B GIO DC V O TO TRNG C KINH T CNG NGH TPHCM KHOA CNG NGH SINH HC *****************

GVGD: ThS. L Thanh Hi TPHCM, thng 03nm 2013 - i - MC LC LI NI U ............................................................................................................................ 1 NI QUY THC TP ............................................................................................................... 2 BI1:NHLNGNITACIDAMINBNGPHNGPHPCHUN FORMOL(PHNG PHP SORENSEN) ............................................................................. 3 1/Nguyn tc ...................................................................................................................... 3 2/Thc hnh ....................................................................................................................... 3 2.1.Nguyn liu v ha cht ........................................................................................... 3 2.2.Dng c v thit b ................................................................................................... 3 2.3.Tin hnh .................................................................................................................. 3 2.4.Kt qu ..................................................................................................................... 4 BI 2: PHN NG BIURE ...................................................................................................... 5 1/Nguyn tc ...................................................................................................................... 5 2/Thc hnh ....................................................................................................................... 5 2.1.Nguyn liu v ha cht ........................................................................................... 5 2.2.Dng c v thit b ................................................................................................... 5 2.3.Tin hnh .................................................................................................................. 5 2.4.Kt qu ..................................................................................................................... 5 BI 3: XC NH IM NG IN CA PROTEIN CASEIN ........................................ 6 1/Nguyn tc ...................................................................................................................... 6 2/Thc hnh ....................................................................................................................... 6 1.1.Nguyn liu v ha cht ........................................................................................... 6 1.2.Dng c v thit b ................................................................................................... 6 1.3.Tin hnh .................................................................................................................. 6 1.4.Kt qu ..................................................................................................................... 6 BI 4: SO SNH TC DNG XC TC CA ENZYME VI XC TC V C ............ 7 1/Nguyn tc ...................................................................................................................... 7 2/Thc hnh ....................................................................................................................... 7 2.1.Nguyn liu v ha cht ........................................................................................... 7 2.2.Dng c v thit b ................................................................................................... 7 2.3.Tin hnh .................................................................................................................. 7 2.4.Kt qu ..................................................................................................................... 8 BI 5: TNH C HIU CA ENZYME ............................................................................... 9 1/Nguyn tc ...................................................................................................................... 