Note: Descriptions are shown in the official language in which they were submitted.
-I,-
21291'~~
'Thermostable Xvlanase DNA, Protein and lyiethods of Use
F'deld' of the Ireventlon
The present invention is in the area of thermostabile enzyzz~.es, and the
use of same. l~speciatly, ttte lnventiou is in the arcs of xylanascs that are
activt at a low pki and high rerttpe.-store. The compositions of the invention
are useful to modify plant biamass properties, especially to reduce the lignin
content. The invention is also directed to a method for biobleacbing using the
enzyme compositions of tlse irvention.
Buck~r~a~r~d of the In~en~i~rs
Xylan, a major component of hemicctlulose, is a polymer consisting
of g backbone of ~(I,4)~~linked D-xylose residues (often acetylated) with a-L
arabinofliranose arid gltacuronic acid side ct~s~ins (Timell, T.E. , et al. ,
Wood
Sci. TeChnad. 1:45-?0 {1967)). AfiCSr cellulose, xylan is the second most
15 abundant carbohydrate fraction of plant biomass. Xylan bas recently
received
increased attention as a renewable bioresource.
Being complex, ~eore than one enzyme iB r~equiz~ed to completely
degrade xylau to soluble monomers. Xylan can be hycimlyzed by rnaay
hcmicelltzlasPS, such as, for example, ~-I,4-xylanases (EC 8.2.1.8), i3-
20 xylosid,ases mnd several debranching enzymes (Biely, P., Trends Biotec~ol
3:28-290 (1980; l3ekker, R.~.H" in Hignelti, T., ~d" igiosynthesis arut
btodegradn:ion of wood components (Academic Press lne., Orlando), pp. 505-
533 {i985); 't~Voodward,1. , Top ,Lrt~me;Fertptent. Biotechnod. &:9-30
(1984)).
Tho activities of these eatzymes play as important role in the decomposition
25 of soil plant litter amd have been extensively studied both.in bacteria amd
fungi
(along, K.K.Y, et tad., MiCr~biol. Bev. 52:305-317 (1988j; Poutanen, K.
SENT BY:S K G & F : ?-2°-94 ~ 13:2E ~ SKG&Fi G.5 & H~tt 7
21~91'~2
-2-
et al., in ~n~ymes irt ~~:amas~s conversion (ACS Symposium series 460),
l,eatham & Himmel, eds., American Chemical Society, Washington, bC
a
(1991), pp.426-436; Gilbect &. Hazlewood, J. Gen. Microbiol. 139:187-I94
(1993)).
Various microorganisms secrete enzymes that are capable of degrading
xyl~s~s, and xylansses have been found in both prokaryotes and eukaryotes
(Dekker, R.~.H., Ftichards, G.N.. Adv. Carhohydrdte Chem. Biochem.
32:277-352 (1970. Xylanolytic micro-organisms often produce multiple
xylanases to attack the different bonds in these molecules. All the xylanases
so far characterized fall into two classes; the high M,llow pI class and the
low
M,lhigh pI class whiclh coincide, respectively, with the families 10 and 11 of
glycosyl hydrolases (Henrissat & Hairoch, Biochem. J. 293;781-788 (1993)).
The cloning of xylanases has been reported firom Actinomadura sp.
FC?' (Ethier, J.-F. et al., in: Industrial Microorganisrrts: Basic and Applied
Molecular Genetics, R. Bale. et al. , eds, (Pros. Sth ASM Conf. Gen. Mol.
Hiol. Ind~.~st. Nlicroorg., Oct 11-I~, 1992, Bloomington, Indiana, posterC2S);
bacteria (e.g. Ghangas, G.S. et al., J. Bacteriol. 171:2963-2969 (19$3); Lin,
L.-L., Thomsan, J.A., Mot. Gen. Genet. 228:55-61 (199i); Sharcck, F.
et aL, tens 1~7:'75-82 (1991); Scheirlinck, T. et al., A~pl Microbial
~iotechnol. 33:534-541 (1990); Whitehead, T.R., Lee, D.A., G~rr.
Microbial. 23:15-19 (1991)); and fungi (Boucher, F. et al. , NucleieAcids Res.
14:9874 (1988); Ito, K. et ad. , Biosci. Biatec. BiochEm. 56:90b-912 (1992);
Maat, 1. et at. , in Visser, J. et al. , eds., Ji~lans and Xylanases (Elsevier
Science, Amsterdam), pp. 349-360 (1992); van den Broeck, H. et al., EP
2S 463,706 A1 (1992), Wa 931256'1 and Wt~ 93f25693).
The xylan-remaining hemicellulases in plant blamaee are tightly bound
to cellulose and lignin. In the pulp and paper industry, in chemical gulping
(cooking) of the wood, the major part of the lignin is e~racted to get
acceptable cellulose pulp product. Howcvcr, the rcsultiag pulp ie brown,
mainly b~au~se of the small portion of the lignin still remaining is the pule
after cooping. 'This residual lignin is traditionally rEmovtd in a multl-stage
SENT BY:S K G & F , ~-2g-g~ ; ~3:pg ; 5KG&F-~ G, S & H;it 8
2129172
bleaching procedure using typically a combination of chlorine ct~emiegls and
extraction stages. Peroxide, oxygen and ozone are also used when the use of
the chlorine cheznical$ is wanted to be reduced or avoided totally.
~Iecnicellulases can be used in enzyme-aided bleaching of pulps to
decrease chemical dosage in subsequent bleaching or to increase brightness of
the pulp (Kantelinen et al. , International Pulp Bleaching Conference, Tappi
Proceedings, I-5 (19$8}; Viikari et al., Paper and Timber 7:384-389 (I991);
and ICantelinen et al., "Enzymes in bleaching of lcraft pulp," Diesertaticjn
for
the degree of Doctor of Technology, Technical Research Centre of Pialand,
VTT Publications 114, Espoo, 1.992). Naturally, in this use, the
hemicellulase should be face of cellulases, which would harm the cellulose
fibers.
The use of hemicellulose hydrolyzing enzymes in different bleaching
sequences is discussed in WO 89108738, F.P 383,999, 't~VO 91102791, F.P
395,792, EP 386,888, EP 473,545, EP 489,104 and WO 91105908.
Other industrial applications for hemicellulolytic enzymes are in the
production of thernto-mechanical pulps, where the aim of the use of
hemicellululytie enzymes is decreased energy consumption. Hemicellulolytic
enrymes can be used to improve drainage of recycled pulp or hemicellulolytic
enzymes can be used in the protiucdon of dissolving pulps ('Yiikari et al.,
"Hemicellulases for Industrial Applications," In: Hioconversion of Forest and
Agri,culrurat Wastes, Saddler, Y., ed., CAB Laternational, USA (1993)).
The use of hemicellu~olytic enzymes for improved water removal from
mechanical pulp is discussed in EP 262,040, 13P 334,739 az~d EP 351,665 and
DE 4,000,558). When the hydmiysis of b:omass to liquid fuels or chemicals
is considered, the conversion of both cellulose and heaticellulose is
essential
to obtain a high yield ('Yiikazi et al. , "Hemicellulases for Industrial
Applications," In: Bioconverslon of Forest anti Agricultural Wastes, Saddler,
J., ed., CAB International, USA (1993)). Also, in the feed industry, tb~re is
a need to use a suitable combination of enzyme activities to degrade the high
i
~-glucan and henvcellulose con.Caining substrate.
SENT BY:S H G ~ F , '?-'2;!--~4 ; 13:27 ~ SKG&F-~ u.5 & H~ti 9
2129172
'f o be ar~onable to enzymatic hydrolysis in vitro, the cellulo5e-
he~icellulc~s:-lignin matrix must be chemically pretreated. One of such
procedures ' izv~rol va a thermo-mecYoanical steam treatment followed by
extraction with hat water (Ck~ahal, D.S. et al. , J. Inriust. Microbial, 1:3SS-
36I
(1~8'~~. ~ mildly acidic liquor is obtained, which contains ~-ater-soluble
hen~icellulose chains and some lignin derivatives.
However, to ens~tre~ further enzymatic hydrolysis of the x~y~lan chains
into oligomelcs or monomers, enzyme systems that are efficient at conditions
combining high temperature (such as 70°C) and moderately a::idie pH
(around
4.0) are . The combination of these rwa parameters seeot~s however to
be harmful for the majority of la~.own xylanases. 1'ar instance, at pH 4,
xylanase II from the mesophilic aetinomycete 5'treptomyces roseiscleratieus (a
low M,,lhig>z pI onzyrre) retains less than 5 ~ of the activity it had at pH
6.0 -
b.5 (Gra'bski ~ Jeffries, ~ppl. Environ. Microbial. 57;387-992 (1.991)). The
1,5 crude xylartase from Aureobasidium prtllulans (Myburgh, 7, et al. , Prac.
Eiochem. 2a:3~3-348 (1991)) is acidophilic, having a pH optimum between
3.5 and 4.0 but its actin.=ity sharply decreases at temperatures higher than
35°C. The thermostable xylar~age from the fungus Tlternmascus
auranttacus
retains at pH 3.~, only 129 of its maximal activity (Tan L.U.L. et ul., Can.
,~. Microbial. 33;68'9-692 {1987)). Another xylauase, a aitgh M~llnw pI
enzyme fr9m the extremophile bacterita~aa, "Caldocellu»~ saccharolyricum" was
shown to 19e Very stable at 64°C but retained little activity below pH
5 (Lfzthi,
E. et al., ~lppt. Environ. Microbial. 56:2677-2b83 (1990); Litthi, B. et al.,
Appl. ~nvdror~. Microbial. 56:1017-102a (1990). Crude xylanases from various
Actinomadura isolates were stable for many hours when incubated at 7Q-75
°C,
but retained less tban 15'~ of their activity at pH ~.0~.5 and 70°C
(I~ioltz, C.
et al., Antonie van Leeuwerthaek 59:1-7 (199I)).
Thus, there is a need for enzyme preparations that contain xylanases
which retain activity under industrial ambient conditions. Fspocially in the
i
pmper mamafacttwing industry; there is a need for xylanase prepartions that
are
SFI~T BY~ ~ K G 8 F . ?-29-94 ~ .3:26 ~ 5ltG&F~ G~ S & Hs3t10
X1291'72
functional in the high temperature, acidic liquor produced by thermo-
mechanical steam treatment aml hot water extraction.
Sunemar,~ of ttee Invention
Recognizing the importance of developing an environmentally safe and
economical method of cheznic.~.atly modifying plant biomass so that it may be
enzymatically treated under harsh conditions of high temperature and towpH,
processes such as those employed by the paper marnifacturing industry, the
inventors have searched for n now mitxobe that might be a source of such
enzymes.
1~ These studies have resulted in the isolation and identification of a navel
strain of thermophilic actinomycete, Actinomaciura sp. FC7. Acrirwmadura sp.
FC7 expz~esses t~vo unique xylanases, X'SfL I and XYL II that retain a large
amount of their enzymatic activity at high temperatures and law pH.
The invention is further directed to DNA encoding XYLI and XYL II,
and to recombinant hosts transformed with such DNA.
The inverctfon is further directed to purified XYL I and XYI, II, and
to emyme preparations containing XYL I, XYL II, or mixtures of XYL I and
XYL n.
The invention is further directed to a method of treating plant biomass
with the enzyme preparations of the invention, especially a method of
biobteaching.
Brief De~cnipdo~ of the Drawings
Figure x shows the restriction map of the inserts clod in plasmid
pJFI. 'The shaded boxes shows the approximate iacations of the xylanase genes
after deletions of their.respective inserts. Top line, restriction sites in
the f~tll-
le~th insert. Top shaded line: plFl (2Ø0 kb; full length insert); Second
shaded tire;: p~'102 (13.5 kb); third shaded line: pJF103 (11.5 kb); and
fourth
SENT 6Y:S H G & F ; 7-29-~4 ~ 13:28 ~ 5KG&F-~ G~ S & H~Ji11
-6-
shaded line: pJF1020 (7.5 kb). Bg, BgIII; H, BamHi; X, XhaI. +, xylanase
positive; -, xylanase negative.
Figure 2 shows the restriction reap of the inserts cloned in plasmid
pJF6. The shaded boxes shows the approximate locations of the xylanase
genes after deletion of the respective insert. Bg, BgliI; N, NruI; No, NorI,
l S, SaII. +, xylanase positive; -, xylanase negative. Tap shaded line: pJF6;
second shaded Line: pJF6l; third shaded tine: g3F62.
Figure 3 shows the effect of pH and temperature on Xylanase I
activity. Purified Xyl I (5 units) was incubated for 10 min at the temperature
1<0 and pH values indicated and the release of reducing sugar was measured by
the Nelson-Somogyi method. (~) 60°C; (~) 70°C; (~) 80°C.
