Process Biochemistry 73 (2018) 117–123

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Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Biotransformation of 4-hydroxyphenylacetonitrile to 4-hydroxyphenylacetic T

acid using whole cell arylacetonitrilase of Alcaligenes faecalis MTCC 12629
⁎
Neerja Thakur, Vijay Kumar, Shikha Thakur, Nikhil Sharma, Sheetal, Tek Chand Bhalla
Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla, 171 005, Himachal Pradesh, India

A R T I C LE I N FO A B S T R A C T

Keywords: The whole cell arylacetonitrilase of Alcaligenes faecalis MTCC 12629 was employed to develop a bioprocess for
Nitrilase the synthesis of 4-hydroxyphenylacetic acid (4-HPAA) from 4-hydroxyphenylacetonitrile (4-HPAN).
Bioprocess Isobutyronitrile emerged as the most appropriate inducer with a 1.2-fold enhanced nitrilase production in in-
4-Hydroxyphenylacetic acid ducer feeding approach. A. faecalis MTCC 12629 nitrilase preferably hydrolyzed 4-HPAN, 4-aminoyphenylace-
tonitrile, mandelonitrile and phenylacetonitrile to their corresponding acids. Thermostability profile revealed
the stability of nitrilase at 40, 45 and 50°C with the half-life of 45 h, 36 h and 13 h, respectively. Michaelis
constant (Km) and reaction velocity (Vmax) of nitrilase were calculated as 20 mM and 2 U/mg dcw, respectively.
This nitrilase exhibited complete conversion of 4-HPAN to 4-HPAA at 45°C in 50 mM K2HPO4/KH2PO4 buffer
(pH 7.0) in 30 min. A fed batch process designed at 500 ml with five feedings of substrate resulted in 150 mM
product formation using 18 U/ml whole cells nitrilase (16 mg/ml dcw) at 45°C. This is the first report on the
production of 4-HPAA through nitrilase mediated pathway with the volumetric and catalytic productivity of
23 g/l and 4.13 g/g dcw/h, respectively.

1. Introduction
Nitrilase mediated hydrolysis of nitriles has been explored ex-
tensively, as nitriles are synthetically important chemical building
blocks which can be hydrolysed to generate high-value acids and
amides [1–3]. Nitrilases catalyse the transformation of myriad of ni-
triles into their corresponding acids and ammonia, under mild reaction
conditions and without the formation of amide intermediates. They
have thus emerged as an important tool in green chemistry [4–6]. They
are acknowledged as useful biocatalysts for the synthesis of pharma-
ceutically important drug intermediates [7,8], degradation of toxic ni-
triles [9] and production of commercially valuable acids [10]. Nitrilases
are also preferred due to their ability to hydrolyse different classes of
nitriles such as aliphatic [11,12], aromatic [13,14], heterocyclic [15]
and arylacetonitriles [16,17]. Arylacetonitriles are the derivatives of
acetonitrile in which the hydrogen of the methyl group substituted by
phenyl or heterocyclic groups (arylacetonitriles). Substituted phenyla-
cetonitriles, such as 4-hydroxyphenylacetonitrile, 4-aminophenylace-
tonitrile and mandelonitrile act as suitable substrates for arylacetoni-
trilases [17]. An arylacetic acid, 4-hydroxyphenylacetic acid (4-HPAA),
finds its application as an intermediate in the chemical synthesis of
atenolol drug (β-receptor blocking agent) which is used in the cure of
cardiovascular diseases. This acid is also an active component of Aster
tataricus and Rhodiola rosea, which are Chinese herbs that are used for
curing pulmonary diseases [18]. Furthermore, several previous studies
have reported 4-HPAA as a potential hypopigmenting agent [19–21], as
well as a good inhibitor of hypertonicity and hypoxia, by lowering the
inflammatory cytokines level [18]. 4-HPAA can be synthesized via
chemical routes such as chloromethylation of anisole, acetylation of p-
cresol and reactions of phenol with different reagents [22], however,
there is poor crystallizability of the intermediates in the anisole and p-
cresol based methods, while the methods involving phenol suffer from
poor yield of the product and generation of unwanted by-products [22].
