Enzym Co Dinh Cua Nga

8/4/2019 Enzym Co Dinh Cua Nga

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Tiu lun ng hc enzyme L Hng Nga

M U

Enzym l cht xc tc sinh hc c bn cht protein, c th ho tan trong

nc v trong dung dch mui long. Enzym c phn t lng ln t 20.000 n

1.000.000 dalton v vy cng nh protein, enzym khng i qua c cc mngbn thm.

Enzym c nhiu tnh cht u vit hn hn cc cht xc tc ho hc. Enzym

c cng lc xc tc rt ln; c tnh c hiu cao nn khng to ra nhng sn

phm ph. Enzym li c th thc hin hot ng xc tc trong iu kin nh

nhng, p sut thng, pH trung tnh, nhit 30C-50C. Enzym c ngun gc

t nhin khng c, c sn xut trn nhng ngun nguyn liu r tin.

Hin nay, Vit Nam s dng rt ph bin enzym trong ngnh cngnghip, song cc ch phm enzym ang s dng phn ln l nhp khu. Do vy,

gim gi thnh s dng, ngi ta nghin cu c th s dng lp li nhiu

ln mt lng enzym, l to ra enzym khng tan (enzym c nh). T nm

1950, vic nghin cu enzym c nh bt u pht trin mnh. n nm 1995,

ngi ta thu c hn 100 cc ch phm enzym c nh[1]. Ngy nay, enzym

c nh ngy cng c s dng rng ri trong cng nghip. V vy, vic nghin

cu ng hc enzym c nh l rt quan trng.ng hc enzym l mn hc nghin cu v tc phn ng xc tc bi

enzym v cc yu t nh hng ln n. Nghin cu ng hc enzym cho php xc

nh c bn cht ca qu trnh phn ng v c ch xy ra phn ng xc tc bi

enzym; thnh phn v cu to ca tm hot ng enzym; vai tr ca mt enzym

no trong mt khu chuyn ho no . Nghin cu ng hc enzym cn c

ngha thc tin l n cho php ch ng iu khin s dng mt ch phm enzym

no trong nghin cu v sn xut.Trong tiu lun ny, chng ta i su nghin cu ng hc ca enzym c

nh, bao gm nhng ni dung chnh:

Phn 1: Tng quan v enzym c nh

Phn 2: ng hc phn ng ca enzym c nh

Phn 3: Lipase t Candida rugosa v mt s phng php c nh

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Tiu lun ng hc enzyme L HngNga

PHN 1: TNG QUAN V ENZYM C NH

1.1. nh ngha:

Enzym c nh (immobilized enzymes) l nhng enzym c gn lncc cht mang khng ho tan hoc cc enzym c lin kt vi nhau bng lin

kt ng ho tr to nn i phn t enzym khng ho tan.

Enzym c nh c nhiu u im hn hn enzym ho tan. Enzym c

nh khng nhng c th s dng lp li nhiu ln vi cng mt lng enzym

m cn c th s dng enzym c nh ln vo trong sn phm m khng lm

nh hng n mu sc, mi v v cht lng thc phm. Enzym c nh c th

tch ra khi phn ng, lm ngng nhanh phn ng. Enzym c nh bn hnenzym t do, do c th kt hp tn dng cc yu t pH v nhit [1]. Mc

d hot ring ca ch phm enzym c nh c b gim xung nhng v n c

th tch ra khi sn phm phn ng dng tr li nn hiu sut s dng

enzym c nh vn ln hn nhiu so vi enzym t do.

1.2. Cc phng php c nh enzym:

1.2.1. Phng php gn enzym bng lin kt ng ho tr

C nh enzym bng lin kt ng ho tr vi cc polyme c hotho l mt trong nhng phng php c nh enzym ph bin nht, m bo

lin kt vng chc ca enzym vi cht mang. Bn cht ca phng php ny l

enzym c gn vi cht mang thng qua cu ni. Cu ni ny c kch thc

khng ln v c hai u, mt u ni vi cht mang polyme, u cn li ni

vi enzym. V d glutaraldehit cng hay c s dng lm cu ni gn

enzym v n cha hai nhm aldehit hai u ca phn t. Hai nhm ny gi

tr pH trung tnh s phn ng vi cc nhm NH2 t do. Nh vy, mt u caglutaraldehit c gn vo cht mang cn u kia s c gn vo enzym[2].

Cht mang thng c s dng l cc polyme khc nhau nh:

celluloza, agaroza, alginic axit, collagen, keratin v cc dn xut ca chng;

hoc cc polyme tng hp nh polyme ca acrylic axit, polyurethan, cc dn

xut N-vinylpyrolidon v cc loi polyme khc[2].

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1.2.2. Phng php gi enzym trong khun gel

Nguyn tc ca phng php ny l gi enzym vo trong khun gel bng

cch trng hp ho gel khi c mt ng thi gel v enzym. Sau khi kt thc qu

trnh trng hp ca gel th enzym c gi trong cc khun gel .

Gel thng s dng l gel polyacrylamit hoc agaroza, Na-alginat. C

nhiu phng php gi enzym vo khun gel: gi di dng ht (vin); gi

enzym trong cc l nh ca gel dng si; gi enzym trong bao vi th

(Microcapsul); phng php tin polyme.

1.2.3.Phng php hp ph vt l trn cc cht mang c cha hoc

khng cha in tch

Qu trnh thc hin hp ph kh n gin: cht hp ph v enzym ctrn ln vi nhau trong mt khong thi gian cho php s hp ph xy ra nh

tng tc ca cc lin kt ion, k nc, hydrogen v lc Van der Wals. Tuy

nhin phng php ny c nhc im l qu trnh gii hp ph enzym d dng

xy ra bi s thay i pH, nhit v thnh phn ion[1].

Nu cht mang khng c l xp, enzym c hp ph trn b mt cht

mang. Nu cht mang c l xp, enzym s c hp ph nh lin kt ion. Cc

cht mang v c thng c s dng l cc loi chitin ca v tm, mai cua,bt san h, than hot tnh, than ci, gch, , si, ct Cht mang hu c

thng s dng l dn xut celluloza.

1.3. Mt s c tnh ca enzym c nh

1.3.1. Enzym c nh c hot ring thp hn hot ring ca enzym

t do. C th do khi gn enzym vo cht mang di nh hng ca in tch

cht mang lm cho hnh th enzym thay i do kh nng xc tc cng b

thay i. Cc enzym c gi trong khun gel, khi mt s c cht c kchthc ln khng tip xc c vi enzym do m hot lc enzym s gim i.

