Expression, purification and characterization of 5′-nucleotidase from caterpillar fungus by efficient genome-mining
Shan Lin, Cuibing Zhou, Hancheng Zhang, Zhiming Cai
Protein Expression and Purification
5′- nucleotidase (5′-NT) is a key enzyme in nucleoside/nucleotide metabolic pathway, it plays an important role in the biosynthesis of cordycepin in caterpillar fungus. In this study, a 5′-NT gene was identified and mined from genomic DNA of caterpillar fungus, which was 1968 bp in length and encoded 656 amino acid residues. The recombinant 5′-NT was first time heterologously expressed in Pichia pastoris GS115, subsequently purified and functionally characterized. The optimal reaction temperature for 5′-NT was 35 °C, and it retained 52.8% of its residual activity after incubation at 50 °C for 1 h. The optimal reaction pH was 6.0 and it exhibited high activity over a neutral pH range. Furthermore, 5′-NT exhibited excellent Km (1.107 mM), Vmax (0.113 μmol/mg·min) and kcat (4.521 S-1) values compared with other typical 5′-nucleotidase. Moreover, substrate specificity analyses indicated that 5′-NT exhibited different phosphatase activity towards the substrates containing different basic groups. The work presented here could be useful to 5′-NT applications and provide more scientific basis and new ideas for the biosynthesis of artificial control cordycepin.
Circular dichroism, Secondary structure, Protein folding, Thermal stability, Biochemistry