enzyme engineering 1.introduction 1.1 history of enzyme engineering 1.2 background of enzyme...
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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
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 metabolites to allow new (catalyzed) pathways for reactions to occur to convert from some certain compound into others (biotransformation)
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)
- 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”
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
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)
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 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 SteinAmino acid sequence of lysozyme and ribonuclease eluciated
1985 Michael SmithSite-directed gene mutagenesis to change enzyme sequence
1988 Kary B. Mullis Invention of PCR
Enzyme Technology vs. Chemical Technology
Advantages DisadvantagesHigh degree of selectivity
Environmentally friendly
Catalyze broad spectrum of reactions
Less byproducts
Non-toxic, non-flammable
Too expensive
Too unstable
Productivities - 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 Enzymes
Production scale Product Enzyme Company
>1,000,000High-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 AmpicillinPenicillin amidase
DSM-Gist Brocades
(S)-methoxyisopropylamine Lipase BASF
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
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
Prof. Dr. B. Hauer, BASF AG
S-MOIPAS-MOIPA
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 O2 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
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
typicalparameter
s1-50 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
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, kinase 4.2 107 88
carbohydrates transferase, amylases,aldolases 3 237 90
-lactamsamidases, acylases,oxidase, lipase,peptidases 18.5 87 94
nucleotides lactamase, deaminase - 65 47
acidslipases, esterases,amidases, hydroxylases,oxygenase 1.7 108 81
epoxides oxygenase 1 7 90
hydroxyaromatics
hydroxylases 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