1 Introduction.ppt [호환 모드] - SNU OPEN COURSEWARE

Enzyme Engineering

1. Introduction

1.1 History of Enzyme Engineering

1.2 Background of Enzyme Engineering

1.3 Fundamentals of Protein Chemistry

1.1 History of Enzyme Engineering

Enzyme

http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Enzyme_engineering

• 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 metabolitesto allow new (catalyzed) pathways for reactions to occurto convert from some certain compound into others (biotransformation)

History of BiotechnologyB.C.

Biotechnology used for bread, beer using yeast (Egypt)

Production of cheese, wine (Sumeria, China and Egypt)

History of Biotechnology1797 – First vaccination

Edward Jenner(1749 – 1823)

- English scientist

- Pioneer of smallpox vaccine

- “Father of Immunology”

1865 – Mendelian inheritanceGregor Johann Mendel(1822 – 1884)

- Austria – Hungarian scientist and Augustinian priest

- Known for discovering genetics

- “Father of Genetics”

History of Biotechnology1877 – 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” modelHermann Emil Fischer(1877 – 1947)

- German chemist

- Proposed the substrate and enzyme interaction

- The winner of the 1902 Nobel Prize in Chemistry

History of Biotechnology1928 – 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 insulinFrederick Sanger(1918 - )

- English biochemist

- Twice a Nobel laureate in chemistry(1958/1980)

History of Biotechnology

1978 – Recombinant DNAStanley Norman Cohen(1935 -)

Herbert W. Boyer(1936 -)

-American geneticist

- Developed the method of genetic engineering technique

1953 – Proposed DNA structureJames D. Watson(1928 -)

Francis Crick(1916 – 2004)

- Proposed DNA structure

- Awarded jointly the 1962 Nobel Prize

for Physiology or Medicine

History of Biotechnology

1985 – Site-directed mutagenesis

1988 – Invention of PCR

Michael Smith(1932 – 2000)

- British-born Canadian biochemist

- Established site-directed mutagenesis

- The winner of 1993 Nobel Prize in Chemistry

Kary B. Mullis(1944 -)

- American biochemist

- Delevoped polymer chain reaction(PCR)

- The winner of 1993 Nobel Prize in Chemistry

History of Enzyme Engineering1893 - Definition of term “catalyst” (Ostwald)

1894 - “Lock-and-key” model was proposed (Fischer)

1897 - Demonstrated that enzymes do not require a cell(Buchner)

1926 - Enzyme is proved to be a protein (Sumner)

1958 - “Induced fit” model was proposed(Koshland)

1963 - The first amino acid sequence of ribonuclease was reported

1965 - “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

History of Enzyme Engineering8 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

Enzyme Technology vs. Chemical Technology

Advantages DisadvantagesHigh degree of selectivityEnvironmentally friendlyCatalyze broad spectrum of reactionsLess byproductsNon-toxic, non-flammable

Too expensiveToo unstableProductivities - too low

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

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 http://www.chem.qmul.ac.uk/iubmb/enzyme

Industrial EnzymesProduction 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

Chemical & Enzymatic ReactionsReaction EC Number Enzyme

Meerwein-Ponndorff-Verley reduction 1.1.1.1 Alcohol dehydrogenase

Baeyer-villiger oxidation 1.14.13.22 BV monooxidase

Ether cleavage 1.14.16.5 Glyceryl etherase

Disproportionation 1.15.1.1 Superoxide dismutase

Etherification 2.1.1.6 COMT

Transamination 2.6.1.x Aminotransaminase

Oximolysis 3.1.1.3 Lipase

Aldol reaction 4.1.2.x Aldolase

Racemization 5.1.2.2 Mandelate racemase

Claisen rearrangement 5.4.99.5 Chorismate mutase

1.2 Background of Enzyme Engineering

Productivity & Biocatalysis

… Selectivity is only one important issue among others, which determine the usefulness of catalysts.

… organic chemists should pay more attention to E. Jacobsencatalyst productivity, activity, and recycling. M. BellerThese are key parameters for application, too. (Adv. Synth. Catal. 346, 2004)

Hydrolases in Industrial Biocatalysis

Prof. Dr. B. Hauer, BASF AG

SS--MOIPAMOIPA

Outlook®

New PlantGeismar/USA

Capacity: 2.500 t/a

OONHNH22

O

O

ClN

S

(Herbicide)

Carbohydrates

Fat derivatives

Steroids

Amino acids

sec-Alcohols

Nucleotides

Other chiral

Other non-chiral

Peptides / §-lactams

A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13:548-556.

