Optimization Of Fermentation Conditions Of Recombinant CpYFP And EC-SOD For The Detection And Scavenging Of Superoxide

Superoxide dismutase(SOD)is the most important enzyme in human body to remove superoxide radicals.There are three major families of SOD,in which the extracellular superoxide dismutase(EC-SOD)is the least in human body.The aim of this study was to express recombinant EC-SOD by yeast Pichia pastoris and optimize the fermentation parameters to obtain high yield of recombinant protein.Detailed experiments include single factor optimization,optimization of YPD medium by response surface methodology,orthogonal experimental design,fermentation with 5L fermentor.The recombinant protein EC-SOD was successfully expressed by Pichia pastoris and the molecular weight was 15 kDa identified by immunoblotting.Compared with YPD medium and BMMY/BMGY medium,we found that there was no significant difference in the yield of EC-SOD proteins between the two groups.Therefore,the lower cost YPD medium was used.The fermentation parameters were finally optimized as follows:fermentation temperature 30 ?,inoculation amount 2%,culture medium initial pH value 6,methanol induction concentration 2%.The activity of the enzyme was the highest in each group.Usihg the Design-Expert design response surface methodology,the optimized formula for fermatation was:Yeast extract 15 g/L,Tryptone peptone 25 g/L.The optimum protein expression level under above optimized parameters was 200.8 U/mL,which was about 6.4 times higher than that of the non-optimized batches.The optimal conditions for the expression of EC-SOD obtained by orthogonal experimental design were listed as follows:initial pH was 6,age 24h,induced concentration 2%,fermentation temperature 30 ?,180 rpm rotation speed.The yield of SOD activity in the optimized group was 235.7 U/mL,which is about 7.7 times higher than that before optimization.Furthermore,EC-SOD protein was also fermented by 5L fermentor.The fermentation parameters were as follows:fermentation temperature 30 ?,age 24 h,inoculation amount 10%,ammonia regulation pH 6,50%glycerol flow and feed,50%methanol flow and inducing.The dissolved oxygen is maintained by 20%and the speed and flow acceleration are adjusted according to the feedback of the dissolved oxygen.The YPG medium was used to replace the FM22 medium.The results showed that the cell growth delay was significantly shorten and the yield of EC-SOD was increased by 79.3%.In addition,yeast Pichia pastoris was also used to express the yellow fluorescent protein(cpYFP),which is a specific fluorescent probe.The results of single factor optimization for cpYFP protein showed as follows:the fermentation temperature 26 ?,the initial pH value 7,inoculation amount 2%,methanol induction concentration 1.5%.Much higher yield of cpYFP protein was gained from the fermentation broth using FM22 medium compared to YPG medium.Moreover,5L fermentation experiment could further improve the yield of cpYFP.The fermentation parameters were as follows:fermentation temperature 26 ?;ammonia regulation pH 7,inoculation amount 10%,50%glycerol flow and feed,50%methanol flow and inducing.The dissolved oxygen is maintained by 20%and the speed and flow acceleration are adjusted according to the feedback of the dissolved oxygen.