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Expression and Characterization of a Novel Nitrilase from Hyperthermophilic Bacterium Thermotoga maritima MSB8
Expression and Characterization of a Novel Nitrilase from Hyperthermophilic Bacterium Thermotoga maritima MSB8
Journal of Microbiology and Biotechnology. 2015. Oct, 25(10): 1660-1669
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : February 12, 2015
  • Accepted : June 09, 2015
  • Published : October 28, 2015
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About the Authors
Zhi Chen
Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China
Huayou Chen
National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, P.R. China
hyc@ujs.edu.cn
Zhong Ni
Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China
Rui Tian
Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China
Tianxi Zhang
Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China
Jinru Jia
Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China
Shengli Yang
Institute of Life Sciences, Jiangsu University, Zhenjiang 212000, P.R. China

Abstract
The present study describes the gene cloning, overexpression and characterization of a novel nitrilase from hyperthermophilic bacterium Thermotoga maritima MSB8. The nitrilase gene consisted of 804 base pairs, encoding a protein of 268 amino acid residues with a molecular mass of 30.07 kDa after SDS-PAGE analysis. The optimal temperature and pH of the purified enzyme were 45℃ and 7.5, respectively. The enzyme demonstrated good temperature tolerance, with 40% residual activity after 60 min of heat treatment at 75℃. The kinetic constants V max and K m of this nitrilase toward 3-cyanopyridine were 3.12 μmol/min/mg and 7.63 mM, respectively. Furthermore, this novel nitrilase exhibited a broad spectrum toward the hydrolysis of the aliphatic nitriles among the tested substrates, and particularly was specific to aliphatic dinitriles like succinonitrile, which was distinguished from most nitrilases ever reported. The catalytic efficiency k cat /K m was 0.44 /mM/s toward succinonitrile. This distinct characteristic might enable this nitrilase to be a potential candidate for industrial applications for biosynthesis of carboxylic acid.
Keywords
Introduction
Biocatalysis, as the core of industrial biotechnology, has been widely investigated to improve the sustainability and efficiency so as to prepare industrial fine chemicals, mainly focusing on intermediates for pharmaceuticals, agrochemicals, materials, and food ingredients [23 , 35] . Nitrile compounds, generally speaking, are synthetically more available for the production of high-value carboxylic acids and amides, which are important intermediates in producing the fine chemicals and pharmaceuticals [32] . Nitrilase-mediated biocatalysis reactions of nitrile compounds to their corresponding carboxylic acids provide an ecofriendly alternative allowing clean and mild synthesis combined with high yield and selectivity when compared with conventional chemical approaches typically requiring harsh basic or acidic reaction conditions and usually producing undesired byproducts [36] . This has increasingly aroused tremendous recognition of their potential due to the possibility of performing such biotransformation under mild condition that would not alter other labile reactive groups [2] .
