CN112779233A - 重组草铵膦脱氢酶、基因工程菌及其在制备l-草铵膦中的应用 - Google Patents
重组草铵膦脱氢酶、基因工程菌及其在制备l-草铵膦中的应用 Download PDFInfo
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- CN112779233A CN112779233A CN202110086335.XA CN202110086335A CN112779233A CN 112779233 A CN112779233 A CN 112779233A CN 202110086335 A CN202110086335 A CN 202110086335A CN 112779233 A CN112779233 A CN 112779233A
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Abstract
本发明公开了重组草铵膦脱氢酶、基因工程菌及其在制备L‑草铵膦中的应用,该重组草铵膦脱氢酶的氨基酸序列如SEQ ID NO.2所示。本发明通过交错延伸pcr基因重排技术,构建获得重组草铵膦脱氢酶的基因文库,并从中筛选获得酶活、催化性能和立体选择性均高的重组草铵膦脱氢酶;该重组草铵膦脱氢酶的活性较之前突变体PPTDHE3‑A164G和突变体PPTDHE0‑V375S分别提高了31%和35%,最终完全催化108g/L的2‑羰基‑4‑(羟基甲基氧膦基)‑丁酸生产L‑草铵膦仅仅需要20分钟(转氨酶通常需要40小时),且ee值大于99.5%。
Description
技术领域
本发明涉及生物化工技术领域,尤其涉及重组草铵膦脱氢酶、基因工程菌及其在制备L-草铵膦中的应用。
背景技术
草铵膦(glufosinate-ammonium)的化学名称为4-[羟基(甲基)膦酰基]-DL-高丙氨酸,是世界第二大转基因作物耐受除草剂,由赫斯特公司(几经合并后现归属拜耳公司)开发生产,又称草铵膦铵盐、Basta、Buster等,属膦酸类除草剂,非选择性(灭生性)触杀型除草剂是谷氨酰胺合成酶抑制剂。
草铵膦有两种光学异构体,分别为L-草铵膦和D-草铵膦。但只有L-型具有生理活性,且在土壤中易分解,对人类和动物的毒性较小,除草谱广,对环境的破坏力小。
目前,市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用,可显著降低草铵膦的使用量,这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
手性纯L-草铵膦的主要制备方法主要由三种:手性拆分法,化学合成法和生物催化法。生物催化法生产草铵膦则具有立体选择性严格、反应条件温和、收率高等优点,是生产L-草铵膦的优势方法。主要包括以下三类:
1)以L-草铵膦的衍生物为底物,通过酶法直接水解获得,主要的优点转化率高,产物ee值较高,但需要昂贵且不易获得的手性原料作为前体,成本加高,不利于工业化生产。例如生物法制备L-草铵膦最简单的方法就是利用蛋白酶直接水解双丙氨膦。双丙氨膦是一种天然的三肽化合物,在蛋白酶的催化下,双丙氨膦脱去2分子L-丙氨酸,生成L-草铵膦。
2)以外消旋草铵膦的前提为底物,通过酶的选择性拆分获得。主要优点为原料相对易得,催化剂活力高,但其理论收率只能达到50%,会造成原料的浪费。例如一种采用α-胰凝乳蛋白酶拆分双丙氨膦乙酯制备L-草铵膦的方法,该法首先经过3步反应将外消旋的草铵膦合成双丙氨膦二乙酯,接着用碱性mesinterico肽酶将其C端脂基水解。再经α-胰凝乳蛋白酶催化水解肽键。在该步反应中,α-胰凝乳蛋白酶能选择性水解L-双丙氨膦乙酯,生成L-草铵膦乙脂。最后利用磷酸二脂酶水解P端酯基得到L-草铵膦。
3)以α-酮酸-2-羰基-4-(羟基甲基膦酰基)丁酸为底物,通过酶的不对称合成获得,主要涉及的酶包括转氨酶与草铵膦脱氢酶。早在研究草铵膦在土壤微生物体内的代谢途径时就已经发现,L-草铵膦在转氨酶的作用下,发生转氨作用被分解成一种α-酮酸-2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)。SchμLz A等(Stereospeciric production orthe herbicide phosphinothricin(glurosinate)by transamination:isolation andcharacterization or a phosphinothricin-speciric transaminase from Escherichiacoli[J].Applied&Environmental Microbiology,1990,56(1):1-6.)在上世纪90年代就利用从大肠杆菌中克隆的转氨酶,以2-羰基-4-(羟基甲基膦酰基)丁酸为底物,L-谷氨酸作为氨基供体,催化转氨反应生产L-草铵膦。该在转氨酶经固定化并安装至生物反应器,催化制备L-草铵膦,其产物浓度可达76.1g/L,最高产率为50g/(L·h),L-草铵膦的ee值超过99.9%。但利用转氨酶制备L-草铵膦有两大缺陷,其一是原料PPO不能完全转化为L-PPT,转化率最高只有90%;其二是要使可逆反应向生成L-PPT的方向进行,需要4倍当量以上的L-谷氨酸作为氨基供体,过量的谷氨酸给L-草铵膦的分离带来了很大的麻烦。
在诸多草铵膦的酶法合成路线中,酮酸中间体的酮羰基是潜手性官能团,能够通过酶法合成途径构建手性中心,酮酸路线也因原料价廉易得且可以避免使用剧毒氰化物,而成为适宜L-草铵膦工业化开发生产的路线。
草铵膦脱氢酶(EC 1.4.1.-,AADH)是一类能将氨基酸可逆的脱氨生成对应酮酸的氨基酸脱氢酶,其反应需要核苷类辅酶的参与(NAD(P)+)。根据其底物特异性,可分为谷氨酸脱氢酶、亮氨酸脱氢酶、丙氨酸脱氢酶、缬氨酸脱氢酶等。因其表现出的优良催化效率与选择性,草铵膦脱氢酶被广泛的应用于天然与非天然α-氨基酸的合成。
发明内容
本发明提供了一种重组草铵膦脱氢酶和包含该重组草铵膦脱氢酶的基因工程菌及其在制备L-草铵膦中的应用,该重组草铵膦脱氢酶不仅活性高,而且催化制备的L-草铵膦转化率高、收率高、产物易于分离提纯且显著缩短了反应进程。
具体技术方案如下:
本发明提供了一种重组草铵膦脱氢酶,所述重组草铵膦脱氢酶的氨基酸序列如SEQ ID NO.2所示。
本发明利用分别来源于假单胞菌(Pseudomonas)、适冷假单胞菌(Pseudomonasextremaustralis)、莫尔氏假单胞菌(Pseudomonas moorei)和沙门氏假单胞菌(Pseudomonas saudiphocaensis)的野生型草铵膦脱氢酶(NCBI登录号为依次为WP_092488511.1、WP_010562566.1、WP_090325311.1或WP_037025837.1),以及草铵膦脱氢酶突变体PPTDHE3-A164G(专利申请号CN201910813762.6)和PPTDHE0-V375S(专利申请号CN201910751249.9),进行交错延伸pcr基因重排,构建获得重组草铵膦脱氢酶的基因文库,并从中筛选获得酶活、催化性能和立体选择性均高的重组草铵膦脱氢酶。
本发明提供了所述的重组草铵膦脱氢酶的编码基因。
作为优选,所述编码基因的核苷酸序列如SEQ ID NO.1所示。
本发明提供的重组草铵膦脱氢酶可用于制备L-草铵膦。
本发明还提供了一种基因工程菌,包括宿主细胞和转入宿主细胞的目的基因,所述目的基因包含如权利要求1所述的重组草铵膦脱氢酶的编码基因。
作为优选,所述目的基因还包含葡萄糖脱氢酶的编码基因或甲酸脱氢酶的编码基因。
本发明还提供了上述基因工程菌在制备L-草铵膦中的应用。
具体地,本发明提供了一种L-草铵膦的制备方法,为以下两种中的任意一种:
(1)以2-羰基-4-(羟基甲基氧膦基)-丁酸或其盐为底物,在无机氨基供体和辅酶循环系统存在的条件下,利用催化剂催化底物发生还原胺化反应,制得L-草铵膦;
(2)以D-草铵膦为底物,在无机氨基供体、D-氨基酸氧化酶和和辅酶循环系统存在的条件下,利用催化剂催化底物反应获得L-草铵膦;
在(1)和(2)中,所述辅酶循环系统为葡萄糖脱氢酶循环系统或甲酸脱氢酶循环系统;所述催化剂为如权利要求1所述的重组草铵膦脱氢酶或其固定化酶,或如权利要求5或6任一项所述的基因工程菌。
进一步地,所述无机氨基供体为硫酸铵或甲酸铵。
与现有技术相比,本发明具有以下有益效果:
(1)本发明通过交错延伸pcr基因重排技术,构建获得重组草铵膦脱氢酶的基因文库,并从中筛选获得酶活、催化性能和立体选择性均高的重组草铵膦脱氢酶;该重组草铵膦脱氢酶的活性较之前突变体PPTDHE3-A164G和突变体PPTDHE0-V375S分别提高了31%和35%,最终完全催化108g/L的2-羰基-4-(羟基甲基氧膦基)-丁酸生产L-草铵膦仅仅需要20分钟(转氨酶通常需要40小时),且ee值大于99.5%。
