Solvent free biocatalytic synthesis of isoniazid from isonicotinamide using whole cell of Bacillus smithii strain IITR6b2

Article abstract of DOI:10.1016/j.molcatb.2013.07.010

A biocatalytic route for the synthesis of isoniazid, an important first-line antitubercular drug, in aqueous system is presented. The reported bioprocess is a greener method, does not involve any hazardous reagent and takes place under mild reaction conditions. Whole cell amidase of Bacillus smithii strain IITR6b2 having acyltransferase activity was utilized for its ability to transfer acyl group of isonicotinamide to hydrazine-2HCl in aqueous medium. B. smithii strain IITR6b2 possessed 3 folds higher acyltransferase activity as compared to amide hydrolase activity and this ratio was further improved to 4.5 by optimizing concentration of co-substrate hydrazine-2HCl. Various key parameters were optimized and under the optimum reaction conditions of pH (7, phosphate buffer 100 mM), temperature (30 C), substrate/co-substrate concentration (100/1000 mM) and resting cells concentration (2.0 mg _dcw/ml), 90.4% conversion of isonicotinamide to isoniazid was achieved in 60 min. Under these conditions, a fed batch process for production of isoniazid was developed and resulted in the accumulation of 439 mM of isoniazid with 87.8% molar conversion yield and productivity of 6.0 g/h/g _dcw. These results demonstrated that enzymatic synthesis of isoniazid using whole cells of B. smithii strain IITR6b2 might present an efficient alternative route to the chemical synthesis procedures without the involvement of organic solvent.

Full text of DOI:10.1016/j.molcatb.2013.07.010

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 67–73
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Solvent free biocatalytic synthesis of isoniazid from isonicotinamide
using whole cell of Bacillus smithii strain IITR6b2
∗
Shilpi Agarwal, Meenu Gupta, Bijan Choudhury
Bioprocess Engineering Laboratory, Department of Biotechnology, Indian Institute of Technology, Roorkee 247667, Uttarakhand, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2013
Received in revised form 27 June 2013
Accepted 13 July 2013
Available online 7 August 2013
A biocatalytic route for the synthesis of isoniazid, an important first-line antitubercular drug, in aqueous
system is presented. The reported bioprocess is a greener method, does not involve any hazardous reagent
and takes place under mild reaction conditions. Whole cell amidase of Bacillus smithii strain IITR6b2
having acyltransferase activity was utilized for its ability to transfer acyl group of isonicotinamide to
hydrazine–2HCl in aqueous medium. B. smithii strain IITR6b2 possessed 3 folds higher acyltransferase
activity as compared to amide hydrolase activity and this ratio was further improved to 4.5 by optimizing
concentration of co-substrate hydrazine–2HCl. Various key parameters were optimized and under the
Keywords:
Isoniazid
Hydrazinolysis
Bacillus smithii strain IITR6b2
Acyltransferase activity
Amidase
◦
optimum reaction conditions of pH (7, phosphate buffer 100 mM), temperature (30 C), substrate/co-
substrate concentration (100/1000 mM) and resting cells concentration (2.0 mgdcw/ml), 90.4% conversion
of isonicotinamide to isoniazid was achieved in 60 min. Under these conditions, a fed batch process for
production of isoniazid was developed and resulted in the accumulation of 439 mM of isoniazid with
8
7.8% molar conversion yield and productivity of 6.0 g/h/gdcw. These results demonstrated that enzymatic
synthesis of isoniazid using whole cells of B. smithii strain IITR6b2 might present an efficient alternative
route to the chemical synthesis procedures without the involvement of organic solvent.
©
2013 Elsevier B.V. All rights reserved.
1
. Introduction
multistep, expensive, hazardous, energy intensive and can lead to
decomposition of desired product.
Hydrazides (R-CO NHNH ), the acylated derivatives of
Yadav et al., [10] explored the possibility of replacing the chem-
ical methods by the enzymatic route, working in non-aqueous
media and under milder reaction conditions using lipase as cat-
alyst. This process resulted in 52% conversion in 24 h. They further
improved method by using microwave irradiation with immobi-
lized lipase and process led to a conversion of 54% in 4 h [11].
