Efficient synthesis of β-primeverosides as aroma precursors by transglycosylation of β-diglycosidase from Penicillium multicolor

Article abstract of DOI:10.1080/07328300500439413

The enzyme activity transferring a β-primeverosyl unit was found from culture filtrates of Penicillium multicolor IAM7153 and was useful for synthesizing a series of β-primeverosides via a β-primeverosyl transfer reaction in an aqueous-organic biphasic system. With the acceptors benzyl alcohol, 2-phenylethanol, and (Z)-3-hexenol, the enzyme induced the transfer products benzyl, 2-phenylethyl, and (Z)-3-hexyl β-primeverosides in high yields of 51% to 70% based on the donor added. When geraniol and eugenol were used as acceptors, the corresponding geranyl and eugenyl β-primeverosides were obtained in lower yields of 8% to 12%. The enzyme was an excellent tool for producing naturally occurring β-primeverosides on a mmol scale. Copyright Taylor & Francis LLC.

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Efficient Synthesis of β‐Primeverosides as
Aroma Precursors by Transglycosylation of
β‐Diglycosidase from Penicillium multicolor
Kazutaka Tsuruhami a , Shigeharu Mori b , Kanzo Sakata c , Satoshi Amarume
d , Shigetaka Saruwatari d , Takeomi Murata d & Taichi Usui a d
a Science of Biological Resource, The United Graduate School of Agricultural
Science , Gifu University , Gifu
b Amano Enzyme Inc. , Gifu R&D Center , Sue‐cho, Kakamigahara, Gifu
c Institute for Chemical Research , Kyoto University , Kyoto
d Department of Biological Chemistry , Shizuoka University , Shizuoka
Published online: 02 Feb 2007.
To cite this article: Kazutaka Tsuruhami , Shigeharu Mori , Kanzo Sakata , Satoshi Amarume , Shigetaka
Saruwatari , Takeomi Murata & Taichi Usui (2005) Efficient Synthesis of β‐Primeverosides as Aroma Precursors
by Transglycosylation of β‐Diglycosidase from Penicillium multicolor , Journal of Carbohydrate Chemistry,
24:8-9, 849-863, DOI: 10.1080/07328300500439413
To link to this article: http://dx.doi.org/10.1080/07328300500439413
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Journal of Carbohydrate Chemistry, 24:849–863, 2005
Copyright # Taylor & Francis LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300500439413
Efficient Synthesis of
b-Primeverosides as
Aroma Precursors by
Transglycosylation of
b-Diglycosidase from
Penicillium multicolor
Kazutaka Tsuruhami
Science of Biological Resource, The United Graduate School of Agricultural Science,
Gifu University, Gifu
Shigeharu Mori
Amano Enzyme Inc., Gifu R&D Center, Sue-cho, Kakamigahara, Gifu
Kanzo Sakata
Institute for Chemical Research, Kyoto University, Kyoto
Satoshi Amarume, Shigetaka Saruwatari, and
Takeomi Murata
Department of Biological Chemistry, Shizuoka University, Shizuoka
Taichi Usui
Science of Biological Resource, The United Graduate School of Agricultural Science,
Gifu University, Gifu and Department of Biological Chemistry, Shizuoka University,
Shizuoka
Received September 28, 2005; accepted October 26, 2005.
Address correspondence to Taichi Usui, Science of Biological Resource, The United
Graduate School of Agricultural Science, Gifu University, Gifu 501-1193. E-mail:
actusui@agr.shizuoka.ac.jp
849

