quantify the non-enzymatic
hydrolysis of the substrate. One enzyme unit (U) is defined as the amount of enzyme
◦
that hydrolyzes 1.0 ␮mol of substrate per minute at 25 C.
Km (Michaelis–Menten constant) and Vmax (maximum velocity) for substrate
and coenzyme were obtained from substrate hydrolysis and were calculated using
the Lineweaver–Burk plot [20]. Data are reported as the mean ± S.D. of triplicate
measurements, which were considered to be statistically significant at P ≤ 0.05.
2.4. Effect of pH and temperature on enzymatic activity
The influence of both pH and temperature on the kinetics of the enzymes was
◦
determined by assaying enzymatic activity from 15 to 55 C at various pH levels
−1
between 4 and 10. To this end, the following 0.1 mol L buffer solutions were used:
acetate buffer (NaAc/HAc) for pH 4–5; phosphate buffer (NaH2PO4/Na2HPO4) for pH
+
2.
Materials and methods
6–7; tris-hydroxymethyl amino methane–HCl (Tris ) buffer for pH 8–10.
2.1. Chemicals
3. Results and discussion
All the reagents were analytical reagent grade and were used without fur-
−
1
ther purification. The enzymes ADH (E.C. 1.1.1.1, initial activity of 331 U mg
)
Once most studies aim at obtaining high enzymatic activity and
−1
and AldDH (E.C. 1.2.1.5, initial activity of 1.02 U mg ), both obtained from Sac-
charomyces cerevisiae lyophilized powder, were purchased from Sigma–Aldrich and
used as received. The coenzyme NAD+ and the polyelectrolyte PAMAM generation
enhanced lifetime for anchored enzymes, evaluating and under-
standing all the parameters influencing the kinetic behavior of
dehydrogenase enzymes is very important. Therefore, evaluation
of how the amount of enzyme, substrate, and coenzyme influences
the enzymatic activity was carried out by always having the kinetic
parameters of the enzymes in solution as reference values.
4
were also purchased from Sigma–Aldrich and used as received. All solutions were
prepared with high-purity water from a Millipore Milli-Q system, and pH measure-
ments were carried out with a pH electrode coupled to a Qualxtron model 8010 pH
meter. All enzyme and coenzyme solutions were freshly prepared and rapidly used.
The initial kinetic results obtained after the immobilization
process curiously showed that the anchored enzymes apparently
displayed enzymatic activity during only one cycle; i.e., the immo-
bilized enzyme had no significant activity after the first kinetic
2
.2. Enzyme immobilization
The enzymes were anchored onto a 1 cm2 carbon platform (carbon fiber paper,
TGP-H-060, Fuel Cell Earth, Stoneham, MA) with a homemade gas diffusion layer
specifically designed to have low Teflon content [13]. The choice of support was
®
assay. This is because the presence of dendrimers combined with
made so that two goals would be achieved, namely a support with the hydrophilicity
required for the LbL process and increase in the diffusional limits on the bioanode
surface. In fact, the presence of a gas diffusion layer tends to increase the total sur-
face area through formation of a more disperse three-dimensional structure, thus
providing sufficient uniformity and enough porosity that culminate in enhanced
kinetics of the substrates and co-enzymes in terms of reaching the active center of
the enzyme.
enzymes and NAD+ species on the carbon platforms probably
imposed some diffusional limits, thereby hindering flow of the
reduced form of the coenzyme from the dendrimers to the bulk
solution. Due to this diffusional obstruction, it seems that the enzy-
matic activity of the anchored enzymes is lost after the first assay;
however, if the NADH species formed during the catalytic reaction
are forced to leave the active site, the system can be regenerated. For
this reason, it was necessary to design a reproducible method and a
reliable system, to ensure NADH species removal from the anchored
+
There are several studies employing NAD -dependent enzymes, such as amper-
ometric sensors and biofuel cells [12–19]. In these devices, the regeneration of the
coenzyme from its reduced form is quite important, and this process requires the
use of an electrocatalyst because of the high overpotential of the reaction. Hence,

S. Aquino Neto et al. / Process Biochemistry 46 (2011) 2347–2352
2349
Table 1
Substrate kinetic parameters for both dehydrogenase enzymes in solution and immobilized onto a carbon platform.
