Purification and characterization of phosphotriesterases from ...

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Purification and characterization of phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11 Subhas Das and Dileep Kumar Singh

Abstract: A microbial biodegradation of monocrotophos was studied in the present investigation. The monocrotophosdegrading enzyme was purified and characterized from two soil bacterial strains. The cells were disrupted and the membrane-bound fractions were studied for purification and characterization. Solubilization of the membrane-bound fractions released nearly 80% of the bound protein. Phase separation further enriched the enzyme fraction 34–41 times. The enzyme phosphotriesterase (PTE) from both the strains was purified to more than 1000-fold with 13%–16% yield. Purified PTE from Clavibacter michiganense subsp. insidiosum SBL11 is a monomeric enzyme with a molecular mass of 43.5 kDa (pI of 7.5), while PTE from Pseudomonas aeruginosa F10B is a heterodimeric enzyme with a molecular mass of 43 and 41 kDa (pI of 7.9 and 7.35). Both purified enzymes are stable enzymes with peak activity at pH 9.0. The enzyme from strain F10B was more thermostable (half-life = 7.3 h) than that from SBL11 (half-life = 6.4 h at 50 °C), while both showed the same temperature optimum of 37 °C. Inhibitors like dithiothreitol and EDTA inhibited the purified enzyme, while p-chloromercuribenzoic acid and indoleacetic acid had a very little effect. Key words: biodegradation, monocrotophos, phosphotriesterase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11. Résumé : Une biodégradation microbienne du monocrotophos fut étudiée dans l’enquête présente. L’enzyme dégradant le monocrotophos fut purifiée et caractérisée à partir de deux souches bactériennes du sol. Les cellules furent désintégrées et les infractions associées aux membranes furent étudiées pour la purification et la caractérisation. La solubilisation des fractions associées aux membranes ont libéré presque 80 % des protéines liées. La séparation de phase a par la suite enrichi d’un facteur de 34 à 41 la fraction des enzymes. L’enzyme phosphotriestérase (PTE) des deux souches fut purifiée jusqu’à plus de 1000 fois avec un rendement de 13 % à 16 %. La PTE purifiée de Clavibacter michiganense subsp. insidiosum SBL11 est une enzyme monomérique d’une masse moléculaire de 43,5 kDa (pI de 7,5) alors que la PTE de Pseudomonas aeruginosa F10B est une enzyme hétérodimérique d’une masse moléculaire de 43 et 41 kDa (pI de 7,9 et 7,35). Les deux enzymes stables purifiées avaient une activité maximale à un pH de 9,0. L’enzyme de la souche F10B était davantage thermostable (T1/2 = 7,3 h) que celle de la souche SBL11 (T1/2 = 6,4 h à 50 °C) alors que les deux démontraient la même température optimale de 37 °C. Des inhibiteurs tels que le dithiothréitol et l’EDTA ont inhibé l’enzyme purifiée alors que le p-chloromercuribenzoic acid et le indoleacetic acid n’ont eu que peu d’effet. Mots clés : biodégradation, monocrotophos, phosphotriestérase, Pseudomonas aeruginosa F10B, Clavibacter michiganense subsp. insidiosum SBL11. [Traduit par la Rédaction]

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Introduction Organophosphorus (OP) triesters and related OP diesters are extremely toxic because of their ability to specifically inactivate the enzyme acetylcholinesterase. Hence, it is imporReceived 15 February 2005. Revision received 9 September 2005. Accepted 16 September 2005. Published on the NRC Research Press Web site at http://cjm.nrc.ca on 11 February 2006. S. Das1 and D.K. Singh.2 Department of Zoology, University of Delhi, Delhi 110 007, India. 1

Present address: Department of Pharmacology, Medical School, 6-120 Jackson Hall, 321 Church St. SE, Minneapolis, MN 55455, USA. 2 Corresponding author (e-mail: [email protected]). Can. J. Microbiol. 52: 157–168 (2006)

tant to know the mode of action of the enzymes involved in detoxification of these chemicals. In general, bacterial isolates have been identified that have the capacity to hydrolyze and detoxify the wide range of OPs, including the most toxic of the chemical warfare agents. OP compounds, such as monocrotophos (dimethyl (E)-1methyl-2-(methylcarbamoyl)vinyl phosphate or MCP), have been used extensively in India as an insecticide to protect the standing crops from pests. This insecticide is one of the most toxic insecticides and acaricide with contact, systemic, and residual modes of action (Beynon et al. 1973). Owing to its extensive use, the degree of exposure to farmers and related personnel is increasing every day. At the same time, there are concerns about residues of OP insecticides in the environment and foodstuff because of the broad-spectrum

doi:10.1139/W05-113

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vertebrate and invertebrate toxicities of these molecules. Much effort has therefore been put into isolating phosphotriesterases (PTEs) that are capable of detoxifying these pesticides. Microorganisms containing hydrolytic enzymes that detoxify these OP insecticides may provide a practical solution to toxic waste disposal. Two types of bacterial PTEs have been studied extensively: PTEs from Flavobacterium sp. ATCC 27551 (Brown 1980) and from Pseudomonas diminuta (Dumas et al. 1989, 1990). Both PTEs are metalloenzymes and hydrolyze a wide variety of phosphotriesters, including MCP. In both the cases, PTE, an OP hydrolase, is encoded by the opd (for OP degradation) gene located on their respective plasmids. The gene sequence from these two sources has more than 99% homology. PTEs are the most implicated enzyme in the detoxification of OP compounds. PTEs are therefore given various names on the basis of the pesticides they hydrolyze. Thus, PTEs hydrolyzing paraoxon and DFP (di-isopropyl fluorophosphate) are called paraoxonase and DFPase, respectively. PTE activities are distributed extensively among a wide variety of organisms from microorganisms to humans. Marked differences in activities exist among species and among different tissues of the same species. These enzymes show stereoselectivity towards their substrate owing to faster hydrolysis of one isomer than of another (Lewis et al. 1988). Since PTEs are metalloenzymes, the activities of most PTEs are largely dependent on the presence within the reaction medium of inorganic cations that may act as either activators, inhibitors, or as cofactors. The most widely studied PTE is from P. diminuta. The enzyme from this source has been studied in detail and has been cloned into various expression vectors. The active site (Omburo et al. 1992) and its three-dimensional structure, hydrolysis mechanism (Donarski et al. 1989), and X-ray crystallography (Benning et al. 1994) have been well investigated. Despite many studies conducted to date, the biological functions of PTEs remain to be established. No physiological substrates have been identified for these enzymes. Studies have also been done of the possible biotechnological application of purified PTEs in the elimination of OP compound residues from water, for the detection of the OP compounds in different media, and even in the destruction of chemical weapon stockpiles (Caldwell and Raushel 1991a, 1991b) At the same time, PTEs from other sources have been neglected that may prove to be better variants than that from the two strains explored. To date, MCP-degrading PTE has not been investigated in detail. In the last published work, we showed that two strains isolated from soil could utilize MCP as a sole source of phosphorus (Subhas and Singh 2003). In the continuation of the work, we have undertaken the purification and characterization of MCP-degrading PTEs from two different soil isolates.

