CN106434587B - 一种葡聚糖蔗糖酶及其应用 - Google Patents

一种葡聚糖蔗糖酶及其应用 Download PDF

Info

Publication number
CN106434587B
CN106434587B CN201610967869.2A CN201610967869A CN106434587B CN 106434587 B CN106434587 B CN 106434587B CN 201610967869 A CN201610967869 A CN 201610967869A CN 106434587 B CN106434587 B CN 106434587B
Authority
CN
China
Prior art keywords
thr
asp
asn
ala
gly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201610967869.2A
Other languages
English (en)
Other versions
CN106434587A (zh
Inventor
姜岷
张敏
古蕾
孔祥平
马江锋
董维亮
吴昊
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Tech University
Original Assignee
Nanjing Tech University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing Tech University filed Critical Nanjing Tech University
Priority to CN201610967869.2A priority Critical patent/CN106434587B/zh
Publication of CN106434587A publication Critical patent/CN106434587A/zh
Application granted granted Critical
Publication of CN106434587B publication Critical patent/CN106434587B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C12P19/08—Dextran
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01005—Dextransucrase (2.4.1.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明公开了一种葡聚糖蔗糖酶，所述葡聚糖蔗糖酶具有如SEQ ID NO.2所示的氨基酸序列，编码所述酶的核苷酸序列如SEQ ID NO.1所示。将该葡聚糖蔗糖酶基因构建重组质粒再导入宿主菌，即获得含有该基因的基因工程菌。本发明还提供了本发明的葡聚糖蔗糖酶在催化蔗糖制备水不溶性葡聚糖的应用，所述葡聚糖蔗糖酶在特定的催化条件下可高效催化蔗糖制备水不溶性葡聚糖，同时葡聚糖得率高达90%以上。

