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© 2025 BUCT-iGEM Team. All rights reserved.

Using synthetic biology to solve real-world problems

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# Engineering Success

### Contents

* [Engineering success](#Engineering-success)
* [Cycle A: Enzyme mining and optimization](#cycle-a)
* [Cycle B: Immobilized enzyme engineering](#cycle-b)
* [Cycle C: Monitoring platform engineering](#cycle-c)
* [Future prospective](#Future-prospective)

## Engineering success

![](https://static.igem.wiki/teams/5566/engineering/cf6a8c5490523d8bde7d7722e00aecb9.webp)

Fig. 1 The Design-Build-Test-Learn (DBTL) Cycle

To overcome the challenge of persistent polyester plastics in marine environments, we adopted a Design–Build–Test–Learn (DBTL) - based engineering strategy to iteratively optimize enzyme function, integration into materials, and whole-cell catalytic platforms. Through three cycles of rational enzyme engineering, immobilization integration, and dual-anchor monitoring platform, we successfully developed enzyme-embedded "active films" that activates the marine degradation of polyester plastics and a dual-anchor microbial monitoring platform system that exhibited significantly enhanced degradation performance compared to free enzyme systems. This demonstrates the viability of combining synthetic biology, materials science, and microbial design into a scalable, modular system for polyester biodegradation and microplastic remediation.

### ACycle A: Enzyme mining and optimization

#### Design

To address the serious persistence problem of polyester plastics (PET, PBAT, PLA) in the marine environment, we aim to explore and develop marine-derived ester hydrolases that can catalyze the cleavage of ester bonds in polyester plastics under marine conditions, and further optimize and transform the wild-type enzymes with the help of computer-assisted and multi-platform collaborative AI-driven methods.

#### Build

1. Thermophilic cutinase (TfCut), which exhibits significant activity against aliphatic-aromatic polyesters, was selected as a reference template. A BLASTP search was performed against the NCBI BLAST database using the TfCut amino acid sequence as a query.

2. The resulting sequences were screened against stringent thresholds (identity ≥ 35%, coverage ≥ 50%, and E-value ≤ 1.0E-58). To further optimize the sequences, we focused on sequences with ≥ 40% identity but only moderate coverage (35-60%) to identify homologous enzymes. Multiple sequence alignment (MSA) was performed on all results using MAFFT to confirm the conservation of essential catalytic motifs (Ser-His-Asp triplet and Gly-x-Ser-x-Gly). Phylogenetic analysis was performed on the conserved sequences, and evolutionary trees were constructed to reveal distinct clades associated with environmental origins, prioritizing enzymes from marine microorganisms naturally adapted to salinity conditions relevant to our application.

3. Based on the marine-derived screening results, dual computational platforms (DeepSoluE and Protein-sol) were used to predict the expression potential of each recombinant protein. Candidate enzymes that met both solubility thresholds (DeepSoluE > 0.48, Protein-sol > 55.00) were retained. Molecular docking and kinetic simulations with multiple substrates (PET, PBAT, PLA) were performed based on the results to identify candidate enzymes for experimental validation.

4. Expression systems were constructed for the candidate enzymes. Optimized gene sequences were obtained through the International Gene Synthesis Consortium (ISGC) and introduced into Escherichia coli BL21 (DE3) for heterologous expression. Proteins were purified using Ni-NTA affinity chromatography, and expression and protein content were verified by SDS-PAGE. Further basic data characterization was performed.

5. To address the common problem of low soluble expression levels of heterologous proteins, ProteinMPNN was used to enhance solubility. Low-energy variants were selected for structural alignment, and variants with active site distortion were eliminated. Variants with excellent structural preservation were selected for solubility prediction, and candidate enzymes that met the criteria were further validated experimentally and characterized based on baseline data.

6. To address the common problem of poor in vitro stability of wild-type enzymes, a multi-platform co-combination strategy was employed for the best-performing variants. Cross-analysis was performed using three independent stability prediction tools: PROSS (global skeleton optimization), FireProt (evolutionary conservation + energy calculation), and PoPMuSiC (machine learning + force field evaluation). Active site protection and evolutionary conservation were used to filter out high-confidence mutation sites that enhanced stability and retained function, which were then further validated and characterized experimentally.

#### Test

![](https://static.igem.wiki/teams/5566/engineering/02.webp)

Fig 2. Characterization of solubility, catalytic activity, salt tolerance, and thermostability data for wild-type enzyme PEH-2013 and variant SEQ4, as well as relative activity testing of the mutants of SEQ4. A. Protein MPNN prediction results and PEH-2013 enzyme SDS-PAGE images. B. Enzyme PEH-2013 enzymatic properties. a. Optimal reaction temperature. b. Salt tolerance. c. Tm. C. Enzyme SEQ4 enzymatic properties. a. Optimal reaction temperature. b. Salt tolerance. c. Tm. D. The relative activity of the mutans of SEQ4.

We measured the enzyme's solubility, catalytic activity, thermostability, and salt tolerance. Variant SEQ4 exhibited significantly enhanced soluble yield in E. coli. The variant exhibited comparable salt tolerance to the wild-type enzyme PEH-2013 and exhibited higher salt tolerance than the common ester hydrolases LCC and ICCG. In temperature-dependent activity testing using nano-PET, both wild-type PEH-013 and variant SEQ4 maintained catalytic activity over the 40-60°C range and exhibited improved thermostability (Tm approximately 58°C compared to approximately 42°C for the wild-type enzyme). Incremental consensus mutations were further evaluated using pNPB assays, identifying S210Y as the best-performing alternative, with an approximately 1.75-fold improvement in catalytic activity. Therefore, we named the variant SEQ4 (S210Y) as polygonase, as a product for further development and application in our project.

#### Learn

Rational computational engineering has effectively improved the solubility, thermal stability, and catalytic efficiency of engineered enzymes. However, despite these enhancements, the enzyme still exhibited limited tolerance to the extreme thermal and processing conditions encountered during polymer manufacturing and environmental exposure, constraining its direct industrial application.

#### Redesign

To overcome these limitations, we aim to further develop enzyme variants through industrial-focused engineering to further enhance their application in industrial processes. We aim to expand their functional scope by enhancing carrier protection and physically enhancing the environmental adaptability of the engineered enzymes. Furthermore, in the redesign of this experimental line, we also hope to further develop the enzymes from this project, promoting their industrial application through both biological functionality and engineering design.

### BCycle B: Immobilized enzyme engineering

#### Design

To overcome these limitations, we aim to further develop enzyme variants through industrial-focused engineering to further enhance their application in industrial processes. We aim to expand their functional scope by enhancing carrier protection and physically enhancing the environmental adaptability of the engineered enzymes. Furthermore, in the redesign of this experimental line, we also hope to further develop the enzymes from this project, promoting their industrial application through both biological functionality and engineering design.

#### Build

1. Using an inorganic nano-calcium carbonate-silica composite carrier, a low-cost and commonly used additive for polyester plastics, as the immobilization material, the sol-gel method was used to encapsulate CaCO₃ and SiO₂ precursors for synthesis. Thermal stability testing of the immobilized enzyme was conducted to develop an immobilized enzyme engineering solution.

2. Polyester plastic film mixing experiments were conducted with the developed immobilized enzyme to explore filming methods for carrying the immobilized enzyme, verify the distribution of the enzyme preparation in the film, and its impact on material properties.

3. Small-scale seawater simulation experiments were conducted on the films containing the immobilized enzyme to investigate their natural degradation properties. Scanning electron microscopy (SEM) was used to elucidate the degradation mechanism.

#### Test

![](https://static.igem.wiki/teams/5566/engineering/03.webp)

Fig3. Degradation performance of nano-calcium carbonate-silicon dioxide composite carrier and enzyme-containing plastic film. A. Thermal protection effect diagram of nano Calcium Carbonate – Silicon Dioxide composite carrier. B. Observation of enzyme containing PBAT plastic film with 1% fluorescein isothiocyanate (FITC) - labeled polygonase inorganic nano-calcium carbonate-silica complex enzyme preparation by confocal microscope. a) Surface of fluorescently labeled enzyme-containing PBAT plastic film, b) Edge of fluorescently labeled enzyme-containing PBAT plastic film, c) Cross-section of fluorescently labeled enzyme-containing PBAT plastic film. C. SEM analysis of surface morphology of enzyme-embedded polyester films after 30-day seawater degradation. (a, d) Control PBAT and PBAT/PLA (1: 1) films immobilized with nano Calcium Carbonate–Silicon Dioxide composite show smooth and intact surfaces with minimal physical damage. (b, e) PBAT and PBAT/PLA (1: 1) films immobilized with nano CaCO₃-SiO₂-immobilized polygonase display clear evidence of microcracks, pitting, and surface erosion. (c, f) PBAT and PBAT/PLA (1: 1) films exhibit more obvious roughness and interface delamination at a viewing angle of 10 μm. Scale bars: 10 μm (a, c, d, f), 100 μm (b, e). D. Comparison of plastic film degradation in seawater environment. a. PBAT - immobilized with polygonase. b. PBAT - immobilized with nano Calcium Carbonate – Silicon Dioxide composite. c. PBAT/PLA (1:1)-immobilized with polygonase. d. PBAT/PLA (1:1) - immobilized with nano Calcium Carbonate-Silicon Dioxide composite. All experiments were conducted at room temperature and seawater.

