Biotransformation of nicotinamide to nicotinyl hydroxamic acid at bench scale by amidase acyl transfer activity of Pseudomonas putida BR-1

Article abstract of DOI:10.1016/j.molcatb.2014.07.001

Acyl transfer activity of amidase of Pseudomonas putida BR-1 has been explored for the conversion of N-substituted aromatic amide (nicotinamide) and hydroxylamine to nicotinyl hydroxamic acid. Nicotinyl hydroxamic acid is an important pharmaceutical compound with enormous biomedical applications. P. putida BR-1 produces maximum amidase acyl transfer activity 138 U/mg dcm at 50 °C, with highest conversion (95%) of nicotinamide to nicotinyl hydroxamic acid. A bioprocess was developed for production of nicotinyl hydroxamic acid in batch reaction (final volume 1 L) by adding 200 mM nicotinamide and 1000 mM of hydroxylamine in 100 mM sodium phosphate buffer (pH 7.5) at 50 °C, using 20 U/ml acyl transfer activity resting cells of P. putida BR-1 in reaction mixture. From 1 L reaction mixture 16 g of nicotinyl hydroxamic acid was recovered with 32 g/L/h volumetric productivity. The amidase acyl transfer activity of P. putida BR-1 and the process developed in the present study are of industrial significance for the enzyme mediated production of nicotinyl hydroxamic acid.

Full text of DOI:10.1016/j.molcatb.2014.07.001

Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biotransformation of nicotinamide to nicotinyl hydroxamic acid at
bench scale by amidase acyl transfer activity of Pseudomonas
putida BR-1
Ravi Kant Bhatia, Shashi Kant Bhatia, Praveen Kumar Mehta, Tek Chand Bhalla∗
Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, HP 171005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Acyl transfer activity of amidase of Pseudomonas putida BR-1 has been explored for the conversion of N-
substituted aromatic amide (nicotinamide) and hydroxylamine to nicotinyl hydroxamic acid. Nicotinyl
hydroxamic acid is an important pharmaceutical compound with enormous biomedical applications. P.
putida BR-1 produces maximum amidase acyl transfer activity 138 U/mg dcm at 50 ◦C, with highest con-
version (95%) of nicotinamide to nicotinyl hydroxamic acid. A bioprocess was developed for production
of nicotinyl hydroxamic acid in batch reaction (final volume 1 L) by adding 200 mM nicotinamide and
1000 mM of hydroxylamine in 100 mM sodium phosphate buffer (pH 7.5) at 50 ◦C, using 20 U/ml acyl
transfer activity resting cells of P. putida BR-1 in reaction mixture. From 1 L reaction mixture 16 g of
nicotinyl hydroxamic acid was recovered with 32 g/L/h volumetric productivity. The amidase acyl trans-
fer activity of P. putida BR-1 and the process developed in the present study are of industrial significance
for the enzyme mediated production of nicotinyl hydroxamic acid.
Received 13 January 2014
Received in revised form 4 June 2014
Accepted 1 July 2014
Available online 9 July 2014
Keywords:
Nicotinyl hydroxamic acid
Amidase
Acyl transfer activity
Anti-tumor
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
suffer from substrate inhibition in the reactions and also produced
by-products [10,12]. In the present study, a bench scale (1 L) pro-
Hydroxamic acid (R CO NHOH) and their derivatives have
tremendous applications in medicine, agriculture, bioremediation,
food additives, antibiotics, antifungal agents, tumor inhibitors [1,2]
siderophores, enzyme inhibitors [3] and in bioremediation [4,5].
