High-capacity and Cost-effective Manufacture of Chloroperoxidase

Bioengineering offers an efficient and robust route for the production of enzymatic catalysts such as chloroperoxidase (CPO). The CPO from Caldariomyces fumago can catalyze non-specific halogenation of electrophilic organic molecules, epoxidation and hydroxylation of olefins and organic sulfides, and generation of free chlorine from chloride ion. The objectives are to: A. Develop a Caldariomyces fumago expression system and purification scheme to produce milligram quantities of CPO at a purity of 90% or greater. Specifcally, we will carry out Experimental Design techniques to develop a more balanced approach to fermentation improvement. We will use screening designs to evaluate the important factors and interactions on the concentration of CPO, rate of production and purity. The important factors can then be evaluated in a statistically designed optimization study. We will also develop a low cost purification scheme to produce CPO at a purity greater than 90% B. Perform detailed biochemical characterization of the purified enzyme (CPO). We will determine the enzyme's catalytic efficiency and stability under reaction conditions suitable for field use. This will entail identification of model substrates and standardized assay conditions. We will also determine the storage stability (shelf life) of CPO preparations under various environmental conditions.