The Screening And Isolation Of Proteases From The Goat Rumen-derived Metagenome Library

Rumen is an important digestive organ of ruminants, which is enriched of many microorganisms including bacteria, fungi, protozoa and so on. And about90%of these microboes are uncultured and unculturable. The protein degradation in the rumen provides40%of nitrogen resource to ruminants. In order to complete the degradation the proteases profiles of rumen are complex. But compared with polysaccharide-degrading enzymes, the studies of rumen proteases are relatively fewer. So we think it is a good resource to be excavated. Therefore, we made use of metagenomic techniques to screen the novel proteases from the the metagenomic library of goat rumen.A goat rumen metagenome library was constructed in our laboratory earlily. In this thesis, based on function-driven screening strategy, we used skim milk as the substrate to screen the library, and got two positive clones:R-P-1and R-P-2. By SDS-PAGE zymography analysis in which casein was used as substrate, we found that R-P-1could produce one protease activity band as well as R-P-2was able to produce3protease bands. Then terminal sequencing was implemented, the amino acid sequences had low similarity with the existing sequences in GenBank, showing its novelty. Full length sequencing and annotation of R-P-1and R-P-2showed that a serine protease-encoding gene (ISP1) in R-P-1and4serine proteases-encoding gene (2SP1-2SP4) and a peptidase-encoding gene (2P) in R-P-2. These protein sequences showed low homology with the known sequences in the database. Protein phylogenetic tree analysis revealed that these serine proteases evoluted as a separate branch, distantly related to the same family known proteases, which further explained the novelty of these proteases. The prediction of the subcellular localization of these proteases showed that most proteases located in the extracellular or outer membrane, which was benefit to extracellular protein degradation.4serine protease genes in R-P-2all encoded protease to belong Peptidase_S8family, however there are some differences in the sequence, suggesting that the function had a certain complementarity of collaborative and efficient degradation of the extracellular complex protein.We prepared the crude protease sample from the culture fluid of R-P-2. The result of analysis showed that the proteases of R-P-2had a wide pH tolerance range. The crude enzyme samples were precipitated with60%saturated ammonium sulfate, then were isolated through ion exchange chromatography with Q-XL column and molecular sieve chromatography with Superdex7510/300GL column. After SDS-PAGE detect, we found two constituents which show protease activity:RP2a and RP2b. RP2a was identified as2SP1and RP2b was identified as2SP3by LC-MS/MS. These results could help us to do further study of enzymatic properties.