tieu luan protease

8/3/2019 tieu luan protease

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Ngi lm:

Nguyn ThMinh Thyo ThSen


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Ngy nay cng vi s pht trin ca cng ngh sinhhc cc ch phm enzim c sn xut ra cng nhiutrn nhiu lnh vc nh:nng nghip,chn nui ,thc

phm ,v vic s dng chng cngc phbin nhiu nc v mang li li ch v kinh t kh

ln.Hng nm lng enzim c sn xut 300.000 tn c gitr 500USD

Trc y enzim dng ngin cu hoc p dng trongsn xut thng thu nhn ng vt thc vt tuy

nhin gn y ngun thu enzim phong ph v rtin hn l ngun thu enzim t vi sinh vt.

Protease c dng kh nhiu trong cn sx nh chbin thc phm nh(ng t sa lm pho mt,lm

mm th

t ..),ch

t t

y r

a,y t

,nng nghi

p


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Ccphngphp tch v tinhch

tch v tinh chenzyme ni ringv protein ni chung thng c mtlotphng php ha-l v ha hckhc nhau. C thchia lm ba nhmphng php sau:

- Phng php ktta

- Phng php sc k- Phng php phn tch h hai phanc


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ENZIM PROTEASE

Enzim xc tc qutrnh thy phn linkt peptit trong phnt protein,polipeptitn sn phm cuicng l cc axitamin.

Nhiu proteaze cngc kh nng thyphn lin kt este vvn chuyn axit amin.


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NGUN THUPROTEASE

Ngunngvt: Tytng trispsin:lngun enzim smnht

lu i nht ,v cnhiu enzim nht .

D dy b :renin bnicazein thnh paracazec khnngkttatrong mi trngsa cCa.nunh trongd day b bnhimpepxin th khnngngtcnggimi.

Ch :ngny c nghincusnxut protease t

vi sinh vt c c tnh reninnh loi eudothiaparasitica v mucorpurillus.


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NGUN THUPROTEASE

Ngunthcvt: Promelain:thu tqu

,chida ,vda.

Papain:thu tnhaca thn ,quu(carica papaya)

Pincin:thu tnhacy c


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NGUN THUPROTEASE

Ngun vi sinh vt:

Vi khun :lng proteazacsnxut 59% lngenzim sdng bacillussubtilis,B.mesentericus

protease ca vi khun ctnh chiurt cao,vchng hotngtt pHtrung tnh v kimyu.

Nm :aspergillusoryza,A.saitoic khnngngtsasdng trong cng nghsnxut phomat.

Xkhun :streptomycesgrieus,chng c ctnh chiurng ,c kh

nngthy phn lin ktpeptido thnh aminoacid.


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Protease thuc lp th 4 ca phn lp th 3

Peptidase(protease)

Exopeptidase

Aminop

eptidas

e

Carbo

xypep

tidase

Endopeptidase

Serine

prinna

seote

Cystei

n

protei

nase

Aspart

ic

protei

nase

Matall

o

protei

nase

Sphnloiproteaze


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Enzyme protease ni bo l nhngenzyme c tit ra t bn ngoi hoc

ngoi bin mng protein v c trch lyvo mi trng bng k thut trch ly

Enzyme protease ngoi bo c thunhn t qu trnh ln men, nh qu trnh

ln men trn mi trng rn.


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Phng php gy t bin

To nhng t bin b gim khnng sinh tng hprepretsor

To nhng t bin tng hp enzim c cu trc bc 1

Gy t bin hot ha promotor lm tng p lc ca

n vi ARN lm tng tc sao chp m .


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Phng php bin np :l s bin i tnh trng ditruyn ca 1 ni vi sinh vt di nh hng caAND trong dnh chit nhn nhn c t t bo cavi sinh vt khc.

Phng php tip hp gene:vt liu di truyn ctruyn t t bo cho n t bo nhn v th vi sinhvt c kh naeng bin hp s khng c khnngtip hp gene na.

