CN115322980B - 酰胺水解酶突变体及其在手性环状化合物合成上的应用 - Google Patents
酰胺水解酶突变体及其在手性环状化合物合成上的应用 Download PDFInfo
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Abstract
本发明公开了酰胺水解酶突变体及其应用。该酰胺水解酶突变体是对野生型酰胺水解酶进行突变得到的蛋白质，所述野生型酰胺水解酶的氨基酸序列如SEQ ID No.1第1‑509位所示，所述蛋白质为下述A)或B)：A)所述蛋白质的氨基酸序列与野生型酰胺水解酶的氨基酸序列相比，如下至少一种氨基酸残基进行突变：所述野生型酰胺水解酶第146位的苯丙氨酸残基、第193位的甘氨酸残基、第198位的异亮氨酸残基、第223位的异亮氨酸残基、第328位的色氨酸残基、第332位的丙氨酸残基和第450位的异亮氨酸残基，B)所述蛋白质为将A)所示蛋白的氨基酸序列末端添加蛋白标签序列且具有酰胺水解酶活性的由A)衍生的蛋白。该酰胺水解酶突变体可被用于合成高光学纯度的手性环状化合物。
Description
技术领域
本发明涉及酶工程技术领域，尤其是涉及酰胺水解酶突变体及其在手性环状化合物合成上的应用。
背景技术
生物催化是迄今为止最具高效、高选择性和环境友好的过程。利用生物催化的方法合成一些具有高附加值的化学品，特别是手性化学品具有重要的应用前景和意义。来源于红球菌Rhodococcus erythropolis AJ270的酰胺水解酶(ReAMI)已被证实能够催化多种酰胺类底物的水解反应生成相应羧酸产物，且具有高效和高选择性的优点，特别是它具有高对映选择性，已被应用于多种手性非天然氨基酸以及药物分子结构骨架的合成。
但是采用生物催化方法进行水解反应通常只能得到单一构型产物(如果想要获得相反构型的产物需要将其进行多步且复杂的官能团转化)，且对一些底物表现出了较低的效率和对映选择性，这大大限制了其应用。为此人们越来越多的采用定向进化等分子生物学手段对现有酶进行改造，通过在实验室条件下模拟自然进化过程，从构建的突变体文库中筛选到能满足特定需求的目标突变体，这无疑极大的拓展了生物催化的应用范围。但其中针对酰胺水解酶的改造研究较少，且都集中在提高酰胺水解酶催化效率和酰胺水解酶的稳定性上，至今没有提高或反转酰胺水解酶对映选择性的改造范例。
手性环状化合物广泛存在于具有生物活性的药物及天然产物结构中，例如聚醚类抗体(polyether antibiotics)、海洋天然产物(marine macrolides)、核苷类似物药物(nucleoside analog drugs)、蛋白质抑制剂Keap I以及血管紧张素转化酶抑制剂。但现有的合成方法仍然具有产率低、原料制备复杂，立体选择性较低的缺点，因此发展新的手性环状化合物的合成方法是很有必要的。
发明内容
本发明的目的之一是提供红球菌Rhodococcus erythropolis AJ270的野生型酰胺水解酶(AMI)突变体，其具有更高的对映选择性或反转的对映选择性，可被用于合成高光学纯度的手性环状化合物。
为达到上述目的，本发明提供蛋白质，所述蛋白质是对野生型酰胺水解酶进行突变得到的蛋白质，所述野生型酰胺水解酶的氨基酸序列如SEQ ID No.1第1-509位所示，所述蛋白质为下述A)或B)：
A)所述蛋白质的氨基酸序列与野生型酰胺水解酶的氨基酸序列相比，如下至少一种氨基酸残基进行突变：
所述野生型酰胺水解酶的第146位(即SEQ ID No.1第146位)的苯丙氨酸残基(F)、第193位的甘氨酸残基(G)(即SEQ ID No.1第193位)、第198位的异亮氨酸残基(I)(即SEQID No.1第198位)、第223位的异亮氨酸残基(即SEQ ID No.1第223位)、第328位的色氨酸残基(W)(即SEQ ID No.1第328位)、第332位的丙氨酸残基(A)(即SEQ ID No.1第332位)和第450位的异亮氨酸残基(即SEQ ID No.1第450位)，
B)所述蛋白质为将A)所示蛋白的氨基酸序列末端添加蛋白标签序列且具有酰胺水解酶活性的由A)衍生的蛋白。
将上述提供的蛋白质命名为酰胺水解酶突变体蛋白。酰胺水解酶突变体蛋白可通过对野生型酰胺水解酶进行单轮或多轮定点突变构建获得。
上述蛋白质中，所述蛋白标签(protein-tag)是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测、示踪和/或纯化。所述蛋白标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
所述酰胺水解酶活性具体可为水解环状二酰胺类化合物生成手性单羧酸单酰胺的催化活性。
可选地，酰胺水解酶突变体蛋白的氨基酸序列与野生型酰胺水解酶的氨基酸序列相比，进行如下至少一种突变：
所述野生型酰胺水解酶的第146位的苯丙氨酸残基突变为丙氨酸残基或甘氨酸残基、第193位的甘氨酸残基突变为丙氨酸残基、第198位的异亮氨酸突变残基为丙氨酸残基、第223位的异亮氨酸残基突变为苯丙氨酸残基、第328位的色氨酸残基突变为丙氨酸残基、第332位的丙氨酸残基突变为色氨酸残基和第450位的异亮氨酸残基突变为丙氨酸残基或甘氨酸残基。
可选地，酰胺水解酶突变体蛋白的氨基酸序列如SEQ ID No.2-7第1-509位中至少一种所示；酰胺水解酶突变体蛋白的氨基酸序列也可如SEQ ID No.2-7第1-521位中至少一种所示；酰胺水解酶突变体蛋白的氨基酸序列还可如SEQ ID No.2-7中至少一种所示。
酰胺水解酶突变体蛋白的氨基酸序列如SEQ ID No.2所示，该酰胺水解酶突变体蛋白(突变体蛋白G193A)具有更高的对映选择性，可以获得ee值高达99.5％的手性产物；酰胺水解酶突变体蛋白的氨基酸序列如SEQ ID No.3所示，该酰胺水解酶突变体蛋白(突变体蛋白I198A)具有更高的对映选择性，可以获得ee值高达99.5％的手性产物；酰胺水解酶突变体蛋白的氨基酸序列如SEQ ID No.4所示，该酰胺水解酶突变体蛋白(突变体蛋白I450GI223F)具有反转的对映选择性，可以获得ee值达-99.5％的手性产物；酰胺水解酶突变体蛋白的氨基酸序列如SEQ ID No.5所示，该酰胺水解酶突变体蛋白(突变体蛋I450GA332W)具有反转的对映选择性，可以获得ee值达-99.5％的手性产物；酰胺水解酶突变体蛋白的氨基酸序列如SEQ IDNo.6所示，该酰胺水解酶突变体蛋白(突变体蛋白I450GA332WF146A)具有反转的对映选择性，可以获得ee值达-99.5％的手性产物；酰胺水解酶突变体蛋白的氨基酸序列如SEQ ID No.7所示，该酰胺水解酶突变体蛋白(突变体蛋白I450AW328YF146A)具有反转的对映选择性，可以获得ee值达-99.5％的手性产物。
本发明还提供了所述的酰胺水解酶突变体蛋白相关的生物材料，所述生物材料为B1)至B8)中的任一种：
B1)编码酰胺水解酶突变体蛋白的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B2)所述表达盒的重组载体；
B5)含有B1)所述核酸分子的重组微生物；
B6)含有B2)所述表达盒的重组微生物；
B7)含有B3)所述重组载体的重组微生物；
B8)含有B4)所述重组载体的重组微生物。
可选地，B1)所述的核酸分子的核苷酸序列为如下至少一种所示：
B11)将野生型酰胺水解酶基因序列第577-579位的GGC替换为GCC，其它核苷酸不变；
B12)将所述野生型酰胺水解酶基因序列第592-594位的ATC替换为GCT，其它核苷酸不变；
B13)将所述野生型酰胺水解酶基因序列第1348-1350位的ATC替换为GGC，第667-669位的ATC替换为TTC，其它核苷酸不变；
B14)将所述野生型酰胺水解酶基因序列第1348-1350位的ATC替换为GGC，第994-996位的GCC替换为TGG，其它核苷酸不变；
B15)将所述野生型酰胺水解酶基因序列第1348-1350位的ATC替换为GGC，第994-996位的GCC替换为TGG，第436-438位的TTC替换为GCC，其它核苷酸不变；
B16)将所述野生型酰胺水解酶基因序列第1348-1350位的ATC替换为GGC，第982-984位的TGG替换为GAA，第436-438位的TTC替换为GCC，其它核苷酸不变；
所述野生型酰胺水解酶基因序列如SEQ ID No.8的第1-1527位所示。
本发明还提供了催化非手性环状二酰胺生成手性环状单酰胺单羧酸的方法，包括采用酰胺水解酶突变体蛋白或上述的重组微生物作为催化剂，催化非手性环状二酰胺生成手性环状单酰胺单羧酸。
可选地，所述非手性环状二酰胺包括：如式I所示化合物：
所述式I结构通式中，两个酰胺基处于顺式构型；
