Characterization of a versatile nitrile hydratase of the neonicotinoid thiacloprid-degrading bacterium Ensifer meliloti CGMCC 7333

Abstract

The nitrogen-fixing bacterium Ensifer meliloti CGMCC 7333 and its nitrile hydratase (NHase) degrade the neonicotinoid insecticides, thiacloprid (THI) and acetamiprid (ACE), to their corresponding amide metabolites. The NHase gene cluster is composed of α-subunit and β-subunit genes and a hypothetical protein gene. The functionality of the hypothetical protein downstream of the NHase coding genes and the characteristics of CGMCC 7333 NHase were explored in this study. Co-expression of the hypothetical protein coding gene with NHase (α- and β-subunit genes) in Escherichia coli Rosetta enhanced NHase hydration of THI and ACE two- and four-fold, respectively, and also significantly improved NHase solubility compared with the absence of the hypothetical protein coding gene. The NHase displayed an optimal reaction temperature of 50 °C for THI hydration and was unstable when the incubation temperature exceeded 40 °C. The optimum reaction pH was 7.0 and the NHase activity was stable in the pH range of 6 to 9. The enzyme activity for THI hydration was slightly inhibited by copper, zinc, and iron, and decreased by 68.6%, 75.7%, and 70.3% when 2% ethanol, ethyl acetate, and acetone were added to the reaction mixture, respectively, whereas dichloromethane and trichloromethane had no effect. The K_m and k_cat values of CGMCC 7333 NHase for THI hydration were 12.39 mmol L⁻¹ and 131.36 s⁻¹, respectively. Substrate specificity analysis indicated that CGMCC 7333 NHase also transformed 3-cyanopyridine, benzonitrile, and indole-3-acetonitrile to the corresponding amide products, with maximum specific activities of 652.52, 255.32, and 263.93 U mg⁻¹ protein, respectively.

---

1. Introduction

Neonicotinoids are a class of N-heterocyclic insecticides that act agonistically on insect nicotinic acetylcholine receptors. These compounds are the newest class of synthetic insecticides to be produced during the past two decades and the biggest selling insecticide class worldwide.¹ Thiacloprid (THI), (Z)-[3-[(6-chloro-3-pyridinyl)methyl]-2-thiazolidinylidene]cyanamide, is a second-generation neonicotinoid insecticide, which has activity not only against sucking insects, such as aphids, whiteflies, and some jassids, but also against weevils, leafminers, and various species of beetle. However, like other neonicotinoids, THI has been reported to adversely affect the integrity of ecosystems. For example, exposure to sublethal doses of THI greatly increases the mortality of honeybees. THI is highly water soluble and 80–98% of residues remaining in the soil of treated crops eventually move into surface water or leach into groundwater and, therefore, produce contamination and delayed mortality in freshwater arthropods.¹

Microbial metabolism is one of the major pathways of pesticide degradation in natural soil environments, and so far, the bacteria Stenotrophomonas maltophilia CGMCC 1.1788,² Variovorax boronicumulans CGMCC 4969,³ and Ensifer meliloti CGMCC 7333,⁴ and the yeast Rhodotorula mucilaginosa strain IM-2,⁵ have been reported to degrade THI. S. maltophilia CGMCC 1.1788 degrades THI via hydroxylation of the thiazolidine ring to form the metabolite 4-hydroxy THI;² R. mucilaginosa strain IM-2, V. boronicumulans CGMCC 4969, and E. meliloti CGMCC 7333 all degrade THI to the major metabolite N-carbamoylimine derivative (THI amide) with a conversion rate greater than 70%.

E. meliloti CGMCC 7333 is a nitrogen-fixing bacterium that produces the highest THI degradation among the THI-degrading microbes. It degraded 86.8% of 200 mg L⁻¹ THI in 60 h with a half-life of 20.9 h, and 90.9% of the reduced THI was converted to THI amide. We further proved that hydrolysis of THI to THI amide by E. meliloti CGMCC 7333 is mediated by a nitrile hydratase (NHase) and that the NHase gene cluster codes a cobalt-type NHase composed of an α-subunit, β-subunit, and hypothetical protein with lengths of 213, 219, and 128 amino acids, respectively.⁴ Because the enzymatic characteristics of the NHase with respect to THI hydrolysis had not been previously reported, purified NHase from CGMCC 7333 was over-expressed in Escherichia coli Rosetta and its substrate diversity, temperature, pH stability, and the effects of metal ions and organic solvents on its enzyme activity were determined. On the other hand, genes corresponding to this type of NHase are found in the genomic DNA sequences of all E. meliloti strains: 2011, GR4, SM11, AK83, BL225C, CCNWSX0020, 1021, and RM41. The NHase gene clusters in these organisms share similar genes, with an α-subunit, β-subunit, and a hypothetical protein coding gene (the GenBank accession numbers of the CGMCC 7333 NHase gene cluster are KF601242, KF601243, and KF601244, respectively). The α- and β-subunit genes have a 4-bp overlapping sequence “ATGA”, and the β-subunit and hypothetical protein genes have a 14-bp overlapping sequence “TTGAACCCGCATAG” (Fig. 1). We speculate that these components within the NHase gene cluster may be associated with important functions of E. meliloti NHase. Therefore, expression plasmids hosting different NHase subunit genes were constructed and over-expressed in E. coli Rosetta to evaluate the contributions of the different NHase subunits to NHase activity. Investigation of the metabolism of THI in the soil revealed that about 70% of THI was converted to THI amide and microbial activity plays a key role in this conversion.⁶ Therefore, the present study will help us to further understand the environmental fate of THI and may suggest potential applications of NHase subunits in the biodegradation of pesticides and in the biocatalysis of high-value organic compounds.

