Purification, Characterization And Construction Of Biosensor By His-tag L-Proline Dehydrogenase From A Hyperthermophilic Archaeon, Thermococcus Profundus

L-proline dehydrogenase is a very useful and well distributed enzyme in living organisms, which catalyzes the oxidation of L-proline in the presence of artificial electron acceptors. Hot spring origin hyperthermophilic archaeon, Thermococcus profundus, was marked by His-tag at the gene fragment of dye-linked L-proline dehydrogenase (Dye-linked L-pro DH). Plasmid represented dye-linked His-tag L-pro DH was extracted and purified, which was mixed by E. coli Rosetta-gami (DE3) Competent Cells to carry out transformation, after fermentation, His-tag L-pro DH was gained through cell free extraction and Ni Sepharose purification. The subunits'molecular weights are of 58.6 kDa,40.2 kDa,22.3 kDa. It proves that His-tag has strong affinity to Ni ion, using Ni Sepharose can reach a good purification effect, as the basis of enzyme sited immobilization. At the experiment of characterization, it was found that the optimal temperature, buffer type and pH value were 50℃,300 mM Tris buffer with pH of 8.0.His-tag L-pro DH is immobilized on a glassy carbon electrode(GCE) modified with multiwall carbon nanotubes by physical adsorption method. It was found that L-pro DH on the modified electrode takes place a reversible redox reaction in 300mM phosphate buffer(pH 7.5) with L-proline. It is obviously proved that bioactivity of L-pro DH on the modified electrode with MWCNTs is kept. QCM was carried out to certify the property of oriented immobilization of His-tag, with a convincible result we believe His-tag L-pro DH can behave a good affinity to Ni ion.