1. Enzyme Engineering 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 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)

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

5. 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”

6. 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

7. 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)

8. 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

9. 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

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
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

12. 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

13. 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

14. 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

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
>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

16. 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

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)

19. 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

20. Products β
A. Straathof, Panke S., Schmid A. (2002) Curr. Opin. Biotechnol. 13:

21. 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:

22. 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%

23. 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

24. 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:

25. (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:

26. 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)

27. What productivity is needed for synthetic applications ?
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
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)
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:

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)