JPH09506003A - Thermostable xylanase - Google Patents

Abstract

(57) Summary The present invention discloses an enzyme having xylanase activity. This xylanase is characterized by being active at a temperature of 80 ° C. or higher. This enzyme is obtained from anaerobic thermophilic bacteria. This enzyme is suitable for use in paper / pulp manufacturing processes. The present invention also discloses cloning and expression of a gene having xylanase activity obtained from the deposited strain.

Description

Detailed Description of the Invention                           Thermostable xylanase                                 Technical field   The present invention relates to novel microorganisms and novel enzymes, especially thermostable xylanase. .   These xylanases are derived from anaerobic thermophilic bacteria and are used in paper and pulp industry. It can be used under the conditions used in industry, that is, pH 9 or higher and temperature 70 ° C or higher. Is particularly active at temperatures of 80 ° C.                                BACKGROUND OF THE INVENTION   Xylan is a constituent of plant hemicellulose and xylan is 1,4- It consists of a glucosidic linked β-D-xylose substrate. Usually, xylan is xylo Side chains containing sugars and other pentoses, hexoses, uronic acids and acetyl groups or Having a group.   Bleaching pulp is the most important step in the paper manufacturing process. Paper / pulper The method used for pulp treatment in the industry is performed diagrammatically as follows.   Pulp has a so-called oxygen delignification process, in order to remove most of the lignin, p Treatment with H10-12 at a temperature of 80 ° C. Treated pulp is 2-5% rig Contains nin. This lignin makes the pulp brown. This pulp is then It is bleached by the stage bleaching method. With this bleaching method, chlorine, chlorine dioxide, hydrogen peroxide and And / or chemicals such as ozone are used to obtain white pulp of high quality paper.   Chlorine and chlorine-containing chemicals are commonly used in this bleaching method. However, The use of these chemicals forms dioxins and other chlorinated organic compounds. And they pose a threat to the environment. Therefore, generate this type of waste product There is a growing tendency to limit the use of certain chemicals.   This is a chlorine-free method, namely complete chlorine-free (TCF) and basic chlorine-free (ECF). ) Promoted the development trend of the method. In these methods, hydrogen peroxide or ozone is bleached. Used for. However, the use of these oxidizing chemicals is not Can have a negative effect on paper strength.   Some enzymes make pulp more acceptable for bleach, which results in bleach levels. Is known to reduce. In particular, xylanase is not suitable for paper and pulp processing. Have proved to be very useful. Xylanase is the lignin from pulp Increased extractability has been reported. In the current method, xylanase They are not active and do not survive under the conditions used during oxygen delignification. It is mostly used after the oxygen delignification step.   Xylanase is a hemicase involved in the adhesion of lignin to the cellulose network structure. Cleave the lulose chain. After treatment with xylanase, the lignin is more Easily removed.   Therefore, the use of xylanase does not consume active chlorine at 25-30% prebleaching. Leads to a decrease in This reduction of chlorine does not affect the quality factor of the resulting paper (Vii kari et al., 1986. Proc. of the third Int. Conf. Biotechnology in Pulp a nd Paper Ind., Stockholm, p.67-69 and Bajpai and Bajpai. 1992. Process B iochemistry. 27: 319-325).   The xylanase treatment also requires the use of other chemicals in the bleaching process, such as hydrogen peroxide. Reduce the need.   The use of xylanase from a bacterial source in bleaching kraft pulp has been reported. They are acid xylanases, the optimum pH and temperature of this enzyme is pH = 3 ~ 5, temperature = 30-50 degreeC. These values are effective conditions when pH ≥ 9 Not ideal for use in bleaching processes where the degree ≧ 70 ° C.   Xylanase from bacterial sources with optimally high pH and / or temperature will It has been reported to be used in the white method. Some of these are from the following species (In parentheses is the reported optimum pH and temperature for xylanase activity):   Bacillus pumilas(Bacillus pumilus) (pH = 7-9, temperature = 40 ℃, Nissen et al., 1992. Progress in Biotechnology7: 325-337),Bacillus stearosa Mophyrus (Bacillus stearothermophilus) (pH = 7, temperature = 65 ° C, international special Permit application WO 91/10724),Dictobogromas thermophilium(Dictvoglomus t hermophirium) (pH = 6-8, temperature = 70 ° C., European patent application EPO 511933),Low Do Thermus ( Rhodothermus) (pH = 5.5-6.5, temperature = 80-100 ° C, European patent issued Wish EPO 538177),Thermo toga(Thermotoga) (pH = 5-6, temperature = 90 ° C, international patent (Application WO 93/19171) andThermoanaerobacter Ethanolicus(Thermoanaeroba cter ethanolicus (Temperature = 68 ℃, Deblois and Wiegel. 1992. Progress in Biote chnology 7: 487-490).   The use of some of these xylanases for bleaching is claimed, ≧ 80 To date, it has been found that at the temperature of ° C it has the desirable property of having outstanding delignification activity. Not reported.                               SUMMARY OF THE INVENTION   The present invention is based on anaerobic thermophilic microbes isolated from New Zealand hot springs in pure culture. A xylanase obtained from an organism is disclosed. This microorganism was dated April 14, 1994 At the Centraal Bureau voor Sch of Baarn, the Netherlands. deposited with immelcultures) (CBS).   Further, the present invention is an enzyme having a considerable xylanase activity at a temperature of at least 80 ° C. Disclose the element. Furthermore, the present invention provides that the xylanase has a temperature of at least 80 ° C. It is disclosed that it has a remarkable delignification activity. The enzyme was obtained from the deposited strain It is.   Furthermore, the present invention discloses a method for preparing a xylanase active at a temperature of 80 ° C. This The method of culturing the deposited microorganism of the present invention in an appropriate medium and then performing the indicated activity. Includes recovery of this enzyme.   The present invention also discloses cloning of a gene encoding the above xylanase. You. Also disclosed is the expression of these genes in host cells. This enzyme is It is almost pure when obtained by law. Thus, also the present invention provides a xylanase An alternative method for expressing recombinant DNA is disclosed.   Furthermore, the present invention provides that at high temperature, under conditions equivalent to industrial application conditions using high pH. The application of xylanase is disclosed.                             