Fig. 1 The composition of the CGMCC 7333 NHase gene cluster and expression plasmid construction.

On the other hand, nitrile-containing compounds have been largely artificially synthesized and widely used in the form of solvents, plastics, synthetic rubber, herbicides, and pharmaceuticals. For example, the benzonitrile herbicides, dichlobenil, bromoxynil, and ioxynil, are important broad-spectrum or selectively used herbicides in agriculture, orchards, and public areas worldwide. The nitrogen-fixing bacteria Rhizobium radiobacter strains 8/4 and DSM 9674 and Rhizobium sp. 11401 have been reported to degrade these benzonitrile compounds through NHase/amidase systems.^7–10 The nitrogen-fixing Mesorhizobium sp. F28 and its NHase degrade the solvent acetonitrile.¹¹ The aim of the present study is to investigate the characteristics of E. meliloti CGMCC 7333 NHase, which will help us to understand the mechanism of nitrile metabolism in nitrogen-fixing bacteria.

2. Experimental

2.1 Chemicals

Indole-3-acetonitrile (IAN), indole-3-acetamide (IAM), 3-cyanopyridine (3-CP), nicotinamide, benzonitrile (BN), benzamide, 2,6-dichloro-benzonitrile (dichlobenil), isobutyronitrile, succinonitrile, hexanedinitrile, and (R)-(+)-4-methylmandelonitrile (4-MMN) were purchased from Sigma-Aldrich (Shanghai, China; each of 98% purity). The nitrile-containing pesticides thiacloprid (THI), acetamiprid (ACE), fipronil, chlorfenapyr, fludioxonil, azoxystrobin, and cyhalofop-butyl were provided by Professor Jue-Ping Ni (Jiangsu Pesticide Research Institute, Nanjing, China; each of 95% purity). THI amide and the ACE hydration metabolite (IM-1-2) were synthesized by the methods described in our previous studies.¹² HPLC grade acetonitrile and methanol were purchased from Tedia Company Inc. (Fairfield, OH, USA). All other reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

2.2 Strains and media

The bacterium E. meliloti CGMCC 7333 was isolated from rhizosphere soil and deposited in the China General Microbiological Culture Collection Center (CGMCC) (Beijing, China). E. coli Rosetta (DE3) pLysS was used as the host for NHase gene expression. Luria–Bertani (LB) medium contained 10.0 g peptone, 5.0 g yeast extract, and 10.0 g NaCl in 1.0 L deionized water, pH 7.2. CoCl₂ at a final concentration of 0.1 mmol L⁻¹ was added to the culture broth to produce active NHase as necessary.

2.3 Plasmid construction

The total genomic DNA from E. meliloti CGMCC 7333 was extracted using a MiniBEST bacterial genomic DNA extraction Kit (Takara, Dalian, China). Primers were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. The primers ABC-F (5′-GCTAGCATGTCCGAGCATCATCATGGGC-3′) with an NheI restriction site and ABC-R (5′-AAGCTTACGCAGAGGATCATTCGCGAG-3′) with an HindIII restriction site were used for amplifying the NHase gene cluster containing the α-subunit gene (642 bp), β-subunit gene (660 bp), and the hypothetical accessory protein coding gene (387 bp). The primers ABC-F and AB-R (5′-AAGCTTTGCGGGTTCAAGATAGCTCTCCC-3′) with an HindIII restriction site were used for amplifying the α-subunit and β-subunit genes. The 14-bp overlapping sequence was part of the β-subunit gene. The primers ABC-F and A-R (5′-AAGCTTTCATCGTACCCCCTCCGGCGATAG-3′) with an HindIII restriction site were used for amplifying the α-subunit gene alone. Polymerase chain reactions (PCR) were performed in 25 μL reaction volumes containing 5 μL each of standard PCR buffer (5×), 2 μL of dNTP (2.5 mmol L⁻¹ each), 0.1 μL of forward primer (10 μmol L⁻¹), 0.1 μL of antisense primer (10 μmol L⁻¹), 0.25 μL of template DNA (300 ng μL⁻¹), 0.25 μL of PrimeSTAR HS DNA polymerase (2.5 U μL⁻¹), and 17.3 μL of autoclaved deionized water. An initial denaturation for 5 min at 95 °C was followed by 32 cycles of denaturation for 40 s at 95 °C, annealing for 15 s at 62 °C, and extension for 90 s at 72 °C, with a final 10 min extension step at 72 °C. The PCR products were cloned into vector pMD19-T (Takara) by a TA (thymine and adenine) cloning method, according to the manufacturer's protocol, and sequenced by Springen Biotech Co. (Nanjing, China). The verified PCR products were excised with restriction enzymes NheI and HindIII and ligated into vector pET28a digested at the same restriction enzyme sites.

