Isolation Of Hemicellulose Degrading Thermophilic Bacteria And Characterization Of Thermostable Xylanase

Xylan is the major component of plant hemicellulose and the second most abundant renewable polysaccharide in nature after cellulose. Due to the heterogeneity of hemicellulose, completely breakdown of it requires the cooperative actions of several hydrolytic enzymes. Among them, endo-β-1,4-xylanases are the crucial enzymes since they initiate the degradation of the xylan backbone to produce short chain xylo-oligosaccharides of various length. In recent years, xylanases have been deserving particular worldwide research interests, because of their biotechnological potential industrial application including biomass conversion and other various industrial processes. Although lots of xylanases have been isolated and characterized from various microorganisms, however, most of them are active at a neutral or acidic pH, and their optimal activity temperature is usually at lower temperatures, considering the applications of xylanase generally prefer higher pH or temperature optima, a better thermostability and a broad pH range. Therefore, it is necessary to search the robust xylanase for industrial applications. Becouse of thermophilic bacteria-producing xylanases are better than other microorganism on the thermostability and alkali stability, so the research of thermophilic bacteria become the focus of attention.In this study, more than70thermophilic bacteria were successfully isolated from Great Basin Hot Spring in Nevada and Yongtai hot spring of Fujian province, many isolates displayed good xylanase activities. Based on the SDS-PAGE of whole-cell proteins and16S rDNA sequences, they are assigned to three different genera. Among them, a species assigned to Geobacillus sp.TC-W7showed the highest xylanase production. The result of fermentation conditions shows when tempreture and pH reached the70℃or7.2, speed of shake was120rpm and culture time was36hours, the total xylanase activity reached its peak.Subsequently, a xylanase gene encoding a protein of407amino-acid with a molecular of47.4kDa from TC-W7was cloned and expressed in Escherichia coli BL21(DE3). sequence analyse this xlyanase has98%and86%similarity with the xylanase gene of Geobacillus thermodenitrificans (NG80-2) and Geobacillus sp.WBI, respectively.The purified recombinant xylanase exhibited maximum activity at75℃and the optimal pH at8.2. It was still active when the tempreture reached up to95℃. Meanwhile, the recombinant xylanase also showed higher thermostable and pH stable, with more than80%of its activity remained after120min incubation at70℃. The activity of recombinant xylanase was enhanced by enzyme inbititors (DDT, Tween-20,2-Me or TritonX-100), while it was inhibited by EDTA or PMSF. In addtion, its function was stable in the presence of Li+, Na+, K+, but inhibited by Hg2+, Ni2+, Co2+, Cu2+, Zn2+, Pb2+, Fe3+, Al3+. The characterization of crude xylanase analyse show that its xylanase exhibited maximum activity at75℃and pH7.2. The crude xylanase also showed good thermostable and pH stable, with more than70%of activity remained after120min incubation at70℃and holded80%actvity after90min at pH9.2. The activity of xylanase was enhanced by enzyme inbititors (DDT, Tween-20,2-Me or TritonX-100), its function was promoted when the metal ions of Li+, Na+, K+, Mg2+, Ca2+, Ba2+, Mn2+, Fe2+appeared.Xylanase homologous models ware constructed through web servers of Geno3d. All this models were evaluated by different tools such as molecular dynamics, stereochemistry and Ramachandran Plot, then a rational model was selected out.