The present invention relates to a novel peptide having β-xylosidase activity, to a nucleotide sequence encoding such a peptide, and to the use of such a β-xylosidase- like peptide.

Background

Beta-xylosidase (1,4-β-D-xylan-xylohydrolase; EC 3.2.1.37) is one of the xylanolytic enzymes. Xylans are a major constituent of the cell walls of plants, and are second only to cellulose. They are abundantly found in most land plants, especially in agricultural by-products such as straw, wheat-bran, corn cobs, cotton seed, etc. Xylan is a complex polymer consisting of a β-l,4-linked xylose polymer with arabino-furanose, glucuronic acid, methylglucuronic acid and acetyl side-groups. Endoxylanase (EC 3.2.1.8) randomly cleaves the β-l,4-bonds in the xylan backbone to yield oligosaccharides, xylobiose and xylose. Beta-xylosidase cleaves terminal xylose units from the non- reducing end of the xylose oligomers resulting from endoxylanase activity. α-Glucuron- idase cleaves glucuronic acid side groups from backbone xylose units, whereas α-L- arabinofuranosidases (EC 3.2.1.55) cleave arabinose units from the xylan backbone and acetylesterases (EC 3.1.1.6) remove the acetyl side-groups.

Beta-xylosidase is also effective in transglycosylation reactions wherein monosaccharide units or alcohols are attached to or cleaved from xylose units. Beta- xylosidase is rate-limiting in xylan hydrolysis (Dekker 1983, Poutanen and Puls 1988).

The hydrolysing and transglycosylating reactions of β-xylosidases are eco¬ nomically important for the breakdown and utilisation of agricultural waste material e.g. in the production of xylose, xylose oligomers and xylitol, which are useful as sweeteners in foodstuffs, candies and medicaments, especially as a sugar substitute. Also, the enzyme or its products can be used as bread improvers and in the beer brewing industry.

Beta-xylosidases have been isolated from various sources including bacteria and fungi. For example, the purification of β-xylosidase from Aspergillus niger was reported by Rodionova et al. (1983); the molecular weight was reported to be 253,000 on the basis of gel filtration and 122,000 on the basis of SDS electrophoresis, whereas its isoelectric point was at pH 4.9.

Three endoxylanases and one β-xylosidase were isolated from Aspergillus

awamori by Kormelink et al. (1993); the β-xylosidase had a molecular weight of

110,000, a pH optimum of 6.5 and a temperature optimum of 70°C.

Beta-D-xylosidase from rumen fungus Neocallimastix frontalis was reported by

Garcia-Campayo and Wood (1993) and had an apparent molecular weight (gel filtration) of 150,000, a pH optimum of 6.4 and a temperature optimum of 37°C. Utt et al (1991) report the sequencing of the xylB of the ruminal bacterium Butyrivibrio fibrisolvens encoding both β-xylosidase and α-arabinofuranosidase activities.

Known β-xylosidases have activity pattems that do not always correspond to the industrial needs. In particular it is often desirable that the enzyme has a high xylosidase specificity and low specificities for other substrates, such as glucosides and galactosides.

Especially fungal β-xylosidases are highly advantageous for their activity levels and specificity pattems.

In order to be able to provide β-xylosidase-like enzymes having the desired activity pattems from the desired production organisms, sequence information of the β- xylosidase gene should be available. Up to now however, no sequence information on fungal β-xylosidases has been reported.

Description of the invention

A novel β-xylosidase has now been found and its amino acid sequence as well as its encoding nucleotide sequence have been determined. The protein is denoted herein as xylD, whereas the encoding gene is denoted as xlnO. The primary structure of the novel β-xylosidase appears to be different form known β-xylosidase-like enzymes. Also, its activity pattem is different form known β-xylosidase-like enzymes, and its xylosidase activity is about two times higher than that of the β-xylosidase reported by Rodionova et al (supra). Accordingly, the invention relates to a nucleotide sequence encoding a peptide having β-xylosidase activity and exhibiting at least 30% amino acid homology with the amino acid sequence shown in SEQ ID NO. 1 or hybridising under stringent conditions with a nucleotide sequence shown in SEQ ID NO. 1, or a part thereof having at least 15, preferably at least 21, more preferably at least 24 or even at least 30 nucleotides encoding an amino acid sequence shown in SEQ ID NO. 1. By amino acid homology is meant here amino acid identity in the primary structure. Amino acid similarity is usually higher than the figures given for identity.

In this context, heterologous hybridisation conditions are as follows: hybridisation in 6 x SSC (20xSSC per 1000 ml : 175.3 g NaCl, 107.1 g sodium citrate.- 5H ₂ 0, pH 7.0), 0.1% SDS, 0.05% sodium pyrophosphate, 5* Denhardt's solution (100 x Denhardt's solution per 500 ml : 10 g Ficoll-400, 10 g polyvinylpyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction V)) and 20 μg ml denatured herring sperm DNA at

56°C for 18-24 hrs followed by two 30 min. washes in 5 x SSC, 0.1 % SDS at 56°C and two 30 min. washes in 2 x SSC, 0.1% SDS at 56°C.

The nucleotide sequence of the invention encodes a peptide having substantial β-xylosidase activity, i.e. it has β-xylosidase activity as its predominant enzymic activity, and thus may be used for the production of β-xylosidases or mutants thereof. The coding sequences may contain mutations (insertions, deletions or both) which serve to modify the structure and/or the activity of the expression product. For an active expression product, the minimum identity and/or the hybridisation characteristic as defined above should preferably be maintained. The nucleotide sequence may also correspond to regulating or signal sequences of β-xylosidases. For these uses, the nucleotide sequence comprises substantially the encoding or regulating sequences of the β-xylosidase. On the other hand, the nucleotide sequence may be used as a primer or probe in detecting β-xylo¬ sidase encoding sequences. For these uses, the sequence comprises at least 15, up to e.g. 60, consecutive nucleotides of the sequence of SEQ ID NO. 1. The invention also relates to an isolated peptide having β-xylosidase activity and exhibiting at least 30% amino acid homology (identity) with the amino acid sequence shown in SEQ ID NO. 1 or a part thereof, said peptide having no β-glucosidase and/or no β-galactosidase activity; essentially no β-glucosidase or β-galactosidase activity means that these activities are less than 2%, in particular less than 1% of the β- xylosidase activity. A peptide exhibiting at least 40%, preferably at least 60%, most preferably at least 75% amino acid identity with the amino acid sequence shown in SEQ ID NO. 1 forms a preferred embodiment of the invention. Also part of the invention are peptides comprising a series of at least 8 contiguous amino acids of the amino acid sequence shown in SEQ ID NO.l. These can be produced by translating a nucleotide sequence as described above. Preferably the peptide has a contiguous series of at least 10, most preferably at least 12 amino acids from the sequence set forth in SEQ ID NO. 1.

The peptide according to the invention is especially from fungal origin, in particular from filamentous fungi, e.g. strains from the genera Aspergillus (especially A.

niger, A. niger var tubigensis, A. niger var awamori, A. niger var kawachii, A. oryzae, A. sydowii, A. japonicus, A. aculeatus, A. ochraceus, A. terreus, A. fumigatus, A. versicolor, A. flavus, A. phoenicis, A. nidulans, A. foetidus and A. carbonarius), Trichoderma (especially T. reesei, T. viride, T. longibrachiatum, T. harzianum, T. kongingii, T. pseudokongii), Penicillium (P. wortmani, P. pinophilum, P. janthinellum, P. citrinum, P. capsulatum, P. oxalicum, P. verruculosum, P. chrysogenum), Humicola (H. thermophilium = Scytalidium thermophilium) and Fusarium (F. oxysporum, F. solani).

Also, the invention concems antibodies raised against a peptide as described above e.g. for purifying β-xylosidases and for determining the presence of β-xylosidases. The antibodies can be produced by immunisation with the peptide described above, using hybridoma techniques which are well-known to the skilled person.

Also claimed are expression vectors and plasmids containing the nucleotide sequences described above under the control of a homologous or heterologous promoter.

Furthermore, the invention is concerned with the use of these sequences for the production of β-xylosidases by different hosts under the control of its own, or heterologous τegulatory sequences, or for the production of other peptides using the β- xylosidase promoter sequence. The expression vectors and host cells may contain multiple copies of the xylD-encoding sequences (altered or not with respect to SEQ ID NO. 1) and of other genes. Host organisms may be homologous production strains or altematively hetero¬ logous hosts. Suitable host organisms include fungi, yeasts, bacteria and plants. Examples are Aspergillus species, Trichoderma species, Bacillus species, Kluyveromyces species, Saccharomyces species and Fusarium species. Particularly preferred are A. niger, A. niger var. tubigensis, A. niger var. awamori, A. oryzae, A. japonicus, A. carbonarius, A. aculeatus, T. reesei, T. viride, T. harzianum, F. oxysporum, B. subtilis, B. licheniformis,

K. lactis and S. cerevisiae. The host organism is preferably a food-grade organism.

Examples of own control regions and heterologous regulatory regions include fungal constitutive and/or inducible promoters such as the pymvate kinase promoter (pkiA) and the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters. Examples of strong yeast promoters are alcohol dehydrogenase, 3-phosphoglycerate kinase and triose phosphate isomerase promoters. Examples of bacterial promoters are α-amylase, spo2 and promoters of extracellular protease genes.

The invention is furthermore concerned wit,* the use of regulatory sequences

contained in the 5'-noncoding part of SEQ ID NO.l (nucleotides 1-854 or a part thereof) for expression of homologous or heterologous genes, e.g. xylanase, amylase, glucanase, oxidoreductases e.g. hexose oxidase, α-glucuronidase, lipase, esterase, ferulic acid esterase, proteases, or human interleukin-6, bovine (pro)chymosin, human lactoferrin, fungal phytase. A signal sequence of xlnD may be used in such constmcts, as well as a suitable terminator, e.g. xlnD or trpC.

Further part of the invention is the use of the nucleotide sequence described above in such a manner as to disrupt the β-xylosidase gene of a host organism. To this end a nucleotide sequence containing a mutation which brings about a defunctionalisation of the β-xylosidase gene is introduced in the host cell. The mutation may be a deletion of one or more nucleotides, an insertion of one or more nucleotides, or a combination thereof.

The host cell - whether altered so as to produce or overproduce β-xylosidase or so as not to produce its β-xylosidase - may advantageously express or overexpress other relevant proteins, including enzymes, in particular other xylanolytic enzymes such as endoxylanases, and/or other enzymes such as amylases, glucanases, oxidoreductases such as hexose oxidase, α-glucuronidase, lipases, esterases, ferulic acid esterase and/or proteases. The corresponding genes may be under the control of homologous control regions or under the control region of the β-xylosidase gene contained in the nucleotide sequence described above.

