Expression of esterase gene in yeast for organophosphates biodegradation

Pesticide Biochemistry and Physiology 94 (2009) 15–20

Contents lists available at ScienceDirect

Pesticide Biochemistry and Physiology journal homepage: www.elsevier.com/locate/pest

Expression of esterase gene in yeast for organophosphates biodegradation Devaiah M. Kambiranda a, Shah Md. Asraful-Islam a, Kye Man Cho b, Renukaradhya K. Math a, Young Han Lee c, Hoon Kim d, Han Dae Yun a,b,* a

Division of Applied Life Science (BK21 Program) and Research Institute of Agriculture and Life Science, Gyeongsang National University, Chinju 660-701, Republic of Korea Department of Food Science, Jinju National University, Chinju 660-758, Republic of Korea c Division of Plant Environmental Research, Gyeongsangnam-do Agricultural Research and Extension Service, Chinju 660-360, Republic of Korea d Department of Agricultural Chemistry, Sunchon National University, Suncheon 540-742, Republic of Korea b

a r t i c l e

i n f o

Article history: Received 2 September 2008 Accepted 18 February 2009 Available online 24 February 2009 Keywords: Rumen metagenome est5S gene Esterase Yeast expression OP degradation

a b s t r a c t Organophosphates are esters of phosphoric acid and can be hydrolyzed and detoxified by carboxylesterase and phosphotriesterase. In this work esterase enzyme (Est5S) was expressed in yeast to demonstrate the organophosphorus hydrolytic activity from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) is 1098 bp in length, encoding a protein of 366 amino acid residues with a molecular weight of 40 kDa. Est5S enzyme was successfully produced by Pichia pastoris at a high expression level of approximately 4.0 g L1. With p-nitrophenol butyrate as the substrate, the optimal temperature and pH for enzyme activity were determined to be 40 °C and pH 7.0, respectively. The esterase enzyme was tested for degradation of chlorpyrifos (CP). TLC results obtained inferred that CP could be degraded by esterase enzyme (Est5S) and HPLC results revealed that CP could be efficiently degraded up to 100 ppm. Cadusafos (CS), coumaphos (CM), diazinon (DZ) dyfonate (DF), ethoprophos (EP), fenamiphos (FM), methylparathion (MPT), and parathion (PT) were also degraded up to 68, 60, 80, 40, 45, 60, 95, and 100%, respectively, when used as a substrate with Est5S protein. The results highlight the potential use of this enzyme in the cleanup of contaminated insecticides. Ó 2009 Published by Elsevier Inc.

1. Introduction Chlorpyrifos (CP, O,O-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate) has been commercially used since the 1960s, particularly for the control of foliar insects of cotton, paddy fields, pasture and vegetable crops. The widespread use of CP in agriculture has increased public concern on potential human health risks that may result from acute or chronic dietary exposure to chlorpyrifos residues in food [1]. Korea regulates the maximum residue limit (MRL) in stored fruits and vegetables at 0.1–2.0 mg kg1. CP is defined as a moderately toxic compound having acute oral LD50: 135–163 mg kg1 for rat and 500 mg kg1 for guinea pig. In addition, Lee et al. [2] reported that individuals in the highest quartile of CP lifetime exposure-days (>56 days) had a relative risk of lung cancer 2.18 times than that of the farmers with no CP exposure. Thus there is a need to monitor and degrade the CP content in vegetable and foods destined for human consumption. CP is characterized by a P–O–C linkage as other organophosphate pesticides, such as diazinon (DZ), parathion (PT), methylparathion (MPT), and fenitrothion (FT). There is an increasing need to develop new methods

* Corresponding author. Address: Division of Applied Life Science (BK21 Program), Gyeongsang National University, Chinju 660-701, Republic of Korea. Fax: +82 55 757 0178. E-mail address: [email protected] (H.D. Yun). 0048-3575/$ - see front matter Ó 2009 Published by Elsevier Inc. doi:10.1016/j.pestbp.2009.02.006

to detect, isolate, and characterize the strains/enzymes playing a part in these degradation processes [3]. Screening novel biocatalysts from isolated microorganisms using traditional cultivation techniques has limits in exploring the vast genetic diversity of environmental microorganisms because more than 99% of microbes present in various environments cannot be cultured [4,5]. The rumen ecosystem is comprised of diverse population of obligatory and anaerobic bacteria, fungi, and protozoa defined by the intense selective pressures of the ruminal environment [6]. The rumen microbial population presents a rich and under-utilized source of novel enzymes. The variety of enzymes present in rumen arises not only from the diversity of the microbial community but also from the multiplicity of fibrinolytic enzymes produced by individual microorganisms [7]. Esterases belonging to the group of carboxylester hydrolases (EC 3.1.1.1) hydrolyze the ester bonds of water soluble fatty acid esters with short-chain acyl groups (C8), whereas lipases (EC 3.1.1.3) hydrolyze long-chain acyl groups (C10) [8]. Even though esterases and lipases have divergent substrate specificity, they commonly contain a catalytic triad composed of Ser, His, and Asp/Glu in the polypeptide chain [8], in which the active site Ser residue is integrated at the center of the conserved pentapeptide sequence motif, GXSXG, where ‘X’ can be any amino acid [9]. Several investigators reported previously that the majority of organophosphates (OP) are esters of phosphoric acid and can therefore be