2.4 Expression and purification of recombinant NHase in E. coli Rosetta (DE3) pLysS

E. coli Rosetta (DE3) pLysS cells were made competent for plasmid transformation using the calcium chloride method.⁴ Over-expression of recombinant NHase in E. coli Rosetta (DE3) pLysS was described in our previous study.^4,12 The purification of the NHase by His-tag affinity chromatography was performed according to the protocol of the manufacturer of the chromatography resin (Novagen, Inc., Madison, WI). To analyze enzyme solubility, the cell pellets were resuspended in 0.2 culture volumes of 0.2 mol L⁻¹ sodium phosphate buffer and disrupted by sonication (10 s) for 3 min at 4 °C. A total protein sample was collected from the cell suspension after sonication, and the soluble proteins were further separated from the supernatant after the insoluble debris was pelleted by centrifugation at 12 000g for 20 min. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was carried out as described by Ge et al.⁴ Gels were stained with Coomassie Brilliant Blue for protein detection.

2.5 Western blot analysis

Protein samples were separated with SDS–PAGE and then blotted for 100 min on a PVDF membrane (Millipore Immobilon-P, Millipore Cor., Billerica, MA, USA) with blotting buffer containing 25 mmol L⁻¹ Tris and 190 mmol L⁻¹ glycine in 20% (v/v) aqueous methanol. The electric field was 0.9 mA cm⁻² and a His-tag monoclonal antibody was employed (Novagn, Inc., Madison, WI, USA).¹³

2.6 Enzyme assay

The NHase activity was determined in a reaction mixture (1 mL) containing 0.2 mol L⁻¹ sodium phosphate buffer (pH 7.5), 200 mg L⁻¹ THI, and an appropriate amount of enzyme. The reaction was conducted for 10 min at 30 °C and stopped by the addition of 1 mol L⁻¹ HCl (20 μL). The samples were centrifuged at 12 000g for 10 min and the supernatants were analyzed using HPLC. One unit (U) of NHase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of product in 1 min. The protein concentration was determined according to the Bradford method¹⁴ using bovine serum albumin as the standard. The NHase activities toward the aliphatic nitrile compounds acetonitrile, isobutyronitrile, succinonitrile, and hexanedinitrile were assessed according to the methods reported by Okamoto and Eltis.¹⁵ The NHase assay was coupled with an aliphatic amidase from Variovorax boronicumulans CGMCC 4969,¹⁶ which was heterologously produced in E. coli Rosetta as a His-tagged amidase and purified using a Ni-NTA resin. Sufficient amidase (9.1 mU) was added to the reaction mixture, and the reaction was conducted as described above.

2.7 Characterization of the CGMCC 7333 NHase

To determine the influence of pH on the NHase activity, the reaction was conducted in 0.2 mol L⁻¹ sodium phosphate buffer at pH 5, 6, 7, 8, and 9. To determine pH stability, the enzyme was incubated at 4 °C for 12 h in different buffers, and the residual activity was measured using the standard enzyme activity assay method described above. The optimal reaction temperature was determined at the optimal pH by conducting the reaction at temperatures from 20 to 60 °C, and the thermal stability was assessed by incubating the enzyme at different temperatures for 1 h, with non-heated enzyme used as the control (100% activity).

The effects of metal ions on NHase activity toward THI were determined by individually adding CoCl₂, CaCl₂, CuCl₂, FeCl₂, MnCl₂, ZnCl₂, and MoCl₂ to the standard reaction mixture at a final concentration of 0.1 mmol L⁻¹. The effects of organic reagents on the NHase activity were investigated by individually adding 2% (v/v) methanol, ethanol, ethyl acetate, dichloromethane, acetone, dimethyl sulfoxide, isopropanol, isoamylol, and trichloromethane to the standard reaction mixture. Enzyme activity without any additive was used as the control and its activity for THI was defined as 100%.

Substrate specificities of the NHase toward the cyano group-containing pesticides, THI, ACE, 2,6-dichloro-benzonitrile (dichlobenil), fipronil, chlorfenapyr, fludioxonil, azoxystrobin, and cyhalofop-butyl, and the organic intermediates, IAN, 3-CP, BN, and 4-MMN, were assayed in a standard mixture (1 mL) containing 0.2 mol L⁻¹ sodium phosphate buffer (pH 7.5), 10 μL of the purified NHase solution with a final concentration of 19 μg mL⁻¹, and 1 mmol L⁻¹ substrate.