We evaluated the immobilized enzyme’s heat resilience, spatial distribution in polymer films, and degradation performance under seawater. Immobilized polygonase retained ~40% activity after 200 °C exposure (lost beyond 240 °C). FITC fluorescence imaging confirmed uniform enzyme embedding across film surfaces, edges, and cross-sections. After 30 days of degradation testing, SEM showed cracks, pores and erosion on the surface of the enzyme-embedded film, confirming its effective hydrolysis and degradation under simulated marine conditions. At the same time, in the simulated marine environment, the enzyme-containing film showed gradually deepening cracks, pore formation and surface roughening, achieving macroscopic degradation in 60 days, which was significantly different from the control group without enzyme.

#### Learn

Immobilization effectively enhanced enzyme thermal robustness, allowing compatibility with polymer processing. The films maintained structural uniformity and biodegradability, validating immobilization as a viable strategy for source-level degradable polyester materials. However, this system alone could not address existing environmental microplastic pollution, motivating further development of a biological microplastic remediation platform.

#### Redesign

In response to the persistent issue of polyester microplastics in the environment, our team aims to design and develop a biological detection device specifically targeting polyester microplastics. By integrating microplastic enrichment, enzymatic degradation, and quantitative detection of the degradation product terephthalic acid (TPA), this system seeks to establish a continuous biodegradation–monitoring platform.

### CCycle C: Monitoring platform engineering

#### Design

To target dispersed polyester microplastics in aquatic environments, we engineered a co-surface display system that combines surface adhesion and enzymatic degradation. Using INPNC–mfp-3 for polyester microplastics adhesion and SpyCatcher/SpyTag coupling for enzyme display, we aimed to achieve synergistic microplastic binding and catalytic hydrolysis on E. coli surfaces. Detection of terephthalic acid (TPA), a polyester degradation product, achieves the development of a monitoring platform.

#### Build

1. Using the SpyCatcher-SpyTag high-efficiency irreversible covalent protein binding system, SpyCatcher was fused to the highly efficient surface anchoring peptide Lpp-OmpA. The optimal variant was fused and covalently bound to SpyTag, and the adhesion protein mfp-3 was displayed using INPNC to construct a surface display anchoring system.

2. A dual-plasmid system was constructed for the surface display system to avoid competition between the protein components. Dual resistance was verified in E. coli BL21 (DE3) by replacing the resistance vector. The commonly used strong constitutive lpp1.2 promoter was replaced in the INPNC-mfp-3 system to achieve low competition and high expression.

3. Fluorescent proteins (sfGFP and mCherry) were expressed instead of the display proteins. Surface display and colocalization of the two anchoring systems were verified using fluorescence microscopy and confocal laser scanning imaging.

4. The adhesion effect of the adhesion protein mfp-3 was verified by conducting adhesion experiments on bacteria in the adhesion system and plastic films; the efficiency of the co-display system was verified by comparing the catalytic activity of inactivated cells with that of co-expression and single-system systems.

5. A standard curve of TPA concentration-fluorescence intensity was established for terephthalic acid (TPA), a characteristic degradation product of PET/PBAT, utilizing its ability to generate a fluorescent signal when reacting with hydrogen peroxide. A chemiluminescence detection method was developed to quantitatively assess degradation efficiency and microplastic contamination levels.

#### Test

![](https://static.igem.wiki/teams/5566/engineering/04.webp)

Fig4. Functional verification of the detection system. A. Adhesion protein MFP-3 adhesion assay results. B. Fluorescence microscopy characterization of surface protein display effects. a. pETlac-Plpp1.2-INPNC-mCherry surface display effect. b. pET28a (+)-Lpp-OmpA-SpyC/SpyT-polygonase surface display effect. c. Surface co-display system effect. From left to right, the fields of view are bright field, red fluorescence channel, green fluorescence channel, and merged image. C. Comparison of nano-PET degradation rate (%) for three E. coli cell systems and free polygonase. D. Standard curve of TPA concentration and fluorescence intensity within 1 min reaction time. The slope of the curve was 30759.251 ± 9571.638, the intercept was 4407384.784 ± 155889.044, and the R² = 0.99813.

We engineered an Escherichia coli strain with an adhesion module (INPNC-mfp-3) and a catalytic module (SpyC/SpyT-polygonase) and verified their specific functions. First, we verified the function of adhesion proteins. Adhesion assays showed that mfp-3 display significantly enhanced bacterial binding to polyester substrates. Then we validated localization via mCherry and sfGFP fluorescence imaging. Nano-PET degradation tests over 6 hours revealed that the dual-anchor strain produced approximately 2.7 times more TPA than single-anchor or free-enzyme controls. And the developed fluorescence-based hydroxyl radical oxidation assay provided sensitive and linear quantification of TPA (R² = 0.998), enabling accurate performance comparison among systems.

#### Learn

Spatial co-localization of adhesion and catalytic modules effectively enhanced substrate accessibility and degradation efficiency. The dual-anchor system established a foundation for developing an autonomous, self-sustaining biological platform for microplastic degradation and monitoring.

#### Redesign

While the detection platform has achieved some success and significantly improved upon the more common HPLC method, its use remains laboratory-based and presents limitations for environmental applications. Our next redesign will focus on industrial scalability and environmental deployment. At this stage, our goal is to develop a product biosensor and design an integrated whole-cell degradation platform system, bridging the gap between laboratory results and practical applications.

## Future prospective

Building on our DBTL engineering framework, future work will integrate biosensing circuits into the co-surface display system to construct a self-regulating whole-cell platform capable of detecting, degrading, and reporting microplastic pollution. On a larger scale, immobilized hydrolase films provide a source-level for polyester plastics and whole-cell biocatalysts could be adapted into microplastic filtration membranes and bioreactors for continuous water remediation. This project thus lays the groundwork for intelligent, sustainable biotechnological applications in environmental protection and green material innovation.

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© 2025 BUCT-iGEM Team. All rights reserved.

Using synthetic biology to solve real-world problems

---

© 2025 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).

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# Experiments

### Contents

* [1. Primer Design](#primer-design)
* [2. Recombinant Plasmid Construction](#recombinant-plasmid)
* [3. Expression of the Target Protein](#protein-expression)
* [4. Protein Purification](#protein-purification)
* [5. Various Parameter Tests](#parameter-tests)
* [6. Calcium Carbonate-Silicon Dioxide Composite Immobilization](#immobilization)

## 1. Primer Design

Retrieve the target gene sequence from databases such as NCBI. Follow these principles for primer design:

1. Primer length is typically 20–30 bp, with 25 bp recommended.
2. Primer GC content should be maintained between 40 and 60%, with a Tm value around 60°C.
3. For genes with high GC content (>60%), use a DNA polymerase with GC buffer.
4. The Tm values of forward and reverse primers should be as close as possible.
5. Avoid complementarity of more than 4 bases between forward and reverse primers to prevent primer dimer formation.
6. Include protective bases (GGGAAA) and a restriction enzyme site at the 5′ end.
7. When designing the reverse primer, replace the stop codons TAG or TGA with TAA to reduce methylation effects (G and C are prone to methylation).
8. Select restriction enzyme sites that are not present in the target gene. Prefer sites with sticky ends for easier ligation (e.g., KpnI, BamHI, XbaI, NdeI, SalI, HindIII, NheI). Avoid isoschizomers.
9. When using the pETduet-1 vector for plasmid construction, ensure the start codon (AT) is removed from the forward primer to prevent frameshift mutations.

