CN116926024A - 一种耐热性增强的谷胱甘肽合成酶及应用 - Google Patents
一种耐热性增强的谷胱甘肽合成酶及应用 Download PDFInfo
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- CN116926024A CN116926024A CN202310888275.2A CN202310888275A CN116926024A CN 116926024 A CN116926024 A CN 116926024A CN 202310888275 A CN202310888275 A CN 202310888275A CN 116926024 A CN116926024 A CN 116926024A
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- glutathione synthetase
- fermentation
- cysteine
- glutathione
- sodium
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Abstract
本发明涉及酶催化技术领域,尤其是一种耐热性增强的谷胱甘肽合成酶及应用,所述谷胱甘肽合成酶的氨基酸序列如SEQ ID NO:1所示。使用时,反应体系pH7.0,含有硫酸镁、六偏磷酸钠、甘氨酸及谷氨酸钠、L‑半胱氨酸盐酸盐、AMP钠盐,加入谷胱甘肽合成酶的粗酶液及耐热型多聚磷酸激酶,30~45℃反应。该谷胱甘肽合成酶与野生型谷胱甘肽合成酶相比,可应用于高温催化NADH,表现出更强的热稳定性。
Description
技术领域
本发明涉及酶催化技术领域,具体领域为一种耐热性增强的谷胱甘肽合成酶及应用。
背景技术
谷胱甘肽(γ-谷氨酰-L-半胱氨酸甘氨酸,GSH)是一种由谷氨酸、半胱氨酸和甘氨酸组成的三肽分子,缩写为GSH,是一种重要的非蛋白质硫醇化合物,广泛分布于生物体中。它在维持细胞氧化还原平衡、活性氧的解毒和抵御氧化应激方面发挥着至关重要的作用。此外,生物体内高浓度的谷胱甘肽可以抵抗重金属离子,并适应环境条件。因此,它在土壤和水的生物修复中具有潜在的应用。谷胱甘肽对氧化损伤的保护作用也已在乳酸菌中得到证实。例如,补充谷胱甘肽增强了乳酸菌对酸、冷和渗透胁迫的耐受性。除了谷胱甘肽作为氧化应激反应一部分的作用外,γ-谷氨酰肽还包括谷胱甘肽、γ-Glu-Cys、γ-Glu-Glu、γ-Glu-Gly、γ-Glu-Gln、γ-Glu-Met和γ-Glu-Leu激活人类钙敏感受体(CasR),增强和扩展对咸味、鲜味和甜味等主要味道的感知。谷胱甘肽在制药、化妆品和食品工业中有各种应用。近年来,对GSH的需求逐年增加。
谷胱甘肽的工业生物合成主要有两种方式,发酵和酶催化。这些过程的关键因素是以三种前体氨基酸(即L-谷氨酸,L-半胱氨酸和甘氨酸)的两次连续ATP消耗反应进行的。谷胱甘肽的合成是一个两步酶反应,每一步消耗一个ATP分子,是一个大量消耗ATP的过程。γ-谷氨酰半胱氨酸连接酶或γ-谷氨酰半胱氨酸合成酶(γ-Gcl,EC 6.3.2.2)将半胱氨酸连接到谷氨酸,谷胱甘肽合成酶(GS,EC 6.3.2.3)催化甘氨酸与γ-谷氨酰半胱氨酸连接,产生三肽谷胱甘肽。只有还原型谷胱甘肽才具有生物活性。两个还原GSH分子脱氢,而后二硫化键形成氧化型GSH(GSSG)。GSSG可以通过消耗NADPH通过GSH还原酶还原为活性还原GSH。众所周知,γ-GCS催化大多数真核和原核细胞中GSH合成的反应被GSH抑制,而双功能GSH合成酶(GshF)即使在20mM时对GSH引起的反馈抑制也不敏感。因此,最近已有学者将新型双功能酶的GshF编码应用于通过发酵或酶促反应产生GSH。传统上,存在于大肠杆菌、产朊假丝酵母和酿酒酵母中的这些酶是γ-l-戊二酸-l-半胱氨酸合成酶(γ-GCS)和l-谷胱甘肽合成酶(GS)。γ-GCS和GS在不同宿主菌株中的过表达可以改善GSH的形成。然而,γ-GCS的活性受到GSH的严重反馈抑制,导致GSH生物合成的速率限制和细胞内GSH积累的减少。
