SUMMARYThe production of a rennin-like milk-clotting enzyme by Penicillium citrinum 805 was investigated. Corn-steep water was the best medium for the production of the enzyme. Dephytinization of corn-steep water lowered production of the enzyme. The extent of mycelial sporulation was correlated with the milk-clotting activity. Precipitation with ammonium sulphate and ethanol was unsuitable, but acetone produced active fractions. The enzyme action was optimal at 60" and at pH 6.0 while the stability of the enzyme was maximal at pH 5-37. The enzyme exhibited feeble proteolytic activity.

SUMMARYOf 48 fungal isolates, 28 (including one Penicillium and three Aspergilli) secreted milk-clotting enzymes. This activity was in general related to the mycelial growth of a given isolate, but not with pH change of the culture filtrate during incubation. It was not found when mucilage was formed by isolates capable of doing so. The ratio of general proteolytic activity to milk-clotting activity varied from one organism to another, and with the age of culture of the same fungal strain.

Electrophoresis of the partially purified milk-clotting enzyme produced by Penicillium citrinum with 0.02 M acetate buffer (pH 3.42) showed four protein components.Fractionation of the enzyme preparation with acetone led to individual isolation of the four electrophoretic components.The course of proteolysis in the first phase of enzymic action of each of the partially purified fungal enzymes and the four pure fractions was conducted and compared with those of trypsin and calf rennin. The fungal enzyme contained two rennin-like fractions constituting most of the milk-clotting activity with limited proteolysis and another two fractions with less clotting activity and more proteolysis.The macroglycopeptide isolated by the action of the fungal rennin-like enzyme fraction comprised 12 amino acids while its carbohydrate moiety consisted of galactose, galactosamine, and N-acetylneuraminic acid. Further studies on the most active rennin-like enzyme fraction showed that calcium chloride enhanced only the primary stage of the enzymic phase but near the end of this phase the rate of NPN release was very slow.

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The influence of some metal ions was investigatedon the milk-clotting activityof the pure rennin-like enzyme of Penicillium citrinum 805. Barium and calcium enhanced the clotting activity while copper, nickel, and sodium ions showed slight inhibitory effects. Above a certain concentration limit, EDTA played a role by which its complexing action with calcium ions was compensated and the milk became readily coagulable by the enzyme. This phenomenon was more significant when EDTA was added to the milk instead of the enzyme. It was suggested that treatment of milk with EDTA led to homogenization and aggregation of the phosphocaseinate particles whereby the clottability of milk was increased. The heating of milk rendered it more coagulable by the rennin-like enzyme. This was attributed to different factors including the increase in the degree of aggregation of casein micelles and the release of NPN-bound NANA by heat.Studies on the action of metal ions on animal rennin enzymes were carried out by many investigators (HOSTETTLER and RUGGER 1950, WAKUI and KAWACHI 1954a, S A S A K I~~ al. 1955, PURI and PARKASH 1962. The effect of calcium concentration on the clotting of milk by microbial rennin and HANSEN'S Cheese rennet was investigated by TSUGO et al. (1964). The latter investigators found that the milk-clotting activity of microbial rennin was increased by the addition of CaCI, but the rate of increase was more pronounced than in the case of HANSEN'S Cheese rennet. Recently, ABDEL-FATTAH and MABROUK (1972a) studied the effect of CaC1, on the milk-clotting enzyme of Aspergillus niger and found that addition of CaCl, to buffered reaction mixtures caused decrease in p H and time of milkclotting.The effect of EDTA as a complexing agent on milk coagulation by rennet was studied by ODAGIRI and NICKERSON (1964). The latter authors found that coagulation time was prolonged in logarithmic proportion to the amount of complexing agent added, but was restored to normal by the addition of calcium. Detailed studies about the action of EDTA on the coagulability of milk by microbial milk-clotting enzymes are, however, scanty.It has long been known that milk, when heated and then cooled immediately, shows an increase in rennet coagulation time (PYNE 1945, ZITTLE et al. 1962, HINDLE and WHEELOCK 1970. However, in a study on the milk-clotting enzyme of the leaves of Streblus asper, RAM and R E D D I (~~~~) reported that the purified enzyme preparation clotted boiled milk more rapidly than raw cow's milk. Furthermore, RAMSDELL and WHITTIER (1953) found that heating of milk rendered it more coagulable by rennet and attributed this t o the denaturation 25*

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