CN112553182B - 具提升活性的α-半乳糖苷酶 - Google Patents

具提升活性的α-半乳糖苷酶 Download PDF

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CN112553182B
CN112553182B CN201910910821.1A CN201910910821A CN112553182B CN 112553182 B CN112553182 B CN 112553182B CN 201910910821 A CN201910910821 A CN 201910910821A CN 112553182 B CN112553182 B CN 112553182B
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郑雅珊
吴姿慧
林正言
赖惠琳
黄婷沅
郑成彬
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Asiapac Dongguan Bio Technology Co ltd
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Abstract

本申请涉及一种具提升活性的α‑半乳糖甘酶，其氨基酸序列是将SEQ ID NO：2第450个位置的精氨酸改为苯丙氨酸、缬氨酸、丝氨酸、丙氨酸、或甘氨酸取代修饰的序列。

Description

具提升活性的α-半乳糖苷酶
技术领域
本申请涉及一种α-半乳糖苷酶，尤指一种具提升活性的α-半乳糖苷酶。
背景技术
α-半乳糖苷酶(α-galactosidase；EC 3.2.1.22)是自然界中广泛存在于微生物、植物以及动物体内的一种外切水解酶。它能够催化像是棉子糖(raffinose)和水苏糖(stachyose)等不同种α-半乳糖苷类寡糖上的1,6-糖苷键的水解，进而释放非还原端的半乳糖。除了能水解广泛存在于豆类植物中的半乳糖苷类寡糖之外，此类糖苷酶还能分解半乳甘露聚糖(galactomannan)、半乳糖脂(galactolipid)、以及糖蛋白(glycoprotein)。根据底物特异性可将半乳糖苷酶分成两大类：第一类是只能作用在小型底物，像是合成底物或是寡糖。第二类则是能从高聚合的半乳甘露聚多糖中释放出半乳糖。另一方面，根据氨基酸序列的相似度可分类成糖苷水解酶(glycoside hydrolase)的4、27、36、57、97与110家族。对于半乳甘露聚糖的完全分解上，半乳糖苷酶能与其他苷酶，像是甘露聚糖酶(mannanase)以及甘露糖苷酶(mannosidase)，具有协同作用。也因此半乳糖苷酶的应用范围十分广泛，像是在饲料、食品、造纸甚至医药方面。
在以豆粕为主的饲料原料中，添加半乳糖苷酶能够分解动物本身无法代谢的半乳糖苷物质，进而增加饲料的利用率，并且减缓抗营养物质所造成胀气或下痢的不良影响。食品工业上，半乳糖苷酶可添加在大豆或其他豆类制品中。它能分解会造成胀气的水苏糖这类的半乳糖苷类寡糖。造纸工业上，半乳糖苷酶能分解在软木里面半乳糖苷的支链，进而帮助后续的其他酶种分解主链多糖以及纤维基质。医药应用方面，半乳糖苷酶可以用于法布里式病(Fabry’sdisease)的治疗以及B-O血型的转换。在这些许多不同的工业应用中，半乳糖苷酶也需要符合不同的工业需求。除了酶蛋白本身的特性以及稳定性之外，其比活性也是改良工业酶的重点。酶蛋白的比活性愈高，代表对于底物分解效率愈好，进而降低工业制程上所花费的成本，也就提高了利润。因此，本申请欲借由基因改造提升酶的比活性，进而增加半乳糖苷酶在工业应用上的经济价值。
发明内容
本申请的目的在于改造现有α-半乳糖苷酶，利用结构分析及点突变技术，以有效提升半乳糖苷酶的活性，进而增加半乳糖甘酶的工业应用价值。
为达上述目的，本申请的一个较广义的实施方案为提供一种α-半乳糖甘酶，其氨基酸序列是将SEQ ID NO：2第450个位置的精氨酸改为苯丙氨酸、缬氨酸、丝氨酸、丙氨酸、或甘氨酸取代修饰的序列。
在一个实施方案中，编码该SEQ ID NO：2的基因是从黑曲霉(Aspergillus niger)所分离出来的AnBgl36基因。
在一个实施方案中，该α-半乳糖甘酶的氨基酸序列如SEQ ID NO：9所示。
在一个实施方案中，该α-半乳糖甘酶的氨基酸序列如SEQ ID NO：11所示。
在一个实施方案中，该α-半乳糖甘酶的氨基酸序列如SEQ ID NO：13所示。
在一个实施方案中，该α-半乳糖甘酶的氨基酸序列如SEQ ID NO：15所示。
在一个实施方案中，该α-半乳糖甘酶的氨基酸序列如SEQ ID NO：17所示。
附图说明
图1显示野生型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。
图2显示点突变技术所采用的引物序列。
图3显示R450F突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。
图4显示R450V突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。
图5显示R450S突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。
图6显示R450A突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。
图7显示R450G突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。
图8显示野生蛋白与突变蛋白R450F、R450V、R450S、R450A及R450G的α-半乳糖甘酶活性分析。
具体实施方式
体现本申请特征与优点的一些典型实施例将在后段的说明中详细叙述。应理解的是本申请能够在不同的方案上具有各种的变化，其均不脱离本申请的范围，且其中的说明及附图在本质上是当作说明之用，而非用以限制本申请。
本申请的α-半乳糖苷酶基因Angal36是从真菌黑曲霉(Aspergillus niger)所分离出来。将此α-半乳糖苷酶基因构建到载体内，并转化入工业常用的毕赤酵母(Pichiapastoris)中表达蛋白。为了增加其催化效率，进而提升应用价值，本申请借由相似度高的已知蛋白结构作为模板，利用软件仿真出此α-半乳糖甘酶的结构模型，再加以分析，并从中挑选出有潜力的氨基酸，利用点突变技术(site-directed mutagenesis)进行基因改造。通过结构分析，本申请从活性区中挑出位于氨基酸序列上第450个位置的精氨酸(arginine)，将其个别突变成苯丙氨酸(phenylalanine)、缬氨酸(valine)、丝氨酸(serine)、丙氨酸(alanine)以及甘氨酸(glycine)，并发现这些突变蛋白均能增加α-半乳糖甘酶的活性。
以下将详述本申请改造α-半乳糖甘酶的方法及其所得到的改良α-半乳糖甘酶。
图1显示野生型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列。如图1所示，野生型α-半乳糖甘酶基因Angal36包含2178个碱基(含终止密码子，核苷酸序列以SEQ IDNO：1标示)以及725个氨基酸(氨基酸序列以SEQ ID NO：2标示)。将Angal36基因利用EcoRI与XbaI构建在pPICZαA载体内，并借由电穿孔技术送入毕赤酵母中。利用zeocin抗生素筛选出成功转染的菌株，之后挑选单一菌落接种到YPD培养基中至隔天。为了放大菌量，再接种到BMGY培养基中至隔天。利用离心将菌液分离之后，把菌体转移到含有0.5％甲醇的BMMY培养基中，以诱导蛋白表达。最后，采集诱导后的样本进行后续实验。
本申请α-半乳糖甘酶基因Angal36的五种突变基因系利用点突变技术取得，以野生型基因Angal36做为模板进行聚合酶连锁反应，当中所用的突变引物列于图2，其中R450F意指α-半乳糖甘酶Angal36第450个位置的氨基酸由精氨酸(arginine)突变成苯丙氨酸(phenylalanine)，且R450F突变引物序列以SEQ ID NO：3标示；R450V意指α-半乳糖甘酶Angal36第450个位置的氨基酸由精氨酸(arginine)突变成缬氨酸(valine)，且R450V突变引物序列以SEQ ID NO：4标示；R450S意指α-半乳糖甘酶Angal36第450个位置的氨基酸由精氨酸(arginine)突变成丝氨酸(serine)，且R450S突变引物序列以SEQ ID NO：5标示；R450A意指α-半乳糖甘酶Angal36第450个位置的氨基酸由精氨酸(arginine)突变成丙氨酸(alanine)，且R450A突变引物序列以SEQ ID NO：6标示；R450G意指α-半乳糖甘酶Angal36第450个位置的氨基酸由精氨酸(arginine)突变成甘氨酸(glycine)，且R450G突变引物序列以SEQ ID NO：7标示。因此，本申请利用点突变技术取得α-半乳糖甘酶Angal36的五种突变基因分别是R450F、R450V、R450S、R450A以及R450G。
图3至图7即显示本申请所构建的五种突变型的核苷酸以及氨基酸序列。图3显示R450F突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列，其中核苷酸序列以SEQID NO：8标示，氨基酸序列以SEQ ID NO：9标示，且其第450个位置的氨基酸由精氨酸(arginine)突变成苯丙氨酸(phenylalanine)。图4显示R450V突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列，其中核苷酸序列以SEQ ID NO：10标示，氨基酸序列以SEQID NO：11标示，且其第450个位置的氨基酸由精氨酸(arginine)突变成缬氨酸(valine)。图5显示R450S突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列，其中核苷酸序列以SEQ ID NO：12标示，氨基酸序列以SEQ ID NO：13标示，且其第450个位置的氨基酸由精氨酸(arginine)突变成丝氨酸(serine)。图6显示R450A突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列，其中核苷酸序列以SEQ ID NO：14标示，氨基酸序列以SEQ ID NO：15标示，且其第450个位置的氨基酸由精氨酸(arginine)突变成丙氨酸(alanine)。图7显示R450F突变型α-半乳糖甘酶Angal36的核苷酸序列以及氨基酸序列，其中核苷酸序列以SEQID NO：16标示，氨基酸序列以SEQ ID NO：17标示，且其第450个位置的氨基酸由精氨酸(arginine)突变成甘氨酸(glycine)。
待突变反应完成后，加入DpnI除去原始模板，再将质粒DNA送入大肠杆菌感受态细胞中，通过抗生素进行筛选。借由DNA定序以确认序列突变的成功与否。最后，如前述蛋白表达步骤所述，将成功突变的基因个别送入毕赤酵母中表达，并进行蛋白量测量及α-半乳糖甘酶活性检测。
α-半乳糖甘酶的活性检测方法是利用α-半乳糖甘酶催化水解作为底物的硝基苯基半乳糖苷(p-nitrophenyl-β-D-galactopyranoside)，释放出具呈色效果的硝基苯酚(nitrophenol)，再进而推算出α-半乳糖甘酶的活性。实验步骤如下：将7mM的底物与稀释过的酶蛋白溶液混合之后，置于37℃的水浴槽中作用15分钟，随后加入0.2M硼酸溶液以终止反应。最后，在OD405波长下测定吸光值，再换算出α-半乳糖苷酶的活性。
为了能贴近工业上的应用，本申请将野生基因与突变基因分别送入工业常用的毕赤酵母系统中表达酶蛋白，并在相同的蛋白浓度下进行α-半乳糖苷酶的活性检测。图8显示野生(Wild type)的Angal36酶蛋白与突变蛋白R450F、R450V、R450S、R450A及R450G的α-半乳糖甘酶活性分析，其中不同的酶活性是以野生蛋白的酶活性做为100％的基准之下进行比较。由图8所示结果可知，五种突变蛋白的活性均明显高于野生蛋白，当中R450G活性大幅提升最高至约207％，其次为R450F也增加至约191％活性。另外三种突变蛋白R450V、R450S及R450A活性也明显增加到约150％。此外，突变蛋白在毕赤酵母系统的表达量与野生蛋白相差不大。因此，突变蛋白的总活性也是显著高于原始蛋白，尤其是R450G。
