CA2129172C - Thermostable xylanase dna, protein and methods of use - Google Patents

Abstract

The present invention is for a method of chemically treating plant biomass with an enzyme system that retains function at low pH and high temperature. Enzyme preparations enriched in xylanase enzymes which retain activity in low pH and high temperature are described. Such preparations may be utilized in a crude unpurified form, and are especially useful in the production of pulp and paper.

Description

-I,-21291'~~
'Thermostable Xvlanase DNA, Protein and lyiethods of Use F'deld' of the Ireventlon The present invention is in the area of thermostabile enzyzz~.es, and the use of same. l~speciatly, ttte lnventiou is in the arcs of xylanascs that are activt at a low pki and high rerttpe.-store. The compositions of the invention are useful to modify plant biamass properties, especially to reduce the lignin content. The invention is also directed to a method for biobleacbing using the enzyme compositions of tlse irvention.
Buck~r~a~r~d of the In~en~i~rs Xylan, a major component of hemicctlulose, is a polymer consisting of g backbone of ~(I,4)~~linked D-xylose residues (often acetylated) with a-L
arabinofliranose arid gltacuronic acid side ct~s~ins (Timell, T.E. , et al. , Wood Sci. TeChnad. 1:45-?0 {1967)). AfiCSr cellulose, xylan is the second most 15 abundant carbohydrate fraction of plant biomass. Xylan bas recently received increased attention as a renewable bioresource.
Being complex, ~eore than one enzyme iB r~equiz~ed to completely degrade xylau to soluble monomers. Xylan can be hycimlyzed by rnaay hcmicelltzlasPS, such as, for example, ~-I,4-xylanases (EC 8.2.1.8), i3-20 xylosid,ases mnd several debranching enzymes (Biely, P., Trends Biotec~ol 3:28-290 (1980; l3ekker, R.~.H" in Hignelti, T., ~d" igiosynthesis arut btodegradn:ion of wood components (Academic Press lne., Orlando), pp. 505-533 {i985); 't~Voodward,1. , Top ,Lrt~me;Fertptent. Biotechnod. &:9-30 (1984)).
Tho activities of these eatzymes play as important role in the decomposition 25 of soil plant litter amd have been extensively studied both.in bacteria amd fungi (along, K.K.Y, et tad., MiCr~biol. Bev. 52:305-317 (1988j; Poutanen, K.

SENT BY:S K G & F : ?-2°-94 ~ 13:2E ~ SKG&Fi G.5 & H~tt 7 21~91'~2 et al., in ~n~ymes irt ~~:amas~s conversion (ACS Symposium series 460), l,eatham & Himmel, eds., American Chemical Society, Washington, bC
a (1991), pp.426-436; Gilbect &. Hazlewood, J. Gen. Microbiol. 139:187-I94 (1993)).
Various microorganisms secrete enzymes that are capable of degrading xyl~s~s, and xylansses have been found in both prokaryotes and eukaryotes (Dekker, R.~.H., Ftichards, G.N.. Adv. Carhohydrdte Chem. Biochem.
32:277-352 (1970. Xylanolytic micro-organisms often produce multiple xylanases to attack the different bonds in these molecules. All the xylanases so far characterized fall into two classes; the high M,llow pI class and the low M,lhigh pI class whiclh coincide, respectively, with the families 10 and 11 of glycosyl hydrolases (Henrissat & Hairoch, Biochem. J. 293;781-788 (1993)).
The cloning of xylanases has been reported firom Actinomadura sp.
FC?' (Ethier, J.-F. et al., in: Industrial Microorganisrrts: Basic and Applied Molecular Genetics, R. Bale. et al. , eds, (Pros. Sth ASM Conf. Gen. Mol.
Hiol. Ind~.~st. Nlicroorg., Oct 11-I~, 1992, Bloomington, Indiana, posterC2S);
bacteria (e.g. Ghangas, G.S. et al., J. Bacteriol. 171:2963-2969 (19$3); Lin, L.-L., Thomsan, J.A., Mot. Gen. Genet. 228:55-61 (199i); Sharcck, F.
et aL, tens 1~7:'75-82 (1991); Scheirlinck, T. et al., A~pl Microbial ~iotechnol. 33:534-541 (1990); Whitehead, T.R., Lee, D.A., G~rr.
Microbial. 23:15-19 (1991)); and fungi (Boucher, F. et al. , NucleieAcids Res.
14:9874 (1988); Ito, K. et ad. , Biosci. Biatec. BiochEm. 56:90b-912 (1992);
Maat, 1. et at. , in Visser, J. et al. , eds., Ji~lans and Xylanases (Elsevier Science, Amsterdam), pp. 349-360 (1992); van den Broeck, H. et al., EP
2S 463,706 A1 (1992), Wa 931256'1 and Wt~ 93f25693).
The xylan-remaining hemicellulases in plant blamaee are tightly bound to cellulose and lignin. In the pulp and paper industry, in chemical gulping (cooking) of the wood, the major part of the lignin is e~racted to get acceptable cellulose pulp product. Howcvcr, the rcsultiag pulp ie brown, mainly b~au~se of the small portion of the lignin still remaining is the pule after cooping. 'This residual lignin is traditionally rEmovtd in a multl-stage SENT BY:S K G & F , ~-2g-g~ ; ~3:pg ; 5KG&F-~ G, S & H;it 8 bleaching procedure using typically a combination of chlorine ct~emiegls and extraction stages. Peroxide, oxygen and ozone are also used when the use of the chlorine cheznical$ is wanted to be reduced or avoided totally.
~Iecnicellulases can be used in enzyme-aided bleaching of pulps to decrease chemical dosage in subsequent bleaching or to increase brightness of the pulp (Kantelinen et al. , International Pulp Bleaching Conference, Tappi Proceedings, I-5 (19$8}; Viikari et al., Paper and Timber 7:384-389 (I991);
and ICantelinen et al., "Enzymes in bleaching of lcraft pulp," Diesertaticjn for the degree of Doctor of Technology, Technical Research Centre of Pialand, VTT Publications 114, Espoo, 1.992). Naturally, in this use, the hemicellulase should be face of cellulases, which would harm the cellulose fibers.
The use of hemicellulose hydrolyzing enzymes in different bleaching sequences is discussed in WO 89108738, F.P 383,999, 't~VO 91102791, F.P
395,792, EP 386,888, EP 473,545, EP 489,104 and WO 91105908.
Other industrial applications for hemicellulolytic enzymes are in the production of thernto-mechanical pulps, where the aim of the use of hemicellululytie enzymes is decreased energy consumption. Hemicellulolytic enrymes can be used to improve drainage of recycled pulp or hemicellulolytic enzymes can be used in the protiucdon of dissolving pulps ('Yiikari et al., "Hemicellulases for Industrial Applications," In: Hioconversion of Forest and Agri,culrurat Wastes, Saddler, Y., ed., CAB Laternational, USA (1993)).
The use of hemicellu~olytic enzymes for improved water removal from mechanical pulp is discussed in EP 262,040, 13P 334,739 az~d EP 351,665 and DE 4,000,558). When the hydmiysis of b:omass to liquid fuels or chemicals is considered, the conversion of both cellulose and heaticellulose is essential to obtain a high yield ('Yiikazi et al. , "Hemicellulases for Industrial Applications," In: Bioconverslon of Forest anti Agricultural Wastes, Saddler, J., ed., CAB International, USA (1993)). Also, in the feed industry, tb~re is a need to use a suitable combination of enzyme activities to degrade the high i ~-glucan and henvcellulose con.Caining substrate.

SENT BY:S H G ~ F , '?-'2;!--~4 ; 13:27 ~ SKG&F-~ u.5 & H~ti 9 'f o be ar~onable to enzymatic hydrolysis in vitro, the cellulo5e-he~icellulc~s:-lignin matrix must be chemically pretreated. One of such procedures ' izv~rol va a thermo-mecYoanical steam treatment followed by extraction with hat water (Ck~ahal, D.S. et al. , J. Inriust. Microbial, 1:3SS-(1~8'~~. ~ mildly acidic liquor is obtained, which contains ~-ater-soluble hen~icellulose chains and some lignin derivatives.
However, to ens~tre~ further enzymatic hydrolysis of the x~y~lan chains into oligomelcs or monomers, enzyme systems that are efficient at conditions combining high temperature (such as 70°C) and moderately a::idie pH
(around 4.0) are . The combination of these rwa parameters seeot~s however to be harmful for the majority of la~.own xylanases. 1'ar instance, at pH 4, xylanase II from the mesophilic aetinomycete 5'treptomyces roseiscleratieus (a low M,,lhig>z pI onzyrre) retains less than 5 ~ of the activity it had at pH
6.0 -b.5 (Gra'bski ~ Jeffries, ~ppl. Environ. Microbial. 57;387-992 (1.991)). The 1,5 crude xylartase from Aureobasidium prtllulans (Myburgh, 7, et al. , Prac.
Eiochem. 2a:3~3-348 (1991)) is acidophilic, having a pH optimum between 3.5 and 4.0 but its actin.=ity sharply decreases at temperatures higher than 35°C. The thermostable xylar~age from the fungus Tlternmascus auranttacus retains at pH 3.~, only 129 of its maximal activity (Tan L.U.L. et ul., Can.
,~. Microbial. 33;68'9-692 {1987)). Another xylauase, a aitgh M~llnw pI
enzyme fr9m the extremophile bacterita~aa, "Caldocellu»~ saccharolyricum" was shown to 19e Very stable at 64°C but retained little activity below pH
5 (Lfzthi, E. et al., ~lppt. Environ. Microbial. 56:2677-2b83 (1990); Litthi, B. et al., Appl. ~nvdror~. Microbial. 56:1017-102a (1990). Crude xylanases from various Actinomadura isolates were stable for many hours when incubated at 7Q-75 °C, but retained less tban 15'~ of their activity at pH ~.0~.5 and 70°C
(I~ioltz, C.
et al., Antonie van Leeuwerthaek 59:1-7 (199I)).
Thus, there is a need for enzyme preparations that contain xylanases which retain activity under industrial ambient conditions. Fspocially in the i pmper mamafacttwing industry; there is a need for xylanase prepartions that are SFI~T BY~ ~ K G 8 F . ?-29-94 ~ .3:26 ~ 5ltG&F~ G~ S & Hs3t10 X1291'72 functional in the high temperature, acidic liquor produced by thermo-mechanical steam treatment aml hot water extraction.
Sunemar,~ of ttee Invention Recognizing the importance of developing an environmentally safe and economical method of cheznic.~.atly modifying plant biomass so that it may be enzymatically treated under harsh conditions of high temperature and towpH, processes such as those employed by the paper marnifacturing industry, the inventors have searched for n now mitxobe that might be a source of such enzymes.
1~ These studies have resulted in the isolation and identification of a navel strain of thermophilic actinomycete, Actinomaciura sp. FC7. Acrirwmadura sp.
FC7 expz~esses t~vo unique xylanases, X'SfL I and XYL II that retain a large amount of their enzymatic activity at high temperatures and law pH.
The invention is further directed to DNA encoding XYLI and XYL II, and to recombinant hosts transformed with such DNA.
The inverctfon is further directed to purified XYL I and XYI, II, and to emyme preparations containing XYL I, XYL II, or mixtures of XYL I and XYL n.
The invention is further directed to a method of treating plant biomass with the enzyme preparations of the invention, especially a method of biobteaching.
Brief De~cnipdo~ of the Drawings Figure x shows the restriction map of the inserts clod in plasmid pJFI. 'The shaded boxes shows the approximate iacations of the xylanase genes after deletions of their.respective inserts. Top line, restriction sites in the f~tll-le~th insert. Top shaded line: plFl (2Ø0 kb; full length insert); Second shaded tire;: p~'102 (13.5 kb); third shaded line: pJF103 (11.5 kb); and fourth SENT 6Y:S H G & F ; 7-29-~4 ~ 13:28 ~ 5KG&F-~ G~ S & H~Ji11 shaded line: pJF1020 (7.5 kb). Bg, BgIII; H, BamHi; X, XhaI. +, xylanase positive; -, xylanase negative.
Figure 2 shows the restriction reap of the inserts cloned in plasmid pJF6. The shaded boxes shows the approximate locations of the xylanase genes after deletion of the respective insert. Bg, BgliI; N, NruI; No, NorI, l S, SaII. +, xylanase positive; -, xylanase negative. Tap shaded line: pJF6;
second shaded Line: pJF6l; third shaded tine: g3F62.
Figure 3 shows the effect of pH and temperature on Xylanase I
activity. Purified Xyl I (5 units) was incubated for 10 min at the temperature 1<0 and pH values indicated and the release of reducing sugar was measured by the Nelson-Somogyi method. (~) 60°C; (~) 70°C; (~) 80°C.
Figure 4 shows the effect of pH and temperature cn Xylanase II
activity. Puri~.ed Xyl 1~ (8 ututs) was incubated for 10 thin at the tetnperatuze and pH values indicated. and the z~lease of reducing sugar was measured by the Nelson-Somogyi method. (') 60°C; (~) 70°C; (~) 80°C.
Figure 5 shows a restriction map of the 2.7 kb insert of clone pJF6 (encoding Xyl In. 'Ihe black line shows the sequenced portion of the insert, starting from the indicated Nrul site. The loners rept~esent the following ' restriction sites: Bg=~gni, S=SadI, N=Nrul, No=NatI.
'i ' ~,p Figure 6 shows the nucleotide sequence of the pJF6 insert (xln2) from the NruI site shown i.n Figure 5, to the Bgla site shown in Figure 5. The amino acid sequence of Xyl H begins at nucleotide 521. The -35 (TTGACG) and -10 (CACAAT) promoter regions, the ribosome binding site (RBS:
(3GAGGA), and the iniation colon (CIT: GTa) are shown in bold.
Figure 7 is a comparison of the R$S of 40 streptornyeetes genes versus that for xlnll as encoded by the pTF6 xylatiase gene. Tlu nucleotides corresporxling to the RBSs are underligned, while those in bold identify the translation initiation c4dan.
Figure 8 shows a partial. amino acid sequence of X'1'I. II on which the 30 signal peptide is located. The long sequence of hydrophobic amino acids is shown in bald. The characteristic arginines (R) usually found in the SENT BY:S ~( G & F ~ 7-29-94 ~ 13:29 ~ 5~5G&F-~ G, S & H:itl2 _7_ hydrophilic region are underlined. The arrow indicates the possible cleavage site of the peptidase signal, bordered by a proline (P).
Figure 9 shows a comparison of nucleotide sequence homology between the streptomycetes promoters having a spacing of 16 nucleotides between regions -35 and -10, and the promoter of the xlnll gene encoded by the pJP6 xyianase. The -35 and the -10 regions are in bold.
Figures 1~1GC show the optimal alignment of the amino acid sequence of XYL II as encoded by pJF6 with other enzymes. The list of enzy inea is as follows; ~ xylanases of Pseudomonas fluoresceos {Psexyna, Psexynbc), pIF6 xylanase (xlttpjf6), xylazvxsc A of Stre~tomyces livlclcuts (Stmxlna), exogluconase of Cellulomonas firm (Cficex), xylanase of CIostrGdlum rhermocellum (Claxylz), xylanase of Bacillus sp. {Bscxynaa), celloaylanase of Clostridium srercoirarium (Pclocxl), acylanase of Caldoc~llurn saccharolytlcum (Cdcxynab), xylanase of Thermoanaerobacter sp. (Teoendxyla), endocellulose iS of Caldocellurn sacctiamlytlcum (Cdccelb), xylanase of Buryrlvibrlo ~'tbrisolvens (Hutxynb) and s xylanase of Rumiococcus flrtvefaciens (Ru.yna). Amino acid consensus is indicated in bold, and those amino acids retained in all. exatnfned enzymes are represented by an asterisk (*).
Hypothetically retained regions arc shoevtt by an underline bracket.
Figure I1 shows the homology among the amino acid derived sequences of xylanase A of Streptomyces livuians and chat of XYL II as encoded by pJF6. The symbols between sequences i~icate that the comparison value is tlx same (~), ~ 0.5 (:), ~ 0.1 (.). An indication of z 0.5 means that the two different amino acids represent conservative changes (ie., there is some structural andlor functional similarity between them,). An indication of ~ 0.1 represents amino acids that have no or weak structtual andlor f~n~rioaal similarity.
Figure I2 shows the seqtteare of nucleotides 1538 to 1572 inclusive, of the xlnll sequence on p3F6. Arrows indicate repeated and inverted sequences.

