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CHNG 1: TNG QUAN

1.1 Tng quan v enzyme -amylase. Gii thiu chung v enzyme -amylase [1, 2]. 1.1.1

- L enzyme ngoi bo thy phn lin kt 1,4 glucoside mt cch ngu nhin v tr bt k nhng ch yu tp trung gia mch amylose v amylopectin nn n thuc loi endo enzyme. Cc -amylase c phn loi theo hot ng v tnh cht ca chng , amylase thy phn tinh bt to thnh sn phm l cc dextrin phn t nh, maltose v glucose. Tuy nhin, ty ngun gc m -amylase t cc vi sinh vt c th c sn phm thy phn theo t l cc thnh phn khng ging nhau (Forgaty 1990) [1]. - Mt s -amylase to maltose: khi s dng -amylase t nm mc Asp.oryzae cc sn phm chnh ca qu trnh thy phn thng l maltose v maltotriose ; -amylase t Streptomyces hygroscopicus l mt endo enzyme c th thy phn tinh bt ti 75 -85 % sn phm cha maltose. Enzyme ny cn c hot tnh transglycosylase, maltotriose c chuyn thnh maltotetraose sau thy phn thnh maltose m khng to ra glucose; amylase ngoi bo ca chng Thermonospora c kh nng thy phn tinh bt cho sn phm chnh l glucose v maltose (Forgaty 1990, Neidleman 1991) [2].

1.1.2

Cu to ca -amylase [1, 2, 3, 4] .

Cu to ca -amylase gm ba tiu n v, A l tiu n v li c ni vi tiu n C c cu trc 8 on sheet, tiu n v B gm 2 on sheet c lng vo tiu n v A. Tiu n v B quyt nh hot tnh enzyme v lin kt enzyme c cht. N cng nh hng n isoform v cc tnh cht c bit ca isoform ny. C mt ion Ca2+ ni gia tiu n v A v B. Tuy nhin, -amylase ca nm mc, lin kt ny yu v c th b hn ch (Brzozowski 1997, 2000, Ferrari 1993, Marco 1996).

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Tm hot ng: - C cha mt s gc acid amin c hiu. Thng thng cc nhm c hiu ca n l nhn imidazol v nhm hydroxyl. - -amylase d tan trong nc, trong cc dung dch mui v ru long. - Protein ca cc -amylase c tnh cht ca acid yu v tnh cht ca globulin. - im ng in nm trong vng pH = 4,2 5,7. Phn t lng ca cc -amylase t cc ngun khc nhau rt gn nhau (ca nm mc 45000 50000, ca malt 59000). Thnh phn acid amin: - -amylase t cc ngun khc nhau c cc thnh phn acid amin khc nhau, mi loi -amylase c mt t hp acid amin c hiu ring, song chng u kh giu tyrosin v tryptophan. - Cc acid glutamic v aspartic chim gn tng lng acid amin cu thnh phn t enzyme. - Trong -amylase rt t methinonine v ch c khong 7- 10 gc cystenie tr amylase ca Bac.subtilis khng c cc lin kt sulfuahydryl v disulfuahydryl. - Trong thnh phn acid amin ca -amylase ca Asp.oryzae c cha t arginine v histidine nn cng cha t nhm amin v hm lng nito, do chng mang tnh acid hn. Phn ln -amylase c phn t lng tng i gn nhau (khong 40000 Da) song c nhng trng hp nh -amylase ca B.stearolthermophilus c phn t lng l 15600 Da (Campbell, Manning1964). Ngi ta nhn thy rng phn t lng ca amylase gim i (ch cn khong 14000 n 15000) cn hm lng Ca li tng theo nhit nui vi sinh. Khng ging cc -amylase khc, -amylase ca Asp.oryzae c cha phn phi protein l polysaccharide. Polyose ny bao gm 8 mol manose, 1 mol glucose, 2 mol hexoseamin trn 1 mol enzyme (Akabori v cng s, 1955). Vai tr ca cc polyose ny

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vn cha r, song ngi ta bit rng n khng tham gia vo thnh phn trung tm hot ng v nm pha trong phn t enzyme. Calcium v vai tr ca n trong phn t -amylase [5]: - -amylase l mt metalloenzyme (enzyme c kim). Cc -amylase u cha t 1 30 nguyn t gam Ca/mol. Khi tch hon ton Ca ra khi phn t enzyme th -amylase mt ht kh nng thy phn c cht. V Ca tham gia vo s hnh thnh v n nh cu trc bc 3 ca enzyme, duy tr cu hnh hot ng ca enzyme (Molodova, 1965). - Calcium cn bo m bn ca -amylase i vi tc ng gy bin tnh v s phn hy bi cc enzyme phn hy protein. - -amylase bn nhit hn so vi cc amylase khc. c tnh ny c th lin quan n hm lng calcium trong phn t ca n. - -amylase ca vi khun a nhit c cha calcium nhiu hn ca -amylase t nm mc nn n bn nhit hn. - Ion Ca2+ lm n nh -amylase ca malt, ca vi sinh vt (trong c c 3 loi amylase t Asp.flavus, Asp.awamori, Asp.oryzae). -amylase t Asp.awamori v t Asp.oryzae c n nh bi Al3+, ion st c ch hot ng ca -amylase t Asp.awamori v t Asp.oryzae. Tt c cc -amylase u b kim hm bi cc kim loi nng ( Cu2+, Hg2+, Ag+). Cc kim loi nh Li+, Na+, Mg2+, Cr3+, Mn2+, Zn2+, Co2+, Cd2+, Sn2+ khng nh hng my n -amylase.

1.1.3

C cht ca enzyme -amylase [6, 7].

C cht ca qu trnh thy phn: gm cc polysaccharide cha cc lin kt 1,4glucoside v ch yu l cc glucan: tinh bt v glycogen (tinh bt ng vt).

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1.1.3.1 Tinh bt. - Tinh bt bao gm hai cu t l amylose v amylopectin, cc cht ny khc hn nhau v tnh cht l hc v ha hc. Amylose to mu xanh vi iod cn amylopectin li to mu tm . Chng cng khc nhau v tnh ha tan. Amylose d ha tan trong nc m v to dung dch c nht khng cao cn amylopectin ch tan trong nc nng v to dung dch c nht cao. V phn t lng cng c s khc bit r rt. Amylose c phn t lng t 3.105 106, cn amylopectin c phn t lng t 5.104 106. Trong tinh bt t l amylose v amylopectin thng l 1:4. - V cu to: hai thnh phn trn u c cha cc n v cu to l glucose. Trong amylose cc gc glucose c gn vi nhau nh lin kt 1,4 glucoside to nn mt chui di gm 200 1000 gc glucose. Phn t amylose bao gm mt s chui xp song song nhau, trong cc gc glucose cun li theo hnh xon c. Mi chui amylose c mt u khng kh v mt u kh. - Trong phn t amylopectin, cc gc glucose khng nhng gn vi nhau nh lin kt 1,4 glucoside m cn nh lin kt 1,6 glucoside. V th m n c cu trc nhnh. Phn t amylopectin ch c mt u kh duy nht. Ngi ta cn tm thy trong thnh phn ca phn t amylopectin cn c mt t phospho. Phospho c gn vo nguyn t cacbon th su ca gc glucose.

1.1.3.2 Glycogen - L polysaccharide d tr ca c th ngi v ng vt. V vy, ngi ta coi n l polysaccharide c trng ca ng vt hay tinh bt ng vt. - Ngoi ra, ngi ta cn pht hin n cc ngun khc nhau nh nm mc, nm men v mt vi loi ht ng. Glycogen c vai tr quan trng trong chuyn ha glucid c th ng vt v nm men khi ln men ru. N ha tan c trong nc nng. Khi tc dng vi iod s cho mu tm hoc mu nu ging mu ca amylopectin vi iod.

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- Phn t lng ca glycogen x dch trong mt khong kh ln v c th t ti 4.106 tng ng vi s gc glucose khong 24000. - Glycogen c cu to gn ging vi amylopectin ca tinh bt nhng mc phn nhnh nhiu hn. Trong phn t ca glycogen cng c lin kt 1,4 glucoside v 1,6 glucoside nhng lin kt 1,6 glucoside nhiu hn amylopectin. Cc on mch pha trong v ngoi vi ca glycogen ngn hn ca amylopectin. Chiu di trung bnh ca cc on mch nhnh ch c 12 14 gc glucose.

1.1.4

C ch xc tc [1, 2, 8].

C ch phn ng thy phn tinh bt: - Khng ph thuc ngun gc ca cht xc tc, mi khi mi lin kt 1,4 glucoside b phn ct th ti ngay im lp tc c lin kt vi cc nhm ion ca nc. - Kho st qu trnh thy phn tinh bt trong mi trng giu nc bi enzyme amylase , ngi ta thy rng, cc cht xc tc b gy mi lin kt glucoside gia cacbon s 1 (C1) vi nguyn t oxy. mch amylose, khi mi lin kt 1,4 glucoside b phn ct th nhm hydroxyl (OH-) ca nc s lin kt vi cacbon s 1 (C1) ca gc glucoside bn tri, cn cation hidro (H+) s lin kt vi O- nguyn t C4 gc glucoside bn phi. i vi mch amylopectin, khi mi lin kt 1,6 glucoside b ct t, th nhm OH- ca nc s lin kt vi nguyn t C1 mch chnh, cn H+ s lin kt vi O nguyn t C6 mch nhnh. - Mt cch tng qut, nu tt c cc mi lin kt glucoside ca tinh bt b phn ct (bao gm n gc) th cn c (n 1) phn t nc lin kt vi chng v s to ra n phn t ng glucose. Nhng v n l i lng ln cho nn hiu s n v (n-1) c th xem l khng ng k. Lc phng trnh thy phn tinh bt c th biu din: ( C6H10N5)n + n H2 O n C6H12O6

- Nu ch tnh cho mt gc glucose th c th vit ngn gn:

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C6H10N5 162

H2 O 18

C6H12O6 180

T y ta c th rt ra kt lun: lng glucose thu hi theo l thuyt t phn ng thy phn hon ton tinh bt bng 111,11% lng c cht tham gia u vo.

- Trong thc t, sn phm thy phn ca tinh bt thng tnh theo lng ng maltose, lc s cn bng sn phm c th tnh theo phng trnh: ( C6H10N5)n + n162 n/2 H2O n/2. 18 n/2 C12H22O11 n/2 . 342

- Mt c im quan trng ca lin kt glucoside trong cc polysaccharide l ch c cc electron m khng c cc electron tham gia. Trong trng hp nh vy, ng vai tr phn cc l electron . Do hiu ng cm ng ca nguyn t oxy trung tm cn gy ra mt s tp trung in tch no trn nguyn t oxy, nn n tch in m. - -amylase t cc ngun khc nhau c rt nhiu im ging nhau. -amylase khng ch thy phn h tinh bt m n cn thy phn c ht tinh bt cha h ha nhng vi tc rt chm. Qu trnh thy phn tinh bt bi -amylase l qu tnh nhiu giai on [1,8]: - giai on u giai on dextrin ha ch mt s lin kt trong phn t c th b thy phn to thnh mt lng ln dextrin phn t thp, nht ca h tinh bt b gim nhanh (c amylose v amylopectin u b dch ha nhanh). - giai on th hai giai on ng ha- cc dextrin phn t thp va mi to thnh b thy phn tip tc to ra cc tetramaltose, trimaltose khng to mu vi iod. Cc cht ny b thy phn rt chm bi amylase cho ti disaccharide v monosaccharide. - Di tc dng ca -amylase, amylose b phn gii kh nhanh thnh oligosaccharide gm 6 -7 gc glucose ( 6 -7 gc glucopyranose mt). Sau cc polyglucose ny li b phn ct tip tc nn cc polyglucose ny c ngn dn li v b phn gii chm n maltotetraose, maltotriose v maltose.

