Abstract
Objective
To identify and characterize a novel aryloxyphenoxypropionate (AOPP) herbicide-hydrolyzing carboxylesterase from Aquamicrobium sp. FPB-1.
Results
A carboxylesterase gene, fpbH, was cloned from Aquamicrobium sp. FPB-1. The gene is 798 bp long and encodes a protein of 265 amino acids. FpbH is smaller than previously reported AOPP herbicide-hydrolyzing carboxylesterases and shares only 21–35% sequence identity with them. FpbH was expressed in Escherichia coli BL21(DE3) and the product was purified by Ni–NTA affinity chromatography. The purified FpbH hydrolyzed a wide range of AOPP herbicides with catalytic efficiency in the order: haloxyfop-P-methyl > diclofop-methyl > fenoxaprop-P-ethyl > quizalofop-P-ethyl > fluazifop-P-butyl > cyhalofop-butyl. The optimal temperature and pH for FpbH activity were 37 °C and 7, respectively.
Conclusions
FpbH is a novel AOPP herbicide-hydrolyzing carboxylesterase; it is a good candidate for mechanistic study of AOPP herbicide-hydrolyzing carboxylesterases and for bioremediation of AOPP herbicide-contaminated environments.
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Introduction
Aryloxyphenoxypropionate (AOPP) herbicides, that have the basic structure of an ester of 4-oxyphenoxypropanoic acid (Fig. 1), are potent inhibitors of acetyl-CoA carboxylase (Nie et al. 2011). The most commonly used AOPP herbicides are fluazifop-P-butyl, cyhalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, haloxyfop-P-methyl and quizalofop-P-ethyl (El-Metwally and Shalby 2007). AOPPs are effective herbicides for the control of annual and perennial grasses in broadleaf crop fields. However, the widespread use of AOPP herbicides has led to large amounts of them entering into the environment where they have high levels of chronic toxicity toward some aquatic organisms, disturb soil bacterial communities, and injure subsequent rotation crops (Aksoy et al. 2007; Dong et al. 2015; Liu et al. 2015; Trabelsi et al. 2015). Therefore, the behavior and degradation mechanisms of AOPP herbicides in the environment have received considerable attention.
Microorganisms are one of the most important factors in the fate of AOPP herbicides in the environment. Several AOPP-degrading strains have been isolated. Nie et al. (2011) isolated five cyhalofop-butyl-degrading strains from rice field soil. Liu et al. (2015) screened a fenoxaprop-P-ethyl-degrading strain from wheat field soil. Hou et al. (2011) and Dong et al. (2015), respectively, isolated the fenoxaprop-ethyl-degrading strains Rhodococcus sp. T1 and Acinetobacter sp. DL-2. In these strains, the initial degradation of AOPP herbicides involves the cleavage of the ester linkage, generating the corresponding acid. Four AOPP herbicide-hydrolyzing carboxylesterases have been reported: ChbH (332 amino acids with molecular mass 36 kDa) from Pseudomonas azotoformans QDZ-1 (Nie et al. 2011), Feh (380 amino acids with molecular mass 40 kDa) from Rhodococcus sp. T1 (Hou et al. 2011) and R. ruber JPL-2 (Liu et al. 2015), and AfeH (309 amino acids with molecular mass 34 kDa) from Acinetobacter sp. DL-2 (Dong et al. 2015).
In this study, we describe the degradation of AOPP herbicides by a bacterial strain Aquamicrobium sp. FPB-1, the cloning of a novel AOPP herbicide-hydrolyzing carboxylesterase gene fpbH, and the exogenous expression, purification and characterization of FpbH.
Materials and methods
Chemicals and bacterial growth medium
Fluazifop-P-butyl, fluazifop-P, cyhalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, haloxyfop-P-methyl and quizalofop-P-ethyl were obtained from Sigma-Aldrich and were of analytical grade. Lysogeny broth (LB) consisted of: 5 g NaCl, 10 g tryptone, and 5 g yeast extract per liter water, pH 7. The mineral salts medium (MSM) consisted of: 1.5 g K2HPO4, 0.5 g KH2PO4, 1 g (NH4)2SO4, 1 g NaCl, and 0.2 g MgSO4·7H2O per liter water, pH 7. To make solid medium, 15 g of agar was added per liter. All media were sterilized by autoclaving at 121°C for 30 min.
