Caseinolytic Specificity of Cardosin, an Aspartic Protease from the Cardoon Cynara cardunculus L.: Action on Bovine αs- and β-Casein and Comparison with Chymosin
Open accessAbstract
The action of cardosin on bovine α s - and β-casein at 30 °C in 50 mM citrate buffer (pH 6.2) was studied. Peptides were isolated by reversed-phase HPLC on C 18 columns and identified from their amino acid composition and N -terminal amino acid sequence. The relative susceptibility of peptide bonds cleaved was Phe 23 -Phe 24 > Trp 164 -Tyr 165 > Tyr 166 -Val 167 > Tyr 165 -Tyr 166 > Phe 153 -Tyr 154 > Phe 145 -Tyr 146 ≈ Leu 149 -Phe 150 ≈ Leu 156 -Asp 157 ≈ Ala 163 -Trp 164 for α s1 -casein and Leu 192 -Tyr 193 > Leu 191 -Leu 192 ≈ Leu 165 -Ser 166 > Phe 190 -Leu 191 ≥ Ala 189 -Phe 190 ≈ Leu 127 -Thr 128 for β-casein. In α s2 -casein, cardosin cleaved the bonds Phe 88 -Tyr 89 and Tyr 95 -Leu 96 . The enzyme shows a clear preference for bonds between hydrophobic, bulky amino acids, cleaving four consecutive peptide bonds in extremely bulky, hydrophobic regions of both α s1 -CN (Ala 163 -Val 167 ) and β-CN (Ala 189 -Tyr 193 ), which was less attacked by chymosin in various experimental conditions. The active site cleft of cardosin accommodates sequences as bulky as Trp-Tyr-Tyr in different subsites (S 1 to S‘ 2, S 2 to S‘ 1, and probably S 3 to S 1 ). Several bitter peptides were identified in the digests. Keywords: α s -Casein; β-casein; cardosin; vegetal rennet; specificity; proteolysis; bitter peptides
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A comparison between chymosin and cardosins shows that cardosins have a greater proteolytic activity, which leads to the onset of bitter flavors.
“However, a comparison between chymosin and cardosins shows that the latter have a greater PA, which leads to the onset of bitter flavors [44,45].”
From Plant Milk-Clotting Enzymes for Cheesemaking (2022) · cites this paper as [44]
The Cheese Quality section (page unavailable) suggests that cardosin’s high activity on a specific β-casein segment may contribute to bitter taste in cheese. It does not establish that cardosin has greater proteolytic activity overall than chymosin or that this activity necessarily causes bitterness; the section also notes similar activity against κ-casein and cautions against directly extrapolating from solution studies to cheese. (LLM, conf=0.88)
Experimental — accuracy not yet measuredIn solution, the fragment bovine αs1-casein (f24-199) was identified as the major peptide produced by the action of proteinases from C. cardunculus, a result confirmed independently by Macedo et al. [32].
“In solution, the fragment bovine α s1 -casein (f24-199) was identified as the major peptide produced by the action of proteinases from C. cardunculus [48], a result confirmed independently by Macedo et al. [32].”
From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [32]
The Results and Discussion identify αs1-casein Phe24–Trp199 as a peptide produced by cardosin, and the proteolysis pathway describes it as a product of the primary cleavage. The paper does not establish that it was the major peptide by abundance in solution; it also describes subsequent cleavage of that fragment. No page number is available in our copy. (LLM, conf=0.82)
Experimental — accuracy not yet measuredαs2-casein is cleaved at Phe88-Tyr89 in bovine, ovine and caprine caseinates.
“Finally, α s2 -casein was cleaved at Phe 88 -Tyr 89 in bovine [32], ovine and caprine caseinates [55] (Fig. 1d).”
From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [32]
Proteinases from C. cardunculus show a clear preference for bonds between hydrophobic regions of both αs1-casein (f163-167) and β-casein (f189-193), which were less attacked by chymosin under various experimental conditions.
“Macedo et al. [32] reported that proteinases from C. cardunculus show a clear preference for bonds between hydrophobic regions of both α s1 -casein (f163-167) and β-casein (f189-193), which were less attacked by chymosin under various experimental conditions.”
From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [32]
The most susceptible bond on bovine β-casein is Leu192-Tyr193, although proteinases from C. cardunculus were reported to cleave Leu127-Thr128, Leu165-Ser166 and every single peptide bond of Ala189-Tyr193 in bovine β-casein as well.
“The most susceptible bond on bovine β-casein is Leu 192 -Tyr 193 , [32,48], although proteinases from C. cardunculus were reported to cleave Leu 127 -Thr 128 , Leu 165 -Ser 166 and every single peptide bond of Ala 189 -Tyr 193 (i.e.Ala 189 -Phe 190 , Phe 190 -Leu 191 , Leu 191 -Leu 192 and Leu 192 -Tyr 193 ) in bovine β-casein as well [32] (Fig. 1b).”
From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [32]
The bond Tyr95-Leu96 is also cleaved in bovine αs2-casein, whereas the bonds Ser9-Ser10 and Tyr179-Leu180 are cleaved in caprine caseinate as well.
“The bond Tyr 95 -Leu 96 was also cleaved in bovine α s2 -casein [32], whereas the bonds Ser 9 -Ser 10 and Tyr 179 -Leu 180 were cleaved in caprine caseinate as well [55].”
From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [32]
The most susceptible site in bovine αs1-casein is Phe23-Phe24.
“The most susceptible site in bovine α s1 -casein is Phe 23 -Phe 24 [32,48] (Fig. 1c).”
From Advances in the role of a plant coagulant ( <i>Cynara cardunculus</i> ) in vitro and during ripening of cheeses from several milk species (2002) · cites this paper as [32]
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- Authors
- Macedo, I. Q., Faro, C. J., & Pires, E. M.
- Affiliations
- University of Aveiro (PT)
- Venue
- Journal of Agricultural and Food Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1996-01-01
- Volume
- 44
- Issue
- 1
- Pages
- 42-47
- ISSN
- 0021-8561 (ISSN-L)
- 1520-5118
- OpenAlex
- W2078792868
- Cited by
- 70
- References
- 20
- Citations by year
- 2026: 1 · 2025: 5 · 2024: 3 · 2022: 1 · 2021: 3 · 2020: 3 · 2019: 1 · 2018: 4 · 2017: 4 · 2015: 2 · 2014: 6 · 2012: 3
- FWCI
- 3.63
- Type
- journal article
- Language
- en
- Open access
- green
- hdl.handle.net
- Locations
- Journal of Agricultural and Food Chemistry (publishedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion, other-oa)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Cynara cardunculus studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Hydrolysis and Bioactive Peptides
- Keywords
- aspartic protease, Cynara cardunculus, bovine casein, αs1-casein, αs2-casein, β-casein, chymosin, substrate specificity, peptide bond cleavage, hydrophobic amino acids, active site subsites, bitter peptides, cheese ripening
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