Expression and characterization of a 9-cis-epoxycarotenoid dioxygenase from Serratia sp. ATCC 39006 capable of biotransforming isoeugenol and 4-vinylguaiacol to vanillin

Article abstract of DOI:10.1016/j.btre.2018.e00253

A 9-cis-epoxycarotenoid dioxygenase gene from Serratia sp. ATCC 39,006 (SeNCED) was overexpressed in soluble form in E.coli. SeNCED showed the maximum activity at 30 °C and pH 8.0, and it was stable relatively at range of pH 5–10 and temperature of 20 °C to 30 °C. SeNCED effectively catalyzes the side chain double bond cleavage of isoeugenol and 4-vinylguaiacol to vanillin. The kinetic constant K_m values toward isoeugenol and 4-vinylguaiacol were 18.92 mM and 6.31 mM and V_max values were 50.73 IU/g and 4.77 IU/g, respectively. Moreover, the SeNCED exhibited an excellent organic solvent tolerance and the enzyme activity was substantially improved at presence of 10% of trichloromethane. The produced vanillin was achieved at an around 0.53 g/L (3.47 mM) and 0.33 g/L (2.17 mM) after 8 h reaction at 4 mM of isoeugenol and 4-vinylguaiacol, respectively, using transformed Escherichia coli cells harboring SeNCED in the presence of trichloromethane.

Full text of DOI:10.1016/j.btre.2018.e00253

Biotechnology Reports 18 (2018) e00253
Contents lists available at ScienceDirect
Biotechnology Reports
journal homepage: www.elsevier.com/locate/btre
Expression and characterization of a 9-cis-epoxycarotenoid
dioxygenase from Serratia sp. ATCC 39006 capable of biotransforming
isoeugenol and 4-vinylguaiacol to vanillin
Jiao Tang, Lei Shi, Lulu Li, Liangkun Long, Shaojun Ding*
The Co-Innovation Center of Efficient Processing and Utilization of Forest Resources, Jiangsu Key Lab for the Chemistry & Utilization of Agricultural and Forest
Biomass, College of Chemical Engineering, Nanjing Forestry University, Nanjing, 210037, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 10 April 2018
Accepted 16 April 2018
A 9-cis-epoxycarotenoid dioxygenase gene from Serratia sp. ATCC 39,006 (SeNCED) was overexpressed in
ꢀ
soluble form in E.coli. SeNCED showed the maximum activity at 30 C and pH 8.0, and it was stable
ꢀ
ꢀ
relatively at range of pH 5–10 and temperature of 20 C to 30 C. SeNCED effectively catalyzes the side
chain double bond cleavage of isoeugenol and 4-vinylguaiacol to vanillin. The kinetic constant K values
Keywords:
m
Serratia sp. ATCC 39006
toward isoeugenol and 4-vinylguaiacol were 18.92 mM and 6.31 mM and Vmax values were 50.73 IU/g and
4.77 IU/g, respectively. Moreover, the SeNCED exhibited an excellent organic solvent tolerance and the
enzyme activity was substantially improved at presence of 10% of trichloromethane. The produced
vanillinwas achieved at an around 0.53 g/L (3.47 mM) and 0.33 g/L (2.17 mM) after 8 h reaction at 4 mM of
isoeugenol and 4-vinylguaiacol, respectively, using transformed Escherichia coli cells harboring SeNCED
in the presence of trichloromethane.
9
-Cis-epoxycarotenoid dioxygenase
Isoeugenol
-Vinylguaiacol
Vanillin
4
©
2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
As we all know, ferulic acid (FA) is an abundant phenolic acid
and can be recovered from agro-industrial wastes [5–7]. Several
Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one of the
metabolism pathways from FA to vanillin have been reported in
microbes [8,9]. Of those, in the coenzyme-dependent deacetyla-
tion pathway, the FA is converted to feruloyl-CoA catalyzed by
feruloyl-CoA-synthetase (Fcs), and subsequently transferred to
vanillin by enoyl-CoA-hydrolase (Ech). The engineered Escherichia
coli and other bacterial cells harboring Fcs and Ech effectively
converted FA to vanillin [10,11]. The engineered strains possess
high potential for biosynthesis of vanillin, but the fact that Fcs
requires expensive ATP and CoA as coenzymes makes the synthetic
route complicate and high-cost. Assuredly, if the vital enzymes
could be substituted by coenzyme independent proteins, the
biosynthesis process of vanillin will be more efficient and
economical.
Isoeugenol is the main constituent of essential oil of clove tree,
and a variety of microbial species that metabolize isoeugenol to
vanillin or vanillic acid have been isolated in succession [12–17].
The Pseudomonas putida IE27 cells produced 16.1 g/L vanillin from
150 mM isoeugenol, with a molar conversion yield of 71% [18].
While in strain I58, the produced vanillin was continuously
converted to vanillic acid with a molar yield of 98%, which leads to
an extremely low accumulation of vanillin [14]. The enzymes
responsible for the transformation of isoeugenol to vanillin have
been characterized, of which the sequence was similar to some of
most important flavor and fragrance compounds, which is widely
used in beverages, confectionery, foods, perfumes, and cosmetics
industry. Historically, its production was almost dependent on
extracting from vanilla beans directly, by which the accessible
vanillin was seriously restrictive and at a high market price
ꢁ
1
calculated as being between 1200 and 4000 $kg [1]. Whereas the
products obtained through chemical synthesis was at the price of
ꢁ
1
less than 15 $kg , which satisfied the constantly increasing
markets. However, many unexpected environmentally unfriendly
by-products often appeared during the chemical synthetic
processes. Besides, the increasing concerns for health and nutrition
stimulated a worldwide demand not for those synthesized
chemically but natural vanillin. The vanillin produced from raw
materials by biotechnology was equal to those extracted directly
from vanilla beans on quality, and it was identified as “nature”
vanillin by the FDA and European legislation [2–4]. Hence,
biotransformation-based approaches for vanillin production in
the filed green chemistry become more and more attractive for
flavor industry to replace conventional chemical syntheses.
*
Corresponding author.