1,4-Dioxane, solvent used in lipase catalysed synthesis is highly
flammable and combines with atmospheric oxygen to form explo-
sive peroxide on prolonged exposure to air. It is a known carcinogen
to animals and is classified as a possible carcinogen to human
[12–14]. Hence an enzymatic synthesis method with high conver-
sion rate in aqueous medium will provide an economic and greener
approach for isoniazid synthesis. Amidases [EC 3.5.1.4] are widely
distributed enzymes in nature, with diverse applications [15,16].
The acyltransferase activity of amidase results in transfer of acyl
group of amide to co-substrate hydroxylamine or hydrazine in
aqueous medium [17–19] and it has been applied for synthesis of
few hydroxamic acids [20–25]. Although the ability of amidase to
transfer acyl group of amide to hydrazine can be an efficient alter-
native for biocatalytic synthesis of hydrazides [18,19], surprisingly
bioprocess development for hydrazides synthesis using amidase
has not been explored till now. To the best of our knowledge,
hydrazinolysis of isonicotnamide by amidase has not been applied
2
hydrazine are important precursor and intermediates in the
synthesis of certain amides, heterocyclic compounds, aldehydes,
pharmaceuticals and polymers. These are reported to possess
antitumor, antibacterial, anticancer, anticorrosive and anti-
inflammatory activities [1–3]. Isonicotinic acid hydrazide, also
known as isoniazid (INH) is the first-line antitubercular drug
and is given in combination with rifampicin and ethambutol
[
4,5]. It is a prodrug and must be activated by KatG enzyme
of Mycobacterium tuberculosis. Isoniazid inhibits the synthesis of
mycolic acid, which is an essential component of the mycobacterial
cell wall [6]. Chemical methods reported for isoniazid synthesis
involves hydrazinolysis of 4-cyanopyridine, isonicotinamide or
ethyl isonicotinate with hydrazine hydrate in aqueous or alcoholic
◦
solution in presence of sodium hydroxide at 100 C under reflux
conditions. These reactions are spontaneous, exothermic and com-
plete in 4–9 h [7–9]. 4-Cyanopyridine is prepared from expensive
-picoline whereas less reactive ester requires longer reaction
␥
time (from few hours to 2–3 days). Generally these reactions are
∗ Corresponding author. Tel.: +91 1332 285297; fax: +91 1332 273560.
E-mail address: bijanfbs@iitr.ernet.in (B. Choudhury).
1
381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.07.010

6
8
S. Agarwal et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 67–73
yet for the preparative scale synthesis of isoniazid. The current work
focused on the synthesis of isoniazid from isonicotinamide and
hydrazine–2HCl in aqueous medium using acyltransferase activity
of whole cell amidase of Bacillus smithii strain IITR6b2. Whole cells
possessed higher acyltransferase to amide hydrolase activity ratio
and this ratio was further improved by optimizing hydrazine–2HCl
concentration. Various reaction parameters as temperature, pH,
substrate/co-substrate concentration, resting cells concentration
and time course of reaction were optimized and a fed batch process
was developed.
HPLC analysis of isoniazid. Reaction mixture without resting cells
was also tested for any possible spontaneous chemical synthesis
of isoniazid. One unit of acyltranferase activity was defined as that
amount of resting cells (mg dry cell = mgdcw) which catalysed the
formation of one ␮mol of isoniazid in 1 min under the assay con-
ditions.
2.5. Amide hydrolase activity assay
The amide hydrolase activity assay was performed in a reaction
mixture (1 ml) of the following composition – 800 ␮l of isonicoti-
namide solution (125 mM in 50 mM phosphate buffer, pH 7), 100 ␮l
of 50 mM phosphate buffer and 100 ␮l of resting cell (1.0 mgdcw/ml)
in phosphate buffer (50 mM, pH 7). Final concentration of isoni-
cotinamide in reaction mixture was 100 mM. Control reaction was
conducted in the absence of enzyme. After incubation of reaction
2
. Materials and methods
2.1. Chemicals
Isonicotinamide and hydrazine–2HCl were purchased from
◦
Sigma Aldrich chemicals Pvt. Ltd. (USA). Isoniazid was obtained
from Loba Chemie Pvt. Ltd. (India). The culture media components
were obtained from S. D. Fine Chem limited (India). All other chem-
icals were of analytical or HPLC grade as per requirement, procured
from various commercial sources.
mixture at 45 C for 20 min, the reaction was terminated by the
addition of 10 ␮l of 2 N HCl and cells were removed by centrifuga-
tion at 10,000 × g for 8 min. Supernatant was collected for HPLC
analysis of isonicotinic acid (INA). One unit of amide hydrolase
activity was defined as that amount of resting cells (mgdcw) which
catalysed the formation of one ␮mol of isonicotinic acid in 1 min
under the assay conditions.
