CA2129171A1

CA2129171A1 - Actinomadura xylanase sequences and method of use

Info

Publication number: CA2129171A1
Application number: CA 2129171
Authority: CA
Inventors: Marja Paloheimo; Pirkko Suominen; Richard Fagerstrom; Arja Mantyla; Jari Vehmaanpera; Tarja Lahtinen; Paula Kristo; Raija Lantto
Original assignee: Individual
Current assignee: AB Enzymes Oy
Priority date: 1994-07-29
Filing date: 1994-07-29
Publication date: 1996-01-30

Abstract

The isolation and cloning of Actinomadura flexuosa xylanases having a molecular weight of 35 kDa and 50 kDa are described. These xylanases are thermostable and useful in biobleaching of wood pulp.

Description

~32'''1254Z ~ G ~ f F-4_3 T-~3 P-13e6,~54 JUL ~ ' g4 14:1~8 A~ om~duna Xyl~nase Sequences and Met~od of ~se Background of the Inv~r~ion The aim of krai:t pulp bleaching is to remo-e dle resi~ual li~ that is left in pulp af~.~r kraft cooking. Tr~1itio~Ally~ this h~s becn dcx using S ~hloriD~o~ n;~ chPn ir~ls Because of en~ir nro~nt~l con~:erns and corL~u~ner ~ n~1s, ~terDa~ive ble~chin~ tech~ologies have been desired.
The first ~iotechni~ oach to this problem was to a~tack ~he 11 directly with lig~in degrading e~rl~-es. However, the ,~.l~.,.i~l, ~ of ~y~ ie 1~ degradation ~ems to be very complicated and difficult to control.
Li~nin c~ be degrad~d, if the whole rnie,oolgal~ hat produces li~nin~ce~c ~ used. However, trea~nent times are relatively long. For example, ~re~ne~ ~mes m~y ~ke days, ar~ the miel~yanis,,,s need supple-rn~nt~l ie~ to work. lt ~an al~ be difficult to con~ol the gro~th of other, un~lesired, microbes. The use of lignill degradatio~ b~ isolated 1i~,J~ o~
by micr~rganism~ is c~ subj~c~ of much ~se,~h. (soc, for e~ample, Fa~Tell, R.L. e~al.. Llgn~ r~losics ~-315 (1~); Jura~ek, L., Ligru~cel~loQcs 317-32~
In addi~on to cellulose and lignin~ ~o~ pulp co~ hemiceIlulo~e.
Another approach is to at~ck hPmi~eilulose - ~ t~ main co~ponent of wood. The h~njeellulose in n~eive hard~vo~d is mainly xylan, while in softwc~ the h~rni~ lose is mainly gluc~msnn~s and some xylan. Durin,~
l~aft coo~, pa~ of the ~yla~ is dissolved int~ ~e coo~ing liquor. Towa~s the er~l of the co~LiIlg period wher~ ~e alkali conl~nt~tioIl dec-eases, pan of the dissol~od ~nd modified xyl~ ~ back omo the cellulose fiber.
~5 Irl 1~8~, it w~ noticed th~t ~ ~O,nt of unbleac_e~ ~afL
pu~p results in a lesselled need for ch~nir~k in the b'~ i~ process (Viikari, 023712540 S ~ G ~ f F-433 T-608 P-0g2i016 JUU 29 '54 15:1~
21~9171 L. et af., Proceedi~ OI the 3r~ Int. Conf. on Pi~te~hn~logy in ~he Pulp Paper Ind. ~ Sto~hnlm (1986), ~p. ~i7-6g). Xylanase plet~ uel~t Of haft pu1p p~ally hydrolyses ~e xylan in kr~fl pulp. This r~es ~e pulp st,~~ e more por~us an~ bles more e~ficient removal of li~ f.~ in th~
S s~lhs~ n~ bk~c1lin~ extr~on st~ges. Lat~, in several labor~lo~ the xylanase p~t~ was rep~rted to be useful i~ co~ju~ lior~ ~ith bl~Ch s~qu~ c4nC;~ of ~, CIO2, ~ 2- z and O3. See reYiewS in V~k~, L. etal., FEAlSMicrobiol. ~ev. 13: (lg~in press); V;~ri, L. eto~., in:
Saddler, J.N., ed., Bioconversion ~ores~ rnl P~ ~csidues, o C-A-B I -rD~tl~n~l (1gg3~, pp. 131-182, Gran~, ~., P~1p and ~r ~.(Sept. 1~3), pp. 5~S7; Senior & ~llon, ~r. Pu~p d~ P~per ~ 114 (Sept.
); Bajpai & Bajpai, Process Brochcm. 27:319-325 (1~), Onysko~ A., Bio~ech. ~dv. 11:179 198 (15193~; and Viikari, L. e~ al., ~. P~et an~ 'rim~er 73:384-38~ ~19gl).
lS As a direct resull of t~2e be~ bleachabili~y of the pulp after such a xylanase ~ t~ , there is ~ ~*lrt;011 of tbc su~seque~t c<~ ;on of b'~l~h;~ cl~ ,.ieqls, which when c*lnrkl~ eo~ .;"~ ~h~rPls ar~ used, leads to a redu~ed fo~matioll of ~ rlro!~ y l~ndesired o.g~ochlorin~
co~ds. Also as a di~ result of the better bl~h~hil~tv ~f p~llp aftsl a xylanasc ~ h is possible to p~oduce a product with a ~ hri~htn~
w~re suc~ b.;~ s~ would olL~vise ~e ~ to ~cllieve ~such as T~F
ble~ in~ us~ peroxide). 13e~e of t~e suL~Lrate s~lrlL;ily of d~e xyla~ase enz~me, cellulose fibers a~e not ~nne~ and the stre-n~ properties of ~e p~duct are well wit~ a~f ~.p1~'.1r lim~ts However, i~ is not as simple as merel~ a~ding ~ ~Lyl~ase tr~l,lLcrlt ~tep. M~st co~mercial xylallases A~si~t~ for pUlp bl~acl~ are not very ~e3rmotc~'~r~nt, especially when n~utral or alkaline pH ~ ons are used.
In praetice, xyla~ are generally in~ffi~i.ent or inactive at t~ UI~S
higher than ~
The cloning~ of ~la~ases ~s been rerpoll~d from Actinoma~l~ra sp.
FC7 (Ethie~, J.-P. e~ cll., in: Indus~n~l Micrwrgar~sms. Basic ~ p~ced 0~371~54~3 5 K G & f F-433 T-~0a P-003/016 JUL 29 ' 94 15 1~

,BS~ITUTE S~E~T

Molecufar G~neti~ c, R. Baltz et al., edst ~oc. 5th ASM Conf. Ge~
Biol. ~ndust. Microorg., OCt 11-155 lg~2, Blo-~min~n~ TT~i~nq, postcr C25), bacte~ (e.g. Gh~ , G.S. et al., ~. Bacte~iol. ~71:2963-2969 (198~; Lin, L.-L., Thomson, J.A., A~ol. Gen. Genet. 228:5~-61 (1!~91); Sh~ecl~, F.
S etal., Gene 107:'7~-8~ 1); SL~ , T. e~al., Appl ~icrobiol Bzo~echnol. 33:534-~41 (1990); W~it~h~ , T.:R., LA~e, D.A., ~urr.
Microbiol. 23:15-19 (1991)~; and fungi (Boucher, F. e~ al., NucfeicAcids~es.
lo g874 (19g8); Ito, ~. et al., Biosci. Biot~c. Bioche7n. 56:90~-91~
~t, J. e~ al., in Visser, J. et Ql., eds., ~ykms and ~yl~nases ~EIsevier Science, ~t~ ), pp. 349-3~0 ~19g2); van dcn Br~cck, H. et aZ" I~P
~63,7~6 A1~1~2), WO 93/25671 an(l ~O g~l25~3).
It is known that T7zennomonospora ~sca p~ c and aLIcaline stabl~ xylanases (EP473,545, Sandoz). ~e use of betni~
hyd~vl~ Rs in ~l;rf ~-A b'---hing c~~ LS ~l.g...~A ill WO
~5 89108738, ~P 383,9g9, WO gl/02791, E~ 3gS,7!~ P 386,8~38, E~
473,545,EP48~,104ar~WO91/0590~ neuseofh~Tnir~lhllolytic~ ",e~
for iu~p~ ter ~oval from ",,~l~,bA1~3,~l pulp is di~l~s~l in EP 2~,040, EP 334,73~ and EP 351,~S a~ E 4,000,~5g. ~ en the hydro1ysis of biom~ss to liquid fuels o~ c~ c is consid~:red, t~e C~ of bo~
cellul~ d hP.mire~ 0se is ~c~nriql to obtam a high yield (Viik~ri et al., rnjre~ 5for IJ..~ pli~ati~ns, r In: Bioc~ .on~fForesta7ul A~ricultural ~a~tes, Saddler, J., ed., C~ TntA~ oq~l USA (1993. Al~o, in the feed i~dus~y, there is a need to use a sui~ble ~omb~tion of eDz~n~
a~ivities to de~rade ~e high ,B-~lucan and h~cellulose umt~inin~ s~bs~Al~.

2~ A ~ylaluse tl~at i~ active at an alk~line pH would dccr~ase ~he ~eed ~
acidify ~e pulp plior to xylanase l~c~lll, .ll In ~AAitjonl the t~,Dlp~a~S of many mode~ aft cooking a~d bJ.e ~ c'- ~ procc~s are relatively high, well above ~e 50ac that is suitable for maIly of ~e c~.,ner~ l ble~h e~ s.
Accord~ngl~, a need exists for t~ hle xy~n~e pr~p~r7~tinn~ that are stable at alkal~e p~I's for ~se i~ wood pulp bl~rhin~ processes.

023712540 ~ K G ~ f F-433 , -608 P-004/016 JUL 29 ' 94 15: 1~

SU3STl~E S~
Figures Pigure 1 s~ws ~ effea of pH ~n A. ~osa lpS~43 186) ~ylar~se Yity Fig~e 2 shows ~e effect of t~J~ JI~ onA. ~e~osa (l~S~43186 ~rla~e a~tivity (cul~re ~lr-n~Q~rl). The four bars at each time poin~
L pH 7, pH 8, p~ g aI~d p~ 9.5, ~ f~n lefe eo r~gh~.
Fig~re 3 shows ~ AE S~pharose C~6B chxomat ,g~p~ elution le of A. J~exuosa (DSM43186) xylanases.
Figure 4 sbows ~ se CL 4B Chl~ to8,r~ y elution profile of D~A~ pool I of Figure 3. The tubes that we~ c4u~in~i to prov~de sa~le DEPS Ill ~re in~ ate~
Figure 4A show~ the P~enyl Sep~rose CLAB ch~otna~ p~ elu~ion profile of 1:)1~ pool Il of Figure 3 . I~ es tb~t were c~n~in~ to provid~e sample Dl~PS Il/l and DEPS W2 a~G shown.
Fi~ure 4B shows the Phe~rl 5e~1~o~e CIAB cb~oma~ogla~ e1ution profile of DEAE pool IlI of Fl~ure 3. T~e tubes ~at we~ h:-~d to provide sample l:)EPS ~IIIl and ~EPS III12 ~re sbown.
Figure 5 s~ows the Goolnassie Bluç prote~n stain~ pauern of the varic~C, ~ aphic p~ols~ Two leftmost lanes~ r weight ~0 markers; la~ --P ~ , lane 2: DEPS ~Pool I/l); lanes 3 and 4: DEPS
(Pool IIIl ~nd II/~, respectively~; lane 5: empty; la~es G and ;': DEPS (Pool mtl and ~IIt2, respectively); lane 8: a:~. D~PS: F~actions after ~e DEAE
chroInato~ ~ of Figu~e 3 a~d the Phenyl S~:p1~ro~e Cl~u~llatOgl~ of Figure 4.
Figure 5~ shows ~Le Wes~n blot analysis of t~e va~ious ch~omatographic pooLs st~i~d n Fi~ure 5. IFftrnost la~e: m~ r weigllt mqrlr~ : m~h~Jn lane ~ ~EPS (Pool I/l); lanes 3 and 4: ~E;PS
(Pool lIJl and IIf2, s~ ely); la~e 5 emp~ es 6 a~ 7; l~EPS ~Pool m/l arld 1~12~ le~Lively); lane 8: empty. l:)E;PS: Fracdons after the O~AE

