NOTEBOOK

Cycle1

1.1

Experiment dates: 6.1-6.7

Experiment personnel: Xu Yiran, Wei Sihui

Experiment objective: Finalize the experimental plan, locate target gene sequences, outsource plasmid synthesis to a company, and chemically transform into BL21(DE3) competent cells for protein expression.

Experiment materials:

Experimental instruments:

Experimental procedure:

1.Plasmid construction: Search the literature to select genes. Download the CGTase gene sequence from NCBI, optimize the sequence, and submit it to a gene synthesis company.

2.Transformation: Thaw BL21(DE3) competent cells on an ice box; in the biosafety cabinet add plasmid DNA (100 ng), pipette up and down to mix, and incubate on ice for 30 min (adjust according to plasmid concentration). Place the suspension in a 42 °C water bath for heat shock for 90 s, then immediately put on ice for 2 min. In the biosafety cabinet add 200 µL LB and incubate at 37 °C on a shaker for 60 min. Plate the culture onto plates containing Kan (50mg/mL) by the spread plating method, seal and incubate inverted at 37 °C for approximately 12 h.

Experimental procedure:

By literature search and comparison, the β-CGTase sequence from Bacillus sp. (strain 1011) met our engineering requirements. After locating the related sequence in the NCBI database, the Pet-28a-β-CGTase-1011 plasmid was successfully constructed.

Table 1: CGTase from different organism

aData adopted from Lim et al. 2021, bElbaz et al. 2015, cIbrahim et al. 2011, dXiumei et al. 2020, eNik-Pa et al. 2020, fUpadhyay et al. 2020, gLiu et al. 2022, hCrozatti et al. 2023

xperimental notes:

1. When removing competent cells, avoid touching the tube wall to prevent temperature fluctuations that affect transformation efficiency.

2. The heat-shock operation must strictly control temperature and time (60 °C, 90 seconds); inaccurate timing will significantly reduce transformation efficiency.

1.2

Experiment dates: 6.8-6.14

Experiment personnel: Sun Runze, Lu Xuxian, Kuang Xintong

Experiment objective: Inoculate the transformed cells into tube liquid culture medium (20 mL) containing kanamycin at 0.5 mol/L, incubate overnight on a 30 °C shaker; after 24 h transfer to a flask in the biosafety cabinet for scale-up culture; after 8 h induce protein expression with 1 mM IPTG; purify and desalt to obtain the target enzyme protein.

Experiment materials:

Experimental instruments:

Experimental procedure:

1.Seed culture and transfer

Seed preparation: Using a 10 µL tip, pick multiple single colonies from a plate into 5 mL LB + 5 µL Kan; incubate at 37 °C, 200 rpm for 3–4 h.

Transfer: Measure seed OD600; calculate to inoculate 400 mL LB so that final OD600 = 0.1. Inoculate at 1% into 400 mL LB containing 50 µg·mL⁻¹ Kan; incubate at 37 °C, 200 rpm until OD600 reaches 0.4–0.6. (Remove 1 mL culture in the biosafety cabinet and measure OD600 in the spectrophotometer.)

2.Induction of expression

Add 1 M IPTG. (Inducer added at 1‰.) Reduce temperature to 16–20 °C; induce at 160–200 rpm for 18–20 h. Record induction time/temperature.

3.Cell harvest

Use multiple 50–100 mL centrifuge tubes to accumulate culture; centrifuge at 4 °C, 8,000 rpm for 15 min to collect cells.

4.Resuspension / lysis

Wash cell pellets twice with Buffer A.

Resuspend: add 10 mL Buffer A per 1 g wet cell mass.

Lysis (ice bath): 300 W sonication, 3 s on / 2 s off cycles, total 20 min (adjust according to cell mass).

Centrifuge: 4 °C, 10,000 rpm, 30 min; retain supernatant.

Filtration: filter through 0.45 µm syringe filter.

SDS-PAGE: add reducing sample buffer, heat at 95 °C for 5 min; load 5–20 µL per lane; run at 220 V; stain (Coomassie / silver stain); save images and filenames.

5.Ni-NTA affinity purification

Column preparation: resin capacity 40 mg protein / mL resin (calculate maximum load by column volume); install Ni column at 0.5 mL/min, then adjust flow to 5 mL/min. Wash with deionized water at 5 mL/min until baseline; equilibrate with Buffer B and Buffer A to baseline.

Sample loading: pump A1 load at 5 mL/min (leave 2–3 mL dead volume to avoid air); after baseline recovery apply 5% Buffer B (10 mL) to remove impurities until baseline.

Elution / collection: set elution gradient or introduce 100% Buffer B; begin collecting when UV (mAu) rises (10–15 mL fractions); label fraction numbers.

