19 IsolatedEnzymesfor theTransformation and DetoxificationofOrganicPollutants LilianaGianfreda UniversityofNaplesFedericoII, Portici,Naples, Italy Jean-Marc Bollag Centerfor BioremediationandDetoxification,ThePennsylvaniaStateUniversity, University Park,Pennsylvania I.INTRODUCTION A.Pollutionof theEnvironment Pollutionoftheenvironmenthasbeenoneofthelargestconcernsfor scienceandthe generalpublicinthelast50years.Therapidindustrializationofagriculture,expansions inthechemicalindustry,andtheneedtogeneratecheap formsofenergyhaveallcaused the continuousreleaseofanthropogenicorganicchemicalsintothebiosphere.Conse- quently,the atmosphere,thehydrosphere,andmanysoilenvironmentshavebecomepol- lutedtoalesserorgreaterextentbyalargevarietyofxenobioticcompounds (1).Some toxiccompoundsareresistantto physical,chemical,orbiologicaldegradationandthus constituteanenvironmentalburdenofconsiderable magnitude(2).Athighconcentration, orafter prolongedexposure,somexenobioticshavethepotentialtoproduceadverseef- fectsinhumansandotherorganisms;theseincludeacutetoxicity,carcinogenesis,muta- genesis,andteratogenesis.Moreover, substances usually found at very low concentrations, and not considered as pollutants, may become contaminants because of their bioaccumula- tion in food chains to high concentrations (3). Some of the principal sources of environmental contamination with organic chemi- cals are current or decommissioned industrial sites where there has been spillage of wastes of various origins. Thus both petroleum- and coal-derived fossil fuel–related materials, effluents from vehicle and equipment cleaning and maintenance, wood-preserving chemi- cals, paper mill effluents, and a host of pesticides are organic chemicals that find their way into the environment (3). In addition, feedlot operations and landfill sites generate potential pollutants of soil, water, and atmosphere. Finally, industrial sites that produce, store, and distribute organic chemicals may be considered one of the largest sources of environmental pollutants. Soils and groundwater are natural and preferred sinks for contamination, and their pollutionrepresentsanimportantconcernforhumanandenvironmentalhealth.Table1 Copyright © 2002 Marcel Dekker, Inc. Table 1 Common Pollutants in Soil and Water Chemicals Potential source Location Pesticides Pesticide manufacture; pesticide Soil, water, sediments application Volatile organic compounds (e.g., Industrial and commercial wastes Soil benzene, toluene, xylenes, di- chloromethane, trichloroethane, trichloroethylene) Polychlorinated biphenyls (PCBs) Insulators in electrical equipment Soil, water (100 different isomers in com- mercial use) Chloroderivatives (e.g., chlorophe- Paper mill effluents; bleached Soil, water nols, chlorobenzene) kraft mill effluents; wood indus- try; solvents, intermediates in pesticide manufacture Polycyclic aromatic hydrocarbons Creosote waste sites; fossil fuel Soil, water (PAHs) (e.g., naphthalene, an- wastes; by-products of old gas thracene, fluorene, phenan- manufacturing threne) summarizes some of the more common pollutants of soil and water and lists their potential origins and possible locations in the environment. Among these chemicals, some, such as the polycyclic aromatic hydrocarbons (PAHs), have long been recognized as a worldwide environmental contamination problem because of their intrinsic chemical stability, high resistance to all types of degradation, and carcinogenic and genotoxic effects (4). PAHs, which occur ubiquitously in the environment as complex mixtures, are formed mainly during industrial combustion such as coke production, catalytic cracking, or power genera- tion by fossil fuels. N-Heterocyclic compounds, which constitute the second largest group of chemicals present in coal-derived material; chlorophenols, largely used as wide-spec- trum biocides for the control of bacteria, fungi, algae, molluscs, and insects (5); and ni- trophenols, which are widely used in the chemical industry, all accumulate in soils, sedi- ments, natural waters, and animals because of their long-term usage and recalcitrant nature. Trichloroethylene (TCE), as well as other halogenated compounds, may be persistent in the environment, partly because of its physical properties (i.e., high density and water solubility and low chemical reactivity) and partly because of its biological recalcitrance (i.e., ability to resist microbial degradation). B. Remediation Technologies The U.S. Environmental Protection Agency (EPA) has classified those pollutants whose removal from soil and water is considered an indispensable priority for environmental cleanup and human health. As a result, research groups, not