r Figure 13 and 1.3A show the MAP progranm prediction of the pioteOlytiG GlCBVage SlteB along the amizio acid derived sequence of X'YL II
as encoded by p3F6 and the xylanase A of SteptomyGes ldvidans. The letters represent the follawiag prciteases: S (Staphylocossus aureus protease), T
(TryPsin) and C (Chymotrypsin). The differences encountered are shown in bold.
l3e~osits _ Plaa~nid pJPI was deposited in E. colt at the American Type Culture Collection, (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on July Z9, 1994 and assigned accession no. ATCC 69670. .
Plaamid pJF6 was deposited in E, colt at the ATCC on July 23, 1994 ~ v and assigned accession no, ATCC 69671.
Act3nomadura sp. FC7 was deposited at iha ATCC on July 24, 1995 and assigned accession no. ATCC 55698.
is ~etoiled ~escri~tio~a of the invention 1, l~r~n8tleans In the description that follows, a number of terms used in recombinant DNA tGChaolo~y are extensively utilized. In order to provide a clearer and consistent ur~deerstanding of tha specification anti claims, including the scope to be given such terms, the following definitions arc provided.
~landse. As used herein, a xylanase is a hemicellutase that cats the . , ~~1,4 bonds within the xylosic chain of xylan, (xylan is a polymer of D-xylose residues that are joinad through S-1.4 linkages. Xylaaase activity is_ synonyatous with xylanolytic activity. .
By a host that is "sul~~ly incapable" of syathesizi~ one or more cellulose et~ymes is meant x host in which the activity of one or more of the 5E~'T BY:S K G & F ; 7-P9-84 ; 13:50 5KG&F-~ G. S & n;i~l4 _g_ eeilulase e~ymes is depressed, deficient, yr absent when compared to the wild-type.
En~yrne preparation. By "enzyme preparation" is meant a composition containing enzymes that have been extracted from (either partially or completely purified from) a microbe or the medium used to grow such microbe. "Extracted frsom" means any method by which the desired enzymes are separated from the cellular mass and in;,ludes breaking cells and also simply removing the culture medium from spent cells. Thezefore, the term "enT,Yrrre preparation" includes coazposirions comprising medium previously used to culture a desired microbes) and any enzymes which the miembe(s) has secreted into such medium during the culture.
Biobleaching. Hy "biobleaching" is meant the extraction of lignin from cetlulase pulp after the action of hemicellulose degrading enzymes with or without lignin degrading enzymes. Removal of the lignin may be restricted by hemicelluloses either physically (through repreeipitativn onto the fiber surface dur~:ng cooking) yr chemically (through lignin-carbohydrate complexes). The hemicellnlase activity partially degrades the hemiceltulosG, which enhances the extractability of lignins by conventional bleaching chemicals (like cblvcine, chlorine dioxide, peroxide, ere.) (Viikari et al., ~0 "Bleaching with Enzymes" in Biotechnology in the Pulp and Paper Industry, Proc. ~z~d Int. Conf. , Stockhoim, pp. b7-69 (1886); Viikari et al. , "Applications of Enrymes in Bleaching" in Proc. 4th Int. S~mp. Wood and Pulping Chemistry, pane, Vol. 1, pp. 151-154 (19$7); Kantelinen et al., "Hemicellulases and their Potential Hole in Bleaching" in International Pulp Bleaching Cc~nf~renee. Tappi Proceedings, pp. i-9 (1988)). The advantage of this improved bleachabiiity is a lower consumption of bleaching chemicals and lower environmental loads or higher final brightness values.
By an enzyme "hot~alogous" to a host of the invention is meant that an untransfarmed strain of the same species as the host spites naturally produces some amount of the native protein; by a gene "homologous" to a host of the invention is meant a gone found in the genome of an SENT 3Y:S K G & F ~ 7-29-94 ~ 13:30 ~ BKG&F-~ G.5 & H.#15 2~~~172 a_ urtransfermed strain of the same spc~;ies as the host species. By an enzyme "heternlogous" to a host of the invention is meant that an unu-ansformed strain , of the same species as the host species does not naturally produce $ome amount of the native protein; by a gene "heterologous" to a host of the invention is meant a gene not found in the genome of an untransformed strain of the same species as the host species.
Clonfrtg~ vehicle. A plasmid or phage DNA or other DNA sequence (such as a linear Dri'Aj which provides an appropriate nucleic acid environment for the transfer of a gene of interest into a host ccll. The cloning vehicles of the invention may be designed to replicate autonomously in prokaryotic and eukaryotic hosts. In fungal hosts such as TYichoderma, the cloning vehicles generally do not autonozaously replicate and instead, merely provide a vehicle for the transport of the gene of interest into the ~ichoderma boat for subsequent insertion :nto the fY~icltodernsa genome. The cloning vehicle may be further characterized by one or a small ntunber of endonuclease recognition sites at which such DNA sequences may be cut in a determinable fashion without loss of an essential biological function of the vehicle, and into which DNA may be splicxd in order to bring about replication and cloning of such DNA. 'r'he cloning vehicle may further contain a marker suitable for use in the identification of cells transformed with the cloning vehicle. Markers, for example, are antibiotic resistance.
Alternatively, such markers may be provided on a cloning vehicle which is separate from that supplying the gene of intere8t. The word "vector" is sometimes used for "cloning vehicle."
~xpresslan vehracde. A vehicle or vector similar to a cloning vehicle but which, is capable of expressing a gone of interest, after transformation into a desired host.
When a fungal host is used, the gene of interest is preferably provided to a fungal host as part of a cloning or expression vehicle that integrates into 3a the fungal chromosome. Seryences which derive from the cloning vehicle or ~Xpre8B101~ YChiClt Iilay ~.l$0 be integrated with the gene of interest during the SENT BY:S K G & F . ~--Zy-94 ~ 1?:31 ~ SKG3F-~ G~ S & ~~~16 integration process. For example, in T. reeSei, the gene of interest can be directed to the cbhl locus, , The gene of interESt may preferably be placed under the control of (i.e., operably linked to) certain conuol sequences such ss promoter sequences provided by the vector (which integrate with the geae of interest). If desired, such control sequences may be provided by the host's chromosome as a recruit of the locus of Insertion.
Expression control sequences an an exp'rcssion vector wilt vary depending oa 'arhether the vector is designed to express a certain gene in a 1Q pmlcaryotic Or euksryotic h~ast {for example, a shuttle vector may provide a gene for selection in lsacterial hosts) and may ad3itiortally contain transcriptional elements such as, enha,nie~cr elements, termination sequences, and~or translatlonal initiation and termination sites.
1. Isoladfo~ of Actlnoniyeetas actlnomadur~ ap. FC'fi The Project had the objective of isolating a microorganism, an actinomycete antler the circumstances, vr~Ch would have an acidcrphilic said thermostable xylanotytic activity. The actlrlomycetes are aerobic gram-positive bacteria founai mainly in the soil. The actinomycetes display a a~ycelial morphology intxrestiugiy reseuibling microscopic fv.ngi, lrmare, they are recognized as excellent enzyme secretors, thereby playing a very important role during biomass degradation.
A aere~ing program was established in order to find actinomycetes that produce xylanases able to hydrolyse xylan chains in a hemicellulose liquor (a by-product of steam treatment of the lignocellulosic biomass) at moderately said pH (4.0) aixl high temperature (70°C), The hunt for such an organism was made based upon places in which xo important periodic heatixig-up c4uld be produced, such as in hay, .compost and manure.

