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6. Industrial Applications of Molecular
Biotechnology
6. Industrial Applications of Molecular
Biotechnology
Bi7430 Molecular Biotechnology
Definition of white biotechnology
Enzymes and applications
Sustainable development
Enzyme sources
Industrial production of proteins
Enzyme and cells immobilisation
Examples of biocatalytic applications
Outline
White (industrial) biotechnology
biotechnology incorporated into production processes and products
that involve chemical reactions - biocatalysis
uses enzymes and micro-organisms to make products and services
in a wide range of industrial sectors
fine chemica ls and pharmac eu ti c a ls
materia ls and polym ers
paper, pulp and texti l es
food and feed
detergen ts
sustainable and environmentally-friendly industry
using biomass rather than traditional petrochemicals
provide energy efficiency, increased productivity and better
safety and environmental characteristics
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natural catalysts (biocatalyst)
catalyse chemical reactions in living systems
o x i d o r e d u c t a s e s - o x i d a t i o n / r e d u c t i o n
t r a n s f e r a s e s - t ra n s f e r o f f u n c t i o n a l g r o u p s
h y d r o l a s e s – h y d r o l y t i c c l e a va g e
l y a s e s - c l e a va g e o f C- C , C- N a n d C- O b o n d s
i s o m e r a s e s - ra c e m i z a t i o n , e p i m e r i z a t i o n
l i g a s e s - f o r m a t i o n o f C- C , C- N a n d C- O b o n d s
Enzymes
Enzyme applications
c el l u l as es
l i gn i n as e
l i pas es
r es t r i c t as es
D N A l i gas es
pol ym er as es
am yl as es
pr ot eas es
c el l u l as es
ph yt as es
l i pas es
ph os ph at as es
per oxi d as es
l i pas es
n i t r i l as es
pept i das es
am i das es
al dol as es
as par agi n as e
D N as e
u r ok i n as es
pr ot eas es
deh al ogen as e s
O PH , BC h E
per oxi d as es
am yl as e
c el l u l as es
c at al as e
Sustainable solution
i n o v a t i v e a n d c o m p e t i t i v e p r o d u c t s a n d p r o c e s s e s
m e e t i n g c r i t e r i a o f s u s t a i n a b i l i t y
t r a n s f e r o f b i o l o g i c a l s o l u t i o n s t o m o d e r n t e c h n o l o g i e s
c r e a t e t h e f u t u r e i n b a l a n c e b e t w e e n e c o n o m y, c l e a n e r
e n v i r o n m e n t a n d b e t t e r l i v e s
“ … d e v e l o p m e n t t h a t m e e t s t h e n e e d s o f t h e p r e s e n t
w i t h o u t c o m p r o m i s i n g t h e a b i l i t y o f f u t u r e g e n e r a t i o n s
t o m e e t t h e i r o w n n e e d s ” ( W C E D , 1 9 8 7 )
r e d u c e e n v i r o n m e n t a l i m p a c t
r e d u c e c o n s u m p t i o n o f r e s o u r c e s
( r a w m a t e r i a l s , e n e r g y, a i r , w a t e r )
u s e o f r e n e w a b l e r a w m a t e r i a l s
r e d u c e w a s t e p r o d u c t i o n
m a x i m i z e w a s t e r e c y c l i n g
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Example of sustainable technology
Bi oc a t a l ys i s ( + 3 0 °C )
1 0 0 0 t pe ni c i l l i n G
4 5 t a mmoni a
1 0 , 0 0 0 m3 w a t e r
1 t ENZ YM E
( 1 $ / k g 6 - A PA )
C he mi c a l pr oc e s s ( - 4 0 °C )
1 0 0 0 t pe ni c i l l i n G
1 6 0 t a mmoni a
3 0 0 t di me t hyl c hl or os i l a ne
8 0 0 t N , N - di me t hyl a ni l i ne
6 0 0 t phos ph o pe nt a c hl or i de
4 , 2 0 0 m3 di c hl or ome t ha ne
4 , 2 0 0 m3 n - but a nol
h y d r o l y s i s o f p e n i c i l l i n G
ADVANTAG ES
high catalyt ic efficiency
broad substrat e specifity
high degree of selectivity
