SK82093A3 - Xylanase bacillus strain producing xylanase and their use - Google Patents

Abstract

A xylanase has a molecular weight of approximately 22 kDA, an isoelectric point of approximately 7.7, is stable at 60 C and has an optimal activity pH of between 4.8 and 7. Said xylanase is secreted by bacillus strains and can be purified by concentrating the supernatant found on the culture of these strains, passage through an ion exchange column and then a hydrophobic interaction column. The xylanase obtained can be used in for bleaching paper pulp and for the preparation of xylo-oligosaccharides from vegetal raw materials.

Description

The invention relates to xylanase and Bacillus strains producing this enzyme.

It further relates to the use of this enzyme and these bacteria in the bleaching of pulp and in the preparation of xylose or xylo-oligosaccharides, in particular from vegetable raw materials.

BACKGROUND OF THE INVENTION

Various uses of xylanases in the field of biotechnology have been suggested, especially in the food industry (White, 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 manufacture of chemical compounds from hemicellulose (Reilly PJ, 1981,

Xylanases: Structure and Function in Trends in Biology of Fermentations for Fuels and Chemicals, A.J. Hollaender (Ed.) Plenum, New York).

Technically, mainly by application, however, the feasibility of such applications has been evaluated by the enzymes produced by mesophilic fungi. These can be facilitated by the use of fungi with better thermal stability.

Various bacteria and enzymes are known for the production of xylanases (see in particular Wing et al., Microbiological Reviews, 52, No. 3, 305317, 1988). So far, the highest yields of enzymes have been achieved using fungi (Yu et al., Enzyme Microb. Technol. 9, 16-24, 1987). Meanwhile, the hyperproduction strains of Bacillus have been described (Okazaki et al., Appl. Microbiol. Biotechnology 19, 335-340, 1984, Okazaki et al., Agric. Biol. Chem. 49, 2033-2039, 1985). Bacillus species that are thermophilic and degrade xylan may be good candidates for the industrial production of xylanases due to their significant growth factor and good knowledge of their genetics.

The xylanases isolated by Okazakim et al. Come from two Bacillus strains called W1 and W2. In each of these strains, two components with xylanase activity, termed Ia II, were shown. Component I degrades xylan to xylobiose and to higher-stage oligomers, while component II produces xylose in addition to the compounds.

Components I (called W1.1 and W2.I) have a molecular weight

21.5 kDa, respectively. 22.5 kDa and isoelectric point 8.5, respectively. 8.3. Components II (W1.II and W2.II) have a molecular weight of 49.5 kDa, respectively. 50 kDa.

Both components I and II are inhibited by Hg ²⁺ ions and to a lesser extent by Cu ²⁺ ions.

Many other xylanases have been isolated, inter alia, from various Bacillus, Clostridium Aspergillus, Streptomyces or Trichoderma species (Wong et al., 1988).

For example, the abstract of Japanese Patent JP 13096 84 (Rikagaku Kenkyusho) relates to a WII type xylanase having a molecular weight of 50 kD or 42 kD. No isoelectric point value is given for this xylanase.

1

i. (Applied Microbiology and October 1990, pp. 141-144) refers to naturally producing xylanase at 60 and 70 ° C and pH in the range of 6-7.

Article by Rajarama

Biotechnology, Vol. 34, no.

strain of Bacillus, isolated with optimal activity between

This enzyme is not characterized by molecular weight or isoelectric point. In addition, this strain produces other enzymes, such as cellulases.

Another Japanese abstract in the name of Rikagaku Kenkyusho (JP 85 118 644) describes xylanase with optimum activity at a pH in the range of 6-7. This enzyme has a molecular weight determined by ultrafiltration of 50-100 kD. There is no isoelectric point in the abstract.

Article by Gruninger et al. (Enzyme Microbiology and Technology, Vol. 8, May 1986, pp. 309-314) relates to another strain of Bacillus isolated from mucilage and producing thermostable xylanase. The enzyme is characterized by optimal activity at 78 ° C and at a pH of 7.5. This enzyme is not characterized by molecular weight or isoelectric pH.

