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WO1994004664A1 - New xylanases having high activity and stability at alkaline conditions and high temperatures - Google Patents

New xylanases having high activity and stability at alkaline conditions and high temperatures Download PDF

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Publication number
WO1994004664A1
WO1994004664A1 PCT/DK1993/000277 DK9300277W WO9404664A1 WO 1994004664 A1 WO1994004664 A1 WO 1994004664A1 DK 9300277 W DK9300277 W DK 9300277W WO 9404664 A1 WO9404664 A1 WO 9404664A1
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WO
WIPO (PCT)
Prior art keywords
enzyme preparation
enzyme
strain
xylanase
bacillus
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Application number
PCT/DK1993/000277
Other languages
French (fr)
Inventor
Arne Agerlin Olsen
Kjeld Ingvorsen
Henrik Bisgård-Frantzen
Shamkant Anant Patkar
Torben Halkier
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Novo Nordisk A/S
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Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU49446/93A priority Critical patent/AU4944693A/en
Priority to EP93919031A priority patent/EP0663949A1/en
Priority to JP6505799A priority patent/JPH08500485A/en
Priority to BR9306980A priority patent/BR9306980A/en
Publication of WO1994004664A1 publication Critical patent/WO1994004664A1/en
Priority to FI950852A priority patent/FI950852L/en

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    • 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
    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT OF FLOUR OR DOUGH FOR BAKING, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/189—Enzymes
    • 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

  • the present invention relates to novel xylanolytic enzymes obtainable from strains of alkalophilic Bacillus sp . Moreover, the invention relates to a method for producing the enzymes and the use of the enzymes in the pulp and paper industry .
  • Xylanases with high activity and stability at alkaline conditions are of great commercial interest, e.g. for applications in the pulp and paper industries, for modification of lignocellulose.
  • very few xylanases are reported which are able to function at pH values 9-12, and the available literature indicates that these enzymes are rapidly inactivated at a pH of more than 10, especially at temperatures exceeding 50°C.
  • the present invention describes new xylanase enzymes obtained from alkaline Bacillus sp.. which are superior to previously described bacterial xylanases with respect to activity and stability in the alkaline region. Furthermore, the xylanases of the present invention are also able to function at high temperature, e.g. 70°C at pH 7-8.
  • the invention provides enzyme preparations having xylanolytic activity, and having more than 50% relative activity in the range pH 6-9 at 50°C and temperature optimum in the range of from 55 to 75°C (at pH 6-10) .
  • the invention provides a process for the preparation of the enzyme preparations comprising cultivation of a strain of Bacillus sp., preferably the strain Bacillus sp., DSM 7197, or a mutant or a variant thereof, in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
  • the invention relates to the use of the enzyme preparation in a process for treatment of lignocellulosic pulp.
  • the invention provides an agent containing an enzyme preparation, 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.
  • Figs. 1-3 show the temperature profiles of the fraction purified according to Ex. 2, in standard Britton & Robinson buffers at pH 7, pH 9, and pH 10, respectively. All reaction mixtures contained 0.013 EXU/ml and were incubated for 30 minutes ( ⁇ sample; A substrate blank);
  • Fig. 4 shows the effect of pH on the activity of the fraction purified according to Ex. 2, in 50 mM Britton & Robinson buffers (0.013 EXU/ml; 30 minutes of incubation; 50oC; ⁇ sample; A buffer; ⁇ enzyme blank); and
  • Figs. 5, 6, and 7 show the effect of temperature and pH on the stability of the fraction purified according to Ex. 2, in the absence of substrate.
  • the fraction was diluted to a concentration of 0.05 EXU/ml in 50 mM Britton & Robinson buffers of pH 7, pH 9, and pH 10, respectively, and incubated at 40°C. At appropriate intervals, 50 ⁇ l samples were removed from each incubation mixtures and transferred to 950 ⁇ l 50 mM Britton & Robinson buffer pH 10. The residual xylanolytic activity was determined at 50°C using Xylazyme TabletsTM (Megazyme, Australia). The incubation time was 30 minutes in all cases ( ⁇ sample; A blind). DETAILED DISCLOSURE OF THE INVENTION
  • the invention provides xylanase preparations having high stability and excellent activity at alkaline conditions.
  • the enzyme preparation of the invention can be further described by the following characteristics.
  • the enzyme preparation of the invention comprises at least 5 xylanolytic enzymes, having pi in the range of from appr. 3 to appr. 9.5.
  • the fraction of the enzyme preparation, purified according to Ex. 2 has more than 50% relative activity in the range pH 6-10, determined after 30 minutes of incubation. No pronounced pH optimum is detectable, but appears to be in the range pH 5.5 to 9.0 (cf. Fig. 4).
