CN112839512A - 用于供体器官上的碳水化合物抗原切割的酶促组合物、与其相关的方法和用途 - Google Patents

用于供体器官上的碳水化合物抗原切割的酶促组合物、与其相关的方法和用途 Download PDF

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CN112839512A
CN112839512A CN201980067904.7A CN201980067904A CN112839512A CN 112839512 A CN112839512 A CN 112839512A CN 201980067904 A CN201980067904 A CN 201980067904A CN 112839512 A CN112839512 A CN 112839512A
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马塞洛·塞普拉
王艾舟
沙菲克·克沙夫吉
斯蒂芬·G·威瑟斯
彼得·拉费尔德
加雅善德兰·基萨科达特胡
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Abstract

本文提供了用于酶促切割来自供体器官的A抗原的灌注流体，以及与其相关的方法、用途。具体地，所述灌注流体包含两种酶GalNAc脱乙酰酶和半乳糖胺酶，并且所述流体还可以包含缓冲的细胞外溶液和/或拥挤剂。此外，发现本文所述的组合物在适于细胞存活的温度和pH水平下具有活性。

Description

用于供体器官上的碳水化合物抗原切割的酶促组合物、与其 相关的方法和用途
相关申请的交叉引用
本申请要求2018年8月17日提交的题为“用于碳水化合物抗原切割的酶促组合物，与其相关的方法、用途、设备和系统(ENZYMATIC COMPOSITIONS FOR CARBOHYDRATEANTIGEN CLEAVAGE,METHODS,USES,APPARATUSES AND SYSTEMS ASSOCIATED THEREWITH)”的美国临时专利申请系列第62/719,272号的权益。
技术领域
本发明涉及酶组合物领域。具体地，本发明涉及用于切割供体器官上的抗原的酶组合物，以及提供使用该组合物切割抗原的方法和用途。
背景
血型的正确匹配是输血医学的主要要求，因为A血型个体的血浆含有针对B抗原的抗体，反之亦然，因此不相容的输血可以导致补体激活和红血细胞(RBC)裂解(Daniels2010)。这些细胞表面抗原是分别终止于A型血和B型血的α-1,3-连接的-N-乙酰基半乳糖胺(GalNAc)或半乳糖(Gal)的碳水化合物结构。另一方面，O型RBC不含有这些末端糖，并且可以被普遍输血(Garratty 2008)。因此，在患者血型未知或不清楚的紧急情况下，在血库中需要O型RBC的良好供应。然而，供应通常是有限的。
Goldstein首次提出并证明了从A RBC或B RBC酶促去除GalNAc或Gal结构作为将ARBC或B RBC转变成O RBC的手段的概念(Goldstein 1982；US4609627和CA2272925)。使用来自绿咖啡豆的α-半乳糖苷酶，将B型RBC转变为O型RBC，随后进行成功的输血(Kruskall2000)。然而，所需的酶量使得该方法不切实际。A型的转变更具挑战性，主要是因为A型血液存在其内部连接不同的许多亚型(Clausen 1989)。类似地，已经使用α-半乳糖苷酶去除B型抗原(例如，参见EP2243793)。通过使用四糖底物筛选具有A和B转变活性的细菌文库，向包括A型在内的实际转变迈出了重要一步。发现两个新的糖苷酶家族在中性pH值下显示出高的抗原切割活性：CAZy GH109α-N-乙酰基半乳糖胺酶(α-N-acetylgalactosaminidases)和GH110α-半乳糖苷酶(Liu 2007)。两种酶都转变了它们相应的RBC，其中完全去除了各自的抗原。然而，转变仍然需要大量的酶，特别是A型(60mg酶/血液单位)，这限制了进一步的发展。在切割来自细胞的碳水化合物抗原方面具有更高效率的酶将是有用的。
概述
本发明部分地基于出人意料的发现，即如本文所述的半乳糖胺酶(Galactosaminidase)和GalNAc脱乙酰酶的组合比先前鉴定的A抗原切割酶更有效几个数量级。例如，在一些条件下，一些GalNAc脱乙酰酶和半乳糖胺酶能够在1μ/ml或低于1μ/ml下切割A抗原。此外，将酶组合的切割效率维持在适于维持红细胞存活力的pH(即pH为约6.5至约7.5)下。另外，发现酶在4℃至37℃的温度下是有活性的，这也适用于血液收集、洗涤和存储方案。此外，通过添加拥挤剂(例如，右旋糖酐)进一步提高了酶的效率。还已经意识到，相同的两步切割过程可以被应用于供体器官。
然而，本领域技术人员将理解，可以使用更多的酶来减少其中可以灌注供体器官的时间，或者可以使用更少的酶，只要供体器官的灌注时间更长。
根据一个实施方案，用于酶促切割来自供体器官的A抗原的灌注流体，其包括：(a)纯化的GalNAc脱乙酰酶蛋白；和(b)纯化的半乳糖胺酶蛋白。
根据另一个实施方案，提供了灌注流体，其中所述灌注流体包括：(a)所述GalNAc脱乙酰酶是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:2；SEQ ID NO:4；SEQ ID NO:5；SEQ ID NO:17；SEQ ID NO:23；SEQ ID NO:29；SEQ ID NO:31；SEQ ID NO:32；SEQ ID NO:33；SEQ ID NO:34和SEQ ID NO:35；以及(b)所述半乳糖胺酶是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:7；SEQ ID NO:9；SEQ ID NO:10；SEQ ID NO:19；SEQ ID NO:21；SEQID NO:36和SEQ ID NO:37。
根据另一个实施方案，提供了灌注流体，其中所述灌注流体包括：具有GalNAc脱乙酰酶活性的纯化酶，其基本上由与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少90％相同的氨基酸序列组成；和具有半乳糖胺酶活性的纯化酶，其基本上由与SEQ IDNO:7、9、10、19、21、36和37中之一所示序列至少90％相同的氨基酸序列组成。
所述酶可以选自以下中的一种或多种：(a)所述纯化的GalNAc脱乙酰酶蛋白是SEQID NO:2、SEQ ID NO:4和SEQ ID NO:5的纯化的普氏梭杆菌(Flavonifractor plautii)的GalNAc脱乙酰酶蛋白；和(b)所述纯化的半乳糖胺酶蛋白是SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白。所述酶可以选自以下中的一种或多种：(a)所述纯化的GalNAc脱乙酰酶蛋白是SEQ ID NO:17或SEQ ID NO:32的纯化的第三梭状芽胞杆菌(Clostridium tertium)的GalNAc脱乙酰酶蛋白；和(b)所述纯化的半乳糖胺酶蛋白是SEQ ID NO:19或SEQ ID NO:36的纯化的第三梭状芽胞杆菌的半乳糖胺酶蛋白。所述GalNAc脱乙酰酶和半乳糖胺酶可能能够在1μg/ml或低于1μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.5至约7.5的pH下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在4℃至37℃的温度下具有A抗原切割活性。所述灌注流体还可以包含缓冲的细胞外溶液。所述缓冲的细胞外溶液可以选自：SteenTM；PerfadexTM；Perfadex PlusTM；EuroCollins溶液；组氨酸-色氨酸-酮戊二酸(HTK)溶液；威斯康星大学溶液(UW)；Celsior溶液；肾脏灌注液(KPS-1)；京都大学溶液；IGL-1溶液；和柠檬酸盐溶液。
根据另一个实施方案，提供了用于离体酶促切割来自供体器官的A抗原的方法，所述方法包括：(a)用包含GalNAc脱乙酰酶蛋白和半乳糖胺酶蛋白的流体灌注展示A型抗原的供体器官一段时间，所述时间足以允许所述酶切割来自所述供体器官的A抗原；或者(b)使展示A型抗原的供体器官与包含GalNAc脱乙酰酶蛋白和半乳糖胺酶蛋白的流体一起孵育一段时间，所述时间足以允许所述酶切割来自所述供体器官的A抗原。
所述GalNAc脱乙酰酶可以是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:2；SEQ ID NO:4；SEQ ID NO:5；SEQ ID NO:17；SEQ ID NO:23；SEQ ID NO:29；SEQ ID NO:31；SEQ ID NO:32；SEQ ID NO:33；SEQ ID NO:34和SEQ ID NO:35；以及所述半乳糖胺酶可以是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:7；SEQ ID NO:9；SEQ ID NO:10；SEQ IDNO:19；SEQ ID NO:21；SEQ ID NO:36和SEQ ID NO:37。
具有GalNAc脱乙酰酶活性的纯化酶可以基本上包括与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少90％相同的氨基酸序列；和具有半乳糖胺酶活性的纯化酶可以基本上包括与SEQ ID NO:7、9、10、19、21、36和37中之一所示序列至少90％相同的氨基酸序列。
所述GalNAc脱乙酰酶可以是SEQ ID NO:4或SEQ ID NO:5的纯化的普氏梭杆菌的GalNAc脱乙酰酶蛋白，和所述半乳糖胺酶可以是SEQ ID NO:9或SEQ ID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白。
所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白可以处于缓冲的细胞外溶液中。所述缓冲的细胞外溶液可以选自：SteenTM；PerfadexTM；Perfadex PlusTM；EuroCollins溶液；组氨酸-色氨酸-酮戊二酸(HTK)溶液；威斯康星大学溶液(UW)；Celsior溶液；肾脏灌注液(KPS-1)；京都大学溶液；IGL-1溶液；和柠檬酸盐溶液。所述供体器官可以是实体器官。所述实体器官可以选自以下之一：肺；肾脏；肝脏；心脏；胰腺和肠。所述实体器官可以是肺。
可以使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外肺溶液混合并循环通过肺，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述肺的脉管系统中基本上清除所述A抗原。可以使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外肾脏溶液混合并循环通过肾脏，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述肾脏的脉管系统中基本上清除所述A抗原。可以使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外肝脏溶液混合并循环通过肝脏，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述肝脏的脉管系统中基本上清除所述A抗原。可以使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外心脏溶液混合并循环通过心脏，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述心脏的脉管系统中基本上清除所述A抗原。可以使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外胰腺溶液混合并循环通过胰腺，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述胰腺的脉管系统中基本上清除所述A抗原。可以使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外肠溶液混合并循环通过肠，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述肠的脉管系统中基本上清除所述A抗原。
从所述脉管系统清除所述A抗原的时间可以为约1小时。从所述脉管系统清除所述A抗原的时间可以少于1小时。从所述脉管系统清除所述A抗原的时间可以为约2小时。
所述方法还可以包括洗涤所述供体器官以去除GalNAc脱乙酰酶、半乳糖胺酶和切割的A抗原。所述GalNAc脱乙酰酶和所述半乳糖苷酶可能能够在1μg/ml或低于1μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖苷酶可以在约6.5至约7.5的pH下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在4℃至37℃的温度下具有A抗原切割活性。
根据另一个实施方案，提供了组合物，所述组合物包括：具有GalNAc脱乙酰酶活性的纯化酶，其基本上由与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少85％相同的氨基酸序列组成；和具有半乳糖胺酶活性的纯化酶，其基本上由与SEQ ID NO:7、9、10、19、21、36和37中之一所示序列至少85％相同的氨基酸序列组成。
根据另一个实施方案，提供了组合物，所述组合物包括：具有GalNAc脱乙酰酶活性的纯化酶，其基本上由与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少80％相同的氨基酸序列组成；和具有半乳糖胺酶活性的纯化酶，其基本上由与SEQ ID NO:7、9、10、19、21、36和37中之一所示序列至少80％相同的氨基酸序列组成。
根据另一个实施方案，提供了组合物，所述组合物包括：具有GalNAc脱乙酰酶活性的纯化酶，其基本上由与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少75％相同的氨基酸序列组成；和具有半乳糖胺酶活性的纯化酶，其基本上由与SEQ ID NO:7、9、10、19、21、36和37中之一所示序列至少75％相同的氨基酸序列组成。
所述组合物可以包括：(a)所述纯化的GalNAc脱乙酰酶和所述纯化的半乳糖胺酶可以是固定化的；(b)所述纯化的GalNAc脱乙酰酶可以是固定化的；或者(c)所述纯化的半乳糖胺酶可以是固定化的。
固定化的酶可以连接到表面上，所述表面可以选自以下中的一种或多种：(a)珠或微球体；(b)容器；(c)管；(d)柱；和(e)基质。所述组合物还可以包括拥挤剂。所述拥挤剂可以选自以下中的一种或多种：右旋糖酐、硫酸右旋糖酐、糊精、普鲁兰多糖、聚(乙二醇)、聚蔗糖TM和惰性蛋白。
根据另一个实施方案，提供了纯化的酶，其包括SEQ ID NO:2、SEQ ID NO:4或SEQID NO:5的普氏梭杆菌的GalNAc脱乙酰酶。
根据另一个实施方案，提供了纯化的酶，其包括SEQ ID NO:7、SEQ ID NO:9或SEQID NO:10的普氏梭杆菌的半乳糖胺酶。
根据另一个实施方案，提供了纯化的酶，其包括SEQ ID NO:17或SEQ ID NO:32的第三梭状芽胞杆菌的GalNAc脱乙酰酶。
根据另一个实施方案，提供了纯化的酶，其包括SEQ ID NO:19或SEQ ID NO:36的第三梭状芽胞杆菌的半乳糖胺酶。
蛋白质标签可以选自以下中的一种或多种：白蛋白结合蛋白(ABP)；碱性磷酸酶(AP)；AU1表位；AU5表位；Avi标签；噬菌体T7表位(T7标签)；噬菌体V5表位(V5标签)；生物素-羧基载体蛋白(BCCP)；蓝舌病毒标签(B标签)；单结构域骆驼科动物抗体(C标签)；钙调蛋白结合肽(CBP或钙调蛋白标签)；氯霉素乙酰转移酶(CAT)；纤维素结合结构域(CBP)；几丁质结合结构域(CBD)；胆碱结合结构域(CBD)；二氢叶酸还原酶(DHFR)；DogTag；E2表位；E标签；FLAG表位(FLAG标签)；半乳糖结合蛋白(GBP)；绿色荧光蛋白(GFP)；Glu-Glu(EE标签)；谷胱甘肽S转移酶(GST)；人流感血凝素(HA)；；HaloTagTM；交替的组氨酸和谷氨酰胺标签(HQ标签)；交替的组氨酸和天冬酰胺标签(HN标签)；组氨酸亲和标签(HAT)；辣根过氧化物酶(HRP)；HSV表位；Isopeptag(Isopep标签)；类固醇异构酶(KSI)；KT3表位；LacZ；荧光素酶；麦芽糖结合蛋白(MBP)；Myc表位(Myc标签)；NE标签；NusA；PDZ结构域；PDZ配体；聚精氨酸(Arg标签)；聚天冬氨酸(Asp标签)；聚半胱氨酸(Cys标签)；聚谷氨酸(Glu标签)；聚组氨酸(His标签)；聚苯丙氨酸(Phe标签)；Profinity eXact；蛋白C；Rho1D4标签；S1标签；S标签；Softag 1；Softag 3；SnoopTagJr；SnoopTag；Spot标签；SpyTag(Spy标签)；Streptavadin结合肽(SBP)；葡萄球菌蛋白A(蛋白A)；葡萄球菌蛋白G(蛋白G)；Strep标签；Streptavadin(SBP标签)；Strep标签II；Sdy标签；小泛素样修饰剂(SUMO)；串联亲和纯化(TAP)；T7表位；四半胱氨酸标签(TC标签)；硫氧还蛋白(Trx)；TrpE；Ty标签；泛素；通用的；V5标签；VSV-G或VSV标签以及Xpress标签。
根据另一个实施方案，提供了用于酶促切割来自供体器官的A抗原的方法，所述方法包括：(a)将GalNAc脱乙酰酶蛋白和半乳糖胺酶蛋白与展示A型抗原的供体器官组合；(b)将所述酶灌注到供体器官血管中一段时间，所述时间足以使所述酶从供体器官的血管腔中切割A抗原。
所述方法还可以包括添加拥挤剂。所述拥挤剂可以选自以下中的一种或多种：右旋糖酐；硫酸右旋糖酐；糊精；普鲁兰多糖；聚(乙二醇)；聚蔗糖TM；超支化甘油和惰性蛋白。所述方法可以包括用包含本文所述的酶组合物的器官灌注或器官保存溶液灌注所述供体器官。
所述方法还可以包括洗涤所述供体器官以去除GalNAc脱乙酰酶、半乳糖胺酶和/或所述拥挤剂。
所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在1μg/ml或低于1μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.5至约7.5的pH下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在4℃至37℃的温度下具有A抗原切割活性。
所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在100μg/ml或低于100μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在90μg/ml或低于90μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在80μg/ml或低于80μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在70μg/ml或低于70μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在60μg/ml或低于60μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在50μg/ml或低于50μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在40μg/ml或低于40μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在30μg/ml或低于30μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在20μg/ml或低于20μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在15μg/ml或低于15μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在14μg/ml或低于14μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在13μg/ml或低于13μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在12μg/ml或低于12μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在11μg/ml或低于11μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在10μg/ml或低于10μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在9μg/ml或低于9μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在8μg/ml或低于8μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在7μg/ml或低于7μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在6μg/ml或低于6μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在5μg/ml或低于5μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在4μg/ml或低于4μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在3μg/ml或低于3μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在2μg/ml或低于2μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在1μg/ml或低于1μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.9μg/ml或低于0.9μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.8μg/ml或低于0.8μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.7μg/ml或低于0.7μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.6μg/ml或低于0.6μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.5μg/ml或低于0.5μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.4μg/ml或低于0.4μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.3μg/ml或低于0.3μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.2μg/ml或低于0.2μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.1μg/ml或低于0.1μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.09μg/ml或低于0.09μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.08μg/ml或低于0.08μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.07μg/ml或低于0.07μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.06μg/ml或低于0.06μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.05μg/ml或低于0.05μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.04μg/ml或低于0.04μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.03μg/ml或低于0.03μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.02μg/ml或低于0.02μg/ml下切割A抗原。所述GalNAc脱乙酰酶和所述半乳糖胺酶可能能够在0.01μg/ml或低于0.01μg/ml下切割A抗原。
所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.5至约7.5的pH下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.0至约8.0的pH下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.8至约7.8的pH下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.9至约7.9的pH下具有A-抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在约6.4至约7.8的pH下具有A抗原切割活性。
所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在4℃至37℃的温度下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在3℃至38℃的温度下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在4℃至40℃的温度下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在4℃至37℃的温度下具有A抗原切割活性。所述GalNAc脱乙酰酶和所述半乳糖胺酶可以在5℃至37℃的温度下具有A抗原切割活性。
根据另一个实施方案，提供了包括SEQ ID NO:2、SEQ ID NO:4或SEQ ID NO:5的普氏梭杆菌的GalNAc脱乙酰酶的纯化酶。
根据另一个实施方案，提供了包含SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:10的普氏梭杆菌的半乳糖胺酶的纯化酶。
根据另一个实施方案，提供了纯化的酶，其包括SEQ ID NO:14的纯化的第三梭状芽胞杆菌GalNAc脱乙酰酶和半乳糖胺酶融合蛋白。.
根据另一个实施方案，提供了包括本文所述核酸和异源核酸序列的载体。
根据另一个实施方案，所述方法可以在体外或离体进行。如本文所用的离体意指该方法在生物体外进行。例如，离体将包括离体肺灌注(EVLP)和捐献血液的处理。如本文所用的，离体是指在来自外部环境中的生物体的组织或细胞(例如，红细胞或供体器官)中或上进行的实验或测量或处理，其中组织或细胞在体内时处于最小或一些条件改变之下。
附图简述
图1显示了终止于α-1,3-连接的N-乙酰基半乳糖胺(GalNAc)或半乳糖(Gal)的A型、H型和B型细胞表面抗原碳水化合物结构的示意图，其中三角形标记了α-N-乙酰基半乳糖胺酶EmGH109和α-半乳糖苷酶BfGal110的切割点。
图2显示了A抗原切割的脱乙酰酶促途径，由此普氏梭杆菌(Fp)的GalNAc脱乙酰酶切割来自A抗原的末端α-N-乙酰基半乳糖胺(-42m/z)的乙酰基，然后半乳糖胺中间体被普氏梭杆菌(Fp)的半乳糖胺酶(-161m/z)切割，其中用相应的质谱(MS)分析。
图3显示了用不同浓度的EmGH109或普氏梭杆菌的GalNAc脱乙酰酶(FpGalNAc脱乙酰酶)加普氏梭杆菌的半乳糖胺酶(Fp半乳糖胺酶)处理的A+RBC或在37℃下处理1h的A+RBC的FACS分析，其中为了可视化，使用抗H抗体(加第二FITC标记的抗体)和APC标记的抗A抗体，其中H抗原出现的区域在左上角的框中。行A-D比较了在5μg/ml(A)；10μg/ml(B)；50μg/ml(C)和50μg/ml+右旋糖酐40k(D)的EmGH109和FpGalNAcDeAc+FpGalNase。
图4显示了在不同温度(即4℃、室温(RT)和37℃)下，在具有(■)和不具有(◆)右旋糖酐的各种酶浓度下，EmGH109和FpGalNAcDeAc+FpGalNase的比较。
图5显示了A+B+和O+红细胞切割产物的HPAE-PAD分析，以及A+红细胞上全长普氏梭杆菌的GalNAc脱乙酰酶(FpGalNAcDeAc)+普氏梭杆菌的半乳糖胺酶(FpGalNase)酶与截短的FpGalNAcDeAc+FpGalNase酶的比较。
图6显示了(A)FpGalNAc脱乙酰酶和(B)Fp半乳糖胺酶中每一种的pH谱。
图7显示了对于(A)A+RBC对照、(B)普氏梭杆菌的GalNAc脱乙酰酶(FpGalNAcDeAc)+普氏梭杆菌的半乳糖胺酶(FpGalNase)(10μg/mL)、(C)FpGalNAcDeAc+第三梭状芽胞杆菌(Ct)Ct57577_GalNase(10ug/mL)和(D)FpGalNAcDeAc+Robinsoniella peoriensis(Rp)的半乳糖胺酶(Rp1021)GalNase(10ug/mL)，经由FACS分析的A RBC上A抗原向H抗原的转变。
图8显示了酶在不同灌注溶液(即PBS、SteenTM和PerfadexTM)中对从A型人红细胞去除A抗原的剂量递增效应。
图9显示了酶在STEEN溶液中对A型人动脉的剂量递增效应，其中通过对获自未经处理的(对照)、经处理的(处理)的A型动脉和作为阴性对照的O型动脉的活检组织进行免疫组织化学分析，对A型抗原的百分比进行定量。
图10显示了测试1小时酶促处理对离体灌注的人供体肺的影响，其中活检人供体肺的免疫组织化学染色比较了肺的右上依赖性(RUD)区域、右上非依赖性(RUND)、右中非依赖性(RMND)区域、右中依赖性(RMD)区域、右下非依赖性(RLND)区域和右下依赖性(RLD)区域的处理前图像与处理后图像，A血型抗原在血管中不存在。
图11显示了测试3小时酶促处理对离体灌注的人供体肺的影响，其中活检人供体肺的免疫组织化学染色比较了肺的右上依赖性(RUD)区域、右上非依赖性(RUND)、右中非依赖性(RMND)区域、右中依赖性(RMD)区域、右下非依赖性(RLND)区域和右下依赖性(RLD)区域的处理前图像与处理后图像，A血型抗原在血管中不存在。
详述
当结合附图阅读时，将更好地理解下面的详细描述。为了说明本发明，附图示出了本发明的实施方案。然而，本发明不局限于所示的精确布置、实例和手段。
本文未直接限定的任何术语应被理解为具有在本发明领域内理解的通常与其相关的含义。
如本文所用的“固定化酶”是连接到表面的酶，所述表面可以是惰性不溶性材料。酶的固定化可以提供对条件(如pH、温度等)变化的增强的抗性，并且有助于它们在使用后的去除和酶的再使用。
酶的固定可以通过各种方式实现(例如，亲和标签结合，在玻璃、树脂、藻酸盐珠或基质上的表面吸附，珠、纤维或微球体截留，与表面或其它酶的交联和与表面的共价结合)。
如本文所用的“亲和标签结合”是指将酶固定到表面(例如，使用非共价或共价蛋白质标签的多孔材料)。亲和标签结合已经被用于蛋白质纯化并且最近已经被用于EziGTM(ENGINZYME ABTM，瑞典-例如，PCT/US1992/010113和PCT/SE2015/050108)的生物催化应用。用于将活性酶连接到表面的可替代系统在本领域中是已知的(参见例如US4088538；US4141857；US4206259；US4218363；US4229536；US4239854；US4619897；US4748121；US4749653；US4897352；US4954444；US4978619；US5154808；US5914367；US5962279；US6030933；US6291582；US6254645；US10,016,490和US10,041,055)。
蛋白质标签是基因移植到重组蛋白质上的肽序列，通常是通过化学试剂或通过酶促方法可去除的，并连接到蛋白质上用于各种目的。表A中列出的蛋白质标签旨在作为实例并且不旨在以任何方式进行限制。一种类型的蛋白质标签是亲和标签，其被添加到蛋白质或肽序列中，使得它们可以使用亲和技术(例如，来自表达系统生物体)从粗生物源中被纯化，或者促进“标记的”蛋白质固定到表面上。亲和标签的一些实例包括几丁质结合结构域(CBD)、麦芽糖结合蛋白(MBP)、Strep标签、谷胱甘肽-S-转移酶(GST)和与金属基质结合的聚组氨酸(His标签)。另一种类型的蛋白质标签是表位标签(例如，包括V5标签、Myc标签、HA标签、Spot标签和NE标签)，它们是为易于产生高亲和力抗体而选择的短肽序列，并且通常衍生自病毒基因序列以改善免疫反应性。表位标签特别用于蛋白质印迹、免疫荧光和免疫沉淀实验，尽管它们也用于蛋白质到表面的纯化和固定化。另一种类型的蛋白质标签是色谱标签(例如，聚阴离子氨基酸，如FLAG标签)，其可以被用于改变蛋白质的色谱性质以帮助分离和纯化或固定化。另外的蛋白质标签是增溶标签(例如，麦芽糖结合蛋白(MBP)、谷胱甘肽S-转移酶(GST)、硫氧还蛋白(TRX)和聚(NANP))和荧光标签(例如，绿色荧光蛋白(GFP))。蛋白质标签可以允许特异性的酶促修饰、化学修饰或将蛋白质连接到其它组分上。然而，取决于添加到蛋白质序列中的标签的类型或数量、蛋白质的天然功能，在这种情况下，酶促功能可能被标签损害。因此，需要选择蛋白质标签以确保酶的活性不被损害，或者可替代地，在使用前可以将蛋白质标签从蛋白上切割下来。
表A：示例性蛋白质标签
Figure BDA0003020590770000141
Figure BDA0003020590770000151
Figure BDA0003020590770000161
Figure BDA0003020590770000171
Figure BDA0003020590770000181
在本申请中，通过使用如SEQ ID NO:5、10、15、17、19、21、23、25、27、29和31中所示的聚组氨酸蛋白质标签(His标签)例证了蛋白质标签的使用，但是本领域技术人员将容易理解，任何数量的其它蛋白质标签可以被用于纯化酶和/或被用于将酶连接于如本文所述的表面上，这取决于所用的纯化方法和/或酶所连接至的表面。此类蛋白质标签可以选自表A中所列的蛋白质标签中任何一种或多种，但其它此类蛋白质标签是本领域已知的。
此外，可以采用一个或多个切割位点(例如，如SEQ ID NO:15、17、19、21、23、25、27、29和31中所用的凝血酶切割位点)来从酶中释放蛋白质标签或以其它方式切割酶。切割位点可以被用于去除N-末端甲硫氨酸、信号肽，和/或将非活性或非功能性蛋白转变为活性蛋白(即酶原(zymogens/proenzymes))。可替代地，切割位点可以被用于分离在同一阅读框中表达的两种或更多种酶。能够切割蛋白质或肽并且将具有序列特异性切割位点的酶的实例可以选自以下中的一种或多种：Arg-C蛋白酶；Asp-N内肽酶；Asp-N内肽酶+N-末端GluBNPS-粪臭素；半胱天冬酶1；半胱天冬酶2；半胱天冬酶3；半胱天冬酶4；半胱天冬酶5；半胱天冬酶6；半胱天冬酶7；半胱天冬酶8；半胱天冬酶9；半胱天冬酶10；糜蛋白酶-高特异性([FYW]的C-末端，不是在P之前)；糜蛋白酶-低特异性([FYWML]的C-末端，不是在P之前)；梭菌蛋白酶(梭状芽胞杆菌肽酶B)；CNBr；肠激酶；因子Xa；甲酸；谷氨酰内肽酶；颗粒酶B；羟胺；亚碘酰基苯甲酸；LysC；LysN；NTCB(2-硝基-5-硫氰基苯甲酸)；嗜中性粒细胞弹性蛋白酶；胃蛋白酶(pH1.3)；胃蛋白酶(pH>2)；脯氨酸-内肽酶；蛋白酶K；葡萄球菌肽酶I；烟草蚀纹病毒蛋白酶；嗜热菌蛋白酶；凝血酶和胰蛋白酶。
本领域技术人员将理解，如本文所述的能够有效切割A抗原的活性半乳糖胺酶和活性GalNAc脱乙酰酶的组合是重要的，并且本领域技术人员还将理解，添加一个或多个切割位点和/或一个或多个蛋白质标签是任选的，并且可以基于具体的表达系统、纯化系统和可能的表面连接策略选择此类修饰。此外，对半乳糖胺酶和GalNAc脱乙酰酶序列的其它修饰也是可能的，只要A抗原的切割活性没有显著受损。另外，对半乳糖胺酶和GalNAc脱乙酰酶的修饰是可能的，只要A抗原切割活性没有显著受损。对半乳糖胺酶和GalNAc脱乙酰酶序列的修饰可以是缺失、插入和/或取代。取代可以是保守性取代或中性取代。例如，半乳糖胺酶和GalNAc脱乙酰酶序列可能与成熟酶共享90％或更高的序列同一性。例如，半乳糖胺酶和GalNAc脱乙酰酶序列可能与成熟酶共享85％或更高的序列同一性。例如，半乳糖胺酶和GalNAc脱乙酰酶序列可能与成熟酶共享75％或更高的序列同一性。可替代地，半乳糖胺酶和GalNAc脱乙酰酶序列可以对5％、10％、13％、15％、20％或高达25％的氨基酸进行修饰。
如本文所用的“吸附在玻璃、藻酸盐珠或基质上”是指将酶连接到惰性材料的外部。通常，这种类型的固定化不是由化学反应引起的，并且固定化酶的活性位点可以被其吸收的表面封闭，这可以降低被吸收的酶的活性。
如本文所用的“截留”是指将酶捕捉在不溶性珠或微球体内。然而，截留可能阻碍底物的到达和产物的离开。一个实例是使用藻酸钙珠，其可以通过藻酸钠溶液和酶溶液的混合物与氯化钙反应来产生。
如本文所用的“交联”是指酶彼此共价键合以产生几乎仅由酶组成的基质。当设计交联酶反应时，结合位点理想地不覆盖酶的活性位点，使得酶的活性仅受固定性的影响，而不受酶活性位点的封闭的影响。然而，间隔分子(如聚(乙二醇))可以被用于降低底物的位阻。
如本文所用的“共价键合”是指酶经由共价键与不溶性支持物或表面(例如硅胶)键合。由于酶和支持物或表面之间共价键的强度，酶从支持物或表面脱离的可能性小得多。
如本文所用的“拥挤剂”是指通过在细胞表面上浓缩酶以改善酶的活性而促进大分子拥挤的任何聚合物或蛋白质。拥挤剂可以例如是右旋糖酐、硫酸右旋糖酐、糊精、普鲁兰多糖、聚(乙二醇)、聚蔗糖TM、超支化甘油和惰性蛋白。(Kuznetsova,I.M等人，Int J MolSci.(2014)“What Macromolecular Crowding Can Do to a Protein”15(12):23090–23140)。
如本文所用的“右旋糖酐”是指分子量≥1,000道尔顿并且具有α-连接的d-吡喃葡萄糖基重复单元的线性主链的多糖。可以基于吡喃糖环结构将右旋糖酐分为3个结构类别(即第1-3类)，其含有五个碳原子和一个氧原子。第1类右旋糖酐含有用具有α(1→2)、α(1→3)和α(1→4)键联的d-葡萄糖分支的小侧链修饰的α(1→6)连接的d-吡喃葡萄糖基主链。第1类右旋糖酐在它们的分子量、空间排列、支化类型和程度以及支链长度方面变化，3-5取决于微生物产生菌株和培养条件。异麦芽糖和异麦芽三糖是具有第1类右旋糖酐主链结构的寡糖。第2类右旋糖酐(交替的)含有具有α(1→3)连接的分支的交替的α(1→3)和α(1→6)连接的d-吡喃葡萄糖基单元的主链结构。第3类右旋糖酐(变聚糖(mutans))具有有α(1→6)连接的分支的连续的α(1→3)连接的d-吡喃葡萄糖基单元的主链结构。
如本文所用的“普鲁兰多糖”是主要由真菌普鲁兰短梗霉(Aureobasidiumpullulans)从淀粉产生的结构多糖，并且由具有偶有麦芽四糖单元的重复的α(1→6)连接的麦芽三糖(D-吡喃葡糖基-α(1→4)-D-吡喃葡糖基-α(1→4)-D-葡萄糖)单元组成。
如本文所用的“糊精”是指链长比右旋糖酐短的D-吡喃葡萄糖基单元，其以单一α(1→6)键开始，但是线性延续有α(1→4)连接的D-吡喃葡萄糖基单元。
如本文所用的“聚蔗糖TM”是中性的、高度支化的、高质量的亲水性多糖，其容易溶解在水性溶液中。
如本文所用的“灌注(perfusion/perfusing)”是指通过使流体循环通过血管而使器官渗透有流体。
器官保存中的重要目标是增加可用的可移植器官的数量。通常，将器官保存在冷库中，但这具有潜在的扩散限制，因此开发了冷灌注系统。此外，近常温系统也被用于增强包括肝脏、肺、心脏和肾脏在内的实体器官的功能保存。许多缓冲的细胞外溶液被用作灌注溶液或保存溶液。许多缓冲的细胞外溶液是已知的。例如，SteenTM、PerfadexTM、PerfadexPlusTM、EuroCollins溶液、组氨酸-色氨酸-酮戊二酸(HTK)溶液、威斯康星大学溶液(UW)、Celsior溶液、肾脏灌注液(KPS-1)、京都大学溶液、IGL-1溶液和柠檬酸盐溶液(Guibert，E.E.等人(2011年))。这些中的许多是可商购获得的，并且对于本领域技术人员而言，这些溶液的变型将是显而易见的。
本文描述了各种可替代的实施方案和实施例。这些实施方案和实施例是说明性的并且不应被解释为限制本发明的范围。
材料和方法
除非另有说明，用于该研究的化学物质和商业酶购自Sigma-AldrichTM。单糖甲基伞形酮糖苷是Hongming Chen博士的慷慨礼物，并且亚型1A抗原五-MU是David Kwan博士的慷慨礼物(Kwan等人，2015)。
人类排泄物宏基因组文库
为了产生人类宏基因组学fosmid文库，从具有AB+血型的健康亚洲男性志愿者采集人类新鲜排泄物样品。直接DNA提取和fosmid文库创建根据MoE方案中所述的程序(Armstrong等人，2017)进行。
Fosmid文库筛选
将51×384孔AB+血液Fosmid文库板在室温下解冻并复制到含有50μL筛选LB培养基(12.5μg/mL的氯霉素、25μg/mL的卡那霉素、100μg/mL的阿拉伯糖、0.2％(v/v)的麦芽糖、10mM的MgSO4)的384孔板中。将板在含有蓄水池的密封容器中于37℃孵育18小时以防止过度蒸发。使用QFillTM仪器[GenetixTM]将45μl反应混合物(100mM的NaH2PO4，pH7.4、2％(v/v)的Triton-X 100、100μM的GalNAc-α-MU、100μM的Galα-MU)添加到生长的筛选板上。然后将板在密封容器中于37℃孵育24h，并且在第1、2、4、8和24小时经由Synergy H1读板器[BioTekTM]测量每个板的荧光(Ex：365nm，Em：435nm，扫描模式，增益80)。对于所有孔，计算Z评分，其由下式给出：Z评分＝(荧光值-中值)/标准偏差。
将高于一定阈值的所有阳性命中物重新排列在新的384孔板中，被称为“简单底物命中”板，并储存在-70℃下。从“简单底物命中”板复制两个筛选板，并重新筛选GalNAc-α-MU或Gal-α-MU活性，以验证和反卷积先前检测到的活性。
为了确定哪种命中物可以切割A抗原或B抗原结构，使用偶联酶测定来确定它们对50μM的亚型1A抗原四-MU或50μM的亚型1B抗原四-MU的活性。先前Kwan(Kwan等人，2015)描述了这种偶联测定的一种版本。通过使用BgaC(Jeong 2009)代替BgaA(Singh 2014)作为偶联酶，我们的测定被修改以也检测亚型1A抗原的切割。潜在的α-N-乙酰基半乳糖胺酶或α-半乳糖苷酶将切割末端糖，释放亚型I H抗原三-MU。随后，α-岩藻糖苷酶(AfcA(Katayarna2004))、β-半乳糖苷酶(BgaC(Jeong 2009))和β-己糖胺酶(SpHex(Williams 2002))将以外向方式切割残余的糖，直到4-甲基伞形酮醇被释放；随着荧光的增加是可检测到的。为了实现这一点，将50μg/mL的每种酶添加到反应混合物中。一式三份地再次筛选高于某一阈值的所有阳性命中物，并将含有缺少任何插入片段的载体的宿主细胞株用作阴性对照。将所有验证的命中物分别于-70℃下储存在LB培养基(12.5μg/mL的氯霉素、25μg/mL的卡那霉素、15％(v/v)的甘油、0.2％(v/v)的麦芽糖、10mM的MgSO4)中。
Fosmid命中测序
为了分离用于测序的fosmid DNA，使用阳性命中的fosmid甘油储备液接种5mL的TB培养基(12.5μg/mL的氯霉素、25μg/mL的卡那霉素、100μg/mL的阿拉伯糖、0.2％(v/v)的麦芽糖、10mM的MgSO4)，于37℃、220rpm下孵育过夜。使用GeneJetTM质粒小量制备试剂盒(Thermo FisherTM)进行Fosmid分离。使用Plasmid-SafeTM ATP依赖性DNA酶(EpicentreTM)从污染的线性大肠杆菌(E.coli)DNA中纯化分离的Fosmid，然后使用GeneJetTM PCR纯化试剂盒(Thermo FisherTM)进行另一轮纯化。在QbitTM荧光计(ThermoFisherTM)上使用Quant-iTTM dsDNA HS测定试剂盒(InvitrogenTM)计算浓度。用1％琼脂糖凝胶验证预期的DNA大小。对于完整的fosmid测序，将2ng每个fosmid送到UBC测序中心(温哥华，BC，加拿大)。使用Illumina MiSeqTM系统将每个fosmid单独条形码化并测序。
使用在GitHubTM上可获得的python脚本(https://github.com/hallamlab/FabFos)对所有Illumina MiSeqTM原始序列数据进行修整和汇编。简言之，Trimmomatic被用于从读取中去除衔接子和低质量序列(Bolger 2014)。使用BWA(Li 2013)筛选这些读取的载体和宿主序列，然后使用SamtoolsTM和bam2fastq脚本过滤以去除污染物。通过MEGAHIT汇编这些高质量和纯化的读取，其中k-mer值在71至241的范围，以10的增量增加(Li 2015)。由于这些文库通常具有超过20,000倍的覆盖率，并且为了防止干扰正确序列程序集的测序错误的累积，通过fosmid的估计覆盖率的1％计算最小k-mer多重性。然后在python脚本程序集之外，使用minimus2(Treangen 2011)python脚本程序集产生多于一个的重叠群。参数化命令可以在GitHubTM页面和python脚本本身的文档中找到。
Fosmid ORF预测和命中验证
使用ProdigalTM(Hyatt 2010)的宏基因组版本鉴定Fosmid ORF，并与使用作为MetaPathwayTM v2.5软件包(Konwar 2015)的一部分的BLASTPTM的CAZyTM数据库进行比较。MetaPathwayTM的参数：长度>60，BLAST评分>20，blast评分比>0.4，E值<1×10-6。
使用Golden GateTM克隆策略(Engler 2008)将具有对GH或CBM家族成员(具有已知或疑似的α-半乳糖苷酶和/或α-N-乙酰基半乳糖胺酶活性)的注释的所有预测的ORF克隆到pET16b质粒中，引物序列列于表B中。蛋白在10mL的ZY5052自动诱导培养基(Studier 2005)中于37℃、220rpm下培养20h的BL21(DE3)中表达。通过离心(4000×g，4℃，10min)收获细胞，并重悬于1mL裂解缓冲液(100mM的NaH2PO4，pH7.4、2％(v/v)的Triton-XTM 100，无EDTA的1×蛋白酶抑制剂[PierceTM])中。用来自与50μL测定缓冲液(100mM的NaH2PO4，pH 7.4、50μg/mL的SpHex、50μg/mL的AfcA、50μg/mL的BgaC、100μM的亚型1A抗原四-MU或100μM的亚型1B抗原四-MU)混合并于37℃下孵育的候选物的50μL的粗细胞裂解物进行偶联测定(Kwan2015)。所有反应在黑色的96孔板上一式三份进行。使用SynergyTM H1读板器[BioTekTM]连续监测荧光(365/435nm)4小时。重复来自显示A或B抗原切割活性的粗提物的测定，这次没有偶联酶，并且经由HF Bond Elut C18柱分离反应产物，以及用LC-MS和/或TLC分析。使用TLC硅胶60F254 TLC板[EMD Millipore Corp.TM，比勒利卡，Ma，美国]进行TLC。
表B：引物序列
Figure BDA0003020590770000241
Figure BDA0003020590770000251
HPAE-PAD测定
半乳糖胺的酶促释放的分析在HPAE-PAD(DionexTM)HPLC系统上进行。在以下底物上测试不同蛋白质的切割活性：在100mM的NaH2PO4(pH7.4)中的7.5μg/μL的来自猪胃的II型粘蛋白；在100mM的NaH2PO4(pH7.4)中的5mM的亚型1A抗原五-MU和在1×PBS(pH7.4)中来的自A+型供体、B+型供体和O型供体的RBC(50％的血细胞比容)。将含有10μg/mL酶的样品于37℃孵育2小时，然后在-80℃存储以用于进一步分析。将反应的小等分试样(10μl)稀释在H2O(100μl)中，并在HPAE-PAD仪器上进行分析。在具有保护柱的CarboPac PA200TM(150mm)柱上进行分离，并且使用聚四氟乙烯(PTFE)电极上的一次性金和四电位波形进行检测。分离条件如下：100mM的氢氧化钠和乙酸钠梯度在分离的前10分钟内从70至300mM。将洗脱剂在最终梯度条件下保持1min，然后在下一分钟内返回到起始条件。流速为1.0ml/min，每27分钟进行一次注射。游离糖GalNAc、Gal1和GalN(10μM)的标准品也被应用于HPAE-PAD以确定用于参考的峰洗脱时间。
动力学测定
使用4-甲基伞形酮作为离去基团的所有动力学测定通过荧光测量进行。为了避免基于内过滤效应(Palmier 2007)的测量误差，使用标准曲线来验证荧光团的线性范围。
Fp半乳糖胺酶
在100mM的NaH2PO4(pH7.4)中于37℃下，确定亚型1GalN抗原五-MU和亚型1A抗原五-MU的Michaelis-Menten参数。反应在100μL中进行，其中具有3.4nM的Fp半乳糖胺酶(5.31nM的FpGalNase_截短的)和0.1mg/mL的SpHex、AfcA、0.2mg/mL的BgaC和不同浓度的底物(5μM-2mM)。反应以一系列四个重复运行，其中对照(无Fp半乳糖胺酶)为一式两份。通过SynergyH1TM平板读数器[BioTekTM]监测由通过水解释放MU产生的荧光信号(365/435nm)，并使用在相同反应条件下确定的MU标准浓度曲线转变成浓度。确定初始速率(μM/s)，并在Grafit7.0TM中做图以确定动力学参数。
在pH7.4和37℃下，确定亚型1/2/4GalN抗原四-MU和亚型1B抗原四-MU的Kcat/KM参数。在黑色96板孔中进行反应(总体积为100μL)，并作为在100mM的NaH2PO4(pH7.4)中的偶联测定进行，其中100mM的NaH2PO4(pH7.4)中具有8.63nM的Fp半乳糖胺酶、0.1mg/mL的SpHex、BgaC(亚型2的BgaA)、AfcA、不同浓度的底物(25μM、20μM、15μM、10μM、7.5μM、5μM)。反应以一系列四个重复运行，其中对照(无Fp半乳糖胺酶)为一式两份。通过Synergy H1TM平板读数器[BioTekTM]监测由通过水解释放MU产生的荧光信号(365/435nm)，并使用在相同反应条件下确定的MU标准浓度曲线转变成浓度。确定初始速率(μM/s)，并在Grafit7.0TM中做图以确定kcat/KM(s-1*mM-1)参数。
在透明96板中于37℃下，在具有863.2nM的Fp半乳糖胺酶(在100mM的NaH2PO4中，pH7.4)或369.9nM的FpGH4(在50mM的Tris/HCl(pH7.4)、100μM的NAD+、1mM的MnCl2中)以及不同浓度的底物(10μM-5mM)的100μl的体积中确定GalN-α-pNP的Michaelis-Menten参数。反应以一系列三个重复运行，其中两个对照(无酶)。通过Synergy H1TM读板器[BioTekTM]监测由水解释放pNP产生的吸收(在405nm处)，并使用在相同反应条件下确定的对硝基苯酚标准浓度曲线转变成浓度。确定初始速率(μM/s)并在Grafit7.0TM中做图以确定动力学参数。
FpGalNAc脱乙酰酶
使用先前描述的偶联测定(Kwan 2015)于37℃下，在100mM的NaH2PO4(pH7.4)中确定亚型1A抗原五-MU的Michaelis-Menten参数。通过使用BgaC(Jeong 2009)代替BgaA(Singh2014)作为β-半乳糖苷酶，修改测定以允许检测亚型1(和稍后4)的切割。此外，由于亚型1A抗原五-MU含有另外的半乳糖，BgaC的浓度增加至0.2mg/mL以补偿其切割Gal-β-1，3-β-GlcNAc-β-1，3-Gal-β-MU和Gal-β-MU两者的需要。此外，还包括Fp半乳糖胺酶以允许切割含半乳糖胺的中间体。在100μ中的反应设置是3nM FpGalNAc脱乙酰酶(4.52nM的FpGalNacDeAc_D1ext、3.55nM的FpGalNacDeAc_D1+2)和0.01mg/mL的Fp半乳糖胺酶、0.1mg/mL的SpHex、AfcA、0.2mg/mL的BgaC和不同浓度的底物(5μM–2.5mM)。反应以一系列四个重复进行，其中对照(无FpGalNac脱乙酰酶)为一式两份。通过Synergy H1TM平板读数器[BioTekTM]监测由通过水解释放MU产生的荧光信号(365/435nm)，并使用在相同反应条件下确定的MU标准浓度曲线转变成浓度。确定初始速率(μM/s)，并在Grafit7.0中做图以确定动力学参数。
在pH7.4，37℃下，确定亚型1/2/4A抗原四-MU的Kcat/KM参数。在黑色96板孔中进行反应(总体积为100μL)，并作为在100mM的NaH2PO4(pH7.4)中的偶联测定进行，其中100mM的NaH2PO4(pH7.4)中具有12nM的FpGalNac脱乙酰酶、0.1mg/mL的SpHex、BgaC(亚型II的BgaA)、AfcA、不同浓度的底物(25μM、20μM、15μM、10μM、7.5μM、5μM)。反应以一系列四个重复运行，其中对照(无FpGalNac脱乙酰酶)为一式两份。通过Synergy H1TM平板读数器[BioTekTM]监测由通过水解释放MU产生的荧光信号(365/435nm)，并使用在相同反应条件下确定的MU标准浓度曲线转变成浓度。确定初始速率(μM/s)，并在Grafit7.0TM中做图以确定kcat/KM(s-1*mM-1)参数。
GH109亚型的动力学
在pH7.4和37℃下，确定亚型1/2/4A抗原四-MU的Kcat/KM参数。在黑色96板孔中进行反应(总体积为100μL)，并作为在100mM的NaH2PO4(pH7.4)中的偶联测定进行，其中100mM的NaH2PO4(pH7.4)中具有86.02nM的BvGH109_1/100.49nM的EmGH109/80.52nM的BvGH109_2/87.4nM的BsGH109和5μM的NAD+、各自0.1mg/mL的SpHex、BgaC(亚型2的BgaA)、AfcA，不同浓度的底物(25μM、20μM、15μM、10μM、7.5μM、5μM)。反应以一系列四个重复运行，其中对照(无α-N-半乳糖胺酶)为一式两份。通过Synergy H1TM平板读数器[BioTekTM]监测由通过水解释放MU产生的荧光信号(365/435nm)，并使用在相同反应条件下确定的MU标准浓度曲线转变成浓度。确定初始速率(μM/s)，并在Grafit7.0TM中做图以确定kcat/KM(s-1*mM-1)参数。
晶体学
在结晶之前，使用制造商建议的方案，用浓度为1mg/mL的凝血酶(NovagenTM)消化FpGalNAcDeAc_D1ext过夜。然后通过HisTrap FF柱纯化蛋白质并收集流通物，缓冲液交换到10mM的Tris(pH8.0)+75mM的NaCl中，并浓缩至12mg/mL。
结晶
使用悬滴扩散法，使用由0.2M的CaCl2、0.1M的MES(pH6)、18％的PEG 4000和20mM的MnCl2组成的储液以1:1的蛋白质：储液比，使FpGalNAcDeAc_D1ext(12mg/mL)结晶。使用快速溴化物浸泡来衍生用于定相的晶体，并且通过将晶体转移至1M的NaBr、25％的甘油、18％的PEG4000、20mM的CaCl2和0.1M的Mes pH的溶液30秒并在液氮中快速冷冻来制备。在与上述相同的条件但省略MnCl2下设置液滴之前，具有B型血抗原三糖(B_三)的晶体复合物通过将蛋白(12mg/mL)与10mM的B_三预孵育2小时来制备。晶体用补充有25％甘油的储液冷冻保护。
数据采集、定相和结构确定
在加拿大光源TM(Canadian Light SourceTM)处采集数据集。使用XDS(Kabsch2010)整合数据并用AimlessTM(Evans 2013)确定比例。使用CRANK2TM(Skubak 2013)在CCP4I2TM程序组(Potterton 2018)中进行定相和自动化结构溶液。使用CootTM(Emsley2004)和RefmacTM(Vagin 2004)的交替循环检查和改进结构。B_三结构复合物通过差值傅里叶法来求解，并且配体在CootTM中手动构建，水和金属离子也在CootTM中手动构建。差异密度图证实了在apo结构中存在Mn2+以及在配体结构中存在Ca2+。通过CootTM和MolprobityTM(Chen 2010)验证模型。apo和B_三复合物的原子坐标和结构因子已经保藏在蛋白质数据库(PDB)中，其中登录号为：
普氏梭杆菌的GalNAc脱乙酰酶蛋白的SEQ ID NO：WP_009260926.1；和
普氏梭杆菌的半乳糖胺酶蛋白的SEQ ID NO：WP_044942952.1
活性位点诱变
基于结构信息(未示出)和序列比对(未示出)，使用QuickchangeTM方案(Zhang2004)，利用表B中所示的引物，使FpGalNAcDeAc_D1min和FpGalNase_截短的突变。如上所述，经由NiNTA和HIC柱纯化突变体。经由CD光谱检查所有突变体的结构完整性；所有经测试的酶在结构上与它们的野生型相似。对于活性相对低的突变体，在用于完全动力学测定的相同条件下进行反应；然而，如先前所述的(Vocadlo 2002)，使用底物消耗方法测定kcat/KM值。简言之：在其中[底物]<KM(相当于Km的～1/5-1/10)的低浓度底物下，kcat/KM值可以通过将反应时间过程非线性拟合到一级曲线并除以酶浓度来近似。
GH36系统发育图
使用SACCHARISTM cazy_提取物.pl脚本(Jones 2018)从CAZyTM数据库下载GH36的参考序列。使用基于系统发育的蛋白质剖析软件TreeSAPPTM(在https://github.com/hallamlab/TreeSAPP处可获得)构建参考树并将序列映射到这些树。简言之，来自dbCAN的HMM被用于从从CAZyTM(Yin 2012)下载的所有全长序列提取蛋白质家族结构域。然后使用UCLUSTTM以70％序列相似性聚类这些序列以去除冗余序列空间并减小树的大小(Edgar2010)。使用RAxMLTM 8.2.0版本来构建具有“--autoMRE”的参考树，以决定在进行1000次重复之前何时退出引导指令，并且PROTGAMMAAUTOTM选择最佳蛋白质模型(Stamatakis 2006和Stamatakis 2008)。
然后使用TreeSAPPTM将查询序列映射到这些参考树上。简言之，使用hmmsearchTM将蛋白质序列与HMM比对，并提取比对的区域(Eddy 1998)。hmmalignTM被用于在参考多重比对中包括新的查询序列，然后TrimA1TM从比对文件中去除非保守位置(Capella-Gutierrez2009)。使用RAxMLTM通过插入来对参考树中的查询序列进行分类。过滤每个查询序列的位置，并串联成单一的查询序列。在iTOLTM中可视化之前的JplaceTM文件(Matsen 2012和Letunic 2016)。
RBC测定
使用不列颠哥伦比亚大学(The University of British Columbia)的临床伦理学委员会批准的方案将来自健康同意供体的全血收集到柠檬酸盐真空采血管中。将管在RT下以1000×g旋转4min，分离RBC，并用1×PBS(pH7.4)洗涤3次。对于在右旋糖酐40k存在下的测定，将洗涤的RBC(200μL，10％血细胞比容)置于管中，部分去除上清液，并用含有或不含右旋糖酐40k(最终浓度为300mg/mL)的1×PBS(pH7.4)替换。此外，在1×PBS(pH7.4)+25％血浆或100％血浆中进行一些测定。小心地混合RBC并置于定轨振荡器上30s。然后加入稀释的酶溶液至最终体积为200μL。将管非常轻地涡旋，并在设定温度下放置在定轨振荡器上限定的时间。
MTS卡
反应后，用过量的1×PBS(pH7.4)洗涤RBC 3次，并使用微型打字系统TM(MicroTyping SystemTM，MTS)卡[MTSTM，佛罗里达，美国]进行分析。将悬浮在稀释剂[MTS，佛罗里达，美国]中的RBC(12μl，5％血细胞比容)小心地加入到微型凝胶柱中，在血液和微型凝胶的内容物之间留下空间。使用如所推荐的具有改进的样品架的Beckman Coulter AllegraX-22RTM离心机，在RT下将MTS卡以156×g离心6min。根据制造商的说明书，从旋转后的微型凝胶中RBC的位置评估从RBC表面去除抗原的程度。具有高表面抗原浓度的RBC在与存在于凝胶柱中的单克隆抗体相互作用时凝集并且不能渗透(MTSTM评分为4)。没有表面抗原的RBC没有凝集并迁移到微型凝胶的底部(MTS评分为0)。根据制造商的说明书，部分去除迁移到这些抗原之间的位置的表面抗原的RBC被赋予0(不存在)至4(存在)的评分。
H抗原凝集测定
为了分析酶促处理后A抗原向H抗原的转变，将经洗涤的A-ECO-RBC以等份与2μg/mL的抗H抗体(抗H ab血型抗原抗体[97-1]：目录号：ab24213(AbcamTM))混合，并且监测在30分钟时间范围内的凝集现象。用抗H抗体进行凝集的RBC被赋予0(在1800sec内没有凝集)至5(在120sec内凝集)的评分。
FACS
将酶处理的RBC用1×PBS(pH7.4)洗涤2次，并将1％血细胞比容的ECO-RBC用1/100APC-抗A抗体(AlexaFluorTM647小鼠抗人A血型：目录号：565384(BD PharmingenTM))和/或抗H抗体(抗H ab血型抗原抗体[97-1]：目录号：ab24213(AbcamTM))在RT下处理30分钟，然后用1×PBS(pH7.4)洗涤2次。为了检测抗H抗体，使用浓度为1/500的第二FITC-标记的抗体(山羊F(ab')2抗小鼠IgM mu链(FITC):目录号：ab5926(AbcamTM))。在用流式细胞仪(CytoFLEXTM(Beckman CoulterTM))重构成1×PBS(pH7.4)(1％血细胞比容)后，评估数据。
酶吸附和抗原性
为了测试处理后，酶是否可以容易地从RBC中去除，评估了潜在的吸附。将太平洋蓝色标记的FpGalNAc脱乙酰酶和FpGalNase(F/P＝1)与RBC在37℃单独孵育1h，并且在几个洗涤步骤之后，然后在流式细胞仪(CytoFLEXTM(Beckman CoulterTM))上测量残留荧光。
通过将RBC与50μg/mL的每种酶一起孵育并将酶处理的RBC与同种异体或自体血清混合，观察潜在的凝集来测试抗原性。此外，为了评估潜在的抗IgG、-C3d暴露，在抗IgG、-C3d MTSTM卡[MTSTM，佛罗里达，美国]上测试经处理的RBC。在37℃下孵育时间为30分钟。
抗原亚型合成
亚型1/2/4A和B抗原四-MU的合成是用如Kwan(Kwan等人，2015)所述的改良的方案进行的。
二步亚型1/2/4H抗原三-MU的合成
所有三种合成均以在10mL的50mM的Tris/HCl、200mM的NaCl，pH7.4，10mM的MnCl2、50U的碱性磷酸酶、1.5当量的UDP-Gal、1.2当量的GDP-Fuc(以LacNAc-MU产物确定比例)的20mg的GalNAc-α-MU/GlcNAc-α-MU的规模上进行。根据所期望的产物，为亚型I CgtB S42和Te2FT、为亚型II HP0826和WbgL、为亚型IV LgtD和Te2FT，添加浓度为100μg/mL的不同糖基转移酶。反应于37℃下进行并且经由TLC(流动相，EtAc:MeOH:H2O，比例为6:2:1)控制进程，4-甲基伞形酮经由10％的H2SO4从化合物水解并且经由UV(360nm)检测。在没有观察到进一步的产物增加之后，将反应施加到HF Bond Elut C18柱上，用几个柱体积的5％甲醇洗涤，并用25％甲醇洗脱产物。然后真空去除溶剂。
亚型1/2/4A抗原四-MU的合成
最终的合成步骤于37℃下，在5mL的50mM的Tris/HCl、200mM的NaCl，pH7.4，10mM的MnCl2、25U的碱性磷酸酶、1.5当量的UDP-Gal和100μg/mL的BgtA中的10mg的亚型1/2/4H抗原三-MU的规模进行。经由TLC追踪进展，在没有观察到进一步的产物增加之后，将反应物施加到HF Bond Elut C18柱上，用几个柱体积的5％甲醇洗涤，并用25％甲醇洗脱产物。然后真空去除溶剂。最终产物在1.5×46cm的HW-40F大小排阻柱上进一步纯化，然后冷冻干燥。
亚型1/2/4B抗原四-MU的合成
最终的合成步骤于37℃下，在5mL的50mM的Tris/HCl、200mM的NaCl，pH7.4，25U的碱性磷酸酶、1.5当量的UDP-Gal和100μg/mL的BoGT6a中以10mg的亚型1/2/4H抗原三-MU的规模进行。经由TLC追踪进展，在没有观察到进一步的产物增加之后，将反应物施加到HF BondElut C18柱上，用几个柱体积的5％甲醇洗涤，并用25％甲醇洗脱产物。然后真空去除溶剂。最终产物在1.5×46cm的HW-40F大小排阻柱上进一步纯化，然后冷冻干燥。
亚型1GalN抗原五-MU合成
将10mg的亚型1A抗原五-MU与在5mL的100mM的NaH2PO4中的1μg/mL的FpGalNAc脱乙酰酶于37℃下孵育30min，然后通过添加1mM的EDTA终止。经由TLC检查底物的完全转变，并将反应物施加到HF Bond Elut C18柱上，用几个柱体积的2％甲醇洗涤，并用10％甲醇洗脱产物。然后真空去除溶剂。
蛋白纯化
经由Golden GateTM克隆(Engler 2008)或PIPE克隆(Klock 2008)将所有蛋白质和其中的截短物克隆到pET16b或pET28a中。引物序列列在表B中。
用于延长表征的蛋白质的产生在BL21(DE3)细胞中进行，在200mL的ZY5052自动诱导培养基(Studier 2005)中于37℃，220rpm下培养20h，接种100μl过夜LB培养物。通过离心(4000×g，40℃，10min)收获细胞，并重悬于10mL裂解缓冲液(50mM的Tris/HCl、150mM的NaCl、1％(v/v)的甘油、40mM的咪唑，pH7.4，2mM的DTT、1×无EDTA的蛋白酶抑制剂(PierceTM)、2U的Benzonase(NovagenTM)、0.3mg/mL的溶菌酶、10mM的MgCl2)中，然后在冰上超声处理(3min的脉冲时间；5sec的脉冲，10sec的暂停，35％的振幅)。通过离心(14000×g，4℃，30min)去除细胞碎片后，收集上清液并使用蠕动泵加载到镍亲和色谱柱(5mL的HisTrap HPTM柱(GETM))上。在AEKTApurifierTM系统(GETM)上进行并监测洗脱，其中使用10-75％梯度的50mM的Tris/HCl、400mM的咪唑，pH7.4，2mM的DTT，经由SDS-PAGE鉴定含有蛋白质的级分，然后合并。在Amicon Ultra-15离心过滤装置TM MWCO 10kDa(MilliporeTM)中，将缓冲液交换成50mM的Tris/HCl、150mM的NaCl，pH7.4，2mM的DTT，并进行浓缩。
FpGalNAc脱乙酰酶、Fp半乳糖胺酶和其中的截短物不得不经历第二轮纯化，在将蛋白质加载到疏水相互作用色谱柱(10mL的苯基琼脂糖高效柱(Pharmacia BiotechTM))上之前，使用Amicon Ultra-15离心过滤装置TM MWCO 10kDa(MilliporeTM)交换缓冲液。柱的加载、洗涤和洗脱(梯度为0-100％)通过AEKTApurifierTM系统(GETM)处理，使用以下缓冲条件：FpGalNAc脱乙酰酶；结合1×PBS，800mM的NH2PO4,pH7.4和洗脱1×PBS(pH7.4)以及Fp半乳糖胺酶；结合25mM的Tris/HCl、1M的NaCl,pH7.4和洗脱25mM的Tris/HCl(pH7.4)。经由SDS-PAGE，鉴定含有蛋白质的级分，然后合并。在Amicon Ultra-15离心过滤装置TM MWCO10kDa(MilliporeTM)中，将缓冲液交换成50mM的Tris/HCl、150mM的NaCl，pH7.4，并进行浓缩。
蛋白质表征
最佳pH值
对于亚型1A抗原五-MU和亚型1GalN抗原五-MU，FpGalNAc脱乙酰酶和Fp半乳糖胺酶的活性的一般pH范围分别通过在TLC板上出现的用于改变pH值的产物来确定。在37℃下，用50μM的底物和1μg/mL的酶在适当的缓冲系统中以100μl的规模进行反应。用于pH 4-6的缓冲液基于50mM的柠檬酸/柠檬酸钠缓冲液，用于pH 6-8的缓冲液基于50mM的磷酸钠缓冲液和用于pH 8-10的缓冲液基于50mM的甘氨酸/氢氧化钠缓冲液。
为了确定最佳pH值，将5μg/mL的Fp半乳糖胺酶在具有不同pH范围(5.8-8.0)和200μM的GalN-α-pNP的100μL 50mM的磷酸钠缓冲液中孵育。由pNP释放产生的吸收(在405nm处)通过Synergy H1TM读板器(BioTekTM)于37℃下监测1h。
将5μg/mL的FpGalNAc脱乙酰酶和50μM的亚型I A抗原五-MU在具有不同pH范围(5.8-10.0)的25mM的磷酸钠缓冲液中于37℃下预孵育10min。用100mM的磷酸钠缓冲液(pH7.5)、100μM的EDTA、5μg/mL的Fp半乳糖胺酶、50μg/mL的SpHex、50μg/mL的AfcA和50μg/mL的BgaC(最终体积为100μl)淬灭反应。由MU通过水解释放产生的荧光信号(365/435nm)通过Synergy H1TM读板器(BioTekTM)于37℃下监测30min。
蛋白稳定性
将FpGalNAc脱乙酰酶和FpGalNase在1×PBS缓冲液(pH7.4)中于4℃下存储。在2周和12周后，如针对FpGalNAc脱乙酰基酶的偶联酶反应中的亚型I A抗原五-MU和针对FpGalNase的偶联酶反应中的GalN-α-pNP的PH最佳条件所述的，测试酶的活性。
FpGalNAc脱乙酰酶的抑制
在96孔板形式中，针对不同的潜在抑制剂对FpGalNAc脱乙酰酶进行测试，作为偶联测定。反应在37℃下以100μL的规模进行，其中在具有10μg/mL的Fp半乳糖胺酶、50μg/mL的SpHex、50μg/mL的AfcA、50μg/mL的BgaC的100mM的NaH2PO4(pH7.4)中的50μM的亚型1A抗原五-MU和5μg/mL的FpGalNAc脱乙酰酶。作为抑制剂，测试了EDTA(1、10、100μM)、马立马司他(1、10、100、1000μM)、DMSO(2％、4％)、无EDTA的蛋白酶抑制剂混合物(PierceTM)(1×、2×和4×)。使用Synergy H1TM读板器(BioTekTM)连续监测荧光(365/435nm)1小时。在没有偶联酶的情况下再次运行显示强效果的添加剂，并经由TLC分析产物形成。
有限的蛋白水解作用