Materials and methods Microorganisms and culture conditions The microorganisms degrading MCP were isolated from soil with a repeated history of MCP spray over a long period of time. The screening and isolation of the isolates have been discussed before and the cultures were grown in basal

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medium as discussed before (Subhas and Singh 2003). The cultures were incubated at 37 °C. Purification of membrane-bound PTEs Enzyme assay The enzyme assay procedure has been discussed elsewhere (Subhas and Singh 2003). Briefly, to 2.88 mL of glycine– NaOH buffer, 20 µL of substrate (5.5 µg Tris – p-nitrophenyl phosphate (TpNPP)/µL in acetone) was added and the reaction mixture was preincubated at 37 °C for 10 min. After the preincubation, 100 µL of the crude enzyme was added to the reaction mixture and incubated in a water bath for 1 h at 37 °C. The final product (p-nitrophenol) was measured spectrophotometrically at 410 nm. All of the experiments were performed in triplicate and control experiments lacking either the substrate or the enzyme were used. The amount of protein was estimated by the method of Lowry et al. (1951) with bovine serum albumin as the standard. Enzyme activity was defined as the amount of enzyme required to release 1 µmol of p-nitrophenol per minute per millilitre under the standard assay conditions. Cell lysis by sonication The harvested cells were suspended in 50 mmol Tris– HCl/L (pH 7.0). This diluted solution was then sonicated at 80% efficiency in a sonicator (SoniPrep 150). After sonication, the solution was centrifuged (10 000g for 10 min) and the extracellular, intracellular, and membrane-bound fractions were tested for PTE activity. After sonication, the membranebound fractions were observed to contain maximum PTE activity. In all further experiments, the membrane-bound fractions were used as a source of PTE unless otherwise stated. Cell solubilization The crude enzyme source was lyophilized and 10 mg of this was mixed with 10.0 mL of the solubilization buffer (10 mmol Tris–HCl/L (pH 7.2), 1 mmol/L EDTA, 1 mol NaCl/L). The reaction mixture contained 1.0 mL of the solubilization buffer with 1.0 mL of the detergent (2% SDS, 2% Triton X-10, and a combination of 4% SDS and 4% Triton X-100). The solutions were left at room temperature for 1 h. After the incubation time, the PTE activity was assessed. Phase separation by Triton X-114 Phase separation was done as described in Bordier (1981). In both phases, protein estimation and enzyme activity were estimated. Removal of Triton X-114 For detergent removal, Bio-Beads® SM hydrophobic and polar interaction adsorbents (Bio-Rad, Hercules, California) were used. In a 10 mL glass vial, 0.2 g of the beads was taken for 1 mL of the sample. The solution was mixed on the stirrer for 2 h at room temperature. The sample was recovered by decanting or by removing supernatant with a pipette. The beads were regenerated by washing in four bed volumes of methanol followed by rinsing with deionized water. © 2006 NRC Canada

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Ultrafiltration The washed enzymes were then subjected to ultrafiltration. The Vivaspin 2 (Vivascience, Lincoln, UK) concentrators (10 kDa molecular mass cut-off) were used. The samples were filled into the concentrators and were centrifuged in a fixed-angle rotor centrifuge (5000g) for 8 min. The filtrate was collected in the lower tube, while the concentrate was retained over the membrane in a pelleted form. Adsorption chromatography The PTE enzyme from the concentrate of the ultrafiltration was subjected to adsorption chromatography. Binding of PTE Three matrices (phenyl-Sepharose, octyl-Sepharose, and butyl-Sepharose) were selected for binding studies. The binding capacity was measured by incubating a known quantity of enzyme with matrices overnight. The enzyme activity and protein concentration were estimated. On the basis of better binding capacity, phenyl-Sepharose was selected for hydrophobic interaction chromatography. Pretreated phenylSepharose was packed onto a glass column (18 cm × 2.5 cm) and equilibrated with three bed volumes of the binding buffer (50 mmol Tris–HCl/L (pH 7.2), 1 mol ammonium sulphate/L). Protein (0.5 mg) in the binding buffer containing 1.0 mol ammonium sulphate/L was loaded onto the column. Elution of PTE Elution was first attempted by reducing the ionic strength of the system by using a decreasing salt gradient. The ionic strength of the system was gradually decreased from 1.0 to 0 mol ammonium sulfate/L, followed by buffer without ammonium sulfate. In the last step, elution was carried out using 10% (v/v) buffered acetone. A flow rate of 1.0 mL/min was maintained throughout the elution. Fractions were collected using a fraction collector (Frac 100; Pharmacia-LKB, Denmark). Protein in each fraction was monitored with a UV detector (Optical unit UV-1; Pharmacia-LKB, Denmark). Background absorbance from acetone was corrected with a blank containing the same concentration of acetone. The fractions were tested for PTE activity and protein concentration. The fractions positive for PTE activity were pooled and concentrated with a Speed-Vac® and subjected to gel filtration. The purity of the samples was checked by nondenaturing PAGE stained with Coomassie blue (C.I. 42660) and silver nitrate (Sambrook et al. 1989). Size exclusion chromatography A column (80 cm × 1.0 cm) was packed with swollen Sephadex G-100. The column was preequilibrated with 0.125 mol ammonium bicarbonate/L in 50 mmol Tris–HCl/L (pH 7.2). The concentrated sample from hydrophobic interaction chromatography was loaded onto this column. The flow rate was maintained at 5 mL/min. A fraction of 4.0 mL was collected using a fraction collector. Protein in each fraction was monitored with a UV detector. Determination of molecular mass The molecular mass of the two PTEs was determined by nondenaturing PAGE and SDS–PAGE (Sambrook et al. 1989). PAGE was carried out in a Hicon (India) gel apparatus. The

