Reagent	Concentration
Tryptone	10g/L
Yeast Extract	5g/L
NaCl	10g/L
Agar Powder (for solid medium)	1.5g/100mL

Add distilled water to a final volume of 1 L, then autoclave at 121°C for 15 minutes.

·Activation of pET28a Strain

1. Prepare Kanamycin (Kana) solution at 100 mg/mL.
2. Inoculate the strain harbouring the pET28a plasmid into 5 mL of liquid LB medium at an inoculation ratio of 1%, and add kanamycin to a final concentration of 100 µg/mL.

3. Incubate at 37°C and 220 rpm in a shaker for 12-16 hours.

4.After the prepared LB solid medium plates cool to room temperature, add kanamycin to a final 100 µg/mL concentration, mix thoroughly, and pour plates under sterile conditions.

1. For high-copy plasmids, take 2 mL of bacterial culture, centrifuge at 8000 x g for 2 minutes at room temperature to collect the cells, and discard the supernatant.    

2. Add 250 μL of Buffer SP1 to the cell pellet and pipette to resuspend the cells thoroughly.
3. Add 250 μL of Buffer SP2, gently invert the tube 10 times to mix immediately, and let it stand at room temperature for 1 minute.  

4. Add 350 μL of Buffer SP3 and gently invert the tube 10 times to mix immediately.
5. Centrifuge at 12000 x g for 10 minutes, transfer the supernatant to a spin column, and centrifuge at 9000 x g for 1 minute. Discard the flow-through and back the spin column into the same collection tube.

6. Add 500 μL of Buffer DW1 to the spin column and centrifuge at 9000 x g for 1 minute. Discard the flow-through and back the spin column into the same collection tube.

7. Add 500 μL of Wash Solution to the spin column and centrifuge at 9000 x g for 1 minute. Discard the flow-through and place the spin column back into the same collection tube.

8. Repeat step 7.
9. Centrifuge the empty spin column and collection tube at 9000 x g for 1 minute, then let it stand with the lid open for 2 minutes to evaporate the ethanol.

10. Add 50 μL of Elution Buffer to the centre of the spin column membrane and let it stand at room temperature for 1 minute, and centrifuge at 9000 x g for 1 minute. Store the resulting plasmid DNA solution at -20°C.

 

·Double Digestion with NdeI/XhoI → Target Gene/pET28a Plasmid

Preparation of the Digestion System:

Reagent	Volume
10×CutSmart Buffer	5 μL
pET28a	5 μL
NdeI	1 μL
XhoI	1 μL
ddH2O	38 μL

1. Incubate the reaction mixture at 37°C for 20 minutes.
2. add 5 μL of 10✖ loading buffer after the reaction for staining.

 

·Agarose Gel Electrophoresis

Component	Amount
Agarose	0.5 g
1×TAE Buffer	50 mL
Nucleic Acid Stain	3 μL

Microwave on high for 2 minutes until completely melted.

2. Add 3 μL of nucleic acid stain to the molten agarose, mix thoroughly, and pour into the mould. Allow to solidify.

3. Perform electrophoresis at 150V for 15 minutes.
4. Use a gel imaging system to photograph the gel and confirm the size of the target gene.

 

·Gel Extraction of DNA Bands: pET28a/EC2.4.1.5/EC2.4.1.9

1. Separate the target DNA fragment from other fragments via agarose gel electrophoresis. Using a scalpel, excise the agarose gel slice containing the target DNA and place it into a 1.5 mL microcentrifuge tube. Weigh the gel slice.

2. Based on the weight and concentration of the gel slice, add Buffer B2 at a ratio of 300 μL per 100 mg of agarose.
3. Incubate the tube in a 50°C water bath for 10 minutes, occasionally mixing until the gel slice is completely dissolved.  

4. Transfer the dissolved solution into a spin column and centrifuge at 8,000 x g for 30 seconds. Discard the flow-Place the spin column back into the same collection tube.

5. Add 300 μL of Buffer B2 to the spin column and centrifuge at 8,000 x g for 30 seconds. Discard the flow-through and place the spin column back into the same collection tube.

6. Add 500 μL of Wash Solution to the spin column and centrifuge at 9,000 x g for 30 seconds. Discard the flow-Place the spin column back into the same collection tube.

7. Repeat step 6 once.
8. Centrifuge the empty spin column and collection tube at 9,000 x g for 1 minute to remove any residual wash buffer.

9. Add 15-40 μL of Elution Buffer to the centre of the spin column membrane, let it stand at room temperature for 1-2 minutes, and then centrifuge at 9,000 x g for 1 minute. Store the obtained DNA solution at -20°C or use it for subsequent experiments.

 

·T4 DNA ligase-mediated ligation of pET28a-2.4.1.5 / pET28a-2.4.1.9

Component	Amount
Linearized pET28a plasmid	0.02 μg
Insert (target gene)	0.2 μg
T4 DNA ligase	1 μL
10× Ligase Buffer	2 μL
ddH₂O	Y μL
total	20 μL

Mix thoroughly and incubate at 37°C for 2 hours.