Phng php ti np :trong qu trnh ti np ccon AND c truyn t t bo cho n tb tiphp vi AND ca tb nhn do lm bin i tnhcht di truyn c tb nhn .


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* Phng php boqun

ging vi sinh vtPhng php cychuyn.* y l phng php phbinnhtdthchinbng

cch giging trn mi trngthch (thch nghing, hppetri,) vi thnh phn mi trng nui cy v iukin nuicy thch hp cho ging vi sinh vt. Sau khi gingmcttcnboqun nhitlnh 3-40C v sau mitunphicychuynli. Khi cychuynchly bo thockhunlc mkhng nn lyc mi trng dinh dngmbo khngchuyn cc snphm trao icht vo mi trngmi (c thgy binibtli khng thlnghtc). Nu l xkhunth khng nn boqunging trn mi trngthch m nn gitrong tkh trng.

* ko di thi gian boqungingt hng thng n

1 nm, ngi ta ph 1 lp paraphin lngtit trng trn bmtginghnchs pht trinca n. Cnlu chphlpdu sau khi cy vi sinh vttn chn sinh l.

* Phng php cychuynrt c hiuquboqun ccgingnm men, vi khun v rthuhiu, d dng trin khaiging ra snxutln, hnch cc tai bin c thdnnhhngginggc.


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Phng php lm kh.

* Bng cch gi ging trn ct, t, silicagen trongiu kin kh ro (tt c u c kh trng cnthn). Trong iu kin nh vy s hn ch s phttrin tip tc ca ging khi bo qun. Phg php nyrt hay c s dng bo qun nm mc, x

khun, mt vi loi nm men, vi khun thi gian giuging c th c 1 nm.

*Phng php lm kh cng thc hin n gin, khngcn dng c t tin. Tuy nhin ging nh phngphp cy chuyn thi gian bo qun tng i ngn.


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Phng php ng kh:*ng kh l qu trnh m nc

c ly ra khi mu khi cc muang trng thi lnh su. yvi sinh vt c huyn ph trongmi trng thch hp v c lmlnh trong mi trng chn khng.Thit bng kh s ht nc vcui cng mu c lm kh n

mc nht nh. Mu c hn kn cho mi trng cha mu lchn khng. y l phng phpph bin c hiu qu cao cho boqun cc i tng vi sinh vtkhc nhau nh nm si, nm men,vi khun v mt s virut. Tuynhin, phng php ny t cng dng i vi to, ng vtnguyn sinh v tbo ng vt.


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Phng php bo qun lnh su:

* i vi phng php bo qun lnh su th visinh vt c bo qun trong mi trng dch thv nc cn cho hot ng sng ca vi sinh vt bbt hot nhit lnh su (-196C -> -80 C).

*Phng php bo qun ny c hiu qu vi nhiu nhm visinh vt khc nhau nh nm si, nm men, vi khun, xkhun v virut

*kinh ph cho thit b v in, nit lng hoc ri ro nh chy

n... c bit phng php ny khng thch hp vi ccchng vi sinh vt thng xuyn dng n. Ni chung phngphp ny thng c dng vi cc chng vi sinh vt cnhng c tnh qu m khng thch hp vi phng phpng kh.


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Cc phn t enzyme khng c kh nng i qua mng ca t bo v mngca cc cu t ca t bo. Do c th chit rt cc enzyme ni bo,

bc u tin l phi ph v cu trc ca cc t bo c cha enzyme vchuyn chng vo dung dch.

Ph v cu trc ca t bo cha enzim:

Nghin nh, nghin vi ct , nghin vi vn thy tinh, nghin bi t bo t phn hy

Dng tc ng ca siu m hoc p sut thm thu ,trch li bng mui,dung dch mui trung tnh ,dung mi hu c.

Kt ta enzim bng cc cht in li thch hp. Sau khi ph v t bo chuyn chng vo dung

dch.


