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WO1997036995A2 - Xylanase - Google Patents

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Publication number
WO1997036995A2
WO1997036995A2 PCT/NZ1997/000042 NZ9700042W WO9736995A2 WO 1997036995 A2 WO1997036995 A2 WO 1997036995A2 NZ 9700042 W NZ9700042 W NZ 9700042W WO 9736995 A2 WO9736995 A2 WO 9736995A2
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WO
WIPO (PCT)
Prior art keywords
enzyme
xylanase
pulp
bleaching
xynb
Prior art date
Application number
PCT/NZ1997/000042
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English (en)
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WO1997036995A3 (fr
Inventor
Peter Leonard Bergquist
Moreland David Gibbs
Daniel Morris
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Pacific Enzymes Limited
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Priority to AU25235/97A priority Critical patent/AU2523597A/en
Publication of WO1997036995A2 publication Critical patent/WO1997036995A2/fr
Publication of WO1997036995A3 publication Critical patent/WO1997036995A3/fr

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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)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
    • 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
    • C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • thermophilic enzymes that is, enzymes which are stable at elevated temperatures, and more particularly to enzymes having xylanolytic activity (classified into EC class 3.2.1.8) likely to be of some use in bleaching paper pulp.
  • a peptide chain as being comprised of a series of amino acids "substantially or effectively" in accordance with a list offering no alternatives within itself, we include within that reference any versions of the peptide chain bearing substitutions made to one or more amino acids by similar amino acids in such a way that the overall structure and the overall function of the protein composed of that peptide chain is substantially the same as - or undetectably different to - that of the unsubstituted version. For example it is generally possible to exchange alanine and valine without greatly changing the properties of the protein, especially if the changed site or sites are at positions not critical to the morphology of the folded protein.
  • thermostable as applied to an enzyme means that the enzyme is relatively unaffected by heat. Normally such enzymes are used in aqueous solutions and the upper limits of the temperature range are determined by the boiling point of water at the relevant environmental pressures. Preferably a thermostable enzyme remains active for a long period at a high temperature and preferably it also has an enhanced Km at a high temperature.
  • Natural paper has a brownish or buff colour, whereas there is a great demand in the Western world for bleached paper.
  • Commercial pulp bleaching operations which normally employ sulphur dioxide or sulphites, or reactive chlorine as chlorine dioxide (hence having the risk of liberation of dioxin into the waste stream), have been the target of much criticism and therefore there is considerable interest within the paper industry in reducing the environmental impact of paper bleaching while still working within the rather narrow chemical and physical constraints that apply to paper processing.
  • Various biomimetic and biological bleaching treatments are known, yet there is still a need for an effective, efficient bleaching treatment.
  • an enzyme derived from a gene contained within Dictyoglomus thermophilum, and contained within the family of enzymes known as G- Xylanases, and having beta-l,4-xylanase activity at elevated temperatures, wherein the enzyme has an amino acid sequence substantially as described herein or an amino acid sequence which would not substantially alter the activity of the enzyme, said enzyme capable of being applied to the bleaching of cellulose products.
  • the enzyme is substantially as isolated from Dictyoglomus thermophilum.
  • the enzyme is a recombinant enzyme.
  • the enzyme has activity within between 60 and 90 degrees Celsius and within the pH range 5 to 7.
  • the enzyme has optimal activity at approximately 85 degrees Celsius and approximately pH 6.5.
  • a recombinant gene encoding the enzyme described above wherein the gene has a nucleotide sequence substantially as herein described, or at least part thererof, or one which is not sufficiently different so as to alter substantially the amino acid sequence of the enzyme expressed therefrom, or at least part thereof.
  • a recombinant vector containing the recombinant gene as described above.
  • micro-organism capable of producing an enzyme as described above wherein the micro-organism contains a recombinant vector as described in the previous paragraph.
  • a preparation of an enzyme as described above wherein the preparation contains an amount of the enzyme and a biologically acceptable carrier.
  • a process for the bleaching of cellulose products wherein the process utilises an enzyme to aid in the bleaching of the cellulose products wherein the enzyme is derived from a gene contained within Dictyoglomus thermophilum, and contained within the family of enzymes known as G-Xylanases, and having beta- 1 ,4-xylanase activity at elevated temperatures, the enzyme having an amino acid sequence substantially as described herein or an amino acid sequence which would not substantially alter the activity of the enzyme.