The kinetic parameters of the CGMCC 7333 NHase were estimated over a range of substrate concentrations (10–4000 mg L⁻¹) at 30 °C under the above conditions. The maximal hydrolysis rate (V_max) and apparent Michaelis–Menten constant (K_m) were calculated from Lineweaver–Burk plots.

2.8 Analytical methods

The various aromatic nitriles and their corresponding amides were analyzed by HPLC. An Agilent 1260 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a reverse-phase HC-C18 guard column (4.6 × 12.5 mm, 5 μm particle size, part number 520518-904; Agilent Technologies) and an HC-C18 column (4.6 × 250 mm, 5 μm particle size, part number 588905-902; Agilent Technologies) was used to analyze each substrate and its metabolites. The signals were monitored using an Agilent G1314A UV detector (part number G1314-60100) and the HPLC conditions are listed in Table 1.

Table 1 HPLC conditions for the substrates used in this study

Substrate	Mobile phase			UV wavelength (nm)	Flow rate (mL min^−1)
	A	B	A : B (v : v)
IAN	Water	Acetonitrile	60 : 40	230	1
THI	Water	Acetonitrile	65 : 35	242	1
ACE	Water	Acetonitrile	70 : 30	235	1
3-CP	Water	Methanol	70 : 30	230	1
BN	Water	Acetonitrile	45 : 45	231	1
2,6-Dichloro-benzonitrile	Water	Acetonitrile	45 : 45	231	1
4-MMN	Water	Acetonitrile	40 : 60	230	1
Fipronil	Water	Acetonitrile	40 : 60	215	1
Chlorfenapyr	Water	Acetonitrile	40 : 60	255	1
Fludioxonil	Water	Acetonitrile	40 : 60	270	1
Azoxystrobin	Water	Acetonitrile	40 : 60	255	1
Cyhalofop-butyl	Water	Acetonitrile	40 : 60	238	1

---

3. Results

3.1 THI hydration by over-expression of different components of the CGMCC 7333 NHase gene cluster in E. coli Rosetta

In a previous study,⁴ we cloned a 1.7-kb DNA fragment from E. meliloti CGMCC 7333 that contained the α-subunit and β-subunit genes and a hypothetical protein coding gene (Fig. 1). This hypothetical protein, with a length of 128 amino acids and a molecular mass of 14 kDa, exhibited 80–99% identity with the NHase accessory proteins of Ensifer spp. and about 66% identity with the NHase accessory protein of Rhizobium sp. (accession number WP_024313557) (another nitrogen-fixing genus). The hypothetical protein had only 13.2% and 21.8% identity with the NHase activator P14K of Pseudomonas putida NRRL-18668 (accession number AAC18420.1) and Bacillus pallidus RAPc8 (accession number AAS84452.1), respectively.

To examine the functionality of the hypothetical protein, a plasmid (pNAB) containing only the α-subunit and β-subunit genes was constructed and over-expressed in E. coli Rosetta. As shown in Table 2, resting cells of E. coli-pNAB exhibited THI hydration activity of 0.21 U mL⁻¹ of culture medium, whereas E. coli-pNABC exhibited an NHase activity of 0.42 U mL⁻¹. Similarly, E. coli-pNABC exhibited NHase activity of 0.41 U mL⁻¹ for ACE hydration, but E. coli-pNAB only exhibited 0.09 U mL⁻¹ NHase activity for ACE hydration. These results indicated that co-expression of the hypothetical protein coding gene with the α-subunit and β-subunit genes significantly increases the NHase activity. Therefore, the protein downstream of the β-subunit gene is an activator of functional NHase, which is similar to the NHase activator P14K of P. putida NRRL-18668 ¹⁷ and of B. pallidus RAPc8.¹⁸ E. coli-pNA, in which only the α-subunit gene was expressed, and control E. coli-pET28a with no NHase expression had almost no NHase activity.

Table 2 Effects of different combinations of NHase coding genes on NHase activity in hydration of THI and ACE^a

Strain	NHase activity (U mL^−1)
	THI	ACE
a Data indicate the means of three replicates. Mean values (±SD) within a column followed by different letters are significantly different at p ≤ 0.05 according to the Duncan test.
E. coli-pNABC	0.42 ± 0.01a	0.41 ± 0.05a
E. coli-pNAB	0.21 ± 0.01b	0.09 ± 0.03b
E. coli-pNA	0.01 ± 0.02c	0.02 ± 0.01c
E. coli-pET28a	0.01 ± 0.01c	0.01 ± 0.01c

---

The expression of different components of NHase from the recombinant plasmids pNABC, pNAB, and pNA was detected by SDS–PAGE after lysing the host E. coli Rosetta cells (Fig. 2; lanes 1, 3, and 7). In the soluble fractions (lanes 2, 4, and 8), the amount of over-expressed pNABC NHase (lane 8) was apparently higher than that of pNAB NHase (lane 4). Although the over-expressed accessory protein could not be observed in lane 8, it could be detected by western blotting analysis (lane 10) and in the purified NHase by His-tag affinity chromatography (lane 9). These results indicate that the presence of the protein expressed from the accessory coding gene improved the protein solubility of the target NHase and consequently increased NHase activity in the conversion of THI and ACE to their respective amide metabolites.

