JP3769655B2 - Thermostable xylanase - Google Patents

Description

  The present invention relates to a novel thermostable xylanase, a production method and use thereof, a thermostable xylanase gene, and a microorganism that produces the thermostable xylanase.

  Xylanase is an enzyme that degrades xylan, and is a useful enzyme used as an enzyme for pre-bleaching treatment of paper pulp or for the production of functional xylo-oligosaccharides. Since Viikari et al. Reported that xylanase improves the bleachability of kraft pulp (Non-patent Document 1), xylanase has begun to attract attention in the pulp and paper industry. Since there are many high temperature conditions, when using a xylanase efficiently in these processes, the xylanase with high heat resistance is calculated | required. This is because the use of a thermostable xylanase enables an enzyme reaction at a high temperature, thereby reducing the cooling equipment and energy and preventing the propagation of various bacteria during the enzyme reaction.

  On the other hand, it is self-evident that the higher the enzyme production capacity of the microorganism is, the more advantageous for the cost reduction of the enzyme production. Therefore, there is a demand for a xylanase with high heat resistance and a microorganism that produces the enzyme at a high level. According to a review of xylanase (Non-patent Document 2) and the like, microorganisms producing xylanase are filamentous fungi such as Aspergillus, Trichoderma, Aureobasidium, Schizophyllum commune, etc. Many microorganisms such as bacteria such as Bacillus genus, Clostridium genus, and Streptomyces genus are known. The reaction pH of xylanase produced from these microorganisms is acidic to neutral, and the reaction temperature is 40 ° C to 80 ° C. Also known are microorganisms that produce an alkaline xylanase having activity on the alkali side, for example, the genus Bacillus (Non-patent Documents 3 and 4), the genus Aeromonas (Non-patent Document 5), or Streptomyces. Microorganisms belonging to the genus (Streptomyces) (Non-patent Document 6) are known.

  Among these xylanase-producing microorganisms, filamentous fungi often produce cellulase in addition to xylanase, which may reduce paper yield and strength when used in the paper pulp manufacturing process. There is a problem. Moreover, filamentous fungi have a longer culture period than bacteria. In addition, bacteria have a problem that xylanase productivity is low.

  Recently, Viikari et al. Produced a high yield (400 U / ml) of xylanase by culturing at pH 8 to 8.5 at 30 ° C. for 2 days using an alkali-resistant Bacillus circulans VTT-E-87305 strain. (Non-patent Document 7). However, the heat resistance of this enzyme is not clear and the culture temperature is as low as 30 ° C., so it is difficult to carry out the enzyme reaction at high temperature.

  On the other hand, for the purification of thermostable xylanase produced by microorganisms belonging to the genus Bacillus, molecular weight 21,500, isoelectric point 8.5, reaction optimum pH 6.0, reaction optimum from alkali thermophilic thermophilic Bacillus W-1 and W-2 A W1-I thermostable xylanase having a temperature of 65 ° C. and a W2-I thermostable xylanase having a molecular weight of 22,500, an isoelectric point of 8.3, an optimum pH of 6.0, and an optimum temperature of 65 ° C. have been reported (non-patented). Reference 8).

  In addition, for Bacillus stearothermophilus, known as thermophilic Bacillus, T. Nanmori et al. From Strain 21 culture filtrate had a molecular weight of 39,500, isoelectric point 5.1, optimum reaction pH 7.0, optimum reaction. It has been reported that a thermostable xylanase having a temperature of 60 ° C. has been purified and produced (Non-patent Document 9). However, its production is only 1.96 U / ml at 55 ° C for 2 days.

  Furthermore, regarding the use of xylanase, several attempts have been reported to reduce bleaching chemicals and AOX (adsorptive organohalogen compounds, especially organochlorine compounds) by causing xylanase to act on pulp (for example, patent documents). 1-7, non-patent documents 10-13).

  However, in these attempts, the bleaching process in the pulp and paper manufacturing process requires a high temperature treatment of 40 to 100 ° C., but an enzyme that does not have heat resistance must be used in order to incorporate the enzyme treatment in the process. In many cases, the pulp must be cooled to the optimum reaction temperature and heated for the next step, which requires a lot of energy.

  Therefore, heat resistance is an enzyme that can be manufactured at a low cost by making cooling equipment unnecessary by making it manufactured under high temperature conditions or making it possible to save cooling water and reducing the possibility of contamination. Xylanases are desired. It is also desired to obtain a large amount of xylanase by isolating a gene encoding a thermostable xylanase by gene recombination technology and expressing the gene.

  Many reports on the xylanase gene have been made so far. For example, regarding bacteria origin, Bacillus circulans (Non-patent document 14), Bacillus subtilis (Non-patent document 15), Pseudomonas fluorescens (Non-patent document 16) and Luminococcus For example, Flabefaciens (Ruminococcus flavefaciens; Non-Patent Document 17) and Clostridium acetobutylicum (Non-Patent Document 18), Aspergillus awamori (Non-patent Document 19) and Streptomyces lividans ( Streptomyces lividans; Non-patent document 20). However, it is not clear whether enzymes produced by recombinants using these genes are suitable for bleaching.

JP-A-2-10085 JP-A-2-10086 JP-A-2-221482 JP-A-2-64087 JP-A-2-93486 Japanese Patent Laid-Open No. 3-40887 JP-A-3-505785 3rd international conference on biotechnology in the pulp and paper industry 1986 Abstracts of lectures p.67 Wong et al., Microbiological Reviews, Sept., 305,1988 Honda et al., System. Appl. Microbiol., 8,152,1986, Okazaki et al., Appl. Microbiol. And Biotechnol., 19,335,1984 Ohkoshi et al., Agric. Biol. Chem., 49, 3037, 1985 Vyas et al., Biotechnol.Let., 12, 225, 1990 Appl. Microbiol. Biotechnol., 37,470,1992 Okazaki et al., Agric.Biol.Chem., 49,2033,1985 J. Bacteriol., 172,6669,1990 L.S.Pederson et al., Production of bleached chemical pulp in the future international pulp bleaching conference, Vol.2,107,1991 Pulp and Paper Technology Times, 5, 20, 1992 S. Hogman et al., Biotechnology in Pulp and Paper Industry, Uni Publishers Co., Ltd., P.107,1992 Viikari et al., Biotechnology in Pulp and Paper Industry, Uni Publishers Co., Ltd., P.101,1992 YANGR.C.A. Et al., NUCLEIC ACIDS RES. 16: 7187-7187 (1988) PAICE M.G. et al., ARCH.MICROBIOL. 144: 201-206 (1986) KELLETT L.E. et al., BIOCHEM.J.272: 369-376 (1990) ZHANG J.-X. et al., MOL.MICROBIOL. 6: 1013-1023 (1992) ZAPPE. Et al., NUCLEIC ACIDS RES. 18: 2179-2179- (1990) ITO K. et al., BIOSCI.BIOTECHNOL.BIOCHEM. 56: 1338-1340 (1992) SHARECK F. et al., GENE 107: 75-82 (1991)

  An object of the present invention is to provide a novel thermostable xylanase, a production method and use thereof, a thermostable xylanase gene, and a microorganism that produces the thermostable xylanase.