16

D.M. Kambiranda et al. / Pesticide Biochemistry and Physiology 94 (2009) 15–20

hydrolyzed and detoxified by carboxylesterase and phosphotriesterase [10,11]. Microbial degradation of OP pesticides is of particular interest because of the high mammalian toxicity of such compounds and their widespread and extensive use. For some OP such as parathion (PT), it has been relatively easy to isolate degrading bacteria: two different strains, Flavobacterium sp. strain ATCC 27551 and Pseudomonas diminuta strain Gm, have been isolated from soils in the Philippines and United States, respectively [12,13]. Molecular basis of degradation of certain organophosphates has been studied extensively [13–15]. A widely distributed organophosphatedegrading gene (opd) was isolated from temporally, geographically, and biologically different species [12,13]. Coumaphos (CM) degrading Nocardioides simplex NRRL B-24074 has been reported to have novel organophosphate-degrading enzyme and gene systems [16]. Expression of opd genes in Escherichia coli has been done to maximize the recombinant protein yield for opd gene [17–20]. 3,5,6-Trichloro-2-pyridinol (TCP) the main transformation product of CP displays antibacterial activity at higher concentrations [21]. To overcome the shortcomings of antibacterial activities and high protein expression, opd gene has been previously expressed in Pichia pastoris [22]. In the present report P. pastoris expression system was used to clone esterase gene est5S that was isolated from cow rumen metagenomic library. The est5S gene was obtained by screening the pBluescript II SK+ clones harboring the partially digested fosmid DNA fragments (3–5 kb) constructed from rumen metagenome for esterase activity. The esterase activity displaying clone est5S was cloned and expressed in P. pastoris so as to produce large amounts of active recombinant esterase enzyme. Cadusafos (CS), CP, CM, DZ, dyfonate (DF), ethoprophos (EP), fenamiphos (FM), MPT, and PT were used as substrate with purified esterase (Est5S) for observation of organophosphate hydrolytic activity.

2. Materials and methods 2.1. Chemicals and reagents All reagents and samples used in the present study were of analytical grade and were used without further purification. Cadusafos (CS; S,S-di-sec-butyl O-ethyl phosphorodithioate), chlorpyrifos (CP; O,O-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothioate), coumaphos (CM; O-3-chloro-4-methyl-2-oxo-2H-1-benzopyran-7-yl O,Odiethyl phosphorothioate), diazinon (DZ; O-3-chloro-4-methyl-2oxo-2H-1-chromen-7-yl O,O-diethyl phosphorothioate), dyfonate (DF; O-ethyl S-phenyl ethylphosphonodithioate), ethoprophos (EP; O-ethyl S,S-dipropyl phosphorodithioate), fenamiphos (FM, RS-ethyl 4-methylthio-m-tolyl isopropylphosphoramidate), methylparathion (MPT; O,O-dimethyl O-4-nitrophenyl phosphorothioate), parathion (PT; O,O-diethyl O-4-nitrophenyl phosphorothioate), and TCP were obtained from Chemservice (West Chester, PA, USA) and DETP was purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLC-grade water, methanol, acetonitrile, and glacial acetic acid were purchased from Fisher Scientific (Fairlawn, NJ, USA). All other reagents were of analytical grade. Tributyrin, qnitrophenol-b-butyric acid (q-NPB), and q-nitrophenol (q-NP) were purchased from Sigma Chemical Co. All other reagents were of analytical grade. Standard procedures for restriction endonuclease digestions, agarose gel electrophoresis, purification of DNA from agarose gels, DNA ligation, and other cloning-related techniques followed Sambrook and Russell [23]. Restriction enzymes and DNA-modifying enzymes were purchased from Gibco-BRL (Gaithersburg, MD, USA) and Boehringer Mannheim (Indianapolis, IN, USA). Other chemicals were purchased from Sigma Chemical Co.

