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Appl Environ Microbiol. 2010 May; 76(9): 2940–2945.
Published online 2010 March 12. doi:  10.1128/AEM.02990-09
PMCID: PMC2863442

Cloning and Biochemical Characterization of a Novel Carbendazim (Methyl-1H-Benzimidazol-2-ylcarbamate)-Hydrolyzing Esterase from the Newly Isolated Nocardioides sp. Strain SG-4G and Its Potential for Use in Enzymatic Bioremediation[down-pointing small open triangle]

Abstract

A highly efficient carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC)-mineralizing bacterium was isolated from enrichment cultures originating from MBC-contaminated soil samples. This bacterium, Nocardioides sp. strain SG-4G, hydrolyzed MBC to 2-aminobenzimidazole, which in turn was converted to the previously unknown metabolite 2-hydroxybenzimidazole. The initial steps of this novel metabolic pathway were confirmed by growth and enzyme assays and liquid chromatography-mass spectrometry (LC-MS) studies. The enzyme responsible for carrying out the first step was purified and subjected to N-terminal and internal peptide sequencing. The cognate gene, named mheI (for MBC-hydrolyzing enzyme), was cloned using a reverse genetics approach. The MheI enzyme was found to be a serine hydrolase of 242 amino acid residues. Its nearest known relative is an uncharacterized hypothetical protein with only 40% amino acid identity to it. Codon optimized mheI was heterologously expressed in Escherichia coli, and the His-tagged enzyme was purified and biochemically characterized. The enzyme has a Km and kcat of 6.1 μM and 170 min−1, respectively, for MBC. Radiation-killed, freeze-dried SG-4G cells showed strong and stable MBC detoxification activity suitable for use in enzymatic bioremediation applications.

Microbial bioremediation is quite widely used for cleaning up contaminated soils, because it often is more efficient and less expensive than physicochemical remediation. However, microbial bioremediation works slowly, taking weeks to months to achieve substantive remediation, and it is not suitable for cleaning up contaminated water because of the low-aeration and low-nutrient state of the water. There has been increasing interest in the concept of using formulated enzymes rather than live microbes as bioremediation agents, a process known as enzymatic bioremediation (2, 23). This is particularly suitable for situations like water treatment, where rapid remediation is needed and/or microbial growth and survival are not desirable.

Carbendazim (methyl-1H-benzimidazol-2-ylcarbamate, or MBC) is a systemic benzimidazole fungicide that is used in many countries around the world to control a broad range of fungal diseases in agricultural crops (8, 20). MBC also is the hydrolytic product and active component of some other widely used systemic fungicides, such as benomyl and thiophenate methyl (9, 17). MBC is quite stable in soil and water, which in turn can lead to the contamination of foodstuffs (4, 6, 7, 12, 24). This is a serious concern, because MBC is a suspected mutagen, carcinogen, and endocrine disruptor, and its use is tightly controlled by regulatory bodies in many countries (18, 21, 27, 30).

Microbial metabolism is responsible mainly for MBC degradation in soil, although it also can be degraded very slowly by physicochemical processes (1, 15, 16, 26). A few individual bacterial isolates with MBC degradation activities have been described (13, 14, 25, 28), but no gene-enzyme system for MBC degradation has been reported previously. Here, we describe the cloning and biochemical characterization of a novel MBC-hydrolyzing esterase from the newly isolated Nocardioides sp. strain SG-4G. We also provide a proof-of-concept demonstration of the potential of radiation-killed, freeze-dried SG-4G cells in the bioremediation of MBC.

MATERIALS AND METHODS

Chemicals.

Analytical-grade MBC, 2-aminobenzimidazole (2-AB), 2-hydroxybenzimidazole (2-HB), methyl salicylate, p-nitrophenyl acetate, α-napthyl acetate, and formic acid were purchased from Sigma-Aldrich, Inc. All other chemicals were used at the highest purity available commercially.

Medium and growth conditions.

