Cng ngh Enzym Protein

i cng
- Xc tc sinh hc
- Enzym c nh
-

i cng
Cng ngh enzym: ng dng enzym hoc xc tc sinh
hc to ra sn phm hay dch v
ng dng ca enzym

Cng nghip:

Thc phm: Lm bnh m, ru bia, cc sn phm sa

Ty ra: protease, lipase
X l tinh bt: cc amylase, isomerase, CGTase
Dt-da: amylase, lipase, cellulase

i cng

ng dng ca enzym

Y Dc

Tr tiu ha: amylase, protease

Chn on: Alcol dehydrogenase, Cholesterol esterase,
Creatinase
iu tr: Asparaginase, Uricase, Streptokinase
Ha dc: Ibuprofen, Salbutamol

Nghin cu: Endonuclease, RNase, DNase, Polymerase,

Ligase

Nhu cu s dng enzym

Ch tiu

Cng
nghip

Phn tch

Dc phm

Lng s
dng

Tn

Milligam
gam

Milligam
gam

tinh
khit

Khng tinh
khit

Tinh th tinh
khit

Tinh th tinh
khit

Ngun
gc

Vi sinh vt,
Vi sinh vt,
thng ngoi ng vt, thc
bo
vt, thng
ni bo

Vi sinh vt,
ng vt, thc
vt, thng
ni bo

Gi sn
xut

Thp

Cao

Trung bnh

Khi nim xc tc sinh hc

Enzym l cht xc tc ca cc qu trnh sinh hc

Bn cht l protein

Gip phn ng t c im cn bng nhanh hn

Gip phn ng xy ra nhanh hn trong iu kin bnh

thng v p sut, nhit , pH

Enzym khng th xc tc P vi s thay i nng

lng t do khng thun li tr khi P song hnh
vi mt P c s thay i nng lng t do thun li

Cng ngh enzym hin i

Ha hc protein
L sinh phn t
Sinh hc phn t
Cu trc, hot ng ca protein-enzym
Can thip thay i phn t protein-enzym cng
ngh protein (protein engineering)

Xc tc sinh hc

Tnh chn lc cao

Chn lc theo v tr nhm ha hc

Chn lc khng gian

Hot ng trn c cht a dng

Hot ng c trong mi trng khng phi l nc
Kh nng m nhn lng c cht cao
C bn cao
Tnh kinh t

Tnh chn lc theo v tr

O
N
HO

O N

CH3

N
N

NH2

OH
HO
Dn xut Purine (Nelarabine)

vinyl acetat
C. antarctica
lipase

CH3

5' O N
O

CH3
N

OH
HO
Sn phm acyl ha

NH2

Tnh chn lc khng gian

Lipase t Candida rugosa

(S)-naproxen

COOH
COOCH3
H3CO
(R,S)-naproxen methyl ester

Lipase t Aspergillus niger

lipase

H3CO

H 2O

COOCH3
H3CO
(R)-naproxen methyl ester

Xc tc sinh hc

Tnh chn lc cao

Chn lc theo v tr nhm ha hc

Chn lc khng gian

Hot ng trn c cht a dng

Hot ng c trong mi trng khng phi l nc
Kh nng m nhn lng c cht cao
C bn cao
Tnh kinh t

Hot ng trn c cht a dng

Hot ng trn c cht a dng

Xc tc sinh hc

Tnh chn lc cao

Chn lc theo v tr nhm ha hc

Chn lc khng gian

Hot ng trn c cht a dng

Hot ng c trong mi trng khng phi l nc
Kh nng m nhn lng c cht cao
C bn cao
Tnh kinh t

Sn xut enzym - Ngun nguyn liu

Chit tch

ng vt
Thc vt

Ln men

Vi sinh vt
Nui cy t bo
Cng ngh gen

Sn xut enzym - Ngun nguyn liu

Chit tch t m: lysozym, papain, bromelin,
Ln men b mt: amylase, protease nm mc
Ln men chm: cc enzym dng trong cng nghip,
phn tch v y hc