Brief description of the drawings   Figure 1 shows the internal common distribution of 6 extremophilic strains. The phylogenetic analysis of an internal consensus sequence is shown. The sequences are named in the following order Included: Microorganisms / forward primers / recombination numbers. The branch number displayed is the block number of this array. This is the value obtained from bootstrap analysis.   FIG. 2 shows the sequential sequence of the internal common sequence of family G.   FIG. 3 shows that TG456 was inserted as a Sph1-BamH1 fragment in pJLA602. The xylanase domain of xynD is shown.   FIG. 4 is prepared from the bleached hardwood pulp TCF disclosed in Example 15. The paper quality of the prepared paper is shown. Ref is enzyme-free, 715 is TG456 xynD, 716 is TG53 xynD It is. A pull on Schoppen-Riegler values The modulus (A), porosity (B), tear (C) and rupture (D) are given.   FIG. 5: Prepared from the bleached hardwood pulp ECF disclosed in Example 15. The paper quality of the prepared paper is shown. Ref is enzyme-free, 715 is TG456 xynD, 716 is TG53 xynD It is. Schoppen-Riegler values pull The modulus (A), porosity (B), tear (C) and rupture (D) are given.   FIG. 6 is prepared from the bleached softwood pulp TCF disclosed in Example 15. The paper quality of the prepared paper is shown. Ref is enzyme-free, 715 is TG456 xynD, 716 is TG53 xynD It is. Schoppen-Riegler values pull The modulus (A), porosity (B), tear (C) and rupture (D) are given.   FIG. 7: Prepared from bleached softwood pulp ECF disclosed in Example 15 The paper quality of the prepared paper is shown. Ref is enzyme-free, 715 is TG456 xynD, 716 is TG53 xynD It is. Schoppen-Riegler values pull The modulus (A), porosity (B), tear (C) and rupture (D) are given.   FIG. 8 shows TCF (A) and ECF (B) bleached hardwood pulp and TCF (C) and Figure 16 shows the refining curves determined in Example 15 for E. coli and ECF (D) bleached softwood pulp. Ref is no enzyme, 715 is TG456 xynD and 716 is TG53 xynD.   Figure 9 shows TG456 xynD xylanase and Pulpzyme HB (Novonor A comparison between the optimum pH (A) and the optimum temperature (B) of Disc Co.) is shown.   Figure 10 shows TG456 xynD xylanase at 80 ° C, pH 7.0 (A) and 65 ° C, pH 9.0 (B), The comparison of the thermal stability of Pulpzyme HB (Novo Nordisk) is shown.                           Detailed Description of the Invention   The present invention discloses microorganisms isolated from New Zealand hot springs. these Microorganisms are characterized by anaerobic thermophilic bacteria, and Rainey (199 2． PhD thesis, University of Waikato, Hamilton, New Zealand) Was done.   This organism was screened sequentially using the Xylan-Agar dispersion assay. Was. Strains showing zona pellucida in this test were selected as potential xylanase-producing strains Was. This strain grows under anaerobic conditions of pH 7.0 and microorganisms at temperatures of 75 or 80 ℃. did. After concentration by ultrafiltration, the xylanase activity of the culture broth was adjusted to pH = 6.5 and 9, Analyzed in assay at degree = 80 ° C. (Example 1).   6 different strains were found to produce xylanase activity under the indicated conditions Was. These microorganisms were deposited on April 14, 1994 with deposit numbers CBS211.94, C By BS212.94, CBS213.94, CBS214.94, CBS215.94 and CBS216.94 Was entrusted.   The present invention also relates to an enzyme having xylanase activity, especially at a temperature of at least 80 ° C. , And an enzyme having a remarkable xylanase activity at pH 6 or higher is disclosed. The enzyme is , Mutant strains and variants of the deposited strain.   The expression “significant activity” means that the enzyme of the present invention is at least at 80 ° C. Also have 40% activity, so these are preferably at 70 ° C. This is at least 60%, more preferably at least about 80%, More preferably, it is about 200%.   Furthermore, the present invention is active at temperatures of about 80 ° C. or significantly under these conditions. A method for preparing xylanase having various activities is disclosed. This method is Includes cultivation of the deposited microorganism of the invention, followed by recovery of the enzyme having the indicated activity . Isolation and purification of xylanase can be performed according to a conventional method.   Partial purification of the enzyme is exemplified in Example 2. In this example, the cells are first First, it is removed by hollow fiber filtration. Then tamper The protein is further purified by the addition of ammonium sulfate to a final concentration of 1M. . The solution was then loaded onto a Phenyl Sepharose column and the protein was Elute with sodium chloride. Finally, the protein was concentrated by ultrafiltration , Salts are removed by diafiltration.   To confirm the xylan-degrading ability of xylanase at alkaline pH, pH 7 and 9, a comparison of the activity of xylanases isolated from the indicated strains using different substrates And 70 ° C. (Example 3). This xylanase is remarkable at alkaline pH It was shown to have activity. Depending on the substrate and assay, at pH 9, this xyla Naseses have about 20% or more of the activity they have at pH 7. A xylana The enzyme was shown to retain 80% of the activity at pH 7 at pH 9.   The thermostability of xylanase isolated from the indicated strains varies significantly. Some Xylanase is highly thermostable. They are pH = 9 and temperature is 80 ℃ at 2:00 It has a half-life greater than or equal to (see Example 4). The pH of the xylanase of the present invention is 9 and the temperature is The half-life at 80 ° C is at least 10 minutes, preferably 20 minutes or less under these conditions. Above, more preferably 30 minutes or more, still more preferably 60 minutes or more, most preferred In other words, the half-life is 120 minutes or more.   The present invention is also characterized by encoding a xylanase obtained from an anaerobic thermophilic strain. Disclosed is the cloning and expression of characteristic DNA sequences. The present invention provides the Vector containing an expression vector characterized in that it contains a DNA sequence coding for Lanase Is disclosed. Microbiota characterized by being transformed with one of those vectors Physical host cells are also disclosed.   Sequence analysis of the DNA sequence encoding the xylanase of the invention reveals that the resulting sequence is Two enzymes, Gilkes et al. (1991, Microbiol. Rev. 55: 303. -315), it was classified into the F-type and G-type previously defined.   The xynA, xynB and xynC sequences obtained from the microorganism of the present invention are F-type xylanase. Belong. The xynD sequences obtained from these microorganisms belong to the G-type xylanase ( See Examples 6, 7, 8 and 9). G-type xylanase derived from thermophilic microorganism Nothing has already disclosed that it is the best in. Source of Xylanase of the Invention This thermophilic microorganism, which is Defined as a living being. Preferably, this optimal growth temperature is above 68 ° C, and more preferably Most preferably, this optimal growth temperature is above 70 ° C, most preferably this optimal growth temperature is 75 ° C. That is all. Of the F-type and G-type two xylanase DNA sequences of the present invention in E. coli. Expression is shown to give the active protein (see Examples 10 and 11). This Protein is active at a temperature of 80 ° C.   