CN109423483A - 葡萄糖氧化酶突变体 - Google Patents

葡萄糖氧化酶突变体 Download PDF

Info

Publication number
CN109423483A
CN109423483A CN201710760957.XA CN201710760957A CN109423483A CN 109423483 A CN109423483 A CN 109423483A CN 201710760957 A CN201710760957 A CN 201710760957A CN 109423483 A CN109423483 A CN 109423483A
Authority
CN
China
Prior art keywords
gly
ala
leu
val
thr
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.)
Granted
Application number
CN201710760957.XA
Other languages
English (en)
Other versions
CN109423483B (zh
Inventor
吴秀秀
邵弨
黄亦钧
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.)
Qingdao Vland Biotech Group Co Ltd
Original Assignee
Qingdao Vland Biotech Group Co Ltd
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 Qingdao Vland Biotech Group Co Ltd filed Critical Qingdao Vland Biotech Group Co Ltd
Priority to CN201710760957.XA priority Critical patent/CN109423483B/zh
Publication of CN109423483A publication Critical patent/CN109423483A/zh
Application granted granted Critical
Publication of CN109423483B publication Critical patent/CN109423483B/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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03004—Glucose oxidase (1.1.3.4)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Polymers & Plastics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Animal Husbandry (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

本发明的目的是提供耐热的葡萄糖氧化酶突变体。包含V104I单个位点突变的葡萄糖氧化酶突变体在70℃处理2.5min后，残余酶活为29.02％，显著高于野生型；而为了进一步提高葡萄糖氧化酶三点突变体GOD‑K(A65R，H275F和A416K)的耐热性，申请人分别引入V104I单点突变，以及V104I与T32L和/或A290L的突变位点组合，结果显示：所获得的突变体的耐热性均显著高于GOD‑K，所述获得的突变体在75℃处理2.5min后，酶活残留率达高达83.10％，比GOD‑K提高了46.42％，因此更适合应用于饲料加工领域。

