Goal: To formulate a nutrient-rich medium capable of supporting the growth, activation, and fermentation of Escherichia coli and other microorganisms. Both liquid and solid forms can be prepared depending on experimental needs.

Materials:
Required Materials and Reagents：
1)Glass conical flask (≥1 L capacity)
2)High-pressure steam sterilizer (autoclave)
3)Tryptone (protein hydrolysate, nitrogen source)
4)Yeast extract (vitamin and growth factor source)
5)Sodium chloride (NaCl, osmotic stabilizer)
6)Double-distilled water (ddH2O)
7)Agar powder (only for solid medium preparation)

Procedures:
1.Weighing of components, For each liter of medium, prepare:
a.10 g tryptone
b.5 g yeast extract
c.10 g NaCl
d.For solid medium: additionally weigh 15 g agar powder (final concentration 1.5%).

2.Dissolution of ingredients
a.Add all dry components into a beaker or flask containing ~800 mL ddH2O.
b.Stir continuously until completely dissolved.
c.Adjust the final volume to 1 L with ddH2O.

3.Distribution into containers
a.Transfer the prepared solution into a 1 L Erlenmeyer flask (leave 1/3 volume empty to prevent overflow during sterilization).
b.If required, dispense into smaller bottles or tubes according to downstream applications.

4.Sterilization process
a.Place the prepared medium into an autoclave.
b.Sterilize at 121℃ under 15 psi pressure for 20 minutes.
c.Allow the medium to cool naturally after sterilization.

5.Final preparation
a.For liquid LB: use directly after cooling.
b.For solid LB: keep the agar medium warm (~50℃), pour into sterile Petri dishes, and let it solidify under aseptic conditions.

pET-28a(+)-AsCDA/Cts2p/FjChiB

Ⅰ. Obtain target gene fragments by PCR
Goal: PCR technology is used to amplify the target gene fragment to provide DNA fragments for subsequent infusion recombination.

Materials:
1)2x PrimeStarMix
2)Gene template
3)Double distilled water (ddH2O)
4)Primer-F
5)Primer-R
6)PCR tube
7)PCR thermal cycler
PrimeStarMix includes buffer and dNTPs for PCR. The extention rate for PrimeStarMix (2x) is 1000bp/10 s.

Procedures:
1)Aseptically prepare the PCR reaction system. The recommended total reaction volume is 50 µL: add 25 µL of 2×PrimeStar Mix, 1 µL of template DNA, 1 µL of Primer-F, and 1 µL of Primer-R. Add ddH2O to the remaining volume to 50 µL. After preparing the reaction solution, briefly centrifuge to remove any bubbles.
2)Place the reaction tube in a PCR instrument and set the reaction program:
3)Initial denaturation: 95℃ for 3 minutes to fully unwind the double-stranded structure of the template DNA;
4)Cycling: Perform 35 cycles, each cycle consisting of:
5)Denaturation at 95℃ for 30 seconds
6)Annealing at 55℃ for 30 seconds (the specific temperature can be adjusted based on primer design);
7)Extension at 72℃ for 2 minutes (the PrimeStar Mix has an extension rate of approximately 1000 bp/10 seconds; the time can be adjusted based on the target fragment size);
8)End extension: 72℃ for 1 minute to ensure complete extension of all fragments.
9)After the reaction, if not used immediately, the product can be stored at 4℃ for a short period of time.
10)To obtain purified DNA fragments, separate the PCR products by electrophoresis on a 1%–1.2% agarose gel. Cut out the target band and use a DNA recovery kit to extract the fragment for subsequent infusion recombination.

Ⅱ. Linearize pET-28a(+) backbone by restriction endonucleases
Goal: The pET-28a(+) plasmid was double-digested with restriction endonucleases EcoRI and XhoI to obtain a linearized vector fragment that can be used for subsequent infusion cloning.

Materials:
1)pET-28a(+) plasmid DNA (approximately 5 µg)
2)10×digestion buffer (matching the enzymes used)
3)EcoRI restriction enzyme
4)XhoI restriction enzyme
5)Sterile double-distilled water (ddH2O)
6)PCR tubes or 1.5 mL centrifuge tubes
7)Micropipette and tips
8)Benchtop low-speed centrifuge
9)PCR instrument or thermostatted metal bath

Procedures:
1. Prepare a 50 µL total reaction mixture on ice. Typical ratios are:
a.Plasmid DNA: approximately 5 µg
b.10×digestion buffer: 5 µL
c.EcoRI: 5 µL
d.XhoI: 5 µL
e.ddH2O: make up to 50 µL
2. Gently pipette the reaction mixture to mix thoroughly, avoiding violent shaking and creating bubbles. If bubbles are present, briefly centrifuge at low speed to allow the liquid to settle to the bottom of the tube.
3. Place the reaction mixture in a PCR instrument or metal bath at 37℃ for 30 minutes to allow the plasmid to be completely digested by both enzymes.
4. After digestion, remove a small sample for electrophoresis to confirm that the plasmid has been linearized. Subsequently, separate the entire reaction product on a 1% agarose gel and extract the corresponding band.
5. Purify the linearized pET-28a(+) fragment using a DNA purification kit according to the kit instructions. Dissolve the fragment in sterile ddH2O or low-salt buffer and set aside.

