Difference between revisions of "Part:BBa K4665120"

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===Biology and Usage===
 
===Biology and Usage===
  
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Biomineralisation is the process by which living organisms synthesise minerals (Dhami et al., 2013). One of the main metabolic pathways of microbial calcium carbonate production utilizes the zinc metallo-enzyme carbonic anhydrase as the catalyst of the reaction (Chaparro-Acuña et al., 2019). This pathway produces no pollutant byproduct (as is often the case with other biomineralisation pathways) and extracts CO<sub>2</sub> from the atmosphere as a substrate for the reaction catalyzed by the carbonic anhydrase.
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Biomineralisation is the process by which living organisms synthesise minerals (Dhami et al., 2013). One of the main metabolic pathways of microbial calcium carbonate production utilizes the zinc metallo-enzyme carbonic anhydrase as the catalyst of the reaction (Chaparro-Acuña et al., 2019). This pathway produces no pollutant byproduct, which is often the case with other biomineralisation pathways, and extracts CO<sub>2</sub> from the atmosphere as a substrate for the reaction catalyzed by the carbonic anhydrase.
  
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SazCA is a thermostable α-carbonic anhydrase derived from the thermophilic bacterium <i>Sulfurihydrogenibium azorense</i> and has been characterised as the fastest known carbonic anhydrase to date, possessing a kcat/KM value of 3.5 × 108 M<sup>−1</sup> s<sup>−1</sup>. SazCA facilitates the reversible hydration of carbon dioxide to bicarbonate and protons (see fig. 1) (De Simone et al, 2015). This process creates alkaline conditions which increase the solubility of Ca<sup>2+</sup> ions trapped on the extracellular matrix (EPS) of bacterial cells, which readily bond to HCO<sup>3-</sup>, facilitating the formation of calcium carbonate crystals (Anbu, et al. 2016).
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SazCA is a thermostable α-carbonic anhydrase derived from the thermophilic bacterium <i>Sulfurihydrogenibium azorense</i> and has been characterised as the fastest known carbonic anhydrase to date, possessing a kcat/KM value of approximately 3.5 × 108 M<sup>−1</sup> s<sup>−1</sup>. SazCA facilitates the reversible hydration of carbon dioxide to bicarbonate and protons (see fig. 1) (De Simone et al., 2015; De Luca et al., 2013). This process creates alkaline conditions which increase the solubility of Ca<sup>2+</sup> ions trapped on the extracellular matrix (EPS) of bacterial cells, which readily bond to HCO<sup>3-</sup>, facilitating the formation of calcium carbonate crystals (Anbu, et al. 2016).
  
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CO<sub>2</sub> and HCO<sup>3-</sup> are small molecules capable of diffusing in and out of cells, yet the intracellular activity of CA is significantly limited by the permeability of the cell membrane, as it restricts both the amount of substrate available for catalysis and the subsequent secretion of the product, reducing overall enzymatic efficiency (Jo, 2013). Therefore, the proposed approach is to express the protein on the surface of the cell, directly exposing it to extracellular concentrations of CO<sub>2</sub>, and bypassing cellular secretion of bicarbonate ions, which ultimately enhances the production of calcium carbonate on the surface of limestone cracks.
 
  
 
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       <img height="250px" width="350px" src="https://static.igem.wiki/teams/4665/wiki/parts-registration-images/bba-k4665005-sazca-reaction.jpg" alt="Image 1">
 
       <img height="250px" width="350px" src="https://static.igem.wiki/teams/4665/wiki/parts-registration-images/bba-k4665005-sazca-reaction.jpg" alt="Image 1">
 
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<b>Figure 1.</b> The reversible hydration reaction of CO<sub>2</sub> into HCO<sub>3</sub><sup>-</sup>, followed by CaCO<sub>3</sub> formation
 
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The SazCA sequence was obtained from research by Zhu et al.(2022). This sequence has undergone codon optimization for <i>E. coli</i> and was employed in the BL21 DE3 chassis. A 6X-His tag is attached to SazCA, enabling detection using anti-His antibodies and purification through nickel immobilized metal affinity chromatography (IMAC).
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<span class='h3bb'><b>Sequence and Features</b></span>
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===Functional Parameters===
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===References===
 