An especially suitable protein to be expressed by the recombinant host cell according to the invention is the activating regulator of the xylanolytic pathway denoted as xylR. The target genes for this regulator comprise the genes xin A, xlnB, xlnC (all three endoxylanase-encoding genes), xlnO and axeA. Thus, the host cells according to the invention containing the xlnR gene, i.e. capable of expressing xylR or an active equivalent thereof, are effective producers of β-xylosidase - in case they contain the active xlnO gene - or of other xylanolyitc enzymes including endoxylanases, excluding β-xylosidase - in case their xlnO gene has been defunctionalised. The nucleotide sequence of the xlnR gene is set forth in SEQ ID NO.2. Also comprised by the invention is the use of the enzyme activity of the peptide in transglycosylation reactions of substrates contained in bread doughs and other bakery products, resulting in improved bread characteristics. This includes the use of the β- xylosidase as a bread improver in a manner known per se for enzymic bread improvers.

The β-xylosidases encoded by the present sequences can also be advantageously used for the production of xylose and xylose oligomers from wood and plant wastes and spent paper pulp, which xylose and oligomers are suitable as sweeteners. They can also be reduced to xylitol, which is also an effective bulk sweetener. The host cells according to the invention, wherein the β-xylosidase gene has been disrupted, can be used e.g. in the production of enzymes and enzyme preparations e.g. to be added to animal feed. Animals, including poultry and pigs, have a poor metabolism for xylose (Schutte, 1991). Xylose which is absorbed over the gut wall occupies the urinary excretion system and thus xylose uptake is energetically dis— advantageous to the animal. Moreover, a high intake of xylose is known to cause cataracts, diarrhoea and anorexia. On the other hand xylose and xylo-oligomers can be fermented to short chain fatty acids, which is an energetic asset. Therefore hemicellulose- degrading enzymes in feed should produce xylo-oligomers and no xylose monomers, and thus the enzymes should have endoxylanase activity and no, or a reduced level of, β- xylosidase activity. Thus the inventions also pertains to the use of host cells, such as fungi, bacteria, yeasts and plants, having a defunctionalised β-xylosidase gene but being capable of effectively producing endoxylanase and optionally other, especially xylanolytic, enzymes, for the production of enzyme preparations free of β-xylosidase. The invention also comprises such xylanolytic enzyme preparations lacking α-xylosidase activity. The β-xylosidase encoded by the nucleotide sequence of SEQ ID NO. 1 differs greatly from the β-xylosidase of A. niger reported by Rodionova et al. (1993), as is shown in table A.

Table A

Activity and inhibition of β-xylosidases of the invention (X-I and X-II), compared to the β-xylosidase according to Rodionova et al.

(1983) substrate X-I X-II β-xyl activity (U/m) invention invention (Rodionova) p-nitrophenyl-β-D-xylopyranoside 60.2 60.9 35.2 p-nitrophenyl-β-D-glucopyranoside 0.2 0.3 7.9 p-nitrophenyl-β-D-galactopyranoside 0.0 0.0 0.6 p-nitrophenyl-α-L-arabinofuranoside 2.8 3.4 5.8 inhibition K- (mM xylose) 9.8 13.2 2.9

Table A summarises the specificity pattem of two β-xylosidases X-I and X-II of the invention - presumably differing only in their glycosylation pattem, not in their amino acid sequence - and of the β-xylosidase reported by Rodionova et al., and their inhibition by xylose. The amino acid composition of these β-xylosidases is given in table

B.

Table B

Amino acid composition of β-xylosidase according to SEQ ID NO. 1, compared to the β-xylosidase according to Rodionova et al.

Amino acid number mole % mole % (Rodionova)

Ala 86 11.1 8.2

Arg 27 3.5 2.1

Asn + Asp 95 12.2 5.6

Cys 7 0.9 1.1

Gin + Glu 74 9.5 12.4

Gly 63 8.0 11.3

His 13 1.7 1.4

Ile 39 5.0 4.8

Leu 69 8.9 8.8

Lys 24 3.1 2.9

Met 6 0.8 3.3

Phe 24 3.1 2.9

Pro 39 5.0 8.6

Ser 53 6.8 8.7

Thr 58 7.5 7.0

Trp 15 2.0 2.6

Tyr 41 5.3 3.4

Val 45 5.8 6.1

Example 1: Purification of niger ^-xylosidase

Mutant strain A. niger NW147, a derepressed derivative of strain NW205::130#2 (cspAl, pyrAό, nicAl, argB13 ,::pIM130) constmcted as described in the copending PCT application filed 96.06.24 (as well as in EP 95201707.7 and EP 95202346.3) was grown in Aspergillus minimal medium (MM) (contains per litre: 6.0 g NaN0 ₃ , 1.5 g KH ₂ P0 ₄ , 0.5 g MgS0 ₄ .7H ₂ 0, 0.5 g KCl, Carbon source as indicated, pH 6.0 and 1 ml Vishniac solution (Vishniac, W. and Santer, M., 1957) (contains per litre 10 g EDTA, 4.4 g ZnS0 ₄ .7H,0, 1.0 g MnCl ₂ .4H ₂ 0, 0.32 g CoCl ₂ .6H ₂ 0, 0.32 g CuS0 ₄ .5H ₂ 0, 0.22 g (NH ₄ ) ₆ Mo ₇ 0 ₂₄ .4H ₂ 0, 1.47 g CaCl ₂ .2H,0, 1.0 g I S0 ₄ .7H ₂ 0, pH 4.0) supplemented

with 1.5 % cmde Wheat arabinoxylan, 10 mM L-arginine, 10 μM nicotinamide. This medium was inoculated with 1 * IO ⁶ spores per ml and mycelium was grown for 96 hours at 30°C and 250 m in an orbital New Brunswick shaker. The culture filtrate was collected after filtration of the mycelium on Myracloth (nylon gauze) using a Buchner funnel and mild suction. The pH of the culture filtrate was adjusted to pH 6.0 with 0.1 M

NaOH after which the culture filtrate was diluted by the addition of 2 volumes of Millipore water.

DEAE-Sephadex A-50 was equilibrated in 50 M sodium acetate buffer pH 5.0 and was added to the culture filtrate. After 30-60 minutes of stirring at 4°C, the DEAE- Sephadex together with the culture filtrate were passed through a funnel with a glass filter holder and the DEAE-Sephadex A-50 was transferred to a column. This column was first eluted with 50 mM sodium acetate buffer pH 5.0, then with 50 M sodium acetate buffer pH 5.0 + 0.5 M NaCl. Fractions containing β-xylosidase activity, as was detected using the chromogenic substrate 4-methylumbelliferyl-β-D-xyloside (detects β-xylosi- dases and endo-xylanases) (Sigma #M7008), were pooled and desalted by dialysis against

Millipore water and subsequently dialysed against 20 mM piperazine-HCl buffer pH 5.0: After dialysis the sample was loaded on a DEAE-Sepharose Fast Flow column. This column was first eluted with 3 volumes 20 M piperazine-HCl buffer pH 5.0 and then with a linear gradient of 0.5 M NaCl in 20 mM piperazine-HCl buffer pH 5.0. Detection of the eluted protein was performed by continuous measurement of the UV absorption at

280 nm (Figure 1). Fractions of 10 ml were collected which were assayed for activity of β-xylosidase on /κjτtf-nitro-phenyl-β-D-xylopyranoside (PNP-X) (Sigma #N2132). The β-xylosidase was found in fractions 11-27, which were pooled and subsequently dialysed against 20 mM piperazine-HCl buffer pH 6.0 (Figure 2). 5 ml of the dialysed sample was applied on a Mono Q HR 5/5 column (Pharmacia). Protein was eluted using 59 ml of a linear gradient of 1 M NaCl in 20 mM piperazine-HCl buffer pH 6.0. Detection of the eluted protein was performed by continuous measurement of the UV absoφtion at 280 nm (Figure 3). Two peaks containing β-xylosidase activity were found; β-xylosidase I was eluting at 0.19 M NaCl, while peak II eluted at 0.29 M NaCl. SDS-PAGE of both peak fractions revealed that the fractions coπesponding with both peaks each contained a single protein band, both having the same apparent molecular weight of 110 kDa. The specific activity of both β-xylosidase I and II towards the artificial substrate PNP-X was determined as described by Rodionova et al, 1983, to be respectively 60.2 and 60.9 U/mg protein. In addition the activity against PNP-β-D-glucopyranoside (Sigma

#N7006) was determined to be 0.2 and 0.3 U/mg, against PNP-β-D-galactopyranoside (Sigma #N1252) 0.0 and 0.0 U/mg and against PNP-α-L-arabinofuranoside (Sigma #N3641) 2.8 and 3.4 U/mg respectively for β-xylosidase I and II.

Example 2: Construction of a cDNA expression library A. niger NW147 was cultivated for 69 and 81 hr on MM containing 2% wheat arabinoxylan after which the mycelium was harvested by filtration and then washed with sterile saline. The mycelium was subsequently frozen in liquid nitrogen after which it was powdered using a Microdismembrator (Braun). Total RNA was isolated from the mycelial powder in accordance with the guanidium thiocyanate/CsCl protocol described in Sambrook et al. (1989), except that the RNA was centrifuged twice using a CsCl gradient. Poly A ⁺ mRNA was isolated from 5 mg of total RNA by oligo(dT)-cellulose chromatography (Aviv and Leder, 1972, Sambrook et al., 1989) with the following modifications: SDS is omitted from all solutions and the loading buffer was supplemented with 9% (v/v) dimethylsulfoxide. cDNA was synthesised from 7 μg poly A ⁺ mRNA and ligated into bacteriophage lambda Uni-ZAP XR using the ZAP -cDNA synthesis kit (Stratagene) according to the manufacturer's instmctions. After ligation of the cDNA into Uni-ZAP XR vector-arms, the phage DNA was packaged using Packagene™ extract (Promega) according to the manufacturer's instructions. Ligation of 120 ng cDNA in 1.2 μg vector arms and subsequent packaging of the reaction mixture resulted in a primary library consisting of

3.5 * IO ⁴ recombinant phages. This primary library was amplified using E. coli XLl - Blue MRF (Stratagene), titrated and stored at 4°C.

Example 3: Preparation of antibodies against ^-xylosidase

250 μg of both β-xylosidase I and II was dialysed against 1 mM phosphate buffer pH 7.0 and freeze-dried. The protein was resuspended in 100 μl sterile PBS (0.136

M NaCl; 2.7 mM KCl; 8 mM Na ₂ HP0 ₄ ; 1.75 mM KH ₂ P0 ₄ ; pH 7.4). To this protein mixture, 100 μl of Freunds' complete adjuvant was added and vortexed for 30 minutes to obtain a stable emulsion. For both proteins this mixture was injected into a mouse subcutaneously. In week 4 a booster was given by injecting 25 μg β-xylosidase in 100 μl sterile PBS to which 100 μl of Freunds' incomplete adjuvant was added. The mice were bled in week 7 and the serum tested. In week 13 the mice was given a second booster of

25 μg followed by a bleeding in week 14. This procedure of boosters with an interval of 6 weeks followed by a bleeding may be repeated several times.

