BC CN Enz Hoan Chinh

tai: THU NHN ENZYME CELLULASE TRONG Aspergillus niger VA NG DUNG TRONG SAN XUT BIOETHANOL
GVHD: Ts. Nguyn Hoai Hng
MC LC MC LC________________________________________________________1 Chng 1: GII THIU______________________________________________3 Chng 2: TNG QUAN____________________________________________4 2.1 Enzyme cellulase____________________________________________4 2.2 Aspergillus Niger ___________________________________________4 2.3 Gii thiu c cht____________________________________________5 2.4 C ch xc tc______________________________________________6 2.5 Tm hoat ng______________________________________________8 2.6 Cac thng s cn xac inh_____________________________________9 Chng 3: QUY TRINH LN MEN, THU NHN VA TINH SACH ENZYME CELLULASE_____________________________________________________10 3.1 S lc quy trinh ln men____________________________________10 3.1.1 Mi trng nui cy___________________________________11 3.1.2 Ging______________________________________________12 3.1.3 Ln men____________________________________________12 3.1.4 Cc yu t nh hng n sinh tng hp enzyme cellulase_____12 3.2 Quy trinh thu nhn va tinh sach________________________________13 3.2.1 Thu nhn enzyme cellulase______________________________13 3.2.2 Tinh sach enzyme cellulase______________________________14 3.3 Phng phap xac inh hoat tinh________________________________15
Lp 08DSH2
Khoa: MT-CNSH
Chng 4: NG DUNG CUA ENZYME CELLULASE TRONG SAN XUT NHIN LIU SINH HOC ETHANOL________________________________________17 4.1 Gii thiu v nhin liu sinh hoc ethanol_________________________17 4.2 S san xut ethanol va thuyt minh quy trinh san xut____________20 4.2.1 S quy trinh san xut________________________________20 4.2.2 Thuyt minh quy trinh__________________________________21 4.2.2.1 Tin x l_____________________________________21 4.2.2.2 Thuy phn_____________________________________22 4.2.2.3 Ln men_______________________________________23 4.2.2.4 Chng ct- kh nc_____________________________24 4.3 Cac thng s ky thut cua h enzyme cellulase____________________24 4.4 Phng php xc nh ng glucose sinh ra_____________________25 Chng 5: KT LUN_____________________________________________26 PH LC________________________________________________________27 1. Phng php xc nh hm lng enzyme cellulase bng phng php Bradford________________________________________________________27 2. Phng php xc nh hm lng ng kh bng phng php DNS____________________________________________________________28 3. Phng php tinh sch enzyme cellulase bng phng php sc k lc gel______________________________________________________________30 TAI LIU THAM KHAO____________________________________________32
Lp 08DSH2
Khoa: MT-CNSH
Chng 1: GII THIU Enzyme cellulase l mt trong nhng enzyme thng mi ha ln nht hin nay c s dng ch yu trong cng nghip dt nh lm mm si bng, ty hay gi mu, chng kt ta; dng tch chit trong thc phm hay cng nghip giy v bt giy. Th trng cellulase dng tin x l nguyn liu cellulose sn xut hng ha nh bioethanol v cc sn phm sinh hc c bn khc trn quy m ln tng t ngt. V d nh th trng cellulase tim nng c c on trn 400 triu USD mi nm, ch ring s dng thy phn bp v rm kh v trong tng lai s chim khong 33% tng gi tr cng nghip enzyme ca M. S tim nng ca th trng enzyme cellulase c vai tr quan trng ca ni bt trong cng nghip nng lng sinh hc v cc sn phm c bn ca sinh hc, l mt ng lc to ln pht trin cng ngh cellulase thy phn vch t bo thc vt. pht trin cng nghip cellulase i hi cn phi c nhng c im nh hiu sut xc tc phn ng c cht cellulose cao trong mi trng khng ha tan, tng s n nh ca mi trng nhit cao, khi pH thay i, v khng b c ch nng sn phm cao. Ngoi ra h enzyme cellulase trong Aspergillus niger cng c nghin cu t lu nhm ng dng sn xut cc ch phm sinh hc nhm phc v nhu cu ch bin thc phm, trong nng nghip s dng d x l t nhm tng mu m.
Lp 08DSH2
Khoa: MT-CNSH
Chng 2: TNG QUAN 2.1 Enzyme cellulase Cellulase l enzyme thuc nhom thy phn cellulose thnh glucose. Enzyme cellulase c thu nhn t cc ngun khc nhau:
Thc vt: trong ht ng cc ny mm nh i mch, yn mch, la m mch
en ng vt: ch yu l do cc vi sinh vt sng cng sinh trong rut ca ng vt nhai li hay mi Vi sinh vt: cc loi x khun, vi khun, nm si, nm men Trong thc t ngi ta thng thu nhn enzyme cellulase t vi sinh vt. Cc chng vi sinh vt thng s dng:
Nm mc: Aspergillus niger, Aspergillus oryzae, Aspergillus candidus,
Trichodesma reesei
X khun: Actinomyces griseus, Streptomyces reticuli Vi khun: Acetobacter xylinum, Bacillus Subtilis, Bacillus pumilis
Trong nm mc v vi khun l 2 ngun to cellulase ch yu, mc t hn l mm malt v ht cy. Trong k thut, cellulase c san xut chu yu t nm Trichodesma reesei, Aspergillus niger v vi khun Cellulomonas. 2.2 Aspergillus Niger
Lp 08DSH2
Khoa: MT-CNSH
