Development and scale-up

of efficient biocatalytic

oxidations using oxygen

EU Horizon2020 demonstration workshop:

New developments in industrial biocatalysis

Frankfurt, 14 February 2019

Dr. Martin Schürmann

Principal Scientist Biocatalysis at InnoSyn B.V.

www.innosyn.com

martin.schuermann@innosyn.com

EU Horizon 2020 Innovation Action “ROBOX”

Expanding the industrial use of Robust Oxidative Biocatalysts

for the conversion and production of alcohols.

“In order to achieve the widening of industrial application of enzymatic

biooxidation processes, ROBOX will demonstrate the techno-economic

viability of biotransformations of four types of oxidative enzymes”

Coordinator: Marco Fraaije, RU Groningen

“The research for this work has received funding from the European Union (EU) project ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1. Any statement made herein reflects only the author’s views. The European Union is not liable for any use that may be made of the information contained herein.”

InnoSyn

A new, independent company since 1 May 2017.

A new name, however, InnoSyn has over 25 years of experience and history as

chemical R&D group at DSM, both fundamental and process research (~50% PhD).

A team of passionate & experienced problem solvers, applying innovative and

cost-efficient technologies (incl. biocatalysis) to advance competitiveness of our customers across the (fine) chemical industries.

Track record in successful development and implementation of new and

improved chemical processes for all market segments:

- Agro - - Pharma - - Flavor & Fragrances -

- Specialty Chemicals - - Food additives -

- Sweeteners - - Dyes - - Fuel Additives -

- Functional Monomers - - Biobased Monomers -

EU Horizon 2020 Innovation Action “ROBOX”

Expanding the industrial use of Robust Oxidative Biocatalysts

for the conversion and production of alcohols.

“In order to achieve the widening of industrial application of enzymatic

biooxidation processes, ROBOX will demonstrate the techno-economic

viability of biotransformations of four types of oxidative enzymes”

Coordinator: Marco Fraaije, RU Groningen

“The research for this work has received funding from the European Union (EU) project ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1. Any statement made herein reflects only the author’s views. The European Union is not liable for any use that may be made of the information contained herein.”

ROBOX Target Enzyme Classes

ROBOX Demonstration Cases at

InnoSyn, DSM & Givaudan

Vitamin intermediate (DSM/InnoSyn)

Vitamin intermediate (DSM/InnoSyn)

API metabolite (DSM/InnoSyn)

Pharma or F&F intermediate (InnoSyn)

F&F product (Givaudan)

ROBOX Demonstration Cases at

InnoSyn, DSM & Givaudan

F&F product (InnoSyn)

Specialty polymer (InnoSyn →

Univ. Maastricht & ChemStream)

Performance polymer (DSM/InnoSyn)

F&F product (Givaudan)

ROBOX Structure & ApproachTechnical Work Packages

WP1

Enzyme Identification &

Engineering

RU Groningen

WP2

Enzyme Production

TU Graz

WP3

Process design & validation

UA Barcelona

WP4

Demonstration (100 L / 1 kg)

DSM/InnoSyn

Feedback e.g. on inhibition/stability/activity Parameters to optimize

Enzyme formulation

DSP

WP5 Techno-economic and environmental Evaluation (Denmark TU, Lyngby)

WP5 Benchmarking & Evaluation: Definition of Process Metrics

Process metrics Definition Unit Effect on

Reaction conversion𝑛𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒,𝑖𝑛𝑖𝑡𝑖𝑎𝑙 − 𝑛𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒

𝑛𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒,𝑖𝑛𝑖𝑡𝑖𝑎𝑙× 100 %

Raw material

costs

Reaction yield𝑛𝑝𝑟𝑜𝑑𝑢𝑐𝑡, − 𝑛𝑝𝑟𝑜𝑑𝑢𝑐𝑡,𝑖𝑛𝑖𝑡𝑖𝑎𝑙

𝑛𝑠𝑢𝑏𝑠𝑡𝑟𝑎𝑡𝑒,𝑖𝑛𝑖𝑡𝑖𝑎𝑙× 100 %

Raw material

costs

Vol. Productivity (STY)𝑚𝑝𝑟𝑜𝑑𝑢𝑐𝑡

τ × 𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟g L-1 h-1 Reactor costs