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gels were stained in Coomassie brilliant blue R-250 and with silver staining (Sambrook et al. 1989). Isoelectric focusing of purified PTEs The purified PTEs were used as a sample. The system used was Phastsystem™ and the gel used was PhastGel® IEF 3-9 (Amersham Pharmacia Biotek AB, Uppsala, Sweden). These media were homogenous polyacrylamide gels (5% T and 3% C; T represents the total percentage concentration (m/v) of monomer (acrylamide plus crosslinker) in gel; C refers to the percentage of the total monomer represented by the crosslinker) containing Pharmalyte® carrier ampholytes. Pharmalyte® generated stable, linear pH gradients in the gels during the run. The sample protein (diluted in 50 mmol Tris–HCl/L (pH 7.2), 1.7 ng of protein/µL) was taken and loaded into the sample applicator. The isoelectric focusing was done according to the manual provided by the supplier. The calibration kit (Pharmacia, Sweden) was also run with the protein samples. Characterization of purified protein Effect of pH The effect of pH on PTE activity was determined by carrying out the reaction at different pHs ranging from pH 3.0 to 12.0 using various activity buffers. Different activity buffers (0.05 mol/L) used were citrate– phosphate (pH 2.0–5.0), phosphate (pH 6.0–8.0), Tris–HCl (pH 9.0), glycine–NaOH (pH 10.0), phosphate (pH 11.0), and hydroxide–chloride (pH 12.0). The activity was expressed as percent relative activity with respect to the maximum activity, which was considered as 100%. pH stability pH stability of the enzyme PTE was determined by incubating the enzyme in the buffers of varying pH (2.0–12.0) for 1 h at room temperature (25–30 °C) and then measuring the residual activity of the enzyme. Effect of temperature Effect of temperature on PTE activity was determined by incubation of the reaction mixture at different temperatures varying from 20 to 50 °C for 10 min at the pH optimum. The relative activity was expressed as percent relative activity in relation to the temperature optimum, which was considered as 100%. Thermostability of PTEs The enzymes were incubated at different temperatures (4– 50 °C) for different incubation periods up to 24 h. At regular intervals, the residual activity was determined at optimum temperature and pH. Effect of inhibitors and metal ions The effect of various inhibitors (p-chloromercuribenzoic acid, indoleacetic acid, N-ethylmaleimide, dithiothreitol (DTT), and EDTA) on enzyme activity was studied by incubating the enzyme with different concentrations (0–5.0 mmol/L) of the inhibitor for 1 h. The residual activity was measured at optimum temperature and pH. The effect of various metal ions (CoCl2, MgCl2, CuCl2, MnCl2, ZnCl2, and CaCl2) on PTE activity was studied by © 2006 NRC Canada

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Can. J. Microbiol. Vol. 52, 2006 Table 1. Purification steps of phosphotriesterase from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11.

P. aeruginosa F10B Membrane bound Sonication Solubilization Triton X-114 (lower phase) Washing with Bio-Beads® Ultrafiltration (retentate) Phenyl-Sepharose G-100

Protein (mg/mL)

Enzyme (U/mL)

Specific activity (U/mg protein)

Fold purification

3.9 8.6 1.9 11.7 4.0 1.0 0.2 0.1

1.8 15.8 43.8 404.2 142.4 76.8 59.8 53.4

0.5 1.8 23.1 34.5 35.6 76.8 299.0 534.0

1.0 3.6 46.2 69.0 71.2 153.6 598.0 1068.0

1.3 17.4 19.1 470.7 221.2 140.3 90.8 78.1

0.4 2.1 38.2 41.3 36.9 73.8 302.7 390.5

1.0 5.3 95.5 103.3 92.3 184.5 756.8 976.3

C. michiganense subsp. insidiosum SBL11 Membrane bound 3.3 Sonication 8.2 Solubilization 0.5 Triton X-114 (lower phase) 11.4 Washing with Bio-Beads® 6.0 Ultrafiltration (retentate) 1.9 Phenyl-Sepharose 0.3 G-100 0.2

incubation of the enzyme with varying concentrations of metal ions (0–10.0 mmol/L) at room temperature for 1 h and then determining the residual activity against the control. Substrate specificity The substrate specificity of the enzyme PTE was studied using substrates, such as TpNPP, paraoxon, parathion, methyl parathion, malathion, and malathion 50% EC. Both organisms were checked for their ability to utilize commercially available insecticides as substrates at pH 9.5 in glycine– NaOH buffer. All of the pesticides were prepared in ethanol. The concentration of each insecticide was taken that gave the best PTE production. The Km for all of the substrates was determined using varying concentrations of the substrates. The assay was carried out at optimum temperature and pH.

Results Purification Cell lysis and solubilization As evident from Table 1, there was an increase to 1.8 and 2.1 in the specific activity after the sonication of cells in Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11, respectively. The effect was obvious as the enzyme was exposed to the substrate and was more amenable than whole cells. After sonication, it was found that the major share of the enzyme was still attached to membrane-bound fractions. The specific activity for the SDS treatment (14.6 U/mg) and for Triton X-100 (13.3 U/mg) showed no significant increase in the specific activity of the crude enzyme (specific activity = 11.3 U/mg protein) after the detergent treatment. The two detergents in combination had a specific activity 1.8 times more than the separate application of detergents. The enzyme from P. aeruginosa

Yield (%)

100.0 35.2 1914.8 13.2

100.0 47.0 29.8 19.3 16.6

F10B showed that nearly 80% of the fraction was solubilized after treatment with Triton X-100. The specific activity in the soluble fraction increased to 12 times that in the sonication step (Table 1). The enzyme from C. michiganense subsp. insidiosum SBL11 showed that after detergent treatment, the major part of the enzyme (87%) was still bound to the cellular fraction. Only 12% of the fraction was solubilized. The specific activity in the soluble fraction increased to 18 times that in the sonication step (Table 1). Phase separation of the sonicated cells by Triton X-114 In the phase separation of the P. aeruginosa F10B enzyme from lysed cells, enzyme activity was greatly enhanced in the lower phase. The enzyme activity was found to be 404 U/mL as compared with 145 U/mL in the control. The lower phase enhanced the specific activity to 34.5 from 11.7 U/mg protein, 5.8-fold increase (Table 2). The enzyme from membrane-bound fractions of C. michiganense subsp. insidiosum SBL11 was also greatly concentrated in the detergent-rich lower phase, but some of the enzyme activity was also observed in the upper aqueous phase. The specific activity was enhanced to 41.3 from 13 U/mg showing a 3.3-fold increase in the specific activity (Table 2). Removal of detergent Triton X-114 by Bio-Beads® The removal of the detergent had a very drastic effect on the enzyme activity of both PTEs. The enzyme from P. aeruginosa F10B was reduced to just 142 from 404 U/mL with a net yield of just 35%, while the enzyme from C. michiganense subsp. insidiosum SBL11 was reduced to 221 from 470 U/mL showing a yield of just 47%. The total protein was also reduced to 52% (Table 2). Purification of enzyme Three matrices phenyl-Sepharose, butyl-Sepharose, and octylSepharose were selected to study the hydrophobic interac© 2006 NRC Canada

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161 Table 2. Phase separation of sonicated Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11 cells with Triton X-114.