Minimal medium (MM) consisted of 1.36 g KH2PO4, 1.78 g Na2HPO4-2H2O, 0.50 g MgSO4-7H2O, and 0.50 g NH4Cl per liter. The pH was adjusted to 7.2 with NaOH, and 1 ml of trace element solution [0.10 g Al(OH)3, 0.05 g SnCl2-2H2O, 0.05 g KI, 0.05 g LiCl, 0.08 g MgSO4, 0.05 g H3BO3, 0.10 g ZnSO4-7H2O, 0.01 g CoCl2, 0.01 g NiSO4-6H2O, 0.05 g BaCl2, and 0.05 g (NH4)6Mo7O24-4H2O per liter] per liter also was added. Unless otherwise stated, MBC, 2-AB, and 2-HB were added at final concentrations of 40 μM to MM in powdered form, and the final solution was filter sterilized (0.22 μm) and stored at 4°C until further use. Nutrient agar (NA) and nutrient broth (NB) were used as rich media for bacterial growth.

Soil enrichment and isolation of an MBC-mineralizing microorganism.

MBC-exposed soil samples were collected from the Murrumbidgee Country Club, Australian Capital Territory (ACT), Australia. MM (100 ml) containing 40 mM MBC was inoculated with 1 g of pooled soil samples and incubated at 28°C for a week on a rotary shaker at 150 rpm. This culture (1%, vol/vol) was transferred to fresh medium and then incubated at 28°C for another week. After five rounds of such enrichments, the growth medium was plated onto the NA plates and incubated at 28°C for 48 h. Individual colonies then were tested for MBC degradation by a plate-clearing assay.

Plate-clearing assay.

MM plates containing 0.7% agarose were sprayed with 0.1% carbendazim as an emulsion in diethyl ether and dried overnight for the complete evaporation of the diethyl ether. A 3-μl drop of bacterial culture, grown overnight in nutrient broth at 28°C, was transferred onto the carbendazim-sprayed plate. Clearing around a culture drop indicated carbendazim hydrolytic activity, because the hydrolysis products of carbendazim are highly soluble compared to those of carbendazim (soluble at 8 ppm only).

Identification of MBC-degrading bacteria.

The nucleotide sequence of the 16S rRNA gene (1,448 bp) was determined by direct PCR sequencing using the universal 16S primers 8F and 1492R (5). The nucleotide sequence was compared to sequences in the nonredundant database using a BLAST search (29).

Growth experiments.

Aerobically grown seed culture of strain SG-4G in NB was inoculated (1%, vol/vol) in MM containing 40 μM MBC, 2-AB, or 2-HB and incubated at 28°C on a rotary shaker at 100 rpm. For quantitative or qualitative analysis, 1-ml samples were collected at different time points and immediately filtered through 0.45-μm filters (Millipore, MA), and formic acid was added to a final concentration of 1%, vol/vol. Samples then were stored at 4°C until analysis by liquid chromatography (LC) or LC-mass spectrometry (LC-MS).

Analytical methods.

An Agilent series LC system (Agilent Technologies, CA) controlled by Agilent time-of-flight (TOF) software (version A.01.00) was used for the quantitative analysis of MBC, 2-AB, and 2-HB. The compounds were separated at 25°C on an Aqua C18 column (5-μm particle size, 250 by 4.60 mm) (Phenomenex, CA) using acetonitrile-water (18:82 [vol/vol], both containing 0.1% [vol/vol] formic acid) as a mobile phase at a flow rate of 0.7 ml min−1. Substrate and products were monitored at 270 nm. The qualitative analysis of MBC, 2-AB, and 2-HB was performed using an LC-MS TOF mass spectrometer (Agilent Technologies, CA) with an electrospray ionization (ESI) source as described previously (19).

Protein purification and sequencing from SG-4G.

The MBC-degrading enzyme MheI was purified from strain SG-4G using a two-step protocol. The first step involved the use of molecular-size cutoff membranes, while the second step involved anion-exchange chromatography. Briefly, SG-4G cells were harvested from two 800-ml flasks of NB by centrifugation at 8,000 × g for 10 min. The cell pellet was washed three times with ice-cold 20 mM sodium phosphate buffer (pH 7.0). The washed pellet was incubated (50 rpm) in 100 ml MM at 37°C. After overnight incubation, the supernatant was filtered (0.22 μM) and passed through a series of Amicon molecular size cutoff centrifugal filters of 100, 50, 30, and 10 kDa (Amicon, Millipore, MA). At each step the retentate (150 μl) was kept and the filtrate passed through the next centrifugal filter, in descending order. Finally, all of the retentates were analyzed for MBC degradation, and the 50-kDa retentate, which had the maximum MBC hydrolysis activity, was loaded onto a Mono Q HR 5/5 anion exchange column (Mono Q HR 5/5; GE Healthcare, United Kingdom) preequilibrated in 20 mM NaPO4 buffer (pH 7.0) and eluted with a gradient of NaCl (0 to 100%) for 30 min. Seventy-eight fractions (0.5 ml) were collected, and each fraction then was tested for MBC degradation activity. Two fractions showing MBC degradation activity were concentrated to ~100 μl using 10-kDa molecular size cutoff membranes. These two fractions then were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and found to contain a pure protein of ~25 kDa.

