Use of a sequential fermentation method for the production of aspergillus tamarii urm4634 protease and a kinetic/thermodynamic study of the enzyme

Microbial proteases are commonly produced by submerged (SmF) or solid-state fermentation (SSF), whose combination results in an unconventional method, called sequential fermentation (SF), which has already been used only to produce cellulolytic enzymes. In this context, the aim of the present study was the development of a novel SF method for protease production using wheat bran as a substrate. Moreover, the kinetic and thermodynamic parameters of azocasein hydrolysis were estimated, thus providing a greater understanding of the catalytic reaction. In SF, an approximately 9-fold increase in protease activity was observed compared to the conventional SmF method. Optimization of glucose concentration and medium volume by statistical means allowed us to achieve a maximum protease activity of 180.17 U mL−1. The obtained enzyme had an optimum pH and temperature of 7.0 and 50 °C, respectively. Kinetic and thermodynamic parameters highlighted that such a neutral protease is satisfactorily thermostable at 50 °C, a temperature commonly used in many applications in the food industry. The results obtained suggested not only that SF could be a promising alternative to produce proteases, but also that it could be adapted to produce several other enzymes