Another method for the synthesis of 4-HPAA utilizes benzyl phenyl
ether or hydroxymandelic acid, but its limitations include requirement
of elevated temperature and pressure for the reaction and the use of
expensive solvents for the extraction of intermediates [22,23]. Thus in
comparison to the chemical routes, the synthesis of 4-HPAA using ni-
trilase is advantageous due to the requirement of mild reaction condi-
tions, easy downstream processing and minimal generation of by-pro-
ducts. The transformation of nitrile to its corresponding acid can be
accomplished using whole cell, cell free extract, purified or im-
mobilized nitrilase. Biotransformation reactions involving whole cell
nitrilase are more economical and technically beneficial, as whole cells
provide a natural environment and stability to the enzyme inside. The
activity of nitrilase for 4-HPAN has earlier been reported in bacteria

⁎
Corresponding author.
E-mail address: bhallatc@rediffmail.com (T.C. Bhalla).

https://doi.org/10.1016/j.procbio.2018.07.012
Received 11 February 2018; Received in revised form 25 May 2018; Accepted 17 July 2018
Available online 19 July 2018
1359-5113/ © 2018 Elsevier Ltd. All rights reserved.
N. Thakur et al.
[17] and in plants [24], however, studies pertaining to the development
of nitrilase-mediated process for the synthesis of 4-HPAA from 4-HPAN
have not been conducted so far. Keeping in view the above, the present
work was undertaken with the aim of developing a bioprocess for the
synthesis of 4-HPAA from 4-HPAN by using whole cell nitrilase of A.
faecalis MTCC 12629. To the best of our knowledge, this is the first
report on the process development for nitrilase mediated synthesis of 4-
HPAA from 4-HPAN.
2. Materials and methods
2.1. Chemicals and media components
The medium components used for the culture of bacterium were
purchased from Hi Media, India. The nitriles, amides and acids used in
the present study were purchased from Lancaster Synthesis, England.
High Performance Liquid Chromatography (HPLC) grade solvents and
water were purchased from Merck, India for performing HPLC.
2.2. Microorganism and culture conditions
The bacterial strain Alcaligenes faecalis MTCC 12629 obtained from
MTCC (Microbial Type Culture Collection and Gene Bank) Institute of
Microbial Technology, Chandigarh, India served as the source of ar-
ylacetonitrilase. The bacterial culture was maintained on nutrient agar.
Pre-culture was prepared by inoculating a loop full of A. faecalis MTCC
12629 culture in the nutrient broth (25 ml). Medium for the production
of arylacetonitrilase of A. faecalis MTCC 12629 comprised yeast extract
(10 g/l), tryptone (11 g/l), NaCl (5 g/l), pH 7.0, inoculum 2.75% (v/v)
and isobutyronitrile (50 mM) as inducer. Production medium (50 ml)
was seeded with 1.37 ml (18 h) of pre-culture in 250 ml Erlenmeyer
flask. This was grown for 24 h at 30°C and the culture was centrifuged
at 10000 g for 10 min, followed by washing of the cells with 100 mM
potassium phosphate buffer (pH 7.0). The cells were suspended in the
same buffer and stored at 4°C.
2.3. Nitrilase assay
Nitrilase activity in the resting cells was assayed in the reaction
mixture (1 ml) containing potassium phosphate buffer (100 mM, pH
7.0), 4-hydroxyphenylacetonitrile (1 mM) and resting cells (0.32 mg
dcw), incubated at 30°C in a water bath. After 30 min, the reaction was
terminated by 0.1 N HCl (1 ml), and ammonia formed in the reaction
was estimated using phenyl-hypochlorite method [25]. One unit of
nitrilase activity is defined as the amount of enzyme (mg dry cell
weight) required to release 1 μmol of ammonia from the hydrolysis of 4-
HPAN per min. All experiments were carried out in triplicates and
average values were calculated for the experiment followed by applying
the standard deviation for the mean values.
2.4. Analytical detection of substrate and product
HPLC system using a LiChrosorb®RP-18 column (4.6 × 150 mm) at a
flow rate of 1.0 ml/min and wavelength of 210 nm with the mobile
phase acetonitrile: water (65:35), was used to detect and quantify 4-
hydroxyphenylacetonitrile (4-HPAN) and 4-hydroxyphenylacetic acid
(4-HPAA) in the reaction mixture. HPLC analysis showed retention
times of 1.28 and 2.53 for 4-HPAN and 4-HPAA, respectively.
