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Bio-Catalysis
Presented by V surya kumar
CA11M005
National Centre for Catalysis Research Indian Institute of Technology, Madras
Contents o Challenges facing mankindo History of bio-catalysiso Enzymes and their classification o Advantages and disadvantages of bio-catalysiso Theories of enzyme action o Statistics of Bio-catalysis in industries o Immobilization o Commercial exampleso Conclusion
challenges facing mankind today
Maintenance and improvement of environment
Maintenance and improvement of human health
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) 1st enzyme crystallized: urease
1944 Linus Pauling 1st attempt to explain catalysis as transition-state 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 synthesisDiscovery of ion transporting enzyme
Aaron Ciechanover, Avram Hershko,Irwin Rose (Nobel price in 2004)
"for the discovery of ubiquitin-mediated protein degradation"
Brief History
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 .
Carboic anhydrase With Zn +2 ion cofactor
Heam as prosthetic group in catallase
IUBMB Classification of enzymes
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
PROCESS Time period (years)
acrylamide process
20
l-carnithineprocess
15
Number of catalyst known Process development periodLack of sufficient knowledge
• What is the source of the energy for the lowering of the activation energies?
• How these enzyme catalyzed reactions are highly selective?
E + S ES EP E + P
‘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 theoryIt 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.
catBuncat GGG
Enzyme optimizes ∆GB by providing functional
groups using amino acidsPresent on proteins in the special cavity Called Active Site (AS) .
Now catalysis is achieved, How specificity is achieved is the next question?
Ref. .Lehninger's 4TH Ed
Factors needed for specificity•Proper alignment of catalytic functional groups•Entropy reduction – decrease in freedom of motion.•Distortion of substrate.•Charge redistribution.•Conformational change .----------------------------------Mechanisms for specificity•general acid-base catalysis• covalent catalysis•metal ion catalysis.
Rate enhancement
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 transferproviding 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.
A X A + XH2O
A XH2O
B: + A + X A + B: + XB
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 ofCollision(non-productive binding and keeps selectivity intact.
Ref. http://course1.winona.edu/sberg/ANIMTNS/ind-fit.htm
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
Statistics of Bio-catalysis in industries
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 two-phase 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.
DL-amino acidimmoblized enzyme
D-amino acid + L-amino acidaminoacylase
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 SepharoseSephalros - 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
Ref -file:///F:/F/IIT/iit%20m%207th%20dec%202011/Third%20sem/SEMINAR/SEMINAR/NOTES/NET/methods%20of%20immoblizatin.html
Unflavored
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 mid-1980’s over a billion (109) kilograms of fructose were produced (for the soft drink market) from corn syrup in the USA alone.
Glucoseimmoblized enzyme
FructoseGlucose isomerase
Acrylamide production•First commercial bulk chemical manufactured bio-catalytically is Acrylamide Acrylamide bio-catalytic processConventional catalyst - Cu-catalysisBio-catalyst - immobilized whole cells of Rhodococcus rhodochrous Enzyme - nitrile hydrataseAmount - 200000 t/aUses - water-soluble polymers and copolymers
Acrylamide by chemical and bio catalytic processAspect 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
removed, difficult No need
Waste Toxic No waste Purity 80% 99.9%
carried out by Nitto Chemical Corp., now part of the Mitsubishi Rayon Corp. japan.
O
NH2
acrylamide
H
O
H
N
acrylnitrile
+ nitrile hydratase
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.
NH
S
O
OH
ON-AC-DL-methionine
NH2
S
O
OH
L- methionine
Amioacylace
Laundry
On June 30, 2013 the European Commission is adopting a ban on phosphates(STPP)in laundry detergents
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.
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.
Na+P
O
O
OO-
S
sodium thio phosphate
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