Industrial production of xylanases encounters the simultaneous presence of contaminating activities, such as cellulases, causing increased cleaning costs. On the other hand, the use of genetically modified germs may pose problems, such as instability of plasmids carrying genes coded for xylanases.

To the best of the applicant's knowledge, the best productivity of endoxylanase, obtained by non-cellulase-producing microorganisms, was achieved by using mutants of Streptomyces lividans, depleted of cellulose activity after plasmid introduction, carrying genes encoding xylanases A and B. 1 / h. However, there are problems associated with non-hydrolysis of xylan, insoluble xylanase A, and thermal stability of xylanase B (Kluepfel et al., Biochem. J. 267: 47-50, 1990).

None of the enzymes described in the prior art, i.e. to the knowledge of the Applicant, have any characteristics which allow industrial application, i. good thermal stability, significant substrate degradation capability, and the ability to prepare by superproduction strains.

SUMMARY OF THE INVENTION

Thus, the Applicant has surprisingly shown that certain Bacillus strains produce thermostable xylanases with significant degradation activity.

Even more surprisingly, the Applicant has demonstrated that these Bacillus strains secrete these enzymes in large quantities without producing a significant amount of cellulases.

The present invention relates to xylanase-producing Bacillus strains, in particular strains deposited under Nos. 1-1017 and 1-1018 in the National Collection of Cultures of Microorganisms of the Pasteur Institute (Pasteur Institute of Cultures des Microorganisms).

These strains were isolated from manure.

The bacteria have the shape of short, straight spore-forming rods of medium size 0.5 to 2.5 μπι. They are usually alone or paired, have gram-positive staining and spores are oval and central.

These strains are strictly aerobic, no growth is observed in the absence of oxygen and nitrate is used only as an electron acceptor.

The optimum pH of growth and xylanase production is 7.8, while the growth limit pH is between 6.5 and 8.5 and the maximum temperature is 63 ° C.

In addition to xylan, these strains utilize xylose, glucose, sucrose, maltose and starch. Fructose, arabinose, cellulose, pectin and chitin are not utilized. In addition, the growth is inhibited by sodium chloride at a concentration of 5%.

The G + C content of the DNA of these strains, determined by thermal denaturation, was 57.5 mol%.

These strains are well distinguished from other xylanase production strains as shown in Table I in terms of different culture time and xylanase productivity.

Strain 1-1018 was obtained by mutagenesis of strain 1-1017. Strain 1-1018 has the same general characteristics as strain 1-1017, but is characterized by hyperproduction of xylanase.

The invention also provides a thermophilic xylanase having a molecular weight of about 22 kDa and an isoelectric point of about 7.7.

An advantage of this enzyme is furthermore a high stability at 60 ° C for at least 24 h and a pH of optimal activity in the range from 4.8 to 7, preferably around 6.

It should be noted that the pHi of this enzyme is sufficiently high but nevertheless lower than the near molecular weight pHi xylanase produced by Bacillus species, mainly described by Okazaki et al. (1985 cited above).

pH 6 corresponds to an optimum pH, but activity remains above 80% over the entire range of 4.8 to 7.

The xylanase of the invention may have the following N-terminal amino acid sequence: Asn-Thr-Tyr-Gln-Tyr-Trp-Thr-Asp-Gly-Ile-Gly-Tyr-Val-Asn-Ala-ThrAsn-Gly-Gln.

It preferably contains in its sequence 15 alanines, 6 arginines, 25 aspartates and asparagines, 19 glutamates and glutamines, 31 glycine, 3 histidines, 6 isoleucines, 7 leucines, 3 lysines, 1 methionine, 4 phenylalanines, 8 proline, 19 serines, 13 threonines , 22 tyrosines and 14 valines.

In addition, this enzyme is inhibited by Hg ²⁺ but not Ag ²⁺ (1 mM) or 0.1% SDS.

