CN116265579A - 环己胺氧化酶突变体及其在制备右美沙芬中间体中的应用 - Google Patents
环己胺氧化酶突变体及其在制备右美沙芬中间体中的应用 Download PDFInfo
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Abstract
本发明属于生物制药和生物工程技术领域,具体为环己胺氧化酶突变体及其在制备右美沙芬合成中间体中的应用。本发明涉及环己胺氧化酶CHAOCCH12‑C2突变体及其编码基因,突变体的获得方法,含有环己胺氧化酶突变体基因的重组表达载体及重组表达转化体,其重组酶和该重组酶的制备方法,以及环己胺氧化酶CHAOCCH12‑C2突变体在制备右美沙芬合成中间体(S)‑1‑(4‑甲氧基苄基)‑1,2,3,4,5,6,7,8‑八氢异喹啉中应用;本发明的环己胺氧化酶突变体与野生酶相比,氧化能力明显提高,可实现对高底物浓度消旋品胺的动态动力学拆分,高效合成右美沙芬合成中间体,具有良好的工业应用前景。
Description
技术领域
本发明属于生物制药和生物工程技术领域,具体涉及环己胺氧化酶CHAOCCH12-C2突变体及其编码基因,并且涉及含有所述环己胺氧化酶突变体基因的工程菌的制备方法,以及该基因工程菌为生物催化剂,结合使用非选择性还原剂,动态动力学拆分消旋品胺制备(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉((S)-I)反应中的应用。
背景技术
(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉是右美沙芬工业生产中的关键合成中间体。相较于化学方法,生物催化合成具有其自身的特点,包括优异的立体选择性、高分离产率、温和的反应条件和环境友好性等。朱敦明课题组使用来自氧化短杆菌IH-35A的环己胺氧化酶CHAOIH-35A的Y321I突变体以及来自马齿菌的亚胺还原酶,在半制备级规模下(0.5-1mmol)不对称合成(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉,均具有较高的立体选择性(>98%ee)(Scientific Report,2016,6,24973-24981;Adv.Synth.Catal.,2019,361,556-561]。然而,这些研究所报道的低底物浓度(10mM)限制了其实际工业应用潜力。我们前期通过基因挖掘及筛选获得一个新环己胺氧化酶CHAOCCH12-C2(J.Org.Chem.,2020,85,5598-5614.中国发明专利申请201910890595.5)。结合非选择性还原剂,该酶亦可实现对(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉的动态动力学拆分合成。然而,酶活测定表明野生型CHAOCCH12-C2对于底物(rac)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉的酶活仅为0.083U/mg,远低于该酶对其天然底物环己胺的底物(2.16U/mg)。因此,提高该环己胺氧化酶对底物(rac)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉的酶活对于实现其工业应用至关重要。
将基于结构的半理性设计与随机突变相结合,对来源于赤杆菌Erythrobacteraceae bacterium CCH12-C2的环己胺氧化酶CHAOCCH12-C2全酶进行分子改造,获得对rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I)氧化能力显著提升的突变体,并将其应用于高底物浓度rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I)的动态动力学拆分,此方法具有良好的工业应用价值。
发明内容
本发明所要解决的技术问题是,针对野生型环己胺氧化酶CHAOCCH12-C2动态动力学拆分反应中底物浓度低的问题,提供一种可有效提高催化活力的CHAOCCH12-C2突变体蛋白质、编码该突变体蛋白质的核酸序列、含有该核酸序列的重组表达载体,以及含有所述环己胺氧化酶突变体基因的工程菌的制备方法,还提供其在动态动力学拆分rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I),制备(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉((S)-I)中的应用。
