CA2240391A1 - Novel xylanases and uses thereof - Google Patents

Abstract

Enzyme compositions containing thermostabile xylanases of Chaetomium thermophilum, purified enzyme preparations of such xylanases, and the use of such compositions and preparations in the bleaching of plant pulp and in feed and baking applications are described.

Description

W O 97/22691 PCT~I96100670 NOVEL XYLANASES AND USES l~l~;~EOF

Background of the Invention Field of the Invention The present invention is in the area of thermostable enzymes, and the use of same.
S Especially, the invention is in the area of C~haetomium xylanases that are active at a high tule. The com~osilions of the invention are useful to modify plant biomass properties.
The invention is also directed to a method for enzyme-aided blç~ hing using the enzyme compositions of the invention. Furthermore, the invention concerns the use of the novel enzyme compositions in feedqh~ and flour compositions.

D~,.~l,on of ~ t~ art Plant biomass is a composite m~t~ri~l cnn~i~ting prim~nly of a matrix of cellulose, hemicellll1Ose, and lignin. Enzymes clegr~-iin~ e.g. the hemicellulose xylan, xyl~n~ces, can be used in animal ~eed compositions which are rich in arabinoxylans and glucoxylans, in baking, and for bleaching of cellulosic pulps.
Thus, when added to feeds for monogastric ~nim~l~ (e.g. poultary or swine) whichcontain cereals (e.g. barley, wheat, maize, rye or oats) or cereal by-products, a h~micelllllolytic enzyme improves the break-down of plant cell walls which leads to better ntili7~tion of the plant nlltrient~ by animal. This leads to improved growth rate and feed conversion. Also, the viscosity of the feeds co..~ g xylan can be reduced.
In baking applications small amounts of xylanases added to the flour impart favorable cl.~ ;t~ . ;stif~s to the dough and to the bread itself. Such char~cteri~tif~s include e.g. increased loaf volume and better textural characteristics (break and shred quality and crumb quality).
In the pulp and paper industry xylanases and other hemicell~ es are used, e.g., to improve the bleachability of the pulp.
The aim of kraft pulp ble~hing is to remove the residual lignin that is left in pulp after kraft cooking. Traditionally, this has been done using chlorine-co~ .g f~h~n ieS~l~. Because WO97/22691 PC~I96/00670 of en~ 1 c~ nr~-rn~ and consumer rl~m~nr1~7 alternative bleaching technologies have been desired.
The first bioterhnic~l approach to this problem was to attack the lignin directly with lignin degrading enzymes. However, the ~h~mi~try of ~l~ylllaLic lignin clegr~ tiQn seems to be very complicated and difficult to control.
Lignin can be degraded, if the whole microorganism that produces li~nin~es is used.
However, tre~trnent times OEe relatively long. For example, trç~fment times may take days, and the microor~ni~m~ need supplement~l mltri~ont~ to work. It can also be difficult to control the growth of other, undesired, microbes. Lignin degradation by using li~ninQ~es or by microor~;~ni~mc is the subject of much rese~.ill. (see, for example, Farrell, R.L. et al., Lignocellulosics 305-315 (1992); Jurasek, L., Lignocellulosics 317-325 (1992~).
In addition to cellulose and lignin, wood pulp cont~in~ hemicellulose. Another approach to lignin removal is to attack hemicellulose - the third main coln~llent of wood.
The h~mi~çlllliose in native hOEdwood is mainly xylan, while in softwood the h~miceliulose is mainly ~ com~nn~n~ and some xylan. During kraft cooking, part of the xylan is dissolved into the cooking liquor. Towards the end of the cooking period when the aLkali con-~n~ti<)n decreases, part of the dissolved and modified xylan l~re.;ipi~les back onto the cellulose fiber.
In 1986, it was noticed ~at xylanase ~ e~ nt of unbleached kraft pulp results ina lee~n~d need for rhemir~lc in the bleaching process (Viikari, L. et al., Procee~iin~s of the 3rd Int. Conf. on Biotechnology in the Pulp Paper Ind., Stockholm (1986), pp. 67-69).
Xylanase pl~ ont of kraft pulp partially hydrolyses the xylan in kraft pulp. This makes the pulp ~L~ ;Lule more porous and enables more efficient removal of lignin fr~pm~nt~ in the subsequent blç~hin~ and extraction stages. Later, in several laboratories, the xylanase ~ llr-ll was reported to be useful in conjunction with hie~rllin~ sequences con~i~tin~ of Cl~, ClO2,H2~2~ ~2 and O3. See reviews in Viikari, L. et al., F~MS Microbiol. Rev. 13: 3 35-350 (1994); Viikari, L. et aL, in: Saddler, J.N., ed., Bioconversion of Forest and Agricultural Plant Residues, C-A-B Int~ tional (1993), pp. 131-182; Grant, R., Pulp and Paper Int.
(Sept. l9g3), pp. 56-57; Senior & Hamilton, J. Pulp & Paper :111-114 (Sept. 1992); Bajpai & Bajpai, Process Biochem. 27:319-325 (1992); Onysko, A., Biotech Adv. Il:179-198 (1993); and Viikari, L. et al., J. Paper and ~imber 73:384-389 (1991).

WO97/22691 PCT~19~ 70 Asadirectresultofthebetterbleachabilityofthepulpaftersuchaxyl~n~ceLlç~
there is a reduction of the subsequent consumption of bles-chin~ chemicals, which when chloride cc ~ ch~omic~l~ are used, leads to a reduced formation of cllviro~ Pnt~lly undesired organo-rhll~rine compounds. Also as a direct result of the better bleachability of pulp after a xylanase trP~trnentJ it is possible to produce a product with a high final hri~htnP~s where such brightnPss would otherwise be hard to achieve (such as totally chl-)rin~ free (TCF) ble~ching using peroxide). Because of the :jUb~Lld~; specificity of the xylanase enzyme, cellulose fibers are not harmed and the strength plupclLies of the product are well within acceptable lirnits.
However, in many of the practical applications, the use of xylanases is not strai~ L~l vvill l; the xylanases must be active in the l~ claLulc and pH conflitione of the process in which they are used. Formnl~ti(m of commercial feed using p~lleting extrusion or ~Yp~ntling, often contains steps involving high temperatures (70 - 180 ~C). Enzymes added to the forrnlll~tion process should with~t~n~l these conditions. On the other hand, the c.,l~ ollding LClll~ dlUIC in the intestin of animals is about 40 ~C. Thus, ideal xylanases for feed c~ o~iLions should Wilh~ the above mPntionf~d extream Icllll~dLul~,s. In ble~hing applications, xylanase application is not as simple as adding a xylanase tre~tmPnt step.
Because the ble~rhing process, and even the sequence of the steps used in the ble~rhing process varies in dirrGlclll pulp mills, there is thus a continous need to find new xylanases active in dirr~G.lt Lc~ ,.aLul~, and pH conditions.
Most comm~orcial xyl~lases ~1P~; nPcl for feed applications and pulp ble~rhin~ are not very the~no-tolerant, ~eperi~lly when neutral or aLkaline pH con-litic ns are used. ~n practice, xylanases are generally inefficient or inactive at tclll~ alulGS higher than 60~C and often these cllGyllles work under acidic conditions. Generally, there are differences in the physical rh~r~rteri~tir~ of xylanases of fungi and b~rt~ri~ (for review, see Wong et al., Microbiol. Rev.
52:305-317 (1988)). Typically, fungal xylanases have tcllly~,laLulG o~Lilllu,l. at about 50 ~C
and lower pH O~Lilll~ll than have those of b~ctt~ri~l origin. Xylanases of bacterial origin generally have a tClll~cl~Lulc upLilllulll in the range of 50 to 70 ~C.
PCT/US90/05933 (WO 91/05908~ proposes the use of xylanase in pulp bleaching together with rhlc~rin~ or chlorine compolln-l~. Chaetomium is proposed as a xylanase source.
Screening for xylanase from Streptomyces and Chainia strains is described. Ble~ching e,.~ ; " ,~ were yrl r()~ o(l using xylanase ylG~ Lions from Chainia sp. culture mediurn.