K. Tsuruhami et al.
850
The enzyme activity transferring a b-primeverosyl unit was found from culture filtrates
of Penicillium multicolor IAM7153 and was useful for synthesizing a series of b-prime-
verosides via a b-primeverosyl transfer reaction in an aqueous-organic biphasic system.
With the acceptors benzyl alcohol, 2-phenylethanol, and (Z)-3-hexenol, the enzyme
induced the transfer products benzyl, 2-phenylethyl, and (Z)-3-hexyl b-primeverosides
in high yields of 51% to 70% based on the donor added. When geraniol and eugenol
were used as acceptors, the corresponding geranyl and eugenyl b-primeverosides
were obtained in lower yields of 8% to 12%. The enzyme was an excellent tool for produ-
cing naturally occurring b-primeverosides on a mmol scale.
Keywords Enzymatic synthesis, Plant aroma precursors, b-Primeverosides, b-Digly-
cosidase, Penicillium multicolor IAM7153
INTRODUCTION
Floral aroma is one of the most important factors in determining the quality of
brewed tea and various fruits and flowers. Monoterpene alcohols (linalool,
geraniol, a-terpineol, etc.) and aromatic alcohols (benzyl alcohol, 2-phenyletha-
nol, etc.) are known to be the major floral aroma constituents of tea.[1] Various
tea aroma constituents, such as geraniol,[2] linalool, benzyl alcohol, and 2-phe-
nylethanol,[3] were shown to be present mainly as b-diglycoside precursors
such as b-primeverosides (6-O-b-D-xylopyranosyl-b-D-glucopyranoside, Psd)
in tea plant leaves used to produce oolong tea. In addition, many naturally
occurring compounds, such as anthraquinones[4–9] and (R)-lucumin,[10] exist
as b-primeverosides in various plants.
Our purpose is to develop an efficient synthetic method of obtaining such
b-Psds in sufficient amounts to study the function. Therefore, we searched
b-primeverosidase-like enzymes among microbes showing transglycosylation
activity so that it transfers the entire primeverose from p-nitrophenyl (pNP)
b-Psd as substrate. If such an enzyme was found, it might make it possible
to catalyze direct transfer of primeverosyl unit to various acceptor aromas.
No study has been reported regarding transglycosylation of a primeverose
unit. We recently reported that a b-Psd hydrolyzing enzyme from Aspergillus
fumigatus AP-20 cleaved in an endo-manner pNP b-Psd into primeverose
and p-nitrophenol.[11] However, the enzyme showed only a limited transferring
activity. Therefore, we searched for another source of b-primeverosidase-like
enzyme among microbes already commonly used in traditional food processing.
The availability of this enzyme from such sources would be useful not only for
the enzymatic synthesis of b-Psds as aroma precursors, but also for its poten-
tial applications in biotechnology, for example, in the control of aroma release
and the high recovery of aromas in extracts.
In the present study, we performed transglycosylation of a primeverose unit
from pNP b-Psd to various alcoholic and phenolic aroma compounds by use of the
partially purified enzyme powder preparation from P. multicolor IAM7153.

Synthesis of b-primeverosides
851
RESULTS
A total of 30 types of cultures were grown in a liquid medium and screened for
secretion of a b-primeverosidase activity, namely the hydrolysis of pNP and
eugenyl b-Psds to primeverose and the corresponding aglycones. Each
culture supernatant was reacted with pNP and eugenyl b-Psds, and the
reaction mixture was analyzed by TLC. Hydrolysis of the substrates were
detected by the release of primeverose. The b-primeverosidase activity was
detected in the supernatant from eight different types of cultures (Aspergillus
oryzae JMC5560, Aspergillus niger IAM2107, Aspergillus awamori IFO4033,
Rhizopus oryzae JCM5560, Talaromyces emersonii IFO9747, Trichoderma
reesei QM9414B, Penicillium camembertii FERMP-10452, Penicillium multi-
color IAM7153), with P. multicolor IAM7153 having the highest enzyme
activity. The supernatant also showed transglycosylation activity with pNP
b-Psd and eugenyl b-Psd.
Partially Purified b-diglycosidase
The supernatant was precipitated with ammonium sulfate at 50% (w/v)
saturation and subjected to Phenyl Sepharose column chromatography
[HiLoad 16/10 Phenyl Sepharose H.P., sodium acetate buffer (pH 5.5) contain-
ing 30% (w/v) ammonium sulfate]. Two fractions of this column (F-1 and F-2)
showed enzyme activity with assays A and B. The major fraction (F-1) was used
as partially purified enzyme preparation for the synthesis of b-primeverosides
as aroma precursors.
Synthesis of Five Different Aroma Precursor b-Psds
b-Psds exist in various kinds of plants,[11–15] but it is not easy to obtain
large amounts of the diglycosides from natural sources. Therefore, we
attempted the mmol scale synthesis of aroma precursor b-Psds by b-primever-
osidase-like enzyme-mediated transglycosylation. Generally, glycosidases have
transglycosylation activity as a reverse reaction of hydrolysis. At first, the
transglycosylation reaction was carried out on a small scale by using the
partially purified enzyme from P. multicolor IAM7153 and products resulting
from transglycosylation from pNP b-Psd donor to various alcoholic aroma
acceptors (benzyl alcohol, 2-phenylethanol, (Z)-3-hexenol, and geraniol), and
phenolic aroma acceptors (eugenol) were analyzed by TLC. A new spot corre-
sponding to a transglycosylation product was detected in each sample on the
TLC plate (data not shown). On the basis of these data, benzyl b-Psd (1) was
synthesized on a preparative scale from pNP b-Psd and benzyl alcohol by trans-
glycosylation. The molar ratio of the donor to the acceptor was 1 : 5. The
transfer product 1 was readily obtained in one step by Toyopearl HW-40S
column chromatography in a 51% yield, based on the amount of donor added.