Notes:
• EcoRI and XhoI must maintain good activity in the same buffer system to avoid incomplete cleavage due to buffer incompatibility.
• During electrophoresis, use a DNA marker to accurately distinguish linearized plasmid from uncut supercoiled forms.
• Avoid prolonged UV exposure during gel excision and DNA recovery to minimize DNA damage.

Ⅲ. Agarose gel construction, deployment and recycle
Goal: Gels suitable for DNA electrophoresis are prepared by dissolving agarose in a buffer and adding a nucleic acid dye.

Materials:
1)Agarose powder (analytical grade, Agarose M is recommended)
2)1×TAE buffer
3)10,000×nucleic acid dye (such as GelRed, GelSafe, etc.)
4)Microwave oven
5)Erlenmeyer flask (≥ 200 mL)
6)Gel casting tray
7)Gel well comb
8)Gloves, pipette, and sterile tips

Procedures:
1. Weigh 1.5 g of agarose powder and add it to a 200 mL Erlenmeyer flask.
2. Add 100 mL of 1×TAE buffer to the flask. Gently swirl to evenly distribute the agarose powder.
3. Heat the mixture in a microwave oven. Heat for 30 seconds at a time, then gently shake the mixture to ensure the agarose is completely dissolved. Avoid excessive boiling and bubbles.
4. Once the agarose is completely dissolved and the solution is clear, cool it to approximately 60℃.
5. Add approximately 3 µL of 10,000×nucleic acid dye to the solution and mix thoroughly. Wear gloves to avoid contact with skin.
6. Pour the prepared agarose solution into the gel casting well. Pour the gel slowly along the sides of the well to reduce bubbles.
7. Insert a comb to create sample wells and let the gel stand at room temperature for approximately 20–30 minutes to allow the gel to completely solidify.
8. After the gel solidifies, gently remove the comb and carefully remove the casting template. This will yield an agarose gel with electrophoresis wells.
9. Place the prepared gel in the electrophoresis tank and cover it with 1×TAE buffer until the gel is completely submerged. Sample loading and electrophoresis can then begin.

Notes:
• Observe the agarose solution carefully as it dissolves to ensure there are no visible particles.
• If bubbles appear in the solution, remove them with a pipette before pouring the gel.
• When adding the dye, ensure the temperature is not too high to prevent inactivation of the dye.

Ⅳ. Agarose gel electrophoresis
Goal: Use agarose gel electrophoresis to detect PCR or enzyme digestion products to confirm whether the DNA band size is as expected, providing a basis for subsequent gel recovery.

Procedures:
1. Take approximately 50 µL of each sample to be tested and add 5 µL of 10× loading buffer. Pipette repeatedly to mix until the solution is uniform in color and to ensure that the sample is fully incorporated into the buffer.
2. Place the solidified agarose gel into a horizontal electrophoresis tank and add enough 1×TAE buffer to completely submerge the gel surface.
3. Before loading the sample, add a DNA molecular weight marker to the first sample well on the gel to facilitate subsequent band length comparison.
4. Carefully pipette the mixed DNA sample into the sample wells of the gel one by one, adding approximately 50 µL to each well. Slowly move the pipette tip close to the bottom of the well to avoid puncturing the gel or spilling the liquid.
5. Connect the electrophoresis instrument, set the voltage to 120 V, and run for approximately 20 minutes. Observe the dye migration at the sample front during electrophoresis to ensure the correct electrophoresis direction (DNA migration toward the positive electrode).
6. After electrophoresis, turn off the power and carefully remove the gel. Then, place the gel under a UV or blue light transilluminator to observe the positions of DNA bands and compare the fragment sizes to molecular weight standards.
7. Based on the size of the target fragment, determine whether gel excision and recovery are necessary and proceed to the subsequent DNA extraction step.

Notes:
• The loading buffer increases sample density, allowing it to sink into the gel wells. It also contains an indicator dye to facilitate monitoring of migration progress.
• Electrophoresis time that is too short will result in unclear DNA separation, while too long may cause small fragments to escape the gel. Therefore, adjustments should be made based on gel concentration and fragment size.
• When observing bands, minimize UV exposure time to avoid DNA damage, which could affect subsequent recovery efficiency.

Ⅴ. DNA gel extraction
Goal: The target DNA fragments (target gene and linearized pET-28a(+) vector) separated by electrophoresis were cut out from the agarose gel and purified to obtain high-purity DNA for subsequent cloning.

