SBIR-STTR Award

Organophosphorus acid anydrases (opa) have enormous potential to be used in the decontamination and demilitarization of extremely toxic chemicals that inhibit cholinesterase. As the number of opas being discovered is expanding rapidly, it is important to produce and purify samples of the more interesting opas for comparision by detailed biochemical and physical analysis and characterization of their efficacy. Eventually opas with promising potential must be produced economically for field tests and full scale application. Chemgen currently is developing several novel enzymes for industrial markets using newly isolated bacteria, including one process in pilot scale production. Achieving a low cost for enzyme manufacture has been a significant factor in chemgen's commercial success and this is accomplished through the application of traditional process improvement techniques or applied molecular biology where applicable. In phase i, chemgen plans to identify and develop bench-scale fermentation and enzyme recovery/purification procedures for the assigned ops(s) in order to produce small research quantities of enzyme. In phase ii, chemgen plans to further test and improve the bench-scale process, refine purification methods, and then scale them up to produce larger quantities of enzyme, as required for test and evaluation studies. In phase iii, chemgen would propose to expand the production system to meet federal and/or commercial production demands for the enzyme.

The goal of this project is to develop highly efficient means to produce OPA anhydrase enzymes and to purify large quantities for test and evaluation studies. For OPA anhydrases of greatest interest, ChemGen proposes to use recombinant DNA technology in E. Coli for large scale production of the bacterial agent-degrading enzymes. This will involve gene cloning, modification for high level expression with inclusion of an innocuous affinity tag consisting of a stretch of six histidine residues. The histidines bind immobilized nickel ion chelate with high affinity allowing one step purification to greater than 95% purity, while in most cases not effecting other biochemical properties of an enzyme. This approach will allow very efficient enzyme production, and also provide useful structural information about the enzymes produced through DNA sequencing. GhemGen will study the biochemistry of the purified enzymes produced in this way to verify the retention of native activity. In this contract work, ChemGen is also prepared to negotiate the production of other OPA anhydrases from non-engineered strains. Fermentation and more traditional purification technology using pilot plant scale equipment and methodology developed during the Phase I SBIR work can be used. ChemGen has fermentation and processing equipment with up to 2,500 Liters working volume (1,000 gallon fermentor) to apply to this problem, but 250 Liter fermentors should be sufficient.