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Enzyme Engineering Introduction 1.1 History of Enzyme Engineering
1.2 Background of Enzyme Engineering 1.3 Fundamentals of Protein Chemistry
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1.1 History of Enzyme Engineering
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Enzyme Enzyme Enzyme Engineering
- Enzymes are proteins that catalyze (i.e. increase the rate of) chemical reactions. Enzyme Engineering - Enzyme engineering is the application of (1) Modifying an enzyme’s structure (2) Modifying the catalytic activity of isolated enzymes to produce new metabolites to allow new (catalyzed) pathways for reactions to occur to convert from some certain compound into others (biotransformation)
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History of Biotechnology
B.C. Biotechnology used for bread, beer using yeast (Egypt) Production of cheese, wine (Sumeria, China and Egypt)
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History of Biotechnology
1797 – First vaccination Edward Jenner(1749 – 1823) - English scientist - Pioneer of smallpox vaccine - “Father of Immunology” 1865 – Mendelian inheritance Gregor Johann Mendel(1822 – 1884) Austria – Hungarian scientist and Augustinian priest Known for discovering genetics “Father of Genetics”
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History of Biotechnology
1877 – 1st alcoholic respiration with cell-free extract Eduard Buchner(1860 – 1917) - German chemist - The winner of the 1907 Nobel Prize in Chemistry for his work on fermentation 1894 – “Lock-and-key” model Hermann Emil Fischer(1877 – 1947) German chemist Proposed the substrate and enzyme interaction The winner of the 1902 Nobel Prize in Chemistry
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History of Biotechnology
1928 – Discovery of antibiotics Sir Alexander Fleming(1881 – 1955) - Scottish biologist & phamacologist - The winner of the 1945 Nobel Prize in Physiology or Medicine 1951 – Sequence determination of insulin Frederick Sanger( ) - English biochemist - Twice a Nobel laureate in chemistry(1958/1980)
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History of Biotechnology
1953 – Proposed DNA structure James D. Watson(1928 -) Francis Crick(1916 – 2004) Proposed DNA structure Awarded jointly the 1962 Nobel Prize for Physiology or Medicine 1978 – Recombinant DNA Stanley Norman Cohen(1935 -) Herbert W. Boyer(1936 -) American geneticist Developed the method of genetic engineering technique
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History of Biotechnology
1985 – Site-directed mutagenesis Michael Smith(1932 – 2000) - British-born Canadian biochemist - Established site-directed mutagenesis - The winner of 1993 Nobel Prize in Chemistry 1988 – Invention of PCR Kary B. Mullis(1944 -) - American biochemist - Delevoped polymer chain reaction(PCR) - The winner of 1993 Nobel Prize in Chemistry
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History of Enzyme Engineering
Definition of term “catalyst” (Ostwald) “Lock-and-key” model was proposed (Fischer) Demonstrated that enzymes do not require a cell(Buchner) Enzyme is proved to be a protein (Sumner) “Induced fit” model was proposed(Koshland) The first amino acid sequence of ribonuclease was reported “Allosteric model” of enzyme was proposed (Monod) 1970 – Immobilzed enzymes , HFCS 1980 – Protein engineering, chiral compounds Enzymes in organic solvent, polymers 1990 – Directed evolution 2000 – Computational designe of enzymes
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History of Enzyme Engineering
8 Nobel prize winners Year Who? What? 1877 Eduard Buchner 1st Alcoholic respiration with cell-free extract 1893 Wilhelm Ostwald Definition of term “catalyst” 1894 Emil Fischer “Lock-and key” concept 1926 James B. Sumner 1st Enzyme crystallized: urease from jack beans 1951 Frederick Sanger & Hans Tuppy Sequence determination of insulin β-chain 1963 Stanford Moore & William Stein Amino acid sequence of lysozyme and ribonuclease eluciated 1985 Michael Smith Site-directed gene mutagenesis to change enzyme sequence 1988 Kary B. Mullis Invention of PCR
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Enzyme Technology vs. Chemical Technology
Advantages Disadvantages High degree of selectivity Environmentally friendly Catalyze broad spectrum of reactions Less byproducts Non-toxic, non-flammable Too expensive Too unstable Productivities - too low
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Nomenclature The International Union of Biochemistry and Molecular Biology developed a nomenclature for enzymes, the EC number; EC number system 1st number – Class of the enzyme 2nd number – Subclass by the type of substrate or the bond cleaved 3rd number – Subclass by the electron acceptor or the type of group removed 4th number – Serial number of enzyme found
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Classification of enzymes
The top-level classification(1st number) EC 1 Oxidoreductases – Catalyze oxidation/reduction reactions EC 2 Transferases – Transfer a functional group EC 3 Hydrolases – Catalyze the hydrolysis of various bonds EC 4 Lyases – Cleave various bonds by means other than hydrolysis & oxidation EC 5 Isomerases – Catalyze isomerization changes within a single molecule EC 6 Ligases – Join two molecules with covalent bonds The complete nomenclature can be browsed at
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Industrial Enzymes Production scale Product Enzyme Company
