Note: Descriptions are shown in the official language in which they were submitted.
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FUNGAL LICHENASE AND CODING SEQUENCES
STATEMENT REGARDING FEDERAL RESEARCH SUPPORT
This invention was made, at least in part) with funding from the United States
Department of
Energy. Accordingly, the United States Government has certain rights in this
invention.
BACKGROUND OF THE INVENTION
The present invention relates to polysaccharide-degrading enzymes, especially
to the enzymes,
in particular, to a lichenase enzyme which is capable of degrading ( 1,3-1,4)-
(~-glucans and sequences
encoding lichenase enzymes.
Hemicellulose (non-cellulosic polysaccharides including glucans, mannans and
xylan) is the
major constituents of plant cell walls. The mixed-linked 1,3-1,4-/3-glucans
form the major part of cell
walls of cereals like oat and barley. {3-Glucans consist of glucose units
jointed by {3-1,4 and /3-1,3
linkages, and include lichenan and barley i3-glucan. ~i -Glucan accounts for
up to 70% of the cell wall
in barley endosperm (Guliga and Brant, 1986).
Endo-1,3-1,4-~i-D-glucanohydrolase (1,3-1,4-(3-glucanase) lichenase) cleaves
~3-1,4 linkages
adjacent to (3-1,3 in glucans yielding chiefly cellobiosyltriose and
cellotriosyltetraose {Fleming and
Kawami, 1977; Anderson and Stone, 1975). ~3-Glucanase is especially
interesting to the brewing
industry because /3-glucans cause problems in filtration processes (Godfrey,
1983). ~i-glucanase also
_. has application in the poultry industry; it has been added to broiler chick
feedstuffs to improve
digestibility (White et al., 1983). a-Glucanases have been cloned from several
Bacillus species,
including Bacillus subtilis (Murphy et al., 1984), B. amyloliquefaciens
{Hofemeister et al., 1986), B.
macerans {Borriss et al 1990)) B. licheniformis (Lloberas et al., 1991), B.
brevis (Louw et al., 1991),
B. polymyxa (Gosalbes et al. , 1991 ), and from other genera, including
Clostridium thermocellum
(Schimming et al. , 1992; Zverlov et al. , 1992), Fibrobacter succinogenes
(Teather and Erfle, 1990),
Ruminococcus flavefaciens (Flint et al., 1993), Rhizobium meliloti (Berker et
al., 1993, and Cellvibrio
mixtus (Sakellaris et al., 1993). A cDNA clone encoding barley a-glucanase has
been isolated and
sequenced from germinating barley (Fincher et al., 1986).
Unlike endo-1,4-~3-D-cellulases which are widely distributed in various
organisms, 1,3-1,4-(3-
D-glucanases are known to be produced only by plants and certain bacteria
(Borriss et al., 1990;
Fincher et al., 1986). No fungal 1,3-1,4-(3-glucanases which lack the ability
to degrade /3-(1,4)-glucans
are believed to have been discovered prior to the present invention.
Obligately anaerobic fungi are part of the natural microflora of the
alimentary tract of many
herbivorous mammals (Orpin and Joblin) 1988). Since the first strictly
anaerobic and filamentous
fungus Neocallimastix frontallis was isolated in 1975 from the rumen of a
sheep (Orpin, 1975), at least
thirteen different anaerobic fungi have been isolated from ruminant and
nonruminant herbivores (Chen
et al. , 1995a). Anaerobic fungi are divided into two groups based on
morphology. One is
CA 02267078 1999-03-29
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monocentric, and it includes Neocallimastix (Orpin, 1975), Caecomyces (Wubah
and Fuller, 1991), and
Piromyces species (Burr et al., (1989) Can. J. Botany 67:2815-2824); the other
is polycentric and it
contains Orpinomyces (Bart et al . , ( 1989) supra), Anaeromyces (Breton et al
. , 1990) ) and Rurninomyces
(Ho and Bauchop, 1990). The anaerobic fungi produce a variety of enzymes that
degrade plant
materials ingested by the host animals (Borneman et al., 1989). The physical
association with the
lignocellulosic tissues of plant fragments, and the ability to penetrate and
weaken the plant tissue in
vivo (Akin et al., 1983) suggest that the fungi are involved in degradation of
digesture and that they
play an important role in the rumen ecosystem. Several cellulases and
xylanases have been cloned and
sequenced from both monocentric Neocallimastix patriciarum (Gilbert et al.,
1992; Zhou et al., 1994;
Black et al., (1994) Biochem.J. 299:381-387; Denman et al.) (1996)Appl. Envir.
Microbiol. 62:1889-
1896; Piromyces sp. (Fanutti et al. , 1995) and polycentric Orpinomyces PC-2.
