CN108588045A - 谷氨酸脱氢酶突变体及其在制备l-草铵膦中的应用 - Google Patents

谷氨酸脱氢酶突变体及其在制备l-草铵膦中的应用 Download PDF

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CN108588045A
CN108588045A CN201810194742.0A CN201810194742A CN108588045A CN 108588045 A CN108588045 A CN 108588045A CN 201810194742 A CN201810194742 A CN 201810194742A CN 108588045 A CN108588045 A CN 108588045A
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杨立荣
尹新坚
吴坚平
徐刚
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Zhejiang University ZJU
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Abstract

本发明公开了一种谷氨酸脱氢酶突变体及其在制备L‑草铵膦中的应用。该谷氨酸脱氢酶突变体的氨基酸序列如SEQ ID NO.1～9、11、13、15、17～19、22所示。本发明通过将谷氨酸脱氢酶底物结合口袋中特定的丙氨酸突变为甘氨酸和/或将缬氨酸突变为丙氨酸的分子改造方法，获得了一类在催化底物2‑羰基‑4‑(羟基甲基膦酰基)丁酸或其盐制备L‑草铵膦工艺中具有高酶活的NADPH依赖型谷氨酸脱氢酶突变体或者对PPO具有催化活力的NADH依赖型谷氨酸脱氢酶突变体，显著提高了底物转化率和产物L‑草铵膦的浓度。

Description

谷氨酸脱氢酶突变体及其在制备L-草铵膦中的应用
技术领域
本发明涉及酶工程技术领域，尤其涉及谷氨酸脱氢酶突变体及其在制备L-草铵膦中的应用。
技术背景
草铵膦(4-[羟基(甲基)膦酰基]-DL-高丙氨酸)是一类含磷的氨基酸类除草剂，作用的靶标是谷氨酰胺合成酶，具有活性高、吸收好、杀草谱广、低毒、环境兼容性好等特点。近些年，作为世界第三大非灭生性除草剂及世界第二大转基因作物耐受除草剂，草铵膦表现出巨大的市场潜力，主要的促因是抗草铵膦转基因农作物的大范围推广及其主要竞品(草甘膦与百草枯)市场的萎缩。
草铵膦的两种构型中，只有L-型具有除草活性，而市售的草铵膦均为其消旋体(Herbicidal compositions[P].Patent application US4265654 A，1981)。L-型草铵膦单体的使用，能够显著减少草铵膦的施用量，减轻环境的压力，同时减缓杂草抗性的产生，表现出非常显著的环保优势。
因此，L-草铵膦制备工艺的开发具有非常重要的意义。在实验室规模下，已经有多条酶法制备L-草铵膦的工艺被开发出来，其中一条比较有潜力的就是利用谷氨酸脱氢酶氨化还原2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)制备L-草铵膦(生物法合成L-草铵膦的研究进展[J],《现代农药》,2009,8(3):1-4.)。
谷氨酸脱氢酶(Glutamate dehydrogenase,EC 1.4.1.2–1.4.1.4)是一类能够可逆的将谷氨酸氧化脱氨生成α-酮戊二酸的氧化还原酶，在生物体内几乎无所不在。根据其辅酶特异性，可将其分为NADH依赖型、NADPH依赖型及NADH/NADPH双辅酶依赖型的谷氨酸脱氢酶。微生物通常会表达NADH依赖型或NADPH依赖型的谷氨酸脱氢酶其中的一种，植物则会在线粒体和叶绿体中分别表达NADH依赖型和NADPH依赖型的谷氨酸脱氢酶，而动物细胞中表达的均为NAD(P)双辅酶依赖型的谷氨酸脱氢酶(The biochemistry and enzymology ofamino acid dehydrogenases[J].Critical reviews in biochemistry and molecularbiology,1994,29(6):415-467.)。与转氨酶等酶法制备L-草铵膦工艺相比，利用谷氨酸脱氢酶氨化还原PPO制备L-草铵膦具有两个显著的优点：1)当辅酶再生系统效率较高时，底物能够实现100％的转化；2)利用无机铵作为氨基供体，无难分离副产物的产生，产物易于提纯精制。
在公布号为CN106978453A的发明专利中，本团队利用基于基因组挖掘的酶库构建策略，获得多株对PPO具有催化活力的谷氨酸脱氢酶，并成功将其应用于L-草铵膦的氨化还原制备，这些谷氨酸脱氢酶的获得证明了该工艺的可行性。但是，从实际应用的角度来讲，这些谷氨酸脱氢酶的酶活均较低，其中酶活最高的谷氨酸脱氢酶重组菌其单位发酵液的酶活也仅为35.54U/L。较低的酶活会带来催化剂制备成本过高、后处理困难等一系列问题。因此，能否提高谷氨酸脱氢酶对PPO的催化活力是该工艺能否实现工业应用的关键。
另外，上述发明专利中获得的对PPO具有催化活力的谷氨酸脱氢酶均为NADPH依赖型，在以NADH为辅酶时并无催化活性。而相对于NADP(H)，NAD(H)的使用无论是在稳定性还是成本方面都具有显著优势。因此，如果能开发对PPO具有催化活力的NADH依赖型的谷氨酸脱氢酶，就可实现L-草铵膦生产成本的近一步降低。
发明内容
本发明针对还原胺化法制备L-草铵膦工艺中谷氨酸脱氢酶酶活低及缺乏对PPO具有催化活力的NADH依赖型谷氨酸脱氢酶的问题，通过对谷氨酸脱氢酶底物结合口袋处的氨基酸进行定点突变，获得了一类在催化底物2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦工艺中具有高酶活的NADPH依赖型谷氨酸脱氢酶突变体或者对PPO具有催化活力的NADH依赖型谷氨酸脱氢酶突变体，显著提高了底物转化率和产物L-草铵膦的浓度。
具体技术方案如下：
本发明提供了一类谷氨酸脱氢酶突变体，该突变体的氨基酸序列如SEQ ID NO.1～9、11、13、15、17～19、22所示。
本发明还提供了权利要求1所述的谷氨酸脱氢酶突变体的编码基因。
本发明还提供了包含所述谷氨酸脱氢酶突变体编码基因的表达载体或转化子。
本发明基于对Pseudomonas putida来源的谷氨酸脱氢酶(NCBI登录号：NP_742836.1)进行理性设计，将其底物结合口袋中的丙氨酸(A167)突变为甘氨酸，或者将缬氨酸(V378)突变为丙氨酸显著提高了该酶对2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)的催化活力。
申请人将该突变方法拓展至其它来源的并且表现出不同辅酶特异性的谷氨酸脱氢酶，通过序列比对找到其它谷氨酸脱氢酶底物结合口袋中与PpGluDH序列中167位丙氨酸与378位缬氨酸对应的氨基酸残基，并通过分子生物学手段分别突变为甘氨酸与丙氨酸，同样可提高这些谷氨酸脱氢酶对PPO的催化活力。
具体分子改造操作步骤包括：
1)以PpGluDH的氨基酸序列为模版，通过序列比对找到微生物谷氨酸脱氢酶中与PpGluDH序列中167位丙氨酸和378位缬氨酸对应的氨基酸；
2)设计突变引物；
3)以带有编码谷氨酸脱氢酶基因的质粒为模版进行全质粒PCR，引入突变；
4)PCR产物利用DPNⅠ消化后，转化进大肠杆菌BL21(DE3)中；
5)挑取单菌落，测序验证其是否为阳性突变株；
6)诱导培养，收集菌体，测定突变后酶活的变化。
本发明所述的谷氨酸脱氢酶为微生物来源的谷氨酸脱氢酶，包括NADH依赖型及NADPH依赖性谷氨酸脱氢酶；其来源于Pseudomonas putida、Corynebacteriumglutamicum、Pseudomonas entomophila、Lysinibacillus sphaericus、Geobacillusstearothermophilus、Bacillus subtilis、Bacillus megaterium、Clostridiumsymbiosum或Brevibacillus thermoruber，具体信息见实施例2的表2。
本发明还提供了所述谷氨酸脱氢酶突变体在催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦中的应用。
本发明还提供了一种催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法，包括：
(1)制备表达谷氨酸脱氢酶突变体的基因工程菌；所述谷氨酸脱氢酶突变体的氨基酸序列如SEQ ID NO.1～9、11、13、15、17～19、22所示；
(2)培养所述基因工程菌，制备酶液；
(3)将所述酶液加入含有底物2-羰基-4-(羟基甲基膦酰基)丁酸、氨基供体及还原型辅酶的混合体系中，进行还原胺化反应，制得L-草铵膦。
步骤(2)中，所述的酶液为基因工程菌的静息细胞悬液或者破胞粗酶液；当然，将该粗酶液进行纯化获得的纯酶也适用本发明所述的L-草铵膦制备方法。
作为优选，步骤(3)中，所述还原胺化反应的温度为15～60℃，反应液的pH值为5～10。
步骤(3)中，所述还原型辅酶为还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)或还原型烟酰胺腺嘌呤二核苷酸(NADH)。
作为优选，步骤(3)的反应体系中，还包括辅酶再生系统；所述辅酶再生系统为：以葡萄糖脱氢酶为辅酶再生酶、以葡萄糖为辅酶再生底物、包含NAD(P)H和NAD(P)+的葡萄糖脱氢酶辅酶再生系统；或，以醇脱氢酶为辅酶再生酶、以异丙醇为辅酶再生底物、包含NAD(P)H和NAD(P)+的醇脱氢酶辅酶再生系统；或者，以甲酸脱氢酶为辅酶再生酶、以甲酸盐为辅酶再生底物、包含NAD(P)H和NAD(P)+的甲酸脱氢酶辅酶再生系统。
更优选，步骤(3)中的反应体系中，所述辅酶再生系统为葡萄糖脱氢酶辅酶再生系统，所述葡萄糖脱氢酶基因(BsGDH-2M)来源于Bacillus subtilis，并通过分子改造提高其稳定性，其编码的氨基酸序列为SEQ ID NO.21；醇脱氢酶(TBADH)来源于Thermoanaerobacter brockii，GenBank登录号为WP_041589967.1。
进一步地，步骤(3)中，所述氨基供体为硫酸铵。
与现有技术相比，本发明具有以下有益效果：
(1)本发明通过将谷氨酸脱氢酶底物结合口袋中特定的丙氨酸突变为甘氨酸，和/或者将缬氨酸突变为丙氨酸的分子改造方法，显著提高了谷氨酸脱氢酶对2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)的催化活力；解决了还原胺化法制备L-草铵膦工艺中谷氨酸脱氢酶酶活低及缺乏对PPO具有催化活力的NADH依赖型谷氨酸脱氢酶的问题；获得的基因工程菌不仅能够表达酶活高的NADPH依赖型谷氨酸脱氢酶，以及对PPO具有催化活力的NADH依赖型谷氨酸脱氢酶；而且在L-草铵膦制备工艺中能够显著提高底物转化率和产物L-草铵膦的浓度。
(2)本发明谷氨酸脱氢酶突变体对PPO的催化活力显著提高，最高的提高倍数达到1641倍，单位发酵液的酶活高达34.47U/mL；同时，也可使对PPO没有催化活力的NADH依赖型的谷氨酸脱氢酶获得对PPO的催化活力；该谷氨酸脱氢酶突变体在L-草铵膦的制备中，均表现出良好的催化效率，底物转化率>99％，产物浓度最高达83.7g/L，ee值>99％，表现出非常大的工业应用前景；本发明成功的解决了氨化还原反应制备L-草铵膦过程中的生物催化剂活力低的关键问题，对于实现该工艺的工业化应用奠定了基础。
(3)本发明提供的谷氨酸脱氢酶突变体，不仅可实现NADPH依赖型谷氨酸脱氢酶对PPO活力的显著提高，同时也可使原本对PPO没有催化活力的NADH依赖型谷氨酸脱氢酶获得对PPO的催化活力，具有非常重要的学术与应用价值。
附图说明
图1为利用谷氨酸脱氢酶与辅酶再生酶双酶耦联制备L-草铵膦的反应式。
图2为原料2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)的质谱图；
其中，A图为PPO的正源质谱图；B图为PPO的负源质谱图。
图3为原料2-羰基-4-(羟基甲基膦酰基)丁酸(简称PPO)的核磁图；
其中，A图为PPO的13C NMR谱图；B图为PPO的1H NMR谱图。
图4为底物2-羰基-4-(羟基甲基膦酰基)丁酸的标样高效液相检测图谱(非手性分析，5mM)；其中，底物PPO的保留时间为9.7min。
图5为外消旋草胺膦标样柱前衍生化高效液相图谱(手性分析，2mM)；
其中，保留时间：L-草铵膦为6.3min，D-草铵膦为7.2min。
图6为实施案例1中PpGluDH的野生型及突变体的蛋白纯化结果；
M:protein marker；1：PpGluDH；2：A167G；3：V378A。
图7为实施案例2中谷氨酸脱氢酶的多序列比对结果；
其中，167位与378位(PpGluDH编码)氨基酸残基用黑色箭头标出。
图8为实施案例7中反应液(反应结束后)柱前衍生化高效液相检测图谱(手性分析)。
具体实施方式
以下结合具体实施例，对本发明做进一步描述。应理解，以下实施例仅用于说明本发明而非用于限制本发明的范围。
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DNA聚合酶(MaxDNA Polymerase)和Dpn I均购自TaKaRa，宝生物工程(大连)有限公司；质粒提取试剂盒购自Axygen杭州有限公司；E.coli BL21(DE3)、质粒等购自Novagen公司；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。
本发明所涉及带有谷氨酸脱氢酶基因的重组大肠杆菌，均由本实验室构建与保藏。所用载体为pET-28a(+)，所用宿主为大肠杆菌BL21(DE3)。蛋白纯化所用NI-NTA树脂(HisPurTMNi-NTA Resin)购买于Thermo ScientificTM，并参考其说明书进行蛋白的纯化。
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)为人工合成，其质谱图与核磁图如图2，3所示；D，L-草铵膦标准品购自Sigma-Aldrich公司；NAD+、NADP+、NADH与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。在本申请文本中所使用的氨基酸三字母或单字母表达方式，采用IUPAC规定的氨基酸代码(Eur.J.Biochem.,138:9-37,1984)。
本发明通过高效液相色谱(HPLC)分析反应液中底物的浓度，监测反应的进行。HPLC分析方法为：色谱柱型号：QS-C18,5μm,4.6mm×250mm。流动相：50mM(NH4)2HPO4,加入1％的10％四丁基氢氧化铵水溶液，用50％磷酸(质量分数)调pH至3.6，加入8％乙腈。检测波长：205nm。流速：1.0mL/min。柱温：40℃。底物出峰情况见附图4。
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,4.6mm×250mm。流动相：50mM乙酸钠溶液:乙腈＝8：0.5。检测波长：338nm。流速：0.85mL/min。柱温：30℃。
(2)衍生化试剂：分别称取0.03g邻苯二甲醛与0.1g N-乙酰-L-半胱氨酸，用400uL乙醇助溶，再加入4mL 0.2mol/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150μL衍生化试剂，混匀后至于25℃保温5min，进样20μL进行分析。
D-草铵膦与L-草铵膦的出峰情况，见附图5。
实施例1 Pseudomonas putida来源的谷氨酸脱氢酶的改造及活力测定
步骤一：重组菌的活化及质粒提取
将带有编码Pseudomonas putida来源的谷氨酸脱氢酶(NCBI登录号：NP_742836.1)基因的大肠杆菌工程菌(PpGluDH)使用LB培养基进行活化与培养。
LB培养基的具体配方为：蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L，用去离子水溶解后定容，121℃灭菌20min，待用。固体培养基为LB培养基加入2％的琼脂。
