Microbial production of neryl-α-D-glucopyranoside from nerol by Agrobacterium sp. M-12 reflects glucosyl transfer activity

Article abstract of DOI:10.1080/09168451.2018.1514250

Terpene alcohol is widely used in perfumes and is known to possess antibacterial activity. Moreover, in its glycosylated form, it can be applied as a nonionic surfactant in food, and in the pharmaceutical, chemical, cosmetic, and detergent industries. Presently, chemical production of terpene glucosides is hampered by high costs and low yields. Here, we investigated the microbial glucosylation of nerol (cis-3,7-dimethylocta-2,6-dien-1-ol), a component of volatile oils, by Agrobacterium sp. M-12 isolated from soil. A microbial reaction using washed cells of Agrobacterium sp. M-12, 1 g/L of nerol, and 100 g/L of maltose under optimal conditions yielded 1.8 g/L of neryl-α-D-glucopyranoside after 72 h. The molar yield of neryl-α-D-glucopyranoside was 87.6%. Additionally, we report the successful transglucosylation of other monoterpene alcohols, such as geraniol, (-)-β-citro-nellol, and (-)-linalool, by Agrobacterium sp. M-12. Thus, microbial glucosylation has potential widespread applicability for efficient, low-cost production of glycosylated terpene alcohols.

Full text of DOI:10.1080/09168451.2018.1514250

Bioscience, Biotechnology, and Biochemistry
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Microbial production of neryl-α-D-glucopyranoside
from nerol by Agrobacterium sp. M-12 reflects
glucosyl transfer activity
Kazuki Takahashi, Issei Terauchi, Marie Ono, Hiroshi Satoh & Makoto Ueda
To cite this article: Kazuki Takahashi, Issei Terauchi, Marie Ono, Hiroshi Satoh & Makoto
Ueda (2018): Microbial production of neryl-α-D-glucopyranoside from nerol by Agrobacterium
sp. M-12 reflects glucosyl transfer activity, Bioscience, Biotechnology, and Biochemistry, DOI:
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BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
https://doi.org/10.1080/09168451.2018.1514250
Microbial production of neryl-α-D-glucopyranoside from nerol by
Agrobacterium sp. M-12 reflects glucosyl transfer activity
a
Kazuki Takahashia, Issei Terauchia, Marie Onoa, Hiroshi Satohb and Makoto Ueda
b
aOyama National College of Technology, Oyama, Japan; Nissei Bio Co., Eniwa City, Hokkaido, Japan
ABSTRACT
ARTICLE HISTORY
Received 26 June 2018
Accepted 9 August 2018
Terpene alcohol is widely used in perfumes and is known to possess antibacterial activity.
Moreover, in its glycosylated form, it can be applied as a nonionic surfactant in food, and
in the pharmaceutical, chemical, cosmetic, and detergent industries. Presently, chemical
production of terpene glucosides is hampered by high costs and low yields. Here, we
investigated the microbial glucosylation of nerol (cis-3,7-dimethylocta-2,6-dien-1-ol), a
component of volatile oils, by Agrobacterium sp. M-12 isolated from soil. A microbial
reaction using washed cells of Agrobacterium sp. M-12, 1 g/L of nerol, and 100 g/L of
maltose under optimal conditions yielded 1.8 g/L of neryl-α-D-glucopyranoside after 72 h.
The molar yield of neryl-α-D-glucopyranoside was 87.6%. Additionally, we report the
successful transglucosylation of other monoterpene alcohols, such as geraniol, (-)-β-citro-
nellol, and (-)-linalool, by Agrobacterium sp. M-12. Thus, microbial glucosylation has
potential widespread applicability for efficient, low-cost production of glycosylated ter-
pene alcohols.