(2)本发明提供的L-草铵膦制备方法原料转化率高、收率高、产物易于分离提纯。
(3)本发明方提供的L-草铵膦制备方法与转氨酶等催化工艺相比,工艺相对简单,原料转化率高,转化率可达100%,且获得的产物易于从反应液中分离提纯。
附图说明
图1为实施例1筛选获得的重组草铵膦脱氢酶的结构示意图。
图2为实施例1利用草铵膦脱氢酶W1与辅酶再生酶双酶耦联催化2-羰基-4-(羟基甲基氧膦基)-丁酸不对称胺化还原制备L-草铵膦的反应示意图。
图3为一锅法制备L-草铵膦的反应示意图。
图4为实施例1第5部分E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH的反应进程图。
图5为实施例1第5部分E.coli BL21(DE3)/pETDuet-1-PPTDHW1-LbFDH的反应进程图。
图6为实施例1第6部分E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH一锅法制备L-草铵膦的反应进程图。
图7为实施例1第6部分E.coli BL21(DE3)/pETDuet-1-PPTDHW1-LbFDH一锅法制备L-草铵膦的反应进程图。
具体实施方式
下面结合具体实施例对本发明作进一步描述,以下列举的仅是本发明的具体实施例,但本发明的保护范围不仅限于此。
本发明中的实验方法如无特别说明均为常规方法,基因克隆操作具体可参见J.萨姆布鲁克等编的《分子克隆实验指南》。
上游基因工程操作所用试剂:本发明实施例中使用的一步克隆试剂盒均购自Vazyme,南京诺唯赞生物科技有限公司;质粒提取试剂盒、DNA回收纯化试剂盒购自Axygen杭州有限公司;E.coli BL21(DE3)、质粒等购自上海生工;DNA marker、FastPfu DNA聚合酶、低分子量标准蛋白、琼脂糖电泳试剂、引物合成与基因测序工作由杭州擎科梓熙生物技术有限公司完成。以上试剂使用方法参考商品说明书。
下游催化工艺所用试剂:2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO),D,L-草铵膦、L-草铵膦标准品购自Sigma-Aldrich公司;NADPH购于邦泰生物工程(深圳)有限公司;其他常用试剂购自国药集团化学试剂有限公司。
下列实施例中高效液相色谱的检测方法如下:
高效液相色谱(HPLC)检测底物浓度,分析方法为:色谱柱型号:QS-C18,5μm,4.6×250mm。流动相:将50mM磷酸二氢胺溶于800mL超纯水中,加入10mL四丁基氢氧化铵(10%)用水稀释并定容至1000mL,用磷酸调pH到3.8,与乙腈以体积比88:12混合。检测波长为232nm,流速:1.0mL/min。柱温:40℃,2-羰基-4-(羟基甲基氧膦基)-丁酸出峰时间为:9.7分钟。
产物的手性分析及浓度分析通过柱前衍生化高效液相色谱进行,具体的分析方法为:
(1)色谱条件:色谱柱型号:QS-C18,5μm,4.6×250mm。流动相:50mM乙酸铵溶液:甲醇=10:1。荧光检测波长:λex=340nm,λem=455nm。流速:1mL/min。柱温:30℃,L-草铵膦出峰时间为8.5min,D-草铵膦出峰时间为10.2min。
(2)衍生化试剂:分别称取0.1g邻苯二甲醛与0.12g N-乙酰-L-半胱氨酸,用10mL乙醇助溶,再加入40mL 0.1moL/L硼酸缓冲液(pH 9.8),振荡使其充分溶解,4℃冰箱保存备用(不超过4天)。
(3)衍生化反应与测定:取100μL样品加入400μL衍生化试剂,在振荡器上500rpm,30℃震荡5min,再加入400μL超纯水混匀,进样10μL进行HPLC分析。
实施例1
1、重组草铵膦脱氢酶基因文库的构建
重组草铵膦脱氢酶的构建主要采用交错延伸pcr的方式:设计引物1、2,同时在引物设计中引入Sac I和NotI酶切位点,对来源于假单胞菌(Pseudomonas)、适冷假单胞菌(Pseudomonas extremaustralis)、莫尔氏假单胞菌(Pseudomonas moorei)和沙门氏假单胞菌(Pseudomonas saudiphocaensis)的野生型草铵膦脱氢酶PPTDHS1、PPTDHS2、PPTDHS3、PPTDHS4(NCBI登录号为依次为WP_092488511.1、WP_010562566.1、WP_090325311.1或WP_037025837.1),以及草铵膦脱氢酶突变体PPTDHE3-A164G(专利申请号201910813762.6)和PPTDHE0-V375S(专利申请号201910751249.9),共6种草铵膦脱氢酶的目的基因,通过交错延伸pcr,进行基因重组,获得带有Sac I和NotI酶切位点的重组草铵膦脱氢酶基因文库。
引物1:5’-GAGCTCATGATCGAATCTGTCGAAAGT-3’;
引物2:5’-ACTTTCGACAGATTCGATCATGAGCTC-3’。
2、表达载体文库及工程菌株文库的构建
构建表达载体文库:利用Sac I和NotI限制性内切酶(TaKaRa)对扩增片段和载体pETDuet-1进行处理,并利用T4 DNA连接酶(TaKaRa)将重组草铵膦脱氢酶基因与载体pETDuet-1进行连接,获得带有重组草铵膦脱氢酶的表达载体pETDuet-1-PPTDH文库。
构建工程菌文库:将储藏于-80℃的大肠杆菌BL21(DE3)(Invitrogen)感受态细胞在0℃冰浴10min,然后在超净台内分别加入5μL的带有重组草铵膦脱氢酶的表达载体pETDuet-1-PPTDH,0℃冰浴30min,42℃水浴中热击90s,0℃冰浴2min,加入600μL的LB培养基,在37℃、200rpm摇床培养1h;涂布于含有50μg/ml氨苄霉素抗性的LB平板,37℃下培养8-12h,获得含有表达重组质粒的重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDH工程菌文库。
感受态细胞的制备方法为:从-80℃冰箱中获得甘油管保藏的E.coli BL21(DE3)菌株,在无抗LB平板上划线,37℃培养10h,获取单菌落;挑取LB平板的单菌落,接种至含5mL的LB培养基的试管中,37℃、180rpm培养9h;从试管中取200μL菌液,接种到50mL的LB培养基中,37℃、180rpm培养OD600至0.4-0.6;将菌液在冰上预冷,取菌液至灭菌的离心管中,冰上放置10min,4℃、5000rpm离心10min;将上清液倒出,注意防止染菌,用预冷的0.1mol/L的CaCl2水溶液重悬沉淀细胞,并在冰上放置30min;4℃、5000rpm离心10min,弃上清,用预冷的含15%甘油的0.1mol/L的CaCl2水溶液重悬沉淀细胞,取100μL重悬细胞分装至灭菌的1.5mL离心管中,保藏于-80℃冰箱,需要时取出。
3、重组草铵膦脱氢酶菌株的高通量筛选
一)高通量筛选方法的建立
配制50mL工作液:邻苯二甲醛0.013g,N乙酰-L-半胱氨酸0.032g,用pH=9.8硼酸缓冲液溶解定容到50mL,作为高通量工作液。吸取50μL样品反应液加入50μL工作液震荡反应30s,再加入100μLddH2O,在λex=340nm,λem=455nm条件下测定荧光值。
二)高通量筛选
将步骤2获取的获得含有表达重组质粒的重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDH工程菌用灭菌的牙签挑取单菌落到灭好菌的96深孔板中,同时每个96孔板加入3-5个孔的E.coli BL21(DE3)/pETDuet-1-PPTDHE3-A164G、E.coli BL21(DE3)/pETDuet-1-PPTDHE0-V375A作为对照。每孔有1mL含有50μg/mL氨苄抗性的LB培养基,在37℃、200rpm摇床上培养8h后,每孔保留500μL的菌液密封保存好,作为菌种保藏。每孔吸取500μL的菌液,转移到另一每孔有500μL的含有终浓度为50μg/mL氨苄和终浓度为24μg/mLIPTG诱导剂的LB培养基的96深孔板中,然后放在18℃、200rpm的摇床上培养16h后,离心,收取菌体于96孔板底。
1)初筛:配置反应液:终浓度50mM底物PPO(2-羰基-4-(羟基甲基膦酰基)丁酸),100mM的硫酸铵,20mM的NADPH以pH=7.5的磷酸缓冲液为反应介质构成反应液。96深孔板每孔加入500μL的反应液,同时用移液枪反复吹打,将96孔板收集的菌体重悬,然后将96深孔板放入40℃、200rpm的摇床上反应0.5h后,离心取上清,测荧光值,λex=340nm,λem=455nm,筛选荧光值高于E.coli BL21(DE3)/pETDuet-1-PPT DHE3-A164G、E.coli BL21(DE3)/pETDuet-1-PPTDHE0-V375A反应液的菌株,找到相对应的保藏菌种,划线培养,做进一步的保藏。
