CN106676090B - 热稳定性提高的头孢菌素c酰化酶突变体及其构建方法 - Google Patents

热稳定性提高的头孢菌素c酰化酶突变体及其构建方法 Download PDF

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CN106676090B
CN106676090B CN201610983326.XA CN201610983326A CN106676090B CN 106676090 B CN106676090 B CN 106676090B CN 201610983326 A CN201610983326 A CN 201610983326A CN 106676090 B CN106676090 B CN 106676090B
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谢渊
刘新花
杨广宇
刘天罡
马富强
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Wuhan New Biocalysis Solution Co ltd
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Abstract

本发明公开了一种热稳定性提高的头孢菌素C酰化酶突变体，其突变位点是SEQ ID NO:1表示的氨基酸序列的第113位谷氨酸、218位精氨酸、226位赖氨酸、334位谷氨酸、454位谷氨酸、547位甘氨酸、632位色氨酸中的任意一个；本发明还提供一种上述突变体的构建方法，通过分析该酶结构上的高B因子位点，以此选取突变热点，采用基因工程方法引入定点饱和突变后筛选目的蛋白。本发明的优点和有益效果是获得半衰期明显长于野生型头孢菌素C酰化酶的突变体，能够更好地适应工业生产；所提供的同源建模方法能够扩大获得头孢菌素C酰化酶突变体的来源，并提高筛选出符合要求的突变体的几率。

Description

热稳定性提高的头孢菌素C酰化酶突变体及其构建方法
技术领域
本发明涉及生物遗传工程技术，具体是涉及一种热稳定性提高的头孢菌素C酰化酶突变体及其构建方法。
背景技术
头孢菌素类(cephalosporins)抗生素是继青霉素之后发现的第二类应用于临床治疗的广谱抗生素，约占全球抗生素市场的40％(Turkish Journal ofBiochemistry,2014,39(1):51-56)。7-氨基头孢烷酸(7-ACA)作为许多半合成头孢菌素类抗生素的关键中间体，在医药工业具有重要价值。目前，7-ACA主要由头孢菌素C(Cephalosporin C，CPC)经化学法或酶法合成。相对于化学法，酶法具有工艺简单，安全，高效，污染小等优势，因此日益受到人们的重视。
7-ACA酶法生产主要分为两步酶法和一步酶法两种。两步酶法在国内外已经成功工业化，并逐步取代化学法。虽然两步酶法避免了环境污染问题，但由于两步酶法需要采用D-氨基酸氧化酶和戊二酰-7-氨基头孢烷酸(GL-7-ACA)酰化酶共同作用(Criticalreviews in biotechnology,1998,18(1):1-12)，导致大量副产物产生，降低了产率、提高了成本，因此无法满足工业生产的进一步需求(Applied microbiology andbiotechnology,2013,97(6):2341-2355)。一步酶法是一种新兴的7-ACA生产方法，它采用CPC酰化酶直接一步酰化CPC高效获得7-ACA(Journal ofmicrobiological methods,2003,54(1):131-135)，一步酶法既具有化学法转化率高和纯度高的优势也具有两步酶法低污染的优势，具有良好的工业应用前景。
来源于Pseudomonassp.SE83的头孢菌素C酰化酶SE83acyII编码83kDa左右的多肽前体，并经翻译后自剪切得到25kDaα亚基和58kDaβ亚基组成的成熟蛋白能够完成从CPC到7-ACA的一步酶法转化(Protein Engineering Design and Selection,2010,23(12):903-909)。韩国学者Shin等对Pseudomonassp.SE83acyII进行了突变研究，得到的多点突变体V122αA/G140αS/F58βN/I75βT/I176βV/S471βC(也被命名为Pseudomonas sp.SE83acyIIS12)相比于野生型酶活提高了8.5倍，并且产物抑制也显著降低(U.S.Patent7,592,168[P].2009-9-22；US Patent20,070,207,519[P].2007)。然而，CPC酰化酶在应用中仍存在对热敏感、稳定性不佳等一些问题(Applied and environmental microbiology,1996,62(8):2919-2925；World Journal of Microbiology and Biotechnology,2011,27(4):823-829)，SE83acyII S12最适温度仅为40℃，生产过程中的发热易使CPC酶失活，导致催化效率降低。因此，提高CPC酶热稳定性使其更好的适应运输、工业生产等条件是推动其进一步工业应用的关键。
本发明通过分析头孢菌素C酰化酶结构上的高B因子位置选择突变热点，应用分子生物学技术构建定点饱和突变库，基于pH指示剂法并利用
Figure BDA0001148514750000021
FXP实验室自动工作站建立高效的筛选程序，快速筛选出稳定性提高的突变体，进一步推进头孢菌素C酰化酶的优良改造，为工业化生产奠定基础。
发明内容
为实现上述发明目的，本发明提供一种热稳定性提高的头孢菌素C酰化酶突变体及其构建方法。