## 2. Recombinant Plasmid Construction

### Plasmid Extraction

1. Streak the glycerol-preserved bacterial strain containing the plasmid onto a plate, pick a single fresh colony, and inoculate into 4 mL of LB medium with the appropriate antibiotic. Incubate at 37°C for 12–16 hours.
2. Column equilibration: Add 500 μL of equilibration buffer BL to the CP3 adsorption column (placed in a collection tube). Centrifuge at 12,000 rpm for 1 minute, discard the flow-through, and return the column to the collection tube.
3. Transfer 4 mL of overnight culture to a centrifuge tube. Centrifuge at 12,000 rpm for 1 minute and carefully remove the supernatant.
4. Resuspend the bacterial pellet in 250 μL of solution P1 (containing RNase A) using a pipette or vortex mixer.
5. Add 250 μL of solution P2 and mix gently by inverting the tube 6–8 times for complete lysis. The solution should become clear and viscous. Do not exceed 5 minutes.
6. Add 350 μL of solution P3 and mix immediately by inverting 6–8 times. Centrifuge at 12,000 rpm for 10 minutes.
7. Transfer the supernatant to the CP3 adsorption column. Centrifuge at 12,000 rpm for 30–60 seconds, discard the flow-through, and return the column to the collection tube.
8. Add 600 μL of wash buffer PW (containing ethanol) to the column. Centrifuge at 12,000 rpm for 30–60 seconds, discard the flow-through, and return the column to the collection tube.
9. Repeat the wash step.
10. Place the column back into the collection tube and centrifuge at 12,000 rpm for 2 minutes. Open the column and air-dry the membrane using a cold blow-dryer to remove residual ethanol.
11. Transfer the column to a clean collection tube. Apply 30 μL of ddH₂O (preheated to 50°C) to the center of the membrane. Incubate at 50°C for 5 minutes, then centrifuge at 12,000 rpm for 2 minutes to collect the plasmid DNA.
12. Measure the DNA concentration using a spectrophotometer.

### PCR

Prepare a 50 μL reaction mixture in a PCR tube:

| Component | Volume |
| --- | --- |
| ddH₂O | 20 μL |
| Upstream primer | 2 μL |
| Downstream primer | 2 μL |
| Template | 1 μL |
| Prime STAR Max | 25 μL |

PCR Thermal Cycling Condition:

| Step | Temperature | Duration | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98°C | 30 s | 1 |
| Denaturation | 98°C | 10 s | 32 |
| Annealing | 56°C | 15 s | 32 |
| Extension | 72°C | 1 kb /5 s | 32 |
| Final extension | 72°C | 5 min | 1 |
| Hold | 4°C | ∞ | 1 |

### Agarose Gel Electrophoresis

50X TAE buffer:

| Component | Weight or volume |
| --- | --- |
| Tris | 242 g |
| EDTA | 18.6 g |
| Acetic acid | 57.1 ml |
| Deionized water | 800 ml |

After adjusting the pH to 8.3 with NaOH, add deionized water to make the volume up to 1L and store it at room temperature. Dilute with deionized water to 1X before use.

1. Prepare 50X electrophoresis buffer and dilute to 1X with deionized water.
2. Weigh 1 g of agarose into a conical flask, add 100 mL of 1X electrophoresis buffer, and mix well. Heat in a microwave until clear and transparent (approximately 3 minutes).
3. Pour the solution into a gel tray, add 1 μL of EB solution, mix thoroughly, and allow to solidify for 20 minutes.
4. Place the gel in the electrophoresis tank and add 1X buffer until the gel is submerged. Gently shake the gel to remove bubbles from the wells.
5. Mix the samples with an appropriate volume of 6X loading buffer and carefully load into the wells.
6. Select a suitable DNA ladder based on fragment size. For standard wells (50 μL), load 5 μL of ladder; for small wells (10 μL), load 2 μL. Use one lane for small gels and two lanes for large gels.
7. Set the voltage to 180 V and start electrophoresis.
8. After approximately 15 minutes (when the bromophenol blue band reaches the middle of the gel), stop electrophoresis and analyze the gel using a gel imaging system.

### Gel DNA Extraction

1. After electrophoresis, excise the target DNA band under UV light. Minimize the gel volume and transfer to a 2 mL EP tube.
2. Add an appropriate volume of Binding Buffer XP2 (typically 100 μL per 0.1 g gel) to dissolve the gel and enhance DNA binding to the column.
3. Incubate the tube at 65°C, shaking every 2 minutes until the gel is completely dissolved.
4. Transfer the solution to a HiBind DNA column placed in a 2 mL collection tube. Let stand for 3 minutes, then centrifuge at 12,000 rpm for 1 minute.
5. Reload the flow-through into the column, incubate for 1 minute, and centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.
6. Add 700 μL of SPW Wash Buffer to the column and centrifuge at 12,000 rpm for 1 minute. Discard the flow-through.
7. Repeat the wash step.
8. Centrifuge the column at 12,000 rpm for 3 minutes to dry. Discard the collection tube and air-dry the column with a cold blow-dryer until no ethanol smell remains.
9. Add 30 μL of preheated ddH₂O to the center of the membrane. Place the column in a 1.5 mL EP tube and incubate for 3–5 minutes. Centrifuge at 12,000 rpm for 5 minutes to collect the eluate.
10. Verify the extraction by running 2 μL of the eluate on a gel.

### Recombinant Plasmid and Chemical Transformation Method

1. Prepare a 10 μL ligation reaction system. The insert-to-vector ratio is determined by calculation: the insert volume is calculated as insert length × 0.04 / c, and the vector volume as insert length × 0.02 / c. The combined volume of insert and vector should be 5 μL (adjusted with ddH₂O if necessary). Add 5 μL of ligase to reach a final reaction volume of 10 μL.
2. React in a 50°C water bath for 30 minutes, then place on ice for 2 minutes.
3. In a sterile environment, add 10 μL of the recombinant plasmid to 50 μL of DH5α competent cells. Incubate on ice for 30 minutes.
4. Heat-shock at 42°C for 90 seconds, then immediately place on ice for 2 minutes. Add 600 μL of sterile LB medium and incubate at 37°C with 200 rpm for 1 hour.
5. Centrifuge at 6,000 rpm for 1 minutes, remove most of the supernatant leaving approximately 100 μL, and resuspend the pellet to concentrate. Spread onto LB solid medium plates with the appropriate antibiotic. Incubate at 37°C for 12 hours.

### Electroporation Method

#### Preparation of electrocompetent cells

1. Streak the preserved bacterial culture from -80°C onto an LB plate without antibiotics using a sterile loop. Incubate at 37°C overnight.
2. Pick a single colony and inoculate it into 4 mL of LB medium without antibiotics. Incubate at 37°C with shaking at 220 rpm for 8 hours.
3. Transfer 1 mL of the culture to 50 mL of LB medium. Incubate at 37°C with shaking at 220 rpm for 1.5 – 2 hours until OD600 reaches approximately 0.6.
4. Pour the culture into a sterile 50 mL centrifuge tube. Centrifuge at 6,000 rpm at 4°C for 5 minutes. For a 100 mL culture, use two tubes.
5. Discard the supernatant and add 20 mL of sterile 10% glycerol (W/V). Resuspend and centrifuge at 6,000 rpm at 4°C for 10 minutes. Repeat twice.
6. Resuspend each pellet in 300 μL of 10% glycerol. Adjust the volume based on bacterial density.
7. Aliquot 90 μL into 1.5 mL centrifuge tubes.

Use a BIO-RAD electroporator to transfer the verified recombinant plasmid into competent cells:

1. Place a cleaned and UV-sterilized 1 mm electroporation cuvette on ice.
2. Thaw the prepared competent cells on ice, add 1–5 μL of recombinant plasmid, mix gently, and transfer to the cuvette.
3. Set the electroporation parameters, ensure the cuvette is dry, and deliver the pulse.
4. Immediately add 600 μL of sterile LB medium to the cuvette, mix thoroughly, and transfer to a 1.5 mL centrifuge tube. Incubate at 37°C for 45–60 minutes for recovery.
5. Spread 200 μL of the culture onto a selective solid medium with the appropriate antibiotic. Incubate inverted at 37°C for approximately 10 hours.
6. Pick 3 single colonies of appropriate size from the selective medium for subsequent experiments.
7. Mix the bacterial culture with an equal volume of glycerol in a cryovial and store at -20°C.

### Colony PCR

Pick 4–6 single colonies from the antibiotic plate and resuspend in 10 μL of ultra-pure water as a template.

Prepare a 20 μL reaction system in a PCR tube:

| Component | Volume |
| --- | --- |
| ddH₂O | 7 μL |
| Upstream primer | 1 μL |
| Downstream primer | 1 μL |
| Template | 1 μL |
| Taq | 10 μL |

PCR Run Protocol:

| Step | Temperature | Duration | Cycles |
| --- | --- | --- | --- |
| Initial denaturation | 98°C | 5 min | 1 |
| Denaturation | 98°C | 10 s | 29 |
| Annealing | 56°C | 30 s | 29 |
| Extension | 72°C | 1 kb/5 s | 29 |
| Final extension | 72°C | 10 min | 1 |
| Hold | 4°C | ∞ | 1 |

Verify by agarose gel electrophoresis and send the correct plasmid for sequencing.