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- Qua mt thi gian di di tc ng ca -amylase, sn phm thy phn ca amylase cha 13% glucose v 87% maltose. - Tc dng ca -amylase ln amylopectin cng tng t, nhng v -amylase khng phn ct c lin kt 1,6 glucoside ch mch nhnh trong phn t amylopectin nn d c tc dng lu th trong sn phm cui cng ngoi cc ng ni trn (72% maltose, 19% glucose) cn c dextrin thp phn t v isomaltose (8%). Tm li, di tc dng ca -amylase, tinh bt c th chuyn thnh maltotriose, maltose, glucose v dextrin phn t thp. Thng thng -amylase ch thy phn tinh bt ch yu to ra dextrin thp phn t khng cho mu vi iod v mt t maltose. V vy, ngi ta thng gi n l amylase dextrin ha hay amylase dch ha. S biu din cho s thy phn ny nh sau: Tinh bt Glycogen
+ H2O

+ Maltose + Glucose nhiu t

Nhn nh ny tn ti trong thi gian kh di. Tuy nhin, sau ngi ta tm thy rng trong nm mc c -amylase va biu l hot tnh dextrin ha cao va to mt lng ng ln glucose v maltose. Pakumuro v cng s cho rng trong s cc -amylase ca vi khun c loi amylase dextrin ha, c loi -amylase ng ha. Khi thy phn tinh bt bng amylase ng ha th c ti 60% (v hn na) tinh bt b phn gii, cn bng -amylase dextrin ha th mc depolymer ha khng vt qu 30 40% (Robyt, Prench, 1977). So vi -amylase ca nm mc, -amylase ca vi khun c hot lc dextrin ha vt tri hn l hot lc ng ha. Theo s liu ca Lifsitx v Braznitsenco, kh nng dextrin ha ca ch phm enzyme t asp.oryzae cao hn kh nng ng ha 1,75 ln, cn Bac.subtilis th cao hn 2 ln (2000). -amylase ca nm mc hu nh ch tn cng nhng ht tinh bt khng cn nguyn, cn -amylase ca vi khun li c kh nng phn hy cc ht tinh bt cha c h ha
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ln h tinh bt (Brovaretx,Popadicts v ng s, 1967). Ch phm -amylase ca B.subtilis phn gii ht tinh bt cha c h ha 2 -2,5 ln nhanh hn so vi -amylase ca nm mc ( Lixink, Popadicts, 2001). cc giai on thy phn cui, tc dng ca -amylase t nm mc khc tc dng ca -amylase t vi khun. Dextrin do -amylase ca vi khun to ra khi phn gii tinh bt c mch di hn dextrin do -amylase ca malt v nm mc. -amylase ca vi khun Bac.subtilis tc dng ln amylose to ra cc dextrin c mch di khong 6 8 gc glucose ( G6, G7, G8). Cc dextrin ny li b phn gii tip tc theo s : G6 G7 G8 G1 G1 G2 + + + G5 G6 G6 Hay Hay G2 G3 + + G5 G5

(Robyt, French) Sn phm cui cng ca s thy phn amylose l glucose v maltose. i vi amylase ca vi khun, t l glucose : maltose thu c l 1 : 5; cn i vi -amylase ca nm mc t l ny l 1 : 3,79 (Hanrahan, Caldwell, 1987). Fenikxova v Ermosina cho bit rng cc maltopentaose v maltohexaose thy phn theo s sau: G5 G6 G1 G2 + + G4 G4 Hay 2G3 Hay G5 + G1

-amylase ca nm mc v vi khun khng tn cng lin kt 1,6 glucoside ca amylopectin nn khi thy phn, n s to thnh cc dextrin gii hn phn nhnh. Sn phm u tin ca s phn gii amylopectin bi -amylase vi khun l dextrin c 6 gc glucose, cn sn phm cui cng l glucose, maltose v cc maltodextrin (saccharide c phn nhnh) t cc maltodiose n maltohexaose. Sn phm thy phn cui cng ca tinh

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bt di tc dng ca -amylase ca nm mc ch yu l maltose, tip n l maltotriose (Sproster, Uhlig, 1987). Nu dng nng -amylase vi sinh vt tng i ln c th chuyn ha 70 -85% tinh bt thnh ng ln men. Vi -amylase vi khun, tinh bt b thy phn thnh glucose v maltose n 70 -75%. Cn vi -amylase ca nm mc th mc ng ha n glucose v maltose c th n 84 87%. Vn tc thy phn tinh bt bi -amylase vi khun Bac.subtilis giai on u cao hn -amylase Asp.oryzae ti 20% v gim mnh khi t c 30 32% s thy phn.

1.1.5

Cc yu t nh hng n hot tnh ca enzyme -amylase [1, 4, 8, 9].

iu kin hot ng ca -amylase t cc ngun khc nhau thng khng ging nhau. pH: - -amylase t nm mc c th hot ng tt trong vng pH 4,5 4,8 ; pH ti thch ca i mch ny mm v thc mm l 5,3 (c th hot ng tt trong vng pH 4,7 5,4) v ca vi khun l 5,8 6,0 ( hot ng tt trong vng pH 5,8 7,0). - Theo s liu ca Liphsis, pH ti thch cho hot ng dextrin ha v ng ha ca ch phm -amylase t Asp.oryzae ging nhau v nm trong vng pH 5,6 6,2 (2000). Cn theo s liu ca Fenixova th pH ti thch cho hot ng dextrin ha ca n l 6,0 7,0 (2001). - bn i vi tc dng ca acid cng khc nhau. -amylase ca nm mc Asp.oryzae bn vng i vi acid tt hn l -amylase ca malt v vi khun Bac.subtilis. pH 3,6 v nhit 00C, -amylase ca malt b v hot hon ton sau 15 30 pht; amylase ca vi khun b v hot 50%; trong khi hot lc ca -amylase t nm mc hu nh khng gim bao nhiu (Fenikxcova, Ermoshina, 2005).

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- Trong dung dch -amylase ca nm mc c bo qun tt pH 5,0 5,5; amylase dextrin ha ca nm mc en c th chu c pH 2,5 2,8. 00C v pH 2,5 n ch b v hot sau mt gi (Bernfeld, Okazaki, Kitahara, Kurushima, Ermoshina, 2005). - Ngoi -amylase khng bn acid ra, cc loi nm mc Asp. Awamori, Asp. Butatea, Asp.usamii cn to ra ti 5 10% -amylase bn acid. Trong khi nm mc Asp.niger 475 li ch to ra cc -amylase bn acid. N khng b mt hot tnh pH 2,5; nhit 300C trong vng 1 gi.

Bng 1.1: bn acid ca -amylase mc en [8].

Hot dextrin Chng ha tng s (v/ml) Asp.awamori Asp.usamii 45 Asp.usamii 45 56 Asp.usamii 45 21 Asp.niger 475 0,5 0,43 0,5 0,4 0,35

Hot dextrin ha bn acid (v/ml) 0,08 0,1 0,11 0,06 0,34

Hot dextrin ha bn acid, % hot tng s 16 23 22 15 97

- pH < 4,0 -amylase ca vi khun b v hot hon ton (Virits, 1996). Theo Uhlig, -amylase ca nm bn vng pH 5,3 8,0 cn -amylase ca vi khun bn khong pH 5,0 10,0 (2000).

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Nhit : - Nhit ti thch cho hot ng xc tc ca -amylase t cc ngun khc nhau cng khng ng nht, -amylase ca nm mc rt nhy vi tc ng ca nhit, nhit ti thch ca n l 500C, -amylase ca thc mm v ca malt bn nhit hn v hot ng ti thch 58 60oC. Trong sn xut ru ngi ta thng ng ha bng canh trng nm mc 50 52oC, v bng malt 60 65oC, -amylase ca vi khun c bn nhit cao hn c. Nhit ti thch ca n l 70 75oC (Liphsis, Bragnitsenko, 1997), amylase ca Bac.stearothermophilus vn gi c hot lc trong mt thi gian di 85oC v cao hn na (Manning Campbell, Karpukina, 2001). - -amylase ca vi khun c th chu c nhit 92oC trong khi -amylase ca nm mc b v hot 70oC (Kozmina, 2002). Trong dung dch h tinh bt, -amylase ca vi khun ch bt u v hot nhanh khi nhit cao hn 770C. 700C, -amylase ca nm mc b mt 50% hot lc, cn -amylase ca malt hu nh cha b mt hot lc.

Bng 1.2: bn nhit ca -amylase t cc ngun khc nhau (Miller, Johnson, 2005) [9]. Nhit
0

Hot -amylase, % so vi hot ban u Nm mc 100 52 3 1 Malt 100 100 58 25 1 Vi khun 100 100 100 92 58 52 8

65 70 75 80 85 90 95

- Trong dung dch m pH 4,7; -amylase ca asp.oryzae rt nhy vi tc ng ca nhit cao, thm ch 400C trong 3 gi hot lc dextrin ha ca n ch cn 22 - 29%,
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hot lc ng ha cn 27 85%. 500C trong 2 gi, -amylase ca n b v hot ha hon ton. - Miller v cng s cn nhn thy rng -amylase ca vi khun c th gi c mt phn hot lc thm ch ngay c khi un si trong nc mt thi gian ngn. Tnh bn nhit ca -amylase vi khun l mt u im ln. Trong sn xut, -amylase ca vi khun c s dng x l nguyn liu cc cng on phi dng nhit cao. Ty c tuyn chn c mt chng Bac.subtilis m -amylase ca n khng khc g ca vi khun thng thng v c tnh tc dng ln tinh bt, song li c bn nhit kh cao (Sprister, Uhlig, 2000). Tnh cht -amylase ca chng bn nhit ny cng c trng cho Bac.suptilis; pH ti thch 6,0; b v hot hon ton pH 4,0 v b v hot mt na pH 8,0. - Nhng khc bit v tnh cht nh nhit v pH ti thch, mc thy phn v c tnh thy phn ca -amylase t cc ngun khc nhau ang m ra nhiu kh nng to ln cho vic ng dng chng mt cch hp l v hiu qu cc giai on khc nhau ca qu trnh sn xut. Nng c cht: - Nng c cht nh hng kh mnh n khi lng v cht lng ca sn phm thy phn do s phn ct ca -amylase. Nhn chung nu nng c cht kh long, lng ng to ra theo c cht cng nhiu, c bit l nhm ng thp phn t c kh nng ln men c. Tuy nhin, tng quan khng phi l t l thun. - Ngoi ra, gn y, cc nh nghin cu tm hiu v thy rng thnh phn amylose , amylopectin v cu trc tinh bt cng l nhng yu t nh hng kh mnh n tc dng thy phn ca enzyme -amylase. Ngun tinh bt khc nhau cng nh hng n qu trnh thy phn khc nhau.

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1.1.6

-amylase t vi khun Bac.licheniformis [3, 10].

- Bacillus c xem l ging vi sinh vt quan trng nht sn xut -amylase trong cng nghip. c nhiu nghin cu sn xut -amylase bn nhit t cc chng Bac.amyloliquefaciens v Bac.licheniformis. - -amylase bn nhit t Bac.licheniformis c nghin cu tch trong h hai pha PEG/dextran, sau cho chy qua h sc k lc gel v sc k trao i ion (Ivanova, 1993). S dng gradient pH l mt phng php thng c s dng trong cng nghip tinh sch enzyme. Dobransky dng phng php ny tinh sch -amylase t cc chng Bac.subtilis khc nhau. Cc pick thu c c tch tt, lng dung dch enzyme ban u c th t ti 1000L, vi nng protein 3g/100mL. tinh sch -amylase sn xut bi chng chuyn gen bng phng php sc k min dch cng ang c quan tm (Pandey, 2000). - Cc tnh cht ca -amylase ngoi bo dng nh phn nh iu kin pH v nhit ca mi trng sng. Bac.flavothermus pht trin trong mi trng pH 6,0 v nhit 65oC tng hp -amylase c pHopt v toopt tng ng l 5,5 6,0 v 60oC. Mt s tnh cht ca -amylase ngoi bn nhit t Bac.subtilis c nghin cu, kh nng bn nhit ca enzyme ph thuc vo hm lng Ca2+, enzyme b v hot mnh bi EDTA v N bromosucinimide. Mt s tc gi nghin cu tnh cht ca -amylase c ngun gc t chng Bac.subtilis (57700 Da); chng b v hot bi cc kim loi nh Hg2+, Fe3+, Al3+ v c kch hot bi Mn2+, Co2+ thu nhn c amylse ni bo ca Bac.subtilis SUH4 2 vi t l monomer/dimer tng ng l 3/2 trong m phosphate pH 7,0. Enzyme ny c cho l c vai tr quan trng trong qu trnh ng ha cacbon cytoplasm. Cc dng -amylase bin tnh v sau khi khi phc hot tnh ca Bac.subtilis cng c nghin cu nh s dng pht x hunh quang (Cho, 2000).

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1.1.7

ng dng ca enzyme -amylase [11].

