Extreme Enzymes

Science is commercializing nature's diehard proteins

Thirteen degrees north of the equator in the East Pacific, a mile and a half below the surface, University of Delaware scientists in a submersible vessel collect worms with feathery bacterial plumage from the sides of hydrothermal vents where the water temperature reaches 185F. Half a world away, in Iceland, a German team collects hardy microbes from a vent spewing sulfuric steam into the air. In Israel, researchers hunt for bugs in the intense salinity of the Dead Sea. And from arctic pools, scientists collect bacteria that grow best at 32F.

Criss-crossing the globe, these scientists are chasing some of nature's oddest enigmas: microbes called extremophiles that populate the world's most inhospitable environments. These roaming scientists share something else, too: All are consultants and advisers to a small company, Recombinant BioCatalysis Inc. (RBI) in Sharon Hill, Pa. Its mission: to discover, clone, and commercialize unusual enzymes secreted by extremophiles.

BRAIN TRUST. For years, common enzymes--the protein catalysts that control the metabolism of all creatures--have been replacing expensive and polluting chemical catalysts used in the production of food, drugs, and paper goods. The trouble is, most of these enzymes are unstable at high heat or in solvents found in many industrial processes. That has limited their usefulness.

Enter the extremophiles--and RBI. Noting that many of the organisms thrive in just the sorts of toxic brews that industry likes to concoct, RBI CEO Barry L. Marrs--a former director of life sciences at DuPont Co.--has recruited top academicians who study these creatures. As consultants, they share data with RBI and license the company to commercialize enzymes from organisms they collect.

The quest carries them to some bizarre locales. One RBI collaborator, University of Delaware marine biologist Craig Cary, has spent hours on the ocean floor in the Alvin, a U.S. Navy research submarine. With grants from the National Science Foundation, he studies the behavior of 4-inch-long Pompeii worms that live in holes on the sides of "black smokers"--underwater chimneys that release jets of superheated water laden with cadmium, zinc, and other metals toxic to most creatures.

Inside the worm's hole, heat rises to 185F. Outside, where the worm ventures for food, the mercury plummets to 36F. "It has got to be one of the harshest environments on the planet, with the most severe temperature gradient," says Cary. On its back, the worm carries a thick fleece of bacteria. The enzymes they secrete, possibly to detoxify surrounding waters, are valuable for their stability at extreme temperatures.

Thus far, the fleece has resisted all efforts to cultivate it in the lab. So to analyze it, Cary is relying on RBI's high-tech wizardry. Bypassing the laborious gene-sequencing steps preferred by top gene companies like Human Genome Sciences Inc., RBI can scoop up a bit ef bacterial soup, enzymes and all, and extract "naked" nucleic acid. The next step is to break the chains into short pieces containing just a few genes and stick them into hosts such as E. coli. High-speed robotic screens then determine if the host produces the desired enzyme, thus leading scientists to the responsible genes. The test doesn't give any hints about the identity or behavior of the organism that the enzyme came from. But RBI's customers don't care--they just want the enzymes.

While RBI isn't the only company targeting extremophiles--Switzerland's Roche Holdings, Denmark's Novo Nordisk, and others are hot on the trail--none has shown the same skill in tapping the world's scientific braintrust. After just 15 months in business, the company expects to break even on $5 million in revenues this year.

RBI's scientific connections explain its rapid launch. The reputation of one of its founders, Human Genome Sciences Chairman and CEO William A. Haseltine, helped attract $10 million from venture capitalists. Other scientists flocked to the startup thanks to the involvement of a second founder, Karl O. Stetter of Germany's University of Regensburg, a top expert in extremophiles.

RBI anticipates applications for its new enzymes across a sweep of industries, from agriculture to oil production (table). Searching for an enzyme that could help extract petroleum from deep in the earth, North Carolina State University chemical-engineering professor Robert M. Kelly, an RBI adviser, turned to an enzyme from a microbe that functions at temperatures up to 248F--above the boiling point of water. He and RBI are talking with several oil field service companies that want to license the technology.

Medicine is another target market. Therapeutic enzymes--including clot-busters such as streptokinase--already account for roughly half the $2.5 billion world market for enzymes. Of course, drugs and routine biomedical tests don't need to function in raging heat or corrosive chemical environments. But the high stability of enzymes from extremophiles could lead to improved versions of enzyme-based therapies.

QUICK STUDY. Such hopes have helped RBI land some big partners, including chemical and equipment giant Hercules Inc. David A. Simpson, Hercules' director of new-technology initiatives, hopes a collaboration with RBI may lead to new techniques for cleaning up industrial pollution, for example. Because 99.9% of all microbial life remains unexplored, Simpson anticipates plenty of startling and useful discoveries.

To tailor enzymes for industry, RBI turned to California Institute of Technology chemical engineering professor Frances H. Arnold. Brought on as a consultant, she tutored RBI's engineers in a technique for mutating specific bits of DNA to produce desired characteristics--a method RBI's Marrs calls "directed evolution." By forcing DNA that codes for a high-temperature enzyme through repeated, rapid mutations, each time selecting offspring that can function in slightly cooler environments, RBI can create enzymes that are active at room temperatures but have the stability of proteins from extremophiles.

In this very technology, though, some see a threat to RBI's long-term agenda. The principles of directed evolution are well-understood by other scientists and engineers. Taken to its logical extreme, the approach could create enzymes that operate in any environment and obviate the need to findnew extremophiles.

A technique known as chemical cross-linking could also rival RBI's approach. By tinkering with the crystalline structure of highly purified enzymes, engineers at Cambridge (Mass.) startup Altus Biologics say they have bestowed stability on molecules similar to those of natural enzymes from extreme settings.

Even if its approach does win out, RBI won't have the field to itself. Scientists at Roche point out that the first commercial enzyme isolated from an extremophile is Taq polymerase--a molecule that helps amplify trace quantities of DNA. This molecule became the basis of a major scientific tool called polymerase chain reaction, which is used, among other things, for DNA "fingerprinting." Roche owns the rights to this enzyme and is exploring other useful proteins isolated from extremophiles.

Nonetheless, RBI's stable of peripatetic academic collaborators gives it an edge. Their message: Keep exploring. After two years scrutinizing the structure of high-temperature enzymes, RBI adviser Michael W.W. Adams, a University of Georgia biochemist, concludes that there are no effective shortcuts to finding them in nature. "If you want highly stable enzymes, you must go to the extreme environments," he says. Nature's bounty, it seems, abides in its black smokers, steam vents, and salt lakes.

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