Team:SZ SHD/Protocol
Protocol
1. TOP 10 plasmid extraction and measurement of concentration and purity
Procedure:
- Column equilibration: Place a Spin Column CP3 in a clean collection tube, and add 500 μl Buffer BL to CP3. Centrifuge for 1 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Discard the flow-through, and put the Spin Column CP3 back into the collection tube. (Please use freshly treated spin column).
- Harvest 2.5 ml bacterial cells in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400 × g) in a conventional, table-top microcentrifuge for 1 min at room temperature (15- 25°C), then remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained (For large volume of bacterial cells, please harvest to one tube by several centrifugation step.)
- Re-suspend the bacterial pellet in 250 μl Buffer P1 (Ensure that RNase A has been added). The bacteria should be resuspended completely by vortex or pipetting up and down until no cell clumps remain. Note: No cell clumps should be visible after resuspension of TIANprep Mini Plasmid Kit Handbook | 4 the pellet, otherwise incomplete lysis will lower yield and purity.
- Add 250 μl Buffer P2 and mix gently and thoroughly by inverting the tube 8 times. Note: Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min. If the lysate is still not clear, please reduce bacterial pellet.
- Add 350 μl Buffer P3 and mix immediately and gently by inverting the tube 6-8 times. The solution should become cloudy. Centrifuge for 10 min at 12,000 rpm (~13,400 × g) in a table-top microcentrifuge. Note: To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer P3. If there is still white precipitation in the supernatant, please centrifuge again.
- Transfer the supernatant from step 5 to the Spin Column CP3 (place CP3 in a collection tube) by decanting or pipetting. Centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through and set the Spin Column CP3 back into the Collection Tube.
- Wash the Spin Column CP3 by adding 600 µl Buffer PW (ensure that ethanol (96%-100%) has been added) and centrifuge for 60 s at 12,000 rpm (~13,400 × g). Discard the flow-through, and put the Spin Colum CP3 back into the Collection Tube.
- Repeat Step 7.
- Centrifuge for an additional 5 min at 12,000 rpm (~13,400 × g) to remove residual wash Buffer PW. Note: Residual ethanol from Buffer PW may inhibit subsequent enzymatic reactions. We suggest open CP3 lid and stay at room temperature for a while to get rid of residual ethanol.
- Place the Spin Column CP3 in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 100 μl ddH2O (A little bit more, may use 80ul next time)to the center of the Spin Column CP3, incubate for 2 min, and centrifuge for 2 min at 12,000 rpm (~13,400 × g).
2. Transformation of plasmid to E.coli BL21 (DE3)
Material:
BL 21 (DE3), ice bath, water bath, 1.5 mL centrifuge tube, LB broth, shaker, centrifuge, incubator.
Procedure:
- Take 50 µL of E.coli BL21 (DE3) cells, mix gently with 5 µL target plasmid (4 types of plasmid, place it on the ice bath for 25 min. *Make sure to defrost E.coli with ice bath and preheat waterbaath before hand.
- Heat shock at 42 °C for 90 s in the water bath, place it immediately in the ice bath for another 2 min. DO NOT SHAKE.
- Add 300 µL LB broth (not containing antibiotics), put it in the shaker to recover at 200 rpm, 37 °C for 60 min.
- Centrifuge at 5,000 rpm for 1 min, keep 100 µL of supernatant and resuspend the bacteria. Coating on the LB broth.
- Place upside down to incubate at 37 °C for 16 hrs in the incubator.
3. PCR and electropherosis
- Mix up the recipe:
25µl 2x T5 super PCR mix
2 µL forward primer
2 µL reverse primer
To 50 µL (21 µL) ddH2O
50 µL in total
- Prepare a new LB medium plate(solid) which contains antibiotics,,and label the plate and PCR tube
- Use sterilized toothpick to obtain a single bacteria colony on the transformation plate. Draw a line on the LB plate on a copy of the original colony, and immense in the PCR tube contains the PCR recipe respectively to the label. Rotate the toothpick and throw it to the waste tank.