Figure 4 shows the effect of pH and temperature cn Xylanase II
activity. Puri~.ed Xyl 1~ (8 ututs) was incubated for 10 thin at the
tetnperatuze
and pH values indicated. and the z~lease of reducing sugar was measured by
the Nelson-Somogyi method. (') 60°C; (~) 70°C; (~) 80°C.
Figure 5 shows a restriction map of the 2.7 kb insert of clone pJF6
(encoding Xyl In. 'Ihe black line shows the sequenced portion of the insert,
starting from the indicated Nrul site. The loners rept~esent the following
' restriction sites: Bg=~gni, S=SadI, N=Nrul, No=NatI.
'i
' ~,p Figure 6 shows the nucleotide sequence of the pJF6 insert (xln2) from
the NruI site shown i.n Figure 5, to the Bgla site shown in Figure 5. The
amino acid sequence of Xyl H begins at nucleotide 521. The -35 (TTGACG)
and -10 (CACAAT) promoter regions, the ribosome binding site (RBS:
(3GAGGA), and the iniation colon (CIT: GTa) are shown in bold.
Figure 7 is a comparison of the R$S of 40 streptornyeetes genes versus
that for xlnll as encoded by the pTF6 xylatiase gene. Tlu nucleotides
corresporxling to the RBSs are underligned, while those in bold identify the
translation initiation c4dan.
Figure 8 shows a partial. amino acid sequence of X'1'I. II on which the
30 signal peptide is located. The long sequence of hydrophobic amino acids is
shown in bald. The characteristic arginines (R) usually found in the
SENT BY:S ~( G & F ~ 7-29-94 ~ 13:29 ~ 5~5G&F-~ G, S & H:itl2
_7_
hydrophilic region are underlined. The arrow indicates the possible cleavage
site of the peptidase signal, bordered by a proline (P).
Figure 9 shows a comparison of nucleotide sequence homology
between the streptomycetes promoters having a spacing of 16 nucleotides
between regions -35 and -10, and the promoter of the xlnll gene encoded
by the pJP6 xyianase. The -35 and the -10 regions are in bold.
Figures 1~1GC show the optimal alignment of the amino acid sequence
of XYL II as encoded by pJF6 with other enzymes. The list of enzy inea is
as follows; ~ xylanases of Pseudomonas fluoresceos {Psexyna, Psexynbc),
pIF6 xylanase (xlttpjf6), xylazvxsc A of Stre~tomyces livlclcuts (Stmxlna),
exogluconase of Cellulomonas firm (Cficex), xylanase of CIostrGdlum
rhermocellum (Claxylz), xylanase of Bacillus sp. {Bscxynaa), celloaylanase of
Clostridium srercoirarium (Pclocxl), acylanase of Caldoc~llurn saccharolytlcum
(Cdcxynab), xylanase of Thermoanaerobacter sp. (Teoendxyla), endocellulose
iS of Caldocellurn sacctiamlytlcum (Cdccelb), xylanase of Buryrlvibrlo
~'tbrisolvens (Hutxynb) and s xylanase of Rumiococcus flrtvefaciens
(Ru.yna). Amino acid consensus is indicated in bold, and those amino
acids retained in all. exatnfned enzymes are represented by an asterisk (*).
Hypothetically retained regions arc shoevtt by an underline bracket.
Figure I1 shows the homology among the amino acid derived
sequences of xylanase A of Streptomyces livuians and chat of XYL II as
encoded by pJF6. The symbols between sequences i~icate that the
comparison value is tlx same (~), ~ 0.5 (:), ~ 0.1 (.). An indication of
z 0.5 means that the two different amino acids represent conservative changes
(ie., there is some structural andlor functional similarity between them,). An
indication of ~ 0.1 represents amino acids that have no or weak structtual
andlor f~n~rioaal similarity.
Figure I2 shows the seqtteare of nucleotides 1538 to 1572 inclusive,
of the xlnll sequence on p3F6. Arrows indicate repeated and inverted
sequences.
r
Figure 13 and 1.3A show the MAP progranm prediction of the
pioteOlytiG GlCBVage SlteB along the amizio acid derived sequence of X'YL II
as
encoded by p3F6 and the xylanase A of SteptomyGes ldvidans. The letters
represent the follawiag prciteases: S (Staphylocossus aureus protease), T
(TryPsin) and C (Chymotrypsin). The differences encountered are shown in
bold.
l3e~osits _
Plaa~nid pJPI was deposited in E. colt at the American Type Culture
Collection, (ATCC), 12301 Parklawn Drive, Rockville, MD 20852 on July Z9,
1994 and assigned accession no. ATCC 69670. .
Plaamid pJF6 was deposited in E, colt at the ATCC on July 23, 1994 ~ v
and assigned accession no, ATCC 69671.
Act3nomadura sp. FC7 was deposited at iha ATCC on July 24, 1995 and
assigned accession no. ATCC 55698.
is ~etoiled ~escri~tio~a of the invention
1, l~r~n8tleans
In the description that follows, a number of terms used in recombinant
DNA tGChaolo~y are extensively utilized. In order to provide a clearer and
consistent ur~deerstanding of tha specification anti claims, including the
scope
to be given such terms, the following definitions arc provided.
~landse. As used herein, a xylanase is a hemicellutase that cats the . ,
~~1,4 bonds within the xylosic chain of xylan, (xylan is a polymer of D-xylose
residues that are joinad through S-1.4 linkages. Xylaaase activity is_
synonyatous with xylanolytic activity. .
By a host that is "sul~~ly incapable" of syathesizi~ one or more
cellulose et~ymes is meant x host in which the activity of one or more of the
5E~'T BY:S K G & F ; 7-P9-84 ; 13:50 5KG&F-~ G. S & n;i~l4
_g_
eeilulase e~ymes is depressed, deficient, yr absent when compared to the
wild-type.
En~yrne preparation. By "enzyme preparation" is meant a composition
containing enzymes that have been extracted from (either partially or
completely purified from) a microbe or the medium used to grow such
microbe. "Extracted frsom" means any method by which the desired enzymes
are separated from the cellular mass and in;,ludes breaking cells and also
simply removing the culture medium from spent cells. Thezefore, the term
"enT,Yrrre preparation" includes coazposirions comprising medium previously
used to culture a desired microbes) and any enzymes which the miembe(s)
has secreted into such medium during the culture.
Biobleaching. Hy "biobleaching" is meant the extraction of lignin
from cetlulase pulp after the action of hemicellulose degrading enzymes with
or without lignin degrading enzymes. Removal of the lignin may be restricted
by hemicelluloses either physically (through repreeipitativn onto the fiber
surface dur~:ng cooking) yr chemically (through lignin-carbohydrate
complexes). The hemicellnlase activity partially degrades the hemiceltulosG,
which enhances the extractability of lignins by conventional bleaching
chemicals (like cblvcine, chlorine dioxide, peroxide, ere.) (Viikari et al.,
~0 "Bleaching with Enzymes" in Biotechnology in the Pulp and Paper Industry,
Proc. ~z~d Int. Conf. , Stockhoim, pp. b7-69 (1886); Viikari et al. ,
"Applications of Enrymes in Bleaching" in Proc. 4th Int. S~mp. Wood and
Pulping Chemistry, pane, Vol. 1, pp. 151-154 (19$7); Kantelinen et al.,
"Hemicellulases and their Potential Hole in Bleaching" in International Pulp
Bleaching Cc~nf~renee. Tappi Proceedings, pp. i-9 (1988)). The advantage
of this improved bleachabiiity is a lower consumption of bleaching chemicals
and lower environmental loads or higher final brightness values.
By an enzyme "hot~alogous" to a host of the invention is meant that
an untransfarmed strain of the same species as the host spites naturally
produces some amount of the native protein; by a gene "homologous" to a
host of the invention is meant a gone found in the genome of an
SENT 3Y:S K G & F ~ 7-29-94 ~ 13:30 ~ BKG&F-~ G.5 & H.#15
2~~~172
a_
urtransfermed strain of the same spc~;ies as the host species. By an enzyme
"heternlogous" to a host of the invention is meant that an unu-ansformed
strain ,
of the same species as the host species does not naturally produce $ome
amount of the native protein; by a gene "heterologous" to a host of the
invention is meant a gene not found in the genome of an untransformed strain
of the same species as the host species.
Clonfrtg~ vehicle. A plasmid or phage DNA or other DNA sequence
(such as a linear Dri'Aj which provides an appropriate nucleic acid
environment for the transfer of a gene of interest into a host ccll. The
cloning
vehicles of the invention may be designed to replicate autonomously in
prokaryotic and eukaryotic hosts. In fungal hosts such as TYichoderma, the
cloning vehicles generally do not autonozaously replicate and instead, merely
provide a vehicle for the transport of the gene of interest into the
~ichoderma
boat for subsequent insertion :nto the fY~icltodernsa genome. The cloning
vehicle may be further characterized by one or a small ntunber of
endonuclease recognition sites at which such DNA sequences may be cut in
a determinable fashion without loss of an essential biological function of the
vehicle, and into which DNA may be splicxd in order to bring about
replication and cloning of such DNA. 'r'he cloning vehicle may further
contain a marker suitable for use in the identification of cells transformed
with
the cloning vehicle. Markers, for example, are antibiotic resistance.
Alternatively, such markers may be provided on a cloning vehicle which is
separate from that supplying the gene of intere8t. The word "vector" is
sometimes used for "cloning vehicle."
~xpresslan vehracde. A vehicle or vector similar to a cloning vehicle
but which, is capable of expressing a gone of interest, after transformation
into
a desired host.
When a fungal host is used, the gene of interest is preferably provided
to a fungal host as part of a cloning or expression vehicle that integrates
into
3a the fungal chromosome. Seryences which derive from the cloning vehicle or
~Xpre8B101~ YChiClt Iilay ~.l$0 be integrated with the gene of interest during
the
SENT BY:S K G & F . ~--Zy-94 ~ 1?:31 ~ SKG3F-~ G~ S & ~~~16
integration process. For example, in T. reeSei, the gene of interest can be
directed to the cbhl locus, ,
The gene of interESt may preferably be placed under the control of
(i.e., operably linked to) certain conuol sequences such ss promoter sequences
provided by the vector (which integrate with the geae of interest). If
desired,
such control sequences may be provided by the host's chromosome as a recruit
of the locus of Insertion.
Expression control sequences an an exp'rcssion vector wilt vary
depending oa 'arhether the vector is designed to express a certain gene in a
1Q pmlcaryotic Or euksryotic h~ast {for example, a shuttle vector may provide
a
gene for selection in lsacterial hosts) and may ad3itiortally contain
transcriptional elements such as, enha,nie~cr elements, termination sequences,
and~or translatlonal initiation and termination sites.
1. Isoladfo~ of Actlnoniyeetas actlnomadur~ ap. FC'fi
The Project had the objective of isolating a microorganism, an
actinomycete antler the circumstances, vr~Ch would have an acidcrphilic said
thermostable xylanotytic activity. The actlrlomycetes are aerobic gram-
positive
bacteria founai mainly in the soil. The actinomycetes display a a~ycelial
morphology intxrestiugiy reseuibling microscopic fv.ngi, lrmare, they
are recognized as excellent enzyme secretors, thereby playing a very important
role during biomass degradation.
A aere~ing program was established in order to find actinomycetes
that produce xylanases able to hydrolyse xylan chains in a hemicellulose
liquor
(a by-product of steam treatment of the lignocellulosic biomass) at moderately
said pH (4.0) aixl high temperature (70°C), The hunt for such an
organism
was made based upon places in which xo important periodic heatixig-up c4uld
be produced, such as in hay, .compost and manure.
CA 02129172 2000-08-17
-12-
The first step involved a selection for xylanolytic actinomycetes having
optimal growth at SO-60°C and that demonstrated a strong degradation
capability
ofRemazol Brilliant Blue(RBB)-xylan on solid medium. A series ofthermostable
and acidophilic actinomycetes with these characteristics were isolated from
compost; mancire and straw and further examined for ;heir ability to produce
zylanolytic enzymes that were relatively active at pH4, and 70°C. In
this
second step of the screcaing> the zylan hydrolysis rates at pH 5160°C
of crude
enzyme preparations secreted from the selected actinomycetcs were compared
to those at pH 4170°C for the same microbe. This was done to determine
the
level of acid- arid thermo-resistence of the xylanase enrymcs being secreted
by each microbe.