only in the United States but also in other countries, are putting great effort into the exploration of new strategies di- rected at remediating contaminated systems. Remediation is a general term that indicates the use of techniques suitable for partial or total recovery of a polluted system. In other words, physical, chemical, and biological treatments are applied to remove as much as possible of the contaminant(s) from the site or to transform it into an innocuous or less- toxic compound. Copyright © 2002 Marcel Dekker, Inc. A complete remediation program usually requires more than one step, including (1) knowledge of the past history of the polluted area and activities leading to the contamina- tion of the site, (2) examination and quantification of the severity of the contamination problem, (3) development of the remediation action program to target the specific contami- nant or group of contaminants, and (4) development of a treatment sequence suited for the wastes and the site. With regard to soil, remediation techniques can be distinguished as hard or soft tech- niques depending on the intensity of chemical and/or physical manipulations required for the contaminated area and the expected cost of the operation. Bioremediation, typically, is thought of as a soft technique requiring less equipment and cost than many other methods. Bioremediation usually refers to the use of biological processes that transform pollutants into innocuous products, and it is the activities of biological agents (microorganisms, plants and their enzymes) that account for most of the transformation. However, physical and chemical processes contribute directly or indirectly to the removal of some compounds under certain environmental conditions (6). In natural systems, bacteria, fungi, and yeasts and their enzymatic components bio- degrade a large variety of hazardous compounds. However, such natural processes can be very slow, and consequently certain chemicals may persist for years. Bioremediation technologies usually help natural biodegradation processes work faster, or they may pro- vide additional, exogenous biological agents to polluted systems and improve the transfor- mation processes. Depending on the type of remediation strategy used, treatments for the recovery and restoration of both aquatic and terrestrial polluted systems may be carried out in situ or ex situ. Where appropriate, in situ strategies usually are preferred because they generally do not require expensive and sometimes dangerous manipulations of the environment. Ex situ treatments require the excavation of the polluted material; its transfer to another loca- tion, where remediation takes place; and its return to the original site. C. Potential for Using Isolated Enzymes As previously stated, microorganisms are among the main agents for the transformation and degradation of organic chemicals. A large number of genera and species of aerobic and anaerobic bacteria (including actinomycetes) and fungi have all been shown to transform, cometabolize, and metabolize many anthropogenic organic compounds. The complete mineralization of organics to simple nontoxic compounds (e.g., CO 2 ,NH 4 ,H 2 O) may occur, and this is usually the desired outcome in bioremediation. One of the most utilized techniques for in situ bioremediation of contaminated sites is the enhancement (or biostimulation) of the indigenous microbial activity by removal of existing constraints (e.g., supplying necessary nutrients, electron acceptors, moisture, or aeration). An alternative methodology is the inoculation of laboratory cultures of known degraders into the contaminated environment. However, this technique, referred to as bio- augmentation, has often been unsuccessful (7) because (1) the concentration of pollutant in the site is too low to sustain the growth of the microbial inoculant; (2) inoculated microor- ganisms are inactivated by toxins or natural predators in the environment; (3) inoculated microorganisms prefer centrally metabolized other (natural) organic substrates rather than the pollutants as their substrates; (4) the movement of microorganisms to sites containing pollutants (which are usually discontinuously distributed) is hindered in solid environments such as soil (8); (5) inoculants are outcompeted by indigenous microorganisms that are highly evolved and well adapted to the nutrient status of the environment; and (6) the Copyright © 2002 Marcel Dekker, Inc. expressionofthepollutant-degradingproperty(demonstratedinvitro)maynotbeinduced inthenaturalenvironment.Theregulatingandcontrolmechanismsoforganismswithnovel degradativeabilitiesarepoorlyunderstood,andunpredictableprocessesmayoccurunder