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

Afsharnezhad, Moslem; Shahangian, S. Shirin; Sariri, Reyhaneh

doi:10.1016/j.ijbiomac.2018.10.006

Cited by 72Enzyme Production and CharacterizationPineapple and bromelain studiesPhytochemistry and biological activities of Ficus species

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

Afsharnezhad, Moslem, Shahangian, S. Shirin, Sariri, Reyhaneh·2019·International Journal of Biological Macromolecules

DOI 10.1016/j.ijbiomac.2018.10.006

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Abstract

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 K m and V max 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.

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Sodium chloride improves the stability of casein micelles.
“Sodium chloride improves the stability of casein micelles; however, excessively high concentrations may cause enzyme denaturation, resulting in loss of activity (Afsharnezhad et al., 2019;Artifon et al., 2023).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Afsharnezhad et al., 2019
Excessively high NaCl concentrations may cause enzyme denaturation, resulting in loss of activity.
“Sodium chloride improves the stability of casein micelles; however, excessively high concentrations may cause enzyme denaturation, resulting in loss of activity (Afsharnezhad et al., 2019;Artifon et al., 2023).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Afsharnezhad et al., 2019

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Background
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“Similar studies were conducted by Afsharnezhad et al. (2019) and Ahmed et al. (2009) to isolate and purify cysteine and serine proteases from Ficus johannis and Bodhi trees, respectively, providing a new avenue for their application in cheese in the dairy industry.”
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Exploiting pitaya cladodes for protease Extraction: Immobilization on nanomaterials and its role in antioxidant production
Alves, A. N., Santos, I. A., Nascimento, P. A., Porfírio, M. C. P., Boas, E. V. D. B. V., Fontan, R. D. C. I., & Bonomo, R. C. F. (2025) · Separation and Purification Technology · doi:10.1016/j.seppur.2025.134994
Additional file 1 of The catalytic and kinetic characterization of Bacillus subtilis MK775302 milk clotting enzyme: comparison with calf rennet as a coagulant in white soft cheese manufacture
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Protease production by Penicillium roqueforti through solid-state fermentation of ora-pro-nóbis (Pereskia aculeata): optimization and characterization
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Pepsin extracted from the proventriculi of geese raised under different fattening techniques and its effect on microbiological quality and cheese production
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Effect of acid protease from Lacticaseibacillus paracasei strain S1P3 on milk proteolysis and milk-clotting properties in cheesemaking
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Enzymes: Types and Uses in Food Processing
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Green extraction of milk-clotting proteases from sweet Indrajao (Wrightia tinctoria) stem utilizing an ethanol-phosphate based aqueous two-phase system - optimization and scalability
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Mazorra-Manzano, M., Moreno-Hernández, J. M., Ramírez-Suárez, J. C., Torres-Llanez, M. D. J., González-Córdova, A. F., & Vallejo-Córdoba, B. (2013) · LWT · doi:10.1016/j.lwt.2013.07.009 · Cited by 53 · see citing works
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de Farias, V. A., Lima, A. D. D. R., Costa, A. S., Freitas, C. D. T., Araújo, Í. M. D. S., Garruti, D. D. S., de Figueiredo, E. A. T., & de Oliveira, H. D. (2019) · Food Research International · doi:10.1016/j.foodres.2019.108689 · Cited by 33 · see citing works

Authors: Afsharnezhad, M., Shahangian, S. S., & Sariri, R.
Affiliations: University of Guilan (IR)
Corresponding author: Shahangian, S. Shirin
ORCID: Shahangian, S. Shirin
Sariri, Reyhaneh
Venue: International Journal of Biological Macromolecules
Publisher: Elsevier BV
Published: 2019-01
Volume: 121
Pages: 173-182
ISSN: 0141-8130 (ISSN-L)
DOI: 10.1016/j.ijbiomac.2018.10.006
OpenAlex: W2895268279
Cited by: 72
References: 61
Citations by year: 2026: 8 · 2025: 7 · 2024: 10 · 2023: 16 · 2022: 13 · 2021: 12 · 2020: 4 · 2019: 2
FWCI: 2.84
Type: journal article
Language: en
Open access: closed
Licence: www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/legal/tdmrep-license (tdm)
doi.org/10.15223/policy-017 (stm-asf)
doi.org/10.15223/policy-037 (stm-asf)
doi.org/10.15223/policy-012 (stm-asf)
doi.org/10.15223/policy-029 (stm-asf)
doi.org/10.15223/policy-004 (stm-asf)
Locations: International Journal of Biological Macromolecules (publishedVersion)
PubMed (publishedVersion)
Funders: University of Guilan
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pineapple and bromelain studies
Life Sciences › Agricultural and Biological Sciences › Plant Science › Phytochemistry and biological activities of Ficus species
Keywords: cysteine protease, milk-clotting enzyme, κ-casein hydrolysis, enzyme purification, enzyme stability, cheese production, enzyme kinetics
MeSH terms: Animals, Enzyme Stability, Hydrogen-Ion Concentration, Hydrolysis, Milk, Cysteine Proteinase Inhibitors, Ficus, Cysteine Proteases

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

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