为了提高GSH产量并改善工艺性能,多个实验室已经研究了野生型菌株的诱变或工艺优化。如有研究者构建了两种分别过表达gshA和gshB的重组大肠杆菌菌株,积累了约5克/升的谷胱甘肽。加入SDS可以显著增强GSH的产生,可能是因为这种处理大大提高了细胞膜结构的通透性,随后GSH被释放到培养基中,对GSH的反馈抑制作用得到部分缓解。此外,有学者提出了使用渗透酵母细胞的两阶段反应,可以有效降低GSH的反馈抑制。
另一种提高谷胱甘肽产量的策略是使用具有较少反馈抑制的新型酶来代替γ-GCS和GS。前者已有实验室分离和研究了几种同时具有γ-GPS和GS功能的双功能谷胱甘肽合成酶(GSH-FS)。此外,一些GshFS被证明对Gsh抑制不敏感。如在组成型启动子的控制下,成功地将嗜热链球菌的GshF表达到转基因烟草中,并在其叶片中积累了极高水平的谷胱甘肽。在毕赤酵母中表达单核细胞增多性李斯特菌GshF可以显著减少γ-GC的积累。在一些革兰氏阳性细菌中,编码双功能蛋白gshAB/gshF的单个基因(由N端的Gcl结构域和C端的GS结构域组成)介导γ-谷氨酰二肽和谷胱甘肽的生物合成。编码多结构域双功能蛋白GshAB/GshF的基因首先在无乳链球菌和李斯特菌中发现,但也存在于嗜热链球菌和肠球菌属中。迄今为止,大多数预测编码gshAB/gshF都是病原体菌株。
华东理工大学对携带嗜热链球菌GshF的重组大肠杆菌菌株进行了Gsh的酶促合成研究,并做出巨大贡献。质粒的不同拷贝数和启动子强度会影响蛋白质的表达水平,并进一步影响酶活性和途径的通量。pET、pTrc和pUC的载体系列具有不同的拷贝数和诱导启动子(分别为T7、pTrc和Plac),已成功用于大肠杆菌中靶蛋白的过量生产和代谢途径的修饰。
发明内容
本发明的目的在于提供一种耐热性增强的谷胱甘肽合成酶及应用。
为实现上述目的,本发明提供如下技术方案:
一种耐热性增强的谷胱甘肽合成酶,所述谷胱甘肽合成酶的氨基酸序列如SEQ IDNO:1所示。
本发明的耐热性增强的谷胱甘肽合成酶可用于催化L-半胱氨酸中的应用,其使用方法为:反应体系pH7.0,含有硫酸镁、六偏磷酸钠、甘氨酸及谷氨酸钠、L-半胱氨酸盐酸盐、AMP钠盐,加入谷胱甘肽合成酶的粗酶液及耐热型多聚磷酸激酶,30~45℃反应。
其中,所述粗酶液的制备方法包括以下步骤:
(1)通过引物拼接的方法,将SEQ ID NO:1所示蛋白的对应编码多核苷酸序列进行组装,并克隆到原核表达载体,以实现在大肠杆菌中的高表达;
(2)采用摇瓶发酵或者分批补料发酵
①摇瓶发酵
挑取含有表达载体的大肠杆菌单菌落接种于10mL高压灭菌后的培养基A中,30℃,250rpm过夜培养;所述培养基A为:胰蛋白胨10g/L,酵母提取物5g/L,磷酸氢二钠3.55g/L,磷酸二氢钾3.4g/L,氯化铵2.68g/L,硫酸钠0.71g/L,七水硫酸镁0.493g/L,六水氯化铁0.027g/L,甘油5g/L,葡萄糖0.8g/L,添加卡那霉素至50mg/L;
次日取1L三角瓶,按1:100的接种比例接入到100mL高压灭菌后的培养基B中,于30℃中培养至菌体OD 5-6,立刻将三角瓶置于25℃摇床中,250rpm培养1小时;加IPTG至终浓度0.1mM,并于25℃,250rpm继续培养16小时;所述培养基B为:胰蛋白胨10g/L,酵母提取物5g/L,磷酸氢二钠3.55g/L,磷酸二氢钾3.4g/L,氯化铵2.68g/L,硫酸钠0.71g/L,七水硫酸镁0.493g/L,六水氯化铁0.027g/L,甘油5g/L,葡萄糖0.3g/L,添加卡那霉素至50mg/L;
培养结束后,将培养液于4℃,12000g下离心20分钟收集湿菌体;然后将菌体沉淀用蒸馏水清洗两次,收集菌体,-70℃保存;同时取少量菌体进行SDS-PAGE检测;
②分批补料发酵
