Bioresource Technology 102 (2011) 6579–6586
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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech
An improved bioprocess for synthesis of acetohydroxamic acid using DTT
(dithiothreitol) treated resting cells of Bacillus sp. APB-6
Deepak Pandey, Rajendra Singh, Duni Chand ⇑
Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171005, India
a r t i c l e
i n f o
Article history:
Received 5 February 2011
Received in revised form 21 March 2011
Accepted 22 March 2011
Available online 12 April 2011
Keywords:
Acyltransferase
Bacillus sp. APB-6
N-methylacetamide
DTT
Acetohydroxamic acid
a b s t r a c t
Acyltransferase activity of amidase from Bacillus sp. APB-6 was enhanced (24 U) by multiple feedings of
N-methylacetamide (70 mM) into the production medium. Hyperinduced whole resting cells of Bacillus
sp. APB-6 corresponding to 4 g/L (dry cell weight), when treated with 10 mM DTT (dithiothreitol) resulted
in 93% molar conversion of acetamide (300 mM) to acetohydroxamic acid in presence of hydroxylamineHCl (800 mM) after 30 min at 45 °C in a 1 L reaction mixture. After lyophilization, a 62 g powder containing 34% (wt wt1) acetohydroxamic acid was recovered. This is the first report where DTT has been used
to enhance acyltransfer reaction and such high molar conversion (%) of amide to hydroxamates was
recorded at 1 L scale.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Amidase or amidohydrolase (E.C. 3.5.1.4) is an interesting member of nitrilase superfamily which catalyzes the hydrolysis of
amides to carboxylic acid and ammonia and is used by prokaryotes
in carbon and nitrogen fixation (Pace and Brenner, 2001). In industries amidases are employed in combination with nitrile hydratases for the production of commercially important organic acids
(acrylic acid, p-aminobenzoic acid, pyrazinoic acid, nicotinic acid
etc.) through biotransformation of nitriles (Banerjee et al., 2002).
They are also utilized as industrial catalysts in effluent treatment
(Madhavan et al., 2005; Nawaz et al., 1996) and wastewater treatment (Chand et al., 2004).
Different wide spectrum amidases exhibit acyl transfer activity
(transfer of R–CO group) in the presence of hydroxylamine (Fournand et al., 1998a) which acts as an acyl acceptor and the amide
acts as an acyl donor. Acyl transfer activity of amide-hydrolysing
enzymes has been described long since. Grossowicz et al. (1950)
reported the formation of hydroxamic acids (RCONHOH) by the enzyme-catalysed replacement of the amide groups of glutamine and
asparagine with hydroxylamine. Thiery et al. (1986) have reported
a wide spectrum amidase from Brevibacterium sp. R312 with acyl
transferase, acid transferase and ester transferase activity.
Maestracci et al. (1986) explained the detailed catalytic action
of the aliphatic amidase from Rhodococcus sp. R312 with acetamide
as acyl group donor and hydroxylamine as the acyl group acceptor
⇑ Corresponding author. Tel./fax: +91 177 2831948.
E-mail address: [email protected] (D. Chand).
0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.03.071
and suggested the mechanism was of ‘‘Ping Pong Bi Bi’’ type where
amides react with the enzyme to give acyl-enzyme complexes,
which then transfer acyl groups to the cosubstrate (water or
hydroxylamine) which lead to formation of carboxylates or
hydroxamates (Fig. 1). In case of group transfer reactions better
yield of product is observed when hydrolases with high ratios of
transferase to hydrolase activity are used (Kasche, 1986).
Acyltransferase activity of amidase has been used for the biosynthesis of a range of hydroxamic acids, which have a high chelating potential. Several hydroxamic acids are used as drugs and have
been reported as tumor inhibitors, anti-HIV and anticancerous
(Fournand et al., 1998b; Ramakrishna et al., 1999). Some hydroxamic acids can conjugate with metal ions and thus find their use to
eliminate metal ions in wastewater treatment and nuclear technology (Koide et al., 1987). Some other hydroxamic acids (a-aminohydroxamic acid, acetohydroxamic acid etc.) have also been
investigated as anti-human immunodeficiency virus agents, antimalarial agents and have also been recommended for treatment
of ureaplasma infections and anaemia (Gao et al., 1995; Holmes,
1996). Some fatty hydroxamic acids have been studied as inhibitors of cyclooxygenase and 5-lipooxygenase with a potent antiinflammatory activity (Hamer et al., 1996).
