Difference between revisions of "Part:BBa K3051005"
 
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<partinfo>BBa_K3051005 short</partinfo>
 
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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 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.  
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<h2>Comparative Enzyme Activity Assay</h2>
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<div align="center"><img height="85%" width="85%" src="https://2019.igem.org/wiki/images/8/83/T--Warwick--2019-EnzymeLipaseGraph.png"></img></div>
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<p><b>Figure 1. Relative Comparaison Lipase Activity Assay.</b> Candida Antarctica Lipase A, Bacillus Subtilis Lipase and Compost Lipase enzyme activity were examined using p-Nitrophenol octanoate as a substrate.</p>
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Figure 1 compares enzyme activity of Candida Antarctica Lipase A <bbpart>BBa_K3051001</bbpart>, Bacillus Subtilis Lipase <bbpart>BBa_K3051005</bbpart> and Compost Lipase <bbpart>BBa_K3051421</bbpart>. The lipase parts were tested and characterized using a [https://2019.igem.org/Team:Warwick/Results p nitrophenol octanoate assay]. The enzyme activity was determined by measuring change in light absorbance at a wavelength of 400nm, which directly correlated to the concentration of p-Nitrophenol (pNP), caused by lipolysis of p-Nitrophenol octanoate (pNPO) substrate. The data from the pNPO lipolysis assay defined the enzyme activity.
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<h2>Background</h2>
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Bacillus Subtilis Lipase A (BSAL) is a lipase from Bacillus Subtilis XF-1; identified during a metagenomic analysis of restaurant wastewater containing high levels of FOGS (fat oil and grease)[1]. The organism appears to express a suitability to thrive in high lipid environments, although there is not yet data to support these assumptions. The lipase contains no secretion tag, and does not express noticeable toxicity when added to E.Coli BL21. The enzyme is not secreted, but cells containing the lipase demonstrated a positive lipase activity in a p-Nitrophenol test. The p-Nitrophenol octanoate (pNPO) substrate undergoes lipolysis via BSAL enzyme action to become p-Nitrophenol (pNP), causing increased absorption of light at a wavelength of 400nm. In order for the lipase activity to be observed the cells had to be lysed using a sonicator.
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Supernatants of cell cultures were not sufficient in providing lipase activity.  
  
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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).  
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The Bacillus Subtilis lipase has a kDa of 24.1, based on the amino acid sequence and has an expected molar absorption coefficent of 24410 M-1 cm-1 (values found through the ProtoPam ExPaSy tool).  
  
 
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<h2>Protein Kinetics</h2>
  
 
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The Km and Vmax of the lipase was tested and characterized using a [https://2019.igem.org/Team:Warwick/Results p nitrophenol octanoate assay].
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The Km and Vmax of the lipase were tested and characterized using a [https://2019.igem.org/Team:Warwick/Results p nitrophenol octanoate assay].
  
 
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<p><b>Figure 2. Simulated 3D structure.</b>Structure of BSAL simulated using Phyre 2 modelling.</p>
 
<p><b>Figure 2. Simulated 3D structure.</b>Structure of BSAL simulated using Phyre 2 modelling.</p>
 
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<div>BSAL contains an EstA triacylglycerol lipase conserved region, which makes up a large proportion of its total length. It also shares similarities with iGEM part (<bbpart>BBa_K258006</bbpart>) (Thermostable Lipase A)</div>
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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>
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Latest revision as of 03:52, 22 October 2019


Bacillus Subtilis Lipase

Comparative Enzyme Activity Assay

Figure 1. Relative Comparaison Lipase Activity Assay. Candida Antarctica Lipase A, Bacillus Subtilis Lipase and Compost Lipase enzyme activity were examined using p-Nitrophenol octanoate as a substrate.

Figure 1 compares enzyme activity of Candida Antarctica Lipase A BBa_K3051001, Bacillus Subtilis Lipase BBa_K3051005 and Compost Lipase BBa_K3051421. The lipase parts were tested and characterized using a p nitrophenol octanoate assay. The enzyme activity was determined by measuring change in light absorbance at a wavelength of 400nm, which directly correlated to the concentration of p-Nitrophenol (pNP), caused by lipolysis of p-Nitrophenol octanoate (pNPO) substrate. The data from the pNPO lipolysis assay defined the enzyme activity.


Background

Bacillus Subtilis Lipase A (BSAL) is a lipase from Bacillus Subtilis XF-1; identified during a metagenomic analysis of restaurant wastewater containing high levels of FOGS (fat oil and grease)[1]. The organism appears to express a suitability to thrive in high lipid environments, although there is not yet data to support these assumptions. The lipase contains no secretion tag, and does not express noticeable toxicity when added to E.Coli BL21. The enzyme is not secreted, but cells containing the lipase demonstrated a positive lipase activity in a p-Nitrophenol test. The p-Nitrophenol octanoate (pNPO) substrate undergoes lipolysis via BSAL enzyme action to become p-Nitrophenol (pNP), causing increased absorption of light at a wavelength of 400nm. In order for the lipase activity to be observed the cells had to be lysed using a sonicator. Supernatants of cell cultures were not sufficient in providing lipase activity.

The Bacillus Subtilis lipase has a kDa of 24.1, based on the amino acid sequence and has an expected molar absorption coefficent of 24410 M-1 cm-1 (values found through the ProtoPam ExPaSy tool).


Protein Kinetics

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.

The Km and Vmax of the lipase were tested and characterized using a p nitrophenol octanoate assay.

Structure and domains of BSAL

Figure 2. Simulated 3D structure.Structure of BSAL simulated using Phyre 2 modelling.

BSAL contains an 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]