Most of the hydroxamic acids are synthesized through various
chemical routes [6–8]. A number of hydroxamic acids viz. aceto-
hydroxamic acid, butyrohydroxamic acid, benzohydroxamic acid
(BHA), succinic hydroxamic acid have been synthesized using bio-
catalytic routes [9], but very little information is available about
N-substituted aromatic hydroxamic acids. Among these, nicotinyl
hydroxamic acid (NHA) finds application as anti-HIV, antimicrobial
and antineoplastic agent. It is also used in the treatment of anemia,
and reported as potential inhibitor of leukemia and ureaplasma
[10]. Nicotinyl hydroxamic acid has been synthesized at test tube
scale using Rhodococcus sp. R312 [9,10] and Bacillus smithii strain
IITR6b2 [11] acyl transfer activity of amidase. However, study of
these earlier reports divulged that the acyl transfer activity of the
amidases of Rhodococcus sp. R312 and Bacillus smithii strain IITR6b2
cess is developed for the synthesis of NHA using the acyl transfer
activity of the amidase of P. putida BR-1 at higher temperature and
with higher purity than reported before.
2. Materials and methods
2.1. Chemicals
All the nitriles and amides used in the present study were pur-
chased from Sigma–Aldrich, USA. The culture media ingredients
were procured from Hi Media (Mumbai, India). All the chemicals
were of analytical grade.
2.2. Microorganism and culture conditions
P. putida BR-1 (isolated in our laboratory and identified at
the Institute of Microbial Technology, Chandigarh, India) is used
as a source of acyl transfer activity. This organism was grown
aerobically in 250 ml Erlenmeyer flask containing 50 ml salt
medium (Na2HPO4·12H2O: 2.5 g, KH2PO4: 2.0 g, MgSO4·7H2O:
0.5 g, FeSO4·7H2O: 0.03 g, CaCl2·2H2O: 0.06 g and yeast extract:
1 g L−1 of distilled water, added 1% (v/v) isobutyronitrile) at 30 ◦C,
∗
Corresponding author. Tel.: +91 177 2831948; fax: +91 177 283154.
E-mail address: bhallatc@rediffmail.com (T.C. Bhalla).
http://dx.doi.org/10.1016/j.molcatb.2014.07.001
1381-1177/© 2014 Elsevier B.V. All rights reserved.

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R.K. Bhatia et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
160 rpm in an incubator shaker for 12 h to prepare preculture.
Preculture (5%) was used as seed and isobutyronitrile 20 mM as
inducer was added at four different intervals to 50 ml of salt
medium in 250 ml Erlenmeyer flask and incubated for 56 h at 25 ◦C
and 160 rpm in an incubator shaker for the production of acyl trans-
fer activity. The cells were harvested by centrifugation at 10,000 × g
for 10 min at 4 ◦C and washed twice with 100 mM sodium phos-
phate buffer (pH 7.0). The cells were suspended in the same buffer
and stored at 4 ◦C until further use [11].
and 500 mM: 25 U/ml). For maximum conversion of 200 mM nic-
otinamide to nicotinyl hydroxamic acid in shortest time, resting
cell concentration of P. putida BR-1 was assessed by varying the
resting cell amount from 5.0 U/ml to 25.0 U/ml while the concen-
tration of substrates was kept constant. The complete conversion
of nicotinamide and hydroxylamine to nicotinyl hydroxamic acid
was analyzed by HPLC.
2.6.1. Fed batch reaction at 50 ml scale (200 mM nicotinamide
and 1000 mM hydroxylamine per feed)
2.3. Acyl transfer activity assay
Fed batch reaction was carried out at 50 ml scale in 100 mM
sodium phosphate buffer (pH 7.5) using 200 mM nicotinamide,
1000 mM hydroxylamine and resting cell equivalent to 20 U/ml acyl
transfer activity at 50 ◦C. The same amount of nicotinamide and
hydroxylamine were fed after 30 min, so that reaction moves in the
forward direction for getting higher yield of the product. Nicotinyl
hydroxamic acid formed in the reaction was periodically analyzed
by HPLC.
