Enzymes in Biotechnology

ThÞ tr­êng enzyme c«ng nghiÖp

th­¬ng m¹i hµng n¨m: 1.6 tû $
 ChÕ biÕn thùc phÈm vµ tinh bét: 45%
 ChÊt tÈy röa: 34%
 DÖt: 11%
 Da: 3%
 GiÊy vµ bét giÊy: 1.2%
ThÞ tr­êng thÕ giíi cho mét sè s¶n phÈm
cña c¸c ph¶n øng enzyme

Sir« ng« cao fructose: 1 tû $

Aspartame: 850 triÖu $
Acrylamide: 300 triÖu $
S¶n xuÊt sir« ng« cao fructose tõ tinh bét

Corn Starch Slurry (30-35% DS, pH 6.0-6.5, Ca2+ 50 ppm)

Liquefaction
Thermostable -Amylase
Gelatinization (105°C, 5 min)
Dextrinization (95°C, 2h)

Liquefied Starch DE 10-15

Saccharification
Glucoamylase
(60°C, pH 4.0-4.5, 24-72 h)

Glucose Syrups DE 95-96

Isomerization
Glucose isomerase
(pH 7.5-8.0, 55-60°C, 5 mM Mg2+)

High Fructose Corn Syrups (42% fructose)

S¶n xuÊt Glucose tõ tinh bét
_______________________________________________________________
Liquefaction Saccharification DE
Glucose
_______________________________________________________________

Acid Acid 92 85
Acid Glucoamylase 95 91
Acid/α-amylase Glucoamylase 96 92
α-Amylase/High pressure Glucoamylase 97 93

cooking/ α-amylase
α-Amylase (thermostable) Glucoamylase 97 94
α-Amylase (thermostable) Glucoamylase 97-98.5 95-
97.5
_______________________________________________________________
 ChuyÓn ®æi Glucose thµnh Fructose

HO
OH HO OH
O glucose O
isomerase
OH HO
OH
HO
OH
OH
 Qu¸ tr×nh Deacylation Penicillin
ho¸ häc & enzyme thµnh 6-APA
H
R C N S CH3 S CH3
Penicillin acylase NH2
CH3 CH3
O
N Alkaline N
O COOH [Enzymatic] O COOH
Penicillin V or G (6-APA)
[R=Ph or PhO]

[Chemical] PCl5
Pyridine
Me3SiCl ROH
H2O
H
R C N S CH3
CH3
O
N
O COOSiMe3
C¸c enzyme:
Penicillin G acylase (PGA)- Escherichia coli, Bacillus megaterium,
Streptomyces lavendulae
Penicillin V acylases (PVA)- Beijerinckia indica var. Penicillium,
Fusarium sp., Pseudomonas acidovorans

C¸c enzyme cè ®Þnh:

§ßi sèng: 500-2880 giê
 Sù biÕn ®æi Penicillin thµnh 6-APA
b»ng enzyme vµ Penicillin b¸n tæng hîp

H
R C N S CH3 S CH3
Penicillin acylase NH2
CH3 CH3
O
N Alkaline N
O COOH [Deacylation] O COOH

Penicillin V or G (6-APA)

Penicillin acylase
[Acylation] Acidic

Semisynthetic Penicillins
 Tæng hî p Acrylamide
Copper-catalysed process

Microbial process

Nitrile hyratase and amidase reactions

Aspartame (L-Asp-L-Phe-Methyl Ester)

Aspartame lµ chÊt ngät dipeptide ®­îc h×nh thµnh b»ng liªn

kÕt methyl ester cña phenylalanine víi aspartic acid

 Sö dông réng r·i trong thùc phÈm vµ ®å uèng

 200 lÇn ngät h¬n sucrose
 Th­¬ng m¹i hµng n¨m: 200 triÖu Ibs, 850 triÖu $
 Nutrasweet Corp. chiÔm lÜnh 75% cña thÞ tr­êng US
Ph­¬ng ph¸p ho¸ häc:
 Nhãm amino cña aspartic acid cÇn b¶o vÖ ®Ó ng¨n c¶n
ph¶n øng cña nã víi ph©n tö kh¸c cña aspartic acid ®Ó cho
s¶n phÈm phô kh«ng mong muèn.

