iGEM 2022 — Team beijing-united

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Notebook

2022.7.21

Team ice-breaking
Introduction to the project
Project discussion

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook01.png)

2022.7.22

Laboratory safety training
Culture medium preparation
Inoculation of E. coli strains containing plasmids pET22b and pRSFDeut1.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook02.png)

2022.7.23

PCR to amplify target genes
Plasmid Extraction and double-enzyme digestion
Gel electrophoresis and gel extraction
Link vector and target gene by Gibson-assembly
Recombinant vector transport target gene into competent cells

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook03.png)

2022.7.24

Colony PCR to identify the correct colony
Gel electrophoresis test
Inoculation of correct recombinant E. coli strains

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook04.png)

2022.7.25

Plasmid Extraction
Plasmids digestion verification
Send the correct plasmids to the company for Sanger sequencing

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook05.png)

2022.7.26

Transport recombinant plasmids into BL21(DE3)
Plate on the agar plate

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook06.png)

2022.7.27

Cultivate the bacteria
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification
Site-direct mutation of SbPETase by PCR and transform DH5α

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook07.png)

2022.7.28

Check the transformation and inoculate the strain into LB liquid medium
Inoculate strains containing SbPETase mutants

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-notebook08.jpg)

2022.7.29-30

Protein purification
SDS-PAGE
Mutant plasmids extraction and send for Sanger sequencing

2022.7.31

In vitro platform to identify the activity of SbPETase towards PET and BHET
HPLC experiments of SbPETase degrade PET and BHET materials
Transport recombinant mutant plasmids into BL21(DE3)

2022.8.1

Cultivate the bacteria
Prepare IPTG for inducing the protein expression
Prepare buffer A and buffer B for protein purification

2022.8.2-3

Protein purification
SDS-PAGE

2022.8.4-5

In vitro platform to identify the activity of SbPETase towards PET and BHET
HPLC experiments of SbPETase degrade PET and BHET materials
Collect the data

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{% block title %}Engineering Success{% endblock %}
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Engineering

Introduction

Polyethylene terephthalate (PET), one of the most widely used plastics in the world, is currently causing serious
environmental pollution. In recent years, more and more researchers have focused on the enzymatic degradation of
PET, which is a more environmentally friendly degradation and recycling method than the traditional chemical and
physical recycling methods. In this study, our team established an efficient secretion system of PET degradation
enzyme SbPETase in E. coli BL21(DE3) using the signal peptide PelB and the colistin-releasing protein Kil, which not
only improved the enzyme yield but also greatly simplified the purification process of PET degradation enzyme
(Figure 1A). This provided a more convenient tool for further study of the enzyme. In addition, our team
rationalized the PET hydrolase SbPETase from Schlegelella brevitalea sp. nov. and used the established secretion
system to rapidly screen for triple mutants with significantly increased activity (Figure 1B).

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-engineering01.jpg)
Figure1. The principle of our project. A. the secretion system of SbPETase in E. coli BL21(DE3), B. shows how
does SbPETase work

Design

The DNA sequence of SbPETase was inserted into the BamHI and XhoI sites of the pET22b
vector (Figure 2A). The gene Kil was amplified by PCR from the plasmid containing the Kil gene and cloned into the
NcoI and XhoI sites of the pRSFduet1 vector (Figure 2B).

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-engineering02.jpg)
Figure 2. Plasmid profiles in this project. A. pET22b-PelB-SbPETase, B. pRSFdeut-1-kil

Build

We amplify SbPETase was amplified from the genome of S. brevitalea sp. nov (Figure3A.) and ligated it to the
double-enzyme digestion pET22b vector, and We amplify Kil by PCR from the plasmid containing the Kil gene
(Figure3B.), ligated to the double-enzyme digestion pRSFduet1 carrier.
To verify if the plasmid is correct, we digest plasmid pET22b-PelB-SbPETase with ApaLI and pRSFdeut-1-Kil with AseI
(Figure 3C, E). We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned
sequencing comparison results showed that there were no mutations in the ORF region (Figure 3D, F.), and the
plasmids were successfully constructed. So far, we have successfully obtained the recombinant plasmids.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-engineering03.jpg)

