Purification and characterization of arylacetonitrile-specific nitrilase of Alcaligenes sp. MTCC 10675
S. K. Bhatia P. K. Mehta R. K. Bhatia T. C. Bhalla∗
Department of Biotechnology, Himachal Pradesh University, Shimla, India
Abstract Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 ◦ C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20 Min at its optimum temperature. The value
for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec− , and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 4, Pages 459–465, 2014
Keywords: Alcaligenes sp. MTCC 10675, arylacetonitrile, mandelonitrile, chromatography
1. Introduction α-Hydroxyphenylacetic acid (mandelic acid) is used in the synthesis of antitumor agents, antiobesity, semisynthetic penicillin, and cephalosporin [1, 2]. There is considerable industrial interest in the enzymatic conversion of nitriles into acid, as enzymatically these processes are regio- and enantioselective, and this has led to the isolation of a number of nitrile-hydrolyzing bacteria [3–5]. Nitrile-metabolizing organisms have nitrilase or nitrile hydratase and amidase enzyme systems that convert nitriles into their corresponding acids. There are different types of nitrilases according to their substrate specificity, e.g., aliphatic, aromatic, and arylacetonitrilases. Arylacetonitrilase
has immense potential for the hydrolysis of arylaliphatic nitriles; however, these biocatalytic processes are not widely adopted by the industry due to high cost, instability, low selectivity, and specificity of the enzyme [6]. Thus, there is a need to search new nitrilases to meet the above-mentioned challenges. Hitherto very few nitrilases have been reported that are substrate specific [6, 7]. A nitrile-degrading bacterium Alcaligenes sp. MTCC 10675 has been isolated in our laboratory, which has high affinity for arylacetonitriles [8]. In this article, purification, characterization, and sequence analysis of the amino acid sequence of arylacetonitrile-hydrolyzing nitrilase of Alcaligenes sp. MTCC 10675 is reported.
2. Materials and Methods Abbreviations: MTCC, microbial type culture collection; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; CFE, cell-free extract; ASF, ammonium sulfate fraction; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PCR, polymerase chain reaction. ∗ Address
for correspondence: Professor T. C. Bhalla, Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171 005, India. Tel: +91-177-2831948; Fax: +91-177-2832154; e-mail: [email protected]. Received 3 July 2013; accepted 11 December 2013 DOI: 10.1002/bab.1192 Published online 25 March 2014 in Wiley Online Library (wileyonlinelibrary.com)
2.1. Chemicals Isobutyronitrile and mandelonitrile were purchased from Lancaster Synthesis (Lancashire, UK). Media components were obtained from Hi-Media (Mumbai, India). All other chemicals were procured from Merck (Mumbai, India).
2.2. Microorganism and cultivation condition Alcaligenes sp. MTCC 10675 (isolated from soils of Shimla District of Himachal Pradesh, India, as a nitrile-metabolizing bacterium and identified at the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India, was grown overnight in a seed medium
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2.3. Arylacetonitrilase assay Arylacetonitrilase assay was carried out in a reaction mixture containing 0.1 M KH2 PO4 /K2 HPO4 buffer (pH 6.5), 20 mM mandelonitrile, and an appropriate amount of arylacetonitrilase at 50 ◦ C for 30 Min. The reaction was stopped by adding an equal volume of 0.1 N HCl to the reaction mixture. The ammonia released by the action of arylacetonitrilase on mandelonitrile was assayed using the phenate-hypochlorite method [9]. In terms of ammonia release, one unit of arylacetonitrilase activity was defined as the amount of enzyme that catalyzed the release of 1 µmol of ammonia/Min by the hydrolysis of nitrile under standard assay conditions.
2.4. Purification of arylacetonitrilase of Alcaligenes sp. MTCC 10675 2.4.1. Preparation of cell-free extract The Alcaligenes sp. MTCC 10675 cells were grown in 1 L of production medium, and after 24 H of incubation cells were harvested from the culture broth by centrifugation at 8,000g and washed twice with 0.1 M K2 HPO4 /KH2 PO4 (pH 7.0) buffer and suspended in the same buffer containing 1 mM DTT and 1 mM EDTA. The cell suspension (800 mg) was added to a bead beater chamber (100 mL capacity) containing half filled 0.1 mm glass beads, and cells were disrupted in eight cycles (each cycle involves agitation for 1.0 Min and 1.0 Min rest) using BeadBeaterTM (BioSpec Products, Bartlesville, OK, USA). The cell-free extract (CFE) was prepared by centrifuging crude extract at 15,000g for 25 Min. The protein content was estimated following the Bradford method [10]. All the purification steps were performed at 4 ◦ C.
2.4.2. Ammonium sulfate precipitation The cell-free extract was subjected to ammonium sulfate saturation (20–60%), and the precipitates collected after centrifugation at 15,000g (25 Min at 4 ◦ C) were suspended and dialyzed against the same buffer. The ammonium sulfate fraction (ASF) having arylacetonitrilase activity was used in a subsequent step of purification.
2.4.3. Gel permeation chromatography The gel permeation chromatography was performed using a column (2.6 × 60 cm) packed with the Sephacryl S-300 High Resolution (GE Healthcare, Little Chalfont, UK) matrix as per manufacturer’s instructions. The 12 mL of dialyzed protein sample having arylacetonitrilase activity was centrifuged at 15,000g for 10 Min at 4 ◦ C and loaded on a gel permeation
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column preequilibrated with buffer. The elution was performed at a flow rate of 1.0 mL/Min with the potassium phosphate buffer having pH 7.0 (0.05 M).
2.4.4. Anion exchange chromatography The fractions having arylacetonitrilase activity were pooled and applied on a DEAE-Sepharose anion exchange column (1.5 × 10 cm). After loading the sample, the column was washed with potassium phosphate buffer pH 7.0 (0.05 M) until there was no further elution of protein. The enzyme was then eluted with a gradient of NaCl (from 0 to 0.5 M) at a flow rate of 1 mL/Min in the same buffer. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions that showed arylacetonitrilase activity was performed in 10% gel [11].
2.4.5. Molecular weight determination The native molecular mass of the purified arylacetonitrilase enzyme was determined by gel permeation chromatography. The 30 µg enzyme was subjected to a SephadexTM G-200 (GE Healthcare) column and eluted with 50 mM KH2 PO4 /K2 HPO4 buffer, pH 6.5 with 1 mM DTT, and 1 mM EDTA containing 0.15 M NaCl and eluted at a flow rate of 0.5 mL/Min. The optical density of effluent was recorded at A280 . The relative molecular mass of the purified arylacetonitrilase was then calculated from the relative mobility compared with those of standard proteins, thyroglobulin (Mr, 669,000), ferritin (Mr, 440,000), catalase (Mr, 232,000), lactate dehydrogenase (Mr, 140,000), and albumin (Mr, 66,000) [12].
2.5. Characterization of purified arylacetonitrilase 2.5.1. Effect of pH The activity of purified arylacetonitrilase of Alcaligenes sp. MTCC was assayed in different buffers, e.g., citrate buffer (4.0– 6.0), sodium phosphate buffer (6.0–8.0), potassium phosphate buffer (6.0–8.0), borate buffer (7.0–9.0), and carbonate buffer (9.0–10.0). The 1.0 mL reaction mixture contained specified buffer and 20 mM mandelonitrile as a substrate. A reaction was carried out at 30 ◦ C for 1 H.