X选自下述基团中的任意一种：-CH2-、-NH-、-o-、--，其中--表示无任何原子；
Y选自下述基团中的任意一种：-CH2CH2CH2CH2-、-CH2CH＝CHCH2-、-CH2CH2CH2-、-CH2CH2-、-CH＝CH-。
可选地，所述手性环状单酰胺单羧酸包括：如式II所示化合物：
所述式II结构通式中，*代表手性，为R或S；
x选自下述基团中的任意一种：-CH2-、-NH-、-O-、--，其中--表示无任何原子；
Y选自下述基团中的任意一种：-CH2CH2CH2CH2-、-CH2CH＝CHCH2-、-CH2CH2CH2-、-CH2CH2-、-CH＝CH-。
上述手性环状单酰胺单羧酸ee值可在82-99.5％之间。
可选地，上述方法中，所述重组微生物与所述非手性环状二酰胺类底物的用量比为0.2g：0.1mmol-100mmol，例如0.2g：0.1mmol
可选地，上述方法中，催化温度为20-40℃，例如37℃，催化时间为5-500分钟，例如5-120分钟。
可选地，所述重组微生物为以包含酰胺水解酶突变体蛋白编码基因的工程菌经发酵培养后获得的湿菌体，以pH值为6.0-8.0的缓冲溶液作为反应介质。所述缓冲溶液可为Na2HPO4-柠檬酸缓冲溶液、K2HPO4-KH2PO4缓冲溶液、Tris缓冲溶液、Hanks’缓冲溶液或PBS缓冲溶液；所述重组微生物与所述缓冲溶液的用量比可为0.2g∶5mL-1L，例如0.2g∶5mL。
上述湿菌体的制备方法例如可为：将包含酰胺水解酶突变体蛋白编码基因的工程菌菌种接入到已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h获得种子液。之后向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述获得的种子液，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
可选地，上述方法中，包括生物催化体系与式I所示非手性环状二酰胺进行催化水解反应，反应完毕得到式II所示手性化合物。所述生物催化体系，由包含酰胺水解酶突变体编码基因的工程菌经发酵培养后获得的湿菌体作为生物催化剂，和pH值为6.0-8.0的缓冲溶液组成。该生物催化体系具体为将所述工程菌接于所述pH值为6.0-8.0的缓冲溶液中37℃活化30分钟而得。所述缓冲溶液为Na2HPO4-柠檬酸缓冲溶液、K2HPO4-KH2PO4缓冲溶液、Tris缓冲溶液、Hanks’缓冲溶液或PBS缓冲溶液，优选K2HPO4-KH2PO4缓冲溶液。所述工程菌与式I所示化合物的用量比为0.2g∶0.1mmol-100mmol，优选0.2g∶0.1mmol。所述工程菌催化体系中，工程菌与所述缓冲溶液的用量比为0.2g∶5mL-1L，优选0.2g∶5mL。所述催化水解反应步骤中，温度为20-40℃，优选37℃，时间为5-500分钟，优选5-120分钟。不同底物与用量优选不同时间，使得反应产物ee值在82-99.5％之间；
本发明还提供了酰胺水解酶突变体蛋白、上述生物材料在催化非手性环状二酰胺生成手性环状单酰胺单羧酸中的应用。
本发明还提供了生产酰胺水解酶突变体蛋白的方法，所述方法包括使酰胺水解酶突变体蛋白的编码基因在生物中进行表达得到所述酰胺水解酶突变体蛋白的步骤，所述生物为微生物、植物或非人动物。
上述方法中，使所述酰胺水解酶突变体蛋白的编码基因在生物中进行表达可包括将所述的蛋白质的编码基因导入受体微生物，得到表达所述蛋白质的重组微生物，培养所述重组微生物，表达得到所述酰胺水解酶突变体蛋白。
上述方法中，所述受体微生物可为C1)-C4)中的任一种：C1)原核微生物；C2)革兰氏阴性细菌；C3)埃希氏菌属细菌；C4)大肠杆菌BL21(DE3)。
本发明通过对来源于红球菌Rhodococcus erythropolis AJ270的野生型酰胺水解酶(AMI)进行改造，获得对映选择性提高或反转的突变体蛋白，从而有利于获得高光学纯度的手性产物。本发明构建的突变体蛋白G193A以及I198A具有更高的对映选择性，可以获得更高ee值的手性产物；构建的突变体蛋白I450GI223F、I450GA332W、I450GA332WF146A、I450AW328YF146A具有反转的对映选择性，可以获得相反构型的手性产物。本发明所构建的突变体蛋白、相关生物材料作为催化剂，具有可发酵培养和/或保存方便的特点。运用本发明提供的方法制备手性酰胺羧酸类化合物，具有操作简便，反应高效，反应条件温和，对映选择性高，产物易分离，产物纯度高的特点，具有重要的应用价值。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
采用高效液相色谱法测定手性化合物对映体过量值(ee值)检验。
下述实施例中所使用的质粒pET22b-AMI为用SEQ ID No.8第1-1563位所示的DNA替换pET22b(+)(优宝生物公司)的NdeI和NotI识别位点间的片段，保持pET22b(+)的其它序列不变，得到酰胺水解酶ReAMI基因重组表达载体。
pET22b-AMI含有ReAMI基因，ReAMI基因的核苷酸序列是SEQ ID No.8所示，ReAMI基因编码SEQ ID No.1所示的蛋白质ReAMI(即酰胺水解酶)。pET22b-AMI表达SEQ ID No.1所示的蛋白质ReAMI。其中，SEQ ID No.8第1-1527位核苷酸是野生型酰胺水解酶AMI基因的核苷酸序列，SEQ ID No.1的第1-509位是野生型酰胺水解酶AMI的氨基酸序列。
酰胺水解酶突变体G193A基因序列为将ReAMI基因序列第577-579位的GGC(G密码子)替换为GCC(A密码子)，其它核苷酸不变。
酰胺水解酶突变体I198A基因序列为将ReAMI基因序列第592-594位的ATC(I密码子)替换为GCT(A密码子)，其它核苷酸不变。
酰胺水解酶突变体I450GI223F基因序列为将ReAMI基因序列第1348-1350位的ATC(I密码子)替换为GGC(G密码子)，第667-669位的ATC(I密码子)替换为TTC(F密码子)，其它核苷酸不变。
酰胺水解酶突变体I450GA332W基因序列为将ReAMI基因序列第1348-1350位的ATC(I密码子)替换为GGC(G密码子)，第994-996位的GCC(A密码子)替换为TGG(W密码子)，其它核苷酸不变。
酰胺水解酶突变体I450GA332WF146A基因序列为将ReAMI基因基因序列第1348-1350位的ATC(I密码子)替换为GGC(G密码子)，第994-996位的GCC(A密码子)替换为TGG(W密码子)，第436-438位的TTC(F密码子)替换为GCC(A密码子)，其它核苷酸不变。
酰胺水解酶突变体I450AW328YF146A基因序列为将ReAMI基因序列第1348-1350位的ATC(I密码子)替换为GGC(G密码子)，第982-984位的TGG(W密码子)替换为GAA(Y密码子)，第436-438位的TTC(F密码子)替换为GCC(A密码子)，其它核苷酸不变。
实施例1：突变体G193A及其在手性环状化合物合成上的应用
1、酰胺水解酶突变体重组菌的构建与表达
构建含有酰胺水解酶(ReAMI)基因的质粒pET22b-AMI，以此质粒为模板，设计定点突变引物序列如下：
G193A-F：5’-CCATCGGCGGGGATCAAGCCGGATCGATCC-3’
G193A-R：5’-GGGATCCGGATCGATCCGGCTTGATCCCCG-3’
通过PCR对模板进行全质粒扩增，PCR体系如下(50uL)：PrimeSTAR Max Premix(2×)25.0ul，Forward primer 2.0ul，Reverse primer 2.0ul，Template DNA＜200ng，ddH2Oup to 50ul。其中PrimeSTAR Max Premix包括PrimeSTAR HS DNA Polymerase，dNTPMixture(each 0.4mM，2×)，Reaction Buffer(2×)，和Mg2+(2mM，2×)。
PCR反应程序为：1)98℃ 2.0min；2)98℃ 10sec，MT℃ 5-10sec，72℃ Et sec，25-30个循环；3)72℃ 5min。其中MT为退火温度，视不同引物而定；Et为延伸时间，以10sec/kb根据目的基因的长度来计算。
PCR产物经过0.9％琼脂糖凝胶电泳分析后，取PCR反应液20uL，加入1uL限制性内切酶DpnI于37℃条件下酶切4小时，随后转化到E.Coli BL21(DE3)感受态细胞中(南京诺唯赞生物科技公司)，复苏后涂布于含有氨苄青霉素的LB平板上培养12小时，挑取单菌落培养于已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h。提取质粒测序。将含有测序正确质粒(名称为pET22b-G193A)的菌株命名为含有酰胺水解酶突变体G193A的大肠杆菌工程菌。该大肠杆菌工程菌为含有酰胺水解酶突变体G193A基因的E.Coli BL21(DE3)感受态细胞。该大肠杆菌工程菌表达酰胺水解酶突变体G193A，酰胺水解酶突变体G193A的氨基酸序列如SEQ ID No.2所示。
