FIELD OF THE INVENTION

The present invention relates to the use of a cellulase preparation in the treatment of paper pulp.

BACKGROUND OF THE INVENTION

In the preparation of pulp for paper making, the drainage properties of the pulp may in some cases be unsatisfactory, whereby the capacity of the paper line may be reduced. The drainage properties of the pulp are commonly determined by the Schopper-Riegler test (a high SR value indicating poor drainage) or by Canadian Standard Freeness (a low CSF value indicating poor drainage) . Unsatisfactory drainage may particularly occur in case of repulping material that has already been through a pulping and drying process, e.g. dried virgin pulp, recycled fibre or waste paper.

EP 262,040 (Cellulose du Pin) and EP 351,655 (Cultor) describe improved drainability by use of cellulase and hemicellulase during pulping. JP-A 59-9299 and JP-A 63-59494 describe the use of cellulase during pulping at a high pH (above 9) to improve ink removal from waste paper.

Kraft pulping, a process widely used in the pulp and paper industry, involves alkaline sulfate cooking of the pulp to remove 95% of the lignin. The remaining 5% of the lignin gives the pulp a dark brown colour which tends to get darker in UV light or by oxidation. In order to obtain a white pulp for high quality paper, the brown colour is removed by a multi-stage bleaching process using chlorine or chlorine dioxide.

To reduce the amount of chlorine used in the bleaching process, it has been suggested to subject the pulp to enzymatic treatment with xylanases (cf. K.E.L. Eriksson, Wood Science and

Technology 24, 1990, pp. 79-101; M.G. Paice et al., Biotechnol. and BioencT. 32, 1988, pp. 235-239; and J.C. Pom ier et al., Tappi Journal, 1989, pp. 187-191) .

SUMMARY OF THE INVENTION

It has been found that a cellulase preparation exhibiting a high degree of endoglucanase activity is may advantageously be used for the treatment of paper pulp, in particular for use in a bleaching, beating or de-inking process or for improving the drainage properties of the pulp.

Accordingly, the present invention relates to the use of a cellulase preparation with a high content of endoglucanase and little or no cellobiohydrolase for the treatment of paper pulp.

In the present context, the term "endoglucanase" is understood to indicate a cellulase which attacks amorphous regions of low crystallinity in cellulose fibres resulting in substantially no loss of crystallinity.

DETAILED DESCRIPTION OF THE INVENTION

According to the invention, it has been found that when the content of endoglucanase in the cellulase preparation used in the pulping process is high, the damage to cellulose fibres in the pulps is less than when a cellulase preparation is used which has a significant content of cellobiohydrolase. Thus, it is preferred to employ a cellulase preparation which contains at least 50%, in particular at least 90%, (by weight of the total cellulase protein content) of endoglucanase.

Several endoglucanases are known and may be used according to the invention. Microbial endoglucanases are preferred for reasons of economy. The endoglucanase should be active and stable at the conditions, especially the pH, of the pulping process. Examples of suitable endoglucanases are those derived

from Aspercfillus (particularly A. niger) , Trichoderma (particularly T. viride, T. reesei and T. konincrii) , Humicola (particularly H. insolens, see US 4,435,307), Fusarium, in particular Fusarium oxysporum, Myceliophthora. Phanerochaete. Penicillium. Geotricum, and alkalophilic Bacillus • (US 3,844,890). The endoglucanase included in 'the cellulase preparation is preferably a monocomponent endoglucanase, and is more preferably one which includes a cellulose-binding domain.

A particularly preferred endoglucanase for use according to the invention is an endoglucanase which is im unoreactive with an antibody raised against a highly purified ^" 43 kD endoglucanase derived from Humicola insolens, DSM 1800. An example of such an endoglucanase is one which has amino acid sequence shown in the appended Sequence Listing ID#1, or a homologue thereof exhibiting endoglucanase activity. In the present context, the term "homologue" is intended to indicate a polypeptide encoded by DNA which hybridizes to the same probe as the DNA coding for the aforesaid endoglucanase under certain specified conditions (such as presoaking in 5xSSC and prehybridizing for 1 h at ^" 40"C in a solution of 20% formaminde, SxDenhardt's solution, 50 mM sodium phosphate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 100 μM ATP for 18 h at ^" 40°C) . The isolation and preparation of this enzyme is described in co-pending Danish patent application No. 1159/90.

The use according to the invention can be applied to any pulp to aid in the bleaching, beating or de-inking thereof, or to improve its drainage properties. This is particularly of interest for pulps with a SR value above 25, and particularly for repulping of previously pulped and dried material such as dried virgin pulp, recycled fibres and waste paper. Other types of pulp which may advantageously be treated with the cellulase preparation are kraft pulp, sulphite pulp or thermomechanical pulp and other high yield pulps.

The Schopper-Riegler number (SR) is determined according to ISO standard 5267 (part 1) on a homogenous pulp with a consistency of 2 g/1. A known volume of pulp is allowed to drain through a metal sieve into a funnel. The funnel is provided with an axial hole and a side hole. The volume of filtrate that passes through the side hole is measured in a vessel- graduated in Schopper-Riegler units.