本发明首先提供一种对rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I)氧化能力提高(即催化活力提高)的环己胺氧化酶CHAOCCH12-C2突变体蛋白质。
野生型CHAOCCH12-C2的氨基酸序列如SEQ ID NO.2所示,对如SEQ ID NO.2所示氨基酸序列的蛋白质进行突变,同时或分别将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸;
进一步地,在此基础上,且保持酶催化活力的前提下,对如SEQ ID NO.2所示的氨基酸序列的蛋白质的200、201及209位之外的一个或几个位点经过氨基酸残基替换,获得的由CHAOCCH12-C2衍生的具有环己胺氧化酶活性的突变体蛋白质。
尤其是,对第68位的组氨酸及第198位的谷氨酸分别或共同进行氨基酸残基替换,获得由CHAOCCH12-C2衍生的具有环己胺氧化酶活性的突变体蛋白质。
优选的,所述环己胺氧化酶突变体,为野生环己胺氧化酶CHAOCCH12-C2的氨基酸残基发生如下任意一种情况的突变所获得的突变体:
(1)野生环己胺氧化酶CHAOCCH12-C2的氨基酸序列中,同时将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸,命名为CHAOCCH12-C2 L200V/I201L/V209S;
(2)野生环己胺氧化酶CHAOCCH12-C2的氨基酸序列中,同时将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸,且第68位组氨酸替换为谷氨酰胺,命名为CHAOCCH12-C2 H68Q/L200V/I201L/V209S;
(3)野生环己胺氧化酶CHAOCCH12-C2的氨基酸序列中,同时将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸,且第68位组氨酸替换为谷氨酰胺,且第198位谷氨酸替换为甘氨酸,命名为CHAOCCH12-C2 H68Q/E198G//L200V/I201L/V209S。
本发明中,突变体CHAOCCH12-C2 L200V/I201L/V209S所示的氨基酸序列组成的蛋白质是从基于CHAOCCH12-C2所构建的组合活性中心饱和突变文库中筛选得到。在筛选获得催化活力提高的突变体CHAOCCH12-C2 L200V/I201L/V209S的基础上,以突变体CHAOCCH12-C2 L200V/I201L/V209S的基因序列为模板构建随机突变文库,以进一步提高该酶的催化活性。
本发明中,野生环己胺氧化酶CHAOCCH12-C2基因,其序列如SEQ ID NO.1所示。
本发明还包括获得所述野生型环己胺氧化酶突变体的方法,具体为:
以含有来源于赤杆菌Erythrobacteraceae bacterium CCH12-C2的野生环己胺氧化酶CHAOCCH12-C2基因的重组载体pET28a-CHAOCCH12-C2为模板,利用含有组合活性中心饱和突变碱基信息的突变引物,通过PCR方法扩增,得到突变文库,转化后筛选得到突变体CHAOCCH12-C2 L200V/I201L/V209S;
再以含有突变体CHAOCCH12-C2 L200V/I201L/V209S基因的重组表达载体pET28a-CHAOCCH12-C2 L200V/I201L/V209S为模板,利用随机突变引物进行易错PCR构建随机突变文库,转化后筛选得到含有突变体CHAOCCH12-C2 H68Q/L200V/I201L/V209S;
再以重组表达载体pET28a-CHAOCCH12-C2 H68Q/L200V/I201L/V209S为模板,进行第二轮随机突变,对随机突变文库进行筛选,得到突变体CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S,随后利用定点突变,将第198位氨基酸替换为其余氨基酸,筛选得到催化活力明显提高的突变体CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S。
