Characterization of a novel polyphenol-specific oligoxyloside transfer reaction by a family 11 xylanase from Bacillus sp. KT12

Article abstract of DOI:10.1271/bbb.80116

A culture filtrate of Bacillus sp. KT12 was used to prepare polyphenyl β-oligoxylosides from xylan and polyphenols in a one-step reaction. One oligoxyloside transfer enzyme was purified from multiple xylanolytic enzymes in the culture filtrate. N-terminal amino acid sequence determination classified the enzyme as a glycosyl hydrolase family 11 (endo-xylanase). The xylanolytic enzyme activities could be markedly altered; its hydrolytic activity was almost entirely inhibited at acidic pH, whereas near constant transxylosylation activity was observed at pH 4-11. Further, metal ions activated transxylosylation and almost completely inhibited hydrolysis. The enzyme specifically induced a β-xylosyl transfer reaction to acceptor molecules, such as divalent and trivalent phenolic hydroxyl groups, and displayed no activity toward alcoholic compounds. The Bacillus sp. KT12 xylanolytic enzyme was a suitable enzyme for the synthesis of polyphenyl β-oligoxylosides.

Full text of DOI:10.1271/bbb.80116

Biosci. Biotechnol. Biochem., 72 (9), 2285–2293, 2008
Characterization of a Novel Polyphenol-Specific
Oligoxyloside Transfer Reaction by a Family 11 Xylanase
from Bacillus sp. KT12
y
Kazuhiro CHIKU,1; Jun UZAWA,2 Hiroko SEKI,2 Seigo AMACHI,1
1;3
Takaaki FUJII,1 and Hirofumi SHINOYAMA
1Department of Advanced Bioresources Science, Graduate School of Science and Technology,
Chiba University, Matsudo, 648 Matsudo-shi, Chiba 271-8511, Japan
2Chemical Analysis Center, Chiba University, Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japan
3School of Art and Design, Meisei University, Nagabuchi, Ome-shi, Tokyo 198-8655, Japan
Received February 25, 2008; Accepted May 24, 2008; Online Publication, September 7, 2008
[doi:10.1271/bbb.80116]
A culture filtrate of Bacillus sp. KT12 was used to
prepare polyphenyl ꢀ-oligoxylosides from xylan and
polyphenols in a one-step reaction. One oligoxyloside
transfer enzyme was purified from multiple xylanolytic
enzymes in the culture filtrate. N-terminal amino acid
sequence determination classified the enzyme as a
glycosyl hydrolase family 11 (endo-xylanase). The
xylanolytic enzyme activities could be markedly altered;
its hydrolytic activity was almost entirely inhibited at
acidic pH, whereas near constant transxylosylation
activity was observed at pH 4–11. Further, metal ions
activated transxylosylation and almost completely
inhibited hydrolysis. The enzyme specifically induced a
ꢀ-xylosyl transfer reaction to acceptor molecules, such
as divalent and trivalent phenolic hydroxyl groups, and
displayed no activity toward alcoholic compounds. The
Bacillus sp. KT12 xylanolytic enzyme was a suitable
enzyme for the synthesis of polyphenyl ꢀ-oligoxylosides.
one-step reaction. An enzymatic approach using glyco-
syltransferase and glycosidase for glycoside synthesis
has been partly successful.5–7)
Some glycosidases not only hydrolyze glycosidic
bonds, but also catalyze the formation of transxylosyl
products with various acceptors. The transglycosylation
(or solvolytic) reaction can progress in the presence of
various alcoholic and phenolic compounds (R-OH)
instead of water (H-OH).8) In recent years, there have
been few reports on the synthesis of oligoglycoside
using endo-type enzymes. The synthesis of alkyl
oligoxylosides using xylanase and the surfactant and
biodegradable capabilities of these alkyl oligoxylosides
have been reported.9,10) In contrast, little attention has
been paid to the fact that some endo-type xylanolytic
enzymes exhibit transxylosylation activity toward poly-
phenols. This prompted us to develop an efficient
transxylosylation system for the production of novel
bioactive polyphenyl oligoxylosides.
Key words: transxylosylation; xylanase; oligoxyloside;
Bacillus; xylan
We screened various enzymatic preparations to
detect new xylanolytic enzymes to synthesize xylosyl
derivatives by transglycosylation. A high population
of xylanolytic enzyme-producing microorganisms was
identified from various botanical origins using xy-
lan.11–13) Moreover, some of the microorganisms capable
of glycosylating lignin-degrading products to reduce
toxicity were isolated there.14) Hence, we assumed
that xylanolytic enzymes exhibiting a high level of
transxylosylation activity can be found in such micro-
organisms.
Polyphenols and related glycosides are widely present
in nature and potentially possess various physiological
functions, including antibacterial and antioxidative
activities, free radical-scavenging,1) and inhibition of
tyrosinase.2–4) There is great interest in developing
synthetic methods of obtaining these glycosides. Chemi-
cal methods of obtaining the glycosides have been
developed, but they involve various elaborate proce-
dures for protection, glycosylation, and deprotection.
Enzymatic glycosylation proceeds stereoselectively by a
We isolated a large number of microorganisms
capable of producing xylanolytic enzymes with trans-
y
To whom correspondence should be addressed. Tel/Fax: +81-47-308-8871; E-mail: chiku06UD7204@graduate.chiba-u.jp
Abbreviations: CBB, coomassie brilliant blue; CX2, 2-hydroxyphenyl ꢀ-xylobioside; CX3, 2-hydroxyphenyl ꢀ-xylotrioside; CX4, 2-hydroxyphenyl
ꢀ-xylotetraoside; DP, degree of polymerization; DPFGSE, double pulse field gradient spin echo; TLC, thin-layer chromatography; NMR, nuclear
magnetic resonance; N-terminal, amino-terminal; HPLC, high pressure liquid chromatography; IEF, isoelectric focusing; PAGE, polyacrylamide gel
electrophoresis; Rf, rate of flow; ROESY, rotating frame nuclear overhanser effect spectroscopy; SDS, sodium dodecyl sulfate; TOCSY, total
correlation spectroscopy; X1, xylose; X2, xylobiose; X3, xylotriose