The selection procedure identified one microbe from manure that was
especially desirable in that it produced xylanolytic enzymes -that were
relatively active at pH4, and ?0°C. This microbe was identified as a
member
IS of the genus Actinomadum by chcmotaxonomic procedures, and was named
Acrinomadura sp. FC7. Pure preparations of ActinornQdura sp. FC7,
produced at least four xylanolytie activities as dersonstrated by zymogram.
The crude enzymes produced by the strain PCl retained 65 9& of their activity
in the more stringent of the two conditions (pH 4170°C).
11. Xylanase Btobleaehing at High Temperature and Acidic pH
The present invrntion comprehends a method for chemically treating
plant biamass under conditions of high tenxperature aar3 low pH. In a
preferred embodiment, plant biomass is bio-bleached with xylanase$ that are
able to hydrolyze xylan chains in a hemicellulose liquor (a by-product of
steam treatment of the lig~ellulor~ biomass) at moderately acid gH (4.0) and
high temperature (70°C).
Plant biomass is a composite material consisting primarily of a mauL~
of cellulose, hcmicellulose, and lignin. Removal of the lignin component is
desirable during the mnaufacturer of paper because of its brown color and
SEPJT 8Y':5 K G & f= ~ x-29-94 s 13:32 ~ 5KG&F-~ G. S & H~#18
212~17~
-13-
tendency to reduce the strength of the paper product. Many processes have
beer, developed for the removAl of lignin. Typically, the wood pulp is treated
,
with chorine or other toxic chen~i;,als in order to remove the lignin
component
and provide for a brightened pulp, However, the toxic by-products of this
chemical treatment negatively impact upon the health and stability of the
environment into which they are released. Consequently there is a great need
for developing alternative, more environmentally protective techniques to
achieve puig bleaching.
A common treatment of plant biomass for paper production involves
a thermo-mechanical steam treatment followed by extraction with hot water.
This process dissociates xylan cornaining hemicxliuloses and some lignin
derivative$ which are otherwise tightly bound to the ceIlulos~e. Under the
method of the present invention, a biobleaching technique is developed
whereby theimoscable xylanases which are active at low pH may be used in
1~ vitro to modify or decrease the lignin in wood pulps, These stringent ;
processing conditions may additionally act to reduce cellulose activity in the
enzyJme preparation or culture medium.
In a preferred embodiment, the process of the invention is carried out
in vitro in the acidic hemicellulose liquor. The process involves placitxg the
2U enzyme preparation, culture medium, or coc»centiated mixture containing
xylanase into contact with the wood pulp, Routine calculations enable those
in the art to determine the optimum treatment time depending upon the result
desired, the concentration and specific activity of the xylsnase enzyme used,
the type and concxrttration of pulp used, pH and temperattue of the acidic
liquor, and other parameter variables.
It is preferred that the process occurs at the ambient temperature and
pH of the liquor with temperatures from 45-90° being preferred and
temperatures of 70° being mast prefe;«ed. It is also preferred that the
pH of
the liquor be less titan 6.0 with a pkli of 4.U being most preferred.
3~ The method of the present invention may be applied alone or as a
supplement to other treatments that reduce the lignin content of wood pulp,
SENT BY:S K 3 & F ~ '~'°~9-94 ~ X3:33 : S~tG&F~ G~ G & H.#19
- ~ 4-
increase its drainabiliy and/or decrease its water retention. In a preferred
embodiment, tEce present invention is used to enhance brightness properties of
,
the wood pulp by treatment of chemical pulps, i.e., those pulps conta;n
lignin that has been chemically modified through chemical treatment.
In a preferred embodiment, The xylanases used in the methods of the
invention are preferably those of.4ctinorncrdura sp. FC7, and especially XYL
I and XYL II. XYL I and X'YL II can be provided by the native ~f ctinomadura
sp. FC7 host (and especially the culture medium from the growth of p'C7
celis) or can be provided by a recombinant bast, for example, as encoded by
expression of the ins~enrs on pdFl and p3P6.
111. Genetic Engtrtaerang of ti3te Hods off' the Invarrtion
The process for g$n~tically engincezing the hosts of the invention is
faellitated through the cloning of genetic sequences that encode the desired
xylanase activity and through the expression of such genetic sequences. As
used herein the tmm "~CnetlC sequences" is intended to refer to a nucleic acid
molecule (preferably IaNA). Genetic sequences that encode the desired
xylanase are derived from a variety of sources. These sources include
~cttnorit~rdura sp, FC7 genomic DNA, cDNA, synthetic DNA and
combinations thereof, Vector systems may be used to produce hosts for the
2Q production of the enzyme preparations of the invention. Such vector
construction (a) may further provide a separate vector construction (b) which
encodes at least one desired gene to tx integrated to the genome of the host
and (c) a selectzble masker coupled to (a) or (b). Alternatively, a separate
vector may be used for the marker.
A nucleic acid molecule, such as DNA, is said to be "capable of
e~~preas$ng" a polypeptade if it contains expression control sequences which
contain transcriptional regulatory information anc3 such se~utnees are
"operably linked" to the tnzeleotide sequencx which encodes the polypeptide.
SENT B1':S K G & F ; 7-~9-94 ; 13:33 ~ SKG~F-~ u, S & H;~20
2129172
An operable linkage is a linkage in which a sequence is connected to
a regulatory sequence (car sequences) in such a way as to place expression of
the sequence under the influence or control of the regulatory sequence. Two
DNA sequences (such as a protein encoding sequence and a promoter region
sequence linked to tl've 5' end of the encoding sequence) are said to be
operably linked if induction of promoter function results in the transcription
of the protein encoding sequence mRNA and if the nature of the linkage
between the iwo DNA sequences does iwt (1) result in the introduction .of a
frame-shift mutation, (2} interfere with the ability of the expression
regulatory
sequences to direct the expression of the trtRNA, antisense RNA, or protein,
or (3) interfere with the ability of the template to be transcribed by the
prpmoter region sequence. Thus, a gmmoter region would be operably linked
to a DNA sequence if the promoter were capable of effecting transcriprion of
that DNA sequence.
The precise nature of the reguiatory regions needed for gone expression
may vary between species or cell types, but shall in general include, as
necessary, 5' non-transcribing and 5' non-translating (non-coding) sequences
involved with initiation of transcription and translation respectively.
Expriession of the protein in the transformed boats requires the use of
regulatory regions functional in such boats. A wide variety of transcriptional
and tranalational regulatory sequence$ can be employed. In eukaryotee, where
transcription is not linkad to translation, such control regions may or may
not
provide an initiator methionine (AUG) colon, depending on whether the
cloned sequence contains such a methionine. Such regions will, in general,
include a promoter region sufficient to direct the initiation of RNA synthesis
in the host cell.
hs is widely known, translation of eukaryotic mltNA is initiated at the
colon which encodes the first methionine. For this reason, i.t is preferable
to
ensure that the linkage betwan n eukaryotic promoter and a x3N~i sequence
which encodes the protein, or a functional derivative thereof, dots not
contain
any intervening codoas which are capable of encoding a methionine. The
SENT BY:S tt a a F ; ~-28-84 ; 13:34 ; 5KG&F-~ G~S & H;#2?
-ls-
presence of such colons results either in a formation of a fusion protein (if
the
AUG colon is in the same reading frame as the protein encodizig DNA
sequence) or a frame-shift mutation (if the ALJG colon is not in the same
reading frame as tk~e protein encoding sequence).
In a preferred embodiment, a desired protein is secreted into the
surrounding malium due to the presence of a secretion signal sequence. If a
desired protein dues not possess its own signal seqsence, or if such signal
sequence does not funetioa well in the host, then the protein' s coding
sequance
may be operably linked to a signal sequence homologous ar heterologous to
the host. The desired ceding acqusncc may be linked to any signal sequence
which will allow secretion of the protein fmm the host. Such signal sequences
may be designed with or without specific protease sites such that the signal
peptide sequence is amenable tv subsequent removal. Alternatively, a host that
leaks the protein into the medium may be used, for example a host with a
mutation in its ~aembeane.
If desired, the non-transcr'bed and/or non-translated regions 3' to the
sequence coding far a protein can be obtained by the above-described cloning
methods. The 3'-non-tt~nscribed region may be retained for its transcriptional
termination regulatory sequence elements; the 3-non-translated region may be
retained for its translational termination regulatory sequence elements, or
for
those elements whiGl3 direct polyadenylation in eukaryotiC cells.
The vectors of the invention may further comprise other vperably
linked regulatory elements such as enhancer sequences.
In a preferred embodiment, genetically stable transformants ate
constructed whereby a desired protein's DNA is integrated into the host
chromosome. The coding sequence for the desired protein may be from any
source. Such integration may occur de novo within the cull or,. in a most
preferred embodiment, be assisted by transformation with a vector which
functionally inserts itself into the host chromosome, for example, DNA
elements which prnmote integration of DNA sequences in chromosomes.
SENT QY:S K G & F , 7-29-94 ~ 13:34 ~ 5KG&F-~ G~ S & H;ti2P
-I 7-
Cells that have sta~ly integr$ted the introduced DNA into their
chromosomes are selected by also introducing one or more markers which
allow for selecson of host cells which contain the expression vector in the
chromosome, for example the marker may provide biocide resistance, e.g.,
resistance to antibiotics, or heavy metals, such as copper, or the like. T'he
selectable. marker gene can either be directly linked to the DNA gene
sequences to be expressed, or introduced inra the same cell by co-
transformation. -
Factors of importance in selecting a particular plasrnid or viral vector
include: the ease will? which recipient cells that contain the vector may be
recognized and selected from those recipient cells which do not comtain the
vector; the number of copies of the vector which are desired in a particular
host; and whether it is desirable to be able to "shuttle" the vector between
host
cells of different species.
Once the vector or DNA sequence containing the eonstritet(s) is
prepared for expression, the DNA constructs) is introduced into an
appropriate host cell by any of a variety of suitable means, including
transformation as described above. After the introduction of the vector,
recipient cells are grown in a selective medium, which selects for the growth
of transformed cells. Expression of tfue cloned gene sequencc(s) results in
the
production of the desired protein, or in. the praiuc;tion of a fragment of
this
protein. This expression can take place in a condntwu~s manu~er in the
transformed cells, or in a controlled manner.
Accordingly, the XYL I and XYL II encoding sequences may be
operably linked to any desired vector and transformed into a selected host, so
as to provide for expression of such proteins in that host,
CA 02129172 2000-08-17
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IV. Tfie En,ryme Preparations of the Invention
According to the invention, there is provided enzyme compositions
useful in a method for bioblcaching and pulp and paper processing. There is
also provided a method for producing an enzyme preparation partially or
completely deficient in cellulolytic activity (that is, in the ability to
completely
degrade cellulose io glucose) and enriched in xylanascs desirable for pulp and
paper processing. By "deficient in cellulolytic activity" is meant a reduced,
lowered, degreased, or repressed capacity to degrade cellulose to glucose.
Such cellulolytic~.activity deficient. preFarations, and the malting of same
by
recombinant DNA methods, are described !n US 5,298,405.,
As described herein, xylanascs may be provided directly
by the hosts of the ,invention (the hosts themselves are placed in .the wood
processing medium). Alternatively, used medium from the growth of the
hosts, or purified rnzytnes therefrom, can be used. Further. if desired
activities are present in more than one recombinant host, such preparations
may be isolated from the appropriate hosts and combined prior to usa in fne
method of the invendan.
The enzyme preparations of the invention satisfy the requirements of
sp~cifie needs in various applications in the pulp and paper industry. For
example, if the intended application is improvement of the strength of the
mechanical mass of the pulp, than the enzyme prCparations of the invention
may provide enzymes that enhance or facilitate the ability of cellulose fibers
to bind together. In a similar manner, in the application of pulp milling, the
enzyme greparations of the invention may provide enzymes that enhance or
facilitate such swelling.
To obtain the enzyme preparations of the invention, the nation or
recombinant hosts describai above having the desired properties (that is,
hosts
capable of eupressiitg large quantities of the desired xylanase enzymes and
optionally, those which are Substantially incapable of e~cpressing one or more
cellulase enzymes) are cultivated under suitable conditions, the desired
SENT BY:S K G & F , r-29-94 : 13:35 ~ SKG&F~ G,S & h~ti24
-19-
enzymes are secreted from the hosts into the culture medium, and the enzyme
preparation is recovered from said culture medium by methods known in the ,
art.
