Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
"RECOMBINANT XYLANASE"
FIELD OF INVENTION
This invention relates to a recombinant xylanase derived from an
5 anaerobic fungus and a method of production of the recombinant
xylanase and clones utilised in the method.
BACKGROUND ART
Xylan is a major component of hemicellulose and the second major
component of plant fibre. Xylan consists of a backbone of ,t~-1,4-linked
xylose units. The enzymic cleavage of 13-1,4-xylosidic linkages is
performed by endo-~-1,4-xylanases (xylanases). Many microorganisms
produce extracellular xylanases. In the past decade, many xylanase
genes were isolated from lignocellulolytic bacteria, but isolation of
xylanase genes from fungi with functional expression in E. coli has not
been documented prior to this invention.
Lignocellulolytic fungi usually produce more active xylanase than
bacteria, in particular, the anaerobic fungus Neocallimastix patriciarum,
isolated from the sheep rumen, has a high capacity for xylan
degradation .
Reference may also be made to other prior art which serves as
background prior art prior to the advent of the present invention. Such
prior art includes:
(i) Reymond et. al. Gene 110 (1992) 57-63;
(ii) Wong et. al. Clin. Reviews in Biotechnology 12 413-435
( 1992);
(iii) Orpin et. al. Current Microbiology Vol 3 (1979) pp 121-
1 24;
(iv) Mountfort and Asher in "The Roles of Protozoa and Fungi
in Ruminant Digestion" ( 1989) Pernambul Books (Australia);
(v) doblin et. al. FEMS Microbiology Letters 65 (1989) 119-
1 22;
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(vi) Lowe et. al. Applied and Environmental Microbiology June
1987 pp 1210-1215; and
(vii) Lowe et. al. Applied and Environmental Microbiology June
1987 pp 1216-1223.
Cloning of xylanase genes from bacteria can be achieved by
isolation of enzymatically active clones from genomic libraries
established in E coli. However this approach for isolation of xylanase
genes from fùngal genomic libraries with functional expression of
xylanase is not possible. This is because fungi are eucaryotic
10 microorganisms. Most eucaryotic genes contain introns and E. coliis
unable to perform post-transcriptional modification of mRNAs in order to
splice out introns. Therefore, enzymatically functional protein cannot
normally be synthesised in clones obtained from a fungal genomic
library.
The cDNA cloning approach can be used to overcome the post-
transcriptional modification problem in E. co/i. However, xylanases in
fungi are usually glycosylated and glycosylation is often required for
biological activity of many glycosylated enzymes. E. coli lacks a
glycosylation mechanism. This problem can be solved if the cloned gene
20 is transferred to an eucaryotic organism, such as yeast. Other problems
which are often encountered in obtaining a biologically functional protein
from a cDNA clone in E. coli are (i) that many eucaryotic mRNAs contain
translational stop codons upstream of the translational start codon of a
gene which prevents the synthesis of the cloned protein from the
25 translational start provided in the vector, and (ii) that synthesis of the
cloned protein is based on fusion proteins and the biological function of
the cloned protein is often adversely affected by the fused peptide
derived from the cloning vector.
Therefore, in the past, researchers in this field employed
30 differential or cross hybridisation, antibody probes or oligonucleotide
probes for the isolation of fungal polysaccharide hydrolase cDNA or
genomic DNA clones. Relevant publications in this regard include
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Reymond et. al. FEMS Microbiology letters 77 (1991) 107- 112; Teeri
et. al., Biotechnology 1 696-699 (1983); Shoemaker et. al.,
Biotechnology 1 691-696 t1983); Sims et. al. Gene 74 411-422
(1988); Morosoli and Durand FEMS Microbiology Letters 51 217-224
(1988); and Azevedo et. al. in J. Gen. Microbiol. 136 2569-2576
(1990). However, these methods are very time-consuming, and quite
often two stages of intensive cloning work are required for isolation of
an enzymatically functional clone. For antibody or oligonucleotide
probes, purification of the fungal xylanase is also required. It usually
takes more than one year to obtain a functional enzyme clone using the
above approaches.
Isolation of fungal xylanase cDNAs by utilising an expression
system in E. coli has not been reported prior to the advent of this
invention probably at least partially due to failure in obtaining
enzymatically functional xylanase clones by using improper expression
vectors. Selection of expression vector systems is important. If plasmid
expression vectors such as pUC vectors are used, and the cloned
enzyme is trapped inside the cell, therefore screening for xylanase clones
by the convenient xylan-agar plate technique is difficult. Bacteriophage
vectors have an advantage in respect to the release of the cloned
enzyme into xylan-agar medium due to cell Iysis. However, commonly
used bacteriophage expression vectors"Igt11 and its derivatives, have
polyclonal sites at the C-terminus of the LacZ peptide. The large part of
LacZ peptide fused to the cloned enzyme often adversely affects the
cloned enzyme activity.
