Enzyme Process Technology 1 - Introduction

Enzyme Process Technology 1 - Introduction

Enzyme Process Technology
1 - Introduction
Antje C Spiess
DAAD Summer School, Mexico City, 27.-28.08.2012
Outline of today
Introduction (9:00 – 10:30)
• Welcome and presentations
• Outline of Enzyme Process Technology
• Enzymes as Protein Catalysts
Basic kinetics (10.45 – 12:30)
Advanced mechanisms (14:00 – 15:30)
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Who am I?
Prof. Dr.-Ing. Antje C. Spiess
RWTH Aachen University
Aachener Verfahrenstechnik – Enzyme process technology
[email protected],de
Professional CV
• 1989 – 1995 Dipl. in Process Engineering from TU Hamburg-Harburg,
Germany
• 1995 – 2000 PhD „Kinetically controlled synthesis of amoxicillin using
Penicillin amidase from E. coli“, TUHH Biotechnology II, Prof. Kasche
• 2000 – 2004 Process Engineer and Equipment Qualification Manager
at Procter & Gamble Pharmaceuticals, Weiterstadt, Germany
• 2004 – 2010 Habilitation „Model-based experimental analysis of
enzyme reaction in non-conventional media“, RWTH Aachen
University, Biochemical Engineering, Prof. Büchs
• Since 2010 Full professor in Enzyme Process Technology at RWTH
Aachen University
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Locations
Berlin
Dortmund
Aachen
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Facts of RWTH Aachen University
Founded in 1870
• 465 Professors
• 4,274 Academic Staff
• 2,364 Non-Academic Staff
• 32,240 Students in 9 Faculties:
- 17,229 in engineering (54 %)
- 7,856 in natural science (24 %)
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Team
Co-workers
Lab impressions
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Research in Enzyme Process Technology
Interaction
Enzyme-Environment
Interactive materials
Polymerenzyme-conjugation
Polymer-based
compartments
Enzyme models
Thermodyn.
models
Reaction kinetics
Modelling
Identification
Integrated
Enzyme processes
Reaction networks
Immobilisation
Online spectroscopy
Downstream processing
Online chromatography
Microreactors
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Our research topics
Quantitative description and prediction of enzyme processes
using non-conventional media
• (Ligno-)cellulose degradation in ionic liquids
Cellulases, laccase
• Gas/solid bioreactor
• Aqueous/organic mono- and biphasic reaction systems
Alcohol dehydrogenases
• Reaction networks in Thiamine catalysis
ThDP-dependent enzymes (Benzaldehyde lyases)
We have (nearly) always topics for
• Master theses, internships (research blocks), student coworkers
• …also for international students….
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Who are you and what are your expectation?
?
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Contents of the two days
Monday, 27.08.2012
Homogeneous enzyme
reactions
Tuesday, 28.08.2012
Heterogeneous enzyme
reactions
1. Enzymes are proteins –
things of beauty and joy
forever
2. Enzyme kinetic models –
basic kinetic analysis
3. Advanced enzyme kinetics –
progress curve analysis
4. Enzyme stability and
immobilization
5. In situ analysis of reactiondiffusion problems
6. Outlook and Enzymes acting
on surfaces
Practical:
- Lecture slides and exercises on USB
- Exercises mixed with lectures, some with computers (Excel)
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(Further) reading
K. Buchholz, V. Kasche, U. Bornscheuer: Enzyme
technology. Wiley-VCH, 2005.
A. Bommarius, B. Riebel: Biocatalysis. Wiley-VCH, 2004.
A. Liese, K. Seelbach, C. Wandrey: Industrial Biotransformations.
Wiley-VCH, 2006.
K. Faber: Biotransformations in Organic Synthesis. SpringerVerlag, 2000.
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Definitions and Objectives
Enzyme process technology – What is that?