Materials:
• Agarose gel containing the target band
• 1.5 mL sterile centrifuge tube (EP tube)
• Agarose gel DNA recovery kit (including gel elution buffer, elution buffer, purification column, etc.)
• Pipette and tips
• Constant temperature water bath or metal bath (50℃)
• Benchtop high-speed centrifuge (supporting ≥12,000 rpm)

Procedures:
1)Confirm the location of the target DNA band using a blue light or UV gel exciter, minimizing prolonged UV exposure to the DNA.
2)Use a sterile scalpel or blade to cut the gel block containing the target band, cutting as close to the edge of the band as possible to avoid any excess agarose. Place the gel block in a 1.5 mL EP tube and record the approximate weight of the gel block (usually 100 mg = 100 µL).
3)Add an appropriate amount of dissolution buffer to the gel block (generally, approximately 3–5 times the volume of the gel block, 500 µL is common). Place the centrifuge tube in a 50℃ water bath, heating with intermittent gentle shaking until the gel block is completely dissolved.
4)Transfer the dissolved mixture to a DNA purification column, place it in a collection tube, and centrifuge at 12,000 rpm for 30 seconds to allow the DNA to bind to the silica membrane. Discard the flow-through.
5)Add 500 µL of wash buffer (containing 70% ethanol) to the column and centrifuge again for 30 seconds. Discard the flow-through.
6)Repeat the wash step to ensure removal of residual impurities and salts.
7)Centrifuge again at 12,000 rpm for 2 minutes to completely remove any residual ethanol from the column. Then, leave the purification column uncapped for approximately 1 minute to allow the ethanol to evaporate completely.
8)Transfer the purification column to a new, clean 1.5 mL EP tube. Slowly add 50 µL of sterile double-distilled water (ddH2O) or low-salt elution buffer to the center of the column membrane and let it sit for 1 minute to facilitate DNA elution.
9)Centrifuge again at 12,000 rpm for 1 minute. Collect the supernatant, which is the recovered DNA fragments.
10)Discard the purification column and store the DNA solution at 4℃ for short-term storage or -20℃ for long-term storage.

Notes:
• Avoid contaminants when cutting the gel. Smaller gel fragments improve purification efficiency.
• Ensure sufficient gel dissolution buffer, otherwise the gel fragments may not be completely dissolved.
• The final elution volume of DNA can be adjusted as needed. A smaller volume results in a higher concentration, but the total amount may be slightly lower.
• During the elution step, add water precisely to the center of the column membrane to ensure maximum recovery.

Ⅵ. Homologous recombination of linearized pET-28a(+) backbone with target gene fragments
Goal: To assemble the recombinant plasmid pET-28a(+) carrying the target gene (AsCDA/Cts2p/FjChiB) through homologous recombination using a linearized vector backbone and gene fragments.

Materials:
• CloneExpress Mix (2×reaction buffer and enzyme mix)
• Purified PCR fragments of target genes (AsCDA, Cts2p, FjChiB)
• Linearized pET-28a(+) backbone DNA
• PCR tubes suitable for thermal cycler use

Procedure:
1.Quantification of DNA
• After gel purification of both the linearized vector backbone and the PCR-amplified gene inserts, measure the DNA concentration of each sample using a spectrophotometer (e.g., NanoDrop) or equivalent method.
• Record concentrations to allow accurate adjustment of fragment ratios in the recombination reaction.

2.Assembly Reaction Setup
• In a sterile PCR tube, prepare the following reaction mixture for each construct:
• 5 µL of CloneExpress Mix (2×)
• 2.5 µL of the linearized pET-28a(+) backbone DNA
• 2.5 µL of the purified gene fragment (either AsCDA, Cts2p, or FjChiB)
• Gently pipette to mix and briefly centrifuge to collect the liquid at the bottom of the tube.

3.Recombination Reaction
• Place the PCR tubes into a thermal cycler.
• Run the recombination program: maintain at 50 ℃ for 30 minutes to allow ligation-free homologous recombination between the overlapping ends of the vector and insert.
• After completion, hold the reaction at 4 ℃ until transformation.

Notes
• Ensure equimolar ratios of insert and vector DNA for optimal recombination efficiency.
• Use freshly prepared, high-purity DNA fragments to maximize success rate.
• The resulting recombination products are directly suitable for transformation into competent E. coli cells.