9 2/Thc hnh ....................................................................................................................... 9 2.1.Nguyn liu v ha cht ........................................................................................... 9 - ii - 2.2.Dng c v thit b ................................................................................................... 9 2.3.Tin hnh ................................................................................................................. 9 2.4.Kt qu ................................................................................................................... 10 BI 6: TC DNG CA CHT KCH THCH V CHT KM HM .............................. 11 1/Nguyn tc ................................................................................................................... 11 2/Thc hnh .................................................................................................................... 11 2.1.Nguyn liu v ha cht ........................................................................................ 11 2.2.Dng c v thit b ................................................................................................. 11 2.3.Tin hnh ............................................................................................................... 11 2.4.Kt qu ................................................................................................................... 12 BI 7: XC NH HOT TNH CA ENZYME LIPASE .................................................. 13 1/Nguyn tc ................................................................................................................... 13 2/Thc hnh .................................................................................................................... 13 2.1.Nguyn liu v ha cht ........................................................................................ 13 2.2.Dng c .................................................................................................................. 13 2.3.Tin hnh ............................................................................................................... 13 2.4.Tnh kt qu ........................................................................................................... 14 BI 8: KHO ST HOT TNH ENZYME ASCOBATOXIDASE .................................... 15 1/Nguyn tc ................................................................................................................... 15 2/Thc hnh .................................................................................................................... 15 2.1.Nguyn liu v ho cht ........................................................................................ 15 2.2.Dng c .................................................................................................................. 15 2.3.Tin hnh ............................................................................................................... 15 2.4.Cch tnh kt qu: .................................................................................................. 16 BI9:XCNHHMLNGPROTEINTRONGDCHCHITTQUDA (PHNG PHP LOWRY) ................................................................................................... 17 1/Nguyn tc ................................................................................................................... 17 2/Thc hnh .................................................................................................................... 17 2.1.Nguyn liu v ho cht ........................................................................................ 17 2.2.Dng c v thit b ................................................................................................. 17 2.3.Tin hnh ............................................................................................................... 17 2.4.Tnh kt qu ........................................................................................................... 19 BI 10: KHO ST HOT TNH ENZYME BROMELIN ................................................. 