Hoc khi gn cc enzym to i phn t enzym khng tan, s tng tc

protein-protein cng c th lm bin dng mt phn cu trc khng gian ca

phn t enzym v lm hot lc enzym gim i.

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1.3.2. Enzym c nh tun theo ng hc Michaelis-Menten.

Hnh 1.Dng chung ng biu din s ph thuc

ca vn tc theo Michaelis-Menten

Tuy nhin cng c nhng sai khc do c s c mt ca cht mang. Khi

c th xy ra hin tng cnh tranh c cht gia cht mang v dung dch;

hin tng khuch tn cng c tc dng hn ch vn tc ca phn ng. Vi

cht mang v c cht c in tch cng du th hng s i lc ca enzym c

nh (kmkt) > hng s i lc ca enzym t do (kmt). Khi c cht v cht mang tri

du th kmkt


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1.4.1. Trong thc phm

Trong cng ngh thc phm enzym c nh c ng dng rt rng ri

v n c th lm tng cht lng sn phm, em li hiu qu kinh t cao.

Catalaza kh trng sa bng H2O2, xenlulaza sn xut glucoza, pectinaza

lm trong nc qu. Hay lipase ci bin cc triacyglycerol, nng cao cht

lng cc loi du thc vt r tin, cht lng thp bng cch thay th cc axit

bo no bi cc axit bo khng no, trong dung mi him nc. i vi thc

phm c ch bin t sa, lipase c s dng rng ri lm tng mi v,

lm tng chn ca phomat, hoc ch bin nhng sn phm nh phomat v

phn gii lipit.

1.4.2. Trong y hc v cng ngh ho hcTrong y hc, c th s dng enzym c nh trong vic lm cc ni quan

gi hoc s dng di dng Microcapsul a enzym vo trong c th cha

bnh thiu enzym v khng gy phn ng min dch. S dng enzym c nh

s trnh c s ph hu do cc proteaza ca c th gy ra. Streptokinaza,

urokinaza c th lm tan cc cc mu ng gy tc nghn ng mch, vim

tnh mch. Urokinaza gn trong microcapsul c ng dng c hiu qu loi

ure mu trong chy thn nhn to. Vi mong mun c th sn xut mt loithuc lm gin ng mch vnh, ngi ta da vo tnh cht chn lc thu phn

ca lipase trong mi trng dung mi hu c.

Ngi ta c th s dng enzym c nh tng hp cc cht dinh

dng, dc liu nh: axit amin, khng sinh... Sn xut L-axit amin s dng

aminoacylaza c nh trn DEAE-sephadex. Khng sinh axit 6-aminopenicillin

(6-APA) l mt loi khng sinh quan trng thuc h ampicillin c th thu nhn

bng cch thu phn penicillinG hoc penicillinV bng enzym penicillinamidaza c nh.

Enzym c nh c ng dng rng ri trong qu trnh sn xut cc loi

ho cht bo v thc vt (thuc tr su, tr c). Gn y hn, trong lnh vc

ho hc tinh khit, ngi ta dng lipase to cc cht trung gian tng hp

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qua qu trnh thu phn lp th chn lc, ng dng trong sn xut thuc tr su

Pyretinoidic.

PHN 2. NG HC PHN NG CA ENZYM C NH

Enzym c c nh ln mt cht mang s dn n to mt pha rn d

th v trong mi trng phn ng lc ny thng c hai pha, mt pha lng

cha c cht, dung dch m v cc cht hiu ng; mt pha rn cha enzym c

nh thng dng khi, mng, mng mng hoc ht trn. V trong pha rn

ny ca enzym c nh cng d th: cc phn t enzym phn b khng ng

u, b bin dng hoc khng ging nhau v b gi vo cht mang cng khc

nhau. V vy khi nghin cu ng hc ca enzym c nh khc vi ng hcca enzym ng th. Mt mt phi nghin cu s lin quan ti chnh enzym: s

bin i cc tnh cht ni ti ca enzym do s c nh to ra. Mt khc nghin

cu s c mt ca mt pha rn, iu ny s dn n nhng hin tng vt l

nh hng n ng hc ca enzym c nh.

Hnh 2. S bin i cc c tnh ca cc enzym do c nh

2.1. S bin i cc tnh cht ni ti ca enzym

Khi enzym c kt gn vo cht mang, s gy ra nhng thay i tnhcht ng hc ca enzym do c s thay i v hnh th khng gian ca enzym,

cng nh s to ra nhng n ng khng gian to ra s km hm hnh th gia

cht mang v tm hot ng hoc gia cht tc ng v tm ca n.

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Cc phn t enzym c nh s khng ging nhau, chng c th c lin

kt vi cht mang bng mt s lin kt khc nhau (ng ha tr hoc phi ng

ho tr) v chu nhng s bin i v hnh th mc khc nhau. Mt v d

l trypsin dng ho tan c th thu phn 15 lin kt peptid ca pepsinoza

protein, nhng enzym c nh ch thu phn c 10 lin kt. Vic li gn tm

xc tc ph thuc vo hng ca cc phn t enzym so vi cht mang; v tr

ca phn t enzym trn b mt hoc trong cc l ca cht mang. Trong pha rn

thng pha ngoi, mt cc phn t enzym cao hn pha trong.

V nhng l do , cc tham s ng hc ca cc enzym c nh bng

cc phng php khc nhau c th rt khc nhau [7].Mi quan h gia cc thng s c biu th nh sau:

Cc thng s bn cht ca enzym ho tan

Cc thng s bn cht ca enzym c nh

Cc thng s c c bi s khuch tn v phn ct

Cc gi tr (nh l km, vmax ...) khi c xc nh bng thc nghim c

th khc vi cc gi tr thc cht. Cc gi tr ny ch tng ng vi gi tr

chung ca ton b cht mang s dng, n cho php nh lng c cc bin

i ca tnh cht bn trong enzym. iu ny c th do nhng thay i v cc

tnh cht ca dung dch bi s tip cn t ngt ca enzym c nh; hoc do

nh hng ca s khuch tn phn t trong mi trng.