2.2. Microorganism
B. smithii strain IITR6b2 was previously isolated from soil sample
2.6. Hydrazide hydrolase activity assay
and preserved in our laboratory. This amidase producing strain with
a high amide acyltransferase activity towards heterocyclic amides
was used in the present study. Strain was identified by morpho-
logical, biochemical tests and 16S rDNA gene sequencing analysis
in our previous reported work [26]. The 16S rRNA sequence has
been deposited in NCBI GenBank database with accession number
JX157878.
The hydrazide hydrolase activity assay was performed in a
reaction mixture (1 ml) of the following composition – 800 ␮l of iso-
niazid solution (125 mM in 50 mM phosphate buffer, pH 7), 100 ␮l
of 50 mM phosphate buffer and 100 ␮l of resting cell (1.0 mgdcw/ml)
in phosphate buffer (50 mM, pH 7). Final concentration of isoniazid
in reaction mixture was 100 mM. Control reaction was conducted
in the absence of enzyme. After incubation of reaction mixture
◦
2.3. Culture media and growth conditions
at 45 C for 20 min, the reaction was terminated by the addition
of 10 ␮l of 2 N HCl and cells were removed by centrifugation at
10,000 × g for 8 min. Supernatant was collected for HPLC analysis
of isonicotinic acid. One unit of hydrazide hydrolase activity was
defined as that amount of resting cells (mgdcw) which catalysed
the formation of one ␮mol of isonicotinic acid in 1 min under the
assay conditions.
B. smithii strain IITR6b2 was cultured in the mineral base
medium having following composition (g/l): tri sodium citrate 0.2,
glycerol 10, K HPO 0.87, KH PO4 1.35; 10× mineral base (g/l)
2
4
2
(
NaCl 10, CaCl2 0.1, MgSO ·7H O 2) and 1 ml/l trace elements
4
2
solution (g/l) (H BO 0.3, Na MoO ·H O 0.03, NiCl ·6H O 0.03,
3
3
2
4
2
4
2
CoCl ·6H O 0.2, ZnSO ·7H O 0.2, CuCl ·2H O 0.01 and MnCl ·4H O
2
2
4
2
2
2
2
2
0
.1). 10 mM phenylacetonitrile was added in the sterilized mineral
2.7. Analytical method
base medium (pH 7) as a sole source of nitrogen. To prepare inocula,
the organism was grown aerobically in 50 ml of the sterile medium
in a 250 ml Erlenmeyer flask for 36 h at 45 C and 200 rpm in an incu-
bator shaker. The inocula (0.5% v/v) was added into 100 ml of same
medium in a 500 ml Erlenmeyer flask and incubated under similar
conditions. The bacterial cells were harvested at mid-exponential
HPLC analysis of substrate, product and by product were
carried out by Varian Prostar HPLC with waters spherisorb®
10 ␮m ODS2, 4.6 × 250 mm analytical column. The standards plots
of commercially available isoniazid, isonicotinic acid and ison-
icotinamide were prepared. The analysis was carried out at a
flow rate of 1.0 ml/min at 210 nm using 20 mM KH2PO4 with
5% (v/v) acetonitrile as mobile phase. 20 ␮l of the sample was
injected.
◦
phase of growth (OD
for 12 min at 4 C and washed twice with 100 mM phosphate buffer
= 0.6–0.9) by centrifugation at 10,000 × g
6
00
◦
(
pH 7). Bacterial cells were suspended in same buffer and referred
as resting cells.
