CN111466511A - 含有去环氧基蛋白酶的饮食品或饲料组合物及其加工方法 - Google Patents

含有去环氧基蛋白酶的饮食品或饲料组合物及其加工方法 Download PDF

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CN111466511A
CN111466511A CN202010147974.8A CN202010147974A CN111466511A CN 111466511 A CN111466511 A CN 111466511A CN 202010147974 A CN202010147974 A CN 202010147974A CN 111466511 A CN111466511 A CN 111466511A
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Abstract

本发明公开含有去环氧基蛋白酶的饮食品或饲料组合物及其加工方法，其包含去环氧基蛋白酶，该蛋白酶在15‑35℃温度下能够使单端孢霉烯族毒素与谷胱甘肽催化反应产生谷胱甘肽化衍生物。本发明通过去环氧基蛋白酶来体外酶催化处理受毒素污染的谷物制品以满足饲料工业、食品工业等对单端孢霉烯族毒素的脱毒需求。弥补了目前尚未有明确的蛋白可通过去环氧基催化单端孢霉烯族毒素进行解毒的空白，在涉及饮食品或饲料单端孢霉烯族毒素脱毒中具有广阔的应用前景。

Description

含有去环氧基蛋白酶的饮食品或饲料组合物及其加工方法
技术领域
本发明涉及饮食或饲料组合物领域，具体地涉及一种含有去环氧基催化活性的蛋白 酶的饮食品或饲料组合物及其加工方法。
背景技术
单端孢霉烯族毒素的基本化学结构是倍半萜烯，因其在第12位碳、第13位碳上形成环氧基，故又称12,13-环氧单端孢霉烯族化合物。自从二十世纪70年代，科研工作者 已经探明单端孢霉烯族毒素的环氧基是主要毒性来源基团。单端孢霉烯族毒素可大量存 在于受镰刀菌属细菌侵染的植物或籽粒中，而镰刀菌属是一类世界性分布的真菌，它不 仅可以在土壤中越冬越夏，还可侵染多种植物，引起植物的根腐、茎腐、茎基腐、花腐 和穗腐等多种病害，寄主植物达100余种，侵染寄主植物维管束系统，破坏植物的输导 组织维管束，并在生长发育代谢过程中产生毒素危害作物，造成作物萎蔫死亡，影响产 量和品质，是生产上防治最艰难的重要病害之一。
由镰刀菌属导致的谷物减产和籽粒霉菌毒素污染已成为我国及世界范围内最亟待解 决的粮食安全问题之一。例如，禾谷镰刀菌在小麦杨花期侵染小麦穗部，分泌大量单端 孢霉烯族毒素从而显著增加病原菌的致病性，导致对产量造成毁灭性的破坏。另外，食用受到该类毒素污染的小麦籽粒后会导致食欲减退或废绝、胃肠炎症和出血、呕吐、腹 泻、坏死性皮炎、运动失调、血凝不良、贫血和白细胞数量减少、免疫机能降低和流产 等，严重威胁人、畜健康。
目前，尽管从动物肠道微生物中已分离出几种厌氧细菌可以对单端孢霉烯族毒素进 行解毒，但由于依赖于厌氧条件，实际工业应用受到很大限制。因此，分离出可高效对单端孢霉烯族毒素去环氧基的基因或酶，通过体外酶催化处理受毒素污染的谷物制品或制成蛋白类解毒药物，将满足饲料工业、食品工业等对单端孢霉烯族毒素的脱毒需求。 遗憾的是，目前尚未报道有明确的基因或蛋白可通过去环氧基催化单端孢霉烯族毒素进 行解毒。
发明内容
针对解决现有技术中的技术问题，本发明提供含有去环氧基蛋白酶的饮食口或饲料 组合物，其能够使具有毒性的单端孢霉烯族毒转化为无毒或低毒的衍生物，从而实现食品或饲料工业中对于单端孢霉烯族毒素解毒脱毒的需求。具体地，本发明包括以下内容。
本发明的第一方面，提供一种饮食品或饲料组合物，其包含去环氧基蛋白酶，所述蛋白酶在15-35℃温度下能够使单端孢霉烯族毒素的环氧基与谷胱甘肽(GSH)在PBS缓冲液中催化反应产生GSH化衍生物。此处，虽然限定了15-35℃的反应温度，但这里仅仅 是表征或鉴定多肽在该条件下具有去环氧催化活性，并不意味着在低于15℃或高于35℃ 时本发明的活性多肽不具有去环氧基催化活性。实际上，本发明的活性多肽催化反应的 条件并不局限于上述温度。催化反应的条件优选为在20-30℃温度下进行催化反应，更优 选为22-27℃。在具体实施方案中，反应条件为25℃。
根据本发明的饮食品或饲料组合物，优选地，所述蛋白酶具有选自由下述(1)-(5)组成 的组中的一种氨基酸序列：
(1)SEQ ID No.:1-35所示的氨基酸序列。其中SEQ ID No.1表示来源于长穗偃麦草十 倍体的氨基酸序列，SEQ ID No.2表示来源于长穗偃麦草二倍体的氨基酸序列，SEQID No.:3-24表示已验证具有原活性的SEQ ID No.1的突变体序列，SEQ ID No.:25-35表示来 源于香柱菌属
Figure BDA0002401430690000011
的不同物种的氨基酸序列。
(2)与(1)中的氨基酸序列的序列同一性为90％以上，且来源于相同属的氨基酸序列。 与(1)中的氨基酸序列的序列同一性优选95％以上，更优选97％以上，还优选98％以上， 进一步优选99％以上，且来源于相同属，优选来源于相同物种的氨基酸序列；进一步优选这些序列组成的多肽仍具有原蛋白酶活性。在某些实施方案中，活性多肽的氨基酸序 列与(1)中的氨基酸序列的序列同一性为95％以上，且均来源于香柱菌属。
(3)与(1)或(2)的氨基酸序列相比具有一个或多个氨基酸突变且序列同一性为90％以 上的氨基酸序列，优选95％以上，更优选97％以上，还优选98％以上，进一步优选99％ 以上的氨基酸序列，此处的氨基酸突变包括氨基酸的插入、缺失或替换。
(4)源自(1)-(3)任一项所述的氨基酸序列的部分连续序列。优选具有该部分连续序列 的多肽(或截短多肽)仍具有原多肽的酶催化活性，更优选具有位于(1)-(3)任一项所述的氨 基酸序列N端的部分连续序列，例如，具有从N端起前200至前250个氨基酸序列的多肽，例如具有N端起前208个氨基酸序列的多肽，或具有从N端起前242个氨基酸序列 的多肽。在某些实施方案中，部分连续序列可以是第92-110位之间的氨基酸序列或第 144-184位之间的氨基酸序列，或者两者的组合。
(5)在(1)-(4)任一项所述的氨基酸序列的N端和/或C端连接其他氨基酸序列的嵌合 序列。即，本发明的活性多肽可以为嵌合多肽。在某些实施方案中，其他氨基酸序列为增强多肽表达或分泌的序列，其实例包括但不限于引导肽、信号肽和转动肽。在某些实 施方案中，活性多肽为全长蛋白的活性片段与其他氨基酸序列的嵌合多肽，其中其他氨 基酸序列为对应于该活性片段之外的来源于同源的其他蛋白的序列，例如结构区或功能 区的序列。例如，当来源于某物种中的蛋白酶全长由A+B两部分组成，与其相同属但不 同物种的另一同源蛋白酶全长对应的由A’+B’两部分组成，且A与A’为同源对应区域， B与B’为同源对应区域的情况下，嵌合多肽可以由A’+B或A+B’组成。在某些实施方案 中，其他氨基酸序列包含非功能序列，例如连接臂或间隔序列。在某些实施方案中，其 他氨基酸序列为具有独立功能的多肽，其通过非功能序列，例如连接臂或间隔序列与本 发明的活性多肽连接。
根据本发明的饮食品或饲料组合物，优选地，当以SEQ ID No.1所示的氨基酸序列作为位置参考时，所述蛋白酶具有选自下述至少之一的保守位点：第98位氨基酸A、第 99位氨基酸A。
根据本发明的饮食品或饲料组合物，优选地，所述饮食品组合物进一步包含谷胱甘 肽。
根据本发明的饮食品或饲料组合物，优选地，所述饮食品或饲料组合物包含选自由 小麦粉、大麦粉、黑麦粉、燕麦粉、玉米粉、谷子粉、大米粉、高粱粉、大豆粉和花生 粉组成的组中的至少一种粮食粉；或者包含粮食加工产品，例如豆皮、豆浆、胚芽粕、 胚芽、植物油、淀粉、葡萄糖、蛋白粉、酒精和发酵物。
根据本发明的饮食品或饲料组合物，优选地，所述饮食品或饲料组合物包含选自苹 果、柑橘和葡萄组成的组中的至少一种果汁成分。
本发明的第二方面，提供一种饮食品或饲料组合物的加工方法，其包括在适于反应 的条件下使饮食品或饲料原料与去环氧基蛋白酶或产生该酶的细胞接触的步骤。
根据本发明的饮食品或饲料组合物的加工方法，优选地，进一步包括向饮食品或饲 料原料添加谷胱甘肽。添加的谷胱甘肽的浓度优选为0.05-1.5mol/L。还优选为 0.08-1.2mol/L。更优选为0.09-1.1mol/L。在具体实施方案中，谷胱甘肽的浓度为0.1mol/L。
根据本发明的饮食品或饲料组合物的加工方法，优选地，其原料中加入水混合均匀 得到反应液，然后向反应液中加入去环氧基蛋白酶或产生该酶的细胞和可选的谷胱甘肽， 在2～40℃下反应5分钟至36小时。其中，产生该酶的细胞可以是例如通过基因工程手段使能够产生第一方面所述的蛋白酶的核酸分子导入到宿主细胞与单端孢霉烯族化合物和GSH接触，从而生成GSH化衍生物的步骤。此类宿主细胞可举例例如原核细胞或真 核细胞。原核细胞的实例包括但不限于大肠杆菌等，真核细胞的实例包括但不限于酵母、 植物细胞或动物细胞。本发明的饮食品或饲料组合物的加工方法的反应条件包括1～45℃， 优选2～40℃，更优选5～35℃，进一步优选10～30℃的反应温度；10分钟至36小时，例 如10～60分钟、1.5～24小时的反应时间。具体的反应条件需根据酶的来源、酶活性大小、 底物浓度、反应量等由本领域人员根据需要而调整，并不作特别限定。
本发明的第三方面，提供一种降低或减少组合物中毒素的方法，其包括在适于反应 的条件下使含有毒素的饮食品或饲料原料与去环氧基蛋白酶或产生该酶的细胞接触的步 骤，其中毒素为单端孢霉烯族化合物。优选地，毒素为脱氧雪腐镰刀菌烯醇、15-乙酰脱氧雪腐镰刀菌烯醇、3-乙酰脱氧雪腐镰刀菌烯醇、雪腐镰刀菌烯醇、镰刀菌烯酮-X、二 乙酰氧基草镰刀菌烯醇、T-2毒素和HT-2毒素中的至少一种。
附图说明
图1A为LC-HRMS(方法1)DON与GSH体外酶促反应的提取离子流谱图EIC。
图1B为LC-HRMS2(方法2)DON与GSH体外酶促反应获得的DON-GSH的高能碰 撞解离产生的子离子质谱图。
