Environ. Sci. Technol. 1994, 28, 306-31 1
Comparison of Substituted 2-Nitrophenol Degradation by Enzyme Extracts and Intact Cells Brian R. Folsom,' Ruth Stierll, R e d P. Schwarzenbach, and Josef Zeyer Swiss Federal Institute for Water Resources and Water Pollution Control (EAWAG), SFLK, CH-6047 Kastanienbaum, Switzerland
The first catabolic pathway enzyme, nitrophenol oxygenase, transforms o-nitrophenol (ONP) to catechol. Thirteen of 16 substituted nitrophenols tested were actively transformed by both enzyme preparations and intact cells yielding a wide range of Km (K,) and Vmax. Individual chemicals in binary mixtures demonstrated competitive inhibition. Chemical and physical characteristics (electron withdrawal, size, and position of substitution on the 2-nitrophenol ring) affected degradation kinetics. The strongest correlations were between Km or Vmax values and electron withdrawal, though there was also evidence for effects relating to position and size of substitution on the aromatic ring. Kinetic parameters determined for enzyme preparations did not correlate to those determined for intact cells. Though enzyme reactivity ultimately determined whether a given chemical would be transformed, the transformation by intact cells was apparently affected by factors other than those directly impacting the initial catabolic enzyme.
Introduction
Nitroaromatics are ubiquitous contaminants which enter the environment as wastes, pesticides, explosives, and dyes (1-4) or are formed through photochemical processes ( 5 ) or biological activity (6,7). Several members of this group pose significant health and environmental rists. 2-Nitrophenol (0-nitrophenol or ONP) and 2,4-dinitrophenol are listed as priority pollutants by the US. EPA (2). Biodegradation of nitroaromatics can be initiated by both reductive and oxidative mechanisms. The reductive pathways involve conversion of the nitro substituent to an amino group through the action of nitroreductases (3, 8-12). Many nitroreductases have been reported to possess broad substrate specificity (3,9),which leads to the rapid formation of aromatic amines in the environment (1,5). In the oxidative pathways, nitrite is directly released from the aromatic ring. This mechanism has been demonstrated with nitrophenols (4, 13-16), nitrotoluenes (17, 181, and nitrobenzoates (19). Pseudomonas p u t i d a B2, isolated from soil, grows on ONP as the sole source of carbon and nitrogen (4, 13). ONP is catabolized first to catechol, which is subsequently degraded via an ortho cleavage pathway (4). Initial enzymatic transformation is by a nitrophenol oxygenase that has been isolated and purified (20). Although both the purified nitrophenol oxygenaseand the cell suspensions of P. putida B2 can act on severalsubstituted nitrophenols, only ONP was shown to be fully mineralized (4). One objective of this study was to further characterize the breadth of substrate specificity for the nitrophenol oxygenase.
* Address correspondence to this author at his present address: Envirogen Inc., 4100 Quakerbridge Rd., Lawrenceville, NJ 08648. 306
Envlron. Scl. Technol., Vol. 28, No. 2, 1994
Previously, we reported chemical and physical constants which influence environmental partitioning for a set of substituted nitrophenols (21). Trends were clearly established between chemical parameters such as electronegativity and physical characteristics such as pK, and octanol/water partition coefficients. The new data presented in this paper attempted to establish whether these chemical characteristics could be related to degradation kinetic constants. The investigation used both purified enzymes, as a simple model system, and intact microorganisms, which represent the environmentally significantly catalytic unit. M e t h o d s and Materials
Chemicals. The physical properties and abbreviations of the nitrophenols used in this study are listed in Table 1. Sources of selected chemicals were reported earlier (4, 20, 21). Additional chemicals were from commercial sources: chloramphenicol (Aldrich Chemical Co., Steinheim, Germany); NADPH and bovine serum albumin, fraction V (Boehringer Manheim AG, Rotkreuz, Switzerland); and 6-Me-ONP (Dr. Braun, Cosmital SA, Marly, Switzerland). All chemicals were of the highest purity available and were used as received. Microorganism, Preparation of Cell Suspensions, and Enzyme Preparations. Isolation and characterization of the ONP-degrading strain P. putida B2 was described previously (13). Batch cultures of organisms induced for nitrophenol degradation were grown on a basal medium (pH 7.5) (20) supplemented with 1mM ONP and 0.02% yeast extract (11). An optimal pH for growth of bacteria and degradation of ONP was previously determined to be at 7.5 (13). As soon as the ONP was depleted, cells were harvested by centrifugation, washed, and suspended in 20 mM phosphate buffer, pH 7.5. Concentrated cell suspensions (1 g of wet weight cells in 12 mL of buffer) were supplemented with 10mM chloramphenicol (to inhibit protein synthesis) and 1 mM succinate (to stabilize the cells) and stored at 4 OC in phosphate buffer. The activity of cell suspensions was determined within 5 h after a 100-fold dilution that significantly lowered the concentrations of both chloramphenicol and succinate in solution. Crude cell extracts were prepared from resting cells by sonication as described previously (20). The nitrophenol oxygenase was partially purified through the gel filtration step (20), stored at 4 "C, and used within 4 days. Assay Conditions. All assays were performed in phosphate buffer (20 mM, pH 7.5) under aerobic conditions. A pH of 7.5 was previously determined to be optimal for enzymatic activity (20). Nitrophenol transformation rates for cell suspensions were determined following dilution to 50-200 pg of protein/mL. Assays were initiated by the addition of a nitrophenol stock solution to a final concentration of 100 FM. Unless otherwise indicated, 0013-936X/94/0928-0306$04.50/0
0 1994 American Chemical Society
Table 1. Constants and Values for Selected Substituted Nitrophenols no.
1 2
3 4 5
6 7 8 9
10 11 12 13 14 15
16
compound 2-nitrophenol 5-fluoro-2-nitrophenol 4-chloro-2-nitrophenol 6-chloro-2-nitrophenol 3-methyl-2-nitrophenol 4-methyl-2-nitrophenol 5-methyl-2-nitrophenol 6-methyl-2-nitrophenol 2,4-dinitrophenol 2,5-dinitrophenol 4-methoxy-2-nitrophenol 4-trifluoromethyl-2-nitrophenol 4-chloro-5-methyl-2-nitrophenol 6-methyl-2,4-dinitrophenol 4-secondary butyl-2-nitrophenol 4-phenyl-2-nitrophenol
abbreviation
€0
pKab
ac
Yvdwd
log K O 2
ONP 5-F 4-C1 641 3-Me 4-Me 5-Me 6-Me &NO2 5-NO2 4-OMe 4-CF3 4-Cl-5-Me 4-NO2-6-Me 4-sBu 4-Ph
3 490 5 000 3 980 4 560 990 2 820 3 560 1980 10 300 3 210 2 830 2 610 4 370 11 530 2 540 2 470
7.23 6.30 6.43 5.35 7.00 7.63 7.34 7.65 3.94 5.13 7.40 5.65 6.84 4.31 7.59 6.69
0.00 0.06 0.37 0.37 -0.13 -0.07 -0.17 -0.07 0.71 0.78 0.12 0.43 0.20 0.35
3.45 5.80 12.00 12.00 13.67 13.67 13.67 13.67 16.87 16.87 16.87 20.48 25.67e 30.47" 44.35 45.84
1.89 1.91 2.46 ndf 2.29 2.37 2.31 nd 1.67 1.80 2.02 2.34 2.93 2.21 3.84 3.71
-0.10
0.06
Molar extinction coefficient determined at 410 nm (20 mM phosphate buffer, pH 7.5,20 -+ 0.5 "C). Values from ref 21, except for 6-Me and 6-C1which were determined as outlined previously (22). Hammett constant for inductive electron withdrawal (29). Value for substituent position relative to 2-NO2 group. van der Waals radius for volume of substituent only (30). e Total volume of both substituents. f nd = value not determined. a
nitrophenol transformation assays using partially purified enzyme included 4 mM MgSOd, 100 pM NADPH, 0.1 mg/ mL bovine serum albumin (BSA). Assays were initiated by the addition of a nitrophenol stock solution to a final concentration of 10 pM. Addition of BSA to the assay mixture minimized irreversible inhibition at high substrate concentrations yet did not affect initial transformation rates at lower substrate concentrations. Therefore, standard assay conditions included BSA as a stabilizing agent. Transformation rates were calculated based only on the final concentration of the partially purified enzyme added to the reaction mixture (excluding BSA additions). Transformation of individual nitrophenols to catechols was monitored spectrophotometrically (UvikonModel 810 spectrophotometer, Kontron, Zurich, Switzerland) at 410 nm in 1-or 5-cm path length quartz cuvettes maintained at 20 f0.5 "C. Transformation rates were calculated using molar extinction coefficients as listed in Table 1. HPLC was used to separate components of mixtures, and concentrations were determined by using a UV detector. Reactions were stopped by transferring 0.5-mL aliquots of the assay mixture to a 1.5-mL microfuge tube containing 0.05 mL of 2 M HC1 plus an internal standard (either 4-OMe or 4-Cl-5-Me). Samples were extracted with 0.5 mL of ethyl acetate. HPLC separations were performed on a C12 reversed-phase column, with methanol:water, 65:35 (v:v), acidified by addition of 1/10 vol of 0.1 M phosphate pH 3 as reported earlier (21). Retention times for the various nitrophenols were as follows: ONP, 2.4 min; 4-C1,4.1 min; 4-Me, 3.8 min; &Me, 3.8 min; 4-OMe, 3.1 min; and 4-C1-5-Me, 7.4 min. Nitrophenol concentrations were determined by comparison to external standards. Nitrophenol transformation rates from either method were expressed as wmol min-l (g of protein)-1 after the determination of protein concentration as outlined previously (23). Theory. The analysis of results from mixtures of two substrates used a basic competitive inhibition relationship (eq 1)where Vm,, Km, and S refer to the first substrate and where KIand I refer to the second substrate or inhibitor (22). The second substrate may or may not be transformed
by the enzyme.
Results and Discussion Transformation Kinetics for Several Substituted Nitrophenols. Nitrophenol transformation kinetics were characterized for both partially purified nitrophenol oxygenase and for cell suspensions of P. p u t i d a B2. The model enzyme system offered the greatest level of experimental control while the cellular system generated environmentally significant degradation rates. Initial transformation of the colored nitrophenol to the colorless catechol is mediated by the nitrophenol oxygenasein both systems. The nitrophenol oxygenase requires nitrophenol, 0 2 , NADPH, and Mg2+for full activity ( 4 ) . Transformation rates for ONP, 4-Me, and 5-Me were determined using enzyme preparations over a range of nitrophenol concentrations at three NADPH concentrations. 0 2 and inorganic ion concentrations were kept constant. Initial transformation rate data were collected during the first 10% loss of substrate, yielding linear plots with correlation coefficients typically greater than 0.90. Secondary plots of transformation rate data (primary slope or primary intercept from Lineweaver-Burk plots versus 1/[NADPHI) yielded apparent Km values of 1.06,0.45, and 16.50 pM and Vm, values of 480,264, and 543 pmol min-1 (g of protein)-l for ONP, 4-Me, and &Me, respectively. Although Km and V, were different for these three nitrophenols, the apparent Km for NADPH remained essentially constant at 68 f 1p M . All subsequent enzyme assays were performed at 100 pM NADPH. This concentration was close to saturation and was 10-100 times higher than the concentrations of nitrophenol used. In general, NADPH concentrations decreased less than 1pM during initial kinetic determinations and would have little effect on the kinetic values determined for comparative purposes. Variations in Kmand V, for this group of nitrophenols suggested a relationship between kinetics and chemical Environ. Sci. Technol., Vol. 28, No. 2, 1994
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Table 2. Enzymatic and Cellular Parameters for Substituted Nitrophenol Transformation
compd ONP 5-F 4-C1 6-C1 3-Me 4-Me 5-Me 6-Me 4-NO2 5-No2 4-OMe 44x3 4-Cl-5-Me 4-NO2-6-Me 4-sBu 4-Ph
nitrophenol oxygenase Vmm [pmol min-l K, (g of protein)-'] (pM) 283 142 85 174 222 188 238 263
