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CN111819278B - 新的阿洛酮糖-6-磷酸磷酸酶及其用途 - Google Patents

新的阿洛酮糖-6-磷酸磷酸酶及其用途 Download PDF

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CN111819278B
CN111819278B CN201880048274.4A CN201880048274A CN111819278B CN 111819278 B CN111819278 B CN 111819278B CN 201880048274 A CN201880048274 A CN 201880048274A CN 111819278 B CN111819278 B CN 111819278B
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CN111819278A (zh
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孙秉三
赵显国
梁成才
金成俌
金承焕
朴炫俊
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CJ CheilJedang Corp
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Abstract

本申请涉及包含基序A和基序B的阿洛酮糖‑6‑磷酸磷酸酶、用于产生阿洛酮糖并且包括所述酶的组合物，以及利用所述酶产生阿洛酮糖的方法。

Description

新的阿洛酮糖-6-磷酸磷酸酶及其用途
技术领域
本申请涉及新的阿洛酮糖-6-磷酸磷酸酶、包括所述酶的用于产生D-阿洛酮糖的组合物，以及利用所述酶产生D-阿洛酮糖的方法。
背景技术
已知D-阿洛酮糖-3-表异构酶(EC 5.1.3.30)和D-塔格糖-3-表异构酶(EC5.1.3.31)是催化D-果糖的3-差向异构化以产生D-阿洛酮糖的酶。当利用该酶通过单一酶促反应产生D-阿洛酮糖时，底物(即D-果糖)与产物(即D-阿洛酮糖)之间的反应平衡以恒定水平(产物/底物＝～20％-35％)存在。因此，高纯度D-阿洛酮糖的产生需要另外的处理以从酶反应产物中分离和去除相对高浓度的D-果糖。
另一方面，Chan等人.(2008.Biochemistry.47:9608-9617)报道了来源于酿脓链球菌(Streptococcus pyogenes)的D-核酮糖-5-磷酸-3-表异构酶(EC 5.1.3.1)和来源于大肠杆菌(E.coli)的D-阿洛酮糖6-磷酸-3-表异构酶(EC 5.1.3.-)，其能够催化D-果糖-6-磷酸和D-阿洛酮糖-6-磷酸的3-差向异构化。然而，由于较差的抗热性，这些酶在工业上不适用。
在这样的情况下，本申请的发明人认真进行了研究以开发一种以经济的方式在工业规模上提高D-阿洛酮糖的转化率的方法。结果，本申请的发明人发现，在将作为廉价原料的蔗糖或淀粉(例如麦芽糖糊精)转化为D-阿洛酮糖-6-磷酸后，使用D-阿洛酮糖-6-磷酸特异性的阿洛酮糖-6-磷酸磷酸酶并参与不可逆的反应途径，使得能够通过利用D-阿洛酮糖产生途径中涉及到的两种或更多种酶的一锅法酶促转化来产生D-阿洛酮糖，并且可以显著提高至D-阿洛酮糖的转化率。基于该发现完成了本申请。
公开内容
技术问题
本申请的一个目标是提供包含基序A和基序B的新的阿洛酮糖-6-磷酸磷酸酶。
本申请的另一个目标是提供编码本文中所述的阿洛酮糖-6-磷酸磷酸酶的核酸和包含所述核酸的转化子。
本申请的另一个目标是提供用于产生D-阿洛酮糖的组合物，其包含肌醇-单磷酸酶、表达肌醇-单磷酸酶的微生物或所述微生物的培养物。
本申请的又一个目标是提供用于产生D-阿洛酮糖的方法，其包括使肌醇-单磷酸酶、表达肌醇-单磷酸酶的微生物或所述微生物的培养物与D-阿洛酮糖-6-磷酸接触，以将D-阿洛酮糖-6-磷酸转化为D-阿洛酮糖。
技术方案
现在将详细描述本申请。同时，本申请中公开的方面和实施方案的说明也可以应用于其他方面和实施方案的说明。另外，本申请中公开的各种要素的所有组合都落入本申请的范围内。此外，不应认为本申请的范围受到以下详细描述的限制。
为了达到本申请的上述和其他目标，本申请的一个方面提供了阿洛酮糖-6-磷酸磷酸酶，其包含由Xa1-Xa2-Xa3-DPLDG-Xa4表示的基序A，其中Xa1是W、F、V、I或A，Xa2是I、F、V、A或空位，Xa3是V、I或L，且Xa4是T或S；以及由Ya1-D-Ya2-Wa1-Ya3-Wa2-Ya4-Wa3表示的基序B，其中Ya1是W、Y、T、L或V，Ya2是V、I、C、F或A，Wa1是AAG、AAS、SAG、APG、APF、AGG、APL或AGA，Ya3是W、I、P、M、V、Y、F、R、L、T或S，Wa2是LLV、LIV、LLI、LII、ILI、FIA、ALV、IIA、VLV、VIL、TIG、NFC或PIF，Ya4是E、R、S、T、L、K或P，且Wa3是EAGG、EGGG、EAKG、KAGG、AAGG、YVDG、EAGA或RLGV。
具体地，在基序A中，Xa1可以是W或F，Xa2可以是I或V，Xa3可以是V或I，且Xa4可以是T。具体地，在基序B中，Ya1可以是W，Ya2可以是V或I，Wa1可以是AAG，Ya3可以是W、I或V，Wa2可以是LLV、LIV、LII或LLI，Ya4可以是E、R或S，且Wa3可以是EAGG或EGGG。
在基序A和基序B之间、在基序A的一端或在基序B的一端可以存在一个或多个氨基酸。可以从已知的肌醇-单磷酸酶的序列(例如，SEQ ID NO:1-20中所示的序列中除基序A和基序B外的序列)定义除基序A和基序B外的氨基酸序列。
基序A和/或基序B是肌醇-单磷酸酶的序列中的活性位点。基序A和/或基序B也被称为作为酶的底物的肌醇磷酸的结合位点(参见Federation of European BiochemicalSocieties，第294卷，期号1,2、16-18，1991年12月)。本申请的发明人已发现肌醇-单磷酸酶表现出阿洛酮糖-6-磷酸磷酸酶活性。本申请的发明人已发现肌醇-单磷酸酶的基序A和/或基序B可以是磷酸酶的底物结合位点。
阿洛酮糖-6-磷酸磷酸酶可以包含例如SEQ ID NO:1-20中所示的氨基酸序列中的任一个。
本申请的阿洛酮糖-6-磷酸磷酸酶可以是具有已知肌醇-单磷酸酶的功能的酶。
本申请的阿洛酮糖-6-磷酸磷酸酶更加选择性地催化D-阿洛酮糖-6-磷酸的去磷酸化，并且可以是D-葡萄糖-1-磷酸、D-葡萄糖-6-磷酸或D-果糖-6-磷酸非特异性的。
可以通过以下方式来产生本申请的阿洛酮糖-6-磷酸磷酸酶：用上述酶本身或表达上述酶的DNA(例如，SEQ ID NO:21-40)转化菌株，培养转化的菌株，破碎培养物，然后纯化。可以通过柱色谱法进行纯化。菌株可以是例如，大肠杆菌(Escherichia coli)、谷氨酸棒杆菌(Corynebacterium glutamicum)、米曲霉(Aspergillus oryzae)或枯草芽孢杆菌(Bacillus subtilis)。
根据本申请的一个实施方案，本申请的阿洛酮糖-6-磷酸磷酸酶可以包括与基序A和/或基序B的序列具有至少90％、至少95％、至少97％、至少99％或100％的同源性、相似性或同一性，并且表现出阿洛酮糖-6-磷酸磷酸酶活性的酶。可选地，本申请的阿洛酮糖-6-磷酸磷酸酶可以是与除基序A和/或基序B外的序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％或100％的同源性、相似性或同一性的酶。在一个实施方案中，本申请的阿洛酮糖-6-磷酸磷酸酶可以包括由以下序列组成的蛋白质，所述序列与SEQ ID NO:1-20中所示的序列中的基序A和/或基序B的序列具有至少90％、至少95％、至少97％、至少99％或100％的同源性、相似性或同一性，并且与SEQ ID NO:1-20中所示的序列中除基序A和/或基序B外的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、至少99％或100％的同源性、相似性或同一性，或者由任何两个上述值所限定的范围内的同源性、相似性或同一性。例如，本申请的范围还包括任何这样的氨基酸序列，其具有以上定义的同源性、相似性或同一性，并且表现出与阿洛酮糖-6-磷酸磷酸酶蛋白质，例如，由SEQ ID NO:1-20中所示的序列中的任一个组成的蛋白质相当的功效，并且为部分缺失的、修饰的、取代的或添加的。
在基序A和基序B的序列的上游和下游添加不相干序列的蛋白质属于本申请的范围内，并且其天然存在的突变或沉默突变不排除在本申请的范围之外，只要它们具有与包含基序A和基序B的阿洛酮糖-6-磷酸磷酸酶相当的活性。特别地，在SEQ ID NO:1-20中所示的氨基酸序列中的任一个的上游和下游添加不相干序列的蛋白质，其天然存在的突变和沉默突变不排除在本申请的范围之外，只要其具有与包含基序A和基序B的阿洛酮糖-6-磷酸磷酸酶相当的活性。包含基序A和基序B的蛋白质或包含SEQ ID NO:1-20中所示的氨基酸序列的蛋白质也属于本申请的范围内，只要其具有与包含基序A和基序B的阿洛酮糖-6-磷酸磷酸酶相当的活性。
本申请的另一个方面提供了编码阿洛酮糖-6-磷酸磷酸酶的核酸。
如本文中所用的，术语“核酸”包括DNA和RNA分子，并且作为核酸的基本单元的核苷酸包括天然的核苷酸以及具有修饰的糖或碱基的类似物(参见Scheit,NucleotideAnalogs,John Wiley,New York(1980)；Uhlman和Peyman,Chemical Reviews,90:543-584(1990))。
具体地，编码阿洛酮糖-6-磷酸磷酸酶的核酸可以包括包含可以被翻译成基序A和基序B的核苷酸的序列。在一个实施方案中，本申请的核酸可以由SEQ ID NO:21-40中所示的核苷酸序列中的任一个组成。更具体地，本申请的核酸可以包括这样的核酸，其与可以翻译成基序A和基序B的核苷酸具有至少90％、至少95％、至少99％或100％的同源性、相似性或同一性，并且在翻译后可以表现出所需的酶活性。具体地，本申请的核酸可以包括与可以被翻译成SEQ ID NO:21-40中所示的序列中的基序A和基序B的核苷酸具有至少90％、至少95％、至少99％或100％的同源性、相似性或同一性的核酸。可选地，本申请的核酸可以是与可以翻译成SEQ ID NO:21-40中所示的序列中除基序A和基序B外的基序的核苷酸具有至少80％、至少90％、至少95％、至少97％或至少99％的同源性、相似性或同一性的核酸。由于密码子简并性可以翻译成包括基序A和基序B的蛋白质的蛋白质，具体地，由SEQ ID NO:1-20中所示的氨基酸序列中的任一个组成的蛋白质，或者可以翻译成与由SEQ ID NO:1-20中所示的氨基酸序列中的任一个组成的蛋白质具有同源性、相似性或同一性的蛋白质的多核苷酸也属于本申请的范围内。
根据本申请的包含基序A和基序B的酶可以来源于耐热或嗜热菌株。具体地，由SEQID NO:1-20中所示的氨基酸序列中的任一个组成的酶可以是来源于以下菌株的酶：海洋红嗜热盐菌(Rhodothermus marinus)、莱廷格热袍菌(Thermotoga lettingae)、红色亚栖热菌(Meiothermus ruber)、Dictyoglomus turgidum、铁还原热棒菌(Pyrobaculumferrireducens)、威吉利热厌氧杆菌(Thermoanaerobacter wiegelii)、嗜热栖热菌(Thermusthermophilus)、海滨嗜热球菌(Thermococcus litoralis)、嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)、嗜热厌氧绳菌(Anaerolinea thermophila)、嗜酸热硫化叶菌(Sulfolobus acidocaldarius)、Thermosulfidibacter takaii、强烈火球菌(Pyrococcus furiosus)、闪烁古生球菌(Archaeoglobus fulgidus)、酸热脂环酸芽孢杆菌(Alicyclobacillus acidocaldarius)、Meiothermus silvanus、Meiothermus rufus、台湾亚栖热菌(Meiothermus taiwanensis)、Meiothermus chliarophilus或Meiothermuscerbereus。
如本文中所用的，术语“同源性”或“同一性”表示两个给定的氨基酸序列或核苷酸序列之间的相关程度，其可以表示为百分比。
术语“同源性”和“同一性”经常可互换使用。
通过标准比对算法确定保守的多核苷酸或多肽的序列同源性或同一性，并且可以与所用程序建立的默认空位罚分一起使用。基本上同源或相同的序列通常均沿目标全长多核苷酸或多肽的至少约50％、60％、70％、80％或90％在中等严格性或高严格性下杂交。还考虑了在杂交多核苷酸中包含简并密码子以代替密码子的多核苷酸。
可以利用已知的计算机算法如“FASTA”程序，采用例如，如Pearson等人.(1988),Proc.Natl.Acad.Sci.USA 85:2444中的默认参数，来确定任何两个多核苷酸或多肽序列是否为同源的、相似的或相同的。可选地，可以利用Needleman-Wunsch算法(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)，如在EMBOSS包(EMBOSS:The European MolecularBiology Open Software Suite,Rice等人.,2000,Trends Genet.16:276-277)(版本5.0.0或更新的版本)的Needle程序中实施的，来确定序列同源性、相似性或同一性。其他的程序包括GCG程序包(Devereux,J.,等人,Nucleic Acids Research 12:387(1984))、BLASTP、BLASTN、FASTA(Atschul,[S.][F.,][ET AL,J MOLEC BIOL 215]:403(1990)；Guide toHuge Computers,Martin J.Bishop,[ED.,]Academic Press,San Diego,1994，和[CARILLO等人.](1988)SIAM J Applied Math 48:1073)。例如，可以使用NCBI数据库的BLAST工具来确定同源性、相似性或同一性。也可以使用其他可商购获得的或公开获得的程序如ClustalW。
可以例如，通过利用GAP计算机程序(例如，Needleman等人.,(1970)J.Mol.Biol.48:443，如由Smith和Waterman所修订的,Adv.Appl.Math(1981)2:482)比较序列信息，来确定多核苷酸或多肽的百分比同源性、相似性或同一性。简言之，GAP程序将相似性定义为相似的比对符号(即核苷酸或氨基酸)的数量除以两条序列中的较短一条中符号的总数。GAP程序的默认参数可以包括：(1)一元比较矩阵(包含对于相同的值1和对于不同的值0)，和Gribskov等人.(1986)Nucl.Acids Res.14:6745的加权的比较矩阵，如Schwartz和Dayhoff编辑,Atlas Of Protein Sequence And Structure,National BiomedicalResearch Foundation,pp.353-358(1979)所描述的；(2)对于每个空位的3.0的罚分，和对于每个空位中的每个符号的另外的0.10的罚分(或者空位开放罚分10.0，空位扩展罚分0.5)；以及(3)对于最后的空位无罚分。因此，如本文中所用的术语“同源性”或“同一性”指示多肽或多核苷酸之间的比较。
本申请的另一方面提供了包含编码本文中所述的阿洛酮糖-6-磷酸磷酸酶的核酸的载体，或者包含编码阿洛酮糖-6-磷酸磷酸酶的核酸或包含编码阿洛酮糖-6-磷酸磷酸酶的核酸的载体的转化子。
如本文中所用的，术语“载体”是指用于将碱基核酸克隆和/或转移到宿主细胞中的任何媒介物。载体可以是复制子，另一个DNA区段可以连接至该复制子，以引起所连接区段的复制。“复制子”是指任何遗传元件(例如质粒、噬菌体、粘粒、染色体或病毒)，其作为体内DNA复制的自主单元发挥功能，即能够在其自身的控制下复制。术语“载体”可以包括用于在体外、离体或体内将核酸引入宿主细胞中的病毒和非病毒媒介物。术语“载体”还可以包括微环DNA。具体地，包含编码根据本申请的阿洛酮糖-6-磷酸磷酸酶的核酸的载体可以是pET21a-CJ_Rma、pET21a-CJ_Tle、pET21a-CJ_Mrub、pET21a-CJ_Dtu、pET21a-CJ_Msi、pET21a-CJ_Mruf、pET21a-CJ_Mta、pET21a-CJ_Mch、pET21a-CJ_Mce、pBT7-C-His-CJ_Pfe、pBT7-C-His-CJ_Twi、pBT7-C-His-CJ_Tth、pBT7-C-His-CJ_Tli、pBT7-C-His-CJ_Gst、pBT7-C-His-CJ_Ath、pBT7-C-His-CJ_Sac、pBT7-C-His-CJ_Tta、pBT7-C-His-CJ_Pfu、pBT7-C-His-CJ_Afu或pBT7-C-His-CJ_Aac。
如本文中所用的，术语“转化”是指将包含编码目标蛋白质的核酸的载体引入宿主细胞中，以在宿主细胞中表达核酸编码的蛋白质。转化的核酸可以插入和位于宿主细胞的染色体中，或者可以在染色体外存在，只要其可以在宿主细胞中表达即可。核酸包括编码目标蛋白质的DNA和RNA。可以以任何形式引入核酸，只要其可以被引入宿主细胞中并在宿主细胞中表达即可。例如，可以将核酸以表达盒(其为包括其自主表达所需的所有元件的基因构建体)的形式引入宿主细胞中，但是其形式不限于此。通常，表达盒包括可操作地连接至核酸的启动子、转录终止信号、核糖体结合域和翻译终止信号。表达盒可以为可自我复制的表达载体的形式。可以将核酸原样引入宿主细胞中，并可操作地连接至在宿主细胞中表达所需的序列。
如本文中所用的，术语“可操作地连接”是指起始和介导编码本申请的目标蛋白质的核酸转录的启动子序列与基因序列的之间的功能连接。
对于在本申请中提及的氨基酸使用了以下缩写和名称。
[表1]
氨基酸的类型 缩写
丙氨酸 A
精氨酸 R
天冬酰胺 N
天冬氨酸 D
半胱氨酸 C
谷氨酸 E
谷氨酰胺 Q
甘氨酸 G
组氨酸 H
异亮氨酸 I
亮氨酸 L
赖氨酸 K
蛋氨酸 M
苯丙氨酸 F
脯氨酸 P
丝氨酸 S
苏氨酸 T
色氨酸 W
酪氨酸 Y
缬氨酸 V
可以使用将核酸引入细胞中的任何转化方法。可以根据宿主细胞的类型，通过本领域已知的合适的标准技术进行转化方法。这样的转化方法的实例包括但不限于：电穿孔、磷酸钙(CaPO4)沉淀、氯化钙(CaCl2)沉淀、逆转录病毒感染、显微注射、聚乙二醇(PEG)方法、DEAE-葡聚糖方法、阳离子脂质体方法和醋酸锂-DMSO方法。
宿主细胞优选是高效引入DNA且引入的DNA以高水平表达的宿主细胞。宿主细胞的实例包括但不限于：属于棒杆菌属(Corynebacterium)、埃希氏菌属(Escherichia)和沙雷氏菌属(Serratia)的微生物的细胞。具体地，宿主细胞可以是大肠杆菌细胞。
本申请的转化子可以是：大肠杆菌BL21(DE3)/pET21a-CJ_Rma(大肠杆菌_P1_CJ_Rma,KCCM12057P)、大肠杆菌BL21(DE3)/pET21a-CJ_Tle(大肠杆菌_P2_CJ_Tle,KCCM12058P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mrub(大肠杆菌_P3_CJ_Mrub,KCCM12059P)、大肠杆菌BL21(DE3)/pET21a-CJ_Dtu(大肠杆菌_P4_CJ_Dtu,KCCM12060P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Pfe(大肠杆菌_P5_CJ_Pfe,KCCM12061P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Twi(大肠杆菌_P6_CJ_Twi,KCCM12062P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tth(大肠杆菌_P7_CJ_Tth,KCCM12063P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tli(大肠杆菌_P8_CJ_Tli,KCCM12064P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Gst(大肠杆菌_P9_CJ_Gst,KCCM12065P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Ath(大肠杆菌_P10_CJ_Ath,KCCM12066P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Sac(大肠杆菌_P11_CJ_Sac,KCCM12067P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tta(大肠杆菌_P12_CJ_Tta,KCCM12068P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Pfu(大肠杆菌_P13_CJ_Pfu,KCCM12069P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Afu(大肠杆菌_P14_CJ_Afu,KCCM12070P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Aac(大肠杆菌_P15_CJ_Aac,KCCM12071P)、大肠杆菌BL21(DE3)/pET21a-CJ_Msi(大肠杆菌_P16_CJ_Msi,KCCM12072P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mruf(大肠杆菌_P17_CJ_Mruf,KCCM12073P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mta(大肠杆菌_P18_CJ_Mta,KCCM12074P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mch(大肠杆菌_P19_CJ_Mch,KCCM12075P)或大肠杆菌BL21(DE3)/pET21a-CJ_Mce(大肠杆菌_P20_CJ_Mce,KCCM12076P)。
本申请的另一方面提供了用于产生D-阿洛酮糖的组合物，其包含肌醇-单磷酸酶、表达肌醇-单磷酸酶的微生物或表达肌醇-单磷酸酶的微生物的培养物。
本申请的肌醇-单磷酸酶可以包含：由Xa1-Xa2-Xa3-DPLDG-Xa4表示的基序A，其中Xa1是W、F、V、I或A，Xa2是I、F、V、A或空位，Xa3是V、I或L，且Xa4是T或S；以及由Ya1-D-Ya2-Wa1-Ya3-Wa2-Ya4-Wa3表示的基序B，其中Ya1是W、Y、T、L或V，Ya2是V、I、C、F或A，Wa1是AAG、AAS、SAG、APG、APF、AGG、APL或AGA，Ya3是W、I、P、M、V、Y、F、R、L、T或S，Wa2是LLV、LIV、LLI、LII、ILI、FIA、ALV、IIA、VLV、VIL、TIG、NFC或PIF，Ya4是E、R、S、T、L、K或P，且Wa3是EAGG、EGGG、EAKG、KAGG、AAGG、YVDG、EAGA或RLGV。即，肌醇-单磷酸酶可以与阿洛酮糖-6-磷酸磷酸酶互换使用。因此，对阿洛酮糖-6-磷酸磷酸酶的说明可以适用于肌醇-单磷酸酶。在一个实施方案中，肌醇-单磷酸酶可以是由SEQ ID NO:1-20中所示的氨基酸序列中的任一个组成的酶。
本申请的组合物还可包含D-阿洛酮糖产生途径(参见图1)中涉及到的酶和/或底物[(i)淀粉、麦芽糖糊精、蔗糖或以上的组合；(ii)磷酸；(iii)D-果糖-6-磷酸-3-表异构酶；(iv)D-葡萄糖-6-磷酸-异构酶；(v)葡萄糖磷酸变位酶或葡萄糖激酶；和/或(vi)α-葡聚糖磷酸化酶、淀粉磷酸化酶、麦芽糖糊精磷酸化酶、蔗糖磷酸化酶、α-淀粉酶、普鲁兰酶、异淀粉酶、葡糖淀粉酶或蔗糖酶]；表达D-阿洛酮糖产生途径中涉及到的酶的微生物；或者表达D-阿洛酮糖产生途径中涉及到的酶的微生物的培养物。另外的酶和底物仅是说明性的，并且不受限制，只要可以利用本申请的阿洛酮糖-6-磷酸磷酸酶产生D-阿洛酮糖即可。
淀粉/麦芽糖糊精磷酸化酶(EC 2.4.1.1)和α-葡聚糖磷酸化酶可以包括在磷酰基转移至葡萄糖以从淀粉或麦芽糖糊精产生D-葡萄糖-1-磷酸中具有活性的任何蛋白质。具体地，在从淀粉或麦芽糖糊精产生D-葡萄糖-1-磷酸中具有活性的蛋白质可以由SEQ IDNO:59中所示的氨基酸序列组成。可以通过SEQ ID NO:60中所示的序列编码在从淀粉或麦芽糖糊精产生D-葡萄糖-1-磷酸中具有活性的蛋白质。
蔗糖磷酸化酶(EC 2.4.1.7)可以包括在磷酰基转移至葡萄糖以从蔗糖产生D-葡萄糖-1-磷酸中具有活性的任何蛋白质。
α-淀粉酶(EC 3.2.1.1)、普鲁兰酶(EC 3.2.1.41)、葡糖淀粉酶(EC 3.2.1.3)和异淀粉酶为淀粉液化酶，并且可以包括在将淀粉或麦芽糖糊精转化为葡萄糖中具有活性的任何蛋白质。
蔗糖酶(EC 3.2.1.26)可以包括在将蔗糖转化为葡萄糖中具有活性的任何蛋白质。
葡萄糖磷酸变位酶(EC 5.4.2.2)可以包括在将D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸中具有活性的任何蛋白质。具体地，在将D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸中具有活性的蛋白质可以包含SEQ ID NO:61中所示的氨基酸序列。可以通过SEQ ID NO:62中所示的序列编码在将D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸中具有活性的蛋白质。
葡萄糖激酶可以包括在磷酰基转移至葡萄糖以将葡萄糖转化为D-葡萄糖-6-磷酸中具有活性的任何蛋白质。具体地，葡萄糖激酶可以是多聚磷酸依赖型葡萄糖激酶。更具体地，葡萄糖激酶可以是由SEQ ID NO:77或78中所示的氨基酸序列组成的蛋白质。D-葡萄糖-6-磷酸-异构酶可以包括在将D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸中具有活性的任何蛋白质。具体地，D-葡萄糖-6-磷酸-异构酶可以是包含SEQ ID NO:63中所示的氨基酸序列的蛋白质。可以通过SEQ ID NO:64中所示的序列编码D-葡萄糖-6-磷酸-异构酶。
D-果糖-6-磷酸-3-表异构酶可以包括在将D-果糖-6-磷酸转化为D-阿洛酮糖-6-磷酸中具有活性的任何蛋白质。具体地，D-果糖-6-磷酸-3-表异构酶可以是包含SEQ IDNO:65中所示的氨基酸序列的蛋白质。可以通过SEQ ID NO:66中所示的序列编码D-果糖-6-磷酸-3-表异构酶。
根据本申请的用于D-阿洛酮糖产生的组合物还可包括在将葡萄糖转化为淀粉、麦芽糖糊精或蔗糖中具有活性的蛋白质。蛋白质可以为例如，4-α-葡聚糖转移酶。在将葡萄糖转化为淀粉、麦芽糖糊精或蔗糖中具有活性的蛋白质可以是包含SEQ ID NO:67中所示的氨基酸序列的酶。具体地，可以通过SEQ ID NO:68中所示的核苷酸序列编码在将葡萄糖转化为淀粉、麦芽糖糊精或蔗糖中具有活性的蛋白质。根据本申请的用于D-阿洛酮糖产生的组合物还可包括本领域已知的任何合适的赋形剂。这样的赋形剂的实例包括但不限于：防腐剂、湿润剂、分散剂、助悬剂、缓冲剂、稳定剂和等渗剂。
根据本申请的用于D-阿洛酮糖产生的组合物还可包括金属离子或金属盐。在一个实施方案中，金属离子可以是二价金属阳离子。具体地，金属离子可以选自：Ni、Mg、Co、Mn、Fe和Zn离子。更具体地，根据本申请的用于D-阿洛酮糖产生的组合物还可包括金属盐。甚至更具体地，金属盐可以选自：NiSO4、MgSO4、MgCl2、NiCl2、CoSO4、CoCl2、MnCl2、FeSO4、ZnSO4和以上的混合物。
