CN113754726A - 一种含多肽标签的重组酶及其在医药化学品合成中的应用 - Google Patents
一种含多肽标签的重组酶及其在医药化学品合成中的应用 Download PDFInfo
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- CN113754726A CN113754726A CN202110936180.4A CN202110936180A CN113754726A CN 113754726 A CN113754726 A CN 113754726A CN 202110936180 A CN202110936180 A CN 202110936180A CN 113754726 A CN113754726 A CN 113754726A
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- nitrilase
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Abstract
本发明公开了一种含多肽标签的重组酶及其在医药化学品合成中的应用,所述重组酶是将多肽标签连接至腈水解酶氨基酸序列的N端所得到的酶;所述多肽标签的氨基酸序列为GKGKGKGKG。本发明重组腈水解酶制备1‑氰基环己基乙酸时活力高达3034.7U/g dcw,多肽标签的引入显著提高了腈水解酶的可溶性表达,相同浓度全细胞催化剂水解1M底物时较母本酶快30min完成,且稳定性优于母本酶。本发明重组腈水解酶还能够催化合成其它医药中间体,并提高反应中全细胞催化剂的活力,提高其它不同种类腈水解酶的可溶性,以及相应的全细胞催化剂的活力。
Description
本申请为“申请号2020112112516,申请日2020年11月3日,名称为多肽标签、高度可溶性的重组腈水解酶及其在医药化学品合成中的应用”专利申请的分案申请
(一)技术领域
本发明涉及一种多肽标签,特别涉及一种可增强腈水解酶等酶可溶性表达的通用多肽标签,及在医药化学品合成中的应用,属于基因工程以及蛋白质工程技术领域。
(二)背景技术
腈水解酶(EC 3.5.5.1)是一类重要的具有Glu-Lys-Cys催化活性中心三联体的水解酶,可在一步反应中有选择地且有效地催化氰基水解为羧基,在有机酸、氨基酸、维生素等化工产品和如加巴喷丁、氯吡格雷、巴氯芬、阿托伐他汀等医药化学品的合成中具有广泛的应用。
迄今为止,已经报道了许多提高蛋白质可溶性表达水平的有效方法,一种是构建重组周质渗漏菌株或共表达周质伴侣蛋白,另一种是添加融合标签(也称为融合伴侣或溶解性标签)进行协同表达。但这些方法或多或少的存在一些缺陷。如多肽标签上氨基酸的选择、序列的长度可能对重组酶的酶活,稳定性,溶解度或选择性有很大副作用,某些标签甚至会改变酶蛋白的结构导致酶完全失去活性。对于不同的酶的结构和蛋白整体在催化体系中的带电属性,不同的多肽标签的引入会带来不同的效果,需要结合动力学模拟、同源建模等技术进行协助标签的设计。但是有效的多肽标签的优点之一是和目的基因连接后表达并不需要去除即可发挥酶蛋白的作用。其中,Sun-Ki Kim开发了一种新的聚阴离子多肽标签,显著提高了南极假丝酵母脂肪酶的表达水平和细胞外转运效率。此外,Han等人开发了一种新的融合标签[HE-MBP(Pyr)]以提高重组蛋白在大肠杆菌中的溶解度,研究表明目的蛋白(如单克隆抗体,抗原蛋白和聚合蛋白)溶解度有不同程度的改善。
在生物合成方法中,涉及到生物催化的过程通常都是使用全细胞催化剂(湿细胞)来进行的。一般认为产酶发酵中发酵液的体积酶活为比酶活和可溶性表达水平两者的结合。若能在不影响原始特性的前提下,提高酶的比酶活或有效蛋白质的数量将促进全细胞催化性能的进一步提高。所以,增加腈水解酶在大肠杆菌中的溶解度是有巨大意义的。然而增加大肠杆菌所产酶的溶解度是总是以牺牲活力为代价的。因此,在不降低催化水平的情况下合理地增加腈水解酶的溶解度是实现加巴喷丁中间体1-氰基环己基乙酸(1-CA)的高效生产仍需进一步研究。
(三)发明内容
本发明目的是提供一种多肽标签、含多肽标签的重组酶及其在医药化学品合成中的应用,所述多肽标签能够使含多肽标签的重组酶的全细胞催化剂活力和热稳定性均得到有效提升。解决酶热稳定性差,酶活不满足工业大规模应用等问题。
本发明采用的技术方案是:
本发明提供一种多肽标签,所述多肽标签两端的氨基酸均为不带电的甘氨酸(G),其余为甘氨酸(G)、组氨酸(H)、谷氨酸(E)、天冬氨酸(D)、赖氨酸(K)、精氨酸(R)中任意一种或多种的随机组合;所述多肽标签的长度为5-11个氨基酸。
进一步,所述多肽标签氨基酸序列为下列之一:GKGKG、GKGEG、GKGHG、GRGRG、GRGGG、GHGHG、GDGDG、GDGEG、GDGRG、GDGKG、GEGEG、GEGKG、GEGGG、GEGRG、GEGDG、GKGKG、GKGDG、GKGEG、GKGGG、GKGHG、GKGRG、GRGRG、GRGDG、GRGEG、GRGKG、GRGGG、GGGKG、GGGEG、GHGHG、GKGKGKG、GKGKGKGKG、GKGKGKGKGKG。
进一步,优选所述多肽标签两端和中间位置均为不带电的甘氨酸(G),其余为甘氨酸(G)、组氨酸(H)、谷氨酸(E)、天冬氨酸(D)、赖氨酸(K)、精氨酸(R)中任意一种或多种的随机组合,更优选构成具有回文元素的氨基酸序列。