Description

葡萄糖氧化酶突变体
技术领域
本发明属于基因改造技术领域，具体涉及一种葡萄糖氧化酶突变体及其应用。
背景技术
葡萄糖氧化酶(glucose oxidase,E.C.1.1.3.4,GOD)在有氧条件下能专一性地催化β-D-葡糖生成葡萄糖酸和过氧化氢。葡萄糖氧化酶是同型二聚体分子，含有2个黄素腺嘌呤二核苷酸(FAD)结合位点。每个单体含有2个完全不同的区域：一个与部分FAD非共价但紧密结合，主要为声折叠；另一个与底物β-D-葡萄糖结合，由4个α-螺旋支撑1个反平行的β-折叠。
葡萄糖氧化酶广泛分布于动植物和微生物体内。但动植物组织中葡萄糖氧化酶含量有限，而微生物由于具有来源广泛、生长周期短等优点被广泛用作生产葡萄糖氧化酶的来源。但通常天然菌株产葡萄糖氧化酶水平不高，难以直接用来生产葡萄糖氧化酶。一方面我们可以通过菌株诱变、优化菌株的发酵条件等传统方法，获得葡萄糖氧化酶高产菌株；另一方面，通过基因重组等方法获得葡萄糖氧化酶高产菌株，采用重组工程菌获得葡萄糖氧化酶。
葡萄糖氧化酶在工业生产中有诸多用途，在食品工业中，能够除氧保鲜，去葡萄糖，经过精制的葡萄糖氧化酶，可以用于医疗诊断,如比色血糖试纸和基于生物传感器的血糖检测仪。在饲料工业中，作为饲料添加剂，葡萄糖氧化酶可以解除肠道霉菌毒素中毒，减少饲料霉菌超标危害；同时，葡萄糖氧化酶催化动物肠道内的葡萄糖产生葡萄糖酸，葡萄糖酸可降低胃内食糜pH值，有效抑制有害菌繁殖，促进有益菌生长，还能激活胃蛋白酶活性，有利于蛋白质、维生素、矿物质等营养成分的消化吸收，从而提高饲料转化效率；添加了葡萄糖氧化酶的畜禽饲料可以通过清除动物因应激反应产生的自由基，保护肠道上皮细胞完整，阻挡病原体入侵。
然而，在饲料加工过程中的短暂高温过程会造成葡萄糖氧化酶的失活，影响葡萄糖氧化酶的应用效果，在葡萄糖氧化酶产量得到保证的前提下，葡萄糖氧化酶的耐温性能越来越受到关注，因此，通过基因工程手段开发耐温性强的葡萄糖氧化酶迫在眉睫。
发明内容
本发明的目的是提供葡萄糖氧化酶突变体，所提供的葡萄糖氧化酶突变体的耐热性得到显著提高，有利于其在饲料领域的广泛应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种葡萄糖氧化酶突变体，其具有(Ⅰ)、(Ⅱ)或(Ⅲ)所示的氨基酸序列中任意一个：
(Ⅰ)与葡萄糖氧化酶的氨基酸序列SEQ ID NO:1具有至少95％同源性的序列；
(Ⅱ)具有所述(Ⅰ)中所述的葡萄糖氧化酶的至少一个免疫表位，且所述葡萄糖氧化酶的氨基酸序列经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列；
(Ⅲ)由如SEQ ID NO:2所示的核苷酸序列或其互补序列或因遗传密码的简并性而与如SEQ ID NO:2所示的核苷酸序列或其互补序列的核苷酸序列不同的序列编码的氨基酸序列；
在本发明的一些实施例中，所述取代为取代1个或1个以上氨基酸。
在本发明的另一些实施例中，所述取代包括氨基酸序列为SEQ ID NO:1的葡萄糖氧化酶的第104位氨基酸发生了取代。
作为实施例的一种具体记载，所述的取代是氨基酸序列为SEQ ID NO:1的葡萄糖氧化酶的第104位氨基酸由V变为I。
在本发明的另一些实施例中，所述突变体的氨基酸序列为SEQ ID NO:3，其一种编码基因的核苷酸序列为SEQ ID NO：4。
在本发明的另一些实施例中，所述的取代还包括第65位、第275位和第416位氨基酸同时发生了取代。
在本发明的另一些实施例中，所述取代还包括第65位氨基酸由A变为R，第275位氨基酸由H变为F,第416位氨基酸由A变为K。
在本发明的另一些实施例中，所述取代还包括第32位或第290位氨基酸发生了取代。
在本发明的另一些实施例中，所述取代还包括第32位氨基酸由T变为L，或第290位氨基酸由A变为L。
在本发明的另一些实施例中，所述取代还包括第32位和第290位氨基酸同时发生了取代。
在本发明的另一些实施例中，所述取代还包括第32位氨基酸由T变为L，第290位氨基酸由A变为L。
在本发明的另一些实施例中，所述葡萄糖氧化酶突变体具有如SEQ ID NO:7或SEQID NO:9或SEQ ID NO:11或SEQ ID NO:13所示的氨基酸序列。
本发明还提供了编码上述葡萄糖氧化酶突变体的DNA分子。
在本发明的一些实施例中，编码上述葡萄糖氧化酶突变体的DNA分子具有如SEQID NO：8或SEQ ID NO:10或SEQ ID NO:12或SEQ ID NO:14所示的核苷酸序列。
本发明还提供了携带上述DNA分子的重组表达载体。
本发明还提供了一种宿主细胞，包含上述重组表达载体。
在本发明的一些实施例中，宿主细胞为毕赤酵母(Pichia pastoris)。
本发明还提供了上述葡萄糖氧化酶突变体在饲料中的应用。
本发明提供的包含V104I单个位点(简称单点)突变的葡萄糖氧化酶突变体GOD104在70℃处理2.5min后，残余酶活为29.02％，显著高于野生型葡萄糖氧化酶GOD；而为了进一步提高葡萄糖氧化酶三点突变体GOD-K(A65R，H275F和A416K)的耐热性，申请人分别引入V104I单点突变，以及V104I与T32L和/或A290L的突变位点组合，结果显示：所获得的突变体的耐热性均显著高于GOD-K，所述获得的突变体在75℃处理2.5min后，酶活残留率达高达83.10％，比GOD-K提高了46.42％，因此更适合应用于饲料加工领域。
具体实施方式
本发明具体实施例所使用的实验材料和试剂如下：
菌株与载体：大肠杆菌DH5α、毕赤酵母GS115、载体pPIC9K、氨苄青霉素(Amp)购自Invitrogen公司。
酶与试剂盒：PCR酶及连接酶购买自Takara公司，限制性内切酶购自Fermentas公司，质粒提取试剂盒及胶纯化回收试剂盒购自Omega公司，GeneMorph II随机诱变试剂盒购自北京博迈斯生物科技有限公司。
培养基配方：
大肠杆菌培养基(LB培养基)：0.5％酵母提取物，1％蛋白胨，1％NaCl，pH7.0)；
LB-AMP培养基：LB培养基加100μg/mL氨苄青霉素；
酵母培养基(YPD培养基)：1％酵母提取物，2％蛋白胨，2％葡萄糖；
酵母筛选培养基(MD培养基)：2％葡萄糖，2％琼脂糖，1.34％YNB，4×10-5生物素；
BMGY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10-5生物素，1％甘油；
BMMY培养基：2％蛋白胨，1％酵母提取物，100mM磷酸钾缓冲液(pH6.0)，1.34％YNB，4×10-5生物素，0.5％甲醇。
下面结合实施例对本发明进行详细的描述。
实施例1葡萄糖氧化酶耐热突变体的获得
1.1葡萄糖氧化酶基因的扩增
以黑曲霉(Aspergillus niger)基因组为模板进行PCR扩增，PCR引物GOD-F1、GOD-R1如下：
GOD-F1：GGTATTGAGGCATCTTTGTTGAC；
GOD-R1：TTATTGCATAGAAGCGTAATC；
胶回收PCR产物，连接pEASY-T载体，转化至大肠杆菌DH5α中，挑取正确的转化子进行测序。测序结果显示，扩增得到的基因片段的核苷酸序列为SEQ ID NO：2，其编码的氨基酸序列为SEQ ID NO：1。通过NCBI BLAST比对发现，SEQ ID NO：1与来源于黑曲霉的葡萄糖氧化酶序列相似性高达100％，从而确定通过PCR获得的基因为葡萄糖氧化酶基因，命名为出发酶GOD。
1.2葡萄糖氧化酶突变体基因的扩增及合成
为了提高上述葡萄糖氧化酶GOD的耐热性，申请人通过定向进化技术对该酶进行了大量突变的筛选，设计PCR引物GOD-F2、GOD-R2如下：
GOD-F2：GGCGAATTCGGTATTGAGGCATCTTTGTTGAC(下划线为限制性内切酶EcoRI识别位点)
GOD-R2：ATAGCGGCCGCTTATTGCATAGAAGCGTAATC(下划线为限制性内切酶Not I识别位点)
以GOD基因为模板，以上述引物用GeneMorph II随机突变PCR试剂盒(Stratagene)进行PCR扩增，胶回收PCR产物，EcoRI、Not I进行酶切处理后与经同样酶切后的pET21a载体连接，转化至大肠杆菌BL21(DE3)中，涂布于LB+Amp平板，37℃倒置培养，待转化子出现后，用牙签逐个挑至96孔板，每个孔中加入150ul含有0.1mM IPTG的LB+Amp培养基，37℃220rpm培养6h左右，离心弃上清，菌体用缓冲液重悬，反复冻融破壁，获得含有葡萄糖氧化酶的大肠杆菌裂解液。
分别取出10μL裂解液至两块新的96孔板，其中一块于70℃处理5min后，两块96孔板都加入40μL底物，于30℃反应30min后，DNS法测定生成的还原糖，计算高温处理的酶液相比于未处理酶液的相对酶活。
进一步地，申请人通过对上述转化子分别进行测序以及序列比对分析，获得了突变体对应的突变位点信息。
实验结果表明，有些突变(例如S13F，A83P，T257K)对葡萄糖氧化酶GOD的耐热性没有影响，有些突变(例如K270D，R334E，T387P，H443D，N470K，D489P)甚至使其耐热性或酶活变得更差了；另外还有些突变，虽然能提高葡萄糖氧化酶对温度的耐受性，但突变后其酶学性质发生了显著的变化，这些均不符合要求，具体结果下表1。
表1葡萄糖氧化酶突变体耐热性比较
最终，申请人筛选到既能显著提高葡萄糖氧化酶GOD的耐热性，又不会影响其酶活及原有酶学性质的新的突变位点：V104I。