E-mail address: dshaojun@hotmail.com (S. Ding).
https://doi.org/10.1016/j.btre.2018.e00253
215-017X/© 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
2

2
J. Tang et al. / Biotechnology Reports 18 (2018) e00253
the carotenoid cleavage oxygenases (CCOs), and they also had been
reported to possess the potential for transforming 4-vinylguaiacol,
a vital intermediate observed in microbial metabolism of FA to
vanillin, but the activity was extremely low [16,19].
2.3. Cloning and expression of SeNCED
According to SeNCED sequence (WP_021015152.1), two oligo-
0
nucleotide primers, SeNCED-F: 5 -CATGCCATGGCCATGAGTCT-
0
Recently, a novel CCO protein from Caulobacter segnis ATCC
GAAATTTCC-3 (the NcoI restriction site underlined) and
0
21,756 (Cso2) was characterized capable of transforming both
SeNCED-R: 5 -CCGCTCGAGTCAATGATGATGATGATGATGCCGGCCG-
0
isoeugenol and 4-vinylguaiacol to vanillin without any coenzymes.
A two-step biosynthetic pathway was constructed in E.coli, in
which FA was firstly decarboxylated to 4-vinylguaiacol by phenolic
acid decarboxylase and then oxidized to vanillin via oxygenase
GATTGGGTAC-3 (the XhoI restriction site underlined) were
designed to amplify the SeNCED gene by PCR. The amplified
DNA fragment digested with NcoI and XhoI was subsequently
inserted into pET-28a (+) digested with the same enzymes. The
constructed plasmid, pET-28a (+)-SeNCED was cloned into E. coli
Top 10 strain and the target gene was confirmed by sequencing.
The pET-28a (+)-SeNCED was then transformed into E. coli BL21
(
Cso2) at an 80% of overall conversion [20]. Furthermore, the
cascade synthesis of vanillin from ferulic acid via 4-vinylguaiacol
was also achieved by the immobilized phenolic acid decarboxylase
and Cso2 [21]. In fact, many organisms such as Paecilomyces
variotii, Pestalotia palmarum [22], Bacillus coagulans [23] and
Enterobacter sp. Px6-4 [24] had been reported to metabolize ferulic
acid to vanillin via 4-vinylguaiacol. The enzymes catalyzing the
first reaction have been well studied but the biotransformation of
(DE3). The recombinant E. coli BL21 (DE3) cells were cultured at
ꢀ
37 C in LB medium supplemented with 100
mg/mL kanamycin for
3 h (OD600 = 0.6–1.0), then a certain amount of IPTG and 1 mM
ꢀ
FeCl
2
was added and cultured at 28 C for 12 h for the over-
expression of SeNCED protein.
4-vinylguaiacol to vanillin had been rarely reported before. The
Cso2 protein was actually able to catalyze this reaction effectively;
however, the insoluble expression reduced its application value. In
order to increase the solubility, a molecular chaperone protein is
indispensable to co-expressed with the target protein, which
consequently led to a complexity of operation.
In order to mine superior catalysts useful for vanillin production
from lignin-related phenylpropanoids, a gene mining method was
carried out in this study and a new CCO protein named SeNCED
from Serratia sp. ATCC 39,006 was functionally cloned and
overexpressed in a large proportion of soluble form in E. coli. It
cleaves isoeugenol, noticeably as well as 4-vinylguaiacol to vanillin
independent on coenzyme, similar as Cso2 mentioned above. The
enzymatic properties were studied and the potential of application
for vanillin synthesis from isoeugenol or 4-vinylguaiacol was
investigated in this study.
2.4. Purification of SeNCED
The expressed cells were harvested by centrifugation (6000 ꢂ g,
ꢀ
30 min, 4 C). The pellets resuspended in lysis buffer (50 mM
NaH
2
4
PO ; 300 mM NaCl pH 8.0) were disrupted by sonication at an
ꢀ
ice bath. After centrifugation (10,000 g, 4 C, 10 min), the superna-
tant was applied onto a Ni-NTA agarose gel equilibrated in the lysis
buffer containing 1 mM imidazole for the recombinant enzyme
purification according to the manufacturer’s manual and the
concentration of obtained protein was measured with BCA Protein
Assay Kit (Sangon, Shanghai, China). The purified recombinant
enzymes were maintained at 4 C and used for the following
research as soon as possible. The enzyme homogeneity and the
molecular of purified SeNCED were estimated using SDS-PAGE.
ꢀ
2
.5. Enzyme assay
2. Materials and methods
The catalytic activity of recombinant SeNCED was assayed with
isoeugenol or 4-vinylguaiacol as substrates. The standard assay
mixture contains 2 mM substrate and 10% v/v of glycerol in
potassium phosphate buffer (100 mM, pH 8.0), and an appropriate
amount of enzyme in a total volume of 0.5 mL. The reactions were
performed at optimal temperature with vigorous shaking for 24 h
and then terminated by adding 0.5 mL methanol. After centrifuga-
tion at 10,000 g for 10 min, the supernatant was analyzed using
high-performance liquid chromatography (HPLC). One unit of
enzyme activity was defined as the amount of enzyme catalyzing
2
.1. Chemicals
Isoeugenol and ferulic acid from Sinopharm Group (Beijing,
China), 4-vinylguaiacol and vanillin from Sigma-Aldrich (St. Louis,
MO, USA) were all dissolved in dimethyl sulfoxide as stock
solutions of 500 mM. The dimethyl sulfoxide, trichloromethane
and other organic compounds were obtained from Sinopharm
Group (Beijing, China). Standard protein marker for sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),
polymerase chain reaction (PCR) buffers, FastPfu DNA polymerase,
and restriction enzymes were all obtained from Takara (Japan). The
kits used for bacterial plasmid extraction, gel extraction and PCR
purification were all purchased from Transgene (Beijing, China). All
other chemicals were analytical grade.
the formation of 1
mmol of vanillin per minute. Kinetic constants
were determined at optimal condition with different substrate
concentrations ranging from 0 to 20 mM. The maximal reaction
rate (Vmax) and apparent Michaelis–Menten constant (Km) were
calculated by Origin 8.0 software (http://www.originlab.com/)
using nonlinear regression.