本申请的另一方面提供了用于产生D-阿洛酮糖的方法，其包括使肌醇-单磷酸酶、表达所述酶的微生物或所述微生物的培养物与D-阿洛酮糖-6-磷酸接触，以将D-阿洛酮糖-6-磷酸转化为D-阿洛酮糖。
肌醇-单磷酸酶可以包括由Xa1-Xa2-Xa3-DPLDG-Xa4表示的基序A和由Ya1-D-Ya2-Wa1-Ya3-Wa2-Ya4-Wa3表示的基序B。在本文中，肌醇-单磷酸酶可与阿洛酮糖-6-磷酸磷酸酶互换使用。
本申请的方法还可包括在将D-阿洛酮糖-6-磷酸转化为D-阿洛酮糖之前，使D-果糖-6-磷酸-3-表异构酶、表达D-果糖-6-磷酸-3-表异构酶的微生物或表达D-果糖-6-磷酸-3-表异构酶的微生物的培养物与D-果糖-6-磷酸接触，以将D-果糖-6-磷酸转化为D-阿洛酮糖-6-磷酸。
本申请的方法还可包括在将D-果糖-6-磷酸转化为D-阿洛酮糖-6-磷酸之前，使D-葡萄糖-6-磷酸-异构酶、表达D-葡萄糖-6-磷酸-异构酶的微生物或表达D-葡萄糖-6-磷酸-异构酶的微生物的培养物与D-葡萄糖-6-磷酸接触，以将D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸。
本申请的方法还可包括在将D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸之前，使葡萄糖磷酸变位酶、表达葡萄糖磷酸变位酶的微生物或表达葡萄糖磷酸变位酶的微生物的培养物与D-葡萄糖-1-磷酸接触，以将D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸。
本申请的方法还可包括在将D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸之前，使葡萄糖激酶、表达葡萄糖激酶的微生物或表达葡萄糖激酶的微生物的培养物及磷酸与葡萄糖接触，以将葡萄糖转化为D-葡萄糖-6-磷酸。
本申请的方法还可包括在将D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸之前，使α-葡聚糖磷酸化酶、淀粉磷酸化酶、麦芽糖糊精磷酸化酶或蔗糖磷酸化酶、表达磷酸化酶的微生物或表达磷酸化酶的微生物的培养物及磷酸与淀粉、麦芽糖糊精、蔗糖或以上的组合接触，以将淀粉、麦芽糖糊精、蔗糖或以上的组合转化为D-葡萄糖-1-磷酸。
本申请的方法还可包括在将葡萄糖转化为D-葡萄糖-6-磷酸之前，使α-淀粉酶、普鲁兰酶、葡糖淀粉酶、蔗糖酶或异淀粉酶、表达α-淀粉酶、普鲁兰酶、葡糖淀粉酶、蔗糖酶或异淀粉酶的微生物，或者表达α-淀粉酶、普鲁兰酶、葡糖淀粉酶、蔗糖酶或异淀粉酶的微生物的培养物与淀粉、麦芽糖糊精、蔗糖或以上的组合接触，以将淀粉、麦芽糖糊精、蔗糖或以上的组合转化为葡萄糖。
本申请的又一个方面提供了用于产生D-阿洛酮糖的方法，其包括使(a)肌醇-单磷酸酶、D-果糖-6-磷酸-3-表异构酶、D-葡萄糖-6-磷酸-异构酶、葡萄糖磷酸变位酶或葡萄糖激酶和α-葡聚糖磷酸化酶、淀粉磷酸化酶、麦芽糖糊精磷酸化酶、蔗糖磷酸化酶、α-淀粉酶、普鲁兰酶、异淀粉酶、葡糖淀粉酶或蔗糖酶，或(b)表达酶(a)的微生物或所述微生物的培养物与淀粉、麦芽糖糊精、蔗糖或以上的组合及磷酸接触。
在本申请的方法中，可以在5.0-9.0的pH，具体地6.0-8.0的pH下进行接触反应。
在本申请的方法中，可以在40℃-80℃的温度，具体地40℃-60℃或50℃-60℃的温度下进行接触反应。
在本申请的方法中，接触反应可以进行2小时至24小时，具体地6-24小时。
在本申请的方法中，接触反应可以在5.0-9.0的pH下、在40℃-80℃的温度下进行，和/或进行2小时至24小时。具体地，接触反应可以在6.0-8.0的pH下、在40℃-60℃或50℃-60℃的温度下进行，和/或进行6小时至24小时。
本申请的方法还可包括纯化反应产物D-阿洛酮糖。对纯化D-阿洛酮糖的方法没有特别限制。可以通过本领域已知的任何合适的方法纯化D-阿洛酮糖。这样的纯化方法的非限制性实例包括：色谱法、分步结晶和离子纯化，其可以单独或组合进行。例如，可以通过色谱法纯化D-阿洛酮糖。在该情形下，可以基于附着在离子树脂上的糖与金属离子之间的结合力的微小差异来分离目标糖。
根据本申请的每种方法还可包括在纯化步骤之前或之后漂白和/或脱矿质。漂白和/或脱矿质使D-阿洛酮糖更纯净而没有杂质。
有利效应
本申请的新的阿洛酮糖-6-磷酸磷酸酶为耐热的肌醇-单磷酸酶。由于耐热性，本申请的酶可以参与D-阿洛酮糖-6-磷酸转化为D-阿洛酮糖的途径中，使得能够在工业规模上生产D-阿洛酮糖。根据本申请的酶的使用允许进行从作为廉价的原料的葡萄糖或淀粉(例如，麦芽糖糊精)合成D-阿洛酮糖的途径。另外，当使用本申请的酶时，可以通过D-阿洛酮糖-6-磷酸的不可逆的去磷酸化产生D-阿洛酮糖。因此，根据本申请的酶的使用极大地提高了D-阿洛酮糖的转化率。
此外，本申请的基于使用肌醇-单磷酸酶的方法能够以高转化率产生高浓度的D-阿洛酮糖，简化或省略了反应产物的分离和纯化。因此，本申请的方法可以以简单的方式进行并且从经济的角度来看是有利的。
附图说明
图1以图示示出了用于从淀粉(例如，麦芽糖糊精)或葡萄糖产生D-阿洛酮糖的酶促反应途径。
图2a、2b和2c显示了用以确定分子量的尺寸标志物(M)、表达的辅酶(S)和纯化的重组酶(E)(pET21a-CJ_Rma、pET21a-CJ_Tle、pET21a-CJ_Mrub、pET21a-CJ_Dtu、pBT7-C-His-CJ_Pfe、pBT7-C-His-CJ_Twi、pBT7-C-His-CJ_Tth、pBT7-C-His-CJ_Tli、pBT7-C-His-CJ_Gst、pBT7-C-His-CJ_Ath、pBT7-C-His-CJ_Sac、pBT7-C-His-CJ_Tta、pBT7-C-His-CJ_Pfu、pBT7-C-His-CJ_Afu、pBT7-C-His-CJ_Aac、pET21a-CJ_Msi、pET21a-CJ_Mruf、pET21a-CJ_Mta、pET21a-CJ_Mch、pET21a-CJ_Mce)的SDS-PAGE凝胶图像，其在蛋白质电泳后采集。
图3a显示了用于去磷酸化的肌醇-单磷酸酶(pET21a-CJ_Rma、pET21a-CJ_Tle、pET21a-CJ_Mrub、pET21a-CJ_Dtu、pBT7-C-His-CJ_Pfe、pBT7-C-His-CJ_Twi、pBT7-C-His-CJ_Tth、pBT7-C-His-CJ_Tli、pBT7-C-His-CJ_Gst、pBT7-C-His-CJ_Ath、pBT7-C-His-CJ_Sac、pBT7-C-His-CJ_Tta、pBT7-C-His-CJ_Pfu、pBT7-C-His-CJ_Afu、pBT7-C-His-CJ_Aac)的相对活性(％，左侧Y轴，柱状图)，和肌醇-单磷酸酶在包含D-葡萄糖-6-磷酸、D-葡萄糖-1-磷酸、D-果糖-6-磷酸和D-阿洛酮糖-6-磷酸的去磷酸化混合物中的选择性去磷酸化速率(％，右侧Y轴，方形点)。
图3b针对去磷酸化比较了肌醇-单磷酸酶(pET21a-CJ_Mrub、pET21a-CJ_Msi、pET21a-CJ_Mruf、pET21a-CJ_Mta、pET21a-CJ_Mch、pET21a-CJ_Mce)的相对活性(％)。
图4a显示了HPLC色谱图，其证实了通过利用α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、D-葡萄糖-6-磷酸-异构酶、4-α-葡聚糖转移酶、D-果糖-6-磷酸-3-表异构酶和阿洛酮糖-6-磷酸磷酸酶的多个酶促反应，从麦芽糖糊精产生了D-阿洛酮糖。
图4b显示了HPLC色谱图，其证实了在根据本申请的肌醇-单磷酸酶存在下，D-阿洛酮糖-6-磷酸在包含D-葡萄糖-1-磷酸、D-葡萄糖-6-磷酸、D-果糖-6-磷酸和D-阿洛酮糖-6-磷酸的反应溶液中通过选择性去磷酸化，产生了D-阿洛酮糖。
发明模式
将参考以下实施例详细说明本申请。然而，提供这些实施例是为了帮助理解本申请，并不限制本申请的范围。
实施例
实施例1：肌醇-单磷酸酶的重组表达载体和转化的微生物的产生
为了提供D-阿洛酮糖产生途径所必需的阿洛酮糖-6-磷酸磷酸酶，筛选了耐热的肌醇-单磷酸酶基因。具体地，从GenBank中登记的以下微生物的基因组序列筛选了肌醇-单磷酸酶基因(Rma、Tle、Mrub、Dtu、Msi、Mruf、Mta、Mch和Mce)：海洋红嗜热盐菌、莱廷格热袍菌、红色亚栖热菌、Dictyoglomus turgidum、铁还原热棒菌、威吉利热厌氧杆菌、嗜热栖热菌、海滨嗜热球菌、嗜热脂肪地芽孢杆菌、嗜热厌氧绳菌、嗜酸热硫化叶菌、Thermosulfidibacter takaii、强烈火球菌、闪烁古生球菌、酸热脂环酸芽孢杆菌、Meiothermus silvanus、Meiothermus rufus、台湾亚栖热菌、Meiothermus chliarophilus和Meiothermus cerbereus。
基于筛选的基因的核苷酸序列(SEQ ID NO:21、22、23、24、36、37、38、39和40，按基因的顺序)和氨基酸序列(SEQ ID NO:1、2、3、4、16、17、18、19和20，按基因的顺序)的信息，设计出了正向引物(SEQ ID NO:41、43、45、47、49、51、53、55和57)和反向引物(SEQ ID NO:42、44、46、48、50、52、54、56和58)。通过聚合酶链式反应(PCR)，利用合成的引物从海洋红嗜热盐菌、莱廷格热袍菌、红色亚栖热菌、Dictyoglomus turgidum、Meiothermus silvanus、Meiothermus rufus、台湾亚栖热菌、Meiothermus chliarophilus和Meiothermuscerbereus的基因组DNA扩增基因。利用限制酶NdeI和XhoI或Sa1I，将扩增的肌醇-单磷酸酶基因插入质粒载体pET21a(Novagen)中用于大肠杆菌表达，以构建重组表达载体，其分别命名为pET21a-CJ_Rma(Nde I/Xho I)、pET21a-CJ_Tle(Nde I/Xho I)、pET21a-CJ_Mrub(NdeI/Xho I)、pET21a-CJ_Dtu(Nde I/Xho I)、pET21a-CJ_Msi(Nde I/Sal I)、pET21a-CJ_Mruf(Nde I/Sal I)、pET21a-CJ_Mta(Nde I/Sal I)、pET21a-CJ_Mch(Nde I/Sal I)和pET21a-CJ_Mce(Nde I/Sal I)。
另外，筛选了来源于铁还原热棒菌、威吉利热厌氧杆菌、嗜热栖热菌、海滨嗜热球菌、嗜热脂肪地芽孢杆菌、嗜热厌氧绳菌、嗜酸热硫化叶菌、Thermosulfidibacter takaii、强烈火球菌、闪烁古生球菌和酸热脂环酸芽孢杆菌的肌醇-单磷酸酶基因(Pfe、Twi、Tth、Tli、Gst、Ath、Sac、Tta、Pfu、Afu和Aac)。基于筛选的基因的核苷酸序列(SEQ ID NO:25、26、27、28、29、30、31、32、33、34和35，按基因的顺序)和氨基酸序列(SEQ ID NO:5、6、7、8、9、10、11、12、13、14和15，按基因的顺序)的信息，请求Bioneer(Korea)进行了DNA合成。将DNA插入载体pBT7-C-His(Bioneer)中，以构建重组表达载体，其被命名为pBT7-C-His-CJ_Pfe、pBT7-C-His-CJ_Twi、pBT7-C-His-CJ_Tth、pBT7-C-His-CJ_Tli、pBT7-C-His-CJ_Gst、pBT7-C-His-CJ_Ath、pBT7-C-His-CJ_Sac、pBT7-C-His-CJ_Tta、pBT7-C-His-CJ_Pfu、pBT7-C-His-CJ_Afu和pBT7-C-His-CJ_Aac。
通过一般的转化技术(参见Sambrook等人.1989)将表达载体转化至大肠杆菌BL21(DE3)菌株中，以产生转化的微生物，其被命名为大肠杆菌BL21(DE3)/pET21a-CJ_Rma(大肠杆菌_P1_CJ_Rma,KCCM12057P)、大肠杆菌BL21(DE3)/pET21a-CJ_Tle(大肠杆菌_P2_CJ_Tle,KCCM12058P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mrub(大肠杆菌_P3_CJ_Mrub,KCCM12059P)、大肠杆菌BL21(DE3)/pET21a-CJ_Dtu(大肠杆菌_P4_CJ_Dtu,KCCM12060P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Pfe(大肠杆菌_P5_CJ_Pfe,KCCM12061P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Twi(大肠杆菌_P6_CJ_Twi,KCCM12062P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tth(大肠杆菌_P7_CJ_Tth,KCCM12063P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tli(大肠杆菌_P8_CJ_Tli,KCCM12064P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Gst(大肠杆菌_P9_CJ_Gst,KCCM12065P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Ath(大肠杆菌_P10_CJ_Ath,KCCM12066P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Sac(大肠杆菌_P11_CJ_Sac,KCCM12067P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tta(大肠杆菌_P12_CJ_Tta,KCCM12068P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Pfu(大肠杆菌_P13_CJ_Pfu,KCCM12069P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Afu(大肠杆菌_P14_CJ_Afu,KCCM12070P)、大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Aac(大肠杆菌_P15_CJ_Aac,KCCM12071P)、大肠杆菌BL21(DE3)/pET21a-CJ_Msi(大肠杆菌_P16_CJ_Msi,KCCM12072P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mruf(大肠杆菌_P17_CJ_Mruf,KCCM12073P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mta(大肠杆菌_P18_CJ_Mta,KCCM12074P)、大肠杆菌BL21(DE3)/pET21a-CJ_Mch(大肠杆菌_P19_CJ_Mch,KCCM12075P)和大肠杆菌BL21(DE3)/pET21a-CJ_Mce(大肠杆菌_P20_CJ_Mce,KCCM12076P)。
于2017年7月10日，根据布达佩斯条约(登陆号：KCCM12057P-KCCM12076P)将转化的菌株保藏在韩国微生物保藏中心(Korean Culture Center of Microorganisms,KCCM)。
实施例2：D-阿洛酮糖产生途径所必需的酶的产生
为了提供作为D-阿洛酮糖产生途径所必需的耐热酶的来源于新阿波罗栖热袍菌(Thermotoga neapolitana)的α-葡聚糖磷酸化酶、葡萄糖磷酸变位酶、D-葡萄糖-6-磷酸-异构酶和D-果糖-6-磷酸-3-表异构酶，筛选了对应于酶的基因(ct1、ct2、tn1和fp3e，按酶的顺序)。
基于筛选的基因的核苷酸序列(SEQ ID NO:60、62、64和66，按酶的顺序)和氨基酸序列(SEQ ID NO:59、61、63和65，按酶的顺序)，设计了正向引物(SEQ ID NO:69、71、73和75)和反向引物(SEQ ID NO:70、72、74和76)。通过聚合酶链式反应(PCR)，利用引物从作为模板的新阿波罗栖热袍菌的基因组DNA扩增酶基因。PCR采用以下条件进行总共25个循环：95℃下变性30sec，55℃下退火30sec，和68℃下聚合2min。利用限制酶NdeI和XhoI将扩增的酶基因插入质粒载体pET21a(Novagen)中用于大肠杆菌表达，以构建重组表达载体，其被命名为pET21a-CJ_ct1、pET21a-CJ_ct2、pET21a-CJ_tn1和pET21a-CJ_fp3e。通过一般的转化技术(参见Sambrook等人.1989)将重组表达载体转化至大肠杆菌BL21(DE3)菌株中，以产生转化的微生物，其被命名为大肠杆菌BL21(DE3)/pET21a-CJ_ct1(KCCM11990P)、大肠杆菌BL21(DE3)/pET21a-CJ_ct2(KCCM11991P)、大肠杆菌BL21(DE3)/pET21a-CJ_tn1(KCCM11992P)和大肠杆菌BL21(DE3)/CJ_tn_fp3e(KCCM11848P)。于2016年6月23日，根据布达佩斯条约将菌株保藏在韩国微生物保藏中心(KCCM)。
实施例3：重组酶的产生
在本实施例中，产生了重组酶。首先，用实施例1和2中产生的每种转化的微生物接种含有5ml LB液体培养基的培养管。将接种物在37℃下于振荡孵化器中培养直至在600nm处的吸光度达到2.0。将培养肉汤添加到培养烧瓶中的LB液体培养基中，然后加入主要培养物。当培养物在600nm处的吸光度达到2.0时，加入1mM IPTG以诱导重组酶的表达和产生。培养温度维持在37℃，并以180rpm搅拌。将培养肉汤在8,000×g和4℃下离心20min以收集细菌细胞。将收集的细菌细胞用50mM Tris-HCl缓冲液(pH 8.0)洗涤两次，并悬浮在相同的缓冲液中。然后，使用超声均质器破碎细胞。将细胞裂解物在13,000×g和4℃下离心20min。通过His标签亲和层析从上清液中纯化重组酶。用50mM Tris-HCl缓冲液(pH 8.0)对纯化的重组酶进行透析，然后用于后续反应。通过SDS-PAGE测定纯化的重组酶的分子量。
利用转化的微生物产生的纯化的酶的名称和分子量如下所示(图2a、2b和2c)：
从大肠杆菌BL21(DE3)/pET21a-CJ_Rma(大肠杆菌_P1_CJ_Rma)产生的酶(RMA)为30.3kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Tle(大肠杆菌_P2_CJ_Tle)产生的酶(TLE)为28.5kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Mrub(大肠杆菌_P3_CJ_Mrub)产生的酶(MRUB)为28kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Dtu(大肠杆菌_P4_CJ_Dtu)产生的酶(DTU)为30.2kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Pfe(大肠杆菌_P5_CJ_Pfe)产生的酶(PFE)为27.2kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Twi(大肠杆菌_P6_CJ_Twi)产生的酶(TWI)为28.8kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tth(大肠杆菌_P7_CJ_Tth)产生的酶(TTH)为28.6kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tli(大肠杆菌_P8_CJ_Tli)产生的酶(TLI)为28kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Gst(大肠杆菌_P9_CJ_Gst)产生的酶(GST)为29.6kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Ath(大肠杆菌_P10_CJ_Ath)产生的酶(ATH)为28.7kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Sac(大肠杆菌_P11_CJ_Sac)产生的酶(SAC)为29.9kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Tta(大肠杆菌_P12_CJ_Tta)产生的酶(TTA)为28.6kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Pfu(大肠杆菌_P13_CJ_Pfu)产生的酶(PFU)为27.9kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Afu(大肠杆菌_P14_CJ_Afu)产生的酶(AFU)为28kDa；
从大肠杆菌BL21(DE3)/pBT7-C-His-CJ_Aac(大肠杆菌_P15_CJ_Aac)产生的酶(AAC)为29kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Msi(大肠杆菌_P16_CJ_Msi)产生的酶(MSI)为28.1kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Mruf(大肠杆菌_P17_CJ_Mruf)产生的酶(MRUF)为28kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Mta(大肠杆菌_P18_CJ_Mta)产生的酶(MTA)为28.1kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Mch(大肠杆菌_P19_CJ_Mch)产生的酶(MCH)为28.4kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_Mce(大肠杆菌_P20_CJ_Mce)产生的酶(MCE)为28.1kDa；
从大肠杆菌BL21(DE3)/pET21a-CJ_ct1(KCCM11990P)产生的酶(CT1)；
从大肠杆菌BL21(DE3)/pET21a-CJ_ct2(KCCM11991P)产生的酶(CT2)；
从大肠杆菌BL21(DE3)/pET21a-CJ_tn1(KCCM11992P)产生的酶(TN1)；和
从大肠杆菌BL21(DE3)/CJ_tn_fp3e(KCCM11848P)产生的酶(FP3E)。
实施例4：肌醇-单磷酸酶的活性分析
4-1.阿洛酮糖-6-磷酸磷酸酶的活性分析
阿洛酮糖-6-磷酸很难购买。因此，发明人直接从D-果糖-6-磷酸产生了D-阿洛酮糖-6-磷酸，并研究了用于D-阿洛酮糖产生的肌醇-单磷酸酶的活性。
具体地，将50mM D-果糖-6-磷酸悬浮在50mM Tris-HCl(pH 7.0)中，然后向其中加入实施例3中产生的D-果糖-6-磷酸-3-表异构酶(FP3E)和0.1单位/ml的20种肌醇-单磷酸酶中的每种。允许混合物在70℃下反应1h。通过HPLC(SP_0810柱(Shodex)，Aminex HPX-87C柱(Bio-RAD)，80℃，流动相流速0.6ml/min，折光率检测器)证实了D-阿洛酮糖的产生。
研究了所有20种肌醇-单磷酸酶对D-阿洛酮糖-6-磷酸的去磷酸化效能(图3a和3b)。
4-2.肌醇-单磷酸酶对D-阿洛酮糖-6-磷酸的特异性去磷酸化的活性分析
测量了在肌醇-单磷酸酶的存在下，包含D-葡萄糖-6-磷酸、D-葡萄糖-1-磷酸、D-果糖-6-磷酸和D-阿洛酮糖-6-磷酸的混合物中D-阿洛酮糖-6-磷酸的特异性去磷酸化速率。
具体地，向1％(w/v)D-葡萄糖-6-磷酸、D-葡萄糖-1-磷酸、D-果糖-6-磷酸和D-阿洛酮糖-6-磷酸的混合物中加入0.1单位/ml的每种肌醇-单磷酸酶和5mM MgCl2。允许反应在50℃下进行12h。通过HPLC(Aminex HPX-87C柱(Bio-RAD)，80℃，流动相流速0.6ml/min)分析反应产物。使用折光率检测器检测D-阿洛酮糖和其他糖类(果糖和葡萄糖)的产生。
结果，酶MRUB显示最高的D-阿洛酮糖-6-磷酸的特异性去磷酸化速率(图3a)。
实施例5：通过多个酶促反应分析酶的活性
对于从麦芽糖糊精产生D-阿洛酮糖，允许酶CT1、CT2、TN1、FP3E和MRUB同时与麦芽糖糊精反应。向0.1单位/ml的每种酶、5mM MgCl2和20mM磷酸钠(pH 7.0)中添加5％(w/v)麦芽糖糊精。允许混合物在50℃的温度下反应12h。通过HPLC(Aminex HPX-87C柱(Bio-RAD)，80℃，流动相流速0.6ml/min，折光率检测器)分析反应产物。
结果，证实了通过多个酶促反应从麦芽糖糊精产生D-阿洛酮糖(图4a)。
尽管已经详细描述了本申请的实施方案，但是本领域技术人员将理解，可以以其他特定形式来实现本申请，而不改变本申请的精神或基本特征。因此，应当注意，前述实施方案在所有方面仅是说明性的，并且不应被解释为限制本申请。本申请的范围由所附的权利要求而不是本申请的详细描述来界定。在权利要求的含义和范围内所做的所有改变或修改或其等效形式都应解释为落入本申请的范围内。
<110> CJ第一制糖株式会社(CJ CHEILJEDANG CORPORATION)
<120> 新的阿洛酮糖-6-磷酸磷酸酶、包括所述磷酸酶的用于产生阿洛酮糖的组合物和利用所述磷酸酶产生阿洛酮糖的方法
<130> P18-6064
<150> KR 2017/0097334
<151> 2017-07-31
<160> 78
<170> KoPatentIn 3.0
<210> 1
<211> 272
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> RMA的氨基酸序列
<400> 1
Met Met Asp Ala Tyr Thr Leu Tyr Glu Glu Ala Arg Glu Val Ala Ala
1 5 10 15
Arg Leu Ala Arg Asp Ala Gly Gln Ile Ala Arg Tyr Tyr Ala Gly Arg
20 25 30
Val Thr Val Arg Glu Lys Gly Tyr Asn Glu Leu Val Thr Gln Ala Asp
35 40 45
Glu Glu Val Gln Arg Phe Leu Ile Glu Gln Ile His Arg His Phe Pro
50 55 60
Glu His Ala Ile Leu Ala Glu Glu Asn Leu Ser Asp Met Gln Asp Gly
65 70 75 80
Arg Glu Gly Ala Ser Phe Arg Trp Ile Ile Asp Pro Ile Asp Gly Thr
85 90 95
Thr Asn Phe Thr His Gly Val Pro Pro Tyr Gly Ile Ser Leu Ala Leu
100 105 110
Gln His Glu Gly Arg Thr Val Val Gly Val Val Tyr Asp Val Pro His
115 120 125
Asp Glu Leu Phe Thr Ala Val Arg Gly Gly Gly Leu Tyr Val Asn Gly
130 135 140
Val Arg Ala Arg Val Ser Gln Thr Glu Thr Leu Arg Glu Ala Leu Ile
145 150 155 160
Thr Thr Gly Phe Pro Tyr Arg Glu Val Val His Leu Glu Glu Tyr Leu
165 170 175
Glu Ala Leu Gly Arg Val Ile Arg Ala Thr Arg Gly Val Arg Arg Pro
180 185 190
Gly Ala Ala Ser Val Asp Leu Ala Trp Val Ala Cys Gly Arg Phe Asp