 ChÝnh x¸c enantiomer ®¬n cña mçi chÊt ph¶n øng ph¶i
®­îc sö dông ®Ó ®­îc ho¸ häc kh«ng gian stereochemistry mong
muèn cña aspartame (beta-aspartame cã vÞ ®¾ng)
Ph­¬ng ph¸p enzyme:

Thermolysin thøc ®Èy ph¶n øng chØ ë alpha-

functionality
§iÒu kiÖn trung b×nh, pH 6-8, 40 oC
Cbz, benzyloxycarbonyl
 Sù s¶n xuÊt xóc t¸ c sinh häc cña Aspartame

HO2C Ph

+ thermolysin
PhCH2OCNH CO2H H2 N CO2Me

O
H2O
N-Cbz-aspartic acid D,L-phenylalanine
Methyl ester HO2C Ph

PhCH2OCNH CNH CO2Me

O O

Cbz-aspartame
Cbz, benzyloxycarbonyl
 L-Carnitine

 ChÊt øc chÕ thyroid

 ChÊt gi¶m bÐo (Slimming agent)

 Thùc phÈm bæ sung cho vËn ®éng viªn ®iÒn kinh
 ChØ mét enantiomer cña thµnh phÇn ®­îc sö dông
 Hai con ®­êng xóc t¸c sinh häc s½n cã ®Ó s¶n xuÊt L-
carnithine.
Saccharomyces cerevisiae
Rhizobiaceae
 Tæng hî p L-Carnitine
O O reductase HO O
H
Cl OC8H17 Cl OC8H17
-chloroacetoacetic (R)--chloro--hydroxybutanoic
acid octyl ester acid octyl ester

HO O
H
Me3N
OH
L-carnitine

O hydroxylase HO O
H
Me3N Me3N
OH OH
-butyrobetaine L-carnitine
 T æn g h î p N a p r o x e n e
C O 2H

b io c a ta ly s ts *

C H 3O C H 3O

C O 2H C O 2H

m u ltis te p * r e s o lu tio n *
(D /L )
C H 3O C H 3O C H 3O

O 1) T a r ta r ic a c id C O 2H
2) B r2
C C H 2C H 3
3) H y d r o ly s is *
C H 3O C H 3O
Tæng hî p Calcium – Antagonist Drug
Diltiazem
OMe OMe
O O
(R,R)
esterase
MeO2C HO2C
racemate

OMe

S (S, S)
O
Diltiazem
N O
H O
T æ n g h î p L -m a lic A c id v µ L -A s p a r tic A c id
T õ F u m a r ic A c id

H O 2C fu m a ra s e H O 2C
H

C O 2H H 2O H O C O 2H

fu m a r ic a c id L -m a lic a c id

H O 2C a s p a rta s e H O 2C
H

C O 2H N H 3 H O C O 2H

fu m a r ic a c id L -a s p a r tic a c id
 T æn g h î p C a t e c h o l t õ G l u c o s e
T ­ ¬ n g t h Ýc h m « i t r ­ ê n g
a c e to n e

h y d r o q u in o n e
a b
HO c
benzene cum ene phenol

OH C O 2H C O 2H
HO
OH OH
O d
d d c a te c h o l
HO OH O OH HO
OH OH OH
D - g lu c o s e 3 -d e h y d r o s h ik im ic p r o to c a te c h u ic
a c id a c id
(a ) p r o p y le n e , s o lid H 3 P O 4 c a t a ly s t , 2 0 0 -2 6 0 ° C , 4 0 0 - 6 0 0 p s i.
(b ) O 2, 8 0 -1 3 0 °C th e n S O 2, 6 0 -1 0 0 °C .
(c ) 7 0 % H 2O 2, E D T A , F e 2+ o r C o 2, 7 0 -8 0 °C .
D ra th s a n d F ro s t, 1 9 9 5
(d ) E . c o li A B 2 8 3 4 /p K D 1 3 6 /p K D 9 .0 6 9 A , 3 7 °C .
Khö ®¾ng c¸c protein thuû ph©n
 Xö lý b»ng carbon ho¹t tÝnh
 TriÕt t¸ch b»ng cån
 KÕt tña ®ång ®¼ng ®iÖn
 T¸ch b»ng s¾c ký
 Bao phñ vÞ ®¾ng
 Thuû ph©n enzyme c¸c peptide ®¾ng
• b»ng aminopeptidase
• b»ng alkaline/neutral protease
b»ng
carboxypeptidase
• C¸c ph¶n øng trïng ng­ng sö dông protease
Xylanase-trî gióp xö lý lµm tr¾ng
____________________________________________________
Sequence after Pulp Total active chlorine
Enzyme treatment consumption
decrease (%)
____________________________________________________
(CD)EDED Softwood kraft 21
(CD)EoDED Pine kraft 18.4
(CD)EpDEpD Birch kraft 18
(CD)EopDEpD Pine kraft 12
DEopDED Softwood kraft 15
___________________________________________________
C, elemental chlorine (Cl2), D, chlorine dioxide (ClO3), E,
alkaline extraction (NaOH), Eo/Ep, oxygen/hydrogen peroxide
 Mannitol
Phô gia thùc phÈm

