The Separation And Purification Of Active Protease From Goat Rumen Metagenomic Library

Herbivores do not secrete cellulases and hemicellulases by themselves, but its rumens have very rich microorganisms, which can secrete cellulases and hemicellulases that help the herbivores digest plant fiber. With the eating of plant fiber, animals also eat some plant protein. Heterologous proteins first reach rumens, then are degraded into ammonia by microorganisms. Part of ammonia is to synthesize microbial protein, and meet the need of microbial life.The rest is absorbed and cycled by rumen reuse, or excluded body outside by nephritic. The degradation of protein in rumen plays a very important role in the stability of microbial community in the rumen. Compared with in-depth study of cellulases and hemicellulases, the study of protease in rumen is a little relatively.We had already established metagenomic library of the goat rumen, screened two positive clones on skimmed milk tablet,namely R-P-land R-P-2. By gene sequence and annotation, we found that R-P-1 only had one protease (1SP) gene. Without one uncomplete protease(2SP4) gene, R-P-2 had 3 complete proteases (2SP1/2SP2/2SP3) gene and one metallopeptidase (2P)gene. The five proteases were serine proteases, belonging to the family of the S8; metallopeptidase belonged to M48 family, depending on Zn2+.On the basis of the above, we set and blasted the four completed proteases sequence and one metallopeptidase sequence in protein data. 1SP was a hypothetical protein with unknown function,which came from Ruminobacter sp. RM87.Three proteases and metallopeptidase also had higher identity with unknown functional hypothetical proteins,which from Ruminobacter sp. RM87.we used SMART to predict the domain of five proteins,and found that they were S8 family protases and M48 family metallopeptidase. Four proteases had long unknown functional amino acid sequence at two flanks of S8 domain.By secondary structure prediction of five protein, disorder ratio of protease was higher, while alpha helix of 2P had higher proportion.To build tertiary structure by I-TASSER tool, only 2SP3 and 2P typical globular structure could form typical globular structure, 1SP/2SP1/2SP2could form typical globular structure, but also had a long N-terminal long arm at the surface of globular structure.We first concentrated extracellular protein of R-P-1 by ammonium sulfate precipitation, then purified 1SP by strong anion exchange chromatography or gel filtration chromatography separation, but we could not get the 1SP because of the little of production. We have expressed soluble and considerable amount of 2P in intracellular escherichia coli, but we failed to measure activity using the casein. We suspected that the catalytic site of 2P had mutated, leading to loss of activity. We set Escherichia coli as host, and expressed active extracellular 2SP3 and intracellular inclusions, respectively. Preliminary separation and LC-MS/MS determined the full-length of 2SP3 gene expressed the active protease and C-terminal played very important role in activity and stability of 2SP3. The little expression of active 2SP3 and low inclusions renaturation rate, caused great difficulties toget a lot of active 2SP3.