1. Optimizing extraction methods to improve utilization of wheat gluten in bio-product manufacturing Project Title: Mentors: Dr. Andrew RossDr. Kaichang Li Crop and Soil Science Wood ScienceOregon State University By: Shannon Williamson

2. Temporary wet strength resins (TWSR) When paper/paper products become wet their tensile strength decreases dramatically. Therefore, resins are added to these products to increase wet strength for a duration of time. Two types of resins: Temporary and permanent that can provide either short or long lasting wet strength. Ren and Li (2005) developed a temporary wet-strength additive from wheat gluten for use in the paper and paperboard industry.

3. Why wheat gluten? Wheat gluten is widely available, inexpensive and bio- degradable. Currently the paper industry uses glyoxal- polyacrylamide (GPA) which is manufactured from petro-chemicals. Wheat gluten is a renewable resource The new temporary wet-strength additive could be used in the industry if the wheat gluten extraction process was refined.

4. Wheat endosperm protein composition Albumins and Globulins~15-20% (Not useful for TWSR) Gliadins and Glutenins~75-80% This new additive has been made with only the gliadins –only using about half of the available protein.

5. Gliadins vs. Glutenins Gliadins are soluble in 70% ethanol Glutenin –is the residue leftover from the extraction of gliadin. Glutenin subunits are not soluble... –due to the large size of the protein subunits. –due to inter-subunit disulfide bonds

6. Approach Chemical Modification of wheat gluten I.Gluten becomes soluble in a variety of chemicals: soaps, detergents, hydrochloric acid, sodium hydroxide, 70% ethanol are just a few possibilities. II.Solubilization by deamidation is also used; this can be in acidic or basic solutions. Removal of the amide group of glutamine results in a change in the potential ionic charge on the protein. III. Enzymatic modification is a process that hydrolyzes peptide bonds and increases the solubility of gluten.

7. Outline of reaction scheme Measure protein content of gluten with LECO and perform HPLC analysis of existing proteins 8% (w/v) ratio of gluten in aqueous solution Addition of enzyme to modify gluten protein The fractions will be separated by centrifugation Remeasure protein content and examine HPLC Sonication of existing residues (insoluble fraction)

8. Enzymes Two enzymes utilized 1. Bacterial protease from Bacillus licheniformis( Subtilisin A) 2. Fungal protease from Aspergillus oryzae Both are endoproteases with broad specificity toward native and denatured proteins Both perform best in an environment pH= 6.0-9.0

9. Chromatogram for untreated gluten Gluten – sonicated 1 min (black) Gluten- sonicated 3 min (green) Gluten – sonicated 5 min (blue) Gliadin Peak Glutenin Peak

10. SE-HPLC Data for Run 1-1 and 1-2 Red- 1 minute sonication of gluten Blue- No sonication of gluten Black-Run - Fungal protease - 1 hr. treatment Green-Run - Fungal protease - 30 minute treatment

11. Results of Enzymatic Treatment ~100% of protein was soluble in water Solubilized protein has a considerably reduced mass due to hydrolysis With the protein in solution the TWSR was then made according the Ren and Li (2005)

12. Resin From Hydrolyzed Gluten Protein Hydrolyzed protein was modified with glycidyltrimethylammonium chloride (GTA) GTA modified protein underwent another reaction with glyoxal to create a GTA-gluten protein-glyoxal resin. When this last reaction was performed between GTA-gliadin and glyoxal, a significant change in viscosity was observed. In the case of my enzymatic treated protein, no change in viscosity was observed.

13. Conclusions and Future goals Total solubilization of gluten protein is possible with enzymatic treatment. Resin made from low molecular weight gluten proteins failed to yield a usable product. Future approaches –Hydrolyze less to keep MW closer to the MW of gliadin –Separate gliadin from glutenin, followed by hydrolysis of glutenin and then recombining with gliadin

14. Acknowledgments Ernest and Pauline Jaworski Fund for Summer Research Experiences for Underserved Undergraduates in Plant Science Howard Hughes Medical Institute Dr. Andrew Ross and Dr. Jae Ohm Dr. Kaichang Li Crop and Soil & Wood Science Department at Oregon State University