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 laboratory’s 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.

SAMPLE COLLABORATIONS
Projects conducted at the CBB laboratories include:

Steroid Biotransformation
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.

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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%.

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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.

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Glycolate Oxidase, a Platform Technology
E. I. DuPont donated patents to the university defining glycolate oxidase (GO) technology. CBB developed the process to produce high-cell-density fermentations of Pichia pastoris providing quantities of biocatalyst. (Recently, CBB added a manifold to supply oxygen from Dewars, enhancing capabilities for 100- and 1,000-liter fermentors to produce cells at high density.) CBB can continuously monitor methanol, and control its addition to Pichia fermentations. The laboratory safely handles solvent additions with oxygen sparging. A large-scale permeabilization process allowed reactants to access the biocatalyst. CBB converted l-lactate to pyruvate to establish benchmark data. CBB then optimized reaction conditions to minimize activity loss, allowing repeated recycling, and maximize conversion. Most of this work was done with pyruvate, a dietary supplement and chemical intermediate. Ion-exchange chromatography purified pyruvate, which was then esterified to ethyl pyruvate in a rotary evaporator. Possibilities for conversions of many alpha-hydroxy acids and diols to keto products suggest this technology is on a fundamental platform. Licensing is available. S. Gough, M. Deshpande, M. Scher and JPN Rosazza. 2001. Permeabilization of Pichia pastoris for glycolate oxidase activity. Biotechnol. Lett. 23:1535-1537.

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Organism-Enzyme-Polymer
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.