Figure 3. Gel electrophoresis results of target gene fragments and the sequencing results map to the recombinant
plasmids. A. The gene fragment of SbPETase, B. The gene fragment of Kil, C. digest plasmid pET22b-PelB-SbPETase
with ApaLI, D. the sequencing result of the recombinant plasmid pET22b-PelB-SbPETase, E. digest plasmid
pRSFdeut-1-Kil with AseI, D. the sequencing result of the recombinant plasmid pRSFdeut-1-Kil.

Activity identification

a) Protein expression and purification

We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the
culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight
induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular
proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results03.png)

Figure 4. SDS-PAGE result of SbPETase

In order to test if Kil signal peptide can improve the yield of SbPETase proteins in the
culture medium, we also co-transformed the recombinant plasmids pET22b-PelB-SbPETase and pRSFDeut1-Kil into E. coli
BL21(DE3), expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached
0.4. After overnight induction and culture, we purified the protein SbPETase we wanted and collected the data
(Figure 5). In Figure5, when co\_transformed pET22b-PelB-SbPETase and pRSFDeut1-Kil, the yield of protein SbPETase in
the extracellular medium is higher than without it.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-engineering05.png)

Figure 5. the yield of protein SbPETase both with and without Kil

b) Biochemical characterization of SbPETase

We set up an in vitro system and confirmed the ability to use the SbPETase to degrade
PET materials. As shown in Figure 6, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small
quantity of TPA through HPLC (Figure 6A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate)
or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.We set up an in vitro system and confirmed
the ability to use the SbPETase to degrade PET materials. As shown in Figure 6, we demonstrated that SbPETase could
degrade PET and BHET into MHET and a small quantity of TPA through HPLC (Figure 6A, B), the optimum conditions
showed that at 30°C, pH 7.0 (BHET as substrate) or pH 8.0 (PET film as substrate), SbPETase showed the highest
activity.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-engineering06.png)

Figure 6. Two different HPLC experiments of SbPETase degrade PET and BHET materials. A. result recorded with an
HPLC system successfully identified the MHET and BHET peak when used BHET as substrate, B. result recorded with an
HPLC system successfully identified the MHET, BHET, and TPA peak when used PET as substrate.Figure 6. Two
different HPLC experiments of SbPETase degrade PET and BHET materials. A. result recorded with an HPLC system
successfully identified the MHET and BHET peak when used BHET as substrate, B. result recorded with an HPLC system
successfully identified the MHET, BHET, and TPA peak when used PET as substrate.Figure 6. Two different HPLC
experiments of SbPETase degrade PET and BHET materials. A. result recorded with an HPLC system successfully
identified the MHET and BHET peak when used BHET as substrate, B. result recorded with an HPLC system successfully
identified the MHET, BHET, and TPA peak when used PET as substrate.

c) Site-direct mutation of SbPETase

To screen more efficiently PETase, we did site-direct mutation of SbPETase. PCR was
performed using the identified correct pET22b-pelB-SbPETase plasmid as a template with the corresponding
site-directed mutant primers. The PCR amplicons were digested with DpnI for 2 h, and the digested products were
transformed into E. coli DH5α competent cells and incubation at 37℃ overnight. We constructed 3 mutants: L61T,
W132H, and R259A. After identification of the correct mutant SbPETase plasmids, we co-transformed plasmids
pET22b-PelB-SbPETase-mutants and pRSFDeut1-Kil into E. coli BL21(DE3) and purified the corresponding mutant protein.

