Part:BBa_K4665005
Usage and Biology
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.
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
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).
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.
Characterisation
In Vitro Mineralization 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
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
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
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
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
Sequence and Features
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