Protocol

Construction of Recombinant Plasmids

(1) Acquisition of Target Gene

The DNA sequences of the target genes were obtained from the NCBI database. Specific primers were designed using SnapGene software to amplify the target genes from the corresponding wild-type strains, adding homologous arms for recombination with the plasmid backbone. PCR was performed according to the reaction system in Table 1 and the thermal cycling program in Table 2 to obtain the target gene fragments with homologous arms.

Note: use 1 µL if the template is a bacterial culture; 0.3–1 µL if it is plasmid DNA.

Note: adjust annealing temperature according to primer design; use 56 °C if not otherwise specified.

After PCR, add 5 µL 10× DNA Loading Buffer to the reaction, mix using a vortex and quick-spin centrifuge, then load into 1.5% agarose gel wells (make sure no leakage and check gel orientation). Load 10 µL DNA marker in one lane. Run electrophoresis at 180 V. Compare to DNA marker to confirm target band size and position. If correct, excise the band using a Fast Gel Extraction Kit following the instructions below. Quantify the recovered DNA using a Gene5™ Take3 Session on a plate reader, and store at −20 °C.

Fast gel extraction steps:

1. Under UV light, locate the desired band, excise it with a clean scalpel, and place it into a 1.5 mL microtube containing 650 µL GSB solution. Heat in a water bath until the gel dissolves completely, then cool on ice to room temperature.
2. Transfer the cooled solution into the spin column of the kit. If the volume is large, load in batches. Let stand 1 min, centrifuge at 1000 rcf for 1 min, discard the flow-through.
3. After the DNA has bound to the column membrane, add 650 µL ethanol-containing Wash Buffer, centrifuge at 1000 rcf for 1 min, discard the flow-through, then centrifuge again at 1000 rcf for 1–2 min to remove residual liquid.
4. Place the column in a clean 1.5 mL tube and incubate at 65 °C for 1–2 min to evaporate residual ethanol.
5. Finally, elute by adding 30 µL pre-warmed ddH₂O or kit EB buffer into the column, centrifuge at 1000 rcf for 1 min. The eluate contains the target DNA fragment; store at −20 °C.

(2) Acquisition of Plasmid Backbone

Using laboratory-stock pWT-021a plasmid as the template, primers were designed in SnapGene. PCR was carried out as in Table 1 and Table 2 to amplify the plasmid backbone.

(3) Homologous Recombination of Target Gene with Plasmid Backbone

Recombination steps:

1. Mix components as in Table 4 and incubate at 50 °C in water bath for 25 min. If plasmid is >8 kb, extend incubation to 30 min.
2. Place reaction on ice for 5 min; when 2 min remain, retrieve E. coli DH5α competent cells from −80 °C freezer and keep on ice.
3. In a biosafety cabinet, add the entire recombination reaction to thawed competent cells, gently mix with pipette tip, and incubate on ice for 30 min.
4. Heat-shock at 42 °C for 90 s, then immediately place on ice for 5 min.
5. Add 600 µL fresh LB (no antibiotics) in biosafety cabinet and recover at 37 °C, 220 rpm for 45–60 min.
6. Centrifuge at 6000 rpm for 1 min, remove 400–500 µL supernatant, and gently resuspend the pellet.
7. Plate the resuspended cells evenly on selective LB agar containing the appropriate antibiotic(s), allow plates to dry, seal with parafilm, and incubate inverted at 37 °C for 12 h.

(4) Screening of Positive Clones

Open the incubated plates in a biosafety cabinet, pick single colonies into 10 µL sterile water. Use 1 µL as the template for colony PCR to verify the presence of the target insert.

Note: use 1 µL bacterial culture or 0.3–1 µL plasmid as template.

(5) Strain Preservation

Single colonies confirmed by sequencing were inoculated into LB liquid medium containing the appropriate antibiotic and cultured for 8–10 h. For long-term storage, mix the culture 1:1 (v/v) with 50% glycerol and store at −80 °C.

Plasmid Extraction

Activated bacterial cultures were subjected to plasmid extraction to obtain sufficient amounts of plasmid for transformation into chassis strains.

Extraction steps:

1. Transfer 5 mL of activated bacterial culture into a centrifuge tube and centrifuge at 12,000 rpm for 1 min, then collect the pellet. (For higher plasmid yield, increase culture volume as needed.)
2. Add colorless RB solution containing RNase A (store at 2-4 °C after addition) to the pellet at a ratio of 250 µL RB per 5 mL culture, and vortex to resuspend the cells thoroughly.
3. Add blue LB solution at a ratio of 250 µL LB per 5 mL culture, invert gently 4-6 times to mix, and let stand for 5 min until the solution turns completely transparent blue, indicating full lysis of the cells.
4. Add yellow NB solution at a ratio of 350 µL NB per 5 mL culture, invert gently 5-6 times to mix, and let stand for 2 min until yellow flocculent aggregates form.
5. Centrifuge at 12,000 rcf for 5 min to pellet cell debris, transfer the supernatant to a spin column provided in the kit, and centrifuge at 12,000 rcf for 1 min. Discard the flow-through.
6. Add 650 µL WB solution (with ethanol added) to the spin column, centrifuge at 12,000 rcf for 1-2 min, remove any residual WB solution, and discard the flow-through.
7. Place the spin column in a clean 1.5 mL microtube, add 30-50 µL EB buffer or pre-heated ddH₂O (60-70 °C) directly to the center of the membrane, let stand for 1 min.
8. Centrifuge at 12,000 rcf for 1 min to elute DNA into the microtube. Store plasmid DNA at −20 °C for later use.

Preparation of E. coli Nissle 1917 (EcN) Competent Cells and Electroporation

Plasmids extracted above were introduced into EcN competent cells to construct the desired recombinant strains. The preparation and transformation steps were as follows:

Preparation of Competent Cells

1. Take EcN stock from −80 °C freezer and streak onto LB agar plates without antibiotics in a biosafety cabinet. Allow plates to dry, then incubate inverted at 37 °C for 10 h for activation.
2. Scrape appropriate amounts of activated EcN colonies and inoculate into 5 mL LB broth, incubate at 37 °C, 220 rpm for 10–12 h for secondary activation.
3. Transfer 500 µL of secondary culture into 100 mL fresh LB broth, grow until OD₆₀₀ = 0.6–0.8.
4. Chill the culture on ice for 30 min, then centrifuge at 4 °C, 3000 rcf for 10 min.
5. Discard the supernatant in a biosafety cabinet, resuspend the cell pellet in 30 mL of ice-cold 10% glycerol, centrifuge again at 4 °C, 3000 rcf for 10 min.
6. Repeat step 5 three times in total. Finally, resuspend the pellet in 800 µL ice-cold 10% glycerol, aliquot 100 µL per sterile microtube.
7. Keep aliquoted competent cells on ice for 30 min, snap-freeze in liquid nitrogen, and store at −80 °C for future use.

Electroporation of Plasmids into EcN

1. Place both plasmid DNA and competent cells on ice for 5 min to thaw. In a biosafety cabinet, gently add 5 µL plasmid DNA to an aliquot of EcN competent cells, mix gently without pipetting up and down, and incubate on ice for 10 min.
2. Retrieve pre-sterilized, UV-treated and dried electroporation cuvettes from −20 °C freezer (if removed earlier, wipe off condensation and keep on ice).
3. Transfer the plasmid–cell mixture into the bottom of the cuvette, close the lid, place in the electroporator, and deliver a single pulse at 2.5 kV, 4 ms. Immediately after pulsing, add 600 µL fresh antibiotic-free LB, mix gently, transfer to a sterile microtube, and recover at 37 °C, 220 rpm for 45–60 min.
4. Proceed with screening of positive clones and preservation as described in steps 6–7 of the homologous recombination protocol.

Protein Expression and Purification

This experiment involves the expression and purification of uricase.

(1) Strain Activation

1. Take EcN from a −80 °C freezer, streak onto antibiotic-free LB agar plate in a biosafety cabinet, air-dry, and incubate inverted at 37 °C for 10 h.
2. Pick appropriate colonies and inoculate into 5 mL liquid LB, incubate at 37 °C, 220 rpm for 10–12 h for secondary activation.

