Salt-tolerant endopeptidases from xerophilic Aspergillus sydowii for potential applications in meat processing

Dashigara koji is a fermented product made from katsuobushi grounds using the fungus Aspergillus sydowii MA0196. It contains bioactive peptides generated through the proteolysis of katsuobushi proteins. However, the enzymatic basis of this proteolysis remains unclear. This study characterized two major endopeptidases secreted by A. sydowii MA0196: a subtilisin-type protease (Oryzin MA0196) and a chymotrypsin-like serine protease (Tryp MA0196).

Recombinant Oryzin (rOryzin MA0196) expressed in Pichia pastoris, and wild-type Tryp MA0196, purified from the native strain, were analyzed biochemically. Both enzymes exhibited broad pH tolerance and stability under high salt conditions, indicating xerotolerance. Tryp MA0196 retained high activity at up to 12.5% (w/v) NaCl (2.14 mol L⁻¹), whereas the activity of both enzymes gradually decreased with increasing glycerol concentration. Neither enzyme was affected by high histidine concentrations, which may reflect adaptation to katsuobushi fermentation. Activity assays showed that Tryp MA0196 acted on a broad range of protein substrates, whereas rOryzin MA0196 displayed high levels of activity toward casein, myofibrillar proteins, and type I collagen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed efficient hydrolysis of katsuobushi proteins, including myosin-derived peptides, by both enzymes.

rOryzin MA0196 showed high levels of activity toward intact myofibrillar and collagen proteins, supporting its potential application as a meat-tenderizing enzyme. Tryp MA0196 showed superior activity toward physicochemically modified myosin-derived proteins in katsuobushi, indicating a specific role in katsuobushi protein degradation and potential applications in the valorization of katsuobushi byproducts. © 2026 Society of Chemical Industry.