Alkalitolerant xylanases

Technical field

The present invention relates to novel microorganisms and to novel enzymes. More specifically the enzymes are alkalitolerant xylanases. These xylanases are obtainable from gram-positive, alkalitolerant microorganisms. The xylanases are applicable under conditions used in the paper and pulp industry i.e. pH = 9 and T = 70°C.

Background of the invention

Xylan is a component of plant hemicellulose. Xylan consists of 1 ,4- glycosidically linked ?-D-xylose. Usually xylans have side chains containing xylose and other pentoses, hexoses and uronic acids.

In the paper production process the bleaching of pulp is an important step. Schematically the steps used in the pulp treatment in paper and pulp industry is performed as follows:

Pulp is treated at pH 10-1 2 at 80°C to remove most of the lignin in the so- called oxygen delignifying step. The remaining pulp contains 2-5% of lignin. This lignin gives the pulp the brown color. Subsequently, the pulp is bleached in a multistage bleaching process. In this bleaching chemicals such as chlorine, chlorine dioxide, hydrogenperoxide and/or ozone are used to obtain a pulp for high quality paper.

Chlorine and chlorine-containing chemicals are often used to remove lignin, which is responsible for the brownish color of the pulp. U se of the indicated chemicals leads to the formation of dioxin and other chlorinated organic compounds. These compounds form a threat to the environment and

there is a growing tendency to omit the use of chemicals giving rise to similar waste products.

This has prompted a tendency to develop chlorine-free processes; total chlorine free (TCF) and elementary chlorine-free (ECF). In these processes hydrogen peroxide or ozone is used for bleaching.

It has been found that the introduction of an enzymatic step in the paper and pulp preparation process has several advantages.

Xylanases have been found to be very useful in the paper and pulp processing. Xylanases have been reported to increase the extractability of lignins from the pulp. Xylanases are mostly used after the oxygen delignifying step.

Xylanases cleave the hemicellulose chain linking the lignin to the cellulose chain. After xylanase treatment the lignin is more easily removed in the subsequent steps.

Therefore the use of xylanases leads to a reduction of the consumption of active chlorine in prebleaching of 25-30%. This reduction of chlorine does not afflict the quality parameters of the resulting paper (Viikari et al. 1 986. Proc. of the third Int. Conf. Biotechnology in Pulp and Paper Ind., Stockholm, p.67- 69 and Bajpai and Bajpai. 1 992. Process Biochemistry. 2_Z : 31 9-325) .

The xylanase treatment also reduces the need for other chemicals in the bleaching process.

The use of xylanases from fungal sources in bleaching of kraft pulp has been reported. The pH and temperature optima of these enzymes are : pH = 3-5 and T = 30-50°C. These values are not ideal for the use in the bleaching process where the prevailing conditions are pH > 9 and temperature > 70°C.

Xylanases from bacterial origin, with higher pH and/or temperature optima have also been reported for use in the bleaching process. Some of these are the following:

Bacillus pumilus (pH = 7-9, T = 40°C, Nissen et al., 1 992. Progress in Biotechnology ] ^~ _ : 325-337), Dictvoqlomus thermophilum (pH = 6-8, T = 70°C, European patent application EP 0 51 1 933), B.stearothermophilus T-6 (pH = 9.0, T = 65 °C, Shoham, Y. et al. ( 1 992) Biodegradation 3, 207-1 8),

B.stearothermophilus (pH = 9, T = 50°C, WO 91 /1 8976) and

Thermoanaerobacter ethanolicus (68°C, Deblois and Wiegel.1 992. Progress in Biotechnology 7 : 487-490).

Even though most of the above cited xylanases show activity at pH > 9 and temperature > 70°C, their effectiveness under industrial application conditions (i.e. during the bleaching of pulp), in terms of e.g. increased brightness of the pulp is only limited and can vary significantly (see e.g. WO 91 /1 8976, highest increase in pulp brightness at pH 9 and 50°C is only 0.5 % ISO brightness).

Summary of the invention

The present invention relates to xylanases having considerable activity at pH 9.0 and at a temperature of 70°C, and which is characterized in that the xylanase is obtainable from a microorganism of which the 1 6S ribosomal DNA sequence shares more than 92 % identity with the 1 6S ribosomal DNA sequence of strain DSM 8721 as listed in SEQ ID NO 20. The present invention also relates to xylanases having considerable activity at pH 9.0 and at a temperature of 70°C, and characterized in that the xylanase is obtainable from a microorganism selected from the group consisting of the strains deposited under the following deposition numbers: CBS 666.93, 667.93, 669.93, and 673.93. The present invention further relates to xylanases having considerable activity at pH 9.0 and a temperature of 70°C further characterized in that the xylanase produces an increase in % ISO brightness of soft-wood pulp over non-enzymatically treated pulp of at least 1 .0, preferably an increase in % ISO brightness of soft-wood pulp between 1 .5 and 5.0, in an ECF pulp bleaching process wherein the enzyme treatment of the pulp is carried out at a pH of 9.0 at a temperature of 65°C.

The present invention also relates to xylanases having considerable activity at pH 9.0 and a temperature of 70°C further characterized in that the xylanase produces an increase in % ISO brightness of soft-wood pulp over non-enzymatically treated pulp of at least 1 .0, preferably an increase in % ISO brightness of hard-wood pulp between 1 .2 and 3.0, in an ECF pulp bleaching process wherein the enzyme treatment of the pulp is carried out at a pH of 9.0 at a temperature of 65 °C.

Detailed description of the invention

The present invention relates to microorganisms which have been isolated from soil and water samples collected in the environment of alkaline soda lakes in Kenya, East-Africa. These microorganisms have been characterized as being alkaliphilic, Gram-positive and belonging to the genus

Bacillus (see below).

The microorganisms have subsequently been screened using a xylan- agar diffusion assay. Strains which showed a clearing zone in this test were isolated as potential xylanase producing strains.

The strains were grown at pH 10, and T = 45 °C. After centrifugation the culture broth was tested for xylanase activity in an assay at pH = 9 and T = 80°C (Example 2) . Eight different strains were found to produce xylanase activity under the indicated conditions. These microorganisms have been deposited at the Centraal Bureau voor de Schimmelcultures in Baarn, the Netherlands under

deposition number CBS 666.93, 667.93, 668.93, 669.93, 670.93, 671 .93, 672.93, 673.93.

Most of these strains have been send to the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) for an independent identification using comparisons of 1 6S ribosomal DNA sequences as described by Nielsen et al. (1 994, FEMS Microbiol. Lett. 1 17. 61 -65). On the basis of this sequence comparison the eight strains can be assigned to the genus Bacillus and are most related to B.alcalophilus (DSM 485 ^τ ) . The sequence comparison further shows that the eight strains fall into two groups. The first group is very similar or almost identical to DSM 8721 and comprises strains 1 -1 6-2, 1 -25-2, and 1 -43-3 (CBS 670.93, 671 .93, 672,93, respectively). The second group is most related to DSM 871 8 and comprises strains 2-47-1 , 2-M-1 , 1 -47-3 and 2-26-2 (CBS 666.93, 667.93, 669.93 and 673.93), respectively. The classification of the deposited strains into these two groups is confirmed by xylanase zymograms.

Surprisingly, we have found that the xylanases obtainable from the first group of strains, i.e. the strains most related to DSM 8721 (comprising 1 -1 6-2, 1 -25-2, and 1 -43-3) show a superb performance in the bleaching of pulp. This performance is exemplified by the increased brightness of both soft-wood and hard-wood pulp when treated with the enzymes of the present invention and is most pronounced on softwood pulp. In this respect, the performance of the xylanases obtainable from most of the strains in the second group, i.e. the group related to DSM 871 8, is much less, although the xylanases obtainable from strain 1 .47.3. shows the best performance on hard-wood pulp as compared to the other strains. The increase in brightness obtained with the enzymes of the present invention is at least 1 .0, expressed as Δ Final ISO Brightness over the non-enzymatically treated control pulp. Preferably the brightness increase in the case of soft-wood pulp is between 1 .5 and 5.0, and in the case of hard-wood pulp between 1 .2 and 3.0.