Nitrilase (E.C. 3.5.5.1), as one kind of valuable biocatalyst, was utilized for the enzymatic biocatalysis of nitrile compounds directly to corresponding carboxylic acids, liberating ammonia. It had drawn sustainable attention to chemical hydrolysis in the organic chemical industry. Over the past few decades, a considerable amount of nitrilases, mainly derived from bacteria, yeasts, fungi, and plants, had been acquired and reviewed in details, some of which had already been applied into the production of carboxylic acids in the chemical industry [8 , 11] . Furthermore, indepth investigations on nitrilases had been widely dwelt upon with respect to their natural sources function mechanisms, enzyme structure, screening pathways, biocatalytic properties, immobilization, purification, cloning and modifications of the nitrilase gene [4 , 8 , 9 , 14 - 16 , 22 , 24 , 28 , 30] . On the basis of the broad substrate spectrum, nitrilases were commonly classified into three major groups, which included aliphatic, aromatic, and arylacetonitrilases, making them useful for the hydrolysis of a large number of nitriles [19] . For example, a new nitrilase from Fusarium proliferatum AUF-2 was characterized to be specific towards aliphatic, heterocyclic, and aromatic nitriles, which exhibited good catalytical efficiency for detoxification of nitriles [29] . The nitrilase from Rhodobacter sphaeroides could enantioselectively hydrolyze aliphatic dinitriles to corresponding cyanocarboxylic acids, which demonstrated great potential for commercial production of various cyanocarboxylic acids by readily available dinitriles [26] . The nitrilase from Pseudomonas putida CGMCC3830 was indentified as an aromatic nitrilase and harbored high conversion efficiency toward cyanopyridine [37] . In addition, some arylacetonitrilases from Burkholderia cenocepacia J2315 and Alcaligenes sp. ECU0401 were reported to harbor the merits of high substrate tolerance, yield, and optical purity while converting mandelonitrile to (R)-(−)-mandelic acid [10 , 25 , 31 , 33 , 34] . Moreover, a few fungal nitrilases from Gibberella intermedia and Aspergillus niger were reported to be highly specific toward 3-cyanopyridine and 2-cyanopridine, respectively [7 , 12] . Although nitrilases to some extent exhibited prominent potential in the chemical industry, some drawbacks including insufficient stability, narrow spectrum, low specific activity, and poor selectivity still exist at present and limit their applications [21] . Seeking for novel nitrilase resources and their potential applications would be a constant demand for researchers for a long period of time.
In this paper, the gene encoding the nitrilase from Thermotoga maritima MSB8 was cloned and overexpressed in E. coli BL21 (DE3). The recombinant nitrilase was purified and biochemically characterized in detail in order to gain a deeper understanding about the properties and application potential of this enzyme.
Materials and Methods
- Material
The genomic DNA of Thermotoga maritima MSB8 was obtained from Professor Weilan Shao at Jiangsu University. The E. coli strains DH5α and BL21 (DE3) (Gene Copoeia, USA) were used as hosts for cloning and expression experiments of the nitrilase, respectively. The plasmid pET-28a (+) (Novagen, Shanghai, China), carrying an N-terminal and a C-terminal His6-Tag sequence, was used for the cloning and expression of the nitrilase. The nitrile substrates were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). All the other chemicals used were commercially available and of analytical grade.
- Sequence Analysis
Amplified DNA fragments were sequenced by Sangon Biotech (Shanghai, China). Nucleotide and protein sequence homology analyses in the NCBI database ( http://blast.ncbi.nlm.nih.gov/Blast.cgi ) were conducted using the BLAST algorithm. Multiple sequence alignment was carried out using MEGA 6.06 software.
- Gene Cloning and Expression ofThermotoga maritimaMSB8 Nitrilase
The primers that were designed based on the reported amino acid sequences of nitrilase in NCBI for the amplification of genes were as follows: Forward: 5’-CGC GGATCC TTGCGAGTGGCGGC AGTACAGAT-3’ ( Bam HI restriction site is underlined) and Reverse 5'-CCG CTCGAG TCATAACCTCCCCTTCTGAAGC-3’ ( Xho lI restriction site is underlined), which incorporated Bam HI and Xho lI restriction sites, respectively. The amplified 804 bp DNA fragment digested with Bam HI and Xho I was ligated into the expression vector pet-28a(+) digested with the same restriction enzymes, and then transformed into the E. coli BL21 (DE3) cells by heat shock. The positive clones were identified by colony PCR and double digestion. Sequencing of the cloned nitrilase gene was subsequently performed at Sangon Biotech.
For the expression of the nitrilase, the resulting recombinant E. coli cells were cultivated in Luria–Bertani liquid medium containing 50 mg/ml kanamycin at 37℃ on a rotary shaker at 220 × g . A final concentration of 0.1 mM isopropyl-β-D-thiogalactoside was added for the induction when the optical density at 600 nm of the culture broth reached between 0.6 and 0.8. The cells were then further incubated at 28℃ and 160 × g for another 20 h. After centrifugation at 8,000 × g for 20 min, the cells were harvested and preserved at -20℃ for further experiments.