图2A为LC-HRMS(方法1)3-ADON与GSH体外酶促反应的提取离子流谱图EIC。
图2B为LC-HRMS2(方法2)3-ADON与GSH体外酶促反应获得的3-ADON-GSH通 过高能碰撞解离产生的子离子质谱图。
图3A为LC-HRMS(方法1)15-ADON与GSH体外酶促反应提取的提取离子流谱图 EIC。
图3B LC-HRMS2(方法2)15-ADON与GSH体外酶促反应获得的15-ADON-GSH的 高能碰撞解离产生的子离子质谱图。
图4A为LC-HRMS(方法1)NIV与GSH体外酶促反应的提取离子流谱图EIC。
图4B为LC-HRMS2(方法2)NIV与GSH体外酶促反应获得的NIV-GSH的高能碰撞 解离产生的子离子质谱图。
图5A为LC-HRMS(方法1)Fus-X与GSH体外酶促反应的提取离子流谱图EIC。
图5B为LC-HRMS2(方法2)Fus-X与GSH体外酶促反应获得的Fus-X-GSH的高能碰 撞解离产生的子离子质谱图。
图6A为LC-HRMS(方法1)DAS与GSH体外酶促反应提取的提取离子流谱图EIC。
图6B为LC-HRMS2(方法2)DAS与GSH体外酶促反应获得的DAS-GSH的高能碰撞 解离产生的子离子质谱图。
图7A为LC-HRMS(方法1)HT-2与GSH体外酶促反应提取的提取离子流谱图EIC。
图7B为LC-HRMS2(方法2)HT-2与GSH体外酶促反应获得的HT-GSH加合物的高 能碰撞解离产生的子离子质谱图。
图8A为LC-HRMS(方法1)T-2与GSH体外酶促反应提取的提取离子流谱图EIC。
图8B为LC-HRMS2(方法2)T-2与GSH体外酶促反应获得的T2-GSH加合物的高能 碰撞解离产生的子离子质谱图。
图9单端孢霉烯族化合物对人体细胞系活力的影响。不同浓度梯度的DON、3ADON、15ADON、FUS-X、NIV、T-2、HT-2、DAS处理细胞48h后，测定OD450nm。
图10FTCD体外纯化蛋白对玉米深加工产品中DON的清除结果。
图11FTCD体外纯化蛋白对两种品牌苹果汁中DON的清除结果。
图12FTCD及同源序列进化树。
图13LC-HRMS(方法1)DON处理FTCD同源序列转基因酵母提取的离子色谱图。 正离子模式提取到DON-GSH加合物，m/z 604.21730(对应于[M+H]+，Δ±5ppm)。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制， 而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。 另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限以及它们之 间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范 围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可 独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技 术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。除非另有说明，否则“％”为基于重量的百 分数。
本文中，术语“多肽”和“蛋白”可互换使用，是指氨基酸残基的聚合物及其变体 和合成的和天然存在的类似物。这两个术语适用于其中一个或多个氨基酸残基是合成的 非天然存在的氨基酸聚合物，以及适用于天然存在的氨基酸聚合物及其天然存在的化学 衍生物。此类化学衍生物包括例如翻译后修饰和降解产物，包括焦谷氨酰、异天冬氨酰、 蛋白水解的、磷酸化的、糖基化的、氧化的、异构化的和脱氨基化的变体。
本文中，术语“活性多肽”是指具有去环氧基酶催化活性的多肽，即将环氧基转化为其他基团或去除该基团的活性多肽。本文有时也称作“蛋白酶”。
本文中，术语“序列同一性”是指基因或蛋白质之间分别在核苷酸或氨基酸水平下的序列同一性。“序列同一性”是蛋白质之间的同一性在氨基酸水平的量度和核酸之间 的同一性在核苷酸水平下的量度。蛋白质序列同一性可以在比对序列时通过比较每个序 列中的给定位置的氨基酸序列来确定。类似地，核酸序列同一性可以在比对序列时通过 比较每个序列中的给定位置的核苷酸序列来确定。用于比对用以比较的序列的方法为本 领域熟知的，此类方法包括GAP、BESTFIT、BLAST、FASTA和TFASTA。BLAST算法 计算序列同一性百分比并且对两个序列之间的相似性执行统计分析。用于执行BLAST分 析的软件可通过国家生物技术信息中心(NCBI)网站公开获得。
本文中，术语“第x位氨基酸”或类似表述均以来源于长穗偃麦草十倍体的去环氧酶 的氨基酸序列为位置参考，即以SEQ ID No.1所示的氨基酸序列为位置参考，除非另有明 确说明。
本文中，术语“单端孢霉烯族毒素”或“单端孢霉烯族化合物”是指基本化学结构为倍半萜烯，在第12位碳、第13位碳上形成环氧基的一类化合物的统称。优选地，单端 孢霉烯族毒素具有下述通式(II)所示的结构：
Figure BDA0002401430690000041
其中，R1、R2和R3各自独立地表示氢原子、羟基或由-OCO-R’表示的酯基，其中R’ 为直链或支链C1-C5烷基，例如CH3、CH2CH3、CH2CH2CH3或CH2(CH3)2，R4表示氢原 子或羟基，R5表示氢原子、＝O、羟基或由-OCO-R”表示的酯基，其中R”为直链或支链 C1-C10烷基，优选为CH3、CH2CH3，还优选为直链或支链C3-C8烷基，更优选CH2CH(C H3)2。在某些实施方案中，单端孢霉烯族毒素包括脱氧雪腐镰刀菌烯醇(DON)、15-乙酰 脱氧雪腐镰刀菌烯醇(15-ADON)、3-乙酰脱氧雪腐镰刀菌烯醇(3-ADON)、雪腐镰刀菌烯 醇(NIV)、镰刀菌烯酮-X(Fus-X)、二乙酰氧基草镰刀菌烯醇(DAS)、T-2毒素(T-2)、HT-2 毒素(HT-2)。
本文中，术语“去环氧基催化活性”是指能够将单端孢霉烯族毒素中的环氧基(优选 在第12位碳、第13位碳上形成的环氧基)去除的活性或功能。具体催化过程如下所示：
Figure BDA0002401430690000051
其中，R1-R5的含义与通(I)和(II)中的含义相同。
实施例1
一、活性多肽FTCD催化单端孢霉烯族毒素去环氧基反应
1.实验方法：
1.1体外酶促反应：
将DON、3-DON、15-ADON、NIV、DAS、HT-2、T-2毒素(1mg)分别溶解于新鲜制 备的GSH(30.7mg，100μmol)的PBS缓冲液中，并加入活性多肽，于20℃水浴锅保温24h。
1.2LC-HRMS(/MS)分析
体外反应液通过0.22μm滤膜过滤后，转移到进样小瓶中准备进行LC-HRMS检测。LC-HRMS(/MS)分析使用Xcalibur 2.1.0分析数据。采用所提出的生物转化产物的色谱峰形状，保留时间(±0.2分钟)和质量(±5ppm)对毒素及其衍生物的提取离子色谱图(EIC)进行 了研究。根据二级图谱以及物质基本结构解析中性丢失，推测化学结构。
2.实验结果
2.1FTCD催化DON毒素并转化成谷胱甘肽加合物DON-GSH
图1A为LC-HRMS1(方法1)DON与GSH体外酶促反应的提取离子流谱图EIC。如 图1A所示，以负离子模式从LC-HRMS(Full scan模式)中得到DON的提取离子流谱图 EIC，m/z355.13984(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到DON-GSH 加合物，m/z604.21707(对应于[M+H]+，Δ±5ppm)。
图1B为LC-HRMS2(方法2)DON与GSH体外酶促反应获得的DON-GSH的高能碰 撞解离产生的子离子质谱图，[M+H]+(m/z 604.21707，Δ±5ppm)。通过对带正电([M+H]+) 的离子进行靶向HRMS2分析，研究了DON-GSH环氧加合物的MS片段。DON-GSH的 离子碎裂产生m/z299.0939的特征离子，对应于C14H19O5S+。该特征离子可归因于在C-6 处侧链的裂解和除S以外GSH部分的损失。该片段还可以进一步裂解产生 m/z281.08482(C14H17O4S+)，263.07425(C14H15O3S+)和231.10218(C14H15O3 +)。m/z 263.07425 处的产物离子是HRMS2质谱图的基峰，该产物离子在m/z 299.0939的基础上脱去两分子 H2O。
DON-GSH在甘氨酸损失之后，可得到碎片离子m/z 529.18503(C23H33O10N2S+)，也可得到损失脱水谷氨酸的碎片离子475.17466(C20H31O9N2S+)。损失了C-6处侧链的m/z574.20717(C24H36O11N3S+)离子碎片从其GSH部分损失脱水谷氨酸可得到m/z445.16389 的特征离子(C19H29O8N2S+)；也可得到脱去谷氨酰胺的428.13733(C19H26O8NS+)。
产物离子为m/z 308.09108(C10H18O6N3S+，对应GSH的[M+H]+)。该碎片离子损失脱水谷氨酸得到m/z 179.04907(C5H11O3N2S+)；损失谷氨酰胺得到m/z162.02251(C5H9O3NS+)。此外，m/z130.05044(C5H8O3N+)、m/z145.06077(C5H9O3N2 +)的产物离子均与GSH相关。
2.2FTCD催化3-ADON毒素并转化成谷胱甘肽加合物3-ADON-GSH
图2A为LC-HRMS(方法1)3-ADON与GSH体外酶促反应的提取离子流谱图EIC。 如图2A所示，以负离子模式从LC-HRMS(Full scan模式)中得到3-ADON的提取离子流 谱图EIC，m/z 397.15041(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到 3-ADON-GSH加合物，m/z 646.22764(对应于[M+H]+，Δ±5ppm)。
