Enzyme Engineering

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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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1.1 History of Enzyme Engineering

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Enzyme

http://en.wikipedia.org/wiki/Enzyme

http://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 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 – 1

^st

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(1918 - ) - English biochemist

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

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History of Biotechnology

1978 – Recombinant DNA

Stanley Norman Cohen(1935 -) Herbert W. Boyer(1936 -)

-American geneticist

- Developed the method of genetic engineering technique

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

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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

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History of Enzyme Engineering

1893 - 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

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History of Enzyme Engineering

8 Nobel prize winners

Year Who? What?

1877 Eduard Buchner 1^stAlcoholic 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 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

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

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

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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 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

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Chemical & Enzymatic Reactions

Reaction 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

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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

Prof. Dr. B. Hauer, BASF AG

S

S--MOIPA MOIPA

Outlook

^®

New Plant Geismar/USA Capacity: 2.500 t/a

O O NH NH

₂₂

O O N Cl

S

(Herbicide)

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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

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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

cosmetics

polymers

Biocatalysis - Product Markets

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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 4^thed. RESEARCH

Oxido- reductases

Transferases

Hydrolases Lyases

Isomerases Reducing

cells

Oxidizing cells

25 %

~ 5%

65%

~ 5%

~ 1%

28%

4%

11%

45%

12%

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H⁺

lignin monomers organics

H⁺ H⁺ 1/2 O₂ H₂O

Thiosulfate, H₂

lignin monomers organics

Chemoautotrophic

Chemoheterotrophic

Photoautotrophic

Photoheterotrophic

ATP

N₂ NH₄

Light

Light H₂

H₂

H⁺

CO₂

H⁺ H⁺ ^1/2 O₂ H₂O Thiosulfate, 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

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Unknown Batch

Fed-batch

Continuous stirred tank

Continuous plug flow

A. Straathof, S. Panke, and A. Schmid (2002)

Curr. Opin. Biotechnol. 13: 548-556

Type of reactors

used in industrial biotransformations

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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

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

Type of biocatalyst

in industrial biotransformations

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Enzyme activity

phosphorylation

expression level

inhibitions (substrate, products, other) stability / inactivation

glycosylation

Enzyme activity

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)

cofactors

(pH, redox, …) mechanism, kinetics

molecular dynamics

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What productivity is needed

for synthetic applications ?

µg - gram / gram - kg / kg - ton

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Space time yields - ranges

Biotech. Processes (g l

^-1

h

^-1

)

Phenylethylamin 400 - 1000 (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 500 - 2000

NH

₃

1000 - 4000

Industrial (bio)processes

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How good do we have to be ?

(annual production is over 1 ton, in each case 1-14 processes evaluated)

Compound class

Biocatalysts / enzymes used

Volumetric productivity

(g L^-1 h^-1)

Final product concentration

(g L^-1)

Yield

% amino acids decarb oxylase,

oxidoreductases,

amidases, lyase 54.6 ¹⁰² ⁸²

alcohols lipase, oxidoreductase,

fumarase, k inase 4.2 107 88

carbohydrates transfe rase, amylases,

aldolases 3 237 90

b-lactams amidases, acylases, oxidase, lipase,

peptidases 18.5 87 94

nucleotides lactamase, deaminase - 65 47

acids lipases, este rases,

amidases, hydr oxylases,

oxygenas e 1.7 108 81

epoxides ^{oxygenas e} 1 7 ⁹⁰

hydroxy

aromatics hydroxy lases 1.4 59 72

amines lipase, oxidoreductase 12.8 80 43.5

amides hydratas e,

oxidoreductases 42 225 96 (44 )

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

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1.3 Fundamentals of Protein Chemistry

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