Protease production by thermophilic Bacillus sp strain SMIA-2 cultivated in liquid cultures containing 1% maltose as a carbon source and supplemented with whey protein (0.1%) and corn steep liquor (0.3%) reached a maximum at 14 h, with levels of 42 U/mg protein. The microorganism was capable of utilizing a wide range of carbon sources, but protease activity varied according the carbon source. Starch and maltose were the best carbon sources in the present study for protease secretion, while lactose and sucrose were less effective. Increasing maltose concentration in the medium until 1%, improved the growth of the organism and the enzyme activity. Regarding the amounts of corn steep liquor and whey protein in the medium, the concentrations of 0.2% and 0.1% respectively, were considered the most effective for protease secretion by the organism. Studies on the protease characterization revealed that the optimum temperature of this enzyme was 70ºC. Thermostability profile indicated that the enzyme retained 80% of the original activity after 2 h heat treatment at 60ºC. At 70ºC, 70% of the original activity was retained after 15 min heat treatment. The optimum pH of the enzyme was found to be 8.5. After incubation of crude enzyme solution at room temperature for 2 h at pH 6.0-10.0, a decreased of about 15% of its original activity at pH 8.5 was observed. At pH 10.0, the decrease was 24%. In the presence of 1.0 M and 5.0 M NaCl, 76% and 37% of protease activity was retained after 2 h incubating at 45ºC respectively.

ResumoBacillus sp. SMIA-2 cresceu e secretou proteases quando cultivado em culturas líquidas contendo citrato trissódico como fonte de carbono e nitrato de amônio como fonte de nitrogênio. A atividade máxima da enzima foi alcançada após 8 horas de incubação do microrganismo, com níveis de 7,2 U.mg -1 proteína. A suplementação do meio com 2,0 mM de CaCl 2 não proporcionou um aumento da atividade da protease, porém aumentou a sua estabilidade de 2 para 4 horas. A redução da concentração do fosfato de potássio no meio de cultura de 11,0 para 5,0 mM promoveu um acréscimo de 82% (13 U.mg -1 proteína) na atividade da protease. A substituição do nitrato de amônio presente no meio de cultura por 0,1% de soro de queijo aumentou a atividade da protease para 25 U.mg -1 proteína. Nestas condições, o tempo requerido para que a enzima atingisse seu pico de atividade máxima, que antes era de 9 horas, passou para 16 horas. A substituição do citrato trissódico do meio de cultura pela água de maceração de milho (0,5%) não só aumentou a atividade da enzima como também retardou o processo de desativação que é típico da produção de proteases. A atividade máxima da enzima obtida quando estes dois resíduos foram utilizados no meio foi 59,5 U.mg -1 proteína. Após alcançar este valor a enzima se manteve estável por mais de 20 horas o que favorece a sua produção em larga escala. Palavras-chave: proteases; Bacillus sp.; água de maceração de milho; soro de queijo. AbstractBacillus sp. SMIA-2 grew and produced proteases when cultivated in liquid cultures containing trisodium citrate as a carbon source and ammonium nitrate as a nitrogen source. The maximum activity of the enzyme was reached after incubating the microorganism for 9 hours, with levels of 7.2 U.mg -1 protein. The supplementation of the medium with 2.0 mM of CaCl 2 did not improve the activity of the protease, but increased its stability from 2 to 4 hours. The reduction in the amount of inorganic phosphate in the medium from 11.0 to 5.0 mM resulted in an improvement of 82% (13 U.mg -1 protein) in the protease activity. Replacing ammonium nitrate in the medium by 0.1% milk whey increased the protease activity to 25 U.mg -1 protein. In these conditions, the time required for the enzyme to reach a maximum activity increased from 9 to 16 hours. Replacing citrate trissodium in the medium by corn steep liquor (0.5%) not only produced much better enzyme activity, but also delayed the deactivation process which is typical for the production of proteases. The maximum activity reached when these wastes were used in the medium was 59.5 U.mg -1 protein. In addition, the enzyme maintained stable for more than 20 hours, which is favorable for its production on a large scale.

RESUMO: As bactérias ácido láticas são de grande importância para a indústria alimentícia, pois promovem a acidificação do meio auxiliando na obtenção das características sensoriais de produtos fermentados e na