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Concentration and Purification of Recombinant Manganese Peroxidase for Biofuel Applications

This project focuses on optimizing methods for concentrating and purifying recombinant manganese peroxidase for biofuel production. By overcoming challenges such as lignin hindrance, the goal is to enhance enzymatic conversion processes crucial in the biofuel industry.

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Concentration and Purification of Recombinant Manganese Peroxidase for Biofuel Applications

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  1. Ian Gluck Mentor: Dr. Christine Kelly OSU Dept. of Chemical, Biological and Environmental Engineering Concentration and Purification of Recombinant Manganese Peroxidase for Application in the Biofuel Industry

  2. The Biofuel Industry

  3. The Conversion Process • Cellulose must be converted to glucose • Performed by cellulases • Process is hindered by lignin • A large, irregular polymer • Surrounds the cellulose

  4. Lignin Cellulose

  5. The Solution

  6. The Solution • White-rot fungi cannot be effectively mass produced • DNA responsible for MnP production was cloned into a yeast (Pichia pastoris) • Yeast is cultivated to create recombinant manganese peroxidase (rMnP) • Performs same lignin-degrading function

  7. The Solution

  8. The Project Purpose To test the effectiveness of a variety of methods in the concentration and purification of recombinant manganese peroxidase

  9. The Initial Substance • White-rot fungi cultivation broth • Filtered • Cell-free broth submitted to: • A concentration process • A purification process

  10. The Concentration Process

  11. The Purification Process • Ion exchange chromatography • Uses ionic interactions to separate molecules of different charge • Diethylaminoethyl (DEAE) column -O-CH2-CH2-N+H(CH2CH3)2•Cl- • Positive charge with negative Cl- ions • Cl- ions are exchanged for MnP, and vice versa

  12. Cl - - - Fungi Culture Protein MnP MnP - - - - - - - - - - - - - - - - - - - - - + DEAE + + + + + + - - - - - - - - - - - - - - - - - - - - - - - - - - - - Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl Cl - - - - - - - - - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - + - - - - - - - - - - - - - - - - - - - - - - - - - - - - +

  13. MnP Measurement Enzyme Activity Assay

  14. MnP Measurement

  15. Purification Measurement Total Protein Assay

  16. The Results • Desired Outcomes • MnP yield > 50% • Dramatic increase in mass fraction

  17. The Goal • Possible outcomes of success • Improve research on MnP transformations of lignin • Cellulosic ethanol is able to be produced more efficiently on an industrial scale • Product of lignin degradation is examined for possible applications • A model system for purifying proteins is developed

  18. Acknowledgments • HHMI • Dr. Kevin Ahern • Dr. Christine Kelly • Kelsey Yee • OSU Department of Chemical, Biological and Environmental Engineering

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