图2B为LC-HRMS2(方法2)3-ADON与GSH体外酶促反应获得的3-ADON-GSH通 过高能碰撞解离产生的子离子质谱图，[M+H]+(m/z 646.22764，Δ±5ppm)。对带正电 ([M+H]+)的3-ADON-GSH环氧加合物离子进行靶向HRMS2分析：3-ADON-GSH的离子 碎裂会产生m/z323.09539的特征离子，对应于C16H19O5S+。该特征离子可归因于在C-6 处连接的侧链的裂解、脱水以及除S以外GSH部分的损失。该片段还可以进一步裂解产 生m/z，263.07425(C14H15O3S+)和231.10218(C14H15O3 +)。m/z 263.07425处的子离子是 HRMS2质谱图的基峰，该产物离子是在m/z 323.09539离子的基础上于C-3处脱去 CH3COOH。
3-ADON-GSH甘氨酸损失之后，可得到碎片离子m/z 571.19560(C25H35O11N2S+)，其C-6处侧链进一步断裂可产生的m/z 541.18503(C24H33O10N2S+)的碎片离子；脱去1分子 H2O的碎片离子m/z 628.21707(C27H38O12N3S+)损失甘氨酸可得到m/z 553.18503(C25H33O10N2S+)；损失脱水谷氨酸可得到499.17466(C22H31O9N2S+)。
2.3FTCD催化15-ADON毒素并转化成谷胱甘肽加合物15-ADON-GSH
图3A为LC-HRMS(方法1)15-ADON与GSH体外酶促反应提取的提取离子流谱图 EIC。如图3A所示，以负离子模式从LC-HRMS(Full scan模式)中得到15-ADON的提取 离子流谱图EIC，m/z 397.15041(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取 到15-ADON-GSH加合物，m/z 646.22764(对应于[M+H]+，Δ±5ppm)。
图3BLC-HRMS2(方法2)15-ADON与GSH体外酶促反应获得的15-ADON-GSH的高 能碰撞解离产生的子离子质谱图，[M+H]+(m/z 646.22764，Δ±5ppm)。通过对带正电 ([M+H]+)的离子进行靶向HRMS2分析，研究了15-ADON-GSH环氧加合物的MS片段。 15-ADON-GSH的离子碎裂产生m/z 311.09475的特征离子，对应于C15H19O5S+。该特征 离子可归因于在C-15处连接的侧链CH3COOH的裂解和除S以外GSH部分的损失。
同3-ADON-GSH一样，15-ADON-GSH甘氨酸损失之后，可得到子离子m/z 571.1956(C25H35O11N2S+)。脱去1分子H2O的m/z 628.21707(C27H38O12N3S+)损失甘氨酸 可得到m/z553.18503(C25H33O10N2S+)。损失脱水谷氨酸的可得到m/z 499.17466(C22H31O9N2S+)。
特征离子m/z 440.13736(C20H26O8NS+)损失脱水谷氨酸可得到m/z 311.09475(C15H19O5S+)的碎片离子；特征离子m/z 450.15471(C17H28O9N3S+)损失甘氨酸得 到m/z375.12267(C15H23O7N2S+)子离子，损失脱水谷氨酸得到m/z 321.1121(C12H21O6N2S +)，此外该离子还可以脱去两分子H2O，形成子离子m/z 414.13295(C17H24O7N3S+)，该特 征离子损失甘氨酸可得到m/z 339.10091(C15H19O5N2S+)，损失脱水谷氨酸得到m/z 285.09035(C12H17O4N2S+)。该片段进一步脱水还可以产生m/z 267.07979(C12H15O3N2S+)。 与GSH相关的m/z145.06077(C5H9O3N2 +，Δ±5ppm)特征离子是质谱图的基峰。
2.4FTCD催化NIV毒素并转化成谷胱甘肽加合物NIV-GSH
如图4A所示，以负离子模式从LC-HRMS(Full scan模式)中得到NIV的提取离子流谱图EIC，m/z 371.13366(对应[M+CH3COO]-形式，Δ±5ppm)；正离子模式提取到 NIV-GSH加合物，m/z 620.21199(对应于[M+H]+，Δ±5ppm)。
图4B为LC-HRMS2(方法2)NIV与GSH体外酶促反应获得的NIV-GSH的高能碰撞 解离产生的子离子质谱图，[M+H]+(m/z 620.21199，Δ±5ppm)。通过对带正电([M+H]+) 的NIV-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。NIV-GSH的离子 碎裂产生m/z229.08652的产物离子，对应于C14H13O3 +。该产物离子可归因于在C-6处 侧链的裂解、3分子H2O的断裂以及GSH部分的损失，该结构保留NIV的基本骨架。
NIV-GSH在甘氨酸损失之后，可得到子离子m/z 545.17995(C23H33O11N2S+)。也可得到损失脱水谷氨酸的子离子491.16938(C20H31O10N2S+)。C-6处侧链断裂后形成m/z590.20142(C24H36O12N3S+)，该离子的GSH部分损失脱水谷氨酸可得到m/z 461.15881的 子离子(C19H29O9N2S+)。
[M+H]+形式的GSH损失谷氨酰胺可得到子离子m/z 162.02251(C5H9O3NS+)；损失脱水谷氨酸得到m/z 179.04907(C5H11O3N2S+)，该离子是HRMS2质谱中最突出的产物离子。 此外，子离子m/z 130.05044(C5H8O3N+)和子离子m/z 145.06077(C5H9O3N2 +)均与GSH相 关。
2.5FTCD催化Fus-X毒素并转化成谷胱甘肽加合物Fus-X-GSH
图5A为LC-HRMS(方法1)Fus-X与GSH体外酶促反应的提取离子流谱图EIC。如 图5A所示，以负离子模式从LC-HRMS(Full scan模式)中得到Fus-X的提取离子流谱图 EIC，m/z377.12069(对应[M+Na]+形式，Δ±5ppm)；正离子模式提取到Fus-X-GSH加合 物，m/z662.22255(对应于[M+H]+，Δ±5ppm)。
图5B为LC-HRMS2(方法2)Fus-X与GSH体外酶促反应获得的Fus-X-GSH的高能碰 撞解离产生的子离子质谱图。通过对带正电([M+H]+)的FusX-GSH环氧加合物离子进行 靶向HRMS2分析，研究其MS片段。FusX-GSH的离子碎裂产生m/z 297.07973的产物 离子，对应于C14H17O5S+。该产物离子可归因于在C-4处侧链的裂解、C-6处侧链的裂解 及除S以外的GSH部分的损失，该结构仅仅保留Fus-X的基本骨架。
FusX-GSH在甘氨酸损失之后，可得到子离子m/z 587.19051(C25H35O12N2S+)。C-6 处侧链断裂后形成m/z 632.21198(C26H38O13N3S+)的特征离子损失脱水谷氨酸可得到m/z503.16937(C21H31O10N2S+)的子离子，损失谷氨酰胺可以得到m/z 486.14281(C21H28O10NS +)。其中m/z 503.16937(C24H36O12N3S+)的子离子是HRMS2质谱中最突出的产物离子。
[M+H]+形式的GSH损失谷氨酰胺可得到子离子m/z 162.02251(C5H9O3NS+)；损失 脱水谷氨酸得到m/z 179.04907(C5H11O3N2S+)，此外，子离子m/z 130.05044(C5H8O3N+) 和子离子m/z 145.06077(C5H9O3N2 +)均与GSH相关。
2.6FTCD催化DAS毒素并转化成谷胱甘肽加合物DAS-GSH
图6A为LC-HRMS(方法1)DAS与GSH体外酶促反应提取的提取离子流谱图EIC。 以正离子模式从LC-HRMS(Full scan模式)中得到DAS的提取离子流谱图EIC，m/z 389.15707(对应[M+Na]+形式，Δ±5ppm)；DAS-GSH加合物，m/z 674.25894(对应于 [M+H]+，Δ±5ppm)。
图6B为LC-HRMS2(方法2)DAS与GSH体外酶促反应获得的DAS-GSH的高能碰撞 解离产生的子离子质谱图，[M+H]+(m/z 674.25894，Δ±5ppm)。通过对带正电([M+H]+) 的离子进行靶向HRMS2分析，研究DAS-GSH环氧加合物的MS片段。DAS-GSH的离 子碎裂产生m/z229.12231的产物离子，对应于C15H17O2 +。该产物离子可归因于C-4、 C-15处连接的侧链CH3COOH的裂解、脱水以及GSH部分的损失。
DAS-GSH在甘氨酸损失之后，可得到子离子m/z 599.22690(C27H39O11N2S+)；损失 谷氨酰胺，可得到m/z 528.18977(C24H34O10NS+)的子离子；也可得到损失脱水谷氨酸的子 离子545.21633(C24H37O10N2S+)；损失了CH3COOH可得到m/z 614.23781(C27H40O11N3S+) 的特征离子。
与GSH相关的m/z 130.05044(C5H8O3N+)、m/z 145.06077(C5H9O3N2 +)、m/z162.02251(C5H9O3NS+)、m/z 179.04907(C5H11O3N2S+)子离子中，失去脱水谷氨酸m/z的特 征离子在179.04907处(C5H11O3N2S+)是质谱图的基峰。
2.7FTCD催化HT-2并转化成谷胱甘肽加合物HT-2-GSH
图7A为LC-HRMS(方法1)HT-2与GSH体外酶促反应提取的提取离子流谱图EIC。 以正离子模式从LC-HRMS(Full scan模式)中得到HT-2的提取离子流谱图EIC，m/z 447.19894(对应[M+Na]+形式，Δ±5ppm)；HT-GSH的加合物，m/z 732.30080(对应于[M+H] +，Δ±5ppm)。