K. Tsuruhami et al.
852
The typical elution pattern was as shown in Fig. 1. Similarly, with the acceptors
2-phenylethanol and (Z)-3-hexenol, the transfer products 2-phenylethyl (2)
and (Z)-3-hexenyl b-Psds (3) were obtained in high yields of 70% and 62%,
respectively. However, geraniol was a much poorer acceptor [8% yield of
geranyl b-Psd, (4)] than the other aroma alcohols. In this case, the transfer
product was isolated in two steps by successive chromatographies on Toyopearl
HW-40S and Sep-pak C18 columns. In the case of eugenol, the rate of formation
of eugenyl b-Psd (5) was too rapid at the standard concentration of enzyme to
be sure of its maximum production. In a separate experiment, compound 5 was
hydrolyzed 25-fold faster than the alcoholic aroma b-Psds (1–4), as mentioned
below. Thus, 5 is a much better substrate than 1–4 under hydrolytic conditions.
In general, the order of formation of transfer products closely corresponds to
that of hydrolytic rate. The present reaction made it possible to synthesize 5
by lowering the enzyme concentration. Therefore, the reaction was performed
at an enzyme concentration one-eighth that used for the preparation of b-Psd
from the alcoholic aromas mentioned above. The desired compound 5 was
obtained in a 12% yield based on the amount of donor added. Structures of
these synthetic transfer products were elucidated by 1H and 13C NMR
(Table 2 and Fig. 5) and FAB-mass analysis and identical to those of the
authentic samples reported previously.[2,3,17,18]
Time Course of the Formation of b-Psd During
Transglycosylation
The time courses of 2 synthesis and pNP b-Psd substrate degradation during
transglycosylation are shown as an example of reaction profile in Fig. 2. The
samples (10 mL) were taken at intervals during the incubation, inactivated by
Figure 1: Isolation of transglycosylated products 1 by Toyopearl HW-40S chromatography.
The transglycosylation products were extracted with chloroform and applied to a Toyopearl
HW-40S column. The fractions corresponding to compound 1 (F-1) were combined and
lyophilized. F-2, the fractions corresponding to the absorbance of p-nitrophenol; F-3, the
fractions corresponding to the absorbance of benzyl alcohol; A, A210; 4, A300; V, A485
.

Synthesis of b-primeverosides
853
Figure 2: The time course of the transglycosylation reaction. As the synthesis of 2 proceeded,
the amount of pNP b-Psd decreased. , pNP b-Psd; O, compound 2.
†
heating in a boiling water bath for 5 min, and then diluted with water (190 mL) for
analysis by HPLC. With 2-phenylethanol as an acceptor, compound 2 was formed
in appreciable amounts. The maximal concentration of 2 was reached in 50 min
and then decreased slightly during the subsequent reaction. The same trend
was also observed for the formation of 1, 3, and 4 (data not shown).
Hydrolytic Activities on Various Substrates
In a separate experiment, hydrolytic activities on a series of b-Psds and
related compounds were analyzed by the partially purified enzyme. Because
hydrolytic activity generally has a close relationship with transglycosylation
activity, the partially purified enzyme was shown to hydrolyze pNP b-Psd
into primeverose and p-nitrophenol. Based on this result, five synthetic
b-Psds (1–5), three pNP b-glucobiosides, and pNP b-Glcd were tested as
substrates for the enzyme (Table 1). In this experiment, the amounts of
p-nitrophenol or aglycones formed from initial substrates were analyzed at
an early stage of incubation with the enzyme (up to 15% to 20% hydrolysis).
The best substrate for the enzyme was the artificial substrate pNP b-Psd;
five other b-Psds were not readily hydrolyzed. The relative rates of attack on
these b-Psds were compared with that on pNP b-Psd. The relative rate of
hydrolysis of b-Psds having alcoholic aglycones (1–4) was 0.1% (1,000-fold
slower than pNP b-Psd). Of the naturally occurring aroma precursors,
compound 5 was the most rapidly hydrolyzed, although with 1/40 activity of
pNP b-Psd. It is notable that the alcoholic aroma precursors were very
poor substrates. The relative rate of hydrolysis of pNP b-Gend was 2.5%,
but two other pNP b-Lamd and pNP b-Celd did not act as substrates. No
detectable hydrolysis of reducing primeverose, other b-linked glucobioses,
and glucooligosaccharides was observed.

