Outline

1. Media Preparation

• 1.1 YPD Liquid Medium
• 1.2 YPD Solid Medium
• 1.3 LB Solid Medium
• 1.4 LB Liquid Medium

2. Plasmid Construction

• 2.1 PCR Amplification of Target Genes (T-Gal/GOD)
• 2.2 Agarose Gel Electrophoresis and DNA Recovery
• 2.3 Plasmid Extraction of Empty Vector
• 2.4 Double Digestion of Empty Vector
• 2.5 Homologous Recombination

3. Transformation and Screening

• 3.1 Transformation into E. coli DH5α
• 3.2 Colony PCR Verification
• 3.3 Recombinant Plasmid Extraction and Sequencing
• 3.4 Transformation into Pichia pastoris GS115

4. Protein Expression and Purification

• 4.1 Expansion Culture of Positive Clones
• 4.2 Methanol-Induced Expression
• 4.3 Protein Extraction
• 4.4 Protein Purification and SDS-PAGE Verification

5. Functional Assays

• 5.1 β-T-galactosidase Hydrolytic Activity Assay
• 5.2 β-T-galactosidase Transglycosylation Activity Assay
• 5.3 Dual-Enzyme Catalysis Analysis
• 5.4 Glucose Oxidase Activity Assay

1. Media Preparation

Reagents: Peptone, Yeast Extract, Glucose, diluted water, Agar, Zeocin (antibiotics)

Instruments: Erlenmeyer flask, Pipette, Autoclave, Laminar flow hood, Graduated cylinder, Disposable gloves.

1.1 YPD liquid medium

Table 1. YPD liquid medium

Steps:

1. Dissolve 20 g peptone/tryptone and 10 g yeast extract into about 800 mL of distilled water;
2. Put 20 g of glucose into 200ml of distilled water;
3. Sterilize the glucose solution and the yeast-peptone solution respectively (121°C, 20 min); (Send them to the autoclave room before 2:00 p.m., including student safety warnings)
4. Cool to room temperature after sterilization and mix the solutions together. Add antibiotics under laminar flow hood for standby use (Zeocin, final concentration: 100 µg/mL)

1.2 YPD solid medium

Table 2. YPD solid medium

Steps:

1. Dissolve 2 g peptone/tryptone, 1 g yeast extract, and 2 g Agar into about 80 mL of distilled water;
2. Put 2 g of glucose into 20ml of distilled water;
3. Sterilize the glucose solution and the yeast-peptone solution respectively (121°C, 20 min), before 2:00 p.m.;

1.3 LB solid medium

Table 3. LB solid medium

Steps:

1. Put all of the powder (All the ingredients except distilled water) and dissolve it in about 80 mL of distilled water, and bring to the volume of 100 mL;

1.4 LB liquid medium

Table 4. LB liquid medium

Steps:

1. Dissolve all the powder (Ingredients other than water) into about 80 mL of distilled water, and bring to the volume of 100 mL;

2. Plasmid Construction

Reagents: Plasmid extraction kit (ex.: Qiagen Plasmid Mini Kit), EcoRI, SalI enzyme, 10×Buffer, dd H₂O, Agarose, 1×TAE Buffer, Nucleic acid strain Gel Red, Buffer B2, Elution Buffer, Exnase II, 5x CE Buffer, ddH₂O

Consumables: Consumables in the plasmid extraction kit, Agarose gel related consumables, Electrophoresis related consumables, Centrifuge tube, Gel extraction kit, PCR tubes, Centrifuge tubes

Instruments: Centrifuge, Microwave (To prepare the agarose gel), Electrophoresis powering supply, blue light transilluminator, Water bath, PCR machine.

