Journal of Biotechnology 243 (2017) 25–28

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Short communication

Biotechnological production of vanillin using immobilized enzymes

Toshiki Furuya a,b,∗ , Mari Kuroiwa a , Kuniki Kino a,∗
a
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, 3-4-1 Ohkubo, Shinjuku-ku, Tokyo 169-8555, Japan
b
Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Vanillin is an important and popular plant flavor, but the amount of this compound available from plant
Received 1 November 2016 sources is very limited. Biotechnological methods have high potential for vanillin production as an alter-
Received in revised form native to extraction from plant sources. Here, we report a new approach using immobilized enzymes for
26 December 2016
the production of vanillin. The recently discovered oxygenase Cso2 has coenzyme-independent catalytic
Accepted 28 December 2016
activity for the conversion of isoeugenol and 4-vinylguaiacol to vanillin. Immobilization of Cso2 on Sep-
Available online 29 December 2016
abeads EC-EA anion-exchange carrier conferred enhanced operational stability enabling repetitive use.
This immobilized Cso2 catalyst allowed 6.8 mg yield of vanillin from isoeugenol through ten reaction
Keywords:
Biocatalysis
cycles at a 1 mL scale. The coenzyme-independent decarboxylase Fdc, which has catalytic activity for
Cascade reaction the conversion of ferulic acid to 4-vinylguaiacol, was also immobilized on Sepabeads EC-EA. We demon-
Ferulic acid strated that the immobilized Fdc and Cso2 enabled the cascade synthesis of vanillin from ferulic acid via
Immobilized enzyme 4-vinylguaiacol with repetitive use of the catalysts. This study is the first example of biotechnological
Vanillin production of vanillin using immobilized enzymes, a process that provides new possibilities for vanillin
production.
© 2016 Elsevier B.V. All rights reserved.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is the major
aroma component of vanilla, giving it a sweet and creamy odor.
Its flavor and fragrance properties have made this compound
important around the world, especially to the foods and cosmet-
ics industries (Rao and Ravishankar, 2000; Walton et al., 2003).
Vanillin is available from the beans of the vanilla orchid through
extraction. However, the yield of vanillin is very low because it
accumulates at low levels in the plant (Rao and Ravishankar, 2000;
Walton et al., 2003). Biotechnological production of vanillin using
microorganisms has attracted much attention as an alternative to
extraction from vanilla beans (Gallage and Møller, 2015; Kaur and
Chakraborty, 2013; Priefert et al., 2001). For example, vanillin can
be produced from isoeugenol, which occurs in some essential oils,
by microorganisms (Priefert et al., 2001; Wangrangsimagul et al.,
2012; Yamada et al., 2007). Ferulic acid is a more practical starting
material for the biotechnological production of vanillin, because
a large amount of this compound can be recovered from agroin-
dustrial wastes including wheat and rice bran (Di Gioia et al., 2007;
Rosazza et al., 1995). Many microorganisms that convert ferulic acid
to vanillin have been isolated and extensively studied for vanillin
∗ Corresponding author.
E-mail addresses: tfuruya@rs.tus.ac.jp (T. Furuya), kkino@waseda.jp (K. Kino).
production (Priefert et al., 2001; Hua et al., 2007; Ma and Daugulis,
2014).
In addition to microorganisms, use of immobilized enzymes
shows promise in methods used for vanillin production. Enzymes
in microbial cells and cell-free enzymes are often unstable when
used as biocatalysts. In contrast, immobilized enzymes may be
more operationally stable due to rigidification of their structure via
direct interaction with proper carriers. Furthermore, immobilized
enzymes can be easily separated from reaction solutions, allowing
repetitive, cost-effective use (Brady and Jordaan, 2009; Hilterhaus
et al., 2008; Sheldon and van Pelt, 2013). Although vanillin pro-
duction using microorganisms has been extensively studied, there
have been no reports concerning production using immobilized
enzymes. This is partly due to the complexity of the enzyme system
in microorganisms that convert ferulic acid to vanillin. A well-
known pathway in microorganisms consists of the conversion of
ferulic acid to feruloyl-CoA, catalyzed by feruloyl-CoA synthetase,
followed by the conversion of feruloyl-CoA to vanillin by enoyl-CoA
hydratase/aldolase; the first step requires ATP and CoA as coen-
zymes (Achterholt et al., 2000; Barghini et al., 2007; Lee et al., 2009;
Narbad and Gasson, 1998). These expensive coenzymes have to be
supplied to continue the coupled enzymatic reactions when the
synthetase and hydratase/aldolase are used as immobilized cata-
lysts.

