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December 1997

Extreme chemistry

by William Wells

The next leap forward in laundry detergents may originate not in a chemist's laboratory, but with bacteria that live on a marine worm, a mile below the ocean surface, and at temperatures of over 80ºC.

Yellowstone
Dr Ron Swanson, Associate Director of Genomics at Diversa, collecting a sample from a geothermal vent in Yellowstone National Park, Wyoming, USA. Photo courtesy of Diversa Corp.
Jeff Stein's main task is to sift through bacterial sludge, but he couldn't be happier. His search for bizarre microbes takes him on dives to the bottom of the ocean, and forays into geothermal areas and tropical rainforests.

Stein is a Principal Scientist in charge of microbial diversity at Diversa Corporation in San Diego, California. Diversa provides its industrial customers with enzymes, and the weirder the source, the better. Enzymes are the cell's version of a chemist, capable of converting one chemical to another with astonishing efficiency and zero toxicity. They are used in the production of everything from cheese, to perfume, to stone-washed jeans Table 1. Enzymes from the single-celled bacteria that live in extreme environments should, says Stein, be able to withstand both the harsh conditions in industrial plants and the wear and tear of everday use by consumers.

The trick to this approach is direct isolation of DNA from the bacterial sample. Scientists then search for genes in the DNA that hold the code for useful enzymes. Going straight for the DNA allows you to skip the intermediate and tricky step of growing the bacteria in the laboratory.

The method seems to be yielding results. "This is reaching out beyond organisms that are well studied," says Norman Pace, of the University of California at Berkeley. "I think that is wonderful."

Boundless diversity

Even in the soil of a temperate garden, there is an enormous flowering of life. One gram of this soil contains the DNA of ~109 bacteria, representing ~104 different species. And that is only in the front yard -- the shady back yard is a whole new universe. "The vastness makes the question of 'How many?' irrelevant," says Pace. "I mean, how many stars are there?"

Table 1: Which enzymes are used to make your favorite product?
Enzyme(s)Application
MannaseDispersal of the galactomannans used to increase the flow of oil in a drilling operation (the other alternative is bleach). Thermophilic mannase can withstand the high temperatures at deep strata.
Endoglucanases and phosphatases Increase the absorption of animal feed by breaking down glucan coatings on grain. Thermophilic enzymes survive the high-temperature extrusion process that creates the feed pellets.
Cellulases and xylanases Bleaching of wood products. The enzymes loosen the structure of wood pulp. Paper manufactures can then use far less bleach, as the lignin can more easily escape from the plant matter.
Endoglucanases, lipases and proteases Washing detergents. Thermophilic enzymes are useful for hospitals that wash at high temperatures for sterilization purposes; psychrophilic enzymes can be used in room-temperature washing detergents.
ProteasesBreak down proteins in cheese to enhance flavor
EsterasesCreate new chemicals for use in fragrances.
CellulasesSoften the fibres in jeans to create stone-washed effect.

Perhaps, then, Diversa need go no further than a San Diego garden. But Diversa scientists have faith in their search for extremophiles, the bacteria that live in harsh environments. Although 104 is a lot, 104 of pretty much the same thing may not be so useful. Isolated populations of bacteria in extreme environments probably have enzymes that can direct novel chemical reactions. And enzymes from thermophilic (heat-loving) bacteria have several potential advantages.

The first is obvious: A number of chemical processes require enzymes to act, or retain their activity, at high temperatures (see the first two entries in Table 1).

But the main stumbling block for those wishing to use enzymes in industry has been lack of stability, not lack of heat-resistance. Enzymes are non-toxic, biodegradable, and great at doing only what they are told, but they go off. Diversa recognised that thermophilic enzymes are stable enzymes. Even if they are to be used at mild temperatures, they last far longer than regular enzymes. They also resist the destructive effects of organic chemicals used in other steps of the industrial process.

Geysers, vents, and searching for whales

image
Alvin Submersible
The two best sources of thermophilic bacteria are where the heat of the earth's interior comes to the surface: deep-sea vents, and geothermal pools. In the ocean, Stein has used the Alvin submersible to access vents at sites like the East Pacific rise, ~800 miles off Costa Rica and ~1.5 miles down. The hottest vents have core temperatures of 350-400 ºC. These black smokers get their name from the plumes of precipitated metals that drift into the surrounding sea water, which is at a temperature of only 2 ºC.

The centers of the vents are devoid of life, but as the temperature decreases in the surrounding chimney, core samples turn up a wealth of thermophiles. Other samples are collected using either a slurp gun, which filters water from around the vent, or collection plates seeded with chemicals that attract bacteria because the microbes see them as lunch.

The bacteria can live at temperatures of up to 113 ºC, and in conditions that are extremely salty, acidic, or alkaline.

