Mn: CNG

NGH ENZYME

Enzyme Papain & Enzyme glucose oxydase

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Thng 3 nm 2012

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GII THIU..................................................................................................................3 ENZYME PAPAIN.......................................................................................................... 4 Tng quan enzyme papain....................................................................................4 1.1. Tnh cht vt l..............................................................................................4 1.2. Tnh cht ha hc..........................................................................................5 ng dng enzyme papain lm trong bia...........................................................10 3. u v nhc ca enzyme papain.......................................................................12 ENZYME GLUCOSE OXYDASE.....................................................................................13 Tng quan enzyme Glucose oxydase.....................................................................13 1.1 Tnh cht ha hc.............................................................................................13 1.1.1 Cu to ha hc v cu trc khng gian....................................................13 1.1.2 Cu trc trung tm hot ng ca Glucose oxydase.................................14 1.1.3 Phn ng ca enzyme Glucose oxydase....................................................15 1.1.4 Cc yu t nh hng................................................................................16 ng dng glucose oxydase trong thc phm v ung......................................16 KT LUN..................................................................................................................18 TI LIU THAM KHO.................................................................................................19

GII THIU Cng ngh enzyme hin nay pht trin rt rng ri v ng dng trong nhiu lnh vc quan trng trong c cng ngh thc phm, mt ngnh khng th tch ri vi enzyme. Nhn dn ta t xa bit dng thm nu canh tht b, dng u hm xng m thc cht l s dng papain, bromelain Ngoi kh nng phn gii lm mn tht, to thc n d tiu ha, cng ngh sn xut cc loi dch thy phn giu protein c p dng mt cch c hiu qu tnh nng ca protease. Khi ni ti protease ngi ta ngh ti kh nng phn gii protein ng dng nhiu trong sn xut thc n nhng t ai ngh ti ng dng trong ung v c bit l enzyme papain c trong nha u li c tc dng lm trong bia. V khi ni ti enzyme dng trong nc gii kht ngi ta li ngh n enzyme peptinase. Th nhng, enzyme dng trong Cng nhip thc ung li rt nhiu trong k enzyme glucose oxydase dng trong loi b oxy trong sn phm. Bi bo co ca nhm tp trung trnh by v lm r hai enzyme papain v glucose oxydase.

ENZYME PAPAIN Tng quan enzyme papain Papain (EC 3.4.22.3) l cysteine protease c bit n nhiu nht v c phn lp ln u tin vo nm 1879 t nha tri u . y cng l enzyme u tin c xc nh cu trc tinh th (Drenth et al., 1968; Kamphuis et al., 1894). Trong nha u ngoi enzyme papain cn c cc loi protease khc nh: chymopapain, caricain, glycyl endopeptidase v mt s enzyme khc (Baines and Brock-lehurst, 1979). Nha u c hm lng v hot tnh papain cao nht tp trung vng c nng nng v m n nh quanh nm. 1.1. Tnh cht vt l Bng 1.1: Tnh cht vt l ca papain Tnh cht vt l im ng in Hng s sa lng S20 Hng s phn tn D20 (10 giy.cm ) Phn t lng trin quang [ ]20D xon Vng hiu ng cotton Th tch ring phn V(mL/g) Tr s ma st f/fo 17% 290nm 0,724 1,16
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Gi tr pI = 8.75 2,42 0,04 10,27 0,13 20,700 66,7

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Bt mu vng hay mu nu nht, ty thuc phng php sy, khng tan trong hu ht cc cht hu c nhng tan mt phn trong H2O hay glycerine. Bn nhit.

1.2. Tnh cht ha hc 1.2.1 Cu to ha hc Theo kt qu phn tch bng tia X, papain l mt endoprotease cha 15,5% N v 1,2% S. Phn t papain c cu to bi 212 acid amin trong khng c cha methionine. Phn t lng khong 23,350 Da, phn t l mt mch polypeptide vi u N l Isoleucine, u C l Asparagine, c 6 gc Cysteine to thnh 3 cu disulfur cc v tr 22-63, 56-95, 153-200 khng c chc nng sinh hc, ch lm tng tnh bn vng ca cu trc v mt nhm SH t do v tr 25. 1.2.2 Cu trc khng gian o Phn t papain c dng hnh cu vi kch thc 36x48x36A v mch chnh b gp thnh hai phn ring bit bi mt khe. Trung tm hot ng nm ti b mt ca khe ny, nhm -SH hot ng ca cysteine 25 nm bn tri khe v nhm histidine 159 nm bn phi khe. Phn xon chim 20% ton b cc amino acid c trong phn t.