2286
K. CHIKU et al.
xylosylation activity, including fungi (Aspergillus, Pen-
icillum, and Pestalotiopsis15)) and bacteria of the genus
Bacillus, from the leaves of Japanese cedar. In partic-
ular, the transxylosylation activity in culture filtrates of
the Bacillus isolates was remarkably high as compared
to the hydrolysis activity. Then, we described a novel
xylanolytic enzyme with transxylosylation activity,
termed oligoxyloside transfer enzyme, from the culture
filtrate of Bacillus sp. KT12, and determined its
enzymatic properties in detail.
Thin-layer chromatography. TLC was performed
on Silica gel-60 plates (Whatman, Maidstone, UK)
and Silica gel-60 F254 (Merck, Darmstadt, Germany)
using ethyl acetate:acetic acid:water (3:1:1) as a solvent.
Spots were visualized at 254 nm using a SLUV-6 UV-
Handy Lamp (As One, Osaka) and spraying with 20%
sulfuric acid in methanol, followed by heating at 120 ꢀC
for 7 min.
Preparation of xylosylated catechols. A mixture
containing 5.0 g of xylan and 5.0 g of catechol as
polyphenyl acceptors in 100 ml enzyme solution (ap-
proximately 2.0 U of hydrolysis activity) in 100 mM
sodium acetate buffer (pH 5.0) was incubated at 30 ꢀC
for 24 h. The reaction mixture was centrifuged at
8;000 ꢁ g for 30 min at 4 ꢀC, and then passed through
a filter paper (no. 131, Advantec, Tokyo). A sample
containing transxylosyl products was applied to an
active carbon column (25 i.d. ꢁ 300 mm) and eluted
with DW, 10% methanol, or 30% isopropanol. Activated
charcoal (Wako, Tokyo) was used as the active carbon.
The eluted xylosylated products were scanned at 254 nm
on TLC, collected, and dried by reduced-pressure
distillation. The sample was resolved in a small amount
of solvent, ethanol:butanol:DW (1:10:2), applied onto a
cellulose column (25 i.d. ꢁ 1000 mm), and eluted with
the same solvent. The eluates were fractionated by
5.0 ml. The separated transxylosyl product was dried and
preserved. The isolated products were identified by acid
hydrolysis, fast atom bombardment-mass spectroscopy
Materials and Methods
Materials and chemicals. Xylan from beech wood
was purchased from Sigma-Aldrich (St. Louis, MO).
Nutrient broth was from Nissui Pharmaceutical (Tokyo).
All other chemicals used were of the highest commer-
cially available grade.
Microorganisms and enzymes. Asperugillus nigar IFO
31628 and Penicillum expansum IFO 8800 was from
the Institute for Fermentation (IFO, Osaka). Xylanase
XL-200 from Tricoderma viride was from Shin-Nippon
Biomedical Laboratories (SNBL, Tokyo).
Isolation of microorganisms producing xylanolytic
enzyme with transxylosylation activity. For the first
screening of xylanolytic microorganisms, an agar plate
of xylan medium was used. The xylan liquid medium
was comprised of xylan (20 g/l) as the main carbon
source, peptone (2.0 g/l), K2HPO4 (1.0 g/l), KH2PO4
1
(FAB-MS), and H-nuclear magnetic resonance (NMR)
.
(2.0 g/l), MgSO4 7H2O (0.40 g/l), and trace elements
(2.5 ml/l). The trace element solution contained NaCl
spectra. Acid hydrolysis of the purified xylosylated
products was carried out with 500 ml of the sample were
set at first at a concentration of about 1%. Xylosylated
products were incubated with 25 ml of trifluoroacetic
acid (TFA) at 100 ꢀC for 5, 10, and 120 min, and
analyzed by TLC.
.
(0.50 g/l), FeC6H5O7 H2O (6.0 g/l), ZnSO4 7H2O
(2.0 g/l), MnCl2 4H2O (0.10 g/l), CoCl2 6H2O (0.10
.
.
.
.
g/l), H3BO3 (0.20 g/l), and CaCl2 H2O (4.0 g/l). There-
after, the liquid medium was adjusted to pH 5.6 (for
fungi) or 7.0 (for bacteria) with 1.0 N HCl, and sterilized
at 120 ꢀC for 20 min. Sources for screening were
collected from Japanese cedar leaves in many parts of
Japan. A small amount of the sample (snippets of
Japanese cedar leaves, approximately 1.0 g) was sus-
pended in 5 ml of sterile normal saline and immediately
left to stand for 30 min. The supernatant of the microbial
suspension (0.10 ml) was collected and appropriately
diluted with sterile normal saline, and spread on a xylan
agar plate. In addition, the snippets of Japanese cedar
leaves were laid directly on the plates of xylan medium.
The plates were subsequently cultivated at 30 ꢀC, and
colonies formed in 3–7 d were randomly selected and
isolated as xylanolytic organisms. The xylanolytic
microorganisms were cultivated in a xylan liquid
medium at 30 ꢀC at 180 rpm for 5–7 d. After cultivation,
the culture solutions were centrifuged at 6;000 ꢁ g at
4 ꢀC for 30 min. To confirm the formation of xylosylated
catechol, a transxylosylation reaction was conducted
using the culture filtrate and the xylosides was analyzed
by thin-layer chromatography (TLC).
Spectrometric analyses. Fast atom bombardment-mass
spectroscopy (FAB-MS) data were obtained with a JMS-
AX500 system (JEOL, Tokyo) when analyzing xylosy-
lated catechols. The samples were added to NaCl or KI
in 3-nitrobenzyl alcohol or thioglycerol. 1H-nuclear
magnetic resonance (NMR) spectra were recorded at
600 MHz using a model JNM ECA600 (JEOL). The
samples were dissolved in deuterium oxide (D2O), and
the chemical shifts were calibrated using the dimethylsi-
lapentanesulphonic acid (DSS) signal in D2O at 30 ꢀC as
an external standard. TOCSY mixing times were from 80
to 100 millisecond and ROESY mixing time were 400
millisecond. The selective 180ꢀ pulses were from 33 to
66 millisecond in length and were Gaussian in shape. The
duration time depended on the target region selected.
Determination of transxylosylation activity toward
catechol. Transxylosylation activity was determinated
under the following conditions: A mixture containing
0.1 g of xylan and 0.1 g of catechol and enzyme solution