Depending e.g. on the use of recycled water, the process may be acidic (e.g. pH 4 - 7) where an Aspergillus, Phanerochaete, Penicillium, Geotricu or Trichoderma endoglucanase is preferred. Or the process pH may be near-neutral (e.g. pH 6.5- 9) where a Humicola, Fusarium or Myceliophthora endoglucanase is preferred, or alkaline (pH > 9) where an alkaline Bacilllus endoglucanase is preferred.

A suitable cellulase dosage will usually correspond to a cellulase activity at pH 6 of 100 - 10,000 EGU (as defined below) per kg of dry pulp. Where the pulping process is at alkaline pH (above 7) , the cellulase dosage should correspond to a cellulase activity at pH 9 of 100 - 10,000 CEVU (as defined below) per kg of dry pulp.

Determination of cellulase activity (EGU) : A substrate solution containing 34.0 g/1 CMC (Hercules 7 LFD) in 0.1 M phosphate buffer, pH 6.0. The enzyme sample to be analysed is dissolved in the same buffer. 10 ml substrate solution and 0.5 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 available from Sofraser, France) thermostated at 40°C. One endoglucanase unit (EGU) is defined as the amount of enzyme that reduces the viscosity to one half under these conditions.

Determination of cellulase activity (CEVU) : A substrate solution containing 33.3 g/1 CMC (Hercules 7 LFD) in Tris- buffer, pH 9.0, is prepared. The enzyme sample to be determined is dissolved in the same buffer. 10 ml substrate solution and

0.5 ml enzyme solution are mixed and transferred to a viscosimeter (Haake VT 181, NV sensor, 181 rpm) thermostated at 40°C. One Cellulase Viscosity Unit (CEVU) is defined as the amount of enzyme which reduces the viscosity to one half under these conditions.

When the cellulase preparation is to be employed according to the invention for pulping waste paper, de-inking may be achieved by carrying out the pulping at high pH (above 9) in the presence of de-inking chemicals (such as sodium hydroxide, sodium silicate, hydrogen peroxide and surfactant) , followed by ink separation (e.g. by flotation and/or rinsing) . This embodiment of the invention is particularly advantageous since the cellulase/endoglucanase will also serve to improve the de- inking, concomitantly with improving drainage. At the high pH used for de-inking, it is preferred to use an endoglucanase from Humicola or alkaline Bacillus.

The duration of the pulping will generally be 5 - 30 minutes, and this may optionally be followed by maceration (i.e. incubation with or without stirring) to let the enzyme action continue. The temperature throughout this treatment will generally be 15-80°C, typically 30-50°C. The total duration of the cellulase action (i.e. pulping + maceration, if any) will generally be 30-180 minutes.

Pulp prepared according to the invention can be used for conventional paper making.

The invention is further described in the follwing example which is not in any way intended to limit the scope of the invention as claimed.

EXAMPLE

Use of ^" 43 kD Humicola endoglucanase for the treatment of paper pulp

The "43 kD endoglucanase derived from Humicola insolens, DSM 1800, was used for the treatment of several types of paper pulp with a view to investigating the effect of the enzyme on pulp drainag .

The experimental conditions were as follows.

Pulps

1. Waste paper mixture: composed of 33% newsprint, 33% magazines and 33% computer paper. With or without deinking chemicals (WPC or WP, respectively)

2. Recycled cardboard containers (RCC) .

3. Bleached kraft: made from pine (BK) .

4. Unbleached thermomechanical: made from fir (TMP) .

Enzymatic treatment

A preparation of the ^" 43 kD endoglucanase was diluted to 7 CEVU/ml and added to each of the pulps indicated above (50 g DS, consistency 3%) . The enzyme dose was 2400 CEVU/kg dry pulp. The enzymatic treatment was conducted at a pH of 7.5 and at 40"C with gentle stirring for 60 minutes. A sample was taken after 30 minutes to monitor the progression of the reaction. After 60 minutes, the pulp was diluted to a consistency of 0.5% with cold water (+4°C) in order to stop the reaction.

Drainage of the wet pulp was determined as described above and given Schopper-Riegler (SR) values. The drainage time (DT) under vacuum was also determined.

The results are summarized in the following tables

Control experiments. Same conditions as the enzyme treatment, except no enzyme addition.

It appears from the table that the -43 kD endoglucanase treatment causes a significant decrease in SR values and significantly improves drainage of pulps used in papermaking.

Paper sheets were made from the various pulps on a Rapid Kδthen device and measured for strength according to different parameters (including breaking length) . No decrease in strength properties due to enzyme action was observed, as shown in the table.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: NOVO NORDISK A/S,

(ii) TITLE OF INVENTION: (iiϊ) NUMBER OF SEQUENCES: <f

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: NOVO NORDISK A/S, Patent Department

(B) STREET: Novo Alle (C) CITY: Bagsvaerd

(E) COUNTRY: DENMARK

(F) ZIP: DK-2880

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION: (vi.i) ATTORNEY/AGENT INFORMATION:

(A) NAME: Thalsoe-Madsen, Birgit

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: +4544448888 (B) TELEFAX: +4544493256 (C) TELEX: 37304

(2) INFORMATION FOR SEQ ID N0:1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1060 base pai s

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Humicol a insolens

(B) STRAIN: DSM 1800

11

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 73..927.

