Résumé:
Six strains of fungi isolated from Algerian soil, five of the genus Aspergillus and one of the genus Penicillium are identified and are used to produce clotting proteases: A.Niger, A.flavus,A.awamori, A.tubingensis,A.tamarii and P.pinophilum. Enzymes are synthesized by fermentation of whey-based media enriched and optimized in two phases by using two experimental designs.The first plan Plackett-Burman(11 factors and 12 experiments) was used to select factors with a positive and significant effect on the production of proteases.7 factors are real [pH,agitation,yeast extract,lactose,peptone ,CaCl2 and salts(MgSO4 and FeSO4)] and four errors.The second plan, Box and Wilson gave the optimum factors selected whose number varies form one strain to another.The optimization showed that each strain is able to produce an extracellular acid protease and coagulating milk with important activities(between 1822.21 U.and 2583.83 U.). The enzymes are then purified in three steps: molecular filtration on Sephadex G25, ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on pepstatin A-agarose.The latter has eliminated up to 92% of non-active protein with purification factors between 2.84 and 6.19. The separation of the purified enzymes by SDS-PAGE showed molecular weight of the acid protease from each strain:30 kDa for P.pinophilum,35kDa for A.flavus and A.tamarii and 55kDa for A.Niger, A.awamori and A.tubingensis.The study of the properties has allwed the determination of an optimum pH of between 4 and 5.5 and a temperature optimum between 30 and 50°C.All enzymes are stable 1 h at the temperature of optimum activity. Coagulation tests are performed on three different milks at 35°C after each purification step.The coagulation time,the appearance and texture of curdled are different depending on the origin of the protease. All enzymes cause a very rapid coagulation of fresh cow's milk in a very short time: 2 min. For the A.tomarii enzyme and 5 min for all other enzymes.
