COÂNG NGHEÄ ENZYME

1. COÂNG NGHEÄ ENZYME Kyõthuaättaùchchieát enzyme

2. Phaùvôõmoâvaøteábaøo – Choise of tissues(Disruption of tissues and cells) Choice of tissues • Döïa vaøo giaù thaønh, tính saün coù vaø söï phong phuù cuûa enzyme • Recombinant DNA technology – xaây döïng host cell (expression system) • Traùnh hoaëc haïn cheá söû duïng caùc moâ giaøu proteinase. Vd: gan, laùch, thaän… ÔÛ vi si vaät taïo mutant strain (doøng ñoät bieán) thieáu moät soá proteinase (E. Coli, Bacillus subtilis)

3. Phaùvôõmoâvaøteábaøo – Choise of tissues(Disruption of tissues and cells) • Vi sinhvaätthöôøngñöôïcchoïnlaøm host organism • Öuñieåm: toácñoäsinhtröôûngnhanh • Nhöôïcñieåm: thieáucôcheácaûibieânsaudòchmaõvaø enzyme thöôøngôû dang inclusion body. • caûibieân gene bieåuhieängiuùpsaûnphaåm enzyme tieátramoâitröôøngngoaøiteábaøo.

4. Phaùvôõmoâ – taùchteábaøo(disruption of tissue and separation of cells) • Nhieàu loaïi teá baøo coù trong cuøng moät moâ. • Cell suspension ñöôïc chuaån bò baèng phöông phaùp cô hoïc (Mechanical method) hoaëc enzyme hoïc (enzymatic method). • Mechanical method: aûnh höông tôùi söï nguyeân veïn teá baøo. • Enzymatic method: collagenase from Clostridium histolyticum, trypsin, estalase…  taùch döïa vaøo ñieän tích, antigenicity, kòch thöôùc, tyû troïng.

5. Disruption of cells – teá baøo ñoäng vaät • Moâ ñöôïc caét nhoû (loaïi boû môõ vaø moâ lieân keát) • Moâ meàm Ñoàng hoùa trong thieát bò ñoàng hoùa • Moâ cöùng caàn ñöôïc xay nhoû trong maùy xay tröôùc khi cho vaøo thieát bò ñoàng hoùa. • Ly taâm.

6. Disruption of cells – teá baøo thöïc vaät • Thu nhaân enzyme töø teá baøo thöïc vaät töông ñoái khoù khaên do söï hieän dieän vaùch teá baøo (cell wall), khoâng baøo (vacuole) vaø phenolic compound • Vieäc phaù vôõ vacuole  giaûi phoùng proteinases, pH dòch chieát thaáp. • Khi coù oxygen, phenol oxidases xuùc taùc chuyeån phenolic compound thaønh daïng polymeric pigments coù theå baát hoaït (inactivate) enzyme trong dòch chieát • boå sung chaát khöû 2 – mercaptoethanol • boå sung polyvinylpolypyrollidone giuùp haáp thu phenolic compound.

7. Disruption of cells – teá baøo naám men • Vaùch teá baøo • Nhieàu proteinase • taïo ñoät bieán gaây ra söï thieáu huït moät vaøi proteinase • ngaên chaën söï taïo proteinase baèng caùch nuoâi treân moâi tröôøng khoâng chöùa cô chaát protein. • Phöông phaùp 1: uû baùnh men trong toluene (6% (v/w)) vaø 2 – mercaptoethonol (0.2 % (v/w)) ôû 370 C trong 1 h  taùch thaønh phaàn vaùch teá baøo  EDTA (15 mM) pH 7.0 chöùa 5 mM 2 – mercaptoethanol, uû qua ñeâm  phaù vôõ vaùch teá baøo  ly taâm 15 000 rpm trong 30 phuùt

8. Disruption of cells – teá baøo naám men • Phöông phaùp 2: shaking with glass bead – laéc vôùi haït thuûy tinh (ñöôøng kính 1 mm)  1ml dòch ñöôïc ly taâm 2500 rpm ôû 100 C. • Yeast Pichi pastoris ñöôïc duøng nhö teá baøo chuû, recombinant protein ñöôïc tieát vaøo moâi tröôøng nuoâi caáy.

9. Disruption of cells – teá baøo vi khuaån • Vaùch teá baøo • Phöông phaùp cô hoïc thöôøng aûnh höôûng ñeán caùc thaønh phaàn cuûa teá baøo. •  Vi khuaån gram döông (Bacillus, Micrococcus, Streptococcus) : duøng enzyme lysozyme phaù huûy vaùch teá baøo, uû vôùi lysozyme töø tröùng gaø (0.2 mg/ml) ôû 370 C trong 15 phuùt. •  Vi khuaån gram aâm (E. Coli, Klebsiella spp., Pseudomonas spp.) : röûa teábaøo vôùi detergent (0.1% (v/v) N-lauroyl-sarcosine)  xöû lyù vôùi vôùi sucrose (0.7 M), Tris (0.2 M), EDTA (0.04 M)

10. Disruption of cells – teá baøo vi khuaån • Dnatrongdòchchieátcoùcoùñoänhôùtcaogaâykhoùkhaêntrongvieäctinhsaïch enzyme • hoaëcxöûlyùvôùideoxyribonuclease 1 (10 μg/ml) • hoaëc xöû lyù vôùi protamine (protein giaøu arginine trong caáu taïo) • hoaëc xöû lyù vôùi positively charged polymer (polyethyleneimine)

11. Enzyme gaén treân maøng teábaøo • Maøng teá baøo caàn ñöôïc xöû lyù vôùi non – ionic detergent nhö Triton hay Tween. Caùc detergent naøy ít gaây aûnh höông leân caáu truùc protein noùi chung vaø enzyme noùi rieâng. • Qui luaät söû duïng detergent: 2 mg detergent duøng cho 1 mg maøng teá baøo. • CD – circular dichroism ñöôïc duøng ñeå kieåm tra caáu truùc baäc 2 cuûa enzyme taùch chieát.