Investigation of structural effects and behaviour of Pseudomonas aeruginosa amidase encapsulated in reversed micelles

Article abstract of DOI:10.1016/j.procbio.2011.11.001

The acetohydroxamic acid synthesis reaction was studied using whole cells, cell-free extract and purified amidase from the strains of Pseudomonas aeruginosa L10 and AI3 entrapped in a reverse micelles system composed of cationic surfactant tetradecyltrimethyl ammonium bromide. The specific activity of amidase, yield of synthesis and storage stability were determined for the reversed micellar system as well as for free amidase in conventional buffer medium. The results have revealed that amidase solutions in the reverse micelles system exhibited a substantial increase in specific activity, yield of synthesis and storage stability. In fact, whole cells from P. aeruginosa L10 and AI3 in reverse micellar medium revealed an increase in specific activity of 9.3- and 13.9-fold, respectively, relatively to the buffer medium. Yields of approximately 92% and 66% of acetohydroxamic acid synthesis were obtained for encapsulated cell free extract from P. aeruginosa L10 and AI3, respectively. On the other hand, the half-life values obtained for the amidase solutions encapsulated in reverse micelles were overall higher than that obtained for the free amidase solution in buffer medium. Half-life values obtained for encapsulated purified amidase from P. aeruginosa strain L10 and encapsulated cell-free extract from P. aeruginosa strain AI3 were of 17.0 and 26.0 days, respectively. As far as the different sources biocatalyst are concerned, the data presented in this work has revealed that the best results, in both storage stability and biocatalytic efficiency, were obtained when encapsulated cell-free extract from P. aeruginosa strain AI3 at w₀ of 10 were used. Conformational changes occurring upon encapsulation of both strains enzymes in reverse micelles of TTAB in heptane/octanol were additionally identified by FTIR spectroscopy which clarified the biocatalysts performances.

Full text of DOI:10.1016/j.procbio.2011.11.001

Process Biochemistry 47 (2012) 264–272
Contents lists available at SciVerse ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Investigation of structural effects and behaviour of Pseudomonas aeruginosa
amidase encapsulated in reversed micelles
∗
Ana Fragoso, Rita Pacheco , Amin Karmali
Centro de Investiga c¸ ão de Engenharia Química e Biotecnologia do Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro, 1, 1959-007 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2011
Received in revised form 24 October 2011
Accepted 2 November 2011
Available online 15 November 2011
The acetohydroxamic acid synthesis reaction was studied using whole cells, cell-free extract and purified
amidase from the strains of Pseudomonas aeruginosa L10 and AI3 entrapped in a reverse micelles system
composed of cationic surfactant tetradecyltrimethyl ammonium bromide. The specific activity of ami-
dase, yield of synthesis and storage stability were determined for the reversed micellar system as well
as for free amidase in conventional buffer medium. The results have revealed that amidase solutions in
the reverse micelles system exhibited a substantial increase in specific activity, yield of synthesis and
storage stability. In fact, whole cells from P. aeruginosa L10 and AI3 in reverse micellar medium revealed
an increase in specific activity of 9.3- and 13.9-fold, respectively, relatively to the buffer medium. Yields
of approximately 92% and 66% of acetohydroxamic acid synthesis were obtained for encapsulated cell
free extract from P. aeruginosa L10 and AI3, respectively. On the other hand, the half-life values obtained
for the amidase solutions encapsulated in reverse micelles were overall higher than that obtained for
the free amidase solution in buffer medium. Half-life values obtained for encapsulated purified amidase
from P. aeruginosa strain L10 and encapsulated cell-free extract from P. aeruginosa strain AI3 were of 17.0
and 26.0 days, respectively. As far as the different sources biocatalyst are concerned, the data presented
in this work has revealed that the best results, in both storage stability and biocatalytic efficiency, were
obtained when encapsulated cell-free extract from P. aeruginosa strain AI3 at w0 of 10 were used. Con-
formational changes occurring upon encapsulation of both strains enzymes in reverse micelles of TTAB
in heptane/octanol were additionally identified by FTIR spectroscopy which clarified the biocatalysts
performances.
Keywords:
Reversed micelles
Pseudomonas aeruginosa amidases
Acetohydroxamic acid
Water content
Stability
©
2011 Elsevier Ltd. All rights reserved.
1
. Introduction
hand, mutant strain AI3 was obtained from P. aeruginosa strain
L10 by chemical mutagenesis which involved the substitution Thr-
103-Ile. The AI3 strain exhibits altered properties such as different
substrate specificity and is less temperature-stable compared with
strain L10 [9].
Pseudomonas aeruginosa amidase has been used by our group in
the study of reactions of hydrolysis and synthesis, either in conven-
tional medium [1] or in reverse micelles [2]. Amidases (E C 3.5.1.4)
catalyse the hydrolysis of amide bonds in small aliphatic amides.
