CN114657164B - (s)-转氨酶及其应用 - Google Patents
(s)-转氨酶及其应用 Download PDFInfo
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- CN114657164B CN114657164B CN202210392967.3A CN202210392967A CN114657164B CN 114657164 B CN114657164 B CN 114657164B CN 202210392967 A CN202210392967 A CN 202210392967A CN 114657164 B CN114657164 B CN 114657164B
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- Prior art keywords
- glufosinate
- transaminase
- ala
- amino
- leu
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- C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y206/00—Transferases transferring nitrogenous groups (2.6)
- C12Y206/01—Transaminases (2.6.1)
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- Biophysics (AREA)
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- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本申请涉及一种利用生物多酶偶联法制备L‑草铵膦的方法，其包括：在(R)‑转氨酶、(S)‑转氨酶、氨基受体和氨基供体的存在下，使D,L‑草铵膦转化为L‑草铵膦。本申请的方法能够实现高浓度D,L‑草铵膦的高效拆分来制备L‑草铵膦。
Description
本申请是于2020年12月17日递交的、申请号为202011496575.9、发明名称为“利用生物多酶偶联法制备L-草铵膦的方法”的中国专利申请的分案申请。
技术领域
本申请涉及生物技术领域，特别是涉及一种利用生物多酶偶联法制备L-草铵膦的方法。
背景技术
草铵膦(又名双丙氨膦、草丁膦，商品名包括保试达、百速顿等，英文名为phosphinothricin(简称PPT)，化学名为2-氨基-4-[羟基(甲基)膦酰基]丁酸)，是世界第二大转基因作物耐受除草剂，由赫斯特公司开发生产。草铵膦属膦酸类除草剂，是谷氨酰胺合成酶抑制剂，非选择性(灭生性)触杀型除草剂。目前，世界三大除草剂分别为百草枯，草甘膦，草铵膦。在市场使用方面，草甘膦独占鳌头，但是由于其长期使用，使得大量杂草产生抗性，而草甘膦也趋于失效；百草枯由于其剧毒性，已被列入《鹿特丹公约》，全球越来越多国家禁用或限用，中国农业部已发布公告说明，百草枯在2014年7月1日停止生产，2016年7月1日禁止使用；而目前草铵膦产量虽小，却具有优异的除草性能和较小的药害副作用，因此，在未来一段时间内拥有巨大的市场潜力。
草铵膦有两种光学异构体，分别为L-草铵膦和D-草铵膦，但只有L-型具有除草活性，且在土壤中易分解，对人类和动物的毒性较小，除草谱广，对环境的破坏力小。
目前，市场上销售的草铵膦一般都是外消旋混合物。若草铵膦产品能以L-构型的纯光学异构体形式使用，可显著降低草铵膦的使用量，这对于提高原子经济性、降低使用成本、减轻环境压力具有重要意义。
现有制备手性纯L-草铵膦的方法主要两种：化学法和生物法。
其中化学法包括化学拆分法和化学合成法。化学拆分法是通过手性拆分试剂拆分外化学法合成的消旋D,L-草铵膦或其衍生物，从而制得光学纯的L-草铵膦。然而该工艺存在需要使用手性拆分试剂、D-草铵膦需要消旋再利用，需要多次拆分等缺点，难以规模工业化生产。化学合成法可以以天然手性氨基酸或者不对称法合成L-草铵膦。但是该过程氨基保护与脱保护过程复杂，步骤较多，多见于实验室研究中。化学合成法还可以采用不对称催化加氢法合成L-草铵膦。但该路线所使用的催化剂价格昂贵，反应路线较长，工业化成本较高。
相比化学法能耗高、污染重、成本高等劣势，生物催化法具有反应条件温和、立体选择性高、收率高等优点，是工业化制备L-草铵膦的重要趋势。