2)复筛:将初筛获得的菌株进行培养,将收集到的菌体作为催化剂,以2-羰基-4-(羟基甲基氧膦基)-丁酸为底物,外源加入NADPH。反应体系选择为10mL,湿菌体用量为20g/L,底物终浓度300mM,硫酸铵终浓度500mM,NADPH终浓度为50mM,40℃、600转/分钟反应10min取样,取反应液100μL,加5μL盐酸终止反应,以超纯水补足至1mL,即反应液稀释10倍,稀释后的样品先经过衍生化处理,取200μL稀释后的反应液加400μL衍生化试剂30℃衍生化5min,再加400μL超纯水补足至1mL,12000转/分钟离心1分钟,取上清,过0.22μM微滤膜,作为液相样品,HPLC检测2-羰基-4-(羟基甲基氧膦基)-丁酸、L-草铵膦、D-草铵膦及ee值。衍生化试剂:分别称取0.1g邻苯二甲醛与0.12g N-乙酰-L-半胱氨酸,用10mL乙醇助溶,再加入40mL 0.1moL/L硼酸缓冲液(pH 9.8),振荡使其充分溶解,4℃冰箱保存备用(不超过4天)。
以产物L-草铵膦和ee值为指标,筛选含有优势重组草铵膦脱氢酶的菌株,部分复筛实验结果示于表1,筛选到的重组草铵膦脱氢酶菌株E.coli BL21(DE3)/PPTDHW1活力较E.coli BL21(DE3)/PPTDHE3-A164G和E.coli BL21(DE3)/PPTDHE0-V375A都有了明显的提高。
通过测序验证,重组草铵膦脱氢酶W1(如SEQ ID NO.2所示)重组序列为:1-100位氨基酸序列来源于Pseudomonas extremaustralis野生草铵膦脱氢酶S2的第1-100位,101-191位氨基酸序列来源于PPTDHE3-A164G的第101-191位,192-273位氨基酸来源于Pseudomonas saudiphocaensis野生草铵膦脱氢酶S4的第192-273位,274-326位氨基酸来源于Pseudomonas moorei野生草铵膦脱氢酶S3的第274-326位,327-392位氨基酸来源于PPTDHE0-V375A的第327-392位,393-445位氨基酸序列来源于Pseudomonas野生草铵膦脱氢酶S1的第393-445位。
表1重组草铵膦脱氢酶的催化性能和立体选择性
注:PPTDHE3-A164G如SEQ ID NO.3所示,PPTDHE0-V375S如SEQ ID NO.4所示,PPTDHA如SEQ ID NO.5所示,PPTDHB如SEQ ID NO.6所示,PPTDHC如SEQ ID NO.7所示,PPTDHD如SEQ ID NO.8所示,PPTDHE如SEQ ID NO.9所示,PPTDHF如SEQ ID NO.10所示,PPTDHG如SEQ ID NO.11所示,PPTDHL如SEQ ID NO.12所示,PPTDHI如SEQ ID NO.13所示,PPTDHJ如SEQ ID NO.14所示,PPTDHK如SEQ ID NO.15所示。
4、重组草铵膦脱氢酶W1-葡萄糖/甲酸脱氢酶辅酶循环系统的构建
为了进一步降低工业化生产成本,构建葡萄糖-葡萄糖脱氢酶、甲酸-甲酸铵辅酶循环系统,采用一步克隆法将葡萄糖脱氢酶(NCBI登录号:KM817194.1)、甲酸脱氢酶(NCBI登录号:WP_013726924.1)分别克隆到表达载体pETDuet-1的第二个多克隆位点上。
带有同源序列的葡糖糖脱氢酶、甲酸脱氢酶基因的获得:将表达载体pETDuet-1的NdeI和PacI酶切位点前面和后面的各20bp左右序列作为同源序列,以E.coli BL21(DE3)/pET28b-ESGDH、E.coli BL21(DE3)/pET28b-LbFDH(根据NCBI登录号,以全基因组为模板,构建菌株)为模板,设计引物1、引物2引物3,引物4,并将同源臂分别添加到基因特异性正/反向扩增引物序列的5’端,以此对带有同源臂的葡萄糖脱氢酶基因、甲酸脱氢酶基因基因利用高保真Pfu DNA聚合酶进行扩增,将消化模板后的PCR产物用DNA回收纯化试剂盒进行纯化回收,并分别测出核酸浓度,得到带有同源序列的葡萄糖脱氢酶、甲酸脱氢酶基因序列。
带有重组草铵膦脱氢酶W1的线性化载体基因的获得:以pETDuet-1-PPTDHW1为模板,NdeI和PacI酶切位点前面和后面各20bp左右序列作为引物,设计引物5、引物6,利用高保真Pfu DNA聚合酶进行扩增,将消化模板后的PCR产物用DNA回收纯化试剂盒进行纯化回收,并分别测出核酸浓度,得到带有重组草铵膦脱氢酶W1的线性化载体。
引物1:5’-TATAAGAAGGAGATATACATATGGGTTATAATTCTCTGAAA-3’;
引物2:5’-GTGGCAGCAGCCTAGGTTAATCAACCACGGCCAGCCTGA-3’;
引物3:5’-TATAAGAAGGAGATATACATATGACCAAAGTTCTGGCCGTG-3’;
引物4:5’-GTGGCAGCAGCCTAGGTTAATTATTTTTCTGCTTCGCCGCTA-3’;
引物5:5’-TTAACCTAGGCTGCTGCCACCGC-3’;
引物6:5’-ATGTATATCTCCTTCTTATACTTA-3’。
单片段同源重组反应:最适克隆载体使用量={0.02*克隆载体碱基对数}ng(0.03pmol);最适插入片段使用量={0.04*插入片段碱基对数}ng(0.06pmol)
反应体系(表2):
表2一步克隆反应体系
注:X表示加入线性化载体的量,Y表示插入片段的量。
将配置好的反应体系使用移液器轻轻吸打混匀,短暂离心后将反应液收集到管底。反应体系放在50℃的水浴中,静置5min,然后立即放在冰上冷却。将3种不同体系分别转化至大肠杆菌BL21(DE3)中(42℃,90s),涂布于含有50μg/mL氨苄霉素抗性的LB平板,37℃下培养12-16h,随机挑取克隆抽提质粒进行测序鉴定,分别筛选获得含有新型草铵膦脱氢酶W1和葡萄糖脱氢酶的重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH,以及含有新型草铵膦脱氢酶W1和甲酸脱氢酶基因的重组大肠杆菌E.coli BL21(DE3)/pETDuet-1-PPTDHW1-LbFDH。
5、不对称催化合成L-草铵膦
(1)草铵膦脱氢酶W1-葡萄糖/甲酸脱氢酶重组工程菌E.coliBL21(DE3)/pETDuet-1-PPTDHW1-EsGDH/LbFDH不对称胺化还原2-羰基-4-(羟基甲基氧膦基)-丁酸。
通过发酵获得重组草铵膦脱氢酶W1-葡萄糖脱氢酶菌体E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH 0.75g,加入终浓度600mM的2-羰基-4-(羟基甲基氧膦基)-丁酸,终浓度700mM的葡萄糖,终浓度800mM的(NH4)2SO4氨基供体构成反应体系30ml,在40℃、磁力搅拌转速为600rpm下进行反应,流加氨水使反应液pH维持在7.5。通过液相方法检测反应过程中产物L-草铵膦的生成和ee值的变化,反应进程曲线如图4所示。
结果如表3所示,产物浓度随时间的推移而逐渐升高,20分钟内反应完成,底物转化率大于99%,产物ee值始终保持在99.5%以上。
通过发酵获得重组草铵膦脱氢酶W1-甲酸脱氢酶重组工程菌湿菌体E.coli BL21(DE3)/pETDuet-1-PPTDHW1-A164G-LbFDH 0.75g,加入终浓度400mM的2-羰基-4-(羟基甲基氧膦基)-丁酸,终浓度800mM的甲酸铵,在45℃、磁力搅拌转速为600rpm下进行反应。通过液相方法检测反应过程中产物L-草铵膦的生成和ee值的变化,反应进程曲线如图5所示。
结果如表3所示,产物浓度随时间的推移而逐渐升高,4h内反应完成,底物转化率大于99%,产物ee值始终保持在99.5%以上。
表3E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH/LbFDH不对称胺化还原PPO反应条件与结果
(2)D-氨基酸氧化酶工程菌、草铵膦脱氢酶W1-葡萄糖/甲酸脱氢酶重组工程菌E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH/LbFDH以D-草铵膦为底物,采用一锅法制备L-草铵膦
通过发酵获得D-氨基酸氧化酶湿菌体1.5g、草铵膦脱氢酶W1-葡萄糖脱氢酶重组工程菌E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH 0.75g,过氧化氢酶(3000units/mg)0.3g,加入终浓度300mM的D-草铵膦,终浓度400mM的葡萄糖,终浓度200mM的硫酸铵构成反应体系30ml,在40℃、磁力搅拌转速为600rpm下进行反应,流加氨水使反应液pH维持在7.5。通过液相方法检测反应过程中产物L-草铵膦的生成和ee值的变化,反应进程曲线如图6所示。
结果如表4所示,产物浓度随时间的推移而逐渐升高,8h内反应完成,底物转化率大于99%,产物ee值始终保持在99.5%以上。
