Team:WFLA YK PAO/Engineering
Engineering
Background
The use of plastic (poly(ethylene terephthalate), PET) greatly facilitates human
life. However, because plastic products are difficult to degrade, the use of a large number of plastic
products eventually not only causes environmental pollution. At the same time, the daily intake of
microplastics by humans is gradually increasing, which damages human health. Study of a variety of model
organisms suggested that the microplastics ingested through diet are mainly concentrated in the digestive
tract. Therefore, the degradation of ingested microplastics through intestinal probiotics is an effective
way to prevent microplastics from spreading to other parts of the body and reduce the content of
microplastics in the intestine.
In 2016, the Science journal first reported the related enzyme (PETase) capable of
degrading PET. In 2018, the PNAS journal reported that MHETase can further degrade the degradation product
MHET into TPA (mono (2-hydroxyethyl) terephthalic acid) and EG (ethylene glycol). In 2021, Nature Catalysis
reported a high-efficiency enzyme (IsPETase) that degrades PET. So far, plastic products can be efficiently
degraded into recyclable monomers.
Design
We intended to express PET degrading enzymes (IsPETase, MHETase) probiotics
bacteria to reduce the content of human microplastics and improve human health.
Build
The genes IsPETase and MHETase were synthesized and inserted into pET28a vector to
obtain plasmids pET28a-IsPETase and pET28a-MHETase, respectively (Figure 1). A recombinant plasmid
pET28a-IsPETase-MHETase, which contains both genes, was also constructed.
In addition, IsPETase and MHETase genes were also cloned to the expression vector
pGEX-6P-1, which produce recombinant protein fusion with Glutathione-S-transferase (GST) tag (Figure 2).
Figure 1. Schematic maps of pET28a-IsPETase, pET28a-MHETase, and pET28a-IsPETase-MHETase
plasmids.
Figure 2. Schematic maps of pGEX-6P-1-IsPETase and pGEX-6P-1-MHETase.
Test
All the expression plasmids were transformed into Escherichia coli BL21(DE3).
Different expression conditions (temperature and inducer concentration) were tested to obtain soluble
enzymes. 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) (Figure 2).
Figure 2. SDS-PAGE and Western blot analysis of IsPETase from pET28a vector. Lane
1: Protein marker; Lane 2: Cell lysate without induction; Lane 3: Cell lysate with induction for 16h at 15
oC; Lane 4: Cell lysate with induction for 4 h at 37 oC; Lane 5: Supernatant of cell lysate without
induction; Lane 6: Supernatant of cell lysate with induction for 16h at 15 oC; Lane 7: Supernatant of cell
lysate with induction for 4 h at 37 oC; Lane 8: Pellet of cell lysate without induction; Lane 9: Pellet of
cell lysate with induction for 16h at 15 oC; Lane 10: Pellet of cell lysate with induction for 4 h at 37 oC;
The primary antibody for western blot is anti-His antibody.
The theoretical molecular weigh of the IsPETase is 35.13 kDa. As seen from the
SDS-PAGE and Western blot (Figure 2), IsPETase was predominantly expressed in insoluble form, and only
little soluble protein was produced at low temperature.
Figure 3. SDS-PAGE and Western blot analysis of MHETase from pET28a vector. Lane 1:
Protein marker; Lane 2: Cell lysate without induction; Lane 3: Cell lysThe theoretical molecular weigh of
the MHETase is 65.17 kDa. As seen from the SDS-PAGE and Western blot (Figure 3), MHETase was also
predominantly expressed in insoluble form, and only little soluble protein was produced at low temperature.
ate with induction for 16h at 15 oC;
Lane 4: Cell lysate with induction for 4 h at 37 oC; Lane 5: Supernatant of cell lysate without induction;
Lane 6: Supernatant of cell lysate with induction for 16h at 15 oC; Lane 7: Supernatant of cell lysate with
induction for 4 h at 37 oC; Lane 8: Pellet of cell lysate without induction; Lane 9: Pellet of cell lysate
with induction for 16h at 15 oC; Lane 10: Pellet of cell lysate with induction for 4 h at 37 oC; The primary
antibody for western blot is anti-His antibody.
The supernatant of cell lysates with pET28a-IsPETase and pET28a-MHETase were
applied for enzymatic degradation of PET. As shown in Figure 4, no obvious change on the surface of PET
particles observed under light microscope.
Figure 4. Microscope of the PET degradation by IsPETase and MHETase. left: blank;
right: IsPETase + MHETase.
Figure 5. IsPETase and MHETase production from pGEX-6T-1 vector.
In order to improve the solubility of IsPETase and MHETase, we tried to expression
the genes using pGEX-6T-1 vector. The supernatant of cell lysate was purified by GST column. As shown in
Figure 5, bits of GST-MHETase was successfully yielded, and no GST-IsPETase was obtained.
MHETase Reactions were performed in triplicate over a 1 d time course at 30°C.
Reaction mixtures contain 50 nM recombinant GST-MHETase, 5 mM MHET in 90 mM NaCl, 10% (v/v) DMSO, 45 mM
sodium phosphate, pH 7.5. Reactions were terminated by adding an equal volume of methanol. The reactions
using inactive GST-MHETase (obtained by boiling for 10 min) were used as blank controls. The activity of
MHETase was indicated by the decline absorbances at a wavelength of 240 nm (Figure 6). 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 2. MHETase activity assay
Learn
In this experiment, we successfully constructed IsPETase and MHETase expression
plasmids wit 6 x His tag and GST tag. Both IsPETase and MHETase were expressed. However, only little
recombinant enzymes were expressed in soluble form. The inactive enzyme hindered us to make a comprehensive
PET degradation experiment.