Identification and magnetic immobilization of a pyrophilous aspartic protease from Antarctic psychrophilic fungus

Aspartic protease is a versatile protease used in the food processing industry. A novel aspartic protease gene (P10) was cloned from an Antarctic psychrophilic fungus Geomyces pannorum and successfully expressed in Aspergillus oryzae when cultured at 20°C. However, purified P10 exhibited optimal activity at 60°C and retained approximately 80% activity at 50–70°C. The K_m and V_max values for this protease toward BSA were 1.01 mg/ml and 4.4 × 10⁻²mg/(ml min), respectively, with a specific activity of 585 U/mg. P10 showed broad substrate specificity, with an increased affinity for hydrolyzing κ-casein than for α-casein and β-casein, indicating a potential value for cheese-making. P10 also presented wide peptide bond specificity toward the oxidized insulin B chain with high affinity for the C-terminus. Furthermore, P10 was immobilized on iron oxide nanoparticles, wherein it displayed improved thermostability and pH tolerance. These results provide novel insights into psychrophilic fungal enzymes, suggesting P10 as a potential biocatalyst. Practical applications: Aspartic protease is one of the most versatile enzymes in food processing. Owing to the increasing demand in cheese products, microbial aspartic proteases are used as beneficial complements for animal-derived milk coagulant. In this study, a novel aspartic protease (P10) was well studied. Its potential application in cheese-making and magnetic immobilization were investigated. Our results potentially provide novel insights into psychrophilic fungal enzymes and suggest their application in the food industry.