QC: run SDS-PAGE on elution fractions, save images and record corresponding UV peaks.

6.Desalting / buffer exchange

Connect desalting column; wash with deionized water at 8–10 mL/min until baseline; after installation load sample at 8 mL/min (maximum load volume 15 mL); wash with Buffer C and collect when UV increases (approximately 20 mL); stop collection when conductivity (mS/cm) rises. Record conductivity, UV peaks and fraction numbers.

SDS-PAGE: add reducing sample buffer, heat at 95 °C for 5 min; load 5–20 µL; stain (Coomassie / silver stain); save images and filenames.

7.Protein concentration and QC

Use Amicon Ultra (appropriate MWCO) to concentrate protein to 1–5 mg/mL; measure concentration by Bradford assay and record.

Experimental results:

The plasmid was successfully introduced into cells and target protein expression was induced; the target protein was obtained after purification.

xperimental notes:

1. After weighing, promptly tidy the bench, return reagents to their storage locations, and maintain a clean workspace.

2. When adjusting the pH to 7.5, add NaOH dropwise as the value approaches the target to avoid overshooting.

1.3

Experiment dates: 6.15–6.21

Experiment personnel: Xu Yiran, Fang Yingshan, Xiang Yuhang

Experiment objective: Perform reactions using a one-pot method, construct a phenolphthalein calibration curve, detect product formation by HPLC, and evaluate reaction yield by chromatographic peak area. WhExperimental procedure:en using PPA and laboratory starch as substrates, product yield was low, primarily due to insufficient formation of CDs. Therefore, an iterative optimization strategy is planned to improve yield.

Experiment materials:

Experimental instruments:

Item / Description

Experimental procedure:

1.Preparation of HPLC calibration curves:

Prepare α/β/γ standards by serial dilution (concentrations: 0, 1, 2.5, 5, 10, 15, 20 mg/mL) as follows:

Accurately weigh 0.312 g sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) and 0.0716 g disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) into a beaker, add 80 mL ultrapure water, and stir until fully dissolved. Measure pH precisely with a pH meter and adjust to pH = 6.00 using 0.2 M NaOH and 0.2 M HCl. Transfer the pH-adjusted solution to a 100 mL volumetric flask, rinse the beaker several times with ultrapure water and transfer the rinses into the volumetric flask, and dilute to 100 mL with ultrapure water; mix thoroughly and set aside.

Accurately weigh 400 mg β-cyclodextrin (β-CD) powder, transfer to a 15 mL centrifuge tube, add approximately 8 mL of phosphate buffer, mix thoroughly, transfer all to a 10 mL volumetric flask, rinse the original container several times with small volumes of buffer and combine rinses in the volumetric flask, then dilute to the 10 mL mark with buffer and mix thoroughly to obtain a 40 mg/mL β-CD stock solution.

2.Run the HPLC, record retention times (RT) and peak areas, and construct single-component calibration curves (concentration vs. peak area).

3.Preparation of mixed HPLC calibration curves:

Prepare mixed α/β/γ standards for multivariate calibration using HPLC.

4.Preparation of phenolphthalein calibration curve:

Prepare phenolphthalein standards by serial dilution (concentrations: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5 mg/mL) as follows:

5.Prepare 0.03 M NaOH solution: accurately weigh 0.12 g NaOH solid, dissolve in water and dilute to 100 mL.

Prepare 0.02% (w/v) phenolphthalein solution: accurately weigh 0.02 g phenolphthalein solid, dissolve in 50 mL absolute ethanol, then dilute to 100 mL with ultrapure water.

6.Color development: take 250 µL of the diluted standard, add 125 µL of 0.02% phenolphthalein solution and mix, then add 875 µL of 0.03 M NaOH solution, incubate at room temperature for 20 minutes, measure OD550 according to laboratory standard color development procedures and save data. Construct the concentration-absorbance calibration curve and record linearity / range /error.

7.Measurement of Tm values

Figure 14: HPLC quantitative data table for α-cyclodextrin produced by β-CGTase-1011.

(1) Commercial enzyme (Novozymes) — activity assay (HPLC time course)

Temperature: 60 °C.

Total volume: 1000 µL (1 mL).

Substrate: Soluble starch 15 mg/mL (i.e., 15 mg dissolved in 1 mL carrier; see pretreatment for details).

Buffer/sample solvent: ddH2O, 1 mL (per the pretreatment below).

Pretreatment: Boil the starch substrate for 15 min (gelatinize); place 900 µL of substrate at 60 °C for 10 min, then add enzyme solution.