Aspergillus niger thuc gii Eukaryota, lp Eurotiomycetes, b Eurotiales, ho Trichocomaceae, la mt dang nm ph bin nht trong chi nm mc Aspergillus. Nm Aspergillus cn gi l mc tng. Si nm c vch ngn, cung mang bo t bi phng ln ngn. Cc chui bo t bi t u phng mc ta khp mi hng. Bo t bi cua Aspergillus niger c mu en. Asp. niger la nguyn nhn gy bnh mc en mt s loai thc vt nhng lai it co kha nng gy bnh cho ngi, no chi co th gy hai cho ngi khi hit vao vi mt lng ln, t o co th gy ra cac bnh v phi va h hp, no cung co th gy nhim trung tai. No c nui cy trong nhiu nganh cng nghip nh san xut acid citric, acid gluconic, hoc thu cac ch phm enzyme amylase, cellulase, -galactosidase. 2.3 Gii thiu c cht Enzyme cellulase c kh nng s dng nhiu loi c cht. Ngi ta phn ra lm 2 loi ch yu sau:
C cht t nhin: gm cc ngun ph phm ca cc ngnh nng nghip nh b ma, b c ph, rm r, si bng, cng khoai m, dy u sau thu hochChng hn nh cellulose l homopolymer mch thng, c cu to khong vi ngn gc glucose ni vi nhau bng lin kt -1,4glucoside. Cellulose c cu to si, cu trc khng ng nht gm vng kt tinh v vng v nh hnh. Khong 40 si lin kt li vi nhau to thnh lin kt microfibrin. Nhiu si microfibrin tp hp li thnh si macrofibrin c ng knh mt ct khong 0,5nm.
C cht nhn to: gm cc c cht dng phn tch hot tnh enzyme cellulase nh CMC (carboxulmethyl cellulose), HEC (hydroxyethyl cellulose CMC (Carboxymethylcellulose) l mt dn xut ca cellulose. Cu trc ca CMC da trn lin kt -(1 4)-D-glucopyranose, c thnh
5
Lp 08DSH2
Khoa: MT-CNSH
phn tng t nhng ch c lin kt ca phn v nh v ngn hn so vi cellulose. Bang: Cac loai c cht tan va khng tan dung t nui cy va c inh hoat tinh enzyme. C cht tan Radio-labeled cellodextrin Dn xut cellodextrin -methyllumbelliferuloligosaccharides p-nitophenol-oligosaccharides C cht khng tan Cellulose kt tinh nh: Cellulose kt tinh Valonia cellulose Cellulose v nh hnh-PASC, RAC Cellulose nhum
Cacboxymethyl cellulose (CMC), Cellulose pht hunh quang hydroxyethyl cellulose (HEC) Dn xut sinh mu Trinotrophenylcarboxymethylcellulose (TNP-CMC) Fluram-cellulose
2.4 C ch xc tc Enzyme cellulase l mt phc h gm nhiu enzyme tham gia vo qu trnh chuyn ha cellulose.
Enzyme endogluconase (endo-1,4- -D-glucanase (EC 3.2.1.4)) hay Cx: tn
khc l CMCase- gi tn theo c cht nhn to s dng xc nh hot tnh ca enzyme. EG bt u thy phn cellulose trn cc v tr v nh hnh trn b
Lp 08DSH2
Khoa: MT-CNSH
mt si microfibrin cc v tr ngu nhin lm xut hin cc ui khc nhau trn phn cht v nh hnh, t cellulose to thnh cc cellobiose, tin cho xc tc ca cellobiosehydrolase trn cc u mt ca chui t do. iu ny cho bit nh hng ca enzyme trn phn carboxymethylcellulose v cellulose kt tinh. Enyme endoglucanase ca nm thng l mt monomer, c pH hot ng trong khong pH=4-5 v nhit t= 50 -70oC.
Enzyme exoglucanase (cellobiohydrolase (EC 3.2.1.91)) hay C1 : s ct v gii
phng cc cellobiose t u khng kh (u mt ca chui) to thnh trong bc u tin. Exocellulase chim khong 40-70% thnh phn h enzyme cellulase ca nm, xc tc bng cch to ra cc cu lin kt gia c cht vi enzyme. Vi Exocellulase pH hot ng t 4-5, nhng khong nhit rng hn so vi endocellulase khong 37-60oC.
Enzyme -glucanase (1,4- - D -glucosidase(EC 3.2.1.21) ): thy phn lin kt
-1,4-glucoside, ct v gii phng glucose t do. glucosidase thy phn cellobiose, mt s trng hp cellodextrin thnh glucose. Enzyme -glucanase cng b c ch cnh tranh bi glucose, enzyme ny c pH ti u da vo ni hot ng ca n v c nhit ti u khong 45-75oC.
C ch xc tc ca enzyme CMCase c cht CMC: Do c cht CMC ch c
vt cht v nh hnh trong phn t nn phc hp 3 enzyme ca cellulose ch c enzyme endoglucanase hot ng. Do CMC c dng ch yu xc nh hot tnh endoglucanase (Cx) nn c gi l CMCase.
Lp 08DSH2
Khoa: MT-CNSH
Hnh: S cc bc thy phn cellulose bi enzyme cellulase 2.5 Tm hoat ng Trung tm hot ng ca enzyme l phn ca phn t enzyme trc tip kt hp vi c cht, tham gia trc tip trong vic to thnh v chuyn ha phc cht trung gian gia enzyme v c cht to thnh sn phm phn ng. Trung tm hot ng bao gm nhiu nhm chc nng khc nhau ca amino acid, phn t nc lin kt v nhiu khi c c cofactor hu c (coenzyme) v v c. cc enzyme mt thnh phn, trung tm hot ng thng bao gm mt t hp cc nhm chc nng ca acid amin khng tham gia to thnh trc chnh ca si polypeptide.
Lp 08DSH2 Khoa: MT-CNSH 8
Gia c cht v trung tm hot ng to thnh nhiu tng tc yu, do c th d dng b ct t trong qu tr.nh phn ng gii phng enzyme v sn phm phn ng. Trung tm hot ng ca cc enzyme c cu trc bc 4 c th nm trn mt phn di n v hoc bao gm cc nhm chc nng thuc cc phn di n v khc nhau. i vi cellulase, tm hot ng l acid amin v khng co cofactor cung nh coenzyme. 2.6 Cac thng s cn xac inh Trong quy trinh ln men, thu nhn va tinh sach enzyme cellulase t Asp. niger, ta cn chu y cac thng s c tinh cua enzyme nh sau:
Khi lng phn t [Asp. niger]: 26000 Da Cht nn: carboxymethylcellulose + H2O Nhit ct gi: -15oC trong khoang thi gian dai hoc 4oC trong 3 ngay. Khoang nhit hoat ng: nhit qu cao hay qu thp thng d lm bin
tnh enzyme do enzyme c bn cht l protein do enzyme hot ng trong khong 20oC 60oC.
Nhit ti u: 45 oC Khoang pH : pH c vai tr quan trng trong vic to mi trng cho hot ng
enzyme, mi enzyme c mt vng hot ng tt nht ring cho mnh nn khi pH thay i s nh hng ti phn ly cc nhm chc cu trc nn trung tm hot ng ca enzym nh OH, SH.. hoat ng ca h enzyme trong nm mc Aspergillus niger thng c mi trng axit: 3 8.
pH ti u: 3.8 4.
Lp 08DSH2
Khoa: MT-CNSH
GVHD: Ts. Nguyn Hoai Hng Nng c cht: c cht va c vai tr l cht cm ng va c vai tr l cht c
ch s sinh tng hp enzyme khi mi trng dinh dng cn kit.