K. Tsuruhami et al.
Table 1: Relative hydrolytic rates of the partially purified
854
b-diglycosidase on various kinds of b-glycosides and
reducing saccharide substrates. Hydrolytic rate on pNP
b-Psd was set at 100%.
Rate of
Substrate
hydrolysis (%)
pNP b-Psda
100
2.5
NDc
ND
15
pNP b-Genda
pNP b-Lamda
pNP b-Celda
pNP b-Glcda
Eugenyl b-Psdb (5)
Benzyl b-Psdb (1)
2-Phenylethyl b-Psdb (2)
(Z)-3-Hexenyl b-Psdb (3)
Geranyl b-Psdb (4)
Gentiooligosaccharide
Laminarioligosaccharide
Cellooligosaccharide
Sophorose
2.5
0.1
0.1
0.1
0.1
ND
ND
ND
ND
ND
Primeverose
aRelease of p-nitrophenol was detected photometrically at 405 nm.
bRelease of each aroma compound was detected by HPLC as the
hydrolytic product.
cNo hydrolytic products were detected.
DISCUSSION
b-Psds occur quite widely in plants,[18] but it is very difficult to obtain large
amounts of the diglycosides from natural sources. A partially purified
enzyme preparation of P. multicolor IAM7153 was directly used for the syn-
thesis of a series of b-Psds without further purification. An enzymatic synthesis
method, involving b-diglycosidase-mediated transglycosylation, facilitated
large-scale preparation of b-Psds by as short a pathway as possible. A series
of b-Psd aroma precursors were synthesized by transglycosylation from pNP
b-Psd (donor) to various aromas (acceptors). Reactions were carried out in an
aqueous-organic biphasic system. The transfer products 1, 2, and 3 were
readily synthesized on an mmol scale in high yields of 51%, 70%, and 62%
based on the amount of donor added, respectively, and were conveniently
isolated by chromatography on a column of Toyopearl HW-40S. In contrast,
when geraniol and eugenol were used as acceptors, the yields of 4 and 5
were very low (8% to 12% based on the amount of donor added). The product
yield might be greatly influenced by the extent of substrate migration from
the organic phase to the aqueous phase in the biphasic system. Indeed,
benzyl alcohol, 2-phenyhlethanol, and (Z)-3-hexenol have a solubility of 2%
to 4% in water and can gradually move into the aqueous layer to undergo

Synthesis of b-primeverosides
855
transglycosylation. In contrast, geraniol and eugenol have only a poor solubi-
lity in water and thus are less likely to be acted on by the enzyme. The trans-
glycosylation reaction in the biphasic system is schematically shown in Fig. 3.
After formation of a primeverosyl-enzyme complex, two reactions are
possible: (i) hydrolysis and (ii) synthesis. The efficiency of glycosidase-catalyzed
transglycosylation is generally low because of an equilibrium shift favorable to
hydrolysis.[19] However, the biphasic system has several characteristics that
may explain the high product concentrations obtained in the present
study.[20] The b-Psd concentration that can be attained is controlled by the
equilibrium of the system. As an example, we monitored a transglycosylation
reaction from pNP b-Psd to 2-phenylethanol, following the time course of 2 pro-
duction and substrate consumption in the biphasic system, by analyzing the
corresponding spots on TLC plates, as in Fig. 4.
We found that the product and the substrate were present in both the
aqueous and organic phases (2-phenylethanol), indicating that some portion
of the transglycosylation product in the aqueous phase migrates to the
organic phase. As a consequence of this product removal, the equilibrium
shifts toward synthesis and the synthesis reaction comes to be more favored.
Furthermore, once 2 production reached its maximum, its concentration
then varied little during the subsequent reaction (Fig. 4). Consequently,
aromas such as benzyl alcohol, 2-phenylethanol, and (Z)-3-hexenol serve as
suitable acceptors for transglycosylation and the target b-Psds accumulate in
the biphasic system. Once the b-Psd formed, not much was hydrolyzed
because it is a poor substrate for the enzyme. In a separate experiment, the
relative rate of hydrolysis of 1, 2, and 3 compared with pNP b-Psd (100) was
Figure 3: Scheme of the transglycosylation reaction in the aqueous-organic biphasic system.
The synthesis of 2 is shown as a representative example.

