Previous Article | Next Article 
Journal of Bacteriology, January 2000, p. 244-247, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
The engL Gene Cluster of
Clostridium cellulovorans Contains a Gene for
Cellulosomal ManA
Yutaka
Tamaru and
Roy H.
Doi*
Section of Molecular and Cellular Biology,
University of California, Davis, California 95616
Received 19 July 1999/Accepted 4 October 1999
 |
ABSTRACT |
A five-gene cluster around the gene in Clostridium
cellulovorans that encodes endoglucanase EngL, which is involved
in plant cell wall degradation, has been cloned and sequenced. As a
result, a mannanase gene, manA, has been found downstream
of engL. The manA gene consists of an open
reading frame with 1,275 nucleotides encoding a protein with 425 amino
acids and a molecular weight of 47,156. ManA has a signal peptide
followed by a duplicated sequence (DS, or dockerin) at its N terminus
and a catalytic domain which belongs to family 5 of the glycosyl
hydrolases and shows high sequence similarity with fungal mannanases,
such as Agaricus bisporus Cel4 (17.3% identity),
Aspergillus aculeatus Man1 (23.7% identity), and
Trichoderma reesei Man1 (22.7% identity). Sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and N-terminal amino acid
sequence analyses of the purified recombinant ManA (rManA) indicated
that the N-terminal region of the rManA contained a DS and was
truncated in Escherichia coli cells. Furthermore, Western
blot analysis indicated that ManA is one of the cellulosomal subunits.
ManA production is repressed by cellobiose.
| |
TEXT |
Clostridium cellulovorans
(ATCC 35296), an anaerobic, mesophilic, and spore-forming bacterium,
utilizes not only cellulose but also xylan, pectin, and several other
carbon sources (18), produces an extracellular cellulolytic
multienzyme complex called the cellulosome (12) with a total
molecular weight of about 1 million, and is capable of hydrolyzing
crystalline cellulose. The C. cellulovorans cellulosome is
comprised of three major subunits that include the scaffolding protein
CbpA (17), the endoglucanase EngE (22), and the
exoglucanase ExgS (13). In addition to these major subunits,
both sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and zymogram analysis indicated the presence of several
minor enzymatic subunits (16). Recently, we have found genes
for two novel enzymatic subunits, mannanase and pectate lyase, lying in
gene clusters of the C. cellulovorans cellulosome (4,
23). Therefore, the identification of the enzymatic subunits
associated with the cellulosome indicated that this enzyme complex can
degrade not only cellulose, xylan, and lichenan (6, 22) but
also mannan and pectin. The present paper provides data that indicate
that a cellulosomal gene cluster (the engL gene cluster) in
which one of the genes codes for a protein homologous to fungal
mannanases exists and that the enzyme encoded by this gene can degrade
mannan. Thus, the versatility of the cellulosome to degrade cell wall
materials in plants is further established.
A C. cellulovorans genomic library constructed in 
ZAPII
(22) was immunoscreened with an anti-P120 (endoglucanase)
antiserum (diluted 1:500) previously prepared from C. cellulovorans and alkaline phosphatase-conjugated goat anti-rabbit
immunoglobulin G secondary antibody (diluted 1:3,000) (Bio-Rad).
Positive clones were further screened for endoglucanase and mannanase
activity by overlaying with 0.7% soft agar containing 0.3%
carboxymethyl cellulose (CMC; low viscosity; Sigma) or 0.25%
glucomannan. Colonies having enzyme activity against CMC or glucomannan
were recognized by the formation of clear haloes on a red background
after staining with 0.1% Congo red and destaining with 1 M NaCl
(1). From approximately 20,000 transformants, four positive
clones were isolated and further characterized. All clones contained a
common 6.8-kb EcoRI insert named pYl-1 (Fig.