The collected blood was incubated for 30 minutes at 37°C and subsequently stored at 4°C for 16 hours. After centrifugation at 5000 φm in a Sorvall High speed centrifuge the semm was collected and stored at -20°C.

Example 4: Immunoscreening of the A. niger NW147 cDNA library with antibodies against ^-xylosidase II

To screen the A. niger NW147 cDNA library, constmcted as described in Example 3, for β-xylosidase expressing cDNA clones 5 * 10 ³ pfu per plate were plated in NZYCM top-agarose containing 0.7% agarose on 85-mm-diameter NZYCM (1.5% agar) plates as described (Maniatis et al, 1982, pp. 64), using E. coli BB4 (Stratagene) as plating bacteria. Screening of the cDNA expression library obtained was basically performed as described by Young and Davies (1983). In short, 5000 pfu of the amplified stock were plated on NZYCM medium using E. coli BB4 cells as a host in 0.7 % top- agarose. Plates were incubated for 5 hrs at 37°C after which they were covered with nitrocellulose filters which were previously soaked in 10 mM IPTG and air-dried. Plates were then further incubated for 6 hrs at 37°C. Plates were cooled to 4°C, the position of the filters on the plates was marked before they are removed. The filters were incubated for 15 min in 0.5 M NaCl, 0.05 % Tween 20 (Biorad), 20 mM Tris/HCl pH 7.5 with gentle shaking, this was repeated once. The bacterial debris was removed by gentle scrubbing with gloved hands. Phages expressing a fusion protein containing a part of the β-xylosidase protein were identified by probing the filters with anti β-xylosidase II antiserum and subsequent detection using an alkaline phosphatase conjugate, according to the procedure described for Westem blots in the appropriate Biorad manual. In two experiments 5 * IO ³ and 5 * IO ⁴ pfu of the amplified library were screened for expression of β-xylosidase cDNA; 4 positives were found. Each positive plaque was removed from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 μl chloroform, as described in Maniatis et al. (1982). The phages obtained were purified by repeating the procedure described above using filter replicas from plates containing 50-100 plaques of the isolated phages.

Example 5: Analysis of ^-xylosidase expressing cDNA clones

The cDNA clones expressing β-xylosidase were converted to Bluescript phagemids using superinfection with the filamentous helper phage ExAssist™, which is included in the ZAP™-cDNA synthesis kit from Stratagene, according to the manufacturer's instmctions.

The phagemid DNA was subsequently isolated as described in Sambrook et al. (1989). The isolated DNA of the 4 cDNA clones was subjected to restriction analysis using the restriction enzymes EcoRI and Xhol. The DNA was digested for 2 hours at 37°C in a reaction mixture composed of the following solutions; 2 μl (*>\ μg) DNA solution; 2 μl of the appropriate 10 * React buffer (Life Technologies); 10 U of each restriction enzyme (Life Technologies) and sterile distilled water to give a final volume of 20 μl. After addition of 4 μl DNA loading buffer the samples were loaded on a 0.7% TAΕ-agarose gel. The DNA fragments were separated by electrophoresis at 80 V for 1.5 hours. The restriction analysis revealed that the cDNA clones had inserts of different sizes of respectively 1.4, 1.5, 2.4 and 2.5 kb. The nucleotide sequences of a part of each of these cDNA's were determined by the dideoxynucleotide chain-termination procedure (Sanger et al, 1977) using the Pharmacia T7 DNA polymerase sequencing kit. The sequences obtained revealed that these cDNA's correspond all four to the same gene.

Example 6: Screening of the A. niger genomic library for the -xylosidase encoding xlnO gene and isolation of the gene

For the screening of the A. niger N400 genomic library, constmcted as described by Harmsen et al, 1990, for the xlnD gene 3 x IO ³ pfu per plate were plated in NZYCM top-agarose containing 0.7% agarose on five 85-mm-diameter NZYCM (1.5% agar) plates as described (Maniatis et al., 1982) using E. coli LE392 as plating bacteria. After overnight incubation of the plates at 37°C two replicas of each plate were made on

HybondN ^"1" filters (Amersham) as described in Maniatis et al. (1982). After wetting the filters in 3xSSC the filters were washed for 60 min. at room temperature in 3xSSC. Hybridisation using a ³² P-labelled 2.5 kb EcoRI/XhoI fragment of cDNA clone #4, prepared as described by Sambrook et al, 1989, was done according the following procedure (Sambrook et al., 1989); prehybridisation in 6 x SSC (20xSSC per 1000 ml :

175.3 g NaCl, 107.1 g sodium citrate.5.5 H ₂ 0, pH 7.0), 0.1% SDS, 0.05% sodium pyrophosphate, 5* Denhardt's solution (100 x Denhardt's solution per 500 ml : 10 g

Ficoll-400, 10 g polyvinylpyrrolidone, 10 g Bovine Serum Albumin (Pentax Fraction V)) and 20 μg/ml denatured herring sperm DNA at 68°C for 3-5 hrs and hybridisation in an identical buffer, which contained the denatured radiolabelled probe at 68°C for 15-18 hrs, followed by two washes in 2 x SSC, 0.1 % SDS at 68°C and two washes in 0.2 x SSC, 0.1% SDS at 68°C. The membrane was covered with Saran wrap and autoradiographed ovemight at -70°C using Konica X-ray films and Kodak X-Omatic cassettes with regular intensifying screens.

This screening resulted in about 50 positive phages, of which ten were purified. Each positive plaque was picked from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 μl chloroform, as described in Maniatis et al. (1982). The phages obtained were purified by repeating the procedure described above using filter replicas from plates containing 50-100 plaques of the isolated phages.

After purification the phages were propagated by plating 5x10 phages on NZYCM medium. After overnight incubation at 37°C confluent plates were obtained, from which the phages were eluted by adding 5 ml SM buffer and storing the plate for 2. h. at 4°C with intermittent shaking. After collection of the supematant using a pipette, the bacteria were removed from the solution by centrifugation at 4,000 x g for 10 min. at 4°C. To the supematant 0.3% chloroform was added and the number of pfu was determined. These phage stocks contain approximately 10 pfu/ml.

DNA of four selected phages G15-G18, isolated as described in Sambrook et al 1989. was analysed by Southern analysis. The DNA was digested for 5 h. at 37°C in a reaction mixture composed of the following solutions; 5 μl ( ^■ * ^■ = 1 μg) DNA solution; 2 μl of the appropriate 10 x React buffer (Life Technologies); 10 U Restriction enzyme (Life Technologies) and sterile distilled water to give a final volume of 20 μl. The samples were incubated for 10 min. at 65 °C and rapidly cooled on ice, before loading on a 0.6% agarose gel in 1*TAE buffer. The DNA fragments were separated by electrophoresis at 25 V for 15-18 h.

After electrophoresis the DNA was transferred and denatured by alkaline vacuum blotting (VacuGene XL, Pharmacia LKB) to nylon membrane (Hybond N, Amserha ) as described in the VacuGene XL instmction manual (pp. 25-26) and subsequently prehybridised and hybridised using the labelled 2.5 kb EcoRI/XhoI fragment of cDNA clone#4 and hybridisation conditions as described. The hybridisation pattem was obtained

by exposure of Kodak XAR-5 X-ray film for 18 h. at -70°C using an intensifying screen. In all four clones fragments originating from the same genomic region were found for which a restriction map was constmcted (Fig. 4).

Based on the restriction map a 3.6 kb PstI fragment was selected for subcloning. 100 ng pEMBL19 PstI digested fragment was mixed with 250 ng 3.8 kb PstI fragment and 4 μl 5 * ligation buffer (composition; 500 M Tris-HCl, pH 7.6; 100 mM MgCl ₂ ; 10 mM ATP; 10 mM dithiothreitol; 25% PEG-6000) and 1 μl (1.2 U/μl) T ₄ DΝA ligase (Life Technologies) was added to this mixture in a final volume of 20 μl. After incubati¬ on for 16 h at 14°C the mixture was diluted to 100 μl with sterile water. 10 μl of the diluted mixture was used to transform E. coli DH5α competent cells, prepared as described by Sambrook et al., 1989. Six of the resulting colonies were grown ovemight in LB medium (LB medium per 1000 ml: 10 g trypticase peptone (BBL), 5 g yeast extract (BBL), 10 g ΝaCl, 0.5 mM Tris-HCl pH 7.5) containing 100 μg/ml ampicillin. From the cultures, plasmid DΝA was isolated by the alkaline lysis method as described by Maniatis et al. (1982), which was used in restriction analysis to select a clone harbouring the desired plasmid pIM200. The strain containing the plasmid pIM200 was deposited at the Centraal Bureau voor Schimmelcultures, Baarn, ΝL, under access number CBS 677.96. Plasmid DΝA was isolated on a large scale from 500 ml cultures E. coli DH5α containing pIM200 grown in LB medium containing 100 μg/ml ampicillin (Maniatis et al., 1982). The plasmid was purified by CsCl centrifugation, ethanol precipitated and dissolved in 400 μl TE. The yield was approximately 500 μg. This plasmid was used to constmct a detailed restriction map (Fig. 5).

Example 7: Transformation of A. niger using the plasmid pIM200

250 ml of culture medium, which consists of MM supplemented with 2 % glucose, 0.5 % yeast extract, 0.2 % casamino acids (Vitamin free), 2 mM leucine, 10 μM nicotinamide, 10 mM uridine, was inoculated with 1 * 10 spores per ml of strain ΝW155 (cspAl, argB13, pyrA6, nicAl, leuAl, prtF28) (derived from NW228, Van den Hombergh et al, 1995) and mycelium was grown for 16 - 18 hours at 30°C and 250 φm in a orbital New Brunswick shaker. The mycelium was harvested on Myracloth (nylon gauze) using a Buchner funnel and mild suction and was washed several times with SP6

(SP6: 0.8 % NaCl, 10 M Na-phosphate buffer pH 6.0). 150 mg Novozyme 234 was dissolved in 20 ml SMC (SMC: 1.33 M sorbitol, 50 mM CaCl ₂ , 20 mM MES buffer, pH

5.8) to which 1 g (wet weight) mycelium was added and which was carefully re¬ suspended. This suspension was incubated with gentle shaking for 1 - 2 hours at 30°C, every 30 minutes the mycelium was carefully resuspended and a sample was taken to monitor protoplast formation using a haemocytometer to count the protoplasts. When sufficient protoplasts were present (more then 1 * 10 ) these were carefully resuspended and the mycelial debris was removed by filtration over a sterile glasswool plug. The protoplasts were collected by 10 minutes centrifugation at 3000 φm and 4°C in a bench centrifuge, the supematant was removed and the pellet was carefully resuspended in 5 ml STC (STC: 1.33 M Sorbitol, 50 mM CaCl ₂ , 10 mM Tris/HCl, pH 7.5). This wash step was repeated twice and the protoplasts were finally resuspended in STC at a density of 1

* 10 ^s per ml.

