Difference between revisions of "Part:BBa K4275009"
 
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<partinfo>BBa_K4275009 short</partinfo>
 
<partinfo>BBa_K4275009 short</partinfo>
  
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As the second enzyme of the PET degradation two-enzyme system found in Ideonella Sakaiensis, the MHETase catalyzes the cleavage of the ester bond in MHET(Monohydroxyethyl terephthalate) and liberates the final products of PET degradation - EG(ethylene glycol) and TPA(terephthalic acid) into the solution. The MHETase amino acid sequence used in this experiment is the same with the wild-type MHETase (GenBank Accession number: GAP38911.1). The enzyme MHETase works in synergy with PETase, the first enzyme in the two-enzyme PET degradation system found in Ideonella Sakaiensis - a PET-degrading and assimilating strain of bacteria isolated from a water treatment plant.
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As the second enzyme of the PET degradation two-enzyme system found in <i>Ideonella sakaiensis</i>, the MHETase catalyzes the cleavage of the ester bond in MHET(Monohydroxyethyl terephthalate) and liberates the final products of PET degradation - EG(ethylene glycol) and TPA(terephthalic acid) into the solution[1]. The MHETase amino acid sequence used in this experiment is the same with the wild-type MHETase (GenBank Accession number: GAP38911.1). The enzyme MHETase works in synergy with PETase, the first enzyme in the two-enzyme PET degradation system found in <i>Ideonella sakaiensis</i> - a PET-degrading and assimilating strain of bacteria isolated from a water treatment plant.
  
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[[File:GreatBay SCIE--3D MHETase.png|950px]]
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<p align="center"><b>Figure 1</b> The 3D structure of the protein predicted by Alphafold2. </p>
  
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===Usage and Biology===
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==Usage and Biology==
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The 3D structure of MHETase consists of a catalytic domain and an extensive lid domain. The catalytic domain adopts the α/β-hydrolase fold typical of a serine hydrolase, with an active site consist the catalytic triad Ser225, Asp492, and His528, which shows significant structural conservation compared to PETase. The lid domain partially covers the active site, hinders its capability to bind with PET fiber, and harbors a well-coordinated calcium cation.
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The 3D structure of MHETase consists of a catalytic domain and an extensive lid domain[1]. The catalytic domain adopts the α/β-hydrolase fold typical of a serine hydrolase, with an active site consist the catalytic triad Ser225, Asp492, and His528, which shows significant structural conservation compared to PETase. The lid domain partially covers the active site, hinders its capability to bind with PET fiber, and harbors a well-coordinated calcium cation[1].
  
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===Design consideration===
 
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1. The first 39 amino acids in the N' terminal (including the leader sequence) is omitted and replaced with a single start codon.
 
  
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2. 6xHis affinity purification tag (HHHHHH) is added to the C' terminal of the codon sequence.
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==Sequence and Features==
  
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3. DNA sequence is codon-optimized based on the codon-usage table of E.Coli Strain K12.MG1655.
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K4275009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4275009 SequenceAndFeatures</partinfo>
  
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==References==
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1. Knott, Brandon C. et al. "Characterization And Engineering Of A Two-Enzyme System For Plastics Depolymerization". Proceedings Of The National Academy Of Sciences, vol 117, no. 41, 2020, pp. 25476-25485. Proceedings Of The National Academy Of Sciences, https://doi.org/10.1073/pnas.2006753117.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 10:34, 13 October 2022


MHETase

As the second enzyme of the PET degradation two-enzyme system found in Ideonella sakaiensis, the MHETase catalyzes the cleavage of the ester bond in MHET(Monohydroxyethyl terephthalate) and liberates the final products of PET degradation - EG(ethylene glycol) and TPA(terephthalic acid) into the solution[1]. The MHETase amino acid sequence used in this experiment is the same with the wild-type MHETase (GenBank Accession number: GAP38911.1). The enzyme MHETase works in synergy with PETase, the first enzyme in the two-enzyme PET degradation system found in Ideonella sakaiensis - a PET-degrading and assimilating strain of bacteria isolated from a water treatment plant.

GreatBay SCIE--3D MHETase.png

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

The 3D structure of MHETase consists of a catalytic domain and an extensive lid domain[1]. The catalytic domain adopts the α/β-hydrolase fold typical of a serine hydrolase, with an active site consist the catalytic triad Ser225, Asp492, and His528, which shows significant structural conservation compared to PETase. The lid domain partially covers the active site, hinders its capability to bind with PET fiber, and harbors a well-coordinated calcium cation[1].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 123
    Illegal NgoMIV site found at 237
    Illegal NgoMIV site found at 693
    Illegal AgeI site found at 190
    Illegal AgeI site found at 307
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Knott, Brandon C. et al. "Characterization And Engineering Of A Two-Enzyme System For Plastics Depolymerization". Proceedings Of The National Academy Of Sciences, vol 117, no. 41, 2020, pp. 25476-25485. Proceedings Of The National Academy Of Sciences, https://doi.org/10.1073/pnas.2006753117.