其中所述组合活性中心饱和突变为本领域常规技术,PCR反应的体系(50μL)为:模板50ng,FastPfu Fly Buffer,4μL dNTP(各2.5mM),一对突变引物各1μL(10μM),10μL5×PCR Stimulant,0.5μLMgSO4(50mM),2.5个单位的TransStart FastPfuFly DNA polymerase,加无菌蒸馏水至50μL。
所述组合活性中心饱和突变PCR扩增程序为:(1)98℃变性3min;(2)98℃变性20s,(3)65℃退火30s,(4)72℃延伸8min,步骤(2)-(4)共进行20个循环,最后72℃延伸10min,4℃保藏产物。
扩增得到的PCR产物经内切酶DpnI 37℃消化1小时后转化E.coli DH5,涂布于含卡那霉素(50μg/mL)的LB固体培养基,于37℃培养过夜,挑选阳性克隆子,接种于含卡那霉素(50μg/mL)的LB培养基,培养约8小时,提取质粒,测序,对突变文库质量进行评估。将质量合格的突变文库保藏于-20℃,用于后续突变文库的筛选。
其中所述的易错PCR扩增为本领域常规技术,PCR反应的体系(50μL)为:模板50ng,5μL10×TaqBuffer,0.5μL dTTP(100mM),0.5μL dCTP(100mM),0.5μL dATP(20mM),0.5μLdGTP(20mM),一对突变引物各1μL(10μM),0.5μLMgCl2(700mM),0.375μLMnCl2(10mM),2.5个单位的Taq DNA polymerase,加无菌蒸馏水至50μL。
所述易错PCR扩增程序为:(1)95℃变性3min;(2)94℃变性1min,(3)65℃退火30s,(4)72℃延伸2min,步骤(2)-(4)共进行30个循环,最后72℃延伸10min,4℃保藏产物。
所述定点突变为本领域常规技术,PCR反应的体系(50μL)为:模板50ng, FastPfu Fly Buffer,4μL dNTP(各2.5mM),一对突变引物各1μL(10μM),10μL5×PCR Stimulant,0.5μLMgSO4(50mM),2.5个单位的TransStart FastPfu Fly DNApolymerase,加无菌蒸馏水至50μL。
所述定点突变PCR扩增程序为:(1)98℃变性3min;(2)98℃变性20s,(3)65℃退火30s,(4)72℃延伸8min,步骤(2)-(4)共进行20个循环,最后72℃延伸10min,4℃保藏产物。
扩增得到的PCR产物经内切酶DpnI 37℃消化1小时后转化E.coli DH5α,涂布于含卡那霉素(50μg/mL)的LB固体培养基,于37℃培养过夜,挑选阳性克隆子,接种于含卡那霉素(50μg/mL)的LB培养基,培养约8小时,提取质粒,测序,将点突变成功的质粒保藏于-20℃,用于后续突变体的筛选。
本发明还包括一种分离的核酸,所述核酸编码前述任一环己胺氧化酶突变体。
本发明还包括含有所述核酸的重组表达载体。
其中所述重组表达载体可通过本领域常规方法获得,即:将本发明所述的环己胺氧化酶突变体基因的核酸分子连接于表达载体上构建而成。本发明所述载体优选地为质粒pET28a。较佳地,可通过下述方法制得本发明得重组表达载体:将表达载体pET28a用限制性内切酶Nde I和Xho I双酶切;利用重组酶Exnase II将易错PCR核酸产物与双酶切处理过的表达载体进行重组,形成含有随机突变位点的重组表达载体,用于后续突变体的筛选。
本发明还包括包含所述的重组表达载体的基因工程菌。
所述基因工程菌的制备方法,较佳地为:将含上述具有突变位点的重组表达载体转化至宿主微生物中,即可得到所述基因工程菌。其中所述宿主微生物较佳地为:大肠杆菌(E.coli),更佳的为大肠杆菌BL21(DE3)或大肠杆菌DH5α。将前述重组表达质粒转化至E.coli BL21(DE3)中,即可得本发明优选的基因工程菌株,记为E.coli BL21(DE3)/pET28a-CHAOCCH12-C2 L200V/I201L/V209S、E.coli BL21(DE3)/pET28a-CHAOCCH12-C2 H68Q/L200V/I201L/V209S、pET28a-CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S等。转化方法可选择本领域常规方法,如热激法、电转法等,较佳地选择热激法进行转化即可,热激条件较佳的是:42℃,热激90秒。
