Enzyme Engineering. 1. Introduction. 1.1 History of Enzyme Engineering. 1.2 Background of Enzyme Engineering. 1.3 Fundamentals of Protein Chemistry

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1 Enzyme Engineering 1. Introduction 1.1 History of Enzyme Engineering 1.2 Background of Enzyme Engineering 1.3 Fundamentals of Protein Chemistry

2 1.1 History of Enzyme Engineering

3 Enzyme Enzyme - 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)

4 History of Biotechnology B.C. Biotechnology used for bread, beer using yeast (Egypt) Production of cheese, wine (Sumeria, China and Egypt)

History of Biotechnology 1797 First vaccination Edward Jenner(1749 1823) -

Mendelian inheritance Gregor Johann Mendel(1822 1884) - Austria Hungarian

5 History of Biotechnology 1797 First vaccination Edward Jenner( ) - English scientist - Pioneer of smallpox vaccine - Father of Immunology 1865 Mendelian inheritance Gregor Johann Mendel( ) - Austria Hungarian scientist and Augustinian priest - Known for discovering genetics - Father of Genetics

History of Biotechnology 1877 1 st alcoholic respiration with cell-free extract Eduard

his work on fermentation 1894 Lock-and-key model Hermann Emil Fischer(1877 1947) - German

6 History of Biotechnology st alcoholic respiration with cell-free extract Eduard Buchner( ) - German chemist - The winner of the 1907 Nobel Prize in Chemistry for his work on fermentation 1894 Lock-and-key model Hermann Emil Fischer( ) - German chemist - Proposed the substrate and enzyme interaction - The winner of the 1902 Nobel Prize in Chemistry

Nobel Prize in Physiology or Medicine 1951 Sequence determination of insulin

7 History of Biotechnology 1928 Discovery of antibiotics Sir Alexander Fleming( ) - 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)

the 1962 Nobel Prize for Physiology or Medicine 1978 Recombinant DNA Stanley Norman

8 History of Biotechnology 1953 Proposed DNA structure James D. Watson(1928 -) Francis Crick( ) - 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

History of Biotechnology 1985 Site-directed mutagenesis Michael Smith(1932 2000) -

1993 Nobel Prize in Chemistry 1988 Invention of PCR Kary B.

9 History of Biotechnology 1985 Site-directed mutagenesis Michael Smith( ) - 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

10 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

11 History of Enzyme Engineering 8 Nobel prize winners Year Who? What? 1877 Eduard Buchner 1 st Alcoholic respiration with cell-free extract 1893 Wilhelm Ostwald Definition of term catalyst 1894 Emil Fischer Lock-and key concept 1926 James B. Sumner 1 st Enzyme crystallized: urease from jack beans 1951 Frederick Sanger & Hans Tuppy Sequence determination of insulin β-chain 1963 Stanford Moore & William Stein 1985 Michael Smith 1988 Kary B. Mullis Invention of PCR Amino acid sequence of lysozyme and ribonuclease eluciated Site-directed gene mutagenesis to change enzyme sequence

12 Enzyme Technology vs. Chemical Technology Advantages High degree of selectivity Environmentally friendly Catalyze broad spectrum of reactions Less byproducts Non-toxic, non-flammable Disadvantages Too expensive Too unstable Productivities - too low

13 Nomenclature The International Union of Biochemistry and Molecular Biology developed a nomenclature for enzymes, the EC number; EC number system 1 st number Class of the enzyme 2 nd number Subclass by the type of substrate or the bond cleaved 3 rd number Subclass by the electron acceptor or the type of group removed 4 th number Serial number of enzyme found

14 Classification of enzymes The top-level classification(1 st 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

15 Industrial Enzymes Production scale Product Enzyme Company >1,000,000 High-fructose corn syrup(hfcs) Glucose isomerase Various >100,000 Lactose-free milk Lactase Various >10,000 Acrylamide Nitrilase Nitto Co. Cocoa butter Lipase(CRL) Fuji Oil >1,000 Aspartame Thermolysin Tosoh/DSM Nicotinamide Nitrilase Lonza >100 Ampicillin Penicillin amidase DSM-Gist Brocades (S)-methoxyisopropylamine Lipase BASF

16 Chemical & Enzymatic Reactions Reaction EC Number Enzyme Meerwein-Ponndorff-Verley reduction Alcohol dehydrogenase Baeyer-villiger oxidation BV monooxidase Ether cleavage Glyceryl etherase Disproportionation Superoxide dismutase Etherification COMT Transamination x Aminotransaminase Oximolysis Lipase Aldol reaction x Aldolase Racemization Mandelate racemase Claisen rearrangement Chorismate mutase

17 1.2 Background of Enzyme Engineering

18 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)

Hydrolases in Industrial Biocatalysis New Plant Geismar/USA Capacity: 2.

