Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01032—Xylan endo-1,3-beta-xylosidase (3.2.1.32), i.e. endo-1-3-beta-xylanase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/248—Xylanases
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
  • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

Definitions

• This invention relates to novel enzymes. More specifically, the invention provides novel xylanases obtainable from microorganisms of the genus Thermotoga. The invention also relates to a process the for preparation of these xylanases, an agent containing these xylanases, and the use of these xylanases for treatment of lignocelluiosic pulp.
• Xylan a major component of plant hemicellulose, is a polymer of D- xylose linked by ⁇ - ⁇ ,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 bleaching chemicals, e.g. chlorine, used in the bleaching stages, and to increase the freeness of pulps in the recycled paper process
• bleaching chemicals e.g. chlorine
• Kraft pulping a process widely used in the pulp and paper industry, involves the alkaline sulfate cooking of pulp to remove most of the lignin. The remaining pulp contains 2 - 5% of lignin, which gives the pulp a dark brown colour that has the tendency to darken in UV light or by oxidation. In order to obtain a white pulp for high quality paper, the brown colour is removed by a multi-stage bleaching process using bleaching chemicals, e.g. oxygen, ozone, hydrogenperoxide, chlorine and/or chlorine dioxide.
• bleaching chemicals e.g. oxygen, ozone, hydrogenperoxide, chlorine and/or chlorine dioxide.
• Characteristic of the above processes is the need for xylanases capable of exerting hydrolytic activity at high temperatures and at alkaline conditions. o The xylanase should exert a substantial amount of its activity at pH values above pH 7.
• thermostable xylanase has been disclosed, vide Simpson H.D.. Haufler U.R, and Daniel R.M. (1991), Biochem J., 277(2) 413 - 418.
• the xylanase is obtained from the strain Thermotoga sp. FJSS3-B.1 , a strain collected s at Fiji, but, however, not available to the public [HuserB ⁇ .. Patel B.K.C. Daniel R.M. and Morgan H.W. (1986), FEMS Mikrobiol. Lett., 37 121 - 127].
• the enzyme has a pH optimum at 5.4, with 50% of activity limits at pH 4.2 and 6.7, respectively.
• T. maritima and T. neapolitana are marine organisms, collected near Italy.
• T. thermarum is not a marine organism, and it is collected in Africa.
• T. maritima Three strains representative of T. maritima, T. neapolitana, and T. 5 thermarum, respectively, have been deposited as type cultures and hence are publicly available from Deutsche Sammlung von Mirkoorganismen und Zellkulturen GmbH, Mascheroder Weg 1b, D-3300 Braunschweig, Germany.
• the strains and accession numbers are T. maritima, DSM 3109, T. neapolitana, DSM 5068, and T. thermarum, DSM 5069. Due to their improved activity at alkaline pH, the novel enzymes of this invention are especially well suited for use in the manufacture of paper pulp.
• the invention provides xylanases having more than 50% of residual activity in the a pH range 4.0 - 7.5, more preferred the pH 5 range 4.5 - 7.5, most preferred the pH range 5.5 - 7.5, when determined after 20 minutes at 90°C, more than 50% of residual activity in a pH range 4.5 - 8.0, more preferred pH range pH 4.5 - 7.5, most preferred the pH range 5.5 - 7.5, when determined after 20 minutes at 70°C, a temperature optimum in the range 80°C - 100°C, more preferred the range 85°C - 95°C, when determined after 20 minutes at o pH 6.0, and being obtainable from a strain of T. maritima, T. neapolitana, or T. thermarum.
• the invention provides xylanases having pH optimum in the pH range 5.5 - 6.5, around pH 6.0, when determined at 90°C, at least
• the invention provides a process for the preparation of the xylanase comprising cultivation of a xylanase producing strain of
• T. maritima T. neapolitana, or 7. thermarum, in a suitable nutrient medium, 0 containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
• the invention provides an agent containing the xylanase, provided in the form of a granulate, preferably a non-dusting granulate, a liquid, in particular a stabilized liquid, a slurry, or a protected enzyme.
• the invention relates to a process for treatment of lignocellulosic pulp, in which the lignocellulosic pulp is treated with an enzyme of the invention.