WO 97/22691 PCT~196/00670 EP-A 0 406 617 proposes the use of xylanase in an ~ y~llaLic delignifying process of li~nocell~ sic m~teri~1, especially after a ligninolytic er~7yme. The xylanase may be derived form various sources, for ~Y~mple from Chaetomium. The use of xylanase from Chainia sp culture m~ m is e~emplified.
~'J~n(lhi, J.P. et al., J: Chem. Tech. Biotechnol. 60:55-60 (19943 reported studies on th~nnost~hjlity and pH stability of crude xylanase ~le~dLions from Chaetomium globosum.
The ~l~ ~llu~ p ~ l, e of the xylanase was found to be in the rarlge of 50 to 60 ~C, while the Optilllulll pH was found to be pH 5Ø The enzyme was reported not to lose any of the original activity between 40 to 60 ~C for a period of 10 min and was reported to retain more than 70% of the original activity in the range of 70 to 100 ~C for 10 min. The pH stability studies indicated that the enzyme retained all activity b~Lw~;ll pH 5 and 6 and more than 70%
of the original activity over a wide range of allcaline pH values (7- 10). It was suggested to use the culture filtrates for the tre~ nt of cellulose pulps without ~rther pl-rificati- n.
The xylanase of Chaetomium cellulolyticum and Chaetomium trilalerale have also been studied (Dubeau, H. et al., Biotechnol. Lett. 9:275-280 (1987) and K~wi.. i.. ,.. "i, T. and Iitzuka, H. J. Ferment. Technol. 48:161-168 (1970), respectively). However, neither the th.ormost~hility nor the pH profile ofthe enzymes was reported. The xylanases were sl~gg~te~
to be of use in clarification of fruit juices. The use of these enzymes in pulp ble~rhin~ or as feed additive was not ~ugge~ed.
Ganju, RK. et al., Can. J. Microbiol. 35:836-842 (1989) reported the p~lrif~ tion and char~-L~ ;on of two xylanases from Chaetomium thermophile var. coprophile. Two xylanases (I and II) out of several extracellular xylanases produced by C thermophile var.
coprophile were purified to homogeneity. These enzymes had molecular weights of 26,000 Daltons (xylanase I) and 7,000 Daltons (xylanase II). The te~ alule optima for xylanase I and II were 70 and 60 ~C, and they were optimally active at pH 4.8-6.4 and 5.4-6.9, respectively. The use of these xylanases in pulp blç~-~hing or as feed additive was not suggested.
Irie et al., Hakko Kogaku Kaishi 70(2): 109-114 (1992) have reported the pllrific~tinn of a xylanase from a Chaetomium gracile mutant. The molecular weight was reported to be 19,000 ~i~ltrrl~, and the xylanase cc .. I~ cl two subunits: one having a molecular weight of 14,400 daltons and the other a molecular weight of 4,800 daltons. The pI was 8.35. The maximal xylobiose forming activity was found at pH 5.0 and 50 ~C. The pH range was WO97/22691 PCTfF196/00670 s reported to be pH 4.0 - pH 7Ø Yoshino et al., Curr. Genet. 29:73-80 (1995) reported the isolation and sequenring of two xyl~ase genes from Chaetomium gracile wild and mutant strains and their ~yr~s~ion in Aspergillus nidulans. The mature Cg-XA and CgXB xylanases contain 189 and 211 amino acids, l~sLe.i~ively, and share 68.5% homology. The cg~ and cgxB genes were introduced into Aspergillus nidulans and l~yul Led to be ~,~ylc;~ed with their own promoters. The use of these C gracile xylanases in pulp bl~clling or as feed additive was not ~u~,ge~led.

Sl~mma~y of ~e Invention Recognizing the i~ v~ ce of developing an t;;llvi~ nt~lly safe and econc-miczll method of modifying plant biomass, the ~lVGlllvl~ have searched for new enzymes that would be useful in such processes.
These studies have resulted in the isolation and itlPntific~ion of novel xylanases and compositions cv-,l~ g the same, that are useful in such pl~cesses. Accordingly, the invention is directed to a cell-free composition cc)mpri.cin~ at least one xylanase of Chaetomium thermophilum var. thermophilum, Chaetomium thermophilum var. coprophilum, Chaetomium thermophilum var. dissitum or Chaetomium thermophilum, especially wherein such species is the same species as that l~lGsGllled by CBS 733.95, CBS 732.95, CBS 731.95 and CBS 730.95, lG*)c~,LivGly.
The invention is further directed to composition co..l~;t~ g a purified xylanase of Chaetomium, wherein such xylanase has a molecular weight of about 54 l~Da, 33 kDa, 30 kDa ~dimeric 60 kDa) or 22 kDa or xylanases having equivalent ployGlLies or llli~ S
cc,..li.i..;.-g more than one such xylanase.
The invention is further directed to methods of treating plant biomass with the cell-free or purified enzyme yl~Lions of the invention. Such uses include the enzyme aided ble~rhing of wood pulp and methods of modifying plant biomass, like uses as feed additive or in baking.
The invention is further directed to a method of treating plant biomass with a cell-free enzyme plGyz~c~Lion that corlt~in~ at least one xylanase of Chaetomium tkermop~ilum var.

coprophilum, especially wherein such species is the same species as that le~._s~.lL~d by CBS
732.95.

Brief Descripfion of the Figllres l,egends to figures S Figure 1: Tc;l~ profiles of the xylanase activity of the culture filtrates of ALKO4123, ALKO4243, ALKO4244, and ALKO4265 at pH 7.2 and 60 min incubation.

Figure 2: The chromatogram of the Phenyl-Sepharose 6FF run of the ALKO4265 culture filtrate. Pools IXl, TX2, TX3 are indicated.

Figure 3: Tt;lll~J~ldlULe and pH profiles of the purified xylanases of ALKO4265. Incubation time 1 h.
A: 22 kDa xylanase, B: 30 kDa xylanase, C: 33 IcDa xylanase, D:54 kDa xylanase.

De~i~e~ Descripfion of the Preferred Embodiment Deposits Ckaetomium thermophilum var. thermophilum ALKO4123 was deposited on November 8, 1995 at tbe Ct ntr~lhureau Voor Sl~himmelcultures at Oo~ t 1, 3742 SK
BAARN, The Neth~rl~nfl~, and ~ n~cl CBS 733.95. Chaetomium thermophilum var.
coprop*ilum ALKO4243 was deposited on November 8, 1995 at the Centraalbureau Voor Schimmelcultures at Oo~lc~ l 1, 3742 SK BAARN, The Netherl~n~l~, and a~ign~(l asCBS 732.95.
Chaetomium thermophilum var. dissitum ALKO4244, was deposited on November 8, 1995 at the Centraalbureau Voor S~himmelcultures at Oosterstraat 1, 3742 SK BAARN, The Netherlands, and ~cci~ne~ as CBS 731.95.

WO g7/22691 PCT/F196/00670 ALKO4265, ~ ntifie~1 as Chaetomium thermophilum by the Tnt~rnQtional Mycological ~n.~tihlt~/Biosystem Services, was deposited on November 8, 1995 at the CentrQQlhureau Voor Sçhimm~]cultures at Oostçrstr~Qt 1, 3742 SK BAARN, The Netherlands, and ~ceign~ CBS 730.95.

S Def~r;fi~ -In order to provide a clearer and consistent understQn~1inp of the specification and claims, including the scope to be given such terms, the following definitions are provided.
Xylanase. As used herein, a xylanase is a hemicellulase that cuts the ~-1,4 bonds within the xylosic chain of xylan, (xylan is a polymer of D-xylose residues that are joined through ~-1,4 linkQg~s). Xylanase activity is synonymous with xylanolytic activity.
By an amino acid sequence that is an "e~uivalent" of a specific amino acid sequence is meant an amino acid se~l.~.n.~.e that is not itl~nti~Ql to the specific amino acid sequence, but rather contQine at least some amino acid changes (deletion, ~ub~LiLuLions, inversions, insertions, etc) that do not f~es~ntiQlly affect the biological activity ofthe protein as col.lparcd to a similar activity of the specific arnino acid sequence, when used for a desired purpose.
Preferably, an "equivalent" amino acid seq~l~nl~e contQine at least 80% homology at the amino acid level to the specific amino acid sequence, most preferably at least 90% and in an especially highly preferable embodiment, at least 95% homology, at the amino acid level.
Enzyme preparation. By "enzyme ~ ud~ion~ is me~t a composition contQining

2() enzymes that have been extracted from (either partially or completely purified from) a microbe or the m~ m used to grow such microbe. "E-~t~.t~dSrom" means any method by which the desired enzymes are se~dLcd from the cellular m_ss and includes breaking cells and also simply removing the culture m.o-lillm from spent cells. Therefore, the term "enzyme ~r~f,..,~l on" in~hl~1Pc compositions C~JII~;1;ll;~g medium previously used to culture a desired microbe(s) ~d any enzymes which the microbe(s) h_s secreted into such medium during the culture.
El ~...c-nided blon< hing. By "enzyme-aided bleQ~hing" is meant the extraction of lignin from cellulose pulp after the action of hemicellulose ~lçgr~1in~ enzymes with or without lignin clegrQ~ling enzymes. Removal of the lignin may be restricted by hemicçll-llos either physically (through lcy.~ iLdLion onto the fiber surface during cooking) or chf-micQlly .

W O 97n2691 PCT~196/00670 (through lignin-carbohydrate complexes). The hemicellulase activity partially ~legr~ s the hemicellulose, which ~onh~ncf~c the extractability of lignins by conventional blearhinp chemicals (like /~hk)l-lne ~.hlorin~ f~inxi~ peroxide, etc.) (Viikari et al., "Bl~ .hing with Enzymes" in Biotechnology in the Pulp and Paper Industry, Proc. 3rd Int. Conf., Stockholm, pp. 67-69 (1986); Viikari et al., "Applic~tioll~ of Enzymes in Blea~hing" in Proc. 4th Int.
~,~ymp. ,Wood ~md Pulpfng ~emistry~ P~is; V~ 1~ pp~l51-15~1g87~ nt~liren et al.,"Hemicellulases and their Potential Role in Blf arhing" in International Pulp Bleaching Conference, Tappi Procee~ings, pp. 1-9 (1988)). The advantage of this improved hlea~ h~hility is a lower co~ ion of b]ea-~hing chemicals and lower ~llvil~nl ~ .ent~l loads or higher final hrightness values.