分批补料发酵在计算机控制的生物反应器中进行,将含有表达载体的大肠杆菌单菌落制备200ml种子摇瓶,当种子摇瓶的培养物为OD2.0时接入所述生物反应器;在整个发酵过程中,温度保持37℃,发酵过程中溶解氧浓度由搅拌速率和通气供应级联自动控制在30%,而培养基的pH值由50%v/v正磷酸和30%v/v氨水维持在7.0;发酵过程中,当出现溶氧回升时,开始补料,补料溶液含有9%w/v蛋白胨、9%w/v酵母提取物、14%w/v甘油;当OD600为50.0时,控制温度为25℃,并用0.1mM IPTG诱导表达16小时,离心收菌体-25℃保存。
其中,分批补料发酵所用的培养基为:酵母抽提物24g/L,蛋白胨12g/L,0.4%w/v葡萄糖,2.31g/L磷酸二氢酶和12.54g/L磷酸氢二钾,pH 7.0。
与现有技术相比,本发明的有益效果是:
本发明耐热性增强的谷胱甘肽合成酶能够高温催化L-半胱氨酸,与野生型谷胱甘肽合成酶相比,表现出更强的热稳定性,可取得较好的社会效益和经济价值。
附图说明
图1为0.5g/L产物标品的图谱。
图2为标准反应图谱及主要出峰时间;
其中,2.8min:甘氨酸和谷氨酸;3.1min:L-半胱氨酸;3.4min:中间体;3.7min:氧化型谷胱甘肽;4.2min:还原型谷胱甘肽;6.1min:ATP;7.6min:ADP;10.9min:AMP。
图3为实施例5反应4小时的检测结果。
图4为对比例1反应4小时的检测结果。
图5为实施例6反应4小时的检测结果。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
在本实施例中所用到的仪器、试剂,除非有特殊说明,均为市售产品。
以下实施例中涉及到的液相检测条件如下:
流动相:A:20mM乙酸铵水溶液;
B:甲醇。
A:B=97%:3%,保留时间:20min(等度洗脱)。
使用柱子:岛津C18 4.6*250mm*5μm。
波长:340nm。
柱温:25℃。
进样体积:10μL。
实施例1野生型谷胱甘肽合成酶基因序列的获得
通过全基因合成的方法,对基因的二级结构以及密码子偏好性进行调整,以实现在大肠杆菌中的高表达。利用Primer Premier(http://primer3.ut.ee/)和OPTIMIZER(http://genomes.urv.es/OPTIMIZER/)进行设计,并保证退火温度(Tm)差异控制在3℃以内,引物长度控制在60base以内,引物序列如表2所示,并将获得的引物加双蒸水溶解后,加到如下的反应体系中,使得各引物的终浓度为30nM,首尾引物的终浓度为0.6μM。
表1
2mMdNTP mix(2mM eachdNTP) | 5μl |
10×Pfubuffer | 5μl |
Pfu DNA polymerase(10U/μl) | 0.5μl |
ddH2O | 使得反应体系总体积至50μl |
将配制好的PCR反应体系置于博日XP cycler基因扩增仪中,按下列程序进行扩增:98℃30s,55℃45s,72℃120s,35x。将PCR得到的DNA片段进行切胶纯化,利用同源重组的方法克隆进pET30a的NdeI/XhoI位点。挑取单克隆进行测序。测序成功的DNA序列为SEQ IDNO:4,命名为CPGSwt,其对应的氨基酸序列为SEQ ID NO:3。
表2
实施例2谷胱甘肽合成酶突变体基因序列的获得
本发明的耐热性增强的谷胱甘肽合成酶,其源于SEQ ID NO:3的野生型谷胱甘肽合成酶。谷胱甘肽合成酶突变体和编码这种突变体的多核苷酸可以使用本领域技术人员通常使用的方法制备。突变体可以通过使编码该酶的体外重组、多核苷酸诱变、DNA改组、易错PCR和定向进化方法等获得。
通过全基因合成的方法,对基因的二级结构以及密码子偏好性进行调整,以实现在大肠杆菌中的高表达。利用Primer Premier(http://primer3.ut.ee/)和OPTIMIZER(http://genomes.urv.es/OPTIMIZER/)进行设计,并保证退火温度(Tm)差异控制在3℃以内,引物长度控制在60base以内,引物序列如表4所示,并将获得的引物加双蒸水溶解后,加到如下的反应体系中,使得各引物的终浓度为30nM,首尾引物的终浓度为0.6μM。
