CN115710594A

CN115710594A - 一种合成尿苷二磷酸-6-叠氮-d-半乳糖的方法

Info

Publication number: CN115710594A
Application number: CN202110968981.9A
Authority: CN
Inventors: 文留青; 罗雅文
Original assignee: Shanghai Institute of Materia Medica of CAS
Current assignee: Shanghai Institute of Materia Medica of CAS
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2023-02-24
Anticipated expiration: 2041-08-23

Abstract

本发明提供了一种合成尿苷二磷酸‑6‑叠氮‑D‑半乳糖的方法。所述方法包括：6‑叠氮‑半乳糖在半乳糖激酶的作用下转变为6‑叠氮‑半乳糖‑1‑磷酸；6‑叠氮‑半乳糖‑1‑磷酸在糖焦磷酸化酶的作用下转变为尿苷二磷酸‑6‑叠氮‑D‑半乳糖。本发明利用双歧杆菌来源的半乳糖激酶(BiGalK)和拟南芥来源的糖焦磷酸化酶(AtUSP)建立了一种合成尿苷二磷酸‑6‑叠氮‑D‑半乳糖的方法，此方法较已报道的化学法更为简单、易操作，且比已报道的化学酶法更为高效，可以用于大量合成尿苷二磷酸‑6‑叠氮‑D‑半乳糖。

Description

一种合成尿苷二磷酸-6-叠氮-D-半乳糖的方法

技术领域

本发明属于尿苷二磷酸-6-叠氮-D-半乳糖的合成技术领域，具体地，涉及一种化学酶法合成尿苷二磷酸-6-叠氮-D-半乳糖的方法。

背景技术

非天然糖供体常被用于蛋白糖基化修饰的化学酶法标记。利用糖基转移酶将带有生物正交基团的核苷酸糖即糖供体转移到特异的糖基化位点，然后与带有荧光基团或生物素的生物正交基团发生反应，从而实现糖基化位点的检测和定位。叠氮和炔烃基团具有稳定性好，体积小(对天然聚糖结构的影响很小)，不参与生物反应等特点，因此叠氮-炔烃环加成反应成为最常见的化学酶法标记糖基化的反应之一。

尿苷二磷酸-6-叠氮-D-半乳糖(UDP-6-叠氮-D-半乳糖，UDP- 6-N₃-D-galactose)是一种已报道的非天然糖供体。文献表明牛源半乳糖基转移酶(GalT)能将它连接至4-甲基琥珀酸苯甲基N-乙酰乙基葡萄糖胺 (MU-GlcNAc)上。此外，尿苷二磷酸-6-叠氮-D-半乳糖能被来源于脑膜炎奈瑟菌(Neisseria meniningitidis)的β1,4-半乳糖基转移酶(NmLgtB)转移至中国仓鼠卵巢细胞Lec8表面N-糖基化的末端的N-乙酰葡萄糖胺残基上，然后与带有生物素的炔基结合，实现N-乙酰葡萄胺糖基化的标记。这表明尿苷二磷酸-6-叠氮-D-半乳糖在N-乙酰葡萄糖胺糖基化标记中具有一定的应用前景。

抗体药物偶联物(antibody-drugconjugate，ADC)是采用特定的连接子将抗体和小分子细胞毒药物连接起来，其主要组成成分包括抗体、连接子和小分子药物。抗体分子发挥靶向投递作用，小分子药物发挥效应。抗体的N-糖基化发生在抗体Fc片段的Asn297残基上，在抗体与靶标结合位点的远端，不会影响抗体的靶标识别作用。因此抗体的糖基化位点是理想的抗体与药物偶联的部位。含有生物正交基团的非天然糖具有成为ADC药物连接子的潜力。

此外，尿苷二磷酸-6-叠氮-D-半乳糖是一种含有生物正交基团的非天然糖供体，可以用于构建非天然糖供体糖库。尿苷二磷酸-6-叠氮-D-半乳糖有α和β两种构型，其中α构型的结构式如下：

目前，尿苷二磷酸-6-叠氮-D-半乳糖的合成方法有化学合成法和化学酶合成法。化学法和化学酶法都利用化学方法合成UDP-6-叠氮-D-半乳糖的前体6-叠氮-半乳糖(化合物6)，其合成过程如以下反应式1所示(Srivastava, G.；Kaur,K.J.；Hindsgaul,O.；Palcic,M.M.,Enzymatic transfer of a preassembled trisaccharide antigen tocell surfaces using a fucosyltransferase.J Biol Chem 1992,267(31),22356-61.)。

反应式1.化学方法合成6-叠氮-半乳糖(6-N₃-半乳糖)(a)浓H₂SO₄,CuSO₄,丙酮,76％；(b) (CF₃SO₂)₂O,吡啶,DCM；(c)NaN₃,DMF；(d)80％TFA.

已报道的化学法有两种，合成过程见如下反应式2(Bosco,M.；Gall,S. L.；Rihouey,C.；Couve-Bonnaire,S.；Bardor,M.；Lerouge,P.；Pannecoucke,X., 6-Azido d-galactose transfer to N-acetyl-d-glucosamine derivative using commerciallyavailableβ-1,4-galactosyltransferase.Tetrahedron Letters 2008, 49(14),2294-2297.)。两种化学方法产率不高，不适用于大量合成。其中法1生成的产物为α和β两种立体异构体的混合物，难以分离。

法1：

法2：

反应式2.化学法合成尿苷二磷酸-6-叠氮-D-半乳糖(e)TMSCl,吡啶,0℃；(f)TMSI,CH₂Cl₂,0℃； (g)(Bu₄N)₂–UDP；(h)(i)Bu₄NF,H₂O；(ii)碱性磷酸酶(alkalinephosphatase)；(iii)HPLC C18,2.5％(4 steps)；(i)AA,三氟化硼-乙醚络合物(boron trifluoroetherate),0℃；(j)CH₃COONH₄,DIEA,DMF, 85％；(k)(i)2-氯-4H-苯并二氧磷-4-酮(2-chloro-4H-benzodioxaphosphorin-4-one，8),Et₃N,二氧六环(dioxane),THF,0℃；(ii)H₂O,74％(2步)；(l)t-BuOOH(2当量),I₂ cat.,THF,74％；(m)10,1H-四唑(1H-tetrazole),吡啶,46％；(n)(i)Et₃N,NH₄HCO₃,CH₃OH；(ii)HPLC C18,53％.

另一种则是化学酶法，以通过化学法合成的6-叠氮-半乳糖(化合物6) 为底物，通过酶催化反应合成尿苷二磷酸-6-叠氮-D-半乳糖。文献(Zou,Y.； Xue,M.；Wang,W.；Cai,L.；Chen,L.；Liu,J.；Wang,P.G.；Shen,J.； Chen,M.,One-pot three-enzyme synthesisof UDP-Glc,UDP-Gal,and their derivatives.Carbohydr Res 2013,373,76-81.)利用一锅三酶法合成尿苷二磷酸-6-叠氮-D-半乳糖，三种酶分别为肺炎链球菌来源的半乳糖激酶(SpGalK)、肺炎链球菌来源的尿苷二磷酸-葡萄糖焦磷酸化酶(SpGalU)和无机焦磷酸酶(PPA)(以下反应式3)，产率较低。

法3：

反应式3.已报道的化学酶法合成尿苷二磷酸-6-叠氮-D-半乳糖。SpGalK：肺炎链球菌来源的半乳糖激酶；SpGalU：肺炎链球菌来源的尿苷二磷酸-葡萄糖焦磷酸化酶；PPA:无机焦磷酸酶。

发明内容

针对现有技术的不足，本发明提供了一种更为高效的大量制备尿苷二磷酸-6-叠氮-D-半乳糖的合成方法。此方法相较于化学法更为简单、易操作，且比已报道的化学酶法更为高效，可以用于大量合成尿苷二磷酸-6-叠氮-D- 半乳糖。

因此，一方面，本发明提供了一种合成尿苷二磷酸-6-叠氮-D-半乳糖的方法，包括以下步骤：

S1，在双歧杆菌来源的半乳糖激酶的作用下，6-叠氮-半乳糖(化合物 6)转变为6-叠氮-半乳糖-1-磷酸(化合物12)，其反应式如下所示：

优选地，其中，所述半乳糖激酶选自：长双歧杆菌(Bifidobacterium longum)来源的半乳糖激酶(BiGalK)或在BiGalK的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有BiGalK活性的由BiGalK衍生的蛋白质，齿双歧杆菌(Bifidobacterium dentium)来源的半乳糖激酶，链状双歧杆菌(Bifidobacterium catenulatum)来源的半乳糖激酶，小鸡双歧杆菌 (Bifidobacterium pullorum)来源的半乳糖激酶，动物双歧杆菌(Bifidobacterium animalis)来源的半乳糖激酶，两歧双歧杆菌 (Bifidobacteriumbifidum)来源的半乳糖激酶，星状双歧杆菌 (Bifidobacterium asteroides)来源的半乳糖激酶，短双歧杆菌(Bifidobacterium breve)来源的半乳糖激酶，假长双歧杆菌(Bifidobacterium pseudolongum)来源的半乳糖激酶，假链状双歧杆菌(Bifidobacteriumpseudocatenulatum)来源的半乳糖激酶；

S2，在拟南芥(Arabidopsis thaliana)来源的糖焦磷酸化酶(AtUSP) 或在AtUSP的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有AtUSP活性的由AtUSP衍生的蛋白质的作用下，6-叠氮-半乳糖-1-磷酸 (化合物12)转变为尿苷二磷酸-6-叠氮-D-半乳糖，其反应式如下所示：

进一步地，本发明方法步骤S1中，BiGalK能够将6-叠氮-半乳糖转变为6-叠氮-半乳糖-1-磷酸(化合物12)，其氨基酸序列可以如SEQ ID NO:1 所示。BiGalK可被Bifidobacterium系列同源序列替代。齿双歧杆菌 (Bifidobacterium dentium)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:4所示。链状双歧杆菌(Bifidobacteriumcatenulatum)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:5所示。小鸡双歧杆菌(Bifidobacterium pullorum)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:6所示。动物双歧杆菌(Bifidobacterium animalis)来源的半乳糖激酶的氨基酸序列可以如SEQID NO:7所示。两歧双歧杆菌(Bifidobacterium bifidum)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:8所示。星状双歧杆菌 (Bifidobacterium asteroides)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:9所示。短双歧杆菌(Bifidobacterium breve)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:10所示。假长双歧杆菌(Bifidobacteriumpseudolongum)来源的半乳糖激酶的氨基酸序列可以如SEQ ID NO:11所示。假链状双歧杆菌(Bifidobacterium pseudocatenulatum)来源的半乳糖激酶的氨基酸序列可以如SEQ IDNO:12所示。