P. aeruginosa F10B Sonicated cells Upper phase Lower phase After washing with Bio-Beads®

Protein (mg/mL)

Enzyme (U/mL)

Specific activity (U/mg protein)

Fold purification

24.4 0.2 11.7 4.0

145.0 — 404.2 142.4

6.0 — 34.5 35.6

— 5.8 1.0

166.3 3.4 470.7 221.2

12.7 13.0 41.3 36.9

— 1.0 3.3 —

C. michiganense subsp. insidiosum SBL11 Sonicated cells 13.0 Upper phase 0.3 Lower phase 11.4 After washing with Bio-Beads® 6.0

tion of the enzyme in question. The PTE sample was applied to the column of phenyl-Sepharose previously equilibrated with 50 mmol Tris–HCl/L (pH 7.2) and 1 mol ammonium sulfate/L. The reducing salt concentration was not efficient in eluting the bound protein. The enzyme was eluted with 10% buffered acetone. PTE activity was detected in fractions 45–54 (Fig. 1A) in P. aeruginosa F10B and in fractions 45–53 in C. michiganense subsp. insidiosum SBL11 (Fig. 1B). These fractions were pooled and dialyzed overnight against double-distilled water at 4 °C. With this purification step, a fold purification of 598 and 14.8% yield were obtained for P. aeruginosa F10B and a fold purification of 756 and 19.3% yield were obtained for C. michiganense subsp. insidiosum SBL11. The PTE fractions obtained from the phenyl-Sepharose column were concentrated with a SpeedVac® and then applied to a column of Sephadex G-100 preequilibrated with 50 mmol Tris–HCl/L (pH 7.2). PTE activity was detected in fractions 45–59 in P. aeruginosa F10B (Fig. 1C) and fractions 48–58 in C. michiganense subsp. insidiosum SBL11 (Fig. 1D). The active fractions were pooled separately, concentrated with a Speed-Vac®, and the purity checked with nondenaturing PAGE stained with Coomassie blue (C.I. 42660) as well as with silver nitrate and with SDS–PAGE. With this step, a final 1068-fold purity and 13.2% enzyme yield were obtained in the case of P. aeruginosa F10B and 976-fold purification and 16.6% yield in C. michiganense subsp. insidiosum SBL11 (Table 1). Molecular mass determination The molecular mass of both enzymes from two potential isolates was determined using SDS–PAGE. The purified enzymes appeared to be homogenous as judged by their migration as a single protein band in SDS–PAGE with an apparent molecular mass of 43.5 kDa in the case of C. michiganense subsp. insidiosum SBL11 (Fig. 2B). In the case of P. aeruginosa F10B (Fig. 2A), SDS–PAGE showed two distinct bands very close to each other even after gel filtration. But these two bands were seen as a single band on native PAGE gels. These two bands corresponded to 43 and 41 kDa on the SDS–PAGE gels. It is apparent from the above results that P. aeruginosa F10B PTE was a heterodimer protein.

Isoelectric focusing of the purified PTEs From the gel, it can be said that the pIs for the PTE from P. aeruginosa F10B were 7.9 and 7.35, while that for the PTE from C. michiganense subsp. insidiosum SBL11 was 7.5 (Fig. 2C). Characterization of PTEs Effect of pH on PTE activity and stability Pseudomonas aeruginosa F10B as well as C. michiganense subsp. insidiosum SBL11 indicated peak activity at pH 9.0, while it was pH 9.5 in the cell-free enzyme preparations. Both strains showed better enzyme activity in the pH range of 8.0–10.0. At pH 7.0, more than 65% relative activity was retained by both specific strains (Fig. 3A). Both purified PTEs showed a wide pH range for the stability of the enzyme. The maximum residual activity was seen in both cases at pH 7.0. The best range for maximum residual activity retained was between 82% and 100% and between pH 6.0 and pH 10.0 for both strains. Thus, in the pH range 6.0–10.0, the PTE was quite stable for both isolates (Fig. 3B). Effect of temperature on PTE activity The purified protein had a temperature optimum at 37 °C. In the extreme range of 20 and 50 °C, both the PTEs showed a loss of 70% in relative activity (Fig. 4A). The thermal stability of both purified enzymes showed relatively the same pattern. From the data, it appeared that the purified enzyme from P. aeruginosa F10B was more thermostable than the enzyme from C. michiganense subsp. insidiosum SBL11. The half-life was 7.3 h at 50 °C (Fig. 4B). In the case of C. michiganense subsp. insidiosum SBL11, the half-life was 23.5 h at 40 °C, while it was just 6.4 h at 50 °C (Fig. 4C). PTE inhibitors and activators Effect of inhibitors on PTE In the case of the purified enzyme of P. aeruginosa F10B, all of the inhibitors used were not very effective. A maximum of 25% residual activity was lost at an inhibitor concentration of 0.5 mmol/L. At the next inhibitor concentration (1.0 mmol/L), only 20% of the residual activity was left when DTT was used. The rest of the three inhibitors had a © 2006 NRC Canada

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Fig. 1. Hydrophobic interaction chromatography (phenyl-Sepharose) of phosphotriesterase from (A) Pseudomonas aeruginosa F10B and (B) Clavibacter michiganense subsp. insidiosum SBL11. The columns were preequilibrated with Tris–HCl buffer containing 1.0 mol ammonium sulfate/L (pH 7.2). Elution was carried out initially with the binding buffer followed successively by Tris–HCl buffer containing 0.5 mol ammonium sulfate/L (a), Tris–HCl buffer containing 0.05 mol ammonium sulfate/L (b), Tris–HCL buffer (c), and 10% acetone in Tris–HCl buffer (d). The time of change of elution solvent is indicated by the arrows. Elution profile of phosphotriesterase by Sephadex G-100: (C) P. aeruginosa F10B and (D) C. michiganense subsp. insidiosum SBL11. The column was preequilibrated with 50 mmol Tris–HCl/L (pH 7.2). The enzyme sample was applied to the column and elution was carried out with same buffer at a constant flow rate of 5.0 mL/min.