'The enzyme preparation can be produced by cultivating the
recombinant host or native strain in a fermentor. For example, the eruyme
preparation of the present invention can be produced in a liquid cultivatifln
medium tha! contains oat spelt xylans as the main carbon source as described
by Morosoli et al., Btochem J. 239.587-592 (1986)). _
'The enzyme preparation is the culture medium with or without the
IO native or transformed host cells, or is recovered from the same by the
application of methods well known in the art. Howevor, because the xylanase
enzymes are s~reted into the culture media and display activity in the
atnbieat
conditions of the hemicellulose liquor, it is an advantage of the invention
that
the enzyme preparations of the invention may be utilized directly from the
culture medium with no fiuther purification. If desired, such preparations
mey be lyophilized or the enzymatic activity otherwise concentrated andlor
stabilized for storage. The eazyme preparations of the invention are very
economics! to provide and use because (1) the enzymes may be used in a
crude form; isolation of a specific enzyme from the culture fluid is
2,0 unnecessary and (2) because tb~e enzymes are secreted into the culture
medium, only the culture medium need be recovered to obtain the desired
enzyme preparation; there is no need to extract an enzyme from the hosts.
If desired, an expressed protein may be farther purified in accordam
with conventional co~itians, such as extraction, precipitation,
chromatography, affynity chromawgraphy, electrophoresis, or the like.
The invention is described in more detail in the following exanagles,
These examples show only a few concrete applications of the invention. It is
self evident for one skilled in the art to create several similar
applications.
Hence the examples should not be interpzeted to narrow the scope of the
invention only to clarify the use of the invention.
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Example 1
i
Materials and Methods
Bacterial strains and vector
The Escherichia evli strain DHSa (F' ~80dlac~MlS ~(lacZYA-
argk~)U169 deoR recAl endAl hsdRl7 supF.r~4 1,- thi-1 gyrA96 relAl; C3~itmo
HRL), was used in routine manipulations. The pesiplzsmic-leaky strain E. colt
4924 N/14 (de Zwaig et al., J. Bacteriol. 9~:I112-1123 (1967)) was used for
the cloning az~d detection of xy(anase genes. Streptomyces livtdans strain
1326
was kindly provided by I7. A. ~Iopwood (John Inner Institute. Norw»t,
ID U.K.). S. lividarrr strain 10-164, a mutanc of S. ll ldans 1326 negative
for
xylanase and cellulose activities (I~ondou, F. et al. , Gene 49:323-329
(x9$6)),
was kindly provided by D. Kluepfel (Centre de Rechcrche en Microbiologie
Appliqu~e, L$val (Qu6bec), Canada), All the a~e~ s~~ were wild-
type isolates from various natural materials. The shuttle E. coli-Streptomyces
vector pFD666 was described previously (Denis & Hrzezinski, Gene 111: I 13-
I18 (I992)),
Growth off' 6acterfial strains
E. colt strains were grown in Luria Ber'.ani (LB} medium (Sambrook,
J. et al. , h~oleeular cloninS. a laboratory manual (2nd edition), Cold Spring
2a Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).
Act~nomyrxte sa~aing were routinely propagated on Tryptic Soy Broth
(Difco). The media for S, lividans protoplast preparation and regeneration
were as described by $opwood et al. (Genetic manipulation of Streptonryces,
The John Inner Fouttdation~ Norwich (I983)). Lang-term storage and
handti~g was as described previously (Fink, b. et al., Biotech. Len. 13:84~-
85a (1991)).
SENT BY:S H G & F s 7-29-94 ~ 13.37 ~ 5KG&F-~ 6~ S & H~it26
~~~~~'~Z
-2L-
Chemotaxonorr:acul procedures
The diaminopunelic acid form in the exll wall and the predominant
sugars in whole-cell hydrolysates were analyzed by thin-layer chromatography
~or~g to Staneck & Roberts (Appl. Microbial. 2$:216-z31 {L974)). The
G + C content of total DNA was estimated by the method of Ulitzur
{Biochim. Biophvs. Acts 2?2: I-11 (1972)x. Fatty acids were analyzed by the
procedure of Sasser (Sasses, M., in Methods in Phytobactertology, I~Iement
& Sands, eds., Akademiai I~ixdo, Budapest (1990), pp. 199-204).
Biochenaicua' assays
Xylanase activity was assayed using the Nelson-Somogyi method
(Spiro, R.G., Mdth. F,rc~ymol. 8:3-26 {1966)) which measures the release of
reducing sugar from 0.$ 96 (w/v) soluble oat spells xylan is citrate-
plaosphate-
borate buffer (Teorell buffer). Tn standard conditions, the pH was 5.0 and
incubation was for 10 min. at ft0°C. The reaction was terminated by the
1S addition of the first reagent of the redctcing sugar assay. ane unit of
enzyme
activiry was defined as the amount of enzyme releasing I .mole of D-xylose
equivalent per minute in standazd assay conditions.
The ~3-xyIosidase activity was measured with 2 mMp-nitrophenyl-~-Ia
xyloside as s'.tbstrate. Incubation was for 10 znin, at 4Q°C in Teorell
buffer
pH 5.0, The release of p-nitrophenol was monitored at 410 nm.
Total protein was measured by the method of Bradford, M.M. Anal,
Biochem. 7x;24$-254 (1976) using the alkaline reagent descr'bed by Stosehecl~
{StasCheck, C.M., Anal. Bioehem. 1$4:111-llb (1990)). The molxular
weights of flue purified enzymes were estimated by SDS-PAGE (L.aenunli,
U.K, ll~ature z276gp~g5 (1970)). Coloration for glycoproteins with the
Schiff reagent was as described in Glossman & Neville {1971). Thin-layer
chromatography of hydrolysis prod<ccts was performed as described by Hiely,
P. et al., Biochtm. Bioplrys. Acts 1162:246-254 (1993).
i
SENT BY:S K a & F ~ t-29-94 ; 13:39 ; SHG&F-~ G~ S & ii;#2~
~1~~~'~~
_22-
~'he procedure of Hertheau, Y. et al. , Anal. Biochem. 739:3 83-389
(1984) was used to analyze crude or purified xylanases by electxofocusing in
an ultrathin polyacrytamide get (pH gradient 5 to 8). Ten p.I of 20 times
concentrated supernatant were applied, Standard proteins (Bio-Itad) were
applied on tliese gels alongside the culture filtraoes to estimate the pI of
xylanases. An agarose-RBB xylan overlay was used to detect xylanase
activities. The overlay gel was prepared from a mixture of 0. $ ~ agazose and
0. 2 ~ RBB-xylan. The agarose_RHg xylan get was overlaid onto - the
elxtrofocasing gel. Incubation was carried out at 50°C for 1 hr. Clear
zones
i0 in the overlay gel indicated xylanase activity.
Tn liquid culture, xytanase-positive actinomycetes were inoculated into
T~ryptic Soy Broth and cultivated with shaking at 50°C. Once an
appropriate
cell density was reached, the mycelium was recovered by centrifugation and
inoculated into xylanase production medium (MorosoLi, R. et al. , Bivchem.
J. 233:587-592 (198x)) contaiair~g oat spelts xytan as tt~e main carbon
source,
Xylanase activity was measured daily using a standard assay (measuring the
release of reducing sugar from oat spells xylan incubated with culture
supernatants samples for i0 min. at 60°C, pH 5.0).
Bacterial, bacteriophage and plaamid preparations
The bacteriophage M13K07 (Vieira and Messing, Methods EnZymal.
153: 3-lI (1987}) was used in the production of single strand DNA. The
vectors pFD665 (Dents and Brzezinsl4., Gene li: 115~11$ (1992)), pIJC118,
pUC119 (Vieira and Messing, Methods Er~ymal. 153: 3-i 1 (1987)), and
pUC21 (Vieira and Messing, Ge,~ 140: 189-194 (1991)) were used for
cloning and sequencing proposes.
Culture media
SENT 6Y:S K G & F , 7-23-94 ; 13:38 , 5KG&F-~ G, S & H;ii28
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LB mediua:~ was used during the preparation of compeCent cells and
thezr transform~axion (Sambreok et al. , Molecular cloning. A laboratory '
manual, Second edition. Coil Spring Harbor Laboratory Press. New Yark.
(1989)}. LB-R8B-xylan (LB+ 0.29 R.13H-xylan + 1.5~ agar) was used to
detect xylanolytic clones. RBB-xylan is a complex deriving from the joining
of a coloring agent, ltemazol Brilliant Blue (RBB) to xylan. This wac
synthesized by following the protocol published by Biely et al. Anal. Biachem.
144: 14x-146 (19$5)).
The medium M13 was used for the production of xylanase (Morosoli
et al, , ,~iociaem. J. Z39: 587-592(1986)). The composition of the medium is
as follows: IOg xylan, 1.4g (NH4~SO,,, 2,5g K2Hp04, I.Og KFizP04, 2.Og of
extract of yeast, 1.0g peptone, 0.3g MgSO, ~?Hz0 per liter of water. The pH
is adjusted to 7.0 after sterilization, then 1.0 ml of a solution of znicro-
elcznents is added (0,28 CoClz~7Hzp, 0.5g FcSOd~7Iiz0, 0.168 MaS04~H~~,
0. l4gZnSQyHiO, in 100 znl of distilled water with the pH adjusted to 3 with
HCI). Olive oil (2 mi/liter) was added to increase the enayme secretion
(Bemand et af., Biotechnod. Bioeng, ~3: '791-7g4 (I989)),
The minimal 1~BB-xylan medium was used to detect xylanolytic
activity. The method was adapted in accordance with Kluepfel'g protocol
(Methods $n.Zyrrrod. 1~:18d-IB6 (I988)). Part A is autoclaved separately,
containing Q. Sg KiHp04, 0.2g MgSO, ~7Ha0, 1.Og (h~~SO~, 158 agar in a
volume adjusted to 70bm1 of v~ater, rhea part B containing 2g RHB-xylan in
300mI of water is autoclaved. ~;,fter cooling and mixing party A and B, 1 tnt
of the micro-element solution is added.
R2YE medium was used for the transformation and re.8ertaratiaa of the
S, livldatas 10-164 protoplasts (Hopwood et aL. , Genetic martipulariart o,~
Streptonrycrs, a laboratory manual, the John Innes Foarndation, Norwich,
1985, 338 pages). TB medium was used for the far the ampli~eation of E.
colt (Sambrook er ad., , Molecular cloning. A labvratvty manual, SecO~
edition. Cold Spring Harbor Laboratory press, New York, (1989)). TSB
medium was need for the growth of S. divldans 10-164 and ~lcti~tamadura sp.
SEiVT BY:S K G 8 F ~ 7-29-9A ~ ?3.39 ~ SK3&F~ G~ S & H;ii29
tj~2~~ S
FC7 preparatio:~. 2xYT medium was used for the groductivn of single strand
DNA with the E. crrle TG1 preparation. 'This medium is composed of 16g
tryptane, lOg extrgct of yeast and Sg of NaCi for a final volume of 1 liter at
pH 4.
r
Hestrfctian endonuc~eaae, ligaee and phospltatase
Restriction eadonucleases and ligases were purchased from Boehrigger
Mannheim and from Pharmacia. Calf Intestine Phosphatase (CIF) comes from
Pharrnaeia. These enzymes were used in accordance with the manufacturer's
instructions.
1Q lPrepaxatlan of cells, protoplaeta, and thefr tt~aatsforntatioa
E'. cvli I)HSc~', TQ1 and 4924 NI14 competent cells were prepar~i
and transformec° in accordance with a protocol from the Imperial Cancer
Research Foundation, and described by Desmarais, D., ~~rioire de maftrtre.
D6partemeut de biologie. Faculty des sciences. Usiiversit~ de She~rooke. 75
1 ~ p. ( I990) . ~3riefly, the following procedure ways used.
~r ~tion of c~~;t cells
1. Starting wittx the frozen cells of the E. cola DI~Sa preparation
preserved in 20~ glycerol, smear the Petri dish with SOB or
LB (Maniatis eg rxl., Molecular cloning< a laboratory manual,
Cold Spring Harbor Laboratory, N.Y'., 1982, 545 page9
(19$2)) and incubate overnight at 37QC.
2. Inoculate S mI of SOB culture using a single colony.
3. Incubate tlar culture at 3740 under agitadan for about 2 hours,
or to the point of A~ is about 0.3 or tin it begins to becornc
cloudy. '
4. Make a 1.:2p . dil:~~n of the cult4ze in 100 ml t~f SO~
(preine~abated to 37~C) and incubate at 37oC to the poi.aat Of
SENT BY:S K G & F . 7-29-84 ; 13:33 ~ SKG&F~ G~S & H;ii30
e~ ,i a
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Asp is 0.48 {a.bou: 2 hours). This optical density
is optanal for
DHSa and may be slightly different for other preparations.
5. Leave on ice far 5 minutes.
6. Centrifuge for IS minutes to pallet cells.
7. Remove the flogting matter aztd once again suspend
the cells in
40 ml of TPB I (defined below).
8. Leave on ice foe 5 minutes.
9. Centrifuge per item number 6.
IG. Remove the floating matter and once again suspend
the cells in
4 ml of 'I'>?H I.
