Production of hydroxamic acids by immobilized Pseudomonas aeruginosa cells: Kinetic analysis in reverse micelles

Article abstract of DOI:10.1016/j.molcatb.2013.03.016

Intact cells from Pseudomonas aeruginosa strain L10 containing amidase were used as biocatalysts both free and immobilized in a reverse micellar system. The apparent kinetic constants for the transamidation reaction in hydroxamic acids synthesis, were determined using substrates such as aliphatic, amino acid and aromatic amides and esters, in both media. In reverse micelles, K _m values decreased 2-7 fold relatively to the free biocatalyst using as substrates acetamide, acrylamide, propionamide and glycinamide ethyl ester. We have concluded that overall the affinity of the biocatalyst to each substrate increases when reactions are performed in the reversed micellar system as opposed to the buffer system. The immobilized biocatalyst in general, exhibits higher stability and faster rates of reactions at lower substrates concentration relatively to the free form, which is advantageous. Additionally, the immobilization revealed to be suitable for obtaining the highest yields of hydroxamic acids derivatives, in some cases higher than 80%.

Full text of DOI:10.1016/j.molcatb.2013.03.016

Journal of Molecular Catalysis B: Enzymatic 93 (2013) 28–33
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Production of hydroxamic acids by immobilized Pseudomonas aeruginosa cells:
Kinetic analysis in reverse micelles
Marisa Bernardoa, Rita Pachecoa,∗, Maria Luísa M. Serralheirob, Amin Karmalia
a Centro de Investigac¸ ão de Engenharia Química e Biotecnologia do Instituto Superior de Engenharia de Lisboa, Área Departamental de Engenharia Química, R. Conselheiro Emídio
Navarro, 1, 1959-007 Lisboa, Portugal
b Centro de Química e Bioquímica da Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Intact cells from Pseudomonas aeruginosa strain L10 containing amidase were used as biocatalysts both
free and immobilized in a reverse micellar system. The apparent kinetic constants for the transamidation
reaction in hydroxamic acids synthesis, were determined using substrates such as aliphatic, amino acid
and aromatic amides and esters, in both media. In reverse micelles, Km values decreased 2–7 fold rela-
tively to the free biocatalyst using as substrates acetamide, acrylamide, propionamide and glycinamide
ethyl ester. We have concluded that overall the affinity of the biocatalyst to each substrate increases
when reactions are performed in the reversed micellar system as opposed to the buffer system. The
immobilized biocatalyst in general, exhibits higher stability and faster rates of reactions at lower sub-
strates concentration relatively to the free form, which is advantageous. Additionally, the immobilization
revealed to be suitable for obtaining the highest yields of hydroxamic acids derivatives, in some cases
higher than 80%.
Received 30 October 2012
Received in revised form 22 March 2013
Accepted 26 March 2013
Available online 11 April 2013
Keywords:
Amidase
Acyltransferase
Pseudomonas aeruginosa
Reverse micelles
Hydroxamic acids
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
promote reversal of hydrolytic reactions or to modify the specificity
or the stability of the biocatalyst [1,4–7,9]. Reversed micelles are
Enzymes are used as biocatalysts in the synthesis of products
mainly due to their versatility catalyzing a wide range of reac-
tions with high reaction rates, regiospecificity and under milder
conditions than their chemical counterparts [1]. Biocatalysis can
be carried out using either whole cells or lysates containing the
enzyme of interest, or using purified enzymes. However, enzyme
purification is costly and time consuming and often enzymes are
less stable in the purified form than in whole cells [2,3].
Biocatalytic processes are often carried out in aqueous environ-
ments. However, observations that biocatalysts are able to catalyze
reactions in organic solvents encouraged an intense research and
development of applications for enzymatic catalysis in nonaque-
ous environments. A wide range of reactions were carried out
in organic media namely oxidation–reductions, hydroxylations,
esterifications, hydrolysis and transamidations reactions [3–8].
Microemulsions are typical non-conventional media suitable
to promote biocatalysis and are often used to obtain higher sol-
ubility of substrates or products, to lower the water content to
microemulsions in which a shell formed by surfactant molecules
protects the biocatalyst against denaturation by the organic sol-
vent. The biocatalyst is solubilized inside this shell in a water pool
maintaining the catalytic activity and often can exhibit an increased
stability [1,10,11].