Product concentration𝑚𝑝𝑟𝑜𝑑𝑢𝑐𝑡

𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟g L-1 DSP costs

Biocatalyst yield𝑚𝑝𝑟𝑜𝑑𝑢𝑐𝑡

𝑚𝑏𝑖𝑜𝑐𝑎𝑡𝑎𝑙𝑦𝑠𝑡gprod gcww

-1 Enzyme costs

Biocatalyst loading𝑚𝑏𝑖𝑜𝑐𝑎𝑡𝑎𝑙𝑦𝑠𝑡

𝑉𝑟𝑒𝑎𝑐𝑡𝑜𝑟g L-1 Enzyme costs

*The biocatalyst yield is given on a g product / g cell wet weight equivalents (cww) basis

WP5 Benchmarking & Evaluation: Target Setting

Product value

category

Low

value

Medium

value

High

value

Very high

value

Price range [€/Kg] 5 20 100 1000

Example industryMonomers,

vitamins

Fine / Specialty

Chemicals

Pharma

Chemicals

Pharma

Metabolites

Reaction yield [%] 80-100 80-100 80-100 80-100

Productivity [g/L/h] 20 10 2 1

Product concentration

[g/L]100 50 10 5

Biocatalyst yield

[g/gcww]*5 5 1 0.1

*The biocatalyst yield is given on a g product / g cell wet weight equivalents basis

Examples of ROBOX

Demonstrations at InnoSyn

1. P450 hydroxylation of Diclofenac

2. BVMO oxidation of Trimethyl-cyclohexanone

3. Alcohol Oxidase oxidation of Vanillyl alcohol

4. Alcohol Dehydrogenase oxidation of Lactol

InnoSyn Reactors for Biooxidations

• Applied reactor setup at InnoSyn from 30 to 1000 mL scale

• Used for reaction characterization and parameter optimization- pH, temperature, solvents, emulsifiers, mixing etc.

- Biocatalyst loading and formulation & oxygen supply

Applied reactor setup on 1 L scale. a) stirrer, b) pH electrode, c) reflux-condenser, d)

controlled air supply, e) reactor, f) automatic titration device connected to pH

electrode, g) gas washing bottle filled with water, h) oxygen sensor in gas outlet,

i) oxygen sensor in reaction mixture, j) cooling trap.

Pilot Plant: From 1 L to100 L

pH electrode

O2 electrode

200 L

reactor

pH & O2

O2

pH Stat

5 M NaOH

O2

dissolved

O2

headspace

Flowmeters O2& N2

→ (Almost) same setup as on 1 L scale

Demonstration case 2:P450 Production of Diclofenac Metabolites

• Demonstrate Cytochrome P450 process on 100 l scale

• Target: Production of > 100 g hydroxylated diclofenac

• Application of P450-BM3 mutant from combinatorial mutant library

• Cofactor regeneration via co-expressed glucose dehydrogenase (GDH)

* A. Mancy et al., “Diclofenac and Its Derivatives As Tools for Studying Human Cytochromes P450 Active

Sites: Particular Efficiency and Regioselectivity of P450 2Cs”. Biochemistry 1999, 38, 14264-14270

Application of P450-BM3 variant 22C02

Conditions: T = 28°C,

aeration: 8 mL/min, Vtotal = 30 mL, 13 mMdiclofenac (72 mMstock solution), 0.5 M D-glucose, 1 mMNADP, 1 mg/mL GDH, 3 mL P450 CFE/WC

Dosing approach beneficial (avoids substrate inhibition)

Whole cell preparation performs properly

1.8 g/l diclofenac converted

Selectivity for 4’-OH-diclofenac: ~ 74%

Scale-up: 800 mL reaction

Substrate addition in portions of 160 mM in 15 mL pre-heated solution

Reaction progress can reasonably be followed by oxygen electrode

Activities up to 0.51 U/mLcell suspension

2103 mg 4’-OH-D and 923 mg 5-OH-D

Conditions: T = 28°C, Vtotal =

800 mL, pH 7.2, 80 mL/min air, n = 300 rpm; 15.6 mmoldiclofenac, 0.5 M D-glucose, 0.5 mM NADP, 160 mL WC22C02_combi

suspension, 5 mM KPi pH 7.5

Aeration with pure O2

Conditions: T = 28°C, Vtotal =

800 mL, pH 7.2, 40 mL/min O2, n = 300 rpm; 10 mmoldiclofenac, 0.5 M D-glucose, 0.5 mM NADP, 160 mL

WC22C02_combi suspension, 5 mM KPi pH 7.5

High initial activity (> 1.0 U/mLcell suspension)

Significantly decreased stability!

