Identification and application of Koji protese to degrade wheat allergen

我们利用患者血清鉴定小麦ω-5麦麦蛋白是小麦依赖性运动诱导过敏反应（WDEIA）的主要过敏原，并确定了该蛋白的ige结合表位序列。此外，我们发现在曲霉提取制剂中ω-5麦胶蛋白表位肽具有蛋白水解活性。本研究从曲霉提取物中纯化了蛋白酶，并对其生化特性进行了研究，以探讨纯化后的蛋白酶在降低小麦产品过敏性方面的应用可能性。通过柱层析、DEAE sepharose、Butyl sepharose、Superdex 75三个步骤对曲霉提取物进行部分纯化。部分纯化的蛋白酶具有碱性蛋白酶的生化特性。SDS-PAGE分析显示蛋白酶部分有多个条带。35 kDa蛋白n端氨基酸序列为EETTEKNAPXGL （X为未知氨基酸），是部分纯化蛋白酶的蛋白之一。该n端氨基酸序列与米曲霉RIB40中Open Reading Flame 000517编码的枯草素样丝氨酸蛋白酶蛋白n端同源性为91%。此外，部分纯化的蛋白酶对小麦面筋和ω-5麦胶蛋白具有水解活性。这些结果表明，曲霉部分纯化的蛋白酶可以通过降解ω-5麦胶蛋白来降低小麦产品的致敏性。

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.