The transformation was performed by adding 20 μg of pIM200 DNA and 5 μg pGW635, containing the A. niger pyrA gene (dissolved in a 10 - 20 μl TE), to 200 μl of protoplast suspension together with 50 μl of PEG buffer (PEG Buffer: 25 % PEG-6000, 50 mM CaCl ₂ , 10 mM Tris/HCl pH 7.2), mixed gently by pipetting up and down a few times, and incubated at room temperature for 20 minutes. After this period 2 ml PEG buffer was added, the solution was mixed gently and incubated at room temperature for another 5 minutes and subsequently 4 ml of STC was added and mixed gently on a vortex mixer. One ml portions of this suspension were then added to 4 ml of 0.95 M sucrose osmotically stabilised top agar and poured on osmotically stabilised plates. As a control A. niger was also transformed using pGW635.

Example 8: Analysis of transformants

The transformants from pIM200 obtained in Example 7 were analysed pheno¬ typically by plating on MM containing 1% oat spelt xylan and 1 mM 4-methy lumbelli- feryl-β-D-xyloside. Of the 26 transformants tested, five had an increased fluorescence.

These transformants, together with a PYR ⁺ transformant as a reference, were grown on MM containing 1% oat spelt xylan for 20, 27 and 42 hrs, after which the β-xylosidase activity towards PNP-X was measured. The results are summarised in Table C.

An increased level of β-xylosidase activity was found in all five transformants selected, the highest level being more then 30 times the wild-type activity. These results were confirmed by Western blot analysis, using the anti β-xylosidase antibody, prepared as described in Example 3, and the Bio-Rad Immui -blot GAR-AP assay kit following the suppliers instructions.

Table C β-xylosidase activities in A. niger transformants activity (mU/ml culture filtrate) after:

20 hr 27 hr 42 hr pGW 635 15 16 17

XlsAl 82 86 51

XlsA4 90 112 78

XlsA8 211 239 384

XlsA9 63 110 74

XlsA12 96 295 527

Example 9: The primary structure of the xlnD gene 9.1: Sequence analysis of the A. niger xlnO gene

The sequence of the A. niger xlnO gene, its promoter/regulation region, the stmctural part of the gene and the termmation region, was determined by subcloning fragments in pEMBL18/19, in combination with the use of specific oligonucleotides as primers in the sequencing reactions. For nucleotide sequence analysis, restriction fragments were isolated which were then cloned in pEMBL18/19 DNA vectors and digested with the appropriate restriction enzymes. The nucleotide sequences were determined by the dideoxynucleotide chain- termination procedure (Sanger et al., 1977) using the Pharmacia T7 DNA polymerase sequencing kit. Computer analysis was done using the PC/GENE program (Intelli- genetics). The sequence determined is given in SEQ ID NO:l.

9.2: Description of the xlnD gene

The sequence comprising the xlnO stmctural gene (SEQ ID NO:l) is preceded by a 854 nucleotide long upstream region. A putative TATA box is found at position 787-794. The stmctural part of the xlnO gene ranges from position 855 till position 3266 and contains no introns, as was certified by sequencing both the cDNA fragment as well as the genomic fragment in pIM200.

The xlnO gene encodes a protein of 804 amino acids. The N-terminal amino acid sequence is preceded by a 26 amino acids long hydrophobic sequence, which presumably corresponds to the signal sequence. The mature β-xylosidase protein is 778 amino acids in length, and has a deduced molecular weight of 84,727 Da.

Example 10: Screening of the A. tubigensis genomic library for the /3-xyIosidase encoding xlnO gene.

For the screening of the A. tubigensis genomic library, constmcted as described by De Graaff et al, 1994, for the A. tubigensis counteφart, 3 x 10 ³ pfu per plate were plated in NZYCM top-agarose containing 0.7% agarose on five 85 mm diameter

NZYCM (1.5% agar) plates as described in Example 6. Hybridisation using a ³² P- labelled 3.6 kb PstI fragment qf pIM200 prepared as described by Sambrook et al, 1989, was done according the following procedure (Sambrook et al., 1989); prehybridisation in 6 x SSC, 0.1% SDS, 0.05% sodium pyrophosphate, 5 * Denhardt's solution (see Example 6) and 20 μg/ml denatured herring sperm DNA at 65 °C for 3-5 hrs and hybridisation in an identical buffer which contained the denatured radiolabelled probe at 65°C for 15-18 hrs, followed by two washes in 5 x SSC, 0.1 % SDS at 65°C and two washes in 0.2 x SSC, 1% SDS at 65°C. The membrane was covered with Saran wrap and autoradiographed overnight at -70°C using Konica X-ray films and Kodak X-Omatic cassettes with regular intensifying screens.

This screening resulted in about 10 positive phages which were all purified.. Each positive plaque was picked from the plate using a Pasteur pipette and the phages were eluted from the agar plug in 1 ml of SM buffer containing 20 μl chloroform, as described in Maniatis et al. (1982). The phages obtained were purified by repeating the procedure described above using filter replicas from plates containing 50-100 plaques of the isolated phages.

After purification the phages were propagated by plating 5x10 phages on NZYCM medium. After overnight incubation at 37°C confluent plates were obtained, from which the phages were eluted by adding 5 ml SM buffer and storing the plate for 2 h. at 4°C with intermittent shaking. After collection of the supematant using a pipette, the bacteria were removed from the solution by centrifugation at 4,000 x g for 10 min. at 4°C. To the supematant 0.3% chloroform was added and the number of pfu is determined. These phage stocks contain approximately 10 pfu/ml.

Example 11: Disruption of the A. niger xlnO gene 11.1: Construction of the disruption plasmids pIM203 and pIM204

The gene disruption plasmids pIM203 and pIM204 were constmcted by generating an internal fragment of the xlnO gene by PCR. The fragment was generated

using the oligonucleotides derived from the xlnO sequence (SEQ ID NO: 1). XylosOOl was derived from positions 1157 till 1176 and xylos004 was derived from positions 3147 till 3164. The fragment was generated by PCR containing 10 μl 10*reaction buffer (100 M Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl ₂ , 0.01% gelatine), 16 μl 1.25 mM of each of the four deoxynucleotide triphosphates, 1 ng of the plasmid pIM200 DNA and 1 μg of each of the oligonucleotides in a final volume of 100 μl. This reaction mixture was mixed and 1 μl TAQ polymerase (5 U/μl) (Life Technologies) was added. The DNA was denatured by incubation for 3 min at 92°C followed by 25 cycli of 1 min 92°C, 1,5 min 52°C and 1,5 min 72°C. After these 25 cycli the mixture was incubated for 5 min at 72°C. Analysis of the reaction products by agarose electrophoresis revealed a fragment of about 2000 bp, which corresponds to the size expected, based on the sequence of the gene. The resulting fragment was subcloned in the vector pGEM-T (Promega) resulting in the plasmid pIM202. Plasmid pIM203 was constmcted by ligation of a SmaVPstl fragment of pILJ16 (Johnstone et al., 1985), containing the A. nidulans argB gene (Upshall et al., 1986), in the EcoKV/Pstl digested pIM202 vector. Plasmid pIM204 was constmcted by ligation of the Nsil/Xbal fragment of pIM130 (EP 95202346.3), containing the pyrA gene under the control of the UAS of the xlnA promoter of A. tubigensis, in the Spel/Nsil digested pIM202 vector. 11.2: Disruption of the xlnO gene in A. niger The plasmids containing the xlnO internal fragment as well as the argB gene

(pIM203) or the pyrA gene (plM204), as described in Example 11.1, as a selection marker in transformation, were used to dismpt the A. niger xlnO gene. For this A. niger N902 (argB15, cspAl, fwnAl, metBlO, pyrAS) was transformed, as described in Example 7, using the plasmids pIM203 and pIM204 selecting for arginine or uridine prototrophy respectively. The resulting transformants were screened for activity on methyl- umbelliferyl-β-D-xyloside on a 1 % xylan plate as described in Example 8. For both groups of transformants twenty were screened. Of these transformants one of each group had a severe decreased level of MUX activity after 24 h of growth. Southern analysis of the selected transformants demonstrated for the pIM203 transformant a multicopy integration at the homologous xlnD locus. In case of the pIM204 transformant a single homologous integration at the xlnD locus had occurred. Analysis for PNP-X activity, as described in Example 8, of these transformants revealed an at least 100-fold decrease in β-xylosidase activity.

113: Effect of overexpression and inactivation of xlnO gene on the expression of xylanolytic system of A. niger.

To determine the effect of xlnD expression on the expression of the xylanolytic spectmm, A. niger N902, two xlnD multicopy-transformants in N902 and the xlnD gene dismption strains were grown in liquid culture. This was done in a transfer experiment into 2% oat spelt xylan or 3 % D-xylose as a carbon source, after a preculture in 1 % fructose for 18 h. Beta-xylosidase activity was determined as PNP-X activity in the culture filtrate. With both C sources a clear overexpression could be seen for the pIM200 transformants against an almost absense of PNP-X activity for both (pIM203 and pIM204) inactivation transformants. The xlnD gene dismption transformants showed an initial decreased level of endo-xylanase expression, which however increased in time finally after 16 hrs resulting in increased activity levels in comparison to the A. niger wild-type, thus resulting in xylanase preparations free of β-xylosidase.

The culture filtrates were subsequently analysed by HPLC analysis, using a Dionex system and Pulsed Amperometric Detection. For this 1 ml of culture filtrate was boiled immediately after harvesting, to inactivate the xylanolytic enzymes, after which the sample was centrifuged for 10 min. (14.000 φm at 4°C, Eppendorf centrifuge). The resulting supematant was diluted 5-fold in bidest and 20 μl was analysed by HPLC using a Dionex CarboPac 100 column. The analysis indicated that, while in the wild-type and in the over-expression transformants only in the initial stage xylose oligomers could be detected in the culture filtrate, in the dismption mutant xylobiose and to a lesser extend xylotriose accumulated in the culture filtrate, thus resulting in a source for xylooligomers, in particular xylobiose and xylotriose.

Example 12: Expression of the A. niger xlnD gene in A. nidulans The plasmid pIM200 was introduced into A. nidulans by cotransformation of A. nidulans G191 (Balance and Turner, 1985) using the A. niger pyrA gene, located on the plasmid pGW635 as a selective marker and the plasmid pIM200 as the cotransforming plasmid. Protoplasts were prepared as described in Example 7 and the transformation procedure was performed using 1.2 M Sorbitol for osmotic stabilisation, 1 μg pGW635 and 25 μg pIM200. The PYR ⁴ obtained were then screened for xylD expression using the plate assay described in Example 8.

From this screening, five transformants were selected to determine β-xylosidase

activity. The A. nidulans wild-type strain and the selected transformants cultivated for 26 h at 37°C on minimal medium containing either 2% Birchwood xylan (Roth) or 3% D- xylose as an inducing carbon source. After removal of the mycelium, the β-xylosidase activity towards PNP-X in the culture filtrate was determined. The results are summarised in table D. The results show that xylD can be expressed in A. nidulans by using the native expression signals.

