A novel milk-clotting cysteine protease from Ficus johannis: Purification and characterization

• Published in: International journal of biological macromolecules 2019-01, Vol.121, p.173-182
• Main Authors: Afsharnezhad, Moslem, Shahangian, S. Shirin, Sariri, Reyhaneh
• Format: Article
• Language: English
• Subjects: Animals; Cysteine protease; Cysteine Proteases - isolation & purification; Cysteine Proteases - metabolism; Cysteine Proteinase Inhibitors - pharmacology; Enzyme Stability; Ficus - enzymology; Ficus johannis; Hydrogen-Ion Concentration; Hydrolysis; Milk - metabolism; Milk-clotting activity
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2018.10.006

Due to the need for calf rennet alternatives, many attempts have been made to find new proteases. A novel cysteine protease with milk-clotting activity was purified from Ficus johannis by cation exchange chromatography. The protease was stable in various pH (3.0–10.5) with the optimum at 6.5 and showed its maximum activity at 60 °C. The Km and Vmax values of the enzyme were obtained to be 0.604 mg/ml and 0.0273 μmol Tyr/min, respectively. The purified protease exhibited considerable activity towards κ-casein in comparison to α-casein and β-casein. The enzyme was almost completely active in the presence of high salt concentrations. Besides, it had high stability against autodigestion. The content of free amino acids was determined by HPLC, where leucine, lysine, valine, γ-aminobutyric acid and tyrosine were the most abundant amino acids. The cheese manufactured by using the purified protease showed similar textural properties and physico-chemical compositions to cheese produced using commercial rennet. Considering the special characteristics, including high milk-clotting activity, considerable stability over wide ranges of pH and temperature, resistance towards solvents, salts, and surfactants, the new protease might be the promising candidate for the dairy industry as well as other food and biotechnological industries. •A novel milk-clotting enzyme from F. johannis latex was purified and characterized.•The enzyme activity at various pH and temperatures was in favor of dairy industry.•Enzyme showed high stability over a wide range of pH and temperatures.•The high MCA/PA ratio of the enzyme makes it useful as a rennet substitute.•The enzyme may be a good candidate for other food and biotechnological industries.