Biotransformation of aromatic and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization and substrate specificity

Article abstract of DOI:10.1080/10242422.2017.1282467

In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid, nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized against benzamide. The enzyme exhibited optimum activity in 50 mM potassium phosphate buffer pH 7.0. The optimum temperature and substrate concentrations for this enzyme were 50 °C and 50 mM, respectively. The enzyme was quite stable for more than 6 h at 30 °C. It showed substrate specificity against different amides, including aliphatic, aromatic and heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50 mM each of benzamide, nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120 min, respectively, using 5 mg dry cell mass (DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44% achieved by whole cells. The amidase in whole cells can withstand as high as 383 mM concentration of product in a reaction mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp. MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.

Full text of DOI:10.1080/10242422.2017.1282467

Biocatalysis and Biotransformation
ISSN: 1024-2422 (Print) 1029-2446 (Online) Journal homepage: http://www.tandfonline.com/loi/ibab20
Biotransformation of aromatic and heterocyclic
amides by amidase of whole cells of Rhodococcus
sp. MTB5: Biocatalytic characterization and
substrate specificity
Mukram Ismailsab, Monisha T. R., Pooja V. Reddy, Santoshkumar M., Anand
S. Nayak & Timmanagouda B. Karegoudar
To cite this article: Mukram Ismailsab, Monisha T. R., Pooja V. Reddy, Santoshkumar M.,
Anand S. Nayak & Timmanagouda B. Karegoudar (2017): Biotransformation of aromatic
and heterocyclic amides by amidase of whole cells of Rhodococcus sp. MTB5: Biocatalytic
characterization and substrate specificity, Biocatalysis and Biotransformation
To link to this article: http://dx.doi.org/10.1080/10242422.2017.1282467
Published online: 30 Jan 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=ibab20
Download by: [University of South Carolina ]
Date: 31 January 2017, At: 01:12

Biocatalysis and Biotransformation, 2017; Early Online: 1–12
RESEARCH ARTICLE
Biotransformation of aromatic and heterocyclic amides by amidase of
whole cells of Rhodococcus sp. MTB5: Biocatalytic characterization
and substrate specificity
MUKRAM ISMAILSAB, MONISHA T. R., POOJA V. REDDY, SANTOSHKUMAR M.,
ANAND S. NAYAK & TIMMANAGOUDA B. KAREGOUDAR
Department of Biochemistry, Gulbarga University, Kalaburagi, India
Abstract
In this study, an amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 has been used for the
biotransformation of aromatic, monoheterocyclic and diheterocyclic amides to corresponding carboxylic acids. Benzoic acid,
nicotinic acid and pyrazinoic acid are carboxylic acids which have wide industrial applications. The amidase of this strain is
found to be inducible in nature. The biocatalytic conditions for amidase present in the whole cells of MTB5 were optimized
against benzamide. The enzyme exhibited optimum activity in 50 mM potassium phosphate buffer pH 7.0. The optimum
temperature and substrate concentrations for this enzyme were 50 ꢀC and 50 mM, respectively. The enzyme was quite stable
for more than 6 h at 30 ꢀC. It showed substrate specificity against different amides, including aliphatic, aromatic and
heterocyclic amides. Under optimized reaction conditions, the amidase is capable of converting 50 mM each of benzamide,
nicotinamide and pyrazinamide to corresponding acids within 100, 160 and 120 min, respectively, using 5 mg dry cell mass
(DCM) per mL of reaction mixture. The respective percent conversion of these amides was 95.02%, 98.00% and 98.44%
achieved by whole cells. The amidase in whole cells can withstand as high as 383 mM concentration of product in a reaction
mixture and above which it undergoes product feedback inhibition. The results of this study suggest that Rhodococcus sp.
MTB5 amidase has the potential for large-scale production of carboxylic acids of industrial value.
Keywords: Biotransformation, amides, carboxylic acids, amidase, Rhodococcus sp. MTB5, fermentation
Introduction
green chemistry. This enzyme finds a place as an
industrial biocatalyst in organic synthesis, in the
treatment of industrial effluents containing toxic
amides and as therapeutic agents (Rahim et al. 2003;
Nampoothiri et al. 2005). These characteristics of
this enzyme made it a good candidate not only for
environmental remediation but also for the biosyn-
thesis of novel compounds.
Amidases (EC 3.5.1.4) are ubiquitous enzymes in
the living world and they have wide biological
functions. These enzymes are involved in the nitro-
gen metabolism of prokaryotic as well as eukaryotic
cells (Banerjee et al. 2002). They display dual
activity, i.e., hydrolytic activity (amidohydrolase) in
aqueous reaction medium and acyltransfer activity
(acyltransferase) in the presence of more nucleo-
philic hydroxylamine (Sharma et al. 2009). The
amidohydrolase activity of amidase hydrolyzes
amides to corresponding carboxylic acids and
ammonia. On the other hand, acyltransferase is
involved in the transfer of acyl group from amide to
hydroxylamine-forming hydroxamic acid (Figure 1).
Amidases act as versatile biocatalysts in white and
Biotransformation is the process wherein a sub-
strate is transformed into a more useful product by
using biological catalysts. It serves a better option
than those of chemical methods because of its
effectiveness, eco-friendly and economic viability.
Biological synthesis and/or microbial transform-
ations have great potential applications in industrial
microbiology (Yamada and Shimizu 1988). These
reactions employ mild conditions of temperature and
Correspondence: Prof. Timmanagouda B. Karegoudar, Department of Biochemistry, Gulbarga University, Kalaburagi –585 106, Karnataka, India. Tel: +91
9448163622. Fax: +91 8472 245632. E-mail: goudartbk@gmail.com
(Received 8 August 2016; revised 16 November 2016; accepted 31 December 2016)
ISSN 1024-2422 print/ISSN 1029-2446 online ꢀ 2017 Informa UK Limited, trading as Taylor & Francis Group
DOI: 10.1080/10242422.2017.1282467

