CN114107266B - 耐热性提高的蛋白酶突变体及其编码基因和应用 - Google Patents
耐热性提高的蛋白酶突变体及其编码基因和应用 Download PDFInfo
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- CN114107266B CN114107266B CN202111432327.2A CN202111432327A CN114107266B CN 114107266 B CN114107266 B CN 114107266B CN 202111432327 A CN202111432327 A CN 202111432327A CN 114107266 B CN114107266 B CN 114107266B
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Abstract
本发明提供了耐热性提高的蛋白酶突变体及其编码基因和应用。本发明通过利用易错PCR方法来构建突变体文库，对Bacillus licheniformis WX‑02来源的蛋白酶基因进行突变，再通过高通量筛选，获得了蛋白酶突变体BLAPR5、BLAPR6、BLAPR7、BLAPR8、BLAPR9、BLAPR10、BLAPR11、BLAPR12，与野生型蛋白酶相比，其酶活分别提高了36.0％、65.5％、35.2％、55.5％、49.0％、84.0％、45.5％、76.0％，因此耐热性得到了显著提升，有利于在饲料、食品等多领域的使用，具有良好的市场应用前景和工业价值。
Description
技术领域
本发明属于基因工程和酶工程领域，具体涉及耐热性提高的蛋白酶突变体及其编码基因和应用。
背景技术
蛋白酶是催化水解蛋白质中肽键的一类酶，广泛存在于动物、植物和微生物中，有着许多不同的生理功能。蛋白酶在各个领域中被广泛应用，例如食品工业、酿酒酿造、洗涤剂行业、饲料工业、制革工业、丝绸行业和医药行业等，所以，这种环境下对蛋白酶的产量和特性改良方面都提出了更高的要求。
目前市场上大多数蛋白酶经70℃处理后仅剩余不到20%的酶活，且总体耐温性能差，限制了蛋白酶的广泛使用。例如在饲料生产时，酶制剂与饲料混匀后要经过高温造粒，在此过程中酶易变性失活。因此，提高蛋白酶的耐热性至关重要。
易错PCR技术是在采用DNA聚合酶进行PCR反应扩增目的片段时，通过调整反应条件，增加扩增过程中的突变频率，从而以一定的频率向目的基因中随机引入突变，构建突变体文库，从而筛选需要的正向突变体。易错PCR技术可以很好地应用于蛋白质的分子改造中。
发明内容
本发明提供了耐热性提高的蛋白酶突变体及其编码基因和应用。通过易错PCR方法构建突变体文库和定向筛选，对Bacillus licheniformis WX-02来源的蛋白酶基因进行改良，筛选得到多种耐热性提高的突变体，有助于提升其在饲料、食品等领域中的应用。
为了实现上述发明目的，本发明采用以下技术方案予以实现：
本发明提供了耐热性提高的蛋白酶突变体BLAPR5，所述蛋白酶突变体BLAPR5的氨基酸序列如SEQ ID NO：3所示，其编码基因的核苷酸序列如SEQ ID NO：4所示；所述蛋白酶突变体BLAPR5是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR6，所述蛋白酶突变体BLAPR6的氨基酸序列如SEQ ID NO：5所示，其编码基因的核苷酸序列如SEQ ID NO：6所示；所述蛋白酶突变体BLAPR6是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第106位的丙氨酸变为天冬氨酸获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR7，所述蛋白酶突变体BLAPR7的氨基酸序列如SEQ ID NO：7所示，其编码基因的核苷酸序列如SEQ ID NO：8所示；所述蛋白酶突变体BLAPR7是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第120位的赖氨酸变为脯氨酸获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR8，所述蛋白酶突变体BLAPR8的氨基酸序列如SEQ ID NO：9所示，其编码基因的核苷酸序列如SEQ ID NO：10所示；所述蛋白酶突变体BLAPR8是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第176位的缬氨酸变为丙氨酸获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR9，所述蛋白酶突变体BLAPR9的氨基酸序列如SEQ ID NO：11所示，其编码基因的核苷酸序列如SEQ ID NO：12所示；所述蛋白酶突变体BLAPR9是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第208位的酪氨酸变为甲硫氨酸获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR10，所述蛋白酶突变体BLAPR10的氨基酸序列如SEQ ID NO：13所示，其编码基因的核苷酸序列如SEQ ID NO：14所示；所述蛋白酶突变体BLAPR10是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第344位的天冬酰胺变为半胱氨酸获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR11，所述蛋白酶突变体BLAPR11的氨基酸序列如SEQ ID NO：15所示，其编码基因的核苷酸序列如SEQ ID NO：16所示；所述蛋白酶突变体BLAPR11是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第369位的赖氨酸变为天冬酰胺获得的。
