CN113930415A - 一种卤醇脱卤酶突变体及其应用 - Google Patents
一种卤醇脱卤酶突变体及其应用 Download PDFInfo
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- CN113930415A CN113930415A CN202111300094.0A CN202111300094A CN113930415A CN 113930415 A CN113930415 A CN 113930415A CN 202111300094 A CN202111300094 A CN 202111300094A CN 113930415 A CN113930415 A CN 113930415A
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- mutant
- halohydrin dehalogenase
- ala
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- halohydrin
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- 229910052736 halogen Inorganic materials 0.000 title description 2
- 150000002367 halogens Chemical class 0.000 title description 2
- 108010013164 halohydrin dehalogenase Proteins 0.000 claims abstract description 37
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- 238000005695 dehalogenation reaction Methods 0.000 claims abstract 2
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Abstract
本发明公开了一种卤醇脱卤酶突变体以及合成抗真菌药物关键手性环氧中间体的应用。本发明涉及基因工程和酶工程领域,具体涉及一种立体选择性翻转的卤醇脱卤酶突变体N176G,其对应的氨基酸序列如SEQ ID No.4所示。该卤醇脱卤酶突变体可应用于催化化合物I不对称脱卤合成手性环氧化物II,分离收率为80%,光学纯度高达98%ee。
Description
技术领域
本发明属于生物技术领域,特别涉及一种卤醇脱卤酶突变体及其应用。
背景技术
近20年来,随着免疫缺陷患者增多、器官移植和介入性诊断与治疗的广泛开展、以及抗菌药物和化疗药物的广泛应用,真菌感染的发生率正逐年上升。真菌感染严重威胁人类的健康生活,对于免疫系统遭到破坏的病人来说,真菌感染尤为严重。目前临床使用的抗真菌药物主要有多烯类、三唑类、咪唑类、棘白菌素类、吗啉类以及氟胞嘧啶等。以氟康唑为代表的三唑类抗真菌药物的作用机制为选择性抑制真菌甾醇-14α-去甲基酶,使细胞膜麦角固醇合成受阻。此外甾醇-14-去甲基酶受抑,14α-甲基甾醇在真菌细胞内浓集而损伤真菌的一些酶(如ATP酶、电子转运有关的酶)的功能。基于以上作用机制,达到治疗真菌感染的目的。
三唑类抗真菌药物对真菌的抗菌谱广,对浅部真菌和深部真菌均有效,很少产生耐药性。与咪唑类抗真菌药物相比,三唑类抗真菌药物对真菌细胞色素P450酶的选择性较咪唑类高,对人的毒性作用较小,疗效较好。如最新上市的三唑类抗真菌药艾沙康唑(isavuconazonium)是由安斯泰来制药公司(Astellas)和巴塞利亚制药公司(Basilea)联合开发,于2015年3月6日被美国FDA批准上市,用于成人侵袭性曲霉菌病和毛霉菌病感染的治疗,在欧盟和美国,艾沙康唑均被授予孤儿药地位。除此之外,其他已上市的三唑类抗真菌药物还有雷夫康唑(Ravuconazole)、伏立康唑(Voriconazole)、伊曲康唑(Itraconazole)、泊沙康唑(Posaconazole)等。
手性环氧化合物II是合成手性三唑类抗真菌药物的关键手性中间体。目前化合物II的合成主要是通过动力学拆分获得,理论收率低,合成工艺涉及的反应步骤冗长,分离纯化过程复杂(Tetrahedron Lett.2001,42:3331–3333;Tetrahedron,2004,60:601-605;Chin.J.Chem.2013,31:1139—1143)。因此,针对三唑类抗真菌药物的关键手性中间体化合物II的合成,开发高效、绿色和简便的制备工艺对于发展药物绿色制造技术以及新型三唑类抗真菌药物的研发具有重要的应用价值。