2.5.2. Buffer molarity and temperature The effect of buffer (potassium phosphate) molarity on the activity of arylacetonitrilase was studied by varying the buffer concentration from 0.025 to 0.125 M in the reaction. The optimum temperature was determined by carrying out the reaction at different temperatures (25–55◦ C).
2.5.3. Study of incubation time, stability, metal ions, and other kinetic parameters The incubation time for the optimum activity of arylacetonitrilase was determined by incubating the reaction mixture from 10 to 90 Min at 50 ◦ C. Thermal stability of the purified enzyme arylacetonitrilase was investigated at 25 –55◦ C for 5 H. The arylacetonitrilase activity was assayed in the presence of various metal ions and chemicals (AgCl, CaCl2 , CdCl2 , CsCl, CoCl2 , CuSO4 , FeSO4, HgCl2 , MgSO4 , MnCl2 , ZnSO4 , KCl, NaCl, EDTA, and DTT) with a final concentration of 1 mM. The Km ,
Arylacetonitrile-Specific Nitrilase of Alcaligenes sp. MTCC 10675
Purification summary of arylacetonitrilase of Alcaligenes sp. MTCC 10675
TABLE 1 Volume (mL)
Protein (mg/mL)
CFE
30
3.3
99
0.9
ASF
12
5.2
63
Sephacryl S -300
6
0.94
DEAE-sepharose
4
0.18
Purification stage
Total protein (mg)
Activity (U/mL)
Specific activity (U/mg)
Purification fold
Yield (%)
27
0.27
1
100
1.6
19
0.30
1
70
5.6
1.8
11
1.96
7
41
0.7
2.2
46
32
Vmax, kcat , and ki of the purified arylacetonitrilase of Alcaligenes sp. MTCC 10675 were determined using mandelonitrile as a substrate. Substrate specificity of purified arylacetonitrilase was studied against various aliphatic, aromatic, heterocyclic, and arylacetonitriles.
2.6. Polymerase chain reaction amplification of arylacetonitrilase gene sequence Genomic DNA of Alcaligenes sp. MTCC 10675 was extracted following the method as described by Sambrook et al. [13]. Primers for the amplification of arylacetonitrilase gene were designed on the basis of already reported arylacetonitrilase sequences retrieved from NCBI using OligoAnalyzer 3.1, i.e., >gi|158023988|gb|EF467660.1|, >gi|216202|dbj|D13419.1,| and >gi|54013472:105241–107199. Primers designed for amplification were as follows: forward primer S31NF 5 -ATG CAG ACA AGA AAA ATC G-3 and reverse primer S31NR 5 -TCA GGA CGG TTC TTG CAC CAG-3 . Alcaligenes sp. MTCC 10675 genomic DNA was used as a template DNA. An amplification reaction was performed using the following conditions: 5 Min hot start at 94 ◦ C; followed by 30 cycles consisting of denaturation (30 Sec at 94 ◦ C), annealing (60 Sec at 52 ◦ C), and extension (90 Sec at 72 ◦ C); and a final extension at 72 ◦ C for 5 Min. The polymerase chain reaction (PCR) product was visualized by the electrophoresis in 1.0% agarose gel containing ethidium bromide. The amplified PCR product was sequenced and used for the analysis.
2.6.1. Homology with other arylacetonitrilase The 1.1-kb PCR product was sequenced, and the NCBI Blast program was performed with the forward and reverse sequences to obtain the homologous arylacetonitrilase gene sequences deposited in the data bank of NCBI. The forward sequence was translated with a translate tool available at Expasy (http://web.expasy.org/translate/), and BLAST was performed to discover the homologous amino acid sequences of arylacetonitrilases. The ClustalW program was used to align the nucleotide and amino acid sequence of the arylacetonitrilase with other arylacetonitrilases and aliphatic nitrilases. A phylogenetic tree was also constructed using the ClustalW algorithm.
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Total activity (U)
8.8
12.5
2.6.2. Nucleotide sequence accession number Alcaligenes sp. S31 was deposited into the Microbial Type Culture Collection, Chandigarh, India, under the accession number MTCC 10675. The nucleotide sequence of the arylacetonitrilase gene was deposited into the GenBank database under the accession number KC019880.
3. Results 3.1. Purification of arylacetonitrilase The arylacetonitrilase was purified using gel permeation and ion exchange chromatography (Table 1). In S-300 gel permeation chromatography, arylacetonitrilase was eluted in the fraction number 19 (1.5 U/mg protein), 20 (2.3 U/mg protein), and 21 (2.1 U/mg protein). Gel permeation chromatography resulted in a sevenfold increase in arylacetonitrilase activity (1.96 U/mg protein). Fractions having arylacetonitrilase activity were pooled and used in anion exchange chromatography. The major fractions of arylacetonitrilase were eluted at 0.4 M NaCl, and most of the contaminating proteins were removed by eluting the column with low molarity NaCl. Ion exchange chromatography step proved to be a very effective step for the purification of arylacetonitrilase as it resulted in a 46-fold increase in specific activity (12.5 U/mg protein) with 32% yield as compared with activity and purity of the enzyme of CFE (Table 1). By gel filtration on a SephadexTM G-200 column, the molecular mass of the native enzyme was estimated to be 520 ± 60 kDa. The purified arylacetonitrilase of Alcaligenes sp. MTCC 10675 showed a single band of 60 ± 14 kDa on SDS-PAGE and consisted of seven or eight subunits (Fig. 1).
3.2. Characterization of purified arylacetonitrilase 3.2.1. Effect of pH and molarity The purified arylacetonitrilase of Alcaligenes sp. MTCC 10675 has maximum mandelonitrile-hydrolyzing activity (10.5 U/mg protein) in 0.075 M potassium phosphate buffer having pH 6.5. In citrate buffer, the activity of enzyme increases with the increase in the pH (4–5.5), whereas in borate and carbonate buffers very little activity (0.6–2.6 U/mg protein) was observed.
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Biotechnology and Applied Biochemistry activity, and at the 50 mM substrate concentration 76% loss in the activity was recorded. The Vmax, Km , Kcat , and ki values for the arylacetonitrilase were 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec,− and 32.4 ± 03 mM, respectively.
3.2.5. Stability of arylacetonitrilase The thermal stability of arylacetonitrilase was studied at different temperatures, and it was observed that arylacetonitrilase was stable at lower temperature (15◦ C–30◦ C). Exposure of the enzyme at 15, 25, and 35 ◦ C for 5 H caused 8%, 15%, and 28% decrease in arylacetonitrilase activity. A further increase in the temperature to 45 and 55 ◦ C resulted in 58% and 90% decreases in the activity, respectively. The half-life of this enzyme at 15, 25, 35, 45, 50, and 55 ◦ C was, respectively 31 H, 16 H, 9 H, 4 H 30 Min, 3 H 20 Min, and 2 H 40 Min.