1). To determine the coding region of
ManA, various subclones were prepared and determined by the formation
of clear haloes around the colonies with CMC and glucomannan. These
results indicated that the coding region for the manA gene
was on the 3.3-kb BamHI-EcoRI (pYl-3) fragment (Fig. 1).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 1.
Restriction enzyme map of the pYl-1 fragment. The
transformants harboring the plasmids with the appropriate deletion were
transferred to a Luria-Bertani agar plate. After the colonies grew,
agar with 0.3% CMC or 0.25% glucomannan (GM) was poured over the
transformants and enzyme activity against CMC or glucomannan was judged
by the formation of clear haloes around the colonies (+, visible halo;
 , no halo). The genes coding for EngK, EngL, HbpA, ManA, and EngM are
shown at the top. The pin-like marks indicate palindromes.
|
|
Nucleotide sequence of the manA gene.
The
nucleotide sequence analysis revealed that the pYl-1 fragment contained
five different open reading frames (ORFs), i.e., engK-hbpA-engL-manA-engM (Fig. 1). The complete nucleotide
and amino acid sequences of manA and ManA, respectively, are
shown in Fig. 2. Downstream of the
engL gene, the ORF of the manA gene consists of
1,275 nucleotides encoding a protein with 425 amino acids with a
predicted molecular weight of 47,156. The putative initiation codon,
ATG, was preceded at a spacing of 7 bp by a typical gram-positive
ribosome-binding sequence, GGAGG. Upstream of the coding region,
possible promoter sequences, TTGATA for the 35 region and
AATAAG for the 10 region, with a 17-bp spacing between
them (7), were found. A possible transcription terminator that consists of a 13-bp palindrome, corresponding to an mRNA hairpin
loop with a 
G of 11.8 kcal/mol (ca. 49 kJ/mol) (15), was found downstream of the TAA termination codon.
View larger version (54K):
[in this window]
[in a new window]
|
FIG. 2.
Nucleotide and deduced amino acid sequences of
manA and ManA, respectively. The putative promoter (35 and
10) and Shine-Dalgarno (SD) sequences are underlined. Palindrome
sequences are indicated by arrows facing each other. The stop codons
are indicated by asterisks. The DSs, or dockerin, in ManA are boxed and
in white letters. The chemically determined amino acid sequence of ManA
is shaded.
|
|
Amino acid sequence similarity of ManA.
The N-terminal amino
acid sequence of ManA revealed a signal peptide and consensus sequence
(Ala-X-Ala-Ala) (24) in which the predicted cleavage site is
located between position 25 (Ala) and position 26 (Ala). Removal of the
signal peptide yields a mature protein with 400 amino acids and a
molecular weight of 44,528. Interestingly, a duplicated sequence (DS,
or dockerin) was found in the N terminus (Fig. 2). The DSs consisting
of 22-amino-acid repeats are well-conserved in cellulosomal enzymatic
subunits in C. cellulovorans and other
Clostridium species. A homology search revealed that a
catalytic domain (residues 88 to 425) located directly downstream of
the DS belongs to family 5 of the glycosyl hydrolases, which include
large and growing families comprising not only endoglucanases but also
bacterial, fungal, and plant 
-mannanases (5). ManA showed
high sequence similarity with fungal -mannanases such as
Agaricus bisporus Cel4 (17.3% identity) (26),
Aspergillus aculeatus Man1 (23.7% identity) (3),
and Trichoderma reesei Man1 (22.7% identity)
(19). Recently, -mannanases have been classified into two
distinct families, glycosyl hydrolase families 5 and 26 (9).
Clostridium thermocellum CelH, one of the cellulosomal
subunits, contains two catalytic domains belonging to both families 5 and 26 (2). Interestingly, C. cellulovorans ManA
included a catalytic domain homologous to those of fungal mannanases
and a DS, or dockerin, found in several Clostridium spp.,
while ManA from Vibrio sp. had a bacterial mannanase and a
fungal dockerin (21), although both ManAs are from bacteria and belong to the same family of hydrolases, family 5.