Description

一种葡聚糖蔗糖酶及其应用
技术领域
本发明涉及基因工程和酶工程领域，具体涉及一种新型的葡聚糖蔗糖酶及其催化制备水不溶性葡聚糖的方法。
背景技术
葡聚糖（Glucan）是一种以葡萄糖为单体的聚合物，在医药、精细化工、石油开采、食品、化妆品等领域有着广泛的应用，其中根据糖苷键的类型可分为α,葡聚糖和β-葡聚糖。α-葡聚糖中研究使用较多的为右旋糖酐（dextran），右旋糖酐具有较高的分子量，主要由D-葡糖吡喃糖以α,1-6键连接，支链点有α,1-2、1-3、1-4糖苷键连接形成的。随着微生物种类和生长条件的不同，其结构也有差别。由于结构的不同，葡聚糖可分为水不溶性葡聚糖和水溶性葡聚糖。水不溶性葡聚糖在代血浆、药物载体、凝胶分离、色谱分离柱等领域都有重要的应用。
葡聚糖蔗糖酶（Glucansucrase，亦称蔗糖-6-葡萄糖基转移酶）（EC.2.4.15）是一种糖苷转移酶，属于糖苷水解酶第70家族。肠膜明串珠菌分泌的葡聚糖蔗糖酶属于诱导型酶，诱导物和底物都为蔗糖，且在一定范围内，酶的产量与蔗糖浓度成正比。葡聚糖的生产方法有两种：微生物直接发酵法和酶合成法。直接发酵生产葡聚糖时，发酵液成分复杂，导致产物分离困难、葡聚糖分子大小难以控制、细胞核蛋白类杂质多等缺点，而利用纯酶催化制备葡聚糖，可以克服这些不足，生产出高产品质量的葡聚糖。
发明内容
本发明的第一目的是提供一种新型的葡聚糖蔗糖酶。
为实现上述目的，本发明采用如下技术方案：
一种葡聚糖蔗糖酶DsrU，其氨基酸序列如SEQ ID NO.2所示。
本发明还提供了编码该葡聚糖蔗糖酶DsrU的基因，其核苷酸序列如SEQ ID NO.1所示。
本发明所述葡聚糖蔗糖酶DsrU获取自肠膜明串珠菌Leuconostoc mesenteroidesM8，所述肠膜明串珠菌Leuconostoc mesenteroides M8为本实验室从泡菜发酵液中，通过自然筛选、鉴定得到的菌株。
本发明对肠膜明串珠菌Leuconostoc mesenteroides M8进行了全基因组测序，通过对其基因组序列分析和已报道的葡聚糖蔗糖酶的基因序列比对，得到一种未见报道的新型葡聚糖蔗糖酶基因。
本发明的另一目的是提供含有上述DsrU编码基因的表达载体及基因工程菌。所述表达载体的出发载体选用pET-28a，构建含DsrU基因的表达质粒pET28a-DsrU作为表达载体。
选用宿主菌E.coli BL21(DE3)构建含有DsrU基因的基因工程菌E.coli BL21(DE3)- pET28a-DsrU，高效表达该葡聚糖蔗糖酶基因。
所述的基因工程菌E.coli BL21(DE3)- pET28a-DsrU的构建方法为：将葡聚糖蔗糖酶DsrU基因导入到载体pET28a中进行外源表达。构建得到pET28a-DsrU表达质粒，然后将表达质粒pET28a-DsrU转化到大肠杆菌E.coli BL21(DE3)中，，通过选择性培养基挑选得到重组菌E.coli BL21(DE3)- pET28a-DsrU。
本发明的又一目的是提供上述葡聚糖蔗糖酶DsrU在制备水不溶性葡聚糖中的应用。
本发明提供了一种葡聚糖蔗糖酶DsrU制备水不溶性葡聚糖的具体方法，其技术路线包括如下步骤：
（1）将葡聚糖蔗糖酶DsrU基因导入到载体pET28a中进行外源表达。构建得到pET28a-DsrU表达质粒，将该质粒转入大肠杆菌感受态细胞中，通过选择性培养基挑选得到正确的基因工程菌。
（2）将重组菌E.coli pET28a-DsrU接种至种子培养基，进行种子培养。
（3）将培养好的种子培养液接种至发酵培养基中，30℃培养0~4 h，加0.1~1.0 mMIPTG，继续在25~30℃下培养24h后停止发酵，离心菌液后弃上清，用磷酸缓冲液重悬沉淀下来的细胞，超声破碎细胞，获得葡聚糖蔗糖酶粗酶液。
（4）向葡聚糖蔗糖酶的粗酶液中加入不同浓度的蔗糖，并调节pH至5~7，反应一段时间后6~12 h后，向反应液中加入冷乙醇，离心得葡聚糖沉淀，40-60℃下干燥得葡聚糖。
（5）将得到的葡聚糖加水复溶，溶解12h后，离心，取上清液，加入冷乙醇，离心，无沉淀，可判断上清液中无葡聚糖，及所述的葡聚糖为水不溶性葡聚糖。
所述步骤（1）中含葡聚糖蔗糖酶基因导入的载体为pET28a，胞内表达葡聚糖蔗糖酶DsrU有利于酶的回收和纯化。
所述步骤（1）中基因工程菌的构建方法：将葡聚糖蔗糖酶基因DsrU连接到质粒pET28a上，得到包含葡聚糖蔗糖酶基因的重组质粒pET28a-DsrU，然后将重组质粒pET28a-DsrU转化到大肠杆菌E.coli BL21(DE3)中，得到重组菌E.coli BL21(DE3)- pET28a-DsrU。
所述步骤（3）IPTG浓度为0.1 mM~1.0 mM。
所述步骤（4）中pH的初始值为7.0。
所述步骤（4）中需控制pH不低于5.0。
所述步骤（4）中加入冷乙醇的量是上清液的2-3倍，离心条件9,000 rpm，10 min。
所述步骤（5）中加入冷乙醇的量是上清液的2-3倍，离心条件9,000 rpm，10 min。
本发明的积极进步效果在于：
本发明提供了一种来自于肠膜明串珠菌Leuconostoc mesenteroides M8的葡聚糖蔗糖酶DsrU，并提供了利用葡聚糖蔗糖酶DsrU合成水不溶性葡聚糖的方法。该酶在特定的催化条件下可高效催化制备水不溶性葡聚糖，同时葡聚糖得率高达90%以上。本发明提供的新型葡聚糖蔗糖酶应用前景广阔，和以其制备的水不溶性葡聚糖的可被引用于药物载体、凝胶分离系统、分离色谱柱制备等领域。
附图说明
图1是E.coli BL21(DE3)- pET28a-DsrU重组菌株鉴定图；
其中，Line 1为DNA marker，Line 2为重组菌菌落PCR产物。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。
实施例1 产葡聚糖蔗糖酶的菌株筛选与鉴定
步骤1：取泡菜发酵液1mL于9mL的灭菌生理盐水中，并梯度稀释为10-4、10-5、10-6、10-7、10-8，取0.1mL的各梯度稀释液接入选择性平板中，于28℃培养2~3d，菌落长出后，挑取典型菌落，在含有万古霉素（30μl/mL）的MRS平板上划线，置于28℃培养箱中厌氧培养2~3d，观察菌落生长状况，若菌落生长，则进一步选取单菌落于含有万古霉素（30μl/mL）的MRS固体培养基上划线培养，重复上述步骤2~3次后，通过镜检确认为纯培养物、并具有典型明串珠菌细胞形态的菌株转入MRS固体培养基，低温保存备用。
步骤2：将步骤1中分离得到各菌株分别划线接种于葡聚糖生成固体培养基表面，置于28℃培养1~3d，随时观察葡聚糖生成情况，选取能够在培养基表面形成粘稠状多糖的菌株。最终挑取一株在能够在葡聚糖生成培养基上产生较多粘稠状的菌株，接入MRS液体培养基，30℃培养8-10 h后，转入含有15%的甘油的MRS中，低温保存，备用，命名为肠膜明串珠菌Leuconostoc mesenteroides M8。
实施例2产葡聚糖蔗糖酶重组大肠杆菌的构建
步骤1：将肠膜明串株菌Leuconostoc mesenteroidesM8进行试管培养活化，试管装液量为5 mL，接种量为1%，培养温度为30℃，培养时间为10-12 h，得到菌株生长旺盛的菌液，10000 rpm离心获取菌泥，利用革兰氏阳性菌的提基因组试剂盒提取肠膜明串株菌Leuconostoc mesenteroidesM8的基因组作为PCR模板。
步骤2：产葡聚糖蔗糖酶DsrU基因的获取及线性化克隆载体的制备
上游引物：
5’-CGCGGATCCGAATTCATGTTAGTAACAGCTGGTATTTTTTC-3’
下游引物：
5’-ACGGAGCTCGAATTCTTATATAGTATTTAAACGCTGTCTCTCTC-3’
以Leuconostoc mesenteroidesM8的基因组DNA为模板，以上述引入PCR扩增DsrU基因片段，反应条件为：94℃，5 min；（94℃ 45s，50℃ 45s，72℃ 300 s，35个循环），72℃，10min，扩增出DsrU基因，将PCR反应液进行纯化回收，备用。
将pET28a质粒用EcoRⅠ酶进行单酶切，酶切产物进行胶回收，作为线性化克隆载体，备用。
步骤3：制备E.coli BL21(DE3)感受态细胞
将取5μL的E.coli BL21菌体加入5mL的LB试管中，37℃，200 rpm，培养12h，取1mL培养液加入装有100mL LB培养基的摇瓶中，37℃，200 rpm，待菌体OD600为0.5-0.6时，取出摇瓶，冰上放置10min，4℃，4100rpm离心10min，弃上清，用2 mL 含有15%甘油的0.05MCaCl2溶液混悬沉淀，分装后于-80℃保存，备用。
步骤4：利用一步克隆技术构建目标菌株
将步骤2中回收的DsrU基因和线性化pET28a质粒载体，通过一部克隆转入E.coliBL21(DE3)中，具体方法如下：将装有200μL制作好的E.coli BL21(DE3)感受态细胞冰盒上解冻5-10 min；加入20 μL冷却的一步克隆反应液：手指轻弹，混匀，冰上放置30min；42℃，水浴90s，冰浴2min；加入900 μL 新鲜LB培养基，复苏细胞，37℃，200 rpm，培养1 h。取100μL涂布在抗性平板上，37℃培养箱中培养10-12 h，挑取菌落，使用步骤2中的引物进行PCR验证，能扩增出目的基因片段的菌落即为pET28a-DsrU质粒成功表达的菌株。如图1所示，DsrU PCR产物大小约为4500 bp与预期一致，说明构建菌株正确。
实施例3 构建菌株发酵制备葡聚糖蔗糖酶DsrU
种子培养基（g/L）：酵母粉5，蛋白胨10，NaCl 10，pH 7.0，121℃灭菌15 min。
发酵培养基（g/L）：酵母粉24，蛋白胨12，KH2PO4 2.31，K2HPO4 16.43，葡萄糖10，121℃灭菌15 min。
生产方法：取平板上单菌落接种于装液量为5 mL的试管中，30℃、200 rpm培养12h。制备一级种子液，将一级种子液接种于装有100mL发酵培养液的500 mL三角瓶中，200rpm，30℃培养0~4h后加入0.1~1.0 mM IPTG诱导DsrU目的蛋白的表达，继续在25~30℃下培养至24h，停止发酵后，将培养液于10000 r/min离心10min，弃上清，用pH为6.2的磷酸缓冲液重悬沉淀，超声破碎细胞获得粗酶液。如表1所示，不同IPTG诱导条件下，DsrU最高酶活可达4.9 U。
表1不同IPTG浓度诱导重组菌株对DsrU酶活的影响
酶活测定方法：
葡聚糖蔗糖酶的活力表示为在pH 5.4，30℃的条件下，1min中内反应生成1 µmol果糖所需的酶量为一个酶活单位U。
酶活反应体系：30℃，20 mM乙酸钠缓冲液（pH 5.4），0.05 g/L CaCl2，1 g/LNaNO3，100 g/L蔗糖。
具体方法为：
（1）取7支比色管编号0-7，分别加入浓度为1 mg/mL的果糖标准液0mL，0.2mL，0.4mL，0.6mL，0.8mL，1.0mL，1.2mL，补足蒸馏水至2.0mL，加入1.5mL DNS试剂，配成不同果糖浓度的反应液，将比色管摇匀后在沸水浴中加热5 min，取出，冷却至室温，蒸馏水定容至20 mL，颠倒混匀，用0号管调零，540 nm下测定吸光值，以吸光值为横坐标，果糖含量（μmol）为纵坐标制定标准曲线。
（2）取500 μL待测酶液与2.5mL反应体系混合，混匀后放入30℃水浴中反应20min，采用DNS测定反应体系中0 min和20min的还原糖（即为葡聚糖蔗糖酶催化反应生成的果糖）含量。
（3）取葡聚糖蔗糖酶反应体系的样品500μL，加蒸馏水至2.0 mL，加入1.5mL DNS，沸水浴5 min，取出冷却至室温，蒸馏水定容至20 mL，颠倒混匀，测定540 nm下测定吸光值，对照标曲计算得到相应还原糖的含量（μmol）。
（4）葡聚糖蔗糖酶活力（U, μmol/min）=(20 min 反应体系中还原糖总量-0 min反应体系中还原糖总量) /(20min*反应体系中加入的酶液体积)。
实施例4葡聚糖蔗糖酶催化蔗糖生产不溶性葡聚糖
步骤一：向葡聚糖蔗糖酶的粗酶液中加入不同浓度的蔗糖，使反应体系中蔗糖终浓度分别为50 g/L，100 g/L，150 g/L，200 g/L，30℃，反应时间为6~12h，优选8 h；反应体系pH为5.0~7.0，优选6.0；反应温度为30℃~40℃，优选30℃；酶添加量为1~10 U，优选10U，利用葡聚糖蔗糖酶催化蔗糖制备葡聚糖的产量如表2所示。
表2利用葡聚糖蔗糖酶催化蔗糖在优选条件下制备葡聚糖
蔗糖浓度 葡聚糖产量 葡聚糖得率*
50 g/L 23.6 g/L 94%
100 g/L 48.2 g/L 96%
