High Expression Of Endo-chitosanase Gene In Pichia Pastoris

In this research paper, in order to high expression, the gene encoding chitosanase from Aspergillus fumigatus had been successfully cloned into Pichia pastoris host GS115 by electroporation.Endo-chitosanase is a specific enzyme degradation of water-solube chitosan and chitooligosaccharide production of the best biocatalyst, which is alternative to the current common use of strong acide and other chemical degradation processes in urgent need of industrial enzymes.In order to obtain high expression of endo-chitosanase, the study had been improved some key problem, especially, which was the optimization of fermentation conditions of the recombinant Pichia pastoris, the result provived a shandpoint for the industrial produce of endo-chitosanse.The first part studies that endo-Chitosanase gene from Aspergillus fumigatus had been electroporated into the Pichia pastoris. The recombinant expression vector pPIC9K-Csn by Sa1I linearized was introduced into Pichia pastoris GS115 by electroporation. It demonstrated that the gene of chitosanase was correctly expressed into P.pastoris GS115.The second part studies multiple recombinants were screened. With different concentrations of antibiotic G418 selection multiple recombinants, the optimum multiple recombinants were screened from antibiotic G418 (4mg/mL). PCR analysis showed that the endo-chitosanase gene was accurately expressed in the Pichia pastoris GS115 and quite high. The positive transformants were characterized by Chitosanase activity determination and SDS-PAGE. It demonstrated that the highest enzyme activity is up to 67.93U/mL in the laboratory shake-flask fermentation, and its molecular weight was 25KD.The last part presents optimization of the fermentation conditions of the recombinant Pichia pastoris. Improved expression level of recombinant chitosanase by Pichia pastoris was achieved by optimization both the growth phase and induction phase culture conditions. The grow results indicated that the optimum fermentation condition of the growth phase is as follows: 1.0% glycerol, 2.5% (NH4)2SO4, 0.00004% Biotin, 10%lmol/L phosphate buffer, pH5.5, 220rpm, 30℃, 48h, and 1% inoculation amount. the optimum desired fermentation conditions were as follows: 0.5% methanol, 0.25% glycerol, 2.0% (NH4)2SO4, 0.00004% Biotin, 10% lmol/L phosphate buffer, pH6.0, 220rpm, 30℃, 48h, 1:1 inoculation ratio, and the same concentration of methanol and glycerol were supplemented every 24 hours. The experiment showed that the establishment of high-density fermentation conditions obtained the more desirable effect of expression. Fermentation experiments of strain under the optimum conditions showed that the average endo-Chitosanase activity was 107.07U/mL.According to above, the construction engineered strain was successfully expressed in the P.pastoris GS115 and screened multiple recombinants. The enzyme activity was up to 67.93 U/mL in the laboratory shake-flask fermentation. The establishment of high-density fermentation conditions obtained the more desirable effect of expression. Therefore, the engineered strain produced by endo-chitosanase has a wide foreground in industrial production and application prospects.