Identifcation and ~.~ol7tion of Chaetomium~lanases Thermostable xylanases have been char~tt-ri7~cl from Chaetomium thermophilum.
It has been found that strains of Chaetomium, and especially Chaetomium thermophilum, express and secrete thPrmost~ble xylanases that are useful e.g. in feed and baking as well as pulp and paper industry. These thermostabile xylanases are useful in impure forms such as an enzyme compositions that contain, or e~stqnti~lly are, the spent culture me~ m from growth of the org~ni~m- For example, the xylanase activity in enzyme compositions cont~ining the spent culture m~ m of Chaetomium thermophilum var. thermophilum, ALKO4123, was ~ x;~ ly active at 70 ~C. In another example, the xylanase activity in enzyme compositions cont~ining the spent culture me~ m of Chaetomium thermophilum var.
coprophilum, ALKO4243, was ~ x;~ ly active at 60 ~C. In another eY~mple, the xylanase activity in enzyme compositions co"l ~ g the spent culture medium of Chaetomium thermophi~um var. dissitum, ALKO4244, was m~x;,,,t,,,,ly active at 60 ~C. In a further another eY.~mrle, the xylanase activity in enzyme compositions C~ ,;,.g the spent culture medium of Chaetomium thermophilum, ALKO4265, was m~x;",l.~.. ly active at 60 ~C.
In acitliti~ n to the icl~ntific~tjon of useful enzyme compositions, four novel xylanases have been purified from the spent culture medium of Chaetomium thermophilum, ALKO4265. The molecular weights of these xylanases as detennin~ by SDS-PAGE are about 22 kDa,30 kDa,33 kDa and 54 kDa. The acc~d~y of the SDS-PAGE molecular weight ~letermin~tion is about ten percent, leading to an estim~tion of about 22 kDa ~ 2.2 kDa, 30 W O 97~2691 PCT~196/00670 kDa + 3.0 kDa,33 ld~a + 3.3kDa and 54 kDa + 5.4 kDa, ~ e-;Liv~ly. Possible glycosylation of these xylanases may cause wider variability in the molecular weights than the above m.ontic n~l values.The 30 kDa xylanase is probably dimeric in its native state as judged from molecular weight dr~ ;one by gel~Yrllleion chromatography. Each of these xylanases o S has an aLkaline pI as rl~ l by cl~ nldLofocusing. The accuracy of the pI ~. . ",;"~ n method is about 0.3 pH units. Each of these xylanases have a pH o~lhllulll and thl-rmostahility that are desirable for the enzyme-aided bleaching of wood pulp.The 54 lcDa, pI 8.9, xylanase shoved ",~x;",-.." activity at pH 6.2 and 80~C in the pH
range 6.2 to 7.9 and ~e~ ule range of 60 to 80~C.
The 33 kDa, pI 8.3, xylanase shoved m~ x; " ~- I I ~ . activity at pH 5.2 and 70~C in the pH
range 5.2 to 7.9 and temperature range of 50 to 80~C.
Surprisingly, both the 54 kDa and the 33 kDa xylanases of the invention are morethf-rmc~st~hle at pH 7.2 in purified form than is the spent culture m~ lm of the native host that secretes these ~l~yll~es.
The 30 kDa, pI 8.7, xylanase shoved ,,,~x;,,,.,.,~ activity b~w~ pH 5.2-6.2 and 70~C
in the pH range 5.2 to 7.9 and l~"~p .~I~,ie range of 50 to 80~C.
T_e 22 kDa, pI 9.3, xylanase shoved ms~imum activity at pH 6.2 and 60~C in the pH
range 5.2 to 7.9 and temperature range of 50 to 80~C.
Purification of a desired xylanase activity from a Chaetomium host of the invention is exemplified with ALKO4265. The four xylanases ~22,30,33 and 54 kDa) are purified from spent culture mloflium by passage through a series of chrom~tographic colurnns. For ex~mrl.o7 Phenyl-Sepharose 6FF can be used to separate xylanase activities in culture medium into difr~ pools.
The present invention comprehends a method for ~h.omic~lly treating plant biomass under cnn~1itione of high t~" ~ e of 50-80 ~C and pH 5-8, and especially 60 - 70 ~C, p~
6-7 and most preferably 70 ~C and pH 7 for a desired time, such as, for example, one hour.
ln a pr~ft;ll.d embodiment, plant biomass or pulp is treated with xylanases that are able to ~ hydrolyze xylan chains in lignocellulosic material at neutral or moderately alkaline pH and high (~ (above 60 ~C) followed by ble~-hing of the pulp with collv~ ;ons~l ble~ching çh~mie~l~ (like chlorine, chloride dioxid, peroxide, etc.).
Plant biomass is a composite m~t~ri~l coneietin~ prim~rily of a matrix of cellulose, hemicellulose, and lignin. Removal of the lignin coll.ponent is desirable during the W 097/22691 PCT~I96/00670 m~mlf~ctllre of paper pulp because of its brown color and tendency to reduce the strength of the paper product. Many processes have been developed for the removal of lignin. Typically, the wood pulp is treated with chlorine or other chPmic~ls in order to remove the lignin component and provide for a bri~htP~PA pulp. However, the toxic by-products of this S chemic~l tre~tmPnt negatively impact upon the health and stability of the cl~vhol.. llent into which they are released. Consequently there is a great need for developing ~ltPrn~tive~ more cllvhu~ .Pnt~lly protective techniques to achieve pulp hle~rhin~
In a ~lcr~ ,d emboAimP-nt, the process of the invention is carried out in vitro in the hemicellulose-cu~ pulp. The process involves placing the enzyme ~lcl,~dLion, culture medium, or concentrated mixture cu.. ~ i.. g xylanase into contact with the wood pUlp.
Routine calculations enable those in the art to determine the oplilllulll trç~tm~nt time depending upon the result desired, the concentration and specific activity of the xylanase enzyme used, the type and con-~Pntr~tion of pulp used, pH and t~ p~ c of the wood pulp, and other parameter variables.
Ihe method of the present invention may be applied alone or as a supplement to other tre~tm~nt~ that reduce the lignin content of wood pulp, increase its drainability and/or decrease its water retention. In a ~-lercll~,d embodiment, the present invention is used to enh~n~e bleachability of the wood pulp by trç~tmpnt of çhPmic~l pulps, i.e., those pulps co. . l ~ g lignin that has been ~hPmi~ ~lly modified through chPmic~ c~ l . . .rnt In a plcrcllcd embo~l;mPnt, the xylanases present in the enzyme compositions of the invention and used in the methods of the invention are preferably those of Chaetomium, and especially Chaetomium thermop*ilum, and in an especially preferred embodiment, one or more of the purified 54 kDa, 33 kDa, 30 kDa or 22 kDa er~ymes are used in the composition.
Therefore, according to the invention, there is provided enzyme compositions useful in feed and baking industry and for pulp and paper ~loce:i:,ing e.g. enzyme-aided ble?chin~
For ble~hing, the enzyme pl~dlions ofthe invention are plGr~,~dbly partially or cl mp]Ptely ~lPfici~Pnt in cellulolytic activity (that is, in the ability to completely degrade cellulose to glucose~ and enriched in xylanases desirable for pulp and paper processing. Such cellulolytic activity deficient ~ udlions, and the making of same by recombinant DNA methods, are described in US 5,2~8,405, incul~,oldlcd herein by reference.
When used to treat plant pulp, the enzyme l.~c~dlions of the invention may be utilized with any or all the usual ble?rhing chemicals, such as chlorine ~ xitlP~ hydrogen W O 97/22691 PCT~196/00670 peroxide, ozone, oxygen, sodium hydroxide, etc. The dosage, pH, tGlllp~,.dlulc, and time of enzyme LlcdLIllcll~ can all be easily varied so as to provide for m~illlu,ll effectiveness of the trÇsltm~nt For example, the pH may range from about pH S to about pH 8, the tGlll~c.dLtllc may range from about 50 ~C to about 80 ~, the time of tr~s~tmt-nt with the enzyme ~G~ ;on from about 0.5 hour to about 24 hours, and the dosage from about 20 to about 200 nkdtlg of pulp dry matter. Enzyme llGdLIllclll can be added to various blestrhinp processes, that are sequences of s~lcce~ive ch~nniGs~l trçsltm~nt stages. Typical blçs~ching processes are: 1) elementsll chlorine co..l~it.;.-P sequences that can be .G~lcsG,lted by e.g. a sequence of X(CtD)EDED, where X in-licsltçs a trçsltmen~ with an enzyme, such as an enzyme of the invention, C/D inflirs~tPs comhin~-(l treatment with çlPrnpntsll chlnrtn~o (C) and rhlnrin~ dioxide (D), E inrlirsltrs an stlksllin~ extraction and D in~ stt~s chlorine dioxide llc~ ; 2~
elemental chlorine-free (ECF) sequences that can be represented by e.g. a sequence of XDEDED (the two D steps used in the XDED ~se-luGnce shown in Table 2 are named Do and Dl); 3) total chlorine-free (TCF) sequences that can be le~lesGl~lGd by e.g. a sequence of XQPPP, where Q stands for chelation, i.e. metal removal stage, and P in~iirs~tps a hydrogen peroxide trçsttmrnt (PPP in-lirslt~c three sllcce~ive peroxide stages, P~P2P3 ). Typical TCF
seq~ nces also include di~r~,.G.~l other stages, like dirr~e~lL extraction stages (E, EO, EOP), ozone (Z), oxygen (O), ~)le~'iLI~;~e~1 peroxide stage (OP) etc.
The enzyme ~lep~ ;nn~ of the invention satisfy the ~G~uhGlllents of specific needs in various applications in the pulp and paper industry, including the ~lçb~rkinp of logs and refining of wood to reduce energy ~lPm~ncl~ in merh~nical pulp production. In pulp beating, the enzyme ~ dldLions of the invention can be used to increase çxtern~l fibrillation, and ~nh~nc-ecl of f~rilit~t~ swelling of the pulp fibers, and thus improve the paper making properlies of the fibers. The xylanases present in the enzyme pl~ udlion of the invention can also be used to improve pulp drainability and/or decrease water retPntion The enzyme ~ ;on~ of the invention may also be used as feed additives, and thus improve animal growth rate and feed conversion. When used in baking, improvement of the dough and the bread ch~r~rtrristics may be obtained.
To obtain the enzyme ~ A. ,-l ;on~ of the invention, the native hosts described above are cultivated under suitable con~1iti~n~ The desired Gn-~ylnes are se.,l~,led from the hosts into the culture medium, and the enzyme plGp~dlion is recovered from said culture medium by methods known in the art. The enzyme ~ ,~dlion may include the native org;~.ni~m, or the W O 97~2691 PCT~l96/OCC70 native organism may be removed from the culture medium by application of methods well known in the art. The enzyme ~IG~lions of the invention may be provided as a liquid or as a solid, for example, in a dried powder or gr~nul~r or liquid form, especially non~ sfing granules, or stabilized liquid, or the enzyme p~ inn may be othen,vise conccllh~l~d or stabilized for storage or use. It is envisioned that enzyme pl~ dlions c~ g one or more of the xylanases of the invention can be further enriched of made partially or compleately deficient in specific enzymatic activities, so as to satisfy the ,e~lu.lG.llents of d specific utility in various applications e.g. in feed, baking and pulp and paper industry. A mixture of enzyme activities secreted by a host and especially a fungus, can be chosen to be advantageous in a 1 û particular industrial applic~tinn, for example enzyme-aided ble~ hin~
The enzyme ~l~aldLions of the invention can be adjusted to satisfy the requirements of specific needs in various applications in the feed, baking and pulp and paper industry.
Blends may be yl~d-~d with other macromolecules that are not all se.;redLed from the same host (for example, other Gl.~yllles such as endogll~c~n~es, proteases, lipases, peroxi~ ee, oxi~1~sec~ amyl~es or cellobiohydrolases) or ch~tnic~l~ that may enhance the pc, r ., ~ nre7 stability, or buffering of the desired enzyme ~ aldlion. Non-dusting g~.~mllr~
may be coated. Liquid enzyme ~ ~dlions can be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid, accc,l.lillg to established methods.
It is an advantage of the invention t'nat the enzyme ~JlG~ 1 inn~ of the invention may be utilized directly from the culture medium with no further pnrifir~tion~
However, if desired, the xylanase of the invention may be further purified in accordance with convrntion~l con~1ition~, such as extraction, ~lc~ ilalion, chromatography, affinity chromatography, electrophoresis, or the like. As exemplified herein, purification of the 54 kDa and the 33 kDa results in an enzyme compositions that surprisingly have Pnh~nre~l thrtmost~hility when colll~d to the culture medium from which the enzyrne was derived.
Accordingly, when thermostability is especially desired, it is an advantage of the invention to use purified pl~l,~dlions of the 54 kDa and /or the 33 kDa enzymes in the enzyme composition.
30 ~ The invention is described in more detail in the following eY~mpl~s, These t~ lples show only a few coll~ L~ ~pplir~ti(!n~ ofthe invention. It is self evident for one skilled in the WO 97/22691 PCT~I96/00670 art to create several simi}ar applications. Hence the examples should not be i~ ,Led to narrow the scope of the invention only to clarify the use of the invention.