The Michaelis constant (Km) against birch xylan is 0.9 g / L.

The neutral oligosaccharides produced by this enzyme from birch xylene (5 g / l birch xylene, 500 U / l, 60 ° C) consist of:

- at a very short enzymatic incubation time (15 min) with xylotriose and xylooligosaccharides of higher polymerisation stages,

- at long incubation times (24 to 72 h) by xylobiosis, xylotriosis and xylotetraosis, which is present in a very low proportion.

This endoxylanase is mainly secreted by Bacillus, in particular by its strains 1-1017 and 1-1018, as described above.

It is secreted in the culture medium.

The culture medium of these strains is characterized mainly by the absence of endo-β-1,4-glucanase activity (cellulase) and the presence of very weak contaminating β-xylosidase activity (about 0.06 U./ml culture supernatant for both strains). It should be noted that the culture supernatant can be lyophilized without observable loss of xylanase activity and stored frozen for at least one year without loss of activity.

Endo-β-Ι, 4-glucanase activities were determined by measuring the production of sugars released from carboxymethylcellulose (CMC, 1%) or Avicel (1%) in 50 mM sodium acetate buffer at pH 6, β-xylosidase activity was determined by measuring release of p-nitrophenol from p-nitrophenyl-β-D-xyloside (0.1%) in 50 mM sodium acetate buffer pH 6.

The unit of xylanase activity (U.I.) is defined as the micromole of reducing equivalent of xylose released per minute at 60 ° C.

The reducing sugar concentration is determined by the method of Kidby and Davidson (Anal. Biochem. 55, 321-325, 1973).

The purified enzyme is stored frozen in 20% ethylene glycol without observable loss of activity for one year.

The present invention further provides a process for the production of the above xylanase comprising:

- concentration of the supernatant of the Bacillus culture by ultrafiltration,

- passage through an ion exchanger column, such as an O-sepharose Fast Flow column (Pharmacia),

passage through a column of hydrophobic interactions such as a phenylseparose column (Pharmacia).

In particular, the supernatant concentration can be performed by ultrafiltration on a polysulfone membrane with an exclusion threshold greater than 10 kDa.

This procedure makes it possible to obtain a substantially pure xylanase preparation.

The invention further relates to a process for the production of the above-described xylanase, comprising:

in the growth of bacteria in a medium containing a nutrient substrate such as glucose and

- production of xylanase induced by continuous feeding of the culture with xylooligosaccharides in appropriate amounts.

The invention also relates to the use of the above-mentioned xylanase in pulp bleaching.

The advantage of this xylanase is that the degree of hydration of the pulp is of minor importance. It is not necessary to dilute the pulp too much to obtain a good enzymatic attack. The use of this xylanase as an aid in the bleaching of pulp is all the more interesting because the preparations are free of cellulase impurities.

This xylanase can also be used to prepare xylose or xylooligosaccharides from raw materials of plant origin which are inexpensive and renewable (for example, corn cobs).

Other uses of xylanases are disclosed in the literature. An overview of Zeikus et al. (Thermostable Saccharidases, New Sources, Uses and Biodesigns, and Enzymes in Biomass Conversion, Leatham and Himmel, ACS Washington, D.C., 1991) summarizes the main applications of xylanases. They are mainly used in food production, where their properties make it possible to improve the production of bread, clarification of fruit juices and wines and the nutritional value of cereal fiber and in the production of food thickeners.

The second field of application relates to the paper and fiber industry where they are used for pulp bleaching, pulp production and fiber cleaning for silk production.

Use in poultry nutrition has also been reported, where xylanases are used to reduce the viscosity of feed (Van Paridon et al., Xylans and Xylanases, International Symposium, Wageningen,

8-11.12.1991; Beford and Classe H. L., Xylans and Xylanases, International Symposium, Wageningen, 8-11 December 1991).

