117677501 thu nhận enzyme amylase từ Aspergillus Oryzae

B CNG THNG
TRNG I HC CNG NGHIP THC PHM TP.H CH MINH KHOA CNG NGH THC PHM
BI BO CO: ng Dng Cng Ngh Sinh Hc Trong Cng Ngh Thc Phm
QUY TRNH SN XUT AMYLASE T VI SINH VT

GVHD: Th.S Nguyn Th Thu Sang
Nhm : 17 Nguyn Th Bch Phng Nguyn Th Kim Ngn Trng Th Thy An Phm Trc Ly
TP.HCM, Thng 12/2012
Bi tiu lun CNSH trong CNTP
Quy trnh sn xut amylase t vi sinh vt
LI M U Enzyme amylase l mt trong nhng enzyme c quan tm nghin cu sm v nhiu nht hin nay. Cc ng dng ca enzyme trong nhiu lnh vc khc nhau ngy cng c quan tm nghin cu. Ngoi nhng ng dng tiu biu c trnh by trn, enzyme amylase cn c ng dng trong sn xut gim, bt ngt, bnh ko, nc tri cy Ngy nay ngi ta ang nghin cu cc ngun amylase c cht lng cao v c th sn xut theo quy m cng nghip ng thi khc phc nhng hn ch ca cc phng php sn xut to enzyme amylase nng sut cao. Vit Nam bc u c nhiu nghin cu ng dng enzyme amylase trong ch bin nng sn, thc phm, nht l trong lnh vc sn xut bia, ru, ch bin tinh bt. Hin nay, -amylase l mt trong nhng enzyme c s dng rng ri trong nhiu lnh vc nh cng nghip thc phm, chn nui th y, chn on bnhTrong cng nghip thc phm, -amylase ng vai tr c bit quan trng Thng qua s thy phn tinh bt, -amylase to ra nhng sn phm c gi tr dinh dng nh dextrin, maltose, glucose Trc y, ngi ta thu nhn -amylase t malt l ch yu. Ngy nay, vi nn cng nghip pht trin mnh ko theo nhu cu v -amylase tng cao nn vic p dng nhng k thut tin b trong nui cy vi sinh vt thu d dng hn vi lng ln -amylase l rt cn thit
NHM - 17
PHN 1
TNG QUAN
1. GII THIU SN PHM 1.1 nh ngha 1.1.1 Enzyme Trong c th sng (cc t bo) lun lun xy ra qu trnh trao i cht. S trao i cht ngng th s sng khng cn tn ti. Qu trnh trao i ca mt cht l tp hp ca rt nhiu cc phn ng ha hc phc tp. Cc phn ng ny c lin quan cht ch vi nhau v iu chnh ln nhau. Enzyme l hp cht protein xc tc cho cc phn ng ha hc . Chng c kh nng xc tc c hiu cc phn ng ha hc nht nh v m bo cho cc phn ng xy ra theo mt chiu hng nht nh vi tc nhp nhng trong c th sng. Enzyme c trong hu ht cc loi t bo ca c th sng. Chnh do nhng tc nhn xc tc c ngun gc sinh hc nn Enzyme cn c gi l cc cht xc tc sinh hc (biocatalysators) nhm phn bit vi cc cht xc tc ha hc Chng l cht xc tc sinh hc khng ch c vai tr quan trng trong qu trnh sinh trng, pht trin ca mi sinh vt m n cn gi vai tr rt quan trng trong cng ngh ch bin thc phm, trong y hc, trong k thut phn tch, trong cng ngh gen v bo v mi trng. 1.1.2 Enzyme Amylase Amylase l mt h Enzyme rt ph bin trong th gii sinh vt. Cc Enzyme ny thuc nhm Enzyme thy phn, xc tc phn gii lin kt ni phn t trong nhm polysaccharide vi s tham gia ca nc. RR + H-OH RH + ROH
Amylase thy phn tinh bt, glycogen v dextrin thnh glucose, maltose v dextrin hn ch. Cc Enzyme Amylase c trong nc bt (cn c gi l ptyalin), trong dch
NHM - 17 3
tiu ha ca ngi v ng vt, trong ht ny mm, nm si, x khun, nm men v vi khun. Trong nc bt ca ngi c ptyalin nhng mt s loi ng vt c v th khng c nh nga, ch, mo... Ptyalin bt u thy phn tinh bt t ming v qu trnh ny hon tt rut non nh Amylase ca tuyn ty (cn c gi l amylopsin). Amylase ca malt thy phn tinh bt la mch thnh disaccharide lm c cht cho qu trnh ln men bi nm men. Amylase l mt trong nhng loi Enzyme c ng dng rng ri nht trong cng nghip, y t, v nhiu lnh vc kinh t khc, c bit l trong ngnh cng nghip thc phm. 1.2 Ngun gc 1814: Kirchoff, Saint Petercburg chng minh ht la mch ny mm c tc dng chuyn ha tinh bt thnh ng nhit t 4000C 6000C. Nm 1833, Payen v Perso (Php) thm cn vo dch chit ny, thu c kt ta c kh nng phn gii tinh bt thnh ng, v t tn l Diastase (xut pht t ting Hy Lp, diastatics, c ngha l phn gii, l Amylase). Sau ny theo ngh ca Duclo, Enzyme phn gii tinh bt c gi l Amylase. Nm 1851: Leuchs pht hin nc bt cng c kh nng phn gii tinh bt thnh ng. Sau , cc Enzyme Amylase trong nc bt, trong dch tiu ha ca ngi v ng vt, trong ht ny mm, nm mc, nm men v vi khun bt u c quan tm nghin cu. Nm 1862, Danilevxki tch c Amylase ca tuyn ty bng phng php hp th chn lc. Nm 1949, Schwimmer xc nh c s chu chuyn ca -Amylase l 19000. Nm 1950, Englard v Singer cho bit s chu chuyn ca -Amylase l 250000. n nm 1952, ngi ta thu c 72 Enzyme trng thi kt tinh trong c 4 Enzyme -Amylase.
NHM - 17
Nm 1971, Uxtinilov v cng s bng phng php in di trn gel poliacrylamid xc nh c s c mt ca mt lng ln -Amylase v glucoamylase trong canh trng nm mc v mt lng nh cc phn on c hot lc dextrinase v transglucosilase. Hin nay cc nc trn th gii sn xut hng trm tn ch phm Enzyme, trong Nht l nc c truyn thng lu i nht, sau n Anh, Php, M, an Mch, Thy in,... c bit hng Novo ca an Mch l 1 trong nhng hng sn xut Enzyme ni ting trn th gii. Bn cnh trong nhng nm gn y cc nc ng u v Trung Quc cng bt u nghin cu v sn xut Enzyme. Nu nh Ty u mch nha t la mch l ngun Enzyme ch yu cho vic chuyn ha tinh bt thnh ng, th Vin ng Amylase thng c sn xut t nm mc trn mi trng nui cy l cc loi ng cc c cha tinh bt. Nh hng Novo c nhiu ch phm Enzyme Amylase ang c s dng rng ri trong cc ngnh cng nghip nh: Cng nghip sn xut ru bia, cng nghip sn xut bt git, cng nghip giy 1.3 Phn loi Hin nay, c su loi Enzyme Amylase c xp vo 2 nhm: Endoamylase ( Enzyme ni bo ) v Exoamylase ( Enzyme ngoi bo ). - Endoamylase : gm c -Amylase (EC 3.2.1.1) v nhm Enzyme kh nhnh. Nhm Enzyme kh nhnh ny c chia thnh hai loi: kh trc tip l pullulanase ( hay -dextrin 6-glucanohydrolase ) (EC 3.2.1.41); kh gin tip l transglucosylase (hay oligo-1,6-glucosidase) (EC 3.2.1.20) v amylo-1,6-glucosidase (EC 3.2.1.10). Cc Enzyme ny thy phn cc lin kt bn trong ca chui polysaccharide. - Exoamylase. y l nhng Enzyme thy phn tinh bt t u khng kh ca chui polysaccharide. Nhm ny gm c: + -Amylase (EC 3.2.1.2) + Amyloglucosidase (glucoamylase hay -Amylase) (EC 3.2.1.3)
NHM - 17 5
Amylase
Exoamylase
Endoamylase
-Amylase
-Amylase
Enzym kh nhnh
-Amylase
Kh trc tip -dextrin 6-glucanohydrolase (pullulanase)
Kh gin tip oligo-1,6-glucosidase (transglucosylase)
Cc loi Enzyme endoAmylase v exoAmylase 1,6-glucosidase V amylo * S khc bit gia cc loi Enzyme Amylase: - Cc loi Enzyme Amylase khng ch khc nhau c tnh m cn khc nhau pH hot ng v tnh n nh vi nhit - Tc phn ng ca Amylase ph thuc vo pH, nhit , mc polyme ha ca c cht. Cc Enzyme Amylase c ngun gc khc nhau s c tnh cht, c ch tc dng v sn phm cui cng ca qu trnh thy phn khc nhau. - Amylase c ngun gc khc nhau s c thnh phn, tnh cht, nhit hot ng, pH ti u v cc c im thy phn khc nhau. 1.3.1 Enzyme -Amylase (-1,4-glucanohydrolase) (EC 3.2.1.1) a) Cu to Enzyme -Amylase l protein c phn t lng thp, thng nm trong khong 50.000 n 60.000 Dal. C mt s trng hp c bit nh -Amylase t loi vi khun Bacillus macerans c phn t lng ln n 130.000 Dal. n nay ngi ta bit rt r cc chui acid amin ca 18 loi -Amylase nhng ch c 2 loi -Amylase l taka-Amylase t Apergillus oryzae v -Amylase ca ty ln c nghin cu k v hnh th khng gian cu trc bc 3. Mi y cc nghin cu v tnh ng nht ca
NHM - 17 6
chui mch acid amin v v vng k nc cho thy cc chui mch acid amin ca tt c cc Enzyme -Amylase u c cu trc bc 3 tng t nhau.
Cu trc khng gian ca -Amylase b) C ch tc dng ca -Amylase -Amylase t cc ngun khc nhau c nhiu im rt ging nhau. -Amylase c kh nng phn ct cc lin kt -1,4-glucoside nm pha bn trong phn t c cht (tinh bt hoc glycogen) mt cch ngu nhin, khng theo mt trt t no c. Amylase khng ch thy phn h tinh bt m n thy phn c ht tinh bt nguyn song vi tc rt chm. Qu trnh thy phn tinh bt bi -Amylase l qu trnh hai giai on: + Giai on u (giai on dextrin ha): Ch mt s phn t c cht b thy phn to thnh mt lng ln dextrin phn t thp (-dextrin ), nht ca h tinh bt gim nhanh (cc amylose v amylopectin u b dch ha nhanh). + Giai on 2 (giai on ng ha): Cc dextrin phn t thp to thnh b thy phn tip tc to ra cc tetra-trimaltose khng cho mu vi iodine. Cc cht ny b thy phn rt chm bi -Amylase cho ti disaccharide v monosaccharide. Di
NHM - 17 7
tc dng ca -Amylase, amylose b phn gii kh nhanh thnh oligosaccharide gm 6 - 7 gc glucose (v vy, ngi ta cho rng -Amylase lun phn ct amylose thnh tng on 6 - 7 gc glucopiranose 1). + Sau , cc polyglucose ny b phn ct tip tc to nn cc mch polyglucose colagen c ngn dn v b phn gii chm n maltotetrose, maltotriose v maltose. Qua mt thi gian tc dng di, sn phm thy phn ca amylose cha 13% glucose v 87% maltose. Tc dng ca -Amylase ln amylopectin cng xy ra tng t nhng v khng phn ct c lin kt -1,6-glycoside ch mch nhnh trong phn t amylopectin nn d c chu tc dng lu th sn phm cui cng, ngoi cc ng ni trn (72% maltose v 19% glucose) cn c dextrin phn t thp v isomaltose 8%. Tm li, di tc dng ca -Amylase, tinh bt c th chuyn thnh maltotetrose, maltose, glucose v dextrin phn t thp. Tuy nhin, thng thng Amylase ch thy phn tinh bt ch yu thnh dextrin phn t thp khng cho mu vi Iodine v mt t maltose. Kh nng dextrin ha cao ca -Amylase l tnh cht c trng ca n. V vy, ngi ta thng gi loi Amylase ny l Amylase dextrin ha hay Amylase dch ha. Cc giai on ca qu trnh thy phn tinh bt ca -Amylase: + Giai on dextrin ha:
-Amylase
Tinh bt
dextrin phn t lng thp
+ Giai on ng ha: Dextrin Amylase Maltose tetra v trimaltose oligosacharide maltotriose di & monosaccharide poliglucose maltotetrose
NHM - 17
c) c tnh -Amylase -Amylase t cc ngun khc nhau c thnh phn amino acid khc nhau, mi loi -Amylase c mt t hp amino acid c hiu ring. -Amylase l mt protein giu tyrosine, tryptophan, acid glutamic v aspartic. Cc glutamic acid v aspartic acid chim khong tng lng amino acid cu thnh nn phn t Enzyme: + -Amylase c t methionine v c khong 7-10 gc cysteine. + Trng lng phn t ca -Amylase nm mc: 45.000-50.000 Da (Knir 1956; Fisher, Stein, 1960 ). + Amylase d tan trong nc, trong dung dch mui v ru long. + Protein ca cc -Amylase c tnh acid yu v c tnh cht ca globuline. im ng in nm trong vng pH = 4,2 - 5,7 ( Bernfeld P, 1951 ). -Amylase l mt metaloEnzyme. Mi phn t -Amylase u c cha 1-30 nguyn t gam Ca/mol, nhng khng t hn 1 - 6 nguyn t gam/mol Ca tham gia vo s hnh thnh v n nh cu trc bc 3 ca Enzyme, duy tr hot ng ca Enzyme (Modolova, 1965). Do , Ca cn c vai tr duy tr s tn ti ca Enzyme khi b tc ng bi cc tc nhn gy bin tnh v tc ng ca cc Enzyme phn gii protein. Nu phn t -Amylase b loi b ht Ca th n s hon ton b mt ht kh nng thy phn c cht. -Amylase bn vi nhit hn cc Enzyme khc. c tnh ny c l lin quan n hm lng Ca trong phn t v nng Mg2+. Tt c cc Amylase u b kim hm bi cc kim loi nng nh Cu2+, Ag+, Hg2+. Mt s kim loi nh : Li+, Na+, Cr3+, Mn2+, Zn2+, Co2+, Sn2+, Cr3+, khng c nh hng my n -Amylase. Mt c im cn lu l hu ht -Amylase kh bn vi tc ng ca protease nh pepsin, trypsin, papain...