The cloning and expression proved in the examples of the present disclosure show that the cloning and expression in homologous microorganisms was successful. Allows expression of genes. In addition, the expression depends on appropriate expression and secretory regulatory sequences (secretion  It can also be performed on other microorganisms using the regulating sequences). use Microorganisms that may be mentioned include yeasts, fungi and bacteria. With specific microorganisms Aspergillus niger, Kluyveromyces la Kluyveromyces lactis and Bacillus licheni formis).   The enzyme of the present invention comprises oat spelt xylan and birch xylan. It was shown to have remarkable activity with respect to. Furthermore, the enzymes of the present invention are not The ability was tested. This bleaching ability depends on the delignification activity of the enzyme of the present invention. Is determined. The present invention provides significant delignification, as measured on some wood pulps. Disclosed are enzymes having activating activity. Enzyme preparation, xylanase, at least 80 ° C It enables delignification of wood pulp at the temperature of. The expression "wood pulp" is It should be understood in a broad sense and is meant to include any lignocellulosic material. It is to be illustrated.   This enzyme tested its bleaching ability on both hardwood and softwood pulps. I was tested. Lignin removal was measured in two ways (Example 5). In the medium Lignin was determined in the A300 test. In this test, all preparations were hardwood All of the softwoods showed remarkable delignification activity at 80 ° C. pH 9 At 80 ° C, the activity on conifers is at least 43% of the activity at pH 6. In the case of hardwood, the three strains at pH9 showed 50% of the activity at pH6. The above three activities were shown, and the other three showed 18 to 30% activity. Also, softwood pulp Bleaching of Pulp Determines Reduction of Pulp Lignin Content Using the Kappa Test Was measured by The kappa number is the xylanase preparation Thus, there was a 0.5-0.4 unit reduction after pulp culture at pH 9 at 80 ° C.   Further, the bleaching capacity of the enzyme of the present invention is, for example, produced by a conventional processed pulp. Paper, further paper made from pulp cultivated with the above enzymes, and paper made without enzyme It can be measured by the increase of its ISO whiteness in comparison with the prepared paper. The conventional method Treatment is performed using conventional techniques such as hydrogen peroxide, ozone, chlorine gas or chlorine dioxide. Includes exposure to bleach.   The cloned F-type and G-type xylanase of the present invention expressed in E. coli are coniferous trees. Their performance was tested in an ECF bleaching experiment on both and hardwood pulp ( See Example 12). Surprisingly, G-type xylanase (encoded in the xynD gene Was the best F-type xylanase, at most 1.2% compared to ISO brightness increase Compared to F-type xylanase, up to 6.8% ISO delta brightness than no enzyme control It was observed that the performance of bleaching of both softwood and hardwood is better. Was done. Furthermore, tests for thermostable G-type xylanase showed that these were coniferous and broadleaf. It was also shown that TCF bleaching experiments on both wood pulp produced excellent results (Examples). 14 and 15). Examination of the properties of papers made from pulp bleached in this way The testimony showed that it was almost the same as the control paper without enzyme.   Cloned G-type xylanase is extremely thermostable even at high pH. 8 A half-life of 30 minutes or longer was measured at 0 ° C and pH 7.0. At 65 ° C and pH 9.0, this The half-life of the enzyme is not significantly reduced even after 120 minutes of incubation (see Example 16).   The G-type xylanase of the present invention has a half-life of at least 10 at pH 9.0 and 65 ° C. The half-life under these conditions is preferably 20 minutes or more, more preferably Is 30 minutes or more, even more preferably 60 minutes or more, and most preferably a half-life of 12 It is 0 minutes or more.   The present invention is based on the fact that its internal consensus fragment (ICF) , Encodes a thermostable G-type xylanase showing 80% or more identity with the ICF of SEQ ID NO: 12. The DNA sequences that convert are disclosed. This G-type ICF is shown here in SEQ ID NO: 12. Between the sequences corresponding to SEQ ID NO: 4 and SEQ ID NO: 5 (nucleotide position 295 ~ 623). Preferably, with this G-type ICF The identity is 87% or more, more preferably this identity is 95% or more, and most preferably, This identity is greater than 99%.   In yet another embodiment, the invention provides the amino acid sequence of SEQ ID NO: 13, Disclosed are G-type xylanases that share at least 70% identity. Preferably, The amino acid identity is 80% or higher, more preferably the amino acid identity is 90%. More preferably, the amino acid identity is greater than 95%, and most preferably Amino acid identity is greater than 99%.   The enzyme of the present invention is used immediately after the oxygen delignification step in the pulp preparation method described above. Can be used for Expensive cooling and pH adjustment as a result of the temperature and pH characteristics of this enzyme. Avoid knots. In another embodiment of the invention, the enzyme is an oxygen delignification engineer. Used before or during the process. Prior to this step the lignin concentration was high and therefore the key The application effect of silanase is even greater.   Furthermore, as an application of the enzyme preparation of the invention, in particular wood is Methods of treating with the preparation are disclosed.   Similarly, fluff pulp can be treated with the enzyme preparation of the present invention.   Furthermore, the invention relates to a paper made from wood pulp treated with the enzyme preparation of the invention. , Board and fluff pulp.                                 