Puri®cation and properties of a milk-clotting enzyme produced by Penicillium oxalicum

Partial puri®cation of milk-clotting and caseinase enzymes, produced by Penicillium oxalicum, was achieved by fractional precipitation with acetone, ethanol and methanol. Of the fractions obtained by three precipitants, the 60±70% ethanol fraction was the most promising enzyme fraction and possessed the highest milk-clotting activity, which reached about ninefold that of the culture ®ltrate. Puri®cation of the milk-clotting enzyme by DEAE-cellulose column chromatography a€orded a rennin-like enzyme component that showed no proteolytic activity. The enzyme activity was maximum at pH 4.0±5.0 and 65°C. In absence of substrate and up to 50°C, the enzyme showed good stability and retained 80% of its original activity after 20 min. Cu2‡, Co2‡ and Mg2‡ had stimulating e€ects on enzyme activity. Ascorbic acid, sodium lauryl sulphate, cysteine hydrochloride, cystine and EDTA had partial inhibitory e€ects on the enzyme.