2.2. Strains and culture conditions Escherichia coli DH5a was cultured in Luria–Bertani broth (LB, Difco, NJ, USA) at 37 °C. For the culture of recombinant E. coli cells ampicillin (50 lg mL1) was added in LB broth. P. pastoris GS115 (his4) was used as a recipient strain for esterase enzyme expression. P. pastoris was maintained on YPD plates (1% yeast extract, 2% peptone, 2% dextrose, and 1.5% agar) at 30 °C. For the culture of recombinant P. pastoris cells zeocin (100 lg mL1) was added in YPD medium. 2.3. Cloning and characterization of esterases Sampling of rumen metagenome and rumen metagenomic library construction was done as per Cho and Yun [6]. Cloning of esterase gene from cow rumen metagenomic library was done by ligating partially Sau3AI-digested fosmid DNA fragments (3–5 kb) with pBluescript II SK+ (Stratagene, CA, USA) and later transformed into E. coli DH5a. To detect esterase activity these bacterial colonies were grown on a esterase activity indicator medium [LB agar plates containing appropriate antibiotics and 1.0% (v/v) tributyrin]. The esterase activity displaying clone was named as est5S. The nucleotide sequence data for est5S have been deposited in GenBank under Accession No. DQ788540 (Korean patent number 100817321-0000) [24]. We designed gene specific primers for consensus nucleotide sequence est5S-FP (sense, 50 -GAA TTC ATG ATC ATG AAA AAA CAG AA-30 ) and est5S-RP (antisense, 50 -GCG GCC GCT TTC TCC AGG AAT GCC TTG ACG-30 ). Polymerase chain reaction (PCR) was conducted with 50 ng of DNA from a pBSK/est5S clone that was displaying esterase activity with the following parameters: initial denaturation (94 °C for 4 min); followed by 35 cycles as follows: denaturation 94 °C for 30 s, annealing 60 °C for 30 s, and extension 72 °C for 70 s followed by extension at 72 °C for 10 min. The 1098 bp PCR product was cloned into pGEMT vector (Promega, WI, USA). The insert of positive bacterial clones was sequenced and compared. For the determination of esterase activity the isolates were inoculated on esterase activity indicator medium. 2.4. Expression and purification of est5S in E. coli For high expression of Est5S, the PCR product generated with primers, 50 -AAA AGG ATC CAT GAT CAT GAA AAA ACA GAA TTT C-30 (sense, est5S-FPE with BamHI underlined) and 50 -AAA AAA GCT TTT TCT CCA GCA ATG CCT TG-30 (antisense, est5S-FPE with HindIII underlined), was cloned into expression vector pET28a(+) (Novagen) using BamHI and HindIII sites, resulting in the addition of a C-terminal (His)6 tag. E. coli BL21 (DE3) carrying pET-28a(+)/Est5S was grown at 37 °C to mid-log phase in LB medium containing 50 lg/mL kanamycin. The cells were harvested by centrifugation (6000g, 10 min) and washed twice with 10 mM Tris–HCl buffer (pH 7.0). The cells were resuspended in the same buffer and stored at 20 °C. The frozen cells were mixed with 50 mM Tris–HCl buffer (pH 7.5) containing 1 mg of bovine DNase I (Sigma) and incubated at 37 °C for 30 min. Triton X-100 was added to the suspension to attain a final concentration of 2.5%. The supernatant was collected and stored at 4 °C. The solubilized recombinant Est5S with His-tag was applied on a HisTrap kit (Qiagen, CA, USA). Purification of expressed His6-tagged protein was carried out accordingly as previously described by Guo et al. [25] and protein (Est5S) was eluted with 100 mM imidazole with 0.1% Triton X-100. The purified protein sample was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The protein concentration was determined by the method of Bradford [26].

D.M. Kambiranda et al. / Pesticide Biochemistry and Physiology 94 (2009) 15–20

2.5. Heterologous expression and purification of est5S in P. pastoris The recombinant plasmids were digested for release of insert, the fragment was cloned using specific restriction enzymes into P. pastoris expression vector, pPICZaA (Invitrogen, CA, USA). The resulting plasmid was named as pPICaA/est5S, used to transform E. coli DH5a, and transformants were selected on low salt LB plates containing 25 lg mL1 of zeocin. The fusion of the mature peptide encoding region of esterase enzyme with the yeast r secretary factor at N-terminus and myc epitope followed by 6His-tag tail at Cterminus was confirmed by DNA sequencing. A total of 10 lg of pPICaA/est5S DNA was linearized by PmeI digestion and transformed into Pichia competent cells. The transformed cells were plated on YPD agar containing 100 lg mL1 of zeocin to screen for integration of the esterase gene into the 50 AOX1 region of the host chromosomal DNA. Genomic DNA was isolated from positive clones and confirmed by PCR using gene specific primers and sequencing was performed on 50 AOX primers. Each transformant was cultured in 100 mL of a buffered complex medium with glycerol (BMGY) for 24 h according to the manufacturer’s instructions (Invitrogen). After the culture reached a density of OD600 = 6.0, the cells were harvested, resuspended, and cultured in 100 mL of 0.5% methanol media (BMMY) to induce expression. The culture filtrates of positive clones after induction were directly screened for esterase activity as described previously, and the secreted enzymes were monitored by SDS–PAGE every 12 h for 72 h and the highest esterase activity showing clone was selected for further experiments. By virtue of the 6His-tag at its C-terminus, the cloned enzyme was purified using a Ni–NTA agarose column chromatography according to the manufacturer’s instructions (Qiagen, CA, USA). At each step, the purified enzyme was evaluated by SDS–PAGE. Fractions of 3 mL each were collected and protein content was measured colorimetrically at 595 nm using the Bio-Rad protein reagent according to the method of Bradford [26]. Cell growth and enzyme activity were measured for 3 days at 24 h intervals. 2.6. Characteristics of the enzymes The esterase activity was determined by a spectrophotometeric method using q-NPB as the substrate. The rate of hydrolysis of qNPB at 35 °C was measured in 50 mM sodium phosphate buffer (pH 7.0) at 420 nm according to the method of Alvarez-Macarie et al. [27]. One unit of esterase was defined as amount of enzyme required to release 1 lmol of q-NP per minute under the assay conditions. The effects of pH and temperature on the esterase activity were examined with the purified recombinant enzyme. The effect of pH on the esterolytic activity was determined by using the protocol described above, to obtain values from pH 3.0 to 11.0; all of the assays were performed at 30 °C. To determine the effect of temperature on the enzymatic activity, samples were incubated at temperatures ranging from 10 to 70 °C for 1 h. Hydrolysis of CS, CP, CM, DZ, DF, FA, EP, MPT, and PT was measured by changes in absorbance at 214 nm by HPLC. The purified Est5S enzyme (50 lL) was added to an assay mixture containing 700 lL 200 mM phosphate-buffered saline (PBS, pH 7.0) and 250 lL CS, CP, CM, DZ, DF, FA, EP, MPT, and PT (200 mg L1). To use the negative control, this enzyme was unactivated at 120 °C for 20 min. All assays were performed in triplicate. 2.7. Thin layer chromatography and high performance liquid chromatography analysis Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) were performed in order to determine the degradation of nine organophosphates (OP) using the purified enzyme source.