将保存有PpGluDH工程菌的甘油管划线至含有LB固体培养基的平皿上，37℃静置培养12h。从平皿上挑取单菌落接入含50μg/mL卡那霉素的5mL LB培养基中，37℃、200rpm培养12h。在获得培养液后，根据质粒提取试剂盒的操作说明书进行质粒的提取。所得质粒可直接用于后续操作也可置于-20℃长期保存。
步骤二：定点突变
设计特异性引物(见表1)，分别将PpGluDH氨基酸序列中167位的丙氨酸突变为甘氨酸(A167G)，378位的缬氨酸突变为丙氨酸(V378A)。
表1 PpGluDH定点突变引物
注：带下划线的密码子编码突变后的氨基酸
以步骤一中提取的质粒为模版，利用Quickchange技术(An efficient one-stepsite-directed and site-saturation mutagenesis protocol[J].Nucleic AcidsResearch,2004,32(14):e115)引入突变，PCR反应体系和反应条件如下：
PCR扩增体系：
PCR扩增条件：
1)预变性：98℃2min；
2)变性：98℃15s；退火：58℃10s；延伸：72℃2min；共循环30次；
3)后延伸：72℃10min；
4)4℃保存。
PCR扩增结束后，扩增产物用DpnⅠ消化3h去除模版质粒，消化产物转化至E.coliBL21(DE3)感受态细胞中，涂平板、挑单菌落至LB液体培养，测序验证突变正确性。验证后无误的阳性突变性置于-80℃保藏备用。
步骤三：菌体的培养及粗酶液的制备
将保藏的工程菌在经平皿划线活化后，挑单菌落接种至含50μg/mL卡那霉素的5mLLB液体培养基中，37℃震荡培养12h。按2％的接种量转接至50mL同样含50μg/ml卡那霉素的新鲜LB液体培养基中，37℃震荡培养至OD600达到0.6左右时，加入IPTG至其终浓度为0.5mM，18℃下诱导培养16h。
培养结束后，将培养液10000rpm离心10min，弃上清，收集菌体。将培养结束后收集到的菌体，用50mM pH 7.5的磷酸盐缓冲液洗涤菌体两次。之后将菌体重悬于pH 7.5的磷酸盐缓冲液中，400W功率超声破碎30次，每次超声持续3s，间歇7s。将此细胞破碎液12000g 4℃离心5min去除沉淀，得到的上清为含重组谷氨酸脱氢酶的粗酶液。
步骤四：酶活的测定
标准酶活检测体系：适量的酶、100mM底物、10mM辅酶(NADH或NADPH)、500mM NH4 +((NH4)2SO4)，总体系为400μL，反应介质为pH 7.5磷酸盐缓冲液。35℃反应10min,反应结束后加入40uL 5M NaOH终止反应。
酶活的定义：在标准的反应条件下，每分钟生成1μmol L-草铵膦所需要的酶量。
按照上述酶活测定方法，测定了PpGluDH野生型及利用上述改造策略改造后的突变株PpGluDH-A167G，PpGluDH-V378A单位体积发酵液的酶活。PpGluDH野生型的酶活为0.11U/mL，PpGluDH-A167G的活力为14.85U/mL，较野生型提高了135倍；PpGluDH-V378A的活力为13.3U/mL，较野生型提高了121倍。
同时，也测定了纯化获得的纯酶(附图6)的比酶活。经测定，PpGluDH野生型纯蛋白的比酶活为0.31U/mg；A167G的比酶活为38.13U/mg，较野生型提高了123倍；V378A的活力比酶活为35.96U/mg，较野生型提高了116倍。
步骤五：组合突变株构建及酶活的测定
将构建好的PpGluDH-A167G突变株按步骤一进行活化及质粒提取，再利用V378A-F/V378A-R这对引物进行全质粒PCR进行突变，具体方法见步骤二，构建了PpGluDH-A167G/V378A组合突变株(SEQ ID NO.22)。按步骤三、四对该组合突变株进行了诱导培养并测定了其酶活。经测定，PpGluDH-A167G/VA78A的活力单位体积发酵液为13.85U/mL，较野生型提高了126倍。
对比例1
分光光度法测定了PpGluDH野生型及突变株对其它酮酸底物的催化活力。相对于野生型，突变株对大部分的底物(2-8a，10-11a，13a)都表现出催化活力的降低。对于2-酮戊二酸(1a)及2-酮己酸(9a)，PpGluDH-A167G及PpGluDH-V378A均表现出催化活力的提高，而对于高苯丙酮酸(12a)，PpGluDH-A167G的催化活力有所提高，而PpGluDH-V378A的催化活力则有所下降。
表2：PpGluDH野生型及突变株对其它酮酸底物的催化活力
实施例2
步骤一：谷氨酸脱氢酶的选择
选取9个不同来源、不同辅酶特异性及与PpGluDH具有不同同源性的谷氨酸脱氢酶进行同样的蛋白质的改造(详细信息见表2)。其中CgGluDH、PeGluDH、LsGluDHp为NADPH依赖型的谷氨酸脱氢酶，GsGluDH、BsGluDH、BmGluDH、LsGluDH、CsGluDH、BtGluDH为NADH依赖型谷氨酸脱氢酶。
表2待拓展谷氨酸脱氢酶
注：同源性比较以PpGluDH的氨基酸序列作为参考。
步骤二：对应突变点的选择及突变体的构建
通过在线软件T-Coffee(http://tcoffee.vital-it.ch/apps/tcoffee/do:regular)进行多序列比对，找到各谷氨酸脱氢酶与PpGluDH的A167及V378相对应的氨基酸残基(附图7)。
通过比对结果可以发现，不同的谷氨酸脱氢酶其与PpGluDH对应的这两个氨基酸残基均为丙氨酸与缬氨酸，表明这两个氨基酸残基非常保守。将这些谷氨酸脱氢酶的这两个氨基酸残基均突变成甘氨酸与丙氨酸，详细的突变株见表3。按实施例一中的步骤一、二进行突变体的构建。
表3谷氨酸脱氢酶待构建突变株
步骤三：突变体的构建及酶活的测定
将构建好的突变株进行诱导表达培养，并制备粗酶液。通过标准酶活测定体系测定了这些突变株对PPO的催化活力，结果如表4所示。
表4分子改造策略拓展突变株酶活检测结果
从酶活测定的结果可以看出，对于NADPH依赖性谷氨酸脱氢酶，无论是167位(以PpGluDH氨基酸序列编码)还是378位(以PpGluDH氨基酸序列编码)的突变株酶活均较野生型显著提高，最高的提高倍数达1641。
对于NADH依赖型的谷氨酸脱氢酶，其167位(以PpGluDH氨基酸序列编码)的突变株均从野生型的没有酶活获得了较为可观的酶活。
实施例3谷氨酸脱氢酶野生型(PpGluDH)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(PpGluDH)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO，600mM葡萄糖，250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.0g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余447mM，转化率10.6％。L-草铵膦的生成浓度为8.7g/L，ee值>99％。
实施例4谷氨酸脱氢酶突变株(PpGluDH-A167G)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(PpGluDH-A167G,SEQ IDNO.1)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO，600mM葡萄糖，250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.0g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余0.9mM，转化率99.8％。L-草铵膦的生成浓度为83.7g/L，ee值>99％。
实施例5谷氨酸脱氢酶突变株(PpGluDH-V378A)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(PpGluDH-V378A,SEQ IDNO.2)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖，250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为1.0g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余1.2mM，转化率99.8％。L-草铵膦的生成浓度为81.9g/L，ee值>99％。
实施例6谷氨酸脱氢酶野生型(LsGluDHp)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(LsGluDHp)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO，600mM葡萄糖，250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为0.5g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余481mM，转化率3.8％。L-草铵膦的生成浓度为0.87g/L，ee值>99％。
实施例7谷氨酸脱氢酶突变株(LsGluDHp-A175G)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(LsGluDHp-A175G,SEQ IDNO.7)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖，250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为0.5g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余0mM，转化率100％。L-草铵膦的生成浓度为82.4g/L，ee值>99％。
实施例8谷氨酸脱氢酶突变株(LsGluDHp-A175G)，醇脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(LsGluDHp-A175G,SEQ IDNO.7)和醇脱氢酶(TBADH,WP_041589967.1)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,750mM异丙醇，250mM(NH4)2SO4与0.5mM NADP+。谷氨酸脱氢酶菌体(干重)浓度为0.5g/L，醇脱氢酶菌体(干重)浓度为2.5g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间12h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余1.3mM，转化率99.7％。L-草铵膦的生成浓度为81.3g/L，ee值>99％。
实施例9谷氨酸脱氢酶野生型(CsGluDH)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(CsGluDH)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO，600mM葡萄糖，250mM(NH4)2SO4与0.5mM NAD+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余491.3mM，转化率1.7％。L-草铵膦的生成浓度为1.63g/L，ee值>99％。
实施例10谷氨酸脱氢酶突变株(CsGluDH-A164G)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(CsGluDH-A164G,SEQ IDNO.17)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖，250mM(NH4)2SO4与0.5mM NAD+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余0mM，转化率100％。L-草铵膦的生成浓度为79.6g/L，ee值>99％。
实施例11谷氨酸脱氢酶突变株(CsGluDH-V378A)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(PpGluDH-V378A,SEQ IDNO.18)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖，250mM(NH4)2SO4与0.5mM NAD+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为35℃，反应过程通过滴加氨水控制pH为7.5。反应时间12h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余1.8mM，转化率99.6％。L-草铵膦的生成浓度为78.5g/L，ee值>99％。
实施例12谷氨酸脱氢酶野生型(BtGluDH)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(BtGluDH)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO，600mM葡萄糖，250mM(NH4)2SO4与0.5mM NAD+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为50℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余493.1mM，转化率1.4％。L-草铵膦的生成浓度为0g/L，ee值>99％
实施例13谷氨酸脱氢酶突变株(BtGluDH-A155G)，葡萄糖脱氢酶双酶耦联制备L-草铵膦
按实施例1中步骤三的方法培养表达谷氨酸脱氢酶(BtGluDH-A155G,SEQ IDNO.19)和葡萄糖脱氢酶(BsGDH-2M,SEQ ID NO.21)的工程菌，离心收集细胞，并超声破胞制备粗酶液。
反应体系为30mL,含有500mM底物PPO,600mM葡萄糖，250mM(NH4)2SO4与0.5mM NAD+。谷氨酸脱氢酶菌体(干重)浓度为1.25g/L，葡萄糖脱氢酶菌体(干重)浓度为1.25g/L。通过水浴控制反应温度为50℃，反应过程通过滴加氨水控制pH为7.5。反应时间6h后利用非手性HPLC检测PPO的残余浓度，同时利用柱前衍生化高效液相色谱检测L-草铵膦的生成量与ee值。
反应结束数据如下：PPO剩余1.4mM，转化率99.7％。L-草铵膦的生成浓度为80.3g/L，ee值>99％。
序列表
<110> 浙江大学
<120> 谷氨酸脱氢酶突变体及其在制备L-草铵膦中的应用
<160> 22
<170> SIPOSequenceListing 1.0
<210> 2
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys
1 5 10 15
Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val
20 25 30
Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln
35 40 45
Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe
50 55 60
Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly
65 70 75 80
Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu
85 90 95
Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe
100 105 110
Glu Gln Val Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly
115 120 125
Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val
130 135 140
Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly
145 150 155 160
Ala Asp Cys Asp Val Pro Ala Gly Asp Ile Gly Val Gly Ala Arg Glu
165 170 175
Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr
180 185 190
Ser Val Leu Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg
195 200 205
Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu
210 215 220
Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly
225 230 235 240
Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly
245 250 255
Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu
260 265 270
Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn
275 280 285
Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu
290 295 300
Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu
305 310 315 320
Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu
325 330 335
Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr
340 345 350
Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala
355 360 365
Pro Gly Lys Ala Ser Asn Ala Gly Gly Ala Ala Val Ser Gly Leu Glu
370 375 380
Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp
385 390 395 400
Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His
405 410 415
Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn
420 425 430
Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val
435 440 445
Val
<210> 3
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Thr Val Asp Glu Gln Val Ser Asn Tyr Tyr Asp Met Leu Leu Lys
1 5 10 15
Arg Asn Ala Gly Glu Pro Glu Phe His Gln Ala Val Ala Glu Val Leu
20 25 30
Glu Ser Leu Lys Ile Val Leu Glu Lys Asp Pro His Tyr Ala Asp Tyr
35 40 45
Gly Leu Ile Gln Arg Leu Cys Glu Pro Glu Arg Gln Leu Ile Phe Arg
50 55 60
Val Pro Trp Val Asp Asp Gln Gly Gln Val His Val Asn Arg Gly Phe
65 70 75 80
Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly Leu Arg
85 90 95
Phe His Pro Ser Val Asn Leu Gly Ile Val Lys Phe Leu Gly Phe Glu
100 105 110
Gln Ile Phe Lys Asn Ser Leu Thr Gly Leu Pro Ile Gly Gly Gly Lys
115 120 125
Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Leu Glu Ile Met
130 135 140
Arg Phe Cys Gln Ser Phe Met Thr Glu Leu His Arg His Ile Gly Glu
145 150 155 160
Tyr Arg Asp Val Pro Gly Gly Asp Ile Gly Val Gly Gly Arg Glu Ile
165 170 175
Gly Tyr Leu Phe Gly His Tyr Arg Arg Met Ala Asn Gln His Glu Ser
180 185 190
Gly Val Leu Thr Gly Lys Gly Leu Thr Trp Gly Gly Ser Leu Val Arg
195 200 205
Thr Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Val Ser Glu Met Ile
210 215 220
Lys Ala Lys Gly Glu Ser Ile Ser Gly Gln Lys Ile Ile Val Ser Gly
225 230 235 240
Ser Gly Asn Val Ala Thr Tyr Ala Ile Glu Lys Ala Gln Glu Leu Gly
245 250 255
Ala Thr Val Ile Gly Phe Ser Asp Ser Ser Gly Trp Val His Thr Pro
260 265 270
Asn Gly Val Asp Val Ala Lys Leu Arg Glu Ile Lys Glu Val Arg Arg
275 280 285
Ala Arg Val Ser Val Tyr Ala Asp Glu Val Glu Gly Ala Thr Tyr His
290 295 300
Thr Asp Gly Ser Ile Trp Asp Leu Lys Cys Asp Ile Ala Leu Pro Cys
305 310 315 320
Ala Thr Gln Asn Glu Leu Asn Gly Glu Asn Ala Lys Thr Leu Ala Asp
325 330 335
Asn Gly Cys Arg Phe Val Ala Glu Gly Ala Asn Met Pro Ser Thr Pro
340 345 350
Glu Ala Val Glu Val Phe Arg Glu Arg Asp Ile Arg Phe Gly Pro Gly
355 360 365
Lys Ala Ala Asn Ala Gly Gly Val Ala Thr Ser Ala Leu Glu Met Gln
370 375 380
Gln Asn Ala Ser Arg Asp Ser Trp Ser Phe Glu Tyr Thr Asp Glu Arg
385 390 395 400
Leu Gln Val Ile Met Lys Asn Ile Phe Lys Thr Cys Ala Glu Thr Ala
405 410 415
Ala Glu Tyr Gly His Glu Asn Asp Tyr Val Val Gly Ala Asn Ile Ala
420 425 430
Gly Phe Lys Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Ile
435 440 445
<210> 4
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Thr Val Asp Glu Gln Val Ser Asn Tyr Tyr Asp Met Leu Leu Lys
1 5 10 15
Arg Asn Ala Gly Glu Pro Glu Phe His Gln Ala Val Ala Glu Val Leu
20 25 30
Glu Ser Leu Lys Ile Val Leu Glu Lys Asp Pro His Tyr Ala Asp Tyr
35 40 45
Gly Leu Ile Gln Arg Leu Cys Glu Pro Glu Arg Gln Leu Ile Phe Arg
50 55 60
Val Pro Trp Val Asp Asp Gln Gly Gln Val His Val Asn Arg Gly Phe
65 70 75 80
Arg Val Gln Phe Asn Ser Ala Leu Gly Pro Tyr Lys Gly Gly Leu Arg
85 90 95
Phe His Pro Ser Val Asn Leu Gly Ile Val Lys Phe Leu Gly Phe Glu
100 105 110
Gln Ile Phe Lys Asn Ser Leu Thr Gly Leu Pro Ile Gly Gly Gly Lys
115 120 125
Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Leu Glu Ile Met
130 135 140
Arg Phe Cys Gln Ser Phe Met Thr Glu Leu His Arg His Ile Gly Glu
145 150 155 160
Tyr Arg Asp Val Pro Ala Gly Asp Ile Gly Val Gly Gly Arg Glu Ile
165 170 175
Gly Tyr Leu Phe Gly His Tyr Arg Arg Met Ala Asn Gln His Glu Ser
180 185 190
Gly Val Leu Thr Gly Lys Gly Leu Thr Trp Gly Gly Ser Leu Val Arg
195 200 205
Thr Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Val Ser Glu Met Ile
210 215 220
Lys Ala Lys Gly Glu Ser Ile Ser Gly Gln Lys Ile Ile Val Ser Gly
225 230 235 240
Ser Gly Asn Val Ala Thr Tyr Ala Ile Glu Lys Ala Gln Glu Leu Gly
245 250 255
Ala Thr Val Ile Gly Phe Ser Asp Ser Ser Gly Trp Val His Thr Pro
260 265 270
Asn Gly Val Asp Val Ala Lys Leu Arg Glu Ile Lys Glu Val Arg Arg
275 280 285
Ala Arg Val Ser Val Tyr Ala Asp Glu Val Glu Gly Ala Thr Tyr His
290 295 300
Thr Asp Gly Ser Ile Trp Asp Leu Lys Cys Asp Ile Ala Leu Pro Cys
305 310 315 320
Ala Thr Gln Asn Glu Leu Asn Gly Glu Asn Ala Lys Thr Leu Ala Asp
325 330 335
Asn Gly Cys Arg Phe Val Ala Glu Gly Ala Asn Met Pro Ser Thr Pro
340 345 350
Glu Ala Val Glu Val Phe Arg Glu Arg Asp Ile Arg Phe Gly Pro Gly
355 360 365
Lys Ala Ala Asn Ala Gly Gly Ala Ala Thr Ser Ala Leu Glu Met Gln
370 375 380
Gln Asn Ala Ser Arg Asp Ser Trp Ser Phe Glu Tyr Thr Asp Glu Arg
385 390 395 400
Leu Gln Val Ile Met Lys Asn Ile Phe Lys Thr Cys Ala Glu Thr Ala
405 410 415
Ala Glu Tyr Gly His Glu Asn Asp Tyr Val Val Gly Ala Asn Ile Ala
420 425 430
Gly Phe Lys Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val Ile
435 440 445
<210> 5
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ile Glu Ser Val Asp His Phe Leu Ala Arg Leu Gln Gln Arg Asp
1 5 10 15
Pro Ala Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Gln Asn Pro His Tyr Leu Glu Ala 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 Gly 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 Leu
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 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
Glu Gln Arg Ile 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 Phe Cys Glu Ala Gly Leu
260 265 270
Thr Asp Glu Gln Trp Asp Ala Leu Met Glu Leu Lys Asn Val Lys Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Gln Thr Pro Trp Ser Leu Ala Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asn Ala Asp 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 Asp
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Tyr 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 His Ile Met Gln Ser Ile His His Val Cys Val His Tyr Gly Glu
405 410 415
Glu Ala 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> 6
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ile Glu Ser Val Asp His Phe Leu Ala Arg Leu Gln Gln Arg Asp
1 5 10 15
Pro Ala Gln Pro Glu Phe His Gln Ala Val Glu Glu Val Leu Arg Ser