 

·Buffer Preparation

1. IPTG (100 mM): Dissolve 2 g of IPTG in 8 mL of distilled water, then bring the volume to 10 mL with distilled water. Filter-sterilize using a 0.22 μm filter. Aliquot into 1 mL portions and store at -20°C.
2. His A Buffer (500 mL): Dissolve 1.42 g of Na₂HPO₄ (20 mM), 14.6 g of NaCl (500 mM), and 0.6808 g of imidazole (20 mM) in distilled water. Adjust pH to 7.4.
3. His B Buffer (250 mL): Dissolve 0.71 g of Na₂HPO₄ (20 mM), 7.3 g of NaCl (500 mM), and 8.5 g of imidazole (500 mM) in distilled water. Adjust pH to 7.4.
4. Citrate-Citric Acid Buffer (0.1 mM): Dissolve 21.01 g of citric acid monohydrate (C₆H₈O₇·H₂O) and 29.41 g of sodium citrate dihydrate (Na₃C₆H₅O₇·2H₂O) in distilled water.

 

Transformation of Recombinant Product into DH5α Cells

1. Place the DH5α competent cells on ice to thaw. Add 10 μL of recombinant product to  100 μL of competent cells. Gently tap the tube to mix (do not vortex). Incubate on ice for 30 minutes.

2. Perform heat shock by placing the tube in a 42°C water bath for 45 seconds, then immediately place the tube on ice to cool for 2-3 minutes.

3. Add 900 μL of LB liquid medium (without antibiotics) to the cells and incubate at 37°C with shaking for 1 hour (200-250 rpm). Centrifuge at 5,000 rpm (2,500 × g) for 5 minutes and discard the 900 μL supernatant. Resuspend the pellet in the remaining LB medium. Spread the resuspended cells onto LB-kanamycin plates using a sterile spreader.

4. Incubate the plates at 37°C in an inverted position for 12-16 hours.

 

·PCR identification of monoclonal/transformed colonies in DH5α

According to the table above, prepare the PCR system. Pick a single colony from the plate using a 10 μL pipette tip and transfer it into the prepared PCR mixture. Mix thoroughly by pipetting up and down, then place the mixture into the PCR machine for amplification.

 

 

·PCR identification of monoclonal/transformed colonies in BL21(DE3)

According to the table above, prepare the PCR system. Pick a single colony from the plate using a 10 μL pipette tip and transfer it into the prepared PCR mixture. Mix thoroughly by pipetting up and down, then place the mixture into the PCR machine for amplification.

 

·Agarose Gel Electrophoresis

Component	Amount
Agarose	0.5 g
1×TAE Buffer	50 mL
Nucleic Acid Stain	3 μL

1. Microwave on high for 2 minutes until completely melted.

2. Add 3 μL of nucleic acid stain to the molten agarose, mix thoroughly, and pour into the mould. Allow to solidify.

3. Perform electrophoresis at 150V for 15 minutes.

4. Use a gel imaging system to photograph the gel and confirm the size of the target gene.

 

·Preparation of seed culture fermentation broth

1. Transfer a verified single colony into 3 mL of LB liquid medium. Add kanamycin antibiotic at a ratio of 1:1000 (3 μ

L) incubate at 37°C with shaking at 220 rpm for 12-16 hours.

2. Inoculate the overnight culture of pET28a-2.4.1.5/2.4.1.9 into a sterile 50 mL centrifuge tube at a 1% inoculation

ratio. Add 40 mL of LB and 40 μL of kanamycin antibiotic at a ratio of 1:100. Incubate at 37°C with shaking at

220 rpm for 3-4 hours until the OD600 reaches 0.6-0.8.

3. Add 0.2 mM IPTG to induce high-level protein expression and incubate at 16°C for over 20 hours.

 

·SDS-PAGE verification of protein expression

SDS-PAGE Gel Preparation

1. Assemble the gel casting apparatus and prepare the separating gel (1.50 mm thick mini-gel).
2. mix 4 mL of separating gel solution (2X) and 4 mL of separating gel buffer (2X) in a gel preparation cup.
3. Add 80 μL of modified catalyst to the mixture from step 2. Stir gently to mix thoroughly, avoiding bubble

formation.

4. Pour the mixed separating gel solution into the gel casting apparatus until the solution reaches 1.5 cm below the

top of the glass plate. Overlay the separating gel solution with a layer of water or alcohol to keep the gel surface flat and free from bubbles.

5. Allow the gel to polymerise at room temperature (25°C) for 6-10 minutes. The gel is ready when a clear

boundary forms between the separating gel and the overlay.

6. Prepare the stacking gel (1.50 mm thick mini-gel).
7. Carefully pour off the overlay. Mix 1.0 mL of stacking gel solution (2X) and 1.0 mL of stacking gel buffer (2X) in a

new gel preparation cup.

8. Add 20 μL of modified catalyst to the mixture from step 7. Stir gently to mix thoroughly, avoiding bubble

formation.

9. Pour the stacking gel solution onto the polymerised separating gel until it reaches the top of the glass plate. Insert

The comb slowly forms wells into the gel to avoid bubble formation.