  • the fraction of the enzyme preparation, purified according to Ex. 2 has a temperature optimum in the range of 60 to 75°C, more specifically around 70°C, determined after 30 minutes of incubation (cf. Fig. 1).
  • the fraction of the enzyme preparation, purified according to Ex. 2 has a temperature optimum in the range of 55 to 75°C, more specifically in the range of 60 to
  • the fraction of the enzyme preparation, purified according to Ex. 2 has a temperature optimum in the range of 50 to 70°C, more specifically around 60°C, determined after 30 minutes of incubation (cf. Fig. 3).
  • the fraction of the enzyme preparation, purified according to Ex. 2 has a relative residual activity after incubation for 6 hours at pH 10 and 40°C of at least 90%, more preferred at least 95%, most preferred at least 99%.
  • a similar relative residual activity was observed after incubation for 6 hours at pH 10 and 55°C, at pH 10 and 50°C; at pH 10 and 40°C; at pH 9 and 40°C; at pH 9 and 50°C; and at pH 7 and 50°C, cf.
  • the enzyme preparation of the invention has immunochemical properties identical or partially identical (i.e. at least partially identical) to those of a xylanase derived from the strain Bacillus sp., DSM 7197.
  • 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 immunoelectrophoresis according to Axelsen N.H.; Handbook of Immunoprecipitation-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.
  • Monospecific antiserum was generated according to the above-mentioned method by immunizing rabbits with the purified xylanase of the invention.
  • the immunogen was mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week.
  • Antiserum was obtained after a total immunization period of 8 weeks, and immunoglobulin was prepared therefrom as described by Axelsen N.H.. supra.
  • the enzyme preparations are obtainable by cultivation of alkalophilic Bacillus sp. in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
  • the enzyme preparations are obtained by cultivation of the alkalophilic species described as Group 3 by Gordon & Hyde [Gordon R.E and Hyde J.L. (1982); Journal of General Microbiology, 128 1109-1116, Table
  • the enzyme preparations are obtained by cultivation of a strain of the alkalophilic species represented by the strain Bacillus sp.. DSM 7197. In a further preferred embodiment, the enzyme preparations are obtained by cultivation of the strain Bacillus sp., DSM 7197, or a mutant or a variant thereof.
  • the enzyme can also be obtained by recombinant DNA-technology.
  • the strain Bacillus sp.. DSM 7197 was deposited on 4 August 1992 according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures, at Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg lb, 3300 Braunschweig, Germany.
  • the xylanolytic activity is measured in endo-xylanase units (EXU), determined at pH 9.0 with remazol-xylan as substrate.
  • EXU endo-xylanase units
  • a xylanase sample is incubated with remazol-xylan substrate.
  • the background of non-degraded dyed substrate is precipitated by ethanol.
  • the remaining blue colour in the supernatant is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions, i.e. at 50.0 +/-0.1°C, pH 9.0, and 30 minutes' reaction time.
  • the absorbance of the filtrate is measured at 590 nM. Blank incubations were run in all cases in order to correct for chemical hydrolysis of AZCL-xylan (cf. also Megazyme Product Information Leaflet).
  • the invention relates to a method for enzymatic treatment of lignocellulosic pulp, comprising employment of an enzyme of this invention.
  • 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 enzyme of the invention may also be applied in a complexing stage of the pulp process, prior to hydrogen peroxide or ozone 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.
  • AF 293.6/1 FXU
  • EXU AF 293.9/1
  • FXU is determined at pH 6.0
  • EXU is determined at pH 9.0.
  • FXU and EXU express enzymatic activity in the same order of magnitude.
  • the folders AF 293.6/1 and 293.9/1 are available upon request to Novo Nordisk A/S, Denmark, which folders are hereby included by reference.
  • the process of the invention is performed at temperatures between 40 and 100oC, more preferred between 50 and 90°C, most preferred between 60 and 80°C.
  • the enzymatic treatment is performed at a pH above 5.0, more preferred above 6.0, most preferred above 7.0.
  • the enzymatic treatment is performed within a period of 5 minutes to 24 hours, more preferred within 15 minutes to 6 hours, most preferred within 20 minutes to 3 hours.
  • 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.
  • the enzymatic treatment takes place at a consistency of 3-35%, more preferred 5-25%, most 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 new xylanase enzymes according to the invention may also be well suited for use as baking agents and as additives to animal fodder as described in EP 0 507 723. They may especially be useful for addition to animal feeds for in vivo breakdown of the pentosan fraction as the pH in the small intestine of e.g. poultry, piglets and pigs typically will be in the area of 5.5 to 7 in which area the new xylanase enzymes have significant activity.