2.8. Determination of effects of temperature and pH on
2.4. Acyltransferase activity assay
acyltransferase activity
The whole cell acyltransferase activity assay was performed in
The effect of temperature on whole cell acyltransferase
activity was determined in 1 ml reaction mixture for a tem-
perature range of 25–70 C under assay conditions (100 mM
isonicotinamide, 500 mM hydrazine–2HCl, 50 mM phosphate
buffer, pH 7) for 15 min. The optimum pH for the reaction
a reaction mixture (1 ml) having following composition – 400 ␮l
of isonicotinamide solution (250 mM in 100 mM phosphate buffer,
pH 7), 500 ␮l of hydrazine–2HCl solution (1000 mM in distilled
water, freshly neutralized with 10.0 N NaOH) and 100 ␮l of res-
ting cells (1 mgdcw/ml) in phosphate buffer (100 mM, pH 7). Final
concentrations of isonicotinamide and hydrazine–2HCl in reaction
mixture were 100 and 500 mM respectively. After incubation of
◦
was determined for
a pH range of (4.0–10.0) in following
buffers (50 mM): sodium acetate buffer (pH 4.0–5.8), potas-
sium phosphate buffer (pH 5.8–8.0), borate buffer (pH 8.0–9.2),
and sodium carbonate buffer (pH 9.2–10.0) under standard
assay conditions. The effect of molarity of potassium phos-
phate buffer at optimum pH 7 was studied for a molarity range
◦
reaction mixture at 45 C for 20 min, the reaction was stopped by
adding 10 ␮l of 2 N HCl and cells were removed by centrifuga-
tion at 10,000 × g for 8 min. Clear supernatant was collected for

S. Agarwal et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 67–73
69
of 25–500 mM under assay conditions. Thereafter all further
studies were carried out at 100 mM phosphate buffer of pH
7
.
2.9. Optimization of substrate and co-substrate concentrations
for acyltransferase activity
To study the effect of isonicotinamide concentration on amide
acyltransferase activity, varied concentrations of isonicotinamide
(
10–500 mM) at constant hydrazine–2HCl concentration (500 mM)
◦
were used under standard assay conditions (55 C, phosphate buffer
1
00 mM, pH 7, 15 min). Effect of hydrazine–2HCl concentration on
acyltransferase activity and by product formation was studied in
the range of 0–1500 mM keeping isonicotinamide concentration
constant (100 mM).
Fig. 1. Enzymatic reactions during Bacillus smithii strain IITR6b2 catalysed isoniazid
synthesis.
2.10. Effect of reaction temperature on bioconversion
3. Results and discussion
To evaluate the effect of temperature on molar conversion of
isonicotinamide to isoniazid and by product formation, enzymatic
reactions with optimized substrate/co-substrate concentrations
3.1. Enzymatic reactions during B. smithii strain IITR6b2
catalysed isoniazid synthesis
(
4
1
100/1000 mM) were performed at various temperatures (30, 35,
5 and 55 C) in 25 ml of 100 mM phosphate buffer (pH 7) using
.0 mgdcw/ml resting cells. Samples were taken at interval of 15 min
◦
Amidase is capable of catalysing transfer of acyl group of
amide, acid and ester to hydroxylamine and water. This trans-
fer of acyl group to hydroxylamine resulted in hydroxamic acid
synthesis whereas transfer to water resulted in acid synthesis
for 120 min for HPLC analysis.
[
17,18,20]. Few amidases have also been reported to catal-
2
.11. Effect of resting cells on conversion and time course
yse the acyl group transfer from amides to hydrazine, leading
to synthesis of acid hydrazides [18,19]. During amidase catal-
ysed hydrazide synthesis an acyl enzyme complex is formed
which is subjected to nucleophilic attack by either co-substrate
hydrazine or water leading to hydrazide or acid synthesis respec-
tively. [18]. Whole cell of B. smithii strain IITR6b2 possessed
amide hydrolase (0.87 ± 0.05 U/mgdcw) and amide acyltransferase
Cell concentration varying from 1.0 to 2.5 mgdcw/ml were
used to find out the optimum concentration of whole cells and
time course of bioconversion of isonicotinamide to isoniazid
under optimized temperature (30 C) and substrate/co-substrate
◦
(
(
100/1000 mM) concentration in 25 ml of 100 mM phosphate buffer
pH 7).
