LI CM N

Kha lun tt nghip

Vin i Hc M

M UNgy nay, cng ngh sinh hc phn t pht trin nhanh chng, t c nhng thnh tu to ln v l thuyt v thc tin. S pht trin khng ngng ca sinh hc phn t gip cho con ngi hiu bit c s khoa hc mt s bnh nan y, tm ra cc loi thuc gip cho con ngi chng li cc bnh tt v nng cao sc khe i sng. Thnh tu cng ngh sinh hc phn t ngy cng c ng dng rng ri trong cng nghip, nng nghip c bit l cc sn phm chc nng.X hi ngy cng pht trin nn vn sc khe ca con ngi ngy cng c quan tm. Do cc sn phm chc nng khng ngng c ci tin nng cao cht lng. V khi nhng thnh tu v y hc, ha sinh hc, sinh l hc v dinh dng hc c ci tin th nhng thc phm chc nng, thc phm iu tr bnh c sn xut nhiu hn.Ngy nay, thc phm chc nng ang c con ngi c bit quan tm v y l nhng thc phm c gi tr dinh dng v mc nng lng thp nhng trong c cha rt nhiu tc dng. Trong nhm thc phm ngi ta quan tm kh nhiu n ng chc nng. C rt nhiu loi ng chc nng nh: ng Maltitol, ng Fructooligosaccharide, ng Sorbitol, ng TrehaloseTrong s ng Trehalose l mt trong nhng loi ng c nhiu cng dng i vi cuc sng con ngi, c bit i vi nhng ngi bo ph, nhng ngi mc bnh tiu ng, su rng, Cng vi s pht trin ca ng chc nng, cc loi enzyme ng dng trong lnh vc ny cng ngy c quan tm nghin cu v ng dng Trehalase l enzyme c s dng sn xut Trehalose l nhn t iu chnh qu trnh ng lnh, ngn nga qu trnh kt tinh, cung cp gi tr dinh dng. Do vy nghin cu sn xut Trehalase l enzyme c s dng sn xut Trehalose l nhu cu cn thit. c bit trn th trng Vit Nam hin nay ch yu nhp ch phm ny t nc ngoi nn gi thnh tng i cao. V vy, vic nghin cu sn xut enzyme Trehalase khng ch c ngha khoa hc m cn c gi tr kinh t x hi.T nhng l do trn chng ti thc hin ti nghin cu sau:Nghin cu biu hin gen Trehalase ti t hp E.coli v xc nh cc tnh cht ca enzymeMc tiu ca ti:1. Biu hin gen Trehalase ti t hp E.coli.2. Tch v tinh ch enzyme.3. Xc nh cc c tnh ca enzyme4. ng dng enzyme tng hp Trehalose.PHN 1:TNG QUAN TI LIUI.1 Cng ngh DNA ti t hpK thut di truyn l thut ng chung cho nhiu k thut c bn trong nghin cu di truyn phn t. Thut ng k thut gen cn gi cng ngh gen hay k thut gen vn ct li ca sinh hc hin i. Nm 1983 Rudleg v Sediel: k thut di truyn l cc k thut dn n kh nng bin i nh hng gen, tn s kiu gen ca mt qun th, hoc lm tng cn bn kh nng sinh sn ca mt s c th nng cao vn gen v tng s lng hp t ca qun th. K thut gen c bt u nm 1977, bao gm cc thao tc k thut trn gen, nhm iu chnh v bin i gen hoc to ra cc gen mi t to ra cc c th mi. K thut gen bao gm mt s k thut c bn l k thut DNA ti t hp, phn lp gen, chuyn ghp gen, dung hp gen vi thao tc gen.DNA ti t hp l DNA c to ra t hai hay nhiu ngun vt liu di truyn khc nhau. Phn t DNA c gp ni t cc on DNA ca cc c th khc nhau trong loi, hoc cc loi khc nhau gi DNA ti t hp.Nguyn tc chung ca cng ngh DNA ti t hp bao gm mt s bc sau. Trc ht genome mc tiu v DNA chuyn ti (vector) c tinh ch v ct thnh tng on nh mt loi Enzyme hn ch. Trong nhiu trng hp (c bit i vi sinh vt bc cao) DNA gen nome c thay th bng DNA tng b (cDNA) c mRNA thnh thc. DNA ny c tng hp t khun RNA thng tin nh xc tc ca enzyme sao chp ngc (reverse transcriptase, rna-dependent DNA-polymerase). DNA vector c th l mt plasmid hoc phage (nu vt mang dng ha gen l vi khun), plasmid Ti ca vi khun Agrobacterrium tumefaciens (nu vt mang dng ha gen l t bo thc vt), l Vius (retrovirus hoc andovirus khng gy bnh, nu vt mang dng ha gen l t bo ng vt). Bc th hai l chuyn vn DNA ti t hp vo c th vt mang, dng ha v bc tip sau l nui cy vt mang, sn xut (chit sut v tinh ch) sn phm mc tiu. Nh c ct cng mt loi enzyme hn ch (thng l enzyme u dnh) cc on DNA mc tiu v cc on DNA vector (ch ct ti mt im ca mch vng xon kp) c th ngu nhin hnh thnh lin kt hidro tng b cc u dnh, ri c ligase hn gn cc u khc. Kt qu trong hn hp hnh thnh th ti t hp ca vector vi mt hoc mt s mnh DNA ca genome mc tiu (bn cnh cc sn phm khng mong mun nh vector t khp vng, cc vector t ti t hp vi nhau v cc mnh DNA khng ti t hp hoc t ti t hp). Tt c cc sn phm trong hn hp u c trn u vi cc vt mang to dng, sau cc vt mang c nui cy trong mi trng dinh dng c yu t chn lc (cht khng sinh i vi vi khun, ha cht gy cht i vi cc t bo ng vt thc vt). Trong iu kin , ch cc t bo vt mang vector sng st. Hn na, do plasmid b enzyme hn ch ct tho vng ti mt v tr xc nh trong mt gen iu khin mt thuc tnh nhn bit khc, nhng plasmid ti t hp mt kh nng to tnh trng vt mang (chng hn, mt kh nng tng hp Beta-galactosidase), nn ch nhng vt mang sng st v khng biu hin tnh trng th c chn nui cy to dng. Bc tip theo l chn nhng dng tnh trng ca gen mc tiu hoc nhng dng gen mc tiu (nh lai phn t: cc phng php th nghim min dch nh ELISA, Westerm blot anlysis, Northern blot anlysis hoc vi cc d DNA hoc RNA southern blot anlysis, Norther blot anlysis), hoc PCR. Nui cy to dng nhng c th vt mang tha mn cc bc th nghim trn gip to ra dng thun cc sinh vt mang c th sn xut cc sn phm mc tiu.Thc cht ca cng ngh DNA ti t hp l tp hp nhiu k thut trn mt gen, ci bin cu trc ca gen, b gen to ra cc gen mi trong b gen. Cng ngh DNA ti t hp bao gm cc bc c bn sau. Tch chit DNA, RNA (phn lp gen) To vector ti t hp (bao gm chun b vector tch dng) Bin np vector ti t hp vo t bo ch v nhn dng. Sng lc, theo di hot ng v biu hin ca gen Cho n nay, k thut di truyn t c nhng bc tin vt bc. Nhiu sn phm c ch c sn xut t cc vi sinh vt ti t hp hay vi sinh vt bin i gen (gentically modified organisms GMO) nh ging ng cc u tng, ht c du, bng, mang gen chng su bnh hoc khng thuc chng c di hoc c cht lng sn phm nng cao nh khoai ty cha nhiu Protein, la nhiu vitamin A v st, To ra c nhng vacxin ti t hp c th d sn xut (d nui cy trn la cy t bo hay phi g) v d s dng cho n ung, hoc nhng ng vt ti t hp sn sinh nhng sn phm hu ch nh thuc cha bnh, thm ch to ra nhng n ln mang khng nguyn ngi, hy vng l vt c th cho tim hoc ni tng khc iu tr nhng trng hp ngi bnh cn thay th ni quan, Tuy nhin, k thut di truyn cng t loi ngi vo tnh trng c th phi ng u vi nhng e da mi lin quan ti sc khe ca ngi v sinh thi mi trng. Cha th chng minh c tnh v hi ca GMO thc phm i vi con ngi, cng nh cha ph nhn kh nng lan truyn gen khng thuc (ca Plasmid vector) trong tp on cc vi khun t nhin ca c th (v d trong ng rut) ngi v ng vt. Chm sc nhng loi cy trng khng thuc dit c v thuc dit su bnh c th dn n lm dng nng dc v nu thoi ha tr thnh c di th thc vt khng thuc v khng su bnh s l mt nguy c mi i vi nng nghip. Tng t, nguy c nhng sinh vt GMO ln t sinh vt t nhin s l rt cao v c th dn n s tuyt dit ca nhiu loi sinh vt hin c gy hu qu nghim trng n mi trng sinh thiI.2 i cng v vi khun E.coliTheo Nguyn Vnh Phc (1973); Nguyn Ln Dng (1976), trc khun Escherchia Coli c tn khoa hc l Bacterium Coli Comunis bnh thng E.coli c tr phn sau ca rut (rut gi v phn cui ca rut non), t khi c d dy hay pha trc rut non ca ng vt (Nguyn Vnh Phc,1978). T l E.coli kt trng l 58.3%, trc trng 55.3%, manh trng l 39.3% E.coli xut hin v sinh sng ng vt ch vi gi sau khi sinh v tn ti n khi con vt cht. Loi n tp v n tht thi nhiu E.coli hn loi n c. Theo Kefman nhng chng E.coli thng gp v huyt thanh c chia thnh nhng Serotype ring. Trong s ny mt s serotype ng vai tr quan trng trong vic gy bnh cho ngi v ng vt.I.2.1 c im hnh thi E.coli l loi trc khun hnh gy gn, hai u trn, c lng, di ng c, khng hnh thnh nha bo, gip m, bt mu Gram m, thng thm hai u gia nht, kch thc vi khun 2-3 uM x 0.4-0.6 um.Trong c th gia sc, vi khun c hnh cu, ng ring l, i khi xp thnh chui ngn.I.2.2 c tnh nui cyE.coli l vi khun hiu kh hay k kh ty tin, d nui cy, c th sinh trng v pht trin thch hp nht 37oC, pH thch hp l 7.2-7.4. Tuy nhin chng vn c th pht trin trong mi trng c pH = 5.5-8.0.Mi trng nc tht: E.coli pht trin rt nhanh, mi trng c u, c lng cn xung y mu tro nht, trn mt hnh thnh mng mng mu ghi nht dnh vo thnh ng nghim, canh trng c mi phn thi.Mi trng tht thng: nui cy 37oC trong 24h hnh thnh khun lc trn, t hi li, khng trong sut, mu tro nht, ng knh khun lc khong 2-3mm.Mi trng Endo: hnh thnh khun lc c nh kim.Mi trng SS: trn mi trng ny sau 24h nui cy hnh thnh khun lc mu hng nht hoc mu cnh sen. Mi trng EMB: E.coli c khun lc mu tm en.Mi trng Macconkey: E.coli hnh thnh khun lc mu .Mi trng Billiant Greenagar: khun lc ca E.coli c dng S, mu vng chanh.I.2.3.c tnh sinh ha Cc chng E.coli u ln men sinh hi mnh cc loi ng Glucose, Galactose, Lactose, MannitolE.coli ln men khng sinh hi cc loi ng nh Saccarose, Ramnose, Xalixin, Valin, Glyxeron.Cc phn ng sinh ha khc: Phn ng indol (+), phn ng VP (=), phn ng MR (+), phn ng H2S (+), hon nguyn Nitrat thnh Nitrit, Urease (-), Gelatin (-).I.2.4 Sc khng ca E.coli E.coli b tiu dit nhit 55oC trong mt gi, 60oC trong 30 pht (Willon v Miles, 1995), (Nguyn vnh Phc, 1973). Cc cht st trng nh: Axit phenic, Clorua thy ngn, Formol c th tiu dit E.coli trong 5 pht.I.3. Tnh hnh sn xut v ng dng enzyme trn th gii v Vit NamI.3.1 Tnh hnh sn xut v ng dng enzyme trn th giiEnzyme c vai tr v cng quan trng, khng ch xc tc cho cc phn ng ha hc xy ra trong h thng sng m sau khi tch khi h thng sng chng vn c th xc tc cho cc phn ng ngoi t bo (intro). Bi vy, cng ngh enzyme ngy cng pht trin. Nhng thnh tu c bn v enzyme l c s pht trin cc nghin cu ng dng enzyme trong thc tin.Enzyme thng c s dng theo hai cch: khng tch enzyme ra khi nguyn liu m to iu kin thun li cho hot ng ca mt s enzyme c sn trong nguyn liu chng chuyn ha cc cht c cng trong nguyn liu theo hng mong mun. Hoc tch enzyme ra khi nguyn liu dng ch phm khi cn thit. Enzyme hot ng sinh hc c th c tch t bt k c quan sng no. V d enzyme thng mi c th c sn xut t Acitinoplanes ti Zymomonas. T rau chn vt (spinach) n nc c ca rn. Do c th tn dng ngun nguyn liu giu enzyme tch enzyme, dng enzyme ch bin cc loi nguyn liu khc nhau hoc s dng vo nhng mc ch khc nhau. Hng trm enzyme thng mi c sn xut