naturalconditions.Furthermore,bioaugmentationmethodologyrequirescontinuousanaly- sisandmonitoringofmicrobialpopulationdynamicstodefinethepersistenceandactivity oftheinoculant(s)frombothefficacyandriskassessmentperspectives.Otherfactorsin- volvingtheintrinsictoxicityandsolubilityofthecompoundsorthetypeornatureofthe microbialstrainmayinfluencetheefficacyofamicrobialinoculum. Onestrategyforovercomingthelimitationstotheuseofmicroorganismsinthedetoxi- ficationoforganic-pollutedsites(andparticularlytheprocessesunderlinedinpoint[6])is theuseofcell-freeenzymepreparations;areviewofresearchaimedatdevelopingtheuse ofisolatedenzymesinsolid,liquid,andhazardouswastetreatmenthasbeenpublished(9). Theseauthorsexaminedthepotentialapplicationofseveralenzymesaccordingtocategories ofspecificwastetypes(e.g.,phenolsandrelatedcompounds,pulpandpaperwastes,pesti- cides,foodprocessingwastes,solidwasteandsludgetreatment,andheavymetals). D.OriginofEnzymes Asoutlinedinthepreviousparagraphs,microorganismsdegradingvariousaliphatic,ali- cyclic,aromatic,andheterocycliccompoundshavebeenidentifiedandisolated.Inmany cases,thedetailedbiochemicalpathwaysandenzymesresponsibleforthemainreactions ofthedegradationpatternhavebeencharacterized.Microorganismsproduceenzymesable toreactwithchemicalsdifferentfromthosebeingutilizedasprimarycarbonandenergy sources(9),thusbecomingapotentialsourceofalargearrayofenzymesusefulforthe transformationofvariousxenobioticcompounds. Althoughmostresearchhascenteredonbacterialprocesses,fungalenzymeshave beenshowntobeinvolvedinthetransformationofmanytoxicorganiccompounds(10). Forexample,manywhiterotfungi,suchasbasidiomycetes,secreteenzymes(e.g.,ligni- nase,manganeseperoxidase,laccase)involvedinlignindegradation.Theseenzymesseem tobenonspecificallyreactivetowardmanyorganicpollutants(11,12). Inareviewdedicatedtofundamentalandappliedresearchinthemicrobialmetabo- lismofxenobiotics,Singleton(13)reportedthatrelativelyfewenzymescapableofdegrad- ingxenobioticshavebeenstudiedandpurified.Apentachlorophenolmonooxygenase,a dichloromethanedehalogenase,anda2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoicacid– (HOPDA)-reducingenzymearecitedasexamplesofthefewenzymespurifiedfrombacte- ria.Ligninasesandligninperoxidasesareusedasexamplesoffungalenzymes.Nonethe- less,thefeasibilityofusingfungalenzymesinthedecontaminationofsoilcontaining phenolicandaniliniccompoundshasbeensuggested(14). II.APPLICABLEENZYMESANDTHEIRPROPERTIES A.Hydrolases Someorganicpollutants,especiallypesticides,maylosetheirtoxicpropertiesafterhy- drolyticreactionscatalyzedbynonspecifichydrolases(Table2).Forexample,thebreak- down of esteric, amidic, and peptidic bonds by esterases, amidases, and proteases may lead to products with little or no toxicity. However, in some cases toxic by-products may be formed. The hydrolysis of halogen-carbon bonds also may occur and is catalyzed by a group Copyright © 2002 Marcel Dekker, Inc. Table 2 Examples of Hydrolases Purified from Microorganisms Active Toward Organic Chemicals Enzyme Microrganism Substrate Reference Acylamidases Fusarium oxysporum Acylanilides 44 2-Ketocyclohexanecarboxyl Rhodopseudomonas palustris 2-Ketocyclohexanocarboxyl coenzyme A 40 Coenzyme-A hydrolase Chlorohydrolase Pseudomonas spp. Atrazine 38, 39 Malathion hydrolase Soil microorganisms Malathion 32 N-methylcarbamate hydrolase Achromobacter spp., Pseudomonas spp. strain CRL- Carbaryl, carbofuran, aldicarb 41,42 OK Parathion hydrolase Pseudomonas, Brevibacterium, Azotomonas, Xantho- Parathion, triazophos, paraoxon, diazinon 17–19 monas spp. Flavobacterium spp. Parathion; methyl parathion 20,29 Pseudomonas spp. Organophosphates 22,24 Nocardia spp. Coumaphos, Parathion 31 Permethrinase Bacillus cereus Pyrethroids 37 Copyright © 2002 Marcel Dekker, Inc. of halidohydrolases. These enzymes are part of a larger group of dehalogenases that are discussed later (see B3. Hydrolytic dehalogenation; b. Halidohydrolases, p. 509). 1. Parathion Hydrolases A large number of aquatic and terrestrial species (microorganisms and animals) are known to produce enzymes capable of hydrolyzing organophosphorus compounds. The enzymes are generally called organophosphorus acid anhydrases, although other names such as phosphotriesterases, parathion hydrolases, somanases, and paraoxonase have been used. Parathion hydrolases are among the most studied classes of enzymes that possess pesticide-hydrolyzing abilities. They have received great attention from researchers be- cause of their presence in organophosphorus insecticide-resistant organisms (15,16). The results obtained in several studies seem to indicate that this enzyme and similar hydrolases