2.5. Selection of inducer
To select the best inducer in terms of generation of biomass and
nitrile hydrolyzing activity, various nitriles, amides and acids (40 mM,
filtered through Millipore filter) were added to the production medium
for the induction of nitrilase. The culture was harvested after 24 h in
order to detect specific activity of nitrilase in the resting cells.
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Process Biochemistry 73 (2018) 117–123
2.6. Isobutyronitrile (inducer) feeding for hyperinduction of nitrilase
The amount of isobutyronitrile was varied in the culture of A. fae-
calis MTCC 12629 in 15 sets of combinations. In set 1 (control), no
isobutyronitrile was added to the culture at all the time intervals. In sets
2, 3, 4, 5 and 6, the inducer was added as follows 25 mM, 50 mM,
75 mM, 100 mM and 200 mM, respectively at 0 h. To sets 7, 8 and 9,
50 mM of the inducer was added in the medium at regular time inter-
vals (4, 8, 12 h). Furthermore, 50 mM of isobutyronitrile was fed in the
culture, at time intervals of 0, 4 h (set 10); 0, 8 h (set 11); 0, 12 h (set
12); 0, 4, 8 h (set 13) and 0, 4, 8, 12 h (set 14). In set 15 (high ex-
ponential level), the isobutyronitrile was fed at concentrations of 25,
50, 75, 100 mM at 0, 4, 8, 12 h of incubation.
2.7. Optimization of reaction conditions for biotransformation of 4-HPAN
to 4-HPAA by whole cell nitrilase of A. faecalis MTCC 12629
To enhance the activity of nitrilase enzyme, various reaction para-
meters were optimized before proceeding to the synthesis of 4-hydro-
xyphenylacetic acid (4-HPAA). 4-HPAN hydrolyzing activity of nitrilase
was determined in various buffer systems (100 mM) i.e. potassium
phosphate (6.0–8.0), sodium phosphate (6.0–8.0), sodium carbonate
(9.0–10.0), citrate (4.0–6.0), tris−HCl (5.0–8.0), borate (7.0–9.0) and
borax buffer (10–11). The effect of temperature (25°C–60°C), thermal
stability and substrate specificity on nitrilase activity was studied. To
study the effect of product on nitrilase activity, 10 to 40 mM of 4-HPAA
was varied in the reaction mixture and the inhibition constant (Ki) was
determined. Lineweaver-Burk plot was constructed to calculate the ki-
netic parameters and to investigate the type of inhibition involved by
adding 0, 10, 15 and 20 mM of 4-HPAA with varied substrate (4-HPAN)
concentration (5 mM–50 mM) in the reaction mixture.
2.8. Bioprocess development
2.8.1. Time course of nitrilase reaction at different substrate concentration
Concentration of 4-hydroxyphenylacetonitrile in the reaction mix-
ture (1 ml) was varied from 5 mM to 150 mM and the enzyme activity
was assayed at intervals of 30 min until 160 min in order to study the
effect of substrate concentration on nitrilase activity.
2.8.2. Effect of enzyme concentration
The whole cell enzyme concentration was varied (6 U/ml–21 U/ml)
with 40 mM substrate concentration and the samples were withdrawn
after every 30 min upto 120 min for HPLC analysis.
2.8.3. Whole cell enzyme and substrate ratio
The effect of increasing whole cell enzyme with respect to the
substrate (4-HPAN) concentration on the formation of product (4-
HPAA) was observed by taking different combinations of substrate
concentration (mM) and biocatalyst amount (U/ml): 40 mM : 18 U/ml,
50 mM : 22 U/ml and 60 mM : 27 U/ml and 80 mM : 36 U/ml in
K2HPO4/KH2PO4 buffer (50 mM, pH 7.0). The reaction took place at
45°C for 160 min and the product formed was estimated at an interval
of 30 min by HPLC.
2.8.4. Fed batch at 50 ml scale (40 mM 4-hydroxyphenylacetonitrile/
30 min)
40 mM substrate concentration was chosen along with 18 U/ml
whole cell enzyme and fed for 5 successive feedings at an interval of
30 min each, under optimized conditions in 50 ml reaction.