150 g/L 72.7 g/L 97%
200 g/L 93.5 g/L 95%
*得率按照生产每g葡聚糖消耗的蔗糖中的葡萄糖量计算得出。
该新型酶具有高葡聚糖催化得率的特性，在50~200 g/L蔗糖条件下，葡聚糖得率均大于90%。
步骤二：向步骤一的反应液中加入冷乙醇，加入冷乙醇的体积为反应液的2-3倍，冰上静置30~60 min，9000 rpm离心10~20 min得沉淀物，弃上清，50-55℃下干燥至恒重（W1）得葡聚糖。
步骤三：将步骤二中干燥所得的葡聚糖，加水于40℃溶解12h，将溶解液9000 rpm离心10 min得沉淀物，50-55℃下干燥至恒重（W2），并收集上清，加入2-3倍体积的冷乙醇，冰上静置30~60 min，9000 rpm离心10~20 min，经比较，W2和步骤三中的W1值基本一致，且离心后并无沉淀产生，由此可得，步骤二中所产生的葡聚糖为水不溶性葡聚糖。
序列表
<110> 南京工业大学
<120> 一种葡聚糖蔗糖酶及其应用
<130> xb16110401
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 4518
<212> DNA
<213> Artificial Sequence
<220>
<223> 1
<220>
<221> CDS
<222> (1)..(4518)
<400> 1
atg tta gta aca gct ggt att ttt tct gct gtg ata ttt ggc gtt tcc 48
Met Leu Val Thr Ala Gly Ile Phe Ser Ala Val Ile Phe Gly Val Ser
1 5 10 15
ata gct aac gta agc gct gat agt att aac aat act agt att gcg gtg 96
Ile Ala Asn Val Ser Ala Asp Ser Ile Asn Asn Thr Ser Ile Ala Val
20 25 30
gcg caa tca aaa aat ata gca gtg gcc aca acg aca gct aca atg gac 144
Ala Gln Ser Lys Asn Ile Ala Val Ala Thr Thr Thr Ala Thr Met Asp
35 40 45
aaa gta act gat acg aca gct aca acg gac aaa gta act gat acg aca 192
Lys Val Thr Asp Thr Thr Ala Thr Thr Asp Lys Val Thr Asp Thr Thr
50 55 60
gct acg aca gat aaa gta act gat acg aca gct aca acg gac aaa gta 240
Ala Thr Thr Asp Lys Val Thr Asp Thr Thr Ala Thr Thr Asp Lys Val
65 70 75 80
act gat acg aca gct acg aca gat aaa gta act gac acg gca gct acg 288
Thr Asp Thr Thr Ala Thr Thr Asp Lys Val Thr Asp Thr Ala Ala Thr
85 90 95
acg gac aaa gtg gct gac acg aca gct aca acg gac aaa gta act gat 336
Thr Asp Lys Val Ala Asp Thr Thr Ala Thr Thr Asp Lys Val Thr Asp
100 105 110
acg aca gct aca acg ggc aaa gta act gac acg aca gct acg acg gac 384
Thr Thr Ala Thr Thr Gly Lys Val Thr Asp Thr Thr Ala Thr Thr Asp
115 120 125
aaa gtg gct gac acg aca gct acg aca gat aaa gtg gct gac acg aca 432
Lys Val Ala Asp Thr Thr Ala Thr Thr Asp Lys Val Ala Asp Thr Thr
130 135 140
gct aca acg gac aaa gta act gac acg gca gct aca acg gat aaa gca 480
Ala Thr Thr Asp Lys Val Thr Asp Thr Ala Ala Thr Thr Asp Lys Ala
145 150 155 160
act gac acg gca gct aca acg gac aaa gta gct gat aca aca gct acg 528
Thr Asp Thr Ala Ala Thr Thr Asp Lys Val Ala Asp Thr Thr Ala Thr
165 170 175
aca gat aaa gca gcg aac aca aca gtt aca cct tca gaa aaa tca aaa 576
Thr Asp Lys Ala Ala Asn Thr Thr Val Thr Pro Ser Glu Lys Ser Lys
180 185 190
agt att aag caa atc gat ggt aaa aca tat ttc att ggt gat gat ggt 624
Ser Ile Lys Gln Ile Asp Gly Lys Thr Tyr Phe Ile Gly Asp Asp Gly
195 200 205
cag ccc aag aaa aat ttt aca gcc att gtg gac ggt caa gta tta tat 672
Gln Pro Lys Lys Asn Phe Thr Ala Ile Val Asp Gly Gln Val Leu Tyr
210 215 220
ttc gac aaa gat acc ggt act ttg aca tca aat agt agt caa tat acc 720
Phe Asp Lys Asp Thr Gly Thr Leu Thr Ser Asn Ser Ser Gln Tyr Thr
225 230 235 240
gat ggt ttg gtc aat ata gga aat gag cat aat gcg gct tat tca ttg 768
Asp Gly Leu Val Asn Ile Gly Asn Glu His Asn Ala Ala Tyr Ser Leu
245 250 255
tct tcg gac agt ttt aca caa gtt gat ggc tat cta acg gct aac agt 816
Ser Ser Asp Ser Phe Thr Gln Val Asp Gly Tyr Leu Thr Ala Asn Ser
260 265 270
tgg tat cga cct aaa gat ata ttg aaa aat ggt aca aca tgg aca gct 864
Trp Tyr Arg Pro Lys Asp Ile Leu Lys Asn Gly Thr Thr Trp Thr Ala
275 280 285
tca aca gca aat gat ttt cga cca ttg ttg atg tct tgg tgg ccg gat 912
Ser Thr Ala Asn Asp Phe Arg Pro Leu Leu Met Ser Trp Trp Pro Asp
290 295 300
aaa gat acc caa gtt tca tac tta aaa tat atg caa tct gtt gga tta 960
Lys Asp Thr Gln Val Ser Tyr Leu Lys Tyr Met Gln Ser Val Gly Leu
305 310 315 320
tta tca gat gac gtt gta cta tca aac aag gat agt atg aat agt ttg 1008
Leu Ser Asp Asp Val Val Leu Ser Asn Lys Asp Ser Met Asn Ser Leu
325 330 335
aca gct atg gca ctg act gtt caa aaa aat att gaa gag aaa ata ggc 1056
Thr Ala Met Ala Leu Thr Val Gln Lys Asn Ile Glu Glu Lys Ile Gly
340 345 350
cta tta ggt acc act gac tgg ctt aag act gat atg gac caa atg gtt 1104
Leu Leu Gly Thr Thr Asp Trp Leu Lys Thr Asp Met Asp Gln Met Val
355 360 365
gac tca caa tca aat tgg aac att agt agt gag tct aaa gga aca gat 1152
Asp Ser Gln Ser Asn Trp Asn Ile Ser Ser Glu Ser Lys Gly Thr Asp
370 375 380
cat ttg caa ggt ggt gcg ctc cta tat gtg aat agt gat ttg aca cca 1200
His Leu Gln Gly Gly Ala Leu Leu Tyr Val Asn Ser Asp Leu Thr Pro
385 390 395 400
aat gct aat tct gat tat cgt tta tta aat cga acg cca acg aac caa 1248
Asn Ala Asn Ser Asp Tyr Arg Leu Leu Asn Arg Thr Pro Thr Asn Gln
405 410 415
aaa ggt caa att aca aca gac ggt aat caa ggt ggc tat gag atg ctg 1296
Lys Gly Gln Ile Thr Thr Asp Gly Asn Gln Gly Gly Tyr Glu Met Leu
420 425 430
ttg gcc aac gat gtt gat aat tct aac cct att gtt caa gct gaa caa 1344
Leu Ala Asn Asp Val Asp Asn Ser Asn Pro Ile Val Gln Ala Glu Gln
435 440 445
ttg aat tgg ttg tac tac atg atg aat atc ggt agt atc gtc caa aat 1392
Leu Asn Trp Leu Tyr Tyr Met Met Asn Ile Gly Ser Ile Val Gln Asn
450 455 460
gat cca acg gca aat ttt gat ggt tac aga gtt gat gct gtt gac aac 1440
Asp Pro Thr Ala Asn Phe Asp Gly Tyr Arg Val Asp Ala Val Asp Asn
465 470 475 480
gtg aat gct gat ttg ttg caa att gct ggc gat tac ttt aag gca gcg 1488
Val Asn Ala Asp Leu Leu Gln Ile Ala Gly Asp Tyr Phe Lys Ala Ala
485 490 495
tat gga acg gat aaa agt gat gct aat gca aac aat cac att tct atc 1536
Tyr Gly Thr Asp Lys Ser Asp Ala Asn Ala Asn Asn His Ile Ser Ile
500 505 510
tta gaa gac tgg gat aat aat gat ccg gcg tat gta aaa tca cag gga 1584
Leu Glu Asp Trp Asp Asn Asn Asp Pro Ala Tyr Val Lys Ser Gln Gly
515 520 525
aat aac caa tca acc atg gat ttt cca atg cat ttg gcg tta aag tat 1632
