Co-Expression of Recombinant Nucleoside Phosphorylasefrom Escherichia coli and its Application

摘要

The genes encoding purine nucleoside phosphorylase (PNPase), uridinephosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12were cloned respectively into expression vector pET-11 a or pET-28a. The recombinantplasmids were transformed into the host strain E. coli BL21(DE3) to construct four co-expression recombinant strains. Two of them had double recombinant plasmids (DUD andDAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Underthe repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymeswere abundantly expressed after induction with IPTG and large amount of target proteinswere expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain,enzyme activity of the recombinant strains had been notably improved. In the trans-glycosylation reaction, yield of 2,6-diaminopurine-2'-deoxyriboside (DAPdR) from 2,6-diaminopurine (DAP) and thymidine reached 40.2% and 51.8% catalyzed by DAD andTDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridinereached 88.2% and 58.0% catalyzed by TDU and DUD respectively.