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Composite-Part

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You can find our composite part via the registry with the part number: BBa_K4665005

Usage and Biology

Biomineralization is the process by which living organisms synthesise minerals (Dhami et al., 2013).^[1] Microbial calcium carbonate production can proceed through two main metabolic pathways, using urease or carbonic anhydrase (CA) as the catalysts of the reaction (Chaparro-Acuña et al., 2019).^[2] However, synthesis through urea hydrolysis produces toxic byproducts which is not observed in the CA catalyzed pathway.

SazCA, derived from the thermophilic bacterium Sulfurihydrogenibium azorense, is the fastest known carbonic anhydrase to date, with an approximate $k_{cat}/{KM}$ value of 3.5 × 108 $M^{−1} s^{−1}$ (De Simone et al, 2015; De Luca et al., 2013).^[3][4] SazCA facilitates the hydration of carbon dioxide to bicarbonate and protons, creating alkaline conditions that aid the formation of calcium carbonate crystals on the extracellular matrix (EPS) of bacterial cells (Fig. 1) (Anbu, et al., 2016).^[5]

$$CO_2 + H_2O \leftrightarrow HCO_3\ ^- + H^+ \newline \newline$$

$$OH^- + HCO_3\ ^- \rightarrow CO_3\ ^{2-} + H_2O \newline$$

$$Ca^{2+} + CO_3\ ^{2-} \rightarrow CaCO_3$$

Figure 1

The reversible $CO_2$ hydration reaction catalysed by SazCA. In presence of $Ca^{2+}$, $CaCO_3$ is formed.

To enhance enzymatic efficiency, this composite part expresses the SazCA enzyme as a fusion protein on the cell surface of E. coli. This approach bypasses cellular limitations and directly exposes the enzyme to extracellular CO2, increasing calcium carbonate production on limestone surfaces. This component is based on the findings of Zhu et al. (2022)^[6], wherein a membrane fusion protein was designed to showcase SazCA on the surface of E. coli cells. This is achieved by linking the E. coli codon-optimized SazCA enzyme (BBa_K4665120) to the integral membrane protein INPN (BBa_K4665001) using a flexible GGGGS linker (BBa_K4665175).

The SazCA-INPN Membrane Display (SIMD) module

1) Ice nucleation protein N-terminal (INPN): This is the N-terminal of ice nucleation protein which will be embedded into the E. coli cell membrane. The sequence coding for the INPN is preceded by a pelB leader sequence as its expression promotes the secretion of the protein via the Sec pathway whilst avoiding hydrolysis by cytoplasmic proteases that might lower the quantity of proteins on the cell’s surface (Mergulhao et al., 2005).^[7] By attaching the pelB signal peptide in front of the INP protein, the fusion protein will be directed towards the bacterial periplasm where it will be anchored in the cell membrane (Singh et al., 2013).^[8] The INPN sequence is followed by two front-end sub-repeat sequences important for the stability of the fusion protein (Zhu et al., 2022).^[8:1]

2) GGGGS linker: The GGGGS flexible linker is composed of a sequence of 4 glycine repeats followed by a serine amino acid. This flexible linker is used to connect the N-terminal of the INP to the carbonic anhydrase which creates an elongated fusion mode that allows for optimal carbonic anhydrase stability (Hartmann et al., 2022; Zhu et al., 2022).^[9][6:1]

3) SazCA: This sequence codes for the carbonic anhydrase derived from Sulfurihydrogenibium azorense (SazCA). This sequence has been codon optimised for E. coli. The SazCA coding sequence is followed by a His-tag which facilitates the purification and detection of the fusion protein.

Figure 2

Visual representation of the SIMD fusion protein, taken from Zhu et al. (2022).^[6:2]

Info

Zhu et al. (2022)^[6:3] were able to show that the surface display of the INP-SazCA fusion protein significantly elevates the enzyme’s stability, optimising whole-cell activity at 25°C and pH 9, retaining minimal metal inhibition.

