oxidoreductases - politecnico di milano: versione italiana
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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