title: Cloning and characterization of purine nucleoside phosphorylase in Escherichia coli and subsequent ribavirin biosynthesis using immobilized recombinant cells.
abstract: With improved enzymatic activity and easy accessibility, the recombinant purine nucleoside phosphorylase (PNPase) could be a very promising alternative for nucleoside biosynthesis. In our work, the deoD gene encoding PNPase was successfully cloned from Escherichia coli MG1665 and overexpressed in E. coli BL 21(DE3). After optimization of expression conditions including temperature, induction timing and isopropyl-thio-β-D-galactoside (IPTG) concentration, over 70% of expressed total protein was His-tagged PNPase, in the soluble and functional form. Followed assays indicated that the recombinant enzyme exhibited similar substrate specificity and pH preference as the wild type PNPase. Furthermore, the immobilization technology was applied to develop the possible application of recombinant enzyme. Agar from four different polymer carriers was selected as a suitable matrix for whole recombinant cell entrapment. Subsequent enzyme assays, kinetic analysis and stability evaluation of free and immobilized recombinant cells were compared. The results indicated that although the immobilization process reduced the substrate affinity and catalytic efficiency of recombinant cells, it could significantly enhance the stability and reusability of these cells. Finally, the immobilized whole cell biocatalyst was applied to produce ribavirin, as a model nucleoside synthesis reaction. The obtained relative high productivity of ribavirin and quick reaction time suggested the great potential and feasibility of immobilized PNPase in efficient and valuable industrial utilizations.
authors: Institute of Biological Engineering, Department of Chemical and Biological Engineering, Zhejiang University, Zheda Road 37#, Hangzhou 310027, Zhejiang, PR China.
authors: Wei
authors: W
authors: Luo
authors: Yunping
authors: Y
authors: Liu
authors: Xiangcheng
authors: X
authors: Zhu
authors: Weijun
authors: W
authors: Zhao
authors: Lei
authors: L
authors: Huang
authors: Jin
authors: J
authors: Cai
authors: Zhinan
authors: Z
authors: Xu
authors: Peilin
authors: P
authors: Cen
mesh_terms: D000362:Agar; D055162:Biocatalysis; D018914:Cells, Immobilized; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005782:Gels; D005798:Genes, Bacterial; D006151:Guanosine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011683:Purine-Nucleoside Phosphorylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012254:Ribavirin / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature; D014230:Triazoles / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D005782:Gels; D011993:Recombinant Fusion Proteins; D014230:Triazoles; D006151:Guanosine; C017957:1,2,4-triazole-3-carboxamide; D012254:Ribavirin; D000362:Agar; D011683:Purine-Nucleoside Phosphorylase