Example 1                   Isolation of a thermostable xylanase activity producing strain   Are xylanase-producing microorganisms in hot springs in various hot areas of New Zealand? I got it. This strain is used as a substrate from hot springs with a pH of between 6.0 and 8.5 at temperatures above 70 ° C. Isolated by in-situ concentration using autospertoxylan as. Of stock The culture should be at a temperature (70 °, 75 ° or 85 ° C.), obtained by further anaerobic culture in a 2/1 medium + xylan laboratory Was. The composition of 2/1 medium + xylan is as follows.Anaerobic 2/1 medium Resazurin (0.1%) 1.0 ml / l SL10 Trace element 1.0 ml / l Ferric chloride solution (0.28 mg / ml) 1.0 ml / l Wolin's Vitamins 0.1 ml / l Salt 0.9 g / l Magnesium chloride hexahydrate 0.2 g / l Dipotassium hydrogen phosphate 1.5 g / l Potassium dihydrogen phosphate 0.75g / l Ammonium chloride 0.9 g / l Cystine 0.75g / l Yeast extract 0.5 g / l Bacto Tryptone 1.0 g / l Auto spelled xylan 2 g / lpH 7.0 Dispensing under nitrogen gas.Warin vitamin mixture                                                 mg / 100ml Biotin 2.0 Folic acid 2.0 Pyridoxine hydrochloride 10.0 Riboflavin 5.0 Thiamin 5.0 Nicotinic acid 5.0 Pantothenic acid 5.0 Vitamin B12 0.1 p-aminobenzoic acid 5.0 Thioctic acid 5.0SL10 Trace element (modified) 5M hydrochloric acid 15ml                                                 mg / l Zinc chloride 70 Magnesium chloride tetrahydrate 100 Boric acid 6 Cobalt chloride hexahydrate 130 Cupric chloride dihydrate 2 Nickel chloride dihydrate 24 Sodium molybdate dihydrate 36   A pure culture of the strain was obtained from a single colony in an agar roll tube. Ultra The culture concentrate obtained in excess was dyed xylan method (see below) and And PAHBAH method (see Example 3) at 70 ° C. and 80 ° C. at pH 6.5. And xylanase activity at 9.0.   The six strains deposited with CBS (Centraal Bureau voor Schimmelcultures) are 1 is shown.   Connected to Remazol Brilliant Blue R (Sigma) A dyed xylan consisting of combined larch tree xylan was prepared using Biily et al. (1985. Anal. Biochem 144, 142-146).   Add larch xylan (6.7 g) to 250 ml of water and stir at room temperature for several hours to dissolve. I understand. Remazole Brilliant Blue R (10.0 g) was added to this mixture. Dissolution After solution, drop 20ml sodium acetate solution (2.7g sodium acetate in 20ml distilled water) drop Added in the form of. Then a solution of 6 g of sodium hydroxide in 70 ml of water was added and stirred. I continued for 18 hours. The dyed xylan formed was precipitated by adding 700 ml of 96% ethanol. I let you. After standing for 6 hours, the precipitate was separated by filtration (Whatman GF / A). fill Turke with 3 liters of a 2: 1 mixture of 96% ethanol / 0.05M sodium acetate, Then, it was washed with 1 liter of 96% ethanol and 3 liter of acetone until the filtrate became transparent. . The yield was 10.5 g stained xylan.   5.1 g of stained xylan was added to 150 ml of 0.1 M MOPS buffer for enzyme assay. It dissolved in pH 6.8. The mixture was stirred at 70 ° C. for 4 hours to dissolve. Cool to room temperature The insoluble matter was then removed by centrifugation at 10,000 xg. For activity assay, 3. Stock solution containing 34% w / v stained xylan diluted in 0.1M MOPS buffer, pH 6.8. Then, a 0.5% dyed xylan working solution was obtained.   Enzyme assays using stained xylan should be mixed with the appropriate buffer while mixing. , 0.9 ml stained xylan solution (0.5%) was added to 0.6 ml enzyme preparation. this Mixed A portion (0.4 ml) of the compound was transferred into 0.8 ml of 96% ethanol as a control (blank). did. The remaining solution was incubated at 70 ° C. for 90 minutes. 0.4 ml of this test mixture is added to 96 The reaction was stopped by transferring to 0.8 ml of% ethanol. This solution is It was left at room temperature for 30 minutes to precipitate the split-stained xylan. This test sample With a Beckman Microfuge E at full speed for 5 minutes Upon heart separation, the absorbance of the supernatant was measured with a spectrophotometer at 595 nm.                                 Example 2                             Isolation of enzyme preparation   Anaerobic fermentation, either at a temperature of 75 or 80 ° C (depending on the microorganism to be cultured) In static culture for 24 hours, 2 liters of 2/1 + xylan medium containing 1800 ml Performed in a Schott bottle. From 10 liters of well growth culture to 0.01 % Triton X100 (Amicon DC 10 LA and Amicon 0.1 μm H5MP01-43 filter ) Was used to remove cells by hollow fiber filtration. Sulfuric acid up to a final concentration of 1M The ammonium was added to the cell-free culture medium. The resulting solution was treated with 1M sulfuric acid Phenyl Sepharose equilibrated with monium (Pharmacia-Fast Flow-low displacement) ) Was pumped into a 9 × 15 cm column containing 1 liter. Flow rate is 150-200ml / min Was. The column was washed with 51 of 1 M ammonium sulfate. 5 liters of xylanase Eluted with 1M sodium chloride. Fractions were collected in 500-1000 ml volumes. Xylanase activity was determined in that fraction (by the PAHBAH method disclosed in Example 3). ), The active fractions were pooled, ultrafiltered to a small volume and Amicon stirred with a YM2 membrane. Use an Amicon Stirred Cell (Model 2000A or 8400) to reduce the salt concentration. The membrane was filtered for lowering.                                 Example 3               Characteristics of xylanase activity of partially purified enzyme preparations. Analysis method   The xylanase activity assay is based on the Sumner assay (Sumner et al.  al., 1921. J. Biol. Chem. 47: 5-9) using a modified method. Apart , Xylanase activity was determined by the method of Lever (1973. Biol. Med. 1: 274-281). Determined using the base, modified PAHBAH assay.Method 1 Sumner assay of xylanase activity for oat spelled xylan   An autospertoxylan substrate solution was prepared as follows.   4 g of oat spelled xylan was suspended in 100 ml of distilled water, Ultrasonic treatment for 6 minutes (ultrasonic device: Sonic and Materials, Vibrase Type VC250B), incubated at 100 ° C. for 10 minutes, and Sorval RC-5B Centrifuged at 10,000 rpm for 10 minutes in a centrifuge. This supernatant Was used as the substrate solution. Contains about 2% oat spelled xylan.   The assay was performed as follows.   Dilute the test tube with 200 μl of autospertoxylan solution and an appropriate buffer. The diluted enzyme preparation (Example 2) was filled with 600 μl. Take this test tube in a water bath Incubated for 15 minutes under measurement conditions. After culturing, 7.2 ml of DNS (dinitrosal) icylic acid, dinitrosalicylic acid) reagent was added. This mixture is placed in a water bath, Heat at 100 ° C. for 10 minutes, then cool the tube on ice. Absorbance is It was measured at 575 nm. To remove the background absorbance of this enzyme sample, The control experiment was conducted as follows.   Test tubes containing the substrate without enzyme preparation were incubated under the same conditions as the test samples above. Was. After 15 minutes of incubation, 7.2 ml of DNS and enzyme sample were added in that order.   One unit of xylanase activity (xU) is 1 μmol measured as reducing sugar It is defined as the amount of enzyme that produces xylose equivalents of.   The actual measurement conditions were pH 7.0, 9.0 and 70 ° C. 50 mM at pH 7 50mN borate / KOH buffer at pH 9 using phosphate buffer Used. Method 2 Sumner assay of xylanase activity for birch xylan   Almost the same method as disclosed in Method 1 was used. Autospeltoxylan solution Instead, a birch xylan suspension was used. The birch xylan substrate solution It was prepared in this manner. 