Selection Of Strains Producing Xylanase, Xylanase Properties And Construction Of A Metagenomic Library To Screen Xylanase Gene

Xylanases can hydrolyze xylans into xylooligosaccharides and D-xylose. Xylanases have wide commercial applications in industrial processes, such as the feed industry, the paper and pulp industry, the food and weave industry. The property, research advancement and application of xylanase were summarized in the present dissertation. This study carried out a series of experiments to increase the number of strains producing xylanases and to guide the industrial processes. The experiments include the selection and identification of xylanase strains, and construction of a metagenomic library to screen xylanase gene.12 bacteria strains with xytanase were isolated using the method of transparent zone from the soil samples collected in "Tengchong" hot spring of Yunnan province. Their xylanase activities were assayed with the method of DNS. The strain named Xylan-12 was the strongest in terms of producing xylanase. The strain was identified by sequencing it's partial 16S rDNA sequence, and found that it was most similar to Bacillus flavothermus by BLAST homology analysis.Orthogonal design was done to optimize the fermentation condition to increase xylanase activity. The best condition for Bacillus flavothermus producing xylanase was hemicellulose+ wheatbran used as carbon, (NH₄)₂SO₄ as nitrogen source, fermentation time 72h and initial pH6.0. It was ascertained the optimum medium was(g/L): hemicellulose 6, wheatbran 4, (NH₄)₂SO₄ 0.5, K₂HPO₄I.O, MgSO₄-7H₂O 0.3, CaCI₂-2H₂O0.2, K₂SO₄0.1, NaCl0.2, Tween-80 0.1%, pH7.0. The xylanase activitywas increased by 21.4% through the optimization. The research was also performed on the properties of xylanase produced by Bacillus flavothermus to ascertain the optimum temperature and pH and resistant of xylanase by changing the working condition. The result was that the optimum temperature and pH were 65°C and 6.0, respectively. It was approved that this xylanase was megatemperature-resistant because it also had 70% enzyme activity at 80°C. The property of megatemperature-resistant was benefit for the industrial processes. The stability was the best at pH7.0 and the range of pH stability was wide.Genomic DNA of uncultured microorganism from the soil samples was extracted, end-blunted and legated with the plasmid vector of pUC18 to construct a metagenomic library containing about 1.2×10⁵ clones. And the capacity of foreign DNA of the library was about 3.0xl05kb. The achievement ratio of insert DNA fragment was 87%. The work of preservation and identification are being done. We had identified 2 clones with transparent zone around them from the clones about 0.4×10⁵ which had been preservated and screened.