Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

In this study: JPL-2, an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain of Rhodococcus ruber, was isolated and characterized; a novel carboxylesterase gene feh was cloned and expressed; and the characteristics of FeH were studied.

Chemicals and media
The fenoxaprop-P-ethyl, cyhalofop-butyl, quizalofop-p-ethyl, diclofop-methyl, haloxyfop-p-methyl, fluazifop-p-butyl, clodinafop-propargyl, and fenoxaprop acid were from Langchem Inc. (Shanghai, China). The Luria-Bertani (LB) medium consisted of the following components (in g L -1 ): 10.0 tryptone, 5.0 yeast extract and 10.0 NaCl. Mineral salts medium (MSM) consisted of the following components (in g L -1 ): 1.5 NH 4 NO 3 , 0.5 NaCl, 1.5 K 2 HPO 4 , 0.5 KH 2 PO 4 and 0.2 MgSO 4 ·7H 2 O, pH 7.0. For solid medium, 20.0 g agar was added. The stock solutions of the above herbicides (10000 mg L -1 , w/v) were prepared in dimethyl sulfoxide and sterilized by membrane filtration. The solutions were added to the sterilized MSM and used as the carbon source when required. Strain JPL-2 could not utilize dimethyl sulfoxide as the sole carbon source for growth in MSM medium.

Isolation and identification of fenoxaprop-P-ethyl -degrading bacteria
To isolate FE-degrading bacteria, a conventional enrichment method was employed. The soil sample was collected from a wheat field in Henan province that had been subjected to the long-term application of FE. Approximately 2.0 g of the soil sample was added to 100 mL MSM, with the addition of FE (50 mg L -1 ) as the carbon source, and was incubated at 30°C and 150 rpm for approximately 2 d. Then, 5 mL of the enrichment culture was transferred into another 100 mL of fresh medium for an additional round of enrichment. After three rounds of transfer, the enrichment culture was diluted and spread on MSM agar plates with 50 mg L -1 FE. After 2 d of incubation at 30°C, colonies that degraded FE, as evidenced by the production of a visible transparent halo, were picked and purified. The ability to degrade FE was verified by high performance liquid chromatography (HPLC).
Strain JPL-2 was characterized and identified by morphological, physiological & biochemical characteristics as well as 16S rRNA gene analysis. The morphological, physiological and biochemical characterizations were analyzed according to Bergey's Manual of Determinative Bacteriology (Holt et al., 1994). The strain was also characterized biochemically using the API CORYNE system according to the manufacturer's instructions (API bioMérieux).
Genomic DNA was extracted by the high-salt precipitation method. The 16S rRNA gene of strain JPL-2 was amplified from the genomic DNA with the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTCCTTGTTACGACTT-3'). The PCR product was purified with a PCR purification kit (Axygen), ligated into the vector pMD18-T (TaKaRa Biotechnology, Dalian, China) and transformed into E. coli DH5á. An automatic sequencer (Applied Biosystems, model 3730) was used to determine the 16S rRNA gene sequence. Pairwise sequence similarity was calculated using a global alignment algorithm, implemented by the EzTaxon-e server. Following multiple alignments of the sequence data using CLUSTAL_X (Thompson et al., 1997), phylogenetic analysis was performed using the MEGA version 5.0 software package (Tamura et al., 2011). The G+C content of the genomic DNA was determined by thermal denaturation, with E.coli K-12 DNA serving as a reference (Mandel and Marmur, 1968). DNA-DNA hybridizations were performed according to the method of Ezaki et al. (1989).

Growth and degradation experiments
Strain JPL-2 was precultured in LB medium for approximately 12 h to mid-log phase, and the cells were harvested by centrifugation (6,000 rpm for 5 min at room temperature) and washed twice with distilled water. After the cell density had been adjusted to approximately 2.0 at OD 600 , a 1%, (v/v) inoculum was added to 20 mL MSM, with the addition of 100 mg L -1 FE, and incubated at 30°C and 150 rpm. After 6 h, three flasks were taken from the incubator. The bacterial growth was monitored via cell density, and the concentration of FE was determined by HPLC. Each treatment was performed in triplicate, and the control experiments were conducted under the same conditions without inoculation.

Cloning of the FE-hydrolyzing carboxylesterase gene (feh)
To clone the FE-hydrolyzing carboxylesterase gene, the shotgun method was used to construct a gene library of strain JPL-2 in E. coli DH5á. Genomic DNA of strain JPL-2 was extracted by the method described above and digested partially with Sau3AI. Fractions containing approximately 4 to 6 kb DNA fragments were pooled, ligated into the BamHI site of the plasmid pUC118 (TaKaRa Biotechnology, Dalian, China), and transformed into competent cells of E.coli DH5|Á. The transformants were plated onto LB agar plates containing 100 mg L -1 ampicillin and 100 mg L -1 FE and incubated at 37°C for 24 h. Colonies that degraded FE produced clear transparent halos and were screened and further tested for their degrading capabilities. Analysis of the nucleotide and deduced amino acid sequence was performed using Omiga 2.0. BlastN and BlastP were used for the nucleotide sequence and amino acid identity comparison, respectively. (www.ncbi.nlm.nih.gov/Blast).

