Note: Descriptions are shown in the official language in which they were submitted.
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Xylanase, oligonucleotidic sequence
encoding it and its uses
The present invention relates to a xylanase and to a nucleotidic
sequence encoding it.
It also relates to the use of this enzyme in the bleaching of
paper pulp and the preparation of xyiose or of xylo-oligosaccharides
from plant raw materials, in particular.
Varied uses have been proposed for xylanases in the
biotechnology field, especially in the foodstuffs field (Biely, Trends
Biotechnol 3 (11): 286-290, 1985), in the paper industry (Mora et al., J.
Wood Chem. Technol, 6: 147-165, 1986) or in the production of
chemical compounds from hemicellulose (Reilly P.J., 1981, Xylanases:
structure and function in trends in the biology of fermentations for fuels
and chemicals. A.J. Hollaender (Ed), Plenum, New York).
The technical feasibility of such applications has been
assessed chiefly using enzymes produced by mesophilic fungi. However,
such applications could be facilitated by the use of fungi possessing
better temperature stability.
Various bacteria and enzymes are known for the production of
xylanases ( see, in particular, Wong et al., Microbiological Reviews, 52,
No 3 305 317, 1988). Hitherto, the highest yields of enzymes have been
obtained from fungi (Yu et al. Enzymo Microb. Technol. 9: 16-24, 1987).
However, hyperproductive strains of Bacillus have already been
described (Okazaki et al. Appl. Microbiol. Biotechnology, 19: 335-340,
1984; Okazaki et al., Agric. Biol. Chem. 49, 2033-2039, 1985). Such
thermophilic species of Bacillus which degrade xylan can be good
CONFIRMATION COPY
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candidates for the industrial production of xylanases on account of their
high growth rate and of their genetics being weli known.
The xylanases isolated by Okazaki et al. emanate from two
Bacillus strains referred to as W1 and W2 by the authors. In each of
these strains, two components of the xylanase activity, referred to as I
and 11, have been demonstrated. The components I degrade xylan to
xylobiose and to oligomers having a higher degree of polymerization,
while the components 11 produce xylose in addition to the above
compounds.
The components I (referred to as W1. 1 and W2. 1) have
respective molecular weights of 21.5 kDa and 22.5 kDa, as well as
isoelectric points of 8.5 and 8.3. The components 11 (W1.11 and W2.11)
have, for their part, respective molecular weights of 49.5 kDa and 50
kDa.
lS The two components I and 11 are inhibited by Hg + ions and, to
a lesser extent, by Cu +.
Many other xylanases have been isolated from various species
of Bacillus, Clostridium, Aspergillus, Sfreptomyces or Trichoderma, inter
alia (Wong et al., 1988, cited above).
Thus, the résumé of Japanese Patent JP 130 96 84
(RIKAGAKU KENKYSHO) relates to a type Wll xylanase having a
molecular weight of 50 kD or 42 kD. No isoelectric point is mentioned for
this xylanase.
A paper by RAJARAM et al., ( Applied Microbiology and
Biotechnology, Vol. 34, n~1, October 1990, pages 141-144) relates to a
Bacillus strain isolated in the natural environment and which produces a
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xylanase having optimal activity at between 60~C and 70~C and at a pH
of between 6 and 7. This enzyme is characterized neither by its
molecular weight nor by its isoelectric point. This strain produces, in
addition, other enzymes such as cellulases.
Another résumé of a Japanese patent in the name of
RIKAGAKU KENKYUSH0 (JP-85 118 644) describes a xylanase having
optimal activity at a pH of between 6 and 7. This enzyme is consideréd
to have a molecular weight, determined by ultrafiltration, of between 50
and 100 kD. No isoelectric point is mentioned in this résumé.
o A paper by GRUNINGER et al. (Enzyme Microbiology and
Technology, Vol. 8, May 1986, pages 309-314) relates to a Bacillus
stearothermoplilus strain isolated from mud and which produces a heat-
stable xylanase. The enzyme is characterised as having optimal activity
at 78~C and at a pH value of 7.5. This enzyme is characterized neither
by its molecular weight nor by its isoelectric pH.
The industrial production of xylanases is impeded by the
simultaneous presence of contaminant activities such as cellulases,
leading to additional purification costs.