20 1/Nguyn tc ................................................................................................................... 20 2/Thc hnh .................................................................................................................... 20 2.1.Dng c .................................................................................................................. 20 2.2.Ho cht ................................................................................................................. 20 - iii - 2.3.Cch tin hnh ........................................................................................................ 20 2.4.Kt qu ................................................................................................................... 21 BI 11: XC NH HOT TNH ENZYM -AMYLASE THEO PHNG PHP SMITH V ROE ................................................................................................................................... 22 1/Nguyn tc .................................................................................................................... 22 2/Thc hnh ..................................................................................................................... 22 2.1.Nguyn liu v ho cht ......................................................................................... 22 2.2.Dng c v thit b ................................................................................................. 22 2.3.Cch tin hnh ........................................................................................................ 23 2.4.Tnh kt qu ............................................................................................................ 25 - 1 - LI NI U Gio trnh Thc hnh cng ngh protein enzyme c bin son nhm phc v cho mn hc thc hnh cng ngh protein enzyme ca sinh vin chuyn ngnh cng ngh thc phm vsinhhcngdngKhoaCngnghSinhhcTrngCaongKinhtCngngh TPHCM. Gio trnh c bin son da trn c s tham kho cc ti liu v sch th nghim v protein enzyme trong v ngoi nc. Trongti liu ny gm 11 bi thc hnh nhm gip sinh vin lm quenvi ccphng tin vquytcantonthnghim,sdngdngc,thitbvthchnhthnghimnhtnh, nh lng acid amin, protein, enzyme t n gin n phc tp.Ccbithnghimnygipsinhvinnmvngtnhchtcaproteinenzymevhiur hn cc kin thc l thuyt hc t mn hc Cng ngh Protein Enzyme. Tc gi ThS. L Thanh Hi - 2 - NI QUY THC TP 1.Sinhvinphic mttiphngthnghimnggi,nunghhcphi cldo chnh ng, phi xin php gio vin. 2.Sinh vin phi c k, nm vng ni dung bi th nghim nh v chun b sn bn bo co kt qu th nghim trc khi th nghim. 3.Trong phng th nghim phi mc o blouse, rt thn trng khi s dng dung mi d chy n v ha cht c hi. 4.pdngtrim20%tngbithnghimtrongcctrnghpsau:khngchunb trc, khng c o blouse, i tr qu 10 pht khng gi trt t, tc phong khng nghim tc, vi phm ni quy phng th nghim, thao tc cu th khng thn trng. 5.Khi lm th nghim phi trt t, cn thn, gi sch ni lm th nghim, tit kim ha cht, lm v hay gy h hng thit b th phi bi thng. 6.Sau khi th nghim, sinh vin phi ra sch dng c, sp xp li dng c, ha cht ng ch, lau sch bn th nghim v bn giao cho cn b ph trch phng th nghim. 7.Cui bui sinh vin phi np bo co kt qu li cho gio vin hng dn. Thc hnh Cng ngh Protein - Enzyme - 3 - BI 1: NH LNG NIT ACID AMIN BNG PHNG PHP CHUN FORMOL(PHNG PHP SORENSEN) 1/Nguyn tc Ccaldehydedkthpvinhmamin.Khichoformaldehydetcdngviacidamin, nhm amin b methylen ha to thnh dn xut methylen imino acid. Hp cht to thnh c tnh acid mnh hn acid amin t do, cc nhm carboxyl ca chng d dng nh phn bng kim, qua gin tip tnh c lng nit amin ca cc acid amin c trong dung dch. 2/Thc hnh2.1.Nguyn liu v ha cht -Nc mm: 1ml -Dung dch NaOH 0,05N: 100ml -Dung dch HCl 0,05N: 30ml -Formol trung tnh: 10ml -Bromthymol blue 0,04%: 20ml -Phenolphtalein 0,5%: 20ml -Dung dch m phosphate pH = 7: 50ml oDung dich A: 27,8 g NaH2PO4 ho tan trong 1000ml oDungdchB:53.05gNa2HPO4.7H2Ohoc71.1gNa2HPO4.12H2Ophatrong 1000ml o pha dung dch m phosphate pH =7 ht dung dch A v dung dch B theo t l sau: Dung dich A: 39 ml + dung dich B: 61ml thm nc va 200ml. -Dung dch m phosphate pH = 9,2: 50ml (Ha tan 456g kali hydro phosphate ngm 