ENZYMES WITH XYLANOLYTIC ACTIVITY

Abstract: This invention relates to novel enzymes possessing xylanolytic activity. More specifically, the invention relates to novel xylanases obtainable from strains of Bacillus pumilus, a process for their preparation, and the use of these xylanases for treatment of lignocellulosic pulp.

Description: ENZYMES WITH XYLANOLYTIC ACTIVITY FIELD OF THE INVENTION This invention relates to novel enzymes possessing xylanolytic activity. More specifically, the invention relates to novel xylanases obtainable from strains of Bacillus pumilus. a process for their preparation, and the use of these xylanases for treatment of lignocellulosic pulp. BACKGROUND ART Members of Bacillus pumilus are known and described in the literature, and it is known that they are able to produce xylanases. A specific isolate from soil in Thailand, B. pumilus IPO, has been thoroughly described by Panbanσred et al. (1983) ; J. Agric. Biol. Chem. ; 42(5), 957-963; and Fukusaki et al. (1984); FEBS Lett.; 171(2), 197-201. In JP Patent Specification No. 86039036, this B. pumilus IPO xylanase is provided by way of recombinant DNA technology. The specification mentions the use of this enzyme in the decomposition of xylan contained in animal feed or in clarification of muddy wines or fruit juices. There is given no indication of its usefulness in the treatment of lignocellulosic pulp. SUMMARY OF THE INVENTION Now a novel xylanase obtainable from a strain of B. pumilus has been elucidated. The xylanase of this invention possesses excellent properties for treatment of lignocellulosic pulp. Accordingly, in its first aspect, the present invention provides xylanases comprising specified partial amino acid sequences, and having immunochemical properties identical or partially identical to those of a xylanase derived from the strain DSM, No. 6124. In another aspect, the invention provides xylanases comprising specified partial amino acid sequences, and being obtainable from strains of Bacillus pumilus, or a mutant or a variant thereof. The invention also provides a method of producing the xylanases, comprising cultivation of a strain of B. pumilus. or a mutant or a variant thereof, in a nutrient medium containing assimilable carbon and nitrogen together with other essential nutrient, followed by recovery of the desired enzyme. In a further aspect, the invention provides a method of producing the xylanases, comprising isolating a DNA fragment encoding the xylanase; combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector; introducing the plasmid vector into an appropriate host either as an autonomously replicating plasmid or integrated into the chromosome; cultivating the host organism under conditions leading to expression of the xylanase; and recovering the xylanase from the culture medium. Moreover, the invention relates to the use of the xylanases in the treatment of lignocellulosic pulp. In a more specific aspect, the invention provides a process for treatment of lignocellulosic chemical pulp, wherein the lignocellulosic pulp is treated with the xylanase at a pH of above 6.5, preferably above 7.5, whereafter the thus treated cellulosic pulp is treated with chlorine at an active chlorine multiple of 0.20 or less in the first chlorination stage. BRIEF DESCRIPTION OF DRAWINGS The present invention is further illustrated by reference to the accompanying drawings, in which: Fig. 1 shows a comparison of the amino acid sequence of the B. pumilus IPO xylanase according to Fukusaki et al. (1984) , op. cit. (the upper sequence; similar to Sequence No. 1, presented in the sequence list below) and the partial amino acid sequence of the xylanase of the invention (the lower sequence; similar to Sequence Nos. 2-3, presented in the sequence list below) ; the amino acids are indicated in the established one-letter code; Fig. 2 shows the pH profile of a xylanase of the invention; Fig. 3 shows the temperature