17

Five milliliters of the culture was collected and centrifuged. Four milliliters of supernatant was extracted with ethylacetate (8 ml  3), organic layer was dried with Na2SO4 and concentrated under vacuum. Extracted sample was spotted on pre-coated silica gel aluminum plate (0.25 mm, Merck, Germany). TLC plate was developed with chloroform and hexane (4:1, v/v) solvent system for detection of CP. Where ethylacetate, isopropanol, and NH4OH (5:3:2, v/v) system was used for detection of TCP. Finally, the target compounds were detected with UV wavelength (254 nm). In TLC, TCP was confirmed as the spot with approximately 0.66 of Rf value, and more polar CP was determined as the spot with about 0.57 of Rf value. One milliliter of supernatant was mixed with 1 mL of methanol for preparation of HPLC sample. Above mixed solution was filtered through a 0.45 lm Minipore PVDF filter (Schleicher & Schuell GmbH, Dassel, Germany) for HPLC analysis. Injection volume was the 10 lL of filtered sample. The analysis of OP and TCP was carried out on HPLC (HPLC, Perkin-Elmer 200 series, Perkin-Elmer Corp., CT, USA) using C18 column (250  4.6 mm, 5 lm, Phenomenx, CA, USA). The mixture of 0.5% acetic acid and methanol (1:4 v/v) was eluted with a flow rate of 1 mL min1 at 30 °C. Target compounds, OP and TCP, were measured on 214 nm of UV detector (Perkin-Elmer UV 200 series, Perkin-Elmer Corp., CT, USA. In HPLC analysis, TCP was detected approximately at 5.5 min and OP were detected between 4.6 and 14.0 min depending on feature of each functionality. The calibration curves for nine OP were made from the serial dilutions of the samples dissolved in 100% methanol. The linear range and the equation of linear regression were obtained sequentially at 0, 10, 25, 50, 75, and 100 lg mL1. Above serial standard solutions were filtered through a 0.45 lm Minipore PVDF filter (Schleicher & Schuell) for HPLC analysis. Injection volume was the 10 lL of serial standard solutions. Mean areas (n = 3) generated from the standard solution were plotted against concentration to establish the calibration equation. The concentrations of the nine OP were determined on the basis of the peak areas in the chromatogram as follows: CS, y = 36.4x185.02, R2 = 1.0; CP, y = 236.21x + 891.84, R2 = 0.99; CM, y = 290.58x + 254.78, R2 = 0.98; DZ, y = 294.4x769.54, R2 = 1.0; DF, y = 236.21x584.31, R2 = 0.99; FA, y = 333.14x + 283.07, R2 = 0.97; EP, y = 31.69x116.34, R2 = 0.99; MPT, y = 275.02x + 577.51, R2 = 0.99; PT, y = 356.03x + 940.09, R2 = 0.97; and TCP, y = 331.85x + 113.5, R2 = 0.98. 2.8. Analysis statistics Data were analyzed statistically by ANOVA and Duncan’s multiple range tests using SPSS software program (SPSS Inc., Chicago, IL, USA). A p value <0.05 was considered significant.

3. Results 3.1. Cloning of est5S gene The double stranded DNA product from PCR was cloned in pGEM-T easy vector, and three recombinant clones containing the target DNA were picked up randomly for sequencing. The correct DNA sequence was ligated into expression vector pPICZaA. The recombinant plasmid was designated as pPICZaA/est5S. The recombinant plasmids were transformed into P. pastoris GS115. Upon transformation to P. pastoris with PmeI linearized pPICZaA/ est5S, approximately 150 clones were generated from the transformation. PCR amplification of genomic DNA isolated from recombinant P. pastoris showed that only one band appeared at the position of about 1100 bp. Sequencing with 50 AOX primers revealed that est5S gene was in frame with the secretary signal sequence.

