Nov 25, 1990 - future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies. The disposal of organophosphate ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, 0099-2240/91/020440-05$02.00/0 Copyright © 1991, American Society for Microbiology
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Purification and Characterization of a Secreted Recombinant Phosphotriesterase (Parathion Hydrolase) from Streptomyces lividans SHARON S. ROWLAND,t MARILYN K. SPEEDIE,* AND BURTON M. POGELL Department of Biomedicinal Chemistry, School of Pharmacy, and Maryland Biotechnology Institute, University of Maryland, Baltimore, Maryland 21201 Received 13 August 1990/Accepted 25 November 1990
A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 ,uM for parathion, 46 ,uM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 ,uM for methyl parathion, and 357 ,uM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45°C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CUSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CUSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.
The disposal of organophosphate pesticides has become of increasing concern to industrialized nations. Microorganisms containing hydrolytic enzymes which detoxify this class of pesticides may provide a practical solution to toxic waste disposal (1, 5, 6, 12, 18). Genetic engineering offers the possibility of producing high levels of such enzymes, which can then be harvested and used, thus avoiding the problems associated with environmental release of recombinant microorganisms. One of the most intensively used organophosphate insecticides in agriculture is parathion. Coumaphos is a related insecticide used to control ectoparasites on cattle in the southwest United States. A native bacterial phosphotriesterase which hydrolyzes both of these compounds was first identified in a Flavobacterium species (17) and purified to homogeneity by Mulbry and Karns (10). Subsequently, the enzyme was purified to homogeneity from three recombinant sources: Streptomyces lividans (19), Escherichia coli (16), and insect cells (4). The structural genes (opd) for this hydrolase were shown to be plasmid coded in both a Flavobacterium sp. and Pseudomonas diminuta (11, 15). Despite an initial conflicting report (8), the two structural genes have now been shown to have identical nucleotide sequences (9, 16). However, controversy concerning the similarity between the characteristics of the expressed enzymes remains (4, 10, 16). In the Flavobacterium sp. and P. diminuta the native parathion hydrolase is membrane bound, whereas in S. lividans, the opd gene is expressed as a secreted soluble
level expression (13). The characteristics of the pure enzyme have been compared with those of the crude enzyme in fermentation broth, with those of the native enzyme in a Flavobacterium sp., and with those of two similar recombinant enzymes. MATERIALS AND METHODS Bacterial strain and culture conditions. S. lividans 66 containing plasmid pRYE1 (19) was used for parathion hydrolase production. Plasmid pRYE1 contains the Flavobacterium opd gene on a 1.5-kb fragment inserted into the BglII site of the vector pIJ702. In this construction, the mel promoter is upstream from the opd promoter and structural gene. Parathion hydrolase was purified from the extracellular fluid of S. lividans 66(pRYE1) grown under fed-batch conditions (as described below) in 2% tryptone plus 3% glucose, pH 7.2 (tryptone-glucose medium), containing thiostrepton (13). A 5% inoculum of S. lividans 66(pRYE1) grown from spores through two transfers in tryptone-glucose medium with thiostrepton (30 ,ug/ml) was inoculated into tryptone-glucose medium with thiostrepton (5 ,ug/ml) in a baffled flask and grown at 30°C in a shaker set at 240 rpm. At the following times, 50% tryptone and 50% glucose were added to make the indicated final concentrations: 24 h, 1.5% tryptone; and every 24 h thereafter, 2% tryptone and 2% glucose. Incubation was terminated at 4 days, with the parathion hydrolase activity in the extracellular fluid attaining an activity between 10 and 15 U/ml. Cells from the Streptomyces culture were removed by centrifugation (10,000 x g, 4°C, 15 min). The extracellular fluid was used for enzyme purification. Measurement of parathion hydrolase activity. The enzyme was incubated at 30°C with 172 F.M parathion (unless otherwise stated) in 0.012% Tween 80 and 10 mM Tris (pH 8.5) in a final volume of 0.5 ml. A410 was determined. Results are expressed as units (micromoles per minute) per milliliter.
enzyme (19). The purpose of the present work was to purify and characterize the secreted recombinant parathion hydrolase produced during growth conditions optimized for high* Corresponding author. t Present address: Department of Medical and Research Technology, School of Medicine, University of Maryland, Baltimore, MD 21201.
