A11a-T014
TITLE: High-capacity and Cost-effective Manufacture of Chloroperoxidase
TECHNOLOGY AREAS: Chemical/Bio Defense, Materials/Processes
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 support 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 47:2997–3003.
[2] Hernandez J., N. R. Bobledo, L. Velasco, R. Quintero, M. A. Pickard and R. Vazquez-Duhalt.
Chloroperoxidase-mediated oxidation of organophosphorus pesticides. Pest. Biochem. Physiol. 61:87–94.
1998.
[3] Ayala M., M. A. Pickard and R. Vazquez-Duhalt. 2008. Fungal enzymes for environmental purposes, a
molecular biology challenge. J. Mol. Microbiol. Biotechnol. 15:172–180.
[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.
KEYWORDS: chloroperoxidase, haloperoxidase, heterologous protein expression, homologous protein expression,
chemical agent, decontamination
TPOC:
Phone:
Fax:
Email:
Stephanie McElhinny
919-549-4240
919-549-4310
stephanie.mcelhinny@us.army.mil