A new regeneration system for oxidized nicotinamide cofactors

Article abstract of DOI:10.1002/adsc.200900033

A novel regeneration system for oxidized nicotinamide cofactors (NAD ⁺ and NADP⁺) is presented. By combining 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS)-catalyzed oxidation of NAD(P)H with laccase-catalyzed utilization of molecular oxygen as terminal oxidant, a simple chemo-enzymatic NAD(P)+ regeneration method is achieved. Thus, the advantages of both worlds, chemical oxidation of reduced nicotinamide cofactors and laccase-catalyzed utilization of oxygen from air are combined in a simple and generally applicable new approach for biooxidation catalysis. This new application of the well-known laccase-mediator system (LMS) is successfully used to promote alcohol dehydrogenase-catalyzed oxidation reactions of primary and secondary alcohols. Already under non-optimized conditions, high turnover numbers of > 300 and > 16000 were obtained for the nicotinamide cofactor and ABTS, respectively. In this communication, we present the proofof-principle and initial characterization of the proposed new regeneration system.

Full text of DOI:10.1002/adsc.200900033

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DOI: 10.1002/adsc.200900033
A New Regeneration System for Oxidized Nicotinamide
Cofactors
Seda Aksu,a Isabel W. C. E. Arends,a,* and Frank Hollmanna,*
a
Biocatalysis and Organic Chemistry, Department of Biotechnology, Delft University of Technology, Julianalaan 136,
2628BL, Delft, The Netherlands
Fax : (+31)-(0)15-278-1415; phone: (+31)-(0)15-278-4423 (IWCEA), (+31)-(0)15-278-1957 (FH);
e-mail: i.w.c.e.arends@tudelft.nl or f.hollmann@tudelft.nl
Received: January 16, 2009; Published online: May 28, 2009
Dedicated to Prof. M.T. Reetz on the occasion of his 65th birthday.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.200900033.
Abstract: A novel regeneration system for oxidized
nicotinamide cofactors (NAD+ and NADP+) is pre-
sented. By combining 2,2’-azino-bis(3-ethylbenz-
thiazoline-6-sulphonic acid (ABTS)-catalyzed oxi-
dation of NAD(P)H with laccase-catalyzed utiliza-
tion of molecular oxygen as terminal oxidant, a
simple chemo-enzymatic NAD(P)+ regeneration
method is achieved. Thus, the advantages of both
worlds, chemical oxidation of reduced nicotinamide
cofactors and laccase-catalyzed utilization of
oxygen from air are combined in a simple and gen-
erally applicable new approach for biooxidation cat-
alysis. This new application of the well-known lac-
case-mediator system (LMS) is successfully used to
promote alcohol dehydrogenase-catalyzed oxidation
reactions of primary and secondary alcohols. Al-
ready under non-optimized conditions, high turn-
over numbers of >300 and >16000 were obtained
for the nicotinamide cofactor and ABTS, respec-
tively. In this communication, we present the proof-
of-principle and initial characterization of the pro-
posed new regeneration system.
reported. Alcohol dehydrogenases (ADHs, E.C.
1.1.1.x, also called ketoreductases) are the preferred
biocatalysts. Mechanistically, ADH-catalyzed oxida-
tion involves reversible hydride-abstraction from the
alcohol C atom to the oxidized nicotinamide cofactor
[NAD(P)+] yielding the carbonyl product as well as
the reduced cofactor [NAD(P)H]. For economic rea-
sons and to shift the unfavourable thermodynamic
equilibrium,[8] regeneration of the catalytically active
oxidized form is inevitable. In contrast to the mani-
fold regeneration approaches for NAD(P)H,[9–11] com-
plementary systems for the regeneration of oxidized
cofactors NAD(P)+ are scarce.[4] Most commonly, a
reducible cosubstrate is coadministered serving as ter-
minal electron sink to drive the desired oxidation re-
action. In this biocatalytic variant of the Oppenauer
oxidation, the reversible character of the ADH-cata-
lyzed transfer hydrogenation is elegantly exploited in
a single-enzyme system. A drawback of this method-
ology however is that, apart from few exceptions,[12]
high molar surpluses of the cosubstrate are necessary
to shift the equilibrium to the side of the desired
products and so generating additional waste. Further-
more, also enzyme stability often is impaired (even
though in recent times, highly solvent-tolerant ADHs
have been reported).[13] More elegantly, the high oxi-
Keywords: aerobic oxidation; cofactors; enzyme
catalysis; laccase mediator system; oxidoreductases
dation potential of the O2/H2O couple [E0
SHE
(pH 7)=0.82 V] can be exploited to efficiently drive
the reoxidation of NAD(P)H [E0
(pH 7)=
SHE
Oxidation of alcohols is one of the most fundamental ꢀ0.315 V]. This approach is also attractive from an
and important reactions in organic chemistry. economic and ecological point of view as molecular
Amongst the catalytic procedures[1] such as using oxygen is cheap, easy to handle and only water is
metal catalysts[2] or organocatalysts,[3] biocatalytic pro- formed as by-product. Recently, water- and hydrogen
cedures are gaining more and more importance as peroxide-forming NAD(P)H oxidases (E.C. 1.6.3.x)
they enable environmentally benign and selective oxi- have acquired some interest.[14–19]
dations.[4,5] For example, oxidative kinetic resolu-
Chemical oxidants such as quinoid structures,[20–22]
tions[6] and selective oxidations of polyols[7] have been transition metals[23,24] are also attractive since they are
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Scheme 1. The novel LMS-based NAD(P)+ regeneration approach (light grey) coupled to an ADH-catalyzed alcohol oxida-
tion (dark grey). NAD(P)H (originating from an ADH-catalyzed oxidation) is oxidized by the (formal) ABTS radical cation
whose regeneration is accomplished in a laccase-catalyzed, aerobic oxidation yielding water as sole by-product.
robust, inexpensive and do not distinguish between hydride acceptors are preferred to avoid the occur-
phosphorylated and non-phosphorylated nicotinamide rence of intermediate NAD radicals which might di-
cofactors. However, to avoid the generation of haz- merize and thereby become catalytically inactive.[11]
ardous hydrogen peroxide, molecular oxygen has to However, using ESR spectroscopy we were unable to
be substituted by an anode as terminal electron ac- detect a putative pyridinium radical suggesting ex-
ceptor, resulting in complicated reaction set-ups.[11] tremely low (<0.1 mM) concentrations of such inter-
Using such an approach Liese and co-workers recent- mediate radical species. This observation is in accord-
ly reported on the fast and efficient combination of ance with the published redox potentials for the first
2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) and second electron transfer steps (see Supporting In-
(ABTS) and electrochemical regeneration to promote formation)[28,29] suggesting the first electron transfer to
ADH-catalyzed oxidation reactions.[25]
be overall rate-limiting; the second electron transfer
ABTS is well-known as a spectrophotometric probe step is significantly easier (as expressed by a lower
for peroxidase and laccase activity determination. redox potential) which probably can also be attribut-
Combinations of ABTS and laccases in the so-called ed to the aromatization energy contribution.
laccase-mediator-system (LMS) have been successful-
Substituting NADH with its phosphorylated pend-
ly applied by Haltrich and co-workers for the oxida- ant NADPH resulted in essentially identical progres-
tive regeneration of flavin-dependent oxidases.[26,27] sion curves albeit at slightly reduced rates (approxi-
We became interested in the question of whether the mately 90% of the rate observed with NADH). We
LMS can be also used to promote ADH-catalyzed ox- attribute this slightly decreased rate to the additional
idation reactions as outlined in Scheme 1. Such a negative charge present in the phosphorylated
system would combine the advantages of both ap- NADPH and the resulting additional electrostatic re-
proaches: laccase-catalyzed regeneration of oxidized pulsion with the ABTS anion. Thus, we concluded
ABTS enables the use of O2 from air while forming that the regeneration system depicted in Scheme 1 is
water as sole by-product and ABTS as mediator generally applicable to NAD+- and NADP+-depen-
should enable regeneration of both NAD+ and dent ADH-catalyzed oxidation reactions.
NADP+. Due to its supposed high thermal robustness
Encouraged by these results, we further character-
we chose the commercial laccase from the thermo- ized the LMS-based NAD(P)+ regeneration system.
philic fungus Myceliophthora thermophila (MTlac- Expectedly, a linear correlation between laccase and
case, Novozymes) as a model system.
ABTS concentration and NAD(P)H oxidation rate
LMS-catalyzed oxidation of NAD(P)H: Upon ad- was observed (Supporting Information). The pH
dition of laccase to an aerated solution of NADH and range was investigated between pH 5 and 8 showing
ABTS, we were pleased to observe a decrease of the an exponential decrease of the NAD(P)H oxidation
characteristic UV absorption band of reduced nicoti- rate with increasing pH (Figure 1). Using syringalda-
namide at 340 nm (Supporting Information). In the zine as spectrophotometric probe maximum MTlac-
absence of either laccase, mediator, or under anoxic case activity is found at neutral to slightly acidic pH
conditions no significant decrease was observed. Hy- values (Supporting Information). Thus we attribute
drogen peroxide was not detectable throughout the the decreasing NAD(P)H oxidation activity to the
course of the reaction. The catalytic mechanism of pH-dependency of the ABTS redox potential. Bear-
the ABTS-mediated oxidation should include two se- ing in mind that ADH-catalyzed oxidations generally
quential one-electron transfer steps from the reduced are most efficient at alkaline pH values, all subse-
nicotinamide to the ABTS radical cation. Generally, quent experiments were (unless indicated otherwise)
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A New Regeneration System for Oxidized Nicotinamide Cofactors
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Table 1. TADH-catalyzed oxidation driven by the LMS-re-
generation system.[a]
Substrate
Conv. 5 h [%]
Conv. 24 h [%]
cyclohexanol
48.2
16.1
18.0
7.8
62.8
33.8
19.6
13.6
19.3
4-methylcyclohexanol
decahydro-2-naphthol
citronellol
2-octanol
n.d.
[a]
Conditions: 100 mM KPi buffer (pH8), T=408C, [sub-
strate]0 =10 mM, [TADH]=2.96 mM, [NADH]0 =
0.1 mM, [ABTS]=50 mM, [MTlaccase]=10 mM.
Figure 1. pH-dependence of the rate of the LMS-catalyzed
oxidation of NAD(P)H. The efficiency is expressed as turn-
over frequency (TOF) of MTlaccase. (closed diamonds:
NADH, open squares: NADPH. Conditions: buffer: KPi
(50 mM adjusted to the appropriate pH value), T=258C,
[NAD(P)H]0 =0.2 mM, [ABTS]=5 mM, [laccase]=10, 40,
100, and 400 nM (for pH 5, 6, 7, and 8, respectively).
concluded that a reaction temperature range of 30–
408C is optimal considering acceptable catalytic activ-
ity and thermal stability of the bioctatalyst.
The coupled LMS-ADH system: As a model
system to investigate the feasibility of the envisioned
performed at pH 8. It is obvious from Figure 1 that O2-LMS-driven and ADH-catalyzed oxidation of al-
the potentially very high catalytic efficiency of MTlac- cohols, we chose the ADH from Thermus sp.
case is not exploited by far. Further studies on identi- (TADH).[31] Especially its facile fermentation/purifica-
fying more suitable mediators than ABTS such as qui- tion procedure, enabling production at scale within 2
nones or phenylenediimines[11] are underway.
days, make it a well-suited model system.[23] In a first
The temperature-dependencies of the activity and set of experiments we compared the productivity of
stability of the LMS at pH8 are shown in Figure 2. the coupled catalytic system (Scheme 1) with the stoi-
Interestingly, a linear increase of the LMS-cata- chiometric addition of NAD+ for a representative se-
lyzed NAD(P)H oxidation rate was observed. One lection of TADH substrates (Table 1). While initial
plausible explanation for this deviation from expo- rates were hardly influenced (Supporting Informa-
nentiality might lie in the decreasing O2 concentra- tion), the conversions observed in the catalytic system
tion. MTlaccase exhibits a comparably low affinity to- were always higher than when stoichiometric amounts
wards O2 [KM(O2)ꢁ50 mM][30] suggesting MTlaccase of NAD+ were applied. We attribute this to the de-
to be increasingly O2-limited at elevated temperatures creased competitive inhibition of TADH by the re-
thus counteracting the exponential activity increase duced nicotinamide cofactor and shift of the equilibri-
with temperature. MTlaccase stability was superb at um.
ambient or slightly elevated temperatures. Thus, we
Interestingly, there was a slight but significant in-
crease of enantioselectivity (E)[32] when the LMS was
applied. For example, EACTHNUTRGNE(UNG TADH) for the oxidation of
racemic 2-octanol increased from 5.8 to 7.1. Currently
we are lacking a plausible explanation for this obser-
vation.
We further investigated the optimal pH- and T-con-
ditions for the coupled enzyme system in the oxida-
tion of cyclohexanol (Figure 3).
Using the chosen conditions, initial productivities of
up to 7.2 mMhꢀ1 are achievable. The T- and pH-de-
pendence observed reflects the catalytic properties of
TADH.[31] Thus, we assume that under these condi-
tions the TADH-catalyzed oxidation was overall rate-
limiting. Product inhibition was identified as the
major limitation of this set-up (Supporting Informa-
tion). As a result, maximum conversion in the range
of 60–70% (6–7 mM of the ketone product) was ach-
ievable thereby also limiting the turnover number
Figure 2. Temperature-dependence of the activity (black dia-
monds) and stability (grey squares) of the LMS. Conditions:
KPi (50 mM, pH 8), [MTlaccase]=0.4 mM; spectrophoto-
metric detemination of activity was initiated by the simulta-
neous addition of [NADH]0 =0.2 mM and [ABTS]=20 mM. (TTN) of the catalysts. To alleviate this inhibition, we
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Figure 4. Time course of the two-liquid phase system for the
oxidation of cyclohexanol. Conditions: T=408C, 1:1 ratio of
reaction buffer and octane. Organic phase: [cyclohexa-
nol]0 =125 mM, [dodecane]=5 mM (internal standard);
aqueous phase: KPi buffer (50 mM, pH 8) [TADH]=
3.4 mM, [NADH]0 =0.1 mM, [ABTS]=2 mM, [MTlaccase]=
10 mM.
Figure 3. Temperature- and pH-dependence of the coupled
system. Conditions: [cyclohexanol]0 =10 mM, [TADH]=
2.96 mM, [NADH]0 =0.1 mM, [ABTS]=50 mM, [MTlac-
case]=10 mM.
applied a two-liquid phase system comprising octane
as substrate reservoir and product sink.
[i.e., balancing the activities of TADH and the LMS-
Indeed, initial experiments using this improved set- NAD(P)+ regeneration system] is currently underway.
up resulted in a significantly prolonged reaction time
and product accumulation (Figure 4).
Nevertheless, more than 30 mM of product were
obtained corresponding to a total turnover number of
It should be mentioned here that the stability of 325 and 16,400 for NAD and ABTS, respectively. A
the combined system (t1/2 approx. 25 h) was somewhat comparison with established NAD(P)+ regeneration
lower than expected from the stabilities of the isolat- approaches demonstrates the potential of our new ap-
ed enzymes [at 408C: t1/2 (MTlaccase)=120 h proach (Table 2). The TTN achieved for NAD with
(Figure 2) and t1/2 (TADH)@180 h[23]]. The reaction our regeneration system so far is somewhat lower
mixtures exhibited the characteristic colour of the oxi- than for the most widely applied enzyme-coupled,
dized ABTS radical which is in accordance with the glutamate dehydrogenase-catalyzed reductive amina-
assumption that TADH may be the overall rate-limit- tion of a-keto acids; but is clearly superior to other
ing factor. ABTS·+ is a known oxidant, for example, state-of-the-art approaches. Furthermore, due to the
for primary alcohols and thereby may oxidatively in- chemo-enzymatic character both NAD+- and
activate one or both enzymes thereby accounting for NADP+-dependent enzymes can be applied. Also, the
the lower robustness of the full system. Optimization use of O2 as terminal oxidant together with the exclu-
Table 2. Comparison of the catalytic performance of the current system with some reported NAD(P)+ regeneration systems.
Regeneration System
Cofactor
Cosubstrate/Coproduct
TTN[a]
Waste Factor[b]
NAD(P)
Mediator
LDH[c],[33]
NAD+
pyruvate/lactate
59
1.600
32
90
125
19
–
–
74
900
1,000
1
90
GluDH[d],[34]
NAD+
a-ketoglutarate/glutamate
147
ABTS/Anode[25]
NAD(P)+
NAD(P)+
NAD(P)+
NAD(P)+
NAD(P)+
NAD(P)+
[e]
[e]
[Ru
(PDon)3][f],[3,22]
O2/H2O2
O2/H2O2
O2/H2O2
O2/H2O
O2/H2O
34
34
34
9
CtXR/PQ[g],[20]
FMN[35]
NAD(P)H oxidase[17]
ABTS/MTlaccase
110
325
–
16,400
9
[a]
Total turnover number: mol (product)ꢁmol (cofactor/catalyst)ꢀ1.
Waste factor: g (by-product)ꢁmol (cofactor)ꢀ1.
Lactate DH.
Glutamate dehydrogenase.
Depending on the method of power generation.
PDon=1,10-phenanthroline-5,6-dione.
[b]
[c]
[d]
[e]
[f]
[g]
CtXR=Candida tenuis xylose reductase, PQ=9,10-phenanthrenequinone.
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A New Regeneration System for Oxidized Nicotinamide Cofactors
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for generous enzyme donations. Dr. K. Bꢀhler and Prof. A.
Schmid are acknowledged for providing the plasmid encod-
ing TADH. This project is financially supported by the
Dutch Ministry of Economic Affairs and the B-Basic partner
organizations (www.b-basic.nl), a public-private NWO-ACTS
programme.
sive formation of water as by-product enables a ther-
modynamically irreversible and environmentally at-
tractive regeneration system (Table 2).
Further investigations aiming at an in-depth under-
standing of the inactivation mechanism and prepara-
tive scale application of the proposed new oxidation
reaction are currently underway in our laboratory.
Also studies substituting ABTS by other mediators
which do not exhibit a pronounced pH-dependence of
their redox potential are underway.
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