Study On The Extraction Of ?-xylosidase From Symbiotic Bacteria In The Gut Of Termites

The symbiotic bacteria which in higher eat-wooding of Globitermes brachyceraste has the ability to efficiently digest these cellulose as foods and provides a reliable basis for exploring the mechanism of cellulose degradation in termites.This thesis aims to screen new heat-resistant ?-xylosidase and provide an enzyme source for industrial.The main content of this thesis is select the intestinal symbiotic bacteria of Globitermes brachyceraste termite Fosmid library as the initial screening material.The results of this thesis are as follows:The sensitivity of the ?-xylosidase positive control to the chromogenic substrate(5-bromine-4-chloride-3-indole ?-D-xyloside)was explored.The results showed that optimum concentration of the chromogenic substrate was 140ng/ml.According to the reaction of chromogenic substrate with ?-xylosidase,22 positive clones were screened out and the positive rate was 1.8‰.The positive clones were sequenced by 454 sequencing and annotation,the results showed that there were eight potential genes encoding new type of ?-xylosidase,some of which belonged to multifunctional enzymes.Primer design and vector construction were used homologous recombination method,the positive clones were successfully constructed by colony PCR validation.Results: the 8 genes were successfully cloned and the vectors were successfully constructed.We explored whether protein expression could be carried out in E.coil BL21,the results showed that ?-xylosidase were successfully expressed in E.coli BL21 and their crude enzyme solution was obtained.The protein was purified by Ni-NAT affinity chromatograph,the results showed that when the imidazole concentration was 100 mM and obtained their single bands.The results of protein analysis by SDS-PAGE showed that the molecular weight of 8 ?-xylosidases were consistent with the theoretical values;the analysis of the eight ?-xylosidases domains showed that except glycosyl hydrolysate family in their main domain,part of them contained CBM(Carbohydrate-binding module,CBM)? BglX and Fn3-like domains and preliminarily speculated that the existence of these domains might have a effect on cellulase.The effects of temperature and pH on 8 ?-xylosidases were investigated,the results showed that the optimum temperature of 8 ?-xylosidases were concentrated at 40??50 ?and 55?,the optimum pH is 5.0?5.5?6.0 and 6.5.The results of enzyme activity assay showed that Xyl2/Xyl7/Xyl8 had significant advantages.The expression of ?-xylosidases in fungi were investigated.The ?-xylosidases genes of Phanerochaete Chrysosporium,Aspergillus oryzae and Aspergillus terreus were fermented in Trichoderma reesei Rut-C30.The results showed that the ?-xylosidases of three fungi was expressed successfully,their activities were higher than the ?-xylosidases activity of Trichoderma reesei itself.