Optimization And Expression Of Alkaline Xylanase Gene In Pichia Pastoris

The xylanase gene from Bacillus sp. was optimized based on Pichia pastoris codon usagepreference. The synthesized gene was cloned in pGEM-T. The xylanase gene was then releasedfrom recombinant plamid pGEMT-xyl after digestion with EcoR I and Not I and was insertedinto pPIC9K. The recombinant plasmid pPIC9K-xyl was obtained. The pPIC9K-xyl waslinearized with Sal Ⅰ and integrated into the genome ofPichia pastoris GS115byelectroporation. The recombinant P. pastoris GS115/xyl was screened by resiatance to G418and the size of transparent zone around the colony.At last, the expression conditions ofxylanase were optimized using single factor tests, Plackett-Burman design, the path of steepestascent and response surface methodology one by one. The main contents were as follows:1.The original gene collected in laboratory was optimized based on Pichia pastoris codonusage preference, then the xylanase gene was amplified by polymerase chain reaction（PCR）.2.The amplified target gene and pPIC9K were digested separately by using EcoRⅠandNotⅠand ligated by T4DNA ligase, resulting in a recombinant plasmid pPIC9K-xyl.3.The pPIC9K-xyl were linearized with SalⅠ, then linearized product were transformedinto Pichia pastoris GS115by electroporation. For the first time,the positive clones werescreened just by Minimal Dextrose Medium (MD). Furtherly, positive clones GS115/xyl wereidentified by PCR. Based on the resistance to G418, transformants with multiple copies ofinterest gene were selected. A transparent zone around the colony was formed on BufferedMethanol-complex Medium(BMMY) containing0.05%xylan, then the high-yield recombinantbacteria with the most obvious transparent zone were picked.4.The characterization of the xylanase from the high-yield recombinant strain wereanalyzed. The optimum reaction pH for the xylanase was8.8, and65℃was the optimumreaction temperature for the xylanase. The rate of residual enzyme activity of the xylanase wasvery low either under high temperature or under strong alkaline conditions for an hour, whichdemonstrated that the xylanase was not very stable under high temperature or strong alkalineconditions.5. According to single factor tests, the fermentation condition of the xylanase optimized inthe conical flask were as follows: pH6.9for initial, soya peptone1.5%, yeast extracts2.5%, methanol1%, MgSO40.1%, CaSO40.05%,(NH4)SO42.5%,28℃cultivation for5d.Plackett-Burman design showed that methanol, yeast extracts and CaSO4were found to bestatistically significant factors influencing the xylanase production. After the optimal regionof response value were approached by path of steepest ascent, Box-Behnken experimental andresponse surface methodology were further used to optimize the concentration of the selectedthree significant factors and the optimal concentration were: methanol26ml/L, yeast extracts48g/L, CaSO41.049g/L. The predicted maximum yield of xylanase was65.732U/mL. Underthe condition of the optimal concentration, the yield of xylanase reached up to66.2421U/ml.Predicted value was very close to actual value and it was found the activity of xylnase was1.15times higher in the recombinant P. pastoris GS115/xyl than that in the original strainunder optimized induction conditions.