The present invention discloses enzymes having xylanase activity and having a considerable xylanase activity at pH 9 and at a temperature of about

70°C. Said enzymes are obtainable from the deposited strains. Said enzymes are also obtainable from mutants and variants of the deposited strains.

With the expression 'considerable activity' is meant that the enzymes of the present invention have at pH = 9, 40% of the activity they possess at pH = 7, preferably this is 60%, more preferably about 80%. In a most preferred embodiment of the present invention the activity of the xylanase is higher at pH = 9 than at pH = 7.

The present invention also discloses a process for the production of subject xylanases, which can be developed using genetic engineering. As a first step the genes encoding the xylanases of the present invention can be cloned using Λ-phage (expression-) vectors and E.coli host cells. Alternatively, PCR cloning using consensus primers designed on conserved domains may be used. On the basis of homology comparisons of numerous xylanases a distinction in different classes has been proposed (Gilkes et al., 1 991 , Microbiol. Rev. 5_5_, 303-31 5). For each class specific conserved domains have been identified. Class F and class G xylanases can be identified based on this determination. DNA-fragments in between two conserved domains can be cloned using PCR. Full length clones can be obtained by inverse PCR or by hybridization cloning of gene libraries. Expression of some of the genes encoding the xylanases of the present invention in E.coli is shown to give an active protein. Said proteins are active at pH 9 at a temperature of 70°C.

After a first cloning step in E.coli, a xylanase gene can be transferred to a more preferred industrial expression host such as Bacillus or Streptomvces species, a filamentous fungus such as Asperqillus. or a yeast. High level expression and secretion obtainable in these host organisms allows accumulation of the xylanases of the invention in the fermentation medium from which they can subsequently be recovered.

The present invention further relates to a process for the preparation of xylanases obtainable from the deposited strains and having considerable activity at a pH of 9 at a temperature of 70°C. The process comprises cultivation of the deposited microorganisms or recombinant host

microorganisms expressing genes encoding the xylanases of the present invention in a suitable medium, followed by recovery of the xylanases.

The enzymes of the present invention have been shown to have a considerable activity on oat spelt xylan and on birchwood xylan. The enzymes of the present invention have further been tested for their bleaching activities. The enzyme preparations, xylanases, are capable of delignifying wood pulp at a temperature of at least 80°C and a pH of at least 9. The expression "wood pulp" is to be interpreted broadly and is intended to comprise all kinds of lignocellulosic materials. The enzymes of the present invention can be used immediately after the oxygen delignifying step in the paper and pulp preparation process described above. Preferably, the enzymes are used before the oxygen delignifying step. In this step the lignin concentration is much higher therefore the effect of the application of the xylanase is much larger. The enzymes of the present invention have been tested for their activity on both hardwood and softwood pulps. Apart from the kappa reduction, also the increase in brightness has been determined on two types of pulp, both soft-wood and hard-wood kraft pulp in ECF bleaching experiments. It follows that the increased brightness produced by the xylanases of the present invention would also allow to reduce the amount of bleaching chemicals while achieving the same brightness as obtained without the use of enzymes.

Furthermore, the inventions relates to the applications of the enzyme preparations of the invention, particularly to a process in which wood pulp is treated with said enzyme preparations according to the invention, and a wood pulp and a fluff pulp treated with the enzyme preparations according to the invention.

The invention further relates to paper, board and fluff pulp made from a wood pulp treated with the enzyme preparations according to the invention.

The enzyme preparations of the present invention have further been shown to have a low cellulase activity.

EXAMPLE 1

Isolation of alkali- and thermotolerant xylanases

Samples

Soil and water samples were collected in the environments of alkaline soda lakes in Kenya, East Africa.

Screening for xylanase producing microorganisms Two methods were applied for the isolation of xylanase-producing microorganisms: i) The soil and water samples were suspended in 0.85% saline solution and directly used in the xylan-agar diffusion assay, ii) The soil and water samples were incubated in a xylan containing liquid minimal medium or GAM-medium for 1 to 3 days at 45, 55 and 70°C respectively. Cultures that showed bacterial growth were analyzed for xylanase activity using the xylan-agar diffusion assay.

Media The minimal medium (pH 9.7) used in the xylan-agar diffusion assay and the enrichment procedure, consisted of KNO ₃ 1 %, Yeast extract (Difco) 0.1 %, KH ₂ PO4 0.1 %, MgSO ₄ .7H ₂ O 0.02%, Na ₂ CO ₃ 1 %, NaCI 4% and a mixture (0.05% each) of four commercially available xylans [Xylan from oat spelts (Sigma X-0376), Xylan from birchwood (Sigma X-0502), Xylan from oat spelts (Serva 38500), Xylan from larchwood (ICN Biochemicals 103298)]. For solidification 1 .5% agar is added.

The complex medium (GAM) used for enzyme production consisted of Peptone (Difco) 0.5%, Yeast extract (Difco) 0.5%, Glucose. H ₂ O 1 %, KH ₂ PO ₄ 0.1 %, MgSO ₄ .7H ₂ O 0.02%, Na ₂ CO ₃ 1 %, NaCI 4%. The pH is adjusted to 9.5 with 4M HCI after which 1 % Xylan (Serva) is added.

Xylan-agar diffusion assay

Cell suspensions in 0.85% saline solution were plated on Xylan containing minimal medium. After incubation for 1 to 3 days at 45 and 55 ° C respectively, the strains that showed a clearing zone around the colony were isolated as potential xylanase producing microorganisms.

Isolation of alkali- and thermotolerant xylanase producing strains Strains that showed clearing zones in the agar diffusion assay were fermented in 25 ml GAM-medium in 100 ml shake flasks in an Incubator Shaker (New Brunswick Scientific, Edison, NJ, USA), at 250 r.p.m. at 45°C for 72 hours. Xylanase activity was determined in the culture broth at pH 9 and 80°C (Example 2).

Isolation of crude enzyme preparations

Shake flask fermentations were carried out in 2 I erlenmeyer flasks containing 500 ml GAM-medium. The flasks were incubated in an orbital incubator at 250 r.p.m. at 45°C for 48 to 96 hours. The cells were separated from the culture liquid by centrifugation (8000 rpm). The cell-free culture liquid was concentrated by ultrafiltration, using an Amicon Stirred Cell Model 8400 with YM5 filter.

EXAMPLE 2 Characterization of alkali- and thermotolerant xylanases

Analytical methods

Assays for xylanase activity are performed using modified procedures of the

Sumner assay (J. Biol.Chem. 1 921 . 47 5-9).

Procedure 1

Xylanase activity on Oat Spelts xylan

A test tube is filled with 200 μ\ 4% Oat spelts xylan suspension, 600 μ\ aliquots of cell-free culture broth (Example 1 ) diluted in the appropiate buffer. The test tube is incubated in a waterbath for 1 5 minutes. After the incubation, 7.2 ml DNS (Dinitrosalicylic acid) reagent is added. The mixture is heated in a waterbath at 100°C for 10 minutes. After heating the mixture the test tube is cooled on ice. The absorbance is measured at 575 nm. To eliminate the background absorbance of the enzyme samples a control experiment was executed as follows: a tube with substrate incubated under the same conditions as the test tube. After incubation 7.2 ml DNS and the enzyme preparation is added (in this order). One unit of xylanase (xU) activity is defined as the amount of enzyme producing 1 //mol of xylose from xylan equivalent determined as reducing sugar per minute.

Actual measuring conditions were pH 7, 9 and 70 and 80 °C. The buffers were Phosphate pH 7 and Borate/KCI pH 9. The results are shown in table 1 as relative activity.

Table 1 : Relative xylanase activities on Oat Spelts xylan

RELATIVE XYLANASE ACTIVITY ON OAT SPELTS XYLAN

strain 70 °C

Nr number pH 7 pH 9

1 1 -47-3 ^■ 100 82

2 2-47-1 100 51

3 2-m-1 100 67

4 1 -1 6-2 100 55

5 1 -25-2 100 40

6 2-16-1 100 63

7 1 -43-3 100 48

8 2-26-2 100 59

The strains -indicated in Tables 1 , 2 and 3 as 1 to 8 have been deposited under the following deposition numbers;

2-47-1 = CBS 666.93, 2-m-1 = CBS 667.93

2-16-1 = CBS 668.93, 1-47-3 = CBS 669.93

1-16-2 = CBS 670.93, 1-25-2 = CBS 671.93

1-43-3 = CBS 672.93, 2-26-2 = CBS 673.93

Procedure 2

Xylanase activity on Birchwood xylan

The same method as described in procedure 1 is used. Instead of a 4% Oat Spelts xylan suspension a 4% Birchwood xylan suspension is used. The test conditions were: pH 7 and 9 and 70 and 80 ° C, respectively. The results are shown in table 2.

Table 2: Relative xylanase activities on Birchwood xylan

Nr strain pH 7 pH 9 pH 7 pH 9 70°C 70°C 80°C 80°C

1 1-47-3 100 72 100 10

2 2-47-1 100 80 100 9

3 2-M-1 100 90 100 8

4 1-16-2 100 40 100 42

5 1-25-2 100 24 100 65

6 2-16-1 100 74 100 11

7 1-43-3 100 23 100 55

8 2-26-2 100 69 100 18

EXAMPLE 3

Delionification assay at 70°C and 80°C Kappa assay The kappa assay's were performed according to the TAPPI T236 protocol with some modifications. The enzyme solution was added at a dose of 1 0 xU/g pulp (based on Oat spelts xylan for the pulp nb 1 and based Birchwood

xylan for pulps 2 and 3) (dry weight) and incubated for 2 hours at pH 9, 70 and 80 °C. The control, was pulp incubated for the same period under the same conditions without enzyme addition. Tree different pulps were used:

1 ] Kraft softwood pulp 2] Kraft softwood pulp after oxygen delignification

3] Kraft hardwood pulp after oxygen delignification

Pulp properties (nb 2 and 3):

Hardwood Softwood Birch 80% spruce, 20% pine

Brightness, % ISO 50.8 35.8

Kappa number 1 1 .0 1 6.7

Viscosity, dm ³ /kg 979 1003

Calcium, ppm 1 900 2600 Copper, ppm 0.3 0.6

Iron, ppm 5.1 1 1

Magnesium, ppm 210 270

Manganese, ppm 25 70

The difference between the kappanumber with enzyme addition and the kappanumber without enzyme addition is called the kappa reduction and is a value for delignification. The kappa reductions are shown in table 3A.

Table 3A: Kappa reductions at pH 9 and 70 °C and 80 °C

pH 9 70°C pH 9 70°C pH 9 80°C

Nr Strain Softwood kraft Softwood 02 Hardwood Number pulp (nb 1 ) delig (nb 2) 02 delig (nb 3) kappa red kappa red kappa red

1 1 -47-3 1 .7 0.3

2 2-47-1 2

3 2-M-1 2

4 1 -16-2 1 .8

5 1 -25-2 1 .6 1.1 0.5

6 2-16-1 0.4

7 1 -43-3 1 .1 1.2 1

8 2-26-2 0.5

blanks were not determined.

Delignification assay at 60°C Kappa assay

The kappa assay's were performed according to the Tappi T236 protocol with some modifications. The enzyme solution was added at a dose of 10 xU/g pulp (based on birchwood xylan) (dry weight) and incubated for 2 hours at pH 9 , 60°C. The control, was pulp incubated for the same period under the same conditions without enzyme addition. Two different pulps were used:

- Kraft hardwood pulp after oxygen delignification (nb 2).

- Kraft softwood pulp after oxygen delignification (nb 4).

Pulp properties (nb 2 and 4)

Hardwood Softwood

Birch 80 %

Brightness, % ISO 50.8 40.0

Kappa number 1 1.0 10.1

Viscosity, drrrVkg 979 940

Calcium, ppm 1900 1800

Copper, ppm 0.3 0.3

Iron, ppm 5.1 5.2

Magnesium, ppm 210 250

Manganese, ppm 25 35

The difference between the kappanumber with enzyme addition and the kappanumber without enzyme addition is called the kappa reduction and value for delignification. The kappa reductions are shown in table 3B.

Table 3B Kappa reductions at pH 9 and 60°C.

Nr Strain Softwood Hardwood number 02 delig 02 delig kappa red kappa red.

1 1 -47-3 0.0

2 2-47-1 0.9

3 2-M-1 0.5 0.6

4 1 -16-2 1.1 0.7

5 1 -25-2 0.9 0.2

6 2-16-1 0.7 0.2

7 1 -43-3 1.1

8 2-26-2 0.7

EXAMPLE 4

Cellulase activity

Assay's for cellulase activity were performed using a modified procedure of the PAHBAH (parahydroxybenzoicacid hydrazide) assay (Anal. Biochem. 1 972. 47 : 273-279)

0.9 ml 0.5% CMC (carboxymethylcellulose) is incubated with 0.1 ml diluted enzyme preparation and incubated for 60 minutes at pH 9 and 70 °C. after the incubation 3 ml PAHBAH reagent (10 ml 5% PAHBAH in 0.5M HCI was mixed with 40 ml 0.5M NaOH = PAHBAH reagent) is added and the reaction mixture is heated for 5 minutes at 100 °C. After cooling on ice the absorbance is measured at 420 nm. To eliminate the background absorbance of the enzyme samples a control experiment was executed as follows: the CMC was incubated for 30 minutes at pH 9, 70 °C and the enzyme solution is added after adding of the PAHBAH reagent. One cellulase unit (cU) is defined as the quantity of enzyme necessary to produce one /Mol glucose per minute (using CMC as substrate) and is related to the xylanase activity. All strains tested showed a cellulase activity less than 10 mU CMCase per unit of xylanase.

EXAMPLE 5

Cloning of xylanase genes and fragments thereof

Chromosomal DNA was isolated from strains mentioned in Example 2 according to methods described (Maniatis et al, Cold Spring Harbor Laboratory Press, 1 989). Genomic libraries were prepared for each of these selected strains using the ZAP Express ^® cloning system available from Stratagene. The host/vector system was used according to the instructions of the supplier (Catalog # 23921 2, June 30, 1 993). For construction either partial Sau3A digest ligated into the BamHI site or randomly sheared DNA supplied with EcoR1 linkers ligated into the EcoR1 site were used.

Recombinant phages were transformed into plasmid vectors as recommended by the supplier. These plasmid vectors were tested for expression of xylanase using RBB xylan indicator plates.

Positive colonies were isolated and tested for production of xylanase using the following medium: Production medium: 4 x L B C :

20 g yeast extract

40 g Bacto trypton 10 g NaCI

4 g casaminoacids fill up to 1 liter with demineralized water ass 0.25 ml antifoam and sterilize 20' at 1 20 °C. Colonies are grown during 24 hr at 30 °C under vigourous shaking. The enzyme was isolated using a heat shock method ( 10' at 65 °C) to lyse the cells. Xylanase activity was measured as described above. The results of the tests of individual clones are summarized in Table 4.

Table 4. Xylanase activities of cloned xylanases expressed in E.coli.

Strain Clone Production level (U/ml)

1-47-3 KEX101 0.6

KEX106 23.7

KEX107 17

2-M-1 KEX202 <0.2

KEX203 4.0

1-43-3 KEX301 40

KEX303 ^• 1.1

KEX304 1.8

2-26-2 KEX401 12

KEX402 12

KEX403 43

KEX404 33

KEX405 <0.2

KEX406 17

KEX407 110

KEX408 0.8

KEX409 36

It can be concluded that all clones produce xylanase. Although the variability in production level might be due to cloning of partial gene fragments, it most

probably can be regarded as a reflection of the diversity of xylanase genes present within the inserts.

EXAMPLE 6

5

Characterization of selected xylanase encoding inserts

The DNA insert of xylanase producing clones can be characterized by DNA sequencing. The insert of KEX106 was analysed and a gene encoding the alkalitolerant xylanase was identified. The DNA sequence of the gene is o shown in SEQ ID NO 1 .

A comparison of the amino acid sequence of the encoding protein (SEQ ID NO 2) revealed an homology to xylanase protein sequences, i.e. 93 % [Hamamoto et al., 1 987, Agric. Biol Chem., 51 , 953-955]. The amino acid sequence of xylanases of the present invention can therefore s share an identity with the amino acid sequence of SEQ ID NO 2 of higher than 93 %, preferably the identity is at least 95 %, more preferably the identity is at least 98 %, and most preferably more than 99 %.

0

EXAMPLE 7

Identification and cloning of internal fragments of genes encoding 5 alkalitolerant xylanases

As an alternative method to the screening of gene libraries we have worked out a method based on PCR cloning. On the basis of a comparison of numerous xylanase sequences we have designed consensus oligonucleotide primers encompassing conserved sequence boxes. Two types of primers have o been designed. One set of primers is for the F-type of xylanase and one set is for the G-type of xylanases. The following consensus primers have been constructed:

FA: 5' CAC ACT/G CTT/G GTT/G TGG CA 3': forward primer, consensus box 1 (SEQ ID NO 3)

FB: 5' CAT ACT/G TTT/G GTT TGG CA 3': forward primer, consensus box 1 (SEQ ID NO 4) FR: 5' TC/AG TTT/G ACC/A ACG/A TCC CA 3': reverse primer, consensus box 2 (SEQ ID NO 5)

Primers FA and FB bind to the same consensus box, but due to slight differences in the nucleotide sequence they exhibit complementary specificity.

PCR conditions were as follows: [94 °C, 1 min], [50 °C, 1 min] and [72 °C, 1 min] for 30 cycles. Fragments originating from amplification with F-type primers were purified on agarose gel and subcloned. Subsequently the DNA sequence was determined.

G _AF : 5' GAA/G TAT/C TAT/C ATT/C/A GTN GA : forward primer, consensus box 1 (SEQ ID NO 6)

G _BF : 5' GAA/G TAT/C TAT/C GTN GTN GA : forward primer, consensus box 1 (SEQ ID NO 8)

G _AR : 5' CG/TN ACN GAC CAA/G TA : reverse primer consensus box 2 (SEQ ID NO 7)

G _BR : 5' CG/TN ACA/G CTC CAA/G TA : reverse primer consensus box 2 (SEQ ID NO 9) G _CR : 5' CCR CTR CTK TGR TAN CCY TC : reverse primer consensus box 3 (SEQ ID NO 10)

PCR conditions were as follows: [94 °C, 1 min], [40 °C, 1 min] and [72 °C, 1 min] for 30 cycles. The first PCR with G-primers was performed with primers constructed on box 1 and box 3. The resulting mixture of fragments of different sizes were subsequently purified from agarose gel (250-340 bp) and subjected to a

second round of PCR, now using primers from box 1 and box 2. Unique fragments were amplified and subcloned. The blunt-end repair of the PCR fragments was performed in the PCR mix by adding 0.5 rtiM ATP (Boehringer Mannheim), _. 10 u T4 DNA kinase (BRL), 1 u T4 DNA polymerase (BRL) and incubation at 37 °C for 1 hour. The mixture was purified using the PCR extraction kit from Qiagen. The fragment was ligated into the pUC18xSmal (CIAP) vector obtained from Appligene according to Maniatis. E. coli HB101 laqlq was transformed with the ligation mixture using electroporation. The DNA sequence of a number of individual clones was determined.

From the analysis it has become apparent that the selected strains harbor several different xylanase genes, some of which may be cloned by the F-type consensus primers and other which may be cloned by the G-type of primers. As an example several different internal xylanase fragments originating from strains 1 -43-3, 1 -47-3, 1 -M-1 , 2-26-2 (all F-type) and 1-43-3 and 1 -25-2 (all G-type) are depicted in the sequence listings (see Table 5). Table 5.

Strain Consensus primers used Sequence listing

1 -43-3 F-type SEQ ID NO 1 1

1 -47-3 F-type SEQ ID NO 12

2-26-2 F-type SEQ ID NO 13

2-M-1 F-type SEQ ID NO 14

1 -25-2 G 1 -type SEQ ID NO 1 5

1 -43-3 G 1 -type SEQ ID NO 16

1 -43-3 G2-type SEQ ID NO 17

The cloned internal fragment are subsequently used as a specific probe to isolated the cloned gene fragments from the lambdaZAP gene library using

stringent hybridisation conditions. All cloned genes can be isolated using this method.

The method is especially advantageous for those genes that do not express well from their native gene regulatory signals in E.coli, since these genes would escape from detection in the method described in example 5. Using subcloning methods and DNA sequence analysis the complete genes encoding the various alkalitolerant xylanases can be isolated and equipped with expression signals for production in E.coli.

EXAMPLE 8 Further characterization of xylanase clones

With the aid of both the consensus primers and specific primers a further characterization of the clones mentioned in example 5 was performed. It became apparent that there is a clustering of xylanase genes on several of the cloned inserts. On the basis of this inventory single genes were subcloned in expression vectors for both E. coli and Bacillus subtilis. Expression of monocomponent xylanases was obtained upon transformation into E.coli and Bacillus respectively. The Bacillus expression system was based on the PlugBug ^® technology [refl ]

EXAMPLE 9

Characterization of selected G-type xylanase encoding insert

The insert of clone KEX301 was analysed and an open reading frame encoding a G-type xylanase was identified. The sequence of this ORF is given in SEQ ID NO 1 8 and the derived amino acid sequence for the xylanase in SEQ ID NO 1 9. A search for homologous genes within the EMBL database (release 39, version 2) showed that the sequence of G 1 xylanase is unique. No DNA homology of more than 68 % was detected. Also the protein sequence was compared to the database sequences. The closest homology (72 %) was found with a xynY xylanase sequence (Yu et al. 1 993, J. Microbiol. Biotechnol. 3, 139-145).

The amino acid sequence of xylanases of the present invention can therefore share an identity with the amino acid sequence of SEQ ID NO 1 9 of at least 72 %, preferably the identity is at least 80 %, more preferably the identity is at least 90 %, still more preferably the identity is at least 95 %, and most preferably more than 99 %.

refl : Quax, W.J. et al, 1 993, in Industrial Microorganisms: Basic and Applied Molecular Genetics, ASM, Washington D.C., p143.

EXAMPLE 10

Pulp bleaching experiments with supernatants from deposited strains

All experiments were elemental chlorine free (ECF) bleaching with a XwDED bleach sequence. Enzyme treatments on pulp were for two ours at pH 9.0 and 65 °C. To ensure proper temperature throughout the experiment the pulp has been heated in the microwave to 65 °C before adding enzyme. Experiments were run at a pulp consistency of 10 %, which was adjusted by adding pH adjusted tap water. A summary of the ECF bleaching data for xylanase containing culture supernatants of the deposited strains is shown in Table 6.

Table 6. Brightness increase expresses as Δ Final ISO Brightness over the non-enzymatically treated control for the supernatants of the deposited strains and for the reference Cartazyme GT 630 (Sandoz).

Strain/Enzyme Softwood Hardwood

1 .43.3 3.55 1 .45

1 .47.3 1 .45 1 .99

2.47.1 1 .8 1 .55

24 -

1.25.2 3.15 1.45

2.M.1 0 0.4 -

1.16.2 1.55 0.5

2.26.2 0 0.9

GT 630 0 0

Before each bleaching experiment every enzyme containing supernatant was assayed for xylanase activity at pH 9.0, 65 °C. In the bleaching experiments 2 o xylanase units per gram of oven dried pulp were used for each supernatant. Supernatant activities were determined the same day the enzyme bleaching stage was run.

EXAMPLE 1 1 s Pulp bleaching with cloned xylanase genes expressed in E.coli

Xylanases obtained from three of the E.coli clones expressing cloned xylanase genes obtained from the deposited strains were tested in pulp bleaching experiments as described in Example 10. The E.coli clones were cultured as described in Example 5. Recombinant enzyme was isolated from the E.coli 0 bacteria in one of three ways:

1 . Whole Ivsate

In this case, the whole cell culture (cells + spent growth medium) was harvested. Cells were disrupted by sonication followed by heat at 65 °C for 10 minutes. The lysates were then clarified by 5 centrifugation.

2. Cell pellet

Cells were separated from spent medium by centrifugation. Cell pellets were resuspended at 10 ml/g wet weight in 50 mM Tris/HCI, pH 7.0 buffer. The cell suspension was then sonicated and heated as o described for "whole lysate".

25

3 Culture supernatant

Spent growth medium was separated from whole cells by centrifugation. The clarified medium was then diafiltered (tangential flow, 10,000 MWCO membrane) to reduce the total volume and exchange the liquid 50 mM Tris/HCI, pH 7.0 buffer.

The results of the bleaching experiments are shown in Table 7.

Table 7. Pulp bleaching with cloned xylanase genes expressed in E.coli

Parental Clone # source of Δ Final ISO Brightness ^* strain enzyme

Soft-wood Hard-wood

1 -47-3 KEX 106 whole lysate decrease decrease

1 -43-3 KEX 301 whole lysate 3.2 n.d. ^{* *}

cell pellet 3.4 0.7

cell pellet n.d. 1 .0

culture sup. 3.6 1 .5

1 -43-3 KEX 303 cell pellet 3.2 1 .6

culture sup. n.d. 1 .0 over non-enzymatically treated control n.d. = not determined

EXAMPLE 12

Identification of the deposited strains

Most of these strains have been send to the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) for an independent identification using comparisons of 16S ribosomal DNA sequences as described by Nielsen et al. (1 994, FEMS Microbiol. Lett. 1 17. 61 -65). The results of this identification are provided in Table 8. On the basis of this sequence comparison the eight strains can be assigned to the genus Bacillus and among the known Bacilli, they are most related to B.alcalophilus (DSM 485 ^τ ). The sequence comparison further shows that the eight strains fall into two groups. The first group is very similar or almost identical to DSM 8721 and comprises strains 1 -16-2, 1 -25-2, and 1 -43-3 (CBS 670.93, 671 .93, 672,93, respectively). The second group is most related to DSM 8718 and comprises strains 2-47-1 , 2-M-1 , 1 -47-3 and 2-26-2 (CBS 666.93, 667.93, 669.93 and 673.93), respectively. The xylanases of the invention are preferably obtainable from the first group of strains, i.e. the strains most related to DSM 8721 (comprising 1 -1 6- 2, 1 -25-2, and 1 -43-3). The xylanases of the present invention are therefore obtainable from Bacillus strains of which the 1 6S ribosomal DNA sequence shares at least 92 % identity with strain DSM 8721 , preferably the identity is at least 93.3 %, more preferably at least 96.6 %, still more preferably at least 99 %, and in the most preferred embodiment the identity is 100%.

Tabel 8. 16S rDNA sequence similarities of the deposited strains to some alkaliphilic Bacilli

Strain 10 11 12 13 14 15 16 17 18 19 20 21 22 23

1. 1 -9A- K93-509) -

2. 1 -43-3(93-510) 99.2

3. 2-47- 1 (93-511 ) 89.4 88.6 -

4. 2-26-2(93-512) 89.5 88.9 99.6 -

5. 1-25 2(93-513) 100.0 98.6 89.3 89.3 -

6. 1 -47-3(93-514 ) 89.6 89.7 99.9 99.5 89.7 -

7. 1 - 16 ^• 2(93-515 ) 100.0 99.2 89.5 89.5 100.0 89.8 -

8. 2-M- 1 (93-516) 89.6 89.5 99.9 99.8 89.5 99.7 89.7 -

9. B. alcatopliilus 91 .3 90.7 95.8 96.1 91 .6 95.6 91 .4 95.8

10. B. colinii 88.0 87.4 92.4 92.1 87.2 92.0 88.0 92.0 93.4 _ t

11. DSM 8714 89.6 88.6 92.6 92.2 89.3 92.5 89.7 92.4 94.9 91.9 - - 12. DSM 8715 90.4 89.4 94.4 94.3 90.6 94.3 90.5 94.3 96.4 94.0 94.8 -

13. DSM 8716 89.6 88.7 93.3 92.8 89.6 93.0 89.8 92.9 95.0 92.0 96.0 94.8 -

ΓTΪ

14. . DSM 8717 90.3 89.4 93.7 93.4 89.6 93.3 90.4 93.5 95.6 93.1 96.8 94.2 96.1

___ - — 1

15. . DSM 8718 88.8 88.0 98.9 99.3 89.5 99.0 88.9 99.1 96.3 93.7 93.6 96.0 94.0 93.8 - gS 16. . DSM 8719 87.9 87.0 92.8 92.6 87.2 92.5 97.8 92.6 93.0 97.2 90.7 92.8 91.7 92.2 93.5 - i m — 17. DSM 8720 95.6 94.6 92.5 92.4 95.6 92.5 95.6 92.5 93.3 91 .7 90.9 93.7 91.5 91.6 93.0 92.1 - ro 18. DSM 8721 100.0 99.4 90.6 90.2 100.0 90.5 100.0 90.2 93.2 91.1 91 .4 93.3 91.9 91 .7 92.3 91 .0 96.6 -

N_2 19. , DSM 8722 90.0 89.1 92.3 92.1 90.3 92.0 90.1 92.1 94.7 92.1 94.6 94.8 94.4 94.4 94.0 91.3 92.1 92.1 -

20. , DSM 8723 87.5 86.7 93.3 92.8 86.8 92.9 87.5 93.0 93.3 97.6 91.3 93.1 92.6 92.9 93.8 98.4 92.0 90.8 91.4 -

21 . , DSM 8724 91 .3 90.3 95.7 96. 1 91 .7 95.6 91 .4 95.8 99.9 93.3 94.8 96.2 95.0 95.5 96.2 92.9 93.3 93.2 94.6 93.2 -

22. . DSM 8725 89.6 89.2 94.6 94.3 90.0 94.3 89.7 94.6 98.1 93.1 94.4 95.9 94.8 94.8 95.4 92.4 93.0 92.2 94.5 92.8 90.1

23. , B. siibtilis 89.2 88.3 91 .9 91.7 89.9 91 .9 89.2 91.7 92.6 93.9 91 .4 94.1 92.7 91 .5 93.5 93.3 91 .5 91.5 91.6 93.7 92.5 92.9

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Gist-brocades B.V.

(B) STREET: Wateringseweg 1

(C) CITY: Delft (E) COUNTRY: The Netherlands

(F) POSTAL CODE (ZIP) : 2611 XT

(ii) TITLE OF INVENTION: Alkalitolerant Xylanases

(iii) NUMBER OF SEQUENCES: 20

(iv) 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 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1191 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (B) STRAIN: 1-47-3

(C) INDIVIDUAL ISOLATE: CBS669.93

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 1..1191

(D) OTHER INFORMATION: /product= "xylanase"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATG ATT ACA CTT TTT ACA AAG CCT TTT GTT GCT GGA CTA GCG ATC TCT 48 Met lie Thr Leu Phe Thr Lys Pro Phe Val Ala Gly Leu Ala lie Ser 1 5 10 15

TTA TTA GTA GGT AGG GGG CTA GGC AAT GTA GCT GCT GCT CAA GGA GGA 96 Leu Leu Val Gly Arg Gly Leu Gly Asn Val Ala Ala Ala Gin Gly Gly 20 25 30

CCA CCA CAA TCT GGA GTC TTT GGA GAG AAT CAC AAA AGA AAT GAT CAG 144 Pro Pro Gin Ser Gly Val Phe Gly Glu Asn His Lys Arg Asn Asp Gin 35 40 45

CCT TTT GCA TGG CAA GTT GCT TCT CTT TCT GAG CGA TAT CAA GAG CAG 192 Pro Phe Ala Trp Gin Val Ala Ser Leu Ser Glu Arg Tyr Gin Glu Gin 50 55 60

TTT GAT ATT GGA GCT CCG GTT GAG CCC TAT CAA TTA GAA GGA AGA CAA 240 Phe Asp lie Gly Ala Pro Val Glu Pro Tyr Gin Leu Glu Gly Arg Gin 65 70 75 80

GCC CAA ATT TTA AAG CAT CAT TAT AAC AGC CTT GTG GCG GAA AAT GCA 288 Ala Gin lie Leu Lys His His Tyr Asn Ser Leu Val Ala Glu Asn Ala 85 90 95

ATG AAA CCT GTA TCA CTC CAG CCA AGA GAA GGT GAG TGG AAC TGG GAA 336 Met Lys Pro Val Ser Leu Gin Pro Arg Glu Gly Glu Trp Asn Trp Glu 100 105 110

GGC GCT GAC AAA ATT GTG GAG TTT GCC CGC AAA CAT AAC ATG GAG CTT 384 Gly Ala Asp Lys lie Val Glu Phe Ala Arg Lys His Asn Met Glu Leu 115 120 125

CGC TTC CAC ACA CTC GTT TGG CAT AGC CAA GTA CCA GAA TGG TTT TTC 432 Arg Phe His Thr Leu Val Trp His Ser Gin Val Pro Glu Trp Phe Phe 130 135 140

ATC GAT GAA AAT GGC AAT CGG ATG GTT GAT GAA ACC GAT CCA GAA AAA 480 lie Asp Glu Asn Gly Asn Arg Met Val Asp Glu Thr Asp Pro Glu Lys 145 150 155 160

CGT AAA GCG AAT AAA CAA TTG TTA TTG GAG CGA ATG GAA AAC CAT ATT 528 Arg Lys Ala Asn Lys Gin Leu Leu Leu Glu Arg Met Glu Asn His lie 165 170 175

AAA ACG GTT GTT GAA CGT TAT AAA GAT GAT GTG ACT TCA TGG GAT GTG 576 Lys Thr Val Val Glu Arg Tyr Lys Asp Asp Val Thr Ser Trp Asp Val 180 185 190

GTG AAT GAA GTT ATT GAT GAT GGC GGG GGC CTC CGT GAA TCA GAA TGG 624 Val Asn Glu Val lie Asp Asp Gly Gly Gly Leu Arg Glu Ser Glu Trp 195 200 205

TAT CAA ATA ACA GGC ACT GAC TAC ATT AAG GTA GCT TTT GAA ACT GCA 672 Tyr Gin lie Thr Gly Thr Asp Tyr lie Lys Val Ala Phe Glu Thr Ala 210 215 220

AGA AAA TAT GGT GGT GAA GAG GCA AAG CTG TAC ATT AAT GAT TAC AAC 720 Arg Lys Tyr Gly Gly Glu Glu Ala Lys Leu Tyr lie Asn Asp Tyr Asn 225 230 235 240

ACC GAA GTA CCT TCT AAA AGA GAT GAC CTT TAC AAC CTG GTG AAA GAC 768 Thr Glu Val Pro Ser Lys Arg Asp Asp Leu Tyr Asn Leu Val Lys Asp 245 250 255

TTA TTA GAG CAA GGA GTA CCA ATT GAC GGG GTA GGA CAT CAG TCT CAT 816 Leu Leu Glu Gin Gly Val Pro lie Asp Gly Val Gly His Gin Ser His ■ 260 265 270

ATC CAA ATC -GGC TGG CCT TCC ATT GAA GAT ACA AGA GCT TCT TTT GAA 864 lie Gin lie Gly Trp Pro Ser lie Glu Asp Thr Arg Ala Ser Phe Glu 275 280 285

AAG TTT ACG AGT TTA GGA TTA GAC AAC CAA GTA ACT GAA CTA GAC ATG 912 Lys Phe Thr Ser Leu Gly Leu Asp Asn Gin Val Thr Glu Leu Asp Met 290 295 300

AGT CTT TAT GGC TGG CCA CCG ACA GGG GCC TAT ACC TCT TAT GAC GAC 960

Ser Leu Tyr Gly Trp Pro Pro Thr Gly Ala Tyr Thr Ser Tyr Asp Asp 305 310 315 320

ATT CCA GAA GAG CTT TTT CAA GCT CAA GCA GAC CGT TAT GAT CAG TTA 1008 lie Pro Glu Glu Leu Phe Gin Ala Gin Ala Asp Arg Tyr Asp Gin Leu 325 330 335

TTT GAG TTA TAT GAA GAA TTA AGC GCT ACT ATC AGT AGT GTA ACC TTC 1056

Phe Glu Leu Tyr Glu Glu Leu Ser Ala Thr He Ser Ser Val Thr Phe 340 345 350

TGG GGA ATT GCT GAT AAC CAT ACA TGG CTT GAT GAC CGC GCT AGA GAG 1104 Trp Gly He Ala Asp Asn His Thr Trp Leu Asp Asp Arg Ala Arg Glu 355 360 365

TAC AAT AAT GGA GTA GGG GTC GAT GCA CCA TTT GTT TTT GAT CAC AAC 1152 Tyr Asn Asn Gly Val Gly Val Asp Ala Pro Phe Val Phe Asp His Asn 370 375 380

TAT CGA GTG AAG CCT GCT TAC TGG AGA ATT ATT GAT TAA 1191

Tyr Arg Val Lys Pro Ala Tyr Trp Arg He He Asp 385 390 395

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 396 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met He Thr Leu Phe Thr Lys Pro Phe Val Ala Gly Leu Ala He Ser 1 5 10 15

Leu Leu Val Gly Arg Gly Leu Gly Asn Val Ala Ala Ala Gin Gly Gly 20 25 30

Pro Pro Gin Ser Gly Val Phe Gly Glu Asn His Lys Arg Asn Asp Gin 35 40 45

Pro Phe Ala Trp Gin Val Ala Ser Leu Ser Glu Arg Tyr Gin Glu Gin 50 55 60

Phe Asp He Gly Ala Pro Val Glu Pro Tyr Gin Leu Glu Gly Arg Gin 65 70 75 80

Ala Gin He Leu Lys His His Tyr Asn Ser Leu Val Ala Glu Asn Ala

85 90 95

Met Lys Pro Val Ser Leu Gin Pro Arg Glu Gly Glu Trp Asn Trp Glu 100 105 li ^' o

Gly Ala Asp Lys He Val Glu Phe Ala Arg Lys His Asn Met Glu Leu 115 120 125

Arg Phe His Thr Leu Val Trp His Ser Gin Val Pro Glu Trp Phe Phe 130 135 140

He Asp Glu Asn Gly Asn Arg Met Val Asp Glu Thr Asp Pro Glu Lys 145 150 155 160

Arg Lys Ala Asn Lys Gin Leu Leu Leu Glu Arg Met Glu Asn His He

165 170 175

Lys Thr Val Val Glu Arg Tyr Lys Asp Asp Val Thr Ser Trp Asp Val 180 185 190

Val Asn Glu Val He Asp Asp Gly Gly Gly Leu Arg Glu Ser Glu Trp 195 200 205

Tyr Gin He Thr Gly Thr Asp Tyr He Lys Val Ala Phe Glu Thr Ala 210 215 220

Arg Lys Tyr Gly Gly Glu Glu Ala Lys Leu Tyr He Asn Asp Tyr Asn 225 230 235 240

Thr Glu Val Pro Ser Lys Arg Asp Asp Leu Tyr Asn Leu Val Lys Asp

245 250 255

Leu Leu Glu Gin Gly Val Pro He Asp Gly Val Gly His Gin Ser His 260 265 270

He Gin He Gly Trp Pro Ser He Glu Asp Thr Arg Ala Ser Phe Glu 275 280 285

Lys Phe Thr Ser Leu Gly Leu Asp Asn Gin Val Thr Glu Leu Asp Met 290 295 300

Ser Leu Tyr Gly Trp Pro Pro Thr Gly Ala Tyr Thr Ser Tyr Asp Asp 305 310 315 320

He Pro Glu Glu Leu Phe Gin Ala Gin Ala Asp Arg Tyr Asp Gin Leu

325 330 335

Phe Glu Leu Tyr Glu Glu Leu Ser Ala Thr He Ser Ser Val Thr Phe 340 345 35 ^' 0

Trp Gly He Ala Asp Asn His Thr Trp Leu Asp Asp Arg Ala Arg Glu 355 360 365

Tyr Asn Asn Gly Val Gly Val Asp Ala Pro Phe Val Phe Asp His Asn 370 375 380

Tyr Arg Val Lys Pro Ala Tyr Trp Arg He He Asp 385 390 395

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE: (C) INDIVIDUAL ISOLATE: FA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

CACACKCTKG TKTGGCA 17

(2). INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

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

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: FB

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

CATACKTTKG TTTGGCA 17

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: FR

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TMGTTKACMA CRTCCCA 17

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: GAF

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GARTAYTAYA THGTNGA 17

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: GAR

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

CKNACNGACC ARTA 14

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs n (B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: GBF

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GARTAYTAYG TNGTNGA 17

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: GBR

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

CKNACRCTCC ARTA 14

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(C) INDIVIDUAL ISOLATE: GCR

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CCRCTRCTKT GRTANCCYTC 20

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 142 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (B) STRAIN: 1-43-3

(C) INDIVIDUAL ISOLATE: CBS672.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

CATAGCCAAG TACCTGAATG GTTTTTCATC GATAAAGACG GTAATCGTAT GGTAGATGAA 60

ACAAATCCAG CGAAACGTGA GGCTAATAAA CAGCTTTTAT TAGAGCGGAT GGAAACACAT 120

ATCAAAACGG TTGTGGAACG TT 142

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

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

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(B) STRAIN: 1-47-3

(C) INDIVIDUAL ISOLATE: CBS669.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

CACACGCTGG TTTGGCATAG CCAAGTACCA GAATGGTTTT TCATCGATGA AAATGGCAAT 60

CGGATGGTTG ATGAAACCGA TCCAGAAAAA CGTAAAGCGA ATAAACAATT GTTATTGGAG 120

CGAATGGAAA ACCATATTAA AACGGTTGTT GAACGTTATA AAGATGATGT GACTTCATGG 180

GACGTGGTAA ACGA 194

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

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

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(B) STRAIN: 2-26-2 (C) INDIVIDUAL ISOLATE: CBS673.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CACACGCTGG TTTGGCACAG CCAAGTACCA GAATGGTTTT TCATCGATGA AGACGGCAAT 60

CGGATGGTGG ATGAAACAGA CCCAGATAAA CGTGAAGCGA ATAAACAGCT GTTATTGGAG 120

CGCATGGAAA ACCATATTAA AACGGTTGTT GAACGTTATA AAGATGATGT GACTTCATGG 180

GACGTGGTCA ACGA 1 ₉₄

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

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

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(B) STRAIN: 2-m-l (C) INDIVIDUAL ISOLATE: CBS667.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

CACACTCTTG TTTGGCATAG CCAAGTACCA GAATGGTTTT TCATCGATGA AAATGGCAAT 60

CGGATGGTTG ATGAAACCGA TCCAGAAAAA CGTAAAGCGA ATAAACAATT GTTATTGGAG 120

CGAATGGAAA ACCATATTAA AACGGTTGTT GAACGTTATA AAGATGATGT GACTTCATGG 180

GACGTGGTAA ACGA 194

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE: (B) STRAIN: 1-25-2

(C) INDIVIDUAL ISOLATE: CBS671.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GAATATTATA TTGTCGACAG TTGGGGCAAC TGGCGTCCAC CAGGAGCAAC GCCTAAGGGA 60

ACCATCACTG TTGATGGAGG AACATATGAT ATCTATGAAA CTCTTAGAGT CAATCAGCCC 120

TCCATTAAGG GGATTGCCAC ATTTAAACAA TATTGGAGCG TCCG 164

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 164 base pairs

(B) TYPE: nucleic acid (O STRANDEDNESS: double (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(B) STRAIN: 1-43-3 (C) INDIVIDUAL ISOLATE: CBS672.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

GAATATTATA TTGTCGACAG TTGGGGCAAC TGGCGTCCAC CAGGAGCAAC GCCTAAGGGA 60

ACCATCACTG TTGATGGAGG AACATATGAT ATCTATGAAA CTCTTAGAGT CAATCAGCCC 120

TCCATTAAGG GGATTGCCAC ATTTAAACAA TATTGGAGCG TCCG 164

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 164 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(B) STRAIN: 1-43-3

(C) INDIVIDUAL ISOLATE: CBS672.93

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GAATATTACA TCGTTGATAG CTGGGGAAGC TGGCGTCCAC CAGGAGCTAA CGCAAAAGGA 60

ACGATTACTG TTGACGGTGG TGTTTACGAT ATTTATGAAA CAACTCGAGT TAACCAACCT 120

TCCATTATTG GAGATGCGAC TTTCCAACAG TACTGGAGTG TGCG 164

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 744 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(B) STRAIN: 1-43-3

(C) INDIVIDUAL ISOLATE: CBS672.93

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

(B) LOCATION: 1..744

(D) OTHER INFORMATION: /product= "xylanase"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

ATG AGC CAA AAG AAA TTG ACG TTG ATT AAC CTT TTT AGT TTG TTT GCA 48

Met Ser Gin Lys Lys Leu Thr Leu He Asn Leu Phe Ser Leu Phe Ala 1 5 10 15

CTA ACC TTA CCT GCA AGA ATA AGT CAG GCA CAA ATC GTC ACC GAC AAT 96

Leu Thr Leu Pro Ala Arg He Ser Gin Ala Gin He Val Thr Asp Asn 20 25 30

TCC ATT GCC ACC CGC GGT GGT TAT GAT TAT GAA TTT TGG AAA GAT AGC 144 Ser He Ala Thr Arg Gly Gly Tyr Asp Tyr Glu Phe Trp Lys Asp Ser 35 40 45

GGT GGC TCT GGG ACA ATG ATT CTC AAT CAT GGC GGT ACG TTC AGT GCC 192 Gly Gly Ser Gly Thr Met He Leu Asn His Gly Gly Thr Phe Ser Ala 50 55 60

CAA TGG AAT AAT GTT AAC AAT ATA TTA TTC CGT AAA GGT AAA AAA TTC 240 Gin Trp Asn Asn Val Asn Asn He Leu Phe Arg Lys Gly Lys Lys Phe 65 70 75 80

AAT GAA ACA CAA ACA CAC CAA CAA GTT GGT AAC ATG TCC ATA AAC TAT 288

Asn Glu Thr Gin Thr His Gin Gin Val Gly Asn Met Ser He Asn Tyr 85 90 95

GGC GCA AAC TTC CAG CCA AAC GGT AAT GCG TAT TTA TGC GTC TAT GGT 336

Gly Ala Asn Phe Gin Pro Asn Gly Asn Ala Tyr Leu Cys Val Tyr Gly 100 105 110

TGG ACT GTT GAC CCT CTT GTT GAA TAT TAT ATT GTC GAC AGT TGG GGC 384

Trp Thr Val Asp Pro Leu Val Glu Tyr Tyr He Val Asp Ser Trp Gly 115 120 125

AAC TGG CGT CCA CCA GGA GCA ACG CCT AAG GGA ACC ATC ACT GTT GAT 432 Asn Trp Arg Pro Pro Gly Ala Thr Pro Lys Gly Thr He Thr Val Asp 130 135 140

