Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein
The action of Cynara cardunculus L. protease on whole bovine kappa-casein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3% trichloroacetic acid (TCA)-soluble fraction of the kappa-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N...
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| Veröffentlicht in: | Journal of agricultural and food chemistry 1993-10, Vol.41 (10), p.1537-1540 |
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| container_title | Journal of agricultural and food chemistry |
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| creator | Macedo, I. Queiroz Faro, Carlos J Pires, Euclides M |
| description | The action of Cynara cardunculus L. protease on whole bovine kappa-casein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3% trichloroacetic acid (TCA)-soluble fraction of the kappa-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis as the 106-169 fragment [caseinomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the Phe105-Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled kappa-casein (FTC-kappa-casein). The values obtained for kcat, km, and kcat/km were 1.04s-1, 0.16 micromolars, and 6.5 micromolars-1s-1,respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the km is significantly lower, which reflects the higher affinity of Cynara protease to kappa-casein |
| doi_str_mv | 10.1021/jf00034a001 |
| format | Article |
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Queiroz ; Faro, Carlos J ; Pires, Euclides M</creator><creatorcontrib>Macedo, I. Queiroz ; Faro, Carlos J ; Pires, Euclides M</creatorcontrib><description>The action of Cynara cardunculus L. protease on whole bovine kappa-casein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3% trichloroacetic acid (TCA)-soluble fraction of the kappa-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis as the 106-169 fragment [caseinomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the Phe105-Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled kappa-casein (FTC-kappa-casein). The values obtained for kcat, km, and kcat/km were 1.04s-1, 0.16 micromolars, and 6.5 micromolars-1s-1,respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the km is significantly lower, which reflects the higher affinity of Cynara protease to kappa-casein</description><identifier>ISSN: 0021-8561</identifier><identifier>EISSN: 1520-5118</identifier><identifier>DOI: 10.1021/jf00034a001</identifier><identifier>CODEN: JAFCAU</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>ACTIVIDAD ENZIMATICA ; ACTIVITE ENZYMATIQUE ; Biological and medical sciences ; CASEINA ; CASEINE ; CYNARA ; Food industries ; Fundamental and applied biological sciences. Psychology ; GINECEO ; GYNECEE ; Milk and cheese industries. Ice creams ; PROTEASAS ; PROTEASE ; PROTEOLISIS ; PROTEOLYSE</subject><ispartof>Journal of agricultural and food chemistry, 1993-10, Vol.41 (10), p.1537-1540</ispartof><rights>1994 INIST-CNRS</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-a388t-f61d51bc2e11cca3f8b48d457d779c66a31468ac05d7c3a72aa59f7825794fb53</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://pubs.acs.org/doi/pdf/10.1021/jf00034a001$$EPDF$$P50$$Gacs$$H</linktopdf><linktohtml>$$Uhttps://pubs.acs.org/doi/10.1021/jf00034a001$$EHTML$$P50$$Gacs$$H</linktohtml><link.rule.ids>314,780,784,2765,27076,27924,27925,56738,56788</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=3931181$$DView record in Pascal Francis$$Hfree_for_read</backlink></links><search><creatorcontrib>Macedo, I. Queiroz</creatorcontrib><creatorcontrib>Faro, Carlos J</creatorcontrib><creatorcontrib>Pires, Euclides M</creatorcontrib><title>Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein</title><title>Journal of agricultural and food chemistry</title><addtitle>J. Agric. Food Chem</addtitle><description>The action of Cynara cardunculus L. protease on whole bovine kappa-casein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3% trichloroacetic acid (TCA)-soluble fraction of the kappa-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis as the 106-169 fragment [caseinomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the Phe105-Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled kappa-casein (FTC-kappa-casein). The values obtained for kcat, km, and kcat/km were 1.04s-1, 0.16 micromolars, and 6.5 micromolars-1s-1,respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the km is significantly lower, which reflects the higher affinity of Cynara protease to kappa-casein</description><subject>ACTIVIDAD ENZIMATICA</subject><subject>ACTIVITE ENZYMATIQUE</subject><subject>Biological and medical sciences</subject><subject>CASEINA</subject><subject>CASEINE</subject><subject>CYNARA</subject><subject>Food industries</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>GINECEO</subject><subject>GYNECEE</subject><subject>Milk and cheese industries. Ice creams</subject><subject>PROTEASAS</subject><subject>PROTEASE</subject><subject>PROTEOLISIS</subject><subject>PROTEOLYSE</subject><issn>0021-8561</issn><issn>1520-5118</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1993</creationdate><recordtype>article</recordtype><recordid>eNptkE1rGzEURUVJoW6aVXdZaRFISxhHGo1GmmUx-QLTBJxAduJZIyWyx9IgjZO6v75yp4QushJP57zL4yL0lZIpJSU9X1lCCKuAEPoBTSgvScEplQdoQjIuJK_pJ_Q5pVXWJBdkgl4WvdHOOu2GHQbf4rXzZnA64WDx8GzwxnXrQndhGJx_wsb_3m0MtjFssIbYhuDxt9nOQ4S_89brbbdNeD79jofwmn_wMrzkSDxdQ9_DtNCQjPNf0EcLXTJH_95D9HB5cT-7Lua3VzezH_MCmJRDYWvacrrUpaFUa2BWLivZVly0QjS6roHRqpagCW-FZiBKAN5YIUsumsouOTtEZ2OujiGlaKzqo9tA3ClK1L4y9V9l2T4Z7R6Shs5G8NqltxXWsFzmXitGzaXB_HrDENeqFkxwdX-3UHc_ry4fyXWp9v7x6FsICp5ijnxYNBVrmpJkeDpC0Emtwjb63Me71_0B8xmPkw</recordid><startdate>19931001</startdate><enddate>19931001</enddate><creator>Macedo, I. 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Queiroz ; Faro, Carlos J ; Pires, Euclides M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a388t-f61d51bc2e11cca3f8b48d457d779c66a31468ac05d7c3a72aa59f7825794fb53</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1993</creationdate><topic>ACTIVIDAD ENZIMATICA</topic><topic>ACTIVITE ENZYMATIQUE</topic><topic>Biological and medical sciences</topic><topic>CASEINA</topic><topic>CASEINE</topic><topic>CYNARA</topic><topic>Food industries</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>GINECEO</topic><topic>GYNECEE</topic><topic>Milk and cheese industries. Ice creams</topic><topic>PROTEASAS</topic><topic>PROTEASE</topic><topic>PROTEOLISIS</topic><topic>PROTEOLYSE</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Macedo, I. Queiroz</creatorcontrib><creatorcontrib>Faro, Carlos J</creatorcontrib><creatorcontrib>Pires, Euclides M</creatorcontrib><collection>AGRIS</collection><collection>Istex</collection><collection>Pascal-Francis</collection><collection>CrossRef</collection><jtitle>Journal of agricultural and food chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Macedo, I. Queiroz</au><au>Faro, Carlos J</au><au>Pires, Euclides M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein</atitle><jtitle>Journal of agricultural and food chemistry</jtitle><addtitle>J. Agric. Food Chem</addtitle><date>1993-10-01</date><risdate>1993</risdate><volume>41</volume><issue>10</issue><spage>1537</spage><epage>1540</epage><pages>1537-1540</pages><issn>0021-8561</issn><eissn>1520-5118</eissn><coden>JAFCAU</coden><abstract>The action of Cynara cardunculus L. protease on whole bovine kappa-casein, over a 3-h period at pH 6.4, was investigated. Rp-HPLC of the 3% trichloroacetic acid (TCA)-soluble fraction of the kappa-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis as the 106-169 fragment [caseinomacropeptide (CMP)]. Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the Phe105-Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled kappa-casein (FTC-kappa-casein). The values obtained for kcat, km, and kcat/km were 1.04s-1, 0.16 micromolars, and 6.5 micromolars-1s-1,respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the km is significantly lower, which reflects the higher affinity of Cynara protease to kappa-casein</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><doi>10.1021/jf00034a001</doi><tpages>4</tpages><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 0021-8561 |
| ispartof | Journal of agricultural and food chemistry, 1993-10, Vol.41 (10), p.1537-1540 |
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| source | American Chemical Society Journals |
| subjects | ACTIVIDAD ENZIMATICA ACTIVITE ENZYMATIQUE Biological and medical sciences CASEINA CASEINE CYNARA Food industries Fundamental and applied biological sciences. Psychology GINECEO GYNECEE Milk and cheese industries. Ice creams PROTEASAS PROTEASE PROTEOLISIS PROTEOLYSE |
| title | Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein |
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