为了研究是否存在Fp半乳糖胺酶的较小的、稳定的亚结构域，进行了有限的蛋白水解作用。在多种温度(20℃、37℃、42℃、50℃和65℃)下用嗜热菌蛋白酶(10:1的蛋白质：蛋白酶的质量比)处理Fp半乳糖胺酶1.5hr。然后将样品在SDS-PAGE凝胶上运行，并且鉴定运行在约70kDa(从初始118kDa下降)的稳定的片段，其中在50℃孵育温度下实现几乎完全的消化。将该片段送至UBC蛋白质组核心设施进行肽鉴定，并确定为在氨基酸690-700之间具有切割位点的全长蛋白的C-末端截短形式。
聚糖阵列筛选
对于聚糖阵列筛选，使用FluorotagTM FITC缀合试剂盒(SigmaTM)以1的F/P比用异硫氰酸荧光素(FITC)标记500μg的FpGalNAcDeAc_D2ext。筛选在CFG蛋白-聚糖相互作用核心设施TM(the CFG's Protein-Glycan Interaction Core FacilityTM)中进行的，其中打印阵列为5.3版，由在蛋白质浓度为5μg/mL和50μg/mL的6个重复样品中的600个聚糖组成。结合基序的分析是使用埃默里大学的网络工具(https://glycopattern.emory.edu/)进行的。
缓冲的细胞外溶液中的酶测试
使用包含纯化的GalNAc脱乙酰基酶(SEQ ID NO:5)和纯化的半乳糖胺酶(SEQ IDNO:10)的组合物，于37℃、37℃和4℃下，分别测试与缓冲的细胞外溶液PBS、SteenTM和PerfadexTM的相容性。在PBS、SteenTM和PerfadexTM中以不同剂量的酶组合物孵育人A型红细胞(RBC)，以确定酶从红血细胞中切割A抗原的能力。用在PBS、SteenTM和PerfadexTM溶液中的各种剂量的酶处理1％的RBC溶液，并通过流式细胞仪分析处理结束时的抗原去除水平。
动脉活检的免疫组织化学分析
为了测试包含纯化的GalNAc脱乙酰基酶(SEQ ID NO:5)和纯化的半乳糖胺酶(SEQID NO:10)的酶组合物的剂量递增效应，以测试STEENTM溶液中的A型人动脉，通过对获自未经处理的(对照)、经处理的(处理)的A型动脉和作为阴性对照的O型动脉的活检组织进行免疫组织化学分析，对A型抗原的百分比进行定量。使用面积定量软件并使用以下公式针对对照组进行标准化：
Figure BDA0003020590770000371
在O型组中定量的残留A型抗原阳性水平可解释处理过程中出现的伪影。
在人肺动脉中测试了对人动脉的酶促处理(静态处理)。所涉及的剂量被制备为相对于STEENTM溶液体积的酶重量的单位。用CD31(对内皮细胞染色呈阳性)和BTA(对A血型抗原染色呈阳性)的双重染色，通过免疫组织化学对动脉进行活检、处理和分析。在人动脉上以1μg/mL和10μg/mL进行了4小时的酶促处理。不用酶处理的动脉(对照)和用酶处理的动脉(处理)的20×放大倍数的动脉活检组织免疫组织化学染色图像。CD31显示内皮细胞(血管)的位置，而BTA显示A型血抗原的位置。未处理的动脉中的BTA与内皮细胞(CD31阳性)共定位，而处理过的动脉中不存在BTA。
人供体肺研究
1小时酶促处理对离体灌注的人供体肺的影响，使用肺组织活检的免疫组织化学分析和面积定量软件对A型抗原的表达水平进行定量，并使用以下公式针对预处理的活检组织进行标准化：
Figure BDA0003020590770000372
测试了1小时和3小时酶促处理(即包含纯化的GalNAc脱乙酰基酶(SEQ ID NO:5)和纯化的半乳糖胺酶(SEQ ID NO:10)的酶组合物)对离体灌注的人供体肺的影响。将活检的人供体肺的免疫组织化学染色以20×的放大倍数成像，以确定用酶组合物处理的肺的效果。CD31显示了内皮细胞(血管)的位置；BTA显示A型血抗原的位置。预处理图像显示血型抗原位于血管和气道内。在后处理图像中，肺的右上依赖性(RUD)区域、右上非依赖性(RUND)、右中非依赖性(RMND)区域、右中依赖性(RMD)区域、右下非依赖性(RLND)区域和右下依赖性(RLD)区域，A血型抗原在血管中不存在。
在该研究中测试了两个单独的离体灌注的人供体肺，并且结果显示在图10和图11中，分别为1hr和3hr。
实施例
实施例1：宏基因组文库构建和筛选
我们构建了宏基因组文库，其含有从AB+血型的男性供体提供的排泄物样品中提取的大(35-65kb)DNA片段。此类文库每种细菌含有多个基因，这增加了这些基因中至少一些基因表达的可能性，并允许多个基因的小“途径”的表达。我们的文库在51×384孔板中包含～19,500个克隆，可能约800,000个基因，因此具有昂贵的A抗原底物的此类文库的初始筛选是不切实际的。相反，我们首先用简单、灵敏的荧光底物-半乳糖和N-乙酰基-半乳糖胺的甲基伞形酮α-糖苷(Gal-α-MU和GalNAc-α-MU)进行筛选。该初始筛选与两种底物的混合物一起产生226次命中的子集。针对每种单独的底物重新筛选这些底物，用GalNACase鉴定44个和用半乳糖苷酶活性鉴定166个。使用图1中所示的A抗原和B抗原四糖糖苷底物，使用偶联酶测定(Kwan 2015)，以及无底物对照，对这些命中进行第二轮筛选：只有当初始Gal或GalNAc被切割时，偶联酶才能起作用并释放MU。这些命中的十一个含有A抗原切割活性，其中一个也切割B抗原，而六个在不存在底物的情况下产生荧光，因此编码产生不相关荧光产物的途径。
实施例2：命中的测序和初始分析
在Illumina MiSeqTM对十一种fosmid进行测序，并且使用MetapathwayTM软件(Konwar 2015)鉴定其中存在于CAZyTM数据库(http://www.cazy.org/)(Lombard 2014)中的ORF。由于现在可获得的人类微生物组测序的深度相当大，可以鉴定所有fosmid所来源的生物体。它们的序列可以被分成五个簇，因为十一个中的八个来源于仅两个拟杆菌(Bacteroides sp.)的基因组的重叠片段。B簇中所有fosmid共有的唯一基因是GH109酶(普通拟杆菌(B.vulgatus))；簇A还含有GH109(粪便拟杆菌(B.stercoris))，而GH109是在其它拟杆菌属来源的fosmid(普通拟杆菌)中发现的唯一CAZy基因。来自专性厌氧菌普氏梭杆菌(Li 2015)的Fosmid No8含有在CAZy中发现的三种ORF：表观碳水化合物结合模块CBM32和两种潜在的糖苷水解酶-GH36和GH4。最后来自柯林斯氏菌(Collinsella sp.)(可能为Collinsella tanakaei)的fosmid K05不含有CAZy相关ORF。这里，fosmid K05的子文库的产生允许鉴定具有A切割活性的ORF，随后被鉴定为GH36(未显示)。
实施例3：GH109酶的分析
GH109家族是基于其几个成员的A抗原切割活性而建立的。这些酶采用不寻常的NAD+依赖性机制，首先在来自GH4 Add Yip Ref(2004)J.Amer.Chem.Soc.,126,8354-8355的酶中发现，因为这是显示该机制的那一个(Varrot 2005；和Liu 2007)。去除信号肽后，用His标签克隆此处鉴定的三个GH109基因，并在大肠杆菌(Escherichia coli)BL21(DE3)中表达。纯化这三种蛋白质(BsGH109、BvGH109_1和BvGH109_2)(未显示)，以及来自脑膜脓毒性菌(Elizabethkingia menosepticum)的经典GH109(EmGH109)(Liu 2007)作为标准品，并确定每种蛋白的动力学参数。这三种新的酶在测试的三种A亚型底物的每一种中都展示出相似的催化效率，这在很大程度上反映了EmGH109标准品的动力学参数。相比之下，当使用经认可的MTS卡在A+RBC上测试它们的A抗原去除活性时，令人失望的是，只有EmGH109是显著有活性的。在作为拥挤剂的右旋糖酐40K存在下进行测试，我们已经显示通过在细胞表面上浓缩酶来增加活性(Chapanian 2014)。在不存在时，即使是150μg/mL的EmGH109也是无效的，而在300mg/mL右旋糖酐40K的存在下，15μg/mL的酶是足够的(参见图3和图4)。先前的研究表明，低离子强度也提高了EmGH109对细胞的活性(Liu 2007)。因此，EmGH109在全血中是无效的。
实施例4：分析来自柯林斯氏菌的Fosmid K05的GH36
Fosmid K05(命名为K05GH36)中经鉴定的GH36蛋白对GalNAc-α-MU和A抗原四糖具有活性。这与GH36家族的成员一致，其主要含有α-半乳糖苷酶和α-N-乙酰基半乳糖胺酶，并且经由涉及共价β-糖基酶中间体的双置换机制进行水解(Comfort 2007)。系统发育分析将其序列比对在GH36亚家族的第4簇内(Fredslund 2011)。有趣的是，这种簇还含有非常接近的来自产气荚膜梭状芽孢杆菌(Clostridium perfringens)的特征性GH36，其也是已知切割A抗原结构(Calcutt 2002)的。然而，当我们测试K05GH36从红血细胞中去除A抗原的能力时，它的活性令人失望，即使当与拥挤剂联合使用时，评分也仅为3。
实施例5：来自普氏梭杆菌的Fosmid No8的分析
由于这些新的酶没有提供优势，我们的注意力转向来自普氏梭杆菌(F.plautii)的No8 fosmid，尤其是因为它的基因产物切割A抗原和B抗原。克隆了三个CAZy相关基因，去除它们的信号肽序列，在大肠杆菌BL21(DE3)中表达，并纯化了所得酶，产量高达140mg/L。令人惊讶地，当我们测试针对A和B四糖底物的单独纯化的蛋白时，观察到的唯一切割是由No8GH36对B抗原的切割，而它们中的任何一个没有切割A抗原。因此，我们成对测试了这些酶的组合，并且令人惊奇地发现No8CBM32和No8GH36的混合物快速切割了A抗原四糖。用单独的酶对反应混合物进行的TLC分析表明，No8CBM32催化A抗原转变成极性更强但仍具有UV活性的产物，而随后加入No8GH36释放与半乳糖胺共迁移的糖产物以及H抗原三糖。反应混合物的MS分析表明，No8CBM32是A抗原脱乙酰酶，因此m/z降低42，并且极性更强，而No8GH36是半乳糖胺酶，是这种家族的新活性(图2)。这通过反应的高效阴离子交换色谱(HPAE-PAD)分析进一步被证实(图5)，其显示了用两种酶处理A抗原释放半乳糖胺，而单独的酶不释放。用胃粘蛋白底物获得了类似的结果，对于胃粘蛋白底物，推测该酶释放了半乳糖胺。因此，这两种酶此后被称为FpGalNAc脱乙酰酶(FpGalNAcDeAc)和Fp半乳糖胺酶(FpGalNase)。
虽然以前未表征过降解A抗原的这种途径，但令人着迷的是，在50多年前就提出了一种解释，以解释所谓的“获得性”B现象，其中感染了第三梭状芽胞杆菌的A型患者的血液类型明显变为B型(Gerbal 1975)，就像淹没在泰晤士河中的人体组织法医样品一样(RefJudd和Annesley https://doi.org/10.1016/S0887-7963(96)80087-3,Transfusionmedicine reviews(1996)10,111-117)。这大概是因为用于分型的抗B抗体不能区分末端Gal和GalN。
对fosmid GH4中的第三种酶的研究表明，尽管它水解Gal-α-pNP、GalN-α-pNP和GlcN-α-pNP，但它不切割任何基于A抗原的底物。因此，似乎在A抗原的转变中不直接发挥作用。然而，这些糖胺酶确实代表GH4家族内的新活性。
实施例6：FpGalNAc脱乙酰酶的表征
用Phyre2TM(Kelley 2015)对该基因进行的更密切的生物信息学分析表明，在N-末端具有先前未知功能的～308个氨基酸的结构域，并且在C-末端附近具有～145个氨基酸的CBM32，在它们之间具有连接子区域。截短分析证实了这种基本结构，因为含有完整脱乙酰酶结构域的所有构建体确实具有催化活性(表2)。因此，这种蛋白质被分类为新的碳水化合物酯酶家族CExx的创始成员。
乙酰胺糖脱乙酰酶已经被证明都是需要二价金属离子的金属酶(Blair 2005)。与此相应，用100μM的EDTA处理大部分消除了酶活性，而添加Mn2+、Co2+、Ni2+或Zn2+增加了酶活性。其它(非金属)酰胺酶抑制剂没有效果。该酶具有较宽的pH分布，在pH 8附近具有最佳值(图6)，并且底物特异性窄，仅限于不同的A亚型及其较短的形式。但是，在那些亚型中，它不是非常有辨识能力的，所有这些亚型之间的比活性仅相差～2倍(表2)。这种pH依赖性和特异性特征对于RBC转变是理想的，因为A的所有亚型都是脱乙酰化的，但没有其它的。
使用功能糖原组学联盟(the Consortium for Functional Glycomics，CFG)的聚糖阵列来探索蛋白质的CBM部分的特异性。优选的靶标是具有重复的N-乙酰基乳糖胺(LacNAc)结构的聚糖；其在CBM32家族的创始成员中；产气荚膜梭状芽胞杆菌的N-乙酰基葡糖胺酶中也可以看到(Ficko-Blean 2006)。然而，与该CBM不同，我们的没有显示出与血液抗原结构的高亲和力结合。重复LacNAc结构是细胞表面的常见组分(Cohen 2009)，作为复杂和杂合N-聚糖以及一些O-聚糖和糖脂的通用组分。在我们的情况下，它们可能用作连接脱乙酰酶结构域的锚点。这将使其催化结构域非常接近A抗原而不竞争其自身的底物。在该模型的支持下，结构域的去除导致RBC活性降低，而对可溶性底物的切割速率没有影响(表2)。
实施例7：FpGalNAc脱乙酰酶的结晶分析
为了提供对这种新的酶活性的结构洞察，对截短的蛋白质进行结晶试验，发现FpGalNAcDeAc_D1ext产生衍射至最佳分辨率的晶体。这种结构的溶液揭示了采用5倍β螺旋桨结构的催化结构域，其具有含有由D100和H252配位的二价金属离子的活性位点。酶与作为反应产物的接近类似物的B抗原三糖的共结晶揭示了其结合模式。在活性位点口袋的基础上，非还原性末端半乳糖基部分作为A抗原和B抗原的区别基团，与H97、E64和两种金属配位水形成氢键相互作用。配体的其余部分是表面暴露的，并且在岩藻糖基与S61和D121侧链之间确定了极性相互作用。还原末端半乳糖基部分的C1-OH基团是溶剂暴露的，因此酶易于适应底物的延伸(即用GlcNAc)。在该结构上模拟A-三糖的N-乙酰基允许我们对可能参与底物脱乙酰化的附近氨基酸进行合理的突变。由于两个突变体都是无活性的，因此残基E64被证明对于活性是关键的，这表明可能在亲核水分子的活化中的直接作用(表1)。配位二价金属的残基D100、Y315和H252也被证明是重要的，其中任何突变导致～5000倍的速率降低，与它们在结合二价金属离子中的表观作用一致。与其它乙酰氨基糖脱乙酰酶类似，我们提出FpGalNAc脱乙酰酶通过以下机理进行水解，其中金属用于极化羰基并活化水分子以亲核攻击羰基来形成四面体中间体。通过His 100对糖氮原子的质子供给促进该中间体的分解。
表1|FpGalNAcDeAc_D1min及其突变体切割2型A抗原四-MU的比活性
Figure BDA0003020590770000431
N.D.＝无可检测的活性
实施例8：FpGalNAcDeAc和FpGalNase的表征
序列的系统发育分析将FpGalNase置于GH36家族的一个新亚组(5)中(Fredslund2011)。390个氨基酸的催化结构域位于该大(1079个氨基酸)蛋白的中心，其中在C-末端具有潜在的碳水化合物结合结构域。去除该C-末端结构域对酶与可溶性底物的动力学参数没有影响(表2)，但导致去乙酰化的A+RBC的切割效率降低。该酶对含半乳糖胺的糖是特异性的，并且在测试的任何上下文中都将不会切割GalNAc残基。然而，它对从简单的芳基糖苷GalN-α-pNP向上的脱-N-乙酰化半乳糖胺的切割具有相当广泛的特异性。实际上(表2)测试的三种A亚型的kcat/KM值彼此以及与脱乙酰酶的那些kcat/KM值都相似。B抗原切割的kcat/KM值比相应的GalN抗原低2000倍以上，但仍然足以在原始筛选中产生阳性命中。这种对脱乙酰化的α半乳糖构型底物的特异性与其～6.5-7.0的pH最佳值相结合，很好地适用于与脱乙酰酶结合的血型转变(图6)。
表2|不同抗原底物的FpGalNAcDeAc和FpGalNase构建体的动力学参数
Figure BDA0003020590770000441
实施例9：从RBC切割A抗原
A+、B+和O+型RBC单独与FpGalNAcDeAc和FpGalNase孵育，并以混合物的形式，以及在HPAE-PAD离子色谱图上分析释放的糖。所用的酶均不单独释放任何糖产物。然而，当使用两者的混合物时，半乳糖胺明显地从A+型RBC释放，而不是从B+或O+释放，从而仅对A抗原具有高特异性。这是非常重要的，因为它显示GalNAc在任何其它情况下不从RBC表面释放。FpGalNase的截短形式也是有效的，但活性稍低。
然后，我们继续使用工业标准MTSTM卡测试从RBC去除抗原。将这些抗体缀合的柱加载有RBC，并在离心机中旋转。无抗原RBC迁移到柱的底部并评分为0，而未处理的RBC在顶部带有相应的抗原棒并评分为4，中间评分则对抗原去除的程度进行了排名。单独用FpGalNase处理在所采用的浓度下不能去除A或B抗原性(表3)，这与其对GalNAc底物的无活性和对Gal的低活性一致。与FpGalNAcDeAc孵育去除了由于乙酰胺转变成胺而导致的抗原性，从而损害了所采用的抗A抗体的结合。在不存在和存在300mg/ml的右旋糖酐作为拥挤剂的情况下，评估了单独的FpGalNAcDeAc和与FpGalNase组合的完全抗原脱乙酰化所需的酶的最小量。没有来自右旋糖酐的辅助，降至3μg/ml的FpGalNase的量是足够的，而包含300mg/ml的右旋糖酐将所需的加载降低至0.5μg/ml(表3)。通过比较最好的先前的酶，EmGH109在不存在右旋糖酐的情况下是无效的，除非采用低盐缓冲液，而在右旋糖酐的存在下最小有效浓度是15μg/ml(高30倍的加载)。缺少CBM的FpGalNAcDeAc形式效率低得多。
表3|用EmGH109、FpGalNAcDeAc和FpGalNase处理A+、B+和AB+RBC的MTS卡结果。
Figure BDA0003020590770000451
由于对MTSTM卡测试没有评估A抗原的完全转变，并且由于没有抗体可用于检测GalN抗原，因此我们关注于在处理的RBC上检测新形成的H抗原。FpGalNase在仅5μg/ml的浓度下是功能性的，这导致与A抗原损失相一致的H抗原水平的增加，如图3中所见的FACS分析所证实的。通过在抗H-抗体的存在下测量凝集时间，我们证明了两种酶对于几种A+RBC供体的功能性，在全血反应条件下也是如此，这是以前其他血液转变酶所无法实现的。因此，这对酶使用比以前最好的酶所需的低得多的酶加载量将A+RBC转变为O-型“通用供体”RBC。然而，在将这些RBC输送到患者体内之前，建议去除转变中所用的所有痕量酶以避免不利的免疫应答，最建议的是通过离心后洗涤细胞。为了证实这是可以实现的，我们用荧光标记的FpGalNAcDeAc和FpGalNase样品处理A+RBC，然后使用FACS分析证实确实简单的洗涤是有效的(图3)。
对所产生的A-ECO RBC的进一步表征可用于评估它们在输血药物中使用的完全存活力，但是可能在收集献血时将酶直接包括在血浆中的可能性可以允许容易、经济高效地将过程从血液收集和存储的现有自动化程序中分离出来。具体而言，如表4中所示的测试酶的稳定性。
表4：半乳糖胺酶和GalNAc脱乙酰酶的存储稳定性
Figure BDA0003020590770000461
实施例10：来自第三梭状芽孢杆菌的GalNac脱乙酰酶和半乳糖胺酶的融合体
在寻找类似的酶时，鉴定了通过CBM(GH36_结构域-CBM-脱乙酰化_结构域)连接的半乳糖胺酶和GalNAc脱乙酰酶的新的第三梭状芽孢杆菌的天然融合体。最初的测试显示该酶切割红血细胞的A抗原(相同的机制，首先是脱乙酰化，然后是半乳糖胺切割)，但效率不高(即类似于EmGH109)。第三梭状芽孢杆菌的脱乙酰化结构域不如普氏梭杆菌的GalNAc脱乙酰化酶有效，但是如果用普氏梭杆菌的GalNAc脱乙酰化酶补足，则第三梭状芽孢杆菌的半乳糖胺酶结构域在红血细胞上显示出与普氏梭杆菌的半乳糖胺酶相似的活性。
实施例11：可替代的GalNAc脱乙酰酶和半乳糖胺酶
数据显示，第三梭状芽孢杆菌的半乳糖胺糖苷酶(Ct5757_GalNAse)和Rp1021对于GalN抗原向H抗原的转变(第二反应步骤)确实具有相当的酶活性。
还收集了可替代的GalNAc脱乙酰酶和半乳糖胺酶的数据，并将可替代的酶与普氏梭杆菌的GalNAc脱乙酰酶和普氏梭杆菌的半乳糖胺酶进行比较。如表5所示的，显示了半乳糖苷酶和GalNAc脱乙酰酶的第三梭状芽孢杆菌的天然融合体的抗A抗体在经处理的A RBC上的MTS评分，这需要右旋糖酐的存在才能有效地切割A抗原，并且当与普氏梭杆菌的半乳糖胺酶(FpGalNase)组合时还显示出第三梭状芽孢杆菌的GalNAc脱乙酰酶(Ct5757_DeAcase)的良好的活性。同样在表6中，数据显示了Robinsoniella peoriensis(Rp)的Rp3672和Rp3671能够使RBC上的A抗原脱乙酰化，但效率低于FpGalNAcDeAase，并且仅在存在拥挤剂(即右旋糖酐40k)时才实现活性。
表5：经处理的A RBC上的抗A抗体的MTS评分
Figure BDA0003020590770000471
表6：Robinsoniella peoriensis(Rp)的3671和3672的MTS评分
样品 抗A MTS评分
A RBC对照 4
Rp3671(50μg/mL)+右旋糖酐40k 3
Rp3672(50μg/mL)+右旋糖酐40k 1
图7显示了对于(A)A+RBC对照、(B)普氏梭杆菌的GalNAc脱乙酰酶(FpGalNAcDeAc)+普氏梭杆菌的半乳糖胺酶(FpGalNase)(10μg/mL)、(C)FpGalNAcDeAc+第三梭状芽胞杆菌(Ct)的Ct57577_GalNase(10ug/mL)和(D)FpGalNAcDeAc+Robinsoniella peoriensis(Rp)的半乳糖胺酶(Rp1021)GalNase(10ug/mL)，经由FACS分选分析的A RBC上的A抗原向H抗原的转变。数据表明，第三梭状芽孢杆菌(Ct)Ct5757_GalNase和Robinsoniella peoriensis(Rp)的半乳糖胺酶(Rp1021)GalNase具有与用于将GalN抗原转变为H抗原的普氏梭杆菌的半乳糖胺酶(FpGalNase)相当的酶活性(第二反应步骤)。
实施例12：酶组合物与灌注/保存流体的相容性
为了确保酶组合物与EVLP系统相容，我们首先测试了在器官灌注/保存流体中(STEENTM和PerfadexTM，XVIVO灌注)中的酶(SEQ ID NO:5的纯化的普氏梭杆菌的GalNAc脱乙酰化酶蛋白和SEQ ID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白)的功能。根据酶组合物在37℃下的STEENTM或在4℃下的PerfadexTM中的去除红细胞上的A型血抗原的能力来评估相容性。将37℃的磷酸盐缓冲盐水(PBS)用作比较组，因为PBS是用于血液处理的标准溶液之一。针对STEENTM和PerfadexTM的温度研究是基于其在临床实践中的工作温度。通过流式细胞术分析抗原去除的水平。在STEENTM和PerfadexTM中进行了剂量递增研究，以帮助预测要在器官中使用的适当剂量(参见图8)。整个研究中使用的剂量单位被限定为相对于溶液的体积(mL)的酶的重量(μg)。
显示了酶组合物与STEENTM和PerfadexTM灌注/保存流体完全相容，与PBS相比，灌注/保存流体提高了酶组合物的效率。该酶组合物能够在1μg/mL的总酶浓度下去除STEENTM和PerfadexTM中90％以上的抗原，而在4μg/mL的剂量下在PBS中也能达到相同的效果(图8)。
实施例13：人动脉的静态处理
为了测试所述酶(SEQ ID NO:5的纯化的普氏梭杆菌的GalNAc脱乙酰酶蛋白和SEQID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白)在组织水平上的功效，使用了人动脉的体外模型。将来自同一个人供体的肺动脉分为对照组(STEENTM溶液)和处理组(酶组合物+STEENTM溶液)，并在37℃下静态孵育4小时。两组均在孵育结束时进行活检。酶组合物的剂量分别为1μg/mL和10μg/mL。通过免疫组织化学分析血型抗原的变化。活检的连续切片用CD31(内皮细胞的标志物)以显示血管内表面的位置和用BTA以显示血型抗原的表达的双重染色。
与对照组相比，处理组中A型血抗原的表达水平显著降低。1μg/mL和10μg/mL的剂量效应与经处理的动脉相似。总酶浓度(剂量)低于1μg/mL时，酶也可能起作用。当比较BTA与CD31的染色图像时，确认了血型抗原的消失(图9)。
实施例14：人肺的离体灌注
在多伦多EVLP设置下测试了含酶的STEENTM溶液在去除人体器官(例如，肺)中的组织血型抗原上的功效。用临床离体肺灌注(EVLP)对供体人的肺进行评估，并确定其不适合移植，因此适合测试酶组合物。肺功能下降后，将酶组合物(SEQ ID NO:5的纯化的普氏梭杆菌的GalNAc脱乙酰酶蛋白和SEQ ID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白)添加到STEENTM灌注液中以开始处理。使用的剂量为1μg/mL。在处理之前和之后进行活检。通过免疫组织化学分析血型抗原表达的变化。在整个实验过程中，每小时都要对肺的功能和生理进行监测，以确保处理不会引起急性副作用。
对于人的肺，单肺EVLP所需的灌注液体积为1.5L，双肺EVLP所需的灌注液体积为2L。在第一测试(图10)中，对于单个右肺EVLP，1.5mg的酶组合物添加到灌注液中，以达到1μg/mL的剂量。肺被处理一(1)小时。免疫组织化学分析显示，处理后A型血抗原水平显著降低(图10)。对预处理的活检切片进行了比较，该切片对血型抗原和血管进行了双重染色，揭示了肺中的抗原不仅位于血管壁表面，而且位于气道中。双重染色后活检的比较表明，血管内抗原已被有效去除。
在第二测试中(图11)，在STEENTM灌注流体中用1.5mg酶组合物以达到1μg/mL的浓度，来处理另一只右肺EVLP。肺处理了三(3)小时。免疫组织化学分析显示，A型血抗原的表达水平显著降低。比较对血型抗原和血管进行双重染色的预处理活检，揭示了肺中的血型抗原不仅位于血管表面，而且位于气道中(图11)。对双重染色的处理后活检的比较表明，血管内抗原已被有效去除(图11)。酶促处理开始后未观察到肺生理和功能方面的急性副作用。
结果显示，在1μg/mL的剂量下，酶在一小时内在灌注的的人肺中起作用。
尽管本文公开了本发明的各种实施方案，但是根据本领域技术人员的公知常识，可以在本发明的范围内进行许多改编和修改。此类修改包括用已知等价物替代本发明的任何方面，以便以基本上相同的方式获得相同的结果。数字范围包括定义该范围的数字。词语“包含(comprising)”在本文中用作开放式术语，其基本上等同于短语“包括但不限于”，并且词语“包含(comprise)”具有相应的含义。如本文所用的，单数形式“一个/种(a/an)”和“所述(the)”包括复数指示物，除非上下文另有明确规定。因此，例如，提及“事物”包括多于一个这样的事物。本文引用的参考文献不是承认这些参考文献是本发明实施方案的现有技术。本发明包括基本上如上文所述并参考实施例和附图的所有实施方案和变型。
序列
从天然存在的DNA序列(GalNAc脱乙酰酶2311/2319nt/半乳糖胺酶3228/3237nt)修饰普氏梭杆菌的DNA序列。特别地，用于蛋白质纯化的序列的长度存在差异，由此除去信号肽并通过载体骨架添加N-末端His标签。
非正式序列表
SEQ ID NO：2
描述:普氏梭杆菌的GalNAc脱乙酰酶(蛋白序列)
MRNRRKAVSLLTGLLVTAQLFPTAALAADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE
SEQ ID NO：4
描述:普氏梭杆菌的GalNAc脱乙酰酶(去除信号肽的蛋白序列)
ADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE
SEQ ID NO：5
描述:具有His标签的普氏梭杆菌的GalNAc脱乙酰酶(pET16a-蛋白序列)
MGHHHHHHHHHHSSGADSSESALNKAPGYQDFPAYYSDSAHADDQVTHPDVVVLEEPWNGYRYWAVYTPNVMRISIYENPSIVASSDGVHWVEPEGLSNPIEPQPPSTRYHNCDADMVYNAEYDAMMAYWNWADDQGGGVGAEVRLRISYDGVHWGVPVTYDEMTRVWSKPTSDAERQVADGEDDFITAIASPDRYDMLSPTIVYDDFRDVFILWANNTGDVGYQNGQANFVEMRYSDDGITWGEPVRVNGFLGLDENGQQLAPWHQDVQYVPDLKEFVCISQCFAGRNPDGSVLHLTTSKDGVNWEQVGTKPLLSPGPDGSWDDFQIYRSSFYYEPGSSAGDGTMRVWYSALQKDTNNKMVADSSGNLTIQAKSEDDRIWRIGYAENSFVEMMRVLLDDPGYTTPALVSGNSLMLSAETTSLPTGDVMKLETSFAPVDTSDQVVKYTSSDPDVATVDEFGTITGVSVGSARIMAETREGLSDDLEIAVVENPYTLIPQSNMTATATSVYGGTTEGPASNVLDGNVRTIWHTNYAPKDELPQSITVSFDQPYTVGRFVYTPRQNGTNGIISEYELYAIHQDGSKDLVASGSDWALDAKDKTVSFAPVEAVGLELKAIAGAGGFGTAAELNVYAYGPIEPAPVYVPVDDRDASLVFTGAWNSDSNGSFYEGTARYTNEIGASVEFTFVGTAIRWYGQNDVNFGAAEVYVDGVLAGEVNVYGPAAAQQLLFEADGLAYGKHTIRIVCVSPVVDFDYFSYVGE
SEQ ID NO：7
描述:普氏梭杆菌的半乳糖胺酶
MRGKKFISLTLSTMLCLQLLPTASFAAAPATDTGNAGLIAEGDYAIAGNGVRVTYDADGQTITLYRTEGSGLIQMSKPSPLGGPVIGGQEVQDFSHISCDVEQSTSGVMGSGQRMTITSQSMSTGLIRTYVLETSDIEEGVVYTATSYEAGASDVEVSWFIGSVYELYGAEDRIWSYNGGGEGPMHYYDTLQKIDLTDSGKFSRENKQDDTAASIPVSDIYIADGGITVGDASATRREVHTPVQETSDSAQVSIGWPGKVIAAGSVIEIGESFAVVHPGDYYNGLRGYKNAMDHLGVIMPAPGDIPDSSYDLRWESWGWGFNWTIDLIIGKLDELQAAGVKQITLDDGWYTNAGDWALNPEKFPNGASDALRLTDAIHEHGMTALLWWRPCDGGIDSILYQQHPEYFVMDADGRPARLPTPGGGTNPSLGYALCPMADGAIASQVDFVNRAMNDWGFDGFKGDYVWSMPECYNPAHNHASPEESTEKQSEIYRVSYEAMVANDPNVFNLLCNCGTPQDYYSLPYMTQIATADPTSVDQTRRRVKAYKALMGDYFPVTADHNNIWYPSAVGTGSVLIEKRDLSGTAKEEYEKWLGIADTVQLQKGRFIGDLYSYGFDPYETYVVEKDGVMYYAFYKDGSKYSPTGYPDIELKGLDPNKMYRIVDYVNDRVVATNLMGDNAVFNTRFSDYLLVKAVEISEPDPEPVDPDYGFTSVDDRDEALIYTGTWHDDNNASFSEGTARYTNSTDASVVFSFTGTSIRWYGQRDTNFGTAEVYLDDELKTTVDANGAAEAGVCLFEALDLPAAEHTIKIVCKSGVIDIDRFAYEAATLEPIYEKVDALSDRITYVGNWEEYHNSEFYMGNAMRTDEAGAYAELTFRGTAVRLYAEMSFNFGTADVYLDGELVENIILYGQEATGQLMFERTGLEEGEHTIRLVQNAWNINLDYISYLPEQDQPTPPETTVTVDAMDAQLVYTGVWNDDYHDVFQEGTARYASSAGASVEFEFTGSEIRWYGQNDSNFGVASVYIDNEFVQQVNVNGAAAVGKLLFQKADLPAGSHTIRIVCDTPVIDLDYLTYTTNA
SEQ ID NO：9
描述:普氏梭杆菌的半乳糖胺酶(去除信号肽的蛋白序列)
AAPATDTGNAGLIAEGDYAIAGNGVRVTYDADGQTITLYRTEGSGLIQMSKPSPLGGPVIGGQEVQDFSHISCDVEQSTSGVMGSGQRMTITSQSMSTGLIRTYVLETSDIEEGVVYTATSYEAGASDVEVSWFIGSVYELYGAEDRIWSYNGGGEGPMHYYDTLQKIDLTDSGKFSRENKQDDTAASIPVSDIYIADGGITVGDASATRREVHTPVQETSDSAQVSIGWPGKVIAAGSVIEIGESFAVVHPGDYYNGLRGYKNAMDHLGVIMPAPGDIPDSSYDLRWESWGWGFNWTIDLIIGKLDELQAAGVKQITLDDGWYTNAGDWALNPEKFPNGASDALRLTDAIHEHGMTALLWWRPCDGGIDSILYQQHPEYFVMDADGRPARLPTPGGGTNPSLGYALCPMADGAIASQVDFVNRAMNDWGFDGFKGDYVWSMPECYNPAHNHASPEESTEKQSEIYRVSYEAMVANDPNVFNLLCNCGTPQDYYSLPYMTQIATADPTSVDQTRRRVKAYKALMGDYFPVTADHNNIWYPSAVGTGSVLIEKRDLSGTAKEEYEKWLGIADTVQLQKGRFIGDLYSYGFDPYETYVVEKDGVMYYAFYKDGSKYSPTGYPDIELKGLDPNKMYRIVDYVNDRVVATNLMGDNAVFNTRFSDYLLVKAVEISEPDPEPVDPDYGFTSVDDRDEALIYTGTWHDDNNASFSEGTARYTNSTDASVVFSFTGTSIRWYGQRDTNFGTAEVYLDDELKTTVDANGAAEAGVCLFEALDLPAAEHTIKIVCKSGVIDIDRFAYEAATLEPIYEKVDALSDRITYVGNWEEYHNSEFYMGNAMRTDEAGAYAELTFRGTAVRLYAEMSFNFGTADVYLDGELVENIILYGQEATGQLMFERTGLEEGEHTIRLVQNAWNINLDYISYLPEQDQPTPPETTVTVDAMDAQLVYTGVWNDDYHDVFQEGTARYASSAGASVEFEFTGSEIRWYGQNDSNFGVASVYIDNEFVQQVNVNGAAAVGKLLFQKADLPAGSHTIRIVCDTPVIDLDYLTYTTNA
SEQ ID NO：10
描述:具有His标签的普氏梭杆菌的半乳糖胺酶(pET16a-蛋白序列)
MGHHHHHHHHHHSSGAAPATDTGNAGLIAEGDYAIAGNGVRVTYDADGQTITLYRTEGSGLIQMSKPSPLGGPVIGGQEVQDFSHISCDVEQSTSGVMGSGQRMTITSQSMSTGLIRTYVLETSDIEEGVVYTATSYEAGASDVEVSWFIGSVYELYGAEDRIWSYNGGGEGPMHYYDTLQKIDLTDSGKFSRENKQDDTAASIPVSDIYIADGGITVGDASATRREVHTPVQETSDSAQVSIGWPGKVIAAGSVIEIGESFAVVHPGDYYNGLRGYKNAMDHLGVIMPAPGDIPDSSYDLRWESWGWGFNWTIDLIIGKLDELQAAGVKQITLDDGWYTNAGDWALNPEKFPNGASDALRLTDAIHEHGMTALLWWRPCDGGIDSILYQQHPEYFVMDADGRPARLPTPGGGTNPSLGYALCPMADGAIASQVDFVNRAMNDWGFDGFKGDYVWSMPECYNPAHNHASPEESTEKQSEIYRVSYEAMVANDPNVFNLLCNCGTPQDYYSLPYMTQIATADPTSVDQTRRRVKAYKALMGDYFPVTADHNNIWYPSAVGTGSVLIEKRDLSGTAKEEYEKWLGIADTVQLQKGRFIGDLYSYGFDPYETYVVEKDGVMYYAFYKDGSKYSPTGYPDIELKGLDPNKMYRIVDYVNDRVVATNLMGDNAVFNTRFSDYLLVKAVEISEPDPEPVDPDYGFTSVDDRDEALIYTGTWHDDNNASFSEGTARYTNSTDASVVFSFTGTSIRWYGQRDTNFGTAEVYLDDELKTTVDANGAAEAGVCLFEALDLPAAEHTIKIVCKSGVIDIDRFAYEAATLEPIYEKVDALSDRITYVGNWEEYHNSEFYMGNAMRTDEAGAYAELTFRGTAVRLYAEMSFNFGTADVYLDGELVENIILYGQEATGQLMFERTGLEEGEHTIRLVQNAWNINLDYISYLPEQDQPTPPETTVTVDAMDAQLVYTGVWNDDYHDVFQEGTARYASSAGASVEFEFTGSEIRWYGQNDSNFGVASVYIDNEFVQQVNVNGAAAVGKLLFQKADLPAGSHTIRIVCDTPVIDLDYLTYTTNA
SEQ ID NO：12
描述:标识为99345757.1-Ct5757的第三梭状芽胞杆菌分离的蛋白质序列(通过CBM连接的半乳糖苷酶和GalNAcDe乙酰基酶的融合体)(原始蛋白质序列)
MKKRILATFITAMCGLGFFSNWTSSNAYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ
SEQ ID NO：14
描述:具有去除了信号肽的第三梭状芽胞杆菌5757(Ct5757)的分离的蛋白序列(标识为099345757.1-Ct5757)
YNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ
SEQ ID NO：15
描述：具有His标签和凝血酶裂解位点的第三梭状芽胞杆菌5757(Ct5757)的融合蛋白序列表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ
SEQ ID NO：17
描述：具有His标签和凝血酶裂解位点的第三梭状芽胞杆菌5757(Ct5757)的GalNAc脱乙酰酶蛋白序列_表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ
SEQ ID NO：19
描述：具有His标签和凝血酶裂解位点的第三梭状芽胞杆菌5757(Ct5757)蛋白序列半乳糖胺酶_表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK
SEQ ID NO：21
描述：具有His标签和凝血酶裂解位点的Robinsoniella peoriensis Rp1021的半乳糖胺酶蛋白表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHGNGLEVKASPREVAQITGNGVSVTFFQEDGTVQLSCIEDDGNTAFMTRNSEVSYPVVGGEEVTDFSDFQCEVQENVTGAAGAGSRMTITSISSGRGIQRSVVIETVDEVKGLLHISSSYRAEEEVDADEFIDSRFSLDNPSDTVWSYNGGGEGAQSRYDTLQKIDLSDGESFYRENLQNQTAAGIPVADIYGKDGGITVGDASVTRRQLSTPVNERNGTAYVSVKHPGAVITQRETEISQSFVNVHRGDYYSGLRGYADGMKQIGFTTLSREQIPESSYDLRWESWGWEFDWTVELIINKLDELKEMGIKQITLDDGWYNAAGEWGLNNWKLPNGALDMRHLTDAIHERGMTAVLWWRPCDGGREDSALFKEHPEYFIKNQDGSFGKLAGPGQWNSFLGSCGYALCPLSEGAVQSQVDFINRAMNEWGFDGFKSDYVWSLPKCYSQDHHHEYPEESTEQQAVFYRAVYEAMTDNDPNAFHLLCNCGTPQDYYSLPYVTQVPTADPTSVDQTRRRVKAYKALCGDYFPVTTDHNEVWYPSTIGTGAILIEKRDLSGWEEEEYAKWLKIAQENQLHKGTFIGDLYSYGYDPYETYTVYKDGIMYYAFYKDGNRYRPSGNPDIELKGLEDGKLYRIVDYVNNQVVATNVTSSNAVFSYPFSDYLLVKAVEISEPDTDGPGPVPDPEGAVTVEENDPELVYTGDWVREENDGYHGGGARYTKEAEASVELAFYGTGAAWYGQHDVNFGSARIYIDGTYVKTVSCMGEPGINIKLFEISGLDLASHRIKIECETPVIDIDRLTYIKGEEVPAKVMTADLRALTVIANQYDMNSFADGNYKDQLGVSLVRANQLLAADDVTQGAVNEEQKYLLNAMLKIRKKVDKSWIGLPGPIPQDIQTENISRDNLAKVISYTGQLDRDEIIPAIKEQLNDSYDKAVSIAERQDASQPEIDRAWAELMNAVQYSSYIRGSKEELLSLLDEYGKVDTTVYKDAALFIESLEAAKKVYQDENAMDGEISDCIKQLRDAKDQLQLKDPVDPPKPDPDPDPKPDPTPDPGPDPKPDPTPDPTPDPKPNPTPTPDPTPEPALKKPEQVSGLKSKAETDYLTVSWKKLNNAESYKVYIYKSGKWRLAGKTTKTSIKIKKLVSGTKYTVKVAAVNKAGQGKYSSQVYTAAKPKKVKLKSVSRYRTSKVKLNYGKVKAGGYEIWMKNGKGSYKKAATSTKTTAIKSGLKKGKTYYFKVRAYVKNKNQVIYGSFSNIKKYKMVL
SEQ ID NO：23
描述：具有His标签和凝血酶裂解位点的Ruthenibacterium lactatiformansRl8755的GalNAc脱乙酰酶蛋白序列表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTGPNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGTPSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDFVQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER
SEQ ID NO：25
描述：具有His标签和凝血酶裂解位点的Robinsoniella peoriensis Rp3671的GalNAc脱乙酰酶蛋白_表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSRPEVEKRIEVNNENPLLIMPLYGKSYSESGSTLDWGDDLVSRWKQVPEDLKENAVIEIHLGGKIGLNESDSHTAKAFYEQQLAIAQENNIPVMMVVATAGQQNYWTGTANLDAEWIDRMFKQHSVLKGIMSTENYWTDYNKVATMGADYLRVAAENGGYFVWSEHQEGVIENVIANEKFNEALKLYGNNFIFTWKNTPAGTNSNAGTASYMQGLWLTGICAQWGGLADTWKWYEKGFGKLFDGQYSYNPGGEEARPVATEPEALLGIEMMSIYTNGGCVYNFEHPAYVYGSYNQNSPCFENVIAEFMRYAIKNPAPGKEEVLADTKAVFYGKLSSLKSAGNLLQKGLNWEDATLPTQTTGRYGLIPAVPEAVDEKTVKAVFGDIEILNQSSAQLANKDAKKAYFEEKYPEQYTGTAFGQLLNDTWYLYNSNVNVDGVQNAKLPLEGNKSVDITMTPHTYVILDDQDGELQIKLNNYRVDKDSIWEGYGTTVTDRWDTDHNTKLQDWIRDEYIPNPDDDTFRDTTFELVGLESEPEVNVTNGLKDQYQEPVVEYDAAAGTAMITVSGNGWVDLTIDTNTAEVPQVDKAKLNSKIAEAKGIRQGNYTDESYKALQEEIGKSQAVSNKTDATQEEVNAQLSRLESAIARLKEKPAVVSKTALNAKIAEAKGIRQGNYTDESYKALQNAIVKAQELSNKTDATQQQVNDLVSALTNAIKNLKIDADKLAAESAKKVAAVKVAVKAVSYKSKEIKLSWKTVADADGYVIRVKTGKKWSTEKTIKNNRIITYTYKKGTPGKKYVFEVKAFKKVNGKTTYSKYKTATKKVVPQTVTAKAKASKNNVVVKWNKVSGASGYVVMKKKGKTWVKAAQVNAKKLYFTDKKVKKGKVYSYKVKAYKVYKGKKVYGSYSKSVNVKTKS
SEQ ID NO：27
描述：具有His标签和凝血酶裂解位点的Robinsoniella peoriensis Rp3672的GalNAc脱乙酰酶蛋白_表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHAETATEENAALEKTVTLHKSDGTELPEDYRNPQRPATMAVDGIIDDTGEYNYCDFGKDGDKAALYMQVDLGGLYDLSRVNMWRYWKDSRTYDATVITTSESGDFTDEAVIYNSDRSNVHGFGAGGDERYAETASGHEFPVPDGTKAQAVRVYVFGSQNGTTNHINELQVWGTPHTENPDVNSYQVTIPQGNGYQVIPYENDPTTVEEGGSFRFQVLIDSDNGYSATSAVKANGVSLEAVDSVYTIENITEDQVITIEGVHKAQYEVKFPENPQGYSVEIQNEGSTTVDYNGSVSFKLIIDEAYNESVPVVKANGGAALGKDELGVYTIANIQDDITVTVEGIQENTVVKTKTMYLSDMDWKSAANAVGATGEKDTPTKDLNHLQQQMKLLVNGAEKSFDKGIGVQTDSSIVYDLEDKGYTSFHTLAGVDYSAMEYVDGEGCDIQFKVYLDDVVVFDSGVVDASDEAQEVNVAITSENKELKLEAKMVKEPYNDWGNWADASFEMAYPEPSNVALNKTVTVKKTADNSDSEVNSSRPGSMAVDGIIGPTSDSNYCDFGQDGDNTSRYLQVDLGDVYELTQINMFRYWADGRVYNGTVIAVSENADFSNPTFIYNSDKADKHGLGAGSDDTYGETQSGKLFEVPAGTMGQYVRVYMAGSNKGTTNHIAELQVMGYNFNTEPKPYEANAFENAEVYLDMPTHFQDLDSNKNDDGSLKHIGGQVTHPDIQVFDQPWNGYKYWMIYTPNTMITSQYENPYIVASEDGQTWVEPEGISNPIEPEPPSTRFHNCDADLLYDSVNDRLLAYWNWADDGGGIDDELKDQNCQIRLRISYDGINWGVPYDKDGNIATTADTVVRMETGDKDFIPAISEKDRYGMLSPTFTYDDFRGIYTMWAQNSGDAGYNQSGKFIEMRWSEDGINWSEPQKVNNFLGKDENGRQLWPWHQDIQYIPELQEYWGLSQCFSTSNPDGSVLYLTKSRDGVNWEQAGTQPVLRAGKSGTWDDFQIYRSTFYYDNQSDSPTGGKFRIWYSALQANTSGKTVLAPDGTVSLQVGSQDTRIWRIGYTENDYMEVMKALTQNKNYEEPELVDAVSLNLSMDKTSISVGEEATVSTAFVPENATDRIVKYTSQDPEIAVIDPTGIVTGVKDGTTTIVAETKSGAKGELSVTVGELQRGEIRFEVSNDHPMYLENYYWSDDAPKKDGLDANKNYYGDERVDSPVMLYNTVPEELKDNTVILLIAERSLNSTDAVRDWIKKNVELCNENKIPCAVQIANGETNVNTTIPLSFWNELATNNEYLVGFNAAEMYNRFAGDNRSYVMDMIRLGVSHGVCMMWTDTNIFGTNGVLYDWLTQDEKLSGLMREYKEYISLMTKESYGSEAANTDALFKGLWMTDYCENWGIASDWWHWQLDSNGALFDAGSGGDAWKQCLTWPENMYTQDVVRAVSQGATCFKSEAQWYSNATKGMRTPTYQYSMIPFLEKLVSKEVKIPTKEEMLERTKAIVVGAENWNNFNYNTTYSNLYPSTGQYGIVPYVPSNCPEEELAGYDLVVRENLGKAGLKSALDTVYPVQKSEGTAYCETFGDTWYWMNSSEDKNVSQYTEFTTAINGAESVKIAGEPHVFGIIKENPGSLNVYLSNYRLDKTELWDGTIPGGLSDQGCYNYVWQMCERMKNGTGLDTQLRDTVITVKNAVEPKVNFVTESPADRSFAEDNYVRPYKYTVAQKEGTTDEWVITVSHNGIVEFNIVTGDEKVPATSVELSTDKVDVIRNRTAVVKATVLPQNAGNKQLTWTIADPEIASVDNKGTVTGLKEGKTVLRAAISGSVYKECEVNVIDRKVTEVNLNKTELSLSAGDSAKLEASIAPEDPSDSSITWTSTNENVATVASNGTVTAHKAGVAQIIAQSAYQAKGIATVTVNYAASVKLDRTGMTATANSEQSKSGGEGPASNVLDGKQDTMWHTSWTDKPELHPHWIKIDLNGTKTINKFAYTPRTGASNGTIYNYVLIITDLEGNEKQVAKGVWAANADVKYAEFDAVEATAIKLQVDGNDDKASKGGYGSAAEINIFEVAQKPSANELAENIKVIAPVKAEDTKVSIPVITGFDIVISNSSNPDVIGIDGSITRPENDTVVTLTLKVKETDAKSVKAAGTEATTNVDVLVTGTKTSDVEAESVTLDQTSADLTVGGELLLNAVVKPDIATNKAVTWSSDKPGTATVENGRVKALAAGEARITAATANGKTADCVINVKEKEEPEVILPAEVRLNIPSAEFTVGDQIQLTASVLPANAADKTITWKSDKPEVATVANGWVKGIAAGTAKITATSVNGKTAVCVITVKAQPQNLPTGVSLNKKTASVKLNKTLTLSAVVQPSNADNKTVKWTSDNTYVATVENGVVKAVNAGTARITAATVNGHKATCTITVPGTKISKAKVSLASSKTHTGKALKPSVKVTYGKNTLKKNTDYTVSYKNNINPGTASVTITGKGKYYGTINKTFAIKAAEGKTYTVGKGKYKVTDASAKNKTVTFMAPVKKTYSSFSVPSKVKIGNDTYKVTAVAKNAFKKNTKLTKLTIGSNVKTIGSYAFYGASQLKTLTLKTTGLNSVGKNAFKKTNAKLTVKVPKSKLADYKKLLKGKGLSGKAKIQK
SEQ ID NO：29
描述：具有His标签和凝血酶裂解位点的Robinsoniella peoriensis Rp3671的GalNAc脱乙酰酶蛋白Rp3671_表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR
SEQ ID NO：31
描述：具有His标签和凝血酶裂解位点的Robinsoniella peoriensis Rp3672_GalNAc脱乙酰酶_蛋白表达构建体(在pET28a载体中)
MGSSHHHHHHSSGLVPRGSHAETATEENAALEKTVTLHKSDGTELPEDYRNPQRPATMAVDGIIDDTGEYNYCDFGKDGDKAALYMQVDLGGLYDLSRVNMWRYWKDSRTYDATVITTSESGDFTDEAVIYNSDRSNVHGFGAGGDERYAETASGHEFPVPDGTKAQAVRVYVFGSQNGTTNHINELQVWGTPHTENPDVNSYQVTIPQGNGYQVIPYENDPTTVEEGGSFRFQVLIDSDNGYSATSAVKANGVSLEAVDSVYTIENITEDQVITIEGVHKAQYEVKFPENPQGYSVEIQNEGSTTVDYNGSVSFKLIIDEAYNESVPVVKANGGAALGKDELGVYTIANIQDDITVTVEGIQENTVVKTKTMYLSDMDWKSAANAVGATGEKDTPTKDLNHLQQQMKLLVNGAEKSFDKGIGVQTDSSIVYDLEDKGYTSFHTLAGVDYSAMEYVDGEGCDIQFKVYLDDVVVFDSGVVDASDEAQEVNVAITSENKELKLEAKMVKEPYNDWGNWADASFEMAYPEPSNVALNKTVTVKKTADNSDSEVNSSRPGSMAVDGIIGPTSDSNYCDFGQDGDNTSRYLQVDLGDVYELTQINMFRYWADGRVYNGTVIAVSENADFSNPTFIYNSDKADKHGLGAGSDDTYGETQSGKLFEVPAGTMGQYVRVYMAGSNKGTTNHIAELQVMGYNFNTEPKPYEANAFENAEVYLDMPTHFQDLDSNKNDDGSLKHIGGQVTHPDIQVFDQPWNGYKYWMIYTPNTMITSQYENPYIVASEDGQTWVEPEGISNPIEPEPPSTRFHNCDADLLYDSVNDRLLAYWNWADDGGGIDDELKDQNCQIRLRISYDGINWGVPYDKDGNIATTADTVVRMETGDKDFIPAISEKDRYGMLSPTFTYDDFRGIYTMWAQNSGDAGYNQSGKFIEMRWSEDGINWSEPQKVNNFLGKDENGRQLWPWHQDIQYIPELQEYWGLSQCFSTSNPDGSVLYLTKSRDGVNWEQAGTQPVLRAGKSGTWDDFQIYRSTFYYDNQSDSPTGGKFRIWYSALQANTSGKTVLAPDGTVSLQVGSQDTRIWRIGYTENDYMEVMKALTQNKNYEE
SEQ ID NO：32
描述：第三梭状芽胞杆菌5757(Ct5757)的GalNAc脱乙酰酶蛋白序列
HSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEKPLQNAETYLNIPTYDGLNQSTHPDVKYFKNGWNGYKYWMIMTPNRTGSSVAENPSILASDDGINWEVPAGVTNPIAPMPQVGHNCDVDMIYNEATDELWVYWVESDDITKGWVKLIKSKDGVNWSSQQVVVDDNRAKYSTLSPSIIFKDNKYYMWSVNTGNSGWNNQSNKVELRESSDGVNWSNPTVVNTLAQDGSQIWHVNVEYIPSKNEYWAIYPAYKNGTGSDKTELYYAKSSDGVNWTTYKNPILSKGTSGKWDDMEIYRSCFVYDEDTNMIKVWYGAVSQNPQIWKIGFTENDYDKFIEGLTQ
SEQ ID NO：33
描述：Ruthenibacterium lactatiformans Rl8755的GalNAc脱乙酰酶蛋白序列
HEETDLLVNGGFETGDSTGWNWFNNAVVDSAAPHSGNYCAKVAKNSSYEQVVTVSPDTKYVLTGWAKSEGSSVMTLGVKNYGGQETFSATLSADYQQLAVTFTTGPNAQTATIYGYRQNSGSGAGYFDDVELTAVQDFAPYQPLANAIAPQAIPTYDGANQPTHPSVVKFEQPWNGYLYWMAMTPYPFNDGSYENPSIVASNDGENWIVPEGVSNPLAGTPSPGHNCDVDLVYVPASDELRMYYVEADDIISSRVKMISSRDGVHWSEPQVVMQDLVRKYSILSPSIEILPDGTYMMWYVDTGNAGWNSQNNQVKYRTSADGIKWSGAVTCTDFVQPGYQIWHIDVHYDTSSGAYYAVYPAYPNGTDCDHCNLFFAVNRTGKQWETFSRPILKPSTEGGWDDFCIYRSSMLIDDGMLKVWYGAKKQEDSSWHTGLTMRDFSEFMKILER
SEQ ID NO：34
描述：Robinsoniella peoriensis Rp3671的GalNAc脱乙酰酶蛋白
HSPLSAAAESGTGTRLVKGQTGYLTEEQAIRNQEQTTEEREQKLTGEETAEVLMEGTKDSGIVQTEEVQTKEMQTEDAQTEEVQTEEMQTEDAQTKEVQTEEMQTEDAQTEEVQTKEEPAEETHMKEIQTQGTKKASDRNGKARVTEILEDAQDPANRIVYLSDLQWKSENHTVDSELPTRKDKSFGGGKITLKVDGTVTEFDKGIGTQTDSTIVYDLEGKGYTKFETYVGVDYSQKENIPGEVCDVKFRVKIDDKIVSETGVLDPLSNAVKISVNIPDTAKTLTLYADKVTETWSDHANWADAKFYQALPEPENVAFKKTVVTRKTSDNSEAPVNPDSAVNSSKAVDGVIDSSSYFDFGDQANSGAVRESLYMEVDLKGSYLLSDIQLWRYWKDGRTYAATAIVVAEDENFENAAVIYNSDTTGEIHHLGAGSDMLYAETESGKTFPVPENTKARYIRVYTYGVNGTSGVTNHIVELKVNAYVFGDEILPEKPDDSKIFPNAVNPLKLQGPGTNDQVTHPDVTVFDEPWNGYKYWMAYTPNKPGSSYFENPCIAASNDGVNWEFPAQNPVQPRYDSEIENQNEHNCDTDIVYDPVNDRLIMYWEWAQDEAVNGKTHRSEIRYRVSYDGINWGVEDKTGVLMTGPTDHGCAIATEGERYSDLSPTVVYDKTEKIYKMWANDAGDVGYENKQNNKVWYRTSQDGISNWSDKTYVENFLGVNEDGLQMYPWHQDIQWVEEFQEYWALQQAFPAGSGPDNSSLRFSKSKDGLHWEPVSEKALITVGAPGTWDAGQIYRSTFWYEPGGAKGNGTFHIWYAALAEGQSHWDIGYTSANYADAMYKLTGSR
SEQ ID NO：35
描述：Robinsoniella peoriensis Rp3672_GalNAc脱乙酰酶_蛋白
HAETATEENAALEKTVTLHKSDGTELPEDYRNPQRPATMAVDGIIDDTGEYNYCDFGKDGDKAALYMQVDLGGLYDLSRVNMWRYWKDSRTYDATVITTSESGDFTDEAVIYNSDRSNVHGFGAGGDERYAETASGHEFPVPDGTKAQAVRVYVFGSQNGTTNHINELQVWGTPHTENPDVNSYQVTIPQGNGYQVIPYENDPTTVEEGGSFRFQVLIDSDNGYSATSAVKANGVSLEAVDSVYTIENITEDQVITIEGVHKAQYEVKFPENPQGYSVEIQNEGSTTVDYNGSVSFKLIIDEAYNESVPVVKANGGAALGKDELGVYTIANIQDDITVTVEGIQENTVVKTKTMYLSDMDWKSAANAVGATGEKDTPTKDLNHLQQQMKLLVNGAEKSFDKGIGVQTDSSIVYDLEDKGYTSFHTLAGVDYSAMEYVDGEGCDIQFKVYLDDVVVFDSGVVDASDEAQEVNVAITSENKELKLEAKMVKEPYNDWGNWADASFEMAYPEPSNVALNKTVTVKKTADNSDSEVNSSRPGSMAVDGIIGPTSDSNYCDFGQDGDNTSRYLQVDLGDVYELTQINMFRYWADGRVYNGTVIAVSENADFSNPTFIYNSDKADKHGLGAGSDDTYGETQSGKLFEVPAGTMGQYVRVYMAGSNKGTTNHIAELQVMGYNFNTEPKPYEANAFENAEVYLDMPTHFQDLDSNKNDDGSLKHIGGQVTHPDIQVFDQPWNGYKYWMIYTPNTMITSQYENPYIVASEDGQTWVEPEGISNPIEPEPPSTRFHNCDADLLYDSVNDRLLAYWNWADDGGGIDDELKDQNCQIRLRISYDGINWGVPYDKDGNIATTADTVVRMETGDKDFIPAISEKDRYGMLSPTFTYDDFRGIYTMWAQNSGDAGYNQSGKFIEMRWSEDGINWSEPQKVNNFLGKDENGRQLWPWHQDIQYIPELQEYWGLSQCFSTSNPDGSVLYLTKSRDGVNWEQAGTQPVLRAGKSGTWDDFQIYRSTFYYDNQSDSPTGGKFRIWYSALQANTSGKTVLAPDGTVSLQVGSQDTRIWRIGYTENDYMEVMKALTQNKNYEE
SEQ ID NO：36
描述：第三梭状芽孢杆菌5757(Ct5757)的半乳糖胺酶蛋白序列
HYNLIDNISVEKLDTDISQANENVFLNGNGIALEVDNRGATCIYLVDENGVKTKATTSLDTADFSGYPIIGGQKIRDFVIISKNLEENINSILGVGNRLTIISKSSSTNLIRKIVFETSNSNPGAIYSTVSYKAESNDLLVDSFHENEYTMSLGQGPFLAYQGCADQQGANTIVNVTNGYNHNSGQNNYSVGVPFSYVYNSVGGIGIGDASTSRREFKLPIIGKDNTVSLGMEWNGQTLKKGAETAIGTSVITTTNGDYYSGLKSYAEVMKDKGISAPASIPDIAYDSRWESWGFEFDFTIEKIVNKLDELKAMGIKQITLDDGWYTYAGDWKLSPQKFPNGNADMKYLTDEIHKRGMTAILWWRPVDGGINSKLVSEHPEWFIKNSQGNMVRLPGPGGGNGGTAGYALCPNSEGSIQHHKDFVTVALEEWGFDGFKEDYVWGIPKCYDSSHKHSSLSDTLENQYKFYEAIYEQSIAINPDTFIELCNCGTPQDFYSTPYVNHAPTADPISRVQTRTRVKAFKAIFGDDFPVTTDHNSVWLPSALGTGSVMITKHTTLSSSDREQYNKYFGLARDLELAKGEFIGNLYKYGIDPLESYVIRKGEDIYYSFYKDNSSYSGNIEIKGLDSNATYRIEDYVNNRVIARGVKGPTATINTSFTDNLLVRAIPDDTPAEVTTFDVGNNTILSSTDSGNSKYLNAVSTTLEKTATIDSLSIYIGNNSENGKLQIAIYDDNNGKPGTKKAYVEEFVPTKNSWNTKKVVNSVTLPSGQYWLVFQPDNDVLQTKTNPSSMKQSANNNPYNYNILPNSFPIGTGYNAYKGDVSFYATFKEASSQAIPQNSWALKYVDSEETTGENGRATNAFDGNNNTIWHTKYSGGNAAPMPHEIQIDLRGVYNINQINYLPRQDGGTNGTIKDYEVYLSLDGVNWGQPISKGTFESNSTEKIVKFNETKSRYVKLKALSEINNKQFTTVADLKVFGWEISKIEK
SEQ ID NO：37
描述：Robinsoniella peoriensis Rp1021的半乳糖胺酶蛋白序列
HGNGLEVKASPREVAQITGNGVSVTFFQEDGTVQLSCIEDDGNTAFMTRNSEVSYPVVGGEEVTDFSDFQCEVQENVTGAAGAGSRMTITSISSGRGIQRSVVIETVDEVKGLLHISSSYRAEEEVDADEFIDSRFSLDNPSDTVWSYNGGGEGAQSRYDTLQKIDLSDGESFYRENLQNQTAAGIPVADIYGKDGGITVGDASVTRRQLSTPVNERNGTAYVSVKHPGAVITQRETEISQSFVNVHRGDYYSGLRGYADGMKQIGFTTLSREQIPESSYDLRWESWGWEFDWTVELIINKLDELKEMGIKQITLDDGWYNAAGEWGLNNWKLPNGALDMRHLTDAIHERGMTAVLWWRPCDGGREDSALFKEHPEYFIKNQDGSFGKLAGPGQWNSFLGSCGYALCPLSEGAVQSQVDFINRAMNEWGFDGFKSDYVWSLPKCYSQDHHHEYPEESTEQQAVFYRAVYEAMTDNDPNAFHLLCNCGTPQDYYSLPYVTQVPTADPTSVDQTRRRVKAYKALCGDYFPVTTDHNEVWYPSTIGTGAILIEKRDLSGWEEEEYAKWLKIAQENQLHKGTFIGDLYSYGYDPYETYTVYKDGIMYYAFYKDGNRYRPSGNPDIELKGLEDGKLYRIVDYVNNQVVATNVTSSNAVFSYPFSDYLLVKAVEISEPDTDGPGPVPDPEGAVTVEENDPELVYTGDWVREENDGYHGGGARYTKEAEASVELAFYGTGAAWYGQHDVNFGSARIYIDGTYVKTVSCMGEPGINIKLFEISGLDLASHRIKIECETPVIDIDRLTYIKGEEVPAKVMTADLRALTVIANQYDMNSFADGNYKDQLGVSLVRANQLLAADDVTQGAVNEEQKYLLNAMLKIRKKVDKSWIGLPGPIPQDIQTENISRDNLAKVISYTGQLDRDEIIPAIKEQLNDSYDKAVSIAERQDASQPEIDRAWAELMNAVQYSSYIRGSKEELLSLLDEYGKVDTTVYKDAALFIESLEAAKKVYQDENAMDGEISDCIKQLRDAKDQLQLKDPVDPPKPDPDPDPKPDPTPDPGPDPKPDPTPDPTPDPKPNPTPTPDPTPEPALKKPEQVSGLKSKAETDYLTVSWKKLNNAESYKVYIYKSGKWRLAGKTTKTSIKIKKLVSGTKYTVKVAAVNKAGQGKYSSQVYTAAKPKKVKLKSVSRYRTSKVKLNYGKVKAGGYEIWMKNGKGSYKKAATSTKTTAIKSGLKKGKTYYFKVRAYVKNKNQVIYGSFSNIKKYKMVL
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序列表
<110> 不列颠哥伦比亚大学(THE UNIVERSITY OF BRITISH COLUMBIA)
马塞洛·塞普拉(CYPEL, Marcelo)
沙菲克·克沙夫吉(KESHAVJEE, Shafique)
王艾舟(WANG, Aizhou)
<120> 用于供体器官上的碳水化合物抗原切割的酶促组合物、与其相关的方法和用途
<130> P1600PC02
<140> NOT YET ASSIGNED
<141> 2019-08-16
<150> US 62/719,272
<151> 2018-08-17
<160> 108
<170> PatentIn version 3.5
<210> 1
<211> 2319
<212> DNA
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 1
atgagaaatc gaaggaaagc tgtttcgctt ctaacgggcc tactcgtgac ggcccagtta 60
tttccaaccg cggcgcttgc ggcagactcc agcgagtccg cattgaacaa ggcccccgga 120
tatcaggatt ttcccgccta ttacagcgac agtgcgcatg ccgatgacca ggtgactcac 180
ccggacgtag ttgtcctgga agaaccgtgg aacggctatc gctattgggc cgtttatacg 240
cccaacgtga tgcggatctc catctacgaa aacccgtcca tcgttgcctc cagcgacgga 300
gtgcattggg tagaaccgga ggggctttcc aatcccattg agccgcagcc gcccagcacc 360
cgctaccaca actgcgacgc tgatatggtc tataacgcgg aatacgatgc catgatggcc 420
tattggaact gggcggatga ccagggcgga ggcgttgggg ccgaagtccg gctgcggatt 480
tcctatgacg gcgtacattg gggcgtcccc gtgacttatg atgagatgac ccgcgtatgg 540
tcgaagccca cctccgacgc ggagcgtcag gttgcggatg gagaggatga cttcattacc 600
gccattgctt ctccagaccg ctacgatatg ctctctccca ctattgtcta cgatgacttc 660
cgggatgtgt tcatcctgtg ggccaacaat accggcgacg tggggtatca gaatggtcag 720
gcgaacttcg tggaaatgcg ttattcggac gacgggatca cctggggtga gccagtccgc 780
gtcaacggct tcctggggct tgacgagaat gggcagcagt tggccccctg gcatcaggat 840
gtccagtatg ttccagattt gaaggagttt gtttgtattt cccagtgctt tgccggccga 900
aatccggatg gctctgtcct gcacctgacc acatcaaagg atggagtcaa ctgggagcag 960
gtgggcacca agcccctgct gtcccccggg ccagacggca gttgggatga tttccagatc 1020
tatcgctcca gtttttacta tgagccaggc agttccgccg gagatggtac catgcgcgtc 1080
tggtacagtg ccctgcagaa ggacaccaat aacaagatgg tcgcggattc ctccgggaat 1140
ctgaccattc aggccaaaag tgaggatgac cgcatctgga ggatcggcta tgcggaaaac 1200
agttttgttg agatgatgcg cgtgctgctg gatgaccccg gctacacgac gcccgccctg 1260
gtttccggca attcccttat gctgagtgct gagaccactt cccttcccac aggggatgtc 1320
atgaagctgg aaaccagttt cgcgcctgtg gacacctctg atcaggtcgt gaaatatacc 1380
tccagtgatc cggatgtggc gacggtggat gagtttggaa ccattacagg cgtttctgtc 1440