Cht c ch : Ag+
Chng 3: QUY TRINH LN MEN, THU NHN VA TINH SACH ENZYME CELLULASE
3.1 S lc quy trinh ln men Quy trinh ln men ging nm mc Asp. niger thuc dang ln men b mt va c thc hin theo s sau:
Mi trng nui cy Kh trng Khong v cht tng trng (nu c)
Ln men Nc + m Canh trng
Ging
Ly tm v ta bng cn lnh
Ch phm enzyme th
3.1.1 Mi trng nui cy Mi trng ban rn co cac thanh phn gm ba mia va cam mi theo ti l 4:6, m 40 50%. Sau khi trn u mi trng ta cho vao cac binh tam giac th tich 250ml, sau o hp kh trung 121oC/30 phut. Lam ngui mi trng va chun bi cy ging.
3.1.2 Ging
Lp 08DSH2
Khoa: MT-CNSH
11
Ging Asp. niger c cung cp bi phong thi nghim trng H Ky thut cng ngh TP.HCM. Cy dich huyn phu t bao vao mi trng nui cy vi mt 106 bao t/g mi trng. 3.1.3 Ln men Ln men ban rn b mt trong cac khay. Nui cy Asp. Niger trong 2 ngay, sau o bt u thu nhn enzyme th. 3.1.4 Cc yu t nh hng n sinh tng hp enzyme cellulase thong mi trng B ma, cm m tc nhn lm cho mi trng thong xp, to iu kin cho nm mc pht trin tt. Ban u nm mc s tn dng nhng cht d tiu trong mi trng nh cc loi ng nhng khi mi trng dinh dng b cn kit th nm mc phi tit ra nhng enzyme phn hy c cht khc thnh ng tip tc tn ti v pht trin. Tuy nhin, cng ty thuc vo nng ca c cht nhiu hay t m nm mc s tit ra lng enzyme khc nhau. m m l khong 40 -50 %, y l yu t quan trng trong mi trng. Nu m qu thp th xy ra hin tng loi nc ra khi t bo nm mc v lm cho t bo b cht. iu ny s lm hn ch s sinh trng v pht trin ca nm mc. Nu m qu cao th cng khng tt cho s sinh trng v pht trin.
Yu t khc
Lp 08DSH2
Khoa: MT-CNSH
12
S tng hp ca enzyme cn ph thuc vo kh nng tit ti a ca tng loi vi sinh vt c ngha l nu tng hm lng c cht vt qu mt gii hn nht nh th Hot tnh enzyme s gim do 2 nguyn nhn, th nht do c cht to p lc lm gim qu trnh sinh tng hp, th hai do c cht tng trong khi nng lc tit ti a khng i lm hot tnh enzyme gim. Khi cy nm mc vo mi trng, chng s khng tit ngay ra enzyme m s tit kim nng lng n mc ti a v s dng ngay nhng cht d s dng nht l cc ng n, khong cht c sn. Sau chng s tit ra enzyme ty vo thnh phn cc cht c trong mi trng. Nm mc pht trin c chia ra thnh 4 giai on nh sau: thch nghi, pht trin, cn bng v t vong. giai on thch nghi v pht trin, nm mc s tm cch thch nghi v tit ra loi enzyme no v s lng bao nhiu c th tn ti v pht trin. Theo thi gian th lng enzyme tit ra s tng dn v n mt mc n nh, sau s gim i khi mi trng Minh dng cn kit dn. 3.2 Quy trinh thu nhn va tinh sach 3.2.1 Thu nhn enzyme cellulase Mu: enzyme cellulase t nm mc Asp. Niger. Cn 5g ch phm ezyme th pha vi nc ty l 1:5 (5g enzyme vi 25 ml nc), dung ua thuy tinh khuy u trong 20 phut ri loc qua bng . em dich loc ly tm vi tc 3000 vong/phut trong vong 10 phut. Thu dich chit nm phia trn, b phn cn pha di.
- Ly 25ml dich chit tua bng cn lanh 96oC ty l 1:3 (25ml dich : 75 ml cn).
(Khng s dng cn nhit thng v c th lm bin tnh v gim hot lc ca enzyme).

