Part:BBa_K3997001

MHETase

promoter

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.

Figure 1. Action and function of MHETase in MHET degradation.

The enzyme MHETase is a hydrolase, and it represents a key step in the process of microbial PET degradation in I. sakaiensis. It cleaves monoterephthalic acid, the PET degradation product by PETase, to ethylene glycol and terephthalic acid, and it is crucial for hydrolysis of MHET.

Figure 1...

We attempt to express the MHETase in E. coli strain to express and purify the protein to test its activity of degradation the MHET (Figure 1). So as to set up a method of environmental-friendly plastic degradation.

Experimental approach

Production, purification, and activity analysis of MHETase

1. Construction of pET28a-MHETase

Figure 2. Gel electrophoresis of MHETase PCR products...

2. Purification of MHETase( ~65.17 kDa)_BL21(DE3)

In order to present the function of the part, the MHETase gene was expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed, The samples of whole expression cell lysate, supernatant and pellet of cell lysate were analyzed using SDS-PAGE and Western blot (only for his-tag proteins from pET28a vector), which is found in the corresponding protein band of approximately 65kDa (Figure 3).

Fig.3 SDS-PAGE (left) and western blot (right) analysis for MHETase cloned in pET28a and expressed in BL21(DE3) strain...

Lane M1: Protein marker

Lane M2: Western blot marker

Lane PC1: BSA (1μg)

Lane PC2: BSA (2μg)

Lane NC: Cell lysate without induction

Lane 1: Cell lysate with induction for 16h at 15oC

Lane 2: Cell lysate with induction for 4 h at 37oC

Lane NC1: Supernatant of cell lysate without induction

Lane 3: Supernatant of cell lysate with induction for 16h at 15oC

Lane 4: Supernatant of cell lysate with induction for 4 h at 37oC

Lane NC2: Pellet of cell lysate without induction

Lane 5: Pellet of cell lysate with induction for 16h at 15oC

Lane 6: Pellet of cell lysate with induction for 4 h at 37oC

The primary antibody for western blot is anti-His antibody

In this project，western blot (right) analysis for MHETase was cloned in pET28a, the primary antibody for western blot is anti-His antibody. MHETase -His protein was successfully expressed.

Figure4. SDS-PAGE of MHETase-his...

Fig.5 SDS-PAGE (left) analysis for MHETase cloned in pGEX-6P-1 and expressed in BL21(DE3) strain...

In addition, MHETase genes were also cloned to the expression vector pGEX-6P-1, which produce recombinant protein fusion with Glutathione-S-transferase (GST) tag.

Lane 1: MHETase Cell lysate without induction for 20 h at 16oC

Lane 2: MHETase Cell lysate with induction for 20 h at 16oC

Lane 3,4,5: GST elution fractions of purification of lane 2 by GST-affinity chromatography

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 2, after 1 d reaction, MHET concentration was dropped by 31.4%.

Figure 6. Detection of residual MHET by measuring the absorbance at 240 nm...

Figure 7. 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 PstI site found at 612
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 612
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 495
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 612
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 612
  Illegal NgoMIV site found at 180
  Illegal NgoMIV site found at 294
  Illegal NgoMIV site found at 750
  Illegal AgeI site found at 364
  
• 1000
  COMPATIBLE WITH RFC[1000]