OXIDOREDUCTASES

DEHYDROGENASESMONOOXYGENASES

DIOXYGENASESOXIDASES

PEROXIDASES

REACTIONS: oxidation of hydroxyl, aldehyde and oxo groups, oxidation of primary and secondary amines, hydroxylation of aromatic or non-activated carbon atoms, dehydrogenation of carbon-carbon single bonds, heteroatom oxygenation, Baeyer-Villiger oxidation, and double bond epoxidation.

HYDROGEN ACCEPTORS: NAD(P), cytochrome, molecular oxygen, disulfide, quinone or similar compounds, nitrogenous groups, iron-sulfur proteins and flavin.

R2CHOH

R2CO

NAD(P)

NAD(P)H NAD(P)

NAD(P)H

R2CHOH

R2CO

E E

N

NN

N

N H 2

O

O H O R 2

O

P

O

P

O

O

O H O H

R 1

OO

OO

_

_

C o f a c t o r R 1 R 2

N

C O N H 2

N

C O N H 2

N A D +

N A D P +

N A D H

N A D P H

H

P O

H

P O

H R H S

3

3

=

=+

Alcohol oxidation Ketone reduction

General formula of NAD(P)(H)

Strategies for NAD(P)(H) regeneration

- Enzymatic: Enzyme-coupled

Substrate-coupled

- Biological: Engineered whole-cells

- Electrochemical

- Chemical

- Photochemical

Coenzyme costs: Euros/Kilogram (Oriental Yeast, 2007)

- NAD 1,500

- NADP 6,000

NAD(P)

NAD(P)H

Substrate red.

Product ox. Co-substrate

ox.

Co-product red.

a)

b)

NAD(P)

NAD(P)H

Enzyme 1 Enzyme 2

Substrate red.

Product ox.

Co-substrate ox.

Co-product red.

Enzyme

Enzymatic regeneration systems of nicotinamide cofactors illustrated in the direction of oxidized product formation.

a) Enzyme-coupled system b) Substrate-coupled system.

Criteria for an ideal enzymatic cofactor regeneration system

1) Inexpensive and stable enzymes;2) High specific activity;3) Simple and inexpensive reagents that do not interfere with the isolation of the product of interest or with enzyme stability; 4) High turnover number (TN, moles of product formed per mole of cofactor per unit time); 5) High total turnover number (TTN, moles of product formed per mole of cofactor during the entire reaction period). Economic considerations require that the TTN is at least 102-104;6) An overall equilibrium for the coupled enzyme system favourable to product formation.

In this way, the total amount of cofactor continuously present in the system can be reduced to a catalytic level and the conversion of substrate to product can be complete.

- Chenault and Whitesides Appl. Biochem. Biotechnol. 14 (1987) 147-197

b)

NAD(P)+

NAD(P)H + H+

Glucose Dehydrogenase

H2O

NAD+

a)

NADH + H+

Formate Dehydrogenase

HCOOH

CO2

OH OOH

OH

OH

OH

OH OOH

OH O

OH

COOHOH OH

OHOH

OH

Enzymatic systems for NAD(P)H regeneration

c)

b)

NAD+

NADH + H+

L-Lactate Dehydrogenase

_

_

NAD+

NADH + H+

Alcohol Dehydrogenase

NAD(P)+

NAD(P)H + H+

Secondary Alcohol Dehydrogenase

a)

NAD(P)+

NAD(P)H + H+

Glutamate Dehydrogenase

+ NH4_ _

_ _

+

+

OOC COO

NH3

O

COO

O

COO

OH

O

OH OH

OOC COO

O

Enzymatic systems for NAD(P) regeneration

K = 1.4 - 11.6 x 1014 M-1

K = 3.6 x 1011 M-1

K = 1.7 x 109 M-1K = 1.2 x 1011 M-1

Oxidation of monosodium glutamate(MSG) to α-ketoglutarate catalyzedby glutamate DH