3 phn t nc (K2HPO4.3H2O) trong 1000ml nc ct) 2.2.Dng c v thit b -Bnh nh mc 100ml: 1 bnh -Pipette 1ml: 2 -Pipette 10ml: 2 -Erlen 100ml: 6 bnh -Burette 25ml: 1 -Becher 100ml: 4 ci 2.3.Tin hnh Chun b thang mu c pH 7 v pH 9,2 Ly 2 erlen 100ml: -Cho vo bnh th nht: 20ml dung dch c pH 7,0 v 5 git bromthymol blue 0,04%. -Cho vo bnh th hai: 20ml dung dch c pH 9,2; 5 git bromthymol blue 0,04% v 3 git phenolphtalein 0,5% Khi dung dch trong bnh 1 c mu xanh lc nht, dung dch trong bnh 2 c mu tm xanh. Mu ca cc dung dch trn gi trong bnh kn c th bn trong na thng. Thc hnh Cng ngh Protein - Enzyme - 4 - Xc nh hm lng nit amin trong nc mm: Ncmmlmtdungdchthyphnproteincthnhlngnitbngphngphp chunformol.Doncmmcmuticnphiphathtlongmicthsomuca cht ch th. Ly 1ml nc mm cho vo bnh nh mc 100ml, dng nc ct nh mc thnh 100ml, lc u. Ly vo bnh erlen c cng dung tch vi 2 bnh mu chun 20ml dch mu pha long t bnh nhmc,thm5gitbromthymolblue.Nudungdchcmuxanhdngththmtng git HCl hay H2SO4 0,05N; nu dung dch c mu vng th thm tng git NaOH 0,05N cho n khi dung dch c mu ng vi mu ca bnh 1 c pH 7,0. Thm 3 git phenolphtalein 0,5%; 4ml formol trung tnh ri chun bng NaOH 0,05N cho n khi hn hp c mu ng vi mu ca dung dch trong bnh 2 c pH 9,2. Song song tin hnh lm th nghim kim chng, thay dung dch nghin cu bng nc ct. 2.4.Kt qu S gram nit acid amin c trong 1 lt nc mm: = 0,0007 100 100020 Trong :x: lng gram nit acid amin c trong 1 lt nc mm a: s ml dung dch NaOH 0,05N dng chun dung dch th nghim b: s ml dung dch NaOH 0,05N dng chun dung dch kim chng T: h s hiu chnh nng ca dung dch NaOH em dng so vi nng chun V: s ml nc mm cho vo bnh nh mc 0,0007: s gram nit vi 1ml NaOH 0,05N Thc hnh Cng ngh Protein - Enzyme - 5 - BI 2: PHN NG BIURE 1/Nguyn tc y l phn ng thng dng pht hin lin kt peptide (-CO-NH-). Phn ng xy ra i viccchtchathailinktpeptidetrln.Phnngnydngnhlngprotein bng cch lp th chun vi cc dung dch protein chun c nng xc nh nh k thut so mu. Nng protein ti thiu nh lng chnh xc l 10mg/ml. Ty thuc vo gc R m mu phn ng c th l mu xanh tm, tm hoc hng. 2/Thc hnh 2.1.Nguyn liu v ha cht -Dung dch CuSO4 1%: 10ml -Dung dch NaOH 10%: 10ml -Ure: 2 3g -Dung dch lng trng trng 1%: 10ml 2.2.Dng c v thit b -ng nghim: 04 ng -Pipette 1ml: 02 pipette -Pipette 5ml: 01 pipette -Becher 50ml: 03 cc 2.3.Tin hnh Lyngnghimkh,schchovitinhthure,unnhtinngchy,khcngtothnh biure khi c NH3 bay hi, nhn bit bng mi hoc th bng giy qu o pH. ngui thm vo 2 ml dung dch NaOH 10%, lc u cho tan ht v thm 2 3 git dung dch CuSO4 1%.Ly ng nghim th hai cho 1ml dung dch lng trng trng, 1ml dung dch NaOH 10%, thm 2 3 git CuSO4 1%, lc u. 2.4.Kt qu Chp hnh, ghi mu v vit phng trnh phn ng ca 2 phn ng trn. Thc hnh Cng ngh Protein - Enzyme - 6 - BI 3: XC NH IM NG IN CA PROTEIN CASEIN 1/Nguyn tc Cc phn t protein l cc polymer c tnh in ly lng cc. Trong dung dch, khi pH thay i n phn ly to thnh cc nhm tch in dng v cc nhm tch in m khc nhau. i vi mi protein s c mt gi tr pH xc nh m ti tng s in tch m bng tng s in tch dng, khi phn t protein trung ha v in, pH gi l im ng in ca protein. Ti im ng in, dung dch protein khng bn, d b kt ta. 2/Thc hnh 1.1.Nguyn liu v ha cht -Dung dch acid acetic (CH3COOH) 0,1N : 100ml -Dung dch natri acetate (CH3COONa) 0,1N: 100ml -Dung dch casein 0,4%: Cn 0,4g casein cho vo becher 100ml v thm 20ml dung dchCH3COONa0,1N.tlnnicchthyunnnghatanhonton. Chuynsangbnhnhmc100mlvnhmcti100mlbngdungdch CH3COONa 0,1N. -Nc ct: 50ml 1.2.Dng c v thit b -ng nghim: 10 ng -Pipette 1ml: 02 pipette -Bnh nh mc 100ml: 01 bnh -Becher 100ml: 03 cc -Ni cch thy: 01 ci 1.3.Tin hnh Ly dung dch CH3COOH 0,1N v nc vo 5 ng nghim theo th t nh bng sau: Lcu,sauthmvoming1mldungdchcaseinvtheodisbinicachng trong mi ng nghim. Bng 1. B tr th nghim xc nh im ng in ca protein casein TT ng nghim0102030405 CH3COOH 0,1N (ml)0,10,21,04,08,0 Nc ct (ml)8,98,88,05,01,0 Dung dch casein 0,4% (ml)1,01,01,01,01,0 pH5,65,34,74,13,8 Mc kt ta 1.4.Kt qu ng nghim c nhiu kt ta nht l im ng in ca casein. Chp hnh v ghi li kt qu xc nh pH ng in ca casein. Thc hnh Cng ngh Protein - Enzyme - 7 - BI 4: SO SNH TC DNG XC TC CA ENZYME VI XC TC V C 1/Nguyn tc Cht xc tc l cht lm cho phn ng xy ra nhanh hn nhng khng b