  • the strain Bacillus sp., DSM 7197 was cultivated at 40°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml of medium of the following composition (per litre):
  • the medium is sterilized by heating at 120 "C for 45 minutes .
  • the pH of the medium is adjusted to 10.0 by addition of approx. 10 ml 1 M sodium sesquicarbonate to each flask.
  • a fraction of xylanases having acidic pl was partially purified by conventional purification techniques involving sample concentration by ultrafiltration and ammonium sulfate precipitation, and conventional chromatographic separation by ionexchange chromatography on S-Sepharose High Load and Q- Sepharose High Load, size exclusion chromatography on Superdex 200 or G-2000 SW, as well as affinity chromatography for specific removal of proteinases.
  • the partially purified enzyme fraction obtained according to Ex. 2, was subjected to kinetic studies, and the activity was found to be linear (zero-order kinetics) for at least 6 hours, when incubated at the conditions stated in Table 1, below.
  • the partially purified enzyme fraction obtained according to Ex. 2, was subjected to experiments concerning the effects of temperature and pH on enzyme activity and stability using AZCL-xylan tablets (Xylazyme TabletsTM, provided by Megazyme, Australia).
  • the assay was performed as follows:
  • the above described fraction was diluted to a concentration of 0.05 EXU/ml in 50 mM Britton & Robinson buffers of pH 7, pH 9, and pH 10, respectively, and incubated at 40°C. At appropriate intervals, 50 ⁇ l samples were removed from each incubation mixture and transferred to 950 ⁇ l 50 mM Britton & Robinson buffer pH 10. The residual xylanolytic activity was determined at 50°C using Xylazyme TabletsTM. The incubation time was 30 minutes in all cases. The results are presented in Figs. 5-7. EXAMPLE 5
  • N-terminal amino acid sequences of the xylanases were determined using standard methods for obtaining and sequencing peptides (Findlay & Geisow (Eds.), Protein Sequencing - a Practical approach. 1989, IRL Press).
  • This amino acid sequence is identical to the amino acid sequence of residues 18 to 29 in a 45 kDa xylanase from the alkalophilic Bacillus sp. C-125 (Hamamoto et al., Agric. Biol. Chem. 51, 1987, pp. 953-955).
  • N-terminal amino acid sequence of another xylanase purified from the fermentation broth of ex. 1 by conventional chromatographic methods and characterized by having a MW of approx. 22 kDa using SDS-PAGE and a pl value of approx. 9 in a 3.5 to 9.5 isoelectric focusing gel was found to be (SEQ ID No.2 of the attached sequence listing):
  • Asn-Thr-Tyr-Trp-Gln-Tyr-Xaa-Thr-Asp-Gly-Gly-Thr-Val-Asn-Ala-Xaa-Asn-GlyXaa designates unidentified residues.
  • This amino acid sequence is homologous to some of the other low molecular weight xylanases characterized so far (e.g. the xylanase from Bacillus subtilis, for reference see Paice et al., Arch. Microbiol. 144. 1986, pp. 201-206).
  • Matrix assisted lase desorption ionisation time-of-flight mass spectrometry was carried out using a ToftSpecTM mass spectrometer from VG Analytical according to the manufacturers instructions.
  • Matrix assisted lase desorption ionisation time-of-flight mass spectrometry gave a mass value of 20532 Da ⁇ 0.1% for the xylanase described in ex.5 having a MW of approx. 22 kDa and a pl of approx. 9.

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Abstract

The present invention relates to novel xylanolytic enzymes obtainable from strains of alkalophilic Bacillus sp. Moreover, the invention relates to a method for producing the enzymes and the use of the enzymes in the pulp and paper industry.

Description

NEW XYLANASES HAVING HIGH ACTIVITY AND STABILITY AT
ALKALINE CONDITIONS AND HIGH TEMPERATURES
TECHNICAL FIELD
The present invention relates to novel xylanolytic enzymes obtainable from strains of alkalophilic Bacillus sp . Moreover, the invention relates to a method for producing the enzymes and the use of the enzymes in the pulp and paper industry .
BACKGROUND ART
Xylanases with high activity and stability at alkaline conditions are of great commercial interest, e.g. for applications in the pulp and paper industries, for modification of lignocellulose. However, very few xylanases are reported which are able to function at pH values 9-12, and the available literature indicates that these enzymes are rapidly inactivated at a pH of more than 10, especially at temperatures exceeding 50°C.
