Difference between revisions of "Team:AHUT China/Demonstrate"

In our project, we have improved the existing CO2 capture technology and optimized the existing carbonic anhydrase by molecular simulation to finally enable E. coli to express more active and stable carbonic anhydrase to capture CO2 from industrial waste gas. Now we have constructed and tested the CO2 capture system based on pET30-a (+) expression vectors and E. coli BL21(DE3) strains under simulated condition in laboratory. Our main achievements are as follows:
1. The CA2 with high and stable catalytic efficiency designed by molecular simulation was selected as the research object of our project, which provided the basis for further development of CO2 capture.
2. Using molecular simulation to optimize CA2 amino acid sequence, design a highly active and stable CA2 mutant with the 203th leucine changed to lysine (named CA2 (L203K))
3. Mutant CA2 (L203K) is more efficient than wild-type CA2(WT).
4. The mutant CA2(L203K) presents improved thermal stability compared to wild-type CA2(WT).

Colorimetric assay of enzyme activity

Detection device:

Fig.1 Detection device:

CA2 activity assay with CO2 as substrate was performed following the procedure described by Capasso. In brief, The CO2-satured solution was prepared by bubbling CO2 into 100 mL distilled water for approximately 3 h. The CO2 solution was chilled in an ice-water bath. Add 1 mL of 25 mM Tris, pH 8.3, containing bromothymol blue as a dye (to give a distinct and visible blue color) to test tubes chilled in an ice bath. Indicated volume of purified enzyme were added to tubes, and an equivalent amount of buffer was added as control. Then, 1 mL of CO2 solution was added very quickly and simultaneously timing began. The time required for the solution to change from blue to yellow was recorded (transition point of bromothymol blue is pH 6-7.6). The time required for the color change is inversely related to the quantity of carbonic anhydrase presented in the sample.

Fig.2 The color changes of the solution after adding 250 μLCO2 saturated solution.

Fig.3 Comparison of color changing time of CO2 saturated solution

Fig.4 Comparison of unit carbonic anhydrase enzyme activity between wild type and mutant type of CA2

As shown in Fig.3 and Fig.4, by the measurement of the enzyme activity, the result showed that the activity of the mutant CA2 was stronger than that of the wild type CA2.

Determination of thermal stability of carbonic anhydrase by esterase activity analysis

In order to verify that our research results can be applied to higher temperature industrial environments in the future, we designed a gradient temperature to test the activity of wide type and mutant CA2 activity at different temperatures.

Fig.5 Activity of wild type and mutant CA2 at different temperatures

As shown in Fig.5, the results exhibited that the thermal stability of mutant carbonic anhydrase was better than that of wild carbonic anhydrase.

Reference

[1] Rahman F A, Aziz M M A, Saidur R, et al. Pollution to solution: Capture and sequestration of carbon dioxide (CO2) and its utilization as a renewable energy source for a sustainable future[J]. Renewable & Sustainable Energy Reviews, 2017, 71:112-126.
[2] Hu G, Smith K H, Nicholas N J, et al. Enzymatic carbon dioxide capture using a thermally stable carbonic anhydrase as a promoter in potassium carbonate solvents[J]. Chemical Engineering Journal, 2017, 307:49-55.
[3] Yong J K J, Stevens G W, Caruso F, et al. The use of carbonic anhydrase to accelerate carbon dioxide capture processes[J]. Journal of Chemical Technology & Biotechnology, 2015, 90(1):3-10.
[4] Capasso C, De L V, Carginale V, et al. Biochemical properties of a novel and highly thermostable bacterial α-carbonic anhydrase from Sulfurihydrogenibium yellowstonense YO3AOP1[J]. Journal of Enzyme Inhibition & Medicinal Chemistry, 2012, 27(6):892.