Engineering Enzymes for Better Bioremediation Efforts to identify and manipulate these active biochemical agents may lead to more effective bioremediation applications. J E A N N E TROMBLY
ncouraged by a growing number of bioremediation successes, researchers are now concentrating on identifying and optimizing the active biochemical agents involved in this process: enzymes. Knowing about the biodegrading enzymes active in bioremediation projects-whether utilizing bacteria, fungi, or p l a n t t m a y become as important as understanding such factors soil pH, temperature, moisture, and the bioavailability of the contaminants. “Wewandered in the darkness for several years about how to engineer the processes until we could identlfy the enzymes,” said Steve McCutcheon, a research environmental engineer at EPAs Environmental Research Laboratory in Athens, GA. By focusing on the catalytic mechanism ofthe enzyme, many researchers think that bioremediation projects can be made more successful.The first step in this process is to identify critical enzymes. Then scientists can take this knowledge and incorporate the genes that express useful enzymes into other organisms. Enzymes that perform well are being incorporated into indigenous plants and microorganisms that can tolerate the often inhospitable conditions ofpolluted environments better than their nonnative counterparts. Pushing the frontier of this approach are researchers who are using protein engineering to help “boost” the enzyme’s catalytic abilities by redesigning the catalyst to increase its degradative capability and transformation rate. “If we could easily transform enzymes for environmental remediation, we would be living in a completely different world.” observed Peter Holk Nielsen. vice president of Copenhagenbased Novo Nordisk, the worlds largest enzyme producer. Scientists hope that this growing body of enzyme research and application will not only increase the success rates of bioremediation projects but also make environmental cleanup possible at sites 8 6 0 A rn VOL. 29. NO. 12, 1995 I ENVIRONMENTAL SCIENCE &TECHNOLOGY
where current methods have failed. “Redesigned enzymes may provide the only opportunity to remediate certain sites, such as those contaminated with deep, dispersed, and recalcitrant subsurface halogenated hydrocarbons,” said Rick Ornstein, technical group leader at the Department of Energy’s Pacific Northwest Laboratory. A critical look at identifying, enhancing, and redesigning enzymes for toxic waste degradation reveals a few commercial star-ups alongside promising research. The costs of these processes are difficult to pinpoint, because only a few companies are offering their services commercially. Some companies daim that enzyme-enhanced bioremediation can be cost competitive with ex situ approaches.
Identifying the enzyme Enzymes are generally the active agents behind biochemical transformations that take place through bioremediation. The transformation takes place as the enzyme encounters its substrate (the target pollutant) and splits the substrate into component pans or removes part of the molecule. This process occurs very rapidly, leaving the enzyme unaltered and ready to deal with funher molecules of substrate. Enzymes are classified broadly as hydrolytic, oxidizing, or reducing, depending on the type of reaction they control. To better understand and enhance these processes, scientists start by identifying the enzyme. Through a so-called “shotgun” approach commonly used, researchers identify an enzyme with desired characteristics within microorganisms isolated from soil or water samples. They then culture the enzyme-producing microorganism in order to increase its yield or extract the enzyme for cell-free applications. Despite its luck-of-the-draw nature, this approach has traditionally led researchers to find effective enzymes for use in a variety of industries. An 0013-936XrJ510929-56OA~O9.OOm 0 1995 American Chemical Society
enzyme found in the soil of an Indonesian temple is now widely used by softdrink manufacturers to change starch into sugar. Another enzyme found at a Copenhagen cemetery is now used in detergents to P help remove protein stains. An equally challenging process is identifying enzymatic activity and then finding an organism that adequately expresses it. JeanMarc Bollag, co-director of Penn State’sCenter for Bioremediation and Detoxification, is now screening plants from around the world that adequately express laccase and tyrosinase, enzymes that he plans to use to remove phenols from wastewater. Bollag has conducted successful laboratory experiments by applying, with peroxide as a cofactor, The molecular structure of the methane monooxygenase IMMO) enzqme is being studied at the Savannah horseradish plants that River Technology Center to see how environmental factors influence the performance af the enzyme in deexpress the peroxidase en- grading contaminants such as trichloroethylene. A strong oxidizer, MMO oxidizes contaminants near dizyme to phenol-contami- iron active center0 found in the subunit shown in orange. Molecular modeling prediction programs evalunated wastewater ( 1 ) . En- ate the impact of parameters such as pH and temperature on the structure of the enzyme. couraaed bv these results. he thinks the laccase will be equally effective withmediation of organic pollutants-a significant step out the cofactor. beyond the more common practice of using plants By contrast, scientists are meeting quicker sucto pull metals out of soil. “Wherever we have found cess by first identifying plants and microorganisms significant natural activity in the transformation of that appear to naturally degrade toxic wastes, and contaminants mixed with sediment and soil, we have then identifymg the enzyme responsible for the hioisolated plant enzymes as the causative agent,” stated transformation. the researchers.The development of innovative phyAlmost five years ago, a team led by Lee