进一步,所述多肽标签还设有连接肽,所述连接肽氨基酸序列为下列之一:GS、GGS、GGGS、GGGGS。
更进一步,优选多肽标签为GKGKG。
本发明提供一种含所述多肽标签的重组酶,所述酶包含腈水解酶,脂肪酶,脱酰基酶。
进一步,所述重组酶优选为重组腈水解酶,所述重组腈水解酶是将腈水解酶氨基酸序列的N端与多肽标签连接获得的。所述连接可以采用PCR扩增、一步克隆等方法,例如以含腈水解酶基因的载体(优选pET-28b(+)/AcN-M)为模板,设计含多肽标签的引物,经PCR扩增,获得含多肽标签的腈水解酶。
进一步,所述多肽标签通过连接肽与腈水解酶连接的方式为下列之一:GKGKG-GS、GKGKG-GGS、GKGKG-GGGS、GKGKG-GGGGS。
本发明所述腈水解酶基因克隆自敏捷食酸菌(Acidovorax facilis ZJB09122),氨基酸序列为SEQ ID NO.1所示,核苷酸序列为SEQ ID NO.2所示。
本发明还涉及含有所述多肽标签的重组酶编码基因的重组质粒(优选重组腈水解酶编码基因的重组质粒),所述重组质粒以pET-28b(+)为载体构建,具体为:以含有所述重组酶基因的质粒作为模板,设计含多肽标签的引物,进行全质粒PCR,核酸凝胶电泳和测序进行验证,最终得到重组质粒。
本发明还提供一种由所述含多肽标签的重组酶编码基因构建的重组基因工程菌,所述重组基因工程菌是将含多肽标签的重组酶编码基因的载体pET-28b(+),导入宿主菌构建而成;所述宿主菌优选为Escherichia coli BL21(DE3)。
本发明还提供一种所述含多肽标签的重组酶在制备加巴喷丁中间体1-氰基环已基乙酸中的应用,所述的应用为:以含多肽标签的重组酶编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体纯化后的纯酶为催化剂(优选重组腈水解酶),以1-氰基环己基乙腈(1-CN)为底物,以0.2M、pH=7.0的Na2HPO4-NaH2PO4缓冲液为反应介质构成转化体系,35℃、200rpm恒温水浴反应完全,获得含1-氰基环己基乙酸(1-CA)的转化液,转化液分离纯化,获得1-氰基环己基乙酸;所述1-氰基环己基乙酸经过后续加氢等步骤获得加巴喷丁;所述转化体系中,底物加入终浓度为1-2M,催化剂加入量以湿菌体重量计为50g/L。
进一步,所述湿菌体按如下方法制备:将含多肽标签的重组酶(重组腈水解酶)编码基因的重组基因工程菌接种至LB(Luria-Bertani)培养基,37℃培养12-14h后,获得单菌落;挑取单菌落接种到含0.5μg/mL卡那霉素的LB培养基中,37℃培养8h后,以体积浓度2%的接种量转接至含0.5μg/mL卡那霉素的发酵培养基中,37℃培养2h后,加入终浓度0.1mM的IPTG,28℃诱导产酶12-14h,12,000转离心10min,沉淀用0.9%的生理盐水清洗2次,收集湿菌体即为催化剂。所述发酵培养基组成:20g/L酵母粉,15g/L蔗糖,5g/L NaCl,0.9g/L三水合磷酸氢二钾,溶剂为水,pH=6.8。
进一步,所述纯酶按如下方法制备:取湿菌体均匀悬浮于0.2M pH 7.0Na2HPO4-NaH2PO4缓冲液中,在冰浴条件下,进行超声波细胞破碎,超声波细胞破碎仪功率设定为40W,工作1s,间隔1s,总破碎时间为20min;随后,将细胞破碎液在12,000×g,4℃离心15min,去除细胞碎片,收集粗酶液;所述缓冲液体积用量以湿菌体重量计为2mL/g;
将Ni柱用上样缓冲液(Binding buffer:50mM NaH2PO4,300mM NaCl,50mM咪唑,pH8.0)进行平衡,流速为2mL/min;随后,以2mL/min的流速将粗酶液上样,使用上样缓冲液洗脱杂蛋白以及弱吸附蛋白;最后,使用洗脱缓冲液(Elution buffer:50mM NaH2PO4,300mMNaCl,500mM咪唑,pH 8.0)进行洗脱,洗脱速度为3mL/min,以蛋白纯化仪(Bio-RadBioLogic LP层析系统)UV参数为准,当UV≥2时,进行酶液的收集,当UV≤2时,结束收集;接着使用透析袋(上海雷布斯生物科技有限公司MD34-3500)在50mM pH 7.0Na2HPO4-NaH2PO4缓冲液中透析过夜,取截留液,即为腈水解酶纯酶。
本发明所述1-氰基环己基乙酸制备加巴喷丁的方法为:
(1)将含1-氰基环己基乙酸(1-CA)的转化液离心(8000rpm 10min)去除菌体细胞,收集的滤液即为1-氰基环己基乙酸,取滤液于加氢反应釜,加入雷尼镍(型号为RTH-4110)、三乙胺(分析纯)和甲酸(分析纯);通入氮气置换空气,如此重复3次,保证釜内无空气存在;再次通入氢气(保持反应过程中压力在2Mpa),1000rpm反应8h后结束;冷却之后过滤回收雷尼镍,得到的滤液加入等体积的二氯甲烷进行萃取,静置分层取有机相40℃下进行旋蒸得到固体即为加巴喷丁内酰胺,二氯甲烷可回收再利用;所述滤液体积用量以雷尼镍重量计为150mL/1.5g;所述三乙胺与滤液体积比为1:150;所述甲酸体积用量以滤液体积比为0.5:150;
