Isolation,Purification,Properties And Modification Of Groups Of The Gluathione Reductase From Baker's Yeast

Gluathione reductase?GR?is an antioxidant enzyme,belonging to the flavoprotein disulfide oxidoreductase family and widely found in eukaryotes and prokaryotes.Glutathione reductase is a homodimer composed of two subunits in many organisms.As a key member of glutathione metabolism,GR plays an important role in redox metabolism.It catalyses the reduction of oxidized glutathione?GSSG?to reduced glutathione?GSH?in the presence of coenzyme NADPH,maintaining the dynamic balance of GSH/GSSG ratio in the cell that is responsible for the supply of reduced glutathione in the cell,and providing for the removal of reactive oxygen species?ROS?.However,the maintenance of redox homeostasis is particularly important for cells to perform normal physiological functions.If the body's oxidation system and reduction system are out of balance,the cells will be damaged and the body's metabolism will be abnormal.In recent years,the research on glutathione reductase in animals has mainly focused on the role of GR in the occurrence and defense of some diseases,and used it as a therapeutic target for some diseases?such as inflammation,fungal infections,cancer and tumors?to guide drug design.In addition,GR can also be used as a predictor of some diseases.In terms of plants,the research on glutathione reductase has mainly focused on the study of its genetic characteristics and its role in the response of plants to abiotic stresses,which are used to transform more resistant plants and crops.In the aspect of microorganism,the research on GR has mainly focused on the role of glutathione reductase in the development of some fungi,bacteria,and malaria parasites,and has used in the development of some anti-malarial drugs.In general,there is relatively little research on the specific structural information of glutathione reductase and the structure-activity mechanism at the molecular level.At present,glutathione reductase has been used in medicine or agronomy,and has broad application prospects.This study used the baker's yeast as the experimental material which is cheap and easy to be cultured on a large scale.Glutathione reductase genes from different sources were selected on NCBI for bioinformatics analysis,and the physical and chemical properties,enzymatic properties and functional groups of glutathione reductase were studied.This study provided a reference for the industrial production and application research of glutathione reductase.The experimental results are as follows:1.The full-length of the gene of GLR1 was 2743 bp,with an open reading frame?ORF?of 1401 bp.GLR1 encoded a protein of 467 amino acids.The amino acid sequence of glutathione reductase mainly contains two conserved domains.Active site 1is located at amino acid sequence 20 to 333,and active site 2 is located at amino acid sequence 356 to 466.Phylogenetic analysis showed that the genetic relationship between the glutathione reductase gene of Dictyostelium discoideum and the glutathione reductase gene of other species was far away,and the glutathione reductase gene of the remaining 9 species Clustering formed two large branches.Pea,tobacco,Arabidopsis and japonica gathered together,indicating that the glutathione reductase gene genes of these several species were closely related.Aspergillus nidulans,Saccharomyces cerevisiae and Schizosaccharomyces pombe were grouped together,indicating that the glutathione reductase gene of these three species was closely related.2.After optimization by response surface method,the optimal extraction conditions were determined to be extraction buffer pH 7.14,liquid-material ratio?V/m?10.7:1,and extraction time 2.05 hours.After grinding extraction,ethanol fractionation,DEAE-Sepharose ion exchange chromatography and Superdex-200 gel filtration chromatography,electrophoretic pure glutathione reductase was obtained.The purification results showed that the specific activity of glutathione reductase was2471.88 U/mg,the purification multiple was 63.76 times,and the recovery rate was2.14%.3.Glutathione reductase of baker's yeast is a homodimer composed of two subunits of about 55.6 kD,and the molecular weight of the whole enzyme is about 120.4 kD;the optimum temperature was 45?,and the optimum pH was 7.2.The enzyme has good stability at 20?45?,pH 6.0?7.5.Under optimal conditions,using oxidized glutathione?GSSG?as the substrate,the measured K_m value was 0.41 mmol/L,and the V_max was0.176 mmol/L.The inhibitory effect of Mn²⁺,Cu²⁺,Zn²⁺,Fe³⁺and Co²⁺on the activity of glutathione reductase was stronger,and the low concentration showed the inhibitory effect.Low concentrations of Ba²⁺,Ca²⁺,Na⁺,Pb²⁺,and Li⁺had no significant effect on the activity of glutathione reductase,and high concentrations showed inhibitory effects.Low concentration?below 1 mmo L/L?Mg²⁺and K⁺promoted the enzyme,and then as the concentration increased,the activation effect continued to weaken.The four organic solvents methanol,ethanol,isopropanol and n-butanol all inhibited glutathione reductase.The four compounds had different effects on the activity of glutathione reductase.Folic acid had a significant activation effect on the enzyme activity.Ascorbic acid had no significant effect on this enzyme.EDTA played a dual role,and it can activate the activity of GR enzyme at a lower concentration.When the concentration was higher than 1.0 mmol/L,it showed an inhibitory effect on this enzyme.SDS had an inhibitory effect on the activity of glutathione reductase,and as the concentration continued to increase,the inhibitory effect gradually increased.4.The chemical modification of glutathione reductase side chain groups with specific chemical modifiers showed that:lysine?-NH,disulfide bond,arginine guanidinium group,cysteine sulfhydryl group,histidine imidazole group,methionine thioether group,glutamic acid/aspartic acid carboxyl group,tryptophan indole group and tyrosine phenolic hydroxyl group were the essential group of the active center of glutathione reductase.