EMBARGOED UNTIL 5/24/2011 1:00:00 PM CDT
(Session 236, Paper 2794)
Ryan Summers
University of Iowa
Coralville, IA, United States
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Phone: 319-335-4355
A new bacterium that uses caffeine for food was found in a flower bed at the University of Iowa. This bacterium, Pseudomonas putida CBB5, uses four newly discovered “digestive” proteins to break down caffeine, which allows it to live and grow. The compounds formed during break down of caffeine are natural building blocks for drugs used to treat asthma, improve blood flow, and stabilize heart arrhythmias. The caffeine digestive proteins could also be used to remove caffeine and related compounds from large quantities of waste generated from coffee and tea processing industries, which pollute the environment. The decaffeinated waste from these industries can be used as animal feed and for production of transportation fuel.
This research was performed primarily by Ryan Summers, a doctoral student in Chemical and Biochemical Engineering at the University of Iowa. Dr. Michael Louie and Dr. Chi Li Yu, two research scientists in Dr. Mani Subramanian’s laboratory at the Center for Biocatalysis and Bioprocessing, also assisted in the work. Funds were provided from Dr. Subramanian’s state grants at the University of Iowa. The research was presented at the 111th General Meeting of the American Society for Microbiology in New Orleans, LA, from May 21-25.
Caffeine is a natural product that is found in many foods and beverages, including coffee, tea, chocolates, and many soft drinks. It is also used in pharmaceutical preparations. High levels of caffeine consumption, however, can lead to headaches, insomnia, and gastrointestinal problems. Pregnant and breastfeeding women are encouraged to abstain from caffeine due to its harmful effects on developing fetuses and newborn children. The widespread use of caffeine results in release of this chemical into the environment from human waste, degradation of plant matter in coffee and tea fields, and wastes from coffee and tea processing industries all over the world. This can have several environmental impacts, including inhibition of seed germination and toxicity to bacteria and insects.
Due to the extensive presence of caffeine in the environment, it is not surprising that there are bacteria that can “eat” this molecule for growth and reproduction. The caffeine molecule itself is composed of carbon, nitrogen, hydrogen, and oxygen, all of which are necessary for cell growth. We have isolated a new caffeine-degrading bacterium, P. putida CBB5, which breaks caffeine down into carbon dioxide and ammonia. The three methyl groups in caffeine, each composed of one carbon and three hydrogen atoms, are effectively removed by this bacterium from the main part of the molecule. Thus, the bacterium is able to “live” on caffeine. Caffeine utilization by bacteria via removal of the three methyl groups (a process known as N-demethylation) has been reported previously; however, the exact mechanism for N-demethylation has been a mystery for many years. This work, for the first time, demonstrates the enzymes and genes utilized by bacteria to live on caffeine. Also for the first time, this work reports the degradation of theophylline, a natural product related to caffeine which is used to treat asthma. Theophylline contains only two methyl groups, as opposed to three in the caffeine molecule.
N-demethylation of all three methyl groups in is accomplished by three N-demethylase genes in CBB5, designated as NdmA, NdmB, and NdmC. These three genes, in combination with a fourth gene called reductase, Ccr, accomplish the complete degradation of caffeine and theophylline to xanthine, a common chemical found in all living organisms. Each N-demethylase removes a specific methyl group from the caffeine molecule. NdmA and NdmB were co-purified from CBB5 and found to be a type of enzyme referred to as Rieske-type [2Fe-2S], non-heme iron oxygenases. Similar enzymes break down many environmental pollutants, such as naphthalene, toluene, biphenyls, and poly aromatic hydrocarbons (which are components of crude oil). NdmA and NdmB use atmospheric oxygen when removing the methyl groups, converting them into formaldehyde and water.
After biochemical purification of the NdmA and NdmB enzymes, molecular biology techniques were used to determine their gene sequences. These genes were separately cloned into a common bacterium, E. coli, which was used to produce large quantities of NdmA and NdmB. When NdmA was tested along with the Ccr enzyme, caffeine was converted to theobromine (a component of chocolate), theophylline was converted to 3-methylxanthine, paraxanthine was converted to 7-methylxanthine , and 1-methylxanthine was converted to xanthine. The compounds produced by NdmA were not degraded further, which signified that NdmA was only able to remove a single, specific methyl group. Testing of NdmB with Ccr resulted in conversion of caffeine to paraxanthine, theophylline to 1-methylxanthine, theobromine to 7-methylxanthine, and 1-methylxanthine to xanthine. This suggested that NdmB was also only able to remove a single, but different, methyl group from the caffeine molecule than the one removed by NdmA. Further work is currently in progress to purify NdmC, which is responsible for removal of the third methyl group in the caffeine molecule.
There are many potential applications for these new N-demethylase enzymes. Perhaps the largest application is green and sustainable technology for the production of pharmaceuticals to treat asthma, increase alertness, stimulate heart rate, increase blood flow, and treat altitude sickness. Currently, these pharmaceuticals are difficult to synthesize chemically. Using CBB5 enzymes would allow for easier pharmaceutical production, thus lowering their cost. These enzymes could also be used for decaffeination of coffee and tea as an alternative to the harsh chemicals currently used. Another potential application is the decaffeination of coffee, tea, and cocoa plant matter, extensively produced in coffee, tea, and chocolate processing plants. This will enable the use of these wastes as animal food and for production of fuel. Also, because formaldehyde is produced when the methyl groups are removed from caffeine, CBB5 enzymes may find use as a biosensor for formaldehyde to detect caffeine pollution.