very mild inhibitory affect on the enzyme PTE (Fig. 5A). The enzyme PTE from C. michiganense subsp. insidiosum SBL11 showed a different pattern of enzyme inhibition in the presence of inhibitors compared with that of P. aeruginosa F10B. Except for the inhibitor DTT, there was a slight inhibition of 10% in the residual activity even at the high inhibitor concentration of 5.0 mmol/L. The sulphydryl inhibitor DTT was a quite strong inhibitor of the enzyme PTE, which showed a total loss of residual activity at an inhibitor concentration of 5.0 mmol/L. Even an inhibitor concentration as low as 0.1 mmol/L affected the residual

activity of the enzyme (Fig. 5B). Sulphydryl and cysteine inhibitors, such as DTT and N-ethyl maleimide, and the metal chelator EDTA had a profound effect on the purified enzyme PTE, with DTT having a prominent inhibition on both purified PTEs. Effect of divalent cations on PTE activity The purified enzyme from P. aeruginosa F10B showed that the metal ions Co2+ and Mg2+ activated the enzyme from 25% to 37%, while Zn2+ and Mn2+ severely inhibited the enzyme with 30% inhibition (Fig. 6A). In the case of © 2006 NRC Canada

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Fig. 2. SDS–PAGE of the different steps used in purification of (A) Pseudomonas aeruginosa F10B phosphotriesterase. Lanes: 1, molecular mass markers; 2, crude membrane-bound fraction; 3, partially purified fraction from phenyl-Sepharose; 4, phaseseparated fraction from Triton X-114; 5, purified fraction from Sephadex G-100; 6, solubilized membrane-bound fraction. The gel was stained with silver nitrate. SDS–PAGE of the different steps used in purification of (B) Clavibacter michiganense subsp. insidiosum SBL11 phosphotriesterase. Lanes: 1, purified fraction from Sephadex G-100; 2, phase-separated fraction from Triton X-114; 3, partially purified fraction from phenylSepharose; 4, solubilized membrane-bound fraction; 5, crude membrane-bound fraction; 6, molecular mass markers. The gel was stained with silver nitrate. (C) Isoelectric focusing of the purified phosphotriesterases from both of the potential isolates. Lanes: 1, pI markers; 2, phosphotriesterase from P. aeruginosa F10B; 3, phosphotriesterase from C. michiganense subsp. insidiosum SBL11; 4, pI markers. The gel used was PhastGel® IEF 3-9 and was stained with silver nitrate.

1

2

3

4

5

6

A 66 kDa 45 kDa 34.7 kDa 24 kDa 18.4 kDa 14.3 kDa 1

2

3

4

5

6

B

66 kDa

Table 3. Substrate specificity of Pseudomonas aeruginosa F10B membrane-bound phosphotriesterase on various organophosphorus insecticides and Tris – p-nitrophenol phosphate. Substrate Tris – p-nitrophenol phosphate Paraoxon Malathion Parathion Methyl parathion Malathion 50% EC

Enzyme activity (U·mL–1·min–1)a 137.8

Substrate concn. (µmol·L–1)b 1.6

122.0 106.0 34.3 17.4 3.2

48.4 399.0 457.0 501.0 201.0

Note: The concentrations of the substrates were selected that aided the best phosphotriesterase production. a Enzyme activity was performed using membrane-bound fractions as a crude enzyme source. b Final concentration of the substrate in the reaction mixture.

PTE from C. michiganense subsp. insidiosum SBL11, metal ions, such as Mg2+, Mn2+, and Co2+, did not activate or inhibit the enzyme activity. Cu2+ and Ca2+ activated the enzyme even at the higher concentration by factors of 40% and 35%, respectively. The only metal ion that inhibited the enzyme activity was Zn2+ where the loss of percent residual activity was up to 45% (Fig. 6B). Substrate specificity Substrate specificity of the purified PTEs was studied towards different OP insecticides in addition to the substrate TpNPP. From Table 3, it was obvious that the purified enzyme PTE could hydrolyze all of the tested substrates to different degrees. TpNPP was the best substrate and the concentration needed for the best enzyme activity was also very low (1.6 µmol/L). Among the insecticides studied, paraoxon was the best substrate, followed by malathion (technical). Malathion 50% EC proved to be the poorest substrate and the substrate concentration needed was also very high. The Michaelis–Menten parameters Km and Vmax were studied for all of the substrates using the Lineweaver–Burk plot

45 kDa 34.7 kDa 24 kDa 18.4 kDa 14.3 kDa

1

C

2

3 4 9.3 8.45 7.35 6.5 5.2

8.65 8.15 6.8 5.8

4.5 3.5

(Lineweaver and Burk 1934). The purified PTE from P. aeruginosa F10B showed affinity for the pesticide paraoxon besides the chromogenic substrate TpNPP. The enzyme had relatively lower affinity for the other pesticides. When the chromogenic substrate was dissolved in ethanol, it reduced Km as well as Vmax. In the case of C. michiganense subsp. insidiosum SBL11, the enzyme had affinity for paraoxon besides TpNPP. It also showed relatively low affinity for the pesticide parathion. Thus, as evidenced from Table 4, the PTE from P. aeruginosa F10B had greater affinity for the chromogenic substrate TpNPP than the PTE of C. michiganense subsp. insidiosum SBL11, while the affinity was lower in case of the pesticide paraoxon. However, as compared with the rest of the tested pesticides, enzymes from both strains had the best affinity for © 2006 NRC Canada

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Fig. 3. Effect of pH (A) and its stability (B) on the phosphotriesterase activity of two potential strains. Assay conditions for Fig. 3A are as follows: glycine–NaOH buffer (50 mmol/L, pH 9.5) using Tris – p-nitrophenyl phosphate as a substrate incubated at 37 °C for 1 h. The maximum activity obtained is considered as 100% (45.32 U/mL). Assay conditions for Fig. 3B are as follows: enzyme was incubated at different pHs for 1 h at room temperature and residual activity was assayed in glycine– NaOH buffer (50 mmol/L, pH 9.5) using Tris – p-nitrophenyl phosphate as a substrate incubated at 37 °C for 1 h. The initial activity was 32.78 U/mL.

(A)

Fig. 4. (A) Effect of temperature on the phosphotriesterase activity of the two potential strains. Assay conditions: glycine–NaOH buffer (50 mmol/L, pH 9.5) using Tris – p-nitrophenyl phosphate as a substrate incubated at 37 °C for 1 h. The maximum activity obtained is considered as 100% (27.5 U/mL). Effect of temperature on the stability of phosphotriesterase from (B) Pseudomonas aeruginosa F10B and (C) Clavibacter michiganense subsp. insidiosum. The enzyme was incubated at requisite temperatures for different time intervals and residual activity was assayed in glycine–NaOH buffer (50 mmol/L, pH 9.5) using Tris – p-nitrophenyl phosphate as a substrate incubated at 37 °C for 1 h. Initial activity was 34.9 U/mL.

