fine enzymes for fine chemicals waste not ant not … · 2018. 8. 13. · evocatal facts...
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FINE ENZYMES FOR FINE CHEMICALS
PASCAL DÜNKELMANN, JUNE, 18TH 2014
WASTE NOT, WANT NOT - PROFIT FROM SUSTAINABLE CHEMISTRY
APPLICATION OF BIOCATALYSIS TO IMPROVE SUSTAINABILITY IN INDUSTRIAL PRODUCTION
evocatal facts Introduction
DEVELOPMENT AND PRODUCTION OF INNOVATIVE ENZYMES FOR PHARMA AND FINE CHEMICALS
Our enzymes and production strains open alternatives to conventional synthetic routes.
• Founded in 2006 as a spin-off from Heinrich-Heine-Universität Dusseldorf
• 38 employees
• > 1.000 m2 of labs + pilot plant and offices
• Labs are in S1- and S2-standard
• Sustainably financed: High-Tech Grunderfonds
Sirius Seed Fonds
Business Angels
Series-B financing round closed in May 2013 (Lanxess, NRW.Bank)
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evocatal facts Business Model
Customized solutions
Standardized solutions
evocatal has a strong technology background in enzyme production and engineering
evoservices evoproducts
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evocatal facts Partners that trust in evocatal
evocatal has a strong technology background in enzyme production and engineering
evoservices evoproducts
[…]
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evocatal facts Products
evoproducts
Standardized solutions
• from screening kit scale…
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evocatal facts Products
evoproducts
Standardized solutions
• …to kg- and industrial (t-) scale
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evocatal facts evocatal technology platforms
Services and Core Technologies
Products
Enzymes - Fine chemicals - Chiral Pharma-Intermediates
Bio-Development internal
Chemical Development internal
Bulk- Production (Partners)
Bulk- Production (Partners)
Pilot- Production (inhouse)
Pilot- Production (inhouse)
Bioprocess- Development
Process- Development
Strain development
Enzyme optimization
Enzyme Discovery
external
external
Enzyme discovery - Metagenomics
- Datebase Mining
- Screening services
Enzyme engineering - Computer Modelling
- Directed Evolution
- Rational Design
Enzyme production - Expression optimization
- bioprocess development
- manufacturing (fermentation)
Process development - biocatalysis
- enzyme cascades
- tech transfer
Strain development - Metabolic engineering
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Synthesis of an API Intermediate Improvement of enantioselectivity
Alcohol Dehydrogenase
NADPNADPH + H+
Co-substrateCo-product
N
O
N
OH
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Synthesis of (R)-3-quinuclidinol (R-Qol)
● Target Drug:
- Solifenacin (Astellas) - becomes generic in 2018 - white spots exist already (e.g. Turkey) - Application: Overactive bladder
● Current synthesis of R-Qol
- Racemic resolution applying chiral auxiliary - Process no longer competitive
N O
O
N
Solifenacin
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Synthesis of an API Intermediate Improvement of enantioselectivity
Enzymatic Synthesis of R-Qol
● Identification of suitable enzyme:
- Wild-type enzymes and promising enzyme mutants from evocatal stock libraries were produced on small scale
- Produced enzymes were used in small scale reaction (1 mL)
- Performance of enzymes concerning conversion and selectivty was tested and analyzed
- Best enzyme: ADH200
- Conversion: 65 % - Enantiomeric excess: 89 %
Fermentation (production) of potential wild-type ADHs on small scale
Screening of enzymes for activity and enantio-selectivity
Analysis of performance
Enzyme X Enzyme Y Enzyme Z
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Synthesis of an API Intermediate Improvement of enantioselectivity
Enzymatic Synthesis of R-Qol ● Optimization of enzyme: Parameters
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First Sphere: • involved in „active Site“ • NO coordination of NAD • not directly involved in catalysis Second Sphere: • Neighbourhood to First Sphere -> remote effects • max. distance 10-15 Angstrom • no interference with important secondary structures (domains, multimer binding etc.) • preferably medium-sized groups for effects in both directions (smaller/larger)
