CN110846291A - 热稳定性提高的胺脱氢酶突变体及其基因工程菌的构建和应用 - Google Patents
热稳定性提高的胺脱氢酶突变体及其基因工程菌的构建和应用 Download PDFInfo
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Abstract
本发明属于生物技术领域，具体涉及一种热稳定性提高的胺脱氢酶突变体及其基因工程菌的构建和应用。本发明提供的胺脱氢酶突变体包括4种单点突变体和11种组合突变体，与野生型胺脱氢酶相比，其单点突变体和组合突变体在42℃下半衰期均更长；尤其是组合突变体，表现出单点突变体热稳定性的叠加效果，其半衰期大约是野生型脱氨氢酶的5倍。通过本发明所提供的构建方法得到的胺脱氢酶突变体的热稳定性更好，在较高的温度下催化合成手性胺时，表现出优良的立体选择性、区域选择性和催化活性，具有较好的应用前景。
Description
技术领域
本发明属于生物技术领域，具体涉及一种热稳定性提高的胺脱氢酶突变体及其基因工程菌的构建和应用。
背景技术
手性胺是一类非常重要的胺类化合物，广泛存在于医药、农药、合成中间体、天然产物和具有生物活性的化合物中，尤其是手性药物领域占有重要地位，目前约40%的光学活性药物含有手性胺结构。手性胺的不同对映异构体有着非常相似的物理性质，但其立体结构的差异会使得不同手性对映异构体具有不同的生物活性，从而在生物体内代谢、转化或活化的途径不同，作用于生命体时表现出来的生理活性和毒害作用不同。因此，获得具有高对映选择性或高非对映选择性的单一异构体具有重要的意义。传统的化学合成方法是通过加入手性试剂对消旋体进行拆分，此方法需要当量的手性试剂，最高产率只有50%，原子经济不高；生物催化法相比于化学合成法具有更佳的催化效应，尤其在识别手性胺两种对映异构体方面具有显著的技术优势。
胺脱氢酶（amine dehydrogenase，AmDH）能够在辅酶的作用下催化前手性酮和游离胺不对称合成手性胺，是一种有效的生物催化剂。然而，胺脱氢酶属于天然生物酶，众所周知，天然酶都是在生物体内比较温和的环境中发挥作用的，但若将胺脱氢酶应用在工业上比较严苛的环境（如高温、极端酸碱度、有机溶剂、非天然底物、产物抑制等）中发挥作用时，其热稳定性较差，从而催化反应活性不高。
蛋白质工程是以蛋白质分子的结构规律及其生物功能的关系作为基础，通过化学、物理和分子生物学的手段进行基因修饰或基因合成，对现有蛋白质进行改造或制造一种新的蛋白质以满足人类对生产和生活的需求。常用的蛋白质工程方法有理性设计（rational design）和非理性设计（irrational design），其中，理性设计需了解蛋白质的结构、功能和机能，但由于蛋白质结构-功能关系过于庞杂，现今人们仍对其缺乏充足的认识，因而准确性较差。如何通过蛋白质工程提升胺脱氢酶的热稳定性成为时下亟待解决的难题。
发明内容
因此，本发明要解决的技术问题在于克服现有的胺脱氢酶热稳定性差的缺陷，从而提供一种热稳定性提高的胺脱氢酶突变体、胺脱氢酶突变体的基因工程菌的构建方法及胺脱氢酶突变体在手性胺制备中的应用。
为解决上述技术问题，本发明采用的技术方案是：
本发明提供一种热稳定性提高的胺脱氢酶突变体，所述胺脱氢酶突变体为如下（a1）或（a2）：
（a1）将SEQ ID NO.2所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸且与SEQID NO.2所示的氨基酸序列具有相同功能的衍生蛋白质；
（a2）将SEQ ID NO.2所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸且与SEQID NO.2所示的氨基酸序列具有至少90%同源性的衍生蛋白质；
所述胺脱氢酶突变体是在SEQ ID NO.2所示的氨基酸序列上进行突变，所述突变位点选自A35D、L53R、S210A、T321P中一种或一种以上组合。
优选地，该热稳定性提高的胺脱氢酶突变体，所述突变位点为A35D、L53R、S210A、T321P、A35D/L53R、A35D/S210A、A35D/T321P、L53R/S210A、L53R/T321P、S210A/T321P、A35D/L53R/S210A、A35D/L53R/T321P、A35D/S210A/T321P、L53R/S210A/T321P或A35D/L53R/S210A/T321P。
本发明还提供一种编码如上述热稳定性提高的胺脱氢酶突变体的基因。
本发明还提供一种包含如上述基因的重组质粒。
本发明还提供一种包含如上述热稳定性提高的胺脱氢酶突变体的可溶性蛋白、固定化酶或工程菌。
本发明还提供一种如上述热稳定性提高的胺脱氢酶突变体的构建方法，包括以下步骤：
通过在Pfam数据库及NCBI数据库中搜索SEQ ID NO.2所示的氨基酸序列，去除重复出现的相同序列，选取与SEQ ID NO.2所示的氨基酸序列一致性大于30%的氨基酸序列，然后通过Clustalx1.83软件进行多序列比对，将剩余氨基酸质序列整理成fasta.文件上传到Consensus Maker v2.0.0服务器，根据需要修改设置参数后，该在线软件将生成可以后期编辑的consensus sequence；
通过swissmodel在线工具对获得的SEQ ID NO.2所示的蛋白质三维结构预测，采用PyMOL观测SEQ ID NO.2所示的蛋白质晶体结构，筛选出热稳定性相关的突变位点为：A35D、L53R、S210A、T321P。
优选地，该热稳定性提高的胺脱氢酶突变体的构建方法，
突变位点A35D的扩增引物序列为SEQ ID NO.20、SEQ ID NO.21；
突变位点L53R的扩增引物序列为SEQ ID NO.22、SEQ ID NO.23；
突变位点S210A的扩增引物序列为SEQ ID NO.24、SEQ ID NO.25；
突变位点T321P的扩增引物序列为SEQ ID NO.26、SEQ ID NO.27。
进一步优选地，该热稳定性提高的胺脱氢酶突变体的构建方法，
A35D对应的单点突变体的氨基酸序列为SEQ ID NO.3；
L53R对应的单点突变体的氨基酸序列为SEQ ID NO.4；
S210A对应的单点突变体的氨基酸序列为SEQ ID NO.5；
