Screening And Diversity Analysis Of Protease And Dipeptidyl Peptidase â…£ Gene From Rumen Microbes Of Dairy Cows

During the degradative processing of diet protein, proteases are the key enzymes which hydrolyzeprotein to peptide or amino acid. However, little genetic information of protease originating from rumenmicrobiota is known due to the limitation of pure-cultured methods. To screen protease clones fromrumen microbial Fosmid library,14positive clones with protease activity were got by screening30000clones from skimmed milk powder’s and soy protein powder’s selective medium of proteases. Thezymolytic ability of protease from fourteen clones was measured by Folin-Phenol reagent method. Theresults showed that each clone had its unique ability of protease decomposition. The enzyme activityranged from0.59to2.74U/mg and from0.70to7.19U/mg with casein and isolated soybean protein asthe substrate respectively. The end sequences of pro10F have54%similarity with metal peptidasebelonged to peptidase family M13, and the optimal pH of the protease was7.0. Inserted fragments fromtwo clones were sequenced using shotgun sequencing method, and53different protein sequences weregained by GenMark analysis, two of which belonged to different peptidases family.To characterize dipeptidyl peptidase â…£ (DPP-â…£), the DPP-â…£ degenerate primers were used toscreen rumen microbial Fosmid library. The peptidase activity of extract crude enzyme from thepositive clones was measured using Gly-Pro-pNA as a substrate. Ten positive clones named DP1-DP10containing DPP-â…£ fragment were obtained.78%of the Fosmid end sequences could match with theknown genes (similarity44%-94%). DPP-â…£ sequences contained N-conservative region(D-W-V-Y-E-E-E) and C-catalytic domain (G-W-S-Y-G-G). The activity of DP7peptidase was highest(6.88U/mg). Plasmid of ten clones were extracted, mixed equal amounts and built screening pool, thensequenced by Illumina HiSeq2000.3591genes were obtained by Glimmer analysis. These genefunctions were annotated, and the results showed that these genes take part in various metabolicpathways. Particularly, two different DPP-â…£ genes participated in protein degradation. These resultsprovided basic information for further study of protein metabolic in the rumen.The DPP-â…£ gene of DP7clone which had the highest peptidase activity of crude enzyme from tenclones was studied. Two primers were designed using DPP-â…£ gene (GenBank: JX466878) from DP7clone, plasmid of DP7clone was direct sequenced. The structural feature of DPP-â…£ gene was analyzedby bioinformatics method and DPP-â…£ gene was expressed in BL21competent cell. The analysis ofDPP-â…£ gene sequence showed it had one ORF with2298bp length (756amino acid residue)containing the characteristic catalytic domain G-W-S-F-G-G found in all known DPP-â…£s and theconserved region D-W-V-Y-E-E-E. The BLASTP analysis was done using the prediction of the DPP-â…£gene sequence comapring with GenBank database, and the results showed the highest similarity ofsequences derived from the sequence of Pontibacter sp DPP-â…£ (46%identity) followed bySphingobacterium sp DPP-â…£ (46%), Solitalea canadensis DPP-â…£ (46%), Marinilabilia sp (45%)andCecembia lonarensis (45%). DPP-â…£ had the identification of the catalytic triad (Ser-633, Asp-708andHis-740), and inserted with a length of amino acid sequence from422to445than that of other organisms. The results demonstrated DPP-â…£ gene obtained from DP7was a new sequence of DPP-â…£.The DPP-â…£ gene expression sequence was obtain by PCR amplification from DP7clones usingsequence expression primer, and the target protein of DPP-â…£ was acquired by prokaryotic expressionand purification. The molecular weight of target protein was consistent with the predicted molecularweight (78kDa) indicating that could proceed with the study of DPP-â…£ enzymatic properties.To understand the diversity of DPP-â…£ gene sequence structure derived from soybean mealdegradation bacteria, soybean meal degradation bacterium was collected at different time points (0,12,24and48h) by nylon bags technique. The total DNA was extracted, and then16S rDNA V3region wasamplified and analyzed using denaturing gel gradient electrophoresis (DGGE). Result showed thatsoybean meal degradation bacteria had obvious change at12,24and48h time points comparing to0h.Bands of bacteria flora at0h was richer than other time points, while bands at12h was larger than that24and48h. DNA of soybean meal degradation bacteria at12h was select as amplify templates ofDPP-â…£ gene degenerate primers. Five sequences of DPP-â…£ gene were obtained by PCR amplification,cloning and sequencing. ClustalW alignment showed that all five sequences had a conserved region(D-W-V-Y-E-E-E), but the characteristic catalytic domain (G-W-S-F-G-G) contained two mutations(Sâ†'T or R). By gene amplification, the full-length sequences of DPP-â…£ gene were acquired fromDNA mixed samples of soybean meal degradative bacteria at different time points. The length ofDPP-â…£ genes in twenty clones were2193bp. It contained the characteristic catalytic domain(G-W-S-F-G-G) and the catalytic triad composition, but the conserved region (D-W-V-Y-E-E-E) hadtwo mutations (Vâ†'A and Nâ†'S). The four mutations needed to be further study on whether affect theactivity of DPP-â…£.To reveal the genetic diversity of anaerobic bacterial DPP-â…£ gene in vitro cultivation and theinfluence of monensin in bacteria including DPP-â…£ gene, there were two group: one group substratewas casein (P), other group added monensin on the basis of casein (M). Anaerobic culture was doneafter inoculated with rumen fluid respectively. And then anaerobic bacterium was collected after12hculture and its DNA was extracted. DPP-â…£ gene library was established through PCR, and sixty-sixsequence of DPP-â…£ gene were obtained by sequencing the picking clones. Twenty-three operationaltaxonomic unit (OTU) were found by Mothur software analysis, and OTU9might be a DPP-â…£ gene ofprotein degradation bacteria sensitising to monensin. The copy number of DPP-â…£ gene in P group wassignificantly higher than that of M group (P<0.01) by quantitative analysis. These results suggested thatmonensin affected the number of bacteria included DPP-â…£ gene.