In addition this enzyme presents the capacity to catalyse transami-
dation reactions with transfer of acyl groups to amides, broadening
its application in the synthesis of different compounds with amide
bonds [3–5]. Amidases from mutant strains of P. aeruginosa, such as
L10 and AI3, have been reported to exhibit differences in substrate
and inhibitor specificities [5,6]. The strain L10 from P. aeruginosa is a
constitutive mutant obtained from the wild type strain 8602 which
catalyses preferentially the hydrolysis of short chain aliphatic
Hydroxamic acid derivatives, with the general formula
RC(O)NHOH, are products of transamidation reactions catalysed
by amidases, when the acyl acceptor is hydroxylamine [10]. These
compounds present a wide spectrum of applications, namely in
food, waste-water treatment and pharmaceutical industries, as
constituents of growth factors, antibiotics, antibiotic antagonists,
tumor inhibitors, and antifungal agents [10–14]. At the present
state, the process of hydroxamic acids derivatives production con-
sists of chemical reactions from esters in solution and on solid phase
[15]. It is noteworthy that biocatalysis using purified amidase from
P. aeruginosa has been successfully used by our group for the syn-
thesis of acetohydroxamic acid using reverse micelles, with a yield
higher than 90% [2]. The reduced water content in reverse micelles
◦
amides and exhibits high heat stability at 55 C [7,8]. On the other
∗ Corresponding author at: Instituto Superior de Engenharia de Lisboa, Área
Departamental de Engenharia Química, Rua Conselheiro Emídio Navarro 1, 1959-
[
16,17] is more suitable for biocatalysis of synthetic reactions than
for hydrolytic reactions, with the additional advantage of enhanced
activity and higher stability of the biocatalyst [18–20].
0
07 Lisboa, Portugal. Tel.: +351 218 317 066; fax: +351 218 317 267.
E-mail address: rpacheco@deq.isel.ipl.pt (R. Pacheco).
1
359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2011.11.001

A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
2.5. Protein assays
265
In previous work, we have used a reverse micellar system
of the cationic surfactant tetradecyltrimethyl ammonium bro-
mide (TTAB) dissolved in heptane/octanol (80/20%) with a purified
recombinant amidase for the synthesis of acetohydroxamic acid
Amidase solutions (whole cells, cell free extract and purified amidase) were
assayed for total protein concentration by the Coomassie blue binding method by
using bovine serum albumin as the protein standard [31].
[
2]. The present work reports on the use of either whole cells,
cell free extract or purified enzyme from two different strains
of P. aeruginosa L10 and AI3, as the source of amidase. There-
fore it addresses the study of the most suitable conditions for
catalytic activity, productivity and biocatalyst stability which are
key parameters for efficiency and improvement of the process
for acetohydroxamic acid synthesis. Moreover, because there is
a correlation between enzyme behaviour and protein structure,
a novel perspective is presented of the conformational modifica-
tions occurring upon amidase encapsulation which clarifies the
obtained results. Reverse micelles are thermodynamically stable
and optically transparent and therefore are suitable for the appli-
cation of spectroscopic techniques in the characterization of solutes
and micelles [21–23]. The application of infrared spectroscopy for
the study of enzymes encapsulated in reverse micelles has been
published in literature [24–26]. However, to the best of our knowl-
edge, this is the first report on amidase from P. aeruginosa strains
encapsulated in reverse micelles.
2.6. Enzyme assays in buffer medium
Amidase acyl transferase activity was determined by using a previously reported
[
32] discontinuous spectrophotometric assay for the detection of acetohydroxamic
acid formation using as hydroxylamine and acetamide as substrates. The reaction
was carried out in 20 mM Tris buffer pH 7.2 containing 1 mM EDTA, 1 mM ␤-
mercaptoethanol and 10% (v/v) glycerol (TMEG buffer). The acetohydroxamic acid
formed was detected through the addition of FeCl3 6% (w/v) in HCl 2% (w/v) to
aliquots of the reaction mixture. The formation of a brown complex was monitored
−1
−1
2
and quantified at 490 nm (ε = 0.083 mM cm ; R = 0.994). One enzyme unit (U) is
defined as the amount of enzyme required to produce 1 ␮mol of acetohydroxamic
◦
acid per min at 25 C under these experimental conditions.
2
.7. Enzyme assays in the reverse micellar system
The reverse micellar system was prepared by injection. Solutions of 5 mL
reverse micelles of 200 mM tetradecyltrimethyl ammonium bromide (TTAB) in hep-
tane/octanol (80/20%, v/v) under constant agitation were prepared by injecting
1
1
5 mM hydroxylamine and 30 ␮l of amidase containing solution (either whole cells
8 mg protein/mL, cell free extract 10 mg protein/mL or 0.05 mg protein/mL purified
amidase). Acetamide in TMEG buffer was injected to a final concentration of 7.5 mM
to initiate the reaction in the reverse micelles. The water content in the system,
usually defined through the parameter w0 = [H2O]/[surfactant] was controlled by
adjusting the buffer volumes added in order to be established the range of 3.5–11.5.
