Analysis of dipeptidyl peptidase IV from microbial metagenomic library in the rumen of dairy cow

Abstract Text: The study for sequence characteristics and enzymatic properties of dipeptidyl peptidases IV (DPP-IV), which is the key enzyme of oligopeptide degradation, will contribute to searching for it inhibit targets, reduce the dipeptide generating speed, and then decrease ammonia generation, thereby improve nitrogen efficiency. The DPP-IV gene of DP7 clone which could reduce the dipeptide activity of crude enzyme from microbial metagenomic library in the rumen of dairy cow was studied. Two primers were designed using DPP-IV gene (GenBank: JX466878) from DP7 clone, and plasmid of DP7 clone was direct sequenced. The structural feature of DPP-IV gene was analyzed by bioinformatics method and DPP-IV gene was expressed in BL21 competent cell. The DPP-IV gene expression sequence was obtained from PCR amplification of DP7 clones using sequence expression primer, and the target protein of DPP-IV was acquired by prokaryotic expression and purification. The analysis of DPP-IV gene sequence showed it had one open reading frame with 2,298 bp length (756 amino acid residue) containing the characteristic catalytic domain GWSFGG found in all known DPP-IVs and the conserved region DWVYEEE. The results of BLASTP analysis showed the highest similarity of sequences derived from Pontibacter sp DPP-IV (46% identity), followed by Sphingobacterium sp (46%), Solitalea canadensis (46%), Marinilabilia sp (45%) and Cecembia lonarensis(45%). DPP-IV gene of DP7 had the identification of the catalytic triad (Ser-633, Asp-708 and His-740), and an inserted amino acid sequence from 422 to 445 compared with other organisms. The results demonstrated DPP-IV gene obtained from DP7 was a new sequence of DPP-IV. The molecular weight of target protein was consistent with the predicted molecular weight (78 kDa) indicating that the enzymatic properties of DPP-IV could proceed with further study.

Keywords: dipeptidyl peptidases IV; gene expression; sequence analysis