KEYWORDS
Nerol; terpene alcohol;
microbial glucosylation;
Agrobacterium sp;
bioconversion
Terpene alcohol glycosides are known components of
grape, tea, rose, and other plants. They are utilized
mainly in perfumes [1], whereas their glycosides have
wider uses as flavor precursors, antimicrobials, anti-
fungal agents, and detergents. Not surprisingly, they
have attracted increasing attention and a search for
efficient production methods [2,3].
represents an attractive method to form glycosidic
bonds without the need for costly cofactors, such
as UDP, or detailed knowledge of chemical synth-
esis [6,7]. Preparation of glycosides by transglyco-
sylation benefits from a short reaction time and
higher yields. Some glycosides, such as hesperidin
glycosides,
2-O-α-D-glucopyranosyl-L-ascorbic
In plants, glycosides are synthesized via phosphor-
ylation of a sugar. The aglycone and the sugar are
biosynthesized separately, and the sugar is phos-
phorylated by adenine triphosphate. Then, the phos-
phorylated sugar reacts with uridine triphosphate to
produce uridine diphosphate (UDP) sugar, which is
characterized by an energy-rich anhydride.
Eventually, this UDP sugar is coupled with the agly-
cone to produce the glycoside and a free UDP. Based
on these mechanisms, several approaches for UDP
sugar-driven glycosylation have been developed,
including those employing quercetin 3-O-rhamnoside
[4] and apigenin 7-O-glucoside [5]. However, these
techniques are hampered by low productivity, which
has limited their practical use.
acid and arbutin, are already produced at an
industrial scale by transglycosylation using bacter-
ial glycosidases [8–10].
A few reports have described the enzymatic produc-
tion of terpene alcohol glycosides by transglycosylation.
Terpene glucosides have been synthesized by
Escherichia coli expressing Pichia etchellsi β-glycosi-
dases starting from terpenes, such as nerol, geraniol,
and citronellol, plus cellobiose [11]. However, the struc-
ture of these terpene glucosides has not been deter-
mined. Gunata et al. [12] have described the
glucosylation of the above terpene alcohols by β-glyco-
sidases from Aspergillus niger, Trichoderma reesei,
Candida molischiana, and almond, using cellobiose as
carbohydrate donor. However, the yield of monoter-
pene glucoside from monoterpene was unattractively
low (< 4%). The low yield occurred due to the nature of
the enzyme and the accumulation of glucose in the
reaction mixture following hydrolysis of cellobiose.
The enzymatic transfer of the glycosyl group from
cellobiose to terpene alcohols is less efficient than that
of phenolic alcohols, such as benzyl alcohol and 2-
phenylethyl alcohol. Donho et al. [13] showed that
Glycosides are also known to be synthesized by che-
mosynthesis, via the Koenigs-Knorr reaction and
Schmidt glycosylation. These techniques, however,
require expensive reagents such as silver (I) carbonate,
as well as complex, multistage protecting and deprotect-
ing steps.
Enzymatic glycosylation (reversed hydrolysis
and transglycosylation) catalyzed by glycosidases
CONTACT Makoto Ueda
mueda@oyama-ct.ac.jp
Supplementary data for this article can be accessed here.
© 2018 Japan Society for Bioscience, Biotechnology, and Agrochemistry

2
K. TAKAHASHI ET AL.
β-galactosidase from Aspergillus oryzae exhibited glu-
cosylation activity towards nerol, using lactose as car-
bohydrate donor and leading to the accumulation of
neryl-β-D-galactopyranoside. However, the amount
and yield of the product obtained by the enzymatic
reaction was not reported.
The α-glucosidase activities of soil bacteria during
the synthesis of α-D-glucopyranoside by transglucosy-
lation have been described previously [14]. We reported
that Ensifer sp. M-26 could glycosylate 6-gingerol, the
active ingredient in ginger, to 5-O-α-glucosylgingerol
using maltose as a less costly glycosyl donor [15]. While
screening for microorganisms with alkyl alcohol glyco-
sylation activities [16], we discovered a strain,
Agrobacterium sp. M-12, capable of glycosylating ter-
pene alcohol.