发明内容
本发明意在提供一种卤醇脱卤酶突变体以及该突变体催化前手性化合物I不对称合成手性环氧化合物II的应用。
一种卤醇脱卤酶突变体N176G,其氨基酸序列如SEQ ID No.4所示。
本方案中的一种卤醇脱卤酶突变体N176G是在来源于放射形土壤杆菌(Agrobacterium radiobacter)卤醇脱卤酶(核苷酸序列如序列SEQ ID No:1所示,氨基酸序列如SEQ ID No:2所示)为亲本酶,利用其基因的表达质粒为模板,设计并合成对应的PCR引物,利用点饱和突变技术,构建突变文库,结合筛选方法,获得立体选择性逐步翻转的优良突变体。经过三轮突变与筛选,最终获得卤醇脱卤酶突变体N176G。具体是将SEQ ID No.2所示氨基酸序列的第176位天冬氨酸突变为甘氨酸既得卤醇脱卤酶突变体N176G。
本申请文本中所用的术语“卤醇脱卤酶突变体N176G”是指一种以SEQ ID No:2所示氨基酸序列为参考序列,将SEQ ID No.2所示氨基酸序列的第176位的天冬酰胺突变为甘氨酸。
本发明所述的卤醇脱卤酶突变体N176G可以以工程菌全细胞形式使用。如果需要,也可以是经部分纯化或完全纯化的酶的形式使用,还可以利用本领域已知的固定化技术将本发明的卤醇脱卤酶突变体N176G制成固定化酶或者固定化细胞形式的固化酶。
本发明还提供一种所述卤醇脱卤酶突变体N176G制备手性环氧化合物II的应用。所述的合成应用为:以含卤醇脱卤酶突变体N176G编码基因,基因序列如SEQ ID No:3所示的重组基因工程菌经诱导培养获得的湿菌体或湿菌体经冷冻干燥获得的干细胞作为催化剂;以前手性化合物I为底物,以pH 7.5,150mM的磷酸盐缓冲液为反应介质,在35℃,250rpm条件下进行反应,反应结束后,将反应液分离纯化,获得对应的S构型的手性环氧化合物II,分离收率为80%,光学纯度为98%ee。
具体的,卤醇脱卤酶突变体N176G催化剂按如下方法制备:将含有卤醇脱卤酶突变体N176G编码基因的重组基因工程菌接种至含有终浓度为50μg/L硫酸卡那霉素的LB液体培养基中,37℃、250rpm培养8h;培养液以体积浓度2%接种量接种到新鲜的含有终浓度50μg/mL卡那霉素的TB液体培养基中,37℃、250rpm振荡培养2.5h左右,至菌液OD600=0.6~0.8后,加入终浓度为0.2mM的IPTG,28℃、250rpm诱导培养12h,取诱导培养后的菌液于4℃、8000rpm离心5min,收集到的湿菌体或湿菌体经冷冻干燥获得的干细胞作为所述催化剂。
与现有技术相比,本发明的有益效果主要体现在:本发明提供了一种高立体选择性的卤醇脱卤酶突变体,并提供以该卤醇脱卤酶突变体为生物催化剂,合成手性三唑类抗真菌药物的关键手性环氧中间体化合物II的新工艺。该酶催化不对称技术路线的反应条件温和,环境友好,选择性高,催化剂廉价易得,分离纯化简便,光学纯度为98%ee,理论产率(100%)与实际分离收率(80%)均较高。
附图说明
图1:以卤醇脱卤酶突变体N176G为催化剂合成手性环氧化合物II的技术路线;
图2:反应制备的手性环氧化合物II的核磁共振氢谱谱图;
图3:反应制备的手性环氧化合物II的核磁共振碳谱谱图;
图4:反应制备的手性环氧化合物II的光学纯度手性气相分析谱图。
具体实施方式
下面通过具体实施方式进一步详细说明:
在本申请文本中所用的氨基酸三字母或单字母表达方式,采用IUPAC规定的氨基酸代码(Eur.J.Biochem.,138:9-37,1984)。
实施例1:卤醇脱卤酶的突变与筛选
设计对第176位天冬酰胺进行点饱和突变体的引物:N176-F(TAATACGACTCACTATAGGG)和N176-R(TCGCTTGGAACATTATTAAC)。提取含有亲本卤醇脱卤酶基因的质粒作为DNA模板,以N176-F和N176-R为引物进行PCR扩增。PCR体系(50μL):5×Plusbuffer试剂10μL,100μM浓度的N176-F和N176-R各2μL,模板质粒2μL,dNTP试剂4μL,PrimeSTAR DNA聚合酶0.5μL,去离子水29.5μL。PCR程序:第一步,95℃/5min;第二步,98℃/10s,60℃/5s,72℃/6min,共30个循环;第三步72℃/20min。PCR产物用DpnI消化后直接通过热击(42℃,90s)方法转化到大肠杆菌BL21(DE3)感受态细胞。经复苏培养(37℃,1h)后,均匀涂布于含有硫酸卡那霉素抗生素的LB固体培养基上,在37℃条件下培养过夜。挑取单克隆菌落,进行发酵培养、诱导,获得含卤醇脱卤酶突变体的细胞,以此作为生物催化剂。考察其催化化合物I合成手性环氧化合物无II的立体选择性,将立体选择性翻转优良的突变体送去基因测序,获得优良卤醇脱卤酶突变体的准确突变信息及氨基酸序列。如下表1所示,在本轮突变与筛选实施例中,获得优良卤醇脱卤酶突变体N176G,其催化化合物I得到化合物II的光学纯度由15%ee(S)提高至98%ee(S),转化率由40%提高至>99%。