T321P对应的单点突变体的氨基酸序列为SEQ ID NO.6；
A35D/L53R对应的组合突变体的氨基酸序列为SEQ ID NO.7；
A35D/S210A对应的组合突变体的氨基酸序列为SEQ ID NO.8；
A35D/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.9；
L53R/S210A对应的组合突变体的氨基酸序列为SEQ ID NO.10；
L53R/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.11；
S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.12；
A35D/L53R/S210A对应的组合突变体的氨基酸序列为SEQ ID NO.13；
A35D/L53R/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.14；
A35D/S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.15；
L53R/S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.16；
A35D/L53R/S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.17。
本发明还提供上述热稳定性提高的胺脱氢酶突变体在催化合成手性胺中的应用。
本发明技术方案，具有如下优点：
1.本发明提供的热稳定性提高的胺脱氢酶突变体包括单点突变体和组合突变体，与野生型胺脱氢酶相比，其单点突变体和组合突变体在42℃下半衰期均更长；尤其是组合突变体，表现出单点突变体热稳定性的叠加效果，其半衰期大约是野生型脱氨氢酶的5倍。基于此，本发明所提供的胺脱氢酶突变体的热稳定性更好，适于在较高的温度下催化合成手性胺。
2.本发明提供的热稳定性提高的胺脱氢酶突变体的构建方法，与理性设计基于蛋白质精确结构-功能关系不同的是，本发明以Consensus Concept理论为指导思想，从进化的角度分析能提高酶热稳定性的信息，对胺脱氢酶家族序列进行整合分析，并结合生物信息学及晶体学方法辅助，获得具高稳定性的新型胺脱氢酶突变体。
3.本发明提供的热稳定性提高的胺脱氢酶突变体在应用于催化合成手性胺时具有优良的立体选择性、区域选择性和催化活性，具有较好的应用前景。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例2提供的ANDD-TDO蛋白模拟晶体结构示意图及突变位点在晶体结构上的分布示意图。
具体实施方式
为了便于理解本发明的目的、技术方案和要点，下面将对本发明的实施方式作进一步详细描述。本发明可以多种不同的形式实施，而不应该被理解为仅限于在此阐述的实施例。相反，提供此实施例，使得本发明将是彻底的和完整的，并且将把本发明的构思充分传达给本领域技术人员，本发明将仅由权利要求来限定。
实施例1
本实施例提供一种热稳定性提高的胺脱氢酶突变体，其中，胺脱氢酶为来源于Thermosediminibacter oceani的野生型胺脱氢酶，命名为ANDD-TDO蛋白，编码该ANDD-TDO蛋白的核酸序列为SEQ ID NO.1，氨基酸序列为SEQ ID NO.2。
本实施例提供的热稳定性提高的胺脱氢酶突变体包括：将SEQ ID NO.2所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸形成与该SEQ ID NO.2所示的氨基酸序列（即ANDD-TDO蛋白）具有相同功能的衍生蛋白，或者将SEQ ID NO.2所示的氨基酸序列经取代、缺失或添加一个或多个氨基酸形成与该SEQ ID NO.2所示的氨基酸序列（即ANDD-TDO蛋白）具有至少90%同源性的衍生蛋白。
具体地，在SEQ ID NO.2所示的氨基酸序列选择某一个位点进行单点突变，分别得到5种胺脱氢酶单点突变体，其突变位点为：A35D、S41V、L53R、S210A、T321P，将该5种胺脱氢酶单点突变体进行活力测定，筛选出4种热稳定性提高的胺脱氢酶突变体，其突变位点为：A35D、L53R、S210A、T321P，其氨基酸序列分别为SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6。
或者在SEQ ID NO.2所示的氨基酸序列选择多个突变位点进行组合，如从上述4个突变位点中选择2个突变位点进行组合，分别得到以下6种热稳定性提高的胺脱氢酶突变体，其组合突变位点为：A35D/L53R、A35D/S210A、A35D/T321P、L53R/S210A、L53R/T321P、S210A/T321P，其氨基酸序列分别为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9、SEQ IDNO.10、SEQ ID NO.11、SEQ ID NO.12。
如从上述5个突变位点中选择3个突变位点进行组合，分别得到4种热稳定性提高的胺脱氢酶突变体，其组合突变位点为：A35D/L53R/S210A、A35D/L53R/T321P、A35D/S210A/T321P、L53R/S210A/T321P，其氨基酸序列分别为SEQ ID NO.13、SEQ ID NO.14、SEQ IDNO.15、SEQ ID NO.16。
如从上述4个突变位点选择4个突变位点进行组合，得到1种热稳定性提高的胺脱氢酶突变体，其组合突变位点为：A35D/L53R/S210A/T321P，其氨基酸质序列为SEQ IDNO.17。
实施例2
本实施例提供一种热稳定性提高的胺脱氢酶突变体的构建方法，包括以下步骤：
1.野生型胺脱氢酶ANDD-TDO基因的克隆
将野生型胺脱氢酶基因以大肠杆菌为宿主细胞进行密码子优化，得到优化的ANDD-TDO基因，其核酸序列为SEQ ID NO.1，表达的氨基酸序列为SEQ ID NO.2；以SEQ ID NO.1作为目的基因，采用上游扩增引物SEQ ID NO.18和下游扩增引物SEQ ID NO.19扩增目的基因；
SEQ ID NO.18核酸序列为：
5’-ACTGCTCATATGGAAAAAATCCGTGTTATCATC-3’（其中下划线为限制性内切酶NdeI识别位点）；
