1CHNG 1ENZYME V MT S PHN NG ENZYME PH BIN TRONG CNG NGH THC PHM1.1. i cng v enzyme1.1.1. Khi nim enzyme1.1.2. Cu to phn t enzyme1.1.3. C ch xc tc ca enzyme1.2. Danh php v phn loi enzyme1.2.1. Danh php1.2.2. Phn loi enzyme, gii thiu mt senzyme thuc mi loi1.1.1. Khi nim enzymeEnzym l cht xc tc c bn cht proteinc c th tng hp v tham gia vo ccphn ng sinh hcEnzyme l cht xc tc sinh hc Enzym c to ra trong t bo sinh vt Enzym tham gia phn ng trong t bo v ckhi tch ra khi c th Enzym tham gia phn ng trong iu kinnhit n ha (nhit c th). Enzyme c th thu nhn t: ng vt, thc vtv vi sinh vtCAU TAO ENZYM Duo voo Ihonh phon hoohoc cuo enzym nguo|Io ch|o enzym Ihonh ho| nhom. Enzym moI cou Iu. Irong Ihonh phon cou Ioo chco proIe|n. Enzymho| couIu.ngoo| proIe|nconco moIphon ph| proIe|n. Fhon proIe|ngo| |oferon hoyopoproIe|n covo| IroquyeI d|nh I|nh doc h|eu cuo enzyme , Fhonnhomngoo| |o Agon hoyCoenzym |o bophon Iruc I|ep Ihom g|o voo phon ung. Nhom ngoo| coIhe |o coc|onk|m|oo| hooc|o cocdonsuoI cuov|Iom|nFhon |on enzyme co dong cou hooc dong so|Coenzyme c cha kim loi Tham gia phn ng vn chuyn in t, vn chuyn oxy in hnh l hemeChuyen e-, H+hoac nguyen t H) A-+ B A + B-Phan ng chuyen nhom chc A-B + C A + B - CPhan ng phan ly nh nc ( thuy giai) (chuyen nhomchc cho phan t nc) : A-B + H2O A-H + B-OHPhan ng chuyen hoa nh bo sung nhom chc vao lien ketoi hoac tao lien ket o i nh lay i nhom chc (phan giaikhong co nc tham gia) X Y A-B A=B + X-YChuyen nhom chc trong phan t tao cac dang ong phan X YY X A-B A-BTong hp lien ket C-C, C-S, C-O va C-N nh phan ngtrung ngng lien hp vi s thuy gia i ATP.OxidoreductaseTransferaseHydrolaseLyaseIsomeraseLigase123456Phan ng xuc tacNhom chnhcua EnzymSTTPHN LOI ENZYME DA VO CHC NNG1. Oxidoreductase L nhm xc tc cho cc phn ng oxy ha khAH2 + B A+ BH2 Nu cht chuyn hay cht nhn r rng th gi tn theo chng. Ex. Dehydrogenase (NAD, FAD, FMN,) oxydase (nhn hydro), oxygenase (kt hp trc tip vi oxy) Nu cht cho hydro khng r rng th gi l reductase Cc enzyme thng gp trong nhm ny l: dehydrogenase, oxydase, peroxidase, catalase,22. TransferaseL nhm enzyme xc tc chuyn nhm, chuyn gc t cht ny sang cht khcPhn ng tng qut l AX + B A + BXCch gi tn: theo cht nhn nhm chuyn vn transferaseTransferases (transfer of functional groups)Transferring C-1 groupsMethyltransferasesHydroxymethyltransferases and formyltransferasesCarboxyltransferases and carbamoyltransferasesTransferring aldehydic or ketonic residuesAcyltransferasesGlycosyltransferasesTransferring N-containing groupsAminotransferasesTransferring P-containing groupsWith an alcohol group as acceptor22.12.1.12.1.22.1.32.22.32.42.62.6.12.72.7.12.8Tn v cc nhm ph xc tc E.C. NumberNhm transferase c chia ra lm 7 nhm ph2.1 chuyn nhm carbon n nh methyl, carboxyl,2.2 chuyn nhm aldehyde hoc cetone, c 2 enzyme in hnh ltransketolase (chuyn 2C) v transaldolase (chuyn 3C)2.3chuyn gc acyl, acyltransferase2.4 