本发明的技术方案是提供一种热稳定性提高的头孢菌素C酰化酶突变体，所述头孢菌素C酰化酶的氨基酸序列如SEQ ID NO:1所示，或者该序列经取代、缺失或添加一个或多个氨基酸且与SED ID NO:1所示蛋白具有相同功能的衍生氨基酸序列；编码该酶的核酸序列如SEQ ID NO:2所示，或者为与SEQ ID NO:2所示序列具有90％以上同源性，且编码具有头孢菌素C酰化酶活性的蛋白质的核酸序列；所述头孢菌素C酰化酶突变体的氨基酸序列是由SEQ ID NO:1所示序列上的氨基酸被另一种氨基酸取代而获得，所述取代位置为SEQID NO:1表示的氨基酸序列的第113位谷氨酸、218位精氨酸、226位赖氨酸、334位谷氨酸、454位谷氨酸、547位甘氨酸、632位色氨酸中的任意一个。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列为所述第113位谷氨酸被天冬酰胺取代而获得如SEQ ID NO:3所示的氨基酸序列。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列为所述第218位精氨酸被苏氨酸取代而获得如SEQ ID NO:4所示的氨基酸序列，或者被谷氨酰胺取代而获得如SEQID NO:5所示的氨基酸序列。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列为所述第226位赖氨酸被缬氨酸取代而获得如SEQ ID NO:6所示的氨基酸序列。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列为所述第334位谷氨酸被精氨酸取代而获得如SEQ ID NO:7所示的氨基酸序列。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列所述第454位谷氨酸被天冬氨酸取代而获得如SEQ ID NO:8所示的氨基酸序列。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列为所述第547位甘氨酸被谷氨酸取代而获得如SEQ ID NO:9所示的氨基酸序列。
进一步地，所述头孢菌素C酰化酶突变体，其氨基酸序列为所述第632位色氨酸被组氨酸取代而获得如SEQ ID NO:10所示的氨基酸序列。
进一步地，通过基因工程手段在所述头孢菌素C酰化酶突变体的N端与C端引入标签，优选为组氨酸标签(His-tag)。
本发明还提供一种热稳定性提高的头孢菌素C酰化酶突变体的构建方法，步骤包括：
1)头孢菌素C酰化酶核酸序列以大肠杆菌为宿主进行密码子优化，并由专业商业公司设计上下游引物；利用上下游引物，通过PCR(聚合酶链式反应)扩增法，采用一定的PCR反应体系和一系列温度控制，扩增头孢菌素C酰化酶基因；
2)使用常规的酶切、连接方式克隆基因至大肠杆菌表达载体；使用常规的大肠杆菌培养基培养工程菌，IPTG(异丙基-β-D-硫代吡喃半乳糖苷)诱导剂诱导目的蛋白的表达；后破碎工程菌并分离纯化头孢菌素C酰化酶；
3)利用专业软件计算头孢菌素C酰化酶结构上各氨基酸残基B因子值，选取高B因子氨基酸残基位置作为突变热点；
4)通过全质粒突变法引入定点饱和突变，采用上述1)、2)中的方法进行突变基因扩增和工程菌的培养，建立头孢菌素C酰化酶突变库；
5)使用常规培养方法将工程菌放大培养，后破碎分离取得上清液，加入底物，通过检测单位时间内吸光值的变化来筛选热稳定性提高的突变体；
6)将筛选获得的突变体通过2)中的方法培养后，测定头孢菌素C酰化酶活力，利用残余活力百分比的ln值和时间计算其在特定温度下的半衰期，确定不同突变体的最适温度。
本发明的最终的核酸序列通常可以用PCR扩增法、基因重组或人工合成中的一种或几种方法获得。之后再将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
本发明的头孢菌素C酰化酶突变体用基因重组方法来制备，具体为，通过PCR方法克隆野生型头孢菌素C酰化酶基因，选用的表达载体包括各种含有启动子、调控基因、筛选基因和克隆位点等元件的质粒，例如选用质粒pET28a(Novagen)，该质粒可在市场上获得；然后将上述表达质粒转化到重组工程细胞宿主内，从而获得表达头孢菌素C酰化酶的重组工程细胞。这些重组工程细胞可以是来源于动物细胞、植物细胞、昆虫细胞、真菌、酵母和细菌等，质粒pET28a适合转化入大肠杆菌中表达酰化酶，因此优选的重组工程细胞宿主为大肠杆菌，例如选择大肠杆菌Escherichia coli BL21(DE3)-CodonPlus-RIL。
本文所用的术语“转化”是指用基因工程领域技术人员熟知的方法：将含有目的基因的表达载体导入宿主细胞内。转化方法因宿主细胞类型而异，通常包括：电转化；采用氯化钙、DEAE-葡聚糖或其它物质的转染；微粒轰击；脂质体转染；感染等。本发明中较佳的方法是CaCl2热击化学转化的方法；随后，在适宜培养条件下繁殖宿主细胞。
本领域技术人员根据常规试验就能选择和确定培养基配方、培养温度、诱导物、诱导剂量和时间等条件。采用本领域常规检测手段，如聚丙烯酰胺凝胶电泳(SDS-PAGE)、酶联免疫吸附实验(ELISA)等，可以检测出本发明酰化酶的表达，本发明采用1％琼脂糖凝胶电泳检测。最后，可用常规的蛋白分离纯化技术，进行酰化酶的纯化，其包括离心、过滤、层析等过程；具体地，层析方法又包括亲和层析、凝胶过滤、离子交换色谱以及疏水层析等；本发明提供的该酰化酶的分离纯化方法为Ni-NTA亲和层析。
本发明的优点和有益效果：
1、本发明提供的头孢菌素C酰化酶突变体，在相同条件下与野生型头孢菌素C酰化酶具有相似的高催化活性和产物抑制趋势，且其半衰期t1/2均在72min以上，长于野生型头孢菌素C酰化酶的61.34min，说明突变体稳定性高于野生型，在运输和工业生产过程中，失活程度较低，其中突变体R218Q最适温度为43℃左右，较野生型提高3℃，半衰期为231.05min，为野生型的3.76倍，该结果表明突变体能更好地适应反应温度，保证生产催化效率。
2、突变体不但具有更好的热稳定性，而且与野生型头孢菌素C酰化酶相比，拥有相似的高催化活性和产物抑制趋势，尤其是R218T，R218Q和K226V，其催化效率kcat/Km都高于野生型，更适合工业生产应用。
3、本发明提供了一种头孢菌素C酰化酶突变体构建方法，通过分析高同源蛋白各氨基酸残基B因子，针对性地对头孢菌素C酰化酶的结构进行改进，通过基因工程方法来获得头孢菌素C酰化酶突变体，所得的突变体纯度、稳定性以及催化效率等特性更加适应工业化生产。