profile of a xylanase of the invention; and Fig. 4 shows a comparison of the amino acid sequence of the B. pumilus IPO xylanase according to Fukusaki et al. (1984) , o . ci . (the upper sequence; similar to Sequence No. 1, presented in the sequence list below) and the partial amino acid sequence of the xylanase of the invention (the lower sequence; similar to Sequence Nos. 5-8, presented in the sequence list below) ; the amino acids are indicated in the established one-letter code. DETAILED DISCLOSURE OF THE INVENTION The present invention provides novel xylanases. These novel xylanases are especially useful for treatment of ligno¬ cellulosic pulp. The enzymes The novel xylanases of the invention are obtainable from Bacillus pumilus. DSM No. 6124 (deposited on July 23, 1990 at Deutsche Sammlung von Mikroorganismen under the conditions of the Budapest Treaty) , or mutants or variants thereof. By an enzyme variant or mutated enzyme is meant an enzyme obtainable by alteration of the DNA nucleotide sequence of the parent gene or its derivatives. The enzyme variant or mutated enzyme may be expressed and produced when the DNA nucleotide sequence encoding the enzyme is inserted into a suitable vector in a suitable host organism. The host organism is not necessarily identical to the organism from which the parent gene originated. The xylanases of invention comprise the amino acid sequences that appear from Fig. 1 and Sequence Nos. 2-4 of the sequence list. On the figure the upper amino acid sequence represents the B. pumilus IPO xylanase described by Fukusaki et al. (1984) , op. cit. (Seq. No. 1) , and the lower amino acid se¬ quences represent sequences that are found to be held within the amino acid sequence of a xylanase of this invention (Seq. Nos. 2-4). The lower sequence was found after proteolytic degradation of the xylanase. Among 163 amino acid residues determined, 26 are found to be different from the B. pumilus IPO xylanase amino acid sequence published. On the figure the positions that differ between the two sequences are shown in bold typing whereas positions with identical amino acid residues are outlined. The xylanase of the invention has immunochemical properties identical or partially identical to those of a xylanase derived from the strain DSM, No. 6124. The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectro- phoresis according to N. H. Axelsen; Handbook of Immunoprecipi- tation-in-Gel Techniques; Blackwell Scientific Publications (1983), chapters 5 and 14. The terms "antigenic identity" and "partial antigenic identity" are described in the same book, chapters 5, 19 and 20. The molecular weight of a xylanase of this invention has been estimated to approximately 22 kDa, as determined by SDS-PAGE. The xylanase possesses xylanolytic activity in a pH range of from below pH 5 to above pH 11. At pH 5 more than 90% relative activity are determined. The pH optimum for the xylanase is in the range of from pH 5 to pH 7, around pH 6. The temperature profile for the xylanase is very broad, and the xylanase has no distinct temperature optimum. The xylanase is found to be nearly as active at 60"C, with around 95% persisting relative activity, as at 40 β C. The temperature optimum is found to be lower than 70 β C, where only around 5% of the xylanase activity persist. The pi for the purified xylanase was found to be within the range of from 8.0 to 9.5, around 8.8. The amino acid composition of the xylanase has been determined after acid hydrolysis and it is found to differ from the amino acid composition of the B. pumilus IPO xylanase, especially in the content of serine and glycine, as shown in Table 1. Table 1 The preparat i on The xylanases of the invention are producible by cultivation of a xylanase producing strain of Bacillus pumilus. preferably B. pumilus. DSM No. 6124, or a mutant or a variant thereof. Xylanase producing B. pumilus strains can be culti¬ vated under