图7B为LC-HRMS2(方法2)HT-2与GSH体外酶促反应获得的HT-GSH加合物的高 能碰撞解离产生的子离子质谱图，[M+H]+(m/z 732.30080，Δ±5ppm)。通过对带正电([M+H] +)的HT-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。HT-GSH碎裂产 生m/z295.10048的产物离子，对应于C15H19O4S+。该产物离子可归因于C-8处 ((CH3)2CHCH2COOH)的断裂，C-15处CH3COOH的裂解、除S以外GSH部分的损失， 该结构保留了HT-2的基本骨架。而m/z 274.10335是[M+H]+形式的GSH的—SH键断 裂，形成H2S的中性丢失所致。
HT-GSH在C-8、C-15处的侧链断裂之后可得到m/z 570.21226(C25H36O10N3S+)的特征离子，该离子损失甘氨酸可得到m/z 495.18022(C23H31O8N2S+)；损失脱水谷氨酸可得 到m/z 441.16965(C20H29O7N2S+)；损失谷氨酰胺可得到m/z 424.14309(C20H26O7NS+)的碎 片离子。m/z 441.16965处是质谱图的基峰。
此外还检测到与GSH相关的m/z 130.05044(C5H8O3N+)、m/z 145.06077(C5H9O3N2 +)、m/z 162.02251(C5H9O3NS+)、m/z 179.04907(C5H11O3N2S+)离子。
2.8FTCD催化T-2并转化成谷胱甘肽加合物HT-2-GSH
图8A为LC-HRMS(方法1)T-2与GSH体外酶促反应提取的提取离子流谱图EIC。 以正离子模式从LC-HRMS(Full scan模式)中得到T-2的提取离子流谱图EIC，m/z 489.20950(对应[M+Na]+形式，Δ±5ppm)；T2-GSH的加合物，m/z 774.31136(对应于 [M+H]+，Δ±5ppm)。
图8B为LC-HRMS2(方法2)T-2与GSH体外酶促反应获得的T2-GSH加合物的高能 碰撞解离产生的子离子质谱图，[M+H]+(m/z 774.31136，Δ±5ppm)。通过对带正电([M+H] +)的T2-GSH环氧加合物离子进行靶向HRMS2分析，研究其MS片段。T2-GSH碎裂产 生m/z337.11105的产物离子，对应于C17H21O5S+。该产物离子可归因于C-8处、C-15处 所连接侧链的断裂、除S以外GSH部分的损失，该结构保留了T-2的基本骨架。而m/z 274.10335是[M+H]+形式的GSH的-SH键断裂，形成H2S的中性丢失所致。
T2-GSH在C-8、C-15处的侧链断裂之后可得到m/z 612.22283(C27H38O11N3S+)的特征离子，是质谱图的基峰。该离子损失甘氨酸可得到m/z537.19079(C25H33O9N2S+)；损失 脱水谷氨酸可得到m/z 483.18022(C22H31O8N2S+)；损失谷氨酰胺得到m/z 466.15366(C22H28O8NS+)的碎片离子。
同HT2-GSH一样，检测到与GSH相关的130.05044(C5H8O3N+)、m/z 145.06077(C5H9O3N2 +)、m/z 162.02251(C5H9O3NS+)、m/z 179.04907(C5H11O 3N2S+)离子。
3.实验结论
本发明的活性多肽在体外可以高效将单端孢霉烯族毒素(包括DON、3DON、15ADON、FUS-X、NIV、T2、H-T2、DAS等)催化成谷胱甘肽加合物，并且由二级谱图可以知该加 合物的形成破坏了在单端孢霉烯的毒性中起主要作用的环氧环结构，可以大大降低毒素 的毒性。
二、单端孢霉烯族毒素GSH衍生物的物质做人体CKK8细胞毒性试验
使用DMEM基础培养基，添加10％的胎牛血清和500μl的青霉素链霉素双抗，在 37℃、5％CO2的恒温培养箱中培养胰腺癌细胞株(PATU8988)、人胚肾细胞293衍生株 (293T)以及人正常食管上皮细胞(HEEC)，待细胞长至瓶壁80％～90％，每2～3d传代一次， 用胰蛋白酶消化收集细胞并传代，根据细胞生长状态，选取对数生长期细胞进行实验。
以细胞浓度5×107L-1种板，CCK-8法酶标仪检测单端孢霉烯族化合物及其对应的经酶 促反应产生的谷胱甘肽加合物作用48h后胰腺癌细胞株、人胚肾细胞293衍生株以及人正 常食管上皮细胞的OD450值。每组均设3复孔观察，各组处理方式如下：空白组(即仅含培养基的调零孔)，对照组(含10％胎牛血清的DMEM培养基)，单端孢霉烯族化合物及其 对应的酶促反应后产生的谷胱甘肽加合物根据文献结果设置相应浓度进行处理。
如图9所示，采用相应浓度的单端孢霉烯族化合物(DON、3-ADON、15-ADON、FUS-X、NIV、T-2、HT-2、DAS)处理48h后，胰腺癌细胞株、人胚肾细胞293衍生株以及人正常 食管上皮细胞活力均急剧下降，说明不同的单端孢霉烯族化合物对细胞均具有较大毒性； 而单端孢霉烯族化合物DON、3-ADON、15-ADON、FUS-X、NIV、T-2、HT-2、DAS 经反应后产生的相应衍生物，在对应相同的浓度下细胞活力与空白对照基本一致，说明 以上8种单端孢霉烯族化合物相应的谷胱甘肽加合物对细胞基本没有毒性作用。
三、饮食品解毒效果试验
1.材料与方法
玉米深加工产品喷浆玉米皮、喷浆胚芽粕、蛋白粉；可口可乐苹果汁、汇源苹果汁(适 量添加标品DON调节果汁中DON含量)；FTCD体外纯化蛋白。呕吐毒素、L-还原型谷 胱甘肽(美国Sigma-Aldrich)。
2.实验方法
2.1FTCD体外纯化蛋白对玉米深加工产品中DON的清除
分别称取10g玉米深加工产品喷浆玉米皮、喷浆胚芽粕、蛋白粉，研磨成粉状，各称取2g样品分装于15ml离心管中，加4ml PBS，制成粉状均液，之后加入100μg FTCD纯 化蛋白以及适量谷胱甘肽，每种产品重复三次。以添加等体积PBS的样品作为空白对照。 于25℃条件下处理12h，分别在0h、1h、3h、12h取样，用于进一步LC-HRMS(/MS)分 析。
2.2FTCD体外纯化蛋白对果汁中DON的清除
两种品牌果汁各吸取1ml分装于2ml离心管中，加入25μg FTCD纯化蛋白以及适量谷胱甘肽，每种产品重复三次。以添加等体积PBS的样品作为空白对照。于25℃条件下 处理12h，分别在0h、1h、3h、12h取样，用于进一步LC-HRMS(/MS)分析。
3.LC-HRMS(/MS)分析
将几种产品体外反应液通过离心及0.22μm滤膜过滤后，转移到进样小瓶中准备进行 LC-HRMS检测。
3.1FTCD体外纯化蛋白对玉米深加工产品中DON的清除效果
根据LC-HRMS的PRM结果对处理前及处理后玉米深加工产品样品中DON毒素的 相对丰度进行量化。结果如图10所示，FTCD体外纯化蛋白对喷浆玉米皮、喷浆胚芽粕、 蛋白粉这三种玉米深加工产品进行处理，分别在处理0h、1h、3h、12h时取样进行 LC-HRMS分析，发现这三种产品中DON的含量随处理时间延长逐渐降低，在处理12h 后产品中DON的含量可减少70％左右。
3.1FTCD体外纯化蛋白对果汁中DON的清除
根据LC-HRMS的PRM结果对处理前及处理两种品牌苹果汁样品中DON毒素的相 对丰度进行量化。结果如图11所示，FTCD体外纯化蛋白对可口可乐苹果汁、汇源苹果 汁(适量添加标品DON调节果汁DON含量)进行处理，分别在处理0h、1h、3h、12h时 取样进行LC-HRMS分析，发现这三种产品中DON的含量随处理时间延长逐渐降低，在 处理12h后产品中DON的含量可减少50％左右。
利用FTCD体外纯化蛋白对玉米深加工产品喷浆玉米皮、喷浆胚芽粕、蛋白粉以及可口可乐苹果汁、汇源苹果汁(适量添加标品DON调节果汁中DON含量)进行处理，通 过LC-HRMS检测，并对结果进行分析，表明该蛋白在在多种产品中均具有较好的呕吐 毒素的脱毒能力，进一步证明其具有重要的实践应用价值。
四、同源序列基因功能分析
在获得来源于长穗偃麦草的去环氧基酶基因的序列(SEQ ID No.:36)的基础上，通过 NCBI进行blastn比对，在默认参数的情况下，未搜到任何注释的高度同源基因。但根据
Figure BDA0002401430690000101
菌属存在同源基因的信息，发明人联合搜索了其他实验室的基因组数据库，获得 了11条来源于该菌属的序列。如图12所示，这些序列与长穗偃麦草十倍体的去环氧基酶基因具有90％以上的序列同一性。另外，发明人还从长穗偃麦草二倍体中分离得到与十 倍体的去环氧基酶基因的序列同一性为98％的基因，其编码的氨基酸序列如SEQ ID No.:2所示。
将这些基因分别转入酵母细胞表达为对应的蛋白，其氨基酸序列分别如SEQ IDNo.:25-35所示。并利用LC-HRMS进行分析。如图13所示，其他12个同源序列转入毕赤酵 母中，经DON处理。LC-HRMS检测显示均有DON-GSH的生成。RT＝1.68min处存在独立 且特异的峰，该峰为C-13处的GSH加合物(通过去环氧基)。在LC-HRMS(方法1)DON 处理转基因酵母提取的离子色谱图，正离子模式提取到DON-GSH加合物，m/z 604.21730(对应于[M+H]+，Δ±5ppm)。
在此述分析的基础上，发明人进一步分析这些同源基因产生的蛋白之间的保守性， 得到具有第92-110位之间的氨基酸序列和第144-184位之间的氨基酸序列的多肽片段。
四、FTCD突变研究
利用诱导基因突变技术，对来源于长穗偃麦草十倍体的去环氧酶基因(SEQ IDNo.:36 所示的序列)进行随机突变，得到22个氨基酸序列发生改变的突变体。这些突变体的氨 基酸序列分别如SEQ ID No.:3-24所示。
经功能分析，在22个突变体中均不同程度地保留了原去环氧基活性。其中存在两个 终止突变，分别终止于209位和243位氨基酸，但这两种终止不会导致该酶的功能完全丢失。因此，表明该酶的功能结构域主要是N端。
尽管本发明已经参考示例性实施方案进行了描述，但应理解本发明不限于公开的示 例性实施方案。在不背离本发明的范围或精神的情况下，可对本发明说明书的示例性实施方案做多种调整或变化。权利要求的范围应基于最宽的解释以涵盖所有修改和等同结构与功能。