- Ngy nay cc amylase t vi sinh vt thay th acid trong cng nghip thy phn tinh bt, ng ha tinh bt trong sn xut ru, bia. Cc enzyme c tinh sch cao v nhng tnh cht ph hp c trin vng to ln trong cng nghip dc v cng ngh ha cht tinh khit. - Nh thnh tu ca cng ngh sinh hc, ng dng ca amylase ang m rng ra nhiu lnh vc khc. Mt s amylase v lipase ng vai tr quan trng trong nghin cu sinh hc. C nhiu nghin cu trong y hc v lm sng hc lin quan n ng dng amylase. - H phn tch Ciba Corning Express da trn dung dch thuc th cha -amylase cho php pht hin cc oligosaccharide phn t lng ln vi nhy rt cao, -amylase c s dng trong phn tch cyclodextrin.

1.2

S lc v enzyme c nh. Gii thiu.

1.2.1

1.2.1.1 nh ngha [12]. Enzyme c nh c th hiu theo 2 ngha: - Ngha hp: enzyme khng ha tan l enzyme c a vo nhng pha ring r, pha ny c th tch ring vi mi trng dung dch phn ng. Pha enzyme khng ha tan trong nc v c gn vi cc polymer a nc c trng lng phn t ln. - Ngha rng: cc cht xc tc c nh l cc enzyme, t bo, c th sng trng thi cho php s dng li. Nh vy, theo ngha rng, enzyme khng ha tan bao gm c enzyme c c nh vo mt cht mang, c enzyme c trong t bo sng, chng c c nh trong cc bnh phn ng sinh hc c s gn kt vo mt cht mang cho php ta s dng nhiu ln.

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- Enzyme khng ha tan hay enzyme c nh thng l nhng enzyme ha tan c gn vo mt cht mang bng nhiu k thut khc nhau. Nh qu trnh gn ny m enzyme t trng thi ha tan chuyn sang khng ha tan.

1.2.1.2 c im ca enzyme c nh [12]. Enzyme c nh c c im nh sau: - Hot tnh ca enzyme c nh thng nh hn hot tnh ca enzyme t do cng loi. S d c s thay i hot tnh ring ca enzyme c nh l do nhng nguyn nhn sau: Do nh hng in tch ca cht mang: khi in tch ca cht mang c s

khc bit vi in tch ca enzyme th khi gn enzyme vo cht mang, cu trc khng gian ca enzyme c s thay i mc nht nh. Chnh v s thay i cu trc ny m s kt hp gia enzyme v c cht s km i, dn n hot tnh ca chng cng gim. Do enzyme b nht vo mng li hoc b lin kt bi cht mang khin cho s tip xc gia enzyme v c cht b km i. - Enzyme c nh hon ton tun theo nh lut Michaelis Menten nhng c mt s sai khc nht nh: phn ng. - Enzyme c nh thng c tnh bn nhit hn enzyme t do nh c tc dng che ch ca cht mang. - Enzyme c nh thng c pH ti u khng trng vi enzyme t do cng loi. - Enzyme c nh do c cht mang che chn nn c bo v tt hn enzyme t do. - Enzyme c nh c th ti s dng nhiu ln v d dng tch ra khi c cht mt cch d dng sau phn ng v bn ca enzyme c nh cao hn enzyme t do.
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C th xy ra s cnh tranh c cht vi enzyme v cht mang. Xy ra hin tng cn tr khuch tn ca c cht v sn phm lm gim tc

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1.2.1.3 u nhc im ca enzyme c nh [12]. u im: - Enzyme c nh c th ti s dng nhiu ln trong thi gian di. - Enzyme c nh khng ln vo trong sn phm cui ca phn ng enzyme, do n khng gy nh hng xu n cht lng sn phm, hay ni cch khc chng ta khng tn chi ph cho vic tch enzyme ra khi sn phm. - C th ngng phn ng khi cn thit bng cch tch h cht mang enzyme ra khi dung dch c cht. - Enzyme c nh tng i bn vi cc tc nhn vt l v ha hc hn enzyme t do. - D dng tin hnh qu trnh sn xut theo phng php lin tc. Nhc im: - S c mt ca cht mang c th lm gim hot tnh ca enzyme. - Trong a s trng hp, enzyme c th b gim hot mt hot tnh sau qu trnh c nh. Tuy nhin, nhng hn ch trn l khng ng k so vi nhng li ch m enzyme c nh mang li. Do vy, ngy cng c nhiu nghin cu v c nh enzyme cng nh ng dng enzyme c nh vo sn xut cng nghip.

1.2.2

Cc phng php c nh enzyme [12].

Cc phng php c nh enzyme c chia thnh 2 nhm: ha hc v vt l.

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Bng 1.3: u Nhc im ca cc phng php c nh enzyme [12]. Phng php ha hc Phng php Phng php gn enzyme ln cht mang bng lin kt cng ha tr Phng php gn cc phn t enzyme li vi nhau bng lin kt cng ha tr u im Lin kt gia enzyme v cht mang l lin kt bn, do hn ch ti a s mt mt enzyme trong qu trnh phn ng. Nhc im Hot tnh enzyme c th b gim do nhng bin i v cu trc ca enzyme trong qu trnh c nh.

To c lin kt rt bn trc Chi ph cao, thao tc thc cc tc nhn pH, nhit hin tng i phc tp. Thng c dng phi hp vi Hot tnh enzyme c th b cc phng php khc. gim do nhng bin i v cu trc ca enzyme trong qu trnh c nh. Phng php vt l

Phng php

u im

Nhc im

Phng php gn Thao tc thc hin n gin. enzyme ln cht mang iu kin tin hnh c nh bng tc nhn vt l enzyme n ha nn khng lm mt hot tnh ca enzyme trong qu trnh c nh.

Do lc tng tc gia enzyme v cht mang yu nn d xy ra hin tng nh hp ph trong qu trnh s dng enzyme c nh do khuy trn hay do thay i C kh nng ti s dng cht nhit , pH ca mi mang. trng. Ch thch hp cho phn ng Khng i hi phi c cc nhm vi c cht c khi lng to lin kt nn ph hp vi phn t nh. nhiu loi enzyme. Hiu qu c nh enzyme cao. C th c nh ng thi nhiu enzyme.

Phng enzyme

php

nht Thao tc n gin.

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Phng php ha hc: - Gn enzyme ln cht mang bng lin kt cng ha tr. - Gn cc phn t enzyme li vi nhau bng lin kt cng ha tr. Phng php vt l: - Hp ph enzyme ln cht mang bng lin kt vt l nh sau: lc mao qun, lin kt ion, lc Van der Walls. - Phng php nht enzyme trong khun gel hoc mng bao vi th.

1.2.3

Vt liu c nh.

Vt liu v phng php c nh l hai yu t c tnh cht quyt nh n hiu qu ca qu trnh c nh enzyme. Trong , khng c c im, tnh cht ca vt liu c nh no thch hp cho tt c cc loi enzyme v cng khng c enzyme no thch hp vi tt c cc loi vt liu c nh [13]. Vt liu c nh enzyme v t bo c th chia lm hai loi: vt liu v c v vt liu hu c [13], [14]. Vt liu v c: - c im: cht mang v c bn vi cc tc ng bn ngoi, khng b vi sinh vt n mn, phn hy, nhng vic gn vi enzyme thng kh khn hn. - Mt s cht mang v c thng dng nh: thy tinh xp, cc oxid kim loi nh oxid nhm, oxid mangan, oxid magie, silicagel

Vt liu hu c: - c im: cht mang hu c thng c cc nhm hot ng ha hc nh: -NH2, COOH, -OH, -SH, nn d kt gn vi enzyme nhng bn vi tc ng ca mi

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trng khng cao, c bit cc polymer sinh hc rt d b vi sinh vt xm nhp v tn cng. - Mt s cht hu c thng dng nh: Polypeptide, dn xut ca sterol, polymer ca acid malic v ca acid metacrylic, polysaccharide, nilon, dn xut cellulose, dextran, tinh bt, agarose. - Cellulose v dn xut ca cellulose l CM-cellulose, DEAE-cellulose, nitrocellulose l nhng vt liu r hn so vi agarose nhng li khng ng nht, thng ch s dng dng si, v dng tinh th (microcrystalline). - Cellulose l vt liu c nghin cu c nh enzyme u tin, cc quy trnh c nh trn vt liu ny c nghin cu kh hon chnh. Cellulose thng c dng hp th, lin kt ion, lin kt cng ha tr vi enzyme hoc nht trong gel. - Chitin l hp cht c Braconnot phn lp t nm 1811 v Odier t tn l chitin t nm 1823. Chitin l mt polymer sinh hc c nhiu trin vng trong nghin cu v ng dng lm vt liu c nh enzyme v t bo. Chitin l mt polymer sinh hc rt ph bin trong t nhin, ch ng sau cellulose. - Chitin c cu trc tng t cellulose, l mt polymer ca cc gc 2-acetoamide-2deoxy--D-glucoza, lin kt vi nhau bi lin kt -1,4-glycoside. Chitin c trong thnh phn cu trc v ca cn trng, v tm cua, s, vch t bo sinh vt,Chitin l mt vt liu c cha nhm chc OH v cho lin kt vi enzyme, c cu trc siu l, c kh nng hp th tt, d to mng. Chitin c cu trc mng tinh th cht ch, khng ch c cc lin kt hydro hnh thnh trong chui m cn c gia cc lp vi nhau trong mng tinh th. - Chitin l mt polymer k nc, trng thp, din tch b mt tip xc nh v bn v mt ha hc. Gn y c rt nhiu nghin cu c nh enzyme trn chitin, ch yu bng phng php hp th v lin kt cng ha tr qua cu ni glutaraldehyde. - Chitin c tm thy trong thnh t bo ca nm si. N l cht hu c chim lng ln hnh thnh nn lp v ngoi ca ng vt khng xng sng (cn trng, gip xc) [13].

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- V mt cu trc, chitin c cu trc tng t nh cellulose, im khc bit ha hc duy nht l nhm acetamido v tr s 2 trn khung carbon ca chitin c thay th cho nhm hydroxyl (-OH) cellulose. - Chitosan l dn xut ca chitin khi c deacetyl ha trong kim mnh. Chitosan d to mng, to ht, c cu trc siu l, c nhm - NH2. Chitosan c th c nh enzyme bng phng php hp ph, phng php cng ha tr, hoc nht enzyme trong gel chitosan. Chitosan khng tan trong nc, tan trong dung mi hu c nh acid formic, acid dipic, acid acetic. Chitosan l mt polymer sinh hc c trng lng phn t cao, c cc nhm amin hot ng, cc nhm hydroxyl c th tham gia phn ng ha hc.

1.3

Enzyme -amylase c nh. Tnh hnh nghin cu trong nc v nc ngoi

1.3.1

- Lch s nghin cu c nh enzyme bt u t nm 1916, khi Nelson v Griffin quan st kh nng thy phn ng saccharose ca invertase nm men hp ph trn than hot tnh. - Sau n nm 1949 Micheal v Iuers s dng phng php acid ha carboxymethyl cellulose c nh nhng chui protein c hot tnh. - Nm 1953 Grubhofer v Schleith c nh mt vi enzyme nh carboxypeptidase, pepsin, ribonuclease bng lin kt cng ha tr trn nha polyaminostyren c diazo ha. - Chang (1954) to ra cc vi tiu cu c gn enzyme chng d ng khi a vo c th. - Mizt (1956) c nh enzyme catalase ln DEAE-celllulose bng lin kt ion. Beerfeld v Wan m t phng php nht cc enzyme trypsin, papain, amylase v ribonuclease trong gel polyacrylamide vo nm 1963. Nm 1964 Quiocho v Richards

