Screening And Characterization Of Urease And Lipase Clones From A Metagenome Library Of Dairy Rumen Microflora

Urease and lipase clones were screened from rumen microbes. Enzymatic properties were studied and ureolytic bacterias were revealed. Three parts were as follows:Part 1: By the way of urease segregation agar, sixteen urease positive clones were obtained from the rumen bacterial artificial chromosome library containing 15 360 clones. They had different urea hydrolysis activities by phenol-hypochlorite urease assay ranging from 30 to 2 466 U/mg. The insert size in urease positive clones was about 60 kb. The electropherogram of Hind III digested urease positive clones revealed that they had different DNA fragments. And the optimum pH and temperature of U1, U3, U4, U9 clone were 8.0 and 60℃respectively.Part 2: All plasmids of sixteen urease positive clones which were identified as ureolytic clones previously were exacted. Polymerase chain reaction degenerate primers based on the ureC gene which was conserved in ureolytic bacterium were designed and used to amplify the ureC genes from extracted plasmids of positives. The universal 16S bacterial primers were used to amplify the 16S rDNA gene of positive clones. PCR products were purified and ligated to pMD19-T vector. Transformants got by transforming ligation product into E.coli JM109 were sequenced. The sequences were analyzed by Blast on GenBank and RDP, and the phylogenetic tree were built by Mega 4.0. In result, four clones (U1, U5, U13 and U15) containing ureC genes were obtained. The phylogenetic tree o fureC revealed that all four clones were clustered into several bacterial phylums. U1, U5, U13 and U15 belonged to Firmicutes,ε-Proteobacteria,β-Proteobacteria and Actinobacteria, respectively. U1, U3 , U7, U10, U11, U12 and U14 which contained the 16S rDNA sequence had the most identity to Staphylococcus, Shigella, Bacillus, Acinetobacter, Achromobacter, uncultured bacterium and Bacillale. The diversity of ureC and 16S rDNA revealed the diversity of ureolytic bacterial in dairy rumen.Part 3: Using lipase segregation agar containing trioleoylglycerol, eighteen lipase positive clones were screened from a metagenome library construction dairy rumen microflora containing 15 360 clones. The average insert size of lipase positive clones was about 60 kb. Lipase enzyme activity analysis by p-NPP method indicated that Lipase6, Lipase7 and Lipase8 had higher lipolytic activities to substrates of p-nitrophenyl palmitate (C16), p-nitrophenyl alaurate (C12) and p-nitrophenyl palmitate (C16) respectively. The optimum pH of Lipase 6, Lipase 7 and Lipase 8 were 7.5. The half life period of Lipase8 with the value of 15 min in 70℃decreased with the increase of temperature. In conclusion, the lipases screened in this study had different substrates specificity and good thermo stability, which laid a basis for large-scale industrial application.