ra, trong hn mt na t nm mc v nm men, qu 1/3 t vi khun, cn li 8% t ng vt, 4% t thc vt.Vi sinh vt l ngun nguyn liu tt nht ca cng ngh enzyme v chng c tc sinh trng nhanh trn cc mi trng dinh dng khng t tin, khng i hi thit b t tin, c th t ng ha hoc iu khin, nng sut caoHn na, ta c th ch ng iu khin qu trnh sinh tng hp enzyme, nng cao hm lng enzyme trong t bo vi sinh vt d dng hn t bo thc vt v ng vt.Hng nm, hng trm tn ch phm enzyme c sn xut phc v cho ngnh cng nghip v y hc. Nm 1980, trn th gii sn xut 530 tn protease t vi khun, 350 tn glucoamylase, 320 tn Amylase, 70 tn glucoisomerase, tt c tr gi 150 triu USD.Hin nay trn th trng c khong 12 hng sn xut enzyme chnh vi trn 60 nh cung cp ln. Hai hng sn xut enzyme ln nht hin nay l Novo Nordisk (an Mch) v Gist Brocades chim 60% lng enzyme trn th trng. Tip l M 15%, trong 2/3 s dng ni a ch yu dng sn xut cn v ng nghch o. Nm 1993, trn th trng M tiu th 50 triu USD i vi renin, 27 triu USD i vi glucoAmylase, 23 triu USD i vi glucoisomerase Khong 50 loi enzyme khc nhau c bn trn th trng M. Nht chim 12-15% th trng tiu th vi cc sn phm a dng. Trung Quc v Nga l hai th trng sn xut ch yu phc v trong nc.n nay ngi ta bit v phn loi khong 3500 loi enzyme, khong vi trm loi c sn xut trn quy m cng nghip ch yu l enzyme Amylase, Protease. Lipase Amylosidase: sn xut vi sn lng 5 tn /nm s dng tch L- acid amin khi hn hp DL axit amin (250 tn L axit amin trn nm) Amyloglucosidase: sn lng 1 tn /nm, sn xut 3000 tn glucose t tinh bt/nm. Glucoisomerase: s dng sn xut xiroglucose fructose 42% (2.150.000 tn /nm) v xiroglucose- fructose 55% (1.450.000 tn/ nm).Phn ln enzyme c s dng mc cng nghip u thuc loi enzyme n cu t xc tc cho phn ng phn hy. Khong 70% ch phm l enzyme thy phn s dng thy phn cc c cht t nhin.Cc protease l enzyme c s dng nhiu nht hin nay dng trong qu trnh ng t sa sn xut fomat, lm mm tht, sn xut cht ty ra, thuc da. Tip n l cc enzyme nhm Hydaratcacbonat nh Amylase, Cellulose, Pectinase S dng nhiu trong ch bin thc phm, thc n gia sc, cng nghip dt, cng nghip sn xut giy v bt giy.Trong cng nghip thc phm cc ch phm enzyme c s dng vi 4 mc ch chnh:- iu chnh nhng khim khuyt ca nguyn liu. Nguyn liu ca cng nghip thc phm l cc sn phm phc tp c ngun gc t ng vt v thc vt c hnh thnh qua nhiu giai on tng hp sinh hc. Do , thnh phn nguyn liu ph thuc vo cc yu t bn trong nh ging loi v cc yu t bn ngoi nh iu kin canh tc kh hu. Cc yu t ny c th lm thay i thnh phn nguyn liu v chuyn ha n. Trong trng hp ny cc ch phm enzyme c s dng mt ph gia lp y nhng thiu ht. Trong cng nghip ch bin ng, tinh bt, bnh m, bia ngi ta b sung Amylase b hot lc enzym yu ca nguyn liu ng cc. Trong sn xut fomat, enzyme lipase v protease c s dng thay th cc enzyme t nguyn liu sa b v hot thanh trng.- Tham gia chuyn ha, ng dng nhiu nht trong sn xut bnh bisque, fomat, nc ung protease cho php ci thin tnh lc ca bia v tnh nng n nh ca n nhit thp. Amylase, pectinase, hemicellulase to iu kin cho vic tch chit v lc nc qu. Protease trung tnh lm mm do khung gluten ci thin n ca bt nho v gin ca bisque.- Tng gi tr nguyn liu t nhin. Enzyme c th tham gia vo vic a dng ha sn phm nhn t nguyn liu t nhin. S pht trin ca cng ngh enzyme cho ra i mt lot cc ch phm enzyme cho php to ra nhiu sn phm: cht lm c, cht thm, cht to ngt, cht to chua vic ci thin c tnh ca enzyme v tnh c hiu v tnh chu nhit l c s sn xut ch phm enzyme mi thch hp mi trng v c hiu hn.- Ci thin cht lng sn phm: cc enzyme s dng loi b cc cht t nhin c nh hng xu ti tnh cht cm quan ca thc phm. Ch phm enzyme cn cho php to hng v, mu sc sn phm nh lipase trong sn xut fomat, lactase trong sn xut sa. Trong cng nghip thc phm, protease c dng lm mm tht, cng nghip bnh m, enzym dch v 9 ch yu l chimosin thu c t d dy b c s dng sn xut fomat, papain t nha u lm mm tht.Enzyme trong cng nghip thc phm ch yu s dng sn xut ng tinh bt. u tin ngi ta s dng hai enzyme - Amylase v - Amylase sn xut mt t tinh bt, sn phm ny cha dextrin, glucose v maltose c dng nhiu trong cng nghip bnh ko. Glucose c sn xut nh - Amylase v - Amylase, tuy nhin glucose dng trong thc phm c nhc im ngt thp ch vo khong 0.8 ln ng knh v vy n c dng ch yu cho y dc.Nm 1970, ngi ta sn xut c Fructose v xirogluco fructose cao loi ng ny c ngt gp 1.73 ln so vi ng knh nhng gi thnh cao hn nhiu.Nm 1998, tng lng ng tiu th trn th gii l 10 triu tn th ring M tiu th 4.2 tn Fructose (bao gm c xirogluco fructose), Nht l 800.000 tn, EU l 2 triu tn, Canada 300000 tn, ng u 200000 tn, cc nc ang pht trin l 500000 tn.Sau cng nghip thc phm cc ch phm enzyme c s dng trong cng nghip, nng nghip, dc phm, y t, ch bin thc n gia sc. c hiu vic s dng enzyme ca cc h vi sinh vt trong nc thi trong cng nghip x l mi trng ngy cng c nghin cu khai thc s dng.Trong cng nghip dt, vic s dng enzyme c p dng t nhng nm 20 ca th k 20. Enzyme t nc chit Malt v Pancrease c s dng r h. Nm 1971, s dng enzyme Amylase ca Bacillus subtilis r h nhit cao, rt ngn thi gian. Enzyme Glucanase, Pectinase, Estrase, Protese c s dng trong qu trnh nu nhm loi b nhng thnh phn khng phi Cellulose ra ngoi.Trong cng nghip thuc da, hai qu trnh s dng enzyme l tch lng v lm mm da. Ngi ta s dng enzyme collagenase, trypsin, chymotripsin, cacboxypeptidase tch cht nhn v ph hy lin kt trong si collagen, nh vy lng c tch khi da v tr nn mm hn.Trong cng nghip bt git v ty ra, cc ch phm enzyme c s dng l protease, lipase, cellulose, amylase, oxydoreductase tng vn tc v hiu qu qu trnh ty ra. Mt s enzyme c b xung vo cc loi x phng v kem dng da, thuc nh rng nh bromelin, collagenase, lipasecc enzyme ny c tc dng ty hi, ty cc vt bn bm trn da kh tt, ng thi lm mn v tng n hi cho da.Trong nng nghip, enzyme dng sn xut thc n cho ng vt nhm tng gi tr thc n th, tng h s s dng cho thc n. Cc enzyme s dng vi mc ch ny l cc enzyme thy phn nh amylase, protease, c hiu l celluloase v hemicelluloase. Chng thy phn cc cht c phn t ln thnh dng d hp th hn thng lm tng hiu qu s dng thc n.iu ny c ngha quan trng khi chun b thc n cho ng vt cn non v h thng tiu ha ca chng cha thch hp vi vic n cc loi thc n th.S pht trin ca y hc phn nh s ln mnh ca tim nng enzyme. Ngay t th k 19 enzyme ty tng c s dng cha bnh ri lon tiu ha. Enzyme Tripsin, Chymotripsin s dng lm tiu vim, lnh vt thng, vt bng, vim phi, vim kh qun. Tripsin cha bnh vim tnh mch huyt khi, vim ty. Chymotripsin cha bnh lot d dy t trng. Mt s enzyme tiu ha c s dng cha bnh suy dinh dng tr em, ngi gi v ngi bnh nh Pepsin (t d dy ln). Ngy nay, mt s thuc tng hp ha hc ln lt c thay th bng cc qu trnh enzyme nh nhng u im ca n. V d vic sn xut Pinicillin bn tng hp nh enzyme penicillin acylase sn xut hai dn xut ban u 6- APA v 7 ADCA, t tng hp ln khng sinh th h II, III, IV, V. Cng vi s pht trin ca k thut gen v lai to t bo, ngi ta sn xut c nhng cht m c th sng ch c th tng hp c vi lng cc nh nh interferon, hormone sinh trng ngi, isulin, vaccine Trong nhng nm gn y mt s cng trnh ni n vic cha bnh ca asparaginaseS gip ch ca enzyme hc i vi y hc hin nay tuy cn hn ch nhng vi pht trin mnh m ca ngnh khoa hc ni chung v pht trin ca enzyme hc ni ring, cng vi s tin b v k thut th ni chung tng lai enzyme s gip ch nhiu hn cho y hc c bit trong lnh vc phn t.Ngoi vic nghin cu enzyme trong y hc lm sng v trong iu tr hc, ngi ta cn nghin cu s pht trin ca enzyme trong c th t giai on bo thai cho n lc gi. Bi vy ngy nay enzyme hc cn ng vai tr quan trng trong di truyn hc phn t, trong nghin cu qu trnh tin ha ca cc ging ni v trong nhiu lnh vc khc. Ngoi cc enzyme thuc nhm trn, enzyme khc c sn xut vi quy m nh v nhiu enzyme c hiu c dng ct, ni gen trong k thut gen, nghin cu phn t protein (endonuclease), s dng enzym Urease nh lng ure trong nc tiuCho n nay, ngi ta vn cho rng 99% kh nng ng dng ca enzyme vn dng tim nng. Trn c s nghin cu c bn v cu trc phn t v c ch sinh tng hp enzyme m ra trin vng ca ngnh enzyme hc:- Tng hp enzyme nhn to: cc cht c hot tnh tng t enzyme c th tng hp bng con ng nhn to thit k v tng hp cc cht gn rt n gin xong vic thit k trung tm xc tc rt kh khn. n nay, mt vi enzyme nhn to c s dng thnh cng nh acid polyglutamic c hot tnh tng t nh estsrase.- Bin i cu trc enzyme: p dng k thut di truyn lm bin i nh hng cu trc enzyme, bin i hot tnh, hiu sut ngi ta s dng cng ngh protein ci bin trung tm hot ng ca enzyme mc ch lm cho c hot tnh cao hn hoc enzyme c hot tnh mi. - Enzyme s dng trong phn tch, chun on, iu tr.- Bo v mi trng: s dng enzyme x l cht thi, ti to vt liu hay s dng vt liu ti ch.I.3.2 Tnh hnh sn xut v ng dng enzyme Vit Nam. Vic nghin cu v ng dng enzyme Vit Nam ch mi c tp trung ch trong vi thp k tr li y. Cho n nay Vit Nam cha c mt enzyme no c sn xut quy m cng nghip m mi ch dng li quy m nh, phm vi phng th nghim ca cc trng i hc, cc vin nghin cu. Enzyme pepsin t d dy b (Trng i Hc Bch khoa H Ni), bromelin t da (i Hc Quc Gia H Ni), a- amylase glucoase amylase, protease t vi sinh vt (vin cng nghip thc phm)Nhng ch phm enzyme c s dng rng ri nht nc ta hin nay l amylase ch yu dng trong cng nghip sn xut ru bia, ng glucose, ng nha, cng nghip dtEnzyme phn gii protein (protease) tng bn ngt ca bia, cng nghip cht ty ra, nc chm, y hcMt s enzyme (ch yu nhp t nc ngoi) c s dng rng ri trong vic nng cao cht lng sn phm v a dng ha sn phm sa.Cc ch phm protease (dng trong cng nghip sn xut nc chm, ch bin tht c) v prozimabo (y hc) l cc ch phm c ngun gc t thc vt.Sn lng ch phm enzyme tiu th hng nm khong 100-300 tn/nm. Tuy nhin, vic s dng enzyme trong cc ngnh cng nghip nc ta cn dng tim nng, do vy trong nhng nm ti kh nng ng dng ca enzyme cn c khai thc v m rng ra nhiu lnh vc khc: x l rc thi sinh hot v rc cng nghip, sn xut bt dinh dng tr em, sn xut ch phm probiotic, prebioticI.4 TrehalaseI.4.1 Gii thiu v enzyme Trehalase