pET22b-G193A与pET22b-AMI的区别仅在于将pET22b-AMI中所含的ReAMI基因替换为酰胺水解酶突变体G193A基因，其它核苷酸完全相同。向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述测序正确的大肠杆菌工程菌，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
同样的，采用上述方法将含有未突变的酰胺水解酶(ReAMI)基因的质粒pET22b-AMI转化入大肠杆菌E.Coli BL21(DE3)，获得含有酰胺水解酶(ReAMI)基因的大肠杆菌表达菌株，作为突变体的催化性能比较。含有酰胺水解酶(ReAMI)基因的大肠杆菌表达菌株为含有目的基因的E.Coli BL21(DE3)感受态细胞，该目的基因序列如SEQ ID No.2所示，该大肠杆菌工程菌表达酰胺水解酶ReAMI，酰胺水解酶的氨基酸序列如SEQ ID No.1所示。
2、酰胺水解酶突变体催化性能检测
利用含有酰胺水解酶突变体G193A的大肠杆菌工程菌催化环状二酰胺底物Ia化合物(x＝-O-；Y＝-CH2CH2-)的水解反应，如表1所示得到相应产物IIa，经检测其ee值大于99.5％；与之对比，含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIa，经检测其ee值为30％，突变以后对映选择性显著提高。
利用含有酰胺水解酶突变体G193A的大肠杆菌工程菌催化环状二酰胺底物Ib化合物(X＝-O-；Y＝-CH＝CH-)的水解反应，如表1所示得到相应产物IIb，经检测其ee值为99％；与之对比，含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIb，经检测其ee值为54％，突变以后对映选择性显著提高。ee值计算公式为ee＝([R]-[S])/([R]+[S])×100％([]表示浓度)。
表1：含有突变体G193A与含有酰胺水解酶的工程菌催化反应比较
具体实施方法为：取0.2克湿重的大肠杆菌工程菌，37℃条件下解冻30分钟，用磷酸氢二钾和磷酸二氢钾的缓冲溶液(0.1M，pH 7.0，5mL)将菌体洗入带螺纹口的Erlenmeyer平底烧瓶中，分散摇匀后放入摇床中37℃条件下活化30分钟，然后一次性加0.1mmol的式I化合物，放入摇床中37℃，220rpm条件下进行催化水解反应。整个反应TLC监测，原料消失后停止反应，所得反应液通过一层硅藻土抽滤除去菌体，依次用水2mL洗涤滤渣三次，用旋转蒸发仪除去溶剂后，所生成的即为单酰胺单羧酸产物II化合物。
实施例2：突变体I198A及其在手性环状化合物合成上的应用
1、酰胺水解酶突变体重组菌的构建与表达
设计引物，通过PCR扩增在酰胺水解酶末端加上His-tag标签，构建含有酰胺水解酶(ReAMI)基因的质粒pET22b-AMI，以此质粒为模板，设计定点突变引物序列如下：
I198A-F：5’-ATCAAGGCGGATCGATCCGGGCACCGGCGGCATTCTG-3’
I198A-R：5’-ACGCCGCAGAATGCCGCCGGTGCCCGGATCGATCCGC-3’
通过PCR对模板进行全质粒扩增，PCR体系如下(50uL)：PrimeSTARMaxPremix(2×)25.0ul，Forward primer 2.0ul，Reverse primer 2.0ul，Template DNA＜200ng，ddH2O upto 50ul。其中PrimeSTAR Max Premix包括PrimeSTAR HS DNA Polymerase，dNTP Mixture(each 0.4mM，2×)，Reaction Buffer(2×)，和Mg2+(2mM，2×)。
PCR反应程序为：1)98℃ 2.0min；2)98℃ 10sec，MT℃ 5-10sec，72℃ Et sec，25-30个循环；3)72℃ 5min。其中MT为退火温度，视不同引物而定；Et为延伸时间，以10sec/kb根据目的基因的长度来计算。
PCR产物经过0.9％琼脂糖凝胶电泳分析后，取PCR反应液20uL，加入1uL限制性内切酶DpnI于37℃条件下酶切4小时，随后转化到E.Coli BL21(DE3)感受态细胞中，复苏后涂布于含有氨苄青霉素的LB平板上培养12小时，挑取单菌落培养于已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h。提取质粒测序。将含有测序正确质粒(名称为pET22b-I198A)的菌株命名为含有酰胺水解酶突变体I198A的大肠杆菌工程菌。该大肠杆菌工程菌为含有酰胺水解酶突变体I198A基因的E.Coli BL21(DE3)感受态细胞。该大肠杆菌工程菌表达酰胺水解酶突变体I198A，酰胺水解酶突变体I198A的氨基酸序列如SEQ ID No.3所示。
pET22b-I198A与pET22b-AMI的区别仅在于将pET22b-AMI中所含的ReAMI基因替换为酰胺水解酶突变体I198A基因，其它核苷酸完全相同。
向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述测序正确的大肠杆菌工程菌，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
2、酰胺水解酶突变体催化性能检测
利用含有酰胺水解酶突变体I198A的大肠杆菌工程菌催化环状二酰胺底物Ic化合物(X＝--，其中--表示无任何原子；Y＝-CH2CH＝CHCH2-)的水解反应，如表2所示得到相应产物IIc，经检测其ee值大于99.5％；与之对比，实施例1制备的含有未突变的酰胺水解酶的大肠杆菌工程菌催化得到产物IIc，经检测其ee值为94％，突变以后对映选择性显著提高。
利用含有酰胺水解酶突变体I198A的大肠杆菌工程菌催化环状二酰胺底物Id化合物(X＝--，其中--表示无任何原子；Y＝-CH2CH2CH2CH2-)的水解反应，如表2所示得到相应产物IId，经检测其ee值大于99％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IId，经检测其ee值为99％，突变以后对映选择性提高。
表2：含有突变体I198A与含有野生型酰胺水解酶的工程菌催化反应比较
具体实施方法为：取0.2克湿重的大肠杆菌工程菌，37℃条件下解冻30分钟，用磷酸氢二钾和磷酸二氢钾的缓冲溶液(0.1M，pH 7.0，5mL)将菌体洗入带螺纹口的Erlenmeyer平底烧瓶中，分散摇匀后放入摇床中37℃条件下活化30分钟，然后一次性加0.1mmol的式I化合物，放入摇床中37℃，220rpm条件下进行催化水解反应。整个反应TLC监测，原料消失后停止反应，所得反应液通过一层硅藻土抽滤除去菌体，依次用水2mL洗涤滤渣三次，用旋转蒸发仪除去溶剂后，所生成的即为单酰胺单羧酸产物II化合物。
实施例3：突变体I450GI223F及其在手性环状化合物合成上的应用
1、酰胺水解酶突变体重组菌的构建与表达
设计引物，通过PCR扩增在酰胺水解酶末端加上His-tag标签，构建含有酰胺水解酶(ReAMI)基因的质粒pET22b-AMI，以此质粒为模板，设计定点突变引物序列如下：
I223F-F：5’-ATACCGGTGCATTTCCCTTCGAGCGAACAA-3’
I223F-R：5’-TGGTCGATTGTTCGCTCGAAGGGAAATGCA-3’
I450G-F：5’-CCAAGGCTCTCGGGATGGGCGCCAACACGG-3’
I450G-R：5’-AATGGTGCCGTGTTGGCGCCCATCCCGAGA-3’
首先进行I450G单点突变质粒的构建，通过PCR对模板进行全质粒扩增，PCR体系如下(50uL)：PrimeSTAR Max Premix(2×)25.0ul，I450G-F 2.0ul，I450G-R 2.0ul，TemplateDNA＜200ng，ddH2O up to 50ul。其中PrimeSTAR Max Premix包括PrimeSTAR HS DNAPolymerase，dNTP Mixture(each 0.4mM，2×)，Reaction Buffer(2×)，和Mg2+(2mM，2×)。
