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Evolution in an afternoon: Scientists speed the development of enzymes that break down toxic chemicals

Nature occasionally swats the curveballs humans throw at her. As scientists refine industrial processes and create new chemicals, compounds never before seen in nature begin to turn up in the environment. The counterattack can be rapid: Within a decade, natural variation in bacterial populations may give rise to strains containing enzymes that turn some toxic chemicals into benign substances.

Yet other chemicals remain untouched by bacteria. In the hope of finding the means to clean up these pollutants, Stanford researchers are moving the hunt for new enzymes into the lab by speeding up the rate of variation and compressing millions of years of evolution.

"What takes nature millions of years to evolve, we can do in an afternoon," says Alfred Spormann, assistant professor of civil and environmental engineering and biological sciences.

Enzymes are the workhorses of chemical reactions. They speed and facilitate tasks that would otherwise require a great deal of energy. For example, Spormann says, chemists take nitrogen from the air and transform it with hydrogen into ammonia, which organisms can use as a nutrient. But to accomplish the trick, chemists need to heat the gas to a sizzling 840 degrees F (450 degrees C) and compress it with several hundred times the pressure of the Earth's atmosphere at sea level. Yet some humble soil bacteria use enzymes to accomplish the same feat in ordinary, outdoor conditions.

In the past, when researchers wanted to harness the power of bacteria for waste cleanup, they had to isolate and test many strains before finding bugs with the inborn capacity to break down the desired chemicals. Once researchers pinpointed the strain, they would breed large quantities of the bug for the cleanup site.

The Stanford group is approaching the problem from a different angle. Rather than looking for natural strains of bacteria, the group is using nature's own tricks to make many variations of bacterial enzymes in the test tube.

"Nature uses trial and error to produce and test these enzymes," says Spormann. "It comes up with good and bad ideas, and selects for the useful ones. Using the same principles in the laboratory, we can enhance variation and selection to find the best form of an enzyme."

Zoom into a bacterium's DNA and you'll find a gene encoding the recipe for each enzyme the creature produces. Grab another bacterium, and you'll see a similar set of recipes, although small random mutations will make the two bugs' DNA slightly different. If the bacteria breed and exchange DNA, they might end up with two copies of the same enzyme gene. If this happens, the two versions may line up, overlap and swap information creating two new, slightly different versions of an enzyme. A similar process occurs in people when chromosomes from maternal and paternal sex cells swap genetic material.

Interbreeding bacteria and mutations don't happen very often, so Spormann and his student Michael Liu are shuffling genetic information in the lab. They find or generate several versions of the same gene from different bacteria and slice each gene into segments. Randomly drawing on the pool of gene parts, they reassemble the gene. This method, as described in a paper by W.P.C. Stemmer, a researcher at the Redwood City, Calif.-based biotechnology firm Maxygen, generates many variations on the original enzyme theme.

The enzymes produced by these shuffled genes look similar but break down chemicals at different rates and points in the chemical architecture. Some of the enzymes sidle up to the toxic compounds the group is interested in chlorinated alkanes and aromatic hydrocarbons and quickly pull them apart, while others fizzle and don't do a thing.

When testing the efficiency of these enzymes, Spormann's group pops the new gene variation into E. coli bacteria, which lack any copy of the gene, and observe the amount of toxic chemicals the bacteria break down. The ability to generate and test many variations of an enzyme enormously speeds up the process of finding efficient catalysts.

"A lot of this work is done for the love of enzymes," Spormann says. "We want to understand how these incredibly small but powerful machines work."


By Katie Greene

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