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

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose-6-P phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E. faecalis lacks a malA gene encoding a 6-phospho-a-glucosidase, which in B. subtilis hydrolyses maltose 6-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B. subtilis mutant accumulating maltose 6-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6-P, and the resulting maltose is converted by the B. subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phosphosubstrates revealed that MapP preferably dephosphorylates disaccharides containing an O-aglycosyl linkageFil: Mokhtari, Abdelhamid. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; Francia. University Mentouri. Faculty of Natural Science and Life. Department of Biochemistry-Microbiology. Laboratory of Environmental Biology; ArgeliaFil: Blancato, Victor Sebastian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Repizo, Guillermo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Henry, Céline. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; FranciaFil: Pikis, Andreas. Center for Drug Evaluation and Research. Food and Drug Administration; Estados UnidosFil: Bourand, Alexa. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; FranciaFil: Alvarez, Maria de Fatima. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Tucumán. Instituto Superior de Investigaciones Biológicas; ArgentinaFil: Immel, Stefan. Technische Universität Darmstad. Institut für Organische Chemie; AlemaniaFil: Mechakra Maza, Aicha. University Mentouri. Faculty of Natural Science and Life. Department of Biochemistry-Microbiology. Laboratory of Environmental Biology; ArgeliaFil: Hartke, Axel. Universite de Caen Basse Normandie; FranciaFil: Thompson, John. National Institutes of Health. Laboratory of Cell and Developmental Biology. Microbial Biochemistry and Genetics Section; Estados UnidosFil: Magni, Christian. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Deutscher, Josef. Institut National de la Recherche Agronomique. Microbiologie de l’Alimentation au Service de la Santé Humaine; Franci

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

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

Statistical optimization of culture medium for neutral protease production by aspergillus oryzae. Comparative study between solid and submerged fermentations on tomato pomace

International audienceA comparative study of the production of neutral protease was carried out by the cultivation of Aspergillus oryzae NRRL 2220 in solid-state fermentation (SSF) and submerged fermentation (SmF) using tomato pomace based medium. For this purpose, medium optimization was achieved using two experimental designs. The first, corresponded to the Plackett and Burman design with N = 8 experiments and k = N − 1 factors; five real (wheat bran, casein, NH4NO3, NaCl and ZnSO4) and two errors. Statistical analysis of the results allowed the selection of two factors having a significant effect on enzyme production (casein and NaCl in SSF, wheat bran and NaCl in SmF). Optima of the selected factors have been determined through a second experimental design, the central composite design of Box and Wilson with two factors; 19.79 g/L casein and 0.92 g/L NaCl for SSF and 17.92 g/L wheat bran and 1.18 g/L NaCl for SmF were found. The optimal production time of the neutral protease determined from the kinetic study was 96 h leading to 21309 U/g in SSF and 2343.5 U/g in SmF. The comparison of the proteolytic activities of the optimized media demonstrated a ratio of 9 between SSF and SmF, showing the efficiency of the solid-state fermentation compared to the submerged fermentation. The results confirmed the high biotechnological potential of this fungal strain for neutral protease production in solid-state fermentation. Additionally, the utilization of tomato pomace constitutes an efficient and inexpensive agro industrial substrate for protease production via the SSF approach and a suitable mean for its valorization and to reduce its ecological impact

Enterococcus faecalis utilizes maltose by connecting two incompatible metabolic routes via a novel maltose 6-phosphate phosphatase (MapP)

Similar to Bacillus subtilis, Enterococcus faecalis transports and phosphorylates maltose via a phosphoenolpyruvate (PEP):maltose phosphotransferase system (PTS). The maltose-specific PTS permease is encoded by the malT gene. However, E.faecalis lacks a malA gene encoding a 6-phospho--glucosidase, which in B.subtilis hydrolyses maltose 6-P into glucose and glucose 6-P. Instead, an operon encoding a maltose phosphorylase (MalP), a phosphoglucomutase and a mutarotase starts upstream from malT. MalP was suggested to split maltose 6-P into glucose 1-P and glucose 6-P. However, purified MalP phosphorolyses maltose but not maltose 6-P. We discovered that the gene downstream from malT encodes a novel enzyme (MapP) that dephosphorylates maltose 6-P formed by the PTS. The resulting intracellular maltose is cleaved by MalP into glucose and glucose 1-P. Slow uptake of maltose probably via a maltodextrin ABC transporter allows poor growth for the mapP but not the malP mutant. Synthesis of MapP in a B.subtilis mutant accumulating maltose 6-P restored growth on maltose. MapP catalyses the dephosphorylation of intracellular maltose 6-P, and the resulting maltose is converted by the B.subtilis maltose phosphorylase into glucose and glucose 1-P. MapP therefore connects PTS-mediated maltose uptake to maltose phosphorylase-catalysed metabolism. Dephosphorylation assays with a wide variety of phospho-substrates revealed that MapP preferably dephosphorylates disaccharides containing an O--glycosyl linkage