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Protein was determined by using A2801260 (20). Specific activity is expressed as units per milligram of protein. Purification of enzyme. To purify parathion hydrolase, ammonium sulfate was added to the crude extracellular fluid to give 30% saturation, and the solution was stirred at 4°C for 30 min. The precipitate was removed by centrifugation at 20,000 x g for 20 min, and the supernatant fluid was brought to 50% ammonium sulfate saturation. After the solution was stirred at 4°C for 30 min and centrifuged at 20,000 x g for 20 min, the precipitate was redissolved in 10 mM Tris-10 mM TES [N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid], mixed to obtain a pH of 7.0 at 25°C (TES-Tris buffer). All subsequent steps were done at room temperature. (i) Ion-exchange chromatography. The enzyme was desalted by passage through a Sephadex G-25 column (PD10; Pharmacia LKB Biotechnology, Inc., Piscataway, N.J.) previously equilibrated in TES-Tris buffer. The desalted enzyme preparation was applied to a monoS (Pharmacia) cation-exchange column (1 by 5 cm) equilibrated in TES-Tris buffer and washed with 40 ml of the same buffer. The enzyme was eluted with a linear 0 to 0.5 M NaCl gradient in TES-Tris buffer, and 1.0 ml fractions were collected. (ii) Gel filtration. Fractions containing enzymatic activity were pooled, and the protein was precipitated by bringing the pool to 65% ammonium sulfate saturation. The ammonium sulfate solution was stirred for 30 min at 4°C. The enzyme was precipitated by centrifugation at 27,000 x g at 4°C for 30 min. The precipitate was redissolved in 0.2 ml buffer (0.15 M NaCl in TES-Tris, pH 7.0) and applied to a Sephadex G-100 column (1 by 50 cm) equilibrated in 0.15 M NaCl in TES-Tris buffer, pH 7.0. One-milliliter fractions were collected. Specific activity was calculated as units per milligram of protein. SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to the method of Laemmli (7). Fractions were analyzed for purity in 0.75-mm-thick 12.5% acrylamide gels stained with silver stain (Integrated Separation Systems, Hyde Park,
Mass.). Determination of N-terminal sequence. The purified parathion hydrolase was blotted onto Immobilon polyvinylidene difluoride membrane (Millipore Corp., Bedford, Mass.) from an SDS-polyacrylamide gel, and protein sequencing was done by Edman degradation in an Applied Biosystems gas sequenator model 470 with on-line phenylthiohydantoin analysis (Microsequencing Laboratory, University of Victoria, Victoria, British Columbia, Canada). Characterization of parathion hydrolase. (i) Substrate specificity. The Km constants were determined by using parathion concentrations between 8 and 165 ,uM, O-ethyl O-pnitrophenyl phenylphosphonothioate (EPN) concentrations between 2 and 50 p.M, p-nitrophenyl ethyl(phenyl)phosphinate (NPEPP) concentrations between 20 and 100 ,uM, and methyl parathion concentrations between 50 and 400 pLM. Lineweaver-Burk plots were used to calculate Km and Vmax values. Parathion and EPN were obtained from Wako Fine Chemicals, Dallas, Tex. NPEPP was a gift from F. R. Lange, Aberdeen Proving Ground, Md. Methyl parathion was a gift from the Environmental Protection Agency. (ii) Temperature studies. For stability studies, the enzyme (1,145 U/ml) was diluted 1:500 in 10 mM Tris containing 0.012% Tween 80, pH 8.5 (Tris-Tween buffer), and placed at the stated temperature. At a given time, a sample was removed, diluted 1:10 in Tris-Tween buffer (30°C), and immediately assayed for activity. Time points were at 0, 1, 2, 4, 6, and 24 h. To determine the temperature optimum,
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TABLE 1. Purification of a secreted heterologous phosphotriesterase from S. lividans Purification fraction
Total activity (U)
Sp act (U/mg)
Fold purification
Crude extracellular Ammonium sulfate MonoS cation G-100
18,870 9,867 3,915 2,957
12 121 1,431
1 10 113
%
Yild ld
100 52 21 16