20 25 30
Leu Trp Pro Phe Leu Glu Gln Asn Pro His Tyr Leu Glu Ala 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 Gly 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 Leu
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 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
Glu Gln Arg Ile 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 Phe Cys Glu Ala Gly Leu
260 265 270
Thr Asp Glu Gln Trp Asp Ala Leu Met Glu Leu Lys Asn Val Lys Arg
275 280 285
Gly Arg Ile Ser Glu Leu Ala Gly Arg Phe Gly Leu Glu Phe Arg Lys
290 295 300
Gly Gln Thr Pro Trp Ser Leu Ala Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asn Ala Asp 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 Asp
340 345 350
Ala Val Asp Ile Phe Ile Glu Ala Gly Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Ala 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 His Ile Met Gln Ser Ile His His Val Cys Val His Tyr Gly Glu
405 410 415
Glu Ala 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> 457
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Gly Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Val Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 8
<211> 457
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Thr Ile Thr Thr Val Ser Asn Glu Gln Leu Ala Lys Glu Tyr Val
1 5 10 15
Asp Gly Val Phe Glu Gln Leu Lys Gln Gln Asn Cys His Gln Ala Glu
20 25 30
Phe Leu Gln Ala Ala Glu Glu Ile Phe Ile Ser Leu Val Pro Val Phe
35 40 45
Val Gln His Pro Glu Tyr Ile Lys Ala Asn Ile Leu Ser Arg Ile Val
50 55 60
Glu Pro Asp Arg Ile Ile Ser Phe Arg Val Ala Trp Gln Asp Asp His
65 70 75 80
Asn Gln Val Gln Val Asn Arg Gly Tyr Arg Val Gln Tyr Ser Asn Val
85 90 95
Met Gly Pro Tyr Lys Gly Gly Leu Arg Phe His Pro Ser Val Asn Glu
100 105 110
Ser Ile Ile Lys Phe Leu Gly Phe Glu Gln Ile Phe Lys Asn Ala Leu
115 120 125
Thr Gly Gln Pro Ile Gly Gly Gly Lys Gly Gly Ser Asn Phe Asp Pro
130 135 140
Lys Gly Lys Ser Asp Ser Glu Ile Met Arg Phe Cys Gln Ala Phe Met
145 150 155 160
Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Val Asp Val Pro Ala Gly
165 170 175
Asp Ile Gly Val Gly Ala Arg Glu Val Gly Tyr Leu Trp Gly Gln Tyr
180 185 190
Lys Arg Leu Thr Lys Ala Ser Glu Ser Gly Val Leu Thr Gly Lys Thr
195 200 205
Pro Gly Tyr Gly Gly Ser Leu Ala Arg Lys Glu Ala Thr Gly Tyr Gly
210 215 220
Thr Val Tyr Phe Val Asn Glu Met Leu Lys Asp Val Asn Asp Ser Phe
225 230 235 240
Glu Gly Lys Thr Val Val Val Ser Gly Ser Gly Asn Val Ser Thr Tyr
245 250 255
Ala Ile Glu Lys Ala Gln Gln Tyr Gly Ala Lys Val Val Ala Cys Ser
260 265 270
Asp Ser Ser Gly Tyr Ile Tyr Asp Pro Glu Gly Leu Asp Leu Asp Val
275 280 285
Ile Lys Glu Ile Lys Glu Val Lys Gly Asp Arg Ile Ser Thr Tyr Val
290 295 300
Ser Tyr Arg Pro Asn Ala Thr Phe Thr Asn Gly Cys Thr Gly Ile Trp
305 310 315 320
Thr Ile Pro Cys Asp Ile Ala Leu Pro Cys Ala Thr Gln Asn Glu Ile
325 330 335
Asn Gly Glu Ser Ala Arg Thr Leu Ile Ser Asn Gly Val Lys Ala Ile
340 345 350
Gly Glu Gly Ala Asn Met Pro Ser Asp Leu Glu Ala Ile Asn Glu Phe
355 360 365
Leu Asn Ala Gly Val Leu Phe Gly Pro Ala Lys Ala Ala Asn Ala Gly
370 375 380
Gly Ala Ala Val Ser Ala Leu Glu Met Ala Gln Asp Ser Ser Arg Val
385 390 395 400
Phe Trp Ser Phe Glu Glu Val Asp Ala Lys Leu His Gln Ile Met Lys
405 410 415
Asp Ile Tyr Ser Asp Ser Lys Ala Ala Ala Glu Lys Tyr Gly Phe Pro
420 425 430
Gly Asn Leu Val Met Gly Ala Asn Ile Ala Gly Phe Ile Lys Val Ala
435 440 445
Asp Gly Met Leu Thr Glu Gly Ile Tyr
450 455
<210> 9
<211> 423
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ala Ala Asp Lys His Thr Glu Glu Lys Gly Gln Gln Asp Asp Val
1 5 10 15
Leu Ala Ser Thr Gln Ile Val Ile His Arg Ala Leu Glu Lys Leu Gly
20 25 30
Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Ile Arg Val Leu
35 40 45
Thr Val Arg Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile Phe
50 55 60
Thr Gly Tyr Arg Ala Gln His Asn Asp Ala Val Gly Pro Thr Lys Gly
65 70 75 80
Gly Val Arg Phe His Pro Asp Val Thr Glu Arg Glu Val Lys Ala Leu
85 90 95
Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Val Asp Leu Pro Tyr Gly
100 105 110
Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Thr Met Ser Phe Arg
115 120 125
Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln Ile
130 135 140
Val Gly Pro Thr Lys Asp Ile Pro Gly Pro Asp Val Phe Thr Asn Ser
145 150 155 160
Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Arg Glu Phe
165 170 175
Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly Ser
180 185 190
His Gly Arg Glu Thr Ala Thr Ala Lys Gly Val Thr Ile Cys Ile Arg
195 200 205
Glu Ala Ala Lys Lys Arg Gly Leu Ser Leu Glu Gly Ala Arg Val Val
210 215 220
Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Leu His
225 230 235 240
Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Val Tyr Gly Ala Leu
245 250 255
Tyr Asp Pro Asn Gly Leu Asp Ile Asp Tyr Leu Leu Glu Arg Arg Asp
260 265 270
Ser Phe Gly Thr Val Thr Lys Leu Phe Lys Asn Thr Ile Ser Asn Lys
275 280 285
Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile Glu
290 295 300
Asn Gln Ile Thr Ala Glu Asn Ala Pro Arg Ile Lys Ala Ser Ile Val
305 310 315 320
Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Ala Thr Glu Ile Leu
325 330 335
Thr Gln Arg Gly Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala Gly
340 345 350
Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly Tyr
355 360 365
Tyr Trp Thr Glu Glu Glu Val Glu Gln Arg Leu Glu Lys Val Met Val
370 375 380
Lys Ala Phe Asn Asn Val Tyr Glu Met Ala Gln Thr Arg Arg Val Asp
385 390 395 400
Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu Ala
405 410 415
Cys Arg Phe Arg Gly Trp Val
420
<210> 10
<211> 423
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Ala Asp Lys His Thr Glu Glu Lys Gly Gln Gln Asp Asp Val
1 5 10 15
Leu Ala Ser Thr Gln Ile Val Ile His Arg Ala Leu Glu Lys Leu Gly
20 25 30
Tyr Pro Glu Glu Val Tyr Glu Leu Leu Lys Glu Pro Ile Arg Val Leu
35 40 45
Thr Val Arg Ile Pro Val Arg Met Asp Asp Gly Ser Val Lys Ile Phe
50 55 60
Thr Gly Tyr Arg Ala Gln His Asn Asp Ala Val Gly Pro Thr Lys Gly
65 70 75 80
Gly Val Arg Phe His Pro Asp Val Thr Glu Arg Glu Val Lys Ala Leu
85 90 95
Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Val Asp Leu Pro Tyr Gly
100 105 110
Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Thr Met Ser Phe Arg
115 120 125
Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln Ile
130 135 140
Val Gly Pro Thr Lys Asp Ile Pro Ala Pro Asp Val Phe Thr Asn Ser
145 150 155 160
Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Arg Glu Phe
165 170 175
Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly Ser
180 185 190
His Gly Arg Glu Thr Ala Thr Ala Lys Gly Val Thr Ile Cys Ile Arg
195 200 205
Glu Ala Ala Lys Lys Arg Gly Leu Ser Leu Glu Gly Ala Arg Val Val
210 215 220
Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ala Lys Phe Leu His
225 230 235 240
Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Val Tyr Gly Ala Leu
245 250 255
Tyr Asp Pro Asn Gly Leu Asp Ile Asp Tyr Leu Leu Glu Arg Arg Asp
260 265 270
Ser Phe Gly Thr Val Thr Lys Leu Phe Lys Asn Thr Ile Ser Asn Lys
275 280 285
Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala Ile Glu