Trehalase l enzyme phn b rng ri trong t nhin. N c dng kh ph bin trong cng nghip, y hc v nhiu lnh vc khc. Trehalase l mt loi enzyme glycoside hydrolase c tc dng xc tc cho qu trnh chuyn i trehalose thnh glucose. N c tm thy trong hu ht cc ng vt. Trehalose l ng i khng kh (-D-glucopyranosyl-1,1--D-glucopyranoside) l mt trong nhng ngun carbohydrate quan trng nht xut hin trong hu ht cc loi sinh vt, tr lp ng vt c v. Disaccharide c thy phn vo 2 phn t glucose bng enzyme Trehalase. C 2 loi Trehalase c tm thy trong Saccharomyces cerevisiae (nm men), l Trehalase trung tnh (NT)v Trehalase axit (AT) c phn loi da vo pH ca chng [4]. NT c pH l 7.0 trong khi AT l 4.5.Theo bo co mi y, hn 90% AT hot ng trong S. cerevisiae l ngoi bo v tch ngoi bo Trehalose thnh glucose trong mi trng cht bao.I.4.2 Trehalase trong vi khunTrehalose c tm thy nh tch ly carbonhydrate trong Pseudomonas, Bacillus, Rhizobium v trong rt nhiu khun tia v l nguyn nhn ca kh nng khng khun. Hu ht cc enzyme Trehalase tch bit vi cc vi khun c pH t 6.5 n 7.5. Enzyme Trehalase ca Mycobacterium l mt mng protein b rng buc. T bo cht Trehalase ca Escherichiacoli K12 c kch thch b s tng cao trong nng thm thu. S thy phn ca Trehalose thnh glucose din ra trong bo cht, v sau glucose c chuyn vo cc t bo vi khun. Mt t bo cht Trehalase khc cng c tm thy t E.coli. Gen m ha t bo cht Trehalase c tnh ng ng cao vi Trehalase t bo cht.I.4.3 Trehalase trong thc vtTrong th gii thc vt, mc d Trehalose c tm thy t rt nhiu loi cy khng hoa bao gm Selaginella lepidophylla v Botrychium lunaria; ng rt him trong cy c mch v ch c tm thy trong qu chn ca cc loi thuc Apiaceae v trong l cy ca thc vt ht kn c kh nng chu kh hn nh Myrothamnus flabellifolius. Tuy nhin enzyme Trehalase li ph bin trong cc loi cy. iu ny rt kh hiu v tuy thiu c cht nhng Trehalase li c mt cc loi thc vt bc cao. Khng c s chng minh r rng no v vai tr hot ng ca Trehalase trong thc vt. Tuy nhin c nhiu kin cho rng Trehalases c th ng vai tr trong c cu khng khun hoc enzyme c th ng vai tr trong vic gim lng Trehalose c ngun gc t vi sinh thc vt I.4.4 Trehalase trong nm menTrong S.cerevisiae c t nht 2 enzyme Trehalase khc bit c tm thy. Mt loi c iu ha bi cAMP-ph thuc phosphorylation. Hot ng ca loi enzyme ny c tm thy trong dch bo tng. Loi enzyme hot ng th hai c tm thy trong cc khng bo ca 12 vi sinh vt tng ng. Nng pH ca Trehalase dch bo tng vo khong 7.0 do n c cho vo cng loi vi Trehalase trung tnh. Trong khi , enzyme Trehalase khng bo c cho rng c kh nng hot ng tt nht ti pH khong 4.5 v c coi l Trehalase axit. Hai loi enzyme ny m ha bi hai loi gen khc nhau l NTH1 v ATH1I.4.5 Thy phn TrehaloseMt phn t Trehalose c thy phn thnh 2 phn t glucose bng enzyme Trehalase. Thy phn enzyme ca Trehalose ln u tin c tin hnh vo nm 1893 ti Aspergillus niger bi Bourquelot. Fischer thy c phn ng ny trong S. cerevisiae vo nm 1895. Trehalase (, -trehalose-1-C-glucohydrolase, EC 3.2.1.28) c tm thy trong rt nhiu sinh vt bao gm ng vt v thc vt. Mc d Trehalose khng c tm thy trong lp ng vt c v nhng enzyme Trehalase c trong mng bin t bo thn v lng nhung ca mng rut. Trong rut, chc nng ca enzyme ny l gip rut hp th c Trehalose. Thy phn Trehalose bng enzyme Trehalase l mt qu trnh sinh l hc quan trng ca nhiu loi sinh vt nh ny mm bo t nm, cn trng bay v tng trng li trong t bo khng hot ng tch cc.

I.5. Tng quan v ng chc nng Trehalose

I.5.1 Trehalose

Trehalose l ng i khng kh (-D-glucopyranosyl-1,1--D-glucopyranoside)Trehalose l mt ng t nhin vi chc nng tng t nh ng sucrose nhng n nh hn v c v ngt nh hn. Trehalose l ng a chc nng, v ngt nh ca n (45% sucrose).

Hnh 1: Biu hm lng ng trehalose, maltose, glucose, sucroseThnh phn cc cht dinh dng trong ng Trehalose:Thnh phnGi tr thnh phn trong 100g

Nng lng628kj/150kcal

Protein0g

Carbonhydrate trong ng100g

Hm lng cht bo0g

Hm lng xenluloza0g

Hm lng natri0g

Bng 1: Thnh phn cc cht trong ng TrehaloseSau y l bng hm lng Trehalose c trong mt s thc phm v lng m chng ta phi hp th mi ngy m chng ti thng k c:Loi thc phmHm lng ng Trehalose (%)Lng thc phm tiu th mi ngy (g/ngy)

Nc p hoa qu v rau1016

Kem10-204.8

Mt ko7-2010

Lp ph kem trn bnh ngt50.1

Thc, go219

Cc sn phm t bt m214

Bnh quy, bnh ngt5-1040

Sn phm t c100.3

Hn hp khc10-201.8

Bng 2: Hm lng Trehalose v lng thc phm tiu th mi ngyCu to Trehalose:

Hnh 2: Cng thc cu to ca trehalose

Hnh 3: S phn ng enzymeI.5.2 nh hng ca Trehalose ln c th sngI.5.2.1 nh hng ca Trehalose n s chuyn ha hydratcacbon

Trehalose khng hoc rt t b thy phn bi h enzyme ng rut nn khi n lng ng ha tan trong mu khng b bin ng. Trong gan, hot lc ca enzyme trn tng ln nu ch n sacaroza nhng s c bnh thng ha bi s cung ng Trehalose. Nh vy, Trehalose c vai tr kh tch cc trong vic phng v cha bnh tiu ng xt v gc bnh l lin quan n s gia tng ca lipoprotein trong mu. V th Trehalose hin nay c dng nhiu nh mt cht thp nng lng c bit dnh cho cc i tng b bnh tiu ng.

T kt qu ca mt nghin cu khc cho thy, i vi nhng ngi mc bnh tiu ng, nu mi ngy mi ngi n 8g trehalose, lng ng trong mu s gim nhanh trong 14 ngy.I.5.2.2 Vai tr thc y qu trnh hp th canxi ca Trehalose

Nhiu nghin cu cho thy, s dng Trehalose c th tng cng s hp th canxi ca t bo. Nh c tnh ny, Trehalose c th gip cho con ngi phng chng cc bnh v chuyn ha v bnh long xng.

Qu trnh thc y hp th canxi ca Trehalose xy ra trong rut gi. C ch thc y trn cha c xc nh r rng nhng cc tc gi u c mt kt lun chung l do ba yu t sau:

+ ng Trehalose trong rut gi c cc vi sinh vt sinh axit ln men, lm gim pH ca mi trng, dn n s ti ha tan ca cc mui canxi

+ Trong rut gi cc vi khun Bifidobacterium ln men mnh khi mi trng c cha Trehalose sinh ra cc axit mch ngn, cc axit ny khuch tn vo t bo biu b thnh rut, t thc y s hp th canxi.

+ S dch chuyn Trehalose trong rut gi s ko theo s dch chuyn ca hp cht canxi protein, nh canxi c tip xc nhiu hn vi cc t bo thnh rut, to iu kin tt cho s hp thu vo mu. Ngoi canxi, Trehalose ng thi cn thc y s hp th c magie. iu ny thc y qu trnh pht trin xng, tng cng hm lng canxi trong xng. I.5.2.3 nh hng ca Trehalose n h vi sinh vt trong khoang mingBnh su rng ch yu l do vi khun streptococci t bin v cc lin cu khun gy nn. Cc vi sinh vt c rt nhiu trong khoang ming ca ngi v ng vt. Khi a thc n vo chng s la chn thnh phn dinh dng thch hp d ln men, pht trin v gy bnh. V th nu trong thnh phn thc n ca ta khng cha hoc t cha cht thch hp cho qu trnh dinh dng ca loi vi sinh vt trn s c th ngn nga c bnh. xt nh hng ca Trehalose n bnh su rng ngi ta tin hnh th nghim trn c th chut. Kt qu cho thy nhm chut th nghim (n ng Trehalose) b su rng t hn nhiu so vi nhm chut i chng (n Saccaroza) Cc th nghim nui cy vi sinh vt phn lp t khoang ming ln mi trng Trehalose chng t c ch v kh nng phng bnh su rng ca n. l do Trehalose khng phi l mi trng thch hp cho cc vi sinh vt trn pht trin.Ngoi ra Trehalose cn c kh nng cha bnh su rng. V th ngy nay trn th gii ngi ta cn dng Trehalose thay th cho ng knh trong thnh phn n hoc trong ch bin bnh ko, c bit l bnh ko cho tr em phng bnh su rng.I.5.2.4 Tnh khng khun ca Trehalose Nh chng ta bit probiotic l nhng ch phm cha nhiu vi sinh vt c li nh lactobacillus v bifidobacteria. Cc vi sinh vt ny c kh nng khng khun gy bnh i ngoi. Hin nay cc ch phm probiotic bn trn th trng thng cha nhiu ng Trehalose, bi ng Trehalose c tc dng l c cht tt cho s pht trin ca cc vi sinh vt c li. Chnh s c mt v ng gp vai tr tch cc trn ngi ta ni rng ng Trehalose c kh nng khng khun (antibiotic) I.5.2.5 Tnh an ton ca Trehalose

khng nh tnh an ton ca Trehalose i vi c th sng c nhiu nghin cu v t bo v gen ca ng thc vt n Trehalose. Cc nghin cu ny ch yu tp trung tr li cc cu hi l khi ng vt s dng Trehalose c nh hng n kh nng khng khun ca t bo hay khng? C gy ra t bin v gen dn n s tng hp AND bt quy tc hay khng? Tt c cc nghin cu cho kt lun l Trehalose khng gy c hai n t bo ch th.I.6. ng dng ca Trehalose

Mt s sn phm ng Trehalose :

Hnh 4: Mt s loi thc phm cha trehalosePh thuc vo tinh khit Trehalose thng phm ch yu c hai loi l Trehalose c tinh khit 45% v Trehalose c tinh khit cao hn 70%. Vi tinh khit 45% thng c dng trc tip nh mt loi thc phm hoc nh mt loi cht ngt b sung trong ch bin cc loi thc phm khc. Cn ng c tinh khit cao hn 70% thng dng cho cc i tng mc bnh, cc i tng n king. Ngoi ra ng tinh khit 100% dng trong vic phn tch ha hc.ng Trehalose c nhiu c tnh sinh hc ng qu, c hng v v tnh cht ha l tng t ng knh nn c s dng thay th ng knh trong sn xut cc loi thc phm cha ng nh ko, bnh v bt dinh dng tr em.v.v Sn phm sau khi ci tin ny s c gi tr cao v mt dinh dng cng nh chc nng phng nga bnh tt ng thi hng v cng c ci tin rt nhiu.Trn th gii vic s dng ng Trehalose trong sn xut cc mt hng chc nng nh sa, bnh ko, thc n cho ngi bnh.v.v Cc sn phm c b sung u tin l bnh quy, sa chua. Ngy nay c dng cho cc sn phm ung.Ngoi ra, Trehalose cn c s dng cho nhiu mc ch khc nhau. ng c tinh khit thp c th lm cht b sung trong mi trng nui cy mt s vi khun, loi c hm lng trung bnh dng cho ngi n trc tip hoc b sung vo thc phm cn loi tinh khit dng trong k thut phn tch.Trehalose cng c s dng nh mt loi dc phm dng nhiu cho cc i tng ngi gi, tr em v ngi bnh trong thi k phc hi sc khe tng cng hot ng tiu ha, gip cho cc i tng trn nhun trng n tt.Trehalose c tnh ha tan cao

Hnh 5: Tnh ha tan ca Trehalose v SucroseI.7 Tnh hnh nghin cu v sn xut Trehalose trn th gii

Trehalose c th thu nhn c bng cch chit sut chng t cc loi cy qu, hoc bng cc phng php ha hc, ha l, ha sinh. Nhng cho ti nay phng php c coi l c hiu qu v c kh nng cng nghip ha nht l phng php cng ngh sinh hc.

Thng thng Trehalose c sn xut theo phng php lin tc v khng lin tc. Trong phng php sn xut lin tc cng ngh c nh enzyme hoc c nh t bo c s dng, cn i vi phng thc sn xut khng lin tc th s dng enzyme chit tch. Gii php c nh enzyme v c nh t bo trong sn xut Trehalose cho hiu qu cao hn v sn xut lin tc tiu hao nng lng t, nh xng nh nhng tnh n nh km v cn my mc thit b hin i, nh xng tiu chun. Tuy vy, y li l phng php c kh nng cng nghip ha cao v c tp trung nghin cu nhiu cc nc c nn cng nghip pht trin nh Nht Bn, Hn Quc. Trung Quc, Php, M,I.8. Tnh hnh nghin cu v sn xut Trehalose Vit NamCng nghip sn xut thc phm chc nng ni chung v cng nghip sn xut ng Trehalose ni ring ang c pht trin mnh trn th gii. Cng vi xu hng pht trin chung ca ton cu. Vit Nam lnh vc ny cng ang c ch rt nhiu do nhu cu th trng ngy cng cp bch. Ngoi vin Cng nghip thc phm ra mt s trng v cc vin khc nh trng i hc Bch Khoa H Ni, trng i hc Nng Nghip, Vin i hc M H Ni, vin nghin cu thc phm thnh ph H Ch Minh cng bt u c nhng nghin cu thm d trong lnh vc nghin cu v sn xut loi ng chc nng ny. Tuy nhin cc nghin cu mi ch quy m phng th nghim.PHN 2: VT LIU V PHNG PHP NGHIN CUII.1 Nguyn vt liu v ha chtII.1.1 Ging vi sinh vt

Chng ging vi sinh vt. Escherichia coli DH5 alpha.E.coli c nui cy trong mi trng LB (Luria Bertani) [1% tryptone, 0.5% yeast extract v 1% NaCl] c b sung 100g/ml ampicillin lm nhn t chn lc.II.1.2 Plasmid:

DNA mang gen Trehalase c cung cp bi Vin Cng Ngh Sinh Hc Hn Quc, SeoulII.1.3 Ha chtTinh bt tan (Trung Quc), bactotryton (Hn Huc), bacto yeast extract (Hn Quc), NaCl (Trung Quc), canxiclorua (Trung Quc), agar (Vit Nam), glycerol (Vit Nam), acrylamide (Trung Quc), N,N-methylene-bis Acrylamide (Trung Quc), glycine (Trung Quc), sodiummetabisulfate (Trung Quc), methanol (Trung Quc), acetic acid (Trung Quc), Amononium persulfate (ASP, USA), Ampicillin (USA), comassie-blue R-250 (USA), axit photphoric (Trung Quc), ethanol (Trung Quc), Tris-base (Sigma, USA), phenol (Trung Quc), NaOH (Trung Quc), potassium Sodium Tartrate (Trung Quc), EDTA (USA), SDS (USA), DNS 3,5 Dinitro salicylic (Trung Quc), Imidazole (Hn Quc), Glucose (Trung Quc), Maltose (Trung Quc), NaH2PO4(Trung Quc), Na2HPO4(Trung Quc), 2-mercaptoethanol (c), Bromophenol blue (c), NaHCO3 (Trung Quc), Na2CO3(Trung Quc), potassium acetate (Hn Quc), Boric axit (Trung Quc), HCl (Trung Quc), n-butanol (Trung Quc), TEMED (Trung Quc), APS (Trung Quc), Dithiotheritol (Hn Quc), Ethidumbromide (Hn Quc)II.1.4 My mc v thit b My li tm (Trung Quc), my so mu quang in, my o pH, my lc (Trung Quc), bnh n nhit (Trung Quc), ni hp (Trung Quc), my khuy t (Trung Quc), my siu m (Trung Quc), cn in t (c), my in di (Trung Quc), t sy, box cy (Trung Quc), cn k thut (M), bung in di DNA (Nht), my li tm lnh siu tc lin tc (c).V cc dng c cn thit khc nh bnh tam gic, ng nghim, ng eppendof, u cn xanh, u cn vng, a peptri, pipet thy tinh v pipet ...II.2 Phng php nghin cuII.2.1 Bin np E.coli DH5 alpha bng phng php sc nhitBin np l qu trnh chuyn DNA plasmid trc tip tch ra t t bo th cho sang th nhn. C nhiu phng php bin np c s dng nh bin np bng xung in, sc nhitTrong bin np bng sc nhit l phng php c s dng ph bin hn c, n gin v cho hiu qu cao. C ch bin np ch yu bao gm vic lm thay i cu trc thnh t bo vi khun vi khun c th tip nhn DNA t bn ngoi, phc hi li cu trc thnh t bo sau khi tip nhn DNA vi khun mang DNA ngoi lai c th pht trin bnh thng. Nhng t bo c kh nng tip nhn DNA ngoi lai c gi l cc t bo kh binNguyn tc: bin np on DNA vo t bo vi khun th b mt t bo c x l CaCl2 tng hot tnh tip nhn DNA. To hn hp vi khun vi DNA trong iu kin lnh sau a hn hp vo tnh trng nhit cao (4oC). S phn chia t bo vi khun xy ra, DNA t tip xc vi t bo vi khun. Ngay lp tc a hn hp vo lnh, sc lnh cc DNA s i vo v c nh trong t bo vi khun. Hn hp ny c chuyn vo a peptri cha mi trng c b sung khng sinh.Quy trnh bin np gm 2 giai on: to t bo kh bin v bin np.Phng php tin hnh nh sau: + To t bo kh bin DH5 alphaHot ha t bo E.coli trn mi trng thch LB (1% w/v Bacto trypton; 0.5% w/v Bactoyeast extract; 0.5 % NaCl; 2% Agar) 37oC, 24 gi. Ly mt khun lc cy vo mi trng 5ml LB lng

Hnh 6: Vi khun E.coli trong LB rn (khun lc)Mi trng LB lng: 1% w/v Bacto trypton; 0.5% w/v Bactoyeast extract; 0.5 % NaClNui trn my lc (37oC, 12 14 gi). Sau ly 1% dch nui cy trn (50l cho 50ml mi trng nui cy LB) tin hnh lc trn my lc 200 250 rpm 37oC t c Abs 600 (khong 2 3 gi). Tip chuyn dch nui cy trn vo hai ng ly tm 50ml v ly tm 4oC tc 5000 7000 rpm trong 5 pht.Sau khi ly tm b dch trong pha trn v cho mt na (12.5 ml) dung dch 50mM CaCl2. Lc nh lm tan cn t bo sau bo qun 30 pht trn . Tin hnh ly tm 6000 rpm trong 5 pht, b dch trong pha trn v cho vo 1/10 th tch (2.5 ml) dung dch 100mM CaCl2 v lm tan hon ton cn. bo qun dch t bo ly 850 l dch t bo vi 150 l 100% glycerol bo qun trong nit lng.+ Bin np Ly 200 l dch t bo trn cho vo ng eppendoff v b sung 1 l DNA. Sau trn 30 pht ri sc nhit 2 pht 42oC. Sau sc nhit b sung thm 1ml LB v 37oC trong 1h. Sau ly 100 200 l dch bin np trn cy trn mi trng thch LB cha khng sinh (100ml LB, 100l Amp) v pht trin qua m 37oC.