Exan~ples F~. , l~ l S Cultivation of C'h/lef ~ thermophile ALK04123, ALK04243, ALR04244 and ALK04265 Chaetomium thermophilum var. thermophilum ALKO4123 (CBS 733.95), Chaetomium t*ermophilum var. coprophilum ALKO4243 ~CBS 732.95), Chaetomium thermophilum var. dissitum ALKO4244 (CBS 731.95) and ALKO4265 (CBS 730.95, i~ ntifi~d as Chaetomium thermophilum by the Tnt~rns3tic~n~1 Mycological Tn~tih1t~/Biosystem Services) were m~int~in~cl on sporulation agar (ATCC medium 5).
For the ble~hin~ tests ALKO4244 was ~ iv~-~d in 750 ml of the following m~linm 1.5 % distiller's spent grain, 0.2 % soy bean meal, 0.15 % (NH4)2HPO4, 0.2 % KH2PO4, 0.05 % MgSO4x7H2O, 0.05 % NaCl, 0.1 % CaCO3, 0.03 % FeSO4x7H2O, 0.001 % MnSO4 pH
was adjusted to 6.5.
ALKO4123, ALKO4243 and ALKO4265 were cultivated each in 750 ml of the following m~-lillm 0.6 % Solka floc, 0.6 % distiller's spent grain, 0.3 % oat spelt xylan, 0.2 % CaC03, 0.15 % soy bean meal, 0.15 % (HN4)2SO4, 0.1 % barley bran, 0.05 % KH2PO4, 0.05 % MgSO4x7H20, 0.05 % NaCl, 0.05 % kace elem~-n1 solution 1, 0.05 % trace elemf-nt solution 2, 0.03 % KNO3, pH was adjusted to 6.5. The trace element solution 1 c-nt~inecl 1.6 g/l MnSO4, 3.45 g/l ZnSO4x7H20, 2 g/l CoCl2x6H20. The trace cl ~ solution 2 c.~ cl 5 g/l Feso4x7H2o-All cultivations were incubated at 45 ~C for 3 days in a rotary shaker at 250 rpm.
For pllrifi~tinn of xyl~na~es of ALKO4265, 1 1 r~ t ~ cultivations (Biostat M, B.
Braun, Mel~l~n~çn, Gerrnany) were p~ led in a cultivation me~illm c~ 0.3 %
Roth xylan, 3 % soy bean meal, 4 % whey, 0.5 % (NH4)2SO4, 0.1 % NaCl, 0.5 % KH2PO4, 0.2 % CaCO3, 0.05 % trace element I (see above), 0.05 % trace element 2 (see above), 0.05 WO97~2691 PCT~196/00670 % MgSO4. A 10 % in(-clllnm of a shake flask cultivation was used. The pH was m~int7~in~(1 at 6.6 +/- 0.4 by addition of ~mmQ}li~ (12.5 %) and ph~srh~ric acid (17 %). The ferm~nter was stirred at 700 rpm and the air flow was 1 liter/min. The growth temperature was 45 ~C
and growth time 1-3 days.

F ~n~~rl~o 2 ur~ Prof les of the Culture Fil~rates of Chaetomium fhermophilum The culture filtrate of ALKO4123, ALKO4243, ALKO4244 and ALKO4265 were tested for thermal stability by inc--h~ting samples at 60, 70 and 80 ~C at pH 7.2 for 60 min with Roth (No. 7500) birch wood xylan. The released xylose was measured as in the xylanase activity measu~ lGll~ method described by Bailey,M et al., J.Biotechnol. 23:257-270 (1992).
The results are shown in Figure 1. ALKO4123 showed ~ xill~ ll activity at 70 ~C, while the other three culture filtrates showed m~xi~ activity at 60 ~C.

F~nmrl~ 3 Exp~ ,.cnts Using Chaetomium thermophilum Culture f ltrates in One Stage Peroxide R/f 7rhi~g Bleaching e~ ent~ were done to fl~l~ "~ e the usefulness of Chaetomium thermophilum var. thermophilum CBS 733.95 (ALKO4123), G thermophilum var. dissitum CBS 731.95 (ALKO4244) and G thermophilum CBS 730.95 (ALKO4265) xylanase activityco,~ g culture filtrates in one-stage peroxide ble~ching 2Q Culture filtrates (Example l) from growth of C thermophilum ALKO4123,ALKO4244 and ALKO4265 were added to Finnish oxygen-~çlignified softwood kraft pulp (kappa number 16, briphtn~oee 33 %) in the arnount of 100 nkat / g of pulp dry matter.
Xylanase activity G~ ed as nkat was measured according to Bailey et al., J. Biotechnol.
23:257-270 (1992) by using Roth birch wood xylan (no. 750Q) as substrate at 70 ~C and pH
7.Q with a 6Q Ill;,.l~les inctlb~ti~n time. The enzyme trç~fment~ were done at 70 ~C and pH 7 for one hour. Reference pulp was treated in the same way but without enzyme addition.

WO 97/22691 PCT~196/00670 Bllo~rhin~ was ~lÇ~ ed with QP~ sequence. The chelation stage (Q~ was p~ ed by adding EDTA to 0.2 % of pulp dry matter and it was carried out at 3 % pu~p consistency.
Ble~ehin~ ch~omic~l~ in the hydrogen peroxide stage (P~) were the following: 3 % H20~, 3 % NaOH, 0.2 ~/O dietnylene ~ yc~ 'etir acid (DTPA) and 0.5 % MgS04. Conditions of the Q and Pl stages as well as the results of the ble~ hin~ nt~ are shown in Table 1 (a and b).