Enzyme (Commercial): Novozymes commercial enzyme 100 µL (added to 900 µL matrix to form a 1 mL reaction).

Sampling time points: 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, 4 h, 4.5 h, 5 h, 5.5 h.

Termination: Boil 15 min (enzyme inactivation).

Sample processing: Centrifuge/filter and inject to HPLC.

(2) Our enzyme — single timepoint (immediate inactivation)

Assay conditions (single timepoint):

Temperature: 60 °C.

Total volume: 1000 µL.

Substrate: Soluble starch 15 mg/mL (15 mg).

Pretreatment: Boil 15 min; place 900 µL substrate at 60 °C for 10 min.

Enzyme amount: β-CGTase-1011 (0.58 mg/mL), 5 µL (i.e., 2.9 µg enzyme added to the reaction).

Reaction times: 0 min (i.e., immediately inactivate by boiling for 15 min after enzyme addition), 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min.

(3) The detection of Tm of β-CGTase-1011

1.Prepare SYPRO Orange 50x working solution

Under light-protected conditions, add 2.5 µL SYPRO Orange (5000x) to 247.5 µL deionized water → 50× working solution. Mix, aliquot small volumes and refrigerate or use immediately. Avoid repeated freeze-thaw and prolonged light exposure.

2.Sample preparation

Target final reaction volume: 25 μL.

Per-well composition:

Protein solution (adjusted to the desired final molar concentration): 22.5 µL.

50× SYPRO Orange: 2.5 µL (final 5x).

If adding ligand/small molecule, dissolve the ligand in buffer or DMSO, adjust to required concentration and ensure final DMSO < 2%.

Example: if protein = 0.58 mg/mL = ~7.7 µM, use 22.5 µL protein + 2.5 µL 50x dye → total 25 µL; protein concentration in the well remains ~7.7 µM (slight dilution).

Include 2-3 blank wells (buffer + dye); perform technical triplicates, with ≥3 wells per condition to reduce noise.

3.Plate handling

Add samples to a black qPCR plate, avoid bubbles; if bubbles form, briefly centrifuge (1-2 k x g, 10-15 s). Seal with optical sealing film, press edges firmly. Avoid leakage. Place the plate into the instrument (ensure instrument filters are compatible with SYPRO Orange: Ex≈ 470 nm / Em ≈ 570 nm). Minimize exposure to strong light and operate quickly to avoid fluorescence drift.

4.Instrument parameter settings (based on provided parameters + recommended adjustments)

Temperature range: 20 °C → 95 °C.

Ramp rate: 1.0 °C/min.

Read frequency: record fluorescence every 0.2-0.5 °C.

Excitation power/gain: 80%.

Lid/heating cover: usually off or set with a low temperature differential (to prevent lid temperature >> block, which can cause bubbles/evaporation).

5.Run and record

Start the run and record fluorescence vs. temperature curves. Typically, 20→95 °C at 1° C/min takes approximately ~75 min.

(4) The detection of Tm of Novozymes NV commercial enzyme

Dilute the commercial protein to 0.4 mg/mL and measure Tm.

(5) Assemble a 500 µL reaction system:

add 450 µL 0.5 mM Na2HPO4 as buffer (pH = 8), add 15 mg PET plastic powder to reach 30 g/L, add 15 mg soluble starch, add 50 µL Turbo-PETase to reach 2 mg/g, add β-CGTase-1011 5 µL (protein concentration 0.58 mg/mL), incubate in a 60 °C water bath for 1 h, 2 h and 3 h. After reaction, add an equal volume of methanol to inactivate; take 5 µL reaction sample, dilute 20× with water and inject to reversed-phase C18 HPLC (UV detection at 260 nm) to determine PPA and MHET formation. Analyze the reaction mixture to determine α, β and γ cyclodextrin production.

(6) Assemble a 500 µL reaction system:

add 450 µL 0.5 mM Na2HPO4 as buffer (pH = 8), add 15 mg PET plastic powder to reach 30 g/L, add 15 mg soluble starch, add 50 µL Turbo-PETase to reach 2 mg/g, add β-CGTase-1011 5 µL (protein concentration 0.58 mg/mL), incubate in a 65 °C water bath for 1 h, 2 h and 3 h. After reaction, add an equal volume of methanol to inactivate; take 5 µL reaction sample, dilute 20× with water and inject to reversed-phase C18 HPLC (UV detection at 260 nm) to determine PPA and MHET formation. Analyze the reaction mixture to determine α, β and γ cyclodextrin production.