Ⅶ. Transformation
Materials:
• Recombinant plasmid DNA
• LB liquid medium (without antibiotics)
• LB plates (with kanamycin)
• Competent E. coli BL21
• Ice
• Micropipette and tips
• Centrifuge
• 37℃ incubator

Procedures:
1. Place 50–100 µL of freshly prepared competent E. coli BL21 culture on ice. Add an appropriate amount of recombinant plasmid (approximately 10–50 ng) to the culture and mix gently.
2. Incubate the mixture on ice for 20 minutes to allow the plasmid to fully contact the cells.
3. Quickly transfer the tube to a 42℃ water bath for 45 seconds to heat shock the cells. This will instantly permeabilize the cell membrane and facilitate DNA entry into the cells. Immediately return the tube to ice for 2–3 minutes to stabilize the cell membrane.
4. Add 400 µL of LB liquid medium (without antibiotics) to the cells and incubate at 37℃ with shaking or allow the mixture to stand for 30 minutes to promote bacterial recovery and plasmid expression.
5. Centrifuge the culture at 5,000 rpm for 3 minutes, discard approximately 300 µL of supernatant (reserve a portion if desired), and gently resuspend the pelleted cells.
6. Spread the resuspended cells evenly onto an LB plate containing kanamycin, ensuring even distribution.
7. Invert the plate and incubate in a 37℃ incubator for 12–16 hours, until a single colony appears.
8. After transformation is complete, single colonies can be selected for liquid culture and subsequent plasmid extraction.

Ⅷ. Colony PCR verification
Goal: Colony PCR was used to identify whether the transformed E. coli BL21 single colonies contained the correctly constructed recombinant plasmid, thereby screening positive clones for subsequent experiments.

Materials:
• Taq DNA polymerase master mix (2×Master Mix)
• Double-distilled water (ddH2O)
• LB plates containing transformed bacteria
• PCR tubes
• Sterile pipette tips
• Specific primers (forward and reverse)
• PCR thermocycler
• Gel electrophoresis equipment and agarose gel

Procedures:

1. PCR Reaction Setup
• Add the following to each PCR tube:
• 10 µL 2×Taq Master Mix
• 1 µL Forward Primer
• 1 µL Reverse Primer
• 8 µL ddH2O
• Gently mix and briefly centrifuge to collect the liquid at the bottom of the tube.

2. Single Colony Sampling
• Use a sterile 10 µL pipette tip to pick up a single colony from the LB plate.
• Gently pipette the colony into the liquid in the PCR tube and mix by pipetting up and down several times to ensure that the colony is fully exposed to the reaction solution. Be careful not to introduce too much agar.

3. PCR Amplification
• Place the PCR tube in a thermal cycler and perform amplification according to the manufacturer's protocol or the temperature cycle designed for the target fragment:
• Initial denaturation
• Cycling of denaturation-annealing-extension
• Final extension
• The number of cycles and temperature parameters are determined by the primers and fragment length.

4. PCR Product Detection
• After amplification, load the PCR product onto an agarose gel for electrophoresis analysis.
• Determine the band size against a molecular weight standard. If a band of the expected size appears, the colony is positive and contains the correctly constructed recombinant plasmid.

3.1 Induction of protein expression for 6xHis-AsCDA/Cts2p/FjChiB

Ⅰ. Amplification culture for correct colony
Goal: The pET-28a(+)-AsCDA/Cts2p/FjChiB recombinant protein was efficiently expressed in Escherichia coli BL21, and bacterial samples were collected at different times and temperatures for SDS-PAGE analysis of protein expression.

Materials:
• LB liquid medium
• E. coli BL21 transformed with pET-28a(+)-AsCDA/Cts2p/FjChiB
• Kanamycin solution
• Isopropyl β-D-thiogalactopyranoside (IPTG) inducer
• Iso-thermic shaker
• Spectrophotometer (for OD600 measurement)
• 1.5 mL centrifuge tubes and sterile pipette tips

Procedures:
1) Inoculation and Preculture
2) Add 100 µL of the recombinant bacterial culture to 100 mL of LB liquid medium. Also add an appropriate amount of kanamycin (e.g., 100 µg/mL) to ensure selective pressure.
3) Incubate the culture flask at 37℃ on a constant-temperature shaker, shaking vigorously to ensure aeration.
4) Growth Monitoring
5) Measure the OD600 value of the culture every 30 minutes until the culture reaches the logarithmic growth phase (OD600 ≈ 0.4–0.6), which usually takes approximately 2–3 hours.
6) At this stage, the bacteria are actively growing and suitable for IPTG induction.
7) Protein Induction
8) Add IPTG to the culture to a final concentration of 1 mM and gently mix to initiate recombinant protein expression.
9) Parallel cultures at different temperatures (e.g., 25℃ and 37℃) can be set up to compare expression efficiency and solubility.
10) Sampling and Monitoring
11) At 0.5 h, 1 h, 3 h, and 6 h after induction, collect equal volumes of bacterial culture samples (e.g., 1–2 mL) and place them in centrifuge tubes.
12) Samples can be frozen immediately or added to lysis buffer for subsequent SDS-PAGE analysis of protein expression.

Notes:
• IPTG solution should be prepared fresh or stored frozen in aliquots to avoid repeated freeze-thaw cycles.
• The induction temperature affects protein solubility and folding. Low temperatures (25℃) generally favor soluble protein expression.
• When sampling, perform the procedure as quickly as possible to prevent continued bacterial growth or protein degradation.