  • the invention comprises die use of the enzyme described above as a means though not necessarily the sole means for an at least partial degradation of the xylans in a mass of pulp, whereby in use the pulp becomes at least partially bleached.
  • Figure 1 shows the nucleotide sequences of the forward (xynGF) and reverse (xynGR) family G xylanase consensus primers used to amplify the family G xylanase consensus fragment (GXCF) from xynB family G xylanase gene residing in the Rt46B.l genomic DNA.
  • GXCF family G xylanase consensus fragment
  • Nucleotides in the alignment which conform to the consensus at each position in the multiple sequence alignment are indicated in white or black.
  • Figure 2 (A) illustrates the relative positions of the DNA fragments which were sequenced to generate the Rt46B.l xynB nucleotide sequence.
  • the xynB GXCF fragment is shown in black; the forward and reverse genomic-walking fragments are shown in grey (the regions of the genomic-walking fragments which overlap the xynB GXCF are highlighted in light grey).
  • Figure 2 (A) illustrates the relative positions of the DNA fragment
  • xynGF/xynGR genomic-walking PCR primers
  • dictGF/dictGR genomic-walking PCR primers
  • FIG. B shows the position of the oligonucleotide primers (PCR primers) which were used in the polymerase chain reaction (PCR) to amplify the xynB DNA fragment which encoded the 229B N- terminal family G xylanase domain.
  • PCR primers oligonucleotide primers
  • the domain structure of the family G xylanase encoded within the R.46B.1 xynB gene (229B) is indicated diagrammatically under the respective region of the xynB open-reading frame.
  • (C) shows the relative positions and sizes of the xynB fragment amplified by the 229BN and 229BC PCR primers.
  • Figure 3 shows the annotated nuleotide sequence of the R.46B.1 xynB gene, and indicates:
  • the putative leader-peptide region (at the N-terminus of the enzyme) and linker-peptide region (delineating the two domains of 229B) are shown in white on black; (iii) the nucleotide sequence and position of the forward and reverse family G xylanases consensus PCR primers (xynGF and xynGR), the forward and reverse Rt46B.l xynB genomic-walking PCR primers (dictGF and dictGR) and the forward and reverse
  • Rt46B.l xynB expression PCR primers (229BN and 229BC).
  • the 5'-end of the primers are indicated by a bullet (•) whilst the 3'-end of the primers are indicated by an arrow (-
  • Figure 4 outlines the strategy used to clone the Rt46B.l xynB PCR-fragment into the pJLA602 protein-expression vector.
  • the schematic of the procedure is shown on the right-hand side, and the detailed illustration of the nucleotides involved for the cloning of the xynB fragment into pJLA602 is shown on the left.
  • the actual peptide sequence expressed from the xynB:pJLA602 expression construct is shown in white in black at the bottom left.
  • Figure 5 shows the pH-dependent activities of a preparation of the 229B enzyme obtained after expression and purification of the enzyme from a recombinant Escherichia coli JMlOl strain harbouring the xynB:pJLA602 plasmid construction.
  • All buffers were pH adjusted at 70°C.
  • the pH optimum assays were performed at 75°C.
  • Figure 6 shows the temperature-dependent activities of the 229B obtained after expression and purification of the enzyme from a recombinant Escherichia coli JMlOl strain harbouring the xynB:pJLA602 plasmid constructions.
  • Figure 7 Shows the activities of the 229B enzyme obtained after expression and purification of the enzyme from a recombinant Escherichia coli JMlOl strains harbouring the xynB:pJLA602 plasmid construction on a 2% kraft-pulp solution at 75°C, pH6.5
  • Figure 8 shows the effect of enzyme dosage on the D(EO) kappa treated with xylanase in a bleaching sequence applied to eucalypt pulp.
  • Figure 9 Comparison of the final pulp brightness using three different xylanase enzymes at differing dosages in a bleaching sequence applied to eucalypt pulp.
  • Figure 10 Shows the effect of xylanase (10 xu/g) treatment on the D(EO)DD brightness at using different amounts of chlorine dioxide in the first D stage of a bleaching sequence applied to eucalypt pulp.
  • Figure 11 Shows the effect of xylanase treatment on D(EO)DD brightness with various total active chlorine charges.