PCR反应程序为：1)98℃ 2.0min；2)98℃ 10sec，MT℃ 5-10sec，72℃ Et sec，25-30个循环；3)72℃ 5min。其中MT为退火温度，视不同引物而定；Et为延伸时间，以10sec/kb根据目的基因的长度来计算。
PCR产物经过0.9％琼脂糖凝胶电泳分析后，取PCR反应液20uL，加入1uL限制性内切酶DpnI于37℃条件下酶切4小时，随后转化到E.Coli BL21(DE3)感受态细胞中，复苏后涂布于含有氨苄青霉素的LB平板上培养12小时，挑取单菌落培养于已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h。提取质粒测序，测序正确的质粒命名为pET22b-I450G。
随后进行迭代多点突变，以pET22b-I450G作为模板，引物为I223F-F和I223F-R，按照上述操作方法进行PCR扩增，随后经限制性内切酶DpnI酶切的PCR反应液转化到E.ColiBL21(DE3)感受态细胞中，对细胞进行培养。提取质粒测序，测序正确的质粒命名为pET22b-I450GI223F。将含有pET22b-I450GI223F的菌株命名为含有酰胺水解酶突变体I450GI223F的大肠杆菌工程菌。该大肠杆菌工程菌为含有酰胺水解酶突变体I450GI223F基因的E.ColiBL21(DE3)感受态细胞。该大肠杆菌工程菌表达酰胺水解酶突变体I450GI223F，酰胺水解酶突变体I450GI223F的氨基酸序列如SEQ D No.4所示。
pET22b-I450GI223F与pET22b-AMI的区别仅在于将pET22b-AMI中所含的ReAMI基因替换为酰胺水解酶突变体I450GI223F基因，其它核苷酸完全相同。向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述测序正确的大肠杆菌工程菌，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
2、酰胺水解酶突变体催化性能检测
利用含有酰胺水解酶突变体I450GI223F的大肠杆菌工程菌催化环状二酰胺底物Ia化合物(X＝-O-；Y＝-CH2CH2-)的水解反应，如表3所示得到相应产物IIa’，经检测其ee值为82％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIa，经检测其ee值为30％，产物IIa’与产物IIa互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GI223F的大肠杆菌工程菌催化环状二酰胺底物Ib化合物(X＝-O-；Y＝-CH＝CH-)的水解反应，如表3所示得到相应产物IIb’，经检测其ee值为94％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIb，经检测其ee值为54％，产物IIb’与产物IIb互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GI223F的大肠杆菌工程菌催化环状二酰胺底物Ie化合物(X＝-CH2-；Y＝-CH2CH2-)的水解反应，如表3所示得到相应产物IIe’，经检测其ee值大于99.5％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIe，经检测其ee值为99％，产物IIe’与产物IIe互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GI223F的大肠杆菌工程菌催化环状二酰胺底物If化合物(X＝-NH-；Y＝-CH2CH2-)的水解反应，如表3所示得到相应产物IIf’，经检测其ee值大于99.5％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIf，经检测其ee值为99％，产物IIf’与产物IIf互为对映异构体，突变以后对映选择性发生反转。
表3：含有突变体I450GI223F与含有野生型酰胺水解酶的工程菌催化反应比较
具体实施方法为：取0.2克湿重的大肠杆菌工程菌，37℃条件下解冻30分钟，用磷酸氢二钾和磷酸二氢钾的缓冲溶液(0.1M，pH 7.0，5mL)将菌体洗入带螺纹口的Erlenmeyer平底烧瓶中，分散摇匀后放入摇床中37℃条件下活化30分钟，然后一次性加0.1mmol的式I化合物，放入摇床中37℃，220rpm条件下进行催化水解反应。整个反应TLC监测，原料消失后停止反应，所得反应液通过一层硅藻土抽滤除去菌体，依次用水2mL洗涤滤渣三次，用旋转蒸发仪除去溶剂后，所生成的即为单酰胺单羧酸产物II化合物。
实施例4：突变体I450GA332W及其在手性环状化合物合成上的应用
1、酰胺水解酶突变体重组菌的构建与表达
设计引物，通过PCR扩增在酰胺水解酶末端加上His-tag标签，构建含有酰胺水解酶(ReAMI)基因的质粒pET22b-AMI，以此质粒为模板，设计定点突变引物序列如下：
A332W-F：5’-ACATCTGGAACGTGATCTGGACGGACGGTG-3’
A332W-R：5’-TAGGCACCACCGTCCGTCCAGATCACGTTC-3’
I450G-F：5’-CCAAGGCTCTCGGGATGGGCGCCAACACGG-3’
I450G-R：5’-AATGGTGCCGTGTTGGCGCCCATCCCGAGA-3’
首先进行I450G单点突变质粒的构建，通过PCR对模板进行全质粒扩增，PCR体系如下(50uL)：PrimeSTAR Max Premix(2×)25.0ul，I450G-F 2.0ul，I450G-R 2.0ul，TemplateDNA＜200ng，ddH2O up to 50ul。其中PrimeSTAR Max Premix，其中包括PrimeSTAR HSDNAPolymerase，dNTP Mixture(each 0.4mM，2×)，Reaction Buffer(2×)，和Mg2+(2mM，2×)。
PCR反应程序为：1)98℃ 2.0min；2)98℃ 10sec，MT℃ 5-10sec，72℃ Et sec，25-30个循环；3)72℃ 5min。其中MT为退火温度，视不同引物而定；Et为延伸时间，以10sec/kb根据目的基因的长度来计算。
PCR产物经过0.9％琼脂糖凝胶电泳分析后，取PCR反应液20uL，加入1uL限制性内切酶DpnI于37℃条件下酶切4小时，随后转化到E.Coli BL21(DE3)感受态细胞中，复苏后涂布于含有氨苄青霉素的LB平板上培养12小时，挑取单菌落培养于已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h。提取质粒测序，测序正确的质粒命名为pET22b-I450G。
随后进行迭代多点突变，以pET22b-I450G作为模板，引物为A332W-F和A332W-R，按照上述操作方法进行PCR扩增，随后经限制性内切酶DpnI酶切的PCR反应液转化到E.ColiBL21(DE3)感受态细胞中，对细胞进行培养。提取质粒测序，测序正确的质粒命名为pET22b-I450GA332W。将含有pET22b-I450GA332W的菌株命名为含有酰胺水解酶突变体I450GA332W的大肠杆菌工程菌。该大肠杆菌工程菌为含有酰胺水解酶突变体I450GA332W基因的E.ColiBL21(DE3)感受态细胞。该大肠杆菌工程菌表达酰胺水解酶突变体I450GA332W，酰胺水解酶突变体I450GA332W的氨基酸序列如SEQ ID No.5所示。
pET22b-I450GA332W与pET22b-AMI的区别仅在于将pET22b-AMI中所含的ReAMI基因替换为酰胺水解酶突变体I450GA332W基因，其它核苷酸完全相同。向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述测序正确的大肠杆菌工程菌，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
2、酰胺水解酶突变体催化性能检测
