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

Abstract

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 degrees 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.

Aspartic proteinases from Mucor spp. in cheese manufacturing

“…An overall purification factor of 15.5 and total enzyme yield of 46% were achieved, fitting very well with the standard procedures found in the literature. A similar strategy was also applied in the recovery and purification of the aspartic proteinase from M. circinelloides where a final yield of 76.2% and specific activity (purified enzyme) of 1,280 U/mg was obtained (Fernandez-Lahore et al 1999). Table 3 summarizes reports on the purification strategies of aspartic proteinases from Mucorales.…”

“…This concept has been proven experimentally: the R. pusillus enzyme retains 71% of its original activity when heated for 10 min at 55°C while, under similar conditions, the calf rennet and the M. bacilliformis aspartic proteinase retain 31% and 10% of the initial milk-clotting activity, respectively (Fraile et al 1981). Fernandez-Lahore et al (1999) have shown that a M. circinelloides strain (M-105) produces a wild-type enzyme with very promising characteristics, being even less heat stable than the enzyme obtained from M. bacilliformis cultures.…”

“…and (b) rennin-like enzymes produced by Mucor spp. (Fraile et al 1981;Venera et al 1997;Fernandez-Lahore et al 1999;Neelakantan et al 1999;Andrade et al 2002;Machalinski et al 2006), Rhizomucor spp. (Thakur et al 1990;Escobar and Barnett 1993;Silveira et al 2005;De Lima et al 2008), and Endothia parasitica (Sardinas 1968).…”

“…Fernandez- Lahore et al (1999) reported the production, purification, and partial characterization of an aspartic proteinase derived from M. circinelloides (M-105). Durum wheat bran was utilized as solid substrate.…”

Partial Purification and Characterization of Extracellular Protease from <i>Pedicoccus acidilactici</i>

“…Acidic proteases have been isolated from various species of Aspergillus (O'Donnell et al, 2001;Tremacoldi et al, 2004), Mucor (Escobar and Barnett, 1993;Fernandez-Lahore et al, 1999) and Rhizopus (Kumar et al, 2005). Fu et al (2008) obtained an acid protease from Bacillus megaterium KLP-98 which had an optimum pH of 5.5 and was stable in the pH range of 4.5 -6.0.…”