  Based on the above-mentioned problems, the present inventors have intensively researched and sought a thermostable xylanase-producing bacterium. Discovered, found thermostable xylanases XP1 and XP2 having excellent thermostability in the culture of the microorganism, and succeeded in cloning and highly expressing the gene encoding the thermostable xylanase. It came to be completed.

  That is, the present invention is a thermostable xylanase selected from thermostable xylanase XP1 or XP2 having the following physicochemical properties.

(1) Thermostable xylanase XP1 (hereinafter simply referred to as XP1) having the following physicochemical properties.
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylo-oligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 3-9.
(4) Range of optimal working temperature: in the range of 50-80 ° C.
(5) Thermal stability: Retains about 90% or more of the enzyme activity after treatment at 50 ° C. for 30 minutes, and shows about 50% or more of the residual activity even after treatment at 60 ° C. for 30 minutes.
(6) Isoelectric point: around 8.1.
(7) Molecular weight: As a result of measurement by SDS polyacrylamide gel electrophoresis, it is about 22,500.
(8) Inhibition: weakly inhibited by iodoacetic acid and EDTA, and strongly inhibited by Hg ² + and SDS.

(2) Thermostable xylanase XP2 (hereinafter simply referred to as XP2) having the following physicochemical properties.
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylose and xylooligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 4.5-9.
(4) Range of optimal working temperature: 60 to 90 ° C.
(5) Thermal stability: about 90% residual activity after treatment at 70 ° C for 30 minutes.
(6) Isoelectric point: around 8.5.
(7) Molecular weight: As a result of measuring by SDS polyacrylamide gel electrophoresis, it is about 32,000.
(8) Inhibition: weakly inhibited by Mn ²⁺ , Co ²⁺ , Cu ²⁺ , EDTA and iodoacetic acid, and strongly inhibited by Hg ²⁺ and SDS.

  Furthermore, the present invention provides the production of the thermostable xylanase, characterized by culturing a microorganism belonging to the genus Bacillus that produces the thermostable xylanase in a medium, and collecting the thermostable xylanase from the obtained culture. Is the method. Here, examples of the microorganism include Bacillus sp 2113 and Bacillus sp 208.

  Furthermore, the present invention is Bacillus sp 2113 or Bacillus sp 208 having the ability to produce thermostable xylase. Furthermore, the present invention is a thermostable xylanase gene substantially comprising the base sequence substantially encoding the amino acid sequence represented by SEQ ID NO: 1 or the base sequence represented by SEQ ID NO: 2.

  Here, “substantially” means that as long as this peptide has thermostable xylanase enzyme activity, or as long as the base sequence encodes a thermostable xylanase, the amino acid or base sequence has a deletion, substitution, or It means that there may be changes such as addition. Furthermore, the present invention is a thermostable xylanase selected from the recombinant thermostable xylanase XP1 or the recombinant thermostable xylanase XP2 having the following physicochemical properties.

(1) Recombinant thermostable xylanase XP1 having the following physicochemical properties
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylo-oligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 3-9.
(4) Range of optimal working temperature: in the range of 50-80 ° C.
(5) Thermal stability: Retains about 90% or more of the enzyme activity after treatment at 50 ° C. for 30 minutes, and shows about 50% or more of the residual activity even after treatment at 60 ° C. for 30 minutes.
(6) Isoelectric point: around 8.1.
(7) Molecular weight: As a result of measurement by SDS polyacrylamide gel electrophoresis, it is about 22,500.
(8) Inhibition: weakly inhibited by iodoacetic acid and EDTA, and strongly inhibited by Hg ²⁺ and SDS.

(2) Recombinant thermostable xylanase XP2 having the following physicochemical properties
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylose and xylooligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 4.5-9.
(4) Range of optimal working temperature: 60 to 90 ° C.
(5) Thermal stability: shows a residual activity of about 90% or more after treatment at 70 ° C. for 30 minutes.
(6) Isoelectric point: around 8.5.
(7) Molecular weight: As a result of measuring by SDS polyacrylamide gel electrophoresis, it is about 32,000.
(8) Inhibition: weakly inhibited by Mn ²⁺ , Co ²⁺ , Cu ²⁺ , EDTA and iodoacetic acid, and strongly inhibited by Hg ²⁺ and SDS.

  Furthermore, the present invention is a recombinant thermostable xylanase substantially comprising the amino acid sequence represented by SEQ ID NO: 1. Furthermore, the present invention is a recombinant vector containing the thermostable xylanase gene. Furthermore, the present invention is a transformant transformed with the recombinant vector.

  Furthermore, the present invention is a method for producing a recombinant thermostable xylanase, comprising culturing the transformant in a medium and collecting the recombinant thermostable xylanase from the resulting culture. Furthermore, the present invention provides a culture obtained by culturing a microorganism belonging to the genus Bacillus that produces the thermostable xylanase or the transformant in a medium, or a thermostable xylanase XP1 or genetic recombination having the following physicochemical properties: Type thermostable xylanase XP1 and / or thermostable xylanase XP2 or recombinant thermostable xylanase XP2 as an active ingredient.

(1) Thermostable xylanase XP1 or recombinant thermostable xylanase XP1 having the following physicochemical properties
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylo-oligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 3-9.
(4) Range of optimal working temperature: in the range of 50-80 ° C.
(5) Thermal stability: Retains about 90% or more of the enzyme activity after treatment at 50 ° C. for 30 minutes, and shows about 50% or more of the residual activity even after treatment at 60 ° C. for 30 minutes.
(6) Isoelectric point: around 8.1.
(7) Molecular weight: As a result of measurement by SDS polyacrylamide gel electrophoresis, it is about 22,500.
(8) Inhibition: weakly inhibited by iodoacetic acid and EDTA, and strongly inhibited by Hg ²⁺ and SDS.

(2) Thermostable xylanase XP2 or genetically modified thermostable xylanase XP2 having the following physicochemical properties
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylose and xylooligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 4.5-9.
(4) Range of optimal working temperature: 60 to 90 ° C.
(5) Thermal stability: shows a residual activity of about 90% or more after treatment at 70 ° C. for 30 minutes.
(6) Isoelectric point: around 8.5.
(7) Molecular weight: As a result of measuring by SDS polyacrylamide gel electrophoresis, it is about 32,000.
(8) Inhibition: weakly inhibited by Mn ²⁺ , Co ²⁺ , Cu ²⁺ , EDTA and iodoacetic acid, and strongly inhibited by Hg ²⁺ and SDS.

