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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