chuyn gc glycosyl, glycosyl transferase2.6 chuyn nhm m, aminotransferase2.7chuyn nhm phosphate, phosphotransferase2.8 chuyn nhm lu hunh, sulphurtransferase3. Hydrolase L nhm enzyme xc tc cc phn ng thy phn cc hp cht hu c vi s tham gia ca nc Phn ng tng qut XY + H2O XOH + YH Cch gi tn: tn ca c cht c tch ra hydrolase Nhm ny c chia ra 11 nhm ph Cc enzyme thng gp l esterase, phosphatase, nuclease, peptidase, lipase4. LyaseL enzyme phn ct cht hu c khng c s tham gia ca ncc chia lm 7 nhm phCch gi tn: tn c cht + tn ca nhm c tch ra + lyase. Ex. Aspactate ammonia lyaseCc enzyme thng gp: decarboxylase, aldolase, hydratase,5. IsomeraseL nhm enzyme xc tc cho chuyn ha gia 2 dng ng phn D vL, cis v trans, aldose v ketoseCc enzyme thng gp l epimerase, mutase, isomeraseIsomeraseV d: Galactose Glucose6. Synthetase (ligase)L nhm enzyme xc tc tng hp cht hu c v cn nng lng tATP, GTP,c chia thnh 4 ph nhm to cc lin kt: - C O -, - C S-, - C N -, - C C Cch gi tn: Tn c cht + ligase (hay synthetase); ex. Glutamin synthetase3DANH PHAP HAY TEN GOI ENZYM 9 uo| Ion cungose.9 Iheo co choI phon ungFhon Ihe h|en co choI + phon Ihe h|en |oo| phonung + phon duo| ose sucrose phon ung sucrose||pose- phon ung||p|d9 Iheo chuc nong cuo enzymox|dose coIo|yzes ox|doI|onhydro|ose coIo|yzes hydro|ys|s9 o| vo| coc enzym xuc Ioc cho coc quo Ir|nhI|eu hoo Ihuc on Ihuong Ion cung |o |npeps|n, Iryps|n1.4 Danh php quc t v phn loienzym Tn gi enzym thng theo c cht c hiuvi kiu phn ng m chng tham gia: VD: Urease (carbamite-amideohydrolase)- Urea: tn c cht- -ase: ch tn enzymNm 1960, Hip hi ha sinh QT (IUB) thngnht phn lai enzym thnh 6 lp: mt lp chialm nhiu t, mi t chia thnh nhiu nhmChnh v th tn ca enzym gm c 4 s. TRUNG TAM HOAT ONG Ch comoI phon nhophon Iuenzym Ihom g|o IrucI|ep||en keI vo| cochoI,quyeI d|nhhooII|nhxucIoc enzym go| |o Irung Iom hooI dong. $oIrung Iom hooI dong cuo phon Iuenzym coIhe|o moI hoy nh|eu hon. Enzym moI cou IuIrung Iom hooI dong gom moI sonhom chuc oc|d om|n. Enzym ho| cou IuIrung Iom hooI dong gom moI sonhom chuc cuo oc|d om|n vo nhom ngoo|.TRUNG TM HOT NG ENZYMEO ONCCNCC NCCNCCRH RChymotrypsin Has A Site for SpecificityO-CSerActive Site Active SiteSpecificitySiteSpecificitySiteCatalytic SiteJuang RH (2004) BCbasicsSpecificity of Ser-Protease FamilyCOO-CAspCOO-CAspActive SiteTrypsin Chymotrypsin Elastasecut at Lys, Arg cut at Trp, Phe, Tyr cut at Ala, GlyNon-polarpocketDeep and negativelycharged pocketShallow andnon-polarpocketO OCNCCNCCCCNH3+O OCNCCNCO OCNCCNCH3Juang RH (2004) BCbasics4Mo hnh Cha va khoa cua Fisher ve s an khp cua enzyme va c chat(Nam1894).TTHC chatC chatEnzymeEnzymeTTHC chatC chat* Hnh 3 Mo hnh Khp camng cuaKoshland ve s an khp cua Enzyme va cchat (Nam1958).Enzyme EnzymeE + SES E + Pk1k-1k2k-2ES + ESE +PC CH XC TC CA ENZYME1.8.Z. Thh k6 hIE0 60k EhZYME9 AC HIEU PHAN NG9 AC HIEU C CHAT AC HIEU TUYET OI AC HIEU TNG OI AC HIEU