Team:XH-China/Experiments
Summary of Experiments
Why we pick Keratinase
At first Molyneux et al. (1959) attempted to isolate some bacteria that are able to degrade keratin. He isolated organisms from the contents of experimentally induced dermoid cysts from mid lateral region of sheep. Examination of wool sample showed degraded wool with numerous cortical and cuticular cells. He found disruption of wool fiber in both in vivo and in vitro. He showed that the organisms belong to genus Bacillus and the organism was capable of attacking native wool protein. The same year Noval et al. (1959) published another article on enzymatic decomposition of native keratin by Streptomyces fradiae. They showed extracellular enzyme secreted by these bacteria capable of degrading the human hair or other tissue in its native state. Many scientists did a lot of research work about keratinase and provide many interesting findings. They also notice that the enzyme-activity was inhibited by reduced glutathione, PMSF and 2-¬Mercaptaethanol..
According to above achievement and clue, we start our project with keratinase. We try to use this amazing bio-enzyme protein in beauty products. Bio-mask with keratinase to help degrade dead Keratinocyte and deep clean skin to make skin looks young, shiny and healthy. Therefore, the starting point and basis of the whole work is to obtain keratinase. As we all know, genetic engineering is an important method for protein synthesis. Therefore, using genetic engineering methods to prepare the keratinase we need is one of the main tasks of our laboratory.
Plan of our experiment
where did we work in?
Our laboratory located in beautiful Qianhai Free Trade Zone, Shenzhen, which belong to Shenzhen Blot Bio-products Ltd. There are very complete and advanced equipment and experienced technicians, which can fully guarantee the implementation of our project, and the teachers here are very friendly and professional, so any difficulties and problems encountered in our work can get their timely help and resolved.
Construction of Keratinase recombinant plamid
Before start our experiment, the first step is to search and select target keratinase gene. We use https://www.ncbi.nlm.nih.gov/ to search Keratinase gene, the search result show a few keratinase gene from different resource. Finally, we pick keratinase gene from Bacillus licheniformis. The gene sequence shows as following:
1 atgatgagga aaaagagttt ttggcttggg atgctgacgg ccttcatgct cgtgttcacg
61 atggcattca gcgattccgc ttctgctgct caaccggcga aaaatgttga aaaggattat
121 attgtcggat ttaagtcagg agtgaaaacc gcatctgtca aaaaggacat catcaaagag
181 agcggcggaa aagtggacaa gcagtttaga atcatcaacg cggcaaaagc gaagctagac
241 aaagaagcgc ttaaggaagt caaaaatgat cccgatgtcg cttatgtgga agaggatcat
301 gtggcccatg ccttggcgca aaccgttcct tacggcattc ctctcattaa agcggacaaa
361 gtgcaggctc aaggctttaa gggagcgaat gtaaaagtag ccgtcctgga tacaggaatc
421 caagcttctc atccggactt gaacgtagtc ggcggagcaa gctttgtggc tggcgaagct
481 tataacaccg acggcaacgg acacggcaca catgttgccg gtacagtagc tgcgcttgac
541 aatacaacgg gtgtattagg cgttgcgcca agcgtatcct tgtacgcggt taaagtactg
601 aattcaagcg gaagcggatc atacagcggc attgtaagcg gaatcgagtg ggcgacaaca
661 aacggcatgg atgttatcaa tatgagcctt gggggagcat caggctcgac agcgatgaaa
721 caggcagtcg acaatgcata tgcaagaggg gttgtcgttg tagctgcagc agggaacagc
781 ggatcttcag gaaacacgaa tacaattggc tatcctgcga aatacgattc tgtcatcgct
841 gttggtgcgg tagactctaa cagcaacaga gcttcatttt ccagtgtggg agcagagctt
901 gaagtcatgg ctcctggcgc aggcgtatac agcacttacc caacgaacac ttatgcaaca
961 ttgaacggaa cgtcaatggc ttctcctcat gtagcgggag cagcagcttt gatcttgtca
1021 aaacatccga acctttcagc ttcacaagtt cgcaaccgtc tctccagcac ggcgacttat
1081 ttgggaagct ccttctacta tgggaaaggt ctgatcaatg tcgaaggtgc cgctcaataa
Translation="MMRKKSFWLGMLTAFMLVFTMAFSDSASAAQPAKNVEKDYIVGF
KSGVKTASVKKDIIKESGGKVDKQFRIINAAKAKLDKEALKEVKNDPDVAYVEEDHVA
HALAQTVPYGIPLIKADKVQAQGFKGANVKVAVLDTGIQASHPDLNVVGGASFVAGEA
YNTDGNGHGTHVAGTVAALDNTTGVLGVAPSVSLYAVKVLNSSGSGSYSGIVSGIEWA
TTNGMDVINMSLGGASGSTAMKQAVDNAYARGVVVVAAAGNSGSSGNTNTIGYPAKYD
SVIAVGAVDSNSNRASFSSVGAELEVMAPGAGVYSTYPTNTYATLNGTSMASPHVAGA
AALILSKHPNLSASQVRNRLSSTATYLGSSFYYGKGLINVEGAAQ"
We chose this gene coding sequence as our target gene fragment for keratinase expression in E.coli. The main strategy is to insert the target gene sequence into an expression plasmid vector by genetic engineering to express keratinase. The technical pathway show as follows:
The DNA synthesis order had been sent the commercial gene synthesis company to made keratinase gene sequence, which put in a regular plasmid with two specific restriction endonuclease BamHI/KpnI.