Growth and degradation experiments
Aquamicrobium sp. FPB-1 was isolated from a legume field in Jiangsu Province, China (Supplementary Fig. 1). It was grown in LB medium for 12 h, collected by centrifugation (6000×g, 10 min), washed twice with fresh MSM, and resuspended in 10 ml MSM as the stock culture. The density was adjusted to 480 mg cell dry weight l−1, and then 1 ml stock culture was inoculated in 100 ml MSM supplemented with 10 mg of each respective substrate (fluazifop-P-butyl, cyhalofop-butyl, diclofop-methyl, fenoxaprop-P-ethyl, haloxyfop-P-methyl, or quizalofop-P-ethyl). Cultures were incubated at 30 °C with shaking at 150 rpm. Experiments were performed in parallel in three flasks, and negative controls inoculated with sterilized cells were carried out in the same conditions. Samples used for detecting cell growth and substrate degradation were collected at 5 h intervals. Bacterial growth was monitored by cell dry weight (Glazyrina et al. 2010), and the concentration of each substrate was determined by HPLC as described below.
Gene cloning and sequence analysis
Total DNA was extracted by the standard phenol/chloroform procedure. The fluazifop-P-butyl carboxylesterase gene was cloned by a shotgun method. A size-fractionated genomic library of strain FPB-1 was constructed according to the method described by Wang et al. (2009). The library was plated onto LB-agar containing 100 mg ampicillin l−1 and 100 mg fluazifop-P-butyl l−1 and incubated at 37 °C for approx. 12 h, and then incubated at 4 °C for about 24 h. Colonies that produced transparent halos were screened and further tested for their ability to degrade fluazifop-P-butyl. The DNA fragment inserted in a positive clone was sequenced using an automatic sequencer. Nucleotide and deduced amino acid sequence analyses were performed using OMIGA 2.0. Sequence identity analysis was carried out using the BLAST programs at the NCBI (Marco et al. 2014; Arnold et al. 2006; Benkert et al. 2011).
Expression and purification of FpbH
The ORF without its translation stop codon was amplified by PCR with upstream primer 5′-GGGACCCATATGACGATGTTGGGTATGACG (NdeI site underlined) and downstream primer 5′-CTGAAAAAGCTTTCGTGCATCGTGCTGCGCG (HindIII site underlined). Then, the amplified fragment was inserted into the NdeI-HindIII sites of expression vector pET-29a(+) to generate plasmid pET-fpbH, which was subsequently transformed into Escherichia coli BL21(DE3). Transformants were cultivated in 500 ml LB containing 50 mg kanamycin at 37 °C to 150 mg cell dry weight l−1 and then induced with 0.5 mM IPTG for 24 h at 18 °C. Crude enzyme extracts of E. coli BL21 (DE3) were prepared by ultrasonication. The recombinant FpbH was purified with Ni2+-NTA resin (Qiagen), collected and dialyzed against 20 mM phosphate-buffered saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 l−1 at pH 7.5) overnight at 4 °C to remove imidazole. SDS-PAGE and the Bradford method were used to quantify the protein concentration using bovine serum albumin as a standard.
Enzymatic characteristics
The hydrolase activity of the purified FpbH was determined in 2 ml PBS containing 0.5 mM of each substrate and 0.5 μg purified FpbH. The mixture was incubated for 10 min at 30 °C; the reaction was terminated by adding 0.02 ml of 30% HNO3 and cooling in liquid N2. The disappearance of substrates was monitored by HPLC as described below. One unit of hydrolase activity was defined as the amount of enzyme that catalyzed the conversion of 1 μM substrate per min. The effects of temperature, pH, metal ions, and reagents on the enzyme activity were tested as described by Chen et al. (2016). Kinetic values were obtained from Lineweaver–Burk plots for different substrate concentrations in PBS at 30 °C.