The present study reports the synthesis of nerol
glucoside via glucosylation of nerol by
Agrobacterium sp. M-12 and describes some of the
resulting glucoside’s characteristics. We also exam-
ine the transglucosylation activity towards other ter-
pene alcohols, such as geraniol, (-)-β-citronellol, and
(-)-linalool.
Culture media and cultivation
Basal medium consisting of 30.0 g maltose, 2.0 g
(NH4)2SO4, 1.0 g KH2PO4, 1.0 g K2HPO4, 0.2 g
MgSO4 · 7H2O, 2.0 g yeast extract, and 2.0 g poly-
peptone in 1000 mL distilled water (pH 7.0), was used
for a jar fermenter culture, unless otherwise stated. In
the case of flask cultures, the maltose content was
50.0 g/L. For the seed culture, Agrobacterium sp.
M-12 was inoculated onto 50 mL of medium in a
200-mL flask and incubated at 30°C for one day with
shaking at 180 rpm. The cell suspension from the
seed culture was then inoculated in 50 mL of medium
in a 200-mL baffled flask and grown on a rotary
shaker at 180 rpm and 30°C. The cells precultured
in flasks were inoculated in a 1-L jar fermenter (Able
Co., Tokyo, Japan) containing 400 mL of basal med-
ium. There, cells were grown under controlled con-
ditions of pH 7.0 adjusted with 2 N NaOH, 30°C, and
400 rpm. The air flow rate was 1 vvm. Bacterial
growth was measured at 600 nm using an
Amersham Biosciences Ultrospec 3300 Pro (GE
Healthcare, Little Chalfont, UK).
Reaction conditions for Agrobacterium sp. M-12
Materials and methods
Cells harvested from a 0.5-mL aliquot samples from
the jar fermenter were washed once with 0.5 mL of
20 mM potassium phosphate buffer (pH 7.0). The
washed cells were incubated with 0.5 g/L terpene
alcohols such as nerol, 100 g/L maltose, and 20 mM
potassium phosphate buffer (pH 7.0) in a total
volume of 0.5 mL. The mixture was incubated at
30°C with vigorous shaking and then an equal part
of methanol was added to the reaction solution and
centrifuged at 15,000 rpm for 4 min. After that, the
supernatant was analyzed by TLC and HPLC. A time
course for the conversion of nerol to neryl-α-D-glu-
copyranoside was carried out using 8.0 mL of har-
vested cells in culture medium and 8.0 mL of reaction
mixture composed of 20 mM potassium phosphate
buffer (pH 7.0), 100 g/L maltose, and 1.0 g/L nerol.
The reaction was performed at 40°C with vigorous
shaking.
Chemicals
α-Glucosidase from yeast, β-glucosidase from almond,
nerol (cis-3,7-dimethylocta-2,6-dien-1-ol), and geraniol
were purchased from Wako Pure Chemical Industries
(Osaka, Japan). (-)-β-Citronellol was purchased from
Tokyo Chemical Industry (Tokyo, Japan). (-)-Linalool
was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Transglucosidase from A. niger and cyclodextrin
glucosyl transferase (CGTase) from Bacillus macerans
were supplied by Amano Enzyme (Nagoya, Japan).
Other chemicals were commercially available products
and were used without further purification. The gluco-
sides of nerol and geraniol used as standards for high-
performance liquid chromatography (HPLC) were pre-
pared by microbial transglucosylation.
Glucosylation of nerol using transglucosidase and
CGTase
Analysis of transglucosylation products
A reaction mixture (300 µL) containing 0.15 mg
nerol, 30 mg maltose, 20 mM potassium phosphate
buffer (pH 7.0), and 2 units of transglucosidase or
0.03 units of CGTase were incubated at 30°C for 24 h.
The enzymatic activity was determined by thin-layer
chromatography (TLC) or HPLC. One unit of trans-
glucosidase was defined as the amount of enzyme that
hydrolyzed 1 µmol of maltose per minute at 30°C.