Qui m ln: enzym th dng trong cng nghip

Qui m nh: enzym dng trong y hc
Chng c chn lc thng qua bin i di truyn

Tng hot tnh

Tnh n nh
Nhit , pH
Chuyn gen sn xut enzym vo cc k ch d nui cy

Phng php sn xut - Tng qut

Chun b nguyn liu
Chit tch
C c
Tinh ch
Hon chnh sn phm

Loi b m tp
Ph v t bo
Ly tm, lng
Ta bng
Lc
Bay hi
Ta
Siu lc
Kt tinh trc tip
Sc k
Hp ph

C c
Kt tinh
ng kh
n nh

Chun b nguyn liu sinh hc

C quan ng vt

Loi cht bo v m lin kt trc khi ng lnh

Bo qun lnh n khi mu x l

Nghin mu v enzym c chit vi dung dch m, hoc

dng enzym ph v t bo

Nguyn liu thc vt

Thc vt c th c nghin v chit vi dung dch m.

T bo cng c th c ph v bng enzym.

Vi sinh vt

Ngoi bo: tch t bo ra khi dung dch ln men.

Ni bo: ph v t bo

Ph v t bo

Cc thnh phn ca thnh t bo:

Peptidoglucan, phospholipid, lipoprotein, liposaccharid

Sp xp cc thnh phn ca thnh t bo

VK Gram + c lp peptidoglucan dy hn VK Gram

T bo nm men c lp polyphosphoryl mannan v
glucan dy
Periplasm
Cell membrane
Cell membrane
Cell wall

Peptidoglycan
Lipopolysaccharides +
proteins

Cc phng php ph v t bo

p sut cao (Manton - Gaulin, French-press)

Nghin (my nghin bi)
Siu m
Lm kh (ng kh, dung mi hu c)
Ly gii:
Vt l: ng lnh, sc thm thu
Ha hc: cht ty, khng sinh
Enzym: lysozyme

Ph v t bo p sut cao

French-press

p dng: quy m nh
C ch: S thay i p sut t ngt
p sut: 10.000 50.000 psi

Manton - Gaulin

Ph v t bo - Nghin
Nghin bng ci chy
Nghin bi:

Bi c kch thc 0,1 6 mm (0,2 0,7 mm)

C ch: l do s va chm ca bi vi t bo
p dng: vi khun, nm, thc vt, ng vt
Quy m nh: Mt vi kg TB nm men/gi
Quy m ln: Hng trm kg/gi.

Rolling
beads

Cells being
disrupted

Ph v t bo Siu m
p dng: thng dng trong PTN
C ch: cc bt kh c to ra di tc dng ca
sng siu m
Tn s: >18 KHz, thng thng 20-50 KHz.

Ultrasound
generator
Ultrasound tip

Cell suspension

Ph v t bo PP khc

Lm kh (ng kh, dung mi hu c)

Ly gii:
Vt l: ng lnh, sc thm thu
Ha hc: cht ty, khng sinh
Enzym: lysozym

Ph v t bo So snh cc PP
Phng php

Stress

Chi ph

p dng

p lc cao

Ln

Va

Quy m ln

Nghin

Va

Thp

Nh v ln

Siu m

Ln

Ln

Quy m nh

Sc thm thu

Nh

Thp

T bo mu

Enzym

Nh

Ln

Quy m nh

Cht ty ra

Nh

Va

E. coli

Sn xut enzym - Chit tch

Tch dch enzym ra khi

cc phn khc

Lng
Ly tm lin tc
Lc

truyn thng
lc p sut, lc chn khng
lc chy qua (cross-flow
filtration).

