CA2132335A1 - Delignifying preparation exhibiting .alpha.-l-arabinofuranosidase activity production and application thereof - Google Patents

Abstract

2132335 9320192 PCTABScor01 A preparation exhibiting enzymatic activity comprising a substantial portion of .alpha.-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65 ·C, is disclosed. Said preparation is obtainable by aerobic fermentation of a Bacillus stearothermophilus strain selected from the deposited strain NCIMB 40494 and mutants and variants thereof. The deposited strain and mutants and variants thereof are comprised by the invention. A method of producing said preparation, a process comprising treatment of wood pulp with said preparation, and wood pulp that has been treated with said preparation, and also said preparation in association with a preparation exhibiting enzymatic activity produced by another microbial strain and having the capability of delignifying wood pulp at a temperature of at least 65 ·C and a pH of at least 9, are disclosed.
Use of an enzyme expressed by the deposited strain and .alpha.-L-arabinofuranosidase which comprises one or two specified partial sequences and homologues thereof, are also included.

Description

WO 93/201~2 213 2 3 35 PC~/SE93/00269 ~E:I.IG~ YING PREPAR~TION EXHTBITING a-L ARz~gINoFuRAlilosIDAsE
ACTIVITY, PRODUCTIOr~ AND APPLICATION q~IEREOF

¦ The invention relates to a preparation exhibiting enzymatic ~: activity comprising a substantial portion of a-L-arabinofuranO-~10 sidase activity having the capability of delignifying wood pulp at a p~ of 8 - 9 and a temperature of 65C. Further, it relates l ~o a me~hod of producing ~aid preparation by aerobic fermenta-tion of a selected Bacillus stearothermophilus strain. The invention also comprises said selected strain. Moreover, it ~15 relates to a process comprising treatment of wood pulp with a ¦ preparation of ~he invention and also wood pulp that has been j ~ trea~ed with said preparation. Additionally, i~ relates to the ,; use of an enzyme expressed by the deposited Bacillus stearother-mQ ~hiLY~ strain NCIMB 40494 in the ~reatment of wood pulp. Also, ~20 ~ ~ it relates to a-L-arabino~uranosidase having an amino acid ~ ~:
sequence whi~h compris~s one or two specified partial amino acid sequences or:homologues thereof.

BP.C~GROU~ID
:25 :;~ ~
:Due to:en~ironmental reasons, the pulp and paper industry has :sh~wn increasing interest in bleaching se~uences aLming at : de;l~ignifi~a~ion and bleaching of pulp without use of any chl~o~ine-containing compounds.
3:0 To ~hat end, many patent applications disclosing new enzymes for delignification o`f pulp'hav~ been fiIed recently. One such application is our W0'31/10724 (Swedish priority application was issued as patent on December ~.9, 1991), which i.aO claims a 35~ `~ preparation exhibiti:ng enzymatic acti~ity and haYing the capabili~y of delignifying wood pulp at a temperature of at leas~ 65C and a p~ of at ~east 9. Said preparation is obtain-able by aerobic fermentation of a Bacillus stearothermophilus strain having the cap~bility of producing said preparation, such ., ~

'3 ~
' ~:

W~93/2~1~2 3~3 PCT/SE93/00269 ~s one of the two ~eposited strains NCIMB 40221 or NCIMB 40222.
Purification of the enzymatic activity resul~ed in a xylanase of MW 41 000 - 42 000 Da.

Efforts were made to isolate another strain of Bacillus stearothermophilus which could be used for purification of other delignifying enzymatic activities than xylanase activity. It was en~isaged that e.g. an a-L-arabinofuranosidase being capable of delignifying wood pulp at a high temperature and a high pH would be an additional useful tool in the de~elopment of a bleaching sequence without any chlorine-containing bleaching chemicals.

Recently WO 91il8976 (filed by Novo Nordisk A/S) was published.
Claim 14 thereof :is directed to an arabinofuranosidase enzyme produced by Bacillus stearothermophilus. On page 9, lines 11 and 12, is stated ~hat the literature does not contain any reference for an arabinofuranosidase from thermophilic Racillus. Even though WO 91i18976 claLms an arabinofuranosidase enzyme produced by Bacillus stearothermophilus, it fails to disclose how it can be obtained.

Three strains have ~een deposited, two isolated and one mutated.
However, none of these have been shown in said application to produce an arabinofuranosidase.
~25 The~only strain .shown to produce an arabinofuranosidase is a non-deposited ~utant strain designated BPS-3-H-17-4. It should further be mentioned th t the preparations used in the experLments shown in the application (cf. top of page 17) have been conducted with fermentation products obtained from said non-deposited strain. On page 8, lines 12-16 is disclosed that said strain derives from one of the deposited s~rains after l~ mutagenesis with ethylmethansulfonate. This mutagen is very I unspecific and no conditions of how to arrive at an arabinofura-~35 nosidase-producing strain is given. Thus, there is still not .

, . -I WO93~20192 213 2 3 3 3 PCT/SE93/00269 " ... .

disclosed in the a~t how to obtain an arabinofuranosidase from l a thermophilic Bacillus.
,'1 DESC:RIPTION OF TEIE INVE~TION
The present invention provides a preparation exhibiting enzymatic activity comprising a substantial portion of ~-L-arabinofuranosidase activity having the capabality of deligni-fying wood pulp at a pH of 8 - 9 and a temperature of 65C.
. 10 In the present speci~ication and the appended claims the J
expressiQn ~wood pulp" is to be interpreted broadly, and thus it is intended ~o comprise all kinds of lignocellulosic materials.
One aspect of the invention is directed to a preparation : exhibi~:ing enzymatic activity, which is ohtainable by aerobic fermentation~ in a suitable medium, of a Bacillus stearother-mophllus~s~rain selected from the strain NCIM~ 40494 and mutants :20 and variants ~hereo having substantially the same capability of~producing s~aid preparation as said strain ~CIMB 40494, the enzy~latic activity of said preparation comprising a substantial porti.on of a-L-arabinofuranosidase activity having the capabili-ty o~ d~31ignifying wood pulp at a pH of g 9 and a te~perature ~:25 of 6_i C.~

In one ~mbodiment of this aspect of the invention the prepara-tion~is~:a cl~rified culture broth. In another embodiment of this ~ , ~
aspect of the invention the preparatiQn is a concentrated fractlon of said culture broth exhibiting a-L-arabinofuranosida-:se activity in a wood pulp medium. In yet another embodiment of this aspect of the invention the a L-arabinofuranosidase activity derives from an a-L-arabinofuranosidase which is composed of ~wo sub-units ha~ing the approximative molecular 35~ w~igh~s of 52 500 ~a and 57 500 Da, respectively.