2
M. Ismailsab et al.
Amidohydrolase
media with high energy consumption. Further,
during these reactions unwanted toxic substances
and large amounts of salts are also formed.
RCONH + H O
RCOOH + NH
2
2
3
In the present work, we have standardized the
various biocatalytic parameters such as different
buffers, pH, ionic strength, substrate concentration
and temperature on the activity of amidase in whole
cells of Rhodococcus sp. MTB5. The substrate spe-
cificity and the thermal stability of amidase are also
investigated. The biotransformation of benzamide,
nicotinamide and pyrazinamide to their correspond-
ing carboxylic acids is carried out by using whole
cells of Rhodococcus sp. MTB5. Further, the pre-
parative scale biotransformation of nicotinamide to
nicotinic acid by the cells of MTB5 is performed in a
fermenter. The effect of accumulated nicotinic acid
on the amidase activity in whole cells was also
investigated.
Acyltransferase
RCONH + NH OH
RCONHOH + NH
3
2
2
Figure 1. Amidohydrolase and acyltransferase reactions of
amidase.
pH with the formation of pure products without any
by-products (Otto and Mei-Xiang 1997; Cantarella
et al. 2008). Whole cell biotransformation has the
advantage of retaining the enzymes in their natural
environment (which could enhance stability),
whereas soluble enzymes are more susceptible to
changes in operating conditions and substrate or
product toxicity (Woodley 2006). Currently, bio-
transformation of amides to corresponding acids by
amidases has gained more attraction because of its
environmental friendly reaction conditions.
A
number of microbial amidases with acyltransferase
activity has been extensively explored for the pro-
duction of hydroxamic acids of commercial value,
such as benzohydroxamic acid, acetohydroxamic
acid and nicotinylhydroxamic acid (Dadd et al.
2001; Pandey et al. 2011; Bhatia et al. 2013,
2014). In the same way, the amidohydrolase activity
was used for the synthesis of corresponding free and
substituted carboxylic acids (Cantarella et al. 2008;
Yang et al. 2011; Mehta et al. 2014). Recently Mei-
Xiang (2015) has highlighted the amidase catalyzed
hydrolysis of amides for asymmetric synthesis of
enantiomeric carboxylic acids and their derivatives.
Benzoic acid is a white crystalline powder partially
soluble in water and completely soluble in most of
the organic solvents. Benzoic acid and its salts are
used in food preservation, in calico printing and as
an important precursor for the industrial synthesis of
many other organic substances (Matar and Hatch
2000; Hazan et al. 2004). From the medicinal point
of view, it is a constituent of Whitfield’s ointment
which is used for the treatment of fungal skin
diseases, such as tinea, ringworm and athlete’s foot
(Wilson et al. 2004). Nicotinic acid is a form of
vitamin B3 and is an essential component of a
balanced diet for humans and widely used as
pharmaceuticals and food additives. It is widely
referred as pellagra-preventing factor and also used
in the treatment of hypercholesterolemia, schizo-
phrenia, diabetes, auto-immune diseases and osteo-
arthritis (Sharma et al. 2011). Pyrazinoic acid is an
antimicrobial agent against Mycobacterium tubercu-
losis and finds applications in the treatment of
tuberculosis (TB). The chemical reactions for the
production of these carboxylic acids require harsh
reaction conditions either strongly acidic or basic
Materials and methods
Chemicals
Benzonitrile, HPLC grade acetonitrile, acetamide,
formamide, N-methylformamide, N,N-dimethylfor-
mamide, N,N-dimethylacetamide, L-glutamine,
L-asparagine and methanol were purchased from
S.D. Fine-Chem Limited, Mumbai, India.
Benzamide, benzoic acid, nicotinic acid, nicotina-
mide and media ingredients were purchased from
Himedia, India. Pyrazinamide, pyrazinoic acid,
isonicotinamide, picolinamide, adipamide, acryl-
amide, bis-acrylamide, N,N’-methylenebisacryla-
mide, butyramide and propionamide were procured
from Sigma-Aldrich, Steinheim, Germany. All other
chemicals used in this study were of analytical grade
and procured from various commercial vendors.
Microorganism
Rhodococcus sp. MTB5 (previously isolated in our
laboratory) having the ability to hydrolyze both
aliphatic and aromatic nitriles (Mukram et al.
2015) was used as a source of amidase and as a
biocatalyst for biotransformation reactions.
Media and culture conditions
The bacterium was maintained in mineral salts (MS)
medium supplemented with 30 mM benzonitrile as
source of carbon, nitrogen and energy. The medium
contained following ingredients (g/L): K2HPO4 6.8;
KH2PO4 1.2; MgSO4ꢁ7H2O 0.1; MnSO4ꢁ4H2O 0.1;
CaCl2ꢁ2H2O
0.1;
FeSO4ꢁ7H2O
0.1;
Na2MoO7ꢁ2H2O 0.006. The pH of the medium

Amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5
3
was adjusted to 7.0 using 2 N NaOH or 2 N HCl
before sterilizing the medium by autoclaving at 15 lb
pressure for 20 min. The culture was grown in
250 mL Erlenmeyer flask with shaking at 180 rpm
in temperature-controlled orbital shaker (LabTech,
Seoul, Korea) at 30 ꢀC. Same conditions were
maintained for the production of amidase and in
this case benzonitrile will serve as an inducer.
mixture. The enzyme activity was determined in
terms of production of ammonia.
Optimization of reaction conditions for amidase assay
in whole cells of Rhodococcus sp. MTB5
The different reaction conditions for the assay of
amidase in whole cells of Rhodococcus sp. MTB5
were optimized. The effect of various buffers, pH,
ionic strength of the buffer, substrate concentration
and the temperature was checked. The reaction pH
for optimization study ranges from 4 to 13 with
citrate phosphate buffer (pH 4.0–6.5), potassium
phosphate buffer (pH 6.0–8.5), carbonate–bicarbon-
ate buffer (pH 9.0–10.0), glycine–NaOH buffer (pH
10.0–11.0) and KCl–NaOH buffer (pH 11.5–13.0).
To work out the optimum molarity, the reaction was
carried out in the buffer concentration ranging from
10 to 100 mM. Different concentrations of substrate
ranging from 10 to 100 mM were tested in the
reaction mixture to determine the optimum concen-
tration for highest amidase activity of Rhodococcus sp.
MTB5. The optimum reaction temperature was
determined by carrying out the amidase reaction at
various temperatures, from 10 to 80 ꢀC. To check
the thermal stability of this enzyme, the resting cells
were pre-incubated at 30, 35, 40 and 45 ꢀC and then
the residual activity was measured for every 1 h
interval up to 6 h. The residual amidase activity was
determined by comparing the initial amidase activity
of unincubated resting cells.
Preparation of Rhodococcus sp. MTB5 resting cells
and dry cell mass determination
Bacterial cells were harvested at the mid-logarithmic
growth phase by centrifugation (8000 rpm, 10 min,
4 ꢀC) and washed twice with 50 mM potassium
phosphate buffer (pH 7.0), then the cells were
suspended in the same buffer and stored at 4 ꢀC
until further use. Dry cell mass (DCM) was
estimated using the resting cell suspension prepared
as mentioned above. This cell suspension was then
dried at 80 ꢀC for 24 h until a constant weight was
achieved (Cantarella et al. 2006).
Amidase assay in whole bacterial cells
Amidase activity was assayed by estimating the
amount of ammonia liberated. The reaction mixture
contained 5 mg DCM of Rhodococcus sp. MTB5,
10 mM benzamide as substrate and 100 mM potas-
sium phosphate buffer (pH 7.0). The reaction
mixture was incubated for 30 min at 37 ꢀC and was
terminated by the addition of 1 N HCl. The amount
of ammonia released was estimated by the method of
Scha¨r et al. (1986) after the removal of cells by
centrifugation at 8000 rpm for 5 min. One unit of
amidase activity was defined as the amount of resting
cells which catalyzed the hydrolysis of benzamide to
produce 1 mmol of ammonia per min under standard
assay conditions. The assay was carried out in
triplicates.
Biotransformation of amides to corresponding acids
Based on optimized reaction conditions for amidase
against benzamide in preceding experiments, the
biotransformation was carried out in 250 mL
Erlenmayer flask containing 50 mL potassium phos-
phate buffer (50 mM, pH 7.0). Rhodococcus sp.
MTB5 resting cells (5 mg DCM per mL of reaction
mixture) were used as biocatalyst. The biotransform-
ation was initiated by adding 50 mM each of
benzamide, nicotinamide and pyrazinamide. The
reaction was carried out in an orbital shaker at 30 ꢀC
and 180 rpm. The reaction mixture (200 mL) was
withdrawn periodically and the reaction was stopped
by adding 1 N HCl. The resting cells were removed
by centrifugation at 8000 rpm for 10 min and the
clear supernatant was subjected to chromatographic
analysis. The biotransformation of benzamide to
benzoic acid was analyzed by gas liquid chromatog-
raphy (GLC), whereas the biotransformation of
nicotinamide and pyrazinamide were analyzed by
high-performance liquid chromatography (HPLC).
After completion of the biotransformation reactions,
Substrate specificity of amidase of Rhodococcus sp.
MTB5
The substrate specificity of amidase of Rhodococcus
sp. MTB5 was studied against 10 mM different
amides such as benzamide, nicotinamide, pyrazina-
mide, isonicotinamide, picolinamide formamide,
N-methylformamide,
N,N-dimethylformamide,
acetamide, N,N-dimethylacetamide, acrylamide,
butyramide, propionamide, adipamide, bisacryla-
mide, N,N0-methylenebisacrylamide, L-glutamine
and L-asparagine. The reaction was carried out at
37 ꢀC for 30 min in 100 mM potassium phosphate
buffer, pH 7 using 5 mg DCM per mL of reaction