Chemical analysis
The samples were initially extracted. Equal volumes of dichloromethane were added to the cultures and then shaken for 1 h on a reciprocal shaker. The dichloromethane phase was dried by adding anhydrous Na2SO4, and then volatilized using a nitrogen stream at room temperature. The residues were dissolved in 200 µl methanol each and filtered through 0.22 µm millipore membranes. For HPLC analysis, a column of Kromasil 100-5-C18 (internal diameter, 4.6 mm; length, 250 mm). The mobile phase was methanol/water (85:15, v/v) at 0.8 ml min−1. Detection was at 230 nm, and the injection was 20 μl. The metabolites were further identified by MS/MS (Finnigan TSQ Quantum Ultra AM thermal triple quadruple mass spectrometer) according to Dong et al. (2015). In MS/MS, the metabolites were separated, confirmed by standard MS, ionized by electrospray with a positive polarity, and scanned in the normal mass range from 200 m/z (mass to charge ratio) to 600 m/z. The capillary temperature was 350 °C, and the source voltage was 4 kV. The sheath gas was set at 35 arb. Characteristic fragment ions were detected using second-order MS.
Nucleotide sequence accession numbers
The nucleotide sequences of the 16S rRNA gene and fpbH gene from Aquamicrobium sp. strain FPB-1 have been deposited in the GenBank database with accession numbers KJ466199 and KJ466200 respectively.
Results and discussion
Degradation of aryloxyphenoxypropionate (AOPP) herbicides by strain FPB-1
Figure 2 shows the degradation of fluazifop-P-butyl and the growth of strain FPB-1. After 40 h of incubation, 96% fluazifop-P-butyl was degraded by strain FPB-1, in which time the cell dry weight increased from ~7 to ~67 mg l−1. These results indicated that fluazifop-P-butyl could be degraded and utilized as the sole carbon source by strain FPB-1. Strain FPB-1 could also degrade and utilize diclofop-methyl (99%), haloxyfop-P-methyl (97%), quizalofop-P-ethyl (98.5%), fenoxaprop-P-ethyl (97%), and cyhalofop-butyl (96%) in the same conditions.
Members of the genus Aquamicrobium are widely distributed in the environment and are known for their abilities to degrade a wide variety of xenobiotic pollutants, such as thiophene-2-carboxylate, 2,4-dichlorophenol, 4-chloro-2-methylphenol, 4-chlorophenol and phenol (Bambauer et al. 1998; Fritsche et al. 1999; Kämpfer et al. 2009; Lipski and Kämpfer 2012; Jin et al. 2013). This is the first report of an Aquamicrobium strain that is capable of efficiently degrading AOPP herbicides.
Metabolite identification after fluazifop-P-butyl degradation by strain FPB-1
A metabolite was detected by HPLC in samples taken after 36 h incubation during the degradation of fluazifop-P-butyl by Aquamicrobium sp. strain FPB-1 (Fig. 3a). The metabolite had a retention time of 5.1 min, which was equal to that of authentic fluazifop-P (Fig. 3b). Tandem mass spectrometry analysis showed a prominent deprotonated molecular ion at m/z = 326 [M − H]− (Fig. 3c) and a fragment ion peak at m/z = 254 (loss of a –CH3COOH group) (Fig. 3d), which were consistent with fluazifop-P. Thus the metabolite was identified as fluazifop-P. The fluazifop-P generated at 40 h (~79 mg l−1, ~0.24 mM) was approx. equivalent to the amount of fluazifop-P-butyl (97 mg l−1, ~0.25 mM) that had disappeared from the medium (Fig. 2), suggesting that fluazifop-P could not be further degraded. These results indicate that fluazifop-P-butyl was converted to fluazifop-P through hydrolysis of the ester bond.
Gene cloning and sequence analysis of fpbH
A positive clone that produced a transparent halo on assay plates, due to insoluble fluazifop-P-butyl being transformed to soluble fluazifop-P, was screened from approx. 40,000 transformants. The inserted fragment in the transformant was 2865 bp and contained five complete ORFs (named orf1–orf4 and fpbH respectively) (Fig. 4). The five ORFs were then subcloned into the linear vector pMD18-T and used to transform E. coli DH5α. fpbH was finally confirmed to be the target gene encoding the fluazifop-P-butyl carboxylesterase (FpbH).