4 g of birch xylan is suspended in 50 ml of 0.2 N NaOH and is clearly dissolved. I was shaken up to. Adjust the pH of the solution to 7.0 with glacial acetic acid and add water to 100 ml. This solution was centrifuged at 10,000 rpm in a Sorvall RC-5B centrifuge. I took it off. This supernatant was used as a substrate solution. About 3% xylan birch Contains. The test conditions were pH 7 and 9 and 70 ° C. The results are shown in Table 3. . Method 3PAHBAH Assay of Xylanase Activity for Autospertoxylan   Modification of the PAHBAH method (Lever, 1973) was performed on the PAHBAH reagent and Is as follows:   0.05M trisodium citrate, 0.1M sodium sulfite, 0.02M calci chloride Um, 0.5M sodium hydroxide and 0.1M p-hydroxybenzoic acid hydrazide (P AHBAH).   For assay of enzyme preparation, 0.05 ml or 0.1 ml enzyme preparation was added to 0.3 ml Substrate buffer (50 mM Bis-Tris-propane, required pH, +0.2 % Oat spelled xylan suspension). Add an appropriate amount of water to a final volume of 0.5 Add to ml. The culture was carried out at 70 ° C. for 30 minutes as usual. reaction To stop, 1.0 ml of PAHBAH reagent was added after incubation and the sample was added to 100 Heat at 6 ° C for 6 minutes. The blank is a test in which the enzyme is added after the PAHBAH reagent. It consists of the substrate buffer which was cultivated in the same manner as the material. Determine the absorbance at 420 nm Beckmaer all samples before removing suspended xylan from the supernatant. Centrifuge for 1 minute at full speed with a Beckman Microfuge E Was. The results are shown in Table 4. From this table, at pH 9.0 and 80 ° C, all strains had pH 6.0. It is concluded that it still has a remarkable activity as compared to 70 ° C.                                 Example 4                       Thermostability of xylanase activity   The half-life of the xylanase activity of the enzyme preparation was determined as follows.   The enzyme preparation was added to 1 mM in 100 mM TAPS buffer (pH 9.0 at 80 ° C). It was diluted to / 10 and cultured at 80 ° C. Samples were tested for 0, 10, 20, 40, 60 and Remove in 120 minutes and place in a convenient 1/20 to 1/100 for this final assay. Add 100 mM MOPS buffer (pH 6.0 at 70 ℃) on ice until the final dilution. Added. This sample was tested for PAHBAH assay as described in Method 3 of Example 3. The Say method was used and kept on ice until assayed at 70 ° C, pH 6.0. Conclusion The results are shown in Table 5.                                 Example 5                             Delignification activity   The delignification performance of xylanase was determined by two methods. Use A300 method Was used to estimate the amount of lignin released from the pulp after enzyme treatment. Kappa The residual lignin content of the pulp after treatment was measured using a kappa assay. Was.Method 1: A300 test   To measure delignification in the A300 assay, the enzyme preparation was used as a conifer or broad-leaved tree. A concentration of 2 PAHBAH units per gram of wet pulp (dry with wood pulp) About 6 PAHBAH units per gram of pulp). Culture at 80 ℃, 2:00 with both MOPS buffer, pH 6.0 and TAPS buffer, pH 9.0 I went for a while. The pulp concentration in the culture was 0.1 g wet weight / ml. Two kinds of pa I used a lupe. That is, Kraft softwood pulp and oxygen delignification after oxygen delignification. It is a kraft hardwood pulp after gnin. The properties of these pulps are shown in Table 6.   The amount of lignin removed from the pulp is supported on a sintered glass filter. The supernatant was separated from the pulp by suction filtration with a Whatman GF / C filter The absorbance of the supernatant was then determined at 300 nm and determined. A300 test The results are shown in Table 7. Delta A300 values of ≧ 0.2 in this assay It was significantly higher than the round level. Therefore, this sample was It has a delignification activity. Method 2: Kappa test   The kappa test is based on the TAPPI protocol T236 (TAPPI, Technology Par (available from K. Atlanta, USA) with some modifications. enzyme The preparation was added at a dose of 10 xU / g pulp (dry weight), pH 9 and 2 at 80 ° C. Incubated for hours. As a control, pulp was added at the same time and under the same conditions without enzyme addition. It was cultured in. Oxygen delignified softwood pulp having the properties shown in Table 6 was used.   The difference between the kappa value with and without enzyme is the kappa reduction. Called, the value of delignification. The kappa reduction is shown in Table 8. In this assay , ≧ 0.5 are values significantly higher than the background level. Therefore, the previous As in the example, this sample shows that the enzyme has a significant delignification activity at 80 ° C. Is shown.                                 Example 6   The codon for the internally consistent fragment of the gene encoding the thermostable F-type xylanase. Rowing and sequencing 6.1   PCR amplification of internal fragments of the xylanase gene   Three PCR primers were added to increase the internal common fragment of the xylanase gene. Used to widen. That is, two kinds of forward primers (xynFA, {5'CAC ACK C TK GTK TGG CA 3 ', SEQ ID NO: 1} and xynFB, {5' CAT ACK TTK GTT TGG CA 3 ', SEQ ID NO: 2}) and one kind of reversion primer (xynR, {TMG TTK ACM ACR TCC CA, SEQ ID NO: 3}). The xynFA and xynFB primers should be bound at the same location. The sequence of the xylanase gene in the forward common region Is slightly different in. PCR conditions were as follows: (94 ° C for 1 min, 50 ° C 1 minute at 72 ° C for 1 minute) x 30. All Family F internal common fragments are abbreviated It was 350 bp.6.2   Sequencing of PCR products   All internal xylanase PCR-products (about 350 bp) were M13mp10. Before cloning into the Sma1 (phosphatase) site of the sequence vector, End repaired (backfilled) as described below.   Step 1-Ammonium acetate precipitation: (A) 50 μl PCR mixture was added to TE buffer (10 mM Tris-Cl, 1 mM EDT A, pH 8.0) to 100 μl. (B) 100 μl of 4M ammonium acetate and 250 Add μl of 100% ethanol. (C) 15 minutes on ice (or one day at -20 ° C) Culture at night). (D) Centrifuge at 16,000 rpm for 15 minutes to obtain a supernatant. Throw away. (E) Wash the pellet in 500 μl of cold 70% ethanol. Re-centrifuge (16,000 rpm for 5 minutes) and discard the supernatant. (F) The pellet is dried under vacuum for 5 minutes and resuspended in 20 μl TE buffer.   