In specific regard to the abovementioned Reymond et. al. ~1991)
reference there is described an attempt of molecular cloning of
polysaccharide hydrolase (ie. cellulase) genes from an anaerobic fungus
which is N. frontalis. In this reference a clone from a cDNA library
30 derived from N. frontalis hybridized to a DNA probe encoding part of the
exo-cellobiohydrolase (CBH 1) gene of Trichoderma reesei. However it
was subsequently revealed by Reymond et. al. in a personal
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communication that the particular cDNA clone obtained from N. frontalis
does not encode any polysaccharidè hydrolase.
Moreover the Reymond et. al. reference did not describe the
production of biologically functional enzymes from these clones.
In relation to isolation of a fungal xylanase gene, the only report
that exists so far prior to this invention is the abovementioned Morosoli
and Durand reference which describes isolation of a xylanase gene from
yeast CrYptococcus albidus using differential hybridization techniques.
However, this reference does not describe the production of biologically
functional enzymes from this xylanase gene.
BROAD STATEMENT OF INVENTION
It is an object of the invention to provide a recombinant xylanase
from an anaerobic rumen fungus which may be of use commercially in
relation to hydrolysis of xylan.
A further object of the invention is to provide a method of cloning
of xylanase cDNAs from an anaerobic rumen fungus which may encode
the recombinant xylanase of the invention.
A further object of the invention is to provide xylanase clones
which may be produced in the abovementioned method.
The method of cloning of the invention includes the following
steps: --
(i) cultivation of an anaerobic rumen fungus;
(ii) isolating total RNA from the culture in step (i);
(iii) isolating poly A+ mRNA from the total RNA referred to in
step (ii);
(iv) constructing a cDNA expression library;
(v) ligating cDNAs to a bacteriophage expression vector
selected from ~IZAP, ~IZAP 11 or vectors of similar
properties;
(vi) screening of xylanase positive recombinant clones in a
culture medium incorporating xylan by detection of xylan
hydrolysis; and
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_
(vii) purifying xylanase positive recombinant clones.
In step (i) above in relation to preparation of the recombinant
xylanase, from anaerobic fungi, particularly alimentary tract fungi, may
be cultivated as described hereinbelow. These fungi are strict anaerobes
5 and may be exemplified by Neocallimastix patriciarum, Neocallimastix
frontalis, Neocallimastix hurleyensis, Neocallimastix stanthorpensis,
Sphaeromonas communis, Caecomyces equi, Piromyces communis,
Piromycesequi, Piromycesdumbonica, Piromyceslethargicus, Piromyces
mai, Ruminomyces elegans, Anaeromyces mucronatus, Orpinomyces
10 bovis and Orpinomyces joyonii. In regard to the above mentioned
anaerobic alimentary tract fungi, Caecomyces equi, Piromyces equi,
Piromyces dumbonica and Piromyces mai are found in horses and thus
are not located in the rumen of cattle like the other fungi described
above.
The cultivation may proceed in appropriate culture media
containing rumen fluid and also may contain cellulose such as Avicel (ie.
a form of microcrystalline cellulose) as a carbon source under anaerobic
conditions. After cultivation of the fungi total RNA may be obtained in
any suitable manner. Thus initially the fungal cells may be harvested by
20 filtration and subsequently Iysed in appropriate cell Iysis buffer by
mechanical disruption. A suitable RNA preserving compound may also
be added to the fungal cells to maintain the RNA intact by denaturing
RNAses which would otherwise attack the fungal RNA. The total RNA
may subsequently be isolated from the homogenate by any suitable
25 technique such as by ultracentrifugation through a CsCI2 cushion or
alternative technique as described by Sambrook et. al. in Molecular
Cloning; A Laboratory Manual 2nd Edition Cold Spring Harbor
Laboratory Press in 1989. An alternative method for preparation of total
fungal RNA to that described above may be based on or adapted from
30 the procedure described in Puissant and Houdebine in Bio-Techniques
148-149 in 1990. Total fungal RNA in this alternative technique may
also be isolated from the above homogenate by extraction with phenol
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chloroform at pH4 to remove DNA and associated protein. Total RNA
obtained was further purified by washing with lithium chloride-urea
solution .
Poly (A)+ mRNA may then be isolated from the total RNA by
5 affinity chromatography on a compound containing multiple thymine
residues such as oligo (dT) cellulose. Alternatively a compound
containing multiple uracil residues may be used such as poly (U)-
Sephadex. The poly (A)+ mRNA may then be eluted from the affinity
column by a suitable buffer.