The first step involved a selection for xylanolytic actinomycetes having optimal growth at SO-60°C and that demonstrated a strong degradation capability ofRemazol Brilliant Blue(RBB)-xylan on solid medium. A series ofthermostable and acidophilic actinomycetes with these characteristics were isolated from compost; mancire and straw and further examined for ;heir ability to produce zylanolytic enzymes that were relatively active at pH4, and 70°C. In this second step of the screcaing> the zylan hydrolysis rates at pH 5160°C
of crude enzyme preparations secreted from the selected actinomycetcs were compared to those at pH 4170°C for the same microbe. This was done to determine the level of acid- arid thermo-resistence of the xylanase enrymcs being secreted by each microbe.
The selection procedure identified one microbe from manure that was especially desirable in that it produced xylanolytic enzymes -that were relatively active at pH4, and ?0°C. This microbe was identified as a member IS of the genus Actinomadum by chcmotaxonomic procedures, and was named Acrinomadura sp. FC7. Pure preparations of ActinornQdura sp. FC7, produced at least four xylanolytie activities as dersonstrated by zymogram.
The crude enzymes produced by the strain PCl retained 65 9& of their activity in the more stringent of the two conditions (pH 4170°C).
11. Xylanase Btobleaehing at High Temperature and Acidic pH
The present invrntion comprehends a method for chemically treating plant biamass under conditions of high tenxperature aar3 low pH. In a preferred embodiment, plant biomass is bio-bleached with xylanase$ that are able to hydrolyze xylan chains in a hemicellulose liquor (a by-product of steam treatment of the lig~ellulor~ biomass) at moderately acid gH (4.0) and high temperature (70°C).
Plant biomass is a composite material consisting primarily of a mauL~
of cellulose, hcmicellulose, and lignin. Removal of the lignin component is desirable during the mnaufacturer of paper because of its brown color and SEPJT 8Y':5 K G & f= ~ x-29-94 s 13:32 ~ 5KG&F-~ G. S & H~#18 212~17~

tendency to reduce the strength of the paper product. Many processes have beer, developed for the removAl of lignin. Typically, the wood pulp is treated , with chorine or other toxic chen~i;,als in order to remove the lignin component and provide for a brightened pulp, However, the toxic by-products of this chemical treatment negatively impact upon the health and stability of the environment into which they are released. Consequently there is a great need for developing alternative, more environmentally protective techniques to achieve puig bleaching.
A common treatment of plant biomass for paper production involves a thermo-mechanical steam treatment followed by extraction with hot water.
This process dissociates xylan cornaining hemicxliuloses and some lignin derivative$ which are otherwise tightly bound to the ceIlulos~e. Under the method of the present invention, a biobleaching technique is developed whereby theimoscable xylanases which are active at low pH may be used in 1~ vitro to modify or decrease the lignin in wood pulps, These stringent ;
processing conditions may additionally act to reduce cellulose activity in the enzyJme preparation or culture medium.
In a preferred embodiment, the process of the invention is carried out in vitro in the acidic hemicellulose liquor. The process involves placitxg the 2U enzyme preparation, culture medium, or coc»centiated mixture containing xylanase into contact with the wood pulp, Routine calculations enable those in the art to determine the optimum treatment time depending upon the result desired, the concentration and specific activity of the xylsnase enzyme used, the type and concxrttration of pulp used, pH and temperattue of the acidic liquor, and other parameter variables.
It is preferred that the process occurs at the ambient temperature and pH of the liquor with temperatures from 45-90° being preferred and temperatures of 70° being mast prefe;«ed. It is also preferred that the pH of the liquor be less titan 6.0 with a pkli of 4.U being most preferred.
3~ The method of the present invention may be applied alone or as a supplement to other treatments that reduce the lignin content of wood pulp, SENT BY:S K 3 & F ~ '~'°~9-94 ~ X3:33 : S~tG&F~ G~ G & H.#19 - ~ 4-increase its drainabiliy and/or decrease its water retention. In a preferred embodiment, tEce present invention is used to enhance brightness properties of , the wood pulp by treatment of chemical pulps, i.e., those pulps conta;n lignin that has been chemically modified through chemical treatment.
In a preferred embodiment, The xylanases used in the methods of the invention are preferably those of.4ctinorncrdura sp. FC7, and especially XYL
I and XYL II. XYL I and X'YL II can be provided by the native ~f ctinomadura sp. FC7 host (and especially the culture medium from the growth of p'C7 celis) or can be provided by a recombinant bast, for example, as encoded by expression of the ins~enrs on pdFl and p3P6.
111. Genetic Engtrtaerang of ti3te Hods off' the Invarrtion The process for g$n~tically engincezing the hosts of the invention is faellitated through the cloning of genetic sequences that encode the desired xylanase activity and through the expression of such genetic sequences. As used herein the tmm "~CnetlC sequences" is intended to refer to a nucleic acid molecule (preferably IaNA). Genetic sequences that encode the desired xylanase are derived from a variety of sources. These sources include ~cttnorit~rdura sp, FC7 genomic DNA, cDNA, synthetic DNA and combinations thereof, Vector systems may be used to produce hosts for the 2Q production of the enzyme preparations of the invention. Such vector construction (a) may further provide a separate vector construction (b) which encodes at least one desired gene to tx integrated to the genome of the host and (c) a selectzble masker coupled to (a) or (b). Alternatively, a separate vector may be used for the marker.
A nucleic acid molecule, such as DNA, is said to be "capable of e~~preas$ng" a polypeptade if it contains expression control sequences which contain transcriptional regulatory information anc3 such se~utnees are "operably linked" to the tnzeleotide sequencx which encodes the polypeptide.

SENT B1':S K G & F ; 7-~9-94 ; 13:33 ~ SKG~F-~ u, S & H;~20 An operable linkage is a linkage in which a sequence is connected to a regulatory sequence (car sequences) in such a way as to place expression of the sequence under the influence or control of the regulatory sequence. Two DNA sequences (such as a protein encoding sequence and a promoter region sequence linked to tl've 5' end of the encoding sequence) are said to be operably linked if induction of promoter function results in the transcription of the protein encoding sequence mRNA and if the nature of the linkage between the iwo DNA sequences does iwt (1) result in the introduction .of a frame-shift mutation, (2} interfere with the ability of the expression regulatory sequences to direct the expression of the trtRNA, antisense RNA, or protein, or (3) interfere with the ability of the template to be transcribed by the prpmoter region sequence. Thus, a gmmoter region would be operably linked to a DNA sequence if the promoter were capable of effecting transcriprion of that DNA sequence.
The precise nature of the reguiatory regions needed for gone expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences involved with initiation of transcription and translation respectively.
Expriession of the protein in the transformed boats requires the use of regulatory regions functional in such boats. A wide variety of transcriptional and tranalational regulatory sequence$ can be employed. In eukaryotee, where transcription is not linkad to translation, such control regions may or may not provide an initiator methionine (AUG) colon, depending on whether the cloned sequence contains such a methionine. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis in the host cell.
hs is widely known, translation of eukaryotic mltNA is initiated at the colon which encodes the first methionine. For this reason, i.t is preferable to ensure that the linkage betwan n eukaryotic promoter and a x3N~i sequence which encodes the protein, or a functional derivative thereof, dots not contain any intervening codoas which are capable of encoding a methionine. The SENT BY:S tt a a F ; ~-28-84 ; 13:34 ; 5KG&F-~ G~S & H;#2?
-ls-presence of such colons results either in a formation of a fusion protein (if the AUG colon is in the same reading frame as the protein encodizig DNA
sequence) or a frame-shift mutation (if the ALJG colon is not in the same reading frame as tk~e protein encoding sequence).
In a preferred embodiment, a desired protein is secreted into the surrounding malium due to the presence of a secretion signal sequence. If a desired protein dues not possess its own signal seqsence, or if such signal sequence does not funetioa well in the host, then the protein' s coding sequance may be operably linked to a signal sequence homologous ar heterologous to the host. The desired ceding acqusncc may be linked to any signal sequence which will allow secretion of the protein fmm the host. Such signal sequences may be designed with or without specific protease sites such that the signal peptide sequence is amenable tv subsequent removal. Alternatively, a host that leaks the protein into the medium may be used, for example a host with a mutation in its ~aembeane.
If desired, the non-transcr'bed and/or non-translated regions 3' to the sequence coding far a protein can be obtained by the above-described cloning methods. The 3'-non-tt~nscribed region may be retained for its transcriptional termination regulatory sequence elements; the 3-non-translated region may be retained for its translational termination regulatory sequence elements, or for those elements whiGl3 direct polyadenylation in eukaryotiC cells.
The vectors of the invention may further comprise other vperably linked regulatory elements such as enhancer sequences.
In a preferred embodiment, genetically stable transformants ate constructed whereby a desired protein's DNA is integrated into the host chromosome. The coding sequence for the desired protein may be from any source. Such integration may occur de novo within the cull or,. in a most preferred embodiment, be assisted by transformation with a vector which functionally inserts itself into the host chromosome, for example, DNA
elements which prnmote integration of DNA sequences in chromosomes.

SENT QY:S K G & F , 7-29-94 ~ 13:34 ~ 5KG&F-~ G~ S & H;ti2P

Cells that have sta~ly integr$ted the introduced DNA into their chromosomes are selected by also introducing one or more markers which allow for selecson of host cells which contain the expression vector in the chromosome, for example the marker may provide biocide resistance, e.g., resistance to antibiotics, or heavy metals, such as copper, or the like. T'he selectable. marker gene can either be directly linked to the DNA gene sequences to be expressed, or introduced inra the same cell by co-transformation. -Factors of importance in selecting a particular plasrnid or viral vector include: the ease will? which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not comtain the vector; the number of copies of the vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
Once the vector or DNA sequence containing the eonstritet(s) is prepared for expression, the DNA constructs) is introduced into an appropriate host cell by any of a variety of suitable means, including transformation as described above. After the introduction of the vector, recipient cells are grown in a selective medium, which selects for the growth of transformed cells. Expression of tfue cloned gene sequencc(s) results in the production of the desired protein, or in. the praiuc;tion of a fragment of this protein. This expression can take place in a condntwu~s manu~er in the transformed cells, or in a controlled manner.
Accordingly, the XYL I and XYL II encoding sequences may be operably linked to any desired vector and transformed into a selected host, so as to provide for expression of such proteins in that host, IV. Tfie En,ryme Preparations of the Invention According to the invention, there is provided enzyme compositions useful in a method for bioblcaching and pulp and paper processing. There is also provided a method for producing an enzyme preparation partially or completely deficient in cellulolytic activity (that is, in the ability to completely degrade cellulose io glucose) and enriched in xylanascs desirable for pulp and paper processing. By "deficient in cellulolytic activity" is meant a reduced, lowered, degreased, or repressed capacity to degrade cellulose to glucose.
Such cellulolytic~.activity deficient. preFarations, and the malting of same by recombinant DNA methods, are described !n US 5,298,405., As described herein, xylanascs may be provided directly by the hosts of the ,invention (the hosts themselves are placed in .the wood processing medium). Alternatively, used medium from the growth of the hosts, or purified rnzytnes therefrom, can be used. Further. if desired activities are present in more than one recombinant host, such preparations may be isolated from the appropriate hosts and combined prior to usa in fne method of the invendan.
The enzyme preparations of the invention satisfy the requirements of sp~cifie needs in various applications in the pulp and paper industry. For example, if the intended application is improvement of the strength of the mechanical mass of the pulp, than the enzyme prCparations of the invention may provide enzymes that enhance or facilitate the ability of cellulose fibers to bind together. In a similar manner, in the application of pulp milling, the enzyme greparations of the invention may provide enzymes that enhance or facilitate such swelling.
To obtain the enzyme preparations of the invention, the nation or recombinant hosts describai above having the desired properties (that is, hosts capable of eupressiitg large quantities of the desired xylanase enzymes and optionally, those which are Substantially incapable of e~cpressing one or more cellulase enzymes) are cultivated under suitable conditions, the desired SENT BY:S K G & F , r-29-94 : 13:35 ~ SKG&F~ G,S & h~ti24 enzymes are secreted from the hosts into the culture medium, and the enzyme preparation is recovered from said culture medium by methods known in the , art.
'The enzyme preparation can be produced by cultivating the recombinant host or native strain in a fermentor. For example, the eruyme preparation of the present invention can be produced in a liquid cultivatifln medium tha! contains oat spelt xylans as the main carbon source as described by Morosoli et al., Btochem J. 239.587-592 (1986)). _ 'The enzyme preparation is the culture medium with or without the IO native or transformed host cells, or is recovered from the same by the application of methods well known in the art. Howevor, because the xylanase enzymes are s~reted into the culture media and display activity in the atnbieat conditions of the hemicellulose liquor, it is an advantage of the invention that the enzyme preparations of the invention may be utilized directly from the culture medium with no fiuther purification. If desired, such preparations mey be lyophilized or the enzymatic activity otherwise concentrated andlor stabilized for storage. The eazyme preparations of the invention are very economics! to provide and use because (1) the enzymes may be used in a crude form; isolation of a specific enzyme from the culture fluid is 2,0 unnecessary and (2) because tb~e enzymes are secreted into the culture medium, only the culture medium need be recovered to obtain the desired enzyme preparation; there is no need to extract an enzyme from the hosts.
If desired, an expressed protein may be farther purified in accordam with conventional co~itians, such as extraction, precipitation, chromatography, affynity chromawgraphy, electrophoresis, or the like.
The invention is described in more detail in the following exanagles, These examples show only a few concrete applications of the invention. It is self evident for one skilled in the art to create several similar applications.
Hence the examples should not be interpzeted to narrow the scope of the invention only to clarify the use of the invention.