Coenzyme TTN 10

NAD regeneration by NADH oxidase

- Odman, Wellborn, Bommarius Tetrahedron: Asymmetry 15 (2004) 2933-2937

Stereoselective oxidation of rac-1-phenylethanol catalyzed by a R-specific alcohol DH

Coenzyme TTN 6

- Geueke, Riebel, Hummel Enz. Microb. Technol. 32 (2003) 205-211

Quasi-irreversible oxidation of rac-sec-octanol catalyzed by Sphingobium yanoikuyae ADH, employing chloroacetone as hydrogen acceptor

Substrate conc. 30 gL-1 Chloroacetone 1.5 equiv

Lavandera, Kern, Resch, Ferreira-Silva, Glieder, Fabian, Wildeman, Kroutil Organic Lett. 10 (2008) 2155-2158

Reactors

1) Native enzymes/Native coenzymes/Batch reactor

2) Immobilized enzymes/ Native coenzymes/Batch reactor

3) Native enzymes/Native coenzymes/Uncharged-membrane reactor

4) Native enzymes/Macromolecular coenzymes/ Uncharged-membrane reactor

5) Native enzymes/Native coenzymes/Charged-membrane reactor

Kuge, Inoue, Ando, Eguchi, Oshiro, Mochida, Uwajima, Sugaya, Kanazawa, Okabe, Tomioka, J. Chem. Soc., Perkin Trans. 1, 1994, 1427.

Diastereoselective reduction of dihydrofolic acid (2) to tetrahydrofolic acid (3), a precursor of the anticancer drug (S)-leucovorin, catalyzed by dihydrofolate reductase

(Native enzymes/Native coenzymes/Batch reactor)

Scale: 10 kilograms (100 L volume)

Conversion ≥ 90%

De 94%

Coenzyme TTN 100

Regio- and enantioselective reduction of diketoester 10 to a statin precursor catalyzed by Lactobacillus brevis alcohol dehydrogenase (LBADH)

(Native enzymes/Native coenzymes/Batch reactor)

MTBE / Buffer biphasic system

Substrate conc. 0.2 MConversion 98%Coenzyme TTN 14,000

- Wolberg, Filho, Bode, Geilenkirchen, Feldmann, Liese, Hummel, Muller Bioprocess Biosyst. Eng. 31 (2008) 183-191

+ NADPH + O2 + H+ + NADP+ + H2OCHMOO

O

O

OO

O

O O

( + )-1 (1R,5S)-2 (1S,5R)-3

CHMO+

1 5 1 5 15

1 2-3

Cyclohexanone monooxygenase (CHMO) catalyzed oxidationof bicyclo[3.2.0]hept-2-en-6-one 1 to lactones 2 and 3

N

NN

N O

O

R

OHH

H

N

NN

N O

O

R

H

N

NN

N O

O

R

H

H

H

N

NN

N O

O

R

OH

H

O

O2

NADPH

O

H2O

4a-hydroxyflavin

FAD FADH2

4a-peroxy-flavin

N

NN

N O

O

R

OH

HOHO

NADP

N

NN

N O

O

R

OH

H

OH

OO

4a-hydroperoxy-flavin

Mechanism proposed for oxygen insertion by CHMO in Baeyer-Villiger reaction

- Sheng et al. Biochemistry 2001, 40, 11156-11167

Protocol for the oxidation of bicycloheptenone with isolated CHMO and ADHTB, with coenzyme recycling and continuous substrate feeding by a

pump (Native enzymes/Native coenzymes/Batch reactor)

Reaction conditions normalized to 1 liter volume

- Buffer: 0.05 M Tris-HCl buffer, pH 8.6- Coenzyme: 0.25 mM NADP- Second substrate: 1 M 2-propanol- CHMO: 5000 units- ADH from T. brockii: 6000 units- Substrate: bicycloheptenone fed at 0.7 g/h for a total of 14 h(final concentration 92 mM, equivalent to 10 g/L)-Total reaction time: 24 h- Final conversion: 100%- Ee ≥ 98%

Volumetric productivity (space/time/yield): 0.47 gL-1h-1

Coenzyme TTN: 370- Carrea et al. Tetrahedron: Asymmetry 11 (2000) 3653-3657- Carrea et al. Biotechnol. Bioeng. 78 (2002) 489-496

OHOH

OH

H

COOMe

OHOH

OH

H

COOH

OAcAcO

OH

H

COOMe

OAcAcO

O

H

COOMe

OHOH

O

H

COOMe

OHOHH

COOMe

OOHH

COOMe

OHOHH

COOMe

MeOH

H+

Ac2O

Py

CrO3 OH-

Huang- Mi nl on NBS Na , n- BuOH

Chemical synthesis of ursodeoxycholic acid

OHOH

OH

H

COOH

OHOH

O

H

COOH

NADP+ NADPH + H+

12α-HSDH

Scheme of enzymatic synthesis of 12-ketochenodeoxycholic acid with NADP regeneration

GlutDHα-ketoglutarate + NADPH + NH3 + H+ L-glutamate + NADP+ + H2O

Synthesis of 12-ketochenodeoxycholic acid with immobilized enzymes

Effectiveness in coenzyme regeneration of: a) Free 12α-HSDH and GlutDH; b) Coimmobilized 12α-HSDH and GlutDH; c) Separately immobilized 12α-HSDH and GlutDH.NADP(H) concentration was: 1 5 µM; 60 µM; 350 µM.The immobilized enzymes retained ~70% of the initial activity after 2 months of continuous use.

- Carrea, Bovara, Longhi, Riva Enzyme Microb. Technol. 7 (1985) 597-600

Substrate

Reactor

Product

Magnetic stirrer

UF-membrane

Scheme of the enzyme-membrane-reactor used for the synthesis of 12-ketochenodeoxycholoic acid.

(Native enzymes/Native coenzymes/ Uncharged-membrane reactor)

Reactor volume: 10 liters. Substrate concentration: 40 g l-1. Conversion: 99.8%.Product purity: 99.8%. Reaction time: ~ 20 h. Production: up to 40 kilograms. Coenzyme TTN: ~1000. - Carrea et al. Unpublished results

NADP

COO

NH3

+

COO

O

OH

H2O

PEG-NADH

PAG-NADHCOO

CO2

E1 E2

H2O

NH4+_ _

_

H++_

Trimethyl-pyruvate

L-Tert-Leucine

E1 Formate Dehydrogenase (FDH)

E2 Leucine Dehydrogenase (Leu-DH)

Enzymatic synthesis of tert-leucine via reductive amination(Native enzymes/Macromolecular coenzymes/ Uncharged-membrane reactor)

- Wichmann, Wandrey, Buckmann, Kula Biotechnol. Bioeng. 67 (2000) 791-804- Kula, Wandrey Methods Enzymol. 136 (1987) 9-21

PEG-NAD

N

O

NH2

R P P R

N

NN

N

NH CH2CH2NH CO

CH2

CH2

CO

NH PEG

+

spacer NAD+

Structure of poly(ethylene glycol)-NAD

Substrate

Reactor

Product

Magnetic stirrer

UF-membrane

Synthesis of L-tert-leucine from trimethyl pyruvate in a continuous-flow enzyme-membrane-reactor

(Native enzymes/Macromolecular coenzymes/ Uncharged-membrane reactor)

Substrate concentration: 0.5 M; Conversion: ~ 95%; Space/time/yield: 640 grams/liter/day; PEG-coenzyme TTN: 125,000.- Wichmann, Wandrey, Buckmann, Kula Biotechnol. Bioeng. 67 (2000) 791- Kragl, Vasic-Racki, Wandrey Ind. J. Chem. 32 (1993) 103

PEG-NAD

Water-soluble macromolecular derivatives of NAD

- Riva, Carrea, Veronese, Buckmann Enzyme Micrb. Technol. 9 (1986) 556-560

Kinetic constants of some dehydrogenases for NAD and PEG-N6-(2-aminoethyl)-NAD

NAD PEG-N6-(2-aminoethyl)-NAD

___________ ___________________________ Enzyme Km (µM) Km (µM) Vmax (rel. to NAD)

_________________________________________________________________________________

Formate DH 15 82 57

Glucose DH 96 2030 3

Glutamate DH (liver) 175 444 53

Lactate DH 182 142 21

Alcohol DH (yeast) 154 1310 64

Alcohol DH (liver) 62 1150 72

3a-Hydroxysteroid DH 29 647 66

20β-hydroxysteroid DH 21 430 35

7α-Hydroxysteroid DH 193 4630 12

β-Hydroxysteroid DH 31 Not detectable

Xylose XylitolXylose reductase

NADH + H+ NAD+

TBADH

NADPH + H+ NADP+

O OH

The NADPH consumed in the synthesis of (S)-sulcatol was regenerated at the expense of 2-propanol (TTN 4,500).The NADH consumed in the synthesis of xylitol was regenerated with the glucose/glucose dehydrogenase system (TTN up to 10,000).- Rothig, T.; Kulbe, K. D.; Buckmann, A. F.; Carrea, G. Biotechnol. Lett. 1990, 12, 353.- Nidetzky, B.; Neuhauser, W.; Haltrich, D.; Kulbe, K. D. Biotechnol. Bioeng. 1996, 52, 387

(S)-sulcatol

Synthesis of (S)-sulcatol and xylitol in a continuous-flow anionic-membrane reactor

(Native enzymes/Native coenzymes/Charged-membrane reactor)

NAD(P)H regeneration by engineered whole-cells

Synthesis of optically active L-amino acids from α-keto acidswith Escherichia coli cells expressing heterologous genes

- Other enzymes such as AlaDH and PheDH were also expressed in E. coli.-Yields ≥ 80% - Ee up to 100 % - Intracellular NAD pool was used

- Galkin, Kulakova, Yoshimura, Soda, Esaki Appl.Environ. Microbiol. 63 (1997) 4651-4656

- Conversion yield 94% - Enantiomeric excess 91%- Product concentration in organic phase 1.6 M- Coenzyme TTN 13,500

- Kataoka et al. Appl. Microbiol. Biotechnol. 51 (1999) 486-490

Stereoselective reduction of ethyl 4-chloro-3-oxobutanoate by Escherichia coli transformant cells coexpressing thealdehyde reductase and glucose dehydrogenase genes

Production of (R)-1,3-butandiol (BDO) from the racemic alcohol by aCandida parapsilosis alcohol dehydrogenase (CpSADH) expressed in E. coli.

- Conversion yield 97 % - Ee 95 % - Product concentration 30 g/l-1

- NAD was regenerated by the recombinant cells

- Matsuyama, Yamamoto, Kobayashi Organic Process Res. Devel. 6 (2002) 558-561

NADPH regeneration byphosphite dehydrogenase (PTDH) / Baeyer-Villiger monoooxygenases (BVMO)

fusion enzymes expressed in E. Coli

Coenzyme TTN 395 - 1,750

- Torres Pazmino, Snajdrova, Baas, Ghobrial, Mihovilovic, Fraaije Angew. Chem. Int. Ed. 47 (2008) 2275-2278

Chemical and photochemical coenzyme regeneration

- Taken from Wichmann, Vasic-Racki Adv. Biochem. Eng. Biotechnol. 92 (2005) 225-260

6-p-Gluconate DH

Oxidation of organic sulfides catalyzed by BVMOs using(b) organorodium complex / formate and (b) adding NADP to organorodium complex / formate

PAMO: phenylacetone monooxygenase; HAPMO: 4-hydroxyacetophenone monooxygenase;CHMO: cyclohexanone monooxygenase; EtaA: ethionamide monooxygenase

- de Gonzalo, Ottolina, Carrea, Fraaije Chem. Commun. (2005) 3724-3726

__________________________________________________________________Rhodium complex NADP+ Conv. (%) Ee (%) TTN(mM) (mM) (NADP)__________________________________________________________________0 a 0.26 88 ≥98 5510.0 0 5 9 - 10.0 0.26 31 91 191.0 1.3 55 94 7__________________________________________________________________

CHMO-catalysed oxidation of methyl phenyl sulfideusing different recycling systems

a Coenzyme regeneration was carried out with theglucose-6-phosphate/glucose-6-phosphate dehydrogenase system

-de Gonzalo, Ottolina, Carrea, Fraaije Chem. Commun. (2005) 3724-3726

Comments on chemical methods for coenzyme regeneration

“It seems that chemical methods at present suffer from cumbersome reaction conditions, expensive and/or toxic reagents, and/or unwanted side products, and therefore has not been preferred for commercial or preparative applications.

This is probably the reason why very few examples of chemical methods of cofactor regeneration have been found in literature.”

- Wichmann, Vasic-Racki Adv. Biochem. Eng. Biotechnol. 92 (2005) 225-260

Electroenzymatic processes employing dehydrogenases and oxygenases

Electroenzymatic processes employing dehydrogenases and oxygenases (continued)

- Ruinatscha, Hollrigl, Otto, Schmid Adv. Synth. Catal. 348 (2006) 2015-2026

Electroenzymatic production of L-glutamate

Glutamate dehydrogenase (L-GDH) is used to produce L-glutamate from α-ketoglutarate.NADH is regenerated at a platinum electrode using a hydrogenase (H-ase).- STY: 3.04 gL-1h-1 - Final product concentration (after 5 h): 15.2 gL-1 - TTN: 1,034

- Cantet, Bergel, Comtat Enzyme Microb. Technol. 18 (1996) 72-79

Electroenzymatic production of (R)-phenylethanol

Alcohol dehydrogenase (Lb-ADH) is used to reduce acetophenone to R-phenylethanol. NADH is regenerated at a carbon felt electrode using a rhodium-complex mediator.- STY: 0.58 gL-1h-1 - Final product concentration (after 6 h): 2.04 gL-1 _ TTN: 35- Hildebrand, Lutz Tetrahedron: Asymmetry 18 (2007) 1187-1193

N N

Rh

OH2

2+

N N

Rh

H

1 1a

+

Rhodium complex mediator

(2,2‘-bipyridyl)(pentamethylcyclopentadienyl) -rhodium

1 Oxidized form

1a Reduced form

Electroenzymatic production of D-lactate

D-Lactate dehydrogenase (D-LDH) is used to transform pyruvate to D-lactate. NADH is regenerated at a carbon felt electrode using a rhodium-complex mediator.- STY: 0.42 gL-1h-1 - Final product concentration (after 3 h): 1.26 gL-1 - TTN: 4

Ruppert, Herrmann, Steckhan Tetrahedron Lett. 28 (1987) 6583-6586

Electroenzymatic production of 2,3-dihydroxybiphenyl

Hydroxybiphenyl monooxygenase (Hbpa) is used for the conversion of 2-hydroxybiphenyl to 2,3-dihydroxybiphenyl.NADH is regenerated at a carbon felt electrode using a rhodium-complex mediator.-STY: 0.20 gL-1h-1 - Final product concentration (after 2 h): 0.40 gL-1 - TTN: 10

- Hollmann, Schmid, Steckhan Angew. Chem. Int. Ed. 40 (2001) 169-171

Comments on electrochemical methods for coenzyme regeneration

-The productivity of the majority of electroenzymatic processes is rather low

-The final product concentration is often too low for practical synthesis

-It will take some time before electroenzymatic processes can be applied on industrial scale

-Productivity might be enhanced by improving electrochemical cell design

-Kohlmann, Markle, Lutz J. Mol. Catal. B Enymatic 51 (2008) 57-72

Indirect electrochemical reduction of NAD(P)

- Vuorilheto, Lutz, Wandrey Bioelectrochemistry 65 (2004) 1-7

Cross-section of the electrochemical cell.

The solution containing NAD(P) + RhMedox flowed vertically through the cathode and the current flowed horizontally.

Principle of the indirect reduction of NAD(P)

- Vuorilheto, Lutz, Wandrey Bioelectrochemistry 65 (2004) 1-7

Productivity for NAD(P) reduction in an electrochemical cell.

The solution volume was 4-70 ml