Biochemical and functional characterization of a <i>Penicillium purpurescens</i> milk‑clotting enzyme as an animal rennet alternative

Fungal milk‑clotting enzymes (MCEs) provide a sustainable and efficient alternative to animal‑derived rennet in cheese production, providing advantages, such as scalability and compatibility with diverse dietary needs. The present study aimed to optimize the production and characterize the enzymatic properties of an extracellular MCE from <em>Penicillium purpurescens</em>. The highest enzyme yield was achieved using solid‑state fermentation with wheat bran as the substrate under conditions of pH 6.0, 60˚C, a 1% inoculum size and supplementation with 1% CaCl₂ on the 5th day of cultivation. The enzyme was purified 17.2‑fold with a 20% recovery rate through acetone precipitation and ion‑exchange chromatography, and SDS‑PAGE analysis revealed a molecular mass of 29 kDa. Kinetic analyses determined a K_m value of 5x10^‑2 M, indicating high substrate affinity. The purified enzyme exhibited optimal activity at 45‑55˚C and a broad pH range (pH 5‑6), along with notable stability under varying conditions. The analysis of metal ion effects revealed the enhancement of activity by Ca²⁺ and Mg²⁺, while it revealed the inhibition of Hg²⁺ and Cu²⁺. These properties, combined with a high specificity for &kappa;‑casein, highlight the potential of the enzyme for industrial dairy applications, particularly in processes requiring robust and adaptable clotting agents. On the whole, the present study demonstrates that <em>Penicillium purpurescens</em> may be a highly promising fungal strain for MCE production, demonstrating superior clotting efficiency compared to other screened strains.