Optimization Of Fermentation Process Of Pichia Pastoris With Glucose As Carbon Source And Its Induced Regulation

Pichia pastoris expression System is one of the most successful eukaryotic expression systems at present.Compared with other expression systems there are obvious advantages in yield processing external secretion and translation modification.In industrial production Pichia pastoris fermentation can be used as a two stage of fungus growth and methanol induction.Generally,the main use of glycerol in the fungus as a carbon source,in the induction phase depends mainly on the value of dissolved oxygen,combined with production experience to induce regulation.The mainly obtains the following research results:(1)The feasibility of fermentation of pichia pastoris with glucose instead of glycerin was investigated.Flask and 10 L fermentation tank results show that In the fermentation of Pichia pastoris with glycerol and glucose as carbon sources,the growth process of the bacteria was synchronized.Therefore,it is feasible to replace glycerol with glucose in Pichia pastoris fermentation.(2)The fermentation results of 10 L fermentation tank showed that the growth rate of somatic cells in fermentation with glycerol as carbon source could be achieved by optimizing the feeding strategy of glucose,and the fermentation cycle was not prolonged while the cost of raw materials was reduced.(3)Using Pichia pastoris as an example,glucose oxidase and lipase were used as the sole carbon source for the amplification of somatic cells,and the wet weight of somatic cells could reach 180~220 g/l after the culture of 48 h.In the process of fermentation,the biosensor was used to detect glucose and methanol quickly,reducing the effect of glucose deterrence,avoiding the cell toxicity caused by improper methanol dosage,and establishing a more accurate control strategy.(1)The concentration of glucose and methanol was monitored closely by biological sensor,the precise control of the growth and induction stage was realized,and the negative effects caused by the calibration and the use error of the dissolved oxygen electrode were avoided.(2)The methanol induction strategy was optimized,and the expression of exogenous protein was realized by shortening "starvation time" or inducing directly or adding methanol in advance.(3)The expression of glucose oxidase and lipase in Pichia pastoris was as an example,and the fermentation period was 82-90 h,Activities are: glucose oxidase 27.3 u/l,32.9 u/l,lipase 5300 u/l,5175 u/l,using glucose as a carbon source fermentation to achieve with the use of glycerol basically consistent level.