195 200 205
Gly Phe Phe Glu Thr Gly Leu Ser Pro Trp Asp Val Ala Ala Gly Ile
210 215 220
Leu Leu Val Glu Glu Gly Gly Gly Arg Val Thr Asp Phe His Gly Arg
225 230 235 240
Pro Asp Pro Ile Phe Ala Arg Gln Met Leu Ala Thr Asn Gly Arg Ile
245 250 255
His Glu Ala Leu Cys Glu Leu Val Ala Pro Leu His His Val Tyr Ala
260 265 270
<210> 2
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TLE的氨基酸序列
<400> 2
Met Asp Arg Met Asp Phe Ser Ile Lys Ile Ala Arg Lys Val Gly Leu
1 5 10 15
Tyr Leu Met Glu His Trp Gly Asn Ala Glu Asn Val Arg Gln Lys Ser
20 25 30
Ser Phe Gln Asp Leu Val Ser Asp Cys Asp Lys Gln Ala Gln Lys Met
35 40 45
Ile Val Gln Lys Ile Lys Asp His Phe Pro Asp Asp Ala Ile Leu Ala
50 55 60
Glu Glu Gly Leu Phe Glu Lys Gly Asp Arg Met Trp Ile Ile Asp Pro
65 70 75 80
Ile Asp Gly Thr Met Asn Tyr Val His Gly Leu Pro Ser Phe Ala Ile
85 90 95
Gly Ile Ala Tyr Val Glu Lys Glu Gln Val Ile Leu Gly Val Ala His
100 105 110
Asp Pro Val Leu Asn Glu Thr Tyr Tyr Ala Ile Lys Gly Gln Gly Ala
115 120 125
Tyr Lys Asn Gly Glu Arg Ile Asn Val Ser Glu Asn Ser Leu Leu Lys
130 135 140
Asp Ser Ile Gly Asn Thr Gly Phe Tyr Thr Asp Phe Thr Gly Ile Phe
145 150 155 160
Ile Ser Ala Ile Glu Lys Lys Val Arg Arg Val Arg Met Thr Gly Ser
165 170 175
Ala Ile Leu Ala Gly Ala Tyr Val Ala Cys Gly Arg Phe Asp Phe Phe
180 185 190
Ile Ala Lys Arg Ala Asn Ser Trp Asp Val Ala Pro Leu Phe Val Leu
195 200 205
Val Pro Glu Ala Gly Gly Ile Val Thr Asp Leu Ser Gly Asn Gln Ala
210 215 220
His Leu Asn Thr Gly Asn Phe Leu Phe Ser Asn Gly Leu Leu His Asp
225 230 235 240
Gln Val Leu Glu Val Ile Arg Glu Val Asn Lys Lys Val Arg Lys
245 250 255
<210> 3
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MRUB的氨基酸序列
<400> 3
Met Asp Leu Arg Ala Tyr Leu Gln Thr Ala Leu Asp Ala Ala Tyr Leu
1 5 10 15
Ala Lys Gly Ile His Gln Tyr Tyr Gln Glu Lys Gly Phe Thr Gln Ser
20 25 30
Thr Lys Ser Thr Pro Thr Asp Leu Val Thr Gln Ala Asp His Glu Ser
35 40 45
Glu Ala Ala Ile Arg Glu Leu Ile Ala Ser Arg His Pro Asp His Val
50 55 60
Val Leu Gly Glu Glu Gln Gly Gln Asp Lys Glu Gly Ala Phe Arg Trp
65 70 75 80
Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr Ala His Gly Phe Pro
85 90 95
Phe Tyr Ala Val Ser Ile Gly Leu Glu Ala His Gly Glu Val Val Leu
100 105 110
Gly Val Val Leu Asp Thr Ala Arg Gly Glu Leu Phe Thr Ala Thr Lys
115 120 125
Gly Gly Gly Ala Tyr Leu Asn Gly Arg Pro Ile Arg Val Ser Thr Arg
130 135 140
Ser Thr Leu Val Gly Ser Leu Leu Ala Thr Gly Phe Pro Tyr Asp Val
145 150 155 160
Ser Lys Asp Thr Glu Asn Leu Val Tyr Phe Gln Arg Ala Leu Thr Lys
165 170 175
Gly Leu Met Val Arg Arg Pro Gly Ala Ala Ala Leu Asp Leu Ala Tyr
180 185 190
Val Ala Ala Gly Arg Leu Asp Gly Phe Trp Glu Val Lys Leu Asn Pro
195 200 205
Trp Asp Val Ala Ala Gly Trp Leu Ile Val Ser Glu Ala Gly Gly Arg
210 215 220
Ile Thr Gly Leu Gln Gly Glu Asp Tyr Arg Leu Gly Asn Arg Tyr Leu
225 230 235 240
Val Ala Ser Asn Gly Leu Ile His Gly Pro Leu Leu Asp Thr Ile His
245 250 255
Gly Arg
<210> 4
<211> 265
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> DTU的氨基酸序列
<400> 4
Met Lys Arg Ile Leu Glu Val Ala Ile Lys Thr Ile Lys Glu Ser Gly
1 5 10 15
Asn Ile Leu Leu Asn Tyr Ile Gly Glu Glu Lys Glu Ile Glu Leu Lys
20 25 30
Gly Ile Ser Asn Leu Val Thr Gln Val Asp Lys Leu Ser Glu Arg His
35 40 45
Ile Leu Lys Ser Ile Glu Glu Asn Phe Pro Asp His Ser Ile Leu Thr
50 55 60
Glu Glu Thr Gly Phe Ile Asn Lys Asn Ser Glu Tyr Thr Trp Ile Val
65 70 75 80
Asp Pro Leu Asp Gly Thr Thr Asn Tyr Ala His Asn Phe Pro Phe Phe
85 90 95
Gly Ile Ser Ile Ala Leu Ile Lys Asn Lys Glu Ile Ile Leu Gly Leu
100 105 110
Ile Tyr Asp Pro Ile Arg Asp Glu Leu Phe Tyr Ala Ile Lys Asn Glu
115 120 125
Gly Ala Tyr Leu Asn Asp Arg Arg Ile Glu Val Ser Lys Thr Glu Ser
130 135 140
Leu Glu Asn Ser Leu Ile Ser Phe Ala Phe Pro Tyr Glu Leu Ser Leu
145 150 155 160
Glu Glu Lys Asn Phe Ile Pro Phe Ile Asn Phe Ser Ser Arg Thr His
165 170 175
Gly Ile Arg Arg Thr Gly Ser Ala Ala Ile Glu Ile Ala Tyr Val Gly
180 185 190
Cys Gly Arg Leu Asp Gly Phe Trp Ala Lys Lys Leu Lys Pro Trp Asp
195 200 205
Ile Ser Ala Gly Ile Leu Ile Val Glu Glu Ala Lys Gly Lys Val Thr
210 215 220
Asp Phe Ser Gly Asn Asn Ile Asp Ile His Thr Asp Asn Ile Leu Phe
225 230 235 240
Ser Asn Gly Lys Ile His Gln Glu Met Ile Lys Ile Leu Asn Leu Gly
245 250 255
Lys Ile Phe Ile Arg Asn Glu Lys Phe
260 265
<210> 5
<211> 251
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> PFE的氨基酸序列
<400> 5
Met Leu Gly Val Leu Glu Ala Val Ala Val Arg Ala Ser His Phe Leu
1 5 10 15
Met Glu Tyr Phe Arg Ala Gly Arg Gly Val Asp Val Val Ser Arg Lys
20 25 30
Glu Asp Asp Val Thr Arg Glu Val Asp Ile Ala Ala Glu Thr Leu Ile
35 40 45
Tyr Lys Met Leu Arg Glu Ala Phe Lys Glu Gly Gly Val Leu Tyr Ala
50 55 60
Glu Glu Gly Gly Ile Tyr Arg Trp Gly Asp Glu Arg Tyr Ile Phe Val
65 70 75 80
Leu Asp Pro Leu Asp Gly Ser Leu Asn Tyr Ala Val Gly Val Pro Phe
85 90 95
Phe Ala Val Ser Ile Ala Ala Gly Lys His Arg Glu Gly Thr Leu Ala
100 105 110
Asp Leu Glu Tyr Ala Val Val Ala Ile Pro Pro Thr Gly Asp Val Tyr
115 120 125
Thr Ala Ala Pro Gly Val Gly Ala Arg Lys Asn Gly Lys Pro Leu Arg
130 135 140
Arg Thr Pro Arg Ser Asn Ile Val Phe Val Ala Val Ser Asn Ser Phe
145 150 155 160
Pro Pro Lys Thr Cys Glu Val Val Arg Arg Leu Gly Leu Arg Gly Arg
165 170 175
Ser Leu Gly Ser Ser Ala Ala Glu Leu Ala Tyr Thr Val Glu Gly Ile
180 185 190
Ala Arg Gly Phe Leu Asp Leu Arg Gly Lys Leu Arg Leu Leu Asp Val
195 200 205
Ala Gly Ala Leu Thr Ile Gly Lys Tyr Val Asp Gly Phe Arg Tyr Val
210 215 220
Val Met Gly Asp Thr Lys Pro His Ser Lys Val Ser Leu Val Ala Gly
225 230 235 240
Asp Val Asp Phe Val Asn Ala Ala Thr Thr Asp
245 250
<210> 6
<211> 257
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TWI的氨基酸序列
<400> 6
Met Lys Asp Glu Lys Gly Ile Val Val Asp Ile Ile Lys Lys Ala Gly
1 5 10 15
Glu Ile Leu Gln Asp Gly Trp Asn Lys Lys Asn Phe Lys Ile Tyr Arg
20 25 30
Lys Gly Thr Ile Asn Leu Val Thr Glu Ile Asp Lys Lys Ile Glu Phe
35 40 45
Leu Ile Ile Gln Leu Leu Lys Gln Tyr Phe Pro Asp Tyr Gly Ile Leu
50 55 60
Thr Glu Glu Ser Lys Glu Ile Asn Ser Lys Ala Asn Val Arg Trp Ile
65 70 75 80
Ile Asp Pro Leu Asp Gly Thr Thr Asn Tyr Ile Lys Gln Tyr Pro Phe
85 90 95
Val Ala Ile Ser Ile Ala Leu Glu Lys Glu Gly Glu Leu Ile Leu Gly
100 105 110
Val Val Tyr Asn Pro Ile Leu Asn Glu Met Phe Ile Ala Gln Lys Gly
115 120 125
Cys Gly Ala Thr Tyr Asn Gly Lys Ser Ile His Val Ser Lys Ile Lys
130 135 140
Glu Leu Gly Ser Ala Val Leu Ala Ser Gly Phe Pro Tyr Asp Ala Trp
145 150 155 160
Glu Asn Pro Asp Asn Asn Ala Lys Gln Trp Arg Gln Phe Leu Thr Arg
165 170 175
Ser Leu Ser Leu Arg Cys Asp Gly Ser Ala Ala Leu Asp Leu Cys Arg
180 185 190
Val Ala Cys Gly Gln Leu Asp Gly Tyr Trp Glu Lys Gly Ile Ser Pro
195 200 205
Trp Asp Val Ala Ala Gly Ile Val Ile Leu Arg Glu Ala Gly Gly Ile
210 215 220
Ile Thr Asp Tyr Leu Gly Glu Glu Asn Phe Phe Lys Arg Gly Glu Val
225 230 235 240
Val Ala Ala Asn Pro Val Leu His Ala Gln Met Leu Lys Val Leu Asn
245 250 255
Asn
<210> 7
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TTH的氨基酸序列
<400> 7
Met Ile Gly Arg Arg His Pro Tyr Tyr Pro Tyr Leu Glu Ala Ala Leu
1 5 10 15
Glu Ala Ala Ser Leu Ala Arg Gly Ile His Leu Tyr Tyr Leu Glu Lys
20 25 30
Gly Phe Thr Glu Gly Thr Lys Ser Gly Pro Thr Asp Leu Val Thr Gln
35 40 45
Ala Asp Arg Glu Ala Glu Glu Ala Val Lys Gly Leu Leu Leu Ser Arg
50 55 60
Phe Pro Glu Ala Gly Phe Leu Gly Glu Glu Gly Gly Ser Glu Gly Gly
65 70 75 80
Lys Ala Leu Arg Phe Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr
85 90 95
Ala His Gly Phe Pro Phe Phe Ala Val Ser Ile Ala Leu Glu Ala Glu
100 105 110
Gly Ala Ile Gln Met Gly Val Val Met Asp Thr Ala Arg Gly Glu Val
115 120 125
Phe Tyr Ala Leu Arg Gly Glu Gly Ala Tyr Leu Asn Gly Arg Pro Ile
130 135 140
Arg Val Thr Gly Arg Glu Ser Leu Val Gly Ser Leu Leu Ala Thr Gly
145 150 155 160
Phe Pro Tyr Asp Val Ala Lys Asp Pro Glu Asn Leu Thr Tyr Phe Glu
165 170 175
Arg Ala Leu Gly Lys Gly Leu Leu Val Arg Arg Pro Gly Ala Ala Ala
180 185 190
Leu Asp Leu Ala Tyr Val Ala Ala Gly Arg Leu Glu Gly Phe Trp Glu
195 200 205
Val Lys Leu Asn Pro Trp Asp Val Ala Ala Gly Trp Leu Leu Val Glu
210 215 220
Glu Ala Gly Gly Arg Val Thr Asp Leu Glu Gly Asn Pro Tyr Arg Leu
225 230 235 240
Gly Ser Arg Tyr Ile Leu Ala Thr Asn Gly Arg Val His Glu Ala Leu
245 250 255
Arg Arg Thr Leu Leu Gly Leu Asp
260
<210> 8
<211> 251
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TLI的氨基酸序列
<400> 8
Met Tyr Glu Trp Asn Glu Ile Ala Leu Asn Leu Ala Lys Asp Ile Glu
1 5 10 15
Arg Glu Val Met Pro Leu Phe Gly Thr Lys Lys Ala Gly Glu Phe Ile
20 25 30
Gly Phe Ser Pro Ser Gly Asp Lys Thr Lys Leu Val Asp Lys Val Ala
35 40 45
Glu Asp Val Val Leu Glu Tyr Leu Arg Pro Leu Gly Val Asn Val Val
50 55 60
Ser Glu Glu Ile Gly Asn Ile Glu Ala Gly Ser Glu Tyr Thr Ile Val
65 70 75 80
Val Asp Pro Ile Asp Gly Ser Phe Asn Phe Ile Gln Gly Ile Pro Ile
85 90 95
Phe Gly Phe Ser Phe Ala Val Phe Lys Asn Glu Lys Pro Val Tyr Ala
100 105 110
Met Ile Tyr Glu Phe Ile Thr Lys Asn Val Tyr Glu Gly Ile Pro Gly
115 120 125
Glu Gly Ala Tyr Leu Asn Gly Glu Arg Ile Arg Val Arg His Leu Asn
130 135 140
Glu Lys Ser Ile Ser Ile Ser Phe Tyr Thr Arg Gly Arg Gly Ala Arg
145 150 155 160
Leu Val Glu Lys Val Lys Arg Thr Arg Val Leu Gly Ala Ile Ala Val
165 170 175
Glu Leu Ala Tyr Leu Ala Arg Gly Ser Leu Asp Gly Val Ile Asp Ile
180 185 190
Arg Asn Tyr Val Arg Pro Thr Asp Ile Ala Ala Gly Tyr Ile Ile Ala
195 200 205
Lys Glu Ala Gly Ala Ile Ile Thr Asp Asp Ser Gly Glu Glu Ile Lys
210 215 220
Phe Arg Leu Asp Ala Arg Glu Lys Leu Asn Ile Ile Ala Val Asn Asp
225 230 235 240
Lys Arg Leu Leu Lys Leu Ile Leu Glu Val Ile
245 250
<210> 9
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> GST的氨基酸序列
<400> 9
Met Ala Glu Lys Trp Glu Glu Ile Asp Arg Tyr Ala Arg Gln Trp Ile
1 5 10 15
Asp Glu Ala Gly Lys Arg Ile Arg Ala Ser Phe Ala Lys Gln Leu Thr
20 25 30
Val Glu Ala Lys Glu Asn Pro Asn Asp Leu Val Thr Asn Val Asp Arg
35 40 45
Ala Ile Glu Gln Phe Phe Ala Glu His Ile Arg Arg Gln Phe Pro Ser
50 55 60
His Arg Leu Leu Gly Glu Glu Gly Phe Gly Asp Arg Ile Asp Ala Leu
65 70 75 80
Asp Gly Val Val Trp Val Ile Asp Pro Ile Asp Gly Thr Met Asn Phe
85 90 95
Val His Gln Arg Arg His Phe Ala Val Ser Ile Gly Ile Phe Glu Asp
100 105 110
Gly Ile Gly Gln Leu Gly Tyr Val Tyr Asp Val Val Phe Asp Glu Leu
115 120 125
Tyr Ala Ala Gln Lys Gly Arg Gly Val Phe Leu Asn Gly Glu Pro Leu
130 135 140
Gly Leu Leu Gln Pro Ala Pro Val Ala Glu Ser Ile Ile Ala Ile Asn
145 150 155 160
Gly Thr Trp Leu Met Glu Asn Lys Arg Leu Asp His Arg Pro Leu Met
165 170 175
Arg Leu Ala Lys Glu Ala Arg Gly Thr Arg Ser Tyr Gly Ser Ala Ala
180 185 190
Leu Glu Leu Ala Tyr Val Ala Ala Gly Arg Leu Asp Ala Tyr Ile Ser
195 200 205
Pro Arg Leu Ser Pro Trp Asp Phe Ala Gly Gly Met Ile Leu Ile Glu
210 215 220
Glu Ala Gly Gly Met Val Thr Thr Leu Asp Gly Lys Pro Leu Asp Leu
225 230 235 240
Leu Gly Arg Asn Ser Val Leu Ala Ala Lys Pro Gly Val His Glu Glu
245 250 255
Ile Leu Arg Arg Tyr Leu His Asp
260
<210> 10
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> ATH的氨基酸序列
<400> 10
Met Thr Thr Ser Leu Arg Asp Leu Leu Asp Phe Ala Val Glu Thr Ala
1 5 10 15
Tyr Leu Ala Gly Arg Thr Thr Leu Ala Tyr Phe Gln Thr Gly Val Gln
20 25 30
Ala Glu Phe Lys Ala Asp Ser Ser Pro Val Thr Leu Ala Asp Arg Ala
35 40 45
Ala Glu Gln Leu Ile Arg Gln Arg Ile Glu Lys Arg Phe Pro His His
50 55 60
Ala Ile Val Gly Glu Glu Phe Gly Val Gln Gly Ser Ala Asp Ala Thr
65 70 75 80
His Arg Trp Phe Ile Asp Pro Ile Asp Gly Thr Lys Ser Phe Leu Arg
85 90 95
Gly Ile Pro Leu Tyr Ala Val Leu Leu Gly Leu Glu Ile Glu Gly Arg
100 105 110
Val Gln Val Gly Val Ala Tyr Tyr Pro Ala Met Asp Glu Met Leu Ser
115 120 125
Ala Ala Asp Gly Glu Gly Cys Trp Trp Asn Gly Arg Arg Ala Arg Val
130 135 140
Ser Thr Ala Ser Arg Leu Ala Glu Ala Trp Val Thr Ser Thr Asp Pro
145 150 155 160
Tyr Asn Phe Gln Lys Thr Gly Lys Asp Ala Ala Trp Gln Arg Ile Gln
165 170 175
Ala Val Ser Tyr His Arg Gly Gly Trp Gly Asp Ala Tyr Gly Tyr Leu
180 185 190
Leu Val Ala Thr Gly Arg Ala Glu Val Met Leu Asp Pro Ile Met Asn
195 200 205
Glu Trp Asp Cys Ala Pro Phe Pro Pro Ile Phe Arg Glu Ala Gly Gly
210 215 220
Phe Phe Gly Asp Trp Gln Gly Asn Glu Thr Ile Tyr Gly Gly Glu Ala
225 230 235 240
Leu Ala Thr Thr Gln Val Leu Leu Pro Glu Val Leu Glu Cys Leu His
245 250 255
Ser Ser Leu
<210> 11
<211> 267
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SAC的氨基酸序列
<400> 11
Met Lys Arg Glu Asp Val Glu Lys Val Ala Ser Glu Ala Ser Lys Tyr
1 5 10 15
Ile Tyr Glu Glu Arg Glu Asn Lys Asp Val Asp Arg Val Ile Asn Val
20 25 30
His Gly Asn Asp Val Thr Arg Ile Ile Asp Lys Arg Ser Glu Asp Phe
