Proteinase K NGS (hupfu)
Katsi Nhamba: HC4507A
NGS Protease K is a stable serine protease ine high enzyme activity uye wide substrate specificity.Iyo enzyme inosarudza kuora ma bond ester uye peptide bond pedyo neC-terminal yehydrophobic amino acids, sulfur-containing amino acids uye anonhuwirira amino acids.Saka, inowanzoshandiswa kusvibisa mapuroteni kuita mapfupi peptides.NGS Protease K yakajairika serine protease ine Asp39-Zvake69-Ser224catalytic triad iyo yakasarudzika kune serine proteases, uye iyo catalytic centre yakakomberedzwa netow Ca.2+kusunga nzvimbo dzekudzikamisa, kuchengetedza yakakwirira enzyme basa pasi pehupamhi hwemamiriro ezvinhu.
Tsanangudzo
| Chitarisiko | Muchena kusvika kune-white amorphous powder, lyophilized |
| Chiitiko chakananga | ≥40U/mg yakasimba |
| DNase | Hapana chaonekwa |
| RNase | Hapana chaonekwa |
| Bioburden | ≤50CFU/g yakasimba |
| Nucleic acid zvakasara | <5pg/mg yakasimba |
Properties
| Source | Tritirachium album |
| EC nhamba | 3.4.21.64(Recombinant kubva kuTtirachium album) |
| Molecular uremu | 29kDa (SDS-PEJI) |
| Isoelectric point | 7.81 Fig.1 |
| Optimum pH | 7.0-12.0 (Vose vanoita basa repamusoro) Fig.2 |
| Optimum tembiricha | 65℃ Fig.3 |
| pH Kugadzikana | pH 4.5-12.5 (25℃,16h) Fig.4 |
| Thermal kugadzikana | Pasi pe50 ℃ (pH 8.0, 30min) Fig.5 |
| Kuchengetedza kugadzikana | Yakachengetwa pa25 ℃ kwe12months Fig.6 |
| Activators | SDS, urea |
| Inhibitors | diisopropyl fluorophosphate;benzylsulfonyl fluoride |
Storage Conditions
Chengetedza iyo lyophilized powder pa -25~-15 ℃ kwenguva yakareba kure nechiedza;Mushure mekuparadzwa, aliquot muvhoriyamu yakakodzera yekuchengetedza kwenguva pfupi pa 2-8 ℃ kure nechiedza kana kuchengetwa kwenguva refu pa -25 ~ -15 ℃ kure nechiedza.
Zvekungwaririra
Pfeka magirovhosi uye magirazi ekudzivirira paunenge uchishandisa kana kuyera, uye gara uine mhepo yakanaka mushure mekushandisa.Ichi chigadzirwa chinogona kukonzera ganda allergic reaction uye zvakanyanya kushatirwa kwemaziso.Kana inhaled, inogona kukonzera allergies kana asthma zviratidzo kana dyspnea.Inogona kukonzera kushatirwa kwekufema.
Tsanangudzo yeyuniti
Imwe yuniti yeNGS Protease K inotsanangurwa sehuwandu hwe enzyme inodiwa ku hydrolyze casein mu 1 μmol L-tyrosine pasi pezvimiro zvekutsunga.
Reagents kugadzirira
| Reagent | Mugadziri | Catalog |
| Casein technicalkubva mumukaka webovine | Sigma Aldrich | C7078 |
| NaOH | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 10019762 |
| NaH2PO4·2H2O | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 20040718 |
| Na2HPO4 | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 20040618 |
| Trichloroacetic acid | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 80132618 |
| Sodium acetate | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 10018818 |
| Acetic acid | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 10000218 |
| HCl | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 10011018 |
| Sodium carbonate | Sinopharm ChemicalIyo kambani Reagent Co., Ltd. | 10019260 |
| Foline-phenol | Sangon Biotech (Shanghai)Co., Ltd. | A500467-0100 |
| L-tyrosine | Sigma | 93829 |
Reagent I:
Substrate: 1% Casein kubva mumushonga wemukaka webovine: nyungudutsa 1g yebovine milk casein mu50ml ye0.1M sodium phosphate solution, pH 8.0, kupisa mumvura yekugezera pa65-70 °C kwemaminetsi gumi nemashanu, kurunga nekunyunguduka, tonhorera nemvura, inogadziriswa ne sodium hydroxide kusvika pH 8.0, uye wedzera kusvika ku100ml.