One unit of CGTase was defined as the amount of
enzyme that produced 1 µmol of β-cyclodextrin per
minute.
The reaction solution was analyzed by TLC on silica
gel 60 F254 plates of 0.2-mm thickness (Merck,
Darmstadt, Germany). Development was performed
with a solvent system consisting of 2:2:1 (v/v/v) 1-
butanol/2-propanol/water. Separated compounds
were detected by spraying 10% H2SO4-methanol on
the TLC glass plate and heating at 160°C for 1–2 min.
Reaction products were analyzed quantitatively by
HPLC using a COSMOSIL® 5C18-AR column
(5 µm, 4.6 mm × 250 mm; Nacalai Tesque, Inc.,

BIOPRODUCTION OF TERPENE ALCOHOLS
3
Kyoto, Japan). Conditions included a solvent:water/
methanol ratio of 3/7, a flow rate of 0.5 mL/min,
temperature of 40°C, and detection at 215 nm.
Under these conditions, nerol and neryl-α-D-gluco-
pyranoside were eluted at 11.3 min and 17.3 min,
respectively. The amount of neryl-α-D-glucopyrano-
side was calculated from a standard calibration curve
made using the compound isolated in this study. The
product was obtained by extraction with ethyl acetate
from the reaction mixture. Ethyl acetate was then
distilled away from the extract layer and the crude
product was dissolved in methanol. The solution was
fractionated by reverse-phase HPLC using a YMC-
Pack ODS-A column (10 µm, 10.0 mm × 250 mm;
YMC co., LTD., Kyoto, Japan) at a solvent:water/
methanol ratio of 3/7, flow rate of 1.0 mL/min,
room temperature, and detection at 215 nm.
Methanol present in the fraction was distilled away,
and neryl-α-D-glucopyranoside contained in water
was extracted with ethyl acetate. The extract layer
was then dehydrated with anhydrous magnesium
sulfate, and ethyl acetate was distilled. Finally, the
glucoside was obtained as a colorless and transparent
liquid. 13C nuclear magnetic resonance (NMR) and
1H NMR spectra of the products were recorded with
a JNM-ECXII (JEOL, Tokyo, Japan) spectrometer at
room temperature in CDCl3 solution and chemical
shifts were recorded in δ (parts per million). 13C
NMR (400 MHz, CDCl3) shifts were as follows:
140.2 (7-C), 131.5 (2-C), 123.7 (4-C), 121.3 (9-C),
97.6 (1′-C), 73.8 (3′-C), 72.5 (2′-C), 72.2 (5′-C), 70.4
(4′-C), 63.4 (6′-C), 61.3 (10-C), 31.8 (6-C), 26.4 (5-C),
24.6 (1-C), 22.4 (8-C), and 16.4 (3-C); 1H NMR
(400 MHz, CDCl3) shifts were as follows: 5.37 (t,
1H; H-9), 5.09 (t, 1H; H-4), 4.78 (d, 1H; H-1′), 4.10
(dd, 2H, H-10), 3.77 (dd, 1H; H-6′a), 3.66 (dd, 1H;
H-2′), 3.61 (t, 1H; H-3′), 3.54 (m, 1H; H-5′), 3.35 (dd,
1H; H-6′b), 3.29 (t, 1H; H-4′), 2.10 (t, 2H; H-5), 2.04
(t, 2H; H-6), 1.73 (s, 3H; H-8), 1.65 (s, 3H; H-3), and
1.59 (s, 3H; H-1).