As far as the Applicant is aware, the best productivity with
respect to endoxylanase obtained with a microorganism not producing
cellulase has been obtained from a Streptomyces lividans mutant devoid
of cellulase activity after introduction of a plasmid carrying genes coding
~or xylanases A and B. Productivities of the order of 6000 to 10,000
lU.I/h were observed in the culture media. It should nevertheless be
noted that, in this case, problems linked to the failure of xylanase A to
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hydrolyse insoluble xylan, and of thermal stability in the case of xylanase
B, were encountered (Kluepfel et al. Biochem. J. 267, 47-50, 1990).
None of these enzymes hence possessed, as far as the
Applicant is aware, features making an industrial application possible,
that is to say good thermal stability, a large capacity for degradation of
substrates, a means of production by hyperproductive strains and
possibilities to modify the aminoacids sequence.
Another xylanase has been isolated from a Bacillus strain
deposited at the CNCM Culture Collection under the number 1-1017. It
o has been described in EP 0.573.536 application filed under the name of
the present applicant. This xylanase displays a temperature stability.
However its sequence has not been determined and it can only be
produced by growing the said Bacillus strain.
Thus it was not possible to modify its protein sequence in the
aim of improving its properties.
The applicant has thus cloned the gene encoding this xylanase
and has sequenced it.
The subject of the present invention is, thus a thermophilic
xylanase having a sequence sharing an homology o~ at least 80%,
preferentially 90%, and more preferentially 95%, with the following one
(SEQ ID N~2):
Asn Thr Tyr Trp Gln Tyr Trp Thr Asp Gly lle Gly Tyr Val Asn Ala Thr
Asn Gly Gln Gly Gly Asn Tyr Ser Val Ser Trp Ser Asn Ser Gly Asn
Phe Val lle Gly Lys Gly Trp Gln Tyr Gly Aia His Asn Arg Val Val Asn
Tyr Asn Ala Gly Ala Trp Gln Pro Asn Gly Asn Ala Tyr Leu Thr Leu
Tyr Gly Trp Thr Arg Asn Pro Leu lle Glu Tyr Tyr Val Val Asp Ser Trp
,
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Gly Ser Tyr Arg Pro Thr-Gly Asp Tyr Arg Gly Ser Val Tyr Ser Asp Gly
Ala Trp Tyr Asp Leu Tyr His Ser Trp Arg Tyr Asn Ala Pro Ser lle Asp
Gly Thr Gln Thr Phe Gln Gln Tyr Trp Ser Val Arg Gln Gln Lys Arg
Pro Thr Gly Ser Asn Val Ser lle Thr Phe Glu Asn His Val Asn Ala Trp
Gly Ala Ala Gly Met Pro Met Gly Ser Ser Trp Ser Tyr Gln Val Leu Ala
Thr Glu Gly Tyr Tyr Ser Ser Gly Tyr Ser Asn Val Thr Val Trp
The degree of homology can be determined using pairwise
alignment methods such as the GAP and the BESTFIT programs of the
Genetics Computer Group, Inc. Package (GCG) Fast database
searching programs such asd FASTA and BLAST (included in the GCG
package) can be used for the comparison of a sequence to all available
sequences of a database.
For the definition of the term "homology" one can refer to
Altschul et al. (1990, J. Mol. Biol. 215, 403410) and Doolittle R.F. (Ed)
(Molecular evolution: computer analysis of protein and nucleic acid
sequences. Methods in Enzymology 183, Academic Press, London,
1 990).
Xylanases falling under this definition are in particular the ones
in which one or a few aminoacids have been changed, compared to the
sequence SEQ ID N~2.
Such changes in the aminoacids are preferentially the ones
which consist in the substitution of one aminoacid by another one which
has substantially the same properties such as defined by Lehninger
(page 7O, 2nd french edition, 1979, Flammarion ed.) or in its more
~ 25 recent re-edition. It is reminded that the twenty basic aminoacids are
classified in four groups depending on their properties:
- the ones having a hydrophobic or non-polar lateral chain,
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- the ones havirig a polar lateral chain not charged,
- the ones having a negatively charged lateral chain, and
- the ones having a positively charged lateral chain.
Such a xylanase can possess a molecular mass of
approximately 22 kDa, determined by SDS-PAGE, or 20.7 kDa
determined by mass spectroscopy and an isoelectric point of
approximately 7.7.
