Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh CHUONG 1: MO D: 1.1, Dat van dé Hign nay ngudn phé thai hu co do cde nhi may cong nghiép ché bién thye pham thai ra 1a rat ln nhu: rom ra, tréu, ba mia, cém mi, agar...Cac phé thai nay cé thanh phan chinh 1a cellulose. Cellulose c6 thé bj thay phin trong mdi truong kiém hode axit, Tuy nhién vige phan hity cellulose bing phuong phap vat ly va héa hge rat phite tap, tén kém va gay dée hai cho mdi trudng. Trong khi dé, vige xit ly cde chat thai hitw co chia cellulose bing céng nghé sinh hoe, die bigt sit dung cée enzyme cellulase ngoai bao tir vi sinh vat s& cé nhiéu uu diém vé ca mat ky thuat, kinh té va ‘méi trudng. Sé Iugng cic loai vi sinh vat tham gia sinh téng hop enzyme cellulase co trong diéu kign ty nhién rat phong phu. Ching thudc nam sgi, xa khudn, vi khuan va trong mét sé tnrdng hgp, cac nha khoa hoc cén thay ca men cing tham gia qiia trinh phan giai nay. Vi vay néu ta sin xuat durge mt lugng enzyme cellulase lon vai mite chi phi thap thi ta c6 thé tn dung duge ngudn phé thai lon tir ede nha may ché bién thyre phim nhu: ba mia, trdu, rom ra, mat cua...g6p phan vio bio vé méi trudng va cung edp mot lugng Ién nguyén ligu cho nghanh céng nghiép ché bién thyc phim. Nam Trichoderma spp hign dién gin nhur trong tit ca cde logi dat va trong mét sé moi trudng séng khdc. Ching hign dign voi mat d6 cao va phat trién manh 6 ving ré cia cay, mot sé gidng cd kha nding phat trién ngay trén ré. Nhimg gidng nay cé thé duge bé sung vo trong dat hay hat giéng bing nhiéu phuong phép. Ngay khi ching tiép xtc véi 8, ching phat trign trén bé mat ré hay vo ré phy thuge vao timg gidng. Cée nha khoa hoc da thanh céng trong vige phin lip dure ching nim Trichoderma Reesei KY-746 dé ting hop nén enzyme cellulase mt céch ¢6 higu qua nhat ma gid thanh lai re.Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Xuat phat tir thye té nay voi sw hung din cua ky su Huynh Van Thanh t6i da thue hign khéa ludn nay: “Khao Sat Quy Trinh Sin Xudt Enzyme Cellulase Tir Nim Trichoderma Reese’” 1.2. Muc tiéu ciia dé tai 1.2.1. Muc tiéu tong qudt > Quy trinh san xuat enzyme cellulase tir ndm Trihoderma Reesei, > Cé dinh enzyme cellulase trén Natrialginate. 1.2.2. Muc tiéu cu thé > Thu nhan enzyme cellulase tir nam Trichoderma Reesei, > Xac dinh ty 1é tia tdi uu ciia cdc tac nhén tia: Mudi ammonium, cén, aceton. > Xée dinh ol 6, pH tdi wu cho sy hoat dong cita enzyme cellulase. > Cé dinh enzyme cellulase trén Natrialginate, a Tinh sach enzyme cellulase,Khoa ludn t6t nghigp nhung chi cé mét sé it vi sinh vat la cé kha nang sinh téng hgp enzyme cellulase véi sé lugng Ion. Sau day la két qué nguyén citu cdc ching vi sinh vat sin xudt enzyme Mac dit cé nhiéu loai vi sinh vat cé kha nang sinh tong hgp enzyme cellulase, cellulase. ‘SVTH; Nguyén Tran Nhat Minh CHUONG 2: TONG QUAN 2.1, Sang loc cate ching vi sinh vat san xudt enzyme cellulase ngogi bao Bang 2.1 Sing Igc cae chiing vi sinh vit sinh enzyme cellul: Hoat tinh 6 STT)Tén ching) —Tén phan loai celulase (unit/ml) nhom 1 (B26 Bacillus subtilis 20 1B505 Bacillus subtilis 95 cra 771 4 Fh 145 ‘i khudn ISM 04 90 6 SM II 192 7 i acillus lichenformis 9,76 [A-62 treptomyces sp 90 a khudin Sarcharomyces “519 1,35 fibuligera Sarcharomyces 10 W¥-529 67 'im men fibuligera archaromyces 1 y-32 181 -apsularis, 12 F273 Spergillus niger 108 iim moe 13 708 [Tricoderma konigii 89 iim soiKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh js hr 1,32 5 2 93 6 3 1,10 7 4 1,07 (Oj: chwa phan loai 2.2. Gi Trichoderma reesei 2.2.1. Lich sit nghién citu vé Trichoderma Gan 200 nam vé trudc, Trichoderma dugc phat hién ra va hién nay loai dé duge biét la Trichodermaviride. Hon 150 nam sau, Trichoderma chi la d6i tung cia vai nha phan loai ndm hoc nhung khéng hap din duge méi quan tim ctia cée nganh khoa hoc hdc. Tinh hinh thay di trong thé chién lan thir Il, khi quén di MY canh bio vé hign turong cic trang bi quin sy bi myc 6 xi nhigt di, dae bigt ld @ Nam Thai Binh Duong. Chuong trinh didu tra cia quan di My chi ra rng Trichoderma" viride" mi sé QM 6a la loai nm phan hily cellulose & khu vuc nay. ‘Su nham lin nay kéo dai suét 20 nam cho dén khi ching Trichoderma QM 6a nay duge nhin dign va dat tén lai la Trichoderma reesei dé t6 ling t6n kinh ngudi a kham pha ra loai nay la Elwyn T.Reese, tac gid lim vige tai vign nghién cttu Ni ‘v6i sur cOng téc ciia Mary Mandels da nghién ctru nhiéu dé tai vé sinh téng hop, co ché phn hiy cellulose va cic hyp chat polysaccharides khae cia ching Trichoderma reesei niy vi cic thé dt bién trén ching d6. Nho nhing cOng trinh dé ma nhiéu phong thi nghigm khdc & MY, Chau Au va. Chau A tiép tuc nghién ciu va kham pha ra hé théng phan giai cellulose ciia Trichoderma vio et 4i thip nién 60. Cing théi diém dé, Rifai va Webster 6 Anh Lin dau tign phan loai va m6 té duge 9 lodi Trichoderma, vide nudi cdy dé ding va khong, tén kém cic chiing Trichoderma dit 16i kéo céc nha nghién citu di vio cée hudng, nghién citu co ban hon 1a nghién eitu ing dung vé phan giai cellulose eiia ching. Mot phat hign quan trong trong nghién citu vé Trichoderma la kh ning kich thich tangKhoa Iwan t6t nghigp SVTH: Nguyén Trin Nhat Minh truéng cho cay trong va kha nang déi khing véi cée lodi nim bénh gilip Trichoderma durge ding nhur 14 tic nhan kiém sodt sinh hoc trong néng nghiép. Ngiy nay, Tinh vye nay da tré thinh huéng nghién cizu cia nhiéu nha khoa hoc trén thé gidi. Hinh 2.1 Anh nhin qua kinh hién v Tricoderma reesei. Trong hinh, céc protein trong c é nhirng sgi to phat trién cia dong nam c 6 bao nm duge danh dau n iu 6 trong khi chit chitin — m6t thanh phan ciia cae thinh té bao — duge dang diu mau xanh duomg. (Anh: Mari Valkonen, VT Finland) 2.2.2. Nudi cdy Trichoderma reesei trén moi truéng ba mia két hop cam mi Trichoderma reesei VTT-D-80133 sinh truéng trén mdi trudng ban rin véi co chit ba mia két hop voi cam mi. Ty 1@ BM:CM (7:3), 8 Kin néng d6 dinh dudng, d6 am ban dau 60%, thoi gian nudi cdy 7 ngay 1a ti uu cho Trichoderma. reesei VTT-D- 80133 sinh tong hop cellulase trén méi truéng lén men ban ran,Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 2.2.3. Hinh thdi cia ndm Trichoderma reesei Trichoderma 1a mot loai nam bat toan, sinh sin v6 tinh bing dinh bao tit tir khudn ty. Khuan ty cia vi nam khéng mau, cudng sinh bao tir phan nhanh nhiéu, & cudi nnhanh phat trién think mt khdi tron mang ede tur trin khéng cé vach ngan, khong miu, lign két nhau thanh chim nho & dau cinh nhé chat nhiy. Bao tir hinh cau, hinh clip ho&c hinh thuén, Khuan lac nam c6 mau tring hodc tir Iuc tring dén Ic, vang xanh, lye xin dén tuc dam. Hinh 2.2 Hinh thai Trichoderma Reesei 2.2.4. Dic diém Nam Trichoderma spp in dign gan nhw trong tat ca cac loai dat va trong mot s6 méi truéng s6ng khac, ching 1a logi nam duge nudi cdy thong dung nhat. Nim Trichoderma hign dign voi mat 46 cao va phat trién manh & viing ré ctia edy, mot sé giéng c6 kha ning phat trién ngay trén ré, nhiing gidng nay c6 thé duge bé sung vio trong dat hay hat giéng bing nhiéu phuong phap. Nam Trichoderma khi ching tiép xtc fy, khi duge ding trong xtr ly hat gidng, nhitng gidng thich hgp nhat sé phat trién trén be mat vi :, chiang phat trién trén bé mat ré hay vo ré phu thude vao timg gidng. way ca hi r8 phat trign dai hon Im phia duréi mat dat va ching c6 thé t6n tai va ednKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh higu Ive cho dén 18 thang sau khi str dyng, tuy nhién khong nhiéu gidng co kha nang nay. Ngoai sur hinh thanh khudn lac trén r8, ndm Trichoderma cn tin céng, ky sinh va ldy chat dinh dutng tir cdc loai nam khac. Béi vi noi Trichoderma phat trién tét nhit 1a noi cé nhigu ré khée manh, va Trichoderma sé hitu nhiéu co ché cho vige tin céng cae loai ndm gay bénh cing nhur co ché cho viée nang cao syt sinh trudng va phat trién cia cy. Nhiéu phutong php mdi trong kiém soit sinh hoc va nding cao sw sinh trudmg, cla cy hién nay da duge ching minh 1d ring, Qua trinh nay duge diéu khién boi nhiéu gen va san phim tir gen khac nhau. Sau day 1a mét s6 co ché chi yéu: Ky sinh nam, khang sinh, canh tranh chat dinh duéng va khéng gian, su chiu dung cdc diéu kién bat Igi bang viée gia ting sy phat trién cia cay va ré, lam hoa tan va cé lap chat dinh dudng v6 co, cam img su khang bénh, bat hoat enzyme gy bénh. ‘Hau hét cdc giéng Trichoderma khong sinh san hau tinh ma thay vao a6 1a co ché sinh san vé tinh. Tuy nhién, c6 mét sé gidng sinh san hitu tinh da duoc ghi nhan nhung nhiing gidng nay khéng thich hop dé str dung trong cae phuong phap kiém soat sinh hoe. Phuong phap phan loai truyén théng dua én su khdc nhau vé hinh thai cht yéu la & bé phan hinh thanh bao tir v6 tinh, gin day nhiéu phuong phép phan loai dura trén cdu tric phan tir da duge sit dung. Hién nay nam Trichoderma cé it nhit 1a 33 loai. Hinh 2.3 CAu trae khong gian cia nm Trichoderma reeseiKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 2.2.5. Ung dung 2.2.5.1. Luong thue va nganh dét Trichoderma 1a nhimg vi sinh vat sin xudt nhiéu enzyme ngoai bao cé higu qua cao. Ching duge thuong mai héa trong viéc san xudt enzyme cellulase va cic enzyme kde phin hity cée polysaccharide phite tap. Nh vay ching thudmg duge sir dung trong céng nghiép ché bién thy phim va nginh cng nghiép dét cho cde myc dich tong tu. 2.2.5.2, Chat kiém sodt sinh hoc Hign nay loai nam nay da duge sir dung mét cach hgp phap ciing nhu durge ding ky trong viéc kiém sodt bénh trén thye vat. Céc ché phim ndm Trichoderma duge sin xuat va str dyng nhu 1a chat kiém soat sinh hoc mét céch c6 higu qua. Hinh thite sit dung duéi dang ché phim riéng biét hode duge phéi tron vio phan hitu co dé bén cho cay wong vira cung cp dinh dung cho cay vira ting kha ning khdng bénh cita cy. 2.2.5.3. Kich thich sue ting truong ciia cay tréng Nhiing Igi ich ma nhitng loai nam nay mang lai da duge biét dén tir nhiéu nam qua bao gbm vige kich thich sur king trudng va phat trién etia thye vat do vige kich thich sir hinh thanh nhiéu hon va phat trién manh hon ciia b9 r8 so v6i thong thug. Nhimng co ché giai thich cho cdc hign tung nay chi méi duge hiéu 10 rang hon trong thoi gian gin day. Hién nay, mot gidng nim Trichoderma da duge phat hign la ching c6 kha ning gia ting s6 Iugng ré mgc sau (sau hon 1 m duéi mat dat), Nhimg ré sau nay gitip cic loai cdy nhu: bip hay edy canh e6 kha ning chiu duge han hin. ‘M6t kha nang c6 1é dang chit ¥ nhat 1a nhiing cay bap c6 sy hién dign ctia nm Trichoderma dong T22 6 r8 cé nhu cu vé dam thip hon dén 40% so vi nhimg cay khong cé su hién dién cia loai ndm nay 6 ré. 2.2.5.4. Nguén gen dé sir dung trong chuyén gen Nhiéu vi sinh vat kiém soit sinh hoc déu c6 chita mét s6 lugng lon gen ma hod cc sin phdm c6 hoat tinh can thiét sir dung trong kiém soat sinh hgc. Nhiéu gen cOKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh nguén gic tit Trichoderma da duge tao dong va cé tiém ning img dung rat lon trong chuyén gen dé tgo ra cy 6 kha ning khang durge nhiéu bénh. Chua e6 gen nao duge ‘thuong mai héa, tuy nhién c6 mot sé gen hign dang duge nghién citu va phat trién. 