University of Rostock

University of Rostock Biocatalysis Department of Chemistry ... - Downstream processing of low molecular weight componds - Renewable resources - Trace ...

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University of Rostock Department of Chemistry Technical Chemistry

Biocatalysis Udo Kragl

Universität Rostock, Institut für Chemie, Lehrstuhl für Technische Chemie Albert-Einstein-Str. 3A, 18059 Rostock, Germany Tel. +49-381-498-6450, Fax +49-381-498-6452 Email [email protected] www.chemie.uni-rostock.de/kragl Universität Rostock

January 2008

Technische Chemie Biocat 08.ppt _ 1

University of Rostock - founded 1419

August Michaelis Paul Walden Universität Rostock Technische Chemie Biocat 08.ppt _ 2

Technical Chemistry - Research Topics

••

Rostock RostockUniversity, University,Department Departmentof ofChemistry Chemistry --Biocatalysis, Biocatalysis,ionic ionicliquids liquids --Downstream Downstreamprocessing processingof oflow lowmolecular molecularweight weightcomponds componds --Renewable Renewableresources resources --Trace Traceanalyis analyis --Optimisation Optimisationof ofreaction reactionconditions, conditions,modelling modelling

••

Leibniz LeibnizInstitute Institutefor forCatalysis Catalysis --Multi Multiphase phasecatalysis catalysis --Polymerisation Polymerisationof ofpropene propeneoxide oxide --Functionalisation Functionalisationof ofrenewable renewableresources... resources... --... ... in total 22 coworker

Universität Rostock Technische Chemie Biocat 08.ppt _ 3

Technical Chemistry – Research Topics



Biocatalysis - (enantio)-selective oxidation, C-C-bonding - biocatalysis in ionic liquids



Downstream processing of low molecular weight compounds - membrane processes for desalting, concentration - combination of extraction, chromatography, scCO2 extraction, membrane processes (product orientated)



Use of renewable resources („Biogene Rohstoffe“) - fermentation (rare sugars, biomaterials) - plant compounds - chemo- and biocatalytic functionalisation



Trace analysis (environment, processes) - Equipment: GC, LC, GC-MS, LC-MS, ion chromatography



Optimisation of reaction conditions - model based or „black box“ (genetic algorithm)

www.ionic-liquids.uni-rostock.de

Universität Rostock Technische Chemie Biocat 08.ppt _ 4

Outline • introduction • examples - industry - academia • take home message

´Enzymes are proteins, things of beauty and joy forever`

Universität Rostock Technische Chemie Biocat 08.ppt _ 5

Opinions & Prejudices - hopefully overcome Biocatalysts - are sensible and not stable operational stability for several months has been achieved - work only in diluted aqueous solutions molar solutions, organic solvents possible - give only low space-time yields several kg/(L×d) possible

Universität Rostock Technische Chemie Biocat 08.ppt _ 6

Facts Biotransformations are used for synthesis of bulk and fine chemicals, such as: high-fructose corn sirup acrylamide 7-aminocephalosporinic acid chiral amines chiral alcohols

Glc-Fru isomerase nitrile hydratase (wc) acylase lipase oxidoreductase (wc)

> 8x106 t/a > 30,000 t/a > 200 t/a > 100 t/a 1 t/a (?)

... today more than 100 biotransformations are applied in industry...

Universität Rostock Technische Chemie Biocat 08.ppt _ 7

Productivities of Biotechnological Processes

Universität Rostock

Roger Sheldon, 1993

Technische Chemie Biocat 08.ppt _ 8

Recent Books on Biocatalysis & Biotransformations

Universität Rostock Technische Chemie Biocat 08.ppt _ 9

Biocatalysis – Established with a Great Future

Growth of sales volume for Biotechnology until 2010 Universität Rostock

Festel Capital, 2004

Technische Chemie Biocat 08.ppt _ 10

Biotechnology • biotransformation

- whole cell - enzyme

educt

biocatalyst

product

• fermentation - de novo synthesis from nutrients

Universität Rostock Technische Chemie Biocat 08.ppt _ 11

Process Development – an Integrated View catalyst & reaction • equilibrium • (product) inhibition • catalyst stability

process process

reactor

downstream proc.

• type • scale up

Universität Rostock Technische Chemie Biocat 08.ppt _ 12

www.brenda.uni-koeln.de

Universität Rostock Technische Chemie Biocat 08.ppt _ 13

Suppliers of Enzymes

• • • • • • •

Fluka, Sigma, Merck... Novo Nordisk Amano Toyobo Unitika Biocatalysts Codexis www.codexis.com



University groups working in Biochemistry, Mikrobiology Universität Rostock Technische Chemie Biocat 08.ppt _ 14

Enzyme Kinetics

• • • •

• •

pH, temperature activity: 1 U = 1 μmol/min; 1 kat = 1 mol/s Michaelis-Menten type saturation kinetics for >1 substrates order of binding to the enzyme has to be considered - ordered - random - ping-pong substrate-surplus & product inhibition may occur references: - Cornish-Bowden - Segel - Biselli, Kragl, Wandrey in Drauz, Waldmann (eds) Universität Rostock Technische Chemie Biocat 08.ppt _ 15

Recovery of Enzymes or Cells

• • •

immobilisation on a heterogeneous support by - adsorption - covalent attachment (eg. Eupergit) cross-linking entrapment in hydrogels (alginate...) covalent attachment often increases thermal stability, but method has to be developed individually, immobilisation yield <100% (acitivity!)

• •

ultrafiltration (enzymes) microfiltration, centrifugation Universität Rostock Technische Chemie Biocat 08.ppt _ 16

Classification of Enzymes

Universität Rostock Technische Chemie Biocat 08.ppt _ 17

Enzyme Classification & Enzyme Source



enzymes with the same EC-number and CASnumber but from different origin may react totally different!



lipase EC 3.1.1.3 - Burkholderia plantarii (BASF-prozess) - Pseudomonas cepacia - Mucor miehei - Pseudomonas fluorescens - Candida antarctica - Serratia marescens

Universität Rostock Technische Chemie Biocat 08.ppt _ 18

Alcohol Dehydrogenase

EC 1.1.1.1

Zygosaccharomyces rouxii O O

O

E O

OH

O

(S)-2 1 = 3,4-methylenedioxyacetophenone 2 = 4-(3,4-methylenedioxyphenyl)-2-propanol

reaction conditions: [1]: pH: T:

< 0.011 M, 2 g L-1 7.0 33 - 35 °C

Eli Lilly

process parameters: yield: ee: reactor type: reactor volume: capacity: space-time-yield:

Anderson, B.A. et al. (1995) J. Am. Chem. Soc. 117, 12358-12359

96 % > 99.9 % batch 300 L kg scale 75 g L-1d-1

Universität Rostock Technische Chemie Biocat 08.ppt _ 19

Integration in Chemical Synthesis Z. rouxii ATCC 14462

O O

O

O

XAD-7

O2N-Ph-CHO OH

O

1

O O

O

2 NO2

NaOH,air DMSO/DMF

O

O N O

N

1) CH3SO2Cl/Et3N 2) t-BuO-Li 3) H2, Pd/C

O

O

O O2NPh

OH N NHAc

H2NNH-Ac

O

O

OH

O2N H2N

3 1 = 3,4-methylenedioxyacetophenone 2 = (S)-4-(3,4-methylene-dioxyphenyl)-2-propanol 3 = LY300164

The product is tested for treatment of amylotropic lateral scleposis. Universität Rostock Technische Chemie Biocat 08.ppt _ 20

Ketone Reduction using Whole Cells • •

whole cells of Zygosaccharomyces rouxii substrate and product are toxic for the cells XAD-resin as substrate reservoir and for product extraction

80

concentration / g/L

resin phase concentration 60

substrate product

40

20

water phase concentration 0 0

2

4

6

8

10

12

time / h

Universität Rostock

Vicenzi et al., Enz. Microbial. Technol. 20, 494 (1997)

Technische Chemie Biocat 08.ppt _ 21

Flow Scheme H2O

E

acetone

E

E

O O

O on resin =

water slurry of cells O OH

O

• XAD-7 resin is retained by 150 µm filter • yeast cells are in filtrate • product is liberated by washing resin with acetone Universität Rostock

Vicenzi, J.T., Zmijewski, M.J., Reinhard, M.R., Landen, B.E., Muth, W.L., Marler, P.G. (1997) Technische Chemie Enzyme Microb. Technol. 20, 494-499 Biocat 08.ppt _ 22

Alcohol Dehydrogenase

EC 1.1.1.1

Neurospora crassa O

OH

E S

S O

O

(6S)-1

S

S O

O

(4S,6S)-2

1 = 5,6-dihydro-6-methyl-4H-thieno[2,3b]thiopyran-4-one-7,7-dioxide 2 = 5,6-dihydro-4-hydroxy-6-methyl-4H-thieno[2,3b]thiopyran-7,7-dioxide

Zeneca Life Science Molecules

reaction conditions:

process parameters:

pH: T: medium: catalyst:

yield: ee: reactor type: capacity: start-up date:

3.8-4.3 33 °C aqueous suspended whole cells

Holt, R. A. and Rigby, S. R. (1996) Zeneca Limited, US 5580764

> 85 > 98 % batch multi t 1994

Universität Rostock Technische Chemie Biocat 08.ppt _ 23

Substitution of Chemical Steps 1 O OH

pyridine/ tosyl chloride

O

OTs

O

OMe

CH3O2C

LiS OMe

HOOC

S HCONH2

OH

S

S

S

O

NHCOCH3 NH3, THF

S O

BH3·DMS, THF

O

SO2NH2

S

S O

O

O

NHCH2CH3 SO2NH2

S

S

S

(trans)-3

4

SO2Cl

HSO3Cl, SOCl2

S

S

S

2

CH3CN, H2SO4

S

(cis)-3 NHCOCH3

S NHCOCH3

H2O2, NaWO4

S

S

OH

H2SO4, 0°C S

O

S

OH

LiAlH4

TFAA, toluene

conc. HCl

O

S

S O

O

TrusoptTM

• TrusoptTM (Merck) is a novel, topically active treatment for glaucoma • inversion of cis alcohol is incomplete in chemical synthesis

Blacker, A.J., Holt, R.A. (1997) in: Chirality In Industry II pp. 246-261, John Wiley & Sons, New York

Universität Rostock Technische Chemie Biocat 08.ppt _ 24

Why Biotransformation at pH 3.8 - 4.3 ? to prevent epimerization that takes place above pH 5! O

OH

E S

S O

O

Neurospora crassa pH 4

pH 4!

S

S O

(6S)-3

O

4

O

O

S HO2S

pH >5!

S

S O

O

(6R)-3 Universität Rostock Technische Chemie Biocat 08.ppt _ 25

Enzymatic Oxidation Reactions



oxidation with enzymes of EC class 1: - oxidoreductases → oxygenation with O2 or H2O2 (→ dehydrogenation)



oxidation with enzymes of EC class 4: - lyases → addition of water

Universität Rostock Technische Chemie Biocat 08.ppt _ 26

Nicotinic Acid Hydroxylase

EC 1.5.1.13

Achromobacter xylosoxidans COO-

COO-

E

N

HO + H2O

1

N

2 [H] ½ O2

2

H2O 1 = niacin = nicotinic acid = pyridine-3-carboxylate 2 = 6-hydroxynicotinate = 6-hydroxy-pyridine-3-carboxylate

reaction conditions: [1]: pH: T: catalyst:

0.533 M, 65 g L-1 7.0 30 °C suspended whole cells

Lonza AG

process parameters: conversion: yield: reactor type: reactor volume: capacity: residence time:

Lehky, P., Kulla, H., Mischler, S. (1995), Lonza AG, EP 0152948 A2 Kulla, H.G. (1991), Chimia 45, 81-85

>90 % > 90 % (overall) batch 12 000 L several tons 12 h Universität Rostock Technische Chemie Biocat 08.ppt _ 27

Flow Scheme

cells

E

acid

COOMg2+ N 2

COOMg2+ HO

N 2

In contrast to the biotransformation the chemical synthesis of 6-substituted nicotinic acids is difficult and expensive due to the separation of by-products. Universität Rostock Technische Chemie Biocat 08.ppt _ 28

D-Amino

Acid Oxidase

EC 1.4.3.3

Trigonopsis variabilis H N

HOOC NH2

O

S N

E

O

O

O COOH

O

1

reaction conditions: 0.02 M, 7.47 g L-1 7.3 25 °C immob. enzyme

S N

O

+H2O +O2 - NH3 - H2O2

1 = Cephalosporin C 2 = α-ketoadipinyl-7-aminocephalosporanic acid

[1]: pH: T: catalyst:

H N

HOOC

O

O COOH

O

2 Hoechst Marion Roussel

process parameters: reactor type: batch reactor volume: 10 000 L capacity: 200 t.a-1 residence time: 1.5 h dsp: direct transfer to 7-ACA production enzyme consumption: 1.1 U.g-1 Universität Rostock

Aretz, W. (1998) Hoechst Marion Roussel, personal information

Technische Chemie Biocat 08.ppt _ 29

D-Amino Acid Oxidase O

H NH2 COOH

+ O2 + H2O

COOH

O

H NH2 COOH

COOH

O

H NH2 H2N

COOH O

+ NH3 + H2O2

H2N

COOH O O

H NH2 HO

HO COOH

COOH

E.C. 1.4.3.3, Trigonopsis variabilis, 25 U/mg Universität Rostock Technische Chemie Biocat 08.ppt _ 30

Substitution of Chemical Synthesis H N

HOOC NH2

O

S O

N O COOH

O

D-aminoacid oxidase

ZnCPC

E

solvent TMSCl HN Si Si

H N

O O

O

O

O

N COOH

Si

O

PCl5

HN Si

single enzyme ?

O

+ H2O2 - CO2 H N

S

S O

O

O

N COOH

HO

H N

O

O

S

O

O

T < 0 °C

H N

HOOC

S

+ H2O + O2 - H2O2 - NH3

Cl

N

O

O

N

O

O

O

COOH COOH

O

O

E hydrolysis T < 0 °C

H2N

glutaryl amidase

S

- HOOC

COOH

O

N O COOH

O

Universität Rostock Technische Chemie Biocat 08.ppt _ 31

Synthesis of 7-ACS - Comparision of Processes fermentation

chemically

enzymatic filtration 1. enzyme

Zn-salt silylation

2. enzyme

hydrolysis CH22Cl2,2, PCl55 Zn-salt T <0 °C

cristallisation drying

H22O 25 °C

Universität Rostock Technische Chemie Biocat 08.ppt _ 32

Advantages of the Enzymatic Process

• • • •

no toxic compounds waste water can be easily degraded biologically no dangerous chemicals reduction of the amount of waste: – 0.3 t compared to 31 t (for 1 t 7-ACA)

reduction of the cost contribution for waste treatment and disposal from 21% to 1% of the overall process costs

Universität Rostock Technische Chemie Biocat 08.ppt _ 33

Oxygen Activation: FAD R

R

H

N N O N O coordinated to Pd (Wacker-process - alkeneNoxidation, → acetaldehyde N

N N

N

H O

Enzym-B:

H

NH2 PdCl 2 + H2C=CH2 + H2O

HOOC R

R COOH

R=H → Riboflavin H O dinucleotide O R = adenine NH Pd + 2 HCl → + FAD

Pd + 2 CuCl2

PdCl2 + 2 CuCl R

- H2O+2 2 HCl + 0.5 O 2 CuCl 2

H

N

H

2NCuClO2 + H2ONH +

N

R

N

N

N

O

+ O2

H2NC=CH2 + 0.5 O2

N

H

PdCl2/CuCl2

H O

R

COOH

O + H2O, - NH3

H

H

O

O

O OH R

COOH

Universität Rostock Technische Chemie Biocat 08.ppt _ 34

Oxygen Activation: FAD R

H

O

N

N

H

N

R N

N

N

N N

H

HOOC R

NH2 R COOH

- H2O2

O

NH

R

H

N

N

O NH +

N

R

N

N

N

R=H → Riboflavin R = adenine dinucleotide → FAD

H H

O Enzym-B:

O

O

+ O2

H

H

R

COOH

O

+ H2O, - NH3

N N

H

H

O

O

O OH R

COOH

Universität Rostock Technische Chemie Biocat 08.ppt _ 35

Chloroperoxidase Catalyzed Oxidation

+ H2O2

EC 1.11.1.10

O

N

+ H2O

N

H

H

.. O 1

R

S

2

R

+ H2O2

R

1

S R

2

+ H2O

O + H2O2 1

R

2

R

+ H2O R

1

2

R

Universität Rostock Technische Chemie Biocat 08.ppt _ 36

Oxygen Activation: Chemo- ↔ Biocatalyst - oxygen transfer

O R'

H

H N O

R

H R'

H R

HOOC

O N Mn

N

O

N

O

N

Fe N

HOOC

Mn-salen complex NaOCl 0.75 min-1

peroxidase (hem) H2O2 75 min-1 Universität Rostock Technische Chemie Biocat 08.ppt _ 37

Benzoate Dioxygenase

EC 1.14.12.10

Pseudomonas putida R

R

OH

E

+ O2

OH 2 NADH

1

+

2 NAD

(R,S)-2

R = H, F, Me, CF3

1 = benzene 2 = 1,2-dihydroxycatechol

ICI

reaction conditions:

process parameters:

catalyst:

selectivity: capacity:

suspended whole cells

> 99.5 % several t a-1

Universität Rostock

Technische Evans, C., Ribbons, D., Thomas, S., Roberts, S. (1990), Enzymatix Ltd., WO 9012798 A2 Chemie

Biocat 08.ppt _ 38

Degradation of aromatics by microbial di-oxygenases

•• 1968: 1968:mutant mutantstrain strainfrom fromP. P.putida putidawas waslacking lackingthe theactivity activityof ofthe the dihydrodiol dihydrodioldehydrogenase. dehydrogenase. •• Pseudomas Pseudomasputida putidaexhibits exhibitsaahigh hightolerance toleranceto tothe thearomatic aromatic substrates substratesthat thatare arenormally normallytoxic toxicto tomicroorganisms. microorganisms. Universität Rostock

taken from K. Faber: Biotransformations in Organic Chemistry

Technische Chemie Biocat 08.ppt _ 39

Product Range of Benzoate Dioxygenase

Hudlicky et al., Aldrichimica Acta 32, 35-62 (1999)

Universität Rostock Technische Chemie Biocat 08.ppt _ 40

Naphthalene Dioxygenase

EC 1.13.11.11

Pseudomonas putida OH

E N H

1

N H

2

1 = 1H-indole 2 = 2,3-dihydro-1H-indole-2,3-diol

Genencor

reaction conditions:

process parameters:

catalyst:

reactor type:

whole cell

OH

+H2O +1/2 O2

Serdar, C. M., Murdock, D. C., Ensley, B. D. (1992), Amgen Inc., US 5173425

batch

Universität Rostock Technische Chemie Biocat 08.ppt _ 41

Classical Route to Indigo ONa NH 2 +

1

NHCH 2COOH

NaOH Cl

COOH

2 NaNH2 KOH-NaOH

2

3 O

N H

4 H N

air N H

O

5 1 = phenylamine 2 = chloro-acetic acid 3 = phenylamino-acetic acid 4 = sodium salt of 1H-indole-3-ol 5 = indigo

three steps are needed starting from aniline Universität Rostock Technische Chemie Biocat 08.ppt _ 42