Relative activity (%)

100 80 60 40 P. aeruginosa F10B

20

C. m. insidiosum SBL11

0 3

4

5

6

7

8 pH

9

10

11

12

11

12

(B) Residual activity (%)

100 80 60 40

P. aeruginosa F10B

20

C.m. insidiosum SBL11

0 3

4

5

6

7

8 pH

9

10

paraoxon but the lowest for malathion and malathion 50% EC. Paraoxon was hydrolyzed at a better rate than TpNPP in the case of P. aeruginosa F10B, while the reverse was true in the case of C. michiganense subsp. insidiosum SBL11. The Vmax for parathion hydrolysis was 12.6 U/mg protein in the case of P. aeruginosa F10B, while there was a more than sixfold increase in the hydrolysis rate in C. michiganense subsp. insidiosum SBL11.

Discussion Purification PTEs have been shown to be associated with membranebound fractions. The increase in enzyme activity after © 2006 NRC Canada

Das and Singh

165

Fig. 5. Effect of inhibitors on phosphotriesterase activity from (A) Pseudomonas aeruginosa F10B and (B) Clavibacter michiganense subsp. insidiosum SBL11. Enzyme samples were incubated in the presence of different concentrations of inhibitors at room temperature for 1 h and residual activity was assayed in glycine– NaOH buffer (50 mmol/L, pH 9.5) using Tris – p-nitrophenyl phosphate as a substrate incubated at 37 °C for 1 h. Initial activity for P. aeruginosa was 23.4 U/mL and for C. michiganense 38.64 U/mL.

(A) Residual activity (%)

100

PCMB

IAA

DTT

EDTA

Fig. 6. Effect of metal ions on the phosphotriesterase activity from (A) Pseudomonas aeruginosa F10B and (B) Clavibacter michiganense subsp. insidiosum SBL11. Enzyme samples were incubated in the presence of different concentrations of metal ions at room temperature for 1 h and residual activity was assayed in glycine–NaOH (50 mmol/L, pH 9.5) using Tris – p-nitrophenyl phosphate as a substrate incubated at 37 °C for 1 h. Initial activity for P. aeruginosa was 19.89 U/mL and for C. michiganense 32.5 U/mL.

NEM

80 60 40 20 0 0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0

0.5

1 1.5 2 2.5 3 3.5 4 4.5 5 Inhibitor concentration (mmol/L)

(B) Residual activity (%)

100 80 60 40 20 0

sonication was attributed to the release of enzyme in the soluble fraction. Brown (1980) isolated the PTE from Flavobacterium sp. and showed that sonication increased the specific activity up to 21 times. In the case of P. aeruginosa F10B, the combination of detergents was the best, probably because these two detergents might have a synergistic effect in extracting the proteins from the membrane complex. Sode and Nakamura (1997) showed that some detergents, including Triton X-100, enhanced the PTE activity towards the hydrolysis of paraoxon moreso than without the detergent. Membrane proteins are anchored to membranes by hydrophobic stretches of amino acids or by amphiphilic groups covalently linked to the polypeptide chains. The treatment with Triton X-114 separates the hydrophilic proteins recovered in the aqueous phase, whereas amphiphilic membrane proteins are found in the detergent phase. The versatility of Triton X-114 phase partitioning is exemplified by the number of analytical and preparative procedures that can be performed with the proteins still in the detergent phase. However, SDS– PAGE analysis of proteins while still in the detergent phase

is complicated by smearing artifacts caused by detergent. Therefore, the removal of detergent was necessary. The removal of the detergent had a very drastic effect on the enzyme yields in both of the potential strains. Bio-Beads® adsorbents are neutral, macroporous polymeric analyticalgrade adsorbents of high surface area with excellent chemical and physical stability. These beads are also reported to separate the metals and trace organics (Wigilius et al. 1987; Libbey 1986) from the suspensions. The loss of the enzymatic activity is likely to be attributed to this property of the beads, as the metals are known to play an important role in enzyme activity. Since PTE is a metalloenzyme, it is most likely to lose the activity after the washing step. For PTE, hydrophobic interaction chromatography is wellknown for its high enzyme yields and high purity, since PTEs are hydrophobic in nature. Here, separation is based on the interaction between the hydrophobic moieties of a sample and an insoluble, immobilized hydrophobic group of the matrix. Further, hydrophobicity of the matrix is also enhanced by increasing the ionic strength. In this case, PTE © 2006 NRC Canada

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Table 4. Michaelis–Menten parameters (Km and Vmax) of the substrates against phosphotriesterases from Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL11.

Substrate Tris – p-nitrophenol phosphate (in ethanol) Tris – p-nitrophenol phosphate (in acetone) Paraoxon Malathion Parathion Methyl parathion Malathion 50% EC

P. aeruginosa F10B

C. michiganense subsp. insidiosum SBL11

Km (µmol/L) 90.6 1.4 18.4 154.9 121.9 423.1 218.7

Km (µmol/L)

Vmax (U/mg protein)

—

— 210.4 189.2 61.8 78.1 93.5 54.6

could be eluted with buffered acetone, which further confirms the PTE to be a highly hydrophobic protein. We could purify the proteins with 13%–16% yield and over 950- to 1000-fold purification. We are reporting for the first time that the PTE from P. aeruginosa F10B is a heterodimer, where the larger subunit is 43 kDa and the smaller subunit is 41 kDa, which is likely to have novel properties. The purification of PTEs from different sources has been extensively studied (Dumas et al. 1989; Brown 1980; Cheng et al. 1993; Omburo et al. 1992; Mulbry and Karns 1989), all with a high fold purification of up to 1500 times with variable yields between 3% and 9%. DeFrank and Cheng (1991) purified OP acid anhydrolase-2 from a halophilic bacterium, JD6.5, a 60 kDa protein but could not purify this OP acid anhydrolase-2 on phenyl-Sepharose CL-4B, which further suggested it to be a very hydrophobic protein. In another investigation by Cheng et al. (1993), the purified OP acid anhydrolase from Alteromonas undina consisted of single polypeptide chain, functional as monomer, and the molecular mass was determined to be 53 kDa. In the purification of secreted parathion hydolase from Streptomyces lividans, Rowland et al. (1991) showed that this protein was a monomer of 35 kDa. The PTE from P. aeruginosa F10B showed two very close bands in SDS–PAGE, but when applied to isoelectric focusing, these two bands separated out giving two different pIs. These two bands corresponded to a pI of 7.9 and 7.35, while the PTE from C. michiganense subsp. insidiosum SBL11 had a pI of 7.5. Various pIs have been reported for PTE from different sources. A study by Zech and Wigand (1974) showed that the DFPase from Escherichia coli had four pIs (5.3, 5.7, 6.1, and 7.8). The free preparative isoelectric focusing of the phosphohydrolases of E. coli extracts showed variable pIs for different enzymes (paraoxonase: 5.3, 5.6, and 6.2; acid phosphatase: 4.9, 5.3, and 6.4; alkaline phosphatase: 4.8 and 5.3; phosphodiesterase: 5.0, 5.3, 5.6, and 6.8). Dumas et al. (1989) reported that the purified PTE from P. diminuta had a pI of 8.3 ± 0.1, while Rowland et al. (1991) showed a pI of 7.7 for the enzyme parathion hydrolase from S. lividans. Characterization The purified PTE from both strains showed peak activity at pH 9.0. Most of the studies performed on bacterial PTEs show broad pH activity. Brown (1980) demonstrated that the pH optimum for the enzyme from Flavobacterium sp. was