Wolfe, retoremediation, they believe, will revolve around dissearch chemist at EPAS Athens, GA, laboratory, set coveringwhich enzyme systems will degrade chemout to find out why some families of toxic organic icals of concern. compounds degraded faster in certain environA similar philosophy is guiding the work of rements. One team member assumed it was because searchers at DuPont Environmental Remediation Serof enzymes and sought to discover their source. Laura vices (Wilmington,DE). “It is always assumed that Carreira, a research biochemist, was contracted by enzymes are at work in our hioremediation service, EPA to detect the presence of certain enzymes by even though we don’t always know what they are,” modifying the standard ELISA test, an antibody techsaid Dave Ellis, a leader of the DuPont hioremedinique prevalent in the medical testing field. “Firstyou ation group that has developed a process, now availnotice that the degradation happens, then you go and able for licensing, in which natural bacteria dehafigure out how. We now have a tool to do this,” said logenate chlorinated solvents in groundwater. His Caneira. colleague, Martin Oden, monitors a research group Using ELISA, the team verified that the enzymes in Germany that is trying to identifythe enzymes that produced by the plants, not the microorganisms,were are expressed in sulfate-reducingbacteria. “Oncewe responsible for the biodegradation, This body of reknow more. we may look at ways to enhance the ensearch (2)is the first example of successful phytorezymatic activity in place,” said Oden.
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According to Steven Aust, professor of biochemistrv at Utah State Universitv. enzvme identification is one of the most important steps undertaken by his biotransformationcompany. “If you don’t understand the biochemical process of the enzymes, you tun a good chance of failing,”commented Aust. His work led to the development of a small company, Intech One-Eighty (NorthLogan, UT), which licenses a patented process whereby white rot fungus is used to degrade a wide variety of toxic pollutants, including TNT and other explosives, creosote and other polycyclic aromatic hydrocarbons, polychlorinated biphenyls (PCBs).cyanide, and DDT (3). Aust points to instances in whch other researche n tried to replicate the remedial powers ofwhite mt fungi and were disappointed. “What they don’t realize is that not all white rot fungi produce the esI.
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sential ingredient in the cleanup process: the enzyme lignin peroxidase,” he said. “If you don’t have biochemists working with engineers, and you don‘t do the preliminary testing, you’re doomed.’’
Genetic manipulation Groundwork set by enzyme identification can then lead to more creative and challenging uses of these catalysts for environmental cleanup. Projects under way include extracting the enzyme for cell-free application, inserting the genetic material of the enzyme into another organism, and figuring out how to get the enzyme to perform better in its original organism. “We have succeeded in improving an organism’s PCB degradative capability by sitedirected alteration of a PCB-degrading enzyme,” said Frank Mondello, a group leader at the General Electric Research and Development Center (Schenectady, Nn.After discoveringthat two nearly identical PCB-degrading enzymes showed dramatic differences in the range of PCB they attacked, Mondello and a co-worker specifically altered several of the amino acids that differed between the two enzymes. “This modification resulted in a novel strain that exhibits the best activities of both enzymes and which can attack a much wider variety of PCBs than nearly all environmental isolates.” Within the coming year, Mondello expects to conduct further mutagenesis and laboratory soil studies to test the effectiveness of these new strains. “The activity of the organism is good, but whether or not it can do the job on heavily contaminated soil remains to be seen,” said Mondello. This cautious optimism is shared by John Glaser, FPA team leader for soil bioremediation at the National Risk Management Research Laboratory in Cincinnati, OH. Glaser recalls instances where the enzyme was not expressed after its genetic material was inserted into another organism. Besides recreating the genetic expression of enzymes in different host organisms, scientists are using other methods to boost enzymatic degradation of toxic wastes. At the Department of Energy’s Savannah River Site in Aiken, SC, which is a test area for environmental remediation processes, scientists are studying an enzyme on the computer screen to better understand how it performs and potentially increase its effectiveness. a s work builds on a patented bioremediation method used at Savannah~Hiverto treat groundwater contaminated with trichloroethvlene ITCEI. . ,A team assembled by Teny Hazen, environmental microbiologist for the Westinghouse Savannah River Company, recognized that injecting methane into the groundwater triggered the oxidizing abilities of the TCE-degrading methane monooxygenase enzyme, which is expressed in naturally occurring bacteria (4). DOE is currently licensing this system to remediation firms. Hazen is using the three-dimensional computer rendering of the enzyme’s crystal structure Io explore possibilities of manipulating other factors involved in bioremediation to improve the enzyme’s reactivity (see photo. p. 561A). “There are several environmental parameterspH. ionic strength, and temperature. for example-that could cause sigmf-
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Plants horn around the world are being sct L: Bollag (left) and Jeny Dee ai Pennsylvania State Univeroilfs Center for Binremediation and Detoxification to find organisms that express specific e w m e s to remove phenols hom wastewater. Minced horseradish root has proven Mective in recent lahoratbry experiments (i).