The N terminus of the purified protein and three internal peptides (from a trypsin digest of the purified protein) were sequenced commercially at the Australian Proteome Analysis Facility (North Ryde, Australia).

Cloning of the complete gene.

PCR primers F1 (5′-ATGGCCAACTTCGTCCTCGTGC-3′) and R2 (5′-GACGAAGGCGTCGAGGTAGACC-3′), corresponding to the N terminus and one of the internal peptide sequences of the carbendazim-degrading enzyme (MheI), were designed based on the codon usage of Nocardioides sp. The partial gene was amplified using these primers and genomic DNA (gDNA) of Nocardioides sp. SG-4G. For PCR amplification, approximately 375 ng gDNA from strain SG-4G, 50 pmol of each primer, 1 μl of 10 mM deoxynucleoside triphosphates (dNTPs), 5 μl of 10× PCR buffer with MgSO4, and 1 U of Deep Vent DNA polymerase (NEB Biolabs) were added to a final volume of 50 μl. The PCR protocol involved denaturation at 98°C for 5 min, followed by 30 cycles of 98°C for 30 s, 48°C for 30 s, and 75°C for 30 s, with a final extension step of 5 min at 75°C. The resulting PCR products were directly sequenced at the Micromon DNA Sequencing Facility (Victoria, Australia). Three outward-facing DNA sequencing primers, namely, F2-214 (5′-ATCCTCGTCGGCCATTCGTAC-3′), F3-277 (5′-AAGATCAGGTCGCTGGTCTACCTC-3′), and R-466 (5′-GTTCACCCAGTCGCGCTTGTC-3′), were then designed based on this sequence for the direct sequencing of the full-length gene from gDNA of strain SG-4G.

Heterologous expression of MheI.

A version of the mheI gene codon optimized for expression in Escherichia coli (synthesized by Geneart AG, Regensburg, Germany) was recombined into the Invitrogen Gateway vector pDEST17 (Invitrogen, CA) using the manufacturer's protocols. Initially, the synthetic mheI gene was PCR amplified with attB1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGGCGAACTTTGTGCTG-3′) and attB2 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTATTATTAGCCCAGCGCGGC-3′) primers (attB sites are underlined, and start and stop codons are in boldface). Two stop codons were added at the end of the gene to avoid any read-through during translation. The PCR mix comprised 20 ng plasmid, 0.2 μM each primer, 5 μl of 10× PCR buffer, 1 μl of 10 mM dNTPs, and 2 U of Deep Vent DNA polymerase (NEB Biolabs, MA). The PCR protocol involved initial denaturation at 98°C for 3 min, followed by 5 cycles of 98°C for 20 s, 48°C for 20 s, and 75°C for 1 min, and then 25 cycles of 98°C for 20 s, 60°C for 20 s, and 75°C for 1 min. A final extension step of 5 min at 75°C was used. The amplicon then was cloned into pDONOR201 and transferred to pDEST17 using BP and LR reactions, respectively, by following the manufacturer's instructions (Invitrogen).

MheI was expressed from pDEST17-mheI in BL-21 AI (arabinose-inducible) cells by following the Invitrogen protocol, except that the expression was carried out at 20°C. The cell pellet (4.26 g) from a 250-ml culture was lysed by being resuspended into 40 ml of Bugbuster solution (Invitrogen), and the lysate then was eluted through an Ni-NTA Superflow Cartridge (Qiagen, GmbH). Eluate fractions showing degradation activity were dialyzed against two changes of 200 mM sodium phosphate buffer, pH 7.2, and the purified protein was stored at 4°C. Protein expression and purification also were monitored by SDS-PAGE.