The use of xylanases in the recovery of by-products of the pulp industry is described in more detail in the White paper (Trends in Biotechnology, Vol. 3, No. 11, 1985).

Mention may also be made of two European patents EP 228 732 and EP 227 159 relating to the use of xylanases to improve the filterability of glucose syrup, respectively. beer.

The possibility of using xylanases to produce chemical compounds from hemicellulose (Reilly, cited above) has also been reported.

These various publications show that the xylanases of the invention can be used in many applications.

Overview of the drawings

The invention is explained in more detail with reference to the accompanying drawings, which do not limit its scope.

Fig. 1 shows the development of dicontinuous cultures of strain Ι-1Ό17. Reducing sugars and growth in mg / ml are plotted on the left vertical axis, xylanase in units / ml on the right vertical axis, time in h and cross on the horizontal axis are proteins, xylanase triangle and sugars reducing square.

Fig. 2 shows the continuous development of a culture of strain I-1017 using xylo-oligosaccharide solutions of various concentrations. On the left vertical axis, growth at 550 nm is plotted, on the right vertical axis, xylanase in units / ml at 60 ° C, on the horizontal axis, time in h cross means growth, empty shaft 0.26 g / h, empty shaft 0, 36 g / h, solid shaft 0.50 g / h and 0.67 g / j solid triangle.

Fig. 3 depicts 1-1017 and 1-1018 in xylo-oligosaccharides. Na in IU / ml at the stick means a stick ring of 0.67 g / h.

° C, m na

1-1017 0? 36 Comparison of xylanase production by strains of the presence of different concentrations of the vertical axis are plotted by the horizontal axis time in h and g / h, the crosshair of the 1-1018 xylanase square 0.36 g / h,

1-1017 0.67 g / h and triangle handpiece 1-1018

Fig. 4 shows the release of xylo-oligosaccharides from unbleached kraft pulp using various concentrations of xylanase. On the vertical axis, the percentage of xylan degradation is plotted, on the horizontal axis, the time in h and the solid triangle means control, the empty square 10 U.I., the empty triangle 50 U.I., the empty circle 200 U.I. and a full square of 1000 U.I.

Fig. 5 shows the release of xylo-oligosaccharides from the crushed corn cob axis. The vertical axis shows the percentage of xylan degradation and the horizontal axis the time in h.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Strain selection 1-1017

Various soil, compost and manure samples were used as an inoculum to enrich the xylanolytic strains. After 24 h incubation, xylanase activity is assayed in the supernate culture and the three enrichments showing the most significant activities are diluted and plated on agar medium containing xylan.

The basic culture medium containing minerals and vitamins (Zeikus and Wolfe, J. Bacteriol. 109, 707-713, 1972) and yeast extract (0.2%) is supplemented with agar (2%) and oat xylan (0.5%). . After sterilization, KHCO 3 (25 mM) from a 1 M sterile stock solution is added to the isolation medium of the mixed strains. The inoculated Petri dishes are incubated at 55 ° C in sealed bottles.

The colonies forming the light zones are cleared. The most productive isolates (strain 1-1017) are selected and characterized.

The enrichment and growth of the cultures is carried out aerobically on a stirred water bath in sealed 125 ml bottles containing 10 ml of medium. The incubation temperature is 55 ° C and the medium is buffered to pH 7 by the addition of 50 mM KHCO ₃ .

Example 2

Growth of the 1-1017 strain

The kinetics of xylanase production is performed at 55 ° C in 2 L fermenters in an initial volume of 1.5 L. After sterilization, 50 ml of 1M KHCO-1 solution are added and the pH adjusted to

7.8 and maintains a saturation degree of 70%.

Fig. 1 shows the growth of strain I-1017 in discontinuous culture on oat xylan at a concentration of 0.5%. Strain 1-1017 produces up to 110 xylanase units / ml. A transient accumulation of reducing sugars is observed during the first hours of cultivation. This accumulation is due to the presence of small amounts of xylanase in the inoculum, where they catalyze rapid hydrolysis of the soluble fraction of xylan and xylo-oligosaccharides.

