Biosci. Biotechnol. Biochem., 73 (5), 1224–1226, 2009
Note
Cofactor Recycling for Selective Enzymatic Biotransformation
of Cinnamaldehyde to Cinnamyl Alcohol
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;2;y
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Paolo ZUCCA,
Maria LITTARRU, Antonio RESCIGNO, and Enrico SANJUST
1
Consorzio UNO (University of Oristano Consortium), 09170 Oristano, Italy
Department of Science and Biomedical Technology (DISTEB), University of Cagliari,
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0
9042 Monserrato (Cagliari), Italy
The enzymatic, selective hydrogenation of cinnamal-
optimum conditions for each, and then operational
conditions at which both can occur simultaneously were
assessed.
dehyde to cinnamyl alcohol is reported here. Yeast
alcohol dehydrogenase was used in a substrate-coupled
process with cofactor recycling. Both 100% selectivity
and aldehyde conversion were achieved within 3 h. The
reaction took place under very mild conditions, in the
absence of toxic organic solvent. The overall process
proved inexpensive and deserves further optimization
studies in order to evaluate industrial applications.
ADH activity determination and preliminary opera-
tional characterization were carried out by spectropho-
tometric assay. In a 3-ml cuvette, 18 ADH E.U.
(Enzyme Units, one unit defined as the enzyme amount
capable of transforming one micromole of substrate per
ꢀ
min) was incubated at 25 C in the presence of 25 mM
buffer, 0.25 mM NADH, and 1 mM CMA. The decrease
in absorbance at 340 nm was recorded using a Cary
Key words: alcohol dehydrogenase; cinnamaldehyde;
cinnamyl alcohol; cofactor recycling; hy-
drogenation
50 UV-Vis spectrophotometer Varian (NADH "
¼
340
3
ꢂ1
ꢂ1
6:22 ꢁ 10 M cm ). NADH and CMA concentrations
were changed over a proper range for determination of
Michaelis-Menten kinetic parameters.
Cinnamaldehyde (CMA) selective hydrogenation to
cinnamyl alcohol (CMO) is an important challenge in
Although reduction of CMA by NADH exhibits a pH
optimum at about 6, whereas oxidation of ethanol and
2-propanol is best accomplished at pH about 9, we have
found that pH 7 represents a satisfactory compromise,
taking into account that ethanol was always present in
large excess. Moreover, the chosen conditions ensured
optimal durability for ADH (no significant activity loss
up to 24 h).
1
,2)
the perfume and flavor industries.
reactions leading to unwanted products are common by
However, side
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)
conventional methods. Therefore selectivity is the
main goal to be reached in CMA hydrogenation. Many
catalysts have been described,4–6) involving different
inorganic catalysts. Those approaches however do not
show acceptable selectivity, and use extreme operational
conditions and hazardous solvents and chemicals. This
usually leads to serious economic concerns.
ADH activity was tested after 1 h of storage at various
ꢀ
temperatures (ranging between 20 and 60 C). The
ꢀ
In order to overcome these drawbacks, we propose
the use of commercial, inexpensive, NAD-dependent,
alcohol dehydrogenase (ADH) from the yeast Saccha-
romyces cerevisiae, whose action towards cinnamalde-
results showed that up to 30 C, ADH retained all initial
catalytic activity. Only 18% of the activity was lost at
ꢀ
40 C. Almost no activity was detected after incubation
ꢀ
ꢀ
at 50 C and 60 C (30 and 0% residual activity
respectively). Moreover, since prolonged incubation of
7
)
hyde have been described. ADHs are inhibited by
þ
8)
ꢀ
high NAD concentrations; moreover this is a rather
expensive reagent. Accordingly, its stoichiometric use
during CMA reduction appears not to be affordable. In
this perspective, the goal of this study was the develop-
ment of a cofactor recycling catalytic system in which
ADH reduces CMA to CMO (oxidizing NADH to
the enzyme at 30 C caused no significant inactivation,
in the perspective of the development of an industrial
process in which all ADH catalytic activity can be
ꢀ
exploited, 30 C was chosen as the operational temper-
ature. In order to exploit maximum ADH activity,
kinetic constants needed to be determined: the KM
þ
þ
þ
NAD ). The NAD produced was then recycled by the
same enzyme at the expense of ethanol, as suggested
values for NAD , ethanol, and 2-propanol are already
9
)
known, while those for CMA and NADH were found
to be 0.46 and 0.071 mM respectively. Accordingly, the
initial concentrations of CMA and cofactor were chosen
only slightly higher than the respective KM values. The
initial 1 mM CMA and 0.1 mM cofactor were then used.
CMA hydrogenation reactions were performed in a
mechanically stirred glass reactor equipped with tem-
perature and pH controls (Fig. 1B). In a final volume of
7)
previously. This acted as co-solvent for both CMA and
CMO, and was present in large excess to force the whole
process toward CMA reduction (Fig. 1A). Also, elimi-
nation of the obtained dehydrogenation product acetal-
dehyde drove CMA reduction toward completion. As
two reactions have to occur at the same time, the two
reactions were studied separately to determine the
y
To whom correspondence should be addressed. Department of Science and Biomedical Technology (DISTEB), University of Cagliari, 09042
Monserrato (Cagliari), Italy; Fax: +39-070-675-4527; E-mail: pzucca@unica.it
Abbreviations: ADH, alcohol dehydrogenase (E.C. 1.1.1.1); CMA, cinnamaldehyde 2-(E)-3-phenyl-2-propenal; CMO, cinnamyl alcohol,
-(E)-3-phenyl-2-propen-1-ol
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