(2) Scale-up Culture and Induction

1. Transfer 5 mL of activated culture into 100 mL LB containing appropriate antibiotics, grow at 37 °C, 220 rpm for 2–3 h until OD₆₀₀ reaches 0.6–0.8.
2. Add 100 µL IPTG (96 mg/mL) to a final concentration of 0.1 mM under aseptic conditions.
3. Induce expression at either 18 °C, 220 rpm for 12–16 h or 37 °C, 220 rpm for 2–3 h.

(3) Cell Collection and Ultrasonic Disruption

1. After induction, centrifuge cells at 4 °C, 8000 rpm for 10 min to collect pellets.
2. Resuspend cells in PBS to remove residual medium and repeat twice.
3. Finally, resuspend pellets in 30–40 mL PBS for ultrasonic disruption.
4. Ultrasonic parameters: 3 s on / 5 s off, 40% power, disrupt until lysate is clear (do not exceed 40 min).
5. Maintain low temperature to avoid enzyme inactivation.
6. Centrifuge disrupted lysate at 4 °C, 8000 rpm for 10 min, collect the supernatant.

(4) Protein Purification

1. Ni-column equilibration: add 20 mL of 10 mM imidazole to the Ni-column, allow it to flow through naturally.
2. Purification: load the clarified supernatant onto the column, let it flow through, then wash with imidazole 20 mM to remove impurities.
3. Elution: close the column outlet, add 5 mL of 250 mM imidazole, incubate for 5 min to elute uricase, collect eluate, and keep on ice.
4. Column regeneration: wash with 20 mL of 400 mM NaOH, allow to flow through, then wash with 20 mL of 10 mM imidazole and store the column in 10 mM imidazole at 4 °C.

Note: All buffers must be pre-chilled and filtered through a 0.45 µm filter before use.

(5) Protein Concentration

1. Ultrafiltration tube cleaning: choose an appropriate MWCO ultrafiltration tube, remove original 20% ethanol, wash with ddH₂O at 4 °C, 3400 rcf until ∼500 µL remains. Replace ddH₂O with PBS and repeat.
2. Concentration: remove residual PBS, add the eluted protein from the Ni-column (add in batches if exceeding capacity, top up with PBS if insufficient). Centrifuge at 4 °C, 3400 rcf until volume is reduced to 500–1000 µL, refill with PBS, repeat until concentrated to ∼500 µL.
3. Store the concentrated protein at 4 °C for short-term or mix with 50% (v/v) glycerol and store at −80 °C for long-term.
4. Ultrafiltration tube storage: fill with 400 mM NaOH, centrifuge at 4 °C, 3400 rcf for 30 min, discard NaOH, wash twice with ddH₂O, finally fill with 20% ethanol and store at 4 °C.

Uricase Secretion Assay

Strain activation: recover uricase-expressing strain from −80 °C, streak on antibiotic-free LB plate, incubate at 37 °C for 10 h, then inoculate into 5 mL liquid LB and incubate at 37 °C, 220 rpm for 10–12 h.

Scale-up culture: inoculate 300 µL seed culture into 30 mL LB with appropriate antibiotics, grow at 37 °C, 220 rpm for 12–14 h.

Cell separation: centrifuge at 4 °C, 3000 rcf for 10 min to separate cells and supernatant, collect both fractions.

Cell lysis: process as described in section 16 (ultrasonic disruption) to obtain intracellular uricase.

Ultrafiltration of secreted protein:
Clean 10 kDa MWCO ultrafiltration tube as described in section 16.
Concentrate secreted uricase from culture supernatant at 4 °C, 3400 rcf, repeating until final volume is approximately 500 µL.
Store concentrated filtrate on ice for immediate purification to prevent enzyme inactivation.
Clean ultrafiltration tube as in section 16.

Western Blot Analysis

Gel preparation: assemble clean 1 mm gel plates.

Lower gel: mix 2.5 mL lower gel solution + 2.5 mL lower gel buffer + 70 µL catalyst, pour and overlay with isopropanol, allow to polymerize for 30 min, then remove isopropanol.

Upper gel: mix 1 mL upper gel solution + 1 mL upper gel buffer + 40 µL catalyst, pour over lower gel, insert comb until gel solidifies.

Sample preparation: mix protein samples with loading buffer, boil at 95 °C for 3–5 min, cool to room temperature.

Electrophoresis: place gel plates in electrophoresis tank filled with running buffer, remove comb, load samples (15-well comb: 15 µL per well; 10-well comb: 25 µL per well), run at 80 V for 30 min, then 120 V for 60 min until protein markers are resolved.

Coomassie staining: for one gel, stain with Coomassie Brilliant Blue for 30 min, wash and boil in water for 2–3 min to visualize bands.

Western blotting:

1. Equilibrate gel, PVDF membrane (pre-activated with methanol), filter papers, and sponge in transfer buffer.
2. Assemble transfer “sandwich”: sponge–filter paper–PVDF–gel–filter paper–sponge, remove air bubbles.
3. Transfer at 1 min per kDa (e.g., 30 min for <30 kDa proteins), then at 100 V constant voltage for 40 min at low temperature.
4. Block membrane in 5% BSA for 2 h at room temperature, add 2 µL Anti-His antibody, incubate overnight at 4 °C.
5. Wash membrane with TBST 3×15 min, incubate with 10 mL TBST + 1 µL Anti-mouse HRP for 1 h at room temperature, wash again 3×15 min.
6. Develop with 1:1 ECL substrate and image using a chemiluminescence analyzer.

Molecular Docking Experiment

Molecular docking was performed using AutoDock to identify potential sites in mutant proteins generated by directed evolution.

(1) Preparation of Ligand Structure File

Search for uric acid on PubChem, download the 3D Conformer (SDF) file. Convert SDF to PDB format using PyMOL: open the SDF file via File → Open, then export as PDB via File → Export Molecule.

(2) Preparation of Protein Structure File

Obtain the amino acid sequence of the target protein from NCBI, input it into AlphaFold for 3D structure prediction. From the AlphaFold output, select model_0 (highest confidence) for docking.

(3) Preparation of Ligand and Protein PDBQT Files

Docking algorithms require atomic charges and atom type definitions that are not contained in PDB files. Thus, preprocess both ligand and protein PDB files to generate PDBQT files that include this information together with atomic coordinates.

(4) Preparation of Ligand PDBQT File

1. Run AutoDock Tools.
2. In File → Preferences, set the working directory path (ensure no Chinese characters in path).
3. Load ligand via Ligand → Input → Open (switch file format to PDB).
4. Select ligand via Ligand → Input → Choose.
5. Define flexible and rigid parts of the ligand:
   • Ligand → Torsion Tree → Detect Root to set the root.
   • Ligand → Torsion Tree → Set Number of Active Torsions to define degrees of freedom (here set to 0 for rigid ligand).
6. Identify aromatic carbons under Ligand → Aromatic Carbons → Aromatic Criterion, typically set deviation angle to 7.5.
7. Save ligand as PDBQT via Ligand → Output → Save as PDBQT.

(5) Preparation of Protein PDBQT File

1. Run AutoDock Tools again.
2. Set working directory under File → Preferences → Set.
3. Load protein via File → Read Molecular.
4. Remove water molecules via Edit → Delete Water.
5. Add polar hydrogens via Edit → Hydrogens → Add Polar Only.
6. Add charges via Edit → Charges → Add Kollman Charges.
7. Save as PDBQT via Grid → Macromolecular → Choose.

(6) Definition of Docking Box and Configuration File

Select both the target protein and ligand PDBQT files in AutoDock Tools, set grid box parameters to enclose all active sites (or entire protein if active site is unknown). Set Vina input parameters as in Table 8.

Save the configuration file to the working directory.