综上所述，为了提升α-半乳糖甘酶AnBgl36的活性，本申请进一步分析其蛋白结构，挑选出活性区中序列第450个位置的精氨酸(Arg450)进行改造，并个别突变成苯丙氨酸(R450F)、缬氨酸(R450V)、丝氨酸(R450S)、丙氨酸(R450A)、以及甘氨酸(R450G)。结果显示，所改造的五种突变蛋白的活性均明显高于野生蛋白。因此，本申请提供的α-半乳糖甘酶AnBgl36的突变蛋白可有效提升α-半乳糖甘酶的活性，故可进一步降低生产成本，并增加其工业应用价值。
纵使本发明已由上述实施例详细叙述而可由熟悉本领域技术人员任施匠思而为诸般修饰，然皆不脱如附申请专利范围所欲保护者。
序列表
<110> 东莞泛亚太生物科技有限公司
<120> 具提升活性的α-半乳糖苷酶
<130>
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 2178
<212> DNA
<213> 黑曲霉 (Aspergillus niger)
<400> 1
gcacccgcaa ttggggcttc taattcacag acgatcgtta cgaatggcac tagtttcgca 60
ttgaacggcg acaatgtctc atatcgattc catgtcaaca gtagcaccgg cgacttgatt 120
tctgatcatt ttggtggtgt cgtctccggc acaatccctt cgccagtgga acctgctgtc 180
aacggctggg tcggcatgcc tggtcgaatc cgccgggagt tccccgacca aggccgtggg 240
gatttccgca ttcccgccgt tcgtattcgg gaatcggcag gttatactgt tagcgatctc 300
caatatgtgt cgcacgaggt gatcgaaggt aaatatgctt tgcctggcct gcctgccaca 360
tttggcgatg cgcaggacgc caccactttg gtagtccatc tgtatgacaa ctatagctcc 420
gtcgcggccg acttgtcata ctccatattt ccgaaatatg atgcgatcgt gaggagtgtc 480
aatgtgacca accagggccc aggtaatatc actatcgagg cccttgcaag cataagtatc 540
gatttcccct atgaagacct cgacatggtc agcctccgag gcgactgggc cagagaggca 600
aatgttcaga gaagcaaagt gcagtatggc gtccagggat ttggaagcag tactggatat 660
tcctctcacc ttcataatcc cttccttgcc atagtagatc cagctactac cgaatcgcaa 720
ggcgaggcat ggggtttcaa ccttgtatat accggctctt tctcggcgca agtagagaaa 780
ggatcgcaag gtttcacccg ggcgctgctc ggcttcaacc cggaccaatt atcgtggaac 840
cttggccctg gcgagacttt aacctccccc gagtgtgttg cagtctactc ggacaaaggc 900
cttggctcag tgtctcgcaa attccaccgg ctatatcgca accacctcat gaagagcaag 960
ttcgccacgt ccgaccggcc ggttctgctg aatagctggg aaggtgttta tttcgactac 1020
aatcaaagca gcatcgaaac tcttgccgaa gagtccgctg ccctgggtgt ccacctcttt 1080
gtcatggacg acggctggtt tggggacaag taccctcgag tgtccgataa cgccggactg 1140
ggcgactgga tgcccaatcc agcgcgcttc ccggacgggt tgaccccggt cgtgcaagac 1200
atcacaaatc tcaccgtcaa tggcacagag tccacaaaac ttcgctttgg tatttgggtg 1260
gagcccgaga tggtcaaccc caattccact ctctaccacg aacacccgga gtgggcgctt 1320
catgccgggc cttacccccg taccgagcgt cggaaccagc tcgtcctcaa cctggcgctt 1380
ccggctgtgc aggacttcat catagacttc atgacgaacc tgttacaaga taccggcatt 1440
tcctacgtca aatgggacaa caaccggggg atacacgaga cgccctctcc gtcgactgac 1500
catcagtaca tgcttggcct ctaccgggtg ttcgacacac tgaccacccg ttttccggat 1560
gtcctgtggg aaggatgtgc ctcgggcggc ggccgctttg atgctggcat gctgcagtat 1620
gtcccccaga tctggacttc cgacaacacc gacgccatcg accgaatcac catccaattt 1680
gggacctcgc ttgcctaccc gccatcagca atgggagccc acctctccgc ggtccctaat 1740
gcacagaccg gtcgcactgt gccctttact ttccgcgcac acgttgctat gatgggtggt 1800
tctttcggct tggagctgga tccggcgacg gtggaagggg acgaaatagt tcccgagctc 1860
cttgcgctgg cggaaaaggt gaaccctatc attttgaacg gagatctgta tcggctacgc 1920
ctacctcaag actcccagtg gcctgccgca ctctttgtgt ctcaggatgg cgcacaggct 1980
gttctgttct acttccaggt ccagccgaat gtcaaccatg ccgtgccgtg ggtcaggctg 2040
caggggttgg accctaaggc ggactatacc gtcgatgggg atcagacgta ttctggagca 2100
acactaatga atctggggtt gcagtatagt tttgacaccg agtatggaag caaggtagtt 2160
ttcctggaga ggcaatga 2178
<210> 2
<211> 725
<212> PRT
<213> 黑曲霉 (Aspergillus niger)
<400> 2
Ala Pro Ala Ile Gly Ala Ser Asn Ser Gln Thr Ile Val Thr Asn Gly
1 5 10 15
Thr Ser Phe Ala Leu Asn Gly Asp Asn Val Ser Tyr Arg Phe His Val
20 25 30
Asn Ser Ser Thr Gly Asp Leu Ile Ser Asp His Phe Gly Gly Val Val
35 40 45
Ser Gly Thr Ile Pro Ser Pro Val Glu Pro Ala Val Asn Gly Trp Val
50 55 60
Gly Met Pro Gly Arg Ile Arg Arg Glu Phe Pro Asp Gln Gly Arg Gly
65 70 75 80
Asp Phe Arg Ile Pro Ala Val Arg Ile Arg Glu Ser Ala Gly Tyr Thr
85 90 95
Val Ser Asp Leu Gln Tyr Val Ser His Glu Val Ile Glu Gly Lys Tyr
100 105 110
Ala Leu Pro Gly Leu Pro Ala Thr Phe Gly Asp Ala Gln Asp Ala Thr
115 120 125
Thr Leu Val Val His Leu Tyr Asp Asn Tyr Ser Ser Val Ala Ala Asp
130 135 140
Leu Ser Tyr Ser Ile Phe Pro Lys Tyr Asp Ala Ile Val Arg Ser Val
145 150 155 160
Asn Val Thr Asn Gln Gly Pro Gly Asn Ile Thr Ile Glu Ala Leu Ala
165 170 175
Ser Ile Ser Ile Asp Phe Pro Tyr Glu Asp Leu Asp Met Val Ser Leu
180 185 190
Arg Gly Asp Trp Ala Arg Glu Ala Asn Val Gln Arg Ser Lys Val Gln
195 200 205
Tyr Gly Val Gln Gly Phe Gly Ser Ser Thr Gly Tyr Ser Ser His Leu
210 215 220
His Asn Pro Phe Leu Ala Ile Val Asp Pro Ala Thr Thr Glu Ser Gln
225 230 235 240
Gly Glu Ala Trp Gly Phe Asn Leu Val Tyr Thr Gly Ser Phe Ser Ala
245 250 255
Gln Val Glu Lys Gly Ser Gln Gly Phe Thr Arg Ala Leu Leu Gly Phe
260 265 270
Asn Pro Asp Gln Leu Ser Trp Asn Leu Gly Pro Gly Glu Thr Leu Thr
275 280 285
Ser Pro Glu Cys Val Ala Val Tyr Ser Asp Lys Gly Leu Gly Ser Val
290 295 300
Ser Arg Lys Phe His Arg Leu Tyr Arg Asn His Leu Met Lys Ser Lys
305 310 315 320
Phe Ala Thr Ser Asp Arg Pro Val Leu Leu Asn Ser Trp Glu Gly Val
325 330 335
Tyr Phe Asp Tyr Asn Gln Ser Ser Ile Glu Thr Leu Ala Glu Glu Ser
340 345 350
Ala Ala Leu Gly Val His Leu Phe Val Met Asp Asp Gly Trp Phe Gly
355 360 365
Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala Gly Leu Gly Asp Trp Met
370 375 380
Pro Asn Pro Ala Arg Phe Pro Asp Gly Leu Thr Pro Val Val Gln Asp
385 390 395 400
Ile Thr Asn Leu Thr Val Asn Gly Thr Glu Ser Thr Lys Leu Arg Phe
405 410 415
Gly Ile Trp Val Glu Pro Glu Met Val Asn Pro Asn Ser Thr Leu Tyr
420 425 430
His Glu His Pro Glu Trp Ala Leu His Ala Gly Pro Tyr Pro Arg Thr
435 440 445
Glu Arg Arg Asn Gln Leu Val Leu Asn Leu Ala Leu Pro Ala Val Gln
450 455 460
Asp Phe Ile Ile Asp Phe Met Thr Asn Leu Leu Gln Asp Thr Gly Ile
465 470 475 480
Ser Tyr Val Lys Trp Asp Asn Asn Arg Gly Ile His Glu Thr Pro Ser
485 490 495
Pro Ser Thr Asp His Gln Tyr Met Leu Gly Leu Tyr Arg Val Phe Asp
500 505 510
Thr Leu Thr Thr Arg Phe Pro Asp Val Leu Trp Glu Gly Cys Ala Ser