(
2.68 ± 0.24 U/mg
) activities but not acid acyltransferase activ-
dcw
ity. Difference in these two activities might be due to the different
affinity of hydrazine and water for acyl enzyme complex and
hydrazine being a more potent nucleophile (due to ␣ effect) attacks
acyl enzyme complex faster than water. Rate of transfer of acyl
group of isonicotinamide to hydrazine (500 mM) was 3 folds higher
as compared to amide hydrolase reaction rate. Higher the ratio
of acyltranferase to hydrolase activity lesser the by product (iso-
nicotinic acid) will be formed. Thus the synthesis of isonicotinic acid
will be limited during the process. One of the objectives of this work
was to explore the synthesis of isoniazid by whole cell amidase with
minimum formation of by product, isonicotinic acid. Whole cells of
B. smithii strain IITR6b2 also showed hydrazide hydrolase activ-
ity (0.15 ± 0.012 U/mgdcw) when isoniazid was used as substrate
in absence of hydrazine–2HCl but it was only 5.6% of amide acyl-
transferase activity. Kobayashi et al., [19] also reported benzoic acid
hydrazide hydrolase activity catalysed by amidase of Rhodococ-
cus rhodochrous J1. Based on these activities, the possible reaction
mechanism during whole cells catalysed isoniazid synthesis from
isonicotinamide is shown in Fig. 1.
2
.12. Fed batch biotransformation at 50 ml scale
Fed-batch biotransformation was carried out in 250 ml Erlen-
meyer flask containing 50 ml of reaction mixture with initial
isonicotinamide and hydrazine–2HCl concentration of 100 and
1
and 2.0 mgdcw/ml resting cells of B. smithii strain IITR6b2. Powdered
isonicotinamide (0.61 g) and highly concentrated solution (1 ml,
5
at an interval of 60 min to restrict the residual isonicotinamide and
hydrazine–2HCl concentration above 100 and 1000 mM respec-
tively. 500 ␮l of sample was withdrawn at every 30 min during the
reaction and monitored for isoniazid, isonicotinic acid and isoni-
cotinamide concentration. Effort was made to maintain the reaction
volume constant around 50 ml. A control experiment was also con-
ducted with same parameters without enzyme for any spontaneous
chemical reaction.
◦
000 mM respectively in phosphate buffer (100 mM, pH 7) at 30 C
M, pH 7) of hydrazine–2HCl were fed in subsequent seven feeds
2.13. Recovery of isoniazid
3.2. Effect of temperature and pH on acyltransferase activity
A fed batch process was performed at 50 ml scale and after 5 h
To study the influence of reaction temperature on acyltrans-
ferase activity the reaction was carried out at various temperature
reaction was stopped by centrifugation at 10,000 × g for 20 min at
◦
◦
4
C. The supernatant obtained was freeze-dried to recover white
range (25–70 C). As depicted in Fig. 2 maximum acyltransferase
◦
powder. Isoniazid was further purified by extraction of this powder
with 120 ml of methanol for 1 h. The methanolic solution was fil-
tered, evaporated and obtained powder was analysed by HPLC for
its purity.
activity was obtained at 55 C, however no significant increase in
activity with temperature was observed at reaction temperature
◦
◦
from 45 to 55 C. Above 55 C, a sharp decrease in activity was
observed due to thermal inactivation of whole cell enzyme. At

7
0
S. Agarwal et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 67–73
Fig. 4. Effect of substrate (isonicotinamide) concentration on acyltransferase activ-
Fig. 2. Effect of temperature on acyltransferase activity. Reaction conditions: isoni-
ity. Reaction conditions: isonicotinamide (10–500 mM), hydrazine–2HCl (500 mM),
cotinamide (100 mM), hydrazine–2HCl (500 mM), phosphate buffer (50 mM, pH 7),
◦
phosphate buffer (100 mM, pH 7), 55 C, 0.1 mgdcw/ml resting cells. The data are
◦
temperature (25–70 C), 0.1 mgdcw/ml resting cells. The data are mean ± SD, where
mean ± SD, where n = 3.
n = 3.