•
•
•
•
•
Molecular biology – tools to vary enzymes
Biochemistry – enzyme characterization and mechanisms
Enzyme Technology – enzyme variation for applications
Bioorganic chemistry – enzyme use for organic synthesis
Technical Chemistry – enzyme use for larger synthesis
EPT Use engineering tools to design enzyme reactions
Objectives for this summer school
• Transfer the fascination for enzymes
• Show some useful tools and concepts
• Show some of our research examples
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Outline of today
Welcome and presentations
Outline of Enzyme Process Technology
Enzymes as Protein Catalysts
•
•
•
•
Proteins
Catalysis
Enzymes as catalysts
Enzyme technology
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Central dogma of molecular biology
Information flow from coding DNA to (desired) protein (Crick,
1958)
Genetic code
Amino acids
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http://en.wikipedia.org/wiki/File:Central_Dogma_of_Molecular_Biochemistry_with_Enzymes.jpg
Genetic code (RNA)
Nucleic acids
A - Adenin
C - Cytosin
G – Guanin
U – Uracil RNA
T – Thymin DNA
yield
to be read from inside out:
AUG Methionine (Met, M)
Amino acids
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http://scienceblogs.com/oscillator/2010/02/expanding_the_genetic_code.php
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Amino acid overview
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http://upload.wikimedia.org/wikipedia/commons/3/37/Aa.svg
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Amino acid overview – cont‘d
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Amino acid properties
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http://www.unc.edu/~bzafer/aminoacids/
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From primary to quarternary structure
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Catalysis – key technology for sustainability
On catalysis depend directly or indirectly:
• > 15 – 20 % of total market
• > 80 % of added value of chemical industry
• > 90 % of all chemical processes
Döbereiner‘s
Lighter
1823
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VCI: Positionspapier Katalyse, 2002
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Catalyst types
+
Catalyst
anorganic
organic
biological
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Principle of catalysis
Low activation energy
Catalytic cycle
Substoichiometric use
Selectivity
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Free energy of un- vs. catalysed reaction
Thermodynamics studies the properties of state
Catalysts increase reaction rate by
- lowering the free energy of the transition state G≠
- lowering the activation energy ∆G≠
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Lehninger, Biochemistry, Figs. 6-2, 6-6
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Stickase thought experiment
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Lehninger, Fig. 6-5. ∆GM is here equal to ∆∆G#
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How do enzymes work?
Principles of enzyme function
1. Reduction of transition state entropy
Or: Reactant molecule orientation
for reaction
2. General acid-base catalysis
3. Covalent catalysis
4. Metal ion catalysis
Enzyme structural terms
• Active site :=
binding site + catalytic triad
• Holoenzyme :=
apoenzyme + cofactor
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Entropy reduction
• Comparison of
measured rates for
inter- and intramolecular catalysis
• Rate enhancement
correspond here to
effective
concentrations
Translated to enzyme
catalysis
• := Restriction of the
relative motions
• := Binding of
substrates to the
enzyme in orienting
them for reaction
• …will enhance rates
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Lehninger Fig 6-7
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General acid/base catalysis
:= Mediation of a proton transfer to stabilise an intermediate
in a biochemical reaction.
pKa
4.1, 3.9
10.5, 12.5
8.2
6.0
very variable
10.1
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Lehninger, Fig. 6-9
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Covalent catalysis
:= Formation of a transient
covalent bond between
enzyme and substrate.
Typical groups involved:
Amino Acids
•
•
•
•
•
Aspartate
Histidine
Cysteine
Lysine
Serine (acyl-groups)
Coenzymes
Vitamin B12
• Thiamine diphosphate
(aldehydes)
• Pyridoxal phosphate
(amino groups)
• Vitamin B12 (alkyl)
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Lehninger
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Metal ion catalysis
:= Orientation of substrate
for reaction via metalsubstrate bond:
Metalloenzymes
:= Mediation of oxidationreduction reactions:
Metal coenzymes
Fe-S cluster of Aconitase:
Metalloenzyme
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Lehninger
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Classification of
(Fast rising) number of
characterised enzymes;
Available enzymes ~10-30 % thereof
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Pro‘s and Con‘s of biocatalysis
Pro:
Mild conditions
Selectivity (chemo-, regio-, stereo-)
Con‘s:
Limited substrate acceptance
Limited availability
Limited number
Limited stability
Limited space-time-yields
Long development cycles
overcome
under investigation
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„The“ Pro: Chirality
88% of biocatalytic processes involve chiral targets.