11. Ixave on ice for :5 minutes.
I2. Distribute 200 Fal via 1.5 microfuge tube (refrigerating
the
microfuge tubes. pipette tips and pipettes tv 4~C
i9 preferred.
13. Freeze in dry lee.
I5 I4. Ivlaintain the aliquotcs at between -60 or -'FO~C.
~B. 'I~assfortt~t~on
I. Defrost the cells to room temperature just enough
to liquefy the
suspension.
z. Txave for IO minutes in ice.
3. Add fINA (up to I!5 vplume of the cells; use no more
than
100 ng of IaNA for 200 pl of cells). Using freshly
prepared
cells, begin the protocol at this stage.
4. Ixave an ice #or 3~0 minutes.
5. Incubate the cells at 42oC for 90 seconds. This stage
may be
optimized ire accordance with the preparation.
b. Put it on ice for 1-2 minutes.
7. Add 4 volumes of SOB or L13 (800 p,l per 200 ~d of
cells).
8. Irtcttbate at 3?o~C for 1 horaa~ (agitation is preferred
buE
unnecessary). .
CA 02129172 2000-08-17
-26-
9, Centrifuge for 1-2 tnitrutes in a microcent3rifuge and resuspend
the residue in 200 ~l of SOB or LB.
10. Spread on a SOH or LB Petri dfsh with antibiotic,
N,B, _ All centrifugings and solutions must be carried out and
conserved at 4oC respectively. It is preferable to delicately
handle the cells during tlu stages of resuspcnsion.
TFB I conaains 30 mM potassium acetate, 100 mM RbCla, 10 mM
CaCIz2HZ0, 50_m_M Mn.C>'~4H~0, and 159 glycerol. Adjust the pH ta5.8
using 0.2M of acetic acid. Use a 11100 acid dilution of glacial acetic acid:
this corresponds to about 50 drops for 200 ml of solution. Use of distilled L.
water i' preferred in a glass system. SteriIite through filtration.
TFH II contains 10 mM MOPS, 75 mM CaClz-2HI0, 10 rnM RbClz,
and 153b glycerol. Adjust the pH to 6.5 with 1M KOH (about 35 drops).
Sterilize through filtration.
The S. iividan.s 10-164 protoplasts were prepared and transformed
according to the protocol of Hopwood et al. Genetic manipulation of
Streptomycea, a laboratory manual, the John Inner Foundation, Norwich, .
1985, 338 pages.
Purification of a DNA fragment on agawr gel
FoLlow9ng DNA band migration on TAE agar gel (Maniatis et- aI. ,
Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory,
N.Y., 1982, 545 gages), the purificadon of DNA fragments was performed
by the method suggested by "Gone Clean" BioICan Scientific, Iac.
Zy~ogram
'Trademark
SENT BY: S K G & F ; ~-2~-94 : ~ 3 ~ 40 ~ SKG~F'' Ge S & H;t#32
212~1'~2
-27-
The procedure is the same as for a polyacrylamidc gel {SDS-PAGE)
except that the sample has not been boiled, and, further, 296 RBB-xylan is
added into the acrylamide mixture. The protein sample is prepared with the
following {3X) swab: 3.0 mI of glycerol, 0.6 g of SDS, 0.228 g of Tris-Base,
and Q.1 mg of brompherol blue.
The development of the xylanalytic activity is achieved by soaking the
gel in 100 mI of Tris-HCl ~0 mM {pH 7.5) - methanol 20~ at room
temperature for ti0 minutes. Thea the gel is washed in 500 m1 of Tris-HCi
50mM {pH 7.5) - EDTA {1 mN~ at 4~ overnight. The visualization of
enrymatic activity is achieved by incubating the geI at SO~C in s McIlvaine
buffer (pHb) until the bands $ppear.
Extraction of genomic and pla~mid DNA
The plasmid DNA extraction protocol used was the one described by
Maniatis et ai. , Molecutur cdontng: a laboratory manual, Cold Spring Harbor
i5 Laboratory. h'.Y., 1982, 545 pages.
The genomic DNA extraction prowcol used to extract Actinornadura
sp. FC7 DNA was that of Rao et a1. Methods err~ymod. 153: 1~6-198 (1987)),
except that the mycelium of the Acttnomariura sp. FC7 (20 tnl) was broken by
passing French's press $t a pt~ssure of 2,000 lblpo~.
Lxample 2
Screenlrtg ,Yr»gram ~'vr the lsalat~on of .Tylaaiotytic Actlnomycetes
In order to find new variants of xylaxtases, ef~ci~nt at pH 4 and
70°C,
a screening procedure was developo<i to identify organisms showing such
activi4~es, The screening was oriented towards actinomycetes as they are
efficient producers of tiny extracellular enzymes and are amenable to genaic
and molecular anaPysis.
SENT BY:S It G 8 F ; 7-2°-5~ ; 13:40 ~ 5KG&F-~ G~ S & H;ii33
~~2~'~~
-2s_
S~unpies of cornpnst, manure, straw as well as sarrzples of biofilm
cieveiaped on tt~~e inside surfaces of pipelines used by the paper industry
were ,
e~~icbed for triercnophilic actinomycetes by several treatments: dry heat
treatment (12,0°C; 6C3 rnin.) (hionomura & Hayaleawa, in Biology of
rz~tinorrry~ceres '88, Olcarni, Y. et al. , eds. , Japmn Scientific Societies
press,
Tokyo (1988), ~rp. 288-293); selection with phenol (30 min. treatmenx in 1.5
wl v pbenGI soiut;on, pH 6.~ at 30°C) followed by centrifugation and
washing
with water (Planotnura & Hayakawa, in Biology of actinorrrycetes '88, 0kami,
~. ea al., eds., Japan Seiezttifie Societies Press, Tokyo (1988), pp. 288-
293);
14 selection on humic acid-vitamin agar ('Fiayakawa ft Nonomura, J,
,~et~rtent.
T'ect~. d5;5f~1.-509 (198'0 ar cultivation on semidry xyian powder, as
described by 1'Naldron et al, (Appl. Micro~tp, ~oteeh. 24;477-48b (1986))
except ttzat xylan was subgtivtuted for cellulose.
If desired, novobiocirt (50 mg!1) may be used to eliminate mobile
15 bacteria in ttie first selection of aetinamyeete eoloniES. Some
thermophilic:
actinc~xnycete strains may be killed wb~en novobiocin is used in this manner.
Howeuer, td'zc strain of the invention, Actarcornadrrra sp. FC7 seems to be
relatively resistant try novabiocitt
A,~ter these treatments, surviving bacteria were plated on Tryptic Soy
Agat and eultivateQ at 5p'C ar 50°C. Individual colonies were
picked and
inoculated an minimal agar contaitai_,n.,g 0.2 ~ xylan. covalently bound to
ltema~ol $riliis~-tt Blue (RH$-xylaa; Biely, P, et at. , Aril. Blochem.
1~4:142-
1.46 (1985)) and incubated at 5t?°C ar 6Q°C. Each day, the
caloaies were
examined for medium clearing and morphology. Xylanolytic actinomycetes
were retained for further studies.
A total of 12 strains growing at temperatures between 50° and
c50°C
t~ show~:ng marked degradation capability of RB&xylan on solid medium
were isolated froze compost, zr~anure or straw (Table 1). Alt these strains
were ctassia~ed in tt~ actinomycete grattp on the basis of their morphology
aztd
30 the huh ( ~ 65 mol 9~) G ~ C content in their total DNA.
SENT BY:S I' G ~ F ~ "r°~a-94 ~ 13:41 . SHG&Fi G. S & H~ti34
21~~~.'~~
-29-
All the strains were examined for their ability to produce ~cylanolytic
enzymes that were relatively active at pH 4, '70°C. For this purpose,
alI the ,
strains were cultivated in tryptie soy broth at 50°C (except the
control strain,
S. lividans 1326 which was grown at 30°C), then inoculated in
xylanase .
production medium. Extracellular xylanase activity ws.s measured by the
release of reducing sugars from xyian in two different conditions; at
60°C, pH
S.0 (the xylanase activity measured in these conditions was taken as 1000,
and at 70°C, pH 4.0 (strnngent conditions) (Table I). Sia strains (as
well as
S. lividans J,326) kept 596 or less of taeir activity in the stringem
conditions;
three strains retained between 596 and 20~ and three strains retained more
than 4Q~. The strain FC7 (originating from manure and isolated on humic v
acid-vitamin agar) retained 65~ of its activity a:.'70°C pH 4. This
strxan was
chosen for fttrther studies since its crude xylanase was also efficient at
hydrolyzing the xylan cotttxined in the hemicellulose liquor.
SENT BY:S K ~ & F ~ °W °-94 r 13:41 ~ 5K~&F~ u~S & H,#35
-30-
'able 1 Summary of the isolation o~ xylanolvtlc thermophilic
actinornycetes
Xylanolytic
Isolate Origin Bnrichtnent methodactivity
kept at pH
4170C t
F 1 manure dry heat 2 14 9~
F2 mature dry heat + phenol296
3
FAA3 manure solid enrichmentI29~ -
4
FC7 ~~ ~_ag~ s _-65~
FY5tl4 manure phenol 3 9b
FPdt35 manure phenol 296
pAl straw solid enrichmeru~9~
CAI compost solid enrichment5796
CCA3 compose solid earichtnent5096
CCA5 cotrtpost solid enrichment2096 i
CCA601 compost solid enrichment296
C604 compost -- 2 96
S, livictanscvnttoi 296
1326 strain
i: Activity xt pH 5lbCr°C was taken as 10096.
2; Dry heat treatment (1?,0°C, 60 min.) (IrTonomtua & Hayakawa, in
Biology of
actinomycetes '88, Ckatui, Y. et al., ods., Japan Scientific Socaetiee press,
Tokyo
(I988), pp. 28&293).
s: Treattnont in 1.596 phenol {30°C, 30 tnln.) (Nonomurs & Hayakawa,
op. cit.).
4: Modified after Vttaldron lr., C.R. et rtl., Appl. Mlctobio, Birxech. 24:477-
486
(1985).
5: Hutriic-acid - vitamin agar selection (Hayakawa & Nonamura, J, Ferment-
T'ecla.
65;501-509 (1987)).
The FC7 strain demonstrated a typical actinomycece morphology with white-
yellow basal mycelitaat when grown on tryptic soy agar. Tn liquid cultu~ts :n
Tryptic
Soy Broth medium, the growth of FC7 (estimated as wet weight of mycelium per
ml
of culture broth) was maximal at 37-50°C, m~odeeate at 30°C and
60°C and very slaw
at 22°G. Spotulation was obsorved only oacc: the FC7 was monosporic,
with spores
produced on very short sporophores in. a poorly developed aerial mycelium.
rrseso-
SENT BY:S K G 8 F . 9-Z9-9C ; 13:42 ; 5K~&F-~ G, S &. !i;id36
X129172
-31 _
diaminop;,molic acid was found in the cell wall peptidogly;..an,. No mycolic
acids were
found. Whole-cell sugars had no diagnostic value as they varied widely with
the
temperature at which the organzsra was cultivated. The relative abundance of
hexadecanoic (26.45 ~ of total fatty acids content), 14-methylpentadecanoic (I
1.28y6)
and 10-mekhyloctadecanoie (10 75 ~) acids in the fatty acid composition
(pattern "3a"
according to Kroppensredt, Ft.M., in Chemical methods in bocteriat
sysrematics,
Gaodfellaw & Minnikin, eds., Academic Press, London (1985), pp. 173-199), in
conjunction with the other taxonomic data, permitted the classification of FG7
in the
"Actinomadura-Thermomonospora curvata" group of the family
Thermomonosporaceae (I~roppenstedt & Croodfellaw, in Tire Prokaryotes, Balows
et at., eds., Springer-Verlag, New York (1992), pp. 1085-1114). The strain
will thus
be referred to as Actirwmadura sp. PC7. No attempts were made to classify this
strain at the species level. This bacteria synthesizes xylanases which
maintain most
of their xylanolytic activity at a temperature of 70oC and at pH 4. By mean$
of a
zymagram it was determined that this preparation would produce up to 4
xylanascs.
Example 3
Clortirtg of Aettnonaeiduna sp, FC7 xylanuse~ genes into E. colt I»Sa
Preparations of Bscherichia coil DHSaF' (Hethesda Research Laboratory) were
used for cloning manipulations. For gem bank construction, fatal DNA was
isolated
from Actinamadura sp. PACT by the method of Rao, R.N. et at., Meth. Ert~ymol.
