CN113755462A - β-氨基酸脱氢酶突变体及其应用 - Google Patents

β-氨基酸脱氢酶突变体及其应用 Download PDF

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CN113755462A
CN113755462A CN202110133031.4A CN202110133031A CN113755462A CN 113755462 A CN113755462 A CN 113755462A CN 202110133031 A CN202110133031 A CN 202110133031A CN 113755462 A CN113755462 A CN 113755462A
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冯进辉
刘娜
陈曦
李键煚
吴洽庆
朱敦明
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Tianjin Institute of Industrial Biotechnology of CAS
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Abstract

本发明提供了一种β‑氨基酸脱氢酶突变体及其在β‑氨基酸类化合物合成中的应用，具体地，所述β‑氨基酸脱氢酶是来源于Candidatus Cloacamonas acidaminovorans的L‑赤式‑3,5‑二氨基己酸脱氢酶。所述β‑氨基酸脱氢酶突变蛋白为非天然蛋白，且所述突变蛋白具有显著提高催化β‑酮酸类化合物生成β‑氨基酸的活性，并且所述突变蛋白在野生型β‑氨基酸脱氢酶的两个或两个以上的与酶催化活性相关的核心氨基酸发生突变。本发明提供的β‑氨基酸脱氢酶突变体底物谱有所拓宽，且可以显著提高β‑氨基酸脱氢酶催化合成β‑氨基酸的活性。

Description

β-氨基酸脱氢酶突变体及其应用
技术领域
本发明涉及酶和酶工程领域，具体地，本发明涉及一种催化活力提高的β-氨基酸脱氢酶及其在催化合成手性β-氨基酸类化合物中的应用。
背景技术
由于手性β-氨基酸是许多复杂药物分子、生物活性物质的重要合成砌块或其本身就具有一定的生理活性，在医药、化工等领域具有重要的应用价值，因此，开发高效、绿色的制备方法具有重大的研究意义。而酶法催化相对于化学合成法而言，一般具有选择性高、反应条件温和、环境友好等特点。
氨基酸脱氢酶(EC1.4.1.X)，是一类依赖于辅因子NAD(H)或NADP(H)的氧化还原酶，催化可逆的氨基酸氧化脱氨和酮酸还原胺化反应。这类酶不仅在氨基酸代谢途径中起着非常重要的作用，而且在合成氨基酸及手性胺类化合物方面具有极大优势和潜在应用价值。β-氨基酸脱氢酶可以不对称还原胺化β-酮酸合成相应的β-氨基酸。但是，文献已报道的β-氨基酸脱氢酶只有赖氨酸降解途径中的L-赤式-3,5-二氨基己酸脱氢酶(3,5-DAHDH,EC.1.4.1.11)，且这类酶只对其天然底物L-赤式-3,5-二氨基己酸((3S,5S)-DAH)表现出明显的催化活性，有较强的底物专一性，可以转化的底物非常有限。来源于CandidatusCloacamonas acidaminovorans的3,5-DAHDH基因在大肠杆菌(Escherichia coli)中实现异源过量表达后，通过分子改造和大量筛选获得了对非天然底物活性明显提高的突变体，并首次利用β-氨基酸脱氢酶与腈水解酶或脂肪酶偶联两步法转化β-酮腈或β-酮酸酯底物，实现了(R)-β-高甲硫氨酸、(R)-β-苯丙氨酸和(S)-β-氨基丁酸的合成(D.Zhang,X.Chen,et.al.,ACS Catal.,2015,5(4),2220-2224.)。
Figure BDA0002924143720000011
但目前已有的β-氨基酸脱氢酶突变体催化效率仍然很低，转化合成手性β-氨基酸的收率只有12-22％，不能满足工业生产的要求。因此，通过定向进化的方法对β-氨基酸脱氢酶进行改造，扩展其底物谱，并进一步提高酶的催化活性，对手性β-氨基酸的合成研究和工业应用具有重要意义。
发明内容
为了解决上述问题，本发明提供了一种通过基因工程手段改造后的β-氨基酸脱氢酶突变体，具体地，改造后的β-氨基酸脱氢酶突变体合成手性β-氨基酸的活力显著提高。
本发明第一方面提供了一种β-氨基酸脱氢酶突变体，所述的β-氨基酸脱氢酶突变蛋白与SEQ ID NO.:2所示的氨基酸序列具有至少90％同一性，且所述突变蛋白具有合成手性β-氨基酸的能力，并且催化活性显著提高。
本发明提供的β-氨基酸脱氢酶突变体获得首先是：在对应于SEQ ID NO.:1的氨基酸1至352中的位点将第310位谷氨酸(E)突变为甘氨酸(G)，其氨基酸序列为SEQ ID NO.:2。
随后进一步获得的突变体是在对应于β-氨基酸脱氢酶突变体氨基酸序列SEQ IDNO.:2的氨基酸1至352中的位点159、179、180、323、324、328在内的一个或多个位点突变获得的β-氨基酸脱氢酶突变体。
在另一优选例中，所述β-氨基酸脱氢酶的催化底物包括β-氨基酸类或β-酮酸类化合物。
在另一优选例中，β-氨基酸类和β-酮酸类化合物选择下组：
Figure BDA0002924143720000021
在另一优选例中，所述β-氨基酸脱氢酶突变体具有显著提高催化β-酮酸类化合物反应或动力学拆分β-氨基酸消旋体类化合物生成手性β-氨基酸类化合物的活性。
本发明第二方面提供了一种β-氨基酸脱氢酶突变体或者编码其基因工程菌在制备手性β-氨基酸类化合物中的应用，包括步骤：
(i)将本发明第一方面所述的β-氨基酸脱氢酶突变体与反应底物接触，进行催化反应，从而获得所述β-氨基酸类化合物；
(ii)任选地，分离并纯化所述β-氨基酸类化合物。
具体地，所述催化反应以含β-氨基酸脱氢酶突变体编码基因工程菌经发酵培养获得的湿菌体为催化剂，以脂肪水解酶水解β-酮酸酯/腈水解酶水解β-酮腈类化合物得到的β-酮酸类化合物为底物，或者以β-氨基酸消旋体类化合物为底物，以pH为6.0-11.0的缓冲液为反应介质，在25℃-50℃条件下进行反应；
在另一优选例中，所述反应具有选自下组的一个或多个特点：
(i)反应体系含有菌体量为10-100g/L，更佳地，10-30g/L；
(ii)反应体系的pH为6.0-11.0，较佳地，7-10，更佳地，8.5；
(iii)反应体系温度为25℃-50℃，较佳地，25℃-35℃，更佳地，30℃。
反应体系助溶剂可以无助溶剂，或者未乙腈、丙酮、甲醇、乙醇、二甲基亚砜、N，N-二甲基甲酰胺、二氯甲烷、1,4-二氧己环，较佳地，无助溶剂、甲醇、乙醇、二甲基亚砜、丙酮，更佳地，二甲基亚砜。
在另一优选例中，所述β-氨基酸脱氢酶突变体具有选自下组的一个或多个特征：
(a)与野生型的β-氨基酸脱氢酶相比，催化底物浓度为1-200g/L，更佳地，底物浓度为10-100g/L；
(b)与野生型的β-氨基酸脱氢酶相比，催化获得的β-氨基酸类化合物的转化率≥50％，较佳地，≥90％，更佳地，≥95％；
(c)与野生型的β-氨基酸脱氢酶相比，催化获得的β-氨基酸类化合物的ee值≥20％，较佳地，≥90％，更佳地，≥99％；
附图说明
图1β-氨基酸脱氢酶(3,5-DAHDHcca)突变体蛋白经过纯化后电泳检测结果；
其中，M表示Marker，1-8为部分突变体蛋白。
具体实施方式
术语
如本文所用，术语“AxxB”表示第xx位的氨基酸A变为氨基酸B，例如“V159A”表示第159位的缬氨酸V突变为丙氨酸A，以此类推。
在本发明的优选实施方式中，本发明的β-氨基酸脱氢酶突变体的制备方法如下：大肠杆菌为表达宿主。
具体地，该制备方法包括以下步骤：(1)β-氨基酸脱氢酶相应突变位点的基因构建到pET-21b(+)表达载体上，获得带有目的酶基因的重组质粒。(2)将重组质粒转入宿主菌细胞(优选大肠杆菌BL21(DE3))，获得相应的工程菌株。(3)将工程菌株接种至LB培养基中，37℃培养6小时，加入0.5mM的异丙基硫代半乳糖苷(IPTG)，25℃培养6-12小时。(4)离心收集菌体。
实施例1:β-氨基酸脱氢酶突变体E310G重组表达质粒和重组表达转化体的制备
将SEQ ID NO.:1的氨基酸序列对应的核苷酸序列全合成并克隆到pET-21b(+)载体的限制性内切酶位点NdeI和HindIII间，得到重组质粒pET-21b(+)-3,5-DAHDHcca，进一步转化至表达宿主E.coliBL21(DE3)，挑取阳性克隆，即获得重组表达转化体E.coliBL21(DE3)/pET-21b(+)-3,5-DAHDHcca。
构建β-氨基酸脱氢酶突变体E310G：以pET-21b(+)-3,5-DAHDHcca作为模板，设计引物E310G-F:CAGGAGGAATTGGTGCCGATG，E310G-R:TTCCTCCTGCGCCCAGAGCTG，采用全质粒PCR的方法构建突变体E310G，用高保真聚合酶KOD-plus进行PCR。PCR反应条件如下：总体积为50μL的PCR反应体系中，加入5μL 10×KODbuffer，5μLdNTP(2mM)，2μLMgSO4(25mM)，模板20～100ng，一对突变引物各1μL(10μM)，1μL KOD聚合酶，加灭菌蒸馏水至50μL。PCR反应程序：(1)94℃变性3min，(2)94℃变性30sec，(3)55℃退火30sec，(4)68℃延伸7min，步骤(2)～(4)共进行20～30个循环。4℃保存PCR产物。PCR产物经琼脂糖凝胶电泳分析验证后，加入限制性内切酶DpnI在37℃消化2h。将消化产物转入E.coliBL21(DE3)感受态细胞并涂布于含有氨苄抗生素的平板中，置于37℃培养箱中静置培养约12h。挑取阳性克隆，即获得重组表达转化体E.coli BL21(DE3)/pET-21b(+)-3,5-DAHDHcca-E310G。
实施例2:β-氨基酸脱氢酶单点饱和突变体库的构建
构建β-氨基酸脱氢酶单点饱和突变体库：根据β-氨基酸脱氢酶3,5-DAHDHcca的晶体结构，选取其底物结合口袋内的残基，以pET-21b(+)-3,5-DAHDHcca-E310G作为模板，分别进行定点饱和突变，突变体重组表达质粒和重组表达转化体的构建方法如实施例1。将测序确证突变成功的突变体挑取到24孔板中进行培养，对表达的蛋白进行活力检测。通过检测340nm下NADPH吸光值的变化来计算β-氨基酸脱氢酶氧化脱氨方向的催化活性。检测底物为(S)-3-氨基己酸(A)、(S)-β-高赖氨酸(B)、(R)-β-高甲硫氨酸(C)、(R)-β-高丝氨酸(D)、(S)-β-高谷氨酸(E)、(S)-β-高谷氨酰胺(F)、(R)-β-苯丙氨酸(G)、(S)-β-高苯丙氨酸(H)和(S)-β-高酪氨酸(I)，结果见表1，获得β-氨基酸脱氢酶催化活性提高的突变位点分别为159L、179H/S、180D、323S/T、324S、328I/F/R/N/D，活力最优的突变体分别为1、2、3、4、5、6、7、8、9、10、11和12，蛋白序列如SEQ ID NO.:3-14所示。
表1以E310G为模板进行的突变结果
Figure BDA0002924143720000051
实施例3:β-氨基酸脱氢酶组合突变体库的构建
构建β-氨基酸脱氢酶的组合突变体：根据单点饱和突变结果，针对实施例2中的不同β-氨基酸底物选取活力提高最优突变体为模板，分别构建组合突变体库。
首先是以E310G/G323S为模板进行突变，突变体重组表达质粒和重组表达转化体的构建方法如实施例1，活性测定方法如实施例2。经对实施例2中九种β-氨基酸底物的活力进行测定，结果见表2。获得β-氨基酸脱氢酶催化活性提高的突变位点分别为179H/S、180D、328I/F/R/N/D，选择出活力最高的突变体13、14、15、16、17、18、19和20，蛋白序列如SEQ IDNO.:15-22所示。
表2以E310G/G323S为模板进行的突变结果
Figure BDA0002924143720000061
随后以E310G/G323T为模板进行突变，突变体重组表达质粒和重组表达转化体的构建方法如实施例1，活性测定方法如实施例2。经对实施例2中九种β-氨基酸底物的活力进行测定，结果显示在E310G/G323T上进行叠加后的突变体针对九种β-氨基酸底物的活力均未超过以E310G/G323S为模板的突变体活力(表3)。
表3以E310G/G323T为模板进行的突变结果
Figure BDA0002924143720000062
Figure BDA0002924143720000071
以E310G/G323S/H328N为模板进行突变，突变体重组表达质粒和重组表达转化体的构建方法如实施例1，活性测定方法如实施例2。经对实施例2中九种β-氨基酸底物的活力进行测定，结果见表4。获得β-氨基酸脱氢酶催化活性提高的突变位点分别为179S/H，选择出活力最高的突变体21和22蛋白序列如SEQ ID NO.:23,24所示。
表4以E310G/G323S/H328N为模板进行的突变结果
Figure BDA0002924143720000072
以A179H/E310G/G323S为模板进行突变，对328位点进行突变，突变体重组表达质粒和重组表达转化体的构建方法如实施例1，活性测定方法如实施例2。经对实施例2中九种β-氨基酸底物的活力进行测定，结果见表5。获得β-氨基酸脱氢酶催化活性提高的突变体分别为H328I/R，为突变体23和24，蛋白序列如SEQ ID NO.:25，26所示。
表5以A179H/E310G/G323S为模板进行的突变结果
Figure BDA0002924143720000073
Figure BDA0002924143720000081
实施例4：β-氨基酸脱氢酶突变体的诱导表达及纯化
将上述突变体1-24的基因工程菌的单菌落分别接入4mL含有氨苄抗生素的LB液体培养基(蛋白胨10g/L，酵母粉5g/L，NaCl 10g/L)中，于37℃，200rpm摇床中培养过夜，即为种子液。将过夜培养的种子液以1％的接种量转接到50mL含有氨苄抗生素的LB培养基，37℃，200rpm培养至OD600为0.6-1.0左右，加入0.5mM的IPTG，并置于25℃，200rpm诱导8～12h。4℃，6000rpm条件下离心收集菌体。用磷酸钠缓冲液(50mM，pH 8.0，含5％甘油，200mMNaCl，30mM咪唑)重悬菌体，并用高压匀浆机破碎，4℃，12000rpm条件下离心留取上清液，然后采用金属亲和层析(镍柱)法纯化回收目的蛋白。其中，洗脱目的蛋白所用磷酸钠缓冲液中的咪唑浓度为120mM。目的蛋白经超滤除盐、浓缩后即得β-氨基酸脱氢酶突变体纯酶液。SDS-PAGE电泳图谱显示纯化所得蛋白条带单一，如图1所示，其中M表示Marker，1为突变体G323S/H328I的纯化蛋白，2为突变体G323S/H328F的纯化蛋白，3为突变体G323S/H328R的纯化蛋白，4为突变体G323S/H328N的纯化蛋白，5为突变体G323S/H328D的纯化蛋白，6为突变体A179H/G323S/H328I的纯化蛋白，7为突变体A179H/G323S/H328R的纯化蛋白，8为突变体A179S/G323S/H328N的纯化蛋白。结果表明，本实施例的方法能够获得较纯的突变体蛋白，单亚基蛋白分子量为39kD，纯度>95％。
实施例5：β-氨基酸脱氢酶突变体重组菌催化β-酮酸类化合物的方法
将底物丁酰乙酸乙酯(2M，DMSO溶解)加入到碳酸钠缓冲液(pH 8.5，100mM)至终浓度为0.5M，加入5mg/mL脂肪水解酶Novozyme 435，于30℃水解3h以获得β-酮酸底物，水解反应期间需用固体碳酸钠调节pH至8.5左右。
(1)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体16(蛋白序列如SEQ ID NO.:18所示)，离心收集菌体，以菌体作为生物催化剂。取0.8g菌体重悬于27mL碳酸钠-碳酸氢钠缓冲液(pH 8.5，100mM，含200mM NH4Cl)，，加入0.5g/L NADP+，3倍当量的葡萄糖，60mg葡萄糖脱氢酶冻干酶粉，共分批补加上述0.5M的β-酮酸底物12mL(终浓度20g/L)，于30℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，产率为95％，分离收率87％，ee值≥99％(S)。
(2)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体19(蛋白序列如SEQ ID NO.:21所示)，离心收集菌体，以菌体作为生物催化剂。取0.8g菌体重悬于27mL碳酸钠-碳酸氢钠缓冲液(pH 8.5，100mM，含200mM NH4Cl)，，加入0.5g/L NADP+，3倍当量的葡萄糖，60mg葡萄糖脱氢酶冻干酶粉，共分批补加上述0.5M的β-酮酸底物12mL(终浓度20g/L)，于30℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，产率为95％，分离收率89％，ee值≥99％(S)。
(3)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体21(蛋白序列如SEQ ID NO.:23所示)，离心收集菌体，以菌体作为生物催化剂。取0.8g菌体重悬于27mL碳酸钠-碳酸氢钠缓冲液(pH 8.5，100mM，含200mM NH4Cl)，，加入0.5g/L NADP+，3倍当量的葡萄糖，60mg葡萄糖脱氢酶冻干酶粉，共分批补加上述0.5M的β-酮酸底物12mL(终浓度20g/L)，于30℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，产率为95％，分离收率83％，ee值≥99％(S)。
实施例6：β-氨基酸脱氢酶突变体重组菌催化合成手性β-氨基酸化合物的方法
将底物4-甲硫-3-氧代-丁腈(2M，DMSO溶解)加入到碳酸钠缓冲液(pH 8.5，100mM)至终浓度为0.5M，加入5mg/mL腈基水解酶，于30℃水解3h以获得4-甲硫-3-氧代-丁腈底物，水解反应期间需用固体碳酸钠调节pH至8.5左右。
(1)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体16(蛋白序列如SEQ ID NO.:18所示)，离心收集菌体，以菌体作为生物催化剂。取0.8g菌体重悬于27mL碳酸钠-碳酸氢钠缓冲液(pH 8.5，100mM，含200mM NH4Cl)，，加入0.5g/L NADP+，3倍当量的葡萄糖，60mg葡萄糖脱氢酶冻干酶粉，共分批补加上述0.5M的β-酮酸底物12mL(终浓度20g/L)，于30℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，产率为95％，分离收率85％，ee值≥99％(S)。
(2)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体20(蛋白序列如SEQ ID NO.:22所示)，离心收集菌体，以菌体作为生物催化剂。取0.8g菌体重悬于27mL碳酸钠-碳酸氢钠缓冲液(pH 8.5，100mM，含200mM NH4Cl)，，加入0.5g/L NADP+，3倍当量的葡萄糖，60mg葡萄糖脱氢酶冻干酶粉，共分批补加上述0.5M的β-酮酸底物12mL(终浓度20g/L)，于30℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，产率为95％，分离收率81％，ee值≥99％(S)。
(3)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体22(蛋白序列如SEQ ID NO.:24所示)，离心收集菌体，以菌体作为生物催化剂。取0.8g菌体重悬于27mL碳酸钠-碳酸氢钠缓冲液(pH 8.5，100mM，含200mM NH4Cl)，，加入0.5g/L NADP+，3倍当量的葡萄糖，60mg葡萄糖脱氢酶冻干酶粉，共分批补加上述0.5M的β-酮酸底物12mL(终浓度20g/L)，于30℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，产率为95％，分离收率81％，ee值≥99％(S)。
实施例7：β-氨基酸脱氢酶突变体重组菌催化β-氨基酸消旋体类化合物的方法
(1)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体17(蛋白序列如SEQ ID NO.:19所示)，离心收集菌体，以菌体作为生物催化剂。取3.3g菌体重悬于30mL碳酸钠-碳酸氢钠缓冲液(pH 9.5，100mM)，加入底物3-氨基-3-苯基丙酸至终浓度8.3g/L，加入0.5g/L NADP+，加入0.18g/L核黄素，加入0.5g黄素还原酶(来源于E.coli)重组表达菌体，于37℃，200rpm的摇床上反应，8h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，转化率约为50％，分离收率20％，ee值≥99％(R)。
(2)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体18(蛋白序列如SEQ ID NO.:20所示)，离心收集菌体，以菌体作为生物催化剂。取3.3g菌体重悬于30mL碳酸钠-碳酸氢钠缓冲液(pH 9.5，100mM)，加入底物3-氨基-3-(4-氟苯基)丙酸至终浓度9.2g/L，0.5g/L NADP+，加入0.18g/L核黄素，加入0.5g黄素还原酶(来源于E.coli)重组表达菌体，于37℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，转化率约为50％，分离收率39％，ee值≥99％(R)。
(3)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体23(蛋白序列如SEQ ID NO.:25所示)，离心收集菌体，以菌体作为生物催化剂。取3.3g菌体重悬于30mL碳酸钠-碳酸氢钠缓冲液(pH 9.5，100mM)，加入底物3-氨基-3-(4-氟苯基)丙酸至终浓度9.2g/L，0.5g/L NADP+，加入0.18g/L核黄素，加入0.5g黄素还原酶(来源于E.coli)重组表达菌体，于37℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，转化率约为50％，分离收率42％，ee值≥99％(R)。
(4)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体24(蛋白序列如SEQ ID NO.:26所示)，离心收集菌体，以菌体作为生物催化剂。取3.3g菌体重悬于30mL碳酸钠-碳酸氢钠缓冲液(pH 9.5，100mM)，加入底物3-氨基-3-(4-氟苯基)丙酸至终浓度9.2g/L，0.5g/L NADP+，加入0.18g/L核黄素，加入0.5g黄素还原酶(来源于E.coli)重组表达菌体，于37℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，转化率约为50％，分离收率40％，ee值≥99％(R)。
(5)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体18(蛋白序列如SEQ ID NO.:20所示)，离心收集菌体，以菌体作为生物催化剂。取3.3g菌体重悬于30mL碳酸钠-碳酸氢钠缓冲液(pH 9.5，100mM)，加入底物3-氨基己酸至终浓度20g/L，0.5g/L NADP+，加入0.18g/L核黄素，加入0.5g黄素还原酶(来源于E.coli)重组表达菌体，于37℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，转化率约为50％，分离收率41％，ee值≥99％(R)。
(6)按照实施例4的方法诱导表达本发明的β-氨基酸脱氢酶突变体24(蛋白序列如SEQ ID NO.:26所示)，离心收集菌体，以菌体作为生物催化剂。取3.3g菌体重悬于30mL碳酸钠-碳酸氢钠缓冲液(pH 9.5，100mM)，加入底物3,6-二氨基己酸至终浓度30g/L，0.5g/LNADP+，加入0.18g/L核黄素，加入0.5g黄素还原酶(来源于E.coli)重组表达菌体，于37℃，200rpm的摇床上反应，4h后停止反应。反应结束后，HPLC进行检测，用强酸性阳离子树脂进行分离纯化。结果表明，转化率约为50％，分离收率45％，ee值≥99％(R)。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> β-氨基酸脱氢酶突变体及其应用
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<170> SIPOSequenceListing 1.0
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<213> Candidatus Cloacamonas acidaminovorans
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<213> Candidatus Cloacamonas acidaminovorans
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Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ser Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 6
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 6
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Asp Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 7
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 7
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 8
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 8
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Thr Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 9
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 9
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Ser Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 10
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 10
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His Ile Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 11
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 11
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His Phe Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 12
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 12
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His Arg Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 13
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 13
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His Asn Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 14
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 14
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Gly Tyr Ala His His Asp Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 15
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 15
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val His Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 16
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 16
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ser Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 17
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 17
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Asp Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His His Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 18
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 18
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Ile Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 19
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 19
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Phe Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 20
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 20
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Arg Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 21
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 21
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Asn Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 22
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 22
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ala Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Asp Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 23
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 23
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val Ser Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Asn Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 24
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 24
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val His Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Asn Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 25
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 25
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val His Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Ile Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350
<210> 26
<211> 352
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 26
Met Lys Ser Asn Gly Cys Arg Tyr Gly Thr His Arg Val Ile Glu Pro
1 5 10 15
Lys Gly Val Leu Pro Gln Pro Ala Lys Ile Leu Asn Asn Asp Met Ser
20 25 30
Glu Ile Trp Asp Asn Glu Met Leu Ile Asp Val Ile Arg Leu Asn Ile
35 40 45
Asp Ser Ala Ser Phe His Gln Ile Lys Asn Lys Leu Ile Ala Gln Gly
50 55 60
His Gln Asp Leu Glu Lys Ala Phe Ala Glu His Ala Ile Glu Leu Thr
65 70 75 80
Asn Arg Thr Gly Lys His Lys Asn Glu Asp Thr Gly Ser Gly Gly Met
85 90 95
Phe Ile Gly Arg Val Ala Ala Ile Gly Asp Lys Phe Glu Met Lys Glu
100 105 110
Glu Val Lys Val Gly Asp Lys Ile Ala Ser Leu Val Ser Leu Ser Leu
115 120 125
Thr Pro Leu Lys Ile Asn Lys Val Lys Lys Val Leu Leu Asp Lys Asp
130 135 140
Gln Met Glu Ile Glu Gly Gln Ala Ile Leu Phe Ser Ser Gly Val Tyr
145 150 155 160
Ala Lys Leu Pro Asp Asp Leu Asp Glu Asn Leu Ala Leu Ser Val Leu
165 170 175
Asp Val His Gly Ala Pro Ala Gln Val Glu Arg Leu Val Lys Pro Asp
180 185 190
Asp Thr Val Val Ile Ile Gly Ala Asn Gly Lys Ser Gly Ile Leu Cys
195 200 205
Asn Ala Val Ala Lys Glu Arg Ala Gly Ile Cys Gly Lys Val Ile Gly
210 215 220
Val Val Arg Asn Glu Asn Tyr Ile Pro Thr Cys Lys Ala Thr Gly Cys
225 230 235 240
Asp Glu Val Ile Leu Ala Gln Ala Thr Asp Ala Ile Thr Ile Gln Lys
245 250 255
Glu Val Ser Arg Leu Thr Asn Gly Lys Met Ala Asp Val Val Ile Asn
260 265 270
Val Val Asn Thr Glu Asp Thr Glu Leu Pro Ser Ile Met Ala Ala Lys
275 280 285
Asp Arg Gly Met Val Tyr Phe Phe Ser Met Ala Thr Ser Phe Thr Lys
290 295 300
Ala Ala Leu Gly Ala Gly Gly Ile Gly Ala Asp Val Asp Met Met Ile
305 310 315 320
Gly Asn Ser Tyr Ala His His Arg Ser Glu Ile Ala Leu Asp Leu Leu
325 330 335
Arg Arg Asn Ser Val Leu Met Lys Ile Phe Lys Glu Arg Tyr Ala Glu
340 345 350

Claims (10)

1.一种β-氨基酸脱氢酶突变蛋白，其特征在于，所述突变蛋白序列与SEQ ID NO.:2具有至少90％同一性。
2.如权利要求1所述的突变体，其特征在于，所述突变蛋白是对应于SEQ ID NO.:2所示氨基酸序列中的第159、179、180、323、324、328位点一个或多个位点突变的β-氨基酸脱氢酶突变蛋白。
3.如权利要求2所述的突变体，其特征在于，包括在对应于SEQ ID NO.:2上的以下任一种位点的突变：
a)在159位上的突变；
b)在179位上的突变；
c)在180位上的突变；
d)在323位上的突变；
e)在324位上的突变；
f)在328位上的突变。
4.如权利要求3所述的突变蛋白，其特征在于，对应于SEQ ID NO.:2上的位点159的氨基酸由缬氨酸(V)突变为谷氨酸(E)、亮氨酸(L)、异亮氨酸(I)，优选亮氨酸(L)；
对应于SEQ ID NO.:2上的位点179的氨基酸由丙氨酸(A)突变为丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、组氨酸(H)、天冬氨酸(D)，优选丝氨酸(S)和组氨酸(H)；
对应于SEQ ID NO.:2上的位点180的氨基酸由甘氨酸(G)突变为天冬氨酸(D)、谷氨酰胺(Q)、天冬酰胺(N)，优选天冬氨酸(D)；
对应于SEQ ID NO.:2上的位点323的氨基酸由甘氨酸(G)突变为丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、天冬氨酸(D)、谷氨酰胺(N)，优选丝氨酸(S)和苏氨酸(T)；
对应于SEQ ID NO.:2上的位点324的氨基酸酪氨酸(Y)突变为天冬氨酸(D)、丝氨酸(S)、亮氨酸(L)，优选丝氨酸(S)和亮氨酸(L)；
对应于SEQ ID NO.:2上的位点328的氨基酸组氨酸(H)突变为天冬酰胺(N)、谷氨酰胺(Q)、苏氨酸(T)、丝氨酸(S)、丙氨酸(A)、缬氨酸(V)、异亮氨酸(I)、亮氨酸(L)、苯丙氨酸(F)、甲硫氨酸(M)、脯氨酸(P)、半胱氨酸(C)、精氨酸(R)、赖氨酸(K)、天冬氨酸(D)、谷氨酸(E)，优选异亮氨酸(I)、苯丙氨酸(F)、精氨酸(R)、天冬酰胺(N)和天冬氨酸(D)。
5.如权利要求2所述的突变体，其包括对应于SEQ ID NO.:2的氨基酸1至352中的位点159、179、180、323、324和328中的两个及以上位点上的突变。
6.如权利要求5所述的突变体，其特征在于，优选下述在对应于SEQ ID NO.:2上的以下任一种位点的突变：
a)在179位和323位上的组合突变；
b)在323位和328位上的组合突变；
c)在179位和323位和328位上的组合突变。
7.如权利要求6所述的突变体，其特征在于，对应于SEQ ID NO.:2上的位点179的氨基酸由丙氨酸(A)突变为缬氨酸(V)、苏氨酸(T)、半胱氨酸(C)、组氨酸(H)、天冬氨酸(D)，优选半胱氨酸(C)和组氨酸(H)；
对应于SEQ ID NO.:2上的位点323的氨基酸由甘氨酸(G)突变为丝氨酸(S)、苏氨酸(T)、半胱氨酸(C)、天冬氨酸(D)、谷氨酰胺(Q)，优选丝氨酸(S)和苏氨酸(T)；
对应于SEQ ID NO.:2上的位点328的氨基酸组氨酸(H)突变为天冬酰胺(N)、谷氨酰胺(Q)、苏氨酸(T)、丝氨酸(S)、丙氨酸(A)、缬氨酸(V)、异亮氨酸(I)、亮氨酸(L)、苯丙氨酸(F)、甲硫氨酸(M)、脯氨酸(P)、半胱氨酸(C)、精氨酸(R)、赖氨酸(K)、天冬氨酸(D)、谷氨酸(E)，优选异亮氨酸(I)、苯丙氨酸(F)、精氨酸(R)、天冬酰胺(N)和天冬氨酸(D)。
8.一种制备β-氨基酸类化合物的方法，其特征在于，包括步骤：
(i)将权利要求1-7任一所述的β-氨基酸脱氢酶突变蛋白与反应底物接触，进行催化反应，从而获得所述β-氨基酸；
(ii)任选地，分离并纯化所述β-氨基酸。
优选地，所述β-氨基酸类化合物为手性化合物。
9.如权利要求8所述的一种制备β-氨基酸类化合物的方法，其特征在于，利用脂肪酶或腈基水解酶水解后获得的酮酸为底物，在辅酶再生体系的存在下，催化获得手性β-氨基酸。
10.如权利要求8所述的一种制备β-氨基酸类化合物的方法，其特征在于，利用β-氨基酸消旋体为底物，在辅酶再生体系的存在下，催化实现β-氨基酸的手性拆分。
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Citations (3)

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Publication number Priority date Publication date Assignee Title
CN110184246A (zh) * 2019-05-15 2019-08-30 浙江大学 谷氨酸脱氢酶突变体及其应用
US20200102546A1 (en) * 2018-03-09 2020-04-02 Zhejiang University Glutamate dehydrogenase mutants and their application in preparation of l-phosphinothricin
CN112280760A (zh) * 2020-11-26 2021-01-29 宿迁市江南大学产业技术研究院 一种谷氨酸脱氢酶突变体及其应用

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US20200102546A1 (en) * 2018-03-09 2020-04-02 Zhejiang University Glutamate dehydrogenase mutants and their application in preparation of l-phosphinothricin
CN110184246A (zh) * 2019-05-15 2019-08-30 浙江大学 谷氨酸脱氢酶突变体及其应用
CN112280760A (zh) * 2020-11-26 2021-01-29 宿迁市江南大学产业技术研究院 一种谷氨酸脱氢酶突变体及其应用

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