4
M. Ismailsab et al.
the reaction mixture was centrifuged at 10,000 rpm
for 20 min. The supernatant was filtered through
0.2 mm filter paper and the carboxylic acids, such as
benzoic, nicotinic and pyrazinoic acids recovered
were analyzed for their purity.
(length 30 m, inner diameter 0.25 mm, 0.25 mm
Elm), with nitrogen as carrier gas (flow rate
2 mL minꢂ1) was used. The chromatographic con-
ditions were: injector temperature 240 ꢀC and
detector temperature 250 ꢀC. The temperature pro-
gram was 160–240 ꢀC with 15 ꢀC rise per minute for
a hold time of 4 min. Sample of 1 mL was injected
into the injector with the splitless mode.
Preparative scale biotransformation of nicotinamide
Preparative scale biotransformation of nicotinamide
to nicotinic acid by resting cells of Rhodococcus sp.
MTB5 was performed in a 4 L capacity fermenter
(New Brunswick Scientific, BIOFLO C-32). The
resting cells of MTB5 were obtained by growing the
culture in a fermenter and then washed twice with
potassium phosphate buffer. The biotransformation
reaction mixture (1 L) consisted of 50 mM potassium
phosphate buffer, 50 mM nicotinamide (6.106 g) and
5 g DCM of Rhodococcus sp. MTB5. The tempera-
ture of the fermenter vessel was maintained at 30 ꢀC
and the impeller speed of 200 rpm was set for the
proper mixing of nicotinamide and resting cells. The
concentration of formed nicotinic acid was estimated
by HPLC. After completion of the biotransform-
ation, the nicotinic acid was recovered from the
reaction mixture and checked for its purity by
HPLC.
High-performance liquid chromatography
The concentration of nicotinamide, pyrazinamide,
nicotinic acid and pyrazinoic acid in biotransform-
ation reaction mixture were estimated by HPLC.
The HPLC (WATERS 2489, Milford, MA) having
515 Binary pumps with C18 Sunfire column
(250 mm ꢃ4.6 mm, particle size 5 mm) and ultravio-
let detector (230 nm) was used. The solvent system
(mobile phase) used was 12% acetonitrile in 10 mM
potassium phosphate buffer of pH 7.0 prepared in
HPLC grade water. The biotransformation reaction
mixture samples collected were centrifuged and
filtered through 0.2 mm filters before injection.
Sample of 10 mL was injected into the injector for
analysis.
Results and discussion
Effect of accumulated nicotinic acid on amidase
To check the effect of the concentration of accumu-
lated product on the amidase activity, the reaction
was carried out in fed-batch mode at 30 ꢀC. The
reaction mixture (50 mL) contained 50 mM phos-
phate buffer (pH 7.0), 250 mg DCM and the
reaction was initiated by the addition of 50 mM
nicotinamide. The substrate-feeding experiment was
designed on the basis of 98% conversion of 50 mM
nicotinamide in 160 min by 5 mg DCM per mL
reaction mixture. Ten such feedings of 50 mM
substrate were made each at an interval of 160 min
by keeping a constant reaction volume (50 ml). A
sample (200 mL) was withdrawn before every feed for
the estimation of nicotinamide and nicotinic acid. A
control experiment was also conducted with the
same parameters without the resting cells for any
spontaneous chemical reaction.
Amidase activity
Strain MTB5 is capable of producing amide hydro-
lyzing enzyme, amidase when it was grown in MS
medium containing benzonitrile. The MTB5 resting
cells prepared from benzonitrile grown cells showed
amidase activity of 0.135 mmol/min/mg of DCM.
The resting cells prepared from cells grown on 1%
glucose (as carbon source) and 0.1% ammonium
nitrate (as nitrogen source) did not show any
amidase activity. This suggested that the enzyme
involved in the biotransformation of benzamide is
inducible in nature. Amidases could be constitutive
or inducible in nature. There are reports for both
constitutive (Egorova et al. 2004; Prasad et al. 2005;
Mehta et al. 2014) as well as inducible (Ciskanik
et al. 1995; Nawaz et al. 1996) nature of amidases.
Substrate specificity of amidase
Analytical methods
To study the substrate specificity of amidase from
Rhodococcus sp. MTB5, different amides were used
as substrates for this enzyme. The substrate specifi-
city of this enzyme was investigated by using 10 mM
various amides. The activities of amidase against
different amides are given in Table 1. The amidase
produced from Rhodococcus sp. MTB5 showed
Gas chromatography
The concentration of benzamide and benzoic acid in
biotransformation reaction mixture was estimated by
GLC. The GLC system (Agilent 7820A) equipped
with split/splitless injector and an FID DB 5 column

Amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5
5
Table 1. Substrate specificity of amidase of Rhodococcus sp. MTB5.
Amide
Structure
Activity
(U/mg DCM)
O
O
NH
2
Benzamide
0.136
0.133
0.030
0.031
NH
2
Nicotinamide
Isonicotinamide
Picolinamide
N
O
NH
2
N
O
NH
2
N
O
N
NH
2
Pyrazinamide
0.207
0.032
0.004
N
NH
O
O
2
Formamide
N
H
N-Methylformamide
N
O
N,N-Dimethylformamide
0.002
0.09
O
O
Acetamide
NH
2
N
N,N-Dimethylacetamide
Acrylamide
0.002
0.09
O
NH
O
2
Butyramide
0.227
NH
2
O
Propionamide
Bis-acrylamide
Adipamide
0.192
ND
NH
2
O
O
−
N
O
H
N
2
ND
NH
2
O
(continued)

6
M. Ismailsab et al.
Table 1. Continued
Amide
Structure
Activity
(U/mg DCM)
O
O
N, N 0-Methylenebisacrylamide
ND
ND
ND
N
H
N
H
O
O
HO
NH
2
L-Glutamine
NH
2
O
NH
2
HO
L-Asparagine
NH
O
2
ND: not detected.
substrate preference towards aliphatic, aromatic,
monoheterocyclic and diheterocyclic amides. The
maximum substrate specificity was observed for
butyramide, pyrazinamide, propionamide, benza-
mide and nicotinamide, whereas low specificity for
very short-chain aliphatic amides, such as forma-
mide, acetamide and acrylamide, and heterocyclic
amides, such as isonicotinamide and picolinamide.
The negligible specificity was noticed for substituted
amides, such as N-methylformamide, N,N-dimethyl-
formamide and N,N-dimethylacetamide. The tested
amino acids did not serve as substrates for amidase.
The enzyme did not exhibit any activity for amides
that are having two amide functional groups, such as
adipamide, bisacrylamide and N,N0-methylenebisa-
crylamide. It has been reported that some amidases
exhibited broad substrate specificity encompassing
aliphatic, aromatic and heterocyclic amides (Ruan
et al. 2016; Wu et al. 2016). Amidases from Klebsiella
pneumoniae NCTR 1 (Nawaz et al. 1996) and
Geobacillus pallidus (Makhongela et al. 2007) are
reported to be active only against aliphatic amides.
Takashi and Tatsuhiko (1990) reported the amidase
of Pseudomonas sp. GDI 211, which is specific for
aromatic amides but not for aliphatic amides. Trott
et al. (2001) reported the enantioselective amidase
from Agrobacterium tumefaciens strain D3 that has the
ability to convert substituted aromatic amides.
from 4.0 to 13.0 were used. Among the buffers
investigated, the maximum activity of amidase from
strain MTB5 (0.135 U/mg DCM) is observed in
potassium phosphate buffer, pH 7.0 (Figure 2).
On the other hand, the activity of amidase
decreased below pH 4.5 (50.074 U/mg DCM) and
above pH 11.0 (50.09 U/mg DCM). This is because
of unfavorable ionic conditions necessary for the
formation of enzyme–substrate complex (Bhatia
et al. 2013). Similar results were reported for
amidases of Klebsiella pneumoniae NCTR 1 (Nawaz
et al. 1996) and Bacillus smithii strain IITR6b2
(Agarwal et al. 2013) showing the maximal activity
at neutral pH.
Potassium phosphate buffer molarity
The potassium phosphate buffer (pH 7.0) molarity
varied from 10 to 100 mM for amidase assay of
Rhodococcus sp. MTB5. Highest amidase activity
(0.180 U/mg DCM) is observed in 50 mM
(Figure 3). At higher molarity of potassium phos-
phate buffer (470 mM), the amidase of Rhodococcus
sp. MTB5 experienced inhibitory effect that
decreases the activity. This may be due to electro-
static interactions between the polar surface groups
of enzymes and the buffer (Bhatia et al. 2013).
Substrate (benzamide) concentration
Optimization of reaction conditions for
amidase assay in whole cells of Rhodococcus sp.
MTB5
For optimization of substrate concentration, the
amidase assay was carried out at different concen-
trations (10–100 mM) and the maximum activity
(0.176 U/mg DCM) was observed at 50 mM benza-
mide. The further increase in the concentration
of substrate decreases the activity of amidase
Buffer systems and pH
To select an appropriate buffer and pH for amidase
activity, various buffers (100 mM) of pH ranging

Amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5
7
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
3
4
5
6
7
8
9
10
11
12
13
14
pH
Figure 2. Effect of buffer system and pH (m) on amidase of Rhodococcus sp. MTB5.
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
10
20
30
40
50
60
70
80
90
100
110
Buffer molarity (mM)
Figure 3. Effect of potassium phosphate buffer molarity (g) on amidase of Rhodococcus sp. MTB5.
0.2
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
0
10
20
30
40
50
60
70
80
90
100
110
Benzamide (mM)
Figure 4. Effect of benzamide concentration (g) on amidase of Rhodococcus sp. MTB5.

8
M. Ismailsab et al.
120
100
80
60
40
20
0
0.25
0.2
0.15
0.1
0.05
0
0
1
2
3
4
5
6
7
0
10
20
30
40
50
60
70
80
90
Time (h)
Temperature (°C)
Figure 6. Thermal stability of amidase of Rhodococcus sp. MTB5:
(m) 30 ꢀC, (i) 35 ꢀC, (g) 40 ꢀC, (œ) 45 ꢀC.
Figure 5. Effect of incubation temperature (g) on amidase of
Rhodococcus sp. MTB5.
(Figure 4). This decreased enzyme activity may be
due to the toxicity of high concentration of benza-
mide (Jin et al. 2013).
completely lost its activity at 5 h. Further, when the
preincubation temperature was reduced to 40 ꢀC and
35 ꢀC, the half-life of the enzyme was 2.5 and 5.5 h,
respectively. The enzyme retained 80.74% residual
activity even after 6 h of preincubation at 30 ꢀC
(Figure 6). Based on these results, the amidase of
Rhodococcus sp. MTB5 is considered a mesophilic
enzyme. Therefore, 30 ꢀC was found to be suitable
for prolonged biotransformation reactions. It was
demonstrated that the thermophilic amidases retain
the stability for prolonged duration at higher tem-
peratures than mesophilic counterparts (Makhongela
et al. 2007; Fu et al. 2014).
Temperature
The amidase assay was carried out for 30 min in the
temperature range from 10 to 80 ꢀC. The optimum
incubation temperature for amidase of Rhodococcus
sp. MTB5 was found to be 50 ꢀC with amidase
activity of 0.19 U/mg DCM (Figure 5). The amidase
activity linearly increased from 10 to 50 ꢀC and then
it decreased due to thermal denaturation of the
enzyme. The lowest activity is noticed at 80 ꢀC
(0.028 U/mg DCM). At higher temperatures, the
non-covalent bonds holding the amino acids of
enzyme starts breaking due to high kinetic energy.
This causes the change in the 3D structure of the
enzyme and ultimately leads to the loss of enzyme
activity (Demirjian et al. 2001). Amidases from
various bacterial strains have showed different tem-
perature optima. For example, 40 ꢀC for amidase of
Rhodococcus sp (Nawaz et al. 1994) and Burkholderia
sp. strain DR.Y27 (Syed et al. 2012), 55 ꢀC for
amidase of Rhodococcus erythropolis MP50 (Hirrlinger
et al. 1996) and Rhodococcus erythropolis No. 7 (Park
et al. 2008). The thermostable amidases are reported
to have optimum activity at 70 ꢀC and above
(Egorova et al. 2004; Fu et al. 2014; Mehta et al.
2014).
Bench scale biotransformation of amides to corre-
sponding carboxylic acids
Since the amidase of Rhodococcus sp. MTB5 is
mesophilic, the biotransformation of amides was
carried out at 30 ꢀC. The biotransformation of
benzamide, nicotinamide and pyrazinamide were
performed over a period of 180 min. The periodic
analysis of the benzamide biotransformation reaction
mixture by GLC showed 95.02% conversion of
benzamide to benzoic acid. On the contrary, the
HPLC analysis of biotransformation reaction mix-
ture of nicotinamide to nicotinic acid and pyrazina-
mide to pyrazinoic acid showed 98.00% and 98.44%
conversion, respectively (Figure 7). After completion
of the biotransformation reaction, the corresponding
carboxylic acids were recovered. The recovered
carboxylic acids were analyzed and found to be
pure by GC or HPLC analysis.
Thermal stability of amidase
The thermal stability of amidase in whole cells of
Rhodococcus sp. MTB5 was examined at various
temperatures (30–45 ꢀC). The enzyme is not stable
at its optimum temperature, 50 ꢀC (data not shown).
When the resting cells were preincubated at 45 ꢀC,
the half-life of enzyme is 42 min. The enzyme
retained 8.17% residual activity after 3 h and
Preparative scale biotransformation of nicotinamide to
nicotinic acid
The preparative biotransformation of nicotinamide
by Rhodococcus sp. MTB5 was performed in fer-
menter with 1 L reaction mixture. The temperature

Amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5
9
50
45
40
35
30
25
20
15
10
5
50
45
40
35
30
25
20
15
10
5
0
0
0
20
40
60
80
100
120
140
160
180
200
Time (min)
Figure 7. Biotransformation of aromatic and heterocyclic amides to corresponding carboxylic acids by Rhodococcus sp. MTB5 resting cells:
(g) benzamide, (œ) benzoic acid, (m) nicotinamide, (i) nicotinicacid, (ꢄ) pyrazinamide, (*) pyrazinoic acid.
55
50
45
40
35
30
25
20
15
10
5
0
0
60
120
180
240
300
360
Time (min)
Figure 8. Preparative scale biotransformation of nicotinamide at 1 L scale: ( ) Nicotinamide, ( ) Nicotinic acid.
of the fermenter vessel was maintained at 30 ꢀC and
the reaction was carried out for 360 min. At the end
of the reaction, 41.66 mM nicotinic acid was
produced from 50 mM nicotinamide with 88.34%
conversion (Figure 8). After fermentation, the whole
cells were separated from reaction mixture by cen-
trifugation and the nicotinic acid was recovered from
the supernatant and is found to be pure by HPLC
analysis. The transformation of nicotinamide in
preparative scale is 9.66% less than that achieved in
50 mL bench scale and the time required for
biotransformation was also increased. This decrease
in % transformation and increased biotransformation
duration may be due to the changes in kinetics with
the increase in reaction volume. Similar observation
was also made for the biotransformation of aceta-
mide by Bacillus sp. APB-6 (Pandey et al. 2011). The
process parameters and reaction kinetics could
further be optimized for purified and immobilized
amidase at commercial scale for better results.
Many studies have been carried out on biotrans-
formation for the greener synthesis of valuable
amides and acids (Graham et al. 2000; He et al.
2010). In this study, we have focused on the amidase
catalyzed biotransformation reactions. Amidases
catalyze two types of reactions: amide hydrolysis
and acyl transfer. Pandey et al. (2011) reported the
synthesis of acetohydroxamic acid using resting cells
of Bacillus sp. APB-6. The acyl transfer activity of
amidase from Alcaligenes sp. MTCC 10674 and

10 M. Ismailsab et al.
400
350
300
250
200
150
100
50
0
0
160
320
480
640
800
960 1120 1280 1440 1600 1760
Time (min)
Figure 9. Fed-batch reaction profile of Rhodococcus sp. MTB5 amidase: (ꢄ) nicotinamide and (*) nicotinic acid. Upper and lower (ꢄ)
denotes the nicotinamide concentration after and before the addition of 50 mM nicotinamide at every 160 min.
Pseudomonas putida BR-1 has been explored for the
production of benzohydroxamic acid and nicotinyl-
hydroxamic acid, respectively (Bhatia et al. 2013,
2014). Microbacterium imperial CBS 498–74 and
Geobacillus subterraneus RL-2a have been used for
the production of nicotinic acid by amidohydrolase
activity of amidase (Cantarella et al. 2008; Mehta
et al. 2014). Amidases of different microbes have
also been widely used in the synthesis of various
industrially valuable (R)- and (S)-carboxylic acids
(Shaw et al. 2002; Yang et al. 2011). Nitrile
hydratase and amidase of Brevibacterium sp. R 312
were exploited for the synthesis of enantiomerically
pure (S)-cyano acids from prochiral dinitriles
(Kerridge et al. 1994). There are reports on the use
of amidases for benzoic acid and few for nicotinic
acid production. Most of the reports available on
nicotinic acid and benzoic acid employ nitrilase
catalyzed reactions (Prasad et al. 2007; Raj et al.
2007). Whereas, there are no reports in the literature
on the use of amidases for the biosynthesis of
pyrazinoic acid.
inhibition, and a total of 383 mM nicotinic acid
was accumulated in the reaction mixture after 10
feeds. There was no further nicotinamide conversion
after 10th substrate feed. The higher product accu-
mulation in the reaction might affect the amidase
activity (product inhibition). Various researchers
have reported the amidase from different micro-
organisms which can withstand different concentra-
tions of accumulated product. The amidase from
Alcaligenes sp. MTCC 10674 was reported to with-
stand 175 mM of product accumulated (Bhatia et al.
2013), Agarwal et al. (2013) used amidase from
Bacillus smithii strain IITR6b2 for the synthesis of
nicotinic acid hydroxamate, which has no inhibitory
effect up to 536 mM product formed. Mehta et al.
(2014) reported amidase of Geobacillus subterraneus
RL-2a, wherein the enzyme has no inhibitory effect
on the rate of product formation until 6 substrate
feeds of 120 mM each against which 870 mM prod-
uct was accumulated.
To check the effect of accumulated product
(nicotinic acid) on amidase activity, the reaction
was carried out in a fed-batch mode at 50 mL scale
with time course substrate feed. The reaction was
carried out at 30 ꢀC while maintaining biocatalyst
amount at 5 mg DCM per mL in the reaction. There
was maximum conversion of nicotinamide to nico-
tinic acid up to the 7th feed of substrate (Figure 9).
Further feedings decreased the rate of nicotinic acid
production which might be due to product
Conclusions
The present study reports the optimization of reac-
tion conditions for amidase activity in whole cells of
Rhodococcus sp. MTB5 and biotransformation of
aromatic and heterocyclic amides to their corres-
ponding acids. The following conclusions can be
drawn based on the investigations:
(i) The amidase from Rhodococcus sp. MTB5 showed
substrate preference towards aliphatic, aromatic