Table D

Strain of Activity on 2% xylan Activity on 3% D-xylose

A. nidulans (mU/ml) (mU/ml)

WG096 (Wt) 16 0

G191::200-5 725 48

G191::200-7 96 11

G191::200-9 249 40

G191::200-13 520 33

G191::200-15 1525 210

Example 13: Screening filamentous fungi for the xlnD gene

To analyse whether it is possible to isolate the xlnD counteφart from other fungi by heterologous hybridisation, using the 2.5 kb Pstl/Nsil fragment of the xlnD gene as a probe, DNA was isolated from the following strains; A. niger N902 (argB15, csp Al, fwnAl, metBlO, pyrAS), A. tubigensis NW184 (αspAl, fwnA\, pyrAll), A. nidulans

WG096 (pabaAl, yA2) of FGSC 187, A. aculeatus NW240 (pyrA3) of CBS 101.43, A. aculeatus NW217 (fwnAl, cspAl, pyrA , lysAV) of CBS 115.80, A. foetidus (awamori) NW183 (csp Al, fwnA\, pyr A13, lysAV) of CBS 115.52 and Trichoderma reesei QM9414. 1-2 μg DNA was digested with BamΑl or with Xhol and subsequently ana- lysed by Southern analysis. The hybridisation conditions used were; hybridisation in 6 x

SSC (20xSSC per 1000 ml : 175.3 g NaCl, 107.1 g sodium citrate.5H ₂ 0, pH 7.0), 0.1% SDS, 0.05% sodium pyrophosphate, 5 * Denhardt's solution (see Example 6) and 20 μg ml denatured herring sperm DNA at 56°C for 18-24 hrs followed by two 30 min. washes in 5 x SSC, 0.1 % SDS at 56°C and two 30 min. washes in 2 x SSC, 0.1% SSC at 56°C. After hybridisation the membrane was covered with Saran wrap and autoradio-

graphed ovemight at -70°C using Konica X-ray films and Kodak X-Omatic cassettes with regular intensifying screens.

As a result hybridising fragments were found for all fungi analysed, very strong hybridisation signals were found in A. niger, A. tubigensis, A. aculeatus, A japonicus, and A. foetidus, while strong hybridisation signals were found in A. nidulans and Tricho¬ derma reesei.

Example 14: Effect of xlnR gene dosage on the expression of the A. niger xylanolytic system

The strain N902::200-18, harbouring multiple copies (about 6) of the A. niger xlnD gene encoding β-xylosidase, was transformed to arginine prototrophy in a co- transformation experiment, as described in Example 11 using 19 μg of the xlnR harbouring plasmid pIM230 and 2μg of the plasmid pIM650 harbouring the A. nidulans argB gene (Johnstone et al., 1985). The transformants obtained were screened for increased endo-xylanase expression, on MM plates containing 1% oat spelt xylan. Four colonies, having the fastest and largest halo formation, were selected to determine xlnR copy numbers. For this DNA of these transformants and the recipient strain, was isolated and serial dilutions were spotted onto Hybond N membrane. The copy number was estimated from the signals found after hybridisation, using a radiolabelled 4.5 kb Smal/Xbal fragment spanning the coding sequence of the xlnR gene. Based on comparison to the recipient strain the xlnR copy number was determined to be 8 in

N902::200-18-R14 and 32 in N902::200-18-R16. For both these transformants the effect of the increased gene dosage of xlnR was analysed by Northern analysis after strains were grown in liquid culture. This was done in a transfer experiment into 2% oat spelt xylan as a carbon source, after a preculture in 1 % fructose for 18 h. Mycelial samples were taken 8 and 24 hrs after transfer, from which total RNA was isolated using TriZol (Life technologies) according to the manufacturers instmctions and analysed by Northern blot analysis (Sambrook et al., 1989). Xylanase B expression levels were strongly increased in these transformants in comparison to the recipient strain, as detected after hybridisation using the radiolabelled 1 kb £coRl/A7ιoI fragment of A. niger xlnB (Kinoshita et al., 1995).

References:

Aviv, H. and Leder, P. (1972) Proc. Natl. Acad. Sci. USA 11: 1408-1412. Balance D.J. and Turner G.C. (1985) Gene 36: 321-331. Dekker, R.F.H. (1983) Biotechnol. Bioeng. 3, 1127-1146. Flipphi, M.J.A., Visser, J., van der Veen, P. and De Graaff, L.H. (1994) Microbiology

140: 2673-2682. Garcia-Campayo and Wood (1993) Carbohydrate Res. 242, 229-245. De Graaff L.H., Van den Broeck, H.C, Van Ooijen A.J.J. and Visser, J. (1994) Mol. Microbiol. 12: 479-490. Harmsen, J.A.M., Kusters-van Someren, M.A., Visser, J. (1990) Curr. Genet. 18: 161—

166. Hombergh van den, J.P.T.W., van de Vondervoort, P.J.I., van der Heijden, N.C.B.A., and

Visser, J. (1995) Curr. Genet. 28:299-308. Johnstone, LL, Hughes, S.G., Clutterbuck AJ. (1985) EMBO J. 4: 1307-1311. Kinoshita K., Takano, M., Koseki, T., Ito. and Iwano, K. (1995). J. of Ferment, and

Bioeng.:79, no 5, 422-428. Kormelink, F., Searle-Van Leeuwen, M.J.F., Wood, T.M. and Voragen, A.G.J. (1993) J.

Biotechnol. 27, 249-265. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Habor, New York: Cold Spring Habor Laboratory Press.

Poutanen and Puls Appl. Microbiol. and Biotechnol. (1988) 28, 425-432. Rodionova, N.A., Tavobilov, I.M. and Bezborodov, A.M. J. Appl Biochem. 5, 300-312

(1983) Sambrook, J., Fritsch, E.F. and Maniatis T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Habor, New York: Cold Spring Habor Laboratory

Press. Sanger, F., Nickelsen, S. and Coulson A.R. (1977) Proc. Nail Acad. Sci. USA 74: 5463-

5467. Schutte, J.B. (1991), Nutritional value and physiological effects of D-xylose and L- arabinose in poultry and pigs. Datapress & Datavisions, Wageningen, 173 pp.

Upshall, A., Gilbert, T, Saari G., O'Hara, P.J., Weglenski, P., Berse, B., Miller, K. and

Timberlake, W.E. (1986) Mol. Gen. Genet. 204: 349-354. Utt, E.A., Eddy, C.K., HEshav, K.F. and Ingram, L.O. (1991) Appl. Environm. Microbiol. 1227-1234. Vishniac, W. and Santer, M. (1957) Bacteriol Rev. 21: 195-213.

Whittington, H., Kerry-Williams, S., Bidgood, K., Dodsworth, N., Peberdy., J., Dobson,

M., Hinchcliffe, E., and Ballance, DJ. (1990). Curr. genet. 18: 531-536. Young, R.A. and Davies, R.W. (1983) Science 222: 778.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Agricultural University Wageningen

(B) STREET: Coster eg 50

(C) CITY: Wageningen

(E) COUNTRY: The Netherlands

(F) POSTAL CODE (ZIP): 6701 BH

(ii) TITLE OF INVENTION: Novel beta-xylosidase, nucleotide sequence encoding it, and use thereof

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4108 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:

(A) ORGANISM: Aspergillus niger (CBS 120.49)

(B) STRAIN: NW147

(ix) FEATURE:

(A) NAME/KEY: TATA_signal

(B) LOCATION: 787.-79

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 855-.3266

(D) OTHER INFORMATION: /EC_number= 3- .1-37

/product= "1,4-beta-D-xylan xylanohydrolase"

/gene= "xlnD"

/standard_name= "beta-xylosidase"

(ix) FEATURE:

(A) NAME/KEY: sig_peptide

(B) LOCATION: 8 ••932

(ix) FEATURE:

(A) NAME/KEY: mat_peptide

(B) LOCATION: 933..3266

(ix) FEATURE:

(A) NAME/KEY: polyA_site

(B) LOCATION: 3383

(ix) FEATURE:

(A) NAME/KEY: polyA_site

(B) LOCATION: 34θ4

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

CTGCAGGCCA TGTATCCTGC GAAGATGGGT GAGTGGAAGA AAATCGTCAA GATTGAGGCG 60

GAGATGGCGA GGGCCGCGAT GAAGAAGGGT GGCTGGGCAC CGGAGAAGCC AGCCACCGCC 120

ACGGCGGCGC AGATGAGTAT ACCGTATGCG GTGGCGTTGC AGGTTCTGGA TGGGGAGATT 180

GTGCCGGGGC AGTTTGCGCC GGGCATGTTG AATCGGGAGG AGTTATGGGA TGTGATTAGG 240

CTGGTGGAAT GTCGGGAGGC CAAGGAGCTG GATAATACGT GGGCGCAGAG GGTCAAGATC 300

ACGTTTGAGG ATGGGGAGGT GGTGGAGAAG TTGTTGAAGG CTCCGAAGGG AGTCCATCCT 360

GGGGTGACGA ATGAGGAGGT GTTGCAGAAG TGGCGGGCTG TGACGAAGGG GGTAATTTCG 420

GAAGAGAGGC AGAAGAAGAT CGAGGAGATT GTGTTGAATT TGGAAGAGGT GGAGGATGTG 480

GCTGGTGTTT TGGGCGAGTT GTTGAGGGAA GAGACGGTGA ATGTGCTGCA GTAGACGGTT 40

ACCCCATTTG GACGGGGATG GCTTCATATT TCCCAAGCGA TGTCACGCCA TAGAAAGGGC 600

ACATTTACCC GGTGCCTGAG CGAAACTCTA CTTCGAAGAC AATGCCAATG TTTAACTATC 660

TTGTTTTAAT TGCTAAATGC AAACATTCCA GGTTCTTCCT AATGCCGGCT AAATCATTCA 7 0

GGCTAAACCC CCGCGATGAA GTCAATCGGT CATTCTCCGG CGCATCTCCG CATCTCCGCA 780

AACCGCTATA AAATCTACCC CAGATTCAGT CCCCGGCCAC CTTTCTATCC CCCCCCCCAC 840

AGACTGGCTC AACC ATG GCG CAC TCA ATG TCT CGT CCC GTG GCT GCC ACT 890 Met Ala His Ser Met Ser Arg Pro Val Ala Ala Thr -26 -25 -20 -15

GCC GCT GCT CTG CTG GCT CTG GCT CTT CCT CAA GCT CTT GCC CAG GCC 938 Ala Ala Ala Leu Leu Ala Leu Ala Leu Pro Gin Ala Leu Ala Gin Ala -10 -5 1

AAC ACC AGC TAC GTC GAC TAC AAC ATC GAA GCC AAC CCG GAC TTG TAT 86 Asn Thr Ser Tyr Val Asp Tyr Asn Ile Glu Ala Asn Pro Asp Leu Tyr 5 10 15

CCT TTG TGC ATA GAA ACC ATC CCA CTG AGC TTC CCC GAC TGC CAG AAT 1034 Pro Leu Cys Ile Glu Thr Ile Pro Leu Ser Phe Pro Asp Cys Gin Asn 20 25 30