  • FIG. 13 Shows is a schematic representation of the xynB gene showing the PCR (polymerase chain reaction) primers (A) used to obtain products of various lengths from the xynB gene (B). The temperature, pH optimums, thermal stability and relative productions of for the enzyme products expressed from constructs containing each PCR product are shown in B. C shows the N- terminal sequence of the PCR products.
  • xylanase gene encoding a family F xylanase (229 A) has been isolated from the Dictyoglomus thermophilum strain Rt46B.l (Gibbs et al. 1995).
  • This invention relates to xylanases from organisms belonging to the species Dictyoglomus thermophilum including Dictyoglomus thermophilum and Dictyoglomus strain Rt46B.l.
  • the organisms have been characterised by Saiki et al (1985), Patel et al., (1987), and Love et al, (1993).
  • Dictyoglomus thermophilum is publicly available from the Deutsche Sammlung von Mirkoorganismen und Zellkulturen GmbH, Mascheroder Weg lb, D-3300 Braunschweig, Germany, under the accession number DSM 3960.
  • Rt46B.l Cultures of Rt46B.l were obtained from H. Morgan, University of Waikato, Hamilton, New Zealand. Genomic DNA from Rt46B.1 was prepared from a culture of the organism grown at 70°C for two days in TYEG medium (Patel et al. 1985). Amplification and Analysis of the Rt46B.l xynB GXCF
  • the Rt46B.l xynB family G xylanase consensus fragment (GXCF) was amplified from Rt46B.l genomic DNA (figure 2 A) by the xynGF and xynGR consensus primers (figure 1) using standard PCR techniques.
  • the PCR conditions were as follows: 94°C DNA denaturation for 60 seconds, 37°C primer-annealing for 60 seconds, 72°C primer-extension for 30 seconds, 35 reaction cycles.
  • the termini of the GXCFs amplified from the R.46B.1 genomic DNA were made blunt-ended by incubation at 37°C for 30 minutes with 0.1U T4-DNA-polymerse, 1.0U T4-polynucleotide kinase and 1.0U E. coli DNA polymerase Klenow fragment in 0.6 mM dNTPs, 6.6 mM Tris-HCl, pH 7.5, 5 mM MgC12, 1 mM dTT and 1 mM ATP.
  • End-repaired Rt46B.l xynB GXCFs were purified from a 1% low-melting temperature agarose gel following separation by gel-electrophoresis using the GeneClean (BiolOl, La Jolla, Ca.) procedure and cloned into the Smal site of the M13mpl0 bacteriophage sequencing vector.
  • M13mpl0 bacteriophage containing the Rt46B.l xynB GXCF were sequenced from the M13F 21-mer sequencing primer.
  • the sequence data obtained from these four M13mpl0 recombinants were identical and found to correspond to genuine family G xylanase fragments upon comparison to the family G xylanase genes in the GenEMBL nucleotide database.
  • dictGF forward genomic-walking primer
  • dictGR reverse genomic-walking primer
  • the genomic- walking PCR protocols were perfomed as described previously by Morris et al. (1995).
  • Seven Rt46B.l genomic-DNA restriction-fragment/DNA linker-libraries were prepared using each of the following restriction endonuc leases: Ncol, Dral, EcoKV, HincU, Pvu , Sspl and Sail.
  • Genomic-walking PCRs were carried out using the dictGR/berg41 and dictGF berg41 primers on each of the seven linker-libraries to amplify various D ⁇ A fragments upstream and downstream of the Rt46B.l xynB GXCF, respectively (figure 2A).
  • Two upstream genomic-walking fragments (an 800bp fragment amplified from the Rt46B.l Ncol linker- library and a 600bp fragment amplified from the R.46B.1 Dral linker-library) and two downstream genomic-walking fragments (an 800bp fragment amplified from the R.46B.1 Sspl library and a lOOObp fragment amplified from the Rt46B.l Ncol library) were sequenced at least on one D ⁇ A strand to generate 1190bp of uninterrupted nucleotide sequence data.
  • the 1083bp open-reading frame of the Rt46B.l xynB gene was identified within the 1190bp of sequence data (figure 2B and figure 3).