3.2.6. Metal ions and inhibitor Metal ions of Hg2+ , Mg2+ , Cu2+ , and Co2+ were a strong inhibitor of arylacetonitrilase activity, which caused inhibition upto the extent of 98%, 91%, 87%, and 77%, respectively. The presence of 1 mM Ca2+ , Cd2+ , Mn2+ , Cs+ , and DTT in the assay mixture decreased arylacetonitrilase activity to its half. The EDTA, Zn2+ , Na+ , and K+ showed very little effect on arylacetonitrilase activity (Fig. 2).
3.2.7. Substrate specificity
FIG. 1
SDS-PAGE of different fractions in 10% gel. lane 1, marker; lane 2, CFE; lane 3, precipitate; lane 4, pooled fraction of sephacry S-300; lane 5, DEAE fraction; and lane 6, marker.
Nitrilase of Alcaligenes sp. MTCC 10675 is highly specific for arylacetonitriles, and 65 ± 0.40 U/mg protein and 26 ± 0.10 U/mg protein activity were recorded for phenylacetonitrile and mandelonitrile, respectively. The purified enzyme has very little activity against heterocyclic and aliphatic nitriles, and no activity was recorded against aromatic nitriles (Table 2).
3.2.8. Homology with other arylacetonitrilase 3.2.2. Temperature The mandelonitrile-hydrolyzing activity of arylacetonitrilase of Alcaligenes sp. MTCC 10675 increased with the increase in temperature, and maximum activity was observed at 50 ◦ C (12.4 U/mg protein). Above 50 ◦ C, a decrease in the activity of arylacetonitrilase was observed.
3.2.3. Incubation time Mandelonitrile-hydrolyzing activity of purified arylacetonitrilase was assayed at intervals of 10 Min until 90 Min; a continuous increase in arylacetonitrilase activity was recorded with the increase in incubation temperature and highest arylacetonitrilase activity (27 U/mg protein) was recorded at 30 Min incubation; thereafter the activity remained more or less constant until it decreased as the enzyme might have undergone product inhibition.
3.2.4. Vmax , Km , kcat , and ki values of arylacetonitrilase Arylacetonitrilase activity increased with the mandelonitrile concentration in the reaction, and maximum activity was recorded at 15 mM of mandelonitrile. A further increase in the substrate concentration resulted in a decrease in enzyme
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Amino acid alignment with other nitrilase superfamily sequences showed that a catalytic triad (Glu–Lys–Cys) comprises Glu48, Lys131, and Cys165 residues (Fig. 3). Amino acid residues in surroundings of the catalytic triad residue Glu-48 and Cys-165 were VFGETWI and CCW, respectively, whereas in aliphatic nitrilase these were AFPEVFI and HCW, respectively. Amino acid residues surrounding of Lys-131 were conserved RRKLKPTHVER both in arylacetonitrilase as well as in aliphatic nitrilase. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence vary from other already reported arylacetonitrilase, e.g., Gly-11, His-21, Asn-149, Thr-170, Arg-171, Ala-179, Asn-180, and Ala-191 in the present arylacetonitrilase whereas Ala-11, Asp-21, Asp-149, Ser-170, Pro-171, Ser-179, Gln-180, and Ser-191 at the corresponding position in other arylacetonitrilase. In arylaliphatic nitrilase, the conserved amino acid residues besides the catalytic triad (Gly, Lys, Cys) were Val-45, Gly-47, Thr-49, Leu-51, Phe-56, Phe-142, C-164, and Leu-168, and these are replaced by Ala-45, Pro-47, Val-49, Iso-51, Tyr-56, Tyr-142, Asn-164, and Phe-168, respectively in aliphatic nitrilases. The amino acid sequence of arylacetonitrilase showed maximum phylogenetic relationship with Alcaligenes sp. ECU0401 and Pseudomonas putida (Fig. 4).
Arylacetonitrile-Specific Nitrilase of Alcaligenes sp. MTCC 10675
FIG. 2
Effect of metal ions and other molecules on purified arylacetonitrilase of Alcaligenes sp. MTCC 10675.
Substrate specificity profile of purified arylacetonitrilase
TABLE 2 Substrate
Specific activity (U/mg protein)
Aliphatic nitrile
Substrate
Specific activity (U/mg protein)
Aromatic nitrile
Acetonitrile
0.2 ± 0.01
Benzonitrile
ND
Acrylonitrile
1.3 ± 0.03
4-Hydroxynitrile
ND
Adiponitrile
2.1 ± 0.00
Tolunitrile
ND
Butyronitrile
6.2 ± 0.02
Heterocyclic nitrile
Isobutyronitrile
2.4 ± 0.00
2-Cyanopyridine
0.1 ± 0.01
Propionitrile
4.70 ± 0.03
4-Cyanopyridine
0.6 ± 0.03
Valeronitrile
ND
Acetone cyanohydrin
ND
Arylaliphatic nitrile Diphenylacetonitrile
2.3 ± 0.01
Mandelonitrile
26 ± 0.10
Phenylacetonitrile
65 ± 0.40
ND = Not detectable.
4. Discussion A number of nitrilases have been purified and characterized, but only few of the nitrilases can hydrolyze arylacetonitriles. The nitrilase of Alcaligenes sp. MTCC 10675 is an intracellular arylacetonitrilase, which hydrolyzed mandelonitrile into mandelic acid very effectively. Alcaligenes sp. MTCC 10675 resting cells were disrupted using BeadBeaterTM , and 11.3% arylacetonitrilase activity was recovered in the supernatant [1, 14]. Arylacetonitrilase of Alcaligenes sp. MTCC 10675 required a higher percentage of ammonium sulfate (60%) for
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precipitation in comparison with arylacetonitrilase of P. putida (40%), which indicates that it has more hydrophilic amino acid residue. Sephacryl S-300 was used for the purification, contaminating proteins were eluted in the starting fractions, and arylacetonitrilase was eluted in later fractions. Arylacetonitrilase was eluted at 0.4 M NaCl, and most of the contaminating proteins were eluted at low molarity NaCl. Arylacetonitrilase was purified up to 46-fold and 32% yield. Arylacetonitrilase from P. putida MTCC 5110 and Aspergillus niger K10 was purified up to 35- and twofold with 10% and 35% yields,
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FIG. 3
FIG. 4
Multiple sequence alignment of the amino acid sequence of arylaceionitrilase of Alcaligenes sp. MTCC 10675 and already reported nitrilase.