进一步地，优选地，编码所述BiGalK的核苷酸序列如SEQ ID NO:13 所示。

进一步地，本发明方法步骤S1中，所述反应在三磷酸腺苷(ATP)和金属Mg²⁺存在条件下进行。

进一步地，本发明方法步骤S1中，所述反应体系的pH值为6至9，例如6.5、7.0、7.2、7.5、7.8、8.0、8.5等，优选为约7.5。

进一步地，本发明方法步骤S2中，AtUSP能够将6-叠氮-半乳糖-1-磷酸(化合物12)转变为尿苷二磷酸-6-叠氮-D-半乳糖，其氨基酸序列可以如 SEQ ID NO:2所示。

进一步地，优选地，编码所述AtUSP的核苷酸序列如SEQ ID NO:14 所示。

进一步地，本发明方法步骤S2中，所述反应在三磷酸尿苷(UTP)和金属Mg²⁺存在条件下进行。

进一步地，本发明方法步骤S2中，所述反应体系的pH值为6至9，例如6.5、7.0、7.2、7.5、7.8、8.0、8.5等，优选为约7.5。

进一步地，本发明方法步骤S2中，可以使用无机焦磷酸化酶(PPA) 或在PPA的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有 PPA活性的由PPA衍生的蛋白质，以催化副产物焦磷酸盐(PPi)分解，从而避免因PPi的积累可能会抑制AtUSP的酶活性，以推动反应进行。PPA的氨基酸序列如SEQ ID NO:3所示。

进一步地，优选地，所述无机焦磷酸化酶(PPA)可以是大肠杆菌 (Escherichiacoli)O157来源的无机焦磷酸化酶(PPA)。

进一步地，优选地，编码所述PPA的核苷酸序列如SEQ ID NO:15所示。

进一步地，本发明方法步骤S2中，得到的产物尿苷二磷酸-6-叠氮-D- 半乳糖(化合物1)为α构型。

优选地，本发明方法步骤S1、S2可以分开进行或者按照一锅法在同一反应体系中进行。

在一些实施方式中，本发明的方法在同一反应体系中进行，此时可以将6-叠氮-半乳糖(化合物6)、三磷酸腺苷二钠(ATP·2Na⁺)、三磷酸尿苷二钠(UTP·2Na⁺)作为底物，在金属Mg²⁺存在下，在步骤S1中所述的双歧杆菌来源的的半乳糖激酶和步骤S2中所述的拟南芥(Arabidopsis thaliana)来源的糖焦磷酸化酶(AtUSP)或在AtUSP的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有AtUSP活性的由AtUSP衍生的蛋白质、和任选的无机焦磷酸化酶(PPA)或在PPA的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有PPA活性的由PPA衍生的蛋白质的作用下，催化合成尿苷二磷酸-6-D-叠氮-半乳糖，其反应式如下：

本发明合成方法中，可以进一步包括分离纯化步骤。具体地，所述纯化步骤包括：在上述催化合成反应结束后，加入乙醇，固液分离(例如， 10000g，离心10分钟)，液体减压脱除溶剂(优选温度低于35℃)，然后用去离子水溶解，P-2凝胶柱分离，TLC检测分离效果；合并包含产物的组分，减压脱除溶剂，用去离子水溶解后，QS-FF离子交换柱分离，去除杂质(例如，腺苷单磷酸(AMP)等)；将含有产物的组分合并，减压脱除溶剂(优选温度低于35℃)，然后用去离子水溶解，P-2凝胶柱分离收集含有产物的组分，优选经浓缩冻干，得到目标产物。

在上文中已经详细地描述了本发明，但是上述实施方式本质上仅是例示性，且并不欲限制本发明。此外，本文并不受前述现有技术或发明内容或以下实施例中所描述的任何理论的限制。

除非另有明确说明，在整个申请文件中的数值范围包括其中的任何子范围和以其中给定值的最小子单位递增的任何数值。除非另有明确说明，在整个申请文件中的数值表示对包括与给定值的微小偏差以及具有大约所提及的值以及具有所提及的精确值的实施方案的范围的近似度量或限制。除了在详细描述最后提供的工作实施例之外，本申请文件(包括所附权利要求)中的参数(例如，数量或条件)的所有数值在所有情况下都应被理解为被术语“大约”修饰，不管“大约”是否实际出现在该数值之前。“大约”表示所述的数值允许稍微不精确(在该值上有一些接近精确；大约或合理地接近该值；近似)。如果“大约”提供的不精确性在本领域中没有以这个普通含义来理解，则本文所用的“大约”至少表示可以通过测量和使用这些参数的普通方法产生的变化。例如，“大约”可以包括小于或等于10％，小于或等于5％，小于或等于4％，小于或等于3％，小于或等于2％，小于或等于1％或者小于或等于0.5％的变化。

附图说明

图1为显示尿苷二磷酸-6-叠氮-D-半乳糖磁共振¹H谱图。

图2为显示尿苷二磷酸-6-叠氮-D-半乳糖核磁共振¹³C谱图。

具体实施方式

在下文中，将通过实施例详细描述本发明。然而，在此提供的实施例仅用于说明目的，并不用于限制本发明。

下述实施例所使用的实验方法如无特殊说明，均为常规方法。

下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。

表1主要实验仪器

表2主要实验试剂

表3茴香醛染色液配方

制备实施例

制备实施例1：BiGalK的制备

依据BiGalK的蛋白序列人工优化并合成基因序列SEQ ID NO:13。目的基因与载体pET-30a经NdeI与XhoI限制性内切酶双酶切后进行连接。连接产物转化至大肠杆菌DH5α感受态细胞，涂布在卡那抗性平板上，37℃培养箱过夜培养。平板挑点至5mL LB培养基，37℃，180rmp摇床过夜培养后提取质粒。酶切和测序对质粒进行鉴定。经鉴定正确的质粒转化至BL21(DE3)感受态细胞，平板挑点至50mL LB培养基，28℃，180rmp 摇床过夜培养。将50mL培养基转入1.8L LB培养基，37℃，200rmp摇床培养6h，降温至16℃，加入异丙基-β-D-硫代半乳糖苷(IPTG，终浓度为0.5mM)诱导过夜。离心收集菌体，用10mM咪唑溶液(10mM咪唑、 50mMTris-HCl、300mM氯化钠；pH7.5)重悬细胞。破碎细胞后，离心将上清过镍柱，用10mM咪唑溶液洗去杂蛋白，再用300mM咪唑溶液 (300mM咪唑、50mM Tris-HCl、300mM氯化钠；pH7.5)洗脱目的蛋白，得到酶溶液，其浓度约为1mg/mL。

制备实施例2：AtUSP的制备

依据AtUSP的蛋白序列人工优化并合成基因序列SEQ ID NO:14。目的基因与载体pET28a经NdeI与XhoI限制性内切酶双酶切后进行连接。连接产物转化至大肠杆菌DH5α感受态细胞，涂布在卡那抗性平板上，37℃培养箱过夜培养。平板挑点至5mL LB培养基，37℃，180rmp摇床过夜培养后提取质粒。酶切和测序对质粒进行鉴定。经鉴定正确的质粒转化至BL21(DE3)感受态细胞，平板挑点至50mL LB培养基，28℃，180rmp 摇床过夜培养。将50mLLB培养基转入1.8L LB培养基，37℃，200rmp 摇床培养6h，降温至16℃，加入IPTG诱导过夜。离心收集菌体，用10mM 咪唑溶液重悬细胞。破碎细胞后，离心将上清过镍柱，用10mM咪唑溶液洗去杂蛋白，再用300mM咪唑溶液洗脱目的蛋白，得到酶溶液，其浓度约为1mg/mL。

制备实施例3：PPA的制备

以E.coli O157为模板，进行PCR获得目的基因SED ID NO:15。目的基因与载体pET28a经NdeI与HindIII限制性内切酶双酶切后进行连接，连接产物转化至大肠杆菌DH5α感受态细胞，涂布在卡那抗性平板上，37℃培养箱过夜培养。平板挑点至5mL LB培养基，37℃，180rmp摇床过夜培养后提取质粒。酶切和测序对质粒进行鉴定。经鉴定正确的质粒转化至 BL21(DE3)感受态细胞，涂布在卡那抗性平板上，37℃培养箱过夜培养。平板挑点至50mLLB培养基，28℃，180rmp摇床过夜培养。将50mL培养基转入1.8L LB培养基，37℃，200rmp摇床培养6h，降温至16℃，加入IPTG诱导过夜。离心收集菌体，用10mM咪唑溶液重悬细胞。破碎细胞后，离心将上清过镍柱，用10mM咪唑溶液洗去杂蛋白，再用300mM 咪唑溶液洗脱目的蛋白，得到酶溶液，其浓度约为1mg/mL。

实施例

实施例1：尿苷二磷酸-6-叠氮-D-半乳糖合成

按下表4所示的反应体系加入6-叠氮-半乳糖(4.0g，12.2mmol)， ATP·2Na⁺(7.0g，12.7mmol)，UTP·2Na⁺(7.0g，13.3mmol)，1M Tris-HCl (pH＝7.5)30mL，200mM MgCl₂30mL于2000mL试剂瓶中，加适量ddH₂O 溶解，用1M NaOH将溶液pH值调至7.5左右，加入BiGalK(10mL)， AtUSP(50mL)，PPA(100μL)。最后补加ddH₂O至600mL，混匀，在37℃水浴锅内进行反应。TLC监测反应，展开剂为乙酸乙酯：甲醇：水＝5：3： 2，茴香醛显色液显色，尿苷二磷酸-6-叠氮-D-半乳糖R_f值为0.62。展开剂为异丙醇：氨水：水＝7：3：2，茴香醛显色液显色，尿苷二磷酸-6-叠氮-D- 半乳糖R_f值为0.40。反应结束后，加入600mL乙醇，摇匀后出现白色沉淀，离心(10000g，10分钟)取上清，减压脱出溶剂(温度低于35℃)，得到目标产物粗品。

表4合成UDP-6-N₃-D-半乳糖的酶反应体系(600mL体系为例)

将得到的目标产物粗品加入80mL去离子水溶解，P-2凝胶柱分离，去离子水洗脱产物。TLC检测分离效果。合并包含产物的组分，减压脱出溶剂(温度低于35℃)，加50mL去离子水溶解后，QS-FF离子交换柱分离，NaCl梯度洗脱(0-300mM)，去除腺苷单磷酸(AMP)等杂质。将含有产物的组分合并，减压脱出溶剂(温度低于35℃)，加70mL去离子水溶解，P-2凝胶柱除盐，收集含有产物的组分，浓缩冻干得到目标产物，白色固体5.8g，产率为75％(底物(6-叠氮-半乳糖)投料12.2mmol，若完全反应生成尿苷二磷酸-6-叠氮-D-半乳糖的钠盐(M＝635.28)质量为7.747g，实施例得目标产物白色固体5.8g，产率为5.8/7.747＝75％)，核磁验证其结构(见图1，图2)。

¹H NMR(600MHz,D₂O)δ7.95(d,J＝8.2Hz,1H),6.00-5.94(m,2H), 5.61(dd,J＝7.2,3.6Hz,1H),4.39–4.32(m,2H),4.30-4.16(m,4H),3.98(d,J ＝2.4Hz,1H),3.90(dd,J＝10.3,3.3Hz,1H),3.78(dt,J＝10.3,3.2Hz,1H), 3.55(dd,J＝12.7,7.3Hz,1H),3.45(dd,J＝12.8,6.0Hz,1H).