NHM - 17
Cu trc phn t tinh bt do Enzyme -Amylase phn ct to thnh dextrin ti hn phn nhnh. Sn phm thy phn cui cng ca tinh bt di tc dng ca Amylase nm si ch yu l maltose, th n l maltotriose. Nng -Amylase ca vi sinh vt tng i ln c th chuyn ha 70 - 85% tinh bt thnh ng ln men. Cn cc Amylase ca nm mc th mc ng ha n glucose v maltose c th ln ti 84 - 87%. bn i vi tc dng ca acid cng khc khc nhau. -Amylase ca Asp.oryzae bn vng i vi acid tt hn l -Amylase ca malt v vi khun Bac.subtilis. pH= 3,6 v 0oC, -Amylase ca malt b v hot hon ton sau 15 - 30 pht; -Amylase vi khun b bt hot n 50%, trong khi hot lc ca -Amylase ca nm si hnh nh khng gim bao nhiu (Fenilxova, Rmoshinoi 1989). Trong dung dch -Amylase nm si bo qun tt pH = 5,0 - 5,5; -Amylase dextrin ha ca nm si en c th chu c pH t 2,5 - 2,8. 0oC v pH = 2,5, n ch b bt hot hon ton sau 1 gi. Bng 1.1: Mt s tnh cht ca -Amylase t vi sinh vt Tn vi sinh vt pHopt Topt Phn t lng (kD) Bacillus acidocaldarius Bacillus stearothermophilus
NHM - 17
3,5 4,5 6,5
75 65 73
68 48
10
Bacillus subtilis Acinetobacter sp.I Acinetobacter sp.II Bacteroides amylophilus Micrococcus halobius Streptomyces hygroscopicus Streptomyces aureofaciens Thermonospora curvata Aspergillus prysee Mucor pusillus Lipomyces kononenkaae Schwaniomyces castellii
5,3 6,4 7,0 7,0 6,3 6,0 7,0 5,0 6,0
50 50 55 50 55 43 50 55 50 55
47 55 65 92 89 48
4,6 5,3 5,5 6,0 5,5 5,9 3,5 4,0 5,5 6,0
40 65 40 65 70 40 60
40 62 52 48 38 40
1.3.2 Enzyme -Amylase (-1,4-glucan-maltohydrolase) (EC 3.2.1.2) a) Cu to -Amylase hin din ph bin thc vt, c bit l ht ny mm. trong cc ht ng cc ny mm, -Amylase xc tc s thu phn cc lin kt 1,4 -glucan trong tinh bt, glucogen v polysaccharide, phn ct tng nhm maltose t u khng kh ca mch . Maltose c to thnh do s xc tc ca -Amylase c cu hnh . ng cc, -Amylase tham gia vo s phn gii ca tinh bt trong qu trnh ny mm ca ht. la, -Amylase c tng hp trong sut qu trnh ca ht v hu nh khng c tng hp ht kh. la mch, Enzyme c mt trong ht kh, n c tch ly trong sut qu trnh pht trin ca ht, khi dng lin kt, Enzyme ny
NHM - 17 11
l mt phn t c trng lng phn t l 64.000 Da v khi b phn ct bi mt protease s c phng thch di dng t do v c khi lng phn t l 59.000 Da . b) C ch tc dng ca -Amylase -Amylase l mt Enzyme ngoi bo (exoenzyme). Tin trnh phn gii bt u t u khng kh ca cc nhnh ngoi cng c cht . -Amylase phn ct cc lin kt -1,4glucoside nhng khi gp lin kt -1,4 glucoside ng k cn lin kt 1,6glucoside th n s ngng tc dng. Phn polysaccharide cn li l dextrin phn t ln c cha rt nhiu lin kt -1,6 glucoside v c gi l -dextrin. C ch tc dng ca -Amylase ln tinh bt
- amylase Tinh bt maltose (54-58%) + -dextrin (42-46%)
(glucogen) Tinh bt b thu phn ng thi bi c v -Amylase th lng tinh bt thu phn ti 95%. c) c tnh ca -Amylase -Amylase l mt albumin, tm xc tc c cha nhm SH , nhm X-COOH v vng imidazol ca cc gc histidine v l Enzyme ngoi bo (exoEnzyme ) -Amylase khng bn khi c Ca2+, -Amylase b km hm bi Cu2+, Hg2+, urea, iodineoacetamide, iodine, ozon -Amylase chu nhit km hn -Amylase nhng bn hn vi acid. -Amylase b bt hot nhit 700C. Nhit ti thch ca -Amylase l 550C, pH 5,1 5,5. Tham gia vo c ch tc dng ca -Amylase thng c mt nhm caboxyl th hin tnh cht i nhn v mt nhm imidazol th hin tnh cht i electron. S nghch o hnh th ca cacbon anome (C1) c thc hin nh vic to thnh hp cht ng ho tr trung gian kiu este axetal gia cacbon anome v nhm cacboxyl ca tm hot ng. Sau este ny b phn hu bi tc ng ca 1 phn t nc ln nhm cacboxyl gii phng ra -maltose v hon nguyn nhm cacbxyl ca Enzyme.
NHM - 17
12
Bng 1.2: Cc c tnh ca -Amylase Ngun gc Enzyme i mch La m tng Khoai lang B.cerus B.polymyxa B.megaterium 5,2 5,2 5,6 5,4 5,0 6,0 7,0 7,5 6,5 55 55 50 55 40 40 40 - 65 pHopt Topt Phn t lng (kD) 56 64,2 57 50 58 42 58
1.3.3 Enzyme -Amylase (glucoamylase) (EC 3.2.1.3) a) Cu to -Amylase (glucoamylase hay -1,4-glucan-glucohydrolase ) l nhng Enzyme c th thu phn c c hai kiu lin kt ca cc mch -glucan gii phng ra dng . Glucoamylase hay -Amylase ch yu c to ra bi cc vi sinh vt. c bit l kiu nm mc Aspergillus, Penicillium v Rhizopus Amyloglucosidase t nm mc l cc protein c khi lng phn t lng dao ng rt ln t 27.000 n 112.000 Dal tu thuc vo ngun gc ca Enzyme Ni chung, cc amyloglucosidase u cha cc gc methioni, tritophan, v mt na gc cystein. Tuy nhin mi quan h gia chui acid amin, cu trc bc 3 v hot ng ca Enzyme vn cha c lm sng t, tt c cc amyloglucosidase t nm mc u l glucoprotein cha t 5 - 20% gluxit trong ch yu l cc monosaccharid, glucose mannose, galactose v glucosamin
NHM - 17
13
Cc amyloglucosidase ch yu c to nn t hai iso-Enzyme I v II khc nhau kh nng thu phn tinh bt trng thi rn v bi bn ca chng. Amyloglucosidase I t hp th v thu phn tinh bt trng thi rn, ngc li amyloglucosidase II khng c c hai tinh cht ny . b) C ch hot ng Amyloglucosidase c th gii phng ra -D-glucose bng cch thu phn lp li nhiu ln cc lin kt -1,4 ca mch -glucan t u khng kh, chng cng thu phn c cc lin kt -1,6 v -1,3 nhng rt chm (10 - 30 ln ). Tc thu phn cng ph thuc vo bn cht ca cc lin kt k cn vi cc lin kt glucozit c thu phn , cng nh kch thuc v cu trc ca c cht b thu phn . Nht l vi cc -glucan mch di (amylose v amylopectin) th b thu phn nhanh hn l vi cc maltodextrin v cc oligosaccharit. c) Tnh cht Glucoamylase c kh nng thu phn cc lin kt -1,4 ln -1,6 glucoside. Khi thu phn lin kt -1,4-glucan trong chui polysaccharide, glucoamylase tch ln lt tng phn t glucose ra khi u khng kh ca mch to ra glucose. Enzyme ny c nhiu tn gi khc nhau: -1,4; -1,6-glucan- 4; 6-glucohydrolase; glucoamylase; amyloglucosidase; taka-Amylase B; -Amylase L Enzyme ngoi bo. Ngoi cc lin kt -1,4 v -1,6 glucoside, glucoamylase cn c kh nng thu phn cc lin kt -1,2 v -1,3 glucoside . Glucoamylase c kh nng thu phn hon ton tinh bt, glucogen, amylopectin, dextrin, panose, iso maltose v maltose thnh glucose, m khng cn c s tham gia cu cc loi Enzyme khc. Glucoamylase thu gii cc polysaccharide c phn t ln nhanh hn so vi cc cht c phn t nh. Cc polisaccharide c nhnh nh amylopectin, glucogen, -dextrin b glucoamylase thy phn kh nhanh. a s glucoamylase c hot lc cao nht vng c pH = 3,5 5,5 v nhit 500C. N bn vi acid hn -Amylase nhng km bn hn trong ru, acetone v khng c bo v bi Ca2+.
NHM - 17 14
1.3.4. Oligo 1,6-glucosidase (dextrinase ti hn) (EC 3.2.1.10) Enzyme ny c th thu phn lin kt -1,6 glucoside trong isomaltose, panose v cc dextrin ti hn thnh ng c th ln men c. Enzyme ny c vi sinh vt nhng ng thi cng c trong cc ht ny mm (i mch, thc ny mm). Ngoi oligo1,6glucosidase, h dextrinase ca ht ng cc, ht ny mm cn c amylopectin1,6glucosidase hay REnzyme v dextrin1,6glucoside hay amylo 1,6glucoside hay dextrin-6-glucocanhydrolase. Hai loi Enzyme ny u thu phn dextrin trit hn -Amylase v -Amylase do trong dung dch thu phn c nhiu maltose hn . Nhit ti thch cho cc hot ng ca cc dextrinase l 400C v pH ti thch l 5,1. 1.3.5. Enzyme pullulanase (-dextrin 6-glucosidase) (EC 3.2.1.41) Enzyme ny c th thu phn cc lin kt -1,6 ca tinh bt, glucogen, pululan v cc dextrin ti hn. iu ng ch l s nh v ca cc lin kt -1,6 c nh hng ln n tc ng ca Enzyme. c bit l s c mt ca hai lin kt -1,4 nm lin k bn lin kt -1,6 l iu kin cn thit cho Enzyme phn ct lin kt ny Pullulanase phn gii cc lin kt -1,6 glucoside b bao quanh t pha bi cc lin kt -1,4. N cn c kh nng thy phn c nhng dextrin phn t thp ch gm c hai gc maltose ni nhau bng lin kt -1,6 glucoside. Tc dng hip ng ca Amylase v pullulanase lm n b thy phn hon ton. 1.3.6. -glucosidase hay maltase (-D,glucoside-glucohydrolase)(EC 3.2.1.20) Nhiu loi nm si sn sinh Enzyme ny. Ging nh glucomylase, n thy phn maltose thnh glucose nhng khng thy phn tinh bt. Maltase v glucozyltranferase l mt Enzyme ng nht va c kh nng thy phn lin kt -1,4, trong cc glucopiranoside va c kh nng chuyn cc gc glucoside sang ng v ru.
NHM - 17
15
2. NGUYN LIU SN XUT Enzyme Amylase c trong cc t bo sinh vt , ng thc vt , cc loi nm mc. Mun thu nhn Enzyme cn chit rt ra khi t bo. Trong c th sinh vt, Enzyme trong t bo cht v cc cu t to nn t bo nh nhn microsom Enzyme khng c kh nng i qua mng t bo, i vo dung dch chit. V vy vic u tin l cn phi ph v mng t bo. Vic ph v cu trc t bo c th s dng mt s cch sau : + Bin php c hc nh nghin xay vi bt thu tinh , ct thch anh hoc my xay ng ho + Bng dung mi hu c nh butanol, axeton, glycerin, etyl axetat + Bng sng siu m, tia X, tia UV Sau khi nghin nh Enzyme c chit rt bng nuc hay dung dch m thch hp, hoc dung dch mui trung tnh Dung dch thu c sau khi chit ngoi Enzyme cn c cc tp cht khc nh protein phi Enzyme, mui, gluxit Chng ta cn loi b thu nhn Enzyme c tinh khit cao. C rt nhiu phng php tinh sch Enzyme sau y l mt s phng php c bn c s dng rng ri v c hiu qu : + loi mui v tp cht c trng lng phn t nh ta dng phng php thm tch qua mng bn thm. Tinh cht ca mng ny s cho cht c trng lng phn t nh i qua . Cc cht c trng lng phn t ln b gi li (Enzyme, protein...). + loi protein l v tp cht c trng lng phn t cao ta dng phi hp nhiu phng php khc nhau nh sc k hp ph, sc k trao i ion, in di lc gel
NHM - 17
16
2.1 T thc vt c ly t nhiu ngun nh: i mch (hodeum sativum): + + Ging 2 hng: sn xut malt lm bia Ging nhiu hng (4,6 hng): lm thc n cho gia sc.
La (oryza sativa L.): ch yu vng ng Nam Ng (zea mays): c nhiu loi ng: ng , ng bt, ng rng nga. Ht ng c mu trng, mu vng hay mu hng. Ng mu vng do c s hin din ca carotenoid v zeaxanthine trong ni nh. Ng mu hng l do trong ni nh ca ng c anthocyanin.
Thu nhn, x l, lm sch, phn loi
i mch
Ngm
Ny mm
Sy kh
Khng kh
Sy kh
Bo qun
Tch mm r
Mm r kh Malt kh bo qun
Malt kh sn xut bia
Cc bc quy trnh sn xut malt kh
NHM - 17
17