A cDNA expression library may then be constructed using a
standard technique based on conversion of the poly (A) + mRNA to cDNA
by the enzyme reverse transcriptase. The first strand of cDNA may be
synthesised using reverse transcriptase and`the second strand of the
cDNA may be synthesised using E. coli DNA polymerase 1. The cDNA
15 may subsequently be fractionated to a suitable size and may be ligated
to the bacteriophage expression vector, preferably AZAP or AZAPII. The
cDNA library may then be amplified after packaging in vitro, using any
suitable host bacterial cell such as a suitable strain of E. coli.
The choice of the bacteriophage expression vector in step (v~ is
20 important in that such expression vector should include the following
features:
(i) having an E. coli promoter;
(ii) having a translation start codon;
(iii) having a ribosomal binding site;
(iv) the fusion peptide derived from the vector should be as
small as possible as the biological function of the cloned
protein is usually adversely affected by the fused peptide
derived from the vector. Therefore the polyclonal sites of
the bacteriophage expression vector are suitably located at
the N-terminus of lacZ peptides such as in AZAPII.
It will be appreciated from the foregoing that if an expression
vector is utilised as described above the chances of obtaining a
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biologically functional enzyme is greatly increased. Isolation of many
enzymatically functional xylanase clones in the present invention as
described hereinafter has proved the efficiency of this approach. To our
knowledge this is the first record of isolation of xylanase cDNA clones
5 with functional enzyme activity from anaerobic fungi based upon the
expression of recombinant bacteriophage in E. coli using an expression
vector such as that described above. AZAP and AZAP ll are examples of
such expression vectors.
Therefore the term "vectors of similar properties" to AZAP or
10 AZAPII includes within its scope expression vectors having the
abovementioned features (i), (ii), (iii) and (iv).
It is also clear from the product summary which accompanies the
AZAPII vector as supplied by the manufacturer that in relation to fusion
protein expression that such fusion proteins may only be screened with
15 antibody probes. Clearly there was no contemplation that the AZAPII
vector could be utilised for screening of clones involving enzymic
expression on a suitable substrate or any direct screening by biological
activity. When it is realised that the present invention involves
expression in a bacterial host cell such as E. coli of a cDNA of eucaryotic
20 origin (ie. fungal origin) then the novelty of the present invention is
emphasised .
The screening of xylanase positive recombinant clones may be
carried out by any suitable technique based on hydrolysis of xylan. In
this procedure the clones may be grown on culture media incorporating
25 xylan and hydrolysis may be detected by the presence of xylanase-
positive plaques suitably assisted by a suitable colour indicator.
Xylanase positive recombinant clones may then be purified and the
cDNA insert in the clones may then be excised into pBluescript (SK(-))
to provide an expression vector of simplified structure when compared
30 to the AZAP ll construct which will enhance expression of the xylanase
in E. coli.
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.
Any suitable E. coli promoter may be used in the expression vector
described above. Suitable promoters include lacZ, Tac, Bacteriophage
T7 and lambda-P,.
The recombinant xylanase enzyme may then be characterised and
5 principal features that have been ascertained are as follows:
(i) the cloned xylanases have high specific activity.
(ii~ the enzyme has no residual activity against cellulose, while many
other xylanases possess some cellulase activity. This property of
the xylanase is particularly useful in its application to pulp and
paper industry to remove xylan and dissociate lignin from plant
fibre without damaging cellulose fibre.
The high specific activity of the cloned xylanases is an excellent
intrinsic property of this fungal xylanases. The expression level of the
present constructs of xylanase cDNAs can be further improved by
manipulating the gene and promoters.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Exnerimental Methods
1. Microbial strains, vectors and culture media.
The anaerobic fungus Neocallimastix patriciarum (type species)
was isolated from a sheep rumen by Orpin and Munn (1986) in Trans.
Br. Mycol. Soc. 86 178-181 and cultivated in the laboratory for many
years under selection by lignocellulose substrates. Host strains for cDNA
cloning and characterisation of the recombinant xylanases were E. coli
PLK-F, XL1-Blue and JM83.
The vectors were ,IZAPII, pBluescript SK(-) (Stratagene). N.
patriciarum culture was maintained in a medium containing 10% rumen
fluid as described by Kemp et. al. (1984). E. coli strains were grown in
L-broth as described by Sambrook et. al. (1989) for general purposes.
The recombinant phage grown in E. coli strains using NZY medium
according to Stratagene's instructions.
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2. General recombinant DNA techniaues.
Agarose-gel electrophoresis, transformation of E coti and
modification of DNA using restriction enzymes and T4 DNA ligase were
as described in Sambrook et. al. above. The alkaline Iysis method of
Birnboim and Doly as described in Nucl. Acids Res. 7 1513- 1523 (1976)
as employed to isolate plasmid. In vitro DNA amplification by
polymerase chain reaction (PCR) was based on the procedure described
by Saiki (1989) in PCR Technology (H.A. Erlich, ed) pp. 7-16, M.
Stockton Press, New York.
