Screening, Cloning And Expression Analysis Of Xylanase Gene Of Fosmid Library From Bovine Rumen Microbia Domesticated By Miscanthus

Nowadays, with the increasingly serious environmental pollution, shortage of mineral resources, the population of the world increases increasingly, everybody have fewer resources, these factors restrict the development style of the high consumption and high pollution, forcing people to reform, and seeking new modes of development. The industrial of lignocellulose into fuel ethanol have been take attention, and developing rapidly, for the raw materials is cheapest. Degradation of lignocellulose requires many cellulase and xylanase to work together, in order to find new xylanase gene, I carrried out a lot of work, and achieved certain results.The materials of this experiment is the Fosmid metagenomic library from Miscanthus domesticated bovine rumen microbial total DNA, and20160clones at all,14high xylanase activity positive clones strains obtained after functional screening method, take the the142-F6, the maximum activity clone, to construct subclone library, and constructed a subclone library containing417clones, at last,8high activitys trains was screened from the subclone by functional screening method, the most active strain will be send to BGI for sequencing, and used to complete the following test corresponding in this test.The sequencing result showed that, the clone contained an insert of2280bp analyzed by ORF Finder software, obtained a2004bp ORF, named xylF6, which encoded667amino acids, the online software prediction, molecular weight of the encoded protein is74.16kDa, pI5.01, unstable coefficient31.81, suggesting that the protein is very stable. The Blast ratioin in NCBI database, the similarities are very low, indicating that the gene is a novel gene.Amplification of the gene after designed the primers, and successfully construct a recombinant expression vector, transferred into the competent cells BL21, induced express the recombinant strain under different conditions, and tested the enzyme activity of crude enzyme solution under various conditions, the optimum induced condition:IPTG induced concentration is0.83mM. induction temperature is30℃, induction time was12h. The enzymatic properties analysis of the protein is obtained after test, the optimum temperature is40℃, the optimum pH value was6.5, it is stability when the temperature is between30℃-50℃, pH value is between5.0-7.0, the activity was still maintained at more than70%after placed30min at this condition, K+, Ca2+, Zn2+and other metal ions had little influence on its activity, residual enzyme activity were maintained at80%, Mn2+, Cu2+, Fe+has greatly influenced the residual enzyme activity, less than20%, the test showed that the enzyme only has decomposition effect on xylan.The results of this study indicate that, we can obtain many new functional genes by constructing a bovine rumen microorganisms Fosmid library, and screening and cloning. The xylF6gene obtained of this test, its expression protein has high xylanase activity, has benefit for the utilize of lignocellulose in the future.