Results and discussion
Glucosylation of nerol
We previously screened for microorganisms in the
soil that could accumulate glucosylated nerol from
nerol and maltose [16]. After screening a large num-
ber of bacteria, Agrobacterium sp. M-12, which can
grow on a medium containing maltose as the sole
carbon source, was found to exhibit high glucosyla-
tion activity towards nerol when maltose was added
to the reaction mixture. Supporting our finding, an
earlier study revealed α-anomer-selective glucosyl
transfer from maltose by a maltose-assimilating bac-
terium [14]. The 16S rDNA sequence (Figure S1)
revealed 99.8% identity to Agrobacteium larrymoorei
strain AF3.10 (type strain). Agrobacterium sp. M-12
was deposited at the National Institute of Technology
and Evaluation (NITE) under accession number
NITE AP-02189.
In the present study, we compared the synthesis of
nerol glycoside using transglucosidase, CGTase, or
Agrobacterium sp. M-12. As shown by TLC analysis,
a clear spot thought to be a transglucosylation pro-
duct (indicated by an arrow in Figure 1) was detected
in the reaction mixture of Agrobacterium sp. M-12.
Other oligosaccharides such as malto-oligosacchar-
ides, or other glucosylated derivatives such as nerol
Properties of neryl-α-D-glucopyranoside
To confirm the glucosidic linkage between glucose
and nerol, 2.9 g/L neryl-α-D-glucopyranoside and
0.5 units of glucosidase were incubated at 30°C for
2 h. The enzymatic activity of glucosidases was deter-
mined by HPLC. One unit of α-glucosidases was
defined as the amount of enzyme that hydrolyzed 1
μmol of maltose per minute at 25°C. One unit of β-
glucosidases was defined as the amount of enzyme
that hydrolyzed 1 μmol of ρ-nitrophenyl-β -D-gluco-
pyranoside per minute at 37°C. To evaluate its solu-
bility, purified neryl-α-D-glucopyranoside was
diluted 10 times with water and agitated at 30°C for
two days. After centrifugal separation, the superna-
tant was analyzed by HPLC.
Figure 1. Thin-layer chromatography showing reaction pro-
ducts obtained from nerol and maltose using CGTase (lane 1),
transglucosidase (lane 2), and washed cells of Agrobacterium
sp. M-12 (lane 3).
The arrow indicates the reaction product derived from nerol. Spots of
lower mobility than maltose indicate malto-oligosaccharides. P, nerol
glucoside; G, glucose; M, maltose.

4
K. TAKAHASHI ET AL.
oligoglucosides (for example, nerol α-maltoside) were
not detected in the reaction mixture, indicating that
Agrobacterium sp. M-12 had strong transglucosyla-
tion activity towards nerol. Transglucosidase pro-
duced only small amounts of the product, as
revealed by TLC analysis. The accumulation of glu-
cose and oligosaccharides was considerably more
pronounced in the reaction with transglucosidase
than with Agrobacterium sp. M-12. Maltose was pre-
ferred over nerol as an acceptor substrate for the
transglucosylation reactions. Glucosylated com-
pounds from nerol were not observed in the reaction
with CGTase.
hydrolysis by α- and β-glucosidase. In that reaction,
the glucosylated nerol was hydrolyzed by α-glucosi-
dase, with the concomitant release of glucose. In con-
trast, β-glucosidase did not catalyze the hydrolysis of
glucosylated nerol (Figure S2).
Optimization of culture and reaction conditions
for Agrobacterium sp. M-12
To obtain active cells of Agrobacterium sp. M-12 for
neryl-α-D-glucopyranoside production, Agrobacterium
sp. M-12 was cultured in a jar fermenter. The time
course of the fermentation is illustrated in Figure 3. As
enzymatic activity appeared similar during growth and
stationary phase, the glucosylation reaction was evalu-
ated on cells harvested when optical density at 600 nm
was nearly 5.0.