3. Cultivation of rumen anaerobic fungus, N. patriciarum for preparation
of RNA.
N. patriciarum was grown in a rumen fluid-containing medium as
described in Kemp et. al. J. Gen. Microbiol. 130 27-37 (1984) in the
present of 1 % Avicel at 39C and under anaerobic conditions for 48hr
(Alternative culture media, such as described by Philips and Gordon in
Appln. Environ. Microbiol.55 1695-1702 in 1989 and Lowe et. al. in J.
Gen. Microbiol. 131 2225-2229 in 1985 can be used).
4 Total RNA isolation.
The frozen mycelia were ground to fine powder under liquid nitrogen
with a mortar and pestle. 5-10 vol of guanidinium thiocyanate solution
(4M guanidinium thiocyanate, 0.5% sodium laurylsarcosine, 25mM
sodium citrate, pH7.0, 1mM EDTA and 0.1 M 13-mercaptoethanol) was
added to the frozen mycelia powder and the mixture was homogenised
for 5 min with a mortar and pestle and for further 2 min at full speed
using a Polytron homogeniser. Total RNA was isolated from the
homogenate by ultracentrifugation through a CsCI cushion (Sambrook et.
al., 1989). (An alternative method for preparation of total fungal RNA,
such as adaptation of the procedure described by Puissant and
Houdebine in Bio-Techniques 148-149 in 1990 can be used).
5. PolY A+ mRNA Purification.
Poly A+ was purified from the total RNA by Oligo (dT) cellulose
chromatography (Sambrook et. al., 1989).
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6. Construction of a cDNA exPression librarv of N. Patriciarum~
The cDNA library was constructed, using Stratagene'sAZAP cDNA
synthesis Kit, basically according to the manufacturer's instructions.
The procedure is described briefly as follows: PolyA+, RNA was
5 converted to the first strand cDNA by reverse transcriptase, using Xhol
linker - oligo (dT) primer and 5-methyl dCTP. Double-stranded cDNA
was synthesised from the first-strand cDNA by the action of RNase H
and DNA polymerase 1. After blunting cDNA ends, the cDNA was ligated
with EcoR I adaptor, phosphorylated and digested with Xho1 to create
10 cDNA with the EcoR I site at 5' region and the Xhol site at 3' region.
The cDNA was size-fractionated by 1% low-melting point agarose gel
electrophoresis and 1 . 2-8Kb sizes of the cDNA were recovered by phenol
extraction (Sambrook et. at., 1989). The size-fractionated cDNA was
then ligated to the EcoRI/Xhol digested ~IZAPII vector.
The cDNA library was packaged in vitro and amplified using E. coli
PLK-F' as plating cells.
7. Screening xvlanase-Positive recombinant bacterior~hage- clones.
Recombinant phage were grown in E. coli XL1-Blue in 0.7% top
agar containing 0.1 % xylan and 1 OmM isopropyl-~-thio-
20 galactopyranoside (IPTG, an inducer for LacZ promoter controlled geneexpression). After overnight incubation at 37C, 0.5% Congo red
solution was added over the top agar. After incubation at RT for 15
min, the unbound dye was removed by washing with 1 M NaCI.
Xylanase-producing phage plaques were surrounded by yellow haloes
25 against a red background.
The xylanase-positive recombinant phage were purified to
homogeneity by replating and rescreening the phage as above for 2-3
times.
The cDNA insert in xylanase-positive phage were excised into
30 pBluescript SK (-) using R408 helper phage.
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8. Xvlanase and related-enzyme assavs.
The cloned enzyme extracts from E. coli harbouring xylanase-
positive recombinant plasmids were prepared by harvesting the cells by
centrifugation. The cell pellet was suspended in 25mM Tris-CI/ 2mM
5 EDTA containing Iysozyme (0.25mg/ml) and incubated on ice for 60
mins. After freezing, thawing and homogenisation, the crude cell Iysate
was used for enzyme assays.
The enzymes were assayed for hydrolysis of xylan or other
substrates at 40 C in 50 mM Na-citrate, pH 6.5, except where
10 otherwise indicated in the text. The reducing sugars released from xylan
or other plant polysaccharides (Avicel) were measured as described by
Lever in Anal. Biochem. 47 273-279 in 1972.
Xylanase activity on Kraft pulp was conducted as follows: Kraft
pulp was suspended in tap water, and pH was adjusted to pH 7 with 1 M
15 H2SO4. The xylanase extract was added to the Kraft pulp suspension
and the reducing sugar released was measured as above.
9. DNA seauencinq.
Single-stranded plasmid DNA was prepared basically according to
Stratagene's protocol. Sequencing of the resultant DNA was based on
20 the protocol recommended by the manufacturer of the T7 DNA
polymerase sequencing kit (Promega).
