苯丙酮酸脱羧酶突变体M538A在生物发酵生产苯乙醇中的应用

摘要

本发明属于基因工程和酶工程技术领域，公开了苯丙酮酸脱羧酶突变体M538A在生物发酵生产苯乙醇中的应用。本发明通过定点突变的方式，将来源于乳酸乳球菌(

说明书

技术领域

本发明属于酶工程和基因工程技术领域，具体涉及苯丙酮酸脱羧酶突变体M538A在生物发酵生产苯乙醇中的应用。

背景技术

苯乙醇是一种具有淡雅、细腻而持久玫瑰花香气的芳香醇。因其具有的特殊气味，苯乙醇作为香料物质被广泛应用于香水、化妆品以及食品工业。苯乙醇还可以作为杀虫剂和新型生物燃料。因此，苯乙醇具有广泛的应用前景。

微生物主要将简单的碳源通过丙酮酮酸途径和Ehrlich途径代谢产生苯乙醇。然而只有少数微生物具有Ehrlich途径，因此利用微生物合成苯乙醇因菌种种类太少而受到极大限制。酮酸脱羧酶(EC4.1.1.43)是苯乙醇合成途径中关键酶，复杂催化苯丙酮酸生成苯丙醛，随后通过氧化还原反应进一步生成终产物苯乙醇。由于其底物作用的高度特异性，苯丙酮酸脱羧酶是苯乙醇生物合成的限速酶。目前，已经有研究主要通过筛选和优化表达酮酸脱羧酶提高其表达量。目前来自酿酒酵母酮酸脱羧酶Aro10，毕赤酵母中的酮酸脱羧酶Kdc,肠杆菌酮酸脱羧酶Kdc4427，固氮螺菌酮酸脱羧酶AbPDC和运动发酵单胞菌脱羧酶PDC和乳酸乳球菌中酮酸脱羧酶KivD被测试用于苯乙醇的合成，其中来自酵母菌种的酮酸脱羧酶Aro10是最优的酶。在大肠杆菌和酿酒酵母菌中被广泛应用于苯乙醇的合成。但是，这些菌株表达Aro10仍然存在底物特异性不高产生其他副产物和催化效率低等问题，因此，上述技术均并不能够真正运用于工业生产。

来源于乳酸乳球菌中酮酸脱羧酶KivD，是一类具有宽泛底物谱的酮酸脱羧酶，能够同时催化支链酮酸(3-甲基-2-氧代丁酸，4-甲基-2-氧代戊酸和3-甲基-2-氧代戊酸)，芳香族酮酸(4羟基苯丙酮酸，苯丙酮酸和苯甲酰甲酸)，和直链酮酸(2-氧代己酸，2-氧代戊酸和丙酮酸)。通过酶学性质分析发现，KivD最适合底物为支链酮酸中的3-甲基-2-氧代丁酸，用于生成异丁醇。乳酸乳球菌的KivD蛋白结构已被解析，研究人员发现在催化活性中心附近的461位缬氨酸，268位的丝氨酸，381位的苯丙氨酸，538位的甲硫氨酸和542位的苯丙氨酸对底物催化发挥重要的作用。为了获得相应的产物，这5个位点氨酸酸成为蛋白改造的关键靶标。研究人员Peter Lindblad等为了提高异丁醇的产量，提高KivD对3-甲基-2-氧代丁酸催化活性，对其中的四个位点进行了系列突变，构建了V461I(VI)，V461L(VL)，V461F(VF)，V461A(VA)，M538W(MW)，S286T(ST)，S286Y(SY)，S286A(SA)，F542L(FL)和F542W(FW)突变体。众多突变体中只有ST和VI的异丁醇产量大幅提高，提高了对3-甲基-2-氧代丁酸催化活性。James C.Liao课题组为提高3-甲基-1-戊醇的合成，对KivD进行双位点的叠加突变，构建四株突变菌种，分别为V461A/M538A，V461A/M538L，V461A/F381A和V461A/F381L，这四个突变体中3-甲基-1-戊醇产量比对照菌株提高了20-50倍，其中V461A/F381L中3-甲基-1-戊醇产量最高，达到384.3±30.3mg/L。由于对苯丙酮酸低的底物亲和力和催化效率，KivD几乎不被用于苯乙醇的合成。