• Fig. 1A shows the relation between temperature and residual activity (% relative) of the xylanase obtained from T. maritima, determined after 20 minutes at pH 6.0;
• Fig. 1 B shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. maritima, determined after 20 minutes at 90°C;
• Fig 2A shows the relation between temperature and residual activity (% relative) of the xylanase obtained from T. neapolitana, determined after 20 minutes at pH 6.0;
• Fig.2B shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. neapolitana, determined after 20 minutes at 90°C;
• Fig 3A shows the relation between temperature and residual activity (% relative) of the xylanase obtained from T. thermarum, determined after 20 minutes at pH 6.0;
• Fig.3B shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. thermarum, determined after 20 minutes at 90°C;
• Fig. 4A shows the relation between pH and residual activity (% relative) of the xylanase obtained from T. neapolitana, determined after 20 minutes at 70°C;
• Fig. 4B shows the relation between pH and residual activity (% relative) of the xylanase obtained from 7. thermarum, determined after 20 minutes at 70°C.
• the xylanases of this invention are obtainable from and may be produced by cultivation of a strain of T. maritima, T. neapolitana, or T. thermarum, preferably the strain T. maritima, DSM 3109, T. neapolitana, DSM 5068, or T. thermarum, DSM 5069, or mutants or variants thereof, in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
• the enzyme may also be obtained by recombinant DNA- technology.
• the xylanases obtainable from T. neapolitana and T. thermarum have xylanolytic activity in the range of from approximately pH 4 to approximately pH 10.
• Optimum pH for activity was detected in the pH range 5.0 - 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 2B and 3B. 7.
• neapolitana showed 50% of relative activity at pH 4.5 and pH 7.5, respectively.
• T. thermarum showed 50% of relative activity at pH 5.5 and pH 7.5, respectively.
• the xylanase obtainable from T. maritima has xylanolytic activity from pH below 4 to pH approximately 11.
• Optimum pH for activity was detected in the pH range 4.0 - 7.0, more specifically the pH range 4.5 - 6.5, around pH 5.0, as presented in Fig. 1 B.
• T. maritima has 50% of relative activity at pH below 4.0 and at pH 7.5, respectively.
• T. neapolitana and T. thermarum When determined after 20 minutes at 70°C, T. neapolitana and T. thermarum have xylanolytic activity in the pH range from below pH 4 to above pH
• Optimum pH for activity was detected in the pH range 5.0 - 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 4A and 4B.
• the xylanases both showed 50% of relative activity at pH 4.5 and pH 8.5, respectively.
• the xylanases obtainable from T. neapolitana and T. thermarum have xylanolytic activity in the range of from below 20°C to above 115°C, as presented in Figs. 2A and 3A.
• the xylanase obtainable from 7. maritima has xylanolytic activity from below 60°C to above 100°C, as presented in Fig. 1A.
• All xylanases have temperature optimum in the temperature range 80°C - 100°C, more specifically 85°C - 95°C, around 90°C, when determined after 20 minutes at pH 6.0.
• the enzymes of this invention are well suited for treatment of lignocellulosic pulp, in order to improve delignification.
• the enzyme of the invention may also be applied in a complexing stage of the pulp process, prior to hydrogen peroxide or ozone bleaching. Therefore, in a further aspect, the invention relates to the use of the xylanases for delignification of lignocellulosic pulp.
• Enzymatic treatment of lignocellulosic pulp improves the bleachability of the pulp and/or reduces the amount of chemicals necessary for obtaining a satisfactory bleaching.
• the xylanase should preferably be provided in the form of a granulate, preferably a non-dusting granulate, a liquid, in particular a stabilized liquid, a slurry, or a protected enzyme.
• the agent contains the xylanase in amounts of at least 20%, preferably at least 30%, of the total enzyme protein.
• the xylanolytic activity can be measured in xylanase units.
• two kinds of units are used: FXU and EXU.
• a suitable xylanase dosage will usually correspond to a xylanase activity of 10 to 5000 FXU/kg or EXU/kg dry pulp, more preferred 100 to 5000 FXU/kg or EXU/kg dry pulp.
• pH should be above pH 7.0 in order to prevent corrosion problems.
• the enzymatic treatment is performed at a pH above 7.0, preferably above pH 8.0, more preferred above pH 9.0.