- Purification ofThermotoga maritimaMSB8 Nitrilase
Nickle affinity chromatography (Ni-NTA) was applied to purify the recombinant nitrilase by exploiting the histidine tag. The obtained cells were suspended and washed twice with 10 ml of phosphate-buffered saline (50 mM, pH 8.0). Then the cells were resuspended in 20 ml of the same buffer and disrupted by sonication on ice at 200 W for 10 min. The soluble fractions of the sonicated solution were obtained by centrifugation at 8,000 × g for 20 min to remove the cell debris. The resulting supernatant was passed through a 0.22 µm filter, and then loaded onto a Ni-NTA column previously equilibrated with a binding buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0). The column was subsequently washed with 10 ml of wash buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 10 mM imidazole, pH 8.0) to wipe out the unbound proteins and eluted with the elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0). The purified enzyme was further analyzed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was determined using the Bradford method [5] with bovine serum albumin as the standard. All purification steps were performed at 4℃.
- Enzyme Assay
The standard enzyme assay was investigated by measuring the production of the released ammonia, which was based on the Bertholet assay [27] . The reaction mixtures (1 ml of final volume) containing the nitrilase in sodium phosphate buffer (50 mM, pH 7.5) and the substrate nitrile (10 mM of final concentration) were initiated by shaking at 150 × g for 60 min, and terminated by the addition of 100 µl of HCl (2 M). The nitrilase activity was defined as the number of µmol of ammonia produced in 1 min by 1 mg of purified enzyme (µmol/min/mg). All measurements were conducted in triplicate and data were means ± standard deviations from three replications.
- Effects of Temperature and pH on and Temperature Tolerance of the Activity of Purified Nitrilase
The effect of temperature on the nitrilase activity was measured under standard enzyme assay conditions with 3-cyanopyridine (10 mM) as substrate at temperatures ranging from 25℃ to 55℃. The effect of pH was examined using the following buffers respectively: saline sodium citrate buffer (50 mM, pH 4, 5, 6), sodium phosphate buffer (50 mM, pH 6, 6.5, 7, 7.5, 8), Tris-HCl buffer (50 mM, pH 8, 8.5, 9). All experiments were conducted under standard enzyme assay conditions with 3-cyanopyridine (10 mM) as substrate. In addition, a reaction mixture without enzyme was used as the blank.
To investigate the temperature tolerance of the purified nitrilase, reaction mixtures containing the purified enzyme and the buffer were heated in a water bath for 30 and 60 min at 45℃, 55℃, 65℃, 75℃, and 85℃, respectively, and then the residual enzyme activity was measured under standard assay conditions mentioned previously. The value acquired without any heat treatment at the beginning was set as 100%.
- Effects of Various Chemical Compounds on the Activity of Purified Nitrilase
The effect of various chemicals, which included different metal ions (Mn 2+ , Ni 2+ , Mg 2+ , Ba 2+ , Co 2+ , Cu 2+ , Zn 2+ , Fe 2+ , and Fe 3+ , 1 mM final concentration), EDTA (1 mM final concentration), reducing reagents (2-mercaptoethanol, DTT, l-cysteine, and l-glutathione, 1mM final concentration), surfactant (SDS, Triton x-100, Tween-20, and Tween-80, 1% (v/v)), protease inhibitor (PMSF, 1 mM final concentration) and a variety of organic solvents (methanol, ethanol, glycerol, isopropanol, DMSO, acetone, ether, ethyl acetate, and chloroform, 5% and 20% (v/v)) were systematically examined on purified nitrilase. Measurements were conducted under the standard assay condition mentioned previously with 3-cyanopyridine (10 mM) as substrate. The activity assayed in the absence of metal ions or other chemical compounds was taken as 100%.
- Measurement of Kinetic Constants
The kinetic constants of the purified enzyme toward 3-cyanopyridine were measured under standard assay condition with different substrate concentrations (1, 3, 5, 7, 9, 10, 20, 30, 40, and 50 mM). The kinetic constants V max and K m were calculated from double-reciprocal plots according to Lineweaver–Burk by non-linear regression fitting to the Michaelis-Menten equation.