制备L-草铵膦的方法目前大多是2-氧代-4-(羟基甲基氧膦基)丁酸(PPO)为底物，经氨化反应，不对称合成L-草铵膦。其中CN1349561A描述了利用筛选的天冬氨酸转氨酶(Asp-TA)，以L-天冬氨酸作为氨基供体，通过转氨作用将PPO合成L-草铵膦。该工艺使用与PPO大约等摩尔量的氨基供体，生成的草酰乙酸在水中水解成丙酮酸，可以经酶促反应去除，不存在可逆反应。然而此工艺中底物PPO转化率仅有52％，效率较低，并且反应条件苛刻(80℃)。专利CN105603015B描述了一种以L-丙氨酸为氨基供体的氨基转移酶生产L-草铵膦的方法。此工艺能够将100mM的PPO完全转化成L-草铵膦，但是为了抑制逆反应，丙氨酸投量过高，反应结束仍有300mM的L-丙氨酸存在体系中，为后续分离纯化造成困难，同时反应温度高达50℃，能耗较高，不利于生产。另外还有的方法在氨基酸脱氢酶的介导下，以无机氨为供体，将PPO还原胺化制备L-精草铵膦，例如CN106978453A，反应过程采用无机氨，转化率能够达到99％以上，然而该工艺需要额外添加辅酶再生系统，产生的副产物提高了分离纯化的难度。
上述工艺都以PPO为原料，但PPO的成本很高，从而导致这些工艺生产L-草铵膦的成本很高，难以实现工业化。因此，国际专利WO2017151573A1描述了一种以D，L-草铵膦为原料生产L-精草铵膦的方法，该工艺采用D-氨基酸氧化酶氧化D-草铵膦获得PPO，再由转氨酶经转氨反应将PPO转化为L-草铵膦。尽管该工艺实现了草铵膦消旋体的动态动力学拆分，但是该工艺存在明显缺陷：一是底物投量难以提升(仅有300mM D,L-草铵膦)；二是转氨酶介导的PPO到L-草铵膦的反应，由于受到可逆反应影响，仅能实现90％的转化率；三是由于胺供体是L-谷氨酸，反应后仍有大量剩余，产物分离纯化困难。
因此迫切需要开发一种能够高效实现动态拆分高浓度D,L-草铵膦来制备L-草铵膦的方法。
发明内容
本申请提供了一种利用生物多酶偶联法制备L-草铵膦的方法，其以D,L-草铵膦为原料，经多酶催化体系获得L-草铵膦，所述多酶催化体系包括：(R)-转氨酶和(S)-转氨酶。该方法原料转化率高、分离精制过程简单、产品收率高、生产成本低，易于工业化。
在一些实施方式中，本申请提供了一种生产L-草铵膦的方法，所述方法包括：在(R)-转氨酶、(S)-转氨酶、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。
本申请方法在初始时仅需加入外消旋草铵膦、微量氨基受体(例如丙酮酸)(用以启动第一步反应)，氨基供体(例如L-天冬氨酸)(用以启动第二步反应)，以及生物催化剂(R)-转氨酶和(S)-转氨酶。本申请的方法涉及两步反应，在第一步反应中，(R)-转氨酶介导外消旋草铵膦中的D-草铵膦的脱氨反应生成中间体酮酸2-羰基-4-(羟基甲基膦酰基)丁酸(PPO)，而L-草铵膦不参与反应而完全保留；在第二步反应中，PPO及时被(S)-转氨酶还原胺化，生成L-草铵膦，生成的副产物例如草酰乙酸自发降解为氨基受体，回补第一步的脱氨化反应。整个反应过程中，氨基受体的含量始终维持在低浓度，从而降低了对反应的抑制程度，而且由于副产物的自发降解，推动反应平衡始终移向产物一侧，打破热力学平衡的限制实现完全转化。本申请方法仅通过两种立体选择性互补的转氨酶的偶联，即可以实现一锅一步的外消旋草铵膦的去消旋化，无需引入辅因子原位再生系统除去氨基受体，仅通过副产物的自发降解实现氨基受体的循环利用，大大降低了反应体系的复杂性。
转氨酶(Amine Transaminase,ATA,EC 2.6.1.X)属于转移酶类，是催化1个氨基供体(氨基酸或简单的胺)上的氨基转移到前手性的受体酮，得到手性胺和副产物酮或者α-酮酸的一类酶，其催化的反应是可逆的。根据氨基被转移到不同位置的氨基受体上，转氨酶又可以被分为α-转氨酶和ω-转氨酶(EC 2.6.1.1)。ω-转氨酶反应过程可分为两步，第一步反应是在ω-转氨酶的作用下将氨基供体上的氨基转移到PLP的羰基上，从而形成5-磷酸吡哆胺(PMP)和与氨基供体对应的酮；第二步反应同样在ω-转氨酶的作用下将PMP上的氨基转移到氨基受体上，PMP又转变为PLP实现循环。转氨酶的立体选择性可在包含手性中心的底物外消旋混合物的外消旋拆分中测定。ω-转氨酶根据立体选择性可分为“(R)-转氨酶”((R)-amine transaminase)和(S)-转氨酶((S)-amine transaminase)。
根据本申请，(R)-转氨酶可以是在存在酮底物(例如丙酮酸)的情况下，优先从外消旋D,L-草铵膦中诱导D-草铵膦的转氨反应的酶。(R)-转氨酶可以为本领域已知的任何具有(R)-转氨酶活性的酶。在一些实施方式中，所述(R)-转氨酶为选自以下中的任一种：APH1(例如NCBI序列号为WP_015938787.1)、HEA-2(例如NCBI序列号为ABX05998.1)、TSP-1(例如NCBI序列号为WP_013128145.1)、DEP-2(例如NCBI序列号为WP_013615256.1)和MPH(例如NCBI序列号为WP_013863226.1)。在一些实施方式中，所述(R)-转氨酶为APH1。在一些实施方式中，所述(R)-转氨酶来自Pseudarthrobacter chlorophenolicus，例如来自Pseudarthrobacter chlorophenolicus的APH1。在一些实施方式中，(R)-转氨酶的序列的NCBI登录号为WP_015938787.1。在一些实施方式中，所述(R)-转氨酶的氨基酸序列与SEQID No.1所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(R)-转氨酶的核苷酸序列与SEQ ID No.2所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(R)-转氨酶的氨基酸序列为SEQ ID No.1。在一些实施方式中，所述(R)-转氨酶的核苷酸序列为SEQ IDNo.2。