Ⅱ: Protein crude extraction and SDS-PAGE
Goal: Crude protein was extracted from the induced expression E. coli BL21 culture medium, and the expression and size of the target protein were analyzed by SDS-PAGE.

Materials for protein crude extraction:
• Induced bacterial suspension (approximately 500 µL per induction condition)
• Centrifuge
• Double-distilled water (ddH2O)
• Sangon 12.5% SDS-PAGE Preparation Kit
• Electrophoresis Buffer (Tris-Glycine)
• Protein Molecular Weight Standard
• 6×Protein Loading Buffer
• Vertical Electrophoresis System
• Coomassie Brilliant Blue Staining Solution
• Destaining Solution

Procedures for protein crude extraction and SDS-PAGE:
1. Crude Protein Extraction
• Transfer 500 µL of bacterial suspension from each induction condition to a centrifuge tube and centrifuge at 12,000 rpm for 1 minute.
• Discard the supernatant and resuspend the bacterial pellet in 50 µL of 1×protein loading buffer (6×loading buffer diluted with ddH2O).
• Heat the resuspension in a PCR thermocycler at 95℃ for 15 minutes to lyse the cells and denature the proteins.
• After completion, cool briefly or proceed immediately to SDS-PAGE analysis.

2. SDS-PAGE Gel Preparation
• Combine 2.2 mL of 2×separating gel solution, 2.2 mL of 2×separating gel buffer, and 44 µL of catalyst in a centrifuge tube. Mix gently.
• Slowly pour the separating gel into the gel mold, avoiding the formation of bubbles.
• Add 1 mL of ddH2O to the top of the separating gel and let it stand for approximately 8 minutes to allow the gel to fully polymerize.
• After the gel solidifies, discard the top layer of water.
• Combine 825 µL of 2×stacking gel solution, 825 µL of 2×stacking gel buffer, and 11 µL of catalyst. Mix gently.
• Pour the stacking gel into the mold until full and slowly insert the comb to avoid creating bubbles.
• Let it stand for approximately 12 minutes to solidify, then carefully remove the comb and rinse the wells with running buffer.

3. Sample Loading and Electrophoresis
• Add 10 µL of protein molecular weight standard to the first well, followed by an appropriate amount of protein sample to each well.
• Place the gel in a vertical electrophoresis tank and add running buffer.
• Set the electrophoresis condition to 120 V and run for approximately 90 minutes until the protein bands are clearly separated.

4. Staining and Destaining
• After electrophoresis, stain the gel in Coomassie Brilliant Blue solution for approximately 10 minutes.
• Rinse the gel repeatedly in destaining solution until the background becomes transparent and protein bands are clearly visible.

Notes:
• Be gentle when preparing the separating and stacking gels to avoid creating bubbles, which can affect protein band resolution.
• Load a moderate amount of sample to avoid excessive sample loading, which can cause band smearing.
• Adjust the staining and destaining times based on band appearance to achieve optimal contrast.

3.2 Protein His-tag purification
Goal: Utilizing the His tag on the recombinant protein, the target protein is recovered through Ni-NTA agarose affinity purification technology to provide high-purity protein for subsequent antigen preparation.

Materials:
• E. coli culture containing the target protein
• 50 mL centrifuge tube
• Ice-filled container for cold operation
• High-speed centrifuge
• Sonicator
• Beyotime Ni-NTA Agarose Affinity Purification Kit
• Pipette and sterile tips

Procedures:
1) Bacterial Cell Collection
2) Transfer the target protein-expressing bacterial suspension to a 50 mL centrifuge tube.
3) Centrifuge at 4000 rpm for 20 minutes to pellet the cells.
4) Bacterial Cell Lysis
5) Chill the centrifuge tube in an ice-water bath to maintain a low temperature and prevent protein denaturation.
6) Insert an ultrasonic probe and sonicate the cells. Each sample should be processed for approximately 15 minutes, with multiple intervals to avoid overheating.
7) All samples should be processed identically to ensure consistency.
8) Cell Debris Removal
9) After sonication, centrifuge the sample at 10,000 rpm for 30 minutes.
10) Discard the cell debris at the bottom and transfer the supernatant (containing the target protein) to a fresh centrifuge tube for later use. 4.Ni-NTA Agarose Pretreatment
11) Add 500 µL of Ni-NTA agarose to a 2 mL centrifuge tube and centrifuge at low speed (approximately 500×g for 5 minutes). Discard the supernatant.
12) Wash the agarose three times with 1.5 mL of PBS buffer, centrifuging at 500×g for 5 minutes each time to ensure removal of impurities from the storage solution.
13) Protein Binding
14) Add the pretreated Ni-NTA agarose to the protein supernatant and incubate at 4℃ with gentle shaking for 60 minutes to allow the His-tagged protein to fully bind to the agarose.
15) Column Packing and Washing
16) Transfer the incubated protein-agarose mixture to the adsorption column and let it stand for approximately 2 minutes to promote binding. Discard the flow-through.
17) Wash the agarose in the column with 5 mL of wash buffer five times to remove non-specifically bound proteins.
18) Protein Elution
19) Elute the target protein five times using elution buffer containing 250 mM imidazole (500 µL/wash).
20) Collect each eluate as a purified protein sample and combine for subsequent analysis.
21) SDS-PAGE Detection
22) Take a 50 µL sample of the eluate, add 6×protein loading buffer, and mix thoroughly.
23) Load the sample onto an SDS-PAGE gel and separate by electrophoresis.
24) Detect protein bands using Coomassie Brilliant Blue staining to verify the expression and purity of the target protein.

