Cloning Of The Gene Encoding Thermotolerant Cellulase From Streptomyces Xylophagus KX6 And Expression In E.coli

Cellulose is the most wide spread and abundant carbon matrix precursor on the earth whose utilization ratio is relatively low. With the development of gene engineering technology, the genes encoding cellulase have been cloned into bacteria, yeast, fungus and plants for obtaining the recombinant protein with high activity.Actinomycetes can produce a more complete cellulase system, and many of them were thermotolerant. The thermotolerant enzyme has become a hot research due to the production of practical significance. Cloning of the cellulase gene from Streptomyces xylophagus and expressing in E. coli, with a view to obtain recombinant bacteria producing high activity cellulase.The degenerate primers were designed according to some cellulase gene sequences downloaded from Genebank, and the interest gene was obtained using PCR amplification method.Gene fragments were recovered to insert the pMD-18T vector for sequencing. The results showed that the length of cellulose gene is 1140bp, encoding 379 amino acids. On-line analysis of amino acid sequen ces showed that a signal peptide be in the N-side.The recombinant vectors pET-28a/celKX-ns (mature peptide fragment) and pET- 28a/celKX(including signal peptide) were constructed according to signal peptide structural features of the cellulase gene,and transformed into E. coli Rosetta (DE3). The transformants were induced by IPTG.. Expression products were analysed by SDS-PAGE electrophoresis, and showed the specific bands about molecular weight of 39kD and 42kD respectively. The cells precipitation and the supernatant were collected after centrifugation., and the cells precipitation were repeatedly froze and thawed after washing with PBS. The enzyme activity of differrent locations were determined using DNS method.The engineering bacteria containing recombinant plasmid PET-28a/ KX6-ns expressed the target protein which existed mainly in the form of inclusion bodies and showed very low activity, although some soluble.. PET-28a/EGKX6 expressed the purpose of protein in the supernatant and showed the high cellulose activity, secreted the cellulase into the medium about 80.3% of the total enzyme products. This study make the extracellular soluble and high activity of the protein expression be true. Optimization of conditions for inducible expression, the extracellular activity was up to 3.211IU / mL.Characterization of the expression product showed that the optimum reaction temperaturewas at 55oC, the optimum pH value was 5.5 and expression product displayed better thermal stability and pH stability.Preliminary research results showed that recombinant E. coli produced neutral, heat-resistant endo-cellulase with higher enzyme activity. so the enzyme showed the potential application significant in textile field. Further study on the engineered strain will be carried out in the future.