Polyphenol-Specific Transxylosylation of Family 11 Xylanase
2287
as polyphenyl acceptors in 2.0 ml of 100 mM sodium
acetate buffer (pH 5.0) was incubated at 40 ꢀC for
15 min. The reaction was terminated with 0.5 ml of 20%
trichloroacetic acid, and the solution was neutralized
with 0.5 ml of 0.55 mM sodium hydrogen carbonate.
Vanillin was added as an internal standard, to a final
concentration of 0.20 mg/ml. The sample was centri-
fuged at 8;000 ꢁ g for 10 min and then passed through
a 0.20-mm membrane filter. The filtered sample was
analyzed by high pressure liquid chromatography
(HPLC) (Hitachi, Tokyo) with an L-7400 UV detector
(Hitachi) and a 4.6 i.d. ꢁ 300 mm YMC-Pack ODS-AQ
column (YMC, Kyoto). HPLC was conducted under the
following conditions: mobile phase, acetonitrile:water
(10:50); flow rate, 1.0 ml/min; detection, 280 nm;
sample volume, 5 ml; temperature, 30 ꢀC. The amount
of 2-hydroxyphenyl ꢀ-oligoxylosides produced in the
solution was measured. One unit of transxylosylation
activity corresponded to 1 mmol of 2-hydroxyphenyl
by 2.0 ml. Fractions were automatically collected,
scanned at 280 nm, and assayed for hydrolysis activity.
The transxylosylation activity in each eluted fraction
was determined by TLC. The active fractions were
pooled and stored at 4 ꢀC.
Electrophoretic analysis. Sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) was
carried out according to the method of Laemmli18) with
a tris-glycine buffer system (pH 8.5) containing 0.2%
SDS using a current of 20 mA per gel for approximately
1 h at room temperature. The isoelectric point of the
native protein was determined using a commercially
available isoelectric focusing (IEF)-PAGE 4% mini-gel
with a pH gradient of 3–10 (Tefco, Tokyo). The gels
were stained with 0.1% coomassie Brilliant Blue R-250
in 10% acetic acid containing 50% methanol to detect
proteins. After staining, they were destained by immer-
sion in 7.5% acetic acid containing 25% methanol.
ꢀ-oligoxylosides (expressed as total amount of CX2-4
)
released per min.
N-Terminal amino acid sequences. N-Terminal amino
acid sequences of the purified enzyme were identified
using a PPSQ-10 protein sequencer (Shimazu, Tokyo).
Approximately 100 pmol of pure proteins was applied
during each run.
Determination of hydrolysis activity toward xylan.
Hydrolysis activity was determined using a mixture
containing 0.05 g of xylan and enzyme solution in
100 mM sodium acetate buffer (pH 5.0) incubated at
40 ꢀC for 20 min. The reaction was terminated by heat
treatment at 100 ꢀC for 10 min. The amount of xyloo-
ligosaccharides released into the solution was measured
by monitoring the absorbance at 660 nm by the
Somogyi-Nelson method.16) One unit of hydrolysis
activity corresponded to 1 mmol of reducing sugar
(expressed as D-xylose) released per min.
Results
Screening of xylanolytic bacteria for synthesis of
polyphenyl xylosides
We isolated 347 colonies of xylanolytic microorgan-
isms using xylan agar plates, and cultivated them in
xylan liquid medium at 30 ꢀC for 7 d. The transxylosy-
lation reactions of the culture filtrates were examined by
TLC analysis of the reaction products. The transfer prod-
ucts to the phenolic compounds were distinguished from
the hydrolysis products, such as xylose (X1), xylobiose
(X2), and xylotriose (X3), by their higher rate of flow (Rf)
values and UV absorption at 254 nm. The culture filtrates
of 103 fungi (79%) and 31 bacteria (27%) exhibited
transxylosylation reactions toward catechol. The culture
filtrates of the three bacterial isolates produced xylosy-
lated catechols at high concentrations and hydrolysis
products at extremely low concentrations (Fig. 1), and
the bacterial strains were characterized as Bacillus sp.
strains KT12, YR12, and B-1 based on 16S rRNA partial
sequences. In addition, performing the same operation as
for the synthesis of xylosylated catechols, the culture
filtrates of the three Bacillus spp. isolates xylosylated
other polyophenolic compounds such as hydroquinone,
resorcinol, and gallic acid as acceptors (data not shown).
The three Bacillus sp. isolates had the ability to
synthesize polyphenyl oligoxylosides.
Purification of the oligoxyloside transfer enzyme.
Bacillus sp. KT12 was cultivated in 200 ml nutrient
broth in a 1 liter flask at 180 rpm for 48 h at 30 ꢀC. The
culture suspension was centrifuged at 8;000 ꢁ g for
10 min at 4 ꢀC. The protein concentration was deter-
mined by the method of Bradford17) or spectrophoto-
metrically by measuring the absorbance at 280 nm. A
standard curve was drawn using bovine serum albumin
as a standard. Purification of xylanolytic enzyme
catalyzed transglycosylation (oligoxyloside transfer
enzyme) was carried out at 4 ꢀC by a procedure
involving 70% ammonium sulfate fractionation, and
the solution was centrifuged at 8;000 ꢁ g for 20 min at
4 ꢀC. The residue was dissolved in 20 mM sodium
acetate buffer (pH 5.0), and the solution was purified by
column chromatography on a phenyl-toyopearl column
(Tosoh, Tokyo) and a sephadex G-75 column (GE
Healthcare, Tokyo). The phenyl toyopearl column was
eluted with a linear gradient (1000–500 mM) of ammo-
nium sulfate dissolved in 20 mM sodium acetate buffer
(pH 5.0) at a flow rate of 0.5 ml/min. The eluates were
fractionated by 5.0 ml. The sephadex G-75 column was
eluted using 20 mM sodium acetate buffer (pH 5.0) at a
flow rate of 0.3 ml/min. The eluates were fractionated
Production and identification of xylosylated catechols
Xylosylation of catechol using culture filtrate (ap-
proximately 2.0 U of hydrolysis activity) from Bacillus
sp. strain KT12 grown in xylan liquid medium occurred