  • the 1083bp Rt46B.l xynB gene was found to encode a peptide (229B) of 360 amino-acids in length (figure3) with a putative molecular weight of 39.8 kilo Daltons.
  • the 229B peptide sequence was comprised of two separate domains (figure 2b and figure 3): a 200 amino-acid ⁇ -terminal G xylanase domain following the putative 24 amino-acid leader peptide, and a 118 amino-acid C-terrninal domain bearing no homology to any sequence in the GenEMBL nucleotide database or the SwissProt protein database.
  • the full nucleotide sequence of nB and resultant amino acid sequence of the 229B peptide are given below:
  • Oligonucleotide primers were designed based on the xynB nucleotide sequence to allow PCR amplification of a xynB gene fragment from Rt46B.l genomic DNA which encoded the 229B family G xylanase domain.
  • An Ncol restriction endonuclease recognition site was incorporated into the forward PCR primer (229BN) and a BamHI restriction endonuclease recognition site was incorporated into the reverse PCR primer (229BC).
  • the recombinant pJLA602 plasmid construction incorporating the 702base-pair Ncol-Bam l digested Rt46B.l xynB PCR fragment encoding the 229B xylanase domain has been named p ⁇ Z2869.
  • the transformed JMlOl strain containing the pNZ2869 plasmid has been named PB6569. Production of the 229B enzyme
  • Purified 229B enzyme for characterisation was produced as follows; 100-500 ⁇ l of an overnight culture of PB6569 (grown at 30°C in L-broth, 60mg/ml Ampicillin) was used to seed a fresh 2000ml culture which was grown to an OD600 of 1.0 then transferred to 42°C to induce 229B production, and grown for a further 2 hours. The bacterial cells were harvested by centrifugation at 5000rpm for 5 minutes. The cell pellet was then resuspended in 50mls of an ice cold solution of TES buffer (0.05M Tris pH8.0, 0.05M NaCl and 0.005 EDTA), spun again at 5000rpm for 5 minutes then the cell pellet resuspended in 10-20mls TES.
  • TES buffer 0.05M Tris pH8.0, 0.05M NaCl and 0.005 EDTA
  • the bacterial cells were lysed by passage through a french pressure cell at 8000 pounds per square inch pressure differential.
  • the resulting PB6569 whole-cell extracts were heat-treated by incubation at 75°C for 30 minutes.
  • the heated cell lysate was centrifuged at 12000rpm for 30 minutes to pellet denatured mesophilic protein with cell debris and leave a relatively pure supernatant containing the 229B enzyme. This purified enzyme was used for all subsequent enzyme assays.
  • Enzyme activity was defined in XU's (Bailey et al., 1992).
  • One XU is defined as the amount of enzyme required to release one micromole of xylose reducing sugar equivalent per minute from xylan.
  • pH assays were carried out using the method of Lever (1973), using a 0.25% solution of oat spelts xylan (Sigma) in distilled H2O. Enzyme was used at a concentration determined not to be substrate limiting over the period of the assay.
  • Appropriate enzyme was mixed with pH adjusted buffer (either sodium acetate, l-3-bis[tris (hydroxymethyl)-methylamino]propane, 3-[cyclo hexylamino]-l-propanesulfonic acid, or 2[N-Morpholino]ethanesulfonic acid, all pH adjusted at the temperature of the assay) and 0.25% oat spelts xylan to a final buffer concentration of 12.5mM and a final substrate concentration of 0.22%. Assay times for pH were 10 minutes. Release of reducing sugar was measured using a modification of the method of Lever (1973).
  • pH adjusted buffer either sodium acetate, l-3-bis[tris (hydroxymethyl)-methylamino]propane, 3-[cyclo hexylamino]-l-propanesulfonic acid, or 2[N-Morpholino]ethanesulfonic acid, all pH adjusted at the temperature of the assay
  • Colorimetric determination was done as follows; 200 microlitres of OSX/enzyme mixture after incubation under appropriate conditions was mixed with 500 microlitres of PABA buffer (p- hydroxybenzoic acid hydrazide (PABA) 0.05M with 0.3M of NaOH, 0.05M of Na 2 S0 3 , 0.02M of trisodium citrate, and 0.02M of CaCl 2 ), boiled for 5 minutes and cooled.