0237125~10 5 1'~ G ~ f F-'123 T-603 P-E~ '054 JUL 29 ! 9~ 14:10 c~rom~tography of FiD~ 3 ~d the Phenyl Sepharose c~ sraphy of Figure 4 Fi~e ~ ws t~e Phenyl Scph~ose F~ el~o~ phy el~tion profi1e of l~AE flow ~ough p~nP~te. The tu1xs that were wmbined to provide sam~le PF1~ PF2 are intlir~ted.
Figurc 6~ .~hows the Phenyl Sepharose P~ chro~tograph~ eluuon profile of D~E flow, through con~..~ . The ~bes that were combined to provide ~mplc ~F1, KF~ and KF3 are in~ie~
Figure 7 shows the Cooma~sie Blue protein staini~g pat~ern of ~e ~arious chls,.La[ographic pools. Abbrev~auons are as in Figures 6 and 6A.
TPf~-ost and rie~ ost I~Des: molecular weight marke~s; lane 1: m~drum;
la~e 2- PF1; l~e 3: PF~; lar~e 4 Kl;1; lane 5: KPZ; 1~: 6: KF3.
Figure 7~ shows the Wes~rn blot ar~lysis of ~e various chr~ graphic po~Jls s~ed ~or protrin in Figure 7. Abbr~viations are as 1~ in ~ res b a:thd 6A. Lef~most and rightmnst lanes: mol~ular wei~t rnarker~;
Iane 1: m~lnlm; ~ 2: PFl; lane 3: PF2, lane ~; KF1; ]ase 5: K~2; lane fi:
KF3.
~igure 8 shows Ihe ef~ect of BSA on 1he ~ennostabil~ of the 35 kDa xylanase. Closç~ s~uar~s: no BSA; opcn squares: wi~h BSA.
2~ Figure 9 shows ~e effoct of BSA on ~e ~ uo~a~itity of t~e 50 kDa xylanase. Closed squ~res no BSA; open squa~es: with BSA.
Pigure lO shows u~2e effect of pH o~ the ac~Yity of tbe 35 kDa xylanase at 80GC.
Figure 11 ~hows ~e effect of p}I on tlle ~ctivity of t~ ~ Id:)~
2S ~laDasc at 60~C (close~ squares), 70~C (ope~ res) and 80~ (c~osed c~les).
P~e 12 is map of p~s~id pA1~185 (4470 ~p).
Figure 13 shows the DNA and arnino aeid se(Iuence of 410 bps of Actinoma~ura sp. DS~43186 xylanase.

~2371254E~ ' G ~ f F-4_3 T-62'. P-~311/054 JUL 29 '9~ 14: ~0 b-Depos~s Pl~ d pALK~2~ (eo~l~i~ t~e ~ene for rhe 35 k~a Actinoma~ra x~as dcpcsited at the DeUlSC~2e S~nmllln~ von Miklov~ n und 7~11h~ln!~en ~mbH, Mascher~der Weg 1 b, D-3300 n~ rk~v~ig, Germany S a~l ~sign~d acce~ion number DSM932~.

Detai~e~ Desenp~vn of the J~n ~, F.~n~d ' ~n~s 1. Dc~ ons In ~e descrip~o,l ~at follo~rs~ a ~umber of telms u~cd i~ recombi~ t DNA t~rhnok-gy a~e e~ sively u~lized. In o~der to provide a clearer and c~n5isten~ uI~dersta~in~ of the specifica~~ a~i claims, inl~lu~ the scope to ~e ~ven ~uch terms, the fo~iowing d~ini~io~c a}e provided.
Xyl~nase. A~ used ~e~ , a xylanase is a h~nlirP~IllT~s~ that cuts the a~ bonds ~i~in thc x~losic chain of xylan, (x~lan is a polymer of D-xylose residu~s th~it are joi~ed ~rou~h ,B-1,4 linlcages. Xyl~se activit~ is synonyn-~ous ~ith xyl~olytic aclivi~.
B~ a host Tb~t is "~hsron~7ny inc~ '` of a,~r.~he .i7;~r o~le or more en~v~es is rr,~ a h~st in ~rhich ~e activi~r of one or more of ~e lis[ed en~ymes is de~lei,scd~ d~rl~ient o~ absent when compa~d to the ~ild-t~rpe.
E~m~ p~G~On. By "e~zym~ ~-G~al-ation" i~ meant a cQmposition Co~ g enzymes t~t h~ve boen ex~ ted from (ei~er parlially or corn~let.el~ purified from) a microbc or ~2e m~nl~n use~ to grow s~ch lDicro~2 "E~Qct~d~rom" meaDs a;:ly method by which the desired enzymes arc scp~r~tr~ f~om ~e oellular mas6 and ir~ s breald~g ~elLs an~ also simply removi~ e cultu~e medium fr~m spe~ cells. Therefo~ the telm ~5 "en~ym4 pJe~ul-on" includec eo~-~si~ions CnmI~I'iCt~ medium prcviously culture a desir~ micro~s) and any e~y~ ich the micr~be(s) has ~ecrete~. illtO 5uch medium du~ing the culture.

254~i - K G ~ f F-43 T-6~3 ~ 12 ~ ~5~ ~UL .- 9 ' 94 14: 11 21~91~1 ~io blA~ ing. By "bi~bl~rh~n~" is l:~leaM rhe e~ ctioll of lignin ~om ceilulose pulp after the actiotl of h~ oelll-lose d~ in~ .y~ wit.h or w~hout li~ d~r~ e~ymes. ~emoval of ~ ligr~in may be ~s~tio~d by b~nir~ o~es either physically ~rough le~ipitd~io~ o~o the ~cr ~ ce dur~g cookin~? or cl-~omic~lly (th~ou~h ligniIl-car~ohydra~e cor~lexes). The ~emicelluLase activi~ p~1y degrades ~e h~micellul~se, which eTlh~nrps t;be c~h~ili~ of ligiins by c~..Y~icr~ b1eaching chP~i~ls (li~ clorine, chlo.ine dio~ide, pcro~ e, etc.~ (Viikari et al~, ~B~PaCh;~ wi~ E~ymes" in BiotecJu~ology in tfie Pulp and P~per Ind~ry, Proc. 3rd Int. Conf., St~-~lm, pp 67~ ~198~ Jiilcari et a~., "Applica~io~s of Fn,~ es in Bkachi~' in ~c. 4t~1 Int. Sym~. Woo~ and rzg C7zem~ , Pa~s, Vol 1, pp 151-1~4 (}~87~; Kan~elinen ct a~., "Hernicellu~ases and ~eir Potential Role ~ ~ h~n~r i~ rnahon~
Bleach~ng CGnference, T~pFz Proceed~ngs, pp. 1-9 (1~88)). The advantage of lhiS improved bl~rh~hi~ity is a lower co~p~on of ~l~o~llin~ ~hf~m~ c and lower e;lvironmental loads or highe~ fin~ h~ Va1UeS.
~ an en~yme ~homalogous" ~o a host of tbe inve~ion ~s m~t ~ha.
an ~ans~omled s~rain of the sarne specie~ as the host species na~rally pro~uces some amo~nt of tke native protein; by a gene "~omologous~ to a host of dle invention is meant a ge~e foun~ in the gencme of an ed stra~ of the same s~ies as ~ host ~pecies. By an enzyme "keterologous" to a host of ~he in~ntion is mea~ ~at an un~r~nsfc~ned strain of tbe s~me species as the host species does nof nanm~ produce some amount of ~e na~ e protein; by a ge~e 'theterologo~s" to a host of the inven~ion is meant a ge~ t found in ~e genome of an u~transft~m ~l strain of rb~ s2me sp~cies ~s ~ie host specie~.
CZon~g ~ek~lc. A p'asm~d or phage DNA or o~h~r DNA ~y~
(such ~ a linear I)N~) which provides all a~r~priak nucleic ~cid envir~"c,~L for ~e transfer of a ge~e of iIlt~rest inLo a host cell. The clonin~3Q vehicles of the ~vcn~on n~y bc ~ n~ to replicate auto~omously i~
pro~ryc~tic an~ euksryo~c ho6ts. In fu~gal hosts such ~s Tnchodermo, the 71254~i C k: G ~ f F-42_` T-6~ 813~L154 ~TUL 29 '94 14:11 clo~ veh~les generally do not aut~nomo~lsly Teplic~te ar~ instead, merely pro~ide a vehicle for ~e h~ of the ~ge~e of }n~e~st inrQ ~he Ihchodenna host f~ subsequen~ inseltion into the Tr~choderrn4 genome. l~e clon~llg vehicle Ir~ay be fi;~r~er cb~ Jized by ~ne or a ~nall number o~
cn~l~m~ ecoglliuoa 6it~s at ~-hich su~h r)~A sequences ~uy be cut in a detem~inable fashion wit:hout loss of an es~nti~ biologica! function of the vehiclc~ and i~to ~bich DNA may be spliced ~ order to brir1g about re~lira~on aDd clo~g of such l~NA. Thc cloning vehicle may filIther conum a ma~er sui~ for use Ul the ide~tifir3~ of cells t~ ~ with the clor~ing vehicle. M~kers, for example, ar~ lic ~,~ .. re.
Alter~i-el~, ~uch rnarkers rnay bc provided on a clo~ vehicl~ w~h is ~eparate fro~ ~at supply~ng the ge~e of interest. The word "vector" i~.
soJ~ -.rs used for "clon~ng vehi.le~' E:~press~on veh~cle. ~ vchicle or vector si~ilar to a cloi~g veh;cl~
bu~ which is capa~le of c~ es~ing a ~ene of in~rest ~er transfo~tion into a dcsise~l host.
When a ~ngal host ~ e ~ene of ~r~st is preferably provi~ed to a fungal host as pan of a ~lo~ or expressi~n veh~clc that int~grates ~nto the nmgal chrorr~n~ome Seq~u ~ces wh~ch derive from ~e clo~ ~hicle or ~0 expressio~ vehicle may also be integr~ted with the gene of interest du~ing thc int~ tihn process. For example, in T. reesei, the ~en~ of i~erest can be direct~d to thc cbhl locus.
The gene of ~ntere~ may pre~era~ly be plac~d under the control of (i .e., operably linked to) ce~tain c4r~1 sc~uenccs such as p~moter sequ~nres pro~ded ~y the ~ector (w~ich i~g~a~c with ~he gene of interest). lf desir~, s~h con$r~ sequenoes may be l l~Jvidcd b~ the host's chr~mosome a~ a ~ lt of ~e l~cus of iDs~rtion.
Exp~es~io~ control s~ue~s on an e~cpression vec~r will var3r depending on wh~t~cr ~e Yector is ~ci~n~ to express a certain gene in a prokaryo~ or e~lkaryotic host (f.or exalnp~e, ~ shuttle ~ctor may provide a gene for select~on i~ ~ar~ri~l ho~ and rnay a~dition~lly cont~in 02~712540 5 K G ~ f F-433 T-~08 P-005/016 JUL 29 '94 15:11 ~U~S~ITUT SH~Er 21~ 9 171 onal elF~ n.~ such as, F "hAnr~el el ,~ , tet~nin~tion se~ nr~s~
a~dlor ~ /;n~l initiation and ~rrnin^tiol~ sites.