发明内容

本发明的目的在于提供了苯丙酮酸脱羧酶突变体M538A在生物发酵生产苯乙醇中的应用，所述的苯丙酮酸脱羧酶突变体M538A的氨基酸序列为SEQ ID NO.2所示。

为了达到上述目的，本发明采取以下技术措施：

苯丙酮酸脱羧酶突变体M538A在生物发酵生产苯乙醇中的应用，包括利用所述的突变体蛋白直接作用于苯丙酮酸以制备苯乙醇；或是将编码该突变蛋白的基因转化进地衣芽胞杆菌中，发酵生产苯乙醇，所述的突变体M538A的氨基酸序列为SEQ ID NO.2所示。

以上所述的应用中，优选的，所述的地衣芽孢杆菌为Bacillus licheniformisDW2。

与现有技术相比，本发明具有以下优点：

本发明通过定点突变的方式，将苯丙酮酸脱羧酶分子催化结构域附近的第461位缬氨酸突变为异亮氨酸(本发明命名为突变体V461I)，第538位甲硫氨酸突变为丙氨酸(本发明命名为突变体M538A)、第542位苯丙氨酸突变为色氨酸(本发明命名为突变体F542W)，显著提高了苯丙酮酸脱羧酶的酶活力，解决了目前苯丙酮酸脱羧酶对苯丙酮酸催化效率不高的问题。改造后的菌株产酶能力和催化效率都得到了提高，通过气相色谱检测突变体中苯乙醇的含量，测定结果表明，V461I、M538A、F542W突变株分别产生490.62、318.23、243.08mg/L的苯乙醇。

附图说明

图1为突变体催化后产物含量。

图2为不同突变体的酶活情况。

具体实施方式

以下实施例是对本发明的进一步说明，而不是对本发明的限制。本发明所述技术方案，如未特别说明，为本领域的常规方案；所述试剂或材料，如未特别说明，均来源于商业渠道。

实验材料和试剂

1、菌株：乳酸乳球菌乳亚种(Lactococcus lactis subsp.lactis)保藏号为CICC6246，菌株E.coli DH5α购自于北京全式金生物技术有限公司。

2、酶类及其它生化试剂：高保真Taq酶购自武汉擎科生物技术有限公司公司。细菌基因组DNA提取试剂盒购自Tiangen公司，苯乙醇购自Sigma公司，ClonExpress II一步克隆试剂盒购自南京诺唯赞生物科技有限公司，其它均为国产试剂(均可从普通生化试剂公司购买得到)。

3、培养基：

(1)、LB培养基配方为：10g/L胰蛋白胨，5g/L酵母粉，10g/L氯化钠，pH 7.0～7.2，121℃灭菌20min。

(2)发酵培养基：10g/L胰蛋白胨，5g/L酵母粉，10g/L氯化钠、60～100g/L葡萄糖、14.136g/L K₂HPO₄、5.168g/L>2PO₄、2.86mg/L>3BO₃,1.81mg/L>2·4H₂O,0.22>4·7H₂O,0.39mg/L>2MoO₄·2H₂O,79μg/L>4·5H₂O,和49.4μg/L>3)₂·6H₂O、pH>

实施例1：

重组质粒pHY-P43-kivD的构建：

用KivD-F和KivD-R从乳酸乳球菌(Lactococcus lactis subsp.lactis，CICC6246)中克隆得到一种α-酮酸脱羧酶kivD基因。以Bacillus subtilis 168基因组为模板，P43-F和P43-R为引物扩增获得P43启动子；TamyL-F和TamyL-R为引物扩增获得淀粉酶终止子TamyL。P43-F和TamyL-R为引物将P43、kivD、TamyL三个片段进行SOE-PCR获得融合片段P43-kivD-TamyL。以质粒pHY300PLK为模板，pHY-T5-F和pHY-T5-R为引物进行全质粒PCR扩增，获得线性化pHY300PLK载体。以上扩增产物经电泳检验后，采用胶回收试剂盒对PCR产物进行纯化和回收。通过ClonExpress II一步克隆试剂盒，将融合片段与线性化载体pHY300PLK进行融合，得到重组质粒pHY-P43-kivD。