compatibility of each other
reusability
sustainablity
pr odu c ed f r om bi om as s
eas i l y bi ode gr a d a bl e
n on - t oxi c , n on - f l am abl e
n o r eqi r em en t of l ar ge qu an t i t y
of t oxi c m et al s an d s ol ven t s
l es s bypr o du c t s an d w as t es
opar at e at m i l d c on di t i on s
LIMITATIONS
cofactor requirem en t
prone to inhibitions
highest activity in water
generaly less stable
insuffic i ent selectivity
often expensive
allergies
Enzyme-based technology
Enzyme sources
ANIMAL AND PLANT TISSUES
c o n t e n t o f t e n u p t o 1 % e n z y m e o f t i s s u e w e i g h t
t h o u s a n d s y e a r s o l d d e v e l o p e d e m p i r i c a l l y
p a n c r e a s ( e a r l i e r t r e a t m e n t o f h i d e s ) , c a l f s t o m a c h ( c h e e s e - m a k i n g )
p a p a ya , p i n e a p p l e ( t e n d e r i z a t i o n o f m e a t ) , s o d o m a pp l e l e a v e s ( m i l k c l o t t i n g )
r i s k o f c o n t a m i n a t i o n w i t h p r i o n s a n d v i r u s e s h a r m f u l l t o h u m a n s
l e s s c o m p e t e t i v e c o m p a r e d t o f e r m e n t a t i o n o f m i c r o o r g a n i s m
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Enzyme sources
WILD-TYPE MICROORGANISMS
e n z y m e s f r o m m i c r o o r g a n i s m s l o n g b e e n s a f e l y u s e d in f o o d i n d u s t r y
f o o d p r o c e s s i n g e n z y m e s - r e g u l a t i o n s t r i c t f o r u s i n g n o n - r e c o m b i n a n t e n z y m es
m i c r o o r g a n i s m s u s e d f o r s c r e e n i n g f o r „ n e w “ c a t a l y t i c e n z y m e s
s c r e e n f o r e n z y m e s a c t i v e a t d e s i r e d p r o c e s s c o n d i t i o n s ( e . g ., p H , t e m p e ra t u r e )
RECOMBINANT MICROORGANISMS
w h e n y i e l d i n w i l d t y p e o r g a n i s m i s l o w o r d e s i r e d e n z y m e i s n o t i n c l a s s I o r g a n i s m
b a c t e r i a , f u n g i a n d y e a s t s ( e . g ., E . c o l i , B a c i l l u s , A s p e r g i l l u s , S a c c h a r o m y c e s)
m o s t t e c h n i c a l e n z y m e s p r o d u c e d u s i n g r e c o m b i n a n t t e c h n o l o g y
Industrial production of enzymes
FERMENTATION
non-recombinan t and recombina nt organisms
steady and safe (class I or GRAS) organisms
up-scale and optimis a ti on
h i g h c e l l d e n s i t y f e r m e n t a t i o n ( 5 0 g c e l l d r y w e i g h t p e r l i t e r )
u p p e r l i m i t o f p r o t e i n c o n c e n t r a t i o n ( 1 0 g . L
- 1
; 4 0 % o f t o t a l c e l l p r o t e i n )
Downstream process
SEPARATION AND HOMOGENISATION
dependen t on applicatio n and required purity
t e c h n i c a l e n z y m e - l o w t o m o d e ra t e p u r i t y
e n z y m e s f o r t h e r a p y a n d d i a g n o s t i c s - h i g h p u r i t y
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Downstream process
SEPARATION AND HOMOGENISATION
dependen t on applicatio n and required purity
t e c h n i c a l e n z y m e - l o w t o m o d e ra t e p u r i t y
e n z y m e s f o r t h e r a p y a n d d i a g n o s t i c s - h i g h p u r i t y
m e c h a n i c a l
h o m o g e n i z e r s - h i g h p r e s s u r e ( 1 5 0 0 b a r ) f o l l o w e d b y e x p a n s i o n
b a l l m i l l s - s m a l l a b r a s i v e p a r t i c l e s
u l t r a s o n i c d i s r u p t i o n - c e l l l y s i s w i t h h i g h f r e q u e n c y s o u n d
b l e n d e r s - b l a d e s r o t a t e a t s p e e d s o f 6 , 0 0 0 - 5 0 , 0 0 0 r p m
f r e e z e f r a c t u r i n g - w a t e r c r y s t a l s a s a b r a s i v e
n o n - m e c h a n i c a l
c h e m i c a l p e r m e a b i l i z a t i o n ( e . g . , s o l v e n t s , s u r f a c t a n t s , a n t i b i o t i c s )
e n z y m a t i c p e r m e a b i l i z a t i o n ( e . g . , g l y c a n a s e s , p r o t e a s e s , m a n n a s e )
o s m o t i c s h o c k ( e . g . , h i g h s u c r o s e m e d i u m )
Downstream process
SEPARATION AND HOMOGENISATION
dependen t on applicatio n and required purity
t e c h n i c a l e n z y m e - l o w t o m o d e ra t e p u r i t y
e n z y m e s f o r t h e r a p y a n d d i a g n o s t i c s - h i g h p u r i t y
Downstream process
ENZYME PURIFICATION
impuritie s (e.g., proteins , DNA and others)
further purific a ti on when safety (e.g., recomb inan t DNA, viruses)
or functional reasons (impur it i es disturbin g cataly t i c function)
basic knowle d ge of protein properti es necesar y
m o l e c u l a r w e i g h t ( M W )
i s o e l e c t r i c p o i n t ( p I )
c o f a c t o r s
p H ra n g e
t e m p e ra t u r e s t a b i l i t y
methods of protein purific a t ion
p r e c i p i t a t i o n a n d d i f f e r e n t i a l s o l u b i l i z a t i o n
( e . g ., a m m o n i u m s u l f a t e , p H , s o l v e n t s )
m e m b ra n e f i l t ra t i o n
c h r o m a t o g ra p h i c m e t h o d s
( s i z e e x c l u s i o n , i o n e x c h a n g e , h y d r o p h o b i c ,
m e t a l a f f i n i t y, b i o s p e c i f i c )
more steps -> higher purity (multi-s t e p manipula t ion , loss >10% of enzyme)
WHAT ARE THE RELEVANT PROTEIN PROPERTIES?
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Immobilisation methods
b i o c a t a l y s t s ( e n z y m e o r c e l l ) l i m i t e d i n m o v i n g d u e t o
c h e m i c a l o r p h y s i c a l t r e a t m e n t
R E A S O N S
r e u s e o f e n z y m e ( r e d u c i n g c o s t )
e a s y p r o d u c t s e p a r a t i o n
c o n t i n o u s p r o c e s s i n g
s t a b i l i z a t i o n b y i m m o b i l i s a t i o n
L I M I TAT I O N S
c o s t o f c a r r i e r s a n d i m m o b i l i s a t i o n
c h a n g e s i n p r o p e r t i e s ( s e l e c t i v i t y )
m a s s t r a n s f e r l i m i t a t i o n s
a c t i v i t y l o s s d u r i n g i m m o b i l i s a t i o n
Immobilisation of enzyme
p o l y a c r y l a t e A m b e r l i t e ®p o r o u s s i l i c a C e l l u l o s e
Immobilisation of cell
A l g i n a t e b e a d s
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Whole cell vs. isolated enzyme
advantages
al l ow m or e en zym es
c of ac t or r egen er at i on
c h eap
disadvantages
s i de- r eac t i on s f r om ot h er en zym es
l ow t ol er an c e t o or gan i c s ol ven t s
l ow pr odu c t i vi t y
advantages
s m al l er r eac t or s
l es s s i de r eac t i on s
h i gh er pr odu c t i v i t y
disadvantages
m or e exp en s i v e
addi t i on of c of ac t or s
l es s s t abl e ou t s i de c el l
Examples of whole cell biocatalysis
synthesis of agrochemical intermediates by microbial
hydroxylat ion of heteroatom ic s (Lonza)
mandelic acid - urinar y antisept ic, skin care cosmetics
(du Pont, Nitto Chemicals, etc.)
large-sc ale production of commodity chemical - acrylamide
(Mitsubishi, Nitto Chemicals)
Alcaligenes
faecalis
Achromobacter
xylosoxidans
Rhodococcus
rhodochrous J1
Examples of enzyme biocatalysis
large scale production of Aspartame, low-calor ie sweetner
(DSM, NutraSweet)
synthesis of atorvast at in, Lipitor
®
, intermed i at e
(Pfizer - sales since 1996 exceed US$ 125 billion)
synthesis of high fructose syrup from corn starch
(10 million tons per year)