2.1 PCR Amplification of Target Genes (T-Gal/GOD)

1. Amplification of T-Gal sequence

(1) T-Gal sequence 783AA 2199 bp

MRPLIDFNSGWLFEGSETVRLPHNAVELPFSYFDERSYQRPFTYEKRFAADPSWEGKEVSIVFDGAMADCKVSLNGNETQSHRDGYTPFEVRLTGKLQPGENVLTVAIDGSENPEIPPFGGQIDYLTYAGIYRDVWLRVTSPIFVENAKIETPNVLSDHKSVRARIDVANPQNLPLSGTLLAILTDSQGNTIASTDASVDGASIEIAFDDLTGISLWDLDNPALYTLAITIDTTHGQDESAFRFGFRSVEFTHEGFRLNGKPLKLRGLNRHQSFPYSGYAMGRAAQERDAEILKHQLHLNIVRTSHYPQSHWFLDRCDELGLLVFEEIPGWQHIGGEAWKDESVENVRRMITRDWNHPSIVIWGVRINESQDDHDFYVRTNKLARELDTTRPTGGVRYITESELLEDVYTMNDFILGNEELGGNRPRTPLRPQQEVTGLDRVVPYMITEYGGHMYPTKVHDEEQRQAEHVRRHLEVMNAAYGDPQIAGCIGWCAFDYNTHKDFGSGDRIVHHGVMDMFREPKFAAYAYASQCEPDEEVILRPVTFWARGERNIGGVLPLIVLTNCDEVELRYGTNPPKRFRPDRDAFPHLPHAPVVMDRKDFTADELGRWGMSWESATITGYVGGKAVKTIDFVADPVPATLQVEADTPDLPGLGETVRVMVRALDQAGNKLPFFSDPVSIEVSGAAERLGPALVPLRAGATGFWVRATGQGPVTVTVINERLGRQSITLTAN

(2) PCR components

Table 5. PCR components

(3) Program Setup

• 95°C 3 min Initial denaturation
• 30 cycles: 95°C 30 s → 55°C 30 s → 72°C 1 min/kb (2 min 10 s)
• 72°C 5 min extension

2. Amplification of GOD sequence

(1) Glucose oxidase sequence 428 AA 1284 bp

MSEDIFDAIIVGAGLAGSVAALVLAREGAQVLVIERGNSAGAKNVTGGRMYAHSLERIIPGFAEQAPIERIITHEKLAFMTDKGAMTIDYCNGEEAASSQVSYSVLRSKFDAWLMEQAEEAGAQLITGIRVDNVVQREGKVVGVEADGDILEAKVVILADGVNSLLAEKLGMAKRVEASHVAVGVKELIELPKSVIEDRFQLQGNEGAACLFAGSPTDGLMGGGFLYTNETTLSLGLVCGLHHLKDAKKSVPQMLEDFKQHPAVAPLIAGGKLVEYAAHVVPEAGMNMQPELVGDGVLIAGDAAGMCMNLGFTIRGMDLAISAGEAAAKTVLSAMKRDDFSKQSLGEYRQHLDDGPMRDMRMYQKLPAFLDNPRMFTAYPEMAISIARDLFTVDGSAPVPMRKKILRHAKKVGFINLMKDGLKGVTVL

(2) PCR Components

Table 6. PCR components

(3) Program Setup

• 95°C 3 min Initial Denaturation
• 30 cycles: 95°C 30 s → 55°C 30 s → 72°C 1 min/kb (1 min 10s)
• 72°C 5 min

2.2 Agarose Gel Electrophoresis and DNA Recovery

1. Preparation of agarose gel: weight and take 0.35 g of Agarose and put it into a Erlenmeyer flask. Add 35 mL of 1×TAE buffer into the flask and microwave it for about 3 min (until all the agarose melts). Take the flask out and shake it well, then take 5 μL of nucleic acid strain Gel Red and add it to the agarose gel. Install the gel plate and insert the gel comb. Pour the liquid gel onto the plate (For about 3-4 mm high, avoid generating bubbles), and wait until the gel solidify. When the gel completely turns into solid, take out the comb, and put the gel into the electrophoresis tank. Add 1×TAE Buffer until it completely immerse the gel;
2. Sample loading: put the PCR samples into those wells in the gel, and add 10 μL of DNA Marker at last in another well, (Be careful not to damage the wells around or pushing the pipette too deep);
3. Electrophoresis: close the electrophoresis chamber, the direction of the wells should be toward the negative electrode of the electric field. Switch the electrophoresis machine on (160 V, 20 min);
4. Blue light observation: shut the instrument down, take the gel out, and observe it under the blue light transilluminator. The substance with orange-red fluorescence is DNA.
5. Cut the part of the gel that contains target DNA sequences off and weight it. Add 3 × (100 μL Buffer B2/100 mg gel cut). Put the sample at 50℃ in a water bath for 10 min, mix by inversion for 3 times during the water bath and let the gel completely dissolve;
6. Recover Fragments (Following the kit protocol): Transfer 700 µL of gel solution into a spin column and centrifuge it at 8000 ×g for 30 s. Remove the liquid from the collection tube; Add 500 µL of Wash Solution, centrifuge it at 9000 ×g for 30 s. Remove the liquid from the collection tube, and repeat it 2 times; Then, centrifuge the column without adding the wash solution at 9000 ×g for 2 min;. Put the column into a clean 1.5 mL centrifuge tube, and add 15-40 µL of Elution Buffer on the center of the absorption membrane. Incubate the tube at room temperature for 1 min, and then centrifuge for 2 min. Lastly, use Nanodrop to measure the DNA concentration.