Amidases (acylamide amidohydrolase E.C.3.5.1.4) from Pseu-
domonas aeruginosa [12], Rhodococcus sp. [13,14] and Arthrobacter
sp. [15] have been used for the catalysis of amides or carboxylic
acids hydrolysis reactions and of amides or esters acyltransferase
reactions (transfer of R CO groups to hydroxylamine) synthesizing
hydroxamic acids derivatives. [3,8,9,16].
Synthesis of acetohydroxamic acid, using amidases from P.
aeruginosa as biocatalysts, has been performed by our group in
aqueous media [17] and in a reverse micellar system [3,8]. The
success of the latter microenvironment, where yields of prod-
uct synthesis higher than 90% were accomplished in the reverse
micellar system of cationic surfactant tetradecyltrimethyl ammo-
nium bromide (TTAB) in heptane/octanol (80/20%), encouraged
the present study in which we investigated the biocatalysed syn-
thesis reaction of several hydroxamic acid derivatives in this
non-conventional medium. Furthermore we hereby use whole cells
from P. aeruginosa L10, containing amidase as biocatalysts, since it
is also well known from our previous work that there is no need for
the enzyme purification steps prior to the synthesis reaction [3].
∗
Corresponding author. Tel.: +351 218317000.
E-mail addresses: bernardo marisa@hotmail.com (M. Bernardo),
rpacheco@deq.isel.ipl.pt, rpacheco@deq.isel.pt (R. Pacheco), mlserralheiro@fc.ul.pt
(M.L.M. Serralheiro), akarmali@deq.isel.ipl.pt (A. Karmali).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.03.016

M. Bernardo et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 28–33
29
Hydroxamic acids have received considerable attention and
are considered very attractive compounds due to their high che-
lating potential with metal ions. Several hydroxamic acids have
been used as drugs and have been reported as tumor inhibitors,
anti-malarial agents, anti-HIV and recommended for treatment
of ureaplasma infections and anemia [14,18,19]. Other hydrox-
amic acids have been used to eliminate metal ions in wastewater
treatment and nuclear technology [20]. The application of these
compounds in food industry, waste water treatment and in phar-
maceutical industry as antimicrobial agents, antibiotic antagonists
and tumor inhibitors was investigated by [21–24]. However chem-
ical synthesis of these compounds is a complex procedure [25] and
therefore the results obtained from this work show great potential
for the simplified synthesis of these highly important products. This
work aims to present the kinetic analysis of the acyltranferase reac-
tion catalyzed by amidase in intact cells of P. aeruginosa strain L10,
using a wide range of substrates (aliphatic, aromatic and amino
acids amides and esters) for the synthesis of several hydroxamic
acid derivatives, both free in aqueous media or immobilized in
reverse micelles.
The rate of reaction was determined by following the increase
in the concentration of the reaction product a, hydroxamic
acid derivative, during the course of the reaction as described
in [3]. Calibration curves with acetohydroxamate, benzohydro-
xamate, butyrylhydroxamate, glycine hydroxamate and alanine
hydroxamate were established and values for the extinction coef-
ficient (ε) were obtained of 0.105 mM−1 (R2 = 0.993), 0.1210 mM−1
(R2 = 0.964), 0.038 mM−1 (R2 = 0.993), 0.0072 mM−1 (R2 = 0.965),
0.0057 mM−1 (R2 = 0.972), respectively. One enzyme unit (U) is
defined as the amount of enzyme required to produce 1 ␮mol of
hydroxamic acid derivative per min under these experimental con-
ditions.
2.3.2. Acyltransferase activity assay of free P. aeruginosa cells in
buffer solution
The acyltransferase activity was determined as described above
but the reaction was carried out in 10 mM HEPES buffer pH 7.2
containing P. aeruginosa cells (0.050 g cells/mL in 10 mM HEPES
buffer at pH 7.2) and hydroxylamine solution (freshly neutralized
to pH 7). The amide or ester substrate solution in 10 mM HEPES
buffer at pH 7.2 was injected in order to initiate the reaction.
The rate of the reaction was determined as described previously
[3] by following the increase in the concentration of the reac-
tion product, hydroxamic acid derivative, during the course of
the reaction. Calibration curves with acetohydroxamate, benzohy-
droxamate, butyrylhydroxamate, glycine hydroxamate and alanine
hydroxamate were established and values for the extinction coef-
ficient (ε) were obtained of 0.063 mM−1 (R2 = 0.993), 0.0885 mM−1
(R2 = 0.989), 0.0328 mM−1 (R2 = 0.988), 0.002 mM−1 (R2 = 0.935),
0.0017 mM−1 (R2 = 0.951) respectively. One enzyme unit (U) is
defined as the amount of enzyme required to produce 1 ␮mol of
hydroxamic acid derivative per min under these experimental con-
ditions.
