Enzyme hoc

Download Enzyme hoc

Post on 26-May-2015

9.684 views

Category:

Technology

11 download

Embed Size (px)

TRANSCRIPT

<ul><li> 1. 4Mc lcTrangLi ni u 3Chng 1M u71.1.nh ngha enzyme 71.2.Lc s nghin cu enzyme 7 1.2.1. Giai on 1 7 1.2.2. Giai on 2 8 1.2.3. Giai on 3 9 1.2.4. Giai on 4121.3.Phng hng nghin cu enzyme 141.4.Nhng vn cn cp khi nghin cu enzyme161.5.Vn nghin cu enzyme nc ta 17Ti liu tham kho 18Chng 2Phng php nghin cu enzyme192.1.Nhng nguyn tc chung khi nghin cu enzyme 192.2.Tch v lm sch (tinh ch) enzyme 21 2.2.1. Chn ngun nguyn liu 21 2.2.2. Chit rt enzyme 26 2.2.3. Cc phng php tch tng phn protein enzyme28 2.2.4. Kt tinh protein enzyme38 2.2.5. nh gi tnh ng th ca protein enzyme392.3.Hot enzyme 41 2.3.1. Phng php xc nh hot enzyme41 2.3.2. n v hot enzyme41Ti liu tham kho 4 43Chng 3Cch gi tn v phn loi enzyme 443.1.Cch gi tn enzyme443.2.Phn loi enzyme 44</li></ul><p> 2. 5 3.2.1. Cc lp enzyme44 3.2.2. Cc phn ng enzyme 46Ti liu tham kho51Chng 4Cu trc phn t enzyme 524.1.Bn cht ha hc ca enzyme 524.2.Thnh phn cu to ca enzyme 534.3.Cu trc bc 4 ca enzyme 544.4.Trung tm hot ng ca enzyme564.5.Phng php thm d v pht hin cc nhm chc nng trong 57trung tm hot ng ca enzyme 4.5.1. Phng php dng cht c ch58 4.5.2. Phng php nh du bng c cht c hiu hoc coenzyme59 4.5.3. Xc nh tr s pK ca cc nhm hot ng 60 4.5.4. Nghin cu cu trc phn t 604.6.Cc dng phn t ca enzyme 614.7.Phc hp multienzyme62Ti liu tham kho63Chng 5Tnh c hiu ca enzyme645.1.Khi nim chung 645.2.Cc hnh thc c hiu64 5.2.1. c hiu kiu phn ng64 5.2.2. c hiu c cht64Ti liu tham kho68Chng 6C ch tc dng ca enzyme696.1.C ch ca phn ng c xc tc ni chung696.2.C ch ca xc tc enzyme 69Ti liu tham kho73 3. 6Chng 7ng hc Enzyme 747.1. ngha ca vic nghin cu ng hc enzyme 747.2.ng hc cc phn ng enzyme74 7.2.1. nh hng ca nng enzyme74 7.2.2. nh hng ca nng c cht [S] 75 7.2.3. nh hng ca cht km hm (inhibitior) 79 7.2.4. nh hng ca cht hot ha (activator)9 7.2.5. nh hng ca nhit 87 7.2.6. nh hng ca pH88 7.2.7Cc yu t khc 89Ti liu tham kho91Chng 8Sinh hc enzyme 928.1 S phn b enzyme trong t bo928.2 iu ha hot v s lng ca enzyme trong t bo94 8.2.1iu ha hot enzyme 94 8.2.2iu ha sinh tng hp enzyme101Ti liu tham kho 108Chng 9Cng ngh enzyme v ng dng 1099.1.Cng ngh enzyme 109 9.1.1. Enzyme vi cng ngh sinh hc109 9.1.2. Cng ngh sn xut enzyme1099.2.ng dng 111 9.2.1. ng dng trong y dc111 9.2.2. ng dng trong ha hc 112 9.2.3. ng dng trong cng nghip 113Ti liu tham kho 116 4. 7Chng 1 M u1.1. nh ngha enzymeTrong c th sng (cc t bo) lun lun xy ra qu trnh trao icht. S trao i cht ngng th s sng khng cn tn ti. Qu trnh traoi ca mt cht l tp hp cc quy lut ca rt nhiu cc phn ng hahc khc nhau. Cc phn ng ha hc phc tp ny c lin quan cht chvi nhau v iu chnh ln nhau. Enzyme l cc hp cht protein xc tccho cc phn ng ha hc . Chng c kh nng xc tc c hiu ccphn ng ha hc nht nh v m bo cho cc phn ng xy ra theo mtchiu hng nht nh vi tc nhp nhng trong c th sng.Chng c trong hu ht cc loi t bo ca c th sng. Chnh donhng tc nhn xc tc c ngun gc sinh hc nn enzyme cn c gil cc cht xc tc sinh hc (biocatalysators) nhm phn bit vi cccht xc tc ha hc. Enzyme hc l khoa hc nghin cu nhng cht xc tc sinh hc cbn cht protein. Hay ni cch khc, enzyme hc l khoa hc nghin cunhng tnh cht chung, iu kin, c ch tc dng v tnh c hiu ca ccenzyme.1.2. Lc s nghin cu enzymeDo enzyme hc c coi nh ct sng ca ha sinh hc nn phnln cc nghin cu ha sinh t trc n nay u lin quan nhiu n enzyme. V s pht trin ca hc thuyt enzyme, c th chia thnh 4 giai on: - Giai on 1: trc th k th XVII - Giai on 2: t th k XVII n na u th k XIX - Giai on 3: t gia th k XIX n 30 nm u ca th k XX - Giai on 4: t nhng nm 30 ca th k XX n nay.1.2.1. Giai on 1Trc th k XVII ngi ta bit s dng cc qu trnh enzymetrong i sng song ch c tnh cht kinh nghim thc t v thng qua hotng ca vi sinh vt. l cc qu trnh ln men ru, mui da, lmtng v nc chm... thi k ny ngi ta cha hiu v bn chtenzyme v cc qu trnh ln men. 5. 81.2.2. Giai on 2 giai on ny cc nh bc hc tin hnh tm hiu bn cht cacc qu trnh ln men. Thi k ny khi qut hin tng ln men nh lhin tng ph bin trong s sng v enzyme l yu t gy nn s chuynha cc cht trong qu trnh ln men. Vo nhng nm 1600 ca th k XVII, Van Helmont l ngi utin c gng i su tm hiu bn cht ca qu trnh ln men. Van Helmont nhn thy thc cht ca s tiu ha l s chuyn ha ha hc ca thcn v gii thch c ch ca n vi s so snh n vi qu trnh ln menru. Danh t ferment (t ch Latinh fermentatio - s ln men) c VanHelmont dng ch tc nhn gy ra s chuyn bin cc cht trong qutrnh ln men ru. Vo na cui th k th XVIII, nh t nhin hc ngi Php lRaumur cng nghin cu bn cht ca s tiu ha. Nh t nhin hcny cho chim qu en nut nhng ming tht t sn trong ng kim loic thnh c c sn v buc vo dy thp. Sau vi gi khng thyg trong ng. Hin tng ny thc y s nghin cu thnh phn dchtiu ha tm hiu kh nng tiu ha ca dch d dy. Sau th nghimny mt thi gian, vo nm 1783, nh bc hc ngi l Spalanzani lp li th nghim bng cch ly dch d dy trn vi tht mi v thy chin tng ha tan xy ra.Vo u th k XIX, cc nh nghin cu tch c cc cht gyra qu trnh ln men. Nm 1814 Kirchoff, vin s Saint Petercburg phthin nc chit ca mm i mch c kh nng chuyn ha tinh bt thnhng nhit thng. y l cng trnh u tin thu c ch phmamylase dng dung dch v lch s enzyme hc thc s c xem nh btu t y.Mi chn nm sau (nm 1833), hai nh khoa hc ngi Php lPayen v Pessoz chng minh cht c hot ng phn gii tinh bt thnhng c th tch c dng bt. Th nghim c tin hnh bng cchcho etanol vo dch chit ca la i mch ny mm th thy xut hin ktta. Kt ta c hnh thnh ny c kh nng chuyn ha tinh bt v nuun kt ta ny s mt tc dng chuyn ha. Danh t diastase (t chLatinh diastasis - phn ct) l do Payen v Persoz dng gi enzymeamylase lc by gi. 6. 9 Tip ngi ta cng tm ra v tch c nhiu enzyme khcnh enzyme phn gii protein ca dch tiu ha trong d dy nh Pepsin(Emberle v Shwan) - nhng nh khoa hc ngi c, nm 1836)... Sau , l thuyt xc tc ra i. Nm 1835, nh khoa hcBerzelius c quan im cho rng tng tc phn ng l hin tng xctc. y l mt quan im ng. Song tht ng tic l nh khoa hc ny coi cc cht xc tc ny hot ng c l do " lc sng" khng theos iu khin ca con ngi. y l quan im duy tm, siu hnh lmtr tr s pht trin ca khoa hc nht l nh hng su sc n sh phttrin ca ngnh enzyme