Caracterização de protease produzida pelo isolado bacteriano Bacillus sp. CL33A

Abstract

The growing concern with environmental issues has fostered the use of microorganisms in biotechnological processes for degrading recalcitrant protein-rich organic materials, such as chicken feathers and feather meal, as an alternative for its management and valorization. Among the value-added products are proteolytic enzymes, which have various commercial and industrial applications. For this purpose, such biocatalysts should be characterized regarding the that may interfere with enzyme activity. In this context, this study aimed to evaluate protease production by the keratinolytic bacterium Bacillus sp. CL33A during submerged cultivation using feathers and FP as organic substrates, and to characterize the crude protease obtained in cultivations showing higher enzyme yields. Results showed that higher yields occurred using FP as growth substrate. For crude protease characterization, a DCCR approach was utilized to determine the best temperature and pH conditions for enzyme activity, which were, respectively 55 °C and pH 8. At these optimal conditions, the rate of enzyme activity was analyzed using different concentrations of azocasein substrate. Through the Michaelis-Menten equation, the apparent values for V max (13,39 U mg protein -1 min -1 ) and K m (0,515 mg mL -1 ) were obtained. Subsequently, the influence of reagents towards the enzyme activity was evaluated. The presence of 5 mmol L -1 of the salts CoCl 2 , CuSO 4 , FeSO 4 , ZnCl 2 and MnCl 2 , and the protease inhibitors PMSF, EDTA and 1,10- phenanthroline decreased the protease activity. The MgCl 2 salt (1 and 5 mmol L -1 ) increased enzyme activity and, similarly, the reagents ethanol, Triton X-100, Tween 20 and DMSO tended to stimulate the enzyme activity. SDS and β-mercaptoethanol resulted in negative effects on enzyme catalysis. In the evaluation of crude protease thermal stability, at 50 to 60 °C, higher inactivation occurred with increasing temperatures. The performance of the crude protease on protein substrates indicated higher hydrolysis of the substrates PIS, casein and FP.