Phng php kt bng

C c

Bay hi

C ch: nhit thp bay hi

B mt bay hi, khuy trn

Kt ta
Siu lc (MWCO)

C c Kt ta

Mui:

Nng mui cao tc ng ln cc phn t nc bao quanh

protein, thay i lc tnh in

Dung mi hu c: gim hng s lng cc ca MT nc

Polymer
im ng in

C c Siu lc

10.000 n 1.000.000 MWCO

Tinh ch
Kt tinh
in di
Sc k

Loi sc k

Nguyn l

Tch theo

Hp ph

Lin kt b mt

i lc b mt

Phn b

Cn bng phn b

Tnh phn cc

Trao i ion

Lin kt ion

in tch

Lc gel

Khuch tn l

Kch thc v hnh dng phn t

i lc

Hp ph c hiu

Cu trc phn t

K nc

Tng tc k nc

Cu trc phn t

ng ha tr

Lin kt ng ha tr Tnh phn cc

nh bt ion kim
loi

S thnh lp phc

Cu trc phn t

Tinh ch Sc k

Nguyn l Sc k

Chn Phng php sc k

GF = Lc gel
AC = Sc k i lc
IEX = Sc k Trao i Ion
HIC = Sc k Tng tc k nc
RPC = Sc k Pha o

V d tinh ch amylase bng hp ph

Dch Enzym th
Natri alginat

Phc Enzym Alginat natri

Calcium chlorid
Lc ly ta
Dch tp cht

Ta Enzym Alginat Calci

Phn hp ph
Lc ly dch

Alginat Calci

Dch Enzym tinh

C c
Thm tch

Sn phm amylase

V d sn xut Taq polymerase

Thermus aquaticus
hay E.coli ti t hp
Mi trng

Ct DEAE
Ln men

Mi trng tha

Ct Hydroxyapatide
Thu t bo

Xc t bo

Ct DEAE
Ph t bo

Dch ni

Ct phosphocellulose
Ta (NH4)2SO4
Taq polymerase tinh khit
Thm phn

Enzym c nh
L enzym c nh v trong khng gian xc nh
Lch s pht trin

Giai on u (19161950),
Giai on pht trin (1960-1980),
Giai on thit k hp l (1990nay)

n nh trong dung mi hu c
Phng php tinh th enzym lin kt cho (cross-linked
enzyme crystals - CLEC)
Kt hp cc cht mang vi nhau.

Enzym c nh

u im

Enzym c th c s dng lp li nhiu ln

Ch phm bn hn enzym t do
Enzym c nh c tc phn ng ln, d t chc sn
xut mc t ng ha cao.
Nh s c nh m enzym khng ln vo sn phm
cui
Enzym c nh bo qun tt hn enzym t do cng loi

Nhc im

Gim hot tnh ca enzym so vi ban u.

Cn tr v khng gian do lin kt vi cht mang lm
hn ch s tip xc gia enzym v c cht.

Enzym c nh

Vt liu c nh

Cht mang phi r tin, d tm hoc d tng hp

Cht mang phi c tnh c l n nh
Cht mang phi bn vng v mt ha hc, khng ha
tan trong mi trng phn ng.
Cht mang phi c din tch b mt ln
C kh nng trng n trong mi trng

Cc phng php c nh enzym

Phng php lin kt vi cht mang
c tnh

Hp ph
vt l

Lin kt
ion

Lin kt
ng ha tr

Phng php khc

Lin kt
cho

Bt gi

K thut

Kh

Kh

Kh

Hot tnh enzym

Thp

Cao

Cao

Trung bnh Cao

Tnh c hiu c
cht

Khng i Khng i Thay i

Thay i

Khng i

Lc lin kt

Yu

Trung
bnh

Mnh

Mnh

Mnh

Kh nng ti c
nh

C th

C th

Khng

Khng

Khng

Kh nng ng dng

Thp

Trung
bnh

Trung bnh

Thp

Cao

Chi ph c nh

Thp

Thp

Cao

Trung bnh Thp

C nh thun nghch
E
Hp ph

Me- E
Lin kt ion kim
loi hoc chelate

E
E

Me- E

+- E
Lin kt ion

+- E
+- E
E

Lin kt i lc

Me- E

E
E

Lin kt disulfit

C nh khng thun nghch

E
Lin kt ng ha tr

To vi ht bao

Bt gi

E
E
E

E
E

E
E

Lin kt cho
E

E
E
E

ng dng enzym trong ngnh dc

Liu php enzym
Enzym trong sn xut thuc

Tr liu bng enzym

Thay th enzym do bnh di truyn

Thay th enzym do mt bnh mc phi

Cung cp tc dng sinh hc c hiu

Dng trong iu tr h tr khi phi hp vi

cc liu php khc, hay gii c mu hay
m.