are responsible for parathion and methyl parathion resistance in some strains of insects. It is possible to obtain parathion hydrolase in large quantities from various bacterial species that help to make the enzyme commercially viable for the detoxification of pesticide- polluted sites. Munnecke (17–19) reported that a crude, cell-free parathion hydrolase preparation isolated from a bacterial mixture that included Pseudomonas sp., Brevibacterium sp., Azo- tomonas sp., and Xanthomonas sp. was able to hydrolyze several organophosphates, such as parathion, triazophos, paraoxon, diazinon, methyl parathion, chlorpyrifos, fenitrothion, and cyanophos. The enzymatic hydrolysis of almost all the pesticides occurred at rates 40 to 1000 times more rapid than those of chemical methods. Furthermore, the enzyme was stable in the presence of the detergents and solvents used to solubilize and prepare pesticide mixtures (19). Three unique parathion hydrolases were purified from Gram-negative bacterial strains (20). Two membrane-bound hydrolases and one cytosolic hydrolase were purified from a Flavobacterium sp. strain ATCC 27551, a strain SC, and a strain B1, respectively. The purified proteins demonstrated similarities in their affinity for ethyl parathion and their broad temperature optimum around 40°C. Conversely, they differed in their composi- tion (e.g., number and molecular mass of proteic subunits), their response to sulfhydryl reagents (dithiothreitol) and to metal salts (CuCl 2 ), and their substrate range. The B1 hy- drolase showed equal affinity for parathion and the related organophosphate insecticide O-ethyl-O-4-nitrophenyl phenylphosphonothionate (EPN), whereas the Flavobacterium sp. enzyme displayed twofold lower affinity for EPN than for parathion and the SC-pro- duced enzyme was not active toward EPN as a substrate. The capability of 18 Gram-negative bacterial isolates to hydrolyze the organophos- phorus compound diisopropyl fluorophosphate (a structural analog of the nerve gas agents soman and sarin) was investigated (21). The authors compared the detoxifying ability of crude enzyme preparations (frozen cell sonicates and acetone powders) by measuring the hydrolytic release of fluoride and the disappearance of acetyl cholinesterase inhibition in vivo. The highest activities were present in acetone powder preparations from a strain with known parathion hydrolase activity. The results demonstrated that parathion hydrolase was not specific with regard to its phosphotriesterase activity and showed a significant detoxifying activity at low concentrations (Ͻ1 enzymatic unit g Ϫ1 protein, e.g., 1 µmol of substrate transformed min Ϫ1 g Ϫ1 protein) and near-neutral pH. A phosphotriesterase (a parathion hydrolase) from Pseudomonas diminuta was char- acterized for its kinetic behavior toward different organophosphorus compounds (22). To obtain the enzyme in a purified form, the organophosphate degradation gene, opd, was Copyright © 2002 Marcel Dekker, Inc. cloned in an Escherichia coli strain. At pH 7.0 and 25°C, the enzyme showed a higher af- finity for paraoxon (K m value ϭ 0.012 mM ) than for diisopropyl fluorophosphate (K m ϭ 0.12 mM ) and behaved at pH 9.0 as a competitive inhibitor of paraoxon with an inhibi- tion constant K i ϭ 0.32 mM. In addition, an enzyme capable of degrading parathion and methyl parathion at pH 8.0 and fenitrothion at pH 8.5 was obtained from an isolated strain, YF11. For the three insecticides, the optimal reaction temperature was 32.5°C and the K m values ranged from 18.7 to 212.4 µM for parathion and fenitrothion, respectively (23). Crude enzyme extracts from a Pseudomonas sp. and a Xanthomonas sp., both iso- lated from a pesticide disposal site in northern Israel, also displayed parathion hydrolase activity (24). Crude enzyme preparations degraded parathion but showed different sensi- tivity to cations such as Cu 2ϩ ,Fe 2ϩ ,Ca 2ϩ ,Mn 2ϩ ,Al 3ϩ ,Zn 2ϩ , and sodium ethylenediamine- tetraacetic acid (EDTA). Cu 2ϩ strongly inhibited the Pseudomonas sp. enzyme, but it had a stimulatory effect on parathion degradation by the Xanthomonas sp. enzyme. A significant inhibition of the Xanthomonas sp. hydrolase but not of the Pseudomonas sp. hydrolase was also reported for NaEDTA. Great efforts have been made to apply recombinant deoxyribonucleic acid (DNA) technology to the production of parathion hydrolase. Typical genetic studies, involving two bacteria, Pseudomonas diminuta and Flavobacterium sp., were carried out by Mulbry et al. (25,26). Cloning experiments, using DNA–DNA hybridization and restriction map- ping techniques, indicated that two discrete plasmids from the encoding