- Lm lnh 30 pht. - Dch chit c ly tm ln 2 cung 3000 vong/phut trong vong 10 pht. Sau ly tm thu ta phia di (l protein) va tach bo lp dung dich phia trn. Pha dich enzyme thu c trong dung dich m acetate pH= 5=> nhm am bao qua trinh bin tinh cua protein khng xay ra.
Ch phm enzyme
Pha vi nc (t l 1:5)
Khuy 20 pht
Thu dch chit
Ly tm 3000 vng/pht (10)
Lc qua bng
Ta bng cn (1:3)
Lm lnh 30pht
Ly tm 3000 vng/pht (10)
Thu cn ta protein Ch phm enzyme 3.2.2 Tinh sach enzyme cellulase th

Dung dch y b Tinh sach enzyme nam ng sc ky loc gel. (chun b gel xem ph lc). pH=5
Nguyn tc: Sc k lc gel l k thut dng tch nhng phn t c kch thc, trng lng phn t khc nhau bng cch cho chng i qua ct gel. Nhng phn t kich thc nh lt vo bn trong l gel s b tr hon v di chuyn
Ct sc k
Lp 08DSH2
Khoa: MT-CNSH
Ch phm enzyme
14
chm qua ct, trong khi nhng phn t ln hn di chuyn bn ngoi cc ht gel nn s di chuyn nhanh v c gii hp (thi) ra khi ct sm hn cc phn t nh.
3.3 Phng phap xac inh hoat tinh C cht dng xc nh hot ng ca h enzyme cellulase l cellulose khng tan nh giy lc, bt cellulose tinh khit Tuy nhin, do lng ng kh sinh ra khng tuyn tnh vi lng enzyme trong phn ng, ngha l lng enzyme tng gp i s khng sinh ra lng ng kh gp i trong cng thi gian phn ng. Do ta s dng c cht l dn xut ca cellulose tan trong nc nh CMC, hay HEC xc nh hot tnh ca CMCase. Vi cc c cht khc nh giy lc dng xc nh hot tnh enzyme exocellulose. n v o hot tnh ring ca cellulase l CU/mg (cellulase units) Xc nh hm lng protein theo phng php Bradford:
Lp 08DSH2
Khoa: MT-CNSH
15
Cac protein khi phan ng vi xanh Coomassie (Coomassie Brilliant Blue-CBB) se hinh thanh hp cht mau co kha nng hp thu anh sang bc song 595 nm, cng mau ty l vi nng protein trong dung dich. Xc nh hm lng ng kh theo phng php DNS: Phng php da vo s thy phn c cht carboxymethyl cellulose bi enzyme carboxymethyl cellulase pH 5.0 v 40oC. Lng ng kh sinh ra c cho phn ng vi acid 2-hydroxy-3,5-dinitrobenzoic (DNS), mu sinh ra sau phn ng c xc nh bng phng php so mu trn quang ph k bc sng 540nm.
Lp 08DSH2
Khoa: MT-CNSH
16
Chng 4: NG DUNG CUA ENZYME CELLULASE TRONG SAN XUT NHIN LIU SINH HOC BIOETHANOL 4.1 Gii thiu v nhin liu sinh hoc ethanol Nhin liu sinh hc (Biofuel hay Agrofuel) l sn phm ca qu trnh ln men k kh t nm men, y l loi cht t ti to sn xut t nguyn liu ng thc vt. Xng-Ethanol (E) thng dng nht hin nay trn th gii v d dng bin ch t ng v tinh bt. Tt c thc vt quang tng hp u c th bin ch thnh xng sinh hc. Cy nng phm cha ng gm ma, c ci ng, sorgho-ng; nng phm cha tinh bt gm ht ng cc nh la m, la, bp, sorgho; c nh khoai ty, khoai m, khoai lang. Ma c hiu qu kinh t nht v cho nng xut thn (khong 170-200 t/ha Brazil, 80-100 t/ha c, Vit Nam khong 35-50 t/ha), bin ch ethanol thng t nc p, b ma dng lm nng lng chy my p v chng ct ethanol. Ma sn xut trung bnh 15,500 lt ethanol/ha/nm, v c 1 tn cht kh ma sn xut c 438 lt ethanol. Brazil sn xut ethanol chnh t ma. Sorgho-ng hin c a chung hn ma mt s vng nhit i kh hn, c hiu qu kinh t hn ma. Sorgho-ng canh tc Hoa K cho 28,500 lt ethanol/ha/v 4 thng. Ngoai ra con co cac ngun nguyn liu khac san xut xng sinh hoc nh:
Thc vt hoang di: to (algae) nc ngt, to bin, lc bnh (Eichornia
crassipes), c Vetiver, c voi (Elephant grass, Pennisetum purpureum, sn xut 13,700 lt ethanol/ha/nm), lc (Cyperus), c tranh (Imperata cylindrica),
Lp 08DSH2
Khoa: MT-CNSH
17
Ph sn thc vt t sn xut cy nng phm v cy k ngh: rm r, b
ma, thn, g, mt ca, tru, ht cao su (sn xut 217 kg du/ha/nm), ht bng vi (sn xut 273 kg du/ha/nm).