aerobic conditions in a nutrient medium containing assimilable carbon and nitrogen together with other essential nutrient. The medium can be composed in accordance with principles known in the art. During cultivation, the cells secrete xylanases extracellularly, so the recovery of xylanases preferably includes separation of cell mass from the cell broth while avoiding cell lysis, e.g. by filtration or centrifugation. The resulting cell-culture can be used as such, optionally after concentration e.g. by evaporation or ultrafil- tration. If desired, the xylanases can be separated and purified to the desired degree by conventional methods, e.g. by column chromatography. The xylanases are also obtainable by recombinant DNA technology by methods known in the art per se. e.g. isolating a DNA fragment encoding the xylanase; combining the DNA fragment with an appropriate expression signal in an appropri¬ ate plasmid vector; introducing the plasmid vector into an appropriate host (i.e. an Escherichia coli. or a member of the genus Bacillus. Asperqillus. or Streptomvces) , either as an autonomously replicating plasmid or integrated into the chromosome; cultivating the host organism under conditions leading to expression of the xylanase; and recovering the xylanase from the culture medium. The xylanases also may be produced essentially as described in JP Pa-. nt Specification No. 86039036. Treatment of lignocellulosic pulp The xylanase of this invention is valuable for treatment of lignocellulosic pulp. Xylan, a major component of plant hemicellulose, is a polymer of D-xylose linked by B-l,4-xylosidic bonds. Xylan can be degraded to xylose and xylo-oligomers by acid or enzymatic hydrolysis. Enzymatic hydrolysis of xylan produces free sugars without the by-products formed with acid (e.g. furans) . The pulp and paper industry is using xylanase compositions in the bleaching process to enhance the brightness of bleached pulps, to decrease the amount of chlorine used in the bleaching stages, and to increase the freeness of pulps in the recycled paper process fEriksson. K.E.L. (1990) ; Wood Science and Technology, 24., 79-101.; Paice. M.G. ; Bernier. R. ; & Jurasek. L. (1988); Biotechnol. and Bioeng. , 32., 235-239; Pommier. J.C. ; Fuentes. J.L. ; & Goma. G. (1989) ; Tappi Journal, 187-191]. Processes for treatment of lignocellulosic pulp are widely described in the literature, vide e.g. Paice. M.G. & Jurasek. L. (1984) ; Journal of Wood Chemistry and Technology, 4.(2) , 187-198; Noe. P. et al. (1986) ; Journal of Wood Chemistry and Technology, 6.(2 ) , 167-184; or EP Patent Application No. 386,888. In relation to the use of the xylanases of this invention for treatment of lignocellulosic pulp, a particular process is described in International Patent Publication No. WO 91/02839, which publication is hereby included by reference. The invention is further illustrated in the following examples which are not intended to be in any way limiting to the scope of the invention as claimed. EXAMPLE 1 Production of Bacillus pumilus xylanase A Bacillus pumilus culture DSM 6124 was maintained on A3-medium, at 37"C. 120 shake flasks with 150 ml XYL-8 medium each, inoculated from A3-agar slants, were cultivated for 4 days, 37°C, 250 rpm with approximately 2 cm amplitude. XYL-8 medium: pH adjustment to 7.0 before autoclaving. Autoclaving 25 min./121 β C. The broth was centrifuged for half an hour at 4,000 rpm (SORVALL RC-3B centrifuge with a 6000 A rotor) . The supernatant, 7.3 1, was filtered through a 10 μm nylon filter, and concentrated by ultrafiltration by means of a Pellicon 5 equipment from Millipore, with a 10,000 MW cut-off membrane and washed 2 times.with one volume of water. This resulted in 540 ml of concentrate. The concentrate was then lyophilized, whereby 8.8 g of powder were generated. EXAMPLE 2 10 Purification example The xylanase was purified in seven steps. Approximately 500 ml of fermentation broth