2.8.5. Fed batch at 50 ml scale (40 > 35 > 25 > 20 mM 4
hydroxyphenylacetonitrile/30 min)
It was observed that nitrilase encountered inhibition after third
feeding when substrate was fed at a concentration of 40 mM in 5 suc-
cessive feedings. Therefore, substrate was added at lower concentration
N. Thakur et al.
Fig. 1. Thermostability profile of whole cell nitrilase of A. faecalis MTCC 12629 (A) Relative activity of the enzyme at 35°C, 40°C, 45°C and 50°C at different time
intervals (B) Plot of ln Eo/E vs time interval, where Eo is the enzyme activity at 0 h and E is the activity after time interval of 6 h. Half-lives (Thalf) of the whole cell
nitrilase at 40°C, 45°C and 50°C were calculated to be 45 h, 36 h and 13 h respectively.
for 5 feedings in decreasing order as follows 40 mM : 18 U/ml, 35 mM :
18 U/ml, 30 mM : 18 U/ml, 25 mM : 18 U/ml and 20 mM : 18 U/ml, at
an interval of 30 min in 50 ml reaction.
2.8.6. Scale up at 500 ml scale (40 > 35 > 30 > 25 > 20 mM 4-
hydroxyphenylacetonitrile/30 min)
Based on the results of the preceding reaction at 50 ml scale, the
reaction was scaled up to 500 ml in a 1.5 l Bioflow C-32 fermenter (New
Brunswick Scientific, USA) under optimized conditions (50 mM
K2HPO4/KH2PO4 buffer, pH 7.0), at 45°C. Five feedings of 4-HPAN
were carried out at lower level, as per the previous experiment, in order
to maximize the product formation.
3. Results
3.1. Effect of inducer
This experiment was conducted to investigate the effect of various
inducers (nitriles, amides, acids) on nitrilase induction. The experi-
mental result revealed aliphatic nitriles as the best inducers for induc-
tion of A. faecalis MTCC 12629 nitrilase. Aromatic nitriles (benzonitrile,
phenylacetonitrile), aryl nitriles (mandelonitrile), mandelic acid, acet-
onecyanohydrin and ε-caprolactum completely inhibited the growth of
A. faecalis MTCC 12629. Aliphatic nitriles such as isobutyronitrile,
butyronitrile, propionitrile, adiponitrile and valeronitrile, as inducers,
resulted in fairly high biomass and induction of nitrilase. Although
certain amides such as acrylamide, N-methylacetamide and 2-cyano-
pyridine supported the growth of the organism, yet these did not induce
nitrilase. Isobutyronitrile emerged as the most appropriate inducer in
terms of biomass yield and nitrilase induction, followed by butyronitrile
and adiponitrile (Supplementary Table S1).
3.2. Isobutyronitrile (inducer) feeding for hyperinduction of nitrilase
Among the different sets of nitrilase induction by isobutyronitrile in
A. faecalis MTCC 12629, set 1 (control, without induction) caused
negligible induction of nitrilase activity, which showed the inducible
nature of this enzyme. In set 1, the growth profile of A. faecalis MTCC
12629 exhibited stationary phase at 20 h of incubation, but addition of
isobutyronitrile at 0 h to the culture medium (set 3) delayed the
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Process Biochemistry 73 (2018) 117–123
stationary phase of this microorganism to 26 h. Furthermore, the ad-
dition of isobutyronitrile in high concentration at 0 h (set 4, 5, 6)
proved to be toxic to the cells of A. faecalis MTCC 12629. However,
addition of isobutyronitrile after 4 h (set 7), 8 h (set 8) and 12 h (set 9)
to the medium enhanced the induction of nitrilase activity in A. faecalis
MTCC 12629. Feeding of isobutyronitrile at high concentration in late
log phase of growth (set 15) inhibited the growth of the microorganism
compared to other sets due to the toxicity of inducer to the cells.
Addition of 50 mM isobutyronitrile at 4 h (set 7) resulted in maximum
induction of nitrilase activity (Supplementary Table S1, Fig. S1 A) and
growth of the present microorganism (Supplementary Fig. S1B).
