CN114958934B - 一种制备l-草铵膦的方法 - Google Patents
一种制备l-草铵膦的方法 Download PDFInfo
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Abstract
本发明提供了一种利用谷氨酸脱氢酶制备L‑草铵膦的方法,包括如下步骤:以4‑(羟基‑(甲基)氧膦基)‑2‑氧代丁酸为底物,在氨基供体存在下,使用谷氨酸脱氢酶SEQ ID NO:2或者其V375突变体催化不对称还原胺化反应,得到L‑草铵膦。本发明还公开了一种合成底物4‑(羟基‑(甲基)氧膦基)‑2‑氧代丁酸的新方法。
Description
技术领域
本发明属于生物催化技术领域,具体地说,涉及一种利用谷氨酸脱氢酶催化制备L-草铵膦的方法。
背景技术
草铵膦是世界第二大转基因作物耐受的广谱非选择性除草剂,化学名为2-氨基-4[羟基-(甲基)膦酰基]-丁酸,是一种谷氨酰胺合成酶抑制剂。目前,世界上的三大非选择性除草剂分别为草甘膦、草铵膦和百草枯。近年来,随着草甘膦的大规模使用以及抗草甘膦作物的推广种植,杂草对草甘膦的抗性也逐渐增强。而百草枯由于毒性极强导致在全球范围陆续被禁用。随着转基因作物推广种植,且草铵膦的抗性基因陆续被导入各种作物中,草铵膦在未来一段时间内市场需求巨大,前景非常广阔。草铵膦存在两种对映异构体,分别为L-草铵膦和D-草铵膦,市场销售的一般为外消旋的草铵膦。但仅L-草铵膦具有除草活性,除草谱广,其活性接近于消旋体的两倍,并且L-草铵膦在土壤中易分解,对人类和动物造成的毒性较小,对环境破坏力小。
使用光学纯的L-草铵膦,不仅能够减少施药量,而且可以有效减缓杂草抗性增长、减轻环境负担,并且降低运输储存需求,具有显而易见的经济和环境优势,更符合现代环保理念。
目前L-草铵膦的制备方法主要有以下三种:手性拆分法、化学合成法以及生物催化合成法。其中,手性拆分法存在以下缺点:需要使用昂贵的手性拆分试剂,理论收率只有50%,工艺繁琐复杂。化学合成法虽然开发了很多路线,但依然存在着很多弊端,如前体去氢氨基酸不对称加氢制备L-草铵膦需要用到较为昂贵的手性催化剂(Journal of OrganicChemistry,1991,56,1783-1788;Pesticide Science,2014,41,269-277);不对称Strecker反应制备L-草铵膦需用到氰化物,且催化剂用量较高,反应条件苛刻(WO2008035687;Chemical Reviews,2011,111,6947-6983);不对称Michael加成反应制备L-草铵膦的立体选择性不理想(CN105131032A;Bulletin of the Chemical Society of Japan,1987,60,1761-1766),因此这些过程多见于实验室研究中,不易放大生产。
与化学合成法相比,生物催化法具有立体选择性专一、反应条件温和、收率高等优点。已经有多条酶法制备L-草铵膦的工艺报道。其中,以酮酸4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO)为底物,通过谷氨酸脱氢酶催化不对称还原胺化合成L-草铵膦前体的工艺路线是比较有潜力。其中,涉及的谷氨酸脱氢酶在生物体内几乎无所不在,可以可逆地催化谷氨酸氧化脱氨生成α-酮戊二酸。与采用转氨酶的L-草铵膦酶法制备工艺相比,利用谷氨酸脱氢酶催化PPO还原胺化制备L-草铵膦具有两个显著的优点:一是当辅酶再生系统效率较高时,底物能够100%地转化生成L-草铵膦;二是利用无机铵作为氨基供体,不会产生难以分离的副产物,因此产物易于提纯精制。专利文献CN106978453A采用谷氨酸脱氢酶催化不对称还原胺化合成了L-草铵膦:
但在该报道中,酶催化底物浓度低,仅为10~100mM,催化效率不高,表明使用的谷氨酸脱氢酶存在底物抑制性和/或产物抑制性。因此有必要开发高活性、高底物耐受性的谷氨酸脱氢酶。
谷氨酸脱氢酶催化合成L-草铵膦需要以酮酸PPO为底物,该底物的绿色、经济合成决定着整条工艺产业化的环保性和工业可行性性。专利文献CN103665032A中公开了一种该前体酮酸的合成方法,利用环磷酸酐与氰化物反应获得酮腈,进而氰基水解得到酮酸中间体。由于该工艺使用了剧毒氰化物,因此危险性大,并且该反应的第一步不易实现。
专利文献CN101665514A中以甲基亚磷酸二烷基酯与丙烯酸为原料制备3-(甲基烷氧基膦酰基)丙酸酯,这步反应需要在无水无氧条件下进行,条件苛刻。美国专利US4399287中以3-(甲基烷氧基膦酰基)丙酸酯与草酸二乙酯为原料,经Claisen缩合、水解脱羧得到4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO),该反应原子经济性差,并且脱羧过程中会有CO2排放,不符合当下的环保理念。
发明内容
为了克服现有技术生产L-草铵膦的上述缺陷,发明人一方面提供了一种路线简洁、副反应少、收率高、相对环保且避免使用氰化物的L-草铵膦前体酮酸PPO的合成新工艺,另一方面从众多微生物来源的谷氨酸脱氢酶库中筛选出一种对PPO还原胺化具有高立体选择性和几乎没有底物/产物抑制性的谷氨酸脱氢酶,其来源于碱湖生菌(Alkalilimnicolaehrlichii AK93),用于催化PPO反应制备L-草铵膦时产物浓度高、原子经济性强、不需要额外添加昂贵的辅酶、操作简便、易于放大且产物易于分离提纯,具有很好的工业应用前景。
为了实现上述目的,本发明所提供的主要技术方案如下:
一种制备L-草铵膦的方法,包括如下步骤:以4-(羟基-(甲基)氧膦基)-2-氧代丁酸为底物,在氨基供体存在下,使用谷氨酸脱氢酶SEQ ID NO:2或者其V375突变体催化不对称还原胺化反应,得到L-草铵膦。
所述谷氨酸脱氢酶来源于碱湖生菌(Alkalilimnicola ehrlichii AK93),氨基酸序列SEQ ID NO:2:
MSDDLQAFLAYVRERDPDQPEFLQAVEETMRSVWPFIERHPRYRDMGLLERLVEPERIITFRVPWVDDQGRVHVNRGYRVQMNSAIGPYKGGLRFHPSVNVSILKFLAFEQVFKNGLTTLPLGGGKGGADFDPKGRSDAEVMRFCQSFMSELYRHIGHDLDVPAGDIGVSRREIGYLYGMYKKLANEFTGVITGKGYNYGGSLIRPEATGYGLIYFVAEMIKPKYANMDGLKVAISGSGNVAQFAAAKAMEFGAKVITLSDSGGTVYMPEGLTDEQWDFLVRLKNELRGRLSTFAEEFGFEFAADKRPWGYPCDIALPCATQNELDDRDALALINNGCFCVAEGANMPSTIGAVDCFQAARILYAPGKASNAGGVAVSGLEMSQNALRLSWTDGEVDEKLHSIMQSIHTTCMHYGAGKGGYVNYVDGANIAGFVKVADAMNEQGIV(SEQ ID NO:2)。
上述V375突变体可以选自下组:
V375S突变体即SEQ ID NO:3:
MSDDLQAFLAYVRERDPDQPEFLQAVEETMRSVWPFIERHPRYRDMGLLERLVEPERIITFRVPWVDDQGRVHVNRGYRVQMNSAIGPYKGGLRFHPSVNVSILKFLAFEQVFKNGLTTLPLGGGKGGADFDPKGRSDAEVMRFCQSFMSELYRHIGHDLDVPAGDIGVSRREIGYLYGMYKKLANEFTGVITGKGYNYGGSLIRPEATGYGLIYFVAEMIKPKYANMDGLKVAISGSGNVAQFAAAKAMEFGAKVITLSDSGGTVYMPEGLTDEQWDFLVRLKNELRGRLSTFAEEFGFEFAADKRPWGYPCDIALPCATQNELDDRDALALINNGCFCVAEGANMPSTIGAVDCFQAARILYAPGKASNAGGSAVSGLEMSQNALRLSWTDGEVDEKLHSIMQSIHTTCMHYGAGKGGYVNYVDGANIAGFVKVADAMNEQGIV(SEQ ID NO:3)。
V375A突变体即SEQ ID NO:4:
MSDDLQAFLAYVRERDPDQPEFLQAVEETMRSVWPFIERHPRYRDMGLLERLVEPERIITFRVPWVDDQGRVHVNRGYRVQMNSAIGPYKGGLRFHPSVNVSILKFLAFEQVFKNGLTTLPLGGGKGGADFDPKGRSDAEVMRFCQSFMSELYRHIGHDLDVPAGDIGVSRREIGYLYGMYKKLANEFTGVITGKGYNYGGSLIRPEATGYGLIYFVAEMIKPKYANMDGLKVAISGSGNVAQFAAAKAMEFGAKVITLSDSGGTVYMPEGLTDEQWDFLVRLKNELRGRLSTFAEEFGFEFAADKRPWGYPCDIALPCATQNELDDRDALALINNGCFCVAEGANMPSTIGAVDCFQAARILYAPGKASNAGGAAVSGLEMSQNALRLSWTDGEVDEKLHSIMQSIHTTCMHYGAGKGGYVNYVDGANIAGFVKVADAMNEQGIV(SEQ ID NO:4)。
V375G突变体即SEQ ID NO:5:
