The Science of the Total Environment, 123/124 (1992) 267-277 Elsevier Science Publishers B.V., Amsterdam
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Degradation of aliphatic halogen-substituted pesticides by dehalogenase isolated from Pseudomonas alcaligenes. Identification and properties of the enzyme M.D. Busto a, P.P. Smith b, M. Perez-Mateos a and R.G. B u r n s b aDepartment of Biochemistry, Molecular Biology and Physiology, Burgos Faculty of Food Science and Technology, University of Valladolid, Apartado 231, E09071 Burgos, Spain bBiological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, UK ABSTRACT Some characteristics of a 2,2-dichloropropionate dehalogenase induced in a bacterial strain capable of degrading high concentrations of the herbicide dalapon were studied. Polyacrilamide gel electrophoresis of the crude cell free extracts identified only one type of dehalogenase. The single enzymatic protein showed activity against a variety of chlorinated aliphatic acids but differed in their activity levels. Thus activity in #mol substrate converted (rag protein) -t min -l was 2-monochloropropionate 0.65, 2,2-dichloropropionate 0.56, 2-monochloroacetate 1.70 and 2,2-dichloroacetate 1.00. In the crude extracts, the enzyme activity against 2,2-dichloropropionate was optimal at a broad pH range with a mid-point at pH 9.5 and apparent Km values were within the range 0.23-0.73 mM.
Key words: 2,2-dichloropropionate; Pseudomonas alcaligenes; dalapon; monochloroacetic acid; dichloroacetic acid; 2-monochloropropionic acid; 2,2-dichloropropionic acid
INTRODUCTION
The extensive use of halogenated aliphatic compounds in agriculture and industry has led to soil pollution in many areas (Alexander, 1965). Halogenated pesticides (often used as herbicides) may be destroyed in soil by several different mechanisms. Adsorption and inactivation by clay and organic colloidal materials or other non-biological degradations may conAbbreviations." MCA, rnonochloroacetic acid; DCA, dichloroacetic acid; 2MCPA, 2-monoehloropropionic acid; 22DCPA, 2,2-dichloropropionic acid (dalapon); TEMED, M-N,N,N',N'-tetramethyl-l,2diaminoethane.
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tribute significantly to the environmental decontamination (Holly, 1964). Volatilization or photochemical degradation can also contribute to the removal of certain chemicals from a particular ecosystem (Day et al., 1963) and leaching may occasionally be responsible for the removal of pesticides from surface zones of soil (Holly, 1964). However, the principal means by which pesticides are removed from an environment is a consequence of microbiological activity, which transforms the xenobiotic substances into harmless products (Slater and Godwin, 1980; Burns and Martin, 1986; Burns, 1987; Perez-Mateos, 1989). Abundant evidence exists that some soil microorganisms can dehalogenate chlorinated herbicides such as 2,2-dichloropropionic acid (dalapon) or trichloroacetic acid (TCA) (Kaufman, 1964; Burge, 1969; Berry and Skinner, 1976). The reactions associated with fragmentation of these halogenated aliphatic acids have been ascribed to catalytic activity of substrate-induced enzymes (Jensen, 1957). Several studies have indicated that some bacteria contain a number of dehalogenases differing in their thermal stability, pH dependence, kinetic parameters, mechanism of dehalogenation, electrophoretic mobility or inhibition by sulphydryl-blocking agents (Little and Williams, 1971; Slater et al., 1985; Kawasaki et al., 1981; Weightman et al., 1982). Furthermore, studies with partially purified dehalogenases, have shown that different halogenated alkanoic acids can serve as substrates for these enzymes. Thus, it has been suggested that these enzymes could be used as experimental systems for examining the acquisition of novel catalytic functions coupled with the capacity to grow on new compounds (Bull et al., 1976). Some previous studies have suggested the presence of inducible dehalogenases in a soil bacterial strain which has been grown using dalapon as its sole carbon and energy source (Busto et al., 1989). The soil inoculation of bacteria expressing dehalogenases has been investigated in order to accelerate the degradation of halogenated herbicides in soil and to evaluate the use of inoculates in the bioremediation of contaminated soils (Smith P.P., Ph.D. Thesis, University of Kent, Canterbury, 1991). We report in this paper the identification and characterization of a dehalogenase from Pseudomonas alcaligenes isolated from soil which is able to use dalapon as a sole source of carbon and energy. EXPERIMENTAL
Growth conditions The organism, a Gram-negative rod was isolated from soil (sand 41%, silt 34.0%, clay 24.6%, pH 6.5) following enrichment for 21 days in minimal salt
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medium plus 10 mM 22DCPA. The isolate, identified as Pseudomonas alcaligenes, was grown in minimal salts medium supplemented with 10 mM 22DCPA as the sole carbon and energy source in batch cultures incubated at 30°C on a orbital shaker at 200 rev.min-L Growth of the cultures was estimated by measuring absorbance at 640 nm.
Preparation of cell-free extracts Bacterial culture (1 1) were harvested by centrifugation at 5000 x g for 15 min, washed twice in 20 ml of 0.02 M Tris-sulfate buffer (pH 7.0) and resuspended in 5 ml of the same buffer. A solution (50 /~1) of 0.1 M dithiothreitol was added to 5 ml of bacterial suspensions before the cell disruption. Ice-cold cell suspensions were then disrupted by two passages through a French pressure cell at 83 MPa. Bacterial debris were removed by centrifugation at 30 000 x g for 45 min at 4°C to yield a supernatant which was assayed for enzyme activity immediately. The supernatant solutions contained 2-4 mg crude bacterial protein ml -~, estimated using Bradford's method (1976) and bovine serum albumin as standard.
Dehalogenase assay Dehalogenase activity was measured in 10-ml tubes containing 1 ml of the cell-free extract, 4.5 ml of 0.2 M Tris-sulfate buffer (pH 7.9) and 165 #mol substrate (MCA, DCA, 2MCPA or 22DCPA). The assay mixtures were incubated at 30°C for 30 min and 1-ml aliquots were assayed for free chloride ions. Substrate dechlorination in both culture media and dehalogenase mediated reactions was monitored by measuring free chloride ions concentration using a Marius Chlor-O-Counter as described by Slater et al. (1979).
Polyacrylamide gel electrophoresis The stacking gel was prepared by mixing 0.15 M Tris-sulfate buffer at pH 8.8 with 1.13 M acrylamide and 20 mM methylenbisacrylamide. Polymerization was achieved chemically by the addition of 4.4 mM ammonium persulfate in the presence of 3.28 mM TEMED (a catalyst accelerator). Cell-free extract (0.5 ml), prepared as described for the enzyme assay, was added to 0.5 ml of a solution of 20% (w/v) glycerol in 0.02 M Tris-sulfate buffer (pH 7.9) and to 50 /~1 0.05% (w/v) bromophenol blue. Samples (50-100 td) were loaded into the preformed wells in the stacking gel and electrophoresed at 30 mA for 3 h at 4°C or until the marker front reached approximately two-thirds of the way down the gel. It was necessary to pre-run the gels at 25 mA overnight before adding the extracts.
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Activity of separated dehalogenases in the gel was visualized by the method described by Weightman and Slater (1980). The gel was plunged in several 0.2 M Tris-sulfate buffer solutions at pH 7.9 each containing 50 mM of the halogenated substrates and then was incubated at 30°C for 60 min. The incubated gel was washed in distilled water and placed in a solution of 0.1 M AgNO3. The bands of AgC1 where free chloride ions were present, precipitated in the gel showing the regions of dehalogenase activity. Lightinduced fogging of the gels and obscuring of the AgCI bands produced by enzyme activity, were prevented by removing the excess AgNO3. This was performed by washing the gels in 5% (v/v) acetic acid solutions for 10 min and by soaking in distilled water for 12 h.
Kinetics The kinetic properties of dalapon dehalogenase were investigated by using different substrate concentrations in the assay method described above. Six solutions of 22DCPA whose concentrations ranged between 0.25 and 25 mM were used. The values of Km and Vr~xwere calculated by linear regression using Lineweaver-Burk equation.
Effect of pH on activity To determine the pH-enzyme activity curve, twelve solutions of 0.2 M Tris-maleate, 0.2 M Tris-sulfate and 0.2 M Na/NaOH borate buffers in the pH range 6.0-10.5 were used. Buffers with overlapping pH were included to assess the effects of the buffer itself upon enzyme activity.
Thermal stability Crude extracts were incubated at 50°C and samples were taken at intervals over 50 min of incubation, cooled rapidly with ice and immediately assayed for 22DCPA dehalogenase activity. The changes in activity with time at 50°C were expressed as percentages of the initial activity. RESULTS AND DISCUSSION
Dehalogenase activity of the isolates Bacterial growth appears to be directly correlated with the dechlorination of the carbon sources since differential plots of culture absorbance against CI- release were linear (Fig. 1). In the exponential phase of bacterial growth, when at least 90% of initial dalapon was hydrolyzed, cells were
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Fig. 4. Kinetics of 2,2-dicloropropionate dehalogenase.
25 (mM)
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Effect of pH The pH-activity profile of dehalogenase when 22DCPA was used as the substrate is shown in Fig. 3. The optimum-pH value for this enzyme was 9.5, but within the range 8.3-10.3, the enzyme expressed more than 85% of its maximum activity. This agrees with the results previously referred to for other haloalkane halidohydrolases (Goldman et al., 1968; Little and Williams, 1971; Sallis et al., 1990). However, dehalogenase activity in P. putida was optimal at more acid pH values (Slater et al., 1979). Activity was very low at pH 6-7 and was rapidly lost below pH 6.0. Kinetics Apparent Km values were derived from computed least-square analyses of Lineweaver-Burk plots. Dichloropropionate dehalogenase followed Michaelis kinetics (Fig. 4), with a Km value of 0.66 mM and a Vm~xvalue of 1.39. The Km values of the dehalogenase using the other halogenated
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substrates (MCA, DCA and 2MCPA) all fall within the range 0.23-0.73 mM. These kinetic constants are within the range of values reported for dehalogenases from other bacterial sources (Sallis et al., 1990).
Polyacrylamide gel electrophoresis (PAGE) Unpolymerized acrylamide completely inactivated the enzyme, whilst other components of the gel system (TEMED and ammonium persulfate) also inhibited dehalogenase activity to a lesser extent. In an attempt to remove these components from the acrylamide gels before electrophoresis, the gels were pre-run without samples at 25 mA for 18 h (Hardman and Slater, 1981). Activity stain PAGE, utilizing Ag to precipitate free halide ions in regions of dehalogenase activity, has been employed to identify electrophoretically distinguishable 2-haloacid halidohydrolases (Slater et al., 1979; Hardman and Slater, 1981). Using this technique, one single dehalogenase was identified in cell-free extracts (Fig. 5). This was achieved by electrophoresing four equivalent samples of extract, slicing the gels into four strips and incubating each slice with one of the four substrates (MCA, DCA, 2MCPA, 22DCPA). Slight darkening of slice gel incubated with dalapon was due to partial substrate hydrolysis. Native PAGE runs showed the same single clearance band for each substrate with an Rf of 0.15. The most frequent number of dehalogenases per organism is two (Hardman and Slater, 1981) but some reports describe organisms with a single dehalogenase (Davies and Evans, 1962; Goldman, 1965; Little and Williams, 1971; Sallis, 1990). In general, the density of the bands could serve satisfactorily as a valid comparison between the same or different enzymes (Hardman and Slater, 1981). Nevertheless, the degree of AgCI precipitation was attempted cautiously since the activity of the enzyme towards each substrate scarcely shows differences (Fig. 5), probably due either to an insufficient activity in the sample loaded onto the gel or to a partial loss of activity during the electrophoretic period. Indeed, storage of cell-free extracts at 4°C for 24 h in the presence of dithiothreitol resulted in a 57.2% loss of activity when 22DCPA was used as the substrate.
ACKNOWLEDGEMENTS
We acknowledge grants from the Acciones Integradas hispano-britfinicas Programme No. 145, Junta de Castilla y Leon No. 601/89 and CICYT AGR91-0555.
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REFERENCES Alexander, M., 1965. Biodegradation: Problems of Molecular Recalcitrance and Microbial Fallibility. Adv. Appl. Microbiol., 7: 35-79. Berry, E.I.M., N. Allison, A.J. Skinner and R.A. Cooper, 1979. Degradation of the selective herbicide 2,2-dichloropropionate (dalapon) by a soil bacterium. J. G-en. Microbiol., 110: 39-45. Berry, E.K and A.J. Skinner, 1976. The bacterial degradation of dalapon. Proc. Soc. Gen. Microbiol., 4: 38-39. Bradford, M.M., 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72: 248-254. Bull, A.T., E. Senior and J.H. Slater, 1976. Concerning the evolution of metabolic capabilities in bacteria: Dalapon metabolic capabilities in bacteria: Dalapon metabolism by Pseudomonas putida. Proc. Soc. Gen. Microbiol., 4: 39. Burge, W.D., 1969. Populations of Dalapon-decomposing bacteria in soil as influenced by additions of dalapon or other carbon sources. App. Microbiol., 17: 545-550. Burns, R.G. and J.P. Martin, 1986. Biodegradation of organic residues in soil. In: M.J. Mitchell and J.P. Nakas (Eds), Microfloral and Faunal Interactions in Natural and Agroecosystems. Martinus Nijhoff/Dr. W. Junk, Netherlands, pp. 137-202. Burns, R.G., 1987. Interactions of humic substances with microbes and enzymes in soil and possible implications for soil fertility. Ann. Edaf. Agrobiol., 46: 1247-1259. Busto, M.D., P.P. Smith, M. Perez-Mateos and R.G. Burns, 1989. Isolation ofa dichloropropionate dehalogenase (degrading dalapon) from soil bacteria. VII Simposio Internacional de plaguicidas en suelos y plantas. II Workshop Pesticides-Soils. Day, B.E., L.S. Jordan and R.C. Russell, 1963. Degradation of dalapon in soils. Soil Sci., 95: 326. Davies, J.I. and W.C. Evans, 1962. The elimination of halide ions from aliphatic halogensubstituted organic acids by an enzyme preparation from Pseudomonas putida. Biochem. J., 82: 50. Goldman, P., 1965. The enzymatic cleavage of the carbon-fluorine bond in fluoracetate. J. Biol. Chem., 240: 3434-3438. Goldman, P., G.W.A. Milne and D.B. Keister, 1968. Carbon-halogen bond cleavage. III. Studies on bacterial halidohydrolases. J. Biol. Chem., 243: 428-434. Hardman, D.J. and J.H. Slater, 1981. Dehalogenase in soil bacteria. J. Gen. Microbiol., 123: 117-128. Holly, K., 1964. In: L.J. Audus (Ed.), Plant Physiology and Biochemistry of Herbicides. Academic Press, New York, p. 453. Jensen, H.L., 1957. Decomposition of chloro-substituted aliphatic acids by soil bacteria. Can. J. Microbiol., 3: 151-164. Kaufman, D.D., 1964. Microbial degradation of 2,2-dichloropropionic acid in five soils. Can. J. Microbiol., 10: 843-852. Kawasaki, H., N. Tone and K. Tonomura, 1981. Purification and properties of haloacetate halidohydrolase specified by plasmid from Moraxella sp. strain B. Agric. Biol. Chem., 45: 35-42. Kearney, P.C., J.S. Karns and W.W. Mulbry, 1987. Engineering soil microorganisms for pesticide degradation. Pestic. Sci. Biotechnol. Proc. Int. Congr. Chem. Abstr., 107: 591-596. Little, M. and P.A. Williams, 1971. A bacterial halidohydrolase. Its purification, some properties and its modification by specific amino acid reagents. Eur. J. Biochem., 21: 99-109.
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Perez-Mateos, M., 1989. Interacci6n entre compuestos agroquimicos y enzimas del suelo. Aplicaciones de la biotecnologia de enzimas inmovilizados. Biotecnologia, 5: 8-15. Sallis, P.J., S.J. Armfield, A.T. Bull and D.J. Hardman, 1990. Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2. J. Gen. Microbiol., 136: 115-120. Slater, J.H. and D. Godwin, 1980. In: D.C. Ellwood, J.N. Hedger, M.J. Lathman, J.M. Lynch and J.H. Slater (Eds), Contemporary Microbial Ecology. Academic Press, London, pp. 137-160. Slater, J.H., A.J. Weightman and B.G. Hall, 1985. Dehalogenase genes of Pseudomonasputida PP3 on chromosomally located transposable elements. Mol. Biol. Evol., 2: 557-567. Slater, J.H., D. Lovatt, A.J. Weightman, E. Senior and A.T. Bull, 1979. The growth of Pseudomonas putida on chlorinated aliphatic acids and its dehalogenase activity. J. Gen. Microbiol., 114: 125-136. Slater, J.H., A.J. Weightman, E. Senior and A.T. Bull, 1976. The dehalogenase from Pseudomonas putida. Proc. Soc. Gen. Microbiol., 4: 39-40. Weightman, A.J., A.L. Weightman and J.H. Slater, 1982. Stereospecifity of 2monochloropropionate dehalogenation by the two dehalogenases of Pseudomonas putida PP3: Evidence for two different dehalogenation mechanisms. J. Gen. Microbiol., 128: 1755-1762. Weightman, A.J. and J.H. Slater, 1980. Selection of Pseudomonas putida strains with elevated dehalogenase activities by continuous culture growth on chlorinated alkanoic acids. J. Gen. Microbiol., 121: 187-193.
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Degradation of aliphatic halogen-substituted pesticides by dehalogenase isolated from Pseudomonas alcaligenes. Identification and properties of the enzyme M.D. Busto a, P.P. Smith b, M. Perez-Mateos a and R.G. B u r n s b aDepartment of Biochemistry, Molecular Biology and Physiology, Burgos Faculty of Food Science and Technology, University of Valladolid, Apartado 231, E09071 Burgos, Spain bBiological Laboratory, University of Kent, Canterbury, Kent CT2 7NJ, UK ABSTRACT Some characteristics of a 2,2-dichloropropionate dehalogenase induced in a bacterial strain capable of degrading high concentrations of the herbicide dalapon were studied. Polyacrilamide gel electrophoresis of the crude cell free extracts identified only one type of dehalogenase. The single enzymatic protein showed activity against a variety of chlorinated aliphatic acids but differed in their activity levels. Thus activity in #mol substrate converted (rag protein) -t min -l was 2-monochloropropionate 0.65, 2,2-dichloropropionate 0.56, 2-monochloroacetate 1.70 and 2,2-dichloroacetate 1.00. In the crude extracts, the enzyme activity against 2,2-dichloropropionate was optimal at a broad pH range with a mid-point at pH 9.5 and apparent Km values were within the range 0.23-0.73 mM.
Key words: 2,2-dichloropropionate; Pseudomonas alcaligenes; dalapon; monochloroacetic acid; dichloroacetic acid; 2-monochloropropionic acid; 2,2-dichloropropionic acid
INTRODUCTION
The extensive use of halogenated aliphatic compounds in agriculture and industry has led to soil pollution in many areas (Alexander, 1965). Halogenated pesticides (often used as herbicides) may be destroyed in soil by several different mechanisms. Adsorption and inactivation by clay and organic colloidal materials or other non-biological degradations may conAbbreviations." MCA, rnonochloroacetic acid; DCA, dichloroacetic acid; 2MCPA, 2-monoehloropropionic acid; 22DCPA, 2,2-dichloropropionic acid (dalapon); TEMED, M-N,N,N',N'-tetramethyl-l,2diaminoethane.
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tribute significantly to the environmental decontamination (Holly, 1964). Volatilization or photochemical degradation can also contribute to the removal of certain chemicals from a particular ecosystem (Day et al., 1963) and leaching may occasionally be responsible for the removal of pesticides from surface zones of soil (Holly, 1964). However, the principal means by which pesticides are removed from an environment is a consequence of microbiological activity, which transforms the xenobiotic substances into harmless products (Slater and Godwin, 1980; Burns and Martin, 1986; Burns, 1987; Perez-Mateos, 1989). Abundant evidence exists that some soil microorganisms can dehalogenate chlorinated herbicides such as 2,2-dichloropropionic acid (dalapon) or trichloroacetic acid (TCA) (Kaufman, 1964; Burge, 1969; Berry and Skinner, 1976). The reactions associated with fragmentation of these halogenated aliphatic acids have been ascribed to catalytic activity of substrate-induced enzymes (Jensen, 1957). Several studies have indicated that some bacteria contain a number of dehalogenases differing in their thermal stability, pH dependence, kinetic parameters, mechanism of dehalogenation, electrophoretic mobility or inhibition by sulphydryl-blocking agents (Little and Williams, 1971; Slater et al., 1985; Kawasaki et al., 1981; Weightman et al., 1982). Furthermore, studies with partially purified dehalogenases, have shown that different halogenated alkanoic acids can serve as substrates for these enzymes. Thus, it has been suggested that these enzymes could be used as experimental systems for examining the acquisition of novel catalytic functions coupled with the capacity to grow on new compounds (Bull et al., 1976). Some previous studies have suggested the presence of inducible dehalogenases in a soil bacterial strain which has been grown using dalapon as its sole carbon and energy source (Busto et al., 1989). The soil inoculation of bacteria expressing dehalogenases has been investigated in order to accelerate the degradation of halogenated herbicides in soil and to evaluate the use of inoculates in the bioremediation of contaminated soils (Smith P.P., Ph.D. Thesis, University of Kent, Canterbury, 1991). We report in this paper the identification and characterization of a dehalogenase from Pseudomonas alcaligenes isolated from soil which is able to use dalapon as a sole source of carbon and energy. EXPERIMENTAL
Growth conditions The organism, a Gram-negative rod was isolated from soil (sand 41%, silt 34.0%, clay 24.6%, pH 6.5) following enrichment for 21 days in minimal salt
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medium plus 10 mM 22DCPA. The isolate, identified as Pseudomonas alcaligenes, was grown in minimal salts medium supplemented with 10 mM 22DCPA as the sole carbon and energy source in batch cultures incubated at 30°C on a orbital shaker at 200 rev.min-L Growth of the cultures was estimated by measuring absorbance at 640 nm.
Preparation of cell-free extracts Bacterial culture (1 1) were harvested by centrifugation at 5000 x g for 15 min, washed twice in 20 ml of 0.02 M Tris-sulfate buffer (pH 7.0) and resuspended in 5 ml of the same buffer. A solution (50 /~1) of 0.1 M dithiothreitol was added to 5 ml of bacterial suspensions before the cell disruption. Ice-cold cell suspensions were then disrupted by two passages through a French pressure cell at 83 MPa. Bacterial debris were removed by centrifugation at 30 000 x g for 45 min at 4°C to yield a supernatant which was assayed for enzyme activity immediately. The supernatant solutions contained 2-4 mg crude bacterial protein ml -~, estimated using Bradford's method (1976) and bovine serum albumin as standard.
Dehalogenase assay Dehalogenase activity was measured in 10-ml tubes containing 1 ml of the cell-free extract, 4.5 ml of 0.2 M Tris-sulfate buffer (pH 7.9) and 165 #mol substrate (MCA, DCA, 2MCPA or 22DCPA). The assay mixtures were incubated at 30°C for 30 min and 1-ml aliquots were assayed for free chloride ions. Substrate dechlorination in both culture media and dehalogenase mediated reactions was monitored by measuring free chloride ions concentration using a Marius Chlor-O-Counter as described by Slater et al. (1979).
Polyacrylamide gel electrophoresis The stacking gel was prepared by mixing 0.15 M Tris-sulfate buffer at pH 8.8 with 1.13 M acrylamide and 20 mM methylenbisacrylamide. Polymerization was achieved chemically by the addition of 4.4 mM ammonium persulfate in the presence of 3.28 mM TEMED (a catalyst accelerator). Cell-free extract (0.5 ml), prepared as described for the enzyme assay, was added to 0.5 ml of a solution of 20% (w/v) glycerol in 0.02 M Tris-sulfate buffer (pH 7.9) and to 50 /~1 0.05% (w/v) bromophenol blue. Samples (50-100 td) were loaded into the preformed wells in the stacking gel and electrophoresed at 30 mA for 3 h at 4°C or until the marker front reached approximately two-thirds of the way down the gel. It was necessary to pre-run the gels at 25 mA overnight before adding the extracts.
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Activity of separated dehalogenases in the gel was visualized by the method described by Weightman and Slater (1980). The gel was plunged in several 0.2 M Tris-sulfate buffer solutions at pH 7.9 each containing 50 mM of the halogenated substrates and then was incubated at 30°C for 60 min. The incubated gel was washed in distilled water and placed in a solution of 0.1 M AgNO3. The bands of AgC1 where free chloride ions were present, precipitated in the gel showing the regions of dehalogenase activity. Lightinduced fogging of the gels and obscuring of the AgCI bands produced by enzyme activity, were prevented by removing the excess AgNO3. This was performed by washing the gels in 5% (v/v) acetic acid solutions for 10 min and by soaking in distilled water for 12 h.