d) Biochemical characterization of SbPETase mutants

We tested the activity of mutant SbPETase towards PET and BHET materials using the in
vitro platform we set up. In Figure 7A we could find that there were two mutants (SbPETaseW132H, SbPETaseR259A) with
improved catalytic efficiency of degrading PET (Figure 7A). The activity of another mutant SbPETaseL61T was only
improved towards degrading BHET. We then combined these three mutants (SbPETaseW132H, SbPETaseR259A, SbPETaseL61T)
to generate three double mutants and one triple mutant. An in vitro activity assay showed that the triple mutant had
the highest catalytic efficiency towards PET and BHET degradation (Figure 7B)

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-engineering07.jpg)

Figure 7. Comparison of the SbPETase mutants activity of degrading PET materials. A. detection of SbPETase mutants
enzyme activity when the substrate is BHET, B. A. detection of SbPETase mutants enzyme activity when the substrate
is PET.Figure 7. Comparison of the SbPETase mutants activity of degrading PET materials. A. detection of SbPETase
mutants enzyme activity when the substrate is BHET, B. A. detection of SbPETase mutants enzyme activity when the
substrate is PET.

What we learned from our project

We have already collected the data from our experiments. From the experiments, we set up an in vitro screen platform
for identifying active PETase. We first find out that SbPETase has an effect on PET and BHET materials degradation.
To improve the yield of protein SbPETase, we fused it with pelB signal peptide and Kil peptide, as a result, we
successfully obtain more protein SbPETase.
On order to develop more candidates for future use, we did the site-direct mutation of SbPETase, and finally found
out several mutants that are different towards different substrates.
Because of the great effect of the SbPETase and its mutants, we believe that if we can use the SbPETase in the
future and promote it in the market, it will become a great power to degrade PET, and reduce the pollution made by
PET or BHET materials, thereby achieving the purpose of protecting the environment.

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Experiments

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-experiments01.png)

1.PCR
amplification of target gene

1. Add 1μL target gene plasmid sbPETase + Pelb into one micro-centrifuge tube. Then add 1 μL target gene plasmid kil
into the second micro-centrifuge tube.
2. Add both 2 μL forward primer and 2 μL reverse primer into each micro-centrifuge tube. This step makes sure DNA
polymerase could bind to the correct position before extension.
3. Add 5 μL Mix into each micro-centrifuge tube. It provides necessary enzymes for the system.
4. Add 10 μL ddH2O micro-centrifuge tube. It creates a liquid environment for the reaction.
5. Shake three micro-centrifuge tubes and insert them into the PCR Thermal Cycler and set the following steps
6. Denaturation at 98℃ for 15 seconds
7. Annealing at 55℃ for 15 seconds
8. Extension at 72℃ for 1 minute
9. Repeat steps 2-4 for 35 times

2.Agarose Gel
Electrophoresis

1. fold the weighing paper along the diagonal, weigh 0.3g agarose, then pour it into the conical flask.
2. pour 30 ml TAE buffer into the flask and put it in the microwave oven for 3 minutes, until the agarose is
completely dissolved.
3. inject 10 μL Ts-GelRed into the liquid.
4. insert the “comb” into the liquid. Let it stand until the gel is solidified.
5. transfer the gel into the electrophoresis chamber, then pull out the “comb” gently. Pour into enough TAE buffer
until the gel is submerged.
6. carefully use the pipette to transfer a 3μL marker into the first hole, then add 2μL template DNA in the rest
holes.
7. turn on the power and wait for 18 minutes.
8. take out the gel and place it into the UV transilluminator and turn on the UV light.
9. compare with the marker, and determine the length of template DNA.
10. Recover the gel.

3.Double
enzymes digest the plasmids

1. add 2 μL NcoI and 2 μL XhoI restriction enzyme into one micro-centrifuge tube, then add 2 μL BamHI restriction
enzyme and 2 μL XhoI restriction enzyme into another micro-centrifuge tube.
2. add 5μL buffer, 10μL ddH₂O, and 1μL DNA into each micro-centrifuge tube.
3. 37℃ incubation for 1h.
4. Agarose Gel Electrophoresis and gel extraction.

4.Recombinant
plasmid

1. add 2 μL Gibson enzyme, 1 μL PCR product, 1 μL plasmid vector, and 6 μL ddH₂O into the micro-centrifuge tube, 37
℃ water bath for 20 minutes.