.~,i,, ~ , ~ . .
, . . .

':~', WOg3/~0192 PCT/S~93/002~9 ~3233~ 4 t~

In another aspect of ~he invention the preparation according to the above disclosed aspect of the invention is in association ' with a preparation exhibiting enæymatic activity produced by .~j another microbial strain and having the capability of deligni-'~'1 S fying wood pulp at a temperature of at least 65C and a pH of at lea~t 9~ In a specific embodiment of this aspect of the . in~ention the preparation comprises an a-L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40494 and a xylanase produced by the Bacillus _tearothermo~hilus :
strain NCIMB 40222.

$ One additional aspect of the invention is dires~ed to an a L-: arabi'nofuranosidase having an amino acid se~uence which comprises the ~-terminal partial amino acid sequence SEQ ID
;~ 15 NO~
Met? Gln Pro Tyr Arg Pro? Glu Glu ~eu j~ or a h~mologue thereof.

Another additional aspect of the in~ention is directed to an ~ ~a-L-arabinofuranosidase ha~ing an amino acid sequence which comprises the N-terminaI partial amino acid sequence SEQ ID
NO: 2: , Ser Met Lys Lys ~la Thr Met Ile Ile ~lu ~ys A~p Phe Lys Ile Alà Glu Ile Asp:Lys~Arg Ile Tyr ~ 25 ~or a~homologue ~hereof.

',~' ::In the~:context of "an a-L-arabino~uranosidase having an amino ; acid sequence which comprises the N-terminal partial amino acid sequence SEQ ID NO: 1:
Met? Gln Pro Tyr Arg Pro? Glu Glu Leu ~:. or a homologue 'thereofl~ and/or ~an a L-ara~inofuranosidase' !~, '~ having an amino acid sequence which comprises the N-terminal ,' ~ partial amino acid sequence S~Q ID NO: 2:
;~ ~ Ser Met hys Lys Ala Thr Met Ile Ile Glu Lys Asp Phe Lys !.'~ ~ 35 Ile Ala Glu Ile Asp Lys Arg Il~ Tyr or a homologue thereof", such a homologue is intended to have an amino acid sequence which, in relation to the sequence SEQ
, . ~

2 213 ~ 3 3~ PCT/SE93/00269 ID NO l and /or the sequence SEQ ID NO: 2, has some amino acid subs~itutions, Pxtensions or deletions which do not lead to the elimimation of the a-L-arabinofuranosidase activity of the entire enzyme/ e.g. in a wood pulp medium, preferably at a temperature of 65C and a pH of 8 ~ 9. Further, such a homologue may e.g. have a sequence which has 80% or more amino acids in common with the sequence SEQ ID NO: 1 and /or the sequence SEQ
ID NO: 2.

An example of a homologue of the sequence SEQ ID NO: 2 is the sequence SEQ ID NO: 3:
Ala Thr Lys ~ys Ala Thr Met Ile Ile Glu Lys Asp Phe Lys Ile Ala Glu Ile Asp Lys Arg Ile Tyr Gly Ser Phe Ile Glu His Leu Gly Arg Ala Val Tyr Gly Gly Ile Tyr Glu Pro Gly His Pro Gln Ala Asp Glu Asn Gly which is the N-texminal sequence of an a-L-arabinofuranOsidase produced by the Bacillus stearothermophilus strain NCIMB 40222.
Said a-L-arabinofuranosid~se is composed of two identical sub-: 20 units~having an~approximate molecular weight of 64 000 Da each ::~ (the native enzyme, 128 000 Da as judged by SDS gel).

Also, a :homologue of ~he sequence SEQ ID NO: l and /or the s,equence SEQ ID NO: 2 is any amino acid sequence which is su~ficiently homologous on the nucleotide le~el to be recogniæed by any DNA or RNA probe derived from said sequencets).

et another aspect of the invention is direc~ed ~o a method of producing a preparation exhibiting enzymatic activity, whereby a Bacillus s~earothermophilus strain selected from the strain - NCIMB 404g4 and mutants and variants thereof having substantial-ly the s~me capability of producing said preparation as said strain NCIMB 40494, is subjected to aerobic fermentation, in a suitable medium, the enzymatic acti~ity of said preparation ~ 35 comprising a subs~antial portion of a-L-arabinOfuranOsidase ! ~ activity having the capability of delignifying wood pulp at a ~ pH of 8 9 and a temperature of 65C.

.~,, , ',;~
,, Still another aspect of the invention is directed to the isolated Bacillus stearothermophilus strain NCIMB 40494 and mutants and variants thereof, said mutants and variants havlng substantially the same capability of producing a preparation exhibiting enzymatic activity as said strain NCIMB 40494, said enzymatic activity comprising a substantial portion of a-L-arabinofuranosidase activity having the capability of deligni-fying wood pulp at a pH of 8 - 9 and a temperature of 65C.
A furthex aspect of the in~ention is directed to the use of an enzyme expressed by the B. stearothermophilus strain NCIMB 40494 for treatment of wood pulp. As is evident from Table 8 of the specification, said strain produces several types of extra-1~ cellular carbohydrate-degrading enzymatic activities which ~ derive frsm the expression of different genes comprised by the I genome of said bacterium.

¦~ Even ~hough these different activities ha~e not all been in~estigated separately yet, it is believed that ~he different activities, separately or- in different combinations, will be useful in ~he treatment of wood pulp.

:
An addit.ional aspect of the invention is directed to a process comprising treatment of wood pulp, whereby wood pulp is treated in at least one step with a preparation according to the r!~ invention. In one embodiment of this aspect of the invention, the wood pulp to be treated is sulphate pulp. In another embodiment the sulphate pulp to be treated is a partially 1 30 delignified sulphate pulp. In yet another embodimen~ of this aspect of the invention the partially delignified sulphate pu~lp to be treated is an oxygen-delignified sulphate pulp.
i ~, A further aspect of the invention is directed to wood pulp which `~35 has been treated in at least one step with a preparation - according to the invention.

,,~

,, .
i,,~, .,= . ~ .

. .
i, , ' WO93/20192 2 1 3 2 3 3 5 PCT/SE93/00269 ; 7 ., ~ 5till ano~her aspect of the invention is directed to a DNA or ..~
~ RNA probe which recognizes the nucleotide sequence coding for :~ the amino acid sequence S~Q ID NO: 1:
:~ Met? Gln Pro Tyr Arg Pro? Glu Glu Leu -~ 5 and~or .' the amino acid sequence SEQ ID NO: 2:
Ser Met Lys Lys Ala Thr Met Ile Ile Glu Lys ~sp Phe Lys Ile Ala Glu Ile Asp Lys Arg Ile Tyr.
,'''~ .
The last mentioned aspect of the invention i5 useful in the ~, finding of a-L-arabinofuranosidases having the capability of ., delignifying wood pulp at a temperature of at least 65C and a .~ pH of at least 8 - 9.
."~
l~i DEPOSITIOl~ OE' MICROORGANISMS

The Bacillus sterothermophilus strain L1 was deposited under the ,:.;
: Budapest Treaty at the National Collections of Industrial and Marine Bacteria Ltd ~NCIMB), Aberdeen, on March 24, 1992 and was ~5 20 given ~he accession number NCIMB 40494.

The ~acillus stearothermophilus strain T-6, given the accession v~ number NCIMB 40222 by the same depository, was deposited earlier `~ in connection with the filing of the priority application for `, 25 WO 91/10724.
::, Isolation of thermostable a-L-arabinofuranosidaSe producing .1 Bacillus stearQthermophilus, strain Ll .A. Preparation of alkaline extracted pulp ~AKP)o Suspensions of K15, oxygen semi-bleached soft wood sulphate pulp, (6-10% dry weight fiber) in 0.02 N NaOH (pH 11.7) were ~: incubated at 60-62C for 18-20 hours. While still hot, the s~ 3~ fluids were separated from the fibers using a sinter-glass - funnel under suction. T~e resultin~ solution was neutralized to ~ p~ 7.0 and concentrated by ultrafiltration (10,000 M~ cut off).

~, ;
ijf~'", .. ..

S

8 t The retentative (AKP-l) contained 5.2 mg/ml lignin1 l.85 mg/ml carbohydrate ~by phenol-sulphuric acid and 0.83 mg/ml pen~oses (by orcinol).
.
S B. Enrichment culture.

AKP-l was supplemented with salts (0.1% MgSO4.7H20, lQ mM K2HPO4, O.l~ urea and trace elements) and inoculated with mud and water samples taken from the water treatment pools at Korsnas. The flasks were incubated with shaking at 65C and pH 9.0 for 48 h.
A drop of turb.id culture was ~hen inoculated into a flask containing l5 ml of 0.2% galactomannan (Locust bean gum, Sigma) medium. A~er incubation for 24 hours at 65C and pH 9.0, the cul~ure was streaked onto LB agar. A~ter incubation, approxima-tely 20 different colony ~ypes were isola~ed and obtained in pure~culture. ~fter preIiminary testing, one of the strains was studied in de~ail: strain Ll that contained a thermophilic ~~L-arabinofuranosidase.l 20~ Strain~Ll was~a Gram-positive, thermophilic bacterium; it grew wéll at 60-65C, but;did not srow at less than 40C. It was rod-shaped~wlth a terminal spore, aerobic and oxldase-positiva.
Strain;~-Ll grew ~n ~the following carbon sources: D-glucose, D
xylose, L-arabinose, D-mannose and xylan. It did not grow on 2S~ melobiose~and galactose as carbon sources. Base~ on these properties Ll was classified as a strain of the heterogenous group,~;~Bacillus stearothermophilus.

Initial ex~riments (with X15 pulp) 30~
C. Thermophilic a-L-arabinofuranosidase from strain Ll Strain~Ll was selected for further study because the crude extracellular fluid of culture grown on galactomannan medium was

3~5 ~ ~ active on delignification of Kl5 pulp. For the estimation of how much liqnin is made extractable by the enzymatic prepara~ion, a sensltive spectrophotometric assay was used. The absorbance , ~, WO93/20l92 PC~/SE93/00269 ~ :

values at 350 nm(A350) were measured on the supernatant liquids of the sample~, and measured values are presented as % lignin released. The growth a~d del1gniica~1.on ac~ivity o~ s~rain I.l grown on diferent carbon sources is summarized in Table 1. The S strain grew to some 0x~en~ on ~he y0ast ex~rac~ and casamino aci~s wikhout addi~ion o~ another carbon source and yielded a net deligniica~ion o 5.1~. ~ddi~ional gxo~h wa~ obs~r~ed with all the sugars added excep~ the galac~o~e, whlch a~o inhibited the production of deligni~ica~ion ackivi~ he bes~ dollgnii ca~ion a~i~itios wer0 ob~a~ned on cul~ures grown on xylose and arabino~e, 7.1~ and 7.6%~ respectivel~.

The extracellular enz~ma~lc ac~i~ities o~ ~train ~1 g~own on xylose and arabinose media are su~mariæed in Table 2. ~ylanase activity was determined ~y incubating a fresh s~lution of xylan with extracellular superna~ant 1uid and as~a~ing or inc~ease in reducing sugars by khe erricyanld0 method (Spi~or R.G. 1966, Methods in Enzymology ~: 7-9)~ Assay buer was S0 mM ~ris. Cl, pH 7.0 and 0.5~ x~lan (oa~ spe~ts, Sigma). Uni~ of actl~ity or xylanase are ~mol reducing sugar genera~ed pe~ minut~ at 65C.
', The arabinofuranosidase assa~ is a modiication o the standa~d assay ~or ~-galactosidase (G. ~abolt and Syguson, Appl.
E~viro. ~icrobiol, vol 56, No. 11/ 1990). Test ~ube contains 0.5 2~ ml of 40 mM T~is-buffer, pH 7Ø 0.4 ml o the enzyme sample and 0.1 ml 10 m~ p-nitrophenyl-a-L-arabinofuranoside (Sigma Chemicals) was incubated at 65C or lS min. The reac~ion ~as terminated by pu~ting the test tube in ice water bath. The release of p-nitrophen~l is ~etermined spec~rophotome~rical~ at 401 nm. 10 micromole of p-nitrophenol per ml has an absorbance of 18.4. A unit of enzyme acting is dèfined as micromolèiof p-nitrophenyl release per min.
.
On xylose medi~m, only two`activities were de~ec~ed: 1.3 units ~ 35 per ml xylanase and 0~004 units per ml of a~L-`arabinOfuranO
:~ sidase. There were no de~ec~able a L-arabinopyranosidase, mannanase, ~-D-galactosidase or a-~-mannosidase activities. It `

WO 93/2019t 213 2 3 3 ~ P~/SE93/00269 appeared, therefore, that the delignif.ication activity of Ll was due to ~ylanase activit~ when ~he cells were grown on xylose medium. Howe~er, when the cells wer~ grown on arabinose m~dium, the major ~ctivi~y was a-L-arabinouranosidase (0.5 units per ml). ~t ~ppeared, there:~ore, tha~ the a~L-arabino~uranoSida~e wa~ x~sponsibl~ for ~elignlPica~ion in ~-medlum.