序列表
<110> 山东农之安生物科技有限公司
<120> 含有去环氧基蛋白酶的饮食品或饲料组合物及其加工方法
<130> BH2000027-1
<141> 2020-03-05
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225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 7
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 7
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Phe Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 8
<211> 208
<212> PRT
<213> Elytrigia ponticum
<400> 8
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
<210> 10
<211> 242
<212> PRT
<213> Elytrigia ponticum
<400> 10
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly
<210> 10
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 10
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Lys Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 11
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 11
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Ser Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 12
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 12
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Lys Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 13
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 13
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Phe Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 14
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 14
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Leu Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 15
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 15
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile His Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 16
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 16
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Thr Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 17
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 17
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Phe Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 18
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 18
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Ile Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 19
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 19
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Val Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 20
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 20
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Val Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 21
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 21
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Val Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 22
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 22
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Val Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 23
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 23
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Met Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 24
<211> 281
<212> PRT
<213> Elytrigia ponticum
<400> 24
Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg His Ala Gly Leu Ser Ser Trp Asp
180 185 190
Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 25
<211> 280
<212> PRT
<213> Epichloe bromicola
<400> 25
Met Ala Thr Ser Thr Ser Ile Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Thr Arg Ala Ser Pro Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Val Arg Ala Cys His Gly Ala Val Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 26
<211> 279
<212> PRT
<213> Epichloe amarillans
<400> 26
Met Ala Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile Ala Gln
1 5 10 15
Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp Lys Ser
20 25 30
Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr Trp Val
35 40 45
Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Leu Pro Ala
50 55 60
Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe Asp Thr Leu Pro Ile Ile
65 70 75 80
His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser Phe Asp Ile Ala
85 90 95
Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp Leu Phe
100 105 110
Pro Pro Gln Lys Leu Asp Tyr Ala Ala Gly Arg Asp Thr Gln Leu Leu
115 120 125
Ile Pro Leu Ser Glu Val Arg Ala Ala Ser Pro Glu Leu Ala Asp Tyr
130 135 140
Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val Gly
145 150 155 160
Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val Thr
165 170 175
Lys Ala Glu Phe Val Arg Arg Ala Ala Val Ser Ser Trp Asp Asp Leu
180 185 190
Asp Met Val Gly Asp Ala Arg Asp Lys Met Met Gln Ser Leu Arg Asn
195 200 205
Thr Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly Pro
210 215 220
Phe Leu Leu Gly Pro Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly Gly
225 230 235 240
Trp Leu Arg Met Met Arg Ala Thr Leu Pro Pro Ser Glu Trp Gln Ala
245 250 255
Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp Ala Leu
260 265 270
Asp Lys Tyr Ala Glu Val Lys
275
<210> 27
<211> 264
<212> PRT
<213> Epichloe baconii
<400> 27
Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile
1 5 10 15
Phe Tyr Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala
20 25 30
Val Asn Pro Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro
35 40 45
Tyr Thr Thr Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala
50 55 60
Ser Leu Asn Leu Pro Ala Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe
65 70 75 80
Asn Thr Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Val Gly
85 90 95
Asp Ser Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Asp Thr Gln Leu
100 105 110
Leu Ile Pro Leu Ser Glu Val Arg Ala Ala Ser Ser Asp Leu Ala Asp
115 120 125
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
130 135 140
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
145 150 155 160
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Asp Asp
165 170 175
Phe Glu Met Ala Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
180 185 190
Asn Thr Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
195 200 205
Pro Phe Leu Leu Gly Arg Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly
210 215 220
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
225 230 235 240
Ala Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp Ala
245 250 255
Leu Asp Lys Tyr Ala Glu Val Lys
260
<210> 28
<211> 286
<212> PRT
<213> Epichloe festucae
<400> 28
Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr
1 5 10 15
Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn
20 25 30
Pro Trp Lys Thr Arg Leu Ala Leu Asn Phe Lys Ala Val Thr Tyr Thr
35 40 45
Thr Thr Trp Val Lys Met Pro Asp Ile Ser Gly Val Arg Ala Ser Leu
50 55 60
Asn Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe Asn Thr
65 70 75 80
Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser
85 90 95
Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala
100 105 110
Gly His Leu Phe Pro Pro Leu Pro Pro Pro Gln Lys Leu Asp Tyr Ala
115 120 125
Val Gly Arg Asp Met Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala
130 135 140
Ser Ser Glu Leu Ala Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala
145 150 155 160
Ala Phe Thr Ala His Val Gly Val Met Val His Gly Leu Pro Leu Asp
165 170 175
Pro Ala Thr Ala Asp Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly
180 185 190
Val Ser Ser Trp Glu Asp Phe Glu Met Val Gly Glu Ala Arg Glu Lys
195 200 205
Met Met Gln Ser Leu Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe
210 215 220
Arg Arg Asp Ala Ser Gly Pro Phe Leu Leu Gly Gln Gln Ala Thr Tyr
225 230 235 240
Ala Asp Leu Ile Val Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu
245 250 255
Pro Ala Ser Glu Trp Gln Glu Val Arg Ala Trp His Gly Ala Val Phe
260 265 270
Gly Arg Leu His Asp Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280 285
<210> 29
<211> 281
<212> PRT
<213> Epichloe gansuensis
<400> 29
Met Ala Thr Ser Thr Ser Thr Ser Ala Ser Thr Pro Ile Ile Phe Tyr
1 5 10 15
Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn
20 25 30
Pro Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr
35 40 45
Thr Thr Trp Val Glu Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu
50 55 60
Asn Leu Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr
65 70 75 80
Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser
85 90 95
Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala
100 105 110
Asp Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Val Val Gly Ser His
115 120 125
Val Gln Pro Phe Ile Pro Leu Ser Asp Ile Arg Ala Ser Glu Phe Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Leu His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu
180 185 190
Asp Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Phe
195 200 205