2288
K. CHIKU et al.
xylopyranosides with degrees of polymerization from 1
KT12 YR-1 B-1
Strain
to 4. The molecular weights of the xylosylated catechols
were determined by FAB-MS, and their molecular
structures were analyzed by 1H-NMR (Table 1). The
FAB-MS results were in agreement with the identi-
fications of compound 1 (M ¼ 242), compound 2
(M ¼ 374), compound 3 (M ¼ 506), and compound 4
(M ¼ 638). The xylosylated catechols were analyzed by
1H-NMR using one-dimensional DPFGSE (Double
Pulse Field Gradient Spin Echo)-TOCSY (Total Corre-
lation Spectroscopy) and DPFGSE-ROESY (Rotating
Frame Nuclear Overhanser Effect Spectroscopy),19) and
two-dimensional methods. DPFGSE-TOCSY was used
to divide the proton signals into groups or coupling
networks in a xylose structure, and DPFGSE-ROESY
was utilized for identifying the glycosidic linkage
between xylosides and catechol. The NMR spectra of
the xylosyl catechols with degrees of polymerization
(DP) of 1–4 corroborated completely their structures.20)
Chemical shifts in 1H proton bound to the acceptors
and the xylose residues by its ꢀ-anomeric proton
M
Front
A
X1
X2
X3
Origin
Fig. 1. Transxylosylating Reaction Using Culture Filtrates of Xyla-
nolytic Microorganisms Grown in Xylan Liquid Medium.
A reaction mixture containing 5.0 g of xylan and 5.0 g of catechol
(+) or without catechol (ꢂ) in 1.5 ml of 100 mM sodium acetate
buffer (pH 5.0) and 0.5 ml of enzyme solution was incubated at
30 ꢀC for 24 h. The symbols used are as follows: M, marker; A,
arbutin; X1, xylose; X2, xylobiose; X3, xylotriose; and arrow,
xylosylated catechols.
1
were observed at ꢁ4:96–5.06 in the H-NMR spectra.
The glycosidic linkages were determined to be in a
ꢀ-configuration based on the coupling constants (Jd ¼
6:2{7:3 Hz) of the anomeric protons. It was evident that
compound 1–4 was 2-hydroxyphenyl O-ꢀ-D-xylopyra-
noside (CX1), 2-hydroxyphenyl O-ꢀ-D-xylopyranosyl-
(1!4)-ꢀ-D-xylopyranoside (CX2), O-ꢀ-D-xylopyrano-
syl-(1!4)-ꢀ-
D
-xylopyranosyl-(1!4)-ꢀ-
D-xylopyranoside
at 30 ꢀC for 24 h. Four unknown products (compounds
1–4) were isolated in two chromatographic steps, as
described in ‘‘Materials and Methods.’’ Next, the xylo-
sides were degraded by acid hydrolysis to analyze of the
numbers of xylopyranoside residues and acceptors. The
purified products were composed of one catechol and
(CX3), and 2-hydroxyphenyl O-ꢀ-D-xylopyranosyl-1!
(4-ꢀ-D-xylopyranosyl)2-4-O-ꢀ-D-xylopyranoside (CX4).
The time course of the yields of CX2-4 at 30 ꢀC was
periodically analyzed by HPLC. It was found that the
yields of CX3;4 simultaneously increased and that, after
12 h of incubation, maximum yields of CX3 and CX4 of
Table 1. FAB-MS and 1H NMR Data for Transxylosylation Products
Chemical shifts (δ(ppm))
Product
[M+Na]+ [M+K]+ Residue
H-1
5.03