were centrifuged at 5000 rpm for 30 min. The supernatant was then precipitated with 50% w/w ammonium sulfate. The precipitate was 15 dissolved in 20 mM Tris-HCl buffer, pH 7.0, and desalted and concentrated until the conductivity was less than 1 mS/cm using a Filtron ultrafiltration module with 3 kD cut off membranes. The concentrate was applied to a 25 ml anionexchanger (Q-sepharose F.F.) equilibrated with 20 mM Tris-HCl buffer, pH 207.0. The flow-through was applied to a 25 ml cationex- changer (S-sepharose F.F.) equilibrated with the same buffer. The column was eluted with a linear gradient consisting of 200 ml of the above buffer and 200 ml added 500 mM NaCl. 25 Most of the remaining proteases of the fractions with xylanase activity were removed by affinity chromatography at pH 7.0. The flow-through was added ammonium sulfate to a concentration of 20% w/w and applied on a 10 ml Phenyl- 30 Sepharose CL-4B. The column was eluted with a linear gradient consisting of 750 ml 20 mM Tris-HCl buffer, pH 7.0, 20% w/w ammonium sulfate, and 750 ml of the same buffer without ammonium sulfate. The fractions with xylanase activity were con¬ centrated using an Amicon ultrafiltration unit 8200 with DDS GR90PP membranes. When the concentration of xylanase was approximately 100 mg/ml and the conductivity was 20 mS/cm, the xylanase started to crystallize. The yield was near 100%. The crystals were dissolved in 20 mM Tris-HCl buffer, pH 7.0, 25% w/w glycerol. EXAMPLE 3 Method for determining; xylanase activity Xylanase was determined by assaying for reducing sugars released from Birch xylan (the XU method) . The assay is performed using 0.5% Birch xylan (ROTH GmbH Atr. 7500) prepared in 40 mM Britton-Robinson buffer (heat treated 30 min. at 100"C before use) as substrate. The assay is run for 20 min. at 40°C using 0.100 ml Enzyme Solution I (35.1 g Na 2 HP0 4 , 2H 2 0; 40.0 g KNaC 4 H 4 0 6 , 4H 2 0, suspended in 500 ml deionized I. 2 0; 42 ml concentrated H 2 S0 4 ; 6.0 g Na 2 HAs0 4 , 7H z O; ad deionized water to a tota__. volume of 1 litre) is added. Add 2.0 ml of deionized water and measure the absorbance on a spectro- photometer (PYE UniCAM PU8600UV/VIS, Phillips) at 250 nm. The reducing sugars are calculated from a standard curve prepared with xylose (40-400 μg/ l) . One XU is equivalent to 1 nmol xylose released per second per millilitre or per gram of culture broth. EXAMPLE 4 pH characterization The pH characterization of the xylanase was determined using the purified xylanase preparation according to Examples 1 and 2, and the XU method described in Example 3. The characterization was performed in the range of from pH 5 to pH 11. All assays were performed for 20 min. at 40 β C. The profile is presented in Fig. 2. It appears from this characterization that the xylanase of the invention possesses xylanolytic activity from below pH 5 to above pH 11. At pH 5 a relative activity of more than 90% has been determined. At pH 9 a relative activity of more than 40% has been determined. At pH 11 a relative activity of more than 10% has been determined. The pH optimum for the xylanase is in the range of from pH 5 to pH 7, around pH 6. EXAMPLE 5 Temperature characterization The temperature characterization of the xylanase was determined using the purified xylanase preparation according to Examples 1 and 2, and the XU method described in Example 3. The characterization was performed in the temperature range of from 40 to 100"C. All assays were performed for 20 minutes at pH 6. The profile is presented in Fig. 3. It appears from this characterization that the curve for temperature profile is very broad, and that the xylanase has no distinct temperature optimum. The xylanase is found to be nearly as active at 60"C, with around 95% persisting relative activity, as at 40 β C. The temperature optimum is found to be lower than 70 β C, where only around 5% of the xylanase activity persist. The optimum is within