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The purpose of this study was to evaluate the caseinolytic and milk-clotting activities of aqueous crude extracts from leaves and latex of the Pergularia tomentosa, to determine their suitability as a rennet substitute. These extracts were subjected to a series of biochemical tests before being used in the production of cheese. The results showed that the enzymatic latex extract had a higher coagulant activity than the leaf extract. However, under different clotting conditions (pH, temperature, and CaCl2 concentration), both coagulants behaved similarly in the coagulation of Berridge substrate. The SDS-PAGE and zymographic analysis revealed identical protein bands with a single active zone in both extracts, corresponding to a molecular weight of 26.98 kDa and 26.03 kDa in the extract of leaf and latex, respectively. Both extracts were stable to different effectors but strongly inhibited by iodoacetamide and Hg, suggesting it to be a cysteine protease. Both extracts were able to hydrolyze casein and generate peptides of 14 kDa, with excessive hydrolysis of the other casein fractions. The physicochemical parameters of cheese made from latex and leaf extract evolved similarly to control cheese. According to the sensory evaluation, cheese made with latex had a mildly bitter flavor but showed a high acceptance rate (>80%).