Vmax (U/mg protein) 33.3 111.5 221.6 41.2 12.6 71.3 23.1

3.1 31.2 182.1 81.3 354.2 311.3

between 8.0 and 10.0, while Rowland et al. (1991) showed that the parathion hydrolase from S. lividans showed high activity at a pH range of 7.5–10.5 with the pH optimum at 9.5. Cheng et al. (1993) purified OP acid anhydrolase from A. undina and observed that the enzyme possessed high activity over a pH range of 6.0–9.5, with the optimum pH being 8.5. DeFrank and Cheng (1991) found the pH optimum for activity of OP acid anhydrase to be 8.5 for a halophilic bacterial isolate, JD6.5. In another study, Cheng et al. (1998) demonstrated that the PTE was quite reactive in the pH range of 7.0–9.0. Both purified PTEs from the two potential strains have broad pH stability with a peak at pH 7.0. The study rendered by Rowland et al. (1991) showed that the purified enzyme PTE from S. lividans remained stable for 6 h at a pH range of 7.5–10.0. Activity was quickly lost below pH 6.5. The temperature study indicated that the optimal temperature for PTE activity was 37 °C. Cheng et al. (1998) found the optimal temperature for PTE activity to be 35 °C for genetically engineered E. coli. In the case of P. aeruginosa F10B, all of the tested inhibitors had an inhibiting effect on the enzyme PTE with marked inhibition in the case of EDTA and DTT. The chelating effect of EDTA could be the possible reason for the inactivation of the enzyme. In the case of C. michiganense subsp. insidiosum SBL11, the DTT had a very drastic effect on PTE activity. The rest of the inhibitors tested had more or less very little effect on PTE activity. The existence of PTE activities insensitive or only slightly sensitive to EDTA has also been reported in cases such as paraoxonase of S. lividans (25% inhibition; Rowland et al. 1991). The OP acid anhydrolase from A. undina JD6.5 (Cheng et al. 1993) was stimulated and stabilized by DTT and β-mercaptoethanol, suggesting the involvement of sulfhydryl groups in the active site. PTE from Flavobacterium sp. (Brown 1980) was inhibited by mercurial thiol reagent 4-hydroxymercuribenzoate and 4-hydroxymercuriphenyl sulphonate and by cysteine. Rowland et al. (1991) showed that metal chelators, such as EDTA, did not show the severe effect, while dipyridyl and o-phenanthroline decreased the activity to 24% and 2%, respectively. At the same time, the sulfhydryl groups in IAA and N-ethylmaleimide showed partial inactivation, while DTT completely inactivated the enzyme. Dumas et al. (1989) demonstrated that the variety of sulfhydryl-containing compounds, such as DTT, DTE, and β-mercaptoethanol, are rapid and reversible competitive inhibitors. The sulfhydryl © 2006 NRC Canada

Das and Singh

groups in these compounds are perhaps coordinating with zinc in the active site and displaced by higher concentrations of the actual substrates. In the case of P. aeruginosa F10B, the metals Co2+ and Mg2+ had an activating effect, while metals, such as Ca2+, Mn2+, and Zn2+, had an inhibiting effect. Cu2+ did not affect PTE activity at all. In the case of C. michiganense subsp. insidiosum SBL11, metals such as Ca2+ and Cu2+ had an activating effect, while Zn2+ alone had an inhibiting effect. Other metals such as Co2+, Mn2+, and Mg2+ had no effect on PTE activity. The fact that PTEs are sensitive to chelating agents and that their activity sometimes depends on the concentration of divalent cations present in the reaction medium supports the idea that PTEs are metalloenzymes (Jarv 1989). Ca2+ has been suggested to play important roles in the hydrolysis of OPs. Abd-Alla (1994) reported maximum PTE production by Rhizobium and Bradyrhizobium strains in the presence of Ca2+ followed by Mg2+. The PTE of P. diminuta possesses two Zn2+ bound to its active site that are necessary for its catalytic activity. These atoms may be replaced by Mn2+, Co2+, Ni2+, and Cd2+ without significant loss of activity. However, the addition of high concentrations of Zn2+ produced significant enzyme inhibition (Omburo et al. 1992). Copper sulfate at a concentration of 1 mmol/L was observed to be a potent inhibitor (64%) of a parathion hydrolase of bacterial origin (Rowland et al. 1991). The enzymatic activity of the PTE from P. diminuta requires at least one divalent cation for full catalytic activity (Dumas et al. 1989; Omburo et al. 1992). Removal of the native Zn2+ from the purified protein with metal chelators results in the loss of enzymatic activity (>99%), but full catalytic activity can be restored upon reconstitution of the apoenzyme with a variety of other divalent cations (Omburo et al. 1992). Brown (1980) indicated that Mn2+, Zn2+, Cu2+, Co2+, Cd2+, and Mg2+ apparently had no effect on the activity of parathion hydrolase. The Lineweaver–Burk plot showed, as evidenced from the results, that the substitution of P—S from P—O decreased the affinity as well as the hydrolysis rate. The identity of any naturally occurring substrate for this enzyme is unknown at the present time, and we were unable to detect any activity with monoesters or diesters of phosphoric acid. Most pesticide hydrolases examined to date seem to have rather relaxed substrate specificities, being able to hydrolyze almost any compound within the same general class of pesticides (Munnecke et al. 1982). Both Flavobacterium and B-1 hydrolases seemed to follow this pattern, as they both hydrolyzed EPN (ethyl p-nitrophenyl thionobenzene phosphonate) as well as parathion. Brown (1980) suggested that Flavobacterium sp. PTE was specific for triesters of phosphoric acid and its structural analogues having an electron withdrawing aromatic or heterocyclic leaving group. The substitution of S for O between an alkyl group and the P atom had little effect on Km and Vmax. In contrast, when a thiono group replaced the oxon group, both Km and Vmax were decreased. Lai et al. (1995) demonstrated that OP hydrolase from P. diminuta MG was a broad-spectrum enzyme capable of hydrolyzing the P—S bonds of a variety of OP thioates. The hydrolysis of the P—S bond rather than the coordinated P—C bond was demonstrated by the free thiol reaction of hydrolysis products. This enzyme showed poor

167

activity for malathion hydrolysis. While there was significant variation in hydrolysis of phosphotriester substrates (P—O bond cleavage) and phosphofluoridate substrates (P—F bond cleavage), the reaction rates for a broad class of OP thioate substrate (P—S bond cleavage) hydrolysis were much slower. Conclusion This work may add some new information on the production of PTE from two soil isolates. The usefulness of this purified enzyme preparation may be exploited for waste treatment, biodegradation, and bioremediation of contaminated soil, especially in those parts in India where the insecticide monocrotophos has been used extensively to protect the standing crop. The extensive use has also been responsible for accidental exposure to farmers as well as contamination of groundwater resources. The PTEs from these two sources with novel properties can be exploited and harnessed for specific industrial applications as well.