• Lµm gi¶m kh¶ n¨ng kÕt tinh cña ®­êng vµ ®­îc sö

dông ®Ó kÐo dµi tuæi thä cña thùc phÈm.

• Sö dông trong kÑo cao su

• T¹o c«ng thøc d­îc phÈm cña c¸c viªn ngËm vµ h¹t
bét.

• Ng¨n ngõa hÊp thô Èm tõ kh«ng khÝ, thÓ hiÖn

tuþet vêi c¸c tÝnh chÊt nÐn c¬ häc, kh«ng t­¬ng t¸c
víi c¸c thµnh phÇn ho¹t ®éng vµ vÞ ngät m¸t cña nã
lµm lu m¬ vÞ khã chÞu cña nhiÒu lo¹i thuèc.
 Mannitol
• Mannitol hexanitrate ®­îc biÕt rÊt râ lµ chÊt b¶o vÖ
m¹ch (vasodilator), sö dông trong ®iÒu trÞ cao huyÕt ¸p
( hypertension)
• Hçn hîp boric acid víi mannitol ®­îc sö dông trong s¶n xuÊt
chÊt ®iÖn dung kh« (electrolytic capacitor)
• Nã ®­îc sö dông nhiÒu polyol cho s¶n xuÊt resin vµ chÊt
bÒ mÆt.
• Nã Ýt tan trong n­íc, chØ 18% (w/w) ë 25 oC
• Trong dung dÞch kiÒm, nã lµ chÊt Èn c­m¹nh
(sequestrant) cña c¸c ion kim lo¹i.
• Nã ngät b»ng nöa sucrose
 Qu¸ tr×nh hydro ho¸ D-Fructose
H2C OH H2 C OH H2 C OH

C O HC OH HO CH

HO CH HO CH HO CH
H2, catalyst +
HC OH HC OH HC OH

HC OH HC OH HC OH

H2C OH H2 C OH H2 C OH

D-Fructose D-Sorbitol D-Mannitol

 Chu tr×nh chuyÓn ®æi Heterofermentative
cña Fructose thµnh Mannitol
Fructose 2 Fructose
ATP
ADP
Fructose – 6-P

Glucose – 6-P
NADP+
NADPH + H+
6 - Phosphogluconate
NADP+
CO2 NADPH + H+
Ribulose – 5-P

Xylulose – 5-P

Glyceraldehyde - 3-P Acetyl - P

NAD+ 2 ADP ADP
NADH + H+ 2 ATP
Pyruvate
NADH + H+
ATP
NAD+
Lactate Acetate 2 Mannitol
 Sù chuyÓn ho¸ Fructose thµnh Mannitol
b»ng enzyme

CH2OH CH2OH

O HO H

HO H Mannitol 2-Dehydrogenase HO H

H OH H OH
NAD(P)H NAD(P)
H OH H OH

CH2OH CH2OH
D-fructose Mannitol
 Sù chuyÓn ®æi Fructose thµnh Mannitol
b»ng enzyme víi sù t¸i t¹o cofactor liªn tôc

Mannitol Dehydrogenase
D-Fructose Mannitol

NADH NAD+

Gluconic acid Glucose + H20

Glucose Dehydrogenase