培养上述基因工程菌,从培养物中获得全细胞生物催化剂。较佳的方法为:将上述重组大肠杆菌接种至含有卡那霉素(50μg/mL)的LB培养基中,37℃,150-200rpm培养,培养液的吸光密度OD600达到0.5-1.0(优选0.6),加入0.05-1.0mM(优选0.1mM)异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导,诱导温度为18℃,诱导时间18小时即可得到高效表达的重组基因工程菌全细胞生物催化剂。
本发明提供以上述基因工程菌为催化剂,用于动态动力学拆分rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I)反应;反应体系包括:底物rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I)、酶催化剂、非选择性还原剂与缓冲液;其中,酶催化剂为上述基因工程菌全细胞,或其对应的粗酶液或固定化酶;非选择性还原剂优选为硼烷氨;反应路线为:
优选地,在起始反应体系中,底物的浓度为10-500mM,更优选的为100-500mM。所述工程菌的用量(按湿菌体重量计算)为缓冲液体积的10%-25%(w/v),更优选的为15%-20%(w/v)。
优选地,在起始反应体系中,非选择性还原剂的用量为底物摩尔当量的500%-1200%,更有选的为500%-900%。所述非选择性还原剂选自硼烷氨、硼氢化钠、硼氢化钾等。
作为优选,所述反应的温度为20-50℃,更优选的为25-37℃。
作为优选,反应液的pH为6-10,更优选的为6.5-7.5。
上述反应结束后,反应液需经后处理才能获得成品,所述后处理为:用HCl(1N)将反应混合物中pH调节至偏酸性,随后用NaOH(3M)调节至偏碱性。将反应液用乙酸乙酯萃取,合并有机层,用Na2SO4干燥后浓缩,得到粗产品。粗产品通过柱层析进行纯化,得到成品。具体地:加1-6M盐酸终止反应,用1-10M氢氧化钠调节pH至10-11,用乙酸乙酯高速离心萃取3-5次;有机萃取物经无水硫酸钠干燥后,柱层析分离纯化得到产物。
本发明的环己胺氧化酶突变体与野生酶相比,对rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(rac-I)的氧化能力明显提高。本发明获得的环己胺氧化酶突变体可用于高底物浓度、高立体选择性、绿色、经济不对称合成(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉((S)-I),具有良好的工业应用前景。
附图说明
图1为环己胺氧化酶突变体CHAOCCH12-C2 L200V/I201L/V209S、CHAOCCH12-C2 H68Q/L200V/I201L/V209S、CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S及CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S的SDS-PAGE电泳图。其中,M:蛋白质Marker;1:纯化得到的环己胺氧化酶突变体CHAOCCH12-C2 L200V/I201L/V209S;2:纯化得到的环己胺氧化酶突变体CHAOCCH12-C2 H68Q/L200V/I201L/V209S;3:纯化得到的环己胺氧化酶突变体CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S;4:纯化得到的环己胺氧化酶突变体CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S。
图2为本发明实施例6中纯化得到的(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉((S)-I)的手性HPLC图。
具体实施方式
下面结合具体实施例对本发明做进一步详细地说明,但本发明并不限于以下实施例。
实施例1,组合活性中心饱和突变
组合活性中心饱和突变采用TransStart FastPfu Fly DNA polymerase完成。首先设计同时含有突变位点L200、I201及V209的饱和突变引物:
上游引物:AATCTNYTNYTGGTACCCGTAGTGGCGCCCAANYTGCTTGGTTCATCGGC,(SEQ IDNO.3)
下游引物:CAAGCARNTTGGGCGCCACTACGGGTACCARNARNAGATTCGATGCCCTC,(SEQ IDNO.4)
模板为:野生型pET28a-CHAOCCH12-C2,基因序列如SEQ ID NO.1。