After we got received plasmid (powder and E.coli Top10 carry this plasmid), firstly we amplify this plasmid by growing above E.coli in LB medium, then isolate plasmid DNA from E.coli by plasmid extraction kit.
1.Digest the plasmid with restriction endonuclease BamHI/KpnI;
2.Confirm the expected size DNA insert by agarose gel electrophoresis;
3.After confirm the right insert DNA band is there, run above enzyme digest reaction on low melting argrose gel in TAE electrophoresis buffer;
4.Cut and purify the insert DNA band, the insert DNA had been purified by agarose DNA purify kit;
5.On the same time, plasmid pTrcHis2c also had been digest by endonuclease BamHI/KpnI and purify liner plasmid as above method;
6.Run ligation reaction above insert DNA and pTrcHis2c, then transfer ligation into E.coli host cell Rossete or BL21(DE3);
7.Inoculate above reaction on LB plate with Ampicillin(50ug/ml), 37℃, over night;
8.Pick single colony from above culture and confirm the recombinant plasmid is in host cell;
9.Inoculate the right colony in to 5ml LB broth (Ampicillin 50ug/ml), when the E. coli grow to 108/ml;
10.Inoculate above culture into 500ml LB broth, when shake in shaking incubator around 3-5hours (log grow phase), induce by adding IPTG, harvest the cell after 5 hours;
11.Check and confirm the recombinant keratinase expression by SDS-PAGE and western blot;
12.Harvest Keratinase by broken cell and Ni ion affinity chromatography to purify keratinase and test for following experiment.
Evaluation of biological activity and safety of Keratinase
Use casein to detect the biological activity of artificial recombinant keratinase
A. Principle
Proteases can cleave peptide bonds. In the laboratory, it is usually necessary to measure or compare the activity of proteases. Sigma's non-specific protease activity test can be used as a standard method to measure protease activity. The test uses casein as the substrate. When the test protease is used to digest casein, tyrosine is released together with other amino acids and peptides. Folin & Ciocalteus phenol or Folin reagent can mainly react with free tyrosine and produce a blue chromophore, which can be quantified and measured by the absorbance value of a spectrophotometer. The more tyrosine released by casein, the more chromophores are produced, indicating that the protease activity is stronger. In the detection, the absorbance value produced by the protease activity is compared with the standard curve, and the standard curve is generated by reacting a known amount of tyrosine with FC reagent, and the change in absorbance value is compared with the amount of micromolar tyrosine Associated and generated. According to the standard curve, the activity of the protease sample can be determined in units, and the unit represents the micromolar amount of tyrosine equivalent released by casein per minute.
B. Material
Reagents:
Protease
Dipotassium hydrogen phosphate trihydrate
Casein
Trichloroacetic acid
Folin & Ciocalteu’s phenol reagent
Anhydrous sodium carbonate
Sodium acetate trihydrate
Calcium acetate
Free base L-tyrosine
equipment:
0.45 um polyether sulfone syringe filter and syringe
Dram sample bottle or polypropylene tube that can hold 15ml solution
Spectrophotometer
CuvettePipette
Stirring/heating plate
Stirring rod
Balance
pH meter
Drawing program
C. Reagent preparation
1. 50mM potassium phosphate buffer, pH 7.5. During the preparation, 11.4 mg/ml of dipotassium hydrogen phosphate trihydrate was added to purified water, and the pH was adjusted with 1M HCl. Keep this solution at 37°C before use.
2. Prepare a casein solution with a weight/volume ratio of 0.65% by mixing 6.5mg/ml of 50mM potassium phosphate buffer. Under gentle stirring, the temperature of the solution was gradually increased to 80-85°C and maintained for about 10 minutes until uniform dispersion was achieved. It must be ensured that boiling does not occur. Then use NaOH and HCl to adjust the pH as needed.
3. 110mM trichloroacetic acid solution, prepared by diluting the 6.1N stock solution 1:55 with pure water. Trichloroacetic acid is a strong acid and should be handled with care.