Enzyme
Parameters
Km (mmol L 1
−
)
Vmax (␮mol NADH min mg
−1
−1
)
−1
Kcat (s
)
−1 −1
Kcat/Km (M s )
ADH
Soluble
Immobilized
18.2 ± 0.1
17.9 ± 0.1
69.4 ± 0.1
172.6 ± 0.1
1.07 ± 0.02
9500 ± 10
59 ± 1
0.45 ± 0.01
17 × 10− ± 1
3
24.1 ± 0.2
80.3 ± 0.2
4.7 × 10 ± 2
6
AldDH
Soluble
Immobilized
16 × 10− ± 1
3
0.13 ± 0.001
0.43 ± 0.001
26.8 × 10 ± 0.1
3
enzymes in all the assays. So, the supported enzymes were regen-
erated by applying a potential of 0.3 V (close to the NADH oxidation
potential) in a Potentiostat/Galvanostat Model 273A – PAR for a few
minutes after each assay, which forced the NADH species to leave
the PAMAM dendrimers.
platforms. The kinetic constant values Km and Vmax were deter-
mined from the double reciprocal graph plotted in the region
in which there was no inhibition (inset of Fig. 2), and values of
−
1
−1
−1
0.14 ± 0.01 mmol L and 70.7 ± 0.2 ␮mol NADH min mg were
−
1
obtained, respectively. Kcat was 176.7 ± 0.2 s
and Kcat/Km was
6
−1 −1
s (see Table 1).
1
.3 × 10 ± 200 mol L
Comparing the results obtained for the kinetic parameters of
3.1. ADH kinetic behavior
both substrate and coenzyme in this paper with literature data,
it can be inferred that all the kinetic constants for soluble ADH
presented here are in agreement with previously published works
Before performing experiments as a function of the substrate
and coenzyme concentration, the influence of the amount of
ADH on the enzymatic kinetics in solution was evaluated in the
[3,4,21,22]. Also, the huge difference in the K data for ethanol
m
and NAD+ corroborated with the ADH structure, in which the NAD+
0.01–0.8 U range. The results evidenced that there is a linear influ-
binding site is easily available to the solution while the substrate
binding site is quite narrow and almost inaccessible to the solution,
thus hindering the access of ethanol to the ADH active site [1].
ence of the ADH load on the initial reaction rate up to 0.2 U, followed
by formation of a plateau; thereafter, this behavior remained con-
stant up to 0.8 U ADH (data not shown). In the case of the anchored
enzymes, the influence of the amount of ADH on the enzymatic
kinetics was evaluated in the range 1–36 ADH bilayers. The mass of
In order to obtain both K and V
m
max
for the anchored ADH, assays
as a function of ethanol and NAD+ concentration were performed
by employing the same conditions used in the experiments with
the enzyme ADH in solution. Considering the substrate variation,
−
2
enzyme deposited per bilayer was about 95 ng cm , which repre-
sented approximately 1.13 U ADH [13]. The results revealed that the
ADH load directly influences the reaction kinetics with loss of lin-
earity above ca. 12 bilayers and subsequent formation of a plateau
were 17.9 ± 0.1 mmol L−1
the values determined for K and V
m
max
and 0.45 ± 0.01 ␮mol NADH min−1 mg−1, respectively. Results as
a function of coenzyme concentration (Fig. 2) furnish values
[
13]. This result indicates that a significant enzymatic activity on
of 0.15 ± 0.01 mmol L−1 and 0.46 ± 0.01 ␮mol NADH min−1 mg−1,
the carbon support is obtained only when a high amount of enzyme
is added, (above 1 U ADH). Although above 12 bilayers the amount
of enzyme does not significantly influence the kinetics, the value
of 36 bilayers was chosen for subsequent experiments due to the
greater stability of this sample.
for K and V
max
, respectively. Comparison between the kinetic
m
data obtained for ADH in solution and results achieved with the
anchored enzymes, demonstrate that, although there is consider-
able loss of enzymatic activity after immobilization, the affinity
between the ADH molecules and the substrate and coenzyme is
retained.
In order to evaluate the stability of the anchored enzymes as a
function of time, kinetic assays were performed for 90 days, using
the same sample with 36 ADH bilayers. The results evidenced that
there was no appreciable enzymatic activity reduction for 30 days
(Fig. 3).
For investigation of the influence of ethanol concentration on
the activity of ADH in solution, the quantity of substrate was var-
−
1
ied from 0.5 to 500 mmol L , while the enzyme load and the
−
1
co-enzyme concentration were kept at 0.2 U and 1.9 mmol L
,
respectively. At low substrate concentrations, there was a lin-
ear increase in the rate of NADH formation; thereafter, the
NADH conversion rate became constant, leading to a typical equi-
lateral hyperbola curve. From the obtained double reciprocal
−
1
graph, both Km and Vmax were determined (18.2 ± 0.1 mmol L
−1
−1
and 69.4 ± 0.1 ␮mol NADH min mg , respectively). In addition,
−1
the Kcat constant was 172.6 ± 0.1 s , and the Kcat/Km ratio was
−1
−1
(see Table 1).
