Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~32'''1254Z ~ G ~ f F-4_3 T-~3 P-13e6,~54 JUL ~ ' g4 14:1~8
2129171
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~
2129171
,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~
2129171
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
2129171
-5-
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
2129171
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
-7-
~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
2129171
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
2129171
~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
-13-
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
-17-
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
-2
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
2129171
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-
2129171
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 ~
2129171
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
-2
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 ~ ~
2129171
-27-
- 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 $.2 2.2
AM~0 74.0 4.1 2.2
AM30 74.4 2.2 2.0
Control 73.3 6.8 2.z
2~0 74.9 4.2 2.1
~ e use of AhI50 and A~30 clearly incleasod t~e l";~ ss obtained
wi~out i~;,ea~ g ~e amoun~ of pero~cidc tb~t was us~d.
~mp~e 8
r~7fr r~ of the ch,r.~,Ds ~DN,4 ~nd cor;s~,~tJ,on of ~e
~e~t . . 'c ~Y
~ ctinom~ra fl~7s~1 6p. DSM43186 was eul~vated i~ 50 ml of
m~Aillrn c4~ of 0.~ % oat spelt ~ylan, 0.5 % p~ptone fronl caseiD, 0.5
yeast e~c~act, 0.5% beef extract~ 0.074~ CaCl2 ~c 2H;20, p~ 7.~7.5, ~
bafTled s~ake ~l~sk for ~.S d~ys at 52C with ~a~ing at ~OQ ~n. 2.5 ml of
~is culbLre was ll~fi llod to 50 ml of fresh medium su~plem~nted with
0.8~ ~lyc~, a~ g~own for 2 days at 50C, 200 Ipm. Cultures were
tr~rc;l~ed into SS-34 tubes, pelleted a~ 8,000 ~pm, aIld was~d with 109
I`OSe., 25m~I T~ HCl (p~ 8.0)-~SmM EDTP,.
The chrnrno~or~ N~ was isolated ~ g to E~opwood Ct al
(~netic ~a~ ulation of Streptomyce~: A ~ m~u~l, The Joh~ i~s
I~ou~ation, ~rwich, UK (1~85). Brielly, the rllycelium was lysod with
Iyso~me ~d 2 x Ki~by ani~e (2 g sodium triisoy~ n~l~h~t~ p
s~ P, 12 g sodium 4-ami~sali~ylate, 5 ml 2 M Tris-~l (pH 8.0), 6
ml of Tris-HCI ~ ~Illr~ ~-J p~wl, made u~ to lO0 ml w~t wa~r). The DNA
was pr~cipitated wi~ isopropanol and d~ssolved in~o TE. RNA was di~ested
with RNase.
The chromosomai DN~ was par~ially ~c,~ t~l ~ith 5~3A
~Bocl~ gcr) and s~æ-~ction-ted in sucrose g~adient (1~, 20, 30, 4~%
~32371--`51el 5 1~: G ~ f F-42_, T-61~3 P~ " ~351 JUL :~S ' 94 1_: 21
2129171
sucrose in 1 h~ NaCI! 20 mM ~ris-HCl, pH 8.û, ~ DTA). DNA of 7-
10 kb was usod to construct a g~ .,;c Ac~n~n~ Libra~y.
Tbe p~ediyested ZA~ E~p:ress~ Bom~lC}AP Vector Clo~ ~it
(St~atagene) was used to co~$~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~
2129171
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~?
2129171
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