Amidohydrolase activity of amidase in whole cells of Rhodococcus sp. MTB5 11
batch and ultrafiltration membrane bioreactors. J Ind Microbiol
Biotechnol 33:208–214.
Cantarella M, Cantarella L, Gallifuoco A, Intellini R, Kaplan O,
Spera A, Mart{nkov L. 2008. Amidase-catalyzed production of
nicotinic acid in batch and continuous stirred membrane
reactors. Enzyme Microb Technol 42:222–229.
Ciskanik LM, Wilczek JM, Fallon RD. 1995. Purification and
characterization of an enantioselective amidase from
Pseudomonas chlororaphis B23. Appl Environ Microbiol
61:998–1003.
and heterocyclic amides rather than diamides and
amino acids glutamine and asparagine.
(ii) 50 mM potassium phosphate buffer of pH 7.0,
benzamide of concentration 50 mM and 50 ꢀC
incubation temperatures were the optimum con-
ditions for amidase reaction. The enzyme was
stable at 30 ꢀC for more than 6 h.
(iii) 95.02, 98.00 and 98.44% biotransformation of
benzamide, nicotinamide and pyrazinamide to
benzoic acid, nicotinic acid and pyrazinoic acid
was achieved in 100, 160 and 120 min, respect-
ively, at optimum parameters.
Dadd MR, Claridge TDW, Pettman AJ, Knowles CJ. 2001.
Biotransformation of benzonitrile to benzohydroxamic acid by
Rhodococcus rhodochrous in the presence of hydroxylamine.
Biotechnol Lett 23:221–225.
´
Demirjian DC, Morıs-Varas F, Cassidy CS. 2001. Enzymes from
extremophiles. Curr Opin Chem Biol 5:144–151.
Egorova K, Trauthwein H, Verseck S, Antranikian G. 2004.
Purification and properties of an enantioselective and thermo-
active amidase from the thermophilic actinomycete
Pseudonocardia thermophila. Appl Microbiol Biotechnol 65:38–
45.
The biotransformation reported in the present
study has potential for the production of different
useful carboxylic acids.
Fu L, Li X, Xiao X, Xu J. 2014. Purification and characterization
of a thermostable aliphatic amidase from the hyperthermophilic
archaeon Pyrococcus yayanosii CH1. Extremophiles 18:429–440.
Graham D, Pereira R, Barfield D, Cowan D. 2000.
Nitrile biotransformations using free and immobilized cells
Acknowledgements
Authors would like to thank the University Grants
Commission (UGC), New Delhi, India, for financial
support in the form of Special Assistance Programme
(SAP) sanctioned to the department [F.4-27/2015/
DRS-II(SAP-II)]. One of the authors, Mukram
Ismailsab, wishes to thank UGC for providing
financial assistance through Basic Scientific
Research (BSR) fellowship (GUK/DEV-I/2015-16/
413).
of a thermophilic Bacillus spp. Enzyme Microb Technol
26:368–373.
Hazan R, Levine A, Abeliovich H. 2004. Benzoic Acid, a weak
organic acid food preservative, exerts specific effects on intra-
cellular membrane trafficking pathways in Saccharomyces
cerevisiae. Appl Environ Microbiol 70:4449–4457.
He Y, Xu J, Su J, Zhou L. 2010. Bioproduction of glycolic acid
from glycolonitrile with a new bacterial isolate of Alcaligenes sp.
ECU0401. Appl Biochem Biotechnol 160:1428–1440.
Hirrlinger B, Stolz A, Knackmuss H. 1996. Purification and
properties of an amidase from Rhodococcus erythropolis
MP50 which enantioselectively hydrolyzes 2-arylpropiona-
mides. J Bacteriol 178:3501–3507.
Jin L, Liu Z, Xu J, Zheng Y. 2013. Biosynthesis of nicotinic acid
from 3-cyanopyridine by a newly isolated Fusarium proliferatum
ZJB-09150. World J Microbiol Biotechnol 29:431–440.
Kerridge A, Parratt JS, Roberts SM, Theil F, Turner NJ, Willettsa
AJ. 1994. Microbial hydrolysis of glutaronitrile derivatives with
Brevibacterium sp. R 312. Bioorg Med Chem 2:447–455.
Makhongela HS, Glowacka AE, Agarkar VB, Sewell BT, Weber
B, Cameron RA, Cowan DA, Burton SG. 2007. A novel
thermostable nitrilase superfamily amidase from Geobacillus
pallidus showing acyl transfer activity. Appl Microbiol
Biotechnol 75:801–811.
Compliance with ethical standards
The manuscript does not contain human studies or
experiments using animals.
Declaration of interest
The authors declare that they have no competing
interests.
Matar S, Hatch LF. 2000. Chemistry of petrochemical processes.
2nd ed. Houston, TX: Gulf Publishing Company.
Mehta PK, Bhatia SK, Bhatia RK, Bhalla TC. 2014. Bench scale
production of nicotinic acid using a versatile amide-hydrolysing
Geobacillus subterraneus RL-2a isolated from thermal spring of
Manikaran, India. J Mol Catal B: Enzym 105:58–65.
Mei-Xiang W. 2015. Enantioselective biotransformations of
nitriles in organic synthesis. Acc Chem Res 48:602–611.
Mukram I, Nayak AS, Kirankumar B, Monisha TR, Reddy PV,
Karegoudar TB. 2015. Isolation and identification of a nitrile
hydrolyzing bacterium and simultaneous utilization of aromatic
and aliphatic nitriles. Int Biodeterior Biodegrad 100:165–171.
Nampoothiri KM, Roopesh K, Chacko S, Pandey A. 2005.
Comparative study of amidase production by free and
immobilised Escherichia coli cells. Appl Biochem Biotechnol
120:97–108.
Nawaz MS, Khan AA, Seng JE, Leakey JE, Siitonen PH, Cerniglia
CE. 1994. Purification and characterization of an amidase from
an acrylamide-degrading Rhodococcus sp. Appl Environ
Microbiol 60:3343–3348.
Nawaz MS, Khan AA, Bhattacharayya D, Siitonen PH, Cerniglia
CE. 1996. Physical, biochemical, and immunological charac-
terization of a thermostable amidase from Klebsiella pneumoniae
NCTR 1. J Bacteriol 178:2397–2401.
References
Agarwal S, Gupta M, Choudhury B. 2013. Bioprocess develop-
ment for nicotinic acid hydroxamate synthesis by acyltransferase
activity of Bacillus smithii strain IITR6b2. J Ind Microbiol
Biotechnol 40:937–946.
Banerjee A, Sharma R, Banerjee UC. 2002. The nitrile-degrading
enzymes: current status and future prospects. Appl Microbiol
Biotechnol 60:33–44.
Bhatia RK, Bhatia SK, Mehta PK, Bhalla TC. 2013. Bench scale
production of benzohydroxamic acid using acyl transfer activity
of amidase from Alcaligenes sp. MTCC 10674. J Ind Microbiol
Biotechnol 40:21–27.
Bhatia RK, Bhatia SK, Mehta PK, Bhalla TC. 2014.
Biotransformation of nicotinamide to nicotinyl hydroxamic
acid at bench scale by amidase acyl transfer activity of
Pseudomonas putida BR-1. J Mol Catal B: Enzym 108:89–95.
Cantarella M, Cantarella L, Gallifuoco A. 2006. Nitrile biocon-
version by Microbacterium imperiale CBS 498-74 resting cells in