MSDDLQAFLAYVRERDPDQPEFLQAVEETMRSVWPFIERHPRYRDMGLLERLVEPERIITFRVPWVDDQGRVHVNRGYRVQMNSAIGPYKGGLRFHPSVNVSILKFLAFEQVFKNGLTTLPLGGGKGGADFDPKGRSDAEVMRFCQSFMSELYRHIGHDLDVPAGDIGVSRREIGYLYGMYKKLANEFTGVITGKGYNYGGSLIRPEATGYGLIYFVAEMIKPKYANMDGLKVAISGSGNVAQFAAAKAMEFGAKVITLSDSGGTVYMPEGLTDEQWDFLVRLKNELRGRLSTFAEEFGFEFAADKRPWGYPCDIALPCATQNELDDRDALALINNGCFCVAEGANMPSTIGAVDCFQAARILYAPGKASNAGGGAVSGLEMSQNALRLSWTDGEVDEKLHSIMQSIHTTCMHYGAGKGGYVNYVDGANIAGFVKVADAMNEQGIV(SEQ ID NO:5)。
V375C突变体即SEQ ID NO:6:
MSDDLQAFLAYVRERDPDQPEFLQAVEETMRSVWPFIERHPRYRDMGLLERLVEPERIITFRVPWVDDQGRVHVNRGYRVQMNSAIGPYKGGLRFHPSVNVSILKFLAFEQVFKNGLTTLPLGGGKGGADFDPKGRSDAEVMRFCQSFMSELYRHIGHDLDVPAGDIGVSRREIGYLYGMYKKLANEFTGVITGKGYNYGGSLIRPEATGYGLIYFVAEMIKPKYANMDGLKVAISGSGNVAQFAAAKAMEFGAKVITLSDSGGTVYMPEGLTDEQWDFLVRLKNELRGRLSTFAEEFGFEFAADKRPWGYPCDIALPCATQNELDDRDALALINNGCFCVAEGANMPSTIGAVDCFQAARILYAPGKASNAGGCAVSGLEMSQNALRLSWTDGEVDEKLHSIMQSIHTTCMHYGAGKGGYVNYVDGANIAGFVKVADAMNEQGIV(SEQ ID NO:6)。
上述反应中所用的氨基供体可以为硫酸铵、磷酸氢二铵、碳酸氢铵、硝酸铵、氯化铵、乙酸铵、甲酸铵或氨水。
优选地,反应体系中还可以添加有辅酶再生系统,所述辅酶再生系统选自下组:
(1)以葡萄糖为辅酶再生底物、以葡萄糖脱氢酶为辅酶再生酶,包含NADPH或NADP+的葡萄糖脱氢酶再生系统;
(2)以异丙醇为辅酶再生底物、以醇脱氢酶为辅酶再生酶,包含NADPH或NADP+的醇脱氢酶再生系统;
(3)以甲酸盐为辅酶再生底物、以甲酸脱氢酶为辅酶再生酶,构成包含NADPH或NADP+的甲酸脱氢酶再生系统。
反应体系中添加辅酶NADP+(烟酰胺腺嘌呤二核苷磷酸,辅酶II)可以促进还原反应。NADP+的作用是,作为氧化剂掠夺电子,辅酶再生酶利用辅酶再生底物比如异丙醇/葡萄糖/甲酸盐将NADP+还原为NADPH,产生充足的NADPH作为生物合成的还原剂,从而促进还原反应。
上述反应体系中,谷氨酸脱氢酶SEQ ID NO:2或者其V375突变体既可以呈酶形式,也可以呈表达微生物菌体形式例如培养后的静息细胞。
在一种实施方式中,底物4-(羟基-(甲基)氧膦基)-2-氧代丁酸可以通过下述工艺制备:
将式III所示4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸酯与浓盐酸反应脱保护,得到式IV所示底物PPO,
其中,R选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、苯甲基。
其中,式III所示化合物可以通过下述工艺制备:
(1)将二氢-2,3-呋喃二酮(式I)加入到氢溴酸的醋酸溶液中,于60~80℃反应至产物不再继续增加后,冷却至室温,加入醇类试剂,继续搅拌一定时间至开环溴代反应完全终止;将反应液浓缩除去溶剂,得到的残余物溶解于水不溶性溶剂中,洗涤,干燥,浓缩除去溶剂,得到4-溴-2-氧代丁酸酯(式II);
其中所述开环溴代反应中氢溴酸的醋酸溶液的质量分数为30~50%;所述水不溶性溶剂包括酯类比如乙酸乙酯、烷烃类比如正己烷等。
(2)在氮气保护下,将4-溴-2-氧代丁酸酯(式II)与甲基亚磷酸二乙酯按一定摩尔比进行混合,加热至100~200℃反应直至产物不再继续增加,反应液蒸馏得到4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸酯(式III),
其中,R选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、苯甲基。相应地,所述醇类选自甲醇、乙醇、异丙醇、正丙醇、正丁醇、异丁醇、叔丁醇、苯甲醇。
上述步骤(1)中氢溴酸的醋酸溶液的质量分数为30~50%。
上述步骤(2)中4-溴-2-氧代丁酸酯(II)与甲基亚磷酸二乙酯的摩尔比为1:1-1.5。
步骤(2)得到的产物4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸酯(III)粗品可以不经分离提纯而直接用于下一步反应,生成化合物IV,从而省去了提纯分离的后处理工序。
从廉价易得的化工原料二氢-2,3-呋喃二酮(式I)出发合成底物IV的整个工艺中都不必使用金属催化剂、毒性强的试剂,不存在严重的三废污染问题,反应条件温和,不需要高温高压容器,且原子经济性高,副反应少,收率较高,工艺简洁,因此符合绿色环保理念,符合现代化学工业发展趋势。
本发明的第二个方面提供了一种微生物,其表达谷氨酸脱氢酶SEQ ID NO:2或者其突变体SEQ ID NOs:3-6中的一种。
该微生物例如可以选自碱湖生菌(Alkalilimnicola ehrlichii AK93)本身和基因工程领域常用的细菌或酵母,包括枯草芽孢杆菌、短乳杆菌、木兰假丝酵母、毕赤酵母、酿酒酵母、大肠杆菌等。优选最常用的大肠杆菌例如大肠杆菌BL21(DE3)。
在一种优选的实施方式中,上述微生物还同时表达辅酶再生酶即葡萄糖脱氢酶、醇脱氢酶或者甲酸脱氢酶,从而实现谷氨酸脱氢酶与辅酶再生酶的共表达。
本发明的另一个方面提供了上述微生物在生产L-草铵膦中的用途。
经过与现有技术的谷氨酸脱氢酶比如CN106978453A中报道的多种谷氨酸脱氢酶Pseud omona sentomophila str.L48来源酶(NCBI登录号为WP_044487662.1)、Pseudomonas putida KT2440来源酶(NCBI登录号为NP_742836.1)、Bordetella prtriiDSM12804来源酶(NCBI登录号为WP_012247444.1)的实验对比,本发明提供的谷氨酸脱氢酶SEQ ID NO:2及其突变体SEQ ID NO:3-6的酶活力更高,几乎没有底物/产物抑制性,能够高立体选择性地催化底物IV发生不对称还原胺化反应合成L-草铵膦。例如,谷氨酸脱氢酶SEQID NO:2及其突变体SEQ ID NO:3-6催化底物PPO反应的底物浓度至少可达800mM以上、甚至高达1.5M以上,大大高于现有技术酶的底物耐受浓度100mM。另一方面,通过本发明的方法制备的L-草铵膦光学纯度高,ee值达到99%以上。由于本发明的生物催化反应本身就绿色环保,且从廉价易得的二氢-2,3-呋喃二酮(I)直至底物IV的合成工艺具有低碳环保性,使得本发明从二氢-2,3-呋喃二酮(I)直至L-草铵膦的整体工艺具备了较高的绿色经济价值,促进了L-草铵膦的绿色工业化生产。
具体实施方式
发明人对两百多种微生物来源的谷氨酸脱氢酶进行了筛选和比较,发现碱湖生菌(Alkalilimnicola ehrlichii AK93)来源的野生型谷氨酸脱氢酶(简写AeGluDH)对于PPO还原胺化不仅具有高立体选择性,产物L-草铵膦的ee值大于99%,而且具有底物/产物耐受性,催化PPO反应制备L-草铵膦时,底物浓度甚至高达1.5M以上。
为了进一步提高野生酶AeGluDH的酶活力,还对其进行了突变尝试,通过对多种突变体的研究比较,包括根据生物信息学技术计算的3D模型分析,发现第375位缬氨酸等几个氨基酸位点属于酶活性中心的关键位点。对于这些位点的突变改造存在获得高酶活的突变体可能性,于是对该位点进行饱和突变。氨基酸的突变包括取代、缺失或添加。其中,氨基酸的取代包括保守取代和非保守取代,“保守取代”是指具有相似侧链的残基的可互换性,并因此通常包括用相同或相似的氨基酸定义类别中的氨基酸取代多肽中的氨基酸。例如但不限于,具有脂肪族侧链的氨基酸可以用另一种脂肪族氨基酸例如丙氨酸、缬氨酸、亮氨酸和异亮氨酸取代;具有羟基侧链的氨基酸用另一种具有羟基侧链的氨基酸例如丝氨酸和苏氨酸取代;具有芳香族侧链的氨基酸用另一种具有芳香族侧链的氨基酸例如苯丙氨酸、酪氨酸、色氨酸和组氨酸取代;具有碱性侧链的氨基酸用另一种具有碱性侧链的氨基酸例如赖氨酸和精氨酸取代;具有酸性侧链的氨基酸用另一种具有酸性侧链的氨基酸例如天冬氨酸或谷氨酸取代;并且疏水性氨基酸或亲水性氨基酸分别用另一种疏水性氨基酸或亲水性氨基酸取代。“非保守取代”是指用具有显著差别的侧链特性的氨基酸取代多肽中的氨基酸。非保守取代可以利用限定组之间而不是它们之内的氨基酸,并且影响:(a)取代区域(例如,脯氨酸取代甘氨酸)中的肽骨架的结构,(b)电荷或疏水性,或(c)侧链体积。例如但不限于,示例性非保守取代可以是用碱性或脂肪族氨基酸取代酸性氨基酸;用小氨基酸取代芳香族氨基酸;和用疏水性氨基酸取代亲水性氨基酸。
例如,第375位缬氨酸替换为丝氨酸(V375S)、丙氨酸(V375A)、甘氨酸(V375G)、半胱氨酸(V375C)都提高了酶活性。