Sequence analysis indicated that fpbH consisted of 798 bp and encoded a protein of 265 amino acids with a deduced molecular mass of 28 kDa. The molecular mass of FpbH was smaller than those of the four previously reported AOPP herbicide-hydrolyzing carboxylesterases (each 309–380 amino acids, molecular masses 34–40 kDa). The deduced protein sequence was searched against the NCBI NR database using the BLASTP program. Aquamicrobium sp. strain FPB-1 FpbH showed 98–100% identities with some hypothetical or putative alpha/beta-hydrolases. However, FpbH shared only very low sequence identities, respectively 21, 29, 31 and 35%, with Feh, ChbH, Feh and AfeH, the four previously reported AOPP herbicide-hydrolyzing carboxylesterases. In a phylogenetic tree constructed based on FpbH and related hydrolases, FpbH formed a subclade with the Fehs from Rhodococcus sp. JPL-2 and Rhodococcus sp. T1, and EstB from Burkholderia gladioli ATCC10248 (Fig. 5); however, the bootstrap value in this node was only 40%. ChbH and AfeH were not clustered with FpbH. The difference in molecular mass and the low sequence identities observed suggest that FpbH differs from the four previously reported AOPP herbicide-hydrolyzing carboxylesterases.
Multiple alignments of amino acid sequences of FpbH with related carboxylesterases showed that FpbH contains the catalytic triad Ser-Asp-His (Ser100, Asp213 and His239) (Fig. 6), which is highly conserved in alpha/beta-hydrolase-fold proteins (Bornscheuer 2002). A model of FpbH (Fig. 6) generated using the SWISS-MODEL server with an alpha/beta-hydrolase template (Rhodopseudomonas palustris CGA009 alpha/beta-hydrolase, PDB: 4psu.1.A) also indicates that FpbH contains this catalytic triad, and has the same arrangement of β-sheets and α-helices (β1-β2-β3-αA-β4-αB-β5-αC-β6-αD-β7-αE-β8-αF) as a typical alpha/beta-hydrolase (Bornscheuer 2002). These analyses further indicated that FpbH is an alpha/beta-hydrolase-fold protein. Most alpha/beta-hydrolase-fold proteins contain a highly conserved pentapeptide, G-X-S-X-G, which forms a tight turn in a β-strand-α-turn-helix motif (Wang et al. 2009). The G-X-S-X-G motif is present in ChbH from Pseudomonas azotoformans QDZ-1 and AfeH from Acinetobacter sp. DL-2. However, it is interesting to note that this G-X-S-X-G motif is not rigorous in FpbH (nor in Feh from Rhodococcus sp. T1 or R. ruber JPL-2), and is present as G-X-S-X-A.
Gene expression and purification of His-tagged FpbH
Recombinant FpbH was induced in E. coli BL21 (DE3) and purified from the cell-free extract using Ni-nitrilotriacetic acid affinity chromatography. The purified enzyme showed a single band on SDS-PAGE (Supplementary Fig. 2). The molecular mass of the denatured enzyme was ~28 kDa, in agreement with that deduced from its amino acid sequence. HPLC and tandem mass spectrometry analysis demonstrated that FpbH catalyzed the hydrolysis of fluazifop-P-butyl to fluazifop-P (Supplementary Fig. 3).
Characterization and kinetic analysis of FpbH
The effect of metal cations at 0.5 and 5 mM, as well as the metal chelating-agent EDTA on the activity of FpbH are shown in Supplementary Fig. 4a. The enzyme activity was not obviously affected by Li+ or Fe2+ and Mg2+. Fe3+, Ca2+ and Zn2+ showed moderate inhibition and Ag+, Al3+, Cu2+, Pb2+, Hg2+, Ni2+, Cr2+, Mn2+ and Co2+ significantly inhibited the activity (5 mM had a much stronger inhibitory effect than 0.5 mM). The effects of Zn2+, Cu2+ and Co2+ on the activity of FpbH are quite different to those on AfeH, whose activity was strongly inhibited by Zn2+, slightly inhibited by Cu2+ and strongly stimulated by Co2+ (Dong et al. 2015). EDTA had little effect on the enzyme activity of FphH, indicating that it has no requirement for metal ions, which is consistent with observations for ChbH (Nie et al. 2011), FeH (Liu et al. 2015), and AfeH (Dong et al. 2015). The FpbH activity was detected at 10–60 °C and at pH values from 4 to 10 in the absence of added metal ions; the enzyme showed optimal activity at 37 °C (Supplementary Fig. 4b), which was lower than AfeH (50 °C) (Dong et al. 2015), and pH 7 (Supplementary Fig. 4c), which was lower than AfeH (pH 9) and Feh (pH 7.5) (Dong et al. 2015; Liu et al. 2015).
FpbH hydrolyzed all AOPP herbicides tested. The catalytic efficiency (k cat /K m ) of FpbH for different AOPP herbicides was in the order: haloxyfop-P-methyl > diclofop-methyl > fenoxaprop-P-ethyl > quizalofop-P-ethyl > fluazifop-P-butyl > cyhalofop-butyl (Table 1). Thus, the alkyl chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicide; the longer the alkyl chain, the poorer the degradation efficiency. It may be that the increase in length of the alkyl chain of the alcohol moiety increases steric hindrance of the enzyme-substrate interaction. The catalytic efficiency order of FpbH for different AOPP herbicides is different from that of AfeH (fenoxaprop-P-ethyl > haloxyfop-P-methyl > quizalofop-P-ethyl > cyhalofop-butyl), FeH (fenoxaprop-P-ethyl > quizalofop-P-ethyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methyl), and ChbH (quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > cyhalofop-butyl ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl) (Nie et al. 2011; Dong et al. 2015; Liu et al. 2015), which indicates that the chain length of the alcohol moiety and substitutions on the aromatic ring could affect the enzyme catalytic efficiencies to different extents. Additionally, the catalytic efficiencies (k cat /K m ) of FpbH for different AOPP herbicides (9.3–15 μM−1 s−1) are higher than those of ChbH (1.9–3.7 μM−1 s−1) and FeH (0.8–3.5 μM−1 s−1). These data suggest that FpbH differs from previously reported AOPP herbicide carboxylesterases and could be a good candidate enzyme for biodegradation, especially when diclofop-methyl and/or haloxyfop-P-methyl are the dominant pollutants.
Conclusion
A novel carboxylesterase gene, fpbH, was cloned from Aquamicrobium sp. FPB-1 by a shotgun method. FpbH shares low sequence similarity with previously reported aryloxyphenoxypropionate (AOPP) herbicide-hydrolyzing carboxylesterases (<35%). FpbH hydrolyzes a wide range of AOPP herbicides, including haloxyfop-P-methyl, diclofop-methyl, fenoxaprop-P-ethyl, quizalofop-P-ethyl, fluazifop-P-butyl and cyhalofop-butyl. This study elucidates the basis of the degradation of AOPP herbicides by strain FPB-1. The AOPP herbicide-hydrolyzing carboxylesterase FpbH has potential applications in bioremediation of AOPP herbicide-contaminated environments.
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Acknowledgments
This work was supported by the National Natural Science Foundation of China (31270157 and 31560033) and the Project of University-Industry Collaboration of Guangdong Province-Ministry (2013B090500017).
Supplementary information
Supplementary Fig. 1—Phylogenetic tree based on the 16S rRNA gene sequences of strain FPB-1 and related species.
Supplementary Fig. 2—SDS-PAGE of His-tagged FpbH.
Supplementary Fig. 3—HPLC and tandem mass spectrometry analysis of metabolite generated during fluazifop-P-butyl degradation by the purified FpbH.
Supplementary Fig. 4—Characterization of purified recombinant FpbH.
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Chenghong Wang and Jiguo Qiu have contributed equally to this study.
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Wang, C., Qiu, J., Yang, Y. et al. Identification and characterization of a novel carboxylesterase (FpbH) that hydrolyzes aryloxyphenoxypropionate herbicides. Biotechnol Lett 39, 553–560 (2017). https://doi.org/10.1007/s10529-016-2276-z
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DOI: https://doi.org/10.1007/s10529-016-2276-z