  Furthermore, the present invention is a method for bleaching a pulp, wherein the pulp is treated with the bleaching agent. Furthermore, the present invention provides a bleaching method of pulp characterized in that chemical bleaching and / or alkali extraction is carried out before, after or during pulp processing when pulp is processed using the bleaching agent. It is.

  According to the present invention, it is possible to provide a novel thermostable xylanase and its gene, a method for producing the thermostable xylanase and its use. The present invention makes it possible to produce a thermostable xylanase, and contributes to the industrial production of the thermostable xylanase. In addition, by treating the heat-resistant xylanase of the present invention and / or the culture of the strain of the present invention with pulp, the bleaching property of the pulp is improved, chemicals such as chlorine are reduced in paper pulp production, AOX in wastewater is reduced, etc. It contributes to.

Hereinafter, the present invention will be described in detail.
(1) Physicochemical properties of enzyme First, the physicochemical properties of thermostable xylanase XP1 and thermostable xylanase XP2 of the present invention will be described as follows.

(1) Action 10 U each of XP1 and XP2 was added to 5 ml of a 1% solution (pH 7.0, 40 mM sodium phosphate buffer) of xylan (derived from hippo, manufactured by Sigma), and reacted at 60 ° C. . After the reaction for a predetermined time, the reaction was stopped by boiling, and 10 μl was subjected to thin layer chromatography. The thin layer was HPLC Cieselgel 60 F254 manufactured by Merck and the developing solvent was n-butanol: acetic acid: water = 10: 5: 1. Color development was performed by spraying diphenylamine-aniline reagent and heating at 120 ° C. for 10 minutes.

  The results are shown in FIG. The time in the figure represents the reaction time of the enzyme. From this result, it is clear that XP1 produces xylooligosaccharide and XP2 produces xylose and xylooligosaccharide by acting on xylan.

(2) Substrate specificity Acts on hardwood kraft pulp and wheat bran containing xylan in addition to xylan preparations such as birch silane and wheat xylan.

(3) Optimum pH and stable pH range The optimum reaction pH and pH stability of each enzyme are determined using glycine-hydrochloric acid buffer (pH 3 or lower), acetate buffer (pH 4-5), sodium phosphate buffer (pH 6- 7) Measured using Tris-HCl buffer (pH 8-9) and glycine-sodium hydroxide buffer (pH 9.1 to 10.1). The enzyme activity of each enzyme was measured at each pH.

  The results are shown in FIG. In the figure, “◯” represents XP1, and “■” represents XP2. From FIG. 2, the optimum pH of the enzyme reaction was 5 to 8 for both XP1 and XP2. In addition, each enzyme was kept in a predetermined buffer solution of 50 mM for 2 nights at 4 ° C., and then the enzyme activity was measured. The results are shown in FIG. In the figure, “◯” represents XP1, and “■” represents XP2. From FIG. 3, XP1 was stable at pH 3.0 to 9.0, and XP2 was stable at pH 4.5 to 9.0.

(4) Method for measuring titer Xylanase activity was measured as follows. 50 μl of a test solution is added to 200 μl of a 1% solution (pH 6.5, 1/10 McIlvaine buffer solution) of xylan (derived from hippo material, manufactured by Sigma), and reacted at 70 ° C. for 5 minutes. Add 500 μl of DNS reagent and boil for 5 minutes. Immediately cool on ice, add 4 ml of distilled water, and measure the absorbance at 500 nm. A calibration curve is prepared using xylose with a known concentration. With respect to the activity unit of xylanase, the amount of enzyme that produces 1 μmol of reducing sugar per minute under the above conditions was set to 1 Unit (unit: U).

(5) Range of temperature suitable for action The enzyme activity of each enzyme was measured by changing the reaction temperature. The results are shown in FIG. In the figure, “◯” represents XP1, and “■” represents XP2. From FIG. 4, the optimum temperature was 70 ° C. for XP1 and 80 ° C. for XP2. In addition, each enzyme was allowed to stand for 30 minutes in a 50 mM Tris-HCl buffer (pH 7.2) at a predetermined temperature, and then the enzyme activity was measured.

  The results are shown in FIG. In the figure, “◯” represents XP1, and “■” represents XP2. From FIG. 5, XP1 retained about 90% or more of the enzyme activity after treatment at 50 ° C. for 30 minutes, and showed about 50% or more of the residual activity even after treatment at 60 ° C. for 30 minutes. On the other hand, XP2 showed a residual activity of about 90% or more after treatment at 70 ° C. for 30 minutes.

(6) Isoelectric point
As a result of isoelectric focusing by PRECOAT pH 3 to 10 manufactured by SERVA, the isoelectric point of XP1 was 8.1 and the isoelectric point of XP2 was 8.5.

(7) Molecular weight As measured by SDS polyacrylamide gel electrophoresis, the molecular weight of XP1 was about 22,500, and the molecular weight of XP2 was about 32,000.

(8) Effects of metal ions and inhibitors After adding various metal salts to the enzyme solution to 1 mM and keeping at 4 ° C overnight, the same kind of metal salt is also to be 1 mM in the reaction solution. After addition, enzyme activities of XP1 and XP2 were measured.

The results are shown in Table 1. From Table 1, XP1 is strongly inhibited by Hg2 +, XP2 is Mn ^2+, Co ^2+, weakly inhibited by Cu ^2+, it was strongly inhibited by Hg ^2+.

  Various substances such as inhibitors are added to the enzyme solution to 1 mM, and kept at 4 ° C. overnight, and then the same kind of substance is also added to the XP1 and XP2 enzyme solutions in the reaction solution to 1 mM. And the activity was measured. The results are shown in Table 2. From Table 2, XP1 was weakly inhibited by iodoacetic acid and EDTA, and strongly inhibited by SDS. XP2 was weakly inhibited by EDTA and iodoacetic acid and strongly inhibited by SDS.

  By the way, the enzyme of the present invention is a thermostable xylanase having an optimum reaction temperature of 70 ° C. or higher, and is a novel thermostable xylanase having physicochemical properties different from the conventional ones. Conventionally known thermostable xylanases are reported as follows. John et al. Reported xylanase IA derived from Aspergillus niger Strain 21 with an optimum pH of 5.5-6 and an optimum temperature of 65-80 ° C. (Can. J. Biochem., 57, 125, 1979), but there is no description of the isoelectric point, and the molecular weight is 50,000.

