Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase

摘要

BackgroundThe extracellular expression of enzymes in a secretion host such asBacillus subtilisis a useful strategy in reducing the cost of downstream processing of industrial enzymes. Here, we present the first report of the successful extracellular expression inBacillus subtilisWB800 ofGeobacillus stearothermophiluslipase (T1.2RQ), a novel industriallydesirable thermostable lipolytic enzyme which has an excellent hydrolytic and transesterification activity. Signal peptides of α-amylase, extracellular protease, and lipase A, as well as two different promoters, were used in the secretion and expression of lipase T1.2RQ.ResultsLipase 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 parentBacillus subtilisWB800 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.ConclusionsThe successful extracellular secretion of lipase T1.2RQ inBacillus subtilisrepresents a remarkable feat in the industrial-scale production of this enzyme.How to cite:Ridwan E, Suwanto A, Thenawidjaja M. Extracellular expression inBacillus subtilisof a thermostableGeobacillus stearothermophiluslipase. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.07.003.