5.Transformation of recombinant plasmids to DH5α competent cell

1. Transformation
1. Prepare a box of ice and operate all the following steps on the ice
2. Thaw 50μL×4 chemically competent cells DH5α on ice for 1-2 minutes.
3. Mix two tubes of DH5α competent cell(50μL) with precooled recombinant plasmid pET22b-PelB-SbPETase (2μL)
4. Mix the other two tubes of DH5α competent cell(50μL) with precooled recombinant plasmid pRSFDeut-1-kil(2μL)
5. Ice bath for 30 minutes
2. Heat shock
1. Heat shock at 42°C for 1 minute
2. Ice bath immediately for 2 minutes
3. Cultivation
1. Add 900μL LB medium to every bacteria tube
2. Mix the solution uniformly, and incubate it in the shaker at 37℃, 200rpm for 60 minutes
4. Incubation
1. Aspirate the transformed competent cells with one pipette
2. Coat DH5α with pET22b-PelB-SbPETase uniformly on the LB solid media containing Ampicillin
3. Coat DH5α with pRSFDeut-1-kil uniformly on the LB solid media with Kanamycin
4. Turn the dishes upside down and incubate the cells at 37℃ for 12-16 hours.

6.Plasmid
extraction

1. Column equilibrium
1. Insert one spin column into the collection tube.
2. Add 500μL buffer S to the tube.
3. Centrifuge the tube at 12,000rpm for 1 minute.
4. Discard the filtrate in the collection tube.
5. Insert the spin column back into the collection tube
2. Bacteria collection
1. Take 1.5-5mL overnight cultured bacteria into a centrifuge tube.
2. Centrifuge the tube at 12,000rmp for 1 minute and discard the supernatant.
3. Suspension of bacteria
1. Add 250μL buffer SP1
2. Use a vortex machine to entirely suspend bacteria
4. Open lipid bilayers
1. Add 250μL buffer SP2
2. Immediately turn it upside down 5-10 times gently to make the solution is uniformly mixed.
3. Keep it at room temperature for 2-4 minutes
5. Precipitation
1. Add 350μL buffer SP3
2. Immediately turn it upside down 5-10 times gently to make the solution is uniformly mixed.
3. Centrifuge the tube at 12,000rpm for 10min.
4. Collect the supernatant and transfer it to the spin column.
5. Centrifuge it at 12,000rpm for 30 seconds.
6. Discard the filtrate in the collection tube.
6. Washing
1. Add 500μL washing buffer to the spin column.
2. Centrifuge it at 12,000rpm for 30 seconds.
3. Discord the fluid in the collection tube.
4. Insert the spin column back into the collection tube.
5. Repeat steps 1-4 again
7. Elution
1. Centrifuge the empty spin tube again
2. Put the spin column into a clean micro-centrifuge tube.
3. Add 50-100μL elution buffer to the center of the spin membrane.
4. Place the tube at room temperature for 1 minute.
5. Centrifuge it at 12,000rpm for 1 minute.
6. Store the plasmid solution in a tube

7.Site-specific mutagenesis

1. PCR
1. Add recombinant plasmid(1μL), site-specific mutagenesis primers(2μL×2), enzyme(5μL), and ddH2O(10μL) to a PCR
tube.
2. Denaturation at 98℃ for 15 seconds
3. Annealing at 55℃ for 15 seconds
4. Extension at 72℃ for 1 minute
5. Repeat steps 2-4 for 35 times
2. Enzyme Digestion
1. Add DpnI buffer(5μL), DpnI(1μL) and ddH2O(13μL) to PCR tube
2. Water bath at 37℃ for 2 hours
3. Water bath at 65℃ to stop the reaction
3. Transformation
Same procedure as the transformation of vector and target gene to DH5α competent cell
4. Plasmid extraction
5. Transformation
1. Prepare a box of ice and operate all the following steps on the ice
2. Thaw 50μL×4 chemically competent cells BL21(DE3) on ice for 1-2 minutes.
3. Mix two tubes of BL21(DE3) competent cell with recombinant plasmid pET22b-PelB-SbPETase (2μL)
4. Mix the other two tubes of BL21(DE3) competent cell with recombinant plasmid pRSFDeut-1-kil(2μL)
5. Ice bath for 30 minutes
6. Heat shock
1. Heat shock at 42°C for 1 minute
2. Ice bath immediately for 2 minutes