2.2.5.5. Bién php canh téec hitu co Hign nay, vige bon phan hitu co cho edy cao su hau nhur cha duge bi con nong dan chi trong dan dén mat can bang sinh thai méi truéng dat. Hé vi sinh vat suy giém a Lim cho ném bénh, dac bigt 1 nim Corynespora cassiicola ¢6 diéu kign phat trién va phat tan rit nhanh trong mia mua, Vi vay, vige bon phan hitu co 1a mét trong nhiing giai phap quan trong can durge quan tam thie dy. Trong khi bénh vang Id, rung la dang c6 chiéu huéng lay lan nhanh viée bén phan hitu co cé chita nam aéi khang Trichoderma li mét giai phap rat hitw am déi khang higu. Ngoai viéc gidp cho bé ré cay phat trién, dat gitt m tét, dng thoi Trichoderma sé tan céng cic mim bénh corynespora va cic loai nim bénh khdc cO trong dit. Viée phun céc ché pham sinh hge 6 chita Trichoderma lén tan cay cing 1a mot ai php rt t nhiim dua es ¢ bao tir ndim déi Kt ing phat tn rOng rai trong mdi trudng cia vudn cao su tao nén mét hé thong phong thi hiru hiéu lau dai Qua nhiéu két qua thuc tién cho thay, nam Trichoderma giét nhiéu loai nim gay théi chit yéu nhu: Pythium, Rhizoctonia vi Fusarium, do vay nam Trichoderma d& duge khuyén cdo str dung rong rai trong sin xuat néng nghiép céng nghé cao tinh Lam Déng nhimg nam qua.Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Hinh 2.4 U phan hau co c6 sir dung nam a Gidi_ thigu so luge ve enzyme Enzyme hay con goi la chat xiic tae sinh hoe c6 ban chat la protein. Enzyme co chang Trichoderma trong moi co thé sinh vat, né khong nhiing lam nhigm vy xtic téc cho cdc phan tng héa ih vat (i bao (invitro). Vi cé ngudn géc tir sinh vat cho nén enzyme thuréng durge goi 1a chat xii hye nhat dinh trong co thé ivo) ma cdn xiie téc cho cdc phan img ngoai t& te sinh hoe (biocatalisateur) nhim phén bigt vi cc chat xtie tic hoa hge Khdc. Chinh nho sy cé mat ca enzyme ma nhiéu phan tng héa hoc rat khé xy ra trong diéu kign thuong & ngoai co thé (do can nhiét d6, ap suat cao, acid manh hay kiém manh...) nhung trong co thé né xy ra hét site nhanh chong, lién tue va nhip nhang véi nhiéu phan img lién hop khac trong diéu kign hét sire ém diu, nhe nhing (37°C, ap sudt thuong, khong kiém manh hay acid manh...).Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Dc tinh quan trong nhat ca enzyme la tinh dic higu. Tinh dic higu la kha nang xctic tie chon loc, xtc tée sur chuyén héa mét hay mét sé chét nhat dinh theo mét kiéu phan img nhat dinh: dic higu cam img va dac higu co chat. Enzyme khéng thé téng hgp bing con dudng héa hoc. Do dé mudn thu nhin. enzyme chi cé con ding duy nhat la thu nhdn tir co thé sinh vat. Tat ca cac t8 bao i, vi sinh vat déu chita enzyme nhung mite d6 enzyme thi hoan toin dong kde nhau, thye > — Hign nay ngudi ta khai thée enzyme tix ba ngudn co ban: = Déng vat (han ehé). = Thue vat (han ché). = Vi sinh vat (phé bién). “ Vigc Khai thic enzyme tir déng vat va thue vat rat phitc tap vi nguén nguyén Jigu thu nhén khé khan, hiéu sudt thap dan dén gi: ché. % Vige san xuat enzyme tir vi sinh vat ¢6 nhing wu diém nhu sau: thanh cao nén han © Visinh vt cé chu ky sinh truéng va phat trién rat ngan. © Hé enzyme cia vi sinh vat v6 eiing phong phi. ? — Vigc cai tao giéng vi sinh vat dé tao ra nhimg ching vi sinh vat c6 kha nang tong hgp ra cac loai enzyme theo y mudn duge thyc hién m6t cach dé dang trong mét thoi gian ngdin vi khé nang thich ting méi trudng ciia vi sinh vt ki rat cao. © Visinh vat e6 tc dé sinh san eye nhanh. * Visinh vat khong di hoi nghiém ngat vé moi trudng dinh duomg nén 6 thé tn dung nhiing phy phé ligu cng ndng nghigp dé san xudt enzyme va co thé diéu khién dé dang cac yéu 16 anh hudng dén qua trinh nudi cay dé c6 thé thu duge higu xudt cao trong san xuat.Khoa Iwan t6t nghigp SVTH: Nguyén Trin Nhat Minh enzyme tir vi sinh vt hodn toan cé thé thye hign theo guy m6 Enzyme cit méi lién két peptit (CO-NH-) 2.4. Gidi thigu sor luge vé protease Nhém enzyme protease (peptit-hidrolase): Xie tc qua trinh thiiy phan lién két peptit (-CO-NH-)n trong phan tir protein, polypeptide dén sin pham cudi ciing la acid amin. Ngoai ra, nhiéu protease eting e6 kha ning thiy phén lién két este va van chuyén acid amin. Protease durge phan logi dua trén cde dc diém riéng ciia ching cling nhu cfu tg0 trung tim hoat dong enzyme, hign tai durge chia Jam 6 nhém chinh: Serine proteases. = Threonine proteases. - Cysteine proteases. - Aspartic acid proteases. = Metalloproteases.Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh ~ Glutamic acid proteases. Hign nay, ching ta sit dung 3 nguén nguyén ligu sinh hoc co ban dé thu nhgn protease: cdc mé va co déng vat, mé va co quan thre vat, té bao vi sinh vat. Protease cn thiét cho céc sinh vat séng, rit da dang vé chite nang tir mute d6 18 bao, co quan dén co thé nén duge phan bé rit rong rai trén nhiéu déi tuong tr vi sinh ‘vat (vi khudn, ndm va vi rut) dén thyc vat (du du, dita...) va déng vat (gan, da day bé). So vai protease déng vit va thuc vat, protease vi sinh vat cé nhimg dac diém khac biét, protease vi sinh vat I mot hé théng rat phite tap bao gdm nhiéu enzyme rat gidng nhaw ‘vé cau tric, khéi Iugng va hinh dang phan tir nén rat khé tach ra dui dang tinh t1 déng nhit, do dé protease vi sinh vit thuéng cé tinh dic hiéu rong rai cho sin phim co tinh thity phan trigt dé va da dang, come rely Fu owe Hinh 2.6 Cau trac khong gian cia protease 2.4.1. Ung dung ciia protease Protease la enzyme duge sir dung nhiéu nhat hign nay trong mét s6 nginh san sxudt nhu: ‘Trong céng nghiép sita: Protease duge ding trong san xuat phomat nh hoat tinh lam déng ty sita ciia ching. Protease tir m6t sé vi sinh vat nhu A. candidus, P. roquerti, B. mesentericus.... duge diing trong sn xudt pho mat. 13Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Trong céng nghigp sin xudt bénh mi, binh quy.... Protease kim gidim thir gian tron, ting d6 déo va lam nhuyén bot, tao d6 xép va né t6t hon. Trong céng nghigp ché bién thit: Protease duge ding kim mém thjt nhé sy thoy. phiin mét phan protein trong thit, két qua lim cho thit 6 m6t d6 mém thich hop va c6 vj tt hon. Protease duge sir dung dé lam mém thit va ting huong vi thit (ngam thit vao dinh dudng protease & pH va nhiét 46 xée dinh - phuong phap nay phé bién va thudn Ii nit), Trong ehé bit thuy san: Khi san xudt nude mim (va m6t sé 1oai mim) thudng thoi gian ché bién thug la dai mhat, higu suat thuy phan (46 dam) Iai phy thude rat nhiéu dia phuong, phuong phap gai nén, nguyén ligu cd. Nén hién nay quy trinh sin xuat nude mim ngin ngay di duge hoan thign trong do sir dung ché phim enzyme thye ‘vat (bromelain va papain) va vi sinh vat dé rat ngan théi gian lam va cai thién huong vi ceva nue mim, ‘Trong san xuat bia: Ché phim protease cé ¥ nghia quan trong trong viée Lim ‘tang d6 bén cia bia va nit ngin thoi gian lc. Protease cla A. oryzae duge ding dé thuy phan protein trong hat ngii céc, tao diéu kign xtr ly bia tt hon. Trong céng ngiép thudc da: Protease dugc sir dung lim mém da nhé sy thiy phan mét phan protein ctia da, chii yéu 1a collagen, thanh phan chinh lam cho da bj déo nhat dinh, cimg. Két qua da loai bé khéi da cdc cht nhét va Lam cho da cé dd, tinh chat d6 duge hoan thign hon sau khi thuge da. Trude day, dé Lim mém da ngudi ta ding protease duge phan Lip tir co quan tiéu ha ciia dong vat. Hién nay, vige dua cde protease tich tir vi Khudn (B. mesentericus, B. subtilis), nim méc (A. oryzae, A. flavus) va xa khuin (S. fradiae, S. griseus, S. rimosus...) vao eng nghiép thude da 4a dem Iai nhiéu két qua va din dan chiém mét vi tri quan trong. Ngoai ra, protease cdn duge tmg dung rong rai trong rat nhiéu nganh khac nu:Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh = Dieu ché dich dam thiy phan ding lam chat dinh dudng, chat tang vj trong thyc: phim va san xudt mot s6 thite an kiéng, - Protease cia nm médc va vi khudn phéi hgp véi amylase tao thanh hén hop enzyme diing lam thie an gia sie c6 46 tiéu héa cao, ¢6 ¥ nghia Ién trong chain nudi gia sive va gia ~ Diéu ché méi truding dinh dudng ctia vi sinh vat dé san xuat vaccine, kang sinh... = San xudt keo ding vit, chat gidt tng hyp dé giat ty ede chit ban protein, san x my phim 2.5. So luge vé enzyme cellulase thu nhjin tir Trichoderma reesei Cellulase 1a nhém enzyme thuy phan cé kha nang cit méi lién két B -1, 4-O- glucoside trong phan tr cellulose va m6t s6 ec chat twong tyr khéc. Dé 1a mot phite hé gém nhiéu loai enzyme khae nhau va duge xép thanh 3 nhém co ban: endo-B-1, 4- glucanase hay carboxymethyl cellulase (CMCase) (EC 3.2.1.4), exo-B-1, 4-ghicanase hay cellobiohydrolase (EC 3.2.1.91) va f-glucosidase hay f-D-glucoside glucohydrolase (EC 3.2.1.21). MGi loai enzyme tham gia thiy phin co chat theo mét co ché nhit dinh va nho- c6 sur phdi hgp hoat dng cia ede enzyme dé ma phan tit co chét duge thiy phan hodn toan tao thanh céc sin phim don gin nhat.Khoa Iwan t6t nghigp SVTH: Nguyén Trin Nhét Minh. Cellulase Enzyme Hinh 2.7 Cu trie khong gian cia enzyme cellu Co ché hogt d6ng ctia enzyme cellulase M6t phtte hg enzyme cellulase gém: endo-P-1,4-glucanase, exo-f-I,4-glucanase, B-glucosidase. Trong dé: Endo- B-1, 4-lucanase xiic téc cho phan img thiy phan cde lign két 6 bén trong phan tir cellulose. Exo- B-1, 4-gluc 6 dau khir inase thity phan ciie li wu khong khit cua pl cellulose. -Glucosidase thity phan cdc phan tir cellodextrin va cellobiose thanh glucose.Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh i : In Oo O ‘ —_ HH 1H HS Ho. 4 Ho- Endocellulase 9 9 ° , : : H0 A , ecm : Cellobiase HO: Ho: (B-glucosidase) oa ba wo a AE NAP Na = Glucose Callobiose oF Callotetrose Hinh 2.8 Co ché hoat dng cia enzyme cellulaseKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 2.5.2. Tinh chit ly héa ciia enzyme cellulase Tuy thud vao cdu tric va nguén géc ciia enzyme, hoat tinh enzyme dat manh nhat 6 nhiét 4, pH nhat dinh. > Anh hudng ciia nhigt d6: van tbe phan img do enzyme xiic tic chi ting lén khi tang nhigt dO trong mot gidi han nhdt dioh, chua anh hudng dén edu tic enzyme. H tinh enzyme dgt cye dai 6 nhigt 46 thich hgp, khoang nhigt 4} thich hgp cua nhiéu enzyme vao khong 40-50°C. G nhiét 46 cao, enzyme bi bién tinh Lam hoat tinh giam manh hoge mat hoat tinh, edn 6 nhiét d6 thap duéi 0°C, hoat tinh enzyme bj giém nhiéu nhung lai cé thé phye hdi khi dua vé nhiét 6 thich hop. Hoat tinh ctia enzyme cellulase tit Trichoderma reesei dat t6i da & 55°C. 7 Anh hung ctia pH: Kha nang hoat dong ciia enzyme con phy thudc vao pH méi truxmg phan img. Tay thude vao ban chat cua enzyme ma pH thich hop dé enzyme hoat déng cé thé trung tinh, kiém hod acid. Theo nghién ctru truéc day cho thiy, pH 161 uu cho hoat dng ciia cellulase tir Trichoderma reesei la 4,0- 5,0. > Anh hucing cia ion kim logi: Cac ion kim loai c6 thé kim ham hoje hogt héa str hoat ddng cilia cdc enzyme. Cac ion kim loai ning 6 ndng do nhit dinh co thé wily Ngoai ra, cde dung moi hitu eo, inh va kim ham khéng thudin nghich enzyme. v ‘ie chat tay ria ciing anh huéng manh mé dén hoat tinh cia enzyme. Tuy thude vio bin chat cia ede chat trén cing nhur bain chat ctia enzyme ma tinh chét vi mite dO anh hudng t6i hoat déng cla enzyme 18 khée nhau, Cae dung méi hitu co methanol, ethanol, isopropanol va acetone déu ite ché hoat déng cia cellulase, dic biét 1 n-butanol ite ché manh nhit, hoat tinh cellulase chi con 33-63%. ic chat tay ria tween 20, tween 80, SDS va, triton X-100 déu lam gidm hogt tinh cellulase & mite dé khdc nhau, trong do SDS lam gidm manh host tinh cellulase chi edn 18-34%. 18Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 2.5.3. Ung dung cia enzyme cellulase Enzym cellulase duoc img dung rat nhiéu trong cong nghiép sin xudt cd trong xir ly méi trudng. Enzym cellulase duge img dung rat it trong thyc phdm (khoang 1%) nhung hau nhu qué trinh ché bién nao lién quan dén ngudn nguyén ligu 1a thue vat néu cé enzym cellulase déu cho higu sudt cao hon. Hon nifa cellulase duge str dung rong rai trong nginh cng nghiép det, b6t giat, nganh céng nghigp gidy, cellulase con duge sit dung cho cae ting dung duge phim. 2 Céc nguén thu nhén enzyme Hign nay trén thé gidi c6 3 nguén dé thu nhjn enzyme la tir thyc vat, dGng vat ‘va vi sinh vat. Do nhig wu diém néi bat vé toc dé sinh trung, sinh san va phat trién ma nguén vi sinh vat duge quan tim nhiéu hon, Mat khac, do kich thude vi sinh vat nho nén ta cé thé co gidi héa va tw dong héa trong qua trinh nudi cay, diéu kién nudi cdy vi sinh vat e6 thé kiém soat duge ma khéng phu thuge vao yéu t6 bén ngoai nhu thu enzym tir ngudn thu vat va dong vat. C6 2 phuong phap dé nudi cay vi sinh vit tao enzyme La nudi cdy be mat va nudi edy bé su, Méi phuong php c6 uu va nhuge diém riéng, tuy nhién ngay nay phuong phap nudi cay bé sdu ngay cing dugc img dung rong rai do kh hhéa va tw dong hoa. 2.6. K¥ thuat co bin chun dich protein tho Sau khi Iya chon duge nguén cung cap protein viée tiép theo la phai dua protein vé dang dich. Cé nhiéu phuong phip: pha té bao bing dp sudt thim thu, nghién bing thiét bi trung tinh, nghién tay, pha té bao biing phuong phap siéu 4m, Cae phurong phip ké trén thich hop dé xir ly cde m6 mém, mé dGng vat, thy vat. Déi véi vi khuan c6 vo bao vé chic chin thi tét nhdt nghién bang cdi thay tinh cé thém vat ligu cha xt nhu cat hay bOt nhém, hoge xir ly v6 lysozi 1e (enzyme pha vach té bio). Déi vai nhimg té bio 6 vach bao vé rit chic nhu nam men trong mét sé trudng hop phai sir dung may épKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh tao Ap suat cao dé pha vo té bao. Mét sé trudng hgp c6 thé str dung may nghién dé pha miu, ‘Sau khi nhan duge sinh khdi té bao bj nghién, céng vige tiép theo 1a phai tach vvich va ede manh v6 té bio ra khdi dich chita protein duge gidi phéng trong qué trinh nghién, Thudng ngudi ta sir dung phuong phip: ly tam, Igc, téch dich 2 1p, tach nucleic acid va lipid. Tét nhat la dich thé duge bao quan & nhiét 46 thap. inh enzyme 2.7.1. Dinh nghia vé enzyme cb dinh Enzyme c6 dinh li enzyme duge gidi han hay khac bigt vé mat vat ly & mét ‘ving khéng gian nhit dinh, nhung van gitt duoc hoat tinh xuc téc va c6 thé tai str dung nhiéu lan sau mOt qua trinh xtic tae. 2.7.2. Uu nhurge diém cia qué trinh cé dink enzyme * Uudiém: ‘thé tai sir dung lp di fap Iai nbiéu Lin 1 tugng enzyme xc dinh trong 1 thai gian dai, do dé Kim gidm gid thanh san phim, higu qua kinh té cao tiét kigm enzyme, dae biét quan trong 44i v6i nhiing enzyme dit tién, thudn lgi trong cde qué trinh tw dong héa va lién tue - Tang d6 tinh sach ca sin phim vi enzyme duge c6 dinh & nhimg pha rigng ré do 6 dé dang tich ra khdi sin pham. Tranh duge anh huéng khéng tét dén sin phim. Diéu nay dac biét la c6 ¥ nghia khi sir dung enzyme cé dinh trong sin xuat thye phim, trong céng nghiép san xuit héa chat tinh khiét va trong phan tich. + C6 thé dig qué trinh phan ing 6 bat ett giai doan nao khi can thiét, chi can tach ‘enzyme cé dinh ra khdi co chat, ~ Kéo dai théi gian bao quan va bén vig véi chat kiém him ciing nhw véi cac tac nhan gay bién tinh so véi enzyme ha tan. 20Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh ~ Hoat tinh én dinh so vai enzyme tyr do khi ¢6 nhimg thay 46i ctia mdi trudmg nhu: PH, nhiét dd... Nho vao cdc lién két cia enzyme voi chdt mang nhu lién két céng héa tri, lién két hydro, lign két ion va cdc lién két khac. Nhuge diém: Han ché kha ning tiép xtc gitta co chit véi enzyme cho nén hoat tinh riéng cia enzyme c6 dinh thudmg thap hon so véi enzyme tyr do. - Mét lugng enzyme dang ké bj mat hoat tinh khi c6 dinh bang phuong phip cong hha trj 18 do chat hoat ha va do phan img gin két khong dic higu etia céc Hién két tren ‘vat ligu c6 dinh vo trung tim hoat déng eta enzyme. 2.7.3. Cée yéu t6 anh hung dén hogt tinh enzyme c6 dink Khi gin lén chat mang, enzyme bj gidi han trong mét pham vi méi trudng xac dinh, céu tao khdng gian ciia phan tir c6 thé bi thay déi, do dé lim bién ddi mét sé tinh chat cia enzyme ban dau (enzyme hoa tan). Mot sé yéu té anh hudng dén hoat tinh, enzyme khéng hoa tan Li > Ban chat va tinh chat héa hoe cla chit mang. > — Cac tinh chat ly hoc eta chat mang nhu tinh hoa tan, tinh bén co hoc, 46 truong dign tich, tinh hao nude va ki nuéc,... déu co anh huéng nhat dinh dén long enzyme duge lién két, tinh bén va hoat tinh sinh hoc cia cde din xuat enzyme 6 dinh, Ban chat héa hoc cia cac chat mang ciing cé anh huéng dang ké dén kha nang tao thanh dan xuat enzyme, va t6i kha nang hap thy khéng dc trung cae chat tir méi truéng phan ing. > — Dan xuat enzyme khéng héa tan thudng 06 tinh bén nhiét cao so véi enzyme tan do két qua cia sy dinh vj enzyme tao nén. Chat mang ciing 6 tac dung han ché su bién tinh cita enzyme trong cac dung méi cé kha nang Lam dirt méi lién két hydro va lién két ky nude, > — Sw khuyéch tin cia co chat, sin phdm va cde phan tit khac. oi ‘Téc do Khuyéch tan cla co chat, sin phim va cate chat phu khée. auKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 2.7.4. Phwong phap cé dinh enzyme Cé dinh enzyme théng qua lién két héa tri Tao lién két héa tri véi vat ligu mang dugc hoat héa théng qua cdc géc tu do — NHb, OH va SH 6 mach bén cia cic amino acid. Theo nguyén tic, lién két hinh thanh_ trong qué trinh tong téc gitta nhém ua nhian (nucleophilic) eta enzyme véi ede nhém chat duge hoat héa truée dé trén bé mat chat mang, Co dinh enzyme bing céich nhot trong gel Tién hanh nhot enzyme vao nén chit tao gel ty nhién, hay tng hgp, 6 bain chat 1 polymer, 1 kh don gid. Céc polymer nhw Na-alginate, carrageenan, carboxymethyl-cellulose (CMC) hode polymer cationic nhur chitosan, thuong duge sit dung dé tao lién két véi cac ion trai déu nh Ca’? hoje polyphosphate. Tuy nl phuong phdp niy cé mét sé han ché. Ion Ca’? déng vai trd Lm chat két néi gel thuéng d8 bj hda tan khi bd sung thém mudi. Gel dang hinh cau lai mém va xdp, do bén co hoc thip, khé c6 thé nhdi vao Ot phan img, nén it durge sir dung. Tuy nhién huéng cong, nghé nay van duge tiép tye nghién ciru, theo huéng lam cimg nén gel bing cach tgo phe gel véi hat epoxide. 22. Uing dung cia enzyme cé djnh 2.7.5.1. Trong cong nghiép Ngay nay, nhiéu quy trinh img dung enzyme cé dinh trong céng nghiép nhu: cOng nghiép san xuat rugu bia, nude gidi khat, ché bién sita, san xuat da, héa chit... Rugu bia: Cac enzyme amylase, 8 bao nim men cé dinh enzyme duge sir dung 6 quy mé In, Ché bién sita: Enzyme lactase cé dinh dé thity phan lactose trong sita. - Nam 1969 Wilson da san xui n tue glucose bing glucoseamylase cé dinh. = Nam 1971 d& ding Chimotrypsin lin két cng héa tr} véi carboximethyl cellulose lam déng tu sita thay cho rennin dat tién. 2.7.5.2 Trong y hve 2Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Enzyme c6 dinh duye tmg dung nhiéu trong y hoc, dé chita ce bénh di truyén do thiéu enzyme hoie hoat 46 enzyme yéu. Nam 1954, Chung a tao duge vi tiéu cu ‘ban thim cé gin enzyme, nhé thé enzyme cé thé tin tai trong co thé lau dai vi enzyme bj bigt Lap voi méi trum xung quanh. Do dé co thé tao duge néng dé cao cia enzyme thiéu mi khong bi anh hudng cia ede phin ting phy. Enzyme duge c6 dinh con duge ding trong chuan don bénh, Ngoai cae tng dung dign cue enzyme trong phin tich cde chi tiéu sinh héa cia miu nhur lugng: glucose, urea, cholesterol...Enzyme Horseradish peroxidase ¢6 dinh trén polystyren cling véi khang thé , gitip chudn doan nhanh va chinh xéc (Ki thudt ELISA), dua Enzyme L-asparaginase cé kha nang tre ché sur phat trién cia u dc tinh, né trye tiép enzyme nay vao co thé sé bj dura ra ngoai nhanh chéng va wiy nén hign tong dij img, nhumg néu dua cac vi tiéu cau c6 gin enzyme vao co thé sé cé higu qua cao hon. 2.7.5.3. Trong nghién citu khoa hoc Nam 1967, dign cye enzyme di duge ché tao dé xac dinh ndng d6 glucose nhir glucoxydase cé dinh, Dign cuc enzyme li dién cuc oxy trén bé mat gel polyacryamide. Nhiing dign cue vio dung dich glucose thi co chat va oxy sé khuéch tan vao gel chira enzyme. Nhu vay su bién déi dong dign trong hé théng dign cue phu thudc vao tbe 46 phan img va néng dé glucose. Kactsu ding dign cye urease trong mau, ding cholesterol oxydase do ning 46 cholesterol. Clark ding dign cye alcohol oxyreductase dé xc dinh néng dé cén. Ngoai ra enzyme c6 dinh edn c6 thé duge ding vio nhiéu mue dich khée -nhau hur: hoat héa enzyme zymogen, nghién ciru céu trie phan tir protein, sit dung trong, phuong phap sic ki ai hrc dé tinh ché mét sé chat c6 kha nang lién két dic biét voi enzyme. Ngay nay cé nhiéu quy trinh sir dung té bao cé dinh dé xir li mrde thai dat higu qua cao, 23Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 2.7.5.4. Trong bao vé méi ruing Trong cong nghiép ché bién thye phim, rou, bia, ché bién dudng...chét thai, phé phy ligu cia nhitng nha may nay li nguén 6 nhiém ning do giau chat hiru co. Do 6 c6 thé sir dung enzyme c6 dinh dé xit li nuée thai sinh hoc nhim lam giam_ ham lugng hitu eo trude khi dua ra ngoai mdi trudng bén ngo: 2.7.6. Tinh hinh nghién ciu trong va ngoai niréc 2.7.6.1. Tinh hinh nguyén cieu trong nese Nghién edu cé dinh enzyme chi méi bat dau & Viet Nam trong vai nim gin day, ‘két qua thu duge cing cdn nhiéu han ché. Nam 1994 -1995, Vién Sinh Hoc Nhiét Déi Thanh Pho H6 Chi Minh nghién ciru enzyme glucoisomerase trén eae hat Silochrom B2 hoat héa bang glutaraldehyde. Lé Van Higp (1995), c6 dinh vi khudn ti (Vibrio cholerea) lén gia thé PHEMA. bang Ki thuat bite xa, Nguyén Quéc Hign (1996) di nghién ciru ché tao ché pham hormone progesterone thai chim bang ki thuat bite xa. ‘Lé Quang Luan (1997) da co dinh vi khuan Pseudomonas mastophlla dé xt li chit hitu co nude Nguyén Quang Tam (2002) 44 nghién cimu cé dinh pectinase thu thap tir cdc ching nam méc, Nguyén Quyét (2004) da nghién ciru c6 dinh a — amylase thu nhan tir vi khuan Bacillus subtilis. 2.7.6.2. Tinh hinh nguyén citu ngoai music Cho dén nam 1979, ngudi ta cdn rit hy vong e6 thé ép dung céng nghé nay cho tit cd cae loai enzyme. Tuy nhién danh gid thye sy cho thay hign mdi chi c6 2 céng nghé sit dung glucose isomerase va penicillin amidase cé dinh la ¢6 higu qua. Hign tai enzyme amino acid aeyelase, aspartase, fumarase cting da bit du duge ting dung thue tién (chit yéu la & Nhat). 24Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Bang 2.2 M6t s6 enzyme cé djnh dang duge sir dyng trong sain xuat Enzyme c6 djnh San lugng Phuong phap cd ‘Hang san xuat (tdn/nam) djnh Aminoacid acylase | 1000 DEAE sepharose | Tanabe Reactor ming | Degussa Eupergit Rohm ‘Aminoglucosidase | 5000 tsir6 glucose | Than host tinh Tate % Lyle ‘Aspartate 1000 t Nhét t& bao trong | Tanabe carrageeman Furmarase Malic acid Nhdt bio trong | Tanabe carrageeman Glucose Isomerase | 6-7 trigu tin Lactase < 1000 t siré lactose | Hat ceramic, irao | Corning Sumomoto déi ion, soi nylon Lipatse ‘Chuyén ester, thiy | Trao ddi ion celite | Novo Unilever phan Nitrilase 6000 t acrylamide Nhot te bao trong | Nitto polyacrylamide RNAase 55001 RNA Thay tinh, cellulose | VR” China, Japan hoat héa, Eugergit | Rohm Penicillin G/4500 t 6-amino- | Eupergit Rohm amidase penicillanic Beecham, Penicillin 500t Polyacrylamide, sgi | Toyo Jozo V amidase nylon Boehringer Novo Te bao nam men ‘San xuat con Na-alginate KyowaHakko_ 25Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N CHUONG 3: VAT LIEU VA PHUONG PH. NGHIEN CUU NUOI CAY 3.1. Thiét bj va héa chat 3.11. Thiét bj Bé én niet Cn phan tich May ly tam May quang phé UV May lc nudi cdy ‘Noi khir tring May do pH Tu say Ong nghigm Binh tam giée Phéu loc, gidy loc Dita khudy Pipet man cic loai Céc thity tinh: 100ml, 250ml, 500ml Gia dung ng nghiém Ong dong 100ml, 250ml Xilanh Hé théng sac ky c6t ap suat thip Bio-rad, My Cée dung cu chay dign di ‘Ti lanhKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Hinh 3.1 May do pH. Hinh 3.2 Can phan tich im Hinh 3.3 May do quang UV Hinh 3.4 May ly tim aKhoa Iwan t6t nghigp SVTH: Nguyén Trin Nhét Minh. Hinh 3.5 Bé 6n nhigt Héa chat = Triton X-100 — Tween-20 = Tweeen-80 Sodium carboxymethyl cellulose — Coomassie Brilliant Blue — H6a chat dé tia enzyme: Cén 96°, mudi ammonium sulphate, acetone Dung dich dém phosphate: NaH:PO,.12H:0, NaHPOs Héa chit xéc dinh ham rong protein: Thude thit Bradfosd = CaCl, 0,02M = Acid acetic 1%Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh 3.2. Quy trinh thu nhan djch chiét enzyme thé (quy mé cong nghigp) Ché pham. T (Tron ty 18 nude) Dich I Khuiy 4 Loc T Thu Dich T Ly tm (8000 vang/p) T Dich T Tila (bing : cdn, aceton, m r Ly tim 4 Enzyme tho 4 Tinh sgch moni sunfat) 3.2.1, Thuyét mink quy trink Ching nim Trichoderma Reesei duge nudi cAy trén méi trang BM:CM (ba ‘mia:cdm mi), sau qua trinh nudi cdy ta thu dugc ché phim enzyme thé tir méi trong. Can 5 g ché phim enzyme thd cho vio ede thity tinh, thém vao 20 ml nude eat. 29Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Khuay déu dich enzyme trong khoang théi gian 30 phitt (c6 thé khudy trong khoang thdi gian 40, 50 phat, nhumg khoaing thoi gian khudy 30 phit a phi hop nhét), Loe lay dich chiét dirge thé tich khoang 17,5 ml. Dem dich loc dutge di ly tim. ‘Thu dich ly tam. ‘Tua dich ly tim (bing cdn, acetone, ho§c mudi amoni sulphate), trong trumg hop nay ta tia dich ly tam bang cén 96°, vai ty 1é 1:3, tite la: 17,5 ml dich chiét enzyme thé v6i 52,5 ml cén 96°, ‘Tach lay phan tia 6 duéi dem di ly tam. ‘Thu dich ly tam dem edn phan tich thu duge khoang 20,1g enzyme thé. trang th tia enzyme cellulase & 40°C cho dén khi d6 am cudi cing dat 5-8% W (thiét bi say 1ay enzyme rit dé bj bién tinh vi cdn nhiéu nude dé dé bao quan ngudi ta sdy két thudng ding la may sdy phun suong). Sau d6 ta dem enzyme thé di tinh sach bing qua trinh loc gel hay sir dung phuong phap 6 dinh enzyme dé thu duge enzyme cellulase thinh pham, 30Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Hinh 3.6 Djeh chiét enzyme tho sau khi ly tam 3.3. Xée dinh ham hrgng, hoat tinh ciia enzyme c julase 3.3.1, Xée dinh ham lugng enzyme cellulase theo phuong phdp Bradford + Cie Protein khi phan tmg xanh vai xanh Coomassie (Coomassie Brilliant Blue~ CBB) sé hinh thanh hgp chit mau cé kha n’ing hap thu dnh sing 6 bude séng 595nm, cudng d6 mau ty 1é voi ndng d6 protein trong dung dich, Phuong php 6 d@ nhay cao cho phép phat hign téi vai ug protein/ml, dé thye hign va tiét kigm thoi gian, Hoa chat, thiét bi: ‘Dung dich mau enzyme cellulase cin xée dinh. - Dung dich albumine 0.1mg/ml: Can chinh x4e 10mg albumin pha trong 100m! 1u cho tan. Gitr 6 20° C. Khi ding pha loang 100 tan, durge dung dich albumine cé ndng d6 0.01mg/ml. - Dung dich thude thir Bradford: nudeKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh © Bthanl tuyét ddi: 47g Acid phosphoric 85%: 85g Pham mau Coomassie Brilliant Blue duge lim tan trong ethanol trong chai dung, c6 nip, bé sung acid phosphoric 85% va chinh téi 100m! bing nude cat. - Thiét bj: Méy quang phd & Lip dé thi chudin Dé dung dich albumin chudn cé cae ndng dé tir 10-50 ug/ml ta thye hién nhu sau: Bang 3.1 Chuan bj dung djch albumin chun ‘Ong sé 1 2 3 4 5 6 Nong d9 albumin (1g) 0 10 20 30 40 50 Dung dich albuine(ml) 0 OL 0.2 03 04 0S Nuée edt (ml) 1 09 08 07 06 [05 Thude thir Bradford (ml) | 2 2 2 2 2 2 Ong 1 img voi théi gian xudt phat ban dau 0 phat. Thyc hign phan img Lan Iuot 6 cde dng edn lai 2, 3, 4, 5, 6 cde Sng céich nhaw 1 phat Miu thi nghiém eGing duge tién hainh 1 cach twong ty nhur trén, Mau duge pha loang sao cho trj so mat d6 quang do duge trong khoang ciia duémg chuan, Do & bude song 595nm, Tinh két qua Tri mit 46 quang (OD) ciia nhing Ong chimg (6ng 2 dén Ong 6) sau Kkhi trir di tri sé ca dng thir khéng (6ng 1) sé x4y dug duoc dé thi biéu mat d6 quang (OD) theo nong dé protein chuan (ug/ml). Twong ty nhu vay, tri so mat 6 quang cia thi nghigm cing trir di trj sé mat d6 quang cia dng thir khéng, roi chiéu vio duéng cong mau dé suy ra lugng protein cé trong dung dich mau thi nghigm (ug/ml). 32Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N Téng ham lugng protein trong M (g) ché pham thé duge tinh theo cong thite: =bx 10°xmxM mg prot Voi: b: ndng 46 protein suy ra tir duéng chudn (ug/ml) m: h s6 pha loang M: khéi lugng ché pham enzyme thé (g) 3.3.2. Xée dinh hoqt tinh enzyme cellulose bing phuong phip durong chudn glucose Phuong phap nay dya vio su thiy phn co chat carboxymethyl cellulose béi enzyme carboxymethyl cellulase & pH 5,0 va 40° C. Luong dung khir sinh ra duge cho phan img véi acid 2-hydroxy-3,5-dinitrobenzoic, miu sinh ra sau phan img drge ° xa dinh bing phuong phap so mau trén quang phé ké & bude s6ng 540 nm. Dung cu, thiét bi May ly tam. Bé dn nhigt, may do quang phd, may Vortex, pH ké, can phan tich. Bép dign, Chau nude tanh. Que khudy thay tinh, Hoa chit Cén 96" Sodium acetate. NaOH IN. 33Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Bang 3.2 Thi nghigm xac djnh hoat tinh enzyme cellulase ‘Ong sé 0 1 2 3 4 5 6 Nong dG glucose (mgiml) 0 [01 (02 [03 (04 [05 [06 ml dd glucose (Img/ml) 0 ol (02 [03 (04 [05 [06 ml dd CMC 1% I I I I I I It MI dd DNS-Lactose 2 2 re 2 2 2 ry MI nude edt 7 09 [08 [07 [06 [05 [0a Lac déu cac éng nghiém nay, dem dun s6i céch thiy trong 15 phiit. Lam lanh dén nhiét dé phong trong mét chau nude mat. Dat d6 hap thu cia éng sé 0 & bude séng 540 nm bing 0. Do 46 hap thy cita cdc dng can Igi 6 bude song $40 nm, ° ‘Xe dinh hoat tinh enzyme carboxymethyl cellulase (CMCase) 6 hip thu bing 0. ‘Ong nghigm 1: Chinh quang phé Hat Iml dung dich dm Na-acetac 50Mm, pH 5.0 cho vio éng nghigm. ‘Thém 2ml dung dich DNS-Lactose, lic déu dé ngimg phan img enzyme. Thém Iml dung dich CMC 1% vao éng nghiém chua enzyme va lac déu. Dem dun soi edch thiy trong 15 phut. Lam lanh dén nhiét d6 phong trong mot chau nude mit, Dat dé hp thu & bude séng $40 nm bing 0. ‘Ong nghigm 2: Phan img enzyme Hat Im dung dich enzyme cho vio éng nghigm va dé 6 40° C/5 phat. Déng thi, ta cing dé chai chia Iugng dung dich CMC 1% thich hgp 6 40° C/5 phut, ‘Thém ml dung dich CMC 1% vao éng nghiém chita enzyme va lc déu. é phan ting 6 400 C chinh xae 10 phi ‘Thém 2ml dung dich DND-Lactose, lic déu dé ngimg phan tmg