Note: Descriptions are shown in the official language in which they were submitted.
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
, . ~
WO93/20192 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.
.~,, ,
',;~
,,
WO93/20192 PCT/SE93/00269
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
WO93/20192 PCT/SE93/00269
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
11
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
12
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
.~
13
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
14
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
2132335
19
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~
21
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
0
~' ,
~.