Hnh 1.1: Cu trc bc 3 ca papain Hot tnh ca papain da trn hai tm hot ng l Cys25 v His159.
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1.2.3 Cu trc trung tm hot ng ca papain Trung tm hot ng ca papain gm c nhm SH ca cysteine 25 v nitrogen bc 3 ca histidine 159. Bn cnh nhm imidazole ca His 159 cng lin kt vi Asp 175 bi lin kt hydrogen. Khu vc trung tm ca phn t l nhn k nc. Ngoi ra trong phn t cn cha mt s cu ni ni phn t c to thnh bi cc nhm guanidine ca gc arginine v nhm carboxyl. Mt cu ni phn b bn trong c bn cu ni phn b pha ngoi phn t. Vng tm hot ng ca papain cha mch polypeptide vi cc amino acid l: Lys-Asp-Glu-Gly-Ser-Cys-Gly-Ser-Cys. Cc nghin cu ca Lowe, chui polypeptid trong trung tm hot ng ca papain gn ging nh ca ficin hay trypsin, mc d chng c ngun gc khc nhau. Ficin: Arg-Glu-Glu-Gly-Glu-Cys-Gly-Ser-Cys. Trypsin: Lys-Asp-Ser-Cys-Glu-Gly-Gly-Asp-Ser.

Hnh 1.2: Cu trc tm hot ng ca papain

1.2.4 Phn ng ca papain Papain thy phn protein thnh cc polypeptide v cc acid amin, n ng vai tr va nh endopeptidase va nh exopeptidase. So vi cc protein c ngun gc ng vt v vi sinh khc, papain c kh nng thy phn su hn, v vy n c dng phn gii tip cc lin kt peptit cn li sau khi thy phn bng tripsine hoc chymotrypsine. Tnh c hiu ca papain rt rng, n c kh nng thy phn hu ht cc lin kt peptit tr cc lin kt vi prolin v axit glutamic c nhm COOH t do. Papain chu c nhit tng i cao. dng nha kh papain khng b bin tnh trong 3h/1000C. Cn dng dung dch b mt hot tnh sau 30 pht / 82,50C. c bit papain c tinh sch v dng tinh th th n c bn nheietj km hn papain dng m nha doa trong m nha cn cha cc protein khc c tc dng bo v n.

Hnh 1.3: C ch phn ng ca enzyme papain.

1.2.5 Cc yu t nh hng n hot tnh xc tc ca papain

- Nhit : Papain l enzyme chu c nhit tng i cao. dng nha

o kh papain khng b bin tnh trong 3 gi 100 C. Cn dng dung dch papain b o o mt hot tnh sau 30 pht 82.5 C v nu nhit tng cao hn (>100 C) th n s b mt hon ton hot tnh k c khi thm lng ln cht hot ha vo dung o dch. iu ny l do dng dung dch khi tng ln n nhit ln hn 100 C th cu trc tm hot ng ca papain b ph hy hon ton. iu ng lu l sau khi c tinh sch v trng thi tinh th th papain c bn nhit thp hn papain trong nha, do trong nha cn cha cc protein khc c tc dng bo v papain. Papain trong dung dch NaCl gi 4oC bn trong nhiu thng. Trong dung dch dn xut thy ngn, papain cng khng mt hot tnh trong nhiu thng. Trong khi hu ht cc enzyme mt hot tnh mi ngy 1 2% do s phn hy hoc oxy ha. Khi thy phn cc protein khc nhau, th ty thuc vo c cht m nhit thch hp cho papain cng khc nhau chng hn i vi c cht l casein th nhit o ti u cho phn ng l 37 C. Papain dng n nh trng thi kh c th chu o nhit sy 115 C trong thi gian 2 gi m hot tnh vn duy tr c 90%.
- pH: Papain hot ng trong khong pH tng i rng t 4.5-8.5 nhng li

d bin tnh trong mi trng acid c pH < 4.5 hoc trong mi trng kim mnh c pH > 12. Khi c cht l casein th hot tnh ti u ca papain trong vng pH t 5.7 o 7.0 v nhit thch hp l 50 57 C.
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Khi phn ng vi c cht th ty thuc vo bn cht ca c cht m pH ti u s khc nhau. Chng hn, papain phn ng vi casein pH ti u l 7-7.5. Papain dng n nh tc l dng m cu trc khng gian ca enzyme c n nh, c th chu c cc pH = 1.5 v pH = 8.5 trong 90 pht.
- Cht hot ha: Papain thng c hot ha bi cc cht kh nh: cystein,

glutanion, axit thioglycolic, Natri thisolfat ( Na2S2O3) cht hot ha b bin nht l cyanide. Papain b kim hm mnh m bi cc thuc th ca thiol nhng li c hot ho c bit bi 2 mercaptoetanol.
- Cht kim hm: Papain b km hm bi cc cht oxy ha nh: oxy, ozon,

hydro peroxyde, ido axetate, ido axetamit, thy ohaan clobenzoat, cystine v cc hp cht disuflua khc. c bit papain d b mt hot tnh khi c mt hydroperoxy.
- Papain rt bn i vi cc tc nhn bin tnh nh: dung mi hu c vn hay