Characterisation

Enzymatic Activity of SazCA

To measure the activity of the SazCA construct, a colorimetric Wilbur Anderson assay was adapted from Kim & Jo (2022).^[17]  The assay measures the ability of carbonic anhydrase to hydrate $CO_2$. Protons released during the hydration reaction cause a decrease in the pH of the solution. Such displacement of $H^+$ can be recorded as a function of time taken for pH to shift from ~8.3 to ~6.3

$$CO_2(a) + H_2O \rightarrow HCO_3\ ^- + H^+$$

Standard activity assays directly measure the pH change of the reaction mixture with electrodes. However, it was soon discovered that this setup would be difficult for us to achieve and the bubbling of $CO_2$ gas would act as a limitation for controlling the concentration of $CO_2$ administered for the reaction.The colorimetric approach taken for the assay indirectly measured the change of pH by recording the color change of phenol red upon the addition of SazCA. A reaction buffer of 20mM Tris pH. 8.4 ($\text{pKa}$=8.1) and 100µM phenol red ($\text{pKa}$=7.9) was used. Phenol red was chosen as the pH indicator as it shifts colors from pink to yellow over a pH range of ~8.4 to ~6.4.

Colorimetric Assay

To derive control values, several ratios of buffer to $CO_2(aq)$ were tested in order to assign the effectiveness of the reaction buffer upon the addition of saturated $CO_2$ solution. This was measured by color change. The objective was to identify the buffer-to-solution ratio that would result in an absorbance value of 1.034 (pH ~7.3), within the chosen experimental duration, which was set as the baseline condition for all experimental samples. For each reaction, 800 µL of reaction buffer and 200 µL of saturated $CO_2$ solution were used. Ranging volumes of SazCA-BL21 liquid culture were used: 10µL, 20µL, 30µL, 40µL and 50µL. All reactions were run in triplo. Saturated $CO_2$ solution was prepared through addition of dry ice into double-distilled water under constant stirring until complete saturation was achieved (no more dissolution of dry ice perceived).

Data collection was performed by UV-Vis spectrophotometry, measuring absorbance change at 560 nm using the kinetics function of the spectrophotometer, recording every 0.1 min for 10 minutes. All reactions were performed ice-cold. Absorbance values for pH-adjusted reaction buffer were obtained as colorimetric reference at 8.4 (abs=2.079), 7.4 (1.034), and 6.4(abs=0.268).

SazCA-BL21 volume (µL)	pH at 8.57 minutes	ΔpH value (Ref. pH 8.4)
10	6.89671641791045	1.50328358208955
20	6.7820895522388	1.6179104477612
30	6.01958208955224	2.38041791044776
40	6.44823880597015	1.95176119402985
50	6.59653731343283	1.80346268656717

Table 1

Time at absorbance 0.268 for different whole cell catalyst volumes

Following the measurement of absorbance values for each SazCA-BL21 volume and the control, the averaged absorbance values were plotted to visualise the trends. The time at which each line reached an absorbance of 0.268 was derived. Qualitatively, it was observed that as the volume of SazCA-BL21 increased, the time required for the absorbance to reach 0.268 decreased. Notably, samples containing bacteria exhibit a rapid decrease in absorbance prior to reaching a plateau, which could potentially be elucidated by the point at which these samples achieve uniform coloration throughout the entire cuvette. Control values failed to reach the target absorbance of 0.268 even after a prolonged 30-minute reaction period, rendering the calculation for Wilbur Anderson units for the quantification of the enzyme’s activity unviable.

However, pH variations attributed to the activity of SazCA were calculated. Using reference colorimetric values for pH, the control solution was estimated to plateau at pH 7.268 (±0.1). pH values for bacterial samples were estimated by obtaining the average absorbance at 8.57 minutes. This time stamp was chosen as it corresponds to the longest time taken for a sample to reach absorbance 0.268 (10 µL). Absorbance values were converted to pH through the following formula

$$\frac{pH_{initial} abs_{final}}{ {abs_{initial}}} = pH_{final}$$

The average pH value was determined at 6.393 for SazCA-BL21 samples, indicating a 0.875 difference in pH (S.D.= 0.0159) attributed to the production of bicarbonate ions produced by SazCA. Individual ΔpH values for each bacterial volume can be found in Table 2.

SazCA-BL21 volume (µL)	Time at abs 0.268 OD560 (min)
10	8.57
20	8.15
30	7.49
40	7.1
50	6.751

Table 2:

pH differences for different SazCA-BL21 volumes with respect to initial pH 8.4

Our results are comparable to Kim and Jo’s (2020).^[17:1] Firstly, both graphs exhibit similar patterns in absolute absorbance (abs) values, emphasising a significant increase in abs corresponding to the closure of the spectrophotometer's door. This initial surge is succeeded by a sustained decrease in abs, reaching a plateau at absorbance values corresponding to a pH lower than 6.4. This trend underscores the reproducibility of the reaction dynamics across both experimental settings.

