Gene Cloning,Expression And Activity Analysis Of The Lactoylglutathione Lyase And Glyceraldehyde-3-Phosphate Dehydrogenase In Corynebacterium Glutamicum

Lactoylglutathione lyases are one of the enzymes degrading pyruvaldehyde.The NCgl0106 gene in the chromosome of C.glutamicum ATCC13032 was predicted as lactoylglutathione lyase gene,which is still not be experimentally verified.In this study,firstly,the predicted lactoylglutathione lyase gene lac of C.glutamicum was amplified by PCR and inserted into the plasmid pET-28a;the recombinant plasmid pET-28a-lac was transformed into Escherichia coli BL21?DE3?,resulting in the engineered strain E.coli BL21?DE3?/pET-28a-lac.Secondly,the recombinant target protein Lac was expressed in E.coli BL21?DE3?/pET-28a-lac by IPTG induction.The SDS-PAGE results showed that the Lac protein was successfully expressed in the form of dissoluble protein in E.coli and its molecular weight is appx.14 kDa.Finally,purification,activity analysis and enzymatic characterizations test of the recombinant Lac protein were carried out.The Lac protein was successfully purified using His-tag and its solution concentration was 135.69?g/mL.The activity analysis result showed the ability of the Lac protein to convert the substrates pyruvaldehyde and glutathione into the product lactoylglutathione,demonstrating that the NCgl0106 expressed protein is lactoylglutathione lyase.The enzymatic characterizations test showed that the optimum temperature of Lac was 60°C,the optimum pH was 7,the metal ions K⁺and Ca²⁺had a slightly enhanced effect on Lac activity,the metal ion Ba²⁺had little effect on enzyme activity,Zn²⁺,Mg²⁺and Fe²⁺had a certain inhibitory effect on enzyme activity and Mn²⁺and Co²⁺completely inhibited enzyme activity;the maximum reaction rate V_maxax of the recombinant enzyme was 0.025mmol/L/min and its Km value was0.2232 mmol/L.In order to verify the correctness of the predicted glyceraldehyde-3-phosphatedehydrogenase?GAPDH?gene in Corynebacterium glutamicum ATCC13032.In this experiment,GAPDH was amplified by PCR and inserted into the plasmid pET-28a followed by digestion with NcoI and SalI.The recombinant plasmid pET-28a-GAPDH was transformed into Escherichia coli BL21?DE3?to obtain correct transformants.The target protein was expressed in E.coli BL2l?DE3?by IPTG induction,the target protein was isolated and purified.The concentration and Enzymatic characterizations of the purified GAPDH protein were determined.The results showed that the recombinant plasmid was successfully constructed.The dissoluble fusion protein successfully expressed in E.coli BL2l?DE3?.The SDS-PAGE and Western Blot showed that the molecular weight of GAPDH is 103 kDa.The purified GAPDH protein solution concentration was 135.69?g/mL.The optimum temperature is 30°C,the optimum pH is 7,the monovalent metal ions K+have a slightly enhanced effect on GAPDH activity.Mg²⁺and Ba²⁺metal ions had little effect on enzyme activity.Zn²⁺,Mn²⁺,and Ca²⁺had a certain inhibitory effect on enzyme activity.Co²⁺and Fe²⁺completely inhibited enzyme activity and Km is 4.73×10^-3mol/L,Vmax is 23.64?mol/L/min.The above results demonstrate that the NCgl0092expressed protein is glyceraldehyde-3-phosphate dehydrogenases.