35 40 45
Ile Val Asp Arg Leu Lys Ser Leu Gly Tyr Asn Ile Leu Ile Val Thr
50 55 60
Glu Glu Ser Gly Val Ile Asp Ser Tyr Gly Lys Asn Tyr Asp Tyr Ile
65 70 75 80
Ala Ile Val Asp Pro Leu Asp Gly Ser Thr Asn Phe Val Ser Gly Ile
85 90 95
Pro Trp Ser Ser Val Ser Ile Ala Ile Tyr Asn Arg Asp Glu Glu Asp
100 105 110
Ile Leu Ser Ser Asn Val Gly Ala Val Ser Ser Ile Phe Thr Pro Tyr
115 120 125
Thr Phe Ser Tyr Asp Glu Gly Ser Ala Tyr Val Asn Gly Val Lys Ile
130 135 140
Ala Glu Ile Lys Lys Pro Glu Lys Ile Leu Leu Leu Ala Tyr Phe Ser
145 150 155 160
Arg Ser Lys Leu Pro Asn Leu Lys Leu Phe Phe Glu Lys Ile Gly Gln
165 170 175
Gly Tyr Lys Ile Arg Ser Leu Gly Ser Ala Ser Leu Asp Met Ile Leu
180 185 190
Val Cys Thr Gly Arg Ala Thr Met Phe Phe Asp Ile Arg Gly Lys Leu
195 200 205
Arg Asn Val Asp Ile Ala Ala Ser Ser Asn Phe Cys Ser Arg Leu Gly
210 215 220
Val Ile Pro Tyr Asp Ile Gly Leu Arg Lys Ile Lys Ser Ser Leu Thr
225 230 235 240
Glu Val Ser Val Val Lys Asp Leu Val Ile Ser Leu Asp Glu Ser Leu
245 250 255
Leu Arg Ser Phe Ser Leu Ala Leu Gln Thr Val
260 265
<210> 12
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TTA的氨基酸序列
<400> 12
Met Leu Glu Leu Leu Lys Asp Thr Ala Lys Lys Ala Gly Arg Leu Leu
1 5 10 15
Lys Ile Arg Phe Leu Glu Leu Leu Asn Gly Met Glu Thr Glu Val Arg
20 25 30
Glu Lys Gly Lys Ser Asp Phe Val Thr Arg Val Asp Val Glu Val Glu
35 40 45
Asn Tyr Ile Lys Glu Leu Leu Ala Asn Thr Asn Ile Ser Val Val Gly
50 55 60
Glu Glu Ser Phe Lys Gly Glu Ile Pro Gly Thr Cys Ile Phe Ile Asp
65 70 75 80
Pro Ile Asp Gly Thr Arg Asn Phe Met Arg Lys Asn Pro His Phe Ala
85 90 95
Ile Asn Leu Ala Tyr Gln Glu Lys Gly Lys Leu Leu Ala Gly Val Thr
100 105 110
Tyr Asp Pro Met Lys Asn Glu Met Phe Ser Ala Ala Phe Ser Lys Gly
115 120 125
Ala Phe Leu Asn Gly Glu Arg Ile Tyr Ala Ser Thr Asn Lys Asp Ile
130 135 140
Gly Lys Ala Ile Ile Ala Ile Gly Leu Pro Tyr Arg Gly Arg Glu Leu
145 150 155 160
Ile Asp Ile Gln Thr Asn Leu Tyr Arg Asn Ile Phe Leu Asn Gly Ala
165 170 175
Ala Thr Arg His Thr Gly Ser Ala Ala Leu Asp Leu Ala Tyr Ile Ser
180 185 190
Cys Gly Arg Tyr Asp Ala Ala Ile Tyr Phe Tyr Leu Ser Pro Trp Asp
195 200 205
Val Ala Pro Gly Ile Leu Leu Val Glu Glu Ala Gly Gly Glu Val Glu
210 215 220
Gly Thr Met Gly Arg Glu Pro Ile Gln Gly Trp Ile Ile Ala Ser Asn
225 230 235 240
Lys Val Ile His Pro Glu Val Lys Asp Ile Leu Glu Gly Ser Leu Lys
245 250 255
Ala Val
<210> 13
<211> 254
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> PFU的氨基酸序列
<400> 13
Met Lys Leu Lys Phe Trp Arg Glu Val Ala Ile Asp Ile Ile Ser Asp
1 5 10 15
Phe Glu Thr Thr Ile Met Pro Phe Phe Gly Asn Pro Asp Gly Gly Lys
20 25 30
Leu Val Lys Ile Ser Pro Ser Gly Asp Glu Thr Lys Leu Val Asp Lys
35 40 45
Leu Ala Glu Asp Leu Ile Leu Ser Arg Ile Thr Glu Leu Gly Val Asn
50 55 60
Val Val Ser Glu Glu Val Gly Val Ile Asp Asn Glu Ser Glu Tyr Thr
65 70 75 80
Val Ile Val Asp Pro Leu Asp Gly Ser Tyr Asn Phe Ile Ala Gly Ile
85 90 95
Pro Phe Phe Ala Leu Ser Leu Ala Val Phe Lys Lys Asp Lys Pro Ile
100 105 110
Tyr Ala Ile Ile Tyr Glu Pro Met Thr Glu Arg Phe Phe Glu Gly Ile
115 120 125
Pro Gly Glu Gly Ala Phe Leu Asn Gly Lys Arg Ile Lys Val Arg Lys
130 135 140
Thr Pro Asp Glu Lys Pro Ser Ile Ser Phe Tyr Ser Arg Gly Lys Gly
145 150 155 160
His Glu Ile Val Lys His Val Lys Arg Thr Arg Thr Leu Gly Ala Ile
165 170 175
Ala Leu Glu Leu Ala Tyr Leu Ala Met Gly Ala Leu Asp Gly Val Val
180 185 190
Asp Val Arg Lys Tyr Val Arg Pro Thr Asp Ile Ala Ala Gly Thr Ile
195 200 205
Ile Ala Lys Glu Ala Gly Ala Leu Ile Lys Asp Ser Ala Gly Lys Asp
210 215 220
Ile Asp Ile Ser Phe Asn Ala Thr Asp Arg Leu Asp Val Ile Ala Val
225 230 235 240
Asn Ser Glu Glu Leu Leu Lys Thr Ile Leu Ser Leu Leu Glu
245 250
<210> 14
<211> 252
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> AFU的氨基酸序列
<400> 14
Met Asp Glu Arg Asp Ala Leu Arg Ile Ser Arg Glu Ile Ala Gly Glu
1 5 10 15
Val Arg Lys Ala Ile Ala Ser Met Pro Leu Arg Glu Arg Val Lys Asp
20 25 30
Val Gly Met Gly Lys Asp Gly Thr Pro Thr Lys Ala Ala Asp Arg Val
35 40 45
Ala Glu Asp Ala Ala Leu Glu Ile Leu Arg Lys Glu Arg Val Thr Val
50 55 60
Val Thr Glu Glu Ser Gly Val Leu Gly Glu Gly Asp Val Phe Val Ala
65 70 75 80
Leu Asp Pro Leu Asp Gly Thr Phe Asn Ala Thr Arg Gly Ile Pro Val
85 90 95
Tyr Ser Val Ser Leu Cys Phe Ser Tyr Ser Asp Lys Leu Lys Asp Ala
100 105 110
Phe Phe Gly Tyr Val Tyr Asn Leu Ala Thr Gly Asp Glu Tyr Tyr Ala
115 120 125
Asp Ser Ser Gly Ala Tyr Arg Asn Gly Glu Arg Ile Glu Val Ser Asp
130 135 140
Ala Glu Glu Leu Tyr Cys Asn Ala Ile Ile Tyr Tyr Pro Asp Arg Lys
145 150 155 160
Phe Pro Phe Lys Arg Met Arg Ile Phe Gly Ser Ala Ala Thr Glu Leu
165 170 175
Cys Phe Phe Ala Asp Gly Ser Phe Asp Cys Phe Leu Asp Ile Arg Pro
180 185 190
Gly Lys Met Leu Arg Ile Tyr Asp Ala Ala Ala Gly Val Phe Ile Ala
195 200 205
Glu Lys Ala Gly Gly Lys Val Thr Glu Leu Asp Gly Glu Ser Leu Gly
210 215 220
Asn Lys Lys Phe Asp Met Gln Glu Arg Leu Asn Ile Val Ala Ala Asn
225 230 235 240
Glu Lys Leu His Pro Lys Leu Leu Glu Leu Ile Lys
245 250
<210> 15
<211> 265
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> AAC的氨基酸序列
<400> 15
Met Asp Lys Ala Trp Leu Asp Gly Leu Ala Glu Val Val Arg Glu Ala
1 5 10 15
Gly Arg Leu Val Glu Glu Ile Ala Arg Gln Gly Phe Asp Thr Gln Phe
20 25 30
Lys His Pro Glu Glu Arg Arg Asp Pro Val Thr Thr Ala Asp Leu Ala
35 40 45
Cys Asp Ala Phe Leu Lys Glu Arg Leu Leu Thr Leu Leu Pro Glu Ala
50 55 60
Gly Trp Leu Ser Glu Glu Thr Lys Asp Arg Pro Asp Arg Leu Glu Lys
65 70 75 80
Arg Trp Val Trp Ile Val Asp Pro Ile Asp Gly Thr Arg Glu Phe Val
85 90 95
Arg Arg Ile Pro Glu Tyr Ala Ile Ser Val Ala Leu Ala Arg Asp Gly
100 105 110
Glu Pro Val Ala Gly Ala Val Val Asn Pro Ala Thr Gly Asp Leu Phe
115 120 125
Leu Gly Ala Val Gly Val Gly Ala Trp Arg Asn Gly Thr Pro Met Val
130 135 140
Cys Ser Arg Ile Arg Gly Glu Arg Leu Thr Ile Leu Gly Ser Arg Ser
145 150 155 160
Glu Met Asn Arg Gly Glu Phe Glu Pro Phe Ala Gly Ile Leu Glu Val
165 170 175
Arg Ala Val Gly Ser Ile Ala Tyr Lys Leu Ala Leu Val Ala Ala Gly
180 185 190
Glu Ala Asp Gly Thr Phe Ser Leu Gly Pro Lys His Glu Trp Asp Ile
195 200 205
Ala Ala Gly Val Ala Leu Val Leu Ala Ala Gly Gly Arg Val His Asp
210 215 220
Gly Ala Gly Arg Pro Phe Arg Phe Asn Gln Pro His Thr Leu Thr Arg
225 230 235 240
Gly Ile Val Ala Ala Thr Arg Glu Ala Tyr Gly Asp Leu Ala Leu Leu
245 250 255
Ile Glu Arg His Ala Pro Arg Arg Ala
260 265
<210> 16
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MSI的氨基酸序列
<400> 16
Met Asp Leu Gln Arg Tyr Leu Glu Ala Ala Leu Asp Ala Ala Tyr Leu
1 5 10 15
Ala Arg Gly Ile His Leu Tyr Tyr Arg Glu Lys Gly Phe Ala Leu Glu
20 25 30
Ser Lys Thr Ser Pro Thr Asp Leu Val Thr Gln Ala Asp Arg Glu Ser
35 40 45
Glu Glu Ala Ile Arg Ser Leu Leu Leu Glu Arg Phe Pro Asp His Val
50 55 60
Val Leu Gly Glu Glu Gly Gly Gln Glu Gly His Ser Asp Phe Arg Trp
65 70 75 80
Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr Ala His Gly Phe Pro
85 90 95
Phe Tyr Ala Val Ser Ile Ala Leu Glu Val Gly Gly Glu Val Val Val
100 105 110
Gly Ala Val Leu Asp Thr Ala Arg Gly Glu Leu Phe Thr Ala Leu Lys
115 120 125
Gly Glu Gly Ala Phe Gln Asn Gly Arg Pro Ile Arg Val Ser Ser Thr
130 135 140
Ala Thr Leu Ile Gly Ser Leu Leu Ala Thr Gly Phe Pro Tyr Asp Val
145 150 155 160
Ala Lys Asp Ala Glu Asn Leu Thr Tyr Phe Gln Arg Ala Leu Ala Lys
165 170 175
Gly Leu Thr Val Arg Arg Pro Gly Ala Ala Ala Leu Asp Leu Ala Tyr
180 185 190
Val Ala Ala Gly Arg Leu Glu Gly Phe Trp Glu Val Lys Leu Asn Pro
195 200 205
Trp Asp Val Ala Ala Gly Trp Leu Leu Ile Thr Glu Ala Gly Gly Thr
210 215 220
Val Ser Gly Ile Gln Gly Glu Pro Tyr Arg Leu Gly Asn Arg Tyr Leu
225 230 235 240
Val Ala Ser Asn Gly Lys Ile His Glu Gln Leu Leu Asp Thr Leu His
245 250 255
Gly Arg
<210> 17
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MRUF的氨基酸序列
<400> 17
Met Asp Leu Arg Pro Tyr Leu Glu Ala Ala Leu Asp Ala Ala Tyr Leu
1 5 10 15
Ala Lys Gly Ile His Gln Tyr Tyr Gln Glu Lys Gly Phe Thr His Ser
20 25 30
Thr Lys Ser Thr Pro Thr Asp Val Val Thr Gln Ala Asp His Glu Ala
35 40 45
Glu Ala Ala Ile Arg Ala Leu Ile Ala Glu Arg Phe Pro Asp His Val
50 55 60
Val Leu Gly Glu Glu Gln Gly Gln Glu Gly Glu Gly Glu Phe Arg Trp
65 70 75 80
Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr Ala His Gly Phe Pro
85 90 95
Phe Tyr Gly Val Ser Ile Gly Leu Glu Val Arg Gly Glu Val Val Leu
100 105 110
Gly Val Val Leu Asp Thr Ala Arg Gly Asp Leu Phe Thr Ala Thr Lys
115 120 125
Gly Gly Gly Ala Tyr Leu Asn Gly Arg Pro Leu Arg Val Ser Gln Arg
130 135 140
Thr Thr Leu Val Gly Ser Leu Leu Ala Thr Gly Phe Pro Tyr Asp Val
145 150 155 160
Ala Arg Asp Pro Glu Asn Leu Thr Tyr Phe Gln Arg Ala Leu Ala Lys
165 170 175
Gly Leu Thr Val Arg Arg Pro Gly Ala Ala Ala Leu Asp Leu Ala Asn
180 185 190
Val Ala Ala Gly Arg Leu Asp Gly Phe Trp Glu Val Lys Leu Asn Pro
195 200 205
Trp Asp Val Ala Ala Gly Trp Leu Leu Ile Thr Glu Ala Gly Gly Gln
210 215 220
Val Thr Gly Phe Gln Gly Glu Ala Tyr Arg Leu Gly Asn Arg Tyr Leu
225 230 235 240
Val Ala Ser Asn Gly Arg Ile His Gln Ala Leu Leu Glu Ala Leu Gln
245 250 255
Gly Arg
<210> 18
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MTA的氨基酸序列
<400> 18
Met Asp Leu Arg Ala Tyr Leu Gln Thr Ala Leu Asp Ala Ala Tyr Leu
1 5 10 15
Ala Lys Gly Ile His Gln Tyr Tyr Gln Glu Lys Gly Phe Thr Gln Ser
20 25 30
Ser Lys Ser Thr Pro Thr Asp Leu Val Thr Gln Ala Asp His Glu Ser
35 40 45
Glu Ala Ala Ile Arg Glu Leu Ile Ala Ala Arg His Pro Asp His Val
50 55 60
Val Leu Gly Glu Glu Gln Gly Gln Asp Lys Glu Gly Ala Phe Arg Trp
65 70 75 80
Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr Ala His Gly Phe Pro
85 90 95
Phe Tyr Ala Val Ser Ile Gly Leu Glu Ala His Gly Glu Val Val Leu
100 105 110
Gly Val Val Leu Asp Thr Ala Arg Gly Glu Leu Phe Thr Ala Thr Lys
115 120 125
Gly Gly Gly Ala Tyr Leu Asn Gly Arg Pro Ile Arg Val Ser Thr Arg
130 135 140
Ser Thr Leu Val Gly Ser Leu Leu Ala Thr Gly Phe Pro Tyr Asp Val
145 150 155 160
Ser Lys Asp Thr Glu Asn Leu Thr Tyr Phe Gln Arg Ala Leu Thr Lys
165 170 175
Gly Leu Met Val Arg Arg Pro Gly Ala Ala Ala Leu Asp Leu Ala Tyr
180 185 190
Val Ala Ala Gly Arg Leu Asp Gly Phe Trp Glu Val Lys Leu Asn Pro
195 200 205
Trp Asp Val Ala Ala Gly Trp Leu Ile Val Ser Glu Ala Gly Gly Arg
210 215 220
Val Ser Gly Leu Gln Gly Glu Asp Tyr Arg Leu Gly Asn Arg Tyr Leu
225 230 235 240
Val Ala Ser Asn Gly Leu Ile His Gln Pro Leu Leu Asp Thr Leu His
245 250 255
Gly Arg
<210> 19
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MCH的氨基酸序列
<400> 19
Met Asp Leu Arg Pro Phe Leu Glu Thr Ala Leu Ala Ala Ala Tyr Val
1 5 10 15
Ala Arg Gly Ile His Gln Leu Tyr Gln Asp Lys Gly Phe Thr Gln Ser
20 25 30
Thr Lys Ser Thr Pro Thr Asp Val Val Thr Gln Ala Asp Lys Glu Ala
35 40 45
Glu Ala Ala Ile Arg Ala Leu Ile Glu Gln Arg His Pro Gly His Val
50 55 60
Val Leu Gly Glu Glu Glu Gly Gln Gln Gly Glu Gly Glu Tyr Arg Trp
65 70 75 80
Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr Ala His Gly Phe Pro
85 90 95
Phe Tyr Cys Val Ser Ile Gly Leu Glu Val Arg Gly Glu Val Met Val
100 105 110
Gly Val Val Leu Asp Thr Ala Arg Gly Asp Leu Phe Thr Ala Thr Arg
115 120 125
Gly Gly Gly Ala Tyr Phe Asn Gly Arg Pro Met Arg Val Ser Arg Ser
130 135 140
Pro Lys Leu Leu Gly Ser Leu Ile Ala Thr Gly Phe Pro Tyr Asp Val
145 150 155 160
Ala Arg Asp Arg Glu Asn Leu Thr Tyr Leu Glu Arg Val Leu Phe Lys
165 170 175
Gly Ile Thr Val Arg Arg Pro Gly Ala Ala Ala Leu Asp Leu Ala Tyr
180 185 190
Val Ala Cys Gly Arg Leu Asp Gly Phe Trp Glu Val Lys Leu Asn Pro
195 200 205
Trp Asp Val Ala Ala Gly Trp Leu Leu Val Glu Glu Ala Gly Gly Arg
210 215 220
Ile Thr Gly Ile His Gly Glu Pro Tyr Arg Met Gly Asn Arg Tyr Leu
225 230 235 240
Met Ala Ser Asn Gly His Ile His Glu Glu Leu Leu Ala Thr Ile His
245 250 255
Gly Arg
<210> 20
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MCE的氨基酸序列
<400> 20
Met Asp Leu Arg Val Tyr Leu Gln Thr Ala Leu Asp Ala Ala Tyr Leu
1 5 10 15
Ala Lys Gly Ile His Gln Tyr Tyr Gln Glu Lys Gly Phe Thr Leu Ser
20 25 30
Ser Lys Ser Thr Pro Thr Asp Leu Val Thr Gln Ala Asp His Glu Ser
35 40 45
Glu Ala Ala Ile Arg Glu Leu Ile Ala Ala Arg His Pro Asp His Val
50 55 60
Val Leu Gly Glu Glu Gln Gly Gln Asp Arg Glu Gly Ala Phe Arg Trp
65 70 75 80
Ile Val Asp Pro Leu Asp Gly Thr Val Asn Tyr Ala His Gly Phe Pro
85 90 95
Phe Tyr Ala Val Ser Ile Gly Leu Glu Ala His Gly Glu Val Val Leu
100 105 110
Gly Val Ile Leu Asp Thr Ala Arg Gly Asp Leu Phe Thr Ala Thr Lys
115 120 125
Gly Gly Gly Ala Phe Met Asn Gly Arg Pro Ile Arg Val Ser Pro Arg
130 135 140
Ala Thr Leu Val Gly Ser Leu Leu Ala Thr Gly Phe Pro Tyr Asp Val
