CN116042545A - 一种酶活性提高的谷胱甘肽双功能合成酶突变体s722a及其应用 - Google Patents
一种酶活性提高的谷胱甘肽双功能合成酶突变体s722a及其应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,具体涉及一种大肠杆菌谷胱甘肽双功能合成酶蛋白突变体、基因工程菌及应用。本发明首先对来源于嗜热链球菌的谷胱甘肽双功能合成酶进行异源表达,构建了重组大肠杆菌,对谷胱甘肽双功能合成酶蛋白进行改造,对其进行定点突变,提高了酶活。本发明成功在大肠杆菌中表达了谷胱甘肽双功能合成酶,并进一步提高了酶活,酶活提高了48.93%,有利于进一步提高谷胱甘肽的产量。通过全细胞催化,突变体S722A的谷胱甘肽产量达到20.10mmol/L,产量和转化率都有了进一步提高。
Description
技术领域
本发明涉及一种酶活性提高的谷胱甘肽双功能合成酶突变体S722A及其应用,属于基因工程和发酵工程领域。
背景技术
谷胱甘肽(glutathione,r-glutamyl cysteingl+glycine,GSH)是一种非编码的三肽硫醇,由L-谷氨酸、L-半胱氨酸和甘氨酸三种氨基酸通过肽键缩合而成,含有γ-酰胺键和巯基,存在于大多数原核生物和真核生物中,浓度为0.2-10mM。GSH的功能主要是解毒,抗氧化,维持细胞内氧化还原平衡。广泛应用于医药、食品和化妆品领域。
GSH的生物合成主要是两步依赖ATP的酶催化,γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶,其中第一个酶是限速酶,受到谷胱甘肽的反馈抑制。2005年,在无乳链球菌中首次发现了谷胱甘肽双功能合成酶(GshF),它能在ATP和Mg2+或Mn2+存在的情况下,同时催化合成GSH的上述两步反应。
从发现GSH开始,GSH的生产主要是从动植物中直接提取,后来又发展到化学合成法、酶法及微生物发酵法生产。目前应用最广泛的就是酶法及微生物发酵法。目前,利用基因工程改造的高产菌虽已初步实现GSH的工业化生产,但仍存在产物胞内抑制、分离纯化成本较高等问题。酶法生产GSH的优势在于体外体系简化了生产工艺,有效地消除了反馈抑制,使GSH的产量达到了较高水平。其中全细胞催化与游离酶或固定化酶催化相比具有以下优点:不需要酶的分离纯化,减少投资;酶的回收率高,稳定性好;不加重下游纯化的负担等等。完整的细胞结构可以增加细胞重复利用的次数,从而提高工业化生产的强度。
目前酶法合成GSH的关键在于筛选获得高性能的GSH合成酶酶系。因此,本发明通过定点突变提高GshF的酶活,提高GSH的产量。
发明内容
发明目的:目前酶法生产谷胱甘肽存在合成酶催化效率低,稳定性差的问题,为了克服现有技术存在的缺陷,本发明提供一种提高谷胱甘肽双功能合成酶活性的方法,即提出一种酶活性提高的谷胱甘肽双功能合成酶突变体S722A,提高谷胱甘肽双功能合成酶活性,并提高其酶法合成GSH的产量,对于促进谷胱甘肽的合成具有普遍的意义。
本发明的第一个目的是提供一种谷胱甘肽双功能合成酶突变体,所述突变体的氨基酸序列如SEQ ID NO.3所示。
进一步的,所述突变体是在氨基酸序列如SEQ ID NO.1所示的亲本嗜热链球菌GshF的基础上,对第722位的氨基酸进行了突变。进一步的,编码所述亲本嗜热链球菌GshF的基因,在本发明的一种实施方式中,其核苷酸序列是SEQ ID NO.2所示的序列。
进一步的,所述嗜热链球菌GshF的基因gshF(Gene ID:GU138096),在本发明的一种实施方式中,经金唯智生物技术有限公司合成并进行大肠杆菌密码子优化。
进一步的,所述突变体,在本发明的一种实施方式中,是将第722位的丝氨酸突变成为丙氨酸。
