WO1997036995A2

WO1997036995A2 - A xylanase

Info

Publication number: WO1997036995A2
Application number: PCT/NZ1997/000042
Authority: WO
Inventors: Peter Leonard Bergquist; Moreland David Gibbs; Daniel Morris
Original assignee: Pacific Enzymes Limited
Priority date: 1996-03-29
Filing date: 1997-03-27
Publication date: 1997-10-09
Also published as: AU2523597A; WO1997036995A3

Abstract

An enzyme preparation for application in the bleaching of cellulose products, containing an enzyme derived Dictyoglomus thermophilum, the enzyme further being contained within the family of enzymes known as G-Xylanases, and having beta-1,4-xylanase activity at elevated temperatures. The enzyme may be isolated from Dictyoglomus thermophilum or alternatively produced from a recombinant vector contained within a host microorganism (such as Escherischia coli strain JM101).

Description

An Xylanase

TECHNICAL FIELD OF THE INVENTION

This invention relates to 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.

REFERENCES

A list of citations is provided at the end of the specification.

DEFINITIONS

In referring to 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.

The term "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.

Other definitions are used in a manner consistent with the art.

BACKGROUND

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.

A desirable bleaching agent or augmentor would be cheap, easily produced, and economically viable, and in particular would be reactive in hot alkaline aqueous conditions which may be (for example) 80 degrees C and pH = 9, and would retain its activity under such conditions for an extended period (e.g. a half life of some hours or days).

OBJECT

It is an object of this invention to provide an improved xylanase enzyme and/or methods for its production, the enzyme being applicable to processes of biological bleaching of cellulose products, or at least to provide the public with a useful choice. STATEMENT OF THE INVENTION

In a first main aspect of the present invention there is provided an enzyme, 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, 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.

Preferably the enzyme is substantially as isolated from Dictyoglomus thermophilum.

More preferably the enzyme is a recombinant enzyme.

Preferably the enzyme has activity within between 60 and 90 degrees Celsius and within the pH range 5 to 7.

More preferably the enzyme has optimal activity at approximately 85 degrees Celsius and approximately pH 6.5.

In a related aspect of the present invention there is provided 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. In a further related aspect of the present invention there is provided a recombinant vector containing the recombinant gene as described above.

In a further related aspect of the present invention there is provided a micro-organism capable of producing an enzyme as described above wherein the micro-organism contains a recombinant vector as described in the previous paragraph.

In another aspect there is provided a preparation of an enzyme as described above wherein the preparation contains an amount of the enzyme and a biologically acceptable carrier.

In a further main aspect of the present invention there is provided 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.

In a yet further main aspect 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.

DESCRIPTION OF DRAWINGS

The following is a description of a preferred form of the invention, given by way of example only, with reference to the accompanying diagrams in which: 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. The appropriate regions of the family G xylanase sequences from the GenEMBL nucleotide database from which the family G xylanase consensus primers were designed are included in multiple sequence format above the consensus primer sequences.

Nucleotides in the alignment which conform to the consensus at each position in the multiple sequence alignment are indicated in white or black. The standard ambiguity nucleotide bases are included where appropriate: N=A,T,C or G; R=A or G; S=C or G; M=A or C; W=A or T; 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). The positions of the family G xylanase consensus primers

(xynGF/xynGR) and genomic-walking PCR primers (dictGF/dictGR) are indicated by arrow-heads.

(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. 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:

(i) the 1190base-pairs of nucleotide sequence which was obtained from both the xynB GXCF amplified by the xylanase consensus primers and the xynB forward and reverse genomic-walking PCR fragments amplified from the dictGF and dictGR genomic-walking PCR primers, respectively;

(ii) the 229B peptide sequence encoded within the Rt46B.1 xynB open-reading frame.

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 (-

>);

(iv) the start residues encoded by the open-reading frame within the 229BN PCR primer is shown below the 229B peptide sequence in white on black.