SEQ ID NO.19核酸序列为：
5’-TCAGCTCTCGAGTTAAGCGTTGTTAACACCG-3’（其中下划线为限制性内切酶XhoI识别位点）。
扩增条件为：在95℃下扩增2min，然后在56℃下扩增20sec、在72℃下扩增90sec，共30个循环，最后在72℃下扩增10min。
待反应结束，采用1.5%琼脂糖凝胶电泳检测PCR扩增产物，得到1.0kb的条带，其长度符合预期结果。按照试剂盒标准操作，回收、纯化该目的片段，使用限制性核酸内切酶XhoI和NdeI对该目的片段以及pET28a质粒进行双酶切，然后采用T4 DNA连接酶进行连接，将得到的连接产物转化至大肠杆菌BL21(DE3)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，提取阳性克隆质粒，进行测序，结果显示克隆的胺脱氢酶ANDD-TDO基因序列正确，且已正确接入pET28a质粒，得到重组质粒pET28a-ANDD-TDO；
其中，野生型胺脱氢酶来源于Thermosediminibacter oceani；
ANDD-TDO基因由苏州金唯智生物科技有限公司提供；
PCR扩增酶为东洋纺提供的KOD高保真聚合酶。
2. ANDD-TDO蛋白的表达和纯化
将甘油管中的工程菌按体积比1%接种到含100μg/mL Kan的4mL LB培养基试管中，在37℃、220rpm条件下培养12h；取菌液4mL转接至含50μg/mL Kan的1L LB培养基摇瓶中，在37℃、220rpm条件下培养2.5h，使OD600达到0.9左右，加入0.1mM IPTG诱导剂，在25℃、200rpm条件下诱导培养14h。将发酵后收获的大肠杆菌菌体悬液超声破碎，再经过一步Ni-NTA亲和层析处理，得到纯度＞95%的ANDD-TDO蛋白，氨基酸序列为SEQ ID NO.2。
3. ANDD-TDO同源蛋白的多序列比对及Consensus分析
3.1.进入Pfam数据库主页（http://pfam.xfam.org/），在SEQUENCE SEARCH工具中输入ANDD-TDO的氨基酸序列进行搜索，服务器将直接反馈该蛋白整个家族的氨基酸序列的比对结果，将各个突变位点的各类氨基酸丰度以柱状图形式显示，该网站也可以自动生成该蛋白家族的consensus sequence；
3.2.在NCBI protein数据库及Pfam数据库中输入SEQ ID NO.2所示的氨基酸序列，利用Blast工具，找出所有与ANDD-TDO蛋白的氨基酸序列（SEQ ID NO.2）一致性大于30%的蛋白质序列，删除其中的重复出现的相同序列，将剩余氨基酸序列整理成fasta.格式，输入Clustalx1.83软件进行多序列比对，比对结果以aln.、dnd.和fasta.格式输出，其中，dnd.文件为构建进化树文件，aln.和fasta.文件为不同形式的序列文件；
将上述fasta.文件上传到Consensus Maker v2.0.0（http://www.hiv.lanl.gov/content/sequence/ CONSENSUS/consensus.html）服务器，根据需要修改设置参数后，该在线软件将生成可以后期编辑的consensus sequence。
3.3.将ANDD-TDO蛋白的氨基酸序列（SEQ ID NO.2）与该家族consensus sequence以及各位点氨基酸丰度图进行对照比较。
4.ANDD-TDO蛋白三维结构的模拟及突变热点的选择
4.1.通过swissmodel在线工具对获得了ANDD-TDO蛋白（氨基酸序列SEQ ID NO.2）的三维结构预测；
4.2.用PyMOL观测ANDD-TDO蛋白（氨基酸序列SEQ ID NO.2）晶体结构，根据结构信息复查上述待选突变位点及突变形式，筛选出最有可能提高ANDD-TDO蛋白热稳定性的突变***点，筛选条件如下：
（1）判断某一位点为待选位点的标准为：
①该家族大多数蛋白在该位点处氨基酸丰度总体高度较高；
②该位点氨基酸保守；
③该位点出现频率较高的氨基酸与ANDD-TDO蛋白在该位点处的氨基酸有较大的理化性质差异，如电荷差异、极性强弱、空间位阻大小等。
（2）除去活性中心附近，即距离催化残基（104位谷氨酸）10Å范围内的氨基酸残基，除去处于包埋或半包埋状态的氨基酸残基。
经过上述两步筛选，此时共剩余28个差异位点，大多数位于ANDD-TDO蛋白分子的表面，如图1所示，箭头所指即为突变位点。
（3）根据ANDD-TDO蛋白晶体结构，逐个详细分析上述28种突变形式，筛选出可能提高ANDD-TDO蛋白热稳定性的突变体。
主要判断准则为：①突变应消除原有的不利于热稳定的作用力形式，如静电排斥作用、电荷聚集等；②突变不应破坏现有的利于热稳定的作用力形式和稳定的蛋白结构；③突变应引入新的利于热稳定的作用力形式，如氢键、盐桥、疏水相互作用等。
共设计单点突变体5个，其突变位点分别为：
A35D、S41V、L53R、S210A、T321P；
将该5种胺脱氢酶单点突变体进行活力测定，筛选出4种热稳定性提高的胺脱氢酶突变体，其突变位点为：A35D、L53R、S210A、T321P，所对应的单点突变体的氨基酸序列分别为SEQID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6。
5.突变体的构建、表达及纯化
5.1. ANDD-TDO蛋白单点突变体的构建
以步骤1中的重组质粒pET28a-ANDD-TDO为模板，以带有突变位点的一对互补的寡核苷酸为扩增引物，用KOD高保真酶进行全质粒PCR扩增，获得具有特定突变位点的重组质粒；
所采用的扩增引物对为：
（1）突变位点A35D的上游扩增引物SEQ ID NO.20和下游扩增引物SEQ ID NO.21的核酸序列分别如下：
SEQ ID NO.20：
5' -GAAATCGTTGGTGCTATCGACTCTCGTCCGGAAAAATCT- 3'；
SEQ ID NO.21：
5'- AGATTTTTCCGGACGAGAGTCGATAGCACCAACGATTTC -3'；
（2）突变位点L53R的上游扩增引物SEQ ID NO.22和下游扩增引物SEQ ID NO.23的核酸序列分别如下：
SEQ ID NO.22：