parathion- hydrolyzing soil bacteria possess a common but limited region of sequence homology within potentially nonhomologous plasmid structures. Genetic engineering was also applied by Coppella et al. (27) to obtain large amounts of parathion hydrolase. When the gene encoding the enzyme was cloned into Streptomyces lividans, the transformed bacterium was able to express and secrete the enzyme. Fermenta- tion conditions were investigated to improve and enhance enzyme production (27,28). The fermentation was first carried out in the presence of large quantities of nutrients supplied throughout the fermentation period and by sparging with oxygen-enriched gas. Enzyme activity and production did not further increase when cultivation was prolonged more than 90 hours. The authors concluded that some enzyme deactivation occurred and that the rates of enzyme synthesis and deactivation were balanced after 90-h periods. In another study, parathion hydrolase cloned from a Flavobacterium sp. into S. liv- idans was produced in large amounts (milligrams of protein) and purified to homogeneity (29). The enzyme, characterized for its structural and catalytic features, was a single- polypeptide chain with an apparent molecular mass of 35 kD. The enzyme had an affinity in the order of O-ethyl-O-p-nitrophenyl phenylphosphothionate Ͼ parathion Ͼ and p- nitrophenyl ethyl(phenyl) phosphinate Ͼ methyl parathion, as assessed by K m values. An optimal pH of 9.0 and an optimal temperature of 45°C were determined. As observed for other parathion hydrolases, the enzyme was inhibited by dithiothreitol and CuSO 4 . These results demonstrated that the purified recombinant enzyme presented the same characteris- tics as those of the protein produced by the donor Flavobacterium sp. strain. Further studies suggested that the use of a native Streptomyces sp. signal sequence may have resulted in more efficient secretion of the heterologous protein (30). In 1998 another bacterium, Nocardia sp. B1, was reported to hydrolyze organophos- phate insecticides, such as coumaphos and parathion (31). The enzyme organophosphorus hydrolase (OPH) was isolated and shown to be active toward organophosphate insecti- cides. However, it was demonstrated, and explained by genetic studies, that OPH activity in Nocardia sp. often was spontaneously lost during growth in the laboratory (31). Copyright © 2002 Marcel Dekker, Inc. 2. Other Hydrolases There is relatively little evidence of other isolated esterases suitable for the detoxification of organic pollutants. In addition, where it is reported, there is little detailed information on the metabolites and their toxicity. For example, Getzin and Rosefield (32) described the properties of a partially purified enzyme extracted from soil that degraded the insecticide malathion to its monoacid derivative. The enzyme had a high resistance to both thermal and microbial deactivation, probably because of a carbohydrate moiety attached to the protein (33). A HOPDA-hydrolyzing enzyme involved in the degradation of biphenyl was puri- fied to homogeneity from Pseudomonas cruciviae S-93-B1 that was grown on biphenyl as the sole carbon source (34–36). The hydrolytic reaction occurred between the C5-C6 bond of HOPDA, to produce benzoic acid and 2-oxopent-4-enoic acid. Further studies showed the production of three HOPDA-reducing enzymes (I,II,III) in the bacterium, hav- ing different catalytic and structural properties. Experiments performed with methylated- HOPDA derivatives and ring-fission products as substrates allowed new metabolic diver- gence of biphenyl and related compounds to be proposed (35,36). An enzyme responsible for hydrolyzing second- and third-generation synthetic pyre- throids and producing noninsecticidal metabolites was isolated from a pyrethroid-trans- forming strain of Bacillus cereus (37). The enzyme, named permethrinase, was purified as a single protein chain of 61-kD molecular mass. It had a pH optimum of 7.5 and a temperature optimum of 37°C. Several characteristics (i.e., no requirement for cofactors or coenzymes; sensitivity to tetraethylpyrophosphate; protection by dithiothreitol against the inhibition effects of sulfhydryl agents, p-chloromercuribenzoate, and N-ethylmalei- mide) suggested that the microbial esterase was a carboxylesterase. The gene, sequence, enzyme purification, and characterization of a novel enzyme involved in the metabolic transformation of atrazine to carbon dioxide and ammonia via the intermediate hydroxyatrazine by a Pseudomonas sp. strain ADP were studied and described by de Souza et al. (38). Genetic studies previously performed on the bacterium allowed the production of