4. 0.5M Folin & Ciolcaltea’s or Folin phenol reagent can react with tyrosine and produce a measurable color change directly related to protease activity. Folin phenol reagent is an acid and needs to be handled with care.
5. A 500mM sodium carbonate solution, prepared at 53mg/ml by adding anhydrous sodium carbonate to pure water.
6. Enzyme dilution solution, composed of 10mM sodium acetate buffer (containing 5mM calcium, pH 7.5, 37°C). This solution can be used to dissolve solid protease samples or dilute enzyme solutions.
7. 1.1 mM L-tyrosine standard stock solution. Add L-tyrosine to pure water to make a concentration of 0.2mg/ml, and slowly heat until tyrosine is dissolved. As with casein, no boiling occurs. After the L-tyrosine standard is cooled to room temperature, the solution can be further diluted to prepare a standard curve.
D. Establishment of standard curve
1. Prepare 6 15ml polypropylene tubes, add 50ul, 100ul, 200ul, 400ul, 500ul 1.1mM tyrosine standard solution to the above-mentioned tubes, and set a blank control tube without adding tyrosine solution ；
2. Fill the tube with pure water to make up the final volume to 2ml;
3. Add 5ml 500mM sodium carbonate solution to the above tube, and immediately add 1ml Folin phenol reagent;
4. Rotate and mix at 37°C for 30 minutes;
5. Use a 0.45um filter to filter 2ml of the above solution for spectrophotometer detection;
6. Detect the absorbance values of the above standard solution and blank solution at a wavelength of 660nm, and calculate the difference between the absorbance values of the standard solution and the blank solution;
7. Take the change in absorbance of the standard as the Y axis, and use the corresponding micromolar amount of the 5 standard solutions as the X axis, and use the drawing program to draw the standard curve;
8. After we input the corresponding data, generate the standard curve and equation.
The fourth power polynomial regression equation is:
Where:
p1 = 5.423
p2 = -11.43
p3 = 8.855
p4 = -1.8
p5 = 0.1558
E. Protease activity detection
1. Take 4 15ml polypropylene tubes, one of which will be used as a blank control, and the other three will be used for three different dilutions of keratinase;
2. Add 5ml of 0.65% casein solution to the above four sample bottles, and let them stabilize in a water bath at 37°C for about 5 minutes;
3. Add 20ul, 50ul and 100ul keratinase solution to the three test sample bottles other than the blank control bottle. Rotate to mix and incubate at 37°C for 10 minutes;
4. After incubating for 10 minutes, add 5ml trichloroacetic acid TCA reagent to each tube to stop the reaction. Then add an appropriate amount of enzyme solution to the above-mentioned tubes including the blank tube so that the final volume of the enzyme solution in each tube is 0.1 ml;
5. Incubate the above solution at 37°C for 30 minutes;
6. After incubating for 30 minutes, use a 0.45um filter to filter each 2ml of the above solution and transfer it to a new polypropylene tube;
7. Add 5ml 500mM sodium carbonate to the above tube, and immediately add 1ml Folin phenol reagent; 8. Rotate the above solution to mix and incubate at 37°C for 30 minutes;
9. Use a 0.45um filter to filter 2ml of the above solution until colorimetric detection and record the corresponding absorbance value.
Result：
Table 2. Spectrophotometer test results after keratinase digested casein
E. Measure absorbance and calculate enzyme activity
Then, we can substitute the difference in absorbance between each test keratinase and the blank sample into the slope formula to calculate the corresponding amount of tyrosine (uMol) in this specific proteolysis reaction. Subsequently, the corresponding enzyme activity value in units/ml is obtained based on the following formula.
11= The total volume of the detection reaction (ml)
10= Detection time per unit (minutes)
0.1= Volume of enzyme used (ml)
2= Volume used in colorimetric measurement (ml)
The number of micromoles of tyrosine divided by the number of minutes of time is the unit value of protease activity we measured.
Result:
Table 4. Keratinase activity
F. Conclusion
Therefore, the activity of the synthesized keratinase is 4.24 units/ml/min
Function evaluation of Bio-mask with Keratinase
Prepare cream mask base. (Bought from Shanghai woyou media Ltd. Total 500g);
2.Prepare Keratinase protein solution 300ug/ml;
3.Mix keratinase solution with cream mask base to make biological cream mask with different concentration keratinase, such as 10ug/ml,5ug/ml,0.5ug/ml and 0.1ug/ml;
4.The highest keratinase concentration 10ug/ml cream mask had been used to test Bio mask’s sfety;
Results showed as below:
Conclution:
The biological mask with keratinase can clean the skin very well, make the skin moisturized, youthful, and full of elasticity, change the appearance of the skin without any harm to the skin.