18

D.M. Kambiranda et al. / Pesticide Biochemistry and Physiology 94 (2009) 15–20

Esterase activity was not detected from the culture supernatant of P. pastoris GS115, nor was it found in the est5S strains without methanol induction (data not shown).

Table 1 The activity of digestion of q-nitrophenol-b-butyric acid (q-NPB) by Est5S enzyme in kinetic parameters, specific activity, and optimum pH and temperature for the hydrolysis. Kinetic parametera

3.2. Heterologous expression of est5S The est5S was expressed in P. pastoris to examine whether the cloned gene encoded a functional enzyme. A total of eight est5S transformants of P. pastoris were selected and assayed for the secreted enzyme activity. Initial esterase enzyme activity was observed using culture supernatant. Culture supernatants of respective clones showing halo zones indicated hydrolysis on tributyrin plate. After 12 h of induction, all recombinant strains reached early stationary phase and the culture supernatant of all transformants showed significant esterase activity. The enzyme activities increased further until 24 h after the induction. At this time, the enzyme activity was estimated to be 30–50 IU mL1 of media. The transformant exhibiting the highest esterase activity (76 IU mL1) and the secretion yield of approximately 4.0 g L1 was selected, cultivated until 48 h after the induction, and used for the enzyme purification (data not shown) and the highest performance showing clone was selected for further work. 3.3. Purification and characterization of Est5S The solubilized recombinant Est5S with His-tag was applied on a HisTrap kit (Qiagen). Protein was eluted with 100 mM imidazole with 0.1% Triton X-100. The purified protein sample was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). A SDS–PAGE analysis showed only one band at the position corresponding to a molecular mass of 40 kDa when using Coomassie blue staining (Fig. 1). The Km and Vmax values were observed to be 1.34 mM and 22 lmol min1, respectively (Table 1). The effect of temperature on the activity and the stability of the enzyme was studied. The enzyme was found to be stable at high temperatures. The optimum temperature for activity of the enzyme was 40 °C (Table 1). More than 40% of the activity was retained

1

2

3

4

116.0 66.2

Est5S

Km (mM)

Vmax (lmol min1)

1.34

22

Specific activity (U/mg)b /pHc/Temperature (°C)d

354/7.0/40

a

The parameter was determined at 40 °C and pH 7.0. Specific activity was expressed as micromoles of q-NPB hydrolyzed min1 mg1 (protein). c Optimum pH was assayed at different pH values and 40 °C for 30 min. d Optimum temperature assayed at different temperature and pH 7.0 for 30 min. b

after heating the enzyme at 80 °C (data not shown). The optimal pH for activity was pH 7.0 (Table 1). The effect of pH on enzyme stability was studied by incubating the enzyme in a solution of 0.05 g mL1 of q-NPB butyrate with different buffers. It has been observed that the enzyme drastically lost more than 50% of the activity when incubated at a pH less than 5. However it was quite stable at higher pH ranges. About 40% of the activity was observed after treatment with pH range of below pH 6 and over pH 11 (data not shown). 3.4. Organophosphates degradation analysis The purified esterase enzyme was tested for degradation of CP using TLC and HPLC (Fig. 2). TLC analysis revealed that CP could be degraded by the isolated Est5 enzyme compared with the standard. The Rf value: approximately 0.57, in TLC and retention times of approximately 14 min in HPLC was observed for standard CP (Fig. 2A and C). TCP, the breakdown product of CP, could only be detected in the samples of Est5S (Fig. 2B and C) with Rf value of 0.66. Preliminary HPLC analysis was done with the E. coli and P. pastoris purified Est5S enzyme. This resulted in degradation of CP up to 100 mg L1 in Est5S samples from P. pastoris purified enzyme (76 IU mL1) but only 50 mg L1 of CP could be degraded when E. coli purified enzyme (27 IU mL1) was used (Fig. 3). The accumulation of TCP in samples of Est5S could be visualized in HPLC analysis at 214 nm when compared with the standard graph. The degradation of CS, CP, CM, DZ, DF, EP, FM, MPT, and PT was observed up to 12 h when it was decomposed to 68, 60, 80, 40, 45, 60, 95, and 100%, respectively (Table 2).