4、而在目前SE83acyII S12晶体结构尚未清晰无法直接进行分析的情况下，提供了同源建模方法，通过分析高同源N176acy结构的B因子来指导确定突变热点，扩大了获得头孢菌素C酰化酶突变体的来源，并提高了筛选出符合要求的突变体的几率。
附图说明
图1头孢菌素C酰化酶(N176acy)高B因子示意图。
图2头孢菌素C酰化酶(SE83acyII S12)突变位点示意图。
图3温度对头孢菌素C酰化酶及其突变体活性的影响。
图4不同的7-ACA底物浓度下头孢菌素C酰化酶，突变体R218T，R218Q，K226V的产物抑制趋势。
具体实施方式
下面结合具体的实施例，并参照数据进一步详细描述本发明。应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制发明的范围。
本发明生物材料的来源的一般性说明：
1、引物合成：本发明中所使用的引物均由南京金斯瑞科技有限公司合成。
2、实验中所使用Dpn I购于美国New England Biolabs公司，PCR产物纯化试剂盒、胶回收产物纯化试剂盒和质粒小量提取试剂盒，购于美国Axygen Scientific公司；DNA限制性内切酶，T4DNA ligase和Prestained Protein Ladder，购于Fermentas公司；PrimeStar Max DNA聚合酶，购于日本Takara公司；DNAMarker、MilliporeAmiconUltrafiltration Centrifuge Tubes、BCAProteinAssay Kit，购于Thermo FisherScientific公司；序列测序为华大基因完成。
实施例1野生型头孢菌素C酰化酶基因的克隆
将来源于Pseudomonas sp.SE83的头孢菌素C酰化酶基因以大肠杆菌为宿主进行密码子优化，设计上游引物和下游引物序列分别如下：
上游引物F：5'-ATATCATATGACGATGGCGGCCAAGACCGATCGCGAGGCCCTGCAGGCGGCGCTGCCGCCGCTTTCCGGCAGCCTCTCCATTCCGGGTTTAAGCGCCCCTG-3'
下游引物R：5'-ATATCTCGAGTTAGGCCGGAACCAGCTCCTGGCTG-3'
其中下划线分别为NdeI和XhoI酶切位点。PCR反应体系：2×PrimeStar Max DNA聚合酶25μL，上下游引物(10μmol/L)各1.0μL，基因模板(50ng/μL)1.0μL，双蒸水为22μL。PCR反应条件为：98℃2min，然后98℃10sec，55℃15sec，72℃30sec，共25个循环；最后72℃10min。反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，得到2.4kb大小的条带，与预期结果相符。DpnI消化模板，回收，纯化该目的片段，将其用限制性内切酶NdeI和XhoI进行双酶切后与经同样酶双酶切的质粒pET28a(Novagen)进行连接，将连接产物转化大肠杆菌Escherichia coli BL21(DE3)-CodonPlus-RIL感受态细胞中，将转化细胞涂布于含有50μg/mL卡那霉素的LB平板上筛选阳性克隆，提取质粒，对其进行测序，测序结果表明克隆的头孢菌素C酰化酶基因序列正确，且正确连入pET28a中，将该重组质粒命名为pET28a-SE83-S12。
实施例2野生型头孢菌素C酰化酶的表达、纯化
工程菌按体积比1％接种到含100μg/mL Kan的4mL LB培养基试管中，37℃220rpm培养12h。将该4mL菌液转接至含50μg/mL Kan的1L LB培养基摇瓶中，37℃220rpm培养约2.5h，使OD600达到0.9左右，加入0.5mM IPTG诱导剂，25℃220rpm诱导培养12-16h。将发酵后收获的大肠杆菌菌体悬液超声破碎，再经过Ni-NTA亲和层析处理，得到95％以上纯度的目的蛋白。
实施例3野生型头孢菌素C酰化酶同源建模
由于野生型头孢菌素C酰化酶SE83acyII S12晶体结构仍未得到解析，难以通过常规的蛋白质理性设计手段进行操作，因此提供一种以高同源N176acy(PDB ID:4HSR)为模板进行同源建模的方法。
1)进入SWISS-MODEL数据库主页(https://www.swissmodel.expasy.org/)，在Target Sequence工具中输入头孢菌素C酰化酶SE83acyII S12的氨基酸序列进行搜索。服务器直接搜索同源性最高的序列4HSR作为模板进行序列比对，并得到目的蛋白结构模型。
2)在Protein Data Bank(PDB数据库)中搜索与头孢菌素C酰化酶SE83acyII S12同源性最高，来源于Pseudomonas diminutaN176的头孢菌素C酰化酶(N176acy，PDB ID：4HSR)的结构，利用Discovery studio软件对SE83acyII S12结构模型与N176acy晶体结构进行比对，进一步验证两者结构上的高同源性。
实施例4野生型头孢菌素C酰化酶突变热点的选择
通过B因子分析的方法确定突变热点。而对于SE83acyII S12，由于其晶体结构未知，因此通过上述同源建模方法，以高同源N176acy(PDB ID:4HSR)晶体结构为指导选择突变热点。
1)N176acy B因子分析：在Protein Data Bank(PDB数据库)中搜索与头孢菌素C酰化酶SE83acyII S12同源性最高，来源于Pseudomonas diminutaN176的头孢菌素C酰化酶(N176acy)的结构。N176acy由α链，β链两个亚基组成，使用Discovery studio软件对N176acy两个亚基中每一个氨基酸按照B因子大小进行排序(表1)，选取每个亚基中B因子较大的残基作为突变热点，分别为ARG218,LYS226,GLU113,ASP547,TRP632,ASP548,GLU454,GLU764和ASP334(图1)。
表1N176acyα，β链氨基酸残基B因子排序
Figure BDA0001148514750000071
2)突变位点的对应：用PyMOL对N176acy晶体结构与SE83acyII S12结构模型进行比对，并根据比对结果，将N176acy中高B因子的位点对应到SE83acyII S12结构模型上，在SE83acyII S12中分别为ARG218,LYS226,GLU113,GLY547,TRP632,SER548,GLU454,GLU764和GLU334(图2)