(7) Running AutoDock Vina

1. Run docking via Run → Run AutoDock Vina.
2. In the Start Vina window, select the saved configuration file under Config Filename, click Launch, and wait for the AutoDock Process Manager window to close automatically—this indicates docking is finished.
3. Docked conformations (modes) are automatically saved as PDBQT files in the working directory.

(8) Visualization of Docking Results with PyMOL

1. Load the desired docked mode PDBQT file, then load the target protein PDBQT file.
2. Save the complex as a PDB file.
3. Reopen the PDB file in PyMOL, select and name the ligand and protein for clarity.
4. Visualize interactions such as hydrogen bonds and adjust colors/transparency to highlight the docking interface.
5. Save the visualization as a PSE file.

Construction of Point-Mutated Strains

Based on the amino acid positions to be mutated in the nucleotide sequence, corresponding primers were designed using SnapGene software. Plasmids carrying the desired point mutations were then constructed following the same procedure described in the recombinant plasmid construction section. After extraction, the mutant plasmids were transformed into E. coli Nissle 1917 competent cells.

Primer sequences for point mutations are listed in the corresponding table (not shown here). Changes in promoter and ribosome binding site (RBS) sequences were also performed following the same recombinant plasmid construction protocol.

Antibiotic Sensitivity Assay

Activated strains were inoculated into media containing different concentrations of various antibiotics at a 1% (v/v) inoculation ratio. Transfer 200 μL of each culture into a clear-bottom 96-well plate and incubate at 37 °C, 1000 rpm in a microplate shaker. Measure OD₆₀₀ every 1 h using a microplate reader. Plot growth curves under different antibiotic conditions to evaluate the antibiotic sensitivity of the strains.

Minimum Inhibitory Concentration (MIC) Assay

Prepare the medium according to the formulation below or use 22 g MH/CAMHB broth per liter of ddH₂O.

(1) Preparation of Sample Working Solutions

Add 190 μL medium to wells A1-H1 of a 96-well plate; add 100 μL medium to all other wells. Add 10 μL sample stock solution to wells A1-H1, mix well. Transfer 100 μL from A1-H1 to A2-H2, mix well. Continue two-fold serial dilution down the plate to wells A11-H11. The final concentration range in the plate is 2.56 mg/mL to 0.0025 mg/mL. In a new 96-well plate, add 20 μL of the corresponding diluted sample solution to each well; leave column 12 (A12-H12) without antibiotic as a growth control.

(2) Inoculation and Incubation

Prepare a suspension of engineered strain YES302 at turbidity equivalent to 0.5 McFarland standard, then dilute 1:1000 with medium. Add 180 μL of the diluted bacterial suspension to each well. The final sample concentration in wells A1-H1 ranges from 256 μg/mL to 0.25 μg/mL. Incubate at 35 °C in a normal-air incubator for 16-20 h. The test is valid only if the negative control wells (no sample) show clear bacterial growth. The lowest antibiotic concentration that results in a clear, transparent well without visible bacterial growth is recorded as the MIC.

Plasmid Stability Test

Grow strains in LB medium with the corresponding antibiotic, and transfer them every 12 h into antibiotic-free LB for serial passaging. After sampling, appropriately dilute the cultures and plate onto both antibiotic-free LB agar and antibiotic-containing LB agar. Calculate plasmid stability using the formula below:

Measurement of Growth Curves

Strains stored at −80 °C were streaked evenly on LB agar plates. Single colonies were inoculated into 5 mL liquid LB medium and cultured overnight at 37 °C, 220 rpm.

The next day, the overnight culture was inoculated into 30 mL liquid M9 medium and 30 mL liquid LB medium, adjusting the initial OD₆₀₀ to 0.05. At hourly intervals, take 200 µL of culture (three replicates per sample) and measure the OD₆₀₀ using a microplate reader. Use these data to plot growth curves.

Eudragit® L100 Coating of Engineered Probiotics

This technique uses calcium phosphate and the L100-55 polymer to coat engineered probiotics at acidic pH.