PCR反应体系(50μL):模板50ng,FastPfu Fly Buffer,4μLdNTP(各2.5mM),一对突变引物各1μL(10μM),10μL5×PCR Stimulant,1μLMgSO4(50mM),2.5个单位的TransStart FastPfu Fly DNA polymerase,加无菌蒸馏水至50μL。
PCR扩增程序:(1)98℃变性3min;(2)98℃变性20s,(3)65℃退火30s,(4)72℃延伸8min,步骤(2)-(4)共进行20个循环,最后72℃延伸10min,4℃保藏产物。
扩增得到的PCR产物经内切酶DpnI 37℃消化1小时后转化于E.coliBL21(DE3),涂布于含卡那霉素(50μg/mL)的LB固体培养基,于37℃培养过夜,构建饱和突变体文库,并进行筛选。筛选得到一个催化活力提高4倍的突变体,经DNA测序发现其突变位点为L200V、I201L和V209S,命名为CHAOCCH12-C2 L200V/I201L/V209S。
实施例2,随机突变
利用易错PCR技术相环己胺氧化酶基因引入随机核苷酸突变,其中所用引物如下:
上游引物:GCAGCGGCCTGGTGCCGCGCGGCAG,(SEQ ID NO.5)
下游引物:GATCTCAGTGGTGGTGGTGGTGGTG,(SEQ ID NO.6)
模板为:突变体pET28a-CHAOCCH12-C2 L200V/I201L/V209S
PCR反应的体系(50μL)为:模板50ng,5μL10×TaqBuffer,0.5μL dTTP(100mM),0.5μL dCTP(100mM),0.5μL dATP(20mM),0.5μL dGTP(20mM),一对突变引物各1μL(10μM),0.5μLMgCl2(700mM),0.375μLMnCl2(10mM),2.5个单位的Taq DNA polymerase,加无菌蒸馏水至50μL。
易错PCR扩增程序为:(1)95℃变性3min;(2)94℃变性1min,(3)65℃退火30s,(4)72℃延伸2min,步骤(2)-(4)共进行30个循环,最后72℃延伸10min,4℃保藏产物。
将PCR扩增产物经1%琼脂糖凝胶电泳进行检测,并利用DNA凝胶纯化试剂盒回收目标片段。用Nde I和Xho I对表达载体pET28a进行双酶切,并回收产物。利用重组酶ExnaseII将易错PCR目标片段及经双酶切处理过的表达载体回收产物于37℃水浴中重组30min,转化至E.coli BL21(DE3),涂布于含卡那霉素(50μg/mL)的LB固体培养基,于37℃培养过夜,构建随机突变体文库,并进行筛选。筛选得到一个催化活力较突变亲本提高130%的突变体,经DNA测序发现其突变位点为H68Q,命名为CHAOCCH12-C2 H68Q/L200V/I201L/V209S。
以突变体pET28a-CHAOCCH12-C2 H68Q/L200V/I201L/V209S为模板,利用易错PCR技术进行第二轮随机突变,通过筛选,得到一个催化活力较CHAOCCH12-C2 H68Q/L200V/I201L/V209S高22%的突变体,其突变位点为E198A,命名为CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S。
实施例3,定点突变
定点突变采用TransStart FastPfu Fly DNA polymerase完成。首先设计含有突变点的突变引物如下:
突变E198F:
上游引物:TGAGGGCATCATGTCTGTTCTTGGTACCCGTAG,(SEQ ID NO.7)
下游引物:CAAGAACAGACATGATGCCCTCACCTTGATGC,(SEQ ID NO.8)
模板为:突变体pET28a-CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S
PCR反应体系(50μL):模板50ng,FastPfu Fly Buffer,4μLdNTP(各2.5mM),一对突变引物各1μL(10μM),10μL5×PCR Stimulant,1μLMgSO4(50mM),2.5个单位的TransStart FastPfu Fly DNA polymerase,加无菌蒸馏水至50μL。
PCR扩增程序:(1)98℃变性3min;(2)98℃变性20s,(3)65℃退火30s,(4)72℃延伸8min,步骤(2)-(4)共进行20个循环,最后72℃延伸10min,4℃保藏产物。
扩增得到的PCR产物经内切酶DpnI 37℃消化1小时后转化于E.coli BL21(DE3),涂布于含卡那霉素(50μg/mL)的LB固体培养基,于37℃培养过夜,挑选单克隆,接种于含卡那霉素(50μg/mL)的LB培养基,培养约8小时,提取质粒,测序。将测序结构与突变亲本基因序列进行比对,确认突变前后基因序列及相应的氨基酸序列的差异。
将突变体CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S中第198位谷氨酸,采用如上述同样的定点突变方法替换为其余16个氨基酸残基,并对突变体进行进一步筛选,得到一个催化活力较野生CHAOCCH12-C2提高11.6倍的突变体,包含突变位点为:H68Q/E198G/L200V/I201L/V209S,命名为CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S。
实施例4,基因工程菌的构建及诱导表达
用实施例1-3中构建的重组表达质粒转化至表达宿主E.coli BL21(DE3),转化条件为45℃热激90秒,涂布于含有卡那霉素的抗性平板上,37℃倒置培养过夜,即得阳性重组转工程菌E.coli BL21(DE3)/pET28a-CHAOCCH12-C2突变体。将工程菌接种到5mL含卡那霉素(50μg/mL)的LB液体培养基活化8h(37℃,200rpm)。取上述活化的培养物,以1/100的接种量转接到500mL含卡那霉素(50μg/mL)的LB液体培养基中培养(37℃,200rpm),当培养液的吸光密度OD600达到0.6,加入0.1mMIPTG进行诱导,诱导温度为18℃,诱导时间18小时。离心收集细胞(湿菌体),即为工程菌全细胞生物催化剂。
实施例5,环己胺氧化酶突变体的纯化和酶活性测定
将实施例4中所得环己胺氧化酶突变体细胞悬浮于重悬于Lysis缓冲液(50Mm,pH7.5NaPi缓冲液,含30mM NaCl和10mM咪唑)中。于冰浴中超声破碎15min,后于4℃下,13500rpm离心30min去除不溶物。将离心后上清液上样至含有2-3mL预先用Lysis缓冲液平衡过的Ni-NTA树脂的柱子上。随后将含有细胞裂解液及树脂的柱子置于冰浴中,于脱色摇床上摇晃吸附约30min后,弃去流出液后用2×20mL Wash缓冲液洗涤树脂(50Mm,pH7.5NaPi缓冲液,含30mM NaCl和20mM咪唑),去除非特异吸附的物质。最后用Elution缓冲液(50Mm,pH7.5 NaPi缓冲液,含30mM NaCl和250mM咪唑)将目的蛋白洗脱下来。随后将目的蛋白质溶液上样至PD-10脱盐柱(用Storage缓冲液预先平衡),用3.5mL Storage缓冲液(50Mm,pH7.5NaPi缓冲液,含30mM NaCl)将目的蛋白洗脱下来。利用消光系数及分子量,通过NanoDropOne分光光度计测量蛋白质浓度后将目的蛋白分装,于-80℃下储存。
用垂直电泳仪进行SDS-PAGE检测收集到的蛋白样品。取20μL样品(浓度为1mg/mL)加入5μL5×SDS上样缓冲液进行处理,上样量均为25μL。SDS-PAGE胶浓度为12%,选用90V电压进行浓缩,再改为120V电压进行分离电泳。结果表明,经过上述方法获得的突变体能够纯化得到高纯度蛋白(纯度>90%),分子量大小在51kDa左右。
通过检测510nm处吸光值变化的方式进行环己胺氧化酶突变体活力测定。底物胺被环己胺氧化酶氧化后可生成当量过氧化氢,在4-氨基安替比林及2,4,6-三溴-3-羟基苯甲酸存在下,辣根过氧化物酶可将其转化为一种棕红色物质,该物质特征吸收波长为510nm(ε=29.4×103mol–1Lcm-1),从而监测510nm下该棕红色物质的生成可测定环己胺氧化酶的活性。标准酶活测定体系(总体积为200μL)包括:184μL 50mM磷酸盐缓冲液,2μL 2,4,6-三溴-3-羟基苯甲酸溶液20mg/mL DMSO溶液),2μL 4-氨基安替比林溶液(15mg/mL水溶液),2μLrac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉溶液(1mol/L DMSO溶液),以及>2U辣根过氧化物酶。在适当温度预热5min后,添加10μL适量稀释后的突变体纯酶启动反应后立即监测510nm下吸光值变化。每个酶活反应重复三次,取平均值。一个酶活单位(1U)定义为每分钟生成1μmol过氧化氢所需的酶量。对野生酶及各突变体进行测活,结果参见表1。
表1CHAOCCH12-C2及其突变体的比活