New Catalytic Route (Mitsui Toatsu Chemicals)

NH2 +

1

OH

HO

2 ROOH Mo(CO)6

Ag catalyst gas phase

2

N H

3

- 2 ROH - H2O

O

H N

N H

O

4

1 = phenylamine 2 = ethane-1,2-diol 3 = 1H-indole 4 = indigo

produces less organic salts than the classical route

Universität Rostock Technische Chemie Biocat 08.ppt _ 43

Biosynthesis of Indigo COOH glucose N H

NH2

naphthalene dioxygenase

tryptophanase

E1

L-1

2

OH

OH OH

spontaneous

1 = L-tryptophan 2 = 1H-indole 3 = 2,3-dihydro-1H-indole-2,3-diol 4 = 1H-indole-3-ol 5 = indigo

O

H N

air

N H

3

E2

N H

N H

4

N H

O

5

NH 2

classical chemical route

starts from cheap glucose with tryptophan as intermediate Universität Rostock

Murdock, D., Ensley, B. D., Serdar, C., Thalen, M. (1993), Bio/Technology, 11, 381-385 Technische Chemie

Biocat 08.ppt _ 44

Oxygenase

EC 1.14.14.1

Nocardia autotropica HO

HO

O O

O O

O

O O

E

O

H

H

OH

1 1 = Simvastatin 2 = 6-β-hydroxy-methyl-simvastatin (major product)

reaction conditions:

[1]: [2]: pH: T: catalyst:

2 Merck Sharp & Dohme

process parameters:

< 0.05 10-3 M, < 0.02 g L-1yield: 1.85 10-3 M, 0.8 g L-1 selectivity: 6.8 reactor type: 27 °C reactor volume: suspended whole cells

Gbewonyo, K., Buckland, B.C., Lilly, M.D. (1991), Biotech. Bioeng. 37, 1101-1107

24 % 70 % fed batch 19,000 L

Universität Rostock Technische Chemie Biocat 08.ppt _ 45

Monooxygenase

EC 1.14.14.1

Nocardia corallina

E

R

1

O R

2 Nippon Mining

reaction conditions:

process parameters:

[1]: two phase system pH: T: 30 °C catalyst: suspended whole cells

yield: 92 % selectivity: 94 % reactor type: batch rate of aeration is raised to strip the short chain toxic epoxide Universität Rostock Technische Chemie Biocat 08.ppt _ 46

Substrate range F F

Cl Me

F

F

OMe

O

O

F

O

O

O

Me

H/Me

H/Me

H

O

n

H/Me

O

_ 0) (n >

H/Me n

H

n

O

(n = 1, 2...)

_ 0) (n > O n

Cl

_ 4) (n >

F F

F

n (n = 4, 5...)

Universität Rostock Technische Chemie Biocat 08.ppt _ 47

Catalase

EC 1.11.1.6

Microbial source O2N Me +

O2 + 2 NO2

1 1 = nitrotoluolene 2 = dinitrodibenzyl DNDB

H2O2

NO2

E

2

H2O + 1/2 O2

Novartis

• removal of side-product • only incomplete conversion with heavy-metal catalysts • following process steps with DNDB are problematic by contamination with heavy-metal catalyst. Universität Rostock Technische Chemie Biocat 08.ppt _ 48

Flow Scheme catalase O2 t-BuOK

acetic acid

pH

H2O (quenching)

E

Me

NO2

N2

7000 ppm H2O2

E 1000 ppm H2O2

< 200 ppm H2O2

O2N

NO2

Universität Rostock Technische Chemie Biocat 08.ppt _ 49

Cyclodextrin Glycosyltransferase

EC 2.4.1.19

Bacillus circulans

OH O

E n

OH n-6

OH

O

= glucose +

HO

1

O HO OH

OH O HO

H O HO

OOH

HO

O

OH O

Problem: adsorption of cyclodextrins for

HO

OH OH O

OH HO O

HO O

O

OH

separation is ineffective at 55 °C 2

reaction conditions:

[1]: pH: T: catalyst:

process parameters:

8.3 w/v % liquified starch yield: 22.3 % (α-cyclodextrin) 5.8 – 6.0 10.8 % (β-cyclodextrin) 55 °C 5.1 % (γ-cyclodextrin) soluble enzyme chemical purity: 94.9 % reactor type: batch

Chin, P.J., Chein, S. (1989), J. Chem. Technol. Biotechnol. 46, 283-294

Universität Rostock Technische Chemie Biocat 08.ppt _ 50

Flow Scheme - Integrated Process n

OH

= glucose

NaOH OH

pH

O O

OH

O

O OH HO

HO OH

HO

H O HO

OOH

O

HO

E

OH O

OH OH OHO

OH O HO

HO O

heat exchanger

O

OH

adsorption

• Temperature is lowered to 30 °C for effective adsorption. (At this temperature almost no cyclodextrins are formed during circulation.) • Before reentering the main reactor the temperature of the solution is again adjusted to 55 °C. • To prevent adsorption of the cyclodextrin glycosyltransferase on the columns 3 w/v % NaCl are added. Tsuchiyama, Y. et al. (1991), J. Ferment. Bioeng. 71, 407-412

Universität Rostock Technische Chemie Biocat 08.ppt _ 51

Enzymatic Synthesis of GDP-Mannose O

OH HO HO

HO

HO HO

O

HO

O

2-

inhibition

-

O O O O O P P O

N

N

H2N -

OPO3

N

N

OH

O

O HO

Man-1-P+ GTP

GDP-Man PP 2+

GDP-Man

OH

+ PPi

Mg

PPase complexation

2 Pi

- GDP-Man-pyrophosphorylase - inorganic pyrophosphatase Universität Rostock

Elling et al., Glycobiology 6, 591 (1996)

Technische Chemie Biocat 08.ppt _ 52

Modelling of Processes

Universität Rostock Technische Chemie Biocat 08.ppt _ 53

Kinetics of GDP-Man Pyrophosphorylase v1 = [ E ] × A GMPP max ×

1 1+

K MMg

⎛ [Mg ] ⎞ ⎜ ⎟ [ ] GTP ⎝ ⎠

2

+

K

[Mg ] × [GTP ]

