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High-capacity and Cost-effective Manufacture of Chloroperoxidase


OBJECTIVE: Develop a fungal protein expression system with integrated purification scheme for low-cost production of purified, functional Chloroperoxidase in kilogram quantities. DESCRIPTION: Chloroperoxidase (CPO) is an enzyme produced by certain fungal species that catalyzes a diversity of biochemical reactions. For example, the CPO produced by the filamentous fungus Caldariomyces fumago catalyzes the non-specific halogenation, including chlorination, bromination and iodation, of electrophilic organic molecules. In the absence of halide, CPO is similar to the cytochrome P450 enzymes in its epoxidation and hydroxylation of olefins and organic sulfides. A CPO recently identified in the fungus Agrocybe aegerita has been shown to carry out both benzylic and aromatic hydroxylation. The versatile catalytic properties of CPO have application in paper bleaching and potential application as active ingredients in cleaning supplies and in detection and inactivation of chemical agents or products on environmental surfaces. C. fumago CPO is ideally suited for the inactivation of chemical agents due to its exceptional stability, broad substrate profile, and high catalytic efficiency. However, the high cost of producing large amounts of CPO endogenously from fungal hosts is a major obstacle precluding the formulation and field deployment of enzyme systems for chemical agent decontamination. Therefore, an advanced technology is needed that will enable the large-scale and economical production of CPO in a highly active form. Expression in heterologous host systems, including Escherichia coli, insect cells, Saccharomyces cerevisiae, and Pichia pastoris, is hindered due to the complex post-translational modifications required for CPO activity. Therefore, the goal of this topic is to develop an expression system in filamentous fungi, either by enhancing endogenous expression or developing an exogenous expression system. Protein production capacity as well as economic enzyme purification technology will be critical in achieving the required kilogram-scale manufacturing capabilities. Field application of a CPO-based decontamination system is projected to require a cost of enzyme production at or below $0.025/1000 enzyme units. PHASE I: Develop a fungal expression system and purification scheme to produce milligram quantities of CPO at a purity of 90% or greater. Perform detailed biochemical characterization of the purified enzyme, including determination of catalytic efficiency and enzyme stability under a range of proposer-defined reaction conditions suitable for use in field technologies. PHASE II: Optimize the fungal expression system and purification scheme to demonstrate production of kilogram quantities of CPO at a purity of 90% or greater. The purified enzyme must exhibit native activity levels. Enzyme production should be achieved for a cost at or below $0.025/1000 enzyme units. Proper batch record documentation and quality control processes should be demonstrated. PHASE III DUAL USE APPLICATIONS: The development of a high-capacity cost-effective fungal production system for chloroperoxidase will suport capabilities in enzymatic-based field technologies. The proposer should identify appropriate transition or collaborative partners who will use this enzyme in development and fielding of technology that supports the warfighter mission (e.g., chemical decontamination technologies). The economic production of chloroperoxidase may also be beneficial in the commercial sector (e.g., remediation technologies with Hazmat teams, paper industry, etc.). REFERENCES: 1) Manoj, K. M. and L. P. Hager. 2008. Chloroperoxidase, a Janus enzyme. Biochemistry 47:29973003. 2) Hernandez J., N. R. Bobledo, L. Velasco, R. Quintero, M. A. Pickard and R. Vazquez-Duhalt. 1998. Chloroperoxidase-mediated oxidation of organophosphorus pesticides. Pest. Biochem. Physiol. 61:8794. 3) Ayala M., M. A. Pickard and R. Vazquez-Duhalt. 2008. Fungal enzymes for environmental purposes, a molecular biology challenge. J. Mol. Microbiol. Biotechnol. 15:172180. 4) Conesa, A., F. van de Velde, F. van Rantwijk, R. A. Sheldon, C. van den Hondel, and P. J. Punt. 2001. Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme. JBC 276:17635-17640.
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