2.2. Strains and culture conditions
2.6. Effects of pH, temperature, and chemicals on SeNCED activity and
stability
Serratia sp. ATCC 39,006 purchased from the American Type
Culture Collection (Manassas, VA, USA) was grown in LB medium
formulated with 10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl
and incubated with shaking (200 rpm) at 37 C. E. coli Top 10 and
Optimal pH for enzyme reactions were estimated using
isoeugenol as the substrate in different buffers at a range of pH
6.0–10.0 (0.1 M potassium phosphate buffer pH 6.0–8.5, glycine-
NaOH buffer pH 8.0–10.0). Optimal temperature for the SeNCED
ꢀ
E. coli BL21 (DE3) strains (TransGene, Beijing, China) were used for
gene cloning and expressing hosts, respectively. The pET-28a (+)
activity was determined by standard assay at a range of
ꢀ
ꢀ
(
Novagen, Copenhagen, Denmark) was used as expression
temperature from 20 C to 45 C in 0.1 M potassium phosphate
buffer (pH 8.0). The results were expressed as percentage of the
activity values determined under either the optimal pH or
temperature.
vector. The recombinant E. coli cells were cultured with shaking
ꢀ
(
200 rpm) at 37 C in LB medium supplemented with 100
m
g/mL
kanamycin.

J. Tang et al. / Biotechnology Reports 18 (2018) e00253
3
Thermal stability was analyzed by determining the residual
24 h in 10-mL sealed bottles. After incubation, the water phase
activity of purified SeNCED to transform isoeugenol to vanillin after
containing SeNCED were sufficiently separated by centrifugation
(10,000 g at 4 C for 10 min), and the residual activity was
determined under standard condition.
ꢀ
ꢀ
ꢀ
preincubation at a range of 20 C–45 C for a specific time as
indicated. To evaluate the pH stability, enzyme was preincubated
at universal buffer containing 50 mM phosphoric acid, acetic acid
and boric acid at a range of pH 3.0 to pH 12.0 for 2 h, and then the
residual activity was measured under the standard reaction
condition. The results were expressed as percentage of the activity
value determined via the purified enzyme without preincubation.
To investigate the effects of various chemicals containing metal
2.8. Vanillin biosynthesis via the purified SeNCED and whole cells
harboring SeNCED
The biotransformtion of isoeugenol and 4-vinylguaiacol to
vanillin was conducted by both purified SeNCED and transformed
E.coli cells harboring SeNCED. The catalytic reaction initiated by
purified SeNCED was proceeded in the reaction mixture contained
100 mM potassium phosphate buffer (pH 8.0), 10% (v/v) glycerol,
4 mM substrate and 0.2 mg/mL purified enzyme. The reactions
2
+
2+
ions, metal chelators and detergents on SeNCED activity, Ca , Co
,
2
+
+
2+
2+
2+
2+
3+
2+
+
Mg , Li , Cu , Zn , Ni , Fe , Fe , Mn , Na , SDS, Triton X-100,
,2-bipyridine, 10-phenanthrolase and ethylene diamine tetracetic
2
acid (EDTA) were assayed at a final concentration of 5 mM in the
reaction mixture. Activity was determined as described above and
was expressed as percentage of the enzyme activity obtained in the
reaction without any chemicals added.
ꢀ
were performed at 30 C with vigorous shaking for 72 h.
The reaction catalyzed by the transformed E.coli cells was
ꢀ
performed in a 50-mL conical flask at 30 C with vigorous shaking
for 72 h. The reaction mixture (2 mL) contained 100 mM potassium
phosphate buffer (pH 8.0), 10% (v/v) glycerol, 4 mM substrate, 5%
2.7. Effect of organic solvents on SeNCED activity and stability
(
v/v) trichloromethane and the E. coli whole cells at a final
The effect of organic solvents on enzyme activity to two
concentration of OD600 3.0. After reaction, the mixtures were
diluted with methanol to constant volume of 4 mL, and the product
was quantified using HPLC. For preparation of the whole cells, the
transformed E. coli cells were harvested by centrifugation (6000 g,
substrates was also analyzed in the presence of various organic
solvents at 10% (v/v). The reaction was performed at standard assay
condition in 10-mL glass bottles with screw caps (Teflon seals), and
the product was quantified using HPLC. To investigate its stability
ꢀ
30 min, 4 C) and washed triples with potassium phosphate buffer
to organic solvent, the purified SeNCED was co-shaked with 20% or
(100 mM, pH 8.0) containing glycerol (10% v/v) after 12 h induction
at 28 C as described above.
ꢀ
ꢀ
50% (v/v) of various organic solvents at 200 rpm at 20 C for 2 h or
Fig. 1. Multiple alignment of the amino acid sequences of SeNCED and other CCOs enzymes from different bacteria. Identical residues are shaded in black and conserved
residues are shaded in other colors. SeNCED: 9-cis-epoxycarotenoid dioxygenase from Serratia sp. ATCC 39,006 (WP_021015152.1), Cso2: CCO from Caulobacter segnis 21,756
(
WP_013079514.1), Iem-IE27 and Iem-JIN1: Isoeugenol monooxygenase (Iem) from Pseudomonas putida strain IE27 (GenBank: BAF62888.1) and strain Jin1 (GenBank:
ACP17973.1), respectively, NovoNCED: 9-cis-epoxycarotenoid dioxygenase from Novosphingobium sp. Rr 2–17 (GenBank: EIZ77617.1).

4
J. Tang et al. / Biotechnology Reports 18 (2018) e00253
2.9. Analytical methods
as shown in the red frame (Fig. 1), which might be responsible for
2
+
combining Fe . To get a further insight into the evolutionary
relationship between SeNCED and other CCOs, phylogenetic trees
of the 20 amino acid sequences were constructed using the
Neighbor-Joining and Maximum Parsimony method (Fig. 2). As a
consequence, a near relationship among SeNCED, NCED from
Novosphingobium, Dioxygenase from Kaistia soil and Rhodobacter-
aceae was observed. When to investigate the ability of Serratia sp.