A mannanase was
cloned and sequenced from Piromyces sp. (Fanutti et al., I995).
SUMMARY OF THE INVENTION
The present invention provides a substantially purified lichenase. As used
herein, a lichenase
is an enzyme which hydrolyzes the 13-I ,4-glucan bonds adjacent to >3-1,3-
linked glucan bonds, but does
not cleave fi-1,4-linked glucans. Substrates for lichenase include) without
limitation, lichenan and
barley 13-glucan. As specifically exemplified, the lichenase is selected from
the group consisting of that
naturally produced by Orpinomyces PC2 (SEQ ID N0:2, amino acids 1 to 216) and
that recombinantly
produced) for example, in Escherichia coli (SEQ ID N0:2) amino acids -8 to
216). The complete
amino acid sequence of the exemplified lichenase, including the signal
sequence, is given in SEQ ID
N0:2, amino acids -29 to 216.
It is a further object of the invention to provide a nucleotide sequence
encoding a mature
lichenase enzyme from Orpinomyces, where that sequence has at least about 80 %
sequence identity
with the exemplified coding sequence (nucleotides 2I0 to 860 of SEQ ID NO:1 )
and encodes a
lichenase enzyme having the same enzymatic specificity as the exemplified
lichenase. Additional
objects of the invention are nucleotide sequences which encode a lichenase
enzyme of the disclosed
specificity and having an amino acid sequence as given in SEQ ID N0:2, amino
acid I to amino acid
216 or as given in SEQ ID N0:2, from amino acid -8 to amino acid 216 or as
given in SEQ ID N0:2
from -29 to 216, for a lichenase with signal sequence. Variations from the
specifically exemplified
sequence are permitted, to the extent that the functionality of the enzyme is
not changed.
Specifically exemplified embodiments of the Orpinomyces PC2 coding sequences
for a mature
natural lichenase is as given in SEQ ID NO:1, nucleotides 2I0-860; for the
recombinantly expressed
lichenase, SEQ ID NO: l , nucleotides I86-860, and for the complete coding
sequence including the
signal peptide, SEQ ID N0:2, nucleotides 123-860, and sequences with at least
about 70 % homology
to the recited Sequences. Synonymous codings are within the scope of the
present invention, and are
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well within the grasp of the ordinary skilled artisan without the expense of
undue experimentation,
given the teachings of the present disclosure taken with what is well known to
the art.
It is a further object of the present invention to provide non-naturally
occurring recombinant
DNA molecules which direct the expression of a lichenase protein of the
present invention. Where
expression and secretion is desired, the complete coding sequence (SEQ ID
NO:1, nucleotides 123-860)
is operably linked downstream of promoter sequences appropriate to the
recombinant host cell in which
expression is desired. If it is preferred that the expressed lichenase protein
be intracellular, then the
coding sequence for lichenase (either as given in SEQ ID NO:1, nucleotides 186-
860 or as given in
SEQ ID NO:1, nucleotides 210-860) is joined inurtediately downstream of a
translation start signal
(ATG) and operably linked downstream of a promoter appropriate to the host
cell of choice.
Recombinant cells which express lichenase, also an object of the present
invention, are cultured under
conditions suitable for the expression of the lichenase coding sequence. Where
an inducible promoter
is used to control the expression of the lichenase coding sequence, the
skilled artisan understands that
it is desirable or essential, depending on the inducible promoter) to add the
cognate inducer to the
culture to effect gene expression. Substitution of alternative signal peptide
coding sequences is also
within the skill of the skilled artisan.
A further object of the present invention is to provide a method for the
expression of a
lichenase protein of the present invention. This method includes the step of
producing a non-naturally
occurring recombinant DNA molecule as described hereinabove) with the
lichenase coding sequence
operably linked to transcriptionai and translational control sequences
suitable for the host cell of choice,
said combination being incorporated within a vector plasmid or virus suitable
for the chosen host cell,
introducing that recombinant DNA molecule into the host cell to produce a
recombinant host cell) and
culturing the recombinant host cells under conditions suitable for expression
of the lichenase coding
sequence. Substantially pure lichenase can be purified from cell-free medium
of such cultures using
the methods provided herein.
It is a further object of the present invention to provide a substantially
pure lichenase, said
lichenase having the ability to cleave fi-1,4 glucan bonds adjacent to 13-1,3
glucan bonds, but having
no hydrolytic activity for 13-1,4 glucans. As specifically exemplified, the
lichenase expressed by
Orpinomyces PC2 has an extracellular (secreted) enzyme having a molecular
weight of about 26 kDa)
while the recombinant enzyme expressed and secreted by Escherichia coli has an
apparent molecular
weight of about 27 kDa. The lichenase of the present invention has no apparent
activity when assayed
with carboxymethylcellulose as substrate.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows protein staining patterns for crude enzyme sample (lane 1) and
purified
enzyme (lane 2) and the iichenase activity patterns for the crude enzyme
sample (lane 3) and purified
enzyme (lane 4) using lichenase as the substrate.
