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

Background Introduction Methods and Results Conclusion Acknowledgements Dedication Questions

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)

Deamidation to improve functionality of gluten in food production

Deamidation to decrease allergenicity of gluten in food

Gluten • High concentration of Glutamine• Low water solubilityDeamidation improves water solubility by

preventing aggregation of glutamine residues via hydrogen bonding

Deamidation has been shown to decrease allergenicity

Protein Glutaminase (PG) from Chryseobacterium proteolyticum

Basic Reaction

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

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

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

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-7Deamidation lowers pI

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.

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

ELISA- Enzyme Linked Immunosorbent Assay

Gluten allergenic human blood serum- primary antibody

Horseradish Peroxidase labeled goat anti-human immunoglobulin

A- Serum from patient with moderate wheat allergyB- Serum from patient with severe wheat allergySolid line square mark- normal glutenDotted line triangle mark- gluten deamidated by PG for 30 hours

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.

Dr. Moffet LMU Department of Chemistry and

Biochemistry Classes of 2013 and 2014 The Academy

For Scott “Celiac” Bosely

White- non modified glutenBlack- Deamidated glutenAmide region- 1600-1700 wavenumber(cm-1)Fewer Beta-Sheets indicate less hydrogen bonding

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

Oxidation of substrate produces light! Absorbance at 405 nm

Infrared spectroscopy using multiple IR waves simultaneously

Deconvolution by Jasco Spectra Manager software

Reduction of Folin reagent and oxidation of aromatic amino acid residues

Absorption at 750 nm Solution centrifuged and supernatant

collected for solubility measurement

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

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

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

7.2 micromoles min-1 mg-1