Part:BBa_K3997008

pET28a-IsPETase-MHETase

Usage and Biology

Polyethylene terephthalate (PET) is the most widely produced polyester plastic and its accumulation in the environment has become a global concern. At the same time, the daily intake of microplastics by humans is gradually increasing, which damages human health. Therefore, researchers believe that it is important to develop an environmental-friendly plastic degradation method by using microorganisms. Recently, a novel bacterial strain called Ideonella sakaiensis 201-F6 has been discovered that produces a couple of unique enzymes, PETase and MHETase, enabling the bacteria to utilize PET as their sole carbon source.

PET hydrolase, known as PETase, is a recently discovered enzyme that has been found to break down PET, or polyethylene terephthalate-(C10H8O4)n. When the PETase-containing bacteria or fungi are administered to PET plastic, they secrete the PETase enzyme, which causes PET polymers to bind to the active site of PETase, allowing the reaction to occur. During the reaction, the enzyme breaks ester bonds in PET.

The enzyme MHETase is a hydrolase, and it is crucial for hydrolysis of MHET. To verify this property, we use E. coli as the starting strain and construct an engineered strain of MHETase to explore its biological activity of the hydrolysis of MHET. To purify the protein, we also transfer the plasmid expressing MHETase into BL21(DE3) with a 6×His tag at it’s N-terminal. The enzyme is under the regulation of T7 promoter and can be induced by adding IPTG.

The T7 promoter is often used for protein overexpression. It is powerful and specific. It is completely controlled by T7 RNAP. When T7 RNAP is present in the cell, the T7 expression system occupies an absolute advantage compared to the host expression system. Its expression The speed is 5 times that of the former.

Figure 1. Action and function of PETase and MHETase in PET degradation...

Construct design

Figure 2. pET28a-IsPETase-MHETase Plasmids diagram...

The genes IsPETase and MHETase were synthesized and inserted into pET28a vector to obtain plasmids pET28a-IsPETase and pET28a-MHETase, respectively. A recombinant plasmid pET28a-IsPETase-MHETase, which contains both genes, was also constructed (Figure 2).

The enzyme is under the regulation of T7 promoter and can be induced by adding IPTG. The T7 promoter is often used for protein overexpression. It is powerful and specific. It is completely controlled by T7 RNAP. When T7 RNAP is present in the cell, the T7 expression system occupies an absolute advantage compared to the host expression system. Its expression The speed is 5 times that of the former.

BBa_K3997001

Usage and Biology

It is an polyhistidine tag, which is used in the purification of recombinant proteins

BBa_K3997000

Properties: hydrolysis of PET.

PET hydrolase, known as PETase, is a recently discovered enzyme that has been found to break down PET, or polyethylene terephthalate-(C10H8O4)n. When the PETase-containing bacteria or fungi are administered to PET plastic, they secrete the PETase enzyme, which causes PET polymers to bind to the active site of PETase, allowing the reaction to occur. During the reaction, the enzyme breaks ester bonds in PET.

Experimental approach

Production analysis of PETase-MHETase fusion protein In order to present the function of the part, the PETase-MHETase fusion gene was PCR amplified and cloned into pET28a plasmids backbone in order to express the fusion protein in E. coli under the control of T7 promoter.

Figure 3. Gel electrophoresis of PETase-MHETase PCR products...

Proof of function

1. Enzyme Activity Tests

The activity of MHETase was indicated by the decline absorbances at a wavelength of 240 nm (Figure 4). The wavelength is the specific absorption of MHET. As shown in figure 5, after 1 d reaction, MHET concentration was dropped by 31.4%.

Figure 4. Detection of residual MHET by measuring the absorbance at 240 nm...

Figure 5. MHETase activity assay...

References

3. Chun-Chi Chen et al. General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis, Nature Catalysis(2021).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 510
  Illegal PstI site found at 1671
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 153
  Illegal NheI site found at 466
  Illegal PstI site found at 1671
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1554
  Illegal XhoI site found at 1033
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 510
  Illegal PstI site found at 1671
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 510
  Illegal PstI site found at 1671
  Illegal NgoMIV site found at 1239
  Illegal NgoMIV site found at 1353
  Illegal NgoMIV site found at 1809
  Illegal AgeI site found at 789
  Illegal AgeI site found at 1423
  
• 1000
  COMPATIBLE WITH RFC[1000]