一种酶催化法合成反式-4-香豆酸的方法
阅读说明：本技术 一种酶催化法合成反式-4-香豆酸的方法 (Method for synthesizing trans-4-coumaric acid by enzyme catalysis method ) 是由 陈江 孙军亭 徐光彩 于 2019-11-25 设计创作，主要内容包括：本发明提供了一种酶催化法合成反式-4-香豆酸的方法。反式-肉桂酸-4-单加氧酶(EC 1.14.14.91)可催化反式-肉桂酸合成反式-4-香豆酸,同时消耗一分子O<Sub>2</Sub>和一个NAD(P)H,生成一个H<Sup>+</Sup>和一个NAD(P)<Sup>+</Sup>。在单水相或水/有机溶剂的双相反应体系中,加入反式-肉桂酸(原料),加入微量NAD(P)<Sup>+</Sup>或NAD(P)H,加入O<Sub>2</Sub>源,加入还原型氢供体,然后加入反式-肉桂酸4-单加氧酶和NAD(P)<Sup>+</Sup>还原酶,滴加碱液控制pH,控制反应温度；反应结束后反应液过滤除酶后浓缩,降温结晶可得反式-4-香豆酸。(The invention provides a method for synthesizing trans-4-coumaric acid by an enzyme catalysis method. Trans-cinnamic acid-4-monooxygenase (EC1.14.14.91) can catalyze trans-cinnamic acid to synthesize trans-4-coumaric acid while consuming one molecule of O 2 And one NAD (P) H to produce one H + And one NAD (P) + . Adding trans-cinnamic acid (raw material) and trace amount of NAD (P) into a single-water-phase or water/organic solvent two-phase reaction system + Or NAD (P) H, O 2 Source, addition of reduced hydrogen donor, followed by addition of trans-cinnamic acid 4-monooxygenase and NAD (P) + Reductase, dripping alkali liquor to control pH and control reaction temperature; after the reaction is finished, the reaction solution is filtered, enzyme is removed, concentrated, cooled and crystallized to obtain the trans-4-coumaric acid.)
技术领域
本发明涉及生物技术和酶催化领域；更具体地，本发明涉及一种酶催化法合成反式-4-香豆酸的方法。
背景技术
对香豆酸即香豆酸(coumarate)、又名p香豆酸(p-coumalic acid)、4-香豆酸、对-羟基肉桂酸等，是羟基肉桂酸类化合物中最主要的一种；系统命名为3-(4-羟基苯基)-2丙烯酸。对香豆酸有顺式和反式两种,反式对香豆酸即反式-4-香豆酸，为白色细针状晶体,熔点212℃-214℃,略溶于冷水,溶于热水、甲醇、乙醇,微溶于苯和氯仿,不溶于二氯甲烷和石油醚。
反式-4-香豆酸可形成共振稳定的酚自由基，因此具有良好的抗氧化活性；它对过氧化氢、超氧自由基、氢自由基、过氧化亚硝基有强烈的清除作用，对单线态氧有淬灭作用；在淬灭自由基的同时，对香豆酸还能抑制产生自由基的酶，促进产生清除自由基的酶。研究表明反式-4-香豆酸对金黄色葡萄球菌、痢疾杆菌、大肠杆菌、绿脓杆菌有不同程度的抑制作用；Bodini等人的研究发现，反式-4-香豆酸能够抑制紫色色杆菌、农杆菌和绿针假单胞菌的群体感应作。反式-4-香豆酸还对博来霉素、过氧化氢和IQ型喳琳致突变物所诱导的沙门氏菌突变有良好的抑制作用；反式-4-香豆酸还能抑制混合功能氧化酶活性，以及环磷酞胺(CP)引起的体细胞和性细胞突变。
反式-4-香豆酸可应用于医药、农业、食品、化妆品等行业。作为药品，反式-4-香豆酸对金黄色葡萄球菌、痢疾杆菌、大肠杆菌、绿脓杆菌有不同程度的抑制作用；反式-4-香豆酸不仅具有降脂作用，还有阻止胃癌发生，减少粥状动脉硬化发生的作用。在农业行业用于植物生长促进剂。在食品行业用于长效杀菌剂和果蔬保鲜防腐剂。在化妆品行业由于反式-4-香豆酸对酪氨酸单酚酶和二酚酶均有抑制作用，所以添加到化妆品里抑制黑色素的生成。
此外，反式-4-香豆酸还是许多具有较高应用价值的化合物的前体或中间体。例如，对香豆酸可以通过氧化，环合反应合成香豆素；反式-4-香豆酸通过降解反应生成对羟基苯甲酸；反式-4-香豆酸还可作为祛痰药杜鹃素、抗肾上腺素药物艾司洛尔等物质合成的中间体，还可以用于临床上治疗轻、中度老年痴呆的药物-加烂他敏的合成。
反式-4-香豆酸的广泛应用，造成了其具有强大的市场需求，但是现有的反式-4-香豆酸工业化生产方法却不能满足这一需求。现有的生产主法主要有以下几种：提取法：从甘蔗渣、玉米皮、麸皮、竹茹等中提取，虽然该方法提取出来的反式-4-香豆酸是纯天然的，但产率低，成本高。化学合成：由对羟基苯甲醛和丙二酸经缩合反应制得，该方法的主要缺陷在于产品非天然，此外，生产过程化学污染大，成本高。
生物转化法生产反式-4-香豆酸的方法有两条途径：一种是酪氨酸/(苯丙氨酸)解氨酶途径，该途径属于EC 4.3.1.23和EC 4.3.1.25下的酶；专利有：一种来源于虎眼万年青的苯丙氨酸解氨酶，其核苷酸序列及应用，专利号为：201310466953.2，目前处于无权-视为撤回状态。一种酵母菌的用途及天然对香豆酸的生产方法，专利号为：201510411910。该途径既有酶(全细胞)催化法也有发酵发生产反式-4-香豆酸，其中201510411910专利为发酵法，产量可达10g/L左右。另外一种是反式-肉桂酸-4-羟化酶途径，该途径属于EC1.14.14.91下的酶；专利有：一种反式肉桂酸-4-羟基化酶及其编码基因与应用，专利号为：201710136604，目前处于审中-实审阶段。该专利所述的方法为发酵法，其辅酶的循环、底物均由宿主菌提供；该方法产率不高，且纯化成本较高。
发明内容
本发明提供了一种酶催化法合成反式-4-香豆酸的方法；反式-肉桂酸-4-单加氧酶(EC1.14.14.91)可催化反式-肉桂酸合成反式-4-香豆酸，同时消耗一分子O2和一个NAD(P)H,生成一个H+和一个NAD(P)+。在单水相或水/有机溶剂的双相反应体系中，加入反式-肉桂酸(原料)，加入微量NAD(P)+或NAD(P)H，加入O2源，加入还原型氢供体，然后加入反式-肉桂酸4-单加氧酶和NAD(P)+还原酶，滴加碱液控制pH，控制反应温度；反应结束后反应液过滤除酶后浓缩，降温结晶可得反式-4-香豆酸反应式如下：
本发明提供了以下技术方案：
一种酶催化法合成反式-4-香豆酸的方法，在单水相或水/有机溶剂的双相反应体系中，加入反式-肉桂酸，加入微量NAD(P)+或NAD(P)H，加入O2源，加入还原型氢供体，然后加入反式-肉桂酸-4-单加氧酶和NAD(P)+还原酶，滴加碱液控制pH，控制反应温度；反应结束后反应液过滤除酶后浓缩，降温结晶可得反式-4-香豆酸；其中，反式-肉桂酸-4-单加氧酶的编号为：EC 1.14.14.91。
当反式-肉桂酸-4-单加氧酶需要的辅酶为NADH时，反应体系中添加微量的NAD+或NADH；当反式-肉桂酸-4-单加氧酶需要的辅酶为NADPH时，反应体系中添加微量的NADP+或NADPH。
所述O2源为空气、氧气或H2O2，向反应体系中通空气或氧气或加入的方式来补充消耗掉的氧；反应体系中氧的浓度可以通过溶氧电极进行监控；可以通过加压的方式提高反应溶液中的氧溶解度。
所述的碱为氨水、NaOH或KOH；pH控制范围为5.0～10.0，优选的，pH控制范围为6.0～8.0，优选的，pH控制范围为6.8～7.5。
所述的还原型氢供体为葡萄糖、异丙醇、甲酸或甲酸盐。
当还原型氢供体为葡萄糖时，反应中加入的NAD(P)+还原酶为葡萄糖脱氢酶，此酶能够通过葡萄糖供氢产生NAD(P)H；当还原型氢供体为异丙醇时，反应中加入的NAD(P)+还原酶为异丙醇脱氢酶，此酶能够通过异丙醇供氢产生NAD(P)H；当还原型氢供体为甲酸或甲酸盐时，反应中加入的NAD(P)+还原酶为甲酸脱氢酶，此酶能够通过甲酸供氢产生NAD(P)H；当反式-肉桂酸-4-单加氧酶所需的辅酶为NADH时，所述的NAD(P)+还原酶为NAD+还原酶，当反式-肉桂酸-4-单加氧酶所需的辅酶为NADPH时，所述的NAD(P)+还原酶为NADP+还原酶。
所述反式-肉桂酸-4-单加氧酶包括但不限于具有以下氨基酸序列的反式-肉桂酸-4-单加氧酶，所述的氨基酸序列可以是SEQ01、SEQ02、SEQ03、SEQ04、SEQ05、SEQ06、SEQ07、SEQ08、SEQ09、SEQ10、SEQ11、SEQ12、SEQ13、SEQ14、SEQ15或SEQ16；或所述反式-肉桂酸-4-单加氧酶是具有所述氨基酸序列的单点和/或多点突变体的反式-肉桂酸-4-单加氧酶；或所述反式-肉桂酸-4-单加氧酶是与所述氨基酸序列有80％以上同源性的氨基酸序列的反式-肉桂酸-4-单加氧酶。
用于表达所述反式-肉桂酸-4-单加氧酶的宿主为大肠杆菌、芽孢杆菌、霉菌或酵母的一种；优选的，其表达体系为大肠杆菌BL21表达体系或者毕赤酵母GS115表达体系。
所述反式-肉桂酸-4-单加氧酶可以是所构建的宿主菌表达后的发酵液或菌泥，也可以是经简单处理的粗酶或经纯化处理的纯酶，还可以是经絮凝剂及陶瓷膜过滤后的液体酶或者是固定化处理后的固定化酶。
与现有技术相比，本发明的有益效果：
本发明的方法属于反式-肉桂酸-4-羟化酶途径，应用酶催化的方法直接以反式-肉桂酸为底物，通过NAD(P)+还原酶完成辅酶的循环，在细胞外高效合成反式-4-香豆酸。与专利201710136604相比，可以构建更加高效的反式-肉桂酸-4-羟基化酶表达系统，而无需考虑细胞物质代谢和能量代谢平衡的问题，单纯提高酶的表达量相对要容易的多；酶催化的方法在相同产量下细胞的用量是发酵法的1/10,甚至1/100或更少；酶催化的方法反应体系简单，底物浓度可高达100g/L以上，摩尔转化率可达98％以上，反应体系简单，杂质少，产品提取纯化成本低。
具体实施方式
以下的实施例仅用于说明本发明的实施方法，本发明的内容包括但不限于以下的实施例。
实施例一
1.反式-肉桂酸-4-单加氧酶(SEQ01)表达菌的构建与发酵表达
将氨基酸序列为SEQ01的反式-肉桂酸-4-单加氧酶进行大肠肝菌密码子优化，构建pET28a-SEQ01(在SEQ01的氮端***麦芽糖酶标签以促进可溶表达)表达载体，将构建好并经过验证正确的表达载体热激转化大肠杆菌BL21(DE3)，转化完成后涂布含Kan的平板，挑取单克隆表达，测酶活性验证；将表达较好的菌株扩增并保存种子库种子；取一支种子库种子扩增，接种发酵罐发酵，OD达到25左右降温至28℃，加入终浓度0.6mM的IPTG诱导，继续补料培养不少于8小时，放罐，离心收集湿菌体，湿菌体可长期冰冻保存。
2.粗酶提取
取100g湿菌体，加入50mM pH7.0±1.0PB缓冲液1L溶解，高压均质机破碎细胞后，加入硫酸铵，使硫酸铵饱和度达到35％，10000rpm离心10min，取上清，继续加入硫酸铵使饱和度达到65％，10000rpm,离心10min，所得固体即为反式-肉桂酸-4-单加氧酶(SEQ01)粗酶，该粗酶可长期-20℃冰冻保存。
3.酶催化反应
使用磁力搅拌，水浴保温25℃，在2L的三口瓶中加入1.0L纯化水，50ml甲醇，反式肉桂酸80g,持续鼓入适量空气，搅拌均匀后***pH计，连接上pH自动控制，设置pH控制范围为7.6±0.3，以氨水做碱源，加入步骤2中的粗酶0.5g,甲醇脱氢酶0.5g,NADP+50mg，启动反应，用HPLC监测反应情况，当转化率不低于98％时结束反应。
4.反式-4-香豆酸氨盐收获
酶催化反应结束，加热至85℃，保温5min，冷却至室温，过滤或离心去除杂质，清液旋蒸浓缩结晶，得反式-4-香豆酸氨盐。
实施例二
1.反式-肉桂酸-4-单加氧酶(SEQ02)表达菌的构建与发酵表达
将氨基酸序列为SEQ02的反式-肉桂酸-4-单加氧酶进行大肠肝菌密码子优化，构建pET22b-SEQ02表达载体，将构建好并经过验证正确的表达载体热激转化大肠杆菌BL21(DE3)，转化完成后涂布含Amp的平板，挑取单克隆表达，测酶活性验证；将表达较好的菌株扩增并保存种子库种子；取一支种子库种子扩增，接种发酵罐发酵，OD达到25左右降温至28℃，加入终浓度0.6mM的IPTG诱导，继续补料培养不少于8小时，放罐，离心收集湿菌体，湿菌体可长期-20℃冰冻保存。
2.酶催化反应
使用磁力搅拌，水浴保温25℃，在2L的三口瓶中加入1.0L纯化水，50ml甲醇，反式肉桂酸80g,持续鼓入适量空气，搅拌均匀后***pH计，连接上pH自动控制，设置pH控制范围为7.6±0.3，以氨水做碱源，加入步骤1中的湿菌体1.0g,甲醇脱氢酶0.5g,NADP+50mg，启动反应，用HPLC监测反应情况，当转化率不低于98％时结束反应。
3.反式-4-香豆酸氨盐收获
酶催化反应结束，加热至85℃，保温5min，冷却至室温，过滤或离心去除杂质，清液旋蒸浓缩结晶，得反式-4-香豆酸氨盐。
实施例三
1.反式-肉桂酸-4-单加氧酶(SEQ03)表达菌的构建与发酵表达
将氨基酸序列为SEQ03的反式-肉桂酸-4-单加氧酶进行大肠肝菌密码子优化，构建pETDuet-SEQ03(MCS1)-GDH(MCS2)(其中MCS1、MCS2分别指多克隆位点1和多克隆位点2，GDH指葡萄糖脱氢酶)，将构建好并经过验证正确的表达载体热激转化大肠杆菌BL21(DE3)，转化完成后涂布含Amp的平板，挑取单克隆表达，测酶活性验证；将表达较好的菌株扩增并保存种子库种子；取一支种子库种子扩增，接种发酵罐发酵，OD达到25左右降温至28℃，加入终浓度0.6mM的IPTG诱导，继续补料培养不少于8小时，放罐，离心收集湿菌体，湿菌体可长期-20℃冰冻保存。
2.粗酶提取
取100g湿菌体，加入50mM pH7.0±1.0PB缓冲液1L溶解，高压均质机破碎细胞后，加入硫酸铵，使硫酸铵饱和度达到35％，10000rpm离心10min，取上清，继续加入硫酸铵使饱和度达到65％，10000rpm,离心10min，所得固体即为反式-肉桂酸-4-单加氧酶(SEQ03)粗酶和葡萄糖脱氢酶的混合酶，该粗酶可长期冰冻保存。
3.酶催化反应
使用磁力搅拌，水浴保温25℃，在2L的三口瓶中加入1.0L纯化水，50ml乙醇，反式肉桂酸80g,持续鼓入适量空气，搅拌均匀后***pH计，连接上pH自动控制，设置pH控制范围为7.6±0.3，以4M NaOH为碱源，加入步骤2中的粗酶1.0g,NADP+50mg，启动反应，用HPLC监测反应情况，当转化率不低于98％时结束反应。
4.反式-4-香豆酸钠盐收获
酶催化反应结束，加热至85℃，保温5min，冷却至室温，过滤或离心去除杂质，清液旋蒸浓缩结晶，得反式-4-香豆酸钠盐。
实施例四
1.反式-肉桂酸-4-单加氧酶(SEQ04)表达菌的构建与发酵表达
将氨基酸序列为SEQ04的反式-肉桂酸-4-单加氧酶进行大肠肝菌密码子优化，构建pETDuet-SEQ04(MCS1)-MDH(MCS2)(其中MCS1、MCS2分别指多克隆位点1和多克隆位点2，MDH指甲醇脱氢酶)，将构建好并经过验证正确的表达载体热激转化大肠杆菌BL21(DE3)，转化完成后涂布含Amp的平板，挑取单克隆表达，测酶活性验证；将表达较好的菌株扩增并保存种子库种子；取一支种子库种子扩增，接种发酵罐发酵，OD达到25左右降温至28℃，加入终浓度0.6mM的IPTG诱导，继续补料培养不少于8小时，放罐，离心收集湿菌体，湿菌体可长期冰冻保存。
2.粗酶提取
取1Kg湿菌体，加入50mM pH7.0±1.0PB缓冲液10L溶解，高压均质机破碎细胞后，以200KD的陶瓷膜除杂后，再以10KD的陶瓷膜浓缩至2L以下体积，所得溶液为反式-肉桂酸-4-单加氧酶(SEQ04)粗酶和甲醇脱氢酶的混合酶液，该粗酶液可长期冰冻保存。
3.酶催化反应
是哟磁力搅拌，水浴保温25℃，在10L的三口瓶中加入6.0L纯化水，350ml甲醇，反式肉桂酸500g,持续鼓入适量空气，搅拌均匀后***pH计，连接上pH自动控制，设置pH控制范围为7.6±0.3，以4M NaOH为碱源，加入步骤2中的粗酶50g,NADP+0.5mg，启动反应，用HPLC监测反应情况，当转化率不低于98％时结束反应。
4.反式-4-香豆酸钠盐收获
酶催化反应结束，以3KD以下的陶瓷膜过滤除杂，除杂后滤液以纳滤膜浓缩或者双效浓缩后至结晶反应釜内真空结晶，得反式-4-香豆酸钠盐。
以上实施例中使用的氨基酸序列表如下：
SEQ01 Organism:Arabidopsis thaliana
SEQ02 Organism:Glycyrrhiza echinata
SEQ03 Organism:Pisum sativum
SEQ04 Organism:Populus tremuloides
SEQ05 Organism:Phaseolus aureus
SEQ06 Organism:Catharanthus roseus
SEQ07 Organism:Helianthus tuberosus
SEQ08 Organism:Petroselinum crispum
SEQ09 Organism:Ruta graveolens
序列表
<120> 一种酶催化法合成反式-4-香豆酸的方法
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Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
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Arg Pro Glu Arg Phe Leu Glu Glu Glu Ser His Val Glu Ala Asn Gly
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Gly Glu Ile Asn Asp Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn
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Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
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Arg Pro Glu Arg Phe Leu Glu Glu Glu Ala His Val Glu Ala Asn Gly
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Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro
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Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Ile Gly Arg Leu
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Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Ile Asp
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Thr Ser Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser
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Thr Ile Val Ala Lys Pro Arg Ala Phe
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Ile Leu Val Ala Ile Leu Val Ser Lys Leu Arg Gly Lys Arg Phe Lys
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Leu Pro Pro Gly Pro Leu Pro Val Pro Val Phe Gly Asn Trp Leu Gln
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Val Gly Asp Asp Leu Asn His Arg Asn Leu Thr Asp Leu Ala Lys Lys
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Phe Gly Asp Ile Leu Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val
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Val Ser Ser Pro Glu Leu Ser Lys Glu Val Leu His Thr Gln Gly Val
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Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln
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Val Lys Lys Asn Pro Gly Ala Ala Thr His Gly Ile Val Leu Arg Arg
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Arg Leu Gln Leu Met Met Tyr Asn Asn Met Tyr Arg Ile Met Phe Asp
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Arg Arg Phe Glu Ser Glu Glu Asp Pro Leu Phe Asn Lys Leu Lys Ala
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Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Gly Tyr Leu Lys
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Ile Cys Gln Glu Val Lys Glu Arg Arg Leu Gln Leu Phe Lys Asp Tyr
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Phe Val Asp Glu Arg Lys Lys Leu Ala Ser Thr Lys Asn Met Cys Asn
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Glu Gly Leu Lys Cys Ala Ile Asp His Ile Leu Asp Ala Gln Lys Lys
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Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn
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Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
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Glu Leu Val Asn His Pro Glu Ile Gln Lys Lys Leu Arg His Glu Leu
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Asp Thr Leu Leu Gly Pro Gly His Gln Ile Thr Glu Pro Asp Thr Tyr
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Lys Leu Pro Tyr Leu Asn Ala Val Val Lys Glu Thr Leu Arg Leu Arg
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Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys
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Leu Gly Gly Phe Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala
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Trp Trp Leu Ala Asn Asn Pro Ala His Trp Lys Asn Pro Glu Glu Phe
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Arg Pro Glu Arg Phe Leu Glu Glu Glu Ala Lys Val Glu Ala Asn Gly
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Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro
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Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Leu Gly Arg Leu
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Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Ile Asp
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Thr Ser Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser
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Thr Ile Val Ala Lys Pro Arg Ser Phe
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Met Asp Leu Leu Leu Leu Glu Lys Thr Leu Leu Gly Leu Phe Leu Ala
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Ala Val Val Ala Ile Val Val Ser Lys Leu Arg Gly Lys Arg Phe Lys
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Leu Pro Pro Gly Pro Leu Pro Val Pro Ile Phe Gly Asn Trp Leu Gln
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Val Gly Asp Asp Leu Asn His Arg Asn Leu Thr Gln Leu Ala Lys Arg
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Phe Gly Asp Ile Phe Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val
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Val Ser Ser Pro Asp Leu Ala Lys Glu Val Leu His Thr Gln Gly Val
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Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly
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Glu Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys
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Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln
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Gln Tyr Arg His Gly Trp Glu Ala Glu Ala Ala Ala Val Val Asp Asp
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Val Arg Lys Asn Pro Asp Ala Ala Val Ser Gly Leu Val Ile Arg Arg
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Arg Leu Gln Leu Met Met Tyr Asn Asn Met Tyr Arg Ile Met Phe Asp
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Arg Arg Phe Glu Ser Glu Glu Asp Pro Leu Phe Gln Arg Leu Lys Ala
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Leu Asn Gly Glu Arg Ser Arg Leu Ala Gln Ser Phe Glu Tyr Asn Tyr
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Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Lys Gly Tyr Leu Lys
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Ile Cys Lys Glu Val Lys Glu Thr Arg Leu Lys Leu Phe Lys Asp Tyr
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Phe Val Asp Glu Arg Lys Asn Ile Gly Ser Thr Lys Ser Thr Asn Asn
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Glu Gly Leu Lys Cys Ala Ile Asp His Ile Leu Asp Ala Glu Lys Lys
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Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn
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Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
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Glu Leu Val Asn His Pro Glu Ile Gln Gln Lys Val Arg Asp Glu Ile
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Asp Arg Val Leu Gly Val Gly His Gln Val Thr Glu Pro Asp Ile Gln
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Lys Leu Pro Tyr Leu Gln Ala Val Val Lys Glu Thr Leu Arg Leu Arg
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Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys
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Leu Gly Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala
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Trp Trp Leu Ala Asn Asn Pro Ala His Trp Lys Lys Pro Glu Glu Phe
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Arg Pro Glu Arg Phe Phe Glu Glu Glu Ser His Val Glu Ala Asn Gly
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Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro
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Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Leu Gly Arg Leu
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Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Gln Ile Asp
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Thr Ser Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser
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Thr Val Val Ala Lys Pro Arg Ser Phe
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<210> 6
<211> 505
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Met Asp Leu Leu Leu Leu Glu Lys Thr Leu Leu Gly Leu Phe Ala Ala
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Ile Ile Val Ala Ser Ile Val Ser Lys Leu Arg Gly Lys Lys Phe Lys
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Leu Pro Pro Gly Pro Ile Pro Val Pro Val Phe Gly Asn Trp Leu Gln
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Val Gly Asp Asp Leu Asn His Arg Asn Leu Ser Asp Tyr Ala Lys Lys
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Phe Gly Glu Ile Phe Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val
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Val Ser Ser Pro Glu Leu Ala Lys Glu Val Leu His Thr Gln Gly Val
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Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly
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Lys Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys
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Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln
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Gln Tyr Arg Tyr Gly Trp Glu Glu Glu Ala Ala Arg Val Val Glu Asp
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Val Lys Lys Asn Pro Glu Ser Ala Thr Asn Gly Ile Val Leu Arg Arg
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Arg Leu Gln Leu Met Met Tyr Asn Asn Met Tyr Arg Ile Met Phe Asp
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Arg Arg Phe Glu Ser Glu Asp Asp Pro Leu Phe Val Lys Leu Lys Ala
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Leu Asn Gly Glu Arg Ser Arg Leu Ala Gln Gly Phe Glu Tyr Asn Tyr
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Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Gly Tyr Leu Arg
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Ile Cys Lys Glu Val Lys Glu Arg Arg Leu Gln Leu Phe Lys Asp Tyr
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Phe Val Asp Glu Arg Lys Lys Phe Gly Ser Thr Lys Ser Met Asp Asn
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Asn Ser Leu Lys Cys Ala Ile Asp His Ile Leu Glu Ala Gln Gln Lys
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Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn
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Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
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Glu Leu Val Asn His Pro Glu Ile Gln Lys Lys Leu Arg Asp Glu Leu
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Glu Thr Val Leu Gly Pro Gly Val Gln Ile Thr Glu Pro Asp Thr Tyr
340 345 350
Lys Leu Pro Tyr Leu Gln Ala Val Ile Lys Glu Thr Leu Arg Leu Arg
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Met Ala Ile Pro Leu Phe Leu Pro His Met Asn Leu His Asp Ala Lys
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Leu Gly Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala
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Trp Phe Leu Ala Asn Asn Pro Glu His Trp Lys Lys Pro Glu Glu Phe
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Arg Pro Glu Arg Phe Leu Glu Glu Glu Ser Lys Val Glu Ala Asn Gly
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Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro
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Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Ile Gly Arg Leu
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Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Lys Ser Lys Ile Asp
465 470 475 480
Thr Ser Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser
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Thr Ile Val Leu Lys Pro Arg Thr Phe
500 505
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<211> 505
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
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Met Asp Leu Leu Leu Ile Glu Lys Thr Leu Val Ala Leu Phe Ala Ala
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Ile Ile Gly Ala Ile Leu Ile Ser Lys Leu Arg Gly Lys Lys Phe Lys
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Leu Pro Pro Gly Pro Ile Pro Val Pro Ile Phe Gly Asn Trp Leu Gln
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Val Gly Asp Asp Leu Asn His Arg Asn Leu Thr Asp Leu Ala Lys Arg
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Phe Gly Glu Ile Leu Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val
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Val Ser Ser Pro Glu Leu Ala Lys Glu Val Leu His Thr Gln Gly Val
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Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly
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Lys Gly Gln Asp Met Val Phe Thr Val Tyr Gly Glu His Trp Arg Lys
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Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln
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Gln Tyr Arg Tyr Gly Trp Glu Ala Glu Ala Ala Ala Val Val Asp Asp
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Val Lys Lys Asn Pro Ala Ala Ala Thr Glu Gly Ile Val Ile Arg Arg
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Arg Leu Gln Leu Met Met Tyr Asn Asn Met Phe Arg Ile Met Phe Asp
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Arg Arg Phe Glu Ser Glu Asp Asp Pro Leu Phe Leu Lys Leu Lys Ala
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Leu Asn Gly Glu Arg Ser Arg Leu Ala Gln Ser Phe Glu Tyr Asn Tyr
210 215 220
Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Asn Tyr Leu Lys
225 230 235 240
Leu Cys Lys Glu Val Lys Asp Lys Arg Ile Gln Leu Phe Lys Asp Tyr
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Phe Val Asp Glu Arg Lys Lys Ile Gly Ser Thr Lys Lys Met Asp Asn
260 265 270
Asn Gln Leu Lys Cys Ala Ile Asp His Ile Leu Glu Ala Lys Glu Lys
275 280 285
Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn
290 295 300
Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
305 310 315 320
Glu Leu Val Asn His Pro Glu Ile Gln Ala Lys Leu Arg His Glu Leu
325 330 335
Asp Thr Lys Leu Gly Pro Gly Val Gln Ile Thr Glu Pro Asp Val Gln
340 345 350
Asn Leu Pro Tyr Leu Gln Ala Val Val Lys Glu Thr Leu Arg Leu Arg
355 360 365
Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala Lys
370 375 380
Leu Gly Gly Phe Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala
385 390 395 400
Trp Trp Leu Ala Asn Asn Pro Asp Gln Trp Lys Lys Pro Glu Glu Phe
405 410 415
Arg Pro Glu Arg Phe Leu Glu Glu Glu Ala Lys Val Glu Ala Asn Gly
420 425 430
Asn Asp Phe Arg Tyr Leu Pro Phe Gly Val Gly Arg Arg Ser Cys Pro
435 440 445
Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Ile Gly Arg Leu
450 455 460
Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Ile Asp
465 470 475 480
Thr Asp Glu Lys Gly Gly Gln Phe Ser Leu His Ile Leu Lys His Ser
485 490 495
Thr Ile Val Ala Lys Pro Arg Ser Phe
500 505
<210> 8
<211> 506
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Met Met Asp Phe Val Leu Leu Glu Lys Ala Leu Leu Gly Leu Phe Ile
1 5 10 15
Ala Thr Ile Val Ala Ile Thr Ile Ser Lys Leu Arg Gly Lys Lys Leu
20 25 30
Lys Leu Pro Pro Gly Pro Ile Pro Val Pro Val Phe Gly Asn Trp Leu
35 40 45
Gln Val Gly Asp Asp Leu Asn Gln Arg Asn Leu Val Asp Tyr Ala Lys
50 55 60
Lys Phe Gly Asp Leu Phe Met Leu Arg Met Gly Gln Arg Asn Leu Val
65 70 75 80
Val Val Ser Ser Pro Glu Leu Ala Lys Asp Val Leu His Thr Gln Gly
85 90 95
Val Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr
100 105 110
Gly Lys Gly Gln Asp Met Val Phe Thr Val Tyr Ser Glu His Trp Arg
115 120 125
Lys Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val
130 135 140
Gln Gln Tyr Arg Phe Gly Trp Glu Asp Glu Ala Ala Arg Val Val Glu
145 150 155 160
Asp Val Lys Ala Asn Pro Glu Ala Ala Thr Asn Gly Ile Val Leu Arg
165 170 175
Asn Arg Leu Gln Leu Leu Met Tyr Asn Asn Met Tyr Arg Ile Met Phe
180 185 190
Asp Arg Arg Phe Glu Ser Val Asp Asp Pro Leu Phe Leu Lys Leu Lys
195 200 205
Ala Leu Asn Gly Glu Arg Ser Arg Leu Ala Gln Ser Phe Glu Tyr His
210 215 220
Phe Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Gly Tyr Leu
225 230 235 240
Lys Leu Cys Gln Glu Ile Lys Asp Lys Arg Leu Lys Leu Phe Lys Asp
245 250 255
Tyr Phe Val Asp Glu Arg Lys Lys Leu Glu Ser Ile Lys Ser Val Asp
260 265 270
Asn Asn Ser Leu Lys Cys Ala Ile Asp His Ile Ile Glu Ala Gln Gln
275 280 285
Lys Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile
290 295 300
Asn Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile
305 310 315 320
Ala Glu Leu Val Asn Asn Pro Glu Ile Gln Lys Lys Leu Arg His Glu
325 330 335
Leu Asp Thr Val Leu Gly Ala Gly Val Gln Ile Cys Glu Pro Asp Val
340 345 350
Gln Lys Leu Pro Tyr Leu Gln Ala Val Ile Lys Glu Thr Leu Arg Tyr
355 360 365
Arg Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu His Asp Ala
370 375 380
Lys Leu Ala Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn
385 390 395 400
Ala Trp Trp Leu Ala Asn Asn Pro Ala His Trp Asn Lys Pro Asp Glu
405 410 415
Phe Arg Pro Glu Arg Phe Leu Glu Glu Glu Ser Lys Val Glu Ala Asn
420 425 430
Gly Asn Asp Phe Lys Tyr Ile Pro Phe Gly Val Gly Arg Arg Ser Cys
435 440 445
Pro Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Val Ile Gly Arg
450 455 460
Leu Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Ile
465 470 475 480
Asp Thr Ala Glu Lys Gly Gly Gln Phe Ser Leu Gln Ile Leu Lys His
485 490 495
Ser Thr Ile Val Cys Lys Pro Arg Ser Leu
500 505
<210> 9
<211> 506
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 9
Met Asp Leu Leu Leu Leu Glu Lys Ala Leu Leu Gly Leu Phe Ala Ala
1 5 10 15
Ala Val Val Ala Ile Ala Val Ser Lys Leu Arg Gly Lys Arg Phe Lys
20 25 30
Leu Pro Pro Gly Pro Leu Gly Phe Pro Val Phe Gly Asn Trp Leu Gln
35 40 45
Val Gly Asp Asp Leu Asn Gln Arg Lys Leu Ala Asn Leu Ser Lys Lys
50 55 60
Phe Gly Asp Val Tyr Leu Leu Arg Met Gly Gln Arg Asn Leu Val Val
65 70 75 80
Val Ser Ser Pro Glu Met Ala Lys Glu Val Leu His Thr Gln Gly Val
85 90 95
Glu Phe Gly Ser Arg Thr Arg Asn Val Val Phe Asp Ile Phe Thr Gly
100 105 110
Lys Gly Gln Asp Met Val Phe Thr Val Tyr Ser Glu His Trp Arg Lys
115 120 125
Met Arg Arg Ile Met Thr Val Pro Phe Phe Thr Asn Lys Val Val Gln
130 135 140
Gln Gln Arg Phe Asn Trp Glu Asp Glu Ala Ala Arg Val Val Glu Asp
145 150 155 160
Val Lys Lys Asp Pro Gln Ala Ala Thr Thr Gly Ile Val Leu Arg Arg
165 170 175
Arg Leu Gln Leu Leu Met Tyr Asn Asn Met Tyr Arg Ile Met Phe Asp
180 185 190
Arg Arg Phe Glu Ser Val Asp Asp Pro Leu Phe Asn Lys Leu Lys Ala
195 200 205
Leu Asn Gly Glu Arg Ser Arg Leu Ala Gln Ser Phe Glu Tyr Asn Tyr
210 215 220
Gly Asp Phe Ile Pro Ile Leu Arg Pro Phe Leu Arg Gly Tyr Leu Lys
225 230 235 240
Leu Val Lys Glu Val Lys Glu Arg Arg Leu Lys Leu Phe Lys Asp Tyr
245 250 255
Phe Val Glu Glu Arg Lys Lys Leu Thr Ser Thr Lys Ser Met Thr Glu
260 265 270
Glu Asn Phe Lys Cys Ala Ile Asp His Val Leu Asp Ala Gln Gln Lys
275 280 285
Gly Glu Ile Asn Glu Asp Asn Val Leu Tyr Ile Val Glu Asn Ile Asn
290 295 300
Val Ala Ala Ile Glu Thr Thr Leu Trp Ser Ile Glu Trp Gly Ile Ala
305 310 315 320
Glu Leu Val Asn His Pro Asp Ile Gln Lys Lys Leu Arg Ala Glu Ile
325 330 335
Asp Arg Val Leu Gly Pro Asp His Gln Ile Thr Glu Pro Asp Thr His
340 345 350
Lys Leu Pro Tyr Leu Gln Ala Val Ile Lys Glu Thr Leu Arg Leu Arg
355 360 365
Met Ala Ile Pro Leu Leu Val Pro His Met Asn Leu Asn Asp Ala Lys
370 375 380
Leu Ala Gly Tyr Asp Ile Pro Ala Glu Ser Lys Ile Leu Val Asn Ala
385 390 395 400
Trp Trp Leu Ala Asn Asn Pro Ala His Trp Lys Asp Pro Gln Val Phe
405 410 415
Arg Pro Glu Arg Phe Leu Glu Glu Glu Ser Gly Val Glu Ala Asn Gly
420 425 430
Asn Asp Phe Arg Tyr Ile Pro Phe Gly Val Gly Arg Arg Ser Cys Pro
435 440 445
Gly Ile Ile Leu Ala Leu Pro Ile Leu Gly Ile Thr Ile Gly Arg Met
450 455 460
Val Gln Asn Phe Glu Leu Leu Pro Pro Pro Gly Gln Ser Lys Ile Asp
465 470 475 480
Thr Ser Glu Lys Gly Gly Gln Phe Ser Leu Phe Ile Leu Asn His Ser
485 490 495
Thr Ile Val Leu Lys Pro Arg Ser Ser Val
500 505
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