- Repeat 1-3 for different bacteria colony
| procedure | temperature/ °C | time/min | repeat |
|---|---|---|---|
| preprocessing | 98 | 10 | 1 cycle |
| denaturation | 98 | 0.5 | 30 cycles |
| annealing | 61 | 0.5 | |
| extension | 72 | 2 | |
| Final extension | 72 | 5 | 1 cycle |
| Hold | 24 | infinity |
- Gel recipe:
50 ml TAE
0.5 g agar
5 µL Gel stain
- Use a microwave oven to heat the solution for about 90 seconds. Note that turn the medium high heat to medium fire when solution start fizzing.
- Pull out the solution quickly to the electrophoresis plate.
- Wait for the solidification
- Drop 5 µL plasmid solution after PCR.
4. Selecting colonies of BL 21 to inoculate and culture
General procedures:
- Add 5ml of liquid LB growth medium to brown medium 2
- Inoculate BL21 cells using a pipette tip (or add 10-50 µl of bacteria solution if the cells are in a solution)
- Add 50 µg/mL of kanamycin (5μl)
- Incubate the culture at 37°D overnight while constantly shaking
Add 25 µL of 2x T5 Super PCR master mix, 2 µL of forward and reverse primer, add ddH2O up to 50 µL in total. Dip the pipette into the bacterial solution to extract enough culture and put it into the PCR tube, repeat twice for every type, 8 in total. Run the PCR program to replicate enough plasmid for electropherosis later on.
5. IPTG induction of keratinases
Concentrations for IPTG induced expression:
KerAVDZ50: 0.67139 mM (160 µL/mL) [1]
KerBIER15: 0.4196 mM (0.01% w/v) [2]
KerBIMKU3: 0.4 mM [3]
KerBteQ7: 5 mM [4]
[Inoculation]
Inoculate 3% bacterium culture in 200ml of liquid medium [500ml flask] at 37°C 220rpm for 4hrs
Check for OD value
| Name of bacteria | Expected OD value | IPTG/mM | Time/h | Molecular weight（kDa） |
|---|---|---|---|---|
| KerBteQ7 | 0.7 | 5 | 12 | 28 |
| KerAVDZ50 | / | 0.67139 | 12 | 20 |
| KerBIER15 | / | 0.4196 | 16 | 28 |
| KerBIMKU3 | 0.5 | 0.4 | 4 | 30 |
[Induction]
Different volumes of 1M IPTG [according to values in the table above] will be added to 200mL of bacterium culture
| Name of bacteria | IPTG volume/ul | Temperature/°C | Time/h | |
|---|---|---|---|---|
| KerBteQ7 | 1000 | / | 12 | Or 16°C，16h of induction for all |
| KerAVDZ50 | 134.278 | / | 12 | |
| KerBIER15 | 83.92 | 37 | 16 | |
| KerBIMKU3 | 80 | 37 | 4 |
Both KerBteQ7 and KerAVDZ50 will be induced for 12hrs at 16°C.
KerBIER15 will be induced for 16hrs at 37°C.
KerBIMKU3 will be induced for 14-16hrs at 16°C.
[Sonification]
Weigh the empty tubes
Bacteria after induction will be collected in 50ml tubes
Centrifugate at 4℃ 8000rpm for 5min, discard the supernatant [ the remaining bacteria can be stored in -80℃]
Wash with 5ml PBS for 2-3 times [in each round, add 5ml PBS, pipette up and down, centrifugate at 4℃ 8000rpm for 1min, and discard the PBS]
Weigh tubes with bacteria and add PBS accordingly [1ml PBS will be added for every 0.1g of bacteria] 25-30ml PBS is recommended for sonification in a 50ml tube.
Sonification for 6s and pause for 6s [at an intensity of 40% and 300w] continue for 4-5min, then check for its clarity. During sonification, add about 5-10ul isopropanol [stored in shelf beside the sink] if too many bubbles are found.
