Identification and application of Koji protese to degrade wheat allergen

我们曾利用患者血清鉴定出小麦ω-5醇溶蛋白是小麦依赖性运动过敏反应（WDEIA）的主要过敏原，并确定了该蛋白的IgE结合表位序列。此外，还发现曲霉菌提取物中ω-5醇溶蛋白的表位肽具有蛋白水解活性。本研究从曲霉提取物中分离纯化蛋白酶，并对其生化特性进行了研究，以探讨该蛋白酶用于降低小麦制品过敏原性的可能性。通过三步柱层析、DEAE-Sepharose、Butyl-Sepharose和Superdex 75，从曲霉提取物中部分纯化了蛋白酶。部分纯化的蛋白酶的生化性质是碱性蛋白酶的特征。SDS-PAGE分析显示蛋白酶组分中有几条带。部分纯化的蛋白酶中的蛋白质之一35 kDa蛋白质的N-末端氨基酸序列被确定为EETTEKNAPXGL（X是未鉴定的氨基酸）。该N-末端氨基酸序列显示与枯草杆菌蛋白酶样丝氨酸蛋白酶蛋白的N-末端具有91%的同一性，所述枯草杆菌蛋白酶样丝氨酸蛋白酶蛋白由AspergillusRIB 40中的Open阅读Flame 000517编码。此外，部分纯化的蛋白酶对谷朊粉和ω-5醇溶蛋白具有一定的水解活性，表明部分纯化的曲霉蛋白酶可用于降低小麦制品中ω-5醇溶蛋白的致敏性。

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.