SUMMARY OF THE INVENTION
The present invention describes new xylanase enzymes obtained from alkaline Bacillus sp.. which are superior to previously described bacterial xylanases with respect to activity and stability in the alkaline region. Furthermore, the xylanases of the present invention are also able to function at high temperature, e.g. 70°C at pH 7-8.
Accordingly, the invention provides enzyme preparations having xylanolytic activity, and having more than 50% relative activity in the range pH 6-9 at 50°C and temperature optimum in the range of from 55 to 75°C (at pH 6-10) .
In another aspect, the invention provides a process for the preparation of the enzyme preparations comprising cultivation of a strain of Bacillus sp., preferably the strain Bacillus sp., DSM 7197, or a mutant or a variant thereof, in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
In a third aspect, the invention relates to the use of the enzyme preparation in a process for treatment of lignocellulosic pulp.
In a further aspect, the invention provides an agent containing an enzyme preparation, 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.
BRIEF DESCRIPTION OF DRAWINGS
The present invention is further illustrated by reference to the accompanying drawings, in which:
Figs. 1-3 show the temperature profiles of the fraction purified according to Ex. 2, in standard Britton & Robinson buffers at pH 7, pH 9, and pH 10, respectively. All reaction mixtures contained 0.013 EXU/ml and were incubated for 30 minutes (⋈ sample; A substrate blank);
Fig. 4 shows the effect of pH on the activity of the fraction purified according to Ex. 2, in 50 mM Britton & Robinson buffers (0.013 EXU/ml; 30 minutes of incubation; 50ºC;★ sample; A buffer; ⋈ enzyme blank); and
Figs. 5, 6, and 7 show the effect of temperature and pH on the stability of the fraction purified according to Ex. 2, in the absence of substrate. The fraction was diluted to a concentration of 0.05 EXU/ml in 50 mM Britton & Robinson buffers of pH 7, pH 9, and pH 10, respectively, and incubated at 40°C. At appropriate intervals, 50 μl samples were removed from each incubation mixtures and transferred to 950 μl 50 mM Britton & Robinson buffer pH 10. The residual xylanolytic activity was determined at 50°C using Xylazyme Tablets™ (Megazyme, Australia). The incubation time was 30 minutes in all cases (⋈ sample; A blind). DETAILED DISCLOSURE OF THE INVENTION
The invention provides xylanase preparations having high stability and excellent activity at alkaline conditions.
The enzyme preparation of the invention can be further described by the following characteristics.
Physical-Chemical Properties
The enzyme preparation of the invention comprises at least 5 xylanolytic enzymes, having pi in the range of from appr. 3 to appr. 9.5.
At 50°C the fraction of the enzyme preparation, purified according to Ex. 2, has more than 50% relative activity in the range pH 6-10, determined after 30 minutes of incubation. No pronounced pH optimum is detectable, but appears to be in the range pH 5.5 to 9.0 (cf. Fig. 4).
At pH 7.0 the fraction of the enzyme preparation, purified according to Ex. 2, has a temperature optimum in the range of 60 to 75°C, more specifically around 70°C, determined after 30 minutes of incubation (cf. Fig. 1).
At pH 9.0 the fraction of the enzyme preparation, purified according to Ex. 2, has a temperature optimum in the range of 55 to 75°C, more specifically in the range of 60 to
75°C, determined after 30 minutes of incubation (cf. Fig. 2).
At pH 10.0 the fraction of the enzyme preparation, purified according to Ex. 2, has a temperature optimum in the range of 50 to 70°C, more specifically around 60°C, determined after 30 minutes of incubation (cf. Fig. 3).
The fraction of the enzyme preparation, purified according to Ex. 2, has a relative residual activity after incubation for 6 hours at pH 10 and 40°C of at least 90%, more preferred at least 95%, most preferred at least 99%. A similar relative residual activity was observed after incubation for 6 hours at pH 10 and 55°C, at pH 10 and 50°C; at pH 10 and 40°C; at pH 9 and 40°C; at pH 9 and 50°C; and at pH 7 and 50°C, cf. Immunochemical Properties
The enzyme preparation of the invention has immunochemical properties identical or partially identical (i.e. at least partially identical) to those of a xylanase derived from the strain Bacillus sp., DSM 7197.
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 immunoelectrophoresis according to Axelsen N.H.; Handbook of Immunoprecipitation-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.
Monospecific antiserum was generated according to the above-mentioned method by immunizing rabbits with the purified xylanase of the invention. The immunogen was mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week. Antiserum was obtained after a total immunization period of 8 weeks, and immunoglobulin was prepared therefrom as described by Axelsen N.H.. supra.
