Researchers First To Directly Increase Enzyme’s Activity

Joshua Sakon, chemistry, other researchers from the University of Arkansas and scientists from the National Renewable Energy Laboratory in Golden, Colo. study the enzyme cellulase, which cleaves cellulose into glucose, a sugar that can be fermented into ethanol.

Intense research has focused on this process because cellulose found in plants accounts for about half the organic material on earth, which makes it potentially a vast renewable energy resource.

However, in the reaction that transforms the organic matter into fuel, the low activity of the enzyme cellulase acts as a bottleneck, slowing the whole procedure down. For at least ten years, scientists have sought ways to increase the enzyme’s activity, and thus its productivity — to no avail.

Sakon’s group increased the enzyme’s activity by 13 percent. The researchers will present their work today (March 21) at the American Chemical Society meeting in Anaheim, Calif.

Cellulase cuts the plant tissue cellulose into glucose — a simple sugar. It does so by attaching to the cellulose, clipping it and releasing the resulting sugar moelcules.

"Many people tried to come up with better scissors," Sakon said. He decided to focus instead on the enzyme structures that hold the cellulose in place while it’s being clipped. If these structures had a looser grip, he reasoned, they would release the sugars faster, increasing the reaction’s activity.

The researchers extracted cellulose from fast-growing yellow poplar trees. They used X-ray crystallography to examine the structure of the cellulase-cellulose interaction.

Then Sakon worked with University of Arkansas physical chemist Lothar Schaffer, using computer models to predict the impact of modifying the enzyme at certain sites.

The researchers then mutated the amino acid tyrosine 245, one of the biochemical "hands" that holds the cellulose in place. They substituted glycine, an amino acid that creates a weaker bond, or grip, at that site.

The resulting mutant increased the enzyme’s activity by 13 percent.

Sakon already is looking at the next experiment — perhaps trying mutations elsewhere in the enzyme’s "hold-and-release" region.

"Our experimental calculations showed we should have got a lot higher activity," Sakon said. "Now something else has become the rate-limiting step."