GGT CCC CTG CGC AGC CAT CTC ATC TGT GAT GAA ACA GCC ACC CCC TAT 1082 Gly Pro Leu Arg Ser His Leu Ile Cys Asp Glu Thr Ala Thr Pro Tyr 35 40 45 50

GAC CGA GCA GCA TCG CTC ATC TCG CTC TTC ACC CTG GAC GAG CTG ATC 1130 Asp Arg Ala Ala Ser Leu Ile Ser Leu Phe Thr Leu Asp Glu Leu Ile 55 60 65

GCC AAC ACC GGC AAC ACC GGC CTC GGT GTC TCC CGA CTG GGC CTC CCT 1178 Ala Asn Thr Gly Asn Thr Gly Leu Gly Val Ser Arg Leu Gly Leu Pro 70 75 80

GCA TAC CAA GTA TGG AGT GAA GCT CTT CAC GGC CTC GAC CGT GCC AAT 1226 Ala Tyr Gin Val Trp Ser Glu Ala Leu His Gly Leu Asp Arg Ala Asn 85 90 95

TTC AGC GAC TCA GGA GCC TAC AAT TGG GCC ACC TCA TTC CCC CAG CCC 1274 Phe Ser Asp Ser Gly Ala Tyr Asn Trp Ala Thr Ser Phe Pro Gin Pro 100 105 no

ATC CTG ACC ACC GCG GCC CTC AAC CGC ACC CTC ATC CAC CAA ATC GCC 1322 Ile Leu Thr Thr Ala Ala Leu Asn Arg Thr Leu Ile His Gin Ile Ala 115 120 125 130

TCC ATC ATC TCT ACC CAA GGC CGC GCC TTC AAC AAC GCC GGC CGC TAC 1370 Ser Ile Ile Ser Thr Gin Gly Arg Ala Phe Asn Asn Ala Gly Arg Tyr 135 140 145

GGC CTC GAC GTC TAC GCC CCC AAC ATC AAC ACC TTC CGC CAC CCC GTC l l8 Gly Leu Asp Val Tyr Ala Pro Asn Ile Asn Thr Phe Arg His Pro Val 150 155 160

TGG GGT CGC GGA CAA GAA ACC CCA GGA GAG GAC GTC TCT CTC GCC GCC 1466 Trp Gly Arg Gly Gin Glu Thr Pro Gly Glu Asp Val Ser Leu Ala Ala 165 170 175

GTC TAC GCC TAC GAA TAC ATC ACC GGC ATC CAG GGT CCC GAC CCA GAA 1514 Val Tyr Ala Tyr Glu Tyr Ile Thr Gly Ile Gin Gly Pro Asp Pro Glu 180 185 190

TCA AAC CTC AAA CTC GCC GCC ACG GCC AAG CAC TAC GCC GGC TAT GAC 1562 Ser Asn Leu Lys Leu Ala Ala Thr Ala Lys His Tyr Ala Gly Tyr Asp 195 200 205 210

ATC GAG AAC TGG CAC AAC CAC TCC CGC CTG GGC AAC GAC ATG AAC ATC lβlO Ile Glu Asn Trp His Asn His Ser Arg Leu Gly Asn Asp Met Asn Ile 215 220 225

ACC CAG CAA GAC CTC TCC GAA TAC TAC ACG CCC CAA TTC CAC GTC GCC I658 Thr Gin Gin Asp Leu Ser Glu Tyr Tyr Thr Pro Gin Phe His Val Ala 230 235 240

GCC CGC GAC GCC AAA GTC CAG AGT GTC ATG TGC GCC TAC AAC GCC GTC 1706 Ala Arg Asp Ala Lys Val Gin Ser Val Met Cys Ala Tyr Asn Ala Val 245 250 255

AAC GGC GTC CCT GCC TGC GCC GAC TCC TAC TTC CTC CAG ACC CTC CTC 174 Asn Gly Val Pro Ala Cys Ala Asp Ser Tyr Phe Leu Gin Thr Leu Leu 260 265 270

CGC GAC ACC TTC GGA TTT GTC GAC CAC GGA TAC GTC TCC AGC GAC TGC 1802 Arg Asp Thr Phe Gly Phe Val Asp His Gly Tyr Val Ser Ser Asp Cys 275 280 - 285 290

GAT GCC GCC TAT AAC ATC TAC AAC CCC CAC GGC TAT GCC TCC TCC CAG I85O Asp Ala Ala Tyr Asn Ile Tyr Asn Pro His Gly Tyr Ala Ser Ser Gin 295 300 305

GCT GCC GCT GCC GCT GAG GCC ATC CTC GCC GGC ACC GAC ATC GAC TGC I898 Ala Ala Ala Ala Ala Glu Ala Ile Leu Ala Gly Thr Asp Ile Asp Cys 310 315 320

GGT ACC ACC TAC CAA TGG CAC CTG AAC GAG TCC ATC GCT GCG GGA GAT 1946 Gly Thr Thr Tyr Gin Trp His Leu Asn Glu Ser Ile Ala Ala Gly Asp 325 330 335

CTC TCT CGC GAT GAT ATT GAG CAG GGT GTG ATT CGT CTC TAC ACG ACC 1 94 Leu Ser Arg Asp Asp Ile Glu Gin Gly Val Ile Arg Leu Tyr Thr Thr 340 3^5 350

CTC GTG CAG GCC GGA TAC TTC GAC TCC AAC ACC ACA AAG GCG AAC AAC 2042 Leu Val Gin Ala Gly Tyr Phe Asp Ser Asn Thr Thr Lys Ala Asn Asn 355 360 365 370

CCC TAC CGC GAC CTC TCC TGG TCC GAC GTC CTT GAG ACG GAC GCA TGG 2090 Pro Tyr Arg Asp Leu Ser Trp Ser Asp Val Leu Glu Thr Asp Ala Trp 375 380 385

AAC ATC TCC TAC CAA GCC GCG ACG CAG GGC ATT GTC CTT CTC AAG AAC 2138 Asn Ile Ser Tyr Gin Ala Ala Thr Gin Gly Ile Val Leu Leu Lys Asn 390 395 400

TCC AAC AAC GTC CTC CCC CTC ACC GAG AAA GCT TAC CCA CCA TCC AAC 2186 Ser Asn Asn Val Leu Pro Leu Thr Glu Lys Ala Tyr Pro Pro Ser Asn 405 ^10 415

ACC ACC GTC GCC CTC ATC GGT CCC TGG GCC AAC GCC ACC ACC CAA CTC 2234 Thr Thr Val Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr Thr Gin Leu 420 425 430

CTG GGC AAC TAC TAC GGC AAC GCT CCC TAC ATG ATC AGC CCC CGC GCC 2282 Leu Gly Asn Tyr Tyr Gly Asn Ala Pro Tyr Met Ile Ser Pro Arg Ala 435 440 445 450

GCC TTC GAA GAA GCC GGA TAC AAA GTC AAC TTC GCC GAG GGC ACC GGT 2330 Ala Phe Glu Glu Ala Gly Tyr Lys Val Asn Phe Ala Glu Gly Thr Gly 455 460 465

ATC TCC TCC ACA AGC ACC TCG GGC TTC GCT GCC GCC TTA TCC GCC GCA 2378 Ile Ser Ser Thr Ser Thr Ser Gly Phe Ala Ala Ala Leu Ser Ala Ala 470 475 480

CAA TCC GCC GAC GTG ATA ATC TAC GCC GGT GGT ATC GAC AAT ACC CTT 2426 Gin Ser Ala Asp Val Ile Ile Tyr Ala Gly Gly Ile Asp Asn Thr Leu 485 490 495

GAA GCG GAG GCA CTG GAT CGA GAG AGT ATC GCG TGG CCG GGT AAC CAA 2474 Glu Ala Glu Ala Leu Asp Arg Glu Ser Ile Ala Trp Pro Gly Asn Gin 500 505 510

CTG GAC TTG ATC CAG AAG CTC GCC TCG GCG GCC GGA AAG AAG CCG CTC 2522 Leu Asp Leu Ile Gin Lys Leu Ala Ser Ala Ala Gly Lys Lys Pro Leu 515 520 525 530

ATC GTC CTC CAA ATG GGC GGC GGA CAG GTC GAT TCC TCT TCG CTC AAG 2570 Ile Val Leu Gin Met Gly Gly Gly Gin Val Asp Ser Ser Ser Leu Lys 535 540 545

AAC AAC ACC AAT GTT TCT GCA CTT CTC TGG GGC GGA TAC CCC GGC CAA 26l8 Asn Asn Thr Asn Val Ser Ala Leu Leu Trp Gly Gly Tyr Pro Gly Gin 550 555 560

TCT GGC GGC TTC GCT TTG CGG GAT ATC ATC ACG GGG AAG AAG AAC CCC 2666 Ser Gly Gly Phe Ala Leu Arg Asp Ile Ile Thr Gly Lys Lys Asn Pro 565 570 575

GCG GGT AGA CTA GTC ACG ACG CAG TAC CCT GCC AGC TAC GCG GAG GAG 2714 Ala Gly Arg Leu Val Thr Thr Gin Tyr Pro Ala Ser Tyr Ala Glu Glu 580 585 590

TTC CCG GCG ACA GAT ATG AAC CTT CGT CCT GAG GGT GAT AAC CCT GGT 2762 Phe Pro Ala Thr Asp Met Asn Leu Arg Pro Glu Gly Asp Asn Pro Gly 595 600 605 610

CAG ACG TAT AAA TGG TAC ACC GGC GAA GCC GTG TAC GAG TTC GGC CAC 2810 Gin Thr Tyr Lys Trp Tyr Thr Gly Glu Ala Val Tyr Glu Phe Gly His 615 620 625

GGG TTA TTC TAC ACG ACC TTC GCG GAA TCC TCC AGC AAT ACC ACT ACA 2858 Gly Leu Phe Tyr Thr Thr Phe Ala Glu Ser Ser Ser Asn Thr Thr Thr 630 635 640

AAG GAA GTT AAG CTC AAC ATC CAG GAC ATT CTT TCC CAG ACA CAC GAA 2906 Lys Glu Val Lys Leu Asn Ile Gin Asp Ile Leu Ser Gin Thr His Glu 645 60 655

GAC CTG GCG TCG ATT ACC CAG CTC CCT GTG CTG AAC TTC ACC GCC AAT 29 Asp Leu Ala Ser Ile Thr Gin Leu Pro Val Leu Asn Phe Thr Ala Asn 660 665 670

ATC AGG AAC ACT GGA AAG CTG GAA TCG GAT TAC ACC GCT ATG GTA TTC 3002 Ile Arg Asn Thr Gly Lys Leu Glu Ser Asp Tyr Thr Ala Met Val Phe 675 680 685 690

GCC AAT ACC TCT GAT GCC GGG CCG GCG CCG TAT CCC AAG AAG TGG CTG 3050 Ala Asn Thr Ser Asp Ala Gly Pro Ala Pro Tyr Pro Lys Lys Trp Leu 695 700 705