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chng minh kh nng khu mch cc enzyme carboxypeptidase A bng glutaraldehyde. Cng nm 1964, Chang tin hnh nht enzyme hydratase trong cc vi ht. - Enzyme c nh u tin c ng dng quy m cng nghip vo nm 1969 do Chibata v cc cng s. Enzyme aminoacylase c c nh trn DEAE-sephadex nh lin kt ion chuyn ha hn hp D, L-acid amin thnh L-acid amin. Mosbach (1970) c nh t hp c 3 enzyme l -galactosidase, hexokinase v gluco-6-phosphat dehydrogenase bng lin kt cng ha tr vi ht sephadex. T bo vi sinh vt u tin c ng dng qui m cng nghip c thc hin bi Chibata (1973), Chibata v cc cng s c nh E.coli trong gel polyacrylamide sn xut acid L-aspartic t ammonium fumarat nh hot tnh aspartase rt cao ca E.coli [13]. - Hin nay trong cng nghip, enzyme c nh c s dng ch yu trong sn xut syrup giu fructose (HFS) v penicillin bn tng hp. Vit Nam, nhng nghin cu c nh enzyme ch mi bt u trong vi nm gn y, kt qu thu c cn rt hn ch. Nm 1994 - 1995 Vin Sinh Hc Nhit i TP. H Ch Minh nghin cu c nh enzyme glucoisomerase trn cc ht silochrom B2 hot ha bng glutaraldehyde. Hin nay Vin cng ang nghin cu s dng ch phm glucoisomerase c nh ca Novo sn xut syrup fructose, tuy nhin ch mi qui m phng th nghim [14]. - Nhng nm gn y, Phng cng ngh bc x, vin nghin cu ht nhn Lt c nhiu cng trnh nghin cu c nh enzyme, t bo v cc cht c hot tnh sinh hc khc bng bc x nh c nh glucoamylase, protease, vi khun t (Vibro cholerae), t bo nm men (Saccaromyces servisase), vi khun x l nc thi trn polymer tng hp c ch to bng k thut bc x. - C mt s cng trnh nghin cu v amylase c nh, tuy nhin cc nghin cu khng c ng dng trong cng nghip. Do amylase thng c c nh trong cc cht mang dng xp, tinh bt li c kch thc ln nn c cht v enzyme kh tip xc vi nhau, qu trnh truyn khi thp v hiu sut s dng enzyme c nh khng cao. Thit b s dng trong cng nghip thng l ct nhi khng ph hp vi dng c cht lin tc l
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tinh bt dng ht, khi c kch thc ln. Trong cc ct nhi kiu ny d xy ra hin tng tc nghn dng li vo, c cht t tp thnh tng m lm tng p sut ct, ph v hnh dng hay gy nn cc ht xp dn n hiu sut qu trnh v cht lng sn phm thp [15]. - Cc nghin cu v c nh amylase tp trung ch yu c nh glucoamylase v mt vi nghin cu v c nh -amylase. Glucoamylase ca hng Novo c nh trn ht silic xp c s dng trong thit b ct nhi v thit b tng si thy phn tinh bt sn sau khi dch ha. V thc hin cc nghin cu v m hnh ha qu trnh phn ng, nhit ng hc phn ng, nghin cu qu trnh truyn khi trong v ngoi ht cng nh cc nghin cu v bn nhit, nng lng v hot v thi gian bn hy ca enzyme c nh v ha tan. Thit b tng si c nhiu u im hn thit b dng ct nhi, khng c hin tng hn ch khuch tn v hiu sut thy phn tinh bt dng ht 98,5% vi thi gian lu ch 10 pht. Glucoamylase cng c gn trn b mt cc ht siu mn polystyrene, cc ch s ng hc nh Vmax, Km, kh nng chu nhit, pH ca enzyme tt hn so vi enzyme t do [8]. - c mt s cng trnh nghin cu c nh -amylase trn polyacrylamide, canxi alginate v sepharose. - Polyacrylamide c tnh u vit, enzyme t b bin i trong gel nhng dimethyaminopropionate cht khi u cho qu trnh trng hp rt c, khng th s dng trong thc phm. Canxi alginate c nhiu trong t nhin ch yu c chit t rong nu gn nh khng mi khng v, nhc im l qu trnh truyn khi gp tr ngi do cu trc tinh bt ln kh i vo cu trc gel. - -amylase cng c nghin cu c nh trn sepharose nhng phng php ny c nhc im cht hot ha CNBr c tnh cao v ch phm khng th s dng trong cc thit b c cnh khuy [16]. - Chin Jen Tien, Been Huang Chiang (1999) c nh -Amylase trn ht keo l sn phm H2SO4 v ZrOCl2 sau khi c x l bi 3-aminopropyltriethoxysilane (3APTES) v glutaraldehyde 0,5%. Ch phm thu c c hot tnh 59,8 v/g [8].
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- Serpil Aksoy, Nesrin Hasirci (2000) c nh -amylase trn 2-hydroxythel metharylate (HEMA), v trn styrene-2-hydroxyethyl methacrylate (St-HEMA) cc ch phm trn c hot ha bi epichlorohydrin (ECH). Enzyme c nh c hot tnh 61,7 67% so vi enzyme t do, sau 30 ngy bo qun nhit 4oC hot tnh cn 38,9% [15]. - Vi nhng c gng tm ra cht mi, Yang (1998) s dng cht mang perfluorpolymer ph poly vinyl - alcohol c nh -amylase. Enzyme c nh kh bn vi nhit v cc dung dch gy bin tnh mnh, c bit n c bn c hc cao, c th ti s dng trong thit b c cnh khuy. Enzyme c nh ch mt 20% hot tnh sau 3 tun 72oC [17, 18]. - Chen (1998) tm ra mt loi mng gm cc ht hydrogel c gn trn nn polyester, rt nhy vi nhit , c kh nng ng m cc l xp, to ra hiu ng c / khng c hot tnh enzyme tng ng vi nhit . Enzyme c nh trn mng c bn nhit cao, c s dng trong thit b lin tc c kh nng tng hiu sut thy phn v kh nng tch sn phm khi c cht [15].

1.3.2 Cc phng php c nh enzyme -amylase [8, 13, 22]. Theo Scouten Gerhartz c 4 phng php c nh enzyme: - Phng php lin kt vi vt liu c nh. - Phng php khu mch. - Phng php nht trong khun gel. - T hp cc phng php trn. 1.3.2.1 Phng php lin kt vi cc vt liu c nh: Phng php lin kt cng ha tr: y l phng php c nh enzyme ph bin, m bo lin kt vng chc ca enzyme v c cht. u im:
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- Do c lin kt cht ch bng lin kt cng ha tr nn enzyme khng b ra gii trong sut qu trnh s dng. - D dng tip xc vi c cht v enzyme c gn trn b mt. Nhc im: - Cu trc ca enzyme c th b thay i bi qu trnh lin kt c th dn n mt hot tnh. - Do lin kt cht nn c th dn n cn tr hot ng ca enzyme, gim kh nng tip xc vi c cht. - Vt liu c nh khng ti s dng c. Tm li, phng php ny ch ph hp cho nhng enzyme mc tin v n nh hot tnh khi c nh, thng ng dng vi mc ch phn tch. Hot ha bng cyanogen bromit. Dng cho cc vt liu c nhm OH nh polysaccharide, cht hot ha CNBr v cc sn phm trung gian to thnh rt c, nn cn c thit b bo v trong khi tin hnh th nghim. Phng php hot ha acid: - Vi cc vt liu c nh c nhm chc COOH nh carboxymetyl, celluloseGi th c chuyn ha thnh dn xut metyl ester, sau thnh hydrade khi phn ng vi hidrazin v cui cng l acid. - Acid s phn ng vi enzyme nhit thp 0 5oC; pH 7,8. Hot ha bng diazo: - i vi nhng vt liu c nh c nhm amin vng thm nh polyaminostyren, aminobenzyl cellulose c th hot ha theo phng php ny. Phn ng lin kt vi enzyme xy ra nhit thng, mi trng trung tnh.

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- Phn ng alkyl ha: Nhm halogen trn vt liu d phn ng vi nhm amino, nhm sunfuahydride ca enzyme. Nhng cht mang halogen acetyl cellulose, cellulose c th hot ha theo phng php ny. Hot ha bng glutaraldehyde: Cc nhm vt liu c nhm amin nh polyacrylamide, aminoetyl, cellulose, chitin, chitosan c th s dng phng php ny. R-NH2 + HOC-(CH2)-COH + H2N-E R-N=CH(CH2)CHO R-N=CH(CH2)CH=N-E

R-N=CH(CH2)CHO

Hot ha bng carbodiimide: Nhng vt liu c nh c nhm carboxyl c th hot ha bng cc dn xut carbodiimide c bit l N,N dicyclohexylcarbodiimide. Khi c mt cht ny enzyme d dng lin kt cng ha tr vi vt liu c nh, phn ng mi trng acid yu.

1.3.2.2 Phng php lin kt ion: Phng php ny c nhiu u im qui m cng nghip v c cc u im sau: u im: - C nh enzyme bng lin kt ion l phng php n gin, vt liu c th ti s dng li. - Enzyme khng b bin i ha hc nn hot tnh enzyme c nh cao. - Vt liu c nh mang in tch lm tng nng ca c cht, do lm tng kh nng tip xc gia c cht vi enzyme v lm tng hot tnh enzyme c nh. Nhc im: - Enzyme v vt liu c nh nh hng nhiu n s thay i pH, nhit , lc ion, dung dch m s dng.

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- Cc vt liu c nh thng s dng l cc dn xut trao i ion nh CM-, DEAE-, AE-, cellulose, sephadex, chitosan

1.3.2.3 Phng php hp thu vt l : - Qui trnh: trn ln dung dch enzyme, mt thi gian ri lc, ra phn enzyme khng hp th. Enzyme c nh trn gi th nh cc lc lin kt yu nh lin kt hydro, lin kt k nc, lc vander Walls. - Enzyme khng chu bt k mt s thay i no v mt ha hc nn hot tnh ca enzyme c nh thng t 50 100% so vi enzyme t do. - Kh nng hp ph ca enzyme ph thuc vo cc iu kin mi trng, nh nhit , nng enzyme, din tch b mt tip xc ca vt liu c nh.

1.3.2.4 Phng php khu mch [21,23]: OHC(CH2)3CHO Glutaraldehyde + (NH2)n E Enzyme -CH=N-E-N=CH(CH2)3CH=NPhn t enyzme khu mch

1.3.2.5 Phng php nht [20,24,25]: u im: - Phng php ny kh n gin, khng ch c nh mt loi enzyme m c th c nh nhiu enzyme. Nhc im: - Hn ch kh nng tip xc gia enzyme v c cht nn hot tnh gia enzyme c nh thng thp, nht l trong trng hp c cht c khi lng phn t ln. - Vt liu c nh khng s dng li.
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- Nht trong cu trc mng gel. - Enzyme nht trong cu trc mng gel ca cc polymer nh polysacharide, protein, polymer tng hp nh polyacrylamideEnzyme hoc t bo s c to hn hp vi dung dch monomer v cc cht khu mch khc, sau s c polymer ha bi cc tc nhn ha hc, hoc bc x. - Trong trng hp polymer ha bng bc x gamma, enzyme v monomer c hot ha bng cch lm lnh nhit -78oC, sau hn hp c chiu x. K thut ny d to ra cu trc siu l trn gi th polymer v hn ch enzyme mt hot tnh bi bc x. - Cc polymer thng c dng trong phng php ny l gel alginate, carrageenan v chitosan. Alginate v carrageenan l cc polyanion d to ht trong dung dch mui Ca, K. Nhc im cc gel ny khng bn trong mi trng phosphate. khc phc mt phn nhc im ny c th cho gel khu mch trong glutaraldehyde. Dng liposome: - Enzyme c nht trong mng lipid, thng l phospholipid. Dng mng siu lc: - Nguyn tc ca phng php ny tng t nh phng php nht trong vi nang. Enzyme c gi trong cc mng siu lc, phng php ny c ng dng c nh enzyme glucoamylase, glutaminate, galactosidase. Dng vi nang: - Enzyme c nht trong mng bn thm, nhng li thm i vi c cht. V enzyme hot ng trong mi trng dung dch nn kh nng tip xc gia enyzme v c cht ln hn, do hot tnh enzyme cao hn trong trng hp nht trong cu trc ca gel polymer. Catalase c nht bng phng php ny [22][50].

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1.3.3

Mt s yu t nh hng n s c nh enzyme.

nh hng ca cht mang [13, 26, 27]: - Cc tnh cht l hc ca cht mang nh tnh ha tan, tnh bn c ha, din tch, tnh ho nc v k nc,u c nh hng nht nh n th tch enzyme c lin kt, n tnh bn v hot tnh sinh hc ca cc cht dn xut enzyme khng tan. - Bn cht ha hc ca cht mang cng nh hng n kh nng to thnh dn xut enzyme v ti kh nng cht mang hp ph khng c trng cc cht t mi trng phn ng. - S nh v enzyme gip cho dn xut enzyme khng tan c tnh bn nhit cao hn enzyme tan. - Cht mang c tc dng hn ch s bin tnh ca enzyme trong cc dung mi, lm t ni lin kt hydro v lin kt k nc nh hng ca pH [13, 28, 29] - in tch ca cht mang c nh hng ng k n pH ti u ca enzyme khng tan, pH ti u ca enzyme khng tan c th chuyn dch pha kim hay pha acid so vi pH ti u ca enzyme khng tan.