根据本发明，(S)-转氨酶是在存在酮底物比如丙酮酸的情况下，优先从外消旋D,L-草铵膦诱导L-草铵膦的转氨反应的酶。本申请所述的(S)-转氨酶可以为本领域已知的任何具有(S)-转氨酶活性的酶。在一些实施方式中，所述(S)-转氨酶为选自以下中的任一种：LMG42(即EN5)(例如NCBI序列号为PZN34824.1)和ATA-0303(例如NCBI序列号为WP_012204645.1)。在一些实施方式中，所述(S)-转氨酶来自Corynebacterium vitaeruminis，例如来自Corynebacterium vitaeruminis的EN5。在一些实施方式中，所述(S)-转氨酶来自Corynebacterium vitaeruminis DSM 20294。在一些实施方式中，所述(S)-转氨酶的氨基酸序列与SEQ ID No.3所示的氨基酸序列具有至少70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(S)-转氨酶的核苷酸序列与SEQ ID No.4所示的核苷酸序列具有至少60％、70％、80％、90％、91％、91％、93％、94％、95％、96％、97％、98％或99％或100％的同一性。在一些实施方式中，所述(S)-转氨酶的氨基酸序列为SEQ ID No.3。在一些实施方式中，所述(S)-转氨酶的核苷酸序列为SEQ ID No.4。
所述(R)-转氨酶和(S)-转氨酶的酶制剂形式可以为纯化的酶；部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其他任何合适形式。因此，在一些实施方式中，(R)-转氨酶和(S)-转氨酶的形式各自独立地选自：部分纯化的酶；无细胞提取物或粗细胞提取物；液体、粉末或固定形式；含有酶的可透化处理的细胞、完整细胞或完整发酵液、冻干细胞或其任何组合。
在一些优选的实施方式中，本申请方法中使用的(R)-转氨酶和(S)-转氨酶由单一重组微生物共表达。因此，本申请方法可以包括：在共表达(R)-转氨酶和(S)-转氨酶的重组微生物、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。可以利用本领域已知的任何方法构建所述重组微生物。例如，所述重组微生物可以如下构建：构建含所述(R)-转氨酶和(S)-转氨酶基因的重组载体，将所述重组载体转化至微生物，对获得的重组微生物进行诱导培养，分离培养液得到含有(R)-转氨酶和(S)-转氨酶基因的重组微生物。优选地，按照10000rpm离心10min后的菌体湿重计，所述重组微生物的添加量为5g/L-200g/L反应液；更优选地，30g/L-100g/L反应液；最优选地，为30g/L反应液。优选地，以干菌体重量计，所述重组微生物的添加量为1-50g干菌体/L反应液。
所述重组微生物可以是任何适用于酶表达的工程菌。在一些实施方式中，所述重组微生物属于以下属中的一种：酵母属(Saccharomyces)、曲霉属(Aspergillus)、毕赤酵母属(Pichia)、克鲁维酵母属(Kluyveromyces)、假丝酵母属(Candida)、汉逊酵母属(Hansenula)、腐质霉属(Humicola)、伊萨酵母属(Issatchenkia)、毛孢子菌属(Trichosporon)、酒香酵母属(Brettanomyces)、管囊酵母属(Pachysolen)、耶氏酵母属(Yarrowia)或埃希氏杆菌属(Escherichia)。在一些优选的实施方式中，所述重组微生物选自：酿酒酵母(Saccharomyces cerevisiae)、解脂耶氏酵母(Yarrowia lipolitica)、克鲁斯假丝酵母(Candida krusei)、东方伊萨酵母(Issatchenkia orientalis)或大肠杆菌(Escherichia coli)。在一些更优选的实施方式中，所述重组微生物是大肠杆菌。
在一些实施方式中，所述氨基受体为丙酮酸。
在一些实施方式中，所述氨基供体为L-天冬氨酸或L-丙氨酸。在一些优选的实施方式中，所述氨基供体为L-天冬氨酸。
在一些实施方式中，所述方法在反应缓冲液的存在下进行，优选地，所述反应缓冲液是pH为7-10，优选8-9的磷酸氢二钠-磷酸二氢钠缓冲液。在这种磷酸氢二钠-磷酸二氢钠缓冲液中进行反应时可以获得更优的反应效率。
在一些实施方式中，氨基受体和D,L-草铵膦的摩尔比为1:500-1:5，例如1:500-1:10；更优选地，为1:50-1:10。