515 520 525
Gly Gly Gly Arg Phe Asp Ala Gly Met Leu Gln Tyr Val Pro Gln Ile
530 535 540
Trp Thr Ser Asp Asn Thr Asp Ala Ile Asp Arg Ile Thr Ile Gln Phe
545 550 555 560
Gly Thr Ser Leu Ala Tyr Pro Pro Ser Ala Met Gly Ala His Leu Ser
565 570 575
Ala Val Pro Asn Ala Gln Thr Gly Arg Thr Val Pro Phe Thr Phe Arg
580 585 590
Ala His Val Ala Met Met Gly Gly Ser Phe Gly Leu Glu Leu Asp Pro
595 600 605
Ala Thr Val Glu Gly Asp Glu Ile Val Pro Glu Leu Leu Ala Leu Ala
610 615 620
Glu Lys Val Asn Pro Ile Ile Leu Asn Gly Asp Leu Tyr Arg Leu Arg
625 630 635 640
Leu Pro Gln Asp Ser Gln Trp Pro Ala Ala Leu Phe Val Ser Gln Asp
645 650 655
Gly Ala Gln Ala Val Leu Phe Tyr Phe Gln Val Gln Pro Asn Val Asn
660 665 670
His Ala Val Pro Trp Val Arg Leu Gln Gly Leu Asp Pro Lys Ala Asp
675 680 685
Tyr Thr Val Asp Gly Asp Gln Thr Tyr Ser Gly Ala Thr Leu Met Asn
690 695 700
Leu Gly Leu Gln Tyr Ser Phe Asp Thr Glu Tyr Gly Ser Lys Val Val
705 710 715 720
Phe Leu Glu Arg Gln
725
<210> 3
<211> 37
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 合成引物
<400> 3
cttacccccg taccgagttt cggaaccagc tcgtcct 37
<210> 4
<211> 37
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 合成引物
<400> 4
cttacccccg taccgaggtt cggaaccagc tcgtcct 37
<210> 5
<211> 37
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 合成引物
<400> 5
cttacccccg taccgagtct cggaaccagc tcgtcct 37
<210> 6
<211> 37
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 合成引物
<400> 6
cttacccccg taccgaggct cggaaccagc tcgtcct 37
<210> 7
<211> 37
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 合成引物
<400> 7
cttacccccg taccgagggt cggaaccagc tcgtcct 37
<210> 8
<211> 2178
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 8
gcacccgcaa ttggggcttc taattcacag acgatcgtta cgaatggcac tagtttcgca 60
ttgaacggcg acaatgtctc atatcgattc catgtcaaca gtagcaccgg cgacttgatt 120
tctgatcatt ttggtggtgt cgtctccggc acaatccctt cgccagtgga acctgctgtc 180
aacggctggg tcggcatgcc tggtcgaatc cgccgggagt tccccgacca aggccgtggg 240
gatttccgca ttcccgccgt tcgtattcgg gaatcggcag gttatactgt tagcgatctc 300
caatatgtgt cgcacgaggt gatcgaaggt aaatatgctt tgcctggcct gcctgccaca 360
tttggcgatg cgcaggacgc caccactttg gtagtccatc tgtatgacaa ctatagctcc 420
gtcgcggccg acttgtcata ctccatattt ccgaaatatg atgcgatcgt gaggagtgtc 480
aatgtgacca accagggccc aggtaatatc actatcgagg cccttgcaag cataagtatc 540
gatttcccct atgaagacct cgacatggtc agcctccgag gcgactgggc cagagaggca 600
aatgttcaga gaagcaaagt gcagtatggc gtccagggat ttggaagcag tactggatat 660
tcctctcacc ttcataatcc cttccttgcc atagtagatc cagctactac cgaatcgcaa 720
ggcgaggcat ggggtttcaa ccttgtatat accggctctt tctcggcgca agtagagaaa 780
ggatcgcaag gtttcacccg ggcgctgctc ggcttcaacc cggaccaatt atcgtggaac 840
cttggccctg gcgagacttt aacctccccc gagtgtgttg cagtctactc ggacaaaggc 900
cttggctcag tgtctcgcaa attccaccgg ctatatcgca accacctcat gaagagcaag 960
ttcgccacgt ccgaccggcc ggttctgctg aatagctggg aaggtgttta tttcgactac 1020
aatcaaagca gcatcgaaac tcttgccgaa gagtccgctg ccctgggtgt ccacctcttt 1080
gtcatggacg acggctggtt tggggacaag taccctcgag tgtccgataa cgccggactg 1140
ggcgactgga tgcccaatcc agcgcgcttc ccggacgggt tgaccccggt cgtgcaagac 1200
atcacaaatc tcaccgtcaa tggcacagag tccacaaaac ttcgctttgg tatttgggtg 1260
gagcccgaga tggtcaaccc caattccact ctctaccacg aacacccgga gtgggcgctt 1320
catgccgggc cttacccccg taccgagttt cggaaccagc tcgtcctcaa cctggcgctt 1380
ccggctgtgc aggacttcat catagacttc atgacgaacc tgttacaaga taccggcatt 1440
tcctacgtca aatgggacaa caaccggggg atacacgaga cgccctctcc gtcgactgac 1500
catcagtaca tgcttggcct ctaccgggtg ttcgacacac tgaccacccg ttttccggat 1560
gtcctgtggg aaggatgtgc ctcgggcggc ggccgctttg atgctggcat gctgcagtat 1620
gtcccccaga tctggacttc cgacaacacc gacgccatcg accgaatcac catccaattt 1680
gggacctcgc ttgcctaccc gccatcagca atgggagccc acctctccgc ggtccctaat 1740
gcacagaccg gtcgcactgt gccctttact ttccgcgcac acgttgctat gatgggtggt 1800
tctttcggct tggagctgga tccggcgacg gtggaagggg acgaaatagt tcccgagctc 1860
cttgcgctgg cggaaaaggt gaaccctatc attttgaacg gagatctgta tcggctacgc 1920
ctacctcaag actcccagtg gcctgccgca ctctttgtgt ctcaggatgg cgcacaggct 1980
gttctgttct acttccaggt ccagccgaat gtcaaccatg ccgtgccgtg ggtcaggctg 2040
caggggttgg accctaaggc ggactatacc gtcgatgggg atcagacgta ttctggagca 2100
acactaatga atctggggtt gcagtatagt tttgacaccg agtatggaag caaggtagtt 2160
ttcctggaga ggcaatga 2178
<210> 9
<211> 725
<212> PRT
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 9
Ala Pro Ala Ile Gly Ala Ser Asn Ser Gln Thr Ile Val Thr Asn Gly
1 5 10 15
Thr Ser Phe Ala Leu Asn Gly Asp Asn Val Ser Tyr Arg Phe His Val
20 25 30
Asn Ser Ser Thr Gly Asp Leu Ile Ser Asp His Phe Gly Gly Val Val
35 40 45
Ser Gly Thr Ile Pro Ser Pro Val Glu Pro Ala Val Asn Gly Trp Val
50 55 60
Gly Met Pro Gly Arg Ile Arg Arg Glu Phe Pro Asp Gln Gly Arg Gly
65 70 75 80
Asp Phe Arg Ile Pro Ala Val Arg Ile Arg Glu Ser Ala Gly Tyr Thr
85 90 95
Val Ser Asp Leu Gln Tyr Val Ser His Glu Val Ile Glu Gly Lys Tyr
100 105 110
Ala Leu Pro Gly Leu Pro Ala Thr Phe Gly Asp Ala Gln Asp Ala Thr
115 120 125
Thr Leu Val Val His Leu Tyr Asp Asn Tyr Ser Ser Val Ala Ala Asp
130 135 140
Leu Ser Tyr Ser Ile Phe Pro Lys Tyr Asp Ala Ile Val Arg Ser Val
145 150 155 160
Asn Val Thr Asn Gln Gly Pro Gly Asn Ile Thr Ile Glu Ala Leu Ala
165 170 175
Ser Ile Ser Ile Asp Phe Pro Tyr Glu Asp Leu Asp Met Val Ser Leu
180 185 190
Arg Gly Asp Trp Ala Arg Glu Ala Asn Val Gln Arg Ser Lys Val Gln
195 200 205
Tyr Gly Val Gln Gly Phe Gly Ser Ser Thr Gly Tyr Ser Ser His Leu
210 215 220
His Asn Pro Phe Leu Ala Ile Val Asp Pro Ala Thr Thr Glu Ser Gln
225 230 235 240
Gly Glu Ala Trp Gly Phe Asn Leu Val Tyr Thr Gly Ser Phe Ser Ala
245 250 255
Gln Val Glu Lys Gly Ser Gln Gly Phe Thr Arg Ala Leu Leu Gly Phe
260 265 270
Asn Pro Asp Gln Leu Ser Trp Asn Leu Gly Pro Gly Glu Thr Leu Thr
275 280 285
Ser Pro Glu Cys Val Ala Val Tyr Ser Asp Lys Gly Leu Gly Ser Val
290 295 300
Ser Arg Lys Phe His Arg Leu Tyr Arg Asn His Leu Met Lys Ser Lys
305 310 315 320
Phe Ala Thr Ser Asp Arg Pro Val Leu Leu Asn Ser Trp Glu Gly Val
325 330 335
Tyr Phe Asp Tyr Asn Gln Ser Ser Ile Glu Thr Leu Ala Glu Glu Ser
340 345 350
Ala Ala Leu Gly Val His Leu Phe Val Met Asp Asp Gly Trp Phe Gly
355 360 365
Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala Gly Leu Gly Asp Trp Met
370 375 380
Pro Asn Pro Ala Arg Phe Pro Asp Gly Leu Thr Pro Val Val Gln Asp
385 390 395 400
Ile Thr Asn Leu Thr Val Asn Gly Thr Glu Ser Thr Lys Leu Arg Phe
405 410 415
Gly Ile Trp Val Glu Pro Glu Met Val Asn Pro Asn Ser Thr Leu Tyr
420 425 430
His Glu His Pro Glu Trp Ala Leu His Ala Gly Pro Tyr Pro Arg Thr
435 440 445
Glu Phe Arg Asn Gln Leu Val Leu Asn Leu Ala Leu Pro Ala Val Gln
450 455 460
Asp Phe Ile Ile Asp Phe Met Thr Asn Leu Leu Gln Asp Thr Gly Ile
465 470 475 480
Ser Tyr Val Lys Trp Asp Asn Asn Arg Gly Ile His Glu Thr Pro Ser
485 490 495
Pro Ser Thr Asp His Gln Tyr Met Leu Gly Leu Tyr Arg Val Phe Asp
500 505 510
Thr Leu Thr Thr Arg Phe Pro Asp Val Leu Trp Glu Gly Cys Ala Ser
515 520 525
Gly Gly Gly Arg Phe Asp Ala Gly Met Leu Gln Tyr Val Pro Gln Ile
530 535 540
Trp Thr Ser Asp Asn Thr Asp Ala Ile Asp Arg Ile Thr Ile Gln Phe
545 550 555 560
Gly Thr Ser Leu Ala Tyr Pro Pro Ser Ala Met Gly Ala His Leu Ser
565 570 575
Ala Val Pro Asn Ala Gln Thr Gly Arg Thr Val Pro Phe Thr Phe Arg
580 585 590
Ala His Val Ala Met Met Gly Gly Ser Phe Gly Leu Glu Leu Asp Pro
595 600 605
Ala Thr Val Glu Gly Asp Glu Ile Val Pro Glu Leu Leu Ala Leu Ala
610 615 620
Glu Lys Val Asn Pro Ile Ile Leu Asn Gly Asp Leu Tyr Arg Leu Arg
625 630 635 640
Leu Pro Gln Asp Ser Gln Trp Pro Ala Ala Leu Phe Val Ser Gln Asp
645 650 655
Gly Ala Gln Ala Val Leu Phe Tyr Phe Gln Val Gln Pro Asn Val Asn
660 665 670
His Ala Val Pro Trp Val Arg Leu Gln Gly Leu Asp Pro Lys Ala Asp
675 680 685
Tyr Thr Val Asp Gly Asp Gln Thr Tyr Ser Gly Ala Thr Leu Met Asn
690 695 700
Leu Gly Leu Gln Tyr Ser Phe Asp Thr Glu Tyr Gly Ser Lys Val Val
705 710 715 720
Phe Leu Glu Arg Gln
725
<210> 10
<211> 2178
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 10
gcacccgcaa ttggggcttc taattcacag acgatcgtta cgaatggcac tagtttcgca 60
ttgaacggcg acaatgtctc atatcgattc catgtcaaca gtagcaccgg cgacttgatt 120
tctgatcatt ttggtggtgt cgtctccggc acaatccctt cgccagtgga acctgctgtc 180
aacggctggg tcggcatgcc tggtcgaatc cgccgggagt tccccgacca aggccgtggg 240
gatttccgca ttcccgccgt tcgtattcgg gaatcggcag gttatactgt tagcgatctc 300
caatatgtgt cgcacgaggt gatcgaaggt aaatatgctt tgcctggcct gcctgccaca 360
tttggcgatg cgcaggacgc caccactttg gtagtccatc tgtatgacaa ctatagctcc 420
gtcgcggccg acttgtcata ctccatattt ccgaaatatg atgcgatcgt gaggagtgtc 480
aatgtgacca accagggccc aggtaatatc actatcgagg cccttgcaag cataagtatc 540
gatttcccct atgaagacct cgacatggtc agcctccgag gcgactgggc cagagaggca 600
aatgttcaga gaagcaaagt gcagtatggc gtccagggat ttggaagcag tactggatat 660
tcctctcacc ttcataatcc cttccttgcc atagtagatc cagctactac cgaatcgcaa 720
ggcgaggcat ggggtttcaa ccttgtatat accggctctt tctcggcgca agtagagaaa 780
ggatcgcaag gtttcacccg ggcgctgctc ggcttcaacc cggaccaatt atcgtggaac 840
cttggccctg gcgagacttt aacctccccc gagtgtgttg cagtctactc ggacaaaggc 900
cttggctcag tgtctcgcaa attccaccgg ctatatcgca accacctcat gaagagcaag 960
ttcgccacgt ccgaccggcc ggttctgctg aatagctggg aaggtgttta tttcgactac 1020
aatcaaagca gcatcgaaac tcttgccgaa gagtccgctg ccctgggtgt ccacctcttt 1080
gtcatggacg acggctggtt tggggacaag taccctcgag tgtccgataa cgccggactg 1140
ggcgactgga tgcccaatcc agcgcgcttc ccggacgggt tgaccccggt cgtgcaagac 1200
atcacaaatc tcaccgtcaa tggcacagag tccacaaaac ttcgctttgg tatttgggtg 1260
gagcccgaga tggtcaaccc caattccact ctctaccacg aacacccgga gtgggcgctt 1320
catgccgggc cttacccccg taccgaggtt cggaaccagc tcgtcctcaa cctggcgctt 1380
ccggctgtgc aggacttcat catagacttc atgacgaacc tgttacaaga taccggcatt 1440
tcctacgtca aatgggacaa caaccggggg atacacgaga cgccctctcc gtcgactgac 1500
catcagtaca tgcttggcct ctaccgggtg ttcgacacac tgaccacccg ttttccggat 1560
gtcctgtggg aaggatgtgc ctcgggcggc ggccgctttg atgctggcat gctgcagtat 1620
gtcccccaga tctggacttc cgacaacacc gacgccatcg accgaatcac catccaattt 1680
gggacctcgc ttgcctaccc gccatcagca atgggagccc acctctccgc ggtccctaat 1740
gcacagaccg gtcgcactgt gccctttact ttccgcgcac acgttgctat gatgggtggt 1800
tctttcggct tggagctgga tccggcgacg gtggaagggg acgaaatagt tcccgagctc 1860
cttgcgctgg cggaaaaggt gaaccctatc attttgaacg gagatctgta tcggctacgc 1920
ctacctcaag actcccagtg gcctgccgca ctctttgtgt ctcaggatgg cgcacaggct 1980
gttctgttct acttccaggt ccagccgaat gtcaaccatg ccgtgccgtg ggtcaggctg 2040
caggggttgg accctaaggc ggactatacc gtcgatgggg atcagacgta ttctggagca 2100
acactaatga atctggggtt gcagtatagt tttgacaccg agtatggaag caaggtagtt 2160
ttcctggaga ggcaatga 2178
<210> 11
<211> 725
<212> PRT
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 11
Ala Pro Ala Ile Gly Ala Ser Asn Ser Gln Thr Ile Val Thr Asn Gly
1 5 10 15
Thr Ser Phe Ala Leu Asn Gly Asp Asn Val Ser Tyr Arg Phe His Val
20 25 30
Asn Ser Ser Thr Gly Asp Leu Ile Ser Asp His Phe Gly Gly Val Val
35 40 45
Ser Gly Thr Ile Pro Ser Pro Val Glu Pro Ala Val Asn Gly Trp Val
50 55 60
Gly Met Pro Gly Arg Ile Arg Arg Glu Phe Pro Asp Gln Gly Arg Gly
65 70 75 80
Asp Phe Arg Ile Pro Ala Val Arg Ile Arg Glu Ser Ala Gly Tyr Thr
85 90 95
Val Ser Asp Leu Gln Tyr Val Ser His Glu Val Ile Glu Gly Lys Tyr
100 105 110
Ala Leu Pro Gly Leu Pro Ala Thr Phe Gly Asp Ala Gln Asp Ala Thr
115 120 125
Thr Leu Val Val His Leu Tyr Asp Asn Tyr Ser Ser Val Ala Ala Asp
130 135 140
Leu Ser Tyr Ser Ile Phe Pro Lys Tyr Asp Ala Ile Val Arg Ser Val
145 150 155 160
Asn Val Thr Asn Gln Gly Pro Gly Asn Ile Thr Ile Glu Ala Leu Ala
165 170 175
Ser Ile Ser Ile Asp Phe Pro Tyr Glu Asp Leu Asp Met Val Ser Leu
180 185 190
Arg Gly Asp Trp Ala Arg Glu Ala Asn Val Gln Arg Ser Lys Val Gln
195 200 205
Tyr Gly Val Gln Gly Phe Gly Ser Ser Thr Gly Tyr Ser Ser His Leu
210 215 220
His Asn Pro Phe Leu Ala Ile Val Asp Pro Ala Thr Thr Glu Ser Gln
225 230 235 240
Gly Glu Ala Trp Gly Phe Asn Leu Val Tyr Thr Gly Ser Phe Ser Ala
245 250 255
Gln Val Glu Lys Gly Ser Gln Gly Phe Thr Arg Ala Leu Leu Gly Phe
260 265 270
Asn Pro Asp Gln Leu Ser Trp Asn Leu Gly Pro Gly Glu Thr Leu Thr
275 280 285
Ser Pro Glu Cys Val Ala Val Tyr Ser Asp Lys Gly Leu Gly Ser Val
290 295 300
Ser Arg Lys Phe His Arg Leu Tyr Arg Asn His Leu Met Lys Ser Lys
305 310 315 320
Phe Ala Thr Ser Asp Arg Pro Val Leu Leu Asn Ser Trp Glu Gly Val
325 330 335
Tyr Phe Asp Tyr Asn Gln Ser Ser Ile Glu Thr Leu Ala Glu Glu Ser
340 345 350
Ala Ala Leu Gly Val His Leu Phe Val Met Asp Asp Gly Trp Phe Gly
355 360 365
Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala Gly Leu Gly Asp Trp Met
370 375 380
Pro Asn Pro Ala Arg Phe Pro Asp Gly Leu Thr Pro Val Val Gln Asp
385 390 395 400
Ile Thr Asn Leu Thr Val Asn Gly Thr Glu Ser Thr Lys Leu Arg Phe
405 410 415
Gly Ile Trp Val Glu Pro Glu Met Val Asn Pro Asn Ser Thr Leu Tyr
420 425 430
His Glu His Pro Glu Trp Ala Leu His Ala Gly Pro Tyr Pro Arg Thr
435 440 445
Glu Val Arg Asn Gln Leu Val Leu Asn Leu Ala Leu Pro Ala Val Gln
450 455 460
Asp Phe Ile Ile Asp Phe Met Thr Asn Leu Leu Gln Asp Thr Gly Ile
465 470 475 480
Ser Tyr Val Lys Trp Asp Asn Asn Arg Gly Ile His Glu Thr Pro Ser
485 490 495
Pro Ser Thr Asp His Gln Tyr Met Leu Gly Leu Tyr Arg Val Phe Asp
500 505 510
Thr Leu Thr Thr Arg Phe Pro Asp Val Leu Trp Glu Gly Cys Ala Ser
515 520 525
Gly Gly Gly Arg Phe Asp Ala Gly Met Leu Gln Tyr Val Pro Gln Ile
530 535 540
Trp Thr Ser Asp Asn Thr Asp Ala Ile Asp Arg Ile Thr Ile Gln Phe
545 550 555 560
Gly Thr Ser Leu Ala Tyr Pro Pro Ser Ala Met Gly Ala His Leu Ser
565 570 575
Ala Val Pro Asn Ala Gln Thr Gly Arg Thr Val Pro Phe Thr Phe Arg
580 585 590
Ala His Val Ala Met Met Gly Gly Ser Phe Gly Leu Glu Leu Asp Pro
595 600 605
Ala Thr Val Glu Gly Asp Glu Ile Val Pro Glu Leu Leu Ala Leu Ala
610 615 620
Glu Lys Val Asn Pro Ile Ile Leu Asn Gly Asp Leu Tyr Arg Leu Arg
625 630 635 640
Leu Pro Gln Asp Ser Gln Trp Pro Ala Ala Leu Phe Val Ser Gln Asp
645 650 655
Gly Ala Gln Ala Val Leu Phe Tyr Phe Gln Val Gln Pro Asn Val Asn
660 665 670
His Ala Val Pro Trp Val Arg Leu Gln Gly Leu Asp Pro Lys Ala Asp
675 680 685
Tyr Thr Val Asp Gly Asp Gln Thr Tyr Ser Gly Ala Thr Leu Met Asn
690 695 700
Leu Gly Leu Gln Tyr Ser Phe Asp Thr Glu Tyr Gly Ser Lys Val Val
705 710 715 720
Phe Leu Glu Arg Gln
725
<210> 12
<211> 2178
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 12
gcacccgcaa ttggggcttc taattcacag acgatcgtta cgaatggcac tagtttcgca 60
ttgaacggcg acaatgtctc atatcgattc catgtcaaca gtagcaccgg cgacttgatt 120
tctgatcatt ttggtggtgt cgtctccggc acaatccctt cgccagtgga acctgctgtc 180
aacggctggg tcggcatgcc tggtcgaatc cgccgggagt tccccgacca aggccgtggg 240
gatttccgca ttcccgccgt tcgtattcgg gaatcggcag gttatactgt tagcgatctc 300
caatatgtgt cgcacgaggt gatcgaaggt aaatatgctt tgcctggcct gcctgccaca 360
tttggcgatg cgcaggacgc caccactttg gtagtccatc tgtatgacaa ctatagctcc 420
gtcgcggccg acttgtcata ctccatattt ccgaaatatg atgcgatcgt gaggagtgtc 480
aatgtgacca accagggccc aggtaatatc actatcgagg cccttgcaag cataagtatc 540
gatttcccct atgaagacct cgacatggtc agcctccgag gcgactgggc cagagaggca 600
aatgttcaga gaagcaaagt gcagtatggc gtccagggat ttggaagcag tactggatat 660
tcctctcacc ttcataatcc cttccttgcc atagtagatc cagctactac cgaatcgcaa 720
ggcgaggcat ggggtttcaa ccttgtatat accggctctt tctcggcgca agtagagaaa 780
ggatcgcaag gtttcacccg ggcgctgctc ggcttcaacc cggaccaatt atcgtggaac 840
cttggccctg gcgagacttt aacctccccc gagtgtgttg cagtctactc ggacaaaggc 900
cttggctcag tgtctcgcaa attccaccgg ctatatcgca accacctcat gaagagcaag 960
ttcgccacgt ccgaccggcc ggttctgctg aatagctggg aaggtgttta tttcgactac 1020
aatcaaagca gcatcgaaac tcttgccgaa gagtccgctg ccctgggtgt ccacctcttt 1080
gtcatggacg acggctggtt tggggacaag taccctcgag tgtccgataa cgccggactg 1140
ggcgactgga tgcccaatcc agcgcgcttc ccggacgggt tgaccccggt cgtgcaagac 1200
atcacaaatc tcaccgtcaa tggcacagag tccacaaaac ttcgctttgg tatttgggtg 1260
gagcccgaga tggtcaaccc caattccact ctctaccacg aacacccgga gtgggcgctt 1320
catgccgggc cttacccccg taccgagtct cggaaccagc tcgtcctcaa cctggcgctt 1380
ccggctgtgc aggacttcat catagacttc atgacgaacc tgttacaaga taccggcatt 1440
tcctacgtca aatgggacaa caaccggggg atacacgaga cgccctctcc gtcgactgac 1500
catcagtaca tgcttggcct ctaccgggtg ttcgacacac tgaccacccg ttttccggat 1560
gtcctgtggg aaggatgtgc ctcgggcggc ggccgctttg atgctggcat gctgcagtat 1620
gtcccccaga tctggacttc cgacaacacc gacgccatcg accgaatcac catccaattt 1680
gggacctcgc ttgcctaccc gccatcagca atgggagccc acctctccgc ggtccctaat 1740
gcacagaccg gtcgcactgt gccctttact ttccgcgcac acgttgctat gatgggtggt 1800
tctttcggct tggagctgga tccggcgacg gtggaagggg acgaaatagt tcccgagctc 1860
cttgcgctgg cggaaaaggt gaaccctatc attttgaacg gagatctgta tcggctacgc 1920
ctacctcaag actcccagtg gcctgccgca ctctttgtgt ctcaggatgg cgcacaggct 1980
gttctgttct acttccaggt ccagccgaat gtcaaccatg ccgtgccgtg ggtcaggctg 2040
caggggttgg accctaaggc ggactatacc gtcgatgggg atcagacgta ttctggagca 2100
acactaatga atctggggtt gcagtatagt tttgacaccg agtatggaag caaggtagtt 2160
ttcctggaga ggcaatga 2178
<210> 13
<211> 725
<212> PRT
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 13
Ala Pro Ala Ile Gly Ala Ser Asn Ser Gln Thr Ile Val Thr Asn Gly
1 5 10 15
Thr Ser Phe Ala Leu Asn Gly Asp Asn Val Ser Tyr Arg Phe His Val
20 25 30
Asn Ser Ser Thr Gly Asp Leu Ile Ser Asp His Phe Gly Gly Val Val
35 40 45
Ser Gly Thr Ile Pro Ser Pro Val Glu Pro Ala Val Asn Gly Trp Val
50 55 60
Gly Met Pro Gly Arg Ile Arg Arg Glu Phe Pro Asp Gln Gly Arg Gly
65 70 75 80
Asp Phe Arg Ile Pro Ala Val Arg Ile Arg Glu Ser Ala Gly Tyr Thr
85 90 95
Val Ser Asp Leu Gln Tyr Val Ser His Glu Val Ile Glu Gly Lys Tyr
100 105 110
Ala Leu Pro Gly Leu Pro Ala Thr Phe Gly Asp Ala Gln Asp Ala Thr
115 120 125
Thr Leu Val Val His Leu Tyr Asp Asn Tyr Ser Ser Val Ala Ala Asp
130 135 140
Leu Ser Tyr Ser Ile Phe Pro Lys Tyr Asp Ala Ile Val Arg Ser Val
145 150 155 160
Asn Val Thr Asn Gln Gly Pro Gly Asn Ile Thr Ile Glu Ala Leu Ala
165 170 175
Ser Ile Ser Ile Asp Phe Pro Tyr Glu Asp Leu Asp Met Val Ser Leu
180 185 190
Arg Gly Asp Trp Ala Arg Glu Ala Asn Val Gln Arg Ser Lys Val Gln
195 200 205
Tyr Gly Val Gln Gly Phe Gly Ser Ser Thr Gly Tyr Ser Ser His Leu
210 215 220
His Asn Pro Phe Leu Ala Ile Val Asp Pro Ala Thr Thr Glu Ser Gln
225 230 235 240
Gly Glu Ala Trp Gly Phe Asn Leu Val Tyr Thr Gly Ser Phe Ser Ala
245 250 255
Gln Val Glu Lys Gly Ser Gln Gly Phe Thr Arg Ala Leu Leu Gly Phe
260 265 270
Asn Pro Asp Gln Leu Ser Trp Asn Leu Gly Pro Gly Glu Thr Leu Thr
275 280 285
Ser Pro Glu Cys Val Ala Val Tyr Ser Asp Lys Gly Leu Gly Ser Val
290 295 300
Ser Arg Lys Phe His Arg Leu Tyr Arg Asn His Leu Met Lys Ser Lys
305 310 315 320
Phe Ala Thr Ser Asp Arg Pro Val Leu Leu Asn Ser Trp Glu Gly Val
325 330 335
Tyr Phe Asp Tyr Asn Gln Ser Ser Ile Glu Thr Leu Ala Glu Glu Ser
340 345 350
Ala Ala Leu Gly Val His Leu Phe Val Met Asp Asp Gly Trp Phe Gly
355 360 365
Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala Gly Leu Gly Asp Trp Met
370 375 380
Pro Asn Pro Ala Arg Phe Pro Asp Gly Leu Thr Pro Val Val Gln Asp
385 390 395 400
Ile Thr Asn Leu Thr Val Asn Gly Thr Glu Ser Thr Lys Leu Arg Phe
405 410 415
Gly Ile Trp Val Glu Pro Glu Met Val Asn Pro Asn Ser Thr Leu Tyr
420 425 430
His Glu His Pro Glu Trp Ala Leu His Ala Gly Pro Tyr Pro Arg Thr
435 440 445
Glu Ser Arg Asn Gln Leu Val Leu Asn Leu Ala Leu Pro Ala Val Gln
450 455 460
Asp Phe Ile Ile Asp Phe Met Thr Asn Leu Leu Gln Asp Thr Gly Ile
465 470 475 480
Ser Tyr Val Lys Trp Asp Asn Asn Arg Gly Ile His Glu Thr Pro Ser
485 490 495
Pro Ser Thr Asp His Gln Tyr Met Leu Gly Leu Tyr Arg Val Phe Asp
500 505 510
Thr Leu Thr Thr Arg Phe Pro Asp Val Leu Trp Glu Gly Cys Ala Ser
515 520 525
Gly Gly Gly Arg Phe Asp Ala Gly Met Leu Gln Tyr Val Pro Gln Ile
530 535 540
Trp Thr Ser Asp Asn Thr Asp Ala Ile Asp Arg Ile Thr Ile Gln Phe
545 550 555 560
Gly Thr Ser Leu Ala Tyr Pro Pro Ser Ala Met Gly Ala His Leu Ser
565 570 575
Ala Val Pro Asn Ala Gln Thr Gly Arg Thr Val Pro Phe Thr Phe Arg
580 585 590
Ala His Val Ala Met Met Gly Gly Ser Phe Gly Leu Glu Leu Asp Pro
595 600 605
Ala Thr Val Glu Gly Asp Glu Ile Val Pro Glu Leu Leu Ala Leu Ala
610 615 620
Glu Lys Val Asn Pro Ile Ile Leu Asn Gly Asp Leu Tyr Arg Leu Arg
625 630 635 640
Leu Pro Gln Asp Ser Gln Trp Pro Ala Ala Leu Phe Val Ser Gln Asp
645 650 655
Gly Ala Gln Ala Val Leu Phe Tyr Phe Gln Val Gln Pro Asn Val Asn
660 665 670
His Ala Val Pro Trp Val Arg Leu Gln Gly Leu Asp Pro Lys Ala Asp
675 680 685
Tyr Thr Val Asp Gly Asp Gln Thr Tyr Ser Gly Ala Thr Leu Met Asn
690 695 700
Leu Gly Leu Gln Tyr Ser Phe Asp Thr Glu Tyr Gly Ser Lys Val Val
705 710 715 720
Phe Leu Glu Arg Gln
725
<210> 14
<211> 2178
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 14
gcacccgcaa ttggggcttc taattcacag acgatcgtta cgaatggcac tagtttcgca 60
ttgaacggcg acaatgtctc atatcgattc catgtcaaca gtagcaccgg cgacttgatt 120
tctgatcatt ttggtggtgt cgtctccggc acaatccctt cgccagtgga acctgctgtc 180
aacggctggg tcggcatgcc tggtcgaatc cgccgggagt tccccgacca aggccgtggg 240
gatttccgca ttcccgccgt tcgtattcgg gaatcggcag gttatactgt tagcgatctc 300
caatatgtgt cgcacgaggt gatcgaaggt aaatatgctt tgcctggcct gcctgccaca 360
tttggcgatg cgcaggacgc caccactttg gtagtccatc tgtatgacaa ctatagctcc 420
gtcgcggccg acttgtcata ctccatattt ccgaaatatg atgcgatcgt gaggagtgtc 480
aatgtgacca accagggccc aggtaatatc actatcgagg cccttgcaag cataagtatc 540
gatttcccct atgaagacct cgacatggtc agcctccgag gcgactgggc cagagaggca 600
aatgttcaga gaagcaaagt gcagtatggc gtccagggat ttggaagcag tactggatat 660
tcctctcacc ttcataatcc cttccttgcc atagtagatc cagctactac cgaatcgcaa 720
ggcgaggcat ggggtttcaa ccttgtatat accggctctt tctcggcgca agtagagaaa 780
ggatcgcaag gtttcacccg ggcgctgctc ggcttcaacc cggaccaatt atcgtggaac 840
cttggccctg gcgagacttt aacctccccc gagtgtgttg cagtctactc ggacaaaggc 900
cttggctcag tgtctcgcaa attccaccgg ctatatcgca accacctcat gaagagcaag 960
ttcgccacgt ccgaccggcc ggttctgctg aatagctggg aaggtgttta tttcgactac 1020
aatcaaagca gcatcgaaac tcttgccgaa gagtccgctg ccctgggtgt ccacctcttt 1080
gtcatggacg acggctggtt tggggacaag taccctcgag tgtccgataa cgccggactg 1140
ggcgactgga tgcccaatcc agcgcgcttc ccggacgggt tgaccccggt cgtgcaagac 1200
atcacaaatc tcaccgtcaa tggcacagag tccacaaaac ttcgctttgg tatttgggtg 1260
gagcccgaga tggtcaaccc caattccact ctctaccacg aacacccgga gtgggcgctt 1320
catgccgggc cttacccccg taccgaggct cggaaccagc tcgtcctcaa cctggcgctt 1380
ccggctgtgc aggacttcat catagacttc atgacgaacc tgttacaaga taccggcatt 1440
tcctacgtca aatgggacaa caaccggggg atacacgaga cgccctctcc gtcgactgac 1500
catcagtaca tgcttggcct ctaccgggtg ttcgacacac tgaccacccg ttttccggat 1560
gtcctgtggg aaggatgtgc ctcgggcggc ggccgctttg atgctggcat gctgcagtat 1620
gtcccccaga tctggacttc cgacaacacc gacgccatcg accgaatcac catccaattt 1680
gggacctcgc ttgcctaccc gccatcagca atgggagccc acctctccgc ggtccctaat 1740
gcacagaccg gtcgcactgt gccctttact ttccgcgcac acgttgctat gatgggtggt 1800
tctttcggct tggagctgga tccggcgacg gtggaagggg acgaaatagt tcccgagctc 1860
cttgcgctgg cggaaaaggt gaaccctatc attttgaacg gagatctgta tcggctacgc 1920
ctacctcaag actcccagtg gcctgccgca ctctttgtgt ctcaggatgg cgcacaggct 1980
gttctgttct acttccaggt ccagccgaat gtcaaccatg ccgtgccgtg ggtcaggctg 2040
caggggttgg accctaaggc ggactatacc gtcgatgggg atcagacgta ttctggagca 2100
acactaatga atctggggtt gcagtatagt tttgacaccg agtatggaag caaggtagtt 2160
ttcctggaga ggcaatga 2178
<210> 15
<211> 725
<212> PRT
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 15
Ala Pro Ala Ile Gly Ala Ser Asn Ser Gln Thr Ile Val Thr Asn Gly
1 5 10 15
Thr Ser Phe Ala Leu Asn Gly Asp Asn Val Ser Tyr Arg Phe His Val
20 25 30
Asn Ser Ser Thr Gly Asp Leu Ile Ser Asp His Phe Gly Gly Val Val
35 40 45
Ser Gly Thr Ile Pro Ser Pro Val Glu Pro Ala Val Asn Gly Trp Val
50 55 60
Gly Met Pro Gly Arg Ile Arg Arg Glu Phe Pro Asp Gln Gly Arg Gly
65 70 75 80
Asp Phe Arg Ile Pro Ala Val Arg Ile Arg Glu Ser Ala Gly Tyr Thr
85 90 95
Val Ser Asp Leu Gln Tyr Val Ser His Glu Val Ile Glu Gly Lys Tyr
100 105 110
Ala Leu Pro Gly Leu Pro Ala Thr Phe Gly Asp Ala Gln Asp Ala Thr
115 120 125
Thr Leu Val Val His Leu Tyr Asp Asn Tyr Ser Ser Val Ala Ala Asp
130 135 140
Leu Ser Tyr Ser Ile Phe Pro Lys Tyr Asp Ala Ile Val Arg Ser Val
145 150 155 160
Asn Val Thr Asn Gln Gly Pro Gly Asn Ile Thr Ile Glu Ala Leu Ala
165 170 175
Ser Ile Ser Ile Asp Phe Pro Tyr Glu Asp Leu Asp Met Val Ser Leu
180 185 190
Arg Gly Asp Trp Ala Arg Glu Ala Asn Val Gln Arg Ser Lys Val Gln
195 200 205
Tyr Gly Val Gln Gly Phe Gly Ser Ser Thr Gly Tyr Ser Ser His Leu
210 215 220
His Asn Pro Phe Leu Ala Ile Val Asp Pro Ala Thr Thr Glu Ser Gln
225 230 235 240
Gly Glu Ala Trp Gly Phe Asn Leu Val Tyr Thr Gly Ser Phe Ser Ala
245 250 255
Gln Val Glu Lys Gly Ser Gln Gly Phe Thr Arg Ala Leu Leu Gly Phe
260 265 270
Asn Pro Asp Gln Leu Ser Trp Asn Leu Gly Pro Gly Glu Thr Leu Thr
275 280 285
Ser Pro Glu Cys Val Ala Val Tyr Ser Asp Lys Gly Leu Gly Ser Val
290 295 300
Ser Arg Lys Phe His Arg Leu Tyr Arg Asn His Leu Met Lys Ser Lys
305 310 315 320
Phe Ala Thr Ser Asp Arg Pro Val Leu Leu Asn Ser Trp Glu Gly Val
325 330 335
Tyr Phe Asp Tyr Asn Gln Ser Ser Ile Glu Thr Leu Ala Glu Glu Ser
340 345 350
Ala Ala Leu Gly Val His Leu Phe Val Met Asp Asp Gly Trp Phe Gly
355 360 365
Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala Gly Leu Gly Asp Trp Met
370 375 380
Pro Asn Pro Ala Arg Phe Pro Asp Gly Leu Thr Pro Val Val Gln Asp
385 390 395 400
Ile Thr Asn Leu Thr Val Asn Gly Thr Glu Ser Thr Lys Leu Arg Phe
405 410 415
Gly Ile Trp Val Glu Pro Glu Met Val Asn Pro Asn Ser Thr Leu Tyr
420 425 430
His Glu His Pro Glu Trp Ala Leu His Ala Gly Pro Tyr Pro Arg Thr
435 440 445
Glu Ala Arg Asn Gln Leu Val Leu Asn Leu Ala Leu Pro Ala Val Gln
450 455 460
Asp Phe Ile Ile Asp Phe Met Thr Asn Leu Leu Gln Asp Thr Gly Ile
465 470 475 480
Ser Tyr Val Lys Trp Asp Asn Asn Arg Gly Ile His Glu Thr Pro Ser
485 490 495
Pro Ser Thr Asp His Gln Tyr Met Leu Gly Leu Tyr Arg Val Phe Asp
500 505 510
Thr Leu Thr Thr Arg Phe Pro Asp Val Leu Trp Glu Gly Cys Ala Ser
515 520 525
Gly Gly Gly Arg Phe Asp Ala Gly Met Leu Gln Tyr Val Pro Gln Ile
530 535 540
Trp Thr Ser Asp Asn Thr Asp Ala Ile Asp Arg Ile Thr Ile Gln Phe
545 550 555 560
Gly Thr Ser Leu Ala Tyr Pro Pro Ser Ala Met Gly Ala His Leu Ser
565 570 575
Ala Val Pro Asn Ala Gln Thr Gly Arg Thr Val Pro Phe Thr Phe Arg
580 585 590
Ala His Val Ala Met Met Gly Gly Ser Phe Gly Leu Glu Leu Asp Pro
595 600 605
Ala Thr Val Glu Gly Asp Glu Ile Val Pro Glu Leu Leu Ala Leu Ala
610 615 620
Glu Lys Val Asn Pro Ile Ile Leu Asn Gly Asp Leu Tyr Arg Leu Arg
625 630 635 640
Leu Pro Gln Asp Ser Gln Trp Pro Ala Ala Leu Phe Val Ser Gln Asp
645 650 655
Gly Ala Gln Ala Val Leu Phe Tyr Phe Gln Val Gln Pro Asn Val Asn
660 665 670
His Ala Val Pro Trp Val Arg Leu Gln Gly Leu Asp Pro Lys Ala Asp
675 680 685
Tyr Thr Val Asp Gly Asp Gln Thr Tyr Ser Gly Ala Thr Leu Met Asn
690 695 700
Leu Gly Leu Gln Tyr Ser Phe Asp Thr Glu Tyr Gly Ser Lys Val Val
705 710 715 720
Phe Leu Glu Arg Gln
725
<210> 16
<211> 2178
<212> DNA
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 16
gcacccgcaa ttggggcttc taattcacag acgatcgtta cgaatggcac tagtttcgca 60
ttgaacggcg acaatgtctc atatcgattc catgtcaaca gtagcaccgg cgacttgatt 120
tctgatcatt ttggtggtgt cgtctccggc acaatccctt cgccagtgga acctgctgtc 180
aacggctggg tcggcatgcc tggtcgaatc cgccgggagt tccccgacca aggccgtggg 240
gatttccgca ttcccgccgt tcgtattcgg gaatcggcag gttatactgt tagcgatctc 300
caatatgtgt cgcacgaggt gatcgaaggt aaatatgctt tgcctggcct gcctgccaca 360
tttggcgatg cgcaggacgc caccactttg gtagtccatc tgtatgacaa ctatagctcc 420
gtcgcggccg acttgtcata ctccatattt ccgaaatatg atgcgatcgt gaggagtgtc 480
aatgtgacca accagggccc aggtaatatc actatcgagg cccttgcaag cataagtatc 540
gatttcccct atgaagacct cgacatggtc agcctccgag gcgactgggc cagagaggca 600
aatgttcaga gaagcaaagt gcagtatggc gtccagggat ttggaagcag tactggatat 660
tcctctcacc ttcataatcc cttccttgcc atagtagatc cagctactac cgaatcgcaa 720
ggcgaggcat ggggtttcaa ccttgtatat accggctctt tctcggcgca agtagagaaa 780
ggatcgcaag gtttcacccg ggcgctgctc ggcttcaacc cggaccaatt atcgtggaac 840