I. Ider~f~ on ~ ~cl~n of A~-t- ~t~t~Jlexzlosa xyl~nases Two ~l~j have bocn i~Pn~ çd~ ifi d a~d c~ed from ,4ct'r~. ~ur~ f~uos~. Bo~ of tl~se xyl~ases halle a pH ~ti~ and th~ s~hility t~t ar~ desirable for the bi~l~a~h;~ of wood pulp. O~e of t~ese xylanases has ~ moleallar weigbt of a~out ~5 kDa (AM30) aD~I tbe o~er has a ~ol~11~r weight of about 50 ~a (A~5~3.
The opti~l ~~ alu~c rarlge for ~c~inoma~raJ~uosa ~rla~ c in clude ~ aLions is 7~80~C ~t pH ~7. At pH 8, t~e u~U~I lv~ld~e range of this ~r~e is 60 70~C. T~i~ is ~ l in kr~ft puIp ~ hin~
because af~r laaft coohng, the pH of the pulp is ~ 1;n~.
In ~d p,~ala~ions, AM30 re~ins 80% of its a~ivity, and AM50 retains ~0~ of i~ activity after 2A hou~s wb~n incuba~:d ~n t~e "~x~e of ~SA. ~t 80CC, both AM30 and AM50 are most a~ve at p~I6 but ~oth exbibit a broad a~tivity plateau between pH S - p~I 7, wherein about 80% of the a~tivity is retained.
For t~le isoJation of AM30 an~ AM50, the host Achno~u~ ws~
is available a~ deposi~ a~ ion m~ r)SM4318~ ~om Deutsche .~ ln~ ~on Mi~ool~Ar.i~ u~d 7~11hl1nmen GmbH, Mascl~oder Weg lb, 1~-3300 Bra~schweig, ~elUldll,~. Both folms can be p~rified by passage through a senes of cln.~ ogr~phic co11imn~. A f~st ~ n step Of I)EAE ~ e CIA~ ~e~ins ~ut half of ~e xyla~se activity when the s~le i~ applied ~r pH g.~9 in 1~.5 mM Na~P0~; the o~er half is found in ~e flo~ ~ugh.
Eluti~ of ~e bound ~yla~ aetiYity widl a salt g~lient results irl a~
elution of a sh~rp, ea~lier eluting peak of activity and a broad, later elutin~
peak of activi~ The s~arp, ea~lier el~lti~ pe~ak retains i~s homogenei~ when a2371254~l 5 K G ~ f F-43~ T-608 P-~36/016 JUL 29 ' g4 15 :1 1 2129~71 S! I~ SHEE~
-10~

subjected to phe~yl æpharosc (~L4B c~-~alography. Samples ta~e~ ~rom t~e latcr, broardpeakof aCtiviy s~ to tWO peaks w~en sul~r~ to pher~rl o~e CL4B chlu~ o~p~. There is onl~ we~ ivily of ~ese xylanases with a polyclonal an~ibody d~te~ agains;t T~".~.: h~WSpora S fi~sca xyla~e.
By Sl:)S-PAGE, ~e mole~lar wei~ of tb~ ~ld~e iD these pool~
from the l:)EAE ,e~,l~ was abollt 50 kDa, while 1he molccula~ weigh~ of ~u xylana~es in the DEA~ fl~w ~ ugh wa~ 30, 35, 4~ and 50 l~I:)a. Thuæ~
A~inom~r~flexuosa contains three or four ~cylanase pr~m ~ands.

11. Xyl~nasc ~io blench~qg us ng ~e Acfinornadur~ osa Xy~ses The present inve~on con~l~,c~l~ a m~hod for ~ nir~lly treating plant bi~mass under con~litions of bi~h lh~ t~..c of 5~80aC and pH 5~, a~ espe~i~tly 60~-70C, pH ~7 a~d most prcferably 60aC a~d pH ~.5 for one houl. In a p~efe~d ~mbo~imer~, pla~ biom~ss ~ bio-blcacbed wi~
~y' that ar~ able to hydrolyæ xy~An chains ill a ~n~icellu4~e liquor (a by-prod~t of steam t~tment of ~e lign~ lcfie biomass) at ~ l or moderately alkaline pH and hi~h kmpcra~e (60C).
Pla~t biomass is a e~ o~ilr~ materi~ c~ p~ ily of a ma~
of a~llulosf~-, h~rniç~]~ nse, an~ ~n. Rem~val of ~he lignin Cu~ Gl~L iS
desirable du~ d~ lI"r~~ G~ of pape~ because of its ~ro~n ~olor and ~end~y to ~ce the strength of the p~per prod~ct. Ma~y ~ b~ bave ~een ~vcl(~cd for ~e removal of lig~. Ty~i~ally~ ~e wood pulp is treatæd with chorine or ot~ cic rh~niçals i~ orde~ to ~move ~e li~ COlnpO~ t and provide for a bri~ht~nAd pulp. However, ~e to~ic by-products of ~is ~S .~ nir~ W~ gativel~ impact ~porl ffLe h~ealth a~l stability of ~e ir~ t into whi~h the~ a~e released. ~on~e~ l~ntly l~Lere is a great ~ced for dc~ lterna~ivc, more ~ ..,---.c.~ y protecl;ive ~hni~IL~ tO
achieve pulp bl~chins~.

0c3712540 S K ~ ~ f F-433 T-62~ P-007~016 JU- 29 '94 15- lc ~f~

A common ~ a~ o~ plan~ biomass for p~per production involYes a the~ ni~l S~M ~tmf:nt followed ~oy extr~ion with hot watel.
This pracess di~u~ ~ xyla~ ct~ inir~ h~nu~ll~Joce~ and some lig~
deriva~ves which are othen~ise tigh~ly bou~d to the cellulose. Under ~e ~cthod of ~he presen~ inve~tion, a ~io bl~eb;..,~ terhn:gue is develo~d ~.hr~1~v th~ le xy~ases which are active at the cc~n~litinn~ o~ ~.e bl~h~ ay bc ~sed in vitro to mod~ o~ decrease the lignin in wood pulps. Thes~ ~LIi~)g~ 1'~` '~ con~iti~ns ma~ addi~io~ally act to r~e c~ ce activi~;y in ~he e~zyme p~p~ ' ;oJ~ or c~ h~
In a p~re~led embo~ ,n, the process of 11~ inven~ion is ~cd out in vitro i~ the b~~ y~c liquor. The proee~s invol~res pl~ g ~e enzyme p-~ion~ cul~r~ mf~ n, os c~ ted mi~ taimnE ~;ylanase in~o contact with dle wood pulp. I~c~utine ~lr~ ort~ e~able those ~ ~he art to ~1f t~ ;"~- ~e o~ L time depe~ding upon th~ result desired, ~
u~ an~ spf~ific acti~ity of t~ ~yla~ e~yme used, the ~pe aDd c/')J~r~n1T~tion of pulp Used7 pE an~ ul~ of the a~idic l~r, and other p~ nf~qr varia~lcs.
~e met~d of the p~sen~ i~vention m~y be applied al~nc or as ,"I.p~ nt tO l~ther L~ nt~ that r~uce ~1~ co~ent of w~od pulp, in~:rease its ~in~njlity an~or de~ase its w~t~r reter~tinn. In a prefe~red e~bodime~t, t~e p~esent i~ ntion is u~ o e~ e bri~htn~s pr~ lies the ~vood pulp ~y ~ t~ of cberni~sl pulp~, i.e., ~ose pulps con~inin~
li~ ~ ~s been chf n ~ Llly m~ d t~ough ~h~mi~l t~
In a ~-efelred embo~imPnt, the xyl~rlases used in 1he me~ods of the ~5 inve~L~on are pre~rably ~ose of Actino~ra~e~sa, and ~Sp~~ 35 kl)a ~dlor SO k~a ~y1anaseg of Acti~ a~ct~osa. Pc~ lly, culo~re mKl~um that c~n~inc ~e en~y~l~s seaeted as ~ result of the gro~h of the cells a~e useful i~ the m~rhodc of ~he invention, as are ~he culture ...fx~ .. that can ~e provided by a ~ecc....~ 3nt h~st ~at ha~ been l,~ro~ with ~e ~y~ ase encodin~ ge~es of the inven~.

Q23''125~1 a 1~ G ~ f 212917~ 23 T-~3 P-~17 ~5~ JUL 2- 'g~ 11:1' 1~. Gene~zc Fngiq~ng of ~c ~o~s of ~e Invcntion The process for gen~tirQlly e~;~ ~ the hosts of the inv~iori is facili~ d thro~gh t~ie clonin~ of ~ c sequer~ that en~ode the desired xylanase activity and throu~h the C*~ i of such ge~etic scquer~es. As U~i hcrein the te~i rgenetic ~qu~nrç5~ le~d to refer t~ a nL~cleic acid mnl~ule ~;preferably DN~). Ge~etic sequeD~es that encode ~e desh~ed xylanase 2re derived from a v~ty of so~. Theæ s~ include Aa~onu~ur~ fle~oso ~nnmir DNA, c~NA, synt:hetic DNA alld combinat~ons ~ereof. Vector systems may ~e u~ed to produce ~o~ts for the production of the en7yme l.lcp~:ations of ~he inven~on ~uch ~ tor constructio~ (a) may ~rther pro~ide a separate vector construction (b) which en~od~ at lea~t one ~esired gene to be intagrated to tbe genome o~ the host and (c) a sele~table marker couple~ to (a) nr (b). Alt~.~ti~ely, a teparate vector ma~ be u~ed for the ~.
1~ A r~cleie ~cid r~olccule, such as ~NA, is said to bc ~capablc of express~" a polypeptide if it co~tauns ex~ression control sequences which contain transeriptiana~ regulatory inf~lmdtion an~ such c~nf..~
"operably lln~en" tO the mlcle~dde sequence wh~ch encodes ~ polype~u~.
An operable linka~e is a linkagc in which a sP~ - is co~t~d to 8 rc~latory sequen~c (or s~ cs) in such a way a~ to placc cxprussion of ~e se~n~e u~er the infl~lence or con~rol of the regulat~r~- sequence. l`wo I)NA sequences (such as a pro~Rin ~n~ rling .5e~ P an~l a promot~ ~o~
se~uen~e Iin~ed to t~ S' e~d of ~e encoding se/q~ e) are said t~ be operably li~ked if illduction of promo~r function resulSs in thc ll~us~ ~ion of ~ pro~ein e~coding seq~cr~e ~R~A and if lhc na~rc of the linkAgc betwce~ t}~ two ~NA s~ n~ docs not (1) rcsult i~ thc i,ntrl~duc~ion of fram~-shift mutatio~, (2) i~terfe~e with the ability of the expressio~ regulatoly sequences to direct the e.~pr~siorl of the mRNA, ~ntic~ncç RNA, or protein, or (3) i~r~.c wilh ~e abiliry of tbe tk~rl~ n~ l~l by the promoter region ~e~uerre. Thus. a promoter re~ioD would be ope~a~ly linked r-..~ ~ T-61~3 P-~15 05 ' Jl 29 ' 9~ 1 :13 0~37125~a 5 ~; G ~ f 212 ~ 1 7 1 to a DNA s~ ~e if the promcter were capable of effec~ nS~ n of that DNA seque~e.
1~ pr~ise na~ of the regulato~y regions needed f~r geQe cxpression may ~rary betwe~ r cell types. ~ut s~ll i~ ge~eral in~:lude, as S r~ess~ur~ S' n~Jn-~birlg and S' non-~ra~ ti~ (l~OD codi~g) c~que~ces involved ~.th initiativn of tra~scription an~ tPnCi~*O~l es~l Exp~qsioil of ~he protc~n in the ~ed hos~s reqllires the use of r~gulator~ regit)n~ ~tional in su~h hosts. A wide variety of tr~n~crr~
a~ t~lation21 regulatory ~ue~ bc enlployed. In eu~aryotes, where t~s~uon i~ DOt linkt~d tO translatio~, such control regions may or may not provide an i~it~ator n~thio~ (ATJG) codorl, ~in~ OD whe~cr th~
clol2ed se~u~;e contai~s such a m~hioninP Such regions will, i~ general, include a promote~ ~egion sufficient tO direct t~ ~tio~ o~ RNA syn~sis in t~ hvst ce!l.
lS As i~ wideiy l;~lo~, tra~lation of eu~aryo~ic mRNA is iD~tiated at ~e codon which enco~ the ~ust met~ionine. For ~is r~on, it is preferablc to e~svre that ~.he lin~age bet~veen ~ eukaryotic prol"~te, and ~ DNA ~equence which eneode~ rote~n, or a functio~al denvati~e ~hereof, does not con~ain any interve~ing c~do~s ~hich a~e capablc of eIIcoding a mc~olline. The 2~ prese~ce of such codons results either in a formation of a fusio~ protein (if ~e AUC; COdOD ~S in the same readiDg ~rame a~ the proteill ~Tr~ing DNA
~u~ne~`) or a frame-s~ mutation ~if ~e AUC~ codon is not in ~e same rea~ ame as the pro~ enc~dinE~ rc).
~n a pFeferred embodime~t, a desired proteLo is secre~ed in~ the 2S surrour~i~g medium due ~ ~ ~es~e of a secre~ion si~ p~c~ If ~
desired pro~ein does ~ot possess its own signal s~ rre, or if such si~al s~ does llOt function well in *~e ~ost, ~en~e protcin's codin~ sequen~e rnay be operably linlced to a signal ~qu~ hom~logolls or heterologous to the ho&t. ~e desired coding ~n~ ~ay be lin~ed to a~y si~ æqUenr~
w~ich will allow s~cretioll of the ~rotei~ from t~ host. Suc~ signal ,ceq~ nr~s may bc ~1~si~n~d vv~th or ~ithout specific pla~a~e si~s such ~hat ~e si~al F-4~ T-~3~ ~-al~ E~, JiJL 2'-7 ~ A 14:14 ~32-Z7~ G ~ f __ 2129~ ~

-1~

peptide sequence is ameDable to subsoque~t remov~l Alten~a~ively, a host ~hat leaks ~ ptotein into the med~ ma~ be used, for exa~ple a host with a t:~nn i~ its menlbrane.
~f desired, ~e no~-~anscribed andJor non-tran~l~t~ regions 3' tO the Se~n~e coding for a protein can ~e obtai~ by the a~ove-described clonu2~g raerhnd~ Thc 3'-w~anscri~ed re~io~may bc retained for its l.,.nc~ tional t~ ;on regulatosy sequel2ce e~ c; t~ 3-no~-~slated region may be r~ ed far ils ~n~ iQrl~l t~nin~tio~ regulatory s~ elements, or for tkose el~7~?nrc w~,ich d~e~t po,7yadenylation in eu~otic cells.
Ihe ~octc7rs of ~e i~vention may f~er comprise other operably l~k~ regulato~y eiem~rlts such as e-~ v~, scquen~.
In a preferr~d em7~o~im~nt, gezleticalIy stable t~ansfo~mants are co~struc~ed ~hereby ~ desi7~ed pr~tein's DNA is ~I~ ..~d into ~e host chromosome l'he co~ing se~ce for the desired protein may be from any source. Such i~ tjon may occur ~e now withi~ the ceII or, m a nlost prefe~d cmb~lmell~7 be assistod by transforma~ion with a vector which fimCtit~ y i~tÇ iLSe~ to t~e ~ost .,hlu~ x~ .c, for ~ ple, D~A
eieme~s which prOmote ~nt~tjC~rl of DNA $~ es ~n chrorno~-~mt.~s.
Cells that ha~e stably i~t~g~alL~ t~le in~o~d O~A in~ their chromosomes are selected by also ~ rod71C~ ODe or more ma~kers which allow fo~ selecti~n ~f ~ !St cells which contain the e~ ;s~iol) vector in ~he chromc~some, for eY~n~p~e the mar~er may provide bi~ide ~s;~ , e.g., l'eSiCt~nre to L~ iotiC~, or hcavy met~ls, su~h as coppcr, or tL~ like. The sel~t~le marker gen~ ~ ei~er be di~ectly linke~ to the D~A gene se~uen~s to be expressed, or in~oduced into t~e s~ne cell by co tr,ansfection Pactors o~ import~oe in sçle~ a particular plasmid or vir~ ~æt~o~
~}ude: t~le ~se Witll which rec;~.,l cclls *lat co~ain the vector may be recog~ d ælec~d from those r~i~ l cdls which do not contain t~e ~eetor, ~e number of copies of the ~e~tor whi~h are desired in a par~cu~r host; an~ wh~ it is desira~le to be able to "s}lutde" ~e ~ector between host cell~ of different species.

023712540 5 ~' G ~ f F-4_3 T-6~3 ~-02~, ~5$ JUL 23 ' 34 14:14 212917~
,5 e the veaor or DNA s~ ~ cQ~ 8 ehe cons~(s~ is ~d for ~?IC$5iOl~, the r~NA co~stluct(s) is in~oduc~ ~
app~opr~ ho~t cell by any of a ~iety of suitable mearls, in~h~
~ion as A~s~nh~d above. After t~e introducrion of lhe vectvr, recipient cells are grown iII a selecti~e medium, wbich sel~cts for the grow~
of tra~s~oImed cells. F~-.,s~iv~ Of tho cloncJ gcnc s~nce(s) ~esults i~ ~e production of the desired prote~n, vr in tLe prodllction of a ~gment of this protein. This expression can take place in a continuoulls r~ in the ~l~Ço,l~.ed cells, or i~ a con~oll~d rnan~er.
Accord~gly, ~e ~ylanase encoding sequer~es may be operably linkcd to any desired vector an~ L~fo~ed into a selected host, so as to pro~de foI expression of suc~ p~tei~s L~ tbat h~st.

he Enyme ~epQ,~lions of the lnvention According to the illvcll~io.l, tlxre is prc~idcd cnzymc compositions useful in a meth~d for bioble~lli~ and pu1p and paper lJlUC~
also provi~ed a method for prod~cing an enzyme preparation par~ly or completely de~lcie~ in celluloly~ic acliviiy (th~l is, in d:le ability to c~ lc~l~
degrade oellulose to glucose) and e~iched in ~ylanases desira~le for pulp and paper p~ocessi~g. By rde~lcie~t ~n ce~luloly~c activi~ is meant a leduced, lowered, deplessed, or l~l~cd capaci~y t;o degrade cellulose to glucose~
Such cellulol~tic ac~ity deficient p,e~alio~s, aI~d the making of s~ne by recomblDam D~A ~e~l~ods, are d~saibed in US S,2g8.40~, incolporated h~rein by leferellce. As described herein, xylanase~ n:~y be providW dircctly by t~e ~ost~ of tlle inve~tio~ e hosts themsel~es are placed in ~e wood ~Jce~ E medium). Al~.~liv~ly, used m~i--m fr~m ~he grow~ of the hosts, or purified e~ .es L~.~fio~, ca~ be used. Furt~er, if desired activitie~ ~re present in more than one recomhinant host, such preF~rations may be isolated i`rom the appropri~te hosts an~ combined prior to use i~ the metho~ of l~e invenrion.

~32371c54~ K G & f F-43 T-6~3 P-~El,'254 JUL 2~ ' g4 14: 15 21291~

The c~zyme ~Ic~dL~Lio~ f the ir~vcnhon ~a~ ~e requi~ el-lb of ~pccific nced~ in various applieatjons ~ the purip and paper in~ y. For ex~mple, if ~e in~.n~1~d app~ ti~ n is improvement of the s~ng~ of tbe m~c~qnir~ s of ~e pulp, the~ the enzyme ~ ralions of d~e invention may pro~ide enzymes ~t cnhance or faei~it~ the ~ility of ccilulose ~
to bind to~ether. In a simil~ m~er, Dl tbe ~pplication of pulp m~ing, ~e enzyme preparations of the inver¢ion m~y provide enzyll,es that er~ or f~ it~te such swelling.
To obtain the en~me p~par~ti~L~s of ~e mie~tio~, the native or recomb~t ho~ts d~liL~ ~bove luving the desired prop~ s (~t is, hosts capable of e~plessi~ lar~e qi~ntitiPs of the desir~ ase en~es a~d optio~ , those which are sn~st2nt~ iDcapable of ~y~ ~ one or more c~llulase enzymes) are cult:iva~d under su~table con~liticms~ the desired enzymes a~e sec~eted from ~ hosts into the cul~re ln~Ai~lm, a~d ~e enzyme ylc~a~n~ion is fflcover~d from s~id culture medillm by ~ known in thc an.
The enz;y~e preparat;or, can be produoed by ~ltivating the r~combinant host or n~tive s~ain ~n a f~ to~. For e~ample, the enzycne prc~ tlon of the present inve~tion ca~l be pLOdU~ed in a liquid cultivatio ~0 medium that conhins oat spelt ~la~s as tbe main carbon ssurce as desc~ibed by ~orosol~ (~iochem J. 239:5~7-592 ~1986)).
The enzyme pl~,paldLion ~s the culture modium with or without the natiYe or t~d~sforl~d host cells, or is recovere~ from the same by ~e Rllplir~tion of n~tho~l5 ~vell know~ in~e art. However, becaus~ the xylanase en7~es are sccrctcd into the cul~rc media and display ac~ivi~ in the smbient condi~ions of ~che h~ k~lytic liquor, it iS an advantage of th~ invention ~at tl3e cnzyme p~e~ ions of ~ ention may b~ utilized dIrectly f~om thc c~ re n~ m with nc fur~er pl~rifr-~ion~ If desired, such preparations }nay be Iyophilized or the enzymatic activity othexwise c~ ndJox stPbilized for ætorage. The enzyme preparations of the in~cntion arc very econo~nieal to provide and use because (1) t~e en~ymes m~y be used in a ~2371251~ ' ~` G ~ f F-423 T-b0-`; ~-a~ 54 JJ~ 29 `94 14:1=
2129i71 crudc form; iso~tion of a spec~ic er zyme from t~e culture fluid is ~eSs~ d (2) b~cause ~c enzymcs arc sccr~d into the culturc m~lr~lm, only ~e cul~rc rn~ium need be recovered to obtain t~e deæi:ed enz~ne prepa~atio~; there is no need t~ e~ ct an enzyme f}om ~e hosts.
S If ~iled, an e~l~ssc~ p~teiD may be ~r~ pU ~led i~l acu)r~e ~ith convenuona1 conllirit;~n~ such ~s cxtraction, ~ ;on, chromato~raphy, aifil~ty chromatogra~hy, elec~ophoPsis, or the like.
Th~ invention is deseribel in m4re detail in tl~e ~ollow~ ~ s, T~2ese ex~mples show only a few concrete aprli-~ti~ n~ of ~e inven~ion. It is self evide~ fcr ouc sl~lled in t~e art to create se~lreral sim~lar a~ ;o~.c ~Icncc the cxamp1cs shou1d not be i~ ;pl~te~l to ~a~row tbe sc~e of t~e ~nven~io~ only to clarify the use of ~e ~vcn~ioIl.