Deep-sea marine animals, or rather the bacteria living on and in their bodies, are also a good source of biodiversity. One intriguing recent find is a worm that keeps its tail in the hot vent and its head in the cool surrounding water. Researchers are interested in the worm as a source of enzymes that can work over a broad range of temperatures.

With their similar temperatures and chemical makeup, geothermal pools are the terrestrial equivalents of deep-sea vents, at least for harvesting bacteria. Deep-sea vents and geothermal pools also share an island-like quality, isolated biologically by their unique chemistry. For example, one pool that Diversa collects from in Costa Rica, at ~95ºC and pH 0, stands in stark contrast to the surrounding rainforest. Diversa signed a bioprospecting agreement in August giving them access to Yellowstone National Park, which has over 60% of the world's geysers, hot springs and boiling mud pots.

Whale skeletons, another source of microbes, are also like isolated islands, so isolated that they are tricky to find. The US Navy helped out scientists by locating one when it was looking for a wayward Triton missile, but in other cases researchers have resorted to towing dead, beached whales out to sea and dumping them.

Isolation is not the only attraction of whale bones. "They ooze complex lipids [greasy molecules that line the outside of cells] over periods of up to 20 years," says Stein. Bacterial mats that grow on the bones are a rich source of the lipases and esterases needed to break down the lipids, and these two classes of enzymes are especially important to industry. The organisms and their enzymes are adapted to the deep-sea cold; such psychrophilic enzymes can be useful for commercial laundry detergents. More psychrophilic enzymes are turning up in earth and lichen samples from Icelandic and Antarctic glaciers.

From DNA to enzyme

Harvesting the DNA is relatively non-destructive to the environment. "A teaspoonful of sample is generally more than we need," says Jay Short, Chief Technology Officer at Diversa. Once they have the DNA, the job of the scientists becomes more routine. The DNA contributions of common and rare bacteria are evened out, then each piece of DNA is cloned into a separate colony of the ubiquitous laboratory bacterium Escherichia coli. Nanograms of DNA are enough to make this DNA 'library'.

Each E. coli in the library can now make a single enzyme, which is encoded by a fragment of DNA from a single bacterium in the isolated sample. Scientists then test whether this enzyme does the chemical conversion that they want it to do. Testing for the final property of the enzyme "gives us the opportunity to select without any prior knowledge of what the genes should look like," says Short.

Applications

The most obvious use for enzymes is the degradation of chemicals found in nature, as those products might be similar to chemicals that the enzymes normally encounter Table 1. The specificity of enzymes also makes them a powerful tool in the synthesis of pharmaceuticals and fine chemicals. Chemical synthesis of pharmaceuticals often gives two stereoisomers, molecules that have identical numbers and types of atoms, but are mirror images of each other. The Food and Drug Administration (FDA) is particularly concerned about stereoisomers of approved drugs, which can either be inactive or, worse, toxic. Enzymes are often the only way to differentiate between stereoisomers, as they usually react with only one stereoisomer.

Scientists have big plans for this market. "We're trying to replace enzymes already in use, and also open the door to using enzymes in other processes where they haven't been used before," says Dan Robertson, Director of Enzymology at Diversa. That approach is needed, says Pace, because the biotechnology industry has, in the past, relied on "stuff that has fallen into our pockets. There has been relatively little seeking out."

Diversa is not alone, however. Thermogen (Chicago, Illinois) has found a number of enzymes in the species of heat-loving bacteria that can be grown in the laboratory, and are now looking for other bacteria in the wild. Altus Biologics, Inc. (Cambridge, Massachusetts) has taken a very different approach to the stability problem. They add a chemical to purified, crystallized enzymes so that many enzyme molecules are linked together to form a stable enzyme pellet. The cross-linked crystals are stable to heat and organic chemicals, and have a long shelf-life.

In an area that Diversa has entered recently the competition is fiercer still. Along with ChromaXome (San Diego, California) and TerraGen Diversity, Inc. (Vancouver, British Columbia), Diversa is looking to discover new drugs made by bacteria. The companies will test natural products, the chemicals that bacteria make either to do a job in the cell, or to kill other bacteria (the antibiotics). "We think that the vast majority of natural product pharmaceuticals have yet to be discovered," says Stein.

The search is based on the fact that many related bacterial genes are clumped together. By isolating huge fragments of DNA, Stein thinks he has a good chance of picking up all the genes he needs to make an antibiotic in one fell swoop. All the current antibiotics are natural products or minor variants of natural products. With so many bacteria undiscovered, it seems all but certain that there are more drugs just waiting to be found, buried in anything from thermal vents to garden soil. And finding them might be easier than trial-and-error testing of chemicals made by humans. "Evolution has been doing a lot of tinkering over the years," says Pace, "and you may as well take advantage of it."

A Related Site on AE:

Life With Toxic Sulfide: A BioForum presentation on hydrothermal vents, seeps, and estuarine mud.


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