CN110592034A - 一种葡萄糖氧化酶突变体及其载体与应用 - Google Patents

Abstract

本发明公开了一种葡萄糖氧化酶突变体，所述葡萄糖氧化酶包括黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的氨基酸序列，并且包括在第34、51、60、90、106、243和561位的氨基酸处的突变，其中，黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的氨基酸序列如SED ID NO:1所示。所述葡萄糖氧化酶突变体采用毕赤酵母作为蛋白表达平台，能显著提升酶活，并且在酸性条件下，也能保持较高的相对酶活。

Description

一种葡萄糖氧化酶突变体及其载体与应用

技术领域

本发明涉及基因工程技术领域，更具体地，涉及一种葡萄糖氧化酶突变体及其载体与应用。

背景技术

葡萄糖氧化酶(glucose oxidase，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。GOD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。葡萄糖氧化酶在自然界分布广泛，具有反应效率高的特点，催化特异性强的特点，在食品行业，饲料添加等方面得到广泛应用。葡萄糖氧化酶是一种以β-D-葡萄糖为底物的脱氢类酶制剂，因其在酶反应过程中消耗大量氧，形成葡萄糖酸具有酸化剂性质等特征被广泛应用于食品、养殖、医药等行业。饲料中添加葡萄糖氧化酶能有效改善动物肠道健康、提高饲料利用率、促进动物生长和增强机体的抗病能力，是一种绿色、安全、无毒副作用的饲料添加剂。随着近年来GOD不断的发展应用研究，其在食品、养殖和医药工业中应用已取得较大进展。

葡萄糖氧化酶最早发现于20世纪初，之后在1928年，Muller在黑曲霉的无细胞提取液中首次发现葡萄糖氧化酶，并对其催化机理进行较为深入的研究；在1936年，Keilin D等人首次于灰绿青霉(Penicillium glaucum)中发现葡萄糖氧化酶。顾磊等人使用毕赤酵母异源表达黑曲霉来源葡萄糖氧化酶，胞外酶活达到52U/mL，同时通过分子伴侣改造，使酶活活力到达1972.9U/mL，为近年来最高。微生物来源的GOD主要以黑曲霉和青霉为主，相对于青霉，黑曲霉产的GOD具有热稳定性好，底物特异性强等优点。野生型黑曲霉GOD比活力低，生产成本高，限制了其产业化应用。CN 105002147A公开了表达量提高的突变葡萄糖氧化酶及其编码基因和应用，通过定点突变，使葡萄糖氧化酶的氨基酸序列突变位点为Y76C和Q279K，提高了表达量，且稳定性好。CN 101348794A公开了一种高活力葡萄糖氧化酶的编码基因及制备方法和应用，通过在黑曲霉GOD结构基因的1752位和2272位通过重叠PCR引入两处突变，连接于表达载体，提高了突变酶的酶活。CN103525778公开一种催化活性提高的葡萄糖氧化酶突变体，比野生型葡萄糖氧化酶提高了2.5倍。

发明内容

本发明要解决的技术问题在于针对来源于黑曲霉的葡萄糖氧化酶进行分子改造，通过定点突变技术方法，为了提高黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的比活，降低生产成本，提供一种葡萄糖氧化酶突变体。

本发明要解决的另一技术问题在于提供编码上所述葡萄糖氧化酶突变体的核苷酸序列。

本发明要解决的还一技术问题在于提供一种插入有上述核苷酸序列的载体。

本发明要解决的还一技术问题在于提供含有上述载体的表达菌株。

本发明要解决的还一技术问题在于提供上述葡萄糖氧化酶突变体在饲料添加剂、食品添加剂方面的应用。

本发明要解决的还一技术问题在于提供上述的葡萄糖氧化酶突变体、核苷酸、载体或表达菌株在葡萄糖酸钠或葡萄糖酸钙的生产中的应用。

本发明所采取的技术方案是：

提供一种葡萄糖氧化酶突变体，包括黑曲霉Aspergillus niger GIM 3.452(CICC2377)的葡萄糖氧化酶的氨基酸序列，并且包括第34、51、60、90、106、243和561位氨基酸处的突变，所述黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的氨基酸序列如SED ID NO:1所示。

所述黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的核苷酸序列如SED ID NO:2所示。

所述黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶根据毕赤酵母密码子偏好优化的核苷酸序列如SED ID NO:3所示。

具体地，所述第34位的苏氨酸突变为丝氨酸或缬氨酸；所述第51位的丝氨酸突变为甘氨酸、赖氨酸或脯氨酸；所述第60位的脯氨酸突变为丙氨酸、甘氨酸或组氨酸；所述第90位的谷氨酰胺突变为甘氨酸、丙氨酸或精氨酸；所述第106位的缬氨酸突变为酪氨酸、半胱氨酸或异亮氨酸；所述第243位的谷氨酰胺突变为谷氨酸、组氨酸或缬氨酸；所述第561位的甲硫氨酸突变为丝氨酸、苏氨酸、赖氨酸或丙氨酸。

进一步地，所述葡萄糖氧化酶突变体的氨基酸序列如SEQ ID NO:4～9任一所示。

提供编码上所述葡萄糖氧化酶突变体的核苷酸序列。

更具体地，所述核苷酸序列如SEQ ID NO:10～15任一所示。

提供一种包括上述核苷酸序列的载体。

提供一种含有上述载体的表达菌株。

提供上述的葡萄糖氧化酶突变体在制备饲料添加剂、食品添加剂方面的应用。

提供上述的葡萄糖氧化酶突变体、核酸、载体或表达菌株在葡萄糖酸钠或葡萄糖酸钙的生产中的应用。

本发明的有益效果是：

1.本发明针对现有技术中构建得到的葡萄糖氧化酶生产菌的发酵能力较低，导致该酶的生产成本居高不下，制约了葡萄糖氧化酶的广泛应用的技术不足，通过对葡萄糖氧化酶进行蛋白质工程改造，提供一系列酶活显著提高的突变体蛋白。所提供的葡萄糖氧化酶突变体可在毕赤酵母中高效表达，并且具有更高的相对酶活，即使在酸性条件下，也能保持较高的相对酶活。

附图说明

图1葡萄糖氧化酶突变体在不同pH条件下的相对酶活。

具体实施方式

实验材料和试剂：

1、菌株与载体

黑曲霉Aspergillus niger GIM 3.452(CICC 2377)购自中国工业微生物菌种保藏管理中心，大肠杆菌菌株Top10、毕赤酵母X33、载体pPICZαA、载体pGAPzαA、抗生素Zeocin购自Invitrogen公司。

2、酶与试剂盒

PCR酶、质粒提取试剂盒、胶纯化试剂盒购于上海生工公司，限制性内切酶购于NEB公司。

3、培养基

大肠杆菌培养基为LB(1％蛋白胨，0.5％酵母提取物，1％NaCL，pH7.0)。LB-Amp为LB培养基加100μg/ml氨苄青霉素。LB-Zeocin为LB培养基加25μg/ml Zeocin。酵母培养基为YPD(1％酵母提取物，2％蛋白胨，2％葡萄糖)。酵母筛选培养基为YPDZ(YPD+100μg/mlZeocin)。酵母诱导培养基BMGY(1％酵母提取物，2％蛋白胨，1.34％YNB，0.00004％Biotin，1％甘油(v/v))和BMMY(除以0.5％甲醇代替甘油，其余成分与BMGY相同)。重组酵母发酵基本盐培养基：磷酸氢二铵5％、磷酸二氢钾0.5％、七水硫酸镁1.5％、硫酸钾1.95％、硫酸钙0.1％、消泡剂0.03％。高压后每升加4.35mlPTM1。PTM1(微量盐溶液)：硫酸铜0.6％、碘化钾0.018％。一水硫酸锰0.3％、二水钼酸钠0.02％、硼酸0.002％、流水氯化钴0.05％、氯化锌2％、七水硫酸铁6.5％、浓硫酸0.5％、生物素0.02％

4、化学试剂：

葡萄糖氧化酶标准品、邻联茴香胺盐酸盐及辣根过氧化物购于Sigma公司，葡萄糖购于OXIOD公司，其他试剂购于广州化学试剂厂。

5、葡萄糖氧化酶测定方法

葡萄糖氧化酶活性测定采用邻—联茴香胺分光光度法。在葡萄糖氧化酶的作用下，葡萄糖和氧反应，生成葡萄糖酸和过氧化氢，过氧化氢和无色的还原型邻联茴香胺在过氧化物酶的作用下，生成水和红色的氧化型邻联茴香胺。在540nm下测定反应液吸光值，依据标准曲线计算葡萄糖氧化酶的酶活。

实施例1、黑曲霉(Aspergillus niger)葡萄糖氧化酶(GOD)基因的优化与载体构建

黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶(GOD)基因(Genebank:FJ979866.1)，其核苷酸序列如SEQ ID NO:2所示，其氨基酸序列如SEQ ID NO:1所示。

通过大量前期研究发现黑曲霉表达工程菌表达量低，操作复杂，难以进行突变高通量筛选。同时发现大肠杆菌表达系统表达葡萄糖氧化酶不具备活性，不能进行突变筛选。而毕赤酵母是一种成熟的真核微生物外源蛋白表达平台，具有以下特点：生长迅速并且培养廉价；简单容易操作，便于实验进行突变和高酶活筛选；作为一种真核生物，具备翻译后蛋白加工能力，在毕赤酵母合成的蛋白更有可能得到正确的加工、折叠与正确组装成有功能的蛋白分子。毕赤酵母表达黑曲霉葡萄糖氧化酶在发酵罐可以达到较高的水平，但是与实际生产还是有距离。通过对葡萄糖氧化酶基因进行针对毕赤酵母密码子优化和突变来提高氧化酶酶活，进而达到工业生产。针对毕赤酵母密码子偏好性进行优化，优化后的核苷酸序列如SEQ IDNO:3所示，分别在葡萄糖氧化酶优化基因(GOX)的5’端和3’端引入EcoRI和XbaI酶切位点，并连接至Puc57-amp载体上，将GOX-Puc57接种至LBA培养基，培养24小时候，提取质粒，用EcoRI和XbaI酶切，切胶回收目的基因片段，产物纯化回收，分别连接到表达载体pPICzαA和pPGAPzαA，得到表达载体pPICzαA-GOX和pGAPzαA-GOX。

实施例2、葡萄糖氧化酶基因定点突变

以上述pGAPzαA-GOX为模板，引入定点突变并转化毕赤酵母X33

根据葡萄糖氧化酶三维结构信息，经大量研究认为氨基酸序列的第34、51、60、90、106、243、420、561位氨基酸对葡萄糖氧化酶酶活具有重要影响，对上述位点进行饱和突变，将所述的位点饱和突变得到的酵母重组转化子用牙签逐个挑至24孔板，每个孔中加入1mL含有BMGY培养基，30℃，220rpm培养24h左右，离心去上清。再分别加入1.6mL BMMY培养基进行诱导培养。培养24h后，离心取上清，将上述上清液分别取出200μL至96孔板，进行高通量筛选。

葡萄糖氧化酶高通量测定方法是通过一种小体系结合96孔板比色测定葡萄糖氧化酶活力的方法.经实验证明,在96孔板内,波长540nm处,反应时间增加到5分钟，氧化型邻联茴香胺与光密度(OD)值有很好的线性关系；通过加样回收实验发现,其回收率为98.5％～103.2％,相对标准差为1.8％；通过精密度分析实验发现该方法测量样品的精密度与普通邻—联茴香胺分光光度法，其测定的结果与现行国家标准测定葡萄糖氧化酶方法所得到的结果差异率小于6％(如下表1所示)。

表1高通量方法测定酶活相对标准方法测定酶活

高通量方法测定酶活	测定酶活相对标准方法测定酶活
		样品1	97.2％
样品2	95.1
		样品3	94.4％
样品4	98.5％

经过高通量筛选得到21个酶活提高的突变位点，有效突变优选T34S,V；S51G,K,P；P60A,G,H；Q90G,A,R；V106Y,C,I；Q243E,H,V；M561S,T,K,A，各位点的突变后的相对酶活如表2所示。

表2突变位点的相对酶活变化

实施例3、葡萄糖氧化酶基因组合突变

将实施例2中酶活提高的有效突变T34S,V；S51G,K,P；P60A,G,H；Q90G,A,R；V106Y,C,I；Q243E,H,V；M561S,T,K,A分别进行组合。通过实验最终得到6个酶活显著提高的组合突变分别命名为GOX-M1、GOX-M2、GOX-M3、GOX-M4、GOX-M5、GOX-M6。

其中GOX-M1包含的突变位点为：T34S，S51K，P60A，Q90A，V106Y，Q243E，M561S其氨基酸序列如SEQ ID NO:4所示，其对应的核苷酸序列如SEQ ID NO:10所示。

其中GOX-M2包含的突变位点为：T34V，S51P，P60A，Q90R，V106Y，Q243H，M561T其氨基酸序列如SEQ ID NO:5所示，其对应的核苷酸序列如SEQ ID NO:11所示。

其中GOX-M3包含的突变位点为：T34V，S51P，P60A，Q90R，V106I，Q243E，M561S其氨基酸序列如SEQ ID NO:6所示，其对应的核苷酸序列如SEQ ID NO:12所示。

其中GOX-M4包含的突变位点为：T34S，S51K，P60H，Q90A，V106I，Q243E，M561S其氨基酸序列如SEQ ID NO:7所示，其对应的核苷酸序列如SEQ ID NO:13所示。

其中GOX-M5包含的突变位点为：T34V，S51G，P60A，Q90R，V106C，Q243H，M561S其氨基酸序列如SEQ ID NO:8所示，其对应的核苷酸序列如SEQ ID NO:14所示。

其中GOX-M6包含的突变位点为：T34S，S51K，P60A，Q90G，V106C，Q243V，M561A其氨基酸序列如SEQ ID NO:9所示，其对应的核苷酸序列如SEQ ID NO:15所示。

这6个组合突变体的相对比活如表3所示。

表3原始葡萄糖氧化酶和组合突变体葡萄糖氧化酶相对比活

实施例4葡萄糖氧化酶突变体在不同pH条件下的相对酶活

检测实施例2中筛选出的6种突变体在不同pH条件下的相对酶活，从pH2～10的相对酶活如附图1所示。结果发现在酸性条件下相对酶活更高，具有更强的耐酸性能。尤其GOX-M1和GOX-M3在酸性条件下具备60％以上酶活，具有较强的耐酸性能。

以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。

SEQUENCE LISTING

<110> 广东溢多利生物科技股份有限公司

<120> 一种葡萄糖氧化酶突变体及其载体与应用

<130>

<160> 15

<170> PatentIn version 3.5

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<211> 583

<212> PRT

<213> 黑曲霉Aspergillus niger GIM 3.452（CICC 2377）

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Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Met Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 2

<211> 1752

<212> DNA

<213> 黑曲霉Aspergillus niger GIM 3.452（CICC 2377）

<400> 2

agcaatggca tcgaagccag cctcctgact gaccccaagg aggttgccgg ccgcactgtc 60

gactacatca tcgctggtgg aggtctgact ggactcacca ctgctgcccg tctgacggag 120

aaccccgata tcactgtgct tgtcatcgaa agtggctcct acgagtctga cagaggtcct 180

atcattgagg acctgaacgc ttacggtgac atttttggca gcagtgtgga ccacgcctac 240

gagactgtcg agctcgccac caacaatcag actgcgctga tccgctccgg aaatggtctc 300

ggtggctcta ccctcgtcaa cggtggcacc tggactcgcc cccacaaggc acaagttgac 360

tcatgggaga ccgtcttcgg aaatgagggc tggaactggg acagcgtggc cgcctactcc 420

ctccaggctg agcgtgctcg cgcaccaaat gccaaacaga ttgctgctgg ccactacttt 480

aatgcatcct gccatggtat caatggtact gtccacgccg gaccccgcga taccggtgat 540

gactactccc ccatcgtcaa ggctctcatg agcgctgtcg aagacagggg cgttcccacc 600

aagaaggact tgggatgcgg tgacccccat ggtgtgtcca tgttccccaa caccttgcac 660

gaagaccaag tgcgctctga tgccgctcgc gaatggctcc tccccaacta ccagcgtccc 720

aacctgcaag tcctcactgg acagtatgtt ggaaaggtcc tgctcagcca gaacgctacc 780

acacctcgtg ccgttggcgt ggaattcggc acccacaagg gcaacaccca caacgtctac 840

gctaagcacg aggtcctcct ggccgctgga tccgctgtct ctcccaccat cctcgaatat 900

tccggtatcg gaatgaagtc cattctagag cctcttggaa ttgacaccgt cgttgacctg 960

cccgttggtc tcaaccttca ggaccagacc acctctaccg tccgctcacg cattacctcc 1020

gccggtgccg gacagggaca ggccgcttgg ttcgctacct tcaacgagac ctttggcgac 1080

tacgccgaaa aggctcacga gctgctcaac accaagctgg agcagtgggc cgaagaggcc 1140

gtcgcccgtg gcggattcca caacaccacc gctttgctca tccagtacga gaactaccgc 1200

gactggatcg tcaaggacaa tgtcgcatac tcggaactct tcctcgacac ggccggagtg 1260

gccagtttcg atgtgtggga tcttctgccc ttcactagag gatacgtaca catcctcgac 1320

aaggacccct acctccgcca tttcgcatac gaccctcagt actttctcaa cgagcttgac 1380

ctgctcggcc aggctgccgc cactcagctg gcccgcaaca tctccaactc cggtgccatg 1440

caaacttatt tcgctggaga gactattccc ggtgacaacc tcgcgtatga tgccgacttg 1500

agcgcctggg ttgagtatat cccgtacaac ttccgcccta actaccatgg tgtgggtact 1560

tgctccatga tgccgaagga gatgggcggt gttgtcgaca atgctgcccg tgtgtatggt 1620

gtgcagggac tgcgagtcat cgatggttct attcccccta cgcaaatgtc gtcccatgtt 1680

atgacggtct tttatgccat ggccttgaag attgcggatg ccatcttggc ggattatgct 1740

tccatgcagt ga 1752

<210> 3

<211> 1752

<212> DNA

<213> 人工序列

<400> 3

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgacca ccgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag tccggttcct acgagtctga ccgtggtcca 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaatcaa actgctttga tccgttccgg taacggtttg 300

ggaggatcca ctttggttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttgcagg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

atgactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

<210> 4

<211> 583

<212> PRT

<213> 人工序列

<400> 4

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

1 5 10 15

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

20 25 30

Thr Ser Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val

35 40 45

Ile Glu Lys Gly Ser Tyr Glu Ser Asp Arg Gly Ala Ile Ile Glu Asp

50 55 60

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

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Ala Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Leu Leu Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu Glu Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

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

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Ser Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 5

<211> 583

<212> PRT

<213> 人工序列

<400> 5

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

1 5 10 15

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

20 25 30

Thr Val Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val

35 40 45

Ile Glu Pro Gly Ser Tyr Glu Ser Asp Arg Gly Ala Ile Ile Glu Asp

50 55 60

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

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Leu Leu Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu His Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

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

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Thr Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 6

<211> 583

<212> PRT

<213> 人工序列

<400> 6

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

1 5 10 15

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

20 25 30

Thr Val Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val

35 40 45

Ile Glu Pro Gly Ser Tyr Glu Ser Asp Arg Gly Ala Ile Ile Glu Asp

50 55 60

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

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Tyr Phe Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu Glu Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

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

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Ser Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 7

<211> 583

<212> PRT

<213> 人工序列

<400> 7

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

1 5 10 15

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

20 25 30

Thr Ser Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val

35 40 45

Ile Glu Lys Gly Ser Tyr Glu Ser Asp Arg Gly His Ile Ile Glu Asp

50 55 60

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

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Ala Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Tyr Phe Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu Glu Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

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

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Ser Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 8

<211> 583

<212> PRT

<213> 人工序列

<400> 8

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

1 5 10 15

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

20 25 30

Thr Ser Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val

35 40 45

Ile Glu Lys Gly Ser Tyr Glu Ser Asp Arg Gly His Ile Ile Glu Asp

50 55 60

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

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Ala Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Tyr Phe Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu Glu Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

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

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Ser Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 9

<211> 583

<212> PRT

<213> 人工序列

<400> 9

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

1 5 10 15

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

20 25 30

Thr Val Ala Ala Arg Leu Thr Glu Asn Pro Asp Ile Thr Val Leu Val

35 40 45

Ile Glu Gly Gly Ser Tyr Glu Ser Asp Arg Gly Ala Ile Ile Glu Asp

50 55 60

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

65 70 75 80

Glu Thr Val Glu Leu Ala Thr Asn Asn Arg Thr Ala Leu Ile Arg Ser

85 90 95

Gly Asn Gly Leu Gly Gly Ser Thr Leu Phe Asn Gly Gly Thr Trp Thr

100 105 110

Arg Pro His Lys Ala Gln Val Asp Ser Trp Glu Thr Val Phe Gly Asn

115 120 125

Glu Gly Trp Asn Trp Asp Ser Val Ala Ala Tyr Ser Leu Gln Ala Glu

130 135 140

Arg Ala Arg Ala Pro Asn Ala Lys Gln Ile Ala Ala Gly His Tyr Phe

145 150 155 160

Asn Ala Ser Cys His Gly Ile Asn Gly Thr Val His Ala Gly Pro Arg

165 170 175

Asp Thr Gly Asp Asp Tyr Ser Pro Ile Val Lys Ala Leu Met Ser Ala

180 185 190

Val Glu Asp Arg Gly Val Pro Thr Lys Lys Asp Leu Gly Cys Gly Asp

195 200 205

Pro His Gly Val Ser Met Phe Pro Asn Thr Leu His Glu Asp Gln Val

210 215 220

Arg Ser Asp Ala Ala Arg Glu Trp Leu Leu Pro Asn Tyr Gln Arg Pro

225 230 235 240

Asn Leu His Val Leu Thr Gly Gln Tyr Val Gly Lys Val Leu Leu Ser

245 250 255

Gln Asn Ala Thr Thr Pro Arg Ala Val Gly Val Glu Phe Gly Thr His

260 265 270

Lys Gly Asn Thr His Asn Val Tyr Ala Lys His Glu Val Leu Leu Ala

275 280 285

Ala Gly Ser Ala Val Ser Pro Thr Ile Leu Glu Tyr Ser Gly Ile Gly

290 295 300

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

305 310 315 320

Pro Val Gly Leu Asn Leu Gln Asp Gln Thr Thr Ser Thr Val Arg Ser

325 330 335

Arg Ile Thr Ser Ala Gly Ala Gly Gln Gly Gln Ala Ala Trp Phe Ala

340 345 350

Thr Phe Asn Glu Thr Phe Gly Asp Tyr Ala Glu Lys Ala His Glu Leu

355 360 365

Leu Asn Thr Lys Leu Glu Gln Trp Ala Glu Glu Ala Val Ala Arg Gly

370 375 380

Gly Phe His Asn Thr Thr Ala Leu Leu Ile Gln Tyr Glu Asn Tyr Arg

385 390 395 400

Asp Trp Ile Val Lys Asp Asn Val Ala Tyr Ser Glu Leu Phe Leu Asp

405 410 415

Thr Ala Gly Val Ala Ser Phe Asp Val Trp Asp Leu Leu Pro Phe Thr

420 425 430

Arg Gly Tyr Val His Ile Leu Asp Lys Asp Pro Tyr Leu Arg His Phe

435 440 445

Ala Tyr Asp Pro Gln Tyr Phe Leu Asn Glu Leu Asp Leu Leu Gly Gln

450 455 460

Ala Ala Ala Thr Gln Leu Ala Arg Asn Ile Ser Asn Ser Gly Ala Met

465 470 475 480

Gln Thr Tyr Phe Ala Gly Glu Thr Ile Pro Gly Asp Asn Leu Ala Tyr

485 490 495

Asp Ala Asp Leu Ser Ala Trp Val Glu Tyr Ile Pro Tyr Asn Phe Arg

500 505 510

Pro Asn Tyr His Gly Val Gly Thr Cys Ser Met Met Pro Lys Glu Met

515 520 525

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

530 535 540

Arg Val Ile Asp Gly Ser Ile Pro Pro Thr Gln Met Ser Ser His Val

545 550 555 560

Ser Thr Val Phe Tyr Ala Met Ala Leu Lys Ile Ala Asp Ala Ile Leu

565 570 575

Ala Asp Tyr Ala Ser Met Gln

580

<210> 10

<211> 1752

<212> DNA

<213> 人工序列

<400> 10

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgacct ctgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag aagggttcct acgagtctga ccgtggtgct 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaatgct actgctttga tccgttccgg taacggtttg 300

ggaggatcca ctttacttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttggaag tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

tctactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

<210> 11

<211> 1752

<212> DNA

<213> 人工序列

<400> 11

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgaccg ttgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag ccaggttcct acgagtctga ccgtggtgct 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300

ggaggatcca ctttacttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttgcatg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

actactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

<210> 12

<211> 1752

<212> DNA

<213> 人工序列

<400> 12

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgaccg ttgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag ccaggttcct acgagtctga ccgtggtgct 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300

ggaggatcca cttattttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttggaag tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

tctactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

<210> 13

<211> 1752

<212> DNA

<213> 人工序列

<400> 13

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgacct ctgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag aagggttcct acgagtctga ccgtggtcat 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaatgct actgctttga tccgttccgg taacggtttg 300

ggaggatcca cttattttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttggaag tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

tctactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

<210> 14

<211> 1752

<212> DNA

<213> 人工序列

<400> 14

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgacct ctgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag aagggttcct acgagtctga ccgtggtcat 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaatgct actgctttga tccgttccgg taacggtttg 300

ggaggatcca cttattttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttggaag tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

tctactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

<210> 15

<211> 1752

<212> DNA

<213> 人工序列

<400> 15

tctaatggta ttgaggcttc cttgttgacc gacccaaaag aggtcgccgg tagaaccgtc 60

gactacatca ttgccggtgg tggtttgacc ggtttgaccg ttgctgctag attgaccgag 120

aatcctgaca tcactgtttt ggttattgag ggtggttcct acgagtctga ccgtggtgct 180

attattgagg atttgaatgc ctacggtgac atcttcggat cttctgtcga ccacgcctat 240

gagaccgttg agttggctac taacaataga actgctttga tccgttccgg taacggtttg 300

ggaggatcca ctttgtttaa cggtggaacc tggactagac cacataaagc ccaagtcgac 360

tcctgggaga ctgtcttcgg aaacgaaggt tggaactggg actctgttgc tgcttactcc 420

cttcaggctg aaagagctcg tgccccaaat gctaagcaga tcgccgctgg tcactacttt 480

aacgcctctt gccacggtat taacggtact gttcacgctg gaccacgtga tactggtgat 540

gactactctc caatcgtcaa ggccttgatg tctgctgtcg aagatcgtgg agtccctacc 600

aagaaggact tgggttgcgg agaccctcat ggtgtctcca tgttcccaaa caccttgcac 660

gaggaccaag ttcgttccga cgctgccaga gaatggttgc ttcctaacta ccagagacca 720

aacttgcatg tcttgactgg tcagtacgtc ggtaaggtct tgttgtctca gaacgctacc 780

accccaagag ctgttggtgt cgagttcggt actcacaagg gtaacaccca caacgtctac 840

gctaagcatg aggtcctttt ggccgccggt tctgccgttt ccccaaccat cttggagtat 900

tctggaattg gtatgaaatc tattttggag cctttgggaa tcgacaccgt tgttgacctt 960

ccagttggtt tgaacttgca ggaccagacc acctccactg tccgttctcg tattacttcc 1020

gctggtgctg gacaaggtca agctgcctgg ttcgctacct tcaatgagac ctttggtgat 1080

tacgccgaga aggcccacga gttgttgaac accaagttgg agcaatgggc tgaagaggct 1140

gtcgctagag gtggattcca taataccacc gccttgttga tccaatacga aaattataga 1200

gattggattg ttaaggacaa tgttgcttac tccgagttgt ttttggatac cgccggagtc 1260

gcttcctttg acgtctggga cttgttgcct ttcacccgtg gttacgttca cattttggac 1320

aaagatcctt acttgcgtca cttcgcctac gacccacagt acttcttgaa cgagttggac 1380

ttgttgggtc aagctgctgc tactcagttg gcccgtaaca tttctaactc tggtgccatg 1440

caaacctact tcgctggaga gaccattcca ggagacaact tggcctacga tgccgacttg 1500

tctgcctggg tcgagtacat cccttacaac ttccgtccaa actatcacgg tgtcggaacc 1560

tgctccatga tgccaaagga aatgggtgga gtcgtcgaca atgccgctcg tgtttacgga 1620

gtccagggtt tgagagtcat cgacggttct atcccaccaa cccaaatgtc ctcccacgtc 1680

tctactgtct tctacgctat ggccttgaag atcgctgacg ctattcttgc tgactacgct 1740

tctatgcagt aa 1752

Claims (10)

1.一种葡萄糖氧化酶突变体，包括黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的氨基酸序列，并且包括第34、51、60、90、106、243和561位氨基酸处的突变，所述黑曲霉Aspergillus niger GIM 3.452(CICC 2377)的葡萄糖氧化酶的氨基酸序列如SED ID NO:1所示。

2.根据权利要求1所述的葡萄糖氧化酶突变体，其特征在于，所述第34位的苏氨酸突变为丝氨酸或缬氨酸；所述第51位的丝氨酸突变为甘氨酸、赖氨酸或脯氨酸；所述第60位的脯氨酸突变为丙氨酸、甘氨酸或组氨酸；所述第90位的谷氨酰胺突变为甘氨酸、丙氨酸或精氨酸；所述第106位的缬氨酸突变为酪氨酸、半胱氨酸或异亮氨酸；第243位的谷氨酰胺突变为谷氨酸、组氨酸或缬氨酸；所述第561位的甲硫氨酸突变为丝氨酸、苏氨酸、赖氨酸或丙氨酸。

3.根据权利要求1或2所述的葡萄糖氧化酶突变体，其特征在于，所述葡萄糖氧化酶突变体的氨基酸序列如SEQ ID NO:4～9任一所示。

4.编码权利要求1至3任一所述葡萄糖氧化酶突变体的核苷酸序列。

5.根据权利要求4所述的核苷酸序列，其特征在于，所述核苷酸序列如SEQ ID NO:10～15任一所示。

6.一种载体，其插入有权利要求4或5所述的核苷酸序列。

7.一种含有权利要求6所述载体的表达菌株。

8.根据权利要求7所述的表达菌株，其特征在于，所述表达菌株为毕赤酵母。

9.权利要求1至3任一所述葡萄糖氧化酶突变体在制备饲料添加剂、食品添加剂方面的应用。

10.权利要求1至3任一所述葡萄糖氧化酶突变体、权利要求4、5所述核苷酸、权利要求6所述载体或权利要求7、8所述表达菌株在葡萄糖酸钠或葡萄糖酸钙的生产中的应用。