Fig. 2 SDS–PAGE of different components of the CGMCC 7333 NHase gene cluster over-expressed in E. coli Rosetta and the purified co-expression NHase with accessory protein and western blotting analysis. Lanes 1, 3, 5, and 7 represent the total protein of E. coli Rosetta (DE3) over-expressed by the plasmids pNA, pNAB, pET28a, and pNABC, respectively; lanes 2, 4, 6, and 8 represent the soluble protein of E. coli Rosetta (DE3) over-expressed by the plasmids pNA, pNAB, pET28a, and pNABC respectively; lane M: standard protein markers (116.0, 66.2, 45.0, 35.0, 25.0, 18.4, and 14.4 kDa from top to bottom); lane 9: purified NHase expressed with pNABC (arrows labelled I and II indicate the NHase and the accessory protein, respectively); lane 10: western blotting of the purified pNABC NHase. All of the over-expressed α-subunits had a 6 histidine tag at the N-terminus. The β-subunit over-expressed by pNAB and the accessory protein over-expressed by pNABC also had a 6 histidine tag in the C-terminus.

3.2 Substrate specificity and the kinetic parameters of E. meliloti CGMCC 7333 NHase

CGMCC 7333 NHase, which can hydrate the neonicotinoid insecticides THI and ACE, was further examined to determine its substrate specificities toward aromatic nitriles such as BN and 4-MMN, the auxin-precursor IAN, the fine chemical intermediate 3-CP, and bactericides fludioxonil and azoxystrobin, herbicides dichlobenil and cyhalofop-butyl, and two other insecticides, fipronil and chlorfenapyr. HPLC analysis showed that IAN, BN, and 3-CP could be transformed by CGMCC 7333 NHase to the metabolites IAM, benzamide, and nicotinamide, respectively. NHase also transformed the aliphatic nitriles acetonitrile, isobutyronitrile, succinonitrile, and hexanedinitrile, although its relative activities were comparatively lower than with the aromatic BN, 3-CP, and IAN (Table 3). CGMCC 7333 NHase could not transform 4-MMN, dichlobenil, and the other tested pesticides.

Table 3 Substrate specificities of the purified CGMCC 7333 NHase^a

Substrate	Relative activity ± SD (%)
a NHase activity was assayed under the standard assay conditions with 1 mmol L^−1 substrate. The specific activity for THI (1.80 U mg^−1) was taken as 100%. ND, not detected.
THI	100 ± 1.22
ACE	22.41 ± 1.94
IAN	479.79 ± 9.53
3-CP	5528.10 ± 43.77
BN	2532.71 ± 37.26
Acetonitrile	29.02 ± 1.03
Isobutyronitrile	3.78 ± 0.06
Succinonitrile	13.17 ± 0.12
Hexanedinitrile	0.43 ± 0.02
4-MMN	ND
Dichlobenil	ND
Fipronil	ND
Chlorfenapyr	ND
Fludioxonil	ND
Azoxystrobin	ND
Cyhalofop-butyl	ND

---

The kinetic parameters for the reactions of CGMCC 7333 NHase with ACE, THI, IAN, 3-CP, and BN were measured (Table 4). E. meliloti CGMCC 7333 was isolated from soil using THI as the sole nitrogen source,⁴ and therefore its NHase had a relatively high THI hydration activity (40.63 ± 2.67 U mg⁻¹ protein), whereas its hydration activity toward ACE was only 0.24 U mg⁻¹. Although the k_cat/K_m values of the NHase for THI and ACE were similar, the k_cat and K_m for THI were two orders of magnitude larger than those for ACE.