The system was continuously stirred for complete entrapment; in this case the solu-
tion becomes transparent indicating formation of reverse micelles [33]. The rate of
formation of acetohydroxamic acid was detected through the addition of FeCl3 6%
(w/v) in HCl 2% (w/v) to aliquots of the reaction mixture at several times for a period
2. Materials and methods
2.1. Chemicals
Heptane, octanol, iron chloride, glycerol and ethylenediaminetetraacetic
acid (EDTA) were purchased from Merck (Darmstadt, Germany). Tetrade-
cyltrimethyl ammonium bromide (TTAB) and 2-mercaptoethanol was obtained
from Sigma–Aldrich (Madrid, Spain). Acetamide and acetohydroxamic acid were
purchased from Fluka (Madrid, Spain). Tris–HCl was acquired in Riedel-deHaën
of 30 min. The amount of hydroxamic acid was quantified at 490 nm by using a cal-
ibration curve (ε = 0.089 mM−1 cm−1; R2 = 0.993). One enzyme unit (U) is defined as
the amount of enzyme required to produce 1 ␮mol of acetohydroxamic acid per min
at 25 ◦C under these experimental conditions.
(
Seelze, Germany). Hydroxilamine was obtained from Panreac (Barcelona, Spain).
All chemicals used were analytical grade.
2
.8. Storage stability of amidase solutions
Storage stability of the amidase solutions from strains L10 and AI3 of P.
2
.2. Bacterial strain
aeruginosa was evaluated both in TMEG buffer and in the reverse micellar system.
Regarding the reverse micellar system, 30 ␮l of amidase containing solution (either
whole cells 18 mg protein/mL, cell free extract 10 mg protein/mL or 0.05 mg pro-
P. aeruginosa L10 and AI3 strains were used in this study as the source of the
aliphatic amidase (E C 3.5.1.4.) [9,27].
tein/mL purified amidase) was injected in 5 mL of 200 mM TTAB in heptane/octanol
◦
(
80/20%, v/v) at constant w0 of 10 and incubated at 24 C under constant agitation
during several hours. Aliquots of 5 mL of the mixture were taken at several time
intervals and enzyme activity was assayed by injecting 15 mM hydroxylamine and
2.3. Production of whole cells from P. aeruginosa strains L10 and AI3
7.5 mM acetamide in order to initiate the reaction. As mentioned previously, the
system was continuously stirred for complete entrapment [33]. Quantification of
enzyme activity in the reverse micellar system was performed by measuring the rate
of hydroxamic acid formation as described in Section 2.7. Storage stability of ami-
dase solutions in TMEG buffer was evaluated by using a similar approach. Therefore,
amidase solutions were incubated at 24 C under constant agitation during several
hours in TMEG buffer. Aliquots were taken from the mixture during incubation and
The strains were grown overnight as described previously [9]. The cells were
harvested by centrifugation at 10,000 × g for 5 min and the pellet washed twice
◦
with NaCl (8.5%, w/v), centrifuged, and stored at −20 C.
Frozen cells from each strain were resuspended in 2 volumes of 20 mM Tris–HCl
buffer, pH 7.2 containing 1 mM ␤-mercaptoethanol, 1 mM EDTA, 10% (v/v) glyc-
erol and 1 mM benzamidine. The obtained suspensions, identified as CL10 and CAI3,
contained the whole cells from strains P. aeruginosa L10 and AI3, respectively.
◦
15 mM hydroxylamine and 7.5 mM acetamide were added in order to initiate the
reaction. Assay of amidase activity was performed by quantification of the rate of
hydroxamic acid formation as described in Section 2.6. The first order rate inactiva-
tion constant, k was determined by the slope of the semilogarithmic plot of residual
activity versus storage time and used to calculate the half-life values, t1/2 [34].
2
.4. Preparation of cell free extract and amidase purification
Part of the cell suspensions CL10 and CAI3 was broken by sonication three times
2
.9. Fourier transform infrared (FTIR) spectroscopy analysis
at 150 W in an iced bath. The suspension was centrifuged at 10,000 × g for 30 min.
The supernatant was used as the source of enzyme and represents the cell free
extract of each strain EL10 and EAI3, respectively.