This enzyme advantageously displays great stability at 60~C
for at least 24 hours, and a pH of optimal activity within the range
o extending from 4.8 to 7, and preferably approximately 6.
It should be noted that the pHj of this enzyme is fairly high but
nevertheless lower than the pHj of the xylanases of similar molecular
mass produced by some bacilli, in particular those described by Okazaki
et al. (198~, publication cited above).
pH 6 corresponds to an optimal pH, but the activity remains
greater than 80% in the range between 4.8 and 7.
Another subject of the present invention is a nucleotidic
sequence coding for the said xylanase. This sequence can be DNA or
RNA sequence and in particular c DNA, plasmidic DNA, genomic DNA or
m RNA.
Preferentially such a nucleotidic sequence is the following one
(SEQ ID N~1):
aacacgtactggcayldlLyyacggatggcatcggylalylyaacgcgacgaacggaca
aggcggcaactacagcgtaagctggagcaacagcggcaacllcyl~.atcygcaagggct
ggcaatacggtgcgcacaaccgg~llylcaactacaacgcCggCgCatggcagccgaa
cggcaacgcylal~,lgacgctgtacggctggacgcgcaaccc~clcatcyaatactacgt
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cgtcgacagctggggcagctaccgcccgacc99c9actacc999gcagcgtgtacagc
gacggcgcatggtatgacctclatcacagctgs~cyctacaac9caccy Iccatcgacggc
acgcagacgttccaacaatactggagcy llcy lcagcagaaacscccgacgggcagcaa
cgtctccatcacgttcgagaaccacgt9~c!lc~tggggcyclyccygcalyccyatgg
s gcagcagctggtcttaccagyly~lcycaaccgaa9y~ ldllacagcagcggatactcca
acgtcacggtttggtaa
The xylanase according to the present invention can thus also
be produced by a microorganism strain, appropriately chosen,
transformed by a vector coding for the said xylanase. The said
o microorganism is grown in an appropriate medium and thereafter the
xylanase is isolated as described below.
Such a microorganism is chosen in order to be able to produce
and to excrete it.
It can be a bacteria such as Escherichia coli or Baci/lus sp.
The vector coding for the xylanase is chosen in order to be
expressed in the said microorganism. It can be a plasmid, such as
pBluescript or preferentially pET
Systems of expression suitable for the production of the
xylanase according to the present invention are in particular listed in
D.V. Goeddel ((Ed). Gene expression technology. Methods in
Enzymology, 185, Academic Press, London, 1990).
The pET E.coli expression system is one of the most widely
used bacterial expression system (Studier et al., 1990, Meth. Enzymol.,
1 85, 60-89).
The expression of recombinant xylanase can be achieved in
particular as following. The DNA fragment encoding the mature
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WO97/14803 PCTAEP96/04485
xylanaxe, i.e. the sequence SEQ ID N~1, is engineered by PCR so as to
generate Ndel and Bamhl terminal restriction sites suitable for
expression in the T7-based vector pET3a. The PCR fragment is cloned
blunt-ended into pBluescript (Stratagene Cloning Systems) before
cloning as a Ndel/BamHI fragment into pET3a.
The recombinant enzyme is expressed from pET3a in the E.
coli strain BL21 (DE3) carrying pLysS. Cultures are grown in L-broth
containing ampicillin (100 ,ug/ml) and chloramphenicol (25 ,ug/ml) until an
A600 ~f 3 was reached, before induction with 0.1 mM isopropyl 13-D-
l0 thiogalactoside (IPTG) for 3 hours. Large-scale cultures for protein
purification are centrifuged and the cells are Iysed in a buffer containing
50 mM Tris-HCI, pH 8.0, 1 mM EDTA by passage through a French
press (10 MPa). The same process described in EP 0.573.536 for
purifying the xylanase from the culture supernatant of the Bacillus can be
used. One can expect about 1 mg of recombinant protein per ml of cell
culture.
An advantage of this way of production of the xylanase is that
the nucleoditic sequence can be mutated before to be introduced in the
microorganism. It is therefore easy to obtain various mutations
corresponding to xylanases having various sequences.
This was not possible with the thermostable xylanases already
described in the prior art, such as the one described by GRUNINGER et
al. (previously cited), since their sequences were not known.
For carrying out the present invention, in particular this way of
production, the man skilled in the art can refer to the following manual
which describes the usual techniques of molecular biology: Maniatis et
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al. 1982- Molecular Cloning: a Laboratory Manual, Cold Spring Harbor
Ed. New York, or one of its more recent editions.