Figure 2 is a photograph of crude (lane 2) and purified recombinant lichenase
(lane 3) from
the extracellular medium of the E. coli culture producing the lichenase. Lane
1 contains the low
molecular weight standards.
Figure 3 shows the effects of pH on the activity of the Orpinomyces lichenase.
Maximal
activity on the curve is defined as 100%.
Figure 4 illustrates the pH stability of the Orpinomyces lichenase.
Figure 5 shows the effect of temperature on Orpinomyces lichenase activity.
Maximal activity
is defined as 100% .
Figure 6 shows thermostability profiles for Orpinomyces lichenase at selected
temperatures.
Figure 7 is a photograph of a thin layer chromatogram of the products of
barley (3-glucan and
lichenan incubated with Orpinomyces lichenase.
Figure 8A shows the protein staining profile for low molecular weight
standards (lane 1 ),
crude recombinant E. coli cell extract (lane 2), purified recombinant LICA
(lane 3), supernatant from
Orpinomyces PC2 grown on CBG (lane 4) and supernatant from Neocallimastix EB
188 grown on CBG
(lane 5). Figure 8B is a lichenan zymogram and Figure 8C is a CMC cellulose
zymogram. Lanes are
as in Figure 8A.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, lichenase is used synonymously with endo-~i-(1-3,1-4)-D-
glucanase; the
enzyme code assigned to enzymes having this activity is EC 3.2.1.73.
The gene and cDNA encoding the lichenase of the present invention is called
licA. As
specifically exemplified, the mature licA gene product (LICA) has an amino
acid sequence as given in
SEQ ID N0:2, amino acids 1-216 as expressed in Orpinomyces PC2, or a
functionally equivalent
amino acid sequence, for example, as given in SEQ ID NO: 2, amino acids -8 to
216, or -29 to 216.
Also encompassed by the present invention are lichenases with about 70 % amino
acid sequence identity
to any of the foregoing sequences. These functionally equivalent sequences
differ in the proteolytic
cleavage site for the removal of the signal peptide.
The lichenase of the present invention is distinguished from prior art
lichenases from rumen
bacteria in that there are no repeated oligopeptide sequence motifs in the
present lichenase. Without
wishing to be bound by theory, it is proposed that the lack of the repeated
motifs contributes to the
efficient expression and secretion, with functionally correct signal peptide
processing in the Escherichia
coli recombinant host cells.
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The lichenase proteins of the present invention is useful for treatment of
animal grain-
containing feeds to improve nutrient availability and for treatment of grain
(e.g. barley or wheat) in
the brewing and fermentation industries to increase carbon substrate
availability and to maximize
production of desired products. The lichenase coding sequences of the present
invention are useful to
direct the recombinant expression (in Orpinomyces or in other host cells,
including, but not limited to,
Escherichia coli, Bacillus subtilis) Aspergillus nidulans, Aspergillus niger,
Saccharomyces cerevisiae,
and Pichia pastoris).
' A cDNA expression library in ZAPII using mRNA isolated from Orpinomyces sp.
strain PC-2
cells cultivated with Avicel and oat spelt xylan as carbon source was screened
for clones with ~3-
glucanase activity on lichenan plates. Initially) 15 positive plaques were
identified after screening
2x10' plaques from the library. Eight of them were randomly choose for further
enrichment and
purification. The remaining lichenase-positive clones were not further
studied. After secondary
screening, the recombinant lambda clones were converted to pBluescript clones.
Then these eight
clones were tested for CMCase (carboxymethylcellulose-degrading activity) and
lichenase activities
after culturing them in LB-ampicillin medium. Three of them showed only
lichenase activity without
detectable CMCase activity; these clones contain the licA coding sequence.
Four of them exhibited
both lichenase and CMCase activities, and these later were further confirmed
as cellulase- positive
clones.
Analysis of the lichenase producer clones by restriction mapping revealed that
they had
similar restriction patterns (inserts of 0.9, 1.0, and 1.7 kbp) respectively).
Sequencing of both ends of
the inserts showed that they were transcripts from the same gene (licA)
differing in length at their 3'
ends.
The complete nucleotide sequence of licA derived from pLIC6 (1.0 kbp) was
determined
(Table 4 , SEQ ID NO :1 ) . The whole sequence was 971 by with a G-C content
of 28 % , and it
contained an open reading frame (ORF) encoding a polypeptide of 245 amino
acids with a calculated
M, value of 27,929 (See 5EQ ID NO: 2). A typical 18-mer poly(A) tail was found
at its 3' end. The
putative start codon (ATG) for licA was identified because there were stop
codons in all three reading
frames preceding the ORF, there was no ATG codon upstream of the identified
ORF, and a typical
signal peptide occurred at the N-terminus of the ORF. In addition, zymogram
analysis and N-terminal
sequencing of the purified LICA enzyme from the recombinant E, coli
supernatant and partially
purified native enzyme from Orpinomyces PC-2 further confirmed this
assignment. Only one potential
N-glycosylation site (Asn~'-Gly-Ser'6) was present near the N-terminus of the
mature enzyme; the
enzyme may not be glycosylated.
The G+C content of the ORF of licA was 3S.5 % while that of the 5' and 3' non-
coding
sequences was extremely low (4.3 % ). The codon usage for licA was similar to
that observed for other
Orpinomyces PC-2 cellulase and xylanase genes. 21 codons were not utilized,
and there was a marked
preference for a T in the third position (S3 % of all codons contained T in
the third position).
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mRNAs of anaerobic fungi do not contain a typical E. coli Shine-Dalgarno-like
sequence for
translation initiation. However, presumably the sequence AGA, 10 by upstream
of the ATG start
codon, acts as a weak ribosome-binding sequence in E. coli. This sequence was
also found in a
xylanase gene (xynA) from N. patriciarum (Gilbert et al.) 1992).
S The deduced amino acid sequence of the protein LICA was compared with other
protein
sequences in the SWISS PROT and GP data banks. A number of /3-glucanases from
mesophilic and
thermophilic bacteria, including anaerobic rumen bacteria, with some identity
to LICA were found.
Greater than 50 % identity was found with (3-glucanases from certain Bacillus
strains, Clostridium
thermocellum) and the carboxy-terminal lichenase domain of the xylD gene of
the anaerobic rumen
bacterium R. flavefaciens. LICA has 30.6% amino acid identity with /3-
glucanase from Fibrobacter
succinogenes (Table 1). In contrast, limited sequence homology was found upon
comparison with
barley ~-glucanase.
Some of the homologous sequences were aligned with LICA sequence (Table 5).
This
alignment revealed that the similarity between these ,Q-glucanases is stronger
in the central and C-
terminal parts of the proteins. The motif DEIDI (SEQ ID N0:6), which is
located in the active site
cleft in lichenase from Bacillus licheniformis formis (Juncosa et al., 1994))
was conserved in the LICA
sequence (133-137 position). According to the classification of Henrissat and
Bairoch (1993), LICA
should be placed in Glycosyl Hydrolase Family 16, which includes most
bacterial lichenases.
In order to study the expression and the distribution of the ~3-glucanase
synthesized in E. coli
harboring pLIC6, extracellular, periplasmic and cellular fractions were
isolated according to the method
described by Cornelis et al. (1982). A major part of the total activity was
found in the extracellular
fraction, with significant periplasmic activity. These two fractions contained
greater than 90 % of the
expressed enzyme activity in E. coli. J3-Galactosidase and alkaline
phosphatase were used as
cytoplasmic and periplasmic markers, respectively. Additionally, the secreted
enzyme encoded by
clone pLIC6 was visualized by the zymogram technique involving renaturation of
enzyme activity
following separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (Fig. 1 ). The
results show that a strong polypeptide band detected at 27 kDa exhibited only
lichenase but not
CMCase activity.
Taken with results for the N-terminal sequence of the mature protein, these
results indicate
that the export mechanism of E. coli accepts export signals from the anaerobic
fungus Orpinomyces
lichenase, correctly processes the protein and transports it to the
periplasmic space. Similar results
have been reported for expression of bacterial extracellular lichenase genes
cloned in E. coli (Lloberas,
1991; Borriss et al. 1990). By contrast, the xylanase, the cellulase and the
mannanase from the fungus
N. partricerum expressed in E. coli were found predominantly in cell-free
extracts, indicating that these
enzymes were not effectively secreted by E. coli (Gilbert et al, 1992; Zhou et
al., 1994, Fanutti et al.,
1995).
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A summary of the purification of the recombinant LICA from the supernatant of
the E, coli
culture is given in Table 2. The enzyme was purified about 24-fold, with a
yield of about 43.5 % .
Based on calculations of the specific lichenase activity of the purified LICA
and the recovery of the
enzyme, the recombinant LICA protein constituted about 4 % of the secreted
protein in the
recombinant E. coli culture. Considering that E. coli XL1 is not considered a
superb expression host
for recombinant proteins, the surprisingly large amount of recombinant LICA
detected in the culture
supernatant indicates that the signal sequence of LICA is very effectively
processed in E. coli. Using
a similar purification strategy and zymogram analysis to monitor lichenase,
the native lichenase from
the supernatant of Orpinomyces PC-2 culture was also partially purified.
Analysis of the amino acid sequence of the N-terminus of the recombinant LICA
isolated from
the supernatant of E. coli culture indicated that the recombinant protein is
processed in E. coli, with
a 21-amino acid signal sequence being removed to give a mature active enzyme
of 224 amino acids
(22 N-terminal residues if the LICA exactly matched the deduced protein
sequence). The presumptive
21-amino-acid signal sequence deduced from the DNA sequence contains all of
the features normally
associated with a signal sequence for secretion (Von Heijne, 1988), including
a positively charged
lysine (-20) terminal n region and a strongly hydrophobic h region from amino
acid residues -18
through -6 (10 out of 13 are hydrophobic; amino acid positions are given
relative to the first residue
of the mature peptide). The c region of the signal peptide conforms to the "(-
3, -1) rule", with small,
uncharged threonine and alanine residue at positions -3 and -1 relative to the
cleavage site, which is
typical for a peptide cleaved by signal peptidase I in E. toll.
The partially purified native lichenase from supernatant of Orpinomyces PC-2
culture was
subjected to N-terminal sequence analysis. The enzyme had a M~ of 26,000 and
an N-terminal
sequence of GTAWNGLHDVMD) (SEQ ID N0:3) which, with the exception of one amino
acid,
matched the corresponding amino acid sequence deduced from the DNA sequence.
Thus, a 29-amino
acid signal sequence was removed to give a natural mature enzyme of 216 amino
acids (Table 4).
These results indicate that there are differences in the substrate
specificities of the prokaryotic E. toll
and eukaryotic (anaerobic fungus) signal peptidases. Normally, proline is
conspicuously absent from -
3 to + 1 regions of prokaryotic signal peptides, but it is not usual to have
proline at the corresponding
region of eukaryotic signal peptide (Von Heijne, 1986). Another reason for the
lichenase N-terminal
signal sequence being processed differently may come from "the Charge-Block
Effect". A region
encompassing the first 10-20 resides of the mature protein is also critical
for the initiation of membrane
translocation in E. toll (Anderson and von Heijne, 1991). This region normally
contains few positively
charged amino acids; hence, the introduction of only one or two extra
positively charged amino acids
can dramatically affect secretion (Li et al., 1988). With much higher numbers
of charged residues,
' 35 a similar blocking effect can be observed in eukaryotic secreted proteins
(Kohara et al., 1991). The
first 20 amino acid residues of the mature recombinant lichenase contain only
one positively charged
amino acid (histidine); this makes the processed enzyme effectively secreted.
If the cleavage site of
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the lichenase processed in E. coli was the same as in the fungus, the mature
recombinant lichenase
would be difficult to export from E. coli, simply because the N-terminal
region of the mature chain
carries too many positively charged amino acids (3 out of 20 animo acid
residues).
The purified recombinant lichenase appeared as single hand with an apparent
molecular mass
of 27 kDa on SDS-PAGE (Fig. 2), which is consistent with the deduced molecular
mass of the mature
LICA (25.7 kDa) after removal of a signal peptide of 21 ammo acid residues.
The lichenase activity
of the enzyme was measured from pH 4.2 to 8.6 using lichenan as substrate. A
typical pH profile was
obtained (Fig. 3), with a broad pH optimum from pH 5.8-6.2, with approximately
80% of maximum
activity at pH 5.4 and pH 7Ø The enzyme was stable for at least 24 h between
pH 3.4 and 9.8 at
4~C (Fig. 4). The lichenase activity was measured in 50 mM sodium citrate at
pH 6.0 from 30 to 65~C.
Maximum activity was observed at 45~C (Fig. 5). The enzyme exhibited at least
70 % of its optimal
activity over the range 35-55~C, and its activity decreased rapidly above
55~C. Thermal stability was
investigated by incubating the enzyme, up to 24 h, at different temperatures
(Fig. 6). Almost no
activity loss was observed at 40~C with incubation in the above buffer for 24
h. 72 % and 59 % of the
enzyme activity was retained after 24 h incubation at 45~C and 50~C,
respectively. Inactivation
occurred at 55~C with only 30 % of the enzyme activity remaining after lh.