Enzyme assays and determination of kinetic constants.

Assays for MBC degradation by heterologously expressed MheI were performed in 96-well microtiter plates at 22°C. The assay mixture typically contained 20 μg bovine serum albumin, 0.022 μg purified MheI, and 1 to 40 μM MBC in a final volume of 200 μl of 200 mM sodium phosphate buffer. The reaction was stopped by adding 5 μl of formic acid after 10 min, and the initial velocities of substrate disappearance were measured using high-performance liquid chromatography (HPLC) as described above. All of the assays were performed in triplicate with appropriate controls. The kinetic parameters, Michaelis constant (Km), maximum reaction velocity (Vmax), and turnover number (kcat) were determined according to the Michaelis-Menten kinetic equation using the Sigma Plot Kinetic Module (version 1.3). The kcat value is calculated by dividing Vmax by the total enzyme concentration ([Et]).

The esterase activities of MheI against methyl salicylate, p-nitrophenyl acetate, and α-naphthyl acetate were determined qualitatively by LC-MS TOF using a previously described method (19).

Bioremediation experiments.

Strain SG-4G cells were grown at 37°C in a 1,000-liter vessel with an initial working volume of 750 liters. Cells were cultured in a mineral salts/yeast extract medium to a final optical density at 600 nm (OD600) of 103 after 47 h. The medium consisted (per liter) of 20.0 g glucose, 5.0 g yeast extract, 5.2 g K2HPO4, 4.1 g KH2PO4, 2.0 g (NH4)2SO4, 0.3 g ferric ammonium citrate, 0.5 g MgSO4-7H2O, 0.015 g CaCl2-H2O. The pH was adjusted to 7.2 with NaOH, and 1.85 ml of TES, consisting of 0.15 M HCl, 10.0 g Na2SO4, 2.0 g MnSO4-H2O, 2.0 g ZnCl2, 2.0 g CoSO4-7H2O, 0.3 g CuSO4-5H2O, and 10.0 g FeSO4-7H2O per liter, also was added. The harvest was supplemented with 13.3 g K2HPO4, 4.1 g KH2PO4, 12.5 g NaCl, 1.0 g Tween 20, and 53.3 g sucrose per liter, chilled to 4°C, and freeze-dried in four separate 200-liter batches at Biotech Freeze-driers (Knoxfield, Australia), yielding 105 kg of dry powder (13.1% solids). The freeze-dried powder then was sterilized by gamma irradiation at 25 kGy (Steritech, Dandenong, Australia).

For bioremediation experiments, this powder (referred to as dead cells above) was resuspended in tap water to a final OD600 of 2.0. In the first set of experiments, this suspension (1%, vol/vol) was inoculated into 40 μM MBC, 2-AB, or 2-HB in tap water. In the second trial, a 0.1% (wt/vol) suspension of the dead cells was incubated with 500 ppm (2.61 mM, if soluble) of MBC, and the mixture then was analyzed as described above. In the third trial, dead cells were mixed at various doses with a wastewater sample containing 207 ppm (1.08 mM, if soluble) MBC obtained from a commercial fruit-dipping operation and incubated and analyzed as above.

Nucleotide sequence accession numbers.

The nucleotide sequences of Nocardioides sp. SG-4C 16S rRNA genes, native mheI, and E. coli codon-optimized mheI have been deposited in the GenBank database with the accession numbers GQ451604, GQ454794, and GQ454795, respectively.

RESULTS AND DISCUSSION

Enrichment, isolation, and characterization of SG-4G.

An MBC-degrading enrichment culture was readily established from an MBC-exposed soil sample. The initial 8 ppm of MBC in this enrichment culture was rapidly degraded within 24 h. When different dilutions of the enrichment were plated onto nutrient agar plates, 10 morphologically distinct bacterial colonies appeared after 3 days of incubation at 28°C. These colonies were picked and checked individually for MBC degradation using a plate-clearing assay. One bacterial colony, designated SG-4G, showed plate clearing within 20 min of inoculation (see Fig. S1 in the supplemental material). Plate clearing in this unusually short time suggested that strain SG-4G was constitutively producing an extracellular MBC-degrading enzyme during its growth on nutrient broth.

