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.

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Further characterization of an aspartyl protease from Mucor bacilliformis with milk-clotting activity was performed. An extinction coefficient, epsilon 278 cm = 1.61 mL/mg/cm, a molecular mass of 35,400 Da and a pI of 5.2 were determined. Proteolytic activity and kinetic parameters were evaluated by using the hexapeptide Leu-Ser-pNO2-Phe-Nle-Ala-Leu-OMe as the substrate. The effect of pH and temperature on peptide cleavage, as well as protease heat stability, was determined. Such properties, taken as a whole, indicate that the M. bacilliformis protease can be considered a potential substitute for bovine chymosin in cheese manufacture.

Among the abnormalities in erythrocyte porphyrin metabolism already described in patients with chronic renal failure on hemodialysis, a decrease in blood aminolevulinate dehydratase activity has been reported, suggesting the presence in uremic plasma of an inhibitor of the enzyme. The aim of this work has been to isolate and characterize such an inhibitor. Blood samples from 105 patients with chronic uremia were collected; plasma was applied to Sephadex G-100 columns and the fraction with the highest inhibiting capacity was identified and purified by subsequent SDS-polyacrylamide gel electrophoresis, followed by electroelution and electroblotting. It was demonstrated that the factor present in plasma of uremic patients inhibited blood aminolevulinate dehydratase in a concentration-dependent manner; its inhibitory properties were abolished after heat, trypsin and TCA treatment indicating its peptidic nature. The purified inhibitor has an apparent molecular mass of 56.2 kD, it inhibits blood aminolevulinate dehydratase in a competitive way and the K_i value is 12×10^–6 M. The amino acid composition of the inhibitor has been determined and it has been found that its N-terminal amino acid is blocked. The isolated peptide may play a role in heme biosynthesis disturbances and in the pathogenesis of uremic anemia.

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