~he a-~-arabino~uranosida~ F) ackivi~y wa~ concenkrated rom a ten liter culture o~ Ll g~own on ~ mcdium (T~ble 3)~ The acti~ity was precipi~aked wi~h ~ a~ura~0d ammonium ~ul~ate, yi~lding a crude enzyme prepara~ion ~th 11 uni~s per ml AF and 0.96 units per ml o x~anase. ~h~ ~F ac~ y dld no~ bind to carbox~l~me~hy~ cellulose ~MC), bu~ did adsorb comple~ely ~o D~AE cellulose or DE~ ephac01.
'rhe concentrated ~F demonskra~ed signi~ican~ deligni~ication ac~i~it~ (Tabl~ 4). ~t should be poln~ed out tha~ we have not yet optimiæad the conditions ~or using ~F to dellgniy pulp. A
pot~ntially use~ul properk~ ~ AF ls tha~t it should break the bond ~lose ~o lignin, ~h~reb~ lea~ing mos~ o~ th~ h~mi-cellulo~e with thQ cellulose ibers. This shou~d gi~e a high~r yield o~
deligni~ied pulp. In addition, i~, appears ~hat the deligniica-tion acti~ity of ~F plus x~lanase T-6 is more ~han addi~i~s~ :

~5 ~ables 5-7 describe some of the enæymatic proper~ies of ~he puri~ied AF~ using PNP-a-~Ar~ as subs~rat~. The enzyme is mos~
acti~e between pH 6.5-8.0, with an op~lmum a~ pH 7Ø The enzyme has low activity at pH 9.0 a~ 70C~ The temperature op~imum was : 70~C at both pH 7.0 and pH 8Ø The enæyme was most active at 20 m~ Na2S04, pH 7.5 in 10 ~ phosphate bu~er at 70C. At pH
7~0, t~e enzyme was ver~ stab~e at 6~C, but lost about 50~ of its activity at 70C durin~ 75 min and was completely i~acti-; vated at 80C for 15 min.

3g .
` ' ' WO 93/20192 213 ~ 3 35 pcr/sEs3/oo26s Extended ~cPer~L~y~u~L

~3xtracellll~ar c:arb~h~rdrat~-degr~din~ enzymaki :: acti~ritlt3s o Bacil~uS stear~h~b~ - Ll Sp~ci~ic extrac~llular carbohydra~0~-degrading enzym~t:ic acti~ities w~re t:ested ~c~.~lowing ~row~h in dierent media ~Table 8 ) . The cells wer~3 grown ~n di~e~nt c~rbc:1n sources, namely: p~ni~xophenyl-~D ~m~nnc: p~rrano~ld~3 ~ p-ni~rophenyl~ D~
galactopyran~ide and p-ni~rophen~ a~binop~rranoslde, Xylanas~ and two other endohemicellula~s ~manna8e an~ arabina-nase) were assayed in 10 ~ pho~phate bu:ee~, pH 8.0 a~ 60~C in 1 ml total assa~r ~rolurne. .~n approprla~6~1y dilu~ed en~yme sample ~ O . 1 ml ) was added to 0 . 25 ml subs t~a~e, O .1 ~1 100 xnM buer and 0 . 5S ml wa~r . The reaction was terlnina~Qd b~r tran~ ~erring t~ an ice wa~er bath~ Reducing suga~ was de~errnined b~ 3, 5-dlni~rosalicylic acid method as d0scribed b~r ~iller ~illex, GL, ~1959 ) Anal Chem 31: 426-4~8 ) . The ~ubs~ral:;es were 4% Oa~ sple~
xylan, 0.~ Locus~ Bean Gum-g~lactomalman, 4~ arab~nogalactan rom Larchwood and 4% mannan fro~n ~,~:~ ~i~

The enzymatio release of lignin ~rom .so~wood pulp was determi-ned by adding 200 mg wet w~igh~ o K-20 pulp ( ~om the Korsn~s paper mill Gavle, Sweden) to 3 ml o enz~ne solu~icn, ad~us~ing to pH 8 . 0 or 9 . n with a conc0ntrated NaOH solution, and incubating at 65C with shaking. A.tsr 2 h, 1.5 ml o~: the lic~uid : was remo~ed and centxi~uged at lO,OOO x g or 5 min in a minifuge ~o xemove residual pulp ~ihers. The cleax liquid ~0.5 30 ~ ml) was dilu~:ed with 1.0 ml of~ 0.1 N NaOH ~or determination of absorbanc~ a~ ~50 nm. The control or each assay was incu~ation of the pulp under the same conditions without ~nzyme. Since 1 mg lignin per ml had an ~350 of 9.1, and pulp used in ~hese experiments had a lignin content o 1.3~ ~Klason, determined by ~3:5 Z. osi~, the % lignin released = ~350 X 3 -- x 1 0 0 P x 0.013 I , .

1 . .

wo 93/201g2 ~3~33~ PCr/SE93/00269 where P is ~he pulp dry weigh~ and h~350 is the ahsorbance after incubation minus abserbance before incuba~ion. ~h~ net lignin released is ~he ~otal minus ~h~ no enzyme ontxol.

The ~wo signi~icank acti~iti~ ~hat were ound ~exe x~lanase a~d a~~arabinofuxano~idas~, Xylana~e wa~ f~und ln lo~ concent~ation when the carbon sources in ~he gr~w~h media wer~ locus~ bean gum (LBG), D-glucose and L-arabin~a. ~he x~lanase ac~ was ampliied wi~h D-~ylos~ as ~he carbon source, reaching 1. 23 ~/ml. ~anno~ inhibikad produc~ion o x~lanase ac~ivi~y. -L~
arabino~uranosidase ~c~ was ~ow, but signiican~ on ~-xylo~e medium and high on ~arabino~e me~ium, x0aching 1.5 U/ml.
No ac~i~itieæ o~ manna~e, ~-D~ga~actosida~e, ~D-mannosidase or u-L-arabinopyranosidase were ~und.
1~
Purlication o~ arabin~rano~ldas0 ~rom ~alll~ s~axo-~h~.