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 JM101 strain harbouring the xynB:pJLA602 plasmid construction.. All buffers were pH adjusted at 70°C. Abbreviations for buffers are as follows: NaOAc=sodium acetate 12.5mM, BTP=12.5mM l-3-bis[tris (hydroxy-methyl)-methylamino]propane,

Caps=12.5mM 3-[cyclo hexylamino]-1-propanesulfonic acid, Mes=12.5mM (2[N- Morpholinojethanesulfonic acid). 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 JM101 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 JM101 strains harbouring the xynB:pJLA602 plasmid construction on a 2% kraft-pulp solution at 75°C, pH6.5

(12.5mM BTP buffer). The release of reducing sugars were measured by the colorimetric assay described by Lever. The 229B xylanase activity remaining in solution at the end of the 7 hour assay in indicated by the bold grey line (showing 40% residual 229B xylanase activity in solution after 7 hours). 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.

Figure 12: Shows an alignment of the SSU rRNA gene sequences of D. thermophilum and

12 A Rt46B.1.. Figure 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.

DETAILED DISCLOSURE OF THE INVENTION

Following is a description of the preferred embodiment of the present invention. It is given by way of example only, and it should be appreciated that a number of modifications may be made to this example without departing from the scope of the invention. Previously, one xylanase gene (xynA) encoding a family F xylanase (229 A) has been isolated from the Dictyoglomus thermophilum strain Rt46B.1 (Gibbs et al. 1995).

Source Organisms

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).

Deposit

The type culture of 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.

Genes and DNA Sequences

Genomic-DNA Preparation

Cultures of Rt46B.1 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.1 xynB GXCF

The Rt46B.1 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 MgCl2, 1 mM dTT and 1 mM ATP. End-repaired Rt46B.1 xynB GXCFs were purified from a 1% low-melting temperature agarose gel following separation by gel-electrophoresis using the GeneClean (Bio101, La Jolla, Ca.) procedure and cloned into the Smal site of the M13mpl0 bacteriophage sequencing vector. Four recombinant M13mp10 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.

Genomic-walking PCR of the Rt46B.1 xynB gene

One forward genomic-walking primer (dictGF) and one reverse genomic-walking primer (dictGR) were designed from the sequence of the Rt46B.1 xynB GXCF (figure 1A and figure (3). The genomic- walking PCR protocols were perfomed as described previously by Morris et al. (1995). Seven Rt46B.1 genomic-DNA restriction-fragment/DNA linker-libraries (henceforth referred to as "linker-library") were prepared using each of the following restriction endonuc leases: Ncol, Dral, EcoKV, Hincll, Pvull, Sspl and Sall. Genomic-walking PCRs were carried out using the dictGR/berg41 and dictGF/berg41 primers on each of the seven linker-libraries to amplify various DΝA fragments upstream and downstream of the Rt46B.1 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 1000bp fragment amplified from the Rt46B.l Ncol library) were sequenced at least on one DΝA 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.1 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 Ν-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. A linker peptide of 17 amino-acids delineated the N- and C-terminal domains of the 229B peptide. The full nucleotide sequence of xynB and resultant amino acid sequence of the 229B peptide are given below:

Cloning of xynB into the controlled expression vector pJLA602

Oligonucleotide primers were designed based on the xynB nucleotide sequence to allow PCR amplification of a xynB gene fragment from Rt46B.1 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). These sites allowed the in-frame directional ligation of the 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 (see Figure 4). Two amino-acid changes were required at the beginning of the 229B peptide sequence to meet

transcriptional-initiation requirements and to allow the introduction of the Ncol site into the xynB PCR fragment 5' terminal sequence: 229B Isoleucine28 (ATA) and Threonine29 (ACA) were changed to Methionine28 (ATG) and Alanine29 (GCA), respectively.

The recombinant pJLA602 plasmid construction incorporating the 702base-pair Ncol-BamHl digested Rt46B.l xynB PCR fragment encoding the 229B xylanase domain has been named pΝZ2869. The transformed JM101 strain containing the pNZ2869 plasmid has been named PB6569.

Production of the 229B enzyme

Purified 229B enzyme for characterisation was produced as follows; 100-500μl 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. 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.