K. Tsuruhami et al.
856
Figure 4: The time course of 2 production and pNP b-Psd consumption in the aqueous-
organic biphasic system. The synthesis of 2 and the consumption of pNP b-Psd in the water
phase (A) and the organic phase (B) were monitored by TLC analysis. The relative intensity of
spots was scanned densitometrically. B, pNP b-Psd; , compound 2.
†
0.1, a 1,000-fold difference (Table 1). It might be meaningless as the hydrolytic
substrate for the enzyme. This is a reasonable explanation for the high yield of
the transglycosylation product b-Psds. From these results, the present enzyme
might be a kind of b-diglycosidase rather than b-primeverosidase, because it
acts only to a limited extent toward naturally occurring series of b-Psds. The
characteristics of the enzyme will be reported in detail in the near future.
CONCLUSION
By screening for microorganisms producing any b-Psd-hydrolyzing enzyme, we
found a P. multicolor IAM7153 strain that produces a unique b-diglycosidase
that hydrolyzes b-Psd in an endo-manner into primeverose and aglycon.
Since the enzyme had a strong transglycosylation activity with pNP b-Psd,
the synthesis of a series of b-Psds as aroma precursors has been achieved by
b-diglycosidase-mediated transglycosylation in an aqueous-organic biphasic
system. This enzyme is an excellent tool for producing b-Psds on an mmol
scale. This synthetic method has great potential for mass production of
useful glycomaterials.
EXPERIMENTAL
General Methods
p-Nitrophenyl (pNP) b-Psd, primeverose, pNP b-gentiobioside (Gend),
and 2-phenylethyl b-Gend were prepared by our method.[21,22] pNP

Synthesis of b-primeverosides
857
b-Glucopyranoside (Glcd) and pNP b-galactopyranoside (Gald) were purchased
from Sigma Chemical Co. pNP b-Cellobioside (Celd) and pNP b-laminaribio-
side (Lamd) were purchased from Yaizu Suisankagaku Industry Co., Ltd. All
other chemicals were obtained from commercial sources.
Hydrolytic Activities
Hydrolytic activities were assayed toward pNP b-Psd, pNP b-Glcd, and
pNP b-Gald (assays A, B, and C, respectively). A mixture containing 80 mL of
each substrate (2 mM) in 20 mM sodium acetate buffer, pH 5.5, and 20 mL of
enzyme solution was incubated in a 96-well microplate for 30 min at 408C.
The reaction was stopped by adding 100 mL of 1 M sodium bicarbonate. The
released p-nitrophenol was measured photometrically at 405 nm in a micro-
plate reader (Biolumin 960, Amersham Pharmacia Biotech). One unit of the
enzyme was defined as the amount releasing 1 mmol of p-nitrophenol per
minute.
Analytic Methods
TLC analysis was carried out on a silica gel 60 F254 plate using a solvent
system of ethyl acetate/acetic acid/water (3 : 1 : 1, v/v). Sugars on the plate
were visualized by heating at 1208C for 10 min after spraying with 20%
sulfuric acid-methanol solution. HPLC analysis was performed using a
Mightysil RP-18(H) column (f 150 ꢀ 4.6 mm) on a JASCO LCS-905 HPLC
System station with an ultraviolet detector (absorbance at 210 nm). Elution
was performed with H2O at a flow rate of 0.6 mL/min at 658C. FAB-mass
analysis was carried out in the positive ion mode using a JEOL JMS
DX-303HF mass spectrometer coupled with the JEOL DA-800 mass data
system. An accelerating voltage of 10 kV and mass resolution of 1,000 was
employed. A sample of 1 mL in H2O or CH3OH was loaded with 1 mL of
1
glycerol as a matrix. H and 13C NMR spectra of each sample in D2O were
recorded on a JEOL JNM-EX 270 spectrometer at 308C. Chemical shifts
were expressed in d relative to sodium 3-(trimethylsilyl)-propionate (TPS) as
an external standard.
Media
The formula for the medium used for seed cultures for screening is as
follows: defatted soy meal [Honen Co. Ltd. (2.0%)], glucose (3.0%), KH2PO4
(0.5%), (NH4)2SO4 (0.4%), and dry yeast powder (0.3%). The medium for the
main cultures used for screening consisted of Gentose 80# [Nihon Shokuhinn
Kako Co., Ltd., Japan (3.0%)], KH2PO4 (2.0%), (NH4)2SO4 (1.0%), and Meast
P1G [Asahi Food & Healthcare, Ltd. (3.1%)]. The medium for enzyme