hydroxyatrazine to be ascribed to a specific DNA fragment (39). Furthermore, sequence analysis of the fragment indicated that a single open-reading frame, named atzA, encoded an activity transforming atrazine to hydroxyatrazine. The protein was purified and characterized and had an oligomeric structure with a molecular mass of 245 kD. Chlorohydrolase rather than oxygenase activity was attributed to the purified enzyme by studies performed with H 2 18 O that was converted to 18 O-hydroxy- atrazine. The enzyme that dechlorinated atrazine, simazine, and desethylatrazine (but not melamine, terbutylazine, or desethyldesisopropylatrazine) was apparently a novel enzyme that also participated in the hydrolysis of atrazine in soils and groundwaters (38). As discussed later, this enzyme could also be considered a dehalogenase acting on haloaro- matic compounds. A hydrolase-catalyzing ring cleavage reaction (which is very uncommon) during anaerobic degradation of benzoate was isolated from the anaerobic bacterium Rhodopseu- domonas palustris and purified (40). The enzyme 2-ketocyclohexanecarboxyl coenzyme A (2-ketochc-CoA) hydrolase catalyzed the hydration of 2-ketochc-CoA to pimelyl-CoA. The native protein was a homotetramer of 34-kD subunits. The enzyme had no sensitivity to oxygen, and its production was induced by growing the bacterium on benzoate and other benzoate intermediates. Enzymes capable of hydrolyzing the carbamate linkage of the pesticides carbofuran and carbaryl were purified from an Achromobacter sp. (41) and Pseudomonas sp. strain Copyright © 2002 Marcel Dekker, Inc. CRL-OK(42),respectively.ThePseudomonassp.–extractedenzymewasdemonstrated tobeauniquecytosolicenzyme,abletohydrolyzecarbofuranandaldicarbbutnotthe phenylcarbamateisopropylm-chlorocarbinilate,thethiocarbamateS-ethyl-N,N-dipropyl- thiocarbamate,orthedimethylcarbamateO-nitrophenyldimethylcarbamate(42). Earlypapersreportedthepurificationandpropertiesofacylamidasesresponsible forthehydrolysisofacylamidesand/orphenylureas(43,44).However,toourknowledge, nofurtherstudieshavebeencarriedoutwiththeseenzymesaimedspecificallyattheir applicationinthedetoxificationoforganicpollutantsintheenvironment. Alotofliteratureisavailableonotherhydrolasessuchasproteases,lipases,and cellulases.Alloftheseenzymeshavebeenisolatedandpurifiedfromseveralsources, theirpropertieshavebeenwellcharacterized,andmostareavailablecommercially.How- ever,thereisnodirectevidencethattheseenzymesareinvolvedinthetransformation oforganicpollutants.Nonetheless,proteasescouldbeconsideredagroupofhydrolases particularlyusefulforthetreatmentofwastesderivedfromfoodprocessing(e.g.,fishand meatwastes).Indeed,theycansolubilizeproteinspresentinwastestreamsandgenerate productswithaddednutritionalvalue(9).Similarly,cellulasesmayhydrolyzelignocellu- loseandcellulosepresentinmunicipalsolidwastesorpaperindustrywastes.Thepossibil- ityofobtainingenergysourcessuchasfermentablesugars,biogas,andendproductssuch asethanolhasattractedtheattentionofseveralresearchers,andnumerousstudieshave beenconductedinthisfield(9). Theinterestinlipasesstemsmainlyfromthecapabilityoftheseenzymestobe agentsofnonconventionalenzymatictransformationssuchassyntheticratherthanhy- drolyticcatalyticreactions.Esterificationandtransesterificationcanbeachievedifthe processisperformedinorganicorwater/organicsolvents.Similarsyntheticreactionscan becatalyzedbyproteasesaswell.Severalinvestigationshavebeenconductedonthe propertiesofproteasesandlipasesandtheirinvolvementintheformationofsynthetic products.Forexample,aproteasefromBacilluslicheniformiswasshowninanhydrous organicsolventstocatalyzethepolytransesterificationofadiesterofglutaricacidwith aromaticdiolssuchasbenzenedimethanol(45). Studiesalsowereaimedatdefiningprochiralselectivityofbothlipasesandproteases wheninvolvedintheorganicsolvent–mediatedtransformationof2-substituted1,3-pro- panedioloritsdiester(46).Amechanisticmodelwasproposedthatpredictedaninverse correlationbetweenlipase’sprochiralselectivityandsolventhydrophobicityaswellas particulareffectsofsubstratestructurevariation. Inthecontextofthischapter,theimportanceoftheseenzymesandtheirvarying activityinsoilhavetobementionedwithregardtodifferentmanagementand/orfertiliza- tiontreatments.Forexample,proteaseactivitiesstronglyincreasedwhensolidurban wasteswereappliedtothreedifferentsemiaridareasoils,thusshowingtheresponseof thesoilmicrobialpopulationtotheappliedorganicmatter(47). B.Dehalogenases Halogenatedcompoundsarepresentintheenvironmentaseithernaturallyoccurringor syntheticintroducedcompounds.Adetailedoverviewofmicroorganismscapableofme- tabolizinghalogenatedcompoundsisprovidedinChapter18. The removal of halogen atoms from aliphatic