2.3 Plasmid Extraction of Empty Vector

1. Open a plasmid extraction kit (ex.: Qiagen Plasmid Mini Kit).
2. Follow the instruction book in it and extract plasmid DNA.
3. Use Nanodrop to measure the plasmid DNA's concentration and purity.

2.4 Double Digestion of Empty Vector

Table 7. Double digestion components

Let the components react under the temperature of 37°C for 30 min, and use the Agarose gel electrophoresis to verify the effect of digestion. Then recover the fragments and verify their size.

2.5 Homologous Recombination

Table 8. Homologous Recombination components

Put them under the temperature of 37°C to react for at least 30 min.

3. Transformation and Screening

Reagents: Plasmid extraction kit (Ex.: Qiagen Plasmid Mini Kit), SacI enzyme, 10×Buffer, dd H₂O, competent cells, YPD medium, Zeocin (antibiotics)

Consumables: Consumables in the plasmid extraction kit, Centrifuge tube

Instruments: Centrifuge, PCR machine, Shaking incubator, Incubator

3.1 Transformation into E. coli DH5α

1. Take 100 μL of competent cells, separated it into 2 equal parts, and add 1-2 μL of the recombination product;
2. Put them in ice bath for 30 min;
3. Heat shock them under the temperature of 42°C for 45-60 s, and immediately put it back into the ice bath for 2 min;
4. Add 800 μL of LB medium (The medium does not contain antibiotics), thaw the samples under the temperature of 37°C for 1 h;
5. Coat them on a LB-medium plate that contains Zeocin, culture them at 37°C for 12-16 h.

3.2 Colony PCR Verification

Pick a single colony from the plate and put it in a 5 mL LB liquid medium (4 groups, so we made 4 samples). Confirm that the final concentration of Zeocin is 100 µg/mL, then put it in the shaking incubator at 30℃ and 220 rpm for 12-18 h

Table 9. Colony PCR Verification components

PCR Program:

• Pre-degeneration: 95°C 3 min
• 30× cycle: 95°C 30 s → 55°C 30 s → 72°C 1 min/kb(6 min)
• Elongation: 72°C 5 min

3.3 Recombinant Plasmid Extraction and Sequencing

1. Open a plasmid extraction kit (Ex.: Qiagen Plasmid Mini Kit).
2. Follow the instruction book in it and extract plasmid DNA.
3. Use Nanodrop to measure the plasmid DNA's concentration and purity.
4. Send to test.

Pick 500μL of sample and go test its sequence of pPICZαA-T-Gal/pPICZαA-GOD

3.4 Transformation into Pichia pastoris GS115

1. Plasmid linearization

Use SacI to digest the recombinant plasmid, then recover the linear fragments.

Table 10. Plasmid linearization components

React at 37℃ for 20 min

2. Transformation

Pick 100 μL of competent cells, and add 1-22 μL of linear plasmid. Ice bath for 30 min, then heat shock it at 42°C for 90s, and immediately put it back in the ice bath for 2min. Add 800 μL of YPD medium (Not including antibiotics), and thaw the samples at 30°C for 1h.

3. YPD-zeocin Plate screening

Spread the sample on a YPD plate with Zeocin (100 µg/mL) and culture the samples at 30°C for 2-3 days.

4. Colony PCR test

Table 11. Colony PCR components

4. Protein Expression and Purification

Reagents: YPD medium, Antibiotics (Zeocin), PBS buffer, Methanol, Glycerol, SDS-PAGE sample buffer (5×), 0.25% Coomassie Brilliant Blue staining solution, Destaining solution (40% Ethanol, 10% acetic acid, 50% distilled water)

Consumables: Centrifuge tubes, Plates, Ni beads, Consumables used to prepare the SDS-PAGE gel (Mold, gel casting cassette, etc.)

Instruments: Shaking incubator, Incubator, Centrifuge, Ultrasonic cell disruptor, 4℃ refrigerator, SDS-PAGE electrophoresis powering supply

4.1 Expansion Culture of Positive Clones

Pick positive colonies and transfer them into 100 mL of YPD medium, put them in a shaking incubator at 30°C, 220 rpm overnight.

