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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
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Background Introduction Methods and Results Conclusion Acknowledgements Dedication Questions
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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)
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Deamidation to improve functionality of gluten in food production
Deamidation to decrease allergenicity of gluten in food
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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
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Protein Glutaminase (PG) from Chryseobacterium proteolyticum
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Basic Reaction
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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
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*Degree of Deamidation expressed as ratio of released ammonia to total glutamine residues.
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Solubility was determined by dissolving 1 mg samples in 1 mL of buffer at various pH levels.
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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
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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.
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White bars- Day 1Black bars- Day 8A- pH=7B- pH= 5C- pH= 3Size determined via laser diffraction
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ELISA- Enzyme Linked Immunosorbent Assay
Gluten allergenic human blood serum- primary antibody
Horseradish Peroxidase labeled goat anti-human immunoglobulin
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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
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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.
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Dr. Moffet LMU Department of Chemistry and
Biochemistry Classes of 2013 and 2014 The Academy
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For Scott “Celiac” Bosely
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White- non modified glutenBlack- Deamidated glutenAmide region- 1600-1700 wavenumber(cm-1)Fewer Beta-Sheets indicate less hydrogen bonding
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Total number of glutamine residues calculated from release of ammonia upon treatment with 3 M sulfuric acid
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Oxidation of substrate produces light! Absorbance at 405 nm
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Infrared spectroscopy using multiple IR waves simultaneously
Deconvolution by Jasco Spectra Manager software
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Reduction of Folin reagent and oxidation of aromatic amino acid residues
Absorption at 750 nm Solution centrifuged and supernatant
collected for solubility measurement
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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
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Deamidation by PG conducted at 40 celsius and pH=7.0 in solution containing 10 mg/ml wheat gluten ad .13 unit/mL PG
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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
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7.2 micromoles min-1 mg-1