F~rrr~nples Frn~,r~
A~tin~. . R~unafl~o~a DSA~43186 Shoke ~os~ ond Fernnenlcr C~u~v6d h7ns The strain A~no~a~rafle~sa ~SM4318~ was s~aked on ~lled oacs mineral medium pl~ I)eutsche S~mmlll~ von Mikroo~an;~
7rllhl1hlrcn Gm~H ~ marl colle~ion of Inicroo~ ~s and c~ll cultures], DSM Ca~ogue af strains, 3rd ed., R~ c-hweig, Germ~ny ~19~3~); 1 liter con~ins 20 g agar, ~0 ~ r~lled oats, 1 ml trace element solution ~o~
100 mg FeSO4 x 7 H2O, 100 mg MDCI2 x 4 ~I2O, lOO mg Z~2SO" X 7 H~O I
100 ml; pH 9.0~ and i~ ,d at 50~C un~ sporulaiing. A spomlating colony ~as in~.ulat~d in 10 ml of XPYB mfY4il-m (Greiner-Mai, E. et al., S~stem. A~pl. Micr~biol. ~:97-109 (1~87); Hol~e, C. etal., An~or~e van Lee~ver~oe~c 59:1-7 (l9gl)); 1 liter co~tains 5 g oats spelt xylan, 5 g pepto~
~om casein. 5 g yeast ext~ct, 5 g beef extract, 0.74 g CaCl2 x 2 H20, pH

023 ~ )5~ r G ~ f F~ T-t,13~ P-~2~ 4 JUL 2q ' ~4 1~: 15 ~129171 -~8-9.0) a~ was in~llbated at 55C in a ~ LV~a] shaker (2~0 rpm~ f~r two t~
~e d~ys. An ~ m of 5 ml w~s tben ~f,~ed to 250 ml of ~he sa~e m~dium arhi ~ubated a~ ~e same c~ itio~$ for tlu~e da~s. Xylanase activi~ obtained was 17 ~at/ml ~Ineasured ~t pH 6.0, 60C, S min ~eaction, Bailey, M. J. er ~1. . J. Biotechnol 23:2~7-270 (19~2~
The proc~dure for two 1 1 î. ~ tlo~s (B~ostat M, B. Braun, Mel en, Ge~nar.y) was prcparod as abo~e. 10% in~~ m was llsed for ~e f~rme~ o~.. The pH w~s m~int~in~ at pH 7.8 :~:0.2 by ~ o!l of amll~OII~ 2.5~o) aIld phf~phoric acid (17~c), ~e f~rm~ n ~
was ~0C. The fennenter was stirr~d at 400 rpm an~ the air flow ~as 1 I/min. The ~l~n~se activities ~btained we~;e 32 and 58 nka~'ml (pH 6.~, 60-C~, S rn~ rcaction, Bailey, ~1. J. el' al~, J. B~otec~u~ol 23:~57-270 (199~J.
~r,.~r~ 2 Determina~on of t~e Opt~nolp~l ~nd ~emperature of A~ro~ ra flexuosa ~ nase Ac~vi~ from the Culture Srpern~ant Xylana~e ~ti~ities thr~ugho~t the ~Y~ c were mea~ure~ accord~
~ailey, M. J. e~ al. ~ J. Biorechnol 23 2S7-270 (19g2j using 1% bitch ~ylan (Roth 7500) as a substrate. Th~ assa~ conditions arc, if not o~erwise stated, are pEl 5.3 and S~C, with a~ cul,atio-~ time of 5 min. (Bailey, M. e~ al., J. Biotec~u~ol. 2~:257-~70 (19~2)). Xylanase hydrolyzes ~e substrate, bir~h ~cyla~ (~o~h ~o. 7~00~. Clne xylanase unit (1 Dkatj i~ defined as the amount of en~yme that pro~uces ~ cil~g carbohydlates ha~ling a reducu~g power correspond~n~ ~o one nm~l of ~ylose in one se~o~l fron~ birch xylan under assay conditions. Si~ce onc Int~nqtlo~l UI~it ~IU) is the amount of en7yme that can split one ~ic~omole of S~ Lrdk~ in one minute, 1 IU = 17 ~at.
To dPt~nninP tb~ optimai pH for t~e Ac~inom~ura xylanase activity, s~nples from the sh~-e fl~k c~tivat~on (culnlre SUPC1L~L) were diluted U
McIlYai~'s ~uffers ~.2~ M citric acid - 0.5 M Na2HPO~) of pH-ran~e 3.
11Ø I~ pHs of the enz;yme buffer u~lu~s were 3.5, 4.5, 5.4, 6.4, ~123712540 5 K G ~ f F-433 T-608 P-008~016 JIJL 2~ 'g4 1':12 SU~SnTUTE SHEEr ~ 1 2 917 1 l~
7.2, 8.0, 8.5, 9.7 and 11.2. X~rl~as~ activity was "~a~ d at each pH at 50~C, 5 mi~ cti~. The xylanase a~ivily ~Yhi~it~ 80 lOO~o of its m~imlnn activity i~ the pEI ~ange of a~out 5.~8Ø The en~yme had its ~illlU~ ac~vi~ at a~out pH 6.4 (Figu~e 1).
~or the ~e~al stabilit~r ~1 t~;n~ti~n~ samples from the cul~e wcre dilllted in McIlvain's ~ers. ~ was added ~o a ~u-r~.~f~ of 100 ~glml and ~p~lA~jn A (10 ~g/ml) and phenyl meth~l s~llfonyl fl~oride (PMSF, 174 ~g~ul~ werc ad~ , inhibhors. The final pHs of the e~yme buff~r ~lules were 6.9. 7.8, 9.0 and 9.4. S~mrle~
~ere i.~uba~ n ~e abæ~cc of tbc ~ l.r~e at 60C, 70C alld 80GC.
~amples were taken at inter~als of 0, 30, 60 and 120 ~s and imm~diqtely cooled on ice prior to the residual xylanase activi~ Y ~ at 50C
(5 ~n reacdon ill ~e coIre ;ponding pH). The enzy~e was ~leIy s~able when inrub~t~ at 60~C and 70C; afte~ 120 minu~s ~ h7t;~ n at 70C as p~I g over 60% of ~ylandse activi~r was retained (Figure 2).

~cu7~plc 3 c~on ~f Ac~con~ta Xyl~rn~ s eati~n of x~lanases f~om Actinoma~r~ g~wd3 mt~d~vm was ~d at ~4C with cL~oy,l~phic column3 coupled to a FPLC
~ qru5 (Pbs~ ia) X~ C~ were pe rv~l~JPd at SOC and a~ pH 6.5. Protein was monitor~d at 2gO nm throu~hout the pUrifi~`~tinfl Sqm~ were ruII on polyac~yl~e slab ~els c~nt~inin~ 0.1%
SPS o~ a Bi~R~d Mini Prot~ II ~ opllo}esis sys~em ~od staine~ with Coomassie BrilliaIxt Blue. A polyclona1 antibody prepared again ~5 ~72ermom~n~s~,0rl fusca ~yla~e A r~, o~t2Lil~l from Prof. David Wilsorl, Co~el~ UniYersity) was used to detect Act~nom~rc~ xyla~e(s) ln Western blo~ the detection, Promega's PtotoBlot0 ~ System was ~sed.
A ~rowth media of the two 1 1 fe"nt~nt~io~s ~lrsc~l~1 above was pooled and cPn~-fi~ed at 8,000 g for ~0 mi~. T~ (1,500 ml) was dil~ed 1 ~ 2 with 12.5 ~LM di~ ~ phosr1l~te pH g and adjusted to 1323~12540 '- K G ~ f 212~45t31T-60' P-~25/~354 JUL 29 '54 14:16 p~l B.6 with 1 M sodium hydroxide. Thi~ sample w~s applied. iD two sets~
on a DEAE S~pl~ose CL~6B ~Ph~ s3 ion exchanger (2.5 x 29 cm) equilibrated with 12.5 mM disodrum ~hnsphqte pH 9 at 100 ml/h. ll2e flow-~rough of bo~ runs w~e~e was cnmhin~ an~ pl~ces~d ~Ldlelr as S d~l ibcd later.
Eluuon of the bound proteins ~om tbe DEAE colum~ was accomplished by a l~near gradicnt ~400 ml + 400 ml) from 25 mM ~i~ocinlm ph~$~h~t.e pH 9 u) 25 mM ~ m l~bo6l~h~t~ pH 9 c~n~inin~ 1 M sodium chl()ride a- a flow rate of 105 mlJh and f~actions of 10 ml were collec~.
Two ~lanase a~ cl~ntAinin~ pealcs could be collcctcd (pool I and Il), as well as a lon~ "hil~" of the se~ond pca~ (pool III~
Ihc ~c pools (comhin~l ~rom both DEAE runs) were "'Iju5t~ tO
con~n 2 M sodium chlonde each an~ applied ~ ltly on a Phenyl ~halv~ CL 4B (Pharmacia) column (2.5 x 15 cm) equilibrated w~ 25 m~l ~ rn rh~-~ph~ pH ~ con~ining 2 M sodium chloride. ~lu~ion was perfolmed at lO0 rnlfh with a n~o step grad~ent of 1009~ buffer A ~25 mM
disodium phosphate pH 9) to 357O buffer B (25 mM s~iiurn rhn~h~te containi~ 60% ethylen~glyc~i~ in 60 min followed by a s~er g~dient from ~SX B to 100% B in 60 min. Fractions of 7 ml (pool I) or ~ ml (pools II and m) wue coll~d The x~lana~e activi~ cor.~ actions of pool I
obtained weK pooled ~nd named DEPS I. Both DEAE pools II and III
resulted in two ~yla~se acti~iq conr~inin~ peaks n~rned DEPS IIIl, DEPS
II/2 an~ DEPS IIIil, 3EPS m!2, ~ e The flow-through of the DEAE runs (see above) was c~ ~d wi~
2~ a cut off membranc of 30 kOa, a~d a~jus~cd to con~ain 2 M sodium chloride.
This sample ~as applied on a Phenyl S~pharose 6 P~stFlo~ ~low sub;
ri~) colum~ (2.5 x 34 cm) eql]i~ te~ with 25 mM disodium phospllate p~ 9. Eluti~n was accomplishe~ at 300 mlh~J [ml/h~ with the sar~e g~dient as was used for DEAE pools ~n Phenyl Sepharose C~6B and fractions of 10 rnl were c~]lected. Xylanase a~vity co~aining peaks obtained were named KFI, KFII aIxl KPIII. The ~ from ~e cQnrentration was Ç~23~12540 5 ~ 5 & t r~123 T-fDE3 P-E~2D,~E5~ ,TUL 29 '94 14: L7 slibjected to an id~n~iCi7l Phenyl Sepharose 6 Fa~tno~r (lo~h sub~ mn, an~ Ihe xyl~r~sc activity containi~ ~actions ~er~ n~med PFI and PFI~.
All the I~EPS, K~: ~d PF pea~s obtain~ ~-ere dialyæ~ against 25 mM di~ m phosphat~ overni~ht.
S Roughly half of th~ xylarlase activi~ was bol~d to DEAE Sepharose in thc ~Irst pllnfic~at~s~n stcp. Elution of the I)EAE p~oteins from this ion-exchanger }c~u~t~ ~n a qui~e ~arp peak follo~ed by a broad "peak"
(Fig~ 3~. This broa~ "peak~ ~as di~ided into two different poo~s. Each of thes~ pools wer fi~er purified OD a hydrophobic in~orflcti~n chromatography lU (H~Cj colu~ ~i~ure ., 4A an~ 4B~. Some ~ ~s cou}d be seen, in that poo~ m DEAE nesul~d in a holl,og~ous peak oll HIC ~Figur~ 4), bu both pools Il (Figure 4A) and m ~ ure 1B~ result~d in at least two peak~.
Sarnpl-os of t~se poo~s were ~un on SDS-PAGE an~ stained for protein with Cw~ie Blue (~igure 5) as well ~s anal~zed by We~ b~ts with ~. Jicsca ~ntibod~ ~Figure 5A). The antibody ~re~cted only wi~ t~o to ~ee ba~ds of smaller molecular mass (below 35 kDa} ~o~ ~e gro~th me~iium and wea~l~
wi~ ~:hc prote~ i~ these po~ls. The ap~ ~ ~olecular masses of the proteins ~ ese pools were 50 kI:~a as e6tim~t~ ~om SDS-PAGE wit~
mole~ular m&~s st~rds. Pools Dl~PS I~2, DEPS II~11 and DEPS III/~
'~0 were the most pur~.
The flow-throug~ of the I~13AE ion~xchanger was COr~Pn~rAt~d with a Cl~t off r~embrar~ of 30 kDa. Roug~ly half of ~e ~yl~e activir~ was found in ~e conr~n1r~te ~d ~If in ~c permeate. Both wele puri~ed fi~er by hydrophobic interactioll c~vl~at~ p~y, r~tlng in two ~yla~se ~cu~ity peaks for th~ p~ ) and three for the co~n~te (Figure 6A).
The~e peaks were analyze~ on SDS-PAGE as well as o~ Western hlots ~Figure 7). T~e f~r~t peak~ KPl, ~om the co~ hdtc sJlowed a band of 40 kDa ~c,.r molecular macs on 51~S-PAGE, l~ut no reacu.on on Western blots. ~owever. this pe~ ~ad ~ hi~hest xyl~ase ac~vi~. KF2 showe~ a band of 50 kl~ on SI~S-PAGE rcacting weakly with the a~abody, but a cleàr b~nd of 30 kr~a could be see~ Western blots. T~ third peak, KF3, S K r & ~ F--433 T-608 P-009/016 JUL ~9 ~ 94 15 13 13;~371r~541~ ~