H-2
3.63

H-3
3.57
7.00
H-4
3.73
7.06
H-5 ax. H-5 eq.
3.43
6.96
H-6
265
281 β-Xylp (X1)
o-Catechol
Compound 1
4.02
HO
7.15
O
HO
HO
O
HO
X1
397
HO
413 β-Xylp (X1)
β-Xylp (X2)
Compound 2
4.96
4.45

3.64
3.30

3.68
3.45
6.97
3.86
3.59
7.01
3.48
3.32
6.90
4.13
3.99
HO
O
O
HO
HO
O
o-Catechol
7.13
7.19
HO
O
HO
X2
X1
529
545 β-Xylp (X1)
β-Xylp (X2)
Compound 3
5.06
4.50
4.47

3.66
3.33
3.28

3.70
3.58
3.45
7.01
3.89
3.81
3.64
7.07
3.52
3.40
3.32
6.96
4.16
4.13
3.99
HO
O
HO
O
O
O
HO
HO
HO
β-Xylp (X3)
o-Catechol
HO
O
O
HO
HO
X3
X2
X1
661
β-Xylp (X1)
β-Xylp (X2)
β-Xylp (X3)
β-Xylp (X4)
o-Catechol
Compound 4
677
HO
5.00
4.47
4.45
4.43
3.65
3.31
3.30
3.26
3.67
3.56
3.55
3.42
6.97
3.85
3.77
3.76
3.61
7.03
3.48
3.37
3.37
3.30
6.92
4.12
4.10
4.10
3.96
HO
HO
O
O
O
HO
O
HO
HO
O
HO
O
HO
O
O
HO
HO
X4
X3
X2
X1
7.14