290 295 300
Asn Gln Ile Thr Ala Glu Asn Ala Pro Arg Ile Lys Ala Ser Ile Val
305 310 315 320
Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Ala Thr Glu Ile Leu
325 330 335
Thr Gln Arg Gly Ile Leu Leu Val Pro Asp Val Leu Ala Ser Ala Gly
340 345 350
Gly Ala Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly Tyr
355 360 365
Tyr Trp Thr Glu Glu Glu Val Glu Gln Arg Leu Glu Lys Val Met Val
370 375 380
Lys Ala Phe Asn Asn Val Tyr Glu Met Ala Gln Thr Arg Arg Val Asp
385 390 395 400
Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala Glu Ala
405 410 415
Cys Arg Phe Arg Gly Trp Val
420
<210> 11
<211> 424
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ser Ala Lys Gln Val Ser Lys Asp Glu Glu Lys Glu Ala Leu Asn
1 5 10 15
Leu Phe Leu Ser Thr Gln Thr Ile Ile Lys Glu Ala Leu Arg Lys Leu
20 25 30
Gly Tyr Pro Gly Asp Met Tyr Glu Leu Met Lys Glu Pro Gln Arg Met
35 40 45
Leu Thr Val Arg Ile Pro Val Lys Met Asp Asn Gly Ser Val Lys Val
50 55 60
Phe Thr Gly Tyr Arg Ser Gln His Asn Asp Ala Val Gly Pro Thr Lys
65 70 75 80
Gly Gly Val Arg Phe His Pro Glu Val Asn Glu Glu Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Thr Leu Lys Cys Gly Ile Ala Asn Leu Pro Tyr
100 105 110
Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp Pro Arg Thr Met Ser Phe
115 120 125
Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Ile Pro Gly Pro Asp Val Tyr Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Leu Arg Glu
165 170 175
Phe Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser Gln Gly Arg Glu Thr Ala Thr Ala Gln Gly Val Thr Ile Cys Ile
195 200 205
Glu Glu Ala Val Lys Lys Lys Gly Ile Lys Leu Gln Asn Ala Arg Ile
210 215 220
Ile Ile Gln Gly Phe Gly Asn Ala Gly Ser Phe Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Ile Gly Ile Ser Asp Ala Asn Gly Gly
245 250 255
Leu Tyr Asn Pro Asp Gly Leu Asp Ile Pro Tyr Leu Leu Asp Lys Arg
260 265 270
Asp Ser Phe Gly Met Val Thr Asn Leu Phe Thr Asp Val Ile Thr Asn
275 280 285
Glu Glu Leu Leu Glu Lys Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Ser Asn Gln Ile Thr Ala Lys Asn Ala His Asn Ile Gln Ala Ser Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Ile Asp Ala Thr Lys Ile
325 330 335
Leu Asn Glu Arg Gly Val Leu Leu Val Pro Asp Ile Leu Ala Ser Ala
340 345 350
Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Tyr Tyr Trp Ser Glu Glu Glu Val Ala Glu Lys Leu Arg Ser Val Met
370 375 380
Val Ser Ser Phe Glu Thr Ile Tyr Gln Thr Ala Ala Thr His Lys Val
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Thr Gly Ile Arg Lys Ser Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Val
420
<210> 12
<211> 424
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ser Ala Lys Gln Val Ser Lys Asp Glu Glu Lys Glu Ala Leu Asn
1 5 10 15
Leu Phe Leu Ser Thr Gln Thr Ile Ile Lys Glu Ala Leu Arg Lys Leu
20 25 30
Gly Tyr Pro Gly Asp Met Tyr Glu Leu Met Lys Glu Pro Gln Arg Met
35 40 45
Leu Thr Val Arg Ile Pro Val Lys Met Asp Asn Gly Ser Val Lys Val
50 55 60
Phe Thr Gly Tyr Arg Ser Gln His Asn Asp Ala Val Gly Pro Thr Lys
65 70 75 80
Gly Gly Val Arg Phe His Pro Glu Val Asn Glu Glu Glu Val Lys Ala
85 90 95
Leu Ser Ile Trp Met Thr Leu Lys Cys Gly Ile Ala Asn Leu Pro Tyr
100 105 110
Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp Pro Arg Thr Met Ser Phe
115 120 125
Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile Ser Gln
130 135 140
Ile Val Gly Pro Thr Lys Asp Ile Pro Ala Pro Asp Val Tyr Thr Asn
145 150 155 160
Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Leu Arg Glu
165 170 175
Phe Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu Gly Gly
180 185 190
Ser Gln Gly Arg Glu Thr Ala Thr Ala Gln Gly Val Thr Ile Cys Ile
195 200 205
Glu Glu Ala Val Lys Lys Lys Gly Ile Lys Leu Gln Asn Ala Arg Ile
210 215 220
Ile Ile Gln Gly Phe Gly Asn Ala Gly Ser Phe Leu Ala Lys Phe Met
225 230 235 240
His Asp Ala Gly Ala Lys Val Ile Gly Ile Ser Asp Ala Asn Gly Gly
245 250 255
Leu Tyr Asn Pro Asp Gly Leu Asp Ile Pro Tyr Leu Leu Asp Lys Arg
260 265 270
Asp Ser Phe Gly Met Val Thr Asn Leu Phe Thr Asp Val Ile Thr Asn
275 280 285
Glu Glu Leu Leu Glu Lys Asp Cys Asp Ile Leu Val Pro Ala Ala Ile
290 295 300
Ser Asn Gln Ile Thr Ala Lys Asn Ala His Asn Ile Gln Ala Ser Ile
305 310 315 320
Val Val Glu Ala Ala Asn Gly Pro Thr Thr Ile Asp Ala Thr Lys Ile
325 330 335
Leu Asn Glu Arg Gly Val Leu Leu Val Pro Asp Ile Leu Ala Ser Ala
340 345 350
Gly Gly Ala Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln Gly
355 360 365
Tyr Tyr Trp Ser Glu Glu Glu Val Ala Glu Lys Leu Arg Ser Val Met
370 375 380
Val Ser Ser Phe Glu Thr Ile Tyr Gln Thr Ala Ala Thr His Lys Val
385 390 395 400
Asp Met Arg Leu Ala Ala Tyr Met Thr Gly Ile Arg Lys Ser Ala Glu
405 410 415
Ala Ser Arg Phe Arg Gly Trp Val
420
<210> 13
<211> 426
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Val Ala Asp Lys Met Gln Asp Ser Lys Asn Ser Gln Glu Glu Lys
1 5 10 15
His Asp Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu
20 25 30
Lys Leu Gly Tyr Pro Asp Glu Val Tyr Glu Leu Leu Lys Glu Pro Leu
35 40 45
Arg Met Met Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val
50 55 60
Lys Ile Phe Thr Gly His Arg Ala Gln His Asn Asp Ala Val Gly Pro
65 70 75 80
Thr Lys Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val
85 90 95
Lys Ala Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Val Asp Leu
100 105 110
Pro Tyr Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asn Met
115 120 125
Ser Phe Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile
130 135 140
Ser Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Gly Pro Asp Val Phe
145 150 155 160
Thr Asn Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile
165 170 175
Asp Glu Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu
180 185 190
Gly Gly Ser His Gly Arg Glu Thr Ala Thr Ala Lys Gly Val Thr Ile
195 200 205
Cys Ile Arg Glu Ala Ala Lys Lys Arg Gly Ile Glu Leu Gln Gly Ala
210 215 220
Arg Val Val Val Gln Gly Phe Gly Asn Ala Gly Ser Phe Leu Ala Lys
225 230 235 240
Phe Met His Asp Ala Gly Ala Lys Ile Val Gly Ile Ser Asp Ala Tyr
245 250 255
Gly Ala Leu His Asp Pro Asn Gly Leu Asp Ile Asp Tyr Leu Leu Asp
260 265 270
Arg Arg Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asn Thr Ile
275 280 285
Ser Asn Lys Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala
290 295 300
Ala Ile Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Gln Ala
305 310 315 320
Ser Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Ala Thr
325 330 335
Arg Ile Leu Ser Glu Arg Gly Ile Leu Leu Val Pro Asp Val Leu Ala
340 345 350
Ser Ala Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn
355 360 365
Gln Gly Tyr Tyr Trp Thr Glu Glu Glu Val Glu Glu Lys Leu Glu Lys
370 375 380
Val Met Val Lys Ser Phe Asn Asn Ile Tyr Glu Thr Ser Thr Thr Arg
385 390 395 400
Lys Val Asp Met Arg Leu Ala Ala Tyr Met Ile Gly Val Arg Lys Met
405 410 415
Ala Glu Gly Ser Arg Phe Arg Gly Trp Ile
420 425
<210> 14
<211> 426
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Val Ala Asp Lys Met Gln Asp Ser Lys Asn Ser Gln Glu Glu Lys
1 5 10 15
His Asp Val Leu Lys Ser Thr Gln Thr Val Ile His Lys Ala Leu Glu
20 25 30