¹³C NMR(151MHz,D₂O)δ166.31,151.85,141.60,102.60,95.71(d,J＝ 6.6Hz),88.42,83.16(d,J＝9.2Hz),73.76,70.11,69.55,69.14,69.11,68.23(d, J＝8.3Hz),64.86(d,J＝5.3Hz),50.24.

序列：

SEQ ID NO:1

MTAVEFIEPLTHEEGVSQATKLFVDTYGAAPEGVWAAPGRVNLIGEHTD YNAGLCLPIALPHRTFIALKPREDTKVRVVSGVAPDKVAEADLDGLKAR GVDGWSAYPTGVAWALRQAGFDKVKGFDAAFVSCVPLGSGLSSSAAMT CSTALALDDVYGLGYGDSDAGRVTLINAAIKSENEMAGASTGGLDQNA SMRCTEGHALLLDCRPELTPLENVSQQEFDLDKYNLELLVVDTQAPHQL NDGQYAQRRATCEEAAKILGVANLRVTADGISKADDQFQALKETLDALPDETMKKRVRHVVTEIERVRSFVRAFAQGDIKAAGRLFNASHDSLAADYE VTVPELDIAVDVARKNGAYGARMTGGGFGGSIIALVDKGQGHEIAQKIA DRFEKEGFNAPRALPAFAAASASREA

SEQ ID NO:2

MASTVDSNFFSSVPALHSNLGLLSPDQIELAKILLENGQSHLFQQWPELG VDDKEKLAFFDQIARLNSSYPGGLAAYIKTAKELLADSKVGKNPYDGFS PSVPSGENLTFGTDNFIEMEKRGVVEARNAAFVLVAGGLGERLGYNGIK VALPRETTTGTCFLQHYIESILALQEASNKIDSDGSERDIPFIIMTSDDTHS RTLDLLELNSYFGMKPTQVHLLKQEKVACLDDNDARLALDPHNKYSIQ TKPHGHGDVHSLLYSSGLLHKWLEAGLKWVLFFQDTNGLLFNAIPASLG VSATKQYHVNSLAVPRKAKEAIGGISKLTHVDGRSMVINVEYNQLDPLL RASGFPDGDVNCETGFSPFPGNINQLILELGPYKDELQKTGGAIKEFVNP KYKDSTKTAFKSSTRLECMMQDYPKTLPPTARVGFTVMDIWLAYAPVK NNPEDAAKVPKGNPYHSATSGEMAIYRANSLILQKAGVKVEEPVKQVL NGQEVEVWSRITWKPKWGMIFSDIKKKVSGNCEVSQRSTMAIKGRNVFI KDLSLDGALIVDSIDDAEVKLGGLIKNNGWTMESVDYKDTSVPEEIRIRGFRFNKVEQLEKKLTQPGKFSVED

SEQ ID NO:3

MGLETVPAGKALPDDIYVVIEIPANSDPIKYEVDKESGALFVDRFMATAM FYPANYGYVNNTLSLDGDPVDVLVPTPYPLQPGSVIRCRPVGVLKMTDE AGSDAKVVAVPHSKLTKEYDHIKDVNDLPALLKAQIQHFFESYKALEAG KWVKVDGWEGVDAARQEILDSFERAKK

SEQ ID NO:4

MTDVEFIEPLSHDEGVKIAVDLFKAVYGEEPTGVWAAPGRVNLIGEHTD YNAGLCLPIALPHRTFIALKPREDTKVRVVSDVAPDAVAEADLDGLTAGG VEGWAAYPVGVAWALREAGFDTVKGFDAAFSSCVPLGSGLSSSAAMTC STALALDDVYGLGYGSTDAGRVTLINAAIKSENDMAGASTGGLDQNAS MRCSFGHAIRLDCKPGLSAVESVEPKEFDLDRYGLELLVLDTRAPHQLN DGQYAQRRSTCEQAAEILGVANLRVTAETVAASADPAAALADVLDRLED GTMKKRVRHVVTEIGRVDRFVDAFAAGDIKTAGDLFNASHDSLRDDYE VTVPELDTAVDVARANGAYGARMTGGGFGGSIIALVDKGQGHEIAQKIA DEFESKGFNAPRALPAFAAAAASREI

SEQ ID NO:5

MTAVEFIEPLSHDEGVKNATDLFRATYGEEPAGVWAAPGRVNLIGEHTD YNAGLCLPIALPHRTFIALKPREDTKVRVVSDVDSENVTEADLDGLQAG GVEGWAAYPVGVAWALREAGFDAVQGFDAAFSSCVPLGSGLSSSAAMT CSTALALDDVYGLGYGASDAGRVTLINAAIKSENDMAGASTGGLDQNA SMRCTFGHALRLDCRPELSPLENVSQQEFDLDKYGLELLVLDTQAPHQL NDGQYAQRRATCEKAAEILGVANLRVVADEIAKSEDPFQALKETLDKLEDDTMKKRVRHVITEIARVNSFVRAFANGKIDEAGRLFNASHDSLAADYE VTVPELDIAVDVARVNGAYGARMTGGGFGGSIIALVDKGQGHEIAQKIA DRFEKEGFNAPRALPAFAAASASREA

SEQ ID NO:6

MSSVEFIEPISREDGVARATELFRATYGEEPAGVWAAPGRVNLIGEHTDY NAGLCLPIALPHRTFLALKPREDTAVRLVSDVNPTAVAEAELDGLKARGV DGWAAYPTGVAWAMREAGYDQVRGFDAAFVSCVPLGSGLSSSAAMTC STALALDDVYGLGYGATDEGRVTLITMAIKSENDMAGASTGGLDQNAS MRCTPGHAIRLDCMPGLSAVDSVSQQEFDLDKYGLELLVVDTQAHHQL NDGQYEQRRRTCEQAAELLGYEHLRAAAEAVAYSTDSEGSLAALLNCLNDETMKKRVRHVITEIGRVDEFVKAFAAGDIAESGRLFNASHDSLRDDY EVTVPELDVAVDMARANGAYGARMTGGGFGGSIIALVDKGRGREVAQLI ADEFEVRGFHAPRALAAVASASASRED

SEQ ID NO:7

MTSVEFIEPMSDAEGAARAAELFKQAYGKEPAGVWAAPGRVNLIGEHT DYNAGLCLPIALPHRTYIALSPRDDTSVRVVSDLASDVIAEADLDGLEAG GVDGWAAYPVGVAWALRNAGFDGVQGFDAAFSSCVPLGSGLSSSAAMT CSTALALDDVYSQGFGDTDEGRVTLINAAIASENDMAGASTGGLDQNA SMRCTPDHAIRLDCRPGLSAVDSVQQEVFDLEGHGLELLVLDTRAPHQL NDGQYAQRRATCEEAARILGVANLREVADLVNAQADPAAALDGVLDRLDDETMRKRVRHVVTEIGRVDDFVRAFAEGDMQTAGELFNASHDSLRDD YEVTVPELDVAVDVARDEGALGARMTGGGFGGSIIALVNAGESQRIAQA ICDEFERRGFVLPRALPAQASASAHRVQ

SEQ ID NO:8

MTAVEFIEPLSREDGVSRATKLFVDMYATAPEGVWAAPGRVNLIGEHTD YNAGLCLPIALPHRTFIALKPREDTKVRVVSDVAPDKVAEADLDGLKAR GVDGWSAYPTGVAWALREAGFSQVKGFDAAFVSCVPLGSGLSSSAAMT CSTALALDDVYGLGYGSSDAGRVTLINAAIKSENDMAGASTGGLDQNA SMRCTEGHALLLDCRPELTPLENVSQQAFDLDKYGLELLVVDTQAPHQL NDGQYAQRRATCEEAARILGVANLRVAADGISKADDQFQALKETLDALPDVTMKKRVRHVVTEIERVRSFVRAFAQGDIEAAGRLFNASHDSLAADYE VTVPELDVAVDVARKNGAYGARMTGGGFGGSIIALVDKGRSQEVAQKIA DEFEARGFHAPRALPAVAAPSASREA

SEQ ID NO:9

MTRTVEFIQPWTQGADGQGATKARELFRKVYGGDPQGVWSAPGRVNLI GEHTDYNAGLCLPIALPTRTYVAASPRTDSRVRLVSTMDPENPVQADLD GLQARGVSGWAAYPVGVAWALRRDGFPQVRGFDLALASCVPVGSGLSS SAAMTCAMALALDDLFGLGLGGDEGGRVRLIQAAITAENDMAGASTGG MDQSAAMRCRSGCALRLDCRPELDAMSNVRQVPFDLRAAGLELLVVD TRAQHQLNDGQYDQRRATCEQAVHLLGVANLRQAADQVNGAADPPSA LAALLEQLPDETMRRRVRHVISEIGRVDRFIEAFGRGDYVLAGRLINASH DSLRDDYEVTCPELDEAVDAARQGGAYGARMTGGGFGGSIIALADAGK GSGLARDIAERFASKGFKAPRALIALPSSAATRES

SEQ ID NO:10

MSAVEFIEPLTHEEGVSQATKLFVDTYGAAPEGVWAAPGRVNLIGEHTD YNAGLCLPIALPHRTFIALKPREDTKVRVVSGVAPDKVAEADLDGLKAR GVDGWSAYPTGVAWALRQAGFDKVKGFDAAFVSCVPLGSGLSSSAAMT CSTALALDDVYGLGYGDSDAGRVTLINAAIKSENEMAGASTGGLDQNA SMRCTEGHALLLDCRPELTPLENVSQQEFGLDKYNLELLVVDTQAPHQL NDGQYAQRRATCEEAAKILGVANLRVTADGISKADDQFQALKETLDALPDETMKKRVRHVVTEIERVRSFVRAFAQGDIKAAGRLFNASHDSLAADYE VTVPELDIAVDVARKNGAYGARMTGGGFGGSIIALVDKGRSQEVAQKIA DEFEKQGFHAPRALAAYAAPSASREA

SEQ ID NO:11

MTTAVEFIEPMGDADGAARAAALFEARFGTAPAGVWAAPGRVNLIGEHT DYNGGLCLPIALPHRTYVALAPRDDTTVRVISDMTPDEMTMVDLDGLA AGGVDGWGAYPIGVAWALREAGFDQVRGFDAVFSSCVPLGSGLSSSAA MTCSTALALDDVYGLGFGGSDEGRITLIDAAVMAENEMAGASTGGLDQ NASMRCAADHAIRLDCMPGLTAAQSVRQEPFDLSAYGLELLVLDTQA PHQLNDGQYEARRTMCEEAAQILGVPNLRVVADQVNAAVDPAAALEDV LSQLDDETMRRRVRHVITEIGRVDDFIGAFGRGDIETAGALFNASHDSLR DDYEVTVPELDVAVDVARDEGAYGARMTGGGFGGSIIALVNAGESRRIA QAIADEFARRGFDAPRALPARASQSAHRVND

