DOI: 10.1002/cbic.201500219
Communications
A Rapid and High-Throughput Assay for the Estimation of
Conversions of Ene-Reductase-Catalysed Reactions
Maria Chiara Forchin,[a] Michele Crotti,[b] Francesco G. Gatti,[b] Fabio Parmeggiani,*[b]
Elisabetta Brenna,[b] and Daniela Monti*[a]
A fast and sensitive colorimetric assay (FRED, fast and reliable
ene-reductases detection) that allows the estimation of levels
of conversion of ene-reductase (ER)-catalysed reactions has
been developed. The activated olefin is reduced by ER at the
expense of NAD(P)H cofactor, whose regeneration is carried
out in situ by the glucose/glucose dehydrogenase system. Sub-
sequently, the consumption of the co-substrate glucose is
determined colorimetrically by a multienzymatic system. The
FRED assay offers a wide range of possible applications, from
enzyme fingerprinting and kinetic analysis, to primary screen-
ing of enzyme libraries and optimisation of ERs’ performances
under different reaction conditions.
Even though the performances of these biocatalysts in
terms of stereoselectivity are often close to optimal from the
very first stages of the investigations, there is still some room
for improvement as far as other relevant aspects of their
synthetic application are concerned. For example, in different
cases the tolerance shown by ERs to high substrate/product
concentrations,[2b–c] as well as the specific activity toward se-
lected target substrates,[2d,5] are inadequate for large-scale ap-
plications.
Enzyme engineering and process development in ER-mediat-
ed applications demand rapid, convenient and reliable activity
assays.[6] If possible, the use of colorimetric assays also suitable
for microscale analyses is preferred because they allow parallel
screening of many samples and reduce time and reagent con-
sumption. In contrast, ER performances are currently most
often evaluated by monitoring the reactions through GC or
HPLC analyses, which in some cases advantageously provides
a simultaneous evaluation of conversion and stereoselectivity.
However, these methods are time-consuming, and a simpler
conversion-based assay might be very useful as a primary
screening method during, for example, evaluation of enzyme li-
braries and optimization of reaction conditions. In the develop-
ment of a conversion-based screening assay, we avoided the
use of substrate- or product-based assays, as well as of synthet-
ic chromogenic substrates that lack general applicability.
Direct spectrophotometric detection of NAD(P)H consump-
tion at 340 nm has previously been used for mechanistic stud-
ies and characterization of ERs,[7a] and more recently for the
development of a high-throughput screening (HTS) method
based on steady-state kinetic analyses.[7b] However, very often
the maximum absorbance peak of the unsaturated substrate
overlaps with that of NAD(P)H (especially for compounds con-
taining extended conjugated systems, such as one or more ar-
omatic rings), so this detection method becomes inapplicable,
except for the rare cases in which the analysis can be carried
out at a slightly higher wavelength.[7b] Overall, this kind of
assay intrinsically suffers from poor flexibility and requires tedi-
ous and time-consuming setup. Alternatively, a fluorescent in-
tensity (FLINT) assay[7c] has been proposed for HTS.
Ene-reductases (ERs) of the Old Yellow Enzyme (OYE) family
catalyse the stereospecific anti hydrogenation of activated ole-
fins by the transfer of a formal hydride from a flavin mononu-
cleotide (FMNH2) prosthetic group (regenerated by NAD(P)H
cofactor) to the b-carbon, followed by the delivery of a proton
from the hydroxy group of a tyrosine residue to the a-carbon
of the resulting enolate.[1] In the last decade, these biocatalysts
have proven to be a real alternative to the classical metal-
based hydrogenation methods for the preparation of enantio-
pure chiral compounds. Thus, the ER-mediated bioreduction of
various prochiral substrates (a,b-unsaturated aldehydes/ke-
tones, nitroalkenes, maleimides) has been intensively investi-
gated during the last years, although mostly on a lab scale.[2]
Many wild-type OYEs isolated from different sources (bacte-
ria, yeasts, plants etc.), showed relatively broad spectra of
accepted substrates, usually combined with good to excellent
stereoselectivity. As well as by screening naturally diverse ERs,
access to optically pure products can be achieved by exploit-
ing different enzyme-based or substrate-based stereocontrol
strategies;[3] this has in some cases led to significant improve-
ment, or even to a switch of the stereochemical outcome.[4]
[a] M. C. Forchin, Dr. D. Monti
Istituto di Chimica del Riconoscimento Molecolare, CNR
Via Mario Bianco 9, 20131 Milano (Italy)
Here we describe a simple and fast colorimetric method
(Scheme 1) for the determination of levels of conversion in ER-
catalysed reductions in a substrate-independent way (FRED,
fast and reliable ene-reductase detection).
[b] M. Crotti, Prof. F. G. Gatti, Dr. F. Parmeggiani, Prof. E. Brenna
Department of Chemistry, Materials and Chemical Engineering “Giulio
Natta”
Politecnico di Milano
Via Mancinelli 7, 20131 Milano (Italy)
The activated olefin is reduced by the ER at the expense of
the NAD(P)H cofactor, regeneration of which is carried out in
situ with the aid of the glucose/glucose dehydrogenase (GDH)
system (Scheme 1, step 1), thus allowing its use in catalytic
Supporting information for this article is available on the WWW under
ChemBioChem 2015, 16, 1571 – 1573
1571
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim