Detoxification of Organophosphate Pesticides Using an Immobilized Phosphot riesterase f rom Pseudomonas diminuta

Transcription

1 Detoxifiation of Organophosphate Pestiides Using an Immobilized Phosphot riesterase f rom Pseudomonas diminuta Steven R. Caldwell Department of Biohemistry and Biophysis, Texas A&M University, College Station, Texas Frank M. Raushel* Department of Chemistry, Texas A&M University, College Station, Texas Reeived Deember 13, 1989/Aepted July 13, 199 A purified phosphotriesterase was suessfully immobilized onto trityl agarose in a fixed bed reator. A total of up to 92 units of enzyme ativity was immobilized onto 2. ml of trityl agarose (65 pmol trityl groups/ml agarose), where one unit is the amount of enzyme required to atalyze the hydrolysis of one miromole of paraoxon in one min. The immobilized enzyme was shown to behave hemially and kinetially similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pestiides, methyl parathion, ethyl parathion, diazinon, and oumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For inreased solubility and redution in the affinity of these pestiides for the trityl agarose matrix, methanol/water mixtures were utilized. The effet of methanol was not deleterious when onentrations of less than 2% were present. However, higher onentrations resulted in elution of enzyme from the reator. With a 1-unit reator, a 1. mm paraoxon solution was hydrolyzed ompletely at a flow rate of 45 ml/h. Kineti parameters were measured with a.1-unit reator with paraoxon as a substrate at a flow rate of 22 ml/h. The apparent K, for the immobilized enzyme was 3-4 times greater than the K, (.1 mm) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 4% of the value observed for the soluble enzyme. The ph-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effetive method for hydrolyzing and thus detoxifying organophosphate pestiides and mammalian aetylholinesterase inhibitors. INTRODUCTION The urrent environmental problems assoiated with pestiide usage and ontamination require serious attention. It is estimated that up to 8, individuals eah year are affeted by pestiide poisoning. Of partiular * To whom all orrespondene should be addressed. interest are the organophosphate pestiides sine they omprise the major proportion of the agriultural pestiides utilized in today s agriultural industry (E. Brandt, EPA, Washington, DC, personal ommuniation). Contamination results from either oupational or inidental exposure where adsorption ours by diret ontat with air, food, and water. This lass of agriultural insetiides dominate as the leading ause of pestiide poisoning ases in the United States.* Of equal importane is the environment, where the need for safe, onvenient, and eonomially feasible methods for pestiide disposal must be developed. There are several methods of disposal urrently available,3s4 but most of these have some undesirable limitations. Inineration offers a reliable method but is eonomially restritive, and the proper treatment of the noxious gaseous effluents are of onern. Chemial methods, although viable, leave the problem of disposal of large volumes of austis and aids. Landfills funtion adequately, but leahing into the soil and water is a problemati onsideration. To avoid some of these limitations, the use of enzymati detoxifiation is one method reeiving serious attention. Biologial methods suh as the use of ativated sludge and anaerobi digestion have proved suessful. However, these methods require the maintenane of a biologial system and rely on the prodution of enzymes for detoxifying ontaminants. Reently, an enzyme has been purified and haraterized as a phosphotriesterase with a very broad substrate speifiity. This enzyme has been shown to hydrolyze a substantial number of the most widely used organophosphate pestiides inluding methyl and ethyl parathion, diazinon, fensulfothion, dursban, and oumapho~.~ In addition, this phosphotriesterase is able to degrade deadly nerve toxins suh as sarin and Biotehnology and Bioengineering, Vol. 37. Pp (1997) Q 1991 John Wiley & Sons, In. CCC /91/213-7$4.