Qu trnh thy phn tinh bt ca cc Enzyme Amylase 2.2 T ng vt Enzyme Amylase c trong ty tng ca ng vt. Ty tng l ng dng chm, di, ln, nm ngang pha sau d dy, gia l lch v t trng. Chiu di ca ty tng khong 30-35cm, v nng khong 80-150g. Bn trong c nhng vch ngn nh chia ty tng thnh nhiu thy nh. Ty tng va c chc nng ni tit va c chc nng ngoi tit. Thu nhn Amylase t ng vt phn ln l t dch ty tng. 98% ty tng c cu to t cc t bo ngoi tit hoc l t bo tuyn. Cc t bo ny tit Enzyme tiu ho vo trong t trng. 2.3 T Mi trng nui cy vi sinh vt 2.3.1 Nguyn liu to mi trng nui cy. Nguyn liu s dng trong nui cy b mt thng l nhng nguyn liu c ngun gc t nhin nh cm m, cm go, go, ng mnh, u nnh v cc loi ht ng cc khc.Trong cc loi nguyn liu trn, cm go, cm m c s dng nhiu hn c. Hai loi ny c y cc cht dinh dng cn thit cho VSV pht trin. Mt khc khi to mi trng, chng thng c tnh cht vt l rt thch hp va m bo khi kt dnh cn thit, va m bo lng khng kh lu chuyn trong khi nguyn liu.
NHM - 17 18
Nguyn liu s dng trong nui cy b su (S dng mi trng hon ton lng): Nguyn liu nui cy ph bin l dch ng nh glucose, fructose, sacarose, pepton, nng thch hp khong 10 - 15%. 2.3.2 Mi trng lng . mi trng lng, VSV s pht trin trn b mt mi trng, to thnh khun lc ngn cch pha lng (mi trng) v pha kh (khng kh). y, VSV s s dngcht dinh dng t dung dch mi trng, O2 t khng kh, tin hnh qu trnh tng hp enzyme. Enzyme ngoi bo s c tch ra t sinh khi v ha tan vo dung dch mi trng. Enzyme ni bo s nm trong sinh khi vi sinh vt.
Hnh 1.1: Ln men trong mi trng lng quy m phng th nghim (5L) (www.khoahoc.com.vn) 2.3.3 Mi trng bn rn Phn ln cc nh my sn xut enzyme, khi nui cy VSV thu nhn enzyme, ngi ta thng s dng mi trng c. tng kh nng xm nhp ca khng kh vo trong lng mi trng, ngi ta thng s dng cm, tru, ht ng cc lm mi trng. Vi sinh vt pht trin trn b mt mi trng, nhn cht dinh dng t ht mi trng v sinh tng hp ra enzyme ni bo v ngoi bo. Cc enzyme ngoi bo s thm thu vo trong cc ht mi trng, cn cc enzyme ni bo nm trong sinh khi vi sinh vt. Vi sinh vt khng ch pht trin trn
NHM - 17 19
b mt mi trng, ni ngn cch pha rn (mi trng) v pha kh (khng kh) m cn pht trin trn b mt ca cc ht mi trng nm hn trong lng mi trng. Mi trng nui cy va c xp cao v va phi c m thch hp. Nu m qu cao s lm bt mi trng li, khng kh khng th xm nhp vo trong lng mi trng, nu c m thp qu s khng thun li cho VSV pht trin. Thng thng ngi ta thng to m khong 55-65% W l hp l. Nu s dng cm lm nguyn liu chnh nui cy vi sinh vt thu nhn enzyme, ngi ta phi cho thm 20-25% tru lm xp mi trng, to iu kin thun li khng kh d xm nhp vo lng mi trng. Phng php nui cy b mt bn rn (mi trng c) ny rt thch hp cho len men nm mc Asp.oryzae.
Hnh 1.2: Ln men trn mi trng rn. A: ln men k kh trong ni bng t nung, B: ln men hiu kh. A. B.
Bng 1.3: Tnh cht ca amylase thu nhn t cc ngun khc nhau. (Nguyn Tin Thng, Gao trnh cng ngh enzyme 2008) Ngun thu nhn B. subtilis B. licheniformis B.stearothermophilus B. cereus Pseudomonas
NHM - 17
Gii hn pH (pH ti u) 4,5 6,5 5,0 9,0 4,0 5,2(3,0) 6,0 7,0 6,7 7,0
Khi lung phn t (Dalton) 48.000 22.500 96.000 90.000 62.000
Nhit ti u (oC) 60 90 80 50 55 45
20
Ty Malt Aspergilus oryzae
6,0 7,0 4,5 9,5 (5,5) 5,0
45.000 52.000 51.000 70 50 60
3. GING VI SINH VT 3.1 Vi sinh vt trong sn xut enzyme Amylase Nhm enzyme amylase, a phn c tng hp bi nm mc v vi khun, mt s t t nm men. Cc chng nm mc nh: Asp. Oryzae, Asp. ninger, Asp. awamori, Asp. Usamiii, Rhizopus neveus, Mucor sp,... v mt s loi vi sinh vt khc nh: Endomycopsis.Fibuliger, Endomyces sp, Saccharomyces diastaticus,... to amylase, glucoamylase, glucozil transferase. T vi khun Bac. Diastaticus, Bac. Subtilis, Bac. Mesentericus, Bac. Amylosolvens... thng thu -amylase chu nhit cao. Mi chng vi sinh c th tng hp nhiu loi enzyme nhng khi lng mi loi enzyme tng hp c khc nhau. V d: chng Asp. Oryzae (mc vng) tng hp nhiu amylase nhng t glucoamylase v glucozil-transferase. Cn chng Asp. ninger , Asp.awamori (mc en) th ngc li. Amylase ca vi khun c kh nng dch ha cao (to dextrin), kh nng ng ha km hn amylase nm mc, nhng c u im l chu nhit cao (900C). Nht, hng nm sn xut 7.000 tn amylase t vi khun. 3.2 c im sinh l, dinh dng v c ch tng hp sn phm trao i cht 3.2.1 Nm mc Aspergillus Oryzae a) c im nm mc Aspergillus Oryzae (Asp. Oryzae) l mt loi nm vi th thuc b Plectascales, lp Ascomycetes (nang khun). C th sinh trng ca n l mt h si bao gm nhng si rt mnh, chiu ngang 5 -7 m, phn nhnh rt nhiu v c vch ngang,
NHM - 17
21
chia si thnh nhiu bao t bo (nm a bo). T nhng si nm ngang ny hnh thnh nhng si ng thng gi l cung nh bo t, c c quan sinh sn v tnh. Cung nh bo t ca Asp.oryzae thng di 1-2 mm nn c th nhn thy bng mt thng. Pha u cung nh bo t phng ln gi l bng. T bng ny phn chia thnh nhng t bo nh, thun, di, gi l nhng t bo hnh chai. u cc t bo hnh chai phn chia thnh nhng bo t nh vo nhau, nn gi l nh bo t. nh bo t ca Asp.oryzae c mu vng lc hay mu vng hoa cau c im ca ging Asp.oryzae l giu cc enzyme thy phn ni bo v ngoi bo (amylase, protease, pectinasa,), ta rt hay gp chng cc kho nguyn liu, trong cc thng cha ng bt, go ht nhng khng c ra sch, cn b bia, b ru, li ng, b sn Chng mc v pht trin c khi thnh lp mc, c mu sc en ,vng Mu do cc bo t gi c mu sc. Cc bo t ny, d b gi cun bay xa v ri vo u khi gp iu kin thun li s mc thnh mc mi
Hnh 1.3: Aspergillus Oryzae (mc vng hoa cau) b)ng dng ca Asp. Oryzae Dch qu sau khi nghin v tch b v thng cha cc thnh phn t bo tht qu v cc thnh phn ca polysaccharide lm cho dch qu c nht cao. tng hiu sut trch ly dch qu, gim bt nht, tng mc cm quan nc qu v gim bt mt s cng on, vic b sung Endoglucanase rt quan trng. Enzyme ny l
NHM - 17 22
im mu cht ci thin hiu sut dch ha. S kt hp ca glucanase v pectinase s ph hy hon ton mng t bo. Trong cng ngh sn xut bia, cc ch phm enzyme amylase, protease v glucanase c s dng ngn chn s to thnh cc diacetyl do gim lng diacetyl c to thnh, rt ngn thi gian cn thit bia. Trong dch ln men c cha mt lng -glucan, cht ny nh hng ti kh nng lc v gy c cho bia. 3.2.2 Baciluus Subtilis a) c im vi khun B. Subtilis l trc khun, Gram (+), c kh nng sinh catalase, hiu kh hay k kh ty . Thng c tm thy trong t. C kh nng di ng, sinh ni bo t. T bo sinh dng c dng hnh que, kch thc chiu rng t 0,7 0,8 m, chiu di t 2,0 3,0 m. Khng kt thnh chui, bt phm nhum ng u, khng to bao nang.
Hnh 1.4: Bacillus subtilis
Hnh 1.5: Vi khun Bacillus subtilis trong phn chia t bo.
Bo t B. Subilis c dng ellip n hnh cu, kch thc chiu rng 0,6 0,9 m, chiu di 1,0 1,5 m, nm gia hay trong khong trung tm n gn cui t bo, phn ln c to thnh 48 gi. Mi c th ch to mt bo t, bo t c kh nng chu nhit, tia bc x, cht st khun, cht ht m.
NHM - 17
23
Khi nui cy trn mi trng thch a, khun lc trn, khng u hay phn tn. ng knh khun lc t 3 5 mm, mu vng xm, ra c hnh rng ca. Sau 1 4 ngy b mt khun lc nhn nheo, mu hi nu.
Hnh 1.6. Khun lc B. subtilis trn thch a Trong mi trng lng sinh khi to mng mng c lp bao ph. Do bo t chiu nhit cao nn B. subtilis c th gy h hng mt s thc phm hp to mi v kh chu. Sinh acid t xylose, arabinose, glucose, sucrose v mannitol nhng khng to kh (s dng ngun nit l mui amonium). C kh nng phn gii nitrate, sinh nitrit t nitrate. Trong iu kin k kh, khng sinh kh t mi trng lng cha nitrate. Mt c im dng phn bit vi cc vi khun khc l lm tan chy gelatine nhanh chng. Bng 1.4. Mt s phn ng sinh ho ca B. subtilis Phn ng sinh ho Hot tnh Catalase Sinh Indol MR
NHM - 17
Kt qu + +
24
VP S dng Citrate Kh Nitrate Tan chy gelatin Phn gii tinh bt Arabinose Xylose Saccharose Manitol Glucose Lactose Maltose (Theo Holt, 1992)
+ + + + + + + + + + +
Nhit ti thch ca B. subtilis vo khong 36 - 50oC. Nhiu loi vn pht trin c 60oC. Nng mui n lm ngng pht trin l 10 15%. Ngi ta chng minh B. subtilis c tp tnh n ln nhau (cannibalism). (phng php n gin thot khi nhng trng hp iu kin sng gii hn). B. subtilis c kh nng sinh tng hp hn 20 loi khng sinh khc nhau nh: subtilin, subtilosin A, sublancin, chlorotetain, mycobacillin, rhizocticins, bacillanene, difficidin(theo ti liu tng hp ca Nguyn Qunh Nam, 2006). Phn b trong t v cc cht hu c b phn hy v l mt i tng dng trong nghin cu kh nng ly bnh trong phng th nghim. B. subtilis khng c xem l mm bnh gy bnh cho ngi. Chng thng c trong thc phm nhng him khi gy ng c thc phm.
NHM - 17 25
b) Phn loi Bng 1.5. Bng phn loi ca Bacillus subtilis Phn loi khoa hc Gii : Bacteria Ngnh : Firmicutes Lp B : Bacilli : Bacillales Tn kp Bacillus subtilis (Ehrenberg 1835 Cohn 1872) H : Bacillaceae
Ging : Bacillus Loi : subtilis
c) B gen Nm 1997, ngi ta hon tt vic nghin cu v trnh t gen ca B.subtilis v ln u tin cng b trnh t gen ca vi khun. B gen cha 4,2 mega-base, xp x 4100 gen. Trong s , ch c 192 gen khng th thiu c, 79 gen c d on l thit yu. Phn ln gen thit yu u c lin quan vi qu trnh trao i cht ca t bo. d) ng dng ca Bacillus subtilis Trong cng nghip sn xut amino acid, thc n gia sc, Bacillus subtilis l mt trong nhng chng vi sinh vt tng hp lysine c hm lng kh ln (15-20%) t tinh bt. Trong y dc, Bacillus subtilis c ng thnh ng thuc Subtilis 10 ml tr bnh tiu chy cho tr em do vi khun Coliform gy ra, bnh ng rut do l trc trng, p cc vt thng l lot ngoi da.
NHM - 17 26
Sn xut cc khng sinh thc vt, ng dng trong phng tr vi sinh vt gy bnh nh nm Rhizoctonia solani, Fusarium sp, Pylicularia oryzae,... Ngi ta thy rng s pht trin ca Bacillus subtilis trong cy lm tng kh nng tng hp cc peptide khng nm ca vi khun nt r (Rhizobacterium). Kh nng ny c ng dng trong kim sot sinh hc. ng dng trong sn xut ch phm sinh hc (probiotic) b sung trong thc n nhm ci thin tiu ha, sc tng trng; gim s ti pht bnh tiu chy trn gia sc; b sung vo ao nui nhm duy tr cht lng nc ao, hn ch bnh cho thy sn nui. H enzyme ca B. subtilis c s dng nhiu trong sn xut cht ty ra. Chng c th bin i cc dng cht thi c hi thnh nhng dng hp cht v hi ca nitrogen, carbon dioxide, v nc. Mt chng Bacillus subtilis c bit trc y l Bacillus natto c dng trong sn xut thc phm thng mi ca Nht tng t nh thc phm cheonggukjang ca Hn Quc. B. subtilis ti t hp c s dng trong sn xut polyhydroxyalkanoates (PHA) v chng c th s dng malt ph thi nh l ngun cacbon, nh vy chi ph sn xut PHA gim.
NHM - 17
27
Phn 2
CNG NGH SN XUT