  Berenger et al. Isolated three types (A, B, C) of xylanases (A, B, C) with an optimum pH of 6-7 and an optimum temperature of 75 ° C. derived from Clostridium stercorarium (Can. J Microbiol., 31, 635, 1985), all of which have an isoelectric point of around 4.5 and a molecular weight of 44,000-72,000. Wang et al. Isolated xylanase I from Streptomyces cyaneus with an optimum pH of 8.5 and an optimum temperature of 72 ° C. (J. Gen. Microbiol., 139, 1987, 1993). However, it has a molecular weight of 37,000 and an isoelectric point of 5.1.

  As for Bacillus, Honda et al., From Bacillus C-125 strain, the optimal reaction pH was 6-7 and the optimal reaction temperature was 70 ° C, and the optimal reaction temperature was 6-10 and the optimal reaction temperature was 6-10. Isolation of xylanase A at 70 ° C. (Can. J. Microbiol., 31,538, 1985) has molecular weights of 16,000 and 43,000, respectively. JP-A-6-506107 describes a xylanase that is stable at 60 ° C. and has an optimum pH of 4.8 to 7 derived from Bacillus, but has a molecular weight of 22,000 and an isoelectric point of 7.7. The optimum temperature is not described.

As described above, these xylanases are different from the xylanase of the present invention in the optimum temperature, optimum pH, molecular weight, isoelectric point, etc., so that both XP1 and XP2 of the present invention are recognized as novel thermostable xylanases. did.
The results of comparing the enzyme of the present invention with a conventional xylanase are shown in Table 3.

(2) Microorganism Next, the microorganism that produces the thermostable xylanase of the present invention will be described. The microorganism used in the present invention is a strain belonging to the genus Bacillus and capable of producing a thermostable xylanase, and specific examples thereof include Bacillus sp 2113 and Bacillus sp 208.

Each microorganism will be described below.
A. Bacillus SP 2113
Bacillus sp. 2113 is a strain isolated by the present inventors from the soil, and 1% of kabukilan or wheat xylan, 0.5% of peptone, 0.5% of yeast extract, 0.1% of K ₂ HPO ₄ , It grows well in 45 ° C culture using MgSO ₄ · 7H ₂ O 0.02%, pH 7.0 medium. The bacteriological properties of this strain are as follows.

(1) Morphological properties
i) Bacillus having a motility of 0.3 to 0.6 × 2 to 5 μm. May show 2 to 3 chain form. A spore is formed in the center of the cell.
ii) Gram stainability is indeterminate and acidity is negative.

(2) The growth conditions and the like in various media are as shown below. The culture temperature was 45 ° C.
i) On the broth agar plate (Difco Beef Extract 1%, Bacto Peptone 1%, NaCl 0.5%, Agar 1.5%, pH 7.0), the colony is almost circular and the periphery is slightly wavy. . A semi-transparent colony with a slight gloss. Meat broth agar slope medium (Difco Beef Extract 1%, Bacto Peptone 1%, NaCl 0.5%, Agar 1.5%, pH 7.0) is slightly glossy and grows thinly in a spread. In a broth liquid medium (Difco Beef Extract 1%, Bacto Peptone 1%, NaCl 0.5%, agar 1.5%, pH 7.0), the liquid surface hardly grows and precipitates. Neither growth is very good. Difco Nutrient Broth broth agar medium (Difco Nutrient Broth 0.8%, agar 1.5%, pH 7.0) grows worse.
ii) It grows in a medium with 2% NaCl added to a broth agar plate medium (Difco Beef Extract 1%, Bacto Peptone 1%, NaCl 0.5%, agar 1.5%, pH 7.0), but grows when 5% is added. do not do.
iii) Do not liquefy in broth gelatin medium (Difco beef extract 1%, peptone 1%, NaCl 0.5%, gelatin 12% pH 7.0).
(3) Table 4 shows the physiological properties.

  Based on the above bacteriological properties, this strain was identified with reference to Bergey's Manual of Systematic Bacteriology. As a result, it is clear that this strain belongs to the genus Bacillus because it is a gram-staining gonococci that forms spores and is positive for catalase. The species is close to Bacillus circulans, but the strain of the present invention is positive for oxidase and can grow at 55 ° C, whereas Bacillus circulans is negative for oxidase, The strain of the present invention is different from Bacillus circulans because it cannot grow above 50 ° C.

  Compared with Bacillus stearothermophilus, which has a report of thermostable xylanase (T. Nanmori et al., J. Bacteriol., 172, 6669, 1990), the strain of the present invention has a spore position. Is in the middle and cannot hydrolyze gelatin and cannot grow at 65 ° C, whereas Bacillus stearothermophilus has a spore position at the end and can hydrolyze gelatin and grow at 65 ° C. Because of this, the strain of the present invention is different from Bacillus stearothermophilus.

  Also, Bacillus No.I-1017 and No.I-1018 producing xylanase described in JP-A-6-506107 have an optimum growth temperature of 62 ° C. and do not use fructose or arabinose. . On the other hand, the strain of the present invention has an optimum growth temperature of 35-50 ° C. and utilizes fructose and arabinose, so that the strain of the present invention is Bacillus No.I-1017 and No.I- Different from 1018.

  From the above, since there is no bacterial species corresponding to the strain of the present invention, the strain was judged to be a new strain and named Bacillus sp 2113. The Bacillus sp 2113 is deposited as FERM BP-5264 at the Institute of Biotechnology, Ministry of International Trade and Industry.

B. Bacillus SP 208
Bacillus sp 208 is cultured at 45 ° C. using a medium of 1% kabukilan or wheat xylan, 0.5% peptone, 0.5% yeast extract, 0.1% K ₂ HPO ₄ , 0.02% MgSO ₄ .7H ₂ O, pH 7.0 It grows well. The bacteriological properties of this bacterium are as follows.

(1) Morphological properties
1) It is a koji mold having a motility of 0.3 to 0.6 × 2 to 5 μm. May show 2 to 3 chain form. A spore is formed in the center of the cell.
2) Gram stainability is indeterminate and acidity is negative.

(2) Growth conditions etc. in various media are as shown below. The culture temperature was 45 ° C.
1) In the broth agar medium (Difco Nutrient Broth 0.8%, agar 1.5%, pH 7.0), the colony shape is almost circular and its periphery is slightly wavy. It is a slightly glossy translucent colony that grows well.
2) It grows in a medium of 2% NaCl added to a broth agar plate medium (Difco Nutrient Broth 0.8%, agar 1.5%, pH 7), but does not grow when 5% is added.
3) Does not liquefy in gravy gelatin medium (Difco Beef Extract 1%, gelatin 12%, pH 7.0).
(3) Table 5 shows the physiological properties.

  The above bacteriological properties were identified with reference to Bergey's Manual of Systematic Bacteriology. As a result, the strain of the present invention is an aerobic Gram-stained gonococcus that forms spores, and is positive for catalase, and thus clearly belongs to the genus Bacillus.