Asn Asn Gln Ser Thr Met Asp Phe Pro Met His Leu Ala Leu Lys Tyr
530 535 540
tca tta aat atg cca agt agt gct cgt agc ggg ttg gaa cca gat att 1680
Ser Leu Asn Met Pro Ser Ser Ala Arg Ser Gly Leu Glu Pro Asp Ile
545 550 555 560
gta aca agt cta gta aat cgc tca gaa gat tca aca gaa aat gaa gca 1728
Val Thr Ser Leu Val Asn Arg Ser Glu Asp Ser Thr Glu Asn Glu Ala
565 570 575
caa ccg aac tat tca ttt att cgg gca cat gat agt gaa gta caa acc 1776
Gln Pro Asn Tyr Ser Phe Ile Arg Ala His Asp Ser Glu Val Gln Thr
580 585 590
gtt att gcg caa att att aaa gat aaa att aat cct agt tct gat gat 1824
Val Ile Ala Gln Ile Ile Lys Asp Lys Ile Asn Pro Ser Ser Asp Asp
595 600 605
gga ttg act gtt tca acg gat gaa att gcc aaa gca ttc gaa ata tat 1872
Gly Leu Thr Val Ser Thr Asp Glu Ile Ala Lys Ala Phe Glu Ile Tyr
610 615 620
aat gcc gac gaa tta aaa gct gat aaa gag tac act gca tat aat ata 1920
Asn Ala Asp Glu Leu Lys Ala Asp Lys Glu Tyr Thr Ala Tyr Asn Ile
625 630 635 640
ccc tca tca tat gca ttg atg tta act aac aaa gat aca att cct cgt 1968
Pro Ser Ser Tyr Ala Leu Met Leu Thr Asn Lys Asp Thr Ile Pro Arg
645 650 655
gtg tat tat ggt gat ttg ttt acg gat gat gga caa tat atg tct gct 2016
Val Tyr Tyr Gly Asp Leu Phe Thr Asp Asp Gly Gln Tyr Met Ser Ala
660 665 670
aag tca cca tat tat gat gca ctt acc tca ttg ctt cag tcg cga gta 2064
Lys Ser Pro Tyr Tyr Asp Ala Leu Thr Ser Leu Leu Gln Ser Arg Val
675 680 685
aaa tat gtt tca ggt ggt caa tca atg aat atg gct tac ctg cat aat 2112
Lys Tyr Val Ser Gly Gly Gln Ser Met Asn Met Ala Tyr Leu His Asn
690 695 700
aat caa ggc ctt ttg aca tct gtc cgc tat ggg aaa ggt gcc atg aca 2160
Asn Gln Gly Leu Leu Thr Ser Val Arg Tyr Gly Lys Gly Ala Met Thr
705 710 715 720
gct act gat act ggt acc agt gaa act cgt aca caa ggt att ggg tta 2208
Ala Thr Asp Thr Gly Thr Ser Glu Thr Arg Thr Gln Gly Ile Gly Leu
725 730 735
att gtc agt aac aaa act gat tta aat ctg aat aat gat gag caa att 2256
Ile Val Ser Asn Lys Thr Asp Leu Asn Leu Asn Asn Asp Glu Gln Ile
740 745 750
gtg ctt aac atg ggc gct gta cac aaa aat caa gct tac cgt gca tta 2304
Val Leu Asn Met Gly Ala Val His Lys Asn Gln Ala Tyr Arg Ala Leu
755 760 765
atg tta agt act aaa gat gga ttg aaa att tat aat agt gat gac gat 2352
Met Leu Ser Thr Lys Asp Gly Leu Lys Ile Tyr Asn Ser Asp Asp Asp
770 775 780
gca ccg ctg gca tat aca gat gac cag ggt cgt ttg act ttt aaa tct 2400
Ala Pro Leu Ala Tyr Thr Asp Asp Gln Gly Arg Leu Thr Phe Lys Ser
785 790 795 800
gac atg gtt ttt ggt gtg agt gat gct cag gtt tct ggt tat tta gca 2448
Asp Met Val Phe Gly Val Ser Asp Ala Gln Val Ser Gly Tyr Leu Ala
805 810 815
gct tgg gtg cca gtt ggc gca aca gat gat caa gat gct agg aat caa 2496
Ala Trp Val Pro Val Gly Ala Thr Asp Asp Gln Asp Ala Arg Asn Gln
820 825 830
agc agt acg ata gct tca aca gat ggt aat acc tat cat tca aac gct 2544
Ser Ser Thr Ile Ala Ser Thr Asp Gly Asn Thr Tyr His Ser Asn Ala
835 840 845
gct ttg gat tct cag gtt att tat gaa ggt ttt tca aac ttt caa gcg 2592
Ala Leu Asp Ser Gln Val Ile Tyr Glu Gly Phe Ser Asn Phe Gln Ala
850 855 860
atg cca act cag act aat gaa tat acc aat gtt aaa att gct caa aat 2640
Met Pro Thr Gln Thr Asn Glu Tyr Thr Asn Val Lys Ile Ala Gln Asn
865 870 875 880
gcg caa cta ttc aaa aat ctc ggt ata act agt ttt gaa tta gca cct 2688
Ala Gln Leu Phe Lys Asn Leu Gly Ile Thr Ser Phe Glu Leu Ala Pro
885 890 895
caa tat cgg tca agc act gat aat agt ttc tta gat gca gtt gtt caa 2736
Gln Tyr Arg Ser Ser Thr Asp Asn Ser Phe Leu Asp Ala Val Val Gln
900 905 910
aat ggt tat gcc ttc acg gat cga tac gac att ggt tat aat acg cct 2784
Asn Gly Tyr Ala Phe Thr Asp Arg Tyr Asp Ile Gly Tyr Asn Thr Pro
915 920 925
aca aaa tac ggt aca gtt gat caa cta tta gac gct tta aga gca tta 2832
Thr Lys Tyr Gly Thr Val Asp Gln Leu Leu Asp Ala Leu Arg Ala Leu
930 935 940
cat gct caa gac att cag gct atc aat gat tgg gtc cca gac caa att 2880
His Ala Gln Asp Ile Gln Ala Ile Asn Asp Trp Val Pro Asp Gln Ile
945 950 955 960
tat aat ttg cct agc gaa gaa atc gtg act gct agt cga aca aat ggg 2928
Tyr Asn Leu Pro Ser Glu Glu Ile Val Thr Ala Ser Arg Thr Asn Gly
965 970 975
tca gga aag ata aac gaa act tcg gtt att aac aat acg tta tat gat 2976
Ser Gly Lys Ile Asn Glu Thr Ser Val Ile Asn Asn Thr Leu Tyr Asp
980 985 990
tct cat act gtc ggt ggc gga gag tat cag gca ttt tat ggt ggt gct 3024
Ser His Thr Val Gly Gly Gly Glu Tyr Gln Ala Phe Tyr Gly Gly Ala
995 1000 1005
ttc tta gat aag tta aaa caa gat ttt cct gag tta ttt gaa aca 3069
Phe Leu Asp Lys Leu Lys Gln Asp Phe Pro Glu Leu Phe Glu Thr
1010 1015 1020
aaa caa att tca acc ggt gaa gca atg aac cct gat gtc aaa atc 3114
Lys Gln Ile Ser Thr Gly Glu Ala Met Asn Pro Asp Val Lys Ile
1025 1030 1035
aca gaa tgg tca gct aag tat ttt aat ggc tca aac att caa ggg 3159
Thr Glu Trp Ser Ala Lys Tyr Phe Asn Gly Ser Asn Ile Gln Gly
1040 1045 1050
cgt ggt gca tgg tat gtt ctc aag gat tgg gcg aca aat caa tac 3204
Arg Gly Ala Trp Tyr Val Leu Lys Asp Trp Ala Thr Asn Gln Tyr
1055 1060 1065
ttt aat gtt tca agt ggt agc aaa ttt tta cct aaa caa tta tta 3249
Phe Asn Val Ser Ser Gly Ser Lys Phe Leu Pro Lys Gln Leu Leu
1070 1075 1080
ggt gaa aaa aca agc aca ggg ttt acc aac gtt gat aat ggt aag 3294
Gly Glu Lys Thr Ser Thr Gly Phe Thr Asn Val Asp Asn Gly Lys
1085 1090 1095
act gag ttt tac tct aca agt ggt tac caa gct aag aat acc ttt 3339
Thr Glu Phe Tyr Ser Thr Ser Gly Tyr Gln Ala Lys Asn Thr Phe
1100 1105 1110
att caa gat aat gac aat tgg tat tat ttt gat aat gat ggt tac 3384
Ile Gln Asp Asn Asp Asn Trp Tyr Tyr Phe Asp Asn Asp Gly Tyr
1115 1120 1125
atg gtt gtt ggt ggt caa gaa att aat ggt aaa aaa tat tac ttc 3429