153:166-198 (1987). Genomic 1~NA of the Actinomadura sp. PC7 preparation was
completely digested with the restriction endornzclease Bgtll. The genome of
the
Acttnomadura sp. FC7 preparation, following a complete digestion by BgilI,
generated fragments with an average size of I2 kb, v
The BgIII fragments were spliced into tlhe pFD664 vector that had first been
cut with Bttm~iI and dephoaphorylated in accordance with the protocol proposed
by
ivianiatis, T., et al. (In: le~olecular Cloning, A Laboratory hlanuad, Cold
Spring
Harbor Laboratories, Cold Spring Harbor, NY (1982)). ~'. coil DHSaF' (200 ~cl
of
qualif'uad cells) was transformed with 100 ng of binding mixture. The cells
were
SEPaT 'ti4':5 't1 G 8 ~_ ; 7-2~-94 ; ? 3:42 ~ 5KG&F-~ G. S & H;~i~7
212172
spread out an solid ~.$-RBB-xylan plus kanarrlycin (50 Icglml) then incubated
at 37~C
foz5to6cays. ,
The effectiveness of resultant recombination was 86 ! (about 9,000
ez;omb:nauts out of 10,500 examined). The number of recombinant preparatior3s
was
S assessed in accordance with the mini-prepazation method Maniatis, T. , et
ad. (In:
Molecular Clo»ir~g, A Labdratoty Manual, Cold Spring Harbor Laboratories, Cold
Spring Harbor, NY (1982)). The gene bank represented more thazt 99~ of the
genome of Actlrao»iadura sp. FC7. This pezcentage was derived from the formula
described by Clarks arui Carbon, Cell 9: 91 (1976).
1Q Six potentially positive xylan~iytic clones were obtained aver 5 to 6 days
of
incubaaor~ following the appearance of degradation zones. Following a
respreadi~g
. on L13-RBB-xylan znedittm, five positive clones, or pJFl, pJF3, pfF6, plFB
and
pJFIO were identified and selected, while the other closes (pJF2, piF4, pJPS,
pJF7
and pJF'9) were eliFninatad as false-positives. Restriction endorniclease
analysis
i
IS conf"~axed that cloa~s pJFl and pJF3 had an insert of an approximate size
of 2Qkb,
white clones pJF~ an;.-l p,TF$ ara,~d hive the same 2.7 kb insert, but in an
opposite
onentat:on. Clone pJFlO ha;3 multiple BgIII inserts, of which one 2.7 kb
insert was
identical to the onr~ found in pJF6 and pJFB.
.~, eoli is a c'~raxn-negative bacteria, and it is not down to be effective
for the
20 section of enzymes. No~theless, positive clones were isolated thanks to the
natural
lysis of bacteria. In other words, as a result of the release of the contents
of the E.
cold past iota the ~nediunx containing RHB-xylan; since the recombinant host
expressed the xy lanase gene, it produces a degradation zone around it,
occurri~ag after
to 6 ~ys of incubation.
25 ~~pte , f
Cloning of Actlnofiaduraa sp. FC7 xylanasB genes trtlo E. call 4924 h'lId
The plasmids from tire gene bank described is exempla 3 were isolated bg~ a
total plasmid preparation as.proposed by Maniatis, T., et al, (In: Molecular
Cloying,
A Laboraa.~ory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY
StNT BY:S K G & F . 7-29-94 ; 14:44 ; SKG&F1 16135639869#-;# 2
ia~a . o ~ ~~'~bs t~=~.
21291'2
-33-
(1982)) and transformed into E, codi 4924 N/14. After ligatiop, the DNA
mixture
was used to transform competent cells of a periplasmic-leaky strain E, codi
4924. The
transformation mixture was plated on LB agar containing SO ~,g/ml of kanamycin
and
0.2 tng/ml of RBB-~cylan. A total of 8850 recombinants was obtained. After 2-3
days
S of incubation at 37°C, colonies surrounded by clear areas were
picked, grown in LH
liquid medium and repIated at low density on RBB-xylan medium. Six of these
recombinants showed clearing of RBB-xylan after 2 days of incubation.
In order to speed up the operations involving the visualization of degradation
zones, we used the E. coli 4924 N/14 preparation. E. cola 4924 N/14 has a
IO periplasmic deficiency which has not been genetically defined (de Zwaig and
Luria,
J. Bacteriol. 94: 1112-1123 (1967)). So this allows the very swift passage of
its
periplasmic contents to the external medium. The main reasons for its use .are
a
better visualization of degradation, and an economy of time. Clone p3F11, for
example, was isolated thanks to this preparation, because the sensitivty of
the method
15 with E. coli 4924 N/14 was probably stronger than with E. coda DHSa. The
appearance of a degradation zone needs only 16 hours instead of 120.
The ability to k~ydrolyze ItBB-xylan was conserved after plasmid purification
fzom all of these recombinants and retransformation into a new host. Since
xylanolytic activities were detected in recombinant E. coli strains and since
E. cold
Z0 is not known to produce xylanolytic activities, the cloned genes should
encode
xylanases and not a regulatory protein involved in xylanase production. **
The clones that appeared to be able to hydrolyze RBB-xylan after this second a
round of plating were retained for fiutlier studies. Their plasmid DNAs were
e~ctracted and mapped with restriction enzymes using standard methods
(Sambrook,
:~5 J, et al. , Modecular cloning, a Cabaratory manual (2nd edition), Cold
Spring Harbor
Laboratory Press, Cold Spring Harbor, New York (1989)).
Plasmids pJFI, pJF3, pJF6, pJF8 and pJFlO were analyzed by restriction
mapping. pJF1 and pJF3 carried the same cloned insert (about 20 ICb) but in
opposite
orientations. The other three plasmids also had a common cloned segment (2.7
kb),
either in two opposite orientations (pJF6 and pJF8) or fused to another
segment
(pJFlO). This segment was differezxt from the insert present in pJFl arid
pJF3. Thus,
SENI BY:S K G 8 F s "-29-94 ~ 13:43 ~ 5KG&F-~ G~ S & H~it39
~12~1'~~
-34-
the transformants foil into two distinct groups. One transformartt from each
group
(pJFI and pJF6) was chosen for f~.~rther studies. The xylanase-encoding
s~gtnents were
mapped by deletion subcloning, transformation and plating on 1RBB-xylan agar.
The
shortest DNA segmenrs still allowing for xylanase expression are shown on
Figures 1
and 2. The differences between the restrictions maps suggested that two
different
xylanase genes were cloned: xlnl carried by pJFI (Figure 1 j and xlnT1 carried
by pJF6
(Figure 2). The corresponding xylanases were named Xyl I and Xy1 II,
respectively,
Clone pJF6 was chosen for sequencing as it presents the interesting
characteristic of
living a relatively short Insert (2.7 kb), and required no extensive
shortening of its
inert by sub-cloning.
The ~aml~ site of pFD666, used for insertion of the xylanase genes, is
localized inside v>F a multiple cloning site flanked by transcriptional
terminator8.
However, it has beext shown that some transcription occurs from one side, most
likely
driven by the neomycin resistance gene (penis, F., ~Construction d'un verteur
is navette pour Eschcrichia cell et les actinomyebtes et clonage d'ur1 gene de
chitosanase
d'actinomyc~te," Ph.D. thesis, UniversitE de Sherbrooke {1994), 120 pp.).
Since
the xylanase genes were cloned in both orientations in the BaneHl site and
xylanolytic
were detected in. E. cell recombinant strains, whatever their orlentatians, it
seems
likely that some Actinomadura promoters can be recognized in E. cell.
Zymogram analysis revealed that ~ xylartases are pzoduced by the clone plFl .
These pJP1 xylanases would correspond to tht the two highest rnoleeular
x~eight
bands produced by Actinomadr~ra sp. FC7. Zymogram analysis of Actinomadura sp.
PC7 culture supernatants in xylan medium revealed two major (slo~vec) arbd two
minor (faster) bands of activity (not shown). The two major (slower) bands ca-
migrated with the two bands obtaaned with the crude preparatio!t from 10-
164(pJFl)
culture supernatant and corresponded most probably to the ~ IGDa and 37 kDa
forms
of Xyl I (sex below). TTfle activify of ~yl II could not be visualized with
this
particul$r zymogram system, probably because of the inability of this protein
to
renatu~ during the post incubation steps. Thus, besides Xyl I ane! Xyl Q, FC7
produces at least one or two other xylan-degrading activities. The ocairtenet
of
SENT BY:S K G 8 E= , 7-28-94 ; 13:44 ; SKG~Fi G.5 & H;ii40
2121'72
-35-
multiple xylanase activities have been reported in numerous microorganisms
(VVo~tg,
K.K.Y, et ad., Ma'crobial, Rev. 52:3~~-3I'7 (1988)).
The pJFI clone insert was reduced, yielding the sub-clone pJF1020. The
latter has an insert of about 7.5 lCh, which is sufficient to contain witfia
it 2 genes
coding for xylanases. The genes) is located in a 5 k13 portion of the inital
fragment.
Example 5
XyXanase producsion by recombdr~ur~t strudns
The plasznids isolated from the E. toll clones that were able to hydrolyze
RICH-xylan were used to txaasform protoplasts of S. ldvidans IO-I64. After
pmtoplast
IO regeneration anal colony selection far kanamycin resistaz~e, the
transformantb, were
te$ted for their xylanase-positive phenotype oa minimal medium (Hogwood, D.A.
et al. , Genetic manipudatdore of StrEptomyces, The John Ynnes Foundation,
N'orwieh
(1985)) containing RHB-xylan.
Plasmids, p~FI and pJF6 were used to transform S. tivadans, as ~'. cold is not
an efficient host for extracellular enzyme production. The mutant strs~in S.
ldvd~Cans
10-164 was ascd because of its inability to produce endogenous xylanass and
cellulose
activities (lvfoa~ou, P. et al., Cxhe 49:323-324 (I9861). Both plasmids
complomcnt~ed the xylanase-negative phenotype when transferred into the IO-164
strain. This host allowed over-production of the xylanases encoded by the
cloned
genes with a low background of other proteins, thus facilitating the
purification
procedure.
Qne transformant of each type bearing tk~e pJFI and p)Fb plasmids were tested
for xylanase production In Iicluid culture. Each transformant was inocuaated
into
Tryptie Soy Broth (1)ifev) containing 50 ~glm1 of kanamycin sad cultivated for
48-72
hours at 30°C on a rotary shaker at 250 rev./min. The mycelium was
recovered riv
centrifugation of the cultux-~es is a benchtop centrifl~,ge (3,000 g; IS min),
suspendc9cl
in 50 ml of 0.99b sterile saline send centrifuged again. 24 ml of mycelial
pellet wore
then inoculated into Z.2 liters of ~xylanaae production medium tMvrosoli, R<
er al. ,
8doche~na. 1. Z3~':587-~~2 (I986)) without kanamyein (the vector pFD666 and
its
CA 02129172 2000-08-17
-36-
derivatives are generally stably maintained in Streptomyces Lividans without
antibiotic
selxtion (Denis ~. Brzez.inski, Gene 111:11~-118 (1992}). After 72 hours of
cultivation, the culture was cen~_rifug~d (11,000 g, 30 min, 4°C) and
the supernatant
was r.-.covered as the crude enzyme preparation.
g . Examplr 6
Purgation of xyla~cares 1 and 11
All the purification steps were, carried out at 4°C. The chilled
supernatant
(0.6 liter) of a cultuzc of S. lividans 10-164 {pJFI) (for xylanase I
purification] or S.
lividaas 10-I64 (pJF6) (for xylanase iI purification) was mixed with three
volumes ,
of ice-coil 95 ~'o ett~.anal. After settling overnight, the precipitate was
recoveteci by
centrifugation (9,000 g, 30 min}. The pellet was .resuspended in 50 W 1 of 20
ml's
Tris-HCl buffer pH 8.0 and loaded on a 0.9 cm x 30 cm DEA~BioGeI A anion-
exchange column (Bio-Rad) eduilibrated with the same buffer. The colutzin was
then
washed with 50 tni of the same buffer and proteins were eluted with a linear
gradient
(0 to 0.6 urn of KCl (tonal volume: 120 ml}. Pz~actions were collected and the
xylanase activity was detected by spotting 20 ~d samples on RBB-xylan agar and
incubating at 37 °C. The active fractions were pooled, wncentrated dawn
to 4 ml by
dialysis against Concentrator Rcain (H3o-Rod? and loaded on a 1.S cm x 100 cm
BioGel A-0.3rz size-exclusion chromatography column (Bio-Rod) equilibrated
with
20 mM K-phosphate buffer pH 6.0 (prtpared by mixinn appzopriatc proportions of
100 mM monobasic potassium phosphate and 100 mM dibasic potassium phosphate,
then diluting with four volumes of distilled water). Fractions were collected
and
xylanase activity was detected as before. After addition of glycerol (final
cotmcentratioa 50~ vlv), enzymes were stored at -20°C. ;
Both xylanases were purified to homogeneity (as Judged frcm Cootuassie Blue-
statt~cd SDS-PAGE gels) by the above protocol involving ethanoi precipitation,
anio~r
exchange chromatography and size-exclusion chromatography. Table 2 summartzes
the enryme purification data. Yields of 27 and 1490 wire obtained and me
specific
'Trademark l
StNT BY:S iE G & r ~ i-29-94 ~ 13~45 ~ 5KG&F-~ G.5 & H:ii42
-37.
activities in startdazd assay wit~'~ oat spelts xylarv were 178 and 126$
Unitslmg for
purified Xyl I and Xyl II, respectively. ,
Durigg the purification of XyI I, ewe peaks of xylanase activity were
separated
by the size-exclusion chromatography step. The major peak corresponded to a
protein of 48 kDa (and this protein was more extensively studied) while the
minor
peak corresponded to a 37 kDa protein. When vazious deletion derivatives of
p~Fl
ppasmid were analyzed for tl?e pattern of their protein production, the
disappearance
of the minor band was always correlated with the disappearance of the msljor
band.