Fol}owing growth of ~LL~g~ ~u~:K~D~o~oh~lu~ L~ ~or Z4 h a~
0 60C on an ~-arabinose containlng medium, ~he cell ~e~
extracellular 1uid con~aine~ 1.2 U/ml o~ a-L-a~abinofurano-sidase activi~y with a speci~ic a~ivity of 1.7 U/mg (Table 9).
Prel.iminary experiments in~ica~ed ~hat the an~me could be concentrated by precipi~a~ion at gO% ammonium sulate satura-~25 tion. Howe~er, there was onl~ a 68~ reco~e~y o ac~i~ity and essentially no increase in ~peciic acti~ity. Thus, ammonium sulfate precipitation was r.~t u~ed, and khe crude enzyme was : adsorbed directly to a DEAE Sephacel c~lumn ~Table 9, anion ;~ exchanger)~ After rinsing the column with 10 mM potassîum phosphate buf~er, pH 8.0, elu~ion was pe~ormed with a linear gradient from 0.1 to 0.9 M NaC1. The a-~-axabinofuranosidase activity eluted as a sharp peak be~ween 0.53 and O.S7 M NaCl.
Following concentra~ion and ~esalting o~ the ackive ~ractions by dialysis against polyethylene glycol, ~he specific acti~ity increased 38~fold with lO0~ reco~ery of the acti~ity. This ` material was applied dixectly to a Sephadex G-lO0 column. The ;~ : active material eluted as a single sharp peak with an apparent .
~ . .
`;"
` ~

WO93/20l92 213 2 3 3 ~ PCT/SE93/00269 .~

molecular weight o 108,000. The ov~rall purifica~ion was 59 kimes with a yield of 80%. The purifiecl enzyme was examin~d by FPLC Superose 12 gel ~ ra~ion in 100 mM potassium phosphake buffer, pH 7.0, and 100 mM NaCl. O~er 95% o ~h~ ac~i~ity and prokein elu~ed as a single sharp peak wi~h an apparent molecu}ar ~eigh~ of 114,800. B~ SDS P~F,I the puriied enz~me showed two bands with molecular weights o 57,500 and 52,500. ~nalysis of these ~wo bands reve~led, a~er blo~ing on P~DF membrane and sequencing on an ~pplied Bios~q~ms model 475~ gas phase gequencer/ the two N~te~m~nal ~equenc~s S~Q ~D NOs 1: and SEQ
ID NO: 2.

Charac~erixati~n o~ ~9111Y~ ~ Ll a~L-a~a~ino-uranc)sldase.
At pH 7.0, the puxîied en~yme was comple~ely st~ble a~ 60~C ~or at least 80 min, retain~d 50~ o~ i~s maximum acti~ity after incubation at 70C ~or 75 minl an~ lost all i~s ac~ y af~er 15 min at 80C. A~ 70C, the optimum pH or ac~ y was 7.0;
::20 a~ pH ~.5 and pH 8.0, ~he acti~ s w~re 55~ and S0~ o ~he optimum acti~ity, respec~ively. ~ pH 7~0 and pH 8.0, ~he optimum temperature ~or acki~i~y was 7 0 C . Th~3 enz~me showed maximum activi~y in 20-50 mM Na2SO4; a~ lOa mM and lSO mM Na2S04, the acti~i~y de~reased 10~ and SO~, xesp~c~ively.
: :

The kinetic parame~ers o~ the en~ 0 were measured using pNP-a-L ara-~ as the substrate . At p~I 7 . 0 and 65C, Km and Vmax were 2.2 x 10-4M and lOl ~mol min~1mg~1, resp~c~i~ely. The enæym~
showed only low acti~ity on high molecular w~lght substrates, such as arabinoxylan and arabinogal~ctan. . , .
Delig~ifica~lon ac~i~ity of BaciLlu~ s~eaEothermQPh~lus L1 a-L-, ~ arabinofuranosidase~
, ~ .
3$ ` During ~h~ purification o~ the enzyme ~Table 9), column eluents `:3 were examined routinely for delignifiation activity using semi-bleached Kraft pulp as the substrate. Delignification activi~y ..~ , :~.
.`'`1~ ~ .
~;~
;~. 1 . ~

WO93/201~2 2 1 3 2 ~ 3 ~ PCT/~93/00269 was associated with (a) the peak o~ a-L-arabinofuranoSidaSe activity, (b~ the peak o a lower mol.ecular weight endoxylanase activity and (c) fractions which con~ained low amounts of both activities. Since ~he highest specific delignification activity S occurred when both enzymes were present, it was decided to e~amine the possible synergistic acti~ities of khe a-L-arabino-~ furanosidase and a previous purified heat-stable xylanase T6 j discl~sed in WO 9l/lO724.

Table lO summarizes a typical axperimen~ in which deligniica-tio~ acti~ity was examined wi~h pure a-L-arabinofuranosidase~
pure xylanase and a mix~ure ~f th~ ~wo enzyme~. At pH 8.0 and 65C or 2 h, the mixture o 38 U/ml -~-arabinofuranosidase and 5 ~/ml xylanase T6 released a net of l9.2% o~ the lignin from the pulp/ whereas the sum o~ each enzyme acting separately was only 16.S%. To achieve 16.5% net release of lignin using only xylanase ~6 under these con~itions required 50 U/ml ~f ~he ~ enzyme. At pH 9.O an~ 65C for 2 h, the mixtuxe o:~ enzymes `~ released 18.~ lignin, compare~ to only 13.7~ for the sum of the:~20: two ac~ivities acting separately. Clearly, the ~wo enæymes acted . synergistically in releasing lignin from the pulp.

An apparent paradox is:that the a-L-arabinofuranosidase enz~me : sh~wed lèss than 1% of its optimal~acti~ity at pH 9.0, u~ing the ~25 model ~ubs~rate P-nitrophenol:a-L-arabino~uranosidase. However, in the bîobleaching process, khe enzyme was almost as eective ; at pH 9.0~as at pH 8Ø It is possible ~hat ~he pulp somehow ~:: :: protected and conserved the enzyme activity at higher pH values.
These results are encouraging since ~he indus~rial enzymatic 3~ deligni~ication process is more easily performed at pH 9.O and 65C than at lower temperatures and pH values.
: : .