2. Experimental
2.1. Chemicals
Heptane, octanol, benzamide and propionamide were pur-
chased from Merck (Darmstadt, Germany). Tetradecyltrimethyl
ammonium bromide (TTAB), benzohydroxamic acid, l-
alaninamide, phenylalaninamide, phenylalaninamide ethyl ester
and glycine ethyl ester were obtained from Sigma–Aldrich (Madrid,
Spain). Acetamide, acetohydroxamic acid and iron chloride were
purchased from Fluka (Madrid, Spain). Hydroxylamine was
obtained from Panreac (Barcelona, Spain). Glycinamide and leuci-
namide were obtained from Bachem (Bubendorf, Switzerland),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
buffer from Fisher Scientific (Leicestershire, United Kingdom).
All chemicals used were of analytical grade. Benzohydroxamate
and butyrylhydroxamate were obtained from Sigma and glycine
hydroxamate and alanine hydroxamate were obtained from Key
Organics.
2.4. Storage stability
Storage stability of the cells in the reverse micellar system
was demonstrated by incubating the cell solution (0.012 g/mL in
10 mM HEPES buffer at pH 7.2) in 200 mM TTAB in heptane/octanol
(80/20%, v/v) at w0 of 10 at 25 ◦C under constant agitation during
several hours. Sampling was performed at time intervals and activ-
ity was assayed as described in Section 2.3.1 by the injection of
30 mM hydroxylamine and 15 mM acetamide in order to initiate
the reaction.
Storage stability of the cells in HEPES buffer was demonstrated
by incubating the cell solution 0.012 g/mL in 10 mM HEPES buffer
at pH 7.2 at 25 ◦C under constant agitation during several hours.
Aliquots were taken from the mixture during incubation, 30 mM
hydroxylamine plus 15 mM acetamide were added in order to ini-
tiate the reaction and activity was assayed as described in Section
2.3.2.
2.2. Pseudomonas aeruginosa strain L10 cell culture
P. aeruginosa L10 strain was used as the source of amidase (E.C.
3.5.1.4.) The strain was grown overnight as previously described
[26,27] and the cells were harvested by centrifugation at 10,000 × g
for 5 min. The pellet containing cells was washed twice with NaCl
(8.5%, w/v) and stored at −20 ◦C.
2.3. Enzyme assays
2.3.1. Acyltransferase activity assay of immobilized P. aeruginosa
cells in the reverse micellar system
The first order rate inactivation constants, k, were determined
by the slope of the semilogarithmic plot of residual activity versus
storage time and used to calculate the half-life values, t1/2 [28].
The activity assays in the reverse micellar medium were per-
formed using intact cells from P. aeruginosa L10 strain containing
amidase and according to a methodology previously described [3],
in a thermostatic water bath at 40 ◦C. Briefly, the acyltransferase
activity was investigated in a 5 mL stirred reverse micellar sys-
tem of 200 mM TTAB in heptane/octanol (80/20%, v/v) containing
P. aeruginosa cells (0.050 g cells/mL in 10 mM HEPES buffer at pH
7.2), hydroxylamine solution (freshly neutralized to pH 7). The
water content in the system, usually defined through the parame-
ter w0 = [H2O]/[surfactant], was controlled by adjusting the added
volume of buffer 10 mM HEPES at pH 7.2 and established to a value
of 10. The amide or ester substrate solution in 10 mM HEPES buffer
at pH 7.2 was injected in order to initiate the reaction.
2.5. Kinetic studies
For the kinetic analysis the activity assays were performed using
a constant P. aeruginosa cell concentration at different concentra-
tions of each of the substrates in 10 mM HEPES buffer at pH 7.2.
The hydroxylamine solution was used in a 2 fold concentration of
the substrate concentration. The activity assays were run in dupli-
cate both in the reverse micellar system and in the buffer solution
according to the methodology described in Sections 2.3.1 and 2.3.2
respectively.

30
M. Bernardo et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 28–33
The intact cells of P. aeruginosa L10 strain containing amidase
exhibited very broad substrate specificity. The biocatalyst, both free
and immobilized, synthesized a variety of hydroxamic acid deriva-
tives using as substrates aliphatic, aromatic and amino acid amides
and esters.