8.Fermentation of recombinant strain and protein purification

1. Cultivation
1. Pick BL21(DE3) transformants and inoculate them in 20mL LB liquid medium containing Amp and Kana, incubated at
37℃, 200rpm overnight
2. Transfer 1% cultured medium to 2L LB (Amp and Kana)
2. Induction
1. When the OD600 of the cultured medium reaches 0.6, add 0.1mM IPTG to LB liquid medium
2. The culture is incubated at 18℃ and 200rpm for 16-18 hours
3. Buffer preparation
1. 50mL Buffer A: 50 mM Tris-HCI，300 mM NaCl，10 mM imidazole，pH7.6
2. 50mL Buffer B: 50 mM Tris-HCI，300 mM NaCl，250 mM imidazole，pH7.6
3. 50mL Buffer C: 50 mM Tris-HCI，300 mM NaCl，10% glycerin，pH7.6
4. Protein purification
1. Use buffer A to balance the Ni-NTA column
2. Centrifuge the bacteria culture medium at 4500rpm, 4℃ for 15 minutes
3. Add supernatant to balanced Ni-NTA column
4. Rinse unbound protein with buffer A
5. Elute target protein with buffer B
6. Store the target protein in Elution Buffer C
7. SDS-PAGE

9.Enzyme activity testing

This step aims to test the effect of SbPETase on PET degradation.
1. Mix 10µg/mL enzyme with 5mg PET granule under 30 ℃.
2. Proceeded to add 50mM Glycine buffer (pH 9.0) and centrifuge under 1000 rpm.
3. The system is heated to 85℃ and kept for 10min.
4. Use a microscope to observe the degradation effect.

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Results

1. Construction of pET22b-PelB-SbPETase and pRSFdeut-1-kil

To construct a high-efficiency secretion system in E. coli, we developed a method that combines signal peptide PelB
(pectate lyase B signal peptide) and colicin release protein Kil. In this project, extracellular PETase was achieved
by E. coli BL21(DE3) using a sec-dependent translocation signal peptide, pelB, for secretion, and the signal peptide
is removed by a signal peptidase. The colicin release protein Kil releases periplasmic protein by inducing membrane
solubilization; as a result, the recombinant proteins can be efficiently secreted into the culture medium when
co-expressed with Kil protein.
The DNA sequence of SbPETase was inserted into the BamHI and XhoI sites of the pET22b vector (Figure 1A). The gene
Kil was amplified by PCR from the plasmid containing the Kil gene and cloned into the NcoI and XhoI sites of the
pRSFduet1 vector (Figure 1B).

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results01.jpg)
Figure 1. Plasmid profiles in this project. A. pET22b-PelB-SbPETase, B. pRSFdeut-1-kil

We amplify SbPETase was amplified from the genome of S. brevitalea sp. nov (Figure2A.) and ligated it to the
double-enzyme digestion pET22b vector, and We amplify Kil by PCR from the plasmid containing the Kil gene
(Figure2B.), ligated to the double-enzyme digestion pRSFduet1 carrier.
The recombinant plasmids pET22b-PelB-Sbpetase and pRSFDeut1-Kil were 6240 bp and 3623bp in length. To verify if the
plasmid is correct, we digest plasmid pET22b-PelB-SbPETase with ApaLI and pRSFdeut-1-Kil with AseI (Figure 2C, E).
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing
comparison results showed that there were no mutations in the ORF region (Figure 2D, F.), and the plasmids were
successfully constructed. So far, we have successfully obtained the recombinant plasmids.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results02.jpg)
Figure 2. Gel electrophoresis results of target gene fragments and the sequencing results map to the
recombinant plasmids. A. The gene fragment of SbPETase, B. The gene fragment of Kil, C. digest plasmid
pET22b-PelB-SbPETase with ApaLI, D. the sequencing result of the recombinant plasmid pET22b-PelB-SbPETase, E.
digest plasmid pRSFdeut-1-Kil with AseI, D. the sequencing result of the recombinant plasmid
pRSFdeut-1-Kil.