(2)取步骤(1)中制备得到的加巴喷丁内酰胺溶解于6M HCl中加热回流2.5h,待冷却至室温后加入等体积的二氯甲烷进行萃取,静置分层后取水相在0-4℃下进行结晶,抽滤得到的白色固体用丙酮研磨,过滤除去丙酮并在40℃烘干得到加巴喷丁盐酸盐;将得到的所有的加巴喷丁盐酸盐溶于水中,加热至40℃,300rpm搅拌下充分溶解,使用6M NaOH调pH至7.0-7.5;后加入甲苯,500rpm搅拌30min;结束搅拌于0-4℃下进行结晶,过滤得到白色固体即为加巴喷丁粗品,粗品用60%甲醇或异丙醇重结晶并烘干即为加巴喷丁。所述HCl体积用量以加巴喷丁内酰胺重量计为500mL/76.7g;所述溶解加巴喷丁盐酸盐的水体积用量以加巴喷丁内酰胺重量计为500mL/76.7g;所述甲苯体积用量以加巴喷丁内酰胺重量计为125mL/76.7g。
本发明还提供一种所述含多肽标签的重组酶在制备氯吡格雷中间体(邻氯扁桃酸)中的应用,所述的应用为:将含所述多肽标签的重组酶(优选重组腈水解酶)的基因工程菌经发酵培养获得的湿菌体为催化剂,以邻氯扁桃腈为底物,以0.2M、pH7.0的Na2HPO4-NaH2PO4缓冲液为反应介质构成反应体系,35℃恒温水浴反应12h,获得含邻氯扁桃酸的反应液,分离纯化,得到邻氯扁桃酸。所述反应体系中,催化剂加入量以湿菌体重量计为50g/L,所述底物加入终浓度为1-2M。
本发明还提供一种所述多肽标签的重组酶在制备ECBN(棘白菌素B母核)中的应用,所述应用为:将含所述多肽标签的重组酶(优选脱酰基酶(NC_001136.10))的基因工程菌经发酵培养获得的湿菌体为催化剂,以棘白菌素B为底物,以0.2M、pH7.0的Na2HPO4-NaH2PO4缓冲液和1.5%β-环糊精(作为助溶剂)构成反应体系,35℃恒温水浴反应24h,获得含未完全反应的底物和部分产物的混合反应液,分离纯化,得到ECBN。所述反应体系中,催化剂加入量以湿菌体重量计为50g/L,所述底物加入终浓度为2g/L。
与现有技术相比,本发明有益效果主要体现在:本发明提供了一种多肽标签,以及含多肽标签的重组腈水解酶及其在加巴喷丁等医药化学品合成中的应用,通过在腈水解酶N端连接多肽标签构建重组腈水解酶。本发明多肽标签为正电荷标签,将多肽标签添加至腈水解酶基因的N端进行融合表达,构建重组菌株,并将此菌株诱导表达12-14h,得到的全细胞催化剂,在制备加巴喷丁中间体1-氰基环己基乙酸时活力高达3034.7U/g dcw,显著提高了腈水解酶的可溶性表达,相同浓度全细胞催化剂水解1M底物时较母本酶快30min完成,且稳定性优于母本酶。本发明提供的方法也可以用于该腈水解酶催化的以其它医药中间体为底物的生物催化反应,并提高反应中全细胞催化剂的活力;还可以用于提高其它不同种类腈水解酶或者其他酶的可溶性,以及相应的全细胞催化剂的活力。
(四)附图说明
图1:含多肽标签的重组质粒pET-28b+/tag-AcN-M图谱。
图2:核酸凝胶电泳图,泳道M为Maker,泳道1为全质粒PCR产物。
图3:重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M细胞破碎上清液和沉淀样品的SDS-PAGE电泳图;其中M为标准蛋白质的分子质量,1、2分别为原始菌株的细胞破碎上清液和沉淀;3、4分别为重组菌株的细胞破碎上清液和沉淀。
图4:原始菌株和重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞在不同温度作用下的相对活力比较;其中原始菌株在标准酶活测定条件下的活力值设置为100%。
图5:原始菌株和重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞在不同pH作用下的相对活力比较;其中原始菌株在标准酶活测定条件下的活力值设置为100%。
图6:原始菌株和重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞在不同底物浓度催化产物积累浓度的比较,A为底物浓度1M,B为底物浓度2M。
(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:
下列实施实例中所涉及的培养基如下:
LB固体培养基质量组成:5g/L酵母粉,10g/L蛋白胨,10g/L NaCl,2%琼脂粉,溶剂为水,pH=7.0。
LB液体培养基:5g/L酵母粉,10g/L蛋白胨,10g/L NaCl,溶剂为水,pH=7.0。
发酵培养基:20g/L酵母粉,15g/L蔗糖,5g/L NaCl,0.9g/L三水合磷酸氢二钾,溶剂为水,pH=6.8。
下列实施实例中所涉及的检测方法如下:
酶活定义:在一定条件下,每分钟催化底物生成1μmol 1-氰基环己基乙酸(1-CA)所需的酶量定义为一个活力单位,记为U。
比酶活是指特定条件下,单位重量(mg)蛋白具有的酶活力单位数。
静息细胞活力测定方法:取0.01g静息细胞悬浮于1mL、0.2M、pH 7.0Na2HPO4-NaH2PO4缓冲液,35℃保温10min,加入0.03g(终浓度为0.2M)底物1-氰基环己基乙腈(1-CN),200rpm,35℃恒温振荡反应10min,反应结束后12,000rpm离心5min,取上清,测定产物浓度。
标准酶活测定条件下原始菌株的比酶活设为100%,重组菌株的比酶活与原始菌株的比酶活之比即为相对细胞活力(%)。
底物1-CN浓度检测方法:气相色谱:Agilent 7890A,色谱柱:Agilent J&WHP-5Column(30m×0.32mm,膜厚0.25μm),进样口及检测器温度为320℃;柱温160℃,保持8min;载气:高纯氦气;载气流量:1.0mL/min;进样量:1μL;分流比为30:1。
