CN110438194B - 一种脂肪酶在制备d-托品酸甲酯中的应用 - Google Patents
一种脂肪酶在制备d-托品酸甲酯中的应用 Download PDFInfo
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- CN110438194B CN110438194B CN201910687634.1A CN201910687634A CN110438194B CN 110438194 B CN110438194 B CN 110438194B CN 201910687634 A CN201910687634 A CN 201910687634A CN 110438194 B CN110438194 B CN 110438194B
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Abstract
本发明提供了一种脂肪酶在制备D‑托品酸甲酯中的应用,所述脂肪酶的氨基酸序列如SEQ ID NO.1所示,其编码基因如SEQ ID NO.2所示。以重组脂肪酶工程菌发酵培养获得的湿菌体经冷冻干燥后得到的冻干菌体为催化剂,外消旋托品酸甲酯为底物,以pH7.0磷酸缓冲溶液为反应介质,进行拆分制备D‑托品酸甲酯,对映体过量值>99%,D‑托品酸甲酯的收率达到95.0%。
Description
(一)技术领域
本发明属于生物酶催化领域,涉及一种脂肪酶立体选择性催化水解外消旋DL-托品酸甲酯合成单一构型的D-托品酸甲酯的应用。
(二)背景技术
托品酸(Tropic acid,TA)是一个重要的药物前体,可用作合成手性药物的原料。它常用于合成抗胆碱药物,如阿托品(也称莨菪碱)和东莨菪碱。这两种药物具有相似的治疗效果,是一类节后抗胆碱药,具有较广泛的药理作用,用于放松平滑肌,抑制唾液和汗腺的分泌以及预防晕眩病。它们是含有手性中心的化合物,其对映体通常具有极为相近的理化性质,但药理和毒理作用却存在着很大差异,往往一种立体异构体有药效而它的镜像分子的药效却很低,甚至完全没有药效或具有副作用,因此,获得用于改善功效的光学对映体是至关重要的。光学纯TA作为莨菪碱等抗胆碱药物的手性结构单元具有关键的作用,单一对映体托品酸及它们的衍生物都是重要的医药原料和医药中间体。因对映体D-托品酸具有低毒性以及很高的镇痛活性等优点,此外,D-托品酸已应用于人工环状缩酚肽的合成。所以在外消旋托品酸甲酯的手性拆分研究中,获得光学纯的D-托品酸甲酯是一些手性抗胆碱药物合成的必然要求。
目前有许多文献报道了托品酸的光学对映体的手性拆分路线,如动力学拆分、溶剂诱导手性转换、色谱技术、毛细管电泳等方法。综合以上几种拆分方法可知在这些工艺途径中均存在着收率低、成本高、步骤繁多、产物纯度较低、污染严重等问题,从而不能成功地进行工业化。酶法拆分能够利用酶的高度立体、位点、区域选择性,催化化学合成的外消旋体或衍生物中的某一对映体进行水解以获得高产率、高选择性的单一对映体,具有选择性高、反应条件温和、反应较稳定及易于工业化等特点。同时,酶法拆分可以解决现有方法易造成环境污染,产生大量无效甚至对环境有害的对映体问题,对于保护人类的自然环境和健康具有极为重要的意义。因此,我们提供了一种脂肪酶催化水解拆分制备DL-托品酸甲酯来高效生产D-托品酸甲酯的有效方法。
(三)发明内容
本发明目的是为了解决现有方法的不足,通过筛选产脂肪酶微生物,提供一种廉价高效的立体选择性脂肪酶在生物法拆分DL-托品酸甲酯制备D-托品酸甲酯中的应用。本发明利用新筛选的脂肪酶催化立体选择性水解反应拆分制备高光学纯度的D-托品酸甲酯,催化剂易得成本低,ee值和转化率高,路线短操作简便,开发成本低廉、适合产业化放大工艺路线,具有很大的社会意义和经济价值。
本发明采用的技术方案是:
本发明提供一种脂肪酶在拆分DL-托品酸甲酯中的应用(如图2所示),所述脂肪酶氨基酸序列如SEQ ID NO.1所示,编码基因序列为SEQ ID NO.2所示,具体的应用方法为:以含立体选择性脂肪酶编码基因的工程菌经发酵培养获得的湿菌体冷冻干燥后得到的脂肪酶粗酶粉为催化剂,以外消旋DL-托品酸甲酯为底物,以pH 7.0缓冲液为反应介质,在25-50℃、200rpm条件下进行拆分反应,反应完全后,将反应液分离纯化,获得D-托品酸甲酯。所述催化剂用量以缓冲液体积计为5g/L-40g/L(优选10g/L),所述底物体积用量以缓冲液体积计为0.5%-3%(V/V)(优选1%)。
进一步,优选反应时间为30min-120min,更优选反应条件为35℃、200rpm反应60min。
进一步,所述缓冲液为pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液。
进一步,所述催化剂按如下方法制备:将含立体选择性脂肪酶编码基因的工程菌(优选大肠杆菌BL21)接种在LB培养基中,37℃培养OD600至为0.4-0.6,加IPTG至终浓度0.02mM,30℃培养10-12h,菌液8000rpm,4℃离心10min,收集菌体,再用PBS缓冲液洗涤菌体2次,8000rpm,4℃离心10min,收集菌体,冻干,得到脂肪酶粗酶粉;所述LB培养基组成:胰蛋白胨10g/L、酵母粉5g/L、NaCl 5g/L,溶剂为去离子水,pH值自然。
进一步,本发明所述反应液分离纯化的方法为:反应结束后,反应液先用2M NaOH水溶液调pH至9.0,然后用等体积正己烷萃取,分液漏斗分离出有机相,再经纯水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到最终产物D-托品酸甲酯。
本发明脂肪酶氨基酸序列如SEQ ID NO.1所示:
MTINYHELETSHGRIAVRESEGEGAPLLMIHGNSSSGAIFAPQLEGAIGKKWRVIAPDLPGHGKSSDAIDPDRSYSMEGYADAMTEVMQQLGIADAVVFGWSLGGHIGIEMITRYPEMRGLMITGTPPVAREEVGQGFKSGPDMALAGQEVFSERDVDSYARSTCGEPFEASLLDIVARTDGRARRIMFEKFAAGTGGNQRDIVAQAKLPIAVVNGRDEPFVELDFVSKVKFGNLWEGKTHVIDDAGHAPFRETPAEFDAYLARFIESCTK。
由于氨基酸序列的特殊性,任何含有SEQ ID NO.1所示氨基酸序列的多肽的片段或其变体,如其保守性变体、生物活性片段或衍生物,只要该多肽的片段或多肽变体与前述氨基酸序列同源性在90%以上、且具有相同的酶活性,均属于本发明保护范围之列。具体的,所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换;其中,对于变体的保守性改变,所替换的氨基酸具有与原氨基酸相似的结构或化学性质,如用亮氨酸替换异亮氨酸,变体也可具有非保守性改变,如用色氨酸替换甘氨酸。
本发明所述蛋白的片段、衍生物或类似物是指基本上保持本发明所述的蛋白酶相同的生物学功能或活性的蛋白,可以是下列情形:(Ⅰ)一个或多个氨基酸残基被保守或非保守氨基酸残基(优选的是保守氨基酸残基)取代,并且取代的氨基酸可以是也可以不是由遗传密码子编码的;(Ⅱ)一个或多个氨基酸残基上的某个基团被其它基团取代;(Ⅲ)成熟蛋白与另一种化合物(比如延长蛋白半衰期的化合物,例如聚乙二醇)融合;(Ⅳ)附加的氨基酸序列融合进成熟的蛋白而形成的蛋白序列(如用来纯化此蛋白的序列或蛋白原序列)。
所述蛋白可以是重组蛋白、天然蛋白或合成蛋白,可以是纯天然纯化的产物,或是化学合成的产物,或使用重组技术从原核或真核宿主(例如:细菌、酵母、高等植物、昆虫和哺乳动物细胞)中产生。根据重组生产方案所用的宿主,本发明的蛋白可以是糖基化的。本发明的蛋白还可以包括或不包括起始的甲硫氨酸残基。
本发明还涉及所述立体选择性脂肪酶的编码基因,具体的,所述大肠杆菌密码子优化后的编码基因核苷酸序列如SEQ ID NO.2所示:
ATGACTATCAATTATCACGAGCTTGAAACCAGCCATGGCCGCATTGCTGTGCGTGAAAGCGAAGGCGAGGGCGCTCCTCTTCTGATGATCCATGGCAATTCAAGTTCGGGCGCAATTTTCGCACCACAACTCGAAGGTGCAATCGGCAAAAAATGGCGCGTGATAGCGCCTGATCTTCCGGGCCATGGCAAATCTTCCGATGCGATTGATCCCGATCGCAGCTATTCGATGGAAGGCTATGCGGATGCGATGACGGAAGTCATGCAGCAGCTCGGCATTGCCGATGCTGTCGTTTTCGGCTGGTCGCTCGGTGGCCATATCGGTATCGAGATGATTACCCGTTATCCTGAAATGCGTGGCCTTATGATTACCGGAACGCCACCCGTAGCGCGCGAGGAAGTGGGGCAGGGGTTCAAGAGTGGACCCGACATGGCGCTTGCCGGACAGGAAGTCTTTTCCGAGCGCGACGTGGATTCTTACGCACGCAGCACCTGTGGCGAACCATTCGAGGCATCGCTTCTCGATATTGTTGCCCGTACCGACGGACGCGCCCGCCGCATCATGTTTGAAAAATTTGCCGCTGGCACCGGCGGCAACCAGCGCGACATCGTCGCTCAAGCAAAGCTTCCCATTGCAGTCGTCAATGGGCGAGATGAACCTTTCGTCGAACTCGACTTCGTCTCCAAGGTAAAATTCGGTAATCTTTGGGAAGGCAAAACCCACGTTATCGACGATGCAGGACATGCGCCTTTTCGCGAAACGCCAGCTGAATTTGATGCTTATCTCGCTCGTTTCATCGAGAGTTGCACAAAATAA。
由于核苷酸序列的特殊性,任何SEQ ID NO.2所示多核苷酸的变体,只要其与该多核苷酸具有70%以上同源性、且具有相同的功能,均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以是天然发生的等位变异体或非天然发生的变异体,包括取代变异体、缺失变异体和插入变异体。如本领域所知的,等位变异体是一个多核苷酸的替换形式,它可能是一个或多个核苷酸的取代、缺失或插入,但不会从实质上改变其编码的氨基酸的功能。
另外,可与SEQ ID NO:2所示多核苷酸序列杂交的多核苷酸(至少具有50%同源性,优选具有70%同源性),也在本发明保护范围之列,特别是在严格条件下可与本发明所述核苷酸序列杂交的多核苷酸。所述“严格条件”是指:(1)在较低离子强度和较高温度下的杂交和洗脱,如0.2 SSC,0.1%SDS,60℃;或(2)杂交时加用变性剂,如50%(v/v)甲酰胺,0.1%小牛血清,0.1%Ficoll,42℃;或(3)仅在两条序列之间的同源性至少在95%以上,更好是97%以上时才发生杂交。并且,可杂交的多核苷酸编码的蛋白与SEQ ID NO:1所示的蛋白有相同的生物学功能和活性。
本发明还涉及含有所述编码基因的重组载体,以及利用所述重组载体转化得到的重组基因工程菌。
与现有技术相比,本发明的有益效果主要体现在:本发明提供了一种脂肪酶基因核苷酸序列;该脂肪酶基因可与表达载体连接构建得到含该基因的胞内表达重组质粒,再转化至大肠杆菌菌株中,获得重组大肠杆菌,再利用重组大肠杆菌或重组脂肪酶为生物催化剂催化拆分DL-托品酸甲酯,可以生成D-托品酸甲酯,对映体过量值>99%,D-托品酸甲酯的收率达到95.0%。本发明生物催化的手性合成反应具有条件温和、效率高,高度的化学选择性、区域选择性以及对映体选择性等优点,而且生物催化过程具有无毒、无污染和能耗低等特点,是一种环境友好的合成方法。
(四)附图说明
图1为有机合成DL-托品酸甲酯的反应示意图;
图2为脂肪酶对映选择性水解拆分DL-托品酸甲酯的反应示意图;
