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

    As we have described in the background, traditional carbon dioxide collection techniques are still in its early stages, characterized by high consumption and low efficiency. We want low-energy, large-scale, efficient collection of carbon dioxide, in order to achieve this goal, we by the carbonic anhydrase gene into E. coli.

  To ensure that E. coli-producing carbonic anhydrase 2(CA2) normally absorbs carbon dioxide in an industrialized environment, we first simulate the protein molecule expressed by a computer to obtain a thermally stable carbonic anhydrase 2(CA2), which produces a thermally stable carbonic anhydrase 2 (CA2), so that it can normally absorb carbon dioxide in a factory-like environment.

    E. Coli's carbonic anhydrase 2(CA2) promotes CO2 hydration to produce CO32-, which binds to the free Ca2+ in the environment to form calcium carbonate deposits, thereby achieving the purpose of absorbing carbon dioxide, and producing inorganic products that can be used.

  Selection of carbonic anhydride enzymes:
  Our team acquired the sequence of carbonic anhydrase from the human body and contacted Biotech to help us synthesize the carbonic anhydrase gene in its entire sequence. The mutant carbonic anhydrase 2(CA2) used by our team absorbs carbon dioxide more strongly than other mammals, plants, algae, and carbonic anhydrase 2(CA2) produced by bacteria. Carbonic anhydrase 2(CA2) in human body has the fastest reaction rate at 37 °C, and is inactivated at 50 °C, but its maximum reaction rate can reach 106 s-1, which is the fastest catalytic carbonic anhydrase 2(CA2).

Carbonic anhydrase structure(Fig. 1):

Fig. 1 CO2 release in human carbonic anhydrase II crystals

     Increased thermal stability of carbonic anhydrase 2(CA2):

    In order to make the carbonic anhydrase 2(CA2) suitable for the industrial environment to absorb carbon dioxide, later we use molecular simulation technology, the amino acid as the basic unit, the mutations of residues on the secondary structure of the carbonic anhydrase, and the influence of molecular conformation, to obtain the best amino acid mutation sites, The thermal stability of enzymes was improved without affecting the enzyme Activity.

    Thermal stability studies of CA2-WT and CA2 (L203K) protein:

    We then investigated the effect of temperature on CA2 activity by esterase activity assay. As shown in (Fig. 2), as the temperature increases, especially at 55 °C and 65 °C, the enzymatic activity of CA2-WT was significantly decreased, while the mutant CA2 still retain relatively high activity, indicating that CA2 (L203K) was more stable at high temperature and retained its activity.

Fig. 2 Activity of purified CA2-WT and CA2 (L203K) protein under indicated temperatures and time points

    Enzymatic properties of carbonic anhydrase:

    Carbonic anhydrase is currently the most efficient enzyme for catalyzing the hydration of CO2. Carbonic anhydrase can catalyze CO2 andH2O produces HCO3-( Fig. 3). Under natural conditions, the reaction rate is extremely low, but the reaction rate is greatly accelerated under the catalysis of carbonic anhydrase, and the reaction rate can reach 104-106 reactions per second. The main rate limiting step is the diffusion rate of the substrate . In animals, carbonic anhydrase is mainly involved in the acid-base balance and CO2 transport in the blood; in plants, carbonic anhydrase mainly helps the chloroplast to absorb CO2.

Fig. 3 The mechanism of hydration of carbon dioxide catalyzed by carbonic anhydrase 2(CA2）

    Application of carbonic anhydrase 2 (CA2) in carbon dioxide concentration：

    There are two kinds of enzymatic trapping techniques: non-immobilized carbonic anhydrase trapping technology and immobilized carbonic anhydrase trapping technology. Non-immobilized Carbonic anhydrase trapping technology is the first carbonic anhydrase capture technology, This method directly using free carbonic anhydrase CO2 capture, at this time the activity of carbonic anhydrase is low, about 30% of the activity, and this method is not conducive to the re-use of enzymes(Fig. 4); in order to compensate for the deficiency of non-immobilized carbonic anhydrase technology, Some people will silica and other inorganic compounds as a carrier of the carbonic anhydrase, thereby curing the carbonic anhydrase, it is found that the activity of carbonic anhydrase at this time to maintain about 60%, and the easy recovery of carbonic anhydrase , so our team is using immobilized carbonic anhydrase capture technology.

Fig. 4 Schematic diagram of immobilized carbonic anhydrase capture CO2

    references:

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Elder I , Han S , Tu C , et al , 2004.Activation of carbonic anhydrase Ⅱ by active-site incorporation of histidine analogs [ J] .Arc Bioch Biophys, 421:283—289
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Supuran C T, Conroy C W, Maren T H. Is cyanate a carbonic anhydrase substrate [J]. Proteins-structure Function & Bioinformatics, 2015, 27(2): 272-8.