hc.1.2.3. Giai on 3Giai on t gia th k XIX n 30 nm u ca th k XX. giai on ny mt s lng rt ln cc enzyme dng ha tan c tch chit.Trong thi k ny, c hai trng phi u tranh vi nhau: ltrng phi Pasteur - nh bc hc v i ngi Php v trng phi Liebig- nh bc hc ni ting ngi c. * Trng phi Pasteur:Nm 1856 Pasteur cp n bn cht ca qu trnh ln men.ng cho rng khng th tch cc enzyme khi t bo. Tc dng v tnhcht ca enzyme gn lin vi s sng ca t bo v qu trnh ln men rul kt qu hot ng sng ca t bo nm men ch khng phi l kt quca tc dng ca enzyme. ng tin hnh th nghim v nhn thy numt dung dch hu c, v d dung dch glucose trong bnh kh trngth khng xy ra qu trnh ln men ru. Chnh v suy ngh y, Pasteur chia cc enzyme thnh 2 loi: "enzyme c t chc" v "enzyme khng ct chc".Theo ng, cc "enzyme c t chc" l nhng enzyme khng th tchkhi t bo, khi tch chng s b mt tc dng xc tc nh cc enzyme cacc t bo nm men thc hin qu trnh ln men ru; cn cc "enzymekhng c t chc" l cc enzyme c th thc hin tnh xc tc ca nngoi c th nh cc enzyme c trong dch tiu ha (v d Pepsin trongd dy, amylase trong tuyn nc bt, trong mm thc...) Quan im sai lm ny ca Pasteur thng tr ngnh enzyme hctrong mt thi gian di. Nm 1878 Kuhne ngh dng danh t"ferment" (t ting Latinh: fermentatio = ln men) gi cc "enzyme c 7. 10t chc" v gi cc cht chit c tc dng xc tc cho phn ng hahc l cc enzyme (t ch Hy Lp: en = bn trong, zyme = men ru, tcl " trong nm men" gi cc enzyme "khng c t chc". Danh tenzyme c xut pht t y. * Trng phi Liebig:Chng li quan im trn ca Pasteur, Liebig (trc c cBerzelius) cho rng c th khng c hot ng ca cc t bo vi sinh vtcng c qu trnh ln men. iu c ngha l ng coi enzyme nh l mtcht ha hc gy nn hiu qu tng t nh cc cht xc tc, tc dng c trong v ngoi t bo, khng ph thuc vo hot ng sng ca vi sinh vt.Nhng nm 1871 Liebig tht bi v thc nghim khng chng minhc quan im trn ca mnh . Cc th nghim c tin hnh bng cchly dch chit t t bo nm men nghin nt u khng c tc dng gyln men ru. Cng vo nm1871 Manatxein l mt bc s ngi Nga dng ct thch anh nghin cc t bo nm men v thu c dch chitkhng cha t bo c kh nng bin i ng thnh ru. Nhng nhngquan st ny khng c ai ch ti. Chnh v vy, quan im siuhnh ca Pasteur hn ch kh nhiu s pht trin ca ngnh enzymehc. n nm 1897, H. Bchner - mt nh khoa hc ngi c nhnc dch chit nm men bng cch phn hu t bo hon thin hn.Trong th nghim ny, cc t bo nm men c nghin nt hon toncng vi bt thy tinh, sau c p bng p sut cao. Dch chit thuc khng cha t bo vn c kh nng gy ra qu trnh ln men (chuynha glucose thnh ru). iu chng t qu trnh ln men ru khngphi l kt qu ca hot ng sng ca t bo nm men m l kt qu tcdng ca cc enzyme vn c trong cc t bo. Do , quan im sai lmv enzme "c t chc" v enzyme "khng c t chc" m thc cht l vbn cht ca enzyme n lc ny mi hon ton b nh , m ra mtthi k pht trin mi ca ngnh enzyme hc. Cng t khng c sphn bit v ni dung gia thut ng "ferment" v "enzyme". C th nirng, cng trnh ca Bchner nh du mt bc ngot quan trngtrong lch s pht trin ca enzyme hc. Sau , nhiu loi enzyme trongc th sng c tm ra. V vy vic phn loi v gi tn cc enzymemt cch thng nht cng cn thit. Nm 1883, Duyclo, nh bc hc Php ra nguyn tc phn loi enzyme theo c cht (substrate) do chngbin i v thm ui tn cng "ase" vo. V d enzyme phn gii tinh bt(amilun) l amylase. Tuy vy, trong thc t cn tn ti nhiu ngoi l vthut ng, v d nhng tn gi enzyme pepsin, trypsin, catalase trc yvn c dng. 8. 