Purification and characterization of a milk-clotting protease from Mucor pusillus : method comparison

Crude enzymatic extract obtained from five fermentations (300 g of wheat bran) was characterized by a clotting activity of 0.34 ± 0.08 UP/ml with a strength ratio of 1/1: 200. The comparative study of the summaries from 2 purification protocols showed that it is possible to recover 6% of the initial proteins with a 44.54% activity after gel filtration (protocol I), which appeared more technically sound when compared to ion-exchange (1.80% of total proteins with a 23% performance) (protocol II). The protein homogeneity (a single electrophoretic band) of the monomeric protease was confirmed by both methods after precipitation with 80% saturated ammonium sulphate. Moreover, the fractional precipitation technique with this salt (40 and 80%) was useless in the experimental conditions employed and an important loss of activity was observed (28.53%) with a 3-fold purification. In another part of the study, without ammonium sulphate precipitation, the gel filtration enabled the elimination of almost 97% of the inactive proteins and improved the activity performance by 55.13%, while multiplying the specific activity of the coagulant by a factor of 20.88 against a 6.75-fold purification with ionexchange and the appearance of a more or less 20 kDa peptide after electrophoresis. The proteolytic activity of the purified extracts had a similar appearance to a more pronounced kinetic when compared with the reference rennet. The purification protocols did not seem to have an impact on the isolated protease activit

Evolution des pectines et des activités polygalacturonases au cours de la maturation de la datte Deglet-nour

National audienceDuring storage, the date undergoes several changes that affect its organoleptic characters. The characterisation of the parameters which induce these evolutions was carried out for understanding the biochemical mechanisms in order to select the conditions assuring the stability of the date during its storage preserving its nutritional value and its commercial value. The extraction and the fractionation of pectic substances were achieved as a function of the ripening of the date which contained about 2% of total pectin. The observed degradation of these pectin was ascribed to the presence of PG, a pectolytic enzyme responsible for the softening of fruit; indeed the PG activity increased during the stages of development with a maximal activity at the last stage (0.53 Ul/g Fresh Matter). The date polygalacturonase will be an exopolygalacturonase which presents an optimal activity at the temperature 40 degrees C and pH 5.Au cours du stockage, la datte est sujette à plusieurs évolutions qui affectent ses caractéristiques organoleptiques. La caractérisation des paramètres inducteurs de ces évolutions doit permettre de maîtriser les mécanismes biochimiques et enzymatiques afin de pouvoir offrir les conditions adéquates pour assurer la stabilité de la datte au cours du stockage préservant sa valeur nutritive et sa valeur marchande. Ce travail a permis l'extraction et le fractionnement des substances pectiques, au cours de la maturation, de la datte qui contient environ 2 % de pectines. La dégradation enzymatique de ces pectines par les polygalacturonases, responsables avec les pectineméthylestérases du ramollissement, est illustrée par l'activité polygalacturonase qui augmente au cours de la maturation pour atteindre une activité maximale au dernier stade de maturation (0,53 UI/g Matière Fraîche). La polygalacturonase de la datte serait une exo-polygalacturonase qui présente une activité optimale à une température de 40 °C et à un pH égal à 5