实施例5野生型头孢菌素C酰化酶突变库建立及突变体筛选
本发明采用简易全质粒PCR突变法(
Figure BDA0001148514750000072
site-directed mutagenesismethod)引入定点饱和突变。设计引物序列如下：
R218 5'TAGCAGCCCTGNNKCCGGCCGTTGATGCATTACTGAAAGC 3'
5'ATCAACGGCCGGMNNCAGGGCTGCTAAATCGGCTTCCAGG 3'
K226 5'GATGCATTACTGNNKGCCATGGGTGGTGACGCCAGT 3'
5'CCACCCATGGCMNNCAGTAATGCATCAACGGCCGGAC 3'
E113 5'GAAGCCCTGGGCGCANNKGCAAAGGATATGCTGC 3'
5'TATCCTTTGCMNNTGCGCCCAGGGCTTCAAAATCG 3'
G547 5'CCTGGGTATTCAGNNKAGCCTGCCGGCAGAAGAACT 3'
5’CCGGCAGGCTMNNCTGAATACCCAGGGCTTCTAAG 3’
W632 5’GGCATGTTAAAAGGCNNKAGCTGGGATGAGGCCCTGAGCGAAG 3’
5’CTCATCCCAGCTMNNGCCTTTTAACATGCCTGCGTCG 3’
S548 5’GGTATTCAGGGTNNKCTGCCGGCAGAAGAACTGCGCCAGACC 3’
5’CTGCCGGCAGMNNACCCTGAATACCCAGGGCTTCTAAG 3’
E454 5’GGTTGGAGTGGTNNKCACGAATGGCGCGGTTGGATTCCTC 3’
5’AACCGCGCCATTCGTGMNNACCACTCCAACCCGGAACC 3’
E764 5’CGTATCGCAGCCNNKGCCGTGACCAGCCAGGAGC 3’
5’GCTGGTCACGGCMNNGGCTGCGATACGATCCCAGCTATACAGC 3’
E334 5’CACGCTTTGGCAATNNKTTCGAACCTGTGGCCTGG 3’
5’ACAGGTTCGAAMNNATTGCCAAAGCGTGCGGTGCG 3’
PCR反应体系(50μL)为：2×PrimeStar Max聚合酶25μL，上下游引物(10μmol/L)各1.0μL，模板(50ng/μL)1.0μL，双蒸水为22μL。PCR反应条件为：98℃2min，然后98℃10sec，55℃15sec，72℃2min，共25个循环；最后72℃10min。反应结束后，对PCR扩增产物进行1％琼脂糖凝胶电泳检测，得到9kb大小的条带，与预期结果相符。DpnI消化模板后，使用Axygen PCRclean up试剂盒回收纯化PCR产物，转化至BL21(DE3)codon plus-RIL感受态细胞，得到饱和突变文库，且库容量大于300个克隆。
在96孔板每孔中加入200μL含100mg/L Kan的LB培养基，用微生物菌落筛选系统Qpix460挑取单菌落于每孔中，放入摇床37℃、220rpm过夜培养，次日在每孔中加入甘油使终浓度为15％，-80℃冻存作为饱和突变文库母版。吸取10μL母版中的母菌液加入含有140μL，100mg/L Kan的LB培养基中，放入摇床37℃、220rpm培养至菌液OD600为0.6-0.8，再加入混有IPTG诱导剂的LB培养基至200μL，使IPTG终浓度为0.5mM。在25℃、220rpm条件下诱导12-16h后，将装有菌液的96孔板用高速冷冻离心机4500rpm，离心1h，取出并弃尽上清液，菌体放入-80℃冰箱过夜冻存。次日将96孔板从-80℃冰箱中取出在室温下自然融化1h后再次放入冰箱2h，如此反复冻融3次，使细胞充分破碎，再加入200μL 5mM Tris-HCl(pH8.0)，放置于震板仪上充分摇匀，用高速冷冻离心机4500rpm离心15min后，所得上清液即为粗酶液。将上清液分别加入两块96孔板，孔中含有100μL粗酶液，将其中一块置于室温下，另一块放置于55℃水浴锅中加热10min，
Figure BDA0001148514750000081
FXP工作站吸取两块板各孔70μL的酶液加至130μL含底物的缓冲溶液中，检测单位时间内吸光值的变化，筛选热稳定性提高的突变体。测序结果表明，热稳定性提高的突变体为：E113N，R218T，R218Q，K226V，E334R，E454D，G547E及W632H。
实施例6突变体的纯化及性质表征和对比
按照实施例2的方法得到头孢菌素C酰化酶突变体E113N，R218T，R218Q，K226V，E334R，E454D，G547E及W632H的纯酶液。
野生型头孢菌素C酰化酶及其突变体的活力测定采用pH指示剂法(参见文献Journal ofindustrial microbiology&biotechnology,2014,41(11):1617-1625)。具体表现为：反应总体系200μL，包含5mmol/L pH8.0Tris-HCl，0.0025％苯酚红，68.6mmol/L CPC，5μL酶液，在556nm测定其动力学曲线。CPC酰化酶酶活单位的定义：每分钟释放1μmol质子所需的酶量定义为一个酶活单位U。测定产物抑制时，反应体系内分别加入浓度为0,5mmol/L,10mmol/L,15mmol/L,20mmol/L,25mmol/L的底物7-ACA。
根据上述pH指示剂法测定酶活并计算野生型和8个突变体各自的比活力，以及测算野生型和8个突变体在不同底物浓度(0.5mmol/L，1mmol/L，2mmol/L，5mmol/L，10mmol/L，15mmol/L，20mmol/L，30mmol/L，40mmol/L和80mmol/L)下的动力学常数Km和kcat；将浓度为0.5mg/ml的野生型和8个突变体的酶液在40℃下保温不同时间(分别是15min，30min，45min，60min，90min，120min，150min，180min，240min和300min)，然后置于冰上冷却10min，再室温放置10min，测定酶的残余活性。根据酶的残余活性计算其半衰期，具体方法为：在一定温度下孵育酶液，在不同处理时间取样测定酶活，测算头孢菌素C酰化酶残余活力百分比；以残余活力百分比的ln值对时间t(min)作图，直线的斜率为失活常数kd，由t1/2＝ln2/kd得到头孢菌素C酰化酶在该温度下的半衰期。