Hnh 7: Vi khun E.coli sau khi bin np trong LB rn c khng sinhTt c cc thao tc u lm trong box cy v trng, cc dng c u c hp kh trng 121oC trong 20 pht.II.2.2 Tch DNA plasmid

DNA ti t hp sau khi c bin np vo chng DH5 alpha s nhn ln thnh nhiu bn sao cng vi qu trnh sinh trng ca vi khun. Sau vector ti t hp c tch chit ra khi DNA genom vi khun bng cch x l vi NaOH, SDS. Trong iu kin ny, DNA genom ca vi khun dng mch thng c phn t lng ln s b t gy cn plasmid dng mch vng c kch thc phn t nh v bn vng nn s khng b t gy. Protein v tp cht c loi b bng hn hp Chloroform: iso amyl alcohol. DNA plasmid thu c nh qu trnh kt ta vi cn v mui CH3COONa. C rt nhiu phng php tch plasmid nh: tch plasmid bng phng php kim, tch plasmid bng phng php boiling.Phng php tin hnh: Nui cy t bo trong mi trng lng, nht mt khun lc E.coli cha plasmid t a petri nui cy t bo qua m vo ng nghim hoc bnh tam gic c cha 5ml mi trng LB lng c b sung cht khng sinh Ampicillin (nng 100g/ml) nui lc qua m nhit 37oC, tc 200 vng/pht. Thu t bo, chuyn dch nui vo trong cc eppendoff 1.5ml. Ly tm 5000 vng/pht trong 10 pht thu sinh khi loi dch ni. ng nht t bo, cho 100l Sol I vo trong ng eppendoff cha t bo, ha tan t bo bng my vortex.Dung dch Sol I: 25mM Tris HCl pH 8.0; 10mM EDTA pH 8.0; 50mM glucose. Dung dch c kh trng 126oC v c ct gi 4oC.Ph mng t bo, cho 200l Sol II vo tip, dng tay o ngc ng eppendoff nhanh, nh nhng t 3 5 ln.Dung dch Sol II: 0.2N NaOH; 1% SDSCho 150l Sol III vo ta protein, dng tay o nh ng t 3 5 ln.Dung dch Sol III: 3M Potasium Acetate pH 5.5; acetic acid 11.5M dung dch c kh trng v ct gi 4oC Sau ly tm 4oC vi tc 10000 rpm/pht trong 20 pht. Dng pipet ly dch trong trn cho vo eppendoff mi (eppendoff c kh trng) b cn. Ly 600 - 800l Isopropanol cho vo v lc ln lc xung nhiu ln. em ly tm 4oC vi tc 12000rpm/pht trong 10pht. Ta b dch pha trn i, ly cn (DNA). Ta cho 500l cn 70% (cn lnh) ra DNA, dng pipet bm ln, bm xung cho DNA tung ra. Ly tm lnh 12000 rpm/pht trong 15 pht sau ly ht cn ra ri thc hin ra cn 1 ln na. Lm kh mu nhit phng.Ha tan DNA plasmid tch c trong 40l dung dch TE hoc nc ct v trng. in di mu kim tra mu DNA plasmid trn gel agarose 0.8%. Bo qun mu -20oC.II.2.3 Xc nh DNA biu hin E.coli trn gel Agarose

Nguyn tc: Phng php in di DNA trn gel agarose da vo c tnh tch in m ca phn t axit Nucleic pH trung tnh nh cc nhm photphat nm trn cc cu ni phosphodiester ca axit Nucleic. Khi c t trong in trng, cc phn t axit Nucleic s dch chuyn v cc dng vi tc khc nhau ty theo kch thc ca chng. Cc phn t c kch thc ln s di chuyn chm hn cc phn t c kch thc b. Kh nng di ng ca cc phn t axit Nucleic ny cn ph thuc vo nng gel. y chng ti s dng gel agarose 0.8%. Cc axit Nucleic trong gel s hin hnh di tia t ngoi (UV) nh ha cht ethidium bromide.

Phng php tin hnh: 0.8% agarose gel trong dung dch m TBE c trn mt gi th nm ngang (chun b gel agarose vo dung dch m un cho agarose tan hon ton). ngui khong 50oC,cho 1l ethidium bromide vo ri dung dch agarose vo khay in di c ci sn rng lc. Sau khong 30 pht, g lc ra, t bn gel vo b in di. dung dch chy in di l m TBE (40mM Tris HCl v 2 mM EDTA, pH 8.) vo b dung dch ngp cch mt gel khong 2mm v tin hnh tra mu. Trn mu vi 1.5l m mu xanh 1X xanh Bromophenol. Qu trnh chy in di c tin hnh di hiu in th 100V, thi gian l 25 30 pht. Sau khi chy xong ly bn gel ra v em soi bng tia t ngoi UV 280 nm, plasmid DNA l nhng vch mu da cam c so snh vi marker DNA ta xc nh c kch thc v tinh sch ca DNA.II.2.4 Phng php ph v t boProtein chit xut t cc t bo vi khun l mt bc quan trng c bit i vi nghin cu trong cc lnh vc ha sinh hc, vi sinh hc v sinh hc phn t cng nh cho s pht trin ca qu trnh sn xut protein trong cng ngh sinh hc v k thut sinh ha. Hin nay, protein chit xut t cc t bo vi khun thng c s dng phng php c hc, i hi dng c tng i t tin v i hi k nng k thut.m lysis i4 c pht trin bng cng ngh Enzyme phng th nghim ca cc quc gia Trung tm K thut di truyn v Cng ngh sinh hc (BIOTEC-Thailand) cung cp mt gii php r tin chit xut protein, DNA. C rt nhiu phng php ph v t bo: phng php ph v t bo bng phng php c hc v phi c hc. Trong , phng php c hc gm c rt nhiu phng php nh phng php ph v t bo bng sng siu m, ph v t bo bng my ng ha cao p, ph v t bo bng phng php lnh ng, Phng php khng bng c hc nh phng php sc thm thu, phng php x l kim, Do enzyme ca chng ti l enzyme ni bo nn y chng ti s dng ha cht ph v b mt t bo. Cc b m lysis c xy dng thu protein vi khun bng cch s dng ha cht ph v b mt t bo. iu quan trng y l chng ti s dng dung dch ph v t bo rt c hiu qu vi nhiu loi t bo vi khun, nm trong khi vn bo ton hm lng v hot tnh ca protein. Dung dch m cng c s dng cho tch chit DNA t vi sinh vt. Cc c im ca m lysis i4:- Dung dch m c th c s dng tch protein mt cch hiu qu t rt nhiu loi vi sinh vt: vi khun, nm men, nm.- Qu trnh n gin v d lm- dng v dng c, my mc khng i hi cao- Dung dch m khng lm phn ng bin tnh v cung cp protein s dng cho th nghim ha hc hay sn xut protein trong cng ngh sinh hc- Ngoi ra dung dch m ny cn c s dng bc b sung cho vic tch DNA mt cch hiu quPhng php tin hnh: Ly 2 ng eppendoff sch em cn ng eppendoff trn cn phn tch v ghi li trng lng.Nui cy t bo trong mi trng lng. Nht mt khun lc t a petri ( bin np, a petri LB thch c khng sinh). Nui cy t bo qua m vo ng nghim cha 5ml mi trng LB lng c b sung khng sinh, nui lc qua m 37oC tc 200v/pSau thu t bo, chuyn dch nui vo trong 2 eppendoff m ta va cn c em ly tm 6000rpm/pht trong 20 pht 4oC thu sinh khi. B dch trong v cn trng lng t bo thu c.B sung 200-300 l/100mg t bo. Sau , ha tan cn bng vortex v em ngm 15 20 pht. Ly tm 10 000 v/pht trong 30 pht ri b cn thu dch ni.II.2.5 Xc nh hm lng protein tng bng phng php BradfordC s cua phng phap: ham lng protein enzyme a c xac inh vao nm 1976 bng phng phap cua bradford. C s cua phng phap la da vao hp thu mau cua phan ng gia enzyme va dung dich Bradford, da vao thi chun BSA-Bradford tinh ham lng protein enzyme.Phng phap tin hanh: cho 100l dung dich protein pha loang vao 900l dung dich Bradford (100mg Coomassie Brillitant Blue G250 hoa tan 500 ethanol, b sung 100ml phosphorich acid 85%, sau o inh mc n 1lt bng nc ct). Chun bi blank (mu i chng) bng cach b xung nc ct thay cho protein. Hn hp phan ng gi nhit phong trong 5 phut, c 1 phut vortex mt ln. Sau o, hn hp c o quang ph bc song 595nm bng may spectrophotometer (may so mau). hp thu quang cua dung dch phan ng da vao thi ng chun BSA-Bradford tinh c nng enzyme protein co trong mu.Da vo th ng chun BSA-Bradford c:Y = 1.0361x + 0.0524R2 = 0.9944Phng php tnh ton hm lng protein enzyme c vit nh sau:X (mg/ml) = (abs - 0.0524): 1.0361 *D*RFTrong o:X: Ham lng protein co trong mu phan ngabs:Gia tri hp thu quang ph cua mu bc song 595nmD: H s pha loang cua enzymeRF: H s phn ng

Hnh 8: th ng chun Bradford vi protein chun ABSII.2.6 Xc nh hot tnh enzyme bng phng php DNS (3.5 Dinitro Salicylic)C s cua phng phap: Da trn c s phan ng tao mau gia ng kh vi thuc acid dinitro-salicylic. Cng mau cua hn hp phan ng ti l thun vi nng ng kh trong mt pham vi nht inh. Bit c mt quang cua dung dich ng kh nghin cu vi thuc th DNS, da theo thi chun cua glucose tinh khit (hoc thi ng chun maltose) vi thuc th nay ta tinh c lng ng kh cua mu vt nghin cu. c c ng chun cho phng php ny chng ti tin hnh theo cc bc sau: Chun b dch ng 1% glucose tinh khit. Chun b 6 ng nghim vi cc thnh phn sau:STTGlucose 0.5% (mg/ml)Glucose 0.5% (l)H2O (l)DNS (l)OD/575nm

100200020000

20.25100197520000,357

30.5200195020000,525

40.75300192520000,824

51400190020001,125

61.25500187520001,425

71.5600185020001,625

cc ng nghim vo nc un si trong 10 pht. Ri lm lnh ngay bng nc my n nhit phng. o OD bc sng 575nm sau v th ng chun