利用含有酰胺水解酶突变体I450GA332W的大肠杆菌工程菌催化环状二酰胺底物Ie化合物(X＝-CH2-；Y＝-CH2CH2-)的水解反应，如表4所示得到相应产物IIe’，经检测其ee值大于99.5％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIe，经检测其ee值为99％，产物IIe’与产物IIe互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GA332W的大肠杆菌工程菌催化环状二酰胺底物Ig化合物(X＝-O-；Y＝-CH2CH2CH2-)的水解反应，如表4所示得到相应产物IIg’，经检测其ee值为82％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIg，经检测其ee值为99％，产物IIg’与产物IIg互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GA332W的大肠杆菌工程菌催化环状二酰胺底物Ih化合物(X＝-CH2-；Y＝-CH2CH2CH2-)的水解反应，如表4所示得到相应产物IIh’，经检测其ee值大于99.5％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIh，经检测其ee值为99％，产物IIh’与产物IIh互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GA332W的大肠杆菌工程菌催化环状二酰胺底物Ii化合物(X＝-NH-；Y＝-CH2CH2CH2-)的水解反应，如表4所示得到相应产物IIi’，经检测其ee值为93％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIi，经检测其ee值为99％，产物IIi’与产物IIi互为对映异构体，突变以后对映选择性发生反转。
表4：含有突变体I450GA332W与含有野生型酰胺水解酶的工程菌催化反应比较
具体实施方法为：取0.2克湿重的大肠杆菌工程菌，37℃条件下解冻30分钟，用磷酸氢二钾和磷酸二氢钾的缓冲溶液(0.1M，pH 7.0，5mL)将菌体洗入带螺纹口的Erlenmeyer平底烧瓶中，分散摇匀后放入摇床中37℃条件下活化30分钟，然后一次性加0.1mmol的式I化合物，放入摇床中37℃，220rpm条件下进行催化水解反应。整个反应TLC监测，原料消失后停止反应，所得反应液通过一层硅藻土抽滤除去菌体，依次用水2mL洗涤滤渣三次，用旋转蒸发仪除去溶剂后，所生成的即为单酰胺单羧酸产物II化合物。
实施例5：突变体I450GA332WF146A及其在手性环状化合物合成上的应用
1、酰胺水解酶突变体重组菌的构建与表达
设计引物，通过PCR扩增在酰胺水解酶末端加上His-tag标签，构建含有酰胺水解酶(ReAMI)基因的质粒pET22b-AMI，以此质粒为模板，设计定点突变引物序列如下：
F146A-F：5’-TGTGTGAGGACCTGTGTGCCTCCGGTTCGA-3’
F146A-R：5’-GTGAAGCTCGAACCGGAGGCACACAGGTCC-3’
A332W-F：5’-ACATCTGGAACGTGATCTGGACGGACGGTG-3’
A332W-R：5’-TAGGCACCACCGTCCGTCCAGATCACGTTC-3’
I450G-F：5’-CCAAGGCTCTCGGGATGGGCGCCAACACGG-3’
1450G-R：5’-AATGGTGCCGTGTTGGCGCCCATCCCGAGA-3’
首先进行I450G单点突变质粒的构建，通过PCR对模板进行全质粒扩增，PCR体系如下(50uL)：PrimeSTAR Max Premix(2×)25.0ul，I450G-F 2.0ul，I450G-R 2.0ul，TemplateDNA＜200ng，ddH2O up to 50ul。其中PrimeSTAR Max Premix包括PrimeSTAR HS DNAPolymerase，dNTP Mixture(each 0.4mM，2×)，Reaction Buffer(2×)，和Mg2+(2mM，2×)。
PCR反应程序为：1)98℃ 2.0min；2)98℃ 10sec，MT℃ 5-10sec，72℃ Et sec，25-30个循环；3)72℃ 5min。其中MT为退火温度，视不同引物而定；Et为延伸时间，以10sec/kb根据目的基因的长度来计算。
PCR产物经过0.9％琼脂糖凝胶电泳分析后，取PCR反应液20uL，加入1uL限制性内切酶DprI于37℃条件下酶切4小时，随后转化到E.Coli BL21(DE3)感受态细胞中，复苏后涂布于含有氨苄青霉素的LB平板上培养12小时，挑取单菌落培养于已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h。提取质粒测序为pET22b-I450G。
随后进行迭代多点突变，以pET22b-I450G作为模板，引物为A332W-F和A332W-R，按照上述操作方法进行PCR扩增，随后经限制性内切酶DpnI酶切的PCR反应液转化到E.ColiBL21(DE3)感受态细胞中，对细胞进行培养。提取质粒测序，测序正确的质粒命名为pET22b-I450GA332W。以pET22b-I450GA332W作为模板，引物F146A-F和F146A-R，按照上述操作方法进行PCR扩增，随后经限制性内切酶DpnI酶切的PCR反应液转化到E.Coli BL21(DE3)感受态细胞中，对细胞进行培养。提取质粒测序，测序正确的质粒命名为pET22b-I450GA332WF146A。将含有pET22b-I450GA332WF146A的菌株命名为含有酰胺水解酶突变体I450GA332WFl46A的大肠杆菌工程菌。该大肠杆菌工程菌为含有酰胺水解酶突变体I450GA332WF146A基因的E.Coli BL21(DE3)感受态细胞。该大肠杆菌工程菌表达酰胺水解酶突变体I450GA332WF146A，酰胺水解酶突变体I450GA332WF146A的氨基酸序列如SEQ IDNo.6所示。
pET22b-I450GA332WF146A与pET22b-AMI的区别仅在于将pET22b-AMI中所含的ReAMI基因替换为酰胺水解酶突变体I450GA332WF146A基因，其它核苷酸完全相同。
向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述测序正确的大肠杆菌工程菌，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
2、酰胺水解酶突变体催化性能检测
利用含有酰胺水解酶突变体I450GA332WF146A的大肠杆菌工程菌催化环状二酰胺底物Ig化合物(X＝-O-；Y＝-CH2CH2CH2-)的水解反应，如表5所示得到相应产物IIg’，经检测其ee值为99％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIg，经检测其ee值为99％，产物IIg’与产物IIg互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GA332WF146A的大肠杆菌工程菌催化环状二酰胺底物Ih化合物(X＝-cH2-；Y＝-CH2CH2CH2-)的水解反应，如表5所示得到相应产物IIh’，经检测其ee值大于99.5％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIh，经检测其ee值为99％，产物IIh’与产物IIh互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450GA332WF146A的大肠杆菌工程菌催化环状二酰胺底物Ii化合物(X＝-NH-；Y＝-CH2CH2CH2-)的水解反应，如表5所示得到相应产物IIi’，经检测其ee值为99％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIi，经检测其ee值为99％，产物IIi’与产物IIi互为对映异构体，突变以后对映选择性发生反转。
表5：含有突变体I450GA332WF146A与含有野生型酰胺水解酶的工程菌催化反应比较
具体实施方法为：取0.2克湿重的大肠杆菌工程菌，37℃条件下解冻30分钟，用磷酸氢二钾和磷酸二氢钾的缓冲溶液(0.1M，pH 7.0，5mL)将菌体洗入带螺纹口的Erlenmeyer平底烧瓶中，分散摇匀后放入摇床中37℃条件下活化30分钟，然后一次性加0.1mmol的式I化合物，放入摇床中37℃，220rpm条件下进行催化水解反应。整个反应TLC监测，原料消失后停止反应，所得反应液通过一层硅藻土抽滤除去菌体，依次用水2mL洗涤滤渣三次，用旋转蒸发仪除去溶剂后，所生成的即为单酰胺单羧酸产物II化合物。
实施例6：突变体I450AW328YF146A及其在手性环状化合物合成上的应用
1、酰胺水解酶突变体重组菌的构建与表达
设计引物，通过PCR扩增在酰胺水解酶末端加上His-tag标签，构建含有酰胺水解酶(ReAMI)基因的质粒pET22b-AMI，以此质粒为模板，设计定点突变引物序列如下：
F146A-F：5’-TGTGTGAGGACCTGTGTGCCTCCGGTTCGA-3’
F146A-R：5’-GTGAAGCTCGAACCGGAGGCACACAGGTCC-3’