  • PABA buffer p- hydroxybenzoic acid hydrazide (PABA) 0.05M with 0.3M of NaOH, 0.05M of Na 2 S0 3 , 0.02M of trisodium citrate, and 0.02M of CaCl 2
  • the OD405 of 200 microlitres of each sample was measured on a 96 well microtitre plate reader. All samples were done at least in duplicate, preferably in triplicate.
  • the pH optimum of 229B was determined to be around pH6.5 (figure 5). Determination of Temperature Optimum
  • Figure 6 depicts the relationship between temperature and activity for 229B in an experimental assay. Assay conditions were as follows. Approximately 0.005 XU of 229B was mixed on ice with a solution of 0.25% oat spelts xylan in 12.5mM BTP buffer, pH6.5, and incubated for 10 minutes at each temperature under consideration in triplicate assays.
  • Figure 6 shows the enzyme has activity at a temperature range between 60°C and 90°C. Optimum activity is seen between 60°C and 90°C under these experimental conditions. Therefore this enzyme may be considered to be a thermophilic enzyme, suggesting some commercial value.
  • Oligonucleotide primers were designed based on the xynB nucleotide sequence to allow PCR amplification of 7 xynB gene fragments from Rt46B.l genomic DNA. The relative position of each primer on the xynB gene is shown in figure 13 A. Each primer was designed to produce a PCR product differing in length at its N-terminal domain ( Figure 13C) and/or its C-terminal domain.
  • Ncol restriction endonuclease recognition site was incorporated into each forward PCR primer (xynB ⁇ 4, xynBN3, xynBN2, xynBNl) and a BamHI restriction endonuclease recognition site was incorporated into each reverse PCR primer (xynBC3, xynBCl). These sites allowed the in-frame directional ligation of me xynB gene fragment into the Ncol and BamHI sites of the controllable heat-inducible expression vector pJLA602, placing the xynB gene fragment in the correct position for optimal expression.
  • Recombinant enzyme products were obtained from each of the 7 cloned xynB fragments using a protocol as described herein under the heading "Production of the 229B Enzyme”.
  • the purified enzyme products were used for the subsequently described pH and temperature optimum assays.
  • Figure 13B indicates the optimum conditions for each of the seven recombinant enzymes. Of particular interest was xynB6 which showed activity at an optimum pH of 6.5 and a temperature of 85°C. This recombinant enzyme is likely to be readily applicable to an industrial bleaching process.
  • ECF Elemental Chlorine Free
  • TCF totally Chlorine Free
  • the mature eucalypt wood sample was a mixture of E. sieberc wood and E. muellerana, E. globoidea, E. aglomerata and E. obliqua woods.
  • Woodchip samples (600 g oven dry basis) were pulped in 3L stainless steel vessels placed in an electrically heated air bath. The liquor to wood ratio was 3.5:1, the sulfidity of the liquor 25%, the time to temperature 110 min and the pulping temperature of 170°C was maintained for 2 h. An active alkali of 16% (as Na2 ⁇ ) was applied to produce a pulp with a kappa number of 19.7.
  • the kraft pulp was oxygen delignified in the 3L pulping vessels similar to those used above but fitted with lids incorporating valves to introduce oxygen into the vessels.
  • the vessels were rotated in an electrically heated air bath.
  • Pulp samples 150 g oven dry basis
  • magnesium carbonate 1%, pulp basis
  • sodium hydroxide 1%, pulp basis
  • the mixtures were placed in the pulping vessels which were pressurised with oxygen (780 kPa) and heated at 115°C for 30 min (time to temperature was 75 min).
  • the oxygen delignified kraft pulp has a kappa number of 10.2.
  • a xylanase preparation with an activity of 500 xu/ml was prepared from freeze-dried enzyme preparations by dissolution in water.
  • One xylanase unit (XU) of activity is the amount of enzyme which catalyses the release of 1 micromole of reducing carbohydrate per minute.
  • Treatment of the pulp with the xylanase was done in plastic bags at a pulp concentration of 6%. The pH of the pulp was adjusted to 7 using a buffer, xylanase added and the mixture heated at 75°C for 2 h in a water bath. As a control, the pulp was treated under identical conditions but without xylanase.
  • the kraft-oxygen pulp was also pretreated with a commercial xylanase Irgazyme-40 and a non ⁇ commercial xylanase DCPX.