Giy ph thi: 1 tn giy c sn xut khong 416 lt ethanol. Rc thnh ph: 1 tn rc sn xut khong 227 lt ethanol. Ph thi chung tri gia sc: phn chung (to methane sinh hc ri ch
methanol). Xng sinh hoc ethanol (C2H5OH) 99.9% c th chy ng c xe hi chy bng xng. Khi chy, mt phn t ethanol sinh mt nhit lng 1409 kJ. Xng pha vi ethanol thi t kh nh knh hn xng thng. Chng hng E85 thi 1 ppm kh NO2 trong khi xng-c-sinh thi 9 ppm. Trn nguyn tc, bt c cht vt liu sinh hc no cha nhiu Carbon, hoc di dng ng, tinh bt, cellulose u c th ch bin thnh ethanol, hoc cha nhiu acid bo u co th ch bin diesel sinh hc c. Thng thng nht l t thc vt c kh nng quang tng hp bin CO2 ca kh quyn thnh cht ng, tinh bt, cellulose, ri protides, lipids, v.v. Trung bnh c mi phn t CO2 cy hp th v bin ch qua quang tng hp thnh sinh khi cha 114 kilocalories. Kh khn k thut hin ti l lm sao bin ton th nng lng C cha trong sinh khi thnh xng sinh hc. Vi k thut hin nay (c in), c 2 phng thc hu hiu:
Cho ln men (nh men v enzymes trong iu kin ym kh) cht ng
(t ma, c ci ng, v.v.), tinh bt [t ht ngu cc (bp, la, la m, v.v.) v c (khoai ty, khoai m, v.v.)], hay cellulose to ra ru Ethanol (CH3OH), Propanol [CH3CH2CH2OH; (CH3)2CHOH] v Butanol (C4H10O).
Lp 08DSH2
Khoa: MT-CNSH
18
Trch du t thc vt giu cht du, hay m t ng vt (p vi p sut cao
v nhit, hay bng dung mi, hay phi hp c hai). V phng din k thut (v kinh t), chia lm 3 loi nguyn liu:
Cng ngh xng-sinh-hc th h 1: ch bin t ng (ma, c ci ng,
sorgho-ng) v tinh bt ca nng phm (t ht ca bp, la m, la, v.v., hay t c nh khoai ty, khoai m, v.v.) to ethanol; hay t du (ca ht da-du, u nnh, u phng, v.v.) bin ch diesel-sinh-hc. K thut n gin v kinh t nht. Cng ngh xng-sinh-hc th h 2: t cellulose, cht x ca d tha thc vt (rm, r, thn bp, g, mt ca, b ma, v.v.), hay thc-vt-hoang (non-crop) (nh c voi, vetiver, lc bnh). Chng hn, mt ha ma cho khong 25 tn b ma (bagasse, xc ma sau khi p), v mi tn b ma sn xut 285 lt ethanol. K thut hin nay cha hon ho, hiu nng cn km, con men cha hu hiu v gi t, ch mt phn cellulose v lignin bin thnh ethanol, nn gi thnh sn xut cn cao. Cng ngh xng-sinh-hc th h 3: t to (algae), k thut ang pht trin.
Lp 08DSH2
Khoa: MT-CNSH
19
4.2 S san xut ethanol va thuyt minh quy trinh san xut 4.2.1 S quy trinh san xut
Ph phm nng nghip Tin x l Cung cp cho qu trnh tin x l
Cellulose Thy phn Hemicellulas es
Hemicellulose
Lignin
Cellulases Lignin
Ci tin mi trng nui cy Tc nhn gy t bin Sinh vt chuyn gen
SSF
Ln men S.cerevisiae
ng hexoses v pentoses
H thng qun l cht thi
CBP
Sn xut nng lng
Bioethanol th Chng ct v cho bay hi Bioethanol
Nng lng in
Lp 08DSH2
Khoa: MT-CNSH
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SSF: qu trnh thy phn thnh cc ng n gin v ln men c tin hnh trong mt thit b. SHF: qu trnh thy phn thnh cc ng n gin v ln men c tin hnh c lp trong nhng thit b khc nhau. CBP: ng nht bng qu trnh sinh hc 4.2.2 Thuyt minh quy trinh Quy trinh c tin hanh theo 4 bc:

X ly c hoc nguyn liu. Thuy phn cellulose thanh glucose. Ln men glucose thanh ethanol. Tch v tinh ch sn phm 4.2.2.1 Tin x l
Ba mia la ngun ph thai cua nganh cng nghip san xut ng. Thanh phn cua ba mia gm 41% cellulose (C6H10O5)n, ngoi ra cn c lignin, pectin, hemicellulose v phn ng cn d. Tin x l lignocelluloses rt cn thit v nguyn liu thy phn khng c qu trnh tin x l xy ra rt chm v kt qu l cho hiu sut to ra sn phm khng cao. Mt vi phng php nhm lm gim kch thc nguyn liu v gim phn cellulose kt tinh. Tin x l nng cao mc tip xc gia cellulose vi enzyme, lm gim lng enzyme s dng do gim v gi thnh ca quy trnh. C nhiu phng php tin x l khc nhau c th phn chia thnh 3 loi: ha hc (dng axit hoc kim),vt l kt hp vi ha hc (bng cch xay xt kt hp vi s dng hi nc) v tin x l bng phng php sinh hc s dng vi sinh vt. Trong tin x l bng phng php ha hc s dng axit, hemicellulose s l i tng tng tc chnh, ngc li qu trnh tin x l s dng
kim ch yu loi b lignin. V th khng nht thit ch s dng mt phng php cho qu trnh tin x l m da vo thnh phn ca nguyn vt liu m c mt phng php x l thch hp.Nhiu enzyme thy phn cellulose t nm lm vic pH axit yu (4-5). Tin x l bng phng php sinh hc s dng vi sinh vt nh dng loi nm nh Cyathus sp, Streptomyces viridosporus, Phelebia tremellosus, Pleurotus florida v enzyme ca chng dng kh lignin trong lignocelluloses nhm gim nng lng s dng, ti thiu sn phm thi ra v tc ng n mi trng. Mt s loi nm m trng c kh nng s dng lignin nhng khng lm gim lng mannan peroxide v laccase. Trong bc x l c hc, cn loi lignin trong nguyn liu, ch cn li thnh phn cellulose, vi ligin la cht kho phn huy. Nu con sot lai trong nguyn liu se anh hng nng sut cua qua trinh. 4.2.2.2 Thuy phn Qu trnh ny gy tiu tn nhiu chi ph trong giai on sn xut cn. Sau qu trnh tin x l, cellulose v hemicelluloses c thy phn to ra cc ng n s dng enzyme cellulase v hemicellulase. Nhiu loi nm nh Tricoderma, Aspergillus, Penicilium c th sn xut ra mt lng ln cellulase ngoi bo v hemicellulase. Nhng enzyme chu nhit cao v pH thp c u tin s dng do qu trnh tin x l thng dng axit v nhit . Hn na, cc enzyme chu nhit c vi c im thun li nh hot tnh ring cao v mc n nh cao dng ci tin qu trnh thy phn. Cui cng, nng cao hiu sut xc tc ca enzyme tc l gim lng enzyme s dng. Nhng chng chu nhit c enzyme hot ng nhit rt cao trn 60oC v nhit pht trin ti u l 35-55 oC. Hin nay, bng k thut di truyn, cc nh nghin cu ang hng n to ra mt t hp enzyme c th thy phn ngun nguyn liu lignocellulose hiu qu nht. Bao gm qu trnh:
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Chuyn i cellulose: Cellulose l loi polysaccharide ng hnh c cu -(1-4) glycoside.-D-glucose thng qua lin kt thnh t cc n phn. Qu trnh thy phn bng t hp enzyme cellulase bao gm -glucosidase to ra -cellobiohydrolase (exoglucanase), endoglucase v sn phm cui cng l glucose. Chuyn i hemicellulose: hemicellulose l thnh phn di do nht th 2 trong ngun nguyn liu lignocellulose (25-30%). Hemicellulose l mt loi polymer d hnh c to bng cc n phn pentose (D-xylose, D-arabinose), n phn hexose (Dmannose, D-glucose, D-galactose) v cc acid ng. Xylan l thnh phn thng thy trong cc thn g cng, tuy nhin glucomanan li l thnh phn chnh trong cc loi thc vt thn mm. T hp enzyme thy phn hemicellulose cng rt phc tp. V d xylanse, thy phn xylan th t hp enzyme cn thit l endo-1,4- -Larabinofuranosidase.-glucuronidase v -xylosidase. Ngun enzyme c s dng ph bin hin nay l t Trichoderma reesei v Aspergillus niger. Hin nay, ngi ta ang thay th dn cc h enzyme chu nhit, chu cc iu kin ha hc qu hn. Hn ht l cc nghin cu v phc hp cellulosome ca cc vi khun k kh ang dn m ra mt con ng mi nhm tng hiu qu thy phn ca t hp trn cc loi nguyn liu lignocellulose. 4.2.2.3 Ln men Trong qu trnh ln men, sn phm sau qu trnh thy phn bao gm cc monomer hexose ( glucose, mannose, galactose) hay pentose (xylan, arabinose) s c ln men to ra cc sn phm c gi tr nh ethanol. Trong s cc sn phm thy phn, glucose c hm lng cao nht sau n xylose v mannose v nhng ng khc c t l thp hn. S.cerevisiae l vi sinh vt ln men truyn thng s dng ln men ethanol t tinh bt trong quy m cng nghip. S.cerevisiae c nhiu thun li nh c nghin cu t lu, hiu sut ln men cao tuy nhin S.cerevisiae c nhc im l khng th s dng xylose lm ngun cacbon sinh trng hay len men. Do S.cerevisiae c ci tin c th s dng c 2 ng glucose v xylose. Hin ti mt s loi nh S. Cerevisiea hay S. unvarum l ging c kh nng to cn cao (12-13%), hay c bit S. oviformis c kh nng to cn 18% c bit loi nm men ny c kh nng ln men c rt nhiu ng khc nhau nh glucose, manose, saccharose, maltose v rafinose, tuy nhin khng c kh nng ln men galactose. Ngoi ra cn c Zymononas mobilis cng thng c s dng trong qu trnh ru ha. Tuy nhin c Saccharomyces v Zymononas sp
u thiu hon ton kh nng chuyn ha cc loi ng pentose. Khuynh hng bin i gen ca 2 ging ny nhm gip biu hin kh nng chuyn ha 2 loi ng pentose ph bin nht l D-xylose, v L arabinose cng c pht trin nhiu. Gn y, ngi ta pht hin thy c mt s loi nm men nh Pichia stipitis, Candida shehatae v Pachyhysolen tannophillus l nhng chng c kh nng chuyn ha xylose mnh v c dng trong sn xut ethanol. Trong P. stipilis li ni bt bi kh nng sn xut hm lng cn cao v nhu cu dinh dng ca chng khng qu phc tp so vi cc ging nm men khc. Ngoi ra, cc chng chu nhit cao nh G. thermoglucosidasius, T.mathranii v T. saccharolyticum cng ang c s dng. Qu trnh ln men cn ca chng c nhiu li ch hn cc qu trnh chuyn ha xy ra nhit trung bnh. Chng c kh nng ln men khng ch ng pentose, hexose m cn c kh nng ln men cellobiose, thm ch trong mt s trng hp nhng c cht polycarbonhydrate phc tp nh cellulose. Qu trnh ln men nhit cao gip qu trnh thu hi sn phm d dng hn, bi v ethanol c cha nc (aqueous ethanol) s bc hi ti nhit 500C, ng thi gim nng cn trong bn ln men nhm gim thiu nh hng ngc li ca nng cn n s pht trin ca t bo, t gim c chi ph sn xut. 4.2.2.4 Chng ct- kh nc Qu trnh tch nc v tinh sch ethanol p ng c im k thut ca nhin liu. 4.3 Cac thng s ky thut cua h enzyme cellulase
Nhit : enzyme cellulase hoat ng trong khoang nhit t 45 n 50oC, nhit
ti u la 48oC.
pH: mi trng kim hoc phosphoric acid, mi trng nay, cn quan st pH
tao mi trng thch hp, cellulose c n ra do o se bi phn giai rt nhanh.
4.4 Phng php xc nh ng glucose sinh ra