K. Tsuruhami et al.
858
production consisted of Solulys A-ST [Roquette (3.0%)], Pinedex No.2 [Matsu-
tani Chemical Industry Co. Ltd. (1.0%)], and KH2PO4 (0.5%). The pH of the
medium was adjusted to 5.5.
Isolation of Microorganisms and Fermentation
The screening for microorganisms producing the b-Psd-hydrolyzing
enzyme was carried out with type cultures of strains traditionally used for
food processing. Each type culture was streaked on a potato dextrose agar
slant and incubated at 27 to 328C for 3 to 14 days. Agar blocks (5 ꢀ 5 mm)
were taken from the potato dextrose agar slant, inoculated into the screening
medium in a glass test tube, and incubated at 278C for 8 days on a reciprocal
shaker (140 strokes/min). The culture was then centrifuged at 10,000 rpm
for 10 min and the supernatant was used to determine b-primeverosidase
activity. The supernatant (100 mL) was mixed with 100 mL of eugenyl b-Psd
(10 mg/mL in 20 mM acetate buffer) or pNP b-Psd (5 mg/mL in 20 mM
acetate buffer) and incubated at 378C for 96 h. The reaction was stopped by
incubating in boiling water for 10 min. Each assay mixture (20 mL) was
analyzed by TLC (Silica gel 60 F254, Merck; ethyl acetate/acetic acid/
H2O ¼ 3 : 1 : 1 (v/v) and detected by the orcinol-sulfuric acid method [23].
Spots of reaction products were compared with those of authentic samples
[primeverose, pNP b-Psd, eugenyl b-Psd, glucose (Glc), and xylose (Xyl)]. A
b-primeverosidase-like enzyme was detected in the supernatant from several
microorganisms. P. multicolor IAM7153 (Institute of Molecular and Cellular
Biosciences The University of Tokyo) had the highest enzyme production of
all the microorganisms tested. The fungus were streaked on a potato
dextrose agar slant and incubated at 278C for 14 days. An agar block
(5 ꢀ 5 mm) taken from the potato dextrose agar slant was inoculated into the
production media in 500 mL shakers and incubated at 278C for 8 days on a
reciprocal shaker (140 strokes/min). The culture broth from multiple
cultures was combined (5 L total) and filtered through a filter paper to
remove the mycelia. The culture filtrate (4 L) was concentrated to 400 mL
with an ultrafiltration module SIP-1010 (Asahi Kasei Corporation). The
enzyme solution was centrifuged to remove insoluble material. The super-
natant was lyophilized to yield crude enzyme powder (1.5 g), giving 0.1 U/mg
protein of specific activity.
Partially Purified b-diglycosidase
The crude enzyme powder (1.5 g) mentioned above was dissolved in 15 mL
of 20 mM sodium acetate buffer (pH 5.0). Proteins were precipitated in 50%
(w/v) ammonium sulfate. The precipitate was dissolved in 15 mL of 20 mM
sodium acetate buffer (pH 5.0) containing 30% ammonium sulfate. After