M. T. Reetz, Angew. Chem. Int. Ed. Engl., Bd. 50, 1, 138–174, 2011.
Group 2
Group 3
Group 1
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Synthesis of an API Intermediate Improvement of enantioselectivity
Customer´s Challenge: Find an enzyme for the synthesis of (R)-3-quinuclidinol (R-Qol)
89 % ee 65 % conv. (24 h) 8 % subst. load
enzyme modeling 6 aa picked for mutagenesis high throughput screening
98 % ee 99% conv. (24 h) 30 % subst. load
99 % ee 99% conv. (24 h) 30 % subst. load
recombination of positive mutations
Alcohol Dehydrogenase
NADPNADPH + H+
Co-substrateCo-product
N
O
N
OH
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Synthesis of an API Intermediate Improvement of enantioselectivity
Process development for technical scale
● Optimization of Enzymatic Conversion
- Reactor type: Batch reactor - Enzyme preparation: Liquid crude extract (evo-1.1.200*) - 30 % substrate loading, 30 °C, pH = 7.0
● Goals achieved - Optimized catalyst (enzyme) could be evolved (CP > 99 %, ee > 99 %) - Process was transferred to CMO for large scale production
● Project successful
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Improvement of Thermostability From natural enzymes to process (bio-) catalysts
Temperature, pH, solvent (water vs. organic solvent), substrate concentration Stability in the „new“ environment is an important issue
Natural environment Process environment
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Improvement of Thermostability From Natural Enzymes to Process (Bio-) catalysts
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Current process • Enzyme is used in animal feed industry • Produced in Yeast • No structure or homology model available • Temperature stability drops rapidly > 44°C (0% activity @ 49°C)
→ Goal: Improvement of thermostability to 65°-75°C (> 50% residual activity)
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Wildtype (0 % at 49 °C)
D62 I159
I312
48
47
50
49
52
51
54
53
56
55
58
57
60
59
62
61
64
63
tem
pe
ratu
re in
cub
atio
n a
t [
°C]
V377
P394
A424
P429
A430 D473
I159/D62 I159/I312
I159/V377 I159/P394 I159/A424
I159/A430
I159/D463
I159/I312/A424 I159/P394/V377
I159/P394/V377
I199/I312/V377
I159/I312/P394
I159/I312/P394/V377
I159/I312/P394/T444/F372 I159/I312/P394/T444/V350/M244
I159/I302/P394/A424/A430/D62
I159/I312/P394/V377/D62/M244/V350/F372/A424/A430/T444/D473 12 mutations:
58 % residual activity at 64 °C (30 % at 66 °C)
+ in silico data (homology) → rational design
Screening of 30.000 clones + 3,5 °C
II: double substitutions + 6,5 °C
III: triple substitutions + 8,5 °C
IV: multiple substitutions + 10,0 °C
V: multiple combinations + 18,0 °C
recombinations
random mutagenesis
rational design
I: First Generation
- random mutagenesis of the whole gene
- high (3-4 substitutions) and low (1-2) rate
- synthetic DNA approach (high quality libaries)
II: Second Generation
- recombination of beneficial positions
III: Third Generation
- random mutagenesis of best variant
- additionally rational design
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Improvement of Thermostability Improvement of thermostability
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→ Improvement of thermostability to 65°-75°C (> 50% residual activity)
Current process • Enzyme is used in animal feed industry • Produced in Yeast • No structure or homology model available • Temperature stability drops rapidly > 44°C (0% activity @ 49°C)
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Enzymes for Bulk Applications Saving Ressources by Biocatalysis
Core technolgies
Services Exclusive development Research networks
Products Enzymes - Fine chemicals - Screenings
Bio-based chemicals & chiral Pharma-Intermediates
Enzyme discovery Enzyme optimisation Catalyst-Production Process development
- Metagenomics
- enzyme engineering
- evocatal Expression-Platform
- Implementation in production
plant
- Development and production
of industrial enzymes
- Chemical and Pharma
- bio-based economy
Value Chain
Agro-, Silvi-culture
Feedstock-Industry
(Bio-)Chemical Industry
Consumer-Industries
Seed
Corn
Sugarbeet
Plant / Biomass
Lignocellulose
Syngas CO, CO2,
H2 …
Sugar
Succinate
Isoprene
Amino Acid
1,4-Butandiol
L-tert.-Leucine
Polyisoprene
Polyester
Pharma Aktive
Rubber
Plastics
Adhesive
Pharmaceuticals
Tire
Bottle
Core Activities evocatal
Farnesene Terpene
Steroide Flavor & Fragrance
Diamine Polyurethane Insulation
Protein
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Enzymes for Bulk Applications Alliance „Functionalization of polymers“
Together with its partners evocatal develops enzymes and peptides that are suitable for functionalization of various polymers for specific applications in the industrial sectors construction chemicals, detergents and textiles.
Coordinator
Partner
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Enzymes for Bulk Applications Functionalization of polymers
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Enzymes for Bulk Applications Natural Polymers
O
OHHO
OH
OO
OH
HOOH
OHO
ORHO
OH
HOO
n
Lignin and Cellulose are the most abundant organic ressources in the world (ca. 50 Mio t/a Lignin are burned by pulp mills)
Wood or annual plants
no competition to feed and food chain
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Both polymers will be modified so that they are feasible as concrete additive
Enzymes for Bulk Applications Natural Polymers
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urb
anfreak.d
e
wikip
edia.d
e
Las Vegas Water consumption in construction projects is very high
53 Mio Liter are needed for a 240 m high skyscraper
Through the application of a high performance additives 15 % could be
saved
Corresponds to the daily water consumption of 60.000 residents (in
Germany ca. 130 L/day → 8 Mio Liter)
Concrete water requirement is covered by drinking water (No brackish
or rain water can be used)
Particularly relevant in arid regions with water deficiency
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Enzymes for Bulk Applications Natural Polymers
During the production process of concrete much CO2 is released. Chemical Reaction of chalk (lime rock) and clay to Calcium-Silicates Release of CO2 from chalk
CO2-Release of concrete industry worldwide (% worldwide CO2-Emissions)
36.7 Gt 4.5 Gt
Quelle: New Energy Finance, IEA
CO2-emissions will increase 2020 > 10%
36.7 Gt
1.8 Gt (4%)
1.8 Gt (28%)
4.5 Gt
CO2-Release of concrete industry worldwide (% worldwide industrial CO2-Emissions)
enhance concrete quality reduce CO2 release and water consumption substitute petroleum based additives
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Enzymes for Bulk Applications Synthetic Polymers - Detergents
Poly(ethylen-terephthalate), PET, Polyester
The impact of PET – Fibers for the
textile industry increase significantly
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2011: 36 Mio t PET
© Oerlikon 2011
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Enzymes for Bulk Applications Synthetic Polymers - Detergents
identification and improvement of enzymes which prevent or reduce pilling
make textiles more durable during washing cycles sustainable substitution of chemical ingredients eco friendly detergent for synthetic fibers
© H
enkel
PET made textiles show pilling and unraveling after washing cycles -> Enzymes to prevent such damage are not industrially applicable
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DWI, RWTH-Aachen Prof. Dr. Böker, Stomps , Brüx
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Enzymes for Bulk Applications Synthetic Polymers - Textiles
Various synthetic threads (e.g. Polypropylene, Polyethylene and others) are very difficult to modify and prevent the application in different products (or make processes harmful to the environment). -> Biotechnological functionalization should enable dying or the sealing of holes after the sewing process (amongst others)
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Enzymes for Bulk Applications Synthetic Polymers - Textiles
development of biotech-processes for functionalization enable the application of various synthetic threads functionalization without harmful chemical supplements functionalization at lower temperatures
Example: Functionalization of propylene in Fusion with Green Fluorescent Protein (eGFP)
Control
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Benefits from Biocatalysis Summary
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Application of biotechnology enables:
● A reduction of raw materials consumption
● A reduction of energy consumption
● A reduction of consumption of hazardous materials
● A reduction of CO2 emission
But the biotechnological tools have to be adapted to the technical and economical requirements. This is evocatal´s mission!
WE´RE LOOKING FORWARD TO YOUR SUCCESS!