Mg i

×

[GTP] ⎛ [GDP − Man] [PPi ] ⎞ ⎟ + [GTP] K GTP × ⎜1 + + M

⎜ ⎝

K iGDP − Man

K iPPi ⎟⎠

×

± 12.2 ± 9

U/mg

± 2 ± 4

μmol/L

KMMg

μmol/L 2.9 -

± 3 ± 2.6

μmol/L -

KiMg

0.1 -

± 0.09 -

AmaxGMPP

15.0 U/mg

KMGTP

40

μmol/L

9

μmol/L

KiPPi

16

μmol/L

KMMan-1-P

15

KiGDP-Man

[Man − 1 − P] − 1− P K Man + [Man − 1 − P] M

μmol/L μmol/L

Universität Rostock Technische Chemie Biocat 08.ppt _ 54

Verification of Kinetic Model

Universität Rostock Technische Chemie Biocat 08.ppt _ 55

Concentration Profiles of Different Reactors

Universität Rostock Technische Chemie Biocat 08.ppt _ 56

Concentration Profiles of Different Reactors

Universität Rostock Technische Chemie Biocat 08.ppt _ 57

Two-Stage CSTR-Cascade to Reduce Product Inhibition

Universität Rostock

Fey et al., Carbohydr. Res. 305, 475 (1997))

Technische Chemie Biocat 08.ppt _ 58

GDP-Man Production in a 2-Stage CSTR-Cascade

Universität Rostock Technische Chemie Biocat 08.ppt _ 59

GDP-Man Production - Enzyme Consumption enzyme consumption (U/g product)

8

Kdes * [E] 5.8

enzyme consumption =

6

STY

4.6 4

2.0 2

0.9 0 batch

batch with EDTA

CSTR

cascade Universität Rostock Technische Chemie Biocat 08.ppt _ 60

Classification of Enzymes

Universität Rostock Technische Chemie Biocat 08.ppt _ 61

Lipase

EC 3.1.1.3

Burkholderia plantarii

O NH2

NH2 O +

2

O

(R,S)-1

E O

O

NH +

- EtOH

2

(S)-1

(R)-3

freeze freeze drying drying of of the the enzyme enzyme solution solution together together with with fatty fatty acids acids to to increase increase enzyme enzyme activity activity 1 = 1-phenylethylamine 2 = ethylmethoxyacetate 3 = phenylethylmethoxyamide

reaction conditions:

[(R/S)-1]:

1.65 M, 200 g⋅L-1

pH: medium:

8.0-9.0 MTBEethylmethoxyacetate immobilized enzyme

catalyst:

Balkenhohl, F. et al. (1995), BASF AG, DE 4329293

BASF

process parameters:

conversion: yield: selectivity: ee: capacity: residence time:

50 % > 90 % > 500 (E-Value) > 99% (S); 93 % (R) > 100 t.a-1 5-7 h Universität Rostock Technische Chemie Biocat 08.ppt _ 62

Flow Scheme

Universität Rostock Technische Chemie Biocat 08.ppt _ 63

Other Starting Materials NH2

NH2

NH2

Cl O 1-p-tolyl-ethylamine

1-(3-methoxy-phenyl)-ethylamine

1-(4-chloro-phenyl)-ethylamine

NH2

NH2

O NH2

3,3-dimethyl-butyl-2-amine

NH2

1-naphthyl-ethylamine

NH2

2-benzyloxy-1-methyl-ethylamine

NH2 OH

1-phenyl-propylamine

1-ethyl-2-methyl-propylamine

1-amino-indan-2-ol Universität Rostock

Balkenhohl, F., Ditrich, K., Hauer, B., Lander, W. (1997), J. prakt. Chem. 339, 381-384 Technische Chemie

Biocat 08.ppt _ 64

Lipase

EC 3.1.1.3

Serratia marescens O

O COOMe

2

E

O COOMe

+ H2O - MeOH MeO

MeO

MeO

(2S,3R)-2

(2R,3S)-1

1

1 = 3-(4-methoxyphenyl) glycidic acid methyl ester = MPGM 2 = 3-(4-methoxyphenyl) glycidic acid

reaction conditions:

[1]: pH: T: medium: catalyst:

COOH

+

< 0.6 M, < 125 g·L-1 8.5 22 °C aqueous/toluene immobilized enzyme

Tanabe Seiyaku Co., Ltd.

process parameters:

yield: ee: reactor type: capacity: start-up date:

40-45 % 100 % batch 40 kg (-)-MPGM /(m2⋅a) 50 t a-1 1993 Universität Rostock

Matsumae, H., Furui, M., Shibatani, T., Tosa, T. (1994), J. Ferment. Bioeng. 78, 59-63 Technische Chemie Matsumae, H., Shibatani, T. (1994), J. Ferment. Bioeng. 77, 152-158 Biocat 08.ppt _ 65

Flow Scheme

A hydrophilic hollow fibre membrane is used (polyacrylonitrile) as reactor unit (developed by Sepracor) (degradation)

Matson, S. L. (1987), PCT WO 87/02381, PCT US 86/02089

Universität Rostock Technische Chemie Biocat 08.ppt _ 66

Aminopeptidase

EC 3.4.11.1

Pseudomonas putida NH2

NH2 NH2

R

E

NH2

R

O

D,L-1

NH2 +

OH

R

O

O

D-1

L-2

1 = α-amino acid amide 2 = α-amino acid

DSM

reaction conditions:

[1]: pH: T: medium: catalyst:

up to 20 g·L-1 8.0 – 10.0 aqueous suspended whole cells

process parameters:

ee: reactor type: capacity: enzyme activity: enzyme supplier: start-up date:

Schoemaker, H.E. et al. (1992), Pure & Appl. Chem. 64, 1171-1175

> 99 % batch ton scale 338,000 U⋅gprotein-1 Novo Nordisk, Denmark 1988 Universität Rostock Technische Chemie Biocat 08.ppt _ 67

“Dynamic Resolution” (not really) NH2 NH2

R O

1) NH3 2) PhCHO 3) OH- (pH 13) racemization 4) H3O+

1) OH- (pH 13)

D,L-3

racemization 2) H3O+

E

Ph NH2 OMe

R

NH2

MeOH H2SO4

NH2 OH

R

+

NH2

R

N

+PhCHO (pH 8-11)

NH2

R

O

O

O

O

L-4

L-5

D-6

D-7

both bothenantiomers enantiomersaccessible accessiblewith with one onetype typeof ofbiocatalyst biocatalystonly only

formation of Schiff base for easy separation (precipitation)