Synthesis of b-primeverosides
859
centrifugation, the supernatant was desalted and concentrated with Amicon
PM10 membrane. The lyophilized powder (178 mg) was dissolved in 20 mM
sodium acetate containing 30% ammonium sulfate (pH 5.0) and applied to a
HiLoad 16/10 Phenyl Sepharose H.P. column (f 1.6 ꢀ 10 cm) equilibrated
with 20 mM sodium acetate buffer (pH 5.5) containing 30% ammonium
sulfate. The column was washed with 350 mL of the equilibration buffer (flow
rate, 2 mL/min) and eluted with a linear gradient of ammonium sulfate from
30% to 0% in 480 mL of the same buffer, followed by 150 mL of 20 mM
sodium acetate buffer (pH 5.0). The elution pattern showed the enzyme
mixture to be fractionated into F-1 (tubes 41–52) and F-2 (tubes 55–60)
showing enzyme activity with assay A. F-1 fraction, which showed the same
elution profile of the activity with assays A, B, and C, was pooled, concentrated
with Amicon PM10 membrane, and lyophilized to yield partially purified
enzyme powder (20 mg) giving 0.5 U/mg protein of specific activity, which
was used for the enzyme synthesis.
Enzymatic Synthesis of Five Different Aroma Precursor
b-Psds
Benzyl b-Psd (1)
A mixture containing 0.21 g (0.5 mmol) of pNP b-Psd, 2.9 g (2.4 mmol) of
benzyl alcohol, 2.1 mL of water, and 1 mL of partially purified enzyme from
P. multicolor IAM7153 (2.8 U/mL of pNP b-Psd hydrolytic activity with
assay A) in 100 mM sodium acetate buffer (pH 5.5) was incubated at 408C for
3.5 h with vigorous shaking. The reaction was terminated by heating in a
boiling water bath for 5 min. Three ml each of water and chloroform were
added to the reaction mixture and the pH was adjusted to 4.0 with 0.1 N
HCl. After the aqueous phase was separated with a separatory funnel, it was
evaporated to dryness and dissolved in 3 mL of water. The solution was
loaded onto a column of Toyopearl HW-40S (f 4.5 ꢀ 85 cm) equilibrated with
25% (v/v) methanol. The column was eluted with the same solvent. The absor-
bance of the eluate was monitored at 485 nm (carbohydrate content, deter-
mined by the phenol-sulfuric acid method), 210 nm (double bond), and
300 nm (p-nitrophenol group). The fractions (tubes 10–16) containing the
transfer product were collected and concentrated to dryness followed by crys-
tallization from EtOH and gave compound 1 (99 mg) with a yield of 51%
based on the amount of donor pNP b-Psd added. Compound 1 had: mp 1948C
1
(EtOH), m/z 403 (M þ H)þ and 425 (M þ Na)þ. H NMR data (in D2O): d 4.62
(1H, d, J 7.6 Hz, H-100), 4.69 (1H, d, J 7.9 Hz, H-10), 7.60-7.63 (5H, m, phenyl).
13C NMR data in D2O are summarized in Table 2 and Fig. 5.
In a similar manner, 2-phenylethyl b-Psd (2, 140 mg) was obtained as
crystalline in a yield of 70% based on the amount of donor added. Compound

K. Tsuruhami et al.
860
Table 2: 13C NMR data of aroma precursor b-Psds synthesized by partially purified
b-diglycosidase.
(Z)-3-
Hexenyl
b-Psd (3)
Eugenyl
b-Psd
(5)
Benzyl
2-Phenylethyl
Geranyl
b-Psd (4)
b-Psd (1)
b-Psd (2)
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-10
C-20
C-30
C-40
C-500
C-600
C-1
C-200
C-300
C-400
C-500
138.4
130.4
130.4
130.2
130.4
130.4
73.2
-
140.3
130.6
130.3
128.2
130.3
130.6
36.8
50.5
-
64.2
28.3
126.0
136.2
21.4
14.8
-
51.7
121.8
146.3
41.8
28.2
127.0
135.8
20.0
27.9
19.0
103.4
75.8
78.7
72.1
77.7
68.4
105.9
75.8
79.7
71.2
68.0
146.4
151.6
116.1
139.2
124.0
119.4
41.8
140.9
118.5
58.7
103.4
75.6
78.4
72.0
78.2
-
-
-
-
-
-
103.0
74.7
77.3
70.9
76.6
70.3
105.3
74.8
77.3
70.9
66.8
103.9
72.5
77.2
71.0
76.5
70.2
105.2
74.6
77.2
70.8
66.8
106.4
73.0
78.5
72.2
77.8
71.4
106.9
75.8
78.5
72.2
68.0
70.9
106.0
75.8
78.3
72.0
67.9
Chemical shifts are shown in parts per million downfield from internal TPS.
2 had: mp 1828C (MeOH), m/z 417 (M þ H)þ and 439 (M þ Na)þ, 1H NMR data
(in D2O): d 2.96 (2H, t, H-7), 4.30 (1H, d, J 7.6 Hz, H-100), 4.57 (1H, d, J 8.3 Hz,
H-10), 7.29–7.38 (5H, m, phenyl). (Z)-3-Hexenyl b-Psd (3, 235 mg) was prepared
as lyophilized powder in a yield of 62% based on the amount of donor added.
1
Compound 3 had: m/z 395 (M þ H)þ and 417 (M þ Na)þ, H NMR data (in
D2O): d 0.97 (3H, t, H-6), 2.10 (2H, m, H-5), 2.42 (2H, m, H-2), 4.47 (1H, d, J
7.6 Hz, H-100), 4.49 (1H, d, J 7.9 Hz, H-10), 5.49 (1H, m, H-4), 5.60 (1H, m,
H-3). 13C NMR data in D2O are summarized in Table 2.
Geranyl b-Psd (4)
A mixture containing 0.83 g (1.9 mmol) of pNP b-Psd, 3.7 g (2.4 mmol) of
geraniol, 0.6 mL of water, and 1 mL of partilly purified enzyme (0.6 U/mL
with assay A) in 100 mM sodium acetate buffer (pH 5.5) was incubated at
408C for 3.5 h with vigorous shaking. After termination by heating in a
boiling water bath for 5 min, the reaction mixture was treated as in the
water extraction described above. The aqueous extract was loaded onto a
column of Toyopearl HW-40S treated as described above. The fractions (tubes
62–70) containing the transfer product were combined and concentrated.