2.2. Cc hin tng vt l lin quan vi s c mt ca pha rnTrong xc tc ng th tt c cc tham s c nh hng n hot ca

enzym ([S], [P], pH, nhit ...) tt c mi im ca dung dch phn ng u

c mt gi tr ng nht. Nhng trong xc tc d th cc tham s c trng cho

hn hp phn ng tt c mi im s khng c cng mt gi tr o. Mt mt,

l do s tng tc gia cc cht phn ng v pha rn; so vi pha rn, cc cht

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phn ng c nng nhiu hoc t rt khc nhau. Ngi ta gi l hin tng

phn bv cc cht phn ng t phn b mt cch khng u gia pha lng v

pha rn. Mt khc, c s khuch tn kh khn ca cc cht phn ng t pha

lng ti enzym do phn ng enzym u c th b gii hn bi nhng lc cn

khuch tn.

Ni chung, trong lng pha lng (mi trng ln) v gn enzym (vi

mi trng) nng c cht s khng ging nhau.

Hnh 3. Mi trng ln v vi mi trng [7]

Trong mi trng d th, s din bin nng khng nhng ph thuc

vo thi gian m cn ph thuc vo c khng gian, c ngha l nng trin

khai thnh cc graien nng .

Graien gin on, nng khng thay i mt cch tun t m thay i

mt cch t ngt do hin tng phn b.

Graien lin tc, nng thay i dn dn v graien s cng ln khi s

bin thin nng cng nhiu do hin tng khuch tn.

2.2.1. Hin tng phn b:

Hin tng phn b l kt qu tng tc ca cc phn t: phn t c

cht, phn t sn phm hoc phn t cht tc ng vi cc nhm ca cht mang

enzym. Cc tng tc c th l tng tc tnh in, tng tc k nc hoc ho

nc. Do chng c th lm tng hoc gim nng ca cc cht phn ng

vi mi trng

mi trngln

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trong cht mang so vi b ngoi. Thng thng l cc tng tc ca cc c

cht, ca cc sn phm hoc ca cc cht tc ng c mang in tch vi mt

cht mang cng mang in tch. Chnh v vy, cht mang l mt mng mang

in tch m th gn cht mang mt cht phn ng tch in dng s c

tp trung vo nhiu hn trong dung dch, trong khi cht phn ng tch in

m li c tch t t hn. Hiu ng phn b ny s c th hin ra bng mt

graien nng gin on.

2.2.2. Hin tng khuch tn:

Cc hin tng vn chuyn vt cht ng vai tr rt quan trng trong

xc tc d th. y vt cht phi c chuyn t pha lng sang mt pha rn

c ngha l phi vt qua mt b mt lin pha, bao gm:Vn chuyn ngoi (hay khuch tn ngoi) t dung dch ti b mt ca

cht mang v ngc li bng khuch tn v bng i lu

Vn chuyn trong (khuch tn trong) vn chuyn vt cht bn trong

mt cht mang c l, bn trong mt gel, bn trong mt mng bng khuch tn.

Hnh 4.Khuch tn trong v khuch tn ngoi

2.3. Cc yu t nh hng n ng hc ca enzym c nh

2.3.1. nh hng ca s phn b cht ho tan n ng hc cc

enzym c nh.Khc vi enzym ho tan, dung dch gn b mt ca mt enzym c

nh thng b nh hng bi c in tch v k nc ca b mt.

*Hng s km ca enzym c nh

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Cc tng tc k nc thng c vai tr quan trng i vi ho tan

tng i ca cc phn t hu c trong cc dung mi nc-hu c. Do cc

tng tc ny m lm cho cc phn t nc sp xp li mt cch c trt t khi

tip cn vi cc b mt k nc. Lc ht gia cc b mt k nc s gim theo

s m vi khong cch gia chng. Cc tng tc k nc lm gim hng s

in mi ca vi mi trng km theo lm thay i cc hng s axit ca cc

nhm axit v kim trong enzym, trong c cht, trong sn phm v trong cc

m, do m nh hng n km ca enzym c nh.

Nu b mt ca cc ht enzym c nh c tnh k nc l ch yu th cc

phn t k nc s phn b vo trong vi mi trng ca enzym cn cc phn t

a nc s b y vo trong dung dch. Trng hp ngc li s xy ra nu bmt c tnh a nc. S phn b s lm thay i nng cc b ca cc phn

t do nh hng n cc hng s ng hc biu kin ca enzym. Chng hn,

enzym alcol-dehydrogenase c nh vi c cht l butanol, s c km = 0,1 mM

nu cht mang l acrylamid, nhng nu cht mang l cht ng trng hp gia

metacrylat v acrylamid c tnh k nc hn th km s gim xung cn

0,025mM.

i lc ca c cht vi cht mang s quy nh gi tr km ca enzym. Mtcht mang thng trn b mt c cha nhiu vng c cc nn s ht c cht

nu c cht cng ho nc, hoc s y c cht nu c cht k nc. bng cch

nh th, khi cht mang v c cht u tch in th trong mi trng cn st vi

enzym s to ra nhng tng tc tnh in ht hoc y v kt qu l thit lp

nn mt graien nng c cht v mt s thay i ca i lc biu kin:

km s tng ln nu cc in tch ca cht mang v c cht l cng du

km s gim nu cc in tch ca cht mang v c cht l tri duS tng lc ion ca mi trng s c xu hng lm cho gi tr km ca

enzym ban u v enzym c nh gn nhau.

Ta thy km ca mt enzym i vi mt c cht c th b gim i r rt

nu nng c cht vng ln cn tm hot ng ca enzym cao hn trong

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dung dch. Bi v mt nng c cht trong dung dch thp hn l cn thit

nhm to cho nng c cht cc b cao hn bn bo ho c enzym.

*H s phn b tnh in (A)

Do tnh cht lng tnh ca cc enzym nn trn b mt ca cc ht

enzym c nh lun c mt cc in tch. S tch in b mt cn c to ra

do khi s dng cc cht trao i ion hoc cc cht mang in tch c nh.

Gia dung dch v vi mi trng lun xy ra s phn b cc phn t tch in

(cc c cht v cc sn phm). Cc phn t ngc du vi cc phn t ca b

mt enzym c nh th c phn b vo trong vi mi trng, cn cc phn t

c in tch cng du vi cc phn t ca b mt enzym c nh th b y ra

vo dung dch. C th nh lng s phn b trong dung dch bng cch avo h s phn b tnh in (A)

Trong : [C0n+] v [A0n-] l nng mi cation v anion trong dung dch

[Cn+] v [An-] l nng cation v anion trong vi mi trng

A nm trong khong t 0.01 n 100

A >1 i vi cc b mt enzym tch in dngA


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trong

kmbiu kin l hng s Michaelis-Menten thng c xc nh bng

thc nghim vi nng dung dch bit.

