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

Geobacillus stearothermophilus LV cadA gene mediates resistance to cadmium, lead and zinc in zntA mutants of Salmonella entérica serovar Typhimurium

Salmonella entérica serovar Typhimurium cells expressing the cadA gene of Geobacillus stearothermophilus LV exhibit a hypersensitive phenotype to cadmium chloride. Deletion of the ORF STM3576 from the Salmonella genome resulted in cadmium, lead and zinc sensitivity, confirming that this ORF is a homologue of the zntA gene. The observed sensitivity was reverted upon expression of the G. stearothermophilus LV cadA gene. These results indicate that the cadA gene product is involved in Cd, Pb and Zn resistance as a classical P-type ATPase and strongly suggest that the observed hypersensitive phenotype to these metals can be related to the function of the host ·zntA gene product.