Simultaneous purification of nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674

Alcaligenes sp. MTCC 10674 has a bienzymatic system for the hydrolysis of nitriles. The nitrile hydratase and amidase have been purified simultaneously to homogeneity using a combination of (NH)4SO4 precipitation, ion exchange chromatography and gel permeation chromatography. Nitrile hydratase and amidase have molecular weight of 47 and 114 kDa, respectively and exist as heterodimer. Optimum temperatures for maximum activity of nitrile hydratase and amidase were 15 °C (2.4 U/mg protein) and 45 °C (2.3 U/mg protein), respectively. Nitrile hydratase showed maximum 7.8 U/mg protein at 50 mM acrylonitrile and amidase has 9.2 U/mg protein at 25 mM propionamide. Nitrile hydratase has Vmax 10 μmol/min/mg and Km 40 mM, while amidase has Vmax 12.5 μmol/min/mg and Km 45.5 mM, respectively. Heavy metal ions Hg2+, Ag+, Pb2+ and Cu2+ were strong inhibitors of nitrile hydratase and amidase activity.


Introduction
Nitrilase (EC 3.5.5.1), nitrile hydratase (EC 4.2.1.84) and amidase (EC 3.5.1.4) constitute an important class of nitrilase superfamily (Branner 2002). Nitrilase hydrolyzes the nitriles to acids and ammonia in one-step reaction (Sorokin et al. 2007). However, nitrile hydratase first hydrates the nitrile to corresponding amide which is subsequently hydrolyzed by amidases into organic acid and ammonia (Bhalla and Kumar 2005;Bhatia et al. 2013). There is considerable industrial interest in the enzymatic conversion of nitriles because of the increasing demand for conducting such conversions under mild conditions that are often compatible with the sensitive structures of many industrially important compounds. This environmental friendly bioconversion allows clean and mild synthesis with high selectivity and yield. Several bioprocesses have already been reported for the conversion of nitriles and amide compounds into their corresponding acids (Raj et al. 2006). Number of amides has been synthesized using nitrile hydratase, e.g., nicotinamide and butyramide (Raj et al. 2006;Prasad et al. 2007). Amidases are used as catalyst in effluent treatment and their acyltransferase activity is harnessed for the synthesis of pharmaceutically important compounds such as acetohydroxamic acids and benzohydroxamic acid (Prasad et al. 2007;Sharma et al. 2012;Bhatia et al. 2012). Despite immense potential of these hydrolyzing enzymes, these are not used for the commercial production of acids because of non availability of desired enzymes vis-à-vis their cost, selectivity and stability. Alcaligenes sp. MTCC 10674 has a nitrile hydratase and amidase bienzymatic system for the hydrolysis of nitriles. Nitrile hydratase and amidase system was used for the production of a-hydroxyisobutyric acid from a-hydroxyisobutyronitrile (Bhatia et al. 2013), which finds its use in the synthesis of polymethyl methacrylate and acrylic glass (Singh et al. 2006) and acyltransferase activity of amidase used for benzohydroxamic acid production (Bhatia et al. 2012). Whole cell has nitrile hydratase activity accompanied with amidase activity which led to carboxylic acid as side product during the conversion of nitriles into amide (Brady et al. 2004), and purified enzyme can be used to overcome this. Therefore, the objective of present study was to develop simple steps for the purification of nitrile hydratase and amidase to reduce the cost of enzyme production and to characterize the nitrile hydratase and amidase using acrylonitrile and propionamide as substrate, respectively.

Chemicals
All chemicals were of analytical grade and they were purchased from Alfa Aesar, Johnson Matthey Company and Sigma (India).

Medium and cultural conditions
Alcaligenes sp. MTCC 10674 previously isolated from the soil sample of orchid garden of Kinnaur District of Himachal Pradesh (India), was cultured in minimal salt media (MSM) having pH 7.0 and containing g/L, Na 2 H-PO 4 Á12H 2 O 2.5 g, K 2 HPO 4 2.0 g, MgSO 4 Á7H 2 O 1.0 g, FeSO 4 Á7H 2 O 0.1 g, CaCl 2 Á2H 2 O 0.6 g, peptone 5.0 g at temperature 25°C for 24 h. MSM was supplemented with 40 mM isobutyronitrile after 6 h of growth as nitrogen source.
Enzyme assay of nitrile hydratase and amidase

Nitrile hydratase assay
The assay mixture contained 0.125 M NaH 2 PO 4 /Na 2 HPO 4 buffer (pH 8.0), 50 mM acrylonitrile and purified nitrile hydratase of Alcaligenes sp. MTCC 10674 at 15°C for 20 min. Acrylamide production was measured spectrophotometrically at 230 nm. One unit of nitrile hydratase activity was defined as amount of enzyme that hydrates the acrylonitrile to release 1 lmol of acrylamide per minute under assay condition.

Amidase assay
Assay of purified amidase was carried out in 0.075 M NaH 2 PO 4 /Na 2 HPO 4 buffer (8.0), 50 mM amide and purified amidase of Alcaligenes sp. MTCC 10674 at 45°C for 20 min. Ammonia assay was performed for the amidase activity. One unit of amidase activity was defined as the amount of enzyme that hydrolyzes the propionamide to release 1 lmol of ammonia per minute under assay condition. Protein estimation was done according to Bradford method (Bradford 1976).