Cn theo doi lng glucose sinh ra sau phan ng. Glucose sau phan ng co th xac inh bng phng phap o Iode theo ky thut chun ngc. Cach tin hanh nh sau: Cho 1 th tich chinh xac glucose tac dung vi 1 th tich chinh xac (ly d) dung dich I2 trong mi trng kim. Qua trinh oxy hoa din ra theo phng trinh: I2 + 2NaOH NaI + NaIO + H2O CH2OH(CHOH)4-CHO +IO- CH2OH(CHOH)4-COOH + ISau khi phan ng xay ra hoan toan, acid hoa mi trng bng H2SO4 Iode d di dang IO- chuyn v lai I2. sau o inh lng I2 d bng dung dich chun Na2S2O3. Tnh kt qu % glucose nh sau:
%G = (VI 2 * N I 2 Vthio * N thio ) * EG VG *1000 * 100 f
Trong : %G l glucose. f: l h s pha long dd C6H12O6 trc khi nh lng.
Chng 5: KT LUN
S bin i ph liu nng nghip thnh cc ngun nhin liu c th l ethanol cn l thch thc ln cho cc nh nghin cu v nh sn xut. Cc chng cn c ci tin tng cng tng hp t hp enzyme thy phn hiu qu hay tng cng kh nng chuyn ha nhiu dng ng thnh ethanol bng cc k thut bin i gen, bin i qu trnh trao i cht nn cn c pht trin to ra ngun nng lng chi ph thp m vn khng nh hng n mi trng trong tng lai.
PH LC
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1. Phng php xc nh hot tnh enzyme cellulase bng phng php Bradford 1.1 Cc bc tin hnh Dung dch albumine 0.1mg/ml: cn chnh xc 10mg albumine pha trong 100ml nc ct. Lc u cho tan, gi 20oC. Khi dng pha long 100 ln, c dung dch albumine c nng 0,01 mg/ml. Dung dch thuc th Bradford: o o o o Coomassie Brilliant Blue: 0,001g Ethanol tuyt i 4,7g Acid phosphoric 85%: 8,5g. Phm mu Coomassie Brilliant Blue c lm tan trong chai ng c np,
b sung acid phosphoric 85% v chnh ti 100 ml bng nc ct. 1.2 Quy trnh thc hin Lp ng ng chun Albumine(ng chun protein): Hm lng protein c kho st thng qua ng chun albumin c lp trc vi cc nng t 10-50g/ml. lp ng chun th hoa cht c cho theo bang sau (trong ng C l ng i chng).
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Bang: Cac hoa cht dung trong thi nghim lp ng chun Albumin ng Nng albumin (g/ml) Dung dch albumin (ml) Nc ct (ml) Thuc th Comassie(ml) C 0 0 1 2 1 10 0.1 0.9 2 2 20 0.2 0.8 2 3 30 0.3 0.7 2 4 40 0.4 0.6 2 5 50 0.5 0.5 2
o hp th ca ng i chng bc sng 595nm, a v 0. Sau o hp th ca cc ng cn li. T xy dng c ng chun.
Tin hnh tng t vi mu, da vo ng chun xc nh lng protein c
trong mu.
2. Xc nh lng ng kh c to ra bng phng php DNS 2.1 Ha cht Cn 96o Sodium acetate(dng lm cht m) CMC(dng lm c cht) DNS(thuc th) Dung dch enzyme Thit b: my quang ph.
2.2 Tin hnh th nghim
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Thc hin mt lot 6 ng nghim theo bng sau y: ng nghim 1 0.1 0.1 0.9 1ml 2ml 2 0.2 0.2 0.8 3 0.3 0.3 0.7 4 0.4 0.4 0.6 5 0.5 0.5 0.5 i chng 0 0 1
Nng glucose (mg/ml) Vml Glucose (ml) VH2O (ml) CMC 1% pH5 (ml) Thuc th DNS (ml)
Lc u cc ng nghim ny, em un si cch thy trong vng 15 pht, lm ngui ri o bc sng 540nm. T xy dng c ng chun. Tin hnh tng t vi mu, da vo ng chun xc nh lng protein c trong mu. Tnh ton: Xc nh hot tnh ca enzyme:
1000 1 1 ( AT AB ) F V xphalong 180 10 1 CMCase CU =
Trong +AT: hp th ca dung dch c phn ng enzyme. +AB : hp th ca dung dch khng c phn ng enzyme. +1000: chuyn mg sang g. +Mglucose=180
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Khoa: MT-CNSH
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+10: thi gian phn ng +1: Th tch dung dch enzyme( ml ) +V: th tich dung dch mu (ml ) +F: tuyn tnh c tnh da vo ng chun glucose. F=(0,1/A1D1+0,2/A0D2++0,5/A0D5 )/5 Xc nh hm lng ca protein: X= hm lng (g/ml) x pha long x th tch Xc nh hot tnh ring ca enzyme cellulase: HTR= (CU/mg)
3. Phng php tinh sch enzyme cellulase bng phng php sc k lc gel 2.1 Chun bi gel Cn 4.4g gel kh (co th nhiu hn mt chut tr hao phn tht thoat) cho vao dung dich m acetate 50mM, pH5 th tich bng gp i th tich lp nn gel cn dung cho mt ct. Sau khi th huyn phu ng nht cua cac hat gel hinh thanh, khng khuy, n inh trong sut qua trinh hydrate hoa. Sau khi qua trinh hydrate hoa din ra hoan tt, gan lp ni trn b mt, chuyn dung dich trn vao binh hut chn khng, kh khi trong dung dich trong khoang 5-10 phut, thinh thoang lc nhe binh nhng khng c khuy. Thm dung dich m kh khi vi th tich gp 2 ln th tich lp nn, lc nhe. gel n inh cho n khi 90-95% s hat n inh, gan hoc loti lp ni trn b mt bng
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cach hut loti cac hat min. Lp lai qua trinh 4 hoc 5 ln loai >90% hat min lam can tr qua trinh loc gel. gel vao ct, ong l thoat cua ct va cho dung dich m lam y 20% th tich ct. Tranh tao bot khi khi gel vao va am bao vic nhi ct u. Khi lp nn trong ct at t 2-5cm, m khoa u racha ct cho n khi ct nap y gel. Khoa u ra cua ct va gn flow adaptor. M khoa u ra cua ct va cho dung dich m vi th tich gp 2 ln th tich lp nn chay qua mt luc diu chinh tc dong chay. ong u ra cua ct va iu chinh flow adaptor xung n lp nn gel. Nap mu vao trn b mt lp nn bng cach bm hoc tim mu vao trn lp nn gel qua flow adaptor. 2.2 Chun bi mu Mu chay sc ky phai sach va hoa tan hoan toan trong dung dich. 2.3 Thu va xac inh mu tach c Dich sau khi chay ra khoi ct c cho vao 20 ng nghim, mi ng (phn oan) cha 4ml. Tc dong chay 4ml/6phut trong 1 ng. Sau khi chay gel xong ta em i o mt quang bc sng 280 nm nhm xc nh ng no cha hm lng cellulase cao va lam cac bc xac inh hoat tinh cua enzyme cellulase a c tinh sach trong cac ng.
TAI LIU THAM KHAO
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[1] PGS TS Nguyn Tin Thng. Giao trinh cng ngh enzyme. H. KTCN TP.HCM, 2010. [2] Pham Quc Thng. Enzyme hoc. [3] PGS TS Nguyn Tin Thng. Tai liu hng dn thc tp mnCng ngh enzyme va protein. Vin sinh hoc nhit i, 2010. Tai liu ting nc ngoai [1] R.A. Nieves, C.I. Ehrman, W.S. Adney, R.T. Elander, and M.E. Himmel. Technical Communication: Survey and analysis of commercial cellulase preparations suitable for biomass conversion to ethanol. 1998. [2] Y.-H. Percival Zhang, Michael E. Himmel, Jonathan R. Mielenz. Outlook for cellulase improvement: Screening and selection strategies. 2006. Website: [1] http://en.wikipedia.org/wiki/Aspergillus_niger [2] http://cnx.org/content/m30247/latest/ [3] http://cnx.org/content/m30247/latest/
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BC CN Enz Hoan Chinh