The unsubstituted aliphatic hydroxamic acids (such as acetohydroxamic acid) are well established as effective inhibitors of plant
and bacterial urease in vitro (Fishbein et al., 1965; Kobashi and
Hase, 1967) and have been shown to effectively inhibit ureolytic
activity and/or to lower blood ammonia levels in mice, rats, sheep,
cows, dogs and men (Brent and Adepoju, 1967; Streeter et al.,
1969).
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D. Pandey et al. / Bioresource Technology 102 (2011) 6579–6586
8000g (4 °C, 5 min) and washed twice with 0.2 M KH2PO4/
K2HPO4 buffer (pH 7.5), finally suspended in the same buffer and
were referred to as resting cells.
2.3. Enzyme assays
Fig. 1. Mechanism of the acyl transfer reaction from amides to hydroxylamine (a)
and the amide hydrolysis reaction (b) catalyzed by the aliphatic amidase from
Rhodococcus sp. R312 (Maestracci et al. 1986).
Keeping in view the novel characteristics of acyltransferase and
medical applications of acetohydroxamic acid (AHA), in the present
work a bioprocess has been developed for synthesis of AHA at 1 L
scale; using DTT (dithiothreitol) treated whole resting cells of
Bacillus sp. APB-6. The amidase of Bacillus sp. APB-6 shows good
acyltransfer activity and the cells were further induced to produce
acyltransferase by multiple feedings of N-methylacetamide in the
production medium. This is the first report where DTT has been
used to enhance acyltransfer reaction to achieve high molar conversion (%) of amide (acyl donor) to hydroxamates in presence of
hydroxylamine-HCl (acyl acceptor) at 1L scale.
2. Methods
2.1. Chemicals
All the chemicals were of analytical grade. The nitriles and
amides were from Alfa Aesar, A Johnson Matthey Company (earlier
Lancaster Synthesis). Media components were from HiMedia
(Mumbai) and the inorganic salts were of analytical grades. For
high performance liquid chromatography (HPLC), solvents were
from Merck, India.
2.3.1. Acyltransferase assay
Acyltransfer activity was determined spectrophotometrically by
a modified version of the method of Brammar and Clarke (1964).
The reaction mixture (2 ml) contained (if not otherwise mentioned) 0.1 M glycine-NaOH buffer (pH 7.5), 100 mM acetamide,
200 mM hydroxylamine HCl (both the substrates freshly neutralized with 10.0 N NaOH) and resting cells. This reaction mixture
was incubated at 45 °C for 5 min in a water bath shaker and the
reaction was stopped by the addition of 4 ml of FeCl3 reagent.
The reaction mixture was centrifuged at 5000g for 10 min, discarded the pellet and clear supernatant was collected for estimation of hydroxamic acid. The absorbance was read at 500 nm.
One unit (U) of acyltransferase activity was defined as that amount
of enzyme which catalyzed the release of one micromole of acetohydroxamic (AHA) acid per min under assay conditions.
2.3.2. Amidohydrolase assay
The amidohydrolase assay was performed in a reaction mixture
(2.0 ml) containing 0.1 M glycine-NaOH buffer (pH 7.5), 100 mM
acetamide and resting cells at 30 °C in a water bath shaker. After
15 min of incubation, reaction was stopped with equal volume of
0.1 N HCl. The amount of ammonia released in the reaction mixture was colorimetrically estimated using phenate-hypochlorite
method (Fawcett and Scott, 1960). One unit (U) of amidase activity
was defined as that amount of enzyme which catalyzed the release
of one micromole of ammonia per min by the hydrolysis of amide
under assay conditions.
2.3.3. HPLC analysis
For the direct quantitative estimation of substrate and product
in the assay mixture, HPLC was performed using series 200 Ic pump
(Perkin Elmer) equipped with Reverse phase Lichrosorb C18–5 lm
(4 125 mm) column (Merck) and 785A Programmable Absorbance
Detector (Applied Biosystem). The standard curves of acetamide
(20–200 mM) and acetohydroxamic acid (1–10 mM) were prepared. The absorbance was monitored using NetWin Software (Netel Chromatographs, India). The analysis of acetamide/
acetohydroxamic acid was done at a flow rate of 1.0 ml per min
at 210 nm using 25 mM Orthophosphoric acid with 1% (v/v) methanol as mobile phase. The volume of sample injected was 5 ll.