  • the conditions of pretreatment were as follows:- (a) Irgazyme-40: pulp concentration 6%, pH 7.5, 60°C for 3 h. (b) DCPX: pulp concentration 6%, pH 7.0, 53°C for 3 h.
  • the initial chlorine dioxide stage was done at 10% pulp concentration in a sealed plastic bag at 70°C for 70 min.
  • the active chlorine multiple applied were 0.05, 0.10, 0.15 and 0.22. There were no adjustment of the pH. The amount of residual chlorine dioxide was determined and in all instances none was detectable.
  • the (EO) stage was done at 10% pulp concentration in stainless steel vessels at 90°C for 30 min.
  • the vessels were pressurised to 780 kPa with oxygen and the charge of sodium hydroxide was 1.5% (pulp basis).
  • the amount of chlorine dioxide applied was 1.32% (as active chlorine) in each stage.
  • the pH in the two D stages was adjusted with sulfuric acid or sodium hydroxide at the beginning so that a final pH of 3.5-4.0 was obtained.
  • the amounts of residual chlorine dioxide were determined after each stage.
  • the residual after the first stage was non-detectable.
  • the second stage filtrates had residual levels in the range non-detectable to 0.2% (active chlorine on a pulp basis).
  • the pulp was treated with 0.3% EDTA (pulp basis) at a pulp concentration of 10% at pH 6 for 2 h at 53°C.
  • Pressurised peroxide bleaching was done at 10% pulp concentration with 3% hydrogen perixode, 1.5% sodium hydroxide, 2% sodium silicate, 0.2% DTPA, and 1% magnesium sulfate (pulp basis).
  • the mixtures were placed in Teflon lined vessels pressurised to 500 kPa with oxygen and heated at 115°C for 2h.
  • the kappa numbers of the pulps were determined according to Australian standard method AS 1301.201 m-86.
  • Residual hydrogen peroxide concentration was determined on a sample of filtrate by iodometric titration.
  • the kraft-oxygen pulp was treated with xylanase at dosages 0, 3, 7, 10, 15 and 30 XU/g pulp prior to bleaching with D(EO)DD sequence.
  • the results from this series of experiments are summarised in Table 1.
  • One way of assessing the result of xylanase treatment is to use the same conditions in the initial D and the (EO) stages and measure the kappa numbers of the pulps after the D(EO) stage. A lower kappa number will indicate a beneficial effect of the enzyme treatment. In Figure 8, the kappa numbers of the pulps are plotted against the enzyme dosage.
  • the xylanase treatment removes xylan from the pulp and this results in a lower bleached pulp yield.
  • a xylanase dosage of 10 XU/g decreased the yield by about 3% (based on unbleached pulp). This loss of yield has to be taken into account when the overall benefits of the use of xylanase are being assessed.
  • Table 1 Effect of xylanase treatment on D(EO)DD bleaching sequence
  • the xylanase 229B-G was compared with two other xylanases, one of which is commercially available (Irgazyme-40) and the other is a non-commercial xylanase (DCPX).
  • the bleaching sequence D(EO)DD was used for this comparison.
  • the data used in Table 3 are from previous
  • Xylanase 229B has a very high activity when used to treat oxygen-delignified eucalypt kraft pulp. As is described in the text of this specification both ECF and TCF bleaching sequences may be enhanced when pulp is treated with 229B.
  • Dictyoglomus thermophilum is the only valid member of its genus and the only published species which resembles the microorganisms of this invention.
  • Dictyoglomus thermophilum is a thermophilic, strictly anaerobic, chemoorganotrophic non-motile and non-sporulating eubacterium that was isolated from a natural hot spring in Japan (Saiki et al., 1985).
  • the organism of this invention and D. thermophilum are rod shaped and form spherical bodies.
  • the guanine/cytosine (G/C) content of D. thermophilum has been reported as 29.5% (Patel et al., 1987, as measured by thermal denaturation techniques), which is significantly different to that of the Dictyoglomus strain B 1 (Mathrani & Ahring, 1992), having a GC content of 34% (as measured by high performance liquid chromatography).
  • the organism Rt46B.1 was isolated from a natural hot spring in Kuirau Park, Rotorua, New Zealand.
  • Regions where sequence was not obtained in either case is denoted by 'N'.