tiu hao trong qu trnh phn ng. Enzyme l cc cht xc tc ca cc h thng sinh hc. Chng c kh nng xc tc c bit, thng mnh hn nhiu so vi cc cht xc tc tng hp. Tc dng xc tc ca chng mang tnh c hiu cao i vi c cht, lm tng ng k tc cc phn ng ha hc xy ra trong mi trng nc iu kin nhit v pH n ha.2/Thc hnh2.1.Nguyn liu v ha cht -Enzyme amylase: 5ml -Dung dch HCl 1%: 5ml -Nc ct: 5ml -Dung dch h tinh bt 10%: 10ml 2.2.Dng c v thit b -ng nghim: 04 ng -Pipette 1ml: 02 pipette -Ni un cch thy : 01 ci -T m: 01 ci -Becher 50ml: 03 cc 2.3.Tin hnh Ly 4 ng nghim ghi s, mi ng 2ml dung dch tinh bt 10%. Sau thm vo: -ng th nht: 1ml nc ct -ng th hai: 1ml dung dch enzyme amylase -ng th ba: 1ml dung dch enzyme amylase -ng th t: 1ml dung dch HCl 1% Sau t cc ng nghip th nht v th hai nhit phng, ng th ba vo t m 60oC. t ng th t vo ni cch thy ang si. Sau 10 pht ly cc ng nghim ra, cho vo mi ng 5ml thuc th Lugol. t c 4 ng vo ni cch thy trong 2 pht.Ly ra lm ngui v quan st hin tng trong cc ng nghim Thc hnh Cng ngh Protein - Enzyme - 8 - 2.4.Kt qu Chp hnh v ghi kt qu vo bng sau: Bng 2. Bng kt qu th nghim so snh tc dng xc tc ca enzyme vi xc tc v c ng nghimC chtXc tc Nhit (oC) Phn ng Lugol 1Tinh btKhng30 2Tinh btAmylase30 3Tinh btAmylase60 4Tinh btHCl100 Nhn xt v tc dng ca tng loi xc tc v nh hng ca nhit . Thc hnh Cng ngh Protein - Enzyme - 9 - BI 5: TNH C HIU CA ENZYME 1/Nguyn tc Tnhchtchiulbiuhinkhnngxctccaenzymeivicchtnhtnh. Enzyme c tnh c hiu rt cao. Tnh cht c hiu ca enzyme cho thy s khc bit rt ln gia enzyme vi cc cht xc tc khc. Miloienzymechckhnngxctcchoschuynhamthaynhiuchtnhtnh theomtkiuphnngnhtnh.Tnh chtchiuca enzymebiuhinmtskiu c hiu sau: chiukiuphnng:mienzymechxctcchomtkiuphnngchuynhanht nh trong cc kiu phn ng nh phn ng oxy ha kh, phn ng chuyn b, phn ng thy phn c hiu kiu c cht: c 3 kiu c hiu c cht -c hiu tuyt i: mi enzyme ch xc tc i vi mt c cht nht nh -c hiu tng i: mi enzyme ch xc tc i vi mt kiu lin kt ha hc nht nh -c hiu quang hc (c hiu lp th): mi enzyme ch xc tc i vi mt dng ng phn quang hc ca c cht (cis hoc trans) 2/Thc hnh2.1.Nguyn liu v ha cht -Enzyme amylase: 5ml -Dung dch HCl 1%: 5ml -Nc ct: 5ml -Dung dch h tinh bt 10%: 10ml 2.2.Dng c v thit b -ng nghim: 04 ng -Pipette 1ml: 02 pipette -Ni un cch thy : 01 ci -T m: 01 ci -Becher 50ml: 03 cc 2.3.Tin hnh Ly 4 ng nghim ghi s, sau thm vo: -ng th nht: 2ml dung dch h tinh bt 5% + 1ml dung dch enzyme amylase -ng th hai: 2ml dung dch h tinh bt 5% + 1ml dung dch enzyme bromelin -ng th ba: 2ml dung dch casein 2% + 1ml dung dch enzyme amylase -ng th t: 2ml dung dch casein 2% + 1ml dung dch enzyme bromelin Lc u v vo t m 37oC trong 15 pht. Ly ra, cho vo ng nghim th nht v th hai mi ng 02 git thuc th Lugol v t vo b iu nhit ang si trong 2 pht th ly cc ng nghim ra, lm ngui ti nhit phng. Cho ng th ba v th t mi ng 02 git thuc th Folin. Thc hnh Cng ngh Protein - Enzyme - 10 - 2.4.Kt qu Chp hnh v ghi kt qu vo bng sau: Bng 3. Bng kt qu th nghim xc nh tnh c hiu ca enzyme ng nghimC chtXc tcPhn ng thKt qu 1Tinh btAmylase 2Tinh btBromelin 3CaseinAmylase 4CaseinBromelin Nhn xt v tnh c hiu ca enzyme. Thc hnh Cng ngh Protein - Enzyme - 11 - BI 6: TC DNG CA CHT KCH THCH V CHT KM HM 1/Nguyn tc Ccchtctcdnglmtnghottnhcaenzymegilccchthothaenzyme.Cc cht hot ha c th l nhng anion, cc ion kim loi (t th 11 n th 55 trong bng h thng tun hon), cc cht hu c c cu trc phc tp. Cc cht hot ha thng lm nhim v chuyn nhm hydrogen hoc nhng cht c kh nng ph v mt s lin kt trong phn t tinenzymehocccchtctcdngphchiccnhmchcnngtrongtrungtmhot ng ca enzyme. Cc cht km hm hot ng ca enzyme thng l cc cht c mt trong phn ng enzyme, lmgimhottnhenzymenhngkhngbenzyme lmthayitnhchthahc,cuto ha hc v tnh cht vt l ca chng. Cc cht gy km hm hot ng ca enzyme bao gm cc ion, cc phn t v c, cc cht hu c v c protein.-Cht km hm cnh tranh: l nhng cht c cu trc tng t nh cu trc ca c cht. Chng thng l cht kim hm thun nghch. Chng c kh nng kt hp vi trung tm hot ng ca enzyme v chim ly v tr ca c cht trong trung tm hot ng.