Acyl transfer activity was assayed using the method developed
by Brammar and Clarke [13]. The assay mixture contained 100 mM
potassium phosphate buffer (pH 7.0), 100 mM nicotinamide, and
500 mM hydroxylamine and acyl transfer activity containing res-
ting cells 4.0 mg dry cell mass (dcm). The reaction mixture was
incubated at 50 ◦C for 60 min and the reaction was stopped by
adding 1 ml FeCl3 reagent containing 6% FeCl3 and 2% HCl. It was
centrifuged at 10,000 × g for 5 min and then absorbance of super-
natant was measured at 500 nm. One unit (U) of acyl transfer
activity was defined as the amount of resting cells (mg dcm) which
catalyzed the release of 1 ␮mol of nicotinyl-hydroxamic acid per
min under the assay conditions.
2.6.2. Bench scale production of nicotinyl hydroxamic acid at 1 L
scale
On the basis of optimized process parameters the conversion
of nicotinamide to nicotinyl hydroxamic acid was scaled up to 1 L
using New Brunswick Scientific (NBS) BIOFLO C-32 fermenter.
2.6.3. Recovery of nicotinyl hydroxamic acid
2.4. Analytical analysis
The reaction mixture was centrifuged at 10,000 × g for 30 min
to remove the cells. The nicotinyl hydroxamic acid was purified by
solvent extraction method [14] and analyzed by HPLC.
The concentration of nicotinamide, nicotinyl hydroxamic acid
and nicotinic acid in the reaction mixture were quantified by
HPLC using series 200 Ic pump (Perkin Elmer) and programmable
Absorbance Detector (Applied Biosystem) equipped with a Nucle-
osil C18 column (25 cm × 4.6 mm, 5 ␮m particle size; GL Sciences,
Japan). The substrate and product was detected at 230 nm, at a flow
rate of 1 ml/min of mobile phase comprised of potassium phosphate
buffer (0.1 M) and methanol in the ratio of 9:1 in HPLC grade water
and pH of the mobile phase adjusted to 3.5 with ortho-phosphoric
acid.
3. Results
3.1. Optimization of reaction conditions for acyl transfer activity
of P. putida BR-1
3.1.1. Effect of buffer system and buffer pH
For the selection of buffer and optimum pH, six different
buffers (citrate, sodium phosphate, potassium phosphate, borate
and glycine NaOH) of 100 mM concentration having different pH
range (2–11) were tested. The acyl transfer activity was higher in
sodium phosphate buffer (104.44 0.03 U/mg dcm, pH 7.5) in com-
parison to potassium phosphate buffer (79.95 0.02 U/mg dcm, pH
7.5) (Fig. 1). Sodium phosphate buffer (0.1 M, pH 7.5) was used in
subsequent experiments. In citrate buffer, the acyl transfer activity
increased only when its pH reached beyond 5.0 whereas at low pH
no activity was observed. In borate and glycine NaOH buffers, P.
putida BR-1 cells showed very small acyl transfer activity.
2.5. Optimization of reaction conditions for conversion of
nicotinamide to nicotinyl hydroxamic acid
Different reaction parameters were varied one by one to
determine the optimal reaction conditions. To work out the
optimum pH and temperature, reactions were carried out at
pH 2.0–11.0 in various buffer systems (borate buffer, potassium
phosphate buffer, sodium phosphate buffer, citrate buffer, carbon-
ate buffer of 100 mM), buffer molarity (20–500 mM), biocatalyst
(0.06–0.6 mg dcm/ml), and reaction temperature (10–80 ◦C). Fifty
different combinations of substrate and co-substrate were tested in
such a manner that the concentration of nicotinamide was varied
from 100 to 1000 mM at different hydroxylamine concentrations
ranging from 200 to 1000 mM in the reaction mixture to determine
the optimum concentration and ratio for highest acyl transfer activ-
ity of amidase of P. putida sp. BR-1. In order to check the stability of
enzyme at different temperatures this enzyme was stored at differ-
ent temperatures (4 ◦C, 15 ◦C, 20 ◦C, 25 ◦C, 30 ◦C, 35 ◦C, 40 ◦C, 45 ◦C,
50 ◦C, 55 ◦C, 60 ◦C) and then acyl transfer activity was measured
after 1 h interval at the standard temperature i.e. 50 ◦C.