Gene expression and purification of FeH
To express the feh gene, the open reading frame (ORF) of feh lacking a stop codon was amplified by PCR with the primer pair feh-F (5'-CTGATTGCATATGTCCGACATCCACGGCGTCT -3', the NdeI site is underlined) and feh-R (5'-ATGCCTCGAGCGGCACGGCGGCGAA-5', the XhoI site is underlined). The amplicon was inserted into the NdeI-XhoI site of pET29a(+) to generate the recombinant plasmid pET-feh, which was transformed into E. coli BL21(DE3). The transformants were subcultured into 100 mL LB medium and allowed to grow until the culture density reached 0.5 (OD 600nm ). To induce the expression of feh, isopropyl-b-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mmol L -1 , and the purification of the recombinant feh was performed according to the methods of Wang et al. (2009). The molecular mass of the denatured protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970), and the protein concentrations were determined by the Bradford method (Bradford, 1976).

Enzyme assay
The enzymatic activity of FeH and its kinetics toward FE and other AOPP herbicides were determined according to the method of Nie et al. (2011). One activity unit was defined as the amount of enzyme required to catalyze the formation of 1 mmol of product per min.

Chemical analysis
Following culture acidification to a pH 2.0 by the addition of 10% HCl, FE and other AOPP herbicide were extracted from the culture with an equal volume of di-chloromethane. The dichloromethane phase was dried over anhydrous Na 2 SO 4 , and the dichloromethane was removed using a stream of nitrogen. The final extract was redissolved in 500 mL methanol and subjected to measurement by HPLC, equipped with a Zorbax C-18 ODS Spherex column (250 mm x 4.6 mm). The mobile phase was 100% methanol, and the flow rate was 1.0 mL min -1 . Using a 20 mL injection volume, column elutions were monitored by measuring at 237 nm with a Waters 2487 wavelength absorbance detector.

Identification of the Metabolite of FE Degradation by FeH
The enzyme mixture was added with 100 mg L -1 FE and incubated for 30 min in 30°C. Control experiments with heat-inactivated enzyme were performed under the same conditions. The solution mixture was extracted as described above, and the metabolite was identified by LC-MS (LC-MSD-Trap-SL, Agilent, USA) as described by Liu et al. (2012).

Nucleotide sequence accession numbers
The nucleotide sequences of the 16S rRNA and feh genes of strain JPL-2 were deposited in the GenBank database under accession numbers JX110619 and KF601763, respectively.

Isolation and identification of the FE-degrading strain
The enrichment procedure obtained a pure culture designated as JPL-2. This strain was gram positive and aerobic. Colonies grown on LB agar were opaque, convex and red. Strain JPL-2 was positive for catalase, hydrolysis of tween-80 and tyrosine, but negative for oxidase, casein, Voges-Proskauer and DNase. It could produce acid from fermentation of D-Fructose, D-Glucose, Glycerol, D-Mannitol and D-Sorbitol but not from L-arabinose, D-cellobiose, D-galactose, inulin, lactose, D-raffinose and Lrhamnose. Growth was observed over a temperature range of 10-42°C (optimum 30°C), a salinity range of 0-5% NaCl (optimum 1% NaCl) and a pH range of 4.0-10.0 (optimum 7.0). The 16S rRNA gene sequence of strain JPL-2 showed 100% similarity to Rhodococcus ruber DSM 43338 T (X80625) and 99.48% to Rhodococcus aetherivorans 10bc312 T (AF447391). The DNA G+C content of strain JPL-2 is 66.5 mol%.
Strain JPL-2 showed a relatively high DNA-DNA relatedness to strain Rhodococcus ruber DSM 43338 T (81.6%), which was above the 70% threshold recommended for the delineation of bacterial species (Wayne et al., 1987). Based on the above characteristics, strain JPL-2 was identified as Rhodococcus ruber and named Rhodococcus ruber JPL-2. A phylogenetic tree based on Cloning a novel carboxylesterase gene (feh) from Rhodococcus ruber JPL-2 the known representatives of the Rhodococcus species is presented in Figure 1.