1. S QUY TRNH CNG NGH SN XUT ENZYME THEO PHUONG PHP NUI CY VI SINH VT Quy trnh sn xut mc ging
Chun b mi trng thch nghing
ng mc ging
Cy truyn Chun b mi trng trong bnh tam gic Ng mnh, hp thanh trng Nui cy 30-320C trong 5-6 ngy
Nui cy 30-320C trong 5-6 ngy
Nc v trng
Ging trong ng thch nghing
Lm ti
Trn u bo t
Mc ging trong bnh tam gic Mc ging cho sn xut
Trn ging 0,5-10%
Nui mc 60h
Sy kh
Bao gi
NHM - 17
28
1.1. Theo Phng Php Nui Cy B Mt Quy trnh cng ngh thu nhn enzyme amylase t Asp. Oryzae Nguyn liu
X l nguyn liu
Hp thanh trng
Ging vi sinh vt
Lm ngui
Nhn ging
Trn ging vi sinh vt
Ging cho sn xut
Nui cy
Thu nhn ch phm enzyme th
Ch phm enzyme th em s dng
Ch phm enzyme th em tinh ch
Nghin mn
Trch ly
Lc
Dng trong chn nui
Kt ta
Cn hoc sulfat amon
Ch phm enzyme tinh khit

NHM - 17 29
Thuyt minh quy trnh Hp thanh trng: di p sut hi 1-1,5 atm trong thi gian 45-60 pht. Trn ging vi sinh vt: Sau khi lm ngui, tin hnh cy ging hoc rc bo t vo mi trng thanh trng, thnh ng vi gi. Khi cy vo mi trng dinh dng, bo t s pht trin thnh t bo nm mc v to ra cc loi enzyme m ta mong mun. K thut nui cy: Sau khi trn ging, mi trng c tri u ra cc khay vi chiu di 2-3cm, ri c a vo phng nui cy 28-32oC, khng cn iu chnh pH. Thi gian nui nm si thu nhn enzyme vo khong 36-60 gi. Qu trnh pht trin ca nm mc trong mi trng bn rn khi nui bng phng php b mt ny tri qua cc giai on sau: Giai on 1: giai on ny ko di 10-14 gi k t thi gian bt u nui cy. o Nhit tng rt chm. o Si nm bt u hnh thnh v c mu trng hoc mu sa. o Thnh phn dinh dng bt u c s thay i. o Khi mi trng cn ri rc. o Enzyme mi bt u c hnh thnh. Giai on 2: giai on ny ko di 14-18 gi. o Ton b bo t pht trin thnh si nm c mu trng xm o Mi trng c kt li kh cht. o m mi trng gim dn. o Nhit mi trng s tng nhanh c th ln ti 40-45oC. o Cc cht dinh dng bt u gim nhanh do s ng ho mnh ca nm si. o Enzyme amylase c tng hp mnh. o Lng O2 trong khng kh gim v CO2 s tng dn
NHM - 17 30
Giai on 3: giai on ny ko di 10-20 gi. o Qu trnh trao i cht yu dn, do mc gim cht dinh dng s chm li. o Nhit ca khi mi trng gim, do lm gim lng khng kh mi trng xung 20-25 th tch khng kh /th tch phng nui cy/ 1gi. 1.1.1. Thu nhn sn phm Ton b khi lng enzym th amylase c em i nghin nh ph v thnh t bo v lm nh cc thnh phn ca ch phm th. S dng nhng cht tr nghin (ct thch anh v bt thy tinh) khi nghin. Trc khi s dng ct thch anh v bt thy tinh phi c ra sch, sy kh nhit ln hn 1000C loi b nc v tiu dit VSV. Sau khi nghin mn, ngi ta cho nc vo trch ly enzym -amylase. C mt phn ch phm enzym th, ngi ta cho 4-5 phn nc, khuy nh v sau lc ly dch, phn b thu ring dng lm thc phm gia sc (ch cn loi b ct thch anh v bt thy tinh ra khi hn hp b ri mi cho gia sc n). Dch thu nhn c vn dng ch phm enzym th v trong c cha nc, cc cht ha tan khc t khi mi trng nui cy. Dng cn v sunfat amon kt ta enzyme.Trong khi tin hnh kt ta, ngi ta phi lm lnh c dung dch enzym th v c nhng tc nhn kt ta trnh lm mt hot tnh enzyme. Khi cho cht kt ta vo dung dich enzyme th, ngi ta tin hnh khuy nh, sau yn trong iu kin nhit 4-70C. Theo thi gian, cc enzyme s c to kt ta v lng xung y, ngi ta tin hnh gn v lc thu nhn kt ta dng paste ( m > 70%W). 1.1.2 u v nhc im u im : Nui cy b mt rt d thc hin. Quy trnh cng ngh thng khng phc tp.
NHM - 17
31
Lng enzyme c to thnh t nui cy b mt thng cao hn rt nhiu so vi nui cy chm. Ch phm enzyme th (bao gm thnh phn mi trng sinh khi vi sinh vt, enzyme v nc). Sau khi thu nhn rt d sy kh v d bo qun. Nui cy b mt khng cn s dng nhiu thit b phc tp. Trong trng hp b nhim cc vi sinh vt l, ta rt d dng x l. Mi trng c l mi trng tnh, khng c s xo trn nn khu vc no b nhim ta ch cn loi b khu vc khi ton b khi nui cy. Nhng khu vc khc s hon ton c an ton. Nhc im : Phng php ny tn kh ln din tch cho nui cy. Trong phng php ny vi sinh vt pht trin trn b mt mi trng (mi trng lng hoc mi trng bn rn) nn rt cn nhiu din tch.
M hnh nui mc trn khay
NHM - 17
32
1.2. Theo Phng Php Nui Cy B Su: Quy trnh cng ngh
Dch nui cy (mt r)
Hp thanh trng
Vi Sinh Vt Trn ging vi sinh vt
Nui cy
Amylase th
Thuyt minh quy trnh Hp kh trng nhit 118 125 0C, thi gian 40 60 pht, ngui dn nhit bnh thng v tip ging vi sinh vt vo mi trng, t l ging a vo l 2 2,5 %. Sau qu trnh nui cy c thc hin theo 2 phng php: nui cy theo chu k hay nui cy lin tc. Nui cy theo chu k l phng php nui cy trong 1 thit b ln men.
Sau 1 chu k nui t 2 4 ngy 28 320C ngi ta thu nhn ton b dch nui cy nh l 1 loi ch phm enzyme th. Phng php ny khng i hi k thut cao nhng nng sut thp. Nui cy lin tc l khc phc tnh trng trn. Qu trnh nui cy lin
tc c th nui cy trong 1 thit b, cng c th thc hin trong nhiu thit b. Phng
NHM - 17 33
php ny c li l nu cht lng sn phm ra cui cng ra ta thu nhn uc cha t yu cu t ra ta c th khc phc bng hai cch. o Cch th nht: lm cho thi gian lu ca dung dch v t bo vi
sinh vt trong thit b lu hn. o Cch th hai l: ta tin hnh hon lu dch ln men ha chung vi
dng mi trng ti ln men. 1.2.1 Thu nhn sn phm Dung dch sau khi nui cy theo phng php b su c tch khi sinh khi v cc thnh phn khng ha tan bng phng php ly tm. Dch thu thng cha 2 3% cht kh ha tan. Hm lng cht ny rt nh, do ta cn phi c c chng cho n khi khi lng dch gim i 5 10 ln iu kin chn khng. 1.2.2 u v nhc im u im: Phng php nui cy hin i d c kh ho, t ng ho, nng sut cao C th nui cy d dng cc chng vi sinh vt t bin c kh nng sinh tng hp enzyme cao v la chn ti u thnh phn mi trng, cc iu kin nui cy, enzyme thu c tinh khit hn, m bo v trng. Nhc im: i hi phi c v trng tuyt i cc khu v sinh tng hp, thanh trng mi trng dinh dng, thao tc nui cy, khng kh cung cp cho qu trnh nui cy. Tn in nng cho khuy trn, nu khng bo m v trng s b nhim hng lot, ton b gy tn thng ln v thu hi enzyme s c gi thnh cao.
NHM - 17
34
2. TINH SCH ENZYME Cc bc c bn Loi tp cht v cht khng tan: lc; ly tm Trch ly: hp ph hay dung mi. Tinh sch: sc k, in di, kt ta phn on. Hon thin: sy hay kt tinh Phng php dim tch : l phng php kt ta phn on enzyme bng mui trung tnh nh : (NH4)2SO4, NaCl, (Na2SO4, MgSO4 ) Bc 1: Loi b tp cht Lc: kch thc + Vn : nht + Thit b: my lc khung bn -Ly tm: kch thc v t trng Thit b: dng ng, a,
NHM - 17 35
Hnh 2.1: Thit b lc khung bn Bc 2: Ly trch Yu cu: khng bin tnh E Ph mng t bo: enzyme ni bo + Ha hc: Ptt; sy kh, lysozyme, acid, kim, cht ty Triton X-100, + C hc: ng ha, nghin, sng siu m, + Ly trch: dung mi chit rt l dung dch m c pH gn vi pHenzym. + Sc k hp ph: vi enzyme long Bc 3: Tinh sch Loi b: protein tp cht Cc phng php tinh sch + Phng php kt ta - Bng dung mi: aceton, etanol to thp -Bng mui: (NH4)2SO4 + Phng php sc k: - Sc k trao i ion - Sc k hp th
NHM - 17 36
- Sc k lc gel
GEL FILTRATION CHROMATOGRAPHY
Sc k trao i ion
ION EXCHANGE CHROMATOGRAPHY