Step 2-end repair of the PCR fragment: (A) 20 μl of the precipitated DNA was added to 3 μl of 10x ligase buffer (verin). (Germanheim) 1 μl of 12.5 mM dNTP's (Pharmacia DNA polymerization mixture) 0.5 μl (5 U) of E. coli DNA polymerase large (Klenow )) Fragment (BRL Technology Ltd.), 0.25 μl (2.5 U) T4 DNA Polymerase (Boehringer Mannheim), 0.25 μl (2.5 U) of T4 polynuclear Add reotide kinase (Boehringer Mannheim) and water to 30 μl . (B) Incubate at 37 ° C for 30 minutes and thermally kill the enzyme by incubating at 70 ° C for 10 minutes Let it. Step 3-Gel purification of the end-repaired xylanase fragment: (A) DNA was added to 2% LM of 1x Tris-acetate buffer (pH 7.8). Pass through P agarose. (B) A 350 bp xylanase band from agarose delete. (C) Gene Clean procedure (Bio 101   Inc. ) Is used to purify DNA from agarose slices. Step 4-ligation into M13mp10 (Sma1-phosphatase): (A) 1 μl of M13mp10 vector DNA (appropriately diluted to approximately 10 ng / μl) 20 to 50 ng of insert DNA (xylanase common primer fragment) Ragment) 1 μl of 10x ligase buffer (Boehringer Mannheim) 1 μl of T4 DNA ligase (Boehringer Mannheim) and 10 μl of water Mix with. (B) Incubate at room temperature overnight. Step 5-Transformation of the ligation mixture into E. coli strain JM101:   (A) Chung et al. (1989. Proc. Natl. Acad. Sci. 86: 2172-217). Using the DMSO-mediated transformation technique of 5.), in a total of 10 μl of the ligation reaction mixture. Transform JM101. (B) M13 / JM101 as a plate Method (Sambrook et al., 1989. Cold Spring Harbor Laborat ory Press) to isolate the recombinant M13 plasmid.   Recombinant M13 phage containing the internal xylanase consensus fragment were stained- Using the primer chemistry (the sequence primer used was a standard M 13 forward (stained and labeled) primer (ABI Ltd.)), Applied by O System 373A automated DNA sequencer, ssDNA (Sambrook et al . 1989). All DNA sequence data are silicon graphic parts Sonar Iris Workstation (Silicon Graphics Personal Iris Workstatio Analysis using G C G sequence analysis software (installed on Iris) running on n) ,It has been processed.Family F xylanase internal fragment sequence results :   Amplified from the 6 strains shown in Table 1 using xylanase common primers. Based on the PCR fragment generated, each microorganism was identified as a family F xylanase gene. It was predicted to include genes between 1 and 3. This result is known as the bootstrap Boot-strap analysis (Wisconsin Molecular Biology Pakage, Devereux , 1984, Nucleic Acids Res. 12, 387-394). In FIG. 1, various The xylanase and the phylogenetic tree of the strain are shown. Family F common fragment The cleotide sequence reveals that each microorganism contains three different family F genes. It's mild. Each of the family F xylanase genes of each microorganism has a cluster Separate xylanase clusters labeled A, Cluster B and Cluster C (Based on nucleotide and primary amino acid sequence homology). TG456 The full-length xylanase amplified from E. coli belongs to cluster A and is later identified as TG45. It was named 6 xynA. Furthermore, TG456 cluster B xylanase (TG456  xynB) and cluster C xylanase (TG456 xynC) Was identified.                                 Example 7   Cleavage of an internal common fragment of the gene encoding the thermostable G-type xylanase. Rowing and sequencing 7.1   PCR amplification of internal fragment of G-type xylanase gene   Family G internal common fragments (ICFs) In the polymerase chain reaction (PCR) using common primers (GF and GR) Isolated. The contents of this PCR were as follows. Note: ℃ means Celsius degree, x means Represents the number of cycles (ie x1 equals one cycle).   (4 minutes at 94 ° C) x1   (94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 1 minute) x35   (94 ° C for 1 minute, 45 ° C for 1 minute, 72 ° C for 6 minutes) x1   PCR was performed using the following components in a 50 μl reaction: 100 ng G F primer, 100 ng GR primer, 0.25 mM dNTPs, 1 unit Taq Polymerase, 0.25 mM magnesium chloride, 10 mM Tris-hydrochloric acid, pH 8.8, 50 mM potassium chloride, 0.001% gelatin, 1 ~ 10 ng template DNA.   Two PCR primers amplify a common fragment of the xylanase gene Used to:   GF: TATNTGRSTNTMTATGGWTGG (forward internal common primer -) SEQ ID NO: 4.   GR: CCGCTNCTTTTGGTANCCTTC (return internal common primer ) SEQ ID NO: 5.   In all 6 strains, PCR fragments were found by amplification with common primers. Was issued.   Two PCR products (DNA fragments) were amplified: as expected 300 bp fragment and an unexpected 600 bp PCR product-this 600 The bp fragment was the head-to-tail dimmer of the 300 bp PCR-product: maybe , This 600 bp species, PCR as a result of homology between the GF and GR primers. It is the result of self-priming during the reaction.7.2   Sequencing of PCR products   The 300 bp fragment amplified from each microorganism was end repaired (Example 6). reference).   The end-repaired fragment was then subjected to the diene clean method (GeneClean proce dure) (Bio 101, La Jolla, Calif.) using 1% low melting temperature agar Purified from rose gel (run in Tris-acetate running buffer). Approximately 10 ng of the end-repaired, gel-purified 300 ICFs were added to BMT in 10 μl reaction. 4 DNA ligase was used to ligate into the Sma1 site of M13mp10.7.3   Sequencing of PCR products   Six independent phage from strains TG457 and TG53 were sequenced. Of these strains The fact that only a single-stranded G-type xylanase gene is present in each of these sequences ( It is clear from FIG. 2). In addition, TG453, TG456, TG479 and TG An M13mp10 recombinant consisting of the family G-ICFs from 631 was sequenced. These microorganisms show up to 13% change in DNA sequence but almost the same xyla It consisted of all the Family G xylanase genes encoding the Nase.                                 Example 8                   Full length F-type xylanase sequence   Three different types of F-xylaner per substrate for internal PCR fragments Ze genes have been identified: xynA, xynB and xynC.   Full-length using Genome Walking PCR (GWPRCR) The xylanase gene of rape has been isolated from most strains.   The complete long sequence of the TG456xynA gene has been determined. Complete distribution of the xynA gene The columns are given in SEQ ID NO: 6. TG456 Xylanase A-encoded amino acid The sequence is given in SEQ ID NO: 7.   For xynB and xynC, the gene has multiple domains with one xylanase domain. It was discovered to encode the in enzyme. These xylanase domains are key Using PCR primers designed on the sequences bordering the silanase domain Subcloned.   Partial sequence information for xynB and xynC from TG456 is SEQ ID NO: 8 and SEQ ID NO: No. 10 given in each. TG456 xylanase B and C encoded The resulting amino acid sequences are given in SEQ ID NO: 9 and SEQ ID NO: 11, respectively.                                 Example 9                   Complete sequence of G-type xylanase gene   Full-length using Genome Walking PCR (GWPRCR) The porcine xynD gene was isolated from most strains.   The complete sequence of the xynD gene of TG456 is given in SEQ ID NO: 12 and encodes T The G456 xylanase D amino acid sequence is given in SEQ ID NO: 13.                               Example 10   Construction of expression vector and host for xylanase production from cloned gene   In the common PCR primer, use the appropriate expression vector to Appropriate restriction sites were designed to allow subcloning.   