## 3. Expression of the Target Protein

### LB Medium Preparation

2.5 g/100ml LB Medium (per liter)

* LB Medium Powder 25 g
* For solid medium, add 2g agar per 100 ml

### Protein Expression

1. Inoculate the verified bacterial culture into 5 mL of LB medium, culture, and extract the plasmid.
2. Thaw BL21(DE3) competent cells on ice.
3. Add 10 μL of plasmid to 100 μL of competent cells in a sterile environment and incubate on ice for 30 minutes.
4. Heat-shock at 42°C for 45 seconds, then place on ice for 2 minutes.
5. Add 500 μL of sterile LB medium and recover at 37°C with shaking for 1 hour.
6. Centrifuge at 6,000 rpm for 5 minutes.
7. Remove most supernatant, leaving approximately 100 μL, resuspend to concentrate, and spread onto LB solid medium with the appropriate antibiotic (ampicillin sodium for FastPETase, kanamycin sulfate for HotPETase, and ampicillin sodium for FastPETase-M4). Incubate at 37°C for 12 hours.
8. Pick a single colony and inoculate it into 10 mL of sterile LB medium with the appropriate antibiotic. Incubate at 37°C for 12 hours until OD600 reaches approximately 0.6.
9. Transfer the entire culture to 1 L of sterile LB medium with the appropriate antibiotic. Incubate at 37°C with shaking until OD600 reaches the appropriate density (approximately 6–8 hours).
10. Add 1 mL of IPTG solution and induce at 18°C with shaking for 18–20 hours.

## 4. Protein Purification

### Lysis Buffer (1L)

| Component | Weight or volume |
| --- | --- |
| Tris | 6.06 g |
| NaCl | 17.53 g |
| Imidazole | 0.68 g |
| ddH₂O | 900 ml |

Dilute with HCl to pH 8.0, then make up to 1 liter with distilled water.

### Wash Buffer System (1L)

| Different imidazole concentrations of Wash Buffer | 30 mM | 50 mM | 300 mM | 500 mM |
| --- | --- | --- | --- | --- |
| NaCl | 17.53 g | 17.53 g | 17.53 g | 17.53 g |
| Tris | 6.06 g | 6.06 g | 6.06 g | 6.06 g |
| Imidazole | 2.06 g | 3.44 g | 20.58 g | 34.38 g |
| ddH₂O | Dilute to a total volume of 1 liter | | | |

1. Centrifuge the induced culture at 4,000 rpm for 40 minutes and discard the supernatant.
2. Resuspend the pellet in a small volume of 10 mM imidazole buffer and disrupt the cells using a high-pressure homogenizer (800 MPa for 10 minutes) to release the target protein.
3. Centrifuge the lysate at 12,000 rpm for 40 minutes, filter the supernatant, and apply to a Ni-NTA agarose 6FF column. Rotate at 4°C for 30 minutes for binding.
4. Wash the column with 50 mM imidazole buffer until the flow-through no longer changes the color of Coomassie Brilliant Blue G-250.
5. Elute the target protein with 300 mM imidazole buffer until the eluate no longer changes the color of Coomassie Brilliant Blue G-250.
6. Clean the column with 500 mM imidazole buffer and store in 20% ethanol at 4°C.
7. Concentrate the eluate using a 10 kDa ultrafiltration tube at 4°C and 4,000 rpm. Exchange the buffer and concentrate again.
8. Prepare an SDS-PAGE gel using a 12% pre-cast gel kit. Prepare the sample by mixing 4 μL of 5X SDS loading buffer with 16 μL of concentrated protein and heating at 100°C for denaturation. Load the sample and an 180 kDa Plus Prestained Protein Marker. Run at 150 V for 1 hour.
9. Stain the gel with Feto SDS-PAGE staining buffer for 3 minutes with shaking, destain with deionized water for 5 minutes, and image using a gel documentation system. Verify the molecular weight and purity by SDS-PAGE.
10. Measure the absorbance at 280 nm using a NanoDrop One. Calculate the extinction coefficient by entering the amino acid sequence into the NovoPro online tool (https://www.novopro.cn/tools/) and determine the protein concentration using the Beer-Lambert law.

### BCA Protein Quantification Method

1. Prepare the BCA working solution by mixing reagents A and B at a 50:1 ratio based on the number of samples (200 μL per sample). For example, for 10 samples, mix 2000 μL of A with 40 μL of B.
2. Prepare protein standards by diluting a 5 g/L stock solution 10-fold with PBS to obtain 0.5 g/L standards. Prepare a dilution series for the standard curve.
3. Add PBS and standards to a 96-well plate according to the table. For samples, dilute accordingly (e.g., for 10-fold dilution, add 18 μL PBS and 2 μL sample; for 20-fold, add 19 μL PBS and 1 μL sample). Add 200 μL of BCA working solution to each well (prepare fresh).
4. Incubate the plate at 37°C for 45 minutes or at room temperature for 2 hours. A purple color will develop, intensifying with protein concentration.
5. Measure the absorbance at 562 nm using a microplate reader. Plot the standard curve with protein concentration on the x-axis and absorbance on the y-axis. Calculate the sample protein concentration.

BCA protein concentration Standard curve:

| Number | PBS/μL | Protein standard /μL | Concentration(g/L) |
| --- | --- | --- | --- |
| A | 20 | 0 | 0 |
| B | 19 | 1 | 0.025 |
| C | 18 | 2 | 0.05 |
| D | 16 | 4 | 0.1 |
| E | 12 | 8 | 0.2 |
| F | 8 | 12 | 0.3 |
| G | 4 | 16 | 0.4 |
| H | 0 | 20 | 0.5 |

### SDS-Polyacrylamide Gel Electrophoresis

10% SDS preparation system:

| Component | Weight |
| --- | --- |
| SDS | 10 g |
| ddH₂O | 100 ml |

Dilute ddH₂O to 100ml and adjust the pH to 7.2 with concentrated hydrochloric acid.

10% APS preparation system:

| Component | Weight |
| --- | --- |
| APS | 0.1 g |
| ddH₂O | 1 ml |

Dilute ddH₂O to 1 ml, store at 4°C, and prepare as needed.

10X electrophoresis buffer preparation system:

| Component | Weight |
| --- | --- |
| Tris | 30.3 g |
| Glycine | 144.2 g |
| SDS | 10 g |

Dissolve H₂O to a total volume of 1000 ml, and store at room temperature.

Dyeing solution preparation system:

| Component | Weight |
| --- | --- |
| Coomassie Brilliant Blue R-250 | 1 g |
| Methanol | 450 ml |
| ddH₂O | 100 ml |
| Glacial acetic acid | 450 ml |

Bleaching liquid configuration system:

| Component | Weight |
| --- | --- |
| Methanol | 100 ml |
| ddH₂O | 100 ml |
| Acetic acid | 800 ml |

A liquid preparation system:

| Component | Weight |
| --- | --- |
| Monomer acrylamide | 29 g |
| Methyl acrylate dimethacrylamide | 0.8 g |
| ddH₂O | 100 ml |

After filtration, store at 4°C in the dark.

B liquid configuration system:

| Component | Weight |
| --- | --- |
| Tris | 18.17 g |
| 10% SDS | 4 ml |
| ddH₂O | 100 ml |

Adjust the pH to 8.8 with concentrated hydrochloric acid and store it in a dark place at 4°C.

C liquid configuration system:

| Component | Weight |
| --- | --- |
| Tris | 6.06 g |
| 10% SDS | 4 ml |
| ddH₂O | 100 ml |

Adjust the pH to 6.8 with concentrated hydrochloric acid and store it in a dark place at 4°C.