ATCC 39,006 for metabolizing aromatic compounds, a small
amount of vanillin was detected in the culture during its metabolic
process of isoeugenol, 4-vinylguaiacol and ferulic acid as
substrates (Figs. S1–S3 in the Supplementary material). Based
on the sequence and metabolite analysis, we supposed that
SeNCED acts as the vital enzyme in the vanillin synthesis
procedures from those aromatic compounds.
The oxidized product was analyzed using HPLC as described
previously by Hu et al. [25]. Identification of the oxidized product
was performed by gas chromatography–mass spectrometry (GC–
MS) (TRACE DSQ, Thermo Fisher Scientific) with a DB-5 column
(
length, 30 m; diameter, 0.25 mm;Agilent), the column tempera-
ꢀ
ture was initially held at 50 C for 3 min, then programmed to
ꢀ
ꢀ
2
60 C at a rate of 10 C/min, with a final hold time of 5 min. The
components were identified based on the comparison of their
relative retention times and mass spectra with those of the
established standards (NIST05 library data of the GC–MS system)
and previous literature data.
3
. Results and discussion
3.1. Cloning and analysis of SeNCED
3.2. Expression and purification of the recombinant SeNCED
By analysis of the genome sequence of the Serratia.sp ATCC
9,006 published on National Center of Biotechnology Information
NCBI) (Aseembly: GCA_000463345.2), a protein, defined as 9-cis-
epoxycarotenoid dioxygenase (SeNCED) (WP_021015152.1), shares
high sequence similarity (59%) with Cso2 from Caulobacter segnis
ATCC 21,756 (WP_013079514.1), belonging to CCO family, consists
of 1488 bp fragment encoding 496 amino acids with theoretic pI
and molecular weight values of 5.04 and 55.59 KDa. It also showed
The E.coli BL21 (DE3) strain containing recombinant plasmid
pET-28a (+)-SeNCED was used as heterologous host to obtain the
overexpression of SeNCED. The recombinant protein was induced
3
(
2
+
ꢀ
by 0.4 mM IPTG and 1 mM Fe
at 28 C for 12 h after a
ꢀ
preincubation at 37 C to a cell concentration of OD600 0.6–1
2
+
(Fig. 3A–C). When 1 mM Fe was added together with 0.4 mM
IPTG in the medium, the transformed E.coli cells showed a 1.8-fold
2
+
higher activity than those cultured without Fe (Fig. 3 D). It was in
good agreement with catalytic mechanism of the Cso2 and other
8
3.67% similarity with the 9-cis-epoxycarotenoid dioxygenase of
2
+
Novosphingobium sp. Rr 2–17 (GenBank: EIZ77617.1), and 39.44%
and 39.03% similarity with the isoeugenol monooxygenases (Iem)
from Pseudomonas putida IE27 (GenBank: BAF62888.1), and
Pseudomonas nitroreducens Jin1 (GenBank: ACP17973.1), respec-
tively. The CCOs were identified as iron-contained proteins and a
CCOs, which regarded Fe as prosthetic group [26,27]. This result
2
+
indicated that SeNCED was also a Fe contained protein.
The recombinant SeNCED was then purified via affinity
chromatography using Ni-NTA agarose gel. In order to verify the
expression of target protein and its solubility, the non-induced
whole cells, the induced whole cells, and its cell debris and
supernatant were applied to SDS-PAGE. It showed a strong band of
SeNCED in the supernatant of induced whole cells, dissimilar to the
Cso2 shown in the cell debris [21], declaring that the recombinant
2
+
Fe -4-His arrangement acting as a vital role at the oxidative
reactions was existed in these proteins [20]. Alignment of the
amino acid sequences of SeNCED and these CCOs showed that four
167
218
283
480
histidines His , His , His and His
was extremely conversed
Fig. 2. The phylogenetic trees resulting from analysis of carotenoid cleavage oxygenases of 20 amino acid sequences using Neighbor-Joining method. Numbers on nodes
correspond to percentage bootstrap values for 1000 replicates.

J. Tang et al. / Biotechnology Reports 18 (2018) e00253
5
Fig. 3. Effects of induction temperature (A), dosage of IPTG (B), induction time (C), and Fe2+ (D) on the recombinant SeNCED production. Filled circles in (C) represent the
variation of cell concentration.
protein was expressed in a large proportion of soluble form
without co-expression of chaperon proteins. The purified protein
was shown as a single band at molecular mass of about 55 KDa,
which coincided perfectly with the theoretical molecular mass as
shown in Fig. 4.
3.3. Effect of temperature and pH on SeNCED activity
To study the effects of temperature and pH on SeNCED activity,
isoeugenol was used as substrate for the enzymatic reactions. The
ꢀ
maximum activity was detected at 30 C (Fig. 5A). The enzyme
presented over 80% of relative activity under a temperature range
ꢀ
ꢀ
from 20 C to 40 C, but almost totally inactivation as the
ꢀ
temperature is above 45 C. The SeNCED was relatively stable at
0 C and 30 C, more than 90% of initial activity residue was
ꢀ
ꢀ
2
detected after 150 min of incubation (Fig. 5C). However, its activity
was decreased to a certain degree when incubated at the
ꢀ
temperatures higher than 40 C, only about 50% of its initial
ꢀ
activity was detected at 40 C after 30 min of incubation and
ꢀ
almost inactivated at 50 C. The optimum pH was observed at 8.0
and over 80% of relative activity was detected under a pH range
from 7.0 to 9.0 (Fig. 5 B), indicating a preference for slight alkaline
environment, which was similar to that seen for other CCOs
[
5
19,28,29]. The SeNCED also showed a high stability at pH between
.0 and 11.0, over 80% of initial activity was maintained after 2 h of
incubation at universal buffers (Fig. 5D).
3
.4. Effect of various chemicals on SeNCED activity
SeNCED activity was examined in the presence of several
chemicals as shown in Table 1. No significant changes of SeNCED
2
+
2+
2+
+
activity were observed with the addition of Ca , Co , Mg , Li ,
2
+
+
Mn and Na or detergents. And a slight activity decrease by over
Fig. 4. SDS-PAGE analysis of SeNCED solubility and purified protein. Lane 1 and 2:
whole E.coli BL21 (DE3) cells without induction and with a 12 h induction,
respectively; Lane 3: supernatant of induced cell lysate; Lane 4: induced cell debris;
Lane 5: purified SeNCED. Lane M: protein marker.