在本文中,术语“野生(型)”、“野生酶”表示相同的意义,都是指氨基酸序列分别为SEQ ID NO:2的谷氨酸脱氢酶AeGluDH。相应地,野生酶的突变体SEQ ID NOs:3-6可以称为“突变酶”。为了表述方便起见,在本文中可以将野生型谷氨酸脱氢酶与其突变体比如PsMAOM18等统称为“谷氨酸脱氢酶”,只要不与野生酶SEQ ID NO:2混淆即可。
谷氨酸脱氢酶催化PPO还原胺化的反应体系优选还包含辅酶再生系统,即包含辅酶再生酶即葡萄糖脱氢酶、醇脱氢酶或者甲酸脱氢酶。这些酶可以作为添加剂加入,也可以与谷氨酸脱氢酶一起表达、同时存在。当谷氨酸脱氢酶与辅酶再生酶在同一个微生物细胞中共表达时,先构建出克隆了谷氨酸脱氢酶基因和辅酶再生酶基因的共表达重组菌体,利用微生物的简单发酵就可以同时地、按比例地提供两种酶,无需根据两种酶的酶活力进行比例调整,这是共表达两种酶的优势所在。例如,与谷氨酸脱氢酶共表达的葡萄糖脱氢酶可以是蜡样芽胞杆菌(Bacillus cereus)来源的葡萄糖脱氢酶(GenBank:AE016877.1),氨基酸序列是SEQ ID NO:7,简称bcGDH,但不限于此。
本发明的谷氨酸脱氢酶及其突变体的氨基酸数量为446个,结构明确,因此本领域技术人员很容易获得其编码基因、包含这些基因的表达盒和质粒、以及包含该质粒的转化体。
这些基因、表达盒、质粒、转化体可以通过本领域技术人员所熟知的基因工程构建方式获得。例如,可以将谷氨酸脱氢酶核酸序列SEQ ID NOs:2-6之一连接于各种市售常规质粒载体上构建而成。所述质粒优选pET序列质粒,更优选质粒pET-28a(+)。可通过下述方法制得重组表达质粒:将PCR扩增所得的核酸产物与表达载体pET-28a分别用限制性内切酶BamH I和Xho I双酶切,形成互补的粘性末端,经T4 DNA连接酶连接,形成所述谷氨酸脱氢酶基因的重组表达质粒pET28a-AeGluDH。
重组表达质粒pET28a-AeGluDH可通过本领域常规方法如热激法、电转法、化学法等转化至合适的宿主细胞中,例如转化入大肠杆菌BL21(DE3)感受态。其中热激法较佳的实施方式为:将质粒溶液与感受态细胞混合,40℃,热激45秒,然后冰浴2min,随后37℃复苏1h,涂布于含卡那霉素的LB琼脂培养基平板,培养得到重组表达转化体E.coli BL21(DE3)/pET28a-AeGluDH。
当谷氨酸脱氢酶(GluDH)与辅酶再生酶比如葡萄糖脱氢酶(GDH)例如bcGDH共表达时,两种重组表达质粒应能稳定地自行复制,且其所携带的谷氨酸脱氢酶和辅酶再生用酶可被有效表达。为此,还构建出重组表达质粒pACYCDuet-bcGDH,分别将两个重组质粒pET28a-AeGluDH和pACYCDuet-bcGDH转入宿主细胞、或者同时将两个重组质粒pET28a-AeGluDH和pACYCDuet-bcGDH转入转化入大肠杆菌BL21(DE3)感受态,得到克隆了谷氨酸脱氢酶基因和辅酶再生酶基因的共表达重组菌体E.coli BL21(DE3)/pACYCDuet-GDH/pET28a-AeGluDH。
当作为生物催化剂用于制备L-草铵膦时,本发明的谷氨酸脱氢酶可以呈现酶的形式或者菌体的形式。所述酶的形式包括游离酶、固定化酶,包括纯化酶、粗酶、发酵液、载体固定的酶等;所述菌体的形式包括存活菌体(静息细胞)和死亡菌体。
本领域技术人员容易理解,菌体内包含有辅酶NADPH/NADP+(烟酰胺腺嘌呤二核苷磷酸,辅酶II)、NADH/NAD+(烟酰胺腺嘌呤二核苷酸,辅酶I),本身就是一种天然的酶固定化形式,而且不需要进行破碎处理、甚至提取纯化处理,就可以作为一种酶制剂用于催化反应。由于反应底物和反应产物可以很方便地穿过菌体的生物屏障--细胞膜,因此不需要对菌体进行破碎处理,这在经济方面是有利的。另一方面,相比分离出的酶的催化,本发明利用微生物的简单发酵就可以源源不断、取之不尽地提供酶或的供应,无需进一步提取、纯化分离酶等操作,经济效益明显,为工业化应用创造条件。
以下结合具体实施例对本发明做进一步详细说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例中涉及到多种物质的添加量、含量及浓度,其中所述的百分含量,除特别说明外,皆指质量百分含量。
实施例
材料和方法
实施例中的全基因合成、引物合成及测序皆由生工生物工程股份有限公司完成。
实施例中的分子生物学实验包括质粒构建、酶切、感受态细胞制备、转化等主要参照《分子克隆实验指南》(第三版),J.萨姆布鲁克,D.W.拉塞尔(美)编著,黄培堂等译,科学出版社,北京,2002)进行。必要时可以通过简单试验确定具体实验条件。
PCR扩增实验根据质粒或DNA模板供应商提供的反应条件或试剂盒说明书进行。必要时可以通过简单试验予以调整。
LB培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L氯化钠,pH7.2。(LB固体培养基另加20g/L琼脂粉。)
限制性内切酶和DNA连接酶均购自生工生物工程股份有限公司(上海生工);质粒提取试剂盒、DNA回收纯化试剂盒、DNA marker、FastPfu DNA聚合酶、低分子量标准蛋白、琼脂糖电泳试剂等购自北京全式金生物技术有限公司;表达宿主E.coli BL21(DE3)、E.coliDH 5α购自上海唯地生物;辅酶再生酶质粒(包括pACYCDuet-bcGDH等)、载体购自苏州金唯智生物科技有限公司。
实施例1:4-溴-2-氧代丁酸甲酯(IIa)的制备
将二氢-2,3-呋喃二酮(式I)(32.5g,325mmol)以及45%的氢溴酸的醋酸溶液(75mL)加入反应瓶中,于75℃反应4h后,冷却至室温,加入甲醇(150mL),继续搅拌过夜,反应结束后,反应液浓缩得到黑色油状液体,将其溶解于乙酸乙酯(150mL)中,依次用饱和碳酸氢钠溶液(2×150mL)和饱和食盐水溶液(150mL)洗涤、无水硫酸钠干燥,减压浓缩得到无色液体4-溴-2-氧代丁酸甲酯(IIa)60.2g,收率95%。
1H NMR(400MHz,CDCl3)δ4.02(t,2H),3.63(t,2H),3.61(s,3H)。
实施例2:4-溴-2-氧代丁酸乙酯(IIb)的制备
将二氢-2,3-呋喃二酮(式I)(32.5g,325mmol)以及45%的氢溴酸的醋酸溶液(75mL)加入反应瓶中,于75℃反应4h后,冷却至室温,加入乙醇(150mL),继续搅拌过夜,反应结束后,反应液浓缩得到黑色油状液体,将其溶解于乙酸乙酯(150mL)中,依次用饱和碳酸氢钠溶液(2×150mL)和饱和食盐水溶液(150mL)洗涤、无水硫酸钠干燥,减压浓缩得到无色液体4-溴-2-氧代丁酸乙酯(IIb)61.8g,收率91%。
1H NMR(400MHz,CDCl3)δ4.17(q,2H),4.02(t,2H),3.63(t,2H),1.24(t,3H)。
实施例3:4-溴-2-氧代丁酸苄酯(IIc)的制备
将二氢-2,3-呋喃二酮(32.5g,325mmol)以及45%的氢溴酸的醋酸溶液(75mL)加入反应瓶中,于75℃反应4h后,冷却至室温,加入苯甲醇(150mL),继续搅拌过夜,反应结束后,反应液浓缩得到黑色油状液体,将其溶解于乙酸乙酯(150mL)中,依次用饱和碳酸氢钠溶液(2×150mL)和饱和食盐水溶液(150mL)洗涤、无水硫酸钠干燥,减压浓缩得到无色液体4-溴-2-氧代丁酸苄酯(IIc)81.9g,收率93%。
1H NMR(400MHz,CDCl3)δ7.42~7.23(m,5H),5.13(s,2H),4.02(t,2H),3.63(t,2H)。
实施例4:4-(羟基-(甲基)氧膦基)-2-氧代丁酸(IV)的制备
在氮气保护下,将4-溴-2-氧代丁酸甲酯(IIa;58.5g,300mmol)与甲基亚磷酸二乙酯(53.3g,360mmol)加入反应瓶中,搅拌混合,加热至115℃,反应3h。随后,通过减压蒸馏回收过量的甲基亚磷酸二乙酯,在得到的残留物(4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸甲酯粗品,III)中加入浓盐酸(150mL),搅拌,加热回流8h,反应液减压浓缩至干,然后加入水(80mL),搅拌混合,再次浓缩至干,残留物用四氢呋喃重结晶得到白色固体4-(羟基-(甲基)氧膦基)-2-氧代丁酸(IV)42.1g,收率78%。m.p.95~97℃。
1H NMR(400MHz,DMSO-d6)δ10.30(s,2H),3.06~2.91(m,2H),1.89~1.71(m,2H),1.33(d,J=14.1Hz,3H)。
实施例5:4-(羟基-(甲基)氧膦基)-2-氧代丁酸(IV)的制备
在氮气保护下,将4-溴-2-氧代丁酸乙酯(IIb;62.7g,300mmol)与甲基亚磷酸二乙酯(48.9g,330mmol)加入反应瓶中,搅拌混合,加热至130℃,反应2h。随后,通过减压蒸馏回收过量的甲基亚磷酸二乙酯,在得到的残留物(4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸乙酯粗品,III)中加入浓盐酸(150mL),搅拌,加热回流8h,反应液减压浓缩至干,然后加入水(80mL),搅拌混合,再次浓缩至干,残留物用四氢呋喃重结晶得到白色固体4-(羟基-(甲基)氧膦基)-2-氧代丁酸(IV)43.7g,收率81%。
实施例6:4-(羟基-(甲基)氧膦基)-2-氧代丁酸(IV)的制备