表2野生型与突变体的性质比较
Figure BDA0001148514750000091
从表2可以看出，突变体的比活力较野生型没有太大的变化；其中R218T较野生型的kcat值有所下降，突变体R218Q和K226V的kcat有所提升，而其余突变体的kcat值无明显变化；相对野生型，突变体R218T，R218Q和K226V的Km值均有所降低，催化效率kcat/Km都高于野生型。除上述三个突变体外，其他五个突变体的kcat值和Km值与野生型相比并没有大的变化。而根据半衰期t1/2可以看出，在相同条件下，突变体较野生型均能够更长时间保持酶活力，说明其热稳定性均高于野生型；其中稳定性最佳的突变体是R218T，R218Q和K226V，在40℃下，半衰期t1/2分别为161.2min，231.0min，169.1min，分别为野生型的2.63倍，3.77倍，2.77倍。
在不同温度下按照上述pH指示剂法测定野生型和3个稳定性最佳的突变体的酶活。结果如图3所示，野生型与大部分突变体的最适温度均为40℃，而突变体R218Q最适温度为43℃左右，较野生型提高3℃。
CPC酰化酶产物抑制性评估：如图4所示，各突变体与野生型(WT)相比，产物抑制趋势一致，其中突变体K226V在低浓度7-ACA环境时，产物抑制较野生型略微减弱。
上述数据结合图3说明突变体尤其是R218T，R218Q和K226V在保持与野生型头孢菌素C酰化酶相似的高催化活性和产物抑制趋势的前提下，具有更好的热稳定性，更适合工业生产应用。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术人员无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
SEQUENCE LISTING
<110> 武汉瀚海新酶生物科技有限公司
<120> 热稳定性提高的头孢菌素C酰化酶突变体及其构建方法
<130> 2016
<160> 30
<170> PatentIn version 3.3
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450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 4
<211> 774
<212> PRT
<213> 人工序列
<400> 4
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Thr Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 5
<211> 774
<212> PRT
<213> 人工序列
<400> 5
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Gln Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 6
<211> 774
<212> PRT
<213> 人工序列
<400> 6
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu
210 215 220
Leu Val Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 7
<211> 774
<212> PRT
<213> 人工序列
<400> 7
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Arg Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 8
<211> 774
<212> PRT
<213> 人工序列
<400> 8
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Asp Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 9
<211> 774
<212> PRT
<213> 人工序列
<400> 9
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Glu Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly Trp Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 10
<211> 774
<212> PRT
<213> 人工序列
<400> 10
Met Thr Met Ala Ala Lys Thr Asp Arg Glu Ala Leu Gln Ala Ala Leu
1 5 10 15
Pro Pro Leu Ser Gly Ser Leu Ser Ile Pro Gly Leu Ser Ala Pro Val
20 25 30
Arg Val Gln Arg Asp Gly Trp Gly Ile Pro His Ile Lys Ala Ser Gly
35 40 45
Glu Ala Asp Ala Tyr Arg Ala Leu Gly Phe Val His Ala Gln Asp Arg
50 55 60
Leu Phe Gln Met Glu Leu Thr Arg Arg Lys Ala Leu Gly Arg Ala Ala
65 70 75 80
Glu Trp Leu Gly Ala Glu Ala Ala Glu Ala Asp Ile Leu Val Arg Arg
85 90 95
Leu Gly Met Glu Lys Val Cys Arg Arg Asp Phe Glu Ala Leu Gly Ala
100 105 110