图3为DL-托品酸甲酯标样液相色谱图;
图4为DL-托品酸标样液相色谱图;
图5为脂肪酶催化DL-托品酸甲酯水解反应60min的液相色谱图;
图6为化学合成底物DL-托品酸甲酯GC-MS检测图。
(五)具体实施方式
下面结合具体实施例对本发明进行进一步描述,但本发明的保护范围并不仅限于此,本领域的普通技术人员根据这些实施方式所做出的方法上的变换均包含在本发明的保护范围内。
实施例1化学合成底物DL-托品酸甲酯
取5g DL-托品酸和5mL甲醇(分析纯,甲醇过量)放入250mL圆底烧瓶中,加入20mL甲苯作为反应溶剂,再加入125uL的浓硫酸(质量浓度98%)作为催化剂。通过油浴在磁力搅拌器80℃,600rpm条件下反应6-8h。反应结束后得到的反应液,先用饱和Na2CO3洗涤,除去其中的未反应酸,然后用等体积的乙酸乙酯进行萃取,分液漏斗分离有机相和水相,水相用乙酸乙酯萃取两次,合并有机相,再经纯水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到产物DL-托品酸甲酯(如图1所示)。产物经过GC-MS检测(如图6所示)和HPLC分析(如图3所示),没有其他杂质,纯度基本可以达到100%,质量收率95%。
实施例2酶催化拆分DL-托品酸甲酯的脂肪酶筛选
大肠杆菌BL21(F11)工程菌构建:通过杭州擎科生物技术有限公司人工合成SEQID NO.2所示核苷酸序列的基因,将该片段连接到pET28b载体上获得克隆载体pET28b-lip并将其转化于大肠杆菌Escherichia coli BL21中,获得重组大肠杆菌,记为大肠杆菌BL21(F11)。对重组质粒测序,并利用软件对测序结果进行分析,该序列含有一个长为816bp的开放阅读框(SEQ ID NO.2),表明大肠杆菌重组脂肪酶工程菌构建成功。其他脂肪酶基因(如表1所示)重组工程菌,按上述方法及宿主细胞和质粒进行工程菌的构建,并进行发酵获得含脂肪酶菌粉。
大肠杆菌BL21(F11)培养条件:LB培养基组成:胰蛋白胨10g/L、酵母粉5g/L、NaCl5g/L,溶剂为水,pH自然。将菌种斜面接种在LB培养基中,37℃培养OD600至为0.5(大概培养2h),加IPTG至终浓度0.02mM,30℃培养10-12h。300mL菌液8000rpm,4℃离心10min,收集菌体,再用PBS缓冲液洗涤菌体2次,8000rpm,10min收集菌体。将收集的菌体使用-80℃真空冷冻冻干机冻干得到脂肪酶F11粗酶粉,放于4℃冰箱保存备用。
根据脂肪酶F11粗酶粉的制备方法,分别制备得到表1中各个脂肪酶。
称取表1中不同来源的具有水解活性的冻干的脂肪酶菌粉0.01g于2mL EP管中,加入1mL磷酸缓冲液(pH 7.0、0.2mM)作为反应溶剂,然后加入0.01uL底物DL-托品酸甲酯(实施例1制备),以不加菌体为空白对照,置于35℃,200rpm恒温混匀仪中反应60min。反应结束后,反应液经4mMHCl酸化后离心取300uL上清液冻干,加入1mL异丙醇溶解,使用液相色谱检测菌体的立体选择性及酶催化水解活性,结果如表1所示,筛选得到底物的对映体过量值>99%产脂肪酶微生物菌株,最终选择源自根瘤菌(Rhizobiales bacterium,GenBank:WP_113158746.1)的蛋白质序列(表1中F11),试验最终选择大肠杆菌BL21(F11)发酵的粗酶粉作为催化拆分反应的生物催化剂。通过大肠杆菌表达系统密码子优化后获得基因序列SEQID NO.2所示核苷酸序列,编码蛋白氨基酸序列为SEQ ID NO.1所示。
表1筛选具有水解活性的脂肪酶
液相分析条件:采用液相色谱仪Waters 1525型液相色谱仪;大赛璐CHIRALPAKAD-H(5μm,4.6×250mm)手性色谱柱,紫外光检测波长220nm,流动相正己烷:异丙醇=12:1(V/V);流速为0.5mL/min,柱温30℃,进样量10μL。如HPLC结果所示,L-托品酸甲酯和D-托品酸甲酯分别在22.765min和24.049min出峰(如图3所示),L-托品酸和D-托品酸分别在36.033min和44.125min出峰(如图4所示)。酶水解拆分反应后,获得D-托品酸甲酯的液相色谱图(如图5所示)。