SENT BY:S K G & F ~ '~-29-94 ~ 13:36 . SKG&F~ G. o & H~i~25 Example 1 i Materials and Methods Bacterial strains and vector The Escherichia evli strain DHSa (F' ~80dlac~MlS ~(lacZYA-argk~)U169 deoR recAl endAl hsdRl7 supF.r~4 1,- thi-1 gyrA96 relAl; C3~itmo HRL), was used in routine manipulations. The pesiplzsmic-leaky strain E. colt 4924 N/14 (de Zwaig et al., J. Bacteriol. 9~:I112-1123 (1967)) was used for the cloning az~d detection of xy(anase genes. Streptomyces livtdans strain was kindly provided by I7. A. ~Iopwood (John Inner Institute. Norw»t, ID U.K.). S. lividarrr strain 10-164, a mutanc of S. ll ldans 1326 negative for xylanase and cellulose activities (I~ondou, F. et al. , Gene 49:323-329 (x9$6)), was kindly provided by D. Kluepfel (Centre de Rechcrche en Microbiologie Appliqu~e, L$val (Qu6bec), Canada), All the a~e~ s~~ were wild-type isolates from various natural materials. The shuttle E. coli-Streptomyces vector pFD666 was described previously (Denis & Hrzezinski, Gene 111: I 13-I18 (I992)), Growth off' 6acterfial strains E. colt strains were grown in Luria Ber'.ani (LB} medium (Sambrook, J. et al. , h~oleeular cloninS. a laboratory manual (2nd edition), Cold Spring 2a Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).
Act~nomyrxte sa~aing were routinely propagated on Tryptic Soy Broth (Difco). The media for S, lividans protoplast preparation and regeneration were as described by $opwood et al. (Genetic manipulation of Streptonryces, The John Inner Fouttdation~ Norwich (I983)). Lang-term storage and handti~g was as described previously (Fink, b. et al., Biotech. Len. 13:84~-85a (1991)).

SENT BY:S H G & F s 7-29-94 ~ 13.37 ~ 5KG&F-~ 6~ S & H~it26 ~~~~~'~Z

Chemotaxonorr:acul procedures The diaminopunelic acid form in the exll wall and the predominant sugars in whole-cell hydrolysates were analyzed by thin-layer chromatography ~or~g to Staneck & Roberts (Appl. Microbial. 2$:216-z31 {L974)). The G + C content of total DNA was estimated by the method of Ulitzur {Biochim. Biophvs. Acts 2?2: I-11 (1972)x. Fatty acids were analyzed by the procedure of Sasser (Sasses, M., in Methods in Phytobactertology, I~Iement & Sands, eds., Akademiai I~ixdo, Budapest (1990), pp. 199-204).
Biochenaicua' assays Xylanase activity was assayed using the Nelson-Somogyi method (Spiro, R.G., Mdth. F,rc~ymol. 8:3-26 {1966)) which measures the release of reducing sugar from 0.$ 96 (w/v) soluble oat spells xylan is citrate-plaosphate-borate buffer (Teorell buffer). Tn standard conditions, the pH was 5.0 and incubation was for 10 min. at ft0°C. The reaction was terminated by the 1S addition of the first reagent of the redctcing sugar assay. ane unit of enzyme activiry was defined as the amount of enzyme releasing I .mole of D-xylose equivalent per minute in standazd assay conditions.
The ~3-xyIosidase activity was measured with 2 mMp-nitrophenyl-~-Ia xyloside as s'.tbstrate. Incubation was for 10 znin, at 4Q°C in Teorell buffer pH 5.0, The release of p-nitrophenol was monitored at 410 nm.
Total protein was measured by the method of Bradford, M.M. Anal, Biochem. 7x;24$-254 (1976) using the alkaline reagent descr'bed by Stosehecl~
{StasCheck, C.M., Anal. Bioehem. 1$4:111-llb (1990)). The molxular weights of flue purified enzymes were estimated by SDS-PAGE (L.aenunli, U.K, ll~ature z276gp~g5 (1970)). Coloration for glycoproteins with the Schiff reagent was as described in Glossman & Neville {1971). Thin-layer chromatography of hydrolysis prod<ccts was performed as described by Hiely, P. et al., Biochtm. Bioplrys. Acts 1162:246-254 (1993).
i SENT BY:S K a & F ~ t-29-94 ; 13:39 ; SHG&F-~ G~ S & ii;#2~
~1~~~'~~
_22-~'he procedure of Hertheau, Y. et al. , Anal. Biochem. 739:3 83-389 (1984) was used to analyze crude or purified xylanases by electxofocusing in an ultrathin polyacrytamide get (pH gradient 5 to 8). Ten p.I of 20 times concentrated supernatant were applied, Standard proteins (Bio-Itad) were applied on tliese gels alongside the culture filtraoes to estimate the pI of xylanases. An agarose-RBB xylan overlay was used to detect xylanase activities. The overlay gel was prepared from a mixture of 0. $ ~ agazose and 0. 2 ~ RBB-xylan. The agarose_RHg xylan get was overlaid onto - the elxtrofocasing gel. Incubation was carried out at 50°C for 1 hr. Clear zones i0 in the overlay gel indicated xylanase activity.
Tn liquid culture, xytanase-positive actinomycetes were inoculated into T~ryptic Soy Broth and cultivated with shaking at 50°C. Once an appropriate cell density was reached, the mycelium was recovered by centrifugation and inoculated into xylanase production medium (MorosoLi, R. et al. , Bivchem.
J. 233:587-592 (198x)) contaiair~g oat spelts xytan as tt~e main carbon source, Xylanase activity was measured daily using a standard assay (measuring the release of reducing sugar from oat spells xylan incubated with culture supernatants samples for i0 min. at 60°C, pH 5.0).
Bacterial, bacteriophage and plaamid preparations The bacteriophage M13K07 (Vieira and Messing, Methods EnZymal.
153: 3-lI (1987}) was used in the production of single strand DNA. The vectors pFD665 (Dents and Brzezinsl4., Gene li: 115~11$ (1992)), pIJC118, pUC119 (Vieira and Messing, Methods Er~ymal. 153: 3-i 1 (1987)), and pUC21 (Vieira and Messing, Ge,~ 140: 189-194 (1991)) were used for cloning and sequencing proposes.
Culture media SENT 6Y:S K G & F , 7-23-94 ; 13:38 , 5KG&F-~ G, S & H;ii28 -z3-LB mediua:~ was used during the preparation of compeCent cells and thezr transform~axion (Sambreok et al. , Molecular cloning. A laboratory ' manual, Second edition. Coil Spring Harbor Laboratory Press. New Yark.
(1989)}. LB-R8B-xylan (LB+ 0.29 R.13H-xylan + 1.5~ agar) was used to detect xylanolytic clones. RBB-xylan is a complex deriving from the joining of a coloring agent, ltemazol Brilliant Blue (RBB) to xylan. This wac synthesized by following the protocol published by Biely et al. Anal. Biachem.
144: 14x-146 (19$5)).
The medium M13 was used for the production of xylanase (Morosoli et al, , ,~iociaem. J. Z39: 587-592(1986)). The composition of the medium is as follows: IOg xylan, 1.4g (NH4~SO,,, 2,5g K2Hp04, I.Og KFizP04, 2.Og of extract of yeast, 1.0g peptone, 0.3g MgSO, ~?Hz0 per liter of water. The pH
is adjusted to 7.0 after sterilization, then 1.0 ml of a solution of znicro-elcznents is added (0,28 CoClz~7Hzp, 0.5g FcSOd~7Iiz0, 0.168 MaS04~H~~, 0. l4gZnSQyHiO, in 100 znl of distilled water with the pH adjusted to 3 with HCI). Olive oil (2 mi/liter) was added to increase the enayme secretion (Bemand et af., Biotechnod. Bioeng, ~3: '791-7g4 (I989)), The minimal 1~BB-xylan medium was used to detect xylanolytic activity. The method was adapted in accordance with Kluepfel'g protocol (Methods $n.Zyrrrod. 1~:18d-IB6 (I988)). Part A is autoclaved separately, containing Q. Sg KiHp04, 0.2g MgSO, ~7Ha0, 1.Og (h~~SO~, 158 agar in a volume adjusted to 70bm1 of v~ater, rhea part B containing 2g RHB-xylan in 300mI of water is autoclaved. ~;,fter cooling and mixing party A and B, 1 tnt of the micro-element solution is added.
R2YE medium was used for the transformation and re.8ertaratiaa of the S, livldatas 10-164 protoplasts (Hopwood et aL. , Genetic martipulariart o,~
Streptonrycrs, a laboratory manual, the John Innes Foarndation, Norwich, 1985, 338 pages). TB medium was used for the far the ampli~eation of E.
colt (Sambrook er ad., , Molecular cloning. A labvratvty manual, SecO~
edition. Cold Spring Harbor Laboratory press, New York, (1989)). TSB
medium was need for the growth of S. divldans 10-164 and ~lcti~tamadura sp.

SEiVT BY:S K G 8 F ~ 7-29-9A ~ ?3.39 ~ SK3&F~ G~ S & H;ii29 tj~2~~ S
FC7 preparatio:~. 2xYT medium was used for the groductivn of single strand DNA with the E. crrle TG1 preparation. 'This medium is composed of 16g tryptane, lOg extrgct of yeast and Sg of NaCi for a final volume of 1 liter at pH 4.
r Hestrfctian endonuc~eaae, ligaee and phospltatase Restriction eadonucleases and ligases were purchased from Boehrigger Mannheim and from Pharmacia. Calf Intestine Phosphatase (CIF) comes from Pharrnaeia. These enzymes were used in accordance with the manufacturer's instructions.
1Q lPrepaxatlan of cells, protoplaeta, and thefr tt~aatsforntatioa E'. cvli I)HSc~', TQ1 and 4924 NI14 competent cells were prepar~i and transformec° in accordance with a protocol from the Imperial Cancer Research Foundation, and described by Desmarais, D., ~~rioire de maftrtre.
D6partemeut de biologie. Faculty des sciences. Usiiversit~ de She~rooke. 75 1 ~ p. ( I990) . ~3riefly, the following procedure ways used.
~r ~tion of c~~;t cells 1. Starting wittx the frozen cells of the E. cola DI~Sa preparation preserved in 20~ glycerol, smear the Petri dish with SOB or LB (Maniatis eg rxl., Molecular cloning< a laboratory manual, Cold Spring Harbor Laboratory, N.Y'., 1982, 545 page9 (19$2)) and incubate overnight at 37QC.
2. Inoculate S mI of SOB culture using a single colony.
3. Incubate tlar culture at 3740 under agitadan for about 2 hours, or to the point of A~ is about 0.3 or tin it begins to becornc cloudy. ' 4. Make a 1.:2p . dil:~~n of the cult4ze in 100 ml t~f SO~
(preine~abated to 37~C) and incubate at 37oC to the poi.aat Of SENT BY:S K G & F . 7-29-84 ; 13:33 ~ SKG&F~ G~S & H;ii30 e~ ,i a Asp is 0.48 {a.bou: 2 hours). This optical density is optanal for DHSa and may be slightly different for other preparations.

5. Leave on ice far 5 minutes.

6. Centrifuge for IS minutes to pallet cells.

7. Remove the flogting matter aztd once again suspend the cells in 40 ml of TPB I (defined below).

8. Leave on ice foe 5 minutes.

9. Centrifuge per item number 6.

IG. Remove the floating matter and once again suspend the cells in 4 ml of 'I'>?H I.

11. Ixave on ice for :5 minutes.

I2. Distribute 200 Fal via 1.5 microfuge tube (refrigerating the microfuge tubes. pipette tips and pipettes tv 4~C
i9 preferred.

13. Freeze in dry lee.

I5 I4. Ivlaintain the aliquotcs at between -60 or -'FO~C.

~B. 'I~assfortt~t~on I. Defrost the cells to room temperature just enough to liquefy the suspension.

z. Txave for IO minutes in ice.

3. Add fINA (up to I!5 vplume of the cells; use no more than 100 ng of IaNA for 200 pl of cells). Using freshly prepared cells, begin the protocol at this stage.

4. Ixave an ice #or 3~0 minutes.

5. Incubate the cells at 42oC for 90 seconds. This stage may be optimized ire accordance with the preparation.

b. Put it on ice for 1-2 minutes.

7. Add 4 volumes of SOB or L13 (800 p,l per 200 ~d of cells).

8. Irtcttbate at 3?o~C for 1 horaa~ (agitation is preferred buE

unnecessary). .

9, Centrifuge for 1-2 tnitrutes in a microcent3rifuge and resuspend the residue in 200 ~l of SOB or LB.