The xynA and xynC genes are mutated in Lambda L for in-flame fusion. And R promoters (lambda L and R promorters) [Gibbs, M .; G. Saul D. J, Lu thi, E., and Bergquist, P. L. (1992). "Cardo Serum Saccharolyticum" (C β-mannanase from aldocellum saccharolyticum is a multidomain enzyme Part. Appl. Environ. Microbiol. 58 (12): 3864-3867], pJLA602 Inserted into Ncol-BamHI of the expression vector of Escherichia coli [Schauder, B., Bluker, H., Fra nk R., and McCarthy, J. E. G. (1987). Inducible Expression Vectors Incor porating the Escherichia coli atpE Transcriptional Initiation Region. Ge ne 52: 279-283].   The xynB and xynD genes were inserted into the unique Sph1-BamHI site of pJLA602.   The construct was constructed in E. coli JM101 and E. coli DH5α using standard transformation techniques. Was transferred to.   Figure 3 shows xynD from TG456 inserted into pJLA602 of Sph1-BamHI fragment. The sequence of the xylanase domain is shown.                               Example 11                   Production and recovery of cloned xylanase   To obtain a protein sample derived from cloned F-type xylanase, E. coli Was fermented in a 10.0 liter capacity LH fermentor (Series 210). This culture The material is continuously aerated (4.8 l / min), shaken (500 rpm), and pH adjusted (pH 7.1). Maintained at 30 ° C until induction (42 ° C). The medium and other additives used are as follows. It is as follows. Luria broth (for inoculation culture):   Trypton 10g   Yeast extract 5g   Sodium chloride 10g   Up to 1000 ml of water BGM2 bulk growth medium for fermentor run (volume is for final medium, ie 8.5 liters of 2 batches) J):   3.22 g of ammonium chloride   Potassium dihydrogen phosphate 1.05g   Magnesium sulfate heptahydrate 0.135 g   Potassium sulfate 0.043g   Ferrous sulfate heptahydrate 0.003 g   SL-10 Trace element 1ml   Trypton 10g   Yeast extract 4.7g   34 ml of glycerol   Up to 1000 ml of water   Fermentation vessel containing about 6 liters of water in it is heated at 121 ° C for 30 minutes under pressure. After sterilization by physical treatment, it was cooled to 30 ° C. Media Concentrate (8.5 Fold Concentrate (fold concentrate)) with a sterilized 0.2 μm cartridge filter (Sartorius) I pumped through. The following additions were made before culturing.   Defoamer (Bevaloid 5901) 10ml (Pressurized heat treatment)   Calcium chloride 1 ml (pressurized heat treatment)   Ampicillin 850mg (filter sterilization treatment)   Thiamine (JM101 culture only) 8.7 mg (filter sterilization)   The liquid volume was then adjusted to 8.5 l. The pH of the medium was adjusted to 7.1. 4.8 l / min The filter-sterilized air was sparged through the fermentor at a flow rate of. Finger pro The fermentor temperature was maintained at 30 ° C. by circulating hot and cold water through the tube. pH adjustment is By addition of sodium hydroxide (5M) or sulfuric acid (2M) under a toclave Was. The stirring speed of the fermentor was 500 rpm.   Culture is 0.2-0.4 OD₆₅₀100 μg / ml ampicillin and luriab Starting with a fresh culture of E. coli clones grown in Ross (approximately 10.0 ml). The cells were subjected to an OD of between 11 and 13 at 30 ° C.₆₅₀To then concentrate in the medium Fed-batced, harvested, OD between 14-16₆₅₀Have grown to. This They were subsequently triggered by raising the temperature to 42 ° C.   Cells have maximum OD₆₅₀Were collected 4 hours after the Hollow this cell Recovered by fiber filtration (0.1 μm, Amicon), 50 mM Tris-HCl (pH 8.0), 7.5 mM EDTA, 7.5 mM β-mercaptoethanol, 0.02% PMSF, 1.0 μ Resuspend in M pepstatin A, 0.02% DNase and 0.02% lysozyme, 4 ° C The cells were cultured for 1 hour (total volume: about 1 liter). Until the cells are lysed (observed under a microscope) Sonicated on ice in a 160 ml batch for 1 to 6 minutes blast for 4 to 6 minutes. Was. The solution of 56 mM PMSF (in isopropanol) was sonicated for 2 minutes each. The final concentration was less than 1 mM PMSF (each Is equivalent to 160 μM). After lysis was complete, cell debris was removed by centrifugation . The supernatant is allowed to stand until a clear precipitation of protein occurs (typically about 20-30 minutes). ) Heat treatment was performed at 70 ° C., and the denatured protein was removed by centrifugation. Phenyl- Prior to Sepharose chromatography, ammonium sulphate was heat treated to Was added to the final supernatant to a final concentration of 1.0 M (this was Name it). Cell pellets from sonicated cells were treated with 50 mM Tris-HCl (p H8.0), 1 mM suspension of 5 mM EDTA, 7 mM β-mercaptoethanol Re-extract with liquid and then second heat treatment at 70 ° C for 15 minutes to precipitate the precipitated protein Was removed again by centrifugation. This extracts an additional 20-40% xylanase I understood that This was named a 2 ° heat-treated extract. Ammonium sulfate, To this 2 ° heat-treated extract, add up to 1.0M and add 1 ° and 2 ° heat-treated extract. Pooled before phenyl-sepharose separation. Xylanase activity, 1.0M chloride 1100 ml bed volume Phenyl-Sepharose column using sodium From the base solution after extensive washing with 1.0 M ammonium sulfate. The eluate protein is concentrated and the ultrafiltration of YM3 diaphragm (Amicon) is performed. The salt was removed in excess. The final concentration of the preparation was 25 mM Tris-HCl (pH 8.0), 5 mMEDTA, 7 mM β-mercaptoethanol, about 250 mM sodium chloride , 20% glycerol and 0.05% sodium azide.   This method for the production of G-type xylanase comprises extraction (pH 8.0) and desalting (p HEPES buffer was used instead of Tris-HCl for step H7.3). Otherwise, it is almost the same as the above method. Final preparation is 50 mM HEPES, pH 7.3, 1 mM EDTA, 7 mM β-mercaptoethanol, 0.05% sodium acetate Buffer composition of zido, 20.0% glycerol and ± 250 mM sodium chloride I had.   A total of 6 F-type preparations (5 xynA and 1 xynB) and 2 G-types The preparation was of sufficient purity. Protein concentration of F-type and G-type preparations The degree was determined by the standard BCA method (Pierce, Rockford, Illinois, USA). Pure sample About the degree of F-type using SDS-PADE gel (Phast-system, Pharmacia, 20% gel) Compare band thickness at 40 kDa and G-type at approximately 25 kDa with impurity band thickness It was estimated by doing. Sample purity varied between 20-70% (Table 9). ).                               Example 12         ECF bleaching results with cloned F- and G-type xylanase   ECF bleaching was performed with oxygen delignified kraft pulp from a Swedish pulp mill. It was done using Lup. This softwood pulp (SW) has a kappa value of 16.0, Hardwood pulp (HW) had a kappa value of 10.6. Under the conditions shown in Table 10 , The order of XDED was used.   The results of these experiments are shown in Table 11. Comparison of enzyme protein substrates Then, the G-type xylanase acts much better than the F-type xylanase. I know what to do.                               Example 13           ECF administration-response curve with cloned G-type xylanase   Using the same XDED bleaching sequence as described in Example 12, two G- The dose of type xylanase was changed. Table 12 shows the results. 