Purification of nitrile hydratase and amidase
Preparation of cell free extract Alcaligenes sp. MTCC 10674 cells were cultured in 1 L minimal salt medium at 25°C. After 24 h of incubation, cells were harvested from the culture broth by centrifugation at 10,000 g and washed twice with 0.1 M K 2 HPO 4 / KH 2 PO 4 (pH 7.0) and suspended in the same buffer. Bacterial culture of Alcaligenes sp. MTCC 10674 (15 mg/ ml) was disrupted using BeadBeater TM . The resultant suspension was centrifuged at 10,000 g at 4°C for 20 min to remove cell debris. The supernatant fluid was designated as the cell free extract (cell lysate) and stored at 4°C.

Ammonium sulfate fractionation
The cell free extract was subjected to ammonium sulfate saturation (20-60 %) and the precipitates collected after centrifugation at 15,000 g (25 min at 4°C) were suspended and dialyzed against the same buffer. The ammonium sulfate fractionate (ASF) having nitrile hydratase and amidase activity was further used for purification.

Ion exchange chromatography
The ASF having nitrile hydratase and amidase activity were subjected to DEAE-ion exchange chromatography. After loading the ASF onto the column, it was washed with potassium phosphate buffer pH 7.0 (0.05 M), until there was no further elution of protein. The column was subsequently eluted with a linear gradient of NaCl (from 0 to 0.5 M) in the same buffer.

Gel permeation chromatography
The protein fraction of ion exchange chromatography having amidase activity were pooled together and applied to gel permeation chromatography column. The gel permeation chromatography was performed using column (2.6 9 60 cm) packed with Sephacryl S-100 high resolution (GE Healthcare) matrix. The gel permeation column was pre-equilibrated with buffer and it was eluted with potassium phosphate buffer pH 7.0 (0.05 M) at a flow rate of 1.0 ml/min. The molecular weights of purified nitrile hydratase/amidase of Alcaligenes sp. MTCC 10674 were determined by SDS/Native-PAGE (Laemmli 1970).

Buffer molarity and temperature
The activity of purified nitrile hydrolyzing enzyme was estimated in K 2 HPO 4 /KH 2 PO 4 . The effect of buffer molarity on the activities of the enzymes was studied by varying the buffer concentration from 0.025 to 0.125 M in the reaction. The temperature optimum was determined by varying reaction temperature from 25 to 55°C.

Incubation time and stability
Incubation time for optimum activity of nitrile hydrolyzing enzyme was studied by varying the incubation time of reaction from 10 to 90 min. Thermal stability of the purified enzymes (nitrile hydratase/amidase) was investigated at 25 to 55°C.

Results and discussion
Purification Nitrile hydratase and amidase of Alcaligenes sp. MTCC 10674 were purified using different chromatography techniques. Acrylonitrile was used as substrate for nitrile hydratase characterization and propionamide was used as substrate for characterization of amidase. DEAE Sepharose ion exchange chromatography was performed and amidase got eluted with 0.125 M NaCl in fraction number 4-9 (Fig. 1) and further increase in NaCl concentration up to 0.15 M resulted in nitrile hydratase elution (Fig. 2). Fraction number 6, 7, 8, 9, 10 and 11 of 0.15 M NaCl elution contained purified nitrile hydratase and 6.3-fold purification was achieved in a single step (Table 1). Nitrile hydratase has been already purified from various organisms Corynebacterium pseudodipthereticu and Rhodococcus rhodochrous PA-34 up to 8.8 and 52-fold, respectively (Li et al. 1992;Prasad et al. 2009). Fraction 4, 5, 6, 7 and 8 obtained with 0.125 M NaCl showed amidase activity, were pooled together and applied in gel permeation column for further purification. Purified amidase was obtained in fraction number 14, 15 and 16 (Fig. 3), and 9.2-fold purification was achieved (Table 2), previously amidase has been purified from Pseudonocordia thermophila and Delftia tsuruhatensis CCTCCM 205114 up to 48 and 105-fold, respectively (Egorova et al. 2004;Wang et al. 2011). The purified amidase consists of two subunits of 52 and 49 kDa (Fig. 4a), while in native PAGE a single band of 114 kDa was observed (Fig. 4b).

Buffer pH and molarity dependence of Nhase and amidase
Nhase and amidase have maximum hydrolysis activity, i.e., 1.75 U/mg protein and 1.2 U/mg protein, respectively in sodium phosphate buffer (pH 8.0). Nitrile hydratase requires 0.125 M buffer for optimum activity (0.14 U/mg protein) and maximum amidase activity was achieved in 0.075 M (1.95 U/mg protein). Both the enzymes are active at neutral pH, as already reported for the nitrile hydratase of Corynebacterium sp. C5 (Tani et al. 1989).
(a) (b) Fig. 6 a Effect of metal ions on amidase activity. b Effect of metals ion on nitrile hydratase activity