NHM - 17 37
Sc k hp th
Hnh 2.2: Sc k hp th
NHM - 17
38
NHM - 17
39
Hnh 2.3: Nguyn tc ca mi quan h sc k
NHM - 17
40
Dialysis thm thm, thm tch
Hnh 2.5: Ti Cellophane (Cellophane bag) Phng php in di: Cht mang: giy, bn mng, SDS-polyacrylamide - Da: khc nhau v in tch, kch thc. - Cht kh: mercaptoetanol, dithithreitol (DTT) Bc 4: Hon thin: To ra: enzym kh, tinh th rn, xp. - Sy, kt tnh. - Lc, ly tm. - Tnh n nh cao - Bo qun: < 20oC.
NHM - 17 41
Phng php xc nh hot tnh Nguyn tc: Hot tnh xc tc ca enzyme tnh trong mt n v thi gian nht nh c xc nh thng qua: lng c cht gim hoc l s tng hm lng sn phm to thnh. - Cc phng php: ho hc, l hc, l ha xc nh s bin i hm lng ca c cht hay sn phm to thnh. n v o * IU = 1 mol / pht = 10-6mol/60s * Katal (kat) = 1 mol / 1s = 60.106 IU -Hot ring: nh gi sch ca ch phm E. S VH/khi lng. Phng php xc nh hot + Quang ph: o lng sn phm to thnh. - Khi xc nh hot tnh xc tc protease, dng phn ng to mu gia tyrozin (do thy phn protein) vi cht nhum mu Folin. V do bin i mt quang (OD) ca dung dch mu ny 650-720nm. - u im: nhy cao, s chnh xc Thi gian xc nh nhanh chng Tin hnh n gin. + o nht: vi sn phm c nht thp hn c cht. - Th d: Thy phn acid nucleic Thy phn tinh bt Thy phn protein. + Phn cc k: sn phm v c cht lm quay mt phng nh sng phn cc.
NHM - 17 42
- Th d: thy phn sucrose Glucose: +52,5o Fructose: -92,4o Sucrose: +65o + o p sut: sn phm l cht kh hay lm mt cht kh. - Th d: H2 O2 H2 O + O 2 Lipit + H2O glycerol + acid bo + Chun : sn phm/c cht c th bt mu vi thuc th. * Phng php sc k: giy, bn mng, HPLC, GC, * o cng mu
NHM - 17
43
PHN 3
NG DNG CA ENZYME AMYLASE TRONG CNG NGHIP