Glu Ala Lys Asp Met Leu Arg Ala Tyr Val Ala Gly Val Asn Ala Phe
115 120 125
Leu Ala Ser Gly Ala Pro Leu Pro Ile Glu Tyr Gly Leu Leu Gly Ala
130 135 140
Glu Pro Glu Pro Trp Glu Pro Trp His Ser Ile Ala Val Met Arg Arg
145 150 155 160
Leu Gly Leu Leu Met Gly Ser Val Trp Phe Lys Leu Trp Arg Met Leu
165 170 175
Ala Leu Pro Val Val Gly Ala Ala Asn Ala Leu Lys Leu Arg Tyr Asp
180 185 190
Asp Gly Gly Gln Asp Leu Leu Cys Ile Pro Pro Gly Val Glu Ala Glu
195 200 205
Arg Leu Glu Ala Asp Leu Ala Ala Leu Arg Pro Ala Val Asp Ala Leu
210 215 220
Leu Lys Ala Met Gly Gly Asp Ala Ser Asp Ala Ala Gly Gly Gly Ser
225 230 235 240
Asn Asn Trp Ala Val Ala Pro Gly Arg Thr Ala Thr Gly Arg Pro Ile
245 250 255
Leu Ala Gly Asp Pro His Arg Val Phe Glu Ile Pro Gly Met Tyr Ala
260 265 270
Gln His His Leu Ala Cys Asp Arg Phe Asp Met Ile Gly Leu Thr Val
275 280 285
Pro Gly Val Pro Gly Phe Pro His Phe Ala His Asn Gly Lys Val Ala
290 295 300
Tyr Cys Val Thr His Ala Phe Met Asp Ile His Asp Leu Tyr Leu Glu
305 310 315 320
Gln Phe Ala Glu Asp Gly Arg Thr Ala Arg Phe Gly Asn Glu Phe Glu
325 330 335
Pro Val Ala Trp Arg Arg Asp Arg Ile Ala Val Arg Gly Gly Ala Asp
340 345 350
Arg Glu Phe Asp Ile Val Glu Thr Arg His Gly Pro Val Ile Ala Gly
355 360 365
Asp Pro Leu Glu Gly Ala Ala Leu Thr Leu Arg Ser Val Gln Phe Ala
370 375 380
Glu Thr Asp Leu Ser Phe Asp Cys Leu Thr Arg Met Pro Gly Ala Ser
385 390 395 400
Thr Val Ala Gln Leu Tyr Asp Ala Thr Arg Gly Trp Gly Leu Ile Asp
405 410 415
His Asn Leu Val Ala Gly Asp Val Ala Gly Ser Ile Gly His Leu Val
420 425 430
Arg Ala Arg Val Pro Ser Arg Pro Arg Glu Asn Gly Trp Leu Pro Val
435 440 445
Pro Gly Trp Ser Gly Glu His Glu Trp Arg Gly Trp Ile Pro His Glu
450 455 460
Ala Met Pro Arg Val Ile Asp Pro Pro Gly Gly Leu Ile Val Thr Ala
465 470 475 480
Asn Asn Arg Val Val Ala Asp Asp His Pro Asp Tyr Leu Cys Thr Asp
485 490 495
Cys His Pro Pro Tyr Arg Ala Glu Arg Ile Met Glu Arg Leu Val Ala
500 505 510
Ser Pro Ala Phe Ala Val Asp Asp Ala Ala Ala Ile His Ala Asp Thr
515 520 525
Leu Ser Pro His Val Gly Leu Leu Arg Ala Arg Leu Glu Ala Leu Gly
530 535 540
Ile Gln Gly Ser Leu Pro Ala Glu Glu Leu Arg Gln Thr Leu Ile Ala
545 550 555 560
Trp Asp Gly Arg Met Asp Ala Gly Ser Gln Ala Ala Ser Ala Tyr Asn
565 570 575
Ala Phe Arg Arg Ala Leu Thr Arg Leu Val Thr Ala Arg Ser Gly Leu
580 585 590
Glu Gln Ala Ile Ala His Pro Phe Ala Ala Val Pro Pro Gly Val Ser
595 600 605
Pro Gln Gly Gln Val Trp Trp Ala Val Pro Thr Leu Leu Arg Asn Asp
610 615 620
Asp Ala Gly Met Leu Lys Gly His Ser Trp Asp Glu Ala Leu Ser Glu
625 630 635 640
Ala Leu Ser Val Ala Thr Gln Asn Leu Thr Gly Arg Gly Trp Gly Glu
645 650 655
Glu His Arg Pro Arg Phe Thr His Pro Leu Ser Ala Gln Phe Pro Ala
660 665 670