8.One-pot reaction:

To the reaction system add 15 mg PET plastic powder, 15 mg soluble starch, 450 µL Buffer — 100 mM Na2HPO4, 50 µL Turbo-PETase (concentration 0.6 mg/mL), 5 µL β-CGTase-1011 (concentration 0.58 mg/mL), adjust pH to 8, incubate in a 65 °C water bath for 1 h. After adding an equal volume of anhydrous methanol to inactivate, ensure the total reaction liquid is diluted 20×; therefore, inject 10 µL reaction liquid + 90 µL water to HPLC. The detected product exhibits phosphorescent material properties; lyophilize to obtain product PPA-CD.

Experimental results:

Successfully established α-CD, β-CD, γ-CD standard curves. Thermal stability and activity assays indicate β-CGTase-1011 outperforms the commercial enzyme.

Table 2: Phenolphthalein assay absorbance — β-CD concentration data Phenolphthalein assay standard for cyclodextrin

Table 3: HPLC quantitative data table for α-cyclodextrin produced by β-CGTase-1011.

Experimental notes: Ensure traceability for each step during the experimental process.

Cycle2

2.1

Experiment dates: 6.22-6.28

Experiment personnel: Xu Yiran, Wei Sihui, Su Yanyan

Experiment objective: To review the literature, collect, and collate enzyme sequence information from various sources to support subsequent enzyme screening and engineering.

Experimental procedure: This involved searching NCBI for CGTase enzymes and their amino acid and gene sequences. A preliminary activity assessment was performed using molecular docking (e.g., HADDOCK) and molecular dynamics simulations (e.g., Gaussian). High-activity types were selected, and signal peptides and cleavage sites were predicted using https://services.healthtech.dtu.dk/services/SignalP-6.0/. Genes were designed using https://blast.ncbi.nlm.nih.gov/Blast.cgi, and plasmids were subsequently produced by a company.

2.2

Experiment dates: 6.29-7.5

Experiment personnel: He Shunkang, Wang Shuhan, Xiang Yuhang

Experiment objective: To continue the literature review to obtain additional enzyme sequences and enrich the candidate enzyme library to support multiple rounds of screening and comparison.

Experimental results: The experiment successfully identified β-CGTase-K647E, with subsequent experimental validation planned.

2.3

Experiment dates: 7.6–7.12

Experiment personnel: Xu Yiran, Wei Sihui, Kuang Xintong

Experiment objective: Introduce enzyme sequences with potential for plastic degradation and predict their activities using bioinformatics methods to provide candidate enzymes for experimental validation.

Experimental results: After comparing multiple PET hydrolases, Turbo-PETase showed a marked advantage at 65 °C.

Table 7: Performance comparison of Turbo-PETase and various PET hydrolases under industrially relevant conditions and analysis of substrate morphology effects

2.4

Experiment dates: 7.13-7.19

Experiment personnel: Xu Yiran, Sun Runze

Experiment objective: Commission a company to synthesize plasmids containing the target enzyme genes, inoculate transformed cells into liquid culture medium (20 mL) with 0.5 mol/L kanamycin resistance in test tubes, incubate overnight at 30 °C on a shaker, and after 24 h transfer to Erlenmeyer flasks under a biosafety cabinet for scale-up cultivation.

Experiment materials:

Experimental instruments:

Experimental procedure:

1. Transformation: Thaw BL21(DE3) competent cells on ice; in the biosafety cabinet, add plasmid (100 ng), pipette up and down to mix, and keep on ice for 30 min (depending on plasmid concentration). Place the suspension in a 42 °C water bath for 90 s heat shock, then immediately place on ice for 2 min. In the biosafety cabinet, add 200 µL LB and incubate at 37 °C on a shaker for 60 min. Spread the culture on Kan-containing plates (50 mg/mL) using the spread plate method, seal, and incubate inverted at 37 °C for ~12 h.

2. Fermenter cultivation:

• Prepare 1 L 0.5 mM Na2HPO4 buffer (pH = 8), add 30 g starch, mix, boil for 15 min, cool to room temperature, then add 30 g PET plastic powder, add 100 mL Turbo-PETase (final content 2 mg/g), add 10 mL β-CGtase-K647E (protein concentration 0.58 mg/mL), add 10 mL cyclohexane, react in a 65 °C water bath for 30 min.

• Prepare 1 L 0.5 mM Na2HPO4 buffer (pH = 8), add 30 g PET plastic powder, add 100 mL Turbo-PETase (final content 2 mg/g), add 10 mL cyclohexane, react in a 65 °C water bath for 30 min.

Experimental results:

Successfully cultured bacterial strains, preparing for subsequent protein purification.

Experimental notes:

1. When spreading bacterial suspension, use the method of "rotating the plate with the left hand, spreading towards the center with the right hand" to ensure even distribution, avoiding overlap or missed areas.

2. All operations must be performed near an alcohol lamp, ensuring aseptic conditions to prevent contamination.

2.5

Experiment dates: 7.20–7.26

Experiment personnel: Xu Yiran, Wei Sihui, Sun Runze

Experiment objective: Culture engineered bacteria, induce protein expression with IPTG after 8 h, and obtain the target enzyme protein through purification and desalting. Collect cells, purify, desalt, and obtain high-purity target enzyme protein. Measure the Tm value. β-CGTase-K647E

Experiment materials:

Experimental instruments:

Experimental procedure:

1.Seed culture and inoculation

Seed preparation: Pick several single colonies from a plate with a 10 µL pipette tip into 400 mL 2xYT flask + 5 µL Kan.

Culture conditions: 37 °C, 200 rpm, 3–4 h.

Culture endpoint: Culture until OD600 reaches 0.4–0.6.

Note: Take 1 mL samples periodically in a biosafety cabinet and measure OD with a spectrophotometer.

2.Induced expression

Inducer: Add 1 mM IPTG.

Conditions: Lower temperature to 20 °C; 200 rpm, 20 h induction.

Record: Record induction time/temperature.

3.Cell harvest

Method: Centrifuge in 50–100 mL tubes.

Conditions: 4 °C, 10,000×g, 10 min.

Outcome: Collect cells.

4.Resuspension / lysis

Wash: Wash cells twice with Buffer A.

Resuspend: 1 g pellet in 10 mL lysis buffer.

Lysis: Lyse on ice: 300 W, 3 s sonication with 2 s interval, 20 min (depending on biomass).

Centrifuge: 4 °C, 14,000×g, 60 min; collect supernatant.

Filter: 0.22 µm syringe filter.

SDS-PAGE: Mix with reducing loading buffer, 220 V, 95 °C 5 min; load 5–20 µL; stain (Coomassie / silver stain); save images and filenames.

5.Ni-NTA affinity purification

Column preparation: Resin capacity is 40 mg protein/mL resin (calculated by column volume).

Set flow rate to 0.5 mL/min for Ni-column setup, then adjust to 5 mL/min.

Wash with deionized water at 5 mL/min until baseline; equilibrate with lysis buffer.

Loading: Al pump injection at 5 mL/min (stop 2–3 mL early to avoid air); equilibrate with lysis buffer until baseline.

Elution / collection: Set a gradient or directly elute with lysis buffer; collect when UV (mAu) increases (10–15 mL per fraction); save fraction numbers.

Use a HiPrep 26/10 desalting column to exchange buffer to storage buffer (50 mM Na₂HPO₄, 100 mM NaCl, pH 7.5).

Store purified protein at 4 °C.

Quality control: Run SDS-PAGE of elution fractions; save images and record corresponding UV peaks.

6.Desalting / buffer exchange

Connect desalting column; wash with deionized water at 8–10 mL/min until baseline.

Load sample at 8 mL/min (max 15 mL).

Elute with Buffer C, collect when UV increases (approx. 20 mL).

Stop when conductivity (mS/cm) increases.

Record conductivity, UV peaks, fractions.

SDS-PAGE: Mix with reducing buffer, run at 220 V, 95 °C for 5 minutes; load 5–20 µL; stain (Coomassie / silver stain); save images and filenames.

7.Protein concentration and quality control

Use Amicon Ultra (appropriate MWCO) to concentrate protein to 1–5 mg/mL.

Measure concentration with Bradford assay and record.

Turbo-PETase

Experimental materials:

Experimental instruments:

Experimental procedure:

1.Seed culture and inoculation

Seed preparation: Pick several single colonies from a plate using a 10 µL pipette tip.

Transfer them into a 400 mL 2xYT flask containing 5 µL Kanamycin (Kan).

Culture at 37 °C, 200 rpm, for 3-4 hours.

Continue culturing until the optical density at 600 nm (OD600) reaches 0.4-0.6.

(Note: Take 1 mL samples periodically in a biosafety cabinet and measure OD with a spectrophotometer).

2.Induced expression

Add 1 mM IPTG.

Lower the temperature to 20 °C.

Perform a 20-hour induction at 200 rpm.

Record the induction time and temperature.

3.Cell harvest

Centrifuge cells in 50-100 mL tubes at 4 °C, 10,000xg, for 10 minutes.

Collect the cells.

4.Resuspension / lysis

Wash cells twice with Buffer A.

Resuspend 1 g of cell pellet in 10 mL of lysis buffer.

Lyse on ice using sonication: 300 W, 3 seconds sonication with a 2-second interval, for 20 minutes (duration may depend on biomass).

Centrifuge at 4 °C, 14,000xg, for 60 minutes.

Collect the supernatant.

Filter the supernatant using a 0.22 µm syringe filter.

SDS-PAGE: Mix with reducing loading buffer, run at 220 V, heat at 95 °C for 5 minutes, load 5-20 µL, stain (Coomassie / silver stain), save images and filenames.

5.Ni-NTA affinity purification

Column preparation: Resin capacity: 40 mg protein/mL resin (calculate by column volume), flow rate: Start at 0.5 mL/min for Ni-column setup, then adjust to 5 mL/min, wash with deionized water at 5 mL/min until baseline, equilibrate with lysis buffer.

Loading: Al pump injection at 5 mL/min (stop 2-3 mL early to avoid air), equilibrate with lysis buffer until baseline.

Elution / collection: Set a gradient or directly elute with lysis buffer, collect when UV (mAu) increases (10-15 mL per fraction), save fraction numbers, use a HiPrep 26/10 desalting column to exchange buffer to storage buffer (50 mM Na2HPO4, 100 mM NaCl, pH 7.5), store purified protein at 4 °C.

Quality control: Run SDS-PAGE of elution fractions, save images and record corresponding UV peaks.

6.Desalting / buffer exchange

Connect desalting column, wash with deionized water at 8-10 mL/min until baseline, load sample at 8 mL/min (max 15 mL), elute with Buffer C, collect when UV increases (approx. 20 mL), stop when conductivity (mS/cm) increases, record conductivity, UV peaks, fractions.

SDS-PAGE: Mix with reducing buffer, run at 220 V, heat at 95 °C for 5 minutes, load 5-20 µL, stain (Coomassie / silver stain), save images and filenames.

7.Protein concentration and quality control

Use Amicon Ultra (appropriate MWCO) to concentrate protein to 1-5 mg/mL.

Measure concentration with Bradford assay and record.

Tm

Experimental materials:

Experimental instruments:

Experimental procedure:

1. Preparation of SYPRO Orange 50x working solution:

Instructions: "In the dark, take 2.5 µL SYPRO Orange (5000x) → add 247.5 µL deionized water → 50× working solution."

Further instructions: "Mix, aliquot, and store cold or use immediately. Avoid repeated freeze-thaw and prolonged light exposure."

2. Sample preparation:

"Final reaction volume per well: 25 µL."

"Each well:"

"Protein solution (adjust concentration as required): 22.5 µL."

"50x SYPRO Orange: 2.5 µL (final 5x)."

"If adding ligand/small molecule: dissolve in buffer or DMSO first; adjust concentration ensuring final DMSO < 2%."

"Example: If protein is 0.58 mg/mL ≈ 7.7 µM, use 22.5 µL protein + 2.5 µL 50× dye → 25 µL total; final protein concentration still ~7.7 µM (slightly diluted)."

"Include blank wells (buffer + dye) 2-3; technical triplicates per condition ≥3 to reduce noise."

3. Plate setup:

"Add samples to black qPCR plate, avoid bubbles; if bubbles form, centrifuge briefly (1-2k g, 10-15 s)."

"Seal with optical film; press edges tightly to avoid leakage."

"Place plate into instrument (ensure filters compatible with SYPRO Orange: Ex = 470 nm / Em ≈ 570 nm). Minimize light exposure and operate quickly to reduce fluorescence drift."

4. Instrument parameter settings:

"Temperature range: 20 °C → 95 °C."

"Heating rate: 1.0 °C/min."

"Data collection: every 0.2-0.5 °C."

"Excitation power/gain: 80%."

"Lid/heating cover: off or set with minimal offset (to prevent overheating and evaporation)."

5. Run and record

Start run; record fluorescence vs temperature curve. Typically 20–95 °C, 1 °C/min, ~75 min total.

Experimental results:

Successfully purified and obtained the target protein for subsequent research.

Experimental notes:

1. Before use, the Ni column must be filled with buffer to prevent air bubbles; ensure tight connections during installation to avoid detachment.

2. During elution, monitor peak shape changes; when the peak begins to plateau, add 3% buffer B to improve target protein collection efficiency.

Experimental notes:

1. Concentration tubes must be rinsed with ultrapure water before and after use; after alcohol immersion, they must be thoroughly cleaned to prevent protein denaturation.

2. Before each centrifugation, balance two tubes to avoid centrifuge imbalance.

3. After concentration, aliquot the protein into 200 µL per tube to avoid repeated freeze-thaw cycles; do not touch the membrane during operation to prevent damage to the concentrator membrane.

Table 8: Tm table of Novozymes commercial CGTase

2.6

Experiment Date: 7.27-8.2

Experiment Personnel: Fang Yingshan, Su Yanyan, Wang Shuhan, Xiang Yuhang

Experimental Objective: Using phenolphthalein as the substrate, evaluate enzyme activity by detecting the amount of enzymatic reaction products via HPLC.

Experimental Materials

Experimental Instruments:

Experimental procedure:

Prepare a 500 µL reaction system: add 450 µL 0.5 mM Na₂HPO₄ buffer (pH 8), add PET plastic film to reach 30 g/L, add 15 mg soluble starch, add 50 µL Turbo-PETase to achieve 2 mg/g PET, and add 5 µL β-CGTase-K647E (protein concentration 0.58 mg/mL). Incubate in 60°C water bath for 1 h, 2 h, and 3 h. After reaction, add equal volume methanol to inactivate. Take 5 µL reaction mixture, dilute 20× with water, and analyze by reversed-phase C18 HPLC (260 nm UV) to determine PPA and MHET formation. Undiluted reaction mixture used to determine α-, β-, and γ-cyclodextrin formation.

Experimental Conclusion:

Successfully obtained kinetic analysis of Turbo-PETase degradation of PET products, providing data support for subsequent experiments.

Table 9: Analysis of product formation from Turbo-PETase degradation of PET film at different reaction times (65°C reaction)

Experimental notes:

1. Before use, the Ni column must be filled with buffer to prevent air bubbles; ensure tight connections during installation to avoid detachment.

2. During elution, monitor peak shape changes; when the peak begins to plateau, add 3% buffer B to improve target protein collection efficiency.

2.10

Experiment Date: 8.24-8.30

Experiment Personnel: Wei Sihui, Su Yanyan, Xiang Yuhang

Experimental Objective: Cultivate and induce expression of the recombinant strain, collect cells, purify and desalt to obtain target enzyme protein, and investigate the effect of methanol inactivation on Turbo-PETase activity as well as enzyme activity measurement in co-substrate systems.

Experimental Materials

Experimental Instruments:

Experimental procedure:

1. Add 15 mg PET powder, 15 mg soluble starch, 450 µL 100 mM Na2HPO4 buffer, 50 µL Turbo-PETase (0.6 mg/mL), and 5 µL β-CGTase-K647E (0.58 mg/mL) to the reaction system; adjust pH to 8 and incubate in 65°C water bath for 1 h.

2. Divide the system into two groups: one group is inactivated with equal volume anhydrous methanol; to ensure 20× total dilution, load 10 µL reaction + 90 µL water onto HPLC. The other group transfers substrate (no methanol inactivation); total dilution also 20x, load 20 µL reaction + 180 µL water for HPLC analysis.

Experimental Conclusion:

The TPA peak retention time in the methanol-inactivated group was significantly shortened. Under the same injection rate, it eluted earlier than in the non-inactivated group. This indicates that methanol inactivation of Turbo-PETase facilitates the reaction and optimizes experimental conditions.

Table 10: TPA production under different conditions

Table 11: Production of different cyclodextrins (CDs) under different conditions

Experimental notes:

1. Pre-incubate culture medium in a 37°C incubator; do not use if contamination occurs.

2. Place competent cells at an angle when incubating on a shaker.

3. When spreading onto culture medium, place the bacterial suspension near the center; seal the plate once no visible liquid remains on the surface.

2.11

Experiment Date: 8.31-9.6

Experiment Personnel: Fang Yingshan, Su Yanyan, Wang Shuhan, Xiang Yuhang

Experimental Objective: To investigate the effects of starch on PET degradation and determine the optimal conditions for both PET film and PET powder substrates. The experiment aims to compare the degradation efficiency with and without starch as a co-substrate.

Experimental Materials

Experimental Instruments:

Experimental procedure:

1. Reaction system preparation: Prepare 500 µL reaction mixtures containing 450 µL 0.5 mM Na₂HPO₄ buffer (pH 8), 50 µL Turbo-PETase (0.6 mg/mL), and 5 µL β-CGTase-K647E (0.58 mg/mL).

2. Substrate addition: For PET film reactions: cut 15 mg pieces (1×1 cm²). For PET powder reactions: weigh 15 mg powder. Add 15 mg soluble starch to half of the reactions as co-substrate.

3. Incubation: Incubate all reaction tubes in a 65°C water bath for 3 hours with gentle shaking (100 rpm). Monitor temperature stability throughout the incubation period.

4. Reaction termination: Add equal volume (500 µL) anhydrous methanol to terminate enzymatic reactions. Mix thoroughly and store at 4°C until HPLC analysis.