SED ID NO:12

MTAVEFIEPLSHDEGVKNATDLFRATYGEEPAGVWAAPGRVNLIGEHTD YNAGLCLPIALPHRTFIALKPREDTKVRVVSDVDSGNVTEADLDGLQAG GVEGWAAYPVGVAWALREAGFNAVQGFDAAFSSCVPLGSGLSSSAAMT CSTALALDDVYGLGYGASDAGRVTLINAAIKSENDMAGASTGGLDQNA SMRCTFGHALRLDCRPELSPLENVSQQEFDLDKYGLELLVLDTQAPHQL NDGQYAQRRATCEKAAEILGVANLRVVADSIAKSGDPFQALKETLDKLEDDTMKKRVRHVITEIARVNSFVRAFANGKIDEAGRLFNASHDSLAADYE VTVPELDIAVDVARANGAYGARMTGGGFGGSIIALVNKGQGHEIAQKIA DRFEKEGFNAPRALPAFAAASASREA

SEQ ID NO:13

CATATGACAGCTGTAGAATTTATAGAGCCCCTAACCCATGAGGAGGGT GTCTCCCAGGCAACCAAGCTGTTTGTCGACACCTATGGTGCTGCTCCG GAGGGCGTGTGGGCTGCGCCGGGTCGTGTAAATCTGATTGGTGAACATACCGATTATAACGCTGGCCTTTGCCTGCCCATCGCGTTGCCGCACAG AACCTTTATTGCGCTTAAGCCGCGCGAAGATACCAAAGTCCGCGTGGT TTCCGGTGTTGCTCCGGATAAGGTGGCTGAGGCTGATCTGGACGGCCT GAAGGCCCGCGGGGTGGACGGTTGGTCTGCGTACCCGACCGGTGTGG CGTGGGCACTGCGTCAGGCCGGCTTCGATAAGGTGAAAGGTTTCGAC GCGGCCTTCGTGAGCTGTGTTCCGTTGGGCAGCGGTCTTTCTTCCTCA GCCGCAATGACGTGCAGCACCGCTTTAGCGCTCGACGATGTTTACGGC CTGGGTTATGGCGATAGCGATGCGGGCCGCGTGACGCTGATTAACGCG GCGATTAAAAGCGAAAATGAAATGGCAGGTGCGTCGACCGGTGGTTT AGACCAAAACGCAAGCATGCGTTGCACCGAGGGCCACGCACTGCTGT TGGACTGCCGTCCGGAGCTGACCCCGCTGGAGAACGTGTCTCAGCAAGAGTTCGACCTGGACAAGTACAACCTGGAACTGCTGGTTGTCGATAC CCAGGCGCCACACCAGCTGAATGATGGCCAATATGCACAACGTCGTG CGACTTGTGAAGAGGCTGCCAAGATCCTGGGCGTGGCGAATTTGCGC GTCACGGCGGATGGCATCAGCAAAGCGGACGACCAGTTTCAGGCGTT GAAGGAAACTCTGGACGCCTTGCCAGATGAGACAATGAAAAAACGT GTTCGTCACGTGGTAACCGAAATCGAACGTGTTAGAAGCTTTGTTCGC GCGTTTGCACAAGGTGATATCAAGGCGGCTGGCCGTCTGTTCAACGC GAGCCATGATTCGCTGGCTGCCGACTACGAAGTTACGGTTCCGGAGCT CGACATCGCGGTTGACGTTGCGCGTAAAAACGGTGCGTACGGCGCGC GCATGACCGGTGGTGGTTTCGGCGGCTCCATTATCGCGCTTGTGGATA AGGGTCAGGGTCACGAGATCGCCCAAAAAATTGCGGATCGTTTTGAA AAAGAGGGGTTCAACGCTCCGCGTGCGCTTCCGGCATTCGCTGCGGC ATCTGCCAGCCGTGAAGCCAAATTGGCCGCCGCGCTGGAGCTCGAG

SEQ ID NO:14

CATATGGCTAGCACCGTTGATAGCAACTTCTTCTCTAGCGTGCCGGCA CTGCATAGCAACCTGGGTCTGCTGTCCCCGGATCAGATTGAACTGGCA AAAATCCTGCTGGAAAACGGCCAGTCCCACCTGTTCCAGCAGTGGCCGGAACTGGGCGTTGACGATAAAGAAAAACTGGCCTTCTTCGATCAGA TTGCTCGTCTGAACTCTTCCTATCCAGGCGGCCTGGCTGCGTACATCA AAACCGCGAAAGAGCTGCTGGCGGATAGCAAAGTTGGTAAAAACCC GTATGATGGTTTTTCTCCGTCTGTTCCGAGCGGCGAAAACCTGACTTT CGGCACCGATAATTTCATTGAAATGGAAAAACGTGGTGTTGTGGAAG CCCGTAACGCAGCGTTTGTGCTGGTTGCAGGTGGCCTGGGCGAACGT CTGGGTTACAACGGTATCAAAGTTGCGCTGCCGCGTGAAACCACCAC CGGCACCTGTTTCCTGCAGCACTATATCGAATCTATCCTGGCTCTGCAG GAAGCGTCTAACAAAATCGATAGCGATGGCTCTGAACGTGACATTCCG TTCATCATCATGACCTCCGATGATACTCACTCCCGTACCCTGGACCTGC TGGAGCTGAACAGCTACTTTGGCATGAAACCGACCCAGGTGCACCTCCTGAAACAGGAAAAAGTTGCTTGCCTGGATGATAACGATGCCCGTCT GGCGCTGGATCCGCACAACAAATATAGCATTCAGACCAAACCACACG GTCACGGTGATGTGCATAGCCTGCTGTACTCTTCTGGTCTGCTGCACA AATGGCTGGAAGCTGGTCTGAAATGGGTGCTGTTCTTCCAGGATACCA ACGGCCTGCTGTTTAACGCTATTCCGGCCTCTCTGGGCGTGAGCGCGA CTAAACAGTACCACGTTAACTCCCTGGCTGTTCCACGTAAAGCTAAAG AAGCGATCGGTGGTATCAGCAAACTGACCCACGTTGATGGTCGTTCTA TGGTGATTAACGTGGAATATAACCAACTCGACCCGCTGCTGCGCGCTT CCGGCTTCCCGGACGGCGACGTGAACTGTGAAACCGGTTTTAGCCCG TTTCCGGGTAACATCAACCAGCTGATCCTGGAACTTGGCCCGTATAAA GACGAACTGCAGAAAACCGGCGGTGCGATTAAAGAATTCGTTAACCC GAAATATAAAGACAGCACTAAAACCGCGTTCAAATCCAGCACCCGCC TGGAATGCATGATGCAGGACTACCCGAAAACTCTGCCGCCGACCGCG CGCGTTGGCTTCACCGTAATGGATATCTGGCTGGCTTACGCGCCGGTT AAAAACAACCCGGAAGATGCTGCTAAAGTTCCGAAAGGTAACCCGTA CCACAGCGCAACCTCTGGTGAAATGGCGATCTATCGTGCGAACTCTCT GATTCTGCAGAAAGCAGGCGTTAAAGTTGAAGAACCGGTTAAACAGG TGCTGAACGGCCAAGAAGTTGAAGTTTGGAGCCGTATCACCTGGAAA CCGAAATGGGGTATGATCTTTTCTGACATTAAAAAGAAAGTGTCTGGT AACTGTGAAGTTTCCCAGCGTTCCACTATGGCGATCAAAGGTCGCAAT GTGTTTATCAAAGATCTGAGCCTGGACGGTGCTCTGATCGTTGATAGC ATCGATGACGCGGAAGTTAAACTGGGCGGTCTGATTAAAAACAACGG CTGGACCATGGAATCTGTAGATTACAAAGATACCTCTGTTCCGGAAGA AATCCGTATCCGTGGCTTCCGTTTCAACAAAGTTGAACAGCTGGAAA AGAAACTGACCCAGCCGGGTAAATTCTCTGTTGAAGATTAACTCGAG

SEQ ID NO:15

ATGAGCTTACTCAACGTCCCTGCGGGTAAAGATCTGCCGGAAGACATC TACGTTGTTATTGAGATCCCGGCTAACGCAGATCCGATCAAATACGAA ATCGACAAAGAGAGCGGCGCACTGTTCGTTGACCGCTTCATGTCCACCGCGATGTTCTATCCGTGCAACTACGGTTACATCAACCACACCCTGTC TCTGGACGGTGACCCAGTTGACGTACTGGTCCCAACTCCGTACCCGCT GCAGCCGGGTTCTGTGATCCGTTGCCGTCCGGTTGGCGTTCTGAAAAT GACCGACGAAGCCGGTGAAGATGCGAAACTGGTTGCGGTTCCGCAC AGCAAGCTGAGCAAAGAATACGATCACATTAAAGACGTTAACGATCT GCCTGAACTGCTGAAAGCGCAAATCGCTCACTTCTTCGAGCACTACA AAGACCTCGAAAAAGGCAAGTGGGTGAAAGTTGAAGGTTGGGAAAA CGCAGAAGCCGCTAAAGCTGAAATCGTTGCCTCCTTCGAGCGCGCAA AGAATAAATAA。

SEQUENCE LISTING

<110> 中国科学院上海药物研究所

<120> 一种合成尿苷二磷酸-6-叠氮-D-半乳糖的方法

<130> DI21-1174-XC37

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 416

<212> PRT

<213> Bifidobacterium longum

<400> 1

Met Thr Ala Val Glu Phe Ile Glu Pro Leu Thr His Glu Glu Gly Val

1 5 10 15

Ser Gln Ala Thr Lys Leu Phe Val Asp Thr Tyr Gly Ala Ala Pro Glu

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Ile Ala Leu Lys Pro Arg Glu Asp Thr Lys Val Arg Val Val Ser

65 70 75 80

Gly Val Ala Pro Asp Lys Val Ala Glu Ala Asp Leu Asp Gly Leu Lys

85 90 95

Ala Arg Gly Val Asp Gly Trp Ser Ala Tyr Pro Thr Gly Val Ala Trp

100 105 110

Ala Leu Arg Gln Ala Gly Phe Asp Lys Val Lys Gly Phe Asp Ala Ala

115 120 125

Phe Val Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Asp Ser Asp Ala Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Lys Ser Glu Asn Glu Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Glu Gly His Ala Leu Leu Leu Asp Cys

195 200 205

Arg Pro Glu Leu Thr Pro Leu Glu Asn Val Ser Gln Gln Glu Phe Asp

210 215 220

Leu Asp Lys Tyr Asn Leu Glu Leu Leu Val Val Asp Thr Gln Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ala Thr Cys Glu