Table 4 Kinetic constants for CGMCC 7333 NHase in the hydrolysis of ACE, THI, 3-CP, and BN^a

Substrate	Vmax (μmol min^−1 mg^−1)	Km (mM)	kcat (s^−1)	kcat/Km (mM^−1 s^−1)
a The enzyme concentrations were 13.2, 13.2, 13.2, 33.1, and 26.5 μg mL^−1 for IAN, 3-CP, BN, ACE, and THI biotransformation, respectively. The kinetic parameters of the NHase were estimated over a range of substrate concentration (10–4000 mg L^−1) at 30 °C in 0.2 mol L^−1 phosphate buffer at pH 7.5. The reaction time for 3-CP and BN was 2 min and for ACE, THI, and IAN was 5 min. The tested concentrations of ACE, THI, 3-CP, IAN, and BN ranged from 10 mg L^−1 to 50 mg L^−1, 80 mg L^−1 to 200 mg L^−1, 100 mg L^−1 to 200 mg L^−1, 1000 mg L^−1 to 4000 mg L^−1, and 500 mg L^−1 to 900 mg L^−1, respectively.
ACE	0.24 ± 0.01	0.14 ± 0.01	1.04 ± 0.13	7.42 ± 0.74
THI	40.63 ± 2.67	12.39 ± 3.60	131.36 ± 36.19	10.60 ± 0.30
3-CP	652.52 ± 48.45	7.34 ± 1.36	1054.89 ± 78.33	146.43 ± 22.90
IAN	263.93 ± 29.48	17.91 ± 1.47	426.68 ± 47.66	23.80 ± 0.71
BN	255.32 ± 3.22	14.81 ± 2.40	1489.82 ± 191.1	100.90 ± 3.48

---

Among the tested substrates, the highest specificity of CGMCC 7333 NHase corresponded to a k_cat/K_m of 146.43 ± 22.90 mM⁻¹ s⁻¹ for 3-CP, which also had the highest V_max of 652.52 ± 48.45 U mg⁻¹ of proteins, which is higher than the values reported for L-NHase (579 U mg⁻¹ protein) and H-NHase (370 U mg⁻¹ protein), two cobalt-type NHases of the industrially used R. rhodochrous J1.¹⁹ However, the K_m value of CGMCC 7333 NHase for 3-CP, 7.34 ± 1.36 mmol L⁻¹, was higher than that of the R. rhodochrous J1 L-NHase (0.30 mmol L⁻¹) and lower than of the H-NHase (200 mmol L⁻¹). Rzeznicka et al.²⁰ reported that the NHase from R. equi TG328-2, an iron-type NHase with an accessory protein of 46 kDa, had a specific activity for 3-CP of 3003 U mg⁻¹ of proteins.

E. meliloti CGMCC 7333 NHase showed a V_max of 263.93 ± 29.48 U mg⁻¹ of proteins for conversion of IAN to IAM, which is much higher than E. meliloti strain 03-03046 of 0.22 mU mg⁻¹ protein, Rhizobium leguminosarum strain 02-10230 of 1.12 mU mg⁻¹ protein and Rhizobium loti strain 02-10101 of 168 mU mg⁻¹ protein reported by Kobayashi et al.²¹

3.3 Effects of pH and temperature on NHase activity and stability in THI hydration

As shown in Fig. 3A, the maximum NHase activity for THI hydration was observed in the temperature range 40–50 °C. When the temperature reached 60 °C, the NHase activity dramatically decreased and only <20% activity was retained. The thermal stability of the purified enzyme was assayed by incubating the NHase for 1 h at 20–60 °C (Fig. 3B). NHase was stable at temperatures below 40 °C, but when the temperature reached 50 °C, only 14% activity was detected and no activity was seen at 60 °C. The pH value of the reaction mixture had a strong effect on NHase activity toward THI (Fig. 3C); NHase had an optimal pH at 7.0, and less than 6% activity was detected at pH 5.0, but >93% activity was retained after storage at pH 6.0–9.0 for 12 h (Fig. 3D).

Fig. 3 Effects of pH and temperature on NHase activity and stability for THI hydration. The concentration of NHase in reaction mixture was 19.26 μg mL⁻¹ and the substrate THI was 200 mg L⁻¹ in 0.2 mol L⁻¹ phosphate buffer. The reaction time was 10 min, and the conditions were pH 7.5 and 30 °C.

3.4 Effects of metal ions and organic solvents on NHase activity in THI hydration

E. meliloti CGMCC 7333 NHase is a cobalt-type NHase, and the addition of cobalt to the cell growth broth significantly stimulates NHase activity.⁴ However, supplementing cobalt to phosphate buffer containing purified enzyme had no effect on NHase activity. Among the tested metal ions, Ca²⁺, Mo²⁺, and Mn²⁺ had little effect, whereas Cu²⁺ showed a 14.8% inhibitory effect on NHase activity (Fig. 4A). Copper inhibition was also observed for the acetonitrile-catabolic NHase from Rhodococcus sp. RHA1,¹⁵ but not for the NHase from Bacillus sp. BR449.²²

Fig. 4 Effects of metal ions and organic solvents on NHase activity on THI hydration. The concentration of NHase in the reaction mixture was 19.26 μg mL⁻¹ and the substrate THI was 200 mg L⁻¹ in 0.2 mol L⁻¹ phosphate buffer. The reaction was conducted at pH 7.5 and 30 °C and the reaction time was 10 min. The volume of organic solvent was 20 μL in 1 mL reaction mixture. Different letters above the columns represent significant differences at p ≤ 0.05 according to the Duncan test.