This or a similar technique, using organisms such as but not limited to strain PB6569 of E. coli strain JMlOl, carrying the Rt46B.1 xynB:pJLA602 (pNZ2869) or a similar construct may be scaled up to commercial levels, or the technique may be modified as a commercial method because the requirements for enzyme purity may be relaxed in given applications. Of course, alternatives such as a technique for manufacturing quantities of enzyme using an unmodified Dictyoglomus strain itself can be considered but the somewhat demanding growth requirements for this organism, and the likely yield of enzyme, especially if purification was required, would be likely to be disadvantages.

pH Assays 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]-1-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).

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 Na₂SO₃, 0.02M of trisodium citrate, and 0.02M of CaCl₂ ), boiled for 5 minutes and cooled. 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.

Cloning of PCR Products Produced from Amplification from a Number of Regions of xynB

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. An Ncol restriction endonuclease recognition site was incorporated into each forward PCR primer (xynBΝ4, 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.

Production of the Recombinant Enzyme Products of Cloned PCR Products Obtained from Amplification from a Number of Regions of xynB 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. pH and Temperature Optimum Assays for the Protein Products of Cloned PCR Products

Obtained from Amplification from a Number of Regions of xynB

The determination of optimum pH and temperature for each enzyme product was carried out in a manner consistent with that described above under "pH Assays" and "Determination of Optimum Temperature".

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. Example of the Application of Xylanase 229b in the Bleaching of Cellulose Products

Below are described examples of the use of xylanase 229B in the bleaching of eucalypt kraft.

The examples study the effect of xylanase in ECF (Elemental Chlorine Free) and TCF (Totally Chlorine Free) bleaching sequences. As oxygen delignification is practised in modem kraft pulp mills, it was used in this example. The ECF sequence D(EO)DD, where D is a chlorine dioxide stage and (EO) is an oxygen reinforced alkaline extraction stage, was chosen as a 100% chlorine dioxide bleaching sequence which would be suitable for eucalypt kraft pulp.

Wood Sample

The mature eucalypt wood sample was a mixture of E. sieberc wood and E. muellerana, E. globoidea, E. aglomerata and E. obliqua woods.

Kraft pulping

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 Na2O) was applied to produce a pulp with a kappa number of 19.7.

Oxygen delienification

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) were mixed with magnesium carbonate (1%, pulp basis), sodium hydroxide (1%, pulp basis), and water to give a pulp concentration of 10%. 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.

Xylanase treatment (X) 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 noncommercial 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.

Bleaching with the D(EO) DP Sequence

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 final two D stages were done in plastic bags at 10% pulp concentration at a temperature of

70°C for 4 h. 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).

Treatment with EDTA (O)

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 hydrogen peroxide bleaching (PO)

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. Chemical tests

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.

Pulp brightness determination

At the end of the treatment period, 4 g o.d. pulp (equivalent to 200 g/m²) was removed for measurement of handsheet brightness. The pulp was disintegrated in purified water using a domestic Bamix mixer at about 0.8% consistency for 2 min, and the suspension adjusted to pH 5 with sulfuric acid or sodium hydroxide. A handsheet for brightness measurement was prepared by means of a sheet machine. The handsheet was pressed in a sheet press and dried overnight at 23°C and 50% rh. The brightness (expressed in ISO units) was measured on a Techbrite micro TB-IC instrument the following day.

Effect of xylanase treatment on D(EO)DD bleaching 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.

There is an appreciable reduction in the kappa number as the enzyme dosage is increased to about 10 XU/g but there is a levelling off as the dosage is increased to higher levels.

Another measure of the effectiveness of the xylanase treatment is a comparison of the final pulp brightness level at various xylanase dosages. This comparison is shown in Figure 9. It can be seen that there is an increase of about 1.5 units in brightness when the dosage is about 10 XU/g but the effect does not become much larger when the dosage is increased above 10 XU/g.

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.

The beneficial effect of xylanase treatment of the pulp at a dosage level of 10 XU/g was quantified by using different amounts of chlorine oxide in the first D stage and comparing the results with those from the use of untreated pulp. The amount of chlorine dioxide used in the subsequent two D stages was constant. The data are given in Table I. In Figure 10 the bleached pulp brightness levels are plotted against the amount of chlorine dioxide used in the first stage

(expressed as chlorine multiple, % active chlorine). With xylanase treatment, the chlorine multiple could be reduced from 0.13 to 0.07 while maintaining a final brightness of 88% ISO.

