The role of fungal proteases in bioactive peptide production and analytical approaches for tracking their activity

Abstract

Fungal proteases catalyse site-specific peptide bond cleavage in complex protein matrices, enabling bioactive peptide (BP) release. Accurate monitoring is vital for optimising hydrolysis efficiency, minimising nonspecific proteolysis, ensuring consistent yields, and preserving peptide biofunctionality. While analytical and computational tools are well-established for non-fungal systems, their adaptation to fungal proteases remains exploratory, with no comprehensive evaluation addressing the unique challenges in this field. This review synthesises current knowledge, identifies methodological gaps, and outlines future directions for developing fungi-specific analytical pipelines and computational frameworks for BP discovery. It explores fungal protease families, their roles in intracellular peptide processing, and fungal survival strategies. Fungal protease production methods are discussed, highlighting applications and challenges in BP production and hydrolysate quality improvement. Analytical strategies – such as agar plate assays, droplet microfluidics, FRET-based assays, and N-terminomic approaches – are evaluated through fungal case studies to illustrate applications, limitations, and innovation opportunities.