Enzyme glucoamylase 1. Gii thiu chung Glucoamylase c cc nh khoa hc Nht tch ra ln u tin t Aspergillus awamori (Katihara, Kurushima, 1966).

Sau c tm thy Rhizopus delemar, Aspergillus niger, Aspergillus oryzae v cc nhm vi sinh vt khc cng nh m ng vt [14]. Glucoamylase t nm mc l cc protein c khi lng phn t lng dao ng rt ln t 27.000 n 112.000 Dal, tuz thuc vo ngun gc ca enzyme. Cc glucoamylase ch yu c to nn t hai iso enzyme I v II khc nhau kh nng thu phn tinh bt trng thi rn v bi bn ca chng. Glucoamylase I t hp th v thu phn tinh bt trng thi rn, ngc li glucoamylase II khng c c hai tinh cht ny. 2. C ch tc ng Enzym glucoamylase c kh nng thy phn mnh m lin kt -1,4 ln -1,6 glucoside ca phn t tinh bt.

S phn gii ca enzyme glucoamylase Kh nng thu phn mnh m lin kt -1,4 ln -1,6 glucoside glucoamylase to thnh glucose a enzyme ny ln hng u v hiu lc xc tc thu phn tinh bt. V th vic dng ch phm glucoamylase t cc chng vi sinh vt trong vic sn xut bia, ru, mt, glucose c mt { ngha trin vng rt ln. Enzyme glucoamylase xc tc tch tun t tng gc glucose t u khng kh ca chui polysaccharide. Gc glucose t u khng kh c gi l glycone, chui oligosaccharide sau khi glucose c tch ra gi l alglycone. C ch xc tc thy phn lin kt O-glucoside l do hot ng ch yu ca mt cp acid amin ng vai tr nh mt cp acid base. Khi tng tc vi c cht, acid glutamic v tr th 179 ng vai tr nh mt cht xc tc acid, n s proton ha oxi glycosidic (phn t oxi v tr

lin kt O-glucoside). Acid glutamic v tr 400 ng vai tr nh mt cht xc tc base, acid amin ny s thu ht v to lin kt vi proton H+ ca phn t nc lm gii phng gc OH-, gc OH- ny s tn cng vo v tr carbon glycosidic v nh vy lin kt O-glucoside ca phn t c cht s b ph v. 3. Tnh cht ca glucoamylase Glucoamylase phn thu phn tinh bt thnh nhng dextrin c phn t thp, lin tc tch gc glucose v cui cng to sn phm l glucose. Glucoamylase thu phn tinh bt, glycogen, polysaccharide ng lot cc lin -1,4 v -1,6 glucoside. N c gi tr c bit trong ngnh sn xut ru, chuyn nhng dextrin c phn t lng cao khng ln men thnh nhng hpcht ln men c v do nng cao hiu sut ln men ru t cc nguyn liu l tinh bt. Cc ch phm enzyme glucoamylase tch chit t cc t bo vi sinh vt tuy c nhng im ging nhau, song chng cng c mt vi im khc nhau. Ngay c cc ch phm c tch t cc chng ca nm mc Aspergillus cng khc nhau, thm ch ngay c cc chng cng loi cng khc nhau v cc c im v tnh cht l, ho, phn t lng, thnh phn acid amin, tnh c hiu vi c cht v iu kin hot ng. Khi nghin cu cc c ch ca s thy phn mt s oligosaccharide v polysaccharide bng glucoamylase ca nm mc, Backer v cng s thy rng s thy phn tinh bt c thc hin theo c ch a mch ch khng phi theo c ch n mch. S thy phn tinh bt bi glucoamylase:

Glucoseamylase kiu Asp.niger khc nhau

Tinh bt hay oligosaccharide Phn ng cho ra 80 85 % glucose

Glucose

Enzyme glucoamylase l enzyme ngoi bo th hin hot ti a vng pH 3.5 5.5. So vi -amylase th glucoamylase bn vi acid hn, nhng li km bn di tc dng ru etylic, aceton, khng c bo v bng Ca+2 c im chng nm mc Aspergillus niger Aspergillus niger thuc: Gii Lp B H Loi Fungi Fungi imperfecti Moniliales Aspergillaceae Aspergillus niger