Therefore, 4 h and 50 mM isobutyronitrile proved to be the most sui-
table time and concentration of inducer, respectively. This approach
caused a 1.2-fold increase in nitrilase production.
3.3. Optimization of reaction conditions for biotransformation of 4-HPAN
to 4-HPAA by whole cell nitrilase of A. faecalis MTCC 12629
3.3.1. Buffer system and ionic strength
The results of nitrilase assay in seven buffer systems revealed po-
tassium phosphate buffer (pH 7) as the most suitable buffer for nitrilase
activity (0.25 U/mg dcw). The nitrilase also retained 50% activity in
citrate buffer at pH 4.0. Optimum strength of potassium phosphate
buffer was found to be 50 mM (0.25 U/mg dcw) for the activity of ni-
trilase. Above and below this strength of buffer, activity of nitrilase was
declined. Thus, 50 mM potassium phosphate buffer (pH 7) was chosen
for successive experiments.
3.3.2. Thermal/operational stability
Assay of nitrilase activity at various temperatures revealed 45°C to
be an optimum temperature for enzyme activity (0.28 U/mg dcw).
Although this enzyme was found to be active in the range of 25°C to
55°C, yet a significant loss in nitrilase activity was observed above 55°C
(Fig. 1A). This enzyme retained its nitrile degrading activity at 35°C,
40°C and 45°C for more than 24 h and was deactivated abruptly at 60°C
within an hour. Half-life (t1/2) of nitrilase was calculated from plots of
log relative activity versus time at 40°C (45 h), 45°C (36 h) and 50°C
(13 h) (Fig. 1B). This enzyme was found to be very stable at 30°C for
more than 15 days without any loss in nitrile hydrolyzing activity.
Maximum activity as well as operational stability of the nitrilase were
N. Thakur et al.
observed at 45°C, therefore subsequent experiments for the develop-
ment of nitrilase mediated process for the conversion of 4-HPAN to 4-
HPAA were carried out at this temperature.
3.3.3. Substrate specificity
Isobutyronitrile induced whole cell nitrilase of A. faecalis MTCC
12629 was used to screen various aliphatic, aromatic, heterocyclic and
arylacetonitriles, which gave an overview of the clear preference of
present nitrilase towards aryacetonitriles. Negligible activity was ob-
served with other classes of nitriles (aromatic, aliphatic, dinitriles,
heterocyclic). 4-hydroxyphenylacetontrile (0.28 U/mg dcw) and 4-
aminophenylacetonitrile (0.29 U/mg dcw) emerged as the best sub-
strates for nitrilase activity followed by mandelonitrile and phenylgly-
cinonitrile. Amide hydrolyzing activity was not exhibited by A. faecalis
MTCC 12629 when reactions were carried out with different amides as
substrate.
3.3.4. Product inhibition and kinetic parameters
Nitrilase enzyme started losing its activity when the product
(10–40 mM) was directly added at 0 h in the reaction mixture (Fig. 2A),
whereas gradual formation of 4-HPAA from 4-HPAN in the reaction did
not show inhibitory effect at the same concentration of product formed
(data not shown). Inhibition constant (Ki) was calculated to be 18 mM
from the plot by taking varied concentration of product versus nitrilase
activity at optimized substrate concentration and 18 U/ml of enzyme
(Fig. 2A). Michaelis constant (Km) and reaction velocity (Vmax) of ni-
trilase with 4-hydroxyphenylacetonitrile were calculated to be 20 mM
and 2 U/mg dcw, respectively from Lineweaver-Burk plot (Fig. 2B).
Study of inhibition kinetics showed the inhibitory effect of 4-HPAA on
enzyme with the apparent value of Km as 40, 45, 50 mM and Vmax as
1.6, 1.5, 1.1 U/mg at 10, 15 and 20 mM product concentration, re-
spectively (Fig. 2B).