3.1.2. Effect of buffer strength
The enzyme activity was increased as the concentration of
sodium phosphate buffer increased from 20 to 100 mM with a
maximum at 100 mM (110.21 0.03 U/mg dcm) (Fig. S1). However,
above 100 mM sodium phosphate buffers the acyl transfer activity
of P. putida BR-1 experienced inhibitory effect and at 500 mM of
sodium phosphate buffer only 34.85 0.01 U/mg dcm acyl transfer
activity was observed.
Supplementary Fig. S1 related to this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.molcatb.2014.07.001.
3.1.3. Amount of biocatalyst
2.6. Process development for the production of nicotinyl
hydroxamic acid
Maximum acyl transfer activity 114.42 0.01 U/mg dcm of ami-
dase of P. putida BR-1 was observed with 0.12 mg dcm/ml resting
cells. Above 0.12 mg dcm/ml cell concentration, no increase in
activity was recorded (Fig. 2). At 0.60 mg dcm/ml cell concentra-
tion a decrease in acyl transfer activity 62.24 0.02 U/mg dcm of
amidase was observed.
To develop an efficient bioprocess for the maximum pro-
duction of nicotinyl hydroxamic acid, different combinations of
nicotinamide and resting cell concentrations were made (100 mM:
5.0 U/ml, 200 mM: 10 U/ml, 300 mM: 15 U/ml, 400 mM: 20 U/ml

R.K. Bhatia et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
91
Fig. 1. Effect of different buffer system and pH on acyl transfer activity of P. putida BR-1.
3.1.4. Incubation temperature
exposed to 60 ◦C exhibited very rapid loss in acyl transfer activity.
The half life of the enzyme at 50 ◦C was 3 h and at 60 ◦C it was 1 h
(Fig. 5).
Amidase of this organism exhibited very little acyl transfer activ-
ity 6.74 0.02 U/mg dcm at 10 ◦C. Above 10 ◦C, a steady increase
in the activity was observed with the rise in temperature up to
50 ◦C. Maximum acyl transfer activity 124.67 0.02 U/mg dcm of P.
putida BR-1 was recorded at 50 ◦C (Fig. 3). At higher temperature
i.e. 80 ◦C there was drastically decrease in acyl transfer activity and
only 5.10 0.03 U/mg dcm activity could be observed.
3.2. Bioprocess development for the conversion of nicotinamide
to nicotinyl hydroxamic acid
On the basis of result of the preceding optimization of reaction
parameters, the acyl transfer activity of amidase from P. putida BR-1
was used for the synthesis of nicotinyl hydroxamic acid.
3.1.5. Effect of substrate combinations
Enzymatic synthesis of hydroxamic acid is a bi-substrate reac-
tion, therefore our main aim was to get the best combination of
both substrates for maximum activity and bioconversion.
It has been observed that when substrate and co-substrate were
used in 1:5 ratio (200 mM nicotinamide and 1000 mM hydroxy-
lamine), maximum acyl transfer activity 128.34 0.05 U/mg dcm
was achieved and this combination proved to be best of all the
combinations tested (Fig. 4).
3.2.1. Time course of nicotinamide conversion to nicotinyl
hydroxamic acid with increase in substrate and resting cell
concentrations
Substrate and resting cell concentrations were varied from
100 mM: 5.0 U/ml to 500 mM: 25 U/ml, whereas the concentration
of hydroxylamine was kept constant i.e. 1000 mM. The reaction
mixture containing 10 U/ml and 200 mM nicotinamide as substrate
showed highest conversion rate and yield of nicotinyl hydroxamic
acid (186 mM) in 1 h and 20 min. On varying the substrate and
cell concentration the percentage yield of NHA was observed i.e.