Isolation and identification of the product
obtained by glucosylation of nerol by
Agrobacterium sp. M-12
The conditions of glucosylation by Agrobacterium
sp. M-12 were examined in detail, including the effect
of maltose concentration and temperature. The pro-
cess of nerol α-glucosylation was positively affected
by increasing the starting concentration of glycosyl
donor (maltose). We also confirmed that the washed
cells of Agrobacterium sp. M-12 could not form
neryl-α-D-glucopyranoside when glucose replaced
maltose as an α-glucosyl donor. After investigating
the rate of the reaction using a range of maltose
concentrations up to 300 g/L, we selected 100 g/L of
maltose as the optimal glycosyl donor concentration,
as it resulted in the highest reaction rate (Figure S3).
Figure 4 shows the effect of reaction temperature. At
60°C, the activity decreased rapidly and therefore
The product obtained by glucosylation of nerol by
Agrobacterium sp. M-12 was a colorless and transpar-
ent liquid; it was identified as neryl-α-D-glucopyrano-
side (Figure 2) via 1H NMR and 13C NMR. The
specific chemical shifts of the product are listed in
Materials and methods. 13C NMR singlet signal at δ
97.6, which is assignable as an anomeric carbon of
glucose, indicated that Agrobacterium sp. M-12 selec-
tively produced a stereoisomer of an anomer.
1
Furthermore, H NMR doublet signal at δ 4.78 was
derived from an anomeric proton of glucose, its small
coupling constant (J = 3.00 Hz) suggested an α-gluco-
syl linkage to nerol. Moreover, we confirmed the gly-
cosidic linkage between glucose and nerol following
Figure 2. Structure of the transglucosylated product of nerol obtained by reaction with Agrobacterium sp. M-12.
Numbers correspond to the assigned NMR signals described in the text.

BIOPRODUCTION OF TERPENE ALCOHOLS
5
Figure 3. Time course showing growth and transglucosylation activity of Agrobacterium sp. M-12 in a 1-L jar fermenter.
Closed circles, turbidity at 600 nm; closed squares, transglucosylation activity. Values represent means of three independent experiments.
Figure 4. Effect of reaction temperature on the production of neryl-α-D-glucopyranoside.
Open circles, 30°C; closed circles, 40°C; open squares 50°C; open triangles, 60°C. Values represent means of three independent experiments. Bars
represent standard derivations.
optimal temperature was set to 50°C, which allowed
the reaction to be completed within 6 h. Nevertheless,
it should be noted that the largest amount of neryl-α-
D-glucopyranoside accumulated at 40°C in 48 h.
These results may be explained by a gradual loss of
activity at temperatures above 50°C.
1.8 g/L of neryl-α-D-glucopyranoside after 72 h, corre-
sponding to a molar yield of 87.6%. Other oligosacchar-
ides such as maltotriose and maltotetraose, or neryl
maltoside were not detected. However, in the time
course monitored by HPLC, other small peaks appeared
in addition to those for nerol and neryl-α-D-glucopyr-
anoside. These were subsequently identified as geraniol
and geraniol glucoside (geranyl-α-D-glucopyranoside)
by HPLC and NMR analyses (data not shown). Zhou
et al. [17] reported endogenous dehydrogenation and
isomerization of geraniol into nerol in E. coli. Thus, we
suspected that the formation of geraniol and the
Production of neryl-α-D-glucopyranoside
Figure 5 shows the time course for the synthesis of
neryl-α-D-glucopyranoside under optimal conditions.
These allowed for 1.0 g/L of nerol to be converted to

6
K. TAKAHASHI ET AL.
Figure 5. Time course showing the conversion of nerol by Agrobacterium sp. M-12.
Cells harvested from an 8.0-mL aliquot sampled from the jar fermenter were washed once with 8.0 mL of 20 mM potassium phosphate buffer (pH 7.0).