Notes
• Maintain low temperature throughout the entire process to avoid protein degradation.
• Intermittent sonication can be used with cooling intervals to prevent protein denaturation.
• The imidazole concentration in the wash and elution buffers can be adjusted based on the binding strength of the target protein to improve purity and recovery.
• The collected eluate can be further concentrated or dialyzed to meet the needs of subsequent antigen preparation or functional experiments.

4.1 Free enzyme activity test
Goal: The enzyme activities of AsCDA, Cts2p, and FjChiB were determined to provide data support for subsequent comparison of enzyme performance and optimization of application conditions.

Materials:
AsCDA enzyme activity assay:
• p-Nitroacetanilide substrate (p-NPAc, 1 mg/mL)
• 50 mM phosphate buffer (pH 7.4)
• AsCDA enzyme solution
• Ice-water bath or stop solution (e.g., 1 M Na2CO3)
• Spectrophotometer (400 nm)
• 1.5 mL centrifuge tube

Cts2p and FjChiB enzyme activity assay:
• Colloidal chitin solution (1% w/v)
• Enzyme solution (Cts2p or FjChiB)
• DNS reagent
• Boiling water bath
• Centrifuge
• 1.5 mL centrifuge tube
• Spectrophotometer (OD540)

Procedures:
a. AsCDA Enzyme Activity Assay
1. Prepare a 200 μL total reaction system consisting of: 150 μL p-nitroacetanilide substrate solution, 40 μL buffer, and 10 μL AsCDA enzyme solution.
2. After mixing, incubate the reaction tube in a 37℃ water bath for 30 minutes.
3. Upon completion of the reaction, immediately terminate the reaction by placing the tube in ice water or adding an equal volume of stop solution.
4. Measure the absorbance at 400 nm using a spectrophotometer and calculate the concentration of p-nitroaniline produced using a standard curve.
5. Calculate enzyme activity according to the formula:

Where C is the p-nitroaniline concentration (μg/mL), V_total is the total reaction volume (0.2 mL), V_enzyme is the volume of enzyme solution added (0.01 mL), and t is the reaction time (30 minutes).
6. Set up a blank control (substrate + buffer only) and a negative control (inactivated enzyme or irrelevant protein + substrate), measure the absorbance, and subtract the background value from the sample.

b. Cts2p and FjChiB Enzyme Activity Assay
1. Combine 250 μL of 1% colloidal chitin solution with 250 μL of enzyme solution and mix thoroughly.
2. Incubate the mixture at 42℃ for 1 hour to allow the enzyme to catalyze the degradation of the substrate and produce reducing sugars.
3. After the reaction is complete, add 2 mL of DNS solution and immediately heat in a boiling water bath for 5 minutes.
4. Remove the mixture and cool the 50 + 50 + 400 μL DNS mixture to room temperature.
5. Centrifuge at 12,000 rpm for 5 minutes. Collect the supernatant and measure the OD540 absorbance.
6. Convert the OD540 value to reducing sugar concentration using a standard curve. Calculate the enzyme activity using defined enzyme activity units: the amount of enzyme required to produce 1 μmol/L reducing sugar per minute.
7. Similarly, set up a blank control (colloidal chitin + buffer, no enzyme) and a negative control (substrate + heat-inactivated enzyme or unrelated protein) to subtract the background absorbance.

Notes
• Maintain a constant temperature throughout the incubation process: Cts2p and FjChiB should be incubated at 42℃, and AsCDA should be incubated at 37℃ in a water bath.
• Controls are essential to correct for spontaneous substrate degradation or nonspecific reactions.
• DNS treatments must be cooled quickly after heating to avoid further degradation of reducing sugars.
• All assays should be repeated at least three times to ensure data reliability.

4.2 Optimal free enzyme activity reaction conditions
Goal: Determine the optimal reaction conditions for three chitin-related enzymes, AsCDA, Cts2p, and FjChiB, including optimal pH, temperature, pH stability, temperature stability, and the effects of metal ions on enzyme activity, to provide experimental basis for subsequent enzymatic applications and optimization.

Materials:
• Purified AsCDA, Cts2p, and FjChiB enzyme samples (0.5 mg)
• Colloidal chitin solution (1% w/v)
• Buffer system:
• 0.1 M glycine-HCl (pH 2.0–3.0)
• 0.1 M citric acid-Na2HPO4 (pH 3.0–8.0)
• 0.1 M Tris-HCl (pH 8.0–9.0)
• 0.1 M glycine-NaOH (pH 9.0–11.0)
• 50 mM Na2PO4-citric acid buffer (pH 4.0–6.0)
• 50 mM Na2PO4 buffer (pH 6.0–8.0)
• 50 mM Tris-HCl buffer (pH 8.0–9.0)
• 50 mM Glycine-NaOH buffer (pH 9.0–11.0)
• Metal ion solution (final concentration 10 mM): Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, Zn²⁺
• Spectrophotometer (OD540, for DNS assay of reducing sugars)
• Centrifuge tube (1.5 mL)
• Water bath

Procedures:
a. Determination of Optimal pH
1. Centrifuge 1% colloidal chitin, discard the supernatant, and resuspend in an equal volume of buffer covering the pH range of 2.0–11.0 (glycine-HCl, citric acid-Na2HPO4, Tris-HCl, glycine-NaOH).
2. Add each enzyme to the colloidal chitin system in different pH buffers and incubate at 37℃ for 1 hour.
3. After the reaction, determine the reducing sugar yield using the DNS method. The highest enzyme activity was set as 100%. A relative enzyme activity versus pH curve was plotted to determine the optimal pH.

b. Optimal Temperature Determination
1. Dissolve the enzyme in the optimal pH buffer.
2. Set a temperature gradient (20, 30, 40, 50, 60, 70, and 80℃) and add colloidal chitin for a 1-hour reaction.
3. Determine reducing sugar production by DNS. Plot a curve of relative enzyme activity versus temperature to determine the optimal reaction temperature.

c. Effect of Metal Ions on Enzyme Activity
1. At the optimal pH, add a single metal ion (final concentration of 10 mM, Mg²⁺, Co²⁺, Fe³⁺, Ni²⁺, Cu²⁺, Na⁺, K⁺, Mn²⁺, or Zn²⁺) to the enzyme solution.
2. Incubate at 37℃ for 1 hour, measure reducing sugar production, calculate relative enzyme activity, and analyze the promoting or inhibiting effect of each metal ion on enzyme activity.

Notes
• When conducting pH or temperature gradient experiments, perform at least three replicates per group.
• When determining reducing sugars using the DNS method, ensure that the sample temperature is consistent to prevent non-enzymatic factors from influencing the results.
• For metal ion experiments, it is recommended to include a blank control (without metal ions) to correct for background.
• The optimal pH and temperature for each enzyme are determined based on the highest relative enzyme activity and are then used for optimization in subsequent experiments.

4.3 Optimal free enzyme synergistic ratio and reaction conditions
Goal: Evaluate the synergistic effects of three chitin-related enzymes: AsCDA, Cts2p, and FjChiB. Compare the total enzyme activity of different pairwise and trio combinations to identify the most active combination. Further optimize the volume ratio of the two enzymes (2:1, 1:1, 0.5:1, 1:0.5, and 1:2) to ensure optimal conditions for subsequent synergistic reactions and substrate degradation efficiency.

Materials:
• Purified AsCDA, Cts2p, and FjChiB enzyme samples
• Colloidal chitin solution (1% w/v)
• Buffer (optimal pH buffer, based on previous measurements)
• DNS reducing sugar assay reagent or commercial reducing sugar assay kit
• 1.5 mL centrifuge tube
• Micropipette and tips
• Water bath or constant temperature incubator
• Spectrophotometer (OD540 or according to kit instructions)

Procedures:
a. Pairwise and Triple Enzyme Combination Testing
1. Prepare the following combinations of the three enzymes in equal amounts (e.g., 20 μL per combination):
• AsCDA + Cts2p
• AsCDA + FjChiB
• Cts2p + FjChiB
• AsCDA + Cts2p + FjChiB
2. Add an equal volume of the optimal pH buffer to each combination and add colloidal chitin (1%) to the system to a uniform total volume (e.g., 500 μL).
3. Incubate at the optimal temperature for 1 hour.
4. After the reaction, determine the amount of reducing sugar produced in the system using the DNS method or a kit and record the absorbance or measured value.
5. Convert the reaction activity of each group to U/mL (or relative enzyme activity percentage) and compare the combination with the highest enzyme activity.

b. Optimizing the Volume Ratio of the Two Best Enzymes
1. Select the two enzyme combinations with the highest activity from the first step.
2. Prepare different volume ratio systems (keeping the total enzyme volume constant):
• 2:1; 1:1; 0.5:1; 1:0.5; 1:2
3. Mix each enzyme ratio with colloidal chitin and add the optimal pH buffer, maintaining the same total reaction volume.
4. Incubate at the optimal temperature for 1 hour.
5. After the reaction, measure reducing sugar production using the DNS method or a kit.
6. Compare enzyme activity at different ratios to determine the optimal synergistic volume ratio.

c. Data Processing
• Perform at least three replicates for each experiment.
• Calculate relative enzyme activity, taking the highest reducing sugar production or highest U/mL value as 100%.
• Create a bar chart or line graph to display the changes in enzyme activity at different combinations and ratios.