通过发酵获得D-氨基酸氧化酶湿菌体1.5g、草铵膦脱氢酶突变体-甲酸脱氢酶重组工程菌湿菌体E.coli BL21(DE3)/pETDuet-1-PPTDHW1-LbFDH 0.75g,过氧化氢酶0.3g,加入终浓度300mM的D-草铵膦,终浓度500mM的甲酸铵构成反应体系30ml,在40℃、磁力搅拌转速为600rpm下进行反应。通过液相方法检测反应过程中产物L-草铵膦的生成和ee值的变化,反应进程曲线如图7所示。
结果如表4所示,产物浓度随时间的推移而逐渐升高,10h内反应完成,底物转化率大于99%,产物ee值始终保持在99.5%以上。
表4E.coli BL21(DE3)/pETDuet-1-PPTDHW1-EsGDH/LbFDH一锅法制备L-PPT的反应条件与结果
序列表
<110> 浙江工业大学
<120> 重组草铵膦脱氢酶、基因工程菌及其在制备L-草铵膦中的应用
<160> 23
<170> SIPOSequenceListing 1.0
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cctcactatt taacatcggg tatcctggaa cgtatctgtg agccagagcg cgctatcatt 180
ttccgcgtca gttgggttga tgaccacggg aaggtgcaag tgaaccgcgg tttccgtatc 240
caaatgaact cggctatcgg tccttacaaa ggaggcttgc gctttcatcc ctcagtaaac 300
ttgggtgtct taaaattctt agcgttcgag caaacattta aaaacagctt aacatcgtta 360
cccatgggtg gaggaaaggg tggtagtgac ttcgacccaa aggggaagag cgatgcggaa 420
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gatgtgccag gtggagatat tggcgtgggt gcgcgcgaga ttggattttt attcggtcag 540
tataagcgtc tgtctaacca gttcacctcg gtattgaccg ggaaaggcct tgcatacgga 600
ggaagcctta ttcgtcctga ggctacgggg tacggctgcg tgtattttgc gcaagaaatg 660
ttgaagagta cacgcagttc cttcgagggg aagcgcgttt caatttcggg tagcggaaat 720
gtcgcccaat atgctgcgca gaaggtcatc gaattaggag gcctggtagt tagcgtgagc 780
gattccggtg gtacattaca cttccccgat ggcatgaccg aagctcaatg gcaagcagtg 840
ttggaactga agaatgtaca acgtggccgt atttcagaat tagccggacg ctttggtctt 900
gaatttttag cgggccaacg cccctggggt ttatcttgcg atatcgccct tccttgcgcg 960
acgcagaacg agcttgacat cgaagatgcg cgcacccttc ttcgcaatgg ttgtatttgc 1020
gtggctgagg gagctaatat gccgacgacg ttggccgcgg tggacctgtt catcgacgct 1080
ggtatccttt atgcaccggg aaaggcaagt aacgctggcg gttctgccgt ttcggggtta 1140
gagatgagtc aaaacgcgat gcgtcttctt tggacagcag gtgaggtcga ctctaagtta 1200
cacaatatca tgcaaagtat ccatcacgca tgtgtacact acggggaaga gaatggccgc 1260
atcaactacg tgaaaggcgc gaatatcgcg ggattcgtaa aggtcgcaga cgctatgctt 1320
gcccagggaa tcgtgtaa 1338
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Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
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Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
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Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
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Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
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Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
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Asp Val Pro Gly Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
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Thr Gly Lys Gly Leu Ala Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
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Thr Gly Tyr Gly Cys Val Tyr Phe Ala Gln Glu Met Leu Lys Ser Thr
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Arg Ser Ser Phe Glu Gly Lys Arg Val Ser Ile Ser Gly Ser Gly Asn
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Val Ala Gln Tyr Ala Ala Gln Lys Val Ile Glu Leu Gly Gly Leu Val
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Val Ser Val Ser Asp Ser Gly Gly Thr Leu His Phe Pro Asp Gly Met
260 265 270
Thr Glu Ala Gln Trp Gln Ala Val Leu Glu Leu Lys Asn Val Gln Arg
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Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Leu Ala
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Gly Gln Arg Pro Trp Gly Leu Ser Cys Asp Ile Ala Leu Pro Cys Ala
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Thr Gln Asn Glu Leu Asp Ile Glu Asp Ala Arg Thr Leu Leu Arg Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Ala
340 345 350
Ala Val Asp Leu Phe Ile Asp Ala Gly Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Ser Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Asn Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
