Engineering Success

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

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.

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

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.

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

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.