[sonification time should not be too long, or it can influence the protein activity]
SDS-PAGE:
1. Dilute Pre-Cassettle Gel Running Buffer 20x
2. Mix the loading and bacteria solution (total volume is about 40uL) and heat to 95 degrees for 10min
3. Fix protein gel to the machine
4. Add pure water to the middle of the machine to check the sealing( if there's no water on the bottom of the machine after 10min or there's no water colume after lifting it up, the machine is OK to use)
5. Heat the marker to 95 degrees for 1min
6. Pour out the pure water and add SDS-PAGE buffer to the middle until the solution overflows
7. Take out the model of pits gently
8. Drop the bacterial solution and marker
9. Cover the lid and connect the circuit
10. Start the machine at 80v for 30min, then change to 120v for 1h (Note that add buffer every 10min to prevent the gel from drying out)
6. Purification of keratinases
Preparation:
PBS: 20mM Na3PO4 300mM NaCl
[Prepare the following solution in 50ml tube]
ELB: PBS 5Oml, imidazole(MW:68.08g/mol) 0.03404g, test for its PH value and then adjust to PH=7.4（10mM imidazole）
WB: PBS 5Oml, imidazole 0.034g, test for its PH value and then adjust to PH=7.4 (10mM imidazole）
EB: PBS 5Oml, imidazole 0.851g, test for its PH value and then adjust to PH=7.4（250mM咪唑）
EB2: PBS 50ml, imidazole 1.1914g（350mM咪唑）
Version 1.
1. Purification by Ni-Resin via centrifugation [5, 6, 7]
Test for PH, and adjust to a PH value of 7.4-7.5
All steps to be performed under 4°C condition (in ice box)
Steps for washing Ni-Resin:
Gently shake the Ni-Resin container before using to prevent layering
Add Ni-Resin into a separate tube (add 50μl Ni-Resin for every 1ml PBS)
(To balance 50 ml PBS later, 1250 μl Ni-Resin is needed)
*Notice to gently shake the NI-Resin container before using*
Centrifugate at 1000rmp *4°C for 2min, then discard the superantant (maintain a low speed during centrifugation)
-> Add 6250ul ELB (5 times of Ni-Resin volume), pipette up and down, then centrifugate at 3000rmp/700g for 2min and discard ELB
Repeat the step above one more time.
Take a sample of 10ul superantant from a total of 25ml for SDS-PAGE later
Add same volume of ELB(25ml) to the superantant(25ml), and mix them together(30min)
(To ensure good centrifugal quality, seperate the solution into two tubes)
Add the pre-washed Ni-Resin into the above liquid, suspend the Resin, pipette up and down every half hour(when you see Resin at the bottom) for about 2-3hrs at 4°C
Centrifugate at 700g for 2min, and transfer 20ul superantant to another 2ml tube [on ice] for SDS-PAGE later.
Add 500ul WB to the Resin tube, pipette up and down, centrifugate at 700g for 2min at 4°C, transfer WB to another new tube x8, take a sample of 20ul superantant for SDS-PAGE later
->Add EB(same volume as Resin) to wash off protein(add 1500ul EB to 1250ul Resin), pipette up and down to suspend Resin, then set it aside for 3-5min to let the Resin sink down, centrifugate at 700g for 2min at 4°C, and transfer (pipette) the superantant to a new tube x3
Repeat the above step for three times (in seperate tubes), and take a sample of 20ul superantant each round for SDS-PAGE later.
Combine the rest of the samples for BCA test for protein concentration (using BCA kit bought from Beyotime)
*Remaining superantant to be stored at 4°C (shorterm)
*-80 for longterm storage
Version 2.
Purification via Ni NTA Beads 6FF Gravity Column [8]
Ni NTA Beads 6FF uses highly cross-linked agarose gel as matrix, and has identical ligand as Ni NTA Beads. Ni NTA Beads 6FF is more suitable for industrial purification of protein on a large scale not only for its ability to tolerate harsh reagent condition, but also for its high pressure resistance (Maximum Pressure: 3 bar or 0.3 MPa); the two combines to allow purification of protein at a high flow rate.
Buffer Preparation (adjust according to actual amount):
Buffer below are recommendations and can be replaced with ones with similar functions. The basic principle for the recommended buffers is to have a low concentration of imidazole (or high pH) in Lysis and Wash Buffer and a high concentration of imidazole (or low pH) in Elution Buffer. It is recommended to filter all buffers through a 0.22 μm or 0.45 μm filter before usage.