buffer was preequilibrated to the appropriate temperature in a temperature-controlled Gilford 2600 spectrophotometer (Ciba-Corning Diagnostics Corp., Oberlin, Ohio). (iii) pH studies. For stability studies, the enzyme was diluted 1:200 in buffer of the appropriate pH and incubated at 24°C. At intervals, samples were removed, diluted 1:10 in Tris-Tween buffer and assayed for activity. Citric acid buffer at 20 mM was used for pH 4.0 to 6.5. Tris HCl buffer at 20 mM was used for pH 7.0 to 8.5. Glycine buffer at 20 mM was used for pH 9.0 to 10.0. For pH optimum studies, enzyme activity was assayed in the buffer of the appropriate pH. (iv) pl determination. The pI was determined using preformed isoelectric focusing gels (FMC BioProducts, Rockland, Maine), for pH 3 to 10, with a Hoefer isoelectric focusing unit (Hoefer Scientific, San Francisco, Calif.). (v) Effect of sulfhydryl reagents, chelators, and metal ions. Dithiothreitol, N-ethylmaleimide, iodoacetamide, EDTA, dipyridyl, and o-phenanthroline were added to each assay reaction in a final concentration of 1 mM or 10 mM. Activity was calculated as a percentage of control activity. For the metal reactivation studies, the enzyme was chelated with 10 mM o-phenanthroline at 4°C for 30 min. The mixture was then put through a Sephadex G-100 column to remove metals chelated to o-phenanthroline. Metals were added to samples of the inactivated enzyme and the mixtures were placed at 4°C for 18 h. Activity was measured relative to a chelated control lacking metal. RESULTS Purification of parathion hydrolase. Table 1 summarizes the results from a typical purification scheme. Because the high levels of tryptone in the growth medium resulted in high A280260 protein values, purification values were calculated after ammonium sulfate fractionation. Purification by cation exchange (monoS) resulted in 9.6-fold purification. A final gel filtration step with Sephadex G-100 gave a further 10-fold purification and resulted in a single band on an SDSpolyacrylamide gel stained with silver stain. In initial purification attempts, application to a Sephadex G-25 gel filtration column with phosphate buffer resulted in retention of 50% of the activity. Replacement of phosphate buffer (an antichaotropic ion) with TES buffer (a dipolar ionic buffer) resulted in less retention by ion-exchange and gel filtration matrices. Characterization of parathion hydrolase. The molecular weight of the S. lividans(pRYE1) parathion hydrolase was estimated to be 35,000 by SDS-PAGE. The purified recombinant parathion hydrolase ran with a mobility identical to that of the purified Flavobacterium hydrolase of Mulbry and Karns (Fig. 1). The native molecular weight of the recombinant hydrolase was calculated as 34,000 by high-performance liquid chromatography size exclusion chromatography (TSK 3000SW) (19), indicating that the hydrolase is a monomer. The pl was calculated to be 7.7 by using pl markers (FMC BioProducts).
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2
TABLE 2. Effect of inhibitors and copper ions on parathion hydrolase activity
3
--95
Concn (mM)
Residual activity (% of control)
o-Phenanthroline
1.0 10.0
21 2
EDTA
1.0 10.0
75 89
Dipyridyl
1.0 10.0
61 24
Dithiothreitol
1.0 10.0
lodoacetamide
1.0 10.0
84 81
N-Ethylmaleimide
1.0
48
CuS04
0.1 1.0
92 36
CuCl
2.0
100
Inhibitor --55
--43
~-3 6 --29 --18. 4
--12.4 FIG. 1. Comparison of the purified parathion hydrolase protein from the native Flavobacterium sp. (lane 1) with the recombinant form from S. lividans (lane 2) by SDS-PAGE. Lane 3, protein standard molecular weight markers.
The optimum temperature for purified parathion hydrolase activity at pH 8.5 was 45°C. The rate at 45°C was 1.7 times higher than at 25°C. Above 50°C, the enzyme was inactivated during the assay. At the end of a 24-h period, 100% of the activity remained at 4°C, 78% of activity remained at 24°C, and 41% activity remained at 37°C (Fig. 2). Parathion hydrolase showed high activity at a pH range from 7.5 to 10.5, with the optimum pH being 9.5. The enzyme remained stable for 6 h at a pH range from 7.5 to 10.0. Activity was quickly lost below pH 6.5. The effect of Tween 80 on the activity of the pure enzyme was examined. Inclusion of Tween 80 in the assay mixture always resulted in a 10-fold increase in activity at concentrations between 0.00025% and 0.1%. The addition of detergent may allow better interaction between the enzyme and the substrate, parathion, which has limited solubility in water (20 ppm).
4~~~~~~~~~~~~
4 0.4
Enzyme activity was measured in the presence of different classes of inhibitors (Table 2). Metal chelators varied in their effect upon parathion hydrolase activity. EDTA (10 mM) reduced activity only slightly, to 89%. Dipyridyl (10 mM) reduced activity to 24%, while o-phenanthroline (10 mM) showed the greatest inhibition and decreased activity to 2% of the original activity. Compounds which act on sulfhydryl groups of proteins (iodoacetamide and N-ethylmaleimide) only partially inactivated the enzyme. Dithiothreitol, an agent which reduces sulfhydryl bonds, completely inactivated the enzyme. Cupric sulfate caused partial inhibition at concentrations above 0.2 mM, while 2.0 mM cuprous chloride had no effect on activity. When metal ions were chelated and removed from the purified hydrolase with 10 mM o-phenanthroline, activity could be partially restored (to 4 to 5% of untreated enzyme activity) by adding 1 mM cuprous or cupric ion but not calcium, cobalt, ferric, ferrous, magnesium, manganese, molybdate, or zinc ions (Table 3). N-terminal structure. The N-terminal sequence was determined to the 24th residue. The major sequence observed was
Ser-Ile-Gly-Thr-Gly-Asp-Arg-Ile-Asn-Thr-Val-Arg-Gly-Pro-
1- 20 i-
00
U-
1
10
2
3
TABLE 3. Reactivation of parathion hydrolase by metal ions after chelation with o-phenathroline Fold
Metal'
40-
increase'
I.z
C)
CaCl2 .........................................
~~~~OR F~60INUATO 80
0 0
20
10
HOURS
OF
30
INCUBATION
FIG. 2. Effect of temperature on parathion hydrolase stability. Enzyme was incubated at 40, 240, 370, 500, or 800C. Portions were removed and assayed for activity at various times. Activity (units per milligram of total protein) was determined, and percent activity relative to activity at time zero was calculated.
CoCl2 ......................................... CoSO2 .......................................... CuCl .......................................... CuS04 ............................................................... FeCl2 .......................................... FeCl3 ......................................... MgCl2 ......................................... MnCl2 ......................................... NaMoO4 ........................................
............
ZnCl2 ................................................ "All metals had a 1.0 mM final concentration. bIncreased activity above that of the chelated control.
1.8 0.3 0.3 3.1 5.4 0.4 0.3 1.8 0.7 1.1 0.9
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TABLE 4. Substrate specificity of recombinant parathion hydrolase from S. lividans Substrate Km (ILM) Vmax (U/ml) Kcat (s-) Parathion 68 1,274 925.8 Methyl parathion 599 508 369.7 EPN 46 1,271 925.0 NPEPP 357 3,030 2,210.0
Ile-Thr-Ile-Ser-Glu-Ala-*-Phe-Thr-Leu. This
was
identical
to the amino acid sequence found for the purified Flavobacterium hydrolase and indicated that the mature recombinant protein has the signal peptide removed after amino acid 29 (glycine), as in the Flavobacterium sp. A minor amount
(10%) of the sample had an N-terminal sequence which began at amino acid 31 (Ile) or amino acid 32 (Gly). Substrate specificity. Substrate specificities for parathion, methyl parathion, EPN, and NPEPP were compared (Table 4). Michaelis-Menten constants were determined by using Lineweaver-Burk plots. The apparent Kms for parathion, EPN, methyl parathion, and NPEPP were, respectively, 68, 46, 599, and 357 ,uM. The turnover numbers (Kcat) were, respectively, 926, 925, 370, and 2,210 s-. DISCUSSION Secretion of the recombinant hydrolase into the extracellular growth medium at high levels allowed purification of the enzyme in milligram amounts. Growth of S. lividans(pRYEl) in tryptone-glucose medium using a fed-batch technique greatly increased the yield of extracellular parathion hydrolase (13). Extracellular broth containing 10 to 30 U of hydrolase per ml (approximately 10 to 30 mg/liter) was routinely obtained. Location of the enzyme in the extracellular fluid was advantageous for purification compared with a membrane-bound or intracellular location, as in the native Flavobacterizm sp., the native P. diminuta, and recombinant E. coli (10, 14, 16), in which a wider variety of proteins is initially present. Purification was accomplished in three steps with a 30 to 50% ammonium sulfate cut, followed by cation exchange at pH 7.0. A final G-100 gel filtration step resulted in one band on a silver-stained SDS-polyacrylamide gel. Cell-free culture fluid of S. lividans(pRYE1) has the potential to be used in waste treatment of organophosphates. Hydrolysis of parathion in waste cattle dip using the cell-free enzyme from S. lividans has been demonstrated (2). A comparison was therefore made between the characteristics of parathion hydrolase in crude fermentation broth (2) and the purified enzyme. In both instances, the addition of Tween 80 at 0.012% resulted in a 10-fold increase in enzyme activity (3). Other characteristics were also similar. The Km of 67 ,uM for parathion determined for the pure enzyme is similar to the Km of 20 ,uM obtained for the crude enzyme. Both preparations exhibited similar temperature and pH inactivation profiles, being inactivated at 50°C and below pH 6.5. This suggests that parathion hydrolase existing in crude broth behaves similarly to the purified enzyme. The native Flavobacterium parathion hydrolase and native Pseudomonas diminuta hydrolase are encoded on plasmids and have been reported to have identical structural (opd) genes (9, 11, 14-16). A sequence different from that determined by other investigators was originally reported by McDaniel et al. (8). Our N-terminal sequencing and molecular weight data support the sequence determined by the