Phylogenetic tree based on amino acid sequences of the arylacetomlrilase sequence derived from the nucleotide sequence of Alcaligenes sp. MTCC 10675 and nitrilases of other organisms.
respectively [1, 15]. Alcaligenes sp. MTCC 10675 has maximum arylacetonitrilase activity at pH 6–7 like the arylacetonitrilase of P. putida and P. fluorescence EBC191 [1, 16]. The activity of this enzyme decreased with the increase in pH, which may be due to instability of hydroxynitriles and arylacetonitrilase at the basic pH [17]. Arylacetonitrilase of Alcaligenes sp. MTCC 10675 was active in the temperature range of 40–50◦ C, similar to the arylacetonitrilase of Aspergillus niger K10, Alcaligenes faecalis JM3, Bradyrhizobium japonicum USDA110, A. faecalis ATCC 8750, and Fusarium solani O1 [15–21]. Arylacetonitrilase of Alcaligenes sp. MTCC 10675 has the potential to hydrolyze mandelonitrile up to 15 mM, and a further increase in the substrate concentration decreased the hydrolyzing activity of the enzyme, which may be due to substrate inhibition. The Vmax , Km , and Kcat of purified arylacetonitrilase were calculated as 50 ± 05 µmol/Min/mg, 13 ± 02 mM, and 26 ± 03 Sec− . Arylacetonitrilase of P. putida has Vmax , Km , and Kcat as 16.5 µmol/Min/mg and 13.4 and 11.8, respectively [1]. Purified arylacetonitrilase was stable at a lower temperature (15 –35◦ C) and lost activity rapidly above a temperature of 45 ◦ C. It has a half-life of 31 H at 15 ◦ C and 3 H 20 Min at 50 ◦ C whereas arylacetonitrilase of Alcaligenes sp. was stable at 50 ◦ C for 1 H 7 Min [22]. Hg2+ , Mg2+ , Cu2+ , and Co2+ were strong inhibitors of arylacetonitrilase activity of this organism, whereas Hg2+ , Mg2+ , and Cu2+ inhib-
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ited arylacetonitrilase activity of Alcaligenes sp. ECU0401 [21]. Heavy metals such as Ag+ , Hg2+ , and Pb2+ have strong affinity for –SH groups and inhibit activity of the nitrile-metabolizing enzyme [23]. Nitrilase of Alcaligenes sp. MTCC 10675 is highly substrate specific and hydrolyzes arylacetonitriles (phenylacetonitriles and mandelonitriles) in comparison with nitrilase of B. japonicum USDA 110 and P. fluoresecens EBC 191, which show activity for arylacetonitriles as well as for aromatic nitriles and aliphatic nitriles [20, 24]. ClustalW analysis of the amino acid sequence of arylacetonitrilase of Alcaligenes sp. MTCC 10675 with other arylacetonitrilase and aliphatic nitrilase sequences showed that the catalytic triad is present at position Glu-48, Lys-131, and Cys-165, as already reported in aromatic and aliphatic nitrilase amino acid sequence comparison [25]. The amino acid sequence of arylacetonitrilase of Alcaligenes sp. MTCC 10675 differs from other arylacetonitrilases, which led to an increase in thermal stability, product tolerance, and activity. Proteins having an increased percentage of arginine and decreased content of serine amino acid residue resulted in the thermal stability of protein [26]. Amino acid residue in the surroundings of catalytic triad residue of aliphatic nitrilase was changed into V49T, Y142F, and H164C in arylaliphatic nitrilase of Alcaligenes sp. MTCC 10675, which may be a possible reason for high substrate specificity toward arylaliphatic nitriles. Yeom and co-workers have reported that exchange of tyrosine into phenylalanine at the 142 position of nitrilase of Rhodococcus rhodochrous ATCC 33278 did not change its substrate specificity toward aliphatic and aromatic nitriles, whereas arylacetonitrilase of Alcaligenes sp. MTCC 10675 has
Arylacetonitrile-Specific Nitrilase of Alcaligenes sp. MTCC 10675
phenyalalanine at the 142 position and it may be a possible reason for its high specificity toward arylacetonitrile [27]. Alcaligenes sp. MTCC has nitrilase activity for arylacetonitrile, and a maximum phylogenetic relationship was observed with P. putida and Alcaligenes sp. ECU0401, as these organisms also have arylacetonitrilase activity [16,22]. High Vmax , Kcat , and low Km values of arylacetonitrilase of Alcaligenes sp. MTCC 10675 make it an efficient enzyme in comparison with the already reported arylacetonitrilase. The amino acid sequence of this nitrilase is different from other already reported nitrilase, and it has a higher thermal stability and hydrolyzes arylacetonitriles (phenylacetonitrile and mandelonitrile) with a high catalytic rate.
5. Acknowledgements The authors acknowledge the Department of Biotechnology and University Grant Commission, New Delhi, India, for financial support in the form of a Senior Research Fellowship to Mr. Shashi Kant Bhatia, Praveen Kumar Mehta, and Ravi Kant Bhatia. The computational facility used at the Bioinformatics Centre, H. P. University, Shimla, India, is also duly acknowledged.
6. References [1] Banerjee, A., Kaul, P., and Banerjee, U. C. (2006) Arch. Microbiol. 184, 407–418. [2] DeSantis, G., Zhu, Z., Greenberg, W. A., Wong, W. A.K., Chaplin, J., Hanson, S. R., Farwell, B., Nicholson, L. W., Rand, C. L., Weiner, D. P., Robertson, D. E., and Burk, M. J. (2002) J. Am. Chem. Soc. 124, 9024–9025. [3] Mylerova, V., and MartI´nkova, L. (2003) Curr. Org. Chem. 7, 1–17. [4] Bhalla, T. C., Miura, M., Wakamoto, A., Ohba, Y., and Furuhashi, K. (1992) Appl. Microbiol. Biotechnol. 37, 184–190. [5] Bhatia, S. K., Mehta, P. K., Bhatia, R. K., and Bhalla, T. C. (2013) Bioprocess. Biosyst. Eng. 36, 613–625. [6] He, Y. C., Xu, J. H., Su, J. H., and Zhou, L. (2010) Appl. Biochem. Biotechnol. 160, 1428–1440.
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[7] Kaul, P., Banerjee, A., Mayilraj, S., and Banerjee, U. C. (2004) Tetrahedron Asymmetry 15, 207–211. [8] Bhatia, S. K., Mehta, P. K., Bhatia, R. K., and Bhalla, T. C. (2014) Appl. Microbiol. Biotechnol. 98(1), 83–94. [9] Fawcett, J. K., and Scott, J. E. (1969) J. Clin. Pathol.13, 156–159. [10] Bradford, M. M. (1976) Anal. Biochem. 72, 248–254. [11] Laemmli, U. K. (1970) Nature 227, 680–685. [12] Mehta, P. K., Bhatia, S. K., Bhatia, R. K., and Bhalla, T. C. (2013) Extremophiles 17(4), 637–648. [13] Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor, NY. [14] Singh, R., Banerjee, A., Kaul, P., Barse, B., and Banerjee, U. C. (2005) Bioprocess. Biosyst. Eng. 27, 415–424. [15] Kaplan, O., Vejvoda, V., Plihal, O., Pompach, P., Kavan, D., Bojarova, P., Bezouska, K., Mackova, M., Cantarella, M., Jirku, V., Kren, V., and Martinkova, L. (2006) Appl. Microbiol. Biotechnol. 73, 567–575. ¨ [16] Sosedov, O., Baum, S., Burger, S., Matzer, K., Kiziak, C., and Stolz, A. (2010) Appl. Environ. Microbiol. 76, 3668–3674. [17] Rustler, S., and Stolz, A. (2007) Appl. Microbiol. Biotechnol. 75, 899–908. [18] Nagasawa, T., Mauger, J., and Yamada, H. (1990) Eur. J. Biochem. 194, 765–772. [19] Yamamoto, K., Fujimatsu, I., and Komatsu, K. (1992) J. Ferment. Bioeng. 73, 425–430. [20] Zhu, D., Mukherjee, C., Yang, Y., Rios, B. E., Gallagher, D. T.N., Smith, N. N., Biehl, E. R., and Hua, L. (2008) J. Biotechnol. 133, 327–333. [21] Vejvoda, V., Kaplan, O., Bezouska, K., Pompach, P., Sulc, M., Cantarella, M., Benada, O., Uhnakova, B., Rinagelova, A., Lutz-Wahl, S., Fischer, L., Kren, V., and Martınkova, L. (2008) J. Mol. Catal. B: Enzyme 50, 99–106. [22] Zhang, Z., Xu, J. H., He, Y. C., Ouyang, L. M., and Liu, Y. Y. (2011) Bioprocess. Biosyst. Eng. 34, 315–322. [23] Shen, W., Chen, H., Kaizhi, J., Jun, N., Yan, X., and Li, S. (2011) Appl. Microbiol. Biotechnol. 4, 1007–1013. [24] Kiziak, C., Conradt, D., Stolz, A., Mattes, R., and Klein, J. (2005) Microbiology 151, 3639–3648. [25] Sharma, N., Kushwaha, R., Sodhi, J. S., and Bhalla, T. C. (2009) J. Proteomics Bioinform. 2, 185–192. [26] Kumar, S., Tsai, C. J., and Nussinov, R. (2000) Protein Eng.13, 179– 191. [27] Yeom, S. J., Kim, H. J., Lee, J. K., Kim, D. E., and Oh, D. K. (2008) Biochem. J. 415, 401–407.
