Purification and Characterization In Vitro of a 78.9 kDa Zn-Metalloprotease from Arthrobotrys oligospora and Molecular Docking Analysis with Chitin

Nematode-trapping fungi have emerged as biological alternatives to address the increasing problem of antiparasitic drug resistance. These fungi develop three-dimensional adhesive networks that capture, kill, and digest nematodes. In addition, they employ other mechanisms to degrade their prey, including proteolytic enzymes. Arthrobotrys oligospora is recognized as one of the most effective biological control agents against both free-living and gastrointestinal parasitic nematodes. However, little is currently known about the proteases associated with its mycelium. The objective of this work was the identification and purification of the 78.9 kDa from A. oligospora. In this study, we provide the first evidence that A. oligospora mycelium exhibits two distinct proteolytic activities, corresponding to 78.9 and 250 kDa, revealed by zymography analysis. These proteases displayed activity over different pH ranges, suggesting distinct biochemical properties. The 78.9 kDa protease was subsequently purified by ion-exchange chromatography and biochemically characterized. Inhibition assays demonstrated that it belongs to the Zn-metalloproteases, a finding subsequently confirmed by mass spectrometry. We also performed a 3D modeling prediction and identified its potential zinc-binding sites. Finally, molecular docking analyses demonstrate that this enzyme exhibits a high binding affinity for chitin, suggesting chitinase activity; thus, it could be a key component in the degradation of nematode structural components during fungal predation.