===References===
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De Simone, G., et al. (May 1, 2015). Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense. Bioorganic & Medicinal Chemistry Letters, 1;25(9): 2002-2006. https://doi.org/10.1016/j.bmcl.2015.02.068
 
De Simone, G., et al. (May 1, 2015). Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense. Bioorganic & Medicinal Chemistry Letters, 1;25(9): 2002-2006. https://doi.org/10.1016/j.bmcl.2015.02.068
  
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Jo, B.H. (October 3, 2013). Engineered Escherichia coli with Periplasmic Carbonic Anhydrase as a Biocatalyst for CO2 Sequestration. Applied and Environmental Microbiology. https://doi.org/10.1128/AEM.02400-13
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Zhu, Y., et al. (December 6, 2021). Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralisation. Synthetic and Systems biotechnology, 7(1): 460-473. https://doi.org/10.1016%2Fj.synbio.2021.11.008

Latest revision as of 11:49, 11 October 2023

SazCA Carbonic Anhydrase

Biology and Usage

Biomineralisation is the process by which living organisms synthesise minerals (Dhami et al., 2013). One of the main metabolic pathways of microbial calcium carbonate production utilizes the zinc metallo-enzyme carbonic anhydrase as the catalyst of the reaction (Chaparro-Acuña et al., 2019). This pathway produces no pollutant byproduct, which is often the case with other biomineralisation pathways, and extracts CO2 from the atmosphere as a substrate for the reaction catalyzed by the carbonic anhydrase.

SazCA is a thermostable α-carbonic anhydrase derived from the thermophilic bacterium Sulfurihydrogenibium azorense and has been characterised as the fastest known carbonic anhydrase to date, possessing a kcat/KM value of approximately 3.5 × 108 M−1 s−1. SazCA facilitates the reversible hydration of carbon dioxide to bicarbonate and protons (see fig. 1) (De Simone et al., 2015; De Luca et al., 2013). This process creates alkaline conditions which increase the solubility of Ca2+ ions trapped on the extracellular matrix (EPS) of bacterial cells, which readily bond to HCO3-, facilitating the formation of calcium carbonate crystals (Anbu, et al. 2016).


Image 1
Figure 1. The reversible hydration reaction of CO2 into HCO3-, followed by CaCO3 formation


The SazCA sequence was obtained from research by Zhu et al.(2022). This sequence has undergone codon optimization for E. coli and was employed in the BL21 DE3 chassis. A 6X-His tag is attached to SazCA, enabling detection using anti-His antibodies and purification through nickel immobilized metal affinity chromatography (IMAC).

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]


References

Anbu, P. et al. (March 1, 2016). Formations of calcium carbonate minerals by bacteria and its multiple applications. Springerplus 5(250). https://doi.org/10.1186/s40064-016-1869-2

Chaparro-Acuña, S.P., et al. (June, 2018). Soil bacteria that precipitate calcium carbonate: mechanism and applications of the process. Acta Agronómica 67(2). https://doi.org/10.15446/acag.v67n2.66109

De Luca, V. et al. (March 15, 2013). An α-carbonic anhydrase from the thermophilic bacterium Sulphurihydrogenibium azorense is the fastest enzyme known for the CO2 hydration reaction. Bioorganic & Medicinal Chemistry Letters, 21(6): 1465.1469. https://doi.org/10.1016/j.bmc.2012.09.047

De Simone, G., et al. (May 1, 2015). Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense. Bioorganic & Medicinal Chemistry Letters, 1;25(9): 2002-2006. https://doi.org/10.1016/j.bmcl.2015.02.068

Zhu, Y., et al. (December 6, 2021). Surface display of carbonic anhydrase on Escherichia coli for CO2 capture and mineralisation. Synthetic and Systems biotechnology, 7(1): 460-473. https://doi.org/10.1016%2Fj.synbio.2021.11.008