100 mM – 70% yield, 200 mM – 93% yield, 300 mM – 80% yield,
400 mM – 78% yield, 500 mM – 75% yield (Fig. 6). The high con-
centration of substrate had inhibitory effect on the formation of
nicotinyl hydroxamic acid, though the enzyme concentration in the
reaction was increased proportionally.
3.1.6. Stability profile of acyl transfer activity of P. putida BR-1
stored at different temperatures
It has been found that the resting cells of P. putida BR-1 stored
at 4 ◦C and 15 ◦C retained acyl transfer activity for a long time (71%
after 10 h), but the cells at 45 ◦C retained only 50% activity after
10 h. Higher temperature exhibited fast decrease in acyl transfer
activity as compared to the cells stored at 4 ◦C and 15 ◦C. The cells
Fig. 2. Effect of biocatalyst concentrations on acyl transfer activity of amidase from P. putida BR-1.

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R.K. Bhatia et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
Fig. 3. Effect of different temperatures on acyl transfer activity of P. putida BR-1.
Fig. 4. Effect of different combinations of nicotinamide and hydroxylamine concentration on acyl transfer activity of P. putida BR-1.
3.2.2. Effect of resting cell concentration on conversion of
200 mM of nicotinamide to nicotinyl hydroxamic acid
Optimum resting cell concentration of P. putida BR-1 for
maximum conversion of 200 mM nicotinamide and 1000 mM
hydroxylamine was assessed by varying the resting cell amount
equivalent to 5.0–25.0 U/ml of acyl transfer activity. On increasing
the amount of biocatalyst (resting cell), conversion rate of substrate
to product also increased. Highest conversion of nicotinamide and
hydroxylamine into nicotinyl hydroxamic acid (188 mM) in 30 min
was observed with resting cells having 20 U/ml acyl transfer activ-
ity. While, resting cells having (15 U/ml) acyl transfer activity
took 50 min for the production of NHA. When there is almost full
Fig. 5. Stability profile of acyl transfer activity of P. putida BR-1 stored at different temperatures.

R.K. Bhatia et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
93
Fig. 6. Effect of increasing cell concentration on varied substrate concentration.
conversion or almost no substrate left, it seems that undesir-
able reactions become operational. Therefore, resting cell having
20 U/ml acyl transfer activity was used for conversion of nicotin-
amide into nicotinyl hydroxamic acid (Fig. 7).
hydroxylamine, 100 mM sodium phosphate buffer (pH 7.5), and
resting cell equivalent to 20 U/ml. The temperature and impeller
speed of the fermenter was maintained at 50 ◦C and 160 rpm
respectively. At the end of the reaction 190 mM nicotinyl hydrox-
amic acid (95% molar conversion yield) was produced, it was 1%
more than that achieved at 50 ml scale and this gain due to the
change in kinetics with the change in reaction volume moreover
enzyme and substrate has more interaction surface area at 1 L.
3.2.3. Fed batch reaction at 50 ml scale (200 mM nicotinamide
and 1000 mM hydroxylamine per feed)
In the fed batch reaction after 30 min (i.e. before second feed-
ing of substrates) 188 mM nicotinyl hydroxamic acid was formed
from 200 mM nicotinamide with 94% conversion without any side
product but after 1 h (i.e. before third feeding of substrates) out of
400 mM of nicotinamide, 256 mM of NHA and 2 mM of nicotinic
acid (side product) was formed. Further feeding did not resulted in
significant enhancement of nicotinyl hydroxamic acid production
(data not shown). This might be due to product inhibition, when
more enzyme gets saturated and is unable to convert the substrate
into product, resulting in longer reaction times and unwanted side
reactions.
3.2.5. Recovery of nicotinyl hydroxamic acid
After completion of the reaction, the resting cells were separated
from the reaction by centrifugation at 10,000 × g for 15 min. In 1 L
batch mode reaction mixture 16 g of nicotinyl hydroxamic acid was
recovered after solvent extraction. This powder was found 98% pure
without any side product in HPLC analysis (Fig. 8). P. putida BR-1
biotransformed nicotinamide into nicotinyl hydroxamic acid with
13 g/h/dcm catalytic productivity and 32 g/L/h volumetric produc-
tivity.