ggttcagcgc gcatcatggc ggagacccgg gagggcctgt ccgacgacct tgaaattgca 1500
gtggtggaga atccgtacac gctgattccc cagtccaata tgacggcaac cgccaccagc 1560
gtctacggcg ggacgacgga gggccccgcc tccaatgtcc tcgatggaaa cgtccgcaca 1620
atatggcata ccaactatgc tcccaaagat gaactgccgc agagtatcac cgtttccttt 1680
gaccagccct ataccgtcgg ccgcttcgtc tataccccac gtcaaaacgg gacaaatggc 1740
ataatttcgg agtatgagct atacgccatc caccaggacg gcagcaagga cctagtcgcc 1800
tccggctcag actgggcgct cgatgccaag gataaaaccg tgagctttgc accggtagaa 1860
gccgtcggcc tggagctcaa ggcgattgcc ggcgcaggtg ggttcggtac tgccgccgaa 1920
ctcaatgtgt atgcgtatgg tccaatcgag cctgcgcccg tatatgtccc ggtggatgac 1980
cgggatgctt ccctggtgtt tacgggtgca tggaatagcg acagcaacgg aagcttttat 2040
gaagggacgg cccgttatac caacgagatc ggcgcgtccg tggagttcac atttgtgggg 2100
acggccattc ggtggtatgg tcaaaatgat gtaaatttcg gcgctgcgga ggtatacgtg 2160
gacggcgttc tggcagggga ggtaaatgtg tatgggccgg cggcggctca gcagcttcta 2220
tttgaggcgg acggtctggc ctatgggaag cataccatcc gcatcgtctg tgtgtctccg 2280
gtggttgact tcgactattt ttcgtatgtg ggagaataa 2319
<210> 2
<211> 772
<212> PRT
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 2
Met Arg Asn Arg Arg Lys Ala Val Ser Leu Leu Thr Gly Leu Leu Val
1 5 10 15
Thr Ala Gln Leu Phe Pro Thr Ala Ala Leu Ala Ala Asp Ser Ser Glu
20 25 30
Ser Ala Leu Asn Lys Ala Pro Gly Tyr Gln Asp Phe Pro Ala Tyr Tyr
35 40 45
Ser Asp Ser Ala His Ala Asp Asp Gln Val Thr His Pro Asp Val Val
50 55 60
Val Leu Glu Glu Pro Trp Asn Gly Tyr Arg Tyr Trp Ala Val Tyr Thr
65 70 75 80
Pro Asn Val Met Arg Ile Ser Ile Tyr Glu Asn Pro Ser Ile Val Ala
85 90 95
Ser Ser Asp Gly Val His Trp Val Glu Pro Glu Gly Leu Ser Asn Pro
100 105 110
Ile Glu Pro Gln Pro Pro Ser Thr Arg Tyr His Asn Cys Asp Ala Asp
115 120 125
Met Val Tyr Asn Ala Glu Tyr Asp Ala Met Met Ala Tyr Trp Asn Trp
130 135 140
Ala Asp Asp Gln Gly Gly Gly Val Gly Ala Glu Val Arg Leu Arg Ile
145 150 155 160
Ser Tyr Asp Gly Val His Trp Gly Val Pro Val Thr Tyr Asp Glu Met
165 170 175
Thr Arg Val Trp Ser Lys Pro Thr Ser Asp Ala Glu Arg Gln Val Ala
180 185 190
Asp Gly Glu Asp Asp Phe Ile Thr Ala Ile Ala Ser Pro Asp Arg Tyr
195 200 205
Asp Met Leu Ser Pro Thr Ile Val Tyr Asp Asp Phe Arg Asp Val Phe
210 215 220
Ile Leu Trp Ala Asn Asn Thr Gly Asp Val Gly Tyr Gln Asn Gly Gln
225 230 235 240
Ala Asn Phe Val Glu Met Arg Tyr Ser Asp Asp Gly Ile Thr Trp Gly
245 250 255
Glu Pro Val Arg Val Asn Gly Phe Leu Gly Leu Asp Glu Asn Gly Gln
260 265 270
Gln Leu Ala Pro Trp His Gln Asp Val Gln Tyr Val Pro Asp Leu Lys
275 280 285
Glu Phe Val Cys Ile Ser Gln Cys Phe Ala Gly Arg Asn Pro Asp Gly
290 295 300
Ser Val Leu His Leu Thr Thr Ser Lys Asp Gly Val Asn Trp Glu Gln
305 310 315 320
Val Gly Thr Lys Pro Leu Leu Ser Pro Gly Pro Asp Gly Ser Trp Asp
325 330 335
Asp Phe Gln Ile Tyr Arg Ser Ser Phe Tyr Tyr Glu Pro Gly Ser Ser
340 345 350
Ala Gly Asp Gly Thr Met Arg Val Trp Tyr Ser Ala Leu Gln Lys Asp
355 360 365
Thr Asn Asn Lys Met Val Ala Asp Ser Ser Gly Asn Leu Thr Ile Gln
370 375 380
Ala Lys Ser Glu Asp Asp Arg Ile Trp Arg Ile Gly Tyr Ala Glu Asn
385 390 395 400
Ser Phe Val Glu Met Met Arg Val Leu Leu Asp Asp Pro Gly Tyr Thr
405 410 415
Thr Pro Ala Leu Val Ser Gly Asn Ser Leu Met Leu Ser Ala Glu Thr
420 425 430
Thr Ser Leu Pro Thr Gly Asp Val Met Lys Leu Glu Thr Ser Phe Ala
435 440 445
Pro Val Asp Thr Ser Asp Gln Val Val Lys Tyr Thr Ser Ser Asp Pro
450 455 460
Asp Val Ala Thr Val Asp Glu Phe Gly Thr Ile Thr Gly Val Ser Val
465 470 475 480
Gly Ser Ala Arg Ile Met Ala Glu Thr Arg Glu Gly Leu Ser Asp Asp
485 490 495
Leu Glu Ile Ala Val Val Glu Asn Pro Tyr Thr Leu Ile Pro Gln Ser
500 505 510
Asn Met Thr Ala Thr Ala Thr Ser Val Tyr Gly Gly Thr Thr Glu Gly
515 520 525
Pro Ala Ser Asn Val Leu Asp Gly Asn Val Arg Thr Ile Trp His Thr
530 535 540
Asn Tyr Ala Pro Lys Asp Glu Leu Pro Gln Ser Ile Thr Val Ser Phe
545 550 555 560
Asp Gln Pro Tyr Thr Val Gly Arg Phe Val Tyr Thr Pro Arg Gln Asn
565 570 575
Gly Thr Asn Gly Ile Ile Ser Glu Tyr Glu Leu Tyr Ala Ile His Gln
580 585 590
Asp Gly Ser Lys Asp Leu Val Ala Ser Gly Ser Asp Trp Ala Leu Asp
595 600 605
Ala Lys Asp Lys Thr Val Ser Phe Ala Pro Val Glu Ala Val Gly Leu
610 615 620
Glu Leu Lys Ala Ile Ala Gly Ala Gly Gly Phe Gly Thr Ala Ala Glu
625 630 635 640
Leu Asn Val Tyr Ala Tyr Gly Pro Ile Glu Pro Ala Pro Val Tyr Val
645 650 655
Pro Val Asp Asp Arg Asp Ala Ser Leu Val Phe Thr Gly Ala Trp Asn
660 665 670
Ser Asp Ser Asn Gly Ser Phe Tyr Glu Gly Thr Ala Arg Tyr Thr Asn
675 680 685
Glu Ile Gly Ala Ser Val Glu Phe Thr Phe Val Gly Thr Ala Ile Arg
690 695 700
Trp Tyr Gly Gln Asn Asp Val Asn Phe Gly Ala Ala Glu Val Tyr Val
705 710 715 720
Asp Gly Val Leu Ala Gly Glu Val Asn Val Tyr Gly Pro Ala Ala Ala
725 730 735
Gln Gln Leu Leu Phe Glu Ala Asp Gly Leu Ala Tyr Gly Lys His Thr
740 745 750
Ile Arg Ile Val Cys Val Ser Pro Val Val Asp Phe Asp Tyr Phe Ser
755 760 765
Tyr Val Gly Glu
770
<210> 3
<211> 2238
<212> DNA
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 3
gcagactcca gcgagtccgc attgaacaag gcccccggat atcaggattt tcccgcctat 60
tacagcgaca gtgcgcatgc cgatgaccag gtgactcacc cggacgtagt tgtcctggaa 120
gaaccgtgga acggctatcg ctattgggcc gtttatacgc ccaacgtgat gcggatctcc 180
atctacgaaa acccgtccat cgttgcctcc agcgacggag tgcattgggt agaaccggag 240
gggctttcca atcccattga gccgcagccg cccagcaccc gctaccacaa ctgcgacgct 300
gatatggtct ataacgcgga atacgatgcc atgatggcct attggaactg ggcggatgac 360
cagggcggag gcgttggggc cgaagtccgg ctgcggattt cctatgacgg cgtacattgg 420
ggcgtccccg tgacttatga tgagatgacc cgcgtatggt cgaagcccac ctccgacgcg 480
gagcgtcagg ttgcggatgg agaggatgac ttcattaccg ccattgcttc tccagaccgc 540
tacgatatgc tctctcccac tattgtctac gatgacttcc gggatgtgtt catcctgtgg 600
gccaacaata ccggcgacgt ggggtatcag aatggtcagg cgaacttcgt ggaaatgcgt 660
tattcggacg acgggatcac ctggggtgag ccagtccgcg tcaacggctt cctggggctt 720
gacgagaatg ggcagcagtt ggccccctgg catcaggatg tccagtatgt tccagatttg 780
aaggagtttg tttgtatttc ccagtgcttt gccggccgaa atccggatgg ctctgtcctg 840
cacctgacca catcaaagga tggagtcaac tgggagcagg tgggcaccaa gcccctgctg 900
tcccccgggc cagacggcag ttgggatgat ttccagatct atcgctccag tttttactat 960
gagccaggca gttccgccgg agatggtacc atgcgcgtct ggtacagtgc cctgcagaag 1020
gacaccaata acaagatggt cgcggattcc tccgggaatc tgaccattca ggccaaaagt 1080
gaggatgacc gcatctggag gatcggctat gcggaaaaca gttttgttga gatgatgcgc 1140
gtgctgctgg atgaccccgg ctacacgacg cccgccctgg tttccggcaa ttcccttatg 1200
ctgagtgctg agaccacttc ccttcccaca ggggatgtca tgaagctgga aaccagtttc 1260
gcgcctgtgg acacctctga tcaggtcgtg aaatatacct ccagtgatcc ggatgtggcg 1320
acggtggatg agtttggaac cattacaggc gtttctgtcg gttcagcgcg catcatggcg 1380
gagacccggg agggcctgtc cgacgacctt gaaattgcag tggtggagaa tccgtacacg 1440
ctgattcccc agtccaatat gacggcaacc gccaccagcg tctacggcgg gacgacggag 1500
ggccccgcct ccaatgtcct cgatggaaac gtccgcacaa tatggcatac caactatgct 1560
cccaaagatg aactgccgca gagtatcacc gtttcctttg accagcccta taccgtcggc 1620
cgcttcgtct ataccccacg tcaaaacggg acaaatggca taatttcgga gtatgagcta 1680
tacgccatcc accaggacgg cagcaaggac ctagtcgcct ccggctcaga ctgggcgctc 1740
gatgccaagg ataaaaccgt gagctttgca ccggtagaag ccgtcggcct ggagctcaag 1800
gcgattgccg gcgcaggtgg gttcggtact gccgccgaac tcaatgtgta tgcgtatggt 1860
ccaatcgagc ctgcgcccgt atatgtcccg gtggatgacc gggatgcttc cctggtgttt 1920
acgggtgcat ggaatagcga cagcaacgga agcttttatg aagggacggc ccgttatacc 1980
aacgagatcg gcgcgtccgt ggagttcaca tttgtgggga cggccattcg gtggtatggt 2040
caaaatgatg taaatttcgg cgctgcggag gtatacgtgg acggcgttct ggcaggggag 2100
gtaaatgtgt atgggccggc ggcggctcag cagcttctat ttgaggcgga cggtctggcc 2160
tatgggaagc ataccatccg catcgtctgt gtgtctccgg tggttgactt cgactatttt 2220
tcgtatgtgg gagaataa 2238
<210> 4
<211> 745
<212> PRT
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 4
Ala Asp Ser Ser Glu Ser Ala Leu Asn Lys Ala Pro Gly Tyr Gln Asp
1 5 10 15
Phe Pro Ala Tyr Tyr Ser Asp Ser Ala His Ala Asp Asp Gln Val Thr
20 25 30
His Pro Asp Val Val Val Leu Glu Glu Pro Trp Asn Gly Tyr Arg Tyr
35 40 45
Trp Ala Val Tyr Thr Pro Asn Val Met Arg Ile Ser Ile Tyr Glu Asn
50 55 60
Pro Ser Ile Val Ala Ser Ser Asp Gly Val His Trp Val Glu Pro Glu
65 70 75 80
Gly Leu Ser Asn Pro Ile Glu Pro Gln Pro Pro Ser Thr Arg Tyr His
85 90 95
Asn Cys Asp Ala Asp Met Val Tyr Asn Ala Glu Tyr Asp Ala Met Met
100 105 110
Ala Tyr Trp Asn Trp Ala Asp Asp Gln Gly Gly Gly Val Gly Ala Glu
115 120 125
Val Arg Leu Arg Ile Ser Tyr Asp Gly Val His Trp Gly Val Pro Val
130 135 140
Thr Tyr Asp Glu Met Thr Arg Val Trp Ser Lys Pro Thr Ser Asp Ala
145 150 155 160
Glu Arg Gln Val Ala Asp Gly Glu Asp Asp Phe Ile Thr Ala Ile Ala
165 170 175
Ser Pro Asp Arg Tyr Asp Met Leu Ser Pro Thr Ile Val Tyr Asp Asp
180 185 190
Phe Arg Asp Val Phe Ile Leu Trp Ala Asn Asn Thr Gly Asp Val Gly
195 200 205
Tyr Gln Asn Gly Gln Ala Asn Phe Val Glu Met Arg Tyr Ser Asp Asp
210 215 220
Gly Ile Thr Trp Gly Glu Pro Val Arg Val Asn Gly Phe Leu Gly Leu
225 230 235 240
Asp Glu Asn Gly Gln Gln Leu Ala Pro Trp His Gln Asp Val Gln Tyr
245 250 255
Val Pro Asp Leu Lys Glu Phe Val Cys Ile Ser Gln Cys Phe Ala Gly
260 265 270
Arg Asn Pro Asp Gly Ser Val Leu His Leu Thr Thr Ser Lys Asp Gly
275 280 285
Val Asn Trp Glu Gln Val Gly Thr Lys Pro Leu Leu Ser Pro Gly Pro
290 295 300
Asp Gly Ser Trp Asp Asp Phe Gln Ile Tyr Arg Ser Ser Phe Tyr Tyr
305 310 315 320
Glu Pro Gly Ser Ser Ala Gly Asp Gly Thr Met Arg Val Trp Tyr Ser
325 330 335
Ala Leu Gln Lys Asp Thr Asn Asn Lys Met Val Ala Asp Ser Ser Gly
340 345 350
Asn Leu Thr Ile Gln Ala Lys Ser Glu Asp Asp Arg Ile Trp Arg Ile
355 360 365
Gly Tyr Ala Glu Asn Ser Phe Val Glu Met Met Arg Val Leu Leu Asp
370 375 380
Asp Pro Gly Tyr Thr Thr Pro Ala Leu Val Ser Gly Asn Ser Leu Met
385 390 395 400
Leu Ser Ala Glu Thr Thr Ser Leu Pro Thr Gly Asp Val Met Lys Leu
405 410 415
Glu Thr Ser Phe Ala Pro Val Asp Thr Ser Asp Gln Val Val Lys Tyr
420 425 430
Thr Ser Ser Asp Pro Asp Val Ala Thr Val Asp Glu Phe Gly Thr Ile
435 440 445
Thr Gly Val Ser Val Gly Ser Ala Arg Ile Met Ala Glu Thr Arg Glu
450 455 460
Gly Leu Ser Asp Asp Leu Glu Ile Ala Val Val Glu Asn Pro Tyr Thr
465 470 475 480
Leu Ile Pro Gln Ser Asn Met Thr Ala Thr Ala Thr Ser Val Tyr Gly
485 490 495
Gly Thr Thr Glu Gly Pro Ala Ser Asn Val Leu Asp Gly Asn Val Arg
500 505 510
Thr Ile Trp His Thr Asn Tyr Ala Pro Lys Asp Glu Leu Pro Gln Ser
515 520 525
Ile Thr Val Ser Phe Asp Gln Pro Tyr Thr Val Gly Arg Phe Val Tyr
530 535 540
Thr Pro Arg Gln Asn Gly Thr Asn Gly Ile Ile Ser Glu Tyr Glu Leu
545 550 555 560
Tyr Ala Ile His Gln Asp Gly Ser Lys Asp Leu Val Ala Ser Gly Ser
565 570 575
Asp Trp Ala Leu Asp Ala Lys Asp Lys Thr Val Ser Phe Ala Pro Val
580 585 590
Glu Ala Val Gly Leu Glu Leu Lys Ala Ile Ala Gly Ala Gly Gly Phe
595 600 605
Gly Thr Ala Ala Glu Leu Asn Val Tyr Ala Tyr Gly Pro Ile Glu Pro
610 615 620
Ala Pro Val Tyr Val Pro Val Asp Asp Arg Asp Ala Ser Leu Val Phe
625 630 635 640
Thr Gly Ala Trp Asn Ser Asp Ser Asn Gly Ser Phe Tyr Glu Gly Thr
645 650 655
Ala Arg Tyr Thr Asn Glu Ile Gly Ala Ser Val Glu Phe Thr Phe Val
660 665 670
Gly Thr Ala Ile Arg Trp Tyr Gly Gln Asn Asp Val Asn Phe Gly Ala
675 680 685
Ala Glu Val Tyr Val Asp Gly Val Leu Ala Gly Glu Val Asn Val Tyr
690 695 700
Gly Pro Ala Ala Ala Gln Gln Leu Leu Phe Glu Ala Asp Gly Leu Ala
705 710 715 720
Tyr Gly Lys His Thr Ile Arg Ile Val Cys Val Ser Pro Val Val Asp
725 730 735
Phe Asp Tyr Phe Ser Tyr Val Gly Glu
740 745
<210> 5
<211> 760
<212> PRT
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 5
Met Gly His His His His His His His His His His Ser Ser Gly Ala
1 5 10 15
Asp Ser Ser Glu Ser Ala Leu Asn Lys Ala Pro Gly Tyr Gln Asp Phe
20 25 30
Pro Ala Tyr Tyr Ser Asp Ser Ala His Ala Asp Asp Gln Val Thr His
35 40 45
Pro Asp Val Val Val Leu Glu Glu Pro Trp Asn Gly Tyr Arg Tyr Trp
50 55 60
Ala Val Tyr Thr Pro Asn Val Met Arg Ile Ser Ile Tyr Glu Asn Pro
65 70 75 80
Ser Ile Val Ala Ser Ser Asp Gly Val His Trp Val Glu Pro Glu Gly
85 90 95
Leu Ser Asn Pro Ile Glu Pro Gln Pro Pro Ser Thr Arg Tyr His Asn
100 105 110
Cys Asp Ala Asp Met Val Tyr Asn Ala Glu Tyr Asp Ala Met Met Ala
115 120 125
Tyr Trp Asn Trp Ala Asp Asp Gln Gly Gly Gly Val Gly Ala Glu Val
130 135 140
Arg Leu Arg Ile Ser Tyr Asp Gly Val His Trp Gly Val Pro Val Thr
145 150 155 160
Tyr Asp Glu Met Thr Arg Val Trp Ser Lys Pro Thr Ser Asp Ala Glu
165 170 175
Arg Gln Val Ala Asp Gly Glu Asp Asp Phe Ile Thr Ala Ile Ala Ser
180 185 190
Pro Asp Arg Tyr Asp Met Leu Ser Pro Thr Ile Val Tyr Asp Asp Phe
195 200 205
Arg Asp Val Phe Ile Leu Trp Ala Asn Asn Thr Gly Asp Val Gly Tyr
210 215 220
Gln Asn Gly Gln Ala Asn Phe Val Glu Met Arg Tyr Ser Asp Asp Gly
225 230 235 240
Ile Thr Trp Gly Glu Pro Val Arg Val Asn Gly Phe Leu Gly Leu Asp
245 250 255
Glu Asn Gly Gln Gln Leu Ala Pro Trp His Gln Asp Val Gln Tyr Val
260 265 270
Pro Asp Leu Lys Glu Phe Val Cys Ile Ser Gln Cys Phe Ala Gly Arg
275 280 285
Asn Pro Asp Gly Ser Val Leu His Leu Thr Thr Ser Lys Asp Gly Val
290 295 300
Asn Trp Glu Gln Val Gly Thr Lys Pro Leu Leu Ser Pro Gly Pro Asp
305 310 315 320
Gly Ser Trp Asp Asp Phe Gln Ile Tyr Arg Ser Ser Phe Tyr Tyr Glu
325 330 335
Pro Gly Ser Ser Ala Gly Asp Gly Thr Met Arg Val Trp Tyr Ser Ala
340 345 350
Leu Gln Lys Asp Thr Asn Asn Lys Met Val Ala Asp Ser Ser Gly Asn
355 360 365
Leu Thr Ile Gln Ala Lys Ser Glu Asp Asp Arg Ile Trp Arg Ile Gly
370 375 380
Tyr Ala Glu Asn Ser Phe Val Glu Met Met Arg Val Leu Leu Asp Asp
385 390 395 400
Pro Gly Tyr Thr Thr Pro Ala Leu Val Ser Gly Asn Ser Leu Met Leu
405 410 415
Ser Ala Glu Thr Thr Ser Leu Pro Thr Gly Asp Val Met Lys Leu Glu
420 425 430
Thr Ser Phe Ala Pro Val Asp Thr Ser Asp Gln Val Val Lys Tyr Thr
435 440 445
Ser Ser Asp Pro Asp Val Ala Thr Val Asp Glu Phe Gly Thr Ile Thr
450 455 460
Gly Val Ser Val Gly Ser Ala Arg Ile Met Ala Glu Thr Arg Glu Gly
465 470 475 480
Leu Ser Asp Asp Leu Glu Ile Ala Val Val Glu Asn Pro Tyr Thr Leu
485 490 495
Ile Pro Gln Ser Asn Met Thr Ala Thr Ala Thr Ser Val Tyr Gly Gly
500 505 510
Thr Thr Glu Gly Pro Ala Ser Asn Val Leu Asp Gly Asn Val Arg Thr
515 520 525
Ile Trp His Thr Asn Tyr Ala Pro Lys Asp Glu Leu Pro Gln Ser Ile
530 535 540
Thr Val Ser Phe Asp Gln Pro Tyr Thr Val Gly Arg Phe Val Tyr Thr
545 550 555 560
Pro Arg Gln Asn Gly Thr Asn Gly Ile Ile Ser Glu Tyr Glu Leu Tyr
565 570 575
Ala Ile His Gln Asp Gly Ser Lys Asp Leu Val Ala Ser Gly Ser Asp
580 585 590
Trp Ala Leu Asp Ala Lys Asp Lys Thr Val Ser Phe Ala Pro Val Glu
595 600 605
Ala Val Gly Leu Glu Leu Lys Ala Ile Ala Gly Ala Gly Gly Phe Gly
610 615 620
Thr Ala Ala Glu Leu Asn Val Tyr Ala Tyr Gly Pro Ile Glu Pro Ala
625 630 635 640
Pro Val Tyr Val Pro Val Asp Asp Arg Asp Ala Ser Leu Val Phe Thr
645 650 655
Gly Ala Trp Asn Ser Asp Ser Asn Gly Ser Phe Tyr Glu Gly Thr Ala
660 665 670
Arg Tyr Thr Asn Glu Ile Gly Ala Ser Val Glu Phe Thr Phe Val Gly
675 680 685
Thr Ala Ile Arg Trp Tyr Gly Gln Asn Asp Val Asn Phe Gly Ala Ala
690 695 700
Glu Val Tyr Val Asp Gly Val Leu Ala Gly Glu Val Asn Val Tyr Gly
705 710 715 720
Pro Ala Ala Ala Gln Gln Leu Leu Phe Glu Ala Asp Gly Leu Ala Tyr
725 730 735
Gly Lys His Thr Ile Arg Ile Val Cys Val Ser Pro Val Val Asp Phe
740 745 750
Asp Tyr Phe Ser Tyr Val Gly Glu
755 760
<210> 6
<211> 3159
<212> DNA
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 6
gcggcgcctg caacggacac cggcaacgca ggactgattg cagaaggtga ttatgccatt 60
gccggcaatg gcgtccgcgt cacttatgac gcggacgggc agacaatcac tctgtaccgc 120
acagagggat ctgggcttat ccagatgagc aagccttctc cattgggagg gccagtgatt 180
ggagggcagg aggttcagga cttcagccat atttcatgtg atgtggagca gagcaccagc 240
ggagtgatgg gcagcggtca gagaatgacc attacctctc agagcatgag cacgggccta 300
attcgtacct atgtgctgga gacctctgat atcgaggagg gtgtggtata tactgcaaca 360
tcctatgagg caggagcttc tgatgtggaa gtgtcttggt tcattggcag tgtgtatgag 420
ctttatggtg cggaagatcg tatctggagt tataacggcg gcggtgaggg gccgatgcac 480
tactatgata cgcttcaaaa gattgacctg accgactctg gcaagttcag tagggagaat 540
aaacaggatg acacggctgc aagtattcct gtgtcagata tttacattgc tgatggaggg 600
attaccgttg gcgatgcttc tgcaaccaga agggaggtac atactccggt tcaggaaacc 660
agtgattcag ctcaagtttc tatcgggtgg ccaggcaaag tcattgccgc cggaagcgtg 720
atcgaaattg gtgagagctt tgctgtagtc catccgggtg actattataa cggcttgaga 780
ggttacaaaa atgcaatgga tcacttgggc gtgattatgc ctgcacctgg ggatattcct 840
gatagcagct atgatctccg atgggaaagc tggggctggg ggtttaactg gacgatcgat 900
ttaataatcg gcaaattgga tgaacttcag gcagccggag tcaagcagat cactttggat 960
gatggttggt ataccaatgc aggagactgg gccttaaatc cagaaaagtt tccaaatgga 1020
gcctccgatg cgttgcggct gacagatgca attcatgagc atggtatgac tgcactcctt 1080
tggtggagac cttgtgacgg cgggatcgat agtatactct atcagcaaca ccctgaatat 1140
ttcgttatgg atgcagatgg aagacctgca aggcttccta ctcctggtgg tgggaccaat 1200
cccagcttgg gatatgcact ttgccctatg gcggatggtg cgattgcaag ccaagttgac 1260
tttgtaaacc gtgcaatgaa tgattggggg ttcgatggct tcaagggaga ttatgtgtgg 1320
agtatgcctg aatgctacaa tcctgcacat aaccacgcct cgccagaaga atccactgaa 1380
aagcaatccg agatataccg cgtctcttat gaggctatgg tggccaacga ccccaatgtg 1440
ttcaatttgt tgtgcaactg cggtacgccc caggactact atagtttacc atatatgaca 1500
cagattgcta cggctgaccc cacttctgtg gatcaaacaa ggagacgcgt gaaagcctac 1560
aaggcactga tgggagatta tttccctgtt acagccgacc acaataacat ctggtatcca 1620
agtgccgtcg gtacgggctc tgttctcatt gaaaaacgtg accttagcgg tactgccaag 1680
gaagaatatg aaaaatggct tgggattgcg gatacagttc agttgcagaa aggccggttt 1740
attggcgatc tttacagtta tggttttgac ccttacgaaa cctatgtggt ggagaaagac 1800
ggggttatgt actatgcctt ctacaaagat gggagcaaat atagccccac tggctatcca 1860
gatattgagt tgaaggggct agatccaaat aaaatgtata ggattgttga ctatgtcaat 1920
gatcgtgtcg tggcaacaaa cctgatgggt gataacgctg tattcaatac acgtttttcc 1980
gactatctac tggttaaagc ggtggaaatt tcggaaccgg atccagaacc tgttgaccct 2040
gattatggtt tcacctctgt tgatgacaga gacgaggctc ttatttacac agggacatgg 2100
catgatgaca ataacgcatc tttcagcgaa gggactgcac gttataccaa cagtacggat 2160
gcttcggttg tattctcctt tactggaact tccattcgct ggtatggcca gagggatacc 2220
aattttggca cggcagaagt ttatttggac gatgaactga aaacaacagt tgatgcgaat 2280
ggggccgcag aagcaggcgt atgtcttttt gaggcgcttg atcttccggc tgccgagcat 2340
accattaaaa ttgtgtgcaa gagcggagtg attgatattg accgctttgc atatgaagct 2400
gctacccttg aacccatcta tgaaaaggtc gatgcgctct cggatcggat cacttatgtt 2460
gggaattggg aagagtatca caacagcgag ttctacatgg gaaacgcaat gcgcacagac 2520
gaagccggcg cttatgctga actgactttc cgtggtacag ccgtacgcct gtatgcagag 2580
atgagcttca attttggcac tgcagatgtc tatttagacg gagagttagt ggaaaacata 2640
atcctatacg gccaggaagc aactgggcag ctaatgtttg agcgtacggg actggaggaa 2700
ggagaacata ccattcgcct tgtacaaaac gcctggaaca tcaatttgga ctatatttct 2760
tatctaccag agcaagatca accaacgccg ccggagacga cggttactgt tgatgcaatg 2820
gacgcccaac tggtgtatac aggcgtatgg aatgatgact atcatgacgt ctttcaggaa 2880
ggaaccgccc gttatgccag tagtgccggc gcctcggtcg agttcgaatt tactggaagc 2940
gaaatccgtt ggtatggaca aaatgattcc aacttcggtg ttgccagcgt ttatatcgat 3000
aatgagtttg tgcagcaggt aaatgttaac ggagctgcgg ctgtgggaaa gcttttgttt 3060
caaaaggctg atctaccagc cggttcgcac acgatccgca ttgtgtgcga tactccggtt 3120
attgatttgg actatttgac ttataccact aacgcataa 3159
<210> 7
<211> 1078
<212> PRT
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 7
Met Arg Gly Lys Lys Phe Ile Ser Leu Thr Leu Ser Thr Met Leu Cys
1 5 10 15
Leu Gln Leu Leu Pro Thr Ala Ser Phe Ala Ala Ala Pro Ala Thr Asp
20 25 30
Thr Gly Asn Ala Gly Leu Ile Ala Glu Gly Asp Tyr Ala Ile Ala Gly
35 40 45
Asn Gly Val Arg Val Thr Tyr Asp Ala Asp Gly Gln Thr Ile Thr Leu
50 55 60
Tyr Arg Thr Glu Gly Ser Gly Leu Ile Gln Met Ser Lys Pro Ser Pro
65 70 75 80
Leu Gly Gly Pro Val Ile Gly Gly Gln Glu Val Gln Asp Phe Ser His
85 90 95
Ile Ser Cys Asp Val Glu Gln Ser Thr Ser Gly Val Met Gly Ser Gly
100 105 110
Gln Arg Met Thr Ile Thr Ser Gln Ser Met Ser Thr Gly Leu Ile Arg
115 120 125
Thr Tyr Val Leu Glu Thr Ser Asp Ile Glu Glu Gly Val Val Tyr Thr
130 135 140
Ala Thr Ser Tyr Glu Ala Gly Ala Ser Asp Val Glu Val Ser Trp Phe
145 150 155 160
Ile Gly Ser Val Tyr Glu Leu Tyr Gly Ala Glu Asp Arg Ile Trp Ser
165 170 175
Tyr Asn Gly Gly Gly Glu Gly Pro Met His Tyr Tyr Asp Thr Leu Gln
180 185 190
Lys Ile Asp Leu Thr Asp Ser Gly Lys Phe Ser Arg Glu Asn Lys Gln
195 200 205
Asp Asp Thr Ala Ala Ser Ile Pro Val Ser Asp Ile Tyr Ile Ala Asp
210 215 220
Gly Gly Ile Thr Val Gly Asp Ala Ser Ala Thr Arg Arg Glu Val His
225 230 235 240
Thr Pro Val Gln Glu Thr Ser Asp Ser Ala Gln Val Ser Ile Gly Trp
245 250 255
Pro Gly Lys Val Ile Ala Ala Gly Ser Val Ile Glu Ile Gly Glu Ser
260 265 270
Phe Ala Val Val His Pro Gly Asp Tyr Tyr Asn Gly Leu Arg Gly Tyr
275 280 285
Lys Asn Ala Met Asp His Leu Gly Val Ile Met Pro Ala Pro Gly Asp
290 295 300
Ile Pro Asp Ser Ser Tyr Asp Leu Arg Trp Glu Ser Trp Gly Trp Gly
305 310 315 320
Phe Asn Trp Thr Ile Asp Leu Ile Ile Gly Lys Leu Asp Glu Leu Gln
325 330 335
Ala Ala Gly Val Lys Gln Ile Thr Leu Asp Asp Gly Trp Tyr Thr Asn
340 345 350
Ala Gly Asp Trp Ala Leu Asn Pro Glu Lys Phe Pro Asn Gly Ala Ser
355 360 365
Asp Ala Leu Arg Leu Thr Asp Ala Ile His Glu His Gly Met Thr Ala
370 375 380
Leu Leu Trp Trp Arg Pro Cys Asp Gly Gly Ile Asp Ser Ile Leu Tyr
385 390 395 400
Gln Gln His Pro Glu Tyr Phe Val Met Asp Ala Asp Gly Arg Pro Ala
405 410 415
Arg Leu Pro Thr Pro Gly Gly Gly Thr Asn Pro Ser Leu Gly Tyr Ala
420 425 430
Leu Cys Pro Met Ala Asp Gly Ala Ile Ala Ser Gln Val Asp Phe Val
435 440 445
Asn Arg Ala Met Asn Asp Trp Gly Phe Asp Gly Phe Lys Gly Asp Tyr
450 455 460
Val Trp Ser Met Pro Glu Cys Tyr Asn Pro Ala His Asn His Ala Ser
465 470 475 480
Pro Glu Glu Ser Thr Glu Lys Gln Ser Glu Ile Tyr Arg Val Ser Tyr
485 490 495
Glu Ala Met Val Ala Asn Asp Pro Asn Val Phe Asn Leu Leu Cys Asn
500 505 510
Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu Pro Tyr Met Thr Gln Ile
515 520 525
Ala Thr Ala Asp Pro Thr Ser Val Asp Gln Thr Arg Arg Arg Val Lys
530 535 540
Ala Tyr Lys Ala Leu Met Gly Asp Tyr Phe Pro Val Thr Ala Asp His
545 550 555 560
Asn Asn Ile Trp Tyr Pro Ser Ala Val Gly Thr Gly Ser Val Leu Ile
565 570 575
Glu Lys Arg Asp Leu Ser Gly Thr Ala Lys Glu Glu Tyr Glu Lys Trp
580 585 590
Leu Gly Ile Ala Asp Thr Val Gln Leu Gln Lys Gly Arg Phe Ile Gly
595 600 605
Asp Leu Tyr Ser Tyr Gly Phe Asp Pro Tyr Glu Thr Tyr Val Val Glu
610 615 620
Lys Asp Gly Val Met Tyr Tyr Ala Phe Tyr Lys Asp Gly Ser Lys Tyr
625 630 635 640
Ser Pro Thr Gly Tyr Pro Asp Ile Glu Leu Lys Gly Leu Asp Pro Asn
645 650 655
Lys Met Tyr Arg Ile Val Asp Tyr Val Asn Asp Arg Val Val Ala Thr
660 665 670
Asn Leu Met Gly Asp Asn Ala Val Phe Asn Thr Arg Phe Ser Asp Tyr
675 680 685
Leu Leu Val Lys Ala Val Glu Ile Ser Glu Pro Asp Pro Glu Pro Val
690 695 700
Asp Pro Asp Tyr Gly Phe Thr Ser Val Asp Asp Arg Asp Glu Ala Leu
705 710 715 720
Ile Tyr Thr Gly Thr Trp His Asp Asp Asn Asn Ala Ser Phe Ser Glu
725 730 735
Gly Thr Ala Arg Tyr Thr Asn Ser Thr Asp Ala Ser Val Val Phe Ser
740 745 750
Phe Thr Gly Thr Ser Ile Arg Trp Tyr Gly Gln Arg Asp Thr Asn Phe
755 760 765
Gly Thr Ala Glu Val Tyr Leu Asp Asp Glu Leu Lys Thr Thr Val Asp
770 775 780
Ala Asn Gly Ala Ala Glu Ala Gly Val Cys Leu Phe Glu Ala Leu Asp
785 790 795 800
Leu Pro Ala Ala Glu His Thr Ile Lys Ile Val Cys Lys Ser Gly Val
805 810 815
Ile Asp Ile Asp Arg Phe Ala Tyr Glu Ala Ala Thr Leu Glu Pro Ile
820 825 830
Tyr Glu Lys Val Asp Ala Leu Ser Asp Arg Ile Thr Tyr Val Gly Asn
835 840 845
Trp Glu Glu Tyr His Asn Ser Glu Phe Tyr Met Gly Asn Ala Met Arg
850 855 860
Thr Asp Glu Ala Gly Ala Tyr Ala Glu Leu Thr Phe Arg Gly Thr Ala
865 870 875 880
Val Arg Leu Tyr Ala Glu Met Ser Phe Asn Phe Gly Thr Ala Asp Val
885 890 895
Tyr Leu Asp Gly Glu Leu Val Glu Asn Ile Ile Leu Tyr Gly Gln Glu
900 905 910
Ala Thr Gly Gln Leu Met Phe Glu Arg Thr Gly Leu Glu Glu Gly Glu
915 920 925
His Thr Ile Arg Leu Val Gln Asn Ala Trp Asn Ile Asn Leu Asp Tyr
930 935 940
Ile Ser Tyr Leu Pro Glu Gln Asp Gln Pro Thr Pro Pro Glu Thr Thr
945 950 955 960
Val Thr Val Asp Ala Met Asp Ala Gln Leu Val Tyr Thr Gly Val Trp
965 970 975
Asn Asp Asp Tyr His Asp Val Phe Gln Glu Gly Thr Ala Arg Tyr Ala
980 985 990
Ser Ser Ala Gly Ala Ser Val Glu Phe Glu Phe Thr Gly Ser Glu Ile
995 1000 1005
Arg Trp Tyr Gly Gln Asn Asp Ser Asn Phe Gly Val Ala Ser Val
1010 1015 1020
Tyr Ile Asp Asn Glu Phe Val Gln Gln Val Asn Val Asn Gly Ala
1025 1030 1035
Ala Ala Val Gly Lys Leu Leu Phe Gln Lys Ala Asp Leu Pro Ala
1040 1045 1050
Gly Ser His Thr Ile Arg Ile Val Cys Asp Thr Pro Val Ile Asp
1055 1060 1065
Leu Asp Tyr Leu Thr Tyr Thr Thr Asn Ala
1070 1075
<210> 8
<211> 3159
<212> DNA
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 8
gcggcgcctg caacggacac cggcaacgca ggactgattg cagaaggtga ttatgccatt 60
gccggcaatg gcgtccgcgt cacttatgac gcggacgggc agacaatcac tctgtaccgc 120
acagagggat ctgggcttat ccagatgagc aagccttctc cattgggagg gccagtgatt 180
ggagggcagg aggttcagga cttcagccat atttcatgtg atgtggagca gagcaccagc 240
ggagtgatgg gcagcggtca gagaatgacc attacctctc agagcatgag cacgggccta 300
attcgtacct atgtgctgga gacctctgat atcgaggagg gtgtggtata tactgcaaca 360
tcctatgagg caggagcttc tgatgtggaa gtgtcttggt tcattggcag tgtgtatgag 420
ctttatggtg cggaagatcg tatctggagt tataacggcg gcggtgaggg gccgatgcac 480
tactatgata cgcttcaaaa gattgacctg accgactctg gcaagttcag tagggagaat 540
aaacaggatg acacggctgc aagtattcct gtgtcagata tttacattgc tgatggaggg 600
attaccgttg gcgatgcttc tgcaaccaga agggaggtac atactccggt tcaggaaacc 660
agtgattcag ctcaagtttc tatcgggtgg ccaggcaaag tcattgccgc cggaagcgtg 720
atcgaaattg gtgagagctt tgctgtagtc catccgggtg actattataa cggcttgaga 780
ggttacaaaa atgcaatgga tcacttgggc gtgattatgc ctgcacctgg ggatattcct 840
gatagcagct atgatctccg atgggaaagc tggggctggg ggtttaactg gacgatcgat 900
ttaataatcg gcaaattgga tgaacttcag gcagccggag tcaagcagat cactttggat 960
gatggttggt ataccaatgc aggagactgg gccttaaatc cagaaaagtt tccaaatgga 1020
gcctccgatg cgttgcggct gacagatgca attcatgagc atggtatgac tgcactcctt 1080
tggtggagac cttgtgacgg cgggatcgat agtatactct atcagcaaca ccctgaatat 1140
ttcgttatgg atgcagatgg aagacctgca aggcttccta ctcctggtgg tgggaccaat 1200
cccagcttgg gatatgcact ttgccctatg gcggatggtg cgattgcaag ccaagttgac 1260
tttgtaaacc gtgcaatgaa tgattggggg ttcgatggct tcaagggaga ttatgtgtgg 1320
agtatgcctg aatgctacaa tcctgcacat aaccacgcct cgccagaaga atccactgaa 1380
aagcaatccg agatataccg cgtctcttat gaggctatgg tggccaacga ccccaatgtg 1440
ttcaatttgt tgtgcaactg cggtacgccc caggactact atagtttacc atatatgaca 1500
cagattgcta cggctgaccc cacttctgtg gatcaaacaa ggagacgcgt gaaagcctac 1560
aaggcactga tgggagatta tttccctgtt acagccgacc acaataacat ctggtatcca 1620
agtgccgtcg gtacgggctc tgttctcatt gaaaaacgtg accttagcgg tactgccaag 1680
gaagaatatg aaaaatggct tgggattgcg gatacagttc agttgcagaa aggccggttt 1740
attggcgatc tttacagtta tggttttgac ccttacgaaa cctatgtggt ggagaaagac 1800
ggggttatgt actatgcctt ctacaaagat gggagcaaat atagccccac tggctatcca 1860
gatattgagt tgaaggggct agatccaaat aaaatgtata ggattgttga ctatgtcaat 1920
gatcgtgtcg tggcaacaaa cctgatgggt gataacgctg tattcaatac acgtttttcc 1980
gactatctac tggttaaagc ggtggaaatt tcggaaccgg atccagaacc tgttgaccct 2040
gattatggtt tcacctctgt tgatgacaga gacgaggctc ttatttacac agggacatgg 2100
catgatgaca ataacgcatc tttcagcgaa gggactgcac gttataccaa cagtacggat 2160
gcttcggttg tattctcctt tactggaact tccattcgct ggtatggcca gagggatacc 2220
aattttggca cggcagaagt ttatttggac gatgaactga aaacaacagt tgatgcgaat 2280
ggggccgcag aagcaggcgt atgtcttttt gaggcgcttg atcttccggc tgccgagcat 2340
accattaaaa ttgtgtgcaa gagcggagtg attgatattg accgctttgc atatgaagct 2400
gctacccttg aacccatcta tgaaaaggtc gatgcgctct cggatcggat cacttatgtt 2460
gggaattggg aagagtatca caacagcgag ttctacatgg gaaacgcaat gcgcacagac 2520
gaagccggcg cttatgctga actgactttc cgtggtacag ccgtacgcct gtatgcagag 2580
atgagcttca attttggcac tgcagatgtc tatttagacg gagagttagt ggaaaacata 2640
atcctatacg gccaggaagc aactgggcag ctaatgtttg agcgtacggg actggaggaa 2700
ggagaacata ccattcgcct tgtacaaaac gcctggaaca tcaatttgga ctatatttct 2760
tatctaccag agcaagatca accaacgccg ccggagacga cggttactgt tgatgcaatg 2820
gacgcccaac tggtgtatac aggcgtatgg aatgatgact atcatgacgt ctttcaggaa 2880
ggaaccgccc gttatgccag tagtgccggc gcctcggtcg agttcgaatt tactggaagc 2940
gaaatccgtt ggtatggaca aaatgattcc aacttcggtg ttgccagcgt ttatatcgat 3000
aatgagtttg tgcagcaggt aaatgttaac ggagctgcgg ctgtgggaaa gcttttgttt 3060
caaaaggctg atctaccagc cggttcgcac acgatccgca ttgtgtgcga tactccggtt 3120
attgatttgg actatttgac ttataccact aacgcataa 3159
<210> 9
<211> 1052
<212> PRT
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 9
Ala Ala Pro Ala Thr Asp Thr Gly Asn Ala Gly Leu Ile Ala Glu Gly
1 5 10 15
Asp Tyr Ala Ile Ala Gly Asn Gly Val Arg Val Thr Tyr Asp Ala Asp
20 25 30
Gly Gln Thr Ile Thr Leu Tyr Arg Thr Glu Gly Ser Gly Leu Ile Gln
35 40 45
Met Ser Lys Pro Ser Pro Leu Gly Gly Pro Val Ile Gly Gly Gln Glu
50 55 60
Val Gln Asp Phe Ser His Ile Ser Cys Asp Val Glu Gln Ser Thr Ser
65 70 75 80
Gly Val Met Gly Ser Gly Gln Arg Met Thr Ile Thr Ser Gln Ser Met
85 90 95
Ser Thr Gly Leu Ile Arg Thr Tyr Val Leu Glu Thr Ser Asp Ile Glu
100 105 110
Glu Gly Val Val Tyr Thr Ala Thr Ser Tyr Glu Ala Gly Ala Ser Asp
115 120 125
Val Glu Val Ser Trp Phe Ile Gly Ser Val Tyr Glu Leu Tyr Gly Ala
130 135 140
Glu Asp Arg Ile Trp Ser Tyr Asn Gly Gly Gly Glu Gly Pro Met His
145 150 155 160
Tyr Tyr Asp Thr Leu Gln Lys Ile Asp Leu Thr Asp Ser Gly Lys Phe
165 170 175
Ser Arg Glu Asn Lys Gln Asp Asp Thr Ala Ala Ser Ile Pro Val Ser
180 185 190
Asp Ile Tyr Ile Ala Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Ala
195 200 205
Thr Arg Arg Glu Val His Thr Pro Val Gln Glu Thr Ser Asp Ser Ala
210 215 220
Gln Val Ser Ile Gly Trp Pro Gly Lys Val Ile Ala Ala Gly Ser Val
225 230 235 240
Ile Glu Ile Gly Glu Ser Phe Ala Val Val His Pro Gly Asp Tyr Tyr
245 250 255
Asn Gly Leu Arg Gly Tyr Lys Asn Ala Met Asp His Leu Gly Val Ile
260 265 270
Met Pro Ala Pro Gly Asp Ile Pro Asp Ser Ser Tyr Asp Leu Arg Trp
275 280 285
Glu Ser Trp Gly Trp Gly Phe Asn Trp Thr Ile Asp Leu Ile Ile Gly
290 295 300
Lys Leu Asp Glu Leu Gln Ala Ala Gly Val Lys Gln Ile Thr Leu Asp
305 310 315 320
Asp Gly Trp Tyr Thr Asn Ala Gly Asp Trp Ala Leu Asn Pro Glu Lys
325 330 335
Phe Pro Asn Gly Ala Ser Asp Ala Leu Arg Leu Thr Asp Ala Ile His
340 345 350
Glu His Gly Met Thr Ala Leu Leu Trp Trp Arg Pro Cys Asp Gly Gly
355 360 365
Ile Asp Ser Ile Leu Tyr Gln Gln His Pro Glu Tyr Phe Val Met Asp
370 375 380
Ala Asp Gly Arg Pro Ala Arg Leu Pro Thr Pro Gly Gly Gly Thr Asn
385 390 395 400
Pro Ser Leu Gly Tyr Ala Leu Cys Pro Met Ala Asp Gly Ala Ile Ala
405 410 415
Ser Gln Val Asp Phe Val Asn Arg Ala Met Asn Asp Trp Gly Phe Asp
420 425 430
Gly Phe Lys Gly Asp Tyr Val Trp Ser Met Pro Glu Cys Tyr Asn Pro
435 440 445
Ala His Asn His Ala Ser Pro Glu Glu Ser Thr Glu Lys Gln Ser Glu
450 455 460
Ile Tyr Arg Val Ser Tyr Glu Ala Met Val Ala Asn Asp Pro Asn Val
465 470 475 480
Phe Asn Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu
485 490 495
Pro Tyr Met Thr Gln Ile Ala Thr Ala Asp Pro Thr Ser Val Asp Gln
500 505 510
Thr Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Met Gly Asp Tyr Phe
515 520 525
Pro Val Thr Ala Asp His Asn Asn Ile Trp Tyr Pro Ser Ala Val Gly
530 535 540
Thr Gly Ser Val Leu Ile Glu Lys Arg Asp Leu Ser Gly Thr Ala Lys
545 550 555 560
Glu Glu Tyr Glu Lys Trp Leu Gly Ile Ala Asp Thr Val Gln Leu Gln
565 570 575
Lys Gly Arg Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Phe Asp Pro Tyr
580 585 590
Glu Thr Tyr Val Val Glu Lys Asp Gly Val Met Tyr Tyr Ala Phe Tyr
595 600 605
Lys Asp Gly Ser Lys Tyr Ser Pro Thr Gly Tyr Pro Asp Ile Glu Leu
610 615 620
Lys Gly Leu Asp Pro Asn Lys Met Tyr Arg Ile Val Asp Tyr Val Asn
625 630 635 640
Asp Arg Val Val Ala Thr Asn Leu Met Gly Asp Asn Ala Val Phe Asn
645 650 655
Thr Arg Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile Ser Glu
660 665 670
Pro Asp Pro Glu Pro Val Asp Pro Asp Tyr Gly Phe Thr Ser Val Asp
675 680 685