NHOM AC HIEU ONG PHAN QUANG HOC AC HIEU PHAN NGOxy hoa nh oxydase: RCHCOOH+ 1/2O2 RCOCOOH + NH3NH2Kh carboxyl nh decarboxylase: RCHCOOH RCH2NH3+ CO2NH2Chuyen Amin nh transaminase: R1CHCOOH + R2COCOOH R1COCOOH + R2CH COOHNH3NH3UreaUreaCO CO22 + 2NH + 2NH3 3Acetamide Acetamide Khong Khong xa xa y y ra raUrease UreaseH2OUrease UreaseH2O 0 04 4t IF t IF00 t0 t0 t04 t04I I f0F f0Fff 0 00 0I IMi mt enzyme ch tc dng lnmt c cht nht nh504t IF0 t0 t04I f006 00I CH2 O CO - 1 CH O CO - Z CH2 O CO - 1HO - HLIPASECH2 O H HOOC 1 CH O H+HOOC ZCH2 O H HOOC 8Enzyme c tcdng ln mtkiu lin ktha hc nhtnh04t IF0 0MR C N CHR C - OH + NH2 CH COOHR RH COOH OCarboxyl peptidaseH2OEnzyme tc dng ln mt kiu lin ktha hc nht nh vi iu kin mttrong hai thnh phn thamgia to thnhlin kt phi c cu to xc nhCOOH FumarathydrataseHOCHCHCOOH CH2-COOHHOOC-CHL malic Acid fumaric k k6 hIE 6 hIE00 0 0h Fhkh 0kh h0 h Fhkh 0kh h06 6Enzyme ch tc dng vi mt trong hai dngng phn quang hc ca c cht1.4. Cc yu t nh hng n vn tcphn ng enzymeE + SES E + Pk1k-1k2k-2-Nng enzyme - Nng c cht-Cc cht km hm - Cc cht hot ha- Nhit - pH mi trngVn tc phn ng enzymeprotein polypeptidestrypsinwhite clearControlled variables:Volume and concentration of substrate (milk)Volume and concentration of enzyme (trypsin)pH (controlled by buffers)TemperatureVn tc phn ng enzyme c xc nh bng thi gianc cht b chuyn ha hoc sn phm c to thnh1.4.1. nh hng ca nng enzymeE + SES E + Pk1k-1k2k-2Trong iu kin d tha c cht, ngha l [S] >>[E] th tc phn ng ph thuc vo [E]V= K[E] c dng y=ax. Nh ngi ta o [E] bng cch o vn tc phn ng do enzyme xc tc.6S ph thuc ca vn tc phn ng vo [E]1.4.2. nh hng ca nng c chtm hnh Michaelis - Mantenk1 k2E+S ES P + Ek-1 k1,k-1,k2:Hang so van toc cua cac phan ng tngng. Phan ng chuyen hoa phc ES P+Ela phan ngquyet nh qua trnh xuc tac chuyen hoa S Pcuaenzyme. Nong o EScang cao th Vphan ng cang ln: v = k2 [ES] Gi V1l vn tc ca phn ng to thnh phc cht ES.V1=k1[E][S] Gi V-1l vn tc ca phn ng to phn ly phc cht ES to thnh E v S.V-1= k-1[ES] Gi V2 l vn tc ca phn ng to thnh E v P (snphm).V2=k2[ES]E + SES E + Pk1k-1k2Khi h thng t trng thi cn bng ta c:k-1[ES]+k2[ES] = k1[E][S](k-1+k2)[ES] = k+1[E][S](2)Gi E0 l nng ban u:[E0]=[E]+[ES]=>[E]=[E0]-[ES] (3)Thay tr s [E] t (3) vo (2) ta c:(k-1+k2)[ES] = k1([E0]-[ES]) [S]k1[E0] [S] [ES] =--------------k-1+ k2+k1[S]Nu t Km= k-1+k2/ k1(Km: gi l hng s Michalis Menten)Ta c: [ES] = [E0][S]/ Km+[S]Mt khc vn tc phn ng to thnh sn phm P l:V = k2[ES]Thay [ES] bng gi tr trn ta thu c:k2[E0] [S]V= ----------------- (4)Km + [S]Vmax=k2[E0]Thay vo phng trnh (4) ta c:[S]V = Vmax ------------- (5)Km+ [S] Michaelis-Menton EquationV =Vmax [S]Km + [S]7Stnglncanngccht2 1 3 4 5 6 7 8 0024 68C cht (mole)Snphm806040200S+EP th biu din quan h gia (V) v nng ccht ([S]). Km - c trng cho i lc gia enzym v c cht:Khi ([S] >> Km), V t cc i (Vmax). Enzymb c cht bo ho.Khi [S] pI9Io| g|o Ir| pH mo| Iruong bong pI (pH| dongd|en) enzyme b| keI IuoANH HUONG CUA pHReactionRateOptimum pH3 5 7911pH Enzymes hot ng tt nht pHopt. Ngoi khong pH thch hp ca n th enzyme s b bin tnh. Tc phn ngpH1 2 3 4 5 6 7 8 9 10 1112 pepsin amylasenh hng ca pH n hot tnh enzymeOptimum pH10ANH HNG CUA pHXc nh hot ca enzyme 1 Kat = 1mol c cht/giy = 60 mol/pht =. S chuyn ha = mol c cht chuyn ha / pht n v enzyme quc t UI = hot tnh xc tc s chuyn ha ca 1 mol c cht trong 1 pht Hot tnh ring = s Katal / 1 kg protein hot ng Hot tnh molar = s Katal / mol enzyme hot ng ring l hot ng ng vi 1 mg protein enzymeCc phng php xc nh c cht mt i hoc sn phm to thnh bng cc phng php ha hc, quang ph, sc k, in di, so mu, 1.5.1. Phn ng thy phn bi enzyme 1.5.1.1. ngha ca phn ng thy phn1.5.1.2. c im ca cc c cht benzyme thy phn1.5.1.3. c im chung ca tm hotng ca enzyme thy phn1.5.1.4. Mt s c im ca phn ngthy phn bi enzyme1.5.1.1. ngha ca phn ng thy phn Phn ng thy phm lm tng thm phmcht cho thc phm- Enzyme amylase thy phn chuyn ha tinh btthnh ng glucose- Enzyme protease thy phn chuyn ha protein thnh nc chm, phomat, chao, hoc lm mntht Phn ng thy phn lm gim cht lng snphm thc phmc im ca cc c cht b enzyme thy phnCc c cht c enzyme thy phn thng l protein, gluxit, lipit c cht ny l c "lin kt b thy phn" Lin kt peptit trong protein: Lin kt glucozit trong cc polysacarit:Mt vi enzymhydrolase in hnh va ng dng ca chng trong thc phm11H enzyme amylase Amylase thuc nhm enzyme thy phn hydrolase C cht tc dng l tinh bt v glycogen Sn phmca s thy phn l dextrin, maltose, glucose Ngun gc: nc bt, dch tiu ha ngi v ng vt, ht ny mm, nmmc, nmmen v vi khun Cc dng chnh: -, - v -amylase pH ti thch cho -amylase mmla nc l 5,3 (thc hnh pH = 5,0) Nhit ti thch 58 60oC (thc hnh to= 50oC) -amylase c hot ha bi Ca2+v Cl-. B c ch biCu2+, Ag+v Hg2+C ch tc dng ca enzyme amylasea-AmylaseAmylose (Amylopectin) oligosaccharidesb-AmylaseAmylose (Amylopectin) b-MaltoseNy mm ht, lm bnh m, bin hnh tinh btProteaseProtein peptides + amino acids Protease phn b : thc vt, ng vt, vi sinh vt+3HN COC COO-N CR1R2HH H+ H2O+3HN C COO-R1H+3HN C COO-R2H+ProteaseProtease l l nh nh m m enzym enzym thu thu phn phn proteinprotein th th nh nh c c c c s s n n ph ph m mn n gi gi n n hn hn peptid peptid m m ch ch ng ng n n,, pepton pepton, acid, acid amin amin Protease Proteasel nhm enzyme xc tc s thu phnlin kt peptide, l loi lin kt ch yu trong phnt protein v peptide. C ch thu phn nh sau:EndopeptidaseHOHHOHNH2 CH CO NHCHNHCHCO NHCHCO NHCHCO NHCHCOOHR1R2 R3R4 R5R6Amino peptidase HO HCarboxy peptidasePepsin Pepsin hot ng trong dch v ca ng vt c v, chim, b st, c. heo, b, dpepsin tp trungch yu phn y d dy Pepsin th l hn hp ca pepsin, gelatinase, cathepsin. Pepsin heo th cha pepsin chnh A vpepsin ph B, C, D v gastricsin. Cu to: Pepsin gm mt mch polypeptide di 329 amino acid, u C l alanin v u N l isoleucine.Pepsin Pepsin d ht m, tan trong nc chodung dch c