Table la Rere~ G ~ ALKO4123 Enzyme ~- G~
Con~i~t~ncy, % 3 5 3 5 Retentinn time, hours Enzyme dosage, nkatlg 0 100 T~ dl~e~oc~s~/end 72/69 68/68 pH, start / end 7.3/7.3 7.3/7.4 1 5 Q-stage Consistency, % 3 3 Ret~ntinn time, hours Temperature at the end, ~C 63 62 pH at the end 4.9 4.9 FnTA, % of dry matter 0.2 0.2 Pl -stage Consistency, % 10 10 pcet~qntiQn time, hours 3 3 T~~ a~ , ~C 80 80 pH, start / end 11/10.5 11.4/11.3 Peroxide dosage, % 3 3 Peroxide con~llm~1 % 2.9 2.9 Brightn~s, % 57.1 58.8 Kappa number 9.7 8.5 WO 97/22691 PCT/F196/0û670 Table lb Rcfel . ~e ALKO4244 AI,KO4265 Enzyme Ll ~ ~, t Con~i~tfncy, % 3 5 3 5 3 5 S Retentiontime,hours Enzyme dosage, nkat/g 0 100 100 T~ LUI C, ~C, start /end 67/70 70/72 70/71 pH, start / end 7.0/6.9 6.9/6.9 7.1/7.0 Q-stage Ct n~i~ene~y~ % 3 3 3 Retentiontime, hours T~ .. ul~ at ~he end, ~C 57 60 58 pH at the end 5.3 5.4 5.2 EDTA, % of dry matter 0.2 0.2 0.2 Pl-stage Con~i~t~n~y, % 10 10 10 Retentio~ time, hours 3 3 3 Tt;~ dLul~:, ~C 80 80 80 pH, start / end 11.7/10.8 11.6/10.7 11.7/10.7 Peroxidedosage, % 3 3 3 Peroxide ct n.c~lm~A % 2.6 2.6 2.6 Bri~htr~e~, % 65.2 67.7 66.7 Kappa number 8.3 8.0 7.4 As can be seen in Table l(a and b) the use of xylanase activity co.l~zl;..i..~ culture filtrates of G tJ~ermophilum ALKO4123, ALKO4244 and ALKO4265 as ~l~;L~ nt in theone-stage peroxide blç~rhing clearly i-lcl~,ased the brigh~nf c~ obtained without increasing the amount of hydrogen peroxide that was co~ -ne-1 Also lignin content of pulps was decreased as evidenced by the reduction of kappa number.

F~ e4 Experiments Using Chaetomium thermophilum Culture Filtr~ltes in Cfilorine Dioxide (ECF) ~ rehing and ThreeStagePeroxide fTCF) Rleac~hing -CA 02240391 1998-06-1~
W 097~2691 PCT~196/00670 Bleaching c;~ ,.ents were done to cletPrmine the llseftlln~s~ of Chaetomium thermop~ilum CBS 730.95 (ALKO4265) and CBS 731.95 (ALKO4244) xylanase activity cu.~ .g culture filkates both in ECF (elementary chlorine free) and in TCF (totally chlorine free) ble2~chin~ of pulp.

S ECFBle(rc hing Culture filtrates (Example 1) were added to Firmish oxygen-~toli nified softwood kraft pulp (kappa number 15.5, viscosity 900 mVg, brightn~ss 43.5 %) in the arnount of 100 nkat / g of pulp dry matter. Xylanase activity ~ylessed as nkat was lnea~u.ed acco.-li..g to Bailey, M. et al., J. Biotechnol. 23:257-270 (1992) by using Roth (no. 7500) birch wood xylan as ~ul~ dLe at 70 ~C, pH 7.0 with a 5 minntes incubation time. The enzyme trl ~tm~nt~ were done at 70 ~C, pH 7 for one hour. Reference pulp was kept under the same con~liticm~
without enzyme ~ liti~n After the enzyrne Llc ~ i bl~ hing was y.,lro~ ed with DoE Dl se~ e, where Do stands for the first chlorine dioxide stage, E aLkali extraction and D I the second chlorine dioxide stage. Ble~ching conditions and results are shown in Table 2.

Table 2 Reference ALKO4244 ALKO4265 Enzyme Ir. ' : t Con~i~tc-nry, % 3 3 ~t~ntion time, hours Enzyme dosage, nkat/g 0 100 100 T~11Y~lU1G, ~C 70 70 70 pH, start / end 7.0/7.4 6.9/7.2 6.9/7.1 D0-stage Conei~t~nry, % 3 3 P~-t~ontion tirne, hours Ten~ldlul~, ~C 60 60 60 ClO2-dosage, % 2.3 2.3 2.3 ClO~, cc.. ~ rl, % 2.3 2.3 2.3 pH at the end 2.5 2.5 2.5 WO 97~2691 PCT~I96/00670 E-stage Conci~t~ncy, % 10 10 10 Retention time, hours Te~ eld~ufe, ~C 70 70 70 S NaOH, % 1.5 1.5 1.5 pH at the end 10.8 10.8 10.8 Bri~~htnt?c~, % 58.8 62.3 62.5 Kappa number 6.6 5.4 5.4 Viscosity, ml/g 860 850 800 D,-stage Con~i~ten,y, % 10 10 10 Retentiontime, hours 3 3 3 Te~ d~ ,oc 60 60 60 C1O7-dosage, % 2.0 2.0 2.0 ClO2, c~ n~l-me-1, % 2.0 2.0 2.0 pH at the end 3.4 3.2 3.2 P.rightn~ , % 79.1 82.4 82.5 Kappa number 2.1 1.5 1.5 As can be seen in Table 2, the enzyme pretre~tm~nt~ enhanced lignin removal, which is evidenced by the re-1nrtinn of kappa ~ b~l~. Also hri~htnt?ss values of the final pulps were _igher coll~cd with the reference although chlorine dioxide co~"" ,l.~ion was not increased.
Reduction in viscosity with ALKO4265 was due to cellulase activity present in the enzyme ~dLion.

TCF Rl~n<~hi~ g Culture filtrates (Example 1) from growth of Chaetomium thermop*ilum ALKO4244 and ALKO4265 were added to Finnish oxygen-~eli~nified softwood kraft pulp (kappanumber 16, brightn~ss 33 %) in the amount of 100 nkat / g of pulp dry matter. Xylanase activity ~ lcssed as nkat was measured according to Bailey et al., J. Biotechnol. 23:257-270 (1992) by using Roth (no. 7500) birch wood xylan as substrate at 70 ~C, pH 7.0 with a 60 mimlf~s incubation time. The enzyme tre~trn~ont~ were done at 70 ~C, pH 7 for one hour.
Reference pulp was treated in the sarne way but without enzyme addition. Bleaching was WO 97/22691 PCT~196/00670 p~lr~)l,llcd with QP~P2P3 sequence. The chelation stage (Q) was l~clr~lllled by adding EDTA
to 0.2 % of pulp dry matter and it was carried out at 3 % pulp consistency. The three c~ i ve hydrogen peroxide stages (PlP2P3) were carried out the same way except that after each stage, one-third of the pulp was removed for testing. Ble~ hing chemicals in P stages S were the following: 3 % H2O2, 3 % NaOH, 0.2 % diethylene tri~l~inG~ retic acid (DTPA) and 0.5 % MgSO4. Conditions of the Q and P stages as well as the results of the ble~chinp;
experiment are shown in Table 3.

Table 3 Reference ALKO4244 ALKO426 Enzyme treatment C~ n~i~t~ncy, % 3.5 3.5 3.5 Retention time, hours Enzyme dosage, nkat/g 0 100 100 Tt;ulp~"dlu~e, ~C 68 68 68 pH, start / end 7.3/7.3 7.1/7.0 7.0/6.9 Q-stage Cnn~i~t~ncy, % 3 3 3 Retentiontime, hours T~ a~ul~at the end, ~c 56 58 58 pH at the end 5.4 5.7 5.3 EDTA, % of dry matter 0.2 0.2 0.2 P~-stage Con~i~t~.ncy, % 10 10 10 Retention time, hours 3.5 3.5 3.5 T~lllp~,.dlul~, ~C 80 80 80 pH, start / end 11.2/10.7 11.4/10.8 11.4/10.7 Peroxide dosage, % 3 3 3 Peroxideconsumed, % 2.3 2.3 2.4 Bri~htn~, % 61.2 62.7 62.5 Kappa nurnber 9.6 9.3 g o P2-stage Cnn~i~tency, % 10 10 10 Ret~ntion time, hours 3 3 3 T~ .aLuL~, ~C 80 80 80 pH, start / end 11.4/11.2 11.3/11.0 11.3/11.0 Peroxide dosage, % 3 3 3 Peroxide con~-lm~-~l % 2.3 2.2 2.2 Bri~htnes~, % 67.7 69.5 69.4 Kappa number 8.5 7.8 8.0 P3-stage Con~i~t~ncy, % 10 10 10 Retention time, hours 3 3 3 Tt;lllpcl~lule, ~C 80 80 80 pH, start / end 11.4/10.9 11.3/10.6 11.3/10.6 Peroxide dosage, % 3 3 3 Peroxide con~llm~.-l, % 2.3 2.2 2.2 Brightn~, % 73.6 74.7 74.6 Kappa number 7.6 6.8 6.7 Viscosity, ml/g 730 760 720 Total peroxide consurnption, % 6.9 6.7 6.8 As can be seen in Table 3 the use of xyianase co~ .g culture filtrates of Chaetomium thermophilum ALKO4244 and ALKO4265 as a l~lc;L~ nt in the peroxidebleaching clearly increased the brightn~ obtained without increasing the arnount of hydrogen peroxide that was con~um~A Enzyme L.~ lf.~ did not affect the viscosity of the pulps.

W O 97/22691 PCT~196/00670 Example 5 P~rif cation of xyfanases from Chaetomium thermophilum S The culture filtrate of 1 -3 days f~rnt-qnt~tions of ALKO4265 (Example 1) were used for purification purposes.

Defermination of profein conc~,.l,ul~on For protein conr~ontr~tion mea~ ents, the standard Bio-Rad assay (based on the method of Bradford, M., Analytical Biochemistry 72:248-254 (1976)) standardized with g~nm~-globulin was used. During gel-exclusion chromatography runs, protein c~ n~ ;
was followed by measurements at A280.

