Optimisation of expression of trypsin in a bioreactor : A study based on Design of Experiment

• Main Author: Rane, Tova
• Format: Bachelor Thesis
• Language: English
• Published: Uppsala universitet, Biokemi 2022
• Subjects: Chemical Process Engineering; Kemiska processer; Medical Biotechnology; Medicinsk bioteknologi; Organic Chemistry; Organisk kemi; atlantic cod
• Online Access: http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-478382

In recent years, medical treatments against respiratory tract infections (RTIs) based on trypsin from Atlantic cod targeting the surface proteins on viral particles have been approved. However, the stability and compatibility in the human body of human trypsin would be expected to be greater than that from cod. In this study, the process of producing human trypsin recombinantly in E. coli in a fed batch bioreactor was optimised using a Design of Experiment (DOE) method with four 2-level factors and two responses. The factors considered were temperature, carbon source, induction time point and feeding rate, and the two responses were yield and purity. The study was done in 8 consecutive runs in the bioreactor, with controlled changes made to the factors for each run. The responses were measured and analysed for significance and optimisation of factors. The optimal settings for the highest yield with a purity of at least 80% were found to be at 18 °C, glucose based feed at a restricting feed rate and induction at a low OD600. Only the induction time has a significant effect on purity, and no factor had a stand-alone significant effect on yield. These findings provide a baseline for further studies on purification and further production of trypsin recombinantly in E. coli at a larger industrial scale. The highest yield achieved was 7.9 g trypsin per litre media, almost 80 times higher than comparable methods using shake flasks. However, the sample with over 80% purity was 6.3 g trypsin per litre media.