Team:AHUT-ZJU-China/Description
AHUT-ZJU-China
Description
Recently, with the rapid development of science and technology, environmental protection has gradually attracted all circles globally, among which the "greenhouse effect" is one of the most prominent problems.
The greenhouse effect is the way in which heat is trapped close to the surface of the earth by “greenhouse gases”. These heat-trapping gases can be thought of as a blanket wrapped around the earth, which keeps it toastier than it would be without them.
Carbon dioxide is the most abundant greenhouse gas, accounting for about 0.03 of the total atmospheric capacity. When the Sun’s energy reaches the earth’s atmosphere, some of it is reflected back to space and the rest is absorbed and re-radiated by greenhouse gases. Without greenhouse gases, the surface temperature would drop by about 33 ℃ or more. Conversely, if the greenhouse effect continues to strengthen, the global temperature will continue to rise year by year.
Since the Industrial Revolution, carbon dioxide and other heat-absorbing greenhouse gases released into the atmosphere by human activities have been increasing drastically, and the greenhouse effect of the atmosphere has also been enhanced, which caused a series of serious problems such as global warming which attracted the attention of countries all over the world.
As a result, various methods to control CO2 emissions have been come up with, such as CO2 capture, which are becoming more critical nowadays. The primary CO2 capture methods include solvent absorption, physical adsorption, membrane separation, and so forth.
The solvent absorption method is mainly based on chemical absorption. Some particular chemical absorbents can react with CO2 to form compounds and separate CO2 from the flue gas containing the absorbent. However, this method has the subsequent disadvantages: the solution is easy to oxidize and decompose; the process is highly corrosive and easy to corrode the instrument; large energy consumption and high operation cost.
The physical adsorption method collects CO2 according to the adsorption characteristics of different gas components to the solid adsorbent. However, this method requires many adsorbents to maintain process's operation, the adsorbent also has poor selectivity, low adsorption capacity, and low efficiency, resulting in high operating costs and few practical applications.
The membrane separation method is based on the different permeability of the polymer membrane to different gas components to achieve the purpose of separating different gas components. The membranes used can be divided into organic membranes and inorganic membranes. The organic membrane has strong selectivity for the gas components and simple assembly ability, but meantime suffered from low heat and corrosion resistance. On the contrary, the inorganic membrane has good heat and corrosion resistance attributes, but its assembly is involved. In general, CO2 captured by this method is of low purity requiring multiple purification processes, which limits its application in industry.
The above carbon dioxide capture method has the disadvantages of high cost, low efficiency, and poor circulability; thus hard to achieve environmentally efficient performance, which hinders its application in industrial operation conditions. Therefore, in 2018, our iGEM team improved the thermal stability of carbonic anhydrase by mutating the amino acid sequence of human carbonic anhydrase, and in this year, we again proposed an improvement by mutating the sequence of thermophilic carbonic anhydrase in order to increase the bioactivity of capturing CO2 over a broader range of temperatures.
Carbonic anhydrase, a metal enzyme-containing Zn2+, with at least five distinct classes: α, β, γ, δ, and ζ, which bear little structural similarity, but all employ an active site divalent zinc ion or related metal for catalysis, Central to the hydration mechanism is the nucleophilic attack of a Zn(II) bound hydroxide on CO2, recently CA enzymes have been identified as having the potential to accelerate CO2 capture from large combustion emitters can catalyze CO2 and H2O to produce HCO3-. Carbonic anhydrase catalyzes faster than other types of enzymes. The range of its catalytic rates is about 10^4 to 10^6 reactions per second. Among various sources of carbonic anhydrase, carbonic anhydrase (OT3-CA-WT) has the highest catalytic efficiency.
Given the high temperature for regeneration in a solvent-based capture system, thermophilic organisms represent a source of stable CAs. Three CAs from thermophilic organisms have been the primary focus of most biochemical studies. A γ-class CA from Methanosarcina thermophila (CAM), a β-class CA from Methanobacterium thermoautotrophicum (CAB), and a γ-class CA from Pyrococcus horikoshii. CAM shows optimal activity at 55℃, with Kcat values approaching 105/s. CAM is a relatively stable CA — it shows a 50% residual activity after 15 min at 70℃ and is inactivated at 75℃. CAB shows optimal activity at 70℃. It is more thermostable than CAM, showing 50% residual activity after 15 min at ~85℃ and inactivation at 90℃, but has a lower Kcat value of 104/s. CA's activity from P. horikoshii has not been fully characterized, but its host organism shows optimal growth at 98℃. By comparison, Human CA II shows optimal activity at 37℃ and is inactivated above 50℃. However, it is an extremely fast enzyme with a Kcat of 106/s.
The molecular weight ofγ-class CA (OT3-CA-WT) from Pyrococcus horikoshii OT3 is about 19.13KDa, composed of a single peptide chain and contains about 173 amino acids. The OT3-CA-WT has a conserved catalytic Zn2+ ion along the β-sheets comprising the side of the prism-shaped monomers. It belongs to lyases, with an atomic number of 1534 and 173 amino acid residues. (As shown in fig. 1).
fig. 1 (Schematic structural diagram of enzyme OT3-CA-WT )
Metal-ion coordination：Zn2+ is coordinated to His65A ND1, His87A NE2, His82B NE2, HOH44, and Tyr159B OH. The OH is at a distance of 2.71Å from Zn2+. (As shown in fig.2). The catalytic site of enzyme (PDB code 2fko) is formed by combining zinc ion and three histidines, which catalyze carbon dioxide and water to generate bicarbonate ion. (as shown in figure 3)
fig. 2 (Zinc ions combine with three histidines to form catalytic sites)
fig. 3 (Catalytic mechanism of carbonic anhydrase(OT3-CA-WT))
Compared with other methods, synthetic biology to capture carbon dioxide has the characteristics of environmental protection and high efficiency. Carbonic anhydrase can convert carbon dioxide into bicarbonate, which can meet plants and microorganisms' growth needs. Moreover, when carbon dioxide is converted into bicarbonate, bicarbonate is combined with calcium ions to form calcium carbonate, which can be stably stored underground, and the products can also be utilized efficiently.
Although carbonic anhydrase capture technology has high efficiency, it still has some limitations. Although the carbonic anhydrase extracted from thermophilic bacteria has good thermal stability, its activity is still far from human carbonic anhydrase. To improve the activity of wild-type carbonic anhydrase from thermophilic bacteria, we simulated mutation according to the method in 2018 and compared their activities, expecting to improve based on the previous two years. The main steps include:
(1) For the consideration of the environmental protection, safety, and the previous project experience, we chose to start with the carbon substitution enzyme (OT3-CA-WT) of the thermophilic bacteria (Pyrococcus horikoshii) and find its gene sequence;
(2) Establish a recombinant plasmid, introduce the target gene into the pET-28a(+) vector, and transfer it to the embryo for positive cloning and reproduction;
(3) Use IPTG inducer to induce expression;
(4) Continue to cultivate and use the HIS nickel column purification method to purify the protease;
(5) Measure the enzyme activity of the thermophilic bacteria carbon doping enzyme (OT3-CA-WT) at different temperatures;
(6) Use molecular simulation technology to mutate the sequence of the thermophilic carbon-doped enzyme to improve its biological activity;
(7) Outlook: Purify the mutant protein and design it into a new type of CO2 capture method to brake in human practice.
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