• the enzymatic treatment is performed at temperatures between 50 and 100°C, preferably between 60 and 95°C, more preferred between 70 and 90°C.
• the enzymatic treatment is performed within a period of 5 minutes to 24 hours, preferably within a period of 15 minutes to 6 hours, more preferred within a period of 20 minutes to 3 hours.
• the enzymatic treatment takes place at a consistency of 3 - 35%, preferably 5 - 25%, more preferred 8 -15%.
• the consistency is the dry matter content of the pulp. A pulp with a consistency above 35% is difficult to mix effectively with the enzyme preparation, and a pulp with a consistency below 3% carries too much water, which is a disadvantage from an economic point of view.
• the xylanases of this invention can be implemented in processes for treatment of lignocellulosic pulp essentially as described in e.g. International Patent Application PCT/DK91/00239, or International Patent Publication WO 91/02839.
• the extracellular enzyme system was harvested at the late- exponential/early stationary phase of growth.
• the supernatant was concentrated to 50 ml and used for further characterization.
• Xylanase is determined by assaying for reducing sugars released from oat spelt xylan (XU-method). The assay is performed on using 0.5% of oat spelt xylan (Sigma-X- 0627) prepared in 40 mM Britton & Robinson buffer as substrate, heat treated 30 minutes at 100°C before use, and adjusted to the desired pH.
• the assay is performed with 0.100 ml of enzyme solution and 0.100 ml 5 of substrate, both preheated to the desired temperature. The mixture is incubated for 20 minutes at the desired pH. Then 0.200 ml solution I (35.1 g Na 2 HP0 4 ;2H 2 0; 40.0 g KNaC 4 H 4 0 ⁇ ;4H 2 0, suspended in 500 ml deionized H 2 0 add 110 ml 1N NaOH; 8.0 g CuS0 4 ,5H 2 o; 180 g Na ⁇ O ⁇ add deionized to a total volume of 1 litre) is added, and the solution is heated to 100°C for 20 minutes.
• the pH related activity of the enzymes was determined at 20 90°C in a pH range 4.0 - 11.0, using soluble xylan (Roth) in 40 mM Britton & Robinson buffer.
• the xylanases obtained from 7. neapolitana and T. thermarum showed xylanolytic activity in the range of from approximately pH 4 to approximately pH 10.
• Optimum pH for activity was detected in the pH range 5.0 - 25 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 2B and 3B.
• 7. neapolitana showed 50% of relative activity at pH 4.5 and pH 7.5, respectively.
• T. thermarum showed 50% of relative activity at pH 5.5 and pH 7.5, respectively.
• the xylanase obtained from 7. maritima showed xylanolytic activity from
• the xylanases both showed xylanolytic activity in the range of from below pH 4 to above pH 11.
• Optimum pH for activity was detected in the pH range 5.0 - 7.5, more specifically the pH range 5.5 - 6.5, around pH 6.0, as presented in Figs. 4A and 4B.
• the xylanases both showed 50% of relative activity at pH 4.5 and pH 8.5, respectively.
• the temperature related activity of the enzymes was determined using soluble xylan (Roth) in a 40 mM Britton & Robinson buffer, pH 6.0.
• the xylanases obtained from 7. neapolitana and T. thermarum showed xylanolytic activity in the range of from below 20°C to above 115°C, as presented in Figs. 2A and 3A.
• the xylanase obtained from 7. maritima showed xylanolytic activity from below 60°C to above 100°C, as presented in Fig. 1A.
• All of the xylanases showed temperature optimum in the temperature range 80°C - 100°C, more specifically of from 85°C to 95°C, around 90°C.
• the substrate specificity was determined using the substrates listed in Table 1 below, and using the XU-Method described above. Table 1

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• 1992
  • 1992-03-16 DK DK34892A patent/DK34892D0/da not_active Application Discontinuation
• 1993
  • 1993-03-15 JP JP5516176A patent/JPH07504819A/ja active Pending
  • 1993-03-15 EP EP93907814A patent/EP0631621A1/de not_active Withdrawn
  • 1993-03-15 WO PCT/DK1993/000093 patent/WO1993019171A1/en not_active Application Discontinuation

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