3.2.4. Conversion of nicotinamide and hydroxylamine to nicotinyl
hydroxamic acid at bench scale (1 L)
Batch reaction was carried out at 1 L scale for 30 min. The
reaction mixture consisted of 200 mM nicotinamide, 1000 mM
3.2.6. Recycling of recovered cells
After bench scale reaction, 80% of resting cells were
recovered, which showed 92% residual acyl transfer activity
126.14 0.03 U/mg dcm. When these cells were again used in 50 ml
Fig. 7. Effect of resting cell concentration on 200 mM nicotinamide conversion into nicotinyl hydroxamic acid (solid line = nicotinyl hydroxamic acid and dotted
line = nicotinamide).

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R.K. Bhatia et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
Fig. 8. HPLC analysis of purified nicotinyl hydroxamic acid RT: 4.35 (nicotinamide RT: 3.60 min, and nicotinic acid RT: 6.45 min).
reaction mixture in the next experiment, 84% conversion of 200 mM
nicotinamide to nicotinyl hydroxamic acid was achieved.
at 50 ◦C similar to G. pallidus RAPc8 [18] reported from thermal
habitat.
In all the different combinations when the co-substrate
hydroxylamine is present in lower concentration in the reaction
mixture the conversion rate is very slow and hydrolysis of amide
takes place at a faster rate due to the lack of an adequate amount of
a strong nucleophile (hydroxylamine). However, as the concentra-
tion of co-substrate increased in comparison to substrate, the rate of
formation of nicotinyl hydroxamic acid also increased and undesir-
able reactions not observed. Fournand et al. [10] have also reported
that the increase in concentration of hydroxylamine (10–500 mM)
considerably reduced the undesirable hydrolysis of amide as well
as hydroxamic acid. As it was clear from all earlier studies (Table 1)
that hydroxylamine is required in excess, so that acyl group of
amide transferred more rapidly on hydroxylamine for the forma-
tion of respective hydroxamic acid [9]. Moreover, instant use of
multiple ratio of nicotinamide (400 mM) and co-substrate hydroxy-
lamine (2000 mM) might cause substrate inhibition and reduced
the conversion rate.
4. Discussion
The amidase of P. putida BR-1 showed acyl transfer activ-
ity through a wide range (5.0–10.0) of pH in different buffer
system, but maximum acyl transfer activity was recorded in
100 mM sodium phosphate buffer of pH 7.5. Acyl transfer activ-
ity in citrate buffer below pH 5.0 was not observed, which might
be due to denaturation of enzyme in acidic pH. Beyond opti-
mum pH (7.5), and at higher buffer molarity (>100 mM) a decline
in acyl transfer activity was observed because these elevated
buffers molarity and pH are not favoring the ionic conditions
responsible for the formation of enzyme-substrate complex at
faster rate which is quite necessary for the product formation.
Most of the amidases reported so far, are active around neutral
pH. A comparative analysis of acyl transfer activity of amidases
from different organisms against P. putida BR-1 showed that this
amidase is more or less similar to earlier reported amidases
(Table 1).
P. putida BR-1 exhibited highest acyl transfer activity of ami-
dase at 50 ◦C for the production of nicotinyl hydroxamic acid
similar to other mesophilic enzymatic systems. This temperature
supported the effective collisions between the enzyme and sub-
strate, which will lead to higher acyl transfer activity, but as
the temperature increased above from its optimum value (50 ◦C),
the non-covalent bonds holding the amino acids at active site of
enzyme starts breaking due to high kinetic energy. This changes
the 3D structure of the enzyme, which ultimately leads to loss of
activity [17]. This organism showed highest acyl transfer activity
The half life of amidase of P. putida BR-1 was 3 h at 50 ◦C and 1 h
at 60 ◦C. The amidase of Geobacillus pallidus RAPc8 showed half life
5 h at 50 ◦C whereas G. subterraneus RL-2a exhibited t1/2 for 9 h at
60 ◦C [18,19]. The amidase from this organism have lesser stability
at higher temperature than thermophilic strains that might be due
to absence of charged amino acids (Arg, Lys, His, Asp, Glu) in the
polypeptide chain [20].
It was observed that fed batch mode of reaction was not so effi-
cient, as there was no absolute biotransformation in each feeding
this might be due to product inhibition or enzyme get saturated and
unable to convert the substrate into product moreover hydrolysis of
amide and hydroxamic acid to nicotinic acid also get operational in
Table 1
Comparative analysis of different properties of P. putida BR-1 with earlier studies.
S. no.
Parameter
P. putida BR-1
Rhodococcus sp.
Alcaligenes sp.
Bacillus smithii
Bacillus sp.
Rhodococcus
G. pallidus BTP-5x
R312 [10]
MTCC 10674 [11]
strain IITR6b2 [12]
APB-6 [14]
erythropolis [15]
MTCC 9225 [16]
1
Buffer and pH
Sodium
Sodium
phosphate,
20 mM, 7.0
Potassium
phosphate,
100 mM, pH 7.0
Potassium
phosphate,
100 mM, pH 7.0
Glycine-NaOH
100 mM, pH 7.5
Potassium
phosphate,
50 mM, pH 7.0
Sodium
phosphate,
100 mM, pH 7.0
phosphate,
100 mM, pH
7.5
2
Reaction
temperature
Substrate
Amide and
hydroxyl
amine
50 ◦
C
30 ◦
C
50 ◦
C
30 ◦
C
45 ◦
C
30 ◦
C
65 ◦
C
3
4
Nicotinamide
200 mM
1000 mM
Nicotinamide
50 mM
100 mM
Benzamide
100 mM
200 mM
Nicotinamide
200 mM
250 mM
Acetamide
300 mM
800 mM
Acetamide
100 mM
200 mM
Acetamide
100 mM
500 mM
5
6
Conversion
rate
Scale up
95%
1 L
40%
66%
94.5%
50 ml
94%
1 L
–
93%
1 L
Test tube scale
500 ml
Test tube scale

R.K. Bhatia et al. / Journal of Molecular Catalysis B: Enzymatic 108 (2014) 89–95
95
long duration reaction. Agarwal et al. [12] reported the production
of nicotinic acid hydroxamate using acyl transfer activity of Bacil-
lus smithii strain IITR6b2 at moderate temperature (30 ◦C), and only
at 50 ml scale with 86.4% purity along with side product i.e. nico-
tinic acid but the present study resulted into production of nicotinyl
hydroxamic acid significantly with high purity and at elevated tem-
perature. P. putida BR-1 with 20 U/ml activity cells in 1 L batch mode
reaction produced 16 g of nicotinyl hydroxamic acid with 32 g/L/h
volumetric productivity.
Mehta. The computational facility availed at Sub-Distributed
Information Centre (SDIC), H.P University, Shimla, is also duly
acknowledged.
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P. putida BR-1 has emerged as a novel source for transformation
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amine to nicotinyl hydroxamic acid as it exhibited acyl transfer
activity at high temperature (50 ◦C) with substrate and product tol-
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mode till date. The bench scale (1 L scale) production of nicotinyl
hydroxamic acid using resting cells of P. putida BR-1 (containing
acyl transfer activity) showed 13 g/h/dcm catalytic productivity and
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Acknowledgements
Authors are highly grateful to University Grant Commission
(UGC) New Delhi and Department of Biotechnology (DBT) Ministry
of Science and Technology New Delhi, India for providing finan-
cial assistance in the form of Senior Research Fellowship to Mr.
Ravi Kant Bhatia, Mr. Shashi Kant Bhatia and Mr. Praveen Kumar
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