Glycobiology vol. 18 no. 4 pp. 325–330, 2008
doi:10.1093/glycob/cwn011
Advanced Access publication February 9, 2008
Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase
Yuji Honda2 , Shinya Fushinobu3 , Masafumi Hidaka4 ,
Takayoshi Wakagi3 , Hirofumi Shoun3 , Hajime Taniguchi2 ,
and Motomitsu Kitaoka1,4
2 Ishikawa
Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa
921-8836; 3 Department of Biotechnology, The University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-8657; and 4 National Food Research Institute,
2-1-12, Kannondai, Tsukuba, Ibaraki 305-8642, Japan
Received on October 3, 2007; revised on December 26, 2007; accepted on
February 3, 2008
The tyrosine residue Y198 is known to support a nucleophilic
water molecule with the general base residue, D263, in the
reducing-end xylose-releasing exo-oligoxylanase (Rex). A
mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity
and a small increase in the F− releasing activity from αxylobiosyl fluoride in the presence of xylose. In contrast,
mutations at D263 resulted in the decreased F− releasing
activity. As a result of the high F− releasing activity and
low hydrolytic activity, Y198F of Rex accumulates a large
amount of product during the glycosynthase reaction. We
propose a novel method for producing a glycosynthase from
an inverting glycoside hydrolase by mutating a residue that
holds the nucleophilic water molecule with the general base
residue while keeping the general base residue intact.
Keywords: general base/glycosyl fluoride/glycosynthase/
inverting glycoside hydrolase/reducing-end xylose-releasing
exo-oligoxylanase
Introduction
Glycoside hydrolases (GHs) are generally categorized into two
types, retaining and inverting enzymes, on the basis of changes
in their anomeric configurations during hydrolytic reactions
(Sinnott 1990; Henrissat 1991; Henrissat and Bairoch 1993,
1996; Davies and Henrissat 1995). Both these enzymes have
two acidic catalytic residues that act as a general acid (proton
donor) and a general base (nucleophile). The mechanism of a
typical retaining GH is a double displacement reaction, in which
the nucleophile directly attacks the anomeric center of the glycoside with the general acid donating a proton to the glycosyl
oxygen atom, producing a covalently bound intermediate of the
nucleophile with a Walden inversion, followed by hydrolysis of
the intermediate with another Walden inversion. In contrast, the
mechanism of a typical inverting GH is a single displacement reaction, in which a water molecule activated by the catalytic base
attacks the anomeric center to hydrolyze the glycoside with the
aid of the general acid, resulting in an anomeric inversion. The
whom correspondence should be addressed: Tel: +81-29-838-8071; Fax:
+81-29-838-7321; e-mail: [email protected]
1 To
transglycosylation activity is often used to prepare oligosaccharides by retaining GHs. However, inverting GHs theoretically
do not possess the transglycosylation activity because the reaction of inverting GHs is irreversible once the linkage is cleaved
with the addition of a water molecule, which is abundant in
the reaction medium. Thus, the use of inverting GHs has been
theoretically limited to hydrolysis and not synthesis reactions.
Glycosynthase is a nucleophile-mutant retaining GH that catalyzes the synthesis of glycosides from the glycosyl fluoride
of the opposite anomer. It was first reported by Mackenzie
et al. (1998) on a retaining exo-β-glycosidase, β-glucosidase
from Agrobacterium sp., with a mutation in its nucleophilic
residue (E358). Then, glycosynthases from endo-β-glycosidase
(1,3-1,4-β-glucanase from Bacillus licheniformis) (Malet and
Planas 1998) and exo-α-glycosidase (α-glucosidase from
Schizosaccharomyces pombe) (Okuyama et al. 2002) followed.
To date, various retaining GHs have been converted into glycosynthases through substitutions in their nucleophilic residues
(Hancock et al. 2006; Faijes and Planas 2007).
We have attempted to convert an inverting hydrolase, the
reducing-end xylose-releasing exo-oligoxylanase (Rex, EC.
3.2.1.156) belonging to GH family 8 into glycosynthase. Rex is a
strict reducing-end-specific exo-lytic enzyme liberating the xylose residue at the reducing end of xylooligosaccharide whose
degree of polymerization is 3 or higher (Honda and Kitaoka
2004; Fushinobu et al. 2005) (Figure 1A). The wild-type Rex
exhibited the Hehre resynthesis-hydrolysis mechanism (Hehre
et al. 1979; Williams and Withers 2000) in which α-xylobiosyl
fluoride (α-X2 F) was hydrolyzed to xylobiose (X2 ) and HF only
in the presence of xylose (X1 ) as an acceptor molecule via the
formation of xylotriose (X3 ) as an undetectable intermediate
(Honda and Kitaoka 2006) (Figure 1B). Then, the saturation
mutagenesis was performed at the general base residue D263
of Rex to weaken the hydrolysis step of the Hehre resynthesishydrolysis mechanism. We found that the D263C and D263N
mutants of Rex accumulated significant amounts of X3 from αX2 F and X1 , indicating that Rex was converted into glycosynthases (Honda and Kitaoka 2006). This finding expanded the
glycosynthase world to inverting GHs, making it possible to use
them in synthesis reactions.
However, the Rex glycosynthase retained the significant hydrolytic activity that decreased the yield of the synthetic product.
Moreover, the F− releasing activities of the mutants were much
lower than that of the wild type (Honda and Kitaoka 2006). The
mutation at the general base residue of an inverting GH could
not remove the hydrolytic activity completely because the water molecule retained some activity as a nucleophilic reagent
without the aid of the residue (Honda and Kitaoka 2006). It
is therefore still important to reduce the hydrolytic activity of
glycosynthase obtained from inverting GHs.
Collins et al. (2002, 2005) found that a mutation at a conserved tyrosine residue (Y203) in GH-8 endo-xylanase from
c The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected]
325
Y Honda et al.
Fig. 1. Schematic description of Rex reactions. (A) Hydrolysis of X3 . Rex hydrolyzes β-anomer of Xn (n ≥ 3) to form β-anomer of X1 and α-anomer of Xn−1 , but
it does not hydrolyze X2 (Honda and Kitaoka 2004). Subsite +2 of Rex is blocked by a barrier formed by a kink in the loop before helix α10 (Fushinobu et al.
2005). (B) Hehre resynthesis-hydrolysis and glycosynthase reaction of Rex. The reaction occurs only in the presence of both α-X2 F and X1 (Honda and Kitaoka
2006). The glycosynthase product (X3 ) was not observed with the wild-type Rex but was observed with its D263 mutants (Honda and Kitaoka 2006).
Pseudoalteromonas haloplanktis (pXyl), which shares 32.6%
amino acid sequence identity with Rex, causes a drastic decrease in the hydrolytic activity of the enzyme toward xylan.
The residue formed a hydrogen bond with the nucleophilic water molecule that had another hydrogen bond with the general
base residue (D281). The pH dependence of the Y203F mutant
indicated that the tyrosine residue was not a general base, but
was important to locate the nucleophilic water at the proper
position (Collins et al. 2005).
We have found Rex to be a suitable tool to examine glycosynthase. As Rex hydrolyzes X3 to release X1 from the reducing
end, it is expected to utilize α-X2 F as the donor and X1 as the
acceptor. It should be mentioned that the hydrolytic product of
α-X2 F, X2 will never act as the acceptor molecule because of
the subsite structure of Rex (Fushinobu et al. 2005) (Figure 1).
Thus, the donor and the acceptor are completely independent
during the reaction (Honda and Kitaoka 2006). The idea led us
to continue glycosynthase research on Rex. In this study, we
describe an alternative design of a glycosynthase from Rex in
which we mutated the corresponding tyrosine residue, which is
not the general base residue.
Results
Determination of the tyrosine residue in Rex
A water molecule was found to form a hydrogen bond with
the catalytic base residue D263 in the structure of Rex (PDB
accession no. 1WU4) (Fushinobu et al. 2005, Honda et al. 2005).