3.4. Bioprocess development
3.4.1. Time course of enzyme at different substrate concentration
Effect of 4-hydroxyphenylacetonitrile concentration revealed in-
hibitory effect on the nitrilase activity at higher concentration, with the
whole cell enzyme concentration of 3 U/ml in the reaction. It was ob-
served that with the increase in substrate concentration
(10 mM–40 mM), formation of product slowly increased with the pro-
gression of time. A sharp decline in nitrilase activity was observed after
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Process Biochemistry 73 (2018) 117–123
Fig. 2. Effect of substrate and product concentration on whole
cell nitrilase activity using 18 U/ml of activity (A)
Determination of inhibition constant (Ki) with the addition of
product (4-HPAA) ranging from 10 to 40 mM in 1 ml reaction
volume (B) Lineweaver-Burk plot: symbol ♦ depicts the KM
20 mM and Vmax 2 U/mg dcw of whole cell nitrilase for 4-
HPAN and symbols ■, ▲ and x represent 4-hydro-
xyphenylacetic acid inhibition kinetics at product concentra-
tion 10 mM, 15 mM and 20 mM, respectively.
50 mM 4-HPAN. Therefore, 40 mM substrate concentration was selected
for further bioprocess experiments. Complete loss of enzyme activity
was observed at 150 mM 4-HPAN in the reaction containing 3 U/ml of
nitrilase activity.
3.4.2. Effect of enzyme concentration
With the increase in biocatalyst amount (6 U/ml-21 U/ml), the rate
of product formation was also increased with shorter incubation time.
The reaction containing 18 U/ml whole cell enzyme and 40 mM sub-
strate (4-HPAN) resulted in maximum conversion of substrate into
product in 30 min of incubation at 45°C. Addition of more biocatalyst
did not enhance the product formation in shorter incubation time
(Fig. 3).
3.4.3. Whole cell enzyme and substrate ratio
The experiment conducted to investigated the effect of increasing
amount of whole cell enzyme with respect to the substrate concentra-
tion deduced 40 mM substrate concentration and 18 U/ml combination
as the most appropriate for the highest conversion. Thus, the sub-
sequent scales up reactions were carried out with 40 mM substrate
concentration and 18 U/ml of nitrilase (Fig. 4). It was also inferred from
this experiment that the proportional increase in biocatalyst amount
with the substrate did not show higher conversion rate due to inhibition
of nitrilase by high substrate loading.
3.4.4. Fed batch at 50 ml scale (40 > 35 > 30 > 25 > 20 mM 4-
hydroxyphenylacetonitrile/30 min)
Fed batch approach applied at 50 ml scale with the addition of 18
U/ml whole cell nitrilase and 40 mM substrate resulted in steady de-
cline in the rate of conversion of substrate after the first feeding of the
substrate (40 mM). Thus, the substrate was added in lower amount in 5
feedings, at an interval of 30 min in order to achieve maximum yield. A
total of 5 feedings of the substrate i.e. 40 mM, 35 mM, 30 mM, 25 mM
and 20 mM, were made which resulted in complete conversion of the
substrate into product with accumulation of 150 mM 4-HPAA.
3.4.5. Scale up at 500 ml scale (40 > 35 > 30 > 5 > 20 mM 4
hydroxyphenylacetonitrile/30 min)
Addition of lower concentration of substrate in five feedings led to
100% conversion of substrate into product (Fig. 5). After complete
conversion in 5 feedings, the product formed (150 mM) was recovered
by lowering the pH of the reaction. The white precipitates were
N. Thakur et al.
separated by filtration and dried using heat. HPLC analysis of recovered
product confirmed the 96% purity of 4-HPAA when compared with the
standard of 4-HPAA. The purified product was also analysed through
analysis which further confirmed the formation of 4-hydro-
xyphenylacetic acid (Supplementary Fig. S2A & S2B). The catalytic and
volumetric productivity were calculated to be 4.13 g/g dcw/h and 23 g/
l, respectively.
4. Discussion
The present work was carried out with the focus on exploring the
arylacetonitrilase activity of A. faecalis MTCC 12629 for the synthesis of
4-hydroxyphenylacetic acid. Growth kinetics and physiological
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Process Biochemistry 73 (2018) 117–123
Fig. 3. Effect of whole cell nitrilase concentration on
the conversion of 40 mM 4-hydroxyphenylacetonitrile
to 4-hydroxyphenylacetic acid in the reaction con-
taining 50 mM potassium phosphate buffer (pH 7.0) at
45°C. Reaction was carried out with specified con-
centration of whole cell nitrilase as per the legend: 6
U/ml (♦), 9 U/ml (■), 12 U/ml (▲), 15 U/ml (x), 18
U/ml (●) and 21 U/ml (●). All the measurements
were done in triplicates and the data shown are
means ± standard deviation (SD).