Reagent II:
TCA mhinduro: 0.1M trichloroacetic acid, 0.2M sodium acetate uye 0.3M acetic acid (inorema 1.64g trichloroacetic acid + 1.64g sodium acetate + 1.724mL acetic acid zvakatevedzana, kuwedzera 50mL mvura yakasvibiswa, gadzirisa ne4.HCl kusvika pH, 4. 100ml).
Reagent III:
0.4m sodium carbonate solution (inoyera 4.24g anhydrous sodium carbonate uye inonyunguduka mu 100mL mvura)
Reagent IV:
Folin phenol reagent: dilute 5 nguva nemvura yakasvibiswa.
Reagent V:
Enzyme diluent: 0.1 M sodium phosphate solution, pH 8.0.
Reagent VI:
L-tyrosine standard solution: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol / ml L-tyrosine yakanyungudutswa ne0.2M HCl.
Maitiro
1. Batidza UV-Vis spectrophotometer uye sarudza photometric kuyerwa.
2. Seta wavelength se660nm.
3. Vhura bhati remvura, isa tembiricha kusvika 37 ℃, simbisa tembiricha isingachinji kwe3-5mins.
4. Preheat 0.5mL substrate mu 2mL centrifuge chubhu pa 37 ℃ mvura yekugezera kwema10mins.
5. Bvisa 0.5mL diluted enzyme solution mu preheated centrifuge chubhu kwemaminitsi gumi.Isa enzyme diluent seboka risina chinhu.
6. Wedzera 1.0 mL TCA reagent pakarepo mushure mekuita.Sanganisa zvakanaka woisa mubhavhu remvura kwemaminetsi makumi matatu.
7. Centrifugate reaction solution.
8. Wedzera zvinotevera zvikamu muhurongwa hwakataurwa.
| Reagent | Volume |
| Supernatant | 0.5 mL |
| 0.4M Sodium carbonate | 2.5 mL |
| Folin phenol reagent | 0.5 mL |
9. Sanganisa zvakanaka usati waisa incubating mumvura yekugeza a 37℃ for 30 mins.
10. OD660yakatemwa seOD1;blank control group: Enzyme diluent inoshandiswa kutsiva enzyme mhinduro kuona OD660seOD2, DOD=OD1-OD2.
11. L-tyrosine yakajairika curve: 0.5mL yakasiyana-siyana L-tyrosine solution, 2.5mL 0.4M Sodium carbonate, 0.5mL Folin phenol reagent mu 5mL centrifuge tube, incubate mu 37 ℃ kwe 30mins, tsvaga OD660nokuda kwehuwandu hwakasiyana hweL-tyrosine, ndokuwana iyo yakajairwa curve Y=kX+b, apo Y ndiyo L-tyrosine concentration, X iri OD.600.
Calculation
2: Yese vhoriyamu yemhinduro mhinduro (mL)
0.5: Vhoriyamu ye enzyme solution (mL)
0.5: Reaction fluid volume inoshandiswa mukutsunga kwechromogenic (mL)
10: Nguva yekuita (min)
Df: Dilution yakawanda
CKuwedzera kwe enzyme (mg/mL)
Figures
Fig.1 DNA zvakasara
| Muenzaniso | Ave C4 | Nucleic Acid Kupora (pg/mg) | Kudzoreredza(%) | Total Nucleic Asidhi ( pg/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-Yemahara H2O | Undetermined | - | - | - |
Fig.2 Optimum pH
Fig.3 Optimum tembiricha
Fig.4 pH Kugadzikana
Fig.5 Thermal kugadzikana
Fig.6 Kuchengetedza kugadzikana pa25 ℃