Met Val Val Gly Gly Gln Glu Ile Asn Gly Lys Lys Tyr Tyr Phe
1130 1135 1140
ctg cca aat ggt gta gag tta caa gat gct tat ttg tcg gat ggt 3474
Leu Pro Asn Gly Val Glu Leu Gln Asp Ala Tyr Leu Ser Asp Gly
1145 1150 1155
act aat gta tat tat tac agt agt aca ggt cgt caa att act aat 3519
Thr Asn Val Tyr Tyr Tyr Ser Ser Thr Gly Arg Gln Ile Thr Asn
1160 1165 1170
caa tat tat cga gat tca gat agc aaa tgg cat tac ttc ttc tca 3564
Gln Tyr Tyr Arg Asp Ser Asp Ser Lys Trp His Tyr Phe Phe Ser
1175 1180 1185
gat ggg cat atg gcc gtt ggt tta aca aca att act gca gat aac 3609
Asp Gly His Met Ala Val Gly Leu Thr Thr Ile Thr Ala Asp Asn
1190 1195 1200
ggc acg gct aat caa caa tat ttt gac gtt aat ggt ata caa ctt 3654
Gly Thr Ala Asn Gln Gln Tyr Phe Asp Val Asn Gly Ile Gln Leu
1205 1210 1215
aag ggt gtt gct gtt aaa gac act gat gga aat gtt cgt tat ttt 3699
Lys Gly Val Ala Val Lys Asp Thr Asp Gly Asn Val Arg Tyr Phe
1220 1225 1230
gat ggt aac aca gga aac ttg gtt gtc agt aac tgg ggt aaa acg 3744
Asp Gly Asn Thr Gly Asn Leu Val Val Ser Asn Trp Gly Lys Thr
1235 1240 1245
gct gat ggt tca tgg tta tac cta aat gat aaa ggg ata gca gta 3789
Ala Asp Gly Ser Trp Leu Tyr Leu Asn Asp Lys Gly Ile Ala Val
1250 1255 1260
acg gga cag caa aat att aat ggc caa aat gtc tat ttt aac gaa 3834
Thr Gly Gln Gln Asn Ile Asn Gly Gln Asn Val Tyr Phe Asn Glu
1265 1270 1275
gat ggt gtt caa gtg aag ggc gaa gcc att acg gat act aat ggc 3879
Asp Gly Val Gln Val Lys Gly Glu Ala Ile Thr Asp Thr Asn Gly
1280 1285 1290
aat gta cat tac tat gat cgt agt act gga aat atg gtg act aat 3924
Asn Val His Tyr Tyr Asp Arg Ser Thr Gly Asn Met Val Thr Asn
1295 1300 1305
tca tgg gca gag tta ccg gac agc tcg tgg atg tat cta gat gtt 3969
Ser Trp Ala Glu Leu Pro Asp Ser Ser Trp Met Tyr Leu Asp Val
1310 1315 1320
aat ggc gtt gct gct att ggt gct caa aaa att aat ggt ctg gaa 4014
Asn Gly Val Ala Ala Ile Gly Ala Gln Lys Ile Asn Gly Leu Glu
1325 1330 1335
ttt tat ttc gat aat aac ggt aaa caa gtt aag aac gac aaa gtc 4059
Phe Tyr Phe Asp Asn Asn Gly Lys Gln Val Lys Asn Asp Lys Val
1340 1345 1350
att aat gat gat gga aca ata aac tat tac aca ggt atg agc ggt 4104
Ile Asn Asp Asp Gly Thr Ile Asn Tyr Tyr Thr Gly Met Ser Gly
1355 1360 1365
gaa aaa cta aaa aat gat ttt ggt gaa ttg cca gac gga tca tgg 4149
Glu Lys Leu Lys Asn Asp Phe Gly Glu Leu Pro Asp Gly Ser Trp
1370 1375 1380
atg tac ttg gat aat caa ggt aat gct gta ata ggc gac aaa aaa 4194
Met Tyr Leu Asp Asn Gln Gly Asn Ala Val Ile Gly Asp Lys Lys
1385 1390 1395
att aat ggt cag aat cta tac ttc aag ata gac gga caa cag gtt 4239
Ile Asn Gly Gln Asn Leu Tyr Phe Lys Ile Asp Gly Gln Gln Val
1400 1405 1410
aag ggt gaa acc tat ata gat gga gtt ggc aag atg cgt ttc tat 4284
Lys Gly Glu Thr Tyr Ile Asp Gly Val Gly Lys Met Arg Phe Tyr
1415 1420 1425
caa gct gcc agc ggt gaa atg gtg acg aat caa ttc gaa caa gtt 4329
Gln Ala Ala Ser Gly Glu Met Val Thr Asn Gln Phe Glu Gln Val
1430 1435 1440
gct gat ggc aaa tgg gct tac ttt ggt gct gat ggt gtg gct gtc 4374
Ala Asp Gly Lys Trp Ala Tyr Phe Gly Ala Asp Gly Val Ala Val
1445 1450 1455
act gga gag caa tat att gat ggt cag gat ctt ttc ttt gac cca 4419
Thr Gly Glu Gln Tyr Ile Asp Gly Gln Asp Leu Phe Phe Asp Pro
1460 1465 1470
act ggt tat caa gtg aag ggt gac aaa cgc aca att gac agc gtt 4464
Thr Gly Tyr Gln Val Lys Gly Asp Lys Arg Thr Ile Asp Ser Val
1475 1480 1485
ctc tat agc ttt gat aaa gac agt gga gag aga cag cgt tta aat 4509
Leu Tyr Ser Phe Asp Lys Asp Ser Gly Glu Arg Gln Arg Leu Asn
1490 1495 1500
act ata taa 4518
Thr Ile
1505
<210> 2
<211> 1505
<212> PRT
<213> Artificial Sequence
<220>
<223> Synthetic Construct
<400> 2
Met Leu Val Thr Ala Gly Ile Phe Ser Ala Val Ile Phe Gly Val Ser
1 5 10 15
Ile Ala Asn Val Ser Ala Asp Ser Ile Asn Asn Thr Ser Ile Ala Val
20 25 30
Ala Gln Ser Lys Asn Ile Ala Val Ala Thr Thr Thr Ala Thr Met Asp
35 40 45
Lys Val Thr Asp Thr Thr Ala Thr Thr Asp Lys Val Thr Asp Thr Thr
50 55 60
Ala Thr Thr Asp Lys Val Thr Asp Thr Thr Ala Thr Thr Asp Lys Val
65 70 75 80
Thr Asp Thr Thr Ala Thr Thr Asp Lys Val Thr Asp Thr Ala Ala Thr
85 90 95
Thr Asp Lys Val Ala Asp Thr Thr Ala Thr Thr Asp Lys Val Thr Asp
100 105 110
Thr Thr Ala Thr Thr Gly Lys Val Thr Asp Thr Thr Ala Thr Thr Asp
115 120 125
Lys Val Ala Asp Thr Thr Ala Thr Thr Asp Lys Val Ala Asp Thr Thr
130 135 140
Ala Thr Thr Asp Lys Val Thr Asp Thr Ala Ala Thr Thr Asp Lys Ala
145 150 155 160
Thr Asp Thr Ala Ala Thr Thr Asp Lys Val Ala Asp Thr Thr Ala Thr
165 170 175
Thr Asp Lys Ala Ala Asn Thr Thr Val Thr Pro Ser Glu Lys Ser Lys
180 185 190
Ser Ile Lys Gln Ile Asp Gly Lys Thr Tyr Phe Ile Gly Asp Asp Gly
195 200 205
Gln Pro Lys Lys Asn Phe Thr Ala Ile Val Asp Gly Gln Val Leu Tyr
210 215 220
Phe Asp Lys Asp Thr Gly Thr Leu Thr Ser Asn Ser Ser Gln Tyr Thr
225 230 235 240
Asp Gly Leu Val Asn Ile Gly Asn Glu His Asn Ala Ala Tyr Ser Leu
245 250 255
Ser Ser Asp Ser Phe Thr Gln Val Asp Gly Tyr Leu Thr Ala Asn Ser
260 265 270
Trp Tyr Arg Pro Lys Asp Ile Leu Lys Asn Gly Thr Thr Trp Thr Ala
275 280 285
Ser Thr Ala Asn Asp Phe Arg Pro Leu Leu Met Ser Trp Trp Pro Asp
290 295 300
Lys Asp Thr Gln Val Ser Tyr Leu Lys Tyr Met Gln Ser Val Gly Leu
305 310 315 320
Leu Ser Asp Asp Val Val Leu Ser Asn Lys Asp Ser Met Asn Ser Leu
325 330 335
Thr Ala Met Ala Leu Thr Val Gln Lys Asn Ile Glu Glu Lys Ile Gly
340 345 350
Leu Leu Gly Thr Thr Asp Trp Leu Lys Thr Asp Met Asp Gln Met Val
355 360 365
Asp Ser Gln Ser Asn Trp Asn Ile Ser Ser Glu Ser Lys Gly Thr Asp
370 375 380
His Leu Gln Gly Gly Ala Leu Leu Tyr Val Asn Ser Asp Leu Thr Pro
