fine enzymes for fine chemicals waste not ant not … · 2018. 8. 13. · evocatal facts...

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)

2 18.06.2014 RSC Symposium, Budapest

evocatal facts Business Model

Customized solutions

Standardized solutions

evocatal has a strong technology background in enzyme production and engineering

evoservices evoproducts


evocatal facts Partners that trust in evocatal

evocatal has a strong technology background in enzyme production and engineering

evoservices evoproducts

[…]


evocatal facts Products

evoproducts


• from screening kit scale…


evocatal facts Products

evoproducts


• …to kg- and industrial (t-) scale


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


Synthesis of an API Intermediate Improvement of enantioselectivity

Alcohol Dehydrogenase

NADPNADPH + H+

Co-substrateCo-product

N

O

N

OH

8

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

18.06.2014 RSC Symposium, Budapest


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



Enzymatic Synthesis of R-Qol ● Optimization of enzyme: Parameters

10

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



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



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


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


Improvement of Thermostability From Natural Enzymes to Process (Bio-) catalysts

14

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)


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

65


Improvement of Thermostability Improvement of thermostability

16

→ 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)


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


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


Enzymes for Bulk Applications Functionalization of polymers


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


Both polymers will be modified so that they are feasible as concrete additive


21

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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



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


Enzymes for Bulk Applications Synthetic Polymers - Detergents

Poly(ethylen-terephthalate), PET, Polyester

The impact of PET – Fibers for the

textile industry increase significantly

23

2011: 36 Mio t PET

© Oerlikon 2011


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

24

DWI, RWTH-Aachen Prof. Dr. Böker, Stomps , Brüx


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)


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


Benefits from Biocatalysis Summary


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!

fine enzymes for fine chemicals waste not ant not … · 2018. 8. 13. · evocatal facts...

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