Grow the engineered bacteria in 4 mL LB medium at 37 °C, 200 rpm for 12 h. Centrifuge at 4000 rcf for 5 min, wash the cells twice with PBS, and resuspend them in 900 μL ice-cold calcium phosphate solution (containing 12.5 mM CaCl₂). Vortex for 5 min, then add 500 μL of 1 mg/mL L100-55 solution and shake for an additional 5 min. Adjust the pH of the suspension to approximately 5.0 with 0.1 M HCl, then collect coated cells by centrifugation. Wash the coated cells twice and resuspend in 1 mL PBS (pH < 5.0). Store the suspension at −20 °C until use.

CFU-OD Standard Curve

Culture preparation: inoculate overnight culture 1:100 into fresh medium, grow at 37 °C, 220 rpm to OD₆₀₀ ≈ 0.5–1.0.

Ten-fold serial dilution: prepare 7–8 sterile tubes each with 900 µL sterile diluent, label 10⁻¹–10⁻⁷. Transfer 100 µL sequentially between tubes, mixing thoroughly each time.

OD measurement: measure OD₆₀₀ of each dilution, record corrected OD.

Plating: plate 100 µL from selected dilutions (targeting 30–300 CFU/plate) onto agar plates, incubate at 37 °C inverted for 12–18 h.

Counting: select countable plates, average colony counts across technical replicates.

Calculation:

CFU/mL = $\frac{\mathrm{average colony count}}{0.1 \mathrm{mL}}\times \mathrm{dilution factor}$

Fit regression of log₁₀(CFU/mL) vs. OD₆₀₀ or CFU/mL vs. OD₆₀₀ for subsequent rapid estimation.

Survival and Xanthine Transport in Simulated Gastrointestinal Fluids

Baseline CFU: prepare and cultivate engineered strains as in the xanthine transport assay. Centrifuge to collect cells, resuspend in M9 + Smr, measure OD₆₀₀, dilute, and plate on Smr-LB agar, incubate overnight to count colonies.

Gastrointestinal challenge: incubate another aliquot of cells in simulated gastric fluid at 37 °C for 5 min, centrifuge to remove gastric fluid, then incubate in simulated intestinal fluid at 37 °C, 220 rpm for 60 min, plate as above to count surviving CFU.

Xanthine transport assay: adjust both untreated and challenged cells to OD₆₀₀ = 1 in M9 medium, add xanthine to final concentration 100 μM (for challenged cells add xanthine together with intestinal fluid). Incubate at 37 °C, 220 rpm for 60 min, then measure residual xanthine as described in the in vitro xanthine transport assay.

Time-course transport: for challenged cells, measure residual xanthine at 15, 30, 45, and 60 min under the same conditions.

In Vitro Xanthine Transport Assay

(1) Activation and Cultivation of Strains

Retrieve engineered strains from −80 °C, streak 10 µL onto LB agar containing streptomycin (Smr) for activation. Incubate at 37 °C for 10–12 h, then inoculate into 700 µL Smr-containing LB in a 24-well plate. Culture at 37 °C, 1000 rpm for 12 h, then transfer at 1% (v/v) inoculation ratio into 30 mL M9 medium supplemented with 30 µL Smr. Incubate at 37 °C, 220 rpm for 12–14 h. Harvest cells by centrifugation at room temperature, 6000 rpm for 10 min, and resuspend in M9 solution.

(2) Whole-Cell Transport Assay

Adjust the whole-cell suspension to OD₆₀₀ = 0.6 or 1.0 in the reaction mixture. Add xanthine to reach the desired final concentration. Incubate at 37 °C, 220 rpm for 1 h. After incubation, centrifuge at 6000 rpm for 2 min, collect the supernatant, filter through a 0.22 µm aqueous filter, and analyze the xanthine concentration in the filtrate by HPLC. For time-course studies at a fixed xanthine concentration, follow the same procedure but sample at different time points. For testing transport efficiency under different incubation conditions (aerobic, microaerobic, and anaerobic), follow the same procedure with the corresponding culture conditions.

In Vitro Hypoxanthine Transport Assay

The activation and cultivation of strains and the whole-cell transport assay were performed as described in the in vitro xanthine transport assay, except that the xanthine solution was replaced with hypoxanthine.