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other investigators (9, 16). The native Flavobacterium hydrolase has been purified (9). The P. diminuta hydrolase has been expressed and purified in a recombinant form in E. coli and in a baculovirus system (4, 16). Comparison of the purified S. lividans(pRYE1) parathion hydrolase with the purified native Flavobacterium hydrolase showed that the two enzymes have very similar characteristics. Both enzymes have molecular weights of 35,000 as determined by SDS-PAGE and occur as monomers when chromatographed under native conditions by high-performance- liquid chromatography. The temperature optima (40°C [Flavobacterium enzyme] versus 45°C [Streptomyces enzyme]) are similar. Both are strongly inhibited by 1 mM dithiothreitol and partially inhibited by 1 mM Cu2". The Kms for parathion are equivalent (91 ,uM [Flavobacterium enzyme] versus 68 ,uM [Streptomyces enzyme]). However, the recombinant parathion hydrolase appears to have a higher affinity for EPN (Km of 46 piM) than that reported for the Flavobacterium enzyme (Km of 211 ,uM). Despite the fact that the native Flavobacterium parathion hydrolase is membrane-bound and the recombinant hydrolase is secreted by a grampositive organism, both mature proteins appear to have been processed similarly, resulting in the same N-terminal amino acid sequence. These results indicate that overexpression and secretion of the Flavobacterium hydrolase in S. lividans result in an enzyme with the same characteristics as the native enzyme. The parathion hydrolase gene from P. diminuta lacking the nucleotide code for the signal sequence has been expressed in E. coli as a soluble intracellular enzyme (16). This resulted in an active parathion hydrolase with an amino acid sequence identical to that of the processed Flavobacterium hydrolase. Addition of 1 mM Co2" to the growth medium increased specific activity by 20-fold, suggesting that the presence of Co2+ may influence the active enzyme either by enhanced expression or by stabilization of the mature protein. Zn2+ also increased the specific activity. The purified recombinant Streptomyces hydrolase was not reactivated by either Co2+ or Zn2+ after chelation by o-phenanthroline, although Cu+ and Cu2' did cause partial reactivation. The P. diminuta parathion hydrolase has also been expressed in and purified from a baculovirus expression system (4). There appear to be a number of differences between this enzyme and the recombinant Streptomyces hydrolase. The baculovirus-expressed enzyme was also reported to be inhibited by o-phenanthroline but, unlike the Streptomyces enzyme, was partially reactivated by Zn2+ rather than Cu2+. The isoelectric point was reported to be 8.3 versus a pI of 7.7 for the Streptomyces hydrolase. The Km for parathion was reported to be an order of imagnitude higher (240 ,uM) than those of the native Flavobacterium and recombinant Streptomyces hydrolases. The molecular weight was reported to be 39,000, as opposed to 35,000 and 36,000 for the previously discussed; hydrolases. No N-terminal amino acid sequence was obtained. These results suggest that the signal sequence has not been cleaved from the baculovirus-expressed enzyme, and this may account for some of the differences in characteristics observed among this parathion hydrolase and the other native and recombinant forms. An alternate explanation is that there may be a difference in posttranslational modification relative to the Pseudomonas and Flavobacterium hydrolases. Our results show that S. lividans can be used to produce high levels of secreted soluble parathion hydrolase which has been processed in the same manner as the native membrane-bound enzyme in the Flavobacterium sp. The
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characteristics of the recombinant enzyme are similar to those of the purified native enzyme. This provides a source of milligram amounts of purified parathion hydrolase for further structure and mechanism studies. In addition, the characteristics of the crude recombinant enzyme compare favorably with those of the purified enzyme. The availability of high levels of parathion hydrolase in extracellular fluid offers the potential for future use in toxic waste treatment strategies.
