Purification and structural elucidation of a protease-inhibiting substance from Penicillium sp. and the influence of fungal morphology on its formation

Pharmaceutical active ingredients can be produced on an industrial scale by cultivating filamentous fungi, which often synthesize bioactive molecules such as antibiotics in their secondary metabolism. It has been found that the filamentous fungus Penicillium sp. (IBWF 040-09) produces a protease-inhibiting compound intracellularly. This inhibitor targets a protease secreted by Trypanosoma brucei , the organism that causes Human African Trypanosomiasis (HAT). Previous studies have shown that enhanced production of the protease inhibitor occurs only under substrate limitation, especially when the fungus exhibits a pellet-like growth. In the present study, the protease-inhibiting compound was isolated in sufficient quantity and purity and structurally characterized. Structural analysis revealed that the metabolite produced by Penicillium sp. (IBWF 040-09) is cathestatin B, a potent cysteine-protease inhibitor. The structure was confirmed by comparison with chemically synthesized cathestatin B. Investigations into the mechanism of action were carried out, showing that cathestatin B selectively and irreversibly inhibits papain-like cysteine proteases. To increase production yields, the influence of fungal morphology on product formation was analyzed. Both the pellet-to-hyphae ratio and the internal structure of the pellets (based on thin-cuts) were evaluated. A shell structure of the pellets was associated with higher productivity. Additionally, upscaling by a factor of five was successfully achieved.