P43-F:TTTTTATAACAGGAATTCTGATAGGTGGTATGTTTTCG

P43-R:TAATCTCCTACTGTATACATTGATCCTTCCTCCTTTAGA

kivD-F:CTAAAGGAGGAAGGATCAATGTATACAGTAGGAGATTA

kivD-R:TCCGTCCTCTCTGCTCTT TTATGATTTATTTTGTTCAG

TamyL-F:CTGAACAAAATAAATCATAAAAGAGCAGAGAGGACGGATT

TamyL-R:AAGCTTCTAGAAGCTTCTAGCGCAATAATGCCGTCGCACT

pHY-T5-F:GAATTCCTGTTATAAAAAAAGGATC

pHY-T5-R:TCTAGAAGCTTGGGCAAAGCGTTTT

将融合的重组质粒pHY-P43-kivD转化至感受态E.coli DH5α，用含氨苄青霉素的LB平板筛选阳性菌落。37℃摇床过夜培养后提取质粒pHY-P43-kivD，并进行测序验证。

实施例2：

苯丙酮酸脱羧酶野生菌(WT)的制备及酶活测定：

将实施例1中测序正确的质粒pHY-P43-kivD，转化Bacillus licheniformis DW2。挑选转化子验证正确后接种到LB培养基中，37℃，培养14h；转接到发酵培养基，接种量为3％，37℃培养24h，离心收集菌体，利用PBS洗涤菌体两次，最后用1毫升50mM磷酸钾缓冲液(pH 6.8)重悬菌体。利用超声破碎仪进行细胞破碎，超声仪设置：150W，20kHz，工作2s；关闭2s,共8分钟，4℃离心收集上清液，即为粗酶液。

将上清液按照下述的体系进行酶活测定，酶促反应体系(200μL)：50mM磷酸钾缓冲液(pH 6.8)，1mM七水硫酸镁，0.5mM硫胺素焦磷酸，5mM苯丙酮酸钠和10μL粗酶液，30℃下进行反应。采用酶标仪测定320nm吸光度测定苯丙酮酸脱羧酶酶活，将每分钟消耗1微摩尔底物苯丙酮酸所需要的酶量定义为一个酶活力单位(U)。酶活测定结果显示，野生KivD的酶活力(酶浓度)为290.05U/mL(图2)。

实施例3：

苯丙酮酸脱羧酶突变株的制备

以已构建的pHY-P43-kivD为模板，设计引物进行全质粒PCR扩增，获得线性化带P43启动子和淀粉酶TamyL终止子的pHY300PLK载体。对kivD催化活性位点进行定点突变，以引物的方式将突变替换到基因序列上，具体为：

1)将苯丙酮酸脱羧酶分子催化结构域的第461位缬氨酸突变为异亮氨酸，把编码kivD的第461位氨基酸的碱基GTC拆分成两部分，引物V461I-AR和V461I-BF，以实施例1质粒为模板扩增kivd突变序列上下两段；再以kivD-F和kivD-R为引物将两段进行SOE-PCR扩增出kivD突变序列V461I；

2)将第538位甲硫氨酸突变为丙氨酸，把编码kivD的第538位氨基酸的碱基GCG拆分成两部分，引物M538A-AR和M538A-BF，以实施例1质粒为模板扩增kivd突变序列上下两段；再以kivD-F和kivD-R为引物将两段进行SOE-PCR扩增出kivD突变序列M538A；