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Glu Asn Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe
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Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Gly Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Pro Ser Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Arg
210 215 220
Gly Glu Thr Val Glu Gly Lys Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Cys Glu Ser Gly Leu
260 265 270
Thr Glu Ala Gln Trp Gln Ala Val Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Leu Ala
290 295 300
Gly Gln Arg Pro Trp Gly Leu Ser Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Glu Ala Ala Arg Ala Leu Leu Arg Asn
325 330 335
Gly Cys Thr Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Leu Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Gly Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ala Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
405 410 415
Glu Asn Gly Gln Val Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe
420 425 430
Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 4
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ile Glu Ser Val Asp Ser Phe Leu Ala Arg Leu Gln Gln Arg Asp
1 5 10 15
Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Thr
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro Arg Tyr Leu Gln Ser Gly Ile
35 40 45
Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Tyr Arg Ile
65 70 75 80
Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe Glu Gln Val
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Cys
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Gln
210 215 220
Gly Leu Arg Val Asp Gly Arg Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu Gly Gly Leu
260 265 270
Thr Glu Ala Gln Trp Glu Ala Val Met Gln Leu Lys Asn Val Ala Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Glu Ala Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Gly Ile Glu Asp Ala Arg Thr Leu Leu Arg Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Ala
340 345 350
Ala Val Asp Leu Phe Ile Asp Ala Gly Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Ser Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Asn Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
405 410 415
Glu Ala Asp Gly Lys Val Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 5
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Ala Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Thr Phe Arg Val Ser
50 55 60
Trp Val Asp Asp His Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Leu Ala Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Cys Val Tyr Phe Ala Gln Glu Met Leu Lys Ser Thr
210 215 220
Arg Ser Ser Phe Glu Gly Lys Arg Val Ser Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Gln Lys Val Ile Glu Leu Gly Gly Leu Val
245 250 255
Val Ser Val Ser Asp Ser Gly Gly Thr Leu His Phe Pro Asp Gly Met
260 265 270
Thr Glu Glu Gln Trp Glu Tyr Leu Met Asp Leu Lys Asn Val Ala Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Glu Ala Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Gly Ile Glu Asp Ala Arg Thr Leu Leu Arg Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ala Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
405 410 415
Glu Asn Gly Gln Val Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe
420 425 430
Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 6
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Thr Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Val Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Gln
210 215 220
Gly Leu Arg Val Glu Gly Lys Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Cys Glu Ser Gly Leu
260 265 270
Thr Glu Ala Gln Trp Gln Ala Val Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Leu Ala
290 295 300
Gly Gln Arg Pro Trp Gly Leu Ser Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Glu Ala Ala Arg Ala Leu Leu Arg Asn