Gel configuration system:

| Component | 8% | 9% | 10% | 12% | 15% | Concentrated glue |
| --- | --- | --- | --- | --- | --- | --- |
| ddH₂O/ml | 2.4 | 2.25 | 2.1 | 1.75 | 1.25 | 1.45 |
| B solution/ml | 1.25 | 1.25 | 1.25 | 1.25 | 1.25 | C solution 0.625 |
| A solution/ml | 1.35 | 1.5 | 1.65 | 2 | 2.5 | 0.42 |
| 10%APS/ul | 50 | 50 | 50 | 50 | 50 | 30 |
| TEMED/ul | 8 | 8 | 8 | 8 | 8 | 5 |
| Separation range/Kda | 30-120 | 30-90 | 18-75 | 15-60 | 15-45 |  |

1. Prepare glass plates by cleaning and assembling them (ensure the frosted sides and bottoms are aligned). Test for leaks by filling with water and waiting 3–10 minutes.
2. Prepare samples by mixing with 5X loading buffer (containing mercaptoethanol). Denature by boiling for 10 minutes, cool, and centrifuge to collect supernatant.
3. Prepare separating gel based on the target protein size (higher concentration for smaller proteins). Quickly pour into the gel assembly (approximately 3.2 mL). Overlay with isopropanol and let polymerize for 10–30 minutes. Remove isopropanol and dry with filter paper.
4. Prepare stacking gel and pour over the separating gel. Insert a comb without bubbles and add excess gel around the comb. Polymerize for 20–30 minutes.
5. After polymerization, place the gel in the electrophoresis tank and fill with buffer. Carefully remove the comb and load 10–20 μL of sample per well. Include a protein ladder in one well.
6. Connect the tank to the power supply (red to red, black to black). Run at 220 V until the bromophenol blue reaches approximately 1 cm from the bottom (approximately 45 minutes).
7. Carefully disassemble the gel, remove the stacking gel, and place the gel in a staining box. Cover with Coomassie Brilliant Blue stain.
8. Stain at room temperature with gentle shaking (<80 rpm) for 20 minutes. Destain with destaining solution until the background is clear. Image the gel using a pre-warmed gel documentation system.

## 5. Various Parameter Tests

### Enzyme Activity Assay

#### Enzyme - substrate reaction

1. Add 5 mg of PLA or PBAT plastic powder to 5 mL EP tubes. Add 1 mL of seawater or PB buffer and an appropriate concentration of free enzyme. Incubate at a temperature gradient of 30–70°C. Include triplicates and a blank (70 tubes total).
2. After 10 and 20 hours, measure PBAT degradation using a NanoDrop and PLA degradation using HPLC.

#### UV-Vis Spectrophotometer:

1. Measure the concentration of PBAT hydrolysis products at 240 nm using a UV-Vis spectrophotometer.
2. The products are identified as TPA analogs with a molar extinction coefficient of 17,000 M⁻¹cm⁻¹ and expressed as TPA equivalents (TPAeq).

#### TPA Standard Curve

1. Mix 700 μL of gradient TPA solutions, 100 μL of 100 mM PB (pH 6.15), 100 μL of 1 mM ammonium ferrous sulfate, and finally 100 μL of 3% hydrogen peroxide. Record data 3 minutes after adding hydrogen peroxide using a fluorescence spectrophotometer.
2. Plot the TPA standard curve using Origin software.

## 6. Calcium Carbonate-Silicon Dioxide Composite Immobilization

1. Prepare 0.15 M calcium chloride and sodium carbonate solutions.
2. Calculate the volume of enzyme solution to add for a final protein concentration of 1 mg/mL.
3. Add the enzyme solution to 20 mL of calcium chloride solution. Rapidly add 20 mL of sodium carbonate solution with vigorous stirring. Continue stirring for 20–30 minutes.
4. Let stand at 4°C for 1–2 hours. Centrifuge at 8,000 rpm for 10 minutes and collect the supernatant.
5. Wash the precipitate 2–3 times with pre-chilled Tris-HCl.
6. Prepare a solution with Tris-HCl buffer and ammonia water (approximately 0.4 M, pH 8–9).
7. Resuspend the calcium carbonate in the above solution. Slowly add tetraethyl orthosilicate (TEOS) solution with gentle stirring. Use two groups: one with 1X and the other with 3X TEOS relative to calcium carbonate.
8. React at room temperature with gentle stirring for 6–24 hours.
9. After the reaction, let it age for 1 hour. Wash 3 times with Tris-HCl buffer by centrifugation.
10. Lyophilization.

### Thermal Stability Testing

1. Place 10 mg of freeze-dried powder in glass tubes, flush with nitrogen, seal, and heat at 160°C, 180°C, 200°C, 220°C, 240°C, 260°C, 280°C, and 300°C for 3 minutes.
2. Add 2.5 mL of 50 mM PB and 1667 μL of 200 mM EDTA for chelation. Shake and let stand at room temperature until the precipitate dissolves.
3. Prepare reaction buffer: 1.4 mL PB + 1 mL surfactant for a 1.5% concentration. Prepare substrate: 1 mL acetone + 4 μL PNPB.
4. Clean cuvettes with alcohol and water 3 times. Add 900 μL reaction buffer and 10 μL chelated enzyme solution. Finally, add 100 μL of substrate and measure using a spectrophotometer.
5. Determine the enzyme concentration in the chelated solution using the BCA method. Calculate the reaction rate per unit enzyme concentration based on spectrophotometric data.

### Film Pressing and Degradation

#### Film Pressing

1. Weigh 1 g of dried PBAT and PLA at ratios of 1:1 and 3:7. Also weigh 1 g of pure PBAT. Add immobilized enzyme at 5% or 3% to each group.
2. Preheat the powder in the film press for 1 minute. Press at 30 kPa for 1.5 minutes. Heat PBAT to 160°C and the PLA/PBAT mixture to 180°C.

#### Degradation Demonstration

Place 3 cm × 3 cm pressed plastic films in seawater and simulated seawater (50 mM PB pH 8 with NaCl added to seawater salinity). Record changes daily.

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# Results

### Contents

* [Introduction](#Introduction)
* [SectionⅠ-Enzyme mining and optimization](#Section-1)
* [SectionⅡ - Immobilization](#Section-2)
* [SectionⅢ - Lab stage degradation platform](#Section-3)
* [Prospective](#Prospective)

## Introduction

This project aims to systematically discover and engineer marine-derived ester hydrolases to address the persistent retention of aliphatic-aromatic polyester plastics (e.g., PET/PLA/PBAT) in the ocean. Combining marine enzyme discovery, computational protein redesign, inorganic immobilization, and cell-based enrichment/degradation modules, we discovered and biochemically characterized a marine polyester hydrolase (PEH-2013). Using ProteinMPNN and AI-driven mutagenesis, we redesigned it to produce a soluble, active variant, polygonase. To address the vulnerability of free enzymes to inactivation during industrial processing and environmental exposure, we developed an enzyme immobilization formulation based on calcium carbonate-silicon dioxide (CaCO₃-SiO₂) composite nanocarriers, balancing process compatibility with environmentally triggered release. Furthermore, we constructed a system for co-displaying an adhesion module and an engineered hydrolase on the surface of Escherichia coli (adhesion + SpyTag/SpyCatcher-tethered hydrolase) to enhance the accumulation and in situ degradation of polyester microplastics in water. To quantitatively assess the degradation process, a chemiluminescent detection method for the key intermediate terephthalic acid (TPA) was established. In the future, efforts will be made to design and develop an intelligent whole-cell biosensor by integrating degradation and detection capabilities using synthetic biology tools, moving toward the construction of an autonomous, controllable, and responsive bioremediation system for plastic pollution.

## SectionⅠ-Enzyme mining and optimization

### Enzyme mining

To identify promising marine-derived ester hydrolases, we used the highly efficient cutinase TfCut as a template and conducted homology mining using NCBI BLAST. We successfully identified two candidate enzymes originating from marine environments and named them PEH-AM and PEH-2013. [*For details, please check the page of MODEL-ENZYME MINING*](https://2025.igem.wiki/buct/model#ENZYME-MINING).

Based on the computer-assisted screening results, we synthesized genes through the International Gene Synthesis Consortium (IGSC) and cloned the selected sequences into the pET-28a(+) vector with an N-terminal His-tag. We heterologously expressed the two target proteins in Escherichia coli BL21 (DE3) and purified them by Ni-NTA chromatography. The results were then verified by SDS-PAGE gel electrophoresis (Fig. 1).

![](https://static.igem.wiki/teams/5566/results/resultnew/10010.webp)

Fig1. Protein precipitation, supernatant, enzyme SDS-PAGE images of enzyme PEH-AM, PEH- 2013

Theoretical molecular weight calculations indicate that the expected molecular weights of PEH-AM and PEH-2013 are 29.41 kDa and 32.71 kDa, respectively. In the SDS-PAGE analysis of the whole protein lysate, pellet, and supernatant samples in Fig. 1, a distinctly darker band, corresponding to the two target proteins, appears in the 25–35 kDa range, highly consistent with the expected size. This band is significantly enriched in both the soluble fraction and Ni-NTA elution/enrichment samples, providing preliminary evidence that PEH-AM and PEH-2013 were successfully expressed in E. coli BL21(DE3) and recovered in a soluble form.

SDS-PAGE analysis revealed that all eluted protein supernatants contained only a small fraction of the target enzyme, along with significant amounts of protein impurities and nonspecific contaminants. This suggests that the soluble expression levels of both PEH-AM and PEH-2013 in the E. coli system were generally low. In particular, even after scaling up PEH-AM to 10 L, its yield (A280 = 0.097) was still significantly lower than that of PEH-2013 (A280 = 1.256). Therefore, we discarded PEH-AM and focused our further analysis on PEH-2013.

