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UNT System: Resource magazine >> Recycling cyanide | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Cyanide is a poison lethal to humans, but in Dan Kunz's laboratory, it's what's for dinner. A University of North Texas biological sciences professor and respected microbiologist with a background in industrial biodegradation, Kunz, Ph.D., has been working for more than 15 years to understand the biology of cyanide and the bacteria that eat it. His research, which is funded by the National Science Foundation, has provided major contributions to the area of cyanide metabolism and enzymology. Natural and unnatural
Eventually, Kunz hopes to completely understand how cyanide can be nutritious to some life forms but deadly to others. "Cyanide was one of the molecules in the primordial soup believed to be present during the formation of the earth," Kunz says. "It is a natural material made by more than 1,000 species of plants, and just as all natural materials do, it recycles in our environment." However, cyanide is also a byproduct of cigarette smoke, car exhaust and many industrial activities, like gold mining, so it is regularly put into our environment unnaturally. In gold mining, for instance, rock is extracted from the earth, ground into tiny bits and heaped into mounds to be soaked with a cyanide solution that dissolves the gold. The solution, now loaded with gold, is collected in a holding pond for further processing. While precautions are taken to stop the cyanide from leaching into the ground during the mining process or while in the pond, leaks do occur.
Through Kunz's work, a way to clean the spilled cyanide from the earth may eventually be improved upon. Likewise, his work could lead to the creation of a biochemical mechanism that would protect soldiers and citizens from cyanide if it were to be used as a biological weapon. His work could also lead to advancements in medicine and agriculture where cyanide is involved — possibly eradicating some infections and diseases, and increasing crop yields for plants. Cyanide as food But the work first must focus on answering fundamental questions. "There are basic questions about the biology, biochemistry and genetics of the kinds of organisms that are able to naturally recycle cyanide, and right now, we don't have answers. "When we understand how these organisms are able to use cyanide as a food, then the possibilities for applying that understanding exist," he says. In order to understand what he didn't know, Kunz started with what he did know. Because every life form in nature seeks to survive, the aerobic organisms living in the soil near plants that create cyanide had to develop a way to detoxify and metabolize the poison. The work in Kunz's lab, which takes place primarily with microscopic bacteria, is concentrated on finding answers to the question of how varying species of Pseudomonas bacteria metabolize cyanide. In a cyclical process much like nature's, Kunz starts and ends his research experiments by growing colonies of bacteria and breaking them down. The colonies are first grown in petri dishes without cyanide present. They are then spread out as single cells in a new petri dish, where they use cyanide to replicate themselves and create new colonies of tissue. The new colonies are grown in a clean water solution and then collected and concentrated. From this concentration of tissue, Kunz has discovered that a single enzyme, with the aid of a pterin chemical, is responsible for the bacteria's ability to digest cyanide. "All living creatures have to have nitrogen to grow," he says. "When we plant these bacteria in a plate that only has cyanide, they're making every bit of their nitrogen from the nitrogen that comes from the cyanide." The enzyme directly responsible for the bacteria's ability to break the nitrogen out of the cyanide is a pterin-dependent hydroxylase that has never been described before.
Isolating the enzyme Kunz is now working to isolate this enzyme as well as the pterin chemical that must also be present to break down cyanide so he can study their molecular structure and evolution. In order to isolate the substances, he will continue doing the same work that has led him to what he knows now. "The whole idea in biochemistry is separation," he says. "We want to divide and conquer, separate and understand, and then put everything back together." So, Kunz returns to the masses of tissues he grows and keeps cryogenically frozen. He breaks the tissue down to single cells and then splits each cell open and removes two separate liquids — one holding the pterin chemical and the other proteins, including the enzyme necessary for cyanide breakdown.
The protein solution then is purified, using a Fast Protein Liquid Chromatography machine, so that it contains only the enzyme involved in the biological reaction that breaks down the cyanide. The machine runs the protein solution over a column, which is kept cold so the materials don't break down and is programmed to put the different proteins in like groups. The groups of proteins are then stripped from the column at different times and collected in separate test tubes. Currently, Kunz has the process fine-tuned to the point that he is able to collect the enzyme he wants with only two or three others present, instead of the original 2,000 types of proteins that were present in the solution from the cell. A similar process using a High Pressure Liquid Chromatography machine is conducted to isolate the pterin chemical. "When we have them completely isolated we'll be able to study the molecular composition and answer questions about how they work," he says. Kunz estimates that isolation will be achieved in about a year. Genetic answers But he must also find some of the answers from the genes responsible for the existence of the enzyme and pterin chemical. Once the gene and its structure are identified, modern genetic tests can be done to find out where else in nature the gene exists.
"Our work is focused on primarily one type of Pseudomonas bacteria, but we believe that other creatures in nature have this capability," he says. Knowing where else the gene structure responsible for the ability to break down cyanide exists will help Kunz understand how best to apply his knowledge. "It may be that plants already have the ability to make this enzyme and pterin chemical, but we don't know that, and we certainly don't know what would happen if we were to increase its production," Kunz says. "But we're getting closer." Once Kunz finds his answers, or at least most of them, he will be able to help others apply what he has learned.
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