4.2 Methanol-Induced Expression

When OD600 reaches 1.0, add 1000× methanol, leave it at 30℃ overnight to let the methanol induce the expression.

4.3 Protein Extraction

1. Ultrasonic disruption of yeast for protein extraction

1. Centrifuge the sample at 4℃, 4000 rpm for 20 min, collect the cells, and abandon the medium.
2. Cell disruption by ultrasonic waves: take out the sample and add 10 mL PBS Buffer and resuspend cells thoroughly. Put the suspension into a small beaker, ultrasonic disrupt on ice (Recommended: 500W (30%), stop for 3 s every 3 s of disrupting, making a total time of 10-15 min). Centrifuge the supernatant at 4℃, 15000 rpm for 10 min.

2. Preparation of SDS-PAGE gel

1. Assemble the gel casting mold, and prepare the lower layer first (Ex.: 0.75 / 1.00 / 1.50 mm thick gel);
2. Extract lower layer gel solution (2×) and lower running buffer (2×) 2.0 / 2.7 / 4 mL each, put them into a gel casting cassette to mix them;
3. Add 40 / 55 / 80 µL of modified coagulant into the cassette in step 2, mix them carefully, make sure that there are no bubbles formed.
4. Add the mixture into the mold until the liquid level is about 1.5 cm less than the highest amount. Then, put water or alcohol in the mold to keep the liquid level smooth. Make sure not to create bubbles;
5. Put the mold under room temperature (25ºC) for 6-10 min. The gel is well solidified when the bound between the overlay phase is clear;
6. Then, prepare the upper layer gel (Ex.: 0.75 / 1.00 / 1.5 mm thick mini gel);
7. Slowly pour the overlay phase out and extract equal amount of upper layer gel solution (2×) and the upper running buffer (2×, with colors) each for 0.5 / 0.75 / 1.0 mL. Put them in a new cassette and mix it;
8. Add 10 / 15 / 20 µL of modified coagulant to the cassette, mix it carefully so that there are no bubbles created;
9. Add the upper layer gel solution onto the lower layer gel until the liquid level reaches the maximum level. Slowly put the comb into the mold (with an angle), avoid creating any bubbles.
10. Let the gel stand for 10-15 min, and wait for the upper layer gel to solidify. Carefully take out the comb, and use pipette tip or syringe to extract some buffer to wash the wells.

4.4 Protein Purification and SDS-PAGE Verification

1. Binding

Extract 200 μL of Ni beads and put it in the supernatant to bind for 1 - 2 h

2. Wash and elution

Use 25 mM of imidazole for washing and 300mM imidazole for elution, store the samples in PBS buffer + glycerol at -20℃, separate them into several 1mL tubes for storing.

3. SDS-PAGE Electrophoresis

(1) SDS-PAGE Electrophoresis

Extract 40 μL of Protein sample and add it into the 10 μL protein loading buffer (5×), then water bath it at 100˚C for 5-10 min. Take it out and cool to room temperature, and centrifuge at 9000rpm for 1 min;

Loading: Extract 10-15 µL of samples, add the samples orderly;

Electrophoresis: After loading the samples, close the electrophoresis chamber, connect it to the power source, and open the power source. Maintain the voltage at 200-120V before the sample enter the gel for about 15-20 min; When the straining indicator reaches the separating gel, increase the voltage to 200 V, and make sure that the voltage is stable. Stop the electrophoresis when the indicator is about 1-2 cm higher than the bottom of the gel (About 1 h later).

(2) Staining

Staining: After the electrophoresis, shut the powering supply down, take out the mold, and use a tool to separate the gel and the mold. Using the copper wire at the center of the indicator band as a marker, put the gel into a large container to strain, and add 0.25% Coomassie brilliant blue straining solution. Strain for 1-2 h, and it can be put overnight if needed (Optional: use a microwave to speed up the process, heat 1-2 min)

(3) Destaining

Destaining: Abandon the straining solution, use distilled water to wash the gel several times, and add destaining solution to destain. Change the destaining solution frequently until the protein band is clear, and destaining process can be overnight. (Destaining solution formula: 40% Ethanol, 10% acetic acid, 50% distilled water)