在一些实施方式中，所述方法在辅酶磷酸吡哆醛的存在下进行。在一些实施方式中，磷酸吡哆醛与底物的摩尔比为1:10-1:200。在一些实施方式中，以摩尔浓度计，磷酸吡哆醛的添加量为0.1-2mM；更优选为1mM。
在一些实施方式中，所述方法进行的温度为20-70℃，时间为6-96小时；更优选，温度为30-50℃，例如30-45℃，时间为48-72小时，最优选的，时间为48h。
在一些实施方式中，所述方法在pH 6-10，例如pH 7-9或pH 8-9的条件下进行。
本申请所述的方法可以在一个或更多个反应容器中进行。优选地，本申请所述的方法在一个反应容器中进行(即“一锅一步法”)。
本申请方法的产率可以通过本领域已知的任何方法测量。例如，可以通过手性HPLC来测量所获得的草铵膦产物中两个构型含量。在一些实施方式中，获得的草铵膦产物的对映体过量(e.e.)至少为80％、85％、90％、95％、96％、97％、98％、99％或99.9％。
本申请方法具有以下有益效果：
(1)D-草铵膦被氧化为2-羰基-4-[羟基(甲基)膦酰基]丁酸，L-草铵膦因不参与反应而被完全保留；产物2-羰基-4-[羟基(甲基)膦酰基]丁酸又可以被转氨酶继续催化还原为L-草铵膦，进而实现D,L-草铵膦的原位去消旋化。而传统的氧化方法则需要将D-草铵膦和L-草铵膦都转化为2-羰基-4-[羟基(甲基)膦酰基]丁酸，造成了原料的浪费。
(2)本发明能够直接以D,L-草铵膦为底物进行拆分，无需昂贵的拆分试剂，也无需合成草铵膦衍生物，更无需对D-草铵膦进行分离、再消旋、再拆分等步骤。
(3)本方法克服了化学法合成L-草铵膦前体2-羰基-4-[羟基(甲基)膦酰基]丁酸的缺陷，是一种绿色，环保，低碳的工艺路线，适合大规模工业化生产应用。
对序列表的描述
SEQ ID NO:1是来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的氨基酸序列。
SEQ ID NO:2是来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的核苷酸序列。
SEQ ID NO:3是来源于Corynebacterium vitaeruminis DSM 20294的注释为(S)-转氨酶(EN5)的氨基酸序列。
SEQ ID NO:4是来源于Corynebacterium vitaeruminis DSM 20294的注释为(S)-转氨酶(EN5)的核苷酸序列。
附图说明
图1为本申请方法的一些实施例中采用的多酶体系拆分法生产L-草铵膦的反应式。
图2示例性显示了APH1-EN5构建体。
图3示例性显示了单菌多酶一锅一步去消旋化制备L-草铵膦的反应进程。
具体实施方式
实施例
材料和方法
基因工程所用材料和试剂：基因组提取试剂盒、质粒提取试剂盒、DNA纯化回收试剂盒购自康宁生命科学(吴江)有限公司；一步克隆试剂盒是诺唯赞有限公司(Vazyme)的MultiS One Step Cloning Kit；E.coli DH5α、E.coli BL21(DE3)、质粒pET-28a(+)、pCDFduet-1载体等购自上海旭冠生物科技发展有限公司；DNA marker、低分子量标准蛋白、蛋白胶等购自北京GenStar有限公司；过氧化氢酶购自宁夏夏盛实业集团有限公司，商品编号为CAT-400。以上试剂使用方法参考商品说明书。
序列合成，序列测序工作由杭州擎科梓熙生物技术有限公司完成。
催化工艺所用试剂2-羰基-4-[羟基(甲基)膦酰基]丁酸(简称PPO)，D,L-草铵膦购自永农生物科学有限公司；其他常用试剂购自国药集团化学试剂有限公司。
通过高效液相色谱(HPLC)检测反应的进行，并对PPO进行分析。HPLC分析方法为：色谱柱/PBr；柱温/30℃；流速/0.8mL/min；检测波长/210nm；流动相：50mM(NH4)2HPO4，加入12％的乙腈。
通过手性HPLC分析方法检查草铵膦的两个构型含量，手性HPLC分析方法为：色谱柱/OA-5000L；流动相/0.5％五水硫酸铜；检测波长/254nm；流速/0.7mL/min；柱温/35℃。
实施例1：工程菌菌体的培养
将工程菌E.coli BL21(DE3)/pCDFduet-1-APH1、E.coli BL21(DE3)/pCDFduet-1-EN5、E.coli BL21(DE3)/pCDFduet-1-APH1-EN5经平皿划线活化后，挑单菌落接种至含有50μg/mL卡那霉素的10mL LB液体培养基中，37℃震荡培养10h。按2％的接种量转接至50mL同样含有50μg/mL卡那霉素的LB液体培养基中，37℃震荡培养至OD600达到0.8左右时，加入终浓度为0.5mM的IPTG，28℃下震荡培养12h。培养结束后，将培养液8000rpm离心10min，弃上清，收集菌体，放到-80℃超低温冰箱中保存，待用。
实施例2：酶序列合成和菌株构建