5' -GAAGTTCTGGACCTGGGTCGTAAAACCGGTGTTACCATC -3'；
SEQ ID NO.23：
5' -GATGGTAACACCGGTTTTACGACCCAGGTCCAGAACTTC -3'；
（3）突变位点S210A的上游扩增引物SEQ ID NO.24和下游扩增引物SEQ ID NO.25的核酸序列分别如下：
SEQ ID NO.24：
5' -GAATCTATCTCTCTGATCGCTGAAGCTCTGGGTCTGGAA -3'；
SEQ ID NO.25：
5' -TTCCAGACCCAGAGCTTCAGCGATCAGAGAGATAGATTC -3'；
（4）突变位点T321P的上游扩增引物SEQ ID NO.26和下游扩增引物SEQ ID NO.27的核酸序列分别如下：
SEQ ID NO.26：
5' -AAAGTTATCTCTGCTAACCCTGGTCTGGTTACCATGAAA -3'；
SEQ ID NO.27：
5' -TTTCATGGTAACCAGACCAGGGTTAGCAGAGATAACTTT -3'；
扩增条件为：在95℃下扩增2min，然后在56℃下扩增20sec、在72℃下扩增90sec，共30个循环，最后在72℃下扩增10min；胶回收PCR扩增产物，采用DpnI酶在37℃条件下消化胶回收产物2h，降解初始模板；将消化产物转化至大肠杆菌BL21（DE3）感受态细胞中，涂布到含有50μg/mL卡那霉素LB琼脂平板上，37℃过夜培养，筛选阳性克隆，测序验证，得到含胺脱氢酶单点突变体的重组菌；
其中，上述KOD高保真酶由TakaRa公司提供；
上述DpnI酶由Fermentas公司提供。
5.2. ANDD-TDO蛋白组合突变体的构建
使用与单点突变体相似的构建方法，将稳定性提高的单点突变体累加组合，在SEQ IDNO.2所示的氨基酸序列选择多个突变位点进行组合，如从上述4个突变位点中选择2～4个突变位点进行组合，分别得到不同的胺脱氢酶组合突变体：
（1）选择2个突变位点进行组合，可构建6种热稳定性提高的胺脱氢酶突变体胺脱氢酶组合突变体，其组合突变位点分别为：
A35D/L53R、A35D/S210A、A35D/T321P、L53R/S210A、L53R/T321P、S210A/T321P，
该6种热稳定性提高的胺脱氢酶组合突变体的氨基酸序列分别为SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12；
（2）选择3个突变位点进行组合，可构建4种热稳定性提高的胺脱氢酶组合突变体，其组合突变位点分别为：
A35D/L53R/S210A、A35D/L53R/T321P、A35D/S210A/T321P、L53R/S210A/T321P，
该4种热稳定性提高的胺脱氢酶组合突变体的氨基酸序列分别为SEQ ID NO.13、SEQID NO.14、SEQ ID NO.15、SEQ ID NO.16；
（3）选择4个突变位点进行组合，可构建1种热稳定性提高的胺脱氢酶组合突变体，其组合突变位点分别为：
A35D/L53R/S210A/T321P，
该1种热稳定性提高的胺脱氢酶组合突变体的氨基酸序列为SEQ ID NO.17。
实施例3
本实施例提供一种编码如实施例1所述的热稳定性提高的胺脱氢酶突变体的基因：
（1）编码突变位点为A35D的胺脱氢酶突变体的核酸序列为SEQ ID NO.28；
（2）编码突变位点为L53R的胺脱氢酶突变体的核酸序列为SEQ ID NO.29；
（3）编码突变位点为S210A的胺脱氢酶突变体的核酸序列为SEQ ID NO.30；
（4）编码突变位点为T321P的胺脱氢酶突变体的核酸序列为SEQ ID NO.31；
（5）编码突变位点为A35D/L53R的胺脱氢酶突变体的核酸序列为SEQ ID NO.32；
（6）编码突变位点为A35D/S210A的胺脱氢酶突变体的核酸序列为SEQ ID NO.33；
（7）编码突变位点为A35D/T321P的胺脱氢酶突变体的核酸序列为SEQ ID NO.34；
（8）编码突变位点为L53R/S210A的胺脱氢酶突变体的核酸序列为SEQ ID NO.35；
（9）编码突变位点为L53R/T321P的胺脱氢酶突变体的核酸序列为SEQ ID NO.36；
（10）编码突变位点为S210A/T321P的胺脱氢酶突变体的核酸序列为SEQ ID NO.37；
（11）编码突变位点为A35D/L53R/S210A的胺脱氢酶突变体的核酸序列为SEQ IDNO.38；
（12）编码突变位点为A35D/L53R/T321P的胺脱氢酶突变体的核酸序列为SEQ IDNO.39；
（13）编码突变位点为A35D/S210A/T321P的胺脱氢酶突变体的核酸序列为SEQ IDNO.40；
（14）编码突变位点为L53R/S210A/T321P的胺脱氢酶突变体的核酸序列为SEQ IDNO.41；
（15）编码突变位点为A35D/L53R/S210A/T321P的胺脱氢酶突变体的核酸序列为SEQ IDNO.42。
测试例1
1.胺脱氢酶突变体的酶学性质表征
将野生型胺脱氢酶以及实施例2提供的多种胺脱氢酶突变体进行热稳定性测试，按照常规胺脱氢酶活力测定方法（参见文献Catal. Sci. Technol. 2016:10.1039.C6CY01625A），具体为：
在一定温度下孵育酶液，在不同处理时间取样，测定胺脱氢酶或胺脱氢酶突变体残余活力百分比，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kinact，由t1/2=ln2/kinact得到该种野生型胺脱氢酶或胺脱氢酶突变体在该温度下的半衰期。
实验结果表明，以上多种胺脱氢酶突变体中，有4种单点突变体和11种组合突变体的热稳定性得到明显改善，如表1所示：
表1. 野生型胺脱氢酶、单点突变体和组合突变体的酶学性质表征
| ANDD-TDO | 42℃半衰期(min) |
| 野生型 | 8 |
| A35D | 10 |
| L53R | 17 |
| S210A | 23 |
| T321P | 19 |
| A35D/L53R | 25 |
| A35D/S210A | 27 |
| A35D/T321P | 29 |
| L53R/S210A | 25 |
| L53R/T321P | 31 |
