The invention relates to a preparation exhibiting enzymatic activity, which preparation has the capability of delignifying wood pulp at a temperature of. at least 65°C and a pH of at least 9. Further, the invention relates to a method of producing said preparation by aerobically fermen- tating a selected Bacillus stβarothermophilus strain. Furthermore, the invention relates to two isolated Bacillus stearothermophilus strains and mutants and variants thereof . The invention also relates to applications of the preparation of the invention, namely to a process comprising treatment of wood pulp with a preparation according to the in ^d ention, and a wood pulp and a fluff pulp treated with a preparation according to the invention, and also a paper, a board and a fluff made from a wood pulp treated with a preparation according to the invention. Background In the pulp and paper industry major efforts are made to reduce the use of chemicals in delignification and bleach¬ ing processes, since such chemicals, and especially organic chlorine compounds, in the effluent from bleach plants, give rise to environmental pollution. One approach to reduce the use of chemicals in pulping and bleaching has been concerned with the use of lignin-de- grading microorganisms. Lignin-degrading or lignin-modifying enzymes have also been suggested. A comprehensive review of lignin biodegradation has been published in Critical Reviews™ in Biotechnology, vol. 6, Issue 1, 1987, pp. 1-60, Ed. Stewart, G.G. and Russell, I . , CRC Press, Inc. Boca Raton, Florida.

Another approach has been concerned with removal of lignin by using he icellulases to break hemicellulose bonds. A review article on Hemicellulases: Their Occurrence, Purifi-

cation, Properties, and Mode of Action by Dekker, R.F.H. and Richards, G.N. has been published in Advances in Carbohydrate Chemistry and Biochemistry, vol. 32, 1976, pp. 277-352.

Among the hemicellulases, xylanases are the ones that have attracted most attention for use in biopulping and bio- bleaching. FR 2 557 894-A1 discloses treatment of paper pulp with an enzymatic solution which does not have any cellulase activity and which contains xylanase. The treatment is effected at 20-60°C, especially 40°C, and Example 6 reveals that the pH should be 5. However, the paper and pulp industry would be interested in treating wood pulp at higher tempera¬ tures and higher pHs for economical reasons and convenience, since some established bleaching processes are conducted at temperatures exceeding 65°C and pH exceeding 9. To our knowledge, no one has reported treatment of wood pulp with a preparation exhibiting enzymatic activity and having the capability of delignifying wood pulp at a tem¬ perature of at least 65°C and a pH of at least 9. Description of the invention The present invention provides a preparation exhibit¬ ing enzymatic activity, which preparation has the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 9.

In the present specification and the appended claims the expression "wood pulp" is to be interpreted broadly, and thus it is intended to comprise all kinds of lignocellulosic materia1s .

One aspect of the invention is directed to a prepara¬ tion exhibiting enzymatic activity, which preparation is ob- tainable by aerobic fermentation, in a suitable medium, of a Bacillus stearothermophilus strain selected from the deposit¬ ed strains NCIMB 40221 and NCIMB 40222 and mutants and va¬ riants thereof having substantially the same capability of producing said preparation as said strains NCIMB 40221 and NCIMB 4022, said preparation having the capability of delig¬ nifying wood pulp at a temperature of at least 65°C and a pH

of at least 9. In one embodiment of this aspect of the inven¬ tion the preparation is obtainable by aerobic fermentation, in a suitable medium comprising glucose as the carbon source, of a mutant strain of the Bacillus stearothermophilus strain NCIMB 40222. In another embodiment the preparation according to the invention comprises an enzyme including the amino acid sequence

-Lys-Asn-Ala-Asp-Ser-Tyr-Ala-Lys-Lys-Pro-His-I le-Ser-Ala- -Leu-Asn-Ala-Pro-Gin-Leu-Asn-Gin-Arg-Tyr-Lys-Asn-Glu-Phe- -Thr-Ile-Gly-Ala-Ala-Val-Glu-Pro-Tyr-Gln-Leu-Gln-Aεn- or a homologue thereof, the entire enzyme exhibiting enzymatic activity in a wood pulp medium. In this context a homologue of said amino acid sequence is a homologous se¬ quence having some amino acid substitutions, extensions or deletions which do not lead to the elimination of the enzyma¬ tic activity of the entire enzyme in a wood pulp medium. Furhter, a homologue of said amino acid sequence is any se¬ quence which is sufficiently homologous on the nucleotide level to be recognized by any DNA or RNA probe derived from said sequence. In still another embodiment of this aspect of the invention the preparation is a clarified culture broth. In yet another embodiment of this aspect of the invention the preparation is a partially purified fraction of the culture broth exhibiting enzymatic activity in a wood pulp medium. The partially purified fraction is obtainable by ammonium sulphate precipitation. In a further embodiment of this aspect of the invention the preparation is a highly purified fraction of the clarified culture both which is obtainable by the use of a cation exchanger, said fraction being in the form of a xylanase exhibiting enzymatic activity in a wood pulp medium. In one particular embodiment the xylanase has an approximate molecular weight between 41000 and 42000 Dalton determined by SDS PAGE and gel filtration and the following approximate amino acid composition determined as amino acid residues per molecule by amino acid analysis: Asx 58; Thr 12; Ser 10; Glx 44; Pro 24; Gly 20; Ala 30; Cys 1; Val 28; Met 2; He 26; Leu 14; Tyr 22; Phe 16; Lys 38; His 6; Arg 12.

Another aspect of the invention is directed to a method of producing a preparation exhibiting enzymatic acti¬ vity, whereby a Bacillus stearothermophilus strain selected from the strains NCIMB 40221 and NCIMB 40222 and mutants and variants thereof having substantially the same capability of producing said preparation as said strains NCIMB 40221 and NCIMB 40222, is subjected to aerobic fermentation in a suit¬ able medium, said preparation having the capability of de¬ lignifying wood pulp at a temperature of at least 65°C and a pH of at least 9. In a particular embodiment of the method according to the invention, a mutant strain of the Bacillus stearothermophilus strain NCIMB 40222 is subjected to aerobic fermentation in a suitable medium comprising glucose as the carbon source. In another embodiment of this aspect of the invention the fermentation broth is clarified, optionally by centri ugation. In yet another embodiment the clarified fer¬ mentation broth is subjected to ammonium sulphate precipita¬ tion, and optionally resuspension in a liquid medium, to yield a partially purified fraction of said clarified culture broth exhibiting enzymatic activity in a wood pulp medium at a temperature of at least 65°C and a pH of at least 9. In a further embodiment of the method according to the invention the clarified fermentation broth is purified and concentrated using a cation exchanger, to yield a highly purified fraction in the form of a xylanase exhibiting enzymatic activity in a wood pulp medium at a temperature of at least 65°C and a pH of at least 9.

Yet another aspect of the invention is directed to the isolated Bacillus stearothermophilus strains NCIMB 40221 and NCIMB 40222 and mutants and variants thereof, said mutants and variants having substantially the same capability of pro¬ ducing a preparation exhibiting enzymatic activity as said strains NCIMB 40221 and NCIMB 40222, said preparation having the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 9.

Still another aspect of the invention is directed to a process comprising treatment of wood pulp, whereby wood pulp is treated in at least one step with a preparation according to the invention. In one embodiment of this aspect of the invention, the wood pulp to be treated is sulphate pulp. In another embodiment the sulphate pulp to be treated is a par¬ tially delignified sulphate pulp. In yet another embodiment of this aspect of the invention the partially delignified sulphate pulp to be treated is an oxygen-delignified sulphate pulp. The process comprising treatment of wood pulp is suit¬ ably effected at a temperature of at least 65°C in a medium having a pH of at least 9.

An additional aspect of the invention is directed to products obtained from wood pulp which has been treated in at least one step with a preparation according to the invention. Thus, this aspect of the invention comprises wood pulp and fluff pulp which have been treated in a least one step with a preparation according to the invention, and paper, board and fluff that have been made from wood pulp which has been treated in at least one step with a preparation according to the invention.

A further aspect of the invention is directed to a DNA or RNA probe which recognizes the nucleotide sequence coding for the amino acid sequence -Lys-Asn-Ala-Asp-Ser-Tyr-Ala-Lys-Lys-Pro-Hiε-Ile-Ser-Ala- -Leu-Asn-Ala-Pro-Gln-Leu-Asn-Gln-Arg-Tyr-Lys-Asn-Glu-Phe- -Thr-Ile-Gly-Ala-Ala-Val-Glu-Pro-Tyr-Gln-Leu-Gln-Asn-

Additlonally, one aspect of the invention is directed to an antibody which binds to the amino acid sequence -Lys-Asn-Al -Asp-Ser-Tyr-Ala-Lys-Lys-Pro-His-Ile-Ser-Ala- -Leu-Asn-Ala-Pro-Gin-Leu-Asn- In-Arg-Tyr-Lys-Asn-Glu-Phe- -Thr-I le-Gly-Ala-Ala-Val-Glu-Pro-Tyr-Gln-Leu-Gln-Asn- These last two aspects of the invention are useful in the finding of enzymes having the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 9.

Isolation of thermostable enzyme producing organisms

The source of the organisms was water and mud samples taken from the water treatment pools at the pulp and paper mill of Korsnas, Gavle, Sweden. The enrichment medium (XM) contained 0.01% yeast extract, 0.04% tryptone and 0.2% xylan at pH 7.0. The same medium at pH 8.1 and 9.6 contained in addition Na2C03. Water and mud samples were added to the XM media and were cultivated aerobically at 65°C. Samples from the cultures were transferred to fresh media every 2-3 days. Following 4 transfers, samples from the cultures were diluted and spread on XM agar plates (XM containing 2% agar) .

Following three independent enrichment procedures, 30 strains were isolated, that were able to grow on MX agar plates at 65°C. No isolates were obtained from the XM medium at pH 9.6. Out of the 30 isolates, 10 produced clear zones around the colony suggesting that they produce an extracellu¬ lar xylanase. Two out of these ten isolates (strains T2 and T6> were selected for deposition. Deposition of microorganisms Two strains, namely Bacillus sp T-2 and Bacillus sp

T-6 were deposited under the Budapest Treaty at the National Collections of Industrial and Marine Bacteria Ltd (NCIMB), Aberdeen, on November 8, 1989. The accession numbers given were NCIMB 40221 and NCIMB 40222 for the strains T-2 and T-6, respectively.

Identi ication of strains T2 and T6

The strains T2 and T6 are aerobic, gram positive, spore forming rods capable of growing at 65°C. These strains are considered to be different strains of Bacillus stearo ^¬ t _he rmophilus by NCIMB, based on the following test results:

Bacillus - second stage tests

Isolate No. T2 (NCIK3 40221) T6 (NCIMB 40222)

Tests at 60°C 60°C

Spore shape E

Sporangium distended distinctly Spore position dominant T

Anaerobic growth Growth in 5% NaCl Growth in 7% NaCl

Cont.

Growth in 10% NaCl Growth in pH 5.7 broth Acid from glucose Gas from glucose

VP (acetoin) Egg yolk agar opacity No growth No growth Casein decomposition + + Gelatin decomposition + (+)

Aesculin

Starch hydrolysis N0 ₃ ^" to N0 ₂ ~ Residual N

Citrate, Koεer's Dextrose azide pH in VP broth 5.5 6.3

Litmus milk coagulation acidification

Catalase + +

Oxidase + +

Growth temperatures °

25°C 37°C 65°C 70°C

E, elliptical or cylindrical; S, spherical or nearly so;

C, central; T, terminal or subterminal; CT, central to terminal; cOn glucose agar; dPeptone wate sugar, Andrade's indicator

- restricted

Fatty acids composition of the strains T2 and T6

The thermophilic xylanase positive isolates T2 and T6 were sent to Microbial ID, INC. Newark, DE, USA for fatty acids composition. The results received were as follows:

Fatty acids composition (%) of xylanase positive thermophiles

Isolate: T2 T6

Fatty acid

9:0 0.73 0.64 12:0 0.46

14:0 iso 14:0 0.85 1.92

15:0 iso 44.41 46.2 15: anteiso 2.59 2.35 15:0 0.53

Growth characteristics of the strains T2 and T6, and pre¬ paration of concentrated crude supernatants from the cultured strains

Growth medium: XMP medium containing in g/liter: vitamine-free casamino acids, 4; yeast extract, 0.2; MgS0 ₄ , 0.01; (NH ₄ ) ₂ S0 ₄ , 2; K ₂ HP0 ₄ , 0.46; KH ₂ P0 ₄ , 0.1; xylose, 5; MOPS (3- [N-morpholino] propane sulfonic acid) 10.4; and trace elements. The pH of the medium was 7.0.

Xylanase activity:

Xylanase activity was determined by incubating a fresh solution of xylan with extracellular supernatant fluid and assaying for increase in reducing sugars by the ferricyanide method (Spiro, R.G. 1966, Methods in

Enzy ology 8_: 7-9). Assay buffer was 50 mM Tris. Cl, pH 7.0 and 0.5% xylan (oat spelts, Sigma). Units of activity for xylanase are μmol reducing sugar generated per minute at 65°C. Cell turbidity of the medium:

The cell turbidity of the medium is given in Klett units. lKU=0.004g of dry cell weight (DCW)/liter. Once the culture reaches its maximum density it tends to lyse as indicated by the sharp decrease of the culture turbidity. The strains T2 and T6 were grown in XMP medium for 24 hours in a New Brunswick Microferm 7.5 liter fermentor. Growth was carried out in a working volume of 4 liter, at 60°C, agitation rate of 600 RPM and aeration rate of 7 liter per minute. Foaming was controlled automatically by adding silicon antifoam (5%, Fluka) and the working volume was maintained by adding sterile water. The initial starting inocula gave turbidities of 10KU (0.04 g DCW/liter). For the first 8 hours of the fermentations cell turbidity increased to 540 and 700 KU for strains T6 and T2, respectively. At 24 hours cell turbidity declined. The culture liquid was cooled to room temperature and centrifuged (8000 g, 10 min, 20°C) to remove the cells. The cell free supernatant contained 1.0-1.4 and 2.0-2.4 units xylanase per ml for strains T2 and T6 respectively and about 0.02 mg protein per ml for both strains. Protein was concentrated by adding to the supernatant solid- ammonium sulfate to 80% saturation at 4°C and centrifuging (9000 g, 15 min) the mixture following 12 hours at 4°C. The precipitate was dissolved in a total volume of 100 ml of 10 mM phosphate buffer pH 7.0 and dialyzed three times against 5 liter of the same buffer at 4°C. The dialyzed

solution ("120 ml) contained about 7 mg/ml protein and 15-20 and 35-40 units of xylanase for strains T2 and T6 respectively.

Growth of strain T6

Time (h) KU Xylanase (U/ml)

0 10 nd 2 25 nd

4 140 nd

6 400 1.48

8 540 1.88

11 540 1.78

14 528 2.26

24.5 280 2.42

Growth of strain T-2

Time (h) KU Xylanase (U/ml)

10 nd

It should be noted that the xylanase activity of the strains T2 and T6 did not decrease at the end of the fermentation.

The preparation exhibiting enzymatic activity according to the invention

The cell-free supernatants from growth of the strains T2 and T6 in XMP medium contain at least one xylanase, since they were shown to exhibit xylanase activity. Said supernatants (also called crude enzyme preparations) and partially purified fractions thereof have been used in the treatment of unbleached and oxygen semi-bleached softwood sulphate pulps. Unbleached and oxygen semi-bleached pulp samples

The pulp samples were softwood sulphate pulp samples taken from the Korsnas mill.

The unbleached pulp sample was a sample taken after alkali digestion in a Kamyr digester, laboratory screened and washed, and said sample was dried over night at 50°C. The semi-dry sample was used in the experiments, and it was designated K8. This material was dried at 105°C to constant weight yielding a water content of 6.35%.

The oxygen semi-bleached pulp sample was a sample that had been treated in the KorsnSs fiber-line 3, and it was taken after the second oxygen treatment, laboratory screened and washed. A general description of the pulp treatment plant of Korsnas has been published in Paper 26, September 1989, p. 20 and Nordisk Cellulosa 1989 No. 6, p. 8-11.

The oxygen semi-bleached pulp was designated Kll. Lignin content of pulps K8 and Kll

The lignin contents of pulps K8 and Kll were determined by the procedure of "The Swedish Association of Pulp and Paper Engineers, Series CCA5. The lignin content of K8 (unbleached) was 2.8% and the lignin content of Kll (oxygen semi-bleached) was 2.0%. Thus the oxygen semi- bleached sample Kll had approximately 25% reduced value for lignin content compared to K8.

Experiments conducted with crude enzyme preparations from Bacillus stearothermophilus strains T2 and T6 on deligni- fication of pulps K8 and Kll

For the estimation of how much lignin is made extractable by the enzymatic preparations of the invention a sensitive spectrophotometric assay was used. The ab- sorbance values at 350 nm(A_ ₅ _) were measured on the supernatant liquids of the samples, and measured values are presented as % lignin released. The experiments and the results are summarized in Tables 1-5.

From the data given in the Tables 1-5 it can be con¬ cluded that the crude enzyme preparations from Bacillus Stereother ophilus strains T2 and T6 are capable of delignifying both unbleached and semi-bleached pulp samples at pH values of at least 9 and at temperatures of at least 65°C. Moreover, it can be concluded that the % lignin released is both time- and concentration-dependant.

TABLE 1

Release of lignin from pulp Kll as a function of crude enzyme preparations from B. stearothermophilus strains T2 and T6

The enzyme preparations were ammonium sulfate preci¬ pitates that were dissolved in 10 mM phosphate buffer, pH 9.0, to final concentrations of protein as indicated. The control was 10 M phosphate buffer, pH 9.0. Pulp Kll was 150 mg in 3 ml.

Incubation was for 18 h at 65°C and pH 9.0. The % lignin released is based on A ₃₅₀ and is corrected for absorbance of the crude enzyme preparation at the appropriate concentration.

TABLE 2

Kinetics of lignin release from pulp Kll by crude enzyme preparation from B. stearothermophilus strains T2 and T6 ^C

% lignin released Total Net

The experiment was conducted as described in Table 1 at 65°C and pH 9.0.

The concentration of crude enzyme T2 and T6 were 2.8 and 1.5 mg protein/ml, respectively.

TABLE 3

Release of lignin as a function of temperature by crude enzyme preparations from B. stearothermophilus strains T2 and T6 ^*

Temperature Enzyme

53 T2 65 T2 75 T2

53 T6 65 T6 75 T6

The experiment was conducted as described in Table 1 at 65°C and pH 9.0.

The concentration of crude enzymes T2 and T6 were 1.7 and 0.7 mg protein/ml, respectively.

TABLE 4

Release of lignin from Kll as a function of pHa

PH Enzyme % lignin released Total Net

•The experiment was conducted as described in Table 1 at 65°C for 18 h.

The concentrations of crude enzyme preparations T2 an T6 were 1.7 and 1.5 mg protein/ml, respectively.

TABLE 5

Release of lignin from pulp K8

The experiment was conducted as described in Table 1 aatt 6655°°(C and pH 9.0 for 18 h, using pulp K8 instead of Kll

Preparation of a highly purified fraction from growth of strain T6

A highly purified fraction (in the form of a xylanase) of the clarified culture broth from growth of strain T6 is obtainable by the use of a cation exchanger in a single con¬ centration and purification step. Thus, xylanase T6 was puri ^¬ fied and concentrated directly from the cell free supernatant by adsorption to the cation exchanger CM-52 (Whatman) . However, with XMP medium, the recovery yield was only 21% presumably because of the high ionic strength of the medium. When the medium was dialysed against low ionic strength buffer or when MOPS was omitted from the medium the recovery yields were about 50%. To determine the minimal amount of CM-52 needed for effective adsoprtion from the clarified culture medium, different amount of CM-52 were tested for purifying xylanase T-6. The results are given in Table 6.

Table 6. Batch adsorption of xylanase T-6 with different amounts of CM-52

CM-52/broth Fraction Volume Xylanase Total Yield (g/g) (ml) (U/ml) units (%)

0.1

0.05

0.02

0.01

2 0

As shown in Table 6, at 5% adsorbent the yield is 50%. Another commercially available cation exchanger is SE-52 (Whatman) (the negative groups are sulfoxyethyl attached to cellulose) . SE-52 is known to have a larger adsorption capa¬ city than CM-52. Since the purification step is also a con¬ centrating step it is advantageous to use as little adsorbent as possible. We therefore tried SE-52 for adsorbing xylanase. The results are given in Table 7.

Table 7. Batch adsorption of xylanase T-6 with different amounts of SE-52

SE-52/ broth Fraction Volume Xylanase Total Yields (ml) (U/ml) units (%)

Supernatant 100 1.41

0.05 Extract 30 2.4 0.02 Extract 21 3.2 0.01 Extract 20 2.9

As shown in Table 7, only 2% of SE-52 was needed to obtain 48% recovery of xylanase T6.

Characterization of xylanase T-6

The molecular weight of xylanase T-6 was determined by SDS PAGE and gel filtration, yielding values between 41,000 and 42,000 Daltons . The fact that both methods gave similar MW indicates that the enzyme is composed of a single polypep- tide chain (SDS PAGE separates the protein subunits) . The pi (the pH where the total charge of the protein is zero) of the enzyme was 9.0 determined by high voltage isoelectric focus¬ ing. The relatively high pi of the enzyme explains the posi-

tive charge of the enzyme at neutral pH . The pH optimum of the enzyme was around 7.0; at pH 9.0 the enzyme had about 50% of its pH 7.0 activity. The temperature profile of xylanase T-6 activity is given in Table 8.

Table 8. The activity of xylanase T6 at different temperatures . *

Temperature Relative activity

°C %

45 28 55 51 60 70 65 81 70 92 75 100 80 73 85 40

* Reaction time: 5 minutes

The thermostability of xylanase T6 at a temperature of 65°C and a pH of 9 is shown in Table 9.

Table 9. Thermostability of xylanase T6 at 65°C and pH 9

Time Relative activity h %

0 100

0.25 82

0.5 78

1 75

2 72

4 63

Additionally, it can be mentioned that at 65°C and pH 7 no loss of activity was detected for over 10 h.

Obtaining xylanase constitutive mutants from strain T6

To obtain the mutants we used the fact that in many cases xylanase and xylosidase are under the same regulatory control (the same repressor) . Strains that produced xylosi- dase can be easily detected on agar plates containing the chromogenic substrate p-nitropheny1 β-D-xylopyranoside (ONPX) . Only constitutive mutants for xylosidase will produce yellow color on agar plates in the absence of xylose. To ob¬ tain xylosidase constitutive mutants, cells were mutagenized with MNNG ( l-methyl-3-nitro-1-nitrosoguanidine) and then plated on agar plates containing ONPX without xylose. About one out of five hundred cells produced colonies which yielded yellow color on the agar plates. Thirty xylosidase constitu¬ tive mutants were isolated in this may, and all of them were also xylanase constitutives . The growth and xylanase produc¬ tion of two of these mutants (M-7 and M-28), growing in the presence of xylose or glucose, are given in Tables 10 and 11. On both media (XMP and GMP) the mutants and T-6 exhibited similar growth rates. However, the mutants produced consider- ably higher levels of xylanase on XMP medium compared to T-6. On GMP medium (glucose is the carbon source) strain T-6 failed to produce detectable levels of xylanase whereas the mutants produced high level of xylanase activity. The xylanase from strain M-7 was purified and showed identical properties to the T-6 xylanase.

24

Table 10. Growth and xylanase production of T-6 xylanase constitutive mutants on XMP

Strain Time Turbidity Xylanase (h) (KU) (U/ml)

0.5 0.8 2.1 2.3

0.9 1.6 2.1 2.3

0.6

2.0 5.5 7.2

Table 11. Growth and xylanase production of T-6 xylanase constitutive mutants on GMP

Strain Time Turbidity Xylanase (h) (KU) (U/ml)

0

0.1 0.1 0.1 0.1

0.3 0.6 1.1

1.4

2.2

0.1

0.9

1.7

3.1 4.8

Fragment sequence and amino acid composition of xylanase T-6.

From a sufficiently long strech of the N-terminal amino acid sequence of a protein, one can completely identify the protein. The sequence of one fragment of the xylanase T-6 was determined twice at the Weiz an Institute, Israel, on an Applied Biosystems gas phase microsequencer ABI 475A. The first protein sample was electroblotted first onto a PVDF mebrane (Millipore) and gave only 20 amino acids. The second protein sample was purified first on a Superose 12 FPLC column (Pharmacia) and was sufficient for obtaining the se¬ quence of 41 amino acids. The obtained sequence is as follows :

-Lys-Asn-Ala-Asp-Ser-Tyr-Ala-Lys-Lys-Pro-His-Ile-Ser-Ala- -Leu-Asn-Ala-Pro-Gin-Leu-Asn-Gln-Arg-Tyr-Lys-Asn-Glu-Phe- -Thr-Ile-Gly-Ala-Ala-Val-Glu-Pro-Tyr-Gln-Leu-Gln-Asn-

To further characterize the enzyme, an amino acid analysis was performed on the purified xylanase T-6. The results from this analysis are shown in Table 12.

Table 12. Amino Acid composition of xylanase T-6

Pk Ret Component Concentration Residues Num Time Name (nmoles) per molecule ^a

58 ^b

12

10

44 ^c

24

20

30

1 28

2 26 14 22 16 38

6 12

^a Based on a molecular weight of 42,000 Dalton; corrections were made for unstable amino acids.

k The aspartic acid is the sum of aspartic acid and aspara- gine (Asx)

^c The glutamic acid is the sum of glumatic acid and gluta- mine (Glx) .

Experiments conducted with enzyme on PUIP K14

The pulp sample K14 was an oxygen semi-bleached soft wood pulp from the Korsnas mill. The enzyme was the purified thermophilic xylanase T6. Various parameters effecting the extent of delignification of the pulp K14 were investigated. The effect of fiber concentration is summarized in Table 13.

Table 13. Effect of fiber concentration on xylanase treatment*

Xylanase Fiber Net Lignin Released (U/ml) (%) (%)

5.1 4.7 4.9 5.1 2.5 6.6

20.4 4.7 9.3 20.4 2.5 11.8

15.0 1.0 15.0

40.8 2.5 13.9 45.0 1.0 16.7

* Conditions: pH 9.0, 65°C, 2h, K14 pulp: the no enzyme control was substracted in each case.

As seen in Table 13, decreasing the fiber concentration from 4.7 to 2.5 and to 1.0% dry weight fiber, enhanced the process. Since the enzymatic treatment is performed without mixing, it is likely that there was poor contact between the enzyme and insoluble substrate. For practical reasons, we chose to work at 2.5% fiber.

Effect of pH is given in Table 14.

Table 14. Effect of pH on xylanase treatment*

pH Buffer Net Lignin Released (%)

8.5 0.01 M Phosphate 8.3

9.0 8.4 9.5 6.3

8.5 0.1 M Na ₂ S0 ₄ 10.9 9.0 10.2 9.5 8.2

* Conditions: 2.5% K14 pulp, 15 U/ml xylanase, 2h, 65°C

As seen in Table 14, the enzyme is most effective at pH 8.5 - 9.0 in both potassium phosphate and sodium sulfate Earlier experiments indicated lower activities below pH 8.0 and above pH 9.5.

Effect of Na2Sθ concentration is shown in Table 15

Table 15. Effect on Na2Sθ4 concentration ⁵

Na ₂ S0 ₄ (M) Net Lignin Released (%)

0 7.2 0.01 8.6 0.05 9.9 0.10 10.8 0.15 11.5 0.20 10.7

* Conditions: 2.5% K14 pulp, 10 U/ml xylanase, pH 9.0,

65°C, 2h.

The treatment was effective from no added Na2Sθ4 up to 0.2 M with optimum activity at 0.15 M Na2S04.

Effect of xylanase concentration is summerized in Table 16

Table 16. Release of lignin as a function of xylanase con¬ centration*

Xylanase Buffer Lignin Release (U/ml) (% Klason)

0 0.01 M K Phosphate 0

5 8

10 10

15 12

45 14

0 0.1M Na ₂ S0 ₄ 0

5 12

10 13

15 14

45 16

* Conditions: 2.5% dry fiber K-14, 2h, pH 9, 65°C

As seen in Table 16, there was a concentration dependent release of lignin up to approximately 15 U/ml in both 0.1 M N 2S04 and potassium phosphate buffer. Higher concentrations of enzyme or a longer period of incubation (4h) increased the net lignin released only slightly.

Preparation of 10 g and 50 g samples of treated K14 for further tests

10 g samples of K14 pulp were treated with 15 U/ml and 45 U/ml xylanase for 2 h and 4 h, together with the appropriate controls. All samples were prepared in duplicate. All incubations were carried out at pH 9.0 and 65°C. The spectrophotometric analysis of lignin release is shown in Tables 17 (phosphate buffer) and 18 (0.1 M Na ₂ S0 ₄ ) . The net release of lignin ranged from 10.3 to 14.9%, with the 45 U/ml enzyme and 4 h incubation periods giving slightly higher values than 15 U/ml and 2 h.

Recently, 50 g samples of pulp were treated in duplicate for additional analyses. The net release of lignin was 10.8% for 15 U/ml enzyme for 2 h and 12.6% for 15 U/ml for 4 h.

Table 17. Xylanase treatment of 10 g K14 pulp in phosphate buffer*

Xylanase Time Lignin Released (%) (U/ml) (h) Total Net

0 2 5.3 0 15 2 15.6 10.3 45 2 17.9 12.6

0 4 6.3 0 15 4 17.4 11.1

45 4 20.1 13.8

* Conditions: 2.5% K14 pulp, pH 9.5, 65°C; average of two experiments

Table 18. Xylanase treatment of 10 g K14 pulp in 0.1 M Na2S0 ₄

Xylanase Time Lignin Released (%) (U/ml) (h) Total Net

0 2 5.9 0 15 2 17.6 11.7

45 2 19.8 13.9

0 4 7.0 0 15 4 19.4 12.4

45 4 21.9 14.9

* Conditions: As in Table 17.

Analysis of Xylanase-treated 10 g samples The results of the analyses are summarized in Tables

19-23. The parameters given in the tables were according to the SCAN Test series, except for the Zero-span values which were measured according to the manufacturers (Pulmac Instru¬ ments Ltd, Canada) instructions. Table 19 presents the data obtained using pulp that had been treated for 2 h at 65°C, pH 9.0, ; . 0.1 M a2S04 with no enzyme (control) and 15 U/ml enzyme. As can be seen, there was excellent reproducibility with the duplicate samples, prepared at two week intervals. The largest effect was on pentosan content, which decreased 17.4%. There were small but significant decreases in Kappa number and tensile indices, and increases in brightness, viscosity and zero-span values.

The values of the above properties for the pulp or handsheets made from the pulp were not very different at 15 U/ml and 45 U/ml enzyme, 2 h and 4 h treatment and use of 0.01 M potassium phosphate and 0.1 M Na2S0 (Tables 20 and 21) .

