Expression And Characterization Of Xylanase1YNA And Its Mutant In Pichia Pastoris

.Xylanases are enzymes which catalyze the hydrolysis of xylan to xylose andoligosaccharide. They are crucial hemicellulases and produced mainly bymicroorganisms, plants, and lower animals. Xylanases have been widely used in manyindustry processes, such as pulp and paper, foodstuff, animal feed, pharmacy,bio-energy, and environmental protection. The expression of xylanase in Pichia pastoriswas based on its expression in Escherichia coli, and the product in Pichia pastoris wasactive. The expressed xylanase was finally characterised.PCR technology was used to clone xylanase1YNA tlx and its mutant dsb fromThermomyces lanuginosus DSM10635. The PCR products were connectde withexpression vector pPIC9to construct recombinational vector, and then transformed intoPichia pastoris.After screening, the recombinant strains, the wild type xylanase(TLX)and its mutant(DSB), that expresses the secretory protein at high level wereobtained,with electrophoresis pure protein products. The activity of the recombinantsxylanase reached566.65IU/mL(TLX) and2496.03IU/mL(DSB), and the proteinexpression level were1.75mg/mL(TLX) and1.82mg/mL(DSB). The enzymaticproperties showed that DSB performed the optimum temperature at75°C, and the TLXperformed the optimum temperature at68°C. The optimum pH of TLX was6.5, TheDSB keepde high relative activity that can reach over85%at pH5-6.5. DSB showed abetter thermostabllity than TLX. After30min inactivation at75°C, DSB still remained60%of the residual activity, whereas TLX only remained less than20%of its activity.Finally, The GS115-DSB strain was cultivated in a20liters fermentor level. Theresults show that the xylanase activity was reached59000IU/mL, and the cell wetweight was115g/L.