10. 11.
12.
ACKNOWLEDGMENTS This study was supported by funding from the Medical Biotechnology Center and the Center for Agricultural Biotechnology of the Maryland Biotechnology Institute. REFERENCES 1. Brown, K. A. 1980. Phosphotriesterases of Flavobacterium sp. Soil Biol. Biochem. 12:105-112. 2. Coppella, S. J., N. DelaCruz, G. F. Payne, B. M. Pogell, M. K. Speedie, J. S. Karns, E. M. Sybert, and M. A. Connor. 1990. Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment. Biotechnol. Prog. 6:76-81. 3. DelaCruz, N. 1989. M.S. thesis. University of Maryland, Balti-
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4. Dumas, D. P., S. R. Caldwell, J. R. Wild, and F. M. Raushel. 1989. Purification and properties of the phosphotriesterase from Pseudomonas diminuta. J. Biol. Chem. 264:19659-19665. 5. Johnson, L. M., and H. W. Talbot. 1983. Detoxification of pesticides by microbial enzymes. Experientia 39:702-706. 6. Kearney, P. C., J. S. Karns, M. T. Muldoon, and J. M. Ruth. 1986. Coumaphos disposal by combined microbial and uvozonation reactions. J. Agric. Food Chem. 3:702-706. 7. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 8. McDaniel, C. S., L. L. Harper, and J. R. Wild. 1988. Cloning and sequencing of a plasmid-borne gene (opd) encoding a phosphotriesterase. J. Bacteriol. 170:2306-2311. 9. Mulbry, W. W., and J. S. Karns. 1989. Parathion hydrolase
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specified by the Flavobacterium opd gene: relationship between the gene and protein. J. Bacteriol. 171:6740-6746. Mulbry, W. W., and J. S. Karns. 1989. Purification and characterization of three parathion hydrolases from gram-negative bacterial strains. Appl. Environ. Microbiol. 55:289-293. Mulbry, W. W., J. S. Karns, P. C. Kearney, J. 0. Nelson, C. S. McDaniel, and J. R. Wild. 1986. Identification of a plasmidborne parathion hydrolase gene from Flavobacterium sp. by Southern hybridization with opd from Pseudomonas diminuta. Appl. Environ. Microbiol. 51:926-930. Munnecke, D. M. 1976. Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method. Appl. Environ. Microbiol. 32:7-13. Payne, G. F., N. DelaCruz, and S. J. Coppella. 1990. Improved production of heterologous protein from Streptomyces lividans. Appl. Microbiol. Biotechnol. 33:395-400. Serdar, C. M., and D. T. Gibson. 1985. Enzymatic hydrolysis of organophosphates: cloning and expression of a parathion hydrolase gene from Pseudomonas diminuta. Bio/Technology 3:567571. Serdar, C. M., D. T. Gibson, D. M. Munnecke, and J. H. Lancaster. 1982. Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta. Appl. Environ. Microbiol. 44:246249. Serdar, C. M., D. C. Murdock, and M. F. Rohde. 1989. Parathion hydrolase gene from Pseudomonas diminuta MG: subcloning, complete nucleotide sequence, and expression of the mature portion of the enzyme in Escherichia coli. Bio/ Technology 7:1151-1155. Sethunathan, N., and T. Yoshida. 1973. A Flavobacterium sp. that degrades diazinon and parathion. Can. J. Microbiol. 19: 873-875. Shelton, D. R., and C. J. Somich. 1988. Isolation and characterization of coumaphos-metabolizing bacteria from cattle dip. Appl. Environ. Microbiol. 54:2566-2571. Steiert, J. S., B. M. Pogell, M. K. Speedie, and J. Laredo. 1989. A gene coding for a membrane-bound hydrolase is expressed as a secreted soluble enzyme in Streptomyces lividans. Bio/Technology 7:65-68. Suelter, C. H. 1985. A practical guide to enzymology, p. 28. John Wiley & Sons, Inc., New York.