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CHNG 2: NGUYN LIU V PHNG PHP C NH

2.1 Nguyn liu. Enzyme -amylase. 2.1.1

- Enzyme -amylase ca hng Novo, dng lng, l amylase bn nhit, c sn xut t vi khun Bacillus licheniformis. - amylase l mt endo amylase, c tc dng thy phn lien kt 1,4 - glucoside trong phn t amylose v amylopectin thnh cc dextrin v oligosaccharide. - Termamyl d dng tan trong nc mi nng iu kin thng. Sn phm ny c khuyn co l khng bc chy, nn trnh tip xc da v ht phi, nu tip xc da phi ra li nhiu ln vi nc.

2.1.2

Vt liu c nh:

- Vt liu c nh: Chitosan. - Ngun gc: i hc thy sn Nha Trang. - Thng s ha l: m: 15,92% tro: 2,0675% ( theo khi lng cht kh ca nguyn liu). nht dung dch 1% : 50,33 cp. Ch s deacetyl ha ( ch s DD) : 75% Phn t lng trung bnh: 5,187.104 vC.

2.1.3

Cht to lin kt.

- Cht to lin kt: Glutaraldehyde.

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- Mc ch ca vic s dng cht to lin kt trong c dnh enzyme -amylase l hnh thnh nn cc cu ni gia phn t enzyme v cht mang nh cc lin kt cng ha tr. - Glutaraldehyde c 2 nhm chc aldehyde 2 u. Hai nhm ny s to lin kt vi nhm amin ca cht mang v ca enzyme, t gi enzyme c nh trn cht mang. Mt s tnh cht ha l ca glutaraldehyde c trnh by c th trong bng sau:

Bng 2.1: Mt s tnh cht ha l ca glutaraldehyde.

Cng thc Phn t khi ( v C) T trng (g/ml) im nng chy ( 0C) im si (0C)

C5H8O2 100,12 1,06 -14 187

- Glutaraldehyt dng lng nng 25%, do hng Merck sn xut.

2.1.4

Ha cht

- Tinh bt: Tinh bt dng trong nghin cu l tinh bt dc phm, mu trng ng. - Cc ha cht khc: Albumin, ng glucose chun, CuSO4, NaOH, NaH2PO4, Na2HPO4, mui tatrat

2.1.5

Thit b s dng.

- My o quang ph hp thu UV Vis Thermo Spectronic. - My khuy t Magnetic Stirrer HANNA, BIBBY.
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- My o pH Mettler Toledo. - B iu nhit Gallenkamp No.WF250. - Cn 4 s l OHAUS PA214. - Cn 2 s l OHAUS Gold Series. - T sy, t lnh.

2.2

Phng php nghin cu S nghin cu Phng php lin kt cng ha tr hay phng php hp ph

2.2.1

CHN PHNG PHP C NH -AMYLASE

CHN IU KIN C NH -AMYLASE

Th tch enzyme Thi gian c nh Vn tc my lc Kch thc chitosan Nng glutaradehyde

TI U HA IU KIN C NH -AMYLASE GHIN CU ENZYME C NH NGHIN CU TNH CHT -AMYLASE C NH nh hng ca pH. nh hng ca nhit . Tnh cht ng hc Hiu qu ti s dng.

Hnh 2.1:S nghin cu c nh enzyme -amylase.

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2.2.2

Phng php c nh enzyme -amylase.

Chng ti s tin hnh c nh enzyme -amylase theo 2 phng php: - C nh -amylase bng phng php lin kt cng ha tr. - C nh enzyme bng phng php hp ph v lin kt gia cc enzyme. 2.2.2.1 C nh -amylase bng phng php lin kt cng ha tr. - Hot ha chitosan: Ly 1g chitosan, cho vo 10ml glutaraldehyde 2%. Lc trong thi gian 2 gi, tc lc 100 vng / pht.

- Tin hnh ra chitosan 3 ln vi nc ct. - Ly 0,5ml enzyme -amylase nh mc thnh 25ml. - Cho dung dch enzyme vo chitosan sau khi ra, lc trong 2 gi, tc lc 100 vng / pht. - Tin hnh ra chitosan loi cc enzyme khng lin kt, nh mc dung dch sau khi ra thnh 50ml. - Xc nh hm lng protein khng lin kt. T tnh ra hiu sut c nh. - Tin hnh xc nh hot tnh cn li ca enzyme c nh.

2.2.2.2 C nh enzyme bng phng php hp ph v lin kt gia cc enzyme. - Ly 0,5ml enzyme -amylase nh mc thnh 25ml. - Cho dung dch enzyme vo chitosan, lc trong 2 gi, tc lc 100 vng / pht. - Tin hnh ra chitosan loi cc enzyme khng lin kt, nh mc dung dch sau khi ra thnh 50ml. - Xc nh hm lng protein khng lin kt. - Cho vo 10ml glutaraldehyde 2%, lc trong thi gian 2 gi, tc lc 100 vng/pht.
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- Tin hnh ra chitosan loi cc enzyme khng lin kt, nh mc dung dch sau khi ra thnh 50ml. - Xc nh hm lng protein khng lin kt v hiu sut c nh. - Xc nh hot tnh ca enzyme c nh.

2.2.3 amylase.

Kho st cc yu t nh hng n qu trnh c nh enzyme -

th nghim ny chng ti kho st 5 yu t nh hng n qu trnh c nh gm c: th tch enzyme, thi gian c nh, vn tc lc, kch thc chitosan v nng glutaraldehyde. kho st nh hng ca tng yu t, ban u chng ti cho 5 yu t cc gi tr sau: - Th tch enzyme: 0,5ml. - Thi gian c nh: 2 gi. - Vn tc lc o: 100 vng/pht. - Kch thc chitosan: 2,8mm. - Nng glutaraldehyde 2% Khi tin hnh kho st nh hng ca mt yu t, chng ti s thay i yu t , cn nhng yu t cn li s c c nh. Sau , khi kho st nh hng ca yu t tip theo, chng ti s thay i yu t , ng thi ly gi tr ti u ca yu t va kho st v gi c nh nhng yu t cn li.

2.2.3.1 Kho st th tch enzyme s dng. - Th tch enzyme thay i nh sau: 0,1; 0,2; 0,3; 0,4; 0,5 ml. - Gi tr cc yu t khc c gi c nh.

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2.2.3.2 Kho st thi gian c nh enzyme. - Ln lt thay i thi gian c nh: 1, 2, 3, 4, 5 gi. - Th tch enzyme ly t th nghim trc. Gi tr ca cc yu t khc vn c gi c nh.

2.2.3.3 Kho st vn tc lc o. - Ln lt thay i vn tc lc: 0, 50, 100, 150, 200 vng/pht. - Th tch enzyme v thi gian ly t th nghim trc. Gi tr ca cc yu t khc vn c gi c nh.

2.2.3.4 Kho st kch thc ca chitosan - Ln lt thay i kch thc chitosan nh sau: 0,35; 0,7; 1,4; 2,8 mm. Gi tr cc yu t khc gi c nh. - Gi c nh yu t v glutaraldehyde. Cc yu t cn li l nhng gi tr ti u ca cc th nghim trc.

2.2.3.5 Kho st nng glutaraldehyde - Nng glutaraldehyde thay i nh sau: 1%, 2%, 3%, 4%, 5%. - Th tch enzyme, thi gian c nh, vn tc lc o v kch thc chitosan l nhng gi tr ti u ca nhng th nghim trc.

2.2.4

Xc nh hiu xut c nh enzyme -amylase.

- Hiu sut ca qu trnh c nh c tnh theo cng thc sau:

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Vi:

m: l hm lng protein enzyme c trong th tch enzyme ban u (mg) m1: l hm lng protein enzyme khng lin kt (mg).

2.2.5

Xc nh hot tnh ring -amylase

- Cng thc tnh: Vi: Ur: l hot tnh ring ca -amylase (n v hot /mg protein trong ch phm t do hay c nh) G: lng ng kh to thnh do phn ng thy phn (mg). m2: lng protein -amylase dng thy phn (mg). Hot tnh cn li ca ch phm enzyme c nh:

Vi:

Urc l hot tnh ring ca -amylase c nh (n v hot /mg protein amylase c nh ).

Urht l hot tnh ring ca -amylase t do (n v hot /mg ch phm amylase).

2.2.6

Kho st tnh cht ca enzyme -amylase c nh.

2.2.6.1 Kho st nh hng ca pH n hot tnh enzyme -amylase. - pH c nh hng rt ln n hot tnh ca enzyme c nh. V vy, trong th nghim ny, mc ch chng ti l kho st nh hng ca pH v tm ra pH ti u cho hot ng ca enzyme c nh. - Chng ti tin hnh th nghim nh sau: c nh cc yu t nh nng enzyme c nh, nhit , thi gian phn ng, nng c cht. Thay i pH ca dung dch tinh

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bt trong khong pH t 5.5 8 ri xc nh hot tnh ca enzyme c nh v enzyme t do ng vi mi pH khc nhau v suy ra pH ti thch.

2.2.6.2 Kho st nh hng ca nhit n hot tnh enzyme -amylase. - T gi tr pH ti u th nghim trn, chng ti tin hnh kho st nh hng ca nhit n hot tnh ca enzyme c nh. Chng ti cng song song kho st enzyme c nh v enzyme t do c s so snh. - Chng ti c nh cc yu t nh nng enzyme, thi gian phn ng.cho nhit dung dch thay i t 60 90oC v xc nh hot tnh ca enzyme -amylase c nh v t do vi mi nhit .

2.2.6.3 Kho st tnh cht ng hc ca enzyme -amylase c nh trn mng chitosan. - Trong nghin cu ny, chng ti tin hnh kho st nh hng ca nng c cht n hot tnh ca enzyme -amylase c nh. kho st nh hng ca nng c cht, chng ti c nh cc yu t nh: nng enzyme, nhit , pH, thi gian phn ng v thay i nng c cht t 50 250 g/l ri so snh hot tnh ca -amylase nhng nng . - T kt qu thc nghim thu c, chng ti tin hnh tnh ton xc nh gi tr cc thng s ng hc ca phn ng enzyme l Km v Vmax. Tin hnh tng t vi enzyme -amylase t do kim chng. - Xc nh hot tnh ca enzyme t do v c nh ng vi tng nng khc nhau. V th Michaelis Menten ( tc thy phn v theo nng c cht [S]), th Lineweaver Burk ( 1/v theo 1/[S]) xc nh Vmax, Km v nng c cht [S] m ti hot tnh enzyme -amylase l cao nht.

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2.2.6.4 Kho st kh nng ti s dng enzyme -amylase c nh. Th nghim kho st kh nng ti s dng ca enzyme -amylase c nh c tin hnh nh sau: enzyme c nh c s dng lp i lp li 10 ln thy phn tnh bt vi thnh phn khng thay i nh sau: - 25ml dung dch tinh bt 1%. - 1g ch phm -amylase c nh. - Nhit thy phn 75oC. - pH = 7. - Thi gian thy phn 10 pht.

2.2.7

Cc phng php phn tch.

2.2.7.1 Phng php xc nh protein tan. Hm lng protein tan c xc nh bng phng php Lowry. Nguyn tc: Hu ht cc protein u cha Tyrosin v Tryptophan khi cho protein tc dng vi thuc th Folin s to thnh mt phc cht c mu, cng mu t l vi hm lng Tyrosin v Tryptophan. Bng cch o mt quang ca phc mu c trng ny ti bc sng = 750nm trn thit b, ta xy ng chun vi mt loi protein tinh khit c sn thng l albumin. T da vo ng chun, khi c gi tr mt quang, s xc nh c nng protein tan. Ha cht: - Dung dch A: 4g NaOH (0,1M) v 20g Na2CO3 (2%) pha trong 1000ml nc ct. - Dung dch B: 0,5g CuSO4.5H2O (0,5%) pha trong dung dch Natri Citrat (1%) hoc trong dung dch Na K tatrat 1%. - Dung dch C: Hn hp ca hai dung dch A v B theo t l 50:1 (pha trc khi dng). - Thuc th Folin pha long vi nc ct theo t l 1:3.
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- Albumin huyt thanh b 1mg/ml. Tin hnh: Xy dng ng chun: - Cn 0,01g (10mg albumin huyt thanh b) ha tan trong 10ml nc, ta c dung dch gc nng 1mg/ml. - Pha long dung dch gc bng nc ct c cc nng 0,2; 0,4; 0,6; 0,8 v 1mg/ml: Bng 2.2: Pha long dung dch albumin trong xy dng ng chun.