Polyphenol-Specific Transxylosylation of Family 11 Xylanase
2289
Table 2. Comparisons between Transxylosylation and Hydrolysis
Activities of Culture Filtrate Grown on Xylan Medium
Table 3. Production of Xylanolytic Enzyme with Transxylosylation
Activity Produced on Culture Medium Containing Various Carbon
Sources
Xylanase Transxylosylation
Transxylosyl
activity
(U/ml)
(A)
activity
(U/ml)
(B)
Carbon source
Xylan
Groth (A660
)
Transxylosylation
coefficient
(B/A)
7.94
1.68
++ꢃ1
+
Xylooligosaccharide
Xylose
Xylitol
Bacteria
Bacillus sp. KT12
(pH 5.0)
1.74
+
0.0400
3.74
3.26
ꢂ
0.010
0.010
0.010
0.39
0.33
0.28
39
33
28
Starch
Maltose
++
++
+
Bacillus sp. YR-1
(pH 5.0)
Glucose
Cellulose
Chitin
2.80
Bacillus sp. B-1
(pH 5.0)
0.280
0.620
0.180
0.620
1.92
ꢂ
ꢂ
Fungi
Chitosan
Pectin
ꢂ
Pestalotiopsis sp.
(pH 5.0)
P. expansum
ꢂ
0.33
0.74
0.084
0.13
0.25
0.18
Fructose
Sucrose
Lactose
+
4.04
2.88
++
++
+
IFO 8800 (pH 7.0)
A. niger IFO 31628
(pH 5.0)
Mannitol
Sorbitol
Ribose
3.44
0.076
1.1
0.17
0.41
2.2
0.220
0.0400
9.34
ꢂ
T. viride (pH 5.0)
0.37
ꢂ
Nutrient broth
++ꢃ2
Bacteria and fungal isolates were cultured for 3 and for 7 d respectively at
30 ꢀC on xylan liquid medium. The culture filtrates were used to determinate
enzymatic activities at 30 ꢀC and pH 5–9. The enzymatic activities exhibited
the highest transxylosyl coefficient at pH 5–9. An extract from Trichoderma
viride was used as a source of commercial xylanase (5,000 U/g protein as
xylanase activity).
Bacillus sp. KT12 was cultured on synthetic medium at 30 ꢀC for 7 d, and a
2.0% corresponding carbon source without xylan was added. TLC detection
of transxylosylation products was strong (++), weak (+), or not detected
(ꢂ). ꢃ1The culture filtrate showed 0.37 U/ml of maximum transxylosylation
activity and 0.12 U/ml of hydrolysis activity after 48 h of incubation on
xylan liquid medium at 30 ꢀC. ꢃ2The culture filtrate showed 2.4 U/ml of
maximum transxylosylation activity and 0.017 U/ml of hydrolysis activity
after 48 h of incubation on nutrient broth at 30 ꢀC.
4.4 and 0.28 mM were obtained, which then slowly
decreased. On the other hand, the yield of CX2 was
rapidly increased after 15 h of incubation, and the
maximum yield of CX2 was 3.0 mM at 34 h.
of the oligoxyloside transfer enzyme are showen in
Table 3. Bacillus sp. KT12 showed significant growth
acceleration on media containing various carbon sources
(xylose, starch, maltose, fructose, sucrose, lactose,
mannitol, and nutrient broth), and several culture
filtrates further exhibited transxylosylation activities.
Compared with the enzymatic activity of the culture
filtrate on the other media, the production of oligoxylo-
side transfer enzyme increased on nutrient broth that did
not contain xylan. The oligoxyloside transfer enzyme
might have been produced constitutively in the culture
medium devoid of xylan as a carbon source.
Comparisons between transxylosylation and hydrol-
ysis activities of the culture filtrates
For comparison of the culture filtrates of isolated
Bacillus spp., we selected three stocked xylanolytic
fungi (Pestalotiopsis sp.,15) Penicillium expansum IFO
8800, and Aspergillus niger IFO 31628) and a commer-
cial enzyme from Trichoderma viride. The culture
filtrates of these three microorganisms grown in xylan
liquid medium were determined to have enzymatic
activities at 30 ꢀC in 100 mM acetate buffer (pH 5.0),
100 mM phosphate buffer (pH 7.0), and 100 mM Tris–
HCl buffer (pH 9.0) respectively. Table 2 summarizes
the enzymatic activities that exhibited the highest
transxylosyl coefficient at pH 5–9. The culture filtrate
of Bacillus sp. KT12 grown on xylan liquid medium
exhibited the highest transxylosylation activity (0.39
U/ml) and the highest transxylosyl coefficient. Com-
pared to that of the bacteria, the culture filtrate of the
fungi showed high levels of hydrolysis and extremely
low transxylosyl coefficients. In view of these results,
we considered Bacillus sp. KT12 to be suitable for the
synthesis of polyphenyl ꢀ-oligoxylosides, and subse-
quent work focused on this organism.
Purification and characterization of the oligoxyloside
transfer enzyme
The extracellular xylanolytic enzyme with transxylo-
sylation activity was purified from the culture filtrate of
Bacillus sp. KT12 grown on nutrient broth. By butyl-
toyopearl column chromatography, four major xylano-
lytic enzymes were detected in the culture filtrate (data
not shown), but only one of these xylanolytic enzymes
exhibited transxylosylation activity. Purification of the
oligoxyloside transfer enzyme was optimized, and the
enzyme was separated from the other proteins by
phenyl-toyopearl and sephadex G-75 chromatography.
After gel filtration column chromatography, a single
protein that coincided with the peak of enzymatic
activity was obtained. This protocol produced a 7.7-fold
purification of the oligoxyloside transfer enzyme from
the culture supernatant, with an yield of 3.3% of total
Production of oligoxyloside transfer enzyme in the
culture filtrate
The growth of Bacillus sp. KT12 on culture medium
containing different carbon sources and the production