Table 1 and Fig. 2 shown that the biocatalyst displays different
kinetic behavior for the different substrates and in the different
media. Higher values of Km were obtained in the free cells than in
the reverse micellar system (Fig. 2). One must consider that the
substrate in the reverse micellar is confined to the same reduced
volume as the intact cells and this could explain the increase in the
immobilized biocatalyst affinity toward the substrate as opposed
to the free biocatalyst.
Aliphatic amides were among the best substrates for acyl trans-
fer reactions as they exhibited the lowest Km (Fig. 2). The values
obtained of Km increased for amino acid amides, aromatic amides
and esters. The highest values of Km were obtained for aromatic
amides or esters such as benzamide and phenylalanine ethyl ester
both in reverse micelles and in conventional buffer medium, and in
the latter medium additionally for glycine ethyl ester.
Generally for aliphatic, amino acids amides or esters substrate
the increase in substrate chain length on the P side [17,29] led to
an increase in Km and a decrease in substrate affinity. However, in
the case of aromatic amides, the increase in substrate chain length
in phenylalaninamide, which contains a phenyl alkyl side chain,
increased biocatalyst affinity, relatively to the benzamide, which
presents a higher Km value. These effects were observed both with
immobilized and free biocatalyst.
Fig. 1. Typical Michaelis–Menten plot obtained for the rate of Pseudomonas
aeruginosa L10 amidase containing cells using acetamide as substrate in the reverse
micellar system (
) and in HEPES buffer (
). The rate of reaction was deter-
mined as described in Sections 2.3.1 and 2.3.2, respectively.
The data analysis for estimation of apparent kinetic parame-
ters V and Km was performed using GraphPad Prism 5 software
(GraphPad Software Inc., San Diego, CA) with an average correlation
coefficient (R2) 0.94 0.04 for all the estimated parameters.
3. Results and discussion
3.1. Stability of free and immobilized aliphatic amidase in P.
aeruginosa L10 cells
These results are in good agreement with previously described
studies [14] and results previously reported for the hydrolysis reac-
tion catalyzed by a purified recombinant amidase [17]. The enzyme
exhibited higher affinity for amides that for the corresponding
esters as demonstrated by the comparison of acetamide to ethyl
acetate or phenylalaninamide to phenylalanine ethyl ester.
Upon immobilization the biocatalyst experiences the highest
increase in the affinity toward very short chain substrates such as
acetamide, acrylamide, propionamide and glycinamide ethyl ester.
Specifically for aliphatic amides, such as acetamide, acrylamide,
propionamide and glycinamide ethyl ester, the Km value can be
decreased by factors of approximately 5, 7, 4 and 2 fold, respec-
tively, in reverse micelles relatively to the conventional buffer
medium. This effect was much less noticeable for other substrates.
It was also perceptible in Fig. 2 that the immobilized and free
biocatalyst demonstrated a very low affinity toward benzamide as
substrate in the acyl transfer reaction, for benzohydroxamic acid
synthesis. This is in agreement with previously reported results
which demonstrated that for benzamide the hydrolytic reaction
rate is higher than the acyltransferase reaction rate [14,17].
One of the advantages of using the biocatalyst enclosed in
intact cells and in reverse micelles is known to be a higher sta-
bility [9] and this was confirmed in this work. Inactivation rate
constants k of 12.2 × 10−3 h−1 and 2.3 × 10−3 h−1 were obtained
for free and immobilized cells, respectively, in HEPES buffer. As
expected, in reverse micelles the rate of biocatalyst activity decay
obeyed first-order kinetics and followed a two stages series mech-
anism of inactivation. The enzyme activity decay followed a two
stages series-type mechanism of inactivation, both in buffer and
in reverse micelles. In HEPES buffer, the biocatalyst decays in the
first step and, in the second step, the activity is almost constant.
In reverse micelles, the activity remains almost constant, during
an initial period of approximately 7 days, and it decays in the sec-
ond step. In comparison with our previous results, with intact cells
resuspended in a TMEG buffer (20 mM Tris–HCl containing 1 mM
EDTA, 1 mM mercaptoethanol and 10% (v/v) glycerol pH 7.2) [3], the
use of HEPES buffer increased the enzyme stability in the reverse
micelles but decreased it in the free cells. Additionally, most of the
conversions presented here were not accomplished in TMEG buffer
(data not shown), consequently the selection of HEPES buffer.
