Proteinase K NGS (phofo)
Nomoro ea katse: HC4507A
NGS Protease K ke serine protease e tsitsitseng e nang le ts'ebetso e phahameng ea enzyme le likarolo tse ngata tse ikhethang.Ensaeme ka ho khetheha e senya li-ester bond le peptide bonds e haufi le C-terminal ea hydrophobic amino acid, sulfur e nang le amino acid le amino acid e nkhang hamonate.Kahoo, hangata e sebelisoa ho theola liprotheine hore e be li-peptide tse khutšoane.NGS Protease K ke serine protease e tloaelehileng e nang le Asp39- Hae69-Ser224catalytic triad e ikhethang ho serine proteases, 'me setsi sa catalytic se pota-potiloe ke tow Ca.2+libaka tse tlamang bakeng sa ho tsitsisa, ho boloka ts'ebetso e phahameng ea enzyme tlas'a maemo a mangata a mangata.
Tlhaloso
| Ponahalo | E tšoeu ho ea ho e tšoeu ea amorphous phofo, e lyophilized |
| Ts'ebetso e khethehileng | ≥40U/mg e tiileng |
| DNase | Ha ho e fumanoeng |
| RNase | Ha ho e fumanoeng |
| Bioburden | ≤50CFU/g e tiileng |
| Nucleic acid mesaletsa | <5pg/mg e tiileng |
Thepa
| Mohloli | Album ea Tritirachium |
| Nomoro ea EC | 3.4.21.64(Recombinant ho tsoa ho albamo ea Tritirachium) |
| Boima ba molek'hule | 29kDa (SDS-PAGE) |
| Ntlha ea Isoelectric | 7.81 Setšoantšo sa 1 |
| Optimum pH | 7.0-12.0 (Bohle ba etsa mosebetsi o phahameng) Fig.2 |
| Optimum mocheso | 65℃ Setšoantšo sa 3 |
| pH botsitso | pH 4.5-12.5 (25℃,16h) Setšoantšo sa 4 |
| Ho tsitsa ha mocheso | Ka tlase ho 50 ℃ (pH 8.0, 30min) Fig.5 |
| Botsitso ba polokelo | E bolokiloe ho 25 ℃ bakeng sa 12months Fig.6 |
| Li-activators | SDS, urea |
| Li-inhibitors | diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Maemo a polokelo
Boloka phofo ea lyophilized ho -25~-15 ℃ nako e telele hole le leseli;Kamora ho qhibiliha, aliquot ka har'a molumo o loketseng bakeng sa polokelo ea nako e khuts'oane ho 2-8 ℃ hole le khanya kapa polokelo ea nako e telele ho -25 ~ -15 ℃ hole le leseli.
Mehato ya tlhokomelo
Apara litlelafo le likhalase tsa tšireletso ha u sebelisa kapa u bekha boima ba 'mele, 'me u lule u e-na le moea o motle ka mor'a hore u li sebelise.Sehlahisoa sena se ka etsa hore letlalo le se ke la hana le ho teneha haholo.Haeba e hema, e ka baka matšoao a allergy kapa asthma kapa dyspnea.E ka baka ho teneha ha moea.
Tlhaloso ea yuniti
Yuniti e le 'ngoe ea NGS Protease K e hlalosoa e le palo ea enzyme e hlokahalang ho hydrolyze casein ho 1 μmol L-tyrosine tlas'a maemo a tloaelehileng a boikemisetso.
Ho lokisoa ha li-reagents
| Reagent | Moetsi | Catalog |
| Casein tekhenikiho tsoa lebeseng la likhomo | Sigma Aldrich | C7078 |
| NaOH | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 20040718 |
| Na2HPO4 | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 20040618 |
| Asiti ea trichloroacetic | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 80132618 |
| Acetate ea sodium | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 10018818 |
| Acetic acid | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 10000218 |
| HCl | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 10011018 |
| Sodium carbonate | Lik'hemik'hale tsa SinopharmSehlopha sa Reagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Substrate: 1% Casein ho tsoa ho motsoako oa lebese la bovine: qhala 1g ea lebese la bovine casein ka 50ml ea 0.1M sodium phosphate solution, pH 8.0, mocheso ka har'a bateng ea metsi ka 65-70 °C bakeng sa metsotso e 15, hlohlelletsa le ho qhibiliha, pholile ka metsi, e lokisoa ke sodium hydroxide ho pH 8.0, 'me e hlapolle ho 100ml.