12 M. Ismailsab et al.
Otto M, Mei-Xiang W. 1997. An in-depth study of the biotrans-
formation of nitriles into amides and/or acids using Rhodococcus
rhodochrous AJ2701. J Chem Soc Perkin Trans 1:1099–1104.
Pandey D, Singh R, Chand D. 2011. An improved bioprocess for
synthesis of acetohydroxamic acid using DTT (dithiothreitol)
treated resting cells of Bacillus sp. APB-6. Bioresour Technol
102:6579–6586.
Park H, Uhm K, Kim H. 2008. R-Stereoselective amidase from
Rhodococcus erythropolis no. 7 acting on 4-chloro-3-hydroxybu-
tyramide. J Microbiol Biotechnol 18:552–559.
Prasad S, Sharma DR, Bhalla TC. 2005. Nitrile- and amide-
hydrolysing activity in Kluyveromyces thermotolerans MGBY 37.
World J Microbiol Biotechnol 21:1447–1450.
Prasad S, Misra A, Jangir V, Awasthi A, Raj J, Bhalla TC. 2007. A
propionitrile-induced nitrilase of Rhodococcus sp. NDB 1165
and its application in nicotinic acid synthesis. World J Microbiol
Biotechnol 23:345–353.
cyanopyridine with hyperinduced Nocardia globerula NHB-2. J
Ind Microbiol Biotechnol 38:1235–1243.
Shaw NM, Naughton A, Robins K, Tinschert A, Schmid E,
Hischier M, Venetz V, Werlen J, Zimmermann T, Brieden W,
et alet al. 2002. Selection, purification, characterisation, and
cloning of
a
novel heat-stable stereo-specific amidase
from Klebsiellaoxytoca, and its application in the synthesis
of enantiomerically pure (R)- and (S)-3,3,3-trifluoro-
2-hydroxy-2-methylpropionic acids and (S)-3,3,3-trifluoro-
2-hydroxy-2-methylpropionamide. Org Process Res Dev
6:497–504.
Syed MA, Ahmad SA, Kusnin N, Shukor MYA. 2012.
Purification and characterization of amidase from acrylamide-
degrading bacterium. Afr J Biotechnol 11:329–336.
Takashi K, Tatsuhiko O. 1990. Purification and properties of an
aromatic amidase from Pseudomonas sp. GDI 211. Agr Biol
Chem 54:2565–2571.
Trott S, Bauer R, Knackmuss H, Stolz A. 2001. Genetic and
biochemical characterization of an enantioselective amidase
from Agrobacterium tumefaciens strain d3. Microbiology
147:1815–1824.
Wilson CO, Gisvold O, Block JH. 2004. Wilson and Gisvold’s
Textbook of organic medicinal and pharmaceutical.
Philadelphia: Lippincott Williams & Wilkins, p. 234.
Woodley JM. 2006. Choice of biocatalyst form for scalable
processes. Biochem Soc Trans 34:301–303.
Wu Z, Zheng R, Zheng Y. 2016. Exploitation and characterization
of three versatile amidase super family members from
Delftia tsuruhatensis ZJB-05174. Enzyme Microb Technol
86:93–102.
Raj J, Singh N, Prasad S, Seth A, Bhalla TC. 2007. Bioconversion
of benzonitrile to benzoic acid using free and agar entrapped
cells of Nocardia globerula NHB-2. Acta Microbiol Immunol
Hung 54:79–88.
Rahim MA, Saxena RK, Gupta R, Sheoran A, Giri B. 2003. A
novel and quick plate assay for acetamidase producers and
process optimization for its production by Aspergillus candidus.
Process Biochem 38:861–866.
Ruan L, Zheng R, Zheng Y. 2016. Mining and characterization of
two amidase signature family amidases from Brevibacterium
epidermidis ZJB-07021 by an efficient genome mining approach.
Protein Express Purif 126:16–25.
Scha¨r HP, Holzman W, Ramos TGM, Ghisalba O. 1986.
Purification and characterization of N,N-dimethylformamidase
from Pseudomonas DMF 3/3. Eur J Biochem 158:469–475.
Sharma NN, Sharma M, Bhalla TC. 2009. Amidases: versatile
enzymes in nature. Rev Env Sci Biotechnol 8:343–366.
Sharma NN, Sharma M, Bhalla TC. 2011. An improved nitrilase-
mediated bioprocess for synthesis of nicotinic acid from 3-
Yamada H, Shimizu S. 1988. Microbial and enzymatic processes
for the production of biologically and chemically useful com-
pounds. Angew Chem Int Ed Engl 27:622–642.
Yang Z, Nia Y, Lub Z, Liaoa X, Zhengc Y, Suna Z. 2011.
Industrial production of S-2,2-dimethylcyclopropanecarboxa-
mide with a novel recombinant R-amidase from Delfti atsur-
uhatensis. Process Biochem 46:182–187.