将含V104I单点突变的葡萄糖氧化酶突变体命名为GOD104，其编码氨基酸序列为SEQ ID NO：3；编码核苷酸序列为SEQ ID NO：4。
用引物GOD-F2、GOD-R2对上述突变体分别进行PCR扩增，引物两端引入EcoRI、NotI位点。PCR反应条件为：94℃变性5min；然后94℃变性30s，56℃复性30s，72℃延伸1min，30个循环后，72℃保温10min。琼脂糖凝胶电泳结果显示，GOD104基因均为大小1800bp左右的片段。
通过上述同样的PCR方法扩增得到野生型葡萄糖氧化酶GOD的基因片段。
1.3毕赤酵母工程菌株的构建
将上述克隆得到的葡萄糖氧化酶突变体基因GOD104，通过EcoRI和NotI位点与表达载体pPIC9K相连接，构建表达载体pPIC9K-GOD104。
将表达载体pPIC9K-GOD104用Sal I进行线性化，表达载体线性化片段通过电穿孔法转化毕赤酵母GS115，在MD平板上筛选得到毕赤酵母重组菌株GS115/pPIC9K-GOD104，然后在含不同浓度遗传霉素的YPD平板上筛选多拷贝的转化子。
将筛选得到的单点突变体的转化子命名为毕赤酵母GOD104(Pichia pastorisGOD104)，将其转接于BMGY培养基中，30℃、250rpm振荡培养1d；再转入BMMY培养基中，30℃、250rpm振荡培养；每天添加0.5％的甲醇，诱导表达4d；离心去除菌体，得到含葡萄糖氧化酶GOD104的发酵上清液；将其进行SDS-PAGE电泳检测分析。结果显示，发酵上清液中葡萄糖氧化酶突变体GOD104的分子量大小约为64kDa，与理论分子量大小相同。
通过上述同样的酶切连接方法将野生型葡萄糖氧化酶基因GOD克隆到毕赤酵母GS115宿主中，构建得到重组表达野生型葡萄糖氧化酶的毕赤酵母工程菌，命名为毕赤酵母GOD(Pichia pastoris GOD)。摇瓶水平发酵毕赤酵母GOD，30℃、250rpm振荡培养；每天添加0.5％的甲醇，诱导表达4d；离心去除菌体，得到含野生型葡萄糖氧化酶GOD的发酵上清液。
(1)葡萄糖氧化酶酶活单位的定义
在pH6.0，30℃条件下，每分钟能把1μmol的β-D-葡萄糖氧化为D-葡萄糖酸
和过氧化氢所需要的酶量，定义为1个酶活力单位(IU)。
(2)酶活测定方法
将粗酶液直接用缓冲液稀释至约10U/mL。取4支150*15的试管，加入2ml缓冲液、0.3ml葡萄糖、0.4ml苯酚、0.1ml 4-氨基安替吡啉、0.1ml辣根过氧化物酶，30℃预热5min。向其中一管加入0.1ml蒸馏水，作为空白调零。水浴锅放在分光光度计旁以方便操作，向样品管中加入0.1ml样品溶液，此时开始计时，涡旋混匀后立即在500nm波长处用1cm比色杯比色。读取0.5min时吸光度值为A0，再反应1min后，读取吸光度值A1，得出ΔA500＝A1-A0。
酶活计算公式：
试样中酶活力X1(U/mL或U/g)按照如下公式计算：
X1＝ΔA500×f×B×1000/(887×t×A×d)＝33.82×ΔA500×f
式中：
f-------------------酶液稀释倍数
B-------------------反应液体积(3ml)
1000----------------消光系数单位转换系数
887-----------------消光系数(L·mol-1·cm-1)
t-------------------反应时间(min)，即读数A1与A0之间的时间差值1min。
A-------------------加入样品体积(0.1ml)
d-------------------比色皿的厚度(cm)
(3)酶活测定结果
按照上述方法进行发酵上清液的酶活测定，结果显示：重组表达野生型葡萄糖氧化酶的毕赤酵母GOD发酵上清液的酶活为115U/ml，而重组表达葡萄糖氧化酶突变体的毕赤酵母GOD104发酵上清液的酶活分别达到103U/mL。
1.4发酵验证
在10升发酵罐上分别进行毕赤酵母GOD和毕赤酵母GOD104的发酵，发酵使用的培养基配方为：硫酸钙1.1g/L、磷酸二氢钾5.5g/L、磷酸二氢铵55g/L、硫酸钾20.3g/L、硫酸镁16.4g/L、氢氧化钾1.65g/L、消泡剂0.05％。
发酵工艺：pH值5.0、温度30℃、搅拌速率300rpm、通风量1.0-1.5(v/v)、溶氧控制在20％以上。
整个发酵过程分为三个阶段：第一阶段为菌体培养阶段，按7％比例接入种子，30℃培养24-26h，以补完葡萄糖为标志；第二阶段为饥饿阶段，当葡萄糖补完之后，不流加任何碳源，当溶氧上升至80％以上表明该阶段结束，为期约30-60min；第三阶段为诱导表达阶段，流加甲醇诱导，并且保持溶氧在20％以上，培养时间在150-180h之间。发酵结束后，发酵液通过板框过滤机处理后获得粗酶液。
采用实施例1中1.3所述葡萄糖氧化酶酶活测定方法对粗酶液进行酶活检测，结果显示，重组表达野生型葡萄糖氧化酶的毕赤酵母GOD最终的发酵酶活为3971U/ml，而重组表达葡萄糖氧化酶突变体的毕赤酵母GOD104最终的发酵酶活达到3550U/ml。
1.5葡萄糖氧化酶耐热性质测定
将上述粗酶液用pH 6.0的乙酸-乙酸钠缓冲液稀释后，在60℃处理10min、65℃处理5min、70℃处理2.5min后，分别测定其残留酶活，以未处理样品的酶活计100％，计算残留酶活率。具体结果见表2。
残留酶活率＝残留酶活/未处理样品的酶活×100％。
表2葡萄糖氧化酶耐热性比较
注：与GOD相比，*示P＜0.05；#示P＜0.01.
由表2的结果可知，与野生型葡萄糖氧化酶相比，本发明提供的包含V104I单点突变的葡萄糖氧化酶突变体GOD104的酶活残留率得到显著提高。从而说明V104I单点突变导致葡萄糖氧化酶的耐热性得到大幅提升，比野生型更适合在工业生产中的应用，因而市场前景广阔。
实施例2葡萄糖氧化酶耐热突变组合的筛选
为了提高葡萄糖氧化酶GOD的耐热性，申请人对筛选获得的第65位、第275位和第416位氨基酸位点进行突变组合研究，第65位氨基酸由A变为R，第275位氨基酸由H变为F，第416位氨基酸由A变为K；这样获得的突变体命名为GOD-K，其氨基酸序列为SEQ ID NO:5，其一种编码核苷酸序列为SEQ ID NO:6。突变体GOD-K在70℃处理2.5min后残余酶活为68.20％，75℃处理2.5min后残余酶活为42.76％，耐热性得到显著提高。
为了提高上述三点突变体GOD-K(A65R，H275F和A416K)的耐热性，申请人进一步引入T32L、V104I、V134L、A136P和A290L五个耐热突变位点中的任意一个或两个或三个进行组合筛选。申请人参照实施例1所述方法分别构建重组表达菌株，并对获得的突变体分别进行75℃热稳定性实验。
结果发现，有些耐热位点或位点的组合对于提高突变体GOD-K的耐热性影响不明显，有些甚至使其耐热性更差了，例如T32L，V134L，A136P，A290L，T32L+V134L，V134L+A290L，具体的耐热性结果如表3所示。
表3葡萄糖氧化酶突变体耐热性比较
最终，申请人筛选出能够大幅度提高突变体GOD-K耐热性的位点或位点的组合，包括V104I，V104I+T32L，V104I+A290L，V104I+T32L+A290L，分别命名为GOD-K-V104I，GOD-K-V104I+T32L，GOD-K-V104I+A290L和GOD-K-V104I+T32L+A290L，其氨基酸序列分别为SEQ IDNO:7，SEQ ID NO:9，SEQ ID NO:11和SEQ ID NO:13，其编码核苷酸序列分别为SEQ ID NO:8，SEQ ID NO:10，SEQ ID NO:12和SEQ ID NO:14。
上述突变体的耐热性的比较结果如表4所示。
表4葡萄糖氧化酶突变体耐热性比较
注：与GOD-K相比，*示P＜0.05；#示P＜0.01.
上述结果表明，与GOD-K相比，本发明引入V104I单点突变，以及V104I与T32L和/或A290L的突变位点组合，所获得的突变体的耐热性均得到显著提高。所述获得的突变体在75℃处理2.5min后，酶活残留率达高达83.10％，比GOD-K提高了46.42％，取得了意料不到的技术效果，因此更适合应用于饲料加工领域。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 青岛蔚蓝生物集团有限公司
<120> 葡萄糖氧化酶突变体
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 581
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 1
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 2
<211> 1746
<212> DNA
<213> 黑曲霉(Aspergillus niger)
<400> 2
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acgcttacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttgg ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctcacaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccgctgg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
