Difference between revisions of "Part:BBa K4724019"
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<i>Is</i>PETase is a hydrolase produced by Ideonella sakaiensis that degrades PET. Q119F/W159H is a double mutant of <i>Is</i>PETase.
 
<i>Is</i>PETase is a hydrolase produced by Ideonella sakaiensis that degrades PET. Q119F/W159H is a double mutant of <i>Is</i>PETase.
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<h1>Constraction</h1>
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<h1>Characterization</h1>
 
After protein purification, an enzymatic reaction is performed to measure enzyme activity. The substrate used is PET powder, which is decomposed into TPA and MHET under the action of the IsPETase double mutant. Determine the volume of purified enzyme solution required for 500 μL of the reaction system based on protein concentration. It reacted with PEF powder at 30 °C, 37 °C and 45 °C for 48h, and after the end the reaction solution was analyzed by high performance liquid chromatography, the liquid phase result of 6min corresponds to TPA, and the liquid phase result of 8min corresponds to MHET. Through the standard curve, the peak area of the product output by the liquid phase instrument is converted into the product concentration, such as in Fig.2.
 
After protein purification, an enzymatic reaction is performed to measure enzyme activity. The substrate used is PET powder, which is decomposed into TPA and MHET under the action of the IsPETase double mutant. Determine the volume of purified enzyme solution required for 500 μL of the reaction system based on protein concentration. It reacted with PEF powder at 30 °C, 37 °C and 45 °C for 48h, and after the end the reaction solution was analyzed by high performance liquid chromatography, the liquid phase result of 6min corresponds to TPA, and the liquid phase result of 8min corresponds to MHET. Through the standard curve, the peak area of the product output by the liquid phase instrument is converted into the product concentration, such as in Fig.2.
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https://static.igem.wiki/teams/4724/wiki/mutation-fig-3-a.png
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https://static.igem.wiki/teams/4724/wiki/mutation-fig-3-b.png
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https://static.igem.wiki/teams/4724/wiki/mutation-fig-3-c.png
  
 
Fig.2 The concentration of TPA and MHET products of 500nM WT, S93_I94insE/W159H and Q119F/W159H that react with PET powder for 48h at different temperatures. (A) is the product concentration obtained by the reaction temperature of 30 °C; (B) is the product concentration obtained by the reaction temperature of 37 °C; (C) is the product concentration obtained by the reaction temperature of 45 °C.
 
Fig.2 The concentration of TPA and MHET products of 500nM WT, S93_I94insE/W159H and Q119F/W159H that react with PET powder for 48h at different temperatures. (A) is the product concentration obtained by the reaction temperature of 30 °C; (B) is the product concentration obtained by the reaction temperature of 37 °C; (C) is the product concentration obtained by the reaction temperature of 45 °C.
  
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Analysis of the results
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<h1>Conclusion</h1>
 
At 30 °C, the concentration of IsPETaseQ119F/W159H product was about 4 times that of WT;
 
At 30 °C, the concentration of IsPETaseQ119F/W159H product was about 4 times that of WT;
 
At 37 °C, the concentration of IsPETaseQ119F/W159H product was about 5 times that of WT;
 
At 37 °C, the concentration of IsPETaseQ119F/W159H product was about 5 times that of WT;

Revision as of 21:15, 10 October 2023


IsPETaseS93_I94insE/W159H-6xHis Tag

IsPETase is a hydrolase produced by Ideonella sakaiensis that degrades PET. Q119F/W159H is a double mutant of IsPETase.

Characterization

After protein purification, an enzymatic reaction is performed to measure enzyme activity. The substrate used is PET powder, which is decomposed into TPA and MHET under the action of the IsPETase double mutant. Determine the volume of purified enzyme solution required for 500 μL of the reaction system based on protein concentration. It reacted with PEF powder at 30 °C, 37 °C and 45 °C for 48h, and after the end the reaction solution was analyzed by high performance liquid chromatography, the liquid phase result of 6min corresponds to TPA, and the liquid phase result of 8min corresponds to MHET. Through the standard curve, the peak area of the product output by the liquid phase instrument is converted into the product concentration, such as in Fig.2. mutation-fig-3-a.png mutation-fig-3-b.png mutation-fig-3-c.png

Fig.2 The concentration of TPA and MHET products of 500nM WT, S93_I94insE/W159H and Q119F/W159H that react with PET powder for 48h at different temperatures. (A) is the product concentration obtained by the reaction temperature of 30 °C; (B) is the product concentration obtained by the reaction temperature of 37 °C; (C) is the product concentration obtained by the reaction temperature of 45 °C.

Conclusion

At 30 °C, the concentration of IsPETaseQ119F/W159H product was about 4 times that of WT; At 37 °C, the concentration of IsPETaseQ119F/W159H product was about 5 times that of WT; At 45 °C, the concentration of IsPETaseQ119F/W159H product is about 23 times that of WT. We analyzed that the two sites Q119F and W159H played a synergistic role in the enzymatic reaction, which greatly enhanced the activity of IsPETase.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 56
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 56
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 793
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 56
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 56
    Illegal AgeI site found at 549
  • 1000
    COMPATIBLE WITH RFC[1000]