Among the tested organic solvents, 2% volumes of dichloromethane and trichloromethane did not inhibit NHase activity in THI hydration (Fig. 3B), but methanol, isoamylol, and isopropanol inhibited 19.2%, 29.9%, and 35.7% of the activity of the NHase, respectively, and in particular ethanol, ethyl acetate, and acetone strongly inhibited 68.6%, 76%, and 70.3% of the activity of the NHase, respectively.

4. Discussion

NHase contains either an iron or a cobalt ion in the active site. The trafficking of metal ions into NHases is mediated by various activator proteins, which are essential for functional NHase biosynthesis.²³ The activators for iron-type NHases have been shown to act as metallochaperones. The activator for cobalt-type NHases forms a complex with the α-subunit of the NHase to assist cobalt exchange between the cobalt-free α-subunit of the cobalt-free NHase and the cobalt-containing α-subunit of the complex.^23,24 Recently, Kuhn et al.²⁵ reported that the iron-type NHase from Comamonas testosteroni Ni1 does not require an activator accessory protein for its expression in E. coli. In the present study, the E. meliloti CGMCC 7333 NHase expressed in E. coli Rosetta in the absence of the activator protein had NHase activity for conversion of THI to THI amide and ACE to IM-1-2, indicating that the cobalt-type NHase of E. meliloti CGMCC 7333 also does not require an activator accessory protein for its bioconversion function. However, the presence of the activator accessory protein improved the solubility of the target NHase and enhanced its activity in THI and ACE conversion. Therefore, the activator protein from CGMCC 7333 is able to improve heterologous expression of NHase, which is in accordance with the function of the activator protein from P. putida NRRL-18668.¹⁷

The molecular structures of ACE and THI have 6-chloro-3-pyridinylmethyl and N-cyanoamidine groups in common, but differ in their cyclic thiazolidine and acyclic methyl moieties (Fig. 5). These molecular differences did not affect the selectivity of CGMCC 7333 NHase, but the V_max, K_m, and k_cat values of the NHase for the conversion of acyclic ACE were two orders of magnitude lower than for the conversion of the cyclic THI (Table 4). The molecular difference between benzonitrile and dichlobenil is that dichlobenil has two chlorine atoms near the cyano moiety. CGMCC 7333 NHase can hydrate benzonitrile, but cannot convert dichlobenil, which may be due to blockage by the two chlorine atoms in dichlobenil of NHase catalysis. It is interesting that the nitrogen-fixing bacteria Rhizobium radiobacter strains 8/4 and DSM 9674 and Rhizobium sp. 11401 have been reported to use an NHase to convert dichlobenil to its amide metabolite,^7–10 and therefore further comparison of the amino acid differences between the NHases of Ensifer and Rhizobium will help us to understand the relationship between NHase function and structure.

Fig. 5 Chemical structures of nitrile-containing compounds hydrated by E. meliloti CGMCC 7333 NHase.

Many leguminous bacteria and plant growth-promoting rhizobacteria (PGPR) are reported to produce indole-3-acetic acid (IAA), an important plant growth hormone. One of the IAA production pathways is synthesis from indole-3-acetonitrile (IAN), mediated by an NHase/amidase. Strains of Agrobacterium, Rhizobium, Ensifer, and Bradyrhizobium have been shown to convert IAN into IAM via an NHase, and subsequently IAM is converted to IAA by an amidase.^21,26 Substrate specificity assays indicated that CGMCC 7333 NHase converts IAN to IAM and the whole cells of CGMCC 7333 also converted IAN to IAM. However, CGMCC 7333 cannot convert IAN to IAA or convert IAM to IAA (data not shown), which indicates that CGMCC 7333 lacks an IAM amidase and IAN nitrilase that could directly transform IAN to IAA. This characteristic is strain-dependent because Kobayashi et al.²¹ proved that E. meliloti strain 03-03046 converted IAN to IAA through an NHase/amidase system. The absence of IAM amidase and IAN nitrilase activity has the advantage of avoiding the production of by-product acid metabolites in the bioconversion of nitriles to amide products. Therefore, CGMCC 7333 has the potential to biotransform nitriles to amide compounds.

Strains of E. meliloti were isolated from contaminated soils with the ability to degrade organic contaminants and to enhance the effects of plants in phytoremediation of organic compounds in contaminated soils.^27–29 In addition to atmospheric nitrogen fixation and degradation of the popular pesticides THI and ACE, E. meliloti CGMCC 7333 has the ability to solubilize phosphate and produce siderophores (data not shown). Therefore, it can also be used as a biofertilizer and pesticide-degrading bioaugmentation agent.^4,12

Acknowledgements

This research was financed by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions, the National Science Foundation of China (31570104), and the Academic Natural Science Foundation of Jiangsu Province (14KJA180004).