- Substrate Specificity
The catalytic activity of purified enzyme toward different nitriles (10 mM) with diverse structure was examined by quantifying the amount of ammonia liberated during the hydrolysis, based on the Bertholet assay described previously. The relative activity for each nitrile was expressed as the percentage of the specific activity of 3-cyanopyridine, which was taken as 100%.
The GenBank accession number of the nucleotide sequence reported in this paper is NC_023151.1.
Results and Discussion
- Sequence Analysis
The sequence analysis showed that the target fragment containing an open reading frame composed of 804 base pairs encoded a protein of 268 amino acid residues with a theoretical molecular mass of 30.07 kDa. As shown in Fig. 1 , the multiple alignment results demonstrated that the nitrilase from Thermotoga maritima MSB8 possessed 98% identity with the putative nitrilase from Thermotoga petrophila . The identities of Thermotoga maritima MSB8 nitrilase amino acid to other nitrilases from Thermosipho africanus , Thermosipho melanesiensis , Kosmotoga olearia , Mesotoga prima , Pseudomonas putida , Bacillus pumilus , Bacillus sp. WP8, and Rhizobium selenitireducens were 55%, 52%, 45%, 42%, 34%, 31%, 31%, and 33%, separately. Highly conserved domains could be identified and E41, K109, and C144 were found to most likely consist of the catalytic triad of recombinant nitrilase according to the multiple alignment of the amino acid. The involvement of a catalytic cysteine was proved by the inhibiting effect of diverse thiol active agents. In addition, DTT could slightly increase the activity of recombinant nitrilase, which is also in agreement with the expectation of an active cysteine [3] .
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Multiple sequence alignment of various nitrilases with close homologs, conducted by MEGA 6.06. Black represents identical amino acids; dark grey represents conserved amino acids; light grey represents weak similarity of amino acids; no color represents no homology. Residues consisting of the proposed catalytic triad are marked with a downward black arrow.
- Overexpression and Purification of Recombinant Nitrilase
The nitrilase gene from the published genomic DNA of Thermotoga maritima MSB8 strain that contains the opening reading frame encoding a putative nitrilase enzyme was multiplied and subsequently heterologously overexpressed in E. coli BL21 (DE3). The encoded protein was then purified from the cell-free extract of the recombinant E. coli strain harboring the plasmid pET-28a (+) due to the Nterminal His6 affinity tag as mentioned previously. The purified protein gave a single band on SDS-PAGE with a molecular mass of about 30.07 kD ( Fig. 2 ), which was consistent with the predicted molecular mass of the protein.
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SDS-PAGE analysis of the recombinant nitrilase. Lane 1: protein marker; Lane 2: whole cell lysates; Lane 3: supernatant of the cell-free extract; Lane 4: nitrilase purified by NI-NTA column.
- Temperature and pH Optima and Temperature Tolerance of Purified Nitrilase
The effect of temperature on the recombinant nitrilase toward 3-cyanopyridine is presented in Fig. 3 A. The reaction rate increased steadily and reached a maximum at 45℃ along with the rising temperature. However, it decreased sharply at higher temperature owing to the protein denaturation, which parallels the nitrilase from Rhodococcus rhodochrous J1 [17] .
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Effects of temperature (A) and pH (B) on and temperature tolerance of (C) the activity of purified nitrilase. (A) Effect of temperature. The reactions were carried out under standard enzyme assay conditions with 3-cyanopyridine (10 mM) as substrate at temperatures ranging from 25℃ to 55℃. (B) Effect of pH. The reactions were conducted under standard enzyme assay conditions with 3-cyanopyridine (10 mM) as substrate in the following buffers that possess different pH: saline sodium citrate buffer (square), sodium phosphate buffer (round), Tris-HCl buffer (triangle). (C) Temperature tolerance. The reaction mixtures containing the purified enzyme and the buffer were heated in a water bath for 30 and 60 min at 45℃, 55℃, 65℃, 75℃, and 85℃, respectively, and then the residual enzyme activity was measured under standard assay conditions. The value acquired without heat treatment was set as 100%. All experiments were performed in triplicate.
