1. Effects of Enzymatic Deamidation by Protein- Glutaminase on Structure and Functional Properties of Wheat Gluten Hui Yong, Shotaro Yamaguchi, and Yasuki Matsumura Laboratory of Quality Analysis and Assessment, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Gokasho, Uji, Kyoto 611- 0011, Japan, and Gifu R&D Center, Amano Enzyme Inc., 4-179-35, Sue-cho, Kakamigahara, Gifu 509-0108, Japan J. Agric. Food Chem., 2006, 54 (16), pp 6034–6040DOI: 10.1021/jf060344u Publication Date (Web): July 19, 2006 Copyright © 2006 American Chemical Society

2.  Background  Introduction  Methods and Results  Conclusion  Acknowledgements  Dedication  Questions

3.  Gluten is a protein composite comprised of the proteins gliadin and glutenin- 80% of protein in wheat  Used as a meat substitute, thickener, and emulsifier in food products  An estimated 3 million Americans suffer from celiac disease (gluten intolerance)

4.  Deamidation to improve functionality of gluten in food production  Deamidation to decrease allergenicity of gluten in food

5. Gluten High concentration of Glutamine Low water solubility Deamidation improves water solubility by preventing aggregation of glutamine residues via hydrogen bonding Deamidation has been shown to decrease allergenicity

6.  Protein Glutaminase (PG) from  Chryseobacterium proteolyticum

7.  Basic Reaction

8.  Kinetic Assay of PG  Protein characterization via SDS-PAGE  FT-IR to determine structural changes  Solubility Determination by Folin phenol reagent  Evaluation of emulsification properties  Allergenicity via ELISA

9. *Degree of Deamidation expressed as ratio of released ammonia to total glutamine residues.

10.  Solubility was determined by dissolving 1 mg samples in 1 mL of buffer at various pH levels.

11. Solid line with hashes represents Degree of Deamidation. Small dotted line is solubility at pH-3. Dashed line is pH-5. Solid line represents pH-7 Deamidation lowers pI

12.  Emulsification by dissolving gluten samples in buffers of various pH. Corn Oil was mixed into the solutions then homogenized and sonicated to produce the final emulsions.

13. White bars- Day 1 Black bars- Day 8 A- pH=7 B- pH= 5 C- pH= 3 Size determined via laser diffraction

14.  ELISA- Enzyme Linked Immunosorbent Assay  Gluten allergenic human blood serum- primary antibody  Horseradish Peroxidase labeled goat anti- human immunoglobulin

15. A- Serum from patient with moderate wheat allergy B- Serum from patient with severe wheat allergy Solid line square mark- normal gluten Dotted line triangle mark- gluten deamidated by PG for 30 hours

16.  Deamidation of gluten increased solubility and emulsification properties. Deamidation of gluten by PG also decreased allergenicity. Potential for more widespread use in food production and possible hypoallergenicity.

17.  Dr. Moffet  LMU Department of Chemistry and Biochemistry  Classes of 2013 and 2014  The Academy

18. For Scott “Celiac” Bosely

20. White- non modified gluten Black- Deamidated gluten Amide region- 1600-1700 wavenumber(cm -1 ) Fewer Beta-Sheets indicate less hydrogen bonding

21.  Total number of glutamine residues calculated from release of ammonia upon treatment with 3 M sulfuric acid

22.  Oxidation of substrate produces light!  Absorbance at 405 nm

23.  Infrared spectroscopy using multiple IR waves simultaneously  Deconvolution by Jasco Spectra Manager software

24.  Reduction of Folin reagent and oxidation of aromatic amino acid residues  Absorption at 750 nm  Solution centrifuged and supernatant collected for solubility measurement

25.  Used Horiba LA500 laser diffraction particle size analyzer  Large particles scatter at smaller angles and greater intensity  Small particles scatter light and great angles and decrease intensity of light absorption

26.  Deamidation by PG conducted at 40 celsius and pH=7.0 in solution containing 10 mg/ml wheat gluten ad.13 unit/mL PG

27. Lane A- Ladder Lane B- No PG Lane C-.5 hour w/ PG Lane D- 1 hr. w/ PG Lane E- 1.5 hours Lane F- 2 hours Lane G- 3 hours Lane H- 5 hours Lane I- 12 hours Lane J- 30 hours

28.  7.2 micromoles min -1 mg -1