Optimization of the immobilization of milk‐clotting proteases to granular bone

Optimization of the immobilization of pepsin and the fungal proteases from Erdothia parasitica and Mucor miehei (MM)) using glutaraldehyde crosslinking and adsorption methods on clean, sized granular chicken bone (BIOBONE ™) was investigated. Parameters studied to determine maximal conditions for protease immobilization included glutaraldehyde concentration, enzyme concentration, pH and temperature. Factors found to significantly influence immobilization were enzyme concentration and pH for pepsin. Only enzyme concentration influenced the immobilization of the fungal proteases. Results showed that the maximum conditions for immobilizing protease on chicken bone using the glutaraldehyde method were 33 mg/mL protein at pH 5.2 for pepsin and 3 mg/mL protein at pH 4.6 for both fungal proteases. Maximum conditions for the adsorption method were 30.4 mg/mL protein at pH 5.2 for pepsin and 5.4 mg/mL protein at pH 4.7 for the proteases.