Asp Arg Asp Glu Ala Leu Ile Tyr Thr Gly Thr Trp His Asp Asp Asn
690 695 700
Asn Ala Ser Phe Ser Glu Gly Thr Ala Arg Tyr Thr Asn Ser Thr Asp
705 710 715 720
Ala Ser Val Val Phe Ser Phe Thr Gly Thr Ser Ile Arg Trp Tyr Gly
725 730 735
Gln Arg Asp Thr Asn Phe Gly Thr Ala Glu Val Tyr Leu Asp Asp Glu
740 745 750
Leu Lys Thr Thr Val Asp Ala Asn Gly Ala Ala Glu Ala Gly Val Cys
755 760 765
Leu Phe Glu Ala Leu Asp Leu Pro Ala Ala Glu His Thr Ile Lys Ile
770 775 780
Val Cys Lys Ser Gly Val Ile Asp Ile Asp Arg Phe Ala Tyr Glu Ala
785 790 795 800
Ala Thr Leu Glu Pro Ile Tyr Glu Lys Val Asp Ala Leu Ser Asp Arg
805 810 815
Ile Thr Tyr Val Gly Asn Trp Glu Glu Tyr His Asn Ser Glu Phe Tyr
820 825 830
Met Gly Asn Ala Met Arg Thr Asp Glu Ala Gly Ala Tyr Ala Glu Leu
835 840 845
Thr Phe Arg Gly Thr Ala Val Arg Leu Tyr Ala Glu Met Ser Phe Asn
850 855 860
Phe Gly Thr Ala Asp Val Tyr Leu Asp Gly Glu Leu Val Glu Asn Ile
865 870 875 880
Ile Leu Tyr Gly Gln Glu Ala Thr Gly Gln Leu Met Phe Glu Arg Thr
885 890 895
Gly Leu Glu Glu Gly Glu His Thr Ile Arg Leu Val Gln Asn Ala Trp
900 905 910
Asn Ile Asn Leu Asp Tyr Ile Ser Tyr Leu Pro Glu Gln Asp Gln Pro
915 920 925
Thr Pro Pro Glu Thr Thr Val Thr Val Asp Ala Met Asp Ala Gln Leu
930 935 940
Val Tyr Thr Gly Val Trp Asn Asp Asp Tyr His Asp Val Phe Gln Glu
945 950 955 960
Gly Thr Ala Arg Tyr Ala Ser Ser Ala Gly Ala Ser Val Glu Phe Glu
965 970 975
Phe Thr Gly Ser Glu Ile Arg Trp Tyr Gly Gln Asn Asp Ser Asn Phe
980 985 990
Gly Val Ala Ser Val Tyr Ile Asp Asn Glu Phe Val Gln Gln Val Asn
995 1000 1005
Val Asn Gly Ala Ala Ala Val Gly Lys Leu Leu Phe Gln Lys Ala
1010 1015 1020
Asp Leu Pro Ala Gly Ser His Thr Ile Arg Ile Val Cys Asp Thr
1025 1030 1035
Pro Val Ile Asp Leu Asp Tyr Leu Thr Tyr Thr Thr Asn Ala
1040 1045 1050
<210> 10
<211> 1067
<212> PRT
<213> 普氏梭杆菌(Flavonifractor plautii)
<400> 10
Met Gly His His His His His His His His His His Ser Ser Gly Ala
1 5 10 15
Ala Pro Ala Thr Asp Thr Gly Asn Ala Gly Leu Ile Ala Glu Gly Asp
20 25 30
Tyr Ala Ile Ala Gly Asn Gly Val Arg Val Thr Tyr Asp Ala Asp Gly
35 40 45
Gln Thr Ile Thr Leu Tyr Arg Thr Glu Gly Ser Gly Leu Ile Gln Met
50 55 60
Ser Lys Pro Ser Pro Leu Gly Gly Pro Val Ile Gly Gly Gln Glu Val
65 70 75 80
Gln Asp Phe Ser His Ile Ser Cys Asp Val Glu Gln Ser Thr Ser Gly
85 90 95
Val Met Gly Ser Gly Gln Arg Met Thr Ile Thr Ser Gln Ser Met Ser
100 105 110
Thr Gly Leu Ile Arg Thr Tyr Val Leu Glu Thr Ser Asp Ile Glu Glu
115 120 125
Gly Val Val Tyr Thr Ala Thr Ser Tyr Glu Ala Gly Ala Ser Asp Val
130 135 140
Glu Val Ser Trp Phe Ile Gly Ser Val Tyr Glu Leu Tyr Gly Ala Glu
145 150 155 160
Asp Arg Ile Trp Ser Tyr Asn Gly Gly Gly Glu Gly Pro Met His Tyr
165 170 175
Tyr Asp Thr Leu Gln Lys Ile Asp Leu Thr Asp Ser Gly Lys Phe Ser
180 185 190
Arg Glu Asn Lys Gln Asp Asp Thr Ala Ala Ser Ile Pro Val Ser Asp
195 200 205
Ile Tyr Ile Ala Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Ala Thr
210 215 220
Arg Arg Glu Val His Thr Pro Val Gln Glu Thr Ser Asp Ser Ala Gln
225 230 235 240
Val Ser Ile Gly Trp Pro Gly Lys Val Ile Ala Ala Gly Ser Val Ile
245 250 255
Glu Ile Gly Glu Ser Phe Ala Val Val His Pro Gly Asp Tyr Tyr Asn
260 265 270
Gly Leu Arg Gly Tyr Lys Asn Ala Met Asp His Leu Gly Val Ile Met
275 280 285
Pro Ala Pro Gly Asp Ile Pro Asp Ser Ser Tyr Asp Leu Arg Trp Glu
290 295 300
Ser Trp Gly Trp Gly Phe Asn Trp Thr Ile Asp Leu Ile Ile Gly Lys
305 310 315 320
Leu Asp Glu Leu Gln Ala Ala Gly Val Lys Gln Ile Thr Leu Asp Asp
325 330 335
Gly Trp Tyr Thr Asn Ala Gly Asp Trp Ala Leu Asn Pro Glu Lys Phe
340 345 350
Pro Asn Gly Ala Ser Asp Ala Leu Arg Leu Thr Asp Ala Ile His Glu
355 360 365
His Gly Met Thr Ala Leu Leu Trp Trp Arg Pro Cys Asp Gly Gly Ile
370 375 380
Asp Ser Ile Leu Tyr Gln Gln His Pro Glu Tyr Phe Val Met Asp Ala
385 390 395 400
Asp Gly Arg Pro Ala Arg Leu Pro Thr Pro Gly Gly Gly Thr Asn Pro
405 410 415
Ser Leu Gly Tyr Ala Leu Cys Pro Met Ala Asp Gly Ala Ile Ala Ser
420 425 430
Gln Val Asp Phe Val Asn Arg Ala Met Asn Asp Trp Gly Phe Asp Gly
435 440 445
Phe Lys Gly Asp Tyr Val Trp Ser Met Pro Glu Cys Tyr Asn Pro Ala
450 455 460
His Asn His Ala Ser Pro Glu Glu Ser Thr Glu Lys Gln Ser Glu Ile
465 470 475 480
Tyr Arg Val Ser Tyr Glu Ala Met Val Ala Asn Asp Pro Asn Val Phe
485 490 495
Asn Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu Pro
500 505 510
Tyr Met Thr Gln Ile Ala Thr Ala Asp Pro Thr Ser Val Asp Gln Thr
515 520 525
Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Met Gly Asp Tyr Phe Pro
530 535 540
Val Thr Ala Asp His Asn Asn Ile Trp Tyr Pro Ser Ala Val Gly Thr
545 550 555 560
Gly Ser Val Leu Ile Glu Lys Arg Asp Leu Ser Gly Thr Ala Lys Glu
565 570 575
Glu Tyr Glu Lys Trp Leu Gly Ile Ala Asp Thr Val Gln Leu Gln Lys
580 585 590
Gly Arg Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Phe Asp Pro Tyr Glu
595 600 605
Thr Tyr Val Val Glu Lys Asp Gly Val Met Tyr Tyr Ala Phe Tyr Lys
610 615 620
Asp Gly Ser Lys Tyr Ser Pro Thr Gly Tyr Pro Asp Ile Glu Leu Lys
625 630 635 640
Gly Leu Asp Pro Asn Lys Met Tyr Arg Ile Val Asp Tyr Val Asn Asp
645 650 655
Arg Val Val Ala Thr Asn Leu Met Gly Asp Asn Ala Val Phe Asn Thr
660 665 670
Arg Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile Ser Glu Pro
675 680 685
Asp Pro Glu Pro Val Asp Pro Asp Tyr Gly Phe Thr Ser Val Asp Asp
690 695 700
Arg Asp Glu Ala Leu Ile Tyr Thr Gly Thr Trp His Asp Asp Asn Asn
705 710 715 720
Ala Ser Phe Ser Glu Gly Thr Ala Arg Tyr Thr Asn Ser Thr Asp Ala
725 730 735
Ser Val Val Phe Ser Phe Thr Gly Thr Ser Ile Arg Trp Tyr Gly Gln
740 745 750
Arg Asp Thr Asn Phe Gly Thr Ala Glu Val Tyr Leu Asp Asp Glu Leu
755 760 765
Lys Thr Thr Val Asp Ala Asn Gly Ala Ala Glu Ala Gly Val Cys Leu
770 775 780
Phe Glu Ala Leu Asp Leu Pro Ala Ala Glu His Thr Ile Lys Ile Val
785 790 795 800
Cys Lys Ser Gly Val Ile Asp Ile Asp Arg Phe Ala Tyr Glu Ala Ala
805 810 815
Thr Leu Glu Pro Ile Tyr Glu Lys Val Asp Ala Leu Ser Asp Arg Ile
820 825 830
Thr Tyr Val Gly Asn Trp Glu Glu Tyr His Asn Ser Glu Phe Tyr Met
835 840 845
Gly Asn Ala Met Arg Thr Asp Glu Ala Gly Ala Tyr Ala Glu Leu Thr
850 855 860
Phe Arg Gly Thr Ala Val Arg Leu Tyr Ala Glu Met Ser Phe Asn Phe
865 870 875 880
Gly Thr Ala Asp Val Tyr Leu Asp Gly Glu Leu Val Glu Asn Ile Ile
885 890 895
Leu Tyr Gly Gln Glu Ala Thr Gly Gln Leu Met Phe Glu Arg Thr Gly
900 905 910
Leu Glu Glu Gly Glu His Thr Ile Arg Leu Val Gln Asn Ala Trp Asn
915 920 925
Ile Asn Leu Asp Tyr Ile Ser Tyr Leu Pro Glu Gln Asp Gln Pro Thr
930 935 940
Pro Pro Glu Thr Thr Val Thr Val Asp Ala Met Asp Ala Gln Leu Val
945 950 955 960
Tyr Thr Gly Val Trp Asn Asp Asp Tyr His Asp Val Phe Gln Glu Gly
965 970 975
Thr Ala Arg Tyr Ala Ser Ser Ala Gly Ala Ser Val Glu Phe Glu Phe
980 985 990
Thr Gly Ser Glu Ile Arg Trp Tyr Gly Gln Asn Asp Ser Asn Phe Gly
995 1000 1005
Val Ala Ser Val Tyr Ile Asp Asn Glu Phe Val Gln Gln Val Asn
1010 1015 1020
Val Asn Gly Ala Ala Ala Val Gly Lys Leu Leu Phe Gln Lys Ala
1025 1030 1035
Asp Leu Pro Ala Gly Ser His Thr Ile Arg Ile Val Cys Asp Thr
1040 1045 1050
Pro Val Ile Asp Leu Asp Tyr Leu Thr Tyr Thr Thr Asn Ala
1055 1060 1065
<210> 11
<211> 3963
<212> DNA
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 11
atgaaaaaaa gaattttagc tacttttatt acagctatgt gtggactggg atttttttca 60
aactggactt caagtaatgc ttataattta attgataata ttagtgttga aaaattagat 120
actgatattt cacaagcaaa tgaaaatgtt tttttgaatg gaaatggaat tgctttagaa 180
gtagataata gaggcgctac atgtatttat ctagtagatg aaaatggagt taaaacaaaa 240
gctacgactt ctttagatac agcagatttt tcaggttatc caataatagg tggacaaaag 300
ataagagatt ttgtaattat atcaaaaaat ctagaagaaa acataaactc gatattaggt 360
gttggaaata gacttactat tatatctaaa agttcatcta ctaatctgat aagaaagata 420
gtatttgaaa catctaacag caatccagga gcaatatatt caacagtaag ttataaagca 480
gaaagtaacg atttattagt agatagcttt catgaaaatg agtatacaat gagtttaggg 540
caaggacctt ttcttgcata tcaagggtgt gcagatcaac aaggagcaaa tactatcgtt 600
aatgttacta atggatataa ccataatagt ggacaaaata attattctgt aggagttcca 660
tttagttatg tttataactc tgtgggggga attggaatag gtgatgcatc aacttcaaga 720
agagaattta agttgcctat tataggaaaa gataatacag tttcattagg aatggagtgg 780
aatggacaaa ctttaaaaaa aggtgctgaa actgctatag gtacaagtgt tataactaca 840
acaaatggtg attattattc tgggctaaag agttacgcag aagttatgaa agataaggga 900
atatctgcac cagcttcaat acctgatata gcatatgatt ctagatggga aagttgggga 960
ttcgaatttg attttacaat agaaaaaata gttaataaat tagatgaact taaagcgatg 1020
gggataaaac aaattactct agatgatggg tggtacactt atgctggtga ttggaaatta 1080
agtcctcaaa agtttccaaa tggaaatgca gacatgaaat atcttacaga tgaaatccat 1140
aaaagaggaa tgacagctat tttatggtgg agaccagtag acggagggat aaatagcaaa 1200
ttagtatctg aacatccaga gtggtttatt aagaactcac aagggaatat ggttaggtta 1260
ccagggcctg gaggtggaaa tggaggaaca gcaggatatg cattatgtcc aaattcagaa 1320
ggttcaattc aacatcataa agattttgta actgtggcat tagaagaatg gggatttgat 1380
ggattcaaag aagattatgt atggggaata cctaaatgct atgatagttc tcataaacac 1440
tcaagtttat cagatacatt agaaaatcaa tataaattct atgaagccat atatgaacag 1500
tccatagcga taaatccaga tacttttata gaattatgta attgcggaac acctcaggat 1560
ttttattcaa caccatatgt gaaccatgca ccaacagcag atccaatttc gagagtacaa 1620
acaagaacaa gagtgaaagc atttaaagct atatttggag atgattttcc agtaacaaca 1680
gatcataatt cagtttggtt accgtcagca ttaggtacag gatcagttat gattactaaa 1740
catacaacat taagtagttc agatagagaa caatataata aatacttcgg acttgcaaga 1800
gatttagaat tagcaaaggg agaatttata ggaaacttat ataaatacgg aatagatcca 1860
ttagagtcat atgttataag aaaaggagaa gatatttatt attcattcta caaagataat 1920
tctagttatt caggaaatat agaaataaag gggttagaca gtaacgccac atatagaatt 1980
gaagattatg ttaacaatag agttattgct agaggagtaa agggaccaac agcgactata 2040
aatacaagct ttactgataa tttattagtt agagcaatac cagatgatac accagcagag 2100
gttactacat ttgatgttgg aaataataca atattatcat caacagatag tggaaattct 2160
aaatatttaa atgctgtttc tactacatta gaaaagacag caacaataga tagtttaagt 2220
atttatatag gaaataattc agaaaatggc aaactacaaa ttgctattta tgacgataat 2280
aacgggaaac ctggtactaa aaaagcttac gtagaagagt ttgttcctac taaaaatagt 2340
tggaatacaa agaaggttgt aaattctgtt acattacctt cagggcaata ttggttagtt 2400
ttccaacctg ataacgatgt actacaaaca aaaactaatc catcatccat gaaacaaagt 2460
gctaacaata atccatataa ttataatata ttaccaaatt catttcctat tggaacagga 2520
tataatgctt ataaaggcga tgtatctttc tatgcaacct ttaaagaagc aagcagtcaa 2580
gcaattcctc aaaattcttg ggctctaaaa tatgtagata gtgaagaaac tacaggcgaa 2640
aatggaagag ctacaaatgc ttttgatggt aataataata ctatttggca cacaaaatat 2700
agtggcggaa acgctgcacc aatgccgcat gagattcaaa ttgatttaag aggagtatat 2760
aatataaatc aaattaatta tctaccaaga caagatggag gaaccaatgg tacaataaag 2820
gactatgaag tttatttaag tttagatgga gtgaactggg gacaacctat atcaaaagga 2880
acctttgaat caaactctac agaaaaaata gtaaaattca acgaaacaaa atctaggtat 2940
gtaaaactta aagctctgtc agaaattaat aataaacaat ttactacagt agctgattta 3000
aaggtatttg gatgggagat atccaaaata gaaaaaccat tacaaaatgc tgaaacttat 3060
ttgaatatac caacttatga tggattaaat caaagtactc atccagatgt caaatatttt 3120
aaaaatggtt ggaatggata taaatattgg atgataatga ctccaaatag aacaggtagc 3180
tcagttgctg aaaatccttc aatactagca tctgatgatg gaataaattg ggaggttcct 3240
gcaggtgtta caaatcctat agctccaatg ccacaagtag gacataattg tgatgttgat 3300
atgatatata atgaagcaac tgatgagtta tgggtgtact gggtagaatc agatgatata 3360
acaaaaggat gggttaaatt aataaaatca aaggatggag taaattggag ttctcagcaa 3420
gtggtagttg atgataatag ggcaaaatat agtactttat caccatctat aatattcaaa 3480
gataataaat actatatgtg gtcagttaat acaggaaata gtggttggaa caatcaaagt 3540
aataaagttg aattaagaga atcaagtgac ggagtaaatt ggtcaaatcc aacagttgta 3600
aacacattag ctcaagatgg ttctcaaata tggcatgtaa atgtagaata tataccatca 3660
aaaaacgaat attgggctat atatccagca tataaaaatg gaacaggtag cgataaaaca 3720
gaattgtatt atgcgaaatc aagtgatgga gtaaattgga caacttataa gaatcctata 3780
ttatcaaaag gaacatctgg taaatgggat gatatggaga tatatagaag ttgttttgtg 3840
tacgatgaag atacaaatat gataaaggtt tggtatggag ctgtgagtca aaatccacaa 3900
atatggaaaa taggttttac tgaaaatgat tatgataagt ttattgaggg tttaacacaa 3960
taa 3963
<210> 12
<211> 1320
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 12
Met Lys Lys Arg Ile Leu Ala Thr Phe Ile Thr Ala Met Cys Gly Leu
1 5 10 15
Gly Phe Phe Ser Asn Trp Thr Ser Ser Asn Ala Tyr Asn Leu Ile Asp
20 25 30
Asn Ile Ser Val Glu Lys Leu Asp Thr Asp Ile Ser Gln Ala Asn Glu
35 40 45
Asn Val Phe Leu Asn Gly Asn Gly Ile Ala Leu Glu Val Asp Asn Arg
50 55 60
Gly Ala Thr Cys Ile Tyr Leu Val Asp Glu Asn Gly Val Lys Thr Lys
65 70 75 80
Ala Thr Thr Ser Leu Asp Thr Ala Asp Phe Ser Gly Tyr Pro Ile Ile
85 90 95
Gly Gly Gln Lys Ile Arg Asp Phe Val Ile Ile Ser Lys Asn Leu Glu
100 105 110
Glu Asn Ile Asn Ser Ile Leu Gly Val Gly Asn Arg Leu Thr Ile Ile
115 120 125
Ser Lys Ser Ser Ser Thr Asn Leu Ile Arg Lys Ile Val Phe Glu Thr
130 135 140
Ser Asn Ser Asn Pro Gly Ala Ile Tyr Ser Thr Val Ser Tyr Lys Ala
145 150 155 160
Glu Ser Asn Asp Leu Leu Val Asp Ser Phe His Glu Asn Glu Tyr Thr
165 170 175
Met Ser Leu Gly Gln Gly Pro Phe Leu Ala Tyr Gln Gly Cys Ala Asp
180 185 190
Gln Gln Gly Ala Asn Thr Ile Val Asn Val Thr Asn Gly Tyr Asn His
195 200 205
Asn Ser Gly Gln Asn Asn Tyr Ser Val Gly Val Pro Phe Ser Tyr Val
210 215 220
Tyr Asn Ser Val Gly Gly Ile Gly Ile Gly Asp Ala Ser Thr Ser Arg
225 230 235 240
Arg Glu Phe Lys Leu Pro Ile Ile Gly Lys Asp Asn Thr Val Ser Leu
245 250 255
Gly Met Glu Trp Asn Gly Gln Thr Leu Lys Lys Gly Ala Glu Thr Ala
260 265 270
Ile Gly Thr Ser Val Ile Thr Thr Thr Asn Gly Asp Tyr Tyr Ser Gly
275 280 285
Leu Lys Ser Tyr Ala Glu Val Met Lys Asp Lys Gly Ile Ser Ala Pro
290 295 300
Ala Ser Ile Pro Asp Ile Ala Tyr Asp Ser Arg Trp Glu Ser Trp Gly
305 310 315 320
Phe Glu Phe Asp Phe Thr Ile Glu Lys Ile Val Asn Lys Leu Asp Glu
325 330 335
Leu Lys Ala Met Gly Ile Lys Gln Ile Thr Leu Asp Asp Gly Trp Tyr
340 345 350
Thr Tyr Ala Gly Asp Trp Lys Leu Ser Pro Gln Lys Phe Pro Asn Gly
355 360 365
Asn Ala Asp Met Lys Tyr Leu Thr Asp Glu Ile His Lys Arg Gly Met
370 375 380
Thr Ala Ile Leu Trp Trp Arg Pro Val Asp Gly Gly Ile Asn Ser Lys
385 390 395 400
Leu Val Ser Glu His Pro Glu Trp Phe Ile Lys Asn Ser Gln Gly Asn
405 410 415
Met Val Arg Leu Pro Gly Pro Gly Gly Gly Asn Gly Gly Thr Ala Gly
420 425 430
Tyr Ala Leu Cys Pro Asn Ser Glu Gly Ser Ile Gln His His Lys Asp
435 440 445
Phe Val Thr Val Ala Leu Glu Glu Trp Gly Phe Asp Gly Phe Lys Glu
450 455 460
Asp Tyr Val Trp Gly Ile Pro Lys Cys Tyr Asp Ser Ser His Lys His
465 470 475 480
Ser Ser Leu Ser Asp Thr Leu Glu Asn Gln Tyr Lys Phe Tyr Glu Ala
485 490 495
Ile Tyr Glu Gln Ser Ile Ala Ile Asn Pro Asp Thr Phe Ile Glu Leu
500 505 510
Cys Asn Cys Gly Thr Pro Gln Asp Phe Tyr Ser Thr Pro Tyr Val Asn
515 520 525
His Ala Pro Thr Ala Asp Pro Ile Ser Arg Val Gln Thr Arg Thr Arg
530 535 540
Val Lys Ala Phe Lys Ala Ile Phe Gly Asp Asp Phe Pro Val Thr Thr
545 550 555 560
Asp His Asn Ser Val Trp Leu Pro Ser Ala Leu Gly Thr Gly Ser Val
565 570 575
Met Ile Thr Lys His Thr Thr Leu Ser Ser Ser Asp Arg Glu Gln Tyr
580 585 590
Asn Lys Tyr Phe Gly Leu Ala Arg Asp Leu Glu Leu Ala Lys Gly Glu
595 600 605
Phe Ile Gly Asn Leu Tyr Lys Tyr Gly Ile Asp Pro Leu Glu Ser Tyr
610 615 620
Val Ile Arg Lys Gly Glu Asp Ile Tyr Tyr Ser Phe Tyr Lys Asp Asn
625 630 635 640
Ser Ser Tyr Ser Gly Asn Ile Glu Ile Lys Gly Leu Asp Ser Asn Ala
645 650 655
Thr Tyr Arg Ile Glu Asp Tyr Val Asn Asn Arg Val Ile Ala Arg Gly
660 665 670
Val Lys Gly Pro Thr Ala Thr Ile Asn Thr Ser Phe Thr Asp Asn Leu
675 680 685
Leu Val Arg Ala Ile Pro Asp Asp Thr Pro Ala Glu Val Thr Thr Phe
690 695 700
Asp Val Gly Asn Asn Thr Ile Leu Ser Ser Thr Asp Ser Gly Asn Ser
705 710 715 720
Lys Tyr Leu Asn Ala Val Ser Thr Thr Leu Glu Lys Thr Ala Thr Ile
725 730 735
Asp Ser Leu Ser Ile Tyr Ile Gly Asn Asn Ser Glu Asn Gly Lys Leu
740 745 750
Gln Ile Ala Ile Tyr Asp Asp Asn Asn Gly Lys Pro Gly Thr Lys Lys
755 760 765
Ala Tyr Val Glu Glu Phe Val Pro Thr Lys Asn Ser Trp Asn Thr Lys
770 775 780
Lys Val Val Asn Ser Val Thr Leu Pro Ser Gly Gln Tyr Trp Leu Val
785 790 795 800
Phe Gln Pro Asp Asn Asp Val Leu Gln Thr Lys Thr Asn Pro Ser Ser
805 810 815
Met Lys Gln Ser Ala Asn Asn Asn Pro Tyr Asn Tyr Asn Ile Leu Pro
820 825 830
Asn Ser Phe Pro Ile Gly Thr Gly Tyr Asn Ala Tyr Lys Gly Asp Val
835 840 845
Ser Phe Tyr Ala Thr Phe Lys Glu Ala Ser Ser Gln Ala Ile Pro Gln
850 855 860
Asn Ser Trp Ala Leu Lys Tyr Val Asp Ser Glu Glu Thr Thr Gly Glu
865 870 875 880
Asn Gly Arg Ala Thr Asn Ala Phe Asp Gly Asn Asn Asn Thr Ile Trp
885 890 895
His Thr Lys Tyr Ser Gly Gly Asn Ala Ala Pro Met Pro His Glu Ile
900 905 910
Gln Ile Asp Leu Arg Gly Val Tyr Asn Ile Asn Gln Ile Asn Tyr Leu
915 920 925
Pro Arg Gln Asp Gly Gly Thr Asn Gly Thr Ile Lys Asp Tyr Glu Val
930 935 940
Tyr Leu Ser Leu Asp Gly Val Asn Trp Gly Gln Pro Ile Ser Lys Gly
945 950 955 960
Thr Phe Glu Ser Asn Ser Thr Glu Lys Ile Val Lys Phe Asn Glu Thr
965 970 975
Lys Ser Arg Tyr Val Lys Leu Lys Ala Leu Ser Glu Ile Asn Asn Lys
980 985 990
Gln Phe Thr Thr Val Ala Asp Leu Lys Val Phe Gly Trp Glu Ile Ser
995 1000 1005
Lys Ile Glu Lys Pro Leu Gln Asn Ala Glu Thr Tyr Leu Asn Ile
1010 1015 1020
Pro Thr Tyr Asp Gly Leu Asn Gln Ser Thr His Pro Asp Val Lys
1025 1030 1035
Tyr Phe Lys Asn Gly Trp Asn Gly Tyr Lys Tyr Trp Met Ile Met
1040 1045 1050
Thr Pro Asn Arg Thr Gly Ser Ser Val Ala Glu Asn Pro Ser Ile
1055 1060 1065
Leu Ala Ser Asp Asp Gly Ile Asn Trp Glu Val Pro Ala Gly Val
1070 1075 1080
Thr Asn Pro Ile Ala Pro Met Pro Gln Val Gly His Asn Cys Asp
1085 1090 1095
Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu Leu Trp Val Tyr
1100 1105 1110
Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val Lys Leu Ile
1115 1120 1125
Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln Val Val Val
1130 1135 1140
Asp Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro Ser Ile Ile
1145 1150 1155
Phe Lys Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn Thr Gly Asn
1160 1165 1170
Ser Gly Trp Asn Asn Gln Ser Asn Lys Val Glu Leu Arg Glu Ser
1175 1180 1185
Ser Asp Gly Val Asn Trp Ser Asn Pro Thr Val Val Asn Thr Leu
1190 1195 1200
Ala Gln Asp Gly Ser Gln Ile Trp His Val Asn Val Glu Tyr Ile
1205 1210 1215
Pro Ser Lys Asn Glu Tyr Trp Ala Ile Tyr Pro Ala Tyr Lys Asn
1220 1225 1230
Gly Thr Gly Ser Asp Lys Thr Glu Leu Tyr Tyr Ala Lys Ser Ser
1235 1240 1245
Asp Gly Val Asn Trp Thr Thr Tyr Lys Asn Pro Ile Leu Ser Lys
1250 1255 1260
Gly Thr Ser Gly Lys Trp Asp Asp Met Glu Ile Tyr Arg Ser Cys
1265 1270 1275
Phe Val Tyr Asp Glu Asp Thr Asn Met Ile Lys Val Trp Tyr Gly
1280 1285 1290
Ala Val Ser Gln Asn Pro Gln Ile Trp Lys Ile Gly Phe Thr Glu
1295 1300 1305
Asn Asp Tyr Asp Lys Phe Ile Glu Gly Leu Thr Gln
1310 1315 1320
<210> 13
<211> 3882
<212> DNA
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 13
tataatttaa ttgataatat tagtgttgaa aaattagata ctgatatttc acaagcaaat 60
gaaaatgttt ttttgaatgg aaatggaatt gctttagaag tagataatag aggcgctaca 120
tgtatttatc tagtagatga aaatggagtt aaaacaaaag ctacgacttc tttagataca 180
gcagattttt caggttatcc aataataggt ggacaaaaga taagagattt tgtaattata 240
tcaaaaaatc tagaagaaaa cataaactcg atattaggtg ttggaaatag acttactatt 300
atatctaaaa gttcatctac taatctgata agaaagatag tatttgaaac atctaacagc 360
aatccaggag caatatattc aacagtaagt tataaagcag aaagtaacga tttattagta 420
gatagctttc atgaaaatga gtatacaatg agtttagggc aaggaccttt tcttgcatat 480
caagggtgtg cagatcaaca aggagcaaat actatcgtta atgttactaa tggatataac 540
cataatagtg gacaaaataa ttattctgta ggagttccat ttagttatgt ttataactct 600
gtggggggaa ttggaatagg tgatgcatca acttcaagaa gagaatttaa gttgcctatt 660
ataggaaaag ataatacagt ttcattagga atggagtgga atggacaaac tttaaaaaaa 720
ggtgctgaaa ctgctatagg tacaagtgtt ataactacaa caaatggtga ttattattct 780
gggctaaaga gttacgcaga agttatgaaa gataagggaa tatctgcacc agcttcaata 840
cctgatatag catatgattc tagatgggaa agttggggat tcgaatttga ttttacaata 900
gaaaaaatag ttaataaatt agatgaactt aaagcgatgg ggataaaaca aattactcta 960
gatgatgggt ggtacactta tgctggtgat tggaaattaa gtcctcaaaa gtttccaaat 1020
ggaaatgcag acatgaaata tcttacagat gaaatccata aaagaggaat gacagctatt 1080
ttatggtgga gaccagtaga cggagggata aatagcaaat tagtatctga acatccagag 1140
tggtttatta agaactcaca agggaatatg gttaggttac cagggcctgg aggtggaaat 1200
ggaggaacag caggatatgc attatgtcca aattcagaag gttcaattca acatcataaa 1260
gattttgtaa ctgtggcatt agaagaatgg ggatttgatg gattcaaaga agattatgta 1320
tggggaatac ctaaatgcta tgatagttct cataaacact caagtttatc agatacatta 1380
gaaaatcaat ataaattcta tgaagccata tatgaacagt ccatagcgat aaatccagat 1440
acttttatag aattatgtaa ttgcggaaca cctcaggatt tttattcaac accatatgtg 1500
aaccatgcac caacagcaga tccaatttcg agagtacaaa caagaacaag agtgaaagca 1560
tttaaagcta tatttggaga tgattttcca gtaacaacag atcataattc agtttggtta 1620
ccgtcagcat taggtacagg atcagttatg attactaaac atacaacatt aagtagttca 1680
gatagagaac aatataataa atacttcgga cttgcaagag atttagaatt agcaaaggga 1740
gaatttatag gaaacttata taaatacgga atagatccat tagagtcata tgttataaga 1800
aaaggagaag atatttatta ttcattctac aaagataatt ctagttattc aggaaatata 1860
gaaataaagg ggttagacag taacgccaca tatagaattg aagattatgt taacaataga 1920
gttattgcta gaggagtaaa gggaccaaca gcgactataa atacaagctt tactgataat 1980
ttattagtta gagcaatacc agatgataca ccagcagagg ttactacatt tgatgttgga 2040
aataatacaa tattatcatc aacagatagt ggaaattcta aatatttaaa tgctgtttct 2100
actacattag aaaagacagc aacaatagat agtttaagta tttatatagg aaataattca 2160
gaaaatggca aactacaaat tgctatttat gacgataata acgggaaacc tggtactaaa 2220
aaagcttacg tagaagagtt tgttcctact aaaaatagtt ggaatacaaa gaaggttgta 2280
aattctgtta cattaccttc agggcaatat tggttagttt tccaacctga taacgatgta 2340
ctacaaacaa aaactaatcc atcatccatg aaacaaagtg ctaacaataa tccatataat 2400
tataatatat taccaaattc atttcctatt ggaacaggat ataatgctta taaaggcgat 2460
gtatctttct atgcaacctt taaagaagca agcagtcaag caattcctca aaattcttgg 2520
gctctaaaat atgtagatag tgaagaaact acaggcgaaa atggaagagc tacaaatgct 2580
tttgatggta ataataatac tatttggcac acaaaatata gtggcggaaa cgctgcacca 2640
atgccgcatg agattcaaat tgatttaaga ggagtatata atataaatca aattaattat 2700
ctaccaagac aagatggagg aaccaatggt acaataaagg actatgaagt ttatttaagt 2760
ttagatggag tgaactgggg acaacctata tcaaaaggaa cctttgaatc aaactctaca 2820
gaaaaaatag taaaattcaa cgaaacaaaa tctaggtatg taaaacttaa agctctgtca 2880
gaaattaata ataaacaatt tactacagta gctgatttaa aggtatttgg atgggagata 2940
tccaaaatag aaaaaccatt acaaaatgct gaaacttatt tgaatatacc aacttatgat 3000
ggattaaatc aaagtactca tccagatgtc aaatatttta aaaatggttg gaatggatat 3060
aaatattgga tgataatgac tccaaataga acaggtagct cagttgctga aaatccttca 3120
atactagcat ctgatgatgg aataaattgg gaggttcctg caggtgttac aaatcctata 3180
gctccaatgc cacaagtagg acataattgt gatgttgata tgatatataa tgaagcaact 3240
gatgagttat gggtgtactg ggtagaatca gatgatataa caaaaggatg ggttaaatta 3300
ataaaatcaa aggatggagt aaattggagt tctcagcaag tggtagttga tgataatagg 3360
gcaaaatata gtactttatc accatctata atattcaaag ataataaata ctatatgtgg 3420
tcagttaata caggaaatag tggttggaac aatcaaagta ataaagttga attaagagaa 3480
tcaagtgacg gagtaaattg gtcaaatcca acagttgtaa acacattagc tcaagatggt 3540
tctcaaatat ggcatgtaaa tgtagaatat ataccatcaa aaaacgaata ttgggctata 3600
tatccagcat ataaaaatgg aacaggtagc gataaaacag aattgtatta tgcgaaatca 3660
agtgatggag taaattggac aacttataag aatcctatat tatcaaaagg aacatctggt 3720
aaatgggatg atatggagat atatagaagt tgttttgtgt acgatgaaga tacaaatatg 3780
ataaaggttt ggtatggagc tgtgagtcaa aatccacaaa tatggaaaat aggttttact 3840
gaaaatgatt atgataagtt tattgagggt ttaacacaat aa 3882
<210> 14
<211> 1293
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 14
Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu Asp Thr Asp Ile
1 5 10 15
Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn Gly Ile Ala Leu
20 25 30
Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu Val Asp Glu Asn
35 40 45
Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr Ala Asp Phe Ser
50 55 60
Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp Phe Val Ile Ile
65 70 75 80
Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu Gly Val Gly Asn
85 90 95
Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn Leu Ile Arg Lys
100 105 110
Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala Ile Tyr Ser Thr
115 120 125
Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val Asp Ser Phe His
130 135 140
Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro Phe Leu Ala Tyr
145 150 155 160
Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile Val Asn Val Thr
165 170 175
Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr Ser Val Gly Val
180 185 190
Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile Gly Ile Gly Asp
195 200 205
Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile Ile Gly Lys Asp
210 215 220
Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln Thr Leu Lys Lys
225 230 235 240
Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr Thr Thr Asn Gly
245 250 255
Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val Met Lys Asp Lys
260 265 270
Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala Tyr Asp Ser Arg
275 280 285
Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile Glu Lys Ile Val
290 295 300
Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys Gln Ile Thr Leu
305 310 315 320
Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys Leu Ser Pro Gln
325 330 335
Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu Thr Asp Glu Ile
340 345 350
His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg Pro Val Asp Gly
355 360 365
Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu Trp Phe Ile Lys
370 375 380
Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro Gly Gly Gly Asn
385 390 395 400
Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser Glu Gly Ser Ile
405 410 415
Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu Glu Trp Gly Phe
420 425 430
Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro Lys Cys Tyr Asp
435 440 445
Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu Glu Asn Gln Tyr
450 455 460
Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala Ile Asn Pro Asp
465 470 475 480
Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln Asp Phe Tyr Ser
485 490 495
Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro Ile Ser Arg Val
500 505 510
Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile Phe Gly Asp Asp
515 520 525
Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu Pro Ser Ala Leu
530 535 540
Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr Leu Ser Ser Ser
545 550 555 560
Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala Arg Asp Leu Glu
565 570 575
Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys Tyr Gly Ile Asp
580 585 590
Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp Ile Tyr Tyr Ser
595 600 605
Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile Glu Ile Lys Gly
610 615 620
Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr Val Asn Asn Arg
625 630 635 640
Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr Ile Asn Thr Ser
645 650 655
Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp Asp Thr Pro Ala
660 665 670
Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile Leu Ser Ser Thr
675 680 685
Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser Thr Thr Leu Glu
690 695 700
Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile Gly Asn Asn Ser
705 710 715 720
Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp Asn Asn Gly Lys
725 730 735
Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val Pro Thr Lys Asn
740 745 750
Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr Leu Pro Ser Gly
755 760 765
Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val Leu Gln Thr Lys
770 775 780
Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn Asn Pro Tyr Asn
785 790 795 800
Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr Gly Tyr Asn Ala
805 810 815
Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys Glu Ala Ser Ser
820 825 830
Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr Val Asp Ser Glu
835 840 845
Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala Phe Asp Gly Asn
850 855 860
Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly Asn Ala Ala Pro
865 870 875 880
Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val Tyr Asn Ile Asn
885 890 895
Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr Asn Gly Thr Ile
900 905 910
Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val Asn Trp Gly Gln
915 920 925
Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr Glu Lys Ile Val
930 935 940
Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu Lys Ala Leu Ser
945 950 955 960
Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp Leu Lys Val Phe
965 970 975
Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro Leu Gln Asn Ala Glu Thr
980 985 990
Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu Asn Gln Ser Thr His Pro
995 1000 1005
Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn Gly Tyr Lys Tyr Trp
1010 1015 1020
Met Ile Met Thr Pro Asn Arg Thr Gly Ser Ser Val Ala Glu Asn
1025 1030 1035
Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile Asn Trp Glu Val Pro
1040 1045 1050
Ala Gly Val Thr Asn Pro Ile Ala Pro Met Pro Gln Val Gly His
1055 1060 1065
Asn Cys Asp Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu Leu
1070 1075 1080
Trp Val Tyr Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val
1085 1090 1095
Lys Leu Ile Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln
1100 1105 1110
Val Val Val Asp Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro
1115 1120 1125
Ser Ile Ile Phe Lys Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn
1130 1135 1140
Thr Gly Asn Ser Gly Trp Asn Asn Gln Ser Asn Lys Val Glu Leu
1145 1150 1155
Arg Glu Ser Ser Asp Gly Val Asn Trp Ser Asn Pro Thr Val Val
1160 1165 1170
Asn Thr Leu Ala Gln Asp Gly Ser Gln Ile Trp His Val Asn Val
1175 1180 1185
Glu Tyr Ile Pro Ser Lys Asn Glu Tyr Trp Ala Ile Tyr Pro Ala
1190 1195 1200
Tyr Lys Asn Gly Thr Gly Ser Asp Lys Thr Glu Leu Tyr Tyr Ala
1205 1210 1215
Lys Ser Ser Asp Gly Val Asn Trp Thr Thr Tyr Lys Asn Pro Ile
1220 1225 1230
Leu Ser Lys Gly Thr Ser Gly Lys Trp Asp Asp Met Glu Ile Tyr
1235 1240 1245
Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr Asn Met Ile Lys Val
1250 1255 1260
Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln Ile Trp Lys Ile Gly
1265 1270 1275
Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile Glu Gly Leu Thr Gln
1280 1285 1290
<210> 15
<211> 1313
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 15
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu
20 25 30
Asp Thr Asp Ile Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn
35 40 45
Gly Ile Ala Leu Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu
50 55 60
Val Asp Glu Asn Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr
65 70 75 80
Ala Asp Phe Ser Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp
85 90 95
Phe Val Ile Ile Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu
100 105 110
Gly Val Gly Asn Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn
115 120 125
Leu Ile Arg Lys Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala
130 135 140
Ile Tyr Ser Thr Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val
145 150 155 160
Asp Ser Phe His Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro
165 170 175
Phe Leu Ala Tyr Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile
180 185 190
Val Asn Val Thr Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr
195 200 205
Ser Val Gly Val Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile
210 215 220
Gly Ile Gly Asp Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile
225 230 235 240
Ile Gly Lys Asp Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln
245 250 255
Thr Leu Lys Lys Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr
260 265 270
Thr Thr Asn Gly Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val
275 280 285
Met Lys Asp Lys Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala
290 295 300
Tyr Asp Ser Arg Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile
305 310 315 320
Glu Lys Ile Val Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys
325 330 335
Gln Ile Thr Leu Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys
340 345 350
Leu Ser Pro Gln Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu
355 360 365
Thr Asp Glu Ile His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg
370 375 380
Pro Val Asp Gly Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu
385 390 395 400
Trp Phe Ile Lys Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro
405 410 415
Gly Gly Gly Asn Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser
420 425 430
Glu Gly Ser Ile Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu
435 440 445
Glu Trp Gly Phe Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro
450 455 460
Lys Cys Tyr Asp Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu
465 470 475 480
Glu Asn Gln Tyr Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala
485 490 495
Ile Asn Pro Asp Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln
500 505 510
Asp Phe Tyr Ser Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro
515 520 525
Ile Ser Arg Val Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile
530 535 540
Phe Gly Asp Asp Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu
545 550 555 560
Pro Ser Ala Leu Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr
565 570 575
Leu Ser Ser Ser Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala
580 585 590
Arg Asp Leu Glu Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys
595 600 605
Tyr Gly Ile Asp Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp
610 615 620
Ile Tyr Tyr Ser Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile
625 630 635 640
Glu Ile Lys Gly Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr
645 650 655
Val Asn Asn Arg Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr
660 665 670
Ile Asn Thr Ser Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp
675 680 685
Asp Thr Pro Ala Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile
690 695 700
Leu Ser Ser Thr Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser
705 710 715 720
Thr Thr Leu Glu Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile
725 730 735
Gly Asn Asn Ser Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp
740 745 750
Asn Asn Gly Lys Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val
755 760 765
Pro Thr Lys Asn Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr
770 775 780
Leu Pro Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val
785 790 795 800
Leu Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn
805 810 815
Asn Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr
820 825 830
Gly Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys
835 840 845
Glu Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr
850 855 860
Val Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala
865 870 875 880
Phe Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly
885 890 895
Asn Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val
900 905 910
Tyr Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr
915 920 925
Asn Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val
930 935 940
Asn Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr
945 950 955 960
Glu Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu
965 970 975
Lys Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp
980 985 990
Leu Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro Leu Gln
995 1000 1005
Asn Ala Glu Thr Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu Asn
1010 1015 1020
Gln Ser Thr His Pro Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn
1025 1030 1035
Gly Tyr Lys Tyr Trp Met Ile Met Thr Pro Asn Arg Thr Gly Ser
1040 1045 1050
Ser Val Ala Glu Asn Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile
1055 1060 1065
Asn Trp Glu Val Pro Ala Gly Val Thr Asn Pro Ile Ala Pro Met
1070 1075 1080
Pro Gln Val Gly His Asn Cys Asp Val Asp Met Ile Tyr Asn Glu
1085 1090 1095
Ala Thr Asp Glu Leu Trp Val Tyr Trp Val Glu Ser Asp Asp Ile
1100 1105 1110
Thr Lys Gly Trp Val Lys Leu Ile Lys Ser Lys Asp Gly Val Asn
1115 1120 1125
Trp Ser Ser Gln Gln Val Val Val Asp Asp Asn Arg Ala Lys Tyr
1130 1135 1140
Ser Thr Leu Ser Pro Ser Ile Ile Phe Lys Asp Asn Lys Tyr Tyr
1145 1150 1155
Met Trp Ser Val Asn Thr Gly Asn Ser Gly Trp Asn Asn Gln Ser
1160 1165 1170
Asn Lys Val Glu Leu Arg Glu Ser Ser Asp Gly Val Asn Trp Ser
1175 1180 1185
Asn Pro Thr Val Val Asn Thr Leu Ala Gln Asp Gly Ser Gln Ile
1190 1195 1200
Trp His Val Asn Val Glu Tyr Ile Pro Ser Lys Asn Glu Tyr Trp
1205 1210 1215
Ala Ile Tyr Pro Ala Tyr Lys Asn Gly Thr Gly Ser Asp Lys Thr
1220 1225 1230
Glu Leu Tyr Tyr Ala Lys Ser Ser Asp Gly Val Asn Trp Thr Thr
1235 1240 1245
Tyr Lys Asn Pro Ile Leu Ser Lys Gly Thr Ser Gly Lys Trp Asp
1250 1255 1260
Asp Met Glu Ile Tyr Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr
1265 1270 1275
Asn Met Ile Lys Val Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln
1280 1285 1290
Ile Trp Lys Ile Gly Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile
1295 1300 1305
Glu Gly Leu Thr Gln
1310
<210> 16
<211> 1584
<212> DNA
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 16
tcagggcaat attggttagt tttccaacct gataacgatg tactacaaac aaaaactaat 60
ccatcatcca tgaaacaaag tgctaacaat aatccatata attataatat attaccaaat 120
tcatttccta ttggaacagg atataatgct tataaaggcg atgtatcttt ctatgcaacc 180
tttaaagaag caagcagtca agcaattcct caaaattctt gggctctaaa atatgtagat 240
agtgaagaaa ctacaggcga aaatggaaga gctacaaatg cttttgatgg taataataat 300
actatttggc acacaaaata tagtggcgga aacgctgcac caatgccgca tgagattcaa 360
attgatttaa gaggagtata taatataaat caaattaatt atctaccaag acaagatgga 420
ggaaccaatg gtacaataaa ggactatgaa gtttatttaa gtttagatgg agtgaactgg 480
ggacaaccta tatcaaaagg aacctttgaa tcaaactcta cagaaaaaat agtaaaattc 540
aacgaaacaa aatctaggta tgtaaaactt aaagctctgt cagaaattaa taataaacaa 600
tttactacag tagctgattt aaaggtattt ggatgggaga tatccaaaat agaaaaacca 660
ttacaaaatg ctgaaactta tttgaatata ccaacttatg atggattaaa tcaaagtact 720
catccagatg tcaaatattt taaaaatggt tggaatggat ataaatattg gatgataatg 780
actccaaata gaacaggtag ctcagttgct gaaaatcctt caatactagc atctgatgat 840
ggaataaatt gggaggttcc tgcaggtgtt acaaatccta tagctccaat gccacaagta 900
ggacataatt gtgatgttga tatgatatat aatgaagcaa ctgatgagtt atgggtgtac 960
tgggtagaat cagatgatat aacaaaagga tgggttaaat taataaaatc aaaggatgga 1020
gtaaattgga gttctcagca agtggtagtt gatgataata gggcaaaata tagtacttta 1080
tcaccatcta taatattcaa agataataaa tactatatgt ggtcagttaa tacaggaaat 1140
agtggttgga acaatcaaag taataaagtt gaattaagag aatcaagtga cggagtaaat 1200
tggtcaaatc caacagttgt aaacacatta gctcaagatg gttctcaaat atggcatgta 1260
aatgtagaat atataccatc aaaaaacgaa tattgggcta tatatccagc atataaaaat 1320
ggaacaggta gcgataaaac agaattgtat tatgcgaaat caagtgatgg agtaaattgg 1380
acaacttata agaatcctat attatcaaaa ggaacatctg gtaaatggga tgatatggag 1440
atatatagaa gttgttttgt gtacgatgaa gatacaaata tgataaaggt ttggtatgga 1500
gctgtgagtc aaaatccaca aatatggaaa ataggtttta ctgaaaatga ttatgataag 1560
tttattgagg gtttaacaca ataa 1584
<210> 17
<211> 547
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 17
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn
20 25 30
Asp Val Leu Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala
35 40 45
Asn Asn Asn Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile
50 55 60
Gly Thr Gly Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr
65 70 75 80
Phe Lys Glu Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu
85 90 95
Lys Tyr Val Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr
100 105 110
Asn Ala Phe Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser
115 120 125
Gly Gly Asn Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg
130 135 140
Gly Val Tyr Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly
145 150 155 160
Gly Thr Asn Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp
165 170 175
Gly Val Asn Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn
180 185 190
Ser Thr Glu Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val
195 200 205
Lys Leu Lys Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val
210 215 220
Ala Asp Leu Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro
225 230 235 240
Leu Gln Asn Ala Glu Thr Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu
245 250 255
Asn Gln Ser Thr His Pro Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn
260 265 270
Gly Tyr Lys Tyr Trp Met Ile Met Thr Pro Asn Arg Thr Gly Ser Ser
275 280 285
Val Ala Glu Asn Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile Asn Trp
290 295 300
Glu Val Pro Ala Gly Val Thr Asn Pro Ile Ala Pro Met Pro Gln Val
305 310 315 320
Gly His Asn Cys Asp Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu
325 330 335
Leu Trp Val Tyr Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val
340 345 350
Lys Leu Ile Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln Val
355 360 365
Val Val Asp Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro Ser Ile
370 375 380
Ile Phe Lys Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn Thr Gly Asn
385 390 395 400
Ser Gly Trp Asn Asn Gln Ser Asn Lys Val Glu Leu Arg Glu Ser Ser
405 410 415
Asp Gly Val Asn Trp Ser Asn Pro Thr Val Val Asn Thr Leu Ala Gln
420 425 430
Asp Gly Ser Gln Ile Trp His Val Asn Val Glu Tyr Ile Pro Ser Lys
435 440 445
Asn Glu Tyr Trp Ala Ile Tyr Pro Ala Tyr Lys Asn Gly Thr Gly Ser
450 455 460
Asp Lys Thr Glu Leu Tyr Tyr Ala Lys Ser Ser Asp Gly Val Asn Trp
465 470 475 480
Thr Thr Tyr Lys Asn Pro Ile Leu Ser Lys Gly Thr Ser Gly Lys Trp
485 490 495
Asp Asp Met Glu Ile Tyr Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr
500 505 510
Asn Met Ile Lys Val Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln Ile
515 520 525
Trp Lys Ile Gly Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile Glu Gly
530 535 540
Leu Thr Gln
545
<210> 18
<211> 2958
<212> DNA
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 18
tataatttaa ttgataatat tagtgttgaa aaattagata ctgatatttc acaagcaaat 60
gaaaatgttt ttttgaatgg aaatggaatt gctttagaag tagataatag aggcgctaca 120
tgtatttatc tagtagatga aaatggagtt aaaacaaaag ctacgacttc tttagataca 180
gcagattttt caggttatcc aataataggt ggacaaaaga taagagattt tgtaattata 240
tcaaaaaatc tagaagaaaa cataaactcg atattaggtg ttggaaatag acttactatt 300
atatctaaaa gttcatctac taatctgata agaaagatag tatttgaaac atctaacagc 360
aatccaggag caatatattc aacagtaagt tataaagcag aaagtaacga tttattagta 420
gatagctttc atgaaaatga gtatacaatg agtttagggc aaggaccttt tcttgcatat 480
caagggtgtg cagatcaaca aggagcaaat actatcgtta atgttactaa tggatataac 540
cataatagtg gacaaaataa ttattctgta ggagttccat ttagttatgt ttataactct 600
gtggggggaa ttggaatagg tgatgcatca acttcaagaa gagaatttaa gttgcctatt 660
ataggaaaag ataatacagt ttcattagga atggagtgga atggacaaac tttaaaaaaa 720
ggtgctgaaa ctgctatagg tacaagtgtt ataactacaa caaatggtga ttattattct 780
gggctaaaga gttacgcaga agttatgaaa gataagggaa tatctgcacc agcttcaata 840
cctgatatag catatgattc tagatgggaa agttggggat tcgaatttga ttttacaata 900
gaaaaaatag ttaataaatt agatgaactt aaagcgatgg ggataaaaca aattactcta 960
gatgatgggt ggtacactta tgctggtgat tggaaattaa gtcctcaaaa gtttccaaat 1020
ggaaatgcag acatgaaata tcttacagat gaaatccata aaagaggaat gacagctatt 1080
ttatggtgga gaccagtaga cggagggata aatagcaaat tagtatctga acatccagag 1140
tggtttatta agaactcaca agggaatatg gttaggttac cagggcctgg aggtggaaat 1200
ggaggaacag caggatatgc attatgtcca aattcagaag gttcaattca acatcataaa 1260
gattttgtaa ctgtggcatt agaagaatgg ggatttgatg gattcaaaga agattatgta 1320
tggggaatac ctaaatgcta tgatagttct cataaacact caagtttatc agatacatta 1380
gaaaatcaat ataaattcta tgaagccata tatgaacagt ccatagcgat aaatccagat 1440
acttttatag aattatgtaa ttgcggaaca cctcaggatt tttattcaac accatatgtg 1500
aaccatgcac caacagcaga tccaatttcg agagtacaaa caagaacaag agtgaaagca 1560
tttaaagcta tatttggaga tgattttcca gtaacaacag atcataattc agtttggtta 1620
ccgtcagcat taggtacagg atcagttatg attactaaac atacaacatt aagtagttca 1680
gatagagaac aatataataa atacttcgga cttgcaagag atttagaatt agcaaaggga 1740
gaatttatag gaaacttata taaatacgga atagatccat tagagtcata tgttataaga 1800
aaaggagaag atatttatta ttcattctac aaagataatt ctagttattc aggaaatata 1860
gaaataaagg ggttagacag taacgccaca tatagaattg aagattatgt taacaataga 1920
gttattgcta gaggagtaaa gggaccaaca gcgactataa atacaagctt tactgataat 1980
ttattagtta gagcaatacc agatgataca ccagcagagg ttactacatt tgatgttgga 2040
aataatacaa tattatcatc aacagatagt ggaaattcta aatatttaaa tgctgtttct 2100
actacattag aaaagacagc aacaatagat agtttaagta tttatatagg aaataattca 2160
gaaaatggca aactacaaat tgctatttat gacgataata acgggaaacc tggtactaaa 2220
aaagcttacg tagaagagtt tgttcctact aaaaatagtt ggaatacaaa gaaggttgta 2280
aattctgtta cattaccttc agggcaatat tggttagttt tccaacctga taacgatgta 2340
ctacaaacaa aaactaatcc atcatccatg aaacaaagtg ctaacaataa tccatataat 2400
tataatatat taccaaattc atttcctatt ggaacaggat ataatgctta taaaggcgat 2460
gtatctttct atgcaacctt taaagaagca agcagtcaag caattcctca aaattcttgg 2520
gctctaaaat atgtagatag tgaagaaact acaggcgaaa atggaagagc tacaaatgct 2580
tttgatggta ataataatac tatttggcac acaaaatata gtggcggaaa cgctgcacca 2640
atgccgcatg agattcaaat tgatttaaga ggagtatata atataaatca aattaattat 2700
ctaccaagac aagatggagg aaccaatggt acaataaagg actatgaagt ttatttaagt 2760
ttagatggag tgaactgggg acaacctata tcaaaaggaa cctttgaatc aaactctaca 2820
gaaaaaatag taaaattcaa cgaaacaaaa tctaggtatg taaaacttaa agctctgtca 2880
gaaattaata ataaacaatt tactacagta gctgatttaa aggtatttgg atgggagata 2940
tccaaaatag aaaaataa 2958
<210> 19
<211> 1005
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 19
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu
20 25 30
Asp Thr Asp Ile Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn
35 40 45
Gly Ile Ala Leu Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu
50 55 60
Val Asp Glu Asn Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr
65 70 75 80
Ala Asp Phe Ser Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp
85 90 95
Phe Val Ile Ile Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu
100 105 110
Gly Val Gly Asn Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn
115 120 125
Leu Ile Arg Lys Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala
130 135 140
Ile Tyr Ser Thr Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val
145 150 155 160
Asp Ser Phe His Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro
165 170 175
Phe Leu Ala Tyr Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile
180 185 190
Val Asn Val Thr Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr
195 200 205
Ser Val Gly Val Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile
210 215 220
Gly Ile Gly Asp Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile
225 230 235 240
Ile Gly Lys Asp Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln
245 250 255
Thr Leu Lys Lys Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr
260 265 270
Thr Thr Asn Gly Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val
275 280 285
Met Lys Asp Lys Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala
290 295 300
Tyr Asp Ser Arg Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile
305 310 315 320
Glu Lys Ile Val Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys
325 330 335
Gln Ile Thr Leu Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys
340 345 350
Leu Ser Pro Gln Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu
355 360 365
Thr Asp Glu Ile His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg
370 375 380
Pro Val Asp Gly Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu
385 390 395 400
Trp Phe Ile Lys Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro
405 410 415
Gly Gly Gly Asn Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser
420 425 430
Glu Gly Ser Ile Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu
435 440 445
Glu Trp Gly Phe Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro
450 455 460
Lys Cys Tyr Asp Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu
465 470 475 480
Glu Asn Gln Tyr Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala
485 490 495
Ile Asn Pro Asp Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln
500 505 510
Asp Phe Tyr Ser Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro
515 520 525
Ile Ser Arg Val Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile
530 535 540
Phe Gly Asp Asp Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu
545 550 555 560
Pro Ser Ala Leu Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr
565 570 575
Leu Ser Ser Ser Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala
580 585 590
Arg Asp Leu Glu Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys
595 600 605
Tyr Gly Ile Asp Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp
610 615 620
Ile Tyr Tyr Ser Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile
625 630 635 640
Glu Ile Lys Gly Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr
645 650 655
Val Asn Asn Arg Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr
660 665 670
Ile Asn Thr Ser Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp
675 680 685
Asp Thr Pro Ala Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile
690 695 700
Leu Ser Ser Thr Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser
705 710 715 720
Thr Thr Leu Glu Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile
725 730 735
Gly Asn Asn Ser Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp
740 745 750
Asn Asn Gly Lys Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val
755 760 765
Pro Thr Lys Asn Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr
770 775 780
Leu Pro Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val
785 790 795 800
Leu Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn
805 810 815
Asn Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr
820 825 830
Gly Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys
835 840 845
Glu Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr
850 855 860
Val Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala
865 870 875 880
Phe Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly
885 890 895
Asn Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val
900 905 910
Tyr Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr
915 920 925
Asn Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val
930 935 940
Asn Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr
945 950 955 960
Glu Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu
965 970 975
Lys Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp
980 985 990
Leu Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys
995 1000 1005
<210> 20
<211> 3786
<212> DNA
<213> Robinsoniella peoriensis
<400> 20
gggaacggat tagaggtgaa agcctcgcca agggaggtgg cacaaataac cggaaacggg 60
gtatcggtga cgttttttca ggaagatggc acggtgcagt tatcctgtat agaggatgat 120
ggcaatactg cttttatgac caggaactca gaggtctctt atccggtggt gggtggggag 180
gaagtaacag acttttcaga ctttcaatgt gaagtacagg aaaacgtaac cggagctgcg 240
ggagccggca gccggatgac aatcacctcc atttccagcg gcagggggat tcagcggtcg 300
gtagtcattg agacggtaga tgaggtaaaa ggcctgctcc atatcagcag ttcttatagg 360
gcagaagaag aggtagatgc agacgaattt attgacagca gattcagcct ggataatccc 420
tcagatacag tctggagtta caatggcggc ggtgaggggg cccagagccg atacgatact 480
ctacagaaaa tagatctgtc ggatggtgaa agcttctata gggagaactt acagaatcaa 540
actgcggcag gtattccggt ggcggatatc tacgggaaag acgggggtat tacggtgggt 600
gatgccagtg tgacccggcg acagctttcc actccggtaa acgagaggaa tggtaccgct 660
tatgtgtccg tgaaacatcc aggtgcagtt attacccaaa gggaaacaga aatcagccag 720
agctttgtca atgtacacag aggcgactat tattcggggc tgcggggtta tgccgatggt 780
atgaagcaga taggatttac cacactctcc cgggaacaga ttcctgaaag cagctatgat 840
ctccgctggg agagctgggg atgggaattt gactggacag tggaactgat tatcaataag 900
ctggacgagt taaaagagat gggaatcaaa cagattaccc tggatgacgg ctggtataat 960
gccgcaggag aatgggggct gaacaactgg aagcttccta atggtgcttt ggacatgcgg 1020
catctgactg atgcaattca tgaaaggggg atgactgcag tattgtggtg gcgtccctgt 1080
gacggtggaa gggaagacag cgcattattt aaagagcatc cagagtattt tataaaaaac 1140
caggacggaa gctttgggaa gctggcagga ccgggacagt ggaacagttt tctgggaagc 1200
tgcggttatg cgctgtgtcc tttgtcagaa ggggcagtac agagccaggt tgattttatt 1260
aaccgtgcta tgaatgaatg gggatttgat ggatttaaaa gtgattatgt atggagcctt 1320
ccaaagtgct acagtcagga ccatcaccat gaatacccgg aagaatccac agaacagcag 1380
gctgtgttct accgggcagt ttatgaggct atgacagaca atgacccgaa tgcatttcac 1440
cttctatgca actgcggaac gccacaggat tattattctc tgccctatgt aacccaggtg 1500
cctactgccg atcccacttc tgtggatcag acaaggagaa gggtaaaggc atataaagca 1560
ctatgcggtg attatttccc tgttacgaca gatcataatg aagtctggta tccttcaacc 1620
ataggaacgg gagccatact gattgaaaaa cgtgacttgt caggctggga agaggaggag 1680
tatgcaaaat ggcttaaaat tgctcaggaa aaccaattgc ataaagggac atttattggg 1740
gatttgtaca gttacggata tgacccttat gaaacctata cggtgtataa agacggaatc 1800
atgtattatg cattctataa agacggaaac cggtaccgtc cgtccggtaa cccggatatc 1860
gaattaaaag ggctggaaga cggaaagctg taccgcatcg tagattatgt aaataatcag 1920
gtagttgcca caaatgtaac cagtagcaat gctgtatttt cttacccttt cagcgattac 1980
ttgctggtaa aagcagtaga aatcagcgaa ccggatacgg atggacctgg acctgtaccg 2040
gatcctgagg gggcggtaac agtagaggaa aatgatcctg aactggtata tacaggggat 2100
tgggtaaggg aagaaaatga cggataccat ggaggaggag cccgttatac aaaagaagca 2160
gaagcttctg tagaattggc attctatgga acaggtgctg cctggtatgg acagcacgac 2220
gttaactttg gtagtgcacg gatatatata gacggaacct atgtcaagac cgtatcatgc 2280
atgggagaac ctggaataaa tattaaattg tttgaaatca gcggcttgga cttggcttcc 2340
cacaggatta aaatagaatg tgagacaccg gtaattgata ttgacaggct gacttacatc 2400
aaaggagaag aagttcctgc taaagtaatg acggcggacc tccgggcttt gactgttata 2460
gcaaaccaat acgatatgaa cagttttgca gatggcaatt acaaagacca gctgggggta 2520
tccttagttc gtgccaacca gcttctggca gcggatgatg taacccaggg ggctgtaaat 2580
gaagaacaga aataccttct gaatgccatg ctgaaaataa gaaaaaaagt tgataagagt 2640
tggatcgggc ttcccggacc aatcccgcag gatatacaga cagaaaatat cagcagagat 2700
aaccttgcta aagtaatatc ttatactggg cagttggaca gagatgagat tattcctgcc 2760
ataaaagaac agctgaacga ttcttatgat aaggctgtct ccatagcaga acgccaggat 2820
gcatcccagc cggaaataga cagagcgtgg gcagagttaa tgaatgcagt gcaatatagc 2880
agctatatca ggggatcaaa agaggaactg ttatcacttc tggatgaata cggaaaggta 2940
gataccaccg tttataaaga cgctgcttta tttatagaat ccttagaagc cgctaaaaag 3000
gtgtatcagg atgaaaatgc aatggatggg gagatcagtg attgtatcaa acaattgcga 3060
gatgcaaaag atcagctaca actaaaggat ccggtagatc cgccgaaacc cgatccggac 3120
cccgatccaa agcctgatcc aacaccagac ccgggaccag atccaaagcc cgatccaaca 3180
cctgacccga cgccagaccc aaagcccaat ccaacaccga cgcccgatcc aacaccagag 3240
ccagctctaa aaaagccgga acaggtatct ggtttgaagt cgaaagcgga gactgattat 3300
ctgacggttt cctggaagaa attgaataat gctgaatcct ataaggtgta tatttataaa 3360
agcggcaaat ggcgcctggc tggaaaaact acaaagacat ccataaagat aaaaaaactg 3420
gtttcgggaa cgaaatacac cgtaaaagtt gctgcggtca ataaagcagg gcaggggaaa 3480
tattcatcac aggtgtatac ggcagcaaag cccaaaaaag tcaaattaaa atccgtcagc 3540
aggtaccgca catcaaaagt aaagttaaac tatggaaaag taaaagcagg cggatatgaa 3600
atatggatga agaatggaaa gggttcttat aagaaggcag ccaccagtac gaagacaaca 3660
gccataaaga gcggattaaa aaaaggaaaa acatattact ttaaagtcag ggcttatgtt 3720
aaaaataaaa atcaggtgat ttacggcagc ttttccaata taaagaaata caaaatggta 3780
ttatga 3786
<210> 21
<211> 1281
<212> PRT
<213> Robinsoniella peoriensis
<400> 21
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Gly Asn Gly Leu Glu Val Lys Ala Ser Pro Arg Glu
20 25 30
Val Ala Gln Ile Thr Gly Asn Gly Val Ser Val Thr Phe Phe Gln Glu
35 40 45
Asp Gly Thr Val Gln Leu Ser Cys Ile Glu Asp Asp Gly Asn Thr Ala
50 55 60
Phe Met Thr Arg Asn Ser Glu Val Ser Tyr Pro Val Val Gly Gly Glu
65 70 75 80
Glu Val Thr Asp Phe Ser Asp Phe Gln Cys Glu Val Gln Glu Asn Val
85 90 95
Thr Gly Ala Ala Gly Ala Gly Ser Arg Met Thr Ile Thr Ser Ile Ser
100 105 110
Ser Gly Arg Gly Ile Gln Arg Ser Val Val Ile Glu Thr Val Asp Glu
115 120 125
Val Lys Gly Leu Leu His Ile Ser Ser Ser Tyr Arg Ala Glu Glu Glu
130 135 140
Val Asp Ala Asp Glu Phe Ile Asp Ser Arg Phe Ser Leu Asp Asn Pro
145 150 155 160
Ser Asp Thr Val Trp Ser Tyr Asn Gly Gly Gly Glu Gly Ala Gln Ser
165 170 175
Arg Tyr Asp Thr Leu Gln Lys Ile Asp Leu Ser Asp Gly Glu Ser Phe
180 185 190
Tyr Arg Glu Asn Leu Gln Asn Gln Thr Ala Ala Gly Ile Pro Val Ala
195 200 205
Asp Ile Tyr Gly Lys Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Val
210 215 220
Thr Arg Arg Gln Leu Ser Thr Pro Val Asn Glu Arg Asn Gly Thr Ala
225 230 235 240
Tyr Val Ser Val Lys His Pro Gly Ala Val Ile Thr Gln Arg Glu Thr
245 250 255
Glu Ile Ser Gln Ser Phe Val Asn Val His Arg Gly Asp Tyr Tyr Ser
260 265 270
Gly Leu Arg Gly Tyr Ala Asp Gly Met Lys Gln Ile Gly Phe Thr Thr
275 280 285
Leu Ser Arg Glu Gln Ile Pro Glu Ser Ser Tyr Asp Leu Arg Trp Glu
290 295 300
Ser Trp Gly Trp Glu Phe Asp Trp Thr Val Glu Leu Ile Ile Asn Lys
305 310 315 320
Leu Asp Glu Leu Lys Glu Met Gly Ile Lys Gln Ile Thr Leu Asp Asp
325 330 335
Gly Trp Tyr Asn Ala Ala Gly Glu Trp Gly Leu Asn Asn Trp Lys Leu
340 345 350
Pro Asn Gly Ala Leu Asp Met Arg His Leu Thr Asp Ala Ile His Glu
355 360 365
Arg Gly Met Thr Ala Val Leu Trp Trp Arg Pro Cys Asp Gly Gly Arg
370 375 380
Glu Asp Ser Ala Leu Phe Lys Glu His Pro Glu Tyr Phe Ile Lys Asn
385 390 395 400
Gln Asp Gly Ser Phe Gly Lys Leu Ala Gly Pro Gly Gln Trp Asn Ser
405 410 415
Phe Leu Gly Ser Cys Gly Tyr Ala Leu Cys Pro Leu Ser Glu Gly Ala
420 425 430
Val Gln Ser Gln Val Asp Phe Ile Asn Arg Ala Met Asn Glu Trp Gly
435 440 445
Phe Asp Gly Phe Lys Ser Asp Tyr Val Trp Ser Leu Pro Lys Cys Tyr
450 455 460
Ser Gln Asp His His His Glu Tyr Pro Glu Glu Ser Thr Glu Gln Gln
465 470 475 480
Ala Val Phe Tyr Arg Ala Val Tyr Glu Ala Met Thr Asp Asn Asp Pro
485 490 495
Asn Ala Phe His Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr
500 505 510
Ser Leu Pro Tyr Val Thr Gln Val Pro Thr Ala Asp Pro Thr Ser Val
515 520 525
Asp Gln Thr Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Cys Gly Asp
530 535 540
Tyr Phe Pro Val Thr Thr Asp His Asn Glu Val Trp Tyr Pro Ser Thr
545 550 555 560
Ile Gly Thr Gly Ala Ile Leu Ile Glu Lys Arg Asp Leu Ser Gly Trp
565 570 575
Glu Glu Glu Glu Tyr Ala Lys Trp Leu Lys Ile Ala Gln Glu Asn Gln
580 585 590
Leu His Lys Gly Thr Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Tyr Asp
595 600 605
Pro Tyr Glu Thr Tyr Thr Val Tyr Lys Asp Gly Ile Met Tyr Tyr Ala
610 615 620
Phe Tyr Lys Asp Gly Asn Arg Tyr Arg Pro Ser Gly Asn Pro Asp Ile
625 630 635 640
Glu Leu Lys Gly Leu Glu Asp Gly Lys Leu Tyr Arg Ile Val Asp Tyr
645 650 655
Val Asn Asn Gln Val Val Ala Thr Asn Val Thr Ser Ser Asn Ala Val
660 665 670
Phe Ser Tyr Pro Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile
675 680 685
Ser Glu Pro Asp Thr Asp Gly Pro Gly Pro Val Pro Asp Pro Glu Gly
690 695 700
Ala Val Thr Val Glu Glu Asn Asp Pro Glu Leu Val Tyr Thr Gly Asp
705 710 715 720
Trp Val Arg Glu Glu Asn Asp Gly Tyr His Gly Gly Gly Ala Arg Tyr
725 730 735
Thr Lys Glu Ala Glu Ala Ser Val Glu Leu Ala Phe Tyr Gly Thr Gly
740 745 750
Ala Ala Trp Tyr Gly Gln His Asp Val Asn Phe Gly Ser Ala Arg Ile
755 760 765
Tyr Ile Asp Gly Thr Tyr Val Lys Thr Val Ser Cys Met Gly Glu Pro
770 775 780
Gly Ile Asn Ile Lys Leu Phe Glu Ile Ser Gly Leu Asp Leu Ala Ser
785 790 795 800
His Arg Ile Lys Ile Glu Cys Glu Thr Pro Val Ile Asp Ile Asp Arg
805 810 815
Leu Thr Tyr Ile Lys Gly Glu Glu Val Pro Ala Lys Val Met Thr Ala
820 825 830
Asp Leu Arg Ala Leu Thr Val Ile Ala Asn Gln Tyr Asp Met Asn Ser
835 840 845
Phe Ala Asp Gly Asn Tyr Lys Asp Gln Leu Gly Val Ser Leu Val Arg
850 855 860
Ala Asn Gln Leu Leu Ala Ala Asp Asp Val Thr Gln Gly Ala Val Asn
865 870 875 880
Glu Glu Gln Lys Tyr Leu Leu Asn Ala Met Leu Lys Ile Arg Lys Lys
885 890 895
Val Asp Lys Ser Trp Ile Gly Leu Pro Gly Pro Ile Pro Gln Asp Ile
900 905 910
Gln Thr Glu Asn Ile Ser Arg Asp Asn Leu Ala Lys Val Ile Ser Tyr
915 920 925
Thr Gly Gln Leu Asp Arg Asp Glu Ile Ile Pro Ala Ile Lys Glu Gln
930 935 940
Leu Asn Asp Ser Tyr Asp Lys Ala Val Ser Ile Ala Glu Arg Gln Asp
945 950 955 960
Ala Ser Gln Pro Glu Ile Asp Arg Ala Trp Ala Glu Leu Met Asn Ala
965 970 975
Val Gln Tyr Ser Ser Tyr Ile Arg Gly Ser Lys Glu Glu Leu Leu Ser
980 985 990
Leu Leu Asp Glu Tyr Gly Lys Val Asp Thr Thr Val Tyr Lys Asp Ala
995 1000 1005
Ala Leu Phe Ile Glu Ser Leu Glu Ala Ala Lys Lys Val Tyr Gln
1010 1015 1020
Asp Glu Asn Ala Met Asp Gly Glu Ile Ser Asp Cys Ile Lys Gln
1025 1030 1035
Leu Arg Asp Ala Lys Asp Gln Leu Gln Leu Lys Asp Pro Val Asp
1040 1045 1050
Pro Pro Lys Pro Asp Pro Asp Pro Asp Pro Lys Pro Asp Pro Thr
1055 1060 1065
Pro Asp Pro Gly Pro Asp Pro Lys Pro Asp Pro Thr Pro Asp Pro
1070 1075 1080
Thr Pro Asp Pro Lys Pro Asn Pro Thr Pro Thr Pro Asp Pro Thr
1085 1090 1095
Pro Glu Pro Ala Leu Lys Lys Pro Glu Gln Val Ser Gly Leu Lys
1100 1105 1110
Ser Lys Ala Glu Thr Asp Tyr Leu Thr Val Ser Trp Lys Lys Leu
1115 1120 1125
Asn Asn Ala Glu Ser Tyr Lys Val Tyr Ile Tyr Lys Ser Gly Lys
1130 1135 1140
Trp Arg Leu Ala Gly Lys Thr Thr Lys Thr Ser Ile Lys Ile Lys
1145 1150 1155
Lys Leu Val Ser Gly Thr Lys Tyr Thr Val Lys Val Ala Ala Val
1160 1165 1170
Asn Lys Ala Gly Gln Gly Lys Tyr Ser Ser Gln Val Tyr Thr Ala
1175 1180 1185
Ala Lys Pro Lys Lys Val Lys Leu Lys Ser Val Ser Arg Tyr Arg
1190 1195 1200
Thr Ser Lys Val Lys Leu Asn Tyr Gly Lys Val Lys Ala Gly Gly
1205 1210 1215
Tyr Glu Ile Trp Met Lys Asn Gly Lys Gly Ser Tyr Lys Lys Ala
1220 1225 1230
Ala Thr Ser Thr Lys Thr Thr Ala Ile Lys Ser Gly Leu Lys Lys
1235 1240 1245
Gly Lys Thr Tyr Tyr Phe Lys Val Arg Ala Tyr Val Lys Asn Lys
1250 1255 1260
Asn Gln Val Ile Tyr Gly Ser Phe Ser Asn Ile Lys Lys Tyr Lys
1265 1270 1275
Met Val Leu
1280
<210> 22
<211> 1347
<212> DNA
<213> Ruthenibacterium lactatiformans
<400> 22
gaagaaaccg atttgcttgt aaacggaggt tttgagaccg gcgacagcac cggatggaat 60
tggttcaata acgccgttgt tgacagcgct gctccgcata gcggaaacta ttgtgctaaa 120
gtagccaaaa acagcagtta tgagcaagtt gttacggtat ctccggatac gaaatatgtt 180
ttaacagggt gggcaaaatc tgagggcagt tccgttatga cgctgggcgt aaaaaattac 240
ggtgggcagg aaactttttc ggctacgctt tcagccgact atcagcagct ggcggttact 300
ttcacaaccg ggcccaatgc gcaaacagcg actatatatg gatatcgaca gaatagtggt 360
tccggtgcag gctatttcga cgatgtagaa cttacagcgg tgcaagattt tgctccatat 420
cagccgttgg caaatgccat agcgcctcaa gcaattccta cctatgacgg cgccaaccag 480
cctacacatc cctcggtggt gaaatttgaa cagccttgga atggttatct gtattggatg 540
gcaatgacac cttatccctt caatgatggg agctacgaaa acccatcgat tgttgcgtca 600
aacgatggag aaaattggat tgtgccagaa ggggtctcga atcctttggc cggcacgcca 660
agtccgggcc acaattgtga cgtggatctt gtatatgttc cagcctcgga tgaattgcgg 720
atgtactacg tagaggcaga tgatatcatc agctcaaggg taaaaatgat aagttcccgt 780
gacggtgtac actggagcga gccgcaggtc gtaatgcagg atctggtaag gaaatacagt 840
attctatcgc cgtctattga gattctgcca gatggcacct atatgatgtg gtatgtggat 900
acggggaatg caggatggaa tagccagaat aaccaagtaa aatatcgtac atctgcggat 960
ggaatcaaat ggtcaggcgc agtcacctgt acggattttg tacaacctgg atatcaaata 1020
tggcacatcg atgtacatta tgacacatca agcggagctt actatgcagt ttatccggct 1080
tatccgaatg gcaccgattg cgaccactgc aatttgtttt tcgcagtgaa tcggacagga 1140
aaacagtggg aaacttttag ccggccaatt ttgaagccgt caacggaagg cggctgggat 1200
gatttctgca tttaccggtc ctctatgctg attgacgacg gaatgttgaa agtgtggtac 1260
ggagcaaaaa agcaagagga ttcttcctgg catactgggc taaccatgcg tgatttttct 1320
gaatttatga aaatattgga acgctaa 1347
<210> 23
<211> 468
<212> PRT
<213> Ruthenibacterium lactatiformans
<400> 23
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Glu Glu Thr Asp Leu Leu Val Asn Gly Gly Phe Glu
20 25 30
Thr Gly Asp Ser Thr Gly Trp Asn Trp Phe Asn Asn Ala Val Val Asp
35 40 45
Ser Ala Ala Pro His Ser Gly Asn Tyr Cys Ala Lys Val Ala Lys Asn
50 55 60
Ser Ser Tyr Glu Gln Val Val Thr Val Ser Pro Asp Thr Lys Tyr Val
65 70 75 80
Leu Thr Gly Trp Ala Lys Ser Glu Gly Ser Ser Val Met Thr Leu Gly
85 90 95
Val Lys Asn Tyr Gly Gly Gln Glu Thr Phe Ser Ala Thr Leu Ser Ala
100 105 110
Asp Tyr Gln Gln Leu Ala Val Thr Phe Thr Thr Gly Pro Asn Ala Gln
115 120 125
Thr Ala Thr Ile Tyr Gly Tyr Arg Gln Asn Ser Gly Ser Gly Ala Gly
130 135 140
Tyr Phe Asp Asp Val Glu Leu Thr Ala Val Gln Asp Phe Ala Pro Tyr
145 150 155 160
Gln Pro Leu Ala Asn Ala Ile Ala Pro Gln Ala Ile Pro Thr Tyr Asp
165 170 175
Gly Ala Asn Gln Pro Thr His Pro Ser Val Val Lys Phe Glu Gln Pro
180 185 190
Trp Asn Gly Tyr Leu Tyr Trp Met Ala Met Thr Pro Tyr Pro Phe Asn
195 200 205
Asp Gly Ser Tyr Glu Asn Pro Ser Ile Val Ala Ser Asn Asp Gly Glu
210 215 220
Asn Trp Ile Val Pro Glu Gly Val Ser Asn Pro Leu Ala Gly Thr Pro
225 230 235 240
Ser Pro Gly His Asn Cys Asp Val Asp Leu Val Tyr Val Pro Ala Ser
245 250 255
Asp Glu Leu Arg Met Tyr Tyr Val Glu Ala Asp Asp Ile Ile Ser Ser
260 265 270
Arg Val Lys Met Ile Ser Ser Arg Asp Gly Val His Trp Ser Glu Pro
275 280 285
Gln Val Val Met Gln Asp Leu Val Arg Lys Tyr Ser Ile Leu Ser Pro
290 295 300
Ser Ile Glu Ile Leu Pro Asp Gly Thr Tyr Met Met Trp Tyr Val Asp
305 310 315 320
Thr Gly Asn Ala Gly Trp Asn Ser Gln Asn Asn Gln Val Lys Tyr Arg
325 330 335
Thr Ser Ala Asp Gly Ile Lys Trp Ser Gly Ala Val Thr Cys Thr Asp
340 345 350
Phe Val Gln Pro Gly Tyr Gln Ile Trp His Ile Asp Val His Tyr Asp
355 360 365
Thr Ser Ser Gly Ala Tyr Tyr Ala Val Tyr Pro Ala Tyr Pro Asn Gly
370 375 380
Thr Asp Cys Asp His Cys Asn Leu Phe Phe Ala Val Asn Arg Thr Gly
385 390 395 400
Lys Gln Trp Glu Thr Phe Ser Arg Pro Ile Leu Lys Pro Ser Thr Glu
405 410 415
Gly Gly Trp Asp Asp Phe Cys Ile Tyr Arg Ser Ser Met Leu Ile Asp
420 425 430
Asp Gly Met Leu Lys Val Trp Tyr Gly Ala Lys Lys Gln Glu Asp Ser
435 440 445
Ser Trp His Thr Gly Leu Thr Met Arg Asp Phe Ser Glu Phe Met Lys
450 455 460
Ile Leu Glu Arg
465
<210> 24
<211> 5277
<212> DNA
<213> Robinsoniella peoriensis
<400> 24
tcaccattga gcgctgcggc agaaagtggc acaggaacca gattagtgaa agggcaaacg 60
gggtatttga cagaggaaca ggctatccgg aaccaggagc agacaaccga agaaagggag 120
cagaagttaa ccggggaaga gacagcagag gttttgatgg aaggtacaaa agacagcggg 180
attgtacaga cagaagaagt acagacaaaa gaaatgcaga cagaagatgc gcagacagaa 240
gaagtacaga cagaagaaat gcagacagaa gatgcgcaga caaaagaagt acagacagaa 300
gaaatgcaga cagaagatgc gcagacagaa gaagtacaga caaaagaaga accggcagaa 360
gaaacacaca tgaaagaaat acagacgcaa gggacaaaga aagcgtcaga taggaacgga 420
aaggcaaggg taactgaaat tctggaagat gcccaggatc cagcaaaccg gattgtgtat 480
ctgtcagacc tgcaatggaa gtcagaaaat catacagtag atagcgagct gcctaccaga 540
aaggataagt cctttggcgg cggaaaaatt acgctaaaag tggatggaac ggtaacagaa 600
tttgataagg ggattggaac acagacagat tccaccattg tgtacgatct ggagggaaag 660
ggatatacaa agtttgaaac ttacgtgggt gtagactaca gccagaaaga aaacattccg 720
ggggaagtct gcgacgtaaa attcagggtg aaaattgatg acaagattgt atcagaaacc 780
ggtgtactgg atccgctttc gaatgcggtt aagatttctg ttaacatacc cgatacagcc 840
aaaactttaa cattatacgc ggataaagta acggaaactt ggtctgatca cgccaattgg 900
gcagatgcaa aattttatca ggcactgccg gaacccgaaa atgttgcatt caaaaaaacg 960
gtagtgacac gaaagacatc agataattcg gaggctcctg ttaatccgga ttcagcagtt 1020
aacagttcta aggctgttga cggtgttatt gacagctcca gttattttga ttttggagat 1080
caggcaaata gcggagccgt aagggagtca ctctatatgg aggtagattt aaaagggagc 1140
tatttactgt ccgatataca actgtggaga tactggaaag atggcagaac ttatgcagct 1200
actgcaattg tagtagctga ggatgagaac tttgaaaatg cagcagttat ctataactcg 1260
gatacgacgg gagaaataca tcacctggga gcaggaagtg atatgctcta tgcagaaaca 1320
gaaagtggca agacatttcc ggtaccggaa aatacaaaag caaggtatat cagagtttat 1380
acatatggtg ttaatgggac atcaggcgta acaaatcaca ttgtagaatt aaaggtgaat 1440
gcttacgtat ttggagatga aatcttaccg gaaaagccgg atgacagcaa gattttccca 1500
aatgcagtta atccgctgaa gctacaggga ccgggcacga atgatcaggt aacccacccg 1560
gatgttacgg tgtttgatga gccgtggaat gggtataaat actggatggc atatacaccg 1620
aataaaccgg gaagttccta ttttgaaaat ccctgtatag ctgcatccaa cgatggcgta 1680
aactgggagt ttcctgccca gaaccctgta cagccgcgct atgacagtga aatagaaaat 1740
caaaatgaac ataactgtga taccgatatt gtatatgacc cggtaaatga ccggttgatt 1800
atgtactggg aatgggcaca ggatgaggcg gttaatggta aaacacatcg ttctgaaatc 1860
agataccgtg tttcttatga tgggattaac tggggagtgg aagacaaaac tggtgttttg 1920
atgactggac caacggatca tggctgcgcc attgccacag aaggcgaaag atattcagac 1980
ctttctccaa ccgtagtata tgataaaaca gaaaaaatct acaaaatgtg ggcaaatgat 2040
gccggagatg taggatatga aaacaaacag aataacaaag tatggtatcg gacatcccaa 2100
gacgggatca gcaattggtc ggataagact tacgtggaga attttcttgg agtaaatgaa 2160
gacgggctgc agatgtatcc atggcaccag gatatccagt gggtagagga atttcaggaa 2220
tattgggcac ttcagcaggc atttccggca ggaagcggac cggataattc ttccctgcgt 2280
ttctcgaaat ccaaagatgg tcttcattgg gagccggtat ctgaaaaagc tttaattaca 2340
gtaggggcac ccgggacctg ggatgcagga cagatatacc gttctacttt ctggtatgag 2400
ccaggtgggg caaaaggaaa cggaacattc catatctggt atgctgcatt ggcggaaggc 2460
cagtctcact gggatatagg atatacatct gcaaactatg cagatgccat gtacaaatta 2520
acgggaagca gaccggaagt ggaaaaaaga atagaggtaa ataatgaaaa tcctctgctg 2580
attatgccgc tttacggaaa gtcttacagt gaatcaggaa gtaccctgga ttggggagat 2640
gatctggttt cacgctggaa acaggttccg gaagatttaa aagaaaacgc agttattgaa 2700
attcatctgg gtggcaagat tggcttaaat gaaagtgatt cccacacggc aaaagcgttt 2760
tatgagcagc agctggcaat tgcccaggaa aataacatcc cggtaatgat ggtggtagct 2820
acggcaggcc agcagaacta ctggacggga acagcgaatc tggatgctga gtggattgac 2880
cggatgttca agcagcacag tgtgttaaaa ggaattatgt ccactgaaaa ttattggact 2940
gactacaata aggttgctac tatgggtgcc gattatctgc gggttgcagc tgaaaacggc 3000
ggatattttg tatggagcga gcaccaggag ggtgttattg aaaatgtaat agcaaatgag 3060
aaatttaatg aagcattgaa actttacggt aataatttta ttttcacctg gaagaacacg 3120
cctgccggta ctaactccaa tgcaggaaca gccagctata tgcagggcct ctggctaacc 3180
ggaatttgtg cacaatgggg cggtctggct gatacctgga aatggtatga aaaaggattt 3240
ggtaaattat ttgatggtca gtattcttat aatccgggtg gggaagaagc aagaccggtt 3300
gcaaccgaac cggaagcact gcttggtatc gaaatgatga gtatctatac aaatggcgga 3360
tgtgtctaca actttgagca cccggcgtat gtatatggtt cttataacca gaattcacct 3420
tgctttgaaa atgtaattgc agagttcatg cgctatgcga ttaagaatcc ggcaccaggt 3480
aaagaggaag tgcttgctga tacaaaagca gtgttctatg gaaaattaag ttctttaaag 3540
agtgcaggaa acttactgca aaaaggtttg aactgggaag atgccacact gccaacccag 3600
actacgggtc gatatggatt aatacctgca gtcccggagg cagtagatga aaaaactgta 3660
aaagcagtat tcggcgatat tgagatattg aatcaatcca gtgcacagct tgcgaataaa 3720
gatgcgaaaa aagcatattt tgaagaaaaa tatccggaac agtataccgg tacggcattt 3780
ggacagctat tgaatgatac ctggtattta tacaacagta atgtgaatgt ggatggggtg 3840
caaaatgcaa aacttccgtt agaaggtaat aaatccgtag atattacaat gacaccgcat 3900
acttatgtga tcctggatga tcaggatggt gagcttcaga ttaaactgaa caattatcgt 3960
gtggataaag acagtatctg ggaaggatac ggcaccacgg tgacggaccg ctgggatacg 4020
gaccacaata ccaaacttca ggactggata cgggatgagt atattccaaa tccggacgat 4080
gataccttca gagatacaac ctttgaactg gttggactgg aaagtgagcc ggaggtaaat 4140
gtaactaatg gcttaaagga tcagtatcag gaaccggttg tggaatatga tgccgctgca 4200
ggtacggcta tgattactgt atccggaaat ggctgggtag atctgacaat tgacacgaac 4260
acggcagaag taccccaggt tgataaagca aagttaaatt ccaaaatagc agaagctaaa 4320
gggatcagac aggggaacta tacggatgaa tcctacaaag ctcttcagga agagattgga 4380
aaatcccagg cggtatcaaa caaaacagat gccacacagg aggaagtaaa tgcacagtta 4440
agcaggttag aaagtgcaat agccagatta aaagaaaaac cggcggtggt atccaaaacg 4500
gcattaaatg caaaaatagc tgaggcaaaa gggatcagac aaggaaacta tacggatgaa 4560
tcctacaaag ccctgcaaaa tgcaatagta aaagctcagg agttatcaaa caaaacagat 4620
gccacacagc agcaggtaaa tgatctggta tcagcattaa caaatgcaat taaaaattta 4680
aaaatagatg cagataagct ggcagcagag tcagcaaaga aagtagcggc agttaaggtt 4740
gccgtaaaag cagtatccta taaatcaaaa gagattaaat tatcctggaa aacggtagca 4800
gatgcggacg gatatgtaat ccgtgtaaag acaggcaaaa agtggagtac ggagaagacc 4860
attaagaaca accgcataat cacttatact tataagaaag gtactcccgg taagaaatat 4920
gtatttgaag taaaagcttt taagaaagta aatggaaaga cgacctatag taaatacaaa 4980
acagccacta aaaaagttgt gccgcaaacg gtgaccgcaa aggcaaaagc ttctaaaaat 5040
aatgtagtgg taaaatggaa caaagtgtct ggcgcatccg gatatgttgt tatgaaaaag 5100
aaagggaaaa catgggtaaa ggctgcgcag gtaaatgcaa agaaactata ctttacggat 5160
aagaaggtca aaaaaggaaa agtatattca tacaaagtaa aggcttacaa agtatataaa 5220
ggtaaaaaag tatatggaag ctatagcaag tctgtaaatg ttaaaacaaa gtcataa 5277
<210> 25
<211> 1778
<212> PRT
<213> Robinsoniella peoriensis
<400> 25
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Ser Pro Leu Ser Ala Ala Ala Glu Ser Gly Thr Gly
20 25 30
Thr Arg Leu Val Lys Gly Gln Thr Gly Tyr Leu Thr Glu Glu Gln Ala
35 40 45
Ile Arg Asn Gln Glu Gln Thr Thr Glu Glu Arg Glu Gln Lys Leu Thr
50 55 60
Gly Glu Glu Thr Ala Glu Val Leu Met Glu Gly Thr Lys Asp Ser Gly
65 70 75 80
Ile Val Gln Thr Glu Glu Val Gln Thr Lys Glu Met Gln Thr Glu Asp
85 90 95
Ala Gln Thr Glu Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala
100 105 110
Gln Thr Lys Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln
115 120 125
Thr Glu Glu Val Gln Thr Lys Glu Glu Pro Ala Glu Glu Thr His Met
130 135 140
Lys Glu Ile Gln Thr Gln Gly Thr Lys Lys Ala Ser Asp Arg Asn Gly
145 150 155 160
Lys Ala Arg Val Thr Glu Ile Leu Glu Asp Ala Gln Asp Pro Ala Asn
165 170 175
Arg Ile Val Tyr Leu Ser Asp Leu Gln Trp Lys Ser Glu Asn His Thr
180 185 190
Val Asp Ser Glu Leu Pro Thr Arg Lys Asp Lys Ser Phe Gly Gly Gly
195 200 205
Lys Ile Thr Leu Lys Val Asp Gly Thr Val Thr Glu Phe Asp Lys Gly
210 215 220
Ile Gly Thr Gln Thr Asp Ser Thr Ile Val Tyr Asp Leu Glu Gly Lys
225 230 235 240
Gly Tyr Thr Lys Phe Glu Thr Tyr Val Gly Val Asp Tyr Ser Gln Lys
245 250 255
Glu Asn Ile Pro Gly Glu Val Cys Asp Val Lys Phe Arg Val Lys Ile
260 265 270
Asp Asp Lys Ile Val Ser Glu Thr Gly Val Leu Asp Pro Leu Ser Asn
275 280 285
Ala Val Lys Ile Ser Val Asn Ile Pro Asp Thr Ala Lys Thr Leu Thr
290 295 300
Leu Tyr Ala Asp Lys Val Thr Glu Thr Trp Ser Asp His Ala Asn Trp
305 310 315 320
Ala Asp Ala Lys Phe Tyr Gln Ala Leu Pro Glu Pro Glu Asn Val Ala
325 330 335
Phe Lys Lys Thr Val Val Thr Arg Lys Thr Ser Asp Asn Ser Glu Ala
340 345 350
Pro Val Asn Pro Asp Ser Ala Val Asn Ser Ser Lys Ala Val Asp Gly
355 360 365
Val Ile Asp Ser Ser Ser Tyr Phe Asp Phe Gly Asp Gln Ala Asn Ser
370 375 380
Gly Ala Val Arg Glu Ser Leu Tyr Met Glu Val Asp Leu Lys Gly Ser
385 390 395 400
Tyr Leu Leu Ser Asp Ile Gln Leu Trp Arg Tyr Trp Lys Asp Gly Arg
405 410 415
Thr Tyr Ala Ala Thr Ala Ile Val Val Ala Glu Asp Glu Asn Phe Glu
420 425 430
Asn Ala Ala Val Ile Tyr Asn Ser Asp Thr Thr Gly Glu Ile His His
435 440 445
Leu Gly Ala Gly Ser Asp Met Leu Tyr Ala Glu Thr Glu Ser Gly Lys
450 455 460
Thr Phe Pro Val Pro Glu Asn Thr Lys Ala Arg Tyr Ile Arg Val Tyr
465 470 475 480
Thr Tyr Gly Val Asn Gly Thr Ser Gly Val Thr Asn His Ile Val Glu
485 490 495
Leu Lys Val Asn Ala Tyr Val Phe Gly Asp Glu Ile Leu Pro Glu Lys
500 505 510
Pro Asp Asp Ser Lys Ile Phe Pro Asn Ala Val Asn Pro Leu Lys Leu
515 520 525
Gln Gly Pro Gly Thr Asn Asp Gln Val Thr His Pro Asp Val Thr Val
530 535 540
Phe Asp Glu Pro Trp Asn Gly Tyr Lys Tyr Trp Met Ala Tyr Thr Pro
545 550 555 560
Asn Lys Pro Gly Ser Ser Tyr Phe Glu Asn Pro Cys Ile Ala Ala Ser
565 570 575
Asn Asp Gly Val Asn Trp Glu Phe Pro Ala Gln Asn Pro Val Gln Pro
580 585 590
Arg Tyr Asp Ser Glu Ile Glu Asn Gln Asn Glu His Asn Cys Asp Thr
595 600 605
Asp Ile Val Tyr Asp Pro Val Asn Asp Arg Leu Ile Met Tyr Trp Glu
610 615 620
Trp Ala Gln Asp Glu Ala Val Asn Gly Lys Thr His Arg Ser Glu Ile
625 630 635 640
Arg Tyr Arg Val Ser Tyr Asp Gly Ile Asn Trp Gly Val Glu Asp Lys
645 650 655
Thr Gly Val Leu Met Thr Gly Pro Thr Asp His Gly Cys Ala Ile Ala
660 665 670
Thr Glu Gly Glu Arg Tyr Ser Asp Leu Ser Pro Thr Val Val Tyr Asp
675 680 685
Lys Thr Glu Lys Ile Tyr Lys Met Trp Ala Asn Asp Ala Gly Asp Val
690 695 700
Gly Tyr Glu Asn Lys Gln Asn Asn Lys Val Trp Tyr Arg Thr Ser Gln
705 710 715 720
Asp Gly Ile Ser Asn Trp Ser Asp Lys Thr Tyr Val Glu Asn Phe Leu
725 730 735
Gly Val Asn Glu Asp Gly Leu Gln Met Tyr Pro Trp His Gln Asp Ile
740 745 750
Gln Trp Val Glu Glu Phe Gln Glu Tyr Trp Ala Leu Gln Gln Ala Phe
755 760 765
Pro Ala Gly Ser Gly Pro Asp Asn Ser Ser Leu Arg Phe Ser Lys Ser
770 775 780
Lys Asp Gly Leu His Trp Glu Pro Val Ser Glu Lys Ala Leu Ile Thr
785 790 795 800
Val Gly Ala Pro Gly Thr Trp Asp Ala Gly Gln Ile Tyr Arg Ser Thr
805 810 815
Phe Trp Tyr Glu Pro Gly Gly Ala Lys Gly Asn Gly Thr Phe His Ile
820 825 830
Trp Tyr Ala Ala Leu Ala Glu Gly Gln Ser His Trp Asp Ile Gly Tyr
835 840 845
Thr Ser Ala Asn Tyr Ala Asp Ala Met Tyr Lys Leu Thr Gly Ser Arg
850 855 860
Pro Glu Val Glu Lys Arg Ile Glu Val Asn Asn Glu Asn Pro Leu Leu
865 870 875 880
Ile Met Pro Leu Tyr Gly Lys Ser Tyr Ser Glu Ser Gly Ser Thr Leu
885 890 895
Asp Trp Gly Asp Asp Leu Val Ser Arg Trp Lys Gln Val Pro Glu Asp
900 905 910
Leu Lys Glu Asn Ala Val Ile Glu Ile His Leu Gly Gly Lys Ile Gly
915 920 925
Leu Asn Glu Ser Asp Ser His Thr Ala Lys Ala Phe Tyr Glu Gln Gln
930 935 940
Leu Ala Ile Ala Gln Glu Asn Asn Ile Pro Val Met Met Val Val Ala
945 950 955 960
Thr Ala Gly Gln Gln Asn Tyr Trp Thr Gly Thr Ala Asn Leu Asp Ala
965 970 975
Glu Trp Ile Asp Arg Met Phe Lys Gln His Ser Val Leu Lys Gly Ile
980 985 990
Met Ser Thr Glu Asn Tyr Trp Thr Asp Tyr Asn Lys Val Ala Thr Met
995 1000 1005
Gly Ala Asp Tyr Leu Arg Val Ala Ala Glu Asn Gly Gly Tyr Phe
1010 1015 1020
Val Trp Ser Glu His Gln Glu Gly Val Ile Glu Asn Val Ile Ala
1025 1030 1035
Asn Glu Lys Phe Asn Glu Ala Leu Lys Leu Tyr Gly Asn Asn Phe
1040 1045 1050
Ile Phe Thr Trp Lys Asn Thr Pro Ala Gly Thr Asn Ser Asn Ala
1055 1060 1065
Gly Thr Ala Ser Tyr Met Gln Gly Leu Trp Leu Thr Gly Ile Cys
1070 1075 1080
Ala Gln Trp Gly Gly Leu Ala Asp Thr Trp Lys Trp Tyr Glu Lys
1085 1090 1095
Gly Phe Gly Lys Leu Phe Asp Gly Gln Tyr Ser Tyr Asn Pro Gly
1100 1105 1110
Gly Glu Glu Ala Arg Pro Val Ala Thr Glu Pro Glu Ala Leu Leu
1115 1120 1125
Gly Ile Glu Met Met Ser Ile Tyr Thr Asn Gly Gly Cys Val Tyr
1130 1135 1140
Asn Phe Glu His Pro Ala Tyr Val Tyr Gly Ser Tyr Asn Gln Asn
1145 1150 1155
Ser Pro Cys Phe Glu Asn Val Ile Ala Glu Phe Met Arg Tyr Ala
1160 1165 1170
Ile Lys Asn Pro Ala Pro Gly Lys Glu Glu Val Leu Ala Asp Thr
1175 1180 1185
Lys Ala Val Phe Tyr Gly Lys Leu Ser Ser Leu Lys Ser Ala Gly
1190 1195 1200
Asn Leu Leu Gln Lys Gly Leu Asn Trp Glu Asp Ala Thr Leu Pro
1205 1210 1215
Thr Gln Thr Thr Gly Arg Tyr Gly Leu Ile Pro Ala Val Pro Glu
1220 1225 1230
Ala Val Asp Glu Lys Thr Val Lys Ala Val Phe Gly Asp Ile Glu
1235 1240 1245
Ile Leu Asn Gln Ser Ser Ala Gln Leu Ala Asn Lys Asp Ala Lys
1250 1255 1260
Lys Ala Tyr Phe Glu Glu Lys Tyr Pro Glu Gln Tyr Thr Gly Thr
1265 1270 1275
Ala Phe Gly Gln Leu Leu Asn Asp Thr Trp Tyr Leu Tyr Asn Ser
1280 1285 1290
Asn Val Asn Val Asp Gly Val Gln Asn Ala Lys Leu Pro Leu Glu
1295 1300 1305
Gly Asn Lys Ser Val Asp Ile Thr Met Thr Pro His Thr Tyr Val
1310 1315 1320
Ile Leu Asp Asp Gln Asp Gly Glu Leu Gln Ile Lys Leu Asn Asn
1325 1330 1335
Tyr Arg Val Asp Lys Asp Ser Ile Trp Glu Gly Tyr Gly Thr Thr
1340 1345 1350
Val Thr Asp Arg Trp Asp Thr Asp His Asn Thr Lys Leu Gln Asp
1355 1360 1365
Trp Ile Arg Asp Glu Tyr Ile Pro Asn Pro Asp Asp Asp Thr Phe
1370 1375 1380
Arg Asp Thr Thr Phe Glu Leu Val Gly Leu Glu Ser Glu Pro Glu
1385 1390 1395
Val Asn Val Thr Asn Gly Leu Lys Asp Gln Tyr Gln Glu Pro Val
1400 1405 1410
Val Glu Tyr Asp Ala Ala Ala Gly Thr Ala Met Ile Thr Val Ser
1415 1420 1425
Gly Asn Gly Trp Val Asp Leu Thr Ile Asp Thr Asn Thr Ala Glu
1430 1435 1440
Val Pro Gln Val Asp Lys Ala Lys Leu Asn Ser Lys Ile Ala Glu
1445 1450 1455
Ala Lys Gly Ile Arg Gln Gly Asn Tyr Thr Asp Glu Ser Tyr Lys
1460 1465 1470
Ala Leu Gln Glu Glu Ile Gly Lys Ser Gln Ala Val Ser Asn Lys
1475 1480 1485
Thr Asp Ala Thr Gln Glu Glu Val Asn Ala Gln Leu Ser Arg Leu
1490 1495 1500
Glu Ser Ala Ile Ala Arg Leu Lys Glu Lys Pro Ala Val Val Ser
1505 1510 1515
Lys Thr Ala Leu Asn Ala Lys Ile Ala Glu Ala Lys Gly Ile Arg
1520 1525 1530
Gln Gly Asn Tyr Thr Asp Glu Ser Tyr Lys Ala Leu Gln Asn Ala
1535 1540 1545
Ile Val Lys Ala Gln Glu Leu Ser Asn Lys Thr Asp Ala Thr Gln
1550 1555 1560
Gln Gln Val Asn Asp Leu Val Ser Ala Leu Thr Asn Ala Ile Lys
1565 1570 1575
Asn Leu Lys Ile Asp Ala Asp Lys Leu Ala Ala Glu Ser Ala Lys
1580 1585 1590
Lys Val Ala Ala Val Lys Val Ala Val Lys Ala Val Ser Tyr Lys
1595 1600 1605
Ser Lys Glu Ile Lys Leu Ser Trp Lys Thr Val Ala Asp Ala Asp
1610 1615 1620
Gly Tyr Val Ile Arg Val Lys Thr Gly Lys Lys Trp Ser Thr Glu
1625 1630 1635
Lys Thr Ile Lys Asn Asn Arg Ile Ile Thr Tyr Thr Tyr Lys Lys
1640 1645 1650
Gly Thr Pro Gly Lys Lys Tyr Val Phe Glu Val Lys Ala Phe Lys
1655 1660 1665
Lys Val Asn Gly Lys Thr Thr Tyr Ser Lys Tyr Lys Thr Ala Thr
1670 1675 1680
Lys Lys Val Val Pro Gln Thr Val Thr Ala Lys Ala Lys Ala Ser
1685 1690 1695
Lys Asn Asn Val Val Val Lys Trp Asn Lys Val Ser Gly Ala Ser
1700 1705 1710
Gly Tyr Val Val Met Lys Lys Lys Gly Lys Thr Trp Val Lys Ala
1715 1720 1725
Ala Gln Val Asn Ala Lys Lys Leu Tyr Phe Thr Asp Lys Lys Val
1730 1735 1740
Lys Lys Gly Lys Val Tyr Ser Tyr Lys Val Lys Ala Tyr Lys Val
1745 1750 1755
Tyr Lys Gly Lys Lys Val Tyr Gly Ser Tyr Ser Lys Ser Val Asn
1760 1765 1770
Val Lys Thr Lys Ser
1775
<210> 26
<211> 7899
<212> DNA
<213> Robinsoniella peoriensis
<400> 26
gctgagactg caacagaaga aaatgcggcg ctggaaaaaa cagttacatt gcataagagc 60
gatggaacag aactgccgga ggattatcga aatccccaaa gaccagctac catggcggta 120
gatggtatta ttgacgatac aggagagtac aactattgcg atttcggtaa agacggtgat 180
aaagcagccc tgtatatgca ggtggacctt ggaggtctgt atgatttaag cagagtcaat 240
atgtggagat actggaaaga cagcagaact tacgatgcaa cagtaattac cacatctgag 300
agcggcgatt tcacagatga agcagtcata tataattcag acaggtcgaa tgtacatgga 360
tttggggcag gaggagatga acgctacgca gagactgcct ccggacatga attcccagta 420
ccggacggta caaaggcaca ggcagtacgc gtatatgtat ttggcagcca aaacggtact 480
acaaaccaca tcaatgaatt gcaggtctgg ggaactcccc atacagagaa tccggatgta 540
aattcttatc aggtgacaat tccacaggga aatggatatc aggtaatacc ttatgaaaat 600
gacccgacga cagtggaaga aggcggttct ttccgttttc aggtactgat tgactccgat 660
aatggttaca gcgcaaccag tgcggtaaaa gcaaatggag taagtctgga ggcagttgac 720
agtgtttata ccattgagaa cattactgaa gatcaggtaa tcaccattga aggcgtacat 780
aaagcacagt atgaagtgaa attcccggaa aatccacagg gatacagtgt tgagattcag 840
aatgaaggaa gtacaacggt agactataat ggttctgtca gttttaagct tattatagac 900
gaagcttata atgaatccgt accggttgta aaagcaaacg gcggtgcagc tttgggaaaa 960
gatgagctcg gtgtatatac aattgcaaat atccaggacg atattacggt tacagttgag 1020
ggtatccagg aaaataccgt agtaaagaca aaaacaatgt acttgtctga tatggattgg 1080
aagagtgctg caaatgcagt aggtgcaaca ggagaaaaag acactccaac aaaggacctg 1140
aatcatttac agcagcagat gaaattattg gtaaacggag cagagaagtc ttttgataaa 1200
ggaattggag ttcagacgga ttcttctatc gtttatgatc tggaagacaa aggctacact 1260
tctttccaca ccctggcagg cgttgattat tcagcaatgg aatatgtaga cggagaaggc 1320
tgtgatatcc agtttaaagt atatctggat gatgtcgtag tatttgacag cggagtagtt 1380
gatgcatctg atgaggctca ggaagttaat gttgctataa catcagagaa taaagaacta 1440
aaactggaag ctaaaatggt taaagagcct tataatgact ggggaaactg ggcagatgcc 1500
agctttgaaa tggcttatcc cgaaccgtct aatgtggctt taaataaaac agttaccgtt 1560
aagaaaacag cggataactc agactctgaa gtaaattcca gcagaccggg atcaatggct 1620
gtagatggaa tcattggacc tacatcagat tctaactatt gtgattttgg acaggatggg 1680
gataatactt cccgttatct gcaggtagat ttaggggatg tttatgaact tacccagatt 1740
aatatgttta gatactgggc agatggcaga gtatataatg gtactgtaat tgcagtttcc 1800
gaaaacgcag actttagtaa tccaactttt atttataatt cagataaagc agacaaacac 1860
ggacttggcg caggcagtga tgacacttat ggagaaaccc agagtggaaa attattcgaa 1920
gttccggcgg gaaccatggg acagtatgtc cgtgtgtata tggctggttc caacaaaggt 1980
acaacgaacc atatcgctga attacaggta atgggttata atttcaatac agaaccaaaa 2040
ccatatgaag caaatgcatt tgaaaatgca gaagtttatt tagatatgcc aactcatttc 2100
caggatctgg attccaataa aaacgacgat ggaagcttaa agcacattgg cggacaggtg 2160
acacatcctg atatccaggt atttgaccaa ccgtggaacg gttataaata ctggatgatt 2220
tacacaccaa atacaatgat cacttcccag tatgaaaatc catatatcgt agcatctgaa 2280
gatggacaga catgggtaga accggaaggg atttccaatc caattgaacc agaaccgcca 2340
tcaaccagat ttcataactg tgatgcagat ctgttatacg actctgtcaa tgaccgttta 2400
cttgcttact ggaactgggc agatgacggc ggcggaattg atgacgaatt aaaagatcag 2460
aactgtcaga ttcgtctgag aatttcttat gatggaatta actggggagt tccttacgac 2520
aaagacggca atattgccac aacagctgat actgtagtaa gaatggaaac aggagataag 2580
gatttcattc ctgcaatcag cgaaaaagac cgttatggta tgctttcccc aacatttacc 2640
tatgacgatt tccgcggcat atatacaatg tgggcacaaa actcgggtga tgcgggatac 2700
aaccagtccg gaaagttcat cgaaatgaga tggtctgagg atggaataaa ctggtctgaa 2760
ccacaaaaag tgaataattt ccttggaaaa gatgagaatg gcagacagct ttggccatgg 2820
catcaggata ttcagtatat ccctgagcta caggaatatt ggggactgtc ccagtgtttc 2880
tctacatcta atcccgatgg atccgtatta tacctgacca agtccagaga tggtgtcaac 2940
tgggagcagg caggaacaca gccggtatta agggcaggaa aatcaggtac ctgggatgat 3000
ttccagattt accgttctac cttctattat gataatcagt cagacagccc tactggtggg 3060
aaatttagaa tctggtacag tgcactgcag gcaaatactt caggcaagac cgttttggct 3120
cctgatggaa cagtgtctct tcaggttgga agccaggata ccaggatctg gcgtatcggg 3180
tatacagaaa atgactacat ggaagtcatg aaagctctga cccagaataa aaactatgaa 3240
gaaccggaat tagtagacgc agtttcctta aatctgtcaa tggataaaac aagcatttca 3300
gtaggtgaag aagcaacggt aagcactgct ttcgtaccgg aaaatgctac cgaccgcatt 3360
gtaaaatata catctcagga tccggaaatt gcagttattg atccaacagg cattgttaca 3420
ggggttaagg atggaaccac aactattgtt gcagaaacaa aatcgggcgc aaaaggtgaa 3480
ttatccgtaa cggttggtga gcttcaaaga ggtgaaattc gatttgaggt cagcaatgac 3540
catccgatgt atctggagaa ttactattgg agtgatgatg caccaaaaaa agacggctta 3600
gacgcaaaca agaactacta tggggatgaa cgtgtcgaca gtccggtaat gctgtataat 3660
accgttcctg aagaattgaa ggataataca gtcatcctgt taattgcaga gagaagctta 3720
aacagcacag atgcagtaag ggattggatt aaaaagaatg ttgaattatg taatgaaaat 3780
aagattccat gtgcagttca gattgcaaat ggagaaacaa atgtaaatac aaccattcca 3840
ttatcgttct ggaatgagct ggcaacgaac aatgaatacc tggttggatt taatgcagcc 3900
gagatgtata accgttttgc aggtgacaac cgcagctatg ttatggatat gatccgttta 3960
ggggtatccc acggcgtatg catgatgtgg accgatacca atatttttgg tacaaacggt 4020
gtgttgtatg actggctgac tcaggatgaa aaactgtccg gtcttatgcg ggaatacaaa 4080
gagtatatct ctctgatgac aaaagagtct tacggcagtg aggcagcaaa tacagatgct 4140
ctgtttaagg gcctgtggat gacagactac tgcgagaact ggggaatcgc ctccgactgg 4200
tggcattggc agttagacag caatggagca ctctttgatg caggcagcgg cggagatgca 4260
tggaaacagt gtctgacatg gccggaaaat atgtatacgc aggacgttgt gcgtgcagta 4320
agccagggtg caacctgctt taaatcagaa gcacagtggt attcaaatgc tacaaaaggc 4380
atgcgtacac cgacatatca gtattccatg attccgttcc tggagaaact ggtaagcaaa 4440
gaggtaaaaa ttcctacaaa agaagagatg ctggaaagaa caaaagcaat tgttgtaggg 4500
gcagaaaact ggaataactt taattataat actacttatt caaatctgta tccaagcaca 4560
ggacaatatg gaatcgtacc ttatgtacct tcaaattgtc cggaagaaga actggcaggc 4620
tatgatctgg tagtaaggga aaaccttggc aaagcaggac tgaagtctgc acttgatacg 4680
gtatacccgg ttcagaaatc agaaggaacc gcatactgtg aaacctttgg agatacctgg 4740
tactggatga attcctcgga agacaaaaac gtaagccagt acactgaatt tacaaccgca 4800
atcaatggag ctgaaagtgt aaagatagcc ggcgaacccc atgtatttgg tattataaaa 4860
gaaaatccgg gatctttaaa tgtatactta agcaactacc gcctggataa aacagaactc 4920
tgggatggta caatccccgg aggattaagc gatcagggct gctataatta tgtatggcag 4980
atgtgtgagc gcatgaagaa tggaacaggg ctggatacac agcttcgtga caccgttatt 5040
accgttaaaa atgcagtaga accgaaagta aactttgtaa cagaatctcc ggcagacaga 5100
agttttgcag aagataatta tgtaagacca tacaaatata cggttgcaca aaaagaaggc 5160
acaaccgatg aatgggtgat tacggtcagc cacaatggta ttgtggaatt caatattgta 5220
acaggcgatg aaaaagtgcc ggcaacaagt gtggaattat caactgataa agttgatgta 5280
atccgtaacc ggacagcagt tgtaaaggca acggtattgc cgcagaatgc aggaaataaa 5340
cagttaacat ggacaatcgc cgatcctgag attgcttctg tagacaacaa aggaaccgta 5400
accggactaa aagaaggaaa aaccgtatta cgtgcagcta tttctggcag tgtttataaa 5460
gaatgcgaag taaatgtaat tgaccgaaaa gtaacggaag taaacttaaa caaaacagag 5520
ttgtctctta gtgcagggga ttctgcgaaa ctggaagcat ccatagcacc ggaagacccg 5580
tctgacagca gcattacctg gacttccaca aatgaaaatg ttgcaacggt tgcatcaaac 5640
ggtaccgtta cagctcataa agcaggtgta gctcagatta tcgcccagtc tgcttaccag 5700
gcaaagggta tcgcaactgt taccgttaat tatgcggctt ccgtaaaatt agaccgtaca 5760
ggaatgacgg ctacagccaa cagcgaacag tctaaatcag gtggagaagg acctgcttcc 5820
aacgtactgg acggtaagca ggacacaatg tggcatacaa gctggacaga taaacctgaa 5880
ttacatcctc actggattaa aattgattta aacggaacaa aaacaattaa caaatttgct 5940
tatacaccaa gaaccggagc atctaacgga acaatttata attatgttct gattatcacc 6000
gatctggaag gaaatgaaaa acaggttgca aagggcgtat gggcagcaaa tgcagatgta 6060
aaatatgctg aatttgacgc agttgaagct acggcgatca agctgcaggt agacggcaac 6120
gatgacaagg catcaaaagg aggatatggt tccgcggcag aaatcaatat ttttgaagtg 6180
gcacagaaac cttccgcaaa tgagcttgcc gaaaatatta aagtaattgc acctgtaaaa 6240
gcagaagata caaaagtatc tatcccagtc attactggat ttgatatcgt aatcagtaat 6300
tccagcaatc cggacgtaat tggtattgat ggcagcatca ccagaccgga aaatgataca 6360
gttgtaactt taacattaaa agtaaaagaa acagacgcaa agagtgtaaa ggcagcagga 6420
actgaagcaa ccacaaatgt ggatgttctg gttaccggta caaagacatc tgatgtagag 6480
gcagaaagcg ttacgttaga tcagacatca gctgatttaa cagttggagg cgaactttta 6540
ttaaatgccg ttgtgaagcc ggacattgca actaataagg ctgttacctg gagctcagat 6600
aagccgggaa ccgctactgt tgaaaatggc agggtaaaag cgttagcggc aggagaggca 6660
cgtattacag cagcaactgc aaatggaaag acagcagact gcgtcattaa cgtaaaggaa 6720
aaagaggagc cggaagtaat tctcccggca gaagtgcgct taaacattcc atcagctgaa 6780
tttacagtag gagatcagat tcagttaact gcttctgtac tgccggcaaa tgcagcagat 6840
aagacaatta cctggaaatc agacaaacct gaagtggcaa ctgtcgcaaa tggatgggta 6900
aaaggtattg cagccggaac tgctaagatt acagcaacat cagtcaatgg aaaaacggct 6960
gtatgtgtga tcacagtcaa agcacagcca cagaatctac caaccggtgt ttcactgaac 7020
aagaaaacag caagtgtaaa actgaataaa acccttacac tttccgctgt agtacagcct 7080
tccaatgcgg ataataagac cgttaaatgg acgtctgaca atacgtatgt tgcaacagtt 7140
gagaatggag tcgtaaaagc agttaatgca ggaacagcca gaatcactgc agctaccgta 7200
aacggacata aggcaacttg tactataaca gtaccgggca caaagatttc caaggcaaaa 7260
gtaagccttg catcatcaaa aacacataca ggcaaagcat taaaaccatc tgtaaaagta 7320
acttacggta agaatacatt aaagaaaaat actgattata ccgtatctta caaaaataat 7380
ataaatcctg gaactgcatc tgttacgatt acgggcaagg gtaaatatta tggtaccatc 7440
aacaaaactt ttgcaatcaa ggcagcagaa ggaaagacct acacggttgg taaaggaaaa 7500
tataaagtta ctgatgcttc agcaaagaac aaaacagtaa cctttatggc tcctgtaaag 7560
aagacctaca gctcattcag cgtaccttct aaggttaaga tcgggaatga tacttacaaa 7620
gtaactgcag ttgcaaaaaa tgcattcaaa aagaatacaa agcttacaaa gttaaccatt 7680
ggttcgaatg taaaaacaat tggttcttat gcattttatg gcgcttccca attaaaaacg 7740
cttaccttaa aaactaccgg acttaacagt gtaggcaaga atgcatttaa gaaaacaaat 7800
gcaaagctga ctgtaaaggt tccaaagtca aaattagcag attataagaa gctgttaaaa 7860
ggaaaaggat tatctggcaa ggcaaaaatt cagaaataa 7899
<210> 27
<211> 2652
<212> PRT
<213> Robinsoniella peoriensis
<400> 27
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Ala Glu Thr Ala Thr Glu Glu Asn Ala Ala Leu Glu
20 25 30
Lys Thr Val Thr Leu His Lys Ser Asp Gly Thr Glu Leu Pro Glu Asp
35 40 45
Tyr Arg Asn Pro Gln Arg Pro Ala Thr Met Ala Val Asp Gly Ile Ile
50 55 60
Asp Asp Thr Gly Glu Tyr Asn Tyr Cys Asp Phe Gly Lys Asp Gly Asp
65 70 75 80
Lys Ala Ala Leu Tyr Met Gln Val Asp Leu Gly Gly Leu Tyr Asp Leu
85 90 95
Ser Arg Val Asn Met Trp Arg Tyr Trp Lys Asp Ser Arg Thr Tyr Asp
100 105 110
Ala Thr Val Ile Thr Thr Ser Glu Ser Gly Asp Phe Thr Asp Glu Ala
115 120 125
Val Ile Tyr Asn Ser Asp Arg Ser Asn Val His Gly Phe Gly Ala Gly
130 135 140
Gly Asp Glu Arg Tyr Ala Glu Thr Ala Ser Gly His Glu Phe Pro Val
145 150 155 160
Pro Asp Gly Thr Lys Ala Gln Ala Val Arg Val Tyr Val Phe Gly Ser
165 170 175
Gln Asn Gly Thr Thr Asn His Ile Asn Glu Leu Gln Val Trp Gly Thr
180 185 190
Pro His Thr Glu Asn Pro Asp Val Asn Ser Tyr Gln Val Thr Ile Pro
195 200 205
Gln Gly Asn Gly Tyr Gln Val Ile Pro Tyr Glu Asn Asp Pro Thr Thr
210 215 220
Val Glu Glu Gly Gly Ser Phe Arg Phe Gln Val Leu Ile Asp Ser Asp
225 230 235 240
Asn Gly Tyr Ser Ala Thr Ser Ala Val Lys Ala Asn Gly Val Ser Leu
245 250 255
Glu Ala Val Asp Ser Val Tyr Thr Ile Glu Asn Ile Thr Glu Asp Gln
260 265 270
Val Ile Thr Ile Glu Gly Val His Lys Ala Gln Tyr Glu Val Lys Phe
275 280 285
Pro Glu Asn Pro Gln Gly Tyr Ser Val Glu Ile Gln Asn Glu Gly Ser
290 295 300
Thr Thr Val Asp Tyr Asn Gly Ser Val Ser Phe Lys Leu Ile Ile Asp
305 310 315 320
Glu Ala Tyr Asn Glu Ser Val Pro Val Val Lys Ala Asn Gly Gly Ala
325 330 335
Ala Leu Gly Lys Asp Glu Leu Gly Val Tyr Thr Ile Ala Asn Ile Gln
340 345 350
Asp Asp Ile Thr Val Thr Val Glu Gly Ile Gln Glu Asn Thr Val Val
355 360 365
Lys Thr Lys Thr Met Tyr Leu Ser Asp Met Asp Trp Lys Ser Ala Ala
370 375 380
Asn Ala Val Gly Ala Thr Gly Glu Lys Asp Thr Pro Thr Lys Asp Leu
385 390 395 400
Asn His Leu Gln Gln Gln Met Lys Leu Leu Val Asn Gly Ala Glu Lys
405 410 415
Ser Phe Asp Lys Gly Ile Gly Val Gln Thr Asp Ser Ser Ile Val Tyr
420 425 430
Asp Leu Glu Asp Lys Gly Tyr Thr Ser Phe His Thr Leu Ala Gly Val
435 440 445
Asp Tyr Ser Ala Met Glu Tyr Val Asp Gly Glu Gly Cys Asp Ile Gln
450 455 460
Phe Lys Val Tyr Leu Asp Asp Val Val Val Phe Asp Ser Gly Val Val
465 470 475 480
Asp Ala Ser Asp Glu Ala Gln Glu Val Asn Val Ala Ile Thr Ser Glu
485 490 495
Asn Lys Glu Leu Lys Leu Glu Ala Lys Met Val Lys Glu Pro Tyr Asn
500 505 510
Asp Trp Gly Asn Trp Ala Asp Ala Ser Phe Glu Met Ala Tyr Pro Glu
515 520 525
Pro Ser Asn Val Ala Leu Asn Lys Thr Val Thr Val Lys Lys Thr Ala
530 535 540
Asp Asn Ser Asp Ser Glu Val Asn Ser Ser Arg Pro Gly Ser Met Ala
545 550 555 560
Val Asp Gly Ile Ile Gly Pro Thr Ser Asp Ser Asn Tyr Cys Asp Phe
565 570 575
Gly Gln Asp Gly Asp Asn Thr Ser Arg Tyr Leu Gln Val Asp Leu Gly
580 585 590
Asp Val Tyr Glu Leu Thr Gln Ile Asn Met Phe Arg Tyr Trp Ala Asp
595 600 605
Gly Arg Val Tyr Asn Gly Thr Val Ile Ala Val Ser Glu Asn Ala Asp
610 615 620
Phe Ser Asn Pro Thr Phe Ile Tyr Asn Ser Asp Lys Ala Asp Lys His
625 630 635 640
Gly Leu Gly Ala Gly Ser Asp Asp Thr Tyr Gly Glu Thr Gln Ser Gly
645 650 655
Lys Leu Phe Glu Val Pro Ala Gly Thr Met Gly Gln Tyr Val Arg Val
660 665 670
Tyr Met Ala Gly Ser Asn Lys Gly Thr Thr Asn His Ile Ala Glu Leu
675 680 685
Gln Val Met Gly Tyr Asn Phe Asn Thr Glu Pro Lys Pro Tyr Glu Ala
690 695 700
Asn Ala Phe Glu Asn Ala Glu Val Tyr Leu Asp Met Pro Thr His Phe
705 710 715 720
Gln Asp Leu Asp Ser Asn Lys Asn Asp Asp Gly Ser Leu Lys His Ile
725 730 735
Gly Gly Gln Val Thr His Pro Asp Ile Gln Val Phe Asp Gln Pro Trp
740 745 750
Asn Gly Tyr Lys Tyr Trp Met Ile Tyr Thr Pro Asn Thr Met Ile Thr
755 760 765
Ser Gln Tyr Glu Asn Pro Tyr Ile Val Ala Ser Glu Asp Gly Gln Thr
770 775 780
Trp Val Glu Pro Glu Gly Ile Ser Asn Pro Ile Glu Pro Glu Pro Pro
785 790 795 800
Ser Thr Arg Phe His Asn Cys Asp Ala Asp Leu Leu Tyr Asp Ser Val
805 810 815
Asn Asp Arg Leu Leu Ala Tyr Trp Asn Trp Ala Asp Asp Gly Gly Gly
820 825 830
Ile Asp Asp Glu Leu Lys Asp Gln Asn Cys Gln Ile Arg Leu Arg Ile
835 840 845
Ser Tyr Asp Gly Ile Asn Trp Gly Val Pro Tyr Asp Lys Asp Gly Asn
850 855 860
Ile Ala Thr Thr Ala Asp Thr Val Val Arg Met Glu Thr Gly Asp Lys
865 870 875 880
Asp Phe Ile Pro Ala Ile Ser Glu Lys Asp Arg Tyr Gly Met Leu Ser
885 890 895
Pro Thr Phe Thr Tyr Asp Asp Phe Arg Gly Ile Tyr Thr Met Trp Ala
900 905 910
Gln Asn Ser Gly Asp Ala Gly Tyr Asn Gln Ser Gly Lys Phe Ile Glu
915 920 925
Met Arg Trp Ser Glu Asp Gly Ile Asn Trp Ser Glu Pro Gln Lys Val
930 935 940
Asn Asn Phe Leu Gly Lys Asp Glu Asn Gly Arg Gln Leu Trp Pro Trp
945 950 955 960
His Gln Asp Ile Gln Tyr Ile Pro Glu Leu Gln Glu Tyr Trp Gly Leu
965 970 975
Ser Gln Cys Phe Ser Thr Ser Asn Pro Asp Gly Ser Val Leu Tyr Leu
980 985 990
Thr Lys Ser Arg Asp Gly Val Asn Trp Glu Gln Ala Gly Thr Gln Pro
995 1000 1005
Val Leu Arg Ala Gly Lys Ser Gly Thr Trp Asp Asp Phe Gln Ile
1010 1015 1020
Tyr Arg Ser Thr Phe Tyr Tyr Asp Asn Gln Ser Asp Ser Pro Thr
1025 1030 1035
Gly Gly Lys Phe Arg Ile Trp Tyr Ser Ala Leu Gln Ala Asn Thr
1040 1045 1050
Ser Gly Lys Thr Val Leu Ala Pro Asp Gly Thr Val Ser Leu Gln
1055 1060 1065
Val Gly Ser Gln Asp Thr Arg Ile Trp Arg Ile Gly Tyr Thr Glu
1070 1075 1080
Asn Asp Tyr Met Glu Val Met Lys Ala Leu Thr Gln Asn Lys Asn
1085 1090 1095
Tyr Glu Glu Pro Glu Leu Val Asp Ala Val Ser Leu Asn Leu Ser
1100 1105 1110
Met Asp Lys Thr Ser Ile Ser Val Gly Glu Glu Ala Thr Val Ser
1115 1120 1125
Thr Ala Phe Val Pro Glu Asn Ala Thr Asp Arg Ile Val Lys Tyr
1130 1135 1140
Thr Ser Gln Asp Pro Glu Ile Ala Val Ile Asp Pro Thr Gly Ile
1145 1150 1155
Val Thr Gly Val Lys Asp Gly Thr Thr Thr Ile Val Ala Glu Thr
1160 1165 1170
Lys Ser Gly Ala Lys Gly Glu Leu Ser Val Thr Val Gly Glu Leu
1175 1180 1185
Gln Arg Gly Glu Ile Arg Phe Glu Val Ser Asn Asp His Pro Met
1190 1195 1200
Tyr Leu Glu Asn Tyr Tyr Trp Ser Asp Asp Ala Pro Lys Lys Asp
1205 1210 1215
Gly Leu Asp Ala Asn Lys Asn Tyr Tyr Gly Asp Glu Arg Val Asp
1220 1225 1230
Ser Pro Val Met Leu Tyr Asn Thr Val Pro Glu Glu Leu Lys Asp
1235 1240 1245
Asn Thr Val Ile Leu Leu Ile Ala Glu Arg Ser Leu Asn Ser Thr
1250 1255 1260
Asp Ala Val Arg Asp Trp Ile Lys Lys Asn Val Glu Leu Cys Asn
1265 1270 1275
Glu Asn Lys Ile Pro Cys Ala Val Gln Ile Ala Asn Gly Glu Thr
1280 1285 1290
Asn Val Asn Thr Thr Ile Pro Leu Ser Phe Trp Asn Glu Leu Ala
1295 1300 1305
Thr Asn Asn Glu Tyr Leu Val Gly Phe Asn Ala Ala Glu Met Tyr
1310 1315 1320
Asn Arg Phe Ala Gly Asp Asn Arg Ser Tyr Val Met Asp Met Ile
1325 1330 1335
Arg Leu Gly Val Ser His Gly Val Cys Met Met Trp Thr Asp Thr
1340 1345 1350
Asn Ile Phe Gly Thr Asn Gly Val Leu Tyr Asp Trp Leu Thr Gln
1355 1360 1365
Asp Glu Lys Leu Ser Gly Leu Met Arg Glu Tyr Lys Glu Tyr Ile
1370 1375 1380
Ser Leu Met Thr Lys Glu Ser Tyr Gly Ser Glu Ala Ala Asn Thr
1385 1390 1395
Asp Ala Leu Phe Lys Gly Leu Trp Met Thr Asp Tyr Cys Glu Asn
1400 1405 1410
Trp Gly Ile Ala Ser Asp Trp Trp His Trp Gln Leu Asp Ser Asn
1415 1420 1425
Gly Ala Leu Phe Asp Ala Gly Ser Gly Gly Asp Ala Trp Lys Gln
1430 1435 1440
Cys Leu Thr Trp Pro Glu Asn Met Tyr Thr Gln Asp Val Val Arg
1445 1450 1455
Ala Val Ser Gln Gly Ala Thr Cys Phe Lys Ser Glu Ala Gln Trp
1460 1465 1470
Tyr Ser Asn Ala Thr Lys Gly Met Arg Thr Pro Thr Tyr Gln Tyr
1475 1480 1485
Ser Met Ile Pro Phe Leu Glu Lys Leu Val Ser Lys Glu Val Lys
1490 1495 1500
Ile Pro Thr Lys Glu Glu Met Leu Glu Arg Thr Lys Ala Ile Val
1505 1510 1515
Val Gly Ala Glu Asn Trp Asn Asn Phe Asn Tyr Asn Thr Thr Tyr
1520 1525 1530
Ser Asn Leu Tyr Pro Ser Thr Gly Gln Tyr Gly Ile Val Pro Tyr
1535 1540 1545
Val Pro Ser Asn Cys Pro Glu Glu Glu Leu Ala Gly Tyr Asp Leu
1550 1555 1560
Val Val Arg Glu Asn Leu Gly Lys Ala Gly Leu Lys Ser Ala Leu
1565 1570 1575
Asp Thr Val Tyr Pro Val Gln Lys Ser Glu Gly Thr Ala Tyr Cys
1580 1585 1590
Glu Thr Phe Gly Asp Thr Trp Tyr Trp Met Asn Ser Ser Glu Asp
1595 1600 1605
Lys Asn Val Ser Gln Tyr Thr Glu Phe Thr Thr Ala Ile Asn Gly
1610 1615 1620
Ala Glu Ser Val Lys Ile Ala Gly Glu Pro His Val Phe Gly Ile
1625 1630 1635
Ile Lys Glu Asn Pro Gly Ser Leu Asn Val Tyr Leu Ser Asn Tyr
1640 1645 1650
Arg Leu Asp Lys Thr Glu Leu Trp Asp Gly Thr Ile Pro Gly Gly
1655 1660 1665
Leu Ser Asp Gln Gly Cys Tyr Asn Tyr Val Trp Gln Met Cys Glu
1670 1675 1680
Arg Met Lys Asn Gly Thr Gly Leu Asp Thr Gln Leu Arg Asp Thr
1685 1690 1695
Val Ile Thr Val Lys Asn Ala Val Glu Pro Lys Val Asn Phe Val
1700 1705 1710
Thr Glu Ser Pro Ala Asp Arg Ser Phe Ala Glu Asp Asn Tyr Val
1715 1720 1725
Arg Pro Tyr Lys Tyr Thr Val Ala Gln Lys Glu Gly Thr Thr Asp
1730 1735 1740
Glu Trp Val Ile Thr Val Ser His Asn Gly Ile Val Glu Phe Asn
1745 1750 1755
Ile Val Thr Gly Asp Glu Lys Val Pro Ala Thr Ser Val Glu Leu
1760 1765 1770
Ser Thr Asp Lys Val Asp Val Ile Arg Asn Arg Thr Ala Val Val
1775 1780 1785
Lys Ala Thr Val Leu Pro Gln Asn Ala Gly Asn Lys Gln Leu Thr
1790 1795 1800
Trp Thr Ile Ala Asp Pro Glu Ile Ala Ser Val Asp Asn Lys Gly
1805 1810 1815
Thr Val Thr Gly Leu Lys Glu Gly Lys Thr Val Leu Arg Ala Ala
1820 1825 1830
Ile Ser Gly Ser Val Tyr Lys Glu Cys Glu Val Asn Val Ile Asp
1835 1840 1845
Arg Lys Val Thr Glu Val Asn Leu Asn Lys Thr Glu Leu Ser Leu
1850 1855 1860
Ser Ala Gly Asp Ser Ala Lys Leu Glu Ala Ser Ile Ala Pro Glu
1865 1870 1875
Asp Pro Ser Asp Ser Ser Ile Thr Trp Thr Ser Thr Asn Glu Asn
1880 1885 1890
Val Ala Thr Val Ala Ser Asn Gly Thr Val Thr Ala His Lys Ala
1895 1900 1905
Gly Val Ala Gln Ile Ile Ala Gln Ser Ala Tyr Gln Ala Lys Gly
1910 1915 1920
Ile Ala Thr Val Thr Val Asn Tyr Ala Ala Ser Val Lys Leu Asp
1925 1930 1935
Arg Thr Gly Met Thr Ala Thr Ala Asn Ser Glu Gln Ser Lys Ser
1940 1945 1950
Gly Gly Glu Gly Pro Ala Ser Asn Val Leu Asp Gly Lys Gln Asp
1955 1960 1965
Thr Met Trp His Thr Ser Trp Thr Asp Lys Pro Glu Leu His Pro
1970 1975 1980
His Trp Ile Lys Ile Asp Leu Asn Gly Thr Lys Thr Ile Asn Lys
1985 1990 1995
Phe Ala Tyr Thr Pro Arg Thr Gly Ala Ser Asn Gly Thr Ile Tyr
2000 2005 2010
Asn Tyr Val Leu Ile Ile Thr Asp Leu Glu Gly Asn Glu Lys Gln
2015 2020 2025
Val Ala Lys Gly Val Trp Ala Ala Asn Ala Asp Val Lys Tyr Ala
2030 2035 2040
Glu Phe Asp Ala Val Glu Ala Thr Ala Ile Lys Leu Gln Val Asp
2045 2050 2055
Gly Asn Asp Asp Lys Ala Ser Lys Gly Gly Tyr Gly Ser Ala Ala
2060 2065 2070
Glu Ile Asn Ile Phe Glu Val Ala Gln Lys Pro Ser Ala Asn Glu
2075 2080 2085
Leu Ala Glu Asn Ile Lys Val Ile Ala Pro Val Lys Ala Glu Asp
2090 2095 2100
Thr Lys Val Ser Ile Pro Val Ile Thr Gly Phe Asp Ile Val Ile
2105 2110 2115
Ser Asn Ser Ser Asn Pro Asp Val Ile Gly Ile Asp Gly Ser Ile
2120 2125 2130
Thr Arg Pro Glu Asn Asp Thr Val Val Thr Leu Thr Leu Lys Val
2135 2140 2145
Lys Glu Thr Asp Ala Lys Ser Val Lys Ala Ala Gly Thr Glu Ala
2150 2155 2160
Thr Thr Asn Val Asp Val Leu Val Thr Gly Thr Lys Thr Ser Asp
2165 2170 2175
Val Glu Ala Glu Ser Val Thr Leu Asp Gln Thr Ser Ala Asp Leu
2180 2185 2190
Thr Val Gly Gly Glu Leu Leu Leu Asn Ala Val Val Lys Pro Asp
2195 2200 2205
Ile Ala Thr Asn Lys Ala Val Thr Trp Ser Ser Asp Lys Pro Gly
2210 2215 2220
Thr Ala Thr Val Glu Asn Gly Arg Val Lys Ala Leu Ala Ala Gly
2225 2230 2235
Glu Ala Arg Ile Thr Ala Ala Thr Ala Asn Gly Lys Thr Ala Asp
2240 2245 2250
Cys Val Ile Asn Val Lys Glu Lys Glu Glu Pro Glu Val Ile Leu
2255 2260 2265
Pro Ala Glu Val Arg Leu Asn Ile Pro Ser Ala Glu Phe Thr Val
2270 2275 2280
Gly Asp Gln Ile Gln Leu Thr Ala Ser Val Leu Pro Ala Asn Ala
2285 2290 2295
Ala Asp Lys Thr Ile Thr Trp Lys Ser Asp Lys Pro Glu Val Ala
2300 2305 2310
Thr Val Ala Asn Gly Trp Val Lys Gly Ile Ala Ala Gly Thr Ala
2315 2320 2325
Lys Ile Thr Ala Thr Ser Val Asn Gly Lys Thr Ala Val Cys Val
2330 2335 2340
Ile Thr Val Lys Ala Gln Pro Gln Asn Leu Pro Thr Gly Val Ser
2345 2350 2355
Leu Asn Lys Lys Thr Ala Ser Val Lys Leu Asn Lys Thr Leu Thr
2360 2365 2370
Leu Ser Ala Val Val Gln Pro Ser Asn Ala Asp Asn Lys Thr Val
2375 2380 2385
Lys Trp Thr Ser Asp Asn Thr Tyr Val Ala Thr Val Glu Asn Gly
2390 2395 2400
Val Val Lys Ala Val Asn Ala Gly Thr Ala Arg Ile Thr Ala Ala
2405 2410 2415
Thr Val Asn Gly His Lys Ala Thr Cys Thr Ile Thr Val Pro Gly
2420 2425 2430
Thr Lys Ile Ser Lys Ala Lys Val Ser Leu Ala Ser Ser Lys Thr
2435 2440 2445
His Thr Gly Lys Ala Leu Lys Pro Ser Val Lys Val Thr Tyr Gly
2450 2455 2460
Lys Asn Thr Leu Lys Lys Asn Thr Asp Tyr Thr Val Ser Tyr Lys
2465 2470 2475
Asn Asn Ile Asn Pro Gly Thr Ala Ser Val Thr Ile Thr Gly Lys
2480 2485 2490
Gly Lys Tyr Tyr Gly Thr Ile Asn Lys Thr Phe Ala Ile Lys Ala
2495 2500 2505
Ala Glu Gly Lys Thr Tyr Thr Val Gly Lys Gly Lys Tyr Lys Val
2510 2515 2520
Thr Asp Ala Ser Ala Lys Asn Lys Thr Val Thr Phe Met Ala Pro
2525 2530 2535
Val Lys Lys Thr Tyr Ser Ser Phe Ser Val Pro Ser Lys Val Lys
2540 2545 2550
Ile Gly Asn Asp Thr Tyr Lys Val Thr Ala Val Ala Lys Asn Ala
2555 2560 2565
Phe Lys Lys Asn Thr Lys Leu Thr Lys Leu Thr Ile Gly Ser Asn
2570 2575 2580
Val Lys Thr Ile Gly Ser Tyr Ala Phe Tyr Gly Ala Ser Gln Leu
2585 2590 2595
Lys Thr Leu Thr Leu Lys Thr Thr Gly Leu Asn Ser Val Gly Lys
2600 2605 2610
Asn Ala Phe Lys Lys Thr Asn Ala Lys Leu Thr Val Lys Val Pro
2615 2620 2625
Lys Ser Lys Leu Ala Asp Tyr Lys Lys Leu Leu Lys Gly Lys Gly
2630 2635 2640
Leu Ser Gly Lys Ala Lys Ile Gln Lys
2645 2650
<210> 28
<211> 2535
<212> DNA
<213> Robinsoniella peoriensis
<400> 28
tcaccattga gcgctgcggc agaaagtggc acaggaacca gattagtgaa agggcaaacg 60
gggtatttga cagaggaaca ggctatccgg aaccaggagc agacaaccga agaaagggag 120
cagaagttaa ccggggaaga gacagcagag gttttgatgg aaggtacaaa agacagcggg 180
attgtacaga cagaagaagt acagacaaaa gaaatgcaga cagaagatgc gcagacagaa 240
gaagtacaga cagaagaaat gcagacagaa gatgcgcaga caaaagaagt acagacagaa 300
gaaatgcaga cagaagatgc gcagacagaa gaagtacaga caaaagaaga accggcagaa 360
gaaacacaca tgaaagaaat acagacgcaa gggacaaaga aagcgtcaga taggaacgga 420
aaggcaaggg taactgaaat tctggaagat gcccaggatc cagcaaaccg gattgtgtat 480
ctgtcagacc tgcaatggaa gtcagaaaat catacagtag atagcgagct gcctaccaga 540
aaggataagt cctttggcgg cggaaaaatt acgctaaaag tggatggaac ggtaacagaa 600
tttgataagg ggattggaac acagacagat tccaccattg tgtacgatct ggagggaaag 660
ggatatacaa agtttgaaac ttacgtgggt gtagactaca gccagaaaga aaacattccg 720
ggggaagtct gcgacgtaaa attcagggtg aaaattgatg acaagattgt atcagaaacc 780
ggtgtactgg atccgctttc gaatgcggtt aagatttctg ttaacatacc cgatacagcc 840
aaaactttaa cattatacgc ggataaagta acggaaactt ggtctgatca cgccaattgg 900
gcagatgcaa aattttatca ggcactgccg gaacccgaaa atgttgcatt caaaaaaacg 960
gtagtgacac gaaagacatc agataattcg gaggctcctg ttaatccgga ttcagcagtt 1020
aacagttcta aggctgttga cggtgttatt gacagctcca gttattttga ttttggagat 1080
caggcaaata gcggagccgt aagggagtca ctctatatgg aggtagattt aaaagggagc 1140
tatttactgt ccgatataca actgtggaga tactggaaag atggcagaac ttatgcagct 1200
actgcaattg tagtagctga ggatgagaac tttgaaaatg cagcagttat ctataactcg 1260
gatacgacgg gagaaataca tcacctggga gcaggaagtg atatgctcta tgcagaaaca 1320
gaaagtggca agacatttcc ggtaccggaa aatacaaaag caaggtatat cagagtttat 1380
acatatggtg ttaatgggac atcaggcgta acaaatcaca ttgtagaatt aaaggtgaat 1440
gcttacgtat ttggagatga aatcttaccg gaaaagccgg atgacagcaa gattttccca 1500
aatgcagtta atccgctgaa gctacaggga ccgggcacga atgatcaggt aacccacccg 1560
gatgttacgg tgtttgatga gccgtggaat gggtataaat actggatggc atatacaccg 1620
aataaaccgg gaagttccta ttttgaaaat ccctgtatag ctgcatccaa cgatggcgta 1680
aactgggagt ttcctgccca gaaccctgta cagccgcgct atgacagtga aatagaaaat 1740
caaaatgaac ataactgtga taccgatatt gtatatgacc cggtaaatga ccggttgatt 1800
atgtactggg aatgggcaca ggatgaggcg gttaatggta aaacacatcg ttctgaaatc 1860
agataccgtg tttcttatga tgggattaac tggggagtgg aagacaaaac tggtgttttg 1920
atgactggac caacggatca tggctgcgcc attgccacag aaggcgaaag atattcagac 1980
ctttctccaa ccgtagtata tgataaaaca gaaaaaatct acaaaatgtg ggcaaatgat 2040
gccggagatg taggatatga aaacaaacag aataacaaag tatggtatcg gacatcccaa 2100
gacgggatca gcaattggtc ggataagact tacgtggaga attttcttgg agtaaatgaa 2160
gacgggctgc agatgtatcc atggcaccag gatatccagt gggtagagga atttcaggaa 2220
tattgggcac ttcagcaggc atttccggca ggaagcggac cggataattc ttccctgcgt 2280
ttctcgaaat ccaaagatgg tcttcattgg gagccggtat ctgaaaaagc tttaattaca 2340
gtaggggcac ccgggacctg ggatgcagga cagatatacc gttctacttt ctggtatgag 2400
ccaggtgggg caaaaggaaa cggaacattc catatctggt atgctgcatt ggcggaaggc 2460
cagtctcact gggatatagg atatacatct gcaaactatg cagatgccat gtacaaatta 2520
acgggaagca gatga 2535
<210> 29
<211> 864
<212> PRT
<213> Robinsoniella peoriensis
<400> 29
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Ser Pro Leu Ser Ala Ala Ala Glu Ser Gly Thr Gly
20 25 30
Thr Arg Leu Val Lys Gly Gln Thr Gly Tyr Leu Thr Glu Glu Gln Ala
35 40 45
Ile Arg Asn Gln Glu Gln Thr Thr Glu Glu Arg Glu Gln Lys Leu Thr
50 55 60
Gly Glu Glu Thr Ala Glu Val Leu Met Glu Gly Thr Lys Asp Ser Gly
65 70 75 80
Ile Val Gln Thr Glu Glu Val Gln Thr Lys Glu Met Gln Thr Glu Asp
85 90 95
Ala Gln Thr Glu Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala
100 105 110
Gln Thr Lys Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln
115 120 125
Thr Glu Glu Val Gln Thr Lys Glu Glu Pro Ala Glu Glu Thr His Met
130 135 140
Lys Glu Ile Gln Thr Gln Gly Thr Lys Lys Ala Ser Asp Arg Asn Gly
145 150 155 160
Lys Ala Arg Val Thr Glu Ile Leu Glu Asp Ala Gln Asp Pro Ala Asn
165 170 175
Arg Ile Val Tyr Leu Ser Asp Leu Gln Trp Lys Ser Glu Asn His Thr
180 185 190
Val Asp Ser Glu Leu Pro Thr Arg Lys Asp Lys Ser Phe Gly Gly Gly
195 200 205
Lys Ile Thr Leu Lys Val Asp Gly Thr Val Thr Glu Phe Asp Lys Gly
210 215 220
Ile Gly Thr Gln Thr Asp Ser Thr Ile Val Tyr Asp Leu Glu Gly Lys
225 230 235 240
Gly Tyr Thr Lys Phe Glu Thr Tyr Val Gly Val Asp Tyr Ser Gln Lys
245 250 255
Glu Asn Ile Pro Gly Glu Val Cys Asp Val Lys Phe Arg Val Lys Ile
260 265 270
Asp Asp Lys Ile Val Ser Glu Thr Gly Val Leu Asp Pro Leu Ser Asn
275 280 285
Ala Val Lys Ile Ser Val Asn Ile Pro Asp Thr Ala Lys Thr Leu Thr
290 295 300
Leu Tyr Ala Asp Lys Val Thr Glu Thr Trp Ser Asp His Ala Asn Trp
305 310 315 320
Ala Asp Ala Lys Phe Tyr Gln Ala Leu Pro Glu Pro Glu Asn Val Ala
325 330 335
Phe Lys Lys Thr Val Val Thr Arg Lys Thr Ser Asp Asn Ser Glu Ala
340 345 350
Pro Val Asn Pro Asp Ser Ala Val Asn Ser Ser Lys Ala Val Asp Gly
355 360 365
Val Ile Asp Ser Ser Ser Tyr Phe Asp Phe Gly Asp Gln Ala Asn Ser
370 375 380
Gly Ala Val Arg Glu Ser Leu Tyr Met Glu Val Asp Leu Lys Gly Ser
385 390 395 400
Tyr Leu Leu Ser Asp Ile Gln Leu Trp Arg Tyr Trp Lys Asp Gly Arg
405 410 415
Thr Tyr Ala Ala Thr Ala Ile Val Val Ala Glu Asp Glu Asn Phe Glu
420 425 430
Asn Ala Ala Val Ile Tyr Asn Ser Asp Thr Thr Gly Glu Ile His His
435 440 445
Leu Gly Ala Gly Ser Asp Met Leu Tyr Ala Glu Thr Glu Ser Gly Lys
450 455 460
Thr Phe Pro Val Pro Glu Asn Thr Lys Ala Arg Tyr Ile Arg Val Tyr
465 470 475 480
Thr Tyr Gly Val Asn Gly Thr Ser Gly Val Thr Asn His Ile Val Glu
485 490 495
Leu Lys Val Asn Ala Tyr Val Phe Gly Asp Glu Ile Leu Pro Glu Lys
500 505 510
Pro Asp Asp Ser Lys Ile Phe Pro Asn Ala Val Asn Pro Leu Lys Leu
515 520 525
Gln Gly Pro Gly Thr Asn Asp Gln Val Thr His Pro Asp Val Thr Val
530 535 540
Phe Asp Glu Pro Trp Asn Gly Tyr Lys Tyr Trp Met Ala Tyr Thr Pro
545 550 555 560
Asn Lys Pro Gly Ser Ser Tyr Phe Glu Asn Pro Cys Ile Ala Ala Ser
565 570 575
Asn Asp Gly Val Asn Trp Glu Phe Pro Ala Gln Asn Pro Val Gln Pro
580 585 590
Arg Tyr Asp Ser Glu Ile Glu Asn Gln Asn Glu His Asn Cys Asp Thr
595 600 605
Asp Ile Val Tyr Asp Pro Val Asn Asp Arg Leu Ile Met Tyr Trp Glu
610 615 620
Trp Ala Gln Asp Glu Ala Val Asn Gly Lys Thr His Arg Ser Glu Ile
625 630 635 640
Arg Tyr Arg Val Ser Tyr Asp Gly Ile Asn Trp Gly Val Glu Asp Lys
645 650 655
Thr Gly Val Leu Met Thr Gly Pro Thr Asp His Gly Cys Ala Ile Ala
660 665 670
Thr Glu Gly Glu Arg Tyr Ser Asp Leu Ser Pro Thr Val Val Tyr Asp
675 680 685
Lys Thr Glu Lys Ile Tyr Lys Met Trp Ala Asn Asp Ala Gly Asp Val
690 695 700
Gly Tyr Glu Asn Lys Gln Asn Asn Lys Val Trp Tyr Arg Thr Ser Gln
705 710 715 720
Asp Gly Ile Ser Asn Trp Ser Asp Lys Thr Tyr Val Glu Asn Phe Leu
725 730 735
Gly Val Asn Glu Asp Gly Leu Gln Met Tyr Pro Trp His Gln Asp Ile
740 745 750
Gln Trp Val Glu Glu Phe Gln Glu Tyr Trp Ala Leu Gln Gln Ala Phe
755 760 765
Pro Ala Gly Ser Gly Pro Asp Asn Ser Ser Leu Arg Phe Ser Lys Ser
770 775 780
Lys Asp Gly Leu His Trp Glu Pro Val Ser Glu Lys Ala Leu Ile Thr
785 790 795 800
Val Gly Ala Pro Gly Thr Trp Asp Ala Gly Gln Ile Tyr Arg Ser Thr
805 810 815
Phe Trp Tyr Glu Pro Gly Gly Ala Lys Gly Asn Gly Thr Phe His Ile
820 825 830
Trp Tyr Ala Ala Leu Ala Glu Gly Gln Ser His Trp Asp Ile Gly Tyr
835 840 845
Thr Ser Ala Asn Tyr Ala Asp Ala Met Tyr Lys Leu Thr Gly Ser Arg
850 855 860
<210> 30
<211> 3246
<212> DNA
<213> Robinsoniella peoriensis
<400> 30
gctgagactg caacagaaga aaatgcggcg ctggaaaaaa cagttacatt gcataagagc 60
gatggaacag aactgccgga ggattatcga aatccccaaa gaccagctac catggcggta 120
gatggtatta ttgacgatac aggagagtac aactattgcg atttcggtaa agacggtgat 180
aaagcagccc tgtatatgca ggtggacctt ggaggtctgt atgatttaag cagagtcaat 240
atgtggagat actggaaaga cagcagaact tacgatgcaa cagtaattac cacatctgag 300
agcggcgatt tcacagatga agcagtcata tataattcag acaggtcgaa tgtacatgga 360
tttggggcag gaggagatga acgctacgca gagactgcct ccggacatga attcccagta 420
ccggacggta caaaggcaca ggcagtacgc gtatatgtat ttggcagcca aaacggtact 480
acaaaccaca tcaatgaatt gcaggtctgg ggaactcccc atacagagaa tccggatgta 540
aattcttatc aggtgacaat tccacaggga aatggatatc aggtaatacc ttatgaaaat 600
gacccgacga cagtggaaga aggcggttct ttccgttttc aggtactgat tgactccgat 660
aatggttaca gcgcaaccag tgcggtaaaa gcaaatggag taagtctgga ggcagttgac 720
agtgtttata ccattgagaa cattactgaa gatcaggtaa tcaccattga aggcgtacat 780
aaagcacagt atgaagtgaa attcccggaa aatccacagg gatacagtgt tgagattcag 840
aatgaaggaa gtacaacggt agactataat ggttctgtca gttttaagct tattatagac 900
gaagcttata atgaatccgt accggttgta aaagcaaacg gcggtgcagc tttgggaaaa 960
gatgagctcg gtgtatatac aattgcaaat atccaggacg atattacggt tacagttgag 1020
ggtatccagg aaaataccgt agtaaagaca aaaacaatgt acttgtctga tatggattgg 1080
aagagtgctg caaatgcagt aggtgcaaca ggagaaaaag acactccaac aaaggacctg 1140
aatcatttac agcagcagat gaaattattg gtaaacggag cagagaagtc ttttgataaa 1200
ggaattggag ttcagacgga ttcttctatc gtttatgatc tggaagacaa aggctacact 1260
tctttccaca ccctggcagg cgttgattat tcagcaatgg aatatgtaga cggagaaggc 1320
tgtgatatcc agtttaaagt atatctggat gatgtcgtag tatttgacag cggagtagtt 1380
gatgcatctg atgaggctca ggaagttaat gttgctataa catcagagaa taaagaacta 1440
aaactggaag ctaaaatggt taaagagcct tataatgact ggggaaactg ggcagatgcc 1500
agctttgaaa tggcttatcc cgaaccgtct aatgtggctt taaataaaac agttaccgtt 1560
aagaaaacag cggataactc agactctgaa gtaaattcca gcagaccggg atcaatggct 1620
gtagatggaa tcattggacc tacatcagat tctaactatt gtgattttgg acaggatggg 1680
gataatactt cccgttatct gcaggtagat ttaggggatg tttatgaact tacccagatt 1740
aatatgttta gatactgggc agatggcaga gtatataatg gtactgtaat tgcagtttcc 1800
gaaaacgcag actttagtaa tccaactttt atttataatt cagataaagc agacaaacac 1860
ggacttggcg caggcagtga tgacacttat ggagaaaccc agagtggaaa attattcgaa 1920
gttccggcgg gaaccatggg acagtatgtc cgtgtgtata tggctggttc caacaaaggt 1980
acaacgaacc atatcgctga attacaggta atgggttata atttcaatac agaaccaaaa 2040
ccatatgaag caaatgcatt tgaaaatgca gaagtttatt tagatatgcc aactcatttc 2100
caggatctgg attccaataa aaacgacgat ggaagcttaa agcacattgg cggacaggtg 2160
acacatcctg atatccaggt atttgaccaa ccgtggaacg gttataaata ctggatgatt 2220
tacacaccaa atacaatgat cacttcccag tatgaaaatc catatatcgt agcatctgaa 2280
gatggacaga catgggtaga accggaaggg atttccaatc caattgaacc agaaccgcca 2340
tcaaccagat ttcataactg tgatgcagat ctgttatacg actctgtcaa tgaccgttta 2400
cttgcttact ggaactgggc agatgacggc ggcggaattg atgacgaatt aaaagatcag 2460
aactgtcaga ttcgtctgag aatttcttat gatggaatta actggggagt tccttacgac 2520
aaagacggca atattgccac aacagctgat actgtagtaa gaatggaaac aggagataag 2580
gatttcattc ctgcaatcag cgaaaaagac cgttatggta tgctttcccc aacatttacc 2640
tatgacgatt tccgcggcat atatacaatg tgggcacaaa actcgggtga tgcgggatac 2700
aaccagtccg gaaagttcat cgaaatgaga tggtctgagg atggaataaa ctggtctgaa 2760
ccacaaaaag tgaataattt ccttggaaaa gatgagaatg gcagacagct ttggccatgg 2820
catcaggata ttcagtatat ccctgagcta caggaatatt ggggactgtc ccagtgtttc 2880
tctacatcta atcccgatgg atccgtatta tacctgacca agtccagaga tggtgtcaac 2940
tgggagcagg caggaacaca gccggtatta agggcaggaa aatcaggtac ctgggatgat 3000
ttccagattt accgttctac cttctattat gataatcagt cagacagccc tactggtggg 3060
aaatttagaa tctggtacag tgcactgcag gcaaatactt caggcaagac cgttttggct 3120
cctgatggaa cagtgtctct tcaggttgga agccaggata ccaggatctg gcgtatcggg 3180
tatacagaaa atgactacat ggaagtcatg aaagctctga cccagaataa aaactatgaa 3240
gaatga 3246
<210> 31
<211> 1101
<212> PRT
<213> Robinsoniella peoriensis
<400> 31
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Ala Glu Thr Ala Thr Glu Glu Asn Ala Ala Leu Glu
20 25 30
Lys Thr Val Thr Leu His Lys Ser Asp Gly Thr Glu Leu Pro Glu Asp
35 40 45
Tyr Arg Asn Pro Gln Arg Pro Ala Thr Met Ala Val Asp Gly Ile Ile
50 55 60
Asp Asp Thr Gly Glu Tyr Asn Tyr Cys Asp Phe Gly Lys Asp Gly Asp
65 70 75 80
Lys Ala Ala Leu Tyr Met Gln Val Asp Leu Gly Gly Leu Tyr Asp Leu
85 90 95
Ser Arg Val Asn Met Trp Arg Tyr Trp Lys Asp Ser Arg Thr Tyr Asp
100 105 110
Ala Thr Val Ile Thr Thr Ser Glu Ser Gly Asp Phe Thr Asp Glu Ala
115 120 125
Val Ile Tyr Asn Ser Asp Arg Ser Asn Val His Gly Phe Gly Ala Gly
130 135 140
Gly Asp Glu Arg Tyr Ala Glu Thr Ala Ser Gly His Glu Phe Pro Val
145 150 155 160
Pro Asp Gly Thr Lys Ala Gln Ala Val Arg Val Tyr Val Phe Gly Ser
165 170 175
Gln Asn Gly Thr Thr Asn His Ile Asn Glu Leu Gln Val Trp Gly Thr
180 185 190
Pro His Thr Glu Asn Pro Asp Val Asn Ser Tyr Gln Val Thr Ile Pro
195 200 205
Gln Gly Asn Gly Tyr Gln Val Ile Pro Tyr Glu Asn Asp Pro Thr Thr
210 215 220
Val Glu Glu Gly Gly Ser Phe Arg Phe Gln Val Leu Ile Asp Ser Asp
225 230 235 240
Asn Gly Tyr Ser Ala Thr Ser Ala Val Lys Ala Asn Gly Val Ser Leu
245 250 255
Glu Ala Val Asp Ser Val Tyr Thr Ile Glu Asn Ile Thr Glu Asp Gln
260 265 270
Val Ile Thr Ile Glu Gly Val His Lys Ala Gln Tyr Glu Val Lys Phe
275 280 285
Pro Glu Asn Pro Gln Gly Tyr Ser Val Glu Ile Gln Asn Glu Gly Ser
290 295 300
Thr Thr Val Asp Tyr Asn Gly Ser Val Ser Phe Lys Leu Ile Ile Asp
305 310 315 320
Glu Ala Tyr Asn Glu Ser Val Pro Val Val Lys Ala Asn Gly Gly Ala
325 330 335
Ala Leu Gly Lys Asp Glu Leu Gly Val Tyr Thr Ile Ala Asn Ile Gln
340 345 350
Asp Asp Ile Thr Val Thr Val Glu Gly Ile Gln Glu Asn Thr Val Val
355 360 365
Lys Thr Lys Thr Met Tyr Leu Ser Asp Met Asp Trp Lys Ser Ala Ala
370 375 380
Asn Ala Val Gly Ala Thr Gly Glu Lys Asp Thr Pro Thr Lys Asp Leu
385 390 395 400
Asn His Leu Gln Gln Gln Met Lys Leu Leu Val Asn Gly Ala Glu Lys
405 410 415
Ser Phe Asp Lys Gly Ile Gly Val Gln Thr Asp Ser Ser Ile Val Tyr
420 425 430
Asp Leu Glu Asp Lys Gly Tyr Thr Ser Phe His Thr Leu Ala Gly Val
435 440 445
Asp Tyr Ser Ala Met Glu Tyr Val Asp Gly Glu Gly Cys Asp Ile Gln
450 455 460
Phe Lys Val Tyr Leu Asp Asp Val Val Val Phe Asp Ser Gly Val Val
465 470 475 480
Asp Ala Ser Asp Glu Ala Gln Glu Val Asn Val Ala Ile Thr Ser Glu
485 490 495
Asn Lys Glu Leu Lys Leu Glu Ala Lys Met Val Lys Glu Pro Tyr Asn
500 505 510
Asp Trp Gly Asn Trp Ala Asp Ala Ser Phe Glu Met Ala Tyr Pro Glu
515 520 525
Pro Ser Asn Val Ala Leu Asn Lys Thr Val Thr Val Lys Lys Thr Ala
530 535 540
Asp Asn Ser Asp Ser Glu Val Asn Ser Ser Arg Pro Gly Ser Met Ala
545 550 555 560
Val Asp Gly Ile Ile Gly Pro Thr Ser Asp Ser Asn Tyr Cys Asp Phe
565 570 575
Gly Gln Asp Gly Asp Asn Thr Ser Arg Tyr Leu Gln Val Asp Leu Gly
580 585 590
Asp Val Tyr Glu Leu Thr Gln Ile Asn Met Phe Arg Tyr Trp Ala Asp
595 600 605
Gly Arg Val Tyr Asn Gly Thr Val Ile Ala Val Ser Glu Asn Ala Asp
610 615 620
Phe Ser Asn Pro Thr Phe Ile Tyr Asn Ser Asp Lys Ala Asp Lys His
625 630 635 640
Gly Leu Gly Ala Gly Ser Asp Asp Thr Tyr Gly Glu Thr Gln Ser Gly
645 650 655
Lys Leu Phe Glu Val Pro Ala Gly Thr Met Gly Gln Tyr Val Arg Val
660 665 670
Tyr Met Ala Gly Ser Asn Lys Gly Thr Thr Asn His Ile Ala Glu Leu
675 680 685
Gln Val Met Gly Tyr Asn Phe Asn Thr Glu Pro Lys Pro Tyr Glu Ala
690 695 700
Asn Ala Phe Glu Asn Ala Glu Val Tyr Leu Asp Met Pro Thr His Phe
705 710 715 720
Gln Asp Leu Asp Ser Asn Lys Asn Asp Asp Gly Ser Leu Lys His Ile
725 730 735
Gly Gly Gln Val Thr His Pro Asp Ile Gln Val Phe Asp Gln Pro Trp
740 745 750
Asn Gly Tyr Lys Tyr Trp Met Ile Tyr Thr Pro Asn Thr Met Ile Thr
755 760 765
Ser Gln Tyr Glu Asn Pro Tyr Ile Val Ala Ser Glu Asp Gly Gln Thr
770 775 780
Trp Val Glu Pro Glu Gly Ile Ser Asn Pro Ile Glu Pro Glu Pro Pro
785 790 795 800
Ser Thr Arg Phe His Asn Cys Asp Ala Asp Leu Leu Tyr Asp Ser Val
805 810 815
Asn Asp Arg Leu Leu Ala Tyr Trp Asn Trp Ala Asp Asp Gly Gly Gly
820 825 830
Ile Asp Asp Glu Leu Lys Asp Gln Asn Cys Gln Ile Arg Leu Arg Ile
835 840 845
Ser Tyr Asp Gly Ile Asn Trp Gly Val Pro Tyr Asp Lys Asp Gly Asn
850 855 860
Ile Ala Thr Thr Ala Asp Thr Val Val Arg Met Glu Thr Gly Asp Lys
865 870 875 880
Asp Phe Ile Pro Ala Ile Ser Glu Lys Asp Arg Tyr Gly Met Leu Ser
885 890 895
Pro Thr Phe Thr Tyr Asp Asp Phe Arg Gly Ile Tyr Thr Met Trp Ala
900 905 910
Gln Asn Ser Gly Asp Ala Gly Tyr Asn Gln Ser Gly Lys Phe Ile Glu
915 920 925
Met Arg Trp Ser Glu Asp Gly Ile Asn Trp Ser Glu Pro Gln Lys Val
930 935 940
Asn Asn Phe Leu Gly Lys Asp Glu Asn Gly Arg Gln Leu Trp Pro Trp
945 950 955 960
His Gln Asp Ile Gln Tyr Ile Pro Glu Leu Gln Glu Tyr Trp Gly Leu
965 970 975
Ser Gln Cys Phe Ser Thr Ser Asn Pro Asp Gly Ser Val Leu Tyr Leu
980 985 990
Thr Lys Ser Arg Asp Gly Val Asn Trp Glu Gln Ala Gly Thr Gln Pro
995 1000 1005
Val Leu Arg Ala Gly Lys Ser Gly Thr Trp Asp Asp Phe Gln Ile
1010 1015 1020
Tyr Arg Ser Thr Phe Tyr Tyr Asp Asn Gln Ser Asp Ser Pro Thr
1025 1030 1035
Gly Gly Lys Phe Arg Ile Trp Tyr Ser Ala Leu Gln Ala Asn Thr
1040 1045 1050
Ser Gly Lys Thr Val Leu Ala Pro Asp Gly Thr Val Ser Leu Gln
1055 1060 1065
Val Gly Ser Gln Asp Thr Arg Ile Trp Arg Ile Gly Tyr Thr Glu
1070 1075 1080
Asn Asp Tyr Met Glu Val Met Lys Ala Leu Thr Gln Asn Lys Asn
1085 1090 1095
Tyr Glu Glu
1100
<210> 32
<211> 528
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 32
His Ser Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val Leu
1 5 10 15
Gln Thr Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn Asn
20 25 30
Pro Tyr Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr Gly
35 40 45
Tyr Asn Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys Glu
50 55 60
Ala Ser Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr Val
65 70 75 80
Asp Ser Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala Phe
85 90 95
Asp Gly Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly Asn
100 105 110
Ala Ala Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val Tyr
115 120 125
Asn Ile Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr Asn
130 135 140
Gly Thr Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val Asn
145 150 155 160
Trp Gly Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr Glu
165 170 175
Lys Ile Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu Lys
180 185 190
Ala Leu Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp Leu
195 200 205
Lys Val Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys Pro Leu Gln Asn
210 215 220
Ala Glu Thr Tyr Leu Asn Ile Pro Thr Tyr Asp Gly Leu Asn Gln Ser
225 230 235 240
Thr His Pro Asp Val Lys Tyr Phe Lys Asn Gly Trp Asn Gly Tyr Lys
245 250 255
Tyr Trp Met Ile Met Thr Pro Asn Arg Thr Gly Ser Ser Val Ala Glu
260 265 270
Asn Pro Ser Ile Leu Ala Ser Asp Asp Gly Ile Asn Trp Glu Val Pro
275 280 285
Ala Gly Val Thr Asn Pro Ile Ala Pro Met Pro Gln Val Gly His Asn
290 295 300
Cys Asp Val Asp Met Ile Tyr Asn Glu Ala Thr Asp Glu Leu Trp Val
305 310 315 320
Tyr Trp Val Glu Ser Asp Asp Ile Thr Lys Gly Trp Val Lys Leu Ile
325 330 335
Lys Ser Lys Asp Gly Val Asn Trp Ser Ser Gln Gln Val Val Val Asp
340 345 350
Asp Asn Arg Ala Lys Tyr Ser Thr Leu Ser Pro Ser Ile Ile Phe Lys
355 360 365
Asp Asn Lys Tyr Tyr Met Trp Ser Val Asn Thr Gly Asn Ser Gly Trp
370 375 380
Asn Asn Gln Ser Asn Lys Val Glu Leu Arg Glu Ser Ser Asp Gly Val
385 390 395 400
Asn Trp Ser Asn Pro Thr Val Val Asn Thr Leu Ala Gln Asp Gly Ser
405 410 415
Gln Ile Trp His Val Asn Val Glu Tyr Ile Pro Ser Lys Asn Glu Tyr
420 425 430
Trp Ala Ile Tyr Pro Ala Tyr Lys Asn Gly Thr Gly Ser Asp Lys Thr
435 440 445
Glu Leu Tyr Tyr Ala Lys Ser Ser Asp Gly Val Asn Trp Thr Thr Tyr
450 455 460
Lys Asn Pro Ile Leu Ser Lys Gly Thr Ser Gly Lys Trp Asp Asp Met
465 470 475 480
Glu Ile Tyr Arg Ser Cys Phe Val Tyr Asp Glu Asp Thr Asn Met Ile
485 490 495
Lys Val Trp Tyr Gly Ala Val Ser Gln Asn Pro Gln Ile Trp Lys Ile
500 505 510
Gly Phe Thr Glu Asn Asp Tyr Asp Lys Phe Ile Glu Gly Leu Thr Gln
515 520 525
<210> 33
<211> 449
<212> PRT
<213> Ruthenibacterium lactatiformans
<400> 33
His Glu Glu Thr Asp Leu Leu Val Asn Gly Gly Phe Glu Thr Gly Asp
1 5 10 15
Ser Thr Gly Trp Asn Trp Phe Asn Asn Ala Val Val Asp Ser Ala Ala
20 25 30
Pro His Ser Gly Asn Tyr Cys Ala Lys Val Ala Lys Asn Ser Ser Tyr
35 40 45
Glu Gln Val Val Thr Val Ser Pro Asp Thr Lys Tyr Val Leu Thr Gly
50 55 60
Trp Ala Lys Ser Glu Gly Ser Ser Val Met Thr Leu Gly Val Lys Asn
65 70 75 80
Tyr Gly Gly Gln Glu Thr Phe Ser Ala Thr Leu Ser Ala Asp Tyr Gln
85 90 95
Gln Leu Ala Val Thr Phe Thr Thr Gly Pro Asn Ala Gln Thr Ala Thr
100 105 110
Ile Tyr Gly Tyr Arg Gln Asn Ser Gly Ser Gly Ala Gly Tyr Phe Asp
115 120 125
Asp Val Glu Leu Thr Ala Val Gln Asp Phe Ala Pro Tyr Gln Pro Leu
130 135 140
Ala Asn Ala Ile Ala Pro Gln Ala Ile Pro Thr Tyr Asp Gly Ala Asn
145 150 155 160
Gln Pro Thr His Pro Ser Val Val Lys Phe Glu Gln Pro Trp Asn Gly
165 170 175
Tyr Leu Tyr Trp Met Ala Met Thr Pro Tyr Pro Phe Asn Asp Gly Ser
180 185 190
Tyr Glu Asn Pro Ser Ile Val Ala Ser Asn Asp Gly Glu Asn Trp Ile
195 200 205
Val Pro Glu Gly Val Ser Asn Pro Leu Ala Gly Thr Pro Ser Pro Gly
210 215 220
His Asn Cys Asp Val Asp Leu Val Tyr Val Pro Ala Ser Asp Glu Leu
225 230 235 240
Arg Met Tyr Tyr Val Glu Ala Asp Asp Ile Ile Ser Ser Arg Val Lys
245 250 255
Met Ile Ser Ser Arg Asp Gly Val His Trp Ser Glu Pro Gln Val Val
260 265 270
Met Gln Asp Leu Val Arg Lys Tyr Ser Ile Leu Ser Pro Ser Ile Glu
275 280 285
Ile Leu Pro Asp Gly Thr Tyr Met Met Trp Tyr Val Asp Thr Gly Asn
290 295 300
Ala Gly Trp Asn Ser Gln Asn Asn Gln Val Lys Tyr Arg Thr Ser Ala
305 310 315 320
Asp Gly Ile Lys Trp Ser Gly Ala Val Thr Cys Thr Asp Phe Val Gln
325 330 335
Pro Gly Tyr Gln Ile Trp His Ile Asp Val His Tyr Asp Thr Ser Ser
340 345 350
Gly Ala Tyr Tyr Ala Val Tyr Pro Ala Tyr Pro Asn Gly Thr Asp Cys
355 360 365
Asp His Cys Asn Leu Phe Phe Ala Val Asn Arg Thr Gly Lys Gln Trp
370 375 380
Glu Thr Phe Ser Arg Pro Ile Leu Lys Pro Ser Thr Glu Gly Gly Trp
385 390 395 400
Asp Asp Phe Cys Ile Tyr Arg Ser Ser Met Leu Ile Asp Asp Gly Met
405 410 415
Leu Lys Val Trp Tyr Gly Ala Lys Lys Gln Glu Asp Ser Ser Trp His
420 425 430
Thr Gly Leu Thr Met Arg Asp Phe Ser Glu Phe Met Lys Ile Leu Glu
435 440 445
Arg
<210> 34
<211> 845
<212> PRT
<213> Robinsoniella peoriensis
<400> 34
His Ser Pro Leu Ser Ala Ala Ala Glu Ser Gly Thr Gly Thr Arg Leu
1 5 10 15
Val Lys Gly Gln Thr Gly Tyr Leu Thr Glu Glu Gln Ala Ile Arg Asn
20 25 30
Gln Glu Gln Thr Thr Glu Glu Arg Glu Gln Lys Leu Thr Gly Glu Glu
35 40 45
Thr Ala Glu Val Leu Met Glu Gly Thr Lys Asp Ser Gly Ile Val Gln
50 55 60
Thr Glu Glu Val Gln Thr Lys Glu Met Gln Thr Glu Asp Ala Gln Thr
65 70 75 80
Glu Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln Thr Lys
85 90 95
Glu Val Gln Thr Glu Glu Met Gln Thr Glu Asp Ala Gln Thr Glu Glu
100 105 110
Val Gln Thr Lys Glu Glu Pro Ala Glu Glu Thr His Met Lys Glu Ile
115 120 125
Gln Thr Gln Gly Thr Lys Lys Ala Ser Asp Arg Asn Gly Lys Ala Arg
130 135 140
Val Thr Glu Ile Leu Glu Asp Ala Gln Asp Pro Ala Asn Arg Ile Val
145 150 155 160
Tyr Leu Ser Asp Leu Gln Trp Lys Ser Glu Asn His Thr Val Asp Ser
165 170 175
Glu Leu Pro Thr Arg Lys Asp Lys Ser Phe Gly Gly Gly Lys Ile Thr
180 185 190
Leu Lys Val Asp Gly Thr Val Thr Glu Phe Asp Lys Gly Ile Gly Thr
195 200 205
Gln Thr Asp Ser Thr Ile Val Tyr Asp Leu Glu Gly Lys Gly Tyr Thr
210 215 220
Lys Phe Glu Thr Tyr Val Gly Val Asp Tyr Ser Gln Lys Glu Asn Ile
225 230 235 240
Pro Gly Glu Val Cys Asp Val Lys Phe Arg Val Lys Ile Asp Asp Lys
245 250 255
Ile Val Ser Glu Thr Gly Val Leu Asp Pro Leu Ser Asn Ala Val Lys
260 265 270
Ile Ser Val Asn Ile Pro Asp Thr Ala Lys Thr Leu Thr Leu Tyr Ala
275 280 285
Asp Lys Val Thr Glu Thr Trp Ser Asp His Ala Asn Trp Ala Asp Ala
290 295 300
Lys Phe Tyr Gln Ala Leu Pro Glu Pro Glu Asn Val Ala Phe Lys Lys
305 310 315 320
Thr Val Val Thr Arg Lys Thr Ser Asp Asn Ser Glu Ala Pro Val Asn
325 330 335
Pro Asp Ser Ala Val Asn Ser Ser Lys Ala Val Asp Gly Val Ile Asp
340 345 350
Ser Ser Ser Tyr Phe Asp Phe Gly Asp Gln Ala Asn Ser Gly Ala Val
355 360 365
Arg Glu Ser Leu Tyr Met Glu Val Asp Leu Lys Gly Ser Tyr Leu Leu
370 375 380
Ser Asp Ile Gln Leu Trp Arg Tyr Trp Lys Asp Gly Arg Thr Tyr Ala
385 390 395 400
Ala Thr Ala Ile Val Val Ala Glu Asp Glu Asn Phe Glu Asn Ala Ala
405 410 415
Val Ile Tyr Asn Ser Asp Thr Thr Gly Glu Ile His His Leu Gly Ala
420 425 430
Gly Ser Asp Met Leu Tyr Ala Glu Thr Glu Ser Gly Lys Thr Phe Pro
435 440 445
Val Pro Glu Asn Thr Lys Ala Arg Tyr Ile Arg Val Tyr Thr Tyr Gly
450 455 460
Val Asn Gly Thr Ser Gly Val Thr Asn His Ile Val Glu Leu Lys Val
465 470 475 480
Asn Ala Tyr Val Phe Gly Asp Glu Ile Leu Pro Glu Lys Pro Asp Asp
485 490 495
Ser Lys Ile Phe Pro Asn Ala Val Asn Pro Leu Lys Leu Gln Gly Pro
500 505 510
Gly Thr Asn Asp Gln Val Thr His Pro Asp Val Thr Val Phe Asp Glu
515 520 525
Pro Trp Asn Gly Tyr Lys Tyr Trp Met Ala Tyr Thr Pro Asn Lys Pro
530 535 540
Gly Ser Ser Tyr Phe Glu Asn Pro Cys Ile Ala Ala Ser Asn Asp Gly
545 550 555 560
Val Asn Trp Glu Phe Pro Ala Gln Asn Pro Val Gln Pro Arg Tyr Asp
565 570 575
Ser Glu Ile Glu Asn Gln Asn Glu His Asn Cys Asp Thr Asp Ile Val
580 585 590
Tyr Asp Pro Val Asn Asp Arg Leu Ile Met Tyr Trp Glu Trp Ala Gln
595 600 605
Asp Glu Ala Val Asn Gly Lys Thr His Arg Ser Glu Ile Arg Tyr Arg
610 615 620
Val Ser Tyr Asp Gly Ile Asn Trp Gly Val Glu Asp Lys Thr Gly Val
625 630 635 640
Leu Met Thr Gly Pro Thr Asp His Gly Cys Ala Ile Ala Thr Glu Gly
645 650 655
Glu Arg Tyr Ser Asp Leu Ser Pro Thr Val Val Tyr Asp Lys Thr Glu
660 665 670
Lys Ile Tyr Lys Met Trp Ala Asn Asp Ala Gly Asp Val Gly Tyr Glu
675 680 685
Asn Lys Gln Asn Asn Lys Val Trp Tyr Arg Thr Ser Gln Asp Gly Ile
690 695 700
Ser Asn Trp Ser Asp Lys Thr Tyr Val Glu Asn Phe Leu Gly Val Asn
705 710 715 720
Glu Asp Gly Leu Gln Met Tyr Pro Trp His Gln Asp Ile Gln Trp Val
725 730 735
Glu Glu Phe Gln Glu Tyr Trp Ala Leu Gln Gln Ala Phe Pro Ala Gly
740 745 750
Ser Gly Pro Asp Asn Ser Ser Leu Arg Phe Ser Lys Ser Lys Asp Gly
755 760 765
Leu His Trp Glu Pro Val Ser Glu Lys Ala Leu Ile Thr Val Gly Ala
770 775 780
Pro Gly Thr Trp Asp Ala Gly Gln Ile Tyr Arg Ser Thr Phe Trp Tyr
785 790 795 800
Glu Pro Gly Gly Ala Lys Gly Asn Gly Thr Phe His Ile Trp Tyr Ala
805 810 815
Ala Leu Ala Glu Gly Gln Ser His Trp Asp Ile Gly Tyr Thr Ser Ala
820 825 830
Asn Tyr Ala Asp Ala Met Tyr Lys Leu Thr Gly Ser Arg
835 840 845
<210> 35
<211> 1082
<212> PRT
<213> Robinsoniella peoriensis
<400> 35
His Ala Glu Thr Ala Thr Glu Glu Asn Ala Ala Leu Glu Lys Thr Val
1 5 10 15
Thr Leu His Lys Ser Asp Gly Thr Glu Leu Pro Glu Asp Tyr Arg Asn
20 25 30
Pro Gln Arg Pro Ala Thr Met Ala Val Asp Gly Ile Ile Asp Asp Thr
35 40 45
Gly Glu Tyr Asn Tyr Cys Asp Phe Gly Lys Asp Gly Asp Lys Ala Ala
50 55 60
Leu Tyr Met Gln Val Asp Leu Gly Gly Leu Tyr Asp Leu Ser Arg Val
65 70 75 80
Asn Met Trp Arg Tyr Trp Lys Asp Ser Arg Thr Tyr Asp Ala Thr Val
85 90 95
Ile Thr Thr Ser Glu Ser Gly Asp Phe Thr Asp Glu Ala Val Ile Tyr
100 105 110
Asn Ser Asp Arg Ser Asn Val His Gly Phe Gly Ala Gly Gly Asp Glu
115 120 125
Arg Tyr Ala Glu Thr Ala Ser Gly His Glu Phe Pro Val Pro Asp Gly
130 135 140
Thr Lys Ala Gln Ala Val Arg Val Tyr Val Phe Gly Ser Gln Asn Gly
145 150 155 160
Thr Thr Asn His Ile Asn Glu Leu Gln Val Trp Gly Thr Pro His Thr
165 170 175
Glu Asn Pro Asp Val Asn Ser Tyr Gln Val Thr Ile Pro Gln Gly Asn
180 185 190
Gly Tyr Gln Val Ile Pro Tyr Glu Asn Asp Pro Thr Thr Val Glu Glu
195 200 205
Gly Gly Ser Phe Arg Phe Gln Val Leu Ile Asp Ser Asp Asn Gly Tyr
210 215 220
Ser Ala Thr Ser Ala Val Lys Ala Asn Gly Val Ser Leu Glu Ala Val
225 230 235 240
Asp Ser Val Tyr Thr Ile Glu Asn Ile Thr Glu Asp Gln Val Ile Thr
245 250 255
Ile Glu Gly Val His Lys Ala Gln Tyr Glu Val Lys Phe Pro Glu Asn
260 265 270
Pro Gln Gly Tyr Ser Val Glu Ile Gln Asn Glu Gly Ser Thr Thr Val
275 280 285
Asp Tyr Asn Gly Ser Val Ser Phe Lys Leu Ile Ile Asp Glu Ala Tyr
290 295 300
Asn Glu Ser Val Pro Val Val Lys Ala Asn Gly Gly Ala Ala Leu Gly
305 310 315 320
Lys Asp Glu Leu Gly Val Tyr Thr Ile Ala Asn Ile Gln Asp Asp Ile
325 330 335
Thr Val Thr Val Glu Gly Ile Gln Glu Asn Thr Val Val Lys Thr Lys
340 345 350
Thr Met Tyr Leu Ser Asp Met Asp Trp Lys Ser Ala Ala Asn Ala Val
355 360 365
Gly Ala Thr Gly Glu Lys Asp Thr Pro Thr Lys Asp Leu Asn His Leu
370 375 380
Gln Gln Gln Met Lys Leu Leu Val Asn Gly Ala Glu Lys Ser Phe Asp
385 390 395 400
Lys Gly Ile Gly Val Gln Thr Asp Ser Ser Ile Val Tyr Asp Leu Glu
405 410 415
Asp Lys Gly Tyr Thr Ser Phe His Thr Leu Ala Gly Val Asp Tyr Ser
420 425 430
Ala Met Glu Tyr Val Asp Gly Glu Gly Cys Asp Ile Gln Phe Lys Val
435 440 445
Tyr Leu Asp Asp Val Val Val Phe Asp Ser Gly Val Val Asp Ala Ser
450 455 460
Asp Glu Ala Gln Glu Val Asn Val Ala Ile Thr Ser Glu Asn Lys Glu
465 470 475 480
Leu Lys Leu Glu Ala Lys Met Val Lys Glu Pro Tyr Asn Asp Trp Gly
485 490 495
Asn Trp Ala Asp Ala Ser Phe Glu Met Ala Tyr Pro Glu Pro Ser Asn
500 505 510
Val Ala Leu Asn Lys Thr Val Thr Val Lys Lys Thr Ala Asp Asn Ser
515 520 525
Asp Ser Glu Val Asn Ser Ser Arg Pro Gly Ser Met Ala Val Asp Gly
530 535 540
Ile Ile Gly Pro Thr Ser Asp Ser Asn Tyr Cys Asp Phe Gly Gln Asp
545 550 555 560
Gly Asp Asn Thr Ser Arg Tyr Leu Gln Val Asp Leu Gly Asp Val Tyr
565 570 575
Glu Leu Thr Gln Ile Asn Met Phe Arg Tyr Trp Ala Asp Gly Arg Val
580 585 590
Tyr Asn Gly Thr Val Ile Ala Val Ser Glu Asn Ala Asp Phe Ser Asn
595 600 605
Pro Thr Phe Ile Tyr Asn Ser Asp Lys Ala Asp Lys His Gly Leu Gly
610 615 620
Ala Gly Ser Asp Asp Thr Tyr Gly Glu Thr Gln Ser Gly Lys Leu Phe
625 630 635 640
Glu Val Pro Ala Gly Thr Met Gly Gln Tyr Val Arg Val Tyr Met Ala
645 650 655
Gly Ser Asn Lys Gly Thr Thr Asn His Ile Ala Glu Leu Gln Val Met
660 665 670
Gly Tyr Asn Phe Asn Thr Glu Pro Lys Pro Tyr Glu Ala Asn Ala Phe
675 680 685
Glu Asn Ala Glu Val Tyr Leu Asp Met Pro Thr His Phe Gln Asp Leu
690 695 700
Asp Ser Asn Lys Asn Asp Asp Gly Ser Leu Lys His Ile Gly Gly Gln
705 710 715 720
Val Thr His Pro Asp Ile Gln Val Phe Asp Gln Pro Trp Asn Gly Tyr
725 730 735
Lys Tyr Trp Met Ile Tyr Thr Pro Asn Thr Met Ile Thr Ser Gln Tyr
740 745 750
Glu Asn Pro Tyr Ile Val Ala Ser Glu Asp Gly Gln Thr Trp Val Glu
755 760 765
Pro Glu Gly Ile Ser Asn Pro Ile Glu Pro Glu Pro Pro Ser Thr Arg
770 775 780
Phe His Asn Cys Asp Ala Asp Leu Leu Tyr Asp Ser Val Asn Asp Arg
785 790 795 800
Leu Leu Ala Tyr Trp Asn Trp Ala Asp Asp Gly Gly Gly Ile Asp Asp
805 810 815
Glu Leu Lys Asp Gln Asn Cys Gln Ile Arg Leu Arg Ile Ser Tyr Asp
820 825 830
Gly Ile Asn Trp Gly Val Pro Tyr Asp Lys Asp Gly Asn Ile Ala Thr
835 840 845
Thr Ala Asp Thr Val Val Arg Met Glu Thr Gly Asp Lys Asp Phe Ile
850 855 860
Pro Ala Ile Ser Glu Lys Asp Arg Tyr Gly Met Leu Ser Pro Thr Phe
865 870 875 880
Thr Tyr Asp Asp Phe Arg Gly Ile Tyr Thr Met Trp Ala Gln Asn Ser
885 890 895
Gly Asp Ala Gly Tyr Asn Gln Ser Gly Lys Phe Ile Glu Met Arg Trp
900 905 910
Ser Glu Asp Gly Ile Asn Trp Ser Glu Pro Gln Lys Val Asn Asn Phe
915 920 925
Leu Gly Lys Asp Glu Asn Gly Arg Gln Leu Trp Pro Trp His Gln Asp
930 935 940
Ile Gln Tyr Ile Pro Glu Leu Gln Glu Tyr Trp Gly Leu Ser Gln Cys
945 950 955 960
Phe Ser Thr Ser Asn Pro Asp Gly Ser Val Leu Tyr Leu Thr Lys Ser
965 970 975
Arg Asp Gly Val Asn Trp Glu Gln Ala Gly Thr Gln Pro Val Leu Arg
980 985 990
Ala Gly Lys Ser Gly Thr Trp Asp Asp Phe Gln Ile Tyr Arg Ser Thr
995 1000 1005
Phe Tyr Tyr Asp Asn Gln Ser Asp Ser Pro Thr Gly Gly Lys Phe
1010 1015 1020
Arg Ile Trp Tyr Ser Ala Leu Gln Ala Asn Thr Ser Gly Lys Thr
1025 1030 1035
Val Leu Ala Pro Asp Gly Thr Val Ser Leu Gln Val Gly Ser Gln
1040 1045 1050
Asp Thr Arg Ile Trp Arg Ile Gly Tyr Thr Glu Asn Asp Tyr Met
1055 1060 1065
Glu Val Met Lys Ala Leu Thr Gln Asn Lys Asn Tyr Glu Glu
1070 1075 1080
<210> 36
<211> 986
<212> PRT
<213> 第三梭状芽孢杆菌(Clostridium tertium)
<400> 36
His Tyr Asn Leu Ile Asp Asn Ile Ser Val Glu Lys Leu Asp Thr Asp
1 5 10 15
Ile Ser Gln Ala Asn Glu Asn Val Phe Leu Asn Gly Asn Gly Ile Ala
20 25 30
Leu Glu Val Asp Asn Arg Gly Ala Thr Cys Ile Tyr Leu Val Asp Glu
35 40 45
Asn Gly Val Lys Thr Lys Ala Thr Thr Ser Leu Asp Thr Ala Asp Phe
50 55 60
Ser Gly Tyr Pro Ile Ile Gly Gly Gln Lys Ile Arg Asp Phe Val Ile
65 70 75 80
Ile Ser Lys Asn Leu Glu Glu Asn Ile Asn Ser Ile Leu Gly Val Gly
85 90 95
Asn Arg Leu Thr Ile Ile Ser Lys Ser Ser Ser Thr Asn Leu Ile Arg
100 105 110
Lys Ile Val Phe Glu Thr Ser Asn Ser Asn Pro Gly Ala Ile Tyr Ser
115 120 125
Thr Val Ser Tyr Lys Ala Glu Ser Asn Asp Leu Leu Val Asp Ser Phe
130 135 140
His Glu Asn Glu Tyr Thr Met Ser Leu Gly Gln Gly Pro Phe Leu Ala
145 150 155 160
Tyr Gln Gly Cys Ala Asp Gln Gln Gly Ala Asn Thr Ile Val Asn Val
165 170 175
Thr Asn Gly Tyr Asn His Asn Ser Gly Gln Asn Asn Tyr Ser Val Gly
180 185 190
Val Pro Phe Ser Tyr Val Tyr Asn Ser Val Gly Gly Ile Gly Ile Gly
195 200 205
Asp Ala Ser Thr Ser Arg Arg Glu Phe Lys Leu Pro Ile Ile Gly Lys
210 215 220
Asp Asn Thr Val Ser Leu Gly Met Glu Trp Asn Gly Gln Thr Leu Lys
225 230 235 240
Lys Gly Ala Glu Thr Ala Ile Gly Thr Ser Val Ile Thr Thr Thr Asn
245 250 255
Gly Asp Tyr Tyr Ser Gly Leu Lys Ser Tyr Ala Glu Val Met Lys Asp
260 265 270
Lys Gly Ile Ser Ala Pro Ala Ser Ile Pro Asp Ile Ala Tyr Asp Ser
275 280 285
Arg Trp Glu Ser Trp Gly Phe Glu Phe Asp Phe Thr Ile Glu Lys Ile
290 295 300
Val Asn Lys Leu Asp Glu Leu Lys Ala Met Gly Ile Lys Gln Ile Thr
305 310 315 320
Leu Asp Asp Gly Trp Tyr Thr Tyr Ala Gly Asp Trp Lys Leu Ser Pro
325 330 335
Gln Lys Phe Pro Asn Gly Asn Ala Asp Met Lys Tyr Leu Thr Asp Glu
340 345 350
Ile His Lys Arg Gly Met Thr Ala Ile Leu Trp Trp Arg Pro Val Asp
355 360 365
Gly Gly Ile Asn Ser Lys Leu Val Ser Glu His Pro Glu Trp Phe Ile
370 375 380
Lys Asn Ser Gln Gly Asn Met Val Arg Leu Pro Gly Pro Gly Gly Gly
385 390 395 400
Asn Gly Gly Thr Ala Gly Tyr Ala Leu Cys Pro Asn Ser Glu Gly Ser
405 410 415
Ile Gln His His Lys Asp Phe Val Thr Val Ala Leu Glu Glu Trp Gly
420 425 430
Phe Asp Gly Phe Lys Glu Asp Tyr Val Trp Gly Ile Pro Lys Cys Tyr
435 440 445
Asp Ser Ser His Lys His Ser Ser Leu Ser Asp Thr Leu Glu Asn Gln
450 455 460
Tyr Lys Phe Tyr Glu Ala Ile Tyr Glu Gln Ser Ile Ala Ile Asn Pro
465 470 475 480
Asp Thr Phe Ile Glu Leu Cys Asn Cys Gly Thr Pro Gln Asp Phe Tyr
485 490 495
Ser Thr Pro Tyr Val Asn His Ala Pro Thr Ala Asp Pro Ile Ser Arg
500 505 510
Val Gln Thr Arg Thr Arg Val Lys Ala Phe Lys Ala Ile Phe Gly Asp
515 520 525
Asp Phe Pro Val Thr Thr Asp His Asn Ser Val Trp Leu Pro Ser Ala
530 535 540
Leu Gly Thr Gly Ser Val Met Ile Thr Lys His Thr Thr Leu Ser Ser
545 550 555 560
Ser Asp Arg Glu Gln Tyr Asn Lys Tyr Phe Gly Leu Ala Arg Asp Leu
565 570 575
Glu Leu Ala Lys Gly Glu Phe Ile Gly Asn Leu Tyr Lys Tyr Gly Ile
580 585 590
Asp Pro Leu Glu Ser Tyr Val Ile Arg Lys Gly Glu Asp Ile Tyr Tyr
595 600 605
Ser Phe Tyr Lys Asp Asn Ser Ser Tyr Ser Gly Asn Ile Glu Ile Lys
610 615 620
Gly Leu Asp Ser Asn Ala Thr Tyr Arg Ile Glu Asp Tyr Val Asn Asn
625 630 635 640
Arg Val Ile Ala Arg Gly Val Lys Gly Pro Thr Ala Thr Ile Asn Thr
645 650 655
Ser Phe Thr Asp Asn Leu Leu Val Arg Ala Ile Pro Asp Asp Thr Pro
660 665 670
Ala Glu Val Thr Thr Phe Asp Val Gly Asn Asn Thr Ile Leu Ser Ser
675 680 685
Thr Asp Ser Gly Asn Ser Lys Tyr Leu Asn Ala Val Ser Thr Thr Leu
690 695 700
Glu Lys Thr Ala Thr Ile Asp Ser Leu Ser Ile Tyr Ile Gly Asn Asn
705 710 715 720
Ser Glu Asn Gly Lys Leu Gln Ile Ala Ile Tyr Asp Asp Asn Asn Gly
725 730 735
Lys Pro Gly Thr Lys Lys Ala Tyr Val Glu Glu Phe Val Pro Thr Lys
740 745 750
Asn Ser Trp Asn Thr Lys Lys Val Val Asn Ser Val Thr Leu Pro Ser
755 760 765
Gly Gln Tyr Trp Leu Val Phe Gln Pro Asp Asn Asp Val Leu Gln Thr
770 775 780
Lys Thr Asn Pro Ser Ser Met Lys Gln Ser Ala Asn Asn Asn Pro Tyr
785 790 795 800
Asn Tyr Asn Ile Leu Pro Asn Ser Phe Pro Ile Gly Thr Gly Tyr Asn
805 810 815
Ala Tyr Lys Gly Asp Val Ser Phe Tyr Ala Thr Phe Lys Glu Ala Ser
820 825 830
Ser Gln Ala Ile Pro Gln Asn Ser Trp Ala Leu Lys Tyr Val Asp Ser
835 840 845
Glu Glu Thr Thr Gly Glu Asn Gly Arg Ala Thr Asn Ala Phe Asp Gly
850 855 860
Asn Asn Asn Thr Ile Trp His Thr Lys Tyr Ser Gly Gly Asn Ala Ala
865 870 875 880
Pro Met Pro His Glu Ile Gln Ile Asp Leu Arg Gly Val Tyr Asn Ile
885 890 895
Asn Gln Ile Asn Tyr Leu Pro Arg Gln Asp Gly Gly Thr Asn Gly Thr
900 905 910
Ile Lys Asp Tyr Glu Val Tyr Leu Ser Leu Asp Gly Val Asn Trp Gly
915 920 925
Gln Pro Ile Ser Lys Gly Thr Phe Glu Ser Asn Ser Thr Glu Lys Ile
930 935 940
Val Lys Phe Asn Glu Thr Lys Ser Arg Tyr Val Lys Leu Lys Ala Leu
945 950 955 960
Ser Glu Ile Asn Asn Lys Gln Phe Thr Thr Val Ala Asp Leu Lys Val
965 970 975
Phe Gly Trp Glu Ile Ser Lys Ile Glu Lys
980 985
<210> 37
<211> 1262
<212> PRT
<213> Robinsoniella peoriensis
<400> 37
His Gly Asn Gly Leu Glu Val Lys Ala Ser Pro Arg Glu Val Ala Gln
1 5 10 15
Ile Thr Gly Asn Gly Val Ser Val Thr Phe Phe Gln Glu Asp Gly Thr
20 25 30
Val Gln Leu Ser Cys Ile Glu Asp Asp Gly Asn Thr Ala Phe Met Thr
35 40 45
Arg Asn Ser Glu Val Ser Tyr Pro Val Val Gly Gly Glu Glu Val Thr
50 55 60
Asp Phe Ser Asp Phe Gln Cys Glu Val Gln Glu Asn Val Thr Gly Ala
65 70 75 80
Ala Gly Ala Gly Ser Arg Met Thr Ile Thr Ser Ile Ser Ser Gly Arg
85 90 95
Gly Ile Gln Arg Ser Val Val Ile Glu Thr Val Asp Glu Val Lys Gly
100 105 110
Leu Leu His Ile Ser Ser Ser Tyr Arg Ala Glu Glu Glu Val Asp Ala
115 120 125
Asp Glu Phe Ile Asp Ser Arg Phe Ser Leu Asp Asn Pro Ser Asp Thr
130 135 140
Val Trp Ser Tyr Asn Gly Gly Gly Glu Gly Ala Gln Ser Arg Tyr Asp
145 150 155 160
Thr Leu Gln Lys Ile Asp Leu Ser Asp Gly Glu Ser Phe Tyr Arg Glu
165 170 175
Asn Leu Gln Asn Gln Thr Ala Ala Gly Ile Pro Val Ala Asp Ile Tyr
180 185 190
Gly Lys Asp Gly Gly Ile Thr Val Gly Asp Ala Ser Val Thr Arg Arg
195 200 205
Gln Leu Ser Thr Pro Val Asn Glu Arg Asn Gly Thr Ala Tyr Val Ser
210 215 220
Val Lys His Pro Gly Ala Val Ile Thr Gln Arg Glu Thr Glu Ile Ser
225 230 235 240
Gln Ser Phe Val Asn Val His Arg Gly Asp Tyr Tyr Ser Gly Leu Arg
245 250 255
Gly Tyr Ala Asp Gly Met Lys Gln Ile Gly Phe Thr Thr Leu Ser Arg
260 265 270
Glu Gln Ile Pro Glu Ser Ser Tyr Asp Leu Arg Trp Glu Ser Trp Gly
275 280 285
Trp Glu Phe Asp Trp Thr Val Glu Leu Ile Ile Asn Lys Leu Asp Glu
290 295 300
Leu Lys Glu Met Gly Ile Lys Gln Ile Thr Leu Asp Asp Gly Trp Tyr
305 310 315 320
Asn Ala Ala Gly Glu Trp Gly Leu Asn Asn Trp Lys Leu Pro Asn Gly
325 330 335
Ala Leu Asp Met Arg His Leu Thr Asp Ala Ile His Glu Arg Gly Met
340 345 350
Thr Ala Val Leu Trp Trp Arg Pro Cys Asp Gly Gly Arg Glu Asp Ser
355 360 365
Ala Leu Phe Lys Glu His Pro Glu Tyr Phe Ile Lys Asn Gln Asp Gly
370 375 380
Ser Phe Gly Lys Leu Ala Gly Pro Gly Gln Trp Asn Ser Phe Leu Gly
385 390 395 400
Ser Cys Gly Tyr Ala Leu Cys Pro Leu Ser Glu Gly Ala Val Gln Ser
405 410 415
Gln Val Asp Phe Ile Asn Arg Ala Met Asn Glu Trp Gly Phe Asp Gly
420 425 430
Phe Lys Ser Asp Tyr Val Trp Ser Leu Pro Lys Cys Tyr Ser Gln Asp
435 440 445
His His His Glu Tyr Pro Glu Glu Ser Thr Glu Gln Gln Ala Val Phe
450 455 460
Tyr Arg Ala Val Tyr Glu Ala Met Thr Asp Asn Asp Pro Asn Ala Phe
465 470 475 480
His Leu Leu Cys Asn Cys Gly Thr Pro Gln Asp Tyr Tyr Ser Leu Pro
485 490 495
Tyr Val Thr Gln Val Pro Thr Ala Asp Pro Thr Ser Val Asp Gln Thr
500 505 510
Arg Arg Arg Val Lys Ala Tyr Lys Ala Leu Cys Gly Asp Tyr Phe Pro
515 520 525
Val Thr Thr Asp His Asn Glu Val Trp Tyr Pro Ser Thr Ile Gly Thr
530 535 540
Gly Ala Ile Leu Ile Glu Lys Arg Asp Leu Ser Gly Trp Glu Glu Glu
545 550 555 560
Glu Tyr Ala Lys Trp Leu Lys Ile Ala Gln Glu Asn Gln Leu His Lys
565 570 575
Gly Thr Phe Ile Gly Asp Leu Tyr Ser Tyr Gly Tyr Asp Pro Tyr Glu
580 585 590
Thr Tyr Thr Val Tyr Lys Asp Gly Ile Met Tyr Tyr Ala Phe Tyr Lys
595 600 605
Asp Gly Asn Arg Tyr Arg Pro Ser Gly Asn Pro Asp Ile Glu Leu Lys
610 615 620
Gly Leu Glu Asp Gly Lys Leu Tyr Arg Ile Val Asp Tyr Val Asn Asn
625 630 635 640
Gln Val Val Ala Thr Asn Val Thr Ser Ser Asn Ala Val Phe Ser Tyr
645 650 655
Pro Phe Ser Asp Tyr Leu Leu Val Lys Ala Val Glu Ile Ser Glu Pro
660 665 670
Asp Thr Asp Gly Pro Gly Pro Val Pro Asp Pro Glu Gly Ala Val Thr
675 680 685
Val Glu Glu Asn Asp Pro Glu Leu Val Tyr Thr Gly Asp Trp Val Arg
690 695 700
Glu Glu Asn Asp Gly Tyr His Gly Gly Gly Ala Arg Tyr Thr Lys Glu
705 710 715 720
Ala Glu Ala Ser Val Glu Leu Ala Phe Tyr Gly Thr Gly Ala Ala Trp
725 730 735
Tyr Gly Gln His Asp Val Asn Phe Gly Ser Ala Arg Ile Tyr Ile Asp
740 745 750
Gly Thr Tyr Val Lys Thr Val Ser Cys Met Gly Glu Pro Gly Ile Asn
755 760 765
Ile Lys Leu Phe Glu Ile Ser Gly Leu Asp Leu Ala Ser His Arg Ile
770 775 780
Lys Ile Glu Cys Glu Thr Pro Val Ile Asp Ile Asp Arg Leu Thr Tyr
785 790 795 800
Ile Lys Gly Glu Glu Val Pro Ala Lys Val Met Thr Ala Asp Leu Arg
805 810 815
Ala Leu Thr Val Ile Ala Asn Gln Tyr Asp Met Asn Ser Phe Ala Asp
820 825 830
Gly Asn Tyr Lys Asp Gln Leu Gly Val Ser Leu Val Arg Ala Asn Gln
835 840 845
Leu Leu Ala Ala Asp Asp Val Thr Gln Gly Ala Val Asn Glu Glu Gln
850 855 860
Lys Tyr Leu Leu Asn Ala Met Leu Lys Ile Arg Lys Lys Val Asp Lys
865 870 875 880
Ser Trp Ile Gly Leu Pro Gly Pro Ile Pro Gln Asp Ile Gln Thr Glu
885 890 895
Asn Ile Ser Arg Asp Asn Leu Ala Lys Val Ile Ser Tyr Thr Gly Gln
900 905 910
Leu Asp Arg Asp Glu Ile Ile Pro Ala Ile Lys Glu Gln Leu Asn Asp
915 920 925
Ser Tyr Asp Lys Ala Val Ser Ile Ala Glu Arg Gln Asp Ala Ser Gln
930 935 940
Pro Glu Ile Asp Arg Ala Trp Ala Glu Leu Met Asn Ala Val Gln Tyr
945 950 955 960
Ser Ser Tyr Ile Arg Gly Ser Lys Glu Glu Leu Leu Ser Leu Leu Asp
965 970 975
Glu Tyr Gly Lys Val Asp Thr Thr Val Tyr Lys Asp Ala Ala Leu Phe
980 985 990
Ile Glu Ser Leu Glu Ala Ala Lys Lys Val Tyr Gln Asp Glu Asn Ala
995 1000 1005
Met Asp Gly Glu Ile Ser Asp Cys Ile Lys Gln Leu Arg Asp Ala
1010 1015 1020
Lys Asp Gln Leu Gln Leu Lys Asp Pro Val Asp Pro Pro Lys Pro
1025 1030 1035
Asp Pro Asp Pro Asp Pro Lys Pro Asp Pro Thr Pro Asp Pro Gly
1040 1045 1050
Pro Asp Pro Lys Pro Asp Pro Thr Pro Asp Pro Thr Pro Asp Pro
1055 1060 1065
Lys Pro Asn Pro Thr Pro Thr Pro Asp Pro Thr Pro Glu Pro Ala
1070 1075 1080
Leu Lys Lys Pro Glu Gln Val Ser Gly Leu Lys Ser Lys Ala Glu
1085 1090 1095
Thr Asp Tyr Leu Thr Val Ser Trp Lys Lys Leu Asn Asn Ala Glu
1100 1105 1110
Ser Tyr Lys Val Tyr Ile Tyr Lys Ser Gly Lys Trp Arg Leu Ala
1115 1120 1125
Gly Lys Thr Thr Lys Thr Ser Ile Lys Ile Lys Lys Leu Val Ser
1130 1135 1140
Gly Thr Lys Tyr Thr Val Lys Val Ala Ala Val Asn Lys Ala Gly
1145 1150 1155
Gln Gly Lys Tyr Ser Ser Gln Val Tyr Thr Ala Ala Lys Pro Lys
1160 1165 1170
Lys Val Lys Leu Lys Ser Val Ser Arg Tyr Arg Thr Ser Lys Val
1175 1180 1185
Lys Leu Asn Tyr Gly Lys Val Lys Ala Gly Gly Tyr Glu Ile Trp
1190 1195 1200
Met Lys Asn Gly Lys Gly Ser Tyr Lys Lys Ala Ala Thr Ser Thr
1205 1210 1215
Lys Thr Thr Ala Ile Lys Ser Gly Leu Lys Lys Gly Lys Thr Tyr
1220 1225 1230
Tyr Phe Lys Val Arg Ala Tyr Val Lys Asn Lys Asn Gln Val Ile
1235 1240 1245
Tyr Gly Ser Phe Ser Asn Ile Lys Lys Tyr Lys Met Val Leu
1250 1255 1260
<210> 38
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNAcDeAc_无信号P_fw
<400> 38
atggtctcgc catgcagact ccagcgagtc cg 32
<210> 39
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNAcDeAc_D1min_rv
<400> 39
atggtctcga ttcttacgtc gtgtagccgg ggtc 34
<210> 40
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNAcDeAc_D1ext_rv
<400> 40
atggtctcga ttcttaatca ctggaggtat atttcacgac c 41
<210> 41
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNAcDeAc_D1+2_rv
<400> 41
atggtctcga ttcttacgca ggctcgattg gaccatac 38
<210> 42
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNAcDeAc_D2ext_fw
<400> 42
atggtctcgc catgatgtgg cgacggtgga tgag 34
<210> 43
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNAcDeAc_rv
<400> 43
atggtctcga ttcttattct cccacatacg aaaaatagtc g 41
<210> 44
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNase_无信号P_fw
<400> 44
atggtctcgc catcgtggta aaaagttcat atcactcac 39
<210> 45
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNase_截短的_rv
<400> 45
atggtctcga ttcttatgcg ttagtggtat aagtcaaata gtc 43
<210> 46
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物FpGalNase_rv
<400> 46
atggtctcga ttcttattcc gaaatttcca ccgctttaac 40
<210> 47
<211> 50
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Ct5757_fw
<400> 47
atggtctcgc cattataatt taattgataa tattagtgtt gaaaaattag 50
<210> 48
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Ct5757_rv
<400> 48
atggtctcga ttcttattgt gttaaaccct caataaac 38
<210> 49
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Ct5757_GalNase_rv
<400> 49
atggtctcga ttcttaatga gtactttgat ttaatccatc ataag 45
<210> 50
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Ct5757_DeAcase_fw
<400> 50
atggtctcgc cattcagggc aatattggtt agttttc 37
<210> 51
<211> 35
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rp1021_fw
<400> 51
atggtctcgc catgggaacg gattagaggt gaaag 35
<210> 52
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rp1021_rv
<400> 52
atggtctcga ttctcataat accattttgt atttctttat attgg 45
<210> 53
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rl8755_fw
<400> 53
atggtctcgc catgaagaaa ccgatttgct tgtaaac 37
<210> 54
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rl8755_rv
<400> 54
atggtctcga ttcttagcgt tccaatattt tcataaattc ag 42
<210> 55
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rp3671_fw
<400> 55
atggtctcgc cattcaccat tgagcgctgc gg 32
<210> 56
<211> 44
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rp3671_rv
<400> 56
atggtctcga ttcttatgac tttgttttaa catttacaga cttg 44
<210> 57
<211> 38
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rp3672_fw
<400> 57
atggtctcgc catgctgaga ctgcaacaga agaaaatg 38
<210> 58
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物Rp3672_rv
<400> 58
atggtctcga ttcttatttc tgaatttttg ccttgccag 39
<210> 59
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列AU1表位
<400> 59
Asp Thr Tyr Arg Tyr Ile
1 5
<210> 60
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列AU5表位
<400> 60
Thr Asp Phe Tyr Leu Lys
1 5
<210> 61
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Avi标签
<400> 61
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15
<210> 62
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列T7标签
<400> 62
Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly
1 5 10
<210> 63
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列V5标签
<400> 63
Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr
1 5 10
<210> 64
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列B标签
<400> 64
Gln Tyr Pro Ala Leu Thr
1 5
<210> 65
<211> 26
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列钙调蛋白标签
<400> 65
Lys Arg Arg Trp Lys Lys Asn Phe Ile Ala Val Ser Ala Ala Asn Arg
1 5 10 15
Phe Lys Lys Ile Ser Ser Ser Gly Ala Leu
20 25
<210> 66
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列C标签
<400> 66
Glu Pro Glu Ala
1
<210> 67
<211> 23
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列DogTag
<400> 67
Asp Ile Pro Ala Thr Tyr Glu Phe Thr Asp Gly Lys His Tyr Ile Thr
1 5 10 15
Asn Glu Pro Ile Pro Pro Lys
20
<210> 68
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列E2表位
<400> 68
Ser Ser Thr Ser Ser Asp Phe Arg Asp Arg
1 5 10
<210> 69
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 亲和标签序列E标签
<400> 69
Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg
1 5 10
<210> 70
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 亲和标签序列FLAG标签
<400> 70
Asp Tyr Lys Asp Asp Asp Lys
1 5
<210> 71
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列EE标签(1)
<400> 71
Glu Tyr Met Pro Met Glu
1 5
<210> 72
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列EE标签 (2)
<400> 72
Glu Phe Met Pro Met Glu
1 5
<210> 73
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列HA标签
<400> 73
Tyr Pro Tyr Asp Val Pro Asp Tyr Ala
1 5
<210> 74
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列HAT
<400> 74
Lys Asp His Leu Ile His Asn Val His Lys Glu Phe His Ala His Ala
1 5 10 15
His Asn Lys
<210> 75
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列HQ标签
<400> 75
His Gln His Gln His Gln
1 5
<210> 76
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列HN标签
<400> 76
His Asn His Asn His Asn His Asn His Asn His Asn
1 5 10
<210> 77
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列HSV表位
<400> 77
Gln Pro Glu Leu Ala Pro Glu Asp
1 5
<210> 78
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Isopep标签
<400> 78
Thr Asp Lys Asp Met Thr Ile Thr Phe Thr Asn Lys Lys Asp Ala Glu
1 5 10 15
<210> 79
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列KT3表位
<400> 79
Lys Pro Pro Thr Pro Pro Pro Glu Pro Glu Thr
1 5 10
<210> 80
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Myc表位
<400> 80
Cys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 81
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Myc标签
<400> 81
Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
1 5 10
<210> 82
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列NE标签
<400> 82
Thr Lys Glu Asn Pro Arg Ser Asn Gln Glu Glu Ser Tyr Asp Asp Asn
1 5 10 15
Glu Ser
<210> 83
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Arg标签
<400> 83
Arg Arg Arg Arg Arg
1 5
<210> 84
<211> 5
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Asp标签
<400> 84
Asp Asp Asp Asp Asp
1 5
<210> 85
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Cys标签
<400> 85
Cys Cys Cys Cys
1
<210> 86
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Glu标签
<400> 86
Glu Glu Glu Glu Glu Glu
1 5
<210> 87
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列His标签
<400> 87
His His His His His His
1 5
<210> 88
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Phe标签
<400> 88
Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe Phe
1 5 10
<210> 89
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Rho1D4标签
<400> 89
Thr Glu Thr Ser Gln Val Ala Pro Ala
1 5
<210> 90
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列S1标签
<400> 90
Asn Ala Asn Asn Pro Asp Trp Asp Phe
1 5
<210> 91
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列S标签
<400> 91
Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser
1 5 10 15
<210> 92
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Softtag 1
<400> 92
Ser Leu Ala Glu Leu Leu Asn Ala Gly Leu Gly Gly Ser
1 5 10
<210> 93
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Softtag 3
<400> 93
Thr Gln Asp Pro Ser Arg Val Gly
1 5
<210> 94
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Spy标签
<400> 94
Ala His Ile Val Met Val Asp Ala Tyr Lys Pro Thr Lys
1 5 10
<210> 95
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列SBP标签
<400> 95
Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly
1 5 10 15
Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro
20 25 30
Gln Gly Gln Arg Glu Pro
35
<210> 96
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Strep标签 (1)
<400> 96
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 97
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Strep标签 (2)
<400> 97
Ala Trp Ala His Pro Gln Pro Gly Gly
1 5
<210> 98
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Strep标签 II
<400> 98
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 99
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Sdy标签
<400> 99
Asp Pro Ile Val Met Ile Asp Asn Asp Lys Pro Ile Thr
1 5 10
<210> 100
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列SnoopTag
<400> 100
Lys Leu Gly Asp Ile Glu Phe Ile Lys Val Asn Lys
1 5 10
<210> 101
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列SnoopTagJr
<400> 101
Lys Leu Gly Ser Ile Glu Phe Ile Lys Val Asn Lys
1 5 10
<210> 102
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Spot标签
<400> 102
Pro Asp Arg Val Arg Ala Val Ser His Trp Ser Ser
1 5 10
<210> 103
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列TC标签
<400> 103
Cys Cys Pro Gly Cys Cys
1 5
<210> 104
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Ty标签
<400> 104
Glu Val His Thr Asn Gln Asp Pro Leu Asp
1 5 10
<210> 105
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列通用的
<400> 105
His Thr Thr Pro His His
1 5
<210> 106
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列VSV标签
<400> 106
Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys
1 5 10
<210> 107
<211> 14
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列V5标签
<400> 107
Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr
1 5 10
<210> 108
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 蛋白质标签序列Xpress标签
<400> 108
Asp Leu Tyr Asp Asp Asp Asp Lys
1 5

Claims (25)

1.用于酶促切割来自供体器官的A抗原的灌注流体，其包含
(a)纯化的GalNAc脱乙酰酶蛋白；和
(b)纯化的半乳糖胺酶蛋白。
2.根据权利要求1所述的灌注流体，其中：
(a)所述GalNAc脱乙酰酶是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:2；SEQID NO:4；SEQ ID NO:5；SEQ ID NO:17；SEQ ID NO:23；SEQ ID NO:29；SEQ ID NO:31；SEQID NO:32；SEQ ID NO:33；SEQ ID NO:34和SEQ ID NO:35；以及
(b)所述半乳糖胺酶是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:7；SEQ IDNO:9；SEQ ID NO:10；SEQ ID NO:19；SEQ ID NO:21；SEQ ID NO:36和SEQ ID NO:37。
3.根据权利要求1所述的灌注流体，其中所述灌注流体包含：具有GalNAc脱乙酰酶活性的纯化酶，其基本上由与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少90％相同的氨基酸序列组成；和具有半乳糖胺酶活性的纯化酶，其基本上由与SEQ ID NO:7、9、10、19、21、36和37中之一所示序列至少90％相同的氨基酸序列组成。
4.根据权利要求1或2所述的灌注流体，其中所述灌注流体包含选自以下中的一种或多种的酶：
(a)所述纯化的GalNAc脱乙酰酶蛋白是SEQ ID NO:2、SEQ ID NO:4和SEQ ID NO:5的纯化的普氏梭杆菌(Flavonifractor plautii)的GalNAc脱乙酰酶蛋白；和
(b)所述纯化的半乳糖胺酶蛋白是SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白。
5.根据权利要求1或2所述的灌注流体，其中所述灌注流体包含以下中的一种或多种：
(a)所述纯化的GalNAc脱乙酰酶蛋白是SEQ ID NO:17或SEQ ID NO:32的纯化的第三梭状芽胞杆菌(Clostridium tertium)的GalNAc脱乙酰酶蛋白；和
(b)所述纯化的半乳糖胺酶蛋白是SEQ ID NO:19或SEQ ID NO:36的纯化的第三梭状芽胞杆菌的半乳糖胺酶蛋白。
6.根据权利要求1-5中任一项所述的灌注流体，其中所述GalNAc脱乙酰酶和所述半乳糖胺酶能够在1μg/ml或低于1μg/ml下切割A抗原。
7.根据权利要求1-6中任一项所述的灌注流体，其中所述GalNAc脱乙酰酶和所述半乳糖胺酶在约6.5至约7.5的pH下具有A抗原切割活性。
8.根据权利要求1-7中任一项所述的灌注流体，其中所述GalNAc脱乙酰酶和所述半乳糖胺酶在4℃至37℃的温度下具有A抗原切割活性。
9.根据权利要求1-8中任一项所述的灌注流体，其中所述灌注流体还包含缓冲的细胞外溶液。
10.根据权利要求9所述的灌注流体，其中所述缓冲的细胞外溶液选自：SteenTM；PerfadexTM；Perfadex PlusTM；EuroCollins溶液；组氨酸-色氨酸-酮戊二酸(HTK)溶液；威斯康星大学溶液(UW)；Celsior溶液；肾脏灌注液(KPS-1)；京都大学溶液；IGL-1溶液；和柠檬酸盐溶液。
11.用于离体酶促切割来自供体器官的A抗原的方法，所述方法包括：
(a)用包含GalNAc脱乙酰酶蛋白和半乳糖胺酶蛋白的流体灌注展示A型抗原的供体器官一段时间，所述时间足以允许所述酶切割来自所述供体器官的A抗原；或者
(b)使展示A型抗原的供体器官与包含GalNAc脱乙酰酶蛋白和半乳糖胺酶蛋白的流体一起孵育一段时间，所述时间足以允许所述酶切割来自所述供体器官的A抗原。
12.根据权利要求11所述的方法，其中
所述GalNAc脱乙酰酶是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:2；SEQ IDNO:4；SEQ ID NO:5；SEQ ID NO:17；SEQ ID NO:23；SEQ ID NO:29；SEQ ID NO:31；SEQ IDNO:32；SEQ ID NO:33；SEQ ID NO:34和SEQ ID NO:35；以及
所述半乳糖胺酶是选自以下中的一种或多种的纯化蛋白：SEQ ID NO:7；SEQ ID NO:9；SEQ ID NO:10；SEQ ID NO:19；SEQ ID NO:21；SEQ ID NO:36和SEQ ID NO:37。
13.根据权利要求11所述的方法，其中所述组合物包含：具有GalNAc脱乙酰酶活性的纯化酶，其基本上由与SEQ ID NO:2、4、5、17、23、29、31和32-35中之一所示序列至少90％相同的氨基酸序列组成；和具有半乳糖胺酶活性的纯化酶，其基本上由与SEQ ID NO:7、9、10、19、21、36和37中之一所示序列至少90％相同的氨基酸序列组成。
14.根据权利要求11所述的方法，其中所述GalNAc脱乙酰酶是SEQ ID NO:4或SEQ IDNO:5的纯化的普氏梭杆菌的GalNAc脱乙酰酶蛋白，和所述半乳糖胺酶是SEQ ID NO:9或SEQID NO:10的纯化的普氏梭杆菌的半乳糖胺酶蛋白。
15.根据权利要求11至14中任一项所述的方法，其中所述GalNAc脱乙酰酶蛋白和半乳糖胺酶蛋白处于缓冲的细胞外溶液中。
16.根据权利要求15所述的方法，其中所述缓冲的细胞外溶液选自：SteenTM；PerfadexTM；Perfadex PlusTM；EuroCollins溶液；组氨酸-色氨酸-酮戊二酸(HTK)溶液；威斯康星大学溶液(UW)；Celsior溶液；肾脏灌注液(KPS-1)；京都大学溶液；IGL-1溶液；和柠檬酸盐溶液。
17.根据权利要求11-16中任一项所述的方法，其中所述供体器官是实体器官。
18.根据权利要求17所述的方法，其中所述实体器官选自以下之一：肺；肾脏；肝脏；心脏；胰腺和肠。
19.根据权利要求18所述的方法，其中所述实体器官是肺。
20.根据权利要求17所述的方法，其中使所述GalNAc脱乙酰酶蛋白和所述乳糖胺酶蛋白与离体缓冲的细胞外肺溶液混合并在肺中循环，由此所述GalNAc脱乙酰酶蛋白和所述半乳糖胺酶蛋白与所述供体器官的脉管系统接触一段时间，所述时间足以从所述肺的脉管系统中基本上清除所述A抗原。
21.根据权利要求20所述的方法，其中从所述肺的脉管系统清除所述A抗原的时间为约1小时。
22.根据权利要求11-21中任一项所述的方法，其中所述方法还包括洗涤所述供体器官以去除GalNAc脱乙酰酶、半乳糖胺酶和切割的A抗原。
23.根据权利要求11-22中任一项所述的方法，其中所述GalNAc脱乙酰酶和半乳糖苷酶能够在1μg/ml或低于1μg/ml下切割A抗原。
24.根据权利要求11-23中任一项所述的方法，其中所述GalNAc脱乙酰酶和半乳糖苷酶在约6.5至约7.5的pH下具有A抗原切割活性。
25.根据权利要求11-24中任一项所述的方法，其中所述GalNAc脱乙酰酶和半乳糖胺酶在4℃至37℃的温度下具有A抗原切割活性。
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