The enzymatic activities of the recombinant lichenase were assayed using
lichenan, barley-(3-
glucan, laminarin, pachyman) CMC, acid swollen cellulose, puslutan and other
polysaccharides and
glycosides as substrates and analyzed by the dinitrosalicylic acid (DNS)
method. The enzyme was
specific for polysaccharides with mixed 1, 3-1,4-/3-D-linkages (lichenan and
barley ,B-glucan) and did
not hydrolyze the other substrates tested (Table 3). K," and V",~ values at
40~C were obtained from
Lineweaver-Burk plots. K," values of the enzyme towards lichenan and barley-~-
glucan were 0.75 %
(w/v) and 0.91 % (w/v) and V",~, values were 3,786 and 5,314 U/mg protein,
respectively.
The nature of products formed during the action of the purified recombinant
lichenase on
lichenan and barley-~3-glucan was studied using silica gel thin layer
chromatography (TLC, Fig. 7).
In extended incubation with both substrates, the reactions proceeded to
apparent completion. With
lichenan as substrate, the major product was a triose which was migrated on
TLC just slightly ahead
of cellotriose, and was considered to be 3-O-(3-cellobiosyl-D-glucose (Huber
and Nevin, 1977; Erfle
et al., 1988). Minor products included pentose (3-O-(3-cellotetraosyl-D-
glucose) and tetraose (3-O-~-
cellotriosyl-D-glucose) which migrated on TLC a little ahead of cellopentose
and cellotriose. An
additional minor component was a biose (laminobiose), which migrated ahead of
cellobiose. Barley
a-glucan treated in the same manner gave distinctly different profiles which
reflected the structural
differences between lichenan and barley (3-glucan (Buliga et al., 1986). The
major products from
barley (~-glucan hydrolysis were triose and tetrose. The products from both
substrates were similar
with those described for the lichenases from B. subtihs (Huber and Nevin,
1977) and R. succinogenes
(Erfle et al., 1988). From these results) the recombinant lichenase appears
similar to other 1,3-1,4-(3-
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WO 98l14595 PCT/US97I17811
D-glucanases in its general pattern of action, with a cleavage site which is a
a-1,4 glucopyranosidic
linkage of a 3-O-substituted /3-D-glucopyranose unit (Buliga et al., 1986).
Lichenase and cellulose activities were detected using zymogram technology
with an overlay
containing lichenan or CMC, respectively. A clear strong band of lichenase
activity was observed at
approximately 27 kDa for the cell extract of the recombinant E. coli) the
purified recombinant LICA,
and the supernatant of Orpinomyces PC-2 culture. No activity was observed at
this molecular weight
when CMC was used as substrate) indicating that the LICA is specific for
lichenan (Fig. 8).
Additionally, the results revealed that the licA gene product was actively
synthesized and secreted into
medium of the Orpinomyces PC-2 culture. There were multiple faint high
molecular mass bands in both
Orpinomyces PC-2 and Neocallimastex EB188 culture supernatant) which reflects
cellulases having
some ability to hydrolyze lichenan. No equivalent lichenase activity band
corresponding to the
Orpinomyces PC-2 lichenase was detected in the monocentric anaerobic fungus
Neocallimastix EB 188
sample. Thus, Neocallimastix EB188 appears to lick a lichenase gene.
1,3-1,4-a-D-Glucanases cleave 1,4-(3-giycosidic linkages that are adjacent to
1,3-/3-
glycosidic linkages in mixed-linked glucans, which comprise an important
component of plant
hemicellulose. The present 1,3-1,4-~3-D-glucanase (lichenase) does not cleave
the ~3-1,4 glycosidic
- bonds in carboxymethylcellulose. To date, 1,3-1,4-~3-D-glucanase has been
found only in certain
bacterial strains and in plants. This is believed to be the first report that
describes the primary
structure and properties of a typical /3-1,3-1,4-D-glucanase from a fungus.
Most hydrolytic enzymes (particularly xylanases or cellulases) cloned from
anaerobic fungi
have a protein docking domain containing 2-3 repeated motifs of 30-40 amino
acids each. The repeated
peptide sequences are highly homologous to each other regardless of
monocentric or polycentric
origins. The repeated domains are not involved in catalysis or cellulose
binding, but in formation of
multienzyme complexes similar to the cellulosomes of anaerobic bacteria (Felex
and Ljungdahl) 1993).
Orpinomyces LICA does not contain a repeated peptide domain, which indicates
that it is a free
enzyme and not a component of the multienzyme complex. While 1,3-1,4-a-D-
glucanases from the
rumen bacteria R. flavefaciens (Flint et al., 1993) and F. succinogenes
(Teather and Airflow, 1990)
have a repeated docking domain, the partial sequence identities between the
lichenases of the rumen
bacteria and the present lichenase are much lower than those between the
present lichenase and
lichenases of Bacillus strains or Clostridium thermocellum.
Although the N-terminal signal sequence of LICA is a secretory signal that is
functional both
in E. coli and in Orpinomyces) the cleavage sites are different. Besides the
difference between E. coli
and eukaryotes with respect to signal peptides and proteases as discussed
hereinabove, the different
cleavage sites in LICA signal sequence may also relate to the cell membrane of
the anaerobic fungi.
Anaerobic fungi lack the ability to synthesize some common cell-membrane
constituents such as sterol
because of the absence of molecular oxygen. Instead) unusual lipids
synthesized by the anaerobic
pathway are incorporated into the anaerobic cell membrane (Kemp et al. ,
1984). In contrast, all
9
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previously described cloned hydrolytic enzyme genes in the fungi which contain
the repeated peptide
domain are neither effectively expressed nor secreted by E. coli. The
expressed enzymes were also
often subjected to extensive proteolysis in E. coli) perhaps due to partial
removal of non-catalytic
repeated domains of the enzymes (Gilbert et al., 1992; Fanutti et al., 1995).
Monocentric and polycentric anaerobic fungi are two different groups based on
their
morphological patterns. Very commonly, hydrolytic enzymes of monocentric
Neocallimastex and
Piromyces species have more than one catalytic domain in a single protein
(Gilbert et al., 1992; Fanutti
et al. , 1995), but no such structure has been found in any cloned and
sequenced genes for polycentric
Orpinomyces hydrolytic enzymes so far. Neocallimastex EB188 does not appear to
have a Iichenase
i 0 with the same properties as the lichenase disclosed herein. Since
cellulases also have activity to
hydrolyze ( 1, 4)-a bonds in lichenan and /3-glucan, the selective advantage
for Orpinomyces to
synthesis lichenase in the rumen ecosystem is not clear .
The LICA signal peptide of this invention may be used to increase yield of
foreign genes in
host cells in which they are expressed. Any host cell in which the signal
sequence is expressed and
processed may be used. The signal peptide sequence (see SEQ ID NOs. 4 and 5
for coding and amino
acid sequences) from the Aureobasidium xylanase can be substituted for the
exemplified LICA signal
sequence. Preferred host cells are Aureobasidium species and S. cerevisiae, as
well as other yeasts
known to the art for fermentation, including Pichia pastoris (Sreekrishna, K.
, "Strategies for optimizing
protein expression and secretion in the methylotrophic yeast Pichia pastoris,"
in Baltz, R.H., et al.
(eds.) Industrial Microorganisms: Basic and Applied Molecular Genetics, ASM
Press, Washington,
D.C. (1993) 119-126; Glick, B.R. and Pasternak, J.J., "Molecular Biotechnology
- Principles and
Applications of Recombinant DNA," ASM Press (1994) Washington, D.C.).
Filamentous fungi such
as Aspergillus, Trichoderma, Penicillium) etc. are also useful host organisms
for expression of the
DNA of this invention. (Van den Handel, et al., "Heterologous gene expression
in filamentous fungi,"
(1991) In: Bennett, J.W, and Lasure, L.L. (eds.), More gene manipulations in
fungi, Academy Press,
Inc., New York, 397-428). When DNA encoding the LICA signal peptide is ligated
to DNA encoding
other proteins expressible in these hosts, the gene products are secreted from
these organisms with the
help of the signal peptide.
In addition the coding region for both the signal peptide and the mature LICA
protein may be
expressed in such hosts. Alternatively ) the LICA mature protein coding region
isolated from the signal
sequence may be expressed in such hosts) or the coding region for the signal
peptide isolated from the
mature protein coding region may be expressed in such hosts.
In a preferred embodiment, vectors suitable for transformation of the host,
preferably S.
cerevisiae, with the licA gene) cDNA encoding the LICA mature protein, or the
LICA signal peptide
cDNA coding sequence in combination with a suitable foreign gene expressible
in S, cerevisiae, are
prepared with the gene under control of a promoter expressible in the host,
preferably S. cerevisiae.
Preferably the promoter is a constitutive promoter such as the yeast enolase
promoter (Sangadala et
CA 02267078 1999-03-29
WO 98I14595 PCTlUS97/17811
al . , ( 1994) "Preparation and characterization of the site-directed E211 Q
mutant of yeast enolase, " In:
Abstracts of University System of Georgia 1994 Research Symposium: Advances in
Biotechnology,
Georgia State University ) Atlanta, GA, USA) or a strong inducible promoter
such as the yeast alcohol
dehydrogenase promoter (Pacitti, et al. ( 1994), "High level expression and
purification of the
enzymatically active cytoplasmic region of human CD45 phosphatase from yeast,"
Biochimica et
Biophysics Acta 1222:277-286). The vector is used to transform the host either
by integration into the
chromosome or otherwise. The host organism is then cultured under conditions
allowing expression
of the gene and the product recovered from the culture medium.