3)将第542位苯丙氨酸突变为色氨酸，把编码kivD的第542位氨基酸的碱基TGG拆分成两部分，引物F542W-AR和F542W-BF，以实施例1质粒为模板扩增kivd突变序列上下两段；再以kivD-F和kivD-R为引物将两段进行SOE-PCR扩增出kivD突变序列F542W。

采用胶回收试剂盒对PCR产物进行纯化和回收，电泳检验回收产物。将融合的突变片段与带P43启动子和淀粉酶TamyL终止子的线性化载体pHY300PLK进行融合，产物转化E.coli DH5α，得到重组质粒分别命名为pHY-V461I、pHY-M538A、pHY-F542W，并进行测序验证。

V461I–AR:GTCCATGAATTTCTCTTTCGATTGTATAACCATCATTATTG

V461I–BF:AATAATGATGGTTATACAATCGAAAGAGAAATTCATGGAC

M538A-AR:TCAGCAAATAGTTTGCCCGCTTTTTTCAGTACTTTTGGTG

M538A-BF:CCAAAAGTACTGAAAAAAGCGGGCAAACTATTTGCTGA

F542W-AR:GATTTATTTTGTTCAGCCCATAGTTTGCCCATTTTTTTC

F542W-BF:GAAAAAAATGGGCAAACTATGGGCTGAACAAAATAAATC。

利用上述方式，获得了四种苯丙酮酸脱羧酶突变体，分别命名为V461I(SEQ IDNO.1所示)、M538A(SEQ ID NO.2所示)、F542W(SEQ ID NO.3所示)。

实施例4：

苯丙酮酸脱羧酶突变菌株的制备及酶活测定：

将实施例3中测序正确的突变体质粒，分别转化Bacillus licheniformis DW2。挑选转化子验证正确后接种到LB培养基中，37℃，培养14h；转接到发酵培养基中，接种量为3％，37℃培养24h。按照实施例3中的方法检测重组菌株的酶活；

实验结果表明与WT菌株比较，酶活力有所提高，WT、V461I、M538A和F542W酶活力(酶浓度)分别为290.05U/mL、2028.51U/mL、1408.99U/mL、1195.18U/mL(图2)，其中V461I、M538A和F542W是WT株酶活的6.99倍、4.86倍和4.12倍。

实施例5：

发酵产物分析

将实施例2和4获得的重组菌株接种到LB培养基中，37℃，培养14h；转接到发酵培养基中，接种量为3％，37℃培养72h，收集发酵液，利用气相色谱仪进行分析发酵产物。

发酵产物测定方法：发酵液12000rpm离心10min取400μL发酵上清液；再加入1.2mL含1g/L内标的乙酸乙酯做进行萃取，振荡混匀5min，10000rpm离心2min；取上层有机相800μL，加入含0.2g无水硫酸钠的离心管中，混匀，室温静置30min；离心取上清用气相色谱法测定发酵产物浓度。发酵产物测定采用安捷伦7890B气相色谱仪(配置FID检测器)，进样量1μL，不分流进样，载气为氢气(1ml/min)。柱温程序为：50℃平衡2min，10℃/min升温至160℃，20℃/min升至220℃并保持3min。

结果如图1所示，含有本发明的苯丙酮酸脱羧酶突变体的重组地衣芽胞杆菌DW2菌株，能高产苯乙醇，其中V461I、M538A、F542W突变株苯乙醇产量分别为490.62、318.23、243.08mg/L，是WT菌株76.04mg/L的6.45、4.18、3.19倍。

实施例6：

对比例：

以pHY-P43-kivD为模板，采用实施例3的方法，分别将苯丙酮酸脱羧酶第382位苯丙氨酸突变为亮氨酸、第461位缬氨酸突变为天冬氨酸；突变引物如下。

最终获得的苯丙酮酸脱羧酶突变体分别命名为F382L(SEQ ID NO.4)和V461D(SEQID NO.5).