<210> 3
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Ala Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Ala
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 4
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acgcttacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttga ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctcacaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccgctgg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
<210> 5
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Arg Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Val Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr Phe Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Lys
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 6
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acagatacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttgg ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctttcaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccaaggg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
<210> 7
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Arg Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr Phe Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Lys
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 8
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acagatacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttga ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctttcaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccaaggg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
<210> 9
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Leu
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Arg Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr Phe Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Lys
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 10
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accttggccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acagatacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttga ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctttcaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctgct gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccaaggg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
<210> 11
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Thr
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Arg Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr Phe Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Leu Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Lys
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 12
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accactgccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acagatacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttga ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctttcaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctttg gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccaaggg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746
<210> 13
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Gly Ile Glu Ala Ser Leu Leu Thr Asp Pro Lys Asp Val Ser Gly Arg
1 5 10 15
Thr Val Asp Tyr Ile Ile Ala Gly Gly Gly Leu Thr Gly Leu Thr Leu
20 25 30
Ala Ala Arg Leu Thr Glu Asn Pro Asn Ile Ser Val Leu Val Ile Glu
35 40 45
Ser Gly Ser Tyr Glu Ser Asp Arg Gly Pro Ile Ile Glu Asp Leu Asn
50 55 60
Arg Tyr Gly Asp Ile Phe Gly Ser Ser Val Asp His Ala Tyr Glu Thr
65 70 75 80
Val Glu Leu Ala Thr Asn Asn Gln Thr Ala Leu Ile Arg Ser Gly Asn
85 90 95
Gly Leu Gly Gly Ser Thr Leu Ile Asn Gly Gly Thr Trp Thr Arg Pro
100 105 110
His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn Glu Gly
115 120 125
Trp Asn Trp Asp Asn Val Ala Ala Tyr Ser Leu Gln Ala Glu Arg Ala
130 135 140
Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe Asn Ala
145 150 155 160
Ser Cys His Gly Val Asn Gly Thr Val His Ala Gly Pro Arg Asp Thr
165 170 175
Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala Val Glu