385 390 395 400
Asn Ala Asn Ser Asp Tyr Arg Leu Leu Asn Arg Thr Pro Thr Asn Gln
405 410 415
Lys Gly Gln Ile Thr Thr Asp Gly Asn Gln Gly Gly Tyr Glu Met Leu
420 425 430
Leu Ala Asn Asp Val Asp Asn Ser Asn Pro Ile Val Gln Ala Glu Gln
435 440 445
Leu Asn Trp Leu Tyr Tyr Met Met Asn Ile Gly Ser Ile Val Gln Asn
450 455 460
Asp Pro Thr Ala Asn Phe Asp Gly Tyr Arg Val Asp Ala Val Asp Asn
465 470 475 480
Val Asn Ala Asp Leu Leu Gln Ile Ala Gly Asp Tyr Phe Lys Ala Ala
485 490 495
Tyr Gly Thr Asp Lys Ser Asp Ala Asn Ala Asn Asn His Ile Ser Ile
500 505 510
Leu Glu Asp Trp Asp Asn Asn Asp Pro Ala Tyr Val Lys Ser Gln Gly
515 520 525
Asn Asn Gln Ser Thr Met Asp Phe Pro Met His Leu Ala Leu Lys Tyr
530 535 540
Ser Leu Asn Met Pro Ser Ser Ala Arg Ser Gly Leu Glu Pro Asp Ile
545 550 555 560
Val Thr Ser Leu Val Asn Arg Ser Glu Asp Ser Thr Glu Asn Glu Ala
565 570 575
Gln Pro Asn Tyr Ser Phe Ile Arg Ala His Asp Ser Glu Val Gln Thr
580 585 590
Val Ile Ala Gln Ile Ile Lys Asp Lys Ile Asn Pro Ser Ser Asp Asp
595 600 605
Gly Leu Thr Val Ser Thr Asp Glu Ile Ala Lys Ala Phe Glu Ile Tyr
610 615 620
Asn Ala Asp Glu Leu Lys Ala Asp Lys Glu Tyr Thr Ala Tyr Asn Ile
625 630 635 640
Pro Ser Ser Tyr Ala Leu Met Leu Thr Asn Lys Asp Thr Ile Pro Arg
645 650 655
Val Tyr Tyr Gly Asp Leu Phe Thr Asp Asp Gly Gln Tyr Met Ser Ala
660 665 670
Lys Ser Pro Tyr Tyr Asp Ala Leu Thr Ser Leu Leu Gln Ser Arg Val
675 680 685
Lys Tyr Val Ser Gly Gly Gln Ser Met Asn Met Ala Tyr Leu His Asn
690 695 700
Asn Gln Gly Leu Leu Thr Ser Val Arg Tyr Gly Lys Gly Ala Met Thr
705 710 715 720
Ala Thr Asp Thr Gly Thr Ser Glu Thr Arg Thr Gln Gly Ile Gly Leu
725 730 735
Ile Val Ser Asn Lys Thr Asp Leu Asn Leu Asn Asn Asp Glu Gln Ile
740 745 750
Val Leu Asn Met Gly Ala Val His Lys Asn Gln Ala Tyr Arg Ala Leu
755 760 765
Met Leu Ser Thr Lys Asp Gly Leu Lys Ile Tyr Asn Ser Asp Asp Asp
770 775 780
Ala Pro Leu Ala Tyr Thr Asp Asp Gln Gly Arg Leu Thr Phe Lys Ser
785 790 795 800
Asp Met Val Phe Gly Val Ser Asp Ala Gln Val Ser Gly Tyr Leu Ala
805 810 815
Ala Trp Val Pro Val Gly Ala Thr Asp Asp Gln Asp Ala Arg Asn Gln
820 825 830
Ser Ser Thr Ile Ala Ser Thr Asp Gly Asn Thr Tyr His Ser Asn Ala
835 840 845
Ala Leu Asp Ser Gln Val Ile Tyr Glu Gly Phe Ser Asn Phe Gln Ala
850 855 860
Met Pro Thr Gln Thr Asn Glu Tyr Thr Asn Val Lys Ile Ala Gln Asn
865 870 875 880
Ala Gln Leu Phe Lys Asn Leu Gly Ile Thr Ser Phe Glu Leu Ala Pro
885 890 895
Gln Tyr Arg Ser Ser Thr Asp Asn Ser Phe Leu Asp Ala Val Val Gln
900 905 910
Asn Gly Tyr Ala Phe Thr Asp Arg Tyr Asp Ile Gly Tyr Asn Thr Pro
915 920 925
Thr Lys Tyr Gly Thr Val Asp Gln Leu Leu Asp Ala Leu Arg Ala Leu
930 935 940
His Ala Gln Asp Ile Gln Ala Ile Asn Asp Trp Val Pro Asp Gln Ile
945 950 955 960
Tyr Asn Leu Pro Ser Glu Glu Ile Val Thr Ala Ser Arg Thr Asn Gly
965 970 975
Ser Gly Lys Ile Asn Glu Thr Ser Val Ile Asn Asn Thr Leu Tyr Asp
980 985 990
Ser His Thr Val Gly Gly Gly Glu Tyr Gln Ala Phe Tyr Gly Gly Ala
995 1000 1005
Phe Leu Asp Lys Leu Lys Gln Asp Phe Pro Glu Leu Phe Glu Thr
1010 1015 1020
Lys Gln Ile Ser Thr Gly Glu Ala Met Asn Pro Asp Val Lys Ile
1025 1030 1035
Thr Glu Trp Ser Ala Lys Tyr Phe Asn Gly Ser Asn Ile Gln Gly
1040 1045 1050
Arg Gly Ala Trp Tyr Val Leu Lys Asp Trp Ala Thr Asn Gln Tyr
1055 1060 1065
Phe Asn Val Ser Ser Gly Ser Lys Phe Leu Pro Lys Gln Leu Leu
1070 1075 1080
Gly Glu Lys Thr Ser Thr Gly Phe Thr Asn Val Asp Asn Gly Lys
1085 1090 1095
Thr Glu Phe Tyr Ser Thr Ser Gly Tyr Gln Ala Lys Asn Thr Phe
1100 1105 1110
Ile Gln Asp Asn Asp Asn Trp Tyr Tyr Phe Asp Asn Asp Gly Tyr
1115 1120 1125
Met Val Val Gly Gly Gln Glu Ile Asn Gly Lys Lys Tyr Tyr Phe
1130 1135 1140
Leu Pro Asn Gly Val Glu Leu Gln Asp Ala Tyr Leu Ser Asp Gly
1145 1150 1155
Thr Asn Val Tyr Tyr Tyr Ser Ser Thr Gly Arg Gln Ile Thr Asn
1160 1165 1170
Gln Tyr Tyr Arg Asp Ser Asp Ser Lys Trp His Tyr Phe Phe Ser
1175 1180 1185
Asp Gly His Met Ala Val Gly Leu Thr Thr Ile Thr Ala Asp Asn
1190 1195 1200
Gly Thr Ala Asn Gln Gln Tyr Phe Asp Val Asn Gly Ile Gln Leu
1205 1210 1215
Lys Gly Val Ala Val Lys Asp Thr Asp Gly Asn Val Arg Tyr Phe
1220 1225 1230
Asp Gly Asn Thr Gly Asn Leu Val Val Ser Asn Trp Gly Lys Thr
1235 1240 1245
Ala Asp Gly Ser Trp Leu Tyr Leu Asn Asp Lys Gly Ile Ala Val
1250 1255 1260
Thr Gly Gln Gln Asn Ile Asn Gly Gln Asn Val Tyr Phe Asn Glu
1265 1270 1275
Asp Gly Val Gln Val Lys Gly Glu Ala Ile Thr Asp Thr Asn Gly
1280 1285 1290
Asn Val His Tyr Tyr Asp Arg Ser Thr Gly Asn Met Val Thr Asn
1295 1300 1305
Ser Trp Ala Glu Leu Pro Asp Ser Ser Trp Met Tyr Leu Asp Val
1310 1315 1320
Asn Gly Val Ala Ala Ile Gly Ala Gln Lys Ile Asn Gly Leu Glu
1325 1330 1335
Phe Tyr Phe Asp Asn Asn Gly Lys Gln Val Lys Asn Asp Lys Val
1340 1345 1350
Ile Asn Asp Asp Gly Thr Ile Asn Tyr Tyr Thr Gly Met Ser Gly
1355 1360 1365
Glu Lys Leu Lys Asn Asp Phe Gly Glu Leu Pro Asp Gly Ser Trp
1370 1375 1380
Met Tyr Leu Asp Asn Gln Gly Asn Ala Val Ile Gly Asp Lys Lys
1385 1390 1395
Ile Asn Gly Gln Asn Leu Tyr Phe Lys Ile Asp Gly Gln Gln Val
1400 1405 1410
Lys Gly Glu Thr Tyr Ile Asp Gly Val Gly Lys Met Arg Phe Tyr
1415 1420 1425
Gln Ala Ala Ser Gly Glu Met Val Thr Asn Gln Phe Glu Gln Val
1430 1435 1440
Ala Asp Gly Lys Trp Ala Tyr Phe Gly Ala Asp Gly Val Ala Val
1445 1450 1455
Thr Gly Glu Gln Tyr Ile Asp Gly Gln Asp Leu Phe Phe Asp Pro
1460 1465 1470
Thr Gly Tyr Gln Val Lys Gly Asp Lys Arg Thr Ile Asp Ser Val
1475 1480 1485
Leu Tyr Ser Phe Asp Lys Asp Ser Gly Glu Arg Gln Arg Leu Asn
1490 1495 1500
Thr Ile
1505