The xylanase as described above can be obtained through a
process, comprising the following steps:
- concentration of the microorganism culture supernatant,
- passage through an ion exchange column such as a column
of Q Sepharose Fast Flow (Pharmacia),
- passage through a hydrophobic interaction column such as a
column of Phenyl-Sepharose (Pharmacia).
Concentration of the supernatant can, in particular, be
performed by ultrafiltration through a polysulphone membrane having an
exclusion threshold above 10 kDa.
This process enables a substantially pure xylanase preparation
to be obtained.
The xylanase described above can be produced through a
process comprising the steps:
- of growth of the bacteria in a medium containing a growth
substrate such as glucose, and
- of production of xylanase induced by feeding the culture
continuously with suitable amounts of xylo-oligosaccharides.
The subject of the present invention is also the use of the
xylanase described above in the bleaching of paper pulp.
An advantage of this xylanase lies in the fact that the degree of
hydration of the paper pulp is of little importance. It is not obligatory to
dilute the pulp greatly in order to obtain good enzymatic attack. The use
of this xylanase as an auxiliary in the bleaching of paper pulp is all the
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more advantageous for the fact that the preparations are devoid of
cellulase contaminants.
This xylanase may also be used for the preparation of xylose or
of xylo-oligosaccharides from raw materials of plant origin, which are
inexpensive and renewable raw materials (for example maize cobs).
Other uses of xylanases have been mentioned in the literature.
The review by Zeikus et al. (Thermostable saccharidases New Sources
uses and Biodesigns in "Enzymes in biomass conversion", Leatham and
Himmel, ACS Washington D.C., 1991) lists the main uses of xylanases.
l0 They are mainly used in food manufacture, where their properties enable
bread-making, the clarification of fruit juices and wines and the
nutritional qualities of cereal fibres to be improved, and in the production
of thickeners for foodstuffs.
The second sphere of application relates to the paper pulp and
fibre industries, where they are used for the bleaching of pulps, the
manufacture of wood pulp and the purification of fibres for rayon
manufacture.
Uses are also noted in poultry feeding, in which uses xylanases
are employed in order to decrease the viscosity of the feeds (Van
Paridon et al. Xylans and Xylanases, Internationai Symposium,
Wageningen, 8-11 December 1991; Bedford and Classen H.L Xylans
and Xylanases, International Symposium, Wageningen, 8-11 December
1991).
The use of xylanase in enhancing the value of by-products of
2~ the paper pulp industry is more specifically mentioned in the paper by
Biely (Trends in Biotechnology, Vol.3, No.11, 1985).
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Mention may also be made of the two European Patents EP
228,732 and EP 227,159, which relate to the use of Xylanases for
improving the filterability of glucose syrup and of beer, respectively.
The possibility of using the xylanases for the production of
chemical compounds from hemicellulose (Reilly, cited above) will also be
noted.
These various publications show that the xylanases which are
the subject of the present invention may be used in a large number of
applications.
The present invention is illustrated, without, however, being
limited, by the application examples which follow.
Fig. 1 illustrates the homology degree between the xylanase
according to the present invention (XYL20) and other xylanases.
Fig.2 represents HCA plots of four xylanases, including the one
of the present invention.
On fig.3 is indicated the prediction of secondary structural
elements for the xylanase of the present invention.
EXAMPLE 1:
Cloninq and sequencinq of the qene encodin~ for the
xylanase.
1. Materials and methods.
- Strains, vectors and culture conditions.
The strain 1-1017 was grown at 55~C in the liquid medium
described in examples 1 and 2 of patent application EP-0.573.536. The
SURE, XL1-Blue and XLOLR Escherichia coli strains, the vectors ZAP
Express and pBluescript and the filamentous helper phage ExAssistTM
-
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WO97/14803 PCT~E~9G~1185
were all purchased from Stratagene Cloning Systems E. coli cells were
grown in LB medium at 37~C. The medium was solidified by addition of
1.5% (w/v) of Bacto-agar.
- Preparation of DNA
Bacterial genomic DNA was extracted from 1-1017 according to
the method of Yang et al. (Appl. Environ. Microbiol., 1988, 54, 1023-
1 029).
- Obtention of a partial qenomic clone codin~ for the
XYL20.