3.1 ng dng ca enzyme amylase trong cng ngh dt Trong cng ngh dt, ngi ta thng tin hnh x l vi bng nhiu loi bt khc nhau nh: bt khoai ty, bt go v mt s cht khc nh: gelatin, guar gum, poly-vinyl alcohol, methacrylate, trong tinh bt c s dng nhiu nht. Sau khi c h ha lm mn vi, ngi ta tin hnh qu trnh r h vi. Phng php lm sch h tinh bt c s dng l enzyme amylase. Trc y, ngi ta s dng enzyme -amylase ca malt hay pancreatic amylase ph hy nhanh lng tinh bt tha, u tin ta a nhit n nhit si sau lm gim nhit xung 500C hay 600C v cho enzyme amylase vo. Ngy nay, ngi ta s dng -amylase ca vi khun thay cho amylase malt v pancreatin. Enzyme -amylase ca vi khun chu nhit , chng hot ng mnh nhit 85 900C. Mt s enzyme amylase ca Bacillus subtilic c kh nng hot ng 105 1150C. Bng 3.1: Bng thng k mt s enzyme amylase c s dng trong cng ngh Dt Loi enzyme Khong pH hot ng -amylase ca malt Amylase pancreatin -amylase nm si -amylase vi khun -amylase vi khun chu nhit
NHM - 17
Nhit ti u 55-65 45-50 55-65 75-85 90-105
Cht hot ha, cht lm n nh Ca2+ NaCl, Ca2+ Ca2+ NaCl, Ca2+ NaCl, Ca2+
44
4,5-5,5 6,7-7,5 4,5-5,5 5,5-7,5 5,0-7,0
Tuy nhin cn ty thuc vo iu kin kinh t, sn xut, ngun amylase m ngi ta tin hnh chn la enzyme r h vi cho ph hp. QUY TRNH R H VI:
1. Giai on lm sch vi c thc hin trong nc un si, vi hp th n 90 100% nc, vi c ra sch cc cht bn. Cc ht tinh bt trng n, gip cho qu trnh phn gii tinh bt nhanh hn. 2. Giai on ngm Ngi ta b sung mt s cht iu chnh pH v lm n nh iu kin mi trng cho amylase hot ng. i vi -amylase, ngi ta thng cho vo 300g NaCl, 50g CaCl 2, khong 50g nhng cht khng phi l anionic trong 100 lt nc nhit 65 700C. Sau ngi ta cho vo 100 200g amylase vi hot tnh 3000SKB/g. i vi enzyme -amylase chu nhit, ngi ta cho vo 100 lt nhit 70 8000C khong 39g CaCl2 v 400g NaCl, pH iu chnh khong 6 8. 3. Giai on phn gii tinh bt Thng s dng dung dch Iodine kim tra qu trnh phn gii. Thi gian: 2 pht 16 gi. Ty thuc vo hot tnh enzyme. 4. Giai on ra dung dch s dng 5 10g NaOH/1 lt nc ra, lm sch va. Nhit : 95 1000C. -amylase
NHM - 17
45
QU TRNH H VI THEO PHNG PHP LIN TC VI
ENZYME AMYLASE CHUN Thnh phn dung dch ngm: 300g NaCl, 200g CaCl2, 50g surfactant, 250-500g enzyme amylase. Trong nc ra vi cho: 20 30g NaOH. 1000C : 15
Nguyn liu ph go v bp 760C :15
Malt ( +La mch) 520C : 70
670C : 60
Phng php r h vi lin tc i vi cc loi la t tm, ngi ta p dng cc iu kin sau: Bicarbonate Natri 5,0g/I, surfactant khng phi anionic 0,5-1,0g/l, ch phm protease kim t B.licheni formic 1Au/1,0-2,5g/lit. Ngoi ra, cn ty thuc vo iu kin nh my, mc ch v yu cu ca tng loi sn phm m thnh phn cc cht cho vo s thay i cho ph hp. 3.2 ng dng ca enzyme amylase trong sn xut cht ty ra Cht ty ra bao gm nhng cht kim, sodium silicate, sodium bicarbonate, sodium tripolyphosphate. Mc ch: Loi b cc cht v c, hu c bm vo qun o nh: protein, lipid, carbohydrate v nhng cht mu.
NHM - 17 46
Enzyme -amylase ca vi khun l mt trong nhng enzyme thng c ng dng trong cng nghip sn xut cht ty ra do enzyme ny c kh nng chu nhit cao (khong 900C), pH cao (pH = 9). Tuy nhin do enzyme ny khng bn pH kim v nhit cao trong thi gian lu nn ngi ta thng bao chng li trc khi phi trn vi cc thnh phn khc ca cht ty ra bo qun c lu v m bo kh nng hot ng ca chng. Enzyme -amylase lm tng kh nng phn gii cc vt bn do cacbohydrate trong qun o.
3.3 ng dng ca enzyme amylase trong cng nghip thc phm 3.3.1 ng dng ca Enzyme Amylase trong sn xut m chnh: Nguyn liu s dng ch yu: tinh bt sn, r ng ma,... Cc chng vi sinh vt: corymebacterium hydrocacboclastus,
bacillus,Brevibacterium... Enzym amylase c s dng trong cng on thu phn tinh bt.
NHM - 17 47
Mc ch ca cng on ny l to iu kin thc hin cc phn ng thu phn tinh bt thnh ng ln men c ch yu l ng glucoza. Ngi ta c th dng -amila , -amila ca cc ht ny mm hay ca nm mc thu phn tinh bt thnh ng. amylase Tinh bt Dextrin + maltose + glucose Thy phn -1,4 ca amylose
amylase Tinh bt (Glucogen) Maltose + Dextrin
Phng php ny c u im l khng dng n ho cht hay thit b chu axt, chu p lc khng c hi cho ngi v thit b. Nhc im ng ho khng trit tinh bt, m cn dng trung gian nh dextrin lm cho vi khun ln men m chnh khng c kh nng s dng. Thi gian ng ho tng i di. Lng ng sau khi ng ho thp, do phi s dng thit b to, cng knh. 3.3.2 ng dng ca Enzyme Amylase trong sn xut bia: Nguyn liu s dng: Ng cc, hoa Houblon, nm men, cht ph gia. Cc chng vi sinh vt:
Saccharomyces cerevisizae ( ln men ni) Saccharomyces cerevisidae (ln men chm).