在氮气保护下,将4-溴-2-氧代丁酸苄酯(IIb;81.3g,300mmol)与甲基亚磷酸二乙酯(66.68g,450mmol)加入反应瓶中,搅拌混合,加热至150℃,反应1.5h。随后,通过减压蒸馏回收过量的甲基亚磷酸二乙酯,在得到的残留物(4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸苄酯粗品,III)中加入浓盐酸(150mL),搅拌,加热回流8h,反应液减压浓缩至干,然后加入水(80mL),搅拌混合,再次浓缩至干,残留物用四氢呋喃重结晶得到白色固体4-(羟基-(甲基)氧膦基)-2-氧代丁酸(IV)43.1g,收率80%。
实施例7:碱湖生菌的培养
培养基:葡萄糖10g/L,酵母膏3g/L,麦芽提取物3g/L,大豆蛋白胨5g/L,121℃灭菌20min。
从预培养的琼脂培养基平板上挑取碱湖生菌(Alkalilimnicola ehrlichiiAK93)菌落,接种于装有50mL培养基的250mL三角烧瓶中,30℃、200rpm摇床振摇培养12h。然后分别取5mL种子液,转接到装有100mL培养基的500mL三角烧瓶中,30℃、200rpm摇床振摇培养24h。培养液经8000rpm离心,收集菌体沉淀。收集的菌体沉淀即可使用,或置于4℃冰箱保存。
实施例8:碱湖生菌静息细胞催化PPO的还原胺化反应
取1g如实施例7所述的菌体沉淀,充分悬浮于10mL磷酸盐缓冲液(100mM,pH 7.4),加入4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO)(0.9g,5mmol),终浓度为0.75M的葡萄糖和终浓度为1.2M的硫酸铵,置于摇床中30℃、200rpm振摇反应,间歇滴加氨水使反应液pH维持在7.4左右,反应12h。
通过HPLC检测底物以及产物的浓度,方法为:使用AQ-C18色谱柱,柱温为40℃;流速为1mL/min;检测波长为205nm;流动相为:50mM(NH4)2HPO4,含有1g/L四丁基氢氧化铵。
产物的光学纯度通过柱前衍生化高效液相色谱来测定:取100μL样品加入150μL衍生化试剂,混匀后置于25℃保温反应5min,然后取20μL样品进行分析。其中衍生化试剂的配方为:称取0.03g邻苯二甲醛和0.1g N-乙酰-L-半胱氨酸,溶解于400μL乙醇中,加入4mL硼酸缓冲液(0.2M,pH 9.8),振荡使溶液澄清,4℃保存备用。HPLC分析条件为:使用PntulipsQS-C18色谱柱(5μm,4.6mm×250mm);流动相为:50mM乙酸钠溶液:乙腈=8:0.5;检测波长338nm,流速0.85mL/min,柱温30℃。
经检测,产物L-草铵膦的光学纯度为99.1%ee(R构型)。
实施例9:谷氨酸脱氢酶AeGluDH的基因克隆
根据谷氨酸脱氢酶AeGluDH基因的开放阅读框,设计上下游引物对。
上游引物:CGCGGATCCATGTCTGACGATTTGCAGGCG,
下游引物:CCGCTCGAGCTAAACAATGCCCTGCTCG。
其中,上游引物下划线部分为BamH I酶切位点,下游引物下划线部分为Xho I酶切位点。
以碱湖生菌(Alkalilimnicola ehrlichii AK93)的基因组DNA为模板,进行PCR扩增。PCR体系为:2×Taq PCR MasterMix 25μL,上游引物和下游引物(10ng/μL)各2.5μL,基因组DNA(100ng/μL)和ddH2O(19μL)。PCR扩增程序为:95℃预变性5min后进行32次如下循环:94℃变性30s,50℃退火30s,72℃延伸8min;最后72℃再延伸10min。PCR扩增产物进行凝胶电泳纯化后,用DNA回收试剂盒回收目的片段。经过DNA测序,该序列中编码的开放阅读框全长1338bp,核苷酸序列为SEQ ID NO:1。
实施例10:谷氨酸脱氢酶重组表达质粒盒重组表达转化体的制备
将实施例9中PCR扩增所得的谷氨酸脱氢酶基因片段以及pET28a空载体同时用限制性内切酶BamH I和Xho I双酶切过夜,然后经琼脂糖凝胶电泳纯化、DNA试剂盒回收。将回收的经酶切目的片段和空载体在T4 DNA连接酶的作用下,于4℃连接12h,得到重组质粒pET28a-AeGluDH。
将所得重组质粒转化至E.coli DH 5α感受态,涂布至含有50μg/mL卡那霉素的LB培养基平板上,37℃培养8h,挑取单个菌落进行菌落PCR验证,经测序验证后,提取相应的质粒,进一步转化至E.coli BL21,挑取阳性克隆,即获得重组表达转化体共表达重组菌体E.coli BL21(DE3)/pET28a-AeGluDH。
按照与上述相同的方法,可以使用质粒pACYCDuet-bcGDH构建得到葡萄糖脱氢酶的重组表达菌体E.coli BL21(DE3)/pACYCDuet-GDH。
实施例11:谷氨酸脱氢酶突变体AeGluDHV375G与葡萄糖脱氢酶共表达细胞的制备
取100μL的感受态E.coli BL21,于冰盒中静置15min后,在无菌操作台中分别吸取1μL重组质粒pACYCDuet-bcGDH和1μL重组质粒pET28a-AeGluDHV375G加入到E.coli BL21中。继续于冰盒中静置30min后,在42℃恒温水槽中热激45s后,立即将其放置于冰盒中静置2min,于无菌操作台中加入500μL LB培养基,然后于37℃中孵育1h。取出离心2min,保留少许上清液,经移液枪吹打混匀后涂布于固体培养基上,然后于37℃生化培养箱11~13h,挑取阳性克隆,即获得谷氨酸脱氢酶突变体AeGluDHV375G与葡萄糖脱氢酶GDH的共表达细胞。
实施例12:谷氨酸脱氢酶AeGluDH的诱导表达
将实施例10中构建所得的重组表达转化体接种至含有50μg/mL卡那霉素的LB培养基中,37℃、200rpm摇床振荡培养12h,之后按1%(v/v)的接种量接种至装有100mL LB培养基的500mL锥形瓶中,37℃、200rpm摇床振荡培养,当培养液的OD600达到0.7时,加入IPTG至终浓度0.2mM进行诱导,18℃诱导18h后,将培养液以8500rpm转速离心,收集细胞,并用生理盐水洗涤,得到静息细胞。将所得细胞用10mL的磷酸盐缓冲液(100mM,pH 7.4)充分悬浮,冰水浴中进行超声破碎(55%功率,工作6s,间歇14s,工作总时长15min),4℃下13800rpm离心15min,收集上清液并进行活力测定,测得的酶活为76.5U/mL裂解液。
实施例13:重组谷氨酸脱氢酶突变体的制备
对实施例9所得的谷氨酸脱氢酶AeGluDH全长基因序列(SEQ ID NO:1)进行碱基突变,将谷氨酸脱氢酶的第375位残基缬氨酸分别突变为丝氨酸(S)、丙氨酸(A)、甘氨酸(G)或半胱氨酸(C),相应的突变谷氨酸脱氢酶分别命名为AeGluDHV375S、AeGluDHV375A、AeGluDHV375G和AeGluDHV375C。按实施例10所述的方法制备含有突变体基因DNA序列的重组表达质粒和重组表达转化体,按实施例12所述的方法,对表达所述谷氨酸脱氢酶突变体的重组表达转化体进行培养并诱导表达,对细胞超声处理后得到裂解液,酶活测定结果见表1。
表1、谷氨酸脱氢酶突变体细胞裂解液的酶活力
对谷氨酸脱氢酶AeGluDH第375位残基缬氨酸突变后,发现V375S、V375A、V375G和V375C属于正向突变,相对于野生酶的酶活76.5U/mL有了不同程度的提高,其中V375S突变体的酶活力提高幅度最大。
实施例14:突变体AeGluDHV375G与葡萄糖脱氢酶共表达细胞催化PPO的还原胺化
参照实施例7的方法,将谷氨酸脱氢酶突变体AeGluDHV375G与葡萄糖脱氢酶共表达细胞进行培养并收集湿菌体。
取5g湿菌体,充分悬浮于100mL磷酸盐缓冲液(100mM,pH 7.4)中,加入4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO)(18g),终浓度为1.5M的葡萄糖,终浓度为1.2M硫酸铵,置于摇床中25℃、200rpm振摇反应,滴加氨水使反应液pH维持在7.4,通过HPLC监控反应进程,反应6h后底物完全转化。将反应液置于8000rpm下离心30min,取上清液加热至90℃,保持10min,然后立即冷却至20℃,8000rpm离心10min。取上清液加入37%的HCl溶液调节pH至1.0,得到预处理液。
分别使用已经预处理好的DOWEX、50WX8阳离子交换树脂100g与预处理液混合30min,然后用过滤器将树脂分离出来,将收集到的树脂用水清洗,然后再用4M氨水溶液洗脱,通过茚三酮显色反应判断产物是否洗脱完全。将收集得到的流出液用300mL水稀释,加入4M氨水溶液调节pH至9,加入处理过的DOWEX树脂50g,搅拌40min,装到玻璃柱上,向柱上加入40mL水,用0.1M醋酸水溶液洗脱,收集全部流出液,减压浓缩至恒重,加入20mL混合溶液(水/丙酮体积比为1:9)和0.2g聚丙烯酰胺,加热至30℃使L-草铵膦溶解,在0℃下缓慢搅拌12h使L-草铵膦结晶析出后,将其置于冷冻真空干燥机内冷冻干燥,获得15.0g L-草铵膦晶体,收率为83%,ee值为99.5%。
IT-TOF(ESI):m/z=180.0435,calcd.For C5H11NO4P-[M]-:180.14。
1H NMR(400MHz,D2O)δ3.67(t,J=5.8Hz,1H),1.96(dtd,J=11.4,6.1,5.7,2.3Hz,2H),1.63~1.37(m,2H),1.14(d,J=13.4Hz,3H).13C NMR(101MHz,D2O)δ174.30,55.29,27.12,24.33,15.06。
