Team:AHUT China/Notebook

    8.29

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     Pick Clon

8.30

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    Pilot Expression of Protein
   “IPTG+ 4h”“IPTG- 4h”

 

 

   After the rocker culture is 4 H, add IPTG(final concentration is 0.5 mm) and then the rocker culture is 4 hours. Each of the two tubes absorbs 200 μL of bacterial fluid, and in the frozen centrifuge, it is 4 °C and 12000 rpm centrifuge. 10 minutes, go to the clear, It is resuspended with 100 μL 1x SDS Loading buffer, then heated in a water bath pot at 100 °C for 10 minutes and stored in a -20 °C refrigerator.

    8.31

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    Protein extraction
    CA2L203K

 

 

    CA2WT
    CA2L203K

    9.01

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    Purification of Protein
     Prior to purification

    Purified

   9.03

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    Western blot identification of proteins
     CA2L203K    CA2WT

   9.04

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    Quantification of protein concentrations

   9.05

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    Colorimetric enzyme activity
     Add 250 μl saturated CO2 solution

   According to the average calculated in the table above, the unit enzyme activity of the mutated carbonic anhydride enzyme is 0.3710(10 5 WAU/μg), and the unit enzyme activity of wild carbonic anhydride enzyme is 0.1755(10 5 WAU/μg).
Test esterase activity
Determination curve without carbonated anhydrase:

   Determination curve with carbonated anhydrase（CA2WT）

   Determination curve with carbonated anhydrase（CA2L203K）

   Wild carbonic anhydrase activity calculated as 0.5098 mol/g × min
   The mutated carbonic anhydrase enzyme activity was calculated to be 1.0392 mol/g * min.