PCR was used to amplify a region of the chromosomal DNA
coding for the XYL20. The nucleotide sequence of the forward primer
(P1 ) (SEQ ID N~3) was AAYACNTAYTGGCARTAYTGGACNGAYGG
(derived from the sequence NTYWQYWTDG in the N-terminus end of
the XYL20); that of the reverse primer (P2) (SEQ ID N~4) was
YTGWCKNACRCTCCARTAYTG (corresponding to the sequence
QYWSVRQ, a conserved region near the C-terminus of other xylanases
from different Bacillus species). PCR was performed with chromosomal
DNA as a template and the primers P1 and P2 on a thermocycler
(Perkin-Elmer. France) with the following temperature profile: 1 min 94~C
-1 min 50~C - 2 min 72~C for 35 cycles. The PCR product was purified
on a 1% agarose gel and was ligated into EcoRV-digested pBluescript.
The chimaeric plasmid (pBX20) was used to transform SURE cells.
Recombinant cells were selected on L-agar plates containing ampicillin
(40 ,ug/ml), isopropyl-~-D-thiogalactoside (0.2 mM) and 5-bromo-4-
chloro-3-indolyl-t3-D-galactoside (40 ,ug/ml).
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- Construction of a B. sp 1-1017 nenomic library in ZAP
Express.
Chromosomal DNA was partially digested with Sau3AI and the
resulting DNA fragments in the size range 1.5-8 kb were purified and
ligated into BamHI-digested ZAP Express. The library was constructed
using XL1-Blue cells as indicated by the manufacturer.
- Screeninq of the qenomic librarY.
pBX20 was digested with BamHI and Hindlll and the DNA
insert was purified and labeled with digoxigenin (Boehringer Mannheim)
o following the instructions of the manufacturer. The labeled DNA was
used to screen the genomic library. After the third screening, positive
lambda plaques were isolated and the recombinant plasmid pBK-CMV
inserted in the vector ZAP Express was excised using the filamentous
phage ExAssist and then recovered by infecting the XLOLR cells in the
presence of kanamycin (10 ,ug/ml).
- DNA sequence analvsis.
Plasmid preparations for sequence determination were
performed using Qiagen tip 100 (Diagen, Coger, France). Double-
stranded DNA sequencing was done by the dideoxy chain termination
method of Sanger et al (Proc. Nat. Acad. Sci. USA, 1977, 74, 5463-
5467), using the SequenaseTM 2.0 DNA sequencing kit from United
States Biochemical . Both universal and specific primers were used to
sequence the sense and antisense strands of inserts in the plasmids.
- Protein sequence analysis and Hydrophobic Cluster
2~ Analysis.
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The sequence Analysis Software Package by Genetics
Computer, Inc. (The GCG Package) was used throughout this work. In
particular, multiple alignments were performed using the Pileup program
and pair~,vise comparisons were done using the Bestfit program.
Hydrophobic Cluster Analysis (HCA) is a method to compare
amino acid sequence (Gaboriaud et al. FEBS Lett., 1987, 224, 149-155)
which is derived from the theory of Lim (J. Mol. Biol, 1974, 88, 857-872).
The method involves the drawing of the sequence of a theoretical A-helix
where the hydrophobic residues form clusters. The shape, size and the
o relative position of the clusters can be compared and the sequence
similarity, when it exists, may be readily revealed. Conversion of the
amino acid sequences into the 2D-helical plot required by the method
was made using the HCA-Plot software.
2) Results.
In a first attempt to determine the xylanase sequence, the
xylanatic activity has been tested in the genomic library. However this
approach has failed to conduct to the isolation of the xylanase according
to the present invention.
In a second and successful attempt, the sequence of this
xylanase has finally been determined.
It is reminded that the amino acid sequence of the N-terminal
region of the xylanase has been determined (example 4 of EP
0.573.536). It exhibits 67% of identity with the N-terminus of the
xylanases produced by Bacitlus subtilis and Bacillus circulans.
2~ Besides, these enzymes which belong to the G family
(according to the classification of Gilkes et al. (Microbiol. Rev., 1991, 55,
_
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303-315)) share some conserved regions along their polypeptide chains.
Among others, one region consisting of 7 amino acids occurs near the
C-terminus.
