ORIGINAL ART ICLE

Molecular cloning, nucleotide sequence and

characteristics of a xylanase gene (xynA) from
Ruminococcus albus 7
Mutsumi NAKAMURA,1 Takafumi NAGAMINE,1 Akio TAKENAKA,2 Rustem I. AMINOV,3 Koretsugu OGATA,1
Kiyoshi TAJIMA,1 Hiroki MATSUI,1 Yoshimi BENNO1,3 and Hisao ITABASHI5
1

Rumen Microbiology Research Team, STAFF-Institute, Tsukuba-shi, Japan, 2Tohoku National Agricultural
Experiment Station, Morioka-shi, Japan, 3Department of Animal Sciences, University of Illinois at UrbanaChampaign, Illinois, USA, 4Japan Collection of Microorganisms, RIKEN, Wako-shi, Japan and 5Tokyo University of
Agriculture and Technology, Fuchu-shi, Japan

ABSTRACT
A 2.6-kb DNA fragment encoding a xylanase gene (xynA) was cloned from the rumen hemicellulolytic bacterium
Ruminococcus albus 7. The deduced primary structure of the protein (XynA) was divided into a signal peptide region and
3 domains. Domain A was identified as a family 11 (G) catalytic domain, but one amino acid residue was replaced by
another in an active site signature 1 of family 11. Domain B is a stabilizing domain for the catalytic domains of families 10
and 11. Deletion of domain B reduced stability of the xylanase at high temperature and at high and low pH. Domain B may
be useful for protein engineering of xylanase. Domain C has sequence similarity to deacetylases and NodB proteins.

KEYWORDS: fiber digestion, hemicellulose, rumen bacteria, Ruminococcus albus, xylanase gene.

INTRODUCTION

We describe the nucleotide sequence and characterization of a new xylanase gene (xynA gene) isolated
from R. albus.

Hemicellulose is the second most abundant structural

polysaccharide in plant cell walls. The largest hemicellulose component, xylan, is a highly branched heteropolymer with acetyl, arabinosyl and glucuronosyl
side chains. Xylan surrounds cellulose microfibrils and
links with lignin in the cell wall. These chemical and
physical complexities of xylan limit the overall digestion of plant cell walls by microorganisms.
Ruminococcus albus is found at high density in the
rumen and appears to be an active fiber degrader in a
wide range of diets. Several cellulase genes from R.
albus have been cloned and characterized (Ohmiya et
al. 1988; Poole et al. 1990; Karita et al. 1993; Takenaka
et al. 1993; Ohara et al. 2000), and R. albus has been
shown to have high hemicellulolytic activity (BenGhedalia et al. 1993). A better understanding of the
xylanase gene of R. albus would provide useful information not only on hemicellulose digestion but also
cell wall digestion in the rumen.

Animal Science Journal (2002) 73, 347352

MATERIALS AND METHODS

Strains and cloning of the xylanase gene
Ruminococcus albus 7 (ATCC 27210) was obtained from
the American Type Culture Collection (Manassa, CO,
USA), and cultivated in RGC medium (Scott &
Dehority 1965) anaerobically at 37C. Chromosomal
DNA was isolated from R. albus cells by the reported
method (Howard & White 1988). The DNA was partially digested with Sau3 AI, ligated into pBluescript
Correspondence: Takafumi Nagamine, Institute of Livestock
Industrys Environmental Technology, Nishigou-mura,
Fukushima-ken 9618061, Japan. (Email: nagt@shirakawa.
ne.jp)
Received 25 December 2001; accepted for publication 7 May
2002.

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M. NAKAMURA et al.

SK+ (Stratagene, La Jolla, CA, USA) at the BamH I

cutting site, and employed for transformation of E. coli
JM109 (ToYoBo, Japan). The genomic library was
screened for digestion of 4-o-methyl-D-glucurono-Dxylan-remazol Brilliant Blue R (RBB-xylan; Nacalai
Tesque, Japan) by the reported method (Sipat et al.
1987).

to temperatures of 30, 40, 50, 60, 70 or 80C for

10 min. After that the xylanase activities were assayed
with oat spelt xylan at 37C and pH 6.7. Activities
were expressed as a percentage of that recorded without heat treatment (4C).