实施例3反应温度对酶动力学水解拆分DL-托品酸甲酯的影响
在pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液1mL中,加入终浓度10g/L的实施例3方法制备的脂肪酶F11,10μL DL-托品酸甲酯,于恒温混匀仪不同温度(25-50℃),200rpm条件下反应60min,取反应液按实施例2方法检测D-托品酸甲酯的对映体过量值和转化率,结果见表2所示。
结果说明,在反应温度为35℃时,D-托品酸甲酯的对映体过量值最高,其ee值>99%。当反应温度高于35℃或者低于35℃时,催化转化率和对映体过量值都会下降,说明温度对脂肪酶F11的光学选择性具有很大的影响。
表2反应温度对反应的影响
实施例4反应时间对酶动力学水解拆分DL-托品酸甲酯的影响
在pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液1mL中,加入终浓度10g/L的实施例3方法制备的脂肪酶F11,10μL DL-托品酸甲酯,于恒温混匀仪35℃,200rpm条件下反应不同时间(30min-120min),取反应液按实施例2方法检测D-托品酸甲酯的对映体过量值和转化率,结果见表3所示。
结果表明,反应60min后,产物D-托品酸甲酯的对映体过量值已达到最高,对映体过量值>99%,转化率为49.92%,当反应时间大于60min,转化率增大,将导致D-托品酸甲酯的收率降低。
表3反应时间对酶催化反应反应的影响
实施例6不同脂肪酶催化DL-托品酸甲酯水解拆分的效果比较
在pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液1mL中,加入终浓度10g/L的不同脂肪酶(实施例3制备的脂肪酶F11粗酶粉、Novozym 435、Lipozyme TL IM、Lipozyme RM IM),10uL DL-托品酸甲酯,于恒温混匀仪35℃,200rpm条件下反应60min,取反应液按实施例2方法检测D-托品酸甲酯的对映体过量值和转化率,结果见表4所示。在35℃反应60min后,Novozym 435对DL-托品酸甲酯的两种构型能进行一定程度水解,但对底物两种构型没有对映体选择性,而LipozymeTL IM、LipozymeRM IM对底物没有水解活性和对映体选择性。而当脂肪酶F11催化反应时,产物D-托品酸甲酯的ee值>99%,转化率为49.92%,说明脂肪酶粗酶粉F11具有良好的立体选择性。
表4不同脂肪酶催化DL-托品酸甲酯的拆分效果
实施例7 D-托品酸甲酯的分离提取
在50mL圆底烧瓶中加入10mL pH 7.0,0.2mM的Na2HPO4/NaH2PO4缓冲溶液,称取0.1g实施例3方法制备的脂肪酶F11,再加入终浓度1%(V/V)的DL-托品酸甲酯,用200mMNaOH水溶液进行流加滴定反应,控制反应pH维持在7.0,在磁力搅拌器35℃,200rpm条件下反应60min。反应结束后,反应液先用2M NaOH水溶液调pH至9.0,反应液用等体积正己烷萃取,分液漏斗分离出有机相,再经纯水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到最终产物,并称重为0.0475g,产物D-托品酸甲酯ee值>99%,质量收率达到95.0%。
序列表
<110> 浙江工业大学
<120> 一种脂肪酶在制备D-托品酸甲酯中的应用
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Ala Arg Ser Thr Cys Gly Glu Pro Phe Glu Ala Ser Leu Leu Asp Ile
