Identification and application of Koji protese to degrade wheat allergen

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Previously we identified wheat ω-5 gliadin as major allergens for wheat-dependent exercise-induced anaphylaxis (WDEIA) by using patient's sera, and defined IgE-binding epitope sequence of this protein. In addition, we found a proteolytic activity for epitope peptide of ω-5 gliadin in the extracted preparation of Koji mold. In this study, we purified the protease from Koji mold extract and examined the biochemical properties in order to investigate a possibility of application for reduction in allergenicity of wheat products using the purified protease.The protease was partially purified from Koji mold extract by three steps of column chromatographies, DEAE sepharose, Butyl sepharose, and Superdex 75. Biochemical properties of the partially purified protease were characteristic for alkaline protease. SDS-PAGE analysis revealed several band in the protease fraction. N-terminal amino acid sequence of 35 kDa protein, which is one of proteins in partially purified protease, was determined to be EETTEKNAPXGL (X is unidentified amino acid). This N-terminal amino acid sequence showed a 91% identity with the N-terminus of subtilisin-like serine protease protein, which is encoded by Open Reading Flame 000517 in Aspergillus oryzae RIB40. Furthermore, partially purified protease showed proteolytic activity for wheat gluten and ω-5 gliadin.These results indicate that partially purified protease from Koji mold may be useful tool for reduction of allergenicity in wheat products by degradation of ω-5 gliadin.