Activity I ~ ,.b During fçrment~ttions and enzyme pllrific~tion~ xylanase activity was measured according to Bailey, M. et al., J.Biotechnol. 23:257-270 (1992) by using Roth (No 7500) birch wood xylan as ~ at 60~C, pH 6.5 McIlvain s buffer with S min in~ ticm The activity is t;~G~ed as nkat.

Polyacrylamide gel electrophoresis (SDS-PAGE~

Polyacrylamide slab gels (12 % or 14 %) were run as described by T ~entmli, U.K.~ Nature 227:680-685 (1970) in the presence of 0.1 % sodiurn dodecyl sulphate and stained with Coomassie Brilliant Blue. Molecular mass standards (Bio-Rad, Low Range p~
SDS-PAGE Standards) were used to çstim~te molecular masses of sample proteins.

T~.,.y~, ul~re and pH - prof les -W O 97~2691 PCT~196/00670 Tc..l~.dl~lie and pH profiles were obtained by inrllh~ting ~mrlçc at defined tt;~ c~dLules and pH, obtained by using McIlvains buffers, for 1 h with Roth (No. 7500) birch wood xylan as described in activity measu,~me.

Det~, ~ti~n of pI

Cllr~ vfocusing of purified xylanases was performed on a mono P column (0.5 x 20 cm, Ph~rm~ri~) cqllilihr~t~o~ with 25 or 70 mM Tris-acetic acid pH 9.5 at 30 ml/h. Elution was accomplished with Polyl,ulr~, 96 ~Ph~3rm5~ri~) diluted 1:10 with distilled water and adjusted to pH 6.2 with acetic acid. Fractions of 0.5 to 1 ml were collected and both the xylanase activity and pH of each fraction was measured. The pI estim~t~s of the purified proteins are means of two s~al~ runs.

Purif cation of x~ylanases from A4265 A~loxilllately 900 ml of ALKO4265 culture filtrate was adjusted to pH 7.5 with 1M NaOH and HCl. Unless otherwise stated, samples were kept at +4~C. In order to run hydrophobic interaction chromatography, ~mmonillm s~ hz-t~? was added to a finalconf~ntr~til~n of 0.5 M. Alternatively culture filtrate was adjusted to pH 7.5 and EDTA added to 1 mM co~ ntr~t;on followed by preciItit~ted with 45 % (wlv) ~mm~niurn s--lrh~t~ The Le was s~h.~ l by centrifugation at 10 000g for 20 min. The ~3reci~iL~L~ was suspended in 20 mM Tris-HCl pH 7.5 col Il ~; l l i, ,g lmM EDTA and ammonium sulphate was added to a final collce~ ion of 0.5M.
Culture filtrate treated as above was applied on a Phenyl-Sepharose 6FF column (Phzlrmz~ 5 cm x 12 cm) eq -ilihr~t~l at room tell~ldl~ with 20 mM Tris-HCl buffer pH
7.5 cont~ining 0.5 M ammonium slllrh~tP (buffer A). Elution was ~lr~ lled at 30 ml/min with a linear gradient of buffer A to buffer A without ammonium sulphate (buffer B) in 10 min. Elution with buffer B was cnntinl~l for a additional 10 min followed by a linear gradient of buffer B to buffer B c~ llt~ining 60 % ethylene glycol (buffer C) in 20 min. Elution with buffer C was prolonged for a fi~ther 10 min. Fractions of 10 ml were collected and assayed for xylanase activity and protein concentration.

W 097/Z2691 PCT~196/0~670 Three separate pools (see Figure 2) co..~ g xylanase activity were obtained fromthe Phenyl-St;~>h~ose 6FF run. Pool TXl eluted with ayy~o~ullately 20 %, pool TX2 with 40 % and pool TX3 with 60 % ethylene glycol. Samples of each pool were cnnrentr~t~d with an Alnicon con~ ur (10 kDa cut offmem~r~tn~s). The concçntr~tinn sllrcee-le~l only with pool TXl, r~ ltin~ in a 10 fold concentration and over 80 % recovery of xylanase activity.
Conr~n~tion of TX2 and TX3 resulted in only about 30 and 15 % ltco~ .es, respectively.

Further purif cation of pool lXl Conf~ d TXl was run on a Superdex G-75 HiLoad column (Ph~nn~ri~t, 2.6 x 60 cm) equilibrated at room t~ y~.dLul~ with 20 mM disodiurn hydrogen phosphate pH 8.6 co.. ~ 100 mM NaCI at 120 ml/h. Fractions of 6 ml were collected and assayed for xylanase activity. Two xylanase activity cu--l;~ pools were obtained and run on SDS-PAGE.
The early eluting peek-pool, eluting close to the 68 kDa BSA used as standard in the Su~;-dc;~ G-75 run, contained a protein of ~p.. ~x; .~ ly 30kDa as f~stim~f~d on SDS-PAGE.
Thus this xylanase seems to be dimeric in its active forrn. The pI of this xylanase is 8.7 as by cl~o..~ ofocltein~
The second, later eluting peek-pool contain mainly a protein of a~.uxillla~ely 54 kDa.
Since there was two main bands in this sample pool, an aliquot was further cu~lr~ rd with a ~n1rirt~n microcf .t~ (JI (10 kDa cut off) and run on a Superose 12 column eqllilihr~ted with 20 mM Tris-HCl pH 7.5 cû.. ~ g 100 mM NaCl at 30 ml/h. The xylanase activity cul .I ;1; . .; . .~ fractions were pooled. A sample of this pool was run on SDS-PAGE. It showed a single band of a~u2~ L~;ly 54 kDa. Elution from the Superose 12 column intlic~tt-d that the enzyme is mc-nnm~ric in its active form. The pI of this 54 kDa xylanase, as cleterminPd by chlulllalofocusing, is 8.9.

Further purif cation of pools IX2 and IX3 Since the collc~ dlion of TX2 and TX3 on Amicon failed, both pools were conc~ d by ammonium sulphate precipitation (45 % w/v) after ~ itinn of EDTA to afinal concentration of 1 mM. The precipitates were suspended in 20 mM Tris-HCL pH 7.5 CA 0224039l l998-06-l5 W 097~2691 PCT~I~CC670 and run on a Superdex G-75 column equilibrated with 20 mM Tris-HCI pH 7.5, c~ ;"~
100 mM NaCI, at 120 ml/h. Fractions of 6 ml were collected. The xylanase activity Cont~inin~
fractions of both runs were pooled separately and assayed on SDS-PAGE.
The TX2 Superdex G-75 pool showed four main bands. Further pl-rific~ti~ n was S accomplished by con~ g an aliquot by USing Centricon micro conc~ aLol~ (10 kDa cut off) and running a Superose 12 column as above. The xylanase activity cont~ininp~
fractions were pooled an assayed on SDS-PAGE. Purified TX2 showed a protein of o~ ately 33 kDa. Elution from the Superose 12 column in~lir~te~l that the xylanase is monomeric in its active form. A pI of 8.3 was estim~t~rl from cl~ lvfocusing runs.
The TX3 Superdex G-75 pool shoved a homogeneous 22 kDa band on SDS-PAGE
and a pI of 9.3 as estim~t~l by cl~u.l.~l~focusing. Also this enzyme is mon--m~ic in its active form, as judged from its elution from the Superdex G-75 column.

Amino acid seqt~ 7g of the 22 kDa and 54 kDa :~lanases The 22 kDa band of a dried SDS-PAGE gel was cut out and subjected to Edman degradation in a gas-pulsed-liquid-phase sequencer (l~lkkin~n & Til~m~nn, J. Prot. Chem.
7: 242-243 (1988)). The 36 amino acid N-termin~l sequence obtained is shown in Table 4 (24kDa). A BLAST search (NCBI, National Center of Biological Inforrnation) revealed sequence i~it?ntiti~s to many xylanases. The highest identity scores were obtained for the putative N-t~rrnin~l seq~l~nres of Emericella nidulans xyna and xynb (both 64 % identity) and Aspergillus kawachii xynb (64 % identity). The sequence identity to the N-t~rrnin~l sequence of Schizophyllum commune xyna was 55 %.
A sample of 54 kDa xylanase was subjected to el~y~ lic digestion with 2 % (w/w) modified trypsin (Promega) in 1 % ~mm~nillm bicarbonate for 3 hours at 37 ~C. The peptides obtained were se~ cl by reversed-phase chromatography on a 0.2l cm x 15 cm Spherisorb SS (5~m particle size; 30 nm pore size) colum using a linear gradient of acetonitrile (3 - 100 % in 100 min) in 0.1 % trifluoroacetic acid at a flow rate of 200 ,ul/min. Selected peptides were subjected to Edman ~ tion as above. Two peptide sequences were obtained from the 54 kDa xylanase. The 15 amino acid peptide 54 kDaA ~Table 4) shoved 67 % identity to a internal seqll~n~e of C~ostridium t~ermocellum xynz and 66 % identity to Cellulomonas~mi xylanase B. This peptide shoved 60 % identity with the thermostable 50kDa xylanase WO 97~2691 PCT~I96/00670 se~uence of Actinomaduraf~exuosa (CA 2,154,945). The 13 amino acid peptide 54kDaB
(Table 4) shoved 76 % identity to the xylanase xynF of Pseudomonasfluorescens arld 69 %
identity to ~e xylanase xynB of Cellovibrio mixtus and xylanase xynB from ~hermotoga neapolitana as well as xylanase xyn33 of Megnaport~ea grisea. A 70 % identity was also S found to the sequence of the th~rm~ stzlhile 50kDa xylanase of ~ctinomadura flexuosa xylanase (CA 2,154,945).