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Phng php ktta phn onbng (NH4)2 SO4da trn css

khc nhau vkhnngkttaca cc protein enzyme mtnng

mui (tnh theophn % nng bo ha) xc nhc dngphbin

loibbcu protein tpca cc dch enzyme. Cc loimui c

th c dng l (NH4)2 SO4, Na2 SO4, MgSO4 ... ngi ta nhn

thymui (NH4)2 SO4 l ttnht v n khng lm hi m lm nnh

(lm bn)huht cc loi enzyme. Loimui ny lir v phbin.

ha tan ca n lirtln (bo ha 767g/l 250C).


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S cc bc trong quy trnh i lc


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Buc 1: Chn mi trng i lc

Chn mi trng c sn (ligand lin kt sn vi matrix). Nu khng c th phi

chn ligand thch hp to mi trng i lc c hiu.

n nh mi trung i lc trong dung dch m lin kt (binding buffer).

Buc 2: Chun bmi trng v dung dch m

Cc dung dch v cht bo qun cn phi ra trc khi s dng. Nc v cc ho

cht s dng phi c cht lng cao. Dung dch phi c lc qua li lc 0.45

m hoc 0.22 m.

Vic ti s dng mi trng i lc cn phi ph thuc vo bn cht ca mu. Lu

trnh nhng cht bo.

Bc 3: Chun b mu v chy mu

Chun b mu: Mu cn c lm sch trc khi chy sc k. Nu c th nn kim

tra i lc ca ligand vi protease cn tch. Sau , hiu chnh pH mu v loi b

cc thnh phn gy gin on lin kt.

Bc 4: Ra gii

Bc 5: n nh li mi trng i lc trongbinding buffer.


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Da vo s phn b khc nhau ca hn hp trong dung dch hai pha

khng ha tan vo nhau. Cc protein v mnh v t bo c kh nngha tan khc nhau gia hai pha, v vy phng php ny c th c

dng cho c hai trng hp: phn tch protein khi mnh v t bo vphn chia cc enzyme trong sut qu trnh tinh sch protein.

H hai pha nc c xem l phng php tt cho vic phn tch vtinh sch cc hn hp, v phn tch cht lng c mt khc nhau ddng hn phn tch cc cht rn ra khi cc cht lng.

So vi cc phng php tinh sch khc th h hai pha nc c mt su im hn nh: c ha tan trong nc ca hai pha ln (70-80%),t tinh sch cao, hiu sut cao, d dng s dng quy m ln vc bit polymer c ti s dng.

Phng php tch h 2 pha nc trong tchv tinh chprotease


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PEG

PPB

Trn u Sau khi

phn tch

tp chtPEG: polyethylene glycolPPB: potassium phosphate buffer

Protease v tp cht


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Thu nhn enzyme thtccnymm.

Malt l loiht ho thonymm trong nhngiukin nhn to(nhit,

m,thi gian) xc nhgi l qui tc malt.

Qu trnh snxut malt bao gm cc khu sau:

Thu nhn,x l, lm sch, phn loi vboqunht.

Ra, st trng v ngm ht.

mmm ta s thu c malt ti.Sy malt ti.

X l vboqun malt kh.

Hot tnh proteaza trong ht ban u khng ngknhng khi nymmtng

ln 48 ln, tng nhanh hnhot tinh amylaza v tcci vo khong ngy ny

mmth 5. Sthu phn proteinbtubng tc dngca proteinaza to thnhalbumoza, polypeptit, pepton v sau di tc dngca peptidaza to thnh cc axit

amin. Tuy nhin sbini ny thng khng hon ton v cc iukinnymm

thng khngphi l iukinti thch cho hotngcah enzyme proteaza ca

ht.


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Thu nhn enzim tthcvt.

Hda (Bromalaceae)

Bao gmttc cc ni datrnglyqu,lysi(kc cc ni dadi).

Cc nghin cuch ra rng cc nguyn liuly kh nhit 4000C sgic

hot tnh enzyme tthn so vi nguyn liucboqunlnhnhit 40C.

Nguyn tc chung thu nhn Bromelain.

Phliuda lm dp chitlc ly tm ktta ly tm lc

chphmkthutsc k trao i ion sythng hoa snphm tinh khit.