实施例15:突变体AeGluDHV375S与醇脱氢酶共表达细胞催化PPO的还原胺化
参照实施例11,构建谷氨酸脱氢酶突变体AeGluDHV375S与醇脱氢酶共表达细胞。参照实施例7,将谷氨酸脱氢酶突变体AeGluDHV375S与醇脱氢酶共表达细胞进行培养并收集湿菌体。
取10g湿菌体,充分悬浮于100mL磷酸盐缓冲液(100mM,pH 7.4)中,加入4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO)(27g),终浓度为2.25M的异丙醇,终浓度为3.6M的硫酸铵,置于摇床中30℃、200rpm振摇反应,滴加氨水使反应液pH维持在7.4,通过HPLC监控反应进程,反应4h后底物完全转化。将反应液置于8000rpm下离心30min,取上清液加热至90℃,保持10min,然后立即冷却至30℃,8000rpm离心10min。取上清液加入37%HCl溶液调节pH至1.0,得到预处理液。按实施例14中的后处理过程,得到23.2g L-草铵膦晶体,收率为86%,ee值为99.7%。
实施例16:突变体AeGluDHV375C与甲酸脱氢酶共表达细胞催化PPO的还原胺化
参照实施例11,构建谷氨酸脱氢酶突变体AeGluDHV375C与甲酸脱氢酶共表达细胞。参照实施例7,将谷氨酸脱氢酶突变体AeGluDHV375C与甲酸脱氢酶共表达细胞进行培养并收集湿菌体。
取2g湿菌体,充分悬浮于100mL磷酸盐缓冲液(100mM,pH 7.4)中,加入4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO)(9g),终浓度1.75M的甲酸铵,置于摇床中35℃、200rpm反应,滴加氨水使反应液pH维持在7.4,通过HPLC监控反应进程,反应4h后底物完全转化。将反应液置于8000rpm下离心30min,取上清液,加热至90℃,保持10min,然后立即冷却至25℃,8000rpm离心10min。取上清液加入37%HCl溶液调节pH至1.0,得到预处理液。按实施例14中的后处理过程,得到6.9g L-草铵膦晶体,收率为76%,ee值为99.0%。
实施例17:突变体AeGluDHV375G催化PPO的还原胺化
参照实施例11,分别制备AeGluDHV375G粗酶液以及GDH粗酶液。取AeGluDHV375G粗酶液(10mL)以及GDH粗酶液(10mL),充分悬浮于80mL磷酸盐缓冲液(100mM,pH 7.4)中,加入4-(羟基-(甲基)氧膦基)-2-氧代丁酸(PPO)(15g),终浓度为1.25M的葡萄糖,终浓度为1.0mM的NADP+,终浓度为1.1M硫酸铵,置于摇床中25℃、200rpm振摇反应,滴加氨水使反应液pH维持在7.4,通过HPLC监控反应进程,反应10h后底物完全转化。将反应液置于8000rpm下离心30min,取上清液加热至90℃,保持10min,然后立即冷却至20℃,8000rpm离心10min。取上清液加入37%的HCl溶液调节pH至1.0,得到预处理液。按实施例14中的后处理过程,得到12.4g L-草铵膦晶体,收率为83%,ee值为99.3%。
以上实验表明,本发明制备PPO的合成工艺操作简单容易;谷氨酸脱氢酶SEQ IDNO:2及其突变体SEQ ID NO:3-6能够催化PPO不对称还原胺化,得到ee值99%以上的高光学纯度L-草铵膦,具有工业化生产L-草铵膦的应用前景。
上述对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
序列表
<110> 苏州百福安酶技术有限公司
<120> 一种制备L-草铵膦的方法
<130> SHPI2210135
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1341
<212> DNA
<213> Alkalilimnicola ehrlichii AK93
<400> 1
atgtctgacg atttgcaggc gtttctcgcc tatgtgcgag aacgcgaccc tgaccagccc 60
gagtttctgc aagcggtgga agagacgatg cggagcgtct ggccttttat cgaacgccat 120
ccacgctatc gcgatatggg tttactggag cggttggtcg agccggagcg gatcatcact 180
tttcgggtgc cctgggtgga cgatcagggg cgggtgcatg tgaaccgagg ctatcgggtg 240
cagatgaata gtgcgatcgg cccttataag ggagggttgc gctttcatcc gtcggtgaat 300
gtgagcattc tcaagttctt ggcgtttgag caggtcttta aaaacgggct taccaccttg 360
ccgctcggcg gcggtaaagg gggggccgac tttgacccga aagggcgcag cgatgccgaa 420
gtcatgcgct tttgtcagtc ttttatgagc gaactctacc gccatatcgg ccatgatctg 480
gatgtgcccg ccggcgatat cggggtcagt cggcgggaga tcgggtattt gtacggaatg 540
tacaagaagc tcgccaacga gtttaccggg gtgattaccg gcaagggcta taactacggc 600
ggtagcttga ttcgcccgga ggcgacgggc tacggtttga tctatttcgt cgccgagatg 660
ataaagccta aatacgccaa tatggacgga cttaaggtcg cgatttccgg ctctggcaat 720
gttgcgcaat tcgcggctgc caaggcgatg gagttcgggg cgaaggtgat tacgctatcc 780
gattcgggcg gtactgtgta tatgccggag ggtttgaccg acgagcagtg ggactttctc 840
gtccgcttga agaacgagct ccgtggtcgg ttgagcacgt ttgccgagga gttcgggttt 900
gagtttgccg ccgataagcg gccttggggc tacccctgcg atattgcctt gccgtgtgca 960
actcaaaacg agctggatga tcgcgatgcg ctggcgctga taaacaacgg ctgcttttgc 1020
gtagccgaag gcgcgaatat gccttcgacg attggcgccg tggactgttt tcaggccgcc 1080
cgcattcttt atgcgccggg taaggcttcc aatgccggcg gcgttgcggt cagcggcctg 1140
gagatgagcc agaatgcctt acggctaagc tggaccgacg gcgaagtgga cgagaagctg 1200
catagcatta tgcagtcgat tcataccacc tgcatgcatt atggcgcagg caaaggaggg 1260
tatgtgaact acgtcgatgg ggcgaatatt gccggctttg tcaaagttgc ggatgcgatg 1320
aacgagcagg gcattgttta g 1341
<210> 2
<211> 446
<212> PRT
<213> Alkalilimnicola ehrlichii AK93
<400> 2
Met Ser Asp Asp Leu Gln Ala Phe Leu Ala Tyr Val Arg Glu Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe Leu Gln Ala Val Glu Glu Thr Met Arg Ser
20 25 30
Val Trp Pro Phe Ile Glu Arg His Pro Arg Tyr Arg Asp Met Gly Leu
35 40 45
Leu Glu Arg Leu Val Glu Pro Glu Arg Ile Ile Thr Phe Arg Val Pro
50 55 60
Trp Val Asp Asp Gln Gly Arg Val His Val Asn Arg Gly Tyr Arg Val
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Val Ser Ile Leu Lys Phe Leu Ala Phe Glu Gln Val
100 105 110
Phe Lys Asn Gly Leu Thr Thr Leu Pro Leu Gly Gly Gly Lys Gly Gly
115 120 125
Ala Asp Phe Asp Pro Lys Gly Arg Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ser Phe Met Ser Glu Leu Tyr Arg His Ile Gly His Asp Leu