the range of from 35 to 65 β C, more specifically from 40 to 60°C. EXAMPLE 6 pi characterization The pi characterization of the xylanase was determined using the purified xylanase preparation according to Examples 1 and 2, and LKB Ampholine PAG plates at pH 3.5 to 9.5. After electrophoresis, gel was washed twice for 15 min. , once in water, c.ice in a Tris-buffer, pH 9, and then overlayered with a thin coat of detection agar consisting of 0.5% of oat spelt xylan, 1% of agarose, pH 9. The overlayered gel was incubated overnight at 50 β C. The xylanase activity was visualised using Congo Red staining (staining for 10 min. with 0.1% Congo Red, and destained for 2 times 15 min. in 1 M NaCl) . The pi for the purified xylanase was found to be above 8, within a range of from 8.0 to 9.5, around 8.8. EXAMPLE 7 Amino acid sequence Using standard methods for obtaining and sequencing peptides fFindlav & Geisow (Eds.) (1989); Protein sequencing - a practical approach; IRL Press] derived from the B. pumilus xylanase a total of 163 amino acid residues has been iden- tified. In addition, the C-terminal amino acid has been iden¬ tified as isoleucine. During direct sequencing of the purified enzyme the N-terminus was four3 to be partially blocked. An alignuient of the partial sequence of the ______ pumilus xylanase to the amino acid sequence of the B. pumilus IPO xylanase r Fu ^ usak ** L et &! . (1984) ; op. cit.] is shown in Fig. 1. A total of 26 differences is found and shown in bold typing. Positions in the two sequences which are occupied by identical amino acid residues are outlined. In addition to the 26 differences mentioned above there are two amino acid residues less at the C-terminus of the B.. pumilus xylanase of the invention than described for the B. pumilus IPO xylanase. EXAMPLE 8 Amino acid composition The amino acid composition of B. pumilus xylanase was determined after acid hydrolysis rMoore & Stein (1963) ; Methods Enzymol. _5 , 819-831] using an Applied Biosystems 420A amino acid analysis system. The amino acids in the hydrolysis mixture were quantified using reversed phase HPLC after precolumn derivatization with phenylisothiocyanate rHeinrikson & Meredith (1984); Anal. Biochem. 136. 65-74]. The amount of tryptophan was determined spectro- photometrically using the method of Edelhoch [Edelhoch (1967) ; Biochemistry ϋ, 1948-1954]. The presence of a single cysteine (Cys) residue in the xylanase was determined through titration with 5,5'- dithiobis-(2-nitrobenzoic acid) *Ri d dles Qt al. (1983); Methods Enzymol. £1, 49-60]. The presence of only small amounts of cysteine after acid hydrolysis precludes the presence of disulfide-bridges. The combined result is shown in Table 1. In addition, a comparison of the amino acid composition of the B. pumilus xylanase with the amino acid composition of the B. pumilus IPO xylanase is shown revealing some differences, especially in the content of serine (Ser) and glycine (Gly) . EXAMPLE 9 Treatment of lignocellulosic pulp An unbleached hardwood brown stock is treated with a hemicellulase preparation from Bacillus pumilus (produced according to Example 1) at the following conditions: Time: t = 3 hours Temperature: T = 50 β C pH: pH •= 8.0 Consistency: DS = 10% Dosage: 715 EXU/kg dry pulp After the enzyme treatment the pulp is washed with water and bleached in a three stage bleaching sequence, (D50 C50)E D. A control is treated in the same way but without addition of enzyme. Table 2 below shows the kappa numbers of the pulps after the enzyme treatment. The (D50 C50)E D bleaching stages are performed under the following conditions: ( DC) -stage Time : t = 20 min Temperature: T = 50 β C Consistency: DS = 5% Substitution: 50% with C10 2 (as active chlorine) Chlorine multiple: 0.20 E-stage Time: t = 1 hr Temperature: T = 60 β C Consistency: DS = 10% NaOH dosage: 2.0% (w/w) on dry pulp D-stage Time: t = 3 hr Temperature: T = 70 β C Consistency: DS = 