将来源于Pseudarthrobacter chlorophenolicus的注释为(R)-转氨酶(APH1)的序列(氨基酸序列为SEQ ID NO.1所示，核苷酸序列为SEQ ID NO.2所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-APH1。测序验证无误后将pET28a-APH1转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
将来源于Corynebacterium vitaeruminis DSM 20294注释为(S)-转氨酶(EN5)的序列(氨基酸序列为SEQ ID NO.3所示，核苷酸序列为SEQ ID NO.4所示)进行全基因合成后，插入表达质粒pET-28a(+)得到pET28a-EN5。测序验证无误后将pET28a-EN3转入表达宿主大肠杆菌E.coli BL21(DE3)中用于后续重组酶的表达。
实施例3：含(R)-转氨酶和(S)-转氨酶系统的共表达菌株的构建：
将实施例2中使用的APH1基因通过一步克隆试剂盒连接到多克隆位点载体pCDFduet-1上，酶切位点为HindIII和XhoI，一步克隆引物为C1-F和C1-R(表1)，构建得到质粒pCDFduet-1-APH1。再在pCDFduet-1-APH1质粒的基础上，通过一步克隆试剂盒将实施例2中使用的EN5连接到多克隆位点载体pCDFduet-1第二个克隆位点上，酶切位点为NdeI和XhoI，一步克隆引物为C2-F和C2-R，构建得到质粒pCDFduet-1-APH1-EN5，构建得到共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-EN5。APH1-EN5构建体如图2所示。
表1：克隆引物序列
| 引物 | 序列 |
| C1-F | CCCAAGCTTAAGGAGATATACATATGACCTCTCCCGCTTCCGT |
| C1-R | CCGCTCGAGCTATTGGATTCCGGCGTAAAGC |
| C2-F | CCCAAGCTTATGACGCCACAGGGCACGTTCTTCCTCCCGGGGCC |
| C2-R | CCGCTCGAG TTATCAGATGACTTCCCCTATCACGGCGAGGAGGGCGT |
实施例4：单菌多酶一锅一步去消旋化制备L-草铵膦
按照实施例3的方法构建和培养能够表达(R)-转氨酶和(S)-转氨酶系统的共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-EN5，离心收集菌体细胞。
30ml的反应体系包含500mM D,L-PPT，10mM丙酮酸，600mM L-天冬氨酸，100mM磷酸盐缓冲液，用氨水将反应体系的pH调节至8.0，加入共表达菌株E.coli BL21(DE3)/pCDFduet-1-APH1-EN5 30g/L干细胞。反应条件为：温度30℃，转速250rpm。每隔一段时间取样(100μl)，加入900μl去离子水，加热终止反应。通过HPLC检测D-PPT的转化情况和L-PPT的生成情况，反应进程曲线如图3所示。
结果显示，反应48小时，液相检测D-PPT为0mM，D-PPT转化率为99.9％，PPO为2mM，L-PPT为488mM，产品草铵膦的e.e.值为99.9％。
序列表
<110> 永农生物科学有限公司
华东理工大学
宁夏永农生物科学有限公司
<120> (S)-转氨酶及其应用
<160> 4
<170> PatentIn版本 3.5
<210> 1
<211> 309
<212> PRT
<213> Pseudarthrobacter chlorophenolicus
<400> 1
Met Thr Ser Pro Ala Ser Val Val Leu Val Phe Leu Asp Pro Ala Phe
1 5 10 15
Pro Asp Gly Arg Leu Ala Asp Ala Ala Gln Pro Gln Leu Met Val Thr
20 25 30
Asp Gln Gly Ala Thr Arg Gly Asp Gly Ile Phe Glu Thr Met Leu Ala
35 40 45
Val Arg Gly Ser Val Arg Lys Ile Gln Ala His Leu Asp Arg Leu Asp
50 55 60
Gly Ser Ala Ala Ala Leu Asp Leu Ser Ile Pro Gly Gln Asp Asp Trp
65 70 75 80
Arg Arg Ala Ile Ala Thr Ala Ile Ala Glu His Gln Ala Gln Tyr Pro
85 90 95
Ala Pro Asp Ala Gly Asp Asp Glu Leu Val Val Lys Leu Val Val Thr
100 105 110
Arg Gly Val Glu Gly Ala Gly Ser Pro Thr Ala Trp Val Gln Val Ser
115 120 125
Pro Ala Pro Ala Ala Gly Arg Arg Gln Arg Glu Thr Gly Ile Asp Val
130 135 140