l, khng tan trong ethanol, ester, chloroform. im ng in capepsin gn pH=1. Pepsin thu phn protein trong vng acid kh rng t 1.0-4.0. pH hot ng ti uca pepsin thay i tu theo bn cht vtrng thi ca c cht, thng pH trongkhong t 1.8-2.2 12RenninRennin l mt emzymephn gii protein (casein) sa v lm ng t sa, c tm thy trong dchtiu ho d dy b. Rennin l mt emzymechuyn gy ng t sann ngi ta cn gi lemzyme ng t sa inhnh.Rennin Rennin tc ng ln c cht casein casa Hot tnh thu phn ti u pH = 3.8. Rennin hot ng mnh trong mi trngaxit, thu phn -casein thnh para- -casein v cc peptide. -casein para- -casein + peptide renninRenninBromelinBromelin c nhiu trong qu, thnv chi da. Bromelin chim50% protein c trong qu da. Bromelin thuc nhmprotease Cystein, trung tmhot ng chanhmSH, do vy cc cht c chanhmSH u l cht hot ho chobromelin.Ch phmbromelin c hot tnh cci trong khong nhit 50 60oC, pH ti thch ca ch phmkhi sdng c cht casein l 8.0 8.5, PAPAIN Papain l mt loi protease c thunhn t nha u . Nhit hot ng ti thch l 80oC, imng in pI = 9.0 Khi phn ng vi c cht th tu thuc vobn cht ca c cht m pH ti u s khcnhau. phn ng vi casein pH ti u l 7.0 7.5, vi albumin pH ti u 4.5 7.1 v vigelatin li c pH ti u 5.2 6.0 Papain thu phn protein thnh ccpolypeptide v cc amino acid FICIN Ficin c tm thy trong nha cythuc h ficus (sung, v). Ficin l mt loi protease thc vt trong cu trcbc mt c cha nhm SH. trng lng phn t khong 23.000 n 27.000 Da nhit ti thch cho hot ng xc tc ca ficin50 65oC pI = 9.0 10.0 Ficin khng tan trong hu ht dung mi hu cnhng ta mt phn trong nc v glycerinePectinase Pectinase l enzyme xc tc s phn hycc polymer pectin. S phn hy pectin trong t nhin thng xy ra khi tri cychn. Pectinase cng c ng dng nhiu trongqua trnh ch bin thc phm, c bit l khnng lm trong nc qu. Vic kim sothot ng ca pectinase cng c th iuchnh c nht ca sn phm.13Pectinase l nhm enzym xc tc cho s thy phn pectin PectinaseC ch phn ng thy phn pectin bi pectinaseCellulaseCellulose oligosaccharides, glucoseCellulase lenzyme xctc cho qutrnh chuynho Cellulose thnh cc snphm ho tan Cellulase Endocellulase: xc tc qu trnh ct lin kt -1,4- glucoside trong cellulose, lignin v -D-glucan mt cch ngu nhin. Sn phm ca qatrnh phn gii l cc cellulose phn t nh, cellobiose v glucose. Exocellulase: ct 2 hoc 4 n v glucose t ukhng kh ca chui cellulose to thnh cccellobiose (disaccharide) v mt s cellotetrose. Cellobiase: tham gia phn gii cellobiose(disaccharide) v cellotetrose thnh glucose. cellulose (t nhin)C1cellobiaseendocellulasecellobiose / cellotetroseCelluloseexocellulaseGlucoseQu trnh phn gii cellulose ca cellulaseLipasesTriacylglycerol diacylglycerol + fatty acidLipase phn nh m trong h tiu ho ngi, bin itriglyceride trong du n thnh monoglyceride v cc axit bot doC ch: Chng xc tc phn ng thu phn ln lt tng lin ktch khng ct t c 3 lin kt este cng mt lc.qu trnh xctc ca lipase thng chmhn so vi qu trnh xc tc cacc enzyme khc nh protease hay amylase.