Optimization The Culture Conditions For Expression Of Mannanase By Recombinant Pichia Pastoris

Mannanases are of interest and application in many biotechnology fields because of their wide range of biological applications.Mannanase is widely present in the biosphere,and most of the biological species that produce the enzyme belong to the microbial source.Pichia pastoris has been favored by the enzyme preparation industry because it has the advantages of simple operation,easy high density fermentation,high level of foreign protein expression,and low production cost.In this paper,we chose to design and optimize the liquid fermentation protocol of a newly constructed mannanase producing recombinant Pichia pastoris,and then get the pilot program.1.First,use shake flask culture to explore out some of the fermentation process parameters to obtain the optimal composition and ratio of the fermentation medium.The most suitable growth temperature is 28.0? and the optimal pH is 4.80.2.Use 30 L fermentor for fermentation to optimize the remaining process parameters.Use the correlation between dissolved oxygen and metabolic pathways to optimize other fermentation conditions.Inoculum size is 0.6-0.9%,maximum volume of fermentation is controlled at 2.5 m~3/h.The maximum stirring speed is 600 rpm/min.3.The fermentation process was divided into three phases: growth enrichment period,transition period,and induction of enzyme production period.Fermentation optimization was performed on feed composition and flow acceleration for each period.The growth and enrichment period is mainly the accumulation of bacterial counts.Feeding with glycerol,the average speed of supplementation of glycerol is 540 g/h,and the flow is added for about 10 h.At the end of this phase,the wet weight of the fermentation broth is controlled within the range of 30% to 40%.The wet weight was less than 30% and the enzyme production efficiency was low.Above 40%,the post-treatment enzyme yield was low.In the transitional period,a 4:1 mixture of glycerol and methanol was used as the feed and the average speed was 240 g/h.About 4 h flow,this stage is the transition from growth to inducing enzyme production,is the conversion process of metabolic pathways,and the smooth transition is the basis for high-yield enzyme production for a long time;the induction period uses methanol as a supplement.The speed was 55-60 g/h,about 140 h flow(the specific time until the enzyme activity no longer grows),speeding up excessive bacteria is easy to poisoning death,low supplemental rate of methanol conversion is not enough,less enzyme production.4.This process scheme was validated using a 30 L fermentor.After fermentation for 183 h,the enzyme activity reached 1650 U/mL,an increase of 335 % compared to the initial shake flask fermentation enzyme activity of 492 U/mL.The amplification method of oxygen concentration was conducted in a 100 L pilot scale.The fermentation process and the fermentation enzyme activity were basically the same as the 30 L fermentation tank.This process scheme can guide the fermentation of 100 L fermentor to produce mannanase.