Ile Leu Leu Asp Arg Gly Tyr Asp Ser Asp Val Ala Glu Arg Ala Pro
145 150 155 160
Trp Leu Leu Met Gly Ala Lys Thr Leu Ser Tyr Ala Val Asn Met Ala
165 170 175
Ala Leu Arg His Ala Arg Arg Gln Gly Ala Asp Asp Val Ile Phe Leu
180 185 190
Ser Ser Asp Gly Arg Val Leu Glu Gly Pro Thr Ser Thr Val Leu Leu
195 200 205
Ala His Val Glu Glu Ser Ala Asp Gly Thr Ala Ile Lys Arg Leu Ile
210 215 220
Thr Pro Gln Leu Asp Ser Gly Ile Leu Pro Gly Thr Ser Gln Gly Ala
225 230 235 240
Leu Phe Thr Ala Ala Lys Ala Ala Gly Trp Glu Leu Gly Tyr Gly Pro
245 250 255
Leu Glu Pro Gln Asp Leu Leu Asp Ala Asp Ala Val Trp Leu Ile Ser
260 265 270
Ser Val Arg Leu Leu Ala Pro Val Asn Thr Ile Asp Gly Lys Gln Ile
275 280 285
Gly Thr Pro Ala Leu Gln Lys Glu Leu Thr Ala Glu Leu Thr Gly Leu
290 295 300
Tyr Ala Gly Ile Gln
305
<210> 2
<211> 930
<212> DNA
<213> Pseudarthrobacter chlorophenolicus
<400> 2
atgacctctc ccgcttccgt ggtactcgtt ttccttgatc ccgccttccc cgacggccgg 60
ctggccgacg ccgcccagcc gcagctgatg gtcacggacc agggcgccac caggggcgat 120
ggcatcttcg aaacgatgct cgccgtgcgc gggtcagtcc gaaaaatcca ggcccacctg 180
gaccgcctgg acggctccgc ggcggcgctg gacctcagca tcccgggcca ggatgactgg 240
cggcgggcca ttgccactgc cattgccgaa caccaggcgc agtacccggc ccccgatgcg 300
ggcgacgatg aactggtggt caagctggtg gtcacccgcg gcgttgaagg tgcgggctcc 360
cccaccgcct gggtgcaggt ctcccctgct ccggccgccg gccgccgcca acgggaaaca 420
ggcatcgacg tcatcctcct tgaccgcggg tacgacagtg acgttgccga gcgtgcgccg 480
tggctgctca tgggcgccaa gacgctctcc tacgccgtca acatggccgc cctgcgccat 540
gcccgcaggc agggcgcaga cgacgtcatc ttcctgtcct cggatggccg cgtgcttgag 600
ggccccacgt ccacggtgct gctggcgcac gtggaggagt ccgctgacgg gacggccatc 660
aagcgcctca tcacgccgca gctggacagc ggcatcctgc ccggaacatc gcagggggcc 720
ctcttcaccg cggcaaaggc ggcgggctgg gaactgggct acggacccct ggaaccgcag 780
gacctgctgg atgccgacgc ggtctggctg atctccagtg tccgcctcct cgccccggtc 840
aacacgatcg acggcaagca gatcggtacc ccggcgctgc agaaggagct gacggctgag 900
ctcacggggc tttacgccgg aatccaatag 930
<210> 3
<211> 353
<212> PRT
<213> Corynebacterium vitaeruminis DSM 20294
<400> 3
Met Thr Pro Gln Gly Thr Phe Phe Leu Pro Gly Pro Thr Glu Val His
1 5 10 15
Pro Asp Val Leu Ala Ala Gln Thr Arg Pro Met Ile Gly His Arg Gly