The washed cells were incubated with 1.0 g/L nerol, 100 g/L maltose, and 20 mM potassium phosphate buffer (pH 7.0) in a total volume of 8.0 mL. The
reaction was carried out in a 15.0-mL conical tube at 40°C with vigorous shaking. Closed circles, nerol; open circles, neryl-α-D-glucopyranoside; open
squares, geraniol.
glucoside was due to endogenous isomerization of nerol
to geraniol and its subsequent glucosylation in the
Agrobacterium sp. M-12 reaction mixture. This hypoth-
esis is supported by the accumulation, after a 72-h
reaction period, of 0.03 g/L of geraniol and 9.3 mg/L
of geraniol glucoside. According to our knowledge,
neither chemical nor enzymatic preparation of neryl-
α-D-glucopyranoside has been published to date.
Recently, various glucosyltransferases identified in
plant genomes, have been expressed in other organisms
by heterologous recombination. β-D-Glucosides of ter-
pene alcohols, such as geraniol, nerol, and citronellol,
were synthesized by a microbial-based whole cell bio-
transformation system [3]; however, the accumulation
of glucosides was low. Selection of the active enzyme
and construction of an efficient UDP-glucose regenera-
tion system are necessary for any future practical
applications.
sweeter smell compared to nerol, which explains its
use in fragrances.
Glucosylation of other alcohols
Agrobacterium sp. M-12 exhibited a high and efficient
transglucosylation activity towards nerol, which
prompted us to investigate the glucosylation of
other terpene alcohols, such as geraniol, (-)-β-citro-
nellol, and (-)-linalool. Transglucosylation products
were detected in all the reactions tested (Figure S4).
When geraniol was used as substrate, nerol and neryl-
α-D-glucopyranoside appeared in the reaction mix-
ture (Figure S5), in agreement with nerol transgluco-
sylation and the consequent accumulation of geraniol
and the glucoside. (-)-Linalool, which has a sterically
hindered hydroxyl group, was used as an acceptor in
this reaction. It should be noted that the order of
reactivity of glycosidases toward hydroxyl groups is
primary alcohols > secondary alcohols > phenols;
whereas tertiary alcohols are unreactive [5].
Therefore, it will be interesting, in the future, to
confirm the substrate specificity of transglucosylation
by Agrobacterium sp. M-12.
Properties of neryl-α-D-glucopyranoside
In general, glycosylation of non-sugar compounds
increases their solubility; this was confirmed in the
present case. The amount of neryl-α-D-glucopyrano-
side dissolved in water was 22.0 g/L, substantially
higher than the 0.53 g/L of nerol (at 25°C) [18].
On the basis of our recent discovery of α-anomer-
selective glucosylation of terpene alcohol by an intra-
cellular enzyme of Agrobacterium sp. M-12, which
Neryl-α-D-glucopyranoside had also
a
slightly

BIOPRODUCTION OF TERPENE ALCOHOLS
7
Arabidopsis glycosyltransferase in Escherichia coli.
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uses maltose as the α-glucosyl donor, we expect
future work to focus on identification and cloning
of the enzyme responsible for transglucosylation in
this species. This will proceed in parallel with char-
acterization of its acceptor specificity for terpene
alcohols and related compounds, including enantios-
electivity of chiral alcohols.
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[7] Divakar S, editor. Glycosidases. In: Enzymatic trans-
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[8] Kometani T, Terada Y, Nishimura T, et al
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[9] Aga H, Yoneyama M, Sakai S, et al Synthesis of 2-O-
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[10] Nishimura T, Kometani T, Takii H, et al Purification
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Acknowledgments
We thank Lecturer Kei Nishii for performing the NMR
studies and Amano Enzyme (Nagoya, Japan) for the kind
gift of transglucosidase and CGTase.
Disclosure statement
No potential conflict of interest was reported by the
authors.
[11] Bachhawat P, Mishra S, Bhatia Y, et al Enzymatic
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Funding
This work was supported by JSPS KAKENHI under Grant
number [17K07739] and the Institute for Fermentation,
Osaka to MU.
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ORCID
Ltd,
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Japan
Kokai
Tokkyo
Koho188,590.1996-188590. 1996 Jul 23.
Makoto Ueda
http://orcid.org/0000-0002-4503-0049
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