and aromatic halogen-carbon-substi- tuted compounds is an essential step in the biochemical transformation of pollutants; the reaction reduces or eliminates toxicity. The cleavage of carbon-halogen bonds may occur Copyright © 2002 Marcel Dekker, Inc. by (1) enzymatic dehalogenation catalyzed by specific enzymes (dehalogenases), (2) a fortuitous reaction catalyzed by enzymes with a broad substrate specificity and acting on halogenated analogs of their natural substrate (discussed later), or (3) spontaneous dehalogenation of unstable intermediate products of unrelated enzymatic reactions. Dehalogenases have received continuous interest because they not only may play an important role in the remediation of the environment, but also may be applied in biotechno- logical transformations for producing biologically active compounds (discussed late). Slater and coworkers (48) classified dehalogenases according to dehalogenation mechanisms in three groups, namely: hydrolytic dehalogenases, haloalcohol dehalogenases (hydrogen ha- lide lyases), and cofactor-dependent dehalogenases. A further classification may be made in relation to substrate specificity. In an exhaustive review of the mechanisms by which halogenated compounds may lose their halogen substituents, the enzymes involved in the reactions, the products, and the biotechnological applications of these enzymes, Fetzner and Lingens (49) identified seven dehalogenation mechanisms, taking into account both the mechanism of the enzymatic reaction and the substrate involved in it. Both types of classification are summarized in Fig. 1 and discussed for the following six dehalogenation mechanisms: 1. Reductive Dehalogenation In the reductive dehalogenation mechanism, halogen-carbon bonds are replaced by hydro- gen-carbon bonds, with a concomitant release of halogen ions. The process can be per- Figure 1 Reactions catalyzed by dehalogenases. Copyright © 2002 Marcel Dekker, Inc. [...]... 2-monochloropropionate (2-MCPA), producing d-lactate as product and being uninhibited by SH-blocking agents (group 2); (2) enzymes using both d- and l-isomers of 2MCPA as substrate, producing compounds with an inverse optical configuration and being unaffected by SH-blocking agents (group 3); (3) enzymes still active toward both d- and l-2-MCPA isomers, but with retention of configuration and inhibition by SH-blocking agents... products with the same optical configuration (fraction I), and the other yielded products with the opposite optical configuration (fraction II) The two dehalogenases showed other differences in their responses to SH-blocking agents and their efficiency in dechlorinating l- and d-2-MCPA isomers Conversely, a unique dl-2-haloacid dehalogenase catalyzing the hydrolytic dehalogenation of both d- and l-2-haloalkanoic... maximum at 50°C and at pH between 8.5 and 10.5 The enzyme was inactivated at temperatures above 50°C and was inhibited with a mixed-type inhibition mechanism by 2-chloroacetic acid and 2,2-dichloroacetic acid Punctual amino acid modification studies indicated the importance of one or more cysteine and arginine residues in the catalysis and the stability of the protein structure (103) The lb enzyme isolated... C-1 to C-4 1Cl or 1-Br n-alkanes, C-2 to C-5 1-halo-n-alkanes, C-3 to C-5 Br-n-alkanes, C-2 to C-6 halogenated alcohols, halomethanes) is produced by haloalkane-degrading microorganisms (49) In the case of dichloromethane, the hydrolysis to formaldehyde and inorganic chloride may also occur through a thiolytic cleavage catalyzed by a dehalogenating glutathione transferase that requires glutathione The. .. on 2,4-dichlorobenzoate, catalyzing the NADPH-dependent ortho-dehalogenation to 4-Cl-benzoyl-CoA of the intermediate 2,4-dichlorobenzoyl-CoA, which had formed in the first step of the metabolic degra- Copyright © 2002 Marcel Dekker, Inc dation by the action of 2,4-dichlorobenzoyl ligase and required magnesium adenosine triphosphate (Mg ATP) and CoA An enzymatic fraction, capable of catalyzing the direct... occurring within the tertiary structure of the protein after immobilization Denaturing effects can be due to the great number of linkages between the enzyme and the support and to the interference of hydrophilic residue of the carrier with the hydrophobic groups of the enzymatic protein The mode of attachment may involve groups that are part of the active site Furthermore, immobilization of the protein... alter the binding between the various subunits of the enzymes (195 ) Steric limitations on the penetration of the substrate into the enzyme active site may be the result of restricted movement of the substrate to the otherwise available