本发明还提供了耐热性提高的蛋白酶突变体BLAPR12，所述蛋白酶突变体BLAPR12的氨基酸序列如SEQ ID NO：17所示，其编码基因的核苷酸序列如SEQ ID NO：18所示；所述蛋白酶突变体BLAPR11是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸、第53位的缬氨酸变为丙氨酸和第166位的天冬酰胺变为天冬氨酸获得的。
本发明还提供了含有所述的蛋白酶突变体编码基因的基因工程菌。
进一步的，所述基因工程菌为枯草芽胞杆菌和地衣芽胞杆菌。
本发明还提供了所述的蛋白酶突变体在用于制备饲料添加剂或食品添加剂中的应用。
与现有技术相比，本发明的优点和有益技术效果是：
本发明以Bacillus licheniformis WX-02来源的蛋白酶基因为基础，分别提供了包含T183E的单点突变体BLAPR5，以及包含T183E/A106D、T183E/K120P、T183E/V176A、T183E/Y208M、T183E/N344C、T183E/K369N的双位点突变体BLAPR6至BLAPR11，包含T183E/V53A/N166D的三位点突变体BLAPR12。
本发明改造后的突变体BLAPR5至BLAPR12在75℃处理3分钟时的耐热性比原始蛋白酶分别提高了36.0％、65.5％、35.2％、55.5％、49.0％、84.0％、45.5％、76.0％，由此说明本发明的蛋白酶突变体的耐热性较野生型蛋白酶得到了显著的提升，这有利于其在饲料、食品等多领域的广泛使用，且具有良好的市场应用前景。
附图说明
图1是本发明中蛋白酶突变体平板筛选，其中编号0是BLAPR0，编号5-12是BLAPR5至BLAPR12。
图2是本发明中蛋白酶突变体在75℃水浴3min耐热性比较结果。
图3是本发明中蛋白酶在30L发酵罐中的发酵数据。
具体实施方式
为了便于理解本发明，以下将结合附图及实施例对本文发明做更全面、详细地说明，但本发明的保护范围并不限于以下具体实施例。
以下实施例中未作具体说明的分子生物学实验方法，可以参照《分子克隆实验指南》(第三版) J.萨姆布鲁克一书中所列的具体方法进行，或者按照试剂盒和产品说明书进行。具体实施例中使用的试剂和生物材料如无特殊说明均可从商业途径获得。
1、菌株和载体
枯草芽胞杆菌WB600、质粒pUB110、大肠杆菌BL21、质粒pET-21a(+)购自Invitrogen公司。
2、试剂与培养基
质粒提取试剂盒、片段纯化回收试剂盒、限制性内切酶等购自宝生物工程(大连)有限公司；GeneMorph II随机突变PCR试剂盒购自Stratagene公司；氨苄青霉素、IPTG等购自生工生物工程(上海)股份有限公司；蛋白Marker：Blue Plus II Protein Marker (14-120 kDa) 购自北京全式金生物技术有限公司。
LB培养基配方：1%胰蛋白胨，0.5%酵母提取物，1% NaCl。
发酵培养基：豆粕粉50-80g/L，玉米淀粉60-100g/L，磷酸氢二钠2-4g/L，碳酸钠1-2g/L，pH自然。
实施例1：易错PCR构建蛋白酶突变体文库
参考Bacillus licheniformis WX-02来源的蛋白酶氨基酸序列(SEQ ID NO:1)和DNA序列(SEQ ID NO:2)设计引物，在5’端设计Xba I限制性酶切位点，3’端设计BamH I限制性酶切位点。
利用GeneMorph II随机突变PCR试剂盒，以SEQ ID NO:2的基因为模版，进行随机突变，使用的引物序列如下：
BLAPR-F：GCTCTAGAATGATGAGGAAGAAATC（SEQ ID NO:19）；
BLAPR-R：CGCGGATCCTTACTGTGCAGCTGCTTCGA（SEQ ID NO:20）。
反应条件为：94℃预变性3min，94℃变性30s，58℃退火30s和72℃延伸1min，共30个循环。
将扩增好的随机突变PCR产物用Xba I和BamH I双酶切，纯化回收后连接到pET-21a(+)载体上，转化大肠杆菌BL21-DE3，氨苄青霉素抗性LB平板筛选阳性克隆，得到pET-BLAPRx。同样方法将合成的原始基因连接到pET-21a(+)载体上并转化大肠杆菌BL21-DE3，得到pET-BLAPR0。
将筛选的单菌落接种到96孔深孔板中。每个平板接入2个表达BLAPR0的单菌落作为对照。每孔装入300μL LB液体培养基(含有100μg/mL氨苄青霉素)， 37℃、200rpm震荡培养4小时后，转移50μL菌液到新的96孔平板保种，在平板剩余菌液中添加200μL含有IPTG的LB-Amp培养基，使IPTG终浓度为1mM，氨苄青霉素终浓度为100μg/mL，37℃、200rpm摇床培养10h诱导表达蛋白酶。
将诱导完成的菌液反复冻融进行破碎，将破碎后的细胞液离心取上清，进行热处理(75℃水浴3min)，然后检测蛋白酶的剩余活性。对剩余酶活高于对照的突变体基因进行测序。