-Cht km hm khng cnh tranh: nhng cht ny khng chim trung tm hot ng ca enzymemlinktvivtrngoitrungtmhotnglmthayicutrckhng gian ca phn t enzyme theo chiu hng bt li cho hot ng xc tc.2/Thc hnh2.1.Nguyn liu v ha cht -Enzyme amylase: 5ml -Dung dch NaBr 0,5%: 5ml -Dung dch CuSO4 0,5%: 5ml -Nc ct: 5ml -Dung dch h tinh bt 1% : 20ml -Thuc th Lugol: 5ml -Thuc th Fehling: 5ml 2.2.Dng c v thit b -ng nghim: 03 ng -Pipette 5ml: 01 pipette -Pipette 1ml: 01 pipette -B iu nhit: 01 thit b -Becher 50ml: 03 cc 2.3.Tin hnh Cho vo 03 ng nghim mi ng 5ml dung dch h tinh bt 1%, sau cho vo: -ng th nht: 1ml nc ct -ng th hai: 1ml dung dch NaBr 0,5% -ng th ba: 1ml dung dch CuSO4 0,5% Sau cho vo c ba ng mi ng 1ml dung dch enzyme amylase. Lc u v vo t m 37oC trong 15 pht. Ly ra, cho vo mi ng 2 git thuc th Lugol, lc u. Tiptcchovoming5mldungdchFehlingvtvobiunhitangsitrong2 pht, lm ngui ti nhit phng Thc hnh Cng ngh Protein - Enzyme - 12 - 2.4.Kt qu Chp hnh v ghi kt qu vo bng sau: Bng 4. Bng kt qu th nghim xc nh tc dng ca cht hot ha v kim hm hot tnh ca enzyme amylase ng nghimC chtXc tcPhn ng thKt qu 1Tinh btAmylase 2Tinh btAmylase 3Tinh btAmylase Nhn xt v tc dng ca cht hot ha v cht kim hm n hot tnh ca enzyme amylase. Thc hnh Cng ngh Protein - Enzyme - 13 - BI 7: XC NH HOT TNH CA ENZYME LIPASE 1/Nguyn tc Lipase l enzym thuc nhm thy phn, phn nhm esterase xc tc phn ng thy gii cc ni ester gia glycerine v cc acid bo trong glyceride. Nhhotngcalipase,ccacidbocgiiphnglmtngchuacamitrng phnng.Lngacidscchunbngkimvchshotcaenzymsl lng kim cn trung ha cc acid bo mi c hnh thnh. 2/Thc hnh 2.1.Nguyn liu v ha cht -Tu tng ng vt : 4g -Sa ti: 100ml -KOH 0,1N : 100ml -Cn 96%: 50ml -Phenolphtalein1%: 15ml -Hn hp nc - glycerin: 40ml (t l 3 nc : 1glycerin) -H2SO4 0,1N chun: 100ml -Vi mn: 1 -Giy lc: 4 -Phu lc 2.2.Dng c -Erlen 100ml: 7 -B iu nhit: 1-Buret 25ml: 1 -Bercher 100ml: 4 -Pipet 5ml: 2 -Pipet 10ml : 2 -Ci s: 1 -Bp in: 1 2.3.Tin hnh Chunbchphmlipasettutngngvt:Cn1gtytngngvt,chovoci nghin 5ml hn hp nc glycerin (t l 3:1), nghin k t 3 5 pht. Sau , li cho thm 5ml hn hp nc glycerin v nghin tip 1 -2 pht. Lc vt hn hp qua vi mn, sau lc li qua giy lc, thu dch lipase lm th nghim Xc nh hot tnh ca enzyme lipase: Ly 02 erlen 100ml, cho vo mi bnh 10ml sa ti -Erlen 1 (erlen th nghim): cho vo 2 ml lipase, lc u, t vo b iu nhit 370C 400C trong 1 gi -Erlen 2 (erlen i chng): un si sa trong bnh, sau cho ngay 2ml lipase vo v un sitip5pht.nguinnhitphng.Sautvobiunhit370C 400C trong 1 gi Sau 1 gi ly erlen ra, thm vo mi bnh 8ml cn 96%, vi git phenolphtalein, chun c hai erlen bng KOH 0,1N.Thc hnh Cng ngh Protein - Enzyme - 14 - Tin hnh th nghim 2 ln. Ly kt qu l gi tr trung bnh ca 2 ln th nghim Cch xc nh h s hiu chnh k:-Chun 10ml dung dch KOH 0,1N bng dung dch H2SO4 0,1N chun, thuc th mu l phenolphtalein cho n khi dung dch va mt mu hng th dng li -Xc nh lng H2SO4 0,1N chun cn dng -Tin hnh th nghim 3 ln. Ly kt qu l gi tr trung bnh ca 3 ln th nghim 2.4.Tnh kt qu H s hiu chnh k c tnh nh sau:-Tnh nng KOH thc t: = 24 24 -H s hiu chnh k: = = Trong :CKOHt: Nng dung dch KOH thc t VKOHt : Th tch dung dch KOH thc t CKOHp : Nng dung dch KOH pha VKOHp : Th tch dung dch KOH pha Hot lipase: c biu th bng s ml KOH dng chun acid bo to thnh t s thy phn lipid c trong 1 lt sa v c tnh theo cng thc sau: = 1 2 100 Trong :X: Hot ca enzyme lipase (UI) V1: S ml KOH 0,1N dng chun erlen th nghim (erlen 1) V2: S ml KOH 0,1N dng chun erlen i chng (erlen 2) k: H s hiu chnh ca dung dch KOH Thc hnh Cng ngh Protein - Enzyme - 15 - BI 8: KHO ST HOT TNH ENZYME ASCOBATOXIDASE 1/Nguyn tc Ascobatoxidase l enzym thuc lp oxi ho kh (oxireductase) c nhiu trong cc loi rau qu ti v nhiu nht lp v qu. Enzym ny oxi ho acid ascorbic thnh acid dehydroascorbic. Trong thnh phn ca enzym ny c s hin din ca ion Cu2+. 