10. OPtimisation of growth conditions of oNX-Tac clone.
E.coli strain JM83 harbouring pNX-Tac plasmid grew in
LBlAmpllOO~,m,~ at 30C overnight. One millilitre of the overnight culture
25 was inoculated into 100ml of media as specified in Table 5. IPTG was
added at different times of growth. The cultures were grown at 30C
for 17hr, 24hr and 30hr. The cells were harvested for measurement of
xylanase yield.
Results and discussion
30 Isolation and partial characterisation of xylanase cDNA clones.
A cDNA library consisting of 106 clones was constructed using
mRNA isolated from N. patriciarum cells grown with Avicel as sole
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carbon source. Thirty-one recombinant bacteriophage, which hydrolysed
xylan, were identified after an initial screening of 5 x 104 clones from the
library and 16 strongly xylanase-positive phage and two weakly
xylanase-positive phage were isolated and purified. Xylanase activity of
these recombinant bacteriophage clones was initially analysed by scoring
xylan-hydrolysis zones ( Fig . 1 and Table 1) .
These 16 strongly xylanase positive clones were originally
forwarded to Dr H J Gilbert and Dr G P Hazlewood of The University of
Newcastle-upon-Tyne and the AFRC Institute of Animal Physiology and
Genetics Research in the United Kingdom who carried out further
analysis of these clones which included restriction mapping and
hybridization analysis as well as sequencing of the longest clone. In this
regard reference should be made to the publication " Homologous
catalytic domains in a rumen fungal xylanase: evidence for gene
duplication and prokaryolic origin" by H J Gilbert, G P Hazlewood, J I
Laurie, C G Orpin and G P Xue which is published in Molecular
Microbiology (1992) 6 (15) 2065-2072. The longest clone referred to
in this reference is designated pNX1 and this corresponds to clone
pNPX21 described hereinafter. In the Gilbert et. al. reference described
above other plasmids pNX2, pNX3, pNX4, pNX5, pNX6 and pNX7 were
produced as a result of truncation of pNX1 by restriction enzymes.
The clone corresponding to clone pNX1 in E. coli strain XL1-Blue
described above has now been deposited at the International Depository
ie. Australian Government Analytical Laboratories on June 22, 1992
under accession number N92/27542.
In an attempt to obtain more highly active xylanase clones, further
screening of 4 x 105 clones from the library was conducted, which
resulted in >200 xylanase-positive clones. Ten highly active clones
were isolated and purified. Two of these recombinant bacteriophage
clones (~INPX29 and ~NPX30) have much stronger xylanase activity than
previously isolated high activity clones (see Table 1).
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The cDNA inserts encoding Neocallimastix patriciarum xylanases
were in vivo excised from bacteriophage (,IZAP11) form into plasmid
pBluescript SK- form. Several clones with high xylanase activity were
analysed for substrate specificity (four clones presented in Table 2). The
xylanases produced by these clones have no activity on carboxymethyl-
cellulose (CMC, a substrate for endo-glucanase) or Avicel (Avicel is
crystalline cellulose and is a substrate for exo-glucanase). The restriction
maps of the representative clones are presented in Fig. 2. It appears
that these four xylanase cDNAs have the same restriction pattern but
differ in length. pNPX13 and pNPX29 have shorter lengths than
pNPX21 but they have much higher activity than pNPX21. Interestingly,
pNPX30 has a similar length to pNPX21 but it has about 15-fold higher
xylanase activity than pNPX21. Because of the remarkable difference
in enzyme activity between pNPX21 and pNPX30, the xylanase cDNA
of pNPX30 clone was sequenced. The result shows that DNA sequence
of pNPX30 shares the same sequence with pNPX21 in a large part of
cDNA, but differ in both the 5' and 3' regions. (Fig. 3). pNPX30 cDNA
is not full-length. Interestingly, the N-terminus of pNPX30 xylanase has
six repeated arginine/glutamic acid residues (Fig. 4).
The pH and temperature optima of xylanases produced by
pNPX21 and pNPX30 were investigated. These enzymes were active in
a wide range of pH and preferably at pH 5 - 8. The thermostability of
these enzymes was tested at temperatures from 30C - 60C. The
enzymes are active at 30C- 55C and preferably at 40C - 50C.
Genetic modification of N. Patriciarum xvlanase cDNA
pNPX30 (and pNPX21) contains two large repeated domains.