+Nu c cht tch in m th

v

Nu enzym c nh cha mt s nhm c kh nng to phc cng cua

vi cc cation th s phn b ca cc cation nh th vo vi mi trng s ln

hn so vi s phn b c m t theo h s phn b tnh in A trn. Chng

hn enzym glucoisomelase ho tan cn mt nng cc ion Mg2+ cao hn so

vi nng m enzym c nh i hi.

Mt s tp trung cc nhm ion ho cao cng c th lm phn b cc kh

khi vi mi trng do s c nh hng n cc tham s ng hc biu kin

ca chng. cng l phng php hay dng bo v nhng enzym nhy

cm vi oxy bng cch mui tch tch oxy khi vng ph cn ca enzym.

*S phn b cc ion H+ l mt trng hp quan trng ca s phn b

cht ho tan. pH ca vi trng c th khc ng k vi pH ca dung dch nu

cc ion H+ c phn b vo pha trong hoc ra pha ngoi ca khung enzym c

nh.

Kh nng lin kt vi c cht v hot ca enzym c nh u ph

thuc vo pH ca vi mi trng, trong khi pH o c bng my o pH l

pH ca dung dch.

2.3.2. nh hng ca s khuch tn cht ho tan n ng hc ca

cc enzym c nh

2.3

2.4

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mt enzym c nh xc tc cho mt phn ng th c cht phi c kh

nng khuch tn khp dung dch ti tm hot ng v sn phm phi c kh

nng khuch tn vo trong dung dch.

Cc cht ho tan chuyn dch t ni c nng cao n ni c nng

thp hn. Cc c cht tip cn vi b mt cc ht enzym qua mt lp dung dch

mng bao quanh, sau mi khuch tn vo cc l ht v tip xc vi enzym

y. S chuyn dch ca cc cht ho tan thng xy ra theo hai bc:

+ Khuch tn bn ngoi. y s chuyn dch cc c cht hng ti b

mt, cn cc sn phm to thnh th chuyn dch ra khi b mt . Bc ny

thng xy ra trn b mt v gm mt chui cc chuyn i c cht thnh

sn phm.+ Khuch tn bn trong. y s vn chuyn cc c cht v sn phm

bn trong cc l ca cc ht enzym c nh xy ra song song vi phn ng

c xc tc

*H s hiu qu (effectiveness factor):Vn tc ca mt phn ng c xc tc bi mt enzym c nh thng

thp hn vn tc phn ng c xc tc bi mt enzym t do l do c s kim

tra ca qu trnh khuch tn c cht t dung dch n b mt xc tc. Do c

cht trong vi mi trng tham gia vo phn ng nn nng c cht trong

vi mi trng thp hn nng c cht trong dung dch. Biu hin nh lng

s thay i vn tc phn ng bng cch a vo h s hiu qu

= v/vt do

trong : v l vn tc phn ng c xc tc bi mt enzym c nh

vt do l vn tc phn ng xc tc bi enzym t do

Ni chung, nm gia 0 v 1 v ph thuc vo nng c trong dung

dch v mt s yu t khc. i khi, >1 do qu trnh khng ng nhit, do

cc hiu ng phn b hoc km hm, hoc nu enzym c nh c lm bn

tng ng vi enzym t do trong sut thi gian s dng.

2.5

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*nh hng ca s khuch tn ngoi n vn tc ca mt phn ng

c enzym xc tc nu gi thit rng:

- Phn ng tun theo ng hc Michaelis-Menten

- Enzym c c nh vi mt cht mang tr phng

- Khng c mt cc hiu ng phn b hoc tnh in

Nu phn ng xy ra trong cc iu kin n nh th vn tc tng ca sn

phm trong dung dch phi bng vn tc ca ba qu trnh ni tip: vn tc ca

qu trnh khuch tn c cht ti b mt xc tc, vn tc xc tc ca enzym v

vn tc ca qu trnh khuch tn ca sn phm khi b mt xc tc. Gi thit

trn l hp l nu th tch ca dung dch l ln v khng tnh n s thay

i ca [S0] theo thi gian.Theo phng trnh Michaelis-Menten, ta c th vit:

trong A l tng din tch b mt xc tc

vmax l vn tc ti a ca phn ng c xc tc bi mt n v din

tch b mt.

Nu tha nhn tt c b mt tip xc u c kh nng tip xc bng nhau

th vn tc dng c cht ti b mt tip xc s t l vi c din tch b mt v

vi hiu nng c cht gia dung dch v vi mi trng xung quanh st b

mt. Ta c:

Hng s t l kL c gi l h s chuyn i khi (n v ms -1 ) thngph thuc vo h s khuch tn ca c cht DS (n v m2s-1) v khong cch

hu hiu ( ) gia b mt v dung dch:

2.6

2.7

2.8

2.9

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DS ph thuc vo khi lng phn t v kch thc ca c cht cng nh

vo nhit , nht v thnh phn ca pha lng. Nhn chung, khi lng v

nht ca dung dch cng ln th h s khuch tn cng thp.

B dy hu hiu ca lp ng c th coi nh mt khong cch hu

hiu, mc d khng xc nh chnh xc bng b dy ca lp ng (v khong

cch b hn ngi ta pht hin c s chuyn ng ca cht lng). Trong cc

iu kin c dng ng th bng bn knh ca cc ht hnh cu. Thng

ph thuc vo cc iu kin thu ng lc hc, gim khi tng vn tc

khuy. Vi cc ht xc tc sinh hc nh thng c s dng th c th giam

ti a bng cch lm tng s cun xoy ca dung dch quanh enzym c nh

khong 10 ln.

