Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures

: The fourth‐day extract of a solid‐state culture of the mesophilic Mucor sp. (M‐105) strain showed a high milk‐clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk‐clotting proteinase was purified in two steps: ion‐exchange followed by size‐exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS‐PAGE and N‐terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size‐exclusion chromatography and SDS‐PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0–3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30‐min incubation period, at pH 5.0 and 50 and 60°C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p‐CMPS or EDTA. The N‐terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.