1-3 μg / g pulp Dose increases with an ISO brightness of at least 2 points.                               Example 14             TCF bleaching results with cloned G-type xylanase   Two G-type xylanases were added to the XQPP sequence described in Example 15 below. Used and tested in TCF bleaching sequence. Oxygen delignified hardwood kraft pulp Was used (30 g o.d./sample). The dose of G-type xylanase is shown in Table 13. I changed it. ISO white obtained for each sample after the X, P1 and P2 steps The chromaticity values are shown in Table 13. This value represents the average value of two samples.   This experiment was repeated using doses of 3 and 6 μg protein / g pulp Was. Only the whiteness values obtained after the P2 stage were determined. The results are shown in Table 14.                               Example 15                     ECF and TCF results and paper properties   Kraft H / W and Kraft S / W parts supplied from the Swedish pulp mill The xylanase samples were tested for bleaching effect on lupus ECF and TCF. TG456xynD and TG53xynD (referred to as 715 and 716 in the embodiment, respectively) (B) was compared with a reference example (Ref) of "no enzyme". Lampen b mill All mill) refinement tests showed that 715 showed a significant improvement in tear modulus and To give the best overall effect without significant reduction in tension and burst coefficient. Indicated. Screening protocol Properties of unbleached pulp Bleach parameters tested Bleaching conditions A) TCF H / W and S / W (same bleaching conditions for both pulps) B) ECF H / W C) ECF S / W The results obtained in the above experiment are shown in Table 15 (hardwood) and Table 16 (softwood). Verification of paper properties   After all sequences are completely bleached, a 30 g sample is taken and placed in a rampen ball mill. , 10,000, 20.000 and 30,000 rpm. Shopper Leaguer (Schopper- Rieglers) and then the tensile modulus, porosity, tear coefficient and rupture coefficient. About 2 g of sheet was made for several measurements. This sheet is mixed at 50 ° C at 23 ° C It was placed in a humidity-resistant condition for 24 hours. The obtained results are shown in FIGS.                               Example 16               Optimal pH, optimal temperature and thermal stability of TG456xynD   Activity is measured by method 1 of Example 2 using autospertoxylan as a substrate. did. All assays are in 50 mM phosphate buffer, pH and temperature are indicated I went as I did. The results are shown in Figures 9 and 10.   TG456 Xylanase D is a reference enzyme Pulpzyme HB (Novo It is more heat stable and has a slightly higher optimum pH than Nordisk). Have.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI D21C 9/10 7633-3B D21C 9/10 Z // C12P 19/14 7432-4B C12P 19/14 A (C12N 1/21 C12R 1:19) (C12N 9/24 C12R 1:19) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, M C, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, JP, KE, KG, KP, R, KZ, LK, LR, LT, LU, LV, MD, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SI, SK, TJ, TT, UA , US (72) Inventor Williams Diane Py, USA Massachusetts 01748 Hopkinton Stonegate Road 8 (72) Inventor Iverson Sara United States Massachusetts 02173 Lexington Chase Avenue 60 (72) Inventor Forster Simon United Kingdom West Yorkshire Els 13 3 Enzette Leeds Bramley Walls Avenue 3 (72) Inventor Moody Dean United Kingdom West Yorkshire Aichi Dee 1 4 J-EL Hudders Field Paddock Branch Street 9 (72) Inventor Farrell Robert Lee Amey Rica Massachusetts 01450 Groton Old Air Road 264 (72) Inventor Bergquist Peter Leonard New Zealand Auckland 1005 Error Three Puke Kelangi Cresent 31 (72) Inventor Daniel Roy McKever New Zealand Hamilton Nevada Road 74 (72) Inventor Morgan Hugh William New Zealand Hamilton Verdon Road 21 (72) Inventor Quax Wilhelms Johannes The Netherlands Enuel-2253 Vebe Forschotten Yefan Haarren Lahn 8 (72) Inventor Helway Yale Margareta Adriana The Netherlands Enuel-1018 Weher Amsteldam Lacht 72) Inventor Jones Brian Edward Holland Nuel-2 263 Weir Raid Shendham Fuller Vin Juliana Fan Stall Belgran 24

Claims (1)

[Claims] A xy resin characterized by having a remarkable delignification activity at a temperature of 1.80 ° C or higher. Lanase. 2. Claim 1 having a half-life longer than 10 minutes at 80 ° C and pH 9.0. The listed xylanase. 3. a) the xylanase is a G-type xylanase, and     b) Xylanase derived from a thermophilic microorganism having an optimum growth temperature of over 65 ° C The xylanase according to claim 1, characterized in that 4. 4. The xylem according to claim 3, characterized in that the thermophilic microorganism is anaerobic. Lanase. 5. The amino acid sequence has at least 70% homology with the amino acid sequence of SEQ ID NO: 13. The xylanase according to claim 3, wherein the xylanase shares the same sex. 6. Xylanase has a half-life longer than 10 minutes at 80 ° C and pH 7.0. Xylanase according to claim 3, characterized in that 7. Xylanase has a half-life longer than 10 minutes at 65 ° C and pH 9.0. Xylanase according to claim 3, characterized in that 8. Xylanase has deposit numbers CBS211.94, 212.94, 213.94. , 214.94, 215.94, and 216.94. Xylaner according to any one of the preceding paragraphs, characterized in that it is derived from a strain Ze. 9. Encoding at least a part of the xylanase according to any one of the preceding paragraphs. Characterized, isolated and purified DNA sequence. Ten. A vector comprising the DNA sequence according to claim 9. 11. A microorganism characterized by being transformed with the vector according to claim 10. Host cells. 12． Anaerobic and thermophilic bacteria with deposit numbers CBS211.94, 212. Characterized by having 94, 213.94, 214.94215.94 or 216.94 The isolated microorganism. 13. The xylanase is the culture of any one of the deposited microorganisms according to claim 12. Or by culturing the transformed host cell according to claim 11 in an appropriate medium. Further, then obtained by recovery of the enzyme having the indicated activity, Method for preparing xylanase. 14. A xyla comprising the use of a xylanase obtained by the method according to claim 13. How to disassemble the engine. 15. 15. The method according to claim 14, wherein the xylanase is used at a temperature of 80 ° C or higher. Method. 16． Wood pal, characterized in that xylanase is used at a temperature of 80 ° C or higher. Method for delignification 17. A wood powder comprising the use of a xylanase obtainable by the method according to claim 13. Method for delignification of lupus. 18． The xylanase according to claim 16, wherein the xylanase is used at a temperature of 80 ° C or higher. Method. 19. It is further noted that xylanase is used before or during the oxygen delignification step. The method according to any one of claims 16 to 18, characterized in that it is characterized. 20. Wood pulp using the method according to any one of claims 16 to 19. The product obtained after the treatment of.