I-proteinase K mNGS (uketshezi)
I-Proteinase K iyi-serine protease ezinzile enokucaciswa okubanzi kwe-substrate.Yehlisa isithunzi samaprotheni amaningi endaweni yendabuko ngisho nalapho kukhona izinto zokuhlanza.Ubufakazi obuvela ocwaningweni lwe-crystal nesakhiwo samangqamuzana bukhombisa ukuthi i-enzyme ingeyomndeni we-subtilisin ene-site catalytic triad esebenzayo (Asp39-Wakhe69-Ser224).Indawo eyinhloko yokuqhekeka isibopho se-peptide esiseduze neqembu le-carboxyl lama-amino acid aliphatic kanye nephunga elimnandi elinamaqembu e-alpha amino avinjiwe.Ngokuvamile isetshenziselwa ukucaciswa kwayo okubanzi.Le proteinase K yakhelwe ngokukhethekile i-mNGS.Uma kuqhathaniswa nenye i-proteinase K, iqukethe ukungcoliswa kwe-nucleic acid okungaphansi nokusebenza okufanayo kwe-enzymatic, okungaqinisekisa kangcono ukusetshenziswa kwe-mNGS engezansi.
Izimo Zokugcina
2-8℃ iminyaka emi-2
Ukucaciswa
| Ukubukeka | Uketshezi olungenambala ukuya kokunsundu ngokukhanyayo |
| Umsebenzi | ≥800 U/ml |
| Ukugxila Kwamaprotheni | ≥20 mg/ml |
| I-Nickase | Akukho okutholiwe |
| DNase | Akukho okutholiwe |
| RNase | Akukho okutholiwe |
Izakhiwo
| Inombolo ye-EC | 3.4.21.64(I-Recombinant evela ku-albhamu ye-Ttirachium) |
| Iphuzu le-isoelectric | 7.81 |
| I-pH engcono kakhulu | 7.0- 12.0 Umfanekiso 1 |
| Izinga lokushisa eliphezulu | 65 ℃ Umfanekiso 2 |
| pH ukuzinza | pH 4.5- 12.5 (25 ℃, 16 h) Fig. 3 |
| Ukuzinza okushisayo | Ngaphansi kuka-50 ℃ (pH 8.0, 30 min) Umfanekiso 4 |
| Ukuqina kwesitoreji | Umsebenzi ongaphezu kuka-90% wezinyanga eziyi-12 ku-25 ℃ |
| Izishoshovu | SDS, urea |
| Ama-inhibitors | I-Diisopropyl fluorophosphate;phenylmethylsulfonyl fluoride |
Izinhlelo zokusebenza
1. Ikhithi yokuxilonga yofuzo
2. I-RNA kanye ne-DNA extraction kits
3. Ukukhishwa kwezingxenye ezingezona amaprotheni ezicutshini, ukuwohloka kokungcola kwamaprotheni, njenge-DNAimigomo kanye nokulungiswa kwe-heparin
4. Ukulungiswa kwe-chromosome DNA nge-pulsed electrophoresis
5. Ibala laseNtshonalanga
6. Ama-enzymatic glycosylated albumin reagent in vitro diagnostic
Izinyathelo zokuzivikela
Gqoka amagilavu okuzivikela kanye nezibuko lapho usebenzisa noma ukala, futhi uhlale unomoya omuhle ngemva kokuwusebenzisa.Lo mkhiqizo ungase ubangele ukungezwani nesikhumba kanye nokucasuka okukhulu kwamehlo.Uma uhogele, kungase kubangele ukungezwani komzimba noma izimpawu zesifuba somoya noma i-dyspnea.Ingabangela ukucasuka kokuphefumula.
Incazelo yeyunithi
Iyunithi eyodwa (U) ichazwa njengenani le-enzyme edingekayo ukuze i-hydrolyze i-casein ukukhiqiza i-1 μmoltyrosine ngomzuzu ngaphansi kwezimo ezilandelayo.
Ukulungiswa kwama-reagents
I-Reagent I: I-1g yobisi i-casein yahlakazwa ku-50ml we-0.1M yesisombululo se-sodium phosphate (pH 8.0), ifakwe emanzini angu-65-70 ℃ imizuzu engu-15, inyakaziswa futhi incibilike, ipholiswe ngamanzi, ilungiswa nge-sodium hydroxide ibe yi-pH 8.0, kanye nevolumu ehleliwe 100ml.