active site of the enzyme or of the orientation of the enzyme in relation to the carrier surface In other words, the active site may be partially or completely inaccessible... cruciviae S-93 B-1 involved in the degradation of biphenyl Agric Biol Chem 50:931–938, 198 6 T Omori, H Ishigooka, Y Minoda Purification and some properties of 2 hydroxy-6-oxo-6phenylhena-24-dienoic-acid reducing enzyme from Pseudomonas-cruciviae S-93B-1 involved in the degradation of biphenyl Agric Biol Chem 50:1513–1518, 198 6 T Omori, H Ishigooka, Y Minoda A new metabolic pathway for meta ring-fission compounds... trichloro-p-hydroquinone and dichloro-p-hydroquinone but was not active in the presence of reduced nicotinamide-adenine dinucleotide phosphate (NADPH), reduced nicotinamide-adenine dinucleotide (NADH), dithiothreitol, or ascorbic acid as a reducing agent A similar dehalogenating activity was isolated and purified from Sphingosomonas chloro-fenolica, a soil bacterium that degrades pentachlorophenol (58,59) The. .. by the bacterium; 2-hydroxy-5-methylquinone is formed as the product The enzyme is a monomeric protein of 65 kD, contains 1 mol FAD/mol protein, and requires NADPH and oxygen (162) A dimeric nitroalkane-oxidizing enzyme, containing flavin mononucleotide rather than flavin dinucleotide as the prosthetic group, was purified to homogeneity from Neurospora crassa (163) The enzyme catalyzes the oxidation of . Inc. expressionofthepollutant-degradingproperty(demonstratedinvitro)maynotbeinduced inthenaturalenvironment.Theregulatingandcontrolmechanismsoforganismswithnovel degradativeabilitiesarepoorlyunderstood,andunpredictableprocessesmayoccurunder naturalconditions.Furthermore,bioaugmentationmethodologyrequirescontinuousanaly- sisandmonitoringofmicrobialpopulationdynamicstodefinethepersistenceandactivity oftheinoculant(s)frombothefficacyandriskassessmentperspectives.Otherfactorsin- volvingtheintrinsictoxicityandsolubilityofthecompoundsorthetypeornatureofthe microbialstrainmayinfluencetheefficacyofamicrobialinoculum. Onestrategyforovercomingthelimitationstotheuseofmicroorganismsinthedetoxi- ficationoforganic-pollutedsites(andparticularlytheprocessesunderlinedinpoint[6])is theuseofcell-freeenzymepreparations;areviewofresearchaimedatdevelopingtheuse ofisolatedenzymesinsolid,liquid,andhazardouswastetreatmenthasbeenpublished(9). Theseauthorsexaminedthepotentialapplicationofseveralenzymesaccordingtocategories ofspecificwastetypes(e.g.,phenolsandrelatedcompounds,pulpandpaperwastes,pesti- cides,foodprocessingwastes,solidwasteandsludgetreatment,andheavymetals). D.OriginofEnzymes Asoutlinedinthepreviousparagraphs,microorganismsdegradingvariousaliphatic,ali- cyclic,aromatic,andheterocycliccompoundshavebeenidentifiedandisolated.Inmany cases,thedetailedbiochemicalpathwaysandenzymesresponsibleforthemainreactions ofthedegradationpatternhavebeencharacterized.Microorganismsproduceenzymesable toreactwithchemicalsdifferentfromthosebeingutilizedasprimarycarbonandenergy sources(9),thusbecomingapotentialsourceofalargearrayofenzymesusefulforthe transformationofvariousxenobioticcompounds. Althoughmostresearchhascenteredonbacterialprocesses,fungalenzymeshave beenshowntobeinvolvedinthetransformationofmanytoxicorganiccompounds(10). Forexample,manywhiterotfungi,suchasbasidiomycetes,secreteenzymes(e.g.,ligni- nase,manganeseperoxidase,laccase)involvedinlignindegradation.Theseenzymesseem tobenonspecificallyreactivetowardmanyorganicpollutants(11,12). Inareviewdedicatedtofundamentalandappliedresearchinthemicrobialmetabo- lismofxenobiotics,Singleton(13)reportedthatrelativelyfewenzymescapableofdegrad- ingxenobioticshavebeenstudiedandpurified.Apentachlorophenolmonooxygenase,a dichloromethanedehalogenase,anda2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoicacid– (HOPDA)-reducingenzymearecitedasexamplesofthefewenzymespurifiedfrombacte- ria.Ligninasesandligninperoxidasesareusedasexamplesoffungalenzymes.Nonethe- less,thefeasibilityofusingfungalenzymesinthedecontaminationofsoilcontaining phenolicandaniliniccompoundshasbeensuggested(14). II.APPLICABLEENZYMESANDTHEIRPROPERTIES A.Hydrolases Someorganicpollutants,especiallypesticides,maylosetheirtoxicpropertiesafterhy- drolyticreactionscatalyzedbynonspecifichydrolases(Table2).Forexample,thebreak- down. seems to indicate that a single enzyme able to react with a broad range of haloalkanes (e.g., C-1 to C-4 1- Cl or 1-Br n-alkanes, C-2 to C-5 1-halo-n-alkanes, C-3 to C-5 Br-n-alkanes, C-2 to C-6 halogenated. l-isomers, producing d-isomers No 3 Active on d- and l-isomers, producing l- and d- isomers No (inversion of configuration) 4 Active on d- and l-isomers, producing l- and d-isomers (re- Yes tention