What The Lab Does
During a typical year, the CBB laboratory collaborates with more than 60 industrial companies and institutions. Weekly meetings usually schedule work for 15 active projects. Although more than 90% of the CBB laboratorys contract income is from the private sector, CBB services federal agencies and institutions. For example, collaborations have produced vaccines, antibiotics, anticancer drugs, polymers, BL2-LS pathogens (used to develop diagnostics and vaccines), biochemicals, enzymes, pharmaceutical intermediates, and derivatives of bioactive compounds.
Projects conducted at the CBB laboratories include:
A SBIR collaboration with a pharmaceutical company involved screening microbes to alpha-hydroxylate a steroid. This step was critical to synthesize Squalamine, a potent anti-tumor agent. CBB selected the most productive organism from shake-flask studies of sixteen microbial cultures. After selection, the process was optimized for temperature, pH and mode of substrate addition. CBB evaluated growth media, addition of glucose and XAD resin. Fermentor studies demonstrated effects of oxygenation and controlled pH on product yield. TLC, HPLC, Mass Spectrometry and NMR confirmed the biotransformed product. Purification of the hydroxylated product involved extraction, concentration by rotary evaporation, and drying under vacuum. W.A. Kinney, X Zhang, J. Williams, S Johnston, RS Michalay, M. Deshpande, L. Dostal, J. Rosazza. 2000. A short formal synthesis of squalamine from a microbial metabolite. Org. Lett. 2:2921.
Bugs Zap Explosives
CBB helped Army Ammunition Plants treat toxic Pink Water, a waste stream contaminated with TNT, RDX and HMX. CBB examined organism viability and determined chemical fate of degrading TNT. After optimizing treatment conditions, CBB provided inoculum. Pilot-scale testing at Milan and Iowa Army Ammunition Plants demonstrated destruction efficiencies in excess of 99%.
Bad Bugs for Good Causes
CBB grows pathogens in our BL-2, large-scale qualified facility. Organisms like Helicobacter pylori and Haemophilus influenzae, are grown at 100-liter scale for researchers interested in diagnostics, vaccines, and therapeutics. CBB also provides anaerobic bacteria, filamentous microorganisms, yeasts and insect cells. CBB has successfully expressed recombinant proteins in baculovirus systems.
E. I. DuPont donated patents related to glycolate oxidase (GO) technology to The University of Iowa. CBB developed the process to produce high-cell-density fermentations of Pichia pastoris yielding large quantities of biocatalyst. CBB has installed all the safety measures to handle solvent additions with oxygen sparging during fermentation.
For more information about (GO) technology, see the Glycolate Oxidase, a Platform Technology page.
See the contact information page for inquiries or for a sample of pyruvate or (R)-Hydroxyl Butyric Acid produced with (GO) technology,.
A European company approached CBB to help develop a multi-step process to produce a polysaccharide. The first step required fermentation of a recombinant organism to produce an intracellular enzyme. Shake-flask experiments improved enzyme yield while developing a medium free of animal-based products. CBB's state-of-the-art suite-of-eight two-liter fermentors developed a glucose-based medium, improved yields, and reduced the fermentation time from 120 hours to 28 hours. The two-liter process was then scaled up to 10 and 100 liters. CBB evaluated variables affecting enzyme recovery from biomass; these included pH, buffers, salts, enzyme aids, and detergents. Process steps involved microfluidizing biomass, centrifugation, and microfiltration of lysate, followed by ultrafiltration. Subsequently, CBB conducted polymerization reactions at 500-liter scale. A model of the process, based on data obtained at 500 liters, assisted in evaluating economics of scale up to 150,000 liters.
Caffeine Metabolism and Diagnostics
Caffeine (1,3,7-trimethylxanthine) is readily found in many plant species. It is also a common human dietary ingredient. An enzyme, caffeine dehydrogenase (Cdh), has been isolated from Pseudomonas sp. Strain CBB1 that catalyzes the reaction shown in Figure 1. Cdh has been characterized as a novel quino-protein that is highly specific for caffeine (Yu, et.al., 2008). The enzyme is highly suitable for developing a diagnostic test for caffeine, using NBT as the indicator dye, which turns dark blue in the presence of caffeine. A sample test with caffeine added to milk is shown in Figure 2. Cdh gene has been completely sequenced and functional cloning of the gene is in progress. This technology is available for licensing for various applications. Please see the contact information.
Several other caffeine degrading Pseudomonas have been isolated from soil that carry out N-demethylation of caffeine as shown in the Figure 3. These enzymes are suitable for production of alkylxanthines.
Ryan Summers, a PhD student in Dr. Subramanian's laboratory, presented a paper at the ASM 2011 Annual Meeting. His paper was selected by the ASM Communications Committee to be highlighted in the press room during the conference. This resulted in more than 60 citations on popular press with respect to “bacterial addiction to caffeine”.
“Purification, Cloning, and Functional Expression of NdmA and NdmB, Two Positional-Specific Methylxanthine N-Demethylases from Pseudomonas Putida CBB5”.