Purification and characterization of recombinant ManA.
Recombinant ManA (rManA) was purified from the periplasmic fraction of
Escherichia coli XL1-Blue harboring pYl-3 with the Q
Sepharose Fast Flow column (2.6 by 22 cm; Pharmacia) and Sephacryl S-200 column (2.6 by 75 cm; Pharmacia). After purification and SDS-PAGE, the final preparation yielded a single band and the molecular
weight of the enzyme was estimated to be around 38,000 (Fig.
3A). The N-terminal amino acid sequence
of recombinant ManA (rManA) was determined by automated Edman
degradation with a protein sequencer (model 477; Applied Biosystems) to
be Asp-Lys-Gly-Val-Val-Asp-Asn, which is in perfect agreement with the
deduced amino acid sequence at positions 94 to 100 (Fig. 2). This
result indicated that purified rManA was truncated in E. coli and lacked the N-terminal sequence of 93 amino acids.
Therefore, the molecular weight of purified enzyme estimated by
SDS-PAGE was in good agreement with the value (37,341), excluding the
N-terminal region of ManA, calculated from the deduced amino acid
sequence.
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 3.
Analysis of the purified ManA by SDS-PAGE (A) and
Western blotting (immunoblotting) (B). SDS-PAGE was performed in a
12.5% polyacrylamide gel by the method of Laemmli (11).
After electrophoresis, the protein was transferred onto an Immobilon-P
transfer membrane (Millipore Corp., Bedford, Mass.) by electroblotting.
Western blot (immunoblot) analysis was performed with an anti-ManA
antiserum (diluted 1:500). Lane M, standard markers (Low-Molecular
Weight SDS Calibration Kit; Pharmacia) (phosphorylase b [94
kDa], albumin [67 kDa], ovalbumin [43 kDa], carbonic anhydrase
[30 kDa], trypsin inhibitor [20.1 kDa], and  -lactalbumin [14.4
kDa]); lanes 1, purified ManA; lane 2, ASC supernatant; lane 3, cellobiose supernatant; lane 4, guar gum supernatant; lane 5, ASC
cellulosome; lane 6, cellobiose cellulosome; lane 7, guar gum
cellulosome.
|
|
C. cellulovorans cells were grown in various media
containing different carbon sources to determine whether these carbon
sources
could affect ManA production. Extracellular ManA production by
C. cellulovorans was compared with cultures containing
either
acid swollen cellulose (ASC), cellobiose, or guar gum.
Cellulosomes
from
C. cellulovorans were prepared as
described previously (
14).
Both culture supernatant and
cellulosome fractions were prepared
for each substrate and subjected to
Western blot analysis. As
shown in Fig.
3B, anti-ManA antibody prepared
from the rManA immunoreacted
with all culture fractions and some
cross-reaction of the antibody
was evident with several cellulosome
subunits over 67 kDa (Fig.
3B, lanes 5 to 7). With the supernatant
fractions (lanes 2 to
4), the immunoreactive bands (45 and 32 kDa) were
detected with
the ASC or guar gum supernatant (lanes 2 and 4), while
only the
32-kDa band was detected with the cellobiose supernatant (lane
3). In contrast to the supernatant fractions, only the 45-kDa
band was
detected with the ASC or guar gum cellulosome (lanes
5 and 7), while no
band was detected on the cellobiose cellulosome
(lane 6). On the other
hand, the 32-kDa band was not detected
on all the cellulosome fractions
(lanes 5 to 7) and is presumably
a noncellulosomal protein that
crossreacts with anti-ManA antibody.