Liu php enzym - Yu cu ca enzym

Phi n c v tr tc ng ca chng trong c th hay m.

Phi c hot tnh iu kin mi trng ti ni tc ng.

bn c c cc thng s dc ng hc cn thit

tan tha mn yu cu nu c dng theo ng tim bp

hay di da.

tinh khit cao

C hiu qu iu tr da trn hot tnh c hiu ca enzym

c dng. Hiu qu ca liu php phi c chng minh

An ton i vi bnh v ch nh iu tr

Dng s dng thun tin

Cc vn ca thuc protein

Khng n nh
Sinh kh dng
Tnh thm t bo
Tnh cht gy min dch
Phn ng d ng
Sn xut v kim nh
Li th

t tc dng ph hn
Thi gian pht trin ngn
T l thnh cng khi xin cp php cao

Thch thc

Ci thin tnh n nh

PEG, lin kt cho

ng in, a nc,
t hot ng
Bao bo v
Ci tin phn t

Thch thc

Gim tnh min dch

nh hng c hiu

Kh nng xuyn mng t bo

Ngun enzym

Chit xut - Tinh ch:

Khng phi ngi: nm, vi khun, ng vt, thc vt

Ngi: yu t ng mu, urokinase

Gy min dch
Dng ngoi
Gii hn cng sut, nguyn liu hn ch
Nhim virus v cc yu t gy bnh khc

Ti t hp

S dng gen ngi ti t hp trong t bo khc

Ci thin hot tnh, c im thng qua ci tin trnh t

H thng sn xut

Nui cy t bo ngi ng vt

Nm men

Cht lng tt
Nng sut gii hn
Nhim virus
Thc hin c mt s bin i hu dch m

Vi khun

Nng sut cao

Khng thc hin c bin i hu dch m

Sinh vt chuyn gen

Liu php gen

Sn xut thuc bng

cng ngh enzym

Thuc i quang

Cu trc khng gian ca phn t sinh hc v th th

Ch mt trong hai ng phn c hot tnh,

Cc ng phn c hot tnh khc nhau
Dc ng hc v chuyn ha khc nhau

Thuc tinh khit quang hc (enantiopure) - racemic

Bn quyn ng phn

Thuc i quang
Tc dng dc l chnh

Khc bit gia cc ng phn

Thuc chn :
propranolol, acebutolol, atenolol, alprenolol,
betaxolol, carvedilol, meto-prolol, labetalol,
pindolol, sotalol,

l > d (d = khng c hot tnh)

V d: S(-)-propranolol > R(+)-propranolol

Thuc chn knh calci:

verapamil, nicardipine, nimodipine, nisoldipine,
felodipine, mandipine

l>d
V d: S(-)-verapamil > R(+)-verapamil

Thuc gin ph qun:

albuterol (salbutamol), salmeterol v terbutaline

l > d (d = khng c hot tnh)

V d: R(-)-albuterol > S(+)- albuterol

An thn:
hexobarbital, secobarbital, mephobarbital,
pentobarbital, thiopental, thiohexital

l>d
V d: S(-)-secobarbital > R-(+)secobarbital

Thuc gy m:
ketamine, isoflurane

d > l (l = khng c hot tnh)

V d: S(+)-ketamine > R(-)-ketamine
S(+)-isoflurane > R(-)-isoflurane

Gim au tc ng trung ng: Methadone

V d: R(-)-methadone > S(+)-methadone

Khng vim khng steroid:

ibuprofen, ketoprofen, benoxaprophen, fenprofen

d>l
V d: S(+)-ibuprofen > R(-)-ibuprofen

An thn: nhm 3-hydroxy-benzodiazepines:

oxazepam, lorazepam, temazepam

d > l (l = khng c hot tnh)

V d: S(+)-oxazepam > R(-)- oxazepam

Thuc i quang - racemic

T nhin/
bn tng hp: 147

Khng bt i: 2
Bt i: 145

Thuc: 668
Bt i: 252
Tng hp: 521
Khng bt i: 269

Ch phm dng
racemic: 8
Ch phm mt
ng phn: 119
Ch phm mt
ng phn: 110
Ch phm dng
racemic: 140