| S210A/T321P | 32 |
| A35D/L53R/S210A | 40 |
| A35D/L53R/T321P | 33 |
| A35D/S210A/T321P | 35 |
| L53R/S210A/T321P | 37 |
| A35D/L53R/S210A/T321P | 38 |
由表1得知，本发明提供的胺脱氢酶突变体包括单点突变体和组合突变体，与野生型胺脱氢酶相比，其单点突变体和组合突变体在42℃下半衰期均更长；尤其是组合突变体，表现出单点突变体热稳定性的叠加效果，其半衰期大约是野生型脱氨氢酶的5倍。基于此，本发明所提供的胺脱氢酶突变体的热稳定性更好，适于在较高的温度下催化合成手性胺。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
序列表
<110> 中国科学院苏州生物医学工程技术研究所
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accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
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ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
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accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
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35 40 45
Leu Asp Leu Gly Leu Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ser Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 7
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 7
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ser Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Thr Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 8
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 8
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Leu Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Thr Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 9
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 9
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Leu Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ser Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 10
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 10
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Ala Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Thr Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 11
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 11
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Ala Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ser Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 12
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 12
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Ala Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Leu Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 13
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 13
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Thr Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 14
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 14
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ser Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 15
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 15
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Leu Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 16
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 16
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Ala Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 17
<211> 350
<212> PRT
<213> 人工合成(artificial sequrence)
<400> 17
Met Glu Lys Ile Arg Val Ile Ile Trp Gly Leu Gly Ala Met Gly Gly
1 5 10 15
Gly Met Ala Arg Met Ile Leu Gln Lys Lys Gly Met Glu Ile Val Gly
20 25 30
Ala Ile Asp Ser Arg Pro Glu Lys Ser Gly Lys Asp Leu Gly Glu Val
35 40 45
Leu Asp Leu Gly Arg Lys Thr Gly Val Thr Ile Ser Cys Asp Pro Glu
50 55 60
Thr Val Leu Lys Gln Pro Ala Asp Ile Val Leu Leu Ala Thr Ser Ser
65 70 75 80
Phe Thr Arg Glu Val Tyr Pro Gln Leu Gln Arg Ile Ile Ala Ser Gly
85 90 95
Lys Asn Val Ile Thr Ile Ala Glu Glu Met Ala Tyr Pro Ala Tyr Arg
100 105 110
Glu Pro Glu Leu Ala Ala Lys Ile Asp Lys Met Ala Lys Asp His Gly
115 120 125
Val Thr Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Thr
130 135 140
Leu Ile Ile Ala Leu Ser Gly Val Cys Met Asp Ile Lys Lys Ile Thr
145 150 155 160
Ala Arg Arg Ile Asn Asp Leu Ser Pro Phe Gly Thr Thr Val Met Arg
165 170 175
Thr Gln Gly Val Gly Thr Thr Val Asp Glu Phe Arg Lys Gly Leu Glu
180 185 190
Glu Gly Thr Ile Val Gly His Ile Gly Phe Pro Glu Ser Ile Ser Leu
195 200 205
Ile Ala Glu Ala Leu Gly Leu Glu Ile Asp Glu Ile Arg Glu Met Arg
210 215 220
Glu Pro Ile Val Ser Asn Val Tyr Arg Glu Thr Pro Tyr Ala Arg Val
225 230 235 240
Glu Pro Gly Met Val Ala Gly Cys Lys His Thr Gly Ile Gly Tyr Arg
245 250 255
Lys Gly Glu Pro Val Ile Val Leu Glu His Pro Gln Gln Ile Arg Pro
260 265 270
Glu Leu Glu Asp Val Glu Thr Gly Asp Tyr Ile Glu Ile Glu Gly Thr
275 280 285
Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Ile Gly
290 295 300
Thr Ile Ala Ile Ala Val Asn Met Ile Pro Lys Val Ile Ser Ala Asn
305 310 315 320
Pro Gly Leu Val Thr Met Lys Asp Leu Pro Val Pro Ala Ala Leu Met
325 330 335
Gly Asp Ile Arg Lys Leu Ala Lys Asp Gly Val Asn Asn Ala
340 345 350
<210> 18
<211> 33
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 18
actgctcata tggaaaaaat ccgtgttatc atc 33
<210> 19
<211> 31
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 19
tcagctctcg agttaagcgt tgttaacacc g 31
<210> 20
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 20
gaaatcgttg gtgctatcga ctctcgtccg gaaaaatct 39
<210> 21
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 21
agatttttcc ggacgagagt cgatagcacc aacgatttc 39
<210> 22
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 22
gaagttctgg acctgggtcg taaaaccggt gttaccatc 39
<210> 23
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 23
gatggtaaca ccggttttac gacccaggtc cagaacttc 39
<210> 24
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 24