After confirming the expression ability of the enzyme PEH-2013, we further evaluated its catalytic performance on actual polyester substrates. We first evaluated the degradation performance of the newly discovered enzyme PEH-2013 on PET substrates with varying degrees of crystallinity. We measured the depolymerization activity of PEH-2013 on PET with varying degrees of crystallinity (LC-PET and HC-PET) under standard reaction conditions (50°C, 24 hours, 500 nM enzyme loading) (Fig. 2).

![](https://static.igem.wiki/teams/5566/results/resultnew/10020.webp)

Fig2. Degradation analysis of PET with different crystallinity

The results showed that PEH-2013 exhibited significant hydrolytic activity against PET, effectively degrading it to form terephthalic acid (TPA). Its efficiency was significantly affected by the crystallinity of the PET. The concentration of TPA in the final product, LC-PET, was significantly higher (Fig. 2), indicating that the amorphous regions were more accessible to the enzyme. This result confirms that PEH-2013 is an active PET-degrading enzyme, and its catalytic efficiency is largely dependent on the crystallinity of the substrate.

To further characterize the enzymatic properties and environmental adaptability of PEH-2013, we systematically determined its optimal reaction temperature, salt tolerance, and thermal stability.

![](https://static.igem.wiki/teams/5566/results/resultnew/10030.webp)

Fig3. Enzyme PEH-2013 enzymatic properties. a. Optimal reaction temperature. b. Salt tolerance. c. Tm.

Using nano-PET as the substrate, the effect of temperature gradient on the enzymatic activity of the enzyme PEH-2013 was investigated. Reactions were carried out at 50 °C for 3 hours with 6 mg/L nano-PET and an enzyme concentration of 500 nM. Results showed that PEH-2013 exhibited catalytic activity across the temperature range of 40°C to 60°C, reaching a peak at 50°C, confirming its optimal reaction temperature (Fig. 3a). This temperature is consistent with the properties of many industrial enzymes derived from mesophilic microorganisms. Given that PEH-2013 originates from a marine environment, we evaluated the effects of various NaCl concentrations on its activity. Comparisons with common PET hydrolases, LCC and ICCG, revealed that PEH-2013 exhibited significant salt tolerance compared to common PET hydrolases (Fig. 3b). To further investigate its thermal stability, we determined its melting temperature using a fluorescent dye-based protein thermostability assay. The Tm value was found to be approximately 42°C, indicating that PEH-2013 maintains its structural integrity at temperatures between 40 and 50°C, suggesting potential applications at slightly higher temperatures (Fig. 3c).

After defining the basic enzymatic properties of PEH-2013, we evaluated its hydrolysis performance against various polyester substrates under gradient temperature conditions to examine its broad-spectrum degradation potential. The experimental conditions were as follows: 500 nM of the purified enzyme was added to a reaction system containing 5 mg·mL⁻¹ of polyester powder (PET, PBAT, PLA). The reaction was incubated for 12 hours at a range of temperatures. After the incubation period, the reaction supernatant was analyzed by high-performance liquid chromatography (HPLC) to quantify the hydrolysis products (Fig 4).

![](https://static.igem.wiki/teams/5566/results/resultnew/10040.webp)

Fig4. Catalytic efficiency of enzyme PEH-2013 on different substrates

The graph (Fig. 4) shows that PEH-2013 produced detectable hydrolysis products for PET, PBAT, and PLA across the tested temperature range, demonstrating its broad-spectrum polyester hydrolysis activity. Significant differences in product amounts were observed between substrates, suggesting that PEH-2013's catalytic efficiency for different polyesters is substrate-dependent. Overall, PEH-2013 demonstrates potential as a candidate enzyme for treating multiple polyester components in complex plastic waste streams.

In summary, the PEH-2013 enzyme exhibits several excellent properties suitable for real-world environments, including salt tolerance, thermal stability, and broad substrate spectrum. However, its low soluble expression level in the E. coli system has become a major bottleneck in the current project's in-depth characterization, high-throughput mutation screening, and large-scale application verification.

### Solubility improvement strategy based on proteinMPNN

To improve enzyme expression activity and stability, we used ProteinMPNN for sequence prediction optimization using enzyme PEH-2013 as a template. [*For details, please click MODEL-ENZYME OPTIMIZATION - Solubility Optimization: ProteinMPNN - Driven Sequence Redesign and Multi-Stage Screening*](https://2025.igem.wiki/buct/model#enzyme-optimization).

During the wet lab validation phase, we performed small-scale protein expression and purification on 13 candidate sequences from the initial computer-assisted screening. Of these, only three mutants, SEQ4, SEQ11, and SEQ59, successfully achieved soluble expression and were purified to high purity via nickel affinity chromatography. SDS-PAGE results showed that, under the same purification volume (1 L of bacterial culture), the protein band of SEQ4 was significantly darker than that of the other mutants, indicating higher expression levels and solubility (Fig. 5).

![](https://static.igem.wiki/teams/5566/results/resultnew/10050.webp)

Fig5. Protein precipitation, supernatant, and enzyme SDS-PAGE images of Protein MPNN prediction results

Compared to the wild-type enzyme PEH-2013, the proteinMPNN-predicted enzyme expression levels were significantly increased (Fig. 6). Therefore, SEQ4 was selected as the template sequence for subsequent stability engineering.

![](https://static.igem.wiki/teams/5566/results/resultnew/10060.webp)

Fig6. Protein MPNN prediction results and PEH-2013 enzyme SDS-PAGE images

Based on the successful acquisition of the highly expressed soluble mutant SEQ4, we further evaluated its enzymatic properties to confirm whether it has improved activity and stability while maintaining the excellent properties of the wild type.

![](https://static.igem.wiki/teams/5566/results/resultnew/10070.webp)

Fig7. Enzyme SEQ4 enzymatic properties. a. Optimal reaction temperature. b. Salt tolerance. c. Tm

Using nano-PET as the substrate, the temperature dependence of the enzymatic activity of SEQ4 was investigated under identical experimental conditions as those used for enzyme PEH-2013. Reactions were carried out at 50 °C for 3 hours, with 6 mg/L nano-PET and an enzyme concentration of 500 nM. The results indicated that SEQ4 exhibited measurable catalytic activity across the temperature range of 40 °C to 60 °C, with maximum activity observed at 50 °C (Fig. 7a). To assess its salt tolerance, SEQ4 was tested under varying NaCl concentrations and compared directly with PEH-2013. The results showed no significant difference between the two enzymes, confirming that SEQ4 also possesses strong salt tolerance and can maintain catalytic performance under marine-like salinity conditions (Fig. 7b). To further investigate its thermostability, we determined the melting temperature (Tm) of SEQ4 using a fluorescent dye-based protein thermostability assay. The Tm of SEQ4 was found to be approximately 58 °C, substantially higher than that of PEH-2013 (42 °C) (Fig. 7c).

This increase in thermal stability suggests that SEQ4 maintains its structural integrity and enzymatic function over a broader temperature range, making it a more robust candidate for industrial polyester degradation and marine environmental applications.

### Stability Enhancement and Activity Improvement of SEQ4 through Consensus Mutation Screening

To further enhance the stability and catalytic efficiency of SEQ4 for industrial application in polyester processing, we performed Multi-Tool Consensus Mutation Screening to identify beneficial amino acid substitutions. [*For details, please check the page of MODEL – Enzyme Optimization – Stability Enhancement: Multi-Tool Consensus Mutation Screening*](https://2025.igem.wiki/buct/model#enzyme-optimization).

Based on the computational simulation results, several potential stabilizing mutations were proposed and subsequently evaluated experimentally. The enzymatic activities of each mutant were quantified using a p-nitrophenyl butyrate (pNPB) hydrolysis assay, monitored by UV spectrophotometry under identical reaction conditions. Among the tested variants, the S210Y mutation exhibited the highest relative activity, showing approximately 1.75-fold improvement compared to the wild-type SEQ4 enzyme (Fig. 8) This mutation likely enhances local hydrophobic packing near the catalytic region, contributing to both increased stability and improved catalytic efficiency.

![](https://static.igem.wiki/teams/5566/results/resultnew/1008.webp)

Fig8. The relative activity of the mutans of SEQ4.

These results demonstrate that consensus-based computational design effectively guides rational enzyme optimization, enabling SEQ4 to maintain superior performance under the elevated temperatures and processing conditions relevant to industrial polyester production. We renamed SEQ4 (S210Y) to polygonase, and employed it as the key enzyme for the following researches and applications.

