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Bio-Catalysis
National Centre for Catalysis Research
Indian Institute of Technology, Madras
Presented by
V surya kumar
CA11M005
Contents
o Challenges facing mankind
o History of bio-catalysis
o Enzymes and their classification
o Advantages and disadvantages of bio-catalysis
o Theories of enzyme action
o Statistics of Bio-catalysis in industries
o Immobilization
o Commercial examples
o Conclusion
challenges facing mankind today
Maintenance and improvement of
human health
Maintenance and improvement of
environment
Brief History
Year
Name s
Topic
1850
Louis Pasteur
Fermentation by yeast is catalyzed by ferments
1877
Eduard Buchner (Nobel prize 1907)
1st alcoholic respiration with isolated enzyme
1893
Wilhelm Ostwald (Nobel prize 1909)
Definition of ‘catalyst’
1894
Emil Fischer (Nobel prize 1902)
“lock-and-key” concept
1926
James B. Sumner (Nobel prize 1946)
1944
Linus Pauling
1st attempt to explain catalysis as transitionstate complementarity
1951
Frederick Sanger and Hans Tuppy
(Nobel prize 1978)
sequence determination of insulin
1963
Stanford Moore and William Stein
(Nobel prize 1972)
amino acid sequence of lysozyme and
ribonuclease elucidated
1985
Michael Smith (Nobel prize 1993)
gene mutagenesis to change enzyme sequence
1988
Kary B. Mullis (Nobel prize 1993)
invention of PCR
Paul D Boyer, John E Walker ----------Jens C skou ---------------------------------(Nobel price in 1997)
Mech. Of ATP synthesis
Discovery of ion transporting enzyme
Aaron Ciechanover, Avram Hershko,
Irwin Rose (Nobel price in 2004)
"for the discovery of ubiquitin-mediated protein
degradation"
1st enzyme crystallized:
urease
Enzymes
• Enzymes(protein + non-protein part) , non-protein
part is called co-factor, when co-factor is organic
compound called coenzyme. A coenzyme/metal ion
bound tightly to protein is called prosthetic group .
Enzyme
cofactor
Carboic anhydrase With
Zn +2 ion cofactor
Heam as prosthetic group
in catallase
Coenzyme
Ex- vitamins
Prosthetic
group
Ex- Heam
Protein part/
apoenzyme
Metal ion
Ex-Zn=2
IUBMB Classification of enzymes
Classification
Reaction
catalyzed
Examples
Sub examples/function
Oxido reductase
Oxidationreduction rex
dehydrogenase,
oxidase,
reductase
Aldehyde dehydrogenases,
Xanthine oxidase
5-alpha reductase
Transferase
Transfer of
functional
groups
acetyltransferase
methylase
Acetylation
Methylation
Hydrolases
Hydrolysis
reaction
Proteases
HIV protease
catalyze the
cleavage of C-C,
C-O, C-S and CN bonds
decarboxylase
,aldolase
Isomerization
rotamase,
Cis – trans
Bond formation
coupled with
ATP hydrolysis
DNA ligase
RNA ligase.
Joining of DNA strands
N-ribonucliotide
Lyases
Isomerases
Ligases
RuBisCO- CO2 fixation
Fructose-bisphosphate
aldolase
Enzymes employed in organic synthesis
Advantages of bio-catalysis
•
•
•
•
•
•
•
•
•
•
Unsurpassed selectivity(substrate and product specific)
High rates (106- 1012 s-1)
Ability to differentiate b/t enantiomers
Active under mild, near ambient conditions of T,P, PH.
Water as a solvent – economi-enviormental. attractive.
Green process.
Low energy process.
Less byproducts
Can be immobilized and re-used
Bio-degradable.
Disadvantages of biocatalysts
 Not sufficiently stable in the desired conditions.
 Few biocatalysts known & known not fully characterized.(510
CUE’s avail IN 2012)
 Development cycles are too long for new and improved
biocatalysts .
Report of enzyme commotion
No of enzymes known
Enzyme Commission (1961)
712
Enzyme Nomenclature (1964)
875
Enzyme Nomenclature (1972)
1770
Enzyme Nomenclature (1978)
2122
Enzyme Nomenclature (1984)
2477
Enzyme Nomenclature (1992)
3196
Number of catalyst known
Lack of sufficient knowledge
PROCESS
Time period
(years)
acrylamide
process
20
l-carnithine
process
15
Process development
period
• What is the source of the energy for the lowering of
the activation energies?
• How these enzyme catalyzed reactions are highly
selective?