Thus, bacterial growth allows the consumption of xylooligosaccharides and the synthesis of xylanase, which begins as soon as their concentration becomes limiting for bacterial growth.

The production of xylanase is approximately constant over a period of 4 h (30 U. ml / h).

In xylanase production, great variability is observed depending on the previous inoculum xylanase activity, the importance of the stationary phase, and the physical characteristics of xylan.

To obtain reproducible culture conditions, xylanase production was tested in solution-fed cultures in the presence of xylo-oligosaccharides. Fig. 2 shows the effect of xylo-oligosaccharide concentration.

The initial sugar concentration is 2 g / l. The substrate is consumed during the exponential phase of growth during which no xylanase production is detected.

As soon as substrate concentration becomes limiting to growth, immediate synthesis of xylanase is observed.

The rate and also the production time of the enzyme is dependent on the substrate supply ranging from 0.26 g / h to 0.67 g / h with a substrate flow rate of 24 ml / h and an initial culture medium volume of 1.5 L.

The arrow in FIG. 2 corresponds to the addition of xylo-oligosaccharides.

The most significant enzyme production (230 U./ml.) Is achieved when the substrate is delivered at a rate of 0.36 g / h. A higher amount of substrate causes a shortening of the xylanase production time.

When glucose is used in place of xylo-oligosaccharides, production of xylanase is significantly reduced (14 U.I./ml) and takes less than 2 hours.

In contrast, the use of glucose as the starting substrate does not alter the xylanase yield compared to the use of xylooligosaccharides.

To prepare xylo-oligosaccharides as substrates in continuous cultures, a 5% oat xylan suspension is incubated for 8 hours at 60 ° C and pH 6 in the presence of 5 units of xylanase per ml. This suspension is then centrifuged and the supernatant obtained is autoclaved.

Example 3

Purification of xylanase from culture 1-1017

The xylanase purification process comprises the following steps:

- concentration of the culture supernatant of the Bacillus culture by ultrafiltration on a polysulfone membrane with an exclusion threshold of 10 kDa,

- passage through the Sepharose Fast Flow (Pharmacia) ion exchange column,

passage through a column of hydrophobic interactions of phenylseparose (Pharmacia).

The results of this purification are summarized in Table II.

This purification shows that the major protein among the proteins secreted in the culture medium is endoxylanase (about 50% of the total secreted proteins).

Example 4

Characterization of the xylanase μιη xylanase purified according to Example 3 was subjected to a gas phase sequence determination using an Applied Biosystems 470 A type. .

First N-terminal amino acid sequence obtained: Asn-Thr-Tyr-Trp-Gln-Tyr-Trp-Thr-Asp-Gly-Ile-Gly-Tyr-Val-Asn-Ala-Thr-Asn-Gly-Gln.

The total amino acid composition was also determined after hydrolysis with 5.6 N hydrochloric acid at 100 ° C for 24 h.

The results are shown in Table III, where the molar percentage of each amino acid is shown, the estimated number of residues of each type in the protein and, for comparison, the number of residues of each type in the N-terminal 20 amino acids.

It should be noted that the asparagine and glutamine residues were calculated together with the aspartate and glutamate residues respectively. glutamate residues and that tryptophan and cysteine residues were not measured.

Example 5

Mutagenesis of strain 1-1017

Cultivation of strain 1-1017 was performed in medium containing xylan and ethyl methanesulfonate (EMS).

The treated cells are washed twice, incubated overnight and spread on agar-xylan medium.

The clones were cultured in glass microtubes (8 x 35 mm), filled with 100 μΐ culture medium supplemented with oat xylan (0.5%). The vials are sealed with silicone stoppers and incubated on a shaker at 55 ° C for 24 h.

