Filamentous fungi have attracted great research interest recently, and several genetically modified organisms have been successfully constructed to express valuable heterologous proteins. Examples include industrial enzymes and gene products from higher organisms. The advantages of using filamentous fungi as hosts include, (1) the ability to secrete large amount of proteins, (2) post-translational modifications which are essential to proper protein function, but may be neglected in prokaryotic hosts, (3) fast growing and inexpensive cultures compared to insect, plant and mammalian cells. In the formation of recombinant protein in fungi, one of the bottlenecks may be the extracellular proteases, which can digest secreted heterologous proteins at a high rate. In the present work, we cultivated a recombinant filamentous fungus, A. niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL), in a 15L fermentor, and investigated the properties of its extracelluar proteases. Our results suggest that extracellular proteases have negative effects on the HEWL production, and the proteases inhibitors studies show that cysteine, serine and metallo-protease type enzyes are present. Finally, the optimal temperature for the acid protease is around 45OC.
Full conference title:
160th Society for General Microbiology
- SGM 160th (2007)