Arg Thr Met Leu Glu Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Thr
210 215 220
Gly Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 30
<211> 281
<212> PRT
<213> Epichloe typhina
<400> 30
Met Ala Thr Ser Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp
1 5 10 15
Ile Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro
20 25 30
Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr
35 40 45
Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn
50 55 60
Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu
65 70 75 80
Pro Ile Met His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe
85 90 95
Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly
100 105 110
Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu
180 185 190
Asp Phe Glu Met Val Gly Glu Val Arg Glu Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 31
<211> 281
<212> PRT
<213> Epichloe uncinata
<400> 31
Met Ala Thr Ser Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp
1 5 10 15
Ile Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro
20 25 30
Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr
35 40 45
Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn
50 55 60
Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu
65 70 75 80
Pro Ile Met His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe
85 90 95
Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly
100 105 110
Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met
115 120 125
Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala
130 135 140
Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His
145 150 155 160
Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp
165 170 175
Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu
180 185 190
Asp Phe Glu Met Val Gly Glu Val Arg Glu Lys Met Met Gln Ser Leu
195 200 205
Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser
210 215 220
Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val
225 230 235 240
Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp
245 250 255
Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp
260 265 270
Ala Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 32
<211> 280
<212> PRT
<213> Epichloe sylvatica
<400> 32
Met Thr Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Lys Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Lys Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Met His Asp Pro Ala Thr Asp Ser Leu Leu Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Leu Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 33
<211> 280
<212> PRT
<213> Epichloe aotearoae
<400> 33
Met Ala Thr Pro Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile
1 5 10 15
Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp
20 25 30
Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr
35 40 45
Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val
50 55 60
Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro
65 70 75 80
Ile Met His Asp Pro Ala Thr Ser Ser Leu Ile Gly Asp Ser Phe Asp
85 90 95
Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp
100 105 110
Leu Phe Pro Ser Gln Lys Leu Asp Tyr Ala Val Ala Arg Asp Thr Gln
115 120 125
Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Ser Glu Leu Ala Asp
130 135 140
Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val
165 170 175
Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp
180 185 190
Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg
195 200 205
Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly
210 215 220
Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val Gly
225 230 235 240
Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln
245 250 255
Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp Ala
260 265 270
Leu Asp Lys Tyr Ala Glu Val Lys
275 280
<210> 34
<211> 275
<212> PRT
<213> Epichloe glyceriae
<400> 34
Met Ala Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile Ala Gln Arg Pro
1 5 10 15
Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp Lys Ser Arg Leu
20 25 30
Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr Trp Val Ser Met
35 40 45
Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val Pro Ala Cys Arg
50 55 60
Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro Ile Ile His Asp
65 70 75 80
Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser Phe Asp Ile Ala Ala His
85 90 95
Leu Gln Arg Ala Tyr Pro Ala Ser Gly Ala Gly Asp Leu Phe Pro Pro
100 105 110
Gln Glu Leu Asp Tyr Val Val Ala Arg Asp Thr Arg Leu Leu Val Pro
115 120 125
Leu Ser Glu Thr Arg Ala Ser Glu Phe Ala Asp Tyr Ala Arg Phe Asn
130 135 140
Ser Asn Val Asp Ala Ala Phe Thr Ala His Val Gly Leu Met Val His
145 150 155 160
Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val Thr Lys Ala Glu Phe
165 170 175
Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp Phe Glu Leu Val Gly
180 185 190
Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg Asn Val Leu Gly Asp
195 200 205
Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly Pro Phe Leu Leu Gly
210 215 220
Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly Gly Trp Leu Arg Met
225 230 235 240
Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln Glu Ala Arg Ala Trp
245 250 255
His Gly Ala Val Phe Gly Gln Leu His Asp Ala Leu Asp Lys Tyr Ala
260 265 270
Glu Val Lys
275
<210> 35
<211> 280
<212> PRT
<213> Epichloe brachyelytri
<400> 35
Met Ala Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Pro Thr Ala Ile
1 5 10 15
Ala Gly Ala Pro Pro Val Thr Gly Thr Cys Cys Ala Val Ala Pro Thr
20 25 30
Leu Ser Ala Leu Ala Leu Ala Pro Leu Ala Val Pro Thr Thr Thr Thr
35 40 45
Thr Val Leu Met Pro Ala Ile Ser Ser Val Ala Ala Ser Leu Ala Val
50 55 60
Pro Ala Cys Ala Leu Pro Ala Ala Gly Ser Ala Pro Ala Thr Leu Pro
65 70 75 80
Ile Ile His Ala Pro Ala Thr Ala Ser Leu Ile Gly Ala Ser Pro Ala
85 90 95
Ile Ala Ala Thr Leu Gly Ala Thr Thr Pro Ala Ser Gly Ala Gly Ala
100 105 110
Leu Pro Pro Pro Gly Leu Leu Ala Thr Ala Val Ser Ala Ala Met Gly
115 120 125
Leu Leu Ile Pro Leu Ser Gly Met Ala Ala Ser Ser Gly Leu Ala Ala
130 135 140
Thr Ala Ala Pro Ala Ser Ala Val Ala Ala Ala Pro Thr Ala His Val
145 150 155 160
Gly Leu Met Val His Gly Leu Pro Leu Ala Pro Ala Thr Ala Ala Val
165 170 175
Thr Leu Ala Gly Pro Val Ala Ala Ala Gly Val Ser Ser Thr Gly Ala
180 185 190
Pro Gly Met Val Gly Gly Ala Ala Gly Leu Met Met Gly Ser Leu Ala
195 200 205
Ala Met Leu Gly Ala Leu Ala Ala Leu Pro Ala Ala Ala Ala Ser Gly
210 215 220
Pro Pro Leu Leu Gly Gly Leu Ala Thr Thr Ala Ala Leu Ile Val Gly
225 230 235 240
Gly Thr Leu Ala Met Met Ala Ala Thr Leu Pro Ala Ser Gly Thr Gly
245 250 255
Gly Val Ala Ala Thr His Gly Ala Ile Pro Gly Gly Leu His Ala Ala
260 265 270
Leu Ala Leu Thr Ala Gly Val Leu
275 280
<210> 36
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 36
atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60
cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120
aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180
agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240
atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300
ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360
tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420
ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480
gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540
gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600
cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660
aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720
ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780
gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840
aagtag 846

Claims (10)

1.一种饮食品或饲料组合物，其特征在于，其包含去环氧基蛋白酶，所述蛋白酶在15-35℃温度下能够使单端孢霉烯族毒素与谷胱甘肽在PBS缓冲液中催化反应产生谷胱甘肽化衍生物。
2.根据要求1所述的饮食品或饲料组合物，其特征在于，所述蛋白酶具有选自由下述(1)-(5)组成的组中的一种氨基酸序列：
(1)SEQ ID No.:1-35所示的氨基酸序列；
(2)与(1)中的氨基酸序列的序列同一性为90％以上，且来源于相同属的氨基酸序列；
(3)与(1)或(2)的氨基酸序列相比具有一个或多个氨基酸突变且序列同一性为90％以上的氨基酸序列；
(4)源自(1)-(3)任一项所述的氨基酸序列的N端部分连续序列，且仍具有原蛋白酶活性；
(5)在(1)-(4)任一项所述的氨基酸序列的N端和/或C端连接其他氨基酸序列的嵌合序列。
3.根据权利要求2所述的饮食品或饲料组合物，其特征在于，所述蛋白酶具有第98位氨基酸A和第99位氨基酸A的保守位点。
4.根据权利要求1-3任一项所述的饮食品或饲料组合物，其特征在于，所述饮食品组合物进一步包含谷胱甘肽。
5.根据权利要求1-3任一项所述的饮食品或饲料组合物，其特征在于，所述饮食品或饲料组合物包含选自由小麦粉、大麦粉、黑麦粉、燕麦粉、玉米粉、谷子粉、大米粉、高粱粉、大豆粉、马铃薯粉、甘薯粉和花生粉组成的组中的至少一种粮食粉；或者包含粮食加工产品，例如豆皮、豆浆、胚芽粕、胚芽、植物油、淀粉、葡萄糖、蛋白粉、酒精和发酵物。
6.根据权利要求1-3任一项所述的饮食品或饲料组合物，其特征在于，其包含选自牛奶、乳制品、苹果、柑橘和葡萄组成的组中的至少一种果汁或饮品成分。
7.一种饮食品或饲料组合物的加工方法，其特征在于，包括在适于反应的条件下使饮食品或饲料原料与去环氧基蛋白酶或产生该酶的细胞接触的步骤。
8.根据权利要求7所述的饮食品或饲料组合物的加工方法，其特征在于，进一步包括向饮食品或饲料原料添加谷胱甘肽的步骤。
9.根据权利要求7或8所述的饮食品或饲料组合物的加工方法，其特征在于，其包括向原料中加入水混合均匀得到反应液，然后向反应液中加入去环氧基蛋白酶或产生该酶的细胞和可选的谷胱甘肽，在2-40℃下反应5分钟至36小时。
10.一种用于降低或减少组合物中毒素的方法，其特征在于，包括在适于反应的条件下使含有毒素的饮食品或饲料原料与去环氧基蛋白酶或产生该酶的细胞接触的步骤，其中所述毒素为单端孢霉烯族化合物。
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