180 185 190
Asp Arg Gly Val Pro Thr Lys Lys Asp Phe Gly Cys Gly Asp Pro His
195 200 205
Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val Arg Ser
210 215 220
Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro Asn Leu
225 230 235 240
Gln Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser Gln Asn
245 250 255
Gly Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His Lys Gly
260 265 270
Asn Thr Phe Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala Ala Gly
275 280 285
Ser Leu Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly Met Lys
290 295 300
Ser Ile Leu Glu Pro Leu Gly Ile Asp Thr Val Val Asp Leu Pro Val
305 310 315 320
Gly Leu Asn Leu Gln Asp Gln Thr Thr Ala Thr Val Arg Ser Arg Ile
325 330 335
Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala Thr Phe
340 345 350
Asn Glu Thr Phe Gly Asp Tyr Ser Glu Lys Ala His Glu Leu Leu Asn
355 360 365
Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly Gly Phe
370 375 380
His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg Asp Trp
385 390 395 400
Ile Val Asn His Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp Thr Lys
405 410 415
Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr Arg Gly
420 425 430
Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu His His Phe Ala Tyr
435 440 445
Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln Ala Ala
450 455 460
Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met Gln Thr
465 470 475 480
Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr Asp Ala
485 490 495
Asp Leu Ser Ala Trp Thr Glu Tyr Ile Pro Tyr His Phe Arg Pro Asn
500 505 510
Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met Gly Gly
515 520 525
Val Val Asp Asn Ala Ala Arg Val Tyr Gly Val Gln Gly Leu Arg Val
530 535 540
Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val Met Thr
545 550 555 560
Val Phe Tyr Ala Met Ala Leu Lys Ile Ser Asp Ala Ile Leu Glu Asp
565 570 575
Tyr Ala Ser Met Gln
580
<210> 14
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ggtattgagg catctttgtt gacagaccct aaggatgttt ctggaagaac cgttgactac 60
attattgctg gtggtggttt gaccggattg accttggccg caagattgac tgaaaatcca 120
aacatctctg ttttggtcat cgagtctggt tcttacgaat ctgacagagg acctattatc 180
gaagacttga acagatacgg tgatattttc ggatcttctg ttgatcatgc ctacgagaca 240
gttgagttgg ctactaacaa tcagactgct ttgattaggt ctggaaatgg tttgggtgga 300
tctactttga ttaatggagg tacttggact agaccacata aggctcaggt tgattcttgg 360
gaaactgttt ttggtaacga aggttggaac tgggataacg ttgcagctta ctctttgcaa 420
gcagaaagag ctagggctcc aaacgctaag caaattgctg ctggtcatta ctttaacgct 480
tcttgtcacg gtgttaacgg aactgtccac gccggaccta gagacactgg agatgattac 540
tctccaattg tcaaggcatt gatgtctgct gttgaagata gaggagtccc aaccaagaag 600
gatttcggtt gtggtgatcc acatggtgtt tctatgttcc caaatacatt gcacgaagat 660
caagttaggt ctgacgctgc tagagaatgg ttgttgccaa attatcaaag accaaacttg 720
caggtcttga ctggtcagta cgtcggtaag gttttgttgt ctcaaaacgg tactactcca 780
agagctgtcg gtgtcgagtt cggtactcat aagggtaata ctttcaacgt ttacgctaag 840
catgaagttt tgttggctgc tggttctttg gtttctccaa ccatcttgga gtattctgga 900
attggtatga agtctatttt ggaaccattg ggtattgata ctgtcgttga tttgccagtt 960
ggtttgaact tgcaggatca gactacagcc actgtcagat ccagaattac ttctgctggt 1020
gctggtcaag gtcaggctgc atggtttgct acttttaacg aaacttttgg tgattactct 1080
gaaaaggctc atgaattgtt gaacactaag ttggaacaat gggctgaaga agctgttgct 1140
agaggtggtt ttcataatac tactgctttg ttgattcaat acgaaaacta cagagactgg 1200
attgttaacc ataacgttgc ctattctgag ttgtttttgg acaccaaggg tgttgcttct 1260
tttgatgttt gggatttgtt gccatttaca agaggttacg ttcacatttt ggataaagat 1320
ccatacttgc atcactttgc atacgatcca caatactttt tgaacgaatt ggacttgttg 1380
ggtcaagctg ctgctactca attggctaga aacatttcta actctggtgc aatgcaaact 1440
tactttgccg gtgaaactat cccaggagat aacttggctt acgatgctga tttgtctgct 1500
tggactgaat acattccata ccatttcaga ccaaactacc acggtgtcgg tacttgttct 1560
atgatgccaa aggaaatggg aggtgttgtc gataacgctg caagagtcta cggagttcaa 1620
ggtttgagag ttattgatgg ttctattcca ccaactcaaa tgtcttctca tgttatgact 1680
gttttttacg ctatggcttt gaagatttct gatgctatct tggaagatta cgcttctatg 1740
caataa 1746