本发明的第二个目的是提供编码所述谷胱甘肽双功能合成酶突变体的基因,所述基因的核苷酸序列如SEQ ID NO.4所示。
本发明的第三个目的是提供携带所述基因的载体或细胞。
本发明的第四个目的是提供一种表达质粒,所述的表达质粒包含以上所述的基因,以pET-28a(+)为载体。
本发明的第五个目的是提供一种表达所述谷胱甘肽双功能合成酶突变体的基因工程菌。
进一步的,所述基因工程菌,在本发明的一种实施方式中,是以大肠杆菌BL21(DE3)为宿主。
本发明的第六个目的是提供一种利用表达所述突变体的基因工程菌发酵生产谷胱甘肽的方法。
进一步的,所述方法,在本发明的一种实施方式中,包括:利用表达以上所述谷胱甘肽双功能合成酶突变体的微生物细胞转化底物生成谷胱甘肽,所述底物为L-谷氨酸,L-半胱氨酸和甘氨酸的组合物。
进一步的,所述方法,在本发明的一种实施方式中,包括:将所述微生物细胞在培养体系中培养至OD600为0.6~0.8,加入诱导剂25℃诱导10~12h,收集菌体;将菌体添加至含有底物的反应体系中反应6~12h。
进一步的,所述方法,在本发明的一种实施方式中,所述反应体系中底物浓度分别为20~40mM,菌株OD600=30±0.5,在pH8±1、37~45℃下反应。
进一步的,所述方法,在本发明的一种实施方式中,具体为:将重组单克隆接种到含有卡那的10mL LB液体培养基中,37℃220rpm培养10h,再将1%的培养液转接新的含卡那的50mL LB液体培养基中培养,培养约2-3h使OD=0.6-0.8,加入过滤除菌的诱导剂IPTG到终浓度为0.2mM,然后25℃220rpm培养10h,离心收集菌体。通过全细胞催化合成谷胱甘肽。
本发明的第七个目的是提供本发明得到的谷胱甘肽。
本发明的第八个目的是提供本发明得到的谷胱甘肽在食品、化妆品、药物制备等领域的应用。
本发明的有益效果:
(1)对谷胱甘肽双功能合成酶722位点进行定点突变,得到了酶活提高的突变体S722A,其相对酶活较亲本提高了48.93%,说明该突变体的酶活得到大大提升;
(2)通过全细胞催化,S722A谷胱甘肽产量为20.10mmol/L,WT为16.57mmol/L,产量提高了21.46%,较之亲本得到显著提升。
附图说明
图1:重组大肠杆菌BL21(DE3)/pET28a-gshFst的构建和表达,其中,A为gshF基因PCR扩增电泳图,B为菌落PCR验证图,C为谷胱甘肽双功能合成酶的SDS-PAGE图,其中,M:180kDa蛋白marker,1:BL21(DE3)/pET28a超声破碎沉淀中的蛋白,2:BL21(DE3)/pET28a-gshFst超声破碎沉淀中的蛋白,3:BL21(DE3)/pET28a-gshFss超声破碎沉淀中的蛋白,4:BL21(DE3)/pET28a超声破碎上清中的蛋白,5:BL21(DE3)/pET28a-gshFst超声破碎上清中的蛋白,6:BL21(DE3)/pET28a-gshFss超声破碎上清中的蛋白。
图2:定点突变菌株的构建和表达,其中,A为突变体菌落PCR验证图:1-4为S27G,5-8为E130G,9-12为E622D,13-16为F719V,17-20S722A,21-24Y724H,B为突变体的SDS-PAGE图:M:180kDa蛋白marker,1:BL21/pET28a,2:BL21/pET28a-gshFst(WT),3:S27G,4:E130G,5:E622D,6:F719V,7:S722A,8:Y724H。
图3:不同定点突变菌株的相对酶活。
图4:S722A与WT全细胞催化合成谷胱甘肽。
具体实施方式
下面结合附图和实施例对本发明作更进一步的说明。
实施例
根据下述实施例,可以更好的理解本发明。然而,本领域的技术人员容易理解,实施例所描述的具体的物料配比、工艺条件及其结果仅用于说明本发明,而不应当也不会限制权利要求书中所详细描述的本发明。