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Val Ala Arg Thr Asp Gly Arg Ala Arg Arg Ile Met Phe Glu Lys Phe
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ggccatggca aatcttccga tgcgattgat cccgatcgca gctattcgat ggaaggctat 240
gcggatgcga tgacggaagt catgcagcag ctcggcattg ccgatgctgt cgttttcggc 300
tggtcgctcg gtggccatat cggtatcgag atgattaccc gttatcctga aatgcgtggc 360
cttatgatta ccggaacgcc acccgtagcg cgcgaggaag tggggcaggg gttcaagagt 420
ggacccgaca tggcgcttgc cggacaggaa gtcttttccg agcgcgacgt ggattcttac 480
gcacgcagca cctgtggcga accattcgag gcatcgcttc tcgatattgt tgcccgtacc 540
gacggacgcg cccgccgcat catgtttgaa aaatttgccg ctggcaccgg cggcaaccag 600
cgcgacatcg tcgctcaagc aaagcttccc attgcagtcg tcaatgggcg agatgaacct 660
ttcgtcgaac tcgacttcgt ctccaaggta aaattcggta atctttggga aggcaaaacc 720
cacgttatcg acgatgcagg acatgcgcct tttcgcgaaa cgccagctga atttgatgct 780
tatctcgctc gtttcatcga gagttgcaca aaataa 816
Claims (8)
1.一种脂肪酶在制备D-托品酸甲酯中的应用,其特征在于所述脂肪酶的氨基酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的应用,其特征在于所述应用的方法为:以含脂肪酶编码基因的工程菌经发酵培养获得的湿菌体冻干后的脂肪酶粗酶粉为生物催化剂,以外消旋DL-托品酸甲酯为底物,以pH 7.0缓冲液为反应介质,在25-50℃、200rpm条件下进行拆分反应,反应完全后,将反应液分离纯化,获得D-托品酸甲酯。
3.如权利要求2所述的应用,其特征在于所述催化剂用量以缓冲液体积计为5g/L-40g/L,所述底物体积用量以缓冲液体积计为0.5%-3%。
4.如权利要求2所述的应用,其特征在于反应时间为30min-120min。
5.如权利要求2所述的应用,其特征在于所述反应条件为35℃、200rpm反应60min。
6.如权利要求2所述的应用,其特征在于所述缓冲液为pH 7.0、0.2 mM的Na2HPO4/NaH2PO4缓冲溶液。
7.如权利要求2所述的应用,其特征在于所述催化剂按如下方法制备:将含脂肪酶编码基因的工程菌接种在LB培养基中,37℃培养OD600至为0.4-0.6,加IPTG至终浓度0.02 mM,30℃培养10-12 h,发酵液8000 rpm,4℃离心10min,收集菌体,再用磷酸缓冲液洗涤菌体2次,8000 rpm,4℃离心10min,收集湿菌体,冻干后得到脂肪酶粗酶粉。
8.如权利要求2所述的应用,其特征在于所述反应液分离纯化的方法为:反应结束后,反应液先用2M NaOH水溶液调pH至9.0,用等体积正己烷萃取,分液漏斗分离出有机相,经去离子水洗涤两次,饱和NaCl洗涤两次,干燥,旋蒸,得到最终产物D-托品酸甲酯。
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