Lysis Buffer 1L:
50 mM NaH2PO4 （7.80 g NaH2PO4·2H2O）
300 mM NaCl （17.54 g NaCl）
10 mM imidazole （0.68 g imidazole）
Adjust pH to 8.0 using NaOH solution
filter buffer through a 0.22 μm or 0.45 μm filter
Wash Buffer 1L:
50 mM NaH2PO4 （7.80 g NaH2PO4·2H2O）
300 mM NaCl （17.54 g NaCl）
10mM imidazole (0.6808g)
Adjust pH to 8.0 using NaOH solution
filter buffer through a 0.22 μm or 0.45 μm filter
Elution Buffer 1L:
50 mM NaH2PO4 （7.80 g NaH2PO4·2H2O）
300 mM NaCl （17.54 g NaCl）
250 mM imidazole （17.0 g imidazole）
Adjust pH to 8.0 using NaOH solution
filter buffer through a 0.22 μm or 0.45 μm filter
Sample Preparation:
- After expression, harvest the cells from an appropriate volume of bacterial cultures by centrifuging at 7,000 rpm (7,500×g) for 15min
- Resuspend the pellet in 1:10 ratio (w/v) with Lysis Buffer add PMSF (final density 1mM, or do not add because of its harm to enzyme activity).
- If there is a high concentration of cell suspension, it is recommended to add 10 μg/ml RNase A and 5 μg/ml DNase I to the sample. Sonicate the cell suspension/lysate on ice.
- Centrifuge the homogenized lysate at 10,000 rpm for 20 minutes at 4 degree celsius. Set the supernatant aside (on ice or store at -20 degree celsius)
- Packing Ni IDA Beads 6FF (with packing reservoir):
- Flush the column with ddH2O to remove the air from the end-piece and the adaptor. Ensure that there is no air trapped under the column bed support. Close the column outlet, while leaving the bed support covered with 1-2 cm deionized water
- Resuspend the medium. Pour the slurry into the column and minimise the introduction of air bubbles
- Open the bottom outlet, wait for buffer to slowly flow pass the column, then gradually increase the flow rate of the pump until reaching final desired flow rate. (to prevent liquid pressure from impacting the column bed and consequent deformation. If the ideal pressure and flow rate cannot be achieved, the maximum flow rate can be adopted to still ensure a fine loading)
- (In subsequent chromatography system stage, limit the flow rate under 75% maximum flow rate)
- Maintain the packing flow rate for at least 3 bed volumes (ddH2O) after obtaining a constant bed height. Mark the bed height on the column.
- Stop the pump and close the column outlet.
- Remove the upper stopper after Ni NTA Beads 6FF is loaded
- Flush the buffer with 3-5x column volume deionized water
- Use at least 5x column volumes Lysis Buffer to balance chromotography system
- Apply the sample into the column (avoid adding too much that exceeds the column capacity; too much sample creates great resistance for the sampler)
- Flush the column with Wash Buffer until ultraviolet absorbance reaches equilibrium (usually more than 10-15x column volumes is required)
- Adding immidazole in sample and buffer can increase sample purity
- Elution to be completed with Elution Buffer, adopting one-step or linear-gradient approach. Only 5x column volumes Elution Buffer is needed in one-step approach, while linear-gradient method requires gradient [e.g. 20x column volumes or more] to separate protein of different intensity.
- The purified samples (including effluent, wash and elute fractions) and the original samples were determined by SDS-PAGE.
Regenaration
- 1) Use deionized water (5 column volumes) to wash the column
- 2) Use 5 column volumes of 100 mM EDTA (pH 8.0) to stripe off nickel ions
- 3) Use deionized water (10 column volumes) to wash the column
- 4) Wash with 5 column volumes of 0.5M NaOH (10-15min)
- 5) Use deionized water (10 column volumes) to wash the column
- 6) Wash the column with 3-5 column volumes of 100 mM NiSO4 (regenerate hanging Ni)
- 7) Use deionized water (10 column volumes) to wash the column
The medium can be used immediately after regeneration.
Medium to be suspended in 20% ethanol of equal volume at 4 degree celsius if to be saved for future usage.
7. Ultrafiltration [9]
Tools:
Centrifuge with swinging-bucket or fix-angle rotor that can hold 17mmx124mm 15ml cone-bottom test tubes, prioritize using a swinging-bucket rotor
Caution:
to prevent damaging the machine during the process of centrifugation, clean and inspect the machine. 200 uL pipette tips are used to recycle concentrated liquid.
Prerinse:
Ultrafiltration film has traces of glycerol and can be prerinsed using buffer or Milli-Q® water to prevent interfering subsequent analysis.
Ultrafiltration
1. Add no more than 4mL samples into the ultrafiltration centrifugal tube
2. Place the capped ultrafiltration centrifugal tube into the rotor (prioritize using a swing-bucket rotor) and balance with a similar tube
3.Use a maximum of 4,000 x g to centrifugate for 10–40 minutes for a swing-bucket rotor; as for a fixed-angle rotor, keep the tube's film side upwards, Amicon® Ultra 3K、10K、30K and 50K tubes are to be centrifugated at a maximum of 500xg for 10–40 minutes; Amicon® Ultra 100K tube should be centrifugated at 5,000 x g for 10–20 minutes.
Note: typical centrifugation time is charted below
4.When recycling concentrated products, place the pipet tip inside the inner tube of ultrafiltration centrifugal tube, move the tip around when drawing up the sample to ensure complete obtainment. Filtered liquid can be stored inside the outer tube.
Note: retrieve the sample right after centrifugation to achieve the ideal recycle rate.
8. BCA test for protein concentration [10]
Protein standard solution preparation:
Add 0.8ml protein standard to 20mg BSA, fully dissolve the solid to get 25mg/ml protein standard sol (can be stored in -20°C)
Dilute some 25mg/ml Protein standard to 0.5mg/ml (eg. take 25ul 25mg/ml protein standard sol, add 980ul diluent to get 0.5mg/ml Protein standard ) Use PBS as diluent (0.5mg/ml Protein standard sol can be stored in -20°C too)
BCA working solution preparation:
Add 1 volume of BCAreagent B to every 50 volume of BCAreagent A(1:50) to get BCA working sol, mix them together (stable in room temperature for 24hrs)
Protein concentration test:
Add protein standard to the 96 pore plate (according to 0, 1, 2, 4, 8, 12, 16, 20ul), add the diluted protein standard to each pore until 20ul (20, 19, 18, 16, 12, 8, 4, 0ul).
Add the 20ul sample( what we want to measure) to the 96pore plate well, record the sample volume (if sample volume is less than 20ul, add diluted protein standard to it until 20ul )
Add 200ul BCA working sol to each pore, set it aside for 20-30min at 37°C/ 2hrs at room temp/ 30min at 60°C
Measure A562 or absorbance at 540-595nm
Calculate protein concentration using the standard curve and sample volume.
9. Enzyme activity assays [11]
Substrates: azocaein, casein, gelatin, BSA, hair
Prepare 50ml 2 mM CaCl2 stock solution (M) by adding: Dissolve 0.0147g of CaCl2.2H2O
Prepare 500 mM Na2CO3: 2655 mg Na2CO3
Add distilled water until volume is 50 mL.
Blank: buffer only, same treatment process as samples.
Control 1: substrate only; Control 2: enzyme only.
Activity test using Casein as substrate:
Take a number of (32) 5mL tubes, add 0.5 mL crude enzyme and 2.5 mL substrate solution of different concentrations (0.05, 0.1, 0.5, 1% W/V) to mix, incubation time is 0min, 10min, 20min, 30min, 12h, 24h, 36h, 48h, temperature 37℃, PH7.5. The control group only contained 2.5 mL substrate solutions of different concentrations (4), and the same conditions were incubated.
After the incubation, take the syringe and fill the syringe, inhale the liquid in 36 tubes respectively (the syringe can be reused), carefully remove the needle, filter with 0.22um filter membrane, put the filtrate back into the original tube, collect 0.5ml, and the reaction will be terminated after filtration.