http://dx.doi.org/10.1016/j.jbiotec.2016.12.021
0168-1656/© 2016 Elsevier B.V. All rights reserved.
26 T. Furuya et al. / Journal of Biotechnology 243 (2017) 25–28
(A)
O2 CH3CHO O
OCH3 Oxygenase OCH3
OH Cso2 OH
Isoeugenol Vanillin
(B) O OH
CO2 O2 HCHO
OCH3 Decarboxylase OCH3 Oxygenase
OH Fdc OH Cso2
Ferulic acid 4-Vinylguaiacol
Fig. 1. Coenzyme-independent synthesis of vanillin. Vanillin is synthesized from
isoeugenol by oxygenase Cso2 (A) and from ferulic acid via 4-vinylguaiacol by decar-
boxylase Fdc and oxygenase Cso2 (B).
Recently, we developed a route to vanillin from ferulic acid that
does not require any coenzymes (Fig. 1). This artificial pathway con-
sists of a coenzyme-independent decarboxylase (Fdc) that converts
ferulic acid to 4-vinylguaiacol (2-methoxy-4-vinylphenol), and a
subsequent step with a coenzyme-independent oxygenase (Cso2)
that converts 4-vinylguaiacol to vanillin (Furuya et al., 2014; Furuya
et al., 2015; Muschiol et al., 2015). The ferulic acid decarboxylase
Fdc from Bacillus pumilus catalyzes the nonoxidative decarboxyla-
tion of aromatic carboxylic acids (Barthelmebs et al., 2001; Yang
et al., 2009). The 4-vinylguaiacol oxygenase Cso2 from Caulobac-
ter segnis belongs to the carotenoid cleavage oxygenase family
and catalyzes the oxidative cleavage of a conjugated C C bond
(Furuya et al., 2014; Kloer and Schulz, 2006). We demonstrated
that Escherichia coli cells expressing Fdc and Cso2 produced vanillin
from ferulic acid via 4-vinylguaiacol (Furuya et al., 2014). Cso2
also converts isoeugenol to vanillin in a coenzyme-independent
manner (Furuya et al., 2014) (Fig. 1). This coenzyme-independent
feature would be advantageous to the production of vanillin
using immobilized enzymes. In the present study, we investigated
biotechnological production of vanillin using immobilized Fdc and
Cso2.
A proper carrier for immobilization of Cso2, a key enzyme
for vanillin production, which is relatively labile, was first exam-
ined. The oxygenase Cso2 exhibits high activity in the pH range
9.0–10.5, whereas the isoelectric point of Cso2 is pH 5.3 (Furuya
et al., 2014). This property indicates that Cso2, with a negative
charge at the optimal pH, would be immobilized on anion-exchange
carriers. Four anion-exchange carriers were tested. The matrix,
functional group and particle size of each carrier are shown in
Table 1. When each carrier was incubated with Cso2 solution,
all were able to adsorb protein (0.04–0.10 mg mg-carrier−1 ) (Fig.
S1A in Supplementary data). In addition, we examined vanillin-
producing activity of these carriers after adsorbing Cso2. When
each was incubated with isoeugenol, Cso2 immobilized on Sepa-
beads EC-EA efficiently converted this substrate to vanillin (Fig. S1B
in Supplementary data). In contrast, Cso2 on the other carriers lost
catalytic activity. These results suggested that Cso2 was compati-
ble with the polymethacrylate matrix and the ethylamine group of
Sepabeads EC-EA (Table 1).
We then attempted to produce vanillin from isoeugenol using
Cso2 immobilized on Sepabeads EC-EA (Fig. 1A). The activity of the
immobilized Cso2 catalyst was compared with that of a whole-
cell catalyst, E. coli expressing Cso2. Each catalyst was incubated
with a solution containing the substrate isoeugenol. The whole
cells produced 8.4 mM vanillin from 10 mM isoeugenol during 24 h
biotransformation (Fig. S2A in Supplementary data), whereas the
immobilized enzyme produced only 4.6 mM vanillin (Fig. S2B in
Supplementary data). We confirmed that a portion of the substrate
isoeugenol and the product vanillin bound to the carrier, which
10 8
8
6
of vanillin (mg)
Vanillin (mM)
Total amount
6
4
4
O
2
2
OCH3
OH 0 0
Vanillin 1 2 3 4 5 6 7 8 9 10
Cycle number
Fig. 2. The vanillin production from isoeugenol by repetitive use of immobilized
Cso2. Isoeugenol (10 mM) was incubated with 50 mg whole cells or 7 mg protein
immobilized on 100 mg of the carrier in the reaction mixture (1 mL) for 24 h in each
cycle. The amount of protein (7 mg) was almost equal to that obtained through
disruption of 50 mg whole cells. Vanillin produced in each cycle by the whole cells
(gray bars) and the immobilized enzyme (white bars) is shown. Total amount of
vanillin produced by the whole cells (gray circles) and the immobilized enzyme
(white circles) is also shown. Data are the average of three independent experiments,
and error bars indicate standard deviation of the mean.
led to lower yield of vanillin by the immobilized enzyme than
the whole cells. After the first reaction cycle, the whole cells and
the immobilized enzyme were collected from the reaction mixture
and added to a fresh reaction mixture (Fig. 2). In the second reac-
tion cycle, the whole cells produced only 4.9 mM vanillin. After the
third reaction cycle, the activity of the whole cells was almost lost.
Although the oxygenase Cso2 exhibits high activity in the reac-
tion pH 9.0, the enzyme is relatively unstable under the alkaline
condition (Furuya et al., 2014). However, the immobilized enzyme
enabled to produce 6.3 mM vanillin during the second reaction
cycle. Furthermore, more than 50% of the activity was maintained
even in the seventh reaction cycle. These results indicated that
the stability of Cso2 was significantly enhanced by immobilizing
on Sepabeads EC-EA. The total amount of vanillin produced from
isoeugenol by the immobilized Cso2 catalyst reached 6.8 mg at the
1 mL scale after ten reaction cycles, but the whole cells produced
only 2.5 mg (Fig. 2).
Since we confirmed that Cso2 immobilized on Sepabeads EC-EA
had superior operational stability, we used the immobilized Cso2
catalyst in the cascade synthesis of vanillin from ferulic acid via
4-vinylguaiacol (Fig. 1B). We found that the first-step Fdc was as
easily immobilized on Sepabeads EC-EA as Cso2 was. We confirmed
that 5 mg of protein immobilized on 50 mg of the carrier efficiently
converted ferulic acid to 4-vinylguaiacol (Fig. S3 in Supplementary
data). Thus, the immobilized Fdc catalyst and the immobilized Cso2
catalyst were added to a solution containing the substrate ferulic
acid. Although a portion of the substrate and the product bound
to the carrier as described above, the immobilized Fdc and Cso2
efficiently converted ferulic acid to vanillin via 4-vinylguaiacol.
The immobilized enzymes produced 2.8 mM vanillin during 24 h
biotransformation (Fig. S4 in Supplementary data). After the first
reaction cycle, the immobilized enzymes were collected from the
reaction mixture and added to a fresh reaction mixture (Fig. 3).
In the second reaction cycle, the immobilized enzymes produced
3.5 mM vanillin. In the third and fourth reaction cycles, the immo-
bilized enzymes maintained their activity, producing 3.1 mM and
2.4 mM vanillin, respectively. In subsequent reaction cycles, the
production of vanillin gradually decreased. We confirmed that the
intermediate 4-vinylguaiacol accumulated in the reaction mixture.
The total amount of vanillin produced from ferulic acid by the
immobilized Fdc and Cso2 catalysts reached 2.5 mg at the 1 mL scale
after ten reaction cycles (Fig. 3). The amount of vanillin produced
from ferulic acid was lower than that produced from isoeugenol.
 T. Furuya et al. / Journal of Biotechnology 243 (2017) 25–28 27