11 thi k ny, da vo thnh tu ca ha hc, c bit l ha l vha keo, cc nh khoa hc hng vo vic nghin cu cc tnh cht hav l hc ca enzyme cng nh hon thin cc phng php lm thunkhit enzyme. Giai on quan trng nht trong thi k ny l cc cng trnh canh bc hc v i ngi c E. Fisher. ng t nn mng cho nhngkhi nim hin i v tnh c hiu ca enzyme, v s tng tc khnggian gia enzyme v c cht. Gi thuyt ni ting ca ng l gia enzymev c cht kt hp vi nhau nh " kha vi cha kha". Ri nhngnghin cu ca Bach v Palladin v cc enzyme xy ha kh to nnc s cho vic xy dng hc thuyt xy ha kh sinh hc. Trong thi gianny ngi ta cng pht hin ra c tnh tc dng thun nghch caenzyme (anilepski, 1894), cc coenzyme cng c pht hin(Harden v Young, 1906). H l nhng ngi khm ph ra rng, dchchit t bo nm men cha hai loi cht cn thit cho qu trnh ln men l"zymase" v "cozymase". H nhn thy dch chit t bo nm men mthot tnh xc tc nu b thm tch hoc b un ln n 50 oC. Nhng dchchit b thm tch khng hot ng s hot ng khi c trn vi dch b un nng khng hot ng. Nh vy hot ph thuc vo s cmt ca hai loi cht: thnh phn khng bn vi nhit (heat - labile);khng c th thm tch c (c gi l zymase) v mt phn on bnvi nhit (heat - stable), c th thm tch c (c gi l cozymase).Ngy nay chng ta bit rng "zymase" bao gm tt c enzyme, cn"cozymase" bao gm cc ion kim loi, ATP, ADP v cc coenzyme nhNAD+. Thi gian ny ngi ta cng hiu bit c tc dng km hmv hot ha ca mt s enzyme (Sorensen 1909). Vo u th k XX, pht sinh ra c s ng hc trong tc ng ca enzyme da vo nhngnghin cu ca nh bc hc Anh l Brown v nh bc hc Php l Henri.n nm 1913, Michaelis v Menten pht trin cc cng trnh trn vnu ln thuyt ng hc ca s xc tc enzyme.Sau i chin th gii ln th nht nh bc hc ni ting ngi cl Willstatter c rt nhiu cng hin trong vic tm hiu bn cht hahc ca enzyme. l cng trnh khoa hc 5 nm ca ng v cc cng s(1922) nhm lm thun khit enzyme bng phng php hp th chn lc.Qua t nhn xt thy l nhng giai on cui ca qu trnh lm thunkhit enzyme, thng b mt i nhng cht cha c bit no do, enzyme b mt tnh xc tc, cho php Willstatter nu ln ln u tingi thuyt v enzyme hai cu t (enzyme hai thnh phn). Nhm hotng (coenzyme, coferment, agon) ch c kh nng xc tc khi kt hp vi 9. 12phn protein c hiu (apoferment, apoenzyme, feron = protein) n xcnh cc c tnh ca enzyme v ng vai tr ch o trong vic th hintc dng xc tc ca enzyme. Willstatter coi feron (protein) l cht trch c tc dng g. Agon l cht c hp ph trn cht ny. V vo nm1926, trong mt dp thuyt trnh, ng cho rng enzyme khng thucmt trong cc hp cht bit, tc l enzyme khng phi l protein,khng phi l glucid, m chng l nhng "cht c bit". chnh l quannim sai lm ca Willstatter. ng l ngi tm ra c nhiu phngphp lm sch enzyme cng nh lm sng t nhiu tnh cht c hiuenzyme. Nhng mc ch chnh l lm sng t bn cht ha hc caenzyme thi ng li khng t c.Ngy nay ngi ta quan nim nu l enzyme hai thnh phn th phncoenzyme quy nh kiu phn ng v chu trch nhim lm bn. Cnapoenzyme