To select mutants, 96 samples are tested in parallel: 20 μΐ of the culture from each microtube is transferred via a multi-channel pipette to polypropylene microtubes where xylan hydrolysis occurs at room temperature. Reactions are initiated by adding 300 μΐ of substrate suspension to each tube and stop 400 μΐ of 3,5-dinitrosalicylic acid. After boiling for 15 min, 50 μΐ of each tube is transferred to a microtiter plate to read the optical density.

Clones showing the most significant xylanase activity are repeatedly tested with five consecutive punctures and compared to the parental strain.

Mutants showing a significant increase in xylanase activity were selected from 500 clones isolated after mutagenesis by EMS.

These mutants show an increase in xylanase activity in discontinuous cultures in xylan-containing medium.

The xylanase productivity of these mutants is also determined in cultures fed continuously. At a xylo-oligosaccharide flow rate of 0.36 g / h, the xylanase yield in mutants

1-1018 does not significantly increase (less than 10%) over wild strain 1-1017, as shown in FIG. 3, which shows different growth of strains 1-1017 and 1-1018 at two concentrations of xylo-oligosaccharides.

For example, if the flow rate of xylo-oligosaccharides increases to 0.67 g / L, strain 1-1018 exhibits a two-fold increase in xylanase production over strain 1-1017 (parent strain).

Obtaining mutants and optimizing the conditions of a continuously fed culture allows an increase in factor 3 xylanase production compared to discontinuous wild-type cultures on xylan. These increased enzyme ratios (up to 392 UI / ml) are obtained after 7 h of culture and the xylanase productivity by 1-1018 is therefore significantly higher than the best fungal and bacterial strains (Bertrand et al. Biotechnol. Bioeng., 33, 791-794 , 1989; Yu et al., 1987, cited above), producing xylanases, which take several days to obtain such a number of enzymes.

Example 6

Treatment of pulp (unbleached sulphate) with xylanase

8.5 g of wet pulp (corresponding to 1 g of dry matter), originating from marine pine and containing 60 mg of xylan, are treated at 60 ° C for 24 hours with 25 ml of buffer (containing different concentrations of xylanase).

The release kinetics of xylo-oligosaccharides are shown in FIG. 4. Co-release of lignin derivatives (by measuring the optical density at 280 nm) was demonstrated.

Example 7

Obtaining xylo-oligosaccharides from crushed corn cob axes g (dry matter) of corn cob powder (granulometry> 100 mesh) is suspended in 100 sodium of pH 6 containing h at 60 ° ml buffer

1000 U.I.

based on 50 mM xylanase acetate and incubated

C.

Fig. 5 shows transform up to (xylo-oligosaccharides of the compound's and arabinose).

that it is possible for xylan under these conditions to be oligosaccharides and arabino-xylo-oligosaccharides. These can be used as such or as xylose precursors%

Table I

Comparison of the production of xylanases by the strains according to the invention and according to the known state

tribe substrate xylanase productivity time reference cultivation υ.ΐ.χαΐ “ ¹ ^ ¹ h

Clostridium thermo1act i cum Clostridium stercorarlum glucose xylan 0.65 0.78 40 72 Brody, B .. E.Samain a P.Debelre 1990 Biotechnol.Lett. 12 <1) .65-70 Berenger.J.F .. C.Frixon. J. Bigllardi and N. Creuzet 1985 Can.J.Microbiol. 31.635-642 Thermoascus xylan 2.40 240 Yu.E.K.C..L.U.L. auriantacus Tan.M.K.H.Chan. L.Deschatelets and J.N.Saddler 1987, Enzyme Microb. 9.16 to 24 Thlelavia Solka -Floc ** 1.4 18 Merchant.R..F. terrestris Merchant and Ä.

Margaritls 1988 Biotechol.

Streptomyces xy ropes

16.76 livldans A

Streptomyces xylose

3.25 livldans B

Bacillus sp. xylose 2.47 alcalophile

10C7>, 513-516

Bertrand, J.L., R. Morosoli, F. Shareck and D. Kluepfel 1989 Biotechnol. Bioeng. 33C6). 791-794

Kluepfel.D., P.