2 soman.6 The most harateristi reation zyme involves the hydrolysis of paraoxon : I CH3 CH2 I1 of this en- CH3CHZO-P-OH + HO- -NO2 (1) I CH3CH 2 u The immobilization of suh an enzyme may provide the means whereby toxi substanes an be effiiently degraded in a ontinuous proess. Immobilization avoids the limitation of maintaining a biologial system and usually inreases the stability of the enzymati ativity. The utilization of an immobilized enzyme for degradation of pestiides has been desribed in one instane and involves the immobilization of a parathion hydrolase from a rude ell extrat onto porous silia for the hydrolysis of parathion. Beause the phosphotriesterase from Pseudomonas dirninuta is apable of hydrolyzing many different organophosphate pestiides, its immobilization may allow a stable system where its substrates an be hydrolyzed in a ontinuous fashion under mild environmental onditions. Of the various methods of protein immobilization, it has been suggested that the physial adsorption of an enzyme is the simplest to perform and least affets enzyme ativity. In this study, the speifi method of immobilization was based on the demonstrated hydrophobi nature of phosphotriesterase? Trityl agarose was hosen as the water insoluble matrix beause of the reported high oupling properties and the high retention of ativity upon the immobilization of many different enzymes. In this report, the immobilization of the phosphotriesterase from P. diminuta is desribed. The harateristis of this immobilized enzyme are defined, and the potential ability of this immobilized phosphotriesterase to hydrolyze and thus detoxify several organophosphate pestiides is investigated. MATERIALS AND METHODS Materials Phosphotriesterase was purified to a speifi ativity of 25 units/mg of protein from Spodoptera frugiperdu (sf9) ells transfeted with a reombinant baullovirus system ontaining the loned gene for the enzyme? Trityl agarose (65 pmol trityl groups/ml of agarose) was obtained from Sigma Chemial Co. [3-Cylohexylamino]-l-propane sulfoni aid (CAPS), (2-[N-ylohexylamino] ethanesulfoni aid (CHES), (N-2-hydroxylethyl piperazine)-n-2 ethanesulfoni aid (HEPES), (( 2-N-morpholino) et hanesulfoni aid (MES), piperazine-n,n-bis [2-ethanesulfoni aid] (PIPES), (tris[hydrox y met hyl] met hylaminopropane sulfoni aid (TAPS), dithiothreitol (DTT), diisopropyl fluorophosphate (DFP), and paraoxon were also purhased from Sigma Chemial Companyl. Methyl parathion, ethyl parathion, diazinon, and oumaphos were purhased from Chem Servie. Preparation of the Immobilized Phosphotriesterase The immobilization of phosphotriesterase was aomplished by a proedure similar to that of Cashion et al.9 To a slurry of trityl agarose in 125 mm CHES, ph 9., was added a predetermined amount of enzyme ativity. After gently swirling for at least 1 min, the mixture was poured into a glass olumn reator. After the reators were prepared, they were washed with at least 3 ml of 125 mm CHES, ph 9., and frations of the effluent were olleted and assayed for the presene of enzyme ativity. The extent of oupling of the free enzyme to the trityl agarose was determined by measuring the amount of enzyme ativity before it was applied to the olumn and then subtrating the atalyti ativity that was reovered in the wash frations during and after the reator preparation. A general shemati of the experimental system is shown in Figure 1. Enzymati hydrolysis was measured in two different paked bed reators. A 28-unit enzyme reator was prepared in a glass olumn (.7 x 8 m) with a final volume of 3. ml. The other reator system was prepared in a glass olumn (.5 x 1 m) with a final volume of 2. ml. Several independent olumns were prepared using the latter of the two reator systems with various amounts of enzyme ativity applied (.1, 1., 1., 1, 1, and 92 units of enzyme ativity). These reators were stored at 4 C in 125 mm CHES, ph 9., between experiments. Ativity Measurements Enzyme ativity is defined in units where one unit is the amount of enzyme required to atalyze the hydrolysis of one miromole of paraoxon in one minute. The amount of enzyme ativity was determined by monitor- Figure 1. A general shemati for the experimental system, (A) substrate reservoir, (B) Rainin peristalti pump, (C) fixed bed reator, (D) Gilson Model HM Holohrome UV-visible detetor, and (E) effluent olletor. 14 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 37,JANUARY 1991

3 ing the absorbane of p-nitrophenol at 4 mm (E = 17, M-') produed by the hydrolysis of paraoxon at ph 9. with a Gilford 26 spetrophotometer. The hydrolysis of the organophosphates by the immobilized enzyme was performed at ontinuous flow rates. In all ases, frations were olleted in tubes ontaining 1. mm dithiothreitol, a ompetitive inhibitor for the enzyme (K, = 8.8 PM),~ to ensure protetion from further hydrolysis of any unreated substrate. The hydrolysis of methyl parathion and ethyl parathion was monitored by measuring spetrophotometrially the onentration of p-nitrophenol produed in the olleted frations. The hydrolysis of diisopropylfluorophosphate was determined using an Orion Model fluoride sensitive eletrode by measuring the onentration of fluoride anion. The frations olleted from the hydrolysis of oumaphos were monitored spetrophotometrially at 348 nm (E = 91M-') for the release of 3-hloro-7-hydroxy-4-methyl oumarin. The produt from the hydrolysis of diazinon was measured spetrophotometrially at 228 nm for the presene of 2-isopropyl-4-methyl-6-hydroxy pyrimidine. Solvent Effets The effet of organi solvent on the affinity of the immobilized enzyme for the trityl agarose matrix was determined by washing a 28-unit reator (.7 x 8 m) with 125 mm CHES, ph 9., with various onentrations of organi solvent. At least 1 ml of effluent were olleted at a flow rate of 22 ml/h at eah onentration and the amount of enzyme ativity eluted was assayed as previously desribed. Thermostability of the Immobilized Phosphot riesterase The effet of temperature on the stability of the immobilized phosphotriesterase was determined using a 1-unit reator whih was eluted with.3 mm paraoxon in 125 mm CHES, ph 9., at a onstant flow rate of 2 ml/h at 25 C. After steady state hydrolysis was ahieved, the temperature was elevated in 5 C inrements. At eah ontrolled temperature the steady state hydrolysis was measured and monitored for at least 1 h. The amount of p-nitrophenol produed at steady state was determined as above. The thermostability of the free enzyme was determined by inubating idential enzyme solutions buffered at ph 9. with 5 mm triethanolamine for a period of at least 1 h at ontrolled temperatures. Aliquots were removed at timed intervals and assayed for enzyme ativity. Kineti Analysis Kineti measurements were made with a.1-unit reator at 25 C using paraoxon with onentrations ranging from.3 to.92 mm in 1% methanol at ph 9.. Steady state hydrolysis was measured by monitoring the onentration of p-nitrophenol in the effluent at eah flow rate for at least 3 min with a Gilson Model HM Holohrome detetor equipped with a 4-pL uvette and a Linear hart reorder prealibrated at 4 and 46 nm. Flow rates were kept onstant with a Rainin peristalti pump. Frations were olleted and the amount of p-nitrophenol at ph 9. was measured at 4 nm with a Gilford 26 spetrophotometer to orroborate the hart reorder data. In all ases, the frations olleted ontained DTT (final onentration 1. mm). ph Profile The extent of steady state hydrolysis of 1. mm paraoxon was determined in the ph range of 6-1 at intervals of approximately.5 ph units utilizing several independently prepared 1.-unit immobilized phosphotriesterase reators. Eah reator was equilibrated with the appropriate buffer (5 mm CHES, ph 9.5, ph 9.; 5 mm TAPS, ph 8.5, ph 8.; 5 mm PIPES, ph 7.5, ph 7.; and 5 mm MES, ph 6.5, ph 6.) before the elution of paraoxon. At a onstant flow rate of 21 ml/h, frations were olleted and the ph measured. The ph of the frations was adjusted to ph 9 and the amount of p-nitrophenol was determined spetrophotometrially at 4 nm. Data Analysis Kineti data were analyzed using the least squares method of C1eland.l' Data were applied to the appropriate equation to give the kineti parameters and estimates of the standard errors. The apparent values for K,,, and V, were alulated from a fit to the data to Equation (2): v = v,a/(k,,, + A) (2) where v is the initial veloity in miromoles per minute, V, is highest possible