The effect of encapsulation of amidase in reverse micelles was studied by FTIR
spectroscopy. Solutions of 5 mL of 200 mM TTAB in of heptane/octanol 80/20% (v/v)
Amidase from P. aeruginosa strain L10 and AI3 were purified to apparent homo-
geneity from cell-free extracts, respectively EL10 and EAI3. As amidase from L10
were prepared with injection of 30 ␮l of purified amidase solutions (0.05 mg pro-
tein/mL). In order to study the effect of enzyme encapsulation at different w , the
0
◦
2
2
strain is heat stable at 60 C [6,28], the cell-free extract EL10 was previously heat
volume of deuterium oxide ( H O) injected was adjusted in the range between 3.5
◦
treated at 60 C for 10 min and clarified by centrifugation. Subsequently amidase was
and 11.5. As previously mentioned the system was continuously stirred for com-
purified by affinity chromatography on epoxy-activated sepharose 6B-acetamide
column followed by gel filtration chromatography on sephacryl S-300HR column as
described previously [9] (28 U/mg protein and purification factor of 3).
plete entrapment [33]. The use of 2H O is compulsory to obtain high quality FTIR
2
spectra, since, unlike water, 2H O does not have strong absorption in the amide I
2
region between 1600 and 1700 cm−1 [35]. Analysis of the FTIR spectra in the amide
Amidase from AI3 strain was purified from cell-free extract EAI3 using the same
procedures although without the previous heat treatment step, as amidase from AI3
strain is not thermal stable at 60 C [9] (182 U/mg protein and purification factor of
I region is a common technique since this region provides information about the
secondary structures of proteins [36] namely for amidase structure as mentioned in
previous work [37].
◦
1
6).
Infrared spectra were recorded by using a Tensor 27 Bruker (Madison, USA)
The homogeneity of the purified amidase solutions, AL10 and AAI3, was analyzed
by native polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE as described
elsewhere [29] and stained for protein with silver nitrate [30].
FTIR spectrometer. Each spectrum was acquired by averaging 400 scans in the spec-
tral range of 400–4000 cm−1 at 4 cm−1 resolution to obtain a spectrum of high
S/N (signal-to-noise) ratio. The spectrum was obtained by using CaF2 infrared cell

2
66
A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
containing 40 ␮l of amidase solutions. The OPUSNT was used for the acquisition
and edition of the FTIR spectra. The obtained spectra were resolution-enhanced by
second derivative methods.
the lowering of the substrates diffusion inside the microenviron-
ment of the micelle as compared with the buffer solution [42].
The yield of acetohydroxamic acid synthesis was additionally
evaluated considering that the best conditions for enzymatic activ-
ity may not be the same as those for obtaining high product yield
3
. Results and discussion
[
2,42]. In Fig. 3A and B it is noticeable, for P. aeruginosa strains L10
and AI3 respectively, that amidase solutions containing whole cells
CL10 and CAI3) and cell-free extract (EL10 and EAI3) exhibited
3.1. Biocatalysis of acetohydroxamic acids
(
Biocatalysis was carried out by using as the source of amidase
higher yields of synthesis compared with purified amidase solu-
tions (AL10 and AAI3). These results may be explained on the basis
that the microenvironment in whole cells and cell-free extract had a
protective effect which delayed biocatalyst inactivation and there-
fore, higher yields of product synthesis were obtained. However, a
detailed study on the stability of amidase preparations was carried
out in order to address properly this issue (Section 3.2).
As previously reported for the specific activity, considerable
higher yields of product synthesis were obtained by using ami-
dase solutions in the reverse micellar system than free in the
conventional buffer medium (bars on the right of Fig. 3A and B).
Moreover, encapsulated enzyme solutions of P. aeruginosa strain
L10 (Fig. 3A) overall have demonstrated higher product yields than
enzyme solutions from P. aeruginosa strain AI3 (Fig. 3B). Namely, a
reaction yield of acetohydroxamic acid synthesis of approximately
92% was achieved using P. aeruginosa strain L10 cell free extract
whole cells, cell free extract or purified enzyme from two different
strains of P. aeruginosa L10 and AI3, for the synthesis of acetohy-
droxamic acid. The specific activity of amidase for acetohydroxamic
acid synthesis and yield of synthesis in TMEG buffer was com-
pared with non-conventional media of reverse micelles of TTAB in
heptane/octanol. The effect of the micellar water content w0 was
additionally investigated in the reverse micellar system in the range
of 3.5–11.5.
The specific activity of amidase for acetohydroxamic acid syn-
thesis was obtained at different experimental conditions for strains
P. aeruginosa strains L10 and AI3, as shown in Fig. 1A and B, respec-
tively. The data in these figures revealed a higher specific activity
for purified amidase solutions (AL10 and AAI3) and cell free extract
(
EL10 and EAI3), whereas the whole cells (CL10 and CAI3) exhibited
the lowest specific activity. These differences may be explained by
different diffusion resistances occurring due to substrates trans-
portation to the active site of the enzyme and removal of products
from the active site. Therefore, lower specific activity was obtained
in whole cells (CL10 and CAI3), which contains the biocatalyst
inside the bacterial cell, whereas the enzyme is freely available
either in the cell-free extract (EL10 and EAI3) or in purified enzyme
(Fig. 3A EL10) encapsulated in a reverse micellar system at w = 9.