WO 93/201~2 21 3 ~ 3 35 P~/sE93/oo269 , Table 1 ~rowth and del~gni.:eicatic)n ac~i~r.ity o:f s~rain Ll on dif~erent media .

Growth l)eligrli~icatic: n ( ~ ) b S:ar~on Sourcea ~l~s~io) Tok~l Net None ().73 7.1 5.1 :1~ 10 0.2 LB 1.30 8.8 6.8 0-2 ~annose 1.83 8.0 6.0 0 ~ 2 Galac~o~e 0 . 84 4, g ~ 9 O . 2 Glucose 2 . 62 8 . 9 ~ ~ 9 ;~ 0.2 X~ s~ ~,04 9.1 7.1 0 . 2 A~raJ~ino3e 1 . 81 10 . 5 7 . 6 ~ w ~
,~ ~ a q!he growth m0dia cc~rlsi~e~ ~ carbon source ( O . 2~ ), O . 1%
~ ~ ~ yea~t exkr~ ::k I O . S~ casamirlo aclds ~Isd E . ~alts ( O . 1 urea, 0-0296 Mg504~7H20, SmM KH~Pt:4, pH 9.0, ~0 mM Tris HCL, ; ~ 20 pH 9~0, 0.1% NaCl ancl ~andard ~race el~3merl1:s mixture).
11 o the s~gars were ~he ~-conigu~ation, excep~ or L-araibinos~3 7 ~R is locust, bean galac~omannane J
b I:)eli~ni~icatic?n o ~15 was carrled out with ~he ex~ra-- cellula~ supernatarl~ (adjus~ed to p~ 9.0), at 65C for 2h.

~, ~ 1 "
,~i -,~' ` ~ ~ :' ` :

'~3:

WO 93~i!0l92 ,; . PCr/SE93/00269 " , .. ~ , " ~ ,. .
213233~ 16 Ta~le 2 Extracellular enzyrnatic ac tivities o~ Ll " ` .

.~ 5 X-medium~ A~mecliumb Substra~e Act . ~ ~/ml ~ .ct . ( U/ml ) "~
X~lan ~,3 0,05 PNP-a L~AF 0 . 00~ ~ 5 PNP-a~Gal c O ~01 ~; PNP-a-Man c O . 001 ,~l PNP-a ~P c Q ~ 001 ~-LB~GM ~ 0 . 001 y 15 a Xylan and L~3-G~ ~ locu~ b~3an galac~omannan ) degxading activi~;le~ ~ere de~errnin~d b~ productiorl ~:e reducing ~u-gars and PNP-X ac~ ie~ were mea~ured by liibera~ion o~
!
PNP (p-ni~roph~nol ) ~
b Cell~ were ~rown or ~4 hr~ in ~3lther X-meclium or ~-medium ylose or arabinose medium descrlbed ln Table 1 ~ and cen~rifuged ~o remoY~ ~e cells.

~ `~l bl~ 3 ^'; ~ 25. ~reliminaxy purifica~ion of a~ arabinourano~idase ~rom Ll ; grown on ~-medium :

: ~ ~rol Pro~ina P~F ~ylanase ~:30 Fraction (ml ) (mg/ml ) ~U/ml ~ (U/ml ) : -- ------~ ----~__________~
Crude sup~rna~ant3100 1.11 0 . 82 û . 03 ~; `~:~ ( N~I4 ) ~sO4 ppt~ O û 13 . 3 11 . 0 û . 9 6 DEAE -Sephacel 20 41.1 46 . 6 0 . 09 ~ 35 ;;`~; a Protein dete.rmi~ed by BioRad~

j4 ~ ' `' ~ ' , : ' '' WO93/20192 2 1 3 2 3 3 ~ PCT/SEg3/0026g ; 17 i; Table 4 D~lignification of K15 by concentrated L-arabinofuranesidase % Lignin releasea Enxyme ~rotal Net ~i (20 ~/ml)b 8.6 3.0 ylanase T-6C (5 U/ml) 11.3 5.7 A~ -~ Xylanase T-6 16.1 11.5 :~", ~
a 10 mM phosphate bu~fer, p~ 8.0, 65C, 2h.
b Concentr~ted by DE~E-Sepharel chromatography; contains 0.1 U/ml xylanase.
~15 c Xylana~e T-6 is produced by NCIMB 40222, disclosed in WO
91/10724, and is capabl~ of delignif~ing wood pulp a~ a pH
of at least 9 and a temperature of a~ least 65C o ~ ``~
i~ ~20 ~ . Table 5 ~ Efect of te~perature on the activity of AF~at pH 7 and 8 s~ ~ ~ .
TemperaturepH 7 pH 8 ~2~5~ C ; ~ AF (Il/ml)a AF (U/ml~)~

: ~ 2.1 1.0 5~0`~ ~ ~ 3 5 2 ~5 : : 10.1 7.6 ;;~ 75 ~ ~ g.~ 7O5 80 ~ ; ; 4.2 ~ ~ 1.0 9:0 : 0.8 3 ~35~ lO0 ~ : ~ o o ~ ; ~
~ a~ - In 20 mM Tris buffer.

WO 93/20192 ~ ~ 3 2 3 3 ~ pcr/sE93/oo269 ., '. 1~1 Table 6 E~fect: of s~lt concen~ration on th~ activity o~ AF.

S ~
q ~rnM) AF ( V/ml ) 0 3.1 1~ ~0 3 . ~
~.4 1~0 ~ ~
150 1 . ~

a In ~0 mM phospha~e bu:e~r, p~ 7.5, a~ 70~.

:~ ~ l'able 7 S~ability o~ ~-L-~rabinouxan~idase (AF) activity.
~20 R~aGtion- ~Fa A~a ~,p d time acti~rity ac~ r act i ~rlty (min ) ~ U/ml ) ~ V/ml ) ( U/ml ) ~;~ " 2 7 O 7 6 . 7 5 . 0 ;~ 5 7 . 5 7 . 0 :: `
~ 15` 8~4 7.0 8.D~ 6.7

4 5 1 7 . 5 7.~ 4.2 8.0 2.7 ~ . .
~ ~:35~: ~ a In 20 mM Tris ~ pH 7 . O .
.

.. ...