Another way of making a comparison is to plot the final pulp brightness levels against the total amounts of chlorine dioxide (expressed as active chlorine) used in the bleaching sequence. This is shown in Figure 11. Xylanase treatment of the pulp enabled a brightness of SS% ISO to be attained with the use of about 3.4% of ClO₂ (as active chlorine compared with 4.0% ClO₂ when the untreated pulp was used. The saving in chlorine dioxide was about 6 kg (active chlorine) per tonne of pulp or 15% of the chemical charge used for the untreated pulp.

An additional benefit from the use of xylanase is that the maximum attainable brightness with the D(EO)DD sequence is increased from about 88.5% TSO to about 90% ISO. This is significant because if a brightness of 90% ISO was required for the particular kraft pulp used in this study the bleaching sequence would have to be modified, either by addition of hydrogen peroxide to the (EO) stage or by the use of a second alkaline extraction stage (i.e. a sequence such as D(EO)DED). Effect of xylanase treatment in TCF bleaching

The pulp was subjected to a pressurise(l hydrogen peroxide bleaching stage (PO) to assess the effect of xylanase treatment on bleaching with a chlorine-free chemical. Heavy metal ions were removed from the pulp with a chelation stage (Q) prior to hydrogen peroxide bleaching. The results from these experiments are given in Table 2. Treatment of the pulp with xylanase at a dosage level of 10 XU/g resulted in the brightness of the bleached pulp increasing from 82.2 to

86.8% ISO. As observed in the ECF bleaching experiments, there was a yield loss of about 3% when a xylanase stage was used. In practice, a higher brightness for bleached pulp would be required. These results should be regarded as indicative that treatment with xylanase 229B-G would be very beneficial with TCF bleaching of eucalypt kraft pulp.

Comparison of xylanase 229B-G with other xylanases 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 bleaching studies and are not directly comparable because different batches of kraft-oxygen pulp were used and the kappa numbers were slightly different. In Figure 11 the brightness levels of the bleached pulps are plotted against the dosages of the three different xylanases. It can be seen that xylanase 229B-G is the most effective of the three enzymes in that it produces the greatest increase in final brightness when a comparison is made at a particular dosage level. The relevant data in Tables 1 and 3 show that all three enzymes lower the bleached pulp yield by about 3% (unbleached pulp basis) when a dosage of 10 XU/g is used.

ADVANTAGES

The advantages of using an enzyme according to this invention in a pulp degradation or pulp bleaching process include (over inorganic bleaching agents) that waste streams will include less toxic material, and that the environment will be less contaminated, and (over other known enzymes) it is believed that the exceptional temperature stability of the enzyme will enhance the relevance of the use of biological bleaching treatments in industry.

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.

PHYLOGENY

Five isolates of Dictyoglomus have been reported in the literature [Patel et al (1987); Saiki et al (1985); Svetlichnii & Svetlichnaya (1988); Mathrani & Ahring (1991); and Mathrani & Ahring(1992).

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. Recently the phylogenetic position of Dictyoglomus thermophilum within the phylum of the Thermatogales has been reported (Love, et al (1993) based on a partial 16SSU RNA sequence, which indicates that Dictyoglomus forms a deep branch within the phylum of the Thermatogales or may even represent its own phylum. The 16SSU RNA sequence of Dictyoglomus strain Rt46B.1 was also reported showing it to be very closely related to D. thermophilum. No significant differences in the growth rates, physiology or fermentation products of A and Rt46B.l were reported by Patel et al. (1987) and Rt46B.l should be regarded as strain of Dictyoglomus thermophilum. This leads to a comparison of the two isolates with respect to the present invention.

