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 T R O M B L Y
Encouraged by a growing number of bioremediation successes, researchers are now concentrating on identifying and optimizing the active b iochemical agents involved in this process: enzymes. Knowing about the biodegrading enzymes active in
bioremediation projects—whether utilizing bacteria, fungi, or plants—may become as important as understanding such factors soil pH, temperature, moisture, and the bioavailability of the contaminants. "We wandered in the darkness for several years about how to engineer the processes until we could identify the enzymes," said Steve McCutcheon, a research environmental engineer at EPA's Environmental Research Laboratory in Athens, GA.
By focusing on the catalytic mechanism of the 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 tha t can tolerate the often inhosp i tab le conditions of polluted 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 Hoik Nielsen, vice president of Copenhagen-based Novo Nordisk, the world's 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
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 halo-genated 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 start-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 claim 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 parts or removes part of the molecule. This process occurs very rapidly, leaving the enzyme unaltered and ready to deal with further 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
enzyme found in the soil of an Indonesian temple is now widely used by soft-drink manufacturers to change starch into sugar. Another enzyme found at a Copenhagen cemetery is now used in detergents to help remove protein stains.
An equally challenging process is identifying enzymatic activity and then finding an organism that adequately expresses it. lean-Marc Bollag, co-director of Perm State's Center for Biore-mediation 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, horseradish plants that express the peroxidase enzyme to phenol-contaminated wastewater (i). Encouraged by these results,
he thinks the laccase will be equally effective without the cofactor.
By contrast, scientists are meeting quicker success by first identifying plants and microorganisms that appear to naturally degrade toxic wastes, and then identifying the enzyme responsible for the biotransformation.
Almost five years ago, a team led by Lee Wolfe, research chemist at EPA's Athens, GA, laboratory, set out to find out why some families of toxic organic compounds degraded faster in certain environments. One team member assumed it was because of enzymes and sought to discover their source. Laura Carreira, a research biochemist, was contracted by EPA to detect the presence of certain enzymes by modifying the standard ELISA test, an antibody technique prevalent in the medical testing field. "First you notice that the degradation happens, then you go and figure out how. We now have a tool to do this," said Carreira.
Using ELISA, the team verified that the enzymes produced by the plants, not the microorganisms, were responsible for the biodégradation. This body of research (2) is the first example of successful phytore-
The molecular structure of the methane monooxygenase (MMO) enzyme is being studied at the Savannah River Technology Center to see how environmental factors influence the performance of the enzyme in degrading contaminants such as trichloroethylene. A strong oxidizer, MMO oxidizes contaminants near di-iron active centers found in the subunit shown in orange. Molecular modeling prediction programs evaluate the impact of parameters such as pH and temperature on the structure of the enzyme.
mediation of organic pollutants—a significant step beyond the more common practice of using plants to pull metals out of soil. "Wherever we have found significant natural activity in the transformation of contaminants mixed with sediment and soil, we have isolated plant enzymes as the causative agent," stated die researchers. The development of innovative phy-toremediation, they believe, will revolve around discovering which enzyme systems will degrade chemicals of concern.
A similar philosophy is guiding the work of researchers at DuPont Environmental Remediation Services (Wilmington, DE). "It is always assumed that enzymes are at work in our bioremediation service, even though we don't always know what they are," said Dave Ellis, a leader of the DuPont bioremediation group that has developed a process, now available for licensing, in which natural bacteria deha-logenate chlorinated solvents in groundwater. His colleague, Martin Oden, monitors a research group in Germany that is trying to identify the enzymes that are expressed in sulfate-reducing bacteria. "Once we know more, we may look at ways to enhance the enzymatic activity in place," said Oden.
Engineering a more effective PCB-degrading organism Researchers are genetically engineering strains of PCB-destroying organisms to maximize the organisms' ability to degrade a broad range of PCB congeners. Frank Mondello and colleagues at the General Electric Research and Development Center have conducted laboratory test tube studies with three strains: Pseudomonaspseudoalcallgenes{KFJQ7), Escherichia co//(FM4560, a recombinant organism containing genes from Pseudomonas strain LB400), and £ co//strain BDE335-5 (a site-directed mutant of FM4560). Strains were incubated for 24 h with two different mixtures of PCBs (each congener at 5 μπι). Percent degradation is indicated by dot size.