As can be seen from Fig. 3 B, the enzyme demonstrated its maximal activity at around pH 7.5, which is comparable to the nitrilase from Alcaligenes faecalis JM3 [18] . Like many other nitrilases, this enzyme possessed a relatively narrow pH optima within 6-8.5, and the nitrilase activity declined remarkably when the pH value was outside this range. The obvious loss of nitrilase activity under high temperatures and extreme pH values might reveal some significant changes of enzyme structure at those conditions [37] .
The temperature tolerance of the recombinant nitrilase was conducted using 3-cyanopyridine as substrate and is demonstrated in Fig. 3 C. Temperature tolerance studies indicated that nitrilase activity could remain nearly 50% and 40% of the control after heat treatment at 75℃ for 30 and 60 min separately, much better than the nitrilase from a metagenomic library whose activity was completely inactive after 10 min of heat treatment at 60℃ [3] , indicating that this nitrilase possesses good temperature tolerance. The enzyme was almost completely inactivated after heat treatment at 85℃ for 60 min owing to denaturation of the enzyme.
- Effects of Metal Ions and Other Reagents on the Activity of Purified Nitrilase
Nitrilase activity in the presence of various compounds was investigated. As shown in Table 1 , the recombinant nitrilase was highly sensitive to thiol-group metals ions, such as Mn 2+ , Zn 2+ , and Cu 2+ , demonstrating that the thiol group was crucial for the catalytic activity. These experimental data were similar to the nitrilase reported before [20] . It coincides well with the catalytic mechanism of nitrilase. A thiol group of cysteine residue, locating in the catalytic active center of nitrilase, could attack the carbon atom in the nitrile molecule [8] . Ni 2+ also resulted in a conspicuous decrease of nitrilase activity. It might be put down to the formation of coordination complexes with catalytically active thiol groups [1] . In contrast, nitrilase activity could to some degree be enhanced in the presence of Mg 2+ and Fe 2+ . Other metal ions and chelating reagent EDTA were found to have no significant inhibitory effects on enzyme activity.
Effects of metal ions and other reagents on the activity of purified nitrilase.
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The reactions were performed under the standard conditions with 3-cyanopyridine (10 mM) as the substrate. The relative activity was expressed at the percentage of the activity without addition of metal ions and other reagents, which was defined as 100%.
The influence of reductants, surfactant, and protease inhibitor was also investigated. As shown in Table 1 , 2-mercaptoethanol, ʟ-cysteine, l-glutathione showed slight influence on the nitrilase activity, and DTT could somewhat increase the nitrilase activity, which is similar to the nitrilase from Pseudomonas putida [37] . The improvement of the nitrilase activity by a low concentration of DTT might be associated with their function of reforming disulfide bonds in protein structures. Nitrilase activity decreased to 43.5%, 45.7%, 42.9%, 37.1%, and 49.2%, respectively, of the control when using Triton X-100, Tween-20, Tween-80, SDS, and PMSF. The inhibitory effect of non-ionic surfactant, including Triton X-100, Tween-20, and Tween-80, could be due to the hydrogen bond and hydrophobic interaction between the non-ionic surfactant and enzyme molecular. The inhibitory effect of the ionic surfactant SDS could be due to the strong electrostatic interaction between charged groups of the ionic surfactant and enzyme molecule. The inhibitory effect of PMSF, a kind of protease inhibitor, might be associated with the modification of some indispensable amino acids or groups of the protein structure on the surface of the enzyme. PMSF could interact with those amino acids containing the oxhydryl, which indicates that those necessary groups containing the oxhydryl located outside the active site could also indirectly lead to the change of enzyme molecular conformation and subsequent decrease of enzyme activity.