References

1. P. Jeschke, R. Nauen, M. Schindler and A. Elbert, J. Agric. Food Chem., 2011, 59, 2897–2908.
2. Y. J. Zhao, Y. J. Dai, C. G. Yu, J. Luo, W. P. Xu, J. P. Ni and S. Yuan, Biodegradation, 2009, 20, 761–768.
3. H. J. Zhang, Q. W. Zhou, G. C. Zhou, Y. M. Cao, Y. J. Dai, W. W. Ji, G. D. Shang and S. Yuan, J. Agric. Food Chem., 2012, 60, 153–159.
4. F. Ge, L.-Y. Zhou, Y. Wang, Y. Ma, S. Zhai, Z.-H. Liu, Y.-J. Dai and S. Yuan, Int. Biodeterior. Biodegrad., 2014, 93, 10–17.
5. Y. J. Dai, W. W. Ji, T. Chen, W. J. Zhang, Z. H. Liu, F. Ge and S. Yuan, J. Agric. Food Chem., 2010, 58, 2419–2425.
6. Z. Liu, Y. Dai, G. Huang, Y. Gu, J. Ni, H. Wei and S. Yuan, Pest Manage. Sci., 2011, 67, 1245–1252.
7. M. S. Holtze, J. Sorensen, H. C. Hansen and J. Aamand, Biodegradation, 2006, 17, 503–510.
8. N. Layh, B. Hirrlinger, A. Stolz and H.-J. Knackmuss, Appl. Microbiol. Biotechnol., 1997, 47, 668–674.
9. M. S. Holtze, S. R. Sorensen, J. Sorensen and J. Aamand, Environ. Pollut., 2008, 154, 155–168.
10. J. Vosáhlová, L. Pavlů, J. Vosáhlo and V. Brenner, Pestic. Sci., 1997, 49, 303–306.
11. Y. S. Feng and C. M. Lee, J. Hazard. Mater., 2009, 164, 646–650.
12. L. Y. Zhou, L. J. Zhang, S. L. Sun, F. Ge, S. Y. Mao, Y. Ma, Z. H. Liu, Y. J. Dai and S. Yuan, J. Agric. Food Chem., 2014, 62, 9957–9964.
13. Y. Yang, T. Chen, P. Ma, G. Shang, Y. Dai and S. Yuan, Biodegradation, 2010, 21, 593–602.
14. M. M. Bradford, Anal. Biochem., 1976, 72, 248–254.
15. S. Okamoto and L. D. Eltis, Mol. Microbiol., 2007, 65, 828–838.
16. Z. H. Liu, Y. M. Cao, Q. W. Zhou, K. Guo, F. Ge, J. Y. Hou, S. Y. Hu, S. Yuan and Y. J. Dai, Biodegradation, 2013, 24, 855–864.
17. M. S. Payne, S. Wu, R. D. Fallon, G. Tudor, B. Stieglitz, I. M. Turner and M. J. Nelson, Biochemistry, 1997, 36, 5447–5454.
18. R. A. Cameron, M. Sayed and D. A. Cowan, Biochim. Biophys. Acta, 2005, 1725, 35–46.
19. M. Wieser, K. Takeuchi, Y. Wada, H. Yamada and T. Nagasawa, FEMS Microbiol. Lett., 1998, 169, 17–22.
20. K. Rzeznicka, S. Schatzle, D. Bottcher, J. Klein and U. T. Bornscheuer, Appl. Microbiol. Biotechnol., 2010, 85, 1417–1425.
21. M. Kobayashi, T. Suzuki, T. Fujita, M. Masuda and S. Shimizu, Proc. Natl. Acad. Sci. U. S. A., 1995, 92, 714–718.
22. S.-H. Kim and P. Oriel, Enzyme Microb. Technol., 2000, 27, 492–501.
23. Y. Liu, W. Cui, Y. Fang, Y. Yu, Y. Cui, Y. Xia, M. Kobayashi and Z. Zhou, BMC Biotechnol., 2013, 13, 48.
24. Z. Zhou, Y. Hashimoto, K. Shiraki and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 14849–14854.
25. M. L. Kuhn, S. Martinez, N. Gumataotao, U. Bornscheuer, D. Liu and R. C. Holz, Biochem. Biophys. Res. Commun., 2012, 424, 365–370.
26. Y.-S. Feng, P.-C. Chen, F.-S. Wen, W.-Y. Hsiao and C.-M. Lee, Process Biochem., 2008, 43, 1391–1397.
27. Y. Teng, Y. Shen, Y. Luo, X. Sun, M. Sun, D. Fu, Z. Li and P. Christie, J. Hazard. Mater., 2011, 186, 1271–1276.
28. C. Tu, Y. Teng, Y. Luo, X. Li, X. Sun, Z. Li, W. Liu and P. Christie, J. Hazard. Mater., 2011, 186, 1438–1444.
29. T. C. Charles, G.-q. Cai and P. Aneja, Genetics, 1997, 146, 1211–1220.

---