NHM - 17 48
Trong cng ngh sn xut bia, ngi ta thng s dng enzym amylase
c trong mm i mch. Ngoi ra, cn s dng cc enzym khc c trong mm i mch thu phn v chuyn ha cc cht khng tan sang trng thi tan nh chuyn protein, cellulose sang amino acid v glucose. Cc qu trnh cng ngh ny c thc hin nhiu nc trn th gii. So vi cc loi enzym amylase t cc ngun khc, amylase t thc i mch ny mm c s dng vi s lng nhiu nht hin nay. Qu trnh ht i mch ny mm l qu trnh sinh tng hp enzym amylase v nhiu enzym khc. Nh s tng hp ra nhng enzym ny, ht tin hnh qu trnh t thu phn tinh bt, protein v cc hp cht khc cung cp nguyn liu v nng lng cho ht ny mm. Enzyme amylaze tc ng vo giai on ng ha: Bin i tinh bt qua giai on dch ha thnh Dextrin v Maltoza. Amylaza tc ng to Dextrin ha, - amylaza tc ng vo mantoza. amylase Tinh bt Dextrin + maltose + glucose Thy phn -1,4 ca amylose
amylase Tinh bt Maltose + Dextrin
Trong giai on ln men tip theo, cc cht ng, dextrin c phn t thp s c chuyn ha thnh ru etylic, CO2 v mt s sn phm khc to thnh bia theo yu cu k thut v cht lng sn phm.
NHM - 17
49
3.3.3 ng dng ca Enzyme Amylase trong CNSX cn Nguyn liu ch yu m cc nh my ru nc ta thng dng l sn, sau l ng v mt phn go hoc tm. i vi sn xut ru th thnh phn quan trng nht l gluxit ln men c gm tinh bt v mt s ng. Trong a s gluxit ni chung th t l gia H v O u tng t nh trong nc. V d ramnoza - C6H12O5 Vai tr ca -amylase v -amylase Trong ni dung phn ny ch trnh by sn xut cn t hydro carbon, y l qu trnh sn xut c ng dng enzyme. Trc y, ngi ta dng enzyme amylase ca malt, nhng ngy nay thay th bng enzyme amylase ca nm si. Trong sn xut cn t nguyn liu cha tinh bt, enzyme amylase c s dng cc giai on ng ha: Chuyn ha tinh bt bng -amylase to thnh dextrin. Chuyn ha dextrin bng -amylase hay amyloglucosidase to ra
ng c kh nng ln men.
Nguyn liu cha tinh bt
Nu chn nhit cao Amyloglucosidase hot ng 550C Amylase hot ng 550C
Lm ngui
ng ha
S qu trnh chuyn ha tinh bt Qu trnh ng ha ng vai tr quyt nh n kh nng ln men v hiu sut cn thu c. Cc ch phm enzyme c s dng trong sn xut cn bao gm nhng ch phm th c thu nhn t phng php nui cy b mt, nui chm hoc
NHM - 17 50
ch phm m c, tinh khit. Ch yu l s dng ch phm dng th v tnh cht kinh t. V s dng ch phm dng th, do trnh cc phn ng khng mong mun trong qu trnh ng ha, trc khi ng ha phi xc inh hot tnh enzyme ca tng loi enzyme t iu chnh hot ng ca chng. Trong giai on dch ha enzyme -amylase tham gia thc hin cc phn ng sinh ha cn thit v quyt nh n hiu sut sn xut cn. Trong khi cng trong giai on ny enzyme -amylase li to ra cc sn phm khng mong mun, lm cn tr qu trnh ln men. Trong giai on ng ha dch ng nm men, enzyme -amylase tham gia thc hin cc phn ng sinh ha cn thit v quyt nh ti hiu sut sn xut ca cn. Cn enzyme -amylase tham gia nhng bin i c bn c cht tng cng qu trnh chuyn ha c bn. Khi thy phn tinh bt, enzym -amylase tc ng vo lin kt -1,4 glucoside v sn phm to ra l mantose v dextrin m mch ca chng gn bng C6. Cc dextrin sau s phn hy ch yu theo: C6 C7 C8 C5 + C1. C6 + C1 hay C5 + C2. C6 + C2 hay C5 + C3.
Enzym -amylase ca vi khun thy phn tinh bt to ra lng glucose v maltose theo 1: 5,45. Enzym -amylase ca nm si thy phn tinh bt to ra lng glucose v maltose theo 1: 3,79. Enzym -amylase ca vi khun v nm si u khng c kh nng phn gii -1,6 glucoside ca c cht. Nh vy, vai tr c bn ca -amylase trong sn xut ru, cn l lm cho dch ha nhanh giai on nu v c giai on u ca s ng ha, dextrin ha v tch t ng.
NHM - 17
51
Vai tr ca glucoamylase Glucoamylase thy phn lin kt -1,4 glucoside trong cc polysaccharide, chng lin tip phn ct cc gc glucose khng kh trong mch polysaccharide. Ngoi ra, glucoamylase cn c kh nng phn ct lin kt -1,6 glucoside. Sn phm cui cng trong hot ng ca glucoamylase l glucose. Bng 3.2: Hot phn gii ca vi sinh vt trong sn xut cn etylic Hot dv/g cht kh Chng vi sinh vt Asp.oryzae KC Asp.oryzae U476 Asp.niger S4-10-111 Asp.awamori 22 Hot amylaza Hot D Hot Gluco Hot Pr 70.0 85.0 0.65 14.5 450.0 665.0 437.0 850.0 30.0 80.0 70.0 30.0 50.0 40.0 0.5 0
3.3.4 ng dng ca Enzyme Amylase trong sn xut siro Cc nguyn liu cha tinh bt khng ch l ngun thc phm quan trng m cn l ngun nguyn liu to ra nhiu loi thc phm khc nhau. ng dng trong sn xut siro v cc sn phm cha ng phi bp c x l bng SO2 v vi khun lactic ht tinh bt mm, ra khi khi bt bng ly tm. Enzyme amylase ch c s dng sau khi tinh bt ha vo nc. Theo cng ngh trn, dung dch bp c iu chnh pH = 6 v c h
ha nhit cao v c thc hin theo phng php lin tc. giai on gia nhit ny, ngi ta cho mt lng nh - amylase dch tinh bt c nht thp, trnh hin tng chy kht.
NHM - 17
52
Tip theo ngi ta cho lng enzyme cn li vo. y l giai on ng ha rt mnh t c gi tr DE (dextrose equivalents) 15 20. Trong cng ngh ng ha lin tc, thi gian t c DE 15 20 phi mt 2 gi.
Da, glutin va c thah pha kha u ca n n c
Pha ba(ngo SO2 n p ),
Tah (e) c p
a-amylase
D ho la 1 ch a n 0 105-150 C pH: 5,5-6,5
a-amylase
DE 2-5
D ho la 2 ch a n 0 90-95 C
DE 15-20
Nit, cation, anion Magiesium,chata ma t y u isomer ho a 0 55-60 C PH: 7-8 Trao oion i than hoa tnh t bo hi c Chabe, prote t o in Amyloglucosidase g ho n a 55-650 C pH: 4-4,5
Lo c
Tax oligosaccharide i ly
La gia m u fructose
Trao oion i than hoa tnh t
Bo hi c
Sa pha n m
Fructose 55%
Qu trnh chuyn ha tinh bt thnh siro fructose Ngoi tinh bt bp, ngi ta cn s dng tinh bt khoai ty, tinh bt m, tinh bt sn (khoai m). Ty theo ngun nguyn liu tinh bt m ngi ta p dng k thut khc nhau cho ph hp. Enzyme c s dng trong cng ngh sn xut siro fructose phi l nhng enzyme chu nhit. Nhng enzyme ny thng c thu nhn t vi khun Bacillus licheniformis, Bacillus stearothermophilus. Ngoi ra, ngi ta cn s dng amylase t nhiu vi sinh vt khc nhau. c im quan trng nht ca enzyme amylase l chng hot ng pH gn trung tnh.
NHM - 17
53
c im ny gip chng ta thc hin cc qu trnh ng ha rt d dng. c im k thut ca tng cng on sn xut siro fructose c trnh by trong bng sau: Bng 3.3: iu kin chuyn tinh bt thnh ng trong cng ngh sn xut siro fructose
Cc cng on
Hm lng
Ch s DE
Nhit (0C)
pH
Hm lng Ca2+, mg/l Mg2+, mg/l
1. Chuyn ha tinh bt dch ha ln 1 dch ha ln 2
-amylase (0.01-0.02) -amylase (0.1-0.2)
2-5
105-115
5.5-6.5 50-100
15-20
90-95
5.5-6.5 50-100
2. ng ha
Amyluco sidase (0.1)
98-99
55-65
4-4.5
50-100
3. Isomer ha
Glucoseisomerase
41% frutose 55-95% frutose
55-60
7-8
25-100
4. Lm giu frutose
Trong , giai on dch ha v ng ha ng vai tr rt quan trng. Cc giai on ny thng quyt nh cc giai on sau v quyt nh n cht lng sn phm. Trong cng ngh sn xut siro fructose, nhiu nc trn th gii c s dng enzyme c nh amyloglycosidase v pullulanase.
NHM - 17 54
Phng php ny thng thc hin nhit 600C v hm lng cht kh trong dch khong 30 35%. Cc c tnh c bn ca cng ngh sn xut siro fructose bng enzyme c nh c trnh by trong bng sau Bng 3.4: Hiu sut chuyn ha tinh bt bng enzyme c nh trong sn xut siro fructose Enzyme c nh Glucose (%) Maltose(%) Maltosetri ose (%) Maltotetraose (%)
-amylase -amylase-pullunase -amylase-pullanase-amylase
51 60
14 8 18
34 32 11
69
Ngoi cng ngh sn xut siro fructose t nguyn liu ban u l bt ng ra, ngi ta cn tin hnh qu trnh isomer ha glucose ha glucose sn xut siro fructose. Qu trnh isomer c tin hnh nh sau:
H C CH O H HO CH CH H CH2 O O O H CH2 CH O H
glucoisomerase
HO
CH CH O O H CH H CH2 O
glucose
D - fructose
NHM - 17
55
3.3.5 ng dng ca Enzyme Amylase trong sn xut bnh m: Cc loi ht ng cc nh la m, la nc, bp, khoai m l ngun nguyn liu chnh lm bnh m. Trong ch c bt t la m mi l nguyn liu sn xut bnh m. Bnh m c coi l ngun lng thc chnh ca cc nc chu M v cc nc chu u, trong vi thp nin gn y bnh m c coi nh l mt khu phn lng thc ca Vit Nam. Trong sn xut bnh m, ngi ta s dng enzyme nhm gii quyt mt s vn sau: Lm tng th tch bnh Lm mu sc ca bnh p hn Lm tng mi thm cho bnh.
Trong sn xut bnh m, ngi ta s dng c hai loi enzyme -amylase v _amylase, cc loi enzyme ny tham gia thy phn tinh bt to thnh ng. Nh , nm men Saccharomyces cerevisiae s d dng chuyn ha chng thnh cn, CO2, lm tng th tch ca bnh v to ra mu sc, hng v tt cho bnh. Ngun amylase thng s dng l malt. Tuy nhin trong nhng nm gn y, malt dn dn c thay th t nm si. Cc ph phm enzyme t nm si khng ch cha amylase m cn cha protease v mt s enzyme khc rt c li cho qu trnh ln men. Ch phm enzyme ny ch yu thu nhn t Aspergillus oryzae. Trong qu trnh s dng enzyme amylase ngi ta cho sulfat amon vo bt nh mt cht m cho amylase hot ng. Ngi ta thng to cht m cho amylase bng cch trn lng enzyme amylase vo tinh bt theo t l 1 : 1. Hn hp nayfcos tc dng n nh hot tnh enzyme amylase khng thay i trong mt nm bo qun. Khi cho hn hp ny vo bt lm bnh th cht lng bnh s tng ln rt nhiu (tng th tch, xp, tng kh nng gi hnh bnh, tng mc n nh cu trc rut bnh v lm gim qu trnh lm kh ca bnh). Ngoi ra khi s dng ch phm enzyme s lm gim tiu hao nm men lng n 20%.
NHM - 17 56
Mt khc cht lng ca bnh m khng ch ph thuc vo lng CO2 to ra nhiu hay t, m ph thuc vo ng thi ca qu trnh to ra n. Nu lng CO2 to ra ch t lng ng c sn trong bt m th lng CO2 ny s t cc i gi u tin v gi th haicuar qu trnh ln men, sau lng kh s gim bnh m s khng m bo cht lng. Trong cng ngh sn xut bnh m, lng kh CO2 phi c to ra lin tc t gi u tin n gi cui cng ca qu trnh sn xut. c bit l qu trnh to CO2 phi n nh 10 15 pht u khi a bnh vo l nng. Khi trong bt c -amylase hot ng th to thnh CO2 trong qu trnh nho bt xy ra theo chiu hng tng v t cc i gi th t ca qu trnh ln men. Nh vy s to thnh lng kh lin tc l nh hot ng ca amylase a vo trong bt trong qu trnh nho bt. Cng ngh sn xut bnh m ca mt s nc c s dng ch phm enzyme nh: + Nga, ngi ta s dng ch phm c tn thng mi l Amilorizin
P810X. Ch phm ny c sn xut t nm si Aspergillus oryzae chng 476 1, ch phm enzyme ny cha c cc loaijamylase v protease. Ch phm ny thng dng c c tinh khit v c dng bt. + M cc ch phm enzyme thng dng dng ht, hng sn xut ch
phm enzyme dng cho bnh m ln nht M l hng Rom v Khaac. Enzyme c tn thng mi l GUMASE NR-150. + Anh ngi ta s dng cc ch phm Enzyme amylase ch yu t nm
si aspergillus oryzae bng phng php nui cy chm, cc enzyme protease v amylase thng c tch ring bit. Khi s dng h thng s dng chnh xc tng loi enzyme. + Nht, tuy bnh m khng phi l lng thc chnh nhng nhng c s
sn xut bnh m thng ng dng enzyme rt c hiu qu. Cc ch phm enzyme s dng l hn hp amylase v protease.
NHM - 17
57
3.3.6 ng dng ca Enzyme Amylase trong sn xut bnh ko Mc ch ca vic s dng enzyme vo sn xut cc loi bnh quy l lm tng mi v v ca bnh, khi ch bin bt thnh cc loi bnh quy cc enzyme protease v amylase ca bt hot lm tng hm lng cc amino acid t do v lm tng lng ng kh. ng kh v cc amino acid t do c trong khi bt s cng tham gia vo cc phn ng oxy ha kh v kt qu to cho bnh m c mi, v, mu hp dn. Tuy nhin nu ch tn dng lng enzyme c sn trong tinh bt th phn ng trn xy ra khng mnh, nht l khi s dng loi bt xu sn xut bnh. Do , ngi ta thng b sung thm Enzyme protease v amylase vo ch bin bt trong qu trnh sn xut bnh quy. Khi , lng ng kh v lng amino acid t do s tng ln, phn ng oxi ha kh cng c tng cng. 3.3.7 ng dng ca Enzyme Amylase trong sn xut glucoze mt Chng ta bit t tinh bt c th thu c cc phm vt ng khc nhau khi thy phn tinh bt bng acid cng nh bng enzyme amylase s thu c mt. Mt glucoza hay mt maltoza thng c dng trong sn xut bnh ko v trong sn xut cc sn phm n king cho tr em v ngi bnh. Hin nay nhiu nc nh: Nht, M, sn xut glucoza i t tinh bt bng enzyme glucoamylase, nhng trong cc ch phm Enzyme glucoamylase lun lun cha c glucozyltransferaza do s lm gim tnh cht ca glucoza, lm cho glucoza kh kt tinh v nh hng xu n cht lng thc phm. V vy, tt hn ht l nn chn nhng chng nm mc khng to ra glucozyltransferaza ( ta c th hp th n bng t st chua hot bng cch kh hot tnh ca n bng acid hoc kim). Glucoamylase bn trong mt khong pH rng, nhng nm mc ch nhiu glucoamilase A. awarnor, Rhizopus, c bit l Rhizopus Nht sn xut ch phm glucoamylase t canh trng b mt ca nm mc ny.
NHM - 17
58
3.4 ng dng enzyme Amylase trong y hc & dc phm 3.4.1 Thuc, ha cht cha hot tnh enzyme amylase phc v trong nui trng thy sn Enzyme amylase thng c b sung trong thnh phn cc hp cht ha hc nhm ci to ao h, kch thch tng trng v pht trin mnh ca ng vt thy sn cc giai on mong mun. i tng: c n thc vt v c n ng vt. C ch
i vi c n thc vt: Enzyme amylase phn gii tinh bt c trong thc n ca c: c, khoai lang, khoai m, i vi c n tht ng vt: Enzyme amylase phn gii glycogen hay glucid t bo ng vt. 3.4.2 Vai tr ca enzyme amylase trong tiu ha v bin dng carbohydrate Trong thnh phn ca carbohydrat, tinh bt, glycogen c xem nh l ngun nguyn liu chnh cung cp nng lng cho con ngi, ng vt, thc vt v vi sinh vt. S tiu ha tinh bt: H thng enzyme thy phn tinh bt nh sau: amylase Tinh bt Thy phn -1,4 ca amylose Dextrin + maltose + glucose
-1,6 glucosidase Dextrin Maltose + glucose Thy phn -1,6 ca amylospectin