Hnh 9: th ng chun GlucosePhng phap xc nh hot tnh ca enzyme c thc hin nh sau: Phan ng enzyme c tin hanh: ly 1,5ml Trehalose 0,5% b sung vo 5l dung dich enzyme th ri em 65oC trong thi gian 3 ting. Sau 3 gi ta em thc hin phn ng DNS, ly 500 l dch thy phn enzyme pha trong 1500 l nc ct v b sung 2000 l dung dch DNS (5.3g DNS, 9.9g NaOH, 153g K- Na- Tartrate, 4.15g Sodium metabisulphate, 708ml nc ct). Hn hp un nong nhit si cua nc trong 5 phut sau o lam mat bng cach t cac ng nghim cha hn hp phan ng di dong nc chay. Nng ng kh nhng mu phan ng khac nhau phu thuc mc hp thu quang cua cac mu bc song 575nm. Khi thay dung dich phn ng bng nc ct ta co mu i chng. tnh c hot enzyme ta da vo phng trnh ng thng c xc nh t th ng chun DNS theo glucose.Phng trinh tinh toan hoat cua enzyme da trn phng trinh cua thi chun DNS theo tinh bt c vit nh sau:X (mg) = (abs 0.0461): 1.0675H = (X/T)*(D/VE)*1000*RFTrong o:X: Ham lng ng glucose co trong phan ngabs: Gia tri thc cua hp thu quang ph cua mu bc song 575nmT: Thi gian phan ngD: H s pha loang cua enzymeVE: Th tich cua enzymeRF: H s cua phan ngII.2.7 Tch v tinh ch enzyme bng sc k ct Ni NTAQua trinh tinh sach enzyme bng ct Ni-NTA co th thc hin iu kin binh thng. Nui cy E.coli a c bin nap trong khoang thi gian 16 gi va thu nhn bng cach ly tm 8000 rpm trong 15 phut. Bo phn dich trong va hoa tan cn trong dung dich m lysis [50 mM tris-HCl pH 7.0, 300mM NaCl, 10mM imidazole] va siu m lanh ( ch cycle 60%, power 40%, thi gian 3 ln mi ln 1 phut, bng may siu m...). Dich t bao th c ly tm 10.000 rpm 4oC trong 15 phut. Ly 2 ml 50% cht nhi Ni-NTA, nhi vao ct sc ki va ra ct vai ln bng 4ml dung dich m ra [50mM Tris-HCl pH 7.0, 300mM NaCl, 20 mM imidazole] loai bo nhng tap cht bam trn ct. Sau o tach enzyme bam trn ct bng 3ml dung dich m [50mM Tris-HCl pH 7.0, 300 mM NaCl, 25mM imidazole].II.2.8 Phn tch protein bng in di trn gel SDS polyacrylamide (SDS-PAGE)in di l qu trnh dch chuyn ca cc phn t tch in trong mt dung dch t trong in trng. Do vn tc dch chuyn ca cc phn t mang in ph thuc vo in tch, hnh dng v kch thc ca phn t. in di c s dng trong vic phn tch cc phn t ca mt hn hp. in di l phng php phn tch n gin, tng i nhanh, thng c dng phn tch v tinh ch cc phn t ln nh protein, acid nucleic. Tuy nhin, in di cng c dng cho phn tch cc phn t mang in n gin hn, nh cc loi ng mang in, acid amin, peptit, cc nucleic v cc ion n gin. K thut SDS PAGE c dng trong nghin cu enzyme, xc nh phn t lng protein, hm lng, tinh sch ca protein.. in di cc cao phn t thng c thc hin bng cch a mt lng nh mu vo mt dung dch c c nh nh mt mng xp. Di tc dng ca mt hiu in th, cc phn t khc nhau ca hn hp mu chuyn ng qua khi mng vi vn tc khc nhau. Kt thc qu trnh tch, cc phn t khc nhau c pht hin cc v tr khc nhau ca mng di dng cc bng (bng cch nhum gel hay chp pht x). Mng xp y c tc dng gi n nh cho mi trng in di, nh m cc bng mu khng b khuch tn v trn ln khi dung dch in di b lm nng ln do tc dng ca dng in i qua. K thut SDS PAGE s dng vt liu mng l gel polyacrylamide, trong trng hp ny mng to ra cn ng vai tr nh mt li lc kch thc trong qu trnh phn tch. C hai phng php in di vi SDS PAGEC hai phng php in di vi SDS PAGE: lin tc v bn lin tc. Phng php lin tc ch s dng mt dung dch m vi duy nht mt bn gen phn tch. Ngc li phng php bn lin tc s dng thm mt bn gen gn pha trn bn gen tch, gi l gel c c (stacking gel) mi bn gel c dng vi mi dung dch m khc nhau. Phng php khng lin tc c u im hn hn phng php lin tc l c s c c mu trong lp stacking gel, nh vy m mu xut hin bng sc nt hn cc cc bng nhn c t phng php lin tc v nh vy phng php bn lin tc c s dng rng ri hn. Theo Laemli (1970), bn in di mu protein trn gel polyacrylamit bn lin tc c cha SDS. Dung dch Acryamide gm c (33.5% acrylamide, 0.3% (w/v) N.N- methylene bis) v gel c chun b phn tch enzyme c trng lng phn t tng i ln thng c dng l 10% acrylamide. chun b gel c nng 10% acrylamide cho SDS PAGE c kch thc bn gel l (75 x 85) mm c tnh ton theo t l sau: Lp gel tchLp gel tch: 1.5 ml dung dch acrylamit/bis acrylamide, 1.9 ml dung dch m 1M tris HCl (PH 9.1), 50l dung dch SDS 10%, 1.4 ml nc ct v trng, 125 l amonium persulfate 3% (AP), 2.5 l N.N.N.N tetramethylene diamin (TEMED) Lp gel c c c chun b t dung dch hn hp acrylamide/ bis acrylamide (33.5% acrylamide, 0.3% bis acrylamide). Lp stacking gel bao gm: 195 l dung dch acrylamit/ bis, 375 l dung dch m 0.5 M tris HCl (pH 6.8), 12.5 l dung dch SDS 10%, 915 l dung dch nc ct, 50 l AP 3%, 10 l TEMED. Trn u dung dch ca lp gel tch sau dng pipet ht v bm dung dch vo bung gel ti khi mc gel cch mp trn bn thy tinh 1.5 2 cm th dng li. Ch trong qu trnh gel nn trnh khng bt kh c trong lp gel, sau ph mt lp nc ln trn b mt lp gel va , qu trnh polyme ha s hon ton t mt n hai gi.Trn u dung dch ca lp gel tch sau dng pipet ht v bm dung dch vo bung gel ti khi mc gel cch mp trn bn thy tinh 1.5 2 cm th dng li. Ch trong qu trnh gel nn trnh khng bt kh c trong lp gel, sau ph mt lp nc ln trn b mt lp gel va , qu trnh polyme ha s hon ton t mt n hai gi. Sau khi lp gel tch ng hon ton, rt b lp nc pha trn ra v tip lp gel stacking ln trn cho ti y bung gel. Sau t lc vo bung gel trnh to cc bt kh di nhng rng lc. Qu trnh polyme ha lp gel ny khong 30 pht. Sau khi lp gel tch ng hon ton, rt b lp nc pha trn ra v tip lp gel stacking ln trn cho ti y bung gel. Sau t lc vo bung gel trnh to cc bt kh di nhng rng lc. Qu trnh polyme ha lp gel ny khong 30 pht. Sau khi bn gel hon thnh, ton b bn gel c t trong bung in di c cha y dung dch in di 1X v sau ta tin hnh nh mu v chy in di cng dng in l 16mA Sau khi bn gel hon thnh, ton b bn gel c t trong bung in di c cha y dung dch in di 1X v sau ta tin hnh nh mu v chy in di cng dng in l 16mA, n khi mu hin th chy qua hon ton bn gel. in di xong, bn gel c nhum bng dung dch 0.1% (w/v) Coomassie Blue R 250 (Methanol: glacial acetic acid: nc = 5: 5:1) c lc qua giy whatman No 50oC trong thi gian 30 pht) v ty mu bng dung dch ty (Methanol: glacial acetic acid: nc = 2:1:7) 55oC. Sau bn gel khi c chuyn vo mt t giy whatman v sy kh 80oC trong thi gian 80 pht.II.2.9 Xc nh sn phm thy phn bng phng php sc k bn mng (Thin layer chromatography TLC)Nguyn tc: Da trn s tng tac c hiu tng i gia cht hoa tan vi cac vi tri gn kt trn b mt tm sc ki (pha tinh) va h dung mi (pha ng). Trn tm sc ki co phu mt lp mong Silicagel 60, chinh cht nay tao ra cac ng mao dn rt nho khi nhung vao dung dich chay tai y cht cn phn tich se tng tac vi cht cn hp thu nh tinh cht phn cc cua chung,dung dich co lc cng b mt ln thi dung dch chay se i ln trn ng thi se keo theo cac ng i ln trn (ng nay lam san phm cua phan ng enzyme thuy phn tinh bt). Phng phap nay co nhay cam rt cao va cho phep xac inh tinh san phm ng (cac loai ng kh) tao thanh mt cach nhanh chong, chinh xac.Phng php tin hanh: Hoat hoa tm sc ki lp mong K6F (Merk) bng cach t no trong tu sy nhit 110oCs trong thi gian 30 phut. Dung dich chun va cac mu phn tich c chm trn ban sc ki vi cung mt loai pipet thanh nhng chm co ng kinh nho hn 0.5mm va mi chm cach nhau 0.7 1.5cm, sau o sy kh bng may sy cm tay. Mt u tm sc ki nhung ngp trong pha ng sc ki TLC [isoprosi] alcohol: ethylene acetate: nc = 3:1:1 (v/v/v)] sao cho b mt dung dich chay cach ch chm khoang 0.5cm, nhit phong. Cac mao dn cua ban sc ki se dn dung mi va a cac ng trong mu phan ng ln cao theo nhng vn tc khac nhau tuy theo ai lc cua chung vi dung mi. Nh vy cac ng trong cac mu thi nghim se c tach trn b mt sc ki. Khi quan sat thy dung dich chay ln cach mep trn ban sc ki khoang 0.5cm thi ly tm sc ki ra va sy kh bng may sy. Khi ban mong c sy kh trit , ta nhung ngp nhanh no trong dung dich c inh [fixing solution: 3 gam metanol, N-1-naphthy-ethylene diamine va 50 ml H2SO4 c trong 1 lit], ri sy kh bng may sy cm tay. Hinh anh cac ng c phn tach trn ban mong c hin thi khi sy nhit 110oC trong thi gian 10 phut. Cac ng tng ng c xac inh theo cac ng i chng (inh tinh) lng ng tao thanh cung co th xac inh mt cach tng i bng m nhat cua cac vt trn ban sc ki. inh lng chinh xac cac san phm c tao thanh do phan ng cn phai s dung cac may moc hin ai hn nh sc ki long cao ap (HPLC) hoc sc ki ion cao ap (HPIC).PHN 3: KT QU V THO LUNIII.1 Kt qu bin np gen Trehalase vo E.coli DH-5 alphaGen mang m ha Trehalase c gn vo vector biu hin pGEX-4T-1 v c bin np vo E.coli DH-5alpha nhn dng v biu hin. Vector biu hin c mang gen khng khng sinh ampicinine nh l mt marker rt tt sng lc cc khun lc c bin np thnh cng. Nhng t bo khng c tip nhn vector nhn dng s khng c kh nng pht trin trn mi trng thch c khng sinh. Nh vy, chng ta c th khng nh rng cc t bo c bin np bi vector nhn dng pht trin thnh cc khun lc trn mi trng c khng sinh. Tuy th, trong qu trnh thao tc vn c th b nhim mt s vi sinh vt khc m chng c kh nng khng li khng sinh. Chnh v th khng nh c vector c nhn dng chng ta cn phi chng minh s c mt ca DNA nhn dng trong cc khun lc thu c.

Hnh 10: Vi khun E.coli sau khi bin np trong LB rn c khng sinhIII.2 Kt qu tch plasmid DNA v in di trn gel agarose.

Sau khi bin np chn cc khun lc mc c trn mi trng LB rn c cha khng sinh nui sau c cy truyn sang mi trng LB lng c cha 100mg/ml ampicillin, nui 37oC qua m, lc 250 vng/pht. Sau em tch plasmid DNA v kim tra DNA ti t hp trn gel agarose. Kt qu c th hin hnh di y.Qua hnh nh in di cho thy rng plasmid DNA ca Trehalase trong vi khun E.coli DH5 alpha th hin r v tng i sch c kch thc khong 1,2 kb. Qua hnh nh in di ta cng c th khng nh rng DNA mang gen Trehalase c bin np vo E.coli DH5 alpha.