enzyme. Dem dun s6i céch thily trong 15 phiit. Lam lanh dén nhigt dO phong trong mot chau nude mat, Bo dé hap thy 6 bude séng 540 nm,Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh - Ong nghiém 3: Khéng cé phan img enzyme ‘© Hit Iml dung dich enzyme cho vao éng nghiém dun s6i tir 5 dén 10 phat dé bat hoat enzyme. © Thém 2ml dung dich DNS-Lactose va lic déu, © Thém Iml dung dich CMC 1% chira dung dich enzyme va lic déu. © Dem dun s6i cach thay trong 15 phit. Lam lanh dén nhiét 46 phong trong mét chau nude mit, Do do hap thy 6 bude séng 540 nm, * Dinh lung don vj host tinh Mt don vi CMCase la lrgng enzyme ma s® giai phéng duimg khir nhur glucose hi thiiy phan CMC v6i van téc 1umol/phiit du6i cde digu kign phan img, 3.4, Khio sit sy anh huéng cia mi pH toi wu 3.4.1. Nghién citu thanh phan méi trong rudng, 46 Am, thdi gian, nhigt 46 tdi wu, a6 ‘Ching téi tién hanh bé tri thi nghiém véi céc thanh phan méi truéng tuong img. nhw sau.Cé dinh thanh phan cam 75% va thinh phin (NHy):SO, 1%, thay déi thanh phan tréi Trdu 1a tac nhan kich thich sinh téng hgp cellulase. Ta cé bang thanh phan. ‘mdi tring thi nghiém duge bé tri nhur sau. Bang 3.3 Thanh phan méi trwong thi nghi¢m theo phan tram Don Vi Tinh Phan Trim (%) Moi 1 2 3 4 5 6 Trung S6 Cam 8 73 5 7B 73 8 Triw 2 20 18 16 4 12 (NH)2S0, | 1 1 I 1 1 1 ‘Thém nude diéu chinh d6 am 60%. iy giéng vao va nudi cdy & nhiét 46 phong khoang 28-30'C. Théi gian khéo sat d6i voi Trichoderma.viride la 72 gids, Sau d6 xc 35Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh inh hoat tinh enzyme cellulase (d6i voi Trichoderma.viride) bing cach do 49 hap thu quang phé 4 bude song 540 nm cho cellulase. Trong thanh phan méi truéng nhan gidng, ching tdi cd dinh ham lugng cam va mudi sunphat amén ((NH,)2S0,), thay di thanh phan triu . Tréu la tic nhin lim cho méi trudng thoang xdp, tao digu kign cho nim méc phat trién tét. Ban dau nam méc sé tin dung nhing chat dé tiéu trong méi trudng nhur cde logi dudng nhung khi méi trudng dinh duéng bj can kiét thi nm méc phai tiét ra nhimg enzyme dé phan hii co chat khac thanh dudng dé tiép tue tn tai va phat trién, Tuy nhik , cling thy thuge vao néng dé ctia co chat nhieu hay it ma nam moc sé tiét ra Ingng enzyme khic nhau. Su téng hop ciia enzyme con phu thude vio kha nding tiét t6i da cia timg loai vi han nhat dinh thi hoat tinh enzyme sé giim do 2 nguyén nhan, thir nhat do co chat tao ap Ie lam gidm sinh vat co nghia la néu ting him lugng co chat vugt qué mot gi qué trinh sinh téng hgp, thir hai do co chat ving trong khi nding lye tiét 16i da khong adi 3.4.2, Nghién citu anh luring ctia thoi gian Két qué cia thi nghigm trén duge Ap dung dé tiép tue cho thi nghigm khi chon duge méi truéng tét nhat cho sinh téng hop enzyme cellulase (4éi voi Trichoderma. viride), ta tién hanh bé tri thi nghiém nudi cdy nim méc va Ky miu 6 Kiide nhau, Thai gian khio sit tir 28 ~ 32 — 36 ~ 40 ~ 44 — 48 — 52 gid, 60%. ther di itt nguyén 46 Khi cdy nim méc vao méi trudng, ching sé khdng tiét ngay ra enzyme ma sé tiét kigm ning lugng dén mite ti da va sir dung ngay nhiing chat dé sit dung nhat 1a. cac duéng don, khoang chit c6 sin, Sau dé ching sé tiét ra enzyme tiy vio thinh phan cdc chat cd trong méi trudng. Nam méc phat trién duge chia ra thanh 4 giai doan nhw sau: thich nghi, phat trién, cén bang va tir vong. G giai doan thich nghi va phat trién, nam mic sé tim cach thich nghi va tiét ra loai enzyme nao va sé Iugng bao nhiéu dé 36Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh co thé ton ti va phat trién. Theo thi gian thi lugng enzyme tiét ra sé ting din va én mét mite 46 én dinh, sau d6 sé giam di khi méi trudng dinh dudng can kiét din, Qué trinh sinh tng hop enzyme cellulase ciing tng tuong img theo thi gian. O Trichoderma. viride, thoi gian ting trudng chim hon nhung tao ra hé enzyme cellulase rat tt. San phdm cia hé enzyme cellulase 14 cellobiose, mét chat kim ham hoat tinh. cia exo - B -glucanase Do dé ma qui trinh sinh tong hop enzyme cellulase & Trichoderma. viride ting trudng manh trong khoang thé ian tir 4 ngay. 3.4.3. Nghién citu anh hudng cia dj Ciing giéng nhw thi nghiém trén, nhiing két qua khao sat duoc 4p dung nhw thanh phin mdi trudng va tha gian cho thi nghiém nay. Thay nhur sau: 50% - 54% -68% - 62% - 66% - 70%. Cée qua trinh sinh truéng va phat trién cua ndm méc déu c6 lién quan dén do 4m. D6 Am 1a yéu t8 quan trong trong m 19 Am theo cic wid i trong. Néu 6 Am qua thip thi xay ra hign tugng logi nude ra khoi té bao nm méc va lam cho té bao bj chét, Diéu nay sé lim han ché su sinh trudng va phat trién ctia ndm méc, Néu 46 4m qué cao thi cing khong tét cho sur sinh truéng va phat trién. G mot d6 am nhat dinh, khi 46 hoat dng trao déi chat ign ra t6t nhat thi sw sinh truéng va phat trién cia nm méc sé dién ra tét nhat. 6 méi trurdng thi nghiém nay, ching t6i nhan thay 6 46 4m 58% thich hgp cho qué trinh sinh tong hgp enzyme cellulase ¢ hoat tinh cao nhat & Trichoderma. viride. Tir cdc két qua trén, ching t6i nhan thdy dé nim méc sinh truéng va phat trién t6t va san sinh ra timg loai enzyme cut dai thi tiy thugc vao tig loai giéng ma ching ta bé tri d6 am thich hop. 3.4.4 Xie dinh dp pH ti wu Dé xac dinh pH phan img tdi wu cho phan img xuc tac cua enzyme nghién citu, dich enzyme tinh sach (0.002 mg protein) dug i vi co chit CMC pha trong dém 100 ‘mM sodium acetate, pH tir 3.5-3.5 va 100 mM potassium phosphate, pH ti 6.0-8.0, 37Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N hin hop phan img duge 6 50°C trong 20 phitt. Dé danh gid do bén pH cla enzyme nghién citu, true hét cellulase (0,002 mg protein) duge a trong cic dém c6 pH thay di trong khong 3.5-8.0, trong 2 gid 6 37°C. Sau dé, chon ra khong pH thich hop dé tiép tuc xc dinh d6 bén pH theo thdi gian: 6, 12, 24, 36, 48, 60, 72 gid. Tét nhdt & pH 5.0. 3.4.5. Xéc dinh nhiét dé toi wu Bé xac dinh nhigt d6 nudi ‘moi trudmg: urea: 0.3, (NH,)sSO.: 1.4, KH:PO,: 2.0, CaCl: 0.3, MgSOs.7H20: 0.3, peptone: I, cao nam men: 10, tween 80: 1, CMC: 10, cae yéu t6 vi lugng: 0.1% (w/v) ‘eSO,: 18 mM, MnSO,: 6.6 mM, ZnSO,: 4.8 mM, CoCl: 15 mM, pH: 7.0 co ban cho sinh téng hgp cellulase, Kc 200 vong/phiit 6 25°C, 28°C; 30°C, tOi uu, ching nam nghién ctu duge nudi trong bao gdm cae mud 32'C va 37°C, pH 7.0. Dich canh truéng duge thu sau 96 gid nudi cay va xdc dinh hoat tinh cellulase, t6t nhat 6 30° C. 3.5. Khao sat tac nhiin trg tia 3.5.1. Tita protein-enzyme bing mudi, dung méi hitu co hogc polymer Thuong duge sir dung nhu 8 bude tinh sach dau tién, béi ching c6 46 chon loc Khéng cao, dé thay déi. G néng d6 mudi thap, d6 héa tan cita protein tang vei néng do mudi gia ting, goi [a staling in. Khi néng 46 mudi ting cao hon, thi 4 hda tan cia protein giim va tao ta, goi la staling out. Chit yéu céc cation va anion ctia mudi trong tée v6i nude bao quanh phan tir protein, lam ting tinh ky nude ca protein, din dén tao tia, Trong d6, céc mudi trung tinh nhu mudi sulphate vi phosphate, dic biét 1a mudi (NH,)2SO, 18 thong dung hon ca. 3.5.2.Tiia enzyme bing mudi (NH):80, 6 cée ning dp khic nhau Ché phim enzyme cellulase thé dang bét cia ndm Trichoderma reesei dug chiét bing nude cét, thu duge dich chiét enzyme thé. Dich chiét thé nay duge tia voi mudi (NH,);SO, so 6 cae néng d6 (% do bao hda) : 50, 55, 60, 65, 70 va con 96"6 cic ty 16 dich chiét enzyme: cOn la 1:1, 1:2, 1:3, 1:4, 1: :6 clin tha thu duge ha vao 38Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N dung dich dém va xée dinh hogt tinh cellulase nhim tim ra néng dG mudi va ty I¢ eén tdi wu cho viée tua cellulase. Tir dé khio sit diéu kign t6i wu cho hoat déng cia cellulase tir dich tia enzyme thu duge tir ndng 46 mudi va ty 1é cén t6i wu. Cudi cing, dich tia pha trong dém duge chay qua sic ky Toe gel vi xi dinh trong lwong phan tir 65% d9 bio hia va cellulase bang dién di qua thu dugc nhu sau: 6 ndng 46 mudi ty Ié cdn 1:4 1A tdi uu dé tia cellulase, Cellulase hoat dng manh nhit 6 pH =7 va nhigt d6 47” C, 3.5.3. Tita protein-enzyme bing phitc hop nuéc va dung méi hitu co Thuong duc sir dung 6 giai doan dau tinh sach enzyme. Co tro ngai la phai thuc hién 6 nhiét d6 thap (khong 20°C), vi dung méi hau co gay bién tinh protein & nhigt d6 phéng. Ethanol va acetone la 2 dung méi thyéng duge sir dung nhiéu nhat. Két tia bang dung mdi hitu co uu diém hon két tia bang mudi, vi cé d6 chon Igc cao hon, it tao tia v6 dinh hinh, dé ding thu nbn bing ly tm, Tda tgo thanh nhé tong t ign gitta cac phan tir protein véi nhau, ké ca sy cé mat tong tac hdp din Van de Waals, lim gidm hing s6 dign méi gidta ching. 3.5.4. Tiia protein-enzyme bang cde polymer trung tinh Cae polymer trung tinh tao tong tic gitta cae protein vi nhau, nhé wu thé logi bd boc mude, din dén tao tia protein vi ich phase. Khée voi dung mdi hiu co, cde polymer trung tinh nhu polyethylene glycol (PEG), khong gay bién tinh protein, nén c6 thé tién hanh 6 nhigt 46 phong. Ngoai 3 phuong phdp tao tia phén doan protein ké trén, mot sé phuong phap tao tila khic ciing duge sit dung. Dé 18 tao tila phin dogn protein bing céch gay bién tinh béi nhigt d6, 46 pH, dung méi hitu co, tao tia logi protein giy nhiém. Dé ting tia dic higu, nguéi ta bé sung céc tay ndi (ligand) dic hiéu vio cc polymer tao “tia ai lye protein” cho phép lam sach dang ké protein-enzyme dich. 39Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N 3.5.5. tia enzyme bing diém ding dign Dua trén tinh chit protein c6 d0 hod tan thép 6 diém ding dién. G diém ding dign d9 hydrate-hod cita protein li eye tigu Lim ting tuong tic gitta cae phan tir protein, din dén tao tia. Khé khiin iia phutong phap nay 1a do protein chi c6 khoang gid tri pH dang dién gidi han. Phuong phap nay cho higu qua khéng cao, tuy nhién cach tién hanh phite tap va thuéng phai két hgp v6i phuong phap tua bang dung méi hitu co. 3.6. Cé dinh enzyme cellulase trén chit mang Natriaginatel > Tao dung dich Natrialginate 3%: Can 0.75g Natrialginate hda tan trong 25 ml dung dich dgm acetate pH 5. Khudy dén khi tit ca Natrialginate hoa tan hoan toan li duge. i. Tién hanh cé dinh: Can 0.5g ezyme cellulase hda tan trong dung dich Natrialginate 3%. Dung éng xilanh cé dau kim tiém, hit dung dich enzyme hoa tan nay nhé tir dd eao 20em vao trong 100ml dung dich 0.2m CaCl dé tao