Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase |
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| Institution: | 1. Biotechnology Study Program, Research Center for Bioresources and Biotechnology (RCBIO), PAU, IPB University, Bogor 16680, Indonesia;2. Biotechnology Research and Development Division, Seed Wilmar Indonesia Ltd, Cikarang, Bekasi 17530, Indonesia;3. Department of Biology, Faculty of Mathematics and Natural Sciences, Jalan Agatis, Dramaga Campus, IPB University, Bogor 16680, Indonesia;4. Department of Food Science, Faculty of Agricultural Technology, IPB University, Bogor 16680, Indonesia |
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| Abstract: | BackgroundThe 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 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 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.ConclusionsThe successful extracellular secretion of lipase T1.2RQ in Bacillus subtilis represents a remarkable feat in the industrial-scale production of this enzyme.How to cite: Ridwan E, Suwanto A, Thenawidjaja M. Extracellular expression in Bacillus subtilis of a thermostable Geobacillus stearothermophilus lipase. Electron J Biotechnol 2021;53. https://doi.org/10.1016/j.ejbt.2021.07.003 |
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| Keywords: | Expression Extracellular Heterologous Industrial enzymes Lipase Signal peptides Thermostable Zymogram analysis |
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