parameters were studied to enhance the expression and activity of ni-
trilase from A. faecalis MTCC 12629. Kinetics of nitrilase production
revealed the inducible nature of the enzyme unlike constitutive nitrilase
of Alcaligenes sp. ECU0401 [26], Bacillus subtilis ZJB-063 [27] and
Klebsiella ozaenae [28]. Short chain aliphatic nitriles proved to be the
potential inducers for nitrilase of A. faecalis MTCC 12629. The pro-
duction of nitrilase of A. faecalis MTCC 12629 was observed to be
maximum with isobutyronitrile as an inducer, which was similar to the
nitrilase from R. rhodochrous PA-34 [29], N. globerula NHB-2 [7], Gor-
donia terrae [30] and R. rhodochrous J1 [31]. Growth of the present
microorganism was retarded in the presence of nitriles in the produc-
tion medium compared to the medium without inducer, due to toxicity
of nitriles. A similar trend was followed by the reported nitrilase of
Fig. 4. Effect of whole cell enzyme and sub-
strate concentration for maximum conversion
of 4-hydroxyphenylacetonitrile to 4-hydro-
xyphenylacetic acid in 50 mM potassium
phosphate buffer (pH 7.0) at 45°C. Symbols ♦,
▲, x and ● represent combination of whole
cell enzyme and substrate concentration: 18 U/
ml:40 mM; 22 U/ml:50 mM; 27 U/ml:60 mM
and 36 U/ml:70 mM, respectively. Dotted lines
represent the conversion of substrate and solid
lines represent the formation of product during
the reaction. Data represent means ± SD of
triplicate determinations.
N. Thakur et al.
Fig. 5. Formation of 4-hydroxyphenylacetic acid in 500 ml fed batch reaction
with 18 U/ml of whole cell biocatalyst and five feedings of substrate 4-hydro-
xyphenylacetonitrile at an interval of 30 min. Extent of formation of 4-hydro-
xyphenylacetic acid and conversion of 4-hydroxyphenylacetonitrile in the re-
action is denoted by the symbols (■) and (▲) respectively. Dotted lines with
symbol ▲ represent 40 mM substrate feeding per 30 min interval and solid
lines with symbol ▲ denote five feedings of substrate: first feeding (40 mM),
second (35 mM), third (30 mM), fourth (25 mM) and fifth (20 mM). Upper and
lower ▲ in the dotted and solid lines depict substrate concentration after and
before the addition of 4-hydroxyphenylacetonitrile per 30 min respectively.
Nocardia globerula NHB-2 [10]. Isobutyronitrile feeding at varied time
intervals resulted in enhanced production of nitrilase from A. faecalis
MTCC 12629 which was decreased with the progression of time, and
high level of nitrilase expression was achieved in 21 h compared to N.
globerula NHB-2 (30 h, isobutyronitrile) [32], Gordonia terrae mutant E9
(36 h, isobutyronitrile) [30], R. rhodochrous K22 (120 h, iso-
valeronitrile) [33], R. rhodochrous J1 (120 h, ε-caprolactam) [31], and
Alcaligenes sp. MTCC 10675 (24 h, isobutyronitrile) [16]. The increase
in nitrilase activity was observed to be 1.2-fold for A. faecalis MTCC
12629, 1.5-fold for Alcaligenes sp. MTCC 10675, 1.2 fold for G. terrae
mutant E9 and 1.1-fold for P. putida nitrilase [16,30,17] from inducer
feeding experiment. A number of nitrilases have been reported for their
substrate specificity as aromatic, aliphatic, heterocyclic, but only a few
of the nitrilases are reported to degrade arylacetonitriles. The nitrilase
of A. faecalis MTCC 12629 in the present study preferably hydrolyzed
arylacetonitriles similar to that of P. fluorescens DSM 7155 [34], Bacillus
subtilis ZJB-063 [27], P. fluorescens EBC191 [35] and Alcaligenes sp.
