Optimization Of High Cell Density Fermentation And Hydrolysis For Pichia Pastoris

There are a great amount of amino acids of rich kinds, which are a good animal feeding protein. Methanol is an oversupply, low-price by-product from chemical industry. Compare with the agricultural and sideline products, yeast proteins with methanol as main raw materials produced by high-density fermentation of Pichia pastoris has a certain price advantage. The subject of optimization of Pichia pastoris by high-density fermentation and hydrolysis, was found to be a low-cost and high efficiency method of producing yeast proteins. Mainly goals were achieved as follows:1. Based on the biomass (wet weight) as the main indicator, we optimized the concentration of metal ion in basic salt medium in a 50 L fermentor. Concentration of K2SO4, MgSO4·7H2O, KOH and CaSO4 fell down to 9.1 g/L,7.45 g/L,2.06 g/L and 0.46 g/L separately, which shortened production time and proved to be an efficiency way. In the case of using methanol as a carbon source, a weight of 1.72 kg yeast yielded, after 69.5 h fermentation with methanol 10 L added.2. A reuse method of Pichia pastoris fermentation medium has been discussed in this reach. When biomass (wet weight based) increased to 250 g/L or more, part of the broth in the fermentation tank was left as the seed solution for the subsequent batch fermentation; the other solution separated by centrifugation to obtain yeast mud and supernatant fluid. Yeast mud were collected as coarse product for the hydrolysis process; supernatant fluid was the supernatant back to the tank after sterilization, with the sterilized fermentation medium to complement the volume of methanol as a carbon source to continue the batch fermentation. At the end of the fifth cycle of batch fermentation, fermented solution was centrifuged to obtain yeast mud.5 cycles of fermentation last for 104 h with methanol 20 L added. Finally, we got 5.29 kg yeast.3. Took the hydrolysis rate and the content of amino acid in the hydrolysis as major index, Pichia pastoris hydrolyze process was optimized through the single factor experiment. Reached the optimal temperature 52℃resulting a pH value 5.0, and Enzyme M2 and NaCl was shown to have a promote effort for the hydrolyze process. Result showed that hydrolyzed rate could reach 40.57% after 32 h with Enzyme M2 controled to be 0.02 wt%. Optimal adding amount of NaCl was 3%, after 24 h, hydrolyzed rate reached 48.78%.