quy nh tnh c hiu ca enzyme cng nh tng hiu sutxc tc. Coenzyme + apoenzyme = (holo) enzyme = (enzyme hon chnh) Coferment + apoferment = (holo) fermentCoenzyme ch dng ch phn khng phi protein ca enzymetrong trng hp khi n d tch khi phn apoenzyme khi cho thm tchqua mng bn thm v c th tn ti c lp. Phn khng phi protein caenzyme c gi l nhm ngoi hay nhm "prostetic" khi n lin kt chtch vi phn protein ca enzyme.1.2.4. Giai on 4Bn cht ha hc ca enzyme ch c xc nh ng n t sau khikt tinh c enzyme. Nm 1926 nh ha sinh M tr tui Sumner (39tui) thnh cng trong vic chng minh protein c kt tinh t ht utng l cht ging enzyme xc tc cho phn ng thy phn ur. ycng chnh l enzyme u tin c kt tinh. Bn nm sau (1930) MNorthrop tch c pepsin dng tinh th, v vo nm 1931 Northropv Kunitz cng tch c trypsin dng tinh th.Trong thi k ny J.B.S Hardane vit quyn "Enzymes". Mc dlc bn cht phn t ca Enzyme hu nh vn cn l b mt, nhng tcgi a ra d on tuyt vi v vai tr ca cc tng tc v lin kt yugia enzyme v c cht trong c ch hot ng ca enzyme. iu ny vngi nguyn tnh thi s trong thi i ca chng ta. 10. 13 Cc cng trnh ca Sumner v Northrop m ra mt chng mitrong lch s pht trin ca enzyme hc hin i. Nhng kt qu t c cho php xc nh c mt cch dt khot bn cht ha hc caenzyme l protein. Phi ni rng bn cht ha hc ca phn ln enzyme lprotein v nh ngha c tnh cht kinh in v Enzyme phi xem li tsau pht hin ca T. R. Cech nm 1981. Cech pht hin mt RNA chot tnh xc tc nh enzyme v gi l ribozyme (xut pht t cc tnribose v enzyme). Ribozyme xc tc cho qu trnh chuyn ha tin cht.RNA thng tin (pre - m RNA) thnh m-RNA. Do enzyme khng nhtthit phi l protein! y l mt pht minh c ngha rt ln. Tc gi capht minh ny c gii Nobel nm 1989. Cho n nay khong 100ribozyme c bit. C th ni rng, nhng cng trnh ni trn m mn cho giai on th t ca lch s pht trin enzyme hc ko dicho n hin nay. T gia th k th XX, nht l thi gian gn y enzyme hc phttrin rt mnh. Nh ng dng cc phng php mi, hin i nh: in di,sc k, quang ph, ng v phng x... cho php nghin cu cu trccng nh c ch tc dng ca nhiu enzyme, c ch ca qu trnh sinhtng hp enzyme v s iu ha hot ng ca enzyme trong t bo.Ngi ta xc nh c cu to ca coenzyme. xc nh cmi lin h ca enzyme v cc vitamin (nhiu vitamin l thnh phn cuto ca coenzyme v phn ln cc vitamin tan trong nc l thnh phncu to ca cc coenzyme).Ngi ta cng xc nh c cc enzyme xc tc cho cc qutrnh trao i cht nh: h thng enzyme ng phn, Embden -Meyerhof - Parnas nm 1933, h thng enzyme ca chu trnh Kreps -Szent Gyorgy nm 1937 (chu trnh citric acid), chu trnh ornithrin trongtrao i cht ca protein nm 1932 (Krebs - Henseleit). Nh nhngphng php mi trong vic tch v lm sch enzyme, ngi ta xcnh c vai tr rt quan trng ca kim loi trong s xc tc ca enzymev tc dng hot ha ca chng. xc nh c s phn b ca ccenzyme trong t bo. nghin cu c ch tc dng cng nh cu to ccprotein enzyme. Bng phng php Rhengen, ngi ta nghin cu cutrc ca ca phn t enzyme, nh cu trc ca ribonuclease (1960, Stein).Trong vng hn 40 nm tr li y nghin cu cc enzyme sinhtng hp nh nucleotide phosphorylase (Greenberg Marago, 1955), DNA- polymesase ( Kornberg,1956), RNA - polymesase (Sp...</p>