CN116622658A - β-氨基酸脱氢酶突变体及其在合成芳香族β-氨基酸类化合物中的应用 - Google Patents
β-氨基酸脱氢酶突变体及其在合成芳香族β-氨基酸类化合物中的应用 Download PDFInfo
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- CN116622658A CN116622658A CN202210131291.2A CN202210131291A CN116622658A CN 116622658 A CN116622658 A CN 116622658A CN 202210131291 A CN202210131291 A CN 202210131291A CN 116622658 A CN116622658 A CN 116622658A
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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芳香族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Marininema halotolerans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芳香族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
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-氨基酸脱氢酶突变体及其在催化合成手性芳香族β-氨基酸类化合物中的应用。
背景技术
手性芳香族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，在医药、化工等领域具有重要的应用价值。例如，(R)-3-氨基-4-(2,4,5-三氟苯基)-丁酸可用于合成治疗II-型糖尿病的重要药物西他列汀；(R)-3-氨基-3-苯基丙酸、(S)-3-氨基-3-(4-羟基苯基)等可用于合成一些抗炎、抗真菌类药物。因此，开发高效、绿色的手性芳香族β-氨基酸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，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。前期研究通过对来源于Candidatus Cloacamonas acidaminovorans的3,5-DAHDH进行分子改造和大量筛选后，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，实现了手性β-氨基酸的合成(专利申请号：202110133031.4)。
然而，目前已有的β-氨基酸脱氢酶突变体对芳香族β-氨基酸的催化效率仍然很低，并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，进一步提高酶的催化活性，对手性芳香族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芳香族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353中的位点将第307位谷氨酸(E)突变为丝氨酸(S)，将第320位甘氨酸(G)突变为丝氨酸(S)，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353中的位点119、155、175、176、321、325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：
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芳香族手性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，从而获得所述芳香族β-氨基酸类化合物；
(ii)任选地，分离并纯化所述芳香族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，30-60g/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-DAHDH)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，“V159X”表示第159位的缬氨酸V突变为任何一种氨基酸，即第159位的饱和突变体库，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E307S/G320S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-DAHDH，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-DAHDH。
构建β-氨基酸脱氢酶突变体E310S：以pET-21b(+)-3,5-DAHDH作为模板，设计引物E307S-F:CCAGCGGCCTGGGTAAAGATG，E307S-R:GGCCGCTGGCGCCCAGTGCTG，采用全质粒PCR的方法构建突变体E307S，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，5μL dNTP(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-DAHDH-E307S。
构建β-氨基酸脱氢酶突变体E307S/G320S：以pET-21b(+)-3,5-DAHDH-E307S质粒作为模板，仍然采取如上所述全质粒PCR的方法构建组合突变体E307S/G320S，最终获得重组表达转化体E.coli BL21(DE3)/pET-21b(+)-3,5-DAHDH-E307S/G320S。
实施例2:β-氨基酸脱氢酶饱和突变体库的构建与筛选
构建β-氨基酸脱氢酶饱和突变体库：L119X/E307S/G320S、P155X/E307S/G320S、C175X/E307S/G320S、G176X/E307S/G320S、E307S/G320S/F321X、E307S/G320S/H325X。以pET-21b(+)-3,5-DAHDH-E307S/G320S作为模板，分别对119、155、175、176、321、325位点进行定点饱和突变，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(R)-β-苯丙氨酸(A)、(S)-β-高苯丙氨酸(B)和(S)-β-高酪氨酸(C)，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119Q、155L/I/W、175W/F/Y/H/S、176F/Y、321S/T、325I/F/R/N/Q/M/T/S/C/A/P/K，活力最优的突变体分别为1、2、3和4，蛋白序列如SEQ IDNO.:3-6所示。
表1以E307S/G320S为模板进行的突变筛选结果
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：根据前期饱和突变与筛选的结果，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E307S/G320S/H325I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中三种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119Q、155L/I/W、175W/Y/H/S、G176Y、F321T，选择出活力最高的突变体5和6，蛋白序列如SEQ IDNO.:7-8所示。
表2以E307S/G320S/H325I为模板进行的突变结果
以E307S/G320S/H325R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中三种β-氨基酸底物的活力进行测定，结果见表3。获得β-氨基酸脱氢酶催化活性提高的突变位点分别为155L/I/W、179H/S、F321T，选择出活力最高的突变体7，蛋白序列如SEQ ID NO.:9所示。
表3以E307S/G320S/H325R为模板进行的突变结果
以E307S/G320S/H325F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中三种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体分别为159L/I、179S、F321T，为突变体8，蛋白序列如SEQ ID NO.:10所示。
表4以E307S/G320S/H325F为模板进行的突变结果
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1-8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C175H/E307S/G320S的纯化蛋白，2为突变体E307S/G320S/H325F的纯化蛋白，3为突变体E307S/G320S/H325I的纯化蛋白，4为突变体E307S/G320S/H325R的纯化蛋白，5为突变体C175H/E307S/G320S/H325I的纯化蛋白，6为突变体C175S/E307S/G323S/H325I的纯化蛋白，7为突变体C175H/E307S/G323S/H325R的纯化蛋白，8为突变体C175S/E307S/G320S/H325F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3-氧-4-苯基-丁酸乙酯(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4(蛋白序列如SEQ ID NO.:6所示)，离心收集菌体，以菌体作为生物催化剂。取1.8g菌体重悬于24mL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，共加入上述0.5M的β-酮酸底物6mL(终浓度约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96％，分离收率86％，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5(蛋白序列如SEQ ID NO.:7所示)，离心收集菌体，以菌体作为生物催化剂。取1.8g菌体重悬于24mL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，共加入上述0.5M的β-酮酸底物6mL(终浓度约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6(蛋白序列如SEQ ID NO.:8所示)，离心收集菌体，以菌体作为生物催化剂。取1.8g菌体重悬于24mL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，共加入上述0.5M的β-酮酸底物6mL(终浓度约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)。
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3-氧-4-(2,4,5-三氟苯基)-丁酸乙酯(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4(蛋白序列如SEQ ID NO.:6所示)，离心收集菌体，以菌体作为生物催化剂。取1.8g菌体重悬于24mL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，共加入上述0.5M的β-酮酸底物6mL(终浓度25g/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97％，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5(蛋白序列如SEQ ID NO.:7所示)，离心收集菌体，以菌体作为生物催化剂。取1.8g菌体重悬于24mL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，共加入上述0.5M的β-酮酸底物6mL(终浓度25g/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7(蛋白序列如SEQ ID NO.:9所示)，离心收集菌体，以菌体作为生物催化剂。取1.8g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，共加入上述0.5M的β-酮酸底物6mL(终浓度25g/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4(蛋白序列如SEQ ID NO.:6所示)，离心收集菌体，以菌体作为生物催化剂。取2.5g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17g/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，6h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％(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5(蛋白序列如SEQ ID NO.:7所示)，离心收集菌体，以菌体作为生物催化剂。取2.5g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18g/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％(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7(蛋白序列如SEQ ID NO.:9所示)，离心收集菌体，以菌体作为生物催化剂。取2.5g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-4-(2,4,5-三氟苯基)丁酸至终浓度23g/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43％，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5(蛋白序列如SEQ ID NO.:7所示)，离心收集菌体，以菌体作为生物催化剂。取2.5g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-4-苯基丁酸至终浓度18g/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6(蛋白序列如SEQ ID NO.:8所示)，离心收集菌体，以菌体作为生物催化剂。取2.5g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-(2-甲基苯基)丙酸至终浓度18g/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％(S)。
(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7(蛋白序列如SEQ ID NO.:9所示)，离心收集菌体，以菌体作为生物催化剂。取2.5g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-(3,4-二甲氧基苯基)丙酸至终浓度30g/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45％，ee值≥99％(S)。
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在催化合成手性芳香族β-氨基酸类化合物中的应用
<130> amino acid sequences
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<170> SIPOSequenceListing 1.0
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Ser
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Ser
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Ser
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<213> Candidatus Cloacamonas acidaminovorans
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Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala
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Ser
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<213> Candidatus Cloacamonas acidaminovorans
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Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val
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Ser
<210> 6
<211> 353
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
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Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val
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Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu
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Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln
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Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly
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Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val
100 105 110
Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser
115 120 125
Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val
130 135 140
Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro
145 150 155 160
Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Cys Gly
165 170 175
Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val
180 185 190
Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala
195 200 205
Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser
210 215 220
Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val
225 230 235 240
Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu
245 250 255
Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn
260 265 270
Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly
275 280 285
Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu
290 295 300
Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser
305 310 315 320
Phe Ala Pro Gly Arg Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe
325 330 335
Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr
340 345 350
Ser
<210> 7
<211> 353
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 7
Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val
1 5 10 15
Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala
20 25 30
Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu
35 40 45
Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln
50 55 60
Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg
65 70 75 80
Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly
85 90 95
Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val
100 105 110
Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser
115 120 125
Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val
130 135 140
Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro
145 150 155 160
Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val His Gly
165 170 175
Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val
180 185 190
Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala
195 200 205
Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser
210 215 220
Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val
225 230 235 240
Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu
245 250 255
Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn
260 265 270
Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly
275 280 285
Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu
290 295 300
Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser
305 310 315 320
Phe Ala Pro Gly Ile Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe
325 330 335
Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr
340 345 350
Ser
<210> 8
<211> 353
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 8
Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val
1 5 10 15
Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala
20 25 30
Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu
35 40 45
Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln
50 55 60
Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg
65 70 75 80
Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly
85 90 95
Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val
100 105 110
Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser
115 120 125
Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val
130 135 140
Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro
145 150 155 160
Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Ser Gly
165 170 175
Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val
180 185 190
Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala
195 200 205
Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser
210 215 220
Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val
225 230 235 240
Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu
245 250 255
Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn
260 265 270
Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly
275 280 285
Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu
290 295 300
Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser
305 310 315 320
Phe Ala Pro Gly Ile Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe
325 330 335
Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr
340 345 350
Ser
<210> 9
<211> 353
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 9
Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val
1 5 10 15
Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala
20 25 30
Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu
35 40 45
Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln
50 55 60
Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg
65 70 75 80
Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly
85 90 95
Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val
100 105 110
Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser
115 120 125
Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val
130 135 140
Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro
145 150 155 160
Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val His Gly
165 170 175
Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val
180 185 190
Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala
195 200 205
Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser
210 215 220
Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val
225 230 235 240
Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu
245 250 255
Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn
260 265 270
Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly
275 280 285
Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu
290 295 300
Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser
305 310 315 320
Phe Ala Pro Gly Arg Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe
325 330 335
Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr
340 345 350
Ser
<210> 10
<211> 353
<212> PRT
<213> Candidatus Cloacamonas acidaminovorans
<400> 10
Met Val Arg Asn Glu Lys Gln Gly His Arg Phe Gly Leu His Arg Val
1 5 10 15
Val Glu Pro Lys Gly Leu Leu Pro Gln Pro Ala Trp Lys Leu Asp Ala
20 25 30
Asn Pro Ile Cys Leu Asp Asn Glu Met Met Ile Asp Val Ser Cys Leu
35 40 45
Asn Ile Asp Ser Ala Ser Phe Asn Gln Leu Lys Glu Ser Cys Glu Gln
50 55 60
Asp Pro Val Arg Ile Lys Glu Arg Ile Leu Gln Ile Val Arg Glu Arg
65 70 75 80
Gly Lys Met His Asn Pro Val Thr Gly Ser Gly Gly Met Leu Ile Gly
85 90 95
Gln Ile Glu Gln Ile Gly Asp Gly Phe Pro Asp Glu Asp Ile Arg Val
100 105 110
Gly Asp Arg Val Ala Thr Leu Val Ser Leu Thr Leu Thr Pro Leu Ser
115 120 125
Leu Glu Glu Ile Lys Ser Ile Asp Met Lys Thr Gly Gln Val His Val
130 135 140
Arg Gly Lys Ala Ile Leu Phe Ala Ser Gly Pro Phe Ala Val Leu Pro
145 150 155 160
Gln Asp Leu Pro Glu Thr Leu Ser Leu Ala Val Leu Asp Val Ser Gly
165 170 175
Ala Pro Ala Gln Thr Asp Arg Leu Val Gln Glu Gly Asp Thr Val Val
180 185 190
Val Leu Gly Ala Gly Gly Lys Ser Gly Leu Leu Ser Leu Cys Arg Ala
195 200 205
Arg Leu Lys Ala Gly Ser Ser Gly Gln Val Ile Ala Leu Glu Ser Ser
210 215 220
Glu Ala Ala Cys Glu Gln Ile Lys Glu Leu Gly Trp Ala Asp His Val
225 230 235 240
Ala Gln Val Asp Ala Arg Asp Pro Val Ala Val Met Ala Met Val Glu
245 250 255
Lys Leu Thr Asp Gly Lys Met Ala Asp Leu Thr Val Asn Cys Val Asn
260 265 270
Val Pro Asp Thr Glu Leu Ser Ala Ile Leu Ala Thr Arg Glu Glu Gly
275 280 285
Ile Ala Tyr Phe Phe Ser Thr Ala Val Lys Phe Thr Ala Ala Ala Leu
290 295 300
Gly Ala Ser Gly Leu Gly Lys Asp Val Arg Met Glu Ile Gly Asn Ser
305 310 315 320
Phe Ala Pro Gly Arg Ala Ala Leu Ala Leu Asp Thr Val Arg Asn Phe
325 330 335
Ser Ser Leu Arg Arg Leu Phe Glu Ala Arg Tyr Ala Ala Thr Ala Thr
340 345 350
Ser
Claims (9)
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119、155、175、176、321、325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119位上的突变；
b)在155位上的突变；
c)在175位上的突变；
d)在176位上的突变；
e)在321位上的突变；
f)在325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119的氨基酸由亮氨酸(L)突变为缬氨酸(V)、异亮氨酸(I)、谷氨酰胺(Q)，优选谷氨酰胺(Q)；
对应于SEQ ID NO.:2上的位点155的氨基酸由脯氨酸(P)突变为色氨酸(W)、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175的氨基酸由半胱氨酸(C)突变为丝氨酸(S)、组氨酸(H)、酪氨酸(Y)、苯丙氨酸(F)、色氨酸(W)，优选丝氨酸(S)和组氨酸(H)；
对应于SEQ ID NO.:2上的位点176的氨基酸由甘氨酸(G)突变为苯丙氨酸(F)、酪氨酸(Y)，优选酪氨酸(Y)；
对应于SEQ ID NO.:2上的位点321的氨基酸由苯丙氨酸(F)突变为丝氨酸(S)、苏氨酸(T)，优选苏氨酸(T)；
对应于SEQ ID NO.:2上的位点325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)、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)，优选异亮氨酸(I)、苯丙氨酸(F)、精氨酸(R)、甲硫氨酸(M)和苏氨酸(T)。
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353中的位点119、155、175、176、321和325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155位和325位上的组合突变；
b)在175位和325位上的组合突变；
c)在176位和325位上的组合突变；
d)在321位和325位上的组合突变；
e)在155位和175位和325位上的组合突变；
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155的氨基酸由脯氨酸(P)突变为色氨酸(W)、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175的氨基酸由半胱氨酸(C)突变为丝氨酸(S)、组氨酸(H)、酪氨酸(Y)、苯丙氨酸(F)、色氨酸(W)，优选丝氨酸(S)和组氨酸(H)；
对应于SEQ ID NO.:2上的位点176的氨基酸由甘氨酸(G)突变为苯丙氨酸(F)、酪氨酸(Y)，优选酪氨酸(Y)；
对应于SEQ ID NO.:2上的位点321的氨基酸由苯丙氨酸(F)突变为丝氨酸(S)、苏氨酸(T)，优选苏氨酸(T)；
对应于SEQ ID NO.:2上的位点325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)、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)，优选异亮氨酸(I)、苯丙氨酸(F)、精氨酸(R)、甲硫氨酸(M)和苏氨酸(T)。
7.一种制备芳香族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-氨基酸类化合物为芳香族手性化合物。
8.如权利要求8所述的一种制备芳香族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。
9.如权利要求8所述的一种制备芳香族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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