Strain SG-4G was found to be a gram-positive, motile, pleiomorphic bacterium that formed a tiny creamish-white colony after 3 days of incubation on nutrient broth agar plates at 28°C. The 16S rRNA gene of strain SG-4G showed 99% sequence identity with the genus Nocardioides, so it was named Nocardioides sp. SG-4G. To test whether MBC degradation is a common property of the genus Nocardioides, nine type strains (N. albus, N. aromaticivorans, N. alkalitolerans, N. jensenii, N. kribbensis, N. lentus, N. luteus [synonym: N. flavus], and N. nitrophenolicus), all obtained from DSMZ Germany, were tested using the plate-clearing assay and subsequently by LC-MS, and all were found to be negative for MBC degradation (data not shown).

Growth on MBC and metabolite identification.

When 41.8 μM MBC (8 ppm, which is the maximum aqueous solubility of MBC) was provided as a sole source of carbon in minimal medium, a seed culture of SG-4G cells (1%, vol/vol) completely degraded MBC in 5 h (Fig. (Fig.1A).1A). Two metabolite peaks (retention times of 5.42 and 12.87 min) appeared in the HPLC chromatogram during the growth of strain SG-4G on MBC, and these were identified as 2-AB and 2-HB by HPLC and LC-MS using authentic standards. 2-AB is the major metabolite of MBC degradation in soils, plants, and mammalian systems, and it is presumed in these cases to be produced by the enzymatic hydrolysis of the MBC. 2-AB and benzimidazole (BI) were reported to be the intermediates of the MBC catabolism by Rhodococcus qingshengii strain dj1-6 (14), but we did not detect any BI during the growth of strain SG-4G on MBC.

FIG. 1.
Degradation kinetics of live (A, B, and C) and dead cells (D, E, and F) of strain SG-4G on MBC (A and D), 2-aminobenzimidazole (B and E), and 2-hydroxybenzimidazole (C and F).

To further dissect the MBC catabolic pathway in strain SG-4G, we grew SG-4G on 2-AB and 2-HB as sole carbon and energy sources and monitored the appearance of intermediates by HPLC and LC-MS. 2-HB transiently appeared as an intermediate during growth on 2-AB (Fig. (Fig.1B).1B). However, we could not detect any metabolite during the growth on 2-HB, even though 2-HB completely disappeared from the culture medium in 17 h (Fig. (Fig.1C).1C). These results indicated that in strain SG-4C, MBC first is hydrolyzed to 2-AB, which then is degraded to 2-HB before complete assimilation.

Enzyme assays then were performed using the crude cell extract of strain SG-4G cells grown in nutrient broth. Crude cell extract converted MBC into 2-AB and 2-HB, and 2-AB into 2-HB, without the need for any additional cofactors (data not shown). No degradative activity was found when heat-inactivated crude cell extract was used. Since the culture from which the crude cell extract was produced had not been exposed to MBC, the gene-enzyme system(s) carrying out the transformation of MBC to 2-AB and 2-AB to 2-HB must have been constitutively expressed (the evidence for inducible growth on 2-AB and 2-HB is apparent in Fig. 1B and C and therefore might reflect the inducibility of other factors, e.g., the transport system, which is required for the growth of strain SG-4G on these compounds). We have not tried to elucidate the downstream metabolites and the complete catabolic pathway for MBC, because 2-AB and 2-HB have relatively benign toxicity profiles (11, 22).

To further test whether the MBC-degrading enzyme is extracellular, the seed culture was filtered through a 0.22-μm filter and the filtrate was inoculated (1%, vol/vol) into MM containing 41.8 μM MBC. Twenty-five percent of the MBC was depleted in 7 h (data not shown), which indicated the presence of an extracellular MBC-degrading enzyme in the culture supernatant. Extracellular MBC-degrading activity was not examined in the earlier reports on other MBC-degrading strains (10, 13, 14, 25, 28).

Enzyme purification and identification.

A two-step purification strategy was employed to purify the MBC-hydrolyzing enzyme. The supernatant of resting cells of strain SG-4G was fractionated using molecular-mass-cutoff membrane filters and then subjected to anion-exchange chromatography. Two fractions from the eluate demonstrated MBC degradation when analyzed by LC-MS. The same two fractions also demonstrated weak esterase activity when naphthyl acetate was used as a substrate (see below). Further analysis of these fractions by SDS-PAGE revealed a substantially pure protein of ~25-kDa (see Fig. S2 in the supplemental material), which we have named MBC-hydrolyzing enzyme (MheI).

