Expression And Characterization Of Pyruvate Ferredoxin Oxidoreductase From Thermotoga Neapolitana

Thermotoga neapolitana is a strictly anaerobic gram-negative hyperthermophilic bacterium with an optimal growth temperature of 80?.T.neapolitana utilizes a wide range of substrates to produce hydrogen and trace amounts of ethanol.One of the key enzymes in its metabolic pathway is pyruvate ferredoxin oxidoreductase?PFOR?.PFOR holoenzyme consisted with?,?,?and?subunit.The PFOR enzyme catalyzes the conversion of pyruvate and coenzyme A?CoA?to acetyl-CoA.It uses thiamine pyrophosphate?TPP?as a coenzyme.T.neapolitana PFOR gene was cloned in this study.PFOR from T.neapolitana was expressed clearly in E.coli.The recombinant PFOR was purified by a heat treatment followed by an ion exchange chromatography,and the properties of PFOR from T.neapolitana have been elucidated.The application of thermostable recombinant PFOR to catalyze the conversion of pyruvate into acetyl-CoA was also evaluated.The main research contents and results are as follows:?1?Construction of PFOR recombinant expression plasmid pET-PFOR:A PCR method was used to amplify the target gene fragment from the whole genome of T.neapolitana.?2?Expression and purification of recombinant PFOR.The recombinant expression plasmid was transformed into host E.coli BL21?DE3?and induced by IPTG inducer.The recombinant PFOR enzyme was purified by a heat treatment followed by an ion exchange chromatography.?3?Properties of recombinant PFOR.The effects of temperature and pH on the activity and enzyme stability of PFOR was evaluted.The optimal reaction of PFOR occurred at 90?,pH 8.0.It is thermostable,has a half-life of more than 1 hour at90?,and retains more than 80%of its activity when incubated at 85?for 1 hour.?4?Applications for acetyl-CoA production using thermostable PFOR.To optimize the acetyl-CoA production,the influence of different reaction conditions on conversion ratio was examined and discussed.At 90°C,pH 8.0,the rate of synthesis of acetyl-CoA by the recombinant PFOR enzyme was the fastest,reaching 15.1?mol min^-1 mg^-1.The rate of acetyl-CoA synthesis increases with increasing pyruvic acid concentration.