Production of milk-coagulating protease by fungus <i>Pleurotus djamor</i> through solid state fermentation using wheat bran as the low-cost substrate

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus <i>Pleurotus djamor</i> PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.