Team:AHUT-ZJU-China/Design
AHUT-ZJU-China
Design
At present, the traditional CO2 capture technology is still in its infancy. Most of the physical and chemical capture technologies do not accord with the characteristics of environment-friendly. Therefore, the goal of our project is to develop a new low-energy, environmentally friendly and efficient method of carbon dioxide capture by using the method of synthetic biology.
By reading a large number of literatures and referring to the experimental projects of the previous two years, in order to achieve the goal of improving the ability of CO2 capture at high temperature, we intend to take Pyrococcus horikoshii OT3 carbonic anhydrase (OT3-CA-WT) as the research object, because this kind of carbonic anhydrase (OT3-CA-WT) can catalyze the hydration of CO2 to HCO3- (Fig. 1) on the basis of high thermal stability and has good thermal stability.
Referring to the carbonic anhydrase (PDB code 1qq0; PDB code 1thj; PDB code 1v3w; PDB code 1v67; PDB code 2fko) of five different thermophilic bacteria, we found that carbonic anhydrase (PDB code 2fko) has potential high activity through molecular simulation technology, so we selected carbonic anhydrase (PDB code 2fko) of thermophilic bacteria to carry out this year's project.
On the basis of the project of the previous two years, we obtained the engineering bacteria expressing OT3-CA-WT through genetic engineering technology, and then purified the OT3-CA-WT protein and tested its thermal stability. At the same time, in order to further improve the catalytic activity of OT3-CA-WT, we used molecular simulation technology to dock OT3-CA-WT, hoping to improve its catalytic activity on the basis of maintaining its original thermal stability. It is expected to obtain efficient and thermally stable OT3-CA-MU, to lay the foundation for follow-up industrial applications.
fig.1(Catalytic mechanism of carbonic anhydrase(OT3-CA-WT))
The overall design of our project is as follows (Fig. 2):
Fig.2 Project design drawing
Construction of OT3 carbonic anhydrase expression plasmids
The coding sequences of OT3-CA-WT (BBa_K3656304) were both synthesized, then cloned into the expression vector pET-28a(+) (Fig.3).
Fig. 3 Map of OT3-CA-WT recombinant plasmid
Induced expression of OT3-CA-WT protein
The OT3-CA-WT expression plasmid was transformed into E. coli TB1 and the positive clones were screened by kanamycin resistance. The recombinant bacteria were cultured and OT3-CA-WT was expressed in E. coli induced by isopropyl-1-thio- β-Dgalactopyrasonide (IPTG). Then the total protein was extracted by lysing bacteria, and the expression of OT3-CA-WT was verified by SDS-PAGE and Western Blot.
OT3-CA-WT purification
After confirming that OT3-CA-WT can be expressed in E. coli TB1, the nickel chloride in the nickel column can bind to the protein labeled with His (histidine). Therefore, we further purified it with a nickel column, and then determined the concentration of the purified OT3-CA-WT protein by Bradford method.
Determination of OT3-CA-WT protease activity
We used esterase activity assay to determine the enzyme activity of OT3-CA-WT protein at 37 ℃, 45 ℃ and 70℃, in order to obtain carbonic anhydrase with good thermal stability under certain high temperature.
Obtain of mutant OT3-CA with enhanced catalytic activity via molecular simulation
We mutate the original OT3-CA-WT by molecular simulation to improve its catalytic activity.
We simulate and design the gene sequence of the mutant OT3-CA-MU, 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.
Fig. 4 yrococcus horikoshii carbonic anhydrase (OT3-CA-WT)
Molecular docking of enzyme-substrate
By using the software Autodock for molecular docking, we studied the docking conformation of the substrate at the catalytic site, and analyzed the interaction between the residue of the catalytic site and the substrate.
Then we use Autodock and PyMOL to further study the influence of the secondary and tertiary structure of catalytic sites on the catalytic process, and select appropriate mutation sites and alternative residues to simulate mutagenesis, hoping to improve its catalytic performance in theory.
Based on the above simulation results, we finally determine the appropriate mutation site in order to enhance its catalytic activity at high temperature