表1:
实施例2:卤醇脱卤酶突变体N176G全细胞生物催化剂的制备
将卤醇脱卤酶突变体N176G工程菌株接种到含有50μg/mL硫酸卡那霉素的30mL LB液体培养基中,37℃培养8h,得到种子液。按2%体系比将种子液转入新鲜无菌的100mL TB液体培养基中,加入硫酸卡那霉素至终浓度为50μg/mL。将培养物置于37℃条件下培养2.5h左右,加入终浓度为0.2mM的IPTG(异丙基-β-D-硫代半乳糖苷),28℃条件下诱导培养12h。使用离心机收集培养物(离心条件为4℃、8000rpm,10min),获得含有卤醇脱卤酶突变体N176G蛋白的重组全细胞,用于后续生物催化反应。
实施例3:卤醇脱卤酶突变体N176G全细胞催化合成手性环氧化合物II
以卤醇脱卤酶突变体N176G为催化剂合成手性环氧化合物II的技术路线如图1所示。
向250mL规格的三角烧瓶中加入100mL含有卤醇脱卤酶突变体N176G重组细胞(5cdw/L)的磷酸盐缓冲液(150mM,pH 7.5),同时加入终浓度为20mM的化合物I。三角瓶在30℃和250rpm条件下搅拌反应5h。将反应液离心10min(4℃、8000rpm),除去菌体。分离的上清液用80mL乙酸乙酯萃取三次,将萃取的有机相分离后合并。有机相经无水硫酸钠干燥后,过滤得到滤液,再经减压蒸馏进行浓缩。浓缩液通过硅胶柱色谱进行分离,最后通过减压蒸馏得到手性环氧产物II,无色油状物,分离产率为80%,[α]D 25=+35.0(c=1.00,CH3OH)。光学纯度经手性GC分析,色谱柱BGB-175,柱温130℃保留10min,S构型II的保留时间为5.27min,R构型II的保留时间为5.93min,如图2所示,结果显示分离得到的化合物II的ee值为98%(S)。如图3和图4所示,NMR表征数据:1H NMR(400MHz,CDCl3)δ7.33(dq,J=8.2,6.4Hz,1H),6.92(t,J=8.0Hz,2H),3.97(d,J=11.7Hz,1H),3.69(d,J=11.7Hz,1H),3.21(d,J=4.7Hz,1H),3.10(d,J=4.7Hz,1H);13C NMR(100MHz,CDCl3)δ161.8(d,J=250.1Hz,1C),161.7(d,J=250.1Hz,1C),131.1(t,J=10.3Hz,1C)111.8(d,J=25.0Hz,2C),11.8(d,J=13.2Hz,1C),53.8,52.3(t,J=2.0Hz,1C),47.4.高分辨质谱表征:HRMS(ESI-TOF)Calcd.for C9H8ClF2O[M+H]+205.0226;found:205.0223.
序列表
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Claims (7)
1.一种卤醇脱卤酶突变体N176G,其特征在于其氨基酸序列如SEQ ID No.4所示。
2.根据权利要求1所述的一种卤醇脱卤酶突变体N176G的制备方法,其特征在于:将氨基酸序列如SEQ ID No.2所示的放射形土壤杆菌卤醇脱卤酶的第176位天冬氨酸突变为甘氨酸所得。
3.一种含有权利要求1所述的卤醇脱卤酶突变体N176G的编码基因,其特征在于:所述编码基因的氨基酸序列如SEQ ID No.3所示。
4.一种含有权利要求3所述编码基因的重组基因工程菌。
6.如权利要求5所述的一种卤醇脱卤酶突变体N176G的应用,其特征在于:将权利要求4所述重组基因工程菌经诱导培养获得的湿菌体或湿菌体经冷冻干燥获得的干细胞作为催化剂;以前手性化合物I为底物,在一定条件的磷酸盐缓冲液反应介质中进行反应,反应结束后,将反应液分离纯化,获得对应的S构型的手性环氧化合物II。
7.根据权利要求6所述的一种卤醇脱卤酶突变体N176G的应用,其特征在于:所述催化剂的制备方法是将含有卤醇脱卤酶突变体N176G编码基因的重组基因工程菌接种至含有硫酸卡那霉素的LB液体培养基中培养得到培养液;再将一定量的培养液接种到含有硫酸卡那霉素的TB液体培养基中,继续培养至菌液OD600=0.6~0.8后,加入一定浓度的异丙基-β-D-硫代半乳糖苷诱导培养,取诱导培养后的菌液经离心后,收集到的湿菌体或湿菌体经冷冻干燥获得的干细胞作为所述催化剂。
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