Professor, students research ethanol production

By Amanda Phipps

phippsa@thejohnsonian.com

Elizabeth Bales works with a grid screen to look at the effects the changing of characteristics of a mutant Xylanase C enzyme has on crystallization. She was one of the students who worked with assistant professor of chemistry, physics and geology Jason Hurlbert studying the enzyme and its ability to be used for ethanol production. Photos by Kathleen Brown • brownk@thejohnsonian.comUsing wastes from the paper industry can be a way to use unused materials, one professor said.

While other scientists have tried fermenting alcohol from plants such as corn, this process caused problems, said Jason Hurlbert, assistant professor of chemistry, physics and geology. Though corn provides a renewable fuel source, the use of these plants caused spikes in food prices.

An alternative fuel source may lay in the use of waste from woody plants, Hurlbert said. 

“Bacteria that normally degrade woody plants express enzymes that allow them to break the hemicellulose that glues the cellulose fibers together into sugars that can be metabolized,” he said. 

The Project

Hurlbert worked with chemistry majors Cameron Waller and Elizabeth Bales to understand how the enzyme breaks down the plant materials and how to make it work more efficiently. 

They worked with an enzyme from a bacterium found in a compost pile, Hurlbert said. 

“We cloned it from the isolate into a strain of Escherichia coli,” he said. “This E. coli makes large amounts of the cloned enzyme, and we can purify the enzyme from the bacterial cultures.”

Normally, using the waste wood materials from the paper and agricultural industries requires expensive chemical pretreatments to break down the hemicellulose. This breakdown produces sugars, which the bacterium ferments to ethanol.

“We are characterizing enzymes from different bacteria to identify the best ones for the job and then clone them into a biocatalyst that is designed to produce high yields of ethanol,” he said. 

Most enzymes cannot break down the hemicellulose in plants because of the presence of chemical groups on the main polymer that act as protections for xylan, the primary component of the hemicellulose in plants, Hurlbert said. Xylanases are enzymes that break down the xylan. 

He said they are trying to understand the structural basis for the requirement the enzyme has for the xylan substitutions, Hurlbert said. 

“If we can understand the molecular architecture of the enzyme, we may be able to transfer it to another more efficient enzyme or modify it to expand the range of materials it can recognize, bind and degrade,” he said.

This will allow them to create a type of “super bug” or biocatylst that can ferment alcohol from more types of plants and maximize the fermentation of ethanol from a variety of substrates.

“There are wastes in the paper industry already,” Hurlbert said. “The goal is to be able to ferment alcohol from anything that is normally wasted.” 

Forming Mutants

A crystal formed from the Xylanase C enzyme in a grid screen. Photos by Kathleen Brown • brownk@thejohnsonian.comLast year, Hurlbert solved the structure of the Xylanase C enzyme, and based on that work, designed mutations to examine the function of several residues in the protein, Hurlbert said. Waller mutated the residues to the amino acid alanine.

They worked together to understand which amino acids were important in the Xylanase C enzyme function, he said. 

If an amino acid that was taken out does not cause any change to the active site, it is not a key player in the enzyme’s ability to function, Waller said. 

Xylanase C will only work on plants that have side chains or brances off the principle polymer chain, Waller said. Normally, these branches protect the plant and the enzyme cannot work. The goal is to understand how the enzyme requires these branches. 

The enzyme is naturally expressed in a bacterium and can already break down wood as a food source. 

“The groundwork is already in place,” he said. “We want to understand how it works and improve the process so we can apply it better.”

Waller said he started this process when he took chemistry 551 last semester, a course that allowed him to do research for credit.

Crystallization Process

After Hurlbert solved the structure of the enzyme and Waller formed the mutated versions, chemistry majors Tyler Couch and Bales expressed and purified the proteins. Bales then began crystallizing them.

Couch said he is currently growing the protein Bales will use for crystallization and Waller will work with next semester. 

Couch worked with the different Xylanase C mutants that were encoded into circular pieces of DNA, or plasmids. These were inserted into E. coli cells.

“Normally, E. coli does not have the DNA that codes for the Xylanase C enzyme,” he said. 

Once the cells multiplied and each had the DNA, he induced them, causing them to release an enzyme that makes the protein. 

“I then have to extract the protein from the cells and purify it so that all we have left is our pure protein,” he said. 

Over the summer, Bales worked on crystallizing the four Xylanase C enzyme mutants.

She worked on purifying the proteins in the mutants to get the enzyme to crystallize, or making the proteins come together in an ordered fashion. The crystals would form distinctive shapes and could be used to predict the structure of the enzyme.

Bales worked with the structure of the wild type Xylanase enzyme that formed crystals. 

“It’s a lot of guesswork,” she said, “but I took what I knew and went around the enzyme to find conditions that will crystallize it.”

Hurlbert said Bales used grid screens to examine the effects of changing the characteristics. 

“The mutants are similar to the nonmutant form of the enzymes, so we don’t expect there to be a significant change in the conditions (for crystallization),” Hurlbert said.

He said the crystallization process requires work.

“It’s a bit of an artwork to identify the crystallization conditions,” he said.

Bales was able to crystallize two of the mutants, Xylanase C s235A and Xylanase C y274A, over the summer. She will work the rest of this year on crystallizing the other two mutants.

Once crystallized, the hope is that the mutants can be sent off for x-ray diffraction, Bales said. By looking at a pattern formed from light hitting the crystals, or the diffraction process, the structure of the enzyme can be calculated. 

This process will help them understand how the enzyme works with the substitution in place, which will help them better understand how it can help the bacterium ferment alcohol more efficiently, she said.

Currently, Hurlbert and the students involved are working on crystallizing a fifth mutant of the Xylanase C enzyme. 

The hope is to use this enzyme to break down wood wastes for ethanol, Bales said.

“It’s a great way to convert waste into fuel,” she said.