Doctor of Philosophy (PhD), Ohio University, 2002, Chemical Engineering (Engineering)

Filamentous fungi are used in the production of both homologous and heterologous proteins due to their desirable growth characteristics. However, the product concentrations of heterologous proteins are usually much lower than those of homologous proteins. Apart from the difficulties in improving the expression and secretion of heterologous proteins in filamentous fungi, the degradation of heterologous proteins by extracellular proteases is also an unsolved problem that hinders the development of filamentous fungal strains as hosts for recombinant proteins.The purpose of this work is to inhibit proteases and thus increase the heterologous protein production by using bioprocessing strategies. Strategies including growth morphology control, pH control, and process parameter optimization were investigated. A recombinant Aspergillus niger strain AB4.1[PgpdAGLAGFP]#11 which carries a glucoamylase - GFP fusion gene was selected as the model system. Experiments investigating the effect of pH on A. niger protease activity indicated a maximum activity at pH 3. The protease activity in the broth at pH 3 was 6.4 times higher than that at pH 6. The highest GFP concentration at pH 6 was 21 mg/L, which was ten times higher than that at pH 3. This work indicates that pH control can be a viable strategy in inhibiting protease activity and increasing GFP concentration. The extracellular protease activity decreased dramatically when the fungal cells grew as pellets instead of free mycelia. As a result, GFP concentration was increased. An inoculum level of 4'106 spores/L led to a culture consisting of an average size of 1.6 mm, which produced the lowest specific protease activity of 158 U/g dry cell weight and the highest specific GFP yield of 0.98 mg/g dry cell weight, which was much higher than the 0.29 mg/g produced in filamentous growth. A fractional factorial design was applied to study the effects of the process parameters - agitation intensity, initial glucose concentration, ini (open full item for complete abstract)

Committee: Darin Ridgway (Advisor) Subjects: Engineering, Chemical