  The species is similar to the nature of Bacillus circulans, but the growth temperature range of Bacillus circulans is 10-40 ° C, whereas oxidase is negative. The growth temperature of the invented bacterial strain Bacillus sp. 208 is capable of growing even at high temperatures (40-60 ° C.) where it cannot grow on Bacillus circulans, and is positive for oxidase. The strain is different from Bacillus circulans.

  In addition, compared with Bacillus stearothermophilus, which has a report of thermostable xylanase production (T. Nanmori et al., J. Bacteriol., 172, 6669, 1990), Is the center and cannot hydrolyze gelatin, whereas Bacillus stearothermophilus has a spore position at the end and can hydrolyze gelatin, so that the strain of the present invention is Bacillus stearothermophilus. (Bacillus stearothermophilus) is different.

  Further, Bacillus NO.I-1017 and NO.I-1018 producing xylanase described in JP-A-6-506107 do not use arabinose, whereas the strain of the present invention uses arabinose. Therefore, the strain of the present invention is different from Bacillus NO.I-1017 and NO.I-1018. Therefore, the strain of the present invention is a strain similar to the Bacillus sp 2113. However, since the Bacillus sp. 2113 strain does not grow well in the broth medium (Nutrient Broth 0.8%, agar 1.5%), this strain is distinguished from the Bacillus sp. Strain 2113 because it grows well in the broth medium. This strain was judged as a new strain and named Bacillus sp 208.

  Bacillus SP 208 is deposited at the Biotechnology Institute of Industrial Technology, Ministry of International Trade and Industry under the accession number FERM BP-5321.

(3) Cloning of thermostable xylanase gene A DNA library is prepared in order to clone the xylanase gene. A DNA library can be prepared by extracting chromosomal DNA from the Bacillus sp. Strain 2113 or 208, treating it with an appropriate restriction enzyme, connecting it to an appropriate vector, and introducing it into a suitable host. .

  In order to extract chromosomal DNA from the Bacillus sp. Strain 2113 or 208, a usual method can be used (for example, Molecular Cloning; Cold Spring Harbor Laboratory (1982) 1 Blin and Stafford method). Next, the obtained chromosomal DNA is treated with an appropriate restriction enzyme, and after partial digestion, a 3 to 5 kbp fragment fraction is obtained by sucrose density gradient centrifugation. The DNA fragment obtained above is inserted into a cloning vector treated with a restriction enzyme that produces the same sticky end. Examples of vectors used for library preparation include plasmid vectors and phage vectors. Examples of hosts include Escherichia coli and yeast.

  As a cloning vector, for example, a pUC cloning vector can be used. In isolation of the target gene from the DNA library obtained above, Escherichia coli is transformed using the DNA library, and then applied to a xylan medium, and halo formation is used as an indicator. The base sequence of the DNA thus cloned can be analyzed by a dideoxy method using a radiolabel or a fluorescent label, a Maxam-Gilbert method, or the like.

(4) Enzyme Production Method Next, the enzyme production method of the present invention will be described.
(1) Purification of the enzyme of the present invention from a culture solution of a thermostable xylanase-producing bacterium The thermostable xylanase can be produced by culturing the Bacillus sp. 2113 strain or 208 strain of the present invention. Any carbon source and nitrogen source can be used as long as they can be assimilated to produce a thermostable xylanase. For example, as the carbon source, xylan or wheat bran containing xylan, pulp, bagasse, corn fiber, rice straw and other agricultural wastes or plant fiber can be used. As the nitrogen source, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, various inorganic nitrogen can be used. Various salts, vitamins, minerals, and the like can be used as appropriate.

  The culture temperature and pH may be any as long as the bacteria grow and produce a thermostable xylanase. The culture temperature is 20 to 55 ° C, preferably 35 to 50 ° C, and the pH is 5 to 9, preferably 6 to 6. 8. After culturing the microorganism of the present invention, the cells are separated, and the culture filtrate can be used as it is as a heat-resistant xylanase crude enzyme solution. Such a thermostable xylanase crude enzyme solution has an optimum reaction temperature of 60 to 80 ° C. and an optimum pH of 5 to 7.

  Further, the thermostable xylanase can be concentrated or solidified by dialysis, salting out, ultrafiltration, lyophilization or the like. Furthermore, the thermostable xylanases XP1 and XP2 can be purified by appropriately combining and repeating the culture filtrate with ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation, and the like. . Specific purification methods are shown in the examples.

(2) Purification of genetically engineered thermostable xylanase by genetic engineering techniques The thermostable xylanase of the present invention can also be purified by expressing a cloned gene (in the present invention, obtained by expressing a gene). The obtained enzyme is referred to as “gene recombinant thermostable xylanase”). According to the technique, the xylanase gene obtained by the above (3) can be highly produced by expressing it using an appropriate host / vector. As vectors used for expression, plasmid vectors, phage vectors and the like are mainly used. As the host, Escherichia coli, Bacillus subtilis, yeast and the like are mainly used. Any carbon source and nitrogen source can be used as long as they can be assimilated to produce a thermostable xylanase. For example, as the carbon source, xylan or wheat bran containing xylan, pulp, bacus, corn fiber, rice straw or other agricultural waste, or plant fiber can be used. As the nitrogen source, nitrogen compounds such as yeast extract, peptone, various amino acids, soybean, corn steep liquor, various inorganic nitrogen can be used. Various salts, vitamins, minerals, and the like can be used as appropriate. The culture temperature and pH may be any as long as the bacteria produce thermostable xylanase. The culture temperature is preferably 37 ° C. and the pH is preferably 7. As an enzyme purification method, it can be purified by appropriately combining and repeating ammonium sulfate fractionation, molecular weight fractionation by gel filtration, various ion exchange resins, hydroxyapatite, isoelectric point fractionation, and the like. Confirmation of whether the obtained purified enzyme is the desired xylanase is based on comparison of the molecular weight, optimum pH, optimum temperature, N-terminal amino acid sequence, etc. of the obtained purified enzyme with the thermostable xylanase produced by Bacillus sp. It can be judged by doing. Specific examples of enzyme acquisition are shown in the Examples.

(5) Pulp Bleaching Method Next, a pulp bleaching method using the enzyme of the present invention will be described. In the chemical pulp and mechanical pulp manufacturing process, the strain Bacillus sp belonging to the genus Bacillus and / or thermostable xylanase XP1 (including gene recombinant type) and / or XP2 (including gene recombinant type) of the present invention Bleaching can be performed by treating the pulp with a culture of 2113 or Bacillus sp 208. Further, the pulp can be bleached by chemical bleaching and / or alkali extraction before, during or after the enzyme treatment.