Figure 11

Wilbur Anderson Units per whole cell catalyst volume.

However, our experimental control reaction failed to reach the pH level of 6.4 (ab s= 0.268). The absence of a time point for the pH endpoint hindered the direct tabulation of Wilbur Anderson units (WAU). Wilbur Anderson Units can be calculated by the following formula, where t0 is the time taken for the blank reaction to reach pH 6.4 and t correspond to experimental bacterial values. WAU=t0-t/t. To account for this, the time taken for the 10uL experimental sample to reach pH 6.4 was taken as t0, and all WA values were calculated from it (Fig. 11). The direct proportionality (R2 = 0.993) between the amount of enzyme and the measured activity is shown.

Overall, results demonstrate that the SazCA fusion protein demonstrates enzymatic activity. The former indicates that the engineered BL21 bacteria possess the ability to catalyse the formation of bicarbonate ions from CO2 and water at a much faster rate than the natural reaction that occurs at ambient conditions.

Further optimization of the assay is necessary to fully characterise the enzymatic activity of SazCA-INPN. Relative to WA values obtained for SazCA by Zhu et al.(2022)^[6:5], our construct presents 27.7% enzymatic activity. The small working volume of the assay and the consequently small volume of bacterial cells used might have caused prolonged pH reduction times, rendering WA values to be extremely low. Furthermore, selecting appropriate buffer-to-CO2 ratios is instrumental for adequately estimating the enzyme's activity. The ratio should be adjusted to be able to reach pH 6.4, and the reaction should be performed under a thermally controlled photospectrometer.

ThermoGravimetric Analysis on Alginate beads

To evaluate the functionality of the fusion protein while entrapped in hydrobeads, TGA was used to screen for the presence of calcium carbonate. Alginate beads were dried at 80°C prior to TGA analysis to remove moisture. The temperature was increased from 26°C to 900°C at a speed of 10°C/min, under nitrogen flow (Wang et al., 2015).^[18] Calcium carbonate has a characteristic sharp weight loss percentage at between 650-800°C (Oniyama & Wahlbeck, 1995)^[19], while the degradation of alginate hydrogels can be observed between 200-400°C (Wang et al., 2015).^[18:1]

Figure 12

TGA Curve of control alginate beads.

Figure 13

TGA Curve of alginate beads with entrapped bacteria.

The sample showed continuous degradation until 187.23°C, indicating the dehydration of the sample. A steep decline in weight percentage between 187.23°C - 287.39 °C (Δ32.994%), indicates the degradation of the alginate bead, corresponding to 47.1% initial weight. The derivative shows another relevant decrease between 533.85°C-676.59°C (Δ20.7951%), which could suggest the degradation of a non-stable phase of $CaCO_3$ (Liu et al., 2021).^[20] However, the lack of a sharp drop at ∼800 °C indicates the absence of calcite. Differences exist between the bacteria entrapped beads and the control. Control samples show no sharp decrease after 247.18°C, and the derivative indicates a continuous weight loss. Further characterisation with X-Ray diffraction can elucidate the phase of $CaCO_3$ formed in the beads, and spectroscopic analysis of the reaction medium is required to test if $CaCO_3$ permeates through the beads.

Future Usage

The SIMD module, which expresses the SazCA enzyme as a fusion protein on the E. coli cell surface, offers a versatile platform for exploring a range of biotechnological applications. It could for example be altered for Carbon Capture and Storage (CCS): The SazCA enzyme can catalyze the hydration of $CO_2$, which is a key step in this biomineralization process. This process is often used in CCS technologies, where $CO_2$ from industrial emissions is captured and stored in the ground to reduce atmospheric carbon dioxide levels. The SazCA-INPN fusion protein could be used to enhance the efficiency of this process by increasing the rate of $CO_2$ hydration and the formation of calcium carbonate crystals. Since the SazCA enzyme can facilitate the formation of calcium carbonate crystals, it can also be used to form bio concrete. This material has potential applications in construction, as it can be used to replace traditional concrete and reduce the environmental impact of the construction industry. Another potential application could be to use the composite part to develop biosensors for monitoring environmental conditions. For example, changes in $CO_2$ concentrations could be detected by monitoring the activity of the SazCA enzyme on the surface of E. coli cells (Bose & Satyanarayana, 2017).^[21]