In Vitro Adenine Transport Assay

The activation and cultivation of strains and the whole-cell transport assay were performed as described in the in vitro xanthine transport assay, except that the xanthine solution was replaced with adenine.

In Vitro Uric Acid Transport Assay

Mix 500 μL standard bacterial suspension with 500 μL 400 μM uric acid solution in a sterile EP tube to conduct the transport assay. Each strain was divided into two time groups (30 min and 60 min), with three biological replicates per group.

Incubate samples at 37 °C, 220 rpm for the specified time. Centrifuge at 6000 rpm for 2 min, and collect the supernatant for further analysis.

High-Performance Liquid Chromatography (HPLC) Analysis

Chromatographic conditions:

All samples were filtered through a 0.22 μm microporous membrane before injection.

Prepare hypoxanthine/xanthine standards at concentrations of 10.0 μM, 20.0 μM, 25.0 μM, 50.0 μM, and 100.0 μM, analyze them by HPLC, and construct a standard calibration curve based on the linear relationship between peak area and concentration.

Uric Acid Content Measurement

Assay Procedure

Sample loading:
Add 0.2 mL standard to each standard tube.
Add 0.2 mL sample to each test tube.

Protein precipitation:
Add 2.0 mL Reagent 2 to each tube, vortex to mix.
Incubate at room temperature for 10 min.
Centrifuge at 1708 × g for 5 min and collect the clear supernatant.

Supernatant collection:
Transfer 1.6 mL supernatant from each tube into a new EP tube.

Color development:
Add in order to each tube:
0.5 mL Reagent 3 (Alkali Reagent)
0.5 mL Reagent 4 (Phosphotungstic Acid Reagent)
Mix thoroughly and incubate at room temperature for 10 min.

Colorimetric measurement:
Use double-distilled water as blank control (zeroing).
Measure OD at 690 nm using a 1 cm path-length quartz cuvette.

Standard curve construction:
X-axis: Uric acid concentration (mg/L)
Y-axis: ΔA₆₉₀ = A₆₉₀(sample or standard) − A₆₉₀(blank)
Fit the regression equation: y = ax + b.

Calculation of sample UA concentration:

Where:
ΔA₆₉₀ = A₆₉₀(sample) − A₆₉₀(blank)
f = dilution factor of the sample

Uricase Activity Assay

Protein quantification: determine protein concentration using the BCA assay.

Standard curve preparation: prepare uric acid solutions at 0, 10, 20, 40, 80, 100, and 200 µM, measure absorbance at 293 nm using a microplate reader to establish the standard curve.

Enzyme kinetics assay: prepare uric acid solutions at 0–500 µM, adjust volume to 0.9 mL with PBS, add 100 µL enzyme solution, mix thoroughly, transfer into a 96-well plate, and incubate at 37 °C for 10 min. Measure absorbance at 293 nm, dilute samples as necessary. Fit data to the Michaelis–Menten equation using GraphPad Prism 8.0.2 to obtain kinetic parameters.

Uric acid degradation assay: add uric acid to the enzyme solution to a final concentration of 500 µM, incubate at 37 °C, measure residual uric acid every 10 min up to 60 min, and plot the degradation curve.

Whole-Cell Uric Acid Degradation Assay

Activate and cultivate engineered strains as described in the xanthine transport assay, harvest cells, and adjust OD₆₀₀ to 1.0.

1. Incubate the cells in solution containing 100 µM uric acid at 37 °C, 220 rpm for 1 h.
2. Centrifuge at 6000 rpm for 2 min, collect the supernatant, and filter through a 0.22 µm aqueous filter.
3. Measure uric acid concentration by HPLC and plot the degradation curve.

Statistical Analysis and Plotting

All data were analyzed and visualized using GraphPad Prism 8.0.2. Student's t-test was used for comparison between two groups. One-way ANOVA was used for data involving three or more groups. Data fitting was also performed with this software. Results are presented as mean ± SEM.

Significance notation:

• ns: p > 0.05
• *: p ≤ 0.05
• **: p ≤ 0.01
• ***: p ≤ 0.001
• ****: p ≤ 0.0001