The Breeding Of Producing Xylanases Strains And Xylanase Property And Expression Of XynA Gene

Xylanases can hydrolyze hemicelluloses, which are the second abundance renewable resource. Xylanases have a broad prospect in animal feed industry, paper industry, food industry and energy bioconversion etc. This study carried out a series of experiments: screening, identification, mutation, properties of xylanase and expression of xynA. The main results are follows:Dozens of strains were screened on hemicellulose plats from soil and bagasse. The topmost activity of xylanase was 2.9 IU. Through analysis of 16S and 18S rDNA sequence, We discovered that the higher yields of xylanase were all Bacillus and three strains were assigned to the genus Acinetobacter, Sphingobacterium and Klebsiella, which have not been reported on xylanase-production strains in these genus. In order to increase the yields of xylanase, we use physical and transposon mutation. The results showed that the strains increased its yields rapidly mutated by UV and (60)~Co , but this ability can't steadily inherit. Transposon mutagensis of XY3401 hadn't found the positive mutants.The study on properties of xylanases by Bacillus subtilis XY1905 and Bacillus pumilus XY1432 were carried out. The results showed that the activityof xylanase by XY1905 was remained 96.44% after 1h at 80℃, and remained 60% after 2h at 40℃ in pH 2-3. Xylanase by XY1432 is stable in alkaline region. Both of them are all insensitive to metalline ions.Response Surface Methodology was used to optimize the fermentation condition of xylanase production of XY1905 and XY1432. The optimized condition allowed the xylanase production by XY1905 to be increased from 2.04 to 17.78IU; XY1432 to be increased from 4.25IU to 12.54 IU.A pair of primers was synthesized according to the sequence of Bacillus pumilus xynA through the GenBank for PCR with XY1432 genomic DNA as the template. The PCR product was inserted to plasmid pSE380. After transforming competent E.coli JM109, the recombinants were screened on the selected plates. Then recombinants were grown and induced with IPTG, low activity of xylanase can be measured.