SU~S~JTlJTE S~i~
-æ-showed a ban~ of 35 k~a OIl Wester~ blots. A~J~e~ly the eo~s~nt~
c~llt~i~d xylanase~ of 50, 40, 35 as well as 30 kDa, ~ ,L mrl~~
ma~s. The flrst pe~k, PF1~ from the permeate re~cted with ~. ~sca an~ibody showing t~o bands of 35 ~)a a~d 30 kDa~ respectively. PF~, on ~ other S hand, showed only one ~d of 3~ a on Westem ~l~ts.
As a ~v~r, A~nomad~ cor~si;ns three to fo~ ~*la~ pro~in ~ds of ~nol~c~lsr mass 50, 40, 35 ~d 30 k~ f t~se, 3~ kl)a a~ 50 kDa ~xe ~e ma30r b~. It îs possible ~at tbe 40 kDa xylanase is n ~-fd~tinr~ prodll~t of ~e so kDa and the 30 k~a & de~ada~ion product of 35 ~ yl~se.
Example 4 *~ nd seqr~nt~i~ o~ des h sam~le (12 ml~ of pool I from thc r)EAE mn was s:ub3ec~d to gel e~el~ on Llllo~ a~hy on a Hi~hT ~c 2C160 S~d~ ~75 colum~
~Plr~ e~ hr~t~ ~ith 25 mM ~lisod;~ ~h~ r pH ~ at 120 mllh.
A sample (25 ml) of t~ ylanase activ~ty Conl~ pe~k fraction vb~i..~l was d;luted (1 + 1) with water and applied on a mono Q ~ph~ ) ion-e~rrh~ r ~lilihratod wi~ 12.5 mM d;s~;.l .~ p~osph~te p~I 9. Elution~ras pelrurL~ at 30 mlJh with a li~ear ~ nt from 12.5 mM ~ 2 21~ phosph~te pH 9 to 12.~ mM dico~ m l?hl~sph~ pH ~ cn.. t~ 0.5 M
sodium chloride m 50 min. Tbe xylanase activity C4~lA;n;ne pea~ (1 ml) was ~on~en~.it~d on a l~en~ricon ~ro c~ f~ dls~l (CUt off 30 l~a) aDd elut~
Wi~ ~ mmot~ m bicar~on~2e. This co~ted ssmple l,vas ev~poldted alld alkyla~ed ~i~ vinylpy~idin. The alkylated sample was ~lJ~St~ h z5 ~ypsin (nlodified tr~psin~ sP~uen~l gr~e, Prome~a V5111). The d~est was applied o~ a reverse pl~se column coupled to ~ ~ d peal~ a~s~
at 214 rLm were eol1~te~ manually. These peaks were each d~ d ~n a gas-pulsed-li~u~d-ph~e ~u~ Plklri~n ~ Tilgn~nn, J. Protein Chem.
7~ -243 ~lg88)) a~d ~e rele~sed PIH amino acids were anal~ oII-line by usin~ ~arro~ ~o~e reverse phas~ HPLC.

Q2371254Q S K G ~ f ,~-433 T-608 P-1313~016 JUL 29 '94 15:13 SU~S~ITLI~ SH~EI -2~- 212 9171 Pepti~s obtai~ed ~rom dle purified 50 ~D~ xy~e are lis~d Table 1.
T~ble 1: Peptide~ f~om Ihe ~fied 50 kl;la ~ ~~ ~ e # 16g~ Ala~ Se~ Leu-Ala-Glu~l~-Ala-AI~-Gln-Hi~-Asn~
ff 16~7 Ty~-Phe~ly-Val-Ala-ll~Ala-Ala-~sn-Arg s~Ser-Val-l~r-Thr~ -lle Al~-As~Arg # 16~ AsnlGlyJX-Thr~ly-Il~Thr-Val-~Gly-Va~
1703 His/Glu/Th~luJPh~Leu/Asu-~aJJSe~-Tyr~Val-Asn/Thr-Met/Ala-V~/GIu-~sDlX-GllllX-Met/~
~ 1704 Glu-Ph~As~Ser-~al-Thr-AI~-Glu-A6n~1u Me:-(Lys) Thecomhin~fi~mof~hcpeptide~nr~#1696, 115g7, 169g~rldl704 e6p~l~ds wi~ 7~ % s~ amino a~ids 42-8~ in 5~rep~o~ces x:yl~e ~:

ra 50 kl~a 1 ~.As~AEG~R lrPavA~ L~D~VY~I~NR ~ErA~V~rA~ 5R 48 S. Ii~id~s Xy~A 42 A~SsTLGAA~AQSG~ YFG~AI~AsGR LSDS~YT5IAGR ~pNMvTAE~MR ~g Exam~l~ 5 ~e p~ propert~es twd tem~t~ G stabil;ty o~ the pun~ed 35 kD~ and 50 - kDa xykuwses T~e t-.. --L~ sta~ility O~ the purified 35 alld 50 kDa ~L~ S
(ilOO ~ml BSA) ~as d~t...,n11-~ by in~ubah~ the cn~e sample~ at 70~, pH 6.0 for a period of 0, 2, 6 and 24 hours after which ~ ylanase activi~y of the sample6 was dPt~ d (at pH ~.5, ~0C, 20 mi~ reaction).
~n ~e samples in~ which BSA ~ad boen added, o~ of th~ oligin~l activity could be measured even after ~4 h of ~ nbati~n (Figure~ R and 9 for ~e 35 l~a and the 50 I~)a ~ylanases, l~live~ ~hen BSA

0237125~0 ~ ~ G ~ f F-433 1-608 L'-011,'016 JiJL 29 '94 15:13 SU~Sn~UrE SHEET ~

was IlOt added, s~ill about 6~% (~ Icl)a) or 70% (~U ~Da) of t~e or~l ac~vi~r was measured af~r ~4 h of in( .l~lotic~n (Fi~ 8 and 9).
The p~ stability was doDe ~y i~ h~ the e~yme samples at ~lirr~e~ p~I v~lues (for 35 ~a, pH S~8, ~or 50 ~d)a~ pH ~ and at S ~ aAhcs of 80C (35 I~)a) a~d 60. 70 and 804C (SU k~a) for 20 min (~5 kDa) or 10 min ~50 ~a). At 80~, tl~c 35 k~a ~Lylallasc ~ad its ~ a~
around pH 6 ha~n~ nearly 9096 of hs a~vity from about pH 5 to 7 (Figure 10). At 60aC and 70C, the 50 kDa xylanase had i~ ulll at pH
5-7; at 80C, ~e pH opliAIu~ wa~ at pH ~7. The en~ne was v~y stable ~rom pH 5-7 (E;i~ure 11~.

~u7~ple 6 ~It~ g Ex~e~ents Us~ng t~e ~wmad~ ~re Supe natan~

A s~uc- ~e of ble?ch;r~ ls we~e do~e to d~ line ~e usefi~l~ess of Actinom4durafl~wsa xylaDase in both ECF ~e~ nt~ty chlorine free) and TCP (to~lly chl~ri~ free) bl~ of h:a~ pulp.

ECF ,~le3~;~

G~ow~ media c~ itun~ Aczi~ l flexuosa ~yla~se ~see FY~rrrl~ 1) was added to Finnish oxygen~ ifi~d ~oftwood kraR pulp (~:appa r~mber = 15) in the amount of 50 or 1~0 nkat~'~ pulp dry matter, at pH 7 ar~ 70C for 1 hour. This cul~re m~ rn is very low in glllr~n~s alld cellulæs. R~r~.e~ pulp was kept ~der the sarne con~i~ons wi~out enzyme addition.
All pulps were ~en similarly ~leach~d in two steps: chl~rine dioxide a~ allc~l~ ex~ction. The a~soll~a~ of ~e fltrate at 280 mn was 2S ~eterrniT~d as a measuré o~ dissolved ligr~in.