Methods of Producing the Enzymes
The enzyme preparations are obtainable by cultivation of alkalophilic Bacillus sp. in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
In a preferred embodiment, the enzyme preparations are obtained by cultivation of the alkalophilic species described as Group 3 by Gordon & Hyde [Gordon R.E and Hyde J.L. (1982); Journal of General Microbiology, 128 1109-1116, Table
4].
In another preferred embodiment, the enzyme preparations are obtained by cultivation of a strain of the alkalophilic species represented by the strain Bacillus sp.. DSM 7197. In a further preferred embodiment, the enzyme preparations are obtained by cultivation of the strain Bacillus sp., DSM 7197, or a mutant or a variant thereof.
The enzyme can also be obtained by recombinant DNA-technology.
The strain Bacillus sp.. DSM 7197, was deposited on 4 August 1992 according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures, at Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg lb, 3300 Braunschweig, Germany.
Assay for Xylanolytic Activity
The xylanolytic activity is measured in endo-xylanase units (EXU), determined at pH 9.0 with remazol-xylan as substrate.
A xylanase sample is incubated with remazol-xylan substrate. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions, i.e. at 50.0 +/-0.1°C, pH 9.0, and 30 minutes' reaction time.
A folder AF 293.9/1 describing the analytical method is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
Unless stated otherwise, experiments concerning the effects of temperature and pH on enzyme activity and stability were performed using AZCL-xylan tablets (Xylazyme Tablets™, provided by Megazyme, Australia). The assay was performed as follows:
An appropriate amount of enzyme is dissolved in 1 ml of temperature-equilibrated Britton & Robinson buffer (50 mM) . The reaction is started by adding one Xylazyme Tablet, mixed briefly on a Whirley mixer, and incubated at the desired temperature for 15 or 30 minutes. The enzymatic reaction is terminated by addition of 9 ml cold (2-3°C) 1% Tris buffer, vortexed vigorously, and filtered through a Whatman No. 1 filter circle.
The absorbance of the filtrate is measured at 590 nM. Blank incubations were run in all cases in order to correct for chemical hydrolysis of AZCL-xylan (cf. also Megazyme Product Information Leaflet).
Processes for the Treatment of Licrnocellulosic Pulp
In a further aspect, the invention relates to a method for enzymatic treatment of lignocellulosic pulp, comprising employment of an enzyme of this invention.
Enzymatic treatment of lignocellulosic pulp improves the bleachability of the pulp and/or reduces the amount of chemicals necessary for obtaining a satisfactory bleaching.
Due to its temperature stability, the enzyme of the invention may also be applied in a complexing stage of the pulp process, prior to hydrogen peroxide or ozone bleaching.
For use of a xylanase of the invention for delignification of lignocellulosic pulp, 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.
In a further preferred embodiment, 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. In this specification two kinds of units are used: FXU and EXU. By an analytical method a xylanase sample is incubated with remazol-xylan substrate. The background of non-degraded dyed substrate is precipitated by ethanol . The remaining blue colour in the supernatant is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions.
The analytical method and the standard reaction conditions are described in two folders: AF 293.6/1 (FXU) and AF 293.9/1 (EXU). FXU is determined at pH 6.0, and EXU is determined at pH 9.0. However, FXU and EXU express enzymatic activity in the same order of magnitude. The folders AF 293.6/1 and 293.9/1 are available upon request to Novo Nordisk A/S, Denmark, which folders are hereby included by reference.
Preferably, the process of the invention is performed at temperatures between 40 and 100ºC, more preferred between 50 and 90°C, most preferred between 60 and 80°C.
In another preferred embodiment of the process according to the invention, the enzymatic treatment is performed at a pH above 5.0, more preferred above 6.0, most preferred above 7.0.
In yet another preferred embodiment of the process according to the invention, the enzymatic treatment is performed within a period of 5 minutes to 24 hours, more preferred within 15 minutes to 6 hours, most preferred within 20 minutes to 3 hours.
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.
In a further preferred embodiment of the process according to the invention, the enzymatic treatment takes place at a consistency of 3-35%, more preferred 5-25%, most 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.
In several other preferred embodiments, 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.
Other Applications
The new xylanase enzymes according to the invention may also be well suited for use as baking agents and as additives to animal fodder as described in EP 0 507 723. They may especially be useful for addition to animal feeds for in vivo breakdown of the pentosan fraction as the pH in the small intestine of e.g. poultry, piglets and pigs typically will be in the area of 5.5 to 7 in which area the new xylanase enzymes have significant activity.
The following examples further illustrate the present invention, and they are not intended to be in any way limiting to the scope of the invention as claimed.