45.0

4. Discussion

35.0

Esterase gene (est5S) was isolated from a rumen metagenomic library for demonstrating degradation of chlorpyrifos. Eight other OP were also tested for hydrolytic activity by Est5S since several investigators reported previously that the majority of OP are esters of phosphoric acid and can therefore be hydrolyzed and detoxified by carboxylesterase and phosphotriesterase [28–30]. Cloning of esterase gene (est5S) in pGEM-T easy vector and transformation of this gene using pPICaA into Pichia expression system were done. Enzyme activity was found to be optimum at 48 h post-induction time with methanol. Esterase and organophosphate hydrolytic activity was detected by initial screening with purified enzyme. A single band of 40 kDa was observed after Coomassie blue staining in SDS–PAGE (Fig. 1). The activity of the enzyme was found to be optimum at 40 °C and pH 7.0. Expression of Est5S enzyme in yeast could ease the purification of the enzyme, high yield and low cost in production overcoming the antibacterial activity with TCP when expressed in bacteria. CP degradation was tested with the purified enzyme using TLC and HPLC. CP could not be detected in samples treated with the

25.0 18.4 14.4 (kDa)

Fig. 1. Electrophoretic analysis of the purified esterase. Separation was performed on a 12.5% (W/V) SDS–polyacrylamide gel. Lane 1: standard marker, lane 2: crude extract from E. coli BL21 (DE3) containing pET-28(+)/Est5S, lane 3: crude extract from IPTG-induced E. coli BL21 (DE3) containing pET-28(+)/Est5S, lane 4: purified esterase from Hi-Trap kit (Amersham). The gel was stained with 0.025% Coomassie blue R-250. Molecular weight markers used were b-galactosidase (116,000), bovine serum albumin (66,200), ovalbumin (45,000), lactate dehydrogenase (35,000), restriction endonuclease Bsp981 (14,400), b-lactoglobulin (18,400), and lysozyme (14,400).

D.M. Kambiranda et al. / Pesticide Biochemistry and Physiology 94 (2009) 15–20

19

Residual CP concentration (100 mg L -1 )

Fig. 2. TLC (A and B) and HPLC (C) profile of chlorpyrifos (CP) and 3,5,6-trichloro-2-pyridinol (TCP) by the purified Est5S enzyme of P. pastoris incubated at temperature 40 °C at pH 7.0 for 12 h. Lane A1, B1, and C1, CP, and TCP standard, respectively; lane A2, B2, and C2, Est5S enzyme; lane A3, B3, and C3, inactivation Est5 enzyme (negative control). The TLC plates were developed with chloroform and hexane (4:1, v/v) solvent system for detection of CP and ethylacetate, isopropanol, and NH4OH (5:3:2, v/v) system for detection of TCP, respectively. The HPLC was eluted with 0.5% acetic acid and methanol (1:4 v/v). The arrowheads indicate the TCP (Rf value: approximately 0.66, retention time: approximately 5.5 min) and CP (Rf value: approximately 0.57, retention time: approximately 14 min) band and peck, respectively.

110 100 90 80 70 60 50 40 30 20 10 0 0

12

24

36

48

60

72

Incubation time (h) Fig. 3. Residual concentration of chlorpyrifos (CP) by the purified Est5S enzymes from E. coli (27 IU mL1) and P. pastoris (70 IU mL1) incubated at temperature 40 °C at pH 7.0 for 0–72 h. A black circle indicates the purified Est5S enzyme of E. coli (27 IU mL1) and white circle indicates the purified Est5S enzyme of P. pastoris (70 IU mL1). Values indicate means of three replications (n = 3). A p value <0.05 was considered significant.

Table 2 Degraded rate of the different organophosphate (OP) pesticides by the purified Est5S enzyme of P. pastoris incubated at temperature 40 °C at pH 7.0 for 1, 4, 6, and 12 h. OP pesticides

Degraded rate (%)a Incubation time (h) 1

4

6

12

Cadusafos (CS) Chlorpyrifos (CP) Coumaphos (CM) Diazinon (DZ) Dyfonate (DF) Ethoprophos (EP) Fenamiphos (FA) Methylparathion (MPT) Parathion (PT)

4c 40bc 7hi 12abc 2j 6bcd 11hi 35cde 38ab

15ab 65hi 18k 25b 9gh 11de 28j 63e 62cde

40de 82g 30f 49c 20h 28ac 45g 85ac 91ab

68ab 100f 60ef 80ab 40i 45cd 60gh 95d 100a

a Values indicate means of three replications (n = 3). A p value <0.05 was considered significant.

