Cloning, Expression, Purification And Immobilization Of Pyruvate Oxidase From Aerococcus Viridans

Pyruvate oxidase (EC 1.2.3.3) is thiamine pyrophosphate (ThPP)-dependent enzyme which catalyzes the oxidative decarboxylation of pyruvate in the presence of phosphate and oxygen, yielding acetyl phosphate, carbon dioxide, and hydrogen peroxide. Pyruvate oxidase plays an important role in biochemical test, including clinic diagnosis, as well as online monitor of pyruvate during various fermentation processes. Therefore, high yield production of stable pyruvate oxidase is of great importance.In this study, pyruvate oxidase was prepared recombinantly and novel procedure of pyruvate oxidase immobilization was developed to facilitate its application in biosensor production. First, the gene encoding pyruvate oxidase (AvPOX) was cloned from Aerococcus viridians ATCC10400, recombinant plasmid was transformed into E. coli BL21 (DE3) and Pyruvate oxidase was expressed. Pyruvate oxidase was purified by Ni-NTA column and assayed for its enzyme activity via 4-AA method. Results showed that Pyruvate oxidase was able to be produced at high yield from E. coli, with apparent specific activity of 1.0 U/mg. To promote utility of Pyruvate oxidase in producing Biosensor enzyme element, AvPOX gene was fused with various cellulose binding domain sequences generating following constructs:pET24d/POX-CBD-His, pETM10/His-POX-CBD, pET24d/POX-CBM3-His, and pET 28a/CBM3-POX-His. The specific activity of CBM3-pyruvate oxidase fusion protein was 0.3 U/mg, meanwhile, Binding efficiency Wt is 11.00mg/(g·h).In this study, we successfully expressed CBM3-pyruvate oxidase fusion protein. The enzyme immobilization with CBM3 would be more advantage than the traditional immobilization methods such as glutaraldehyde due to its easier operation and lower activity loss. The new immobilization method would be potential in biosensor-used enzymes.