Difference between revisions of "Part:BBa K3051005"

Revision as of 21:04, 16 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 characterized. 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 - however the lipase is not excreted - as the cell extracts of E.Coli BL21 containing the lipase were positive for lipid activity (using a p nitrophenol test - outlined in the wiki of iGem 2019) 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:

From this it was extrapolated that BSAL has a Km of 0.349mM and a Vmax of 0.00375 mM/min at pH 7 using p nitrophenol octanoate as a substrate.

Structure and domains of BSAL

3D structure of BSAL simulated using Pyhre modelling

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)

Sources

1. Sutar, V.P. and Kurhekar, J.V., 2017. ISOLATION AND CHARACTERIZATION OF LIPASE PRODUCING BACTERIA FROM RESTAURANT WASTE WATER.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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-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 characterized. Although it contains no secretion tag, it did not appear toxic when added to BL21 E.Coli, therefore it is not capable of destroying lipid based cell machinery. The lipase is not excreted as the cell extracts of E.Coli BL21 containing the lipase were positive for lipid activity (using a p nitrophenol test - outlined in the wiki of iGem 2019) only when the cells were lysed using a sonicator.
+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 characterized. 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 - however the lipase is not excreted - as the cell extracts of E.Coli BL21 containing the lipase were positive for lipid activity (using a p nitrophenol test - outlined in the wiki of iGem 2019) 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).