2. Protein expression and purification

We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3),
expanded the culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After
overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the
intracellular proteins. Next, we used nickel column (Ni-NTA) purification to purify the protein SbPETase (Figure
3).We transferred the plasmid pET22b-pelB-SbPETase into E. coli BL21(DE3), expanded the culture in the LB medium and
added IPTG to induce protein expression when the OD600 reached 0.4. After overnight induction and culture, we
collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used
nickel column (Ni-NTA) purification to purify the protein SbPETase (Figure 3).

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results03.png)
Figure 3. SDS-PAGE result of SbPETasev

In order to test if Kil signal peptide can improve the yield of SbPETase proteins in the
culture medium, we also
co-transformed the recombinant plasmids pET22b-PelB-SbPETase and pRSFDeut1-Kil into E. coli BL21(DE3), expanded the
culture in the LB medium and added IPTG to induce protein expression when the OD600 reached 0.4. After overnight
induction and culture, we purified the protein SbPETase we wanted and collected the data (Figure 4). In Figure4,
when co\_transformed pET22b-PelB-SbPETase and pRSFDeut1-Kil, the yield of protein SbPETase in the extracellular
medium is higher than without it.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results04.png)
Figure 4. the yield of protein SbPETase both with and without Kil

3. Biochemical characterization of SbPETase

We set up an in vitro system and confirmed the ability to use the SbPETase to degrade PET
materials. As shown in Figure 4, we demonstrated that SbPETase could degrade PET and BHET into MHET and a small
quantity of TPA through HPLC (Figure 5A, B), the optimum conditions showed that at 30°C, pH 7.0 (BHET as substrate)
or pH 8.0 (PET film as substrate), SbPETase showed the highest activity.

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results05.png)
Figure 5. Two different HPLC experiments of SbPETase degrade PET and BHET materials. A. result recorded with
an HPLC system successfully identified the MHET and BHET peak when used BHET as substrate, B. result recorded with
an HPLC system successfully identified the MHET, BHET,and TPA peak when used PET as substrate.

4. Rational design of SbPETase by site-direct mutation

Due to the differences in substrate binding sites between SbPETase, and since reported
mutants showed improved catalytic efficiency, we performed the following site-directed mutagenesis: Y60A, L61T,
W132H, W132A, V181I, T212F, T212S, and R259A. All the SbPETase mutant proteins were obtained from the cultured
medium directly and an in vitro activity assay was performed, which generated two mutants (SbPETaseW132H,
SbPETaseR259A) with improved catalytic efficiency of degrading PET (Figure 6A). The activity of another mutant
SbPETaseL61T was only improved towards degrading BHET (Figure 6A). We then combined these three mutants
(SbPETaseW132H, SbPETaseR259A, SbPETaseL61T), to generate three double mutants and one triple mutant. An in vitro
activity assay showed that the triple mutant had the highest catalytic efficiency towards PET and BHET degradation
(Figure 6B)

![](https://static.igem.wiki/teams/4290/wiki/t-beijing-united-results06.jpg)
Figure 6. Comparison of the SbPETase mutants activity of degrading PET materials. A. detection of SbPETase
mutants enzyme activity when the substrate is BHET, B. A. detection of SbPETase mutants enzyme activity when the
substrate is PET

We biochemically characterized a PET-hydrolyzing SbPETase from
Schlegelella brevitalea sp. nov. using a high-efficiency secretion system. Overall, our study provides a foundation
for accelerating the discovery of novel PETase variants screening platforms for industrial applications.

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