title: Co-expression of recombinant nucleoside phosphorylase from Escherichia coli and its application.
abstract: The genes encoding purine nucleoside phosphorylase (PNPase), uridine phosphorylase (UPase), and thymidine phosphorylase (TPase) from Escherichia coli K12 were cloned respectively into expression vector pET-11a or pET-28a. The recombinant plasmids 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 and DAD) and the other two had tandem recombinant plasmid (TDU and TDA) in them. Under the repression of antibiotic, recombinant plasmids stably existed in host strains. Enzymes were abundantly expressed after induction with IPTG and large amount of target proteins were 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 transglycosylation 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 and TDA respectively; yield of 2,6-diaminopurine riboside (DAPR) from DAP and uridine reached 88.2% and 58.0% catalyzed by TDU and DUD respectively.
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Chongtao
authors.initials: C
authors.lastname: Ge
authors.forename: Liming
authors.initials: L
authors.lastname: OuYang
authors.forename: Qingbao
authors.initials: Q
authors.lastname: Ding
authors.forename: Ling
authors.initials: L
authors.lastname: Ou
mesh_terms: D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D010430:Pentosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D010430:Pentosyltransferases