Three main constructs were produced from pNPX30.
pNXD-Tac
pNPX30 plasmid (pNPX21 can also be used) was used as a
template for in vitro DNA amplification by PCR for construction of pNXD-
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Tac using primer I and primer IV (Fig.5). The amplified DNA was
digested with EcoR1 and Hindl 11 and ligated to EcoR1 and Hind111
digested pBTac2 (Boehringer) to produce pNXD-Tac.
pNXS-Tac
pNXD-Tac plasmid was digested with Hind 1 1 1 and blunted by
filling-in with Klenow followed by partial digestion with Scal. After
fractionation on LMT agarose gel, the 5.3Kb band was recovered from
the gel and ligated to produce the pDGXS construct, which has xylanase
activity. pDGXS plasmid was used as a template for in vitro DNA
amplification for construction of pNXS-Tac using primer I and primer ll
(Fig.5). The amplified DNA was digested with EcoR1 and Hindl 11 and
ligated to EcoR1 and Hind 1 1 1 digested pBTac2 vector to produce pNXS-
Tac.
pNX-Tac
pNPX30 plasmid (pNPX21 or other xylanase cDNAs listed in Fig.2
can be used) was digested with Rsal and a 709bp fragment as indicated
in Fig.5 was isolated after fractionation on agarose gel electrophoresis.
The 709 fragment was ligated to Sma1 and Pst1 digested pUC18 (Pstl
end was blunted with T4 DNA polymerase). This construct is designated
pNXP2 and the xylanase activity of this construct with the right
orientation of truncated xylanase cDNA from pNPX30 confirmed that this
fragment of the cDNA encodes a caterlytically functional domain.
Two oligonucleotide primers, primer lll and primer IV, (Fig.5) were then
designed for PCR amplification of the pNXP2 xylanase cDNA insert. The
PCR amplified fragment was digested with EcoR1 and Hind111 and
ligated to EcoR1 and Hindl 1 1 digested pBTac2 vector to produce pNX-
Tac.
These constructs are all modified at the N-terminal sequence of
the truncated xylanase cDNA and a translational stop codon (TAA) was
introduced into the end of the truncated xylanase coding region. The
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expression of xylanase was controlled by the Tac promoter (Fig.6) and
xylanases in these constructs are synthesised as nonfusion proteins. The
modified xylanase cDNA sequence in pNX-Tac is shown in Fig 7.
The specific activity of crude xylanase preparations of pNXD-Tac,
- 5 pNXS-Tac and pNX-Tac clones were 228, 124 and 672 U/mg of total
cellular protein of E.coli respectively, measured in 50mM Na-citrate
buffer (pH6) and at 50C (Fig.5). The xylanase synthesised bythe clone
pNX-Tac was found mainly in the cell pellet, but a small amount of
xylanase (about 5%) was released into the culture medium (Table 3).
The pNX-Tac xylanase has a temperature optimum at 50C and retained
>80% of the maximum activity from 40C to 55C, and 55% of the
activity at 60C (Fig.8). pNX-Tac xylanase has a broad pH range (Fig
9) and is most active at pH5-7.5, 50% at pH8.5 and 20% at pH9.5.
The pNX-Tac xylanase has a high activity in the release of reducing
sugar from Kraft pulp at 55 C and in tap water (pH was adjusted to pH7
with H2S04, see Fig.8) and remains active in the hydrolysis of xylan from
the pulp at 55C and pH7 for at least 3hr (Fig.10) The pNX-Tac
xylanase is able to hydrolyse a significant amount of xylan from Eucalypt
and Pine Kraft pulps (Table 4).
O~timisation of growth conditions pNX-Tac clone. -
In order to reduce the cost of xylanase production, growth
conditions of E. coli strain JM83 harbouring pNX-Tac plasmid were
investigated. Table 5 shows that on a laboratory scale pNX-Tac clone
preferably grows in LBMG medium at 30C for 24 hr, which produced
2-fold higher xylanase yield than LBS. IPTG is preferably added at the
beginning of the cultivation (Table 6).
Xylanase has many industrial applications, such as the pulp and
paper industry, food processing, the feed industry and animal production
industry. The enzymes produced by these recombinant xylanase clones
have no cellulase activity and have the pH and temperature profile
(especially the genetically modified xylanase clone, pNX-Tac) fitted to
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16
conditions used for the enzymatic pre-treatment of pulp. Therefore it is
beiieved that the xylanases of the present invention are applicable to the
paper and pulp industry.
Sandoz Products Pty Ltd, in the USA, have conducted practical
trials using their product, Cartazyme, which is a fungal xylanase (crude),
active at 30C-55C, pH 3 to 5, and contains 2 xylanases, and have
found that a 25-33% reduction in chlorine is possible using 1 U
xylanase/g pulp. Also the product is brighter than when chemicals alone
are used. Another advantage of the xylanase is that it is specific
whereas chemicals can attack the cellulose at low lignin contents,
leading to reduced fibre strength and other undesirable physical
characteristics. It is therefore clear that xylanases could become more
important in pulp bleaching and recombinant ones particularly so because
of their specificity and high level of expression. In particular, the pNX-
Tac xylanase is very active in hydrolysing of xylan from Kraft pulps.
It is also believed that the xylanase of the invention could find a
valuable application in the sugar industry and in relation to the treatment
of bagasse or other products containing xylan for more efficient disposal
as well as for the treatment of feedstock to improve nutritional value.