1101 mg 4’-OH-D and 442 mg 5-OH-D (regioselectivity 70%)

The space-time yield 0.56 g/l/h

Final product concentration of around 3 g/l or 10 mmol/l

Total turnover number of 2750 moldiclofenac/molP450

Conditions: T = 28°C,

Vtotal = 100 L, pH 7.2, 4 L/min air, n = 300 rpm; 1.05 mol diclofenac in portions, 0.5 M D-glucose, 0.5 mMNADP, 2x ~ 5 L 22C02_combi CFE, 24 g antifoam, 5 mM KPi

pH 7.5

Pilot plant: Batch 2

1. Addition of 50 vol.-% methanol @ 55°C overnight

2. Addition of dicalite as body feed

3. Filtration over Seitz filter to precipitate enzyme/cells

4. Evaporation of methanol

5. H2SO4 addition until pH 2.0

6. Extraction with ethyl acetate (volume ratio: ~ 1:1)

7. Rotating evaporator

→ Product yield Batch 1: ~ 88%

→ Product yield Batch 2: ~ 92%

Pilot plant: Product Isolation

Demonstration case 4:Cyclohexanone derivative oxidation with BVMO

• BVMO: TmCHMO from Thermocrispum municipale

• Identified by Univ. Groningen

• 3D-structure solved at Univ. Pavia

• Screening and reaction optimisation by Univ. Maastricht

• Demonstration by InnoSyn to produce kg quantities

• Evaluation in polymer applications by Univ. Maastricht & ChemStream

• GDH: GDH-01 from InnoSyn

Conditions 30 mL scale: enzyme load 5% (v/v) of TmCHMO CFE or Broth or Whole Cells and 0.1 mg/mL of GDH-105 (Codexis); temperature 30°C; stirring rate 1200 rpm; air flow 16 mL/min; pH 8.0; [TMCH] 15mM h-1 (240 mM final); Methanol 0.625% (v/v) h-1 (10% (v/v) final); [D-Glucose] 375 mM; [NADP+] 0.25 mM; titration solution 1 M NaOH.

0

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CFE Whole Cells Broth Sonicated Broth

Initial rate (m

mo

l h-1)C

on

vers

ion

& Y

ield

(%

)

Conversion Yield Titration (%) Max. Rate

TmCHMO Biocatalyst Formulation

Conditions 30 ml scale: enzyme load 5% (v/v) of TmCHMO Broth and 0.1 mg/mL of GDH-105 (Codexis) or 0.5% (v/v) of GDH-01 (InnoSyn) ; temperature 30°C; stirring rate 1200 rpm; air flow 16 mL/min; pH 7.0 or pH 8.0; [TMCH] 15mM h-1 (240 mM final); Methanol 0.625% (v/v) h-1 (10% (v/v) final); [D-Glucose] 375 mM; [NADP+] 0.25 mM; titration solution 1M NaOH.

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100In

itial rate (mm

ol h

-1)

Co

nve

rsio

n &

Yie

ld &

Tit

rati

on

(%

)

Conversion Yield Titration (%) Initial rate

Comparison GDH-105 vs. GDH-01

GDH-105 pH 8.0 GDH-01 pH 7.0

Fermentative Biocatalyst Production TmCHMO and GDH-01

• From batch to high cell-density fermentations (HCDF) on 15 L scale

Mode Enzyme Fermentation time Biomass production Enzyme Activity

Batch TmCHMO 24 h 35 g/kg (cell wet weight) 0.16 U/mg

HCDF TmCHMO 100 h 375 g/kg (cell wet weight) 0.12 U/mg

HCDF GDH-01 96 h 310 g/kg (cell wet weight) 70-100 U/mg NAD(P)+

→ Sufficient amounts of active enzyme for 100 L demonstration

→ Some activity losses in last TmCHMO fermentation for pilot plant

GDH-01TmCHMO

Batch

HCDF

Pilot plant – 100 L reaction

Pilot plant: Demonstration 100 L

Conditions: enzyme load 10% (v/v) of TmCHMO Broth and 0.2% (v/v) of GDH-01 CFE temperature 30°C; stirring rate 150 rpm; O2 flow 3 L/min; pH7; [TMCH] 34.44 mM h-1(183.35 mM final); methanol 2.47% (v/v) h-1 (10% (v/v) final); [D-Glucose] 375 mM; [NADP+] 0.5 mM; titration solution 5 M NaOH.