Aspergillus l loi hin nay c s dng rng ri trong vic sn xut enzyme v acid hu c. Loi Aspergillus phn b rt nhiu trong t nhin: t, tri cy, xc thc vt, v c bit l chng rt pht trin trong vng kh hu m p. Aspergillus c th bnh hai tng, khun lc trn mi trng Czapek c mu en hoc nu . nh bo t trng thnh c hnh cu. Aspergillus l mt trong nhng loi nm mc c kh nng sinh nhiu enzyme trong qu trnh pht trin ca n nh: enzyme protease, pectinase, cellulase, amylase.tuz theo iu kin nui cy m n c th to ra cc loi enzyme nhiu t khc nhau.

Cng ngh ln men Phng php nui cy b mt Trong phng php nui cy b mt, ngi ta cho vi sinh vt pht trin v bao ph ln b mt cc ht cht dinh dng rn, c lm m, dng lm mi trng(cm go, cm m ). i

vi mt s mc ch c bit, ngi ta nui cy vi sinh vt trc tip trn b mt ca ht go, ht u c hp chn. Ni chung trong tt c mi trng hp, vi sinh vt ly thc n t nhng cht c trong mi trng v s dng oxy phn t ca khng kh h hp. mi trng nui cy nm mc v mt s vi khun bng phng php b mt, thng dng l cm m, cm go v trong ny c cha nhiu cht dinh dng nhng ch yu l to cho mi trng c c cu trc cn thit trong trng thi m. trong iu kin , h nm si ca vi sinh vt s pht trin nhanh chng v ng b, nhng nm mc s khng thu c s nui dng qu di do, do s tn to ra enzim chng s c hon thnh mt cch nhanh chng- sau khong 36-48 gi. tng hot ca mt s enzim, ngi ta cn thm vo cm nhng vt liu c cha nhng cht cm ng cn thit. Mun cho cu trc ca mi trng c tt hn, ngi ta c th thm tru, mn ca, nht l khi dng cm mn lm mi trn. nhng ph liu ny c th l nhng cht cm ng ca xellulaza v hemixelluaza, v a phn nhng cht trong trng thi khng ha tan v t nhiu c hinlin ha. Thng nhng ph liu ny thng lm ngho mi trng i, do nu thm vo nhiu hn 15-20% th s lm gim hot ca cc enzim thy phn trong canh trng b mt. nu trng hp lng cht dinh dng trong cm go b gim i do thm cc ph liu th c th b sung thm ngun nit v c, phospho cng nh cc vt liu giu cht hu c qu nh malt, nc chit ng, dch khoai ty Vic tit trng mi trng c tin hnh trong cc thit b thanh trng vi p sut 1-1,5 atm trong thi gian 45-60 pht. Sau khi lm ngui mi trng bng khng kh lnh, sch hoc bng nc sch xung nhit 38-400C th gieo cy vi sinh vt vo. Qu trnh nui cy nm mc thng ko di t 35-48 gi ty thuc vo thi gian hon thnh vic to ra enzim. mt s loi Aspergillus, s to thnh lng enzim ti a s trng vi s bt u to ra nh bo t, do cho ngng qu trnh pht trin vo lc ny l thch hp. trong mt s trng hp khc, s tch ly enzim cn tip tc c ko di trong c thi gian to ra nh bo t mt cch mnh m. v canh trng nm mc hot ng kiu ny thng c nhiu nh bo t. do khi sy, nghin v bao gi s lm cho khng kh b nhim bi nh bo t ca chng . trnh hin tng ny, tt c cc thit b phi kn v c c cu ht bi. tt hn c l trong sn xut enzim, chn nhng bin chng to nh bo yu. ton b chu trnh pht trin ca nm mc trn cm c th chia thnh ba thi kz sau: Trong 10-14 gi u, xy ra s trng nh bo t v bt u mc nm. trong thi kz ny phng nui cy cn gi nhit khng di 28-300C; khng kim hm s pht trin ban u ca chng.