## SectionⅡ - Immobilization

### Thermal stability test of immobilized enzyme

To evaluate the thermal stability of the engineered enzyme polygonase immobilized on a calcium carbonate–silicon dioxide (CaCO₃–SiO₂) nanocomposite support via a sol-gel method, we subjected the immobilized preparation to high-temperature incubation and activity testing [*For details, please check the page of Experiments - Calcium Carbonate-Silicon Dioxide Composite Immobilization - Thermal Stability Testing*](https://2025.igem.wiki/buct/experiments#immobilization) . As shown in Fig. 9, after a brief high-temperature treatment, the immobilized enzyme retained approximately 40% of its relative catalytic activity at temperatures up to 200°C. No residual activity was detected after calcination at 240 °C for only 3 minutes, demonstrating that immobilization significantly improved the enzyme's thermal stability. This thermal stability enables the enzyme preparation to maintain partial functionality within the typical industrial thermoforming temperature range of PBAT and PBAT/PLA blends (approximately 160–200°C), providing a promising approach for integrating bioactive components into plastic matrices to create process-compatible "functional plastics." Direct adaptation to PET-related high-temperature processes still requires further evaluation and process optimization.

![](https://static.igem.wiki/teams/5566/results/resultnew/1009.webp)

Fig 9. Thermal protection effect diagram of nano Calcium Carbonate – Silicon Dioxide composite carrier

### Distribution of Immobilized Enzymes in Polymer Films

To verify whether the lamination process can achieve uniform distribution of immobilized enzymes within the film, we first focused on PBAT films, the most common and strongest type of film. In this project, fluorescein isothiocyanate (FITC) was used as a tracer surrogate. FITC solution and enzyme solution were coupled and mixed at a ratio of 50 µl:1 ml. Sample preparation and immobilization and lamination were performed using the same method. The surface, edge, and cross-section of the fluorescently labeled enzyme-containing PBAT plastic film were observed under a confocal microscope. As shown in the figure, confocal laser scanning microscopy reveals that FITC-stained polygonase fluoresces green. The distribution of the fluorescent green patches on the film surface reflects the distribution of the polygonase inorganic nano-calcium carbonate-silica complex enzyme within the film. The enzyme exhibits uniform distribution across the surface, edge, and cross-section of the film, with no apparent aggregation or distribution loss (Fig. 10). These results indicate that the adopted sol-gel immobilization and lamination process can uniformly integrate the immobilized enzyme with the CaCO₃ - SiO₂ composite carrier into the polyester matrix, providing an experimental basis for the preparation of spatially uniform and repeatable enzyme-plastic composite films.

![](https://static.igem.wiki/teams/5566/results/resultnew/1010.webp)

Figure 10 Observation of enzyme containing PBAT plastic film with 1% fluorescein isothiocyanate (FITC) - labeled polygonase inorganic nano-calcium carbonate-silica complex enzyme preparation by confocal microscope. a) Surface of fluorescently labeled enzyme-containing PBAT plastic film, b) Edge of fluorescently labeled enzyme-containing PBAT plastic film, c) Cross-section of fluorescently labeled enzyme-containing PBAT plastic film.

### Effect of immobilized enzyme addition amount on the mechanical properties of enzyme-containing PBAT plastic films

This project tested the mechanical properties of enzyme-containing PBAT plastic films with different addition amounts of polygonase calcium carbonate-silicon dioxide complex enzyme preparation. The Young's modulus, elongation at break, and tensile strength values obtained are shown in Table 1.

Table 1 Material mechanical parameters of enzyme-containing PBAT plastic films prepared with different addition amounts of polygonase calcium carbonate-silica complex enzyme preparation

| Enzyme addition percentage (%) | Young's modulus（MPa） | Elongation at break（%） | Tensile strength（MPa） |
| --- | --- | --- | --- |
| 0 | 1.62 | 615.71 | 22.84 |
| 0.5 | 1.92 | 425.57 | 20.16 |
| 1.0 | 1.58 | 307.80 | 14.80 |
| 2.0 | 1.80 | 213.80 | 15.70 |
| 5.0 | 1.99 | 44.86 | 11.60 |

Mechanical analysis revealed that increasing the loading of CaCO₃–SiO₂–immobilized polygonase in PBAT films introduced interfacial microgaps that reduced tensile strength and elongation while the Young’s modulus remained largely unchanged, leaving stiffness largely unaffected, indicating that with enzyme concentrations between 0.5% and 2% providing the optimal balance between mechanical integrity and biodegradability.

### Degradation Behavior of Films Containing Immobilized Enzymes in Real Seawater

PBAT and PBAT/PLA blend films embedded with a 1% enzyme preparation were cut into 3 cm × 3 cm samples and placed in an 80 cm × 50 cm × 50 cm aquarium. Real seawater collected approximately 10 km from the coast was added, and an oscillator was used to simulate seawater flow. Degradation experiments were conducted at room temperature for 60 days.

During the experiment, the degradation of the film samples was macroscopically observed, and structural damage and surface erosion processes were systematically evaluated. The results showed that in the simulated marine environment, the enzyme-containing films exhibited progressively deeper cracking, pore formation, and surface roughening, which were significantly different from the control group without enzyme (Figure 11).

![](https://static.igem.wiki/teams/5566/results/resultnew/10110.webp)

Fig 11. Comparison of plastic film degradation in seawater environment. A. PBAT - immobilized with polygonase. B. PBAT - immobilized with nano Calcium Carbonate – Silicon Dioxide composite. C. PBAT/PLA (1:1) – immobilized with polygonase. D. PBAT/PLA (1:1) - immobilized with nano Calcium Carbonate – Silicon Dioxide composite. All experiments were conducted at room temperature and seawater.

![](https://static.igem.wiki/teams/5566/results/resultnew/1012.webp)

Fig 12. SEM analysis of surface morphology of enzyme-embedded polyester films after 30-day seawater degradation. (A, D) Control PBAT and PBAT/PLA (1: 1) films immobilized with nano Calcium Carbonate–Silicon Dioxide composite show smooth and intact surfaces with minimal physical damage. (B, E) PBAT and PBAT/PLA (1: 1) films immobilized with nano CaCO₃ – SiO₂ – immobilized polygonase display clear evidence of microcracks, pitting, and surface erosion. (C, F) PBAT and PBAT/PLA (1: 1) films exhibit more obvious roughness and interface delamination at a viewing angle of 10 μm. Scale bars: 10 μm (A, C, D, F), 100 μm (B, E).

Scanning electron microscopy (SEM) was also performed on the samples to analyze their surface microstructure (Fig. 12). Comparison of plastic films that degraded over 30 days. High-resolution SEM imaging further revealed distinct microstructural differences between the enzyme-embedded membranes and the control membranes. While the surfaces of the control PBAT and PBAT/PLA (1:1) membranes remained smooth and intact, the membranes containing immobilized polygonase exhibited distinct morphological degradation, with the formation of irregular cavities visible upon further magnification. Together, these SEM observations confirm that immobilized polygonase effectively triggers localized polymer hydrolysis, thereby accelerating surface fragmentation under marine-like conditions.

Together, these results demonstrate the successful development of an innovative strategy for uniformly embedding heat-stable immobilized esterases into a degradable polyester film matrix. This type of "active film" exhibits significant and progressive degradation ability under conditions close to real ocean conditions, providing a strong experimental basis and methodological foundation for the development of a new generation of degradable polyester materials with controllable life cycles.

## SectionⅢ - Lab stage degradation platform

![](https://static.igem.wiki/teams/5566/results/resultnew/10130.webp)

Fig13. Fluorescence microscopy characterization of surface protein display effects. A. pETlac-Plpp1.2-INPNC-mCherry surface display effect. B. pET28a (+)-Lpp-OmpA-SpyC/SpyT-polygonase surface display effect. C. Surface co-display system effect. From left to right, the fields of view are bright field, red fluorescence channel, green fluorescence channel, and merged image.

### Adhesion Protein Display on the Escherichia coli Cell Surface

To obtain a functional module with ideal adhesion to polyester microplastics, we selected the mussel foot protein mfp-3, reported in the literature for its excellent overall performance and strong environmental adaptability, as our experimental adhesion protein. Using a truncated form of the ice nucleation protein INPNC as the membrane anchoring domain, mfp-3 was fused to the cell surface of E. coli BL21(DE3). To directly visualize and verify the system, we constructed a reporter plasmid, pETlac-Plpp1.2-INPNC-mCherry, containing the red fluorescent protein mCherry. Observations under fluorescence microscopy showed that the cell surface of the engineered bacteria expressing the induced protein showed a clear red fluorescent signal, indicating that the anchoring system was effectively displayed on the cell surface (Fig. 13A).