Claims (7)

1.一种葡聚糖蔗糖酶，其特征在于，其氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述葡聚糖蔗糖酶的基因，其特征在于，其核苷酸序列如SEQ IDNO.1所示。
3.含有权利要求2所述核苷酸序列的表达载体。
4.含有权利要求2所述核苷酸序列的基因工程菌。
5.权利要求1所述一种葡聚糖蔗糖酶在制备水不溶性葡聚糖中的应用。
6.根据权利要求5所述的应用，其特征在于，利用葡聚糖蔗糖酶催化蔗糖制备水不溶性葡聚糖，反应条件为pH 5.0～7.0；反应温度为30～40℃；酶添加量为1～10U；反应时间为6～12h。
7.根据权利要求5或6所述的应用，其特征在于，利用葡聚糖蔗糖酶催化蔗糖制备水不溶性葡聚糖，反应条件为pH 6.0；反应温度30℃；酶添加量10U；反应时间8h。
CN201610967869.2A 2016-11-04 2016-11-04 一种葡聚糖蔗糖酶及其应用 Active CN106434587B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201610967869.2A CN106434587B (zh) 2016-11-04 2016-11-04 一种葡聚糖蔗糖酶及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201610967869.2A CN106434587B (zh) 2016-11-04 2016-11-04 一种葡聚糖蔗糖酶及其应用