2/Thc hnh 2.1.Nguyn liu v ho cht -Da chut : 100g -Dung dch m photphat 0,15MpH7: 150ml -Acid ascorbic (nng 1mg/ml): 100ml -Dung dch KI 10%: 50ml -Dung dch HCl 5%: 100ml -Dung dch h tinh bt 1% : 50ml -Dung dch KIO3 0,01N: 150ml -Dung dch Na2S2O3 0,01N: 100ml -Cch pha dung dch m phosphate pH 7: oDung dich A: 27,8 g NaH2PO4 ho tan trong 1000ml oDungdchB:53.05gNa2HPO4.7H2Ohoc71.1gNa2HPO4.12H2Ophatrong 1000ml o pha dung dch m phosphate pH =7 ht dung dch A v dung dch B theo t l sau: Dung dich A: 39 ml + dung dich B: 61ml thm nc va 200ml. 2.2.Dng c -Ci s: 1 -Becher 50ml: 2 -Becher 100ml: 2 -Erlen 100ml: 3 -Bnh nh mc 50ml: 2 -Erlen 250ml: 4 -Buret 25 ml: 1 -Pipet 5ml: 2 -Pipet 10ml : 2 -Giy lc: 4 -Phu lc: 2 2.3.Tin hnh Chit tch enzym ascobatoxidase: Cn 30g da chut (b ht) cho vo ci s, nghin vi 10 15ml dungdchm phosphat 0,15M pH 7. Dch chit c cho vo bnhnh mc 50ml v thm dung dch m n vch nh mc. Lc u nhiu ln v yn trong 1 gi. Lc thu dch lc. Dch lc l ch phm enzym ascobatoxidase. Kho st hot tnh enzym ascobatoxidase -Ht10mldungdchenzymchovobecher100ml.Sauchothm10mldungdch acid ascorbic. Lc u, yn nhit phng 30 pht. Tip theo un si trc tip trn bp ngng phn ng, ri cho vo bnh nh mc 50 ml v lm y bng nc ct. Thc hnh Cng ngh Protein - Enzyme - 16 - -Ly 10ml dch trong bn trn cho vo erlen 250ml, thm 5ml dung dch KI 10%, 10 ml dung dch HCl 5%, 10 git h tinh bt 1% v 10 ml dung dch KIO3 0,01N. Lc u, yn 5 pht ri chun bng dung dch Na2S2O3 0,01N n khi mt mu xanh. -Lm mt mu i chng tng t nh trn vi 10ml dung dch enzym c un si trong 2 pht v lm lnh. -Tin hnh th nghim 2 ln. Kt qu l gi tr trung bnh Xc nh h s hiu chnh ca Na2S2O3 0,01N bng dung dch KIO3 0,01N chun (tng t nh cch xc nh h s hiu chnh ca dung dch KOH 0,1N)2.4.Cch tnh kt qu:Hot tnh ca enzym ascobatoxidase c hin th bng s mg acid ascorbic b oxi ho di tc ng ca enzym c trong 1g nguyn liu trong thi gian 1 pht.Hot tnh ca enzym c tnh theo cng thc: = 1 2 0,088 Trong X: Hot ca enzyme ascorbatoxidase (UI) V1 : Th tch Na2S2O3 dng erlen th nghim V2 : Th tch Na2S2O3 dng erlen i chng k: H s hiu chnh ca dung dch Na2S2O3 0,01N 0,088: S mg acid ascorbic tng ng vi 1 ml dung dch Na2S2O3 0,01N m: Khi lng nguyn liu chit enzym (g) t: Thi gian phn ng (pht) Thc hnh Cng ngh Protein - Enzyme - 17 - BI 9: XC NH HM LNG PROTEIN TRONG DCH CHIT T QU DA (PHNG PHP LOWRY) 1/Nguyn tc HuhtccproteinuchaTyrosinvTryptophan.Hmlngcanhngacidaminny ty thucvo tng loi protein. Vvy nhng protein cng mt loivi nhau chm lng cc acid amin ny ging nhau. Khi cho cc protein tc dng vi thuc th Folin s to thnh mt phc cht c mu. Cng mu ca chtny t l vi hm lng Tyrosin vTryptophan(tng ngvi hm lng protein). V vy c th dng phng php so mu xc nh hm lng protein. 2/Thc hnh 2.1.Nguyn liu v ho cht -Da (thm) xanh: 1 tri -Dung dch albumin 0,1%: 100ml -Dung dch A: : 100ml (Cn 2g Na2CO3 ha tan trong NaOH 0,1N va 100ml) -Dung dch B: : 100ml (Cn 0,5g CuSO4.5H2O ha tan trong dung dch citrat natri 1% va 100ml) -Dung dch C : 50ml (L hn hp ca 2 dung dch A v B theo t l 49:1 (ch pha dng trong ngy)) -Thuc th Folin: 0,2ml 2.2.Dng c v thit b -ng nghim : 20 -Pipet 1ml: 2 -Pipet 10ml : 1 -My o mt quang: 1 -Curvet: 20 -Erlen 100ml: 4-Becher 250ml: 2 -My xay sinh t: 2 -Vi mn: 1 -Giy lc: 4 -Phu lc: 2 2.3.Tin hnhChun b dch protein mu t qu da: Chn tri thm xanh ti, gt b v ngoi, ct nh vxaynttrongmyxaysinht.Lcquatmvimnvvtnc.Lcbnggiylcthu dch lc trong. Dch lc l dung dch protein mu. Thc hnh Cng ngh Protein - Enzyme - 18 - Xy dng ng chun albumin: Bng 5. B tr th nghim xc nh ng chun albumin ng nghim s012345 Nng protein (g/ml)050100150200250 Dungdchalbumin0,1% (ml) 00,51,01,52,02,5 Nc ct (ml)109,59,08,58,07,5 T mi nng khc nhau ht ra 0,4 ml cho vo cc ng nghim khc nhau Dung dch protein (ml)0,40,40,40,40,40,4 Dung dch C (ml)222222 Lc u v yn nhit phng 5 pht Thuc th Folin (ml)0,20,20,20,20,20,2 Lc u v yn nhit phng 10 pht Nc ct (ml)2,42,42,42,42,42,4 o mt quang bc sng 500 nm Vthbiudinsbinthincamtquang(OD)theonngproteinchun (g/ml) vi trc tung l OD, trc honh l nng protein. Thc hnh Cng ngh Protein - Enzyme - 19 - Xc nh hm lng protein trong mu Bng 6. B tr th nghim xc nh hm lng protein trong mu da ng nghim s0123 Dung dch protein mu (ml)0,40,40,40,4 Dung dch C (ml)2222 Lc u v yn nhit phng 5 pht Thuc th Folin (ml)0,20,20,20,2 Lc u v yn nhit phng 10 pht Nc ct (ml)2,42,42,42,4 o mt quang bc sng 500 nmsao cho gi tr OD nm trong khong 0,05 0,25 Tin hnh th nghim 2 ln. Kt qu l gi tr trung bnh. 2.4. Tnh kt qu T ng chun so snh mt quang ca ng nghim cha mu protein. T suy ra hm lng protein ca nguyn liu l a (g/ml) Lng protein (M) c trong 1 g nguyn liu c tnh theo cng thc: = 103 /1 Trong a: hm lng protein (g/ml dung dch) D: h s pha long m: khi lng nguyn liu ly phn tch (g) Thc hnh Cng ngh Protein - Enzyme - 20 - BI 10: KHO ST HOT TNH ENZYME BROMELIN 1/Nguyn tc Bromelin l 1 enzyme thuc loi protease c nhiu trong tri thm. kho st hot tnh ca bromelin, ngi ta cho enzyme ny thu phn cc c cht protein (hemoglobin, casein) v do cc peptid c gii phng ra c cha Tyrosin bng cch cho tc dng vi thuc th Folin. 