文献类型：期刊文献

中文题名：基于能量循环再生系统酶法生产谷胱甘肽

英文题名：Enzymatic Synthesis of Glutathione Based on Energy Regeneration System

作者：张星[1];崔向伟[1];李宗霖[1];李志敏[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2020

卷号：46

期号：5

起止页码：688

中文期刊名：华东理工大学学报（自然科学版）

外文期刊名：Journal of East China University of Science and Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2017】；CSCD:【CSCD_E2019_2020】；

基金：上海市科委产学研合作项目(13DZ1930202)。

语种：中文

中文关键词：谷胱甘肽;谷胱甘肽双功能合成酶;多聚磷酸激酶;ATP再生

外文关键词：glutathione;bifunctional glutathione synthetase;polyphosphate kinase;ATP regeneration

摘要：以多聚磷酸(polyP)为能量供体,通过构建双酶级联反应,以及偶联多聚磷酸激酶(PPK)和谷胱甘肽双功能合成酶(GshF),酶法合成了谷胱甘肽(GSH)。对GshF和PPK诱导表达条件进行了优化,表明低温18℃有利于蛋白的可溶表达。将GshF和PPK纯化后,建立纯酶反应系统,对反应条件进行优化,结果表明,当polyP和MgCl2的浓度比为30:45、腺苷二磷酸(ADP)浓度为0.5 mmol/L时,反应效率较高;45℃为反应最优温度;在PPK质量浓度为4 g/L,GshF质量浓度为1 g/L时,3 h内GSH产量达到了(58±3.3)mmol/L。
Glutathione(GSH), one of the thiol short peptide in organisms, is widely used in medicine, food additive, health care and cosmetic industries. In this study, a two-enzyme cascade consisting of bifunctional glutathione synthetase(GshF) and polyphosphate kinase(PPK) was constructed for ATP-regenerating synthesis of GSH. As the catalytic substrate of PPK, polyphosphate(polyP) was used to provide energy in the reaction. The efficient soluble expressions of GshF and PPK were achieved when induced at a low temperature of 18℃. After purification, the two enzymes were coupled together to synthesize GSH in one pot. It was found that high concentration of MgCl2 inhibited the production of GSH due to the chelation with polyP. The optimal concentration ratio of polyP to MgCl2 was 30∶45.At 0.5 mmol/L ADP, the reaction achieved the highest efficiency for GSH formation. The optimal temperature for the two-enzyme reaction was 45℃, achieving a high yield of 78% based on added L-cysteine. The optimal temperature for GSH synthesis was relatively high, because the activities of GshF and PPK could increase with temperature. The enzyme ratio in the system was further investigated. The production of GSH was improved at higher concentration of PPK, indicating that the ATP-regenerating reaction was a limit step in the reaction. At the optimal ratio of 4 g/L PPK and 1 g/L GshF, the titer of GSH reached(58 ± 3.3) mmol/L with a productivity of(19.3 ± 1.1) mmol/L/h at 3 h, which achieved the reported highest production of GSH. Using the low-cost substrate of polyP to supply energy, the efficient cascade reaction provided a potential application for large-scale enzymatic production of GSH.