Studies On High Density Cultivation Of Recombinant Pichia Pastoris

In this paper, condition for high density cultivation of recombinant pichia pastoris wasstudied. A kind of cheap medium was selected and its components was optimized. Also, thephytase expression condition, the effect of nutrition on cell growth and heterologous proteinexpression by recombinant pichia pastoris were studied.Then, high density cultivationcondition of recombinant yeast was explored in a 5L fermenter. Finally,mensurement ofphytase activity was primarily studied.First, the shaking flask medium for high density cultivation of recombinant pichiapastoris was selected. In this medium,instead of using traditional glycerol, cheaper glucosewas used as the carbon resource and ammonia as the nitrogen resource. The other componentin this medium were also studied. By single factors and orthogonal test,economic and largescaled medium was obtained as follows:glucose concentration 5%,ammonia single feedamount of 20μL,KH2PO4 concentration 0.7%,MgSO₄·7H₂O 0.03%,FeSO₄·7H₂O 0.05%,MnSO₄·H₂O 0.05%.Furthermore,cultivating condition of recombinant pichia pastoris in thisshaking flask medium was also optimized and summaried as follows:using YPD as the seedmedium,culturing seed broth for 24h,inoculation size 10%(V/V),initial pH 5.5 cultured at30℃ on a rotary shaker at 220r/m for 64h. Under this optimized medium and cultureconditions, a maximum cell density could reach 64.3 OD600 ,which was corresponding to acell dry weight of 15g/L.Second,phytase expression condition of the recombinant pichia pastoris was optimizedand the optimal expression condition was determined as harvest time for phytase 3d,methanol inducement concentration 10g/L,pH for cell growth and inducement at 5.5 and 6.0respectively.Finally, effect of different nutrition on phytase expression was studied. The resultindicated that 0.4% PTM1 and 0.1% casamino acid could accelerate the phytase expressionobviously.On the basis of the above studies, high density cultivation condition of recombinant yeastwas explored in a 5L fermenter. Phosphate concentration in fermentation medium wasreduced greatly by batch cultivation optimization. The effect of dissolved oxygen on pichiapastoris growth was studied and the result showed that controlling dissolved oxygen above20% saturation leval is propitious to cell growth. Three feeding methods were tested and theresult indicated that exponential method is propitious to yeast cell growth. The exponentialmethod was further optimized:setting the specific growth rate at 0.20h^-1 and 0.15h^-1 in thephases of feeding prophase and anaphase respectively. The feeding strategy could solve theproblem that the dissolved oxygen in fermenter reaches zero after feeding for 32h and thenenhance cell productivity.Finally, methods of reducing phosphor disturbance and raising the veracity in phytaseactivity mensurement were studied primarily in this paper. After the culture broth wascentrifuged, the supernatant was dialysed in dialytic sack for 2h to remove the phosphor. Thenthe phytase activity was determined and the resulting stability was raised greatly. In addition,Molybdenum-Vanadium method to measure phytase activity was studied primarily and theresult showed that its sensitivity is 0.025mmol/L and its linearity range is0.025-0.60mmol/L.To reduce measurement error, OD415 must be measured within 4h after thecolor reaction finished.