Table 1
Anion-exchange carriers tested in this study.

Sepabeads EC-EA Sepabeads EC-Q1A Diaion HPA25L Diaion WA21J

H2 CH3
C C H2 H H2 H
C O C C C C H2 H
O C C
CH2 CH3 CH3
CH2 CH2N+CH3Cl- CH2N+CH3Cl-

Structure NH2 CH3 CH3 CH2NH(CH2CH2NH)nH

Matrix polymethacrylate styrene-divinylbenzene styrene-divinylbenzene styrene-divinylbenzene

Functional group ethylamine quaternary alkylamine quaternary alkylamine polyamine
Particle size (␮m) 100–200 200–600 300–1200 300–1200

5 3 Author contributions

4 T.F., M.K. and K.K. designed research; T.F. and M.K. performed
research; M.K. analyzed data; and T.F., M.K. and K.K. wrote the

of vanillin (mg)
2
Vanillin (mM)

Total amount
3 paper.

2 Funding
1

1 This work was supported by a Novozymes Japan Research Fund

2016 to T.F.
0 0
1 2 3 4 5 6 7 8 9 10 Acknowledgements
Cycle number

We kindly thank H. Kawabata (Mitsubishi Chemical Group Sci-

Fig. 3. The vanillin production from ferulic acid by repetitive use of immobilized Fdc
and Cso2. Ferulic acid (10 mM) was incubated with the immobilized Fdc (5 mg pro- ence and Technology Research Center, Inc.) for the gift of samples
tein on 50 mg carrier) and the immobilized Cso2 (7 mg protein on 100 mg carrier) in of anion-exchange carries.
the reaction mixture (1 mL) for 24 h in each cycle. Vanillin produced in each cycle by
the immobilized enzymes (white bars) is shown. Total amount of vanillin produced
by the immobilized enzyme (white circles) is also shown. Data are the average of Appendix A. Supplementary data
three independent experiments, and error bars indicate standard deviation of the
mean. Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.12.
021.

It is possible that the reaction time was not enough for the com-
pletion of the two-step reaction in each cycle (Figs. S2B and S4 in
Supplementary data), or that the substrate ferulic acid and/or the
intermediate 4-vinylguaiacol had unfavorable effect on Cso2.
In conclusion, we demonstrated that a coenzyme-independent
cascade consisting of the decarboxylase Fdc and the oxygenase Cso2
was easily applicable to an immobilized enzyme process for vanillin
production. Although a lot of studies have been devoted to biotech-
nological production of vanillin, this study is, to our knowledge, the
first for vanillin production using immobilized enzymes. The immo-
bilized enzyme process provides new possibilities for the practical
bioproduction of vanillin, because vanillin can be prepared by only
mixing isoeugenol or ferulic acid with the immobilized enzyme(s)
that can be used repetitively. In the present study, as the activity
of Cso2 was not so high, a large amount of the enzyme was added
to the reaction mixture for the conversion of isoeugenol to vanillin.
Accumulation of the intermediate 4-vinylguaiacol after the repeti-
tive reaction with ferulic acid indicates that the second-step Cso2
reaction limited further increase production of vanillin. We are
now investigating scale-up of the process as well as genetic engi-
neering of Cso2 for higher activity and stability toward industrial
application.
Competing interests
All authors declare no competing or financial interests.
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