| The thermostability of xylanase (E.G. 3.2.1.8) is critical to its activity. The feed reaches temperatures between 70~80℃ during the pelleting process by stem addition in the conditioner, and the process can, therefore, lead to losses in activity of added xylanase. It becomes a very important issue to increase the thermostability of xylanase. The purpose of this study was to screen the wild strains of xylanase producing microbes, to clone xylanase genes or cDNA, to express these genes in the E. coli using pET 30(a)+ as expression vector, to assess the properties of differently expressed xylanases. Furthermore, the hybrid xylanase gene HX, which can express thermostable xylanase, was designed and constructed using Splicing by Overlap Extension (SOE) technique, and expressed in E. coli. The biochemical properties of purified hybrid xylanase, particularly in the enhanced stability at higher temperature as compared to those of the parents, were also investigated in this study. The main results obtained are as follows:Four wild strains, Bacillus subtilis 2, Bacillus circulans BC, Aspergillus niger FS6 and Thermomonospora fusca TF, were screened for xylanase activity on RBB-xylan (4-O-methyl-D-glucurono-D-xylan-Remazol Brilliant blue) plates. The yields of xylanase from the top down, produced by different strains in liquid cultures, were A. niger FS6 (xylanase ANXW), B. subtilis 2 (xylanase BSXW), B. circulans BC (xylanase BCXW) and T. fusca TF (xylanase TfxW). The optimal pHs and temperatures for activity of TfxW, BSXW, BCXW and ANXW were 6.0 and 70 ℃, 6.0 and 60℃, 5.0 and 50℃~ 60℃, 5.0 and 50℃, respectively. Higher xylanase activity of BSXW, BCXW and ANXW was observed at lower temperatures, but TfxW had higher activity at higher temperatures, compared with their optimal temperature, respectively.The xylanase genes of B. subtilis 2, B. circulans BC and T. fusca TF and cDNA of A. niger FS6 were cloned and sequenced. Each of the PCR fragments was cloned into pGEM-T Easy Vector, generating pGEM-T Easy BSX Vector, pGEM-T Easy BCX Vector, pGEM-T Easy Tfx Vector and pGEM*-T Easy ANX Vector. The genomic DNA fragments of B. subtilis 2 and B. circulans BC consisted of 702 bp, containing an open reading frame from 39bp to 680bp encoding for a protein of 213 amino acids, respectively. The genomic DNA fragment of T. fusca TF consisted of 1067bp, containing an open reading frame from 31bp to 1047bp encoding for a protein of 338 amino acids. Comparison of the cDNA sequence of A. niger FS6 to other published xylanases revealed that ti was a primary transcript, and the xylanase of 211 amino acids was encoded by two exons interrupted by an intron of 49bp from 265bp to 313bp.Each of the DNA fragments comprised of the xylanase encoding region flanked with restriction enzyme sites at both ends was amplified using PCR method with primers containing restriction sites and each of pGEM-T Easy BSX Vector, pGEM-T Easy Tfx Vector and pGEM-T Easy ANX Vector as a template. The PCR fragments were cloned into pGEM-T Easy Vector, generating subcloned vectors of pGEM-T Easy BSXE Vector, pGEM-T Easy TfxE Vector and pGEM-T Easy ANXE Vector. Each of vectors was double digested with restriction endonucleases, and a target fragment was recovered, then it was cloned to pET-30(a)+, an expression vector double digested with the same restriction enzymes. The xylanase gene in pET-30(a)+ was transformed into E. coli BL21, and E. coli BSX, E. coli Tfx and E.coli ANXE which the recombinants producing xylanases, were obtained. The expression product of E.coli ANXE was enzymatically inactive inclusion body, it showed activity after recovered from the inclusion body.Transformants of E. coli BSX and E. coli Tfx were incubated in LB medium at 37℃ for 12h, then induced with isopropyl 0 -D-thiogalactoside (IPTG) for 4h. After cultivation, the cell were harvested, and homogenized by sonication. The activity and molecular weight of xylanase BSX and xylanase Tfx was 10.18U/ml and 20.31KD , 27.63U/ml and 31.98 KD, respectively. Two enzymes were puri... |
PDF Full Text Request