c dng trong ha hc protein ( quay cc ca papain hu nh khng bin i trong etanol 70% hoc urea 6 8 M/l) Ngi ta thng dng papain bin hnh cc protein u tng bng phn ng plastin cng nh lm mm tht. ng dng enzyme papain lm trong bia Bia l sn thc ung c a chung nhng trong qu trnh sn xut bia vn b c do cc nguyn nhn sau: - Nhim vi sinh vt nhng mi nguy ny c loi b. - Mi nguy ha hc l vn nghim trng. S to thnh cc kt ta l lng dng sng m (chill haze) do cc nguyn nhn: + S kt hp protein v tanin trong bia c lm lnh n sduwois 100C. + Polyme ha cc phn t Tanin t hoa hublon, gm cc acid caffeic, acid gallic. Leucoanthocyanin to thnh cc chui polyme hot ng. + Phn ng polyme hot ng vi cc phn t polypeptit. Enzyme protease c dng ngn nga hin tng ny v cc polypeptit b thy phn bi cc enzyme protease, khng cn nguyn liu kt hp vi tanin. Cc
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enzyme ny gm papaine, pepsine, ficin, bromeline, protease vi khun v phaari c hot ha pH acid bnh thng ca bia. Enzyme papain c ng dng rng ri nht.

Hnh 1.4: Cu trc peptid

Hnh 1.5: Cu trc tanin

Hnh 1.6: Lin kt gia tanin v peptid

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Enzyme papain ct ngay v tr OH ca tanin lin kt v tr COOH ca peptit bng lin kt hydro lm chng khng lin kt c vi nhau. Chng khng th ti hp li c v mun ti hp li cn c lin kt hydro nhng lin kt hydro b ct nn kh nng ti hp khng xy ra. 3. u v nhc ca enzyme papain - u im: r, d tm thy trong t nhin, nng sut - Nhc im: nh hng ti bt bia v papain l enzyme phn gii protein trong khi bt bia c cu to lp mng ngoi cng l protein bao ly cc cht kh bn trong cho nn nu s dng enzyme papain nhiu th nh hng lm v bt bia, nh hng hng ti cht lng bia. khc phc nhc im ca papain S dng acid proline-specific endoprotease t nm Aspergillus niger & Foochemistry. Brewers Clarex l tn thng mi ca acid proline-specific endoprotease. S d enzyme Brewers Clarex thay th enzyme papain l v Brewers Clarex khng thy phn protein m ch ct lin kt gia polyphenol vi cc amino acid y polyphenol ra khi vng lin kt v acid proline thay th v tr ca polyphenol to thnh chui acid amino. Chui acid amino gi l haze active protein, polyphenol b loi ra vng lin kt khng cn to ra dng hall chill v haze active protein khng lm c bia.
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Hnh 1.7: Haze active protein

theo nghin cu ca Mchel Lopez & Luppo Edens t tp ch khoa hc Argicultural

ENZYME GLUCOSE OXYDASE Tng quan enzyme Glucose oxydase Glucose oxydase (GOD, -D-glucose: oxygen, 1-oxydoreductase, EC 1.1.3.4) Glucose oxydase c tit ra bi nm mc, ch yu l Aspergillus niger hay Penicillium amagaskinense. N c phn b gia dch ngoi bo, vch t bo, v trong cht dch nhy ca nm mc. 1.1 Tnh cht ha hc 1.1.1 Cu to ha hc v cu trc khng gian L mt phn t oxy ha kh, vi trng lng phn t 192.000 Dalton. L mt protein lng phn hnh thnh t 2 tiu n v ging nhau. Mi tiu n v hoc n phn, cun vo trong 2 domain: mt domain gn vi c cht -D-glucose, trong khi domain khc lin kt khng ng ha tr vi mt nhn t ph flavin adenine dinucleotide (FAD), m n s dng nh mt tc nhn oxy ha mnh.

Hnh 2.1: Cu trc khng gian Glucose oxydase

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Hnh 2.2: Flavin adenine dinucleotide (FAD)

1.1.2 Cu trc trung tm hot ng ca Glucose oxydase V tr hot ng ca glucose oxydase cha 3 amino acid quan trng lin quan n s xc tc: His 516, v Glu 412 m lin kt hydro vi His 559.

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Hnh 2.3: Cu to trung tm hot ng ca Glucose oxydase

1.1.3 Phn ng ca enzyme Glucose oxydase FAD hot ng nh mt cht nhn in t m lm cho n b kh thnh FADH2; FADH2 sau b oxy ha bi cht nhn in t cui cng, phn t oxy, oxy s b kh thnh hydrogen peroxyde (H2O2). N cng l enzyme xc tc qu trnh oxy ha -D-glucose thnh acid gluconic vi s tham gia ca phn t oxy nh cht nhn in t, ng thi gii phng ra hydro peroxyde (H 2O2). Nhm ngoi ca enzyme ny l FAD. Cc sn phm ca phn ng ny l gluconic acid v hydrogen.