EXAMPLE 1
Cultivation Example
The strain Bacillus sp., DSM 7197, was cultivated at 40°C on a rotary shaking table (300 r.p.m.) in 500 ml baffled Erlenmeyer flasks containing 100 ml of medium of the following composition (per litre):
Xylan (Beechwood) 2.5 g
Yeast extract 5 g
Polypeptone 5 g
N WaaCnl 10 g
K2HPO4 1.0 g
MgS04 ; 7H2O 0.4 g
CaCl2; 2H2O 0.1 g
Trace element solution* 2 ml * Trace mineral solution for Desulfotomaculum acetoxidans medium; Medium 124; DSM Catalogue of Strains 1983.
The medium is sterilized by heating at 120 "C for 45 minutes .
After sterilization the pH of the medium is adjusted to 10.0 by addition of approx. 10 ml 1 M sodium sesquicarbonate to each flask.
After 2 days of incubation at 40°C several xylanolytic compounds were produced and excreted into the culture broth.
By isoelectric focusing combined with standard zymogram techniques on gels overlayered with xylan, at least 5 xylanolytic enzymes were detected, having pl in the range of from appr . 3 to appr. 9.5.
EXAMPLE 2
Purification Example
A fraction of xylanases having acidic pl was partially purified by conventional purification techniques involving sample concentration by ultrafiltration and ammonium sulfate precipitation, and conventional chromatographic separation by ionexchange chromatography on S-Sepharose High Load and Q- Sepharose High Load, size exclusion chromatography on Superdex 200 or G-2000 SW, as well as affinity chromatography for specific removal of proteinases.
EXAMPLE 3
Characterization Example
The partially purified enzyme fraction, obtained according to Ex. 2, was subjected to kinetic studies, and the activity was found to be linear (zero-order kinetics) for at least 6 hours, when incubated at the conditions stated in Table 1, below.
Figure imgf000011_0001
It appears from the table that the xylanolytic enzymes of the invention exhibit an extraordinary stability and activity even at strong alkaline conditions.
EXAMPLE 4 Characterization Example
The partially purified enzyme fraction, obtained according to Ex. 2, was subjected to experiments concerning the effects of temperature and pH on enzyme activity and stability using AZCL-xylan tablets (Xylazyme Tablets™, provided by Megazyme, Australia). The assay was performed as follows:
An appropriate amount of enzyme is dissolved in 1 ml of temperature-equilibrated Britton & Robinson buffer (50 mM) . The reaction is started by adding one Xylazyme Tablet, mixed briefly on a Whirley mixer, and incubated at the desired temperature for 15 or 30 minutes. The enzymatic reaction is terminated by addition of 9 ml cold (2-3°C) 1% Tris buffer, vortexed vigorously, and filtered through a Whatman No. 1 filter circle. The absorbance of the filtrate is measured at 590 nM. Blank incubations were run in all cases in order to correct for chemical hydrolysis of AZCL-xylan (cf. also Megazyme Product Information Leaflet). The results are presented in Figs. 1-4.
The above described fraction was diluted to a concentration of 0.05 EXU/ml in 50 mM Britton & Robinson buffers of pH 7, pH 9, and pH 10, respectively, and incubated at 40°C. At appropriate intervals, 50 μl samples were removed from each incubation mixture and transferred to 950 μl 50 mM Britton & Robinson buffer pH 10. The residual xylanolytic activity was determined at 50°C using Xylazyme Tablets™. The incubation time was 30 minutes in all cases. The results are presented in Figs. 5-7. EXAMPLE 5
N-terminal amino acid sequence analysis
N-terminal amino acid sequences of the xylanases were determined using standard methods for obtaining and sequencing peptides (Findlay & Geisow (Eds.), Protein Sequencing - a Practical approach. 1989, IRL Press).
The N-terminal amino acid sequence of a xylanase obtained according to ex. 2 and characterized by having a MW of approx. 43 kDa using SDS-PAGE and a pi value of approx. 4.5 in a 3.5 to 9.5 isoelectric focusing gel was found to be (SEQ ID
No.l of the attached sequence listing):
Asn-Asp-Gln-Pro-Phe-Ala-Trp-Gln-Val-Ala-Ser-Leu-
This amino acid sequence is identical to the amino acid sequence of residues 18 to 29 in a 45 kDa xylanase from the alkalophilic Bacillus sp. C-125 (Hamamoto et al., Agric. Biol. Chem. 51, 1987, pp. 953-955).
