Banca de DEFESA: CAMILA BIANCA FERREIRA DA ROCHA

CHARACTERIZATION OF PROTEASE PRODUCED BY TRIBOLIUM CASTANEUM LARVAE AND INDUSTRIAL 

BIOTECHNOLOGICAL APPLICATION IN MILK COAGULATION

Protease. Milk coagulation. Red flour beetle. Enzymes

A pest, in a broad sense, can be defined as an insect (or other organism) that causes direct damage to humans, their livestock, their crops or even their property. The species Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) stands out as one of the main pests, which, in particular, represents 9% of economic losses of stored grains in developed countries, so that, in developing countries, these losses can represent 20% worldwide. Due to its wide distribution around the globe and its characteristics, the intestine of this species can be used as a source of enzymes for studies on pest control and biotechnological applications. This study aimed to characterize and biotechnologically apply a protease from Tribolium castaneum larvae in the coagulation of milk, aiming at the production of cheese and other derivatives. For this purpose, preparations containing reconstituted powdered milk were subjected to the coagulation process in the presence of the crude enzyme extract. Furthermore, the crude extract was characterized in relation to parameters such as optimum temperature and thermal stability, optimum pH and behavior in the presence of some metal ions, as well as in the presence of specific protease inhibitors. Spectrophotometry was used as an analytical method for quantifying total proteins in order to demonstrate the hydrolysis of casein in the presence of the crude extract, as well as qualitative presentations of the coagulated mass were used to demonstrate the coagulant capacity. It was observed that the protease enzyme under study presented relative activity and stability in a wide pH range, namely, from 6 to 12, with the optimum pH being 8.0. The optimum temperature was identified at 40°C and residual activity of 55% at 70°C, thus demonstrating good thermal stability. In the presence of some metal ions, the highest inhibition rates were observed, in relation to the concentration of 10 mM, it was observed that zinc chloride represented 100% inhibition, and copper sulfate represented, in turn, 80% inhibition, respectively. In the presence of the specific 2-mercaptoethanol, there was a complete absence of residual proteolytic activity, as well as detection of only 20% of residual activity in the presence of DTT, thus indicating a probable protease of the cysteine protease class. Regarding the potential for coagulant application, it was observed that the crude enzymatic extract had the capacity to hydrolyze milk casein, under the best conditions of 50°C and 1 hour, as well as it was possible to verify that the minimum concentration of crude extract capable of hydrolyzing casein was 50 µg and a positive relationship with the increase in calcium ions and proteolytic activity, thus demonstrating intense biotechnological applicability when compared to other milk coagulant proteases, thus implying an important ally and potential adjuvant enzyme to be incorporated in the cheese production industry and other milk derivatives.