Qua thi gian trn, h si nm bt u pht trin nhanh. Thi kz ny ko di trong khong 14-18 gi. Nm mc pht trin mnh m, s dng mt lng ln cc cht dinh dng, h hp rt mnh v mt lng ln lng nhit ta ra nhiu. v vy trong lp canh trng nhit tng ln n 37-400C v c th cn cao hn ty thuc vo dy ca lp. do cn phi thi khng kh iu tit c nhit khng thp hn 28-290C v c m tng i cc i vo. Thi kz th 3 ko di trong 10-20 gi. Trong thi kz ny, qu trnh trao i cht xy ra khng mnh m lm v dn dn yu i, lng nhit thot ra b hn, nhng s to thnh enzim c th cn xy ra. Ph thuc vo tnh cht sinh l ca chng v s kt thc sinh tng hp enzim, m c th lm ngng s pht trin ca nm mc vo mt thi gian bt kz. Canh trng b mt ca nm mc sau em nghin nh ( trong my nghin c bit ) m sy kh n m 8-12% s dng. ch phm enzim di dng ny l ch phm th. Mun thu c ch phm enzim sch th em canh trng nghin nh trch ly bng nc.

Cc yu t nh hng n vic to ra enzim khi nui cy bng phng php b mt Tnh cht l ha ca cm, cu trc ca mi trng c mt { ngha nht nh. Chng hn thu ch phm enzim amilaza th hm lng tinh bt trong cm khng c t hn 20-23%. Ngi ta thy raawngfkhi hm lng tinh bt trong cm b gim i th hot ca enzim cng s gim. Hm m ca mi trng cng c { ngha quan trng. thng thng trong iu kin sn xut, hm m ti u ca mi trng cm l 58-60%. Khi nm mc c nui cy trong iu kin tit trng nghim ngt, th hot ca enzim s tt nht khi hm m cao hn 65-68%. V vy vic bo v cho hm m c ti u trong sut qu trnh pht trin ca vi sinh vt s rt quan trng. thc ra ngay c khi thi khng kh bo ha hi nc vo bung nui cy th canh trng vn b mt m v kh. Gim hm m th lng enzim to ra cng b gim i. Do , trong bung nui cy, vic b tr cc ngn, cc gi, phi tnh ton sao cho hp l. Ngoi ra, trong sut thi kz pht trin ca nm mc cn phi thng gi t nhin hoc bng cch thi gin on khng kh vo bung( mi gi 10-15 pht ). Vic tng nhit ln do h hp trong thi kz pht trin mnh m ca nm mc s c nh hng khc nhau n s tn to ca cc enzim. Chng hn s tn to ra -amilaza nm mc Aspergilus oryzae s khng b kim hm ngay c khi nhit trong lp canh trng ln n 40450C v ko di trong 10-12 gi. Ngc li to ra proteaza th khng cho php nhit tng

ln qu 300C. v hot ca proteaza s b gim i hai ln v gim hn na nu khng duy tr c nhit mc ti u.

Phng php thu nhn cc ch phm enzim sch Trong thc t sn xut, cc ch phm enzim c s dng di cc dng khc nhau. Trong mt s lnh vc cng nghip c th dng di dng canh trng sy kh, nu cht lng thnh phm khng b nh hng. chng hn trong sn xut ru, ngi ta c th dng canh trng nm mc ng ha tinh bt m cht lng ca ru vn khng b xu i, ngc li hiu sut ca ru li c tng ln do s ng ha hon ton hn. a s ngnh sn xut thc phm cng nh cng nghip nh th phi i hi s dng ch phm enzim sch, ngha l ch phm trong phn ln tp cht c tch ra. Chng ta u bit khi nm mc pht trin trn go th 30-35% cht kh cha trong b ha tan v b thy phn. Tinh bt, hemixelluloza v pectin b thy phn hon ton, mt s loi nm mc c th ha tan v thy phn xenlluloza. Cc pectin cng b thy phn trong qu trnh pht trin ca nm mc. cc sc t do nm mc to thnh cng ha tan trong nc. nh vy, trong nc chit t canh trng b mt ca nm mc s cha mt lng ln cc tp cht ha tan phn t. phn ln cc enzim thy phn ha tan rt tt trong nc, do c th d dng chit rt ra khi canh trng nm mc. v trong cc t bo gi ca h si, cc qu trnh trao i cht kt thc, tnh thm ca mng tng ln, nn cc enzim chuyn t trong t bo ca cc h si ra mi trng xung quanh hoc mi trng nc mt cch t do. Mc chit rt cc enzim t canh trng b mt ph thuc vo cc phng php khc nhau. qu trnh chit rt c nhanh ngi ta thng nghin nh canh trng nm mc bng my nghin ( trong iu kin th cng c th nghin trong ci vi ct, thch anh, thy tinh). Tuy nhin vic x l ph ny thng lm cho nc chit ra b c v vic tch cc tiu phn l lng ny cng khng phi d dng. Thng th cc enzim thy phn nh proteinaza, pectinaza, xenllulaza, c chit rt ra kh hon ton. Dung mi tt nht chit enzim l nc. tuy nhin trong iu kin sn xut cn trnh pha long nc chit, v s lm gim nng enzim v tn dung mi kt ta sau ny. gii quyt c vic ny, tt hn c l dn phng php ngm nhiu ln, kt qu l chit rt c hon ton cc cht ha tan trong nc , m khng cn pha long. Chng ta u bit do s khc nhau v nng ca enzim ( cht ha tan) trong nm mc v trong mi trng nc m xy ra s khuch tn enzim vo dung dch nc. s khuch tn thng c tin hnh trong mt dy (b) gm nhiu thit b khch tn. Thit b khuch tn