icant changes in the enzyme’s structure,” said HaZen. “We use computer modeling prediction programs to go through various scenarios and see how the structure changes.” Hazen and his colleague Ralph Wolf are trying to uncover the details of the oxidative reaction mechanisms at the enzyme’s active site. “Once we better understand how the enzyme performs under different control variables, we can fine-tune the process.” Hazen’s work may one day benefit from research under wav that has identified another methane monooxygenase enzyme that oxidizes TCE at least 50 times faster than other known TCEdegrading enzymes (5).Thomas Wood, assistant pmfessor of biochemical and environmental engineering at the University of California at Irvine, has identified a promising methane monooxygenase enzyme expressed in a slow-growing bacterium. Although to insert the e m - he is exploring-options . matic expression into a faster-growingmicroorganism, Wood admitted that the process is “probablyfive years from commercialization.”
Redesigning enzymes While some scientists push the frontiers of screening or selecting for living organisms to express useful enzymes, at least one is trying to redesign enzymes based on the direct use of fundamental struchu&unction4ynamics relationships. Rick Omstein at Pacific Northwest Laboratory is motivated by the idea of redesigning an enzyme and devising answers for environmental problems that currently have no solutions. If successful, Ornsteids work will lead to an environmental cleanup method for degrading recalcitrant halogenated hydrocarbons in
deep soils or under environmental conditions that are too harsh for any known dehalogenating microorganisms. In one project, Ornstein started with a common soil bacterium cytochrome P450 enzyme that is specific for camphor hydroxylation. His collaborators have recently shown that the native enzyme and mutants can break down certain heavily halogenated ethanes under anaerobic conditions, but 1,l.ltrichloroethane is not affected. A series of comvuter simulations that beean with the X-rav,crvstal , structure of this cytochrome P450 has led to a recent prediction of a double mutant form of cytochrome P450 that is expected to increase 1,l.ltrichloroethane dehalogenation (6). If this prediction is successful,the gene for the redesigned dehalogenating enzyme will be inserted into indigenous microflora that can exist in extreme subsurface conditions before the organisms are returned to their niche. ‘“Presumably,such cells returning to their familiar niche will have a greater than reasonable chance of survival and he able to increase biodegradation of the target compound(s),” stated Ornstein (7). As in situ bioremediation using microorganisms gains acceptance, a bandlid of companies has started offering phytoremediation services, and at least two U.S. companies are commercializing fungal-based systems for environmental cleanup. The capacity of these living organisms to degrade organic and other toxic compounds is expected to increase as their enzymatic bioconversion mechanisms are better understood. But a fundamental debate continues among researchers about the scope of future applications.