0
Whereas, the highest yield obtained using P. aeruginosa strain AI3
amidase solutions was approximately of 66% for cell free extract
encapsulated at w0 of 10 (Fig. 3B, EAI3).
Once again, a bell shape profile of acetohydroxamic acid syn-
thesis yield relatively to the micellar water content (Fig. 3A and
B) can be seen with the maximum of product yield approxi-
(
AL10 and AAI3) solutions.
However, it is clear from Fig. 1A and B that all enzyme solutions
whole cells, cell free extract and purified amidase) from both P.
mately at w = 9–10 for the encapsulated enzyme solutions of both
0
(
P. aeruginosa strains. Again a more defined bell shape tendency
was obtained for amidase solutions from P. aeruginosa L10 strain
(Fig. 3A) than for P. aeruginosa strain AI3 which displayed a broader
bell shape tendency of product yield as a function of micellar water
content (Fig. 3B). The variations in the micellar water content have
been known to modify the size of the reverse micelles [16,17]
resulting in modifications of substrate diffusion and/or of the bio-
catalyst structure, which may affect the biocatalyst performance.
The amidase from mutant strain AI3 exhibits a change Thr-103-
Ile, this can reduce the interaction with water molecules, due to
the presence of hydrophobic side chain amino acid Ile1 [29].
Whereas, amidase from mutant strain L10 contains in the same
position, Thr1 , a polar side chain amino acid, which suggests that
water variations in this case could more easily modify the enzyme
structure.
aeruginosa strains exhibit superior activity encapsulated in reverse
micelles than free in the conventional buffer medium (bars on the
right of Fig. 1A and B). Therefore, specific activity of amidase is lower
in the conventional buffer medium due to the high water content
which favors the amidase hydrolysis reaction as opposed to the
acetohydroxamic acid synthesis [2]. Nevertheless, the microenvi-
ronment in which the enzyme is solubilized in reverse micelles has
physico-chemical properties distinct from a bulk aqueous solution
which can alter protein structure and therefore with the biocat-
alytic activity [16,24–26]. Structure–function relationships of the
enzyme in both media were further evaluated in order to fully
understand these results.
03
03
Both figures exhibit a typical bell shape profile of specific activ-
ity of amidase for the micellar water content [38,39], whereas a
more defined bell shape tendency was obtained for amidase solu-
tions from P. aeruginosa L10 strain (Fig. 1A). However both strains
Therefore, we can conclude from these data that the different
performance of both these biocatalysts (L10 and AI3), and the differ-
ent performance in buffer conventional media and reverse micelles,
may be due to the modifications in enzyme structure. Such changes
are accountable for different substrate specificity and enzyme sta-
bility which affected the specific activity of amidase and product
yield. Therefore, we decided to investigate the differences in bio-
catalyst’s performances by studying storage stability of amidase
solutions and enzyme structure both in conventional buffer media
and reverse micelles for both strains.
exhibited the highest specific activity of amidase for w = 10. There-
0
fore, at w = 10 the enzyme solutions exhibit an enhanced catalytic
0
activity. The data presented clearly demonstrated that the synthe-
sis of acetohydroxamic acid can be successfully carried out by using
either whole cells, cell-free extract and purified enzyme. There are
some reports in literature about the use of reverse micelles, which
despite having sizes of nanometers solubilize and maintain viable
whole cells of sizes ranging in several microns [40,41].
The increase in specific activity of amidase upon encapsulation
compared with the free enzyme solution in the conventional buffer
media is clearly seen in Fig. 2A and B for enzyme solutions from P.
aeruginosa strain L10 and AI3, respectively. The results presented
in these figures are expressed as the ratio between specific activity
in reverse micelles and specific activity in TMEG buffer at different
3.2. Amidase solutions storage stability
In general, enzymes are not stable during storage in solution and
their activities are gradually reduced or lost as a function of time.
The investigation on storage stability becomes extremely impor-
tant when considering the significance of enzymes as biocatalysts
in several industrial processes [44].
water content (w ). Both Fig. 2A and B exhibit the highest ratios
0
obtained by using whole cells, CL10 and CAI3. This increase in spe-
cific activity upon encapsulation of whole cells can be explained by

A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
267
Fig. 1. Specific activity of acetohydroxamic acid synthesis in TTAB reverse micelles at different w0 values and in TMEG buffer with different amidase solutions: (A) CL10
black), EL10 (gray) and AL10 (white) represent whole cells, cell free extract and purified amidase from Pseudomonas aeruginosa strain L10 and (B) CAI3 (black), EAI3 (gray)
and AAI3 (white) represent whole cells, cell free extract and purified amidase from P. aeruginosa strain AI3. Amidase activity was assayed as described in Sections 2.6 and 2.7.