Add 2.5 ml Na2CO3 and 0.5ml Folin reagent to 0.5ml filtered and clarified liquid for incubation at room temperature for 30min. Turn on the microplate reader for absorbance measurement.
Prepare 96-well plates or colouring cuvettes. Fill each tube of 32 3.5ml samples with 3 parts of 200ul solution to the 96-well plates to measure the OD (A660). A total of 101 results were recorded by taking one OD from each tube of 4 controls (one blank).
- Azocasein (triplicates):
120 µL of an appropriate dilution of enzyme (ultrafiltrated crude enzyme/dilutes) was added to 480 µL of azocasein (1% w/v) in Tris buffer (50 mmol/L, pH 7.5) and the mixture was incubated at 37°C for 30 min. The reaction was terminated by filtering through a 0.22um filter. 100ml of the supernatant was neutralized (~pH 7.5)by adding 100ml of 0.5N NaOH. Absorbance (A440) was measured using a spectrophotometer.
One unit of protease activity was defined as the amount of enzyme required to yield an increase in absorbance (A420) of 0.01 in 30 min at 37°C.
- Casein (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme/dilutes) solution (0.5 ml) was mixed with 2.5 ml 100 mM Glycine-NaOH buffer supplemented with 2 mM CaCl2 containing 1% w/v (500mg/50ml) casein (prepare 50ml stock), and incubated for 15 min at suitable pH and temperature (pH7.5, 37°C). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control. One unit (U) of activity was defined as the amount of enzyme that hydrolyzed the substrate and produced 1 mg of amino acid equivalent to tyrosine per minute under the above-mentioned conditions.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
- Gelatin (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme) solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) gelatin (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
- BSA (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme) solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) BSA (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
- Hair (triplicates):
A suitably diluted enzyme (ultrafiltrated crude enzyme) solution (0.5 ml) was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) hair (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
10. Quantification of Sulfhydryl Groups in hydrolysate [12]
Catalytic Mechanism
The mechanism by which microorganisms degrade keratin varies, so the product during degradation is not the same. Some fungi reduce the disulfide bonds through the sulfites secreted on the surface of the mycelia and the acidic environment, while Streptomyces through the production of intracellular reductase. However, water-insoluble keratin can only exist extracellularly in the form of particles. Therefore, the reduction of disulfide bonds can only occur outside the whole cell with strong metabolic ability, most likely in the cell-bound redox system on the cell surface because it requires insoluble keratin in close contact with cells. Observations of pure white high-temperature actinomycetes revealed that the disulfide bond reduction was performed by a cell-linked redox system. No sulfhydryl groups were detected during keratolysis of S. freundii and B. licheniformis. This may be due to the fact that the cysteine (-SH) produced by the reduction of the cystine disulfide bond was quickly converted to other product.
Keratinase actually has the activity of polypeptide hydrolase and disulfide reductase. At present, it is generally believed that the degradation process of keratinase is divided into three steps, namely denaturation, hydrolysis and transamination. First, the disulfide reductase acts on the keratin disulfide bond to reduce cystine (-S-S-) to cysteine (-SH), so that the high-level structure of keratin disintegrates to form degenerative keratin protein. The degenerative keratin protein is gradually hydrolyzed into polypeptides, oligopeptides and free amino acids by the action of polypeptide hydrolase. Finally, ammonia and sulfide are produced by transamination to completely hydrolyze keratin.
Procedure for Quantitating Sulfhydryl Groups Based on Molar Absorptivity
A. Material Preparation
• Reaction Buffer: 0.1M sodium phosphate, pH 8.0, containing 1mM EDTA (50ml stock)
• Ellman’s Reagent Solution: Dissolve 4mg Ellman’s Reagent in 1mL of Reaction Buffer.
B. Measure Absorbance
1. For each unknown sample to be tested, prepare a tube containing 50μL of Ellman’s Reagent Solution and 2.5mL of Reaction Buffer.
2. Add 250μL of each unknown to the separate test tubes prepared in step 1. As a blank, add 250μL of Reaction Buffer to a separate test tube prepared in Step 1.