1) H3O+ - CO2 - NH3 2) OH-

NH2 OH

R O

D-5 Universität Rostock Technische Chemie Biocat 08.ppt _ 68

Product Application DSM uses the amino acids produced by this biotransformation in different product syntheses NH 2

H N

COOH

COOH

O COOMe

NH 2 L-phenylalanine

Aspartame (DSM)

NH2

NH2

H N

S

COOH HO

HO

O

N O COOH

D-(p-hydroxy)phenylglycine

NH2 COOH

Ampicillin (Amoxicillin) (DSM)

EtOOC N H

N O

COOH

L-homophenylalanine

Quinapril (Warner-Lambert) Universität Rostock Technische Chemie Biocat 08.ppt _ 69

D-Hydantoinase

EC 3.5.2.2

Aspergillus oryzae O HN

HN

NH

NH

HN

HO

L-1

NH2 COOH

E

O

O

HO

key raw material for semisynthetic penicillins and derivatives

O

O

HO

D-1

D-2

• unreacted L-hydantoins racemize easily under these conditions. • L-Specific hydantoinases are also known. real realdynamic dynamicresolution resolution 1 = 5-(p-hydroxybenzyl)-hydantoin 2 = D-N-carbamoyl amino acid

Kanegafuchi

reaction conditions:

process parameters:

pH: catalyst:

conversion: capacity: enzyme activity: start-up date:

8.0 immobilized whole cells

Ikemi, M. (1994), Bioprocess Technology 19, pp. 797-813

100% 200 t⋅a-1 17.14 U⋅g-1 1983 Universität Rostock Technische Chemie Biocat 08.ppt _ 70

Hydroxynitrile Lyase (S-Oxynitrilase)

EC 4.1.1.11

Havea brasiliensis O

HO H

O

O + HCN

1

hydroxynitrile lyase

2

CN

(S)-3

DSM Linz (R-Oxynitrilase: (R-Oxynitrilase: Rosenthaler Rosenthaler 1908) 1908) reaction conditions:

process parameters:

[1]: [2]: catalyst:

reactor type: ee:

30% w/w 1.3 equ. soluble enzyme (rec.) two phase system

P. Pöchlauer, Chem. today 16, 15, 1998

batch >97%

Universität Rostock Technische Chemie Biocat 08.ppt _ 71

Tyrosine Phenol Lyase

EC 4.1.99.2

Erwinia herbicola HO

O + NH3

+

E

COOH

HO

1

2

HO

COOH +

NH2

HO

H2O

L-3

L-DOPA 1 = catechol 2 = pyruvic acid 3 = dopa

Ajinomoto Co., Ltd.

reaction conditions:

[3]: catalyst:

0.558 M, 110 g⋅L-1 suspended whole cells

process parameters:

reactor type: reactor volume: capacity:

fed batch 60 000 L 250 t⋅a-1 Universität Rostock

Tsuchida, T. et al. (1993) Ajinimoto Co., Ltd., JP 5123177A Technische Chemie Biocat 08.ppt _ 72 Yamada, H. (1998), pp. 13-17, Studies in Organic Chemistry 53, Elsevier, Amsterdam

Competing with Monsanto’s Chemical Process O HO

CHO +

Ac2O AcHN

COOH

AcO O

NaOAc AcO

HO

2

1

Δ H2O

N

HO

COOH NHAc

HO

[(Rh(COD)(R,R-DIPAMP)]+BF4H2

HO

COOH NH2

HO

L-4

3

for the treatment of Parkinson 1 = vanillin 2 = hydantoin 3 = Z-enamide 3 = dopa

Ager, D.J. (1999) Handbook of Chiral Chemicals, Marcel Dekker, New York

Universität Rostock Technische Chemie Biocat 08.ppt _ 73

Nitrile Hydratase

EC 4.2.1.84

Rhodococcus rhodochrous E

NH2

CN + H2O

1 1 = acrylonitrile 2 = acrylamide

2 Nitto Chemical Industry

reaction conditions:

[1]: [2]: pH: T: catalyst:

O

0.11 M, 6 g·L-1 (fed batch) 5.6 M, 400 g·L-1 7.0 5 °C immobilized whole cells

process parameters:

conversion: yield: selectivity: reactor type: capacity: residence time: space-time-yield: start-up date:

Yamada, H., Kobayashi, M (1996), Biosci. Biotech. Biochem. 60 (9), 1391-1400 Yamada, H., Tani, Y. (1987), Nitto Chemical Industry Co., Ltd., US 4637982

> 99.99 % > 99.99 % > 99.99 % fed batch > 30,000 t·a-1 5h 1.920 g·L-1·d-1 1991

Universität Rostock Technische Chemie Biocat 08.ppt _ 74

Chemical Synthesis was Substituted • The chemical synthesis uses copper salt as catalyst for the hydration of acrylonitrile and has several disadvantages: 1) The rate of acrylamide formation is lower than the acrylic acid formation, 2) The double bond of educt and products causes by-product formations such as ethylene, cyanohydrin and nitrylotrispropionamide and 3) at the double bonds occur polymerization. • The biotransformation has the advantages that recovering of unreacted nitrile is not necessary since the conversion is 100 % and the no copper catalyst removal is needed.

Universität Rostock

Shimizu, H., Ogawa, J., Kataoka, M., Kobayashi, M. (1997), in: New Enzymes for Technische OrganicChemie Synthesis; Adv. Biochem. Eng. Biotechnol. 58 (Ghose, T. K., Fiechter, A., Blakebrough, N. eds.), pp. 56-59Biocat 08.ppt _ 75

Product Spectrum

F NH2 N

O

N N

NH2

NH2

O

O F

1,465 g

S

L-1

O

985 g

L-1

306 g L-1

H N O

NH2 NH2

210 g L-1

1,045 g L-1

Universität Rostock Technische Chemie Biocat 08.ppt _ 76

Xylose Isomerase

EC 5.3.1.5

Bacillus coagulans ... CH 2OH O

E

CH2OH

CH 2OH

O HO

OH OH

OH

OH OH

OH

1

2 Novo-Nordisk Gist-brocardes Miles Kali-Chemie Finnsugar Nagase

1 = glucose 2 = fructose

reaction conditions:

process parameters:

[1]: pH: T: reaction type: catalyst:

reactor type: capacity: residence time: start-up date:

> 95 % dry matter 7.5 - 8.0 50 – 60 °C isomerization immobilized whole cell or isolated enzyme

Blanchard, P. H., Geiger, E. O. (1984) 11, Sugar Technol. Rev. 1-9

continuous, fixed bed > 7⋅106 t⋅a-1 0.17 – 0.33 h 1967 by Clinton Corn Processing Co. (USA) Universität Rostock Technische Chemie Biocat 08.ppt _ 77

Remarks • Since these isomerases belong to the group of metallo enzymes, Co2+ and Mg2+ is required. • The reaction enthalpy is slightly endothermic and reversible. The equilibrium conversion is about 50 % at 55 °C. • Several reactors are operated in parallel or in series, containing enzymes of different ages. The feed to a single reactor is controlled by the conversion of this reactor. • Plants proceeding more than 1000 t of HFCS (based on dry matter) per day typically use at least 20 individual reactors. • The product HFCS contains 42 % fructose (53 % glucose) or 55 % fructose (41 % glucose)(dry matter).

Straatsma, J., Vellenga, K., Witt, H. G. J. de, Joosten, G. E. (1983) Ind. Eng. Chem. Process Des. Dev. 22, 356-361

Universität Rostock Technische Chemie Biocat 08.ppt _ 78

Flow Scheme Na2CO3 MgSO4 CH2OH O

purification

OH

heat exchanger deaeration

OH

OH

analytics

analytics

analytics

analytics

analytics

CH2OH

CH2OH

O HO

Universität Rostock Technische Chemie

OH OH

charcoal

acid

ion exchanger

several parallel isomerization reactors

OH

Biocat 08.ppt _ 79

Other Applications of Enzymes purpose

type

washing powder

proteases, lipases etc.

food technology

pectinases, amylases

textile industry

leather, „biobleaching“

biosensors

glucose oxidase

Universität Rostock Technische Chemie Biocat 08.ppt _ 80

Process Devopment & Improvement •

modification of biocatalyst by genetic engineering - rational protein design - directed evolution Review: M. Pohl, U. Bornscheuer, Curr. Opin. Chem. Biol. 5, 37 (2001)



biochemical engineering - reactor design (to overcome product inhibition) - outline of process

– novel approaches such as Ionic Liquids Universität Rostock Technische Chemie Biocat 08.ppt _ 81

Alcohol Dehydrogenase for Production of Chiral Alcohols O

2-octanone

ADH

NADPH

H

NADP+

OH

(R)-2-octanol OH

O

ADH acetone

• rec ADH from: Lactobacillus brevis Thermoanaerobium spec.

2-propanol

→ (R)-alcohol → (S)-alcohol

• used for production of chiral alcohols on 10 - 100 kg scale eg (R)-ethyl 3-hydroxybutyrate or (R)-2-octanol Universität Rostock Technische Chemie Biocat 08.ppt _ 82

Solubility of 2-Octanone / 2-Octanol O

ADH

H

OH

addition of watersoluble organic solvent → one phase system

water-solubility: 1.1 g/L

pure organic solvent (mainly lipases) water + organic solvent → two phase system Universität Rostock Technische Chemie Biocat 08.ppt _ 83

MTBE for Two-Phase Enzymatic Reduction Partition coefficients MTBE / H2O 2-propanol 1.0 acetone 1.1

O

O

↓↑

2-octanone

> 100

2-octanol

> 100

OH

MTBE

H

↑↓

ADH

O

NADPH

H

OH

NADP+ OH

O

buffer

OH

ADH

• thermodynamic equilibrium • distribution between phases • excess of 2-propanol is necessary Universität Rostock Technische Chemie Biocat 08.ppt _ 84

High Excess of Regeneration Substrate Necessary two phase

100 60 min 2 days

MTBE / buffer 10 mM 2-octanone 2 M 2-propanol 30 °C 2.5 ml each phase

50

ADH

25 100

0

1:0

1:2

1:10

1:20

1:100

ratio (2-octanone : 2-propanol)

max. conversion = 98 % in 24 h

1:200

conversion / %

conversion / %

75

75

50

25

1 : 200

0 0

400

800

1200

1600

time / min Universität Rostock Technische Chemie Biocat 08.ppt _ 85

Favorable Partition Coefficient when Using an IL O

Partition coefficients solvent / H2O MTBE

OH

solvent O

H

OH

IL

2-propanol

1.0

0.4

acetone

1.1

2.0

↓↑

O

↑↓

ADH

NADPH

buffer

OH

NADP+ OH

O

[BMIM] [(CF3SO2)2N] [BMIM] [BTA]

H

ADH

• regeneration substrate stays in water phase • regeneration product removed from water phase → reduction of product inhibition Universität Rostock

Eckstein et al.; Chem. Comm. 1084 (2004)

Technische Chemie Biocat 08.ppt _ 86

Increased Productivity of Octanone Reduction 100

conversion / %

75

50

25 [BMIM] [(CF3SO2)2N] / buffer MTBE / buffer

0 0

100

200

300

400

500

600

time / min

• higher reaction velocity due to reduced product inhibition • in both cases max. conversion 95-98 % Universität Rostock

Eckstein et al.; Chem. Comm. 1084 (2004)

Technische Chemie Biocat 08.ppt _ 87

Second enzyme for cofactor regeneration

two phase

ADH

O

OH

H 2-octanone OH HO HO

(R)-2-octanol

NADP+

NADPH

OH O OH

HO HO

GDH

O

OH

gluconolactone non -enz yma tic H2 O

O OH

glucose OH HO HO

OH CO2OH gluconate

Marrit Eckstein

Universität Rostock Technische Chemie Biocat 08.ppt _ 88

Enzyme-coupled cofactor regeneration MTBE

Partition coefficients MTBE / H2O

O

glucose

-

gluconate

-

2-octanone

↓↑

H

> 100

↑↓

ADH

O

> 100

H

OH

NADP+

NADPH

glucose

gluconolactone

2-octanol

OH

GDH

non -enz yma tic H2 O

buffer

gluconate

• irreversible cofactor regeneration

• glucose / gluconate stay in aq. phase Universität Rostock Technische Chemie Biocat 08.ppt _ 89