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0 2 4 6 8

Mass B

alance (%

)[TM

CH

& C

HL]

(m

ol)

(Time)

TMCH CHL TMCH added Mass Balance

0

5

10

15

20

25

0 2 4 6 8 10

CH

L &

NaO

H (

mo

l)

Time (h)

NaOH CHL

+ 1.29% TmCHMO+ 0.11% GDH-01

Process Metrics & Conclusions

• Good product recovery in DSP of ~90%

• 2.2 kg of trimethyl-caprolactones synthesized and isolated

• Supplied to Univ. Maastricht & ChemStream for polymer applications

• Technology is ready for replication in industrial environments

Conversion[%]

Yield[%]

Product conc. [g/L]

STY[g/L/h]

Biocatalyst loading[gcww/L]

Biocatalyst yield

[gprod/gcww]

1 L 97 97 36 6.0 31.5 1.1

100 L 85 85 24 2.7 34.5 0.7

Demonstration case 6:Alcohol Oxidation with Alcohol Oxidase (AOX)

• Eugenol Oxidase (EUGO) from Rhodococcus jostii RHA1

• Identified at Univ. Groningen

• 3D-structure solved at Univ. Pavia

• Enzyme production in E. coli by InnoSyn

• Immobilisation and recycling studies at Auton. Univ. Barcelona

• Initial reaction characterisation & optimisation at Denmark TU

• Final optimisation & pilot plant demonstration on kg scale by InnoSyn

Fermentative production of EUGO

• E. coli fermentations on 15 L scale:

– Batch fermentation: ~24 h to ~30 g cell wet weight / L broth

– Fed-batch high cell-density: ~100 h to ~300 g cell wet weight / L broth

• Better overexpression in HCDF

• Activity: 0.95 U/mg total cell-free protein

on vanillyl alcohol (at pH 7.5 and 30°C)

• fermentation yields:

– 43 kU / L fermentation broth

– 625 kU / kg cell dry weight

• Enzyme available from

– RU Groningen / GECCO (small scale, purified)

– InnoSyn (large scale HCDF, non-purified) B: CFE from batch fermentation

S1-3: time samples of CFE from high

cell-density fermentation

• Acetone and hydrogen peroxide is not a safe combination

• TATP: Tri-acetone-triperoxide: explosive compound !

• Not easy scalable: too many safety issues/risks

• Acetone and vanillin can give aldol condensation with catalytic amounts OH-

• Solubility vanillyl alcohol and vanillin is dependent on pH

Safety & other considerations

• Increase in initial activity with increasing pH

• Higher conversion at higher pH; >8.5

• 85-90% conversion overnight

• Decrease in mass balance; especially at high; pH>8.5

No co-solvent: reactions at various

pH values

Possible side reaction

Dakin oxidation Vanillin

• Possible further polymerization

• high Km of catalase (90 mM)

• H2O2 will always be present

7 7.5 8 8.5 9 9.5

Color pH dependent

No catalase: Sodium sulfite added

30 mL scale

Vanillyl alcohol: 1.0 g

Water: 24 g

Sodium sulfite: 1.0 g

pH adjusted: 8.5 to 9.0

EUGO CFE: 3 g

pH = 9.0

T = 25°C

Titrant: 1 M NaOH

Conversion: >95%

mass balance: 94%

No brown/black color: reaction mix turns yellow overnight

500 mL experiments using oxygen5% (w/w) vanillyl alcohol & 5% (v/v) EUGO

• After 3.2 h increase in titration; brownish color formed; side product

• All sulfite consumed, additional sulfite added.

• Competition between enzyme and sulfite for oxygen

Reaction on 100 L pilot plant scale

• After 4.5 h increase in titration: all sulfite consumed

• Brownish color formed; side product(s) due to presence of hydrogen peroxide

• Additional sulfite added.

• Conversion after 9 h: > 95%

85 L water

5.5 kg sodium sulfite

5.0 kg vanillyl alcohol

5.0 kg EUGO CFE

pH = 9.5

Temp. 25°C

Titrant: 5 M NaOH

Process Metrics Vanillin with EUGO

• More than 4 kg of vanillin produced by vanillyl alcohol oxidation

• Use of co-solvent could be avoided

• Most catalases have a low affinity for H2O2

• Too low in case of sensitive reactants

• Chemical quenching can be a viable alternative (dependent on pH)

ScaleConversion

(%)

Yield

(%)

[Product]

(g L-1)

STY

(g L-1 h-1)

