Team:AHUT-ZJU-China/Results
AHUT-ZJU-China
Result
As we have explained in the background, traditional CO2 capture technologies are still in their infancy,and these capture technologies tend to have high energy consumption but low efficiency. Therefore, our project goal is to develop a new type of low-energy, environmentally friendly, and efficient carbon dioxide capture method by using synthetic biology methods. Based on this goal, we intend to use Pyrococcus horikoshii OT3 carbonic anhydrase (OT3-CA-WT) as the research object because this carbonic anhydrase (CA) can catalyze the hydration of CO2 to HCO3- based on high thermal stability. (Fig. 1). This year, based on the previous two years of projects, we obtained engineered bacteria expressing OT3-CA-WT through genetic engineering technology and then purified OT3-CA-WT protein to test its thermal stability. Simultaneously, to further improve the catalytic activity of OT3-CA-WT, we used molecular simulation technology to do molecular docking of OT3-CA-WT, hoping to improve its catalytic activity while maintaining its original thermal stability, and finally obtained efficient thermally stable carbonic anhydrase, laying the foundation for subsequent industrial applications.
Engineered E. coli TB1 (DE3) with high OT3-CA-WT expression
Construction of OT3-CA-WT expression plasmids
The coding sequences of OT3-CA-WT (BBa_K3656304) were both synthesized , then cloned into the expression vector pET-28a(+) (Fig.2). The length of digested OT3-CA-WT was 546 bp, confirmed by the band above 500 bp in electrophoresis as indicated in Fig. 3, which indicated that the pET-28a(+) OT3-CA-WT recombinant plasmid was successfully constructed.
Induced expression of OT3-CA-WT protein
The OT3-CA-WT expression plasmid was transformed into E. coli TB1, and positive clones were screened for kanamycin resistance. The recombinant bacteria were expanded and cultured, and isopropyl-1-thio-β-Dgalactopyrasonide (IPTG) was used to induce the expression of OT3-CA-WT in E. coli TB1. Then the bacteria were lysed to extract the total protein, and SDS-PAGE and Western Blot were applied to verify the expression of OT3-CA-WT. As shown in Fig. 4 and Fig. 5, it showed that after induction by IPTG, a band about 19.5 KDa with a molecular weight similar to CA was appeared, as indicated by the arrow. This indicates that we have successfully constructed an engineered strain of E. coli TB1 expressing OT3-CA-WT.
Fig. 5 Induced expression of OT3-CA-WT in TB1 strain verified by Western blot
OT3-CA-WT purification
After confirming that OT3-CA-WT can be expressed in E. coli TB1, due to the nickel chloride in the nickel column can bind to the protein with His (histidine) tag, we used nickel column to further purify the OT3-CA-WT,then we used the Bradford method to test the concentration of the purified OT3-CA-WT protein. The analysis showed that the purified protein concentration of OT3-CA-WT was 7.7 mg·mL-1(Fig. 6).
Enzyme activity assay of OT3-CA-WT
We measured the enzymatic activity of OT3-CA-WT protein at 37°C, 45°C and 70°C by esterase activity assay.As shown in Fig. 7, the result showed that the enzyme activity at 45°C is almost or slightly higher than that at 37°C. Although the enzyme activity of OT3-CA-WT at a higher temperature of 70°C slightly decreases, it still has a higher activity. This shows that we have obtained carbonic anhydrase with specific high-temperature resistance and good thermal stability, which is also in line with our previous assumptions.
Obtain of mutant OT3-CA with enhanced catalytic activity via molecular simulation
The obtained OT3-CA-WT protein has high thermal stability, but its catalytic activity is not as good as human carbonic anhydrase. Therefore, we decided to mutate the original OT3-CA-WT by the molecular simulation toimprove its catalytic activity.
3.1
Through searching a large number of references about carbonic anhydrase, especially literature about OT3-CA-WT (Fig.8) and human carbonic anhydrase 2. We found that lysine at some sites can significantly enhance the activity of OT3-CA-WT, while proline and leucine may reduce the activity of OT3-CA-WT. Therefore, we designed the gene sequence of the mutant OT3-CA-MU via molecular simulation. The specific process is as follows:
1) Maintain the 3D structure of enzyme;
2) Modify the interactions between residues around active sites;
3) Improve the rigidity of active sites;
4) Shorten the distance of proton transfer.
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3.2 Perform enzyme-substrate molecular docking.
Using the software Autodock for molecular docking, the docking conformation of the substrate at the catalytic site was studied, and the interaction between the residues at the catalytic site and the substrate was analyzed. We used Autodock and PyMOL to further study the effect of the catalytic site's secondary and tertiary structure on the catalytic process. After simulation, we set the mutation site and substitution residue, carry out molecular docking of the recombinase, and then compare the enzyme-substrate docking conformation before and after the recombination. Finally, we selected suitable mutation sites and substitution residues to simulate, which improved its catalytic performance in theory.
3.3
Based on the above simulation analysis, we finally determined that the appropriate mutation sites are: position 122: P→K (the 122nd proline is mutated to lysine), position 123: P→K (the 123rd proline is mutated to Lysine), position 128: P→K (mutation of the 128th proline to lysine), position 132: L→K (mutation of 132nd leucine to lysine), position 134: L→K ( Leucine at position 134 was mutated to lysine), and position 137: P→K (leucine at position 137 was mutated to lysine), and finally a mutant OT3-CA (OT3-CA-MU, BBa_K3656307, Fig.9 and 10) to enhance its catalytic activity under high-temperature conditions was obtained (Table 1).
Table 1 Molecular docking results of OT3-CA-WT and OT3-CA-MU
| Name | OT3-CA-WT | OT3-CA-MU |
|---|---|---|
| Part Number | BBa_K3656305 | BBa_K3656309 |
| binding_energy | -5.08 | -5.37 |
| ligand_efficiency | -1.27 | -1.34 |
| inhib_constant | 187.83 | 116.58 |
| inhib_constant_units | uM | uM |
| intermol_energy | -5.68 | -5.96 |
| vdw_hb_desolv_energy | -1.76 | -1.92 |
| electrostatic_energy | -3.92 | -4.04 |
| total_intermal | 0.05 | 0.02 |
| torsional_energy | 0.6 | 0.6 |
| unbound_energy | 0.05 | 0.02 |
Outlook
In the future, based on the mutation results of molecular simulations, we will continue to express and purify the OT3-CA-MU protein and test the activity, to obtain an environmentally friendly CO2 capture device that will benefit humanity and the earth.