10% Both pulps are bleached to a kappa number of 3.5 after the (D50 C50)E stages. For the control 2.8% (w/w) active chlorine are needed in the (D50 C50)-stage to reach this kappa number. For the enzyme treated pulp only 2.38% (w/w) active chlorine are needed to reach a kappa number of 3.5. This corresponds to a reduction in active chlorine (aCl) of 14.5% for the enzyme treated pulp compared to the control in order to reach the desired kappa number. In the final D-stage both pulps having a kappa number of 3.5 after (D50 C50)E are bleached to their respective brightness ceilings. For the enzyme treated pulp a chlorine dioxide dosage of 0.99% (w/w) is needed to bring the pulp to a bright¬ ness of 87.2% (ISO) brightness. For the control a dosage of 1.2% (w/w) chlorine dioxide is needed to reach a final bright¬ ness of 85% (ISO) . This shows that the enzyme treatment makes it possible to reduce the dosage of chlorine dioxide in the final D-stage by 17.5% and at the same time elevates the brightness ceiling of the pulp by a 2.2% ISO brightness. The enzyme treatment of the pulp neither causes strength nor yield loss. EXAMPLE 10 Treatment of lignocellulosic pulp An 0 2 delignified kraft pine pulp is treated with the purified hemicellulase preparation from Bacillus pumilus (produced according to Example 1, and purified according to Example 2) at the following conditions: Time: t = 3 hours Temperature: T = 50°C pH: pH = 8.5 Consistency: DS = 10% W/W Dosage: 815 EXU/kg dry pulp The pH was controlled using a Britton-Robinson buffer. After the enzyme treatment the pulp is washed with water and bleached in a two stage bleaching sequence, (D50 C50)E. A control is treated in the same way but with addition of demineralized water instead of enzyme solution. Table 3 below shows the kappa numbers of the pulps after the enzyme treatment. Table 3 kappa number reduction % Control 14.9 Bacillus pumilus 14.1 5.4 The (D50 C50)E bleaching stages are performed using 50% substitution with chlorine dioxide at the following conditions: (D50 C50)-Stage E-Stage Temperature 40 β C 60°C Time 45 min. 60 min. Consistency 5 % 12 % The chlorine multiple in the (D50 C50)-Stage was 0.20, similar to 2.98 % active chlorine (aCl) . The amount of NaOH applied in the E-stage is adjusted according to the dosage of active chlorine (aCl) in the (D50 C50)-Stage, i.e. 0.5(%C1+%C10 2 )+0.3 = 1.8 % After the (D50 C50)E stages, the kappa number of the pulp is determined. The results are presented in Table 4 below. Table 4 The enzyme treatment of the pulp neither causes strength nor yield loss. This Example demonstrate the bleach boosting effect of a purified enzyme preparation of this invention. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: NOVO NORDISK A/S, NN (ii) TITLE OF INVENTION: NOVEL ENZYMES (iii) NUMBER OF SEQUENCES: 8 (2) INFORMATION FOR SEQ ID N0:1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 201 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus pumilus (B) STRAIN: IPO (x) PUBLICATION INFORMATION: (A) AUTHORS: Fukusaki, E. Panbangred, W. Shinmyo, A. Okada, H. (B) TITLE: The complete nucleotide sequence of the xylanase gene (xynA) of Bacillus pumilus (C) JOURNAL: FEBS Lett. (D) VOLUME: 171 (E) ISSUE: 2 (F) PAGES: 197-201 (G) DATE: 1984 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: Arg Thr He Thr Asn Asn Glu Met Gly Asn His Ser Gly Tyr Asp Tyr 1 5 10 15 Glu Leu Trp Lys Asp Tyr Gly Asn Thr Ser Met Thr Leu Asn Asn Gly 20 25 30 Gly Ala Phe Ser Ala Gly Trp Asn Asn He Gly Asn Ala Leu Phe Arg 35 40 45 Lys Gly Lys Lys Phe Asp Ser Thr Arg Thr His His Gin Leu Gly Asn 50 55 60 He Ser He Asn Tyr Asn Ala Ser Phe Asn Pro Ser Gly Asn Ser Tyr 65 70 75 80 Leu Cys Val Tyr Gly Trp Thr Gin Ser Pro Leu Ala Glu Tyr Tyr He 85 90 95 Val Asp Ser Trp Gly Thr Tyr Arg Pro Thr Gly Ala Tyr Lys Gly Ser 100 105 110 Phe Tyr Ala Asp Gly Gly Thr Tyr Asp He Tyr Glu Thr Thr Arg Val 115 120 125 Asn Gin Pro Ser He He Gly He Ala Thr Phe