2290
K. CHIKU et al.
Origin
160
140
120
100
80
(kDa)
97.2
66.4
45.0
29.0
60
40
20
21 kDa
0
20.1
14.3
1
3
5
7
9
11
pH
Fig. 3. Effect of pH on Enzymatic Activities.
Transxylosylation (filled symbols) and hydrolytic activity (open
symbols) were measured under standard assay conditions in the
following buffers: closed circle, 50 mM KCl-HCl buffer ( or
,
Front
pH 1.3); 50 mM sodium citrate buffer ( or , pH 3.0 to 5.0); 50 mM
sodium acetate buffer ( or , pH 5.0); 50 mM sodium phosphate
buffer ( or , pH 6.0 to 8.4); 50 mM Tris–HCl ( or , pH 7.0 to
9.0); and 50 mM sodium bicarbonate buffer ( or , pH 11.0).
Line 1
Line 2
Fig. 2. SDS–PAGE of the Purified Xylanolytic Enzyme from
Bacillus sp. KT12.
Electrophoresis of the denatured enzyme was conducted on a 14%
SDS polyacrylamide gel. Line 1 shows protein standards, including
phosphorylase b (94.0 kDa), bovine serum albumin (66.4 kDa),
ovalbumin (45.0 kDa), carbonic anhydrase (29.0 kDa), soybean
trypsin inhibitor (20.1 kDa), and lysozyme (14.3 kDa). Line 2 shows
the purified enzyme.
Table 4. Effect of Various Reagents on the Activity of the
Xylanolytic Enzyme from Bacillus sp. KT12
Relative enzyme activity (%)
Transxylosylation activity Hydrolysis activity
Ion added
(20 mM)
None
CaCl2
MnCl2
BaCl2
CoCl2
MgCl2
ZnCl2
CuCl2
FeCl2
EDTA
100
121
128
127
124
103
99
100
0
activity, 20.1 U/mg protein of transxylosylation activity,
and 1.47 mg of total protein. The enzyme produced a
single protein band corresponding to a molecular weight
of approximately 20 kDa as determined by SDS–PAGE
(Fig. 2) and HPLC, and a pI of 7.5 as determined by
IEF-PAGE. The first 12 N-terminal residues of the
oligoxyloside transfer enzyme were identified as Ala-
Gly-Thr-Asp-Tyr-Trp-Gln-Asn-Trp-Thr-Asp-Gly- by N-
terminal amino acid sequencing, and complete identity
of the enzyme was observed with the family 11 glycosyl
hydrolase (endo-1,4-ꢀ-xylanhydrolase) from certain
bacteria: Bacillus sp. (DDBJ accession nos. AY170624
and AAZ17392), and Paenibacillus macerans (DDBJ
accession no. AAZ17386).21) The oligoxyloside transfer
enzyme from Bacillus sp. KT12 possessed N-terminal
peculiar sequences of xylanase from a number of
Bacillus spp., and thus was assigned to the family 11
enzyme group.
29
25
3
40
0
135
235
96
45
15
0
After the enzyme was preincubated at 4 ꢀC for 24 h with 20 mM of each
compound, the assay was carried out under standard assay conditions.
observed in pH range 4–11 (Fig. 3). Thermal stability
testing showed that transxylosylation and hydrolysis
activities decreased markedly when the temperature
increased to more than 50 ꢀC. Transxylosylation and
hydrolysis activities were optimal at temperatures of
60 ꢀC and 40 ꢀC respectively (data not shown). The
effects of metal ions and EDTA on the transxylosylation
and hydrolysis activities of the enzyme were determined
(Table 4). Transxylosylation activity was strongly acti-
vated by Fe2þ but was unaffected by other metal ions.
On the other hand, hydrolysis was inhibited by all the
selected metal ions and by EDTA.
Influence of temperature, pH, and chemical com-
pounds on enzymatic activities
The effects of temperature, pH, and chemical com-
pounds on the purified oligoxyloside transfer enzyme
were examined. The enzyme had high stability at pH 3–
11 after 24 h of incubation. Moreover, more than 60%
activity was maintained after 24 h of incubation at
pH 1.3. The optimum pH for hydrolysis activity was 11,
whereas a near constant transxylosylation activity was
Transglycosylation of xylan and catechols by purified
xylanase
Transxylosylation of catechol with purified xylano-
lytic enzyme (100 mU of transxylosylation activity)