The genetically modified xylanase gene can also be used for modification
of rumen bacteria to improve plant fibre utilization by ruminants.
It therefore will be apparent from the foregoing that the invention
includes within its scope not only the recombinant xylanase described
above but also xylanases derived from other anaerobic fungi as described
above which may be prepared by the methods described herein.
The invention also includes within its scope:
(i) DNA sequences derived from these xylanase cDNAs (particularly
the sequences in pNPX30, pNXD-Tac, pNXS-Tac and pNX-Tac)
and DNA sequences capable of hybridising thereto using a
standard nucleic acid hybridisation technique as described in
Sambrook et. al. ( 1 989);
W O 93/25671 2 1 3 8 3 9 9 PC~r/A U93/00294
(ii) a DNA construct containing a DNA sequence as in (i) operably
linked to regulatory regions capable of directing the expression or
over-expression of a polypeptide having xylanase activity in a
suitable expression host;
5 (iii) a transformed microbial host capable of the expression or over-
expression of the fungal xylanase, harbouring the above
mentioned xylanase constructs;
(iv) a polypeptide having xylanase activity produced by expression
using a microbial host as in (iii);
10 (v) amino acid sequence derived from these xylanases, truncations
and modifications therefrom, by one skilled in the art.
Plasmid pNX-Tac in E. coli strain JM83 has been deposited at the
International Depository ie. Australian Government Analytical
Laboratories 17 March 1993 under accession number N93/1221 1.
In summary the cloning method of the invention is based upon
obtaining a large number of recombinant xylanase clones with strong
xylanase activity from an anaerobic rumen fungus such as N. I~atriciarum
which were functionally expressed in E. coli. This approach for isolation
of fungal xylanase or other plant polysaccharide hydrolases such as
20 cellulases has not been documented prior to this invention. The
approach used in this invention is very efficient and requires only a single
cloning step to obtain biologically functional recombinant xylanases from
an anaerobic fungus. Therefore it takes much less time to obtain
biologically functional xylanase clones from a fungal source compared to
25 previous approaches for isolation of plant polysaccharide hydrolases from
fungi which are described in the prior art discussed above.
The term "essentially" as used in the appended claims includes
within its scope sequences having 70-100% identity to those sequences
- shown in Figs. 3, 4, 5 and 7.
21~8399
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18
Table 1
Xylanase activity of rec<~mbinant Bacteriophage clones on Xylan - plate
assay
Xylan - clearing zone
~NPX1 1 L
~NPX1 2 S
~NPX13 L+++ (9mm)
ANPX1 4 L
~NPX15 L+
~NPX1 6 L
~NPX17 S (4mm)
~NPX1 8 L+
~NPX1 9
~NPX20 L
~NPX~l L+ (7mm)
~NPX~2 L
~NPX23 L
~NP~24 L
~NPX25 L+
~NPX26 L++ (8.5mm)
~NPX27 L
~NPX28 L+
~NPX29 L++++(1 O.5mm)
.NPX30 L++++ (1 0.5mm)
L: Large size
S: Small size
Values in parenthesis is diameter of zone.
~NPX11-28 were isolated from in-~ial seening.
~NPX29 and ~NPX30 were isolated after further screening of N.patriaarum
cDNA library.
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Table 2
Speafic activny of the cloned xylanases from N. patriciarum
Spedfic activity (U/mg protein)
Xylan CMC~ Crystalline cellulose
pNPX13 41.6 0 0
pNPX21 7.B 0 0
pNPX29 73.5
pNPX30 113 0 0
Analysed by CMC plate assay.
Crude enzyme extracts were used ~or en~yme assay. The reactions were carr~ed
out at 40C in 50 mM Na-citrate, pH6.5, conlaining 0.25% xylan or 1% Avioel.
¦ SUts~ UTE SHEET ¦
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WO 93/2~671 PCI'/AU93/00294
Table 3
Specific activity of pNX-Tac xylanase.
Cell pellet
Culture supernant
Substrate U/mg protein U/ml cultu-e U/ml cutture
Xylan 672 726 23
CMC- 0
Crystalline cellulose 0
(Avioel)
Analysed on CMC - plate.
E.co~ strain JM83 harbouring pNX-Tac plasmid was grown in L-broth at 30C for
17hrs.
Xylanase ac:tivity was measured in 50mM Na~itrate pH6 containing 0.25% Xylan at
50C and the redudng sugar released was measured as described in the method.
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pcr/Aus3/oo294
Table 4
Reducing sugar rele~ce~l from Kraft pulp.
