Utilizing Engineering Strain In Yeast To Produce Soybean MnSOD With Optimizing Of Fermentation Condition

Superoxide Dismutase (SOD) are ubiquitously expressed antioxidant enzyme in aerobic organis-ms and catalyze dismutation of superoxide anion to hydrogen and molecular oxygen. The first step i-n active oxygen-scavenging systems.SOD play a central role in protecting cells against the toxic effe-cts of reactive oxygen species generated during normal cellular metabolic activity or as a result of v arious environmental stresses. The metallic enzyme superoxide dismutase are classified as the copper-zinc type(Cu,Zn-SOD),the manganese type(MnSOD),the iron type(Fe-SOD) and nickel type (Ni-SOD), iron-zinc type(Fe,Zn-SOD),according to the metal ion cofactor required for their activity. The MnSOD has been gradually recognized as suppression factor of a new type tumor. It is probably application in the treatment of malignant tumor in the future. In addition, The MnSOD has also been recognize d as inflammation drug of a new type tumor.Based on the published known partial sequence of MnSOD gene, we design primers, extract plasmid DNA from the strain of constructed clone plasmid PMD18-T-MnSOD through primer designing, we obtain the interest gene fragment of MnSOD using PCR digestion, the fragment separately into the S.pombe fission yeast expression plasmid, pREP5N with a high-effective promoter Pnmtl, clones fusion expression plasmid pREP5N and obtain recombinant expression plasmid pREP5N. Using electroporation, we transform the expression plasmid into cells of (S.pombe) fission yeast, through plasmid PCR and double enzyme digestion, we prove that the transformation is successful, until now, we get the transformant of S.pombe with Soybean MnSOD gene. SDS-PAGE detection on transformant and SDS-PAGE after IPTG induction prove that there is a specific band of 26KDa of the plasmid pREP5N-MnSOD, the molecular weight is coincident with expecting. Using Native-PAGE detect the activity enzyme of transformant. There emerge clear transparent activity band of SOD on the gel with blue background which is dyed by NBT solution. Afterward, assay the activity of Soybean MnSOD by NBT method. The activity was 120U/g,6 times higher than control. It is further indicated MnSOD gene has expressed successfully in the S.pombe and it also has biological activity.Optimization of the fermentation process for recombination S. pombe strain having Soybean MnSOD. Screening the medium component and element of fermentation condition by biomass determination and activity of SOD measured by NBT method. The results showed maltose and lactose as carbon sources, peptone as organic nitrogen source, potassium acid phosphate as mineral salt, manganese sulfate as metal ion in the medium, culture temperature is 31℃, initial PH is 4, ventilatory capacity is 250ML conical flask have 40ML culture medium; inoculum size is 5%; revolution per minute is 220r/min, incubation time 96H. The finally activity of SOD was attained in the validation experiment under optimized condition, which was 2.94 times the activity of before optimization. The results obtained in this study lay the foundation of the industrial manufacturing.This experiment consummated the entire process of Soybean expression in yeast and optimizing fermentation condition on this strain. The results obtained in this study lay the foundation of the industrial manufacturing.