产物1-CA浓度检测方法:液相色谱:色谱柱类型为C18-H,250mm×4.6mm,J&KScientific Ltd.,China;色谱条件为柱温40℃,紫外检测波长为215nm,流动相为76%缓冲液(0.58g/L NH4H2PO4和1.83g/L NaClO4,pH 1.8)和24%乙腈。
所述腈水解酶基因克隆自敏捷食酸菌(Acidovorax facilis ZJB09122),氨基酸序列为SEQ ID NO.1所示,核苷酸序列为SEQ ID NO.2所示。敏捷食酸菌(Acidovoraxfacilis ZJB09122)保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO.M209044,已在专利CN101629192B中披露。
实施例1:含多肽标签的重组质粒的构建
1、设计原则为:蛋白质的溶解度与残基的疏水性密切相关,同时也受到蛋白质净电荷或螺旋残基比例的影响。极性氨基酸对蛋白质的溶解度有重要影响。回文元素序列通常由含有一个或两个极性氨基酸的多个重复单元组成,具有正电荷或负电荷,有报道称其能够促进蛋白质折叠,且通常小于15个残基。
基于以上原则我们首先设计了一种五肽标签,其中两端和中间(即1、3、5位氨基酸)均为不带电的甘氨酸(G),其余(即2、4位氨基酸)为甘氨酸(G)、组氨酸(H)、谷氨酸(E)、天冬氨酸(D)、赖氨酸(K)、精氨酸(R)中任意一种或两种的随机组合,具体为下列之一:GDGDG、GDGEG、GDGRG、GDGKG、GDGGG、GEGEG、GEGKG、GEGGG、GEGRG、GEGDG、GKGKG、GKGDG、GKGEG、GKGGG、GKGHG、GKGRG、GRGRG、GRGDG、GRGEG、GRGKG、GRGGG、GGGKG、GGGEG、GHGHG、GGGKG。
其次,设计连接肽,氨基酸序列为下列之一:GS、GGS、GGGS、GGGGS。
最后设计下列肽链延长的多肽标签及含有连接肽的多肽标签:GKGKGKG,GKGKGKGKG,GKGKGKGKGKG,GKGKG-GS,GKGKG-GGS,GKGKG-GGGS,GKGKG-GGGGS。
2、根据专利申请(CN104212784A)从敏捷食酸菌(Acidovorax facilis ZJB09122)中得到含表达载体pET-28b(+)的重组大肠杆菌BL21(DE3)/pET28b(+)-AcN2,再根据专利申请(CN107177576A)制备E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G。
提取E.coli BL21(DE3)/pET28b(+)-AcN-T151V/C223A/C250G中的重组质粒pET28b(+)-AcN-T151V/C223A/C250G,即为重组质粒pET-28b(+)/AcN-M,其中AcN-M核苷酸序列为SEQ ID NO.2所示,氨基酸序列为SEQ ID NO.1所示。
以含有SEQ ID NO.1所示腈水解酶(AcN-M)的编码基因的重组质粒pET-28b(+)/AcN-M为模板,以含步骤1所述多肽标签的上下游引物(核苷酸序列如下表1所示),进行PCR扩增反应,步骤1设计的多肽标签通过PCR扩增直接加到腈水解酶基因的N端,PCR扩增产物通过凝胶电泳验证PCR是否成功,随后以1μL/50μL扩增体系的量加入dpn I,37℃反应30min消化模板,取25μL测序验证(杭州擎科生物科技有限公司),获得含重组质粒pET-28b(+)/tag-AcN-M的PCR产物,tag代表多肽标签,见表1。
PCR体系为:25μL 2×Phanta Max Buffer(PCR体系缓冲液),1μL d NTP Mix(dATP,dCTP,dGTP,dTTP),1μL模板,1μL上游引物,1μL下游引物,1μL Phanta Max Super-Fidelity DNAPolymerase(高保真DNA聚合酶),20μL dd H2O,总体积为50μL。
PCR反应条件为:预变性95℃,5min;30个循环:变性95℃,30s,退火55-65℃,1min,延伸72℃,5.5min;72℃10min。
表1含多肽标签的引物
实施例2:重组大肠杆菌的构建
采用Axygen clean-up试剂盒(购自康宁生命科学(吴江)有限公司)对实施例1中含重组质粒pET-28b(+)/tag-AcN-M的PCR产物进行纯化(clean-up),具体操作为:向实施例1的5μL PCR产物中加入三倍体积的PCR-A buffer充分混匀,移到制备管中,12000rpm离心1min,弃滤液,向制备管中加入700μL W2 buffer,12000rpm离心1min,弃滤液,W2 buffer洗涤2次;取5μL加到预先解冻的感受态细胞E.coli BL21(DE3),冰浴30min,之后42℃热击90s,再次冰浴3-5min,加入700μL LB液体培养基,37℃孵育1h。取500μL培养液接种到具有0.5μg/mL卡那抗性的LB固体培养基上,涂布均匀为止,37℃培养12-14h,挑菌测序验证,获得重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M。同样条件下,构建原始菌株E.coli BL21(DE3)/pET-28b(+)/AcN-M。
实施例3:重组大肠杆菌表达腈水解酶