Degradation of FE by strain JPL-2
The growth of strain JPL-2 on liquid MSM supplemented with 100 mg L -1 FE and its ability to degrade fenoxaprop-P-ethyl are shown in Figure 2. The growth curve showed a steady increase in the bacterial population. Simultaneously, the HPLC analysis showed a substantial reduction in the concentrations of FE. After incubation for 54 h, approximately 94.6% of the FE was degraded by strain JPL-2. Correspondingly, the OD 600 increased to 0.35. 428 Hongming et al.  No significant change in FE concentration was observed in cultures that were not inoculated with strain JPL-2, and no growth was observed for strain JPL-2 when it was inoculated into the culture without the addition of FE. Thus, we concluded that strain JPL-2 was able to degrade and utilize FE as its sole carbon source for growth. For degradation of FE, the optimal temperature and initial pH of the medium were 30°C and 7.0, respectively (data not shown).

Cloning and sequence analysis of the feh gene
On the plate containing 100 mg L -1 FE, a positive clone that produced a transparent halo was screened from approximately 6,000 transformants in the gene library. The inserted fragment in the transformant was 5,428 bp and contained three complete ORFs. The three ORFs were subcloned into the linear vector pMD18-T and transformed into E. coli DH5á. One ORF was confirmed to be the target gene encoding the FE-hydrolyzing carboxylesterase. This gene was named feh. Sequence analysis indicated that the length of the feh gene was 1,140 bp, encoding a protein of 380 amino acids, with a 68.94% G+C content. The feh gene showed the highest similarity (78%) with feh from Rhodococcus sp. T1 (Hou et al., 2011).

Expression of the feh gene in E. coli BL21 (DE3)
The FeH produced in E. coli BL21(DE3) was purified from crude extract using Ni-nitrilotriacetic acid affinity chromatography, and the purified FeH showed a single band on SDS-PAGE (Figure 4). The molecular mass of the denatured enzyme was approximately 39 kDa, which coincided with the molecular mass calculated from the amino acid sequence.

Identification of the metabolite
When FeH was added to the enzyme reaction mixture, a new metabolite with a retention time of 2.22 min in Cloning a novel carboxylesterase gene (feh) from Rhodococcus ruber JPL-2 429   the HPLC spectrum was produced ( Figures 5A and 5C). Its retention time was similar to that of the authentic standards FA ( Figure 5B). In the MS spectrum, this metabolite showed a protonated molecular ion at m/z 331.9 (Figure 5D), which was consistent with the corresponding mass spectrum of authentic standards FA. Based on these results, we confirmed that FeH catalyzed the hydrolysis of the carboxylic acid ester bond in FE and produced ethanol and FA.
The optimal pH of FeH was observed to be approximately 7.5, but FeH was stable at pH 6.0-8.0. The enzyme was fairly stable up to 45°C, had 30% residual activity at 60°C, and was completely inactivated at 70°C. Many metal ions (Ni 2+ , Hg 2+ and Ag + ; 1 mM) as well as the surfactants SDS and Tween80 (10 mM) inhibited enzymatic activity, and the chelating agents EDTA and 1,10-phenanthroline (10 mM) had little effect on the enzyme activity, indicating that FeH had no requirement for metal ions (data not shown).

Kinetic analysis of FeH
FeH could hydrolyze these AOPP herbicides with different hydrolysis rates in descending order as follows: fenoxaprop-P-ethyl > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy (Table 1). This result indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, while the catalytic efficiency of the herbicides with methanol moiety was the lowest. We inferred that the production of methanol would be harmful to the enzyme.
In general, the initial step of the metabolism of AOPP herbicides involves the breakdown of the carboxylic acid ester bond. Strain JPL-2 hydrolyzed FE to FA and ethanol, although the latter was not detectable by HPLC chromatography because ethanol cannot absorb UV light. Ethanol could also be further used by strain JPL-2 (data not show). Currently, two genes encoding carboxylesterase have been cloned: chbH, from Pseudomonas azotoformans QDZ-1 (Nie et al., 2011), which was also able to hydrolyze the AOPP herbicides, and feh. The feh gene was first cloned from Rhodococcus sp. T1 (Hou et al., 2011), but the characteristics of FeH were not extensively investigated. In the present study, another feh gene was cloned from strain JPL-2, and the characteristics of FeH were studied in detail. FeH from strain JPL-2 has 78% similarity to that from Rhodococcus sp. T1, but it only exhibited a 10% similarity to ChbH. The Vmax of FeH was approximately 300 times that of ChbH toward the substrate FE, and it has a broader substrate spectrum. However, FeH and ChbH share a common characteristic: against the tested substrates, their enzymatic activity decreased as the length of the aliphatic chain flanking the ester bond of the AOPP herbicides increased.

Conclusion
Strain JPL-2 was isolated from the soil of a wheat field and identified as Rhodococcus ruber. A novel carboxylesterase gene (feh), enabling the strain to efficiently degrade AOPP herbicides, was cloned from JPL-2. FeH from strain JPL-2 has a 78% similarity to that from Rhodococcus sp. T1, but it only showed a 10% similarity to ChbH. FeH had a broader substrate spectrum and higher catalysis efficiency than ChbH.