  About the culture | cultivation or enzyme amount processed to a pulp, what is necessary is just to add 0.1-5U per 1g of dry weight of a pulp as a thermostable xylanase unit, Preferably 0.5-3U should be added. In the case of a culture filtrate (crude enzyme solution), the reaction conditions are a reaction temperature of 50 to 90 ° C. and pH 5 to 8, and in the case of purified enzyme, the reaction temperature is 50 to 80 ° C. and pH 5 to 8 for XP1. In XP2, the reaction temperature is 60 to 90 ° C. and the pH is 5 to 8. The reaction time is 0.2 to 24 hours, preferably 0.5 to 8 hours. Reagents used for chemical bleaching include chlorine, chlorine dioxide, nitrogen dioxide, hypochlorite, oxygen, hydrogen peroxide, ozone and the like. In addition, many alkaline compounds known to those skilled in the art can be used for alkali extraction. For alkali extraction, 0.5 to 3% (absolute dry pulp) alkali in terms of sodium hydroxide can be used, and alkali treatment can be performed while adding oxygen, hydrogen peroxide, or the like.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

[Example 1] Preparation of crude enzyme solution (1)
10 ml of liquid medium containing 0.6% xylan (derived from hippo wood, manufactured by Sigma), 0.5% peptone, 0.5% yeast extract, 0.1% K ₂ HPO _{4 and} 0.02% MgSO ₄ · 7H ₂ O pH 7.0) was taken in a test tube having an inner diameter of 25 mm, sealed with a paper stopper, and steam sterilized at 121 ° C. for 15 minutes. One platinum ear of Bacillus sp 2113 was inoculated into this, and reciprocally shake-cultured at 45 ° C. (amplitude 25 mm, 300 reciprocations / min). After completion of the culture, the culture supernatant was separated by centrifugation (10,000 rpm × 10 minutes) to obtain a crude enzyme solution of thermostable xylanase.

  50 μl of enzyme solution was added to 200 μl of a 1% xylan solution (beech wood xylan, Sigma 1/10 McIlvaine Buffer pH 6.5) and reacted at 70 ° C. for 5 minutes. After 500 μl of DNS reagent was added and boiled for 5 minutes, it was immediately ice-cooled, 4 ml of distilled water was added, and the absorbance at 500 nm was measured. A calibration curve was prepared using xylose with a known concentration.

  For xylanase activity, the amount of enzyme that produced 1 μmol of reducing sugar per minute under the above conditions was defined as 1 unit. As a result, the thermostable xylanase activity in the culture supernatant was 400 U / ml at 24 hours and 500 U / ml at 48 hours after the start of the culture.

[Example 2] Preparation of crude enzyme solution (2)
Xylan (from birch, Sigma) 0.6%, 0.5% peptone, yeast extract 0.5%, K ₂ HPO ₄ 0.1% of MgSO ₄ · 7H liquid medium (pH 7) containing ₂ O 0.02% 10 ml The sample was taken in a test tube having an inner diameter of 25 mm, sealed with a paper stopper, and then steam sterilized at 121 ° C. for 15 minutes. One platinum ear of Bacillus sp 208 was inoculated into this, followed by reciprocal shaking culture at 45 ° C. (amplitude 25 mm, 300 reciprocations / min). After completion of the culture, the culture supernatant was separated by centrifugation (10,000 rpm × 10 minutes) to obtain a crude enzyme solution of thermostable xylanase. The thermostable xylanase activity in the culture supernatant was measured by the following method.

  Add 50 μl of enzyme solution to 200 μl of 1% xylan solution (beech wood xylan, Sigma 1/10 McIlvaine Buffer pH 6.3), and react at 70 ° C. for 5 minutes. After 500 μl of DNS reagent was added and boiled for 5 minutes, it was immediately ice-cooled, 4 ml of distilled water was added, and the absorbance at 500 nm was measured. A calibration curve was prepared using xylose with a known concentration.

  For xylanase activity, the amount of enzyme that produced 1 μmol of reducing sugar per minute under the above conditions was defined as 1 unit. As a result, the thermostable xylanase activity in the culture supernatant was 400 U / ml after 24 hours and 500 U / ml after 48 hours.

[Example 3] Purification of thermostable xylanase Xylan (derived from hippo wood, manufactured by Sigma) 0.6%, peptone 0.5%, yeast extract 0.5%, K ₂ HPO ₄ 0.1%, MgSO ₄ · 7H ₂ O 0.02%, pH 7 0.0 ml of liquid medium (0.0 ml) was placed in a 500 ml Sakaguchi flask, stoppered with cotton, and then steam sterilized at 121 ° C for 15 minutes. To this, 1 ml of the culture solution obtained in Example 1 was added, followed by reciprocal shaking culture at 45 ° C. for 3 days (amplitude 10 cm, 100 reciprocations / min). After completion of the culture, the culture supernatant was obtained by centrifugation (8,000 rpm × 10 minutes). The culture supernatant was fractionated with ammonium sulfate, and the 20-60% fraction was collected by centrifugation (20,000 rpm × 10 minutes), followed by dialysis using 20 mM Tris-HCl buffer (pH 7.2) as an external solution. It was. The resulting crude enzyme solution was subjected to ion exchange chromatography using CM Toyopearl 650-C (diameter 2.5 cm × 30 cm) equilibrated with 20 mM acetate buffer (pH 5.0).

  The adsorbed fraction was eluted with the buffer containing NaCl at a concentration gradient from 0 M to 0.3 M, and fractionated by 5.3 ml. As a result, the xylanase activity was separated into two peaks and eluted, and the active fraction eluted in the first half was designated as xylanase XP1, and the active fraction eluted in the second half was designated as xylanase XP2.

  Each active fraction was collected, and ammonium sulfate fractionation was performed again. After collecting 20 to 60% fraction by centrifugation (20,000 rpm × 10 minutes), 50 mM was added to 20 mM Tris-HCl buffer (pH 7.2). Gel filtration was performed by elution with Sephacryl S-200 (diameter 2.5 cm × 93 cm) equilibrated with NaCl-containing buffer. For XP1, the flow rate was 34 ml / hr, and 5 ml was fractionated. XP2 was fractionated by 6.7 ml at a flow rate of 34 ml / hr.

  The active fractions were collected and subjected to SDS polyacrylamide gel electrophoresis, confirming that they were uniformly purified. The yield of each purified enzyme with respect to the culture filtrate was 47.8% for XP1 and 7.8% for XP2. The specific activity was 1420 U / mg for XP1 and 919 U / mg for XP2.

[Example 4] Cloning of thermostable xylanase gene
(1) Preparation of chromosomal gene library 0.6% xylan (derived from hippo wood, manufactured by Sigma), 0.5% peptone, 0.5% yeast extract, K ₂ HPO ₄ 0.1%, MgSO _4. 50 ml of a liquid medium of 7H ₂ O 0.02%, pH 7.0 was placed in a 500 ml Sakaguchi flask and sealed with a cotton plug and steam sterilized at 121 ° C. for 15 minutes. Bacillus sp. 2113 strain was inoculated with 1 platinum ear, and reciprocal shaking culture was performed at 45 ° C. overnight (amplitude 10 cm, 100 reciprocations / min). After completion of the culture, the cells were obtained by centrifugation (10,000 rpm × 10 minutes).