A part of the gene coding for the xylanase has been amplified
s by PCR using two degenerate primers, P1 and P2, corresponding to the
N-terminus end of the xylanase and to a conserved region near the C-
terminus, respectively. A 450 bp DNA fragment was obtained and
cloned into the vector pBluescript. The sequence of the resultant
plasmid pBX20 can be attributed without any doubt to the xylanase. To
10get the complete gene of xylanase, a genomic library of B. sp 1-1017 was
prepared in E. coli XL1-blue using the phage vector ZAP Express. This
library was screened with the insert of the plasmid pBX20. One positive
plaque, designated pBX52A2, was shown to contain the complete gene
of the xylanase.
15The nucleotidic sequence of this clone is indicated in the
sequence list hereunder as SEQ ID N~ 1.
The complete protein sequence of the xylanase is shown as
SEQ ID N~2 is the sequence list hereunder.
The results of a FASTA search in the protein data bases PIR
and Swiss-Prot yielded 36 xylanase sequences. As shown in the table
the xylanase shares sequence homology with other xylanases of the G
family. The best scores (73% of identity) are observed as expected with
the xylanases from B. subtilis and B. circulans. This shows
unambiguously that the xylanase according to the present invention is a
new protein which possesses a unique amino acid sequence.
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WO 97/14803 PCT~P96/04485
For comparative purposes, only representative xylanases from
different organisms (the ones in bold types on the table ) are listed in the
multiple sequence alignment shown in figure 1. The analysis of a primary
sequence alignment of 14 xylanases of the G family indicates the
residues which are conserved throughout the family. As reported
previously by Wakarchuk et al., (Protein Sci., 1988, 3, 467475), 2
glutamic acid residues are absolutely conserved in this family of
xylanases. The present multiple alignment suggests that Glu76 and
Glu169 are the catalytic residues of the xylanase.
o Alignment was then reconsidered by the HCA method
(Gaboriaud et al., 1987, previously cited), which allows for a rapid
identification of the clusters and an easy alignment (figure 2). The
identification of the clusters is straighfforward even if there are some
variations in cluster shapes. The alignment was checked on crystalline
S structures for three xylanases indicated in figure 1, and the extensions of
the 13-strands are indicated on the HCA plots. Vertical lines have been
inserted to delimit the extension of 13-strands. The most conserved
hydrophobic clusters have been shadowed for better visualization.
For the HCA plot of xylanase according to the present invention
(XYL20), the extension of the ~-strands was deduced from the other
plots. It appears clearly that the secondary structure of XYL20 consists
essentially of ~-strands and only one A-helix. These elements are so
organized to display the characteristic folding of a greek key. We can
also inferred that the following aromatic residues: Tyr 67 and Tyr 78 on
2~i one ~-strand and Tyr 163 on another 13-strand are likely to be involved in
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WO 97/14803 PCT~EP96/04485
the orientation and binding of xylan polysaccharides, prior their
hydrolysis.
The figure 3 summarizes the prediction of the occurrence of
secondary structural elements which can be proposed for the xylanase
according to the present invention on the basis of its primary structure
and a thorough protein sequence analysis. These structural predictions
can be translated into a putative three-dimensional model to be used in
Molecular Isomorphism Replacement in view of solving the crystalline
structure of this xylanase.
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18
T:lble Summa~y of !;~ lanases ceri~ed fro~n -t ~-~ ST-~ se,~rch ir ~ilr- rrotein Data Bases S~vi5s
Prol (release 31.0) and PIR (release 4~.0j.