20 25 30
Ala Ala Ile Gln Thr Leu Met Ala Asn Ile Ala Thr Gly Met Arg Ser
35 40 45
Val Phe Gln Thr Glu Arg Pro Val Ile Val Ser Thr Ser Ser Ala Ser
50 55 60
Gly Leu Met Glu Gly Ala Ile Arg Asn Gly Val Gly Thr Gly Pro Val
65 70 75 80
Leu Cys Leu Val Asn Gly Ala Phe Ser Gly Arg Phe Ala Thr Ile Ala
85 90 95
Lys Ala Cys Gly Lys Asp Thr Glu Ile Trp Glu Val Glu Trp Gly Thr
100 105 110
Thr His Ser Val Asp Glu Leu Ala Arg Arg Leu Ser Glu Arg Arg Tyr
115 120 125
Asp Ala Val Thr Leu Ser Gln Ser Glu Thr Ser Thr Gly Ala Leu His
130 135 140
Asp Leu Glu Arg Val Ala Ala Val Val Ala Glu His Pro Gly Thr Leu
145 150 155 160
Leu Leu Val Asp Ser Val Thr Gly Ile Gly Gly Val Glu Thr Arg Pro
165 170 175
Asp Asp Trp Gly Ile Asp Phe Ile Leu Thr Gly Ser Gln Lys Ala Leu
180 185 190
Ala Leu Pro Pro Gly Leu Ala Phe Gly Ala Ala Ser Glu Ala Met Met
195 200 205
Glu Arg Ala Ala Ser Ile Pro Asp Arg Gly Trp Tyr Phe Asp Leu Val
210 215 220
Lys Leu Ala Glu Gly Ile Glu Lys Asn Gln Thr Pro Ala Thr Pro Ala
225 230 235 240
Ile Ser Leu Leu Tyr Ser Leu Glu Tyr Gln Leu Glu Arg Ile Leu Ala
245 250 255
Glu Gly Ile Glu Ala Arg Trp Lys Arg His Leu Gln Met Gln Ala Arg
260 265 270
Val Leu Glu Trp Val Glu Asp Ala Gly Leu Asp Val Leu Ala Val Glu
275 280 285
Gly His Arg Ser Pro Thr Val Thr Cys Val Val His Pro Glu Pro Gly
290 295 300
Arg Val Val Gly Ala Met Ala Glu Arg Gly Trp Val Ile Gly Ala Gly
305 310 315 320
Tyr Gly Gln Val Ser Asp Arg Thr Phe Arg Ile Gly His Met Gly Asp
325 330 335
His Thr Leu Glu Glu Val Asp Ala Leu Leu Ala Val Ile Gly Glu Val
340 345 350
Ile
<210> 4
<211> 1062
<212> DNA
<213> Corynebacterium vitaeruminis DSM 20294
<400> 4
atgacgccac agggcacgtt cttcctcccg gggcctaccg aggtccaccc cgacgtactc 60
gccgcccaga ctcgaccgat gatcggtcat cgcggtgcgg ccatccagac cctgatggcg 120
aacatcgcca ccggcatgag gtctgtattc cagaccgagc ggcccgtgat cgtctcgacc 180
tcgtcggcgt cggggctcat ggaaggggcc atccgcaacg gcgtgggaac gggcccggtg 240
ctgtgtctcg tcaacggcgc gttctcgggt cgcttcgcca cgatcgccaa ggcctgtggc 300
aaggacaccg agatctggga ggtggagtgg ggaacgacgc actccgtcga cgaacttgcc 360
cgtcggctgt ccgagcgacg ctacgacgcg gtgaccctct cccagtccga gacgtccacc 420
ggtgcgctcc acgacctgga gagggtcgct gccgtggtcg cagagcaccc cggcacactg 480