Reagent II:
Tharollo ea TCA: 0.1M trichloroacetic acid, 0.2M sodium acetate le 0.3M acetic acid (ka boima ba 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid ka tatellano, eketsa 50mL metsi a deionized, fetola le 4.HCl ho pH 4. 100ml).
Reagent III:
0.4m sodium carbonate solution (e boima ba 4.24g anhydrous sodium carbonate mme e qhibilihe ka metsing a 100mL)
Reagent IV:
Folin phenol reagent: hlapolla makhetlo a 5 ka metsi a deionized.
Reagent V:
Mokelikeli oa enzyme: 0.1 M tharollo ea sodium phosphate, pH 8.0.
Reagent VI:
Tharollo e tloaelehileng ea L-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine e qhibilihisitsoeng ka 0.2M HCl.
Tsamaiso
1. Bulela UV-Vis spectrophotometer ebe u khetha tekanyo ea photometric.
2. Beha bolelele ba maqhubu joalo ka 660nm.
3. Bulela ho hlapa metsi, beha mocheso ho 37 ℃, etsa bonnete ba hore mocheso o sa fetohe bakeng sa 3-5mins.
4. Preheat 0.5mL substrate ka 2mL centrifuge tube ka 37 ℃ metsi a ho hlapa bakeng sa 10mins.
5. Ntša 0.5mL tharollo ea enzyme e hlapolotsoeng ka har'a tube ea centrifuge e futhumetseng ka metsotso e 10.Beha diluent ea enzyme e le sehlopha se se nang letho.
6. Eketsa 1.0 mL TCA reagent hang ka mor'a karabelo.Kopanya hantle 'me u e etsetse ka bateng ea metsi metsotso e 30.
7. Centrifugate reaction solution.
8. Kenya likarolo tse latelang ka tatellano e boletsoeng.
| Reagent | Bolumo |
| Supernatant | 0.5 mL |
| 0.4M Sodium carbonate | 2.5 mL |
| Folin phenol reagent | 0.5 mL |
9. Kopanya hantle pele u e kenya ka bateng ea metsi ka 37℃ bakeng sa metsotso e 30.
10. OD660e ile ea khethoa e le OD1;sehlopha sa taolo se se nang letho: Mokelikeli oa enzyme o sebelisoa ho nkela tharollo ea enzyme ho fumana OD660joalo ka OD2, ΔOD=OD1-OD2.
11. L-tyrosine standard curve: 0.5mL e fapaneng ea mahloriso ea L-tyrosine tharollo, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent ka 5mL centrifuge tube, incubate ka 37 ℃ bakeng sa 30mins, lemoha bakeng sa OD660bakeng sa mahloriso a fapaneng a L-tyrosine, joale a fumana lekhalo le tloaelehileng la Y=kX+b, moo Y e leng mahloriso a L-tyrosine, X ke OD600.
Palo
2: Kakaretso ea bolumo ea tharollo ea karabelo (mL)
0.5: Bophahamo ba tharollo ea enzyme (mL)
0.5: Bophahamo ba mokelikeli o sebetsang o sebelisitsoeng qetong ea chromogenic (mL)
10: Nako ea ho arabela (metsotso)
Df: Dilution multiple
CKhatiso ea enzyme (mg/mL)
Lipalo
Fig.1 masalla a DNA
| Mohlala | Ave C4 | Nucleic Acid Ho hlaphoheloa(pg/mg) | Khutlisa(%) | Kakaretso ea Nucleic Asiti ( p/mg) |
| PRK | 24.66 | 2.23 | 83% | 2.687 |
| PRK+STD2 | 18.723 | 126.728 | - | - |
| STD1 | 12.955 |
- |
- |
- |
| STD2 | 16 | |||
| STD3 | 19.125 | |||
| STD4 | 23.135 | |||
| STD5 | 26.625 | |||
| RNA-Mahala H2O | E sa ikemisetsa | - | - | - |
Fig.2 Optimum pH
Fig.3 Mocheso o motle ka ho fetisisa
Fig.4 pH botsitso
Setšoantšo sa 5 Thermal botsitso
Setšoantšo sa 6 Ho boloka botsitso ho 25 ℃