Lys Gin Tyr Trp Ser 130 135 140 Val Arg Gin Thr Lys Arg Thr Ser Gly Thr Val Ser Val Ser Ala His 145 150 155 160 Phe Arg Lys Trp Glu Ser Leu Gly Met Pro Met Gly Lys Met Tyr Glu 165 170 175 Thr Ala Phe Thr Val Glu Gly Tyr Gin Ser Ser Gly Ser Ala Asn Val 180 185 190 Met Thr Asn Gin Leu Phe He Gly Asn 195 200 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (v) FRAGMENT TYPE: N-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus pumilus (B) STRAIN: DSM 6124 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:2: Glu Thr He Tyr Asp Asn Arg He Gly Arg His Thr Gly Tyr Asp Phe 1 5 10 15 Glu Leu Trp Lys Asp Tyr Gly Asn Thr Ser Met He 20 25 (2) INFORMATION FOR SEQ ID N0:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (v) FRAGMENT TYPE: internal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus pumilus (B) STRAIN: DSM 6124 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: Ser Ala Ser Trp Asn Asn He Gly Asn Ala Leu Phe Arg Lys Gly Lys 1 5 10 15 Lys Phe Asp Ser Thr Lys Thr His His Gin Leu Gly Asn He Asp 20 25 30 (2) INFORMATION FOR SEQ ID N0:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 104 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (v) FRAGMENT TYPE: C-terminal (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus pumilus (B) STRAIN: DSM 6124 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: He Val Glu Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Trp 1 5 10 15 Ser Phe Tyr Ala Asp Gly Gly Thr Tyr Asp He Tyr Glu Thr Leu Arg 20 25 30 Val Asn Gin Pro Ser He He Gly Asp Ala Thr Phe Lys Gin Tyr Val 35 40 45 Ser Val Arg Gin Thr Lys Arg Thr Ser Gly Thr Val Ser Val Ser Glu 50 55 60 His Phe Lys Lys Trp Glu Gly Leu Tyr Met Pro Met Gly Lys Met Tyr 65 70 75 80 Glu Thr Ala Leu Thr Val Glu Gly Tyr Arg Ser Thr Gly Ser Ala Asn 85 90 95 Val Met Thr Asn Gin Leu Met He 100 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( i i ) MOLECULE TYPE : peptide (v) FRAGMENT TYPE : i nternal (xi ) SEQUENCE DESCRIPTION : SEQ ID N0: 5 : H e Val Gl u Ser Trp Gly Thr Tyr Arg Pro Thr Gly Thr Tyr Lys Gly 1 5 10 15 (2) INFORMATION FOR SEQ ID N0:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single. (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6: Val Asn Gin Pro Ser He He Gly Asp Ala Thr Phe Lys Gin Tyr Val 1 5 10 15 Ser Val Arg Gin Thr Lys Arg 20 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7: His Phe Lys Lys Xaa Glu Gly Leu 1 5 (2) INFORMATION FOR SEQ ID N0:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:8: Thr Val Ala Gly Tyr 1 5 International Application No: PCT/ MICROORGANISMS Optional ShMt in connection with tha mlcroorginlam referred to on ptga. 3 , Una 30 ol ttia daacrlption < A. IDINTiπCATtON OF DIPO»IT • Furthar deposits ara Identified on an additional sheet [__•» Nam* ol depositary Inet-tutlon « DEUTSCHE SAMMLUNG VON IKROORGANISMEN UND 2ELL- KULTUREN GmbH Addraaa of depoeltary Inatthrtion (Including postal coda and country) • Mascheroder eg lb, D-3300 Braunschweig, Federal Re¬ public of Germany Data ol dapoalt ' Accaaalon Number • 23 July 1990 DSM 6124 AQOITIONAL INDICATIONS » (leave blank II not applicable). Thla Information la continued on a separata attached sheet f~\ In respect of those designations in which a European patent is sought, a sample of the deposited micro¬ organism will be made available only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28 (4) EPC) until the publication of the mention of the grant of the Euro¬ pean patent or until the date on which the appli¬ cation has been refused or is deemed to be withdrawn . C. DtSlβNATID STATIS FOR WHICH INDICATIONS ARC HAOI > (II tha Indications ara not lor all deelβnated SUtea) O. SIPARATI FURNISHINS OF INDICATIONS • (leave blank II not applicable) The Indications Hated below will be submitted to the International Bureau later > (Specify Ih* general nature ol tha Indications e.g., " Accession Number ol Deposit ") I. Thla sheet waa received with the International application whan filed (to be checked by tha receiving Office) (~| The date el receipt (from the applicant) by the International Bureau '• (Authorised Officer) Farm PCT/RO IM (January IM1)