Lys Leu Gly Tyr Pro Asp Glu Val Tyr Glu Leu Leu Lys Glu Pro Leu
35 40 45
Arg Met Met Thr Val Lys Ile Pro Val Arg Met Asp Asp Gly Ser Val
50 55 60
Lys Ile Phe Thr Gly His Arg Ala Gln His Asn Asp Ala Val Gly Pro
65 70 75 80
Thr Lys Gly Gly Ile Arg Phe His Pro Asn Val Thr Glu Lys Glu Val
85 90 95
Lys Ala Leu Ser Ile Trp Met Ser Leu Lys Cys Gly Ile Val Asp Leu
100 105 110
Pro Tyr Gly Gly Gly Lys Gly Gly Ile Val Cys Asp Pro Arg Asn Met
115 120 125
Ser Phe Gly Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Ile
130 135 140
Ser Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Ala Pro Asp Val Phe
145 150 155 160
Thr Asn Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile
165 170 175
Asp Glu Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys Pro Leu Val Leu
180 185 190
Gly Gly Ser His Gly Arg Glu Thr Ala Thr Ala Lys Gly Val Thr Ile
195 200 205
Cys Ile Arg Glu Ala Ala Lys Lys Arg Gly Ile Glu Leu Gln Gly Ala
210 215 220
Arg Val Val Val Gln Gly Phe Gly Asn Ala Gly Ser Phe Leu Ala Lys
225 230 235 240
Phe Met His Asp Ala Gly Ala Lys Ile Val Gly Ile Ser Asp Ala Tyr
245 250 255
Gly Ala Leu His Asp Pro Asn Gly Leu Asp Ile Asp Tyr Leu Leu Asp
260 265 270
Arg Arg Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Asn Asn Thr Ile
275 280 285
Ser Asn Lys Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala
290 295 300
Ala Ile Glu Asn Gln Ile Thr Glu Glu Asn Ala His Asn Ile Gln Ala
305 310 315 320
Ser Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Ala Thr
325 330 335
Arg Ile Leu Ser Glu Arg Gly Ile Leu Leu Val Pro Asp Val Leu Ala
340 345 350
Ser Ala Gly Gly Ala Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn
355 360 365
Gln Gly Tyr Tyr Trp Thr Glu Glu Glu Val Glu Glu Lys Leu Glu Lys
370 375 380
Val Met Val Lys Ser Phe Asn Asn Ile Tyr Glu Thr Ser Thr Thr Arg
385 390 395 400
Lys Val Asp Met Arg Leu Ala Ala Tyr Met Ile Gly Val Arg Lys Met
405 410 415
Ala Glu Gly Ser Arg Phe Arg Gly Trp Ile
420 425
<210> 15
<211> 414
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ser Glu Asn Leu Asn Leu Phe Thr Ser Thr Gln Asp Val Ile Gln
1 5 10 15
Asp Ala Leu Asn Lys Leu Gly Tyr Asp Glu Ala Met Tyr Glu Leu Leu
20 25 30
Lys Glu Pro Leu Arg Met Leu Gln Val Arg Ile Pro Val Lys Met Asp
35 40 45
Asp Gly Thr Thr Lys Val Phe Thr Gly Tyr Arg Ala Gln His Asn Asp
50 55 60
Ala Val Gly Pro Thr Lys Gly Gly Val Arg Phe His Pro Gln Val Ser
65 70 75 80
Glu Glu Glu Val Lys Ala Leu Ser Met Trp Met Thr Leu Lys Cys Gly
85 90 95
Ile Val Asp Leu Pro Tyr Gly Gly Gly Lys Gly Gly Val Ile Cys Asp
100 105 110
Pro Arg Gln Met Ser Met Gly Glu Ile Glu Arg Leu Ser Arg Gly Tyr
115 120 125
Val Arg Ala Val Ser Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Gly
130 135 140
Pro Asp Val Phe Thr Asn Ala Gln Ile Met Ala Trp Met Met Asp Glu
145 150 155 160
Tyr Ser Arg Met Asp Glu Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys
165 170 175
Pro Leu Val Leu Gly Gly Ser Gln Gly Arg Asp Arg Ala Thr Ala Gln
180 185 190
Gly Val Thr Ile Val Ile Glu Glu Ala Ala Lys Lys Arg Gly Ile Asp
195 200 205
Ile Lys Gly Ala Arg Val Val Ile Gln Gly Phe Gly Asn Ala Gly Ser
210 215 220
Phe Leu Ala Lys Phe Met His Asp Leu Gly Ala Lys Val Ile Gly Ile
225 230 235 240
Ser Asp Ala Tyr Gly Ala Leu His Asp Pro Glu Gly Leu Asp Ile Asp
245 250 255
Tyr Leu Leu Asp Arg Arg Asp Ser Phe Gly Thr Val Thr Thr Leu Phe
260 265 270
Glu Asn Thr Ile Ser Asn Lys Glu Leu Leu Glu Leu Asp Cys Asp Ile
275 280 285
Leu Val Pro Ala Ala Ile Glu Asn Gln Ile Thr Ala Asp Asn Ala His
290 295 300
Asn Ile Lys Ala Asp Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr
305 310 315 320
Ala Glu Ala Thr Lys Ile Leu Thr Glu Arg Gly Ile Leu Leu Val Pro
325 330 335
Asp Val Leu Ala Ser Ala Gly Gly Val Thr Val Ser Tyr Phe Glu Trp
340 345 350
Val Gln Asn Asn Gln Gly Tyr Tyr Trp Thr Glu Glu Glu Val Glu Glu
355 360 365
Arg Leu Tyr Lys Lys Met Val Glu Ala Phe Asp Asn Val Tyr Thr Thr
370 375 380
Ala Thr Thr Arg Asn Ile Asn Met Arg Leu Ala Ala Tyr Met Val Gly
385 390 395 400
Val Arg Arg Thr Ala Glu Ala Ser Arg Phe Arg Gly Trp Val
405 410
<210> 16
<211> 414
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Ser Glu Asn Leu Asn Leu Phe Thr Ser Thr Gln Asp Val Ile Gln
1 5 10 15
Asp Ala Leu Asn Lys Leu Gly Tyr Asp Glu Ala Met Tyr Glu Leu Leu
20 25 30
Lys Glu Pro Leu Arg Met Leu Gln Val Arg Ile Pro Val Lys Met Asp
35 40 45
Asp Gly Thr Thr Lys Val Phe Thr Gly Tyr Arg Ala Gln His Asn Asp
50 55 60
Ala Val Gly Pro Thr Lys Gly Gly Val Arg Phe His Pro Gln Val Ser
65 70 75 80
Glu Glu Glu Val Lys Ala Leu Ser Met Trp Met Thr Leu Lys Cys Gly
85 90 95
Ile Val Asp Leu Pro Tyr Gly Gly Gly Lys Gly Gly Val Ile Cys Asp
100 105 110
Pro Arg Gln Met Ser Met Gly Glu Ile Glu Arg Leu Ser Arg Gly Tyr
115 120 125
Val Arg Ala Val Ser Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Ala
130 135 140
Pro Asp Val Phe Thr Asn Ala Gln Ile Met Ala Trp Met Met Asp Glu
145 150 155 160
Tyr Ser Arg Met Asp Glu Phe Asn Ser Pro Gly Phe Ile Thr Gly Lys
165 170 175
Pro Leu Val Leu Gly Gly Ser Gln Gly Arg Asp Arg Ala Thr Ala Gln
180 185 190
Gly Val Thr Ile Val Ile Glu Glu Ala Ala Lys Lys Arg Gly Ile Asp
195 200 205
Ile Lys Gly Ala Arg Val Val Ile Gln Gly Phe Gly Asn Ala Gly Ser
210 215 220
Phe Leu Ala Lys Phe Met His Asp Leu Gly Ala Lys Val Ile Gly Ile
225 230 235 240
Ser Asp Ala Tyr Gly Ala Leu His Asp Pro Glu Gly Leu Asp Ile Asp
245 250 255
Tyr Leu Leu Asp Arg Arg Asp Ser Phe Gly Thr Val Thr Thr Leu Phe
260 265 270
Glu Asn Thr Ile Ser Asn Lys Glu Leu Leu Glu Leu Asp Cys Asp Ile
275 280 285
Leu Val Pro Ala Ala Ile Glu Asn Gln Ile Thr Ala Asp Asn Ala His
290 295 300
Asn Ile Lys Ala Asp Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr
305 310 315 320
Ala Glu Ala Thr Lys Ile Leu Thr Glu Arg Gly Ile Leu Leu Val Pro
325 330 335
Asp Val Leu Ala Ser Ala Gly Gly Ala Thr Val Ser Tyr Phe Glu Trp
340 345 350
Val Gln Asn Asn Gln Gly Tyr Tyr Trp Thr Glu Glu Glu Val Glu Glu
355 360 365
Arg Leu Tyr Lys Lys Met Val Glu Ala Phe Asp Asn Val Tyr Thr Thr
370 375 380
Ala Thr Thr Arg Asn Ile Asn Met Arg Leu Ala Ala Tyr Met Val Gly
385 390 395 400
Val Arg Arg Thr Ala Glu Ala Ser Arg Phe Arg Gly Trp Val
405 410
<210> 17
<211> 450
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Ser Lys Tyr Val Asp Arg Val Ile Ala Glu Val Glu Lys Lys Tyr
1 5 10 15
Ala Asp Glu Pro Glu Phe Val Gln Thr Val Glu Glu Val Leu Ser Ser
20 25 30
Leu Gly Pro Val Val Asp Ala His Pro Glu Tyr Glu Glu Val Ala Leu
35 40 45
Leu Glu Arg Met Val Ile Pro Glu Arg Val Ile Glu Phe Arg Val Pro
50 55 60
Trp Glu Asp Asp Asn Gly Lys Val His Val Asn Thr Gly Tyr Arg Val
65 70 75 80
Gln Phe Asn Gly Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe Ala
85 90 95
Pro Ser Val Asn Leu Ser Ile Met Lys Phe Leu Gly Phe Glu Gln Ala
100 105 110
Phe Lys Asp Ser Leu Thr Thr Leu Pro Met Gly Gly Ala Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Asn Gly Lys Ser Asp Arg Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Ile
145 150 155 160
Asp Val Pro Gly Gly Asp Leu Gly Val Gly Ala Arg Glu Ile Gly Tyr
165 170 175
Met Tyr Gly Gln Tyr Arg Lys Ile Val Gly Gly Phe Tyr Asn Gly Val
180 185 190