Bng:H s khuch tn ca cc phn t trong dung dch nc 20C [6]

Cc chtKhi lng phn t

H s khuch tn

(m2 s-1)

Oxygen 32 21.0 x10-10

Glucose 180 6.7 x10-10

Sucrose 342 4.5 x10-10

Inulin 5,200 2.3 x10-10

Albumin 67,000 0.7 x10-10

Urease 480,000 0.3 x10-10

Bushy stunt virus 10,700,000 0.1 x10-10

* Do tnh cht ni tip ca qu trnh, nn vn tc phn ng caenzym (v) phi bng vn tc khuch tn ca c cht ti b mt xc tc

Phng trnh ny l bc 2 i vi nng c cht [S] trong vi mi

trng do rt kh xc nh c gi tr ca phng trnh bng cc cch c

2.10

15


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lp nhau. Phng trnh ny c th c n gin ho vi cc cc tr ca [S]

lin quan ti km ca enzym i vi c cht

+ Nu [S]>> km th (2.10) tr thnh:

y, vA l vn tc phn ng c xc tc bi mt n v din tch b

mt ca enzym c nh. Do m vn tc phn ng c xc tc bi enzym c

nh bng vn tc cc i ca phn ng c xc tc bng enzym t do khi

[S]>>km.

+ Thng th ngi ta nhn thy [S]


17/33


C th so snh bng cch a vo moun ca c cht bn ngoi (

l t s khng th nguyn ca vn tc phn ng/vn tc dch chuyn)

vmax l vn tc cc i ca phn ng c xc tc bng mt n v din

tch b mt.

Th vo (2.14), ta c

+ Khi kL>> vmax/km th vn tc chung ca qu trnh l do xc tc enzym

quyt nh

+ Tuy nhin khi kL


18/33


km tng theo moun c cht bn ngoi, cng c ngha l lm gim tnh

c hiu biu kin.

* Thng thng ngi ta cn phi xc nh cc tham s ng hc ca

mt enzym c nh khi c mt cc hiu ng khuch tn ngoi nhm nghin cu

nh hng ca vic c nh n hot ca enzym. C th xc nh c v max

thc nu to c nng c cht vi mi trng cao. Cn xc nh km

thc s nh vo nng c cht bit vi mi trng. C th xc nh cc

tham s ng hc ny bng phng php th nu bit c h s chuyn

khi.

Nng c cht trong dung dich [S0]

Hnh 5: S minh ho mt phng php xc nh cc tham s ng hc thc(vmax v km) [6]

ng a- gi tr kL c th c xc nh t ng tip tuyn ca ng

cong thc nghim

ng b- vn tc phn ng vi nng c cht trong dung dch

2.20

2.21

18


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ng c- vn tc phn ng vi nng c cht trong vi mi trng

2.3.3. nh hng ca hiu ng khng gian

Cc hin tng cng knh khng gian, to cc n ng khng gian cng

ng mt vai tr rt quan trng trong vic xem xt ng hc ca enzym c

nh. Vic gn kt enzym vo mt cht mang cng thng lm gim s tip

cn ca c cht, c bit khi c cht l cao phn t th s dn n lm gim i

lc biu kin ca enzym vi c cht. Cc hin tng cng knh khng gian i

khi cng dn n nhng im phn ct khc nhau trn c cht cao phn t bi

cng mt enzym trng thi t do v trng thi c nh.

Nhiu dn chng v s hn ch khng gian n hot tnh ca cc enzym

c nh c m t. Chng hn, ribonuclease c c nh trong nhng vinagarose b gim hot 10-20% so vi enzym t do nu c cht l

citidinmonophosphat vng (CMP-vng) v gim i 50-75% nu c cht l mt

ARN c khi lng phn t cao. Hot ca glucoamilase c nh trn CM-

cellulose ch bng 77% hot ca enzym ban u nu c cht l amylose c

khi lng phn t l 8,000 v s gim xung ch bng 15-17% hot ca

enzym ban u nu nh amylose c khi lng trn 500.000Da.

Tuy nhin, c th ci tin hiu qu ca xc tc bng cch cch xaprotein enzym vi cht mang ca n qua mt cnh tay trung gian lm tng

t do ca h thng, do s lm cho enzym c vy quanh bng mi

trng phn ng.

2.3.4. nh hng ca tnh cht dung dch v cht mang.

*Hiu ng phn bc th lm thay i pH ti u biu kin cng nh

cc hng s ng hc ca enzym c nh. pH ti u c th khc nhau ti hai

n v tu theo enzym c nghin cu trng thi t do hay c nh. C haitrng hp xy ra:

+ Khi cht mang tch in m, gn cc ht rn s to ra mt s tp

trung cc proton ca mi trng, pH vi mi trng ca enzym s axit hn ca

19


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dung dch bn ngoi. Mc d trong thc t, pH ti u thc vn khng thay i

v pH o c mi trng ln c mt s chuyn dch v vng baz.

+ Khi cht mang tch in dng, cc ion H+ b y ra nn enzym c

bao bc trong mt mi trng kim hn mi trng trong thit b phn ng v

pH ti u biu kin s chuyn dch v pha axit hn. Chng hn nh enzym

chimotripxin ho tan c pH ti u l 8,4 s gim xung 7,0 sau khi c nh n

trn mt polyme cation (poliornitin) v s tng ln 9,6 sau khi c nh n trn

mt copolyme etylen.

Thc t, s suy lun ny ch ng i vi nhng mi trng c lc ion

yu. Khi lc ion tng ln, cc in tch ca cht mang c th b trung ho bi

cc ion mui tri du nn cc khc bit v pH gia vng ln cn cht mang vdung dch ngoi s t i.

Trong mt s trng hp, pH ti u bng pH ti u ca enzym dng ban

u nhng enzym c nh vn c kh nng lm vic trong mt ph pH rng

hn.

* Tr lc cho s vn chuyn vt cht l do thit lp nn mt graien

nng ca cc loi phn t ho tan trong mi trng gia dung dch bn

ngoi v b mt lin pha rn-lng ca cht mang v dung dch. Tr lc ny sph thuc vo khi lng v nng ca cc cht phn ng, vo nht ca

mi trng v vo cc c tnh ca cht mang (kch thc cc ht, xp... ).

C th lm tng vn tc vn chuyn bng cch tng lu lng cung cp ca

thit b phn ng, bng cch khuy mi trng mnh hn hoc bng cch gim

kch thc ca cc ht... s gp phn lm gim chiu dy ca lp gii hn. S

vn chuyn vt cht bn trong hoc bn ngoi s ph thuc ch yu vo mi

tng quan gia vn tc phn ng enzym v vn tc vn chuyn.- Nu nng c cht b mt lin pha lng rn l b hn hn nng

c cht trong dung dch Sm < SM ta s thy c s tng biu kin km ca enzym.