- mg redudng sugar released/g dry pulp
Xylanase Eucalypt pulp Pine pulp
~1/9 dry pulp
O O O
1 O,ul 1 1.9 6.97
100111 28.9 9.53
The cn~de xytanase extract ~rom pNX-Tac clone was inPl~t~i with 6%~Y/V) pulp
sus~uension in tap ~ate~ at pH 7Ø The hydrolysis w.as carried out at 52C ~or 3
hour_
¦ SUBSTITUTE SHEET
2138399
WO 93/25671 , ~ PCr/AU93/00294
,
22
Table 5
Optimisation of growth conditions of E.coli JM83 harbouring pNX-Tac plasmid.
Xylanase yield
IPTG oell mass (Relative activity~
at 24hr
(g~re) 17hr 24hr 30hr
LBS 0.5mM 10 100% 100%
LBSG 11 55~O 55%
LBMG 0.1mM Z 168% 168%
0.5mM 22 151% 200% 200%
2.5mM 22 190% 190%
LBMHG 0.5mM 20 110% 110%
Ecofi strain JM83 harbouring pN~Y-Tac plasmld was grown in the sf~fi~d me~la
conlaining 5011g/ml Amp at 30C and IPTG was added at the beginning of the
cultivation.
Composition of Media, per l~re.
LBS: Bacto-tryptone 109LBSG: LBS plus
Bacto-yeast ext 590.4% Glucose
NaCI 1 09
Suose (0.4%) 49
pH 7.2
LBMG: Bacto-tryptone 59LBMHG: L8MG plus
Bacto-yeast ext. 39glucose increased to 1% by
NaCI 0.59adding an extra 69 glucose.
Na2HPO4 1 2H2O 15.19
KH2PO4 39
NH4CI 1 9
Casamino acids 59
Sucrose~ 69
CaC12 (100mM) 1ml
MgSO4 (1 M) 2ml
Glucose 49
pH 7.2
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Table 6
Oplir"isa~ion of Induct~on time of pNX-Tac clone.
IPTG added at Xyl~ase yield (relative activity)
Ohr 1 00%
8hr 82%
1 6hr 40%
E.co~ strain JM83 harbouring pNX-Tac plasmid was grown in LBMG containing
5011g/ml Amp and 0.5 mM IPTG at 30C for 24 hours.
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LEGENDS
Figures 1 (a), 1 (b~, 1 (c) and 1 (d)
Xylan-clearing zones of recombinant bacteriophage clones containing
xylanase cDNAs for N. patriciarum concerning clones A NPX l 3"1 NPX l 7,
5 ~NPX21 and ANPX26 respectively.
Figure 2
Restriction maps of the highly active xylanase clones isolated from
Neocall,;"aslix pal,;~;~,.Jm cDNA library.
Abbreviations for restriction enzymes:
10 B, BstXI; E, EcoRI; H, Hpal; K, Kpnl; P, Pvull; S, Sacl; Sc, Scal; X, Xhol.
Figure 3
The DNA sequence of pNPX30 xylanase cDNA. The sequence typed in
small letters comes from the pBluescript SK vector.
Figure 4
15 The amino acid sequence of pNPX30 xylanase. The amino acid residues
underlined come from the N-terminus of LacZ peptide and encoded by
polylinker sequence in the pBluescript SK vector.
Figure 5
The genetically modified constructs of the xylanase cDNA
20 vector: pBTac2
primers:
Pl:5'-CGGAATTCATG GCT AGC AGA TTA ACC GTC GGT AAT GGT C
P11:5'-ATACG TAAGC TTAAA CAGTA CCAGT GGAGG TAG
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WO 93/25671 213 8 3 9 9 PCI'/AU93/00294
-
Plll: 5'-CGGAA TTCAT GGCTA GCAAT GGTAA AAAGT TTACT G
PIV: 5'-ATACG TAAGC TTAAC GAGGA GCGGC AGAGG TGG
Abbreviations for restriction enzymes:
B, BstX l; E, EcoR l; H, Hpa l; K, Kpn l; P, Pvu ll; S, Sac l; Sc, Sca l; X,
5 Xho 1.
Figure 6
pNX-Tac construct
Figure 7
The sequence of the modified xylanase cDNA in pNX-Tac
10 Figure 8
Effect of incubation temperature on the activity of pNX-Tac xylanase.
Xylanase assays were performed in 50 mM Na-citrate (pH7) and 0.25%
(w/v) xylan at the various temperatures for 30 min.
Figure 9
15 Effect of pH on the activity of pNX-Tac xylanase.
Xylanase assays were performed at 50 C in 50 mM Na-citrate (pH5-7)
or 25 mM Tris-CI / 50 mM NaCI (pH7.5-9.5) with 0.25% xylan for 30
min. The pHs of the buffers were measured at room temperature.
Figure 10
20 Time course of pNX-Tac xylanase activity on eucalypt Kraft pulp.
Hydrolysis was carried out at 55C in tap-water suspended pulp at pH
7Ø
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