3.3. Free and immobilized biocatalyst catalytic efficiency
3.2. Substrate affinity of free and immobilized aliphatic amidase
in P. aeruginosa L10 cells
In kinetic studies, when evaluating for the same enzyme the
reaction using different substrates, conclusions should not be taken
about the relative rates of product formation by simply comparing
V values or Km of the individual substrates.
Typical Michaelis–Menten curves, as exemplified in Fig. 1, were
obtained when initial velocity of the product formation was plot-
ted against the concentration of the various substrates, both using
intact cells in the reverse micellar system and the conventional
buffer medium. From these results, and for each substrate, the
apparent kinetic parameters Km and V were determined in the
reverse micellar system and in conventional HEPES buffer medium.
The obtained constants are apparent and not intrinsic of amidase
since they were obtained using as biocatalyst the enzyme in the free
cells or in the reverse micellar system, these cells were microen-
capsulated in reverse micelles. Additionally the catalytic turnover
(V/Km) was calculated in order to evaluate at low substrate concen-
tration the biocatalyst efficiency.
Just as the rate at high substrate concentration is largely deter-
mined by V, the rate at low substrate concentration is largely
determined by the catalytic turnover as a measure of the biocat-
alyst efficiency V/Km [30,31]. When comparing the value of V/Km
acting on the same substrate, the catalyst with the higher value
will give the faster reaction at low substrate concentration [30]. It
is noticeable in Table 1, that V/Km was much higher in the reverse
micellar system than in the conventional medium for substrates
such as aliphatic amides and glycinamide. Therefore for these sub-
strates the biocatalyst demonstrates better efficiency in the reverse
micellar system than in the conventional buffer medium.

M. Bernardo et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 28–33
31
Table 1
Apparent kinetics parameters, limiting rate (V) and catalytic turnover (V/Km) for the acyltransferase reaction of immobilized (reverse micelles) and free (HEPES buffer)
Pseudomonas aeruginosa L10 cells using different substrates. Kinetic studies were performed in duplicates varying substrate concentrations as described in Section 2.5.
V (U)
V/Km (s−1
)
Reverse micelles
HEPES buffer
Reverse micelles
HEPES buffer
Aliphatic amides
Acetamide CH3 CONH2
Acrylamide CH2 CH CONH2
Propionamide CH3 CH2 CONH2
Butyramide CH3 CH2 CH2 CONH2
Amino acid amides
7.88 0.34
5.20 0.14
1.66 0.13
0.27 0.03
6.96 0.03
9.49 0.01
1.10 0.00
0.24 0.05
26.57 0.54
12.30 0.74
1.71 0.75
0.08 0.00
4.71 0.17
3.03 0.02
0.25 0.01
0.06 0.01
Glycinamide NH2 CH2 CONH2
Alaninamide NH2 CH(CH3) CONH2
Leucinamide (CH3)2CH CH2 CH(NH2) CONH2
Aromatic amides
10.83 1.29
1.39 0.02
0.91 0.16
3.76 0.82
1.59 0.37
5.04 0.57
3.33 0.20
0.35 0.04
0.27 0.07
0.90 0.18
0.35 0.05
1.01 0.01
Benzamide C6H5CONH2
Phenylalaninamide C6H5 CH2 CH(NH2) CONH2
Esters
0.22 0.0
0.06 0.01
0.16 0.02
0.33 0.05
0.04 0.00
0.02 0.00
0.02 0.00
0.07 0.01
Ethylacetate CH3 COO CH2 CH3
Glycine ethyl ester NH2 CH2 COO CH2 CH3
Phenylalanine ethyl ester C6H5 CH2 CH(NH2) COO CH2 CH3
0.25 0.012
1.39 0.08
0.09 0.02
0.27 0.03
5.61 0.39
0.06 0.03
0.09 0.00
0.42 0.02
0.02 0.00
0.08 0.01
0.90 0.05
0.03 0.01
This was not observed for substrates such as long chain
amino acid amides alaninamide and leucinamide, aromatic amides,
glycine ethyl ester and phenylalanine ethyl ester. This can be
explained on the basis of substrate hydrophobicity and partitioning.
In reverse micelles, the non-polar and more hydrophobic substrates
have more affinity to the organic solvent than to the aqueous core
of the micelles which hinders the interaction with the biocatalyst.