Biocatalyst loading

(gcww L-1)

Biocatalyst yield

(gprod/gcww)

0.5 L >97 94 43 6.0 16 2.8

100 L >95 85.7 38 4.2 16 2.4

Demonstration case 7a:Lactol Oxidation with ADH

• High activity and stability of ADH-99 from c-LEcta (identified in WP1)

• High activity NOX produced in high-cell density fermentation (WP2)

• Low operational stability of NOX at substrate concentrations > 35 g/l

• Potential NOX stability improvements:

– Engineering of (alternative) NOX enzymes (c-LEcta)

– Water-miscible co-solvents to solubilize lactol substrate → 1 phase

– Immiscible organic solvents to protect NOX from substrate → bi-phasic

ADH-99 from c-LEcta

NOX from InnoSyn

Effect of non-miscible solvents on

NOX stability and productivity

• Strong increase in activity and conversion using 2-octanone and 2-ethyl-hexanol

• Improved stability in presence of relatively polar solvent to dissolve the lactol

Scale-up to 1 L scale (WP4)5% (w/w) lactol in 2-ethyl-hexanol/buffer (50/50)

• 1 g/l ADH-99 (lyoph. powder)

• 4% (v/v) liquid NOX formulation

• Crude lactol from DERA rxn.

• Oxygen supply using pure O2

• product formation fits oxygen

consumption almost 100%

• 50 g/l product reached

• almost quantitative assay yield

Ready for demonstration

Demonstration on 75 L scale (WP4)5% (w/w) lactol in 2-ethyl-hexanol/buffer (50/50)

• 75 L reaction in 200 L reactor

• 1 g/l ADH-99 (lyoph. powder)

• 4% (v/v) liquid NOX formulation

• Crude lactol from DERA rxn.

• Oxygen supply using pure O2

• Delayed oxygen figures

• 50 g/l product reached

• > 95% assay yield

• > 3.5 kg lactone produced

(non-isolated)

• STY: 6.3 g/(L x h)

Successful demonstration of ADH oxidation technology

Techno-economic evaluation (WP5)

Process metrics Substrate

conv.[%]

Yield[%]

STY[gprod/L x h]

Product

conc. [g/L]

Biocat

yield [gprod/gcww]

Biocat

load [gcww/L]

ADH Lactol

oxidation demo96 95 6.3 47 1.3 36

Raw material

(variable) costs

Efficient use of

installations

(fixed costs)

Biocatalyst cost

price contribution

(variable costs)

Techno-economic evaluation (WP5)

Process metrics Substrate

conv.[%]

Yield[%]

STY[gprod/L x h]

Product

conc. [g/L]

Biocat

yield [gprod/gcww]

Biocat

load [gcww/L]

ADH Lactol

oxidation demo96 95 6.3 47 1.3 36

Process metric Target 1 L Demo at 75 L

Reaction yield [%] 80-100 >99 95

Product concentration [g/l]

10 29 47

STY [g l-1 h-1] 2 7.3 6.3

Biocatalyst yield [gproduct/gcww]

1 4.4 1.3

Overview of Evaluations

Target reactions Targets setBest Yield

[%]

Best Product

concentration

[g/L]

Best biocatalyst

yield [g/gcww]

1a: p-Xylene hydroxylation

(P450/GDH)

Yield: 80-100 %

Product: 100 g/L

Biocat: 5 g/g

22 3.3 0.08

1b: Pseudocumene

hydroxylation (P450/GDH)

Yield; 80-100 %

Product: 100 g/L

Biocat: 5 g/g

2.2 0.29 0.005

2: Diclofenac hydroxylation

(P450/GDH)

Yield: 80-100 %

Product: 5 g/L

Biocat: 0.1 g/g

90 3 0.09

3: α-Isophorone hydroxylation

(P450/GDH)

Yield: 80-100 %

Product: 100 g/L

Biocat: 5 g/g

60 6 0.08

4: 3,3,5-trimethyl-

cyclohexanone oxidation

(BVMO/GDH)

Yield: 95-100 %

Product: 10-20 g/L

Biocat: 0.26-1.9 g/g

99 17 4.5

5a: Cyclopentadecanone

oxidation (BVMO/GDH)

Yield: 80-100 %

Product: 100 g/L

Biocat: 5 g/g

97 40 0.8

6: Vanillyl alcohol oxidation

(AOX)

Yield: 80-100 %

Product: 50 g/L

Biocat: 5 g/g

85 52 1.2

7a: C5-lactol oxidation

(ADH/NOX)

