关键词: 土壤假单胞菌, 亚磷酸盐脱氢酶, 基因克隆, 原核表达

Abstract: Phosphite dehydrogenase（PTDH）catalyzes the oxidation of phosphite to produce phosphate and NADH,using NAD⁺ as the cofactor. This enzyme is of large application potentials in the areas of coenzyme regeneration and phosphite-based phosphorus utilization,and etc. Herein,the full-length of a putative PTDH gene（PsPtx）was obtained by two rounds of PCR from the soil metagenome as DNA template,which was then subcloned into plasmid pET32a（+）by enzyme digestion to generate the prokaryotic expression vector pET（PsPtx）. Sequence analysis showed that the PsPtx gene had an entire coding region of 1 011 bp,encoding a protein of 336 aa and 36.5 kD. The results of the conserved domain prediction demonstrated that the deduced protein PsPtx was a member of the PTDH family,and had the conserved NAD⁺-binding motif as well as the crucial catalytic residues. Furthermore,phylogenetic analysis indicated that the PsPtx gene was originated from an undefined soil Pseudomonas species. In addition,PsPtx efficiently expressed in Escherichia coli strain BL21（DE3）by IPTG induction. The recombinant protein PsPtx was purified by His-tag affinity chromatography,and then detected to have a specific activity of 3.75 U/mg towards its substrate,sodium phosphite. Certainly,this cloned PsPtx gene with verified activity is of fundamental importance for its future application studies.

Key words: soil Pseudomonas species, phosphite dehydrogenase, gene cloning, prokaryotic expression