Production de protéases thermostables par des moisissures thermophiles isolées à partir de sources thermales terrestres de l’Est algérien.

Abstract

The exploitation of the different samples collected from terrestrial thermal springs distributed between the wilayas of Guelma, Khenchela and Mila made it possible to obtain 53 thermophilic fungal isolates. A screening on agar medium based on skimmed milk showed that 23 isolates have a proteolytic activity, of which four (1, 6, 34 and 37) among them are considered as potential producers of proteases, were retained. The isolate 37 was selected for the rest of the work due to the thermostability of its proteases. Molecular characterization by PCR, using the ITS1/ITS4 primer, has shown that isolate 37 has a 99% similarity with Mycothermus thermophilus. The sequence of the amplification product has been deposited in the Gen-Bank database (NCBI) under accession number MK770356. The production of proteases in solid culture was much higher than in both liquid and submerged cultures. Various agro-food wastes were initially explored for the production of thermostable proteases at a lower cost, including wheat bran which proved to be the best substrate for better enzyme production. In addition, a statistical approach consisting of two designs was used to determine the optimal culture conditions allowing the greatest production of these enzymes. The use of the Plackett-Burman plan made it possible to screen the variables; temperature, spore concentration and humidity which have the most significant effect on protease production. Box-Behnken analysis showed a 6.17-fold improvement in protease production (1187.03 U/mL). This improvement was observed under the optimum conditions of 47% moisture content, 5 × 105 spores/g inoculum concentration and temperature of 42°C. The zymography technique demonstrated that the selected strain produced four proteases. These proteases are very active over a wide pH range of 8.0 to 12.0 with an optimum of activity at pH 9.0, at an optimum temperature of 60°C and they retained more than 60% of their activity at the same temperature after heat treatment for 90 min. Likewise, these thermostable alkaline proteases showed great stability towards nonionic surfactants and oxidizing agents, after a pre-incubation of 1 h at 40°C, and extreme stability towards surfactants anionic. The extracted enzymes showed excellent stability and compatibility with some commercial laundry detergents. The results of the washing performance analysis revealed considerable removal of stains from human blood and egg yolk by the enzyme extract. The eco-friendly raw protease hair removal treatment of cow and goat skin has shown very high levels of hair removal. The protein hydrolysates as well as the chitosan resulting from the valorization of shrimp waste by the proteases of the studied strain, showed an important biological activity. Finally, the purification report shows that the protease was purified with a yield of 1.73%, a purification factor of 1.027 and a specific activity of 3124.53 U/mg. Characterization of the partially purified enzyme reveals a pH of 9 and an optimum temperature of 60°C.