Screen Of High-Producation Bacterium Of Alkali-Resistant Xylanase, Optimize Of Xylanase Producing And Clone Of Xylanase Genes

The aim of this experiment is to sceen a high-xylanase producing bacterium, reaserch the properties of it, get the genes coding the xylanase and achieve high expression of the xylanase in E.coli,then satisfy the need of practice.(1) It is based on the oligoses produced by the xylanases breaking down xylan substrates can't be dye by Kongo red, so a transparent circle will be formed among the bacterium,the gathered samples,at first,are dealed with the processing of spore disposing process,then through the first sceening and the second sceening on the filtrate culture medium,at last acquire a high-xylanase producing bacterium ,naming BYG5-20N.It was confirmed BYG5-20N is Bacillus pumilus on the common dye ,Gram staining and 16S rDNA identify.(2) Research on the growth curve of the bacillus and the properties of xylanse:BYG5-20N was inoculated in the LB culture medium,it appear the logarithm growth period at 6h after inoculation,and have the fastigium at 24h.the growth decrease into the neap at 36h,later it have a second growth.the optimum pH is 7.0-8.0.In order to supply the effiency nutrition for the bacterium,the nitrogen source and carbon source in the culture medium were optimized.On the basis of the adding of the inorganic nitrogen(NH₄NO₃),L₉(3⁴) latin design was used to compare the organic nitrogen among beef grease,peptone and yeast,it is ascertained the optium compounding is beef grease 1.0%,peptone 0.5%,yeast 0.5%. The result is xylan is the best induction for xylanase producing,xylose is next,but glucose and maltose depress the xylan deposing.It is exlained that the xylanse of BYG5-20N belong to induce enzyme.and it was double-induced by xylan and xylose. The activity of the xylanase was highly obtained as 150IU/ml,it is enhanced 52.54% than selections of nitrogen and carbon source before.The research was also performed on the properties of xylanase,to adfirm the tempreture and pH optimum and resistant of xylanase.Both in the time of xylanase working are changed.the result is that the optimum tempreture of xylanse is 50℃,it also have 55.36% enmezy activity at 60℃,it is proved this xylanase is midtempreture-resistant.but it hasn't the resisitant at higher tempreture.The optimum pH is 7.0, When PH is 8.0,the remain of enzyme activity is 91.1%,Even if the pH rise to 9.0 and 10.0,the remain of enzyme activity is 75%...