Mu Albumin Nc ct

1 0,2 ml 0,8 ml

2 0,4 ml 0,6 ml

3 0,6 ml 0,4 ml

4 0,8 ml 0,2 ml

5 1 ml 3 ml

- Ly 1ml mu pha long cho vo ng nghim. - Thm vo mi ng 5ml dd C nhit phng 10 pht. - Thm vo 0,5ml thuc th Folin (1N). - Mu trng: cho vo ng nghim 1ml nc ct, 5ml ddC nhit phng. Ri thm vo 0,5ml thuc th Folin (1N). - Sau em o mu bc sng 750nm. T lp ng chun C = f(A)

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0.4
0.3 0.2 0.1 0

y = 0.763x - 0.084 R = 0.992

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Hnh 2.2: ng chun albumin.

Xc nh nng protein ca mu th nghim: Mu th nghim: - Ly 1ml dch cha protein vi hm lng thch hp cho vo ng nghim. - Thm vo 5ml ddC, lc u, yn 10 pht. - Thm vo 0,5ml Foline (1N), lc u, yn 30 pht. - em o hp thu bc sng = 750nm. Mu trng: thay 1ml dch cha protein bng 1ml nc ct. Cc bc sau tng t.

2.2.7.2 Phng php nh lng ng kh: bng acid 3,5 dinotrosalisylic (DNS) Nguyn tc: hm lng ng kh c xc nh bng cch o mt quang ti bc sng = 520nm ca phc mu nu c trng to ra khi cho ng kh tc dng vi 3,5 dinitro salicillic acid.

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Ha cht: - Dung dch chun glucose: 1 mg/ml - Thuc th DNS: Ha tan 1g acid dinitrosalicylic (C7H4N2O7) DNS + 30g mui seignette Chun 3ml mu vi HCl 0,1N n im kt thc ca phenolphtalein.

(KNaC4H406.4H20 Kali naitri tatrat) v 1,6g NaOH trong 100ml nc ct. Lng HCl thng cn l 5 6ml, c th thm NaOH nu cn thit (2g = 1ml HCl 0,1N). Tin hnh: - Pha long dung dch glucose chun 1mg/ml: Ha tan 50mg glucose trong 50ml nc ct. - Pha long dung dch nh sau: Bng 2.3: Pha long dung dch glucose chun.

1 Dung dch Nc ct 1ml 4ml

2 2ml 3ml

3 3ml 2ml

4 4ml 1ml

5 5ml 0ml

- Cho dung dch vo ng nghim theo th t trong bng. - Cho vo mi ng nghim: 1ml rt t mi ng trn + 2ml DNS, lc u. - Dng ming nylon hoc nhm bt kn ming ng nghim, tin hnh un cch thy nhit 100oC trong 5 pht. - Lm ngui nhanh dung dch, lc u n khi dung dch khng cn phn lp. - Thm vo 10ml nc ct lc u. - o hp thu bc sng = 540nm. - Xy dng ng chun C = f(A).

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A

Y = 1.8X + 0.036 R = 0.996

Hnh 2.3: ng chun glucose.

2.2.7.3 Xc nh hot tnh amylase ha tan. Ha cht: - Dung dch tinh bt 1%: Cn 1g tinh bt cho vo 10ml nc ct v x l nhit 100oC cho n khi tinh bt c ha tan. Sau nh mc dung dch trn thnh 100ml bng dung dch m phosphate pH 6. - Dung dch m: Dung dch mononatri orthophosphate 0,2M (a): 31,2g NaH2PO4 ha tan v

nh mc n 1000ml. Dung dch dinatri hydrophosphate 0,2M (b): 53,05g Na2HPO4.7H2O hoc 71,6g Na2HPO4.12H2O ha tan v nh mc n 1000ml.

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Bng 2.4: T l pha dung dch m.

Cch tin hnh xc nh hot tnh enzyme ha tan: - Pha long enzyme. - Cho vo erlen: 25ml h tinh bt 1% pH 6 50oC. Sau khi mu t 50oC cho vo 1ml enzyme pha long, 10 pht. - Sau khi thy phn, rt 1ml mu cho vo ng nghim cha sn 2ml DNS. - Mu trng: 1ml nc v 2ml DNS. - un cch thy cc ng nghim 100oC trong 5 pht. Sau lm ngui nhanh bng nc lnh, ri thm vo mi ng nghim 10ml nc ct. - Lc u v o mt quang bc sng 540nm. Cch tin hnh xc nh hot tnh enzyme c nh: - Cho vo erlen: 1g enzyme c nh 50oC. Sau cho vo 25ml h tinh bt 1% pH 6 10 pht. - Sau khi thy phn, lc thu dung dch ng, nh mc ln 100ml. - Rt 1ml mu cho vo ng nghim cha sn 2ml DNS.
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- Mu trng: 1ml nc v 2ml DNS. - un cch thy cc ng nghim 100oC trong 5 pht. Sau lm ngui nhanh bng nc lnh, ri thm vo mi ng nghim 10ml nc ct. - Lc u v o mt quang bc sng 540nm. Da vo ng chun tnh c hm lng glucose to thnh.

Cng thc tnh hot tnh enzyme:

Vi: G l lng ng kh tnh theo glucose to ra sau phn ng thy phn, mg F l h s pha long enzyme V l th tch dung dch enzyme dng trong phn ng, ml (hoc khi lng ch phm enzyme, g)

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CHNG 3: KT QU V BN LUN
3.1 Ch phm -amylase t do. Bng 3.1: Hm lng protein v hot tnh ring ca -amylase t do.

Hm lng protein (mg/ml) 50,09

Hot tnh ring (n v hot /mg protein) 20,86

3.2

Kho st cc yu t nh hng n qu trnh c nh enzyme -amylase. Trong nghin cu ny chng ti kho st 2 phng php khc nhau c nh -

amylase: - Phng php 1: c nh -amylase bng phng php lin kt ngang. - Phng php 2: c nh -amylase bng phng php hp ph v lin kt gia cc enzyme. Bng 3.2: So snh cc phng php c nh.

Phng php c nh Hiu sut c nh (%) Hot tnh (v/mg) Hot tnh cn li (%)

Phng php 1 20,71 10,24 49,09

Phng php 2 21,12 6,07 29,11

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60 50 40 30 20,71 21,12 20 10 0 Hiu sut c nh (%) Hot tnh cn li (%) 29,11 Phng php 1 Phng php 2 49,09

Hnh 3.1: So snh hiu qu cc phng php c nh - Kt qu th nghim bng trn cho thy hiu sut ca phng php lin kt ngang thp hn phng php hp ph nhng khng ng k. Tuy nhin, hot tnh cn li ca enzyme c c nh theo phng php lin kt ngang cao hn hn phng php hp ph. - iu ny c l gii nh sau: glutaraldehuyde c s dng lm cu ni gia cht mang v enzyme. Bn cht ca lin kt ny l lin kt gia nhm CHO ca glutaraldehyde v nhm - NH2 ca enzyme. - Nh ta bit, chitosan c bn cht l mt polysaccharide cha cc nhm NH2 nn khi cho glutaraldehyde vo chitosan trc th cc lin kt cng ha tr gia nhm NH2 ca nhm chitosan v nhm CHO ca glutaraldehyde s hnh thnh nhiu hn v khng b cn tr. T s hnh thnh cc lin kt tng i cht ch gia cht mang v enzyme thng qua cu ni glutaraldehyde. Ngc li, khi cho enzyme vo trc glutaraldehyde th s lng nhm -NH2 t do khng nhiu, dn n kh nng to lin kt vi glutaraldehyde khng cao.

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3.3

Kho st cc yu t nh hng n qu trnh c nh enzyme -amylase. nh hng ca th tch enzyme s dng.

3.3.1

Hiu sut c nh (%) 45 40 35 30 25 20 15 10 5 0 0

40.92 37.02 29.85 24.85 20.68

0.1

0.2

0.3

0.4

0.5

0.6

Th tch enzyme s dng (ml) Hnh 3.2: th biu din hiu sut c nh ng vi th tch enzyme khc nhau. Bng 3.3: Hot tnh ring ca cc mu -amylase c nh tng ng vi nhng th tch enzyme s dng khc nhau.

Enzyme s dng (ml) 0,1 0,2 0,3 0,4 0,5

Hot tnh ring (n v hot /mg protein) 10,05ab 0,1 10,19ac 0,15 9,97b 0,13 10,18ac 0,05 10,25c 0,09

- T kt qu thu c hnh 3.2, chng ti nhn thy rng hiu sut c nh cao nht t c l 40,92% khi th tch enzyme s dng l 0,2ml.
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- T kt qu phn tch ANOVA bng 3.3, chng ti nhn thy hot tnh ring ca cc mu khc nhau khng c ngha. - Vi nhng kt qu trn, chng ti quyt nh chn th tch enzyme l 0,2ml. Th tch enzyme ny c s dng cho cc th nghim tip theo.

3.3.2

nh hng ca thi gian c nh.

Trong nghin cu ny, chng ti tin hnh kho st nh hng ca thi gian c nh n hiu qu ca qu trnh c nh enzyme -amylase vi cc gi tr 1 gi, 2 gi, 3 gi, 4 gi, 5 gi. Sau khi thc hin th nghim, chng ti thu c kt qu nh sau: Hiu sut c nh (%) 45 40 35 30 25 20 15 10 5 0 0 1

41.87
35.95 27.46 27.63 25.4

4 5 Thi gian c nh (gi)

Hnh 3.3: th biu din hiu sut c nh ng vi thi gian c nh khc nhau.

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Bng 3.4: Hot tnh ring ca cc mu -amylase c nh tng ng vi nhng thi gian c nh khc nhau.

Thi gian c nh (gi) 1 2 3 4 5

Hot tnh ring (n v hot /mg protein) 10,08a 0,15 10,09a 0,07 10.17a 0,08 9.87a 0,16 7.85b 0,38

- Kt qu th nghim hnh 3.3 cho thy khi tng thi gian c nh, hiu sut c nh tng dn v hiu sut c nh t c cc i l 41,87%, sau khi c nh 2 gi. Tip theo, nu tip tc tng thi gian c nh, hiu sut c nh enzyme li gim. - Nguyn nhn ca hin tng ny c gii thch: nu thi gian c nh ngn, s lng lin kt to thnh gia glutaraldehyde v nhm NH2 trn mng chitosan cng t, dn n kh nng to lin kt vi emzyme khng cao, lm hot tnh ca enzyme c nh s thp. Ngc li, nu thi gian c nh di, s dn n hin tng to lin kt ngang gia glutaraldehyde v mng qu cht, lm mng d v hiu sut c nh khng cao. - Mc khc, vi kt qu thu c t phn tch ANOVA bng 3.4, chng ta thy rng hot tnh ring ca mu -amylase c nh 5 gi khc cc mu khc v thp hn, cn 4 mu cn li khc nhau khng c ngha. - Nguyn nhn ca hin tng ny l do khi thi gian c nh ko di nh hng n cu hnh khng gian ca enzyme nh hng n trung tm hot ng; dn n gim hot tnh ca enzyme c nh. - T cc kt qu trn, chng ti chn thi gian c nh enzyme l 2 gi. Thi gian c nh ny s c dng kho st nhng yu t tip theo.

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3.3.3

nh hng ca tc vng quay.

Sau khi thc hin th nghim, chng ti thu c kt qu nh sau:

Hiu sut c nh (%)
50 45 40 35 30 25 20 15 10 5 0 0 50 100 150 200 250 22.23 39.87 40.51 35.83 45.16

Vn tc vng quay (vng/pht)

Hnh 3.4: th biu din hiu sut c nh ng vi vn tc vng quay khc nhau. Bng 3.5: Hot tnh ring ca cc mu -amylase c nh tng ng vi nhng vn tc lc o khc nhau. Vn tc lc o (vng/pht) 0 50 100 150 200 Hot tnh ring (n v hot /mg protein) 9,98a 0,11 10,07ab 0,14 10,11ab 0,05 10,15ab 0,14 10,23b 0,08

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- Kt qu th nghim hnh 3.4 cho thy khi tng vn tc vng quay, hiu sut c nh tng dn v hiu sut c nh t c cc i l 45,16% vn tc 150 vng/pht. Tuy nhin, nu tip tc tng vn tc vng quay, hiu sut c nh enzyme gim. - Khi c nh, lc o cht mang v enzyme nhm mc ch enzyme phn b u v d dng tip xc v gn vo cht mang. Vi vn tc lc o ph hp, th hiu sut c nh s tng ln ng k. - Vi kt qu phn tch ANOVA bng 3.5, ta nhn thy rng hot tnh ring ca cc mu khc nhau khng c ngha. - T cc kt qu trn, chng ti quyt nh chn tc lc o l 150 vng / pht. Tc lc o ny c dng kho st nhng yu t tip theo.