gel. Qiia trinh tg0 gel xdy ra trong 2 gid, & mhigt 49 phong. sau cd dinh, ding gidy lgc, loc enzyme da duge cé dink. Két qua thu duge hat gel enzyme cellulase da duge nhét trong khudn gel. Dich loc thu duge dem di xe dinh ham lung theo phuong phip Bradford va xc dinh hoat tih theo phuong phap cia cng ty Shin Nihon Chemical Co, Ltd. > Cich tinh: HL Protein c6 dinh (mg) = HL Protein tric 6 dinh — HL Protein con lai Hiéu sudt gin Protein duoc tinh theo céng thtre sau: HS ¢é dinh Protein = HL, Protein cé dinh/HL Protein trude 66 dinh x 100 Higu suat hogt tinh enzyme cellulase c6 dinh so véi hogt tinh enzyme cellulase bban dau: Y DVHT E cellulase cé dinh HS hoat tinh enzyme c6 dinh (%) =~ xX 100 Y DVHT E cellulase true 5 dinh 40Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N Trong do: ¥ DVHT E cellulase 6 dinh: $6 don (uve). ¥ DVHT E cellulase truvéc 6 dinh: Sé don vi hoat tinh enzyme cellulase true 6 dinh (U/g). > Khao sat sé lan tai sir dung enzyme cellulase c6 dinh trén Natrialginate ig Khao sat hoat tinh va ham Iugng lan 1 va rita lai bling CaCl>, tiép tue khdo sat hoat tinh joat tinh enzyme cellulase cé dinh ¥y loc, thu dich nude tién hanh Sau khi hat gel da duge c6 dinh ta boc ‘va ham luong lan 2 cir nhur thé ta khao sat duge lin 3, Kin 4,.... Vé dé thj sé Lin tai sir dung gidm 50% so véi enzyme ban diu. 3.7. Tinh sgeh enzyme 3.7.1. Chudin bj héa chat va dung ew 3.7.1.1. Dung cu vis thiét bj © Phéu dé gel © Binh hit chin khong © Cét sic ky Bio-Rad, kich thudc 30 x 1,5 cm, thé tich 53 ml (thé tich = chiéu cao ct x (during kinh/2)’ x 3,14 () = 30x (1.5/2)' x 3,14 ‘© Binh dung dung dich dm © Ong nghiém: 20 cai Voi hit chan khong 48 khit bot khi cho e: 3.7.1.2. Héa chat dung dich, Gel “Sephadex G-100”, e6 ede dae tinh: dang hat min, kich thude hat 45-90 um, kha Dém Acetate 50 Nm, pH 5.0: khir bot khi truée Khi ding 1g ngim muse: 12ml/1 g gel khé, pham vi phin tich: 5.000-100.000 Daltons. 3.7.1.3. Tinh sach enzyme Dich enzyme sau khi duge ly tim 10000 vong/phit trong 10 phit, duge dra Lén 6t slic ki loc gel Sephadex G-100 véi tng thé tich 10 ml, Cot e6 41Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh kich thude 2,6 x 60 em duge cdn bing vai dém 50 mM potassium phosphate, pH 7.5. Téc d6 ding chy 1a 25 ml/gid. Thu 20 phan doan, thé tich mdi phan doan 2 ml. Hm long protein va hoat tinh enzyme due xie dinh trong mdi phan doan. Dich enzyme sau khi qua c6t sic ki loc gel Sephadex G-100, chon nhiing phan doan cé hoat tinh endo- B -1,4-glucanase cao duge dua tiép lén c6t sic ki trao déi ion DEAE v6i tng thé tich 12 ml. Cét c6 kich thude 2,6 x 60 cm duge cin bing vi dém 50 mM Tris-HCl, pH 8,0, 50 mM NaCl, thoi mau bing dém 50 mM Tris-HCl, pH 8.0, 1000 mM NaCl. Toc d6 dong chay 18 3320 ml/gié. Thu 20 phan doan, thé tich méi phan doan 2 ml. Him luong protein va hoat tinh enzyme durge xac dinh cho méi phan dogn. Qua trinh tinh sach endoglucanase e6 thé duge tom tat nhur so dé: Dich enzyme tho 1 Ly tém 10000 ving/10 phiit 4 Dich trong, J ‘Cot Sephadex G100 tv Cot DEAE v Enzyme tinh sach 42Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N 3.8. Xée dinh trong lurgng phan tir bing dign di trén gel Polyacrylamide 3.8.1. Gi6i thigu vé gel Polycrylamide Gel polycrylamide Li gel duge tao thanh do sw polymer héa cée phan tit acrylamide vi N, N’-methylene-bis-acrylamide. Qiia trinh polymer héa duge xtic tie bai hé théng ammoniumpersulfate (APS), N, N,N’ ,N’-tetramethylethylenedi (vel IED). Sy di chuyén cha cdc phan tir trén gel phy thuge vio dign trudng va thude cia 6 gel. Kha nang phan téch cing nhur giéi han trong Iugng phan tir cia cde phin ur sinh hoc ma gel ¢6 thé phan téch tay thude vao ndng do acrylamide va bis- acrylamide: ng d@ thap sé tao ra 18 gel Ién, cho phép phan tach cac phan tir sinh hoe cé kich thuéc Idn va ngugc lai. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) 1 mot Ki thudt dign di trén gel polyacrylamide khi c6 sw hign dién cia SDS, day la mot tée nhain lam bién tinh va dm tinh héa cde phén ti protein. Trong Ki thudt nay protein duge xir ly véi chat tay SDS va tic nhan khir cdu néi disulfite i mercaptoethanol hodc dithiotheitol (DTT). Véi cdc tic nhan nay protein tir edu tric bae 2, 3, 4 duge bién di thinh chudi polypeptide (bac 1) va tit ca cde protein déu duge tich dign am. Nho 46, su di chuyén trong gel ca cac phan tir protein chi phy thude vao kich thurée, nhing phan tir 66 kich thuée Ién s@ di chuyén cham hon nhimg phan tr e6 kich thuge nho Khi di qua mét 16 gel c6 kich thuée nhat dinh. Dudi tac dung cia dign trudng, ic phan tir tich dign am sé di chuyén vé cye duong, va céc phan tir tich dign duong sé di chuyén vé cue dm cia dign trong, Dé x4c dinh phn tir Iegng cia mOt protein, ngudi ta thudng so sinh vi mot thang phan tir tugng chun, 18 m6t hdn hop cdc protein c6 trong long phn tit khic nhau da biét. Dé dua cac protein trong mau vé cing mét vach xuat phat déng thdi tao ra sy phan tach t6t hon, ngudi ta thuong sir dung phuong phap dién di trén gel khéng lién tuc (discontinuous). Trong phuong phap nay gel gom 2 lip: 4BKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Lép gel gom (stacking gel) (Iép trén): cde protein trong mau duge din Iai va tao ‘mot lip bang méng. Lép gel phan tach (separating gel) (Iép duréi): tao ra cc bang protein cé trong, Iwong phan tir, kich thuée khdc nhau tir m6t hén hp protein e6 xuat phat ban dav. Hai l6p gel niy duge phan bigt nhau béi dung dich dém, néng d6 acrylamide va vi ti Ki thudt SDS-PAGE cé thé xe dinh duge nhiing phan tir protein c6 trong lngng phn tir tir 10.000-200.000 Daltons. Nhing phan tir protein cé trong long Ién hon 200.000 Daltons thudng duge xée dinh trén gel c6 ndng d6 acrylamide it hon 2.5%. Ngoai ki thuét SDS-PAGE 1a phuong phap dign di trong diéu kign lam bién tinh protein (Denaturing condition), nguoi ta con ding mét sé ki thudt dién di trén gel polyacrylamide khac nhim phan tich protein trong diéu kign khéng bién tinh (Nondenaturing condition) khong c6 su hign dién ctia SDS hodc dé khic phyc mét vin dé vé SDS gay ra mOt sé anh hudng dén dc tinh cia protein nhu tao lien két (cross-linkes) ira cc protein véi nhau. Mét sé protein khdng cé su ti Ié gidta dign tich va khdi lung véi nhiing protein khde nguisi ta sit dung ki thudt dign di tp trung diém ding dign (Isoelectric focusing gel electrophoresis). 3.8.2. Véit ligu 3.8.2.1. Dung cu va thiét bj ~ Bé dign di chimg ~ BO nguén chay dign di (electrophoresis power supply) Biorad - Pipetteman 1000 ul. = Pipetteman 200 ul. = Pipetteman 10u1. Tip xanh: | hép. - Tip vang: 1 hop. = Tip trang: | hop. = Gidy Ige.Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Gidy tham. Gang tay: 1 di, Phao. Eppendorf. pH ké 3.8.2.2. Héa chat NN, N, N’, N’-tetramethylenediamide [CoH,sN:-TEMED] Dung dich Acryamide/Bisacryamide 40% (29:1) (3,3 % C) f-Mercaptocthanol [HSCH;CH,0H] Thang protein chuin SDS-PAGE (Bio-Rad), g6m cdc protein chun c6 kich thuée xe dinh sau Phosphorylase b 97.400 Daltons Bovine serum albumin 66.200 Daltons Ovalbumin 45.000 Daltons Carbonic anhydrase 31.000 Daltons Soybean trypsin inhibitor 21,500 Daltons Lysozyme 14.400 Daltons Sodium Dodecy! Sulfate [SDS] Ammonium persulfate ((NH,)2S2Os][APS] int Blue G 250 dang vién (Bio-Rad) Cén tuyét d4i [99,5 49] Methanol [CH;OH] Tris (hydroxymethyl) aminomethane Coomassie Bri Axit acetic Bromophenol blue Glycerol [CsH,O5] Glycine [C2Hs02N] 45Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N + Axit Clohydrie [HCI] - Sodium dodecyl sulfate (SDS) 10%: edn 10g SDS cho 100ml nude edt vio khudy cho tdi khi tan hét. = Dung dich dém gel phan tach (separating gel buffer) Tris-Cl 1.5 M, pH 8.8 0.4% SDS: in 36,3 g Tris pha thém 100 ml nude cat thém dan HCl 6 N va Khudy dung dich trén cho téi khi pH ké chi 8.8, Hit 8 ml dung dich SDS 10% cho dit 200 ml, lac déu git 64°C. cho vo. Thém nude - Dung dich gel gom (stacking gel buffer) Tris-Cl 0,5M, pH 6.8-0,4% SDS: en 6.05g Tris pha trong 100ml nude cat thém din HCl 6N va khudy dung dich trén cho téi khi pH ké chi 6.8. Hat 4ml dung dich SDS 10% cho vao. Thém nude cat cho di 100ml, déu git 6 4 C. = Ammoniumperslfate 10% (chi pha truéc khi ding): can 0.1g cho vao mét eppendorf 100041 thém Iml nude edt, lac déu, 42 6 nhié 4 phong, = Dung dich dém dign di Tris-glycine pH 8.3: pha dung dich me 10X chira 30g ‘Tris, 144g Glycine, 10g SDS hda trong 100ml nuvie cat, - Dung dich nap miu 2X (2X treatment buffer) ¢6 thainh phan nhur sau: 2.5m! dung dich dém Tris-Cl 0.5M, pH 6.8, 4ml 10% SDS, 2ml Glycerol, 2mg Bromophenol Blue, 0.2 ml mercaptoethanol, thém nu6e dit 10m = Dung dich nhuém gel: chuan bi 250ml dung dich nhuém gel chira 0.625g Coomassie Blue G 250, 112.Sml ethanol tuygt 461, 112.Sml muie edt vi 25ml axit acetic, lic déu, gitr trong chai mau tdi. ~ Dung dich gidi nhudm: 30ml methanol: 10ml acid acetic: 60ml nurée cat. 3.8.3. Phwong phép 3.8.3.1. Dé gel Chuan bj 46 mét gel co kich thuse 100 x 100 x 0.75mm cé néng 46 acrylar 14 10%. > Gel phan tich (separating gel) 46Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Cho cdc thanh phan sau theo thir ty vio becher 50ml sach: 40% Acrylamide/Bis (29:1) 2.5m! Tris-HCI 1.5M pH 8.8-0.4% SDS 2.5m Nude cat 4.445ml TEMED Sul Ammonium persulfate 10% 50 ql ‘Téng thé tich 10ml Sau khi cho Ammonium persulfate 10% vito, lic nhe Sng Faleon vai Kin (tranh tgo bot khi), diing pipette Pasteur hofie pipetteman bom dung dich gel vao khudn, 46 gel sao cho mite dung dich gel cao hon Tem (trinh tao bot khi trong khudn gel). Sau 6, nhe nhang dat mot lop nude edt 1én trén Ksp gel dé mat gel duoc phiing. Cho gel dong (khoang 20-30 phut). > Gel gom (Stacking gel) Cho cc thinh phin sau vao m6t Becher 50ml khic: 40% Acrylamide/0.85 bisacryla 0.5m1 Tris-HCl 0.5M pH 6.8-0.4% SDS Iml Nude cat 2.476ml TEMED 4ul Ammonium persulfate 10% 20u ‘Téng thé tich 4m1 Sau khi gel phan téch d3 tring ngung hoin toin, dé hét nude bén trén, Tién hanh dé l6p gel gom bang cach ding pipette Pasteur hoc pipetteman bom dung dich gel gom vio khudn. Dong thi dua luge vao gel dé tao cic giéng mau (tranh tao bot khi trong khuén gel), Sauk hi gel tring ngung > thio luge > lip dia gel vio b9 phan dign di > cho dung dich dign di vao budn dign di. aKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran N 