It formed another hydrogen bond with the phenolic oxygen atom
of Y198 (Figure 2). Because this is the only water molecule that
directly interacts with D263, it was considered as a nucleophile
in the hydrolysis. Y198 of Rex was found to correspond to Y203
326
Fig. 2. Superposition around the general base residues of Rex and pXyl. Rex
(PDB, 1WU4) and pXyl (PDB, 1H12) are illustrated in black and gray,
respectively. This figure was created by superimposition of their structures
(Fushinobu et al. 2005) using PyMol Ver. 0.99 (DeLano WL 2002) (PyMOL
Molecular Graphics System, DeLano Scientific, Palo Alto, CA).
http://www.pymol.org). The numbers of residues and water molecules written
out and in parentheses are for Rex and pXyl, respectively.
of pXyl (Collins et al. 2005) in both sequence alignment (data
not shown) and structural comparison (Figure 2). Then, Y198
of Rex was replaced by phenylalanine with and without the
displacement of the general base residue (D263).
Glycosynthase from an inverting hydrolase
Table I. F− releasing activity and hydrolysis activity of Rex and its mutants
Wild type
D263N
D263C
Y198F
D263N/Y198F
D263C/Y198F
F− release (s−1 )
Hydrolysis (s−1 )
F/H ratio
3.1
0.32
0.29
4.7
0.23
0.03
31.2
0.09
0.014
0.06
0.0016
0.0006
0.1
3.6
20.7
78.3
143.8
50.0
The glycosynthase reaction was carried out in a 0.1 M MOPS buffer (pH 7.0)
at 30◦ C. In the α-X2 F and X1 reactions, the other substrate was fixed at
10 mM. The X3 (2.6 mM) hydrolysis reaction catalyzed by the enzymes was
carried out in a 0.1 M MOPS buffer (pH 7.0) at 30 ◦ C. F/H ratio indicates F−
releasing activity divided by hydrolytic activity.
Table II. Kinetic parameters of the wild-type enzyme and its mutants toward
α-X2 F and X1
Enzyme
Substrate α-X2 F
Wild type
Y198F
D263N/Y198F
Substrate X1
Wild type
Y198F
D263N/Y198F
kcat (s−1 )
Km (mM)
kcat /Km (s−1 mM−1 )
nd∗
nd
nd
nd
nd
nd
0.30 ± 0.01
0.48 ± 0.01
0.028 ± 0.001
5.04 ± 0.24
11.9 ± 0.49
0.42 ± 0.02
6.6 ± 1.1
14.8 ± 1.6
6.0 ± 1.7
0.77 ± 0.01
0.80 ± 0.04
0.07 ± 0.01
The enzymatic reaction was carried out in a 0.1 M MOPS buffer (pH 7.0) at
30◦ C. In the α-X2 F and X1 reactions, the other substrate was fixed at 10 mM.
The reaction rates were determined by quantifying fluoride ions.
∗ nd, not determined because of the linear relationship of the Sv curve.
Activity of Y198F mutants
The F− releasing activity and hydrolytic activities of the mutants at Y198 and/or D263 are summarized in Table I. The single
mutation at Y198 drastically decreased the hydrolytic activity
(from 31.2 to 0.06 s−1 ), with a small increase in the F− releasing activity. The F− releasing activity of Y198F was more
than 10 times that of the mutants at the general base residue
(D263). The ratio of F− releasing activity by the hydrolytic activity (F/H) was 78, approximately four times that of the best
glycosynthase mutant at the general base residue (D263C). Double mutation was also tested. Y198F/D263N showed 20 times
less F− releasing activity than Y198F, which was comparable to
that of the corresponding single mutant, D263N. The F/H ratio
(144) of D263N was two times higher than that of Y198F. The
Y198F/D263C mutant showed only faint F− releasing activity.
The wild type, Y198F, and Y198F/D263N showed no activity
on α-X2 F in the absence of X1 , even though significant amounts
of α-X2 F were consumed in the presence of X1 under the same
conditions (Figure 3).
Kinetic analyses of the glycosynthase reaction
The apparent kinetic parameters of the glycosynthase reaction
on α-X2 F and X1 in the presence of 10 mM X1 and α-X2 F,
respectively, are summarized in Table II. The kcat and Km values
of Y198F and Y198F/D263N as well as that of the wild type on
α-X2 F were not calculated because their Sv curves were linear
up to 20 mM. The kcat /Km of Y198F was 1.5 times that of the
wild type. On the other hand, the kcat /Km of Y198F/D263N was
1/10 times that of the wild type. The kcat and Km values of Y198F
Fig. 3. Activity on α-X2 F in the absence and presence of X1 . The reactions
were performed at the substrate concentration of 20 mM α-X2 F in the absence
(odd lanes) and presence (even lanes) of 20 mM X1 with 1 µM each of
enzyme for 2 h. Other conditions are described in the text. Lanes 1 and 2, no
enzyme (negative control); lanes 3 and 4, wild type; lanes 5 and 6, Y198F; and
lanes 7 and 8, Y198F/D263N. M is standard of Xn (n = 1–3).
on X1 were approximately double that of the wild type, resulting
in a similar kcat /Km . For Y198F/D263N, the kcat was 1/10 times
that of the wild type, with a similar Km value.