Claims (10)

1.一种葡萄糖氧化酶突变体，其具有(Ⅰ)、(Ⅱ)或(Ⅲ)所示的氨基酸序列中任意一个：
(Ⅰ)与葡萄糖氧化酶的氨基酸序列SEQ ID NO:1具有至少95％同源性的序列；
(Ⅱ)具有所述(Ⅰ)中所述的葡萄糖氧化酶的至少一个免疫表位，且所述葡萄糖氧化酶的氨基酸序列经修饰、取代、缺失或添加一个或几个氨基酸获得的氨基酸序列；
(Ⅲ)由如SEQ ID NO:2所示的核苷酸序列或其互补序列或因遗传密码的简并性而与如SEQ ID NO:2所示的核苷酸序列或其互补序列的核苷酸序列不同的序列编码的氨基酸序列。
2.权利要求1所述的葡萄糖氧化酶突变体，其中所述取代是氨基酸序列为SEQ ID NO:1的葡萄糖氧化酶的第104位氨基酸发生了取代。
3.如权利要求2所述的葡萄糖氧化酶突变体，其中第104位氨基酸由V变为I。
4.如权利要求1-3任一项所述的葡萄糖氧化酶突变体，其中所述取代还包括第65位、第275位和第416位氨基酸发生了取代。
5.如权利要求4所述的葡萄糖氧化酶突变体，其中第65位氨基酸由A变为R，第275位氨基酸由H变为F,第416位氨基酸由A变为K。
6.如权利要求1-5任一项所述的葡萄糖氧化酶突变体，其中所述取代包括第32位和/或第290位氨基酸发生了取代。
7.如权利要求6所述的葡萄糖氧化酶突变体，其中第32位氨基酸由T变为L，和/或第290位氨基酸由A变为L。
8.编码权利要求1-7任一项所述的葡萄糖氧化酶突变体的DNA分子。
9.携带有权利要求8所述的DNA分子的重组表达载体。
10.权利要求1-7任一项所述的葡萄糖氧化酶突变体在饲料领域中的应用。
CN201710760957.XA 2017-08-30 2017-08-30 葡萄糖氧化酶突变体 Active CN109423483B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710760957.XA CN109423483B (zh) 2017-08-30 2017-08-30 葡萄糖氧化酶突变体