筛选到以BLAPR0为出发模板的耐热性提高的突变体T183E (命名为BLAPR5)，其氨基酸序列如SEQ ID NO:3所示，其编码基因的核苷酸序列如SEQ ID NO:4所示。
实施例2：第二轮易错PCR构建蛋白酶BLAPR1的突变体文库
以实施例1中筛选到的蛋白酶基因BLAPR5为模版，进行第二轮随机突变，突变体文库的构建过程以及使用的材料试剂、引物和操作条件等，同实施例1。突变体培养和筛选时均以BLAPR5为对照，经过热处理(75℃水浴3min)后检测蛋白酶突变体的剩余活性。对剩余酶活高于BLAPR5的突变体基因进行测序。
最终筛选到以下耐热性提高的突变体：
BLAPR6突变方式为T183E/A106DC（第183位的苏氨酸变为谷氨酸、第106位的丙氨酸变为天冬氨酸），其氨基酸序列如SEQ ID NO:5所示，编码基因的核苷酸序列如SEQ IDNO:6所示；
BLAPR7突变方式为T183E/K120P（第183位的苏氨酸变为谷氨酸、第120位的赖氨酸变为脯氨酸），其氨基酸序列如SEQ ID NO:7所示，编码基因的核苷酸序列如SEQ ID NO:8所示；
BLAPR8突变方式为T183E/V176A（第183位的苏氨酸变为谷氨酸、第176位的缬氨酸变为丙氨酸），其氨基酸序列如SEQ ID NO:9所示，编码基因的核苷酸序列如SEQ ID NO:10所示。
BLAPR9突变方式为T183E/Y208M（第183位的苏氨酸变为谷氨酸、第208位的酪氨酸变为甲硫氨酸），其氨基酸序列如SEQ ID NO:11所示，编码基因的核苷酸序列如SEQ ID NO:12所示。
BLAPR10突变方式为T183E/N344C（第183位的苏氨酸变为谷氨酸、第344位的天冬酰胺变为半胱氨酸），氨基酸序列如SEQ ID NO:13所示，编码基因的核苷酸序列如SEQ IDNO:14所示。
BLAPR11突变方式为T183E/K369N（第183位的苏氨酸变为谷氨酸、第369位的赖氨酸变为天冬酰胺），其氨基酸序列如SEQ ID NO:15所示，编码基因的核苷酸序列如SEQ IDNO:16所示。
BLAPR12突变方式为T183E/V53A/N166D（第183位的苏氨酸变为谷氨酸、第53位的缬氨酸变为丙氨酸、第166位的天冬酰胺变为天冬氨酸），其氨基酸序列如SEQ ID NO:17所示，编码基因的核苷酸序列如SEQ ID NO:18所示。
实施例3：耐热性提高的蛋白酶突变体在枯草芽胞杆菌中的表达验证
分别将实施例2的突变体以及BLAPR0分别克隆入质粒pUB110的Xba I和BamH I位点；参考Spizizen创立的枯草芽胞杆菌转化方法，将上述重组质粒转化入枯草芽胞杆菌WB600中，获得重组菌。经过平板筛选，获得BLAPR5至BLAPR12的突变体如图1所示，其中编号0是BLAPR0，编号5-12是BLAPR5至BLAPR12。重组菌在发酵培养基中进行摇瓶发酵78h后，将培养液离心获得上清，测定每个突变体发酵液上清的平均酶活，取每个突变体中酶活最高的转化子发酵上清液，在75℃水浴处理3min后比较酶活保留率。
结果如图2，突变后得到的蛋白酶BLAPR5至BLAPR12在75℃处理3分钟时的耐热性比原始蛋白酶分别提高了36.0％、65.5％、35.2％、55.5％、49.0％、84.0％、45.5％、76.0％。
以上结果表明，将BLAPR0第183位的Thr突变为Glu，能够在保持原有酶活力的同时提高其耐热性。在此基础上，将其第106位的Ala突变为Asp得到的突变体，或将其120位Lys突变为Pro得到的突变体；或将其176位Val突变为Ala得到的突变体；或将其208位Tyr突变为Met得到的突变体；或将其344位Asn突变为Cys得到的突变体；或将其369位Lys突变为Asn得到的突变体；或将其第53位的Val突变为Ala，同时将其第166位的Asn突变为Asp得到的突变体，其耐热性又进一步提高。由此可见，突变后的蛋白酶的耐热性与野生型相比有了较大提高，更加有利于其在饲料、食品或洗涤领域中的应用。
实施例4：蛋白酶突变体在30L发酵罐中发酵和制备
将实施例3中表达蛋白酶BLAPR0以及蛋白酶突变体BLAPR5、BLAPR6、BLAPR7、BLAPR8、BLAPR9、BLAPR10、BLAPR11、BLAPR12的重组菌分别划线于含有卡那霉素（终浓度为20μg/mL）抗性的LB平板上，37℃培养至长出单菌落，挑取长势良好的单菌落继续在含有卡那霉素（终浓度为20μg/mL）抗性的LB平板上划线培养，如此活化三代得到的重组枯草芽胞杆菌菌落，接种于50mL含有卡那霉素（终浓度为20μg/mL）的LB培养基中，37℃、200rpm培养24h。以2％的接种量接种到1L含有卡那霉素（终浓度为20μg/mL）的LB培养基中，37℃、200rpm培养至OD600为5左右，用作种子液接种发酵罐。
发酵生产工艺：豆粕5-10%、玉米粉1-5%、PPG-20000 0.1-1.0%、蛋白酶0.1-1.0%、淀粉酶0.1-1.0%、磷酸氢二钠（12水）0.2-0.5%、pH自然、温度37℃、搅拌速率600rpm、通风量1.5(v/v)，溶氧控制在20％以上。发酵过程pH自然，发酵24h后开始测定酶活力，每4h测定一次，直到发酵结束（48h）。
酶活结果如图3所示，随着发酵时间的增长，蛋白酶酶活均持续提高，蛋白酶突变体的酶活和野生型蛋白酶BLAPR0相当，但蛋白酶突变体的耐热性较野生型更好。