Trong trng hp ny, vn chuyn vt cht s l giai on gii hn ca phn

ng (ch khuch tn).

20


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- Nu vn tc ca phn ng c xc tc l b hn vn tc vn chuyn

th nng c cht ln cn enzym thc t s bng nng trong dung dch

Sm=SM. Trong trng hp ny, chnh s xc tc ca enzym s l giai on gii

hn (ch ng hc).

2.3.5. Cc enzym c nh thng b nh hng ca cc cht km hm

khc vi enzym ho tan

Cht km hm (I) l nhng cht lm cho enzym t trng thi hot ng

thnh trng thi khng hot ng hoc t trng thi hot ng mnh chuyn

sang trng thi hot ng yu. Vi enzym c nh ta cng c th bit c nh

hng ca cc cht km hm cnh tranh, phi cnh tranh v khng cnh tranh

bng cch xem xt s thay i ca cc hng s ng hc biu kin kmbk vvmaxbk.

Theo (2.16) gi l gi tr moun c cht bn ngoi khi c cht km

hm, ta c:

*Km hm cnh tranh xy ra khi cu to ca c cht v cht km hm

ging nhau hoc gn ging nhau; cht km hm v c cht cng tc ng lnmt v tr ca enzym v enzym khng c tnh c hiu tuyt i.

*Km hm khng cnh tranh xy ra khi c cht v cht km hm khc

nhau; c cht v cht km hm tc dng ln hai v tr khc nhau.

*Km hm phi cnh tranh xy ra khi cht km hm khng gn kt vi

enzym m ch duy nht gn kt c vi phc ES; ch phc ES hot ng;

nng cht km hm rt nh so vi nng ca enzym.

2.22

2.23

21


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Ta thy c hai cht km hm cnh tranh v phi cnh tranh u c igim so vi . Vy l s cn khuch tn c cht hai qu trnh b km hm

ny s t hn so vi enzym c nh khng b km hm. l do cc phn ng

b km hm vn chm hn v t c kh nng c kim sot bi tc

khuch tn c cht. Cn trong trng hp km hm khng cnh tranh th khng

c nh hng no ng k nht l khi nng c cht thp.

* Km hm bi sn phm c th cn nghim trng hn do sn phm

khuch tn khi tm phn ng, do lm cho nng sn phm trong vi mitrng c th cao hn trong dung dch nhiu. giai on u ca phn ng,

nng sn phm s tch li cho n khi to c graien nng i vi

dung dch ln cho php sn phm khuch tn ra ngoi vi tc ngang

bng vi tc to thnh n t phn ng.

Nu gi kLS v kLP l h s chuyn khi c cht v h s chuyn khi sn

phm, [P] v [P0] l nng sn phm trong vi mi trng v nng sn

phm trong dung dch. T phng trnh (2.8) ta c:

Do s kim sot ca khuch tn nn [P] v [S0] ln hn [P0] v [S] rt

nhiu, nn

Ta bit, cht km hm cnh tranh c cu to ging c cht v thng

l sn phm ca mt phn ng. T phng trnh vn tc ca phn ng c kmhm cnh tranh, ta c th vit c phng trnh vn tc ca phn ng c km

hm bi sn phm:

2.24

2.25

2.26

2.27

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Vy, nh hng ca s km hm bi sn phm ph thuc vo nng c

cht trong dung dch v t s kmkLS/(kPkLP) th hin s cnh tranh gia c cht

v sn phm i vi b mt enzym. Vic tch t sn phm b mt enzym s

tng theo t s ny v s lm gim c vn tc phn ng v h s hiu lc.

*Km hm bi c chtl mt qu trnh phc tp hn.

- Nu nng c cht thp va to c mt vn tc phn ng tng

i nhanh khng c s km hm ca c cht, va to c mt vn tc khuch

tn c cht vo bn trong nhanh, u nh mt graien nng cao

- Nu nng c cht rt cao va to c mt vn tc phn ng tng

i chm nh c s km hm bi c cht va to c mt vn tc khuch tn

c cht vo trong chm u do mt graien nng tng i thp.- Mt kh nng th ba l c mt nng trung gian, th hin mt trng

thi khng bn, khng c hnh thnh mt cch t nhin v khng c hiu qu

thc t.

PHN 3. LIPASE TCANDIDA RUGOSA V MT S PHNG

PHP C NH

3.1. i cng v lipase:

* Lipase (EC 3.1.1.3) l enzym xc tc thu phn triglyxerit thnh di,

mono glyxerit hoc glyxerol v cc axit bo nh hot ng trn b mt phn

pha du nc; l enzym linh hot c th s dng xc tc nhiu loi phn ng

khc nhau, c kh nng chu kim, chu nhit

lipase

Triglyxerit + H2O ------> iglyxerit + axit bo* Cng nh cc enzym khc, lipase c th thu t m, c quan ng, thc

vt v vi sinh vt. Nm men Candida rugosa l mt ngun sn xut lipase quan

trng. C t nht 7 ging lipase t Candida rugosa trong c 4 ging c

xc nh c tnh v 3 ging c dng sn xut enzym thng phm[3].

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Hnh 6: Cu trc khng gian ca lipase t Candida rugosa [8]

Mu xanh lc l cc chui xon (quy c biu din hnh xon c) nm

cnh cc cu trc gp np (quy c hnh mi tn). Cc phn kt ni biu din

dng si. Nhng chui xon khng quan trng l mu xm. Chui xon

quan trng l phn np, khi np m th mu , khi np ng l mu vng.

Nhn hot tnh (b ba xc tc) l mu nm chnh gia.

Cc lipase u c nhng tnh cht chung, song chng cng khc nhau v

mt s tnh cht nh: tnh c hiu c cht, mc phn gii c cht, khi

lng phn t, thnh phn axit amin v iu kin hot ng. S khc nhau ny

khng nhng gia cc loi m ngay c gia cc chng trong cng mt loi[5].

* Khi lng phn t ca lipase dao ng kh nhiu gia cc chng vi

sinh vt: Candida rugosa c 2 ng phn l LipA (58kDa) v LipB (62kDa).