325 330 335
Gly Cys Thr Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His His Ala Cys Val Ala Tyr Gly Glu
405 410 415
Glu Glu Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 7
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro Arg Tyr Leu Thr Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Val Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Asp
210 215 220
Gly Gln Arg Phe Glu Gly Lys Arg Val Ser Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Gln Lys Val Ile Glu Leu Gly Gly Leu Val
245 250 255
Val Ser Val Ser Asp Ser Gly Gly Thr Leu His Phe Pro Asp Gly Met
260 265 270
Thr Glu Glu Gln Trp Glu Tyr Leu Met Asp Leu Lys Asn Val Arg Arg
275 280 285
Gly Arg Leu Glu Glu Met Gly Ala His Phe Gly Val Thr Tyr Met Pro
290 295 300
Asp Gln Arg Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Gly Asp Asp Ala Arg Thr Leu Leu Lys Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Leu Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Gly Gly Glu Val Asp Ser Ser Lys
385 390 395 400
Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly
405 410 415
Glu Glu Ala Asp Gly Lys Val Asn Tyr Val Lys Gly Ala Asn Ile Ala
420 425 430
Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 8
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Ala Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Thr Phe Arg Val Ser
50 55 60
Trp Val Asp Asp His Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Leu Ala Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Cys Val Tyr Phe Ala Gln Glu Met Leu Lys Ser Thr
210 215 220
Arg Ser Ser Phe Glu Gly Lys Arg Val Ser Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Gln Lys Val Ile Glu Leu Gly Gly Leu Val
245 250 255
Val Ser Val Ser Asp Ser Gly Gly Thr Leu His Phe Pro Asp Gly Met
260 265 270
Thr Glu Glu Gln Trp Glu Tyr Leu Met Asp Leu Lys Asn Val Arg Arg
275 280 285
Gly Arg Leu Glu Glu Met Gly Ala His Phe Gly Val Thr Tyr Met Pro
290 295 300
Asp Gln Arg Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Gly Asp Asp Ala Arg Thr Leu Leu Lys Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Ser Thr Leu Glu
340 345 350
Ala Val Asp Leu Phe Leu Glu Ala Gly Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Cys Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ser Met Arg Leu His Trp Thr Ala Gly Glu Val Asp Thr Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His His Ala Cys Val Ala Tyr Gly Glu
405 410 415
Glu Glu Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 9
<211> 444
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Ala Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Thr Phe Arg Val Ser
50 55 60
Trp Val Asp Asp His Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Pro Ser Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Arg
210 215 220
Gly Glu Thr Val Glu Gly Lys Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu Ser Gly Leu
260 265 270
Thr Glu Ala Gln Trp Ser Ala Leu Leu Glu Leu Lys Asn Val Arg Arg
275 280 285
Gly Arg Leu Glu Glu Met Gly Ala His Phe Gly Val Thr Tyr Met Pro
290 295 300
Asp Gln Arg Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Gly Asp Asp Ala Arg Thr Leu Leu Lys Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Ala
340 345 350
Ala Val Asp Leu Phe Ile Asp Ala Gly Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Ser Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Lys Leu His
385 390 395 400
Ala Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu Glu
405 410 415
Asn Gly Gln Val Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe Val
420 425 430
Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440
<210> 10
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Thr Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Ile Phe Arg Val Ser
50 55 60
Trp Val Asp Asp His Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Asp
210 215 220
Asp Gln Arg Val Glu Gly Lys Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu Ser Gly Leu
