A Novel Chymotrypsin-like Protease from Trichoderma koningii FFT13 with Efficient Milk-Clotting Activity

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in peptides and proteins, have widespread industrial applications, particularly in milk coagulation for cheese production. Microbial enzymes have been employed as alternatives to animal rennet, offering advantages such as cost-effectiveness, availability, and compliance with dietary, cultural, and religious requirements. Solid-state fermentation (SSF) is widely employed for microbial enzyme production because of its low operational costs, reduced water and energy requirements, high product concentrations, and the ability to utilize agro-industrial residues as low-cost substrates, thereby contributing to both process sustainability and waste valorization. We report the production and characterization of a novel milk-clotting protease produced by Trichoderma koningii FFT13. The protease was produced by SSF using wheat bran as the substrate, an agro-industrial residue. It was classified as a chymotrypsin-like serine protease and exhibited a specific caseinolytic activity of 9861 U/mg. The enzyme coagulated both reconstituted skim milk and pasteurized whole milk in the presence or absence of calcium. Coagulation was enhanced by increasing temperature, reaction time, enzyme concentration, and calcium levels. Scanning electron microscopy revealed destabilization of casein micelles, their progressive aggregation, and the formation of a well-defined gel network, confirming the effectiveness of the protease in milk coagulation. Therefore, these results demonstrate that the chymotrypsin-like protease from T. koningii is a promising enzyme for milk coagulation, with potential application in cheese production. The enzyme obtained constitutes an alternative to traditional coagulants, overcoming limitations related to animal rennet while potentially offering additional advantages in terms of process sustainability and industrial scalability.