Screening And Identification Of An Alkali-Tolerate Strain Producing Xylanase, Optimization Of Xylanase Production Conditions And Characterization Of Xylanase And Expression Of Xylanase Gene

Second only to cellulose in natural abundance, xylan is a major component of hemicellulose fraction of the plant cell walls. Depending on the sources, its backbone may carry various substitutents like arabinose and glucuronic acid. Typically, backbone depolymerization is accomplished by the action of endo-xylanases andβ-xylosidases. Xylanase can hydrolyzeβ-1,4-glycosidic linkages of the xylan backbone to produce short chain xylo-oligosaccharides of various length, hence Endo-β-xylanase is the crucial enzyme components of microbial xylanolytic systems.The aim of this experiment is that we sceen a high-xylanase producing bacterium according to the method of Kongo red dye, reaserching the optimization of xylanase production conditions and characterization of xylanase, obtaining the genes coding the xylanase and achieving high expression of the xylanase in E.coli, This thesis was described in the following three sections.1. It is based on the oligoses produced by the xylanases breaking down xylan substrates can't be dyed by Kongo red, consequently, lots of transparent circle will be formed around the bacterium, a bacteria strain BYG6-6 which overproduced alkali-tolerant xylanase was isolated from a waste water treatment plant of pulp paper. It was preliminary identified as Bacillus.Subtilis according to the common dye, Gram staining, its morpHology characters and 16S rDNA sequence.2. Researching in the optimization living conditions of BYG6-6 and enzyme characterization producing by strain BYG6-6, The results showed: the optimization living environments was pH7.0 and temperature 33℃.In order to supply the effiency nutrition for the bacterium, the nitrogen source and carbon source in the culture medium were optimized. When used Birchwood-xylan as carbon source and beef extract as nitrogen source, The activity of the xylanase was relatively higher than before.The further research was performed on the properties of xylanase, we got through changed temperature and PH values when xylanase reacted with the relevant substrate, in order to determine the optimal reaction temperature and PH optimal, The activities of the crude enzymes were measured at different temperature and various PH, The result demonstrated that the optimal temperature of xylanase is at 55℃. And still retained about 73.07% enzyme activity at 65℃, this pHenomenon is proved that bacteria strain BYG6-6 belonged to midtemperature-resistant enzymatic classify, however, when it existed in a high temperature, enzymatic activities dropped dramatically. The optimal PH is at 6.74, and still retained about 91.8% activity when PH is 7.22, even if the PH rise to 9.0 and 10.0, the remain of enzyme activity is approximately 70% upwards, This results revealed that bacteria strain BYG6-6 was slightly more alkaline resistant.3. Amplified a DNA fragment from B. subtilis BYG6-6 genome DNA through designed primers, which was designed according to the reported xylanase gene of Bacillus subtilis. Ligated this fragment to pGEM -T Vector, then sequenced. The homology of this sequence and other reported endo-1,4-xylanase gene was reached 99% through blast online. This xylanase gene was consist of 642 base pairs, and was an integral reading frame, start codon was ATG, and terminal codon was TAG, coding 214 amino acids continuously, the molecular weight is approximately 25kDa.We have cloned a gene that encodes xylanase enzyme from Bacillus.sp. BYG6-6 by using primers B3-up and B3-down, then the gene was cloned into a PET-28(a) expression vector and transformation of Escherichia coli BL21 (DE3), This recombined strain was denominated PYG-6 in our experiment after screening. In 37℃, the xylanase gene was expressed in E. coli after inducement. The xylanase activity of E. coli was ten times higher than that of wide type bacterium. such a pHenomenon showed that the gene realized to heterogenous expression in Escherichia coli and synthesized xylanase which have the activity of the enzyme under the control of the T7 promoter. At the same time we also can prove that the gene was indeed belonging to encoding xylanase gene via SDS-PAGE and zymogram.