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Bi7430 Molecular Biotechnology
8. Molecular Biotechnology in Industry
Outline
 Enzymes and applications
 Definition of white biotechnology
 Sustainable development
 Enzyme sources
 Industrial production of proteins
 Enzyme and cells immobilization
 Examples of biocatalytic applications
Enzymes
 natural catalysts (biocatalyst)
 catalyze chemical reactions in living systems
 oxidoreductases - oxidation/reduction
 transferases - transfer of functional groups
 hydrolases – hydrolytic cleavage
 lyases - cleavage of C-C, C-N and C-O bonds
 isomerases - racemization, epimerization
 ligases - formation of C-C, C-Nand C-O bonds
Enzyme applications
cellulases
ligninase
lipases
restrictases
DNA ligases
polymerases
amylases
proteases
cellulases
phytases
lipases
phosphatases
peroxidases
lipases
nitrilases
peptidases
amidases
aldolases
asparaginase
DNase
urokinases
proteases
dehalogenases
choline esterase
peroxidases
amylase
cellulases
catalase
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White (industrial) biotechnology
 biotechnology incorporated into production processes and products
that involve chemical reactions - biocatalysis
 sustainable and environmentally-friendly industry
 using biomass rather than traditional petrochemicals
 provide energy efficiency, increased productivity and better safety
 uses enzymes and micro-organisms to make products and services
in a wide range of industrial sectors
Sustainable solutions
 i n n o v a t i v e a n d c o m p e t i t i v e p ro d u c t s a n d p ro 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 st a i n a b i l i t y
 t ra 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 re a te t h e f u t u re 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 nv i ro n m e n t a n d b e tte 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 re s e n t
w i t h o u t c o m p ro m i s i n g t h e a b i l i t y o f f u t u re g e n e ra 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 ro 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 re s o u rc 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 re n e wa b l e m a t e r i a l s
 r e d u c e wa st e p r o d u c t i o n
 m a x i m i z e w a s t e re c y c l i n g
Example of sustainable technology
Biocatalysis (+30°C)
1000 t penicillin G
45 t ammonia
10,000 m3 water
1 t ENZYME
(1 $/kg 6-APA)
Chemical process (-40°C)
1000 t penicillin G
160 t ammonia
300 t dimethylchlorosilane
800 t N,N-dimethylaniline
600 t phosphopentachloride
4,200 m3 dichloromethane
4,200 m3 n-butanol
Enzyme-based technologies
ADVANTAGES
 high catalytic efficiency
 broad substrate specificity
 high selectivity
 compatibility of each other
 reusability
 sustainability
 produced from biomass
 non-toxic and biodegradable
 operate at mild conditions
 less byproducts and wastes
LIMITATIONS
 cofactor requirement
 prone to inhibitions
 highest activity in water
 less stable
 low selectivity
 expensive
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Enzyme sources
 animal and plant tissues
 thousands years old developed empirically
 pancreas (treatment of hides), calf stomach (cheese -making)
 papaya, pineapple (meat tenderization)
 content up to 1% enzyme of tissue weight
 less competitive compared to fermentation of microorganism
 risk of contamination with prions and viruses harmful to humans
Enzyme sources
 wild-type microorganisms
 enzymes from microorganisms long been safely used in food industry
 food processing regulation - strict for non-recombinant enzymes
 microorganisms used for screening for „new“ catalytic enzymes
 screen for enzymes active at desired process conditions
(e.g., pH, temperature)
 recombinant microorganisms
 most technical enzymes produced using recombinant technology
 when yield in wild type organism is low or desired enzyme
is not in class I organism
 bacteria, fungi and yeasts
(e.g., E.coli, Bacillus, Aspergillus, Saccharomyces )
Industrial production of proteins
 fermentation
 non-recombinant and recombinant organisms
 steady and safe (class I or GRAS) organisms
 up-scale and optimization
 high cell density fermentation (50 g cell dry weight per liter)
 upper limit of protein concentration (10 g.L-1; 40% of total cell protein)
Downstream process
 separation and homogenization
 dependent on application and required purity
 technical enzymes - low to moderate purity
 enzymes for therapy and diagnostics - high purity
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Downstream process
 enzyme purification
 impurities (e.g., proteins, DNA and others)
 further purification when safety (e.g., recombinant DNA, viruses)
or function reasons (impurities disturbing catalytic function)
 basic knowledge of protein properties necessary
o molecular weight (MW)
o isoelectric point ( pI)
o cofactors
o pH range
o temperature stability
 methods of protein purification
o precipitation and differential solubilization
(e.g., ammonium sulfate, pH, solvents)
o membrane filtration
o chromatographic methods
(e.g., size exclusion, ion exchange, hydrophobic, metal affinity, biospecific)
 more steps -> higher purity (multi-step manipulation, loss >10% of enzyme)
Whole cell vs. isolated enzyme
 advantages
 allow more enzymes
 cofactor regeneration
 cheap
 disadvantages
 side-reactions
 low tolerance to solvents
 low productivity
 advantages
 smaller reactors
 less side reactions
 higher productivity
 disadvantages
 more expensive
 addition of cofactors
 less stable outside cell
Immobilisation methods
 b i o c a ta 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 re a t m e n t
 b e n e f i t s
 s t a b i l i z a t i o n b y i m m o b i l i z a t i o n
 e a s y s e p a r a t i o n o f p r o d u c t
 r e p e a t e d u s e o f b i o c a t a l y s t
 c o n t i n u o u s b i o p r o c e s s i n g
 l i m i t a t i o n s
 e x p e n s e s o f c a r r i e r s a n d i m m o b i l i z a t i o n
 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 z a t i o n
 c h a n g e s i n p r o p e r t i e s o f b i o c a t a l y s t
 m a s s t r a n s f e r l i m i t a t i o n s
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
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Immobilisation of cell
A l g i n a t e b e a d s PVA lens (LentiCats)
Examples of whole cell biocatalysis
 mandelic acid - urinary antiseptic, skin care cosmetics
(du Pont, Nitto Chemicals, etc.)
Alcaligenes
faecalis
 synthesis of agrochemical intermediates by microbial
hydroxylation of heteroatomics (Lonza)
Achromobacter
xylosoxidans
 large-scale production of commodity chemical - acrylamide
(Mitsubishi, Nitto Chemicals)
Rhodococcus
rhodochrous J1
Examples of enzyme biocatalysis
 large scale production of Aspartame, low-calorie sweetener
(DSM, NutraSweet)
 synthesis of atorvastatin, Lipitor
®
, intermediate
(Pfizer - sales since 1996 exceed US$ 150 billion)
 synthesis of high fructose syrup from corn starch
(10 million tons per year)
Let´s make world better
 sustainable and environmentally-friendly industry
 biomass rather than traditional petrochemicals
 energy efficient, increased productivity and better safety