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

H.M. FERNÁNDEZ LAHORE; R.M. AUDAY; E.R. FRAILE; M.J. BISCOGLIO DE JIMÉNEZ BONINO; L. PIRPIGNANI; C. MACHALINSKI; O. CASCONE

INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH

Munskgaard

Lugar: Copenhage; Año: 1999 vol. 53 p. 599 - 605

0367-8377

  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 60ºC 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.