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S. K. Bhatia P. K. Mehta R. K. Bhatia T. C. Bhalla∗
Department of Biotechnology, Himachal Pradesh University, Shimla, India
Abstract Arylacetonitrile-hydrolyzing nitrilase (E.C. 3.5.5.5) of Alcaligenes sp. MTCC 10675 has been purified by up to 46-fold to homogeneity and 32% yield using ammonium sulfate fractionation, Sephacryl S-300 gel permeation, and anion exchange chromatography. The molecular weight of the native enzyme was estimated to be 520 ± 60 kDa. The subunit has a molecular weight of 60 ± 14 kDa in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature of the purified enzyme were 6.5 and 50 ◦ C, respectively. The purified arylacetonitrilase has a half-life of 3 H 20 Min at its optimum temperature. The value
for Vmax, Km , kcat , and ki of enzyme for mandelonitrile as a substrate was 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec− , and 32.4 ± 03 mM, respectively. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence has variations from other reported arylacetonitrilase, namely, A11G, N21H, D149N, S170T, P171R, S179A, Q180N, and S191A, and it has a high thermal stability and catalytic rate as compared with the already purified arylacetonitrilase. C 2013 International Union of Biochemistry and Molecular Biology, Inc. Volume 61, Number 4, Pages 459–465, 2014
Keywords: Alcaligenes sp. MTCC 10675, arylacetonitrile, mandelonitrile, chromatography
1. Introduction α-Hydroxyphenylacetic acid (mandelic acid) is used in the synthesis of antitumor agents, antiobesity, semisynthetic penicillin, and cephalosporin [1, 2]. There is considerable industrial interest in the enzymatic conversion of nitriles into acid, as enzymatically these processes are regio- and enantioselective, and this has led to the isolation of a number of nitrile-hydrolyzing bacteria [3–5]. Nitrile-metabolizing organisms have nitrilase or nitrile hydratase and amidase enzyme systems that convert nitriles into their corresponding acids. There are different types of nitrilases according to their substrate specificity, e.g., aliphatic, aromatic, and arylacetonitrilases. Arylacetonitrilase
has immense potential for the hydrolysis of arylaliphatic nitriles; however, these biocatalytic processes are not widely adopted by the industry due to high cost, instability, low selectivity, and specificity of the enzyme [6]. Thus, there is a need to search new nitrilases to meet the above-mentioned challenges. Hitherto very few nitrilases have been reported that are substrate specific [6, 7]. A nitrile-degrading bacterium Alcaligenes sp. MTCC 10675 has been isolated in our laboratory, which has high affinity for arylacetonitriles [8]. In this article, purification, characterization, and sequence analysis of the amino acid sequence of arylacetonitrile-hydrolyzing nitrilase of Alcaligenes sp. MTCC 10675 is reported.
2. Materials and Methods Abbreviations: MTCC, microbial type culture collection; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetate; CFE, cell-free extract; ASF, ammonium sulfate fraction; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PCR, polymerase chain reaction. ∗ Address
for correspondence: Professor T. C. Bhalla, Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla 171 005, India. Tel: +91-177-2831948; Fax: +91-177-2832154; e-mail: [email protected]. Received 3 July 2013; accepted 11 December 2013 DOI: 10.1002/bab.1192 Published online 25 March 2014 in Wiley Online Library (wileyonlinelibrary.com)
2.1. Chemicals Isobutyronitrile and mandelonitrile were purchased from Lancaster Synthesis (Lancashire, UK). Media components were obtained from Hi-Media (Mumbai, India). All other chemicals were procured from Merck (Mumbai, India).
2.2. Microorganism and cultivation condition Alcaligenes sp. MTCC 10675 (isolated from soils of Shimla District of Himachal Pradesh, India, as a nitrile-metabolizing bacterium and identified at the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, India, was grown overnight in a seed medium
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Biotechnology and Applied Biochemistry (nutrient broth), and 1 mL of the seed culture was transferred into a 250 mL Erlenmeyer flask containing 50 mL of production medium (pH 7.0) consisting of (g/L) peptone 5.5 g, malt extract 3.0 g, yeast extract 5.5 g, and isobutyronitrile 4.0 g (60 mM) at 30 ◦ C for 24 H [8]. After 24 H, cells were separated from the culture broth by centrifugation at 10,000g, washed twice with 0.1 M K2 HPO4 /KH2 PO4 (pH 7.0), and suspended in the same buffer containing 1 mM dithiothreitol (DTT) and 1 mM ethylenediaminetetraacetate (EDTA).
2.3. Arylacetonitrilase assay Arylacetonitrilase assay was carried out in a reaction mixture containing 0.1 M KH2 PO4 /K2 HPO4 buffer (pH 6.5), 20 mM mandelonitrile, and an appropriate amount of arylacetonitrilase at 50 ◦ C for 30 Min. The reaction was stopped by adding an equal volume of 0.1 N HCl to the reaction mixture. The ammonia released by the action of arylacetonitrilase on mandelonitrile was assayed using the phenate-hypochlorite method [9]. In terms of ammonia release, one unit of arylacetonitrilase activity was defined as the amount of enzyme that catalyzed the release of 1 µmol of ammonia/Min by the hydrolysis of nitrile under standard assay conditions.
2.4. Purification of arylacetonitrilase of Alcaligenes sp. MTCC 10675 2.4.1. Preparation of cell-free extract The Alcaligenes sp. MTCC 10675 cells were grown in 1 L of production medium, and after 24 H of incubation cells were harvested from the culture broth by centrifugation at 8,000g and washed twice with 0.1 M K2 HPO4 /KH2 PO4 (pH 7.0) buffer and suspended in the same buffer containing 1 mM DTT and 1 mM EDTA. The cell suspension (800 mg) was added to a bead beater chamber (100 mL capacity) containing half filled 0.1 mm glass beads, and cells were disrupted in eight cycles (each cycle involves agitation for 1.0 Min and 1.0 Min rest) using BeadBeaterTM (BioSpec Products, Bartlesville, OK, USA). The cell-free extract (CFE) was prepared by centrifuging crude extract at 15,000g for 25 Min. The protein content was estimated following the Bradford method [10]. All the purification steps were performed at 4 ◦ C.
