1. Ian Gluck Mentor: Dr. Christine Kelly OSU Dept. of Chemical, Biological and Environmental Engineering

2. Cellulosic (wood-based) ethanol has several advantages Lower greenhouse gas emissionsBiomass is cheap to produce Majority of American ethanol is corn-based InefficientConflicts with food demands The United States is currently the world’s largest producer of ethanol

3.  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. Manganese peroxidase (MnP) Found in white-rot fungi Degrades lignin

6.  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. Crude yeast broth has many other proteins, as well as rMnP For rMnP biocatalysis pathways to be investigated, it must be: ConcentratedPurified

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

9.  White-rot fungi cultivation broth  Filtered  Cell-free broth submitted to:  A concentration process  A purification process

10. Acetone precipitationCentrifugation/ResolubilizationOvernight freezingLyophilization (freeze-drying)Dialysis

11.  Ion exchange chromatography  Uses ionic interactions to separate molecules of different charge  Diethylaminoethyl (DEAE) column -O-CH 2 -CH 2 -N + H(CH 2 CH 3 ) 2 Cl -  Positive charge with negative Cl - ions  Cl - ions are exchanged for MnP, and vice versa

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

14. Sample of subject solution is mixed with buffers, DMP and H2O2 MnP in solution reacts with peroxide and DMP, forming an orange color Absorbance of orange solution is measured Enzyme Activity Assay

15. Absorbance is used to calculate MnP concentration Concentration * Volume Mass Mass of MnP/ *100 = Percent Yield Previous mass of MnP

17. Similar process to MnP assay Absorbance is measured and used to find protein concentration Concentration * Volume = Mass Mass of MnP is divided by protein mass Mass Fraction Total Protein Assay

18. Step Sample Volume (L) MnP Conc. (mg/L) MnP Mass (mg) Yield (%) Protein Conc. (mg/L) Protein Mass (mg) Yield (%) Mass Fraction (*10 -5 ) Crude Broth0.10.1391.39*10 -2 -1503150.3-9.30 Precipitation/ Centrifugation 0.030.3501.23*10 -2 88.5285585.6556.921.6 Lyophilization0.0043.001.19*10 -2 96.710174.0684.7251 Dialysis0.0081.098.68*10 -3 72.9---- Ion Exchange0.0150.4336.50*10 -3 74.95938.888-73.1  Desired Outcomes  MnP yield > 50%  Dramatic increase in mass fraction

19.  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

20.  HHMI  Dr. Kevin Ahern  Dr. Christine Kelly  Kelsey Yee  OSU Department of Chemical, Biological and Environmental Engineering