下面实施例涉及的测定方法或培养方法如下:
谷胱甘肽产量测定方法:
GSH的产量用HPLC检测。用0.22μm水系混合纤维素酯微孔滤膜过滤处理。HPLC用C18柱,流动相为含有0.01mol/L庚烷磺酸钠和0.05mol/L的磷酸二氢钾溶液95%以及甲醇5%,流速0.6mL/min,进样量10μL,紫外检测器的波长设置为210nm,柱温为30℃。以GSH的浓度与峰面积形成的曲线为标准曲线,GSH浓度梯度分别0.1g/L、0.2g/L、0.3g/L、0.4g/L、0.5g/L、0.6g/L、0.7g/L和1g/L。
生物量的测定方法(紫外可见光光度计):将各个取样点的样品稀释合适倍数,至OD600值为0.2-0.8,量取200μL,在波长600nm处测取吸光度。
培养基:LB培养基(g/L):氯化钠10,胰蛋白胨10,酵母提取物5,固体培养基加琼脂粉2%。每250mL三角瓶中装液50mL,121℃灭菌20min。
大肠转化方法(质粒):(1)一管感受态加入10μL连接产物,冰上放置30min;(2)42℃水浴锅中热击90s,放入冰浴中2min;(3)每管加入800μL 37℃LB培养基(液体),至37℃摇床220rpm温育1h;(4)8000rpm离心1min,弃上清,重悬菌体涂布在Kan抗性LB平板,37℃培养箱培养,一般培养10-12h。
实施例1:GshF在大肠杆菌中的构建和表达
1、来源于嗜热链球菌的谷胱甘肽双功能合成酶基因,由GENEWIZ(苏州金唯智生物科技有限公司)进行基因合成,并进行大肠杆菌密码子优化,该基因的核苷酸序列如SEQ IDNO:2所示,氨基酸序列如SEQ ID NO:1所示,合成的基因位于pUC57-Kan标准载体上。
2、以载体为模板,用表1引物扩增目的片段(如图1A),通过同源重组连接至质粒pET28a,构建重组质粒pET28a-gshFst;将该重组质粒pET28a-gshFst转化入大肠杆菌BL21(DE3),挑取转化子进行菌落PCR验证并测序,验证正确(如图1B),重组菌株BL21(DE3)/pET28a-gshFst构建成功。
表1本研究中用得到的引物
实施例2:突变体的构建和表达
通过反向PCR法引入定点突变位点,以实施例1获得的重组质粒pET28a-gshFst为模板进行单点突变。基本流程为首先设计突变引物(引物见表2),以S27G-F/S27G-R、E130G-F/E130G-R、E622D-F/E622D-R、F719V-F/F719V-R、S722A-F/S722A-R和Y724H-F/Y724H-R为引物,在引物上引入突变位点,进行反向PCR,然后用Dpnl酶识别甲基化位点并将其酶切消化模板,Dpnl酶处理过的PCR产物转化,最后进行挑菌测序验证(如图2A),验证正确者提质粒备用。
选择测序正确的突变体,进行发酵实验,培养条件:将37℃、220rpm下培养10h的基因工程菌种子以1%的接种量转入发酵培养基于25℃、220rpm条件下培养10h。
取2mL发酵液离心,重悬洗涤后,菌体用1mL的Tris-HCl缓冲液重悬,用超声破碎仪破碎,破碎条件为:电压350V,电击时间3s,停息时间7s,超声18次。破碎后菌液于4℃、12000r/min离心10min,收集上清,为粗酶液。如图2B所示,SDS-PAGE结果表明突变体成功表达。
GSH合成反应体系:0.1mol/L Tris-HC1(pH8.0),40mmol/L Glu,20mmol/L Cys,40mmol/L Gly,20mmol/L MgCl2,20mmol/L ATP,每1mL反应体系中加入100μL粗酶液,于37℃下水浴反应20min。取出500μL与等体积的10%TCA混匀(终止反应),再在冰上放置30min后,12000r/min离心10min,收集上清测定GSH浓度。
酶活力单位(U)定义为每分钟产生1μmol谷胱甘肽所需GshF酶量,用体积比酶活(U/mL)来衡量。结果如图3所示,S722A酶活较原始菌株WT提高了48.93%,而其他突变菌株的酶活都较原始菌株WT降低。
表2本研究中用得到的引物
注:小写为突变碱基
实施例3:重组大肠杆菌及突变体全细胞催化合成谷胱甘肽
将重组单克隆接种到含有卡那的10mL LB液体培养基中,37℃220rpm培养10h,再将1%的培养液转接新的含卡那的50mL LB液体培养基中培养,培养约2h使OD=0.6-0.8,加入过滤除菌的诱导剂IPTG到终浓度为0.2mM,然后25℃220rpm培养10h,离心收集菌体。
使用收集的菌体,通过全细胞催化合成谷胱甘肽,反应体系:菌体控制OD600=30。反应液:Tris-HCl Buffer 200mmol/L pH 8,L-Glu 40mmol/L,L-Cys 20mmol/L,Gly40mmol/L,MgCl2 20mmol/L,ATP 20mmol/L,220r/min,37℃反应,分别于0h,2h,4h,6h,8h取样,检测GSH的产量。将-20℃保存的菌体在室温下解冻,如此反复三次。结果如图4所示,在2h时,S722A的产量为20.10mmol/L,WT为16.57mmol/L,产量提高了21.46%。S722A基于Cys摩尔转化率100.5%,WT基于Cys摩尔转化率82.85%。产量高于理论值,主要是冻融细胞含有细胞本身产生的GSH。
综上,本发明对来源于嗜热链球菌的谷胱甘肽双功能合成酶进行异源表达,构建了重组大肠杆菌,对谷胱甘肽双功能合成酶蛋白进行改造,对其进行定点突变,提高了酶活。通过全细胞催化,突变体S722A的产量达到20.10mmol/L。本发明成功地构建了谷胱甘肽的外源表达途径,并进一步提高了酶活,节省了原料和成本,提高了经济效益,为工业化生产奠定了基础。
以上所述仅是本发明的优选实施方式,应当指出:对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.一种谷胱甘肽双功能合成酶突变体,其特征在于,所述突变体的氨基酸序列如SEQID NO.3所示。
2.根据权利要求1所述的一种谷胱甘肽双功能合成酶突变体,其特征在于,所述突变体是在氨基酸序列如SEQ ID NO.1所示的谷胱甘肽双功能合成酶的基础上,将第722位的丝氨酸突变成为丙氨酸。
3.编码权利要求1或2所述突变体的基因,其特征在于,所述基因的核苷酸序列如SEQID NO.4所示。
4.携带权利要求3所述基因的载体或细胞。
5.一种表达质粒,其特征在于,所述的表达质粒包含如权利要求3所述的基因,以pET-28a(+)为载体。
6.表达权利要求1或2所述谷胱甘肽双功能合成酶突变体的基因工程菌,其特征在于,以大肠杆菌BL21(DE3)为宿主。
7.一种生产谷胱甘肽的方法,其特征在于,利用表达权利要求1或2所述谷胱甘肽双功能合成酶突变体微生物细胞转化底物生成谷胱甘肽,所述底物为L-谷氨酸,L-半胱氨酸和甘氨酸的组合物。
8.根据权利要求7所述的方法,其特征在于,将所述微生物细胞在培养体系中培养至OD600为0.6~0.8,加入诱导剂25℃诱导10~12h,收集菌体;将菌体添加至含有底物的反应体系中反应6~12h。
9.根据权利要求8所述的方法,其特征在于,所述反应体系中底物浓度分别为20~40mM,菌株OD600=30±0.5,在pH8±1、37~45℃下反应。
10.权利要求1-2任一所述突变体或权利要求4所述载体或细胞在食品、医药、化妆品领域的应用。
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