1、静息细胞:将保藏在-80℃冰箱中由实施例2构建的重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M菌株取出,接种至含0.5μg/mL卡那抗性的LB平板划线,37℃培养12-14h后,获得单菌落。挑取单菌落到10mL含0.5μg/mL卡那霉素的LB试管培养基中,37℃培养8h后,转接2mL至100mL含0.5μg/mL卡那霉素的发酵培养基中,37℃培养2h后,加入100μL IPTG(终浓度0.1mM),28℃诱导产酶12-14h。12,000转离心10min,收集得到的菌体,用0.9%的生理盐水清洗2次后悬浮,获得静息细胞悬液,测定相对酶活。
2、纯酶:取步骤1方法制备的1g静息细胞均匀悬浮于10mL、0.2M pH 7.0Na2HPO4-NaH2PO4缓冲液中,在冰浴条件下,进行超声波细胞破碎,超声波细胞破碎仪功率设定为40W,工作1s,间隔1s,总破碎时间为20min。随后,将细胞破碎液在12,000×g,4℃离心15min,去除细胞碎片,收集粗酶液,采用BCA试剂盒检测蛋白含量,即为总蛋白量。
将Ni柱用上样缓冲液(Binding buffer:50mM NaH2PO4,300mM NaCl,50mM咪唑,pH8.0)进行平衡,流速为2mL/min。随后,以2mL/min的流速将粗酶液上样,使用上样缓冲液洗脱杂蛋白以及弱吸附蛋白。最后,使用洗脱缓冲液(Elution buffer:50mM NaH2PO4,300mMNaCl,500mM咪唑,pH 8.0)进行洗脱,洗脱速度为3mL/min,以蛋白纯化仪(Bio-RadBioLogic LP层析系统)UV参数为准,当UV≥2时,进行酶液的收集,当UV≤2时,结束收集。接着使用透析袋(上海雷布斯生物科技有限公司MD34-3500)在50mM pH 7.0Na2HPO4-NaH2PO4缓冲液中透析过夜,取截留液,即为腈水解酶纯酶,冰浴保存备用,采用BCA试剂盒检测蛋白含量,即为上清液蛋白量。
溶解度(%)=上清液蛋白量/总蛋白量×100%。
表2含不同多肽标签的重组菌株的相对酶活及溶解度的对比
根据表2,相对酶活结果显示重组大肠杆菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的酶活是原始菌株的2.37倍,且标签的插入几乎不影响大肠杆菌的正常生长。蛋白电泳实验如图3所示,重组菌株的可溶性表达(上清液蛋白量/总蛋白量)显著增强,原始菌株的溶解度仅为53.6%,而重组菌株溶解度达到87.9%。
4、多肽标签序列的长度对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/tag-AcN-M溶解度的影响
表3含不同长度多肽标签重组菌株的相对酶活及溶解度的对比
根据表3,多肽标签中的极性氨基酸对溶解度的影响改变较为明显,其中含有3个赖氨酸的多肽标签的腈水解酶溶解度达到了90.9%,而其他长度标签的腈水解酶溶解度变化不大。
5、多肽标签与目的基因之间的连接肽(Linker)对原始菌株及重组菌株E.coliBL21(DE3)/pET-28b(+)/tag-AcN-M溶解度的影响
表4含不同Linker的重组菌株的相对酶活及溶解度的对比
根据表4,Linker对多肽标签和目的基因的作用是很重要的,其中Linker的长度越长,腈水解酶的溶解度随之增加,但对其催化活力的抑制作用越来越强,且最长的Linker和多肽标签组合的重组菌株细胞的催化活力低至59.6%。
根据表2-表4,选取重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M进行后续试验。
实施例4:温度对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M细胞酶活的影响
将200mM pH 7.0的Na2HPO4-NaH2PO4缓冲液900μL与100μL实施例3方法制备的静息细胞悬液混合后,构成1ml反应体系,使得反应体系中静息细胞加入量为10g/L,置于设定温度的振荡反应器上保温10min,温度分别为20℃、25℃、30℃、35℃、40℃、45℃、50℃、55℃、60℃、65℃和70℃,之后加入终浓度为0.2M的1-CN,在转速为800rpm的振荡反应器上反应10min后,取样离心,取上清,使用HPLC分析上清液中1-CA浓度。同样条件下,以原始菌株E.coli BL21(DE3)/pET-28b(+)/AcN-M为对照,结果如图4所示,重组菌株的最适温度为55℃,相比于原始菌株并没有发生改变,并且在相同温度下的,重组菌株的细胞酶活高于原始菌株。
实施例5:pH对原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M细胞酶活的影响
将实施例3方法制备的100μL静息细胞悬液加入900μL不同pH值的缓冲液(0.1M pH为4.0-6.0的柠檬酸-柠檬酸钠缓冲液;0.2M pH为6.0-8.0的Na2HPO4-NaH2PO4缓冲液;50mMpH为9.0-10.0的Glycine-NaOH缓冲液)中,构成1mL反应体系,使得反应体系中静息细胞加入浓度为10g/L,在35℃振荡反应器上预热10min,之后加入终浓度为0.2M的1-CN,800rpm,35℃反应10min。取样12,000rpm离心5min,取上清,使用HPLC分析上清液中1-CA浓度。同样条件下,以原始菌株E.coli BL21(DE3)为对照,结果如图5所示,重组菌株的最适pH为8.0,相比于原始菌株并没有发生改变,并且在相同pH下的,重组菌株的细胞酶活均高于原始菌株。
