Improvement of β-glucosidase activity through adding sugars in high density fermentation of recombinant Pichia pastoris

• Main Authors: SHANG-CHENG LIAN, 連上程
• Other Authors: C. Will CHEN
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/5g5849

碩士 === 大同大學 === 生物工程學系(所) === 106 === In this study, the production of recombinant protein β-glucosidase through high cell density fermentation by exogenous gene expression strain of Pichia pastoris （carrying with GAP promoter system）. Metabolism of the carbon source by P. pastoris could produce glyceraldehyde-3-phosphate dehydrogenase （GAPDH） to initiate foreign gene expression and continuous expression of the exogenous protein. Therefore, the design of carbon source will have a significant impact on the expression of the system. This study used fed-batch fermentation with glycerol as the primary carbon source with the addition of different sugars for evaluation. The fermentation mode adopts a two-stage fermentation strategy. In the pre-fermentation stage, the culture temperature and pH value were adjusted to facilitate the rapid growth of the cells, then the temperature and pH were adjusted after the fermentation at 48 hrs to facilitate enzyme production. The results showed that the maximum enzyme activity of the five groups of fermentation experiments from low to high values were 16.2 U/ml （control group, without adding sugar）, 30 U/ml （adding molasses）, 51 U/ml （adding sugar cane liquid）, 58.3 U/ml （adding maltodextrin）, and 104 U/ml （adding brown sugar）, respectively. Among of all sugars additions, brown sugar achieved the highest enzyme activity, which also reached the maximum cell concentration of 125 g/L at 240 hrs and the maximum protein amount of 590 mg/L at 432 hrs. The maximum enzyme activity of this result was 104 U/ml,that was 6.4 times of the control group （without adding sugar）, and the maximum specific enzyme activity was 176 U/mg, which was 6.3 times of the control group.