3.8.3.2. Chuan bj mau va chay dign di > Xtrly miu Hut 30u1 dung dich nap mau (2X treatment buffer) va 30u1 dung dich miu protein vao ‘mot eppendorf sach, dy nip va bing nhe cho déu, dun s6i cach thiiy 5-10 phat. Ly tam 10.000 vong/phiit trong 1-5 D6i vi nhiing thang chuan protein (BioradO hut 2y11 vao eppendorf Im! chita 8 yl nude: ct vi 10ul dung dich nap miu & Tién hanh chay dign di giéng (20ul/giéng). hanh chay dign di én dinh dong: 100V © Ding pipetteman 1011 nap mau vio eT © Sau khi dign di, nhudm gel voi dung dich nhudm da chuan bj truée (2-4 gid) | gid, Sau dé, tién hanh gidi nhuém bing cach ngim gel trong dung dich gidi nhugm cho dén khi nén gel tr nén trong sudt khong mau, Sauk hi nhudm xong, protein duge phat hign nhi céc vach miu xanh lam trén nén gel trong suét. 3.8.33, Xe dinh trong long phan tic cia protein - Bo khodng eich di chuyén ctia eéc band protein tir gel phan tach dén cde band protein va khong cach di chuyén tir gel phan tich t6i vach mau cudi cing. - Tinh gid tri RF Khoing cach di chuyén cia protein RE Khoang cach di chuyén cua vach mau Brommophenol Blue ~ Ve biéu dé logio ciia cdc gif tri trong Iuong phin tir cia cde protein chuiin va cic gid tri RE cia ching - Trong Iugng phan tir cita cc vach protein chua biét trong Iugng phan tit c6 the xae dinh thong sé gid tri Rf cla chiing thong qua phuong phdp ngoai suy tir dd. thi. 48‘Khéa ludn tot nghigp ‘SVTH: Nguyén Trin Nhat Minh 3.9. Khao sat s6 lan tai sir dyng enzyme cellulase trén Natrialginate Can 1 g enzyme cé dinh dem xdc dinh s6 lin sit dung ciia enzyme cellulase trén Natrialginate theo phuong phép myc 3.6. Xéc dinh hoat tinh enzyme cellulase theo phuong phép muc 3.2.2. Bang 3.4 Két qua khao sat hogt inh enzyme cellulase qua cac Lin tai sir dung S6 lin Hip lai Hoat tinh (U/g) ‘Phan trim hoat tinh gitr Iai (%) 7 136.15 100 2 104.97 7.09 3 73.76 34.18 4 31.08 37.52, 3 39.75 29.20 6 31.25 22.96 7 1421 10.44 Hinh 3.7 Dé thi biéu dién hoat tinh cia enzyme cellulase qua cc Hn tai sir dung 49Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Nhan xét: Vie tai sir dung enzyme li dae tinh quan trong eta enzyme e6 dinh. Tuy nhién qua cac lan tai str dung hoat tinh cia enzyme bj gidm so véi hoat tinh ban diu cla enzyme cé dinh. Su gidm hoat tinh cia enzyme 6 dinh phu thudc nhiéu yéu t6.Theo 45 thi thi hoat tinh cellulase ¢6 dinh trén Natrialginate gidm di rét nhiéu so v6i ban dau. G lan tai str dung thir 4 hoat tinh cellulase chi con 37.52%. Higu suat tai sir dung cia cellulase c6 dinh trén Natrialginate khéng cao c6 thé 1a do co chat Natrialginate kém bén v6i nhigt d6 va céc diéu kién phan img. Sau mdi lin tai str dung, enzyme bi that thot nhiéu, do dé hoat tinh cellulase c6 dinh giam rat nhanh, Tuy nhién sau 4 lan tai sir dung hoat tinh cellulase linh gidm di it hon so voi nhing lin dau tién, C6 thé 1a do durdng kinh hat gel Natrialginate kha Ion nén véi nhimg diéu kign phan img chi téc dng dén mét phin hat gel lam cho nhiing lan tai sit dung sau lvong enzyme trong hat gel it thdt thoat hon nén hogt tinh cellulase ¢6 dinh nhimng lan sau giam it hon va én dinh hon. Tuy vay hoat tinh cita nhiing Lan tai str dung sau van thap (hoat tinh con gidt Iai 10.44% sau 7 lan tai sir dung). 50Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh (CHUONG 4: KET LUAN VA DE NGHI 4.1. Két luan Tir nhitng nghién ciru vé quy trinh téch chiét enzyme cellulase tit Trichoderma reesei xc dinh duge cae yéu t6 nhigt 49, pH, d6 Am co cht anh huéng dén qua trinh sinh téng hop cing nhw tich chiét enzyme cellulase. Xae dinh duoc tée nhiin cia enzyme higu qua nhit cho quy trinh phan hay co chat va cing nhw gidip cho quy trinh thu héi enzyme tét nhat. Xay dyng quy trinh tich chiét enzyme cellulase bing phuong phap sic ky loc gel déng th 4.2. DE nghj Nghién ettu, phan lap va tuyén chon cde ddng ndm Trichoderma Reesei bj d6t xe dinh trong lugng eta phan tir enzyme to thinh, bién xem déng nao mang lai higu qua sin xuat enzyme cellulase 14 cao nhit, ma gia thanh san xuat lai it t6n kém Ngoai chiing ndm Trichoderma Reesei ra, thi chiing ta nén tién hanh khao sat thém kha ning sinh téng hgp enzyme cellulase cia cae ching nim méc Khéc nhu : Bacillus subtilis, Apergilus, vi khudn, xq khudn...xem khd ning tong hop enzyme cellulase eta chiing nao 1a manh nhit, mang lai higu qua kinh té cao nha ‘Tan dung va xit ly 6 higu qua ngudn phé thai hitu co tir cée nha may : mat cua, ‘ba mia, cdim mi, ba diu nanh... N6 khéng chi gop phin vio bio vé moi trugng ma edn tgo ra mt long Ién enzyme cellulase ing dung trong cng nghigp ché bién thyc pham, céng nghiép sin xudt b6t gidc, trong duc phim. Bén canh tng dung cia cée ching nim dé san xudt enzyme cellulase thi ta cing nén nghién ctu thém nhing tg dung khdc ctia ching nam dé phyc vy cho khoa hge, cling nhur trong doi sdng hing ngay. slKhoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh TAL LIEU THAM KHAO. Tai ligu tiéng vide [1] Pham Thi Anh Héng, Ky thuat sinh héa, nha xudt ban Dai hoc quéc gia thanh phd H6 Chi Minh,113 - 114, 2003. [2] New nghigm héa sinh hge, nha xuat ban Dai hge quéc gia thanh phé Hé Chi Minh, 128 - 129, 2003. [3] Lé Héng Phi, Nghién ctu sinh téng hop Enzyme Pectinase va Cellulase tir Due Lugng, Cao Cuimg, Thi nghiém céng nghé sinh hoc, tap 1- Thi Aspergillus niger va img dung dé xit ly vo ca phé trong san xudt phan hitu co, Lugn vin thac si nginh sinh hoc, Truong Dai hoc Khoa hoc Tu Nhién thanh phé Hé Chi Minh, 2003, [4] Nguydn Van Tuan, tuyén chon nudi cay ching Aspergillus Awamori sinh téng hgp Endo- f-1,4-glucanase va danh gid tinh chat ciia Endo-B-1,4-glucanase, lun van Thge hoa Hoc Sinh Hoe, Truong Dai Hoc Thai Nguyén . [5] Trin Thanh Phong, Hoing Quée Khanh, VO Thi Hanh, Lé Bich Phugng, Nguyén Duy Long, Lé Tan Hung, Truong Thi Hdng Van, Thu nhan enzyme cellulase cia Trichoderma Reesei trén mdi truong ban rn, Vign Sinh Hoc Nhigt Déi-Vign Khoa Hoe Va Cong Nghé Vigt Nam. [6] PGS TS Nguyén Tién Thing, Giaé trinh céng nghé Enzyme, TP HCM 2010 52Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh ‘Tai ligu tiéng anh Okada G., The growth of Trichoderma viride on me Biochem, trang 591 — 607, 1963 containing cotton fibres, J Ruy D.D.Y. And Mandels M. ~ Cellulase, Biosynthesis and applications - Enzyme Microbiology Technology, 1980. Gary J. Samuels, Trichoderma a guide to identification and biology. Unit States Deparment of Agriculture Agricultural Research service Systermatic Botany and Mycology Laboratory 304, B-011A Belisville, MD 20705-2350, USA, 2004. Science & Technology Development, Vol 12, No.13 ~ 2009. 33Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh PHY LyC Phy Iyc A. Bang 1.1: Dai dign vi sinh vat sin xudt enzyme cellulase ‘Vi Sinh Vat Vi Khuan ‘Nim ‘Actinomycetes ‘Chrysosporium Acremonium cellulolyticus ‘Thermoactinomyces sp. Aspergillus acculeatus ‘Aetinomycetes Schizophytlum x% ‘Thermomonospora curvata Aspergillus fumigates Selerotium rolfii Aspergillus niger Sporoirichum cellulophilum Fusarium solani Talaromyces emersonii Trpex lacteus Thielavia terrestris Penicillium funmiculosum: Trichoderma koningii Phanerochaete Trichoderma reesei Clostridium thermocellum: Trichoderma viride Ruminococeus albus Streptomyces sp. Phy luc B. ‘Méi trudng nudi cdy cde ching Vi Sinh Vat M6i trudng nudi cdy; Méi trudng (MT) nudi cay vi khudn: CMC 1%, peptone 0,05%, cao thit 0,1%, NaCl 0,1%). MT nudi cay xa khuan: CMC 1%, tinh bét 0.1%, cao ndm men 0,02%, MT nudi cay ndm sgi: CMC 1%, cao malt 0.02%, MT nudi cay nim men: CMC 1%, glucose 0,1%, peptone 0,05%, cao nim men 0,03%, cao malt 0,03%. MT nudi edy cae ching vi sinh vat sinh cellulase due swu tép tir phong thi nghigm cia truémg Bai hoe khoa hoc Tw nhién: CMC 1%, tinh bot 0.1%, MgSO4 0,02 34Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran N %, NaHCO3 0.02%, K2HPO4 0,5%, CaCl2 0.02%, NaCI 0,02%, peptone 0,02%. Mai trugng AN: KH2PO40,1g,NaNO30.3g, MgSO4.H20 0,05g, KCI 0.05g, FeSO4 7H20 0,01g, Cao bip 0.5g, gidy loc 2g, mic cat 11. MT Zapek, Cell Phy Iyc C. Gel “Biogel-P-“Hang Bio-Rad C.L.Gidi thigu CAc loai gel “Bio-Gel P” li cae hgt polyacrylamide tao 15, duge diéu ché bing qua trinh déng héa polymer héa acrylamide va N, N’ ~methylene-bis-aerylamide. Cac gel nay rit va nude va khong mang dign tinh, gitip viée loc cde hop chat nhay cam qua gel dat higu qua. 6 phan giai cao duge xac dinh bang 2 yéu t6 (1) su phan bd hep va consistent cia during kinh hat, (2) su phan tich t6t theo trong. rong phan ttt. Gel “Bio-Gel P” 1 ch hgp véi cae acid hitu co yéu, uré 8M, guanidine 6M, cdc tic nhan chatropic, cac tac nhan khir nhu dithiothreitol va mercaptocthanol, céc tac nhan tiy nh SDS, CHAPS, vi Triton X-100, Bio-Gel P e6 thé duge ding v6i nude cat, tuy nhién cac dung dich 48m cé cumg a6 ion > 50Mm sé mang lai higu qua phan tach protein t6t nhat, C6 thé cho dung mdi hitu co miscible vao chat giai hap duge ding véi gel “Bio-Gel P”. Cin (c6 thé dén 20%) khong Lam thay d6i de tinh phan tach ca gel, va trong mét vai tring hgp né cén Lim t&ng qua trinh phan tach céc hén hop phite tap eta ede phan tir hoa tan kém trong nude nhu ede nucleotide, peptide, tamin, C6 thé diing dung méi formamide & eudng 46 manh vi gel “Bio-Gel P” swell hoan to’n boi dung moi nay, Gel “Bio-Gel P” ¢6 thé khir tring 6 pH 5.5-6.0 trong cde dung dich dém nhur HEPES 50Mm, MES, hoe citrate & 120° C trong 15-30 phiit, G nhigt 4 phong, pham vi pH nén diéu chinh tix 2 dén 10. Gel “Bio-Gel P” nhay cam voi sw thay phan cic nhém amide 6 pH cao hodc thap. Téc dé dong va d6 phan gidi gia tang khi nhiét do trong phgm vi 4-80° C. G.2. Cae thong sé ki thugt Bang 3.1 Cac thong sé ciia sin pham gel “Bio-Gel-P* 35Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh ‘Thong Sé ‘Dic Tinh ‘Chit nén (Matrix) Gel-Bio-Gel polyacrylamide [Kichthude hat [Trungbinn = [90-180um——t—CSCS~=«s@ Min 45-90 um Rat min <45 um Shipping medium Shipped dry Pham vi hoat | pH 210 dong t6t ‘Ap suat 15 psi Cac dung méi hiru |< 20"C co Pham vi nhigt a6 | 4-80°C van hinh Pham vi nhigt d6/ Khir tring bing autoclave, cho phép pH 5.5-6.5 & nhiét dé 120°C trong 30 phit Bao quin Kho, 6 nhigt d> phing, trong nude cét ho’ic dung dich 4ém 6 4° © véi_ sodium azide 0.02%. Bang 3.2 Cac die tinh cia gel “Bio-Gel P” 56Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Pham