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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
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
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