CN110484580A - 谷胱甘肽的合成方法 - Google Patents
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Abstract
本发明涉及一种谷胱甘肽的合成方法,将活化后的酵母菌在发酵培养基中进行发酵培养,然后在所述的酵母菌的生长对数期或稳定期加入十二烷基硫酸钠(SDS),继续发酵培养得到所述的谷胱甘肽。本发明能够明显提高谷胱甘肽的生产量,提高了原料利用率,降低了生产成本。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种谷胱甘肽的合成方法。
背景技术
谷胱甘肽(γ-L-glutamyl-cysteinyl-glycine,GSH),是由L-谷氨酸、L-半胱氨酸和甘氨酸组成的生物活性三肽化合物,是细胞内重要的抗氧化剂和主要的非蛋白巯基化合物(>90%)。在生物组织中,谷胱甘肽通过谷胱甘肽还原酶保持氧化型和还原型的动态平衡并发挥细胞保护、物质代谢、信息调控、调节细胞及组织的发育、对抗或诱发疾病等作用,因此,大量应用于医药保健、护肤美容、食品添加等行业。
目前GSH的生产方法主要有溶剂萃取法、化学合成法和生物酶催化法和生物发酵法。相比之下,生物发酵法具有反应条件温和、反应步骤简单、成本低、转化效率高、生产速率快等优势,是今后生产谷胱甘肽的主要趋势,但目前大部分研究还停留在实验室阶段,实现商业化生产的国家主要是日本。
分子生物学时常见的提高谷胱甘肽产量的方法,但是工程菌需要加入大量的抗生素,这在操作上是不允许的。此外,抗生素一般比较贵,这在经济上也是不允许的。最严重的是,工程菌在发酵过程中容易出现菌株退化,很难长期使用。因此,寻求其他替代的,经济的方法至关重要。表面活性剂作为一种去污剂应用于谷胱甘肽的生产已经有很多报道,其工作原理是改变细胞膜通透性使胞内的谷胱甘肽释放出来,从而,可以减弱谷胱甘肽对其合成酶的反馈抑制,最终提高产量。本方法不涉及基因操纵,且操作方便,经济适用性强。同时,由于添加的表面活性剂可以使产物释放,也降低了后续分离纯化的难度。
发明内容
本发明所要解决的技术问题是提供一种能够更加高效的合成谷胱甘肽的方法。
为解决以上技术问题,本发明采取如下技术方案:
一种谷胱甘肽的合成方法,将活化后的酵母菌在发酵培养基中进行发酵培养,然后在所述的酵母菌的生长对数期或稳定期加入十二烷基硫酸钠(SDS),继续发酵培养得到所述的谷胱甘肽。
优选地,所述的发酵培养基的配方为:葡萄糖20~40 g/L,酵母提取物2~8 g/L,(NH4)2SO4 2~8 g/L, KH2PO4 3~9 g/L, K2SO4 3~4 g/L,MgSO4 1~2 g/L,FeSO4 0.006~0.01g/L,MnSO4 0.006~0.01 g/L。
优选地,所述的发酵培养基的pH为5.5~6.5。
优选地,进行所述的发酵培养的温度为25~35℃。
优选地,所述的十二烷基硫酸钠的添加量为5~10 mg/L。
优选地,所述的活化后的酵母菌的活化方法为:将先酵母菌活化培养至长出单菌落,然后将所述的单菌落进一步活化培养25~35h得到所述的活化后的酵母菌。
进一步优选地,进行所述的活化培养的温度为25~35℃。
进一步优选地,培养至长出单菌落的培养基的配方包括:0.5~1.5% w/v 葡萄糖,0.2~0.8% w/v 蛋白胨,0.1~0.5% w/v 酵母提取物,以及使培养基呈固体的琼脂。
进一步优选地,进一步活化培养的培养基的配方包括:0.5~1.5% w/v 葡萄糖,0.2~0.8% w/v 蛋白胨,0.1~0.5% w/v 酵母提取物。
优选地,所述的酵母菌为酿酒酵母Saccharomyces cerebisiae。
由于以上技术方案的实施,本发明与现有技术相比具有如下优点:
本发明能够明显提高谷胱甘肽的生产量,提高了原料利用率,降低了生产成本。
附图说明
图1是摸索的酵母菌的生长曲线。
图2是不同时间点添加SDS的谷胱甘肽的产量。
具体实施方式
以下结合具体实施例,对本发明作进一步说明。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
以下实施例中为注明具体条件的实验方法,通常按照常规条件,如《分子克隆:实验室手册》(New York: CoLd Spring Harbor Laboratory Press,1989)中所述的条件或厂商提供的方案进行。
在本发明的下述实施例中,使用的试剂,培养基等均购自生工生物公司。
在本发明的下述实施例中,使用的菌株为酿酒酵母Saccharomyces cerebisiae,此菌株已于2007年1月12日保藏于中国普通微生物菌种保藏管理中心,保藏号是:CGMCCNo.1917。
在本发明的下述实施例中,使用的摇瓶发酵培养基的配方为:YPD 培养基包括(1.0% w/v 葡萄糖, 0.5% w/v 蛋白胨, 0.3% w/v 酵母提取物)。发酵培养基配方为(g/L):葡萄糖30,酵母提取物 5, (NH4)2SO4 5, KH2PO4 6, K2SO4 3.6, MgSO4 1.5, FeSO40.008, MnSO4 0.008, 培养基pH 调整到 6.0。
实施例1:酵母菌的生长曲线的摸索
1.1菌种活化及培养基的配制
按照如下体积比配制YPD 培养基,并通过添加琼脂粉获得固体培养基。
YPD培养基配方:1.0% w/v 葡萄糖, 0.5% w/v 蛋白胨, 0.3% w/v 酵母提取物。
从-80℃冰箱取出保藏的菌株,在无抗固体培养基上划线并放在30℃培养箱中培养约24小时。待长出清晰可见的斑点(单菌落),用灭菌牙签挑取单菌落入YPD液体培养基中培养。同上述培养条件培养,单菌落培养约30 h后,转移到发酵培养基中继续发酵以进行后续实验。
发酵培养基配方为(g/L):葡萄糖30,酵母提取物 5, (NH4)2SO4 5, KH2PO4 6,K2SO4 3.6, MgSO4 1.5, FeSO4 0.008, MnSO4 0.008, 培养基pH 调整到 6.0。
1.2酵母菌的生长曲线的摸索
如上操作进行酵母菌的生长曲线的摸索。具体方法如下:
(1)按照1.1方法培养酵母菌,放置30℃下240rpm培养;
(2)每隔3小时取样一次,连续取样10次以上;
(3)培养物离心,烘干至恒重后称量,测定细胞干重。
(4)根据不同时间下的细胞干重绘制生长曲线,结果如图1所示,确定添加试剂的时间点。
实施例2:摸索SDS添加时间对谷胱甘肽的影响
通过单个添加计量添加的方法确定添加时间。由于发酵初期菌体量比较少,因此,选择对数及稳定期几个时间点作为待定的添加点添加相关试剂。主要技术流程如下:
(1)按照1.1方法培养酵母菌;
(2)在不同的时期加入5 mg/L计量的SDS (根据前期实验所得),并在加入后继续培养30小时收菌;
(3)测定培养结束的菌体的细胞干重,同时,测定谷胱甘肽在细胞内外的含量,测试结果如图2所示。
(4)分析实验结果,确定添加时间。
实施例3
按照如下体积比配制YPD 培养基,并通过添加琼脂粉获得固体培养基。
YPD培养基配方:1.0% w/v 葡萄糖, 0.5% w/v 蛋白胨, 0.3% w/v 酵母提取物。
从-80℃冰箱取出保藏的菌株,在无抗固体培养基上划线并放在30℃培养箱中培养约24小时。待长出清晰可见的斑点(单菌落),用灭菌牙签挑取单菌落入YPD液体培养基中培养。同上述培养条件培养,单菌落培养约30 h后,转移到发酵培养基中继续发酵,然后在培养20h后加入10 mg/L计量的SDS,并在加入后继续培养30小时收菌;经测定谷胱甘肽在细胞内外的含量为487.35 mg/L。
以上对本发明做了详尽的描述,其目的在于让熟悉此领域技术的人士能够了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,凡根据本发明的精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围内。
Claims (10)
1.一种谷胱甘肽的合成方法,其特征在于:将活化后的酵母菌在发酵培养基中进行发酵培养,然后在所述的酵母菌的生长对数期或稳定期加入十二烷基硫酸钠,继续发酵培养得到所述的谷胱甘肽。
2. 根据权利要求1所述的谷胱甘肽的合成方法,其特征在于:所述的发酵培养基的配方为:葡萄糖20~40 g/L,酵母提取物2~8 g/L, (NH4)2SO4 2~8 g/L, KH2PO4 3~9 g/L,K2SO4 3~4 g/L,MgSO4 1~2 g/L,FeSO4 0.006~0.01 g/L,MnSO4 0.006~0.01 g/L。
3.根据权利要求1所述的谷胱甘肽的合成方法,其特征在于:所述的发酵培养基的pH为5.5~6.5。
4.根据权利要求1所述的谷胱甘肽的合成方法,其特征在于:进行所述的发酵培养的温度为25~35℃。
5. 根据权利要求1所述的谷胱甘肽的合成方法,其特征在于:所述的十二烷基硫酸钠的添加量为5~10 mg/L。
6. 根据权利要求1所述的谷胱甘肽的合成方法,其特征在于:所述的活化后的酵母菌的活化方法为:将先酵母菌活化培养至长出单菌落,然后将所述的单菌落进一步活化培养25~35 h得到所述的活化后的酵母菌。
7.根据权利要求6所述的谷胱甘肽的合成方法,其特征在于:进行所述的活化培养的温度为25~35℃。
8. 根据权利要求6所述的谷胱甘肽的合成方法,其特征在于:培养至长出单菌落的培养基的配方包括:0.5~1.5% w/v 葡萄糖,0.2~0.8% w/v 蛋白胨,0.1~0.5% w/v 酵母提取物,以及使培养基呈固体的琼脂。
9. 根据权利要求6所述的谷胱甘肽的合成方法,其特征在于:进一步活化培养的培养基的配方包括:0.5~1.5% w/v 葡萄糖,0.2~0.8% w/v 蛋白胨,0.1~0.5% w/v 酵母提取物。
10. 根据权利要求1所述的谷胱甘肽的合成方法,其特征在于:所述的酵母菌为酿酒酵母Saccharomyces cerebisiae 。
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