DNA sequencing and computer analysis

The genomic library was screened by a hydrolyzing

activity assay of RBB-xylan. Eight clones with RBBxylan-degrading activity were isolated from approximately 5000 clones in the genomic library, and then
four transformants maintained the recombinant plasmid. Two transformants had strong activity and the
other two had weak activity. Plasmids in two strongly
active transformants had the same restriction enzyme
map patterns and the same inserted DNA fragment
size (2.6 kb). One of the plasmids, pRA16, was chosen
for further analysis and the encoding gene was named
xynA. Two plasmids in the weakly active transformants encoded the different open reading frames
(ORF), which were designated as xynB and xynC. The
xynB (3948-bp) and xynC (6,654-bp) sequences have
been deposited in the GenBank database under accession numbers AB057588 and AB057589, respectively.
Substrate specificity of the XynA protein was examined on a range of substrates (Table 1). This xylanase
hydrolyzed oat spelt xylan and birchwood xylan, but
did not hydrolyze other glucans. XynA degraded pnitrophenyl (pNP) -b-D-xylopyranoside and pNP-b-Dgalactopyranoside. Although the enzyme hydrolyzed
pNP-b-D-glucopyranoside, the activity was much
lower than in the other pNP-derived substrates used
in this study. The enzyme released RBB from RBBstarch, but the activity was low. These low activities

RESULTS AND DISCUSSION

DNA sequencing was performed with an ABI 373

Sequencer (Perkin-Elmer Applied Biosystems, USA).
The analyzes of DNA and amino acid sequences
were performed by GENETYX-Mac 9.0 (Software
Development Co. Ltd, Japan) and GeneWorks 2.5
(Oxford Molecular Group Inc., USA). Homology
search was carried out by BLAST. The signature of the
amino acid was scanned by Prosite Protein Site and
Patterns Database.

Deletion mutants
The mutants deleted from the C-terminal side of
XynA were constructed from the R. albus chromosome
with a TA Cloning Kit (Invitrogen Co., USA) after PCR
amplification between the nucleotide positions shown
in Fig. 2a. The sequences of the mutants were verified
by DNA sequencing.

Enzyme assay
The crude enzyme solution was prepared by ultrasonication of transformant cultures and mixed with
substrate; oat spelt xylan (Nacalai Tesque), birchwood
xylan (Sigma Chemical Co., St Louis, MO, USA), carboxymethylcellulose (CMC; Nacalai Tesque), lichenan
(Sigma) laminarin (Nacalai Tesque), p-nitrophenyl
compounds (Nacalai Tesque), RBB-xylan and RBBstarch (Nacalai Tesque). The reducing sugars released
from native substrates were measured by the
Somogyi-Nelson method. The p-nitrophenol released
from p-nitrophenyl substrates was measured spectrophotometrically at 400 nm. One unit of enzyme
activity was defined as 1 mmol glucose-equivalent of
reducing sugar, 1 mmol p-nitrophenyl, or 1 mg RBB,
liberated per minute by 1 mL of culture. To investigate
the effect of pH on the xylanase activity, oat spelt
xylan was dissolved in 100 mmol/L acetic acid
buffer (pH 4.05.6), 100 mmol/L sodium phosphate
buffer (pH 5.77.7), and 50 mmol/L tris-HCl buffer
(pH 7.28.8). The xylanase activity was assayed at
37C for 10 min and expressed as a percentage of that
recorded at pH 6.5. For the effect of heat treatment on
the xylanase activity, enzyme solutions were exposed

Animal Science Journal (2002) 73, 347352

Table 1 Substrate specificity of XynA of Ruminococcus albus

Substrate

Enzyme activity

Oat spelt xylan

Birchwood xylan
Carboxymethylcellulase
Lichenan
Laminarin
p-nitrophenyl-b-D-xylopyranoside
p-nitrophenyl-b-D-glucopyranoside
p-nitrophenyl-b-D-galactopyranoside
RBB-xylan
Remazol Brilliant Blue-starch

23.12
6.95
ND
ND
ND
4.87
0.20
9.49
4.13
0.21

Enzyme activity was defined as 1 mmol glucose equivalent of

reducing sugar, 1 mmol p-nitrophenyl or 1 mg remazol Brilliant Blue
liberated per min by 1 mL of culture. ND, not detected. RBB-xylan,
4-o-methyl-D-glucurono-D-xylan-remazol Brilliant Blue R.