The 45-kDa immunoreactive band is
in good agreement with the value
(44,528), excluding the putative
signal peptide, calculated from
the deduced amino acid sequence of
ManA. These results indicated
that the 45-kDa band is identified as
ManA and that ManA is one
of the cellulosomal subunits. Furthermore, it
is likely that ManA
production by
C. cellulovorans can be
repressed by cellobiose
(lanes 3 and 6). Thus, Western blot analysis
indicated that ManA
is one of the cellulosomal subunits and that ManA
production is
repressed by cellobiose. The cellulosome fractions from
ASC and
guar gum contained the 45-kDa immunoreactive band, which is in
good agreement with the molecular mass calculated from the deduced
amino acid sequence of mature ManA (lanes 5 and 7). In addition,
it is
possible that the 32-kDa protein that exists in all the
culture
supernatants may be another noncellulosomal mannanase,
since it
cross-reacts strongly with anti-ManA (lanes 2 to
4).
The optimum pH and temperature of purified ManA for activity were 7.0 and 45°C, respectively. As shown in Table
1, the purified
rManA revealed greatest
activity with glucomannan, followed by
about 80% activity on locust
bean gum, 20% on guar gum, and 10%
on
-mannan, but it did not
hydrolyze CMC. These results indicated
that ManA could not hydrolyze
the
-1,4-cellulosidic linkages
but could only hydrolyze the
-1,4-mannosidic linkages. As shown
in Fig.
4, thin-layer chromatography (TLC)
analysis revealed that
purified ManA cleaved M4 to form M3 and M1, but
it did not completely
hydrolyze it. The main products from M5 were M3
and M1 rather
than M4 and M2. Mannohexose (M6) was completely
hydrolyzed to
produce M4 to M1, but the enzyme did not act on M2 and
M3. ManA
seems to preferentially hydrolyze the
-1,4-mannosidic
linkages
situated at the fourth position, followed by the third
position
from the nonreducing end. Furthermore, the hydrolysis patterns
of ManA on TLC analysis showed that the main products from
mannooligosaccharides
(M4 to M6) produced mostly M1 and M3, followed by
M2 and M4, and
did not act on M2 and M3. Thus, the properties of ManA
differed
from those of EngB (
6), EngE (
22), EngD
(
6,
8), and
EngF (
10) in substrates degraded and
the products that were
formed, although all the enzymes belonged to
family 5.
View larger version (55K):
[in this window]
[in a new window]
|
FIG. 4.
TLC of the hydrolysis products of mannooligosaccharides
with purified ManA. TLC analysis was performed as previously described
(20). Mannooligosaccharides (mannobiose to mannohexose [M2
to M6]) were purchased from Megazyme Ltd. (Sydney, Australia). The
reaction mixture contained 20 µg of purified ManA and 20 µg of each
mannooligosaccharide (mannobiose to mannohexose [M2 to M6]) in 1 mM
sodium acetate buffer (pH 6.0) at 37°C for 16 h.
|
|
Nucleotide sequence accession number.
The nucleotide sequence
data reported in this paper have been submitted to GenBank under
accession no. AF132735.
| |
ACKNOWLEDGMENTS |
We thank T. Araki for providing us with -mannan and glucomannan.
The research was supported in part by grant DE-DDF03-92ER20069 from the
U.S. Department of Energy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Molecular and Cellular Biology, University of California, Davis, CA
95616. Phone: (530) 752-3191. Fax: (530) 752-3085. E-mail:
rhdoi{at}ucdavis.edu.
| |
REFERENCES |
| 1.
|
Ali, B. R. S.,
M. P. M. Romaniec,
G. P. Hazlewood, and R. B. Freedman.
1995.
Characterization of the subunits in an apparently homogeneous subpopulation of Clostridium thermocellum cellulosomes.
Enzyme Microb. Technol.
17:705-711[CrossRef][Medline].
|
| 2.
|
Bayer, E. A.,
L. J. W. Shimon,
Y. Shoham, and R. Lamed.
1998.
Cellulosomes structure and ultrastructure.
J. Struct. Biol.
124:221-234[CrossRef][Medline].
|
| 3.
|
Christgau, S.,
S. Kauppinen,
J. Vind,
L. V. Kofod, and H. Dalbøge.
1994.