These experiments also support the earlier evidence that MheI is extracellular in nature. Contaminated sites often contain insoluble crystals of MBC because of its low aqueous solubility (8 ppm), and in such a situation the extracellular nature of MheI could be advantageous to strain SG-4G; MheI could diffuse away from the cells and hydrolyze the insoluble MBC into the much more soluble 2-AB, which could again diffuse back to the cells and be utilized as a carbon and energy source.

Cloning, expression, and kinetic characterization of MheI.

The N terminus of the purified enzyme and three internal peptides generated by tryptic digestion were sequenced by Edman degradation. The N terminus and one of the three internal peptides aligned extremely well with motifs in known/predicted esterases and other members of the α/β hydrolase fold superfamily of proteins (data not shown). PCR primers corresponding to the N-terminal fragment (MANFVLVHGAWHGGWC) and the most conserved of the three internal fragments (LVYLDAFVPE) were designed, taking account of the codon usage of the genus Nocardioides. The PCR resulted in the amplification of a 318-bp fragment that subsequently was sequenced and analyzed by Blastx (searching nonredundant protein sequence databases using a translated nucleotide query) (3). The closest Blastx match was to a putative esterase of alphaproteobacterium strain BAL199 (GenBank accession no. ZP_02187615.1). This result further supported our hypothesis that MheI is an esterase.

To obtain the complete open reading frame (ORF) encoding MheI, outward-facing sequencing primers (F2-214, F3-277, and R-466) based on the partial gene sequence were designed. The direct sequencing of the genomic DNA from strain SG-4G using these primers provided the complete sequence of mheI (GenBank accession no. GQ454794). This 729-bp gene (including the stop codon) was found to have a G+C content of 69.6% and encoded a 242-amino-acid protein with a predicted molecular mass of 26.327 kDa. The predicted amino acid sequence included the N terminus and all three internal peptide fragments obtained from the tryptic digests described above. A conserved-domain database search showed full-length homology to a predicted hydrolase domain (COG0596) and the α/β hydrolase fold (pfam00561). Its nearest relative was again the putative esterase from strain BAL199 (discussed above), with which it showed 40% amino acid identity. The phylogenetic analysis of MheI and presumptive esterases from the α/β hydrolase fold superfamily, including predicted salicylate esterases (see below), revealed no close relatives with empirically determined functions (data not shown).

The codon-optimized form of the mheI gene then was synthesized for heterologous expression in E. coli and recombined into vector pDEST17 for the overexpression of N-terminally His-tagged protein. A substantially purified enzyme was obtained using nickel-affinity chromatography, which appeared at ~30 kDa in SDS-PAGE (see Fig. S2 in the supplemental material). The quantitative analysis of MBC degradation by His-tagged MheI was monitored by HPLC using authentic standards. The enzyme showed Km and kcat values of 6.1 μM (standard error, 1.0) and 170 min−1 (standard error, 4.1), respectively, at 20°C (see Fig. S3 in the supplemental material). MheI also hydrolyzed methyl salicylate, α-naphthyl acetate, and p-nitrophenyl acetate, so it has quite a wide substrate range. These results were confirmed by LC-MS (data not shown).

Implications for in situ bioremediation with radiation-killed SG-4G cells.

Strain SG-4G cells could be fermented readily to an OD600 of 103 in a pilot-scale (1,000 liter) fermentation vessel (data not shown). A radiation-killed and freeze-dried preparation of strain SG-4G cells from this ferment then was evaluated in three proof-of-concept bioremediation trials. First, the degradation capacity of this preparation, here referred to as dead cells, was compared to those of live SG-4G cells (Fig. (Fig.1).1). In this set of experiments, the dead cells were mixed with tap water to make a suspension with an OD600 of 2.0. This gave a biomass of 2.54 g liter−1, which was approximately three times the biomass (0.82 g liter−1) of freshly harvested live SG-4G cells at an OD600 of 1.0 (as was used in the experiments shown in Fig. 1A, B, and C). This suspension was inoculated (1%, vol/vol) into MM containing 41.8 μM (8 ppm) of MBC, 2-AB, or 2-HB in individual flasks. Samples were collected at different time points and analyzed quantitatively and qualitatively. The dead cells of strain SG-4G degraded MBC completely in less than 1 h, compared to 5 h for live cells (Fig. 1B and D). This is consistent with the fact that the effective concentration of dead cells was three times the concentration of the live cells. 2-AB appeared transiently during the incubation; a maximum concentration of 20.4 μM was reached in 1.5 h, but it completely disappeared during the next 20 h (Fig. (Fig.1D1D).