2.4.2. Ammonium sulfate precipitation The cell-free extract was subjected to ammonium sulfate saturation (20–60%), and the precipitates collected after centrifugation at 15,000g (25 Min at 4 ◦ C) were suspended and dialyzed against the same buffer. The ammonium sulfate fraction (ASF) having arylacetonitrilase activity was used in a subsequent step of purification.
2.4.3. Gel permeation chromatography The gel permeation chromatography was performed using a column (2.6 × 60 cm) packed with the Sephacryl S-300 High Resolution (GE Healthcare, Little Chalfont, UK) matrix as per manufacturer’s instructions. The 12 mL of dialyzed protein sample having arylacetonitrilase activity was centrifuged at 15,000g for 10 Min at 4 ◦ C and loaded on a gel permeation
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column preequilibrated with buffer. The elution was performed at a flow rate of 1.0 mL/Min with the potassium phosphate buffer having pH 7.0 (0.05 M).
2.4.4. Anion exchange chromatography The fractions having arylacetonitrilase activity were pooled and applied on a DEAE-Sepharose anion exchange column (1.5 × 10 cm). After loading the sample, the column was washed with potassium phosphate buffer pH 7.0 (0.05 M) until there was no further elution of protein. The enzyme was then eluted with a gradient of NaCl (from 0 to 0.5 M) at a flow rate of 1 mL/Min in the same buffer. The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of different fractions that showed arylacetonitrilase activity was performed in 10% gel [11].
2.4.5. Molecular weight determination The native molecular mass of the purified arylacetonitrilase enzyme was determined by gel permeation chromatography. The 30 µg enzyme was subjected to a SephadexTM G-200 (GE Healthcare) column and eluted with 50 mM KH2 PO4 /K2 HPO4 buffer, pH 6.5 with 1 mM DTT, and 1 mM EDTA containing 0.15 M NaCl and eluted at a flow rate of 0.5 mL/Min. The optical density of effluent was recorded at A280 . The relative molecular mass of the purified arylacetonitrilase was then calculated from the relative mobility compared with those of standard proteins, thyroglobulin (Mr, 669,000), ferritin (Mr, 440,000), catalase (Mr, 232,000), lactate dehydrogenase (Mr, 140,000), and albumin (Mr, 66,000) [12].
2.5. Characterization of purified arylacetonitrilase 2.5.1. Effect of pH The activity of purified arylacetonitrilase of Alcaligenes sp. MTCC was assayed in different buffers, e.g., citrate buffer (4.0– 6.0), sodium phosphate buffer (6.0–8.0), potassium phosphate buffer (6.0–8.0), borate buffer (7.0–9.0), and carbonate buffer (9.0–10.0). The 1.0 mL reaction mixture contained specified buffer and 20 mM mandelonitrile as a substrate. A reaction was carried out at 30 ◦ C for 1 H.
2.5.2. Buffer molarity and temperature The effect of buffer (potassium phosphate) molarity on the activity of arylacetonitrilase was studied by varying the buffer concentration from 0.025 to 0.125 M in the reaction. The optimum temperature was determined by carrying out the reaction at different temperatures (25–55◦ C).
2.5.3. Study of incubation time, stability, metal ions, and other kinetic parameters The incubation time for the optimum activity of arylacetonitrilase was determined by incubating the reaction mixture from 10 to 90 Min at 50 ◦ C. Thermal stability of the purified enzyme arylacetonitrilase was investigated at 25 –55◦ C for 5 H. The arylacetonitrilase activity was assayed in the presence of various metal ions and chemicals (AgCl, CaCl2 , CdCl2 , CsCl, CoCl2 , CuSO4 , FeSO4, HgCl2 , MgSO4 , MnCl2 , ZnSO4 , KCl, NaCl, EDTA, and DTT) with a final concentration of 1 mM. The Km ,
Arylacetonitrile-Specific Nitrilase of Alcaligenes sp. MTCC 10675
Purification summary of arylacetonitrilase of Alcaligenes sp. MTCC 10675
TABLE 1 Volume (mL)
Protein (mg/mL)
CFE
30
3.3
99
0.9
ASF
12
5.2
63
Sephacryl S -300
6
0.94
DEAE-sepharose
4
0.18
Purification stage
Total protein (mg)
Activity (U/mL)
Specific activity (U/mg)
Purification fold
Yield (%)
27
0.27
1
100
1.6
19
0.30
1
70
5.6
1.8
11
1.96
7
41
0.7
2.2
46
32
Vmax, kcat , and ki of the purified arylacetonitrilase of Alcaligenes sp. MTCC 10675 were determined using mandelonitrile as a substrate. Substrate specificity of purified arylacetonitrilase was studied against various aliphatic, aromatic, heterocyclic, and arylacetonitriles.
2.6. Polymerase chain reaction amplification of arylacetonitrilase gene sequence Genomic DNA of Alcaligenes sp. MTCC 10675 was extracted following the method as described by Sambrook et al. [13]. Primers for the amplification of arylacetonitrilase gene were designed on the basis of already reported arylacetonitrilase sequences retrieved from NCBI using OligoAnalyzer 3.1, i.e., >gi|158023988|gb|EF467660.1|, >gi|216202|dbj|D13419.1,| and >gi|54013472:105241–107199. Primers designed for amplification were as follows: forward primer S31NF 5 -ATG CAG ACA AGA AAA ATC G-3 and reverse primer S31NR 5 -TCA GGA CGG TTC TTG CAC CAG-3 . Alcaligenes sp. MTCC 10675 genomic DNA was used as a template DNA. An amplification reaction was performed using the following conditions: 5 Min hot start at 94 ◦ C; followed by 30 cycles consisting of denaturation (30 Sec at 94 ◦ C), annealing (60 Sec at 52 ◦ C), and extension (90 Sec at 72 ◦ C); and a final extension at 72 ◦ C for 5 Min. The polymerase chain reaction (PCR) product was visualized by the electrophoresis in 1.0% agarose gel containing ethidium bromide. The amplified PCR product was sequenced and used for the analysis.
2.6.1. Homology with other arylacetonitrilase The 1.1-kb PCR product was sequenced, and the NCBI Blast program was performed with the forward and reverse sequences to obtain the homologous arylacetonitrilase gene sequences deposited in the data bank of NCBI. The forward sequence was translated with a translate tool available at Expasy (http://web.expasy.org/translate/), and BLAST was performed to discover the homologous amino acid sequences of arylacetonitrilases. The ClustalW program was used to align the nucleotide and amino acid sequence of the arylacetonitrilase with other arylacetonitrilases and aliphatic nitrilases. A phylogenetic tree was also constructed using the ClustalW algorithm.
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Total activity (U)
8.8
12.5
2.6.2. Nucleotide sequence accession number Alcaligenes sp. S31 was deposited into the Microbial Type Culture Collection, Chandigarh, India, under the accession number MTCC 10675. The nucleotide sequence of the arylacetonitrilase gene was deposited into the GenBank database under the accession number KC019880.