260 265 270
Thr Glu Ala Gln Trp Ser Ala Leu Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Tyr Gly Leu Glu Phe Arg Ala
290 295 300
Gly Lys Thr Pro Trp Glu Leu Ala Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Glu Ala Ala Arg Thr Leu Leu Arg Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Asn Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
405 410 415
Glu Asn Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe
420 425 430
Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Ile Val
435 440 445
<210> 11
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Ala Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Thr Phe Arg Val Ser
50 55 60
Trp Val Asp Asp His Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Asp
210 215 220
Gly Gln Arg Val Glu Gly Lys Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu Ser Gly Leu
260 265 270
Thr Glu Ala Gln Trp Ser Ala Leu Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gln Arg Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Lys Thr Pro Trp Glu Leu Ala Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Gln Ala Ala Arg Thr Leu Leu His Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Asn Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
405 410 415
Glu Asn Gly Arg Val Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe
420 425 430
Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Ile Val
435 440 445
<210> 12
<211> 445
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Ala Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Val Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Gly Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Gln
210 215 220
Gly Leu Arg Phe Glu Gly Lys Arg Val Ser Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Gln Lys Val Ile Glu Leu Gly Gly Leu Val
245 250 255
Val Ser Val Ser Asp Ser Gly Gly Thr Leu His Phe Pro Asp Gly Met
260 265 270
Thr Glu Glu Gln Trp Glu Tyr Leu Met Asp Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gln Arg Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Lys Thr Pro Trp Glu Leu Ala Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Gln Ala Ala Arg Thr Leu Leu His Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ala Ile Met Gln Ser Ile His His Ala Cys Val His Tyr Gly Glu
405 410 415
Glu Asn Gly Gln Val Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly Phe
420 425 430
Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 13
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ile Glu Ser Val Asp Ser Phe Leu Ala Arg Leu Gln Gln Arg Asp
1 5 10 15
Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Thr
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro Arg Tyr Leu Gln Ser Gly Ile
35 40 45
Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Val Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Gly Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Asp
210 215 220
Gly Gln Arg Val Glu Gly Lys Arg Val Ala Val Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu Ser Gly Leu
260 265 270
Thr Glu Glu Gln Trp Ser Ala Leu Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gln Arg Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Lys Thr Pro Trp Glu Leu Ala Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Gln Ala Ala Arg Thr Leu Leu His Asn
325 330 335
Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Leu Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Gly Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His His Ala Cys Val Ala Tyr Gly Glu
405 410 415
Glu Glu Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 14
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro Arg Tyr Leu Thr Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Val Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Cys
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Asp
210 215 220