  The cells were suspended in 5 ml of glucose-lysozyme solution (50 mM glucose, 10 mM EDTA, 25 mM Tris-HCl buffer (pH 8.0), 4 mg / ml lysozyme) and left at room temperature for 15 minutes. 5 ml of alkaline solution (02N NaOH, 1% SDS) was added, mixed gently and cooled in ice for 15 minutes. Thereafter, phenol extraction and chloroform extraction were performed, and ethanol was gradually added to the extracted aqueous layer portion. When DNA was precipitated, the chromosomal DNA was wound with a glass rod and suspended in a TE solution. 100 μg of the obtained chromosomal DNA was partially digested with the restriction enzyme EcoRI and fractionated by 3-20% sucrose density gradient ultracentrifugation (22,500 rpm, 16 hours), and the 3-5 kbp fragment fraction was recovered by ethanol precipitation. did.

(2) Transformation of heat-resistant gene into Escherichia coli E. coli cloning vector pUC19 (Takara Shuzo) 1 μg was completely digested with restriction enzyme EcoRI, dephosphorylated with alkaline phosphatase (derived from Calf intestine), and after ethanol precipitation The gene of the present invention and the vector were ligated in a ligation buffer containing 500 ng of DNA and 2.5 units of T4 DNA ligase at 16 ° C. for 16 hours.
Escherichia coli JM109 strain was transformed by the calcium chloride method using the obtained DNA library.

(3) Isolation of xylanase gene from DNA library Cloning of XP1 gene Selection of clones containing XP1 xylanase gene from the chromosomal DNA library was carried out by transforming Escherichia coli with the DNA library, and then xylan medium. The halo formation around the colony was used as an index. That is, after transforming Escherichia coli using the DNA library, xylan medium (1% xylan (from wheat; manufactured by Sigma)), 1% peptone, 0.5% yeast extract containing 100 μg / ml ampicillin. , 0.5% NaCl, 2% agar (pH 7.0)) and cultured at 37 ° C. overnight, and halo formation was observed. A large amount of plasmid DNA was prepared from the obtained clones by alkaline extraction, purified by ultracentrifugation (16 hours, 20 ° C.), and the nucleotide sequence was determined. The nucleotide sequence was determined using a Sequenase kit manufactured by United States Biochemical.

  The result is shown in SEQ ID NO: 2. The result of estimating the amino acid sequence of XP1 xylanase from the base sequence of the XP1 xylanase gene is shown in SEQ ID NO: 1. FIG. 6 shows a restriction enzyme map of a DNA fragment containing a gene encoding xylanase (XP1) obtained by the above cloning.

  The obtained E. coli JM109 / pUCXP1 containing the XP1 xylanase gene was deposited as FERM BP-5320 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology.

(4) Determination of the N-terminal sequence of XP1 The N-terminal amino acid sequence of XP1 was determined using a protein sequencer (Applied Biosystems (Perkin Elmer) 477A) and PTH using XP1 purified from the culture supernatant of Bacillus sp 2113 as a sample. An analyzer (Applied Biosystems (Perkin Elmer) 120A) was used. As a result, the N-terminal sequence of mature xylanase XP1 was as shown in SEQ ID NO: 3.

[Example 5] Production of recombinant thermostable xylanase In this example, recombinant thermostable xylanase XP1 was produced. The plasmid pUCXP1 was completely digested with EcoRI fragment 500 ng of the present invention obtained by digestion with EcoRI and the restriction enzyme EcoRI, and then dephosphorylated with alkaline phosphatase (derived from Calf Intestine) pH / 3000PLK (Takara Shuzo) 1 μg) and T4 ligase 2.5 unit were reacted in a ligation buffer at 16 ° C. for 2 hours and ligated. Using this, Escherichia coli JM109 was transformed by the calcium chloride method.

  The cells were inoculated in L medium (1% peptone, 0.5% yeast extract, 0.5% NaCl (pH 7.0)) containing 50 μg / ml tetracycline and cultured at 37 ° C. overnight. A large amount of plasmid DNA was prepared from the obtained transformant by an alkali extraction method. This is designated as pHYXP1, and the results are shown in FIG. Bacillus subtilis ISW1214 strain was transformed with the plasmid DNA by the protoplast method. The obtained transformant was placed in a 50 ml Sakaguchi flask in a 50 ml liquid xylan medium (0.6% Kabukilan (Sigma), 1% peptone, 0.5% yeast extract, 0.5% NaCl (pH 7. 5) containing 50 μg / ml tetracycline. 0)) Inoculated and cultured at 37 ° C for 3 days. After completion of the culture, the culture supernatant was collected by centrifugation (10,000 rpm × 10 minutes). The activity of thermostable xylanase in the culture supernatant was 160 U / ml 48 hours after the start of the culture.

  The culture supernatant was fractionated with ammonium sulfate, and the 20-60% fraction was collected by centrifugation (20,000 rpm x 10 minutes), and then dialyzed using 20 mM Tris-HCl buffer (pH 7.2) as an external solution. It was. The resulting crude enzyme solution was subjected to ion exchange chromatography using CM Toyopearl 650-C (diameter: 1.0 cm × 17 cm) equilibrated with 20 mM acetate buffer (pH 5.0).

  The adsorbed fraction was eluted with a 20 mM acetate buffer solution (pH 5.0) containing NaCl at a concentration gradient from 0 M to 0.3 M, and fractionated in 1.5 ml portions. The active fractions were collected and subjected to SDS polyacrylamide electrophoresis, and confirmed to be uniformly purified. This enzyme has a molecular weight of about 22,500, an optimum reaction pH of 5 to 8, a stable pH range of 3 to 9, an optimum temperature range of action of 50 to 80 ° C., and was determined to be XP1.

[Example 6] Bleaching of pulp (1)
The culture supernatant of Bacillus sp 2113 obtained in Example 1 (containing 500 U / ml thermostable xylanase) on hardwood oxygen-bleached kraft pulp (kappa number 8.5, whiteness 46.0%) 2 μl per gram was added and reacted at a pulp concentration of 3%, pH 7.0, and 70 ° C. for 2 hours. After completion of the reaction, the pulp concentration was adjusted to 10% and bleaching was performed in the order of chlorine-alkali-hypochlorite-chlorine dioxide. In addition, what was processed similarly without adding culture supernatant was made into the control | bleached bleached pulp. The standard conditions for bleaching are as follows.
Chlorine treatment: Addition rate is 1.6% per pulp dry weight at 40 ° C for 30 minutes Alkali extraction: Alkali addition rate is 1.0% per absolute dry pulp at 60 ° C for 100 minutes Hypochlorite treatment : Addition rate 0.5% at 45 ° C for 120 minutes Chlorine dioxide treatment: Addition rate 0.2% at 70 ° C for 180 minutes

  Bleaching was performed with the addition amount of chlorine and alkali or hypochlorite reduced with respect to this standard bleaching condition. As a result, it was possible to reduce the amount of chlorine and alkali necessary to obtain the same whiteness (85.6%) as that of the control bleached pulp by 25%. In addition, hypochlorite was reduced by 50%.