Table 4 24kDa GLY-GLY-THR-PRO-SER-SER-THR-GLY-TRP-HIS-GLY-GLY-TYR-TYR-TRP-ASN-GLY-ASN-ASN-GLY-ASN-TYR-GLY-54kD~A GLY-ALA-PRO-ILE-ASP-GLY-VAL-X-PHE-GLN-X-HIS-LEU-ILE-VAL-54kDaB LEU-TYR-TYR-ASN-ASP-TYR-ASN-LEU-GLU-TYR-X-ASN-ALA-T~ ... h~re and pH Prof les of Purif ed Xylanases The te~ el~lLu~e and pH profiles of the purified xylanases (inrmh~tion time 60 min) are shown in Figure 3 (A to D). A~p~t:lllly the 22 kDa xylarase (Figure 3A) is less thl~rmost~-hle than the 30 (Figure 3B), 33 (Figure 3C) and 54 kDa (Figure 3D) xyl~n~ePc, especially at higher pH values. The 54 kDa xylanase showed an ms~X;~ activity during the lh inrllh~tinn at 80 ~C and pH 6.2. The ~ e~ e values for the 33 kDa xylanase was 70 ~C and pH 5.2 and for the 30 kDa xylanase 70 "C and pH 5.2 - 6.2. The 22 kDa xylanase showed m:.x;."~".. act-ivity at 60 ~C and pH 6.2.

W O 97/2Z691 PCT~I96/00670 F.~n~rl~6 An ECF Rl~n(~hi~tg E~f~ t Using 22 kDn, 33 kDa and 54 kDa Xylanases purif ed from Chaetomium thermophilum AL1~04265 A ble~ehing ~ ent was carried out to det( rmine the usefulness of 22 kDa, 33 S kDa and 54 kDa xylanases purified from Chaetomium thermophilum Al,KO4265 (Fx~mple S) in ECF (elPment~ry chlorine free) bleaching of kraft pulp.
Purified 22 kDa, 33 kDa and 54 kDa xylanases (Example S ) were added to Firmish oxygen-c1elignified softwood kraft pulp (kappa number 15, viscosity 930 ml/g and brightness 33 %) in the arnount of 150 nkat / g of pulp dry matter. The enzyme tre~tment~ were done at pH 7 and 70 ~C for one hour. Re~l~.. ce pulp was kept under the same conditions without enzyme addition. After the enzyme tr~tment.~ ble~rlling was perfrrmed with DoED,sequence, where Do means the first chlorine dioxide stage, E means ~lk~lin~ extraction and Dl means the second chlorine dioxide stage. Blearhin~ conditions and results are shown in Table 5.

Table 5 R~ .,c~ 22 kDa 33 kDa 54 kDa Enzyme ll ~ I
Cnn~ nry, % 3 3 3 3 l~etenti~n time, hours Enzyme dosage, rlkat / g 0 150 150 150 T~ Lult;,~C 70 70 70 70 pH, start/end 6.9/7.3 7.1/7.4 7.1/7.4 6.9/7.3 D0-stage Consistency, % 3 3 3 3 Retention time, hours Tt;lllpcl~lLult;, ~C 60 60 60 60 ClOz-dosage, % 2.25 2.25 2.25 2.25 ClO~ con~l-me~, % 2.24 2.24 2.23 2.Z3 pH, start / end 3.0/2.5 3.0/2.5 2.9/2.5 2.9/2.5 W 097/22691 PCT~lg6/00670 E-stage C~on~iet~.ncy, % 10 10 10 10 p~et ntion time, hours Tc~ claLul~:, ~C 70 70 70 70 NaOH, % 1.4 1.4 1.4 1.4 pH at the end 10.8 10.8 10.8 10.9 Bri~htn~, % 55.8 59.6 60.1 60.2 Kappa number 6.3 5.3 S.1 5.0 Dl-stage Con~i~t~on~y, % 10 10 10 10 Retention time, hours 3 3 3 3 Tt;ll~e~Lu~ ~C 60 60 60 60 ClO2-dosage,% 2.0 2.0 2.0 2.0 Cl02 c~ n~lm~-1 % 2.0 2.0 2.0 2.0 pH at the end 2.9 2.7 2.7 2.7 Bri~h1nto,sc, % 78.3 81.5 82.2 82.4 Viscosity, ml/g 900 900 840* 730*

As can be seen in Table 5, after the ~lcllC;t~ nt at p~ 7 and 70~C with Chaetomfum thermophil~m ALKO4265 xylanases 22 IcDa, 33 kDa and 54 kDa, lignin removal in pulps 20was enh~n~e~l (as evidenced by the reduction of kappa ~ lh~l~) when compared with the reference pulp. Also the brightn~o~c values of the final pulps were 3 - 4 units higher than reference, where enzyme was not used, although the cnn~lmrtion of chlorine dioxide stayed on the sarne level. The reduction in viscosity with 33 kDa (*) and 54 kDa (*) xylanase ellL was due to co,-f;~ ;"g cellulase activity present in these enzyme ~ ualions.

F~ , * 7 A TCF ~le~e~ g E~_,.".~.~t using ~lanases from thefungus Ch~etomium thermophilum A ble~chinf~ ~-Xl~r- ;lllfnt can be carried out to clet~rrninf~ the usefulness of 30C~aetomium thermophilum xylanases in TCF (totally chlorine free) bleaching of pulp.

WO 97/226gl PCT~196/00670 Xylanase plG~ Lions are added to softwood or hardwood pulp in the amount of 20-200 nkatlg of pulp dry matter. The enzyme treatrnents are performed at pH 5 - 8 and at 50 -80~C for one to ~ree hours. Reference pulp is kept under the same conditions without enzyme addition. After the enzyrne L~ bl~ .hing can be performed for example with QP, QPP
or QPPP sequ~ nre (also other suitable sequences can be used), where Q stands for chelation stage and P stands for hydrogen peroxide stage.

WO 97/22691 PCT~I96/00670 ~U~N~ LISTING

( 1 ) ~N~ ~T ~ lN~'~.~ ~TION:

(i) APPLICANT:
(A) NAME: Primalco Ltd S tB) STREET: Valta-akseli (C) CITY: Nurmijarvi (E) C'UUN 1 ~Y: Finland (F) POSTAL CODE (ZIP): FIN-05200 (G) TELEPHONE: +3S8 9 13311 ~) TELEFAX: +358 9 133 1546 lii) TITLE OF l~v~LlON: Novel xylanases and uses thereo~

(iii) NUMBER OF S~U~N~S: 3 (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. 30 (EPO) (2) lN~o~--TIoN FOR SEQ ID NO: 1:

(i) ~QU~N~ CHARACTERISTICS:
(A) LENGTH: 36 amino acids (B) TYPE: amino acid (C) STP~AN~ )N-'-c S: 8ingle (D) TOPOLOGY: linear ~ ii ) MOT.~TTT.~ TYPE: peptide -(vi) ORIGINAL SOURCE:
(A) ORGANISM: Chaetomium thermophilum (B) STRAIN: CBS730.95 (ix) FEATURE:

WO 97/22691 PCT~I96/00670 (A) NAME/KEY: Peptide (B) LOCATION:l..36 (D) OTHER lN~-O~ ~TION:/label= 24kDa_peptide_A

(Xi) ~QU~N~ DESCRIPTION: SEQ ID NO: l:

S Gly Gly Thr Pro Ser Ser Thr Gly Trp Eis Gly Gly Tyr Phe Tyr Ser l 5 l0 15 Phe Trp Thr Asp Xaa Gly Gly Glu Val Asn Tyr Trp Asn Gly Asn Asn Gly Asn Tyr Gly (2) lN h'O~ ~TION FOR SEQ ID NO: 2:

( i ) ~U~N-~ CHARACTERISTICS:
~A) LENGTH: l5 amino acids (B) TYPE: amino acid (C) STRP~nFn~5S: single ~D) TOPOLOGY: linear lii) MOLECULE TYPE: peptide (vi) O~T~T~L SOURCE:
(A) ORGANISM: Chaetomium thermophilum (B) STRAIN: CBS730.95 ¦ix) FEATURE:
(A) NAME/KEY: Peptide (B~ LOCATION:l..15 (D) OTHER INFORMATION:/la~el= 54kDa_peptide_A

CA 02240391 1998-06-lS
W O 97/22691 PCT~I96/00670 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gly Ala Pro Ile Asp Gly Val Xaa Phe Gln Xaa Hi~ Leu Ile Val (2) INFORMATIQ~ FOR SEQ ID NO: 3:

(i) ~UUKN~ CHARACTERISTICS:
~A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STR ~n~nN~fi ~: 8 ingle (D) TOPOLOGY: linear ~ii) MOLECULE TYPE: peptide (~i) ORIGINAL SOURCE:
(A) ORGANISM: Chaetomium thermophilum (B) STRAIN: CBS730.95 (ix) FEATURE:
(A) NAME/KEY: Peptide (B) LOCATION:1..13 (D) OTHER INFORMATION:/label= 54kDa peptide_B