Thu nhn Bromelain bngphng php nhanh sdng CMC CMC c tmtbng dung dchm photphat 0,05M, pH= 6,1. Cho dchchi

da vo, thnhthongkhuytrn. Enzyme ny lnbmtca CMC. Sau 2 gily

ra vtncloibcnbn bm vo CMC, ra tri cc protein khngphi enzyme

bngm photphat pH= 6,5. Sau chophnhpthln 1. Dung dchphnhp

th l m photphat pH= 7,1, NaCl 0,5 N khuytrn. Sau 2 gily ra vtc dung

dchmccha Bromelain. Tip tcphnhp thnh ln th 2 rigp chung

dchchitcac hai lnktta enzymebng axeton hay cn lnh.


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Nhau (Carica Papaya. L)

T qu ti hoc thn thu c nha (latex) chnh l ch phm papain th

t tinh ch thnh papain thng phm. Papain c M= 20.700, Top=

800C, pHop= 55,5. B c ch v mt hot tnh bi H2O2, Iodoaxetat, I2,

fericianua. c hot ho bi CN, cystein, H2S v glutation.

Thu nhn papain th: Dng cc loiquu cn non, u gi (cha

chn), dng khn lau schv,ly dao coschnhngng khng qu su,

hngnha vo ccri lm kh. Ktqu cho thyhot tnh papain sau khi

chit tch cao hn khi 3 thng.

Thu nhn papain thngphm: Ngm papain th ha tan nha ti

(catex) trong ncct cb sung glyxerin tng ha tan, lc qua vi

mn. Kttabng axeton lnhvitl 2:1 so vith tch dchlc. Ly tm

lnhlyktta,sy 45- 500C, emnghin thnhbt


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Trong cng ngh ch bintht:protease lm mm tht vtng hng v cho tht bngcch ngm tht vo dung dchprotease thik hp,tm hnhp lm mmtht(enzim,mui ,m chnh)

u imcavicthuphn protease bi enzymel bo ton c vitaminca nguyn liu, khng to

ra cc snphmph,khng lm sm mu dchthu phn.


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Trong ch bin thy sn:trong ch bin nc mms dng enzim protease thc vt(promelanin

,papain) v vi sinh vt rt ngn thi gian lm vci thin hng v ca nc mm.

Tuy nhin vn cn mt s tn ti cn phi honthin thm v cng ngh.


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Trong cng nghip sa :sn xut phomatnh hot tnh lm ng t sa.vd:A.candidus,P.roquerti,B.mensentericus.


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Trong cng nghip sn xut bnh m ,bnh quy:proteaselm gim thi gian o trn tng do v lm nhuynbt,to xp v ntt hn.


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Trong snxut bia :chphm protease lm tngbncabia v rt ngnthi gian lc,proteaseca A.orirae dng thyphn protease trong h ttt ngctoiukins l bia tt

hn.


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Trong cng nghipda:protease cs dng lmmm da loi b dakhi cc cht nhtnh thy phn mt

phn protein chyu l collagenthnh phn lm dadng.Proteasec dng tch

vi khun(B.mensertericus,B.subtilis),nm mc v

x khun.


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Trong cng nghip dt: Proteinase vi sinh vtc s dng lm sch t tm, ty t nhn to

(cc si nhn to c bng cc dung dchcazein, gelatin) si c c bng, dnhum. Protease c tc dng thy phn lpprotein serisin lm dnh bt cc si t tnhin, lm bong v tch ri cc loi t tm, do lm gim lng ho cht ty trng


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iuchdchmthy phn dng lm chtdinh dng, chttng v trong thcphm vsnxutmtsthcn king. Protease canmmc v vi khunphihp

vi amylase to thnh hnhp enzyme dnglm thcn gia sc c tiu ha cao, c

nghalntrong chn nui gia sc v gia cm.


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Snxut keo ngvt, chtgittnghpgit tycc chtbn protein, snxutmphm,

iuch mi trng dinh dngca vi sinh vtsnxut vaccine, khng sinh,


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tieu luan protease

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