We conclude that the smallez protein, is not encoded by a separate gene but is
a
derivative of the 48 kDa Xyl I protein.
The biochemiE;al properties of Xyl I and Xyl II are summarized in Table 3.
Xyl I resembles other high analecular mass/low pI xylanases (along, K.I~.Y, et
al.,
hlicrobiol. Rev. 52:305-317 (19$8)), s>.~h as XInA from S. ltvidarts
(Morosoli, R.
et al. , Biochem. J. 239:587-X92 (1986); Shareclc, P. ,et al. , Gene i0~:75-82
(1991)).
ar XynA from "Caldocellurn saccharolyticurn" (L,dthi, E, et al., .~lpPt.
Envtron.
Microbiol. 55:2677-2683 (1990); Liithi, E. et at. , Appl. Environ. ~ticrobtol.
~6: l017-
1Q?~ (1990): its molecular mass is higher than 40,000; it shows a low bttt
significant
aryl-d-D-Xylosidase activity and it is able to hydrolyze efficiently
xylooligosaccharides, as shown by the appearance of short oligomers
(xylobiose,
xylotriose) among the reaction products early in the hydrolysis (Table 3). In
co~r~t,
Xyl II has no detectable aryl-~-D-xylosidase activity and hydrolyzes
xyloollgosscharides much siawer than Xyi I. In this situation, short oligomers
appear
in the reaction mixture only after very gong incubation time.
The classification of Xyl II on the beeps of the data presented in Table 4 is
not
2~ straightforward. This protein has a neutral pI but its molecular mass is
much lower
than the p~:, of the majority of the ~high M,.Ilow pI~ xylanases. Also, its
high specific
activity against oat spelt xylan and its decd ability to hydrolyze short
xylooligotners classifies Xyp II nearer the low-molecular-mass enzymes with
similar
biochemical properties, such as XInB and XInC of ,~. Ilvidarzs (Bieiy, P. et
al. ,
~iochlm. 8iophys. Acts 1162:246-2S4 (i993)). Ilowevez, in Western blotting
experiments (unpabpished) Xyl iT gave a positive reaction with a rabbit
antibody
SENT BY:S K ~ & F ~ 7-2y-84 ~ 13:46 ; SK~BF~ G,S & H;ti43
~129I72
_3S_
against Xylanase A from ,~. diviciarzs (a high M,Jlow pI xylanasc), which
~3oes not
cross-react wittx the Iow hirlhigh pI xylanases Xin$ and XInC from thG same '
organism ('Jots-Mehta, S, et al. , Gene, 84;119-122 (1990)). ConseqiZently, we
assume that Xyi II is either a low M~ zylanase with an unusual neatra! pl or,
more
probably, a truncated protein, originating fZOm a high \~I~ xylanase gene or
protein.
The effect of pH on both xylanases was studied at three different temperatures
(Figures 3 and 4). The optimal pH lies between 5.2 and 5.7 for Xyl I as well
as for
XyI II. At pH 4 and 70°C (the temperature used in the screening
proceduze), Xyl
I retained 67~ of its rnaximal activity while Xyl II retained only 26'~ of its
activity
in these conditions, Clearly, the Ievel of activity observed at 70°C/pH
4 with the
crude vulture supernatant of Acrinnmadura sp. FC7 was due to the predominance
of
XyI I among the xylanase forms secreted, by this wild~type strain,
Remarkably, at its optimum pH, Xyl I retained foil activity even at
80°C
(Figure 3). At this higher temperature, the decrease of activity in acidic pH
was
faster, but still less marked than for the majority of known xylanases: 41. &
of. the
maximal activity persisted at pH 4. In contrast, even at optimal pH, Xyl II
was 8.1-
times less active at 8Q°C than aL 70°C (Figure 4).
Ta estimate the rh~rn~ ~bilit~, of XyI I, the enzyme was incubated in Tcorell
t;!uffcr in the abse~ er presence of 100 pg/mI of bovine serum albumin at
different
temperatures. Periodically, samples were withdrawn and the residual activity
was
measured by standard assay. When preincubated at pH 6l50°C, Xyl I
conserved full
activity for at least 96 hours, At pH 6l7Q°C, the half-Iife was 6 hours
in the absence
of BSA and 18 hours in the presence of BSA. At pH 4170°C. the half ~ifp
~9° ~n
hours in the absence of B.~,A and 22 hours in the presence of SSA. These
values are
2S within the range of stabilities obtained for trade thermoresistant
xylan$ses from other
~ctirrornadura species {Holtz, C. et al., Antonio vcan f,eeuwenhoek 59:1-7
(1991));
however, tticy are clearly shifted towards mere acidic pHs.
In conclusion, the screening procedure developed for the invention, based on
the simultaneous application of two stringent parameters (low gH and high
~mFere~) resulted in the isolation of a xylanoIytie actinomycete which
produces
SENT SY:E h( G 8 F ; 7-28-94 ; 13:46 ; SKSB~F-~ u, S & H;Ji44
212~I ~2
at least one xyl~.nase that rerrtains almost folly active and is very stable
irl these
conditions.
Table 2: Puri~acaki~n a~f xyl~nase x ~a a ~.om culture
supernatants of recombinant Str~ptornyces tividans 1.0-I64~
stralrls
Teta1 ~c
Protein Yiold ~~ca-
activity activity(~)
(units) (~~ lion
t~tsJmg) factor
A: Xylaaase ,
I produced
by Strtpr4myces
dividons
10-1G4 (pJFl)
Culture brothx,20 ~~ 38 ? 1.0
F_tharloi 325(? 58 56 00 1.5
ptecip. 1265 4.3 135 95 3.6
DEA&BiotJel 37
BioC3e1 A-0.5m910 5.1 178 27 4.7
B: Xylatxace
II produced
by S,trcptorrayces
tiv~dans
IO-164 (pJF6)
G~tltute 11464 Sh.8 132 100 1.0
broth
Irthauol 94X2 37.5 251 82 1.9
precip.
DEA~BioCiel 41 i5 6.7 615 38 4.6
BioGt1 A-0. 1581 1.25 1268 I4 9.6
Sm
Table 3: ~i~x;hemica~ properties of Xpl I and Xyl ~
Y..1 T
_ _" _ _ n~a ax
Molecular weigrt (afder48 I
SI3S-PAGIr; D
c 34 itDa
a
--
s-._._
Isoelectric point ~
$
. 7.I
Optimal temperitcure 75C 74C
at pH 5 1, 2
Optimal pH at 50C I, 5.2
2 5.7
'~ hy~olys~ p~u~ xylobiose, xylotriose_
r ~~er oli~oxylosidea
after 34 miu. reaction higher oligoxylo$ides.;
'
Main hydrolysis producetracos of xylose,
afier 18 h. t~actiou xylobiose, aylotrioaeXYlobiose~
t xytotriose
Azyl-p-D-xylosidase 0.13 U/mg undetectable
specific activity
SEfT BY:S K a ~ F 9-29-5d ; 13:~~ ~ SKG~Fi 3~5 & h;#45
~1~91'~~
Staining with Schiff reagent ~ negative negative
1: Detertnincd with oat spelt xylan as substrate
2: The reaction time wag 10 min,
example T
Sequence of the Insert in pJFd
A restriction map was, drawn up to allow sub-cloning of fragments -thereby
facilitating seduen~cing (Figure 5). Several DNA fragnnenis were sub-cloned
and
sequenced.
The plasmid DNA of the positive clones of the gene bank of the Actinomadura
sp. RC7 preparation contained in vector pl?D666 was digested by the Chosen
restriction endonucleases. The DNA fragments thus produced were purified by
"Gene
Clean" for subsequent ligation to vectors pUC118. pUCIlg and pUC2l. The
unidirectional exonuclease IIllnuclease S1 deletion method described by
Hsnikoff,
Methvdr enzyrnct, 155: 156-165 (lg8'7) was selected in obtaining additional
sub-
clones.
The 'Jieira and Messing protocol (Gene 104:189-194 (198'T)) modified by
Patent J-L., T6e JH.T-~ aetinophage: sequencing and promotional study.
Master's
thesis, Departrnent of Hialogy. Faculty of Sciences. University of Sherbrooke.
8l p,
(1992) was chosen foz preparation of sinigle strand DNA. Double strand DNA
preparation was completed in accordance with the "T7 Quick krime Kit" of
Phatmacia LKB Biotechnology, Single and double strand DNA sequencing was
aehievrd accozding to the method of Sanger et at. , Proc. Ncrtl. Acad. Sci.
U5:4, 74;
~4b3-5467 (19?7) fzom the °Sequenase and 7-deaza-dC,TP" set of United
States
Biochemical.
A prelinxinary cotnput,~ ~yg~ ~~ it possible to prove a very strong
seque~ing homology to Streptomyces lividans xylanase A. This made it possible
to
Iccaiizc the beginning of the OfLF' ~i~ for a xylanase by clone pHrb_ Thus,
the
xlnll gene is Localized at, and stquoncing was directed to, only a portion of
the pIP6
SENT BY:S K G & F ~ 7-29-94 ~ 13:47 ~ SitG&F-~ G~ S 8 H~ii46
21291'2
-~r-
insert, that is, from the NruI site to the BgIII site to the right of the
restriction map
for pJF6 (Figure 5).
The nucleotide sequence of the insert in p,TF6 is presented in Figure 6 and is
Genback Accession Na. U08894. An open reading frame (ORF} begins at nucleotide
521 by a codon GTG and ends probably through an end of translation codou
located
in phase next to the vector, since no terminal radon was found inside the
cloned
fragmeni. The gene would therefore be truncated and coded as active xylanase.
A
Shine-Dalg;arno sequence (GGAGGA) specific to the attachment to ribosarnes was
found at nucleotide 509, pooordi~ to Sttwhl, W.R., Nucleic Acids Research. 20:
I~ 9~1-974 (1992}, this RB$ is completely homologous to the consensus sequence
produced from 40 streptomycete genes (Figure 7). The coding region of this
gene
has a nucleotide content rich in G+C, on the order of 6$9~. Pttrthermore, the
percentage of nucleotide type (G or C) found at position 3 of the radon is
over 906,
which corresponds with the results reported by Bibb et cal. (1984).
IS According to'Wo~ et al., Microbial. Rev. S2(3); 305-317 (198$), xylanases
can be classified into two classes, either class A, which regroups the
xylansses having
a a~oleeular weight over 3~ kDa and an acid pI, while class B brings together
xylanases with a molecular weighs below 35 kDa and a basic pI. Thus this ORI~
of
1527 nucieotides codes for a xylanase of about 43 kDa, and would therefore
belong
2~ to class A.
The signal peptide of the pre-protein of this xylanase bas the characteristics
normally found in such amino acid sequences (Perlman and Halvorson, J. ~4tol.
Blot.
td7: 391-4(yg (1983}: that is, a positively charged N-terminal extremity
containing
~8s (R) followed by a long sequence of hydrophobic amino acids and a C-
25 terminal segment iztchidang a praline (P} localized near the cleavage site
(AXA) of the
peptidase signal (Figure 8).