gaatctatct ctctgatcgc tgaagctctg ggtctggaa 39
<210> 25
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 25
ttccagaccc agagcttcag cgatcagaga gatagattc 39
<210> 26
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 26
aaagttatct ctgctaaccc tggtctggtt accatgaaa 39
<210> 27
<211> 39
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 27
tttcatggta accagaccag ggttagcaga gataacttt 39
<210> 28
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 28
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 29
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 29
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 30
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 30
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 31
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 31
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 32
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 32
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 33
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 33
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 34
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 34
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 35
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 35
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 36
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 36
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 37
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 37
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 38
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 38
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
accggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 39
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 39
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatctct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 40
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 40
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtctga aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 41
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 41
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgcttctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
<210> 42
<211> 1053
<212> DNA
<213> 人工合成(artificial sequrence)
<400> 42
atggaaaaaa tccgtgttat catctggggt ctgggtgcta tgggtggtgg tatggctcgt 60
atgatcctgc agaaaaaagg tatggaaatc gttggtgcta tcgattctcg tccggaaaaa 120
tctggtaaag acctgggtga agttctggac ctgggtcgta aaaccggtgt taccatctct 180
tgcgacccgg aaaccgttct gaaacagccg gctgacatcg ttctgctggc tacctcttct 240
ttcacccgtg aagtttaccc gcagctgcag cgtatcatcg cttctggtaa aaacgttatc 300
accatcgctg aagaaatggc ttacccggct taccgtgaac cggaactggc tgctaaaatc 360
