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High production of glutathione by in vitro enzymatic cascade after thermostability enhancement
AIChE Journal ( IF 3.7 ) Pub Date : 2020-09-13 , DOI: 10.1002/aic.17055
Xiangwei Cui 1 , Zhimin Li 1, 2
Affiliation  

The cell free system has been paid more attention due to its potential of facilitating efficient catalysis of multistep reactions. In this study, an efficient enzymatic cascade of glutathione (GSH) production was developed through the evolution of bifunctional glutathione synthetase (GshF), coupled with polyphosphate kinase (PPK). First, the stability and activity of GshF were enhanced by loop interchange and site‐directed mutagenesis. As a result, the GshF half‐value period increased 163.32‐fold, and its activity raised 18.16%. PPK from Jhaorihella thermophile (PPKJT) was characterized and used to regenerate ATP in the GSH synthesis, with hexametaphosphate (PolyP (6)) as the phosphate donor. After the process optimization, 99.92 mM GSH and 7.61 mM oxidized glutathione (GSSG) were produced within 2 hr. The molar yield was 0.96 mol/mol based on the amino acid added, while the productivities of GSH achieved 49.95 mM/hr, which were the highest yield and productivity ever reported about GSH synthesis.

中文翻译:

增强热稳定性后,通过体外酶促级联反应高产谷胱甘肽

无细胞系统由于其促进多步反应的有效催化的潜力而受到更多关注。在这项研究中,通过双功能谷胱甘肽合成酶(GshF)与多磷酸激酶(PPK)的结合,开发了一种高效的谷胱甘肽(GSH)酶促级联反应。首先,通过环互换和定点诱变增强了GshF的稳定性和活性。结果,GshF半值期间增加了163.32倍,其活动增加了18.16%。嗜热芽孢杆菌PPK(PPKJT)的特性已被表征并用于在GSH合成中再生ATP,其中六偏磷酸盐(PolyP(6))为磷酸盐供体。经过工艺优化后,在2小时内产生了99.92 mM GSH和7.61 mM氧化型谷胱甘肽(GSSG)。基于添加的氨基酸,摩尔产率为0.96mol / mol,而GSH的生产率达到49.95mM / hr,这是关于GSH合成的最高的产率和生产率。
更新日期:2020-09-13
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