Obtenção e caracterização de proteases proveniente de cepas de bactérias isoladas do intestino do inseto adulto Rhynchophorus Palmarum

Abstract

The food industry is a cornerstone of the Brazilian economy, with dairy products standing out as one of the most significant sectors. Within this context, enzymes play a crucial role in dairy production, particularly in the coagulation of milk for cheese manufacturing. The search for alternatives to animal-derived enzymes has propelled research into microbial coagulants, highlighting the importance of studying these microorganisms for the production of biotechnologically relevant enzymes in the food industry. In this regard, the present study aimed to use strains of Enterococcus faecalis and Klebsiella variicola extracted from the gut of the adult insect Rhynchophorus palmarum for the production and characterization of proteases capable of coagulating milk. Enzymatic extracts from bacterial strains were prepared in 50 mM Tris-HCl buffer, pH 8.0, and assays for pH, temperature, inhibition, effects of different substrates, electrophoresis, zymography, and coagulation of skimmed milk powder were conducted. The strains of K. variicola and E. faecalis displayed differences in their biochemical parameters, with distinct activities and characteristics. While K. variicola showed activity in AZOCASEIN and had an optimal pH of 8 and an optimal temperature of 50ºC, the E. faecalis strain exhibited activity in AZOCASEIN and SAAPFpNA, with an optimal pH between 7, 8, and 9, and an optimal temperature of 50ºC. Regarding inhibitors, K. variicola is inhibited by EDTA and DTT, whereas E. faecalis is inhibited by PMSF. Both enzymes have an optimal temperature of 50ºC for coagulant activity, but differ in the minimum protein concentration (0.5 µg for K. variicola and 10 µg for E. faecalis) and minimum calcium concentration (2 mM for K. variicola and 8 mM for E. faecalis) for coagulant activity. Thus, the study concludes that it is possible to obtain proteases from bacterial strains of the microbiota of the adult insect R. palmarum capable of coagulating milk, underscoring the viability of these alternative sources of coagulants in the dairy industry.