MTCC 10675 [16]. However, nitrilase of A. faecalis MTCC 10757 and
Alcaligenes sp. ECU0401 are reported to degrade aliphatic and aromatic
nitriles, respectively [26,36]. To harness the maximum activity of en-
zyme for transformation of 4-HPAN to 4-HPAA, the present enzyme was
characterized by manipulating various reaction parameters. The ar-
ylacetonitrilase activity of present nitrilase was recorded as maximum
in potassium phosphate buffer pH 7, and was found to be stable from
pH 6 to 8, compared to the optimum pH and stability of arylacetoni-
trilase of Alcaligenes sp. MTCC 10675 (pH 6, 6-7), A. faecalis ATCC 8750
(pH 7.5, 6.5–8) and P. putida (pH 7, 6.5–8) [16,37,17]. Nitrilase of A.
faecalis MTCC 12629 exhibited highest activity at 45°C whereas the
temperature optima of 40°C, 55°C, 45°C and 40–45°C have been re-
ported for arylacetonitrilase of P. putida [17], P. fluorescens DSM 7155
[34], Alcaligenes sp. MTCC 10675 [16] and A. faecalis ATCC 8750 [37],
respectively. Thermostability is an important criteria and a desirable
requirement for biotransformation reactions as it is related to the sta-
bility of biocatalyst at a particular temperature for longer time dura-
tions [38]. The previous reports on bacterial arylacetonitrilases from
Alcaligenes sp. MTCC 10675, Bradyrhizobium japonicum USDA110, and
P. putida revealed their stability between 30 to 50°C [16,39,17].
The whole cell nitrilase in the present work was active and stable
122
Process Biochemistry 73 (2018) 117–123
within the temperature range of 30–55°C with the half-life of 45 h, 36 h,
13 h and 2 h at 40°C, 45°C, 50°C and 55°C respectively. The ar-
ylacetonitrilase of P. putida had half-life of 76 min at 40°C and 9 min at
50°C [17]. Substrate inhibition at higher concentration and product
inhibition of nitrilases are well reported earlier [13,10,40,41] which
are similar to the results of present study. Therefore, the fed batch
method was preferred for the synthesis of 4-HPAA from 4- HPAN and a
similar strategy has been considered in the studies on nitrilase reported
by [16,13,10,42]. Most of the studies on arylacetonitrilases are related
to their characterization and exploration to form commercially im-
portant arylacetic acid such as mandelic acid [18,43–47]. Previously,
the bacterial strains such as P. putida MTCC 5110 (0.39 g/g dcw) [17],
A. faecalis ECU0401 (3.8 g/g dcw) [26] and Alcaligenes sp. MTCC 10675
(3.9 g/g dcw) [13,16], possessing arylacetonitrilases, were used to
synthesize mandelic acid. Nitrilase mediated biotransformation process
was also developed for the synthesis of phenylacetic acid from pheny-
lacetonitrile by using recombinant Escherichia coli M15 harbouring a
double mutant MG nitrilase (I113 M/Y199 G) from Burkholderia cen-
ocepacia J2315 and whole cell nitrilase of Alcaligenes sp. MTCC 10675
[48,49]. However, fed batch synthesis of 4-hydroxyphenylacetic acid
has not been carried out so far. Fed batch synthesis at 500 ml scale
resulted in 23 g/l 4-HPAA production, with a catalytic productivity of
4.13 g/g dcw/h by utilizing whole cell nitrilase of A. faecalis MTCC
12629. This is the first report on the synthesis of 4-HPAA by employing
whole cells nitrilase enzyme with better thermal and operational sta-
bility which can be very useful for industrial scale biotransformation
processes. Thus, nitrilase mediated route can be an alternative to che-
mical routes for synthesis of such an important acid as mentioned in the
present investigation. Furthermore, various strategies can be applied to
enhance the product formation through immobilization of enzyme and
chemical mutagenesis.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors acknowledge the University Grant Commission (UGC),
New Delhi for financial support in the form of Senior Research
Fellowship to Ms. Neerja Thakur under UGC-Special Assistance
Programme and Faculty Fellowship to Prof. T.C. Bhalla under UGC-BSR
scheme. The computational facility availed at Bioinformatics Centre,
Himachal Pradesh University is also duly acknowledged.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.procbio.2018.07.012.
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