(4) Gel imaging

5. Functional Assays

Reagents: ONPG (2-Nitrophenyl β-D-galactopyranoside), 1 M Na2CO3, PBS buffer, Lactose, PBS buffer, milk, Glucose, o-Dianisidine solution, Horseradish peroxidase solution (HRP solution),

Consumables: Centrifuge tubes, 96-well plate (optional, used for microplate reader measurements), TLC plate (Kieselgel 60 F254), Mobile phase (N-butanol : ethanol : acetic acid : water = 2 : 1 : 1 : 1), Chromogenic agent (5% Sulfuric acid-ethanol)

Instruments: microplate reader (optional), Spectrophotometer, Water bath, Oven, Spectrophotometer,

5.1 β-T-galactosidase Hydrolytic Activity Assay

1. Preparation of ONPG solution (1 mg/mL)

Use 20 mM phosphate buffer (PBS, pH 7.0) to dissolve ONPG until the concentration is 1 mg/mL. Preheat at 50°C for 10 min before use

2. Prepare 1 M Na₂CO₃ (100 mL)

• Na₂CO₃: 10.6 g
• Distilled water: Add until the solution reaches 100mL

3. Substrate: 1 mg/mL ONPG (2-Nitrophenyl β-D-galactopyranoside)

4. Reaction system

3 mL of ONPG solution (50˚C preheated), 0.1 mL of enzyme template(T-Gal), put it in 40°C water bath and let it react for 15 min, add 2 mL 1M Na2CO3 solution to stop the reaction, and test the absorbency at 420nm.

Unit definition: 1 U = 1 µmol / 1 min.

5.2 β-T-galactosidase Transglycosylation Activity Assay

1. Components

• 5% w/v = 0.5 g Lactose / 10 mL PBS solution (Heat it to help it dissolve)
• 10% w/v = 1g Lactose / 10 mL PBS solution (Heat it to help it dissolve)
• 15% w/v = 1.5 g Lactose / 10 mL PBS solution (High, needed to stirred and heated sufficiently)
• 20% w/v = 2 g Lactose / 10 mL PBS solution (High, needed to stirred and heated sufficiently)

2. Preparation

1. Weight the corresponding lactose powder into a beaker;
2. Add about 8 mL of PBS buffer;
3. Heat the samples in a water bath at 40-50˚C, and stir it when heating until the powder completely dissolve;
4. Wait for the sample to cool to room temperature and then adjust the final volume to 10 mL;

3. Experimental procedure

Substrate: 5%-20% lactose solution

Reaction condition: 2 mL of lactose solution + 0.1mL liquid enzyme (T-Gal), and put it into 40˚C water bath for 15-20 min → 65°C to inactivate for 5 min.

TLC Steps:

1. First, prepare the mobile phase (N-butanol : ethanol : acetic acid : water = 2 : 1 : 1 : 1), and pour it into the developing chamber (About 0.5-1 cm thick). Pre-satiated the filter paper on the chamber walls, and wait 15-30 min to let it form a stable gas phase.
2. Spotting: First, cut a piece of silica gel plate, and use a pencil to mark a starting line (about 1cm from the bottom edge); After sampling, centrifuge the sample at 10,000 × g for 1 min and spot with the supernatant (If the concentration is high, dilute it); Use a small pipette or 2 µL applicator to add the samples on different locations on the line; Every spot is about 1-2 mm in diameter, and quickly dry the samples by using a hair dryer (To prevent streaking).
3. Development: Put the TLC plate into the developing chamber (And perpendicular to the chamber), then add mobile phase (Make sure that the liquid level is lower than the starting line); Close the chamber, and wait until the solvent front is about 5-8 cm higher than the starting line; When the development is over, use a hair dryer to dry the plate quickly or wait in room temperature until it is dry.
4. Visualization: Immerse the plate quickly in the straining solution (5% Sulfuric acid-ethanol); Use a hair dryer or let it dry naturally; Put the plate in an oven and heat it at 120˚C for 2 min; The Rf value of lactose is 0.12 – 0.18 (Optional);

5.3 Dual-Enzyme Catalysis Analysis

Table 12. Dual-Enzyme Catalysis components

Use 5% lactose solution and fresh milk as reaction mixture, and add the 2 enzymes into the mixture. Then, test the carbohydrate distribution using TLC test.

Substrate: 5% lactose solution/fresh milk.

Enzyme ratio: T-Gal : GOD = 1:1 to 1:3 (Used for gradient optimization).