  Moreover, as a result of quantifying the amount of AOX in the bleaching wastewater using a halogen analyzer TOX-10 (manufactured by Mitsubishi Chemical), AOX could be reduced by 25% by the culture supernatant treatment. The reduction of chlorine, hypochlorite, alkali, AOX, etc. indicates that the bleaching property of the pulp has been improved by the action of this enzyme, which can reduce the chemical cost and organic This is useful in that the production of chlorine compounds can be suppressed.

[Example 7] Bleaching of pulp (2)
The culture supernatant of Bacillus sp 208 obtained in Example 2 (containing 500 U / ml of heat-resistant xylanase) is added to kraft pulp exposed to hardwood oxygen (kappa number 8.5, whiteness 46.0%) at 2 μl per gram of dry pulp weight. The mixture was added and reacted at a pulp concentration of 3%, pH 7.0, 70 ° C. for 2 hours. After completion of the reaction, the pulp concentration was adjusted to 10%, and bleaching was performed in the order of chlorine-alkali extraction-hypochlorous acid-chlorine dioxide in the usual manner. In addition, what was processed similarly without adding culture supernatant was made into the control | bleached bleached pulp. The standard conditions for bleaching are as follows.
Chlorine treatment: chlorine addition rate is 1.6% per pulp dry weight at 40 ° C for 30 minutes Alkali extraction: alkali addition rate is 1.0% per dry pulp at 60 ° C for 100 minutes Hypochlorous acid treatment: hypochlorous acid Acid addition rate 0.5% at 45 ° C for 120 minutes Chlorine dioxide treatment: Chlorine dioxide addition rate 0.2% at 70 ° C for 180 minutes

  In contrast to this standard bleaching condition, in the bleaching of enzyme pretreated pulp, the amount of chlorine and alkali or hypochlorous acid added was reduced. As a result, it was possible to reduce the amount of chlorine and alkali required to obtain the same whiteness (85.6%) as the control bleached pulp without enzyme treatment by 27%. Hypochlorite was reduced by 53%.

  The amount of AOX in the bleaching effluent was quantified using a halogen analyzer TOX-10 (manufactured by Mitsubishi Chemical). As a result, AOX was reduced by 28% by treatment with the culture supernatant.

[Comparative Examples 1-5] Bleaching of pulp (comparison with commercially available enzyme)
The pulp was subjected to enzyme treatment and bleaching treatment using various commercially available enzymes, and the reduction rates of chlorine, hypochlorite and AOX were compared with the results of Examples 6 and 7. Comparative Example 1 is Irgazyme 40-X4 manufactured by Chiba-Geigy, Comparative Example 2 is Irgazyme 10A-X4, Comparative Example 3 is Pulpzyme HC manufactured by Novo, and Comparative Example 4 is Alko. The same treatment as in Examples 6 and 7 was performed using Ecopulp made by the company and Cartazyme HS made by Sandoz as the comparative example 5. Table 6 shows the results obtained in Examples 6 and 7 and the results of the comparative examples.

  Table 6 shows that the enzyme of the present invention has a higher reduction rate of chlorine, chlorine dioxide, and AOX under the conditions of pH 7.0 and 70 ° C. than the conventional enzyme. In particular, some enzymes have little activity at such high temperatures, and it has been found that the enzyme of the present invention is excellent in heat resistance.

It is a figure which shows the result of the thin-layer chromatographic analysis of the reaction product when the enzyme of this invention is made to act on birch silane. It is a figure which shows reaction optimal pH of the enzyme of this invention. It is a figure which shows the pH stability of the enzyme of this invention. It is a figure which shows the optimal reaction temperature of the enzyme of this invention. It is a figure which shows the thermostability of the enzyme of this invention. It is a restriction enzyme map of the DNA fragment containing the gene which codes xylanase XP1. FIG. 5 is a construction diagram of plasmid pUCXP1. FIG. 5 is a construction diagram of plasmid pHYXP1.

SEQ ID NO: 1
Sequence length: 211
Sequence type: Amino acid Topology: Linear sequence type: Protein origin Organism name: Bacillus sp.
Stock name: Characteristics of the 2113 strain
1-23 S sig peptide
24-211 S mat peptide
SEQ ID NO: 2
Sequence length: 1207
Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: Genomic DNA
Origin organism name: Bacillus sp.
Strain name: Characteristics of the 2113 strain sequence Symbol representing the characteristics: P CDS
Location: 379..1029
Method for determining characteristics: E
SEQ ID NO: 3
Array length: 10
Sequence type: Amino acid Topology: Linear sequence type: Peptide origin Organism name: Bacillus sp.
Stock name: 2113 shares

Claims (3)

A thermostable xylanase XP2 derived from a microorganism belonging to the genus Bacillus having the following physicochemical properties:
(1) Action: Hydrolyzes 1,4-β-D-xyloside bond of xylan to produce reducing sugar of xylose and xylooligosaccharide.
(2) Substrate specificity: Preparation of birch silane, wheat xylan, etc. In addition to xylan, it acts on hardwood kraft pulp, wheat bran and the like containing xylan.
(3) Optimal pH and stable pH range: The optimal pH range for the reaction is pH 5-8, and the stable pH range is 4.5-9.
(4) Range of temperature suitable for action: in the range of 60 to 90 ° C.
(5) Optimal temperature: 80 ° C.
(6) Thermal stability: shows a residual activity of about 90% or more after treatment at 70 ° C. for 30 minutes.
(7) Isoelectric point: around 8.5.
(8) Molecular weight: As a result of measuring by SDS polyacrylamide gel electrophoresis, it is about 32,000.
(9) Inhibition: weakly inhibited by Mn ²⁺ , Co ²⁺ , Cu ²⁺ , EDTA, iodoacetic acid, and strongly inhibited by Hg ²⁺ , SDS.   The method for producing thermostable xylanase XP2 according to claim 1, wherein microorganisms belonging to the genus Bacillus are cultured, and thermostable xylanase XP2 is collected from the resulting culture. Bacillus sp 208 (FERM BP-5321) having the ability to produce thermostable xylanase XP2 according to claim 1.

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