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Xylanase gene from R. albus

may be related to E. coli enzymes in the crude enzyme

solution.
The optimum temperature (Fig. 1), optimum pH
(Fig. 2b; RA16), and thermostability (Fig. 2c; RA16)
were characterized on oat spelt xylan. The optimum
temperature and the optimum pH were 50C and 6.5,
respectively. Relative activity was maintained at
greater than 50% in the pH range of 5.68.8. The
xylanase lost 80% of its activity upon exposure to
70C for 10 min.
The inserted DNA fragment of pRA16 was completely sequenced. This 2,558-bp DNA sequence
containing xynA has been deposited in the GenBank
database under accession number U43089. GENETYXMac estimated the promoter sequence for E. coli to
be at nucleotide position 315 in the 35 region and
at nucleotide position 343 in the 10 region. In the
downstream region, a putative SD sequence (GAGGT)

Fig. 1 Effect of temperature on the xylanase activity of

XynA. Xylanase activity was assayed with oat spelt xylan at
pH 6.7 for 10 min.

Fig. 2 Effect of pH and heat treatment on the xylanase activity of XynA and deletion mutants. (a) Construction of deletion
mutants. DNA fragments were amplified between nucleotide position 355 and 3-ends of each domain coding region by PCR
amplification and cloned. (b) Effect of pH on xylanase activity. (c) Effect of heat treatment on xylanase activity. With () RA16,
() RA16a, () RA16b and () RA16c.

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M. NAKAMURA et al.

Fig. 3 Amino acid sequence

alignment of domain B of XynA
(U43089) and similar regions of
xylanases of R. flavefaciens
(AJ272430, Z35226 and S61204),
Ruminococcus sp. (Z49970),
Thermotoga maritima (AE001693),
Celdicellulosiruptor sp. (AF036923),
Thermotoga neapolitana (Z46945),
Clostridium thermocellum (X83269).
Residues conserved in all sequences
are boxed, and shading indicates
residues conserved within the ranges
of the following strong groups: STA,
NEQK, NHQK, NDEQ, QHRK, MILV,
MILF, HY, FYW. Dashes have been
inserted to maximize alignment. The
arrow indicates the conserved
phenylalanine residue used in
Table 2.

was found at nucleotide position 486 and an ORF was

followed with initiation codon ATG at nucleotide position 501. The putative protein XynA is composed of
680 amino acid residues. The 28 amino acid sequence
of XynA at the N-terminal consists of three basic
amino acid residues, a hydrophobic region of 16
amino acid residues and a hydrophilic region, in that
order. The amino acid sequence seems to have the
typical pattern of a signal peptide sequence. The
following region after the signal peptide region
was divided into three domains, A, B, and C. The
domains and the signal peptide sequence were connected by highly hydrophilic regions, the amino acid
positions of which were 2954, 263291 and 434
474, respectively.
Domain A had a high similarity to many xylanase
genes in GenBank: Clostridium stercorarium (77%
positive of amino acid homology in BLAST:
GenBank accession number is D13325), Bacillus
sp. (76%: AB029319), Clostridium thermocellum (75%:
AB010958), Clostridium acetobutylicum (76%: M31726).
Domain A had consensus sequences for the glycoside hydrolase family 11 catalytic domain. The

Animal Science Journal (2002) 73, 347352

amino acid sequence in XynA, PLVEFYIVESW, starting at amino acid position 144 matched the active site
signature 1 of family 11 ([PSA]-[LQ]-x-E-Y-Y[LIVM](2)-[DE]-x-[FYWHN]), although the 5th amino
acid residue was substituted with phenylalanine
instead of tyrosine. The amino acid sequence,
LNIEGYQSNGQA at position 241 matched the
active site signature 2 ([LIVMF]-x(2)-E-[AG]-[YWG][QRFGS]-[SG]-[STAN]-G-x-[SAF]).
Domain B, adjoining the catalytic domain, was
similar to the stabilizing domain of xylanases in R.
flavefaciens. The xynE (accession number AJ272430)
was 65% positive (Aurilia et al. 2000). The xynB
(Z35226) and xynD (S61204) were 64% and 61%
positive, respectively (Zhang et al. 1994). The other
xylanases of Ruminococcus sp. (62%: Z49970),
Thermotoga maritima (52%: AE001693), T. neapolitana
(52%: Z46945), Caldicellulosiruptor sp. (55%:
AF036923) and Clostridium thermocellum (49%:
X83269) also had a similar sequence to Domain B.
Many amino acid residues were conserved (Fig. 3). In
almost all genes, the distance was similar (244278
amino acid residue) between the glutamic acid residue

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Xylanase gene from R. albus

Table 2 Distance between catalytic domain and stabilizing domain

Accession

U43089
AJ272430
Z35226
Z49970
S61204
AE001693
AF036923
Z46945
X83269

Amino acid position

Catalytic proton donor (E)

Stabilizing domain (F)

Distance number (EF)

147 (Family 111)

121 (Family 111)
124 (Family 111)
123 (Family 111)
124 (Family 111)
603 (Family 10)
770 (Family 10)
604 (Family 10)
460 (Family 10)

425
391
394
400
394
342
492
338
704

278
270
270
277
270
261
278
266
244

E, Amino acid position of glutamic acid residue acting as proton donor.