245 250 255

Glu Ala Ala Lys Ile Leu Gly Val Ala Asn Leu Arg Val Thr Ala Asp

260 265 270

Gly Ile Ser Lys Ala Asp Asp Gln Phe Gln Ala Leu Lys Glu Thr Leu

275 280 285

Asp Ala Leu Pro Asp Glu Thr Met Lys Lys Arg Val Arg His Val Val

290 295 300

Thr Glu Ile Glu Arg Val Arg Ser Phe Val Arg Ala Phe Ala Gln Gly

305 310 315 320

Asp Ile Lys Ala Ala Gly Arg Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Ala Ala Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Ile Ala Val Asp

340 345 350

Val Ala Arg Lys Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asp Lys Gly Gln Gly His Glu

370 375 380

Ile Ala Gln Lys Ile Ala Asp Arg Phe Glu Lys Glu Gly Phe Asn Ala

385 390 395 400

Pro Arg Ala Leu Pro Ala Phe Ala Ala Ala Ser Ala Ser Arg Glu Ala

405 410 415

<210> 2

<211> 614

<212> PRT

<213> Arabidopsis thaliana

<400> 2

Met Ala Ser Thr Val Asp Ser Asn Phe Phe Ser Ser Val Pro Ala Leu

1 5 10 15

His Ser Asn Leu Gly Leu Leu Ser Pro Asp Gln Ile Glu Leu Ala Lys

20 25 30

Ile Leu Leu Glu Asn Gly Gln Ser His Leu Phe Gln Gln Trp Pro Glu

35 40 45

Leu Gly Val Asp Asp Lys Glu Lys Leu Ala Phe Phe Asp Gln Ile Ala

50 55 60

Arg Leu Asn Ser Ser Tyr Pro Gly Gly Leu Ala Ala Tyr Ile Lys Thr

65 70 75 80

Ala Lys Glu Leu Leu Ala Asp Ser Lys Val Gly Lys Asn Pro Tyr Asp

85 90 95

Gly Phe Ser Pro Ser Val Pro Ser Gly Glu Asn Leu Thr Phe Gly Thr

100 105 110

Asp Asn Phe Ile Glu Met Glu Lys Arg Gly Val Val Glu Ala Arg Asn

115 120 125

Ala Ala Phe Val Leu Val Ala Gly Gly Leu Gly Glu Arg Leu Gly Tyr

130 135 140

Asn Gly Ile Lys Val Ala Leu Pro Arg Glu Thr Thr Thr Gly Thr Cys

145 150 155 160

Phe Leu Gln His Tyr Ile Glu Ser Ile Leu Ala Leu Gln Glu Ala Ser

165 170 175

Asn Lys Ile Asp Ser Asp Gly Ser Glu Arg Asp Ile Pro Phe Ile Ile

180 185 190

Met Thr Ser Asp Asp Thr His Ser Arg Thr Leu Asp Leu Leu Glu Leu

195 200 205

Asn Ser Tyr Phe Gly Met Lys Pro Thr Gln Val His Leu Leu Lys Gln

210 215 220

Glu Lys Val Ala Cys Leu Asp Asp Asn Asp Ala Arg Leu Ala Leu Asp

225 230 235 240

Pro His Asn Lys Tyr Ser Ile Gln Thr Lys Pro His Gly His Gly Asp

245 250 255

Val His Ser Leu Leu Tyr Ser Ser Gly Leu Leu His Lys Trp Leu Glu

260 265 270

Ala Gly Leu Lys Trp Val Leu Phe Phe Gln Asp Thr Asn Gly Leu Leu

275 280 285

Phe Asn Ala Ile Pro Ala Ser Leu Gly Val Ser Ala Thr Lys Gln Tyr

290 295 300

His Val Asn Ser Leu Ala Val Pro Arg Lys Ala Lys Glu Ala Ile Gly

305 310 315 320

Gly Ile Ser Lys Leu Thr His Val Asp Gly Arg Ser Met Val Ile Asn

325 330 335

Val Glu Tyr Asn Gln Leu Asp Pro Leu Leu Arg Ala Ser Gly Phe Pro

340 345 350

Asp Gly Asp Val Asn Cys Glu Thr Gly Phe Ser Pro Phe Pro Gly Asn

355 360 365

Ile Asn Gln Leu Ile Leu Glu Leu Gly Pro Tyr Lys Asp Glu Leu Gln

370 375 380

Lys Thr Gly Gly Ala Ile Lys Glu Phe Val Asn Pro Lys Tyr Lys Asp

385 390 395 400

Ser Thr Lys Thr Ala Phe Lys Ser Ser Thr Arg Leu Glu Cys Met Met

405 410 415

Gln Asp Tyr Pro Lys Thr Leu Pro Pro Thr Ala Arg Val Gly Phe Thr

420 425 430

Val Met Asp Ile Trp Leu Ala Tyr Ala Pro Val Lys Asn Asn Pro Glu

435 440 445

Asp Ala Ala Lys Val Pro Lys Gly Asn Pro Tyr His Ser Ala Thr Ser

450 455 460

Gly Glu Met Ala Ile Tyr Arg Ala Asn Ser Leu Ile Leu Gln Lys Ala

465 470 475 480

Gly Val Lys Val Glu Glu Pro Val Lys Gln Val Leu Asn Gly Gln Glu

485 490 495

Val Glu Val Trp Ser Arg Ile Thr Trp Lys Pro Lys Trp Gly Met Ile

500 505 510

Phe Ser Asp Ile Lys Lys Lys Val Ser Gly Asn Cys Glu Val Ser Gln

515 520 525

Arg Ser Thr Met Ala Ile Lys Gly Arg Asn Val Phe Ile Lys Asp Leu

530 535 540

Ser Leu Asp Gly Ala Leu Ile Val Asp Ser Ile Asp Asp Ala Glu Val

545 550 555 560

Lys Leu Gly Gly Leu Ile Lys Asn Asn Gly Trp Thr Met Glu Ser Val

565 570 575

Asp Tyr Lys Asp Thr Ser Val Pro Glu Glu Ile Arg Ile Arg Gly Phe

580 585 590

Arg Phe Asn Lys Val Glu Gln Leu Glu Lys Lys Leu Thr Gln Pro Gly

595 600 605

Lys Phe Ser Val Glu Asp

610

<210> 3

<211> 175

<212> PRT

<213> Escherichia coli

<400> 3

Met Gly Leu Glu Thr Val Pro Ala Gly Lys Ala Leu Pro Asp Asp Ile

1 5 10 15

Tyr Val Val Ile Glu Ile Pro Ala Asn Ser Asp Pro Ile Lys Tyr Glu

20 25 30

Val Asp Lys Glu Ser Gly Ala Leu Phe Val Asp Arg Phe Met Ala Thr

35 40 45

Ala Met Phe Tyr Pro Ala Asn Tyr Gly Tyr Val Asn Asn Thr Leu Ser

50 55 60

Leu Asp Gly Asp Pro Val Asp Val Leu Val Pro Thr Pro Tyr Pro Leu

65 70 75 80

Gln Pro Gly Ser Val Ile Arg Cys Arg Pro Val Gly Val Leu Lys Met

85 90 95

Thr Asp Glu Ala Gly Ser Asp Ala Lys Val Val Ala Val Pro His Ser

100 105 110

Lys Leu Thr Lys Glu Tyr Asp His Ile Lys Asp Val Asn Asp Leu Pro

115 120 125

Ala Leu Leu Lys Ala Gln Ile Gln His Phe Phe Glu Ser Tyr Lys Ala

130 135 140

Leu Glu Ala Gly Lys Trp Val Lys Val Asp Gly Trp Glu Gly Val Asp

145 150 155 160

Ala Ala Arg Gln Glu Ile Leu Asp Ser Phe Glu Arg Ala Lys Lys

165 170 175

<210> 4

<211> 416

<212> PRT

<213> Bifidobacterium dentium

<400> 4

Met Thr Asp Val Glu Phe Ile Glu Pro Leu Ser His Asp Glu Gly Val

1 5 10 15

Lys Ile Ala Val Asp Leu Phe Lys Ala Val Tyr Gly Glu Glu Pro Thr

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Ile Ala Leu Lys Pro Arg Glu Asp Thr Lys Val Arg Val Val Ser

65 70 75 80

Asp Val Ala Pro Asp Ala Val Ala Glu Ala Asp Leu Asp Gly Leu Thr

85 90 95

Ala Gly Gly Val Glu Gly Trp Ala Ala Tyr Pro Val Gly Val Ala Trp

100 105 110

Ala Leu Arg Glu Ala Gly Phe Asp Thr Val Lys Gly Phe Asp Ala Ala

115 120 125

Phe Ser Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Ser Thr Asp Ala Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Lys Ser Glu Asn Asp Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Ser Phe Gly His Ala Ile Arg Leu Asp Cys

195 200 205

Lys Pro Gly Leu Ser Ala Val Glu Ser Val Glu Pro Lys Glu Phe Asp

210 215 220

Leu Asp Arg Tyr Gly Leu Glu Leu Leu Val Leu Asp Thr Arg Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ser Thr Cys Glu

245 250 255

Gln Ala Ala Glu Ile Leu Gly Val Ala Asn Leu Arg Val Thr Ala Glu

260 265 270

Thr Val Ala Ala Ser Ala Asp Pro Ala Ala Ala Leu Ala Asp Val Leu

275 280 285

Asp Arg Leu Glu Asp Gly Thr Met Lys Lys Arg Val Arg His Val Val

290 295 300

Thr Glu Ile Gly Arg Val Asp Arg Phe Val Asp Ala Phe Ala Ala Gly

305 310 315 320

Asp Ile Lys Thr Ala Gly Asp Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Arg Asp Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Thr Ala Val Asp

340 345 350

Val Ala Arg Ala Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asp Lys Gly Gln Gly His Glu

370 375 380

Ile Ala Gln Lys Ile Ala Asp Glu Phe Glu Ser Lys Gly Phe Asn Ala

385 390 395 400

Pro Arg Ala Leu Pro Ala Phe Ala Ala Ala Ala Ala Ser Arg Glu Ile

405 410 415

<210> 5

<211> 416

<212> PRT

<213> Bifidobacterium catenulatum

<400> 5

Met Thr Ala Val Glu Phe Ile Glu Pro Leu Ser His Asp Glu Gly Val

1 5 10 15

Lys Asn Ala Thr Asp Leu Phe Arg Ala Thr Tyr Gly Glu Glu Pro Ala

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Ile Ala Leu Lys Pro Arg Glu Asp Thr Lys Val Arg Val Val Ser

65 70 75 80

Asp Val Asp Ser Glu Asn Val Thr Glu Ala Asp Leu Asp Gly Leu Gln

85 90 95

Ala Gly Gly Val Glu Gly Trp Ala Ala Tyr Pro Val Gly Val Ala Trp

100 105 110

Ala Leu Arg Glu Ala Gly Phe Asp Ala Val Gln Gly Phe Asp Ala Ala

115 120 125

Phe Ser Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Ala Ser Asp Ala Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Lys Ser Glu Asn Asp Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Phe Gly His Ala Leu Arg Leu Asp Cys