Expression cloning, purification and characterization of a -1,4-mannanase from Aspergillus aculeatus.
Biochem. Mol. Biol. Int.
33:917-925[Medline].
|
| 4.
|
Doi, R. H.,
J.-S. Park,
C.-C. Liu,
L. M. Malburg,
Y. Tamaru,
A. Ichi-ishi, and A. Ibrahim.
1998.
Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans.
Extremophiles
2:53-60[CrossRef][Medline].
|
| 5.
|
Ethier, N.,
G. Talbot, and J. Sygusch.
1998.
Gene cloning, DNA sequencing, and expression of thermostable -mannanase from Bacillus stearothermophilus.
Appl. Environ. Microbiol.
64:4428-4432[Abstract/Free Full Text].
|
| 6.
|
Foong, F., and R. H. Doi.
1992.
Characterization and comparison of Clostridium cellulovorans endoglucanase-xylanase EngB and EngD hyperexpressed in Escherichia coli.
J. Bacteriol.
174:1403-1409[Abstract/Free Full Text].
|
| 7.
|
Goldstein, M. A., and R. H. Doi.
1995.
Prokaryotic promoters in biotechnology.
Biotechnol. Annu. Rev.
1:105-128[Medline].
|
| 8.
|
Hamamoto, T.,
F. Foong,
O. Shoseyov, and R. H. Doi.
1992.
Analysis of functional domains of endoglucanases from Clostridium cellulovorans by gene cloning, nucleotide sequencing and chimeric protein construction.
Mol. Gen. Genet.
231:472-479[CrossRef][Medline].
|
| 9.
|
Henrissat, B., and A. Bairoch.
1993.
New families in the classification of glycosyl hydrolases based on amino acid sequence similarities.
Biochem. J.
293:781-788.
|
| 10.
|
Ichi-ishi, A.,
S. Sheweita, and R. H. Doi.
1998.
Characterization of EngF from Clostridium cellulovorans and identification of a novel cellulose binding domain.
Appl. Environ. Microbiol.
64:1086-1090[Abstract/Free Full Text].
|
| 11.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[CrossRef][Medline].
|
| 12.
|
Lamed, R., and E. A. Bayer.
1988.
The cellulosome concept: exocellular and extracellular enzyme factor centers for efficient binding and cellulolysis, p. 101-116.
In
J.-P. Aubert, P. Béguin, and J. Millet (ed.), Biochemistry and genetics of cellulose degradation. Academic Press, Inc., San Diego, Calif.
|
| 13.
|
Liu, C.-C., and R. H. Doi.
1998.
Properties of exgS, a gene for a major subunit of the Clostridium cellulovorans cellulosome.
Gene
211:39-47[CrossRef][Medline].
|
| 14.
|
Matano, Y.,
J.-S. Park,
M. A. Goldstein, and R. H. Doi.
1994.
Cellulose promotes extracellular assembly of Clostridium cellulovorans cellulosomes.
J. Bacteriol.
176:6952-6956[Abstract/Free Full Text].
|
| 15.
|
Rosenberg, M., and D. Court.
1979.
Regulatory sequences involved in the promotion and termination of RNA transcription.
Annu. Rev. Genet.
13:319-353[CrossRef][Medline].
|
| 16.
|
Shoseyov, O., and R. H. Doi.
1990.
Essential 170 kDa subunit for degradation of crystalline cellulose of Clostridium cellulovorans cellulase.
Proc. Natl. Acad. Sci. USA
87:2192-2195[Abstract/Free Full Text].
|
| 17.
|
Shoseyov, O.,
M. Takagi,
M. Goldstein, and R. H. Doi.
1992.
Primary sequence analysis of Clostridium cellulovorans cellulose binding protein A (CbpA).
Proc. Natl. Acad. Sci. USA
89:3483-3487[Abstract/Free Full Text].
|
| 18.
|
Sleat, R.,
R. A. Mah, and R. Robinson.
1984.
