Production of a Milk-clotting Enzyme Preparation by Aspergillus niger and the Effect of Various Factors on its Activity

Abstract

SUMMARYAmong 20 locally isolated fungi, Aspergillus niger isolate no. 58 proved to be suitable for the production of active extracellular milk-clotting enzyme. The addition of acetate buffer to the reaction mixture enhanced the clotting of milk by the enzyme whereas citrate +phosphate buffer hindered the process. The general properties of the crude enzyme were studied. Precipitation with ammonium sulphate, ethanol, acetone and tannin showed that ammonium sulphate was unsuitable for precipitation while the other precipitantgproduced sufficiently active fractions. I N T R O D U C T I O NStudies on the production of milk-clotting enzymes from microbial sources have gained interest during the last decade; the production of milk-clotting enzymes by different types of micro-organisms has been reported (Arima & Iwasaki, I 964; Veselov, Tipograf & Petina, I 965 ; Knight, I 966). The increasing demands on rennin for cheese making in Egypt and the prohibition of the slaughter of calves justified a study aiming to search for rennin substitutes from sources other than animals and in particular from micro-organisms. Cultivation. Transfers were made from the stock slants to Dox agar plates which were then incubated at 30' for 7 days. Two discs (0.5 cm. diam.) were cut from the 7-day culture and inoculated into 50 ml. sterile medium in 250 ml. flat bottomed flasks. Unless otherwise specified, the flasks were incubated for 3 to 4 days at 3 0 ' . At the end of the incubation period the culture medium was filtered off and its volume adjusted to 50 ml.

Production and Some Properties of Rennin-like Milk-clotting Enzyme from Penicillium citrinum

“…The milk-clotting activity of the enzyme was maximal at 60" but nearly identical activity was achieved at 55'. This result is similar to those found for other microbial milk-clotting enzymes (Osman et al 1969;Kawai & Mukai, 1970; Arnold, Stefan & Wanda, 1970). The enzyme showed maximal milk-clotting activity at …”

“…The association of milk-clotting activity with spore formation was also observed by Osman et al (1969). respectively, of that of the culture filtrate.…”

Role of Physical Properties of Glycidyl Methacrylate- Pentaerythritol Triacrylate Copolymers on Immobilization of Milk Clotting Protease from Aspergillus niger MC4

“…Optimum pH values for the milk clotting protease from Aspergillus candidus, Irpex lacteus, Mucor pusillus, Penicillium citrinum , Physarum polycephalum , and Bacillus steorothephilus were 5.6, 5.4, 5.6−5.9, 6.0, 5.0, and 6.1, respectively, and optimum temperatures for milk clotting protease from Aspergillus ustus SBT 567, A. niger No. 58, I. lacteus, M. pusillus, P. citrinum, P. polycephalum , and B. stereothermophilus were 63, 52, 55−60, 56, 50, 35 and 65−70 °C, respectively (Iwasaki et al, 1967; Osman et al, 1969; Kawai, 1970; Abdel‐Fattah et al, 1972; Farr et al, 1974; Sardinas, 1976; Selim et al, 1991; Kikuchi et al, 1994). Rennet, pepsin, and Emporase immobilized on wax exhibited maximum activity at pH 5.5 (Mashaly et al, 1987a,b).…”

“…The enzymes from Endothia parasitica, I. lacteus , and A. niger No. 58 were totally inactivated when heated at 60 °C for 5−10 and 30 min, respectively (Osman et al, 1969; Kawai, 1970; Sardinas, 1976). Many proteases and milk clotting proteases have been immobilized on sand.…”

Purification and Some Properties of Milk-clotting Enzyme from Aspergillus niger

“…This was done according to the method of Bergkvist (1963) for proteases of Aspergillus oryzae (see Osman et al 1969). The electropherogram was cut longitudinally and one part was stained with Amido black 10 B, the corresponding portion of the protein band on the unstained part was cut off and eluted with citrate+phosphate buffers of different pH values.…”