9
500 ± 10 mol L
To evaluate the effect of the coenzyme concentration on the
kinetics of ADH in solution, experiments were performed by vary-
s
+
−3
−1
ing the amount of NAD from 0.12 to 7.6 × 10 mol L . The
−
1
final concentration of reagents was kept at 0.2 U ADH, 0.1 mol L
EtOH, pH 7.2. Fig. 2 shows that the expected equilateral hyper-
−3
−1
+
bola curve occurred only up to 2.6 × 10 mol L NAD . After this
point, there was a decrease in enzymatic activity. The obtained
profile suggests possible inhibition due to excess substrate. This
behavior is supported by the ADH kinetic mechanism, which indi-
cates that the crucial stage of the process is dissociation of the
+
NADH species from the formed complex. In fact, at high NAD
concentration, there might be a competition between the formed
+
NADH and the NAD species in solution, since both compete
Fig. 2. NADH conversion rate as a function of the coenzyme concentration. (ꢀ) ADH
for the active site of the enzyme. This is an important result
that must be considered for maximization of the performance of
the enzymatic system both in solution and anchored onto solid
−
1
in solution, 0.2 U; (᭹) immobilized ADH, 36 bilayers, 0.1 mol L EtOH, phosphate
buffer, pH 7.2. Inset: double reciprocal graph obtained for (ꢀ) soluble ADH and (᭹)
immobilized enzyme.

2
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S. Aquino Neto et al. / Process Biochemistry 46 (2011) 2347–2352
Fig. 3. Enzymatic activity of anchored ADH as a function of time. 36 bilayers of
−1
immobilized ADH, 0.1 mol L EtOH, phosphate buffer, pH 7.2.
3.2. AldDH kinetic behavior
Prior to the assays as a function of substrate and coenzyme con-
Fig. 5. NADH conversion rate as a function of the acetaldehyde concentration. (ꢀ)
centrations, the influence of the presence of potassium ions and
thiols was evaluated. This is important to evaluate a wide range
of parameters that could influence the final performance of the
immobilized enzymes. Our results gave evidence that the yeast
enzyme is highly dependent on both species (Fig. 4). The assays
were carried out as a function of the potassium concentration from
−
3
−1
+
AldDH in solution, 0.05 U; (᭹) immobilized AldDH, 0.8 U. 1.67 × 10 mol L NAD ,
−
1
−1
KCl 0.1 mol L , 2-mercaptoethanol 0.01 mol L , pH 7.2. Inset: double reciprocal
graph obtained for (ꢀ) soluble AldDH and (᭹) immobilized enzyme.
same architecture employed in the study on ADH (carbon supports
containing 36 AldDH bilayers) was utilized in all the subsequent
assays.
To evaluate the effect of coenzyme concentration on AldDH
kinetics in solution, experiments were performed by vary-
−
1
1
0 to 100 mmol L , and as a function of 2-mercaptoethanol con-
−1
centration from 1 to 10 mmol L . The best results were achieved
in the assays employing 100 and 10 mmol L potassium ions and
-mercaptoethanol, respectively. These data reveal that, besides
−1
2
+
−3
−1
ing the amount of NAD from 0.1 to 5 × 10 mol L . The
anchoring the enzyme properly, it is important to control other
experimental parameters, so that a good final performance is
achieved.
Once the assay conditions were established, the influence
of the amount of AldDH on the enzymatic kinetics in solution
was evaluated in the 0.025–0.1 U range. In a final volume of
final concentration of reagents was kept at 0.05 U AldDH,
−
3
−1
−1
−1
0
.5 × 10 mol L acetaldehyde, 0.1 mol L KCl, and 0.01 mol L
2
-mercaptoethanol, pH 7.2. Contrary to the results obtained with
+
ADH, data as a function of the NAD concentration demon-
strated that no inhibition process occurred in the evaluated
concentration range. Indeed, the expected kinetic behavior was
verified; i.e., the initial rate increased linearly at low NAD+
1
0
mL, the following conditions were maintained: acetaldehyde
−
3
−1
−3
−1
+
−1
KCl,
.5 × 10 mol L , 1.67 × 10 mol L
NAD , 0.1 mol × L
−3
−1
concentrations, followed by formation of
a plateau, which
and 2-mercaptoethanol 0.01 × 10 mol L . There was a linear
increase in the initial rate, which was followed by formation of
a plateau up to 0.05 U AldDH (data not shown). Similarly to the
results obtained with ADH, the assays as a function of the load
of AldDH anchored by the LbL technique showed that consider-
able enzymatic activity was achieved up to 20 bilayers only. The
resulted in typical equilateral hyperbola curve. From the
a
obtained double reciprocal graph, Km and Vmax were determined
−
1
−1
−1
as 0.41 ± 0.01 mmol L
and 23.2 ± 0.1 ␮mol NADH min mg
,
−
1
respectively. The Kcat constant was 77.3 ± 0.1 s
and the Kcat/Km
5
−1 −1
ratio was 1.9 × 10 ± 10 mol L
s (see Table 1).