W328Y-F：5’-TGCATGCTTTCCACATCTACAACGTGATCG-3’
W328Y-R：5’-TCCGTGGCGATCACGTTGTAGATGTGGAAA-3’
I450A-F：5’-CCAAGGCTCTCGGGATGGCCGCCAACACGG-3’
I450A-R：5’-AATGGTGCCGTGTTGGCGGCCATCCCGAGA-3’
首先进行I450A单点突变质粒的构建，通过PCR对模板进行全质粒扩增，PCR体系如下(50uL)：PrimeSTAR Max Premix(2×)25.0ul，I450A-F 2.0ul，I450A-R 2.0ul，TemplateDNA＜200ng，ddH2O up to 50ul。其中PrimeSTAR Max Premix，其中包括PrimeSTAR HS DNAPolymerase，dNTP Mixture(each 0.4mM，2×)，Reaction Buffer(2×)，和Mg2+(2mM，2×)。
PCR反应程序为：1)98℃ 2.0min；2)98℃ 10sec，MT℃ 5-10sec，72℃ Et sec，25-30个循环；3)72℃ 5min。其中MT为退火温度，视不同引物而定；Et为延伸时间，以10sec/kb根据目的基因的长度来计算。
PCR产物经过0.9％琼脂糖凝胶电泳分析后，取PCR反应液20uL，加入1uL限制性内切酶DpnI于37℃条件下酶切4小时，随后转化到E.Coli BL21(DE3)感受态细胞中，复苏后涂布于含有氨苄青霉素的LB平板上培养12小时，挑取单菌落培养于已灭菌的含有1‰氨苄青霉素(终浓度为50mg/L)的LB培养基中，放入摇床中37℃条件下培养10-16h。提取质粒测序为pET22b-I450G。
随后进行迭代多点突变，以pET22b-I450G作为模板，引物为W328Y-F和W328Y-R，按照上述操作方法进行PCR扩增，随后经限制性内切酶DpnI酶切的PCR反应液转化到E.ColiBL21(DE3)感受态细胞中，对细胞进行培养。提取质粒测序，测序正确的质粒命名为pET22b-I450AW328Y突变体质粒。以pET22b-I450AW328Y作为模板，引物为F146A-F和F146A-R，按照上述操作方法进行PCR扩增，随后经限制性内切酶DpnI酶切的PCR反应液转化到E.ColiBL21(DE3)感受态细胞中，对细胞进行培养。提取质粒测序，测序正确的质粒命名为pET22b-I450AW328YF146A。将含有pET22b-I450AW328YF146A的菌株命名为含有酰胺水解酶突变体I450AW328YF146A的大肠杆菌工程菌。该大肠杆菌工程菌为含有酰胺水解酶突变体I450AW328YF146A基因的E.Coli BL21(DE3)感受态细胞。该大肠杆菌工程菌表达酰胺水解酶突变体I450AW328YF146A，酰胺水解酶突变体I450AW328YF146A的氨基酸序列如SEQ IDNo.7所示。
pET22b-I450AW328YF146A与pET22b-AMI的区别仅在于将pET22b-AMI中所含的ReAMI基因替换为酰胺水解酶突变体I450AW328YF146A基因，其它核苷酸完全相同。向灭菌后的LB培养基中加入1‰氨苄青霉素(终浓度为50mg/L)，然后接入上述测序正确的种子，37℃摇床培养至OD600为0.6-0.8时将其取出降温，加入IPTG溶液诱导(1‰含量)，25℃条件下220rpm摇床培养4-6h。8000rpm离心4min，弃去上清。菌体用磷酸缓冲溶液洗2次(每次离心弃去上清溶液)，以0.2g/瓶分装于小样品瓶中。
2、酰胺水解酶突变体催化性能检测
利用含有酰胺水解酶突变体I450AW328YF146A的大肠杆菌工程菌催化环状二酰胺底物Ic化合物(x＝--，其中--表示无任何原子；Y＝-CH2CH＝CHCH2-)的水解反应，如表6所示得到相应产物IIc’，经检测其ee值为99％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IIc，经检测其ee值为94％，产物IIc’与产物IIc互为对映异构体，突变以后对映选择性发生反转。
利用含有酰胺水解酶突变体I450AW328YF146A的大肠杆菌工程菌催化环状二酰胺底物Id化合物(X＝--，其中--表示无任何原子；Y＝-CH2CH2CH2CH2-)的水解反应，如表6所示得到相应产物IId’，经检测其ee值为98％；与之对比，实施例1制备的含有未突变的野生型酰胺水解酶的大肠杆菌工程菌催化得到产物IId，经检测其ee值为99％，产物IId’与产物IId互为对映异构体，突变以后对映选择性发生反转。
表6：含有突变体I450AW328YF146A与含有野生型酰胺水解酶的工程菌催化反应比较
具体实施方法为：取0.2克湿重的大肠杆菌工程菌，37℃条件下解冻30分钟，用磷酸氢二钾和磷酸二氢钾的缓冲溶液(0.1M，pH 7.0，5mL)将菌体洗入带螺纹口的Erlenmeyer平底烧瓶中，分散摇匀后放入摇床中37℃条件下活化30分钟，然后一次性加0.1mmol的式I化合物，放入摇床中37℃，220rpm条件下进行催化水解反应。整个反应TLC监测，原料消失后停止反应，所得反应液通过一层硅藻土抽滤除去菌体，依次用水2mL洗涤滤渣三次，用旋转蒸发仪除去溶剂后，所生成的即为单酰胺单羧酸产物II化合物。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
序列表
<110> 中国科学院化学研究所
<120> 酰胺水解酶突变体及其在手性环状化合物合成上的应用
<130> 210645
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glu His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Phe Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Ser Thr Gly Ala Phe Pro Ile Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Trp Asn Val Ile Ala Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Ile Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 2
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glx His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Phe Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Ala Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Ile Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Trp Asn Val Ile Ala Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Ile Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 3
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glx His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Phe Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Gly Gly Ser Ile Arg Ala Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Ile Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Trp Asn Val Ile Ala Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Ile Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 4