Base on this, we further evaluated the binding ability of the engineered INPNC–mfp-3 bacteria to polyester substrates through adhesion experiments. The engineered bacteria were incubated with 1 cm × 1 cm polyester plastic sheets at room temperature for 12 hours. Quantitative analysis of bacterial cell density revealed that the mfp-3 surface-displaying strain exhibited significantly increased adhesion to the plastic sheet compared to the empty vector control strain (Fig. 14).

![](https://static.igem.wiki/teams/5566/results/resultnew/1014.webp)

Fig 14. Adhesion protein MFP-3 adhesion assay results.

In summary, these results validated the successful display of the INPNC–mfp-3 fusion protein on the cell surface and clearly demonstrated its ability to significantly enhance cell adhesion to polyester substrates, laying a solid experimental foundation for the subsequent development of dual-anchor systems with both adhesion and degradation capabilities.

### E. coli cells display protein hydrolysis by polygonase using a SpyCatcher/SpyTag coupling.

![](https://static.igem.wiki/teams/5566/results/resultnew/10150.webp)

Fig 15. Protein purification results of SpyTag-polygonase and SpyTag-sfGFP

We constructed a polyester hydrolase degradation system for assembly on the surface of E. coli using the SpyC (SpyCather)/SpyT (SpyTag) proteins. Using a lipoprotein signal peptide (Lpp) outer membrane protein A (OmpA) fusion system as a vector, we constructed the pET28a (+)-Lpp-OmpA-SpyC plasmid for presentation of the SpyC protein on the surface of E. coli BL21 (DE3) cells. Furthermore, we constructed a SpyT-sfGFP fusion protein plasmid using super folded green fluorescent protein (sfGFP) as a visual reporter gene. Recombinant SpyT-sfGFP was overexpressed in E. coli and purified. SDS-PAGE analysis confirmed the purity and integrity of the isolated protein, revealing a single band at the expected molecular weight (Both are approximately 30 kDa) (Fig. 15). To test covalent binding, we incubated purified SpyT-sfGFP with E. coli cells expressing SpyC at room temperature for 1 hour. After the reaction, unbound proteins were completely removed by multiple centrifugation washes and observed using fluorescence microscopy. Observations under fluorescence microscopy analysis revealed a strong green fluorescence signal surrounding the bacterial cells, indicating that SpyT-sfGFP successfully and specifically bound to the cell surface (Fig. 13B). These results demonstrate that SpyC is functionally expressed on the E. coli surface and can effectively form a covalent bond with the SpyT-sfGFP fusion protein, confirming that the generation of the fluorescence signal depends on the specific interaction between SpyC and SpyT, successfully validating the effectiveness of our anchoring system.

### Synergistic Verification of Nano-PET Degradation Efficiency by the Dual-Anchor System

To verify the synergistic effect of the adhesion protein and the ester hydrolase polygonase, we constructed a dual-anchor fluorescent strain. First, the pET28a (+)-Lpp-OmpA-SpyC plasmid was introduced into Escherichia coli BL21 (DE3) and cultured. Then the engineered bacteria were prepared into electroporation competent cells and electroporated with the pETlac-Plpp1.2-INPNC-mCherry plasmid. The bacteria were then cultured on ampicillin-kanamycin dual-resistance plates. Single colonies were selected for induction. After induction, purified SpyT - sfGFP was incubated with the dual-anchor engineered E. coli cells at room temperature for 1 hour. After the reaction, the cells were washed several times by centrifugation to completely remove unbound proteins, and the cells were observed by fluorescence microscopy. Observations under fluorescence microscopy showed that strong green and red fluorescence signals were observed around the same bacterial cell perspective (Fig. 13C). This result indicates that the E. coli dual surface display system was successfully and simultaneously localized on the surface of the engineered bacteria.

We replaced the fluorescent protein SpyT-sfGFP with SpyT- polygonase and used INPNC carrying mfp-3 to evaluate the degradation efficiency of this dual-anchor system using nano-PET as a substrate and compared it with the single-anchor strain and the free enzyme. After 6 hours of degradation at room temperature, the dual-anchor strain produced the highest concentration of TPA (Fig. 16).

![](https://static.igem.wiki/teams/5566/results/resultnew/10160.webp)

Fig16. Comparison of nano-PET degradation rate (%) for three E. coli cell systems and free polygonase.

This result clearly demonstrates that co-localizing the adhesion module and the catalytic module on the surface of a single bacterial cell can produce significant synergistic effects. We analyzed that the strong adhesion mediated by mfp-3 tightly anchors the bacteria to the polyester microplastic surface, creating a localized high-concentration substrate environment for the polygonase hydrolase, thereby significantly improving catalytic efficiency, thereby significantly improving the catalytic efficiency by 2.7 times.

### Quantitative Detection of the Degradation Product TPA Based on Hydroxyl Radical Oxidation

To accurately evaluate the degradation efficiency of polyester substrates by polygonase ester hydrolase, we developed a highly sensitive fluorescence detection method for TPA. Based on the specific hydroxylation of terephthalic acid (TPA) by hydroxyl radicals (·OH) to produce the highly fluorescent substance 2-hydroxyterephthalic acid (HTPA), we optimized the reaction conditions and established a standard curve between TPA concentration and fluorescence intensity within a 1-minute reaction time (Fig. 17). The curve showed a good linear relationship between TPA concentration and fluorescence intensity (R² > 0.99) within the range of 0.00 to 0.10 mol/L, making it suitable for the quantitative detection of TPA in aquatic environments such as marine environments.

![](https://static.igem.wiki/teams/5566/results/resultnew/1017.webp)

Fig17. A standard curve comparing TPA concentration and fluorescence intensity within a 1-minute reaction time was constructed. The slope of the curve was 30759.251 ± 9571.638, the intercept was 4407384.784 ± 155889.044, and the R² = 0.99813.

After a 6-hour degradation experiment of nano-PET using the dual-anchor system, 1 ml of the reaction supernatant was added to an equal volume of Fenton's reagent (containing Fe(II) and H₂O₂). The reaction was incubated at room temperature for 1 minute, and the fluorescence intensity was immediately measured. By comparing with a standard curve, the amount of TPA generated in each experimental group was successfully quantified.

The laboratory-scale degradation platform successfully demonstrated the feasibility of constructing a dual-anchor whole-cell system that integrates both adhesion and catalytic functions on the E. coli surface. The INPNC–mfp-3 fusion enabled strong and specific adhesion of engineered cells to polyester substrates, while the SpyCatcher/SpyTag-mediated display of polygonase provided efficient enzymatic degradation capability. When combined in a single E. coli strain, the dual-anchor system exhibited a synergistic degradation effect on nano-PET, yielding a 2.7-fold increase in TPA production compared with single-anchor or free-enzyme systems. Furthermore, the fluorescence-based hydroxyl radical oxidation assay for TPA quantification achieved high linearity (R² = 0.998), enabling accurate and sensitive measurement of polyester degradation products.

These results systematically validate the effectiveness of our constructed dual-anchor whole-cell catalytic platform. By spatially colocalizing the adhesion protein module (mfp-3) and the hydrolase module (polygonase) on the same cell surface, this system significantly enhances the interfacial interaction between the cell and polyester microplastics, making the substrate more accessible and significantly improving the overall hydrolysis efficiency and reaction rate. Furthermore, the dual-anchor system achieves synergistic adhesion and catalysis while maintaining the integrity of the cell structure, demonstrating superior degradation capacity and system stability, providing a solid experimental foundation for the development of an efficient and sustainable biodegradation platform.

## Prospective

Building on the successful establishment of a marine-derived polyester biodegradation system, our future work will focus on advancing this platform toward practical, intelligent, and sustainable applications across multiple dimensions.

At the molecular level, we plan to integrate biological sensing modules with our current dual-anchor system to develop a fully biological monitoring and degradation platform. By coupling the detection of degradation intermediates (such as terephthalic acid, TPA) to gene circuit responses, we aim to construct a cell-based biosensor capable of autonomously quantifying plastic degradation efficiency and dynamically regulating enzyme expression, achieving closed-loop control of microplastic degradation in aquatic environments.

At the systems level, we envision expanding this approach into microplastic purification and environmental restoration technologies. By combining immobilized hydrolase films with water filtration units, we plan to construct microplastic purification membranes that can continuously capture and degrade polyester contaminants in marine and freshwater systems. This concept can be integrated with real-time biosensing units for in situ detection of plastic degradation products, forming an intelligent, self-reporting remediation network.

In summary, our project establishes a robust scientific foundation for next-generation biodegradable polyesters and biological microplastic remediation systems. By integrating synthetic biology, material science, and environmental engineering, we aim to create a scalable, intelligent, and ecologically responsible biotechnological solution for global plastic pollution—advancing toward a sustainable circular bioeconomy.

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Using synthetic biology to solve real-world problems

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