145 150 155 160
Ser Lys Asp Thr Glu Asn Leu Val Tyr Phe Gln Arg Ala Leu Thr Arg
165 170 175
Gly Leu Met Val Arg Arg Pro Gly Ala Ala Ala Leu Asp Leu Ala Tyr
180 185 190
Val Ala Ala Gly Arg Leu Asp Gly Phe Trp Glu Val Lys Leu Asn Pro
195 200 205
Trp Asp Val Ala Ala Gly Trp Leu Ile Ile Arg Glu Ala Gly Gly Thr
210 215 220
Val Thr Gly Met Glu Gly Glu Ala Tyr Arg Leu Gly Asn Arg Tyr Leu
225 230 235 240
Val Ala Ser Asn Gly Leu Ile His Gln Pro Leu Leu Asp Thr Ile His
245 250 255
Gly Arg
<210> 21
<211> 819
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> RMA的核苷酸序列
<400> 21
atgatggatg cctacacgct ttacgaagaa gcccgtgagg tggcggcccg gctggcccgc 60
gatgccggac agatcgcccg ctactatgcc ggtcgggtga ccgtccgcga aaaaggctat 120
aacgagctgg tcacgcaggc cgacgaagaa gtacagcgct tcctgatcga gcagatccac 180
cggcatttcc ccgagcacgc gattctggcc gaggagaacc tgtccgacat gcaggacggc 240
agggaagggg cgtcgtttcg atggatcatc gatccgatcg acggcacgac gaacttcacg 300
cacggtgtgc cgccctatgg catcagtctg gcactccagc atgaaggccg gacggtcgtg 360
ggggtcgtct acgacgtgcc ccacgacgag ctgttcaccg cagtgcgggg cggcgggctg 420
tacgtcaacg gggtgcgcgc ccgggttagc cagaccgaaa cgctccggga ggcgctcatc 480
acgaccggct tcccctaccg ggaagtcgtg catctggaag aatatctgga ggcgctcggg 540
cgtgtgattc gagcgacgcg aggggtgcgt cggccgggcg cggcttcggt cgatctggcc 600
tgggtggcct gcggacgctt cgacggattc ttcgagacgg gcctgagtcc ctgggatgta 660
gcggccggca tcctgctggt cgaagaaggc ggaggacggg tgacggactt tcacgggcgg 720
cccgatccga tctttgcccg ccagatgctg gccacgaacg ggcgcatcca cgaggcgctc 780
tgcgagctgg tcgcgccgct gcaccacgtc tacgcctga 819
<210> 22
<211> 768
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TLE的核苷酸序列
<400> 22
atggacagaa tggatttttc aataaagatc gccagaaaag tcggtctcta cctgatggaa 60
cactggggca atgcagaaaa tgttcggcaa aaaagctctt ttcaggacct ggtaagtgac 120
tgcgacaagc aagcacagaa aatgattgtt cagaagataa aagaccattt tccagatgac 180
gctattttag ccgaggaagg gctattcgaa aaaggagaca gaatgtggat aattgaccca 240
atagatggca caatgaacta cgtccatggt ctaccttctt ttgcgattgg aatcgcctac 300
gttgaaaaag agcaagttat ccttggcgta gcacacgatc cagttctcaa cgagacatac 360
tatgcaatca aagggcaggg tgcgtataaa aatggggaaa gaataaatgt ttcagaaaat 420
tcgcttttga aagactctat tggaaatact ggcttttata cagattttac agggatcttc 480
atcagcgcaa tcgaaaagaa agtcaggcgg gtgagaatga caggcagtgc tatacttgct 540
ggcgcttatg ttgcctgtgg aaggtttgat ttctttatag caaaaagagc caattcttgg 600
gatgtagctc ctttattcgt cctcgtccca gaagctggtg gaatcgtaac ggacctgtcc 660
ggaaaccagg cacatctcaa caccggtaat tttcttttca gtaacggtct attacacgat 720
caggtcttag aagtcataag agaggtgaat aaaaaagtaa gaaaatga 768
<210> 23
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MRUB的核苷酸序列
<400> 23
atggacttgc gtgcctatct gcaaaccgcc ctcgacgccg cctatttagc caaaggcatc 60
caccagtact accaggaaaa aggctttacc cagagcacca agtccacccc caccgatcta 120
gtcacccagg ccgaccacga gtcggaggcg gccatccgag aactcattgc ttcgcgccac 180
cccgaccacg tggtgctggg cgaggaacaa ggccaggaca aagagggggc cttccgctgg 240
attgtagacc ccctggacgg caccgtaaac tacgcccatg gcttcccttt ttatgcggtg 300
agcatcggcc tggaagccca cggcgaggtg gtgctggggg tggttctgga taccgcccgg 360
ggcgagctat ttaccgctac caaggggggc ggggcctacc tgaatggtcg tcccattcgg 420
gtatccaccc gttcaaccct ggtgggcagc cttctagcca ccggattccc ctacgatgtg 480
agcaaggaca ccgaaaacct ggtctacttt cagcgggccc tgaccaaggg gctcatggtg 540
cggcgaccgg gcgcggcagc cctcgacctg gcttatgtgg ccgctgggcg cctggacggc 600
ttctgggaag tgaagctcaa cccctgggat gtggccgcgg gctggctcat cgttagcgag 660
gccggagggc ggataaccgg ccttcaaggg gaggattacc ggctgggcaa ccgctaccta 720
gtggcctcca acggcctgat ccacgggccg ctgctggaca ccatccacgg gcgatag 777
<210> 24
<211> 798
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> DTU的核苷酸序列
<400> 24
atgaagagaa tacttgaagt agctataaaa acaattaaag aatctggtaa tatacttttg 60
aactacatag gcgaggaaaa agaaatagaa cttaaaggaa tttccaattt agtcactcaa 120
gtagataaac tctcagaaag acatatacta aaatccatag aagaaaattt tccggatcat 180
tcaatcttga cagaagaaac aggctttata aataaaaatt ctgaatatac ctggattgta 240
gatcccttag atggaaccac aaattatgcc cataatttcc ctttttttgg aattagtatt 300
gccctcataa aaaataaaga aataatactt ggattaatct atgatcccat aagagacgag 360
ttattttatg ccataaaaaa tgaaggtgca tacctaaatg ataggagaat agaggtatca 420
aaaacagaaa gtcttgaaaa ttctctcatt agttttgcct ttccttacga attaagtctt 480
gaggagaaaa attttattcc ttttataaat ttttcctcta gaactcatgg tataagaagg 540
acaggttcgg cagcaataga aatagcctat gtagggtgtg gaagacttga tggattttgg 600
gcaaagaaac taaaaccatg ggatattagt gcaggcattc taatagtgga agaggcaaaa 660
ggtaaagtta cagattttag tggaaacaat attgatattc atacagacaa tattttgttt 720
tcaaacggta aaatacacca agagatgata aaaatcttaa atttaggtaa aatattcatt 780
agaaatgaaa aattttga 798
<210> 25
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PFE的核苷酸序列
<400> 25
atgctcggcg tcttggaggc cgtggcggtc cgcgcctcgc attttttaat ggagtacttc 60
agagccggca gaggcgtaga cgtcgtgtcg cgtaaagagg acgacgtcac cagggaggtg 120
gacatcgccg cggagacgct catatacaaa atgctcagag aggcctttaa agaaggcggc 180
gtcctatacg cagaggaagg cggcatctac aggtggggag acgagcgcta catcttcgtc 240
ctagacccac tcgacgggtc gctgaactac gccgtgggcg tgcccttctt cgccgtatct 300
atcgcggcag ggaaacatag agagggcacc ctggccgatt tagaatacgc cgtcgtggcg 360
atacccccca cgggagacgt ctacacggcg gcccccggcg tcggcgcccg caaaaacgga 420
aagccgttga ggaggacccc gaggagcaat atagtattcg tagcggtgag caacagcttc 480
cccccaaaga cctgcgaagt cgtgaggcgg ctggggctca gggggaggag cctcggcagc 540
tccgccgccg agctggcgta cacggtggaa ggcatagccc gcggcttcct agacctccgg 600
ggcaagctca gactgctcga cgtggcgggg gcattgacga taggcaaata cgtagacggc 660
ttccggtacg tggtgatggg cgacaccaag ccgcactcca aggtgtctct agtggcgggc 720
gacgtagatt tcgtaaatgc cgcaaccaca gattga 756
<210> 26
<211> 774
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TWI的核苷酸序列
<400> 26
atgaaagatg agaaaggaat tgtagtagat attataaaaa aagcaggaga gattcttcaa 60
gatgggtgga ataaaaaaaa ttttaaaatt tatcggaaag gaacaattaa tttagttaca 120
gaaatagata agaaaattga atttcttata atacaacttt taaaacaata ttttcctgat 180
tatggtattc ttacagaaga aagcaaagaa attaacagca aagcaaacgt gcgatggatt 240
atagatcccc tagatggtac tactaattat ataaaacaat atccgtttgt agccatatca 300
attgctttag agaaagaagg agaattaatt ttgggagttg tttataatcc gattttaaat 360
gaaatgttta tagctcaaaa aggatgtggt gcgacttata atgggaagtc aattcatgtt 420
tctaaaataa aagaattggg atcggcagtt ctagcatcgg gctttcctta tgatgcgtgg 480
gaaaatcctg ataataatgc taaacaatgg aggcaatttc ttactcgtag tttatctctt 540
cgatgcgatg gatcagcagc tctcgatctt tgtagagtag cttgtggaca attggatggt 600
tattgggaaa agggaattag cccgtgggat gttgcagctg gtattgtaat attgcgcgaa 660
gcaggaggaa taattactga ttatttgggt gaggaaaatt tctttaaacg tggagaagtt 720
gttgctgcaa atcctgttct ccacgcacaa atgttgaaag ttttaaataa ttag 774
<210> 27
<211> 795
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TTH的核苷酸序列
<400> 27
atgatcggga ggcgccaccc ctactacccg tacctggagg cggccctgga ggcggcgagc 60
ctcgcccggg gcatccacct ctactacctg gaaaagggct tcaccgaggg gaccaagtcc 120
ggccccaccg acctggtgac ccaggcggac cgggaggcgg aagaggcggt gaagggcctc 180
ctcctctccc gcttccccga ggcggggttt ctgggggagg aagggggaag cgagggcggg 240
aaggccctcc gcttcatcgt ggacccgctg gacgggacgg tgaactacgc ccacggcttc 300
cccttcttcg ccgtatccat cgccctcgag gccgagggcg ccatccagat gggcgtggtg 360
atggacaccg cccgggggga ggtcttctac gccctgaggg gggaaggggc ctacctcaac 420
ggccgcccca tccgggtcac ggggcgggag agcctcgtgg ggagcctcct cgccacgggc 480
ttcccctacg acgtggccaa ggacccggaa aacctcacct actttgaacg ggccctgggc 540
aagggcctgc tcgtccgcag gcctggggcg gcggccctgg acctggccta cgtggccgcg 600
gggcggctgg agggcttctg ggaggtgaag ctgaacccct gggacgtggc ggcggggtgg 660
ctcctcgtgg aagaggcggg aggaagggtg acggacctcg aggggaaccc ctaccgcctg 720
ggcagccgct acatcctcgc caccaacggg cgggtccacg aggccctgcg ccggaccctc 780
ctgggcctgg actga 795
<210> 28
<211> 756
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TLI的核苷酸序列
<400> 28
atgtatgaat ggaatgaaat agccctaaat ctcgcaaagg atattgaaag agaagtaatg 60
cctctctttg gaacaaagaa ggctggagaa ttcatcgggt ttagcccgag cggggacaaa 120
actaaactcg tagataaagt tgcagaagac gttgttctgg agtatctcag accccttggc 180
gtaaacgtag taagtgagga gattgggaac atagaggctg gaagtgaata cactattgtt 240
gtcgacccca tcgatggttc tttcaacttt atccaaggca tccctatctt tggattcagc 300
tttgcagttt tcaaaaatga aaaacccgtc tatgctatga tttacgagtt catcacaaag 360
aacgtttatg aaggaattcc cggtgaggga gcctatctaa acggggagag aatacgagtt 420
aggcatttaa atgaaaagtc catctcaata agcttctaca caagaggtag gggagcgagg 480
cttgttgaaa aagttaagag aacgagggtt ttaggtgcta tagcagttga gcttgcttat 540
ctcgctagag gttctctaga cggtgtaata gacataagga actatgtaag gccgacagac 600
atagcagcag gttacataat cgcaaaagaa gctggagcta ttataaccga cgatagcgga 660
gaggaaataa agtttagatt agatgcaagg gaaaagctca acataatagc ggtaaatgat 720
aagagactcc ttaagctcat acttgaagtt atttaa 756
<210> 29
<211> 795
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> GST的核苷酸序列
<400> 29
atggcagaaa aatgggaaga aatcgaccga tacgcccggc aatggatcga cgaagcagga 60
aagcgcatcc gcgcctcgtt tgccaaacag ctgacggtgg aagcgaaaga gaacccgaac 120
gacttagtga cgaatgttga ccgcgccatt gagcagtttt tcgccgagca catccgtcgc 180
cagttcccta gccaccgcct gttgggggaa gaaggatttg gcgaccggat cgatgccttg 240
gacggcgttg tttgggtgat tgacccgatc gacggcacga tgaatttcgt tcatcagcgg 300
cgccattttg ctgtatcgat cggcattttt gaagatggca tcgggcagct cggctacgtg 360
tatgacgtcg tgtttgacga actgtatgcg gcgcaaaaag ggcggggggt gtttttgaac 420
ggggagccgc tcggcctcct gcagcccgcg ccagtggcgg agtcgatcat cgccatcaac 480
gggacatggc tcatggaaaa caagcgcctc gaccatcgcc cgctcatgag gctggcgaaa 540
gaggcgcgcg gcacgcgctc gtatggttca gcggcgcttg agctcgcgta tgttgccgcc 600
ggccgtttgg acgcctacat ttcgccgcgc ctgtcgccgt gggatttcgc cggcgggatg 660
attttgattg aagaagcggg cggaatggtg acgacccttg acgggaagcc gctggatctg 720
cttggccgca attcggtgct tgccgcgaaa cctggggtgc acgaagaaat tttgcggcgc 780
tatcttcacg attga 795
<210> 30
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ATH的核苷酸序列
<400> 30
atgaccactt cccttcgcga tttgctcgat tttgctgtgg aaacggcgta tctggcagga 60
cgaaccaccc tggcgtattt tcagacgggt gttcaggcgg agttcaaagc cgattcttcc 120
cccgtgactc ttgccgaccg cgccgccgag caattgattc gtcaacgcat tgaaaaacgc 180
ttccctcatc atgccattgt gggtgaggaa tttggtgttc agggaagcgc tgatgctacc 240
caccgctggt tcattgaccc gattgacggc accaaatcct tcctgcgcgg cattcccctg 300
tacgctgtgc tgttgggctt ggagattgaa ggcagggtgc aggtgggagt ggcttattac 360
cccgctatgg acgagatgct gtctgccgcc gatggcgagg gatgctggtg gaacgggcgg 420
cgggcgcgtg tctccacggc gagccgcctg gcagaagcct gggtgaccag caccgacccc 480
tacaatttcc agaaaaccgg caaagatgcc gcctggcagc gcattcaggc ggtgtcttat 540
caccgcggcg gctgggggga tgcctacggc tatctgctgg ttgctactgg gagagccgaa 600
gtgatgctcg atcccatcat gaacgagtgg gattgcgcgc cgttcccgcc catcttccgc 660
gaagccggtg gttttttcgg agactggcag ggtaacgaaa ccatctatgg cggcgaggcg 720
ctggcaacaa ctcaggttct tctccctgag gtgctggagt gtttacattc ttccctttga 780
780
<210> 31
<211> 804
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> SAC的核苷酸序列
<400> 31
atgaaaagag aagatgtaga gaaggtagct agcgaagctt ctaaatatat atatgaagag 60
agagaaaaca aggacgtcga cagagtaatt aatgtacatg gtaatgacgt gactagaatt 120
atagataaga gatctgaaga ctttattgtg gacaggttaa aaagtctagg atataatata 180
ctcatagtta cagaagaatc aggtgtaatt gacagttatg ggaaaaatta cgattacatt 240
gcaattgtag atccactgga tggaagtaca aattttgtat caggaatacc atggtcttca 300
gtatctattg ctatatacaa tagggacgaa gaagatatac tcagttcaaa tgtaggtgct 360
gtaagcagta ttttcactcc ttacacattt tcctacgatg aaggaagcgc ttacgtaaat 420
ggcgtaaaga tagcagagat aaaaaagcct gaaaaaatac tattattagc ctatttctcc 480
aggtcaaagc tacctaatct gaaattattt tttgagaaga ttggtcaggg ttataaaata 540
agaagtttag gaagtgcatc cttagatatg atattagtgt gtacaggcag ggcaacaatg 600
ttttttgata taaggggaaa actgagaaat gtggatattg cagcctcatc taacttctgt 660
tccagattag gggtaattcc atacgacata ggtttaagaa aaattaaaag tagtttgacg 720
gaagtaagtg ttgtaaaaga tttagttata agtcttgacg aaagtcttct aagatctttc 780
tctcttgctt tacagacggt ttaa 804
<210> 32
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TTA的核苷酸序列
<400> 32
atgctagaac tgctaaagga tacagcaaaa aaagcaggac gactgctaaa aataagattc 60
ttggaacttt tgaacggcat ggaaaccgag gtaagagaaa agggaaaaag cgattttgta 120
acaagggtag atgttgaagt ggaaaactac ataaaggaac tgcttgctaa cacaaatata 180
tcggtggtgg gagaagaaag ctttaaaggc gagataccgg gcacctgcat cttcatagac 240
cccatagatg gcacaagaaa cttcatgaga aagaaccccc acttcgccat taaccttgct 300
tatcaggaga agggaaagct tttggctgga gtgacctatg accctatgaa gaatgaaatg 360
ttttctgctg catttagcaa aggtgcattt ttaaacggag agcgcatcta cgcttccaca 420
aacaaggata ttggcaaagc catcatcgct ataggacttc catacagagg aagagaactc 480
atagatattc aaaccaacct ttacaggaac atcttcctaa acggcgctgc tacccgccac 540
acaggctcag cagcccttga tctggcctac atatcctgcg gaagatacga tgctgctatc 600
tatttttatc tttccccctg ggacgtagct ccgggcatac tgctggtaga agaggcaggc 660