Acknowledgements The authors sincerely thank the Indian Council for Agricultural Research, Government of India, for financial assistance. One of the authors (Subhas Das) acknowledges the University Grants Commission, New Delhi, for granting fellowship.

References Abd-Alla, M.H. 1994. Phosphodiesterase and phosphotriesterase in Rhizobium and Bradyrhizobium strains and their roles in degradation of organophosphorus pesticides. Lett. Appl. Microbiol. 19: 240–243. Benning, M.M., Kuo, J.M., Raushel, F.M., and Holden, H.M. 1994. Three-dimensional structure of phosphotriesterase: an enzyme capable of detoxifying organophosphate nerve agents. Biochemistry, 33: 15001–15007. Beynon, K.I., Hutson, D.H., and Wright, A.N. 1973. The metabolism and degradation of vinyl phosphate insecticides. Residue Rev. 47: 55–142. Bordier, C. 1981. Phase separation of integral membrane proteins in Triton X-114. J. Biol. Chem. 256: 1604–1607. Brown, K.A. 1980. Phosphotriesterases of Flavobacterium sp. Soil Biol. Biochem. 12: 105–112. Caldwell, S.R., and Raushel, F.M. 1991a. Detoxification of organophosphate pesticides using a nylon-based immobilized phosphotriesterase from Pseudomonas diminuta. Appl. Biochem. Biotechnol. 31: 59–73. Caldwell, S.R., and Raushel, F.M. 1991b. Detoxification of organophosphorus pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta. Biotechnol. Bioeng. 37: 103–109. Cheng, T.-C., Harvey, S.P., and Stroup, A.N. 1993. Purification and properties of a highly active organophosphorus acid anhydrolase from Alteromonas undina. Appl. Environ. Microbiol. 59: 3138– 3140. Cheng, Y.-D., Karns, J.S., and Torrents, A. 1998. Characterization of a phosphotriesterase from genetically engineered Escherichia coli. J. Environ. Sci. Health Part B Pestic. Food Contam. Agric. Wastes, 33: 347–367. DeFrank, J.J., and Cheng, T.C. 1991. Purification and properties of an organophosphorus acid anhydrase from a halophilic bacterial isolate. J. Bacteriol. 173: 1938–1943. © 2006 NRC Canada

168 Donarski, W.J., Dumas, D.P., Heitmeyer, D.P., Lewis, V.W., and Raushel, F.M. 1989. Structure–activity relationships in the hydrolysis of substrates by the phosphotriesterase from Pseudomonas diminuta. Biochemistry, 28: 4650–4655. Dumas, D.P., Caldwell, S.R., Wild, J.R., and Raushel, F.M. 1989. Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J. Biol. Chem. 264: 19659–19665. Dumas, D.P., Durst, H.D., Landis, W.G., Raushel, F.M., and Wild, J.R. 1990. Inactivation of organophosphorus nerve agents by phosphotriesterase from Pseudomonas diminuta. Arch. Biochem. Biophys. 277: 155–159. Jarv, J. 1989. Insight into putative mechanism of esterase acting simultaneously on carboxyl and phosphoryl compounds. In Enzymes hydrolysing organophosphorus compounds. Edited by E. Reiner, W.N. Aldridge, and F.C.G. Hoskin. Ellis Horwood Limited, Chichester, England. pp. 221–225. Lai, K., Stolwich, N.J., and Wild, J.R. 1995. Characterization of P—S bond hydrolysis in organophosphotrithioate pesticides by organophosphorus hydrolase. Arch. Biochem. Biophys. 318: 5964. Lewis, V.E., Donarski, W.J., Wild, J.R., and Raushel, F.M. 1988. Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase. Biochemistry, 27: 1591–1597. Libbey, A.J. 1986. Determination of ethylene dibromide in aquatic environments. Analyst, 3: 1221–1222. Lineweaver, H., and Burk, D. 1934. The determination of enzyme dissociation constants. J. Am. Chem. Soc. 56: 658–666. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265–275. Mulbry, W.W., and Karns, J.S. 1989. Purification and characterization of three parathion hydrolases from Gram-negative bacterial strains. Appl. Environ. Microbiol. 171: 6740–6746.

Can. J. Microbiol. Vol. 52, 2006 Munnecke, D.M., Johnson, L.M., Talbot, H.W., and Barik, S. 1982. Microbial metabolism and enzymology of selected pesticides. In Biodegradation and detoxification of environment pollutants. Edited by A.M. Chakrabarty. CRC Press, Boca Raton, Florida. pp. 1–32. Omburo, G.A., Kuo, J.M., Mullins, L.S., and Raushel, F.M. 1992. Characterization of the zinc-binding site of bacterial phosphotriesterase. J. Biol. Chem. 267: 13278–13283. Rowland, S.S., Speedie, M.K., and Pogell, B.M. 1991. Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans. Appl. Environ. Microbiol. 57: 440–444. Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Sode, K., and Nakamura, H. 1997. Compatibility of phosphotriesterase from Flavobacterium sp. with detergents. Biotechnol. Lett. 19: 1239–1242. Subhas and Singh, D.K. 2003. Utilization of monocrotophos as phosphorus source by Pseudomonas aeruginosa F10B and Clavibacter michiganense subsp. insidiosum SBL 11. Can. J. Microbiol. 49: 101–109. Wigilius, B., Boren, H., Carlberg, G.E., Grimvall, A., Lundgren, B.V., and Savenhed, R. 1987. Systemic approach to adsorption on XAD-2 resin for the concentration and analysis of trace organics in water below the µg/L level. J. Chromatogr. A, 391: 169–182. Zech, R., and Wigand, K.D. 1974. Organophosphate detoxicating enzymes in E. coli. Gel filtration and isoelectric focusing of DFPase, paraoxonase and unspecific phosphohydrolase. Experientia, 31: 157–158.

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