2+
2+
2+
3+
2
0% was detected in the presence of Zn , Ni , Fe and Fe .
2
+
Moreover, a severe inhibition was appeared due to the Cu
addition. The loss of SeNCED activity might indicate some critical

6
J. Tang et al. / Biotechnology Reports 18 (2018) e00253
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Fig. 5. Effects of temperature (A) and pH (B) on SeNCED activity, and thermal (C: &; 20 C; *, 30 C; b, 35 C; ~, 40 C; !, 45 C) and pH (D) stability. Values shown are means of
duplicate means ꢃ standard error (SE).
activity at some level, which might verified that the Fe2+ was
typically involved in catalytic reaction of SeNCED.
Table 1
Effect of various chemicals on the recombinant SeNCED activity.
Chemicals (5 mM)
None
Relative activity (%)
3
.5. Kinetic parameters of SeNCED and ability for vanillin synthesis
Control
Metal ions
99.84 ꢃ 0.23
93.77 ꢃ 2.46
82.38 ꢃ 4.16
91.11 ꢃ 3.82
94.53 ꢃ 2.73
3.17 ꢃ 0.17
2
+
Ca
Co
2
+
Kinetic constants of SeNCED were tested under the optimized
2
+
Mg
condition. K
V
m
values were 18.92 ꢃ 3.26 and 6.31 ꢃ1.98 mM, while
+
Li
2
+
max values were 50.73 ꢃ 5.28 and 4.77 ꢃ 0.73 IU/g toward
Cu
2
+
isoeugenol and 4-vinylguaiacol respectively. Apparently, the
ability for transforming isoeugenol is over 10-folds higher than
4-vinylguaiacol, which indicated that the SeNCED showed a
substrate preference for isoeugenol rather than 4-vinylguaiacol.
It was similar to the Iem from Pseudomonas putida IE27, an enzyme
also capable of catalyzing those two substrates to vanillin, but its
activity to 4-vinylguaiacol is low at only 1% of that to isoeugenol
Zn
77.07 ꢃ 5.38
74.61 ꢃ 0.64
62.51 ꢃ 0.01
70.55 ꢃ 5.62
98.24 ꢃ 0.44
97.27 ꢃ 0.44
90.28 ꢃ 0.67
66.54 ꢃ 1.46
86.05 ꢃ 0.13
26.53 ꢃ 2.99
64.77 ꢃ 2.16
2
+
Ni
Fe
Fe
3
2
+
+
2
+
Mn
Na
+
Detergents
SDS
Triton X-100
2,2-bipyridine
Metal chelators
[
28]; while to the Cso2, the ability for transforming those two
substrates was roughly equal [21]. The precursors isoeugenol and
-vinylguaiacol were catalyzed by purified SeNCED at the optimal
1,10-phenanthrolase
EDTA
4
The relative activity was expressed at the percentage of the activity without
addition of compound. All values shown are duplicate means ꢃ standard error (SE).
condition and the vanillin was synthesized from those two
substrates during a given time. The recombinant SeNCED produced
approximately 0.57 g/L and 0.2 g/L of vanillin after 72 h reaction
from 4 mM isoeugenol and 4-vinylguaiacol, respectively. Approxi-
mately 92% of isoeugenol and 32% of 4-vinylguaiacol was finally
converted to vanillin (Fig. 6).
changes of enzyme structure in the presence of these metal ions.
2
+
Unexpectedly, the Fe cannot stimulate the SeNCED activity when
it was added in the reaction mixture, which was not similar to the
CCD4 from Osmanthus Fragrans [29]; but an obvious increase of
enzyme activity was detected when the Fe2 was added during the
incubation of transformed E.coli cells (Fig. 3D). These results might
indicate that the Fe was involved in the formation of SeNCED
enzyme activity center and it was no more needed if the protein is
folded rightly. In addition, metal chelators 2, 2-bipyridine, 10-
phenanthrolase and EDTA showed inhibition to the enzyme
+
3.6. Effects of organic solvents on activity and stability of SeNCED
2
+
The effects of various water-immiscible organic solvents on
activity and stability of SeNCED were investigated. The enzyme
activity was substantially increased at the presence of trichloro-
methane by 1.7 and 2.0 folds of its initial activity for both

J. Tang et al. / Biotechnology Reports 18 (2018) e00253
7
Table 3
Effect of various organic solvents on recombinant SeNCED stability.
Organic solvents
Concentration (%,v/v)
Relative activity (%)
2
h
24 h
Dichloromethane
Trichloromethane
n-Caprylic alcohol
Toluene
20
85.88 ꢃ 1.4
87.94 ꢃ 2.39
85.73 ꢃ 1.66
95.30 ꢃ 1.52
112.34 ꢃ 0.34
84.99 ꢃ 2.36
84.73 ꢃ 0.44
86.42 ꢃ 2.82
81.33 ꢃ 1.47
78.11 ꢃ 2.51
70.46 ꢃ 2.04
97.47 ꢃ 0.82
85.96 ꢃ 5.89
90.54 ꢃ 2.30
83.78 ꢃ 1.34
68.68 ꢃ 1.67
68.92 ꢃ 0.59
57.61 ꢃ 1.26
54.27 ꢃ 0.36
84.37 ꢃ 1.03
85.51 ꢃ 1.60
5
0
88.64 ꢃ 0.64
88.74 ꢃ 2.15
85.81 ꢃ 0.18
87.14 ꢃ 1.32
87.78 ꢃ 1.69
84.45 ꢃ 2.18
90.75 ꢃ 1.24
85.54 ꢃ 3.66
80.27 ꢃ 1.61
86.20 ꢃ 3.25
98.79 ꢃ 0.55
84.01 ꢃ 1.34
86.63 ꢃ 0.36
79.81 ꢃ 0.59
85.06 ꢃ 4.88
46.41 ꢃ 0.87
64.01 ꢃ 6.74
80.78 ꢃ 1.39
86.18 ꢃ 3.15
20
5
0
20
5
0
20
5
0
Ethyl acetate
Cyclohexane
n-Hexane
20
5
0
20
5
0
20
5
0
Petroleum ether
n-Butyl alcohol
n-Octane
20
5
0
Fig. 6. Vanillin synthesis from isoeugenol (filled bars) and 4-vinylguaiacol (blank
bars) by purified SeNCED.