实施例6,工程菌E.coli BL21(DE3)/pET28a-CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S全细胞催化不对称合成(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉((S)-I)(200mM底物浓度,克级)。
称取新鲜制备的工程菌E.coli BL21(DE3)/pET28a-CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S湿菌体6.2g(预先用磷酸盐缓冲液洗涤)加入31.1mL磷酸盐缓冲液(50mM,pH 7.0),搅拌均匀制成全细胞悬浮液。于250mL锥形瓶中称取1.60g rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉以及非选择性还原剂NH3·BH31.0g,立即加入31.1mL全细胞悬浮液。将上述反应混合物于35℃,200rpm下震荡反应3d。后用HCl(1N)将反应混合物中pH调节至5.0,随后用NaOH(3M)调节至9.0。将反应液用乙酸乙酯(3×200mL)萃取,合并有机层,用Na2SO4干燥后浓缩,得到粗产品。粗产品通过柱层析进行纯化,得到1.22g产物(S)
-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(分离收率为76%,ee值为97%)。旋光测定结果为[α]20 D=-137(c=1.0,MeOH),(R)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉的文献值为[α]20 D=139(c=1.0,MeOH)(Helv.Chim.Acta,1956,39,1376-1386.)。用手性HPLC测得产物ee值为97%,检测条件为:OJ-H色谱柱,流动相为正己烷:异丙醇:乙醇胺=95:5:0.5的溶液,流速0.5mL/min,柱温25℃,波长230nm(附图2)。1HNMR(400MHz,CDCl3)δ/ppm 7.16(d,J=8.6Hz,2H),6.87(d,J=8.6Hz,2H),3.81(s,3H),3.27(d,J=9.3Hz,1H),3.09-2.94(m,2H),2.79-2.67(m,1H),2.49(dd,J=13.6,10.6Hz,1H),2.26-2.08(m,1H),2.06-1.83(m,5H),1.83-1.48(m,5H).13C{1H}NMR(100MHz,CDCl3)δ/ppm 158.0,131.9,130.1,130.0,128.4,113.9,58.8,55.3,40.7,37.9,31.0,30.4,27.0,23.2,22.8。
实施例7,工程菌E.coli BL21(DE3)/pET28a-CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S全细胞催化不对称合成(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉((S)-I)(500mM底物浓度,克级)。
称取新鲜制备的工程菌E.coli BL21(DE3)/pET28a-CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S湿菌体3.0g(预先用磷酸盐缓冲液洗涤)加入14.8mL磷酸盐缓冲液(50mM,pH 7.0),搅拌均匀制成全细胞悬浮液。于125mL锥形瓶中称取1.90g rac-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉以及非选择性还原剂NH3·BH31.3g,立即加入14.8mL全细胞悬浮液。将上述反应混合物于35℃,200rpm下震荡反应4d。后用HCl(1N)将反应混合物中pH调节至5.0,随后用NaOH(3M)调节至9.0。将反应液用乙酸乙酯(3×200mL)萃取,合并有机层,用Na2SO4干燥后浓缩,得到粗产品。粗产品通过柱层析进行纯化,得到1.5g产物(S)-1-(4-甲氧基苄基)-1,2,3,4,5,6,7,8-八氢异喹啉(分离收率为79%,ee值为97%)。
以上所述仅为本发明的较佳实施例,只为说明本发明的技术构思和特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的任何修改、等同替换、改进等,都应涵盖在本发明的保护范围之内。
序列表
<110> 复旦大学
<120> 环己胺氧化酶突变体及其在制备右美沙芬中间体中的应用
<130> 001
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Claims (9)
1.一种环己胺氧化酶突变体,其特征在于,是对如SEQ ID NO.2所示的野生型CHAOCCH12-C2氨基酸序列的蛋白质进行如下突变得到:(a)同时或分别将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸。
2.根据权利要求1所述的环己胺氧化酶突变体,其特征在于,还包括:在(a)基础上、且保持酶催化活力的前提下,对如SEQ ID NO.2所示的氨基酸序列的蛋白质的第68位的组氨酸及第198位的谷氨酸分别或共同进行氨基酸残基替换,获得由CHAOCCH12-C2衍生的具有环己胺氧化酶活性的突变体蛋白质。
3.根据权利要求2所述的环己胺氧化酶突变体,其特征在于,为野生环己胺氧化酶CHAOCCH12-C2的氨基酸残基发生如下任意一种情况的突变所获得的突变体:
(1)野生环己胺氧化酶CHAOCCH12-C2的氨基酸序列中,同时将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸,命名为CHAOCCH12-C2 L200V/I201L/V209S;
(2)野生环己胺氧化酶CHAOCCH12-C2的氨基酸序列中,同时将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸,且第68位组氨酸替换为谷氨酰胺,命名为CHAOCCH12-C2 H68Q/L200V/I201L/V209S;
(3)野生环己胺氧化酶CHAOCCH12-C2的氨基酸序列中,同时将第200位亮氨酸替换为缬氨酸,第201位异亮氨酸替换为亮氨酸,第209位缬氨酸替换为丝氨酸,且第68位组氨酸替换为谷氨酰胺,且第198位谷氨酸替换为甘氨酸,命名为CHAOCCH12-C2 H68Q/E198G//L200V/I201L/V209S。
4.一种权利要求1-3之一所述环己胺氧化酶突变体的获得方法,其特征在于,具体为:
以含有来源于赤杆菌Erythrobacteraceae bacterium CCH12-C2的野生环己胺氧化酶CHAOCCH12-C2基因的重组载体pET28a-CHAOCCH12-C2为模板,利用含有组合活性中心饱和突变碱基信息的突变引物,通过PCR方法扩增,得到突变文库,转化后筛选得到突变体CHAOCCH12-C2 L200V/I201L/V209S;
以含有突变体CHAOCCH12-C2 L200V/I201L/V209S基因的重组表达载体pET28a-CHAOCCH12-C2 L200V/I201L/V209S为模板,利用随机突变引物进行易错PCR构建随机突变文库,转化后筛选得到含有突变体CHAOCCH12-C2 H68Q/L200V/I201L/V209S;
再以重组表达载体pET28a-CHAOCCH12-C2 H68Q/L200V/I201L/V209S为模板,进行第二轮随机突变,对随机突变文库进行筛选,得到突变体CHAOCCH12-C2 H68Q/E198A/L200V/I201L/V209S,随后利用定点突变,将第198位氨基酸替换为其余氨基酸,筛选得到催化活力明显提高的突变体CHAOCCH12-C2 H68Q/E198G/L200V/I201L/V209S。
5.一种分离的核酸,其特征在于,所述核酸编码如权利要求1或2中所述的任一环己胺氧化酶突变体。
6.一种包含如权利要求5所述的核酸的重组表达载体。
7.一种包含如权利要求6所述的重组表达载体的基因工程菌。
9.如权利要求8所述的应用,其特征在于:
在起始反应体系中,底物的浓度为10-500mM;所述工程菌的用量,按湿菌体重量计算为缓冲液体积的10%-25%(w/v);非选择性还原剂的用量为底物摩尔当量的500%-1200%,非选择性还原剂选自硼烷氨、硼氢化钠、硼氢化钾;反应温度为20-50℃;反应液的pH为6-10;反应结束后,对反应液进一步进行后处理,所述后处理为:加1-6M盐酸终止反应,用1-10M氢氧化钠调节pH至10-11,用乙酸乙酯高速离心萃取3-5次;有机萃取物经无水硫酸钠干燥后,柱层析分离纯化得到产物。
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