,~C (I) d.~le ori~in CT~-st ~- nb M~
P1842J t~l.ll.)ll B-~cillrr.sstlh~ili.s XYNr'~ 73 213 23,31.-7
S39 1 7 1 8.02. )~
PQY8~rl tll 1)3.8J Ba~illus L-ir~u/ons ,~ 73 Z13 23,31.6
S017;~ 07.0G.91)
p 6220 1)1.()2.90 Strrptomvcc~- fi~;dans .YYLC rio 2~U 2~,673
JSO j9 1 1~.n7.9~
S47r71213.01.Y:- Stt~p~olr~!c~.~.. sp EC3r78 2~U
JSOj9010.0;.9~ Srrcplom~rLs li-itrntr.s XYLB ~8 î3;
P 6jlj01.08.92 XYNE7 j8;;3 ;j,~26
S~391920.10.9~ ln~ic~la in.s~llrlls ~227
9~1~.t)6.9~ Tricl~ rll~o ~ ;ri~lc .';YL 11 A r7 1 19(J
A~lj9j27.06.)1 XYL n B j~190
P36217(tl.(K.9 I Tricll~lLlcrllla rrcsri XYN2 j~222 2~,172
S~91 j-l06.03.9~
S;988327.0j.9 1 XYL n *
P3r7809(1l.n6.9~ .Sol~ pll!l61lr~ r~ r 5~ 197 20,978
A~ j9727.0G.9
S ~897;1 8.02.9~
Q(lfi~-762 Itl ()2 y j ~;-cl~h-lllLs curhmlllllr :~3 221 23,728
A44 j9327.t,6.9 I T~icl~o~/c~rrrn Im~ ionrrnr j; 190
poo69~21.(17.9~ r~ rnlr~ .r .YYNA ,t ~U228 2r7,j21
~VF7SXP O.t)g.8~
JQ19350.1)8.3~ Cl~s~ri~ nl.s~rct~ rirllrl Y-LA ~8 ~11
P;;.7j8t)l.O2.9j ~8 ~ 6,jl9
P17137()1.()8.9~ Ch~.srri~i;al/~acct~S,li!lic~,,,, ~6 261 29,032
S127 Ij~O.t~g.g;
P33:;~701.()2.J~- .L~prr"~ l.sa--olllr~ri ~ C ~o211 2.76(J
JC 1 1 )8 ~ r~r~il/rrsrri~cr XYNC 1() 1 1
PiC218t)l.OG.9~ Triolr~7~l rnrn l-~csci ~ 9 29 ~.r78;
S;gl jj()G.O;.)~ XYL2
5-1886 jl; .O l . ) j .\;~OL al I inrasl ix l i orrrnlis t 7 266
5~9 i280.02. ~ ~ ~ j607
~;-47~.09.); ~c(rrnllinrnsri~pnrlic*rnr YYLA ~j G07
P_9127()1.1_.)_ YYNA .~G07 66,17
Pi SI It)l.t)_ 9j l;f-r~-ht~cr~-r.~ 6()8 66.~1
AC: A-cr:;:iiol) Numhcr
~_): cr~ cr~ ollo~r;ll~l)lc ~i;lt;~ oil:~hlr
crc.:l)t;~ ~c ol i~lrlltlt~
Vl~l!- tl)~ I)c.~ hol~lll;l~rh~rl)ll.~ r;lli~ tlt~l~t:;t~ .ol~ lr~ 2
- SUL.~ 1 1 1 UTE SHEET~RULE 26
CA 02234998 1998-04-16
W O 97/14803 PCT~EP96/04485
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: INSTITUT NATIONAL DE LA RECHERCHE
AGRONOMIQUE(INRA)
(B) STREET: 147, rue de l'Universite
(C) CITY: PARIS
(E) COUNTRY: FRANCE
(F) POSTAL CODE (ZIP): 75341
(G) TELEPHONE: 42 75 90 00
(H) TELEFAX: 42 75 94 28
(ii) TITLE OF INVENTION: XYLANASE, OLIGONUCLEOTIDIC SEQUENCE ENCODING
IT AND ITS USES
(iii) NUMBER OF SEQUENCES: 4
(iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC colllpdlible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO)
(v) CURRENT APPLICATION DATA:
APPLICATION NUMBER: US 08/543.956
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 549 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: I~NA (genomic)
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Bacillus sp
(B) STRAIN: 1-1017
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:1..547
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
AAC ACG TAC TGG CAG TAT TGG ACG GAT GGC ATC GGG TAT GTG AAC GCG 48
Asn Thr Tyr Trp Gln Tyr Trp Thr Asp Gly lle Gly Tyr Val Asn Ala
- SUBSTITUTE SHEET (RULE 26)
CA 02234998 1998-04-16
WO 97/14803 PCT/EP96/04185
ACG AAC GGA CAA GGC GGC AAC TAC AGC GTA AGC TGG AGC AAC AGC GGC 96