Interestingly, however, under similar conditions of incubation, the flasks of dead cells set up with 40 μM 2-AB required 48 h to completely degrade the 2-AB (Fig. (Fig.1E),1E), whereas only 16 h was needed for complete 2-AB degradation by the live cells. Also, the dead cells did not transform 2-HB (Fig. (Fig.1F),1F), whereas the live cells completely degraded 40 μM 2-HB in 17 h. The transformation of MBC to 2-AB by the MheI esterase would be a cofactor-independent reaction, and the second transformation of 2-AB to 2-HB would be a hydrolytic deamination, which also should be catalyzed by an enzyme not requiring a diffusible cofactor. However, diffusible cofactors might be required for the further degradation of 2-HB, explaining why it was not further transformed by the dead cells.

To test the efficacy of the dead cells to detoxify higher MBC concentrations, 500 ppm (2.61 mM, if soluble) MBC in tap water was inoculated with 1 g liter−1 of dead cells, and the reaction was monitored by LC-MS. MBC was completely and stoichiometrically transformed to 2-AB and 2-HB in 24 h (data not shown).

To further test the efficacy of the dead cells under field conditions, an MBC-contaminated wastewater sample was obtained from a commercial fruit-dipping operation and treated with various doses of the SG-4G dead cells, ranging from 0.05 to 1 g liter−1. The wastewater contained an initial MBC concentration of 207 ppm (1.08 mM, if soluble). Increasing degradation rates were observed with increasing enzyme dose, and the quickest degradation (80% degradation within 3 h at 20°C) was at a dose rate of 1 g liter−1 (Fig. (Fig.2).2). Greater than 80% of the MBC was degraded within 16 h using dose rates of 0.6 g/liter and above.

FIG. 2.
Degradation kinetics of various doses of dead cells on an MBC-contaminated wastewater sample from a commercial fruit-dipping operation.

Conclusion.

Enzymatic bioremediation potentially is a rapid method of removing pesticide residues from contaminated liquids and wettable surfaces. Applications include the treatment of residues resulting from agricultural production and processing industries, such as the treatment of irrigation waters, surface-contaminated fruit and vegetables, and spent livestock and horticultural dip liquors (2, 23). For an enzyme to be suited to these applications, it must be able to degrade the target pesticide to substantially less toxic products and should not require cofactors, such as NAD(P)H, reduced flavin, or glutathione, which would be prohibitively expensive to provide in a commercial setting. We have isolated Nocardioides sp. SG-4G, which mineralizes MBC via 2-AB and 2-HB. We have purified MheI, the first enzyme of the pathway that detoxifies MBC by hydrolyzing it to 2-AB, without the requirement of any cofactor. The cognate gene was then cloned using a reverse genetics approach. A radiation-killed, freeze-dried culture of strain SG-4G not only retained the enzymatic detoxification activity of the MheI enzyme but also actively detoxified MBC-contaminated wash water from a commercial fruit-processing plant. The ability to grow the source Nocardioides strain to high titer would obviate elaborate downstream processing to satisfy the requirements of gene technology-regulating agencies regarding the open-field use of recombinantly produced MheI. Moreover, the irradiation process we have used to sterilize the harvest from the fermenter could avoid regulatory issues associated with live bacterial cells in the product.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported in part by Horticulture Australia Ltd.

We gratefully thank Hans-Peter E. Kohler, Matthew Taylor, and Rinku Pandey for critically reading the manuscript. Special thanks to Colin Scott, Chris Coppin, Peter Campbell, and Mark Teese for their input in experimental design and data analysis and to Kaiyan Liu and Vinko Momiroski for their technical assistance.

Footnotes

[down-pointing small open triangle]Published ahead of print on 12 March 2010.

Supplemental material for this article may be found at http://aem.asm.org/.

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