3.3.4

nh hng ca kch thc chitosan.

- Sau khi thc hin th nghim, chng ti thu c kt qu nh sau:

Hiu sut c nh (%)

60 50 40 30 20 10 0 0

52.02

50.98 44.26 41.92

0.5

1.5

2.5

Kch thc chitosan (mm)

Hnh 3.5: th biu din hiu sut c nh ng vi nhng kch thc chitosan khc nhau.
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Bng 3.6: Hot tnh ring ca cc mu -amylase c nh tng ng vi nhng kch thc chitosan khc nhau. Kch thc chitosan (mm) 0,35 0,7 1,4 2,8 Hot tnh ring (n v hot /mg protein ) 9,96ab 0,07 10,21c 0,1 9,87a 0,23 10,18bc 0,05

- T kt qu thu c hnh 3.5, chng ti nhn thy rng cng gim kch thc chitosan th hiu sut c nh li cng tng. - iu ny d dng gii thch, khi kch thc chitosan gim, din tch b mt li tng ln, to iu kin cho enzyme gn vo chitosan. Do , s nhm lin kt gia glutaraldehyde v chitosan s tng, dn n kh nng lin kt vi enzyme cng tng. - Khi phn tch ANOVA, hiu sut c nh cc mu chitosan kch thc 0,35mm v 0,7mm khc nhau khng khng c ngha - Vi kt qu phn tch ANOVA bng 3.6, cc mu -amylase c nh trn cc kch thc chitosan khc nhau c hot tnh ring khc nhau khng c ngha. - T cc kt qu trn, chng ti quyt nh chn kch thc chitosan l 0,7 mm. V khi p dng quy m cng nghip, vic sn xut ra cht mang c kch thc ln s gim c mt phn chi ph, ng thi khi ng dng to ct thy phn tinh bt sn xut syrup s khng cn tr c cht tinh bt i qua. - Kch thc chitosan l 0,7mm c s dng cho cc th nghim sau.

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3.3.5

nh hng ca lng glutaradehyde.

Nh chng ti cp trn, lin kt gia cht mang v enzyme thng qua cu ni l glutaraldehyde. V vy, trong th nghim ny chng ti kh st nh hng ca nng glutaraldehyde n kh nng c nh enzyme -amylase. Sau khi thc hin th nghim, chng ti thu c kt qu nh sau:
Hiu sut c nh (%)
54 52 50 48 46 44 42 40 0 1 2 3 4 5 48.28 49.85 50.25 52.15 52.43

Glutaraldehyde (%)

Hnh 3.6: th biu din hiu sut c nh ng vi nng glutaradehyde khc nhau.

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Bng 3.7: Hot tnh ring ca cc mu -amylase c nh tng ng vi nhng nng glutaraldehyde khc nhau.

Nng glutaraldehyde (%) 1 2 3 4 5

Hot tnh ring (n v hot /mg protein) 9,97ab 0,15 9,68b 0,34 10,21a 0,20 10,16a 0,15 8,53c 0,10

- Kt qu th nghim hnh 3.6 cho thy khi tng lng glutaraldehyde, hiu sut c nh tng, nhng sau hiu sut thay i khng ng k. - iu ny c gii thch nh sau: do glutaraldehyde l tc nhn gn kt enzyme ln cht mang nn khi nng glutaraldehyde tng, s th tch enzyme lin kt cng tng, dn n hot tnh eyme c nh tng. nng glutaraldehyde thp, lin kt cng ha tr c hnh thnh nhng cha nhiu. Khi nng glutaraldehyde qu cao th vic to lin kt bo ha nn s lin kt c hnh thnh thm khng ng k. - Kt qu phn tch ANOVA, chng ti nhn thy hiu sut c nh cc mu glutaraldehyde 4% v 5% khc nhau khng c ngha. - ng thi kt qu phn tch ANOVA bng 3.7 cho thy mu c nh s dng glutaraldehyde nng 5% c hot tnh ring khc 4 mu cn li v hot tnh thp hn. - Nguyn nhn ca hin tng ny l do nng glutaraldehyde tng dn n lin kt ha tr tng, m lin kt ny d bin i trung tm hot ng ca enzyme. V vy, lm gim hot tnh ca enzyme -amylase c nh. - Vi cc kt qu trn, chng ti chn nng glutaraldehyde l 4%.
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- Sau khi tin hnh xong cc th nghim kho st bng phng php c in, chng ti tm ra c gi tr ph hp c nh -amylase vo chitosan vi hiu sut t c l 52,15%. V nhng gi tr l: Th tch enzyme: 0,2ml. Thi gian c nh: 2 gi. Vn tc lc: 150 vng/pht. Kch thc chitosan: 0,7mm. Nng glutaraldehyde: 4%.

3.4

Ti u ha qu trnh c nh. - qu trnh c nh t hiu qu cao hn, nn chng ti thc hin ti u qu trnh

c nh. Chng ti tin hnh th nghim theo phng php quy hoch thc nghim trc giao hai yu t, vi hm mc tiu l hiu sut c nh. - T nhng yu t nh hng trn, chng ti thy rng hai yu t v th tch enzyme v thi gian c nh c nh hng nhiu n hiu sut c nh, nn chng ti chn ti u hai yu t ny. V nhng yu t cn li chng ti chn nhng gi tr ph hp c c t nhng th nghim trn. - Chng ti s thay i ln lt gi tr ca hai yu t th tch enzyme v thi gian c nh, ri xc nh quy lut nh hng ca hai yu t ny ln hiu sut c nh, sau xy dng quy trnh hi quy v xc nh thng s ti u. - S th nghim c thc hin nh mc 4 chng II vi gi tr ca hai yu t nh sau: Th tch enzyme s dng: X1 thuc [0,1; 0,3], v tm Xo1 = 0,2 ml. Thi gian c nh: X2 thuc [1; 3], v tm Xo2 = 2 gi. Hiu sut c nh: Y (%)

- Sau khi tin hnh th nghim, chng ti thu c kt qu sau:

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Bng 3.8 Xy dng phng trnh hi quy trong ti u ha. X1 2 - X2 2 -

S th nghim 1 S th nghim nhn: 2k 2 3 4 5 S th nghim cc im giao vi trc 2k 7 8 S th nghim tm S th nghim tm lm thm 9 10 11 6

Xo

X1

X2

X1 X2

+1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1

-1 +1 -1 +1 - + 0 0 0 0 0

-1 -1 +1 +1 0 0 - + 0 0 0

+1 -1 -1 +1 0 0 0 0 0 0 0

1- 1- 1- 1- 2- 2- - - - - -

1- 1- 1- 1- - - 2- 2- - - -

35,24 20,06 45,98 32,78 37,23 49,13 47,95 39,46 52,92 51 53,06

- Phng trnh hi quy ca hm mc tiu c dng nh sau: Y = bo + b1X1+ b2X2 + b12X1X2 + b11X12 + b22X22 - Sau khi gii bi ton quy hoch thc nghim, chng ti thu c kt qu sau:

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Bng 3.9: Kt qu cc gi tr trong phng trnh hi quy.

H s bo b1 b2 b12 b11 b22 - Trong :

Gi tr ca b 52,55 -6,71 5,33 0,5 -9,7 -9,17

sb 0,67 0,53 0,53 0,66 0,82 0,82

t 78,43 12,66 10,06 0,78 11,83 11,18

ttra 2,57 2,57 2,57 2,57 2,57 2,57

iu kin t > ttra Nhn Nhn Nhn Loi Nhn Nhn

sb: l phng sai ca h s torng phng trnh hi quy. t: ngha ca cc h s phng trnh hi quy kim nh theo chun Student. ttra: gi tr tra bng phn b student vi = 0,05.

- T ta c phng trnh hi quy sau Y = 52,55 6,71X1 + 5,33X2 9,7X12 9,17X22 - Khi s dng phn mm Modde 5 chy kt qu, chng ti cng thu c kt qu tng t. Khi dng phng trnh hi quy trn h trc khng gian ba chiu bng phn mm Modde 5 ta c:

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Hnh 3.7: Phng trnh hi quy trn h trc khng gian ba chiu. - Ti u ha hm 2 bin, chng ti thu c gi tr cc i nh sau: Y = 54,11% ti X1 = 0,17 v X2 = 2,3 - T cho thy: nh vo phng php ti u ha bng phng php quy hoch thc nghim m chng ti thu c hiu sut c nh tng 1,66% khi gim i th tch enzyme s dng l 0,3ml v tng thi gian c nh thm 0,3 gi. - Nh vy, hiu sut c nh t cao nht ti nhng gi tr sau: Th tch enzyme: 0,17ml. Thi gian c nh: 2,3 gi. Vn tc lc: 150 vng/pht. Kch thc chitosan: 0,7mm. Nng glutaraldehyde: 4%.
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3.5

Kho st tnh cht ca -amylase c nh. nh hng ca pH.

3.5.1

Trong nghin cu ny, chng ti thc hin th nghim xc nh pH ti thch cho enzyme -amylase c nh. ng thi tin hnh mu i chng l enzyme t do. - Kt qu thu c:
Hot tnh tng i (%)
110 100 90 80 70 60 50 5 5.5 6 6.5 7 7.5 8 8.5

Enzyme t do Enzyme c nh

pH

Hnh 3.8: th biu din nh hng ca pH ln hot tnh enzyme t do v c nh. - T hnh trn, cho thy pH ti thch ca enzyme t do l 6 v ca enzyme c nh l 7. pH ti thch ca enzyme c nh ln hn pH ti thch ca enzyme t do. Ngoi ra, khong pH hot ng ca enzyme c nh rng hn enzyme t do. - Nguyn nhn ca hin tng ny c th gii thch nh sau: trong cng mt dung dch m phosphate, s c hai pH nh hng n hot tnh ca enzyme -amylase c nh. l pH vng trung gian gia chitosan, enzyme c nh v dung dch m phosphate. Chitosan l mt polycation (pKa< 6.5), mang in tch dng, v vy s c nhiu ion OH- xung quanh n. iu ny cho thy vng trung gian gia mng chitosan

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v enzyme c nh s c t ion H+ hn so vi trong dung dch m phosphate, ngha l pH vng ny s cao hn so vi dung dch m phosphate. - Ta nhn thy, pHopt ca enzyme c nh dch chuyn v pha kim so vi enzyme t do. - Kt qu nghin cu ca chng ti cng tng t vi cc nghin cu ca cc tc gi khc [30,31]: Viara (1993), pH ti thch ca enzyme -amylase t do l 6 6,5. Min Yun Chang v Ruey Shin Juang ( 2004), pH ti thch ca enzyme

-amylase c nh l 7.

3.5.2

nh hng ca nhit .

- Cng nh enzyme t do, nhit l mt yu t nh hng rt ln n hot tnh ca enzyme -amylase c nh. Nh lin kt vi cht mang m -amylase c nh c kh nng chu c nhit cao tt hn enzyme t do. - Vi gi tr pHopt ca enzyme c nh th nghim trn, ta kho st s nh hng ca nhit ln hot tnh enzyme.

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Hot tnh tng i (%)
110 100 90 80 70 60 50 40 50 60 70 80 90 100

Enzyme t do Enzyme c nh

Nhit (oC)

Hnh 3.9: th biu din nh hng ca nhit ln hot tnh enzyme t do v c nh.

- T kt qu trn, cho thy khi tng nhit , hot tnh ca enzyme -amylase c nh tng v t cc i ti 75 - 80oC. Trong khi , hot tnh cc i ca enzyme t do l trn 90oC. - Nguyn nhn ca hin tng ny c gii thch nh sau: i vi -amylase c nh, khi nhit tng, cc phn t -amylase v c cht nhn c nng lng hot ha cao hn, do s chuyn ng nhiu hn. Kh nng tip xc vi nhau tt hn. Kt qu l kh nng phn ng ca -amylase s cao, hiu sut thy phn tt. - Nhng sau nu tip tc tng nhit th hot tnh ca enzyme c nh gim. - Nguyn nhn: hin tng enzyme b v hot bi nhit cao c gii thch l khi tng nhit , cc nguyn t trong phn t enzyme tch ly nng lng v bt u c xu hng chuyn ng nhanh hn. n mt lc no khi cc nguyn t ny tch ly nng lng chng s tch ri ra khi phn t enzyme, lm thay i cu trc phn t enzyme, lm cho enzyme mt hot tnh.

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- Kt qu nghin cu ca chng ti cng tng t vi cc nghin cu ca cc tc gi khc [30, 31]: Viara (1993), nhit ti thch ca -amylase t do l 100oC. Min Yun Chang v Ruey Shin Juang ( 2004), nhit ti thch ca

enzyme -amylase c nh l 70 80oC.