NHM - 17 59
glucosidase (maltase) Maltose 2 glucose
glucosidase (lactase) Lactose Glucose + galactose
fructofuranisidase (sucrase) Suctose Glucose + fructose
Enzyme amylase phn gii carborhydrat c trong thc phm nhm cung cp nng lng cho hot ng sng ca c th. Amylase thy phn tinh bt, lm cc lin kt glucoside b ct t, thng qua 2 giai on: dch ha to sn phm trung gian l dextrin v giai on ng ha tip theo to sn phm l maltose v glucose. Amylase phn gii glycogen, ct t lin kt -1,4- glycoside v - 1,6 glycoside. Gii phng nng lng, y l ngun nng lng d tr ca c th. i vi ngi v ng vt:
Amylase c trong dch tiu ha tuyn ty. i vi thc vt:
Amylase c ch yu vng anoron trong phi ht. i vi vi sinh vt:
Amylase ch yu c s dng trong qu trnh thy phn, trao i cht thng qua hai qu trnh: ng ha v d ha ngoi t bo. Enzyme tiu ha carbohydrat ca ng vt thy sn Wilder (1994) cho bit c nc ngt v c vng nc m c kh nng tiu ha tinh bt tt hn c bin v c vng nc lnh. S khc nhau ny c lin quan n hot
NHM - 17 60
lc ca enzyme amylase ca loi. Hot lc ca enzym tiu ha carbohydrat ca c chp cao hn 80 ln so vi c ui vng v 10-30 ln so vi c hi. Nhm c n thc vt c enzym tiu ha carbohydrat mnh hn so vi c n ng vt. Kh nng tiu ha carbohydrate ph thuc rt nhiu vo trng lng phn t v cu to cc ni ca carbohydrate. Cc loi ng n d tiu ha hn cc loi ng a v nhm khng ng nh tinh bt, dextrin. ng n c th hp thu trc tip qua thnh rut trong khi cc nhm khc phi qua qu trnh tiu ha, c bit l qu trnh ny xy ra chm VTS. Khi thy phn cc loi tinh bt dn n lm gia tng tiu ha ca tinh bt, v vy, vic nu chn hay h tinh bt u gip ci thin tiu ha thc n tinh bt. 3.4.3 ng dng enzyme amylase trong chun on vim ty cp tr em Ty l mt c quan sau phc mc, nm sau d dy st thnh sau ca bng. Chc nng : Tuyn ty sn xut cc men tiu ha c kh nng tiu ha gn nh tt c cc thnh phn thc n. Ty c bao bc bi bao ty. Bao ty cng c tc dng phn chia ty thnh cc tiu thy. Nhu m ca ty c cu to bi cc t bo ty ngoi tit. Cc t bo ny cha ng rt nhiu cc ht nh cha enzyme tiu ha di dng tin cht (ch yu l trypsinogen, chymotrysinogen lipase ty v amylase). Vim ty cp l mt trong nhng bnh l ca tuyn ty: Da vo cc c tnh biu hin ca enzyme amylase ngi ta tin hnh nghin cu phng php chun on bnh vim tuyn ty. i tng: enzyme s-amylase v p- amylase. Theo nghin cu ta nhn thy: Vi im ct: -AM =400 u\ l; P-AM=150 u\ l th gi tr ca enzyme S-amylase, P- amylase, lipre mu trong chun on vim ty cp tr em ng tin cy v t gi tr cao nht. Khi chn la im ct thch hp v phi hp c 2 enzyme s-amylase + lipre hoc p-amylase + lipre trong chun on vim ty cp tr em th gi tr chun on chnh xc cao nht.
NHM - 17 61
KT LUN
Vi sinh vt c tc sinh trng v tng hp nhanh, do cho php thu c khi lng sn phm ln trong thi gian ngn. Enzyme vi sinh vt c hot tnh cao, p ng c kh nng phn gii, tng hp nhanh cc cht cn cho sinh trng ca t bo vi sinh vt. S lng, tnh a dng v hot tnh enzyme c vi sinh vt m thc vt khng th no so snh c. v vy m vi sinh vt c th s dng c rt nhiu dng dinh dng kh ng ha v r tin nui cy sn xut. Quy trnh cng ngh sn xut enzyme d thc hin, hiu sut thu hi cao, do enzyme d ha tan trong mi trng v t l enzyme sinh ra l kh ln so vi kch thc v khi lng t bo. Do vy vi sinh vt l i tng chnh sn xut enzyme.
NHM - 17
62
TI LIU THAM KHO

1. Cng ngh Enzyme Nguyn c Lng . Nh xut bn i hc Quc Gia Tp.HCM 2008 2. Cng ngh vi sinh-Tp 2: Vi sinh vt Cng Nghip Nguyn c Lng. Nh xut bn i Hc Quc Gia Tp.HCM 2008 3. Gio trnh Cng Ngh Enzyme Nguyn Tin Thng, Trng i Hc K Thut Cng Ngh Tp.Hcm, 2008. 4. www.congnghesinhhoc24h.com
NHM - 17
63
MC LC
LI M U ................................................................................................................ 2 PHN 1 : TNG QUAN ................................................................................................ 3 1. GII THIU SN PHM ......................................................................................... 3 1.1 nh ngha ..................................................................................................... 3 1.1.1 Enzyme ............................................................................................ 3 1.1.2 Enzyme Amylase ............................................................................. 3 1.2 Ngun gc ..................................................................................................... 4 1.3 Phn loi ........................................................................................................ 5 1.3.1 Enzyme -Amylase (-1,4-glucanohydrolase) (EC 3.2.1.1) ........... 6
1.3.2 Enzyme -Amylase (-1,4-glucan-maltohydrolase) (EC 3.2.1.2) ......... 11
1.3.3 Enzyme -Amylase (glucoamylase) (EC 3.2.1.3) ........................ 13 1.3.4. Oligo 1,6-glucosidase (dextrinase ti hn) (EC 3.2.1.10) ............ 15 1.3.5. Enzyme pullulanase (-dextrin 6-glucosidase) (EC 3.2.1.41) ..... 15 1.3.6. -glucosidase hay maltase (-D,glucoside-glucohydrolase)(EC 3.2.1.20) ............................................................................................................. 15 2. NGUYN LIU SN XUT .................................................................................. 16 2.1 T thc vt................................................................................................... 17 2.2 T ng vt.................................................................................................. 18 2.3 T Mi trng nui cy vi sinh vt ............................................................. 18 2.3.1 Nguyn liu to mi trng nui cy ........................................... 18 2.3.2 Mi trng lng .........................................................................................................19 2.3.3 Mi trng bn rn ....................................................................... 19 3. GING VI SINH VT ............................................................................................ 21
NHM - 17 64
3.1 Vi sinh vt trong sn xut enzyme Amylase ............................................... 21

3.2 c im sinh l, dinh dng v c ch tng hp sn phm trao i cht .......... 21
3.2.1 Nm mc Aspergillus Oryzae ....................................................... 21 3.2.2 Baciluus Subtilis ........................................................................... 23 Phn 2: CNG NGH SN XUT ............................................................................ 28 1. S QUY TRNH CNG NGH SN XUT ENZYME THEO PHNG PHP NUI CY VI SINH VT ............................................................................... 28 Quy trnh sn xut mc ging ........................................................................... 28 1.1. Theo Phng Php Nui Cy B Mt ........................................................ 29 1.1.1. Thu nhn sn phm ............................................................................ 31 1.1.2 u v nhc im ............................................................................... 31 1.2. Theo Phng Php Nui Cy B Su ........................................................ 33 1.2.1 Thu nhn sn phm ........................................................................... 34 1.2.2 u v nhc im ............................................................................. 34 2. TINH SCH ENZYME ........................................................................................... 35 PHN 3: NG DNG CA ENZYME AMYLASE TRONG CNG NGHIP ...... 44 3.1 ng dng ca enzyme amylase trong cng ngh dt .................................. 44 3.2 ng dng ca enzyme amylase trong sn xut cht ty ra ...................... 46 3.3 ng dng ca enzyme amylase trong cng nghip thc phm ................. 47 3.3.1 ng dng ca Enzyme Amylase trong sn xut m chnh ........... 47 3.3.2 ng dng ca Enzyme Amylase trong sn xut bia .................... 48 3.3.3 ng dng ca Enzyme Amylase trong CNSX cn ...................... 50 3.3.4 ng dng ca Enzyme Amylase trong sn xut siro ................... 52 3.3.5 ng dng ca Enzyme Amylase trong sn xut bnh m ............ 56 3.3.6 ng dng ca Enzyme Amylase trong sn xut bnh ko ........... 58
NHM - 17 65
3.3.7 ng dng ca Enzyme Amylase trong sn xut glucoze mt ... 58 3.4 ng dng enzyme Amylase trong y hc & dc phm .............................. 59 3.4.1 Thuc, ha cht cha hot tnh enzyme amylase phc v trong nui trng thy sn ............................................................................................ 59 3.4.2 Vai tr ca enzyme amylase trong tiu ha v bin dng carbohydrate ...................................................................................................... 59 3.4.3 ng dng enzyme amylase trong chun on vim ty cp tr em ...................................................................................................................... 61 KT LUN .................................................................................................................. 62 TI LIU THAM KHO ............................................................................................ 63
NHM - 17
66

117677501 thu nhận enzyme amylase từ Aspergillus Oryzae

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117677501 thu nhận enzyme amylase từ Aspergillus Oryzae

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B CNG THNG

QUY TRNH SN XUT AMYLASE T VI SINH VT

TP.HCM, Thng 12/2012

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Kh trc tip -dextrin 6-glucanohydrolase (pullulanase)

Kh gin tip oligo-1,6-glucosidase (transglucosylase)

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

dextrin phn t lng thp

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

3,5 4,5 6,5

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

5,3 6,4 7,0 7,0 6,3 6,0 7,0 5,0 6,0

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Malt kh sn xut bia

Cc bc quy trnh sn xut malt kh

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Khi lung phn t (Dalton) 48.000 22.500 96.000 90.000 62.000

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Ty Malt Aspergilus oryzae

6,0 7,0 4,5 9,5 (5,5) 5,0

45.000 52.000 51.000 70 50 60

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Hnh 1.4: Bacillus subtilis

Hnh 1.5: Vi khun Bacillus subtilis trong phn chia t bo.

Bi tiu lun CNSH trong CNTP

Quy trnh sn xut amylase t vi sinh vt

Bi tiu lun CNSH trong CNTP