Hnh 11: Hnh nh in di plasmide DNA mang gen trehalase trn gel agarose 0.8%Bng 1: DNA markerBng 2: Plasmid DNA mang gen Trehalase t E.coliIII.3 Xc nh cc tnh cht ca enzyme Nh vy qua cc th nghim trn chng ti xc nh c mt cch r rng rng vector bin np c cha gen Trehalase c bin np vo E.coli c hai mc t bo v gen. khng ch c rng cc vector c th biu hin c dch m thnh protein hot ng, th chng ta phi cn chng minh c mc protein v tnh hot ng ca enzyme.Hm lng protein tng c xc nh theo phng php Bradford Y= 1.0361x+ 0.0524R2= 0.9944Hot ca enzyme c tnh da vo phng trnh ng chun Glucose nh sau: Y= 1.0675x + 0.0461

R2 = 0.9936Ta c kt qu sau:EnzymeTh tch (ml)Protein tng (mg/ml)Hot (U/ml)Hot ring (U/mg)H s ca phn ng

Trehalase th100293.023561,2151

Mt n vi hoat cua enzyme (U) c inh nghia la lng enzyme cn thit chuyn hoa 1 mg Trehalose tao thanh glucose trong thi gian 1 phut.III.4 Tinh ch Trehalase bng ct sc k Ni NTA

Theo s trn, chng ti thu c enzyme Trehalase t t bo vi khun E.coli ti t hp. Dch enzyme th thu c em i tinh ch qua ct sc k Ni NTA thu nhn enzyme tinh sch phc v cho vic xc nh tnh cht l ha v ng dng ca enzyme. tinh sch ca enzyme sau khi tinh ch c th kim tra trn gel polyacrylamide c cha SDS (phng php SDS PAGE)

Da vo phng php SDS PAGE ta c th xc nh mt cch tng i trng lng phn t v tinh sch ca enzyme. Di y l kt qu in di Trehalase tinh ch.

Hnh 12: SDS PAGE ca Trehalase tinh sch. Bng 1- trng lng phn t protein ca mu i chng Bng 2 - Trehalase tinh sch nh ct sc k

Nhn hnh nh in di ta thy: trn bng 1 l mu i chng c trng lng phn t cc protein khc nhau, phn b t trn xung di ln lt l: (250, 150, 100, 75, 50, 37) kDa. Nhng protein c trng lng phn t, kch thc nh s chy nhanh hn. Protein ca Trehalase c khi lng phn t nm di vch c trng lng phn t 75 kDa, c tnh trng lng phn t ca Trehalase khong 70 kDa.T bn gel c th thy Trehalase kh tinh sch. Bng phng php trn, chng ti thu v tinh sch c Trehalase t t bo vi khun E.coli ti t hp. Lng enzyme ny c xc nh cc c tnh v hot lc ca enzyme.III.5 Xc nh mt s tnh cht ca Trehalase

Mi enzyme u c mt nhit , pH v dung dch m ti u l nhng iu kin m enzyme th hin hot tnh mnh nht. Xc nh c iu kin ti u cho phn ng l vic lm khng th thiu c v l bc c bn khm ph tnh cht enzyme, cng nh cc ng dng ca enzyme.III.5.1 nh hng ca nhit ti hot lc ca enzymeNhit l mt trong cc yu t tc ng mnh m ti hot ng ca enzyme c vng nhit hot ng khc nhau. xc nh nhit ti u ta tin hnh phn ng enzyme vi Trehalose 0.5% em cc di nhit khc nhau (35-80oC) mi bc nhit l 5oC trong 3 gi. Sau thc hin phn ng DNS ri o OD bc sng 575nmGi tr tng ng vi nhit m phn ng c gi tr cao nht v ta coi gi tr l hot ng tng i cc i (100%), v cng l nhit ti u ca enzyme. Ta c bng sau:Nhit (oC)OD/575nmT l phn trm (%)

370.65440

400.70743.22

450.73745.05

500.96358.86

551.14770.11

601.27377.81

651.636100

701.05564.48

800.43626.65

Kt qu c th hin hnh sau:

Nhit Hnh 13: nh hng ca nhit ti hot ng ca Trehalase

T th trn ta c th thy: s thay i nhit c nh hng r rt n qu trnh thy phn ca enzyme i vi c cht l ng Trehalose. Hot tnh enzyme tng dn khi nhit th nghim tng ln t 37oC n 60oC v t gi tr cc i 65oC. nhit trn 65oC th enzyme b bin tnh mnh v hot tnh gim i rt nhanh nhit 70oC v 80oC.

T kt qu th nghim trn ta c th kt lun nhit ti u cho hot ng ca enzyme Trehalase l 65oC. nhit ny enzyme cho hot cao nht.III.5.2 nh hng ca pH v dung dch m ln hot ng ca EnzymeTheo cc nh nghin cu c bn v enzyme, pH s nh hng ti trung tm hot ng ca enzyme, khi pH thay i th kh nng ion ha ca cc nhm chc cng thay i, do s nh hng n s kt hp ca enzyme v c cht. Mt khc, pH c nh hng kh ln n vn tc phn ng ca enzyme, mi enzyme hot ng trong mt di pH khc nhau. Gi tr tng ng vi pH m enzyme hot ng mnh nht v ta coi gi tr l hot ng tng i cc i (100%), v cng l pH ti u ca enzyme. Thc hin phn ng enzyme vi c cht l Trehalose 0.5% pha trong cc dung dch m vi pH khc nhau (di pH t 4-11),Cc dung dch m 50mM natri acetate ( pH 4-6.5), 50mM natri phosphate (pH 6.5-8.5) 50mM Tris-HCl (pH 8.5-10.5), em 65oC trong 3 gi.Sau thc hin phn ng DNS ri o OD bc sng 575nm.Ta c bng s liu sau:pHNatri acetatePhosphateTris HCl

40.93

4.51.04

51.16

5.51.37

61.64

6.51.471.38

71.21

7.51.17

81.03

8.50.980.95

90.84

9.50.61

100.56

10.50.36

Kt qu bng trn c biu din bng th sau: Hnh 14: nh hng ca pH ln hot tnh ca TrehalaseQua hnh 20 ta nhn thy rng enzyme hot ng trong khong pH axit yu hoc kim yu (pH4.5-6). Trong khong pH axit, hot enzyme khi thy phn Trehalose thp, thi gian thy phn lu, pH 4.5 hot enzyme thy phn ch khong 63.4%, pH 5.0 hot enzyme thy phn ch khong 70.7%. pH kim (pH=8-10) hot enzyme rt thp v nghch o vi pH, pH cng cao th hot enzyme cng gim. pH 9.0 hot enzyme thy phn khong 51.2%, pH 10.0 hot thy phn ch khong 34.1%Qua hnh trn ta thy gi tr pH ti u ca enzyme Trehalase gi tr pH l 6.0. pH ny enzyme cho hot lc cao nht. Nh vy, enzyme ny thch hp trong mi trng axit yu.III.5.3 Kh nng chu nhit ca Trehalase

ng dng c enzyme vo sn xut chng ta cn phi kho st rt k kh nng n nh ca enzyme cc nhit 60, 70, 80 v 90oC. nhit trn 60oC th enzyme ny rt km chu nhit v hot tnh gim i rt nhanh.

Hnh 15: Biu din kh nng chu nhit ca TrehaloseIII.6. nh hng ca ion kim loi v dung mi kim loi ln hot ca enzymeIII.6.1 nh hng ca ion kim loi ln hot ca enzymeTrong phn ng enzyme, s c mt ca mt s ion kim loi hoc ha cht khc thng c nh hng n hiu sut ca phn ng. i vi mt enzyme nht nh mt s ion kim loi c tc dng thc y, ngc li c mt s khc li c tc dng c ch. Xc nh nh hng ion kim loi n hot ng ca enzyme gip chng ta c th tng cng hiu sut phn ng bng cch iu tit cc cht trong sut qu trnh phn ng. xc nh nh hng ca ion kim loi n hot ng ca enzyme, chng ti tin hnh b sung cc mui cha ion kim loi khc nhau. Th nghim c tin hnh bng cch ngm enzyme trong Trehalose 1% v b sung dung dch m c cha ion: Ca2+, Mg2+, Mn2+ (CaCl2, MgCl2, MnCl2) vi nng 5mM. Sau thc hin phn ng enzyme trong 3h 65oC, thc hin phn ng DNS ri o OD/575nm v tin hnh xc nh hot lc tng i ca enzyme. a s cc kim loi th nghim u c tc ng xu ln tnh hot ng ca enzyme.Ion kim loi km c ch gn nh hon ton hot ng ca Trehalase. Trong khi Mg2+ lm tng hot ng enzyme 17,61% so vi hot ng ban u. Mg2+ c tc ng lm tng kh nng thy phn tinh bt ca enzyme. Mn2+, Ca2+ th hot ng enzyme gim 18-65%. III.6.2 nh hng ca dung mi hu c ln hot ca enzyme nh hng ca dung mi hu c c th nghim trong 10% dung mi hu c EtOH, MeOH, DMF, DMSO. Enzyme b c ch bi dung mi hu c nh: DMSO, DMF, Ethanol v Methanol t 10-63%. Phn t protein trong dung dch cha nc c to bi v hydrat ha, n bao gm cc phn t nc c bm vo cc phn t protein v bo v n v lm cho n c th tan c trong mi trng nc. khi c s hin din ca dung mi hu c, n s c khuynh hng thay th cc phn t nc c trong v hydrat ha v c bn trong cu trc protein. Do m lm sai lch bn cht ca protein (Bachar et.al.1999). V vy kt qu ny cho thy rng s thay i cu trc protein do mt dung mi hu c c th gy nh hng dn vic mt hot tnh enzyme. Ta c bng sau:Ion kim loi v dung mi hu cOD/575nmHot lc (%)

i chng1,425100

Ca2+0.92865.12

Mg2+1.676117.61

Mn2+0.26118.31

EDTA1.40398.46

EtOH0.7653.33

MeOH0.90663.60

DMF0.29520.7

DMSO0.15310.74

T kt qu bng trn ta c th biu din bng thi di y:

Hnh 16: nh hng ca ion kim loi v dung mi hu c ln hot tnh ca enzyme T kt qu nghin cu trn chng ta thy rng hu ht cc ion kim loi v dung mi th nghim u nh hng n hot tnh ca enzyme loi tr ion km c nh hng tt n hot tnh ca enzyme v lm cho hot tnh ca n tng ln khong 17% so vi i chng.III.7 Xc nh sn phm sinh tng hp trehalose bng enzyme ti t hp (Thin layer chromatography TLC)Trehalase c th thy phn Trehalose thnh 2 phn t glucose nhng iu kin nht nh v ngoi ra enzyme ny cng c th thc hin phn ng ngc, ngha l c kh nng kt hp 2 phn t Glucose thnh Trehalose. Theo mt s ti liu qu trnh tng hp ny xy ra nhiu hn khi mt trong hai phn t c cht c gn vi UDP, ADP hay CDP (Uracine Diphosphate, Adenosin Diphosphate, Cytosin Diphophate). Th nghim c tin hnh vi cc c cht sau: Glucose, Fructose, Galactose v Mannose nng l 0,5% trong 50 mM natri axetat (pH 6.0), 65oC trong 12 gi. Sau phn ng c dng bng nhit 100oC trong 10 pht v cc sn phm phn ng c phn tch TLC. iu cho ta thy rng vic tng hp ng Trehalose thu c kt qu tt i vi c cht l glucose, cn cc c cht khc phn ng khng xy ra hoc c th xy ra i vi Manose v to ra sn phm ng dng.