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glx His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Phe Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Phe Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Trp Asn Val Ile Ala Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Gly Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 5
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glx His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Phe Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Ile Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Trp Asn Val Ile Trp Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Gly Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 6
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glx His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Ala Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Ile Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Trp Asn Val Ile Trp Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Gly Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 7
<211> 532
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Thr Ile Arg Pro Asp Asp Lys Ala Ile Asp Ala Ala Ala Arg
1 5 10 15
His Tyr Gly Ile Thr Leu Asp Lys Thr Ala Arg Leu Glu Trp Pro Ala
20 25 30
Leu Ile Asp Gly Ala Leu Gly Ser Tyr Asp Val Val Asp Gln Leu Tyr
35 40 45
Ala Asp Glu Ala Thr Pro Pro Thr Thr Ser Arg Glx His Ala Val Pro
50 55 60
Ser Ala Ser Glu Asn Pro Leu Ser Ala Trp Tyr Val Thr Thr Ser Ile
65 70 75 80
Pro Pro Thr Ser Asp Gly Val Leu Thr Gly Arg Arg Val Ala Ile Lys
85 90 95
Asp Asn Val Thr Val Ala Gly Val Pro Met Met Asn Gly Ser Arg Thr
100 105 110
Val Glu Gly Phe Thr Pro Ser Arg Asp Ala Thr Val Val Thr Arg Leu
115 120 125
Leu Ala Ala Gly Ala Thr Val Ala Gly Lys Ala Val Cys Glu Asp Leu
130 135 140
Cys Ala Ser Gly Ser Ser Phe Thr Pro Ala Ser Gly Pro Val Arg Asn
145 150 155 160
Pro Trp Asp Arg Gln Arg Glu Ala Gly Gly Ser Ser Gly Gly Ser Ala
165 170 175
Ala Leu Val Ala Asn Gly Asp Val Asp Phe Ala Ile Gly Gly Asp Gln
180 185 190
Gly Gly Ser Ile Arg Ile Pro Ala Ala Phe Cys Gly Val Val Gly His
195 200 205
Lys Pro Thr Phe Gly Leu Val Pro Tyr Thr Gly Ala Phe Pro Ile Glu
210 215 220
Arg Thr Ile Asp His Leu Gly Pro Ile Thr Arg Thr Val His Asp Ala
225 230 235 240
Ala Leu Met Leu Ser Val Ile Ala Gly Arg Asp Gly Asn Asp Pro Arg
245 250 255
Gln Ala Asp Ser Val Glu Ala Gly Asp Tyr Leu Ser Thr Leu Asp Ser
260 265 270
Asp Val Asp Gly Leu Arg Ile Gly Ile Val Arg Glu Gly Phe Gly His
275 280 285
Ala Val Ser Gln Pro Glu Val Asp Asp Ala Val Arg Ala Ala Ala His
290 295 300
Ser Leu Thr Glu Ile Gly Cys Thr Val Glu Glu Val Asn Ile Pro Trp
305 310 315 320
His Leu His Ala Phe His Ile Tyr Asn Val Ile Ala Thr Asp Gly Gly
325 330 335
Ala Tyr Gln Met Leu Asp Gly Asn Gly Tyr Gly Met Asn Ala Glu Gly
340 345 350
Leu Tyr Asp Pro Glu Leu Met Ala His Phe Ala Ser Arg Arg Ile Gln
355 360 365
His Ala Asp Ala Leu Ser Glu Thr Val Lys Leu Val Ala Leu Thr Gly
370 375 380
His His Gly Ile Thr Thr Leu Gly Gly Ala Ser Tyr Gly Lys Ala Arg
385 390 395 400
Asn Leu Val Pro Leu Ala Arg Ala Ala Tyr Asp Thr Ala Leu Arg Gln
405 410 415
Phe Asp Val Leu Val Met Pro Thr Leu Pro Tyr Val Ala Ser Glu Leu
420 425 430
Pro Ala Lys Asp Val Asp Arg Ala Thr Phe Ile Thr Lys Ala Leu Gly
435 440 445
Met Ala Ala Asn Thr Ala Pro Phe Asp Val Thr Gly His Pro Ser Leu
450 455 460
Ser Val Pro Ala Gly Leu Val Asn Gly Leu Pro Val Gly Met Met Ile
465 470 475 480
Thr Gly Arg His Phe Asp Asp Ala Thr Val Leu Arg Val Gly Arg Ala
485 490 495
Phe Glu Lys Leu Arg Gly Ala Phe Pro Thr Pro Ala Glu Arg Ala Ser
500 505 510
Asn Ser Ala Pro Gln Leu Ser Pro Ala Ala Ala Ala Leu Glu His His
515 520 525
His His His His
530
<210> 8
<211> 1599
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggcgacaa tccgacctga cgacaaagca atagacgccg ccgcaaggca ttacggcatc 60
actctcgaca aaacagcccg gctcgagtgg ccggcactga tcgacggagc actgggctcc 120
tacgacgtcg tcgaccagtt gtacgccgac gaggcgaccc cgccgaccac gtcacgcgag 180