ggagaagttg aaggcaccat gggacgagaa cccatccagg ggtggataat agcatctaac 720
aaagtcatcc accccgaagt taaagacatc cttgaaggct ctttaaaagc ggtataa 777
<210> 33
<211> 765
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PFU的核苷酸序列
<400> 33
atgaagctta agttctggag ggaagtagcc attgacataa tctcagattt cgaaactaca 60
ataatgcctt tctttggaaa ccctgatggg ggaaagctcg taaagataag ccccagtgga 120
gatgagacaa agctggttga taagctcgcc gaggatttaa tactttcaag aatcacagag 180
cttggagtca acgttgtgag cgaggaagtt ggtgttattg ataacgagag tgaatacacc 240
gttatagtag acccactaga tggttcatat aacttcattg ctggaattcc tttttttgct 300
ttgagtctag cagtatttaa aaaagataag ccaatatatg caattatcta tgaacctatg 360
acagagagat tctttgaggg aatcccagga gagggagctt ttttaaatgg aaaaagaata 420
aaggtaagaa aaaccccgga cgagaagcca tcaataagct tttattctcg aggtaaaggc 480
catgaaatcg ttaaacatgt aaagagaact agaaccttag gggcaatcgc tctagaattg 540
gcatacctag ctatgggtgc attagatgga gtagttgatg tgaggaaata cgtaaggcca 600
acggacatag ctgctggaac gataattgca aaagaggcag gagcccttat taaggactcc 660
gcgggaaagg atatagatat ttcatttaat gcaactgata ggcttgatgt gatagccgtg 720
aacagcgaag agttgctaaa aacaatttta agcttactgg agtag 765
<210> 34
<211> 759
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> AFU的核苷酸序列
<400> 34
atggatgaaa gggatgcgct gagaatttcg agggagattg caggagaggt cagaaaggcc 60
attgcgtcaa tgcccttgag ggagagagta aaggacgtgg gaatggggaa ggatggcact 120
ccaaccaagg cagccgacag agttgcggaa gatgctgcgc ttgaaatttt gagaaaggag 180
agggttacgg tggttacaga ggagtcggga gttttggggg agggagatgt ctttgttgcc 240
ctcgaccccc tcgatggaac cttcaatgct acgagaggga ttccagttta ttcagtcagc 300
ctctgcttct cctattcgga taaactgaaa gacgccttct ttggctacgt ttacaacctc 360
gcaacagggg atgaatacta cgcggactcc agcggggctt acagaaacgg ggagaggatt 420
gaggtgagcg atgctgagga gctttactgc aacgccataa tctactatcc cgacaggaag 480
tttcccttta agaggatgag gatttttgga agtgctgcaa cggagctttg cttctttgct 540
gacggctcct ttgactgctt cctcgacatc cgccccggaa agatgcttag aatctacgat 600
gccgctgcgg gtgtttttat tgcagaaaag gcaggaggaa aggttaccga acttgatgga 660
gaaagcttgg ggaataaaaa atttgatatg caggaaaggc tcaatatcgt cgccgcaaat 720
gaaaaactcc atccaaagct gctggagctg attaaatga 759
<210> 35
<211> 798
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> AAC的核苷酸序列
<400> 35
atggacaagg catggctcga tggcctggcg gaggtcgtgc gggaggctgg ccgcctggtg 60
gaggagatcg ccaggcaggg cttcgatacg caattcaagc atccggaaga gaggcgagat 120
ccggtcacga cagccgatct cgcctgtgac gcgtttctga aagagcggct gctcacgctg 180
cttccggagg cgggctggct gtccgaggag acgaaggatc ggccggatcg attggagaaa 240
aggtgggtgt ggatcgtcga tcccatcgac ggcacgcggg agttcgtccg ccgcattccc 300
gagtacgcca tctcggtggc gctcgcgcgg gacggcgagc cggtggcggg cgcggtggtg 360
aatcccgcga cgggggatct gtttctcggc gccgtgggcg tcggcgcatg gcggaatgga 420
acgcccatgg tttgttcccg catccgcggc gagcggctca cgattctcgg cagccggtcg 480
gagatgaacc gcggcgagtt cgagccgttt gccggcattc tcgaggtccg ggcggtgggc 540
tccatcgcgt ataagctcgc gctcgtcgcg gcgggcgagg cggatggcac gttcagcctc 600
ggcccgaaac acgagtggga tatcgcggcc ggcgtcgcgc tcgtcctggc ggcgggcggc 660
cgggtacacg atggcgccgg gcgcccgttt cgcttcaacc agccgcacac gctcacccgt 720
ggcatcgtcg cggccacgcg cgaggcgtat ggcgatctcg ccctgctcat cgagcggcac 780
gccccacggc gcgcctag 798
<210> 36
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MSI的核苷酸序列
<400> 36
atggacttgc aacgctactt ggaagccgca ctcgatgctg cgtacttggc tcgaggcatc 60
cacctctact accgcgaaaa aggcttcgcc ctggagagca aaaccagtcc caccgacttg 120
gtcacccaag ccgaccggga gagcgaagaa gctatccgga gtttactgct cgagcgcttc 180
cccgatcatg tggtgctggg tgaagaaggt gggcaggaag gccacagcga ttttcgctgg 240
atcgtggacc cgctcgacgg cacggtcaac tacgcccacg gcttcccctt ctacgcggta 300
agcatcgccc tcgaggtggg gggcgaggtc gtggtggggg cagtgctgga caccgcacgg 360
ggcgagctgt ttaccgccct caaaggagag ggagcattcc aaaatggacg ccccattcgc 420
gtttcgagca ccgcgaccct catcggaagc ctgctggcaa ccggcttccc ctacgatgtg 480
gccaaggacg ccgagaacct gacctacttc cagcgggctc ttgccaaagg ccttaccgtg 540
cgccgccccg gagccgccgc gctggatttg gcttacgtgg cggcagggcg gctggagggt 600
ttctgggagg tcaagctcaa cccctgggac gtggccgcgg gctggctcct catcaccgag 660
gcgggcggaa ccgtgagcgg catccagggc gagccttacc ggctaggaaa ccgctacttg 720
gtagccagca acgggaagat tcatgagcag ctcctcgaca cgctccacgg gcgatga 777
<210> 37
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MRUF的核苷酸序列
<400> 37
atggatctgc gtccctacct cgaggccgct ctcgatgcgg cctacctagc caaaggcatt 60
caccagtact accaggaaaa aggcttcacc cacagcacca aatctacccc taccgatgtg 120
gtcacccagg ccgaccacga ggccgaggca gccatccggg ctcttatcgc cgagcgcttc 180
cccgaccacg tggtgttagg cgaggagcag ggccaggaag gggagggcga gtttcgctgg 240
attgtagacc ccttggatgg cacggtgaac tacgcccacg gctttccctt ctatggggtg 300
agcatcgggc tggaggtacg aggagaggtg gtgttggggg tggtgctgga taccgcccgg 360
ggcgatctct tcaccgctac caaaggcggc ggggcctacc tgaatggccg cccgctgcgg 420
gtttcccagc gaaccacttt ggtgggcagt ctgctggcta ccggttttcc ctacgatgtg 480
gcacgagacc cggagaacct cacctacttc cagcgggctt tggccaaggg gctgaccgtg 540
cggcggcccg gggcagcggc cctcgacctg gccaatgtgg ccgcgggccg gctggacggt 600
ttctgggagg ttaagctcaa cccctgggat gtggctgcgg gctggctcct catcaccgag 660
gcaggcggcc aggtcaccgg gttccagggc gaggcctacc gcctgggtaa ccgctattta 720
gtggcctcca atgggcggat tcaccaggcg ctgctcgagg cccttcaggg ccgatag 777
<210> 38
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MTA的核苷酸序列
<400> 38
atggacttgc gtgcctacct gcaaaccgcc ctcgacgccg cctatttagc caaaggcatc 60
caccagtact accaggaaaa aggctttacc cagagcagca agtccacccc caccgatctg 120
gtcacccagg ccgaccacga gtcggaggcg gccatccgag aactcatcgc cgcgcgccac 180
cccgaccacg tggtgctggg cgaggagcag ggccaggaca aagaaggggc cttccgctgg 240
attgtagacc ccctggacgg caccgtaaac tacgcccatg gttttccttt ttatgcggtg 300
agcattggcc tggaagccca cggcgaggtg gtgctggggg tggttctgga taccgctcga 360
ggcgagctgt ttaccgctac caaagggggt ggggcttacc tgaacggtcg tcccatccgg 420
gtttccactc gttcaaccct ggtgggcagc ctcctggcca ccggatttcc ctacgacgta 480
agcaaggata ccgaaaacct gacctacttc cagcgggcat tgaccaaagg gctcatggta 540
cggcgccccg gcgcggcggc cctcgacctg gcctatgtgg ccgccgggcg cctggacggc 600
ttctgggagg tgaagctgaa cccctgggac gtggccgcgg gctggctcat cgtcagtgag 660
gcgggggggc gggtaagcgg ccttcagggc gaggattacc ggctgggcaa ccgctacctg 720
gtggcctcca acggcctaat tcatcagccc ttgctggaca ccctccacgg gcgatag 777
<210> 39
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MCH的核苷酸序列
<400> 39
atggacctgc gccccttcct cgagaccgcg ctcgccgcgg cttacgtggc tcgaggcatc 60
caccagctct accaggacaa aggcttcacc cagagcacca agtccacccc caccgacgtc 120
gtgacccaag ccgacaagga ggccgaggcc gccatccgcg ccctcatcga gcagcggcac 180
cccggccacg tggtgctcgg cgaggaggag ggccagcagg gcgagggcga gtaccgctgg 240
atcgtggacc cgctcgacgg caccgtgaat tacgcccacg gcttcccctt ctactgcgtg 300
agcatcgggc tcgaggtgcg cggcgaggtg atggtgggcg tggtgctcga caccgcccgc 360
ggcgacctct tcaccgcgac ccgcggcggc ggggcctact tcaacgggcg gcccatgcgg 420
gtctcgcggt cgcccaagct gctgggcagc ctcatcgcca ccggcttccc ctacgacgtc 480
gcccgcgacc gcgaaaacct gacctacctc gagcgcgtcc tcttcaaggg catcaccgtg 540
cgccgccccg gcgccgccgc cctcgacctc gcctacgtgg cctgcgggcg gctcgacggc 600
ttctgggagg tcaagctcaa cccctgggac gtggcggcgg gctggctgct ggtcgaggag 660
gcgggcggca ggatcaccgg catccacggc gagccctacc gcatgggcaa ccgctacctc 720
atggcctcca acggccacat ccacgaggaa ttgctggcga cgatccacgg ccggtag 777
<210> 40
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MCE的核苷酸序列
<400> 40
atggacttgc gtgtatacct gcagaccgcc ctcgatgcag cctatctggc caaaggcatt 60
caccagtact accaggagaa gggttttacc ctaagcagca agtccacccc cacagacctg 120
gttactcagg ccgaccacga gtcggaagcg gccatccgag aactcattgc cgcgcgccac 180
cccgaccacg tggtgctggg agaggagcag ggccaggaca gagaaggggc ttttcgctgg 240
attgtagacc ccctggatgg caccgtaaac tacgcccacg gttttccctt ttatgcggtg 300
agtatcggcc tcgaggccca cggcgaggtg gtgctgggag tgattctcga taccgcccgg 360
ggcgacctat tcaccgctac caaggggggc ggggccttca tgaatggccg ccccatccgg 420
gtttcccctc gagccaccct ggtgggaagc ctgctggcca ccggcttccc ctatgacgtt 480
agcaaagaca ccgaaaacct ggtctacttc cagcgggccc tgaccagagg gcttatggtg 540
cggcgtcccg gcgctgccgc cctcgacctg gcctacgtgg ccgccgggcg tctggatggt 600
ttctgggagg tgaagctcaa cccctgggat gtggccgcag gctggctcat catccgcgag 660
gccgggggta cggttacggg catggagggg gaggcctacc ggctgggcaa ccgctacctg 720
gtagcctcca acggcctgat tcaccagcct ctgctggaca ccatacacgg gcgatag 777
<210> 41
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Rma的正向引物DNA序列
<400> 41
ctccgccata tgatggatgc ctacac 26
<210> 42
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Rma的反向引物DNA序列
<400> 42
gccacctcga gggcgtagac gtggt 25
<210> 43
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Tle的正向引物DNA序列
<400> 43
gtgcaaatca tatggacaga atggattttt caa 33
<210> 44
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Tle的反向引物DNA序列
<400> 44
gatattctcg agttttctta cttttttatt cacc 34
<210> 45
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mrub的正向引物DNA序列
<400> 45
tgaccccata tggacttgcg tgcctat 27
<210> 46
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mrub的反向引物DNA序列
<400> 46
ttctgcctcg agtcgcccgt ggatg 25
<210> 47
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Dtu的正向引物DNA序列
<400> 47
taagctcata tgaagagaat acttgaagta gcta 34
<210> 48
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Dtu的反向引物DNA序列
<400> 48
acaaaagact cgagaaattt ttcatttcta atga 34
<210> 49
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Msi的正向引物DNA序列
<400> 49
atatacatat ggacttgcaa cgctacttgg aagc 34
<210> 50
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Msi的反向引物DNA序列
<400> 50
tggtggtcga ctcgcccgtg gagcgtgtcg a 31
<210> 51
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mruf的正向引物DNA序列
<400> 51
atatacatat ggatctgcgt ccctacctcg aggc 34
<210> 52
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mruf的反向引物DNA序列
<400> 52
tggtggtcga ctcggccctg aagggcctcg 30
<210> 53
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mta的正向引物DNA序列
<400> 53
atatacatat ggacttgcgt gcctacctgc aaac 34
<210> 54
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mta的反向引物DNA序列
<400> 54
tggtggtcga ctcgcccgtg gagggtgtcc a 31
<210> 55
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mch的正向引物DNA序列
<400> 55
atatacatat ggacctgcgc cccttcctcg agac 34
<210> 56
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mch的反向引物DNA序列
<400> 56
tggtggtcga cccggccgtg gatcgtcgcc a 31
<210> 57
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mce的正向引物DNA序列
<400> 57
atatacatat ggacttgcgt gtatacctgc agac 34
<210> 58
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Mce的反向引物DNA序列
<400> 58
tggtggtcga ctcgcccgtg tatggtgtcc a 31
<210> 59
<211> 823
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> CT1的氨基酸序列
<400> 59
Met Leu Lys Lys Leu Pro Glu Asn Leu Glu His Leu Glu Glu Leu Ala
1 5 10 15
Tyr Asn Leu Trp Trp Ser Trp Ser Arg Pro Ala Gln Arg Leu Trp Arg
20 25 30
Lys Ile Asp Pro Glu Gly Trp Glu Glu His Arg Asn Pro Val Lys Ile
35 40 45
Leu Lys Glu Val Ser Asp Glu Arg Leu Glu Glu Leu Ser Lys Asp Asp
50 55 60
Asp Phe Ile Ser Leu Tyr Glu Leu Thr Ile Glu Arg Phe Lys Asp Tyr
65 70 75 80
Met Glu Lys Glu Asp Thr Trp Phe Asn Val Asn Tyr Pro Glu Trp Asp
85 90 95
Glu Lys Ile Val Tyr Met Cys Met Glu Tyr Gly Leu Thr Lys Ala Leu
100 105 110
Pro Ile Tyr Ser Gly Gly Leu Gly Ile Leu Ala Gly Asp His Leu Lys
115 120 125
Ser Ala Ser Asp Leu Gly Leu Pro Leu Ile Ala Ile Gly Leu Leu Tyr
130 135 140
Lys His Gly Tyr Phe Thr Gln Gln Ile Asp Arg Asp Gly Lys Gln Ile
145 150 155 160
Glu Ile Phe Pro Asp Tyr Asn Pro Glu Asp Leu Pro Met Lys Pro Leu
165 170 175
Lys Asp Glu Lys Gly Asn Gln Val Ile Val Glu Val Pro Leu Asp Ser
180 185 190
Thr Val Val Lys Ala Arg Val Phe Glu Val Lys Val Gly Arg Val Ser
195 200 205
Leu Tyr Leu Leu Asp Pro Asp Ile Glu Glu Asn Glu Glu Arg Tyr Arg
210 215 220
Lys Ile Cys Asn Tyr Leu Tyr Asn Pro Glu Pro Asp Val Arg Val Ser
225 230 235 240
Gln Glu Ile Leu Leu Gly Ile Gly Gly Met Lys Leu Leu Arg Ala Leu
245 250 255
Asn Leu Lys Pro Gly Val Ile His Leu Asn Glu Gly His Pro Ala Phe
260 265 270
Ser Ser Leu Glu Arg Ile Lys Asn Tyr Met Glu Glu Gly Tyr Ser Phe
275 280 285
Thr Glu Ala Leu Glu Ile Val Arg Gln Thr Ser Val Phe Thr Thr His
290 295 300
Thr Pro Val Pro Ala Gly His Asp Arg Phe Pro Phe Asp Leu Val Glu
305 310 315 320
Lys Lys Leu Ser Lys Phe Phe Glu Gly Phe Glu Lys Arg Asn Leu Leu
325 330 335
Met Asp Leu Gly Lys Asp Glu Thr Gly Ser Phe Asn Met Thr Tyr Leu
340 345 350
Ala Leu Arg Thr Ser Ser Phe Ile Asn Gly Val Ser Lys Leu His Ala
355 360 365
Glu Val Ser Arg Arg Met Phe Lys Asn Val Trp Gln Gly Val Pro Val
370 375 380
Glu Glu Ile Pro Ile Glu Gly Ile Thr Asn Gly Val His Met Gly Thr
385 390 395 400
Trp Ile Asn Arg Glu Met Arg Lys Leu Tyr Asp Arg Tyr Leu Gly Arg