Leu Thr Gly Lys Ala Arg Ser Phe Gly Gly Ser Leu Val Arg Pro Glu
195 200 205
Ala Thr Gly Tyr Gly Ser Val Tyr Tyr Val Glu Ala Val Met Lys His
210 215 220
Glu Asn Asp Thr Leu Val Gly Lys Thr Val Ala Leu Ala Gly Phe Gly
225 230 235 240
Asn Val Ala Trp Gly Ala Ala Lys Lys Leu Ala Glu Leu Gly Ala Lys
245 250 255
Ala Val Thr Leu Ser Gly Pro Asp Gly Tyr Ile Tyr Asp Pro Glu Gly
260 265 270
Ile Thr Thr Glu Glu Lys Ile Asn Tyr Met Leu Glu Met Arg Ala Ser
275 280 285
Gly Arg Asn Lys Val Gln Asp Tyr Ala Asp Lys Phe Gly Val Gln Phe
290 295 300
Phe Pro Gly Glu Lys Pro Trp Gly Gln Lys Val Asp Ile Ile Met Pro
305 310 315 320
Cys Ala Thr Gln Asn Asp Val Asp Leu Glu Gln Ala Lys Lys Ile Val
325 330 335
Ala Asn Asn Ile Lys Tyr Tyr Ile Glu Val Ala Asn Met Pro Thr Thr
340 345 350
Asn Glu Ala Leu Arg Phe Leu Met Gln Gln Pro Asn Met Val Val Ala
355 360 365
Pro Ser Lys Ala Val Asn Ala Gly Gly Val Leu Val Ser Gly Phe Glu
370 375 380
Met Ser Gln Asn Ser Glu Arg Leu Ser Trp Thr Ala Glu Glu Val Asp
385 390 395 400
Ser Lys Leu His Gln Val Met Thr Asp Ile His Asp Gly Ser Ala Ala
405 410 415
Ala Ala Glu Arg Tyr Gly Leu Gly Tyr Asn Leu Val Ala Gly Ala Asn
420 425 430
Ile Val Gly Phe Gln Lys Ile Ala Asp Ala Met Met Ala Gln Gly Ile
435 440 445
Ala Trp
450
<210> 18
<211> 450
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Ser Lys Tyr Val Asp Arg Val Ile Ala Glu Val Glu Lys Lys Tyr
1 5 10 15
Ala Asp Glu Pro Glu Phe Val Gln Thr Val Glu Glu Val Leu Ser Ser
20 25 30
Leu Gly Pro Val Val Asp Ala His Pro Glu Tyr Glu Glu Val Ala Leu
35 40 45
Leu Glu Arg Met Val Ile Pro Glu Arg Val Ile Glu Phe Arg Val Pro
50 55 60
Trp Glu Asp Asp Asn Gly Lys Val His Val Asn Thr Gly Tyr Arg Val
65 70 75 80
Gln Phe Asn Gly Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe Ala
85 90 95
Pro Ser Val Asn Leu Ser Ile Met Lys Phe Leu Gly Phe Glu Gln Ala
100 105 110
Phe Lys Asp Ser Leu Thr Thr Leu Pro Met Gly Gly Ala Lys Gly Gly
115 120 125
Ser Asp Phe Asp Pro Asn Gly Lys Ser Asp Arg Glu Val Met Arg Phe
130 135 140
Cys Gln Ala Phe Met Thr Glu Leu Tyr Arg His Ile Gly Pro Asp Ile
145 150 155 160
Asp Val Pro Ala Gly Asp Leu Gly Val Gly Ala Arg Glu Ile Gly Tyr
165 170 175
Met Tyr Gly Gln Tyr Arg Lys Ile Val Gly Gly Phe Tyr Asn Gly Val
180 185 190
Leu Thr Gly Lys Ala Arg Ser Phe Gly Gly Ser Leu Val Arg Pro Glu
195 200 205
Ala Thr Gly Tyr Gly Ser Val Tyr Tyr Val Glu Ala Val Met Lys His
210 215 220
Glu Asn Asp Thr Leu Val Gly Lys Thr Val Ala Leu Ala Gly Phe Gly
225 230 235 240
Asn Val Ala Trp Gly Ala Ala Lys Lys Leu Ala Glu Leu Gly Ala Lys
245 250 255
Ala Val Thr Leu Ser Gly Pro Asp Gly Tyr Ile Tyr Asp Pro Glu Gly
260 265 270
Ile Thr Thr Glu Glu Lys Ile Asn Tyr Met Leu Glu Met Arg Ala Ser
275 280 285
Gly Arg Asn Lys Val Gln Asp Tyr Ala Asp Lys Phe Gly Val Gln Phe
290 295 300
Phe Pro Gly Glu Lys Pro Trp Gly Gln Lys Val Asp Ile Ile Met Pro
305 310 315 320
Cys Ala Thr Gln Asn Asp Val Asp Leu Glu Gln Ala Lys Lys Ile Val
325 330 335
Ala Asn Asn Ile Lys Tyr Tyr Ile Glu Val Ala Asn Met Pro Thr Thr
340 345 350
Asn Glu Ala Leu Arg Phe Leu Met Gln Gln Pro Asn Met Val Val Ala
355 360 365
Pro Ser Lys Ala Val Asn Ala Gly Gly Ala Leu Val Ser Gly Phe Glu
370 375 380
Met Ser Gln Asn Ser Glu Arg Leu Ser Trp Thr Ala Glu Glu Val Asp
385 390 395 400
Ser Lys Leu His Gln Val Met Thr Asp Ile His Asp Gly Ser Ala Ala
405 410 415
Ala Ala Glu Arg Tyr Gly Leu Gly Tyr Asn Leu Val Ala Gly Ala Asn
420 425 430
Ile Val Gly Phe Gln Lys Ile Ala Asp Ala Met Met Ala Gln Gly Ile
435 440 445
Ala Trp
450
<210> 19
<211> 425
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Met Val Thr Glu Asn Thr Gln Gly Lys Glu Gln Lys Gln Glu Ser Met
1 5 10 15
Asn Leu Leu Gln Ser Thr Gln Thr Val Ile Lys Glu Ala Leu Glu Lys
20 25 30
Leu Gly Tyr Gln Glu Ser Met Tyr Glu Leu Leu Lys Glu Pro Leu Arg
35 40 45
Val Leu Thr Val Arg Ile Pro Val Arg Met Asp Asn Gly Glu Val Lys
50 55 60
Val Phe Thr Gly Tyr Arg Ala Gln His Asn Asp Ala Val Gly Pro Thr
65 70 75 80
Lys Gly Gly Ile Arg Phe His Pro Asp Val Thr Glu Asp Glu Val Lys
85 90 95
Ala Leu Ser Ile Trp Met Ser Leu Lys Ala Gly Ile Val Asp Leu Pro
100 105 110
Tyr Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp Pro Arg Glu Met Ser
115 120 125
Phe Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Val Ser
130 135 140
Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Gly Pro Asp Val Phe Thr
145 150 155 160
Asn Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Arg
165 170 175
Glu Phe Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Ile Ala Leu Gly
180 185 190
Gly Ser His Gly Arg Glu Thr Ala Thr Ala Lys Gly Val Thr Ile Cys
195 200 205
Ile Arg Glu Ala Ala Lys Arg Arg Gly Ile Asp Leu Lys Gly Ala Arg
210 215 220
Val Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ser Lys Phe
225 230 235 240
Met His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly
245 250 255
Ala Leu Tyr Asp Pro Asn Gly Leu Asp Ile Asp Tyr Leu Leu Asp Arg
260 265 270
Arg Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Thr Asn Thr Ile Thr
275 280 285
Asn Lys Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala
290 295 300
Ile Glu Asn Gln Ile Thr Ala Ala Asn Ala His Asn Ile Lys Ala Lys
305 310 315 320
Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Ala Thr Lys
325 330 335
Ile Leu Thr Glu Arg Gly Ile Leu Leu Val Pro Asp Val Leu Ala Ser
340 345 350
Ala Gly Gly Val Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln
355 360 365
Gly Tyr Tyr Trp Ser Glu Glu Glu Val Gln Glu Lys Leu Glu Lys Val
370 375 380
Met Val Lys Ala Phe Glu Asn Val Tyr Ser Leu Ala Gln Thr Arg Arg
385 390 395 400
Val Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala
405 410 415
Glu Ala Ser Arg Phe Arg Gly Trp Val
420 425
<210> 20
<211> 425
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Val Thr Glu Asn Thr Gln Gly Lys Glu Gln Lys Gln Glu Ser Met
1 5 10 15
Asn Leu Leu Gln Ser Thr Gln Thr Val Ile Lys Glu Ala Leu Glu Lys
20 25 30
Leu Gly Tyr Gln Glu Ser Met Tyr Glu Leu Leu Lys Glu Pro Leu Arg
35 40 45
Val Leu Thr Val Arg Ile Pro Val Arg Met Asp Asn Gly Glu Val Lys
50 55 60
Val Phe Thr Gly Tyr Arg Ala Gln His Asn Asp Ala Val Gly Pro Thr
65 70 75 80
Lys Gly Gly Ile Arg Phe His Pro Asp Val Thr Glu Asp Glu Val Lys
85 90 95
Ala Leu Ser Ile Trp Met Ser Leu Lys Ala Gly Ile Val Asp Leu Pro
100 105 110
Tyr Gly Gly Gly Lys Gly Gly Ile Ile Cys Asp Pro Arg Glu Met Ser
115 120 125
Phe Arg Glu Leu Glu Arg Leu Ser Arg Gly Tyr Val Arg Ala Val Ser
130 135 140
Gln Ile Val Gly Pro Thr Lys Asp Ile Pro Ala Pro Asp Val Phe Thr
145 150 155 160
Asn Ser Gln Ile Met Ala Trp Met Met Asp Glu Tyr Ser Arg Ile Arg
165 170 175
Glu Phe Asp Ser Pro Gly Phe Ile Thr Gly Lys Pro Ile Ala Leu Gly
180 185 190
Gly Ser His Gly Arg Glu Thr Ala Thr Ala Lys Gly Val Thr Ile Cys
195 200 205
Ile Arg Glu Ala Ala Lys Arg Arg Gly Ile Asp Leu Lys Gly Ala Arg
210 215 220
Val Val Val Gln Gly Phe Gly Asn Ala Gly Ser Tyr Leu Ser Lys Phe
225 230 235 240
Met His Asp Ala Gly Ala Lys Val Val Gly Ile Ser Asp Ala Tyr Gly
245 250 255
Ala Leu Tyr Asp Pro Asn Gly Leu Asp Ile Asp Tyr Leu Leu Asp Arg
260 265 270
Arg Asp Ser Phe Gly Thr Val Thr Lys Leu Phe Thr Asn Thr Ile Thr
275 280 285
Asn Lys Glu Leu Leu Glu Leu Asp Cys Asp Ile Leu Val Pro Ala Ala
290 295 300
Ile Glu Asn Gln Ile Thr Ala Ala Asn Ala His Asn Ile Lys Ala Lys
305 310 315 320
Ile Val Val Glu Ala Ala Asn Gly Pro Thr Thr Leu Glu Ala Thr Lys
325 330 335
Ile Leu Thr Glu Arg Gly Ile Leu Leu Val Pro Asp Val Leu Ala Ser
340 345 350
Ala Gly Gly Ala Thr Val Ser Tyr Phe Glu Trp Val Gln Asn Asn Gln
355 360 365
Gly Tyr Tyr Trp Ser Glu Glu Glu Val Gln Glu Lys Leu Glu Lys Val
370 375 380
Met Val Lys Ala Phe Glu Asn Val Tyr Ser Leu Ala Gln Thr Arg Arg
385 390 395 400
Val Asp Met Arg Leu Ala Ala Tyr Met Val Gly Val Arg Lys Met Ala
405 410 415
Glu Ala Ser Arg Phe Arg Gly Trp Val
420 425
<210> 21
<211> 261
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Tyr Pro Asp Leu Lys Gly Lys Val Val Ala Ile Thr Gly Ala Ala
1 5 10 15
Ser Gly Leu Gly Lys Ala Met Ala Ile Arg Phe Gly Lys Glu Gln Ala
20 25 30
Lys Val Val Ile Asn Tyr Tyr Ser Asn Lys Gln Asp Pro Asn Glu Val
35 40 45
Lys Glu Glu Val Ile Lys Ala Gly Gly Glu Ala Val Val Val Gln Gly
50 55 60
Asp Val Thr Lys Glu Glu Asp Val Lys Asn Ile Val Gln Thr Ala Ile
65 70 75 80
Lys Glu Phe Gly Thr Leu Asp Ile Met Ile Asn Asn Ala Gly Leu Glu
85 90 95
Asn Pro Val Pro Ser His Glu Met Pro Leu Lys Asp Trp Asp Lys Val
100 105 110
Ile Gly Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
115 120 125
Lys Tyr Phe Val Glu Asn Asp Ile Lys Gly Asn Val Ile Asn Met Ser
130 135 140
Ser Val His Glu Val Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Ile Lys Leu Met Thr Arg Thr Leu Ala Leu Glu Tyr
165 170 175
Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Gln Lys Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Glu Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Ala Trp Leu Ala Ser Lys Glu Ala Ser Tyr Val Thr
225 230 235 240
Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe
245 250 255
Gln Ala Gly Arg Gly
260
<210> 22
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys
1 5 10 15
Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val
20 25 30
Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln
35 40 45
Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe
50 55 60
Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly
65 70 75 80
Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu
85 90 95
Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe
100 105 110
Glu Gln Val Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly
115 120 125
Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val
130 135 140
Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly
145 150 155 160
Ala Asp Cys Asp Val Pro Gly Gly Asp Ile Gly Val Gly Ala Arg Glu
165 170 175
Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr
180 185 190
Ser Val Leu Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg
195 200 205
Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu
210 215 220
Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly
225 230 235 240
Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly
245 250 255
Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu
260 265 270
Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn
275 280 285
Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu
290 295 300
Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu
305 310 315 320
Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu
325 330 335
Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr
340 345 350
Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala
355 360 365
Pro Gly Lys Ala Ser Asn Ala Gly Gly Ala Ala Val Ser Gly Leu Glu
370 375 380
Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp
385 390 395 400
Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His
405 410 415
Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn
420 425 430
Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val
435 440 445
Val
<210> 23
<211> 449
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Ser Thr Met Ile Glu Ser Val Asp Asn Phe Leu Ala Arg Leu Lys
1 5 10 15
Gln Arg Asp Pro Gly Gln Pro Glu Phe His Gln Ala Val Glu Glu Val
20 25 30
Leu Arg Thr Leu Trp Pro Phe Leu Glu Ala Asn Pro His Tyr Leu Gln
35 40 45
Ser Gly Ile Leu Glu Arg Met Val Glu Pro Glu Arg Ala Val Leu Phe
50 55 60
Arg Val Ser Trp Val Asp Asp Gln Gly Lys Val Gln Val Asn Arg Gly
65 70 75 80
Tyr Arg Ile Gln Met Ser Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu
85 90 95
Arg Phe His Pro Ser Val Asn Leu Ser Val Leu Lys Phe Leu Ala Phe
100 105 110
Glu Gln Val Phe Lys Asn Ser Leu Thr Ser Leu Pro Met Gly Gly Gly
115 120 125
Lys Gly Gly Ser Asp Phe Asp Pro Lys Gly Lys Ser Asp Ala Glu Val
130 135 140
Met Arg Phe Cys Gln Ala Phe Met Ser Glu Leu Tyr Arg His Ile Gly
145 150 155 160
Ala Asp Cys Asp Val Pro Gly Gly Asp Ile Gly Val Gly Ala Arg Glu
165 170 175
Ile Gly Phe Met Phe Gly Gln Tyr Lys Arg Leu Ala Asn Gln Phe Thr
180 185 190
Ser Val Leu Thr Gly Lys Gly Met Thr Tyr Gly Gly Ser Leu Ile Arg
195 200 205
Pro Glu Ala Thr Gly Tyr Gly Cys Val Tyr Phe Ala Glu Glu Met Leu
210 215 220
Lys Arg Gln Asp Lys Arg Ile Asp Gly Arg Arg Val Ala Val Ser Gly
225 230 235 240
Ser Gly Asn Val Ala Gln Tyr Ala Ala Arg Lys Val Met Asp Leu Gly
245 250 255
Gly Lys Val Ile Ser Leu Ser Asp Ser Glu Gly Thr Leu Tyr Ala Glu
260 265 270
Ala Gly Leu Thr Asp Ala Gln Trp Asp Ala Leu Met Glu Leu Lys Asn
275 280 285
Val Lys Arg Gly Arg Ile Ser Glu Leu Ala Gly Gln Phe Gly Leu Glu
290 295 300
Phe Arg Lys Gly Gln Thr Pro Trp Ser Leu Pro Cys Asp Ile Ala Leu
305 310 315 320
Pro Cys Ala Thr Gln Asn Glu Leu Gly Ala Glu Asp Ala Arg Thr Leu
325 330 335
Leu Arg Asn Gly Cys Ile Cys Val Ala Glu Gly Ala Asn Met Pro Thr
340 345 350
Thr Leu Glu Ala Val Asp Ile Phe Leu Asp Ala Gly Ile Leu Tyr Ala
355 360 365
Pro Gly Lys Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu
370 375 380
Met Ser Gln Asn Ala Met Arg Leu Leu Trp Thr Ala Gly Glu Val Asp
385 390 395 400
Ser Lys Leu His Asn Ile Met Gln Ser Ile His His Ala Cys Val His
405 410 415
Tyr Gly Glu Glu Ala Asp Gly Arg Ile Asn Tyr Val Lys Gly Ala Asn
420 425 430
Ile Ala Gly Phe Val Lys Val Ala Asp Ala Met Leu Ala Gln Gly Val
435 440 445
Val

Claims (10)

1.谷氨酸脱氢酶突变体，其特征在于，氨基酸序列如SEQ ID NO.1～9、11、13、15、17～19、22所示。
2.如权利要求1所述的谷氨酸脱氢酶突变体的编码基因。
3.包含权利要求1所述谷氨酸脱氢酶突变体编码基因的表达载体或转化子。
4.如权利要求1所述的谷氨酸脱氢酶突变体在催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦中的应用。
5.一种催化2-羰基-4-(羟基甲基膦酰基)丁酸或其盐制备L-草铵膦的方法，其特征在于，包括：
(1)制备表达谷氨酸脱氢酶突变体的基因工程菌；所述谷氨酸脱氢酶突变体的氨基酸序列如SEQ ID NO.1～9、11、13、15、17～19、22所示；
(2)培养所述基因工程菌，制备酶液；
(3)将所述酶液加入含有底物2-羰基-4-(羟基甲基膦酰基)丁酸、氨基供体及还原型辅酶的体系中，进行还原胺化反应，制得L-草铵膦。
6.如权利要求5所述的方法，其特征在于，步骤(3)中，所述还原胺化反应的温度为15～60℃，反应液的pH值为5～10。
7.如权利要求5所述的方法，其特征在于，步骤(3)中，所述还原型辅酶为NADPH或NADH。
8.如权利要求5所述的方法，其特征在于，步骤(3)的反应体系中，还包括辅酶再生系统；所述辅酶再生系统为：以葡萄糖脱氢酶为辅酶再生酶、以葡萄糖为辅酶再生底物、包含NAD(P)H和NAD(P)+的葡萄糖脱氢酶辅酶再生系统；或，以醇脱氢酶为辅酶再生酶、以异丙醇为辅酶再生底物、包含NAD(P)H和NAD(P)+的醇脱氢酶辅酶再生系统；或者，以甲酸脱氢酶为辅酶再生酶、以甲酸盐为辅酶再生底物、包含NAD(P)H和NAD(P)+的甲酸脱氢酶辅酶再生系统。
9.如权利要求8所述的方法，其特征在于，反应体系中，所述辅酶再生系统为葡萄糖脱氢酶辅酶再生系统，所述葡萄糖脱氢酶的氨基酸序列如SEQ ID NO.21所示。
10.如权利要求5所述的方法，其特征在于，步骤(3)中，所述氨基供体为硫酸铵。
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CN113088501A (zh) * 2019-12-23 2021-07-09 浙江大学 一种用于生产l-草铵膦的谷氨酸脱氢酶突变体及l-草铵膦生产方法
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