Reduced ecxess of regeneration substrate

two phase

100

MTBE / buffer 10 mM 2-octanone 20 mM glucose 30 °C 2.5 ml each phase

60 min 1 day

50

GDH

25

100 0

1:0

1:0,2

1:1

1:2

ratio (2-octanone : glucose)

max. conversion >99 % in 6 h

1:4

75

conversion / %

conversion / %

75

ADH

50

25

1:2 0 0

100

200

300

400

500

600

time / min Universität Rostock Technische Chemie Biocat 08.ppt _ 90

Increasing the substrate concentration

two phase

conversion / %

100

MTBE / buffer ADH 100 mM 2-octanone GDH 200 mM glucose 30 °C 2.5 ml each phase

75

50

25 conversion (GC)

0 0

100

200

300

400

500

time / min

Universität Rostock Technische Chemie Biocat 08.ppt _ 91

Increasing the substrate concentration

two phase

conversion; pH rel [%]

100

MTBE / buffer ADH 100 mM 2-octanone GDH 200 mM glucose 30 °C 2.5 ml each phase

75

50

25

end of reaction

pH rel conversion (GC)

pH ~ 3.8

0 0

100

200

300

400

500

time [min]

Universität Rostock Technische Chemie Biocat 08.ppt _ 92

Scale up

two phase

100

MTBE / buffer 400 mM 2-octanone 500 mM glucose 30 °C 100 ml each phase

conversion / -

75

ADH GDH

50

pH - control pH ~ 7.0

25

0 0

25

50

75

100

time / h

max. max.conversion conversion==99.9 99.9% % ~~4.5 4.5ml mlproduct product isolated isolatedyield yieldafter afterdistillation distillation~72 ~72% % space spacetime timeyield yield 50 50g/(L×d) g/(L×d)

Universität Rostock Technische Chemie Biocat 08.ppt _ 93

Take Home Message



Biotransformations are gaining importance due to their better selectivity and „cleaner“ reaction conditions. They replace and complement chemical processes.



Measures have to taken to meet special properties and requirements of biocatalysts (product inhibition, toxicity).



More than 100 industrial biotransformations known - most of them use hydrolases. - oxidoreductases often as whole cells (cofactor!).

• •

Biotransformations are possible in organic solvents & IL´s. Protein metabolic engineering for improved - availability. - selectivity and specificty Universität Rostock Technische Chemie Biocat 08.ppt _ 94

`Bacteria are capable of bringing about chemical reactions of amazing variety and sublety in an extremely short time... Many bacteria are of very great importance to industry where they perform tasks which would take much time and trouble by ordinary chemical methods.´ (The New Scientist, 1st issue 22 Nov. 1956)

Universität Rostock Technische Chemie Biocat 08.ppt _ 95

UNIVERSITÄT ROSTOCK Mathematisch-Naturwissenschaftliche Fakultät FB Chemie – Abt. für Analytische, Technische und Umweltchemie Technische Chemie - Prof. Dr. Udo Kragl

.

U. Kragl, Universität Rostock, D-18051 Rostock

Albert-Einstein-Str. 3A D-18059 Rostock Tel. Fax. Email

Biocatalysis - References

. _

0381-498-6450 0381-498-6452 [email protected]

10.01.2008

Bailey, J. E.; Ollis, D. F.; 1986; Biochemical Engineering Fundamentals, McGraw-Hill, New York. Bisswanger, H. 1994; Theorie und Methoden der Enzymkinetik. Verlag Chemie, Weinheim. 2. Aufl. Bornscheuer, U.; Kazlauskas, R. J.; 1999; Hydrolases in Organic Synthesis, Wiley-VCH. Chmiel, H. (Ed.); 1991; Bioprozeßtechnik 1 & 2, Gustav Fischer Verlag, Stuttgart und neuere Auflage. Collins, A. N.; Sheldrake, G. N.; Crosby, J. (Eds.); 1992; Chirality in Industry; John Wiley & Sons, Chichester. Cornils, B.; Herrmann, W. A.; Schlögl, R.; Wong, C.-H., Catalysis from A to Z, Wiley-VCH, Weinheim, 2000 und neuere Ausgaben. Cornish-Bowden, A. 1995; Fundamentals of enzyme kinetics. 2nd ed. Portland Press. Drauz, K.; Waldmann, H. (Eds.); 2002; Handbook of Enzyme Catalysis in Organic Synthesis; VCH, Weinheim. Faber, K.; 2000; Biotransformations in organic chemistry, 4th ed. Springer Verlag, Berlin. Fluka, Spezialbroschüre Enzymes; Fluka, Buchs, CH. Godfrey, T.; West, S. (Eds.); 1996, Industrial Enzymology - Application of Enzymes in Industry; 2nd Edition, Macmillan, London. Holland, H. L.; 1992; Organic synthesis with oxidative enzymes; VCH Weinheim. Karlson, P.; 1984; Kurzes Lehrbuch der Biochemie; Georg Thieme Verlag, Stuttgart. Liese, A.; Seelbach, K.; Wandrey, C., Industrial Biotransformations, VCH-Wiley, Weinheim, 2000, 2004. Patel, R. N., Stereoselective Biocatalysis, Marcel Dekker, New York, 2000. Roberts, S. M.; Casy, G.; Nielsen, M.-B.; Phythian, S.; Todd, C., Wiggins, K.; 1999; Biocatalysts for fine chemicals synthesis, John Wiley & Sons; previously published as Preparative biotransformations: Whole cells and isolated enzymes in organic chemistry. Wiley, Chichester, 1997. Schmid, R. D. 2001, Taschenatlas der Bio- und Gentechnologie, Wiley-VCH. (Engl. Version 2003) Schomburg, D.; 1993; Enzyme Handbook. Springer, Berlin; www.brenda.uni-koeln.de Segel, I. H.; 1975; Enzyme kinetics. John Wiley & Sons, New York. Sheldon, R. A.; 1993; Chirotechnology: industrial synthesis of optically active compounds; Marcel Dekker, New York. A. J. J. Straathof, P. Adlercreutz (Eds.), 2000: Applied Biocatalysis, 2nd. Edition, Harwood Academic Publishers, Amsterdam, ISBN 90-5823-023-6 Tanaka, A.; Tosa, T.; Kobayashi, T. (Eds.); 1993; Industrial application of immobilized biocatalysts; Marcel Dekker, New York. Wong, C.-H.; Whitesides, G. M. 1994; Enzymes in synthetic organic chemistry. Pergamon Press, Oxford. Selected Chapters/Keywords in (print, online) Roempp´s Chemie Lexikon Ullmann´s Encyclopedia of Industrial Chemistry Winnacker-Küchler Winnacker-Küchler: Chemische Technik 5. Aufl. pp 1377-1416 (2005) Handbok of Heterogenous Catalysis, 2. Aufl. (2008). 1