Vats-Mehta, F. Äumont.F.Shareck and R. Morosol! 1990 Biochem.J.267,45-50

Okazaki V..T.Akiba K. Horikoshi and R. Akahoshi 1984 Appl.Microbiol. Biotechnol. 19, 335-340

Bacillus sp. xylo 44 by oligosaccharides invention mutant xylo 78.4

Bacillus sp. oligosaccharides of the invention * Solka-Floc: purified cellulose containing 5 to 7% hemicellulose

Table II

Degree of i fraction volume total proteins (Mg) total xylanase activity (U.I.) specific activity (U.I./mg.) yield (%) culture supernatant 1500 216 232000 1071 100 I concentration 150 197 225000 1142 97 II Q Sepharose Fast Flow 250 142 204250 1438 88 III phenyl sepharose 205 72 143500 1993 62

Table III

Total amino acid composition of xylanase

amino acid % molar estimated sequencing count number of residues residues (in partial sequence 1-20)

alanine 7.5 15 1 arginine 3.0 6 0 asparagine (A) (A) 3 acid. aspartic 14.5 29 1 cysteine (C) (C) 0 acid. glutamic 9.5 19 0 glutamine (B) (B) 2 glycine 15.5 31 3 histidine 1.5 3 0 isoleucine 3.0 6 1 leucine 3.5 7 0 lysine 1.5 3 0 methionine 0.5 1 0 phenylalanine 2.0 4 0 proline 4.0 8 0 serine 9.5 19 0 threonine 6.5 13 3 tryptophan (C) (C) 2 tyrosine 11.0 22 3 valine 7.0 14 1 (a) calculated as acid aspartic (b) calculated as acid glutamic (c) not measured

PV 820-73

Claims (13)

1. Bacillus strain producing xylanase deposited under number 1-1017 in the National Collection of Cultures of Microorganisms of the Pasteur Institute. 2. Bacillus strain producing xylanase deposited under number 1-1018 in the National Collection of Cultures of Microorganisms of the Pasteur Institute. 3. A thermophilic xylanase having a molecular weight of about 22 kDa and an isoelectric point of about 7.7. 4. The xylanase of claim 3, which is stable at about 60 ° C. Xylanase according to either of Claims 3 and 4, characterized in that its pH at optimal activity is in the range of 4.8 to 7 and is preferably about 6. Xylanase according to any one of claims 3 to 5, characterized in that it has the following N-terminal amino acid sequence: Asn-Thr-Tyr-Trp-Gln-Tyr-Trp-Thr-Asp-Gly-Ile-Gly-Tyr- Val-Asn-Ala-Thr-Asn-Gly-Gln. Xylanase according to any one of claims 3 to 6, characterized in that it comprises in its sequence 15 alanines, 6 arginines, 29 aspartates and asparagines, 19 glutamates and glutamines, 31 glycine, 3 histidines, 6 isoleucines, 7 leucines, 3 lysines , 1 methionine, 4 phenylalanines, 8 proline, 19 serine, 13 threonine, 22 tyrosine and 14 valine. Xylanase according to any one of claims 3 to 7, characterized in that it is secreted by Bacillus. Xylanase secreted by one of the claims of claim 8, from Bacillus strains, characterized in that it is according to one of the claims 1 or 2. A process for preparing purified xylanase according to any one of claims 8 and 9, characterized in that it comprises the steps of: - supernatant concentrations of Bacillus culture, - passages through the ion exchange column, - passages through a column of hydrophobic interactions. A method for producing a xylanase according to any one of the claims 8 and 9, comprising steps the growth of bacteria in a medium containing a nutrient substrate such as glucose and - production of xylanase induced by continuous feeding of the culture with xylo-oligosaccharides in appropriate amounts. 12. Use of xylanase according to some pulp bleaching. of claims 3 to 9 in the Use of a stage xylanase according to any one of claims 3 to 9 in the preparation of xylo-oligosaccharides from raw materials of plant origin.