定点突变改造谷胱甘肽合成相关酶及高效催化生产谷胱甘肽  

Modification of glutathione synthesis-related enzymes by targeted mutagenesis and efficient catalytic production of glutathione

作　　者：张秀春 王骏之 徐华栋[1] 沈美华[1] ZHANG Xiuchun;WANG Junzhi;XU Huadong;SHEN Meihua(School of Pharmacy&School of Biological and Food Engineering,Changzhou University,Changzhou 213164,China;College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211816,China)

机构地区：[1]常州大学药学院生物与食品工程学院,江苏常州213164 [2]南京工业大学生物与制药工程学院,江苏南京211816

出　　处：《食品与发酵工业》2025年第6期91-96,共6页Food and Fermentation Industries

基　　金：国家自然科学基金面上项目(22177015);江苏省自然科学基金面上项目(BK20211334)。

摘　　要：谷胱甘肽(glutathione,GSH)是一种含有γ-酰胺键和巯基的三肽化合物,广泛应用于医药、食品和化妆品等领域。谷胱甘肽的生物合成主要由γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase,GSHA)和谷胱甘肽合成酶(glutathione synthetase,GSHB)两步催化得到。在利用天然酶催化生产谷胱甘肽的过程中,发现天然酶的催化效果较差,其中GSHA为反应过程中的限速酶,该酶会受到产物的反馈抑制。该文对来自大肠杆菌(Escherichia coli)的GSHA进行分子改造,对GSHA中参与转化的活性位点进行分析,通过丙氨酸扫描对关键氨基酸进行替换。对蛋白质表面非活性区域的氨基酸进行分析,引入带负电荷的氨基酸提高稳定性。将酶活力提升的突变位点进行组合,获得了催化效果较高的突变体Y131A-A511D,相比于野生型酶活力提高了6倍,并对最佳突变体的酶学性质进行分析。将突变体Y131A-A511D用于催化合成谷胱甘肽,在8 h内合成了76.8 mmol/L的谷胱甘肽,达到了高效催化生产谷胱甘肽的目的。Glutathione(GSH)is a tripeptide compound that contains anγ-amide bond and a sulfhydryl group.It is widely used in the fields of medicine,food,and cosmetics.The biosynthesis of glutathione involves two main steps,includingγ-glutamylcysteine synthetase(GSHA)and glutathione synthetase(GSHB).The production of glutathione using natural enzymes was found to be inefficient due to the rate-limiting enzyme GSHA being poorly catalysed and inhibited by feedback from the product.This study analysed GSHA from Escherichia coli,molecularly modified it,and identified the active sites involved in the transformation of GSHA,making key amino acid substitutions through alanine scanning.This study analysed amino acids located in inactive regions on the protein surface and introduced negatively charged amino acids to improve stability.The Y131A-A511D mutant with higher catalytic activity was obtained by combination of mutation sites with increased enzyme activity,which exhibited a higher catalytic effect and a 6-fold increase in enzyme activity compared to the wild type.The enzymatic properties of the best mutant were analysed.The mutant Y131A-A511D was used to catalyse the synthesis of glutathione,resulting in the production of 76.8 mmol/L of glutathione within 8 hours.This demonstrates efficient the catalytic production of glutathione.

关 键 词：谷胱甘肽 γ-谷氨酸半胱氨酸连接酶 定点突变 丙氨酸扫描 酶催化 

分 类 号：O629.72[理学—有机化学] TQ426.97[理学—化学]