veloity in miromoles per minute, A is the initial paraoxon onentration, and K,,, is the apparent Mihaelis onstant. The pk, value from the phrate profile was determined from a fit to Equation (3): logy = log[/(l + H/Kn)] (3) where y is the amount of paraoxon hydrolyzed, is the ph independent value of y, H is the hydrogen ion onentration, and Kn is the dissoiation onstant of the group that ionizes. RESULTS AND DISCUSSION Immobilization of Phosphotriesterase Phosphotriesterase was suessfully immobilized onto trityl agarose with a very simple and easily performed proedure. A series of olumns ontaining from.1 to 92 units of enzyme ativity was prepared. These CALDWELL AND RAUSHEL: DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES 15

4 o. olumns were defined by the ativity of the soluble enzyme before immobilization and in no manner infer the ativity of the bound enzyme. During the preparation and washing with 125 mm CHES buffer, eah olumn lost an insignifiant amount of enzyme ativity (<.6 units) in the effluent, exept for the 92-unit reator, whih lost 1.2 units of enzyme ativity. The large amount of enzyme applied to the reator may be approahing the funtional limits of this partiular reator (65 pmol trityl groups/ml agarose). However, the high enzymati ativity of this enzyme7 does not neessarily require the preparation of suh a high ativity olumn. Organophosphate pestiides are not very soluble in aqueous systems and ihus require an organi solvent to ahieve higher onentrations. Previous kineti analysis with a variety of organophosphate pestiides hydrolyzed by phosphotriesterase neessitated the use of at least 1% methanol to improve ~olubility.~ Sine the oupling of phosphotriesterase is based on hydrophobi interations, it was neessary to determine the effet of organi solvent on the oupling of the enzyme to the trityl agarose. Figure 2 shows that with the 28-unit fixed bed reator, inreasing the methanol onentration by 1% inrements in the eluting buffer results in approximately a 1-fold inrease in enzyme leakage from the reator. The use of dimethylformamide (DMF) had an even more severe effet, as shown in Figure 2. Any onentration of DMF over 5% led to signifiant losses greater than.3 units/ml. The use of organi solvents imposes some restritions in the use of the immobilized phosphotriesterase for the hydrolysis of substrates with extreme low solubility. With this partiular reator system, limits are set at 2% methanol in the eluting buffer before deleterious effets are observed while DMF is not a suitable solvent. Hydrolysis of Organophosphate Pestiides The appliation of the immobilized phosphotriesterase as a method for degrading various pestiides was evaluated. Shown in Table I are the initial experiments per- o o perent solvent Figure 2. The effet of solvent on the elution of the immobilized phosphotriesterase from trityl agarose (28-unit reator). Methanol (O), DMF (). Additional details are given in the text. formed with a 28-unit reator whih demonstrates the ability of the immobilized phosphotriesterase to degrade the same substrates as the soluble enzyme, namely paraoxon, DFP, methyl parathion, ethyl parathion, and oumaphos at a flow rate of 22 ml/h. This table reflets the extent of hydrolysis for a limited amount of substrate applied to the immobilized enzyme olumn. The reovered volume is the amount of effluent required to ollet the indiated perentage of the reovered produt. Near quantitative hydrolysis of paraoxon is obtained. However, the kinetially slower substrate? were not ompletely hydrolyzed under the onditions imposed. In the ase of oumaphos and ethyl parathion only a low onentration of the unhydrolyzed substrate was olleted and measured in the frations. Moreover, progressively larger volumes of effluent were required to ollet the hydrolysis produts. Therefore, trityl agarose either showed an affinity for the substrates or to the hydrolysis produts. The latter possibility appears very unlikely sine, at ph 9., p-nitrophenol (pk, 7.2) exists primarily in its ionized form and the fluoride anion produed from DFP hydrolysis should not be retarded hydrophobially. The affinity of the substrates and the produts for the olumn matrix were analyzed by eluting paraoxon or p-nitrophenol from a trityl agtrose olumn ontaining no bound enzyme. The results are shown in Table I1 and learly indiate that p-nitrophenol is not retarded. Paraoxon did show a signifiant affinity for the trityl agarose as indiated by the large el, L n volumes required for omplete reovery of the substrate. Furthermore, the addition of methanol to the elution buffer failitates a smaller volume for reovery of this substrate where methanol serves to diminish the hydrophobi interation. These experiments illustrate the affinity of the hydrophobi substrates for the trityl agarose matrix whih results in greater elution volumes required for reovery of the hydrolysis produts. The lak of a signifiant reoverable unhydrolyzed substrate also supports the affinity of the substrates to the matrix. The affinity of a hydrophobi substrate on a hydrophobi matrix is not unusual and has been observed in other systems. l This affinity does not pose a serious threat to the utilization of this system for pestiide degradation sine in a ontinuous proess of hydrolysis, a steady state woilld eventually be realized. However, this substrate partitioning may be expeted to affet the kinetis of the immobilized enzyme. Beause some of the substrates shown in Table I were not ompletely hydrolyzed due to the affinity of the ompounds for the matrix, experiments were onduted using the higher enzyme ativity olumns. Using the 1-unit reator at a flow rate of 11.5 ml/h, 54% of a.25 mm methyl parathion solution ould be hydrolyzed in a ontinuous proess for at least 2 h. This is a marked improvement in hydrolysis over the 34% hydrolyzed and reovered with the 28-unit reator. Diazinon (.7 mm) at ph 9. in 1% methanol was hydrolyzed to greater than 99% ompletion at a flow 16 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 37. JANUARY 1991

5 Table I. Enzymati hydrolysis of substrates with immobilized phosphotriesterase (28- unit reator).a Applied Hydrolyzed Reovery Reovered Conentration volume produt volume produt Substrate (mm) (ml) (%I (ml) Paraoxon Paraoxonb Methyl parathionb Ethyl parathionb Diisopropyl f luorophosphate' Coumaphosd a Substrates were added at a onstant flow rate of 22 ml/h. Elution of the produt and/or unreated substrate was performed with 125 mm CHES, ph 9, exept where otherwise noted. 1% Methanol. ' 1% Methanol in 125 mm HEPES, ph 7. 2% Methanol. Table 11. The affinity of substrates for the trityl agarose matrix. 5% Reovery Total reovery Conentration Volume Reovery volume volume Substrate (mm) (ml) (ml) (ml) p-nitrophenol Paraoxon Paraoxon" a 1% Methanol. rate of 55.5 ml/h. These results indiate that by inreasing the amount of enzyme ativity or hanging the flow rates, improved hydrolysis of these organophosphate pestiides an be realized with little diffiulty. To further define the properties of the immobilized phosphotriesterase, different onentrations of paraoxon were loaded onto a.1-unit reator. Figure 3 illustrates that as the flow rate and the initial paraoxon onentration are inreased, the perentage of hydrolysis dereases. Only 5% hydrolysis ould be ahieved at 11.5 ml/h with.3 mm paraoxon. Similar experiments were performed with the 1. and 1-unit reators. Esalating to the higher immobilized enzyme reator results in a notieable improvement in effetive hydrolysis. For example, near quantitative hydrolysis is observed at onentrations of.97 mm paraoxon at a muh faster flow rate of 45 ml/h with the 1-unit reator. Under these onditions, the omplete hydrolysis of paraoxon was effetively ahieved for at least 2 h at steady state. As would be expeted, the 1.-unit reator is intermediate in its effetiveness toward the hydrolysis of paraoxon. These experiments demonstrate the minimal requirements for near quantitative hydrolysis of paraoxon with this immobilized phosphotriesterase. Thermostability of the Immobilized Enzyme Figure 4 shows the effet of inreased temperature on the ativity of the enzyme. At 55"C, the half-life of 95 min for the immobilized enzyme is signifiantly greater than 16 min for the soluble enzyme. Temperatures at or below 45 C did not result in the inativation e a v N - x P v I x C al e a &-----A Flow rate (rnl/hour) Figure 3. The hydrolysis of paraoxon in 125 mm CHES, 1% methanol at varied flow rates with an immobilized phosphotriesterase fixed bed reator (.1 unit) (A,.92 mm,,.1 mm,,.46 mm); (1. unit) (W,.17 mm,,.87 mm); (1. unit) (*,.97 mm). I CALDWELL AND RAUSHEL: DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES 17

6 ,-. 1 E > Y._ 3 v.-._ > 9 al $ w C al i m [L I lo Time (minutes) Figure 4. Thermostability of () soluble and (A) immobilized enzyme at 45 C (open symbols) and 55 C (shaded symbols). of either the soluble or immobilized enzyme over periods of at least l h. Immobilization of phosphotriesterase has resulted in inreased stability, whih is onsistent with previous observations that immobilization of enzymes an inrease stability." However, even with this relative inreased stability at 55"C, there is a ontinued deline in ativity making the utilization of this immobilized enzyme unfeasible at this temperature. Kineti Analysis The kineti analysis of the immobilized phosphotriesterase is shown in the Lineweaver-Burk plots in Figure 5. Linear plots are observed over a wide range of paraoxon onentration ( mm) at varied flow rates. The linearity of these data suggest that the immobilized enzyme follows Mihaelis-Menten kinetis in this onentration range. Utilizing the FORTRAN programs of Cleland, a best fit analysis gave the apparent Mihaelis onstant and K values shown in Table 111. The apparent K, values of mm are only 3-4 times greater than that of the free soluble enzyme (K, =.1 mm). These higher K, values are in aord with many other immobilized enzyme systems where higher apparent K, values an be attributed to diffusional limits on the substrate by the matri~.""~ This ould also be the result of steri or onformational ef- z E V N x - 2 V I x Y \ b - A / [Paraoxon] mm Figure 5. Lineweaver-Burk plots for the immobilized phosphotriesterase (.1 unit) with varying onentrations of paraoxon at different flow rates: (A), 11.5 ml/h; (O), 22 ml/h; (m), 34 ml/h. Table 111. The apparent kineti parameters and the effet of flow rates on the hydrolysis of paraoxon with the immobilized phosphotriesterase (.1 unit). Flow K, (ml/h) (mm) Fmol/min k fets. As shown in Table 111, the differenes in the K, values are not substantial, but there is a slight inrease in the apparent K,,, with dereasing flow rates, suggesting external diffusional effet~.'~ Internal diffusional effets have been attributed to enzymes possessing high atalyti turnover rates.12*14 Sine the soluble phosphotriesterase exhibits a high atalyti rate at diffusionlimited rates for paraoxon hydrolysis (unpublished results of S.R. Caldwell and F.M. Raushel), a larger apparent K, relative to the soluble enzyme would be expeted. However, this effet on K, may have been minimized by substrate partitioning and the affinity of the substrates for the trityl agarose.14 Shown in Table I11 is the effetiveness of the immobilized enzyme (.1-unit reator) in degrading paraoxon. Regardless of the flow rate, the maximum performane of this reator is about 4% that of the free soluble enzyme. The value of Vmax for the enzymati hyarolysis of paraoxon in the free soluble system is 23 s-'.~ This lower effetiveness may be attributed to steri or onformational effets imposed by the immobilization of the enzyme. The lower effetiveness may also be a result of internal diffusional limits and/or the inherent high atalyti effiieny of the soluble phosph~triesterase.'~ ph Profile The ph ativity profile for the immobilized phosphotriesterase when ompared to the kineti ph profile for the soluble enzyme' is shown in Figure 6. In this figure the values for log V,,, are plotted in arbitrary veloity units.7 Sine the ph ativity urve at saturating onentrations of paraoxon for the soluble enzyme is similar to the orresponding log V,,, and log V/K profiles for the soluble enzyme, suh a omparison is justified. The kineti pk, for the group that must be unprotonated for ativity has been shifted from 6.1 k.1 for the soluble enzyme7 to 6.8 k.1 for the immobilized enzyme. Shifts of the pk, value in the ph profiles of immobilized enzymes are generally expeted with a harged matrix upp port.'^ However, in the experiments reported here there is no harged support, and the substrate solution is buffered. The shift might be explained on the basis of the highly hydrophobi environment to whih the enzyme has been immobilized where the miroenvironment of the essential amino aids have been affeted. The slight variation might also be attributed to possible onformational effets on the ative site. 18 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 37, JANUARY 1991