Note: For the unknown(s), make dilutions so that the 250μL sample applied to the assay reaction has a sulfhydryl concentration less than 1.0mM. Concentrations exceeding 1mM free sulfhydryl will result in high absorbance values and less accurate estimation of the concentration based on the extinction coefficient.
3. Mix and incubate at room temperature for 15 min.
4. With a spectrophotometer set to 412nm, zero the instrument on the blank and then measure absorbance of each sample.
5. Calculate the amount and concentration of sulfhydryls in the sample from the molar extinction coefficient of TNB.
11. Packaging in hydrogel
The solution configuration:
MES buffer: dissolve 2-N-morpholine ethane sulfonate (MES 9.76g) and nacL 14.61g, constant volume to 500mL PH6.0.
Cross-linking system:0.00125% 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide 1-ethyl- (3-dimethylaminopropyl) carbonimide (EDC),A mixture of 0.000375% N-hydroxysuccinimide, and 0.00075% adipic acid dihydrazide (ADH), dissolved in MES buffer.
Preparation methods:
Keratinase-alginate core:
1. Alginate is dissolved in ultra-pure water (deionized water) and sterilized at 120℃ to obtain 5% alginate solution.
2. Mix the keratinase solution with 5% alginate solution 1:1, and the final concentration of alginate solution is 2.5%.
3. Take the mixture from the syringe and drop it on the sealing film to form bean-shaped balls (50-100 micro).
4. Cure by soaking it in 5%CaCl2 for 1 min (derived from NDNF team).
Preparation of gel shell (4℃):
Preparation of 2% alginate, 30% acrylamide, 0.046% ammonium persulfate, 0.015%N, n-methylenebiscrylamide methylenebiscrylamide preparation solution, vacuum degassed
2. The preparation solution was mixed with tetramethylenediamine (TEMED) solution at a ratio of 1000:1 to form a pre-gel.
3. The alginate core is immersed in a pre-gel to form an adjustable film of 100-1000μm in nitrogen.(Depending on soaking time).
4. Soak the gel in Step 3 into the MES buffer
5. The gel in Step 4 was immersed in the crosslinking system, and the crosslinking system was shaken by the shaking table at 4℃ for 3h
12. Verification of enzyme activity after packaging
- After 12h, 24h, 36h, and 48h of packaging at 4℃ and room temperature,the keratinase-alginate core should be taken out of the gel coat, which will remain liquid.
- The packaged keratinase-alginate core was mixed with 2.5 ml 80 mM tris buffer supplemented with 2 mM CaCl2 containing 1% (w/v) gelatin (500mg/50ml), and incubated for 15 min at suitable pH and temperature (pH7.5). The reaction was stopped by filtering through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
An increase in 0.01 OD was considered as 1 unit of enzyme activity.
13. Porcine (pig) skin tests [13, 14]
Preparation of porcine skin:
- Hides from freshly killed adult pigs with hairs were obtained from a local slaughterhouse. The hides were washed with cold water and de-fatted with a scalpel. The skin section was then rinsed in cold tap water and cut into pieces (3 by 3 cm) with a scalpel.
- The whole piece of porcine skin was cut into 2 x 2 cm pieces for a test.
- Each piece was treated with 70% ethanol and then washed with detergent to get rid of the fatty acid on the surface, then wash with cold water and dried with filter paper to clean up the water.
- The procine pieces were placed skin side down in individual petridishes separately for hair removal tests.
- 3 groups were set up to be treated respectively with 3ml of 94 mg/ml KU3, 91 mg/ml Q7, 90 mg/ml DTT and one group without treatment for control. Set up 2 copies for different incubation times of 12h and 24h.
Hair removal tests:
Hair on each piece of porcine skin was removed by the blunt side of a scalpal.
The liquid after treatment was filtered through a 0.22um filter. After that, 500ul of the clear supernatant was mixed with 2.5 ml 500 mM Na2CO3 and 0.5 ml Folin-Ciocalteu's phenol reagent, followed by incubation at room temperature for 30 min. The absorbance of the resulting supernatant was measured at 660 nm against a blank control.
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