Isolation and characterization of an anaerobic, cellulolytic bacterium, Clostridium cellulovorans sp. nov.
Appl. Environ. Microbiol.
48:88-93[Abstract/Free Full Text].
|
| 19.
|
Stålbrand, H.,
A. Saloheimo,
J. Vehmaanperä,
B. Henrissat, and M. Penttilä.
1995.
Cloning and expression in Saccharomyces cerevisiae of a Trichoderma reesei -mannanase gene containing a cellulose binding domain.
Appl. Environ. Microbiol.
61:1090-1097[Abstract].
|
| 20.
|
Tamaru, Y.,
T. Araki,
H. Amagoi,
H. Mori, and T. Morishita.
1995.
Purification and characterization of an extracellular -1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.
Appl. Environ. Microbiol.
61:4454-4458[Abstract].
|
| 21.
|
Tamaru, Y.,
T. Araki,
T. Morishita,
T. Kimura,
K. Sakka, and K. Ohmiya.
1997.
Cloning, DNA sequencing, and expression of the -1,4-mannanase from a marine bacterium, Vibrio sp. strain MA-138.
J. Ferment. Bioeng.
83:201-205[CrossRef].
|
| 22.
|
Tamaru, Y., and R. H. Doi.
1999.
Three surface layer homology domains at the N terminus of the Clostridium cellulovorans major cellulosomal subunit EngE.
J. Bacteriol.
181:3270-3276[Abstract/Free Full Text].
|
| 23.
|
Tamaru, Y.,
C.-C. Liu,
L. Malburg, and R. H. Doi.
1999.
The Clostridium cellulovorans cellulosome and non-cellulosomal cellulases, p. 488-494.
In
K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita, and T. Kimura (ed.), Genetics, biochemistry and ecology of cellulose degradation. Uni Publishers, Tokyo, Japan.
|
| 24.
|
von Heijne, G.
1985.
Signal sequences. The limits of variation.
J. Mol. Biol.
184:99-105[CrossRef][Medline].
|
| 25.
|
Wood, W. A., and K. M. Bhat.
1988.
Methods for measuring cellulase activities.
Methods Enzymol.
160:87-112.
|
| 26.
|
Yagüe, E.,
M. Mehak-Zunic,
L. Morgan,
D. A. Wood, and C. F. Thurston.
1997.
Expression of CEL2 and CEL4, two proteins from Agaricus bisporus with similarity to fungal cellobiohydrolase I and -mannanase, respectively, is regulated by the carbon source.
Microbiology
143:239-244[Abstract/Free Full Text].
|
Journal of Bacteriology, January 2000, p. 244-247, Vol. 182, No. 1
0021-9193/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Han, S. O., Yukawa, H., Inui, M., Doi, R. H.
(2005). Molecular Cloning and Transcriptional and Expression Analysis of engO, Encoding a New Noncellulosomal Family 9 Enzyme, from Clostridium cellulovorans. J. Bacteriol.
187: 4884-4889
[Abstract]
[Full Text]
-
Koukiekolo, R., Cho, H.-Y., Kosugi, A., Inui, M., Yukawa, H., Doi, R. H.
(2005). Degradation of Corn Fiber by Clostridium cellulovorans Cellulases and Hemicellulases and Contribution of Scaffolding Protein CbpA. Appl. Environ. Microbiol.
71: 3504-3511
[Abstract]
[Full Text]
-
Han, S. O., Yukawa, H., Inui, M., Doi, R. H.
(2005). Effect of carbon source on the cellulosomal subpopulations of Clostridium cellulovorans. Microbiology
151: 1491-1497
[Abstract]
[Full Text]
-
Demain, A. L., Newcomb, M., Wu, J. H. D.
(2005). Cellulase, Clostridia, and Ethanol. Microbiol. Mol. Biol. Rev.