:

W093/20i~2 PCT/SE93/0026g Table 8 Extracellular carbohydrate-degrading enzymatic activities o~
Bacillus stearothermophilus Lla _______________ _______________ _____________________________ ;~ 5 Carbohydrate subskrates used f~:
:~ __,__ _____ _______________ Growth Enz~rne Enzyme Ac~i~7ity : ac~i~ityb type (U/ml) ~10 ___. ____________________~______________________________~______ ~ LsGC LBG Mannanase ~0.01 il LBG Xylan Xylanase 0.136 LBG pNP-a-~-gal-p a-D-galactosidase 0.008 D-Glucose LBG Mannanase <0.01 D-Glucose Xylan Xylanase 0.319 , D-Xylo~e Xylan Xylanase 1.2S
D-Xylose LBG Mannanase <0.0 20X-Xylose Axabinosalactan Galactanase/arabinosidase ~0.01 D-Xylose pNP-a-D-gal-p ~-D-Galactosidase ~0. aol :D-Xylose pNP-~-D-man-p ~-D-Mannos:ida~e ~0.001 . D-Xylose pNP-a-L-ara p ~-~-arabino-pyr~ osidase ~0.001 D-Xylose pNP-~-L-ara-f a-L-arabino-furanosldase 0.01 D-~annose Mannan ~ Mannanase ~0.al -Mannose Xylan~ Xylanase : ~0.01 ;~ D-Mannose LBG Mannanase/gal~cto~idase ~0.01 : D-Manno~e pNP-~-D-Man-p; ~-D-Mannosidase ~0.001 L-Araninose ~rabinogalactan a-L-arabinosidaSe ~0 . 01 L-Ara~inose Xylan ~ Xylanase 0.11 :~ L-Arabinose pNP-a-L-ara-f ~-L-arabino-uranosidase 1.5 ; ~: ~ ___ ________ ~,_______ __________________ _____~_________ _____ . . ~: :
~35~ ; a Cells were grown for 24 h at 60~ and pOH 98.0 iII E salts c~ntaining 0.1~ yeast:extract, 0.1% casamino acids and t,he : carbon source~at 0~2%.
; ~
b ~ ~ After incubation ~or 24 h, the culture was centrifuged and ~4~0 . the extracellul,ar~carbolhydrate-de~rading enzymes assayed as described in the specification.

: c ~LGB is~Locust Bean Gum, a galactomannan; pNP is p-ni~ro-pheno:l. :
.~45 .3~

WO93/20192 j PCT/SE~3/0026~
.~ 213233~ 20 ,, Table 9 :Summary o~ ~-L arabino~uranosidase puriication pxocedure -arabl~ouranosi~dase '.-1 S Purification ~olume Pro~ein (U/ml) Sp.~c~.
st~p (ml) (mg/ml) (U/mg) ~ec~y :- ~
,l ~rudea 1~00 0~70 ~.21 1.72 Anion ~xchan~0xb 4.S 5~ a 377 65 100 ~;l ;lO Gel filtra~ionC 18 0.74 75.6 lal 80 ~ _ ~ ~
~,~ aA 1.4 liter cul~u~e ~ skrain Ll was prepa~ed as described ~ . in ~he specificatlon. ~he culkure was centri~uged ~or 30 i~; min a~ 10,000 x g. T~e superna~ant ~lu.~d was khen pas~ed ~ 15 ~hrough a a. 8 ~m ~ilter ~o r~mo~ residual c~lls . ~his s`'j crude sup~3rrla~axl~ was t~e s~arking ma~rial or the puri~ication procedur~.

~: ~ bDEAE ~eph~c~l column ollowed. by dialysi~ agalnst pol~-20; ethylene gl~rcol.

Sephadex G-100.

.~

~; `

~!' i~` : ~

.1.' ~ :
'~: ~: ~
~; ~

''` '~ : `

~ WO93/20192 PCT/SE93/00269 21 ~233~

Table 10 Delignification ac~ivity of ~-L-arabi.nofuranosidase and xylanase T6 on sem~-bleached pulp SD01iq~.~$i~ o Enzym~a p~I To~al Ne~

No enx~me 8.0 5.3 ¦ No enz~m~ 9,0 S.2 ~10 a-L-ar~binofuranosidase ~.0 7.~ 2~3 ~'I a-L~ara~in~uxanosida~ g~0 7.3 2.1 Xylanase ~.0 19.5 14.2 Xylanase ~0 16.8 11.6 a-L-a~abinouranosidas~x~1anase 8.~ ~4.5 19.2 ~-L~arabinouranosida~e~ylana~e g.0 23.6 18.4 a Purl~ied a-~arabinouranosld~se (38 uni~s per ml, speci-:ic activi~y 126 V/mg~ an~ x~lanas~ T6 ~g U/ml~ 1~2 U~'mg) were u~ed~ The enzymes were in .l a mM ph~sphat~ b~fer.
~20 b The condi~ions used ~ere as ~ollowss ~0 mM each of Na2SO4 a~d (NH4)2S~4, 6S~C, 2h at pH 8.0 or 9.Q.
~;
i ,~
il:~`; .
~, ~

~ , .:
:~

:
"~
~ : ' `1 :
~.

4~;~

WO 93/20192 ,~ P~/SE93/00269 213 2 3 3 ~ SEQUENCE LISTING ~
( 1 ) GENERAL INFO~MATION:
"~'7 ( i ) APPLICANT:
.:~ (A) NAME: Korsnas AB
( B ) STR~ET: none (C) CIT~: Gavle ( E ) COUNTRY: SWEDEN
: (F) POST~ O~E (ZIP) ~ 801 81 (G) TELEPHONE: 026-151000 ". (H) TELEF~: 026-lg5253 1! (P~.) M~: The TaahrliQn ~ e~rch alld De~relopm~n~
.~ Foundatic:)n ~td ., ( ~3 ) ST~EET: Teahnion ki~y ( C ) ~I'rY: ~aia ( E ) COUNT}7~: ~s~ael ( F ) POST~ COD~ ~ Z ~P ): non~
(A) NAME: ~amo~ - Uni~r~rsl~ Au~hori~y ;~QX ~pplied Res . & ~nd . D~3~r . Ltd ( B ) STREET: 3 2 H . Le~anon S~r~t ( C ) CXT~: TeL-Avi~r . ~ E ) CQUNTRY: I~rael ~4 ( F) PO&TAL ~ODE (Z~P): none ~! ~ ( ii ) TITLE OF INVENTION: ~eligni~ylng Prepar~ion Exhibiting :~: Alpha-L-Arabino-.~ur~nosidas0 ac~ y, Produckion and Appliaation Thereof ~;~ (iii) NVMBER OF SEQUENCES: 3 ~ : (i~) COMPUTER READABLE FaRM:
; ~ (A) MEDIUM TYPE: Floppy disk tB) COMPUTER: IBM PC aompati.bla 't' ~ C ) OPE~ATING 5YSTEM: PC-DOS~MS-DOS
(D) SOFTW~RE: Paten~In ~elease ~1.0, Ver~ion ~ 5 (~PO) .~ .~
( 2) INFORMATION FOR SEQ ID NO: 1:
,1. : (i) SEQUENCE CHARACTERISTIC5$
(A) L~GTH: 9 amino aclds . ~ ~ ~ (B) TYPE: amino acid (D) TOPOLOGY: unknown MoLECULE TYPE:~ proltei~
~ (iii) HYP~THETICAL: NO
!~, `; ( V ) FRA~MENtr TYPE: N-~erminal :
: : ~ (vi) ORIGIN~L ~OURCE:
(A) ORG~NISM: Bacillus stearothermophilus xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
~: ~ . Xaa ~ln Pro Tyr Arg Xaa Glu Glu I.eu ,i . . . . .. .. .. ... . . ...

WO93/20l92 213 2 3 3 ~ 23 PC~/SE93/00269 ~ .
: ~2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) I.ENGTH: 23 amino acids ~B) TYP~: amino acid l (D) TOPOLOG~: unknown I (ii) ~9LECULE TYPE. pxotein :, .1' (iii) HYPOTHETIC~L: NO
(v) FR~G~ENT TYPE: N-t~rminal (vi) ORIGIN~L SO~CE~
(~) ORG~NI5M: Bacillus s~ea~o~h~rmophilu~

~ i) S~QUENCE DESCRIPTION: SEQ ~D NO: 2:
;1 S~r ~et Lys Lys Ala rl~hr M~ Ile ~le Glu ~s ~sp Phe ~ys I.Le ~la 1 5 lQ 15 ., Glu Ile ~sp Lys Arg Il~ Tyr ~2) INFO~MATION FOR SEQ ID NO: 3:
~i) 5EQUENCE CH~R~CTER~SllICS:
(A) LENGTH: 50 ~mino acids (B) TYPE: amino acid (D) TOPO~OGY: unknown (ii) MOLEC~LE T~PE: prot~in . ~iii) HYPOTHETIC~L: NO
~, tv) FRAGMENT TYPE: N~terminal .
;i~ ( vi ~ ORIGINAI- SOURCE:
~A) O~l~ISM: Bacillus stearothermophilus , (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 3:
'i Ala Thr ~ys Lys ~la Thr Met Ile Ile Glu Lys Asp Phe ~ys Ile ~la l 5 ~0 15 ~l Glu Ile Asp Lys Arg Ile Tyr ~ly Ser Phe Ile Glu His Leu Gly Arg .~ 20 25 30 ?~ Ala Yal Tyr Gly Gly Ile Tyr Glu Pro ~ly His Pro Gln Ala ~sp vlu '.!~, ~: Asn Gly ~' , ~.

Claims (17)

24

1. A preparation exhibiting enzymatic activity, c h a r a c t e r i s e d in that it is obtainable by aerobic fermentation, in a suitable medium, a of Bacillus stearother-mophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantially the same capability of producing said preparation as said strain NCIMB
40494, the enzymatic activity of said preparation comprising a substantial portion of .alpha.-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

2. A preparation according to claim 1, wherein said preparation is a clarified culture broth.

3. A preparation according to claim 2, wherein said preparation is a concentrated fraction of said culture broth exhibiting .alpha.-L-arabinofuranosidase activity in a wood pulp medium.

4. A preparation according to claim 3, wherein said .alpha.-L-arabinofuranosidase activity derives from an .alpha.-L-arabino-furanosidase which is composed of two subunits having the approximative molecular weights of 52 500 Da and 57 500 Da, respectively.

5. A preparation according to any one of claims 1-4 in asso-ciation with a preparation exhibiting enzymatic activity produced by another microbial strain and having the capability of delignifying wood pulp at a temperature of at least 65°C and a pH of at least 9.

6. A preparation according to claim 5, wherein said preparation comprises an .alpha.-L-arabinofuranosidase produced by the Bacillus stearothermophilus strain NCIMB 40494 and a xylanase produced by the Bacillus stearothermophilus strain NCIMB 40222.

7. An .alpha.-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence (SEQ
ID NO:1) or a homologue thereof.

8. An .alpha.-L-arabinofuranosidase having an amino acid sequence which comprises the N-terminal partial amino acid sequence (SEQ
ID NO:2) or a homologue thereof.

9. A method of producing a preparation exhibiting enzymatic activity, c h a r a c t e r i s e d in that a Bacillus stearothermophilus strain selected from the strain NCIMB 40494 and mutants and variants thereof having substantially the same capability of producing said preparation as said strain NCIMB
40494, is subjected to aerobic fermentation, in a suitable medium, the enzymatic activity of said preparation comprising a substantial portion of .alpha.-L-arabinofuranosidase activity having the capability of delignifying wood pulp at a pH of 8 - 9 and a temperature of 65°C.

10. The isolated Bacillus stearothermophilus strain NCIMB
40494 and mutants and variants thereof, said mutants and variants having substantially the same capability of producing a preparation exhibiting enzymatic activity as said strain NCIMB 40494, said enzymatic activity comprising a substantial portion of .alpha.-L-arabinofuranosidase activity having the capabi-lity of delignifying wood pulp at a pH of 8 - 9 and a tempera-ture of 65°C.

11. Use of an enzyme expressed by the Bacillus stearothermophi-lus strain NCIMB 40494 in the treatment of wood pulp.

12. A process comprising treatment of wood pulp, c h a r a c t e r i s e d in that wood pulp is treated in at least one step with a preparation according to any one of claims 1-6.

13. A process according to claim 12, wherein said wood pulp is sulphate pulp.

14. A process according to claim 13, wherein said wood pulp is a partially delignified sulphate pulp.

15. A process according to claim 14, wherein said partially delignified sulphate pulp is an oxygen-delignified sulphate pulp.

16. A wood pulp, c h a r a c t e r i s e d in that it has been treated in at least one step with a preparation according to any one of claims 1-6.

17. A DNA or RNA probe which recognizes the nucleotide sequence coding for the amino acid sequence (SEQ ID NO: 1) and/or the amino acid sequence (SEQ ID NO: 2) .