GTC GGG TGG GAT CGG CTT GGG GAG GTG AAG GTC GGG GAG ACG AGG GAG 3098 Val Gly Trp Asp Arg Leu Gly Glu Val Lys Val Gly Glu Thr Arg Glu 710 715 720

TTG AGG GTC CCC GTT GAG GTG GGG AGC TTT GCG AGG GTG AAT GAG GAT 3146 Leu Arg Val Pro Val Glu Val Gly Ser Phe Ala Arg Val Asn Glu Asp 725 730 735

GGC GAT TGG GTG GTG TTT CCG GGA ACG TTT GAG TTG GCG TTG AAT TTG 3194 Gly Asp Trp Val Val Phe Pro Gly Thr Phe Glu Leu Ala Leu Asn Leu 740 74 750

GAG AGG AAG GTT CGG GTG AAG GTT GTT CTT GAG GGT GAG GAG GAA GTC 3242 Glu Arg Lys Val Arg Val Lys Val Val Leu Glu Gly Glu Glu Glu Val 755 760 765 770

GTG CTG AAG TGG CCG GGG AAG GAG TAGAAAATAC TATTCTGTTG ATGGCTCTAG 3296 Val Leu Lys Trp Pro Gly Lys Glu 775

GGGATGAGAG TCAGCCTATT ACTGGATATG CATAGTGGTG ATACGATGTA TATAGCTCTA 3356

TGAAGTAATT AGTTCAAGTG GGAATACCCC TTTCACACAT ATAGTATGCT GTTATTCCGA 3416

AATAGGGATC ATTTCTGATT AATAGTAGCG GTAGCGATGG TCACACACGA CTTAATGTTC 3476

CCCATTGTAC CGGAAGTAAC AATTCCAGTG ACCTCTTAGA AGAAAGACAG CAAGAAAAAG 3536

TAAGAAAGGG AAATTGATCA AAAAATAAGG CCATCTACAG CCTATTCACA TTTAGCCGGA 3596

TCTGCAATAC AGCTACAGAA ATAAAGTTTG TTAGGCTGCT TGCTAGCATA GCTCCTACTA 3656

TACTAAACCA ACACAATGGG ACAATACCCC AATTAACCAG CCCTCACTCA ACACAAGTGA 3716

ATCCTACCGA CAACATGCAT AAACCACTGC TTCCCCACCC AGCACCCTTC TTCACGATCA 3776

GATCACGGAG AATTACCAAC TACTCTTCGC ATAAAACGTA AACAACGGCC TCGGGCCAGG 3836

ATCCGTCCGA CTCAAAAGCA ACAAATCCCT CGTTCGCATA CTAGCCACAT GAACCTGTTG 3896

CTCCGAGACC TCCTCAACTG GGTCTTCAAA TGCCCAGAAG ACGCTTTCTT CTCGATATCC 39?

ATCGGATACT CGCTGOCCGC TTAGACATAT GAACGATGAG TCTCGTCTGC CAAAGGAAAC 4016

AACCGTGTTC CCGAATCCAG TGTCAAAGTC GTAGGTCTGG AATTTGAAAA GTGTTCGGGC 4076

GTTTCCTTGG AGGGTCGGGA GTGCGACTGC AG 108

WO 97/00964 ~

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4173 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:

(A) ORGANISM: Aspergillus niger

(B) STRAIN: CBS 120.49

(C) INDIVIDUAL ISOLATE: N400

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: join(948..1173. 1238..3495. 3550.-3690)

(C) IDENTIFICATION METHOD: experimental

(D) OTHER INFORMATION: /function= "Transcriptional activator of xylanolytic genes"

/product= "Binuclear Zn finger DNA binding protein"

/gene= "xlnR"

/standard_name= "XYL R"

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 948..1173

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 1174..1237

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 1238.-3495

(ix) FEATURE:

(A) NAME/KEY: intron

(B) LOCATION: 3 6.-35 9

(ix) FEATURE:

(A) NAME/KEY: exon

(B) LOCATION: 3550.-3690

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CCCGGGCTTG GTTGGTCTCC GTCTGGCTTC CCCGCCTTTT TCCCCTGCAA TTCTGCATCC 60

CCAATCCTTC TTTTTTCTTT GCCTCGCCAG GCTGTGTCTT TTTTCCCCCT CCCCCTCCTC 120

CCTCGTCAGC TTCTCTTCGA CAGCATGCGT GAGGGTCTGC TACCAACTAC AATCCTTGTT 180

CTCACTGTCT GATGGTCTGA CCCGACCGTG GTGTCTGTGG TGTGTGTGTG AGAGAGAAAG 240

GAAAGCTAGT CAGTCCAGTC ACTCTTTCTC GTGGGTTCTT CACCTTCCCC GGACCTGCCC 300

TCCGACACTA AAAAGCCACT TCCCCCCAAC TGGTTAGTTG CTGCTAGTCT CCTTAGTTCA 360

TGGTCGGCCT TGTCGCTTCT CCGGCTGACA TTCTCCTCTT CTGCTGCCTT CTAGGTCCCT 420

GTTTTTTAGT CCCTGTTTTA GTTGCCCCGC AGACTGAATC GGCAATGCCG TGGAGTTGAT 480

CGTTCCGTGG TTTCCTTGCG ACCGCTCCTC TGCTTCATCA TCTTTTTCCT CCTGCCCTCC 540

TGGTCTTGAA TCGCCTGGCC CTCGTCTAGG ATCTGTTGCG CCAGTGTCGC CTTAATCTCC 600

TTTCCCGCTA GCGTAGTGCC CTTTCACGCT TGGGGCCTTA CGGCCCTTCC ATTCGCCAGC 660

GGTCTGAATA CCTCACTTTC CCCCCCAACG ACCGGGGTCT TCATGACCCG CTGGGGTGAT 720

TGTTCCGCCC GGTGAGGATG TCAACCCCCT CGATTCCTCA ATTCACCAGT CCTTTCTCTC 78O

CCTTCTCTTC CGGATCGCAC TCGACTGGCA TGGCGCCGTC TCAGACTGTC GGGTTGGATA 84θ

CGCTCGCCGA GGGCTCGCAG TACGTCCTGG AACAATTGCA GCTGTCGCGA GACGCTGCGG 900

GAACCGGTGC CGGCGATGGC GCGACCTCCA CTTCCTTGCG AAATTCC ATG TCG CAT 956

Met Ser His 1

ACG AAG GAT CAA CCA CCC TTT GAT AAT GAG AAG AAC CAG AGC ACT GGC 1004 Thr Lys Asp Gin Pro Pro Phe Asp Asn Glu Lys Asn Gin Ser Thr Gly 5 10 15

TCG GGT TTT AGG GAC GCT CTG CAA AGA GAT CCC CTC GTG GAG GCT CGC 1052 Ser Gly Phe Arg Asp Ala Leu Gin Arg Asp Pro Leu Val Glu Ala Arg 20 25 30 35

TCT GCC GTC CGC AAA ACC TCG TCT TCA GCT CCG GTT CGC CGC CGA ATC 1100 Ser Ala Val Arg Lys Thr Ser Ser Ser Ala Pro Val Arg Arg Arg Ile 40 45 50

AGC CGT GCG TGT GAC CAG TGT AAC CAA CTC CGA ACG AAA TGC GAC GGG 1148 Ser Arg Ala Cys Asp Gin Cys Asn Gin Leu Arg Thr Lys Cys Asp Gly 55 60 65

CAG CAT CCG TGC GCT CAT TGC ATT G GTAGGCTTCC GCTCTTTCTC 1193

Gin His Pro Cys Ala His Cys Ile 70 75

CGATGCCGGC GATGAGGCGG ACGCTTGACT GACCTGTTCT GTAG AA TTC GGA CTG 1248

Glu Phe Gly Leu

ACC TGC GAG TAT GCG CGA GAA CGC AAG AAG CGT GGA AAA GCG TCG AAG 1296 Thr Cys Glu Tyr Ala Arg Glu Arg Lys Lys Arg Gly Lys Ala Ser Lys 80 85 90 95

AAG GAT CTG GCG GCG GCA GCT GCG GCG GCT ACC CAA GGG TCG AAT GGT 134 Lys Asp Leu Ala Ala Ala Ala Ala Ala Ala Thr Gin Gly Ser Asn Gly 100 105 no

CAT TCC GGG CAG GCC AAC GCG TCG CTA ATG GGC GAG CGA ACG TCG GAA 1392 His Ser Gly Gin Ala Asn Ala Ser Leu Met Gly Glu Arg Thr Ser Glu 115 120 125

GAC AGC CGG CCA GGA CAA GAC GTG AAC GGC ACA TAC GAC TCG GCT TTT l44θ Asp Ser Arg Pro Gly Gin Asp Val Asn Gly Thr Tyr Asp Ser Ala Phe 130 135 140

GAG AGC CAC CAT CTT AGC TCG CAG CCA TCG CAT ATG CAG CAT GCA AGC 1488 Glu Ser His His Leu Ser Ser Gin Pro Ser His Met Gin His Ala Ser 145 150 155

ACT GCA GGG ATA TCC GGC CTG CAC GAG TCT CAG ACG GCA CCG TCG CAT 1 36 Thr Ala Gly Ile Ser Gly Leu His Glu Ser Gin Thr Ala Pro Ser His 160 165 170 175

TCG CAA TCA TCG CTA GGA ACG ACT ATC GAT GCG ATG CAT TTG AAT CAT 1584 Ser Gin Ser Ser Leu Gly Thr Thr Ile Asp Ala Met His Leu Asn His 180 185 190

TTC AAC ACG ATG AAC GAT TCC GGT CGC CCG GCA ATG TCC ATA TCC GAT 163 Phe Asn Thr Met Asn Asp Ser Gly Arg Pro Ala Met Ser Ile Ser Asp 195 200 205

CTG CGT TCG CTA CCC CCG TCC GTC TTA CCA CCG CAA GGA CTA AGC TCC 1680 Leu Arg Ser Leu Pro Pro Ser Val Leu Pro Pro Gin Gly Leu Ser Ser 210 215 220

GGG TAC AAC GCG AGC GCC TTC GCT TTG GTG AAC CCG CAA GAG CCG GGC 1728 Gly Tyr Asn Ala Ser Ala Phe Ala Leu Val Asn Pro Gin Glu Pro Gly 225 230 235

TCA CCA GCT AAC CAG TTT CGC TTG GGA AGC TCA GCG GAA AAC CCA ACC 1776 Ser Pro Ala Asn Gin Phe Arg Leu Gly Ser Ser Ala Glu Asn Pro Thr 240 245 250 255

GCA CCG TTT CTT GGT CTC TCG CCT CCA GGA CAG TCG CCT GGA TGG CTC 1824 Ala Pro Phe Leu Gly Leu Ser Pro Pro Gly Gin Ser Pro Gly Trp Leu 260 265 270