(
The inactivation of amidase preparations followed a two stage
series-type mechanism of inactivation both in buffer and reverse
micelles [45]. However, amidase solutions in reverse micelles fol-
lowed a two first-order steps in which the biocatalyst decays in
the first step and in the second step the activity remained almost
constant. In buffer, the mechanism accounts for a grace period, the
biocatalyst decays after an initial period of activation (∼2 days)
with a more active specie of biocatalyst prior to decay. The rate of
may have contributed to deactivation and the reduction of the bio-
catalyst storage stability.
In buffer medium, whole cells from P. aeruginosa strain L10
(CL10) exhibited a 2-fold higher t1/2 values than those of the whole
cells of P. aeruginosa strain AI3 and CAI3. However, the purified
enzyme AL10 from P. aeruginosa strain L10 revealed lower t1/2
in buffer (4.6 days) than the purified amidase from AAI3 from
P. aeruginosa strain AI3 (7.0 days). Apparently amidase from P.
aeruginosa strain L10 has superior storage stability in TMEG buffer
when the biocatalyst is maintained in the stable cellular environ-
ment, mainly as whole cells (CL10). As previously mentioned in
Section 3.1 for the bell shape profile of Fig. 3A, amidase from P.
aeruginosa strain L10 is susceptible to water interference. Therefore
the cell content possibly shields the enzyme from the water inter-
actions and effect on enzyme inactivation during storage [20]. The
t1/2 value of whole cells of P. aeruginosa strain L10 (CL10) was 3-fold
activity decays obeyed first-order kinetics. The half-life values (t1/2
)
and the inactivation rate constants (k) for the amidase solutions in
buffer and encapsulated in reverse micelles were determined as
described in Section 2 and are shown in Table 1.
Considering the conventional TMEG buffer medium, the lowest
t1/2 values were obtained using cell free extract from both strains,
EL10 (3.8 days) and EAI3 (3.0 days). These results can be explained
by the presence of proteolytic enzymes in cell-free extract which
Table 1
Inactivation constants (k) and half-life values (t1/2) in TTAB reverse micelles at w0 = 10 and in TMEG buffer of different amidase solutions: CL10, EL10 and AL10 represent
whole cells, cell free extract and purified amidase from Pseudomonas aeruginosa strain L10, and CAI3, EAI3 and AAI3 represent whole cells, cell free extract and purified
amidase from P. aeruginosa strain AI3.
CL10
EL10
AL10
CAI3
EAI3
AAI3
k (h 1
−
)
2.7 × 10
−3
−3
3.4 × 10
8.5
−3
−3
1.7 × 10−3
1.7 × 10−3
1.1 × 10−3
4.8 × 10
6.0
−3
−2
Reverse micelles
TMEG buffer
t1/2 (days)
9.0
17.0
17.0
26.0
−1
−3
−3
−2
k (h
)
2.0 × 10
7.6 × 10
6.3 × 10
4.0 × 10
1.0 × 10
4.1 × 10
t1/2 (days)
14.4
3.8
4.6
7.2
3.0
7.0

2
68
A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
Fig. 2. Ratio between specific activity of acetohydroxamic acid synthesis in TTAB reverse micelles at different w0 values and the specific activity in TMEG buffer with different
amidase solutions. (A) CL10 (black), EL10 (gray) and AL10 (white) represent whole cells, cell free extract and purified amidase from P. aeruginosa strain L10. (B) CAI3 (black),
EAI3 (gray) and AAI3 (white) represent whole cells, cell free extract and purified amidase from P. aeruginosa strain AI3.
higher than for the purified amidase (AL10) in TMEG buffer. This
effect was less noticeable for amidase solutions from P. aeruginosa
strain AI3 since whole cells (CAI3) and purified amidase (AAI3) pre-
sented a t1/2 value of 7.2 days a similar value of 7.0 days in TMEG
buffer, respectively.
It can be seen from Table 1 that t1/2 values of the enzyme
solutions considerably increased with the encapsulation, except
for whole cells from P. aeruginosa strain L10 (CL10) and purified
enzyme from P. aeruginosa strain AI3 (AAI3). In these cases, lower
storage stability was obtained for amidase solutions encapsulated
in reverse micelles than free in buffer medium, possibly because
these enzyme solutions in reverse micelles experience the highest
specific activity of all the amidase solutions (Fig. 1A and B). Consid-
erations about enzyme stability and activity suggest that catalytic
efficient enzymes are often less stable [44] since these enzymes
have a more flexible enzyme structure [46].
amidase solutions of P. aeruginosa strain L10, the highest t1/2 value
of 17.0 days was obtained for encapsulated purified amidase (AL10).