F, Amino acid position of conserved phenylalanine residue at the C-terminal side indicated in Fig. 3.

charides and various xylans (Charnock et al. 2000).

However, the domain B of XynA in R. albus did not
bind to xylans (data not shown), which was in accordance with that of xynB in R. flavefaciens (Zhang et al.
1994).
Domain C had a similarity to the deacetylases of
Streptococcus pyogenes (52%: AE006574), the xylanase
of Bacillus subtilis (53%: Z99110), the deacetylase of S.
pneumoniae (50%: AE008503) and the deacetylase of
Streptomvces coelicolor (51%: Al034492). Domain C was
also similar to the NodB proteins that are produced in
all rhizobia and play a part in the synthesis of Nod
factor, which acts as a host-specific symbiotic signal
during infection of and nodule formation in legume
roots (Mergaert et al. 1997), and deacetylates the
non-reducing end of the chitooligosaccharide (John
et al. 1993). Xylan in forage is acetylated at more than
50% of the xylose residues at the C2 or C3 positions
(Chesson et al. 1983). The acetylation seems to impede
fiber digestion in the rumen (Morris & Bacon 1977).
Domain C may also contribute to improving accessibility to substrates of xylanases by deacetylation, so that
xylan degradation proceeds.
Although R. albus is a hemicellulolytic bacterium in
the rumen, little information about its xylanase gene
has been reported. We cloned, sequenced and partly
characterized the xylanase gene from R. albus. The
xylanase A gene encodes a protein consisting of three
domains. Domain A has a consensus sequence for
the glycoside hydrolase family 11 catalytic domain.
Domain B is a stabilizer for the catalytic domains and
could be used for protein engineering of xylanase. We
could not determine the function of domain C and
understanding its function would be necessary to
design a high-performance enzyme.

acting as the proton donor in the catalytic domain

and the conserved phenylalanine residue at the Cterminal side of the region (Table 2).
It has been reported that deletion of the thermostabilizing domain in C. thermocellum, which is similar to
domain B of XynA, decreased stability at high temperature (Fontes et al. 1995). Therefore, three deletion
mutants (RA16a, RA16b, RA16c; Fig. 2a) were constructed and examined for pH and thermostability to
clarify the function of domain B. Figure 2b shows the
effect of pH on the xylanase activity. RA16 and RA16a
maintained high activity over a wide range of pH.
Figure 2c shows the effect of exposure to high temperature. The activity of RA16c was completely lost
after exposure to 70C and was lower than the activities of other transformants in the range from 40 and
60C. The activities of RA16b and RA16c were higher
than that of RA16 at 50 and 60C, but lower at 70C.
The activity of RA16a was higher than that of RA16 in
a range from 50 to 70C. These results show that
deletion transformants expressing XynA lacking the
domain B of XynA had reduced stability at high and
low pH and at high temperature.
The inside of the rumen is maintained at approximately 40C and so stability at high temperature is
unnecessary for ruminal bacteria. The feeding of an
easily digestible diet often decreases the pH in the
rumen and ruminal cellulolytic bacteria are sensitive
to low pH (Russell & Dombrowski 1980). Because
R. albus ceases growth at less than pH 6.0 and continues to ferment glucose until pH 5.5 (Thurston et al.
1993), it seems that the domain B enables effective
functioning at lower pH. It was reported that the thermostabilizing domain module X6b of C. thermocellum
had the capacity to bind specifically to xylooligosac-

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M. NAKAMURA et al.

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ACKNOWLEDGMENTS
We are grateful to Professor K. Ohmiya (Mie
University, Japan) and Dr M. Mitsumori (National
Institute of Livestock and Grassland Science, Japan)
for cloning the xylanase gene.
This work was funded by the National Institute of
Livestock and Grassland Science, Japan.

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