20
5
0
20
5
0
isoeugenol and 4-vinylguaiacol, respectively (Table 2). Meanwhile,
a light increase of enzyme activity was detected in the presence of
dichloromethane, n-caprylic alcohol or cyclohexane when 4-
vinylguaiacol was used as substrate, but a mild decrease was
observed when isoeugenol was used as substrate. However, an
obvious decrease of SeNCED activity was observed by approxi-
mately 20% in the presence of n-hexane, and even over 60% in the
presence of toluene, ethyl acetate or n-butyl alcohol using either
isoeugenol or 4-vinylguaiacol as substrates. Moreover, the activity
was decreased by about 50% when the n-octane and petroleum
ether was added using isoeugenol as substrate, but only 10%–30%
reduction was observed to 4-vinylguaiacol. The effect mechanisms
of those organic solvents on SeNCED activity were not clear now
due to the complexity arising from different organic solvents. As
reported, the three-dimensional structure and flexibility of the
enzyme active site might be affected by organic solvents, leading to
an enzymatic activity and specificity variation [30,31].
The biphasic organic/aqueous system has many advantages for
enzymatic reactions or biotransformations [32], therefore, those
enzymes or microbes exhibiting remarkable organic solvent
tolerance can be effectively used as catalysts in the organic-
aqueous biphasic reaction system [25,33]. However, most native
enzymes exhibit lower activities or stabilities as the organic
solvents were co-presented in the reaction mixture. Thus, the
sufficient organic solvent tolerance for the enzymes is highly
demanded in the biphasic system reactions. Several enzymes
mostly lipases, proteases, and esterases have been reported to be
tolerant to organics, but rarely for CCOs [34–37]. In our study, an
The relative activity was expressed at the percentage of the activity without co-
incubation with any solvents. All values shown are duplicate means ꢃ standard
error (SE).
admirable stability of SeNCED to organic solvent was observed
ꢀ
(Table 3). After co-incubation with solvents in 20 C for 24 h, the
enzyme still remained more than 80% of its initial activity in most
used solvents even at the concentration up to 50%. This result
implied that SeNCED can be effectively used for the vanillin
synthesis from aromatic compounds in organic-aqueous biphase
or even organic phase. The phenolic acid decarboxylase from
Bacillus licheniformis [25] and Bacillus amyloliquefaciens [38]
showed high solvent tolerant and efficient in conversion of
hydroxycinnamic acids to vinyl phenol derivatives when using
the organic-aqueous biphasic system. The SeNCED might be a
potential candidate for vanillin synthesis from ferulic acid by
combined using with these phenolic acid decarboxylases in a two-
step biosynthetic reaction system, owing to its wonderful
tolerance to organic solvents.
3.7. Bioconversion of isoeugenol and 4-vinylguaiacol to vanillin by
transformed E.coli
Bioconversions of isoeugenol and 4-vinylguaiacol using trans-
formed E.coli cells harboring SeNCED were investigated. According
to our experimental results, trichloromethane was added as
organic solvent for vanillin synthesis. The molar conversion was
achieved up to 90% with an 8 h reaction in the presence of 5%
trichloromethane using isoeugenol as substrate; but only about
Table 2
3
6% was obtained in the reaction without trichloromethane.
Effect of various organic solvents on recombinant SeNCED activity.
Similarly, the molar conversion was achieved up to 54% after 8 h
incubation in the presence of 5% trichloromethane but only around
Organic solvents
Relative activity (%)
Isoeugenol
16% was detected in trichloromethane non-contained reactions
4-Vinylguaiacol
when used 4-vinylguaiacol as substrate. This data indicated that
the addition of trichloromethane could significantly reduce the
reaction time which was beneficial for the biosynthesis of vanillin.
The produced vanillin was achieved at around 0.53 g/L and 0.33 g/L
after 8 h, and 0.60 g/L and 0.43 g/L after 72 h reaction at 4 mM of
isoeugenol and 4-vinylguaiacol, respectively, in the presence of
trichloromethane (Fig. 7). Water immiscible organic solvents
might be beneficial for the enzyme reactions by alleviating the
inhibitory and toxic effects of the substrate and product on
enzymes due to the extraction of hydrophobic substrate or product
[26]. There have been several studies exhibited the improvement
Dichloromethane
Trichloromethane
n-Caprylic alcohol
Toluene
Ethyl acetate
Cyclohexane
n-Hexane
Petroleum ether
n-Butyl alcohol
n-Octane
93.83 ꢃ 1.94
176.16 ꢃ 8.37
74.73 ꢃ 3.5
121.97 ꢃ 2.21
208.47 ꢃ 1.82
144.66 ꢃ 5.48
29.36 ꢃ 0.58
35.06 ꢃ 0.13
122.72 ꢃ 6.81
86.15 ꢃ 4.22
92.95 ꢃ 2.89
21.44 ꢃ 1.91
70.45 ꢃ 2.61
39.56 ꢃ 0.75
19.35 ꢃ 0.39
91.01 ꢃ 3.6
84.29 ꢃ 6.15
46.05 ꢃ 1.27
38.19 ꢃ 0.20
55.34 ꢃ 2.86
Therelativeactivitywasexpressedatthepercentageoftheactivitywithoutadditionof
any solvents. All values shown are duplicate means ꢃ standard error (SE).

8
J. Tang et al. / Biotechnology Reports 18 (2018) e00253
References
[
[
[
1] S. Serra, C. Fuganti, E. Brenna, Biocatalytic preparation of natural flavours and
fragrances, Trends Biotechnol. 23 (2005) 193–198.