195 200 205

Arg Pro Glu Leu Ser Pro Leu Glu Asn Val Ser Gln Gln Glu Phe Asp

210 215 220

Leu Asp Lys Tyr Gly Leu Glu Leu Leu Val Leu Asp Thr Gln Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ala Thr Cys Glu

245 250 255

Lys Ala Ala Glu Ile Leu Gly Val Ala Asn Leu Arg Val Val Ala Asp

260 265 270

Glu Ile Ala Lys Ser Glu Asp Pro Phe Gln Ala Leu Lys Glu Thr Leu

275 280 285

Asp Lys Leu Glu Asp Asp Thr Met Lys Lys Arg Val Arg His Val Ile

290 295 300

Thr Glu Ile Ala Arg Val Asn Ser Phe Val Arg Ala Phe Ala Asn Gly

305 310 315 320

Lys Ile Asp Glu Ala Gly Arg Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Ala Ala Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Ile Ala Val Asp

340 345 350

Val Ala Arg Val Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asp Lys Gly Gln Gly His Glu

370 375 380

Ile Ala Gln Lys Ile Ala Asp Arg Phe Glu Lys Glu Gly Phe Asn Ala

385 390 395 400

Pro Arg Ala Leu Pro Ala Phe Ala Ala Ala Ser Ala Ser Arg Glu Ala

405 410 415

<210> 6

<211> 416

<212> PRT

<213> Bifidobacterium catenulatum

<400> 6

Met Ser Ser Val Glu Phe Ile Glu Pro Ile Ser Arg Glu Asp Gly Val

1 5 10 15

Ala Arg Ala Thr Glu Leu Phe Arg Ala Thr Tyr Gly Glu Glu Pro Ala

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Leu Ala Leu Lys Pro Arg Glu Asp Thr Ala Val Arg Leu Val Ser

65 70 75 80

Asp Val Asn Pro Thr Ala Val Ala Glu Ala Glu Leu Asp Gly Leu Lys

85 90 95

Ala Arg Gly Val Asp Gly Trp Ala Ala Tyr Pro Thr Gly Val Ala Trp

100 105 110

Ala Met Arg Glu Ala Gly Tyr Asp Gln Val Arg Gly Phe Asp Ala Ala

115 120 125

Phe Val Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Ala Thr Asp Glu Gly Arg Val Thr Leu Ile Thr Met Ala Ile

165 170 175

Lys Ser Glu Asn Asp Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Pro Gly His Ala Ile Arg Leu Asp Cys

195 200 205

Met Pro Gly Leu Ser Ala Val Asp Ser Val Ser Gln Gln Glu Phe Asp

210 215 220

Leu Asp Lys Tyr Gly Leu Glu Leu Leu Val Val Asp Thr Gln Ala His

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Glu Gln Arg Arg Arg Thr Cys Glu

245 250 255

Gln Ala Ala Glu Leu Leu Gly Tyr Glu His Leu Arg Ala Ala Ala Glu

260 265 270

Ala Val Ala Tyr Ser Thr Asp Ser Glu Gly Ser Leu Ala Ala Leu Leu

275 280 285

Asn Cys Leu Asn Asp Glu Thr Met Lys Lys Arg Val Arg His Val Ile

290 295 300

Thr Glu Ile Gly Arg Val Asp Glu Phe Val Lys Ala Phe Ala Ala Gly

305 310 315 320

Asp Ile Ala Glu Ser Gly Arg Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Arg Asp Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Val Ala Val Asp

340 345 350

Met Ala Arg Ala Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asp Lys Gly Arg Gly Arg Glu

370 375 380

Val Ala Gln Leu Ile Ala Asp Glu Phe Glu Val Arg Gly Phe His Ala

385 390 395 400

Pro Arg Ala Leu Ala Ala Val Ala Ser Ala Ser Ala Ser Arg Glu Asp

405 410 415

<210> 7

<211> 416

<212> PRT

<213> Bifidobacterium animalis

<400> 7

Met Thr Ser Val Glu Phe Ile Glu Pro Met Ser Asp Ala Glu Gly Ala

1 5 10 15

Ala Arg Ala Ala Glu Leu Phe Lys Gln Ala Tyr Gly Lys Glu Pro Ala

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Tyr Ile Ala Leu Ser Pro Arg Asp Asp Thr Ser Val Arg Val Val Ser

65 70 75 80

Asp Leu Ala Ser Asp Val Ile Ala Glu Ala Asp Leu Asp Gly Leu Glu

85 90 95

Ala Gly Gly Val Asp Gly Trp Ala Ala Tyr Pro Val Gly Val Ala Trp

100 105 110

Ala Leu Arg Asn Ala Gly Phe Asp Gly Val Gln Gly Phe Asp Ala Ala

115 120 125

Phe Ser Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Ser Gln Gly

145 150 155 160

Phe Gly Asp Thr Asp Glu Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Ala Ser Glu Asn Asp Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Pro Asp His Ala Ile Arg Leu Asp Cys

195 200 205

Arg Pro Gly Leu Ser Ala Val Asp Ser Val Gln Gln Glu Val Phe Asp

210 215 220

Leu Glu Gly His Gly Leu Glu Leu Leu Val Leu Asp Thr Arg Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ala Thr Cys Glu

245 250 255

Glu Ala Ala Arg Ile Leu Gly Val Ala Asn Leu Arg Glu Val Ala Asp

260 265 270

Leu Val Asn Ala Gln Ala Asp Pro Ala Ala Ala Leu Asp Gly Val Leu

275 280 285

Asp Arg Leu Asp Asp Glu Thr Met Arg Lys Arg Val Arg His Val Val

290 295 300

Thr Glu Ile Gly Arg Val Asp Asp Phe Val Arg Ala Phe Ala Glu Gly

305 310 315 320

Asp Met Gln Thr Ala Gly Glu Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Arg Asp Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Val Ala Val Asp

340 345 350

Val Ala Arg Asp Glu Gly Ala Leu Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asn Ala Gly Glu Ser Gln Arg

370 375 380

Ile Ala Gln Ala Ile Cys Asp Glu Phe Glu Arg Arg Gly Phe Val Leu

385 390 395 400

Pro Arg Ala Leu Pro Ala Gln Ala Ser Ala Ser Ala His Arg Val Gln

405 410 415

<210> 8

<211> 416

<212> PRT

<213> Bifidobacterium bifidum

<400> 8

Met Thr Ala Val Glu Phe Ile Glu Pro Leu Ser Arg Glu Asp Gly Val

1 5 10 15

Ser Arg Ala Thr Lys Leu Phe Val Asp Met Tyr Ala Thr Ala Pro Glu

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Ile Ala Leu Lys Pro Arg Glu Asp Thr Lys Val Arg Val Val Ser

65 70 75 80

Asp Val Ala Pro Asp Lys Val Ala Glu Ala Asp Leu Asp Gly Leu Lys

85 90 95

Ala Arg Gly Val Asp Gly Trp Ser Ala Tyr Pro Thr Gly Val Ala Trp

100 105 110

Ala Leu Arg Glu Ala Gly Phe Ser Gln Val Lys Gly Phe Asp Ala Ala

115 120 125

Phe Val Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Ser Ser Asp Ala Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Lys Ser Glu Asn Asp Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Glu Gly His Ala Leu Leu Leu Asp Cys

195 200 205

Arg Pro Glu Leu Thr Pro Leu Glu Asn Val Ser Gln Gln Ala Phe Asp

210 215 220

Leu Asp Lys Tyr Gly Leu Glu Leu Leu Val Val Asp Thr Gln Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ala Thr Cys Glu

245 250 255

Glu Ala Ala Arg Ile Leu Gly Val Ala Asn Leu Arg Val Ala Ala Asp

260 265 270

Gly Ile Ser Lys Ala Asp Asp Gln Phe Gln Ala Leu Lys Glu Thr Leu

275 280 285

Asp Ala Leu Pro Asp Val Thr Met Lys Lys Arg Val Arg His Val Val

290 295 300

Thr Glu Ile Glu Arg Val Arg Ser Phe Val Arg Ala Phe Ala Gln Gly

305 310 315 320

Asp Ile Glu Ala Ala Gly Arg Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Ala Ala Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Val Ala Val Asp

340 345 350

Val Ala Arg Lys Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asp Lys Gly Arg Ser Gln Glu

370 375 380

Val Ala Gln Lys Ile Ala Asp Glu Phe Glu Ala Arg Gly Phe His Ala

385 390 395 400

Pro Arg Ala Leu Pro Ala Val Ala Ala Pro Ser Ala Ser Arg Glu Ala

405 410 415

<210> 9

<211> 420

<212> PRT

<213> Bifidobacterium asteroides

<400> 9

Met Thr Arg Thr Val Glu Phe Ile Gln Pro Trp Thr Gln Gly Ala Asp

1 5 10 15

Gly Gln Gly Ala Thr Lys Ala Arg Glu Leu Phe Arg Lys Val Tyr Gly

20 25 30

Gly Asp Pro Gln Gly Val Trp Ser Ala Pro Gly Arg Val Asn Leu Ile

35 40 45

Gly Glu His Thr Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu

50 55 60

Pro Thr Arg Thr Tyr Val Ala Ala Ser Pro Arg Thr Asp Ser Arg Val

65 70 75 80

Arg Leu Val Ser Thr Met Asp Pro Glu Asn Pro Val Gln Ala Asp Leu

85 90 95

Asp Gly Leu Gln Ala Arg Gly Val Ser Gly Trp Ala Ala Tyr Pro Val

100 105 110

Gly Val Ala Trp Ala Leu Arg Arg Asp Gly Phe Pro Gln Val Arg Gly

115 120 125

Phe Asp Leu Ala Leu Ala Ser Cys Val Pro Val Gly Ser Gly Leu Ser

130 135 140

Ser Ser Ala Ala Met Thr Cys Ala Met Ala Leu Ala Leu Asp Asp Leu

145 150 155 160

Phe Gly Leu Gly Leu Gly Gly Asp Glu Gly Gly Arg Val Arg Leu Ile

165 170 175

Gln Ala Ala Ile Thr Ala Glu Asn Asp Met Ala Gly Ala Ser Thr Gly

180 185 190

Gly Met Asp Gln Ser Ala Ala Met Arg Cys Arg Ser Gly Cys Ala Leu

195 200 205

Arg Leu Asp Cys Arg Pro Glu Leu Asp Ala Met Ser Asn Val Arg Gln

210 215 220

Val Pro Phe Asp Leu Arg Ala Ala Gly Leu Glu Leu Leu Val Val Asp

225 230 235 240

Thr Arg Ala Gln His Gln Leu Asn Asp Gly Gln Tyr Asp Gln Arg Arg

245 250 255

Ala Thr Cys Glu Gln Ala Val His Leu Leu Gly Val Ala Asn Leu Arg

260 265 270

Gln Ala Ala Asp Gln Val Asn Gly Ala Ala Asp Pro Pro Ser Ala Leu

275 280 285

Ala Ala Leu Leu Glu Gln Leu Pro Asp Glu Thr Met Arg Arg Arg Val

290 295 300

Arg His Val Ile Ser Glu Ile Gly Arg Val Asp Arg Phe Ile Glu Ala

305 310 315 320

Phe Gly Arg Gly Asp Tyr Val Leu Ala Gly Arg Leu Ile Asn Ala Ser

325 330 335

His Asp Ser Leu Arg Asp Asp Tyr Glu Val Thr Cys Pro Glu Leu Asp

340 345 350

Glu Ala Val Asp Ala Ala Arg Gln Gly Gly Ala Tyr Gly Ala Arg Met

355 360 365

Thr Gly Gly Gly Phe Gly Gly Ser Ile Ile Ala Leu Ala Asp Ala Gly

370 375 380

Lys Gly Ser Gly Leu Ala Arg Asp Ile Ala Glu Arg Phe Ala Ser Lys

385 390 395 400

Gly Phe Lys Ala Pro Arg Ala Leu Ile Ala Leu Pro Ser Ser Ala Ala

405 410 415

Thr Arg Glu Ser

420

<210> 10

<211> 416

<212> PRT

<213> Bifidobacterium breve

<400> 10

Met Ser Ala Val Glu Phe Ile Glu Pro Leu Thr His Glu Glu Gly Val

1 5 10 15

Ser Gln Ala Thr Lys Leu Phe Val Asp Thr Tyr Gly Ala Ala Pro Glu

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Ile Ala Leu Lys Pro Arg Glu Asp Thr Lys Val Arg Val Val Ser

65 70 75 80

Gly Val Ala Pro Asp Lys Val Ala Glu Ala Asp Leu Asp Gly Leu Lys

85 90 95

Ala Arg Gly Val Asp Gly Trp Ser Ala Tyr Pro Thr Gly Val Ala Trp

100 105 110

Ala Leu Arg Gln Ala Gly Phe Asp Lys Val Lys Gly Phe Asp Ala Ala

115 120 125

Phe Val Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Asp Ser Asp Ala Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Lys Ser Glu Asn Glu Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Glu Gly His Ala Leu Leu Leu Asp Cys

195 200 205

Arg Pro Glu Leu Thr Pro Leu Glu Asn Val Ser Gln Gln Glu Phe Gly

210 215 220

Leu Asp Lys Tyr Asn Leu Glu Leu Leu Val Val Asp Thr Gln Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ala Thr Cys Glu

245 250 255

Glu Ala Ala Lys Ile Leu Gly Val Ala Asn Leu Arg Val Thr Ala Asp

260 265 270

Gly Ile Ser Lys Ala Asp Asp Gln Phe Gln Ala Leu Lys Glu Thr Leu

275 280 285

Asp Ala Leu Pro Asp Glu Thr Met Lys Lys Arg Val Arg His Val Val

290 295 300

Thr Glu Ile Glu Arg Val Arg Ser Phe Val Arg Ala Phe Ala Gln Gly

305 310 315 320

Asp Ile Lys Ala Ala Gly Arg Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Ala Ala Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Ile Ala Val Asp

340 345 350

Val Ala Arg Lys Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asp Lys Gly Arg Ser Gln Glu