69: 124-154
[Abstract]
[Full Text]
-
Han, S. O., Yukawa, H., Inui, M., Doi, R. H.
(2004). Isolation and Expression of the xynB Gene and Its Product, XynB, a Consistent Component of the Clostridium cellulovorans Cellulosome. J. Bacteriol.
186: 8347-8355
[Abstract]
[Full Text]
-
Perret, S., Belaich, A., Fierobe, H.-P., Belaich, J.-P., Tardif, C.
(2004). Towards Designer Cellulosomes in Clostridia: Mannanase Enrichment of the Cellulosomes Produced by Clostridium cellulolyticum. J. Bacteriol.
186: 6544-6552
[Abstract]
[Full Text]
-
Cho, H.-Y., Yukawa, H., Inui, M., Doi, R. H., Wong, S.-L.
(2004). Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800. Appl. Environ. Microbiol.
70: 5704-5707
[Abstract]
[Full Text]
-
Han, S. O., Cho, H.-Y., Yukawa, H., Inui, M., Doi, R. H.
(2004). Regulation of Expression of Cellulosomes and Noncellulosomal (Hemi)Cellulolytic Enzymes in Clostridium cellulovorans during Growth on Different Carbon Sources. J. Bacteriol.
186: 4218-4227
[Abstract]
[Full Text]
-
Han, S. O., Yukawa, H., Inui, M., Doi, R. H.
(2003). Regulation of Expression of Cellulosomal Cellulase and Hemicellulase Genes in Clostridium cellulovorans. J. Bacteriol.
185: 6067-6075
[Abstract]
[Full Text]
-
Han, S. O., Yukawa, H., Inui, M., Doi, R. H.
(2003). Transcription of Clostridium cellulovorans Cellulosomal Cellulase and Hemicellulase Genes. J. Bacteriol.
185: 2520-2527
[Abstract]
[Full Text]
-
Murashima, K., Kosugi, A., Doi, R. H.
(2003). Synergistic Effects of Cellulosomal Xylanase and Cellulases from Clostridium cellulovorans on Plant Cell Wall Degradation. J. Bacteriol.
185: 1518-1524
[Abstract]
[Full Text]
-
Tamaru, Y., Ui, S., Murashima, K., Kosugi, A., Chan, H., Doi, R. H., Liu, B.
(2002). Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans. Appl. Environ. Microbiol.
68: 2614-2618
[Abstract]
[Full Text]
-
Murashima, K., Kosugi, A., Doi, R. H.
(2002). Determination of Subunit Composition of Clostridium cellulovorans Cellulosomes That Degrade Plant Cell Walls. Appl. Environ. Microbiol.
68: 1610-1615
[Abstract]
[Full Text]
-
Murashima, K., Chen, C.-L., Kosugi, A., Tamaru, Y., Doi, R. H., Wong, S.-L.
(2002). Heterologous Production of Clostridium cellulovorans engB, Using Protease-Deficient Bacillus subtilis, and Preparation of Active Recombinant Cellulosomes. J. Bacteriol.
184: 76-81
[Abstract]
[Full Text]
-
Kosugi, A., Murashima, K., Doi, R. H.
(2001). Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates. J. Bacteriol.
183: 7037-7043
[Abstract]
[Full Text]
-
Tamaru, Y., Doi, R. H.
(2001). Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome. Proc. Natl. Acad. Sci. USA
10.1073/pnas.071045598v1
[Abstract]
[Full Text]
-
Tamaru, Y., Karita, S., Ibrahim, A., Chan, H., Doi, R. H.
(2000). A Large Gene Cluster for the Clostridium cellulovorans Cellulosome. J. Bacteriol.
182: 5906-5910
[Abstract]
[Full Text]
-
Tamaru, Y., Doi, R. H.
(2001). Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome. Proc. Natl. Acad. Sci. USA
98: 4125-4129
[Abstract]
[Full Text]
Top
Abstract
Text