Comparison of the SSU rRNA gene sequences of D. thermophilum and Rt46B.l

Love et al. (1993) recently published findings on the partial SSU rRNA gene sequences of Dictyoglomus thermophilum and Rt46B.l and showed that the genus Dictyoglomus forms a deep branch within the phylum of the Thermatogales or may even represent its own phylum. Figure 12 shows an alignment of the SSU rRNA gene sequences of D. thermophilum and Rt46B.l. These sequences have been updated by sequences obtained for the purposes of this specification. SSU rRNA genes for both organisms were obtained and partially sequenced by the method of Saul et al. (1992), and used to complement the sequence of Love et al. (1993). Regions where sequence was not obtained in either case is denoted by 'N'. Apart from a 51 base pair region where sequence was not present for Rt46B.l and could not be compared, the SSU rRNA gene sequences of Dictyoglomus thermophilum and Rt46B .1 were identical.

CITATIONS:

I . Bailey et al., 1992 Journal of Biotechnology 23 257-270

2. Gilkes et al. 1991, Microbiological Reviews 55 303-315

3. Lever, 1973, Biochem. Med. 7 274-281

4. Love, C. A., Patel, B. K. C, Ludwig, W. and Stackebrant, E. (1993); FEMS Microbiology

Letters 107 317-320

5. Mathrani, I. M., Ahring, B. K. (1991); Arch. Microbiol. 156 13-17;

6. Mathrani, I. M., Ahring, B. K. (1992); Appl. Microbiol. Biotechnol. 38 23-27.

7. Patel, B. K., Morgan, H. W., Wiegel, J., and Daniel, R. M. (1987); Arch. Microbiol.

147 21-24;

8. Patel et al. 1985; Arch Microbiol 141 63-69.

9. Saiki, T., Kobayashi, Y., Kawagoe, K., and Beppu, T. (1985); Int J. Syst. Bacteriol.

35 253-259;

10. Saul D et al. 1989, Nucl. Acids Res. 17 439

I I . Saul D et al. 1992, Int J. Syst. Bacteriol. 43 754-760

12. Svetlichnii, V. A. and Svetlichnaya, T. P. (1988); Mikrobiologiya 57 364-370

14. Morris D et al. 1995; Appl. Environ. Microbiol. 61 2262-2269

15. Gibbs et al. 1995; Appl. Environ. Microbiol. 61 4403-4408

VARIATIONS

It will be appreciated that vectors and micro-organisms other than those specified in this description may be employed to produce the enzyme of the present invention without departing from the scope of the invention as claimed.

Variations in the 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.

In addition, it will be appreciated that modifications (which are based on common biochemical considerations) to the basic enzyme preparation described herein, such as the addition of components to increase the stability of the enzyme in solution, do not depart from the scope of the present invention. Further, it will be appreciated that it is within the scope of the present invention to employ the enzyme of the present invention in alternative industrial bleaching process to the examples described herein.

Finally it will be appreciated that various alterations and modifications may be made to the foregoing without departing from the scope of this invention as set forth.

Claims

CLAIMS:

1 An enzyme, 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, 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.

2 An enzyme as claimed in claim 1 wherein the enzyme is substantially as isolated from

Dictyoglomus thermophilum.

3 An enzyme as claimed in claim 1 wherein the enzyme is a recombinant enzyme. 4 An enzyme as claimed in any of claims 2 to 3 wherein the enzyme has activity within between

60 and 90 degrees Celsius and within the pH range 5 to 7.

5 An enzyme as claimed in claim 4 wherein the enzyme has optimal activity at approximately 85 degrees Celsius and approximately pH 6.5.

6 A recombinant gene encoding the enzyme as claimed in claim 1 wherein said gene has a nucleotide sequence substantially as herein described, or at least part thereof, or one which is not sufficiently different so as to alter substantially the amino acid sequence of the enzyme expressed, or at least part thereof.

7 A recombinant vector containing the recombinant gene as claimed in claim 6.

8 A micro-organism capable of producing an enzyme as claimed in claim 1 wherein the micro- organism contains a recombinant vector as claimed in claim 6.

9 A preparation of an enzyme as claimed in any of claims 1 to 5 wherein the preparation contains an amount of the enzyme and a biologically acceptable carrier.

10 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.