The N-terminal amino acid sequence of another xylanase purified from the fermentation broth of ex. 1 by conventional chromatographic methods and characterized by having a MW of approx. 22 kDa using SDS-PAGE and a pl value of approx. 9 in a 3.5 to 9.5 isoelectric focusing gel was found to be (SEQ ID No.2 of the attached sequence listing):
Asn-Thr-Tyr-Trp-Gln-Tyr-Xaa-Thr-Asp-Gly-Gly-Gly-Thr-Val-Asn-Ala-Xaa-Asn-GlyXaa designates unidentified residues. This amino acid sequence is homologous to some of the other low molecular weight xylanases characterized so far (e.g. the xylanase from Bacillus subtilis, for reference see Paice et al., Arch. Microbiol. 144. 1986, pp. 201-206). EXAMPLE 6
Mass Spectrometry
Matrix assisted lase desorption ionisation time-of-flight mass spectrometry was carried out using a ToftSpec™ mass spectrometer from VG Analytical according to the manufacturers instructions.
Matrix assisted lase desorption ionisation time-of-flight mass spectrometry gave a mass value of 20532 Da ±0.1% for the xylanase described in ex.5 having a MW of approx. 22 kDa and a pl of approx. 9.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: NOVO NORDISK A/S
(B) STREET: Novo Alle
(C) CITY: DK-2880 Bagsvaerd
(E) COUNTRY: Denmark
(F) POSTAL CODE (ZIP) : DK-2880
(G) TELEPHONE: +45 44 44 88 88
(H) TELEFAX: +45 44 49 32 56
(I) TELEX: 37304
(ϋ) TITLE OF INVENTION: NOVEL ENZYMES
(iii) NUMBER OF SEQUENCES: 2
(iv) ∞MPOTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA:
APPLICATION NUMBER:
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Asn Asp Gln Pro Phe Ala Trp Gln Val Ala Ser Leu
1 5 10 (2) INPORMATTON FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) rlYPOTHETICAL: NO
(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2 :
Asn Thr Tyr Trp Gln Tyr Xaa Thr Asp Gly Gly Gly Thr Val Asn Ala
1 5 10 15
Xaa Asn Gly
Figure imgf000017_0001

Claims

1. An enzyme preparation having xylanolytic activity and characterized by having the following properties:
(a) more than 50% relative activity in the range pH 6-9 at 50°C;
(b) temperature optimum in the range of from 55 to 75 "C (at pH 6-10); and
(c) immunochemical properties identical or partially identical to those of a xylanase derived from the strain Bacillus sp., DSM 7197.
2. An enzyme preparation according to claim 1, the enzyme preparation being obtainable from a strain of Bacillus sp., or from another host organism carrying the gene encoding a xylanase having immunochemical properties identical or partially identical to those of the xylanase derived from the strain Bacillus sp.. DSM 7197.
3. An enzyme preparation according to either of claims 1-2, the enzyme preparation being obtainable from a strain belonging to the species represented by the strain Bacillus sp. DSM 7197.
4. An enzyme preparation according to any of claims 1-3, the enzyme preparation being obtainable from the strain Bacillus sp., DSM 7197, or a mutant or a variant thereof.
5. A process for the preparation of an enzyme preparation according to any of claims 1-4, which process comprises cultivation of a strain of Bacillus sp., preferably the strain Bacillus sp.. DSM 7197, or a mutant or a variant thereof, in a suitable nutrient medium, containing carbon and nitrogen sources and inorganic salts, followed by recovery of the desired enzyme.
6. The use of the enzyme preparation according to any of claims 1-4 in a process for treatment of lignocellulosic pulp.
7. A process according to claim 6 for treatment of lignocellulosic chemical pulp, wherein the lignocellulosic pulp is treated with the enzyme preparation 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.
8. The use of the enzyme preparation according to any of claims 1-4 in a process for treatment of animal feed.
9. The use of the enzyme preparation according to any of claims 1-4 as a baking agent in the production of bread.
10. An agent containing an enzyme preparation according to any of claims 1-4, 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.
11. An agent according to claim 10, in which the xylanase preparation constitutes at least 20%, preferably at least 30%, of the total enzyme protein.
