Complete Enzymatic Oxidation of Methanol to Carbon Dioxide: Towards More Eco-Efficient Regeneration Systems for Reduced Nicotinamide Cofactors

Article abstract of DOI:10.1002/adsc.201500173

A novel system for in situ regeneration of reduced nicotinamide cofactors (NADH) is proposed: through a cascade of alcohol dehydrogenase (ADH), formaldehyde dismutase (FDM) and formate dehydrogenase (FDH) complete oxidation of methanol to carbon dioxide (CO₂) is coupled to the regeneration of NADH. As a consequence, from one equivalent of methanol three equivalents of NADH can be obtained. The feasibility of this cascade is demonstrated at the examples of an NADH-dependent reduction of conjugated C=C-double bonds (catalysed by an enoate reductase) and the NADH-dependent hydroxylation of phenols (catalysed by a monooxygenase). The major limitation of the current regeneration system is the comparably poor catalytic efficiency of the methanol oxidation step (low k_cat and high K_M value of the ADH used) necessitating higher than theoretical methanol concentrations.

Full text of DOI:10.1002/adsc.201500173

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DOI: 10.1002/adsc.201500173
Very Important Publication
Complete Enzymatic Oxidation of Methanol to Carbon Dioxide:
Towards More Eco-Efficient Regeneration Systems for Reduced
Nicotinamide Cofactors
a,b,
a,c
a
a
Selin Kara, * Joerg H. Schrittwieser, Serena Gargiulo, Yan Ni,
d
e
f
Hideshi Yanase, Diederik J. Opperman, Willem J. H. van Berkel,
and Frank Hollmann *
Department of Biotechnology, Delft University of Technology, Julianalaan 136, 2628BL Delft, The Netherlands
Fax : (+31)-(0)1527-81415; phone: (+31)-(0)1527-81957; e-mail: f.hollmann@tudelft.nl
a,
a
b
c
d
e
f
Institute of Microbiology, Chair of Molecular Biotechnology, Technische Universitꢀt Dresden, 01062 Dresden, Germany
Fax : (+49)-(0)351-463-39520; phone: (+49)-(0)351-463-39517; e-mail: selin.kara@tu-dresden.de
Present address: Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28,
8010 Graz, Austria
Tottori University, Graduate School of Engineering, Department of Chemistry and Biotechnology, 4-101 Koyamacho
Minami, Tottori 6808552, Japan
Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O. Box 339,
Bloemfontein, 9300, South Africa
Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands
Received: February 26, 2015; Revised: March 18, 2015; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201500173.
Abstract: A novel system for in situ regeneration of
reduced nicotinamide cofactors (NADH) is pro-
posed: through a cascade of alcohol dehydrogenase
tionalisation reactions such as hydroxylation, epoxida-
tion, Baeyer–Villiger oxidation and heteroatom oxy-
genations. Not least due to the development of effi-
[
1]
(
ADH), formaldehyde dismutase (FDM) and for-
cient in situ regeneration systems for the reduced
[
2]
mate dehydrogenase (FDH) complete oxidation of
methanol to carbon dioxide (CO ) is coupled to the
nicotinamide cofactors [NAD(P)H] oxidoreductases
have become practical catalysts currently taking the
2
[
3]
regeneration of NADH. As a consequence, from
one equivalent of methanol three equivalents of
NADH can be obtained. The feasibility of this cas-
cade is demonstrated at the examples of an
NADH-dependent reduction of conjugated C=C-
double bonds (catalysed by an enoate reductase)
and the NADH-dependent hydroxylation of phe-
nols (catalysed by a monooxygenase). The major
limitation of the current regeneration system is the
comparably poor catalytic efficiency of the metha-
nol oxidation step (low kcat and high KM value of
the ADH used) necessitating higher than theoreti-
cal methanol concentrations.
step to industrial application.
Today, the aforementioned regeneration systems
are mainly evaluated with respect to ease of applica-
bility and efficiency in terms of NAD(P)H regenera-
tion rates. However, when it comes to large industrial
applications also questions concerning the eco-effi-
ciency need to be addressed.
Simple alcohols are attractive sacrificial electron
donors for promoting the in situ regeneration of
NAD(P)H. They are cheap and readily available and
may even serve as co-solvents increasing the solubility
of the reactants. Furthermore, a broad range of alco-
hol dehydrogenases (ADHs) catalysing the oxidation
of alcohols are available. The majority of the ADH-
based regeneration systems utilise isopropyl alcohol
as sacrificial electron donor. As a consequence,
Keywords: cofactor regeneration; complete metha-
nol oxidation; enzymatic cascades; oxidoreductases
5
8 grams of acetone by-product are formed per mol
of desired product and only one of the three oxidisa-
ble carbon atoms is actually used for the regeneration
reaction. Some time ago, Berkowitz and co-workers
[4]
Oxidoreductases enable a broad range of synthetically reported a cascade of ADH and aldehyde dehydro-
useful reactions for the organic chemist, ranging from genase for the double oxidation of ethanol to acetic
reduction of C=O, C=N and C=C bonds to oxyfunc- acid while regenerating two equivalents of NADH
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Selin Kara et al.
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Scheme 1. Complete oxidation of methanol using alcohol dehydrogenase (ADH), formaldehyde dismutase (FDM) and for-
mate dehydrogenase (FDH) to regenerate three equivalents of the reduced nicotinamide cofactor (NADH).
per mol of ethanol used. Hence, the NADH yield was
doubled and the amount of by-product formed was
reduced to 30 grams per mol. More recently, we have
established the double oxidation of 1,4-butanediol to
the corresponding lactone as NADH regeneration
[
5]
system yielding 43 grams per mol of by-product. De-
spite these improvements in eco-efficiency, both
methods still leave principally oxidisable carbon-
atoms in the co-substrates unused.
Therefore, we became interested in the oxidation of
methanol to CO to regenerate NADH, which ideally
2
would result in less than 15 grams of a volatile (CO2) Scheme 2. Application of the proposed methanol oxidation
cascade (Scheme 1) to promote a monooxygenase-catalysed
coproduct per mol of the desired product and would
aromatic hydroxylation. 3HB6H: 3-hydroxybenzoate-6-hy-
droxylase.
make full use of the reducing power of methanol
(
Scheme 1). Furthermore, methanol as bulk chemical
is readily available and cheap. Today, it is mostly ob-
tained from fossil resources but fermentative (biore-
[
6]
newable) routes are gaining importance. To achieve
To test the proposed NADH regeneration cascade,
this goal, we envisioned a three-enzyme cascade com- we chose the regioselective hydroxylation of 3-hy-
prising an alcohol dehydrogenase (ADH), formalde- droxybenzoate (3-HB) yielding 2,5-dihydroxyben-
hyde dismutase (FDM) and formate dehydrogenase zoate (2,5-DHB) as catalysed by 3-hydroxybenzoate-
(
FDH).
6-hydroxylase (3HB6H) from Rhodococcus jostii
[
8]
While ADHs and FDH are well-established RHA1 (Scheme 2).
[
2]
NAD(P)H regeneration catalysts, FDM is somewhat
less well-known. FDMs have been reported as early conditions are shown in Figure 1.
as the 1980s as useful catalysts for the disproportiona- The first observation we made was that the overall
tion of aldehydes (Cannizzaro reaction). Mechanisti- reaction rate significantly increased the more com-
cally, the overall redox-neutral disproportionation re- plete the regeneration cascade was. For example,
The results of a systematic variation of the reaction
[
7]
+
action is a sequence of NAD -dependent oxidation of using only YADH as NADH regeneration enzyme (in
an aldehyde (to the corresponding carboxylic acid) the presence of 200 mM MeOH) gave only 34% con-
followed by an NADH-dependent reduction of the version of the starting material after 48 h. This value
second aldehyde to the alcohol (Scheme SI1, Support- increased to 41% in the presence of FDM and to
ing Information).
60% if all three enzymes (YADH, FDM and FDH)
Overall, by designing an ADH–FDM–FDH cas- were present under otherwise identical reaction con-
cade, a complete oxidation of methanol to CO2 ditions. Our current explanation for this observation
should be coupled to the regeneration of three equiv- is that with a more complete cascade the overall
alents of NADH (Scheme 1).
NADH regeneration rate increased thereby alleviat-
2
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Complete Enzymatic Oxidation of Methanol to Carbon Dioxide
Scheme 3. Application of the proposed methanol oxidation
cascade (Scheme 1) to promote an enoate reductase-cata-
lysed C=C-bond reduction (TsER: enoate reductase from
Thermus scotoductus).
Figure 1. Oxyfunctionalisation of 3-HB to 2,5-DHB cata-
lysed by 3HB6H using methanol as a cosubstrate using
YADH only (light grey), YADH and FDM (grey), YADH,
FDM and FDH (dark grey and black). General conditions:
MOPS buffer (200 mM, pH 8), T=308C, c(3-HB)=60 mM,
+
c(NAD )=0.5 mM.
For
c(MeOH)=20–400 mM:
À1
À1
c(3HB6H)=0.5 gL ,
c(YADH)=3 gL ,
c(FDM)=
c(FDM)=
À1
À1
1
.0 gL , c(FDH)=1.0 gL ; for c(MeOH)=500 mM:
À1
À1
c(3HB6H)=0.75 gL ,
c(YADH)=3 gL ,
À1
À1
1
0
.0 gL , c(FDH)=1.0 gL ; black bar, c(3HB6H)=
À1
À1
À1
.75 gL ,
c(YADH)=4.5 gL ,
c(FDM)=1.5 gL ,
À1
c(FDH)=1.5 gL . Reaction times: 48 h (20/200 mM
MeOH) and 72 h (400/500 mM MeOH).
ing the NADH limitation of the production reaction.
Further experiments investigating the in situ NADH
concentration are currently ongoing to substantiate
this assumption.
Even more pronounced, however, was the effect of Figure 2. Reduction of ketoisophorone (KIP) to (R)-levo-
c(MeOH). Fairly high concentrations of MeOH dione using methanol as a cosubstrate using YADH only
(
light grey), YADH and FDM (grey), YADH, FDM and
FDH (dark grey). General conditions: MOPS buffer
200 mM, pH 8, 5 mM CaCl , 1% v/v ACN), T=308C,
(
500 mM) were necessary to achieve (near) complete
conversion using the full regeneration cascade. Under
these conditions complete conversion of the starting
material could be achieved with slightly increased bio-
catalyst concentrations (Table SI3, Supporting Infor-
mation). A thorough determination of the steady-
state kinetics of YADH, FDM and FDH revealed
that maximum activity of enzymes were not explored
(
2
+
À1
c(KIP)=60 mm, c(NAD )=0.5 mm, c(TsER)=0.5 gL ,
c(YADH)=3.0 gL , c(FDM)=1.0 gL , c(FDH)=1.0 gL
Black
c(FDM)=1.5 gL , c(FDH)=1.5 gL . Reaction times: 48 h
(
À1
À1
À1
À1
À1
bar,
c(TsER)=1.0 gL ,
c(YADH)=4.5 gL ,
À1
À1
20 mM MeOH) and 72 h (200/400 mM MeOH).
(
Figures SI1, 2 and 3, Supporting Information). Over-
all, poor catalytic efficiencies on substrates (low kcat
and high KM values) were responsible for this obser-
Qualitatively, the same results were obtained using
vation. This, together with the relatively poor stability TsER as model enzyme (Figure 2). Again, a very sig-
of YADH, may also explain why (so far) we were nificant influence of the methanol concentration on
unable to establish the theoretical 1:3 stoichiometry the overall product formation (rate) was observed
of MeOH to 2,5-DHB. With respect to ADH, future and, again, the more complete the regeneration cas-
development will have to concentrate on the identifi- cade, the better were the reaction rates. The product
cation/evolution of an ADH with more favourable ki- (R)-levodione was obtained with a high enantiomeric
[9e]
netic properties towards MeOH.
excess (ee) value of 92% as previously reported,
We also applied the proposed NADH regeneration hence high concentrations of MeOH did not signifi-
cascade to promote an enoate reductase-catalysed re- cantly affect the ee. However, the product racemised
[10]
duction of conjugated C=C-double bonds (e.g., keto- over time, as also documented in the literature.
isophorone). As a model enzyme we used the enoate Representative time courses for both model reac-
reductase 9f]rom Thermus scotoductus (TsER) tions under ꢂoptimisedꢃ conditions (i.e., high concen-
ACHTUNGTRENNUNG
[
(
Scheme 3).
tration of methanol) are given in Figure 3.
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Acknowledgements
The authors thank Deutsche Bundesstiftung Umwelt (DBU)
AZ 13261) for financial support of the project. Maarten
(
Gorseling is acknowledged for technical support.
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6
Complete Enzymatic Oxidation of Methanol to Carbon
Dioxide: Towards More Eco-Efficient Regeneration
Systems for Reduced Nicotinamide Cofactors
Adv. Synth. Catal. 2015, 357, 1 – 6
Selin Kara,* Joerg H. Schrittwieser, Serena Gargiulo,
Yan Ni, Hideshi Yanase, Diederik J. Opperman,
Willem J. H. van Berkel, Frank Hollmann*
6
asc.wiley-vch.de
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