Fermentation Optimization Of Candida Antarctica Lipase B-displaying Pichia Pastoris

As a eukaryotic display system,yeast surface display technology has gradually become a very effective display technology. Over the last decade, yeast surface display technology continues to improve and perfect,and have been widely used in many fields. Pichia surface display technology has advantages in presents a variety of functional proteins ,which has been made great study progress home and abroad.Candida antarctica lipase B is the most widely used one in a number of lipase , which has a strong catalytic activity to Hydrophilic and hydrophobic substances. Displayed on the pichia surface, CALB not only achieved self-immobilization, also has it's heat resistance and catalytic activity improved. But study on fermentation optimization of enzyme-displaying pichia, especially for fermentation optimization of Candida antarctica lipase B-displaying Pichia pastoris, has not been reported at home and abroad.The idea of this study is, According to Invitrogen "Pichia experiment Manual" and the "Pichia fermentation process Manual"respectively, first has the main components of culture medium and fermentation conditions optimized using single level optimization in flask,to find the optimal level and key factors.Then scale-up to 2L tank, to find out the best glycerol and methanol fed-batch mode.At last scale-up to 50L tank fermentation.In this study, the BMGY/BMMY medium and fermentation conditions of CALB -displaying Pichia pastoris mutant KFS-CALB-GS115 were optimized in single factor shake flask levels .It was found that the optimum medium and fermentation conditions of KFS-CALB-GS115 were: glycerol 2% (w/v), methanol 2% (v/v), inoculum 4% (v/v), volume 12.5mL, speed 300rpm ,induced initial pH=6.5. KFS-CALB-GS115 hydrolase activity was significantly higher to 916.3U/g, increased by 65%compared to the original conditions of 556.9 U/g. By comparing the whole cell catalytic synthesis esterification reaction efficiency before and after optimization, 3h after reaction started, the conversion rate of substrate from optimum conditions CALB was 70%, while only 28% of the original one. 4 hours after reaction , the conversion of optimizing conditions can reach 80%, 1.5h in advance than the original one. Comparing the KFS-CALB-GS115 protein expression before and after optimization , protein band of optimum conditions was slightly better than the original condition.The study also shows BSM medium and fermentation conditions optimization of another CALB-displaying pichia mutant strain KNS-CALB-GS115 (higher synthesis activity but low hydrolytic activity than KFS-CALB-GS115) in high-density fermentation. First, several important components of BSM medium content was investigated by flask simulated glycerol batch culture fermentation.Then , the optimum pH and optimum temperature of KNS-CALB-GS115 in the growth and enzyme production were investigated using multi-batch shaker to automaticly control fermentation pH online .The optimal level of BSM medium conditions and fermentation conditions of KNS-CALB-GS115 were found: glycerol 40g/L, ammonium sulfate 4 g/L, calcium sulfate 10 mmol/L, PTM1 4 mL/L, the optimum growth and enzyme production temperature at 30℃, the optimum growth pH=5.5, the optimum induce pH=7.0. After Optimization, utilization rate of glycerol reaching 95.3% ,6.4% higher.Dry cell weight reached 19.92g/L,increased by 4.5%.CALB hydrolysis activity reached to 100.3 U/g ,increased by 60.3%.The optimization of BSM medium and culture conditions of KNS-CALB-GS115 were scale-up in the 2L fermentation tank, the cell growth efficiency was effectively improved and the enzyme production was also improved latter. The maximum enzyme activity up to 305.9U/g, increased by 27.2% compared to original ones.The biomass rapidly enriched effectively when the glycerol fed-batch phase flow rate was setted at 9.6mL/(L?h) and the flow time of glycerol was extended to 12h , which effectively increase the latter induce expression.Optimal CALB enzyme activity reached 317.3U/g.The CALB expression was improved when methanol fed-batch stage using constant feeding rate at 6.4mL/(L?h) than the DO-stat method, the optimal enzyme activity reached 348.4U/g. Comparing the fluorescence intensity on cell surface before and after optimization, the optimized fluorescence peaks shift markedly to the right, shows the improvement of enzyme activity due to the expression of cell surface proteins; Comparing the CALB whole cell catalytic efficiency of ethyl hexanoate before and after optimization, conversion rate was improved by 18% about 1h after the reaction began. The improve of CALB expression resulted in the improve of the catalytic synthesis activity. Scale-up to 50L fermenter tank, the enzyme activity was 256.5U/g, less than 2L tank level.