Claims: CLAIMS 1. A xylanase comprising the partial amino acid sequences according to Fig. 1 and Seq. Nos. 2-4 of the sequence list, and having immunochemical properties identical or partially identical to those, of a xylanase derived from the* strain DSM, No. 6124. 2. A xylanase comprising the partial amino acid sequences according to Fig. 1 and Seq. Nos. 2-4 of the sequence list, and being obtainable from a strain of Bacillus pumilus, or a mutant or a variant thereof. 3. The xylanase of claim 2, being obtainable from the strain DSM, No. 6124, or a mutant or a variant thereof. 4. The xylanase according to any of claims 1-3, characterized by having a molecular weight of approximately 22 kDa. 5. The xylanase according to any of claims 1-4, further characterized by having pH optimum in the range of from pH 5 to 7; a temperature optimum below 70°C; and pi in the range of from 8.0 to 9.5. 6. A xylanase comprising the amino acid sequences according to Fig. 4 and Seq. No. 5-8 of the sequence list. 7. The xylanase of claim 6, obtainable from a xylanase producing strain of Bacillus pumilus. 8. The xylanase of either of claims 6-7, obtainable from 3. pumilus, DSM No. 6124, or a mutant or a variant thereof. 9. A method of producing a xylanase of any of claims 1-8, comprising cultivation of a strain of B. pumilus. or a mutant or a variant thereof, in a nutrient medium con¬ taining assimilable carbon and nitrogen together with other essential nutrient, followed by recovery of the desired enzyme. 10. The method of claim 9, comprising cultivation of the strain B. pumilus. DSM No. 6124, or a mutant or a variant thereof. 11. A method of producing a xylanase according to any of claims 1-8, comprising isolating a DNA fragment encoding the xylanase; combining the DNA fragment with an appropriate expression signal in an appropriate plasmid vector; introducing the plasmid vector into an appropriate host either as an autonomously replicating plasmid or integrated into the chromosome; cultivating the host organism under conditions leading to expression of the xylanase; and recovering the xylanase from the culture medium. 12. The method of claim 11, in which the host organism is an Escherichia coli, or a member of the genus Bacillus, Aspercrillus. or Streptomvces. 13. The use of the xylanase according to any of claims 1-8 in the treatment of lignocellulosic pulp. 14. A process according to claim 13 for treatment of lignocellulosic chemical pulp, wherein the lignocellulosic pulp is treated with the xylanase at a pH above 6.5, preferably above 7.5, whereafter the thus treated cellulosic pulp is treated with chlorine at an active chlorine multiple of 0.20 or less in the first chlorination stage. 15. The process of claim 14, wherein the xylanase treatment is performed at temperatures between 40 and 100 " C, preferably between 40 and 80"C, more preferably between 50 and 70 β C. 16. A process of either of claims 13-15, wherein the xylanase treatment is performed over a period of 15 minutes to 24 hours, preferably between 30 minutes and 5 hours, most preferably between 30 minutes and 3 hours. 17. A process of any of claims 13-16, wherein the xylanase treatment takes place at a consistency of 5-35%, preferably 8-25%, most preferably 8-15%. 18. A process of any of claims 13-17, wherein an (EOP) treatment is introduced between the xylanase treatment and the chlorine treatment. 19. A process of any of claims 13-18, wherein the chlorine multiple is between 0.10 and 0.20.

Inventors: ANKER LISBETH, BISGAARD-FRANTZEN HENRIK, HALKIER TORBEN, ANKER, LISBETH, BISGAARD-FRANTZEN, HENRIK, HALKIER, TORBEN
Applicants: NOVONORDISK AS, NOVO NORDISK A/S
Application year: 1991
Main classification: C12S3
IPC codes: C12N9, C12N15, C12R1, C12S3, D21C5, D21C9