Difference between revisions of "Part:BBa K3051005"
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The Km and Vmax of the lipase was tested and characterized using a p nitrophenol octanoate assay. Details of this assay are available on the iGem 2019 wiki page. The assay produced the following lineweaver burk plot: | The Km and Vmax of the lipase was tested and characterized using a p nitrophenol octanoate assay. Details of this assay are available on the iGem 2019 wiki page. The assay produced the following lineweaver burk plot: | ||
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| + | <div align="center"><img height="50%" width="50%" src="https://2019.igem.org/wiki/images/f/ff/T--Warwick--BSAL_lineweaver_burk_plot.png"></img></div> | ||
| + | <p><b>Figure 1. BSAL Kinetics Characterisation via Lineweaver-Burk Plot.</b>Bacillus Subtilis lipase has a BSAL has a Km of 0.349mM and a Vmax of 0.00486 mM/min at pH 7 using p-nitrophenol octanoate as a substrate.</p> | ||
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<b>Structure and domains of BSAL</b> | <b>Structure and domains of BSAL</b> | ||
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| − | < | + | <div align="center"><img height="50%" width="50%" src="https://2019.igem.org/wiki/images/archive/9/95/20191020223527%21T--Warwick--BSAL_3D_structure.png"></img></div> |
| + | <p><b>Figure 2. Simulated 3D structure.</b>Structure of BSAL simulated using Phyre 2 modelling.</p> | ||
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<div>BSAL contains a EstA triacylglycerol lipase conserved region, which makes up a large proportion of its total length. It also shares similarities with iGem part BBa_K258006 (Thermostable Lipase A)</div> | <div>BSAL contains a EstA triacylglycerol lipase conserved region, which makes up a large proportion of its total length. It also shares similarities with iGem part BBa_K258006 (Thermostable Lipase A)</div> | ||
Revision as of 23:21, 21 October 2019
Bacillus Subtilis Lipase
Bacillus Subtilis Lipase A (BSAL) is a lipase from Bacillus Subtilis XF-1. It was found during a metagenomic analysis of restaurant wastewater containing high levels of FOG (fat oil and grease)[1]; we therefore believe that it works very well in high lipid environments, however, that is left to be tested. Although it contains no secretion tag, it did not appear toxic when added to BL21 E.Coli, therefore it is not toxic to the cell. The lipase is not naturally excreted, E.Coli BL21 cells containing the lipase were positive for lipid activity using a p nitrophenol test (more details can be found in the Warwick iGem 2019 wiki) only when the cells were lysed using a sonicator, supernatants of cell cultures likewise did not show any lipase activity.
The Bacillus Subtilis lipase has a kDa of 24.1, based on its amino acid composition it has an expected molar absorption coefficent of 24410 M-1 cm-1 (values found using the ProtoPam ExPaSy tool).
The Km and Vmax of the lipase was tested and characterized using a p nitrophenol octanoate assay. Details of this assay are available on the iGem 2019 wiki page. The assay produced the following lineweaver burk plot:
Figure 1. BSAL Kinetics Characterisation via Lineweaver-Burk Plot.Bacillus Subtilis lipase has a BSAL has a Km of 0.349mM and a Vmax of 0.00486 mM/min at pH 7 using p-nitrophenol octanoate as a substrate.
Structure and domains of BSAL
Figure 2. Simulated 3D structure.Structure of BSAL simulated using Phyre 2 modelling.
Sources
1. Sutar, V.P. and Kurhekar, J.V., 2017. ISOLATION AND CHARACTERIZATION OF LIPASE PRODUCING BACTERIA FROM RESTAURANT WASTE WATER.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]