3. Results 3.1. Purification of arylacetonitrilase The arylacetonitrilase was purified using gel permeation and ion exchange chromatography (Table 1). In S-300 gel permeation chromatography, arylacetonitrilase was eluted in the fraction number 19 (1.5 U/mg protein), 20 (2.3 U/mg protein), and 21 (2.1 U/mg protein). Gel permeation chromatography resulted in a sevenfold increase in arylacetonitrilase activity (1.96 U/mg protein). Fractions having arylacetonitrilase activity were pooled and used in anion exchange chromatography. The major fractions of arylacetonitrilase were eluted at 0.4 M NaCl, and most of the contaminating proteins were removed by eluting the column with low molarity NaCl. Ion exchange chromatography step proved to be a very effective step for the purification of arylacetonitrilase as it resulted in a 46-fold increase in specific activity (12.5 U/mg protein) with 32% yield as compared with activity and purity of the enzyme of CFE (Table 1). By gel filtration on a SephadexTM G-200 column, the molecular mass of the native enzyme was estimated to be 520 ± 60 kDa. The purified arylacetonitrilase of Alcaligenes sp. MTCC 10675 showed a single band of 60 ± 14 kDa on SDS-PAGE and consisted of seven or eight subunits (Fig. 1).
3.2. Characterization of purified arylacetonitrilase 3.2.1. Effect of pH and molarity The purified arylacetonitrilase of Alcaligenes sp. MTCC 10675 has maximum mandelonitrile-hydrolyzing activity (10.5 U/mg protein) in 0.075 M potassium phosphate buffer having pH 6.5. In citrate buffer, the activity of enzyme increases with the increase in the pH (4–5.5), whereas in borate and carbonate buffers very little activity (0.6–2.6 U/mg protein) was observed.
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Biotechnology and Applied Biochemistry activity, and at the 50 mM substrate concentration 76% loss in the activity was recorded. The Vmax, Km , Kcat , and ki values for the arylacetonitrilase were 50 ± 05 µmol/Min/mg, 13 ± 02 mM, 26 ± 03 Sec,− and 32.4 ± 03 mM, respectively.
3.2.5. Stability of arylacetonitrilase The thermal stability of arylacetonitrilase was studied at different temperatures, and it was observed that arylacetonitrilase was stable at lower temperature (15◦ C–30◦ C). Exposure of the enzyme at 15, 25, and 35 ◦ C for 5 H caused 8%, 15%, and 28% decrease in arylacetonitrilase activity. A further increase in the temperature to 45 and 55 ◦ C resulted in 58% and 90% decreases in the activity, respectively. The half-life of this enzyme at 15, 25, 35, 45, 50, and 55 ◦ C was, respectively 31 H, 16 H, 9 H, 4 H 30 Min, 3 H 20 Min, and 2 H 40 Min.
3.2.6. Metal ions and inhibitor Metal ions of Hg2+ , Mg2+ , Cu2+ , and Co2+ were a strong inhibitor of arylacetonitrilase activity, which caused inhibition upto the extent of 98%, 91%, 87%, and 77%, respectively. The presence of 1 mM Ca2+ , Cd2+ , Mn2+ , Cs+ , and DTT in the assay mixture decreased arylacetonitrilase activity to its half. The EDTA, Zn2+ , Na+ , and K+ showed very little effect on arylacetonitrilase activity (Fig. 2).
3.2.7. Substrate specificity
FIG. 1
SDS-PAGE of different fractions in 10% gel. lane 1, marker; lane 2, CFE; lane 3, precipitate; lane 4, pooled fraction of sephacry S-300; lane 5, DEAE fraction; and lane 6, marker.
Nitrilase of Alcaligenes sp. MTCC 10675 is highly specific for arylacetonitriles, and 65 ± 0.40 U/mg protein and 26 ± 0.10 U/mg protein activity were recorded for phenylacetonitrile and mandelonitrile, respectively. The purified enzyme has very little activity against heterocyclic and aliphatic nitriles, and no activity was recorded against aromatic nitriles (Table 2).
3.2.8. Homology with other arylacetonitrilase 3.2.2. Temperature The mandelonitrile-hydrolyzing activity of arylacetonitrilase of Alcaligenes sp. MTCC 10675 increased with the increase in temperature, and maximum activity was observed at 50 ◦ C (12.4 U/mg protein). Above 50 ◦ C, a decrease in the activity of arylacetonitrilase was observed.
3.2.3. Incubation time Mandelonitrile-hydrolyzing activity of purified arylacetonitrilase was assayed at intervals of 10 Min until 90 Min; a continuous increase in arylacetonitrilase activity was recorded with the increase in incubation temperature and highest arylacetonitrilase activity (27 U/mg protein) was recorded at 30 Min incubation; thereafter the activity remained more or less constant until it decreased as the enzyme might have undergone product inhibition.
3.2.4. Vmax , Km , kcat , and ki values of arylacetonitrilase Arylacetonitrilase activity increased with the mandelonitrile concentration in the reaction, and maximum activity was recorded at 15 mM of mandelonitrile. A further increase in the substrate concentration resulted in a decrease in enzyme
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Amino acid alignment with other nitrilase superfamily sequences showed that a catalytic triad (Glu–Lys–Cys) comprises Glu48, Lys131, and Cys165 residues (Fig. 3). Amino acid residues in surroundings of the catalytic triad residue Glu-48 and Cys-165 were VFGETWI and CCW, respectively, whereas in aliphatic nitrilase these were AFPEVFI and HCW, respectively. Amino acid residues surrounding of Lys-131 were conserved RRKLKPTHVER both in arylacetonitrilase as well as in aliphatic nitrilase. Alcaligenes sp. MTCC 10675 arylacetonitrilase amino acid sequence vary from other already reported arylacetonitrilase, e.g., Gly-11, His-21, Asn-149, Thr-170, Arg-171, Ala-179, Asn-180, and Ala-191 in the present arylacetonitrilase whereas Ala-11, Asp-21, Asp-149, Ser-170, Pro-171, Ser-179, Gln-180, and Ser-191 at the corresponding position in other arylacetonitrilase. In arylaliphatic nitrilase, the conserved amino acid residues besides the catalytic triad (Gly, Lys, Cys) were Val-45, Gly-47, Thr-49, Leu-51, Phe-56, Phe-142, C-164, and Leu-168, and these are replaced by Ala-45, Pro-47, Val-49, Iso-51, Tyr-56, Tyr-142, Asn-164, and Phe-168, respectively in aliphatic nitrilases. The amino acid sequence of arylacetonitrilase showed maximum phylogenetic relationship with Alcaligenes sp. ECU0401 and Pseudomonas putida (Fig. 4).
Arylacetonitrile-Specific Nitrilase of Alcaligenes sp. MTCC 10675
FIG. 2
Effect of metal ions and other molecules on purified arylacetonitrilase of Alcaligenes sp. MTCC 10675.