10. Spread on a SOH or LB Petri dfsh with antibiotic, N,B, _ All centrifugings and solutions must be carried out and conserved at 4oC respectively. It is preferable to delicately handle the cells during tlu stages of resuspcnsion.
TFB I conaains 30 mM potassium acetate, 100 mM RbCla, 10 mM
CaCIz2HZ0, 50_m_M Mn.C>'~4H~0, and 159 glycerol. Adjust the pH ta5.8 using 0.2M of acetic acid. Use a 11100 acid dilution of glacial acetic acid:
this corresponds to about 50 drops for 200 ml of solution. Use of distilled L.
water i' preferred in a glass system. SteriIite through filtration.
TFH II contains 10 mM MOPS, 75 mM CaClz-2HI0, 10 rnM RbClz, and 153b glycerol. Adjust the pH to 6.5 with 1M KOH (about 35 drops).
Sterilize through filtration.
The S. iividan.s 10-164 protoplasts were prepared and transformed according to the protocol of Hopwood et al. Genetic manipulation of Streptomycea, a laboratory manual, the John Inner Foundation, Norwich, .
1985, 338 pages.
Purification of a DNA fragment on agawr gel FoLlow9ng DNA band migration on TAE agar gel (Maniatis et- aI. , Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory, N.Y., 1982, 545 gages), the purificadon of DNA fragments was performed by the method suggested by "Gone Clean" BioICan Scientific, Iac.
Zy~ogram 'Trademark SENT BY: S K G & F ; ~-2~-94 : ~ 3 ~ 40 ~ SKG~F'' Ge S & H;t#32 212~1'~2 The procedure is the same as for a polyacrylamidc gel {SDS-PAGE) except that the sample has not been boiled, and, further, 296 RBB-xylan is added into the acrylamide mixture. The protein sample is prepared with the following {3X) swab: 3.0 mI of glycerol, 0.6 g of SDS, 0.228 g of Tris-Base, and Q.1 mg of brompherol blue.
The development of the xylanalytic activity is achieved by soaking the gel in 100 mI of Tris-HCl ~0 mM {pH 7.5) - methanol 20~ at room temperature for ti0 minutes. Thea the gel is washed in 500 m1 of Tris-HCi 50mM {pH 7.5) - EDTA {1 mN~ at 4~ overnight. The visualization of enrymatic activity is achieved by incubating the geI at SO~C in s McIlvaine buffer (pHb) until the bands $ppear.
Extraction of genomic and pla~mid DNA
The plasmid DNA extraction protocol used was the one described by Maniatis et ai. , Molecutur cdontng: a laboratory manual, Cold Spring Harbor i5 Laboratory. h'.Y., 1982, 545 pages.
The genomic DNA extraction prowcol used to extract Actinornadura sp. FC7 DNA was that of Rao et a1. Methods err~ymod. 153: 1~6-198 (1987)), except that the mycelium of the Acttnomariura sp. FC7 (20 tnl) was broken by passing French's press $t a pt~ssure of 2,000 lblpo~.
Lxample 2 Screenlrtg ,Yr»gram ~'vr the lsalat~on of .Tylaaiotytic Actlnomycetes In order to find new variants of xylaxtases, ef~ci~nt at pH 4 and 70°C, a screening procedure was developo<i to identify organisms showing such activi4~es, The screening was oriented towards actinomycetes as they are efficient producers of tiny extracellular enzymes and are amenable to genaic and molecular anaPysis.

SENT BY:S It G 8 F ; 7-2°-5~ ; 13:40 ~ 5KG&F-~ G~ S & H;ii33 ~~2~'~~
-2s_ S~unpies of cornpnst, manure, straw as well as sarrzples of biofilm cieveiaped on tt~~e inside surfaces of pipelines used by the paper industry were , e~~icbed for triercnophilic actinomycetes by several treatments: dry heat treatment (12,0°C; 6C3 rnin.) (hionomura & Hayaleawa, in Biology of rz~tinorrry~ceres '88, Olcarni, Y. et al. , eds. , Japmn Scientific Societies press, Tokyo (1988), ~rp. 288-293); selection with phenol (30 min. treatmenx in 1.5 wl v pbenGI soiut;on, pH 6.~ at 30°C) followed by centrifugation and washing with water (Planotnura & Hayakawa, in Biology of actinorrrycetes '88, 0kami, ~. ea al., eds., Japan Seiezttifie Societies Press, Tokyo (1988), pp. 288-293);
14 selection on humic acid-vitamin agar ('Fiayakawa ft Nonomura, J, ,~et~rtent.
T'ect~. d5;5f~1.-509 (198'0 ar cultivation on semidry xyian powder, as described by 1'Naldron et al, (Appl. Micro~tp, ~oteeh. 24;477-48b (1986)) except ttzat xylan was subgtivtuted for cellulose.
If desired, novobiocirt (50 mg!1) may be used to eliminate mobile 15 bacteria in ttie first selection of aetinamyeete eoloniES. Some thermophilic:
actinc~xnycete strains may be killed wb~en novobiocin is used in this manner.
Howeuer, td'zc strain of the invention, Actarcornadrrra sp. FC7 seems to be relatively resistant try novabiocitt A,~ter these treatments, surviving bacteria were plated on Tryptic Soy Agat and eultivateQ at 5p'C ar 50°C. Individual colonies were picked and inoculated an minimal agar contaitai_,n.,g 0.2 ~ xylan. covalently bound to ltema~ol $riliis~-tt Blue (RH$-xylaa; Biely, P, et at. , Aril. Blochem.
1~4:142-1.46 (1985)) and incubated at 5t?°C ar 6Q°C. Each day, the caloaies were examined for medium clearing and morphology. Xylanolytic actinomycetes were retained for further studies.
A total of 12 strains growing at temperatures between 50° and c50°C
t~ show~:ng marked degradation capability of RB&xylan on solid medium were isolated froze compost, zr~anure or straw (Table 1). Alt these strains were ctassia~ed in tt~ actinomycete grattp on the basis of their morphology aztd 30 the huh ( ~ 65 mol 9~) G ~ C content in their total DNA.

SENT BY:S I' G ~ F ~ "r°~a-94 ~ 13:41 . SHG&Fi G. S & H~ti34 21~~~.'~~

All the strains were examined for their ability to produce ~cylanolytic enzymes that were relatively active at pH 4, '70°C. For this purpose, alI the , strains were cultivated in tryptie soy broth at 50°C (except the control strain, S. lividans 1326 which was grown at 30°C), then inoculated in xylanase .
production medium. Extracellular xylanase activity ws.s measured by the release of reducing sugars from xyian in two different conditions; at 60°C, pH
S.0 (the xylanase activity measured in these conditions was taken as 1000, and at 70°C, pH 4.0 (strnngent conditions) (Table I). Sia strains (as well as S. lividans J,326) kept 596 or less of taeir activity in the stringem conditions;
three strains retained between 596 and 20~ and three strains retained more than 4Q~. The strain FC7 (originating from manure and isolated on humic v acid-vitamin agar) retained 65~ of its activity a:.'70°C pH 4. This strxan was chosen for fttrther studies since its crude xylanase was also efficient at hydrolyzing the xylan cotttxined in the hemicellulose liquor.

SENT BY:S K ~ & F ~ °W °-94 r 13:41 ~ 5K~&F~ u~S & H,#35 'able 1 Summary of the isolation o~ xylanolvtlc thermophilic actinornycetes Xylanolytic Isolate Origin Bnrichtnent methodactivity kept at pH
4170C t F 1 manure dry heat 2 14 9~

F2 mature dry heat + phenol296 FAA3 manure solid enrichmentI29~ -FC7 ~~ ~_ag~ s _-65~

FY5tl4 manure phenol 3 9b FPdt35 manure phenol 296 pAl straw solid enrichmeru~9~

CAI compost solid enrichment5796 CCA3 compose solid earichtnent5096 CCA5 cotrtpost solid enrichment2096 i CCA601 compost solid enrichment296 C604 compost -- 2 96 S, livictanscvnttoi 296 1326 strain i: Activity xt pH 5lbCr°C was taken as 10096.
2; Dry heat treatment (1?,0°C, 60 min.) (IrTonomtua & Hayakawa, in Biology of actinomycetes '88, Ckatui, Y. et al., ods., Japan Scientific Socaetiee press, Tokyo (I988), pp. 28&293).
s: Treattnont in 1.596 phenol {30°C, 30 tnln.) (Nonomurs & Hayakawa, op. cit.).
4: Modified after Vttaldron lr., C.R. et rtl., Appl. Mlctobio, Birxech. 24:477-(1985).
5: Hutriic-acid - vitamin agar selection (Hayakawa & Nonamura, J, Ferment-T'ecla.
65;501-509 (1987)).
The FC7 strain demonstrated a typical actinomycece morphology with white-yellow basal mycelitaat when grown on tryptic soy agar. Tn liquid cultu~ts :n Tryptic Soy Broth medium, the growth of FC7 (estimated as wet weight of mycelium per ml of culture broth) was maximal at 37-50°C, m~odeeate at 30°C and 60°C and very slaw at 22°G. Spotulation was obsorved only oacc: the FC7 was monosporic, with spores produced on very short sporophores in. a poorly developed aerial mycelium.
rrseso-SENT BY:S K G 8 F . 9-Z9-9C ; 13:42 ; 5K~&F-~ G, S &. !i;id36 -31 _ diaminop;,molic acid was found in the cell wall peptidogly;..an,. No mycolic acids were found. Whole-cell sugars had no diagnostic value as they varied widely with the temperature at which the organzsra was cultivated. The relative abundance of hexadecanoic (26.45 ~ of total fatty acids content), 14-methylpentadecanoic (I
1.28y6) and 10-mekhyloctadecanoie (10 75 ~) acids in the fatty acid composition (pattern "3a"
according to Kroppensredt, Ft.M., in Chemical methods in bocteriat sysrematics, Gaodfellaw & Minnikin, eds., Academic Press, London (1985), pp. 173-199), in conjunction with the other taxonomic data, permitted the classification of FG7 in the "Actinomadura-Thermomonospora curvata" group of the family Thermomonosporaceae (I~roppenstedt & Croodfellaw, in Tire Prokaryotes, Balows et at., eds., Springer-Verlag, New York (1992), pp. 1085-1114). The strain will thus be referred to as Actirwmadura sp. PC7. No attempts were made to classify this strain at the species level. This bacteria synthesizes xylanases which maintain most of their xylanolytic activity at a temperature of 70oC and at pH 4. By mean$
of a zymagram it was determined that this preparation would produce up to 4 xylanascs.
Example 3 Clortirtg of Aettnonaeiduna sp, FC7 xylanuse~ genes into E. colt I»Sa Preparations of Bscherichia coil DHSaF' (Hethesda Research Laboratory) were used for cloning manipulations. For gem bank construction, fatal DNA was isolated from Actinamadura sp. PACT by the method of Rao, R.N. et at., Meth. Ert~ymol.
153:166-198 (1987). Genomic 1~NA of the Actinomadura sp. PC7 preparation was completely digested with the restriction endornzclease Bgtll. The genome of the Acttnomadura sp. FC7 preparation, following a complete digestion by BgilI, generated fragments with an average size of I2 kb, v The BgIII fragments were spliced into tlhe pFD664 vector that had first been cut with Bttm~iI and dephoaphorylated in accordance with the protocol proposed by ivianiatis, T., et al. (In: le~olecular Cloning, A Laboratory hlanuad, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)). ~'. coil DHSaF' (200 ~cl of qualif'uad cells) was transformed with 100 ng of binding mixture. The cells were SEPaT 'ti4':5 't1 G 8 ~_ ; 7-2~-94 ; ? 3:42 ~ 5KG&F-~ G. S & H;~i~7 spread out an solid ~.$-RBB-xylan plus kanarrlycin (50 Icglml) then incubated at 37~C
foz5to6cays. , The effectiveness of resultant recombination was 86 ! (about 9,000 ez;omb:nauts out of 10,500 examined). The number of recombinant preparatior3s was S assessed in accordance with the mini-prepazation method Maniatis, T. , et ad. (In:
Molecular Clo»ir~g, A Labdratoty Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY (1982)). The gene bank represented more thazt 99~ of the genome of Actlrao»iadura sp. FC7. This pezcentage was derived from the formula described by Clarks arui Carbon, Cell 9: 91 (1976).
1Q Six potentially positive xylan~iytic clones were obtained aver 5 to 6 days of incubaaor~ following the appearance of degradation zones. Following a respreadi~g . on L13-RBB-xylan znedittm, five positive clones, or pJFl, pJF3, pfF6, plFB
and pJFIO were identified and selected, while the other closes (pJF2, piF4, pJPS, pJF7 and pJF'9) were eliFninatad as false-positives. Restriction endorniclease analysis i IS conf"~axed that cloa~s pJFl and pJF3 had an insert of an approximate size of 2Qkb, white clones pJF~ an;.-l p,TF$ ara,~d hive the same 2.7 kb insert, but in an opposite onentat:on. Clone pJFlO ha;3 multiple BgIII inserts, of which one 2.7 kb insert was identical to the onr~ found in pJF6 and pJFB.
.~, eoli is a c'~raxn-negative bacteria, and it is not down to be effective for the 20 section of enzymes. No~theless, positive clones were isolated thanks to the natural lysis of bacteria. In other words, as a result of the release of the contents of the E.
cold past iota the ~nediunx containing RHB-xylan; since the recombinant host expressed the xy lanase gene, it produces a degradation zone around it, occurri~ag after to 6 ~ys of incubation.
25 ~~pte , f Cloning of Actlnofiaduraa sp. FC7 xylanasB genes trtlo E. call 4924 h'lId The plasmids from tire gene bank described is exempla 3 were isolated bg~ a total plasmid preparation as.proposed by Maniatis, T., et al, (In: Molecular Cloying, A Laboraa.~ory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY

StNT BY:S K G & F . 7-29-94 ; 14:44 ; SKG&F1 16135639869#-;# 2 ia~a . o ~ ~~'~bs t~=~.
21291'2 (1982)) and transformed into E, codi 4924 N/14. After ligatiop, the DNA
mixture was used to transform competent cells of a periplasmic-leaky strain E, codi 4924. The transformation mixture was plated on LB agar containing SO ~,g/ml of kanamycin and 0.2 tng/ml of RBB-~cylan. A total of 8850 recombinants was obtained. After 2-3 days S of incubation at 37°C, colonies surrounded by clear areas were picked, grown in LH
liquid medium and repIated at low density on RBB-xylan medium. Six of these recombinants showed clearing of RBB-xylan after 2 days of incubation.
In order to speed up the operations involving the visualization of degradation zones, we used the E. coli 4924 N/14 preparation. E. cola 4924 N/14 has a IO periplasmic deficiency which has not been genetically defined (de Zwaig and Luria, J. Bacteriol. 94: 1112-1123 (1967)). So this allows the very swift passage of its periplasmic contents to the external medium. The main reasons for its use .are a better visualization of degradation, and an economy of time. Clone p3F11, for example, was isolated thanks to this preparation, because the sensitivty of the method 15 with E. coli 4924 N/14 was probably stronger than with E. coda DHSa. The appearance of a degradation zone needs only 16 hours instead of 120.
The ability to k~ydrolyze ItBB-xylan was conserved after plasmid purification fzom all of these recombinants and retransformation into a new host. Since xylanolytic activities were detected in recombinant E. coli strains and since E. cold Z0 is not known to produce xylanolytic activities, the cloned genes should encode xylanases and not a regulatory protein involved in xylanase production. **
The clones that appeared to be able to hydrolyze RBB-xylan after this second a round of plating were retained for fiutlier studies. Their plasmid DNAs were e~ctracted and mapped with restriction enzymes using standard methods (Sambrook, :~5 J, et al. , Modecular cloning, a Cabaratory manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).
Plasmids pJFI, pJF3, pJF6, pJF8 and pJFlO were analyzed by restriction mapping. pJF1 and pJF3 carried the same cloned insert (about 20 ICb) but in opposite orientations. The other three plasmids also had a common cloned segment (2.7 kb), either in two opposite orientations (pJF6 and pJF8) or fused to another segment (pJFlO). This segment was differezxt from the insert present in pJFl arid pJF3. Thus, SENI BY:S K G 8 F s "-29-94 ~ 13:43 ~ 5KG&F-~ G~ S & H~it39 ~12~1'~~

the transformants foil into two distinct groups. One transformartt from each group (pJFI and pJF6) was chosen for f~.~rther studies. The xylanase-encoding s~gtnents were mapped by deletion subcloning, transformation and plating on 1RBB-xylan agar.
The shortest DNA segmenrs still allowing for xylanase expression are shown on Figures 1 and 2. The differences between the restrictions maps suggested that two different xylanase genes were cloned: xlnl carried by pJFI (Figure 1 j and xlnT1 carried by pJF6 (Figure 2). The corresponding xylanases were named Xyl I and Xy1 II, respectively, Clone pJF6 was chosen for sequencing as it presents the interesting characteristic of living a relatively short Insert (2.7 kb), and required no extensive shortening of its inert by sub-cloning.
The ~aml~ site of pFD666, used for insertion of the xylanase genes, is localized inside v>F a multiple cloning site flanked by transcriptional terminator8.
However, it has beext shown that some transcription occurs from one side, most likely driven by the neomycin resistance gene (penis, F., ~Construction d'un verteur is navette pour Eschcrichia cell et les actinomyebtes et clonage d'ur1 gene de chitosanase d'actinomyc~te," Ph.D. thesis, UniversitE de Sherbrooke {1994), 120 pp.).
Since the xylanase genes were cloned in both orientations in the BaneHl site and xylanolytic were detected in. E. cell recombinant strains, whatever their orlentatians, it seems likely that some Actinomadura promoters can be recognized in E. cell.
Zymogram analysis revealed that ~ xylartases are pzoduced by the clone plFl .
These pJP1 xylanases would correspond to tht the two highest rnoleeular x~eight bands produced by Actinomadr~ra sp. FC7. Zymogram analysis of Actinomadura sp.
PC7 culture supernatants in xylan medium revealed two major (slo~vec) arbd two minor (faster) bands of activity (not shown). The two major (slower) bands ca-migrated with the two bands obtaaned with the crude preparatio!t from 10-164(pJFl) culture supernatant and corresponded most probably to the ~ IGDa and 37 kDa forms of Xyl I (sex below). TTfle activify of ~yl II could not be visualized with this particul$r zymogram system, probably because of the inability of this protein to renatu~ during the post incubation steps. Thus, besides Xyl I ane! Xyl Q, FC7 produces at least one or two other xylan-degrading activities. The ocairtenet of SENT BY:S K G 8 E= , 7-28-94 ; 13:44 ; SKG~Fi G.5 & H;ii40 2121'72 multiple xylanase activities have been reported in numerous microorganisms (VVo~tg, K.K.Y, et ad., Ma'crobial, Rev. 52:3~~-3I'7 (1988)).
The pJFI clone insert was reduced, yielding the sub-clone pJF1020. The latter has an insert of about 7.5 lCh, which is sufficient to contain witfia it 2 genes coding for xylanases. The genes) is located in a 5 k13 portion of the inital fragment.
Example 5 XyXanase producsion by recombdr~ur~t strudns The plasznids isolated from the E. toll clones that were able to hydrolyze RICH-xylan were used to txaasform protoplasts of S. ldvidans IO-I64. After pmtoplast IO regeneration anal colony selection far kanamycin resistaz~e, the transformantb, were te$ted for their xylanase-positive phenotype oa minimal medium (Hogwood, D.A.
et al. , Genetic manipudatdore of StrEptomyces, The John Ynnes Foundation, N'orwieh (1985)) containing RHB-xylan.
Plasmids, p~FI and pJF6 were used to transform S. tivadans, as ~'. cold is not an efficient host for extracellular enzyme production. The mutant strs~in S.
ldvd~Cans 10-164 was ascd because of its inability to produce endogenous xylanass and cellulose activities (lvfoa~ou, P. et al., Cxhe 49:323-324 (I9861). Both plasmids complomcnt~ed the xylanase-negative phenotype when transferred into the IO-164 strain. This host allowed over-production of the xylanases encoded by the cloned genes with a low background of other proteins, thus facilitating the purification procedure.
Qne transformant of each type bearing tk~e pJFI and p)Fb plasmids were tested for xylanase production In Iicluid culture. Each transformant was inocuaated into Tryptie Soy Broth (1)ifev) containing 50 ~glm1 of kanamycin sad cultivated for hours at 30°C on a rotary shaker at 250 rev./min. The mycelium was recovered riv centrifugation of the cultux-~es is a benchtop centrifl~,ge (3,000 g; IS min), suspendc9cl in 50 ml of 0.99b sterile saline send centrifuged again. 24 ml of mycelial pellet wore then inoculated into Z.2 liters of ~xylanaae production medium tMvrosoli, R<
er al. , 8doche~na. 1. Z3~':587-~~2 (I986)) without kanamyein (the vector pFD666 and its derivatives are generally stably maintained in Streptomyces Lividans without antibiotic selxtion (Denis ~. Brzez.inski, Gene 111:11~-118 (1992}). After 72 hours of cultivation, the culture was cen~_rifug~d (11,000 g, 30 min, 4°C) and the supernatant was r.-.covered as the crude enzyme preparation.
g . Examplr 6 Purgation of xyla~cares 1 and 11 All the purification steps were, carried out at 4°C. The chilled supernatant (0.6 liter) of a cultuzc of S. lividans 10-164 {pJFI) (for xylanase I
purification] or S.
lividaas 10-I64 (pJF6) (for xylanase iI purification) was mixed with three volumes , of ice-coil 95 ~'o ett~.anal. After settling overnight, the precipitate was recoveteci by centrifugation (9,000 g, 30 min}. The pellet was .resuspended in 50 W 1 of 20 ml's Tris-HCl buffer pH 8.0 and loaded on a 0.9 cm x 30 cm DEA~BioGeI A anion-exchange column (Bio-Rad) eduilibrated with the same buffer. The colutzin was then washed with 50 tni of the same buffer and proteins were eluted with a linear gradient (0 to 0.6 urn of KCl (tonal volume: 120 ml}. Pz~actions were collected and the xylanase activity was detected by spotting 20 ~d samples on RBB-xylan agar and incubating at 37 °C. The active fractions were pooled, wncentrated dawn to 4 ml by dialysis against Concentrator Rcain (H3o-Rod? and loaded on a 1.S cm x 100 cm BioGel A-0.3rz size-exclusion chromatography column (Bio-Rod) equilibrated with 20 mM K-phosphate buffer pH 6.0 (prtpared by mixinn appzopriatc proportions of 100 mM monobasic potassium phosphate and 100 mM dibasic potassium phosphate, then diluting with four volumes of distilled water). Fractions were collected and xylanase activity was detected as before. After addition of glycerol (final cotmcentratioa 50~ vlv), enzymes were stored at -20°C. ;
Both xylanases were purified to homogeneity (as Judged frcm Cootuassie Blue-statt~cd SDS-PAGE gels) by the above protocol involving ethanoi precipitation, anio~r exchange chromatography and size-exclusion chromatography. Table 2 summartzes the enryme purification data. Yields of 27 and 1490 wire obtained and me specific 'Trademark l StNT BY:S iE G & r ~ i-29-94 ~ 13~45 ~ 5KG&F-~ G.5 & H:ii42 -37.
activities in startdazd assay wit~'~ oat spelts xylarv were 178 and 126$
Unitslmg for purified Xyl I and Xyl II, respectively. , Durigg the purification of XyI I, ewe peaks of xylanase activity were separated by the size-exclusion chromatography step. The major peak corresponded to a protein of 48 kDa (and this protein was more extensively studied) while the minor peak corresponded to a 37 kDa protein. When vazious deletion derivatives of p~Fl ppasmid were analyzed for tl?e pattern of their protein production, the disappearance of the minor band was always correlated with the disappearance of the msljor band.
We conclude that the smallez protein, is not encoded by a separate gene but is a derivative of the 48 kDa Xyl I protein.
The biochemiE;al properties of Xyl I and Xyl II are summarized in Table 3.
Xyl I resembles other high analecular mass/low pI xylanases (along, K.I~.Y, et al., hlicrobiol. Rev. 52:305-317 (19$8)), s>.~h as XInA from S. ltvidarts (Morosoli, R.
et al. , Biochem. J. 239:587-X92 (1986); Shareclc, P. ,et al. , Gene i0~:75-82 (1991)).
ar XynA from "Caldocellurn saccharolyticurn" (L,dthi, E, et al., .~lpPt.
Envtron.
Microbiol. 55:2677-2683 (1990); Liithi, E. et at. , Appl. Environ. ~ticrobtol.
~6: l017-1Q?~ (1990): its molecular mass is higher than 40,000; it shows a low bttt significant aryl-d-D-Xylosidase activity and it is able to hydrolyze efficiently xylooligosaccharides, as shown by the appearance of short oligomers (xylobiose, xylotriose) among the reaction products early in the hydrolysis (Table 3). In co~r~t, Xyl II has no detectable aryl-~-D-xylosidase activity and hydrolyzes xyloollgosscharides much siawer than Xyi I. In this situation, short oligomers appear in the reaction mixture only after very gong incubation time.
The classification of Xyl II on the beeps of the data presented in Table 4 is not 2~ straightforward. This protein has a neutral pI but its molecular mass is much lower than the p~:, of the majority of the ~high M,.Ilow pI~ xylanases. Also, its high specific activity against oat spelt xylan and its decd ability to hydrolyze short xylooligotners classifies Xyp II nearer the low-molecular-mass enzymes with similar biochemical properties, such as XInB and XInC of ,~. Ilvidarzs (Bieiy, P. et al. , ~iochlm. 8iophys. Acts 1162:246-2S4 (i993)). Ilowevez, in Western blotting experiments (unpabpished) Xyl iT gave a positive reaction with a rabbit antibody SENT BY:S K ~ & F ~ 7-2y-84 ~ 13:46 ; SK~BF~ G,S & H;ti43 ~129I72 _3S_ against Xylanase A from ,~. diviciarzs (a high M,Jlow pI xylanasc), which ~3oes not cross-react wittx the Iow hirlhigh pI xylanases Xin$ and XInC from thG same ' organism ('Jots-Mehta, S, et al. , Gene, 84;119-122 (1990)). ConseqiZently, we assume that Xyi II is either a low M~ zylanase with an unusual neatra! pl or, more probably, a truncated protein, originating fZOm a high \~I~ xylanase gene or protein.
The effect of pH on both xylanases was studied at three different temperatures (Figures 3 and 4). The optimal pH lies between 5.2 and 5.7 for Xyl I as well as for XyI II. At pH 4 and 70°C (the temperature used in the screening proceduze), Xyl I retained 67~ of its rnaximal activity while Xyl II retained only 26'~ of its activity in these conditions, Clearly, the Ievel of activity observed at 70°C/pH
4 with the crude vulture supernatant of Acrinnmadura sp. FC7 was due to the predominance of XyI I among the xylanase forms secreted, by this wild~type strain, Remarkably, at its optimum pH, Xyl I retained foil activity even at 80°C
(Figure 3). At this higher temperature, the decrease of activity in acidic pH
was faster, but still less marked than for the majority of known xylanases: 41. &
of. the maximal activity persisted at pH 4. In contrast, even at optimal pH, Xyl II
was 8.1-times less active at 8Q°C than aL 70°C (Figure 4).
Ta estimate the rh~rn~ ~bilit~, of XyI I, the enzyme was incubated in Tcorell t;!uffcr in the abse~ er presence of 100 pg/mI of bovine serum albumin at different temperatures. Periodically, samples were withdrawn and the residual activity was measured by standard assay. When preincubated at pH 6l50°C, Xyl I
conserved full activity for at least 96 hours, At pH 6l7Q°C, the half-Iife was 6 hours in the absence of BSA and 18 hours in the presence of BSA. At pH 4170°C. the half ~ifp ~9° ~n hours in the absence of B.~,A and 22 hours in the presence of SSA. These values are 2S within the range of stabilities obtained for trade thermoresistant xylan$ses from other ~ctirrornadura species {Holtz, C. et al., Antonio vcan f,eeuwenhoek 59:1-7 (1991));
however, tticy are clearly shifted towards mere acidic pHs.
In conclusion, the screening procedure developed for the invention, based on the simultaneous application of two stringent parameters (low gH and high ~mFere~) resulted in the isolation of a xylanoIytie actinomycete which produces SENT SY:E h( G 8 F ; 7-28-94 ; 13:46 ; SKSB~F-~ u, S & H;Ji44 212~I ~2 at least one xyl~.nase that rerrtains almost folly active and is very stable irl these conditions.
Table 2: Puri~acaki~n a~f xyl~nase x ~a a ~.om culture supernatants of recombinant Str~ptornyces tividans 1.0-I64~
stralrls Teta1 ~c Protein Yiold ~~ca-activity activity(~) (units) (~~ lion t~tsJmg) factor A: Xylaaase , I produced by Strtpr4myces dividons 10-1G4 (pJFl) Culture brothx,20 ~~ 38 ? 1.0 F_tharloi 325(? 58 56 00 1.5 ptecip. 1265 4.3 135 95 3.6 DEA&BiotJel 37 BioC3e1 A-0.5m910 5.1 178 27 4.7 B: Xylatxace II produced by S,trcptorrayces tiv~dans IO-164 (pJF6) G~tltute 11464 Sh.8 132 100 1.0 broth Irthauol 94X2 37.5 251 82 1.9 precip.