l nhng b hnh tr c y gi v c ni vi nhau bng nhng ng ng nh th no phn di ca b khuch tn ny n thng v phn trn ca b khuch tn k bn. Qu trnh khch tn c tin hnh nh th no c nc (dung mi) i vo t pha b khuch tn cha vt liu nm mc c hm lng enzim (v cc cht ha tan ) t nht, cn dung dch nc giu enzim hn th i ra t b khuch tn c vt liu nm mc mi np. mun vy, ngi ta np vt liu nm mc vo b khuch tn th nht, xong cho nc vo ngm trong mt thi gian nht nh. Sau np vt liu nm mc vo b th hai ri bm vo y dch chit t b khuch tn th nht v ngm trong mt thi gian nht nh. Thao tc np liu vo bm dch chit c tin hnh tun t vi tt c b khuch tn ca dy. Nh vy , khi chuyn t b khuch tn ny sang b khuch tn khc dch chit s c bo ha thm enzim v t b cui cng c bm vo b thu. T b khuch tn th nht, sau khi tho b nm mc c chit rt ht enzim ra, ngi ta ni vo v tr cui cng ca dy khuch tn. V b khuch tn th hai s chim v tr th nht. Trch ly enzim bng phng php khuyn tn hin nay c p dng trong nhiu nh my sn xut cc ch phm enzim sch t canh trng b mt ca nm mc. bng phng php ny trch ly c trn 90-95% enzim. Dch chit thu c khng cha cc to cht khng ha tan v bn thn lp canh trng nm mc cng l vt liu lc gi c cc ht khng ha tan li. Ngi ta thng tin hnh khuch tn enzim bng nc nhit 25-280C. trnh nhim trng, ngi ta thm vo nc mt t formalin hoc cht st trng khc. Dch chit rt c thng trong sut, c mu xanh v cha 10-15% cht kh v tt c enzim ca nm mc. dch chit thu li trong bnh thu v c lm lnh tc khc xung nhit 10-120C. i vi mt s ngnh cng nghip nh thuc da- th ch cn loi ch phm khng cha cc ht khng ha tan (nh bo t, si, cn mi trng), cn cc tp cht ha tan th khng n hng n cht lng ca da.do vi cng nghip da ngi ta thng dn ch phm enzim dng dch c c hoc canh trng b su c c (khng cn tch cc tp cht ha tan). c loi ch phm ny, ngi ta ly dch chit thu c sau khi khuch tn c c li di p sut thp trong chn khng dch c c c hm lng cht kh khng b hn 50-55%. Dch nh vy c kh nng ha tan hon ton, d dng nh lng v c th bo qun trong mt thi gian di m khng mt hot . Trong cng nghip dt, r h cng c th dng ch phm enzim di dng ny. Trong sn xut phomat ngi ta dng dch chit renin c c v tiu chun ha.