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tai: THU NHN ENZYME CELLULASE TRONG Aspergillus niger VA NG DUNG TRONG SAN XUT BIOETHANOL

GVHD: Ts. Nguyn Hoai Hng

ch s sinh tng hp enzyme khi mi trng dinh dng cn kit.

Ln men Nc + m Canh trng

(Khng s dng cn nhit thng v c th lm bin tnh v gim hot lc ca enzyme).

Thu dch chit

Ly tm 3000 vng/pht (10)

Ly tm 3000 vng/pht (10)

Thu cn ta protein Ch phm enzyme 3.2.2 Tinh sach enzyme cellulase th

Thc vt hoang di: to (algae) nc ngt, to bin, lc bnh (Eichornia

Ph sn thc vt t sn xut cy nng phm v cy k ngh: rm r, b

Cho ln men (nh men v enzymes trong iu kin ym kh) cht ng

Trch du t thc vt giu cht du, hay m t ng vt (p vi p sut cao

Cellulose Thy phn Hemicellulas es

Ci tin mi trng nui cy Tc nhn gy t bin Sinh vt chuyn gen

H thng qun l cht thi

Bioethanol th Chng ct v cho bay hi Bioethanol

tao mi trng thch hp, cellulose c n ra do o se bi phn giai rt nhanh.

4.4 Phng php xc nh ng glucose sinh ra

Trong : %G l glucose. f: l h s pha long dd C6H12O6 trc khi nh lng.

o hp th ca ng i chng bc sng 595nm, a v 0. Sau o hp th ca cc ng cn li. T xy dng c ng chun.

Tin hnh tng t vi mu, da vo ng chun xc nh lng protein c

2.2 Tin hnh th nghim

TAI LIU THAM KHAO

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