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Ser Arg Arg Glu Ile Gly Tyr
165 170 175
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Ile Thr Leu Ser Asp Ser Gly Gly Thr Val Tyr Met Pro Glu Gly Leu
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Thr Asp Glu Gln Trp Asp Phe Leu Val Arg Leu Lys Asn Glu Leu Arg
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290 295 300
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305 310 315 320
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Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Ser Thr Ile Gly
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Ala Ser Asn Ala Gly Gly Val Ala Val Ser Gly Leu Glu Met Ser Gln
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His Ser Ile Met Gln Ser Ile His Thr Thr Cys Met His Tyr Gly Ala
405 410 415
Gly Lys Gly Gly Tyr Val Asn Tyr Val Asp Gly Ala Asn Ile Ala Gly
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435 440 445
<210> 3
<211> 446
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<213> 人工序列(Artificial Sequence)
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Met Ser Asp Asp Leu Gln Ala Phe Leu Ala Tyr Val Arg Glu Arg Asp
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Pro Asp Gln Pro Glu Phe Leu Gln Ala Val Glu Glu Thr Met Arg Ser
20 25 30
Val Trp Pro Phe Ile Glu Arg His Pro Arg Tyr Arg Asp Met Gly Leu
35 40 45
Leu Glu Arg Leu Val Glu Pro Glu Arg Ile Ile Thr Phe Arg Val Pro
50 55 60
Trp Val Asp Asp Gln Gly Arg Val His Val Asn Arg Gly Tyr Arg Val
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Val Ser Ile Leu Lys Phe Leu Ala Phe Glu Gln Val
100 105 110
Phe Lys Asn Gly Leu Thr Thr Leu Pro Leu Gly Gly Gly Lys Gly Gly
115 120 125
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130 135 140
Cys Gln Ser Phe Met Ser Glu Leu Tyr Arg His Ile Gly His Asp Leu
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Ser Arg Arg Glu Ile Gly Tyr
165 170 175
Leu Tyr Gly Met Tyr Lys Lys Leu Ala Asn Glu Phe Thr Gly Val Ile
180 185 190
Thr Gly Lys Gly Tyr Asn Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Leu Ile Tyr Phe Val Ala Glu Met Ile Lys Pro Lys
210 215 220
Tyr Ala Asn Met Asp Gly Leu Lys Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Phe Ala Ala Ala Lys Ala Met Glu Phe Gly Ala Lys Val
245 250 255
Ile Thr Leu Ser Asp Ser Gly Gly Thr Val Tyr Met Pro Glu Gly Leu
260 265 270
Thr Asp Glu Gln Trp Asp Phe Leu Val Arg Leu Lys Asn Glu Leu Arg
275 280 285
Gly Arg Leu Ser Thr Phe Ala Glu Glu Phe Gly Phe Glu Phe Ala Ala
290 295 300
Asp Lys Arg Pro Trp Gly Tyr Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Asp Arg Asp Ala Leu Ala Leu Ile Asn Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Ser Thr Ile Gly
340 345 350
Ala Val Asp Cys Phe Gln Ala Ala Arg Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Ser Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Leu Arg Leu Ser Trp Thr Asp Gly Glu Val Asp Glu Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His Thr Thr Cys Met His Tyr Gly Ala
405 410 415
Gly Lys Gly Gly Tyr Val Asn Tyr Val Asp Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Asn Glu Gln Gly Ile Val
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<210> 4
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ser Asp Asp Leu Gln Ala Phe Leu Ala Tyr Val Arg Glu Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe Leu Gln Ala Val Glu Glu Thr Met Arg Ser
20 25 30
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35 40 45
Leu Glu Arg Leu Val Glu Pro Glu Arg Ile Ile Thr Phe Arg Val Pro
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Trp Val Asp Asp Gln Gly Arg Val His Val Asn Arg Gly Tyr Arg Val
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Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Val Ser Ile Leu Lys Phe Leu Ala Phe Glu Gln Val
100 105 110
Phe Lys Asn Gly Leu Thr Thr Leu Pro Leu Gly Gly Gly Lys Gly Gly
115 120 125
Ala Asp Phe Asp Pro Lys Gly Arg Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ser Phe Met Ser Glu Leu Tyr Arg His Ile Gly His Asp Leu
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Ser Arg Arg Glu Ile Gly Tyr
165 170 175
Leu Tyr Gly Met Tyr Lys Lys Leu Ala Asn Glu Phe Thr Gly Val Ile
180 185 190
Thr Gly Lys Gly Tyr Asn Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Leu Ile Tyr Phe Val Ala Glu Met Ile Lys Pro Lys
210 215 220
Tyr Ala Asn Met Asp Gly Leu Lys Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Phe Ala Ala Ala Lys Ala Met Glu Phe Gly Ala Lys Val
245 250 255
Ile Thr Leu Ser Asp Ser Gly Gly Thr Val Tyr Met Pro Glu Gly Leu
260 265 270
Thr Asp Glu Gln Trp Asp Phe Leu Val Arg Leu Lys Asn Glu Leu Arg
275 280 285
Gly Arg Leu Ser Thr Phe Ala Glu Glu Phe Gly Phe Glu Phe Ala Ala
290 295 300
Asp Lys Arg Pro Trp Gly Tyr Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Asp Arg Asp Ala Leu Ala Leu Ile Asn Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Ser Thr Ile Gly
340 345 350
Ala Val Asp Cys Phe Gln Ala Ala Arg Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Ala Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Leu Arg Leu Ser Trp Thr Asp Gly Glu Val Asp Glu Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His Thr Thr Cys Met His Tyr Gly Ala
405 410 415
Gly Lys Gly Gly Tyr Val Asn Tyr Val Asp Gly Ala Asn Ile Ala Gly
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Phe Val Lys Val Ala Asp Ala Met Asn Glu Gln Gly Ile Val
435 440 445
<210> 5
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ser Asp Asp Leu Gln Ala Phe Leu Ala Tyr Val Arg Glu Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe Leu Gln Ala Val Glu Glu Thr Met Arg Ser
20 25 30
Val Trp Pro Phe Ile Glu Arg His Pro Arg Tyr Arg Asp Met Gly Leu
35 40 45
Leu Glu Arg Leu Val Glu Pro Glu Arg Ile Ile Thr Phe Arg Val Pro
50 55 60
Trp Val Asp Asp Gln Gly Arg Val His Val Asn Arg Gly Tyr Arg Val
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Val Ser Ile Leu Lys Phe Leu Ala Phe Glu Gln Val
100 105 110
Phe Lys Asn Gly Leu Thr Thr Leu Pro Leu Gly Gly Gly Lys Gly Gly
115 120 125
Ala Asp Phe Asp Pro Lys Gly Arg Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ser Phe Met Ser Glu Leu Tyr Arg His Ile Gly His Asp Leu
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165 170 175
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Thr Gly Lys Gly Tyr Asn Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
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210 215 220
Tyr Ala Asn Met Asp Gly Leu Lys Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Phe Ala Ala Ala Lys Ala Met Glu Phe Gly Ala Lys Val
245 250 255
Ile Thr Leu Ser Asp Ser Gly Gly Thr Val Tyr Met Pro Glu Gly Leu
260 265 270
Thr Asp Glu Gln Trp Asp Phe Leu Val Arg Leu Lys Asn Glu Leu Arg
275 280 285
Gly Arg Leu Ser Thr Phe Ala Glu Glu Phe Gly Phe Glu Phe Ala Ala
290 295 300
Asp Lys Arg Pro Trp Gly Tyr Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Asp Arg Asp Ala Leu Ala Leu Ile Asn Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Ser Thr Ile Gly
340 345 350
Ala Val Asp Cys Phe Gln Ala Ala Arg Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Gly Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Leu Arg Leu Ser Trp Thr Asp Gly Glu Val Asp Glu Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His Thr Thr Cys Met His Tyr Gly Ala
405 410 415
Gly Lys Gly Gly Tyr Val Asn Tyr Val Asp Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Asn Glu Gln Gly Ile Val
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<210> 6
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ser Asp Asp Leu Gln Ala Phe Leu Ala Tyr Val Arg Glu Arg Asp
1 5 10 15
Pro Asp Gln Pro Glu Phe Leu Gln Ala Val Glu Glu Thr Met Arg Ser
20 25 30
Val Trp Pro Phe Ile Glu Arg His Pro Arg Tyr Arg Asp Met Gly Leu
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50 55 60
Trp Val Asp Asp Gln Gly Arg Val His Val Asn Arg Gly Tyr Arg Val
65 70 75 80
Gln Met Asn Ser Ala Ile Gly Pro Tyr Lys Gly Gly Leu Arg Phe His
85 90 95
Pro Ser Val Asn Val Ser Ile Leu Lys Phe Leu Ala Phe Glu Gln Val
100 105 110
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115 120 125
Ala Asp Phe Asp Pro Lys Gly Arg Ser Asp Ala Glu Val Met Arg Phe
130 135 140