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

Leu Tyr Tyr A6n Asp Tyr Asn Leu Glu Tyr Xaa Asn Ala WO97~Z691 PCT~196/00670 INDICi~l~ONS RUEIAIING TO A DEPOSI1lED ~,~CROORGAU~ISM
(PCTRule13b~3 A. The rn_dc beiow rei~le lo toe ~uu~ reiezred ~o ~n ~he on page 6 . Iine 1 ~i .
B. IDEI~ICATION OF DEPOSlTFurmer deposils are idemified on an qrtAi.jrm_l sheel ~3 zne of ..~
Centraalbureau Voor Schimmelcultures Address of d~.,.o ..~. ~ d . ~inc~ tn~ postal coaic ar~ ccur~
Oosterstraat 1, 3752 SK BAARN, The Netherlands Dale of ocwsit I Acce~islon ~umDer 8 November 1g95 ! CBS 733.95 C.ADDITIONALlNDICATIONS~1cwcblank;fno~aDI~caD1c~ This in;r~ m is~ onan~t~ ~q;sheel Cl Regarding those designations in which a European patent is sought, a sample of the deposited microorganism will be made available only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC~
until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or is deemed to be withdrawn.This request also applies to other designated countries in which similar or corresponding provisions are in force.
. .
D. DESIGNATED STATES FOR W9tlCII INDICATIONSAREMADE ~ifIn ir~icaaonsarcnotforall~e~lS~s~

. SEPARATE FIJRNISE~NG OF TNDICA'IIONS (Ieav DiarL' Jf r~ appiicaalel Ihe ' - ilstedbe~owwiliblesuDm~ttedto-he ~ al~u~al Bureauia~er~spcc~ivLncrcr~cra~na t~rcof~ c~., ~ccer~on ~umKr afl;i~si~') For recelv~ng Office use onlv For l. ~ t Bureau use only 5~ This sheel w~s receiYed wilb the ~ ; 0 Tbis sheel w~ts received b~ lhe l ~ J Bure~u on:

~ h -~ofL~iCer ~ ~ rd officer ~ P~T~rt~ u~viss2) WO 97/22691 PCT~196/00670 INDIC;~I~ONSRUEIAIlNG TO A DEPOSIl~ED ~CROORGAUNISM
(PCT Rulc13b~) A. The made beiow rei1le Io tne ~ u~ w reterred lo m thc .j~
on page 6 . Iint 18 B. IDENT~ICAnON OF DEPOSlT Funner deposi~s ~re identified on ~n 7dA;~n~t~l sheet N~me of .i~
Centraalbureau Voor Schimmelcultures Addressofd.,..,s.~.~ . /mcica~ny po5alcoaican~icoun~
Oosterstraat 1, 3752 SK BAARN, The Netherlands Da~e of der osil Ac~sslon NumDcr 8 November 1995 CGS 732.95 C. ADDlTlONAL INDICATIONS /lcavcblanlci n<xappiicaok) ~bis jnfnT~ jnn IS _ - on ~n Irtrt;~ir~n~l sheet Regarding those designations in which a European patent is sought, a sample of the deposited microorganism will be made available only by the issue of such a sample to an expert nominated by the person requesting the sample (Rule 28(4) EPC-until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or is deemed to be withdrawn.This request also applies to other designated countries in which similar or corresponding provisions are in force.
D. I~ESIGNATED STATES FOR WE~CII ~l)ICATIONS ARE MADE (iflr~ imfica~ons arc ~ o~ jor ail ~cn~a-c~ atcs) E. SEPARATE FURN~S~ING OF INDICATIONS (Icavc biank i~ no~ appiicaolcl ~e-- A-- ~ sledbc~owwiil besuDmlnedlo be J-- - - ~ - I Bureau laler~spcc~nmcrcncra~ Yrcot- - c.~. ~cccsswn t~amocr of J~a~osi~

For recelvmg Of ~lce use oniy i-or l- .. ~ l Burcau use only j This sheer w;~s re~ elved with tbe ,,,~ ,t~ bis sbcel was rcceivcd bv tbe l ~ i3ure u on:

A--' i ~t~icer -~7 ~ d officer Fo~n PCr/ROJ134 ~Julv 19g2) .

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

CA 0224039l l998-06-l5 WO 97J2269I PCT~196/00670

3~
INDIC;~I~ONSRE~L~1~NG TO A DEPOSI1lED ~CROORGA~NISM
~CT ~ulc13~) A. rne m-dc below rciate to nc ~._.,u.~.n sm reierred lo m tbe .I~.. ~,.. u., on p~ge 7 line B. ID~ NT~CATION OF DEPOSlT Funher depos~ts are identified on an 1ddition~1 sheet O
N~me of dL~
Centraalbureau Voor Schimmelcultures Address of d. ., ~ /incirramg pcrstLrl coac arui cr.~Lrr.~
Oosterstraat 1, 3752 SK BAARN, The Netherlands D~te of ~ier~sit Acccss~on NUmDer 8 November 19~5 CBS 730.95 C. ADDlTlONAL~NDICATIONS (lrDc bl4nJ~iimxcpplic4blc) This i r ~ is -i on~n~dditiorlal shcet n Regarding those designations in which a European patent is sought, a sample of the deposited microorganism will be made available only by the issue of such a ~ample to an expert nominated by the person requesting the sample (~ule 28(4~ EPC, until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or is deemed to be withdrawn.This request also applies to other designated countries in which similar or corresponding provisions are in force.
. n. DESIGNATED STA~:S FOR WE~CII INDICATIONS ARE MADE (iflrcirulica ionsarcnar foraU4'cr.r~c4'5ta~cs) E SEPAR~TE F'URI~ISE~NG OF INDICATIONS (lc4r~c blan~ i,r'r~ appiicablc~
Thcr ~h ~ its~edbciu.rl.~lllbesuDmlnedtotbci~ i8urcaui;l~er~s~cclrvu~rcncr41n~urcL~rr ' c~ krlss.ron l~rumocr of Dcposil ') For rcce~ving O~ficc usc onlY For i ~ 1 Bureau usc oni,v This sDect W~s cceived wilh the ~ A~ a~ ! This shect ~as received b~ the i ~ i3ut~u on:

Autnon7 d o~ ~ ,,? A~n~r~ o~ficcr Forrn PCIIR0~134 ~ iul~r 19~2) -

Claims (26)

What Is Claimed Is:

1. A cell-free composition comprising at least one xylanase of a fungal species,wherein said fungal species is selected from the group consisting of the same species as that represented by Chaetomium thermophilum var. thermophilum CBS 733.95, Chaetomium thermophilum var. dissitum CBS 731.95 or Chaetomium thermophilum CBS 730.95.

2. The cell-free composition of claim 1, wherein said species is of the same species as that represented by Chaetomium thermophilum var. thermophilum CBS 733.95.

3. The cell-free composition of claim 1, wherein said species is of the same species as that represented by Chaetomium thermophilum var. dissitum CBS 731.95.

4. The cell-free composition of claim 1, wherein said species is of the same species as that represented by Chaetomium thermophilum CBS 730.95.

5. A cell-free composition comprising a Chaetomium thermophilum xylanase having a molecular weight of about 54 kDa, 33 kDa, 30 kDa and/or 22 kDa and a pI value of about 8.9, 8.3, 8.7 and/or 9.3, respectively.

6. The cell-free composition of claim 1, wherein said Chaetomium thermophilum xylanase has a molecular weight of about 54 kDa on SDS-PAGE and a pI value of about 8.9.

7. The cell-free composition of claim 1, wherein said Chaetomium thermophilum xylanase has a molecular weight of about 33 kDa on SDS-PAGE and a pI value of about 8.3.

8. The cell-free composition of claim 1, wherein said Chaetomium thermophilum xylanase has a molecular weight of about 30 kDa on SDS-PAGE and a pI value of about 8.7.

9. The cell-free composition of claim 1, wherein said Chaetomium thermophilum xylanase has a molecular weight of about 22 kDa on SDS-PAGE and a pI value of about 9.3.

10. Purified Chaetomium thermophilum xylanase having a molecular weight of about 54 kDa on SDS-PAGE and a pI value of about 8.9.

11. Purified Chaetomium thermophilum xylanase having a molecular weight of about 33 kDa on SDS-PAGE and a pI value of about 8.3.

12. Purified Chaetomium thermophilum xylanase having a molecular weight of about 30 kDa on SDS-PAGE and a pI value of about 8.7.

13. Purified Chaetomium thermophilum xylanase having a molecular weight of about 22 kDa on SDS-PAGE and a pI value of about 9.3.

14. A method for treating wood-derived pulp or fiber, comprising the step of adding the cell-free composition of any one of claims 1-9 or at least one xylanase of claims 10-13 or at least one xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95 to wood-derived mechanical or chemical pulp or secondary fiber.

15. A method for bleaching pulp, said method comprising contacting said pulp with the cell-free composition of any one of claims 1-9 or with at least one xylanase of claims 10- 13 or with at least one xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.

16. A method for treating animal feedstuffs, said method comprising contacting said feedstuff with the cell-free composition of any one of claims 1-9 or with at least one xylanase of claims 10-13 or with at least one xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.

17. A method for treating flour compositions, said method comprising contacting said flour compositions with the cell-free composition of any one of claims 1-9 or with at least one xylanase of claims 10-13 or with at least one xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.

18. Use of a composition of any one of claims 1- 9 or at least one xylanase of claims 10-13 in the degradation of xylan-containing substrate.

19. Use of a composition of any one of claims 1-9 or at least one xylanase of claims 10-13 or at least one xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95, for treating wood-derived pulp or fiber.

20. Use of a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 to 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.
in the production of wood pulp.

21. Use of a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 to 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.
in bleaching of wood pulp.

22. Use of a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 to 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.
in the preparation of animal feedstuffs.

23. An animal feed composition comprising a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 - 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.

24. Use of a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 - 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95 in a dough for the preparation of bread.

25. A flour composition comprising a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 - 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS
732.95.

26. A dough composition comprising a composition according to any one of claims 1 to 9 or a xylanase according to any one of claims 10 - 13 or a xylanase of a fungal species that is the same as that represented by Chaetomium thermophilum var. coprophilum, CBS 732.95.