For the AldDH in solution, the assays as a function of sub-
strate concentration were performed by varying the amount of
−
3
−1
acetaldehyde from 0.005 to 10 × 10 mol L . The final concen-
−
3
−1
tration of reagents was kept at 0.05 U AldDH, 1.67 × 10 mol L
+
−1
−1
NAD , 0.1 mol L KCl and 0.01 mol L 2-mercaptoethanol, pH 7.2.
The results from Fig. 5 show that at relatively low acetaldehyde
concentrations, the rate of NADH formation follows the typical
−
3
−1
equilateral hyperbola curve. Thereafter (above 1 × 10 mol L ),
there is a sharp decrease in the initial NADH conversion rate,
suggesting another possible inhibition due to excess substrate.
It is noteworthy that acetaldehyde molecules can also bind
to the formed ternary complex, thus diminishing the enzy-
matic activity [23]. By tracing the double reciprocal graph
(inset of Fig. 5) in the region in which there was no inhi-
bition, the kinetic constants Km and Vmax, were determined
−
1
−1
−1
,
as 16.9 ± 0.2 ␮mol L
and 24.1 ± 0.2 ␮mol NADH min mg
−
1
respectively. The Kcat constant was 80.3 ± 0.2 s
and the Kcat/Km
6
−1 −1
ratio was 4.7 × 10 ± 200 mol L
s (see Table 1).
The kinetic parameters obtained in this work for AldDH in solu-
tion are in agreement with literature reports, for both substrate
and coenzyme [8,24,25]. In addition, the results clearly show the
great affinity between enzyme/substrate as well as the very easy
access of aldehyde molecules to the active site, as compared to the
Fig. 4. Influence of the presence of K+ ions (0.1 mol L ) and thiols (0.01 mol L
−1
−1
)
on AldDH activity in solution. 0.05 U AldDH, 0.5 × 10−3 mol L
−1
acetaldehyde,
.67 × 10− mol L NAD in phosphate buffer, pH 7.2.
3
−1
+
+
1
interaction AldDH/NAD .

S. Aquino Neto et al. / Process Biochemistry 46 (2011) 2347–2352
2351
Fig. 6. Influence of temperature (A) and pH (B) on the enzymatic activity of ADH and AldDH in solution. (᭹) ADH 0.2 U, 0.1 mol L EtOH, 1.67 × 10−3 mol L NAD , phosphate
−
1
−1
+
buffer, pH 7.2. (ꢀ) AldDH 0.05 U, 1.67 × 10− mol L NAD , KCl 0.1 mol L , 2-mercaptoethanol 0.01 mol L
3
−1
+
−1
−1
.
By keeping the same assay conditions described in the study
using soluble AldDH, both Km and Vmax were determined for
the anchored enzyme as a function of acetaldehyde (Fig. 5)
and NAD+ concentrations. Once again, the kinetic values indi-
cated that, despite the large decrease in enzymatic activity after
the immobilization process, the affinity between the anchored
enzyme and its substrate and coenzyme was preserved. The val-
ues determined for Km and Vmax from the substrate variation assay
enzymes onto a solid platform in cascade [29,30]. Despite the lower
kinetic values obtained with AldDH compared with ADH, the large
affinity between AldDH and acetaldehyde enables construction of
an efficient integrated system employing both enzymes; i.e., as
soon as ADH catalyzes the first step of ethanol oxidation, AldDH
is able to rapidly catalyze the oxidation in a second step.
So, in order to simulate these conditions in the kinetic assays,
the LbL technique was employed for immobilization of both ADH
and AldDH onto carbon platforms. To this end, a sample with 36
bilayers containing both dehydrogenase enzymes was prepared
by anchoring the enzymes onto separate layers, which furnished
a final architecture sequence of ADH/PAMAM/AldDH. The kinetic
results obtained with both enzymes anchored onto the carbon plat-
form by using the self-assembly methodology, evidence enhanced
−1
−1
−1
,
were 16.3 ± 0.1 ␮mol L and 0.13 ± 0.01 ␮mol NADH min mg
respectively. As for the coenzyme assays, Km and Vmax were
−
1
−1
−1
.