PCT/DK1993/000277 1992-08-26 1993-08-25 New xylanases having high activity and stability at alkaline conditions and high temperatures WO1994004664A1 (en)

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JP6505799A JPH08500485A (en) 1992-08-26 1993-08-25 Novel xylanase with high activity and stability under alkaline conditions and high temperature
BR9306980A BR9306980A (en) 1992-08-26 1993-08-25 Enzyme preparation having xylanolitic activity Process for preparing an enzyme preparation using the enzyme preparation and agent containing the same
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WO1996002632A1 (en) * 1993-03-12 1996-02-01 Showa Denko K.K. Novel xylanase, process for producing the same, method for the treatment of pulp, and production of xylo-oligosaccharides
EP0698667A1 (en) * 1994-07-26 1996-02-28 SOLVAY & Cie (Société Anonyme) Xylanase, microorganisms for its production, DNA molecules, process of preparation and use thereof
WO1996013574A1 (en) * 1994-10-26 1996-05-09 Biotech International Limited Bacterial protein with xylanase activity
BE1008570A3 (en) * 1994-07-26 1996-06-04 Solvay Xylanase, micro-organisms producing same, DNA molecule, preparation methodsof said xylanase and uses thereof
WO1997009423A1 (en) * 1995-09-07 1997-03-13 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Non-starch polysaccharide hydrolysing enzymes
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WO2001030984A3 (en) * 1999-10-27 2001-11-15 Univ Aarhus Novel halotolerant and halophilic enzymes and the use of halotolerant and halophilic enzymes
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WO2010089302A1 (en) 2009-02-06 2010-08-12 University Of Chile Protein and dna sequence encoding a cold adapted xylanase
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WO1996002632A1 (en) * 1993-03-12 1996-02-01 Showa Denko K.K. Novel xylanase, process for producing the same, method for the treatment of pulp, and production of xylo-oligosaccharides
WO1995027779A1 (en) * 1994-04-11 1995-10-19 Biotech International Ltd. Bacterial xylanase
US6346407B1 (en) 1994-07-26 2002-02-12 Genencor International, Inc. Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter
EP0698667A1 (en) * 1994-07-26 1996-02-28 SOLVAY & Cie (Société Anonyme) Xylanase, microorganisms for its production, DNA molecules, process of preparation and use thereof
US8148104B2 (en) 1994-07-26 2012-04-03 Danisco Us Inc. Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter
BE1008570A3 (en) * 1994-07-26 1996-06-04 Solvay Xylanase, micro-organisms producing same, DNA molecule, preparation methodsof said xylanase and uses thereof
BE1008751A3 (en) * 1994-07-26 1996-07-02 Solvay Xylanase, the producing microorganisms, dna molecules, methods of preparation of this xylanase and uses thereof.
US7638613B2 (en) 1994-07-26 2009-12-29 Genencor International, Inc. Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter
AU711105B2 (en) * 1994-07-26 1999-10-07 Genencor International, Inc. Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter
US7022827B2 (en) 1994-07-26 2006-04-04 Genencor International, Inc. Xylanase, microorganisms producing it, DNA molecules, methods for preparing this xylanase and uses of the latter
US6506593B2 (en) 1994-07-29 2003-01-14 Rohm Enzyme Finland Oy Production and secretion of proteins of bacterial origin in filamentous fungi
US6300114B1 (en) 1994-07-29 2001-10-09 Rohm Enzyme Finland Oy Sequences of xylanase and xylanase expression vectors
US6667170B1 (en) 1994-07-29 2003-12-23 Röhm Enzyme Finland OY Sequences of Xylanase and Xylanase expression vectors
US6548283B1 (en) 1994-10-26 2003-04-15 Agenix Limited Bacterial protein with xylanase activity
US6200797B1 (en) 1994-10-26 2001-03-13 Biotech International Limited Bacterial protein with xylanase activity
WO1996013574A1 (en) * 1994-10-26 1996-05-09 Biotech International Limited Bacterial protein with xylanase activity
WO1997009423A1 (en) * 1995-09-07 1997-03-13 Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno Non-starch polysaccharide hydrolysing enzymes
WO2001030984A3 (en) * 1999-10-27 2001-11-15 Univ Aarhus Novel halotolerant and halophilic enzymes and the use of halotolerant and halophilic enzymes
US7547534B2 (en) 2002-06-14 2009-06-16 Verenium Corporation Methods for making a composition to treat a wood, a pulp or a paper
US7504120B2 (en) 2002-06-14 2009-03-17 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
US8728769B2 (en) 2002-06-14 2014-05-20 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US9765319B2 (en) 2002-06-14 2017-09-19 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
US8043839B2 (en) 2006-02-14 2011-10-25 Verenium Corporation Xylanases, nucleic acids encoding them and methods for making and using them
USRE45660E1 (en) 2006-02-14 2015-09-01 Bp Corporation North America Inc. Xylanases, nucleic acids encoding them and methods for making and using them
WO2010089302A1 (en) 2009-02-06 2010-08-12 University Of Chile Protein and dna sequence encoding a cold adapted xylanase
US8679814B2 (en) 2009-02-06 2014-03-25 University Of Chile Protein and DNA sequence encoding a cold adapted xylanase

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