F-4~, T-b0~ P-~3~2, 1~54 JUL 2C ' 94 11~
0~3~ S~:G~ f 2129171 TABLE`~
O r~t/g 50 r~a~/g ~ /g ~me S~age C~ t~ , % 3 3 3 Th~ c.C 70 70 70 p}~ a~ sta~'end 7.0n 1 7.0~7.2 7.2/7.4 12et~t~ ~ime, mi~. 60 ~0 60 A230 ~dil. 1/10) 0.22 0.49 0.6 C~'2 S~a~e C~n~;~le ~ 3 3 3 ln Cl02dosa~e, % 2.3 2.3 2.
're~l~, oc ~o 6Q ~o pH at end 2.4 2.~ 2.5 Re~e~on ~ime, min 60 60 60 E~c~o~ e CQ~ ~iet~ , 9b 10 15 10 NaOE~ ~osage, % 1.5 l.S 1.5 Tempera~re,'JC 70 70 ~0 pH a~ en~ 10.9 lO.g lO.g Relen~n time. miE. 60 ~u~ ~p hm~ss, ~ ~SO 56.7 59.9 60.6 K~ppa number 6.6 5.6 5.4 sity dm31~g g2Q 910 900 As caII be seen iD Tabk~, afoer plcllc~l~ l1vith ~e xyla~ase m~re ~--~as removed (zs evide~ by ~e change in ~e A2~. The fin~l pul~s had 3~ tm~ts ~igher bri~tness ~ithout losing ~e s~en~ of the pulp (thc viscosity c~ e of ~CI units is inside the normal vanation of the met~od).

02_,7125~1 5 K G & f F-42' 1-~3' p~ 54 JUL 2~ ' 3~ 14:1~
2129~71 T~F Rlf~ - L -ng FiImish ~>xygen-del;~nifi~d softwood kraft pulp, ~vith kappa ~er of 15, wae ~ea.ted w~th Acnnoma~ro fle~uosa x~lanase using en~yme dosages of 50 and 10~ at/g pulp d~ er. The t~ea~nent was done at pH 7 at 70~ for 1 hour. Referen~e pulp was ~ept un~er the same c~n~i~ionc widlout enzyme addition.
After this, ,311 the pulps were ~i~ul~rl~ bl~açhP~ in two steps: metal temo~ al b~ eh~l~tinn ~th El)TA an~ ~Y~lloa~ ~ peroxide. The abs~rban~e of t~e fil~.rate at 280 nrn w~ determine~ as a measure of dissolved l~gn~n.

F-4~ T-6~33 P-~ 354 JJL 25 !94 14:19 ~3c~7 1 254i3 _ 1~. G ~ ~

- T~ ~
O n~t/g 50 r~ g 100 n~/g me Sta~
C.o~ C~. % 3 3 3 T.,.,9~la~,1,c, C 70 70 7Q
pH a; s~art/end 7.0i7.4 7.0/ 7.3 7.0J7.3 Retention ~, min. 60 60 ~0 Abs ~80 nm ~dil lllO) 0.27 0.43 0.57 on Sta~e Collsistenc~, % 3 3 3 o E~lrA, % 0.2 0.2 02 T~ lure, C 70 7(~ 70 pH a~ end 5.5 5.6 5.E
p~nri~-n time, min 60 60 ~0 Abs 280 nm ~dil. I/1O 0.~4 0.44 0.64 t~u,.~ , % 10 10 lO
H2O~ ~osage, ~ 3 0 3.0 3.0 H20~ s~Llon~ % 0.87 0.85 0.91 ~TPA, % 0.2 0.2 0.2 h~gS~,. % o,5 o;~ ~5 NaOH, ~ 3.t) 3.0 T~ C 80 80 80 pH at elld 10.6 10.6 10.6 Rc~iont~ne, m~n. 180 180 1~0 P~ P~P
~,5 R, ;~J~r,~ .0 71.9 72.g r~.o K3~pa rlu~er 9.0 8.3 7.9 Vi~osi~ dm~kg 870 ~0 8gO

Table Z ahows ~t accordin~ ~ the AZ~ measurement an~ kappa number, sig~ific~tly more l~in w~s rrmoved after ~lanase ~lCt~ ..P~.
~hile ~e sfreng.h o~ Ihe pulp (aecor.li~g ~e viscosi~ nf~ ~ood.

1~23712540 5 ~ G ~ f F-433 T-6~8 P-1312/016 JUL 29 ' q4 15:1~

~lfBs~l7~TE ~h~ . .
-2~- 2129171 E~4mple 7 Rl~ ,; experunen~s ~ fhe ptmfud 35 kl)~ and 50 kDa x)~ rs The purified larger 50 kl)a (AMS0) ~l~n~se a~ the ~m~ller 35 l~Pa (~M30) ~yl~dse ~irrill~;n~ also the 30 kD~ were used for ~l~a.~ ."~ s in a ~-~t~ge pe~oxide bl~hi~. T~e ~fied e~me pl~ ,~ were ~e same as used in the d~ tinn of ~e pH and ~e p~op~ies ~or ~ purified e~zymes.
A co~ol sample ~out any en~me ~ea~ent was also inrl~ the dry wei~ht content and kappa number of ~e startin~ twood pulp (V ~41-18 ~21Z9)~ we~e 2g.8% and 13.S, lw~Liv~ly. The s~ng ~ f the pulp was 37. 1.

En~yrne trea~nent ~d dos~e The e~yme k~t~ were ~ed o~t at 3.5% c~ t ~ at 60~C
for one hou~ at pH 6.5, q~ith b~c~wood ~cyl~n (Roth No. 7~00) as substr~t~
The en;cyme dosa~e was 100 nka~lP of d~y pulp. Th~ ~4~in~;~ a A~rlal~s werc dis~olved in 25 mM ~l1so~ n rh- ~h~t~ buffer inf l~ i~ 50 mM NaCl arld ~e same ~o~ of ~is huffer was add~d to ~e conJrol sarnple. Tl~ pH
of the pulp was adjusted with ~Iphar~c acid.

~0 C~

T~e c}le1s~k~n was performed by addin~ EDTA to 0.2% ~f dry weight and it was ca~ied out at 3.0% co~ci~rry at 50C for one hour.

E32371'540 5 k G & , F-427 T-503 P-E34-'~54 JUL 29 ' ~4 14: 2EI

-29- 2l29l 71 Peroxid~ 7:~t.'.g Tl~e three peroxide ~'~ , hin~ stages (80C, 160 tnin~ ~ere carried out the same way e~ccept thal after each stage, one-~ird of the pulp was removed for te~g. The con~i.~ions u~ed were the follow~:

Concic~ryL~%
H~O2 3 %
N~UH 3 9~
DTPA 0.2%
MgSO~ 0.5 %

Resu~s The results wi~ Ecopwlp ~-200~ enzyme preparation (Alko Biotechnology, Re,am~ki, Finl~d~ containing T. recse~ xy~e II a~e also includ~. T~e sta~ting pulp ar~ all other tl~t~ J~tS are the same except that the e~yme ~at~e~ kat~'g of dIy weight) was canied out in water, pH
lS 5.0 at ~0C, usin~ ~e ~ sub~trate as above. This is close t~ tbe optimum of ~ T. reesei ~yl ~ ~oi sample wss treated in the same way but without the e~ne.
The reduein~ ars (% dry wei~) were analyæd ~r the en7.ym~
trea~ment and were ~.e following:

2~ C~ntrol 0.19 O 1.18 ~30 1.64 Cont~o~ 0.20 Ecopul~ ~-2(~ 1.32 The results from the bl~q~ hin~s are shown in Table ~323712~0 5 ~ G ~ f F-423 T-b03 F'-035~354 JJL 29 '~4 14:20 _3~ 21291 71 Table ,~
ISO B~ig~5 Kappa P .u~id~
Pl stage Co~trol 59.~ - 2 7 AM50 62.3 6.3 2.7 S ~ O 63.7 5 3 2.6 Con-ro~ 62.2 8.0 2.2 E~o~ulpX-~ 4.1 7.7 2.3 P2 Stage Control 67.2 6.8 2.2 AM~0 ~9.7 4.8 2.4 AM30 70.7 4.9 2.2 Con~rol ~.8 7. 7 2.2 ~cop~lp X-20~ JO.6 ~ 3 ~2~712540 5 K5 8. t F-4~3 T-6E3~ P-1313/015 JUL 29 ' 94 15:14 ~29171 Sll~STlTUr~ SHE7 -31-ISO '.~.,' ' Kappa P~ ~ ~~ ~d (%) P3 Stage Co~trol 71.3 $.2 2.2 AM~0 74.0 4.1 2.2 AM30 74.4 2.2 2.0 Control 73.3 6.8 2.z 2~0 74.9 4.2 2.1 ~ e use of AhI50 and A~30 clearly incleasod t~e l";~ ss obtained wi~out i~;,ea~ g ~e amoun~ of pero~cidc tb~t was us~d.
~mp~e 8 r~7fr r~ of the ch,r.~,Ds ~DN,4 ~nd cor;s~,~tJ,on of ~e ~e~t . . 'c ~Y
~ ctinom~ra fl~7s~1 6p. DSM43186 was eul~vated i~ 50 ml of m~Aillrn c4~ of 0.~ % oat spelt ~ylan, 0.5 % p~ptone fronl caseiD, 0.5 yeast e~c~act, 0.5% beef extract~ 0.074~ CaCl2 ~c 2H;20, p~ 7.~7.5, ~
bafTled s~ake ~l~sk for ~.S d~ys at 52C with ~a~ing at ~OQ ~n. 2.5 ml of ~is culbLre was ll~fi llod to 50 ml of fresh medium su~plem~nted with 0.8~ ~lyc~, a~ g~own for 2 days at 50C, 200 Ipm. Cultures were tr~rc;l~ed into SS-34 tubes, pelleted a~ 8,000 ~pm, aIld was~d with 109 I`OSe., 25m~I T~ HCl (p~ 8.0)-~SmM EDTP,.
The chrnrno~or~ N~ was isolated ~ g to E~opwood Ct al (~netic ~a~ ulation of Streptomyce~: A ~ m~u~l, The Joh~ i~s I~ou~ation, ~rwich, UK (1~85). Brielly, the rllycelium was lysod with Iyso~me ~d 2 x Ki~by ani~e (2 g sodium triisoy~ n~l~h~t~ p s~ P, 12 g sodium 4-ami~sali~ylate, 5 ml 2 M Tris-~l (pH 8.0), 6 ml of Tris-HCI ~ ~Illr~ ~-J p~wl, made u~ to lO0 ml w~t wa~r). The DNA
was pr~cipitated wi~ isopropanol and d~ssolved in~o TE. RNA was di~ested with RNase.
The chromosomai DN~ was par~ially ~c,~ t~l ~ith 5~3A
~Bocl~ gcr) and s~æ-~ction-ted in sucrose g~adient (1~, 20, 30, 4~%

~32371--`51el 5 1~: G ~ f F-42_, T-61~3 P~ " ~351 JUL :~S ' 94 1_: 21 sucrose in 1 h~ NaCI! 20 mM ~ris-HCl, pH 8.û, ~ DTA). DNA of 7-10 kb was usod to construct a g~ .,;c Ac~n~n~ Libra~y.
Tbe p~ediyested ZA~ E~p:ress~ Bom~lC}AP Vector Clo~ ~it (St~atagene) was used to co~$~uct ~e libraly an~l the instruc~ions of ~he m~m~ .c, we~e followed In all the ~ u~-~ steps. Bncfly, abou~ 200 ng of si~e-fractionatcd ~NA was liga~ to I ~g of ZAP Express prepare~
a~ns, a~ packag~d using Gigapack II ~ gin~ e~tract ~S~tagcne~. The titer of the Li~rary was d~t~",linel by i~fæt~ng E. col~ XL1-~lue MRF ceUs with serial dihltions of the packaged phage and platin~ ~n NZY pla~s. The li~rary ~vas used for sotee"i~ ~out amplifi~ion.