Trp Ala Ala Leu Leu Asn Pro Val Ser Arg Pro Ile Gly Gly Asp Gly
675 680 685
Asp Thr Val Leu Ala Asn Gly Leu Val Pro Ser Ala Gly Pro Glu Ala
690 695 700
Thr Tyr Gly Ala Leu Ser Arg Tyr Val Phe Asp Val Gly Asn Trp Asp
705 710 715 720
Asn Ser Arg Trp Val Val Phe His Gly Ala Ser Gly His Pro Ala Ser
725 730 735
Pro His Tyr Ala Asp Gln Asn Ala Pro Trp Ser Asp Cys Ala Met Val
740 745 750
Pro Met Leu Tyr Ser Trp Asp Arg Ile Ala Ala Glu Ala Val Thr Ser
755 760 765
Gln Glu Leu Val Pro Ala
770
<210> 11
<211> 101
<212> DNA
<213> 人工序列
<400> 11
atatcatatg acgatggcgg ccaagaccga tcgcgaggcc ctgcaggcgg cgctgccgcc 60
gctttccggc agcctctcca ttccgggttt aagcgcccct g 101
<210> 12
<211> 35
<212> DNA
<213> 人工序列
<400> 12
atatctcgag ttaggccgga accagctcct ggctg 35
<210> 13
<211> 40
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (12)..(13)
<223> n is a, c, g, or t
<400> 13
tagcagccct gnnkccggcc gttgatgcat tactgaaagc 40
<210> 14
<211> 40
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 14
atcaacggcc ggmnncaggg ctgctaaatc ggcttccagg 40
<210> 15
<211> 36
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 15
gatgcattac tgnnkgccat gggtggtgac gccagt 36
<210> 16
<211> 37
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 16
ccacccatgg cmnncagtaa tgcatcaacg gccggac 37
<210> 17
<211> 34
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 17
gaagccctgg gcgcannkgc aaaggatatg ctgc 34
<210> 18
<211> 35
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (12)..(13)
<223> n is a, c, g, or t
<400> 18
tatcctttgc mnntgcgccc agggcttcaa aatcg 35
<210> 19
<211> 36
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 19
cctgggtatt cagnnkagcc tgccggcaga agaact 36
<210> 20
<211> 35
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (12)..(13)
<223> n is a, c, g, or t
<400> 20
ccggcaggct mnnctgaata cccagggctt ctaag 35
<210> 21
<211> 43
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (16)..(17)
<223> n is a, c, g, or t
<400> 21
ggcatgttaa aaggcnnkag ctgggatgag gccctgagcg aag 43
<210> 22
<211> 37
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 22
ctcatcccag ctmnngcctt ttaacatgcc tgcgtcg 37
<210> 23
<211> 42
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 23
ggtattcagg gtnnkctgcc ggcagaagaa ctgcgccaga cc 42
<210> 24
<211> 38
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (12)..(13)
<223> n is a, c, g, or t
<400> 24
ctgccggcag mnnaccctga atacccaggg cttctaag 38
<210> 25
<211> 40
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 25
ggttggagtg gtnnkcacga atggcgcggt tggattcctc 40
<210> 26
<211> 38
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (18)..(19)
<223> n is a, c, g, or t
<400> 26
aaccgcgcca ttcgtgmnna ccactccaac ccggaacc 38
<210> 27