2/Thc hnh 2.1.Dng c -Becher 100ml, 250ml, 500ml -Chu thu tinh -Pipet 0,2ml, 1ml, 2ml, 5ml -My ly tm -Phu lc -Quang ph k -ng nghim 2.2.Ho cht -Dung dch NaOH 0,5N 1N -Dung dch HCl 2N 0,2N -Dung dch acid tricloracetic 5% -Dung dch Tyrosin mu: 45mg pha trong 100ml dung dch HCl 0,2N -Dung dch m KH2PO4 1M: 13,6g pha vi nc ct thnh 100ml -Dung dch hemoglobin 2%: cn 2g Hb + 36g ure + 8ml dung dch NaOH 1N ho trong 40ml nc ct. 25oC trong 60 pht, thm 10ml dung dch m KH2PO4 1M, iu chnh li pH 6 vi dungdch HCl 2N. Thm nc y ti vch mc 100ml.Bo qun trong t lnh. -Thuc th Folin 2.3.Cch tin hnh Chit dch nc thm cha enzyme Chn tri thm xanh ti, gt b v ngoi, ct nh v xay nt trong my xay sinh t. Lc quatmvimnvvt nc.Nc thmemlytm6000vng/phttrong10pht.Ly dch bn trn c cha enzyme bromelin. Thc hnh Cng ngh Protein - Enzyme - 21 - Kho st hot tnh enzyme bromelin: Thc hin 3 loi ng nghim (mi loi lm 3 ng ly trung bnh) Bng 7. B tr th nghim kho st hot tnh ca enzyme bromelin ng nghimTyrosinMuKhng DungdchHb2%trongm photphat ml2,52,52,5 35oC trong 5 pht Dung dch tricloracetic 5%ml505 Dung dch HCl 0,2Nml00,50,5 Dung dch tyrosin muml0,500 Dungdchnctrithmc cha enzyme bromelin ml00,20 Lc u yn 35oC trong 10 pht Dung dch tricloracetic 5%ml050 Dchncthmchaenzyme bromelin ml0,200,2 Lc u mi ng v yn trong khong 10 pht 25oC, em lc. Htvoccngnghimkhcming2,5mldchlc,5mldungdchNaOH0,5Nv1ml thucthFolin.Lck,ynkhong15phtvemcmtquangdisng 578nm hay 620nm. 2.4.Kt qu Hot tnh ca enzyme bromelin c biu th l s g tyrosin sinh ra do s thu phn Hb ca enzyme c trong 1ml dung dch hay 1mg hn hp cha bromelin trong 1 pht. Hot tnh ca enzyme bromelin: 450 11012/ Vi 1: ml dung dch tyrosin 2: ml dung dch cha enzyme Thc hnh Cng ngh Protein - Enzyme - 22 - BI 11: XC NH HOT TNH ENZYM -AMYLASE THEO PHNG PHP SMITH V ROE 1/Nguyn tc Hottnh-amylasebiuthkhnngca enzymamylasexctcphnngthyphntinh bt n dextrin trong 1 pht 50oC v c th hin bng s n v ca enzym trong 1g mu. Khiphnngthyphntinhbtxyra,lngtinhbtcnlichacthyphnsto phn ng mu vi Iod v c so mu bng my quang ph bc sng 620 nm. 2/Thc hnh 2.1.Nguyn liu v ho cht -La ny mm: 100 ht -Dung dch m Sorensen 1/15M, pH = 5,6: 100ml (Trn chung 5 ml dung dch Na2HPO4 1/15M v 95 ml dung dch KH2PO4 1/15M) -Dung dch Na2HPO4 1/15M: 5ml (Ha tan 23,876 g Na2HPO4.12H2O trong nc va 1 lt) -Dung dch KH2PO4 = 1/15M: 95ml (Ha tan 2,2681 g KH2PO4 trong nc va 250 ml)) -Dung dch tinh bt 1%: 25ml (1 g tinh bt trong 100 ml dung dch m pH = 5,6) -Dung dch HCl 1N: 25ml (85 ml dung dch HCl m c trong nc thnh 1 lt) -Dung dch NaCl 3%: 25ml (3 g NaCl trong nc thnh 100 ml) -Thuc th Lugol: 25ml (0,5 g I2 v 5 g KI trong nc thnh 200 ml) 2.2.Dng c v thit b -Ci s: 1 -ng nghim: 20 -ng ong 50ml: 1 -B iu nhit: 1 -Pipet 1ml: 2 -Pipet 10ml: 2 -Becher 100ml: 3 -Erlen 100ml: 3 -My quang ph: 1 -Curvet: 6 Thc hnh Cng ngh Protein - Enzyme - 23 - -Giy lc : 4 -Phu lc : 3 2.3.Cch tin hnh Ly trch amylase: cho khong 100 ht la ny mm cho vo ci s nghin nhuyn vi 3-5 ml ncct.Tiptcnghinvthmdnnht50mlnccthatanenzym.Lcdch bng giy lc, thu dch lc c cha enzym amylase. Thc hnh Cng ngh Protein - Enzyme - 24 - Tin hnh th nghim theo bng sau: Bng 8. B tr th nghim xc nh hot tnh ca enzyme amylase Dung dch ha cht ng nghim ng mu (t)ng i chng (o) Nc ct (ml)01 Dch chit -amylase (ml)10 Dung dch m (ml)11 Dung dch tinh bt 1% (ml)11 Dung dch NaCl 3% (ml)0,50,5 Dung dch HCl 1N (ml)01 em 50oC trong 30 pht Dung dch HCl 1N (ml)10 Nc ct (ml)5,55,5 Thuc th Lugol (ml)0,50,5 Lc u hn hp trong ng nghim ri mang i so mu bc sng 620 nm sao cho gi tr OD nm trong khong 0,05 0,25 Tin hnh th nghim lp li 3 ln. Kt qu l gi tr trung bnh. Thc hnh Cng ngh Protein - Enzyme - 25 - 2.4.Tnh kt qu Hot ca enzyme amylase: = 0 Trong : ODo : mt quang ca ng i chng. ODt : mt quang ca ng mu th. m: lng tinh bt ban u tham gia phn ng (mg). t: thi gian phn ng (pht). D: h s pha long mu enzym.