Asp Gln Arg Val Glu Gly Lys Arg Val Ala Val Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu Ser Gly Leu
260 265 270
Thr Glu Glu Gln Trp Ser Ala Leu Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Leu Ala
290 295 300
Gly Gln Arg Pro Trp Gly Leu Ser Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Glu Ala Ala Arg Ala Leu Leu Arg Asn
325 330 335
Gly Cys Thr Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Leu Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Gly Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His His Ala Cys Val Ala Tyr Gly Glu
405 410 415
Glu Glu Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 15
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ile Glu Ser Val Glu Ser Phe Leu Ala Arg Leu Lys Lys Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Ala Asn Pro Arg Tyr Leu Thr Ser Gly Ile
35 40 45
Leu Glu Arg Ile Cys Glu Pro Glu Arg Ala Ile Val Phe Arg Val Ser
50 55 60
Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly Phe Arg Ile
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Leu Gly Val Leu Lys Phe Leu Ala Phe Glu Gln Thr
100 105 110
Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly Ala Asp Val
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu Ile Gly Phe
165 170 175
Leu Phe Gly Gln Tyr Lys Arg Leu Ser Asn Gln Phe Thr Ser Val Leu
180 185 190
Thr Gly Lys Gly Pro Ser Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Phe Gly Cys Val Tyr Phe Ala Glu Glu Met Leu Lys Arg Arg
210 215 220
Gly Glu Thr Val Glu Gly Lys Arg Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly Gly Lys Val
245 250 255
Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Cys Glu Ser Gly Leu
260 265 270
Thr Glu Ala Gln Trp Gln Ala Val Leu Glu Leu Lys Asn Val Gln Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Leu Ala
290 295 300
Gly Gln Arg Pro Trp Gly Leu Ser Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Ala Glu Ala Ala Arg Ala Leu Leu Arg Asn
325 330 335
Gly Cys Thr Cys Val Ala Glu Gly Ala Asn Met Pro Thr Thr Leu Glu
340 345 350
Ala Val Asp Leu Phe Ile Glu Ala Gly Ile Leu Phe Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Met Arg Leu Leu Trp Thr Gly Gly Glu Val Asp Ser Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His His Ala Cys Val Ala Tyr Gly Glu
405 410 415
Glu Glu Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Val
435 440 445
<210> 16
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gagctcatga tcgaatctgt cgaaagt 27
<210> 17
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
actttcgaca gattcgatca tgagctc 27
<210> 18
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
tataagaagg agatatacat atgggttata attctctgaa a 41
<210> 19
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gtggcagcag cctaggttaa tcaaccacgg ccagcctga 39
<210> 20
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tataagaagg agatatacat atgaccaaag ttctggccgt g 41
<210> 21
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
gtggcagcag cctaggttaa ttatttttct gcttcgccgc ta 42
<210> 22
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ttaacctagg ctgctgccac cgc 23
<210> 23
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
atgtatatct ccttcttata ctta 24
Claims (9)
1.一种重组草铵膦脱氢酶,其特征在于,所述重组草铵膦脱氢酶的氨基酸序列如SEQID NO.2所示。
2.如权利要求1所述的重组草铵膦脱氢酶W1的编码基因。
3.如权利要求2所述的编码基因,其特征在于,所述编码基因的核苷酸序列如SEQ IDNO.1所示。
4.如权利要求1所述的重组草铵膦脱氢酶在制备L-草铵膦中的应用。
5.一种基因工程菌,包括宿主细胞和转入宿主细胞的目的基因,其特征在于,所述目的基因包含如权利要求1所述的重组草铵膦脱氢酶的编码基因。
6.如权利要求5所述的基因工程菌,其特征在于,所述目的基因还包含葡萄糖脱氢酶的编码基因或甲酸脱氢酶的编码基因。
7.如权利要求5或6任一项所述的基因工程菌在制备L-草铵膦中的应用。
8.一种L-草铵膦的制备方法,其特征在于,为以下两种中的任意一种:
(1)以2-羰基-4-(羟基甲基氧膦基)-丁酸或其盐为底物,在无机氨基供体和辅酶循环系统存在的条件下,利用催化剂催化底物发生还原胺化反应,制得L-草铵膦;
(2)以D-草铵膦为底物,在无机氨基供体、D-氨基酸氧化酶和和辅酶循环系统存在的条件下,利用催化剂催化底物反应获得L-草铵膦;
在(1)和(2)中,所述辅酶循环系统为葡萄糖脱氢酶循环系统或甲酸脱氢酶循环系统;所述催化剂为如权利要求1所述的重组草铵膦脱氢酶或其固定化酶,或如权利要求5或6任一项所述的基因工程菌。
9.如权利要求8所述的L-草铵膦的制备方法,其特征在于,所述无机氨基供体为硫酸铵或甲酸铵。
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