405 410 415
Val Trp Arg Asp His Thr Asp Leu Glu Gly Ile Trp Tyr Gly Val Asp
420 425 430
Arg Ile Pro Asp Glu Glu Leu Trp Gln Ala His Leu Arg Ala Lys Lys
435 440 445
Arg Phe Ile Glu Tyr Ile Lys Glu Ser Val Arg Arg Arg Asn Glu Arg
450 455 460
Leu Gly Ile Asp Glu Asp Val Pro Asn Ile Asp Glu Asn Ser Leu Ile
465 470 475 480
Ile Gly Phe Ala Arg Arg Phe Ala Thr Tyr Lys Arg Ala Val Leu Leu
485 490 495
Leu Ser Asp Leu Glu Arg Leu Lys Lys Ile Leu Asn Asp Pro Glu Arg
500 505 510
Pro Val Tyr Val Val Tyr Ala Gly Lys Ala His Pro Arg Asp Asp Ala
515 520 525
Gly Lys Glu Phe Leu Lys Arg Ile Tyr Glu Val Ser Gln Met Pro Glu
530 535 540
Phe Lys Asn Arg Ile Ile Val Leu Glu Asn Tyr Asp Ile Gly Met Ala
545 550 555 560
Arg Leu Met Val Ser Gly Val Asp Val Trp Leu Asn Asn Pro Arg Arg
565 570 575
Pro Met Glu Ala Ser Gly Thr Ser Gly Met Lys Ala Ala Ala Asn Gly
580 585 590
Val Leu Asn Ala Ser Val Tyr Asp Gly Trp Trp Val Glu Gly Tyr Asn
595 600 605
Gly Arg Asn Gly Trp Val Ile Gly Asp Glu Ser Val Leu Pro Glu Thr
610 615 620
Glu Val Asp Asp Pro Arg Asp Ala Glu Ala Leu Tyr Asp Leu Leu Glu
625 630 635 640
Asn Glu Ile Ile Pro Thr Tyr Tyr Glu Asn Lys Glu Lys Trp Ile Phe
645 650 655
Met Met Lys Glu Ser Ile Lys Ser Val Ala Pro Arg Phe Ser Thr Thr
660 665 670
Arg Met Leu Lys Glu Tyr Thr Glu Lys Phe Tyr Ile Lys Gly Leu Val
675 680 685
Asn Lys Glu Trp Leu Glu Arg Lys Glu Asn Ala Glu Arg Phe Gly Ala
690 695 700
Trp Lys Glu Arg Ile Leu Arg Asn Trp Ser Ser Val Ser Ile Glu Arg
705 710 715 720
Ile Val Leu Glu Asp Thr Arg Ser Val Glu Val Thr Val Lys Leu Gly
725 730 735
Asp Leu Ser Pro Asp Asp Val Leu Val Glu Leu Leu Ile Gly Arg Gly
740 745 750
Glu Ser Met Glu Asp Leu Glu Ile Trp Lys Val Ile Gln Ile Arg Lys
755 760 765
His Arg Arg Glu Gly Asp Leu Phe Ile Tyr Ser Tyr Val Asn Gly Ala
770 775 780
Leu Gly His Leu Gly Ser Pro Gly Trp Phe Tyr Ala Val Arg Val Leu
785 790 795 800
Pro Tyr His Pro Lys Leu Pro Thr Arg Phe Leu Pro Glu Ile Pro Val
805 810 815
Val Trp Lys Lys Val Leu Gly
820
<210> 60
<211> 2472
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> CT1的核苷酸序列
<400> 60
atgctgaaga aactcccgga gaatctggag catctggaag aactcgccta caacctctgg 60
tggagctggt ctaggcccgc tcagagactc tggagaaaga tagatccgga aggctgggag 120
gaacacagaa accccgttaa aatactgaaa gaagtttctg atgaaaggct cgaagaactt 180
tcaaaagatg atgatttcat atccctctac gaactcacca ttgaaaggtt caaggattac 240
atggagaaag aagacacctg gttcaacgtg aactaccccg aatgggacga gaagatcgtc 300
tacatgtgta tggagtacgg tttgaccaaa gcccttccga tctactccgg tggtcttgga 360
atcctcgcgg gagaccatct caaatccgca agcgatcttg gacttcctct catagcgatc 420
ggacttctct acaaacatgg atatttcacc cagcagatcg acagagatgg aaaacagata 480
gagattttcc ctgattacaa cccagaggac ttacccatga agcccctgaa ggatgaaaag 540
ggaaaccagg tgatcgtgga ggttcctctc gacagtaccg tggtgaaggc acgtgttttt 600
gaagtgaagg taggaagggt gagtctgtac ctgctcgatc cggacatcga ggaaaacgag 660
gaacgataca gaaagatctg caactacctt tacaacccgg aacccgatgt gagggtctcc 720
caggagatac tcctcggaat tgggggaatg aagcttctca gggctctgaa cctgaaacca 780
ggagtcatcc atctgaacga aggacatccg gcgttctctt ccctcgaaag gataaagaac 840
tacatggaag aaggatattc cttcacagag gcccttgaga tcgtgagaca gacgagtgtg 900
tttacaaccc acacacccgt tcccgctgga cacgacagat ttccctttga cctcgtggaa 960
aagaaacttt cgaaattctt cgaaggattc gaaaagagaa atcttctcat ggatcttggg 1020
aaagatgaaa caggcagttt caacatgacg tatcttgccc tgagaacgtc ctctttcata 1080
aacggcgtga gcaaactgca tgcggaagtt tccagaagga tgttcaaaaa cgtgtggcag 1140
ggtgttcccg tggaggaaat accgatcgaa gggataacga acggcgttca catgggaacc 1200
tggatcaacc gtgagatgag aaaactgtac gacagatatc tcggaagggt atggagagat 1260
cacaccgacc ttgagggtat ctggtacggt gttgacagga ttccagatga agaactctgg 1320
caggctcacc tgagggcaaa gaagagattc atcgagtaca taaaagaatc ggtaagaaga 1380
agaaacgaga gactgggaat cgacgaagat gtgccgaaca tcgatgaaaa ttcgctcatc 1440
ataggttttg caagaaggtt tgccacttac aagagggcag ttctcctgct cagcgatctg 1500
gagagactca agaagatcct caacgatcca gaaagacccg tttacgtggt ctatgcgggg 1560
aaggcccatc caagggacga tgcggggaag gaatttttga aacgcatcta cgaagtctcg 1620
cagatgcctg agttcaaaaa caggatcatc gtactggaaa actacgacat tggaatggca 1680
cggctcatgg tgtcgggagt ggatgtgtgg ctgaacaacc cgagaagacc catggaagca 1740
agtggaacaa gcggaatgaa ggcagcagcc aacggagttc ttaacgcgag tgtttacgat 1800
ggatggtggg ttgaagggta caacggcaga aacggctggg tcataggcga tgaaagcgtt 1860
cttccagaga cggaagtgga cgatcccagg gacgcagaag cactctacga tctcctcgaa 1920
aacgaaatca tcccaaccta ctacgaaaac aaagaaaagt ggatcttcat gatgaaagag 1980
agcataaaga gtgttgctcc aagattcagc accaccagaa tgctcaaaga atacacggag 2040
aagttctaca taaagggact tgtgaacaaa gaatggcttg aaagaaaaga aaacgccgaa 2100
aggtttggtg catggaagga aaggatcctc agaaactgga gcagcgtttc catagaaaga 2160
atcgtccttg aggacacaag gagtgttgag gtgacggtga aactgggaga cctttcacct 2220
gatgatgtac tggttgaact tttgattgga agaggagaaa gcatggaaga tctggagatc 2280
tggaaggtga tacagataag aaagcacaga agggaagggg atctgttcat ctacagttat 2340
gtcaacggtg ccctcggtca tcttggctct ccgggatggt tctacgcggt gagggtgcta 2400
ccttatcatc cgaaacttcc caccagattc ttgccggaga tacctgtggt gtggaaaaag 2460
gttctcgggt ga 2472
<210> 61
<211> 441
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> CT2的氨基酸序列
<400> 61
Met Ile Gly Tyr Gln Ile Tyr Val Arg Ser Phe Arg Asp Gly Asn Phe
1 5 10 15
Asp Gly Val Gly Asp Phe Lys Gly Leu Lys Gly Ala Ile Ser Tyr Leu
20 25 30
Lys Glu Leu Gly Val Asp Phe Val Trp Leu Met Pro Val Phe Ser Ser
35 40 45
Ile Ser Phe His Gly Tyr Asp Val Val Asp Phe Tyr Ser Phe Lys Ala
50 55 60
Glu Tyr Gly Asp Glu Lys Asp Phe Arg Glu Met Ile Glu Ala Phe His
65 70 75 80
Asp Asn Gly Ile Lys Val Val Leu Asp Leu Pro Ile His His Thr Gly
85 90 95
Phe Leu His Val Trp Phe Gln Lys Ala Leu Lys Gly Asp Pro His Tyr
100 105 110
Arg Asp Tyr Tyr Val Trp Ala Ser Glu Lys Thr Asp Leu Asp Glu Arg
115 120 125
Arg Glu Trp Asp Asn Glu Arg Ile Trp His Pro Leu Glu Asp Gly Arg
130 135 140
Phe Tyr Arg Gly Leu Phe Gly Pro Leu Ser Pro Asp Leu Asn Tyr Asp
145 150 155 160
Asn Pro Gln Val Phe Glu Glu Met Lys Lys Val Val Tyr His Leu Leu
165 170 175
Glu Met Gly Val Asp Gly Phe Arg Phe Asp Ala Ala Lys His Met Arg
180 185 190
Asp Thr Leu Glu Gln Asn Val Arg Phe Trp Arg Tyr Phe Leu Ser Asp
195 200 205
Ile Glu Gly Ile Phe Leu Ala Glu Ile Trp Ala Glu Ser Lys Val Val
210 215 220
Asp Glu His Gly Arg Ile Phe Gly Tyr Met Leu Asn Phe Asp Thr Ser
225 230 235 240
His Cys Ile Lys Glu Ala Val Trp Lys Glu Asn Phe Lys Val Leu Ile
245 250 255
Glu Ser Ile Glu Arg Ala Leu Val Gly Lys Asp Tyr Leu Pro Val Asn
260 265 270
Phe Thr Ser Asn His Asp Met Ser Arg Leu Ala Ser Phe Glu Gly Gly
275 280 285
Leu Ser Glu Glu Lys Val Lys Leu Ser Leu Ser Ile Leu Phe Thr Leu
290 295 300
Pro Gly Val Pro Leu Ile Phe Tyr Gly Asp Glu Leu Gly Met Lys Gly
305 310 315 320
Ile Tyr Arg Lys Pro Asn Thr Glu Val Val Leu Asp Pro Phe Pro Trp
325 330 335
Ser Glu Asn Met Cys Val Glu Gly Gln Thr Phe Trp Lys Trp Pro Ala
340 345 350
Tyr Asn Asp Pro Phe Ser Gly Val Ser Val Glu Tyr Gln Arg Arg Asn
355 360 365
Arg Asp Ser Ile Leu Ser His Thr Met Arg Trp Ala Gly Phe Arg Gly
370 375 380
Glu Asn His Trp Leu Asp Arg Ala Asn Ile Glu Phe Leu Cys Lys Glu
385 390 395 400
Glu Lys Leu Leu Val Tyr Arg Leu Val Asp Glu Gly Arg Ser Leu Lys
405 410 415
Val Ile His Asn Leu Ser Asn Gly Glu Met Val Phe Glu Gly Val Arg
420 425 430
Val Gln Pro Tyr Ser Thr Glu Val Val
435 440
<210> 62
<211> 1326
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> CT2的核苷酸序列
<400> 62
atgataggct accagatcta cgtgagatca ttcagggatg gaaacttcga tggtgtgggg 60
gatttcaaag gattgaaagg tgcgatttcc tacctgaaag aactgggtgt tgattttgtc 120
tggctcatgc ccgtcttttc ctccatttcc ttccacgggt atgacgtggt ggatttttat 180
tctttcaaag ccgagtacgg agacgagaaa gactttagag agatgatcga ggcgttccac 240
gacaacggta taaaagtcgt tctcgatctt cccatccatc atactggttt cctccatgtg 300
tggtttcaga aagccctgaa aggagatcca cactacaggg attattacgt atgggcgagt 360
gaaaaaacgg atctggacga aagaagagag tgggacaacg aaaggatctg gcatcctctg 420
gaggacggaa ggttctacag aggacttttc ggtcccctct cacccgatct gaactacgat 480
aacccgcagg tttttgaaga gatgaagaag gtggtttatc accttcttga aatgggagtg 540
gacggattca gattcgacgc agcaaagcac atgagagata ctctggaaca gaacgttcgc 600
ttttggaggt atttcctctc cgatattgag ggaatattcc ttgcggaaat ctgggcagaa 660
tccaaagttg tggatgaaca cggcaggata ttcggctaca tgctaaattt cgatacctca 720
cactgtatta aggaagcggt gtggaaggaa aacttcaaag tgttgatcga gtcgatcgaa 780
agggccctgg ttggaaaaga ttatctgccg gtgaacttca catcgaacca tgatatgtca 840
aggcttgcga gtttcgaagg agggttgagt gaagagaagg tgaaactctc actttccatt 900
ctgttcacgc ttcccggggt tcctctcata ttctacggag acgaactggg aatgaaagga 960
atctatcgaa aaccgaacac ggaagtcgtg ctggatccgt tcccctggag cgaaaacatg 1020
tgtgttgaag gccagacatt ttggaaatgg cccgcgtata acgatccatt ctccggtgtt 1080
tctgttgagt atcagaggag aaatcgtgat tcgattctct cacacacgat gaggtgggca 1140
ggattcagag gggaaaatca ctggctggac agggcaaaca tcgaatttct gtgcaaagaa 1200
gaaaaactgc tcgtgtacag actggtcgat gaagggcgtt ctctgaaagt gatacacaac 1260
ctgtcgaatg gtgaaatggt gtttgaggga gtgcgcgtac aaccctacag cacggaggtg 1320
gtttga 1326
<210> 63
<211> 451
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TN1的氨基酸序列
<400> 63
Met Lys Lys Met Ala Leu Lys Phe Asp Phe Ser Asn Leu Phe Glu Pro
1 5 10 15
Asn Ile Ser Gly Gly Leu Arg Glu Glu Asp Leu Glu Ser Thr Lys Glu
20 25 30
Lys Val Ile Glu Ala Ile Lys Asn Phe Thr Glu Asn Thr Pro Asp Phe
35 40 45
Ala Arg Leu Asp Arg Lys Trp Ile Asp Ser Val Lys Glu Leu Glu Glu
50 55 60
Trp Val Val Asn Phe Asp Thr Val Val Val Leu Gly Ile Gly Gly Ser
65 70 75 80
Gly Leu Gly Asn Leu Ala Leu His Tyr Ser Leu Arg Pro Leu Asn Trp
85 90 95
Asn Glu Met Ser Arg Glu Glu Arg Asn Gly Tyr Ala Arg Val Phe Val
100 105 110
Val Asp Asn Val Asp Pro Asp Leu Met Ala Ser Val Leu Asp Arg Ile
115 120 125
Asp Leu Lys Thr Thr Leu Phe Asn Val Ile Ser Lys Ser Gly Ser Thr
130 135 140
Ala Glu Val Met Ala Asn Tyr Ser Ile Ala Arg Gly Ile Leu Glu Ala
145 150 155 160
Asn Gly Leu Asp Pro Lys Glu His Ile Leu Ile Thr Thr Asp Pro Glu
165 170 175
Lys Gly Phe Leu Arg Lys Val Val Lys Glu Glu Gly Phe Arg Ser Leu
180 185 190
Glu Val Pro Pro Gly Val Gly Gly Arg Phe Ser Val Leu Thr Pro Val
195 200 205
Gly Leu Phe Ser Ala Met Ala Glu Gly Ile Asp Ile Glu Glu Leu His
210 215 220
Asp Gly Ala Arg Asp Ala Phe Glu Arg Cys Lys Lys Glu Asp Leu Phe
225 230 235 240
Glu Asn Pro Ala Ala Met Ile Ala Leu Thr His Tyr Leu Tyr Leu Lys
245 250 255
Arg Gly Lys Ser Ile Ser Val Met Met Ala Tyr Ser Asn Arg Met Thr
260 265 270
Tyr Leu Val Asp Trp Tyr Arg Gln Leu Trp Ala Glu Ser Leu Gly Lys
275 280 285
Arg Tyr Asn Leu Lys Gly Glu Glu Val Phe Thr Gly Gln Thr Pro Val
290 295 300
Lys Ala Ile Gly Ala Thr Asp Gln His Ser Gln Ile Gln Leu Tyr Asn
305 310 315 320
Glu Gly Pro Asn Asp Lys Val Ile Thr Phe Leu Arg Leu Glu Asn Phe
325 330 335
Asp Arg Glu Ile Ile Ile Pro Asp Thr Gly Arg Glu Glu Leu Lys Tyr
340 345 350
Leu Ala Arg Lys Arg Leu Ser Glu Leu Leu Leu Ala Glu Gln Thr Gly
355 360 365
Thr Glu Glu Ala Leu Arg Lys Asn Asp Arg Pro Asn Met Lys Val Ile
370 375 380
Phe Asp Arg Leu Thr Ser Tyr Asn Val Gly Gln Phe Phe Ala Tyr Tyr
385 390 395 400
Glu Ala Ala Thr Ala Phe Met Gly Tyr Leu Leu Glu Ile Asn Pro Phe
405 410 415
Asp Gln Pro Gly Val Glu Leu Gly Lys Lys Ile Thr Phe Ala Leu Met
420 425 430
Gly Arg Glu Gly Tyr Glu Tyr Glu Ile Lys Asp Arg Thr Lys Lys Val
435 440 445
Ile Ile Glu
450
<210> 64
<211> 1356
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TN1的核苷酸序列
<400> 64
atgaaaaaga tggctttgaa atttgatttt tcaaatcttt ttgaaccgaa catctccggt 60
ggactgagag aggaagatct ggaaagcaca aaagaaaagg tgatagaggc gataaagaat 120
ttcactgaga acacaccgga ttttgccaga ctggacagaa aatggatcga ttcggtgaag 180
gaactcgagg agtgggtggt gaacttcgac acggtggtcg ttctgggaat tgggggatcc 240
ggtcttggaa accttgccct tcattattcg ttgagaccac tgaactggaa cgagatgtcg 300
agagaggaaa gaaacggtta tgcgagagtc ttcgtggtgg acaacgtaga tcccgatctc 360
atggcctccg tccttgatag gatagatctg aagacaacgc tgttcaacgt gatctcaaaa 420
tctggatcca cggctgaggt tatggcgaat tactcgatcg caaggggaat cctggaggct 480
aatggtctgg acccgaaaga acacatcctc atcacaacag atccagagaa gggctttttg 540
agaaaagtag tgaaagaaga gggcttcaga agtcttgagg tccctcccgg cgttggagga 600
aggttcagcg tgctgacgcc cgttggcctc ttctctgcca tggcggaggg tatcgacata 660
gaagaactcc acgacggtgc ccgggatgcg ttcgagagat gcaagaagga agacctgttc 720
gaaaatccag cggcgatgat cgccctcaca cactatctct atctgaagag aggaaagagc 780
atctccgtca tgatggccta ctccaacagg atgacctacc tcgtggactg gtacagacag 840
ctgtgggcag aaagtctggg aaagagatac aacctgaaag gagaggaggt cttcacgggt 900
cagaccccgg tgaaggcaat aggagccacc gatcagcact ctcagataca gctttacaac 960
gagggcccaa acgacaaagt gataacgttt ttgcggttgg aaaacttcga tagagagatc 1020