3.5.3

Tnh cht ng lc hc ca -amylase c nh.

3.5.3.1 nh hng ca nng c cht. Bng 3.10: nh hng ca nng dung dch tinh bt n vn tc thy phn. Nng c cht (g/l) 50 100 150 200 250 Vc nh (g/l/pht) 0,37 0,57 0,58 0,55 0,46 Vt do (g/l/pht) 0,6 0,75 0,88 0,88 0,85

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V (g/l/pht)
1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 50 100 150 200 250 300

Enzyme t do

Enzyme c nh

[S] (g/l)

Hnh 3.10: th biu din nh hng ca nng dung dch tinh bt n vn tc thy phn. - T th trn, chng ti nhn thy rng, vi nng tinh bt di 150 g/l th phng trnh ng hc ca phn ng thy phn tun theo phng trnh Michaelis Menten. - Kt qu th nghim cho thy khi tng nng c cht trong dung dch th vn tc phn ng thy phn bng enzyme -amylase t do v enzyme -amylase c nh u tng. Vn tc phn ng thy phn bi enzyme -amylase c nh v t do u t gi tr cc i ti nng dung dch tinh bt l 150g/l. Khi nng dung dch tinh bt tng dn, vn tc phn ng s gim. - Hin tng vn tc phn ng enzyme gim khi nng c cht qu cao c gii thch do khi tha c cht, cc phn t c cht c th kt hp vi phc enzyme c cht, to phc cht khng hot ng, lm gim hot tnh ca enzyme.

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3.5.3.2 Xc nh h s phng trnh ng hc Michaelis Menten. - Phng trnh ng hc Michaelis Menten c dng

- Trong :

Km c gi l hng s Michaelis. V: l vn tc phn ng. Vmax: l vn tc cc i. [S]: l nng c cht.

- V mt l thuyt, theo phng trnh ng hc Michaelis Menten, trong mt gii hn nng xc nh, khi tng nng c cht th vt tc phn ng enzyme s tng, vt qua gii hn nng vn tc phn ng enzyme tng thm khng ng k. - V biu din theo phng trnh ny cn tin hnh nhiu th nghim t s chnh xc, nn Lineaweaver v Burk ci tin phng trnh ny nh sau:

- Phng trnh c dng ng thng v ct trc honh ti im ti im .

v ct trc tung

- xc nh hng s Km v Vmax chng ti tin hnh th nghim s dng enzyme c nh v ha tan thy phn cc dung dch tinh bt c nng thay i: 60, 70, 80, 90, 100g/l. - Chng ti thu c kt qu sau:

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Bng 3.11: nh hng ca nng dung dch tinh bt n vn tc thy phn.

Nng c cht (g/l) 60 70 80 90 100

1/V

Vc nh (g/l/pht) 0.42 0.46 0.48 0.5 0.54

Vt do (g/l/pht) 0.65 0.67 0.73 0.76 0.78

2.5 2 1.5 1 0.5 0 0.009

y = 74.11x + 1.141 R = 0.98

C nh

y = 41.415x + 0.865 R = 0.9631

0.011

0.013

0.015

0.017 .

0.019

1/S

Hnh 3.11: th biu din mi lin h gia v

- Bng phng php bnh phng cu tiu, chng ti thu c kt qu sau: Bng 3.12: Gi tr Vmax v Km ca enzyme t do v enzyme c nh. Enzyme t do Vmax Km 1.12 44.07 Enzyme c nh 0.9 70.1
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- T kt qu cho thy i lc ca enzyme c nh cao hn enzyme t do 1,6 ln. iu ny chng t enzyme c nh kh c th tip xc c cht thy phn hn so vi enzyme t. iu ny xy ra c th do cht mang cn tr s khuch tn ca c cht. - ng thi trong trng hp thy phn c cht bng enzyme c nh , vn tc thy phn ng t gi tr cc i thp hn so vi trong trng hp s dng enzyme t do. Nguyn nhn hin tng ny c th l: Enzyme -amylase b thay i cu trc phn t khi c nh, v vy kh nng

phn t c cht tip cn vi trung tm hot ng ca enzyme c nh gp nhiu kh khn dn n i lc gia c cht b enzyme gim, ngha l Km tng. Do enzyme l mt protein, c cha nhng gc a nc, kt hp ci cc

phn t nc to ra nhng lp hydrat bao xung quanh nhng phn t enzyme c nh, dn n kt qu l s gy cn tr cho s tip xc gia enzyme v c cht. Khi Km cng s tng. - Khi enzyme -amylase b c nh ln cht mang, cc phn t c cht phi mt mt khong thi gian di chuyn t mi trng bn ngoi vo bn trong mng tip xc vi enzyme. Trong khi , nu s dng enzyme -amylase t do thy phn, cc phn t enzyme c phn b u trong dung dch v kh nng tip xc trc tip vi c cht cao hn so vi enzyme c nh. Chnh v vy m vn tc cc i ca phn ng thy phn tinh bt s dng enzyme c nh thp hn trong trng hp s dng enzyme t do.

3.6

Kho st hiu qu ti s dng ca -amylase c nh. - Enzyme c nh c s dng lp i lp li 10 ln thy phn tnh b vi thnh

phn khng thay i nh sau: 25ml dung dch tinh bt 1%. 1g ch phm -amylase c nh. Nhit thy phn 75oC.
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pH = 7. Thi gian thy phn 10 pht.

- Chng ti thu c kt qu sau:

Hot tnh tng i (%)

110 100

90
80

70
60

50
0 1 2 3 4 5 6 7 8 9 10 11

S ln ti s dng ( ln)

Hnh 3.12: th biu din hiu qu ti s dng ca enzyme -amylase c nh. - T kt qu th nghim, chng ti nhn thy enzyme sau khi ti s ln 1 th hot tnh cn li ch c khong 80% so vi hot tnh ban u v gim dn nhng ln s dng tip theo. - Nguyn nhn c th c gii thch nh sau: trong phn ng c xc tc enzyme, nh s to thnh phc hp trung gian enzyme c cht m c cht c hot ha. Khi c cht kt hp vi enzyme, do kt qu ca s cc ha, s dch chuyn ca cc electron v s bin dng ca cc lin kt tham gia trc tip vo phn ng dn n lm thay i ng nng cng nh th nng ca phn t c cht, kt qu lm cho phn t c cht tr nn hot ng hn, nh tham gia phn ng d dng hn.
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- iu ngha l khi kt hp gia enzyme c cht, hnh thnh cc lin kt trong qu trnh phn ng nh tng tc tnh in, lin kt hidro, lin kt Van der Walls. - Bn cnh , trc khi bin i phc ny thnh sn phm, xy ra s bin i c cht dn n s ko cng v ph v cc lin kt ng ha tr tham gia phn ng lm nh hng mnh n cu trc phn t enzyme. - Nh vy, sau nhiu ln s dng, cu trc ca enzyme c nh s b thay i, hot tnh s b gim. - V kha cnh hiu qu s dng, tng lng ng do enzyme c nh to ra sau 4 ln ti s dng bng vi lng ng kh do enzyme t do to ra. iu chng t, khi s dng nhiu ln, hiu qu thy phn tinh bt ca enzyme c nh cao hn. Nht l trong nhng qu trnh s dng lin tc hoc nhiu ln.

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CHNG 4: KT LUN V KIN NGH

4.1 Kt lun. Qua qu trnh nghin cu, chng ti thu c kt qu nh sau: - Phng php c nh -amylase trn cht mang chitosan rn c chn la l phng php lin kt cng ha tr. - iu kin c nh : Th tch enzyme -amylase: 0,17ml. Thi gian c nh: 2,3h. Vn tc lc: 150 vng/ pht. Kch thc chitosan: 0,7mm. Nng glutaradehyde: 4%.

- Enzyme -amylase c nh c khong pH hot ng ti thch rng hn v c pH ti u l 7 so vi ca -amylase t do l 6 6,5. - Emzyme -amylase t do c nhit ti u cao hn enzyme c nh (enzyme c nh c toopt l 75 - 80oC; trong khi enzyme t do c toopt hn 90oC) - Km v Vmax ca enzyme -amylase c nh c gi tr ln lt l 70,1 mmol v 0,9 mol/mg.pht. Km v Vmax ca enzyme -amylase t do c gi tr ln lt l 44,07 mmol v 1,12 mol/mg.pht. - Enzyme -amylase ti s dng sau 9 ln s dng hot tnh cn 70% v hiu qu thy phn tinh bt cao hn enzyme t do.

4.2

Kin ngh. Do hn ch v mt thi gian nn trong phn nghin cu ca chng ti chua kho st

c mt s vn lin quan n qu trnh thy phn tinh bt v ng dng trong sn xut syrup. T chng ti a ra kin ngh sau:

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- Nghin cu thm phng php lm tng hiu sut c nh enzyme -amylase trn chitosan. - Thi gian v phng php bo qun hiu qu enzyme -amylase c nh. - Ti u qu trnh thy phn tinh bt. - nh gi cht lng sn phm.

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TI LIU THAM KHO

1. Babu K.R et al Enzyme and Microbial Technology 1993 2. Alan Wiseman Handbook of Enzyme Biotechnology Third edition, Ellis Hoord 3. Ali M.B et al Enzyme and Microbial Technology Third edition, Ellis 4. Brzozowski A.M et al Biochemistry 2000 5. Nguyn Hu Chn Vai tr ca cc ion kim loi trong hot ng xc tc enzyme Nh xut bn Y hc H Ni 1992. 6. Nguyn Trng Cn, Nguyn Th Hin, Th Giang, Trn Th Luyn Cng ngh enzyme Nh xut bn nng nghip thnh ph H Ch Minh 1998. 7. Nguyn Hu Chn Enzyme v cht xc tc sinh hc Nh xut bn Y hc H Ni 1996 8. Johnwilley anh son Immobilized enzyme 1980 9. Frersht A.R Enzyme strucrure and mechanism New York Freeman, W.H and company 1985 10. Nguyn Vn t v cc tc gi - Phn lp v nghin cu cc vi khun nhm Bacillus a kim sn xut -amylase kim 1999. 11. Zanin G.M and de moraes F.F Applied Biochemistry and Biotechnology 1997 12. Nguyn c Lng Cng ngh enzyme Nh xut bn i hc Quc gia TPHCM 13. Nguyn Anh Dng - Nghin cu ch to vt liu c nh enzyme t polymer sinh hc bng k thut bc x kt hp vi k thut sinh ha hc 2000
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14. Nguyn Hu Chn Nhng vn sinh ha hin i Nh xut bn khoa hc k thut H Ni 1992 15. Chen et al Immobilization -amylase to a composite temperature sensitive membrane for starch hydrolysis, Biotechnology and progress 14 1998 16. Phan Th Trn Chu, Trn Th nh Ha sinh hc Trng i hc s phm TPHCM 1992 17. Wang J. et al Trend in Biotechnology and Bioengineering 1999 18. Withers J.K Trend in Biotechnology and Bioengineering 1998 19. Riccardo. A. A. Muzzarelli Immobilization of enzymes on chitin and chitosan 1980 20. S. Schwimmer - Kinectics of malt -amylase action J. Biol. Chem 1950 21. Leng Hong Lim, Douglas G. Macdonald, Gordon A, Hill Hydrolysis of starch particles using immobilized barley -amylase 2002 22. Jolanta Bryjak Glucoamylase, -amylase and -amylase immobilization on acrylic cariers 2003 23. Pefecto Q. Flor and Shinsaku Hayashida Continuous production of hight glucose syrup by Chitin Immobilized -amylase 1982 24. F. Sharmin, S. K. Rakshit, H. P. Jayasuriya enzyme immobilization on galss surface for the development of phosphate detection biosensor 2007 25. P.J Worsfold Characteristics of immobilized enzymes 1995 26. O.R. Zaborsky Immobilized Enzymes CRC Press, Cleveland 1973 27. I. Chibata Immobilized enzymes. Research and Development Wiley, New York 1978

28. P.W. Car and L.D. Bower Immobilized enzymes in analytical and clinical chemistry Fundamentals and Application Wiley, New York 1980
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29. M.D Trevan Immobilized enzymes. An introduction and Application in biotechnology Wiley, New York 1980 30. D. R. Walt, V.I. Agayn The chemistry of enzyme and protein immobilization with glutaraldehyde Trends Anal. Chem 1994 31. Min Yun Chang, Ruey Shin Juang Activities, stabilities, and reaction kinetics of three and chitosan clay composite immobilized enzyme 2004

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