To analyse the significance of these data, the values of pulp and paper properties are presented in Tables 22 and 23 as percent changes compared to the appropriate controls . The conclusions are as follows: 1. Pentosans are partially removed (10.4 - 22.2%); higher levels of enzyme remove more pentosans.

2. There is a small but significant increase in viscosity; this can be explained by the removal of pentosans (about 1% of the dry weight of K14). There is no evidence for cellulose degradation, even at the highest concentration of enzyme at 4 h at 65°C, pH 9.

3. The Kappa number decreased in all eight conditions examined (range: 5.5 - 9.1%) . Since the values did not decrease further at higher enzyme concentrations (whereas pentosans were), the higher levels of enzyme appeared to remove pentosans that were not linked to lignin.

4. In all eight conditions tested, the handsheets showed small increases in brightness and zero-span values and decreases in tensile indices.

Table 19. Xylanase treatment of K14 pulp*

Parameter Control Enzyme Average treated Change (%)

Kappa No. 17.3, 16.8 16.0, 15.5 - 7.6, (± 0.1)

Viscosity 1066, 1045 1072 1059 + 0.8, (± 0.1) (dm ³ /Kg)

Pentosan (%) 9.1, 9.3 7.7, 7.5 -17.4, (± 1.8)

Handsheets (75 g/m ² )

Brightness 32.5, 32.8 33.4, 33.8 + 2.9, (± 0.1)

(% ISO)

Tensile Index 20.9, 20.4 19.0, 19.3 - 7.2, (± 1.8)

(Nm/g)

Zero-span (Wet), 95.2, 97.8 101.5,104.3 + 6.3, (± 0.3) (Nm/g)

* Conditions: 15 U/ml enzyme, pH 9.0, 2h, 0.1 M Na2S0 ₄

Table 20. Xylanase treatment of K14 pulp in 0.1 M Na ₂ S0 ₄ *

0/ml 15 U/ml 45 U/ml 0/ml 15 U/ml 45 U/ml Parameter 2h 2h 2h 4h 4h 4h

Kappa 17.1 15.8 16.0 17.0 15.4 15.8 Viscosity 1055 1066 1072 1061 1075 1075

Pentosan 9.2 7.6 7.3 9.0 7.5 7.0

Brightness 32.6 33.6 34.1 32.6 34.1 34.4 Tensile 20.7 19.2 20.7 21.2 19.6 19.8 Zero-span 97 103 104 100 104 103

Average of two experiments; conditions as in Table 19.

Table 21. Xylanase treatment of K14 pulp in 0.01 phosphate buffer*

0/ml 15 U/ml 45 U/ml 0/ml 15 U/ml 45 U/ml Parameter 2h 2h 2h 4h 4h 4h

* Average of two experiments

Table 22. Xylanase treatment of K14 pulp'

Change (%)

3.7 7.1 3.0

* 0.1 M N 2S04; Data from Table 20

Table 23. Xylanase treatment of K14 pulp*

Change (%)

15 U/ml 15 U/ml 45 U/ml 45 U/ml Parameter 2h 4h 2h 4h

0.01 M phosphate; Data from Table 21

Test of bleachability

The pulp K14 with kappa number 18,5 was used to test the effect of enzyme treatment on bleachability. The pulp K14 was exposed to enzyme treatment at a concentration of 15 units/ml of xylanase produced from the strain T6. After the enzymatic treatment the pulp was bleached with the bleaching sequence used in the Korsnas mill: Chlorine dioxide (D0) - Alkali extraction reinforced with oxygen (EO) - Chlorine dioxide (Dl) - Chlorine dioxide (D2) .

The enzymatic treatment reduced the lignin content of the pulp to a kappa number of 15,9. A pulp treated in the same way as the enzyme treated pulp, i.e. at 65°C and pH 9 in 100 mM Na2S04~buffer, but without addition of enzymes, was delignified to a kappa number of 17,2. The net effect of the enzymatic treatment was a delignification from kappa number 17,2 to 15,9, i.e. a delignification of 8%.

The pulp treated with enzymes in the sulfate buffer system consumed 47 kg chlorine dioxide (active chlorine) /ton of pulp when bleaching to an ISO-brightness of 83% with the bleaching sequence D0(E0)D1D2. The corresponding consumption for the pulp treated merely with buffer solution was 59 kg chlorine dioxide (a CD/ton of pulp, i.e. the enzyme treated pulp consumed 20% less chlorine dioxide.

International Application No: PCT/