2.4. Hyperinduction of amidase for acyltransferase activity
2.2. Microorganism and culture conditions
Bacillus sp. APB-6, a nitrile metabolizing bacterium was isolated
from the soil samples of Shimla (Himachal Pradesh, INDIA). This bacterium has been identified and deposited as Bacillus sp. APB-6 at
Microbial Type Culture Collection (MTCC), Institute of Microbial
Technology, Chandigarh (India) with accession number MTCC-7540.
Preculture was prepared by transferring a single colony of Bacillus sp. APB-6 grown over nutrient agar for 48 h at 30 °C to 50 ml
medium containing peptone (0.5%), beef extract (0.3%), yeast extract (0.1%) and 1% glucose (pH 7.5) in 250 ml Erlenmeyer flask
and incubated at 30 °C, 160 rpm in an incubator shaker till
OD600 15. The preculture (4 ml) was inoculated into 50 ml of
medium (pH 9.0) containing peptone (2.6%), NaCl (0.3%), arabinose
(0.5%), yeast extract (0.9%), beef extract (0.7%) and (NH4)2HPO4
(0.66%) in 250 ml Erlenmeyer flask and incubated at 30 °C,
160 rpm in an incubator shaker. After specific incubation time
the cells from the culture were harvested by centrifugation at
2.4.1. Effect of various nitriles and amides on enzyme induction
Various inducers (nitriles and amides) were added at a concentration of 50 mM in the production medium (Table 1) to select
appropriate inducer for hyperinduction of acyltransferase activity
in the whole cells of Bacillus sp. APB-6. Both amidohydrolase and
acyltransferase activities were measured. The efficiency of selected
inducer for acyltransferase activity was tested by adding it in various concentrations (10–100 mM).
2.4.2. Time course of acyltransferase induction in presence of inducer
The growth curve and acyltransferase activity profile of Bacillus
sp. APB-6 was studied in 50 ml production medium supplemented
with inducer. In control, inducer was omitted from the production
medium. The samples (2.0 ml) were withdrawn after every 6 h up
to 72 h. The acyltransferase activity was assayed using acetamide
and hydroxylamine–HCl as substrates and the growth [mg dry cell
weight (dcw)/ml] of cells was determined turbidometrically (OD600).
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D. Pandey et al. / Bioresource Technology 102 (2011) 6579–6586
Table 1
Effect of nitriles/amides on growth and acyltransferase induction in Bacillus sp. APB-6.
Class
None (control)
Aromatic nitriles
Aliphatic nitriles
Hydroxynitriles
Amides
a
b
c
Namesa
Benzonitrile
Phenylacetonitrile
Acetonitrile
Propionitrile
Isobutyronitrile
Valeronitrile
Acrylonitrile
Adiponitrile
Glutarsurdinitrile
Acetonecyanohydrin
Mandelonitrile
Acetamide
Acrylamide
Methacrylamide
Butyramide
e-Caprolactam
Cyanoacetamide
Urea
Nicotinamide
Isonicotinamide
Glycinamide
Propionamide
Formamide
Lactamide
N-methylacetamide
di-methylformamide
N-ethylacetamide
Growth
(mg dcw/ml)
Acyl transferase
activityb (U)
Amido hydrolase
activityc (U)
3.85 ± 0.12
0
0
3.17 ± 0.12
2.94 ± 0.04
2.57 ± 0.06
1.86 ± 0.02
2.82 ± 0.05
3.52 ± 0.14
2.70 ± 0.08
0
0
3.36 ± 0.21
3.86 ± 0.17
3.15 ± 0.03
2.84 ± 0.11
3.47 ± 0.13
3.53 ± 0.18
3.81 ± 0.15
3.09 ± 0.16
3.43 ± 0.42
2.58 ± 0.29
2.49 ± 0.15
3.33 ± 0.14
3.54 ± 0.28
3.69 ± 0.22
4.00 ± 0.44
3.55 ± 0.23
6.00 ± 0.52
0
0
11.31 ± 0.55
9.96 ± 0.42
7.63 ± 0.32
6.98 ± 0.46
2.19 ± 0.12
3.86 ± 0.19
7.14 ± 0.32
0
0
10.11 ± 0.74
1.25 ± 0.04
3.08 ± 0.13
6.73 ± 0.28
5.50 ± 0.51
6.00 ± 0.62
6.88 ± 0.22
7.21 ± 0.63
5.94 ± 0.44
7.42 ± 0.63
9.67 ± 0.82
3.50 ± 0.28
4.50 ± 0.54
15.00 ± 0.92
3.50 ± 0.16
9.50 ± 0.48
4.23 ± 0.36
0
0
7.64 ± 0.16
8.28 ± 0.72
6.64 ± 0.22
4.94 ± 0.63
2.62 ± 0.45
2.89 ± 0.18
5.84 ± 0.34
0
0
12.22 ± 0.54
0.94 ± 0.14
3.68 ± 0.21
7.89 ± 0.62
3.72 ± 0.42
4.69 ± 0.23
5.18 ± 0.16
4.84 ± 0.34
4.20 ± 0.29
5.22 ± 0.14
10.47 ± 0.58
3.12 ± 0.22
3.79 ± 0.66
7.82 ± 0.36
3.21 ± 0.14
5.23 ± 0.29
Fifty millimolar inducer was supplemented in the production medium.