~sol~on of the gene co~ng for ~he 35 kD ~o~se The polyclonal a~ against Te~r.ornonospc)ra fi~sca 32 kD
~ylan~se, 'rf~A. was used to scrccn ~e Actino~ ura genomic ll~ry.
S~atagenes XL1-Blue MR~ cel1s were grown in L~ + 0.2% m~ltose ~ 10 nL~ MgSO, a~d diluted to O[~600=~ 5 The cells we~e i~fected wi~h the recombinant library for 15 min at 37~C an~ plated ~Yi~ N2:Y to~ agar o~ thc N~Y plates. Plates we~ iru~ ted for 3.5 ho~rs a~ 42C, overlaid wilh r~itrocellulos~ filters saturat;ed with 10 Ir~ IPrG. ar~ ~ o~emight at room lern~ tection was p~lÇ~ e 1;1500 dilu~ed ~ 5C~
Tfx~ antibo~y us~ Pro~egas ProtoBlot~ AP System. Twelve positi~e clones, of which ~e c~one 1.1 clearly gave the strongest s~al, were picked in S~ buffer/chlorofolm, and ~urific~l witl~ a secon~ round of SC~c~
The Z~P Express vector ha~ been desig~d to allow s~le, efflri~nt ~5 in ~ o excision and r~ireula~iza~io!l of any cloned insert contained withi~ the lambda vector to form a phagemid cvn~a~ ~e cloned insert. Briefly, the posi~ve cl~es were in~lbaT~ with XI,l Blue MRP cell~ with t~e E~LAssiss he~ pha~c. A~ter hsat dena~ra~ion ~70C, 15 m-n), ar~d ;~ tion, the excised pha~eTr~iA pBK-CM~ ~s p~k~Pd as fil~m~ntous phage particles in the ~32~71254~ 5 ~¢ G ~ f =-423 ~ 3 P-~38~354 JUL 2~, '94 14~

D~ nt. The ~scued pl~,c~d was mixed with XLO~ cells, snd plated on LBlk~lly~ (50 ~/ml) ~ccor~ to the m~n-lf.~n~rer. ~he r~combmant pBK-CMV plasmid DNA was i~olated from ei~t clon~s using the G~ager procedure (l:~iagen GmbH,~.

S E:~ample 10 Ana~ysls of the 35 kl) clones The Actinom~r~ cll.o~nos~l,al D~A a~ dle plasmid DNA from eight clones was digested with Ec~ aDd PslI to release the Ac~inomadz~ra insert from ~e pBK-CMV vector~ elætrophorese~ and blotted onto the nylon filter. The filter was hybridized with d~goksigenin-labeled 1.1~ kb PstI
fragmcnt ~om p~l~18S ~Figune 12). T~e plasmid pAlK185 contaiD.s ~e ~.
J~sc~ x/n A ge~e from pT~101 ((~h~s, G. S. et a~., J. Bacr. I71 :2963-296g (1994)). Tbe EcoRl~PslI digested pA1~185-DNA was used as a positive control for the ~ybridization. O~ly the Actinom~ clo~e 1.1 and ~e co~ol gave stro~g positive s~ls, ~ clone 1.1 was seleeted to be sequenced. The T. Ji~sn~ ~InA pr~be hybridi~ 4 kb Eco~-PstI
fra~mPnt in ~e Acnnomadur~ sp. DSM431~6 chr(~m~som-l DNA.
Six of the E. coli clo~s co~A~t-inin~ the Acnnom~ru i~serl, ar~ gi~n~
diff~ent rcstriction patte~s, were s~d on RB~-xylan + P~m plate. The plate has hvO la~ers; lower layer of 1~ ml regular LB + K~n (40 ~g/ml) ar~
upper layer of 5 ml of RBB ~ (0.5% ~BB ~lan, 1~; oat spelts ~c,vlan in LB ~ Km, ~uffe~d with 50 mM K-p~.~srhA~ (pH 6.8~ e E. coli strain DH5~JpALK1~, produ~ing Trichodennll reesei ~ryl~lase II p~otein was used as a positi~c control. After an overnig~t in~ubation at 37C, a cle~ halo ~ as s~ing to develop arouDd the. 1.1 clo~e ~ e co~trol.

0237125~0 5 K E ~ F--~;23 T-6~:33 P~ 5 05~. J~IL 2C ~ 54 14: c' ~
21~9171 Sequencing t*e ~y~e gene co~ng for the 35 kD pro~-n lhe clo~e 1.1 was named to pA~23. T3 aGd T7 pr~rs were use~
to s~enre the Act~nom~dura insert fr~m both ends in ~e pBK;-CMV
S recombinant plasrnid. Addition~ally. pAL~C923 was digesled wit~ EcoRI-Pstl, an~ the 2.5 kb fragment hybridizing to the T. fusca ~InA probe was ~l~,lon~
into M13mpl8 and 1~13mpl9 phage vcctors to 211ow se~encing in both orie~a~io~. Because of thc hi~h GC conte~t in the DNA ~es of ~ermoph~lic or~ni~nc~ eaetiol~s were p~lÇol~ed witl DMSO, at ~nn~ n~ t~,n~cla~ure of 58-60C. The se~Up~r~r~ of the 410 bps of Acnnoma~ra sp. DSM4~,186 xylanase ge~e is pre~,eMe~l in Figure 13. The sçql~enre shows high homology to xylana5cs ~m d;ffclc~t os~,~ui~ . At am~no acid levels, the u~de~ ed s~ nce shows B0 % homology to ~. ~sca xlnA.
Exa~nple ~2 I~Q~7a~; of the ~0 kD Actinom~ xyl~ gene 1~ g~nomic library of Ac~znomadl ro ~7CXI osa sp. DSM43186 D~A
in ~AP Expressn' vector ~Ivas sc~e~d using a r)NA probe.
Oligonucleotide p~imers were ~l~si~d based on the peptide s~Ucllres derived from the purified 50 ~D protein. The pr~r ~quenr~s are ~
in Table 3. Bouuse the comb~Datio~ of peptide s~qLPnr~ #1696, ~1697, ~16g8 alld ~1704 colr~o~ds ~ith 7~9~ similaTi~ to arnino a~i~s 42-89 in StrePTOmYCeS livi~ans xyla~ase ~, a 39 bp oli~o was 5yntht-ci7f~d, from bases 331 to 3~9 in ~e S. ~iv~d~ns ~ynaS Sequcl~re. The S. Ii~ ans ~,~naS probe 2~ and the prir~ers #1704&c, ~1703as"Yl6~s were labeled w~th digoksigenin and te~m~l Lldnsf~.~e~ a~d used as pro~es ill hybri~li7~tion at 5~C ~L~l.li~
tc Boel ril g~r, DIG ~)NA ~abe1ing and ~etection NonradioactiYe, Apylireti~nc Manual.

02371254~ 5 K G ~ f F-433 T-60~ P-~14~01b JUL 2~ '94 15 14 SUBSri~UTE SH~?

Ihe #1704as an~ ~e 5. Iivid~nsxynaS probe ~ognized ~e s~me 1.2 k~ Eca~-PstI ~ in Actinoma~ura ~NA. The r.r.~ " is LfÇ~
~om the 4 kb fr~ ecognized by the T. ff~ ynA probe l~ased on ~se results, rhe 3~ mer 5. iivi~ ynaS probe wa~ use~ to screen ~e Ach~lamadur~ lib~y for the SO kr) ~lan~ coding gene.
Twelve plagues gi~ lear po~i~ve signal witih the S. livtd~ ynaS
oli~omer pro~e were picked into 1 ml SM ~uffer-~ lorofoxm, and storcd at +4C. The cl~nes we~ ~amed A~t.~yl.S0 f~om 1 to 12 ~ct.~yl.50/1;
Act.xyl.50/2; Act.xy1.50/3; Act xyl.50/4; Act.xyl.5015; A~t.xyl.5016;
Act.~yl.S0/7; ~Lct.~yl.50~8; Act.xyl.SO/g; Act.xyl.50/10; Act.xy1.50/11; an~
~ct.~yl.50/12).

T~ble 3: Oligonud~tide ~ u~ ' .. o tbe ga~e ~oding for ~ ra ~0 I~ e Primer DN~ sequence A~tir ~ p. DS1~ 186 #1696s GCAII~/(iJ~l~CA/CM/TC~V~ TA/C:Ci #17~ ACCATA/GTT~GTAf~C/GfTAC~C/~A
~l7~ TTCATC~C~GTTC ~ CA/C/~C
S.Ii~d~ ~S CGTGAGTTCAA~TGGTGACGGCCG~GAAC&A~ATG~A~
. .
~- ~ e;~ z~mi~An~e

Claims

What Is Claimed Is:

1. Isolated DNA encoding the amino acid sequence of Actinomadura flexuosa xylanase amino acid sequence of Figure 13 (SEQ ID NO. _).

2. The isolated DNA of Claim 1, wherein said DNA sequence is that of SEQ
ID _) .

3. Plasmid or the fragment of said plasmid that encodes the Actinomadura flexuosa 35 kDa xylanase.

4. Plasmid or the fragment of said plasmid that encodes the Actinomadura flexuosa 50 kDa xylanase.

5. A recombinant vector comprising the xylanase encoding sequence of the DNA of any one of claims 1-2 or the plasmid of claims 3-4.

6. A recombinant host transformed with the recombinant vector of claim 5.

7. The recombinant host of claim 6, wherein said host is T. reesei.

8. A recombinant vector comprising the isolated DNA sequence of any one of claims 1-2, wherein said isolated DNA sequence is operably linked to the homologous xylanase promoter or to the T. reesei cbh 1 promoter.

9. A recombinant host transformed with the recombinant vector of claim 8.

10. The recombinant host of claim 9, wherein said host is T. reesei.

1 1. A recombinant vector comprising the plasmid or fragment thereof of any one of claims 3-4, wherein said isolated DNA sequence is operably linked to the homologous xylanase promoter to the T. reesei cbh 1 promoter.

12. A recombinant host transformed with the recombinant vector of claim 1 1 .

13. The recombinant host of claim 12, wherein said host is T. reesei.

14. Culture medium comprising the enzymes secreted from the host of any of claims 9-10 or 12-13.

15. A method for biobleaching, said method comprising adding the culture medium of claim 14 to wood pulp.

16. The method of claims 15 wherein the temperature is 50-80°C.

17. The method of claim 1 6, wherein the temperature is 60°C.

18. A method for chemically treating plant biomass which comprises contacting said biomass with the culture medium of claim 14 at a temperature above 50°C and a pH above 6Ø

19. The method of claims 18 wherein the temperature is 50-80°C.

20. The method of claim 19, wherein the temperature is 60°C.

21. Purified AM50 xylanase.

22. Purified AM30 xylanase.

23. A method for biobleaching, said method comprising adding the xylanase of any one of claims 21 or 22 to wood pulp.

24. A method of chemically treating plant biomass which comprises contacting said biomass with the xylanase of any one of claims 21 or 22 at a temperature above 50°C and a pH above 6Ø

25. The method of claim 24, wherein the temperature is 60°C.