cttggccctg gcgagacttt aacctccccc gagtgtgttg cagtctactc ggacaaaggc 900
cttggctcag tgtctcgcaa attccaccgg ctatatcgca accacctcat gaagagcaag 960
ttcgccacgt ccgaccggcc ggttctgctg aatagctggg aaggtgttta tttcgactac 1020
aatcaaagca gcatcgaaac tcttgccgaa gagtccgctg ccctgggtgt ccacctcttt 1080
gtcatggacg acggctggtt tggggacaag taccctcgag tgtccgataa cgccggactg 1140
ggcgactgga tgcccaatcc agcgcgcttc ccggacgggt tgaccccggt cgtgcaagac 1200
atcacaaatc tcaccgtcaa tggcacagag tccacaaaac ttcgctttgg tatttgggtg 1260
gagcccgaga tggtcaaccc caattccact ctctaccacg aacacccgga gtgggcgctt 1320
catgccgggc cttacccccg taccgagggt cggaaccagc tcgtcctcaa cctggcgctt 1380
ccggctgtgc aggacttcat catagacttc atgacgaacc tgttacaaga taccggcatt 1440
tcctacgtca aatgggacaa caaccggggg atacacgaga cgccctctcc gtcgactgac 1500
catcagtaca tgcttggcct ctaccgggtg ttcgacacac tgaccacccg ttttccggat 1560
gtcctgtggg aaggatgtgc ctcgggcggc ggccgctttg atgctggcat gctgcagtat 1620
gtcccccaga tctggacttc cgacaacacc gacgccatcg accgaatcac catccaattt 1680
gggacctcgc ttgcctaccc gccatcagca atgggagccc acctctccgc ggtccctaat 1740
gcacagaccg gtcgcactgt gccctttact ttccgcgcac acgttgctat gatgggtggt 1800
tctttcggct tggagctgga tccggcgacg gtggaagggg acgaaatagt tcccgagctc 1860
cttgcgctgg cggaaaaggt gaaccctatc attttgaacg gagatctgta tcggctacgc 1920
ctacctcaag actcccagtg gcctgccgca ctctttgtgt ctcaggatgg cgcacaggct 1980
gttctgttct acttccaggt ccagccgaat gtcaaccatg ccgtgccgtg ggtcaggctg 2040
caggggttgg accctaaggc ggactatacc gtcgatgggg atcagacgta ttctggagca 2100
acactaatga atctggggtt gcagtatagt tttgacaccg agtatggaag caaggtagtt 2160
ttcctggaga ggcaatga 2178
<210> 17
<211> 725
<212> PRT
<213> 人工序列（Artificial）
<220>
<221>
<222>
<223> 突变体
<400> 17
Ala Pro Ala Ile Gly Ala Ser Asn Ser Gln Thr Ile Val Thr Asn Gly
1 5 10 15
Thr Ser Phe Ala Leu Asn Gly Asp Asn Val Ser Tyr Arg Phe His Val
20 25 30
Asn Ser Ser Thr Gly Asp Leu Ile Ser Asp His Phe Gly Gly Val Val
35 40 45
Ser Gly Thr Ile Pro Ser Pro Val Glu Pro Ala Val Asn Gly Trp Val
50 55 60
Gly Met Pro Gly Arg Ile Arg Arg Glu Phe Pro Asp Gln Gly Arg Gly
65 70 75 80
Asp Phe Arg Ile Pro Ala Val Arg Ile Arg Glu Ser Ala Gly Tyr Thr
85 90 95
Val Ser Asp Leu Gln Tyr Val Ser His Glu Val Ile Glu Gly Lys Tyr
100 105 110
Ala Leu Pro Gly Leu Pro Ala Thr Phe Gly Asp Ala Gln Asp Ala Thr
115 120 125
Thr Leu Val Val His Leu Tyr Asp Asn Tyr Ser Ser Val Ala Ala Asp
130 135 140
Leu Ser Tyr Ser Ile Phe Pro Lys Tyr Asp Ala Ile Val Arg Ser Val
145 150 155 160
Asn Val Thr Asn Gln Gly Pro Gly Asn Ile Thr Ile Glu Ala Leu Ala
165 170 175
Ser Ile Ser Ile Asp Phe Pro Tyr Glu Asp Leu Asp Met Val Ser Leu
180 185 190
Arg Gly Asp Trp Ala Arg Glu Ala Asn Val Gln Arg Ser Lys Val Gln
195 200 205
Tyr Gly Val Gln Gly Phe Gly Ser Ser Thr Gly Tyr Ser Ser His Leu
210 215 220
His Asn Pro Phe Leu Ala Ile Val Asp Pro Ala Thr Thr Glu Ser Gln
225 230 235 240
Gly Glu Ala Trp Gly Phe Asn Leu Val Tyr Thr Gly Ser Phe Ser Ala
245 250 255
Gln Val Glu Lys Gly Ser Gln Gly Phe Thr Arg Ala Leu Leu Gly Phe
260 265 270
Asn Pro Asp Gln Leu Ser Trp Asn Leu Gly Pro Gly Glu Thr Leu Thr
275 280 285
Ser Pro Glu Cys Val Ala Val Tyr Ser Asp Lys Gly Leu Gly Ser Val
290 295 300
Ser Arg Lys Phe His Arg Leu Tyr Arg Asn His Leu Met Lys Ser Lys
305 310 315 320
Phe Ala Thr Ser Asp Arg Pro Val Leu Leu Asn Ser Trp Glu Gly Val
325 330 335
Tyr Phe Asp Tyr Asn Gln Ser Ser Ile Glu Thr Leu Ala Glu Glu Ser
340 345 350
Ala Ala Leu Gly Val His Leu Phe Val Met Asp Asp Gly Trp Phe Gly
355 360 365
Asp Lys Tyr Pro Arg Val Ser Asp Asn Ala Gly Leu Gly Asp Trp Met
370 375 380
Pro Asn Pro Ala Arg Phe Pro Asp Gly Leu Thr Pro Val Val Gln Asp
385 390 395 400
Ile Thr Asn Leu Thr Val Asn Gly Thr Glu Ser Thr Lys Leu Arg Phe
405 410 415
Gly Ile Trp Val Glu Pro Glu Met Val Asn Pro Asn Ser Thr Leu Tyr
420 425 430
His Glu His Pro Glu Trp Ala Leu His Ala Gly Pro Tyr Pro Arg Thr
435 440 445
Glu Gly Arg Asn Gln Leu Val Leu Asn Leu Ala Leu Pro Ala Val Gln
450 455 460
Asp Phe Ile Ile Asp Phe Met Thr Asn Leu Leu Gln Asp Thr Gly Ile
465 470 475 480
Ser Tyr Val Lys Trp Asp Asn Asn Arg Gly Ile His Glu Thr Pro Ser
485 490 495
Pro Ser Thr Asp His Gln Tyr Met Leu Gly Leu Tyr Arg Val Phe Asp
500 505 510
Thr Leu Thr Thr Arg Phe Pro Asp Val Leu Trp Glu Gly Cys Ala Ser
515 520 525
Gly Gly Gly Arg Phe Asp Ala Gly Met Leu Gln Tyr Val Pro Gln Ile
530 535 540
Trp Thr Ser Asp Asn Thr Asp Ala Ile Asp Arg Ile Thr Ile Gln Phe
545 550 555 560
Gly Thr Ser Leu Ala Tyr Pro Pro Ser Ala Met Gly Ala His Leu Ser
565 570 575
Ala Val Pro Asn Ala Gln Thr Gly Arg Thr Val Pro Phe Thr Phe Arg
580 585 590
Ala His Val Ala Met Met Gly Gly Ser Phe Gly Leu Glu Leu Asp Pro
595 600 605
Ala Thr Val Glu Gly Asp Glu Ile Val Pro Glu Leu Leu Ala Leu Ala
610 615 620
Glu Lys Val Asn Pro Ile Ile Leu Asn Gly Asp Leu Tyr Arg Leu Arg
625 630 635 640
Leu Pro Gln Asp Ser Gln Trp Pro Ala Ala Leu Phe Val Ser Gln Asp
645 650 655
Gly Ala Gln Ala Val Leu Phe Tyr Phe Gln Val Gln Pro Asn Val Asn
660 665 670
His Ala Val Pro Trp Val Arg Leu Gln Gly Leu Asp Pro Lys Ala Asp
675 680 685
Tyr Thr Val Asp Gly Asp Gln Thr Tyr Ser Gly Ala Thr Leu Met Asn
690 695 700
Leu Gly Leu Gln Tyr Ser Phe Asp Thr Glu Tyr Gly Ser Lys Val Val
705 710 715 720
Phe Leu Glu Arg Gln
725

Claims (1)

1.一种α-半乳糖苷酶，其氨基酸序列是将SEQ ID NO：2第450个位置的精氨酸改为苯丙氨酸、缬氨酸、丝氨酸、丙氨酸或甘氨酸取代修饰的序列。
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WO2011150410A3 (en) * 2010-05-28 2012-04-19 Solazyme, Inc. Tailored oils produced from recombinant heterotrophic microorganisms
CN106676085A (zh) * 2017-03-17 2017-05-17 中国农业大学 具有α‑半乳糖苷酶活性的蛋白质及其应用

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