| With the rapid development of society, human beings are facing to the social economic growth and environmental protection, survival development and energy consumption of the multiple contradictions and pressure. Traditional energy mining is increased continuously, many non-renewable resources constantly reduced and the pollution problem is more serious, at present the most urgent problem is trying to explore and develop new energy, and as one of the new generation of energy, biomass energy will make outstanding contributions to solving energy and environmental issues for human society. This research point is constructing the macro genomic library by extracting rumen content total DNA from miscanthus domestic xiangxi scalpers, and search for new cellulose enzyme gene from the library by using the function screening method, the detailed results of our study were as follows:One CMCase functional clone was obtained from the metagenome library, named185C10.Through the sub-cloning experiment, a total length of2162bp nucleic acid sequence which could code active enzyme were obtained, The sequence was analysed by the ORF Finder of NCBI, the results indicated that the longest ORF is1731bp which located in from107bp to1837bp, the gene was named uncel185C10, the content of （G+C） was48.94%, it could code a protein contains576amino acid residue. Thought the forecast of ProtParam online tools, the molecular mass of the protein that were coded by Uncel185C10is62654.2Daltons, the isoelectric point pI is4.90, in the composition of amino acid, the propotion of Gly reached up to10.9%（63/576）, the lowest amino acid is His which is only0.7%（4/576）, this protein instability index is22.86, the predicted value showed that the protein is stable. Through the analysis of SMART （http://smart.embl-heidelberg.de/）, the predicted result is that the amino acids from1to300are the highly conservative structure function domain of glycosyl hydrolases family eight. In the NCBI database comparison, the results show that high homology between the uncel185C10’s predictive coding protein and the glycoside hydrolase family protein|YP003249877.1|come from Fibrobacter succinogenes subsp, the consistency is79%, the similarity is89%. The uncel185C10’s predictive coding protein compared with the putative carbohydrate-active enzyme （gb|ADX05710.1|） come from cultured organism was indicated that the consistency is57%, the similarity is71%. We inferred that Uncel185C10protein may be some specie of from bovine rumen bacteria bacterium. According to the known similar protein sequence for further analysis of system evolution, phylogenetic analysis showed that Uncel185C10had the closest relationship with glycoside hydrolase family protein of Fibrobacter succinogenes subsp genetic in the evolutionary tree. Thus the protein could well be come from Fibrobacter succinogenes. The study of enzymatic properties of Uncle185C10indicated its optimal reaction temperature was40℃（specific activity:6.258×103U/mg）. At30℃for1h, it can keep above80%enzyme activity, at37℃and50℃for30min, the enzyme activity can maintain above80%,When keep it at above60℃, the enzyme activity would drops rapidly. The Optimum pH of enzemy is4.5, and in the environment of pH4.5or pH5.5the enzemy has good stability and relative enzyme activity remained at above80%, when pH less than3.5or more than7, the relative enzyme activity would declined.The impact of K+on Uncel185C10’s enzyme activity was acceleration, but the rest of the metal ions reduced its enzyme activity, among them, the Fe3+and Cu2+inhibition were the most significant. In addition, the metal ion chelating agent EDTA make enzyme activity reduced to60%.The findings suggested that the cellulolytic microbiome in the local cattle’s rumen could be induced by sole diet M. sinensis, and one CMCase encoding gene uncel185C10was screened from the metagenome library of ruminal microbes of M. sinensis feding cattle. Uncel185C10will be benefit to futher utilization of cellulose in M. sinensis. |
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