• “Let a biocatalyst do a job that no chemocatalyst can.” (Ikunaka
M. Catal. Today 96:93-102 (2004))
Market value of drugs based on chiral building blocks:
• 100 Mrd. US$ (Chem Eng News 2005)
Sales value of chiral building blocks
• 9.5 Mrd. US$ (2005), 14.9 Mrd. US$ (2009) (Frost & Sullivan
2005)
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Diversity of biochemical pathways
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http://www.expasy.ch/cgi-bin/show_thumbnails.pl
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Things of beauty and joy forever…
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Sanderson K. Enzyme expertise. Nature 471:397-398 (2011).
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Some citations therein
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Sanderson K. Enzyme expertise. Nature 471:397-398 (2011).
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Colours of biotechnology
Red Biotechnology
(Medicinal biotechnology)
Healthcare:
• Diagnostics
• Therapeutics
• Vaccines
Green Biotechnology
(Plant biotechnology)
Agriculture:
• Plant protectants
• Feed
• Seeds
Bioeconomy
Blue Biotechnology
(Marine biotechnology)
Marine organisms & processes
• Building blocks
• Deep sea biocatalysts
• Silicate materials
Brown (grey) Biotechnology
(Environmental biotechnology)
• Land reclamation
• Waste water treatment
Biotechnology
(Industrial biotechnology)
Chemistry:
• Fine chemicals
• Building blocks
• Amino acids
• Vitamins
Yellow Biotechnology
(Food biotechnology)
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http://www.chemie.de/news/d/87323/
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Biocatalysts used in industrial biotransformations
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Straathof et al. Curr. Opin. Biotechnol. 13:548-556, 2002
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Which type of biocatalyst to choose?
Cells
• self-replicating
• side-products
• mass transfer limitations
Enzymes
• purification necessary
• better control possible
• cofactor regeneration
Free enzymes
Immobilised enzymes
• single use / complex reactor
• limited stability
• reuse
• diffusion limitations
• oxidoreductions
• macromolecules
• hydrolysis reactions
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Reactors used in industrial biotransformations
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Straathof et al. Curr. Opin. Biotechnol. 13:548-556, 2002
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Important products from enzyme processes
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Buchholz et al. Wiley, 2005
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Example 1: Production of acrylamide building blocks
Mitsubishi-Rayon process: Biocatalytic process (~30.000 t/a)
Patent ascribed to
Nitton Chemical Industry:
Plastics, additives
Nitrile hydratase
(Rhodococcus rhodochrous)
CN
Acrylonitrile
+H2O
NH2
O
Acrylamide
•
•
•
•
Elimination of sulphuric acid and copper catalysts
Increase of yield (> 99 %) and purity
Facilitation of downstream processing
Decrease of energy costs (process temperature: 5°C):
0.4 MJ/kg instead of 1.9 MJ/kg
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Example 2: Non-natural amino acids
Degussa-Hüls process: Reductive amination of keto acids
Leucine dehydrogenase
(Bacillus sphaericus)
COOH
+ NH3,
O
Trimethyl
pyruvic acid
H2O
+
NADH+H+
COOH
NH2
L-tert.-leucine
STY: 65.07 g*L-1
Alternative products:
•
•
•
•
Neopentylglycine
3,3-Dimethylpropane glycine
3-Ethyl-3-methyl-propane glycine
5,5-Dimethyl-butyl glycine
Enzyme Membrane Reactor,
Deutsches Museum Berlin
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Example 3: Sitagliptin
Codexis: Feasibility of biocatalytic route to
antidiabetic pharmaceutical Sitagliptin
• Stepwise increase of binding pocket
in existing transaminase functionality
No rhodium catalyst residuals
Stereoselectivity
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Example 4: De-novo design of a stereoselective Diels-Alderase
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Siegel, J.B. et al. Science 329: 309-313 (2010)
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Example: De-novo design of a stereoselective Diels-Alderase
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Siegel, J.B. et al. Science 329: 309-313 (2010)
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Example: De-novo design of a stereoselective Diels-Alderase
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Siegel, J.B. et al. Science 329: 309-313 (2010)
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In a nutshell…
What are enzymes?
• Catalytically active proteins
• Act via transition state stabilisation
• Use acid/base, covalent, metal catalysis
Why would you want to use enzymes?
• High chemo-, regio- and stereoselectivity
• Broad versatility and variability (manipulation of genetic code)
Where and how can you apply enzymes?
• Free or in cells, diverse reactors
• Application range: Biotechnology (bulk and fine chemicals,
pharmaceuticals, food, materials)
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