◦
2
5 and 70 C relative acyltransferase activities were 56% and 51%
hydrolysis). Thus there was
a need to study the effect of
respectively.
substrate/co-substrate concentrations on acyltransferase activity
and optimize their concentrations. In first set of experiments, con-
centration of isonicotinamide was varied from 10-500 mM keeping
the concentration of hydrazine–2HCl constant (500 mM). Rise in
acyltransferase acivity was observed as the concentration of isoni-
cotinamide increased from 10 to 100 mM with maximum acyltrans-
ferase activity achieved at 100 mM of isonicotinamide. Increasing
the amide concentration increased acyltransferase activity because
of more acyl-enzymes complex formation. A further increase in
the amide concentration led to decrease in the enzyme activ-
ity and 76% reduction in acyltransferase activity was observed
at 500 mM suggesting the inhibition effect of substrate (amide)
The effect of reaction pH on acyltransferase activity was investi-
gated in a pH range of 4–10 using various buffers (50 mM). Results
suggested that the whole cell enzyme was active at narrow range
and activity increased gradually with increasing pH reaching the
maximum at pH 7 (Fig. 3). It was found that it retain 69% and
4
2% of its maximum activity at pH 6 and 10 respectively. The
molarities of potassium phosphate buffer at optimum pH 7 were
varied from 25 to 500 mM to find out its optimum value. Potassium
phosphate buffer of 100 mM (pH 7) was found to show maximum
acyltransferase activity (Supplementary Fig. S1). In earlier studies,
the highest acyl transfer activity of amidase of R. rhodochrous J1 for
benzoic acid hydrazide synthesis was reported at pH 8.5 [19].
(
Fig. 4). The kinetic constants for isonicotinamide substrate were
−
1
−1
Km,isonicotinamide = 4.5 mM and Vmax = 2.87 ␮mol min mg
resting cells of B. smithii strain IITR6b2.
of
dcw
3.3. Optimization of substrate and co-substrate concentrations
for acyltransferase activity
The effect of hydrazine–2HCl concentration on acyltransferase
activity was studied at various hydrazine–2HCl concentrations
B. smithii strain IITR6b2 catalysed hydrazinolysis of isonicoti-
namide which is a bi-substrate reaction involving an acyl group
donar (isonicotinamide) and acyl group acceptor (hydrazine–2HCl).
It was expected that concentrations of both substrates may
influence the acyltransferase activity and side reaction (amide
(0–1500 mM) keeping isonicotinamide concentration (100 mM)
constant. It was reported that higher concentration of co-
substrate was inhibitory to hydrolysis activity [18]. Hence effect
of hydrazine–2HCl concentration on by product formation was
also studied. Higher hydrazine–2HCl concentration resulted in
increased acyltransferase activity up to 1000 mM and above
this concentration decline in activity was observed due to inhi-
bition by hydrazine–2HCl (Fig. 5). The acyltransferase activity
was highest at 1000 mM (4.30 ± 0.33 U/mgdcw). The kinetic con-
stant for hydrazine substrate was K
= 1318 mM and
m,hydrazine
−
1
−1
Vmax = 9.8 ␮mol min mg
dcw
of resting cells of B. smithii strain
IITR6b2.
Higher concentration of co-substrate was found to be inhibitory
to hydrolysis of amide and thus by product formation decreased
with increasing hydrazine–2HCl concentration (data not shown).
At optimum concentrations of isonicotinamide (100 mM) and
hydrazine–2HCl (1000 mM), acyltransferase to hydrolase activity
ratio further improved from 3 to 4.5 and corresponding reduction
in by product formation was 54%. Fournand et al., [18] reported
2
6% of residual hydrolysis with propionamide substrate at 800 mM
hydrazine concentration. This reduction in hydrolysis might be due
to the fact that hydrazine being a more potent nucleophile than
water had higher affinity for acyl enzyme complex and consid-
erably reduced the undesirable amide hydrolysis. In summary, a
high ratio of acyl acceptor (hydrazine–2HCl) to acyl donor (ison-
icotinamide) improved acyltransferase (hydrolzinolysis) reaction,
Fig. 3. Effect of pH on acyltransferase activity. Reaction conditions: isonicoti-
namide (100 mM), hydrazine–2HCl (500 mM), phosphate buffer (50 mM, pH 7),
◦
5
5
C, 0.1 mgdcw/ml resting cells. pH profile: acetate buffer (filled diamonds), phos-
phate buffer (filled squares), borate buffer (filled triangles) and carbonate buffer (filled
circles). The data are mean ± SD, where n = 3.