Substrate specificity profile of purified arylacetonitrilase
TABLE 2 Substrate
Specific activity (U/mg protein)
Aliphatic nitrile
Substrate
Specific activity (U/mg protein)
Aromatic nitrile
Acetonitrile
0.2 ± 0.01
Benzonitrile
ND
Acrylonitrile
1.3 ± 0.03
4-Hydroxynitrile
ND
Adiponitrile
2.1 ± 0.00
Tolunitrile
ND
Butyronitrile
6.2 ± 0.02
Heterocyclic nitrile
Isobutyronitrile
2.4 ± 0.00
2-Cyanopyridine
0.1 ± 0.01
Propionitrile
4.70 ± 0.03
4-Cyanopyridine
0.6 ± 0.03
Valeronitrile
ND
Acetone cyanohydrin
ND
Arylaliphatic nitrile Diphenylacetonitrile
2.3 ± 0.01
Mandelonitrile
26 ± 0.10
Phenylacetonitrile
65 ± 0.40
ND = Not detectable.
4. Discussion A number of nitrilases have been purified and characterized, but only few of the nitrilases can hydrolyze arylacetonitriles. The nitrilase of Alcaligenes sp. MTCC 10675 is an intracellular arylacetonitrilase, which hydrolyzed mandelonitrile into mandelic acid very effectively. Alcaligenes sp. MTCC 10675 resting cells were disrupted using BeadBeaterTM , and 11.3% arylacetonitrilase activity was recovered in the supernatant [1, 14]. Arylacetonitrilase of Alcaligenes sp. MTCC 10675 required a higher percentage of ammonium sulfate (60%) for
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precipitation in comparison with arylacetonitrilase of P. putida (40%), which indicates that it has more hydrophilic amino acid residue. Sephacryl S-300 was used for the purification, contaminating proteins were eluted in the starting fractions, and arylacetonitrilase was eluted in later fractions. Arylacetonitrilase was eluted at 0.4 M NaCl, and most of the contaminating proteins were eluted at low molarity NaCl. Arylacetonitrilase was purified up to 46-fold and 32% yield. Arylacetonitrilase from P. putida MTCC 5110 and Aspergillus niger K10 was purified up to 35- and twofold with 10% and 35% yields,
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FIG. 3
FIG. 4
Multiple sequence alignment of the amino acid sequence of arylaceionitrilase of Alcaligenes sp. MTCC 10675 and already reported nitrilase.
Phylogenetic tree based on amino acid sequences of the arylacetomlrilase sequence derived from the nucleotide sequence of Alcaligenes sp. MTCC 10675 and nitrilases of other organisms.
respectively [1, 15]. Alcaligenes sp. MTCC 10675 has maximum arylacetonitrilase activity at pH 6–7 like the arylacetonitrilase of P. putida and P. fluorescence EBC191 [1, 16]. The activity of this enzyme decreased with the increase in pH, which may be due to instability of hydroxynitriles and arylacetonitrilase at the basic pH [17]. Arylacetonitrilase of Alcaligenes sp. MTCC 10675 was active in the temperature range of 40–50◦ C, similar to the arylacetonitrilase of Aspergillus niger K10, Alcaligenes faecalis JM3, Bradyrhizobium japonicum USDA110, A. faecalis ATCC 8750, and Fusarium solani O1 [15–21]. Arylacetonitrilase of Alcaligenes sp. MTCC 10675 has the potential to hydrolyze mandelonitrile up to 15 mM, and a further increase in the substrate concentration decreased the hydrolyzing activity of the enzyme, which may be due to substrate inhibition. The Vmax , Km , and Kcat of purified arylacetonitrilase were calculated as 50 ± 05 µmol/Min/mg, 13 ± 02 mM, and 26 ± 03 Sec− . Arylacetonitrilase of P. putida has Vmax , Km , and Kcat as 16.5 µmol/Min/mg and 13.4 and 11.8, respectively [1]. Purified arylacetonitrilase was stable at a lower temperature (15 –35◦ C) and lost activity rapidly above a temperature of 45 ◦ C. It has a half-life of 31 H at 15 ◦ C and 3 H 20 Min at 50 ◦ C whereas arylacetonitrilase of Alcaligenes sp. was stable at 50 ◦ C for 1 H 7 Min [22]. Hg2+ , Mg2+ , Cu2+ , and Co2+ were strong inhibitors of arylacetonitrilase activity of this organism, whereas Hg2+ , Mg2+ , and Cu2+ inhib-
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ited arylacetonitrilase activity of Alcaligenes sp. ECU0401 [21]. Heavy metals such as Ag+ , Hg2+ , and Pb2+ have strong affinity for –SH groups and inhibit activity of the nitrile-metabolizing enzyme [23]. Nitrilase of Alcaligenes sp. MTCC 10675 is highly substrate specific and hydrolyzes arylacetonitriles (phenylacetonitriles and mandelonitriles) in comparison with nitrilase of B. japonicum USDA 110 and P. fluoresecens EBC 191, which show activity for arylacetonitriles as well as for aromatic nitriles and aliphatic nitriles [20, 24]. ClustalW analysis of the amino acid sequence of arylacetonitrilase of Alcaligenes sp. MTCC 10675 with other arylacetonitrilase and aliphatic nitrilase sequences showed that the catalytic triad is present at position Glu-48, Lys-131, and Cys-165, as already reported in aromatic and aliphatic nitrilase amino acid sequence comparison [25]. The amino acid sequence of arylacetonitrilase of Alcaligenes sp. MTCC 10675 differs from other arylacetonitrilases, which led to an increase in thermal stability, product tolerance, and activity. Proteins having an increased percentage of arginine and decreased content of serine amino acid residue resulted in the thermal stability of protein [26]. Amino acid residue in the surroundings of catalytic triad residue of aliphatic nitrilase was changed into V49T, Y142F, and H164C in arylaliphatic nitrilase of Alcaligenes sp. MTCC 10675, which may be a possible reason for high substrate specificity toward arylaliphatic nitriles. Yeom and co-workers have reported that exchange of tyrosine into phenylalanine at the 142 position of nitrilase of Rhodococcus rhodochrous ATCC 33278 did not change its substrate specificity toward aliphatic and aromatic nitriles, whereas arylacetonitrilase of Alcaligenes sp. MTCC 10675 has
Arylacetonitrile-Specific Nitrilase of Alcaligenes sp. MTCC 10675
phenyalalanine at the 142 position and it may be a possible reason for its high specificity toward arylacetonitrile [27]. Alcaligenes sp. MTCC has nitrilase activity for arylacetonitrile, and a maximum phylogenetic relationship was observed with P. putida and Alcaligenes sp. ECU0401, as these organisms also have arylacetonitrilase activity [16,22]. High Vmax , Kcat , and low Km values of arylacetonitrilase of Alcaligenes sp. MTCC 10675 make it an efficient enzyme in comparison with the already reported arylacetonitrilase. The amino acid sequence of this nitrilase is different from other already reported nitrilase, and it has a higher thermal stability and hydrolyzes arylacetonitriles (phenylacetonitrile and mandelonitrile) with a high catalytic rate.
5. Acknowledgements The authors acknowledge the Department of Biotechnology and University Grant Commission, New Delhi, India, for financial support in the form of a Senior Research Fellowship to Mr. Shashi Kant Bhatia, Praveen Kumar Mehta, and Ravi Kant Bhatia. The computational facility used at the Bioinformatics Centre, H. P. University, Shimla, India, is also duly acknowledged.
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