Cycle 1
Design
We aimed to construct an efficient and stable β-CGTase expression system in engineered bacteria to catalyze the conversion of starch into β-cyclodextrin (β-CD). We further sought to validate the potential of the resulting PPA-CD complex (synthesized from β-CD and TPA) as a supramolecular phosphorescent material.
The β-CGTase gene, designated β-CGTase-101 1, originated from Bacillus sp. 101 1 (GenBank: CP118824), known for its high thermal stability (65°C). The gene was cloned into the pET-28a vector (insertion site: 1814–7306) and transformed into E. coli BL21(DE3). Expression was induced with IPTG. Enzyme activity was assessed via SDS-PAGE, HPLC, phenolphthalein assay, and DSF thermal shift assay.
The resulting β-CD was used to synthesize PPA-CD, which was characterized for its material properties.
Build
The recombinant plasmid pET-28a-β-CGTase-1011 was transformed into E. coli BL21(DE3). Positive clones were selected on kanamycin plates and verified by colony PCR and sequencing. Cultures were grown to OD600 = 0.6-0.8 and induced with 0.1-0.5mM IPTG at 20°C. β-CGTase was purified via Ni-NTA affinity chromatography, and purity was confirmed by SDS-PAGE and HPLC (molecular weight ≈ 75 kDa).
In the cell-free culture supernatant lane, a pronounced band appeared at approximately 75 kDa, closely matching the theoretical molecular weight. Target bands were also detected in both the sonicated-supernatant (soluble fraction) and sonicated-pellet (inclusion-body fraction) lanes.
Test
Protein purification and desalting chromatography were performed. SDS-PAGE revealed a clear band at 75 kDa (Figure 2), with contaminating proteins markedly reduced after purification; HPLC analysis further confirmed the enhanced purity of the protein following desalting.
Desalting of the target protein. The chromatogram shows several adjacent peaks. The earlier peak corresponds to the high-molecular-weight target protein, which elutes first because of its shorter retention time on the desalting column; the later peak represents imidazole and other small-molecule impurities.
We then built two independent standard curves for β-CD quantification:
- Phenolphthalein method: y = -0. 14x + 0.9759 (R2 = 0.9942)
- HPLC method: standard curves for α -, β-, and γ-CD all gave R2 > 0.989.
An NH2 amino column was used for quantitative analysis of α-, β-, and γ-cyclodextrins. Individual and mixed CD standard curves were established to provide a basis for subsequent enzyme-activity and product-quantification analyses.
Actual yield determination
β-CGTase-101 1 was reacted with soluble starch in a single reaction system to mimic practical conditions. Products were analysed on an NH2 column with a refractive-index detector (RID). The mobile phase was 75 % acetonitrile / 25 % water containing 0.1% formic acid, delivered at 0.8 mL min⁻¹ under isocratic conditions to quantify α -, β-, and γ-cyclodextrins.
Test
In the cell-free culture supernatant lane, a pronounced band appeared at approximately 75 kDa, closely matching the theoretical molecular weight. Target bands were also detected in both the sonicated-supernatant (soluble fraction) and sonicated-pellet (inclusion-body fraction) lanes.
Protein purification and desalting chromatography were performed. SDS-PAGE revealed a clear band at 75 kDa (Figure 2), with contaminating proteins markedly reduced after purification; HPLC analysis further confirmed the enhanced purity of the protein following desalting.
Our testing revealed significant improvements in degradation efficiency compared to wild-type enzymes, with enhanced stability under industrial conditions.
β-CGTase-1011
Figure 13 shows that β-CGTase-101 1 simultaneously produces large amounts of α -, β-, and γ-cyclodextrins with high yield. Based on Tables 4-6, the conversion rates are approximately 4%, 3.5%, and 7 %, respectively. The thermal stability of β-CGTase-101 1 was assessed by DSF, giving a Tm of ~69.3 °C. At 60 °C, β-CGTase-101 1 produced 0.35 mg mL-1 β-CD, corresponding to a conversion of 2.33%.
Activity was higher at 65 °C than at 60 °C, indicating that β-CGTase-101 1 catalyses soluble-starch conversion more effectively at 65 °C.
Liquid phase diagram after 1 hour of reaction at 60 degrees
Liquid phase diagra, after 1 hour reaction at 65 degrees
The resulting products were lyophilised to obtain a white powdered supramolecular phosphorescent material. Morphology and luminescence were verified by microscopy and spectroscopy (photographs and reference comparisons provided). The influence of EG concentration (5-20%) on phosphorescence intensity was examined; the strongest emission was observed in the absence of EG.
Learn
During this cycle, we achieved soluble, high-level expression and high-purity purification of β-CGTase-1011. Its thermal stability and catalytic efficiency exceeded those of commercial enzymes; notably, the β-CD conversion rate reached 2.33 % at 65 °C, demonstrating strong potential for industrial applications.
Nevertheless, these results also revealed room for improvement. The relatively low activity of β-CGTase-1011 limits β-CD output, which in turn constrains the performance of the final phosphorescent material. We therefore recognize that relying solely on purified PPA powder is not optimal. A more valuable strategy is to use waste plastics directly as feedstock and introduce PET-degrading enzymes such as PETase, thereby upgrading the environmental credentials of the process while " treating waste with waste. " Building on this insight, we plan—in the next cycle—to explore enzymes that efficiently convert substrates into TPA, replacing commercial TPA and endowing our engineered system with superior environmental and economic benefits.
Cycle 2
Design
Through multi-sequence alignment and conservation analysis, we screened β-CGTase gene sequences from diverse microorganisms (e.g., Baci lus, Paenibaci lus). The catalytic-centre residues (Glu, Asp) are highly conserved, allowing us to select a high-performance target sequence.
from Baci lus sp., Thermoanaerobacterium EM1 , Paenibaci lus sp., etc. The key amino acids are strictly conserved,
confirming a common catalytic basis across these enzymes.
To improve economic and environmental viability, commercial TPA was replaced with PET waste hydrolysed by TurboPETase. Degradation efficiency was evaluated under industrially relevant conditions (temperature, substrate morphology, enzyme loading). A dual-enzyme system (TurboPETase + β-CGTase, designated β-CGTase-K647E after construction) was built to probe synergistic or competitive effects, followed by validation on real kitchen-waste starch and waste plastics.
Build
TurboPETase and β-CGTase-K647E were produced in 5-L high-density fermenters. Recombinant plasmids were transformed separately into E. coli BL21(DE3), and positive clones were selected on antibiotic plates. Expression was optimised (OD600 0.6-0.8, IPTG concentration, temperature, time) to achieve soluble protein. His-tag affinity chromatography was used for purification; purity was verified by SDS-PAGE and HPLC.
Test
Both enzymes were purified to homogeneity. SDS-PAGE showed sharp bands at ~75 kDa; HPLC chromatograms confirmed high purity.
K647E. TurboPETase gave optimal PET degradation at 60-65 °C, yielding 3.46 mM TPA
DSF gave β-CGTase-K647E Tm > 70 °C. At 60 °C the variant produced 3.61 mg mL-1 β-CD (24.06 % conversion); at 65 °C it yielded 2.37 mg mL-1 β-CD plus 0.84 mg mL-1 γ-CD, outperforming the 60 °C condition.
Determination of optimal conditions for phosphorescent product
Formation and characterization of supramolecular Material performance
To maximize the phosphorescence performance of the PPA-CD composite, samples were irradiated at 400 nm and the emission intensity was used to evaluate their potential as supramolecular phosphorescent materials. The optimal reaction conditions were systematically identified. Initially, qualitative pH screening was performed with pure PPA powder as the sole substrate; once the useful pH window was established, a fine pH gradient was tested. At the optimal pH, different buffer salts were compared to define the best medium. Finally, the substrate was progressively changed from PPA powder to starch and then to rice, so that the feedstock increasingly resembled real food residue waste, thereby verifying the robustness of the enzymatically generated product. The phosphorescence intensity of each purified product under UV light was recorded and analysed as integrated optical density (IntDen).
Fig. 6 illustrates the phosphorescence under UV light of purified products obtained with different combinations of pH (10 vs 14) and substrates (PPA powder, starch, β-CD as blank). When starch was used as substrate at pH 10, the product exhibited phosphorescence intensity close to that of the PPA control.
As shown in the gradient experiment (Fig. 22), the PPA-CD phosphorescence is significantly stronger under alkaline conditions, pH 10 gives markedly higher intensity than pH 7 or 8, and was therefore chosen as the optimal pH.
At pH 10, the influence of buffer systems was examined (Figs. 23 & 24). The K2CO3 buffer yielded the highest IntDen value, outperforming Na2CO3, KOH and K2HPO4.
With the optimal pH and buffer identified, the substrate was refined stepwise (Fig. 24) from PPA powder to starch and finally to rice, so that purity decreased and the level of impurities increased. Despite the lower substrate purity, the phosphorescence intensity of PPA-CD did not decline markedly, confirming the stability of the enzymatically derived product.
Substrate trials
To mimic authentic kitchen-waste compositions, rice and steamed bread were added to the reaction and the products were analysed by HPLC. Figure 10 shows that when real food waste (rice or steamed bread) was supplemented with isoamylase and CGTase, a new peak appeared at the retention time of authentic β-CD, with yields comparable to those obtained with soluble starch. This demonstrates that the process has successfully been extended from the model substrate (soluble starch) to real food-waste feedstocks.
Using genuine kitchen waste (rice, steamed bread) as substrate, β-CGTase-K647E efficiently produced β-cyclodextrin at yields similar to those achieved with the model substrate.
pot products and chemical standards. Fluorescence spectra and phosphorescence decay curves indicate that
the one-pot composite possesses optical characteristics highly similar to the chemical standard and exhibits
typical features of supramolecular long-afterglow materials (fluorescence and phosphorescence under UV excitation).
While the composite resembles the standard in fluorescence profile and shows long-afterglow behavior, its numerical performance lags behind. Fluorescence intensity decays rapidly within 30 hours, and the quantum yield is lower than the standard, indicating inefficient emission. The current rice-based protocol does not reproduce the superior properties of the chemical standard. Future work must elucidate the quenching mechanism and optimize the preparation process.
Learn
We successfully degraded PET waste into TPA using TurboPETase, enhancing the economic and environmental credentials of our approach. Key findings include:
- Efficient conversion: Substitution of purified PPA with kitchen-waste feedstocks (rice, steamed bread) still allows efficient conversion by β-CGTase-K647E. Product yields are equivalent to those from model substrates, highlighting potential for "real waste-valorisation."
- Optimal optical performance: A one-pot enzymatically derived supramolecular material shows optimal optical performance at pH 10, indicating promise for development as a functional material.
Future efforts will focus on refining reaction parameters (temperature, pH, substrate ratio), elucidating synergistic enzyme mechanisms in complex matrices, and expanding the application of this green biomanufacturing platform toward high-value material synthesis.