10. Allow the stacking gel to polymerise for 10-15 minutes. Carefully remove the comb and rinse the wells with

electrophoresis buffer using a pipette or syringe. The gel is now ready for SDS-PAGE electrophoresis.

 

 

SDS-PAGE Separating Gel Concentrations and Optimal Separation Ranges:

SDS-PAGE Gel Concentration	Optimal Separation Range
6% PAGE gel	70-300 kD
8% PAGE gel	30-200 kD
10% PAGE gel	20-80 kD
12.5% PAGE gel	15-60 kD
15% PAGE gel	10-45 kD

1. Centrifuge the overnight culture at 12,000 r/min for 10 minutes to collect the cell pellet. Resuspend the pellet in an appropriate amount of PBS buffer (pH 7.4). 

2. Sonicate the cell suspension on ice with a power of 500 W (40%) in pulses of 3 seconds on and 3 seconds off for 15 minutes. Centrifuge at 12,000 rpm for 30 minutes at 4°C, and collect the supernatant and pellet.

3. Nickel Affinity Purification (His-tag Protein Purification Kit)
4. Take 1 ml of well-mixed 50% His-tag Purification Resin, centrifuge at 4°C (1,000g for 10 seconds), and discard the storage buffer. Add 0.5 ml of non-denaturing lysis buffer to the resin, mix well to equilibrate, centrifuge at 4°C (1,000g for 10 seconds), and discard the buffer. Repeat the equilibration 1-2 times, discarding the buffer each time. Add 4 ml of bacterial lysate to the resin and incubate on a rotator at 4°C for 60 minutes.

5. Transfer the mixture of lysate and His-tag Purification Resin into the empty affinity column provided in the kit.
6. Open the bottom cap of the purification column to allow the liquid to flow out by gravity. Collect 20 μl of the flow-through for subsequent analysis.

7. Wash the column 5 times with 0.5-1 ml of non-denaturing wash buffer each time. Collect approximately 20 μl of the wash buffer for each wash for later analysis.

8. Elute the target His-tagged protein 6-10 times with 0.5 ml of non-denaturing elution buffer each time. Collect the eluates into different centrifuge tubes. The collected eluates are the purified His-tagged protein samples.

9. Mix the cell lysate supernatant with 5x SDS-PAGE loading buffer, boil at 100°C for 5-10 minutes, and centrifuge at 12,000 r/min for 2 minutes. Perform 6% SDS-PAGE, ensuring equal protein loading. Stain the gel with Coomassie Brilliant Blue will visualise the protein bands.

1. Use sucrose as the substrate. In a buffer system containing 50 mM acetate-acetate sodium (pH 5.8) and 1 mM CaCl2, add 2% sucrose and an appropriate amount of recombinant enzyme. Conduct the reaction at 37°C to ensure the recombinant enzyme exhibits catalytic activity. The activity can be determined by analysing the reduction of sucrose and the formation of glucose, fructose, and oligosaccharides using thin-layer chromatography (TLC). 

2. Analyze the hydrolysis products using Kieselgel 60 silica gel plates (Merck). The developing solvent is n-butanol acid (2:1:1, v/v/v). After centrifuging the samples at 10,000×g for 1 minute, spot 2 µL of the supernatant onto the silica gel plate. Develop the silica gel plate twice with the developing solvent; air dry it, then immerse it evenly in a colour development reagent composed of sulfuric acid (5:95, v/v). Finally, bake the plate at 130°C in an oven for 5 minutes to visualise the spots.

·Construction of Digestion Model and Detection of Sucrose Changes and Product Formation in Food Digestion Mixture

A conical flask was used as the container. A food product rich in sucrose (or a mixture of a specific amount of food and sucrose) was pulverised to prepare a simulated food suspension. Subsequently, a particular amount of two recombinant enzyme solutions was added, and the mixture was incubated at 37°C with shaking for 1 hour. The sucrose content and the amount of oligosaccharides produced in the reaction products were quantitatively analysed using High-Performance Liquid Chromatography (HPLC).

 

2% Sucrose Treatment		Trehalose Treatment
Number	Sample Amount - Time	Number	Sample Amount - Time
1	1-5	26	1-5
2	1-10	27	1-10
3	1-15	28	1-15
4	1-30	29	1-30
5	1-60	30	1-60
6	5-5	31	5-5
7	5-10	32	5-10
8	5-15	33	5-15
9	5-30	34	5-30
10	5-60	35	5-60
11	10-5	36	10-5
12	10-10	37	10-10
13	10-15	38	10-15
14	10-30	39	10-30
15	10-60	40	10-60
16	15-5	41	15-5
17	15-10	42	15-10
18	15-15	43	15-15
19	15-30	44	15-30
20	15-60	45	15-60
21	20-5	46	20-5
22	20-10	47	20-10
23	20-15	48	20-15
24	20-30	49	20-30
25	20-60	50	20-60

 

Chromatographic Conditions:

Column: Waters Sugar-Pak I column (6.5 × 300 mm)

Detector: Differential Refractive Index (RI) detector

Detector Temperature: 40°C

Flow Rate: 0.5 mL·min⁻¹

Mobile Phase: 100% deionised water

Injection Volume: 20 μL

Column Temperature: 80°C