‘Weak Interactions between Enzyme and Substrate
are responsible for enzyme catalysis’
J.B.S Haldane
• Formation of transient covalent bonds b/w Enzyme &
Substrate provides alternative routes wherein ∆G‡ is low.
• Non-covalent interactions such as H-bonding, ionic and
hydrophobic interactions between enzyme and substrate
provides free energy.
How enzyme manage these week interaction is next question?
Fischer Lock & key model(1894)
• Fisher suggested enzyme specificity is due to
complementary structural features b/t enzyme and
substrate, substrate fits into the complementary
features of enzyme as key.
Limitation of theory
It considers enzyme as rigid body and
do not explain flexibility of protein
Ref. file:///F:/F/IIT/iit%20m%207th%20dec%202011/Third%20sem/SEMINAR/SEMINAR/NOTES/NET/enzymes%20full%20chapter.htm
Transition state stabilization
by Pauling(1944)
Poor enzyme
Efficient enzyme – optimal interaction
Ref. .Lehninger's 4TH Ed
 Few weak interactions are formed in the ES complex.
 full complement of interactions between S and E is
formed only in transition state, but still TS is not
stable on enzyme hence P are formed.
G

uncat
 GB  G
Enzyme optimizes ∆GB by providing
functional groups using amino acids
Present on proteins in the special
cavity Called Active Site (AS) .
Ref. .Lehninger's 4TH Ed
Now catalysis is achieved, How specificity is achieved is the next question?

cat
Factors needed for specificity
• Proper alignment of catalytic functional groups
• Entropy reduction – decrease in freedom of motion.
• Distortion of substrate.
Rate
enhancement
• Charge redistribution.
• Conformational change .
---------------------------------Mechanisms for specificity
• general acid-base catalysis
• covalent catalysis
• metal ion catalysis.
Ref. .Lehninger's 4TH Ed
General Acid base catalysis
In uncatalyssed rex unstable charged intermediates are
stabilized by donation or acceptance of H+, which is done by
water , normal acids or bases .
 In the active site of an
enzyme, a number of amino
acid side chains act as
proton donors and acceptors
These groups are precisely
positioned in enzyme active
site to allow proton transfer
providing rate enhancement
of the order of 102 to 105 .
Ref. .Lehninger's 4TH Ed
Covalent Catalysis
Hydrolysis of a bond b/w A and B
In the presence of covalent catalyst (nucleophile),
transient covalent bond is formed between the enzyme
and the substrate, leading to new path which has small
activation energy.
Ref. .Lehninger's 4TH Ed
Metal ion catalysis
• Ionic interactions between an enzyme-bound metal and
a substrate help orient the substrate for reaction or
stabilize charged reaction transition states.
• Metals also mediate oxidation-reduction reactions by
reversible changes in the metal ion’s oxidation state.
• For example – in hemoglobin Fe in ferric and ferrous have
different activities
Ref. Wikipedia
Koshland induced-fit model(1958)
• Enzyme is complementary to substrate in ES complex
but not in free enzyme state.
• Enzymes are flexible and substrate brings conformational
change in 3-D structure of enzyme
• In lock & key model the active sites are always open and
freely accesble, so any molecule can access it.
• In induced-fit model
Minimizes the risk of chance of
Collision(non-productive binding
and keeps selectivity intact.
Ref. http://course1.winona.edu/sberg/ANIMTNS/ind-fit.htm
Statistics of Bio-catalysis in industries
Cumulative no. of biotransformation processes
that have been started on an industrial scale.
compounds produced using biotransformation
processes
Industrial sectors in which the products of industrial
Biotransformation's are used (based on 134 processes).
Ref. Current Opinion in Biotechnology 2002, 13:548–556
Enzyme types used in industrial biotransformation's
Bio-catalysis in chemical industry
Mitsubishi Rayon, Japan - acrylamide production
ChiPros®-BASF - (R)-1-Phenylethylamine, (R)-Isobutyllactate raw,
(R)-Mandelic acid, (S)-1-Phenylethylamine
Problems in bio-catalysis
• Enzymes are expensive.
• lack of established process.
• Enzyme wastage - enzyme cannot be economically
recovered for re-used.
• Contaminate the product - The enzyme residue
remains , hence involves extra purification.
Immobilization is
solution
Definition of immobilization
Separation of enzyme and product using a twophase system, One phase containing the enzyme
the other phase containing the product, This is
known as IMMOBILISATION
Origin of Immobilization
• In 1920’s Nelson and Griffin initiated this idea, than
imaginatively Katchalkski and his colleagues at the
Weizmann Institute in Israel exploited this idea
• First commercial immobilized process was Resolution
of racemic mixtures of amino acid by hydrolysis using
immoblized enzyme aminoacylase in japan by Tanabe
Seiyaku comp.