<211> 34
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 27
cgtatcgcag ccnnkgccgt gaccagccag gagc 34
<210> 28
<211> 43
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (14)..(15)
<223> n is a, c, g, or t
<400> 28
gctggtcacg gcmnnggctg cgatacgatc ccagctatac agc 43
<210> 29
<211> 35
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (15)..(16)
<223> n is a, c, g, or t
<400> 29
cacgctttgg caatnnkttc gaacctgtgg cctgg 35
<210> 30
<211> 35
<212> DNA
<213> 人工序列
<220>
<221> misc_feature
<222> (13)..(14)
<223> n is a, c, g, or t
<400> 30
acaggttcga amnnattgcc aaagcgtgcg gtgcg 35

Claims (1)

1.一种热稳定性提高的头孢菌素C酰化酶突变体，所述头孢菌素C酰化酶的氨基酸序列如SEQ ID NO:1所示；所述头孢菌素C酰化酶突变体的氨基酸序列是由SEQ ID NO:1所示序列上的氨基酸被另一种氨基酸取代而获得，其特征在于，取代位置为SEQ ID NO:1表示的氨基酸序列上的第218位精氨酸，所述第218位精氨酸被苏氨酸取代而获得如SEQ ID NO:4所示的氨基酸序列，或者被谷氨酰胺取代而获得如SEQ ID NO:5所示的氨基酸序列。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108239630B (zh) * 2016-12-27 2021-07-16 武汉臻智生物科技有限公司 一种改造萜类合酶的方法
CN108841801A (zh) * 2018-05-29 2018-11-20 江南大学 一种筛选酶中与酶活力相关的氨基酸残基的方法
CN111172142B (zh) * 2020-02-14 2021-09-28 上海陶宇晟生物技术有限责任公司 一种热稳定性高的头孢菌素c酰化酶突变体
CN112662655B (zh) * 2020-12-29 2022-05-03 山东金城柯瑞化学有限公司 头孢菌素c酰化酶突变体及其制备方法和应用
CN113215130B (zh) * 2021-05-11 2022-12-06 集美大学 磷脂酶c突变体、制备方法及其应用
CN116144517A (zh) * 2021-11-22 2023-05-23 中国科学院天津工业生物技术研究所 一种7-氨基头孢烷酸生产菌及其制备方法与应用
CN115232856A (zh) * 2022-07-27 2022-10-25 河南省健康元生物医药研究院有限公司 一种基于固体发酵的产黄支顶孢霉高通量筛选方法

Non-Patent Citations (9)

* Cited by examiner, † Cited by third party
Title
Cloning and nucleotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains;Ichiro Aramori等;《Journal of Fermentation and Bioengineering》;19911231;第72卷(第4期);第232-243页 *
Determination of the second autoproteolytic cleavage site of cephalosporin C acylase and the effect of deleting its flanking residues in the α-C-terminal region;Zhang J等;《J Biotechnol》;20140527;第184卷;第138-145页 *
Engineering of a CPC acylase using a facile pH indicator assay;Xiao Y等;《J Ind Microbiol Biotechnol》;20140914;第41卷(第11期);第1617-1625页 *
Evolution of an acylase active on cephalosporin C;Pollegioni L等;《PROTEIN SCIENCE》;20051231;第14卷(第12期);第3064-3076页 *
High-throughput screening of B factor saturation mutated Rhizomucor miehei lipase thermostability based on synthetic reaction;Zhang JH等;《Enzyme Microb Technol》;20120320;第50卷(第6-7期);第325-330页 *
Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel;Wang Y等;《J Biosci Bioeng》;20111002;第114卷(第1期);第36-41页 *
Pollegioni L等.Evolution of an acylase active on cephalosporin C.《PROTEIN SCIENCE》.2005,第14卷(第12期),第3064-3076页. *
登录号：AOT80792.1;Aramori I等;《GenBank》;20161008;第1-774位 *
登录号：P15558.2;Matsuda A等;《UniProtKB》;20161005;第1-774位 *

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