Fermentation Process Of Recombinant Pichia Pastoris For The Production Of SOD

SOD (Superoxide dismutaes) is an important radical scavenger, and its great value in health care attracts our attention.In early stage, SOD is mostly obtained from animal blood and plants. However, this way is limited by immunogenicity and the difficulty of obtaining from raw materials resource. Therefore, it is necessary to study the production of recombinant SOD as a replace.Methylotrophic Pichia pastoris is a excellently expressive system of heterologous protein. Because it can express heterogeneity protein by secretion, with fewer background protein and the price of inducer is low. In this experiment, based on Pichia pastoris GS115 combing pPICZa (eukaryotic expressive vector with human SOD gene in it), we studied different factors, including contain inducer concentration, medium composition and other factors which influenced the expression of the heterologous protein level in shake culture. And then shake flask experiments served as a guide to fermentation of 5 L tank. In 5 L tank fermentation we study the factors affecting the expression of the heterologous protein level. Such as, feeding way at growth period, WCW (wet cell weight) of the starting of inducing, mixture of carbon sources and supplemented nitrogen source. In addition, by co-culture of Pichia pastoris, we study that whether SOD is helpful to strengthen the ability of resisting adversity.The following results were found in this study:1. Among these factors, different composition of medium was the major factor in all. Strains cultured in FM22 (include histidine and PTM4) had a huge increase about 5.5 folds than YPG medium in expression of purpose protein level.2. The results are as follows:inducer concentration of 1%, induction temperature 27 ℃, FM22 medium (containing histidine and PTM4), initial pH 6.0. In this finally optimized condition, the level of activity in shake flask was 895U/ml, increased by 7.5 folds compared with the initial.Comparied to previous, results-625 U/ml in shake flask, the enzyme activity increased by 40%.3. DO-stat strategy (DO was controlled about 20%-35%) was used in 5 L tank fermentation, at growth stage, feeding with glycerol to WCW 190 gL, and inducing by mixture carbon sources. In induction period,decreasing glycerol and increasing methanol gradually, keeping the activity of Pichia pastoris, so that it can take up methanol as much as possible. Enzyme activity in 5 L fermentor reaches up to 16000U/ml, and was 17.8 folds than the flask culture. The co-culture of GS115/αA-SOD and X33 with expression of cpYFP protein and dye and pry test of DCFH-DA had shown a result that the SOD enzyme intracellular cleaned ROS in the cell and strengthened the ability of resisting adversity. Comparing to previous experiental results-5835 U/ml, our enzyme activity increased by 170%.4. In conclusion, results above show that the optimization of fermentation is helpful to enhance biomass, so that the expression of protein can reach a high level. Inducible expression of SOD can improve the ability of anti-aging, and then we can obtain larger amount of target protein. These results provide important theoretical basis for arge scale production of genetic recombination SOD.