The promoter region is typieai; that is, a spacing of 16 nucieotide$ separates
ttte -35 region (T°I'GACG} from the -10 regioa (CACAAT). This pmuwter
is
comparable to those illusarated by Sr<ohl, NucleicAcicts Research. 2Q: 961-974
(i992)
(Figure 9}. Ft~cthermore, this promoter would be quite horncalogous to the
promorrr
SE~JT BY:S K G & F ; 7-~9-94 ; 14:44 ; 5KG&F~ 16135639869#-;# 3
l Cz~o ~ a'~1~ l ~Sl,~ b s'-~~ 'f~-t'~ ~'
~129~7~
-42-
consensus sequence (TTGAC...TATAAT) found in Escherichia cola (Lewin, Genes,
John Wiley & Sons, Inc. USA, 1983, 715 pages).
The restriction map studies suggested that the 2.7 kb fragment found in clones
pJF6, pJFB, pJFlo and pJFll were identical. The extrenuties of these
fragrnEnts
were sequenced and compared with one another in order to verify this. The
results
indicated that the 2.7 kb fragment present in clones pJF8 was identical to the
one
found in pJF6. Furthermore, the fragments adjoining the 2.7 kb fragment in
pJFlO
and pJFl l appear to be the result of a multiple ligation, since the sequences
obtained
represent no significant homology to xylanase A of S. lividans or each other.
,o No translation termination codon was found in the xlnTl sequence of pJF6's
insert. The implication is that the cloned gem is truncated in its 3' part.
'This is
further suggested by comparing the coding sequence of the xylanase A of S.
livadans
with that encoded by pJF6. About 185 nucleotides appear to be truncated or
missing
for the sequence encoded by pJFd.
~5 pJF6's coding sequence has the potential for coding a 4.4 lcDa xylanase.
However, the MW of the xylanase produced is on the order of 34 kDa. There are
three possibilities to explain this. The first hypothesis is that the RNA
polynnerase is
stopped during transcription. The second is that a terminator sequence is
present and
thereby stops the translational mechanism. The third possibility is that the
protein is
:?0 naturally cleaved proteolytically after synthesis.
According to Akino et al., Appl. Environ Mtcrobiol. 55: 3178-3183 ((1989),
it is possible for the transcription to be stopped by several inverse repeated
sequences.
These authors have described a gene coding for two ~i-mannanases having MWs of
54 lcDa and 37 lcDa. The production of the 37 kDa mannanase would be due to
the
'~5 stoppage of RNA polymerase as the result of the combined presence of
repeated and
inverse sequences and a rare codon. I~ez~e, such repeated and inverse
sequences
appear between nucleotides number X538 and 1672.
These sequences also have the potential to form several secondary structures,
which could be very stable in terms of energy, for example, between
nucleotides 1538
.'30 and 1672 (Figure 12). A hairpin loop between nucleotides 1538 and 1610
has a
calculated internal energy (~G) of -55.2 Kcal (Tinoco et al., Nature. 24b: 40-
41
SENT BY:S K G & F . 7-?9-94 ; s3:48 ; SKGB~F-~ G' S & ~i;#4fi
t1973). 'This might produce a prvtyin of about 34 lcDa, but no rare eodon ktas
been
found near the latter. ,
'The second i:ypathesis requires the presence of a sequence region in the
~A. allowing the creation of a second stable structure. Ire the same area
prGniously shown, there is the potential of formit~ such a secondary
structure. This
secondary structure might possibly have the ability to slow down tine
progression c~f
ribosomes oa the mRNA so as to finally stop the entire translation mechanism,
in
order trr ultimately yield a protein on the order of 34 kDa. The third
possibility is
discussed below.
~xarreFle 8
Com~son of the sequence derived from PJFd xydunas~e
amino acids with atfier pr~otxins
I3NA sequences were analyzed with programs of the UWGCG system:
PASTA, T'FASTA, IiF.SFIT, FILEUh, PRETTY, STEMLOgP, REP$AT, MAP and
15 PROTEINSTItUCTURE upon sequences obtained from the "Genliank" and
"F~1~IHI,'~
databases (Devereux et al., Nucleic Acids Rep. 12: 387-395 1984).
The TFASTA program was used to study the degree of homology encountered
~ ~ tea acid derived sequence of xdnll as sncoded by clone p3'F~, as compared
to the sequeaices derived from proteins present in databanks.
20 The PILEUP program then made it possible to align the protein sequ~ces
derived (Figures 10-lOC). A signi,ieant homology was obszrved with tHc
fottowir~
genes: the xylanase genes of B~tyrivibrio fibrisoivens (Lin et ai. , Genbank.
Accession no.: X61485 {1991), Rurninococcus flauefactens (2hang et ol., Mal.
.ll~ierobiol. 6: 1U13-1023 1992), ?nernsoaruxerabacter sal.~charotyticum (b.ee
et al.,
~5 Gcnbanlc. Aceessioa No.: M9'1882, the C-125 alkalophile preparation
ofBaclILus sp.
{Hamamoto et ai., Ag:~zc, Blot. Chem. Sl: 953-955 (1987), Cla.stri~tum
ther»aocelium
(Grr~piz~t et u:. , .i. Bacterloi. I70; 4582-45$8 (1988) as well as two
xylanases of
Pseudomorsasfluvrescens (I~al? et~al., Moi. Microbial. 3: 1211-1219 (I989);
Kelleue
er ai., Bioche~. J. 272: 369-376 (1990), ~t~rmore, hmnologies have been found
SENT BY~S K G & F ; 7-go_g4 ; i3~49 ~ SKG&F-~ G~ S & H;#49
2191 ~2
-44-
m protejn sequences derived. fmm proteins coding for exoglucanase genes of
Cellutornonas ~tarr~i (~'Neill et al., Gene. 44: 325-330 (1986) , for
Clostridium '
.rcerGOirarium celloxylanase (Pukumura et al., I992), and lastly, with a
cellulase and
a xylanase of Caldocellum saccharol)~ticum (Saul et al. , ~spl. ~nviron.
Microbial. 56:
x117-3124 (1940); ~~ et al., Appt. Errviron. Microbial. Sb: 1017-1024 (1990),
A hoxr~alo~y of over 80~ has been observed in the xylanase A of Streptomyces
lividans (Shareck er al., Gene 107: 75-82 (1991) (Figure 11).
The alignment of sequences of proteins derived from the 13 genes mentioned
above reveals a total of 66 amino acids which were maintained with a
similarity of
la over 759~, 22 of which a~~c identical at 1009b, and therefore the possible
presence of
7 regions of retained amino acids (Figures 10-lOC).
Example 9
~~P~er ~~ction of Protease sites
The MAF Program was used to evaluate the potential cleavage sites of
proteases in an amino acid sequence. Figure I3 and 13A shows the analysis
obtained
far the sequences derived from, the amino acids of S, lividans and pJF6
xylanase.
'Y'he significant differenfie that exists between the two analyzed amino acid
derived sequences is as follows: in the vicinity of amino acid 318 encoded by
the
p,TF6 sequence, no cleavage site by Staphylococcr~r aurer~s protease was
found. In
ZQ contrast, such a site is present in the analysis of the xylanase A sequence
of S.
livlrians.
In order to approach the third hypothesis discussed above con~ecrning a
possible proteolytic mechanism for the post-translational shortening of the
protein, it's
necessary to illustrate this last point by comparing the xyianolytic proteins
groduccd
z5 by the xylanase A v f Srreptornyces lividans to that of pJF6. It must be
noted that the
xylanase A gene codes for a 4'7 kDa protein, and moreover, a second protein on
the
order of 31kI3a is visible on a polyacrylamide gel (Moreau, A., Doctoral
thesis,
Department of Microbiology and, immunology, Faculty of Medicine, Upiversity of
Montt'ssal, 1992, 140 pies). A post-translational maturation process might
explain
CA 02129172 2000-08-17
-45-
the production of this second 31 kDa molecular form. Comparisons will be
brought
to bear on This last type of xylanase.
First off, the results show that the sequences derived from the amino acid
sequence encoded by plF6 ~;ylanase and the xylanase A of S. lividans az~e
quite
homologous. It's normal to expect a practically identical computer analysis
regarding
the poasil3le protease cleavage sites known .from the two sequences derived
from
amino acids. And yet, a significant difference is revealed in the one
proteolytic site.
In comparing the analysis of the likely proteolytic cleavage sites of the
xylanase A of
S. lividanJ, the plP6 xylanasc would have one cleavage site less for a
Staphylococcus
protease. This protease would recognize glmamic acid (E), wish 318 amino;acids
for
xylanase A and 3d5 amino acids for pJF6 xylanase, to ultimately cleave in the
C-
terminal portion of the amino acid. This difference in proteolytic cleavage
pattern
might then explain the production of 31 kDa of xylanolytic protein in S.
livtdarss and
that of 34 kDa in pJF6. h is known that the pordons of a protein exposed to
IS protcolytic cleavage are prvieases that tend to cleave the protein in a
loop for example
situated between two alpha helices, or one alpha helix and a beta shot, or yet
between
two beta sheets.
Thanks to the PROTFZNSTRUCTURE program, it was possible to prove the
potentially cleavable areas using the professes on the xyianase A of S.
lividans and
2o the xylanase of pJF6. These resultsinterestinglyeolncide with the protease
cleavage
site of the Staptrylococcus discussed earlier. Tliercfore the involvement of
protease
may explain the maturation mechanism of the xylanase of pTF6 as well as the
xylanase A of S. llvidcuu.
FiEurea 10-lOC demonstrate that there is a eigni~cant homology between the
25 xylanases and eellulases. Gilkes et al., Micmbiol. Rev. SS: 303-315 (1991),
after
analyzing amino acid soquences for more than 70 cellulase and zylanases,
proposed
the creation of nitre fazniliea of enzymes. According to these researchers,
the
observation of cellulase .isoaazynaes and the xylanasea of several
nucroorganisms
would prove that these proteins would not have evolved from a single gene, but
zathsr
30 came from a large muitigenic family. Furthermore, the enzymes with a
predominant
xylan~olytic activity are classified into two distinct families. This bFings
up the
S~NT E7Y:S K G & F ~ 7-2q-94 : 13:50 ~ 5KG&F-~ G. S & ~-i~i~51
21291'2
following hypothesis: true cellulases anti true xylanases v~ould therefore
have evolved
from different genes.
Given that the xylanase A of Streptomyces lividans is so similar to the
xylanase of p3F6, the thermostability and acid stability of the xylanase of
p3F6 is
surprising. According tv lvloresu, A., Doctoral thesis, Department of
Microbiology
and Immunology, Faculty of Medicine, University of Montreal, 1992, 140 pages,
the
xylanase A of S. lividans retains only 1p9~ of its activity following
incubation at a
temperature of 60oC for 8 hours in the absence of its substrate, while the
pJF6
xylanase under the same conditions keeps alinost 95 ~b of its activity. TtAe
snnall
differences found ixt amino acid sequences of these two xylanases seems to
have
imparted a much more stable consistency for the pJF6 xylanase at high
temperature.
Example 10
Biobltachzng Using F~C'7
Approximately one liter of spent culture medium per ton of pulp Es added to
pine lQaft pt=lp; the culture medium is talcan from Actinomadura sp. FC7
cultivatians
and contains XYL I and XYL II aedvities as described in Table 3. The pulp is
incubated at a relatively high temperature such as 70°C and acidic pH
such as pH 4
for a period of time sufficient to aFlow degradation of the XYL I and XYL II
susceptible bonds in the xylan that is present. If necessary, the culturre,
medium is
filtered before use or concentrated using techniques Imown in the art. After
incubation
at the desired temperature and pH, the product is a pine kraft pulp
preparation
wherein the kappa number (the amottnl; of lignin) in T.he pine kraPt pulp is
lower
without affecting the mechanical propezties of the pulp. Additionally, the
preparation
requires less chlorine comsumption in any subsequent chemical bleaching.
SENT BY:S K G 8 F . 7-2q-9~ ; 13:51 5KG&F-~ G~ S & H;ti52
~;_
~,~~pt~ rr
Biobte~c~ti~ag Using Recam~inar~fly Produced XYd. I ar~~'or X~'L It' a
Approxiirately one liter of spent culture medium per ton 6f pulp is added to
pine kraft pulp; the culture medium is taken from cultivations of reombizant
host
cells that erpress recombinant XYL i a~/or recombinant XYJ... II activities as
described in Table 3. The pulp is incubated as described in Example 1.0, at a
relatively high temperature such as '?0°C and acidic pH such as pH 4
for a period of
time sufficient to allow degradation of the XYL I and XYL II sasceptable bonds
in
the xylan that is present. If necessary, the Culture medium is faltered before
use or
coneentrateti using techniques itnown in the art. .After incubation at the
desired
temperature and pH, the product is a pine kraft pulp preparation wherein the
kappa
number (the amount of lignin) in the pine kraft pulp is lower without
affecting the
mechanical properties of the pulp. Additionally, the preparation requires less
chlorine
comsumptian in any subsequent chemical bleaching.
