Physical and chemical properties of the acid protease from Onopordum acanthium: Comparison between electrophoresis and HPLC of degradation casein profiles

A protease was extracted by grinding, precipitation and gel filtration from Onopordum acanthium flowers. The physicochemical study of the enzyme showed an optimum pH of 4, a temperature of 40°C and kinetic parameters of 12.25 mM-1 for KM and 1329.6 UmL-1 for Vmax. The inhibition by pepstatin indicated that it is an aspartyl-protease (APs). Zymogram showed that the protease has a monomeric structure and a molecular mass (MM) of 45 kDa. The hydrolysis of α, β and Κ- and whole casein by the protease was evaluated using electrophoresis and HPLC; the profiles showed many similarities between the vegetal protease action and that of industrial chymosin. So, the properties of the protease studied and the quality of its action showed its effectiveness and relevance of its use as a milk clotting enzyme which leads to a better use of extract of flowers O. acanthium as a locally substitute for rennet.Keywords: Aspartic protease, Onopordum acanthium, purification, characterization, casein hydrolysi

Milk-clotting properties and specific hydrolysis of caseins of the acid protease extracted from Scolymus maculatus flowers

A milk-clotting acid protease was extracted from the flowers of an Asteraceae; a widespread plant traditionally used in Algeria, Scolymus maculatus. The enzyme was purified 40 fold using ammonium sulfate fractionation followed by exclusion chromatography. The estimation of the molecular mass of the protease by SDS-PAGE electrophoresis gave a weight of 45 kDa and Zymogram analysis revealed only one proteolytic band. The enzyme inhibition at 99% by pepstatin-A 5 mM proved that it is an aspartic proteinase; EDTA and iodoacetamide had no effect. The milk coagulation activity of this protease was optimal at pH 5 and 60°C, the rennet strength (RS) of the extract increased with calcium concentrations and was saturated at 50 mM. The enzyme exhibited hydrolytic activity toward κ-casein, α s -and β-casein. These results indicated that Scolymus maculatus protease has technological effects similar to that of the chymosin; the enzyme could be used in cheese making as a substitute for rennet

Optimisation à l’aide d’un plan d’expériences de la production d’une protéase fongique sur milieu à base de déchets agro-industriels

The production of neutral protease was carried out by the cultivation of Aspergillus oryzae Ahlburg (Cohen) 1042.72 in submerged fermentation using orange waste resulting from juice processing as a basal medium enriched with agro-industrial wastes. Optimization of the enzyme synthesis was achieved using the statistical method of Plackett-Burman design with N = 8 experimentsand N-1 factors; five real (corn-steep liquor, pH, salt, decommissioned dates, whey) and two errors. The results were modeled using a linear regression of the type: β0 + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + e The statistical results (correlations and significance level) made it possible to select the factors allowing the maximum production of protease (2016 U) obtained on a medium containing orange waste enriched in corn-steep liquor in 72 h and at pH 6. A large production is therefore obtained on a cheap medium.La production d’une protéase neutre a été réalisée par fermentation d’une moisissure mésophyle (Aspergillus oryzae Ahlburg (Cohen)1042.72) en erlenmeyers sur milieux à base de déchets d’oranges enrichis à l’aide de sous-produits agro-industriels. L’optimisation de la synthèse de l’enzyme a été effectuée en utilisant une méthode statistique de planification expérimentale (les matrices de Plackett et Burman à N = 8, soit à 8 expériences et N-1 = 7 variables). Les variables utilisées sont les 5 facteurs de production (lactosérum, déchets de dattes ou dattes déclassées, corn-steep liquor, sels minéraux et pH) et 2 erreurs. Les résultats ont été modélisés selon une régression linéaire multiple de type : β0 + β1X1 + β2X2 + β3X3 + β4X4 + β5X5 + e Les résultats statistiques (corrélation et seuil de signification) ont permis de sélectionner les facteurs permettant la meilleure production de l’enzyme. Celle-ci (2016 U) est obtenue sur un milieu à base de déchets d’oranges enrichis en corn-steep liquor après 72 h de fermentation à pH 6. Ainsi, le milieu optimal permet une production importante de protéase neutre sur un milieu à moindre coût