Kinetics The kinetic properties of dalapon dehalogenase were investigated by using different substrate concentrations in the assay method described above. Six solutions of 22DCPA whose concentrations ranged between 0.25 and 25 mM were used. The values of Km and Vr~xwere calculated by linear regression using Lineweaver-Burk equation.
Effect of pH on activity To determine the pH-enzyme activity curve, twelve solutions of 0.2 M Tris-maleate, 0.2 M Tris-sulfate and 0.2 M Na/NaOH borate buffers in the pH range 6.0-10.5 were used. Buffers with overlapping pH were included to assess the effects of the buffer itself upon enzyme activity.
Thermal stability Crude extracts were incubated at 50°C and samples were taken at intervals over 50 min of incubation, cooled rapidly with ice and immediately assayed for 22DCPA dehalogenase activity. The changes in activity with time at 50°C were expressed as percentages of the initial activity. RESULTS AND DISCUSSION
Dehalogenase activity of the isolates Bacterial growth appears to be directly correlated with the dechlorination of the carbon sources since differential plots of culture absorbance against CI- release were linear (Fig. 1). In the exponential phase of bacterial growth, when at least 90% of initial dalapon was hydrolyzed, cells were
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Fig. 4. Kinetics of 2,2-dicloropropionate dehalogenase.
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Effect of pH The pH-activity profile of dehalogenase when 22DCPA was used as the substrate is shown in Fig. 3. The optimum-pH value for this enzyme was 9.5, but within the range 8.3-10.3, the enzyme expressed more than 85% of its maximum activity. This agrees with the results previously referred to for other haloalkane halidohydrolases (Goldman et al., 1968; Little and Williams, 1971; Sallis et al., 1990). However, dehalogenase activity in P. putida was optimal at more acid pH values (Slater et al., 1979). Activity was very low at pH 6-7 and was rapidly lost below pH 6.0. Kinetics Apparent Km values were derived from computed least-square analyses of Lineweaver-Burk plots. Dichloropropionate dehalogenase followed Michaelis kinetics (Fig. 4), with a Km value of 0.66 mM and a Vm~xvalue of 1.39. The Km values of the dehalogenase using the other halogenated
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substrates (MCA, DCA and 2MCPA) all fall within the range 0.23-0.73 mM. These kinetic constants are within the range of values reported for dehalogenases from other bacterial sources (Sallis et al., 1990).
Polyacrylamide gel electrophoresis (PAGE) Unpolymerized acrylamide completely inactivated the enzyme, whilst other components of the gel system (TEMED and ammonium persulfate) also inhibited dehalogenase activity to a lesser extent. In an attempt to remove these components from the acrylamide gels before electrophoresis, the gels were pre-run without samples at 25 mA for 18 h (Hardman and Slater, 1981). Activity stain PAGE, utilizing Ag to precipitate free halide ions in regions of dehalogenase activity, has been employed to identify electrophoretically distinguishable 2-haloacid halidohydrolases (Slater et al., 1979; Hardman and Slater, 1981). Using this technique, one single dehalogenase was identified in cell-free extracts (Fig. 5). This was achieved by electrophoresing four equivalent samples of extract, slicing the gels into four strips and incubating each slice with one of the four substrates (MCA, DCA, 2MCPA, 22DCPA). Slight darkening of slice gel incubated with dalapon was due to partial substrate hydrolysis. Native PAGE runs showed the same single clearance band for each substrate with an Rf of 0.15. The most frequent number of dehalogenases per organism is two (Hardman and Slater, 1981) but some reports describe organisms with a single dehalogenase (Davies and Evans, 1962; Goldman, 1965; Little and Williams, 1971; Sallis, 1990). In general, the density of the bands could serve satisfactorily as a valid comparison between the same or different enzymes (Hardman and Slater, 1981). Nevertheless, the degree of AgCI precipitation was attempted cautiously since the activity of the enzyme towards each substrate scarcely shows differences (Fig. 5), probably due either to an insufficient activity in the sample loaded onto the gel or to a partial loss of activity during the electrophoretic period. Indeed, storage of cell-free extracts at 4°C for 24 h in the presence of dithiothreitol resulted in a 57.2% loss of activity when 22DCPA was used as the substrate.
ACKNOWLEDGEMENTS
We acknowledge grants from the Acciones Integradas hispano-britfinicas Programme No. 145, Junta de Castilla y Leon No. 601/89 and CICYT AGR91-0555.
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