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/proteol ytic activity ratio similar to that of commercial preparations from microbi al origin used in cheese manufacture. After ultrafiltration of the crude ex tract, 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-exc lusion chromatography and SDS-PAGE data. The optimum pH for proteolytic act ivity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retai ned 26 and 13% of its proteolytic activity after a 30-min incubation period , at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower h eal stability than that of the thermophilic enzymes currently used in the c heese 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 ED TA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compa red with those from other fungal enzymes.