7 TI I I al N x - P I mn Q r x - I - T PH.1 Figure 6. The effet of ph on the hydrolysis of saturating paraoxon by the soluble enzyme () and the immobilized phosphotriesterase (). The values for V,,, of the soluble enzyme are in arbitary units. Regardless, a omparison of the two profiles serve to underline the similarity of the atalyti performane of the immobilized enzyme in relation to the free enzyme. The immobilized phosphotriesterase on trityl agarose provides an effetive system for the degradation of some organophosphate pestiides and a mammalian aetylhohesterase inhibitor. The hemial and kineti parameters of the immobilized phosphotriesterase are omparable to those of the free enzyme. However, one of the primary limitations of the immobilized phosphotriesterase system is the negative effet of high onentrations of organi solvent, whih results in the elution of enzyme ativity from the reator. The effet of organi solvents (>l%) was also shown to be deleterious to the immobilized parathion hydrolase desribed by Munneke. In this ase the negative impat was the inhibition of the enzyme, not the diret loss of enzyme ativity from the reator, and restoration of the enzymati ativity was possible by removing the solvent from the reator. To realize the effetive hydrolysis of high onentrations of organophosphate pestiides by the immobilized phosphotriesterase, a solubilizing agent with minimal effets on the retention of the immobilized enzyme or the inhibition of enzyme ativity will be required. At present, these types of reagents are being evaluated. Another limitation of the immobilized phosphotriesterase system involves eonomi onsiderations. Though the immobilization of this enzyme onto trityl agarose is quite simple, the ost of using trityl agarose as a support matrix on a very large sale may beome ost preventative. For this reason, other methods of immobilizing phosphotriesterase are being developed. Preliminary experiments have indiated that the immobilization of this phosphotriesterase onto a nylon mem- brane is funtional and has the extra advantage of being relatively ost effetive. Current studies are now being direted at this system. This work was supported in part by the Army Researh Offie (DAAL ) and the Texas Advaned Tehnology Program. FMR is the reipient of NIH Researh Career Development Award (DK-1366). Referenes 1. J. Feldman, Subommittee of Department Operations, Researh and Foreign Agriulture, Committee on Agriulture, US. House of Representatives, Otober 6, 1983 (US. Government Printing Offie, Washington, DC, 1983). 2. U.S. Environmental Protetion Ageny, Offie of Pestiides Programs, Pestiide Regulation Division (1976), National Study of Hospital Admitted Pestiide Poisonings. 3. D. M. Munneke, Pro. Biohem., 13, 14 (1978). 4. D. M. Munneke, R. Day, and H.W. Trask, Review of Pestiide Diposal Researh, U.S. Environmental Protetion Ageny, Washington, DC (1976). 5. D. P. Dumas, S. R. Caldwell, J. R. Wild, and F.M. Raushel, J Biol. Chem., 264, (1989). 6. D. P. Dumas, H. D. Durst, W.G. Landis, F. M. Raushel, and J. R. Wild, Arh. Biohem. Biophys. 227, 155 (199). 7. W. J. Donarski, D.P. Dumas, D. H. Heitmeyer, V. E. Lewis, and F. M. Raushel, Biohemistry, 28, 465 (1989). 8. D. M. Munneke, Biotehnol. Bioeng., 21, 2247 (1979). 9. P. Cashion, A. Javed, D. Harrison, J. Seeley, V. Lentini, and G. Sathe, Biotehnol. Bioeng., 23, 43 (1982). 1. W.W. Cleland, The Enzymes, 2, 1 (197). 11. A. Johansson and K. Mosbah, Biohim. Biophys. Ata, 37,348 (1974). 12. L. Goldstein, in Methods in Enzymology (K. Mosbah, Ed.) (Aademi, New York, 1976), p D. Thomas and G. Brown, Biohimie, 54,229 (1972). 14. M.D. Trevan, in Immobilized Enzymes, (Wiley, New York, 198), p L. Goldstein, Y. Lewin, and E. Kathalaski, Biohemistry, 3, 1913 (1964). CALDWELL AND RAUSHEL: DETOXIFICATION OF ORGANOPHOSPHATE PESTICIDES 19