Publications (2)

Publication Number Publication Date
CN106434587A CN106434587A (zh) 2017-02-22
CN106434587B true CN106434587B (zh) 2019-07-02

Family

ID=58181048

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201610967869.2A Active CN106434587B (zh) 2016-11-04 2016-11-04 一种葡聚糖蔗糖酶及其应用

Country Status (1)

Country Link
CN (1) CN106434587B (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107083371A (zh) * 2017-04-27 2017-08-22 南京工业大学 一种新型葡聚糖蔗糖酶及其在催化制备水溶性葡聚糖中的应用
CN108969879B (zh) * 2018-06-05 2021-06-22 南京工业大学 一种复合微针及微针贴片
CN114574461A (zh) * 2022-03-23 2022-06-03 南京工业大学 一种酶法制备不溶葡聚糖并进行化学修饰的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100716797B1 (ko) * 2004-12-03 2007-05-14 전남대학교산학협력단 당전이 효소를 이용한 당전이 화합물의 유도체 제조방법 및 이로부터 제조된 유도체
KR101048355B1 (ko) * 2009-01-07 2011-07-14 전라남도 클로로제닉산 배당체의 제조방법
KR101091138B1 (ko) * 2009-03-26 2011-12-09 한국생명공학연구원 류코노스톡 락티스로부터 유래된 글루칸수크라제 및 그 제조방법
US9644190B2 (en) * 2014-02-06 2017-05-09 The United States Of America, As Represented By The Secretary Of Agriculture Modified glucansucrase and related methods

Also Published As

Publication number Publication date
CN106434587A (zh) 2017-02-22

Similar Documents

Publication Publication Date Title
CN109486786A (zh) 一种环糊精葡萄糖基转移酶突变体
TW200914610A (en) Isopropyl alcohol-producing bacteria and method for producing isopropyl alcohol using the same
CN106434587B (zh) 一种葡聚糖蔗糖酶及其应用
CN107058200B (zh) 制备l-抗坏血酸-2-葡萄糖苷的方法
CN113652385B (zh) 一种高产乳酰-n-四糖的微生物的构建方法及应用
CN107603938A (zh) 过表达异源谷氨酰胺合成酶的基因工程菌及其构建方法
CN102191208A (zh) 高产多杀菌素的基因工程菌及其制备方法
CN113930347B (zh) 一种能够合成褪黑素的绿色木霉工程菌及其构建方法与应用
CN105950590B (zh) 一种ι-卡拉胶酶的制备方法
CN114480465B (zh) 一种产生2’-岩藻糖基乳糖的枯草芽孢杆菌及其应用
CN107083371A (zh) 一种新型葡聚糖蔗糖酶及其在催化制备水溶性葡聚糖中的应用
CN109576239A (zh) 耐热磷酸化酶及其应用
CN110343654A (zh) 一种产蔗糖磷酸化酶的基因工程菌
CN109321508A (zh) 产heparosan的基因工程菌及其应用
CN109456950A (zh) 一种环糊精葡萄糖基转移酶的突变体及其应用
CN104611284A (zh) 一种环糊精葡萄糖基转移酶生产菌株及其应用
CN108330113B (zh) 一株高底物专一性酰基转移酶的珍贵束丝放线菌及其应用
CN105969751A (zh) 一种β-葡萄糖苷酶基因及其应用
Jin et al. Cephamycin C production is regulated by relA and rsh genes in Streptomyces clavuligerus ATCC27064
CN107760609A (zh) 一种适宜在碱性条件下生产高分子、无色素普鲁兰多糖的菌株及其应用
US10465177B2 (en) Maltooligosyl trehalose trehalohydrolase (MTHase) mutant and application thereof
CN105907816A (zh) 一种酶法生产大元环糊精的方法
CN105087515B (zh) 一种嘌呤核苷磷酸化酶的制备方法及应用
RU2731289C2 (ru) Способ конструирования на основе бактерий рода Rhodococcus штамма-биокатализатора, обладающего нитрилазной активностью и повышенной операционной стабильностью, рекомбинантный штамм бактерий Rhodococcus rhodochrous, полученный таким способом, способ синтеза акриловой кислоты с использованием этого штамма в качестве биокатализатора
CN111548978B (zh) 一种产甘露聚糖的枯草芽孢杆菌及其应用

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant