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Activity of the MtLac-15 system towards NADH and NADPH
Conclusions
The performance of the MtLac-15 system towards NAD(P)H was
determined by using the spectrophotometric assay described
above (308C, pH 8) with final concentrations of NAD(P)H, 15, and
MtLac of 0.5 mm, 0.2 mm, and 2 mm, respectively.
The well-known laccase-mediator system (LMS) has recently
been explored to promote alcohol dehydrogenase (ADH)-cata-
lysed oxidation reactions.[4g,6b] In this study, we have demon-
strated that the mediator scope goes significantly beyond the
mediators reported so far.
pH-dependent activity and stability
All mediators used in this study are commercially available
from various suppliers at reasonable quantities and costs.
From a sustainability point of view, especially natural media-
tors such as caffeic acid, chlorogenic acid, syringaldehyde and
acetosyringone may be attractive. Other lignin-derived phenol-
ics may also prove to be efficient mediators as well.
By using acetic acid and triethylamine solutions (100 mm), buffers
of pH 5, 6, 7, 8, 9 and 10 were prepared. For each pH, the laccase
activity was determined as described for the mediator screening.
Upon the activity assay, the reaction solutions were incubated at
308C and 1000 rpm. After 24 h, a sample of the reaction solution
(150 mL) was used to determine the residual activities under similar
conditions.
To demonstrate the practical usefulness of these LMSs for
in situ nicotinamide adenine dinucleotide (phosphate) NAD(P)+
regeneration, we have coupled the LMS based on the laccase
from Myceliophthora thermophila MtLac and acetosyringone
with two ADH-catalysed oxidation reactions. In this study, we
have focussed on acetosyringone because of the superior ac-
tivity found in the screening and MtLac because of its availabil-
ity at scale. This selection should, however, not exclude the
evaluation of the remaining mediators to promote ADH-cata-
lysed oxidations. Especially if bi-phasic reaction conditions are
envisioned, highly polar or charged mediators may prove su-
perior to acetosyringone as a result of more favourable parti-
tioning coefficients. Moreover, new laccases are reported fre-
quently, which may be even more suitable than MtLac.
Overall, LMSs represent an interesting approach for the aero-
bic regeneration of oxidised nicotinamide co-factors (NAD(P)+)
to promote ADH-oxidation catalysis. The performance of all
catalytic components observed in this preliminary study is al-
ready very promising, which justifies further exploration of this
reaction concept.
KM and maximum rate of MtLac towards 15
The KM value and maximum rate of MtLac at saturating substrate
concentration (Vmax) were determined by fitting the experimental
data from the correlation between the concentration of 15 and ini-
tial reaction rate to the Michaelis–Menten equation by non-linear
regression. The activities were determined in a direct and an in-
direct assay. The in-direct assay was based on the NADH-oxidation
activity as determined by the assay described for the mediator
screening by using MtLac (1 mm) at pH 7.5.
The KM and Vmax values of MtLac towards 15 were determined
photo-spectrometrically in the absence of NADH under otherwise
identical conditions. The rates were quantified by using the ab-
sorbance of oxidised 15 at 360 nm for which a calibration curve
was made.
Oxidation of d-glucose
A 10 mL reaction solution was prepared in MilliQ water that con-
tained d-glucose (25 mm), NAD+ (100 mm), MtLac (0.4 mm) and 15
(100 mm). The solution was equilibrated to a temperature of 308C
in a jacked reaction vessel as part of a Metrohm Dosimat setup.
After adjusting the pH to 7.0, the reaction was started by the addi-
tion of GDH (50 mg, 1750 U). Throughout, the reaction the pH was
maintained at pH 7.0 by means of titration with NaOH solution
(100 mm). The reaction progress over time was in-directly deter-
mined from the titration volume. The reaction mixture was centri-
fuged (3000 ꢁ g, 5 min), after which the supernatant was isolated.
The product was dried to a powder, dissolved in 10% ND3 in D2O
and subsequently analysed by NMR spectroscopy: 1H NMR
(250 MHz) d=3.50–3.83 (4H, m), 3.93 (1H, s, broad), 4.02 ppm (1H,
d); 13C NMR (250 MHz) d=65.37 (CH2), 73.72 (CH), 73.94 (CH), 75.32
(CH), 76.85 ppm (CH).
Experimental Section
Materials
The chemicals applied in this study were obtained commercially at
analytical-grade quality and used as received. The enzymes used
were laccase from Myceliophthora thermophila (MtLac, Novozym
51003, Novozymes), laccase from Trametes pubescens (TpLac), pre-
pared as described by Ferrandi et al.,[6b] glucose dehydrogenase
(evo-1.1.060, Evocatal) and horse liver alcohol dehydrogenase iso-
enzyme E (evo-1.1.210, Evocatal).
Mediator screening
The performance of different mediators was determined by means
of a spectrophotometric assay. Reaction solutions were prepared
that consist of NADH (1 mm) and mediator (50 mm) in triethyl-
amine/acetate buffer pH 7.0 or sodium citrate buffer pH 5.0
(50 mm, 1.0 mL final volume). After 5 min of incubation at 308C
and 1000 rpm, the reactions were started by addition of MtLac or
TpLac to a final concentration of 2 mm MtLac or 4.2 mm TpLac. In
time, samples (150 mL) were withdrawn, and the absorbance at
340 nm was determined upon addition of 1250 mL MilliQ water
with mixing. The recorded linear slope was used to calculate the
turnover frequency (molreduced NADH mollaccaseÀ1 sÀ1).
Oxidation of 1,4-butanediol to g-butyrolactone
Stock solutions of HLADH (10 gLÀ1), NAD+ (25 mm), 15 (2 mm) and
1,4-butanediol (500 mm) were prepared in reaction buffer (50 mm,
Tris-HCl, pH 8.0). A mixed stock solution of 1,4-butanediol stock
(200 mL), NAD+ stock (40 mL), 15 (200 mL) and reaction buffer
(940 mL) was incubated at 308C for 5 min in a 10 mL reaction
vessel. The reaction was started by the addition of MtLac (20 mL,
0.2 mm stock) and HLADH (600 mL, 10 gLÀ1). The initial concentra-
tions were: 50 mm 1,4-butanediol, 200 mm 15, 0.5 mm NAD+, 2 mm
MtLac and 3 gLÀ1 HLADH (20 UmLÀ1) in 2 mL. Aliquots (50 mL) were
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ChemCatChem 2013, 5, 3027 – 3032 3031