Thr Asn Gly Gln Gly Gly Asn Tyr Ser Val Ser Trp Ser Asn Ser Gly
AAC TTC GTC ATC GGC AAG GGC TGG CAA TAC GGT GCG CAC AAC CGG GTT 144
Asn Phe Val lle Gly Lys Gly Trp Gln Tyr Gly Ala His Asn Arg Val
GTC AAC TAC AAC GCC GGC GCA TGG CAG CCG AAC GGC AAC GCG TAT CTG 192
Val Asn Tyr Asn Ala Gly Ala Trp Gln Pro Asn Gly Asn Ala Tyr Leu
ACG CTG TAC GGC TGG ACG CGC AAC CCG CTC ATC GAA TAC TAC GTC GTC 240
Thr Leu Tyr Gly Trp Thr Arg Asn Pro Leu lle Glu Tyr Tyr Val Val
GAC AGC TGG GGC AGC TAC CGC CCG ACC GGC GAC TAC CGG GGC AGC GTG 288
Asp Ser Trp Gly Ser Tyr Arg Pro Thr Gly Asp Tyr Arg Gly Ser Val
TAC AGC GAC GGC GCA TGG TAT GAC CTC TAT CAC AGC TGG CGC TAC AAC 336
Tyr Ser Asp Gly Ala Trp Tyr Asp Leu Tyr His Ser Trp Arg Tyr Asn
100 105 110
GCA CCG TCC ATC GAC GGC ACG CAG ACG TTC CAA CAA TAC TGG AGC GTT 384
Ala Pro Ser lle Asp Gly Thr Gln Thr Phe Gln Gln Tyr Trp Ser Val
115 120 125
CGT CAG CAG AAA CGC CCG ACG GGC AGC AAC GTC TCC ATC ACG TTC GAG 432
Arg Gln Gln Lys Arg Pro Thr Gly Ser Asn Val Ser lle Thr Phe Glu
130 135 140
AAC CAC GTG AAC GCA TGG GGC GCT GCC GGC ATG CCG ATG GGC AGC AGC 480
Asn His Val Asn Ala Trp Gly Ala Ala Gly Met Pro Met Gly Ser Ser
145 150 155 160
TGG TCT TAC CAG GTG CTC GCA ACC GAA GGC TAT TAC AGC AGC GGA TAC 528
Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Tyr Ser Ser Gly Tyr
165 170 175
TCC AAC GTC ACG GTT TGG T AA 549
Ser Asn Val Thr Val Trp
180
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 182 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
SU~5 ~ JTE SHEET (RULE 26)
CA 02234998 1998-04-16
W O 97/14803 PCT~P96/04485
21
Asn Thr Tyr Trp Gln Tyr Trp Thr Asp Gly lle Gly Tyr Val Asn Ala
5 10 15
Thr Asn Gly Gln Gly Gly Asn Tyr Ser Val Ser Trp Ser Asn Ser Gly
20 25 30
Asn Phe Val lle Gly Lys Gly Trp Gln Tyr Gly Ala His Asn Arg Val
35 40 45
Val Asn Tyr Asn Ala Gly Ala Trp Gln Pro Asn Gly Asn Ala Tyr Leu
50 55 60
Thr Leu Tyr Gly Trp Thr Arg Asn Pro Leu lle Glu Tyr Tyr Val Val
65 70 75 80
Asp Ser Trp Gly Ser Tyr Arg Pro Thr Gly Asp Tyr Arg Gly Ser Val
85 90 95
Tyr Ser Asp Gly Ala Trp Tyr Asp Leu Tyr His Ser Trp Arg Tyr Asn
100 105 110
Ala Pro Ser lle Asp Gly Thr Gln Thr Phe Gln Gln Tyr Trp Ser Val
115 120 125
Arg Gln Gln Lys Arg Pro Thr Gly Ser Asn Val Ser lle Thr Phe Glu
130 135 140
Asn His Val Asn Ala Trp Gly Ala Ala Gly Met Pro Met Gly Ser Ser
145 150 155 160
Trp Ser Tyr Gln Val Leu Ala Thr Glu Gly Tyr Tyr Ser Ser Gly Tyr
165 170 175
Ser Asn Val Thr Val Trp
180
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "synthetic,degenerate
oligonucleotide"
(iii) HYPOTHETICAL: NO
SUBSTITUTE SHEET (RULE 26)
CA 02234998 1998-04-16
W O 97/14803 22 PCT/EP96/04485
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
AAYACNTAYT GGCARTAYTG GACNGAYGG 29
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "synthetic,degenerate
oligonucleotide"
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
YTGWCKNACR CTCCARTAYT G 21
SUBSTITUTE SHEET(RULE 26