References

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1. 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 ↩︎

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 ↩︎

3. 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 ↩︎

4. 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 ↩︎

5. 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 ↩︎

6. 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 ↩︎ ↩︎ ↩︎ ↩︎ ↩︎ ↩︎

7. Mergulhao, F.J.M. et al. (January 8, 2005). Recombinant protein secretion in Escherichia coli. Biotechnology Advances, 23(3): 177-202. https://doi.org/10.1016/j.biotechadv.2004.11.003 ↩︎

8. Singh, P., et al. (2013). Effect of signal peptide on stability and folding of Escherichia coli thioredoxin. PloS one, 8(5), e63442. https://doi.org/10.1371/journal.pone.0063442 ↩︎ ↩︎

9. Hartmann, S., et al. (January 22, 2022). Structure and protein-protein interactions of Ice Nucleation Proteins drive their activity. BioRxiv. https://doi.org/10.1101/2022.01.21.477219 ↩︎

10. Pan, S. H., & Malcolm, B. A. (2000). Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). BioTechniques, 29(6), 1234–1238. https://doi.org/10.2144/00296st03 ↩︎

11. National Institute of Standards and Technology. (n.d.). Calcium carbonate (precipitated). https://webbook.nist.gov/cgi/cbook.cgi?ID=C471341&Mask=80#IR-Spec ↩︎ ↩︎

12. Zhou, G., et al. (2004). Sonochemical synthesis of aragonite-type calcium carbonate with different morphologies. New Journal of Chemistry, 28(8), 1027. https://doi.org/10.1039/b315198k ↩︎

13. Levi, Y. et al. (December 14, 1998). Control Over Aragonite Crystal Nucleation and Growth: An In Vitro Study of Biomineralization. Chemistry – A European JournalVolume 4,(3). Pp. 389-39. https://oi.org/10.1002/(SICI)1521-3765(19980310)4:3<389::AID-CHEM389>3.0.CO;2-X ↩︎ ↩︎

14. Ivanova, L.A. et al. (February 28, 2023). Matrix is elsewhere, extracellular DNA is a link between biofilm and mineralization in Bacillus cereus planktonic lifestyle. Npj Biofilms and Microbiomes 9(9). https://doi.org/10.1038/s41522-023-00377-5 ↩︎

15. Ivanova, L.A. et al. (May 30, 2023). Structure Evolution of CaCO3 Precipitates Formed during the Bacillus cereus Induced Biomineralization. Minerals, 13(6). https://doi.org/10.3390/min13060740 ↩︎

16. Siva, T., et al. (2017). Enhanced polymer induced precipitation of polymorphous in calcium carbonate: calcite aragonite vaterite phases. Journal of Inorganic and Organometallic Polymers and Materials, 27(3), 770–778. https://doi.org/10.1007/s10904-017-0520-1 ↩︎

17. Kim, J. H., & Jo, B. H. (2022). A Colorimetric CO2 Hydration Assay for Facile, Accurate, and Precise Determination of Carbonic Anhydrase Activity. Catalysts, 12(11), 1391. MDPI AG. http://dx.doi.org/10.3390/catal12111391 ↩︎ ↩︎

18. Wang, J., et al. (2015). Application of modified-alginate encapsulated carbonate producing bacteria in concrete: a promising strategy for crack self-healing. Frontiers in Microbiology, 6. https://doi.org/10.3389/fmicb.2015.01088 ↩︎ ↩︎

19. Oniyama, E., & Wahlbeck, P. G. (1995). Application of transpiration theory to TGA data: Calcium carbonate and zinc chloride. Thermochimica Acta, 250(1), 41–53. https://doi.org/10.1016/0040-6031(94)01935-a ↩︎

20. Liu, R. et al. (April 19, 2021). Bio-mineralisation, characterisation, and stability of calcium carbonate containing organic matter. RSC Advances, 11: 14415-14425. https://doi.org/0.1039/d1ra00615k ↩︎

21. Bose, H., & Satyanarayana, T. (2017). Microbial Carbonic Anhydrases in Biomimetic Carbon Sequestration for Mitigating Global Warming: Prospects and Perspectives. Frontiers in microbiology, 8, 1615. https://doi.org/10.3389/fmicb.2017.01615 ↩︎