Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

BACKGROUND The extracellular expression of enzymes in a secretion host such as Bacillus subtilis is a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression in Bacillus subtilis WB800 of Geobacillus stearothermophilus lipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of a-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ. RESULTS Lipase activity assay using p-nitrophenyl laurate showed that all three signal peptides directed the secretion of lipase T1.2RQ into the extracellular medium. The signal peptide of lipase A, resulted in the highest extracellular yield of 5.6 U/ml, which corresponds to a 6-fold increase over the parent Bacillus subtilis WB800 strain. SDS-PAGE and zymogram analysis confirmed that lipase T1.2RQ was correctly processed and secreted in its original size of 44 kDa. A comparison of the expression levels of lipase T1.2RQ in rich medium and minimal media showed that the enzyme was better expressed in rich media, with up to an 8-fold higher yield over minimal media. An attempt to further increase the lipase expression level by promoter optimization showed that, contrary to expectation, the optimized promoter exhibited similar expression levels as the original one, suggesting the need for the optimization of downstream factors. CONCLUSIONS The successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme

Influence of the oxygen and growth rate on the metabolic activity of bacillus stearothermophilus grown in glucose-limited cultures

O aumento da taxa de diluiçäo de uma cultura quimiostática de Bacillus stearothermophilus var non-diastaticus, contendo glicose como fonte de carbono para 0,89 h-1 a 50 p/cento de saturaçäo com ar, resultou em aumentos lineares nas taxas de consumo de glicose e oxigênio. Estes dados foram analisados de acordo com a equaçäo de Pirt e foi observado que quando o nível de oxigênio dissolvido diminuiu em funçäo da diminuiçäo da saturaçäo com ar de 50 p/cento para 5 p/cento, o coeficiente da manutençäo (m) para o consumo de oxigênio aumentou e o valor Ymax (produçäo máxima) decresceu. O coeficiente de manutençäo para o consumo de glicose e o valor da produçäo máxima a 50 por cento de saturaçäo com ar foram também calculados, mas quando o nível de oxigênio dissolvido foi abaixado para 5 p/cento de saturaçäo com ar, estes valores näo puderam ser calculados, uma vez que o aumento da taxa de diluiçäo da cultura näo resultou em aumentos lineares no consumo de glicose