The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

Verı́ssimo, Paula; Esteves, Cristina L.; Faro, Carlos; Pires, Euclides

doi:10.1007/bf00129389

Cited by 137 publications

(127 citation statements)

References 6 publications

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“…Those proteinases were reported to have a pH optimum of 5.1 and they were classified as aspartic proteinases [27]. Sousa [48] indicated that only two of the three original peaks (peaks 2 and 3) possessed clotting and proteolytic activities; these two proteinases were later named cardosin A (peak 2) and cardosin B (peak 3) [63]. These authors also reported that each cardosin occurs in dimeric form, with apparent molecular masses of 31 and 15 kg .…”

Section: Purification Of Proteinasesmentioning

confidence: 99%

“…mol -1 for those in cardosin B. The kinetic parameters associated with hydrolysis of the synthetic peptide Leu-SerPhe-(NO 2 )-Nle-Ala-Leu-oMe were determined, and together with their specificity (see Section 4) they were compared to those of chymosin and pepsin [63]; it was thus shown that cardosin A is similar to chymosin, whereas cardosin B resembles pepsin. These proteinases share pH optima in the acid range, inhibition by pepstatin and preferential cleavage of peptide bonds between hydrophobic residues (as happens with other aspartic proteinases) [64].…”

Section: Purification Of Proteinasesmentioning

confidence: 99%

“…The drying process decreases the proteolytic activity, especially that of cardosin B (which is the stronger enzyme in the crude extract), and thus has a considerable effect on the observed overall proteolytic activity [17]. More recent studies [21,63,64] have indicated that those two enzymes appear chiefly in the female part of the flowers of C. cardunculus, i.e. in the upper portion (where the ratio of concentrations of cardosin A to cardosin B is also higher); in the lower portion, only cardosin B is present [9].…”

Section: Purification Of Proteinasesmentioning

confidence: 99%

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Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheeses from several milk species

Sousa

Malcata

2002

Lait

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-Extracts from dried flowers of Cynara cardunculus have been employed successfully for many centuries in Portugal and some regions of Spain for the manufacture of traditional cheeses. Several studies have been performed in vitro and in actual cheeses focusing on the activity and specificity of proteinases from C. cardunculus, in attempts to further characterise this plant coagulant as a proteolytic agent in cheese ripening. In vitro studies encompassed extraction conditions, storage of extracts, proteolysis and specificity on caseins using experimental conditions that parallel milk and cheese during ripening. In vivo studies encompassed the effect of the type of milk (bovine, ovine and caprine) in terms of proteolysis, specificity of those plant proteinases during ripening, comparison of microbiological, chemical, proteolytic and lipolytic characteristics in ovine milk cheeses relative to animal rennet counterparts, and effect of native microflora, thermal processing and addition of starter cultures in ovine cheeses. milk clotting enzyme / plant coagulant / cheese / ewe's milk / cow's milk / goat's milk / ripeningResumé -Rôle de l'extrait coagulant de chardon (Cynara cardunculus) in vitro et au cours de l'affinage de fromages fabriqués à partir de laits de plusieurs espèces. L'extrait de fleurs séchées de Cynara cardunculus a été employé avec succès depuis plusieurs siècles au Portugal et dans quelques régions d'Espagne pour la fabrication de fromages traditionnels. De nombreuses études ont été réalisées in vitro et en fabrication fromagère sur l'activité et la spécificité des protéases de C. cardunculus, dans le but de mieux caractériser ce coagulant végétal comme agent protéolytique dans l'affinage du fromage. Les études in vitro ont concerné les conditions d'extraction, la conservation des extraits, la protéolyse et la spécificité sur les caséines en utilisant des conditions expérimentales qui 151

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Section: Purification Of Proteinasesmentioning

confidence: 99%

Section: Purification Of Proteinasesmentioning

confidence: 99%

Section: Purification Of Proteinasesmentioning

confidence: 99%

See 1 more Smart Citation

Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheeses from several milk species

Sousa

Malcata

2002

Lait

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“…a higher proteolytic activity [19,20]. The localisation of cardosin A has been studied in detail [13].…”