Cys Gln Ser Phe Met Ser Glu Leu Tyr Arg His Ile Gly His Asp Leu
145 150 155 160
Asp Val Pro Ala Gly Asp Ile Gly Val Ser Arg Arg Glu Ile Gly Tyr
165 170 175
Leu Tyr Gly Met Tyr Lys Lys Leu Ala Asn Glu Phe Thr Gly Val Ile
180 185 190
Thr Gly Lys Gly Tyr Asn Tyr Gly Gly Ser Leu Ile Arg Pro Glu Ala
195 200 205
Thr Gly Tyr Gly Leu Ile Tyr Phe Val Ala Glu Met Ile Lys Pro Lys
210 215 220
Tyr Ala Asn Met Asp Gly Leu Lys Val Ala Ile Ser Gly Ser Gly Asn
225 230 235 240
Val Ala Gln Phe Ala Ala Ala Lys Ala Met Glu Phe Gly Ala Lys Val
245 250 255
Ile Thr Leu Ser Asp Ser Gly Gly Thr Val Tyr Met Pro Glu Gly Leu
260 265 270
Thr Asp Glu Gln Trp Asp Phe Leu Val Arg Leu Lys Asn Glu Leu Arg
275 280 285
Gly Arg Leu Ser Thr Phe Ala Glu Glu Phe Gly Phe Glu Phe Ala Ala
290 295 300
Asp Lys Arg Pro Trp Gly Tyr Pro Cys Asp Ile Ala Leu Pro Cys Ala
305 310 315 320
Thr Gln Asn Glu Leu Asp Asp Arg Asp Ala Leu Ala Leu Ile Asn Asn
325 330 335
Gly Cys Phe Cys Val Ala Glu Gly Ala Asn Met Pro Ser Thr Ile Gly
340 345 350
Ala Val Asp Cys Phe Gln Ala Ala Arg Ile Leu Tyr Ala Pro Gly Lys
355 360 365
Ala Ser Asn Ala Gly Gly Cys Ala Val Ser Gly Leu Glu Met Ser Gln
370 375 380
Asn Ala Leu Arg Leu Ser Trp Thr Asp Gly Glu Val Asp Glu Lys Leu
385 390 395 400
His Ser Ile Met Gln Ser Ile His Thr Thr Cys Met His Tyr Gly Ala
405 410 415
Gly Lys Gly Gly Tyr Val Asn Tyr Val Asp Gly Ala Asn Ile Ala Gly
420 425 430
Phe Val Lys Val Ala Asp Ala Met Asn Glu Gln Gly Ile Val
435 440 445
<210> 7
<211> 261
<212> PRT
<213> Bacillus cereus
<400> 7
Met Tyr Ser Asp Leu Ala Gly Lys Val Val Val Ile Thr Gly Ser Ala
1 5 10 15
Thr Gly Leu Gly Arg Ala Met Gly Val Arg Phe Ala Lys Glu Lys Ala
20 25 30
Lys Val Val Ile Asn Tyr Arg Ser Arg Glu Ser Glu Ala Asn Asp Val
35 40 45
Leu Glu Glu Ile Lys Lys Val Gly Gly Glu Ala Ile Ala Val Lys Gly
50 55 60
Asp Val Thr Val Glu Ser Asp Val Val Asn Leu Ile Gln Ser Ala Val
65 70 75 80
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85 90 95
Asn Ala Val Pro Ser His Glu Met Pro Leu Glu Asp Trp Asn Arg Val
100 105 110
Ile Asn Thr Asn Leu Thr Gly Ala Phe Leu Gly Ser Arg Glu Ala Ile
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Ser Val His Glu Lys Ile Pro Trp Pro Leu Phe Val His Tyr Ala Ala
145 150 155 160
Ser Lys Gly Gly Ile Lys Leu Met Thr Glu Thr Leu Ala Leu Glu Tyr
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Ala Pro Lys Gly Ile Arg Val Asn Asn Ile Gly Pro Gly Ala Ile Asn
180 185 190
Thr Pro Ile Asn Ala Glu Lys Phe Ala Asp Pro Lys Lys Arg Ala Asp
195 200 205
Val Glu Ser Met Ile Pro Met Gly Tyr Ile Gly Asn Pro Glu Glu Ile
210 215 220
Ala Ala Val Ala Thr Trp Leu Ala Ser Ser Glu Ala Ser Tyr Val Thr
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Gly Ile Thr Leu Phe Ala Asp Gly Gly Met Thr Leu Tyr Pro Ser Phe
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Gln Ala Gly Arg Gly
260
Claims (9)
1.一种制备L-草铵膦的方法,其特征在于,包括如下步骤:以4-(羟基-(甲基)氧膦基)-2-氧代丁酸为底物,在氨基供体存在下,使用氨基酸序列为SEQ ID NO:2的谷氨酸脱氢酶或者其V375突变体催化不对称还原胺化反应,得到L-草铵膦,其中所述V375突变体选自下组:
V375S突变体即SEQ ID NO:3,V375A突变体即SEQ ID NO:4,V375G突变体即SEQ ID NO:5,V375C突变体即SEQ ID NO:6。
2.如权利要求1所述的方法,其特征在于,所述氨基供体为硫酸铵、磷酸氢二铵、碳酸氢铵、硝酸铵、氯化铵、乙酸铵、甲酸铵或氨水。
3.如权利要求1所述的方法,其特征在于,反应体系中添加有辅酶再生系统,所述辅酶再生系统选自下组:
(1)以葡萄糖为辅酶再生底物、以葡萄糖脱氢酶为辅酶再生酶,包含NADPH或NADP+的葡萄糖脱氢酶再生系统;
(2)以异丙醇为辅酶再生底物、以醇脱氢酶为辅酶再生酶,包含NADPH或NADP+的醇脱氢酶再生系统;
(3)以甲酸盐为辅酶再生底物、以甲酸脱氢酶为辅酶再生酶,构成包含NADPH或NADP+的甲酸脱氢酶再生系统。
4.如权利要求1所述的方法,其特征在于,反应体系中,所述谷氨酸脱氢酶或者其V375突变体呈酶形式或者呈表达所述酶的微生物菌体形式添加。
5.如权利要求1所述的方法,其特征在于,底物4-(羟基-(甲基)氧膦基)-2-氧代丁酸通过下述工艺制备:
将式III所示4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸酯与浓盐酸反应脱保护,得到式IV所示底物,
其中,R选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、苯甲基。
6.如权利要求5所述的方法,其特征在于,式III所示化合物通过下述工艺制备:
(1)将二氢-2,3-呋喃二酮(式I)加入到氢溴酸的醋酸溶液中,于60~80℃反应至产物不再继续增加,加入醇类试剂,继续搅拌一定时间至反应终止,得到4-溴-2-氧代丁酸酯(式II);
(2)在氮气保护下,将4-溴-2-氧代丁酸酯(式II)与甲基亚磷酸二乙酯混合,加热至100~200℃反应直至产物不再继续增加,得到4-(乙氧基-(甲基)膦酰基)-2-氧代丁酸酯(式III),
其中,R选自甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、苯甲基。
7.一种微生物,其特征在于,其表达如权利要求1中所述的氨基酸序列为SEQ ID NO:3-6中一种的谷氨酸脱氢酶。
8.如权利要求7所述的微生物,其特征在于,还同时表达如权利要求3中所述的辅酶再生酶。
9.如权利要求7或8所述的微生物在制备L-草铵膦中的用途。
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