- Glucose oxydase c th oxy ha -D-glucose s dng cc cht oxy ha khc

bn cnh phn t oxy, bao gm cc quinine v cc cht nhn 1 electron. D-glucono1,5-lactone sau t ng thy phn to ra gluconic acid. C6H12O6 + H2O + 1/2O2 C6H12O7 + H2O2 Glucose
- E: Glucose oxydase

gluconic acid

Hnh 2.4: Phn ng to gluconic acid v D-glucono-1,5 lactone

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1.1.4 Cc yu t nh hng
pH v nhit : phn ng ti u iu kin nhit gia 30 n 50 oC ti

pH gia 4,5 n 6,5. Glucose oxydase chuyn i glucose thnh acid gluconic, do lm gim pH.

Hnh 2.5: nh hng pH

Cht hot ha: Qu trnh tng hp glucose oxydase c th c kch thch bi cc

cht khc nhau, bao gm phn t oxy, m n kch thch s dch m enzyme.. Glucose Oxydase bn thn n khng hot ng, nhng vi s hin din ca nhng c cht c trng, enzyme c s dng oxy ha trc tip phn t glucose hoc oxygen. ng dng glucose oxydase trong thc phm v ung
- Kh oxy trong nc ng chai
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Glucose oxydase c th c s dng loi b oxy u chai nc ung trc khi chng c ng chai. Ngoi ra glucose oxydase loi b oxy trong nc p tri cy, thc ung ng hp.
- Gim cn ca ru: vi sinh vt s dng oxy tip tc ln men lm tng

cn trong sn phm. Glucose oxydase c kh nng s dng trong cng nghip lm ru, m n lm gim cn trong ru thng qua vic loi b mt s glucose (chuyn n thnh dng D-glucono-1,5-lactonea nu khng n s chuyn thnh cn). Hn na glucose oxydase c th ngn chn s h hng ru thng qua tc ng dit vi khun ca n ln vi khun acetic acid v vi khun lacitc acid trong sut qu trnh ln men. Enzyme ny s c b sung thm vo ru tng hiu qu dit vi sinh vt. Glucose oxydase s dng oxy c ch ngc li sng ca vi sinh vt ln men nn cn s gim.
- Ko di thi gian s dng sn phm: Glucose oxydase c s dng thnh

cng ct cu ni glucose v oxy trong thc phm v thc ung ko di thi gian bo qun cho thc phm v ung. Hydrogen peroxyde c sn sinh ra t enzyme ny hot ng nh mt cht dit vi khun rt tt.
- Chng oxy ha sn phm

E Glucose O2 Gluconic acid

Khi phn ng xy ra nng O2 gim, do gim qu trnh oxi ha kh lm h hng sn phm. Gluconic acid Glucose oxidase cng c s dng nh l mt ngun thng mi ca gluconic acid, m c th c sn sinh bi s thy phn D-glucono-1,5-lactone, sn phm cui ca s xc tc glucose oxidase. Gluconic acid c s dng
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trong ph gia thc phm hot ng nh mt cht iu ha acid, dung dch v trng hoc cht lm trng trong sn xut thc phm.

KT LUN Ngoi tc dng lm trong bia ca enzyme papain ra th nhng tc dng nh bin hnh cc protein u tng bng phn ng plastein cng nh lm mm tht. Enzyme papain cn c ng dng trong lnh vc dc phm, ha cht
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Nhng tc dng ca papain cn ang c ng dng rt nhiu khng ring g cng ngh thc phm. Enzyme glucose oxydase l enzyme oxy ha kh ng dng rng ri ngoi thc phm ra cn c ng dng rng ri nh Ngn cn phn ng maillard gia glucose v amino acid nh loi b glucose t lng trng trc khi chng c sy kh dng trong cng nghip lm bnh. Chng r mt trong cc bao b kim loi. Trong y dc s dng glucose oxydase trong phng php o hm lng glucose trong mu. Trong dc phm dng lm m phm ty trng da C hai enzyme papain v glucose oxydase ang c ng dng trong nhiu lnh vc v c nhiu nghin cu v hai enzyme ny.

TI LIU THAM KHO 1. 2.

3.

Chim Lm Phc Dim, lun vn thc s khoa hc trng i hc Ng Th Ty Na, bi ging Cng ngh enzyme trng i hc Nng http://www.rcsb.org/pdb/101/motm.do?momID=77.
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Khoa hc t nhin. Lm tp. HCM.

4.

http://en.wikipedia.org/wiki/Glucose_oxidase. http://www.m-biotech.com/fig2.html.

5.

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