|Tilley, K - KANSAS STATE UNIV|
Submitted to: Inter Assoc for Cereal Science & Technology Jubilee Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: July 21, 2004
Publication Date: January 3, 2005
Citation: Tilley, M., Tilley, K.A. 2005. Modifying tyrosine crosslink formation in wheat dough by controlling innate enzymatic activity. p. 142-146. Using cereal science and technology for the benefit of consumers. Proceedings of the 12th ICC Cereal and Bread Congress. S.P. Cauvain, S.B. Salmon, and L.S. Young, eds. CRC Press. Boca Raton, FL. Interpretive Summary: Amino acid side chains of proteins are very important. They can interact for crosslinking within a protein and between different proteins. Crosslinking of the side chains of two tyrosines form dityrosine (DY). DY occurs in many extensible proteins such as those found in insect eggs, fibers such as silk and wool and may have a functional role in food processing. The water-soluble extract (WSE) of wheat flour was shown to catalyze the formation of DY from tyrosine. The objective of this project was to identify the active component(s) of the WSE that are involved in catalyzing dityrosine. The WSE of flour was fractionated and tested for dityrosine forming activity. The fraction causing the greatest formation of the crosslink was further divided into single components. The major find was the presence of a peroxidase enzyme that is specifically found in the endosperm in the fraction that makes the most DY. The enzyme was purified from that fraction and the gene that codes for the enzyme was found.
Technical Abstract: Dityrosine (DY) is one of several crosslinks found in biological protein polymer systems, including plants and food matrices. Protein crosslinking via DY formation is initiated by free radical oxidation and/or enzymatic methods. Peroxidase is commonly used to catalyze DY and related bonds. DY has recently been found to form during mixing and baking of wheat flour. The water-soluble extract (WSE) from wheat flour, contain several biologically active enzyme systems and has the ability to catalyze DY formation. The albumin fraction (water soluble extract -- WSE) was fractionated via preparative isoelectric focusing and resulting fractions were collected and tested for ability to form DY from free tyrosine. Proteins in the most reactive fractions were purified by cation exchange chromatography and subjected to N-terminal amino sequencing. The fraction that catalyzed the greatest amount of DY contained a predominant 38 kDa protein that was determined to have the N-terminal sequence: AEPPVARGLSFDFYRRT?PRAES. cDNA libraries from developing wheat kernels and Aegilops tauschii were screened and isolated cDNAs were sequenced. The resulting cDNAs of 1197 (T. aestivum) and 1191 (Ae. tauschii) nucleotides both have an open reading frame of 1077 nucleotides and encode a protein of 358 amino acids with a 26 amino acid signal sequence. The sequence has 90.4% identity at the nucleotide level and 89% identity at the amino acid level with barley endosperm-specific cationic peroxidase BP1. Comparison to other peroxidase sequences from non-endosperm tissues of wheat display 40-45% similarity, however amino acid residues of the active site are highly conserved. The identification of endogenous components that catalyze DY may provide a means of predicting and controlling breadmaking quality.