3.2. C ch ng hc xc tc ca lipase

Theo cc nghin cu lipase c nhm serin, histidin v axit aspastic

trung tm hot ng ca n. Vi cc c cht khng tan trong nc, hot tnh

ca lipase t c cc i ch khi n c phn tn vo gia b mt phn phadu nc. Qu trnh c gi l qu trnh hot ho phn pha[9]

P

SEis* Es* Es*S

24


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-------------------------------------------------------------------Mt phn pha

Sw

E E* E*Sw Nc

Pw

Hnh 7.M hnh hot ng ca lipase trn c cht ho tan v khng tan trong

nc. M hnh ny do Verger xut (1980). [9]

E: Lipase ho tan khng hot ng

E*: Lipase ho tan dng hot ng

Es*: Lipase hot ng c hp ph

Eis*: Lipase khng hot ng c hp ph

Sw: C cht tan trong nc

S: C cht khng tan trong nc

E*Sw v Es*S: Phc hp lipase- c cht

Khi khng c mt nc hoc khi c nc vi lng nh, phn ng este

ho v phn ng este c u tin. Trong trng hp ny, nhng c tnh nh

tc xc tc v tnh c hiu ph thuc nhiu vo iu kin phn ng v bn

cht c cht [Paul Woolley & Steffen B. Petersen, 1992].

3.3. C nh lipase trn cht mang Nylon

* Nylon l cht nha k thut ni ting l c cng cao v sc

khng cao i vi cht ho hc v du m. Nylon c l hng nh c s dng

lm cht mang cho vic c nh lipase. Nylon c nhit chuyn bin nha

thp v nhit bin dng thp.

H2-N-(CH2)5-CO-[-NH-(CH2)5-CO-]n-NH-(CH2)5-COOH

Nylon

* Nguyn l: C nh lipase bng hp ph vt l. S dng phng php

ny, c th c nh lipase trn 30g Nylon. Trong qu trnh c nh s dng

nng HCl 3,5N x l cht mang 45C trong 15 pht v nng enzym

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l 0,5 mg/ml trong m photphat 0,1M (pH 7,0)[4]. Sau hn hp c

4C qua m. Ri c ra nhiu ln bng nc ct, mi ln 20ml loi

nhng enzym khng c gn kt.

* Mt s kt qu:

Sau khi c nh, hot ca lipase c nh l 120 mol/h/g; trong

khi , vi enzym t do hot l 210 mol/pht/g. R rng hot ng

ca lipase t do l cao hn nhng s dng lipase c nh vn c u im

hn do c th s dng nhiu ln mt lng enzym.

So snh hot ca lipase t do v c nh trn cng mt c cht l du

oliu, theo Michaelis-Menten c c nhng s liu[4]:

Vi enzym c nh: vmax= 58,7 mol/pht/mg

km= 17,4

vmax/km= 3,37.105

Vi enzym t do: vmax/km= 0,97.105

Nh vy, t s vmax/km ca lipase c nh ln hn so vi lipase t do, th

hin kh nng xc tc ca enzym c nh ln hn so vi enzym ho tan.

Lipase t Candida rugosa n nh trong mi trng kim pH t 7-10

v c hot mnh nht pH 8,5. Theo nghin cu ny, lipase c nh trn

Nylon hot ng mnh pH 7,4. iu ny cng ph hp vi l thuyt, lipase

c c nh ln cht mang polyamit tch in dng nn pH ti u chuyn v

vng axit.

3.5. C nh lipase trn chitosan

* Chitosan l chit sut ca chitin (polysacarit v nh hnh, mu trng

hoc khng mu, to thnh lp v ngoi cng ca tm, cua...)

* Qu trnh thc hin hp ph kh n gin: cht hp ph v enzym

c trn ln vi nhau trong mt khong thi gian cho php s hp ph xy ra

nh tng tc ca cc lin kt ion, k nc, hydrogen v lc Van der Wals.

Tuy nhin phng php ny c nhc im l qu trnh gii hp ph enzym d

dng xy ra bi s thay i pH, nhit v thnh phn ion.

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Tien- Chieh Hung v cng s tin hnh c nh lipase t Candida

rugosa trn cht mang v c chitosan, s dng cu ni glutaraldehit trong

20 pht v sau c nh 6ml lipase nng 0,5mg/ml trong 1 gi v

pH=7,0. [10]. y, dng EDC [1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide] hot ho nhm hydroxyl (OH-) ca chitosan qua gn

nhm amino (NH-) ca lipase vo chitosan.

* Kt qu l vmax ca lipase c nh (96,1 U/mg) cao hn vmax ca lipase

t do (92,5U/mg) v km =18,1 cng cao hn km ca lipase t do (1,67).

Lipase sau khi c nh trn chitosan hot ng mnh trong vng pH 5-8

v ti u pH 8, trong khi lipase dng t do li c pH ti u ch l 7. Nh

vy, lc ny pH ti u ca lipase c nh li chuyn v vng baz.

3.6. ng dng ca lipase

Lipase c ng dng rng ri trong cng nghip sn xut cc cht ty

ra, trong cng nghip thc phm, dc phm v cc cng nghip khc.

3.6.1.Trong cng nghip sn xut cc cht ty ra

Enzym dng trong bt git vn l mt th trng ln nht, c ch tm

nhiu nht do khng c nhiu enzym nh vy: enzym c y tnh n nh v

hot lc trong iu kin kim tnh. Hin nay trn th gii c xu hng nghing

v cc bt git c nhit thp (v hay git nhit lnh hn nhit

nng), do phi tm c enzym hot ng c nhit lnh; nhng khi

ty ra cc cht du m th rt kh sch trong iu kin nhit thp cho nn

phi tm c enzym nh vy.

3.6.2. Trong cng nghip thc phm

Trong cng nghip thc phm ngi ta s dng lipase ci bin cc

triacyglycerol, nng cao cht lng cc loi du thc vt r tin, cht lng

thp bng cch thay th cc axit bo no bi cc axit bo khng no, trong dung

mi him nc.

i vi thc phm c ch bin t sa, lipase c s dng rng ri

lm tng mi v, lm tng chn ca phomat, hoc ch bin nhng sn phm

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nh phomat v phn gii lipit [Bech, 1992]. Nhng axit bo t do mch ngn

(ch yu l C4, C6) c thu phn ra do tng lipase vo sa bo dn n s

tng hng v c trng cho sa. Trong khi nu cc axit bo mch trung

bnh (C12, C14) c to ra s c xu hng to ra mi v ta nh x phng cho

sn phm. Thm vo nhng axit bo t do ny c th tham gia vo cc phn

ng ho hc n gin vi vi sinh vt dn n vic tng hp nhng gia v c

mi thm khc nhau. Nhng ngun lipase dng cho vic tng hng v

phomat trn m ca nhng loi ng vt nhai li (d non, cu, b).