- Effects of Different Organic Solvents on the Activity of Purified Nitrilase
It is necessary to determine the resistant degree of nitrilase to organic solvents in nitrilase-mediated biocatalytic reactions owing to the poor solubility of substrates (nitriles) or products (carboxylic acids) in the reaction process. As is presented in Fig. 4 , the nitrilase activity could remain at 68.1% of the control in 5% (v/v) methanol, which is similar to the nitrilase from Gibberella intermedia whose remaining enzyme activity was 60% in 5% (v/v) methanol. Relative activity values of 91.1%, 59.5%, 70.6%, 55.6%, 43.1%, 30.1%, 15.1%, and 11.8% were determined in 5% (v/v) ethanol, glycerol, isopropanol, DMSO, acetone, ether, ethyl acetate, and chloroform, respectively. Among them, hydrophilic organic solvents had slight inhibitory effect on enzyme activity, whereas hydrophobic organic solvents such as ether, ethyl acetate, and chloroform had a strong effect, which might be ascribed to the deactivation by them. In addition, when 20% (v/v) of organic solvent was utilized, the nitrilase activity declined sharply and only very low activity could be detected, except that in methanol, ethanol, and isopropanol with 32.4%, 40.6%, and 37.3% of relative activity retained, respectively.
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Effects of different organic solvents on the activity of purified nitrilase. The reactions were performed under the standard conditions with 3-cyanopyridine (10 mM) as the substrate. The activity assayed in the absence of different organic solvents was taken as 100%.
- Kinetic Constants and Substrate Specificity
As is presented in Fig. 5 , kinetic constants were detected with different 3-cyanopyridine concentrations as the substrate in the present study so as to obtain a better estimation of the catalytic properties of the recombinant nitrilase. As we can see, the reaction rate increased with the increase of substrate concentration up to 40 mM, and then the reaction rate declined rapidly, which can be explained by substrate inhibition. It was calculated that the V max and K m were 3.12 µmol/min/mg and 7.63 mM, respectively, through the non-linear plotting. The K m value of this nitrilase is much smaller than that from Pseudomonas putida CGMCC3830 and Rhodobacter sphaeroides LHS-305 toward 3-cyanopyridine, whose K m values are 27.9 mM and 73.1 mM [26 , 37] , respectively. The K m value represents the affinity between the enzyme and its substrate, and a smaller K m value indicates stronger affinity between the enzyme and substrate, and vice-versa . Thus, the kinetic constants obtained in this study indicate a better substrate affinity compared with nitrilases from Pseudomonas putida CGMCC3830 and Rhodobacter sphaeroides LHS-305. On the contrary, the nitrilase from Alcaligenes faecalis whose K m and V m values were 4.36 mM and 3.15 µmol/min/mg, respectively [30] , exhibited better activity than T. maritima nitrilase. P. fluorescens nitrilase also showed a high activity with dinitriles. However, differences existed. Both of them showed the highest activity when succinonitrile was used as substrate for T. maritima nitrilase ( Table 2 ) and P. fluorescens nitrilase [13] . After calculation, the K m value was 17.9 mM and catalytic efficiency k cat /K m was 0.5 /mM/s for P. fluorescens nitrilase, whereas the K m value was 10.58 mM and catalytic efficiency k cat /K m was 0.44/mM/s for T. maritima nitrilase, which represented that T. maritima nitrilase had a higher affinity toward succinonitrile. However, P. fluorescens nitrilase had a better catalytic efficiency than T. maritima nitrilase toward the hydrolysis of succinonitrile. Not all nitrilases harboring the ability to hydrolyze dinitriles could show similar catalytic efficiency toward the same substrate.
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Influence of substrate concentrations on the reaction rate of the purified nitrilase.
Substrate spectrum of the recombinant nitrilase fromThermotoga maritimaMSB8.
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Nitrilase activity toward different nitriles was examined under the standard assay conditions with 10 mM substrate. The specific activity with 3-cyanopyridine was set at 100%. ND means not detected.