According to Steven Aust, professor of biochemistry at Utah State University, enzyme identification is one of the most important steps undertaken by his biotransformation company. "If you don't understand the biochemical process of the enzymes, you run a good chance of failing," commented Aust. His work led to the development of a small company, In-tech One-Eighty (North Logan, 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 which other researchers tried to replicate the remedial powers of white rot fungi and were disappointed. "What they don't realize is that not all white rot fungi produce the es
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 capabil i ty by site-directed alteration of a PCB-degrading enzyme," said Frank Mondello, a group leader at the General Electric Research and Development Center (Schenectady, NY). After discovering that 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, EPA 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.
This work builds on a patented bioremediation method used at Savannah River to treat groundwater contaminated with trichloroethylene (TCE). A team assembled by Terry 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 to explore possibilities of manipulating other factors involved in bioremediation to improve the enzyme's reactivity (see photo, p. 561A). "There are several environmental parameters—pH, ionic strength, and temperature, for example—that could cause signif-
Plants from around the world are being screened by Jean-Marc Bollag (left) and Jerzy Dec at Pennsylvania State University's Center for Bioremediation and Detoxification to find organisms that express specific enzymes to remove phenols from wastewater. Minced horseradish root has proven effective in recent laboratory experiments (/).
icant changes in the enzyme's structure," said Ha-zen. "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 way that has identified another methane monooxygenase enzyme that oxidizes TCE at least 50 times faster than other known TCE-degrading enzymes (5). Thomas Wood, assistant professor 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 he is exploring options to insert the enzymatic expression into a faster-growing microorganism, Wood admitted that the process is "probably five 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 structure-function-dynamics relationships. Rick Orn-stein 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, Ornstein's 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 1,1,1-trichloroethane is not affected. A series of computer simulations that began with the X-ray crystal structure of this cytochrome P450 has led to a recent prediction of a double mutant form of cytochrome P450 that is expected to increase 1,1,1-trichloroethane 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 be able to increase biodégradation of the target compound(s)," stated Ornstein (7).
As in situ bioremediation using microorganisms gains acceptance, a handful 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.
Cell-free enzymes: Antidotes to toxic terrorism? Extracting enzymes from bacteria and other enzyme-expressing organisms and applying them in their cell-free state for 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 U.S. Army Chemical Research and Development Center 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. "Purified 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 remediation research because of the nontoxic nature of these catalysts. "We don't want to spray something that is
Limitations of enzymes Researchers recognize that the extreme 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. McCutch-eon 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. "A 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 EPA's Glaser.
While debates continue, some scientists are ea-
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 biore-mediation for environmental cleanups. According to White, the Army's 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 of Biochemistry at Texas A&M University, works with the U.S. Army on several approaches. One project would genetically modify microorganisms with enhanced enzymatic capacity to break down organophosphate neurotoxins; another would encapsulate immobilized organophos-phate-hydrolyzing enzymes in a bioreactor for degradation. —JEANNE TROMBLY
ger to showcase the strengths of enzyme-based bio-degradation through an integrated approach. "Bacteria versus plants versus fungi is not an either/or situation. These systems don't have to happen exclusively of each other," stated Milton Gordon, professor of biochemistry at the University of Washington. 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 remediation of Superfund sites topping $1 trillion, we simply can't afford this highly elaborate genetic engineering research for every single problem we have," insisted McCutcheon.
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2473. (6) Manchester, J. I.; Ornstein, R. L.J. Biomol. Struct. Dyn., in
press. (7) Ornstein, R. In Structural Biology: The State of the Art;
Sarma, R. H.; Sarma, M. H., Eds.; Adenine Press: Albany, NY, 1993; Vol. 1, pp. 59-76.
Jeanne Trombly is a freelance science writer based in San Francisco, CA. She is program director for the Materials for the Future Foundation.