vikich | Thé tich nén | Tée d6 dong Pham vi thude hat, | dahydrated | dién hinh phan tach Gel cic hat dabj | diénhinh, | (em/hr) | tiéu biéwgi ngém millg gel kho han phan nurde(uM) tach thong thudng (Dattons) Bio-Gel P-2 [45-90 3 3.0-10 100-1.800 Gel, min Bio-Gel P-2, | < 45 <10 100-1.800 rdt min Bio-Gel P-4 | 90-180 4 15-20 800-4.000 Gel, trung binh Bio-Gel P-4 | 45-90 10-15 800-4.000 Gel, min Bio-Gel P-4 | < 45 <10 800-4.000 Gel, rat min Bio-Gel _p-6 | 90-180 65 15-20 1.000-6.000 Gel, trung binh Bio-Gel P-6 | 45-90 10-15 1,000-6.000 Gel, min Bio-Gel P-6 | < 45 <10 1,000-6.000 Gel, rit min Bio-Gel P- | 90-180 65 15-20 1.000-6.000 37Khoa ludn t6t nghigp 6DG Gel ‘SVTH; Nguyén Tran Nhat Minh Bio-Gel P-10 Gel, trung binh 90-180 73 15-20 1.500-20.000 Bio-Gel P-10 Gel, rat min 45-90 10-15 1.500-20.000 Bio-Gel P-30 Gel, trung binh Bio-Gel P-30 Gel, min 90-180 45-90 15-20 10-15 2.500-40.000 2.500-40.000 Bio-Gel P-60 Gel, trung binh Bio-Gel P-60 Gel, min 90-180 45-90 15-20 10-15 3.000-60.000 3.000-60.000 Bio-Gel P- 100 Gel, trung binh, Bio-Gel P- 100 Gel, min 90-180 45-90 15-20 10-15 5.000-100.000 5,000-100.000 * Lny: Téc d6 dong xde dinh & c6t 1.5 x 70 em, Pham vi phan tach trén 40.000 daltons déi véi cdc phan té hinh cau. 58Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh ‘Doi voi muc dich kiém soat chit lrgng, gidi han phin tach duge xéc dinh bing cach tinh gid tri Kd, hode hé sé phan 66, Hé s6 phan bé 18 gid tri thoi gian lau cia phan tir trong céc 18 gel, durge biéu dién nhu sau: (Ve~Voy/ (Vt Vo) Trong dé: Ve 1a thé tich giai hap (thé tich thi = elution volumn). Voli thé tich tréng (void volumn). ‘Vt la thé tich téng xée dinh bing mét phan tir nhé nhw vitamin By. C.3. Hwéng dan sir dung Bio-Gel P C.3.1. Chon cot Kich thude cét ly tuéng 14 nhing kich thuéc cho phép phan giai vach ranh gidi cic chat phan tich ma khéng can pha loang miu nhiéu. Dién hinh, chiéu dai c6t so voi ty 1é duong kinh 6 khoang pH 5-10, va thé tich 1ép nén (bed volumn) gp 4-20 lan thé tich cua mau, Hé sé pha lodng ti thiéu co thé thu duge d6i voi mot chét d3 duge téch (excluded substance) khoang 1,25. Nhin chung, 46i voi qua trinh phan tach khé thi cn chigu dai lép nén so véi ty 1 dudng kinh li 25-100 hode 1én hon, va thé tich lp nén gip 25-100 lin thé tich mau. * Chon chat tich Chat tach (chat giai hdp) 1a cht long ding dé chiét mét chat khdi chat khée trong sic ky. Chat téch chon cn tgo tinh dn dinh tdi da cho cae chat tan khong 6n dinh trong miu, Nong 46 ion it nhat 1& 20mM dé loai trir anh hudng cia s6 long nhd cée nhém mang dign 4m trén gel. Vige sir dyng cde dung dich mudi & néng d6 cao cé thé giy ra nhing thay déi nnhé trong thé tich I6p nén gel va gidi han phan tach (exclusion limits). Bio-Gel P tuong thich voi céc trang thai héa tan va lam bién tinh (solubilizing and denaturing condition) durge sit dung trong vige xde dinh trong long phan tit nhw 59Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh guanidine-HC] 6M, céc téc nhan chaotropic, cde tae nhan khir nhu dithiothreitol va mercaptoethanol, va cac thude tiy nhu SDS, CHAPS, va Triton X-100. Co thé sit dung céc mudi dém dé bay hoi nhu pyridine, acetic acid, ammonium formate hoc ammonium bicarbonate néu sin phim cudi cing cin phai & trang th khong e6 mudi dgm. Cé thé loai ce chat nay ra Khoi phan doan ding chay (effluent fractions) dé dang bing déng khé. ‘Nén logi cdc khi hoa tan nhu CO, dé ngan chan sy tao bot trong hé thong. Thyc hign diéu nay bing cach hit dung dich dém trong mot lo chin khdng bling mot vdi nude tao chan khéng hoi bing may tao chan khong. Nén trnh sir dung cde chit téch e6 pH trén 10 hoge nhd hon 2 dé ngan su thay phan gel. Trinh ding cac tac nhan Oxy hoa manh lim gia ting him lugng ede hm tich din trén chat lop. % Chuan bi gel ‘ic chat nay sé phan ting véi gel va Cho tir tir mdi trating Bio-Gel P vao dung dieh dm da duge dyng trong ede mo (beaker). Cé thé ude tinh lang gel Bio-Gel P cin cho vao cét c6 thé tich da biét bing cach sir dung bang 2, bing nay cho biét thé tich lap neenfkhi duge hydrat héa. Lua ¥ sur mat gel trong suét qué trinh tién hanh c6ng vige. Ding dung dich dém nhiéu gap 2 Lin so véi thé tich Isp nén cn cho trong cét. Gi véi cae gel tir Bio-Gel P-2 dén Bio-Gel P-10 can hydrat héa trong vong 4 gid & nhiét 46 phng (1 gié néu dung dich dém da duoc lam néng dén 100° C va réi lam lanh sau khi da cho gel vao dung dich dm). Déi véi cdc gel tir Bio-Gel P-30 dén Bio-Gel P-100 cin 12 gid 6 20° C dé hydrat hoa hodc 4 gid néu bat diu 6 100° C Sauk hi thé huyén phi déng nhit cua cac hat gel hinh thanh, khéng can phai khudy, hay dé dn dinh trong suét qua trinh hydrat héa, Sau khi qué trinh hydrat xay ra hoan toan, gan Idp néi trén bé mat. Chuyén dung dich vao mét binh loc va gin véi nguén chan khong. Khir khi cua dung dich trong 60Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh khoang 5-10 phit, thinh thoang lic nhg (xoay) binh. Khéng ding diia khudy vi n6 co thé lam hur hai gel. ‘Thém dung dich dém khir khi vdi thé tich gap hai lan thé tich Iép nén va lic nhe binh. Dé gel én dinh cho dén khi 90-95% s6 hat én dinh. Gan hoac loai 6p ndi trén be mat bing cach hit dé loai cae hat min. Lap lai céng vige trén 4 lan dé loai > 99% hat su trén dinh e6t, déng 16 thodt ciia 6t vi cho dung dich dém lam diy 20% thé tich c6t. Rot déu chat pha tron loang (slurry) vao cét thanh mét dong di chuyén nhe. Tranh lam ban (tung tée) chat pha trén loing dé dim bio viée nhéi cét déu va dé tranh viée bay cate bot khi, Khi 2-5 cm lép nén da hinh thah, cho c6t chay cho dén khi c6t duge nap day (packed). Khi cét di duge nap diy, déng dau ra (outlet) ctia Ot va gin flow adaptor. M& dau ra ciia c6t va cho dung dich dém véi thé tich gp 2 lin thé tich trén lép nén chay qua c6t hic didu khién tc d6 chay. ‘Dong dau ra ciia cét va diéu chinh flow adaptor xuéng dén lép nén gel. Nap mau vao trén bé mat lép nén bing céch bom hod tiém miu vao trén lop nén gel qua flow adaptor. Néu tiém miu thi téc 46 dong tiém khéng nén vugt qua toc 46 ding tach dé nghj, Néu khdng diing flow adaptor thi lay 1ép gel thira cho dén khi dat chiéu cao lop nén mong muén khi ma ct di due nap xong va gin ct vao mot reservoir. Cho dung dich dém véi thé tich gap hai lan thé tich lép nén chay qua c6t lic diéu khién toc 46 dong chiy. Réit dung dich dém xuéng bing 1ép nén gel va miu nim trén lép nén gel, sau dé cho thém dung dich dém dé lay mau vao lép nén. Thay lop dung dich dm néi trén bé mat va gin cét vio reservoir. 61Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Thu cae phan doan dé phan tich hoc theo doi bing mét thiét bj dong lién tuc (continuous flow equipment) nhu UV/ ‘ac may do d6 din (conductivity) va chi sé Kinic xa (refractive index), Mau Loe gel phan 1én déc lap véi ndng 46 miu nhung thé tich mau tuong tng voi thé tich lép nén Iai kha quan trong. Véi muc dich phan tich, miu khéng nén 1én hon 1-5% thé tich lop nén. ‘V6i muc dich khir mudi, miu c6 thé [8 30-35% so véi thé tich 16p nén, D6 nhét cia mau cé thé gidi han néng 46 miu ma co thé sir dung. Miu cé d6 nhét cao cé thé pha loang dé 1 giam d6 nhét. Co thé dat duge két qua tét hon bing cach 4p dung mau c6 d6 nhét cao 6 tc d6 chay thap. Mau can sach va héa tan hoan toan trong dung dich dém chay ma khéng bi nhiém ban va khong cé cdc hat rin. Loc mu sé lam gia ving 9 bén/thdi gian sir dung cia c6t. Néu vi tinh chat cia miu ma khéng thé dem loc duge thi nén ly tam miu cho dén khi n6 sach, Trinh bay anh huéng theo gia thuyét vé cde diéu kign sie ki khéic nha. % Nac dinh thé tich tring (void volumn) va higu chuén (calibration) ‘Thé tich tréng (Void volumn) = Vo cia lép nén bang v6i thé tich phan tach (elution volumn = Ve) cita ce nguyén ligu da phan tach. Nén xde dinh thé tich chan khdng cua Iép nén va nén kiém tra tinh dong nhat cla chét phan tich cia Iép nén trude khi ap dung mau thi nghiém. Cac protein c6 mau nhwr hemoglobin, ferritin phit hgp véi phuong phip xac dinh, thé tich chan khdng nay. Dextran xanh khong duge 48 nghj xée dinh thé tich chan khéng vi né khéng déng nhit (heterogeneous) va cé thé cho ra nhiing két qua khic hau, Né cing c6 thé lién két khéng dic higu véi gel 62Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Sir dung cée protein chuan cho phép kiém tra vieej nap e6t (colum packing) va sur phan tich protein (protein elution). Né cing cho phép so sinh ede loai edt v5i nhaw va cde nguyén Tigu dé nap e6t khéc nhau mi khong Lim lang phi miu. ‘Chuan loc gel ciia hing Bio-Rad la mét hén hyp 5 protein c6 trong lrgng phan tir da bide: (Mr 670.000), gamma globulin bo (Mr 158.000), ovalbumin cia ga (Mr 44,000), myoglobin ctia ngya (Mr 17.000), va vitamin By (Mr 1350). thyroglob ‘Vitamin B,, va myoglobin ¢6 thé nhin thay duge khi ching di chuyén qua cot & Ve sink va Khe ring Gel Bio-Gel P cé thé duoc khir tring bén trong c6t bing cach sir dung 3% hydrogen peroxide trong nuréc, dung dich ethanol, diethyl pyrocarbonat hode thimerosal 1: 10.000. Gel Bio-Gel P da hydrat héa ¢6 thé duge khir tring bing autoclave 6 pH 5.5-6.5 & 120° C trong 30 phit. Khi khir trung bing autoclave, gel c6 thé bj phdng lén tir 4-20 lan thé tich ban dau. Sy phdng lén cang nhiéu thi im cho kich thudc 18 cang lén. © Baio quéin Cae edt da durge nap gel Bio-Gel P 6 thé bio quin khdng gidi han néu duge duy tr] 6 pH trung tinh khi c6 mgt chit khing khudn nhur 0.02% sodium azide. Nén bio quan cae e6t dé 64°C. © Xéic dinh toc dé dong Loe gel 1a m6t qua trinh ¢6 kiém soat chat khyéch tin : hiéu qua phan tach tly thudc vao téc 46 va d6 déng nhat cia kich thuéc hat gel. D6 phn tach cao nht nhan duoc Khi téc 46 dong duge duy tri trong pham vi 2-10 em/hr. Déi voi tbe do dong tuyén tinh Semvhr (nghia 1a tbe do dong theo chiéu dai cita et), dé xée dinh tbe 46 dong cia cét ta lay tée d6 dong tuyén tinh nhan véi dign tich thiét dign (cross sectional area) cita cét. 63Khoa ludn t6t nghigp ‘SVTH; Nguyén Tran Nhat Minh Bang 3.3 Xac djnh téc 46 dong ‘Toc dO dong tuyén | Dudngkinh cht | Dign tich thiét dign | Toe do dong qua tinh (em/hr) (cm) (em’) ‘eft mV/hr 5 07 0.385 19 5 1.0 0.785 39 3 LS 177 89 3 25 491 3B 3 50 19.6 98 = Dai voi e6t 66 kich thude 1.5 x 70 em. Téc dG dong sé gidm néu chiéu dai c6t tang. = ml/hr/em? (dign tich thiét dién) = cm’ fhr/em? = cm/hr.