Advantages of immobilization
• stabilization of enzymes, as translational motion
restricted in immobilized mode
• Easy separation from reaction mixture, providing the
ability to control reaction time.
• minimize the enzymes lost in the product.
• Re-use of enzymes for many reaction cycles, lowering
the total production cost of enzyme mediated
reactions.
• Ability of enzymes to provide pure products.
• continuous process
• Possible provision of a better environment for enzyme
activity
METHODS OF IMMOBILISATION
•
•
•
•
Adsorption
Covalent binding
Entrapment
Membrane confinement
Methods of immobilizations
Immobilised enzyme systems. (a) enzyme non-covalently adsorbed to an
insoluble particle.(b) enzyme covalently attached to an insoluble particle
(c) enzyme entrapped within an insoluble particle by a cross-linked polymer
(d) enzyme confined within a semipermeable membrane.
Ref . file:///F:/F/IIT/iit%20m%207th%20dec%202011/Third%20sem/SEMINAR/SEMINAR/NOTES/NET/methods%20of%20immoblizatin.html
Adsorbents used for immobilization
•
•
•
•
•
•
Ion-exchange matrices
Porous carbon
Clays
Hydrous metal oxides
Glasses
Polymeric aromatic resins
Immobilizing enzymes on Sepharose
Sephalros - agarose (poly-{β-1,3-D-galactose-α-1,4-(3,6-anhydro)-L-galactose}) gel
Unflavored
Cellulose as support and ethyl chloroformate as activator
Matrix as support and carbodiimide as activator
Unflavored
Ref -file:///F:/F/IIT/iit%20m%207th%20dec%202011/Third%20sem/SEMINAR/SEMINAR/NOTES/NET/methods%20of%20immoblizatin.html
List of commercial process
• Pharmaceutical companies Beecham (in the UK) and
Bayer (in Germany), in association with Lilly and
Dunnill at University College, London, immobilized
enzyme amidase which cleaves penicillin G or V to
penicillin nucleus.
• Isomersation of glucose to fructose
with
immobilized enzyme glucose isomerase , in the mid1980’s over a billion (109) kilograms of fructose were
produced (for the soft drink market) from corn syrup
in the USA alone.
Acrylamide production
• First commercial bulk chemical manufactured biocatalytically is Acrylamide
Acrylamide bio-catalytic process
Conventional catalyst - Cu-catalysis
Bio-catalyst - immobilized whole cells of Rhodococcus
rhodochrous
Enzyme - nitrile hydratase
Amount - 200000 t/a
Uses - water-soluble polymers and copolymers
Acrylamide by chemical and bio catalytic process
Aspect
Chemical process Bio catalytic process
Catalyst
Cu salt
nitrile hydratase whole
cells, immobilized on
poly(propenamide) gel
Energy input High
pH – 7, Temp 5 OC
Separation
Cu ion must be
No need
removed, difficult
Waste
Toxic
No waste
Purity
80%
99.9%
carried out by Nitto Chemical Corp., now part of the Mitsubishi Rayon Corp. japan.
chemical routes lack stereo-selectivity, bio-catalysis
are highly steroselective and enantioselective.
• Example – production of L-Methionine
separation of racemates of N-acetyl-dl-amino acids by
aminoacylase
• commercialized by Tanabe Seiyaku (Japan) in 1969
using the very first immobilized enzyme reactor
system using enzyme from Aspergillus oryzae fungus
which was immobilized on amino-exchange resin.
Laundry
On June 30, 2013 the
European Commission is
adopting a ban on
phosphates(STPP)in laundry
detergents
Laundry is normal household activity but washing at 30 oC rather than
60 or 40 oC the CO2 savings potential in Europe and the US is around
32 million tons – equivalent to the emission of 8 million cars.
Around 40 years ago, proteases were first added to detergents to
remove recalcitrant stains, cellulases in 1983 against grass, lipases in
1988 Against grease stains, and amylases against starchy residues.
Conclusion
• The quest for sustainable production (chemicals and
energy) favors biocatalysis.
• Can be Representative of Green Chemistry.
• Chiral intermediates made through biocatalysis are a
growing business.
• Major chemical companies have built up groups and
have arrived successfully at products.
• Biocatalysis is a dynamic area of research providing
many chances for innovation