Est5S enzyme. TCP, the primary degradation product of CP, could be detected in the CP treated samples (Fig. 2). Degradation of TCP was previously reported by Feng et al. [31] and Yang et al. [32], but Est5S was not able to break down TCP since it was detected in CP treated samples. Nine OP were tested for degradation activity by using Est5S purified enzyme. Highest degradation was observed for three OP, PT, MPT, and CP after 12 h of incubation (Table 2). Lowest degradation was observed with DF and EP (Table 2). These compounds have the diethyl (or dimethyl) phosphorothionate side chain which explains their degradation. Esterase enzymes are remarkable for its extremely broad substrate profile. It can catalyze the hydrolysis of many neurotoxic agents and OP insecticides [28]. Hydrolysis of OP compounds by carboxylesterase/phosphotriesterase reduces mammalian toxicity by several orders of magnitude. Different species of Enterobacter have been reported to degrade phosphonate [33], glyphosate [34], pentaerythritol tetranitrate [35], and trinitrotoluene [36]. However, this is the first report of OP degradation by an esterase enzyme from unculturable bacteria of cow rumen. Because of the low frequency of finding desirable genes from a metagenomic library of diverse microbial genomes, cloning efficiency is an important factor in constructing large clone library, which should include most of the microbial DNA [6]. Some species of bacteria have been isolated that can utilize OP as a source of carbon or phosphorus [37] from the hydrolysis products [38]. However the enzyme expressed in this study had very strong OP hydrolytic activity (OPH) and hydrolyzed 100 mg/L concentrations of CP within 12 h compared to E. coli harboring the est5S gene (Fig. 3). Same esterase enzyme was collected from est5S harboring P. pastoria and E. coli, but enzyme concentration was higher in P. pastoria than that of E. coli, so degradation was higher by enzyme collected from P. pastoris. This could overcome the factor that whole bacterial cells in the presence of other carbon sources stopped degrading chlorpyrifos. When these carbon sources were depleted, they then degraded CP as a source of carbon, signifying the environmental adaptation of this bacterium. In conclusion, Est5S enzyme isolated from rumen metagenomic library was active against a range of OP compounds, the isolated Est5S possesses a great potential to provide a versatile enzyme system that may be used for the remediation of highly toxic OP nerve agents and also further could be expressed in plants for demonstrating OP hydrolytic activity. The rumen ecosystem is comprised of diverse population of bacteria, fungi, and protozoa that produce

20

D.M. Kambiranda et al. / Pesticide Biochemistry and Physiology 94 (2009) 15–20

a vast range of enzymes which may help in detoxifying toxic compounds in the rumen. Further screening of rumen metagenomes may also reveal novel enzymes that could be useful in bioremediation. Acknowledgments This work was supported by Grant No. R01-2008-000-20220-0 from the Basic Research Program of KOSEF, Republic of Korea. Devaiah M Kambiranda was supported by a postdoctoral fellowship and Shah Md. Asraful Islam by scholarships from the BK21 Program, Ministry of Education & Human Resources Development, Republic of Korea. References [1] K.B. Singh, A. Walker, Microbial degradation of organophosphorus compounds microbial degradation of organophosphorus compounds, FEMS Microbiol. Rev. 30 (2006) 428–471. [2] W.J. Lee, A. Blair, J.A. Hoppin, J.H. Lubin, J.A. Rusiecki, Cancer incidence among pesticide applicators exposed to chlorpyrifos in the agriculture health study, J. Natl. Cancer Inst. 96 (2004) 1781–1789. [3] T. Vallaeys, R.R. Fulthorpe, A.M. Wright, G. Soulas, The metabolic pathway of 2,4-dichlorophenoxycetic acid degradation involves different families of tfdA and tfdB genes according to PCR-RFLP analysis, FEMS Microbiol. Ecol. 20 (1996) 163–172. [4] P.D. Schloss, J. Handelsman, Biotechnological prospects from metagenomics, Curr. Opin. Biotechnol. 14 (2003) 303–310. [5] W.R. Streit, R. Daniel, K.E. Jaeger, Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms, Curr. Opin. Biotechnol. 15 (2004) 285–290. [6] S.J. Cho, H.D. Yun, Cloning of a-amylase gene from unculturable bacterium using cow rumen metagenome, J. Life Sci. 15 (2005) 1013–1021. [7] L.B. Selinger, C.W. Forsberg, K.J. Cheng, The rumen: a unique source of enzymes for enhancing live stock production, Anaerobe 2 (1996) 263–284. [8] R. Verger, Interfacial activation of lipases: facts and artifacts, Trends Biotechnol. 15 (1997) 32–38. [9] U.T. Bornscheuer, Methods to increase enantio selectivity of lipases and esterases, Curr. Opin. Biotechnol. 13 (2002) 543–547. [10] D.P. Weston, E.L. Amweg, Whole-sediment toxicity identification evaluation tools for pyrethroid insecticides: II. esterase addition, Environ. Toxicol. Chem. 26 (2007) 2397–2404. [11] S. Whyard, V.K. Walker, Characterization of malathion carboxylesterase in the sheep blowfly Lucilia cuprina, Pestic. Biochem. Physiol. 50 (1994) 198–206. [12] N.N. Sethunathan, T. Yoshida, A Flavobacterium that degrades diazinon and parathion, Can. J. Microbiol. 19 (1973) 873–875. [13] C.M. Serdar, D.T. Gibson, D.M. Munnecke, J.H. Lancaster, Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta, Appl. Environ. Microbiol. 44 (1982) 246–249. [14] T.D. Horne, R.L. Sutherland, R.J. Harcourt, R.J. Russell, J.G. Oakeshott, Identification of an opd (organophosphate degradation) gene in an Agrobacterium isolate, Appl. Environ. Microbiol. 68 (2002) 3371–3376. [15] W.W. Mulbry, P.L.D. Valle, J.S. Karns, Biodegradation of the organophosphate insecticide coumaphos in highly contaminated soils and in liquid wastes, Pestic. Sci. 48 (1996) 149–155. [16] W.W. Mulbry, Characterization of a novel organophosphorus hydrolase from Nocardioides simplex NRRL B-24074, Microbiol. Res. 154 (2000) 285–288.