3.6.3. Trong cng nghip dc

Trong nhng nm gn y enzym ngy cng c s dng nhiu v rng

ri trong y hc, sn xut mt s thuc v dng trc tip cha bnh. nhiu nc trn th gii, c bit Nht Bn hng nm ngi ta sn xut

mt lng ln ch phm enzym vi sinh vt c tinh khit cao trong c mt

ca lipase.

Cc lipase c th xc tc este ho, chuyn i este ho v este ho tng

tc trong cc dung mi hu c vi lng nc thp [Kazlaus kas v

Bornscheuer, 1998; Godberg v cng s, 1989], ng dng chnh ca cc lipase

trong ho hu c l s phn gii cc hn hp enantiomeric. C mt vi nhmphn t lin quan n s truyn tnh trng du hiu lm gia tng nhng cn

bnh d ng, vim l nhng lipit hay lipit aqnalogues. S truyn tn hiu gia

v trong ni bo sinh vt lin quan gn gi vi nhng loi lipit n l nh

phosphatidylinositol, lipase cng nh photpholipase u c th c s dng

tng hp trn phm vi ln.

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KT LUN

Ngy nay ngi ta thng s dng enzym c nh trong cc ngnh cng

nghip v n c nhiu u im hn hn enzym ho tan. Mc d hot ring

ca ch phm enzym c nh c b gim xung nhng v n c th tch ra khi

sn phm phn ng dng tr li nn hiu sut s dng enzym c nh vn

ln hn nhiu so vi enzym t do.

Ngi ta c nh enzym ln cc cht mang c th bng nhiu phng

php khc nhau: phng php gn enzym bng lin kt ng ho tr; phng

php gi enzym trong khun gel hoc bng phng php hp ph vt l trn

cc cht mang.

V vy khi nghin cu ng hc ca enzym c nh ngi ta nghin

cu s c mt ca hai pha l pha lng v pha rn. Mt mt nghin cu s lin

quan ti chnh enzym: s bin i cc tnh cht ni ti ca enzym do s c nh

to ra. Mt khc nghin cu s c mt ca pha rn, iu ny s dn n nhng

hin tng vt l nh hng n ng hc ca enzym c nh. l:

* nh hng ca s phn b cht ho tan

* nh hng ca s khuch tn cht ho tan

* nh hng ca hiu ng khng gian

* nh hng ca tnh cht dung dch v cht mang

* nh hng ca cc cht km hm

T vic bit c cc nh hng m ngi ta bit c s thay i

cc tham s (nh vmax, km...) t c nhng ng dng thc t ph hp s

dng c enzym c nh mt cch c hiu qu nht.

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TI LIU THAM KHO

TI LIU TING VIT

1. ng Th Thu (ch bin) v cng s Cng ngh enzym. NXB Khoa

hc v K thut, H Ni.

2. L Ngc T (ch bin) v cng s Ho sinh cng nghip.2000. NXB

Khoa hc v K thut, H Ni.

TI LIU TING ANH

3. Antonio sanchez, Pau ferrer, Alicia serrano. Characterization of the

lipase and esterase mutiple from in an enzyme preparation from a

Candida rugosa piot-plant scale fed-batch fermentation. Enzyme and

Microbial Technology 1999, 25, 214-223.

4. Benita Braun and Elias Klein. Immobilization of Candida rugosa

Lipase to Nylon Fibers Using Its Carbonhydrate Groups as the Chemical

Link. Biotachnology and Bioengineering 1996, 51, 327-341.

5. Didier Rotticci, Torbjorn Norin, Karl Hult, Mats martinelle. An

active-site titration method for lipases. Biochimica et Biophysica Acta

2000, 1483, 132-140.

6. Matin Chaplin Effects of solute diffusion on the kinetics of

immobilised enzymes London South Bank University

7. Matin Chaplin Kinetics of immobilised enzymes London South Bank

University8. Miroslaw Cygler, Joseph D. Schrag. Structure and conformational

flexibility of Candida rugosa lipase. Biochimica et Biophysica Acta

1999, 1441, 205-214.

9. Paul Woolley & Steffen B. Petersen. Lipase- their structure,

biochemistry and application- Cambridge University.

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10. Tien- Chieh Hung et al. Binary immobilization of Candida rugosa

lipase on chitosan. Journal of Molecular catalysis B: Enzymatic 2003,

26, 69-78.

11. http://www.lsbu.ac.uk/biology/enztech/index

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MC LC

M U

PHN 1: TNG QUAN V ENZYM C NH

1.1. nh ngha:

1.2. Cc phng php c nh enzym:

1.2.1. Phng php gn enzym bng lin kt ng ho tr

1.2.2. Phng php gi enzym trong khun gel

1.2.3.Phng php hp ph vt l trn cc cht mang c cha hoc

khng cha in tch

1.3. Mt s c tnh ca enzym c nh

1.4. ng dng ca enzym c nh

PHN 2. NG HC PHN NG CA ENZYM C NH

2.1. S bin i cc tnh cht ni ti ca enzym

2.2. Cc hin tng vt l lin quan vi s c mt ca pha rn

2.2.1. Hin tng phn b

2.2.2. Hin tng khuch tn

2.3. Cc yu t nh hng n ng hc ca enzym c nh

2.3.1. nh hng ca s phn b cht ho tan n ng hc cc

enzym c nh.

2.3.2. nh hng ca s khuch tn cht ho tan n ng hc ca

cc enzym c nh

2.3.3. nh hng ca hiu ng khng gian

2.3.4. nh hng ca tnh cht dung dch v cht mang.2.3.5. Cc enzym c nh thng b nh hng ca cc cht km

hm khc vi enzym ho tan

PHN 3. LIPASE TCANDIDA RUGOSA V MT S PHNG

PHP C NH

3.1. i cng v lipase

1

3

3

3

3

4

4

4

6

8

8

9

10

11

11

11

14

20

21

22

25

25

32


33/33


3.2. C ch ng hc xc tc ca lipase

3.3. C nh lipase trn cht mang Nylon

3.5. C nh lipase trn chitosan

3.6. ng dng ca lipase

KT LUN

TI LIU THAM KHO

26

27

28

29

31

32

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