Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus

Heimgartner, Urs; Pietrzak, Maciej; Geertsen, Ralf; Brodelius, Peter E.; Figueiredo, A C da Silva; Pais, M. S.

doi:10.1016/0031-9422(90)80090-4

Cited by 178 publications

(168 citation statements)

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“…A partial purification of the proteolytic enzyme from fig tree latex was carried out between 50 to 85% saturation (Oner and Akar, 1993). The study of proteases from Cynara cardunculus showed three proteases with milk clotting activity after fractional ammonium sulphate precipitation between 30 and 80% saturation (Heimgartner et al, 1990). Ammonium sulphate precipitation was chosen in this study because of its simplicity and the economy of the method, but results showed that there was no significant increase in the milk clotting activity.…”

Section: Protease Purificationmentioning

confidence: 98%

“…2 can be observed the SDS-PAGE gel, with one single major band, which revealed a protein with a molecular weight of approximately 33 kDa. These results can be compared with study of milk clotting protease from flowers of Cynara cardunculus with three proteases with milk clotting activity after two steps of the chromatography (DEAE-Sepharose and MonoQ column) (Heimgartner et al, 1990). The purified proteases showed only one band for each protease, with molecular weights of 41, 42 and 45 kDa.…”

Section: Protease Purificationmentioning

confidence: 99%

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Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Duarte

Moreira

et al. 2009

Braz. arch. biol. technol.

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The partial characterization and purification of milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions and among the fractions obtained, the 40-60% fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.

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Section: Protease Purificationmentioning

confidence: 98%

Section: Protease Purificationmentioning

confidence: 99%

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Duarte

Moreira

et al. 2009

Braz. arch. biol. technol.

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“…Dried flowers of this cardoon have been used since ancient times for the production of high quality sheep-milk cheese. The clotting enzyme, cardosin, is an aspartic protease that was isolated from these flowers, purified, and partly characterized (Heimgartner et al, 1990;Faro et al, 1992). The flowers of Cynara cardunculus L. are not used for the production of cheese of cow milk.…”

Section: Introductionmentioning

confidence: 99%

Caseinolytic Specificity of Cardosin, an Aspartic Protease from the CardoonCynara cardunculusL.: Action on Bovine α_s- and β-Casein and Comparison with Chymosin

Macedo

Faro

Pires

1996

J. Agric. Food Chem.

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The action of cardosin on bovine R s -and -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. ), 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.

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“…Scots pine [lo], soybean [Ill, barley and wheat [12], but also in leaves of tomato [I 31 and barley [14]. Aspartic proteinases have been purified and partly characterized from a number of seeds, including rice [15], cucumber and squash [16], wheat [I71 and barley [18], as well as from the leaves of tomato [13] and the flowers of thistle [19]. These enzymes are all inhibited by pepstatin.…”

mentioning

confidence: 99%

Primary structure of a barley‐grain aspartic proteinase

Runeberg-Roos

Törmäkangas

Östman

1991

European Journal of Biochemistry

127

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Two enzymatically active heterodimeric forms of an aspartic proteinase, a putative 32 kDa + 16 kDa precursor form and a putative 29 kDa + 11 kDa mature form, are present in resting barley grains (Sarkkinen, P., Kalkkinen, N., Tilgmann, C., Siuro, J., Kervinen, J. & Mikola, L., 1990, in the press). The cDNA corresponding to this enzyme has been cloned and sequenced. The full-length 1863-bp cDNA sequence codes for an open reading frame of 508 amino acids. The open reading frame consists of a 66-amino acid preprosequence and a 442-amino acid mature protein.Comparison of the N-terminal amino acid sequences of the enzyme subunits with the sequence of the cDNA clone indicates that the heterodimeric enzyme is translated as a proenzyme which is processed into two subunits. The localisation of the experimentally determined N-terminal amino acid sequences of all four subunits (32 kDa + 16 kDa and 29 kDa + 11 kDa) in the same transcript, as well as the detection of only one 2.0-kb mRNA on Northern blots from resting seeds, clearly indicates that the larger (32 kDa + 16 kDa) enzyme is an intermediate precursor form of the smaller (29 kDa + 11 kDa) enzyme. The processing pattern of the barley enzyme, which is the first sequenced plant aspartic proteinase, differs from that of all other known aspartic proteinases. The barley enzyme is highly similar to mammalian and yeast aspartic proteinases, especially to human and porcine cathepsin D. This similarity is clearly dispersed over two regions, separated by a dissimilar, barley-specific region of 104 amino acids.In animals and yeast, members of the family of aspartic proteinases, including cathepsin D, renin, chymosin and proteinase A, are very substrate-specific enzymes with well-defined physiological functions. This class of proteinases is involved in the activation of other enzymes, e.g. the lysosomal acid phosphatase and carboxypeptidase Y [l -31 as well as extracellular proteins like procollagen and the prohormonal angiotensinogen [4, 51. Aspartic proteinases are also involved in the digestion and inactivation of specific proteins like the protcinase-inhibitor cystatin and the IC chain of the milk Kcasein [6, 71. A common feature of aspartic proteinases is an active cleft where two aspartate residues play a central role in the catalytic mechanism [S, 91. The amino acid sequences around these aspartate residues are generally conserved. The Note. The novel nucleotide acid sequence data published here has been dcposited with the EMBL sequence data bank and i s available under accession number X56136.Dediwtion. To the memory of Juhani Mikola who initiated this work as a continuation of his research on storage proteins and their mobilisation during germination. activity of most aspartic proteinases is inhibited by the specific inhibitor pepstatin. Different aspartic proteinases differ in their cellular localisation and processing pattern, as well as in their substrate specificity.In plants, aspartic proteinase activity has been localised in seeds from a broad variety of plant sp...

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Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus

Cited by 178 publications

References 11 publications

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Caseinolytic Specificity of Cardosin, an Aspartic Protease from the CardoonCynara cardunculusL.: Action on Bovine α_s- and β-Casein and Comparison with Chymosin

Primary structure of a barley‐grain aspartic proteinase

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Purification and partial characterization of milk clotting proteases from flowers of Cynara cardunculus

Cited by 178 publications

References 11 publications

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Jacaratia corumbensis O. Kuntze a new vegetable source for milk-clotting enzymes

Caseinolytic Specificity of Cardosin, an Aspartic Protease from the CardoonCynara cardunculusL.: Action on Bovine αs- and β-Casein and Comparison with Chymosin

Primary structure of a barley‐grain aspartic proteinase

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Caseinolytic Specificity of Cardosin, an Aspartic Protease from the CardoonCynara cardunculusL.: Action on Bovine α_s- and β-Casein and Comparison with Chymosin