For substrates such as leucinamide, phenylalaninamide and
glycine ethyl ester higher values of V were obtained in buffer
medium (Table 1) than using the biocatalyst immobilized in reverse
micelles meaning that a better biocatalyst performance could be
achieved in buffer medium, however at the expense of using high
substrate concentrations.
3.4. Hydroxamic acid derivatives production in different reaction
media
The above mentioned kinetic results are merely indicative of
the biocatalyst performance regarding the initial rate of the cat-
alyzed acyl transfer reaction. We additionally determined the
production of the several hydroxamic acid derivatives in the
reactions in reverse micelles and buffer medium. Table 2 shows
Fig. 2. The apparent Michaelis constant (Km) for each of the used substrates, in reverse micellar system (gray bars) and in HEPES buffer (white bar). Kinetic studies were
performed in duplicates varying substrate concentrations as described in Section 2.5.

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M. Bernardo et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 28–33
Table 2
Hydroxamic acid derivatives concentration obtained in the reverse micellar system and in HEPES buffer medium.
Substrate
Substrate concentration (mM)
Reaction time (min)
Hydroxamic acid derivative (mM)
Reverse micelles
HEPES buffer
Acetamide
Acrylamide
Propionamide
Butyramide
Glycinamide
Alaninamide-
Leucinamide
Benzamide-
Phenylalaninamide
Ethylacetate
Glycine ethyl ester-
Phenylalanine ethyl ester
12.50
15
15
45
30
30
30
9
30
30
30
30
8–10
15
25
180
60–120
180
150
210
120–150
120
180
12.50 0.79
12.43 0.54
13.68 0.14
5.72 0.67
15.87 4.21
15.52 2.01
18.07 3.68
0.64 0.003
1.52 0.38
2.20 0.10
20.85 2.34
0.91 0.17
3.36 0.05
7.59 0.11
1.11 0.15
2.88 0.50
16.00 0.50
16.18 0.29
21.76 2.35
0.46 0.10
2.71 0.37
2.16 0.12
17.00 3.50
0.92 0.21
180
the higher products concentrations obtained from all the tested
conditions.
intact cells of P. aeruginosa strain with different substrates. It is
noticeable that higher yields of hydroxamates production were
obtained using immobilized biocatalyst than free biocatalyst,
except for phenylalaninamide.
Again it was noticeable that overall product concentrations were
higher in reverse micellar medium than in conventional medium,
particularly in the case of using as substrates aliphatic amides.
Considering other substrates minor alterations in the obtained
product concentrations were observed using the immobilized bio-
catalyst compared with the free biocatalyst.
Another advantage of the reverse micellar medium was that
the yields of hydroxamic acid derivatives production were often
over 80%. Our previous results, using this immobilized biocata-
lyst, have demonstrated these high yields for acetohydroxamate
synthesis [3,8,17]. Additionally this work reveals that this can be
accomplished for the bioconversion of other hydroxamates, using
as substrates aliphatic amides acetamide, acrylamide and propi-
onamide; amino acid amides such as glycinamide, alaninamide,
and leucinamide and an ester, glycine ethyl ester. These substrate
conversions where achieved since the biocatalyst continues active
3.5. Yield of production of hydroxamic acid derivatives in both
media
Fig. 3 represents the highest yields of synthesis of several
hydroxamic acid derivatives obtained using free and immobilized
Fig. 3. Yield of hydroxamic acid derivatives production in both media. Percentage of conversion of the different used substrates in the reverse micellar system (gray bars)
and in HEPES buffer (white bars) at the indicated substrate concentration and reaction time.

M. Bernardo et al. / Journal of Molecular Catalysis B: Enzymatic 93 (2013) 28–33
33
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This work analyses the transamidation reaction for the synthe-
sis of numerous hydroxamic acid derivatives using as biocatalyst
intact cells of P. aeruginosa. The determination of the apparent
kinetic parameters is of major importance, in order to define the
most appropriate substrates and the best reaction conditions for
obtaining high reaction rates and high substrate conversion. In this
work, intact cells proved to be suitable for these syntheses, enzyme
purification being unnecessary. Our results demonstrated a broad
substrate specificity of the biocatalyst and the importance of using
the reverse micellar medium for higher biocatalyst stability and
greater yields of hydroxamic acid production.
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Acknowledgement
The authors would like to acknowledge Fundac¸ ão para a Ciência
e Tecnologia for the financial support to the project PTDC/EBB-
BIO/111236/2009.
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