2] H. Priefert, J. Rabenhorst, A. Steinbüchel, Biotechnological production of
vanillin, Appl. Microbiol. Biotechnol. 56 (2001) 296–314.
3] L. Yan, P. Chen, S. Zhang, S.Y. Li, X.J. Yan, N.B. Wang, N. Liang, H.Y. Li,
Biotransformation of ferulic acid to vanillin in the packed bed-stirred
fermenters, Sci. Rep. 6 (2016) 34644.
[
[
[
[
4] C. Fleige, F. Meyer, A. Steinbuchel, Metabolic engineering of the Actinomycete
Amycolatopsis sp strain ATCC 39116 towards enhanced production of natural
vanillin, Appl. Environ. Microbiol. 82 (2016) 3410–3419.
5] H.D. Shin, S. Mcclendon, T. Le, F. Taylor, R.R. Chen, A complete enzymatic
recovery of ferulic acid from corn residues with extracellular enzymes from
Neosartoryaspinosa NRRL185, Biotechnol. Bioeng. 95 (2006) 1108–1115.
6] S.Q. Zhao, S.W. Yao, S.Y. Ou, J. Lin, Y. Wang, X.C.A.J. Li, B. Yu, Preparation of
ferulic acid from corn bran: its improved extraction and purification by
membrane separation, Food Bioprod. Process. 92 (2014) 309–313.
7] K.K. Jiang, L.L. Li, L.K. Long, S.J. Ding, Comprehensive evaluation of combining
hydrothermal pretreatment (autohydrolysis) with enzymatic hydrolysis for
efficient release of monosaccharides and ferulic acid from corn bran, Ind. Crop
Prod. 113 (2018) 348–357.
[8] S. Mathew, T.E. Abraham, Bioconversions of ferulic acid, an hydroxylcinnamic
acid, Crit. Rev. Microbiol. 32 (2006) 115–125.
Fig. 7. Biotranformationsofisoeugenoland4-vinylguaiacoltovanillinbywholecellsof
recombinantE.coliBL21(DE3)harboringSeNCED.Symbols:reactionsusedisoeugenol
as substrate in the presence of 5% (v/v) of trichloromethane (&) or not (&) and used
[
9] B. Kaur, D. Chakraborty, Biotechnological and molecular approaches for vanillin
production: a review, Appl. Biochem. Biotechnol. 169 (2013) 1353–1372.
[
10] W. Yang, H. Tang, J. Ni, Q. Wu, D. Hua, F. Tao, P. Xu, Characterization of two
Streptomyces enzymes that convert ferulic acid to vanillin, PLoS One 8 (2013)
e67339.
4
-vinylguaiacol as substrate with 5% of trichloromethane (*) or not (ꢀ).
[
11] D. Chakraborty, A. Selvam, B. Kaur, J.W.C. Wong, O.P. Karthikeyan, Application
of recombinant Pediococcus acidilactici BD16 (fcs (+)/ech (+)) for bioconversion
of agrowaste to vanillin, Appl. Microbiol. Biotechnol. 101 (2017) 5615–5626.
of productivity in the presence of specific organic solvents
32,38,39]. In previous study, the isoeugenol at a concentration
of 200 mM was transformed to vanillin by Pseudomonas putida cells
18]; while 4-vinylguaiacol at concentration of 75 mM was
transformed to vanillin efficiently using recombinant E.coli cells
26]. In the future, the substrate feeding strategies or an organic/
[
[12] H. Furukawa, H. Morita, T. Yoshida, T. Nagasawa, Conversion of isoeugenol into
vanillic acid by Pseudomonas putida I58 cells exhibiting high isoeugenol-
degrading activity, J. Biosci. Bioeng. 96 (2003) 401–403.
[
[
[
[
[
13] L.Q. Zhao, Z.H. Sun, P. Zheng, L.L. Zhu, Biotransformation of isoeugenol to
vanillin by a novel strain of Bacillus fusiformis, Biotechnol. Lett. 27 (2005)
1505–1509.
[
14] H. Furukawa, H. Morita, T. Yoshida, T. Nagasawa, Conversion of isoeugenol into
vanillic acid by 32 Pseudomonas putida I58 cells exhibiting high isoeugenol-
degrading activity, J. Biosci. Bioeng. 96 (33) (2003) 401–403.
15] M. Ashengroph, I. Nahvi, H. Zarkesh-Esfahani, F. Momenbeik, Conversion of
isoeugenol to vanillin by Psychrobacter sp. strain CSW4, Appl. Biochem.
Biotechnol. 166 (2012) 1–12.
16] J.Y. Ryu, Seo J, S. Park, J.H. Ahn, Y. Chong, M.J. Sadowsky, H.G. Hur,
Characterization of an isoeugenol monooxygenase (Iem) from Pseudomonas
nitroreducens Jin1 that transforms isoeugenol to vanillin, Biosci. Biotechnol.
Biochem. 77 (2013) 289–294.
aqueous biphasic system still need to be optimized for a further
improvement of vanillin synthesis.
4. Conclusion
In this study, SeNCED from Serratia sp. ATCC 39,006 was
successfully overexpressed and characterized. The recombinant
protein was expressed in a large proportion of soluble form
independent on chaperon proteins. Moreover, it showed an
excellent organic solvent tolerance especially for trichlorome-
thane. The addition of trichloromethane could significantly
increase the enzyme activity and reduce the reaction time
beneficial for vanillin biosynthesis. The transformed E.coli cells
converted isoeugenol and 4-vinylguaiacol at molar conversions of
[17] M. Ashengroph, J. Amini, Bioconversion of isoeugenol to vanillin and vanillic
acid using the resting cells of Trichosporon asahii, 3 Biotech 7 (2017) 358.
[18] M. Yamada, Y. Okada, T. Yoshida, T. Nagasawa, Biotransformation of isoeugenol
to vanillin by Pseudomonas putida IE27 cells, Appl. Microbiol. Biotechnol. 73
(
2007) 1025–1030.
[19] M. Yamada, Y. Okada, T. Yoshida, T. Nagasawa, Purification, characterization
and gene cloning of isoeugenol-degrading enzyme from Pseudomonas putida
IE27, Arch. Microbiol. 187 (2007) 511–517.
[
20] T. Fufuya, M. Miura, K. Kino,
oxygenase cascade for the efficient synthesis of vanillin, ChemBioChem 15
2014) 2248.
A coenzyme-independent decarboxylase/
9
0
0% and 65% and the maximum vanillin concentrations were up to
.60 g/L and 0.43 g/L, respectively. This study provided a promising
(
[21] T. Furuya, M. Kuroiwa, K. Kino, Biotechnological production of vanillin using
CCO enzyme, which may be used for the “nature vanillin”
immobilized enzymes, J. Biotechnol. 243 (2017) 25–28.
production from isoeugenol and 4-vinylguaiacol.
[22] M. Rahouti, F. Seigle-Murandi, R. Steiman, K.E. Eriksson, Metabolism of ferulic
acid by Paecilomyces variotii and Pestalotia palmarum, Appl. Environ. Microbiol.
5
5 (1989) 2391–2398.
Conflicts of interest
[
23] B. Karmakar, R.M. Vohra, H. Nandanwar, P. Sharma, K.G. Gupta, R.C. Sobti, Rapid
degradation of ferulic acid via 4-vinylguaiacol and vanillin by a newly isolated
strain of Bacillus coagulans, J. Biotechnol. 80 (2000) 195–202.
None declared.
[
24] X. Li, J. Yang, X. Li, W. Gu, J. Huang, K. Zhang, The metabolism of ferulic acid via
4
-vinylguaiacol to vanillin by Enterobacter sp. Px6-4 isolated from Vanilla root,
Acknowledgements
Process Biochem. 43 (2008) 1132–1137.
[
25] H. Hu, L. Li, S. Ding, An organic solvent-tolerant phenolic acid decarboxylase
from Bacillus licheniformis for the efficient bioconversion of hydroxycinnamic
acids to vinyl phenol derivatives, Appl. Microbiol. Biotechnol. 99 (2015)
The Key Research and Development Program of Jiangsu
(
BE2015759) and the National Forestry Science and Technology
5
071–5081.
Promotion Project [(2016)41], and the Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD)
supported this study.
[26] T. Fufuya, M. Miura, M. Kuroiwa, K. Kino, High-yield production of vanillin from
ferulic acid by a coenzyme-independent decarboxylase/oxygenase two-stage
process, New Biotechnol. 32 (2015) 335–339.
[
27] G. Moiseyev, Y. Takahashi, Y. Chen, S. Gentleman, T.M. Redmond, R.K. Crouch, J.
X. Ma, RPE65 is an iron (II)-dependent isomerohydrolase in the retinoid visual
cycle, J. Biol. Chem. 281 (2006) 2835–2840.
Appendix C. Supplementary data
[
28] J.Y. Ryu, J. Seo, S. Park, J.H. Ahn, Y. Chong, M.J. Sadowsky, H.G. Hur,
Characterization of an isoeugenol monooxygenase (Iem) from Pseudomonas
nitroreducens Jin1 that transforms isoeugenol to vanillin, Biosci. Biotechnol.
Biochem. 77 (2) (2013) 289–294.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.btre.2018.e00253

J. Tang et al. / Biotechnology Reports 18 (2018) e00253
9
[
29] X. Zhang, J. Pei, L. Zhao, F. Tang, X. Fang, J. Xie, Overexpression and
characterization of CCD4 from Osmanthus fragrans and -ionone biosynthesis
from -carotene in vitro, J. Mol. Catal. B Enzym. 134 (2016) 105–114.
[35] H. Guo, Y. Zhang, Y. Shao, W. Chen, F. Chen, M. Li, Cloning, expression and
characterization of a novel cold-active and organic solvent-tolerant esterase
from Monascus ruber M7, Extremophiles 20 (2016) 451–459.
b
b
[
30] T. Gerhards, U. Mackfeld, M. Bocola, E.V. Lieres, W. Wiechert, M. Pohl, D. Rother,
Influence of organic solvents on enzymatic asymmetric carboligations, Adv.
Synth. Catal. 35 (2012) 2805–2820.
[36] V.U. Yele, K. Desai, A New thermostable and organic solvent-tolerant lipase
from Staphylococcus warneri ; Optimization of media and production
conditions using statistical methods, Appl. Biochem. Biotechnol. 175 (2015)
855–869.
[37] T. Maruthiah, A. Palavesam, Characterization of haloalkalophilic organic
solvent tolerant protease for chitin extraction from shrimp shell waste, Int. J.
Cast Met. Res. 97 (2017) 552–560.
[38] D.H. Jung, W. Choi, K.Y. Choi, E. Jung, H. Yun, R.J. Kazlauskas, B.G. Kim,
Bioconversion of p-coumaric acid to p-hydroxystyrene using phenolic acid
decarboxylase from B. amyloliquefaciens in biphasic reaction system, Appl.
Microbiol. Biotechnol. 97 (2013) 1501–1511.
[39] J. Yang, S. Wang, M.J. Lorrain, D. Rho, K. Abokitse, P.C. Lau, Bioproduction of
lauryl lactone and 4-vinylguaiacol as value-added chemicals in two-phase
biotransformation systems, Appl. Microbiol. Biotechnol. 84 (2009) 867–876.
[
31] A.M. Klibanov, Improving enzymes by using them in organic solvents, Nature
09 (2001) 241–246.
4
[
[
[
32] N. Wangrangsimagul, K. Klinsakul, A.S. Vangnai, J. Wongkongkatep, P.
Inprakhon, K. Honda, Bioproduction of vanillin using an organic solvent-
tolerant Brevibacillus agri 13, Appl. Microbiol. Biotechnol. 93 (2) (2012)
5
55–563.
33] Z.Q. Liu, S.C. Dong, H.H. Yin, Y.P. Xue, X.L. Tang, X.J. Zhang, J.Y. He, Y.G. Zheng,
Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase
coupled with glucose dehydrogenase in an aqueous-organic solvent system,
Bioresour. Technol. 229 (2017) 26–32.
34] N. Doukyu, H. Ogino, Organic solvent-tolerant enzymes, Biochem. Eng. J. 48
(
2010) 270–282.