19 Hydrolases in Industrial Biocatalysis New Plant Geismar/USA Capacity: t/a S-MOIPA NH 2 O Outlook (Herbicide) S N O O Cl Prof. Dr. B. Hauer, BASF AG

20 Products Other non-chiral Other chiral Nucleotides sec-alcohols Amino acids Carbohydrates Fat derivatives Steroids Peptides / -lactams β A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13:

21 Biocatalysis - Product Markets cosmetics food polymers feed pharma agro several sectors A. Straathof, S. Panke, and A. Schmid (2002) The production of fine chemicals by biotransformations. Curr. Opin. Biotechnol. 13:

22 Biotransformations: What enzymes are used as catalysts? 28% 25 % Oxidizing cells Oxidoreductases Transferases ~ 5% 11% Reducing cells 4% ~ 1% Isomerases Lyases ~ 5% 12% Hydrolases 65% 45% A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13: IND. K. Faber (2000) Biotransf. in Org. Synthesis, Springer 4 th ed. RESEARCH

23 Genome analysis, Rhodopseudomonas palustris (Larimer, Chain, Harwood et al Nature Biotechnol. 22, 1:55-61) Chemoautotrophic H + Photoautotrophic Light CO 2 CO 2 ATP ATP CH 2 O CH 2 O N 2 NH 4 H 2 Thiosulfate, H 2 H + H + 1/2 O 2 H 2 O Thiosulfate, H 2 + O 2 - O 2 lignin monomers organics H + lignin monomers organics Light ATP ATP CH 2 O CH 2 O N 2 NH 4 H 2 H + H + H 2 O 1/2 O 2 Chemoheterotrophic Photoheterotrophic

24 Type of reactors used in industrial biotransformations Fed-batch Continuous plug flow Continuous stirred tank Batch Unknown A. Straathof, S. Panke, and A. Schmid (2002) The production of fine chemicals by biotransformations. Curr. Opin. Biotechnol. 13:

25 Type of biocatalyst in industrial biotransformations immobilized cells free cells immobilized enzymes free enzymes not reported Number of processes A. Straathof, S. Panke, and A. Schmid The production of fine chemicals by biotransformations. (2002) Curr. Opin. Biotechnol. 13:

26 Enzyme activity mechanism, kinetics cofactors (ph, redox, ) molecular dynamics Enzyme activity phosphorylation stability / inactivation expression level glycosylation inhibitions (substrate, products, other) Cofactor dep. enzymes k cat K m 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)

27 µg - gram / gram - kg / kg - ton What productivity is needed for synthetic applications?

28 Space time yields - ranges Industrial (bio)processes Biotech. Processes (g l -1 h -1 ) Phenylethylamin (enzyme) Acrylamide 400 (enzyme) Acetate (ferment.) 5 Citric acid (ferm.) 1 Riboflavin (ferm.) 0.2 Chem. Processes heterogeneous catalysis (g l -1 h -1 ) Acrylonitrile 10 Methanol NH

29 How good do we have to be? (annual production is over 1 ton, in each case 1-14 processes evaluated) Compound class amino acids Biocatalysts / enzymes used Volumetric productivity (g L -1 h -1 ) Final product concentration (g L -1 ) Yield % decarb oxylase, oxidoreductases, amidases, lyase alcohols carbohydrates b-lactams nucleotides acids lipase, oxidoreductase, fumarase, k inase transfe rase, amylases, aldolases amidases, acylases, oxidase, lipase, peptidases lactamase, deaminase lipases, este rases, amidases, hydr oxylases, oxygenas e epoxides oxygenas e hydroxy aromatics amines amides hydroxy lases lipase, oxidoreductase hydratas e, oxidoreductases (44 ) Straathof, Panke, Schmid 2002 Curr. Opin. Biotechnol. 13:

30 1.3 Fundamentals of Protein Chemistry

34 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)

Paper No.: 01. Paper Title: FOOD CHEMISTRY. Module 22: Enzymes: General nature and Kinetics of. enzyme reactions

Paper No.: 01. Paper Title: FOOD CHEMISTRY. Module 22: Enzymes: General nature and Kinetics of. enzyme reactions Paper No.: 01 Paper Title: FOOD CHEMISTRY Module 22: Enzymes: General nature and Kinetics of enzyme reactions Enzymes: General nature and kinetics of enzyme reactions INTRODUCTION Enzymes are defined as