Cc loi phn ng c xc tc bi enzym

Lp enzym theo IUBMB

Phn ng xc tc

Oxi ha/kh i vi CHOH, C=O, C=C,

EC 1 Oxidoreductases
Khng i hi ng c cht
EC 2 Transferases
Khng i hi ng yu t

Chuyn cc nhm chc nh halogen,

aldehyde, keto, acyl, glycosyl,

EC 3 Hydrolases
Khng i hi ng yu t

Thy phn/ngng t cc ester, glycosid, nitril,

amid, halogen,

EC 4 Lyases
Khng i hi ng yu t

Thm/loi b; ct cc lin kt CC, CO, CN

EC 5 Isomerases
Khng i hi ng yu t

ng phn ha, chuyn dng cis-trans, epime

ha

EC 6 Ligases
Cn ng c cht ATP

To cc lin kt CO, CS, CN, CC

Mt s vn cng ngh

Chi ph c nh phi b p c bi s gia tng tnh n nh

hoc hot tnh.
Gim thiu s mt hot tnh trong qu trnh c nh.
Khi enzym c c nh bng phng php c tnh thun
nghch, s r r enzym phi c nghin cu v c bin php
thch hp.
S gii hn v lun chuyn vt cht trong cht mang cn c
tnh ton khi iu chnh pH hay nhit trong qu trnh phn
ng.
Vi enzym t do, hot tnh chuyn ha c th t c cao
hn nng enzym cao, do vic chuyn ha cc c cht
kh c th thc hin c.
Tnh v trng ca phn ng cn c quan tm.
S tng thch ca dung mi vi cht mang v nh hng ca
dung mi n hot tnh cn c xem xt.

La chn ni phn ng
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A: vn hnh theo l; B: vn hnh theo l c tun hon; C: thng khuy c siu lc;
D: thng khuy vn hnh lin tc; E: thng nhi lin tc; F: tng si lin tc

Oxidoreductase
Ph

HN
N Ph

N
5'
HO
HO

O N
OH

Cl

Ph

HN
Nucleoside Oxidase

H2O, pH 6, 25 oC, 12-24h HO

HO

N Ph

N
O N

Cl

OH

Nucleoside oxidase ly t Stenotrophomonas maltophilia

(FERM BP-2252)
Sn xut cc dn xut 5-carboxylic acid ca cc
nucleoside ng ng.
Enzym th t dch chit t bo hoc c nh ln Eupergit-C

Lipase Transferase
F

O
F

OH

OAc

OH

Lipase
37oC, 20h

+
O

N N
N N

N N
N N (S)-acetat

racemic
F

Thuc h cholesterol

N N
N N

O
F

Lipase
H2O

N N
N N (R)
F

OH

OH

COOH
OH

O
F

N N
N N (S)

Lipase - Hydrolase
O

OH

O
Lipase

H2O, 25oC, 4h

(3R,4S)-phenol

racemic cis-hexanoate
O

Thuc nga thai tc dng ko di (1 vin/tun)

(-)-Ormeloxifene

Lipase
CH3 O
OH
CH3 O
OH
(R,S)-ibuprofen

Novozym 435
1-Dodecanol

(S)-(+)-ibuprofen
+

CH3 O
OC12H25

(R)-(-)-ibuprofen ester
Thuc khng vim khng steroid

Amidase
O

O
NH

rac-lactam

-lactamase
H2O, 70oC

O-

NH

NH3+

+
(+)-lactam

(-)-acid amin

OH
N
HO

NH
N

Thuc khng HIV

Carbovir

NH2

ng ng Carbovir

Deaminase
Cytidine Deaminase

N
O

HO
S

NH2

NH2

NH

H2O, pH 7, 32oC, 35-70h

HO
S

HO

S
Lamivudine

Thuc khng virus (HBV, HIV)

Lyase
CN T bo R. rhodochrous J1
c nh
N

H2O, pH 8, 25oC, 18h

CONH2
N
Nicotinamide

Vitamin B3