CCT CTT CCC TCG CCA TCT CCT GCC AAC TTT CCT TCT TTC AGC TTG CAT 1872 Pro Leu Pro Ser Pro Ser Pro Ala Asn Phe Pro Ser Phe Ser Leu His 275 280 285

CCG TTT TCC AGC ACT TTA CGA TAC CCT GTT TTG CAG CCG GTC CTG CCT 1920 Pro Phe Ser Ser Thr Leu Arg Tyr Pro Val Leu Gin Pro Val Leu Pro 290 295 300

CAC ATC GCC TCC ATT ATT CCG CAG TCG CTA GCG TGT GAC CTT CTG GAT 1968 His Ile Ala Ser Ile Ile Pro Gin Ser Leu Ala Cys Asp Leu Leu Asp 305 310 315

GTT TAC TTC ACT AGT TCC TCT TCG TCC CAC CTG TCT CCC TTG TCC CCA 2016 Val Tyr Phe Thr Ser Ser Ser Ser Ser His Leu Ser Pro Leu Ser Pro 320 325 330 335

TAC GTG GTG GGC TAC ATC TTC CGC AAG CAG TCT TTC CTT CAC CCG ACA 2064 Tyr Val Val Gly Tyr Ile Phe Arg Lys Gin Ser Phe Leu His Pro Thr 340 345 350

AAA CCC CGA ATA TGC AGC CCC GGT CTC CTG GCG AGT ATG CTC TGG GTA 2112 Lys Pro Arg Ile Cys Ser Pro Gly Leu Leu Ala Ser Met Leu Trp Val 355 360 365

GCC GCA CAA ACG AGT GAA GCT GCG TTT CTG ACA TCG CCG CCC TCG GCT 2160 Ala Ala Gin Thr Ser Glu Ala Ala Phe Leu Thr Ser Pro Pro Ser Ala 370 375 380

CGG GGG CGT GTA TGC CAG AAA CTG CTA GAA CTG ACC ATT GGT TTG CTC 2208 Arg Gly Arg Val Cys Gin Lys Leu Leu Glu Leu Thr Ile Gly Leu Leu 385 390 395

CGA CCG TTG GTC CAT GGT CCT GCT ACC GGA GAA GCG TCG CCC AAC TAT 2256 Arg Pro Leu Val His Gly Pro Ala Thr Gly Glu Ala Ser Pro Asn Tyr 400 405 410 415

GCG GCG AAT ATG GTC ATC AAT GGC GTC GCT CTG GGC GGA TTT GGG GTC 2304 Ala Ala Asn Met Val Ile Asn Gly Val Ala Leu Gly Gly Phe Gly Val 420 425 430

TCC ATG GAT CAG CTG GGC GCG CAA AGT AGC GCC ACC GGC GCC GTG GAT 23 2 Ser Met Asp Gin Leu Gly Ala Gin Ser Ser Ala Thr Gly Ala Val Asp 435 440 445

GAT GTA GCA ACT TAT GTG CAT CTT GCG ACA GTA GTA TCC GCC AGC GAG 2400 Asp Val Ala Thr Tyr Val His Leu Ala Thr Val Val Ser Ala Ser Glu 450 455 460

TAC AAG GCG GCC AGC ATG CGC TGG TGG ACT GCG GCG TGG TCT CTA GCG 2448 Tyr Lys Ala Ala Ser Met Arg Trp Trp Thr Ala Ala Trp Ser Leu Ala 465 470 475

CGT GAG CTG AAG CTA GGC CGT GAG CTG CCA CCC AAT GTT TCC CAC GCA 2496 Arg Glu Leu Lys Leu Gly Arg Glu Leu Pro Pro Asn Val Ser His Ala 480 485 90 495

CGG CAA GAT GGA GAG CGA GAT GGG GAT GGC GAG GCG GAC AAA CGA CAT 2 Arg Gin Asp Gly Glu Arg Asp Gly Asp Gly Glu Ala Asp Lys Arg His 500 50 510

CCT CCG ACC CTC ATC ACG TCA CTG GGT CAT GGA TCG GGA AGC TCC GGC 2592 Pro Pro Thr Leu Ile Thr Ser Leu Gly His Gly Ser Gly Ser Ser Gly 515 520 52

ATT AAT GTC ACC GAA GAG GAG CGT GAG GAG CGT CGA CGC CTA TGG TGG 2640 Ile Asn Val Thr Glu Glu Glu Arg Glu Glu Arg Arg Arg Leu Trp Trp 530 535 540

CTC TTA TAT GCG ACC GAT CGG CAC CTG GCG CTG TGC TAC AAC CGG CCC 2688 Leu Leu Tyr Ala Thr Asp Arg His Leu Ala Leu Cys Tyr Asn Arg Pro 545 550 555

CTC ACG CTG CTG GAC AAG GAA TGT GGC GGG CTG CTG CAG CCG ATG AAC 2736 Leu Thr Leu Leu Asp Lys Glu Cys Gly Gly Leu Leu Gin Pro Met Asn 560 565 570 575

GAT GAT CTG TGG CAG GTC GGC GAC TTT GCA GCG GCT GCC TAC CGC CAG 2784 Asp Asp Leu Trp Gin Val Gly Asp Phe Ala Ala Ala Ala Tyr Arg Gin 580 585 590

GTC GGA CCG CCC GTC GAG TGT ACG GGT CAC AGC ATG TAT GGA TAC TTT 2832 Val Gly Pro Pro Val Glu Cys Thr Gly His Ser Met Tyr Gly Tyr Phe 595 600 605

CTA CCG CTG ATG ACG ATT CTT GGA GGG ATC GTC GAT CTG CAC CAC GCT 2880 Leu Pro Leu Met Thr Ile Leu Gly Gly Ile Val Asp Leu His His Ala 610 615 620

GAG AAT CAT CCG CGC TTT GGC CTG GCG TTC CGC AAT AGC CCG GAG TGG 2928 Glu Asn His Pro Arg Phe Gly Leu Ala Phe Arg Asn Ser Pro Glu Trp 625 630 635

GAG CGT CAG GTA CTG GAC GTT ACG CGG CAG CTG GAC ACA TAT GGG CGC 2976 Glu Arg Gin Val Leu Asp Val Thr Arg Gin Leu Asp Thr Tyr Gly Arg 640 645 650 655

AGC TTG AAG GAA TTC GAG GCC CGC TAC ACC AGC AAC TTG ACT CTG GGG 3024 Ser Leu Lys Glu Phe Glu Ala Arg Tyr Thr Ser Asn Leu Thr Leu Gly 660 665 670

GCT ACG GAT AAC GAG CCT GTC GTC GAA GGT GCC CAC TTG GAT CAC ACG 3072 Ala Thr Asp Asn Glu Pro Val Val Glu Gly Ala His Leu Asp His Thr 675 680 685

AGT CCT TCG GGG CGC TCC AGC AGC ACC GTG GGA TCG CGG GTG AGC GAG 3120 Ser Pro Ser Gly Arg Ser Ser Ser Thr Val Gly Ser Arg Val Ser Glu 690 695 700

TCC ATC GTC CAC ACG AGG ATG GTG GTC GCC TAC GGG ACG CAT ATC ATG 3168 Ser Ile Val His Thr Arg Met Val Val Ala Tyr Gly Thr His Ile Met 705 710 715

CAC GTC CTG CAT ATT TTG CTC GCG GGA AAA TGG GAC CCG GTG AAT CTG 3216 His Val Leu His Ile Leu Leu Ala Gly Lys Trp Asp Pro Val Asn Leu 720 7 5 730 735

TTG GAA GAT CAT GAT CTG TGG ATC TCC TCG GAG VCG TTT GTC TCG GCC 3264 Leu Glu Asp His Asp Leu Trp Ile Ser Ser Glu Ser Phe Val Ser Ala 740 7 750

ATG AGC CAT GCG GTC GGT GCC GCA GAA GCA GCG GCA GAA ATC TTG GAG 3312 Met Ser His Ala Val Gly Ala Ala Glu Ala Ala Ala Glu Ile Leu Glu 755 760 765

TAC GAC CCG GAT CTC AGC TTC ATG CCG TTC TTC TTC GGG ATT TAC CTA 3360 Tyr Asp Pro Asp Leu Ser Phe Met Pro Phe Phe Phe Gly Ile Tyr Leu 770 775 780

CTA CAG GGC AGT TTC TTG CTG CTA CTG GCG GCG GAC AAG TTG CAG GGC 34θ8 Leu Gin Gly Ser Phe Leu Leu Leu Leu Ala Ala Asp Lys Leu Gin Gly 785 790 795

GAT GCC AGT CCC AGT GTC GTG CGG GCA TGC GAG ACG ATC GTG CGG GCG 3456 Asp Ala Ser Pro Ser Val Val Arg Ala Cys Glu Thr Ile Val Arg Ala 800 805 810 815

CAT GAA GCG TGC GTC GTG ACC TTG AAC ACG GAG TAC CAG GTAGGTTTTC 3505 His Glu Ala Cys Val Val Thr Leu Asn Thr Glu Tyr Gin 820 825

TTGTTTCTCT CCCTAGCTTG GCAATAGTAG CTAACACAAT GTAG AGG ACA TTC CGC 3561

Arg Thr Phe Arg 830

AAG GTC ATG CGA TCG GCG CTG GCA CAG GTT CGA GGA CGC ATC CCA GAG 3609 Lys Val Met Arg Ser Ala Leu Ala Gin Val Arg Gly Arg Ile Pro Glu 835 840 845

GAC TTT GGG GAG CAG CAG CAG CGC CGA CGC GAA GTG CTT GCG CTA TAC 3657 Asp Phe Gly Glu Gin Gin Gin Arg Arg Arg Glu Val Leu Ala Leu Tyr 850 855 860

CGC TGG AGC GGC GAT GGC AGT GGG CTG GCA CTG TAGTTTTGCA GTAACACGGC 3710 Arg Trp Ser Gly Asp Gly Ser Gly Leu Ala Leu 865 870 875

TGATGATGAG ATGCGATTTA TGGCGGTGCA TTGACCGGTC AATGGCTTCT TACATTCTGA 3770

TTTGATACTA CTTTTGGATT CGCTATTTCA CTCCGGGCTT ATGCTGGCTT CATTGTCAAG 383O

AGGGGTGGCA TGGCGAATGG AAATATGCTT ACTTCGTGTT GATACGGATT CGTACATATA 389O

CTTTGGTGAT ATATGTGGAT ATTTGTGGCA TGTACACTAT GCGTGATCTT TGGACATGAT 3950

ACTTTGATAC CAGGTCAATC TAATTGCGTT CTTTTCATTT GTTGCGCAAC AGCCGAGGTA 4010

TGACGCCATG GCTGAGATAA GCTGCCGATA AGCATTCGCA TTCCATCCTC CATCGAAGCA 4070

CCAAAATCTT CTTCATATAA CCAATCCATC AATTCAACAT TCGTAATGAC AATAGTATAA 4130

TCCCCAAAAT GCCCTCCCTA TTACACTCCC TCCGCACTTC CCC 4173