Difference between revisions of "Part:BBa K4665120"
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===Usage and Biology===
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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 CO2 from the atmosphere as a substrate for the reaction catalyzed by the carbonic anhydrase.
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<partinfo>BBa_K4665120 short</partinfo>
  
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SazCA  is a thermostable α-carbonic anhydrase derived from the thermophilic bacterium Sulfurihydrogenibium azorense. SazCA has been characterised as the fastest known carbonic anhydrase to date, possessing a kcat/KM value of 3.5 × 108 M−1 s−1
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===Biology and Usage===
  
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SazCA facilitates the reversible hydration of carbon dioxide to bicarbonate and protons (CO2 + H2O -><- HCO3 + H+) (De Simone et al, 2015). 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).
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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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CO2 and HCO3- 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 CO2, 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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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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===Characterisation===
 
  
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---In Vitro Mineralization---
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To test the ability of engineered BL21(DE3) E. coli strain to precipitate CaCO3, we performed an in vitro mineralisation assay, adapting Zhu, et al. 's technique. Bacteria were cultured overnight in 30mL of LB +Kanamycin medium and 0.5 mM ZnSO4 at 25℃. IPTG induction was performed 3 hours prior to experimentation. The assay was run on 8 mL Tris-HCl buffer 8.3 and 50mL of saturated CO2 aqueous solution at 0℃. 3 mL of cell pellet were introduced into the solution, and the reaction was allowed to proceed on ice for an hour. At this point, the bacteria should have been able to produce bicarbonate ions. Cells were removed from the solution by centrifugation (15 min x 5000g). 25mL of a 0.3M solution of CaCl2 was added to the remaining supernatant as a calcium source. The reaction was left to run at 25℃ for 12h. Samples were filtered using vacuum filtration and dried at 50℃ to evaporate the solvent. Solid mass was weighed and recorded as “Wet Weight”. Upon preliminary analysis of FT-IR data, it was concluded that the mineral sample contained a large amount of water, elucidated by the stretching O-H peak at 3400 cm.1. Hence, the sample was dried further in liquid nitrogen for 48 hours, final weight was recorded at 2.3 g (yield=306.17%). Precipitated dry crystals were analysed using ATR-IR and 3C NMR
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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">
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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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===References===
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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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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
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<span class='h3bb'><b>Sequence and Features</b></span>
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<partinfo>BBa_K4665120 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K4665120 parameters</partinfo>
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===References===
  
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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
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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
  
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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
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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
  
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Dhami, N.K., et al. ( May 2013). Biomineralization of calcium carbonate polymorphs by the bacterial strains isolated from calcareous sites. Journal of Microbiology and Biotechnology, 23(5): 707-714. https://doi.org/10.4014/jmb.1212.11087
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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
 
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