0
.53 ± 0.01 mmol L and 0.12 ± 0.01 ␮mol NADH min mg
Table 1 summarizes all the kinetic parameters determined for
both dehydrogenase enzymes in solution and anchored onto carbon
platform.
−
1
−1
enzymatic activity behavior (Vmax = 0.62 ␮mol NADH min mg )
3
.3. Influence of temperature and pH on the enzymatic activity
as compared to the individual systems (Table 1). By comparing the
results between the double enzymatic system with those obtained
in the case in which only ADH was anchored, it is clear that the use
of multiple dehydrogenase enzymes enhances the kinetic perfor-
mance of the whole system, without causing damage to the first
oxidation step. In addition, these results evidence that a very good
arrangement of the anchored enzymes on the carbon support is
provided by the layered structure, which facilitates the diffusional
processes during the catalysis is and contributes to the overall per-
formance of the system.
Although all the kinetic results indicate that the use of the LbL
technique provides good control of enzyme disposition on the car-
bon platform, thus providing good kinetic rates for the anchored
enzymes, a large decrease in enzymatic activity is still observed
when one compares the results from the tridimensional condition
in solution and the two-dimensional situation on the carbon sup-
port (Table 1). In this context, the question that arises is related to
the effect of immobilization employing dendrimers on the enzy-
matic activity. In fact, a few factors must be considered in order
to understand the kinetic behavior of dehydrogenase enzymes
anchored with dendrimers onto carbon platforms. The first factor
involves the possibility of enzyme inactivation after the immo-
bilization step. Normally, this risk is pronounced when chemical
bonds are formed during the immobilization step, and also when
sudden changes in temperature occur during the anchoring pro-
cess. These two situations have not been reported for dendrimers
yet. In fact, no enzymatic denaturation was observed in a previ-
ous investigation using PAMAM dendrimers for the immobilization
of Cl-cathecol 1,2 deoxygenase [31]. However, denaturation may
occur after protein immobilization and, considering such reduc-
tion in activity after enzyme anchoring, this possibility cannot be
disregarded.
Fig. 6 depicts the kinetic behavior of both dehydrogenase
enzymes in solution as a function of pH and temperature. The
obtained curves allowed determination of the optimum temper-
ature (Fig. 6A) and pH (Fig. 6B) for ADH and AldDH. The highest
activity was achieved in the 7.0–8.0 pH range for ADH, and around
7
.0 for AldDH. The optimum temperature for ADH lay between 35
◦
◦
and 40 C and at about 35 C for AldDH.
The results in terms of pH and temperature coincide with data
reported in the literature for both enzymes in solution [24,26,27].
Moreover, the results also show that, in order to maintain an
environment in which both enzymes display good activity and to
provide conditions for future technological applications, physio-
logical conditions and ambient temperature can satisfactorily be
applied to an enzymatic system involving dehydrogenase enzymes.
In order to verify product formation after the kinetic tests,
product yields were followed by high performance liquid chro-
matography (HPLC) employing the best conditions obtained in all
the assays utilizing the anchored ADH and AldDH. A highly specific
behavior was verified for both dehydrogenase enzymes. Consider-
ing the ADH kinetic behavior, an average of acetaldehyde recovery
of 92% was obtained after the assay (aldehyde leakage may have
occurred during the experiment), but acetic acid was not detected,
probably due to the low activity of ADH for acetaldehyde oxida-
−1
tion, Kcat = 2.3 s at pH 8.8 [28]. In the case of AldDH, an acetic acid
recovery of about 90% was achieved.
3
.4. Double enzymatic system
Aiming at the complete oxidation of fuels, multiple immobi-
lized dehydrogenase enzymes are generally used by anchoring the

2
352
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with PAMAM dendrimers, the effects of mass transfer should be
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pared to the individual cases is a clear indication that good mass
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parameters (mainly the concentration of substrates and coenzyme,
which act as inhibitors of the enzymes) should be considered,
for achievement of maximum enzymatic activity for the anchored
enzymes. Additionally, the immobilization process seems to be cru-
cial for preparation of a viable anchored system, making a careful
choice of immobilization process for each type of anchored system
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the possible inhibition due to substrate excess. The kinetic rates
obtained for the anchored enzymes showed that the choice of
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PAMAM dendrimers provide a good environment for the immobi-
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Financial support from FAPESP and CNPq is gratefully acknowl-
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