Reaction conditions:

• Temperature: 25°C, 35°C, 45°C
• Time: 0, 20, 40, 60 min. Take a sample every 20 min.
• Water bath every sample for 5 min at 65˚C to stop the reaction; Centrifuge the samples at 5000 × g for 5 min;

TLC:

1. First, prepare the mobile phase (n-butanol : acetic acid : ethanol : water = 2:1:1:1), and pour it into a developing chamber (For about 0.5-1 cm thick). Pre-satiated the filter paper on the chamber walls, and wait 15-30 min to let it form a stable gas phase.
2. Spotting: First, cut a piece of silica gel plate, and use a pencil to mark a starting line (about 1cm from the bottom edge); After sampling, centrifuge the sample at 10,000 × g for 1 min and spot with the supernatant (If the concentration is high, dilute it); Use a small pipette or 2 µL applicator to add the samples on different locations on the line; Every spot is about 1-2 mm in diameter, and quickly dry the samples by using a hair dryer (To prevent streaking).
3. Development: Put the TLC plate into the developing chamber (And perpendicular to the chamber), then add mobile phase (Make sure that the liquid level is lower than the starting line); Close the chamber, and wait until the solvent front is about 5-8 cm higher than the starting line; When the development is over, use a hair dryer to dry the plate quickly or wait in room temperature until it is dry.
4. Visualization: Immerse the plate quickly in the straining solution (5% Sulfuric acid-ethanol); Use a hair dryer or let it dry naturally; Put the plate in an oven and heat it at 120˚C for 2 min;

HPLC:

1. Sample Collection: At the preset time points of the dual-enzyme synergistic reaction, accurately aspirate 1.0 mL of the reaction solution as the test sample using a pipette.
2. Centrifugation for Impurity Removal: Transfer each aspirated sample to a clean centrifuge tube separately, and centrifuge at 15,000 rpm and 4°C for 15 minutes. Collect the supernatant.
3. Filtration for Clarification: Aspirate the centrifuged supernatant, pass it through a 0.22 μm cellulose acetate filter membrane, collect the clarified filtrate into a clean HPLC vial, cap and seal the vial, and set it aside for subsequent HPLC analysis.
4. HPLC System Equilibration: Connect the Prevail Carbohydrate ES chromatographic column for glucose detection system equilibration, pump the 65% (v/v) acetonitrile-water solution at a flow rate of 1.0 mL/min. Set the column oven temperature to 35°C and the refractive index detector (RID) temperature to 35°C. Continue equilibrating the chromatographic column for at least 30 minutes until the detector baseline becomes stable. Replace with the Aminex HPX-87H ion-exchange column for lactose detection system equilibration. Pump the 5 mmol/L H₂SO₄ aqueous solution at a flow rate of 0.6 mL/min. Set the column oven temperature to 65°C and the detector temperature to 35°C. Continue equilibrating the chromatographic column for at least 60 minutes until the baseline becomes stable.
5. Standard Curve Preparation: Inject the glucose and lactose standard working solutions into the HPLC system sequentially in the order of "from low concentration to high concentration", and record the chromatogram of each concentration of the standard. Draw the glucose and lactose standard curves respectively using the linear regression method, and calculate the regression equations.
6. Sample Detection: Inject each dual-enzyme reaction sample into the HPLC system separately. First, perform analysis under the glucose detection conditions, then switch to the lactose detection conditions for analysis. Record the chromatogram of each sample and the corresponding peak area.
7. Quantitative Calculation and Reaction Efficiency Analysis: Complete the quantitative calculation of glucose and lactose concentrations in the samples based on the standard curves, and analyze the efficiency of the dual-enzyme synergistic reaction through the time-concentration relationship.

5.4 Glucose Oxidase Activity Assay

1. Formula of 1.5% Glucose solution

Weight 5 g of glucose and dissolve it into 100 mL of deionized water.

2. Experimental procedure

Add 150 µL of 5% glucose solution, 150µL of o-dianisidine solution, and 10µL of horseradish peroxidase solution to the 96 well in sequent. Put the system in water bath at 25˚C to let it react for 10 min, and add 10 µL of sample (Test solution) into the system. After the reaction, test the mixture using a microplate reader at 460 nm wave length. Test the sample 1 time per 1 min, and test for 5 times. Under the conditions of 25 ˚C and PBS buffer (pH = 6.5), we define that 1 GOD unit is needed to convert 1µmol of glucose to gluconic acid and hydrogen peroxide per minute. People can also use spectrophotometer or some kit instead of microplate reader.