[17] S. Dayananda, E.R. Raju, P.V.E. Paul, M. Merrick, Overexpression of parathion hydrolase in E. coli stimulates the synthesis of outer membrane porin OmpF, Pestic. Biochem. Physiol. 86 (2006) 146–150. [18] J. Zang, W. Lan, C. Qia, H. Jiang, Bioremediation of organophosphorus pesticides by surface expressed carboxylesterase from mosquito on E. coli, Biotechnol. Prog. 20 (2004) 1567–1571. [19] C.F. Wu, J.J. Valdes, G. Rao, W.E. Bentley, Enhancement of organophosphorus hydrolase yield in Escherichia coli using multiple gene fusions, Biotechnol. Bioeng. 75 (2001) 100–103. [20] D.G. Kang, J.Y.H. Kim, H.J. Cha, Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin, Biotechnol. Lett. 24 (2002) 879–883. [21] K.D. Racke, D.A. Laskowski, M.R. Schultz, Resistance of chlorpyrifos to enhanced biodegradation in soil, J. Agric. Food Chem. 38 (1990) 1430–1436. [22] X.Y. Chu, N.F. Wu, M.J. Deng, J. Tian, B. Yao, Y.L. Fan, Expression of organophosphorus hydrolase OPHC2 in Pichia pastoris: purification and characterization, Protein Expr. Purif. 49 (2006) 9–14. [23] J. Sambrook, D.W. Russel, Molecular Cloning: A Laboratory Manual, in: D. Seghers, L. Wittebolle, E.M. Top (Eds.), third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA, 2001. [24] K.M. Cho, H.D. Yun, October 2006, Esterase gene (est 5S) encoding a new group esterase enzyme identified from cow rumen metagenome and amino acid sequence encoded there form, Korean patent number 10-0817321-0000. [25] P. Guo, L. Zhang, Z. Qi, R. Chen, G. Jing, Expression in Escherichia coli, purification and characterization of Thermoanaerobacter tengcongensis ribosome recycling factor, J. Biochem. 138 (2005) 89–94. [26] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [27] E. Alvarez-Macarie, V. Augier-Magro, J. Baratti, Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis, Biosci. Biotechnol. Biochem. 63 (1999) 1865–1870. [28] D.P. Dumas, S.R. Caldwell, J.R. Wild, E.M. Raushel, Purification and properties of the phosphotriesterase from Pseudomonas diminuta, J. Biol. Chem. 264 (1989) 19659–19665. [29] D.P. Dumas, J.R. Wild, F.M. Raushel, Expression of pseudomonas phosphotriesterase activity in the fall armyworm confers resistance to insecticides, Experientia 46 (1990) 729–731. [30] J. Huang, C. Qiao, X. Li, J. Xing, Cloning and fusion expression of detoxifying gene in E. coli, Acta Genetica Sinica 28 (2001) 587–588. [31] Y. Feng, K.D. Racke, J.M. Bollag, Isolation and characterization of a chlorinatedpyridinol-degrading bacterium, Appl. Environ. Microbiol. 63 (1997) 4096– 4098. [32] L. Yang, Y.H. Zhao, B.X. Zhang, C.H. Yang, X. Zhang, Isolation and characterization of a chlorpyrifos and 3,5,6-trichloro-2-pyridinol degrading bacterium, FEMS Microbiol. Lett. 251 (2005) 67–73. [33] K.S. Lee, W.W. Metcalf, B.L. Wanner, Evidence for two phosphonate degradative pathways in Enterobacter aerogenes, J. Bacteriol. 174 (1992) 2501–2510. [34] R.E. Dick, J.P. Quinn, Glyphosate-degrading isolates from environmental samples: occurrence and pathways of degradation, Appl. Microbiol. Biotechnol. 43 (1995) 545–550. [35] P.R. Binks, C.E. French, S. Nicklin, N.C. Bruce, Degradation of pentaerythritol tetranitrate reductase by Enterobacter cloacae PB2, Appl. Environ. Microbiol. 62 (1996) 1214–1219. [36] C.E. French, S. Nicklin, N.C. Bruce, Aerobic degradation of 2,4,6-trinitrotoluene by Enterobacter cloacae PB2 and by pentaerythritol tetranitrate reductase, Appl. Environ. Microbiol. 64 (1988) 2864–2868. [37] A. Rosenberg, M. Alexander, Microbial cleavage of various organophosphorus insecticides, Appl. Environ. Microbiol. 37 (1979) 886–891. [38] D.M. Munnecke, D.P.M. Hsieh, Pathways of microbial metabolism of parathion, Appl. Environ. Microbiol. 31 (1976) 63–69.