DEA~BioCiel 41 i5 6.7 615 38 4.6 BioGt1 A-0. 1581 1.25 1268 I4 9.6 Sm Table 3: ~i~x;hemica~ properties of Xpl I and Xyl ~
Y..1 T
_ _" _ _ n~a ax Molecular weigrt (afder48 I
SI3S-PAGIr; D

c 34 itDa a --s-._._ Isoelectric point ~
$

. 7.I

Optimal temperitcure 75C 74C
at pH 5 1, 2 Optimal pH at 50C I, 5.2 2 5.7 '~ hy~olys~ p~u~ xylobiose, xylotriose_ r ~~er oli~oxylosidea after 34 miu. reaction higher oligoxylo$ides.;
' Main hydrolysis producetracos of xylose, afier 18 h. t~actiou xylobiose, aylotrioaeXYlobiose~
t xytotriose Azyl-p-D-xylosidase 0.13 U/mg undetectable specific activity SEfT BY:S K a ~ F 9-29-5d ; 13:~~ ~ SKG~Fi 3~5 & h;#45 ~1~91'~~
Staining with Schiff reagent ~ negative negative 1: Detertnincd with oat spelt xylan as substrate 2: The reaction time wag 10 min, example T
Sequence of the Insert in pJFd A restriction map was, drawn up to allow sub-cloning of fragments -thereby facilitating seduen~cing (Figure 5). Several DNA fragnnenis were sub-cloned and sequenced.
The plasmid DNA of the positive clones of the gene bank of the Actinomadura sp. RC7 preparation contained in vector pl?D666 was digested by the Chosen restriction endonucleases. The DNA fragments thus produced were purified by "Gene Clean" for subsequent ligation to vectors pUC118. pUCIlg and pUC2l. The unidirectional exonuclease IIllnuclease S1 deletion method described by Hsnikoff, Methvdr enzyrnct, 155: 156-165 (lg8'7) was selected in obtaining additional sub-clones.
The 'Jieira and Messing protocol (Gene 104:189-194 (198'T)) modified by Patent J-L., T6e JH.T-~ aetinophage: sequencing and promotional study.
Master's thesis, Departrnent of Hialogy. Faculty of Sciences. University of Sherbrooke.
8l p, (1992) was chosen foz preparation of sinigle strand DNA. Double strand DNA
preparation was completed in accordance with the "T7 Quick krime Kit" of Phatmacia LKB Biotechnology, Single and double strand DNA sequencing was aehievrd accozding to the method of Sanger et at. , Proc. Ncrtl. Acad. Sci.
U5:4, 74;
~4b3-5467 (19?7) fzom the °Sequenase and 7-deaza-dC,TP" set of United States Biochemical.
A prelinxinary cotnput,~ ~yg~ ~~ it possible to prove a very strong seque~ing homology to Streptomyces lividans xylanase A. This made it possible to Iccaiizc the beginning of the OfLF' ~i~ for a xylanase by clone pHrb_ Thus, the xlnll gene is Localized at, and stquoncing was directed to, only a portion of the pIP6 SENT BY:S K G & F ~ 7-29-94 ~ 13:47 ~ SitG&F-~ G~ S 8 H~ii46 21291'2 -~r-insert, that is, from the NruI site to the BgIII site to the right of the restriction map for pJF6 (Figure 5).
The nucleotide sequence of the insert in p,TF6 is presented in Figure 6 and is Genback Accession Na. U08894. An open reading frame (ORF} begins at nucleotide 521 by a codon GTG and ends probably through an end of translation codou located in phase next to the vector, since no terminal radon was found inside the cloned fragmeni. The gene would therefore be truncated and coded as active xylanase.
A
Shine-Dalg;arno sequence (GGAGGA) specific to the attachment to ribosarnes was found at nucleotide 509, pooordi~ to Sttwhl, W.R., Nucleic Acids Research. 20:
I~ 9~1-974 (1992}, this RB$ is completely homologous to the consensus sequence produced from 40 streptomycete genes (Figure 7). The coding region of this gene has a nucleotide content rich in G+C, on the order of 6$9~. Pttrthermore, the percentage of nucleotide type (G or C) found at position 3 of the radon is over 906, which corresponds with the results reported by Bibb et cal. (1984).
IS According to'Wo~ et al., Microbial. Rev. S2(3); 305-317 (198$), xylanases can be classified into two classes, either class A, which regroups the xylansses having a a~oleeular weight over 3~ kDa and an acid pI, while class B brings together xylanases with a molecular weighs below 35 kDa and a basic pI. Thus this ORI~
of 1527 nucieotides codes for a xylanase of about 43 kDa, and would therefore belong 2~ to class A.
The signal peptide of the pre-protein of this xylanase bas the characteristics normally found in such amino acid sequences (Perlman and Halvorson, J. ~4tol.
Blot.
td7: 391-4(yg (1983}: that is, a positively charged N-terminal extremity containing ~8s (R) followed by a long sequence of hydrophobic amino acids and a C-25 terminal segment iztchidang a praline (P} localized near the cleavage site (AXA) of the peptidase signal (Figure 8).
The promoter region is typieai; that is, a spacing of 16 nucieotide$ separates ttte -35 region (T°I'GACG} from the -10 regioa (CACAAT). This pmuwter is comparable to those illusarated by Sr<ohl, NucleicAcicts Research. 2Q: 961-974 (i992) (Figure 9}. Ft~cthermore, this promoter would be quite horncalogous to the promorrr SE~JT BY:S K G & F ; 7-~9-94 ; 14:44 ; 5KG&F~ 16135639869#-;# 3 l Cz~o ~ a'~1~ l ~Sl,~ b s'-~~ 'f~-t'~ ~' ~129~7~