ataataccgg acaccggaag agaagagctc aaataccttg caagaaaaag actctctgaa 1080
cttctccttg cagaacagac aggaacagag gaagccctaa ggaaaaacga cagaccgaac 1140
atgaaggtga tcttcgacag actcacctct tacaatgtgg gccagttctt cgcttattat 1200
gaagccgcaa ctgctttcat ggggtatctc ctcgagatca acccgtttga tcagccgggt 1260
gtggaacttg gaaagaagat cacgtttgcc ctcatgggaa gggaaggtta cgaatacgaa 1320
ataaaagatc gcaccaagaa ggtgatcata gaatga 1356
<210> 65
<211> 221
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> FP3E的氨基酸序列
<400> 65
Met Met Val Lys Ile Ala Ala Ser Ile Leu Ala Cys Asp Leu Ala Arg
1 5 10 15
Leu Ala Asp Glu Val Lys Arg Val Glu Glu His Ile Asp Met Val His
20 25 30
Phe Asp Val Met Asp Gly His Phe Val Pro Asn Ile Ser Phe Gly Leu
35 40 45
Pro Val Leu Lys Ala Leu Arg Lys Glu Thr Ser Leu Pro Ile Ser Val
50 55 60
His Leu Met Ile Thr Asn Pro Glu Asp Tyr Val Asp Arg Phe Val Glu
65 70 75 80
Glu Gly Ala Asp Met Val Ala Val His Tyr Glu Thr Thr Pro His Leu
85 90 95
His Arg Ile Val His Arg Ile Lys Asp Leu Gly Ala Lys Ala Phe Val
100 105 110
Ala Leu Asn Pro His Thr Pro Val Phe Leu Leu Ser Glu Ile Ile Thr
115 120 125
Asp Val Asp Gly Val Leu Val Met Ser Val Asn Pro Gly Phe Ser Gly
130 135 140
Gln Arg Phe Ile Ala Arg Ser Leu Glu Lys Ile Arg Ser Leu Lys Lys
145 150 155 160
Met Ile Arg Asp Leu Gly Leu Glu Thr Glu Ile Met Val Asp Gly Gly
165 170 175
Val Asn Glu Glu Asn Ala Ser Ile Leu Ile Lys Asn Gly Ala Thr Ile
180 185 190
Leu Val Met Gly Tyr Gly Ile Phe Lys Asn Glu Asn Tyr Val Glu Leu
195 200 205
Val Arg Ser Ile Lys Gln Glu Arg Gly Glu Ser Ala Gly
210 215 220
<210> 66
<211> 666
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> FP3E的核苷酸序列
<400> 66
atgatggtaa agatcgccgc ttcaatcctt gcgtgtgatc ttgcaagact cgccgatgag 60
gtaaaaaggg tggaagaaca catagacatg gttcacttcg atgtcatgga tggacacttc 120
gttccgaaca tctcgttcgg attgcccgtt ctcaaagccc tgagaaaaga aaccagcctt 180
cctataagtg ttcatctgat gatcacaaat ccagaggact atgtggaccg tttcgtggaa 240
gagggagcgg acatggtggc ggtccactac gagacaacgc cgcaccttca caggatagtg 300
cacaggataa aggatctcgg ggcgaaggcg ttcgtcgccc tcaacccaca cacaccggtt 360
tttctcctgt ctgagatcat aacggatgtg gatggcgtac tcgtgatgag tgtgaacccg 420
ggcttttctg gtcagagatt cattgcaagg agtctggaaa aaataaggag tctgaagaag 480
atgataaggg atctgggact cgaaacggag atcatggtcg atggtggtgt caacgaagaa 540
aacgcttcta tcttaataaa gaacggtgcg acgatccttg taatggggta cggtatcttc 600
aaaaacgaaa actatgtgga actggtgaga tccatcaagc aggaaagagg ggaatctgct 660
ggctga 666
<210> 67
<211> 441
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TN2的氨基酸序列
<400> 67
Met Ile Gly Tyr Gln Ile Tyr Val Arg Ser Phe Arg Asp Gly Asn Phe
1 5 10 15
Asp Gly Val Gly Asp Phe Lys Gly Leu Lys Gly Ala Ile Ser Tyr Leu
20 25 30
Lys Glu Leu Gly Val Asp Phe Val Trp Leu Met Pro Val Phe Ser Ser
35 40 45
Ile Ser Phe His Gly Tyr Asp Val Val Asp Phe Tyr Ser Phe Lys Ala
50 55 60
Glu Tyr Gly Asp Glu Lys Asp Phe Arg Glu Met Ile Glu Ala Phe His
65 70 75 80
Asp Asn Gly Ile Lys Val Val Leu Asp Leu Pro Ile His His Thr Gly
85 90 95
Phe Leu His Val Trp Phe Gln Lys Ala Leu Lys Gly Asp Pro His Tyr
100 105 110
Arg Asp Tyr Tyr Val Trp Ala Ser Glu Lys Thr Asp Leu Asp Glu Arg
115 120 125
Arg Glu Trp Asp Asn Glu Arg Ile Trp His Pro Leu Glu Asp Gly Arg
130 135 140
Phe Tyr Arg Gly Leu Phe Gly Pro Leu Ser Pro Asp Leu Asn Tyr Asp
145 150 155 160
Asn Pro Gln Val Phe Glu Glu Met Lys Lys Val Val Tyr His Leu Leu
165 170 175
Glu Met Gly Val Asp Gly Phe Arg Phe Asp Ala Ala Lys His Met Arg
180 185 190
Asp Thr Leu Glu Gln Asn Val Arg Phe Trp Arg Tyr Phe Leu Ser Asp
195 200 205
Ile Glu Gly Ile Phe Leu Ala Glu Ile Trp Ala Glu Ser Lys Val Val
210 215 220
Asp Glu His Gly Arg Ile Phe Gly Tyr Met Leu Asn Phe Asp Thr Ser
225 230 235 240
His Cys Ile Lys Glu Ala Val Trp Lys Glu Asn Phe Lys Val Leu Ile
245 250 255
Glu Ser Ile Glu Arg Ala Leu Val Gly Lys Asp Tyr Leu Pro Val Asn
260 265 270
Phe Thr Ser Asn His Asp Met Ser Arg Leu Ala Ser Phe Glu Gly Gly
275 280 285
Leu Ser Glu Glu Lys Val Lys Leu Ser Leu Ser Ile Leu Phe Thr Leu
290 295 300
Pro Gly Val Pro Leu Ile Phe Tyr Gly Asp Glu Leu Gly Met Lys Gly
305 310 315 320
Ile Tyr Arg Lys Pro Asn Thr Glu Val Val Leu Asp Pro Phe Pro Trp
325 330 335
Ser Glu Asn Met Cys Val Glu Gly Gln Thr Phe Trp Lys Trp Pro Ala
340 345 350
Tyr Asn Asp Pro Phe Ser Gly Val Ser Val Glu Tyr Gln Arg Arg Asn
355 360 365
Arg Asp Ser Ile Leu Ser His Thr Met Arg Trp Ala Gly Phe Arg Gly
370 375 380
Glu Asn His Trp Leu Asp Arg Ala Asn Ile Glu Phe Leu Cys Lys Glu
385 390 395 400
Glu Lys Leu Leu Val Tyr Arg Leu Val Asp Glu Gly Arg Ser Leu Lys
405 410 415
Val Ile His Asn Leu Ser Asn Gly Glu Met Val Phe Glu Gly Val Arg
420 425 430
Val Gln Pro Tyr Ser Thr Glu Val Val
435 440
<210> 68
<211> 1326
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> TN2的核苷酸序列
<400> 68
atgataggct accagatcta cgtgagatca ttcagggatg gaaacttcga tggtgtgggg 60
gatttcaaag gattgaaagg tgcgatttcc tacctgaaag aactgggtgt tgattttgtc 120
tggctcatgc ccgtcttttc ctccatttcc ttccacgggt atgacgtggt ggatttttat 180
tctttcaaag ccgagtacgg agacgagaaa gactttagag agatgatcga ggcgttccac 240
gacaacggta taaaagtcgt tctcgatctt cccatccatc atactggttt cctccatgtg 300
tggtttcaga aagccctgaa aggagatcca cactacaggg attattacgt atgggcgagt 360
gaaaaaacgg atctggacga aagaagagag tgggacaacg aaaggatctg gcatcctctg 420
gaggacggaa ggttctacag aggacttttc ggtcccctct cacccgatct gaactacgat 480
aacccgcagg tttttgaaga gatgaagaag gtggtttatc accttcttga aatgggagtg 540
gacggattca gattcgacgc agcaaagcac atgagagata ctctggaaca gaacgttcgc 600
ttttggaggt atttcctctc cgatattgag ggaatattcc ttgcggaaat ctgggcagaa 660
tccaaagttg tggatgaaca cggcaggata ttcggctaca tgctaaattt cgatacctca 720
cactgtatta aggaagcggt gtggaaggaa aacttcaaag tgttgatcga gtcgatcgaa 780
agggccctgg ttggaaaaga ttatctgccg gtgaacttca catcgaacca tgatatgtca 840
aggcttgcga gtttcgaagg agggttgagt gaagagaagg tgaaactctc actttccatt 900
ctgttcacgc ttcccggggt tcctctcata ttctacggag acgaactggg aatgaaagga 960
atctatcgaa aaccgaacac ggaagtcgtg ctggatccgt tcccctggag cgaaaacatg 1020
tgtgttgaag gccagacatt ttggaaatgg cccgcgtata acgatccatt ctccggtgtt 1080
tctgttgagt atcagaggag aaatcgtgat tcgattctct cacacacgat gaggtgggca 1140
ggattcagag gggaaaatca ctggctggac agggcaaaca tcgaatttct gtgcaaagaa 1200
gaaaaactgc tcgtgtacag actggtcgat gaagggcgtt ctctgaaagt gatacacaac 1260
ctgtcgaatg gtgaaatggt gtttgaggga gtgcgcgtac aaccctacag cacggaggtg 1320
gtttga 1326
<210> 69
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ct1的正向引物DNA序列
<400> 69
aggagaaact catatgctga agaaactccc ggag 34
<210> 70
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ct1的反向引物DNA序列
<400> 70
agccccctcg agcccgagaa c 21
<210> 71
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ct2的正向引物DNA序列
<400> 71
aaagggcata tgatcctgtt tggaac 26
<210> 72
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ct2的反向引物DNA序列
<400> 72
ataccagtct cgagcagttt caggatc 27
<210> 73
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> tn1的正向引物DNA序列
<400> 73
ttactgaggg catatgaaaa agatggcttt gaaa 34
<210> 74
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> tn1的反向引物DNA序列
<400> 74
aagacgcgtc gacttctatg atcaccttct 30
<210> 75
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> fp3e的正向引物DNA序列
<400> 75
ggaacatatg atggtaaaga tcgccgcttc aatc 34
<210> 76
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> fp3e的反向引物DNA序列
<400> 76
catactcgag cttcccctct cctatct 27
<210> 77
<211> 270
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来源于嗜热异常球菌(Deinococcus geothermalis)的多聚磷酸依赖型葡萄糖激酶
<400> 77
Met Leu Ala Ala Ser Asp Ser Ser Gln His Gly Gly Lys Ala Val Thr
1 5 10 15
Leu Ser Pro Met Ser Val Ile Leu Gly Ile Asp Ile Gly Gly Ser Gly
20 25 30
Ile Lys Gly Ala Pro Val Asp Thr Ala Thr Gly Lys Leu Val Ala Glu
35 40 45
Arg His Arg Ile Pro Thr Pro Glu Gly Ala His Pro Asp Ala Val Lys
50 55 60
Asp Val Val Val Glu Leu Val Arg His Phe Gly His Ala Gly Pro Val
65 70 75 80
Gly Ile Thr Phe Pro Gly Ile Val Gln His Gly His Thr Leu Ser Ala
85 90 95
Ala Asn Val Asp Lys Ala Trp Ile Gly Leu Asp Ala Asp Thr Leu Phe
100 105 110
Thr Glu Ala Thr Gly Arg Asp Val Thr Val Ile Asn Asp Ala Asp Ala
115 120 125
Ala Gly Leu Ala Glu Ala Arg Phe Gly Ala Gly Ala Gly Val Pro Gly
130 135 140
Glu Val Leu Leu Leu Thr Phe Gly Thr Gly Ile Gly Ser Ala Leu Ile
145 150 155 160
Tyr Asn Gly Val Leu Val Pro Asn Thr Glu Phe Gly His Leu Tyr Leu
165 170 175
Lys Gly Asp Lys His Ala Glu Thr Trp Ala Ser Asp Arg Ala Arg Glu
180 185 190
Gln Gly Asp Leu Asn Trp Lys Gln Trp Ala Lys Arg Val Ser Arg Tyr
195 200 205
Leu Gln Tyr Leu Glu Gly Leu Phe Ser Pro Asp Leu Phe Ile Ile Gly
210 215 220
Gly Gly Val Ser Lys Lys Ala Asp Lys Trp Gln Pro His Val Ala Thr
225 230 235 240
Thr Arg Thr Arg Leu Val Pro Ala Ala Leu Gln Asn Glu Ala Gly Ile
245 250 255
Val Gly Ala Ala Met Val Ala Ala Gln Arg Ser Gln Gly Asp
260 265 270
<210> 78
<211> 253
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 来源于嗜热厌氧绳菌(Anaerolinea thermophila)的多聚磷酸依赖型葡萄糖激酶
<400> 78
Met Gly Arg Gln Gly Met Glu Ile Leu Gly Ile Asp Ile Gly Gly Ser
1 5 10 15
Gly Ile Lys Gly Ala Pro Val Asp Val Glu Thr Gly Gln Leu Thr Ala
20 25 30
Glu Arg Tyr Arg Leu Pro Thr Pro Glu Asn Ala Leu Pro Glu Glu Val
35 40 45
Ala Leu Val Val Ala Gln Ile Val Glu His Phe Gln Trp Lys Gly Arg
50 55 60
Val Gly Ala Gly Phe Pro Ala Ala Ile Lys His Gly Val Ala Gln Thr
65 70 75 80
Ala Ala Asn Ile His Pro Thr Trp Ile Gly Leu His Ala Gly Asn Leu
85 90 95
Phe Ser Glu Lys Cys Gly Cys Pro Val Ser Val Leu Asn Asp Ala Asp
100 105 110
Ala Ala Gly Leu Ala Glu Met Ile Phe Gly Ala Gly Lys Gly Gln Lys
115 120 125
Gly Val Val Leu Met Ile Thr Ile Gly Thr Gly Ile Gly Thr Ala Leu
130 135 140
Phe Thr Asp Gly Ile Leu Val Pro Asn Thr Glu Leu Gly His Ile Glu
145 150 155 160
Ile Arg Gly Lys Asp Ala Glu Gln Arg Ser Ser Glu Ala Ala Arg Gln
165 170 175
Arg Lys Asp Trp Thr Trp Gln Gln Trp Ala Lys Arg Leu Asn Glu His
180 185 190
Leu Glu Arg Leu Glu Ala Leu Phe Trp Pro Asp Leu Phe Ile Leu Gly
195 200 205
Gly Gly Ala Val Lys Asn His Glu Lys Phe Phe Pro Tyr Leu Lys Leu
210 215 220
Arg Thr Pro Phe Val Ala Ala Lys Leu Gly Asn Leu Ala Gly Ile Val
225 230 235 240
Gly Ala Ala Trp Tyr Ala His Thr Gln Glu Thr Gln Ala
245 250

Claims (16)

1.阿洛酮糖-6-磷酸磷酸酶用于产生D-阿洛酮糖的用途，其中所述阿洛酮糖-6-磷酸磷酸酶由SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示的氨基酸序列组成。
2.如权利要求1所述的阿洛酮糖-6-磷酸磷酸酶的用途，其中所述阿洛酮糖-6-磷酸磷酸酶分别由SEQ ID NO:21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40所示的核苷酸序列编码。
3.核酸用于产生D-阿洛酮糖的用途，其中所述核酸编码阿洛酮糖-6-磷酸磷酸酶，所述阿洛酮糖-6-磷酸磷酸酶由SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示的氨基酸序列组成。
4.转化子用于产生D-阿洛酮糖的用途，其中所述转化子包含编码阿洛酮糖-6-磷酸磷酸酶的核酸，并且所述阿洛酮糖-6-磷酸磷酸酶由SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示的氨基酸序列组成。
5.用于产生D-阿洛酮糖的组合物，其包含肌醇-单磷酸酶、表达所述酶的微生物或所述微生物的培养物，其中所述肌醇-单磷酸酶由SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示的氨基酸序列组成。
6.如权利要求5所述的组合物，其中所述肌醇-单磷酸酶是阿洛酮糖-6-磷酸磷酸酶。
7.如权利要求6所述的组合物，其还包含：(a)(i)淀粉、麦芽糖糊精、蔗糖或以上的组合；(ii)磷酸，(iii)D-果糖-6-磷酸-3-表异构酶，(iv)D-葡萄糖-6-磷酸-异构酶，(v)葡萄糖磷酸变位酶或葡萄糖激酶，以及(vi)α-葡聚糖磷酸化酶、淀粉磷酸化酶、麦芽糖糊精磷酸化酶、蔗糖磷酸化酶、α-淀粉酶、普鲁兰酶、异淀粉酶、葡糖淀粉酶或蔗糖酶；或者(b)表达所述项(a)中的酶的微生物或所述微生物的培养物。
8.用于产生D-阿洛酮糖的方法，其包括使阿洛酮糖-6-磷酸磷酸酶或肌醇-单磷酸酶、表达所述酶的微生物或所述微生物的培养物与D-阿洛酮糖-6-磷酸接触，以将所述D-阿洛酮糖-6-磷酸转化为D-阿洛酮糖，其中所述阿洛酮糖-6-磷酸磷酸酶或所述肌醇-单磷酸酶由SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示的氨基酸序列组成。
9.如权利要求8所述的方法，其还包括在将D-阿洛酮糖-6-磷酸转化为D-阿洛酮糖之前，使D-果糖-6-磷酸-3-表异构酶、表达所述D-果糖-6-磷酸-3-表异构酶的微生物或所述微生物的培养物与D-果糖-6-磷酸接触，以将所述D-果糖-6-磷酸转化为D-阿洛酮糖-6-磷酸。
10.如权利要求8所述的方法，其还包括在将D-果糖-6-磷酸转化为D-阿洛酮糖-6-磷酸之前，使D-葡萄糖-6-磷酸-异构酶、表达所述D-葡萄糖-6-磷酸-异构酶的微生物或所述微生物的培养物与D-葡萄糖-6-磷酸接触，以将所述D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸。
11.如权利要求10所述的方法，其还包括在将D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸之前，使葡萄糖磷酸变位酶、表达所述葡萄糖磷酸变位酶的微生物或所述微生物的培养物与D-葡萄糖-1-磷酸接触，以将所述D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸。
12.如权利要求11所述的方法，其还包括在将D-葡萄糖-6-磷酸转化为D-果糖-6-磷酸之前，使葡萄糖激酶、表达所述葡萄糖激酶的微生物或所述微生物的培养物和磷酸与葡萄糖接触，以将所述葡萄糖转化为D-葡萄糖-6-磷酸。
13.如权利要求12所述的方法，其还包括在将D-葡萄糖-1-磷酸转化为D-葡萄糖-6-磷酸之前，使α-葡聚糖磷酸化酶、淀粉磷酸化酶、麦芽糖糊精磷酸化酶或蔗糖磷酸化酶，表达所述磷酸化酶的微生物或所述微生物的培养物和磷酸与淀粉、麦芽糖糊精、蔗糖或以上的组合接触，以将所述淀粉、麦芽糖糊精、蔗糖或以上的组合转化为D-葡萄糖-1-磷酸。
14.如权利要求12所述的方法，其还包括在将葡萄糖转化为D-葡萄糖-6-磷酸之前，使α-淀粉酶、普鲁兰酶、葡糖淀粉酶、蔗糖酶或异淀粉酶，表达所述α-淀粉酶、普鲁兰酶、葡糖淀粉酶、蔗糖酶或异淀粉酶的微生物或者所述微生物的培养物与淀粉、麦芽糖糊精、蔗糖或以上的组合接触，以将所述淀粉、麦芽糖糊精、蔗糖或以上的组合转化为葡萄糖。
15.用于产生D-阿洛酮糖的方法，其包括使(a)阿洛酮糖-6-磷酸磷酸酶；D-果糖-6-磷酸-3-表异构酶；D-葡萄糖-6-磷酸-异构酶；葡萄糖磷酸变位酶或葡萄糖激酶；以及α-葡聚糖磷酸化酶、淀粉磷酸化酶、麦芽糖糊精磷酸化酶、蔗糖磷酸化酶、α-淀粉酶、普鲁兰酶、异淀粉酶、葡糖淀粉酶或蔗糖酶，或者使(b)表达(a)中的酶的微生物或所述微生物的培养物与淀粉、麦芽糖糊精、蔗糖或以上的组合以及磷酸接触，
其中所述阿洛酮糖-6-磷酸磷酸酶由SEQ ID NO:1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19或20所示的氨基酸序列组成。
16.如权利要求8-15中任一项所述的方法，其中接触反应在5.0-9.0的pH下、在40℃-80℃的温度下进行，和/或进行2小时至24小时。
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