转化子由生工生物工程(上海)股份有限公司进行测序，转化子命名为pHY-F382L和pHY-V461H，分别转化Bacillus.licheniformis DW2。F382L突变菌株的酶活力相比WT菌株下降，为230.26U/mL，而V461D突变菌株几乎检测不到酶活(图2)。

F382L-AR:AAAATTGATGAAGCGCCAAGGAATGATGTCCCTTGTTCA

F382L-BF:TGAACAAGGGACATCATTCCTTGGCGCTTCATCAATTTTC

V461D-AR:TCCATGAATTTCTCTTTCATCTGTATAACCATCATTATTGA

V461D-BF:CAATAATGATGGTTATACAGATGAAAGAGAAATTCATGGA。

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Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu

202530

Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn

354045

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys

505560

Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val

65707580

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

859095

Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His

100 105 110

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln

180 185 190

Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro

195 200 205

Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr

210 215 220

Val Ser Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn

225 230 235 240

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

245 250 255

Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser

260 265 270

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

275 280 285

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn

290 295 300

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

305 310 315 320

Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys

325 330 335

Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala

340 345 350

Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln

355 360 365

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

370 375 380

Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu

385 390 395 400

Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile

405 410 415

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

420 425 430

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

435 440 445

Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu

450 455 460

Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr

465 470 475 480

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

485 490 495

Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala

500 505 510

Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys

515 520 525

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

530 535 540

Gln Asn Lys Ser

545

<210> 3

<211> 548

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 3

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

1 5 1015

Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu

202530

Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn

354045

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys

505560

Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val

65707580

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

859095

Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His

100 105 110

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln

180 185 190

Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro

195 200 205

Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr

210 215 220

Val Ser Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn

225 230 235 240

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

245 250 255

Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser

260 265 270

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

275 280 285

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn

290 295 300

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

305 310 315 320

Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys

325 330 335

Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala

340 345 350

Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln

355 360 365

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

370 375 380

Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu

385 390 395 400

Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile

405 410 415

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

420 425 430

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

435 440 445

Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu

450 455 460

Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr

465 470 475 480

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

485 490 495

Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala

500 505 510

Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys

515 520 525

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

530 535 540

Gln Asn Lys Ser

545

<210> 4

<211> 548

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 4

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

1 5 1015

Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu

202530

Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn

354045

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys

505560

Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val

65707580

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

859095

Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His

100 105 110

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln

180 185 190

Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro

195 200 205

Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr

210 215 220

Val Ser Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn

225 230 235 240

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

245 250 255

Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser

260 265 270

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

275 280 285

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn

290 295 300

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

305 310 315 320

Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys

325 330 335

Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala

340 345 350

Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln

355 360 365

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

370 375 380

Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu

385 390 395 400

Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile

405 410 415

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

420 425 430

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

435 440 445

Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Val Glu Arg Glu

450 455 460

Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr

465 470 475 480

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

485 490 495

Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala

500 505 510

Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys

515 520 525

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

530 535 540

Gln Asn Lys Ser

545

<210> 5

<211> 548

<212> PRT

<213> 人工序列(Artificial Sequence)

<400> 5

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

1 5 1015

Ile Glu Glu Ile Phe Gly Val Pro Gly Asp Tyr Asn Leu Gln Phe Leu

202530

Asp Gln Ile Ile Ser Arg Lys Asp Met Lys Trp Val Gly Asn Ala Asn

354045

Glu Leu Asn Ala Ser Tyr Met Ala Asp Gly Tyr Ala Arg Thr Lys Lys

505560

Ala Ala Ala Phe Leu Thr Thr Phe Gly Val Gly Glu Leu Ser Ala Val

65707580

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

859095

Val Gly Ser Pro Thr Ser Lys Val Gln Asn Glu Gly Lys Phe Val His

100 105 110

His Thr Leu Ala Asp Gly Asp Phe Lys His Phe Met Lys Met His Glu

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Ser Leu Pro Leu Lys Lys Glu Asn Ser Thr Ser Asn Thr Ser Asp Gln