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710760957.XA CN109423483B (zh) 2017-08-30 2017-08-30 葡萄糖氧化酶突变体

Publications (2)

Publication Number Publication Date
CN109423483A true CN109423483A (zh) 2019-03-05
CN109423483B CN109423483B (zh) 2021-10-29

Family

ID=65502119

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710760957.XA Active CN109423483B (zh) 2017-08-30 2017-08-30 葡萄糖氧化酶突变体

Country Status (1)

Country Link
CN (1) CN109423483B (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628738A (zh) * 2019-09-27 2019-12-31 华东理工大学 提高葡萄糖氧化酶活性的方法、突变体及其应用
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1229139A (zh) * 1999-01-28 1999-09-22 中国农业科学院生物技术研究中心 葡萄糖氧化酶基因在培育抗病转基因植物中的应用
WO2014173822A2 (en) * 2013-04-24 2014-10-30 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Novel glucose oxidase variants
CN105950578A (zh) * 2016-07-06 2016-09-21 青岛红樱桃生物技术有限公司 耐热的葡萄糖氧化酶突变体及其编码基因和应用
CN105950577A (zh) * 2016-07-06 2016-09-21 青岛红樱桃生物技术有限公司 热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1229139A (zh) * 1999-01-28 1999-09-22 中国农业科学院生物技术研究中心 葡萄糖氧化酶基因在培育抗病转基因植物中的应用
WO2014173822A2 (en) * 2013-04-24 2014-10-30 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Novel glucose oxidase variants
CN105950578A (zh) * 2016-07-06 2016-09-21 青岛红樱桃生物技术有限公司 耐热的葡萄糖氧化酶突变体及其编码基因和应用
CN105950577A (zh) * 2016-07-06 2016-09-21 青岛红樱桃生物技术有限公司 热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
J. TODD HOLLAND等: "Rational Redesign of Glucose Oxidase for Improved Catalytic Function and Stability", 《PLOS ONE》 *
JULIA MARÍN-NAVARRO等: "Identification and Structural Analysis of Amino Acid Substitutions that Increase the Stability and Activity of Aspergillus niger Glucose Oxidase", 《PLOS ONE》 *
NCBI: "GenBank登录号：ACB30370.1", 《NCBI GENBANK》 *
NCBI: "GenBank登录号：P13006.1", 《NCBI GENBANK》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110628738A (zh) * 2019-09-27 2019-12-31 华东理工大学 提高葡萄糖氧化酶活性的方法、突变体及其应用
CN110628738B (zh) * 2019-09-27 2021-01-12 华东理工大学 提高葡萄糖氧化酶活性的方法、突变体及其应用
WO2023225459A2 (en) 2022-05-14 2023-11-23 Novozymes A/S Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections

Also Published As

Publication number Publication date
CN109423483B (zh) 2021-10-29

Similar Documents

Publication Publication Date Title
CN112725306B (zh) 热盐性改变的菊粉酶突变体MutY119T及其应用
CN112831485B (zh) 一种低温活性改良的外切菊粉酶突变体MutDR121EH9
CN112813052B (zh) 一种低温活性提高的外切菊粉酶突变体MutDP121ET6
CN112980813B (zh) 低温改良的外切菊粉酶突变体MutS117G
CN112725307B (zh) 一种耐热性降低的低温外切菊粉酶突变体MutG169Δ4及应用
CN112725309B (zh) 一种中温下稳定的低温外切菊粉酶突变体MutP126R
CN112646792A (zh) 一种热稳定性降低的低温外切菊粉酶突变体MutA122Δ5及应用
CN105296456B (zh) 一种pH稳定性提高的谷氨酸脱羧酶突变体及其应用
CN108251389A (zh) 一种耐热性提高的葡萄糖氧化酶突变体
CN108251391A (zh) 新型葡萄糖氧化酶突变体
CN112725310A (zh) 一种不耐热的低温外切菊粉酶突变体MutG360Δ9
CN105950578A (zh) 耐热的葡萄糖氧化酶突变体及其编码基因和应用
CN105950577A (zh) 热稳定性提高的葡萄糖氧化酶突变体及其编码基因和应用
CN108118037A (zh) 一种耐热性提高的葡萄糖氧化酶突变体
CN108118038A (zh) 一种葡萄糖氧化酶突变体
CN108118036A (zh) 新型葡萄糖氧化酶突变体
CN113430181B (zh) 一种来源亚洲象肠道宏基因组的细菌漆酶及其基因
CN109423483A (zh) 葡萄糖氧化酶突变体
CN108251390A (zh) 一种葡萄糖氧化酶突变体
CN109207446A (zh) 葡萄糖氧化酶突变体
CN110592084B (zh) 一种rhtA基因启动子改造的重组菌株及其构建方法与应用
CN107988177A (zh) 葡萄糖氧化酶突变体
CN114736880B (zh) 酸稳定性提高葡萄糖氧化酶GoxM10的突变体D497N及其衍生突变体和应用
WO2023231547A1 (zh) NCgl2747基因突变体及其在制备L-赖氨酸中的应用
CN109355274A (zh) 一种对胰蛋白酶和胃蛋白酶抗性提高的β-葡萄糖苷酶

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20221118

Address after: 273400 west side of Yan Bin Road, Feixian County Economic Development Zone, Linyi, Shandong

Patentee after: SHANDONG KDN BIOTECH Co.,Ltd.

Patentee after: QINGDAO VLAND BIOTECH GROUP Co.,Ltd.

Address before: 266061 Shandong 12A07 Shandong High Speed Building, 29 Miaoling Road, Laoshan District, Qingdao City, Shandong Province

Patentee before: QINGDAO VLAND BIOTECH GROUP Co.,Ltd.

TR01 Transfer of patent right
TR01 Transfer of patent right

Effective date of registration: 20230506

Address after: No. 596-1, jiushui East Road, Laoshan District, Qingdao City, Shandong Province 266100

Patentee after: QINGDAO VLAND BIOTECH GROUP Co.,Ltd.

Address before: 273400 west side of Yan Bin Road, Feixian County Economic Development Zone, Linyi, Shandong

Patentee before: SHANDONG KDN BIOTECH Co.,Ltd.

Patentee before: QINGDAO VLAND BIOTECH GROUP Co.,Ltd.