One unit (U) of acyltransferase activity was defined as one micromole of AHA formed per minute under assay conditions.
One unit (U) of amidase activity was defined as one micromole of ammonia released per minute under assay conditions.
2.4.3. Multiple feedings of inducer in the growth medium
Once the time of incubation for maximum enzyme production
was achieved, a study was carried out to find if the application of
inducer in multiple feedings (at different time intervals) could enhance the induction of enzyme. For this experiment sixteen different combinations were designed (Table 2).
dithiothreitol (DTT), phenyl methyl sulphonyl fluoride (PMSF) and
polyethyleneglycol (PEG) on the acyltransferase activity, these
were added to the reaction mixture at 1 mM final concentration.
Enzyme activity was assayed as described earlier in Section 2.3
but the reaction was carried out at 60°C in the presence of 0.2 M
KH2PO4/K2HPO4 buffer (pH 7.5) as these were the optimized reaction conditions for acyltransferase activity.
2.5. Bioprocess development for production of acetohydroxamic acid
2.5.1. Effect of metal ions and inhibitors
To work out the effect of various metal ions, carbonyl reagents
and chelators like Ethylene di amine tetra acetic acid (EDTA), Urea,
2.5.2. Optimization of resting cells’ concentration
Varied amounts of resting cell suspension [0.5–5 mg dry cell
weight (dcw) per ml of reaction mixture] were used to study optimum concentration of enzyme.
Table 2
Effect of multiple feedings of inducer into the production medium.
a
b
Combination No.
N-methylacetamidea
Growth (mg dcw/ml)
Enzyme activityb (U)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Only once at 0 h for 36 h
0h+6h
0 h + 12 h
0 h + 18 h
0 h + 24 h
0 h + 30 h
0 h + 6 h + 12 h
0 h + 6 h + 12 h + 18 h
0 h + 6 h + 12 h + 18 h + 24 h
0 h + 6 h + 12 h + 18 h + 24 h + 30 h
0 h + 12 h + 18 h
0 h + 12 h + 18 h + 24 h
0 h + 12 h + 18 h + 24 h + 30 h
0 h + 18 h + 24 h
0 h + 18 h + 24 h + 30 h
0h + 24h + 30h
3.13 ± 0.21
2.35 ± 0.17
2.50 ± 0.05
2.60 ± 0.02
3.08 ± 0.18
3.10 ± 0.12
2.23 ± 0.06
2.38 ± 0.19
2.28 ± 0.07
2.30 ± 0.09
2.45 ± 0.14
2.28 ± 0.11
2.78 ± 0.23
2.60 ± 0.08
2.40 ± 0.12
3.05 ± 0.17
17.93 ± 0.62
19.90 ± 0.87
21.66 ± 0.53
18.40 ± 0.39
20.33 ± 0.46
20.49 ± 0.21
21.19 ± 0.18
21.60 ± 0.57
22.50 ± 0.49
20.65 ± 0.31
23.40 ± 0.24
21.68 ± 0.22
23.72 ± 0.41
22.63 ± 0.36
22.57 ± 0.63
24.02 ± 0.44
Seventy millimolar N-methylacetamide was supplied in every single feed.
One unit (U) of enzyme activity was defined as one micromole of AHA formed per minute under assay conditions.
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2.5.3. Optimization of substrates’ concentration
Since the reaction for acyltransferase activity is a bi-substrate
reaction so the aim was to get the best combination of both the
substrates’ concentration (acetamide and hydroxylamine-HCl) for
maximum activity and bioconversion. This experiment was performed in such a manner that concentration of acetamide was varied (50, 100, 150, 200, 250, 300, 350 400, 450 and 500 mM) at
different fixed concentrations of hydroxylamine-HCl (100, 200,
300 400, 500, 600, 700, 800, 900 and 1000 mM). Total 100 combinations of both the substrates were available and the one with
maximum enzyme activity was used for biotransformation
reaction.
2.5.4. Time course of enzyme reaction at different temperatures for
bioconversion
To find out the best time and temperature combination for
maximum conversion of substrates into product, time course of enzyme reaction at different temperatures (45, 50, 55 and 60 °C) was
investigated by terminating the reaction at different intervals of
time viz. 5, 10, 20, 30, 40, 60, 80, 100 and 120 min. Optimized concentrations of substrates and resting cells were used in the reaction
mixture. The amount of product formed (mM) was calculated by
comparing with the AHA standard prepared by HPLC (Section
2.3.3).
2.5.5. Effect of DTT on conversion of acetamide to acetohydroxamic
acid at 50 ml scale
In the earlier experiments, the presence of DTT in the reaction
mixture had shown significant increase in the activity of enzyme,
so its role in the product formation was investigated. Reaction
was carried out at 50 ml scale in the presence of 0.2 M KH2PO4/
K2HPO4 buffer (pH 7.5), 300 mM acetamide, 800 mM hydroxylamine HCl and 4 mg cells dcw/ml reaction mixture at 45 °C for
30 min. DTT was used at a concentration of 1 mM in the reaction
mixture. A control was also run i.e. without DTT in the reaction
mixture. AHA formed was estimated by HPLC as described earlier
in Section 2.3.3.
2.5.6. Optimization of concentration of DTT for conversion of
acetamide to AHA at 50 ml scale
Again, the reactions were carried out at 50 ml scale. DTT was
used at different concentrations (1, 5, 10, 20, 30, 40 and 50 mM)
in the reaction mixtures. A control was also run i.e. without DTT
in the reaction mixture. Acetohydroxamic acid formed was estimated by HPLC.
2.5.7. Bioconversion of acetamide and hydroxylamine to AHA at 1 L
scale
The reaction was carried out in a 1.5 L fermenter (BioFlow C-32
New Brunswick Scientific, USA). The reaction mixture consisted of
0.2 M KH2PO4/K2HPO4 buffer (pH 7.5), 300 mM acetamide,
800 mM hydroxylamine HCl, 10 mM DTT and 4 g cells (dry
weight). The temperature of the vessel was maintained at 45 °C
and the impeller speed of 200 rpm was set for the proper mixing
of substrates and resting cells. The reaction was carried out for
30 min. AHA formed was estimated by HPLC as described earlier
in Section 2.3.3.
3. Results and discussion
3.1. Hyperinduction of amidase for acyltransferase activity
3.1.1. Effect of nitriles and amides on acyltransferase induction
The amidase of Bacillus sp. APB-6 shows high acyltransferase
activity as compared to amidohydrolase activity (Table 1). The
effect of supplementation of medium with different inducers on
the growth and acyltransferase expression of Bacillus sp. APB-6 is
summarized in Table 1. The growth of the organism was completely inhibited in presence of aromatic nitriles and hydroxynitriles. The amidase of this organism was expressed in presence of
most of the aliphatic nitriles and amides, however, in their presence there was some suppression of growth. Highest acyltransferase
activity
(15.00 ± 0.92 U)
was
observed
with
Nmethylacetamide whereas amidohydrolase activity was maximum
(12.22 ± 0.54 U) with acetamide. In the control medium (without
nitriles and amides) the amidohydrolase activity was
4.23 ± 0.36 U, acyltransferase activity was 6.00 ± 0.52 U and the
growth was 3.85 ± 0.12 mg dcw/ml. Although growth was highest
(4.00 ± 0.44 mg dcw/ml) in the medium supplemented with dimethylformamide but it repressed the enzyme activity
(3.50 ± 0.16 U) so N-methylacetamide was selected as the inducer.
Further optimization of inducer concentration showed that
70 mM N-methylacetamide was optimum for induction of acyltransferase from Bacillus sp. APB-6 (data not shown). Kelly and
Clarke (1962) have also reported N-methylacetamide as non-substrate inducer for amidase (with acyltransferase activity) from P.
aeruginosa.
3.1.2. Time course of acyltransferase induction in presence of inducer
The organism was a fast growing bacterium and attained stationary phase in 30 h of incubation, both in the presence and absence of inducer. There was suppression of growth in the
presence of inducer. Final pH of the culture increased with the
growth of the bacterium both in the presence and absence of inducer (data not shown) which means that ammonia was being produced as the organism utilized the organic and inorganic
nitrogen sources from the production medium. Maximum enzyme
activities, 8.92 ± 0.32 U (after 18 h) and 19.32 ± 0.52 U (after 36 h)
were recorded in the absence and presence of inducer respectively
(Fig. 2). Henceforth the organism was cultured for 36 h at 35 °C in
presence of 70 mM N-methylacetamide.
3.1.3. Multiple feedings of inducer in the growth medium
Out of the 16 different combinations designed (Table 2) for the
application of inducer in multiple feedings, sixteenth combination
in which inducer was added to the culture medium at 0, 24 and
30 h of incubation showed maximum enzyme activity
(24.02 ± 0.44 U). There was not much variation in growth of the
bacterium in the different combinations used; they almost showed
similar growth although multiple feedings of inducer resulted in
enhanced induction of enzyme.
3.2. Bioprocess development for production of acetohydroxamic acid
3.2.1. Effect of metal ions and inhibitors
The effect of various metal ions, chelators and carbonyl reagents
on the acyltransferase activity using resting cells of Bacillus sp.
APB-6 is shown in Fig. 3. Among the metal ions CuSO45H2O substantially inhibited acyltransferase activity at 1 mM concentration.
Acyltransferase of this bacterium was strongly inhibited by AgNO3
and HgCl2 which are known blockers of free thiols (Goil, 1978).
This indicates the presence of free thiol residues at the active site
of the enzyme. Presence of DTT showed a significant increase in
the acyltransferase activity (63 ± 2.17 U) as compared to Control
(without metal ion/inhibitor) that had an activity of 45 ± 2.22 U.
Mercaptoethanol has also been tested with the purified enzyme
(data not shown) and significant enhancement of enzyme activity
was recorded which shows that disulfide reductants play a major
role in increasing the amount of free thiols. Kotlova et al. (1999)
investigated the effect of metal ions and inhibitors on amidase of
Rhodococcus rhodochrous M8, and reported that heavy metal ions
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D. Pandey et al. / Bioresource Technology 102 (2011) 6579–6586
4.5
25
Growth without inducer
Growth with inducer
Activity without inducer
Activity with inducer
Growth (mg dcw/ml)
3.5
20
3
15
2.5
2
10
1.5
1
Enzyme activity (U)
4
5
0.5
0
0
0
6
12
18
24
30
36
42
48
54
60
66
72
Time (hours)
Fig. 2. Effect of incubation time on growth and induction of acyltransferase of Bacillus sp. APB-6.
70
Enzyme activity (U)
60
50
40
30
20
10
M Fer
ag ric No
ne
c n
siu hlo e
m rid
c
Zi hlo e
nc
r
C
s id
ob ulp e
a
h
C lt c ate
op
h
pe lor
So r s ide
di ulp
um h
at
c
e
Si hlo
lv
r
Ba er ide
n
ri
um itr
a
ch te
lo
ri
de
D
TT
ED
M
TA
er
cu
P
ri M
c c SF
So hlo
C diu rid
al
ci m a e
um z
i
ch de
lo
C
r
id
ad
e
m
iu
m Ure
ch a
lo
ri
de
M
L
P
an ea
EG
ga d n
ne
i
se tra
ch te
lo
ri
de
0
Metal ions and inhibitors (1mM)
Fig. 3. Effect of metal ions and inhibitors on acyltransferase activity of Bacillus sp. APB-6.
(characteristic sulfhydryl reagents) and Fe2+ inhibited enzyme
completely. Chelators (EDTA and o-phenanthroline) and serine
protease inhibitor PMSF did not influence the enzyme activity;
where as the presence of DTT caused a 1.5-fold increase in the
amidase activity (Kotlova et al., 1999).
This study shows that enzyme has free sulphydryl groups at its
active site, which might be due to the presence of cysteine residue,
as in case of aliphatic amidases that are related to nitrilases (Novo
et al., 2002).
3.2.3. Optimization of substrates’ concentration
The effect of substrates’ concentration on acyltransferase activity of Bacillus sp. APB-6 has been shown in Fig. 4. Out of the 100
combinations of substrates tested, maximum enzyme activity
(104 ± 1.98 U) was recorded with 300 mM acetamide and
800 mM hydroxylamine–HCl.
3.2.2. Optimization of resting cells’ concentration
Varied amounts of resting cell suspension [0.5–5 mg cells (dry
weight) per ml of reaction mixture] were used to study optimum
concentration of enzyme. Maximum acyltransferase activity
(90 ± 1.84 U) was recorded when 4.0 mg cells (dry weight) per ml
of reaction mixture were used above this concentration there
was no further increase in enzyme activity probably due to de-
3.2.4. Time course of enzyme reaction at different temperatures for
bioconversion
Maximum amount of AHA (261 ± 2.8 mM) was formed after
30 min. of reaction at 45 °C (Fig. 5). There was a decrease in concentration of acetohydroxamic acid with time at all the temperatures which might be due to the fact that hydroxamic acids also
act as substrates for amidases; Fournand et al. (1998c) have reported that amidases have very high affinity for hydroxamic acids.
crease in substrate enzyme ratio with increasing concentration of
resting cells.
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D. Pandey et al. / Bioresource Technology 102 (2011) 6579–6586
120
Enzyme activity (U)
100
80
100 mM hydroxylamine
200 mM hydroxylamine
300 mM hydroxylamine
400 mM hydroxylamine
500 mM hydroxylamine
600 mM hydroxylamine
700 mM hydroxylamine
800 mM hydroxylamine
900 mMhydroxylamine
1000 mM hydroxylamine
60
40
0
50
100
150
200
250
300
350
400
450
500
Acetamide concentration (mM)
Fig. 4. Optimization of substrates’ concentration for maximum acyltransferase activity.
280
45°C
50°C
55°C
60°C
Concentration of AHA (mM)
260
240
220
200
180
160
140
120
100
0
10
20
30
40
50
60
70
80
90
100
110
120
Time (minutes)
Fig. 5. Time course of enzyme reaction at different temperatures for maximum product formation.
This is one reason why it is difficult to achieve 100% conversion of
amide to hydroxamic acid.
Formation of AHA was more at higher temperatures (as compared to 45 °C) for the first 10 min but after 20 min the concentration began to decline due to the hydrolysis of AHA by the enzyme.
At 60 °C the amount of product became constant after 40 min of
incubation, perhaps due to the inactivation of enzyme, the thermostability study (data not shown) of enzyme had shown that at this
temperature enzyme rapidly loses activity and therefore there was
no further hydrolysis of AHA.
3.2.5. Effect of DTT on conversion of acetamide to acetohydroxamic
acid at 50 ml scale
The presence of DTT at 1 mM concentration did not show much
difference in the amount of product formed with respect to control.
In control 263 ± 3.2 mM AHA was synthesized whereas in presence
of DTT the amount of product formed was 272 ± 2.6 mM from
300 mM acetamide. It might be due to the fact that large amount
of resting cells were now being used for the conversion of substrates to product and 1 mM concentration of DTT was not able
produce significant effect, so DTT was used again at higher
concentrations.
3.2.6. Optimization of concentration of DTT for conversion of
acetamide to AHA at 50 ml scale
Increased concentration of DTT resulted in enhanced production of acetohydroxamic acid. The results are summarized in Table
3; 10 mM concentration of DTT resulted in 95% molar conversion of
Table 3
Optimization of concentration of DTT.
Concentration of DTT (mM)
AHA formed (mM)
Molar conversion (%)
Control (without DTT)
5
10
20
30
40
50
263 ± 2.14
276 ± 3.16
284 ± 2.22
285 ± 2.28
284 ± 3.32
286 ± 3.06
285 ± 2.34
88
92
94.7
95
94.7
95.3
95
D. Pandey et al. / Bioresource Technology 102 (2011) 6579–6586
acetamide to acetohydroxamic acid whereas in control (without
DTT) the molar conversion was 88%. Further increase in the concentration of DTT did not increase the % conversion.
This was an important finding, since it is very difficult to
achieve such high molar conversion yield when working on acyltransferase activity of amidase. Perhaps the method employed for
optimizing substrates’ concentration and the use of DTT for bioconversion played an important role in providing such high molar
conversion. Fournand et al. (1998a) have worked extensively on
acyltransferase activity of amidase from Rhodococcus sp. R312
and they could get only 86% molar conversion yield while working
with purified amidase by using acetamide (0.1 M) as acyl donor
and hydroxylamine–HCl as acyl acceptor (0.5 M). Since the amidase of Bacillus sp. APB-6 exhibits high ratio of transferase to
hydrolase activity, the better product yield up to 95% was achieved,
similar activity ratio effect has been hypothesized by Kasche, 1986.
Fournand et al. (1998c) have reported that the increase in concentration of hydroxylamine–HCl (10–500 mM) considerably reduced
the undesirable hydrolysis of amide as well as hydroxamic acid.
Since in present investigation we have already used 800 mM
hydroxylamine–HCl (optimized), it resulted in high yield of product (AHA) due to inhibition of hydrolysis of AHA.
3.2.7. Bioconversion of acetamide and hydroxylamine to AHA at 1 L
scale
Batch reaction was carried out at 1 L scale. The temperature of
the vessel was maintained at 45 °C and the reaction was carried
out for 30 min. At the end of the reaction 280 ± 3.14 mM acetohydroxamic acid (93% molar conversion yield) was produced, it was
2% less than that achieved at 50 ml scale but that loss can be due
to the change in kinetics with the change in reaction volume; still
it is more than that reported by earlier workers. Fournand et al.
(1997) have also synthesized AHA at 1 L scale by using purified
amidase of Rhodococcus sp. R312 immobilized on Duolite A-378 resin and they reported 61% molar conversion of acetamide to AHA.
Since the amidase of Bacillus sp. APB-6 shows a great potential
for synthesis of AHA at bench scale. In future the process parameters and reaction kinetics could be optimized for purified and
immobilized enzyme at commercial scale for the better yield of
AHA. Further, the synthesis of other pharmaceutically important
hydroxamic acids through biotransformation reactions catalyzed
by amidase of Bacillus sp. APB-6 can be explored.
3.3. Recovery of acetohydroxamic acid
After completion of the reaction, the resting cells were separated from the reaction mixture through centrifugation at
10,000g for 15 min. The supernatant obtained was collected and
freeze-dried (Flexi-Dry MP™, FTS USA) to recover the powder of
acetohydroxamic acid. Total 62 g of white powder of acetohydroxamic acid was obtained after lyophilization. When this powder was
compared with the commercial acetohydroxamic acid by HPLC
analysis, it showed that 34% (w/w) AHA was present in it.
Inorder to characterize the product, method reported by Fournand et al. (1997) was employed, 2 g of this powder was resuspended in 20 ml acetone after acidification with 0.6 ml 12.5 N
HCl. The solution was filtered to remove salts. The filtrate was allowed to evaporate and after evaporation slightly stained oil was
obtained which was subjected to HPLC analysis and showed only
one peak of AHA (RT: 1.35 min).
3.4. Recycling of recovered cells
There was 30% loss of the resting cells during reaction due to
disruption. The recovered cells from the reaction showed 84%
residual (88.2 ± 2.02 U) acyltransferase activity. These cells when
6585
used at in 50 ml reaction mixture for the next experiment resulted
in 94% conversion of the 300 mM acetamide to AHA.
4. Conclusion
The results obtained in this investigation show that Bacillus sp.
APB-6 efficiently expresses acyltransferase activity that has been
successfully used for the bench scale molar conversion (93%) of
acetamide (300 mM) to acetohydroxamic acid. Biotransformation
at commercial scale has a very high potential in contrast to chemical processes for the synthesis of AHA, which is a key compound
for a variety of applications including the use as pharmaceutical,
e.g., in the treatment of tumors, HIV and other health threats.
More over, to the best of our knowledge, no one has so far reported this much synthesis of AHA through biotransformation in
the presence of DTT. Therefore, we conclude that Bacillus sp.
APB-6 or the appropriate enzymes extracted from this microbe
could be successfully used for the commercial synthesis of AHA.
Acknowledgements
The authors acknowledge the Indian Council of Medical Research, New Delhi and Department of Biotechnology, Ministry of
Science and Technology, New Delhi for the financial support in
the form of Senior Research Fellowships to Mr. Deepak Pandey
and Mr. Rajendra Singh, respectively. The computational facility
availed at Bioinformatics Centre, H P University Shimla is also duly
acknowledged.
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