The kinetic parameters of X3 hydrolytic activity by Y198F
were also determined. The Km value of Y198F (4.3 mM) was
similar to that of wild type (6.4 mM), whereas its kcat value
(0.22 s−1 ) was 0.27% that of wild type (83 s−1 ).
Time course of the glycosynthase reaction by Y198F and
Y198F/D263N
The reaction products from α-X2 F and X1 produced by Y198F
and Y198F/D263N gave the 1 H- and 13 C-NMR spectra identical with those of authentic X3 (see Supplementary data). Time
courses of the reactions with 5 mM α-X2 F and X1 by Y198F
and Y198F/D263N were compared with that catalyzed by the
best base mutant D263C (Figure 4). Much greater accumulation
of X3 (glycosynthase product) and much less formation of X2
(hydrolytic product from X3 ) were observed in the reactions catalyzed by Y198F and Y198F/D263N than in that catalyzed by
D263C. The ratio of X3 in the products {X3 /(X2 + X3 )} when
half of α-X2 F was consumed was 0.59 (D263C), 0.93 (Y198F),
and 0.96 (Y198F/D263N). Although Y198F/D263N showed a
slightly larger X3 ratio than Y198F, the F− releasing activity
of Y198F was 20 times that of Y198F/D263N. Note that the
experiments shown in Figure 2B and C were performed with
0.1 µM Y198F and 7.7 µM Y198F/D263N, respectively. These
327
Y Honda et al.
Fig. 4. Time courses of α-X2 F and X1 reaction by glycosynthase mutants of Rex. Enzymatic reaction was carried out in a 0.1 M MOPS buffer (pH 7.0) at 30◦ C.
Both substrate concentrations were 5.0 mM. A: D263C (enzyme concentration was 7.2 µM); B: Y198F (0.1 µM); and C: Y198F/D263N (7.7 µM). Symbols
indicate F− (closed diamonds), X2 (open circles), and X3 (closed circles).
results suggest that Y198F was the best glycosynthase of the
mutants.
Discussion
Glycosynthase is useful for converting GHs into a catalyst for
sugar chain synthesis (Mackenzie et al. 1998). To date, all glycosynthases derived from retaining GHs have been generated by
mutating their nucleophilic acidic residues (Malet and Planas
1998; Okuyama et al. 2002; Trincone et al. 2005; Faure et al.
2006; Hancock et al. 2006; Kim et al. 2006; Mullegger et al.
2006; Sugimura et al. 2006; Faijes and Planas 2007; Hommalai
et al. 2007). Prior to this report, the glycosynthase of Rex, the
only glycosynthase derived from an inverting GH, was also
created by mutating the general base residue D263 (Honda and
Kitaoka 2006). However, our observations showed that the wildtype Rex exhibited the highest F− releasing activity among the
mutants at the nucleophilic residue and concluded that it was
important to minimize the decrease in the F− releasing activity while maximizing the decrease in the hydrolytic activity to
generate a glycosynthase from an inverting GH. Thus, we proposed that a mutation at a residue other than the general base
D263 that the decreased hydrolytic activity would generate an
effective glycosynthase of Rex.
Collins et al. (2005) reported that the Y203F mutant of pXyl
has drastically decreased the hydrolytic activity toward xylan.
The tyrosine residue holds a nucleophilic water molecule with
the general base aspartic acid residue. Mutation of the tyrosine
residue in Rex (Y198) into phenylalanine resulted in 1/500 of
the hydrolytic activity toward X3 without a decrease in the F−
releasing activity as expected. de vos D et al. (2006) also pointed
out that the residue in pXyl might be important for binding the
xylan chain in the correct direction by forming an indirect hydrogen bond through another water molecule to the endocyclic
O5 in the xylosyl residue binding at subsite −2. Such a role is
negligible for Rex because the mutation at Y198 did not significantly change the Km value toward X3 in the hydrolytic reaction and the kcat /Km value toward α-X2 F in the glycosynthase
reaction.