Yield: 80-100 %

Product: 10 g/L

Biocat: 1 g/g

95 47 1.3

Biocatalyst production & formulation

Demonstration case Production host Fermentation

type

Biocatalyst

formulation

1. P450 Aromatics Escherichia coli Batch Cell-free extract (CFE)

2. P450 API E. coli / Pichia pastoris

Batch / fed-batch CFE / cells

3. P450 Alkenes E. coli Batch/fed-batch CFE

4. Cyclohexanone BVMO E. coli High cell-density Ferm. broth / CFE

5a. Macrocyclic BVMO E. coli High cell-density CFE

6. Alcohol AOX E. coli High cell-density Homogen. broth

7a. Lactol ADH E. coli High cell-density (freeze-dried) CFE

Biooxidation enzymes can in general be produced efficiently in E. coli

P450s (bacterial, eukaryotic incl. human) require more attention & effort

Limitations and Engineering AspectsReaction or Enzyme Engineering ?

Demonstration case Enzymes Main limitation Solution by

1. P450 AromaticsP450-BM3

GDH-02

P450 Activity &

Selectivity

Enzyme

Engineering RWTH

2. P450 APIP450-BM3

GDH-02

P450 Activity &

Selectivity

Enzyme

EngineeringDSM/InnoSyn

3. P450 AlkenesP450-BM3

GDH-02

P450 Activity &

Selectivity

Enzyme

EngineeringRWTH

4. Cyclohexanone BVMOTmCHMO

GDH-01Substrate solubility Cosolvent Univ. Maastricht

5a. Macrocyclic BVMORrCDMO

GDH-01Substrate solubility Cosolvent InnoSyn

6. Alcohol AOXEUGO

(catalase)

H2O2 derived by-

product

Chemical

quenchingInnoSyn

7a. Lactol ADHADH-99

NOX-01

operational

stability NOX-01Biphasic reaction InnoSyn

7b. Alcohol ADHADH

NOX

operational

stability NOX

Enzyme

Engineeringc-LEcta

• Both Reaction and Enzyme engineering contributed to successful demos

• Efficient / industrial processes with wild-type enzymes are possible

• Ideally both engineering approaches go hand-in-hand

Oxygen Supply, Reactors and

Productivities in Demonstrations

Demonstration case Reactor type Oxygen supply STY [g L-1 h-1]

1. P450 Aromatics Stirred tank reactor (STR)

Oxygen 0.15

2. P450 API STR Air 0.4

3. P450 Alkenes STR Oxygen 1.5

4. Cyclohexanone BVMO STR Oxygen 2.7

5a. Macrocyclic BVMO STR Oxygen 4.0

6. Alcohol AOX STR Oxygen 4.2

7a. Lactol ADH STR Oxygen 6.3

7b. Alcohol ADH STR Oxygen 14.0

Sufficient oxygen for an efficient biocatalytic reaction can be supplied

conventional stirred tank reactors are suited for a safe operation with O2

Technology summary and outlook

ROBOX demonstrations

The good news after a lot of red lights:

→ There is a role for P450s in industrially applied biocatalysis

Dark green: proven at demonstration scale

Light green: potential improvements through biocatalyst engineering required prior to additional optimization

Orange: potential improvements; primarily process optimization, improvements in biocatalyst yield (enzyme activity and stability)

Yellow: same as orange but with higher probability of success

Summary & Conclusions

• Major steps achieved to establish oxidative biocatalysis on pilot scale

– Techno-economically viable demonstration of 4 oxidative enzyme classes

– Generation of new robust enzyme & technology platforms

• Oxygen supply can become limiting for efficient oxidative enzymes

• Use of pure O2 instead of air in a stirred tank reactor is sufficient

• Enzyme and reaction engineering are key for successful implementation of industrial biocatalytic processes

• Enzyme production in high-cell density fermentations is a prerequisite

• More efforts needed to routinely apply biooxidations for bulk chemicals

• Especially for P450s: higher operational stability and coupling efficiency

Acknowledgments

The research for part of this work has received funding from the European Union project ROBOX (grant agreement n° 635734) under EU’s Horizon 2020 Programme Research and Innovation actions H2020-LEIT BIO-2014-1. Any statement made herein reflects only the author’s

views. The European Union is not liable for any use that may be made of the information contained herein.

InnoSyn B.V.

Jan Brummund

Monika Müller

Harrie Straatman

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