5 (ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 10..72

(ix) FEATURE: 0 (A) NAME/KEY: CDS

(B) LOCATION: 10..927

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1: 5

GGATCCAAG ATG CGT TCC TCC CCC CTC CTC CCG TCC GCC GTT GTG GCC 48 Met Arg Ser Ser Pro Leu Leu Pro Ser Ala Val Val Ala -21 -20 -15 -10 0 GCC CTG CCG GTG TTG GCC CTT GCC GCT GAT GGC AGG TCC ACC CGC TAC 96 Ala Leu Pro Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr -5 1 5

TGG GAC TGC TGC AAG CCT TCG TGC GGC TGG GCC AAG AAG GCT CCC GTG 144 5 Trp Asp Cys Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val 10 15 20

AAC CAG CCT GTC TTT TCC TGC AAC GCC AAC TTC CAG CGT ATC ACG GAC 192 Asn Gin Pro Val Phe Ser Cys Asn Ala Asn Phe Gin Arg He Thr Asp 0 25 30 35 40

TTC GAC. GCC AAG TCC GGC TGC GAG CCG GGC GGT GTC GCC TAC TCG TGC 240 Phe Asp Ala Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys 45 50 55

_>

GCC GAC CAG ACC CCA TGG GCT GTG AAC GAC GAC TTC GCG CTC GGT TTT 288 Ala Asp Gin Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe 60 65 70 GCT GCC ACC TCT ATT GCC GGC AGC AAT GAG GCG GGC TGG TGC TGC GCC 336 Ala Ala Thr Ser He Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala 75 80 85

TGC TAC GAG CTC ACC TTC ACA TCC GGT CCT GTT GCT GGC AAG AAG ATG 384 Cys Tyr Glu Leu Thr Phe Thr Ser Gly pro Val Ala Gly Lys Lys Met

90 95 100

GTC GTC .CAG TCC ACC AGC ACT GGC GGT GAT CTT GGC AGC AAC CAC TTC 432 Val Val Gin Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe 105 110 115 120

GAT CTC AAC ATC CCC GGC GGC GGC GTC GGC ATC TTC GAC GGA TGC ACT ^' 480 Asp Leu Asn He Pro Gly Gly Gly Val Gly He Phe Asp Gly Cys Thr 125 130 135

CCC CAG TTC GGC GGT CTG CCC GGC CAG CGC TAC GGC GGC ATC TCG TCC 52 Pro Gin Phe Gly Gly Leu Pro Gly Gin Arg Tyr Gly Gly He Ser Ser 140 145 150

5 CGC AAC GAG TGC GAT CGG TTC CCC GAC GCC CTC AAG CCC GGC TGC TAC 576 Arg Asn Glu Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr 155 160 165

TGG CGC TTC GAC TGG TTC AAG AAC GCC GAC AAT CCG AGC TTC AGC TTC 624 10 Trp Arg Phe Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe 170 175 180

CGT CAG GTC CAG TGC CCA GCC GAG CTC GTC GCT CGC ACC GGA TGC CGC 672 Arg Gin Val Gin Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg 15 185 190 195 200

CGC AAC GAC GAC GGC AAC TTC CCT GCC GTC CAG ATC CCC TCC AGC AGC 720 Arg Asn Asp Asp Gly Asn Phe Pro Ala Val Gin He Pro Ser Ser Ser 205 210 215

20

ACC AGC TCT CCG GTC AAC CAG CCT ACC AGC ACC AGC ACC ACG TCC ACC 768 Thr Ser Ser Pro Val Asn Gin Pro Thr Ser Thr Ser Thr Thr Ser Thr 220 225 230

25 TCC ACC ACC TCG AGC CCG CCA GTC CAG CCT ACG ACT CCC AGC GGC TGC 816 Ser Thr Thr Ser Ser Pro Pro Val Gin Pro Thr Thr Pro Ser Gly Cys 235 240 245

ACT GCT GAG AGG TGG GCT CAG TGC GGC GGC AAT GGC TGG AGC GGC TGC 864 30 Thr Ala Glu Arg Trp Ala Gin Cys Gly Gly Asn Gly Trp Ser Gly Cys 250 255 260

ACC ACC TGC GTC GCT GGC AGC ACT TGC ACG AAG ATT AAT GAC TGG TAC 912 Thr Thr Cys Val Ala Gly Ser Thr Cys Thr Lys He Asn Asp Trp Tyr 35 265 270 275 280

CAT CAG TGC CTG TAGACGCAGG GCAGCTTGAG GGCCTTACTG GTGGCCGCAA 964 His Gin Cys Leu

285 0

CGAAATGACA CTCCCAATCA CTGTATTAGT TCTTGTACAT AATTTCGTCA TCCCTCCAGG 1024

GATTGTCACA TAAATGCAAT GAGGAACAAT GAGTAC 1060 5