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confidence: 99%

Identification and proteolytic processing of procardosin A

Ramalho-Santos

Verı́ssimo

Cortes

et al. 1998

European Journal of Biochemistry

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Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.Keywords : aspartic protease ; milk-clotting enzyme; cardosin ; proteolytic processing; saposin.Aspartic proteinases (AP) are a widely distributed class of kingdom [5]. In the majority of plants AP are located in seeds, endoproteases that share significant similarities at the amino-whether quiescent or germinating. They are believed to particiacid-sequence level and at the structural level [1, 2]. The typical pate in storage-protein cleavage, which is necessary for germinacharacteristics of the family are an acidic pH optimum, inhibi-tion [5,10]. AP have also been found in leaves of some plants. tion by pepstatin A, preference for bonds between hydrophobic In leaves they have been implicated in mechanisms of defense amino acids, and sequence similarities, in particular the conser-against pathogens [11,12]. Species of the genus Cynara contain vation of the catalytic triads Asp-Thr-Gly or Asp-Ser-Gly [1Ϫ considerable amounts of AP in flowers. Recent data indicate that 4]. AP have been implicated in diverse physiological processes, the major AP from cardoon (Cynara cardunculus L.), cardosin such as digestion or blood-pressure regulation, and in some A, may be involved in the sexual reproduction of the plant [6] pathological conditions, including infection by fungi and retro-and may have a defensive role as well [13]. viruses or cancer [5, 6].A few primary structures deduced from the cDNAs of AP Like many other proteases, A...

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“…The clotting activity of this extract was first attributed to the presence of three proteolytic enzymes synthesized by the stigmae of those flowers [3], and such (tentatively termed) cynarases 1,2 and 3 were purified and partially characterized [6]. Two new aspartic acid proteinases, (tentatively named) cardosins A and B, were later isolated and are believed to be genetically different from the aforementioned cynarases [17]; in terms of activity and specificity, cardosins A and B are remarkably similar to chymosin and pepsin, respectively [16].…”

Section: Introductionmentioning

confidence: 99%

Proteolysis of ovine caseins by cardosin A, an aspartic acid proteinase from Cynara cardunculus L.

Silva

Malcata

1998

Lait

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-The breakdown of as-caseins and~-caseins (in the form of as-caseins, the form of -caseins, and the form of a mixture of a s -and~-caseins in Na-caseinate) by cardosin A, one of the major two proteinases present in the flowers of Cynara cardunculus L., was experimentally studied via urea polyacrylamide gel electrophoresis. In Na-caseinate, a s -and~-caseins were degraded up to 46 and 76 %, respectively, by 10 h of hydrolysis. In separated form, as-caseins reached a level of degradation up to 67 % while~-caseins were quickly and extensively degraded up to 76 %. In general,~-caseins seemed to be more susceptible to proteolysis than as -caseins. © InralElsevier, Paris. milk protein / enzyme / plant rennet / electrophoresis Résumé -Protéolyse des caséines de brebis par la cardosine A, une protéase acide aspartique de Cynara cardunculus L. La dégradation des caséines as et des caséines~(sous la forme de caséines as' de caséines~, et d'un mélange de caséines as et~d'un caséinate du sodium) par la cardosine A, une des protéinases majeures présentes dans les fleurs de Cynara cardunculus L., a été étu-diée par électrophorèse en gel de polyacrylamide à l'urée. Dans le caséinate de sodium, les caséines as et~ont été respectivement dégradées jusqu'à 46 et 76 %, après 10 h d'hydrolyse; sous forme sépa-rée, les caséines as ont atteint des niveaux de dégradation de 67 %; pourtant, les caséines~ont été dégradées plus rapidement et extensivementjusqu'à 76 %. En général, les caséines~doivent être plus sensibles à la protéolyse que les caséines as' © InralElsevier, Paris.protéine laitière / enzyme / présure végétale / électrophorèse

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The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

Cited by 137 publications

References 6 publications

Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheeses from several milk species

Advances in the role of a plant coagulant (Cynara cardunculus) in vitro and during ripening of cheeses from several milk species

Identification and proteolytic processing of procardosin A

Proteolysis of ovine caseins by cardosin A, an aspartic acid proteinase from Cynara cardunculus L.

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