表3
将配制好的PCR反应体系置于博日XP cycler基因扩增仪中,按下列程序进行扩增:98℃30s,55℃45s,72℃120s,35x。将PCR得到的DNA片段进行切胶纯化,利用同源重组的方法克隆进pET30a的NdeI/XhoI位点。挑取单克隆进行测序。测序成功的DNA序列为SEQ IDNO:2,命名为CPGS1,其对应的氨基酸序列为SEQ ID NO:1。与CPGSwt的序列相比,具有三处突变K72S,Q323D,E409Q。
表4
实施例3摇瓶表达测试
挑取含有表达载体的大肠杆菌单菌落接种于10ml高压灭菌后的培养基中:胰蛋白胨10g/L,酵母提取物5g/L,磷酸氢二钠3.55g/L,磷酸二氢钾3.4g/L,氯化铵2.68g/L,硫酸钠0.71g/L,七水硫酸镁0.493g/L,六水氯化铁0.027g/L,甘油5g/L,葡萄糖0.8g/L,添加卡那霉素至50mg/L。30℃,250rpm过夜培养。
次日取1L三角瓶,按1:100的接种比例接入到100ml高压灭菌后的培养基中:胰蛋白胨10g/L,酵母提取物5g/L,磷酸氢二钠3.55g/L,磷酸二氢钾3.4g/L,氯化铵2.68g/L,硫酸钠0.71g/L,七水硫酸镁0.493g/L,六水氯化铁0.027g/L,甘油5g/L,葡萄糖0.3g/L,添加卡那霉素至50mg/L。于30℃中培养至菌体OD 5-6,立刻将三角瓶置于25℃摇床中,250rpm培养1小时。加IPTG至终浓度0.1mM,并于25℃,250rpm继续培养16小时。
培养结束后,将培养液于4℃,12000g下离心20分钟收集湿菌体。然后将菌体沉淀用蒸馏水清洗两次,收集菌体,-70℃保存。同时取少量菌体进行SDS-PAGE检测。
实施例4分批补料发酵
分批补料发酵在计算机控制的生物反应器(上海国强)中进行,反应器容量为15L,工作体积为8L,所用到的培养基为酵母抽提物24g/L,蛋白胨12g/L,0.4%w/v葡萄糖,2.31g/L磷酸二氢酶和12.54g/L磷酸氢二钾,pH 7.0。
将含有表达载体的大肠杆菌单菌落制备200ml种子摇瓶,当种子摇瓶的培养物为OD2.0时接入所述生物反应器。在整个发酵过程中,温度保持在37℃,发酵过程中溶解氧浓度由搅拌速率(rpm)和通气供应级联自动控制在30%,而培养基的pH值由50%(v/v)正磷酸和30%(v/v)氨水维持在7.0。发酵过程中,当出现大幅的溶氧回升时,开始补料。补料溶液含有9%w/v蛋白胨、9%w/v酵母提取物、14%w/v甘油。当OD600约为50.0(湿重约为100g/L)时,控制温度为25℃,并用0.1mM IPTG诱导表达16小时,离心收菌体-25℃保存,使用时按每公斤湿菌体加入2公斤纯水使用。
实施例5突变体高温反应
反应体系pH7.0,70mM硫酸镁,25g/L六偏磷酸钠,160mM甘氨酸及160mM谷氨酸钠,64mM L-半胱氨酸盐酸盐,2g/L AMP钠盐,10ml体系,加入制备的CPGS1粗酶0.6mL,耐热型多聚磷酸激酶LE496(源自南京朗恩生物科技有限公司)0.2mL。45℃摇床。中间不调pH、不补六偏磷酸钠,取4小时样检测。过夜测pH约6.3。HPLC图谱见图3,此时L-半胱氨酸摩尔转化率约为90%。
对比例1野生型蛋白高温反应
反应体系pH7.0,70mM硫酸镁,25g/L六偏磷酸钠,160mM甘氨酸及160mM谷氨酸钠,64mM L-半胱氨酸盐酸盐,2g/L AMP钠盐,10ml体系,加入制备的CPGSwt粗酶0.6mL,耐热型多聚磷酸激酶LE496(源自南京朗恩生物科技有限公司)0.2mL。45℃摇床。中间不调pH、不补六偏磷酸钠,取4小时样检测。HPLC图谱见图4,此时L-半胱氨酸摩尔转化率约为60%。此时仍残余较多的L-半胱氨酸。证明野生蛋白并不适应高温反应体系,从而造成转化率偏低。