4.4 Preparation and immobilized enzyme efficiency
Goal: Prepare immobilized chitinases (including exo-chitinase, endo-chitinase, and chitin deacetylase), characterize the immobilization efficiency by SDS-PAGE, and evaluate the binding of the enzymes to the epoxy support LX-1000EP.

Materials:
• Epoxy carrier LX-1000EP (0.5 g/tube)
• Purified enzyme
• PBS buffer (0.1 M, pH 7.0)
• 15 mL empty centrifuge tube
• Centrifuge
• Mixer
• SDS-PAGE reagents and equipment
• ImageJ software (for band grayscale analysis)

Procedures:
a. Resin Pretreatment
1. Transfer 0.5 g of LX-1000EP epoxy support to a 15 mL empty centrifuge column tube.
2. Add 3 mL of PBS buffer, gently mix, and wash three times. Centrifuge at 4000 rpm for 1 minute each time, and discard the supernatant.

b. Enzyme Immobilization
1. Adjust the purified enzyme solution to 5 mg/mL with PBS buffer. Add 10 mg of enzyme/g of resin to the resin. Add 2 mL of enzyme solution, which is the pre-immobilization supernatant.
2. Place the centrifuge tube horizontally on a shaker to allow the resin to shake freely. Incubate at 25°C for 12 hours.

c. Collecting the Post-Immobilization Supernatant
1. After the reaction is complete, transfer the resin-enzyme mixture to a 15 mL centrifuge tube and centrifuge at 4000 rpm for 1 minute. Collect the supernatant and record it as the post-immobilization supernatant.

d. Resin Washing
1. Add 2 mL of PBS buffer to a centrifuge tube containing 0.5 g of resin. Gently mix. Centrifuge at 4000 rpm for 1 minute. Discard the supernatant.
2. Repeat the wash twice to remove unbound enzyme from the surface.

e. SDS-PAGE Analysis and Calculation of Immobilization Efficiency
1. Take the pre- and post-immobilization supernatants, dilute them 5-fold if necessary, and perform SDS-PAGE analysis.
2. Calculate the grayscale value of each sample band using ImageJ software.
3. Calculate the Immobilization Efficiency

4.5 Number of reuses of immobilized enzymes
Goal: Evaluate the reusability of immobilized chitinase by measuring the amount of reducing sugars produced during each reaction cycle to determine the enzyme's activity retention after multiple reactions.

Materials:
• Immobilized chitinase (immobilized on LX-1000EP resin)
• 1% colloidal chitin solution
• PBS buffer (0.1 M, pH 7.0)
• DNS reducing sugar assay reagent or commercial reducing sugar assay kit
• Centrifuge tubes
• 37°C water bath or incubator
• Centrifuge
• Spectrophotometer or microplate reader
• Pipette and tips

Procedures:
a. Enzyme Cycling Reaction
1. Add an appropriate amount of immobilized chitinase (e.g., 0.5 g resin) to a reaction system containing 1% colloidal chitin solution (total volume adjusted according to experimental requirements, e.g., 2 mL).
2. Incubate the reaction in a 37°C water bath or on a constant-temperature shaker for 1 hour.
3. After the reaction, remove the immobilized enzyme from the reaction solution with a pipette and gently wash twice with PBS buffer to remove any residual substrate or product.
4. Collect the reaction supernatant for reducing sugar determination.

b. Reducing Sugar Determination
1. Take an appropriate amount of the reaction supernatant (e.g., 200 μL) and add an equal volume of DNS reagent. Incubate in a boiling water bath for 5 minutes. After the reaction, cool to room temperature.
2. Alternatively, use a commercial reducing sugar kit according to the instructions.
3. Measure the absorbance at 540 nm (or the wavelength recommended by the kit) using a spectrophotometer or microplate reader and calculate the reducing sugar concentration in the product.

c. Recycling
1. Add the washed immobilized enzyme back to fresh 1% colloidal chitin solution and repeat steps 1-2.
2. Record the amount of reducing sugar in the product after each cycle and calculate the relative enzyme activity (assuming the yield of the first reaction is 100%).
3. Continue until the enzyme activity significantly decreases. Plot a curve comparing the number of times the immobilized enzyme is reused versus the relative enzyme activity.