consensus sequence (TTGAC...TATAAT) found in Escherichia cola (Lewin, Genes, John Wiley & Sons, Inc. USA, 1983, 715 pages).
The restriction map studies suggested that the 2.7 kb fragment found in clones pJF6, pJFB, pJFlo and pJFll were identical. The extrenuties of these fragrnEnts were sequenced and compared with one another in order to verify this. The results indicated that the 2.7 kb fragment present in clones pJF8 was identical to the one found in pJF6. Furthermore, the fragments adjoining the 2.7 kb fragment in pJFlO
and pJFl l appear to be the result of a multiple ligation, since the sequences obtained represent no significant homology to xylanase A of S. lividans or each other.
,o No translation termination codon was found in the xlnTl sequence of pJF6's insert. The implication is that the cloned gem is truncated in its 3' part.
'This is further suggested by comparing the coding sequence of the xylanase A of S.
livadans with that encoded by pJF6. About 185 nucleotides appear to be truncated or missing for the sequence encoded by pJFd.
~5 pJF6's coding sequence has the potential for coding a 4.4 lcDa xylanase.
However, the MW of the xylanase produced is on the order of 34 kDa. There are three possibilities to explain this. The first hypothesis is that the RNA
polynnerase is stopped during transcription. The second is that a terminator sequence is present and thereby stops the translational mechanism. The third possibility is that the protein is :?0 naturally cleaved proteolytically after synthesis.
According to Akino et al., Appl. Environ Mtcrobiol. 55: 3178-3183 ((1989), it is possible for the transcription to be stopped by several inverse repeated sequences.
These authors have described a gene coding for two ~i-mannanases having MWs of 54 lcDa and 37 lcDa. The production of the 37 kDa mannanase would be due to the '~5 stoppage of RNA polymerase as the result of the combined presence of repeated and inverse sequences and a rare codon. I~ez~e, such repeated and inverse sequences appear between nucleotides number X538 and 1672.
These sequences also have the potential to form several secondary structures, which could be very stable in terms of energy, for example, between nucleotides 1538 .'30 and 1672 (Figure 12). A hairpin loop between nucleotides 1538 and 1610 has a calculated internal energy (~G) of -55.2 Kcal (Tinoco et al., Nature. 24b: 40-SENT BY:S K G & F . 7-?9-94 ; s3:48 ; SKGB~F-~ G' S & ~i;#4fi t1973). 'This might produce a prvtyin of about 34 lcDa, but no rare eodon ktas been found near the latter. , 'The second i:ypathesis requires the presence of a sequence region in the ~A. allowing the creation of a second stable structure. Ire the same area prGniously shown, there is the potential of formit~ such a secondary structure. This secondary structure might possibly have the ability to slow down tine progression c~f ribosomes oa the mRNA so as to finally stop the entire translation mechanism, in order trr ultimately yield a protein on the order of 34 kDa. The third possibility is discussed below.
~xarreFle 8 Com~son of the sequence derived from PJFd xydunas~e amino acids with atfier pr~otxins I3NA sequences were analyzed with programs of the UWGCG system:
PASTA, T'FASTA, IiF.SFIT, FILEUh, PRETTY, STEMLOgP, REP$AT, MAP and 15 PROTEINSTItUCTURE upon sequences obtained from the "Genliank" and "F~1~IHI,'~
databases (Devereux et al., Nucleic Acids Rep. 12: 387-395 1984).
The TFASTA program was used to study the degree of homology encountered ~ ~ tea acid derived sequence of xdnll as sncoded by clone p3'F~, as compared to the sequeaices derived from proteins present in databanks.
20 The PILEUP program then made it possible to align the protein sequ~ces derived (Figures 10-lOC). A signi,ieant homology was obszrved with tHc fottowir~
genes: the xylanase genes of B~tyrivibrio fibrisoivens (Lin et ai. , Genbank.
Accession no.: X61485 {1991), Rurninococcus flauefactens (2hang et ol., Mal.
.ll~ierobiol. 6: 1U13-1023 1992), ?nernsoaruxerabacter sal.~charotyticum (b.ee et al., ~5 Gcnbanlc. Aceessioa No.: M9'1882, the C-125 alkalophile preparation ofBaclILus sp.
{Hamamoto et ai., Ag:~zc, Blot. Chem. Sl: 953-955 (1987), Cla.stri~tum ther»aocelium (Grr~piz~t et u:. , .i. Bacterloi. I70; 4582-45$8 (1988) as well as two xylanases of Pseudomorsasfluvrescens (I~al? et~al., Moi. Microbial. 3: 1211-1219 (I989);
Kelleue er ai., Bioche~. J. 272: 369-376 (1990), ~t~rmore, hmnologies have been found SENT BY~S K G & F ; 7-go_g4 ; i3~49 ~ SKG&F-~ G~ S & H;#49 2191 ~2 m protejn sequences derived. fmm proteins coding for exoglucanase genes of Cellutornonas ~tarr~i (~'Neill et al., Gene. 44: 325-330 (1986) , for Clostridium ' .rcerGOirarium celloxylanase (Pukumura et al., I992), and lastly, with a cellulase and a xylanase of Caldocellum saccharol)~ticum (Saul et al. , ~spl. ~nviron.
Microbial. 56:
x117-3124 (1940); ~~ et al., Appt. Errviron. Microbial. Sb: 1017-1024 (1990), A hoxr~alo~y of over 80~ has been observed in the xylanase A of Streptomyces lividans (Shareck er al., Gene 107: 75-82 (1991) (Figure 11).
The alignment of sequences of proteins derived from the 13 genes mentioned above reveals a total of 66 amino acids which were maintained with a similarity of la over 759~, 22 of which a~~c identical at 1009b, and therefore the possible presence of 7 regions of retained amino acids (Figures 10-lOC).
Example 9 ~~P~er ~~ction of Protease sites The MAF Program was used to evaluate the potential cleavage sites of proteases in an amino acid sequence. Figure I3 and 13A shows the analysis obtained far the sequences derived from, the amino acids of S, lividans and pJF6 xylanase.
'Y'he significant differenfie that exists between the two analyzed amino acid derived sequences is as follows: in the vicinity of amino acid 318 encoded by the p,TF6 sequence, no cleavage site by Staphylococcr~r aurer~s protease was found. In ZQ contrast, such a site is present in the analysis of the xylanase A sequence of S.
livlrians.
In order to approach the third hypothesis discussed above con~ecrning a possible proteolytic mechanism for the post-translational shortening of the protein, it's necessary to illustrate this last point by comparing the xyianolytic proteins groduccd z5 by the xylanase A v f Srreptornyces lividans to that of pJF6. It must be noted that the xylanase A gene codes for a 4'7 kDa protein, and moreover, a second protein on the order of 31kI3a is visible on a polyacrylamide gel (Moreau, A., Doctoral thesis, Department of Microbiology and, immunology, Faculty of Medicine, Upiversity of Montt'ssal, 1992, 140 pies). A post-translational maturation process might explain the production of this second 31 kDa molecular form. Comparisons will be brought to bear on This last type of xylanase.
First off, the results show that the sequences derived from the amino acid sequence encoded by plF6 ~;ylanase and the xylanase A of S. lividans az~e quite homologous. It's normal to expect a practically identical computer analysis regarding the poasil3le protease cleavage sites known .from the two sequences derived from amino acids. And yet, a significant difference is revealed in the one proteolytic site.
In comparing the analysis of the likely proteolytic cleavage sites of the xylanase A of S. lividanJ, the plP6 xylanasc would have one cleavage site less for a Staphylococcus protease. This protease would recognize glmamic acid (E), wish 318 amino;acids for xylanase A and 3d5 amino acids for pJF6 xylanase, to ultimately cleave in the C-terminal portion of the amino acid. This difference in proteolytic cleavage pattern might then explain the production of 31 kDa of xylanolytic protein in S.
livtdarss and that of 34 kDa in pJF6. h is known that the pordons of a protein exposed to IS protcolytic cleavage are prvieases that tend to cleave the protein in a loop for example situated between two alpha helices, or one alpha helix and a beta shot, or yet between two beta sheets.
Thanks to the PROTFZNSTRUCTURE program, it was possible to prove the potentially cleavable areas using the professes on the xyianase A of S.
lividans and 2o the xylanase of pJF6. These resultsinterestinglyeolncide with the protease cleavage site of the Staptrylococcus discussed earlier. Tliercfore the involvement of protease may explain the maturation mechanism of the xylanase of pTF6 as well as the xylanase A of S. llvidcuu.
FiEurea 10-lOC demonstrate that there is a eigni~cant homology between the 25 xylanases and eellulases. Gilkes et al., Micmbiol. Rev. SS: 303-315 (1991), after analyzing amino acid soquences for more than 70 cellulase and zylanases, proposed the creation of nitre fazniliea of enzymes. According to these researchers, the observation of cellulase .isoaazynaes and the xylanasea of several nucroorganisms would prove that these proteins would not have evolved from a single gene, but zathsr 30 came from a large muitigenic family. Furthermore, the enzymes with a predominant xylan~olytic activity are classified into two distinct families. This bFings up the S~NT E7Y:S K G & F ~ 7-2q-94 : 13:50 ~ 5KG&F-~ G. S & ~-i~i~51 21291'2 following hypothesis: true cellulases anti true xylanases v~ould therefore have evolved from different genes.
Given that the xylanase A of Streptomyces lividans is so similar to the xylanase of p3F6, the thermostability and acid stability of the xylanase of p3F6 is surprising. According tv lvloresu, A., Doctoral thesis, Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, 1992, 140 pages, the xylanase A of S. lividans retains only 1p9~ of its activity following incubation at a temperature of 60oC for 8 hours in the absence of its substrate, while the pJF6 xylanase under the same conditions keeps alinost 95 ~b of its activity. TtAe snnall differences found ixt amino acid sequences of these two xylanases seems to have imparted a much more stable consistency for the pJF6 xylanase at high temperature.
Example 10 Biobltachzng Using F~C'7 Approximately one liter of spent culture medium per ton of pulp Es added to pine lQaft pt=lp; the culture medium is talcan from Actinomadura sp. FC7 cultivatians and contains XYL I and XYL II aedvities as described in Table 3. The pulp is incubated at a relatively high temperature such as 70°C and acidic pH
such as pH 4 for a period of time sufficient to aFlow degradation of the XYL I and XYL II
susceptible bonds in the xylan that is present. If necessary, the culturre, medium is filtered before use or concentrated using techniques Imown in the art. After incubation at the desired temperature and pH, the product is a pine kraft pulp preparation wherein the kappa number (the amottnl; of lignin) in T.he pine kraPt pulp is lower without affecting the mechanical propezties of the pulp. Additionally, the preparation requires less chlorine comsumption in any subsequent chemical bleaching.

SENT BY:S K G 8 F . 7-2q-9~ ; 13:51 5KG&F-~ G~ S & H;ti52 ~;_ ~,~~pt~ rr Biobte~c~ti~ag Using Recam~inar~fly Produced XYd. I ar~~'or X~'L It' a Approxiirately one liter of spent culture medium per ton 6f pulp is added to pine kraft pulp; the culture medium is taken from cultivations of reombizant host cells that erpress recombinant XYL i a~/or recombinant XYJ... II activities as described in Table 3. The pulp is incubated as described in Example 1.0, at a relatively high temperature such as '?0°C and acidic pH such as pH 4 for a period of time sufficient to allow degradation of the XYL I and XYL II sasceptable bonds in the xylan that is present. If necessary, the Culture medium is faltered before use or coneentrateti using techniques itnown in the art. .After incubation at the desired temperature and pH, the product is a pine kraft pulp preparation wherein the kappa number (the amount of lignin) in the pine kraft pulp is lower without affecting the mechanical properties of the pulp. Additionally, the preparation requires less chlorine comsumptian in any subsequent chemical bleaching.

Claims (37)

1. Purified Actinomadura sp. FC7 XYL I wherein the DNA encoding said xylanase has a restriction endonuclease map as shown in Figure l and said xylanase has biochemical properties as provided in Table 3.

2. Purified Actinomadura sp. FC7 XYL II having the amino acid sequence as shown in Figure 10 for XYL pJF6.

3. Culture medium comprising XYLII secreted from a recombinant host that has been transformed with a vector that comprises a DNA sequence encoding said XYLII, said XYLII having the amino acid sequence of Actinomadura sp. FC7 XYLII
(Figure 10), or an enzymatically active fragment thereof.

4. A method for treating plant biomass, which comprises contacting said biomass with Actinomadura sp. FC7 XYL I, Actinomadura sp. FC7 XYL II, or both said XYL
I
and said XYL II.

5. The method of claim 4, wherein said method is biobleaching.

6. The method of claim 4, wherein the temperature is above 50° C.

7. The method of claim 6, wherein the temperature is 50°-80° C.

8. The method of claim 7, wherein the temperature is 70° C.

9. The method of claim 4, wherein the pH is below 6Ø

10. The method of claim 9, wherein the pH is between 4.0 and 6Ø

11. The method of claims 10, wherein said pH is 4Ø

12. The method of claim 4, wherein the temperature is above 50° C and the pH is below 6Ø

13. The method of claim 12, wherein said method is biobleaching.

14. The method of claim 12, wherein the said temperature is 50°-80° C.

15. The method of claim 14, wherein the said temperature is 70° C.

16. The method of claim 12, wherein the pH is between 4.0 and 6Ø

17. The method of claim 16, wherein said pH is 4Ø

18. The method of any one of claims 4-17, wherein said biomass is contacted with said XYL I.

19. The method of any one of claims 4-17, wherein said biomass is contacted with said XYL II.

20. The method of any one of claims 4-17, wherein said biomass is contacted with both said XYL I and said XYL II.

-49a-~

21. A method for hydrolyzing xylan, said method comprising contacting said xylan with Actinomadura sp. FC7 XYL I, Actinomadura sp. FC7 XYL II or both said XYL I and said XYL II.

22. A method for treating xylan-containing plant biomass byproducts to hydrolyze the xylan therein, said method comprising contacting said xylan in said xylan-containing plant biomass byproduct with Actinomadura sp. FC7 XYL I, Actinomadura sp. FC7 XYL II or both said XYL I and said XYL II.

23. The method of any one of claims 21 or 22, wherein the temperature is above 50° C.

24. The method of claim 2 3, wherein said temperature is 50°-80°
C.

25. The method of claim 24, wherein said temperature is 70° C.

26. The method of any one of claims 21 or 22, wherein the pH is below 6Ø

27. The method of claim 26, wherein said pH is between 4.0 and 6Ø

28. The method of claim 27, wherein said pH is 4Ø

29. The method of any one of claims 21 or 22, wherein the temperature is above 50° C and the pH is below 6Ø

30. The method of claim 29, wherein said temperature is 50°-80°
C.

-49b-

31. The method of claim 30, wherein said temperature is 70° C.

32. The method of claim 29, wherein said pH is between 4.0 and 6Ø

33. The method of claim 32, wherein said pH is 4Ø

34. The method of any one of claims 21 or 22, wherein said xylan is contacted with said XYL I.

35. The method of any one of claims 21 or 22, wherein said xylan is contacted with said XYL II.

36. The method of any one of claims 21 or 22, wherein said xylan is contacted with both said said XYL I and said XYL II.

37. The method of any one of claims 21 or 22, wherein said xylan is in a hemicellulose liquor.