However, considering possible industrial applications it should
be selected the enzyme solution in which a high t1/2 is associated
to a favorable product yield (Fig. 4). Fig. 4 shows that this condition
was obtained for cell free extract from P. aeruginosa strain AI3 (EAI3)
encapsulated in reverse micelles, with a half-life value of 26.0 days
and a yield of 66%. High half-life values and product yields higher
than 50% were additionally exhibited in reverse micellar solution
for whole cells of P. aeruginosa strain AI3, CAI3, and purified amidase
from P. aeruginosa strain L10, AL10.
3.3. Fourier transform infrared (FTIR) spectroscopy analysis of
amidase solutions
The conformation of purified amidase from both strains in aque-
ous solution and in TTAB reverse micellar solution was studied
by using FTIR spectroscopy. The amide I region between 1600
The highest t1/2 values (17.0 and 26.0 days) were obtained for
whole cells and cell-free extract of P. aeruginosa strain AI3 encap-
sulated in reverse micelles, CAI3 and EAI3, respectively. For the
−
1
and 1700 cm is the vibrational mode most used for secondary

A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
269
Fig. 3. Yield of acetohydroxamic acid synthesis in TTAB reverse micelles at different w0 values and in TMEG buffer with different amidase solutions: (A) CL10 (black), EL10
gray) and AL10 (white) represent whole cells, cell free extract and purified amidase from P. aeruginosa strain L10; (B) CAI3 (black), EAI3 (gray) and AAI3 (white) represent
(
whole cells, cell free extract and purified amidase from P. aeruginosa strain AI3.
structure analysis of proteins [35]. The amide I band of proteins
consists of many overlapping component bands that represent
different structural elements such as ␣-helices, ␤-sheets, turns
and non-ordered or irregular structures. These individual com-
ponent bands cannot be resolved in the experimental spectra
therefore separation of the overlapping bands has to be achieved
by analysis procedures like Fourier self-deconvolution or second
derivative [35]. Fig. 5 represents the second derivative spectra
of purified amidase from P. aeruginosa strain L10 and AL10, in
spectrum of the free enzyme in aqueous solution (black line spec-
trum). However there were visible shifts in other band position to
lower frequencies in the amidase AL10 spectra going from water
−
1
to micellar solution at w = 10. Namely the band at 1663 cm , in
0
2
−1
,
H O, has shifted to, or been replaced by a band at 1658 cm
2
at w = 10, this latter vibration is often attributed to ␣-helical
0
structure [35]. Therefore upon encapsulation enzyme increases the
␣-helical structures with a concomitant loss of the turn structure.
−
1
2
Additionally the component at 1678 cm , in H O, has shifted
2
2
−1
H O and in TTAB reverse micelles at w = 10 and 4, black full,
to 1676 cm , at w = 10, suggesting an increase in the hydrogen
2
0
0
dotted and gray line spectrum respectively. From Fig. 5 it is
clear that the second derivative FTIR spectra of amidase, AL10,
in aqueous solution (black full line spectrum) shows five absorp-
bonding in the ␤-turn structures when the enzyme AL10 is encap-
sulated [49]. These modifications occurring in enzyme structure
upon encapsulation have considerably improved enzyme specific
activity, product yield and storage stability in reverse micelles, as
previously seen in Sections 3.1 and 3.2.
Some modifications were additionally detected in the enzyme
AL10 structure when in reverse micelles of lower water content.
The second derivative FTIR spectrum of the enzyme AL10 in the
−1
−1
−1
−1
tions located at 1628 cm , 1641 cm , 1663 cm , 1678 cm and
−
1
−
1
1
692 cm in the frequency region 1600–1700 cm . The bands in
−1
the region 1620–1640 cm are often assigned to ␤-sheet struc-
−
1
−1
−1
ture (1628 cm and 1641 cm ) and turn structure (1663 cm ,
−
1
−1
1
678 cm and 1692 cm ) [35,36,47,48].
The band shape of the components at 1628 cm and 1641 cm
in the FTIR spectrum (Fig. 5) of purified amidase AL10 in the micel-
lar solution at w = 10 (dotted line spectrum) is very similar to the
−
1
−1
micellar solution at w = 4 is represented in Fig. 5 top gray line
0
spectrum. The comparison between the enzyme AL10 spectrum at
w = 10 (Fig. 5, dotted line) with the spectrum at w = 4 (Fig. 5, gray
0
0
0

2
70
A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
Fig. 4. Relationship between half-life values and yield of acetohydroxamic acid syn-
thesis in TTAB reverse micelles at w0 = 10 (ꢀ) and in TMEG buffer (᭹) for different
amidase solutions: CL10, EL10 and AL10 represent whole cells, cell free extract and
purified amidase from P. aeruginosa strain L10, and CAI3, EAI3 and AAI3 represent
whole cells, cell free extract and purified amidase from P. aeruginosa strain AI3.
Fig. 6. Comparison of second derivative FTIR spectrum in amide I region of P.
aeruginosa strain AI3 purified amidase in H2O (full line), in TTAB reverse micelles
at w0 = 10 (dotted line) and in TTAB reverse micelles at w0 = 4 (gray line).