I-Reagent II: 0.1M trichloroacetic acid, 0.2M sodium acetate, 0.3M acetic acid.
I-Reagent III: 0.4M Na2CO3isisombululo.
I-Reagent IV: I-Forint reagent ihlanjululwe ngamanzi ahlanzekile izikhathi ezi-5.
I-Reagent V: I-enzyme diluent: 0.1M isisombululo se-sodium phosphate (pH 8.0).
I-Reagent VI: isixazululo se-tyrosine: 0, 0.005, 0.025, 0.05, 0.075, 0.1, 0.25 umol/ml tyrosine ehlakazwe ngo-0.2M HCl.
Inqubo
1. I-0.5ml ye-reagent I ifudunyezwa ngaphambilini ibe ngu-37℃, engeza u-0.5ml wesisombululo se-enzyme, uhlanganise kahle, futhi ufukamele37℃ imizuzu engu-10.
2. Engeza i-1ml ye-reagent II ukuze umise ukusabela, uhlanganise kahle, futhi uqhubeke nokufukamela imizuzu engu-30.
3. Isixazululo se-Centrifugate reaction.
4. Thatha i-0.5ml supernatant, engeza u-2.5ml reagent III, 0.5ml reagent IV, hlanganisa kahle futhi ufukamele ku-37℃imizuzu engama-30.
5. OD660kwanqunywa njenge-OD1;iqembu lokulawula elingenalutho: I-0.5ml reagent V isetshenziselwa ukufaka esikhundleni se-enzymeisisombululo sokunquma i-OD660njengoba OD2, ΔOD=OD1-OD2.
6. Ijika elijwayelekile le-L-tyrosine: 0.5mL isixazululo se-concentration esihlukile se-L-tyrosine, 2.5mL Reagent III, 0.5mL Reagent IV ku-5mL centrifuge tube, incubate ku-37℃ imizuzu engu-30, thola i-OD660ngokugxila okuhlukile kwe-L-tyrosine, kwase kutholwa ijika elijwayelekile elingu-Y=kX+b, lapho u-Y ewugxilisano lwe-L-tyrosine, u-X engu-OD600.
Ukubala
2: Isamba sevolumu yesixazululo sokusabela (mL)
0.5: Umthamo wesixazululo se-enzyme (mL)
0.5: Ivolumu yoketshezi yokusabela esetshenziswa ekunqumeni kwe-chromogenic (mL)
10: Isikhathi sokuphendula (imizuzu)
Df: Dilution multiple
CUkugxila kwe-enzyme (mg/mL)
Izikhombo
1. Wieger U & Hilz H. FEBS Lett.(1972);23:77.
2. Wieger U & Hilz H. Biochem.I-Biophys.I-Res.Umphakathi.(1971);44:513.
3. Hilz, H.et al.,I-Eur.J. Biochem.(1975);56:103–108.
4. Sambrook Jet al., I-Molecular Cloning: Incwadi Yelabhorethri, uhlelo lwesi-2, i-Cold Spring HarborLaboratory Press, Cold Spring Harbour (1989).
Amanani
Fig.1 Okungcono kakhulu pH
Isixazululo sebhafa esingu-100mM:pH6.0-8.0, Na-phosphate;pH8.0-9.0, Tris-HCl;pH9.0-12.5, Glycine-NaOH.Enzyme concentration:1mg/mL
Fig.2 Izinga lokushisa eliphezulu
Ukusabela ku-20 mM K-phosphate buffer pH 8.0.Ukugxila kwe-enzyme: 1 mg/mL
Fig.3 pH Ukuzinza
25 ℃, 16 h-ukwelashwa ngesixazululo sebhafa esingu-50 mM: pH 4.5- 5.5, Acetate;pH 6.0-8.0, Na-phosphate;pH 8.0-9.0, Tris-I-HCl.pH 9.0- 12.5, Glycine-NaOH.Ukugxila kwe-enzyme: 1 mg/mL
Fig.4 Thermal ukuzinza
Ukwelashwa kwamaminithi angu-30 nge-50 mM Tris-HCl buffer, pH 8.0.Ukugxila kwe-enzyme: 1 mg/mL
Fig.5 Isitoreji uzinzilety at 25℃