ctactcgtcg actccgtcac cggaatcggc ggtgtggaga cgcggccgga cgactggggc 540
atcgacttca tcctgaccgg ctcccagaag gccctggcac tgccgcccgg gctcgcattc 600
ggcgccgcct cggaggccat gatggagcgg gccgcctcga tccccgaccg tggctggtac 660
ttcgacctgg tcaagctcgc cgagggcatc gagaagaacc agaccccggc gactcctgcg 720
atctcgctcc tctattcgct ggagtaccag ctggagagga tcctcgccga gggcatcgag 780
gcgcgctgga agcggcacct gcagatgcag gcccgggtgc tcgagtgggt cgaggatgcc 840
ggcctcgatg ttctcgctgt cgaaggccat cggtccccga ccgtgacctg cgtggtccac 900
ccggagccgg gacgcgtggt cggggcgatg gccgagcgcg ggtgggtgat cggggcgggc 960
tacggccagg tgagcgaccg gacgttccgg atcgggcata tgggcgacca cacgctcgaa 1020
gaggtcgacg ccctcctcgc cgtgataggg gaagtcatct ga 1062
Claims (12)
1.一种(S)-转氨酶，所述(S)-转氨酶的氨基酸序列如SEQ ID No.3所示。
2.分离的核酸，其编码权利要求1所述的(S)-转氨酶。
3.根据权利要求2所述的核酸，其核苷酸序列如SEQ ID No.4所示。
4.含权利要求2或3所述核酸的重组载体。
5.重组微生物，其表达i)，或同时表达i)和ii)：
i)权利要求1所述的(S)-转氨酶；
ii)(R)-转氨酶，其氨基酸序列如SEQ ID No.1所示。
6.根据权利要求5所述的重组微生物，其含有1)，或同时含有1)和2)：
1)权利要求4所述的重组载体；
2)含能够编码所述(R)-转氨酶的核酸的重组载体。
7.根据权利要求1所述的(S)-转氨酶在制备L-草铵膦中的应用。
8.L-草铵膦的制备方法，包括：
在氨基供体存在的条件下，2-羰基-4-(羟基甲基膦酰基)丁酸被权利要求1所述的(S)-转氨酶还原胺化，生成L-草铵膦；所述氨基供体为L-天冬氨酸或L-丙氨酸。
9.根据权利要求8所述的制备方法，所述2-羰基-4-(羟基甲基膦酰基)丁酸的制备方法包括：
在氨基受体存在的条件下，(R)-转氨酶介导D,L-草铵膦中的D-草铵膦的脱氨反应生成2-羰基-4-(羟基甲基膦酰基)丁酸；所述氨基受体为丙酮酸。
10.根据权利要求9所述的方法，其中所述氨基受体和所述D,L-草铵膦的摩尔比为1:500-1:5。
11.根据权利要求9所述的方法，其中所述方法包括：在共表达所述(R)-转氨酶和所述(S)-转氨酶的重组微生物、氨基受体和氨基供体的存在下，使D,L-草铵膦转化为L-草铵膦。
12.根据权利要求9所述的制备方法，所述(R)-转氨酶的氨基酸序列如SEQ ID No.1所示。
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| CN111139271A (zh) * | 2019-03-21 | 2020-05-12 | 浙江工业大学 | 一种单一转氨酶催化级联反应不对称合成l-草铵膦的方法 |
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| US5587319A (en) * | 1988-12-14 | 1996-12-24 | Hoechst Aktiengesellschaft | Process for the preparation of L-phosphinothricin using transaminases of different specificities in a linked process |
| CN101384723A (zh) * | 2006-02-13 | 2009-03-11 | 罗扎股份公司 | 旋光手性胺的制备方法 |
| CN111139271A (zh) * | 2019-03-21 | 2020-05-12 | 浙江工业大学 | 一种单一转氨酶催化级联反应不对称合成l-草铵膦的方法 |
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| CN111321193A (zh) * | 2020-03-18 | 2020-06-23 | 浙江工业大学 | 一种生物多酶偶联法氧化还原不对称制备l-草铵膦的方法 |
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