实施例6突变体常温反应
反应体系pH7.0,70mM硫酸镁,25g/L六偏磷酸钠,160mM甘氨酸及160mM谷氨酸钠,64mM L-半胱氨酸盐酸盐,2g/L AMP钠盐,10ml体系,加入制备的CPGS1粗酶0.6mL,耐热型多聚磷酸激酶LE496(源自南京朗恩生物科技有限公司)0.2mL。30℃摇床。中间不调pH、不补六偏磷酸钠,取4小时样检测。HPLC图谱见图5,此时L-半胱氨酸摩尔转化率约为70%。可见,同样的反应体系,45℃对比30℃在反应4小时后转化率有明显提升,证明适应了高温反应的突变体CPGS1在高温下反应速度更快且转化率更高。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (5)
1.一种耐热性增强的谷胱甘肽合成酶,其特征在于:所述谷胱甘肽合成酶的氨基酸序列如SEQ ID NO:1所示。
2.权利要求1所述耐热性增强的谷胱甘肽合成酶在催化L-半胱氨酸中的应用。
3.根据权利要求2所述的耐热性增强的谷胱甘肽合成酶在催化L-半胱氨酸中的应用,其特征在于:反应体系pH7.0,含有硫酸镁、六偏磷酸钠、甘氨酸及谷氨酸钠、L-半胱氨酸盐酸盐、AMP钠盐,加入谷胱甘肽合成酶的粗酶液及耐热型多聚磷酸激酶,30~45℃反应。
4.根据权利要求3所述的耐热性增强的谷胱甘肽合成酶在催化L-半胱氨酸中的应用,其特征在于:所述粗酶液的制备方法包括以下步骤:
(1)通过引物拼接的方法,将SEQ ID NO:1所示蛋白的对应编码多核苷酸序列进行组装,并克隆到原核表达载体,以实现在大肠杆菌中的高表达;
(2)采用摇瓶发酵或者分批补料发酵
①摇瓶发酵
挑取含有表达载体的大肠杆菌单菌落接种于10mL高压灭菌后的培养基A中,30℃,250rpm过夜培养;所述培养基A为:胰蛋白胨10g/L,酵母提取物5g/L,磷酸氢二钠3.55g/L,磷酸二氢钾3.4g/L,氯化铵2.68g/L,硫酸钠0.71g/L,七水硫酸镁0.493g/L,六水氯化铁0.027g/L,甘油5g/L,葡萄糖0.8g/L,添加卡那霉素至50mg/L;
次日取1L三角瓶,按1:100的接种比例接入到100mL高压灭菌后的培养基B中,于30℃中培养至菌体OD 5-6,立刻将三角瓶置于25℃摇床中,250rpm培养1小时;加IPTG至终浓度0.1mM,并于25℃,250rpm继续培养16小时;所述培养基B为:胰蛋白胨10g/L,酵母提取物5g/L,磷酸氢二钠3.55g/L,磷酸二氢钾3.4g/L,氯化铵2.68g/L,硫酸钠0.71g/L,七水硫酸镁0.493g/L,六水氯化铁0.027g/L,甘油5g/L,葡萄糖0.3g/L,添加卡那霉素至50mg/L;
培养结束后,将培养液于4℃,12000g下离心20分钟收集湿菌体;然后将菌体沉淀用蒸馏水清洗两次,收集菌体,-70℃保存;同时取少量菌体进行SDS-PAGE检测;
②分批补料发酵
分批补料发酵在计算机控制的生物反应器中进行,将含有表达载体的大肠杆菌单菌落制备200ml种子摇瓶,当种子摇瓶的培养物为OD2.0时接入所述生物反应器;在整个发酵过程中,温度保持37℃,发酵过程中溶解氧浓度由搅拌速率和通气供应级联自动控制在30%,而培养基的pH值由50%v/v正磷酸和30%v/v氨水维持在7.0;发酵过程中,当出现溶氧回升时,开始补料,补料溶液含有9%w/v蛋白胨、9%w/v酵母提取物、14%w/v甘油;当OD600为50.0时,控制温度为25℃,并用0.1mM IPTG诱导表达16小时,离心收菌体-25℃保存。
5.根据权利要求4所述的耐热性增强的谷胱甘肽合成酶在催化L-半胱氨酸中的应用,其特征在于:粗酶液制备方法中,分批补料发酵所用的培养基为:酵母抽提物24g/L,蛋白胨12g/L,0.4%w/v葡萄糖,2.31g/L磷酸二氢酶和12.54g/L磷酸氢二钾,pH 7.0。
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