>1,000,000 High-fructose corn syrup(HFCS) Glucose isomerase Various >100,000 Lactose-free milk Lactase >10,000 Acrylamide Nitrilase Nitto Co. Cocoa butter Lipase(CRL) Fuji Oil >1,000 Aspartame® Thermolysin Tosoh/DSM Nicotinamide Lonza >100 Ampicillin Penicillin amidase DSM-Gist Brocades (S)-methoxyisopropylamine Lipase BASF
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Chemical & Enzymatic Reactions
EC Number Enzyme Meerwein-Ponndorff-Verley reduction Alcohol dehydrogenase Baeyer-villiger oxidation BV monooxidase Ether cleavage Glyceryl etherase Disproportionation Superoxide dismutase Etherification COMT Transamination 2.6.1.x Aminotransaminase Oximolysis Lipase Aldol reaction 4.1.2.x Aldolase Racemization Mandelate racemase Claisen rearrangement Chorismate mutase
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1.2 Background of Enzyme Engineering
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Productivity & Biocatalysis
… Selectivity is only one important issue among others, which determine the usefulness of catalysts. … organic chemists should pay more attention to E. Jacobsen catalyst productivity, activity, and recycling M. Beller These are key parameters for application, too (Adv. Synth. Catal. 346, 2004)
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Hydrolases in Industrial Biocatalysis
S-MOIPA Outlook® New Plant Geismar/USA Capacity: t/a O NH2 Cl N S (Herbicide) Prof. Dr. B. Hauer, BASF AG
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Products β A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13:
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Curr. Opin. Biotechnol. 13:548-556
Biocatalysis - Product Markets A. Straathof, S. Panke, and A. Schmid (2002) The production of fine chemicals by biotransformations. Curr. Opin. Biotechnol. 13:
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Biotransformations: What enzymes are used as catalysts?
A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13: IND. K. Faber (2000) Biotransf. in Org. Synthesis, Springer 4th ed. RESEARCH 25 % ~ 5% 65% ~ 1% 28% 4% 11% 45% 12%
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Chemoautotrophic Photoautotrophic Chemoheterotrophic
Genome analysis, Rhodopseudomonas palustris (Larimer, Chain, Harwood et al Nature Biotechnol. 22, 1:55-61) Chemoautotrophic Photoautotrophic H+ Light CO2 CO2 ATP ATP N2 NH4 CH2O CH2O H2 Thiosulfate, H2 Thiosulfate, H2 H+ H+ 1/2 O2 H2O + O2 - O2 lignin monomers organics H+ lignin monomers organics Light Carrie Harwood Rhodopseudomonas palustris example Diversity of metabolism Chemo - lito - autotrophy etc Link to genomics -->> Genomics--> accessability of new functionality Where do we get enzymes --> today ? Commercial suppliers, Culture collections, own production, nature --> tomorrow? Understanding of structure Synthesis from scratch (Steve Mayo, current limit : 150aa?) ATP ATP N2 NH4 CH2O CH2O H2 H+ H+ O2 H2O 1/2 Chemoheterotrophic Photoheterotrophic
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Curr. Opin. Biotechnol. 13: 548-556
Type of reactors used in industrial biotransformations A. Straathof, S. Panke, and A. Schmid (2002) The production of fine chemicals by biotransformations. Curr. Opin. Biotechnol. 13:
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(2002) Curr. Opin. Biotechnol. 13:548-556
Type of biocatalyst in industrial biotransformations A. Straathof, S. Panke, and A. Schmid The production of fine chemicals by biotransformations. (2002) Curr. Opin. Biotechnol. 13:
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Enzyme activity kcat Km STY [S, P] stability cofactors (pH, redox, …)
mechanism, kinetics molecular dynamics phosphorylation Enzyme activity stability / inactivation expression level glycosylation inhibitions (substrate, products, other) Cofactor dep. enzymes kcat Km STY [S, P] stability typical parameters 1-50 s-1 µM-mM < 1 g L-1 h-1 (10 g L-1 h-1) µM -mM (M) sec. - hours ( >> days)
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What productivity is needed for synthetic applications ?
µg - gram / gram - kg / kg - ton What productivity is needed for synthetic applications ?
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Space time yields - ranges
Industrial (bio)processes Biotech. Processes (g l-1 h-1) Phenylethylamin (enzyme) Acrylamide Acetate (ferment.) 5 Citric acid (ferm.) 1 Riboflavin (ferm.) 0.2 Chem. Processes heterogeneous catalysis (g l-1 h-1) Acrylonitrile 10 Methanol NH
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How good do we have to be ? (annual production is over 1 ton, in each case 1-14 processes evaluated)
Message: This is how good boicatalysts have to be in order to be interesting for technical applications !!! --> this makes the difference between biochemical characterizations of enzymatic activities (about enzyme reactions are described!) and rapplications of enzymes as biocatalysts in industry (about 140 processes are known , see earlier slides) !!!! Straathof, Panke, Schmid 2002 Curr. Opin. Biotechnol. 13:
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1.3 Fundamentals of Protein Chemistry
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Critical Thinking * Criteria of novel enzyme?
Examples of finding new function of enzymes? Relationship between the optimum temperature for growth and enzyme activity In vivo stability of enzymes World top enzyme producer - Novo (Denmark) - Genenco (USA)
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