cacgcggtgc caagtgcgag cgaaaatcct ttgagcgctt ggtatgtgac caccagcatc 240
ccgccgacgt cggacggcgt cctgaccggc cgacgcgtgg cgatcaagga caacgtgacc 300
gtggccggag ttccgatgat gaacggatct cggacggtag agggatttac tccgtcacgc 360
gacgcgactg tggtcactcg actactggcg gccggtgcaa ccgtcgcggg caaagctgtg 420
tgtgaggacc tgtgtttctc cggttcgagc ttcacaccgg caagcggacc ggtccgcaat 480
ccatgggacc ggcagcgcga agcaggtgga tcatccggcg gcagtgcagc actcgtcgca 540
aacggtgacg tcgattttgc catcggcggg gatcaaggcg gatcgatccg gatcccggcg 600
gcattctgcg gcgtcgtcgg gcacaagccg acgttcgggc tcgtcccgtc taccggtgca 660
tttcccatcg agcgaacaat cgaccatctc ggcccgatca cacgcacggt ccacgatgca 720
gcactgatgc tctcggtcat cgccggccgc gacggtaacg acccacgcca agccgacagt 780
gtcgaagcag gtgactatct gtccaccctc gactccgatg tggacggcct gcgaatcgga 840
atcgttcgag agggattcgg gcacgcggtc tcacagcccg aggtcgacga cgcagtccgc 900
gcagcggcac acagtctgac cgaaatcggt tgcacggtag aggaagtaaa catcccgtgg 960
catctgcatg ctttccacat ctggaacgtg atcgccacgg acggtggtgc ctaccagatg 1020
ttggacggca acggatacgg catgaacgcc gaaggtttgt acgatccgga actgatggca 1080
cactttgctt ctcgacgcat tcagcacgcc gacgctctgt ccgaaaccgt caaactggtg 1140
gccctgaccg gccaccacgg catcaccacc ctcggcggcg cgagctacgg caaagcccgg 1200
aacctcgtac cgcttgcccg cgccgcctac gacactgcct tgagacaatt cgacgtcctg 1260
gtgatgccaa cgctgcccta cgtcgcatcc gaattgccgg cgaaggacgt agatcgtgca 1320
accttcatca ccaaggctct cgggatgatc gccaacacgg caccattcga cgtgaccgga 1380
catccgtccc tgtccgttcc ggccggcctg gtgaacgggc ttccggtcgg aatgatgatc 1440
accggcagac acttcgacga tgcgacagtc cttcgtgtcg gacgcgcatt cgaaaagctt 1500
cgcggcgcgt ttccgacgcc ggccgaacgc gcctccaact ctgcaccaca actcagcccc 1560
gccgcggccg cactcgagca ccaccaccac caccactga 1599
Claims (7)
1.蛋白质，其特征在于：所述蛋白质是对野生型酰胺水解酶进行突变得到的蛋白质，所述野生型酰胺水解酶的氨基酸序列如SEQ ID No.1第1-509位所示，所述蛋白质的氨基酸序列与野生型酰胺水解酶的氨基酸序列相比，第193位的甘氨酸残基突变为丙氨酸残基。
2.与权利要求1所述的蛋白质相关的生物材料，其特征在于：所述生物材料为B1)至B4)中的任一种：
B1)编码权利要求1所述蛋白质的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体；
B4)含有B1)所述核酸分子的重组微生物。
3.根据权利要求2所述的生物材料，其特征在于：B1)所述的核酸分子的核苷酸序列为将野生型酰胺水解酶基因序列第577-579位的GGC替换为GCC，其它核苷酸不变；
所述野生型酰胺水解酶基因序列如SEQ ID No.8的第1-1527位所示。
4.催化非手性环状二酰胺生成手性环状单酰胺单羧酸的方法，其特征在于：包括采用权利要求1所述的蛋白质或权利要求2中所述的重组微生物作为催化剂，催化非手性环状二酰胺生成手性环状单酰胺单羧酸；
所述非手性环状二酰胺为式I所示化合物：
所述式I结构通式中，两个酰胺基处于顺式构型；
X为-O-；
Y为-CH2CH2-、-CH＝CH-。
5.权利要求1所述的蛋白质在催化非手性环状二酰胺生成手性环状单酰胺单羧酸中的应用；
所述非手性环状二酰胺为式I所示化合物：
所述式I结构通式中，两个酰胺基处于顺式构型；
X为-O-；
Y为-CH2CH2-、-CH＝CH-。
6.权利要求2或3所述的生物材料在催化非手性环状二酰胺生成手性环状单酰胺单羧酸中的应用；
所述非手性环状二酰胺为式I所示化合物：
所述式I结构通式中，两个酰胺基处于顺式构型；
X为-O-；
Y为-CH2CH2-、-CH＝CH-。
7.生产权利要求1所述的蛋白质的方法，其特征在于：所述方法包括使权利要求1所述的蛋白质的编码基因在生物中进行表达得到所述蛋白质的步骤，所述生物为微生物、植物或非人动物。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110513923.7A CN115322980B (zh) | 2021-05-11 | 2021-05-11 | 酰胺水解酶突变体及其在手性环状化合物合成上的应用 |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110513923.7A CN115322980B (zh) | 2021-05-11 | 2021-05-11 | 酰胺水解酶突变体及其在手性环状化合物合成上的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115322980A CN115322980A (zh) | 2022-11-11 |
| CN115322980B true CN115322980B (zh) | 2024-04-02 |
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| WO2008054821A2 (en) * | 2006-10-30 | 2008-05-08 | Promega Corporation | Mutant hydrolase proteins with enhanced kinetics and functional expression |
| CN107164350A (zh) * | 2017-06-26 | 2017-09-15 | 湖北大学 | 一种吡嗪酰胺水解酶与其编码基因和应用 |
| CN110857276A (zh) * | 2018-08-22 | 2020-03-03 | 中国科学院化学研究所 | 一类手性β-羟基酰胺类化合物及其制备方法与应用 |
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| WO2008054821A2 (en) * | 2006-10-30 | 2008-05-08 | Promega Corporation | Mutant hydrolase proteins with enhanced kinetics and functional expression |
| CN107164350A (zh) * | 2017-06-26 | 2017-09-15 | 湖北大学 | 一种吡嗪酰胺水解酶与其编码基因和应用 |
| CN110857276A (zh) * | 2018-08-22 | 2020-03-03 | 中国科学院化学研究所 | 一类手性β-羟基酰胺类化合物及其制备方法与应用 |
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