National Institute for Water Resources, Water Resources Research Institute Program
3/1/2013 - 2/28/2014
Horseradish peroxidase (HRP) is a widely used enzyme in water pollution monitoring and wastewater treatment. However, its poor stability has limited its applications, particularly for wastewater treatment. Much research efforts have been focused on searching for a HRP substitute. Palm peroxidases show exceptional stability, high activity and unique catalytic properties. The goal of this project is to develop an enabling system to produce highly active and stable engineered palm peroxidases and to collect preliminary data on wastewater treatment and water pollution monitoring for grant applications.
Priority Issues (Significance):
This proposal addresses the priority of wastewater reuse and innovative monitoring technologies. Novel technologies are highly needed for treatment of sewage wastewater, environmental and animal wastewater in Hawaii. We will develop a perxodase-catalyzed wastewater treatment technology and integrate it with our recently patented wastewater treatment technology (Rima, Aouezova, Li. 2011. US patent 8,048,317).
The objectives are (a) to express windmill palm peroxidase and its mutant derivatives in a bioreactor at a gram scale; (b) to test the recombinant and engineered peroxidases for degradation of wastewater pollutants; (c) to immobilize the peroxidases on magnetic nanoparticles for wastewater treatment.
Experimental Design and Methods:
We have cloned the peroxidase genes from windmill palm leaves. Two mutants have been made via site-directed mutagenesis. Additional mutants will be made to further improve the stability and catalytic properties. Small amounts of the peroxidase and mutants have been expressed in bacteria Escherichia coli and yeast Pichia pastoris at a milligram scale in batch fermentors. The engineered peroxidases have shown excellent stability and catalytic properties. An adequate amount of the peroxidases will be produced with a bioreactor. After purification, the engineered peroxidases will be studied for removal of phenols, bisphenol A and pesticides. Purified peroxidases will also be immobilized on magnetic nanoparticles that have been made in this lab, which will be tested for wastewater treatment. We will also express the peroxidases on the yeast cell surface. Due to the high pH and thermal stabilities of the peroxidases, the collected cells will be killed by pasteurization at 65 ºC while the peroxidases on the cell surface remain active. After inactivation, the yeast cells will be directly used to clean up wastewater. Purified peroxidases will be used to replace HRP to develop biosensor for water pollution monitoring.
A prototype bioreactor system will be developed to express highly stable and active engineered palm peroxidases and swill be potentially scalable. One gram of novel engineered peroxidases is expected to produce for exploratory studies of wastewater treatment and as a reporter enzyme in immunoassays for pollutant analysis. Water pollutants that will be studied include industrial phenols, aryl, amines and environmental estrogens. Protein and genetic data of the peroxidases gained through this study will be released into public databases.
Dr. E. Kan is an assistant professor in the Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa. Dr. Kan will serve as a co-PI on this project. He has extensive experiences in bioreactor design, setup and operation as well as bioremediation research. He will assist on the bioreactor operation and wastewater treatment. Dr. Zhao is a post-doctoral fellow. He will be mentored in gene cloning and expression, enzyme characterization, wastewater treatment, scientific writing and presentations, career advancement and federal grant applications. One undergraduate will work on the project to receive training in bioreactor operation and degradation studies of pollutants in water.