Study Of Pilot Process Conditions Of Antibacterial Peptide Spinigerin α

The appearance of the super bacteria made antibiotic era is gone, and The uniquemechanism of action of antimicrobial peptides slowly Become the substitute of antibiotics,Domestic and foreign researchers have attached great importance to its research, early researchlimitied to Cloning and Expression of Antibacterial Peptide and Later research how to improvethe expression level and realize large-scale production is seldom, Pichia yeast because of itsgenetic stability, high expression level, Protein translation after processing, products secretely,high density fermentation etc, and It is easy to expand from the shake flask culture to highdensity fermentation, so it is a important expression system to realize the high densityfermentation express recombinant protein, the pilot fermentation is an important step to achievethe research development to the production.The purpose of this study is to use genetic engineering recombinant bacteria, Through theSpinigerin a antibacterial peptides scale pilot fermentation in50L, and preliminary purificationtechnology research, finally, provide the scientific basis for industrial production.In shake flask experiment, firstly, optimize Spinigerin α antimicrobial peptides cultureconditions affecting antimicrobial peptides production in the growth phase, which includes thesix factors of seed inoculation time, inoculation,rotation speed, initial pH, the initial temperature,the initial concentration of glycerol; After use the response surface study the initial glycerolconcentration,temperature, pH affected the antibacterial activity, finally, Determine the optimumculture conditions in the shake flask cultures stage.use the results of optimized shake flask cultures trial found Spinigerin α antimicrobialpeptide yield up16mg/L, compared with before optimization up to twice.on the basis of the shake flask culture determining the parameters, optimize the pilotproduction process condition glycerol feed mode, induction temperature, induction pH,methanol feeding mode, the ratio of methanol mixed with sorbitol, induction time in the50Lfermenter, and use the orthogonal optimze the three factors effecting productions,whichincludes the ratio of methanol mixed with sorbitol, induction temperature and induction pH, andultimately the optimum fermentation conditions in50L fermenter pilot production processconditions is: after irrigation Seed cultivation18h,10%inoculum into the50L fermentor,initial pH5.5, initial temperature26℃, and initial concentration of glycerol1.0g/100mL,fermenter speed is250r/min and cultured until initial concentration of glycerol runs out in theoriginal tank, Start additional glycerol with Variable speed mode, after20h, enter the methanolinduction phase, Set up induction temperature24℃, induction pH5.0and the ratio of methanoland sorbitol mixed1:1, induction time96h. Under optimum pilot conditions,50L fermenter Spinigerin α antimicrobial peptidesproduction is19.2mg/L, which improved22.48percent compared with shake flasks.Subsequently, explore Spinigerin α antimicrobial peptides initial purification conditions,pilot obtain the expression supernatant through four purification steps, Which includesammonium sulfate precipitation, Dialysis membrane filtration, SephadexG-25chromatography,nickel affinity chromatography, and ultimately Tricine-SDS-PAGE verify the purpose proteinSpinigerin α antimicrobial peptides obtaining a single band, the results laid a theoretical andpractical foundation for the future using of Pichia pastoris scale Spinigerin α peptidesproduction.