180 185 190

Glu Ile Leu Asn Lys Ile Gln Glu Ser Leu Lys Asn Ala Lys Lys Pro

195 200 205

Ile Val Ile Thr Gly His Glu Ile Ile Ser Phe Gly Leu Glu Lys Thr

210 215 220

Val Ser Gln Phe Ile Ser Lys Thr Lys Leu Pro Ile Thr Thr Leu Asn

225 230 235 240

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

245 250 255

Tyr Asn Gly Lys Leu Ser Glu Pro Asn Leu Lys Glu Phe Val Glu Ser

260 265 270

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

275 280 285

Gly Ala Phe Thr His His Leu Asn Glu Asn Lys Met Ile Ser Leu Asn

290 295 300

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

305 310 315 320

Glu Ser Leu Ile Ser Ser Leu Leu Asp Leu Ser Glu Ile Glu Tyr Lys

325 330 335

Gly Lys Tyr Ile Asp Lys Lys Gln Glu Asp Phe Val Pro Ser Asn Ala

340 345 350

Leu Leu Ser Gln Asp Arg Leu Trp Gln Ala Val Glu Asn Leu Thr Gln

355 360 365

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

370 375 380

Ser Ser Ile Phe Leu Lys Pro Lys Ser His Phe Ile Gly Gln Pro Leu

385 390 395 400

Trp Gly Ser Ile Gly Tyr Thr Phe Pro Ala Ala Leu Gly Ser Gln Ile

405 410 415

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

420 425 430

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

435 440 445

Pro Ile Cys Phe Ile Ile Asn Asn Asp Gly Tyr Thr Asp Glu Arg Glu

450 455 460

Ile His Gly Pro Asn Gln Ser Tyr Asn Asp Ile Pro Met Trp Asn Tyr

465 470 475 480

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

485 490 495

Lys Ile Val Arg Thr Glu Asn Glu Phe Val Ser Val Met Lys Glu Ala

500 505 510

Gln Ala Asp Pro Asn Arg Met Tyr Trp Ile Glu Leu Ile Leu Ala Lys

515 520 525

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

530 535 540

Gln Asn Lys Ser

545

<210> 6

<211> 38

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

tttttataac aggaattctg ataggtggta tgttttcg 38

<210> 7

<211> 39

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 7

taatctccta ctgtatacat tgatccttcc tcctttaga 39

<210> 8

<211> 38

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 8

ctaaaggagg aaggatcaat gtatacagta ggagatta 38

<210> 9

<211> 38

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 9

tccgtcctct ctgctctttt atgatttatt ttgttcag 38

<210> 10

<211> 40

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 10

ctgaacaaaa taaatcataa aagagcagag aggacggatt 40

<210> 11

<211> 40

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 11

aagcttctag aagcttctag cgcaataatg ccgtcgcact 40

<210> 12

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 12

gaattcctgt tataaaaaaa ggatc 25

<210> 13

<211> 25

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 13

tctagaagct tgggcaaagc gtttt 25

<210> 14

<211> 41

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 14

gtccatgaat ttctctttcg attgtataac catcattatt g 41

<210> 15

<211> 40

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 15

aataatgatg gttatacaat cgaaagagaa attcatggac 40

<210> 16

<211> 40

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 16

tcagcaaata gtttgcccgc ttttttcagt acttttggtg 40

<210> 17

<211> 38

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 17

ccaaaagtac tgaaaaaagc gggcaaacta tttgctga 38

<210> 18

<211> 39

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 18

gatttatttt gttcagccca tagtttgccc atttttttc 39

<210> 19

<211> 39

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 19

gaaaaaaatg ggcaaactat gggctgaaca aaataaatc 39

<210> 20

<211> 39

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 20

aaaattgatg aagcgccaag gaatgatgtc ccttgttca 39

<210> 21

<211> 40

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 21

tgaacaaggg acatcattcc ttggcgcttc atcaattttc 40

<210> 22

<211> 41

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 22

tccatgaatt tctctttcat ctgtataacc atcattattg a 41

<210> 23

<211> 40

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 23

caataatgat ggttatacag atgaaagaga aattcatgga 40