370 375 380

Val Ala Gln Lys Ile Ala Asp Glu Phe Glu Lys Gln Gly Phe His Ala

385 390 395 400

Pro Arg Ala Leu Ala Ala Tyr Ala Ala Pro Ser Ala Ser Arg Glu Ala

405 410 415

<210> 11

<211> 418

<212> PRT

<213> Bifidobacterium pseudolongum

<400> 11

Met Thr Thr Ala Val Glu Phe Ile Glu Pro Met Gly Asp Ala Asp Gly

1 5 10 15

Ala Ala Arg Ala Ala Ala Leu Phe Glu Ala Arg Phe Gly Thr Ala Pro

20 25 30

Ala Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His

35 40 45

Thr Asp Tyr Asn Gly Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg

50 55 60

Thr Tyr Val Ala Leu Ala Pro Arg Asp Asp Thr Thr Val Arg Val Ile

65 70 75 80

Ser Asp Met Thr Pro Asp Glu Met Thr Met Val Asp Leu Asp Gly Leu

85 90 95

Ala Ala Gly Gly Val Asp Gly Trp Gly Ala Tyr Pro Ile Gly Val Ala

100 105 110

Trp Ala Leu Arg Glu Ala Gly Phe Asp Gln Val Arg Gly Phe Asp Ala

115 120 125

Val Phe Ser Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala

130 135 140

Ala Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu

145 150 155 160

Gly Phe Gly Gly Ser Asp Glu Gly Arg Ile Thr Leu Ile Asp Ala Ala

165 170 175

Val Met Ala Glu Asn Glu Met Ala Gly Ala Ser Thr Gly Gly Leu Asp

180 185 190

Gln Asn Ala Ser Met Arg Cys Ala Ala Asp His Ala Ile Arg Leu Asp

195 200 205

Cys Met Pro Gly Leu Thr Ala Ala Gln Ser Val Arg Gln Glu Pro Phe

210 215 220

Asp Leu Ser Ala Tyr Gly Leu Glu Leu Leu Val Leu Asp Thr Gln Ala

225 230 235 240

Pro His Gln Leu Asn Asp Gly Gln Tyr Glu Ala Arg Arg Thr Met Cys

245 250 255

Glu Glu Ala Ala Gln Ile Leu Gly Val Pro Asn Leu Arg Val Val Ala

260 265 270

Asp Gln Val Asn Ala Ala Val Asp Pro Ala Ala Ala Leu Glu Asp Val

275 280 285

Leu Ser Gln Leu Asp Asp Glu Thr Met Arg Arg Arg Val Arg His Val

290 295 300

Ile Thr Glu Ile Gly Arg Val Asp Asp Phe Ile Gly Ala Phe Gly Arg

305 310 315 320

Gly Asp Ile Glu Thr Ala Gly Ala Leu Phe Asn Ala Ser His Asp Ser

325 330 335

Leu Arg Asp Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Val Ala Val

340 345 350

Asp Val Ala Arg Asp Glu Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly

355 360 365

Gly Phe Gly Gly Ser Ile Ile Ala Leu Val Asn Ala Gly Glu Ser Arg

370 375 380

Arg Ile Ala Gln Ala Ile Ala Asp Glu Phe Ala Arg Arg Gly Phe Asp

385 390 395 400

Ala Pro Arg Ala Leu Pro Ala Arg Ala Ser Gln Ser Ala His Arg Val

405 410 415

Asn Asp

<210> 12

<211> 416

<212> PRT

<213> Bifidobacterium pseudocatenulatum

<400> 12

Met Thr Ala Val Glu Phe Ile Glu Pro Leu Ser His Asp Glu Gly Val

1 5 10 15

Lys Asn Ala Thr Asp Leu Phe Arg Ala Thr Tyr Gly Glu Glu Pro Ala

20 25 30

Gly Val Trp Ala Ala Pro Gly Arg Val Asn Leu Ile Gly Glu His Thr

35 40 45

Asp Tyr Asn Ala Gly Leu Cys Leu Pro Ile Ala Leu Pro His Arg Thr

50 55 60

Phe Ile Ala Leu Lys Pro Arg Glu Asp Thr Lys Val Arg Val Val Ser

65 70 75 80

Asp Val Asp Ser Gly Asn Val Thr Glu Ala Asp Leu Asp Gly Leu Gln

85 90 95

Ala Gly Gly Val Glu Gly Trp Ala Ala Tyr Pro Val Gly Val Ala Trp

100 105 110

Ala Leu Arg Glu Ala Gly Phe Asn Ala Val Gln Gly Phe Asp Ala Ala

115 120 125

Phe Ser Ser Cys Val Pro Leu Gly Ser Gly Leu Ser Ser Ser Ala Ala

130 135 140

Met Thr Cys Ser Thr Ala Leu Ala Leu Asp Asp Val Tyr Gly Leu Gly

145 150 155 160

Tyr Gly Ala Ser Asp Ala Gly Arg Val Thr Leu Ile Asn Ala Ala Ile

165 170 175

Lys Ser Glu Asn Asp Met Ala Gly Ala Ser Thr Gly Gly Leu Asp Gln

180 185 190

Asn Ala Ser Met Arg Cys Thr Phe Gly His Ala Leu Arg Leu Asp Cys

195 200 205

Arg Pro Glu Leu Ser Pro Leu Glu Asn Val Ser Gln Gln Glu Phe Asp

210 215 220

Leu Asp Lys Tyr Gly Leu Glu Leu Leu Val Leu Asp Thr Gln Ala Pro

225 230 235 240

His Gln Leu Asn Asp Gly Gln Tyr Ala Gln Arg Arg Ala Thr Cys Glu

245 250 255

Lys Ala Ala Glu Ile Leu Gly Val Ala Asn Leu Arg Val Val Ala Asp

260 265 270

Ser Ile Ala Lys Ser Gly Asp Pro Phe Gln Ala Leu Lys Glu Thr Leu

275 280 285

Asp Lys Leu Glu Asp Asp Thr Met Lys Lys Arg Val Arg His Val Ile

290 295 300

Thr Glu Ile Ala Arg Val Asn Ser Phe Val Arg Ala Phe Ala Asn Gly

305 310 315 320

Lys Ile Asp Glu Ala Gly Arg Leu Phe Asn Ala Ser His Asp Ser Leu

325 330 335

Ala Ala Asp Tyr Glu Val Thr Val Pro Glu Leu Asp Ile Ala Val Asp

340 345 350

Val Ala Arg Ala Asn Gly Ala Tyr Gly Ala Arg Met Thr Gly Gly Gly

355 360 365

Phe Gly Gly Ser Ile Ile Ala Leu Val Asn Lys Gly Gln Gly His Glu

370 375 380

Ile Ala Gln Lys Ile Ala Asp Arg Phe Glu Lys Glu Gly Phe Asn Ala

385 390 395 400

Pro Arg Ala Leu Pro Ala Phe Ala Ala Ala Ser Ala Ser Arg Glu Ala

405 410 415

<210> 13

<211> 1278

<212> DNA

<213> 人工序列

<220>

<223> 编码BiGalK的核苷酸序列

<400> 13

catatgacag ctgtagaatt tatagagccc ctaacccatg aggagggtgt ctcccaggca 60

accaagctgt ttgtcgacac ctatggtgct gctccggagg gcgtgtgggc tgcgccgggt 120

cgtgtaaatc tgattggtga acataccgat tataacgctg gcctttgcct gcccatcgcg 180

ttgccgcaca gaacctttat tgcgcttaag ccgcgcgaag ataccaaagt ccgcgtggtt 240

tccggtgttg ctccggataa ggtggctgag gctgatctgg acggcctgaa ggcccgcggg 300

gtggacggtt ggtctgcgta cccgaccggt gtggcgtggg cactgcgtca ggccggcttc 360

gataaggtga aaggtttcga cgcggccttc gtgagctgtg ttccgttggg cagcggtctt 420

tcttcctcag ccgcaatgac gtgcagcacc gctttagcgc tcgacgatgt ttacggcctg 480

ggttatggcg atagcgatgc gggccgcgtg acgctgatta acgcggcgat taaaagcgaa 540

aatgaaatgg caggtgcgtc gaccggtggt ttagaccaaa acgcaagcat gcgttgcacc 600

gagggccacg cactgctgtt ggactgccgt ccggagctga ccccgctgga gaacgtgtct 660

cagcaagagt tcgacctgga caagtacaac ctggaactgc tggttgtcga tacccaggcg 720

ccacaccagc tgaatgatgg ccaatatgca caacgtcgtg cgacttgtga agaggctgcc 780

aagatcctgg gcgtggcgaa tttgcgcgtc acggcggatg gcatcagcaa agcggacgac 840

cagtttcagg cgttgaagga aactctggac gccttgccag atgagacaat gaaaaaacgt 900

gttcgtcacg tggtaaccga aatcgaacgt gttagaagct ttgttcgcgc gtttgcacaa 960

ggtgatatca aggcggctgg ccgtctgttc aacgcgagcc atgattcgct ggctgccgac 1020

tacgaagtta cggttccgga gctcgacatc gcggttgacg ttgcgcgtaa aaacggtgcg 1080

tacggcgcgc gcatgaccgg tggtggtttc ggcggctcca ttatcgcgct tgtggataag 1140

ggtcagggtc acgagatcgc ccaaaaaatt gcggatcgtt ttgaaaaaga ggggttcaac 1200

gctccgcgtg cgcttccggc attcgctgcg gcatctgcca gccgtgaagc caaattggcc 1260

gccgcgctgg agctcgag 1278

<210> 14

<211> 1854

<212> DNA

<213> 人工序列

<220>

<223> 编码AtUSP的核苷酸序列

<400> 14

catatggcta gcaccgttga tagcaacttc ttctctagcg tgccggcact gcatagcaac 60

ctgggtctgc tgtccccgga tcagattgaa ctggcaaaaa tcctgctgga aaacggccag 120

tcccacctgt tccagcagtg gccggaactg ggcgttgacg ataaagaaaa actggccttc 180

ttcgatcaga ttgctcgtct gaactcttcc tatccaggcg gcctggctgc gtacatcaaa 240

accgcgaaag agctgctggc ggatagcaaa gttggtaaaa acccgtatga tggtttttct 300

ccgtctgttc cgagcggcga aaacctgact ttcggcaccg ataatttcat tgaaatggaa 360

aaacgtggtg ttgtggaagc ccgtaacgca gcgtttgtgc tggttgcagg tggcctgggc 420

gaacgtctgg gttacaacgg tatcaaagtt gcgctgccgc gtgaaaccac caccggcacc 480

tgtttcctgc agcactatat cgaatctatc ctggctctgc aggaagcgtc taacaaaatc 540

gatagcgatg gctctgaacg tgacattccg ttcatcatca tgacctccga tgatactcac 600

tcccgtaccc tggacctgct ggagctgaac agctactttg gcatgaaacc gacccaggtg 660

cacctcctga aacaggaaaa agttgcttgc ctggatgata acgatgcccg tctggcgctg 720

gatccgcaca acaaatatag cattcagacc aaaccacacg gtcacggtga tgtgcatagc 780

ctgctgtact cttctggtct gctgcacaaa tggctggaag ctggtctgaa atgggtgctg 840

ttcttccagg ataccaacgg cctgctgttt aacgctattc cggcctctct gggcgtgagc 900

gcgactaaac agtaccacgt taactccctg gctgttccac gtaaagctaa agaagcgatc 960

ggtggtatca gcaaactgac ccacgttgat ggtcgttcta tggtgattaa cgtggaatat 1020

aaccaactcg acccgctgct gcgcgcttcc ggcttcccgg acggcgacgt gaactgtgaa 1080

accggtttta gcccgtttcc gggtaacatc aaccagctga tcctggaact tggcccgtat 1140

aaagacgaac tgcagaaaac cggcggtgcg attaaagaat tcgttaaccc gaaatataaa 1200

gacagcacta aaaccgcgtt caaatccagc acccgcctgg aatgcatgat gcaggactac 1260

ccgaaaactc tgccgccgac cgcgcgcgtt ggcttcaccg taatggatat ctggctggct 1320

tacgcgccgg ttaaaaacaa cccggaagat gctgctaaag ttccgaaagg taacccgtac 1380

cacagcgcaa cctctggtga aatggcgatc tatcgtgcga actctctgat tctgcagaaa 1440

gcaggcgtta aagttgaaga accggttaaa caggtgctga acggccaaga agttgaagtt 1500

tggagccgta tcacctggaa accgaaatgg ggtatgatct tttctgacat taaaaagaaa 1560

gtgtctggta actgtgaagt ttcccagcgt tccactatgg cgatcaaagg tcgcaatgtg 1620

tttatcaaag atctgagcct ggacggtgct ctgatcgttg atagcatcga tgacgcggaa 1680

gttaaactgg gcggtctgat taaaaacaac ggctggacca tggaatctgt agattacaaa 1740

gatacctctg ttccggaaga aatccgtatc cgtggcttcc gtttcaacaa agttgaacag 1800

ctggaaaaga aactgaccca gccgggtaaa ttctctgttg aagattaact cgag 1854

<210> 15

<211> 531

<212> DNA

<213> 人工序列

<220>

<223> 编码PPA的核苷酸序列

<400> 15

atgagcttac tcaacgtccc tgcgggtaaa gatctgccgg aagacatcta cgttgttatt 60

gagatcccgg ctaacgcaga tccgatcaaa tacgaaatcg acaaagagag cggcgcactg 120

ttcgttgacc gcttcatgtc caccgcgatg ttctatccgt gcaactacgg ttacatcaac 180

cacaccctgt ctctggacgg tgacccagtt gacgtactgg tcccaactcc gtacccgctg 240

cagccgggtt ctgtgatccg ttgccgtccg gttggcgttc tgaaaatgac cgacgaagcc 300

ggtgaagatg cgaaactggt tgcggttccg cacagcaagc tgagcaaaga atacgatcac 360

attaaagacg ttaacgatct gcctgaactg ctgaaagcgc aaatcgctca cttcttcgag 420

cactacaaag acctcgaaaa aggcaagtgg gtgaaagttg aaggttggga aaacgcagaa 480

gccgctaaag ctgaaatcgt tgcctccttc gagcgcgcaa agaataaata a 531

Claims

1.一种合成尿苷二磷酸-6-叠氮-D-半乳糖的方法，包括以下步骤：

S1，化合物6在双歧杆菌来源的半乳糖激酶的作用下转变为化合物12，其反应式如下所示：

优选地，其中，所述半乳糖激酶选自：长双歧杆菌来源的半乳糖激酶(BiGalK)或在BiGalK的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有BiGalK活性的由BiGalK衍生的蛋白质，齿双歧杆菌来源的半乳糖激酶，链状双歧杆菌来源的半乳糖激酶，小鸡双歧杆菌来源的半乳糖激酶，动物双歧杆菌来源的半乳糖激酶，两歧双歧杆菌来源的半乳糖激酶，星状双歧杆菌来源的半乳糖激酶，短双歧杆菌来源的半乳糖激酶，假长双歧杆菌来源的半乳糖激酶，假链状双歧杆菌来源的半乳糖激酶；

S2，在拟南芥来源的糖焦磷酸化酶(AtUSP)或在AtUSP的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有AtUSP活性的由AtUSP衍生的蛋白质的作用下，化合物12转变为尿苷二磷酸-6-叠氮-D-半乳糖1α，其反应式如下所示：

2.根据权利要求1所述的方法，其中，所述BiGalK的氨基酸序列如SEQ ID NO:1所示；所述齿双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:4所示；所述链状双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:5所示；所述小鸡双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:6所示；所述动物双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:7所示；所述两歧双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:8所示；所述星状双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:9所示；所述短双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:10所示；所述假长双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:11所示；所述假链状双歧杆菌来源的半乳糖激酶的氨基酸序列如SEQ ID NO:12所示。

3.根据权利要求1所述的方法，其中，编码所述BiGalK的核苷酸序列如SEQ ID NO:13所示。

4.根据权利要求1所述的方法，

其中，步骤S1中，所述反应在三磷酸腺苷和金属Mg²⁺存在条件下进行，和/或

其中，步骤S1中，所述反应体系的pH值为6至9，优选为7.5。

5.根据权利要求1所述的方法，步骤S2中，所述AtUSP的氨基酸序列如SEQ ID NO:2所示，优选地，编码所述AtUSP的核苷酸序列如SEQ ID NO:14所示。

6.根据权利要求1所述的方法，

其中，步骤S2中，所述反应在三磷酸尿苷和金属Mg²⁺存在条件下进行，和/或

其中，步骤S2中，所述反应体系的pH值为6至9，优选为约7.5。

7.根据权利要求1所述的方法，步骤S2中，进一步使用无机焦磷酸化酶(PPA)或在PPA的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有PPA活性的由PPA衍生的蛋白质。

8.根据权利要求1所述的方法，步骤S2中，所述PPA的氨基酸序列如SEQ ID NO:3所示，优选地，编码所述PPA的核苷酸序列如SEQ ID NO:15所示。

9.一种合成尿苷二磷酸-6-叠氮-D-半乳糖的方法，包括以下步骤：

将化合物6、三磷酸腺苷、三磷酸尿苷作为底物，在金属Mg²⁺存在下，在权利要求1中步骤S1中所述的双歧杆菌来源的半乳糖激酶和步骤S2中所述的拟南芥来源的糖焦磷酸化酶(AtUSP)或在AtUSP的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有AtUSP活性的由AtUSP衍生的蛋白质、和任选的无机焦磷酸化酶(PPA)或在PPA的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有PPA活性的由PPA衍生的蛋白质的作用下，催化合成尿苷二磷酸-6-叠氮-D-半乳糖，其反应式如下：

10.根据权利要求1-9中任一项所述的方法，进一步包括分离纯化步骤，优选地，所述纯化步骤包括：在上述催化合成反应结束后，加入乙醇，固液分离，液体减压脱除溶剂，然后用去离子水溶解，聚丙烯酰胺凝胶柱(P-2)分离，薄层色谱法(TLC)检测分离效果；合并包含产物的组分，减压脱除溶剂，用去离子水溶解后，琼脂糖凝胶(QS-FF)离子交换柱分离，去除杂质；将含有产物的组分合并，减压脱除溶剂，然后用去离子水溶解，P-2凝胶柱分离收集含有产物的组分，优选经浓缩冻干，得到目标产物。