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Cell-Ire Extracting enzymes from bacteria and other enzymeexpressing organisms and applying them in their cellfree state tor environmental remediation is a prohibitively expensive process. Although not economically feasible for remediation, cell-free purified enzymes may one day be essential in combating a different type of environmental threat: toxic terrorism. In the wake of the recent Japanese subway attack involving the lethal gas Sarin, the U S . Army acknowledged that enzymes may be effective tools in responding to such civilian threats. According to William White, a senior investigator at the US. Army Chemical Research and Development Canter in Aberdeen, MD, enzymes are probably the best treatment to use in responding to chemical attacks. ”If an airport were hit, you would have to turn the job around as fast as possible,’‘ he said. ”Puiiied enzymes would work rapidly and catalyze the chemical reaction faster and safer than any other known chemical or microbial process.’’ The Army had initially pursued enzymatic remedia9 research because of the nontoxic nature of these talysts. ‘‘We don’t want to spray someihing that is
Limitations of enzymes Researchers recognize that the e m m e specificity that characterizes most enzymes is both a weakness and a strength. Where substrate and enzyme match precisely, enzymes operate with astounding speed and efficiency. “The problem is that toxic waste is rarely a pure stream.” said Nielsen of Novo Nordisk. “But where you have one very poisonous pollutant in pure wastewater streams, enzymes can deal with it.” Many researchers challenge this view. McCutcheon and his team have identified a few nonspecific enzymes, especially those evolved from fairly ancient plants, that are expected to efficiently and simultaneously break down mixes of chemicals such as TNT. “Having plants that contain three or more effective enzyme systems known to degrade classes of compounds hints at the marvelous natural diversity that can be harnessed,” he remarked. Carreira concurs, having found that nitroreductase enzymes, present in about 20% of the plants she tested, are capable of reducing just about any nitro group bound to almost any aromatic ring to an amine. ‘X whole consortium of enzymes can work on sites with multiple pollutants.” Carreira asserted. Another challenge facing the field is understanding the pathway analysis and making sure that the enzyme completes its job. “If an enzyme-based system breaks down one compound into a product which is more toxic than the original substance,you’re worse off than before,” said EPKs Glaser. While debates continue, some scientists are ea-
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going to corrode the metal and pose harm to soldiers,” said Joseph DeFrank who heads the Center‘s environmental research. The Army is currently expanding its enzyme research program to improve its own efforts to use bioremediation for environmental cleanups. According to White, the Armvs initial goal for its bioremediation program was to develop a series of bacteria to treat specific pollutants. However, nonnative bacteria were crowding out the indigenous microorganisms. To work around this, the Army is studying how the genes coded for enzymes that would hydrolyze chlorinated compounds could be incorporated into the indigenous bacteria. James Wild, head of the Department ot Biochemistly at Texas A&M Universw, works with the U.S. Army on several approaches. One project would genetically modify microorganisms with enhanced enzymatic capacky to break down organophosphate neurotoxins; another would encapsulate immobilized organophosphate-hydrolyzing enzymes in a bioreactor for degradation. -JEANNE TROMBLY
ger to showcase the strengths of enzyme-based biodegradation through an integrated approach. “Bacteria versus plants versus fungi is not an eitherlor situation. These systems don’t have to happen exclusively of each other,” stated Milton Gordon, professor of biochemistry at the University of W a s h g ton. Gordon is working with Occidental Petroleum to remediate a large TCE-contaminated site using poplar trees. Other researchers, however, think that resources would be better spent on first understanding the basics of enzyme-based remediation. “With costs of Emediation of Superfund sites topping $1trillion, we simply can’t afford this highly elaborate genetic engineering research for every single problem we have,” insisted McCutcheon.
References I 1 Rollag. I.. Dec. I.RioIeclinoL BiaPng. 1994. 44. 1132-39. 12) Schnaor. I. el. al. Ennuimn. Sn.Terhnol. 1995.29l7).31RA. (31 Barr, D.; Aurl. S. E n w o n . Sci. TPclmol. 1994. 28t21. 78A. .41 Hazen. T. Fnuirnnn~~nlal h ~ e c r i o n . A p r i l1995. 12 15, lahng. 0.; Wood.’I.Appl. Enuim,r Mirmb!ol. 1994.60(71, 2473 (6) Manchester, 1. I.; Omstein.R. L.I. BiomoL Snuct Dyn. in (7)
press. Omstein, R. In Srructuml Biology: The Sfare of the Arc
Sarma, R. H.; Sarma, M. H., Eds.; Adenine Press: Albany, Ny,1993; Vol. 1, pp. 59-76.
leanne Tmmbly is nfreelnncescience writer based in San Francisco, CA. She is program director for the Materials for the Future Foundation.
