The Expression And Function Research Of Pyruvate Kinase ? Monomer

Adenosine triphosphate(ATP)is widely used in medical field,which has a good curative effect on hepatitis,stroke sequelae,myocardial diseases and other diseases.However,due to the enzyme of the synthesis of adenosine triphosphatase is expensive,it not widely used in industry.In the presence of Mg2+ inside and outside the living body,Pyruvate kinase(PK)is able to catalyze the transfer of phosphate group from phosphoenolpyruvate(PEP)to adenosine diphosphate(ADP)to produce pyruvate(PYR)and ATP.This reaction provides a new way for the synthesis of ATP enzyme method;and the ATP regeneration system based on PK and PEP,using recycled ATP cheap substrate instead of ATP,and can effectively improve the economic efficiency of the production process of useful substances.In this study,the Pyk A amino acid sequence was analyzed by DNAMAN and MEGA7 software.It was found that The amino acid residues of Pyk A involves that make up ATP and PEP binding sites are highly conserved.Then in this study,the expression vector pET28a(+)-pyk A was successfully constructed by using genetic engineering technique,and the expression of Pyk A was highly expressed in E.coli BL21:from the 1L bacteria liquid can be extracted 34 mg protein;Pyk A was purified by nickel column and the purified protein was quantified at a concentration of 2.3 mg/m L.The PykA's catalytic function was detected by high performance liquid chromatography(HPLC)and it turns out that at 30?,pH 8 environment,the concentration ratio of ADP and PEP was 1:1,no matter the reaction is carried out in 0.5h or 1H,0.05mg/mL Pyk A could not completely convert ADP to ATP,ATP accumulation has an inhibitory effect on Pyk A-catalyzed phosphorylation of ADP;when the concentration of ADP and PEP is1:2,0.05mg/m L Pyk A can completely convert ADP to ATP for only 0.5h.The results of this study can achieve the better application of pyruvate kinase in ATP regeneration system,and provide new ideas for enzymatic synthesis of ATP.