GGA GGA ACA TAT GAT ATC TAT GAA ACT CTT AGA GTC AAT CAG CCC TCC 480 Gly Gly Thr Tyr Asp He Tyr Glu Thr Leu Arg Val Asn Gin Pro Ser 145 150 155 160

ATT AAG GGG ATT GCC ACA TTT AAA CAA TAT TGG AGT GTC CGA AGA TCG 528 He Lys Gly He Ala Thr Phe Lys Gin Tyr Trp Ser Val Arg Arg Ser 165 170 175

AAA CGC ACG AGT GGC ACA ATT TCT GTC AGC AAC CAC TTT AGA GCG TGG 576 Lys Arg Thr Ser Gly Thr He Ser Val Ser Asn His Phe Arg Ala Trp 180 185 190

GAA AAC TTA GGG ATG AAC ATG GGG AAA ATG TAT GAA GTC GCG CTT ACT 624 Glu Asn Leu Gly Met Asn Met Gly Lys Met Tyr Glu Val Ala Leu Thr 195 200 205

GTA GAA GGC TAT CAA AGT AGC GGA AGT GCT AAT GTA TAT AGC AAT ACA 672 Val Glu Gly Tyr Gin Ser Ser Gly Ser Ala Asn Val Tyr Ser Asn Thr 210 215 220

CTA AGA ATT AAC GGA AAC CCT CTC TCA ACT ATT AGT AAT AAC GAG AGC 720 Leu Arg He Asn Gly Asn Pro Leu Ser Thr He Ser Asn Asn Glu Ser 225 230 235 240

ATA ACT CTA GAT AAA AAC AAT TAG 744

He Thr Leu Asp Lys Asn Asn 245

(2) .INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 247 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

Met Ser Gin Lys Lys Leu Thr Leu He Asn Leu Phe Ser Leu Phe Ala 1 5 10 15

Leu Thr Leu Pro Ala Arg He Ser Gin Ala Gin He Val Thr Asp Asn 20 25 30

Ser He Ala Thr Arg Gly Gly Tyr Asp Tyr Glu Phe Trp Lys Asp Ser 35 40 45

Gly Gly Ser Gly Thr Met He Leu Asn His Gly Gly Thr Phe Ser Ala 50 55 60

Gin Trp Asn Asn Val Asn Asn He Leu Phe Arg Lys Gly Lys Lys Phe 65 70 75 80

Asn Glu Thr Gin Thr His Gin Gin Val Gly Asn Met Ser He Asn Tyr 85 90 95

Gly Ala Asn Phe Gin Pro Asn Gly Asn Ala Tyr Leu Cys Val Tyr Gly 100 105 110

Trp Thr Val Asp Pro Leu Val Glu Tyr Tyr He Val Asp Ser Trp Gly 115 120 125

Asn Trp Arg Pro Pro Gly Ala Thr Pro Lys Gly Thr He Thr Val Asp 130 135 140

Gly Gly Thr Tyr Asp He Tyr Glu Thr Leu Arg Val Asn Gin Pro Ser 145 150 155 160

He Lys Gly He Ala Thr Phe Lys Gin Tyr Trp Ser Val Arg Arg Ser 165 170 175

Lys Arg Thr Ser Gly Thr He Ser Val Ser Asn His Phe Arg Ala Trp 180 185 190

Glu Asn Leu Gly Met Asn Met Gly Lys Met Tyr Glu Val Ala Leu Thr 195 200 205

Val Glu Gly Tyr Gin Ser Ser Gly Ser Ala Asn Val Tyr Ser Asn Thr 210 215 220

Leu Arg He Asn Gly Asn Pro Leu Ser Thr He Ser Asn Asn Glu Ser 225 230 235 240

He Thr Leu Asp Lys Asn Asn 245

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1521 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus sp.

(C) INDIVIDUAL ISOLATE: DSM 8721

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GACGAACGCT GGCGGCGTGC CTAATACATG CAAGTCGAGC GCAGGAAGCC GGCGGATCCC 60

TTCGGGGTGA ANCCGGTGGA ATGAGCGGCG GACGGGTGAG TAACACGTGG GCAACCTACC 120

TTGTAGACTG GGATAACTCC GGGAAACCGG GGCTAATACC GGATGATCAT TTGGATCGCA 180

TGATCCGAAT GTAAAAGTGG GGATTTATCC TCACACTGCA AGATGGGCCC GCGGCGCATT 240

AGCTAGTTGG TAAGGTAATG GCTTACCAAG GCGACGATGC GTAGCCGACC TGAGAGGGTG 300

ATCGGCCACA CTGGAACTGA GACACGGTCC AGACTCCTAC GGGAGGCAGC AGTAGGGAAT 360

CATCCGCAAT GGGCGAAAGC CTGACGGTGC AACGCCGCGT GAACGATGAA GGTTTTCGGA 420

TCGTAAAGTT CTGTTATGAG GGAAGAACAA GTGCCGTTCG AATAGGTCGG CACCTTGACG 480

GTACCTCACG AGAAAGCCCC GGCTAACTAC GTGCCAGCAG CCGCGGTAAT ACGTAGGGGG 540

CAAGCGTTGT CCGGAATTAT TGGGCGTAAA GCGCGCGCAG GCGGTCTCTT AAGTCTGATG 600

TGAAAGCCCA CGGCTCAACC GTGGAGGGTC ATTGGAAACT GGGGGACTTG AGTGTAGGAG 660

AGGAAAGTGG AATTCCACGT GTAGCGGTGA AATGCGTAGA TATGTGGAGG AACACCAGTG 720

GCGAAGGCGA CTTTCTGGCC TACAACTGAC GCTGAGGCGC GAAAGCGTGG GGAGCAAACA 780

GGATTAGATA CCCTGGTAGT CCACGCCGTA AACGATGAGT GCTAGGTGTT AGGGGTTTCG 840

ATACCCTTAG TGCCGAAGTT AACACATTAA GCACTCCGCC TGGGGAGTAC GGCCGCAAGG 900

CTGAAACTCA AAGGAATTGA CGGGGGCCCG CACAAGCAGT GGAGCATGTG GTTTAATTCG 960

AAGCAACGCG AAGAACCTTA CCAGGTCTTG ACATCCTCTG ACACCTCTGG AGACAGAGCG 1020

TTCCCCTTCG GGGGACAGAG TGACAGGTGG TGCATGGTTG TCGTCAGCTC GTGTCGTGAG 1080

ATGTTGGGTT AAGTCCCGCA ACGAGCGCAA CCCTTGATCT TAGTTGCCAG CATTCAGTTG 1140

GGCACTCTAA GGTGACTGCC GGTGATAAAC CGGAGGAAGG TGGGGATGAC GTCAAATCAT 1200

CATGCCCCTT ATGACCTGGG CTACACACGT GCTACAATGG ATGGTACAAA GGGCAGCGAG 1260

ACCGCGAGGT TAAGCGAATC CCATAAAGCC ATTCTCAGTT CGGATTGCAG GCTGCAACTC 1320

GCCTGCATGA AGCCGGAATT GCTAGTAATC GCGGATCAGC ATGCCGCGGT GAATACGTTC 1380

CCGGGTCTTG TACACACCGC CCGTCACACC ACGAGAGTTT GTAACACCCG AAGTCGGTGC 1440

GGTAACCTTT TGGAGCCAGC CGNCGAAGGT GGGACAGATG ATTGGGGTGA AGTCGTAACA 1500

AGGTATCCCT ACCGGAAGGT G 1521