Etude de la production de la protéase par aspergillus oryzae sur milieu solide en fermenteur FUJIWARA

Abstract

Agro-industrial residues are rich in organic matter. This makes such wastes very appropriate for transformation by biotechnological processes. Among these, the solid-state fermentation (SSF) have renewed interest worldwide because they provide significant production of various substances compared to submerged cultures. Thus, the objective of this thesis is the use of tomato waste in SSF for production of fungal protease. This substrate is composed of more than 52 % of insoluble fiber, more than 20 % of proteins, 6.52 % ash, and other organic materials. This composition is used in this type of fermentation as support and nutrients for the microorganism and covers the requirements for inducing production of proteases. Among five strains of Aspergillus grown on tomato pomace SSF in Erlenmeyer flasks, two strains were selected for the development of culture conditions, A. oryzae 5777 and A. oryzae 2220. The study of various parameters on neutral protease production gave a significant effect of moisture, initial pH, age of inoculum and protein content. Two factors (spores rate and particle size) were found no significant effect on enzyme synthesis. The fermentations performed on a pilot scale by A. oryzae strain 2220, at 50 % initial moisture of substrate and the enrichment of soy flour in an amount of 15 mg / g of substrate, more than 12 U / gds is achieved after 42 hours. Culture with tomato pomace without adding additional protein source allows higher production with 12 % more activity, of 13.55 U / gds. Among the solutions studied for protease extraction, the 3% NaCl (w / v) gives the maximum activity. The results also showed that the wet fermented substrate provides more neutral protease relative to the dry substrate. The effect of the contact time of the fermented substrate with water gave a maximum activity after stirring for 4 hours with the wet substrate and after 7 hours for the dry one. On the other hand, the pH between 7.6 and 8 and a temperature of 45 ° C allow to obtain maximum catalyst activity. The enzyme retains this activity until a temperature of 55 ° C. The produced protease has a residual activity of 25 % at 65 ° C after 45 minutes and 34 % at 60 ° C after 60 minutes; it is inhibited at 70% by EDTA and activated by divalent cations of zinc and copper. This indicates that it is a metallo-protease. The enzyme was purified 4-fold by SA precipitation in the 40-80% fraction. The efficiency of this separation is 30%. These results show the potential of tomato pomace as a culture substrate for protease production by SSF. This would make it very economical synthesis and could meet the demand of growing market for this group of hydrolases with very diverse technological interests