  • SSU rRNA gene sequences of Dictyoglomus thermophilum and Rt46B .1 were identical.
  • nucleotide sequence which do not substantially alter the amino acid sequence of the enzyme of the present invention should be appreciated as being contained within the scope of the invention as claimed. It will also be appreciated that modifications may be made to the amino acid sequence of the enzyme which do not substantially alter the morphology or activity of the enzyme of the present invention; such modifications not departing from the scope of the invention as claimed.

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Abstract

Cette invention concerne une préparation enzymatique destinée au blanchiment de produits cellulosiques et contenant un enzyme dérivé de Dictyoglomus thermophilum, ledit enzyme appartenant à la famille des enzymes connus sous le nom de G-Xylanases et étant doté d'une activité de 1,4-xylanase à températures élevées. Cet enzyme peut être isolé à partir de Dictyoglomus thermophilum ou il peut également être produit à partir d'un vecteur recombiné contenu dans un micro-organisme hôte (tel que la souche Escerischia coli JM101).
PCT/NZ1997/000042 1996-03-29 1997-03-27 Xylanase WO1997036995A2 (fr)

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AU25235/97A AU2523597A (en) 1996-03-29 1997-03-27 A xylanase

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NZ286296 1996-03-29
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002057541A3 (fr) * 2001-01-18 2003-04-03 Iogen Bio Products Corp Procedes permettant un traitement de blanchiment a l'aide de xylanases
US7718411B1 (en) 2004-08-05 2010-05-18 Danisco Us Inc. Trichoderma reesei G/11 xylanases with improved stability
US8426181B2 (en) 1999-10-12 2013-04-23 Danisco Us Inc. Method to improve the stability and broaden the pH range of family G/11 xylanases
US8927248B2 (en) 2006-04-12 2015-01-06 National Research Council Canada Modification of xylanases to increase thermophilicity, thermostability and alkalophilicity
CN107723309A (zh) * 2009-11-06 2018-02-23 谷万达公司 转基因植物和动物饲料
US10196623B2 (en) 2009-11-06 2019-02-05 Agrivida, Inc. Intein-modified enzymes, their production and industrial application
US10407742B2 (en) 2009-11-06 2019-09-10 Agrivida, Inc. Intein-modified enzymes, their production and industrial applications
US10988788B2 (en) * 2009-11-06 2021-04-27 Agrivida, Inc. Plants expressing cell wall degrading enzymes and expression vectors

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK69591D0 (da) * 1991-04-18 1991-04-18 Novo Nordisk As Nye mikroorganismer

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8426181B2 (en) 1999-10-12 2013-04-23 Danisco Us Inc. Method to improve the stability and broaden the pH range of family G/11 xylanases
US8846364B2 (en) 1999-10-12 2014-09-30 Danisco Us Inc. Method to improve the stability and broaden the pH range of family G/11 xylanases
US9481874B2 (en) 1999-10-12 2016-11-01 Danisco Us Inc. Method to improve the stability and broaden the pH range of family G/11 xylanases
WO2002057541A3 (fr) * 2001-01-18 2003-04-03 Iogen Bio Products Corp Procedes permettant un traitement de blanchiment a l'aide de xylanases
US7320741B2 (en) 2001-01-18 2008-01-22 Iogen Bio-Products Corporation Method of xylanase treatment in a chlorine dioxide bleaching sequence
US7718411B1 (en) 2004-08-05 2010-05-18 Danisco Us Inc. Trichoderma reesei G/11 xylanases with improved stability
US8927248B2 (en) 2006-04-12 2015-01-06 National Research Council Canada Modification of xylanases to increase thermophilicity, thermostability and alkalophilicity
CN107723309A (zh) * 2009-11-06 2018-02-23 谷万达公司 转基因植物和动物饲料
US10196623B2 (en) 2009-11-06 2019-02-05 Agrivida, Inc. Intein-modified enzymes, their production and industrial application
US10407742B2 (en) 2009-11-06 2019-09-10 Agrivida, Inc. Intein-modified enzymes, their production and industrial applications
US10988788B2 (en) * 2009-11-06 2021-04-27 Agrivida, Inc. Plants expressing cell wall degrading enzymes and expression vectors
CN107723309B (zh) * 2009-11-06 2022-02-01 谷万达公司 转基因植物和动物饲料

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