以上实施例仅用以说明本发明的技术方案，而非对其进行限制；尽管参照前述实施例对本发明进行了详细的说明，对于本领域的普通技术人员来说，依然可以对前述实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或替换，并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 青岛尚德生物技术有限公司
<120> 耐热性提高的蛋白酶突变体及其编码基因和应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Thr Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 2
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacgacgg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 3
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 4
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 5
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Asp Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 6
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgatca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 7
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Pro Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 8
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagatcca 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 9
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Ala
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 10
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggccgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 11
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Met
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 12
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc tatgtcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 13
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Cys Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 14
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatcctt gtttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 15
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Val Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asn Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Asn Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 16
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgtga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcaacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaatgga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 17
<211> 379
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Met Arg Lys Lys Ser Phe Trp Leu Gly Met Leu Thr Ala Phe Met
1 5 10 15
Leu Val Phe Thr Met Ala Phe Ser Asp Ser Ala Ser Ala Ala Gln Pro
20 25 30
Ala Lys Asn Val Glu Lys Asp Tyr Ile Val Gly Phe Lys Ser Gly Val
35 40 45
Lys Thr Ala Ser Ala Lys Lys Asp Ile Ile Lys Glu Ser Gly Gly Lys
50 55 60
Val Asp Lys Gln Phe Arg Ile Ile Asn Ala Ala Lys Ala Lys Leu Asp
65 70 75 80
Lys Glu Ala Leu Lys Glu Val Lys Asn Asp Pro Asp Val Ala Tyr Val
85 90 95
Glu Glu Asp His Val Ala His Ala Leu Ala Gln Thr Val Pro Tyr Gly
100 105 110
Ile Pro Leu Ile Lys Ala Asp Lys Val Gln Ala Gln Gly Phe Lys Gly
115 120 125
Ala Asn Val Lys Val Ala Val Leu Asp Thr Gly Ile Gln Ala Ser His
130 135 140
Pro Asp Leu Asn Val Val Gly Gly Ala Ser Phe Val Ala Gly Glu Ala
145 150 155 160
Tyr Asn Thr Asp Gly Asp Gly His Gly Thr His Val Ala Gly Thr Val
165 170 175
Ala Ala Leu Asp Asn Thr Glu Gly Val Leu Gly Val Ala Pro Ser Val
180 185 190
Ser Leu Tyr Ala Val Lys Val Leu Asn Ser Ser Gly Ser Gly Ser Tyr
195 200 205
Ser Gly Ile Val Ser Gly Ile Glu Trp Ala Thr Thr Asn Gly Met Asp
210 215 220
Val Ile Asn Met Ser Leu Gly Gly Ala Ser Gly Ser Thr Ala Met Lys
225 230 235 240
Gln Ala Val Asp Asn Ala Tyr Ala Arg Gly Val Val Val Val Ala Ala
245 250 255
Ala Gly Asn Ser Gly Ser Ser Gly Asn Thr Asn Thr Ile Gly Tyr Pro
260 265 270
Ala Lys Tyr Asp Ser Val Ile Ala Val Gly Ala Val Asp Ser Asn Ser
275 280 285
Asn Arg Ala Ser Phe Ser Ser Val Gly Ala Glu Leu Glu Val Met Ala
290 295 300
Pro Gly Ala Gly Val Tyr Ser Thr Tyr Pro Thr Asn Thr Tyr Ala Thr
305 310 315 320
Leu Asn Gly Thr Ser Met Ala Ser Pro His Val Ala Gly Ala Ala Ala
325 330 335
Leu Ile Leu Ser Lys His Pro Asn Leu Ser Ala Ser Gln Val Arg Asn
340 345 350
Arg Leu Ser Ser Thr Ala Thr Tyr Leu Gly Ser Ser Phe Tyr Tyr Gly
355 360 365
Lys Gly Leu Ile Asn Val Glu Ala Ala Ala Gln
370 375
<210> 18
<211> 1140
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atgatgagga agaaatcatt ttggttaggg atgctgacgg cgtttatgtt agtgtttacg 60
atggcgtttt cagatagcgc ttctgctgca caacctgcga aaaatgttga aaaagattat 120
atcgtggggt ttaaatctgg agttaaaacg gcgtctgcga aaaaagatat tattaaagaa 180
tcaggcggca aagtcgataa acagtttcgg attatcaatg ctgcgaaagc gaaacttgat 240
aaagaagcat tgaaagaagt caaaaatgat ccggatgttg cttacgtcga agaagatcat 300
gtcgcacatg cacttgctca gacggtgccg tatggcatcc ctcttatcaa agcagataaa 360
gtccaagcac aaggctttaa aggcgctaat gtcaaagtcg cggtccttga tacgggaatc 420
caagcaagtc atccggatct taatgtggtt gggggtgcgt catttgtcgc gggagaagca 480
tataatacag atggcgacgg tcatggaaca catgttgcgg gaacggtcgc agcgttagat 540
aatacggagg gtgtgcttgg tgttgcaccg tctgtctcac tgtatgcggt gaaagtcctt 600
aattctagcg gatctggatc ttattcagga attgtgtctg gaatcgaatg ggctacaacg 660
aatggcatgg atgtcatcaa tatgagcctg ggaggcgcga gcggctctac agctatgaaa 720
caagcagtcg ataatgcgta tgcgcgcggt gttgtggtgg tcgcagctgc gggcaattca 780
ggctcatctg gcaatacgaa tacgatcggc tatccggcta aatatgattc agtcattgct 840
gtgggcgcgg tcgattctaa ttctaatcgt gcttctttta gctcagtggg cgcagaactt 900
gaagtgatgg caccgggcgc tggagtgtat agcacctatc cgacaaatac ctatgctaca 960
ctgaatggca cgtctatggc ttcacctcat gttgcaggcg ccgccgctct tatcctgagc 1020
aaacatccta atttgagcgc gagccaggtt cgtaatagac tttcttcaac agcgacgtat 1080
ttgggctcta gcttttatta tggcaaagga ctgatcaatg tcgaagcagc tgcacagtaa 1140
<210> 19
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gctctagaat gatgaggaag aaatc 25
<210> 20
<211> 29
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cgcggatcct tactgtgcag ctgcttcga 29
Claims (10)
1.耐热性提高的蛋白酶突变体BLAPR5，其特征在于，所述蛋白酶突变体BLAPR5的氨基酸序列如SEQ ID NO：3所示，所述蛋白酶突变体BLAPR5是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸获得的。
2.耐热性提高的蛋白酶突变体BLAPR6，其特征在于，所述蛋白酶突变体BLAPR6的氨基酸序列如SEQ ID NO：5所示，所述蛋白酶突变体BLAPR6是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第106位的丙氨酸变为天冬氨酸获得的。
3.耐热性提高的蛋白酶突变体BLAPR7，其特征在于，所述蛋白酶突变体BLAPR7的氨基酸序列如SEQ ID NO：7所示，所述蛋白酶突变体BLAPR7是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第120位的赖氨酸变为脯氨酸获得的。
4.耐热性提高的蛋白酶突变体BLAPR8，其特征在于，所述蛋白酶突变体BLAPR8的氨基酸序列如SEQ ID NO：9所示，所述蛋白酶突变体BLAPR8是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第176位的缬氨酸变为丙氨酸获得的。
5.耐热性提高的蛋白酶突变体BLAPR9，其特征在于，所述蛋白酶突变体BLAPR9的氨基酸序列如SEQ ID NO：11所示，所述蛋白酶突变体BLAPR9是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第208位的酪氨酸变为甲硫氨酸获得的。
6.耐热性提高的蛋白酶突变体BLAPR10，其特征在于，所述蛋白酶突变体BLAPR10的氨基酸序列如SEQ ID NO：13所示，所述蛋白酶突变体BLAPR10是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第344位的天冬酰胺变为半胱氨酸获得的。
7.耐热性提高的蛋白酶突变体BLAPR11，其特征在于，所述蛋白酶突变体BLAPR11的氨基酸序列如SEQ ID NO：15所示，所述蛋白酶突变体BLAPR11是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸和第369位的赖氨酸变为天冬酰胺获得的。
8.耐热性提高的蛋白酶突变体BLAPR12，其特征在于，所述蛋白酶突变体BLAPR12的氨基酸序列如SEQ ID NO：17所示，所述蛋白酶突变体BLAPR11是由氨基酸序列为SEQ ID NO:1的蛋白酶的第183位的苏氨酸变为谷氨酸、第53位的缬氨酸变为丙氨酸和第166位的天冬酰胺变为天冬氨酸获得的。
9.含有权利要求1-8任一项所述的蛋白酶突变体BLAPR5、BLAPR6、BLAPR7、BLAPR8、BLAPR9、BLAPR10、BLAPR11、BLAPR12分别对应的编码基因的基因工程菌。
10.权利要求1-8任一项所述的蛋白酶突变体在用于制备饲料添加剂或食品添加剂中的应用。
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| CN114774396B (zh) * | 2022-06-20 | 2022-08-30 | 山东龙昌动物保健品有限公司 | 角蛋白酶突变体及与胆汁酸的复配制剂和在添加剂的应用 |
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