实施例6:原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的细胞催化效率比较
将实施例3方法制备的重组菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的湿菌体5g悬浮于100mL Na2HPO4-NaH2PO4缓冲液中(0.2M,pH=7.0),分别加入1.48g和2.2g 1-CN(终浓度分别1M和2M),35℃恒温水浴反应12h。每隔30min取样,12,000rpm离心5min,取上清,使用HPLC分析上清液中1-CA浓度,并检测1-CN的浓度。同样条件下,以原始菌株E.coliBL21(DE3)-AcN-M为对照,结果如图6所示,在催化1M 1-CN时,重组菌株所需的反应时间为2h,而原始菌株的反应时间需2.5h,且转化率两者都达到了99%以上;然而在2M底物时,由于底物浓度过高,反应12h后,原始菌株对底物的转化率为77.5%,而重组菌株由于有效蛋白量的增加,相同条件下转化率达到了81.7%。
实施例7:利用全细胞催化生产的转化液中1-氰基环己基乙酸制备加巴喷丁内酰胺
将实施例6中获得转化液进行离心(8000rpm 10min)去除菌体细胞,收集的滤液即为1-氰基环己基乙酸,取150mL滤液于500mL加氢反应釜,加入1.5g雷尼镍(型号为RTH-4110)、1mL三乙胺(分析纯)和500μL甲酸(分析纯);通入氮气置换空气,如此重复3次,保证釜内无空气存在;再次通入氢气(保持反应过程中压力在2Mpa),1000rpm反应8h后结束;冷却之后过滤回收雷尼镍,得到的滤液加入等体积的二氯甲烷进行萃取,静置分层取有机相40℃下进行旋蒸得到固体即为加巴喷丁内酰胺,二氯甲烷可回收再利用。实验结果表明全细胞催化得到的转化液可直接用于下一步加氢反应,底物转化率达到了99.6%,加巴喷丁内酰胺得率得到95.8%,底物转化率和产物得率得到工业生产化学品要求。
实施例8:利用加氢得到加巴喷丁内酰胺制备医药化学品-加巴喷丁
取实施例7中制备得到的加巴喷丁内酰胺76.7g固体溶解于500mL 6M HCl中加热回流2.5h,待冷却至室温后加入等体积的二氯甲烷进行萃取,静置分层后取水相在0-4℃下进行结晶,抽滤得到的白色固体用丙酮研磨,过滤除去丙酮并在40℃烘干得到加巴喷丁盐酸盐,将得到的所有的加巴喷丁盐酸盐溶于500mL水中,加热至40℃,300rpm搅拌下充分溶解,使用6M NaOH调pH至7.0-7.5;后加入125mL甲苯,500rpm搅拌30min;结束搅拌于0-4℃下进行结晶,过滤得到白色固体即为加巴喷丁粗品,粗品用60%甲醇或异丙醇重结晶并烘干即为加巴喷丁。以上实验涉及到萃取、抽滤、过滤操作中未用到的样品、使用后的试剂均可进行回收利用。实验结果表明加巴喷丁盐酸盐的收率达到了81%,重结晶得到的加巴喷丁收率达到73.6%,并且经过母液重复回收3-5次之后的加巴喷丁收率达到了93.2%。中间体及最终加巴喷丁的收率均达到了一个较高的水准,并且多次样品及试剂回收步骤降低了成本和废水的产生,满足绿色化学的理念,实现了医药化学品的化学-酶法高效生产。
实施例9:多肽标签在其他来源腈水解酶上的应用效果
将实施例1优选的多肽标签GKGKG按照实施例1的方法连接到腈水解酶LNIT5(Accession No.:AAR97494.1),腈水解酶No.385,386(Accession No.:AY487562)及来源于R.rhodochrous K22(Accession No.:Q02068.1)的腈水解酶(以下简称RrNit)的N末端。按实施例3方法测定溶解度及相对细胞酶活。
实验结果如下表5,三种不同的腈水解酶的溶解度均有不同程度的提升,其中LNIT5提升最多,达到了1.9倍;并且三种腈水解酶以1-CN为底物时,细胞酶活提升水平均超过150%,充分说明该多肽标签对其他来源的腈水解酶的普适性。
表5不同来源腈水解酶相对酶活及溶解度的对比
实施例10原始菌株及重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M催化合成氯吡格雷中间体(邻氯扁桃酸)的效率比较
将实施例3方法制备的重组菌株E.coli BL21(DE3)/pET-28b(+)/GKGKG-AcN-M的湿菌体5g悬浮于100mL Na2HPO4-NaH2PO4缓冲液中(0.2M,pH=7.0),分别加入终浓度分别1M和2M邻氯扁桃腈,35℃恒温水浴反应12h。每隔30min取样,12,000rpm离心5min,取上清,使用HPLC分析上清液中邻氯扁桃酸浓度,并检测邻氯扁桃腈的浓度。同样条件下,以原始菌株E.coli BL21(DE3)-AcN-M为对照,结果如表6所示,在催化1M邻氯扁桃腈时,重组菌株所需的反应时间为3h,而原始菌株的反应时间需4h,且转化率两者都达到了99%以上;然而在2M底物时,由于底物浓度过高,反应12h后,原始菌株对底物的转化率为60.4%,而重组菌株由于有效蛋白量的增加,相同条件下转化率达到了79.4%。
表6原始菌株及重组菌株催化合成氯吡格雷中间体的效率比较
实施例11优选多肽标签在脱酰基酶中转化棘白菌素B制备棘白菌素B母核的应用效果
将实施例1优选的多肽标签GKGKG按照实施例1的方法连接到脱酰基酶(NC_001136.10)上,构建重组菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-DEA(脱酰基酶),对其溶解度及酶活进行测定,含多肽标签的脱酰基酶较不含多肽标签的脱酰基酶溶解度提升2.8倍且比酶活提升358.5%。
将实施例10中催化剂改为50g/L的重组菌E.coli BL21(DE3)/pET-28b(+)/GKGKG-DEA(脱酰基酶)静息细胞,底物改为终浓度2g/L棘白菌素B,反应时间改为24h,其他同实施例10,底物转化率达到60.6%,而不含多肽标签的脱酰基酶的转化率仅为35.7%。说明此多肽标签具有一定的扩展性,但在其他酶上增溶效果的优良与否需要进行深入的探讨。
虽然本发明已以较佳实例公开如上,但其并非用以限定本发明,任何熟悉此项技术的人,在不脱离本发明的精神和范围内,都可做各种的改动和修饰,因此本发明的保护范围应该以权力要求书所界定的为准。
序列表
<110> 浙江工业大学
<120> 一种含多肽标签的重组酶及其在医药化学品合成中的应用
<160> 2
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 敏捷食酸菌(Acidovorax facilis )
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Met Val Ser Tyr Asn Ser Lys Phe Leu Ala Ala Thr Val Gln Ala Glu
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Pro Val Trp Leu Asp Ala Asp Ala Thr Ile Asp Lys Ser Ile Gly Ile
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Val Phe Ile Pro Gly Tyr Pro Tyr Trp Ala Trp Leu Gly Asp Val Lys
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Tyr Ser Leu Ser Phe Thr Ser Arg Tyr His Glu Asn Ser Leu Glu Leu
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Gly Asp Asp Arg Met Arg Arg Leu Gln Leu Ala Ala Arg Arg Asn Lys
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Ile Ala Leu Val Met Gly Tyr Ser Glu Arg Glu Ala Gly Ser Arg Tyr
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Leu Ser Gln Val Phe Ile Asp Glu Arg Gly Glu Ile Val Ala Asn Arg
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Arg Lys Leu Lys Pro Thr His Val Glu Arg Thr Ile Tyr Gly Glu Gly
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Asn Gly Thr Asp Phe Leu Thr His Asp Phe Ala Phe Gly Arg Val Gly
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Gly Leu Asn Cys Trp Glu His Phe Gln Pro Leu Ser Lys Phe Met Met
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Tyr Ser Leu Gly Glu Gln Val His Val Ala Ser Trp Pro Ala Met Ser
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Pro Leu Gln Pro Asp Val Phe Gln Phe Ser Ile Glu Ala Asn Ala Thr
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Val Thr Arg Ser Tyr Ala Ile Glu Gly Gln Thr Phe Val Leu Cys Ser
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Thr Gln Val Ile Gly Pro Ser Ala Ile Glu Thr Phe Cys Leu Asn Asp
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Glu Gln Arg Ala Leu Leu Pro Gln Gly Cys Gly Trp Ala Arg Ile Tyr
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Lys