S. Agarwal et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 67–73
71
Fig. 5. Effect of co-substrate (hydrazine–2HCl) concentration on acyltransferase
activity. Reaction conditions: isonicotinamide (100 mM), hydrazine–2HCl (0-
Fig. 6. Effect of reaction temperature on conversion and by product formation.
Reaction conditions: isonicotinamide (100 mM), hydrazine–2HCl (1000 mM), 25 ml
phosphate buffer (100 mM, pH 7), 1.0 mgdcw/ml resting cells, temperatures (30, 35,
◦
1
500 mM), phosphate buffer (100 mM, pH 7), 55 C, 0.1 mgdcw/ml resting cells.
Relative acyltransferase activity (filled diamonds) and acyltransferase to amide
hydrolase activity ratio (filled squares). The data are mean ± SD, where n = 3.
◦
◦
◦
◦
4
5 and 55 C). Isoniazid (mM) at 30 C (filled diamonds), 35 C (open squares), 45 C
◦
◦
(
filled triangles) and 55 C (open circles), isonicotinic acid (mM) at 30 C (open dia-
◦
◦
◦
monds), 35 C (closed squares), 45 C (open triangles) and 55 C (closed circles). The
data are mean ± SD, where n = 3.
and suppressed hydrolysis. During the determination the optimum
concentrations of isonicotinamide and hydrazine–2HCl, only one
substrate concentration was varied whereas the other substrate
was kept constant. The possibility of determined concentrations of
isonicotinamide and hydrazine–2HCl may be suboptimal cannot be
ruled out. Thus to find the true optimum mole ratio of substrates
3.5. Effect of resting cells on conversion and time course of
reaction
The amount of biocatalyst used and time of process is crucial for
development of an economic bioprocess. The effects of whole cell
concentration on the molar conversion and time course of reaction
were studied in the range of 1.0–2.5 mgdcw/ml under optimized
reaction conditions. The concentration of by product (isonicotinic
acid) formed was also determined simultaneously. The conversion
rate increased with increase in cell concentration and attained a
conversion of 90.4% at 2.0 mgdcw/ml while the overall conversion
time reduced to 60 min from 90 min (Fig. 7). At higher cell con-
centration (2.5 mgdcw/ml), however the initial conversion rate was
marginally higher but maximum conversion achieved was nearly
same and no reduction in reaction time was observed. It indi-
cated that an excess amount of cells would not increase the molar
(
isonicotinamide and hydrazine–2HCl); reactions were performed
with several possible ratios of amide and hydrazine–2HCl. Highest
acyltransferase activity was obtained with 100 mM of isonicoti-
namide and 1000 mM of hydrazine–2HCl (Supplementary Fig. S2).
Therefore all the subsequent experiments were performed with
1
00 and 1000 mM of isonicotinamide and hydrazine–2HCl respec-
tively.
3
.4. Effect of reaction temperature on molar conversion and by
product formation
◦
It was already found that 55 C is the optimum temperature
for whole cell acyltransferase activity. The objective of this work
was to develop a biocatalytic process for optimum synthesis of iso-
niazid with minimum by product formation. Thus the effects of
temperature on molar conversion of isonicotinamide to isoniazid
and isonicotinic acid were studied for 120 min at various temper-
◦
atures (30, 35, 45 and 55 C). Initial conversion rates were found
◦
to increase with an increase in temperature from 30 to 55 C for
first 45 min (Fig. 6). However maximum conversion of 89.6% was
◦
achieved at 30 C in 90 min. There was a marginal difference in
◦
the rate of conversion at 45 and 55 C and maximum conversions
achieved were 81.9% and 78.7% respectively in 75 min and there-
after the concentration of isoniazid decreased with time probably
due to product hydrolysis. This lower conversion rate at higher tem-
perature can be explained by isonicotinic acid (by product) analysis.