Hnh 17: Sc k bn mng tng hp Trehalose v Trehalose tng t bng enzyme vi glucose v UDP-G cc thi gian khc nhau

Hnh 18: Sc k bn mng tng hp Trehalose bng enzyme vi glucose v ADP-G 12h1. ADP-G + Glucose5. ADP-G + Fructose

5. ADP-G + Fructose2. ADP-G + Glucose vi enzyme 12h6. ADP-G + Fructose vi enzyme 12h

6. ADP-G + Fructose vi enzyme 12h3. ADP-G + Galactose7. ADP-G + Mannose

7. ADP-G + Mannose4. ADP-G + Galactose vi enzyme 12h

8. ADP-G + Mannose vi enzyme 12h

Hnh 19: Sc k bn mng tng hp Trehalose bng enzyme vi glucose v CDP-G 12h1. CDP-G + Glucose5. CDP-G + Galactose

5. CDP-G + Galactose2. CDP-G + Glucose vi enzyme 12h6. CDP-G + Galactose vi enzyme 12h

6. CDP-G + Galactose vi enzyme 12h3. CDP-G + Fructose7. CDP-G + Mannose

7. CDP-G + Mannose4. CDP-G + Fructose vi enzyme 12h8. CDP-G + Mannose vi enzyme 12h

8. CDP-G + Mannose vi enzyme 12hKt qu cho thy: Trehalose c tng hp bng enzyme vi glucose l c cht v c s hin din ca UDP, ADP, CDP. Ngoi ra, ngun c cht Fructose v Mannose cng c th dng tng hp ng Trehalose tng tKT LUNSau qu trnh tin hnh th nghim, chng ti thu nhn c cc kt qu sau:1. Plasmid mang gen Trehalase c bin np vo E.coli DH5-alpha v c biu hin tt trong vi khun ny c mc DNA v protein.2. C th tinh sch c enzyme ny bng sc k ct Ni-NTA.3. xc nh tnh cht ha l ca enzyme Trehalase cng nh cc yu t nh hng n kh nng xc tc ca enzyme: nhit ti u l 65oC, pH ti u l 6.0, Trehalase l enzyme km bn nhit.4. Cc ion kim loi v mt s dung mi hu c c nh hng nhiu n hot tnh ca enzyme, ngoi tr ion km.5. Trehalase ti t hp c th s dng tng hp Trehalose vi s c mt ca UDP, ADP, CDP. TI LIU THAM KHO1.Van Dao Nguyen, Mutagenesis Studides on the Quaternary Structure and Physicochemical Properties of Maltogenic Amylase from Thermus strain2. Nguyn Trng Cn, Nguyn Th Hin, Th Giang, Trn Th Luyn, cng ngh enzyme, nh xut bn (NXB) Nng Nghip, 19983. Nguyn Hu Chn, Enzyme v xc tc sinh hc, NXB Y hc, 19964. Khut Hu Thanh, c s di truyn vi sinh vt v cng ngh gen, Trng i Hc Bch Khoa H Ni, 2001.5. ng Th Thu, L Ngc T, T Kim Anh, Phm Thu Thy, Nguyn Th Xun Sm, Cng Ngh Enzyme, NXB Khoa hc k thut.6. ng Th Thu, Nguyn Th Xun Sm, T Kim Anh, Th Nghim Ha Sinh Cng Nghip, Trng i Hc Bch Khoa H Ni7. L Ngc T v cng s, Ha sinh cng nghip, NXB khoa hc k thut, 2000.8. Nguyn Th Hng, molecular cloning and characterization of Trehalose synthesizing Glycosyltransferase Enzyme from Thermotoga maritime, The Graduate School Yonsei University, Department of Food and Nutrition9. V Th Thanh Phng, Biu hin ca Baciluss sterothermophiluss Maltogenic Amylase trn vi khun E.coli DH5- Alpha, kha lun tt nghip, 2009.10. Sambrook, Fritsch, Maniatis, Molecular Cloning, Second Edition.MC LCLI CM NBNG CC K HIU VIT TT1M U

PHN 1:TNG QUAN TI LIU3I.1 Cng ngh DNA ti t hp3I.2 i cng v vi khun E.coli5I.2.1 c im hnh thi6I.2.2 c tnh nui cy6I.2.3 c tnh sinh ha7I.2.4 Sc khng ca E.coli7I.3. Tnh hnh sn xut v ng dng enzyme trn th gii v Vit Nam7I.3.1 Tnh hnh sn xut v ng dng enzyme trn th gii7I.3.2 Tnh hnh sn xut v ng dng enzyme Vit Nam.13I.4 Trehalase14I.4.1 Gii thiu v enzyme Trehalase14I.4.2 Trehalase trong vi khun14I.4.3 Trehalase trong thc vt15I.4.4 Trehalase trong nm men15I.4.5 Thy phn Trehalose15I.5. Tng quan v ng chc nng Trehalose16I.5.1 Trehalose16I.5.2 nh hng ca Trehalose ln c th sng19I.5.2.1 nh hng ca Trehalose n s chuyn ha hydratcacbon19I.5.2.2 Vai tr thc y qu trnh hp th canxi ca Trehalose19I.5.2.3 nh hng ca Trehalose n h vi sinh vt trong khoang ming20I.5.2.4 Tnh khng khun ca Trehalose21I.5.2.5 Tnh an ton ca Trehalose21I.6 ng dng ca Trehalose22I.7 Tnh hnh nghin cu v sn xut Trehalose trn th gii24I.8. Tnh hnh nghin cu v sn xut Trehalose Vit Nam25PHN 2: VT LIU V PHNG PHP NGHIN CU26II.1 Nguyn vt liu v ha cht26II.1.1 Ging vi sinh vt26II.1.2 Plasmid:26II.1.3 Ha cht26II.1.4 My mc v thit b27II.2 Phng php nghin cu27II.2.1 Bin np E.coli DH5 alpha bng phng php sc nhit27II.2.2 Tch DNA plasmid29II.2.3 Xc nh DNA biu hin E.coli trn gel Agarose31II.2.4 Phng php ph v t bo31II.2.5 Xc nh hm lng protein tng bng phng php Bradford33II.2.6 Xc nh hot tnh enzyme bng phng php DNS (3.5 Dinitro Salicylic)34II.2.7 Tch v tinh ch enzyme bng sc k ct Ni NTA37II.2.8 Phn tch protein bng in di trn gel SDS polyacrylamide (SDS-PAGE)37II.2.9 Xc nh sn phm thy phn bng phng php sc k bn mng (Thin layer chromatography TLC)40PHN 3: KT QU V THO LUN42III.1 Kt qu bin np gen Trehalase vo E.coli DH-5 alpha42III.2 Kt qu tch plasmid DNA v in di trn gel agarose.43III.3 Xc nh cc tnh cht ca enzyme43III.4 Tinh ch Trehalase bng ct sc k Ni NTA44III.5 Xc nh mt s tnh cht ca Trehalase46III.5.1 nh hng ca nhit ti hot lc ca enzyme46III.5.2 nh hng ca pH v dung dch m ln hot ng ca Enzyme47III.5.3 Kh nng chu nhit ca Trehalase49III.6. nh hng ca ion kim loi v dung mi hu c ln hot ca enzyme5050III.6.1 nh hng ca ion kim loi ln hot ca enzyme50III.6.2 nh hng ca dung mi hu c ln hot ca enzyme51III.7 Xc nh sn phm sinh tng hp trehalose bng enzyme ti t hp (Thin layer chromatography TLC)52KT LUN55TI LIU THAM KHO56

LI CM N

hon thnh kho lun tt nghip ny, em xin by t lng knh trng v bit n su sc ti TS NGUYN VN O, ph ch nhim khoa Cng Ngh Sinh Hc, Vin i Hc M - H Ni, ngi thy vch ra phng hng, phng php nghin cu v hng dn ch bo em tn tnh chu o em c th hon thnh kha lun tt nghip ny.

Em cng chn thnh cm n ti thc s V KIM THOA l ngi tn tnh ch bo cho em nhng thao tc k thut trong sut qu trnh thc tp ti phng th nghim.

c s gip tn tnh ca cc thy c gio khoa Cng Ngh Sinh Hc - Vin i Hc M H Ni sau thi gian hc tp v nghin cu ti khoa em mi t c kt qu nh ngy hm nay. Em cng xin gi li cm n ti cc thy c trong khoa Cng Ngh Sinh hc - i Hc M H Ni gip v to iu kin cho em trong thi gian hc tp ti khoa.

Cui cng, em xin gi li cm n su sc ti gia nh, bn b lun bn gip v ng vin em trong qu trnh hc tp v hon thnh kha lun ny.

Mt ln na, cho php em c cm n tt c s gip tn tnh v qu bu .

H Ni, thng 5 nm 2010

Sinh vin

V Th HngBNG CC K HIU VIT TTAmpAmpicillin

AmpicillinDNAD

Deoxyribonucleic acidARN

Axit RibonucleicEDTAEthyl

Ethylene Diamine Tetraacetic AcidEtBrEthydium Bromine

Ethydium BromineKbKilo base

Kilo baseLBLuria-Bertani (Mi tr

Luria-Bertani (Mi trng LB)ODOptical d

Optical densitySDSSodium

Sodium Dodecyl SulphateTAETr

Tris-Acetate-EDTATaqTh

Thermus aquaticus polymeraseTETr

Tris-EDTATLCThin la

Thin layer chromatography TLCW/v

Weight/Volume (khi lng/th tch)kDaKilo Dalton

Kilo DaltonUDP-G

Uracine Diphosphate GlucoseADP-GAdenosin Diphosphat

Adenosin Diphosphate GlucoseCDP-GCytosin Diphophat

Cytosin Diphophate GlucoseDMFDimethylamide Formic Acid

Dimethylamide Formic AcidOD/595nm

T bo E.coli bin np

Nui cy trong LB 37oC, 16h

Ly tm 8000rpm, 15 pht

Ph v t bo

Ly tm 10.000 rpm, 4oC, 15 pht

Tinh ch qua ct sc k Ni NTA

Thu enzyme tinh ch

Hot lc tng i (%)

Trehalase

Hot Enzyme

Hot lc tng i (%)

1Khoa Cng Ngh Sinh Hc

V Th Hng - 0604

_1336504273.xlsChart1

0.357

0.525

0.824

1.125

1.425

1.625

OD/575nm

phng trnh ng chun DNS theo Glucose

Hm lng Glucose (mg/ml)

y = 1.0675x + 0.0461R2 = 0.9936

Sheet1

0.250.357

0.50.525

0.750.824

11.125

1.251.425

1.51.625

Hm lng tinh bt (mg/ml)

Sheet1

0

0

0

0

0

0

Sheet2

0

0

0

0

0

0

OD/575nm

phng trnh ng chun DNS theo Glucose

Hm lng Glucose (mg/ml)

y = 1.0675x + 0.0461R2 = 0.9936

Sheet3

00

0.250.356

0.50.525

0.750.802

11.12

1.251.326

1.51.625

Sheet3

0

0

0

0

0

0

0

0

0

0

0

0

0

_1336803251.xlsChart2

0.356

0.525

0.802

1.12

1.326

1.625

Phuong trinh duong chuan Protein bang phuong phap Bardford

Hm lng Protein (mg/ml)

y = 1.0361x + 0.0524R = 0.9944

Sheet1

00

0.250.356

0.50.525

0.750.802

11.12

1.251.326

1.51.625

Sheet1

0

0

0

0

0

0

Phuong trinh duong chuan Protein bang phuong phap Bardford

Hm lng Protein (mg/ml)

y = 1.0361x + 0.0524R = 0.9944

Sheet2

Sheet3