2
a four-layer ␣␤␤␣ sandwich structure [43] which becomes more
compact with the increasing ␤-sheets content and the interactions
in the enzyme interiors [20], and less flexible enzymes often expe-
rience lower biocatalytic activity [46].
line) revealed band shifts in this frequency region, which evidence
structural changes with water content variation inside reverse
micelles, as previously suggested in Sections 3.1 and 3.2. Specifi-
−
1
cally, the component in the amidase AL10 spectrum at 1658 cm
in w = 10 (dotted line spectrum) is absent in the spectrum at w = 4
Upon encapsulation of the purified amidase from P. aeruginosa
strain AI3 (AAI3), the second derivative of the spectra represented
0
0
(
1
gray line spectrum) and in the former appears a component band at
649 cm often assigned to ␤-sheet antiparallel components [49].
in Fig. 6 also shows visible shifts in band positions from 2H O
2
−1
(
full line spectrum) to the micellar solutions at w = 10 (dotted
0
Moreover, amidase AL10 spectrum at w = 4 (gray line spectrum)
0
1
line spectra) and at w = 4 (gray line spectrum) which suggests
0
−
−1
displays component bands at 1620 cm and 1684 cm suggest-
ing a rearrangement of the ␤-sheets structure of the enzyme by
formation of intermolecular hydrogen bonding [49] as a result of
the encapsulation in lower water content micelles. However the
additional ␤-sheets content and the increase in intermolecular
interaction in the amidase AL10 structure accounted for a lower
enzyme conformation rearrangements. The second derivative FTIR
spectrum of amidase AAI3, in aqueous solution (full line spec-
−1
−1
−1
trum) shows bands located at 1630 cm , 1641 cm , 1661 cm ,
−1
−1
2
1
676 cm and 1691 cm . In H O (full line spectrum) the band
at 1661 cm , attributed to turn structure, has shifted to or been
replaced, at w = 10 (dotted line spectrum), by a band at 1655 cm
2
−1
−1
0
activity and product yield at w = 4 as previously demonstrated
0
(␣-helical structure). And there are visible band components aris-
in Section 3.1. One possible explanation is that amidase AL10 has
ing in the amidase AAI3 at w = 10 (dotted line spectrum) at
0
−
1
−1
1
622 cm and 1690 cm . In this case, enzyme AAI3 encapsula-
tion at w = 10 caused an increase in the ␣-helices content with an
0
additional formation of strong intermolecular hydrogen bonding
−
1
−1
on the ␤-sheets (1622 cm and 1690 cm ), as previously verified
in enzyme AL10 encapsulation (Fig. 5, dotted line spectrum). How-
ever, the spectrum of the enzyme AAI3 in reverse micelles solutions
at w = 10 (Fig. 6, dotted line) showed a shift to a lower frequency
0
−
1
in the band component often assigned to ␣-helices (1655 cm
relatively to the spectrum of enzyme L10 at w = 10 (Fig. 5, dotted
line), which shows this band component at 1658 cm . This shift
)
0
−
1
suggests an increase in hydrogen bonding on the ␣-helical con-
tent of enzyme AAI3 in reverse micelles at w = 10. On account of
0
these effects, encapsulation of enzyme AAI3 in reverse micelles at
w = 10 considerably improved the biocatalytic performance rela-
0
tively to the buffer media and with a higher specific activity than
AL10, as seen in Section 3.1. But, as mentioned in Section 3.2, puri-
fied enzyme AAI3 storage stability decreased in reverse micelles.
One possible explanation is that the increase in the hydrogen bond-
ing of the enzyme AAI3 ␣-helices upon encapsulation at w = 10
0
possibly increased the enzyme dynamics for effective catalysis yet
the enzyme will become less stable [46].
The second derivative FTIR spectrum of purified enzyme AAI3 in
reverse micelles at lower water content w = 4 can be seen in Fig. 6
0
Fig. 5. Comparison of second derivative FTIR spectrum in amide I region of P.
aeruginosa strain L10 purified amidase in H2O (black full line), in TTAB reverse
micelles at w0 = 10 (black dotted line) and in TTAB reverse micelles at w0 = 4 (gray
(gray line spectrum). Again, there are visible shifts in band compo-
2
nents positions suggesting enzyme AAI3 structural rearrangements
line).
upon encapsulation at w0 = 4. Nevertheless these modifications

A. Fragoso et al. / Process Biochemistry 47 (2012) 264–272
271
were responsible for a poorer catalytic efficiency of amidase AAI3 at
these water contents. When comparing the spectrum of the enzyme
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[
the previously mentioned (Section 3.1) minor effects in enzyme
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4
. Conclusion
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[
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As seen in this study the use of a reverse micellar system of TTAB
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L10 and AI3 revealed to be advantageous in acetohydroxamic acid
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