The specific activities of the nitrilase from Thermotoga maritima MSB8 was examined for its catalytic activity toward various nitrile compounds by quantifying the amount of ammonia liberated during the hydrolysis. For the convenience of the comparison, the relative activity toward 3-cyanopyridine was defined as 100%, and the results for these nitriles are shown in Table 2 . As can be seen from Table 2 , this nitrilase exhibited a broad spectrum for the tested nitriles and showed highest activities toward the hydrolysis of aliphatic dinitriles such as malononitrile, succinonitrile, and hexanedinitrile. The maximum activity was observed with succinonitrile, which was approximately 7-fold higher than that toward acetonitrile. Lower activity was also observed on some aromatic nitriles, including phenylacetonitrile and 4-aminobenzonitrile. No detectable activity was observed when mandelonitrile, benzonitrile, and glycolonitrile were utilized as a substrate of the present nitrilase. The activity of the nitrilase toward favorite substrates such as succinonitrile was likely due to the structure of dinitriles, which possess a double cyano group. It appeared possible that the conversion of this type of compounds could be further increased in the presence of a double cyano group. The conversion of the cyano group located on the one side might affect another on the other side, which might result in a positive effect on the enzymatically catalytic reaction. Consequently, the T. maritima nitrilase might prefer certain nitriles like the dinitriles. In the catalytic triad (CEK) of nitrilase, cysteine attacks the cyano group of nitrile as a nucleophile, glutamate plays as a general base, and lysine is involved in stabilization of the tetrahedral intermediate. When dinitrile was used as a substrate, dinitrile was bound into the binding site through H-bond with Cys, Glu, and Lys. After the first cyano group was bound with the Cys, another cyano group was bound with Glu, which probably results in the stable transition state intermediate and proper orientation for nucleophilic attack to the first cyano carbon by the –SH group of Cys. Here, the distance between the cyano group and sulfur of Cys played a prominent role in the enzymatically catalytic reaction. That some nitrilases showed no or very low activity on dinitriles might be due to the long distance between the cyano group and sulfur of Cys [13] . The distance between sulfur of Cys of the T. maritima nitrilase and the cyano group of dinitriles is much shorter than that of some other nitrilases. That might be the main reason that T. maritima nitrilase has a higher affinity toward dinitriles than other nitrilases. For some other substrates harboring benzene or oxhydryl, such as mandelonitrile, benzonitrile, phenylacetonitrile, and glycolonitrile, no activity or only very low activity could be detected by T. maritima nitrilase. It might be attributed to the negative effect by the benzene or oxhydryl in the presence of another side of the cyano group. For the aliphatic nitriles, such as acetonitrile, propionitrile, butyronitrile, and pentanenitrile, the activities decreased as the chain lengths became longer. Therefore, the structure of substrates might play a dominant role in determining the activity of T. maritima nitrilase. To the best of our knowledge, only a few nitrilases reported before possess the ability for hydrolyzing aliphatic dinitriles [6 , 26 , 36] . According to the classification method by Banerjee A et al . [2] , this enzyme could be classified as an aliphatic dinitrile nitrilase based on the substrate preference.
In conclusion, in the present study, a novel nitrilase was cloned from Thermotoga maritima MSB8 and successfully overexpressed in E. coli (DE3). The nitrilase was purified with a molecular mass of 30.07 kDa. After biochemical characterization, the optimum temperature and pH toward 3-cyanopyridine were observed to be around 45℃ and 7.5, respectively. This nitrilase aslo showed good temperature tolerance, with 40% residual activity after 60 min of heat treatment at 75℃. Furthermore, this recombinant nitrilase displayed a broad substrate spectrum and especially good catalytic efficiency on aliphatic dinitriles, which is distinguished from most nitrilases ever reported. It is expected that this novel nitrilase might be a potential candidate for industrial applications for biosynthesis of carboxylic acid.
Acknowledgements
This study was supported by the Open Funding Project of National Key Laboratory of Biochemical Engineering, the National Key Basic Research Program of China (973 Program, No. 2011CBA00800) and the Key Agriculture Support Project of Jiangsu Province, China (No. BE2013400).
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