Base Pairs: 1752 bp
Origin: Ideonella sakaiensis 201-F6
Properties: hydrolysis of MHET.
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.
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.
Production, purification, and activity analysis of MHETase
1. Construction of pET28a-MHETase
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).
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.
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%.
1. Shosuke Yoshida et al. A bacterium that degrades and assimilates poly(ethylene terephthalate), Science (2016).
2. Harry P Austin. et al. Characterization and engineering of a plastic-degrading aromatic polyesterase, PNAS(2018)
3. Chun-Chi Chen et al. General features to enhance enzymatic activity of poly(ethylene terephthalate) hydrolysis, Nature Catalysis(2021).
Sequence and Features
- 10Illegal PstI site found at 612
- 12Illegal PstI site found at 612
- 21Illegal BamHI site found at 495
- 23Illegal PstI site found at 612
- 25Illegal 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
- 1000COMPATIBLE WITH RFC