Difference between revisions of "Part:BBa K3051001"
 
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<partinfo>BBa_K3051001 short</partinfo>
 
<partinfo>BBa_K3051001 short</partinfo>
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<h2>Comparative Enzyme Activity Assay</h2>
  
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Candida Antartica Lipase A (CALA) is a lipase extracted from the yeast Candida Antartica. It has been shown to work at very low temperatures. CALA has been shown to be very receptive to C8 p nitrophenol esters, compared to C4 and >C12 esters.
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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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It has also been shown to be very sensitive to phenylmethylsulfonyl fluoride and high alcohol contents, causing a decrease in activity of as low as 50% compared to its control.[1]
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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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It has a kDa of 49.3 and has been shown to react with p nitrophenol octanoate, to give p nitrophenol. Its molar extinction coefficient in water has been calculated to be 54570 M-1cm-1 (using ProtoPram ExPaSy tool). Its 3D structure has been calculated to be as follows: 
 
  
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https://2019.igem.org/wiki/images/archive/1/14/20191020221459%21T--Warwick--CALA_3D_structure.png
 
  
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<h2>Protein 3D Structure Prediction</h2>
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Candida Antartica Lipase A (CALA) is a lipase extracted from the yeast Candida Antartica - the enzyme has expressed function at extremely low temperatures. CALA shows high affinity to C8 p-Nitrophenol esters, compared to C4 and >C12 esters (where C denotes the number of carbons in the fatty acid tail bound to the p-Nitrophenol group.)
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The protein has shown sensitivity toward phenylmethylsulfonyl fluoride and high alcohol contents, causing a drop in activity to as low as 50% compared to the control.[1]
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The enzyme has a kDa of 49.3 and has been shown to hydrolyse p-Nitrophenol octanoate into  p-Nitrophenol. The molar extinction coefficient in water has been calculated to be 54570 M-1cm-1 (using ProtoPram ExPaSy tool). A 3D structure of the enzyme has been calculated.</div>
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<div align="center"><img height="50%" width="50%" src="https://2019.igem.org/wiki/images/archive/1/14/20191020221459%21T--Warwick--CALA_3D_structure.png
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"></img></div>
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<p><b>Figure 2. Simulated 3D structure.</b>Structure of Candida Antartica lipase simulated using Phyre 2 modelling.</p>
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<b>Sources</b>
 
<b>Sources</b>

Latest revision as of 03:52, 22 October 2019


Candida Antarctica Lipase A

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.


Protein 3D Structure Prediction

Candida Antartica Lipase A (CALA) is a lipase extracted from the yeast Candida Antartica - the enzyme has expressed function at extremely low temperatures. CALA shows high affinity to C8 p-Nitrophenol esters, compared to C4 and >C12 esters (where C denotes the number of carbons in the fatty acid tail bound to the p-Nitrophenol group.)

The protein has shown sensitivity toward phenylmethylsulfonyl fluoride and high alcohol contents, causing a drop in activity to as low as 50% compared to the control.[1]

The enzyme has a kDa of 49.3 and has been shown to hydrolyse p-Nitrophenol octanoate into p-Nitrophenol. The molar extinction coefficient in water has been calculated to be 54570 M-1cm-1 (using ProtoPram ExPaSy tool). A 3D structure of the enzyme has been calculated.</div>


Figure 2. Simulated 3D structure.Structure of Candida Antartica lipase simulated using Phyre 2 modelling.

Sources 1. Lan, Dong-Ming et al. “A novel cold-active lipase from Candida albicans: cloning, expression and characterization of the recombinant enzyme.” International journal of molecular sciences vol. 12,6 (2011): 3950-65. doi:10.3390/ijms12063950


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]