Products

β

A. Straathof, S. Panke, and A. Schmid (2002) The production of fine chemicals by biotransformations.

Curr. Opin. Biotechnol. 13:548-556

pharma

several sectors

agro

feed

food

cosmeticspolymers

Biocatalysis - Product Markets

Biotransformations:What enzymes are used as catalysts?

A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13:548-556. IND.K. Faber (2000) Biotransf. in Org. Synthesis, Springer 4th ed. RESEARCH

Oxido- reductases

Transferases

HydrolasesLyases

Isomerases

Reducing cells

Oxidizing cells

25 %

~ 5%

65%

~ 5%

~ 1%

28%

4%

11%

45%

12%

H+lignin monomersorganics

H+ H+ H2O1/2 O2

Thiosulfate, H2

lignin monomersorganics

Chemoautotrophic

Chemoheterotrophic

Photoautotrophic

Photoheterotrophic

ATPN2 NH4

N2 NH4

Light

Light

H2

H2

H+

CO2

H+ H+ H2O1/2 O2Thiosulfate, H2

ATP

CH2O CH2O

CH2O CH2O

ATP ATP

CO2

- O2+ O2

(Larimer, Chain, Harwood et al. 2004 Nature Biotechnol. 22, 1:55-61)Genome analysis, Rhodopseudomonas palustris

UnknownBatch

Fed-batch

Continuous stirred tank

Continuous plug flow

A. Straathof, S. Panke, and A. Schmid (2002) The production of fine chemicals by biotransformations.

Curr. Opin. Biotechnol. 13: 548-556

Type of reactors used in industrial biotransformations

0 10 20 30 40 50 60

not reported

free enzymes

immobilized enzymes

free cells

immobilized cells

Number of processes

A. Straathof, S. Panke, and A. Schmid The production of fine chemicals by biotransformations.

(2002) Curr. Opin. Biotechnol. 13:548-556

Type of biocatalystin industrial biotransformations

Enzyme activity

phosphorylation

expression level

inhibitions (substrate, products, other)

stability / inactivation

glycosylation

Enzyme activity

Cofactor dep. enzymes

kcat Km STY [S, P] stability

typicalparameters

1-50 s-1 µM-mM< 1 g L-1 h-1

(10 g L-1 h-1)µM -mM

(M)sec. - hours( >> days)

cofactors(pH, redox, …)mechanism, kinetics

molecular dynamics

What productivity is needed

for synthetic applications ?

µg - gram / gram - kg / kg - ton

Space time yields - ranges

Biotech. Processes (g l-1 h-1)

Phenylethylamin 400 - 1000(enzyme)Acrylamide 400(enzyme)Acetate (ferment.) 5Citric acid (ferm.) 1Riboflavin (ferm.) 0.2

Chem. Processesheterogeneous catalysis (g l-1 h-1)

Acrylonitrile 10

Methanol 500 - 2000

NH3 1000 - 4000

Industrial (bio)processes

How good do we have to be ?(annual production is over 1 ton, in each case 1-14 processes evaluated)

Compoundclass

Biocatalysts /enzymes used

Volumetricproductivity

(g L-1 h-1)

Final productconcentration

(g L-1)Yield

%

amino acidsdecarboxylase,oxidoreductases,amidases, lyase 54.6 102 82

alcohols lipase, oxidoreductase,fumarase, k inase 4.2 107 88

carbohydrates transferase, amylases,aldolases 3 237 90

b-lactamsamidases, acylases,oxidase, lipase,peptidases 18.5 87 94

nucleotides lactamase, deaminase - 65 47

acidslipases, este rases,amidases, hydroxylases,oxygenase 1.7 108 81

epoxides oxygenase 1 7 90hydroxyaromatics

hydroxy lases 1.4 59 72

amines lipase, oxidoreductase 12.8 80 43.5

amides hydratase,oxidoreductases 42 225 96 (44 )

Straathof, Panke, Schmid 2002 Curr. Opin. Biotechnol. 13:548-556

1.3 Fundamentals of Protein Chemistry

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)