Polyphenol-Specific Transxylosylation of Family 11 Xylanase
2291
Xylosyl acceptor specificity of transxylosylation
reactions
16
14
12
10
8
Acceptor specificity was studied by incubating the
purified enzyme with various phenolic and alcoholic
compounds as xylosyl acceptors at a shorter (60 min) and
a longer reaction time (24 h). The products were
distinguished from the hydrolysis products by their
higher Rf values and UV absorption at 254 nm. Catechol,
hydroquinone, pyrogallol, gallic acid, and catechin were
strongly xylosylated. Resorcinol, hydroxyl-hydroqui-
none, phloroglucinol, caffeic acid, ascorbic acid, and
4-methoxyphenol were weakly xylosylated. Coumarin,
o-nitrophenol, coumarin, vanillin, methanol, ethanol,
ethylene glycol, and glycerol were not detected by TLC
(Table 5).
6
4
2
0
0
6
12 18 24 30 36 42 48
Reaction time (h)
Fig. 4. Course of Transxylosylation to Catechol Catalyzed by the
Purified Enzyme.
Discussion
A reaction mixture (2 ml) containing 5% of xylan, 5% of catechol,
and the purified enzyme (100 mU of transxylosylation activity) in
100 mM acetate buffer (pH 5.0) was incubated at 40 ꢀC, and the
2-hydroxyphenyl xylosides produced were analyzed by HPLC.
The symbols used are as follows: , CX2; , CX3; , CX4.
There have been a number of reports on the glyco-
sylation of fatty alcohols using xylanolytic enzymes
aimed at providing material for the food and pharma-
ceutical industries.5,6,9,10,22–29) Our group has further
focused on various surface-active alkyl ꢀ-xylosides
and has reported their efficient synthesis using the
two-phased transxylosylation reaction with xylanolytic
enzyme in high yield.27) In contrast, there is little
information on the synthesis of phenolic xylosides using
xylosidase.5,15) Further, the acceptor specificity of these
xylanolytic enzymes and the precise mechanism of
transglycosylation are not yet fully understood. In this
study, we isolated an oligoxyloside transfer enzyme
from Bacillus sp. KT12, and its xylosyl acceptor
specificity was limited to polyphenols. There have been
no reports of a family 11 xylanase having polyphenol-
specific transxylosylation capability.
occurred in 2.0 ml of 100 mM sodium acetate buffer
(pH 5.0) with the addition of various chemical com-
pounds: 20 mM metal (CaCl2 or FeCl2) and 20 mM
EDTA. These reaction mixtures were incubated at
40 ꢀC for 0.50–48 h, and the time course of the
synthesis yield of CX2-4 using the purified enzyme
was periodically analyzed by HPLC (Fig. 4). After 8 h
of incubation, the yields of CX2, CX3, and CX4 were
12, 5.5, and 0.28 mM respectively, and the molar
conversion yield based on the amount of catechol
supplied (transfer rate) reached 4.2%. In the reaction
mixture with FeCl2, the final concentration of CX2-4
greatly increased. The maximum yields of CX2, CX3,
and CX4 were 35, 2.9, and 0.50 mM respectively, and
the transfer rate reached 8.5% at 24 h. The other metals
and EDTA had insignificant effects on the final
The reaction mechanism of transglycosylation has
been explained in terms of a double-displacement
reaction involving a glycosyl enzyme intermediate,
similar to the mechanism of glycosidases,8) but this
oligoxyloside transfer enzyme requires two or more
accumulation of CX2-4
.
Table 5. Xylosyl Acceptor Specificity of the Xylanolytic Enzyme from Bacillus sp. KT12 for Various Phenolic and Alcoholic Compounds
Acceptor
Transxylosylation
Acceptor
Transxylosylation
+
Phenolic hydroxyl groups
Phenol
DOPA
Trihydric phenols
Pyrogallol
ꢂ
+
ꢂ
ꢂ
ꢂ
4-Methoxyphenol
Guaiacol
++
+
Hydroxy-hydroquinon
Phloroglucinol
Gallic acid
o-Nitrophenol
Vanillin
+
++
Dihydric phenols
Catechol
Polyhydric phenols
Catechin
++
++
+
++
Hydroquinone
Resorcinol
Alcoholic hydroxyl groups
Methanol
Ethanol
ꢂ
ꢂ
ꢂ
ꢂ
Caffeic acid
Ascorbic acid
Dopamine
+
+
Ethylene grecol
Grecerol
+
TLC detection of transxylosylation products was strong (++), weak (+), or not detected (ꢂ). Xylosylated products of the phenolic compounds were distinguished
from the hydrolysis products by their larger Rf values and UV absorption at 254 nm.

2292
K. CHIKU et al.
adjacent hydroxyl groups on the aromatic ring for
more effective transxylosylation. These results suggest
that transxylosylation using the enzyme is relatively
limited as compared with the transxylosylation (solvo-
lytic) reaction toward alcohols using other reported
xylanases,9,10) and the transglycosylation reaction toward
alcohols and phenols using xylanase from Hypsizigus
marmoreus30) and galactanase from B. subtilis31) and
P. citrinum.32) Similarly, Nishimura et al.33) reported
acceptor specificity during transglucosylation toward
polyphenols using B. subtilis X-23 amylase. These
enzymes might recognize the hydroxyl group of poly-
phenols by the active site. Possibly, one hydroxyl group
is required to interact with the enzyme to ensure the
proper acceptor orientation in the active site necessary
for the second hydroxyl group to react with the C1 atom
of xylose. Molecules with only one hydroxyl group
interacting with the enzyme do not have a second
hydroxyl group available for transxylosylation. This
xylanolytic enzyme is a key enzyme for understanding
transxylosylation with polyphenols.
improving the reactivity of the catechol and stabilizing
the generated 2-hydroxyphenyl oligoxylosides.
Here we report that a novel and specific oligoxyloside
transfer reaction to polyphenols was catalyzed by a
xylanolytic enzyme from Bacillus sp. KT12. Trans-
xylosylation using the enzyme enabled direct synthesis
from xylan and produced polyphenyl ꢀ-oligoxylosides
with a high degree of polymerization. We expect these
oligoxylosides to exhibit biological activities. Studies of
the biological activities of the synthesized polyphenyl
ꢀ-oligoxylosides are in progress and will be reported at a
later stage.
Acknowledgments
We thank Professor Akikazu Ando, Professor
Yoshihiro Nishida, Dr. Akihiro Saito, and Dr. Hirofumi
Dohi of Chiba University for valuable discussion and
comments on the manuscript.
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