By product formation rates were 1.8 and 1.6 folds higher at 55 and
◦
◦
4
5 C as compared to 30 C respectively. It may be due to higher
rate of side reactions (amide and hydrazide hydrolysis) at higher
temperatures. This study suggested that the final conversion of
isonicotinamide to isoniazid was higher at 30 C with less by prod-
Fig. 7. Effect of resting cells (DCW) on conversion and time course of reaction.
◦
Reaction conditions: isonicotinamide (100 mM), hydrazine–2HCl (1000 mM), 25 ml
◦
uct (7.6%) formation. Whole cell amidase of B. smithii strain IITR6b2
phosphate buffer (100 mM, pH 7), 30 C. Isoniazid (mM) at 1 mg/ml (filled diamonds),
◦
1.5 mg/ml (open squares), 2.0 mg/ml (filled triangles) and 2.5 mg/ml (open circles),
isonicotinic acid (mM) at 1 mg/ml (open diamonds), 1.5 mg/ml (closed squares),
was reported to be relatively stable at 30 C with half life of 29 h
◦
[
26]. As a result the optimal temperature was chosen to be 30 C
2
.0 mg/ml (open triangles) and 2.5 mg/ml (closed circles). The data are mean ± SD,
for bioprocess development.
where n = 3.

7
2
S. Agarwal et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 67–73
isonicotinic acid. Further purification was done by solvent extrac-
tion to recover isoniazid as white powdery solid (3.1 g) with 94%
purity.
4. Conclusions
Fed batch bioprocess for isoniazid synthesis has been suc-
cessfully developed for high molar conversion with limited side
reactions using acyltransferase activity of B. smithii strain IITR6b2.
By optimizing various parameters as hydrazine–2HCl concentra-
tion, temperature and resting cells concentration, reaction was
directed to the preferred hydrazinolysis hence synthesis of by prod-
uct (isonicotinic acid) was limited. During this fed batch process
after four subsequent feedings a molar conversion (87.8%) of ison-
icotinamide (500 mM) to isoniazid (439 mM) was achieved in 5 h
with only 7.8% by product, isonicotinic acid. Till now the biocat-
alytic route to isoniazid synthesis using lipase enzyme has been
reported, however this process occurs at a slow rate in 1,4-dioxane
and resulted in lower conversion (54%). The advantages of present
method include aqueous medium, higher conversion in shorter
reaction time and mild reaction conditions. These results suggest
that B. smithii strain IITR6b2 has potential for the commercial pro-
duction of isoniazid in future. These findings can contribute to
industrial production of isoniazid via a biocatalytic route. Further
improvements in process can be expected by immobilization of
biocatalyst.
Fig. 8. Time course of production of isoniazid during fed batch biotransformation
process. Reaction conditions: 50 ml phosphate buffer (100 mM, pH 7), 30 C, resting
cells concentration (2.0 mgdcw/ml). Isoniazid (mM) (filled triangles), isonicotinic acid
◦
(mM) (open squares) and isonicotinamide (mM) (filled circles), residual whole cell
activity (%) (filled squares). The data are mean ± SD, where n = 3.
conversion of isonicotinamide, which might be due to increase in
concentration of catalyst above the saturated substrate concentra-
tion or mass transfer limitations. A lower amount of the biocatalyst
and shorter reaction time will reduce the bioprocess development
cost. Hence whole cell concentration of 2.0 mgdcw/ml was used
for fed batch process development with intermittent addition of
substrates (100/100 mM) at 60 min interval.
Acknowledgement
The authors acknowledge the University Grant Commission,
New Delhi for financial support in the form of Senior Research
Fellowship to Ms. Shilpi agarwal.
3
.6. Fed batch bioprocess development at 50 ml scale
Acyltransferase activity of whole cell enzyme was inhibited
by isonicotinamide concentration above 100 mM, hence fed batch
biotransformation can be a useful strategy in order to obtain
high yield of isoniazid. Previous study suggested that under opti-
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
mized conditions of pH (7, phosphate buffer 100 mM), temperature
◦
(
30 C), substrate/co-substrate concentration (100/1000 mM) and
2
013.07.010.
cell concentration (2.0 mgdcw/ml), excellent molar bioconversion
yield (90.4%) was achieved with in 60 min. Feedings of substrates
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to isoniazid up to third feeding and during 4th feed 79% conversion
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