The double mutant, Y198F/D263N, had a better F/H ratio
than did Y198F; however, its F− releasing activity was much
less than that of Y198F. This result suggests that the general
base residue should not be substituted in order to obtain high
F− releasing activity. We propose a novel concept for creating
328
a glycosynthase from an inverting GH by mutating a residue
holding the nucleophilic water molecule with the general base
residue while keeping the general base residue intact.
Materials and methods
Reagents
Xylooligosaccharides (Xn , where n = degree of polymerization)
were purchased from Megazyme (Wicklow, Ireland). α-X2 F
was synthesized by reacting hexa-O-acetyl xylobiose with pyridinium poly(hydrogen fluoride), followed by O-deacetylation
with sodium methoxide in methanol according to the standard
procedure (Hayashi et al. 1984; Yokoyama 2000). Other analytical grade reagents were obtained from various commercial
sources.
Construction of Rex mutants
Genes encoding wild type, D263N, and D263C Rex from Bacillus halodurans C-125 containing the His × 6 sequence at the
C-terminal end were constructed as reported previously (Honda
and Kitaoka 2004). To construct Y198F, D263N/Y198F, and
D263C/Y198F Rex, site-directed mutagenesis was performed
by PCR with a KOD polymerase (Toyobo, Osaka, Japan). In the
reaction, pET28b plasmids of wild type, D263N, and D263C
were used as the templates. The following mutagenic primers
were used (the mismatched base is underlined): 5 -AGT GAT
CCC TCT TTT CAT CTC CCC CAT TTT-3 (forward primer)
and 5 -G GAG ATG AAA AGA GGG ATC ACT GAA CTC
CA-3 (reverse primer). The reaction solution was digested with
DpnI. Next, pET28b plasmids for Y198F, D263N/Y198F, and
D263C/Y198F were transformed into Escherichia coli BL21
(DE3) cells, and positive colonies were selected.
Preparation of Rex mutants
Each transformant was incubated in Luria broth (100 mL) containing 0.05 mg/mL kanamycin at 37◦ C until the optical density,
at 600 nm, reached 0.6. Isopropyl-β-D-thiogalactopyranoside
was then added to give a final concentration of 1 mM, and the
cultures were incubated for 22 h at 25◦ C. The expressed Rex
mutant proteins were extracted from the wet cells using Bug
Buster(TM) HT (Novagen, Darmstadt, Germany). The cell-free
extract was loaded onto a Ni-NTA agarose (Qiagen, Hilden,
Germany) column (1 × 3 cm), and the enzyme was eluted
with a stepwise gradient of imidazole (1, 10 mM; 2, 20 mM;
Glycosynthase from an inverting hydrolase
3, 250 mM) in a 50 mM sodium phosphate buffer (pH 8.0)
containing 0.3 M NaCl.
Finally, Y198F, D263N/Y198F, and D263C/Y198F were desalted using an Amicon Ultra-4-10k (Millipore,Carrigtwohill,
Ireland). The purity of the proteins was checked by SDS–PAGE
(Laemmli 1970). Protein concentrations were determined by
measuring absorbance at 280 nm based on the theoretical molar absorption coefficients (106,210 M−1 cm−1 ) as described
previously (Pace et al. 1995).
Enzyme assay
The enzymatic F− release reaction was performed in a 0.1 M
MOPS buffer (pH 7.0) containing 10 mM α-X2 F and X1 at
30◦ C. The reaction was monitored using an F− -selective electrode (9609 BN, Thermo Orion, Beverly, MA) interfaced with
a portable pH/ISE Meter model 290A+ (Thermo Orion). A
F− standard curve was determined using sodium fluoride solution under the same reaction conditions without enzymes and
substrates. The rate constant of the spontaneous hydrolysis of
α-X2 F was determined to be 2.6 × 10−6 s−1 and the half-life
of α-X2 F was 2.7 × 105 s under the above conditions. All the
glycosynthase experiments in this report were designed to make
the spontaneous hydrolysis of α-X2 F negligible.