gacaaaatgg ctaaagacca cggtgttacc gttctgggta ccggtatcaa cccgggtttc 420
gttctggaca ccctgatcat cgctctgtct ggtgtttgca tggacatcaa aaaaatcacc 480
gctcgtcgta tcaacgacct gtctccgttc ggtaccaccg ttatgcgtac ccagggtgtt 540
ggtaccaccg ttgacgaatt ccgtaaaggt ctggaagaag gtaccatcgt tggtcacatc 600
ggtttcccgg aatctatctc tctgatcgct gaagctctgg gtctggaaat cgacgaaatc 660
cgtgaaatgc gtgaaccgat cgtttctaac gtttaccgtg aaaccccgta cgctcgtgtt 720
gaaccgggta tggttgctgg ttgcaaacac accggtatcg gttaccgtaa aggtgaaccg 780
gttatcgttc tggaacaccc gcagcagatc cgtccggaac tggaagacgt tgaaaccggt 840
gactacatcg aaatcgaagg taccccgaac atcaaactgt ctatcaaacc ggaaatcccg 900
ggtggtatcg gtaccatcgc tatcgctgtt aacatgatcc cgaaagttat ctctgctaac 960
cctggtctgg ttaccatgaa agacctgccg gttccggctg ctctgatggg tgacatccgt 1020
aaactggcta aagacggtgt taacaacgct taa 1053
Claims (9)
1.一种热稳定性提高的胺脱氢酶突变体，其特征在于，
所述胺脱氢酶突变体是在SEQ ID NO.2所示的氨基酸序列上进行突变，所述突变位点选自A35D、L53R、S210A、T321P中一种或一种以上组合。
2.根据权利要求1所述的热稳定性提高的胺脱氢酶突变体，其特征在于，所述突变位点为A35D、L53R、S210A、T321P、A35D/L53R、A35D/S210A、A35D/T321P、L53R/S210A、L53R/T321P、S210A/T321P、A35D/L53R/S210A、A35D/L53R/T321P、A35D/S210A/T321P、L53R/S210A/T321P或A35D/L53R/S210A/T321P。
3.一种编码如权利要求1或2所述的热稳定性提高的胺脱氢酶突变体的基因。
4.一种包含如权利要求3所述的基因的重组质粒。
5.一种包含如权利要求1或2所述的热稳定性提高的胺脱氢酶突变体的可溶性蛋白、固定化酶或工程菌。
6.一种如权利要求1或2所述的热稳定性提高的胺脱氢酶突变体的构建方法，其特征在于，包括以下步骤：
通过在Pfam数据库及NCBI数据库中搜索SEQ ID NO.2所示的氨基酸序列，去除重复出现的相同序列，选取与SEQ ID NO.2所示的氨基酸序列一致性大于30%的氨基酸序列，然后通过Clustalx1.83软件进行多序列比对，将剩余氨基酸质序列整理成fasta.文件上传到Consensus Maker v2.0.0服务器，根据需要修改设置参数后，该在线软件将生成可以后期编辑的consensus sequence；
通过swissmodel在线工具对获得的SEQ ID NO.2所示的蛋白质三维结构预测，采用PyMOL观测SEQ ID NO.2所示的蛋白质晶体结构，筛选出热稳定性相关的突变位点为：A35D、L53R、S210A、T321P。
7.根据权利要求6所述的热稳定性提高的胺脱氢酶突变体的构建方法，其特征在于，
突变位点A35D的扩增引物序列为SEQ ID NO.20、SEQ ID NO.21；
突变位点L53R的扩增引物序列为SEQ ID NO.22、SEQ ID NO.23；
突变位点S210A的扩增引物序列为SEQ ID NO.24、SEQ ID NO.25；
突变位点T321P的扩增引物序列为SEQ ID NO.26、SEQ ID NO.27。
8.根据权利要求7所述的热稳定性提高的胺脱氢酶突变体的构建方法，其特征在于，
A35D对应的单点突变体的氨基酸序列为SEQ ID NO.3；
L53R对应的单点突变体的氨基酸序列为SEQ ID NO.4；
S210A对应的单点突变体的氨基酸序列为SEQ ID NO.5；
T321P对应的单点突变体的氨基酸序列为SEQ ID NO.6；
A35D/L53R对应的组合突变体的氨基酸序列为SEQ ID NO.7；
A35D/S210A对应的组合突变体的氨基酸序列为SEQ ID NO.8；
A35D/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.9；
L53R/S210A对应的组合突变体的氨基酸序列为SEQ ID NO.10；
L53R/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.11；
S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.12；
A35D/L53R/S210A对应的组合突变体的氨基酸序列为SEQ ID NO.13；
A35D/L53R/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.14；
A35D/S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.15；
L53R/S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.16；
A35D/L53R/S210A/T321P对应的组合突变体的氨基酸序列为SEQ ID NO.17。
9.如权利要求1或2所述的热稳定性提高的胺脱氢酶突变体在催化合成手性胺中的应用。
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