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<213> 敏捷食酸菌(Acidovorax facilis )
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atggtatctt acaactccaa atttctggct gctaccgtac aggctgaacc ggtttggctg 60
gacgcggacg caactatcga taaatctatt ggtatcatcg aggaggcggc ccagaaaggt 120
gcgtctctga ttgccttccc ggaagttttc atccctggtt acccgtattg ggcctggctg 180
ggtgacgtaa agtactccct gtccttcacc tcccgttacc acgaaaactc cctggaactg 240
ggtgacgacc gtatgcgccg tctgcaactg gctgcgcgtc gtaacaaaat cgcgctggtt 300
atgggttaca gcgagcgtga ggcaggcagc cgctacctgt cccaggtctt tatcgacgaa 360
cgtggtgaaa tcgttgctaa ccgtcgtaaa ctgaaaccaa ctcacgttga acgtacgatt 420
tatggtgaag gcaacggtac cgactttctg acgcatgact tcgcatttgg tcgtgttggt 480
ggtctgaact gctgggagca ctttcagccg ctgtccaaat tcatgatgta ctccctgggt 540
gaacaggtac acgtcgcttc ttggccggct atgtccccgc tgcaaccgga cgtgtttcaa 600
ttttccatcg aggctaatgc gaccgtaacc cgctcctatg ctattgaagg ccaaaccttc 660
gttctgtgct ctacgcaggt tatcggtccg tctgcaattg aaaccttctg tctgaacgat 720
gagcaacgtg cactgctgcc gcagggttgc ggttgggcgc gtatctacgg cccggacggc 780
agcgaactgg ccaagccgct ggctgaagac gcagagggta ttctgtacgc agaaatcgat 840
ctggaacaga ttctgctggc caaggctggc gctgatccgg ttggtcacta cagccgccct 900
gatgtcctgt ccgtgcagtt cgacccgcgt aaccacaccc cggtacaccg cattggtatc 960
gatggccgtc tggatgttaa cacgcgttcc cgtgtagaaa actttcgcct gcgtaaagca 1020
gcagaaaaag aacgtcaggc cagcaaacgt ctgggcacga aactgtttga acagtctctg 1080
ctggcggagg agccggtacc agccaaatag 1110
Claims (6)
1.一种含多肽标签的重组酶,其特征在于所述重组酶是将多肽标签连接至腈水解酶氨基酸序列的N端所得到的酶;所述多肽标签的氨基酸序列为GKGKGKGKG。
2.如权利要求1所述含多肽标签的重组酶,其特征在于所述腈水解酶的氨基酸序列为SEQ ID NO.1所示。
3.一种含权利要求1所述多肽标签的重组酶的编码基因构建的重组基因工程菌。
4.一种含权利要求1所述多肽标签的重组酶在制备加巴喷丁中间体1-氰基环已基乙酸中的应用,其特征在于所述的应用为:以含多肽标签的重组腈水解酶的编码基因的重组基因工程菌经发酵培养获得的湿菌体或湿菌体纯化后的纯酶为催化剂,以1-氰基环己基乙腈为底物,以0.2M、pH=7.0的Na2HPO4-NaH2PO4缓冲液为反应介质构成转化体系,35℃、200rpm恒温水浴反应完全,获得含1-氰基环己基乙酸的转化液,转化液分离纯化,获得1-氰基环己基乙酸;所述转化体系中,底物加入终浓度为1-2M,催化剂加入量以湿菌体重量计为50g/L。
5.如权利要求4所述的应用,其特征在于所述湿菌体按如下方法制备:将重组基因工程菌接种至LB培养基,37℃培养12-14h后,获得单菌落;挑取单菌落接种到含0.5μg/mL卡那霉素的LB试管培养基中,37℃培养8h后,以体积浓度2%的接种量转接至含0.5μg/mL卡那霉素的发酵培养基中,37℃培养2h后,加入终浓度0.1mM的IPTG,28℃诱导产酶12-14h,12,000转离心10min,沉淀用0.9%的生理盐水清洗2次,收集湿菌体。
6.一种含权利要求1所述多肽标签的重组酶在制备氯吡格雷中间体邻氯扁桃酸中的应用,其特征在于所述的应用为:将含多肽标签的重组腈水解酶的基因工程菌经发酵培养获得的湿菌体为催化剂,以邻氯扁桃腈为底物,以0.2M、pH7.0的Na2HPO4-NaH2PO4缓冲液为反应介质构成反应体系,35℃恒温水浴反应12h,获得含邻氯扁桃酸的反应液,分离纯化,得到邻氯扁桃酸;所述反应体系中,催化剂加入量以湿菌体重量计为50g/L,所述底物加入终浓度为1-2M。
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