Synthesis of b-primeverosides
861
Figure 5: Structures of benzyl b-Psd (1), 2-phenylethyl b-Psd (2), (Z)-3-hexenyl b-Psd (3),
geranyl b-Psd (4), and eugenyl b-Psd (5).
The crude products were separated by a column of Sep-pak C18 Cartridges Vac
35 cc equilibrated with water. The fractions (tubes 10–62) were combined, con-
centrated, and lyophilized to afford 70 mg of 4 in a yield of 8% based on the
amount of donor added. Compound 4 had: m/z 449 (M þ H)þ and 471
1
(M þ Na)þ, H NMR data (in D2O): d 1.63 (3H, s, H-8), 1.69 (3H, s, H-9), 1.70
(3H, s, H-10), 2.15 (4H, m, H-4, 5), 4.34 (2H, d, J 7.9 Hz, H-1), 4.52 (1H, d, J
7.6 Hz, H-100), 4.60 (1H, d, J 7.9 Hz, H-10), 5.20 (1H, t, H-6), 5.39 (1H, t, H-2).
13C NMR data in D2O are summarized in Table 2.
Eugenyl b-Psd (5)
A mixture containing 0.2 g (0.08 M) of pNP b-Psd, 3.9 g (0.2 M) of eugenol,
1.1 mL of water, and 0.3 mL of partially purified enzyme (0.2 U/mL with assay
A) in 100 mM sodium acetate buffer (pH 5.5) was incubated at 408C for 3.5 h
with vigorous shaking. After termination by heating in a boiling water bath
for 5 min, the reaction mixture was treated as in the water extraction described
above. The water extract was loaded onto a column (f 4.5 ꢀ 85 cm) of Toyopearl
HW-40S treated as described above. The fractions (tubes 21–33) were
combined, concentrated, and lyophilized to afford 26 mg of 5 (12% yield based
on the amount of donor added). Compound 5 had: m/z 459 (M þ H)þ and 481

K. Tsuruhami et al.
862
1
(M þ Na)þ, H NMR data (in D2O): d 3.86 (3H, s, H-10), 4.37 (1H, d, J 7.9 Hz,
H-100), 5.05 (1H, d, J 7.3 Hz, H-10), 5.99 (1H, m, H-8), 6.82 (1H, d, H-5), 6.95
(1H, s, H-3), 7.12 (1H, d, H-6). 13C NMR data in D2O are summarized in Table 2.
The structural data for these synthetic products 1–5 were identical to
those reported previously.[2,3,17,18]
Relative Rate of Hydrolysis of Various Substrates by the
Partially Purified Enzyme
The relative rates of hydrolysis catalyzed by the partially purified enzyme
from P. multicolor IAM7153 of a series of b-Psds and related compounds were
investigated by incubating a mixture (0.5 mL) containing 20 mM substrate in
20 mM acetate buffer pH 5.5 with 8 mU of enzyme for pNP b-glycosides, or
80 mU for aroma precursors (1–5), with assay A at 408C. Samples (50 mL)
were taken at 5-min intervals (0, 5, 10, 15, 20, and 25 min) and inactivated
by adding 50 mL of 1 M sodium carbonate or by boiling for 5 min. The amount
of hydrolysates formed at an early stage of the incubation (up to 15% hydroly-
sis) was analyzed by spectrophotometry (405 nm) for p-nitrophenol, or by
HPLC for aroma components, and a determination kit. From these results,
the relative rate of hydrolysis for each substrate was calculated with the rate
of pNP b-Psd hydrolysis taken as 100%.
ACKNOWLEDGMENT
This work was supported by a research grant (Agribusiness) from the Ministry
of Agriculture, Forestry, and Fisheries of Japan.
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