To determine the apparent kinetic parameters of the glycosynthase reaction, α-X2 F and X1 were subjected to F− release in
a 0.1 M MOPS buffer (pH 7.0) at 30◦ C. The initial rates were
measured as fluoride ions increased, as described above. The
kinetic parameters were calculated by regressing the experimental data (substrate concentration range: 0.2–3 Km ) with the
Michaelis–Menten equation by the curve-fitting method using
Kaleidagraph(TM) ver. 4.00 (Synergy Software, Reading, PA).
To determine the apparent kinetic parameters of X3 hydrolytic
activity, X3 was subjected to hydrolysis in a 0.1 M MOPS
buffer (pH 7.0) at 30◦ C. The initial rates were determined by
measurements of the increase in xylose by high-performance
ion-exchange chromatography (HPIC), as described above. The
kinetic parameters were calculated by regressing the experimental data (substrate concentration range: 0.2–3 Km ) with the
Michaelis–Menten equation by the curve-fitting method.
Analysis of the reaction products
The reaction products from α-X2 F and X1 were separated by
thin-layer chromatography on silica gel 60 F254 plates (5.0 × 7.5
cm; Merck, Darmstadt, Germany) with a solvent system of acetonitrile/water (4:1, v/v). Sugars were detected by baking after
dipping the plates in 5% sulfuric acid in methanol. When necessary, the amounts of the products were quantified by HPIC on a
CarboPac(TM) PA1 column (2 × 250 mm, Dionex, Sunnyvale,
CA) equipped with a pulsed amperometric detector (ICS-3000,
Dionex). Chromatography was performed with a linear gradient
of 0–0.2 M sodium acetate in 0.1 M NaOH for 20 min at a flow
rate of 0.25 mL/min.
GEL Amido-80 column (4.6 × 250 mm, Tosoh, Japan) and
eluted with acetonitrile/water (7:3 v/v) at a flow rate of 1.5 mL/
min at 25◦ C. The products were detected with a refractive index monitor (RID 10A, Shimadzu, Kyoto, Japan). The fractions
containing the products were evaporated and lyophilized to obtain white powders (Y198F: yield, 1.1 mg, 2.7 µmol, 27%;
Y198F/D263N: yield, 1.3 mg, 3.1 µmol, 31%). The 1 H-NMR
and 13 C-NMR spectra of the products were taken in D2 O using the Bruker Avance800 spectrometer and Bruker Avance500
spectrometer, respectively, and were compared with those of
authentic X3 .
Time course of the glycosnthase reaction
The enzymatic reactions by D263C, Y198F, and D263N/Y198F
were performed in a 0.1 M MOPS buffer (pH 7.0) containing
5.0 mM α-X2 F and X1 at 30◦ C. At appropriate reaction times,
aliquots of the reaction solution were withdrawn and diluted
with distilled water (1:100). The solutions were subjected immediately to HPIC and analyzed as described above. In the
enzymatic reaction, fluoride ions in the reaction solution were
also detected using a fluoride electrode as described above.
Supplementary Data
Supplementary data for this article is available online at
http://glycob.oxfordjournals.org/.
Funding
Ministry of Education, Culture, Sports, Science, and Technology, Japan (17780086 to Y.H.; and 19380062 to Y.H. and H.T.),
Research Fellowships of the Japan Society for the Promotion
of Science for Young Scientist (17-182 to M.H.), and the Program for Promotion of Basic Research Activities for Innovative
Biosciences (PROBRAIN).
Conflict of interest statement
None declared.
Abbreviations
GH, glycoside hydrolase; HPIC, high-performance ionexchange chromatography; MOPS, 3-morpholinopropanesulfonic acid; pXyl, GH8 endo-xylanase from Pseudoalteromonas haloplanktis; Rex, reducing-end xylose-releasing
exo-oligoxylanase; Xn , xylooligosaccharide with n degrees of
polymerization; X1 , xylose; α-X2 F, α-xylobiosyl fluoride.
References
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with Y198F (2.7 nmol) or Y198F/D263N (5.9 nmol). The reaction mixture was incubated for 2 h at 30◦ C, followed by
deionization with Amberlite MB-3 (Organo, Tokyo, Japan), and
lyophilized. The products were isolated by HPLC using a TSK-
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