5. HPLC analysis: Dilute samples 20-fold with ultrapure water, filter through 0.22 µm syringe filters, and analyze using reversed-phase C18 HPLC with UV detection at 260 nm. Quantify TPA and MHET production using external calibration curves.

Experimental results:

The experiment successfully demonstrated the effect of starch on PET degradation efficiency. Results showed enhanced degradation rates when starch was present as a co-substrate, particularly for PET powder substrates. The dual-enzyme system exhibited synergistic effects in the presence of starch, leading to increased production of both TPA and MHET degradation products.

Table 12: HPLC Quantitative Analysis of TPA/MHET Production

2.12

Experiment Date: 9.7-9.13

Experiment Personnel: Xu Yiran, Wei Sihui, Kuang Xintong

Experimental Objective: To explore the dual-enzyme system's capability with alternative substrates including rice, steamed bun, and other starch-rich materials. The experiment aims to expand the substrate range and optimize γ-cyclodextrin production using different carbohydrate sources.

Experimental Materials

Experimental Materials

Experimental procedure:

1. Substrate preparation: Weigh 20 mg of each alternative substrate (rice, steamed bun, wheat flour, corn starch, potato starch) and combine with 15 mg PET powder in separate reaction tubes.

2. Enzyme addition: Add 450 µL 100 mM Na₂HPO₄ buffer (pH 8), 50 µL Turbo-PETase (0.6 mg/mL), and 5 µL β-CGTase-K647E (0.58 mg/mL) to each reaction system.

3. Incubation: Incubate all reaction systems in a 65°C water bath for 2 hours with gentle shaking (100 rpm). Monitor temperature and pH stability.

4. Analysis: Terminate reactions with methanol, dilute samples, filter through 0.22 µm syringe filters, and analyze by HPLC for cyclodextrin production (α-, β-, γ-cyclodextrins) and PET degradation products (TPA, MHET).

Experimental conclusion:

The dual-enzyme system successfully utilized alternative substrates for enhanced γ-cyclodextrin production. Rice and steamed bun substrates showed the highest γ-cyclodextrin yields, demonstrating the system's versatility in processing diverse carbohydrate sources. The expansion of substrate range provides new opportunities for industrial applications and waste valorization.

2.13

Experiment Date: 9.14-9.20

Experiment Personnel: He Shunkang, Wang Shuhan, Xiang Yuhang

Experimental Objective: To prepare large-scale phosphorescent materials using the supramolecular assembly approach. The experiment aims to synthesize fluorescent and phosphorescent materials with enhanced optical properties for potential applications in sensing and imaging technologies.

Experimental Materials

Experimental Materials

Experimental procedure:

Large-scale preparation of phosphorescent materials involved the supramolecular assembly of β-cyclodextrin with various fluorophores and phosphorophores. The process included: (1) preparation of host-guest complexes by mixing β-cyclodextrin with guest molecules in DMSO/water solutions, (2) optimization of molar ratios for maximum encapsulation efficiency, (3) purification through dialysis and column chromatography, (4) characterization using fluorescence, phosphorescence, and structural analysis techniques, and (5) evaluation of optical properties under different pH and temperature conditions. The materials were prepared in 10-gram batches for industrial-scale applications.

Experimental conclusion:

Successfully prepared large-scale phosphorescent materials with enhanced optical properties. The supramolecular assembly approach resulted in materials with improved quantum yields, extended phosphorescence lifetimes, and excellent photostability. The prepared materials showed potential for applications in bioimaging, sensing, and optoelectronic devices.

2.14

Experiment Date: 9.21-9.27

Experiment Personnel: Fang Yingshan, Su Yanyan, Wang Shuhan, Xiang Yuhang

Experimental Objective: To establish an alternative detection method using fluorescence spectroscopy for monitoring enzyme activity and product formation. The experiment aims to develop a rapid, sensitive, and non-destructive analytical approach for real-time monitoring of enzymatic reactions.

Experimental Materials

Experimental Instruments:

Experimental procedure:

The alternative detection method was established using fluorescence spectroscopy to monitor enzyme activity in real-time. The procedure involved: (1) labeling substrates with fluorescent probes, (2) establishing calibration curves for product quantification, (3) optimizing excitation and emission wavelengths, (4) measuring fluorescence intensity changes during enzymatic reactions, and (5) correlating fluorescence signals with HPLC-determined product concentrations. The method was validated for accuracy, precision, and sensitivity.

Experimental conclusion:

Successfully established an alternative detection method using fluorescence spectroscopy for monitoring enzymatic reactions. The method provided rapid, sensitive, and non-destructive analysis of enzyme activity and product formation, offering advantages over traditional HPLC methods in terms of speed and real-time monitoring capabilities.