Additionally, it will be recognized by those skilled in the art that allelic
variations may occur
in the licA coding sequence from different strains of Orpinomyces or other
fungi which will not
significantly change activity of the amino acid sequences of the proteins
which these sequences encode.
All such equivalent DNA sequences are included within the scope of this
invention and the definition
of the LICA mature protein coding region and signal sequence coding region.
The skilled artisan
understands that the amino acid sequence of the exemplified LICA polypeptide
and signal peptide can
be used to identify and isolate additional, nonexemplified nucleotide
sequences which will encode
functional equivalents to the polypeptides defined by the amino acid sequences
given in SEQ ID N0:2)
or an amino acid sequence of greater than 90 % identity thereto and having
equivalent biological
activity. DNA sequences having at least about 70 % , 80 % and/or 85 % homology
to the DNA
sequences of 5EQ ID NO:1 {nucleotides 210 to 857) and encoding polypeptides
with the same function
are considered equivalent to the sequences of SEQ ID NO:1 and are included in
the definition of "DNA
encoding the LICA raature protein" and the "licA gene. " Following the
teachings herein, the skilled
worker will be able to make a large number of operative embodiments having
equivalent DNA
sequences to those listed herein.
It is further understood that codons for conservative amino acid substitutions
can change the
primary amino acid sequence of a lichenase protein without significantly
affecting the function of that
protein. Such conservative amino acid substitutions are well known to the art
(See, e. g. , Dayhoff et
al. (1978) in Atlas of Protein Sequence and Structure, Vol. 5, Supplement 3,
Chapter 22, pages 345-
352). Dayhoff et al.'s frequency tables are based on comparisons of amino acid
sequences for proteins
having the same function from a variety of evolutionarily different sources.
As used herein, a recombinant DNA molecule is not naturally occurnng; it is
produced by the
hand of man in the laboratory. DNA segements or sequences from different
sources can be joined by
chemical synthesis, enzymatic ligation or by directed recombination.
Monoclonal or polyclonal antibodies, preferably monoclonal) specifically
reacting with a
lichenase encoded by a particular coding sequence may be made by methods known
in the art. See,
e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratories;
Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d ed., Academic
Press, New York.
11
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Standard techniques for cloning, DNA isolation, amplification and
purification, for enzymatic
reactions involving DNA ligase) DNA polymerase, restriction endonucleases and
the like, and various
separation techniques are those known and commonly employed by those skilled
in the art. A number
of standard techniques are described in Sambrook et al. ( 1989) Molecular
Cloning, Second Edition,
Cold Spring Harbor Laboratory, Plainview, New York; Maniatis et al. ( 1982)
Molecular Cloning, Cold
Spring Harbor Laboratory, Plainview, New York; Wu (ed.) (1993) Meth. Enrymol.
218, Part I; Wu
(ed. ) ( 1979) Meth Enrymol. 68; Wu et ai. (eds. ) ( 1983) Meth. Enzymol. 100
and 101; Grossman and
Moldave (eds.) Meth. Enrymol. 65; Miller (ed.) (1972) Experiments in Molecular
Genetics, Cold
Spring Harbor Laboratory, Cold Spring Harbor, New York; Old and Primrose (
1981 ) Principles of
Gene Manipulation, University of California Press, Berkeley; Schleif and
Wensink (1982) Practical
Methods in Molecular Biology; Glover (ed.) (l985) DNA Cloning Vol. I and II)
IRL Press, Oxford,
UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridi2ation, IRL Press,
Oxford, UK; and Setlow
and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4,
Plenum Press, New
York. Abbreviations and nomenclature, where employed) are deemed standard in
the field and
commonly used in professional journals such as those cited herein.
All references cited in the present application are incorporated by reference
herein.
- The following examples are provided for illustrative purposes, and are not
intended to limit
the scope of the invention as claimed herein. Any variations in the
exemplified articles which occur
to the skilled artisan are intended to fall within the scope of the present
invention.
EXAMPLES
Example 1: Fungal Strain, Culture Condition, and Vectors
Orpinomyces sp strain PC-2 was isolated and described by Borneman et al. (
1989);
Neocallimastix sp. EB188 was provided by Dr. Calza (Washington State
University). For enzyme
production, the fungi were grown at 39~C for 7 day in 2 L round bottles, each
containing 1 L of basic
medium (Barichievich and Calza, 1990) and 0.3 % of coastal Bermudagrass (CBG).
The medium was
autoclaved for 30 min, and then cooled under a stream of CO2. Penicillin (334
U/ml), streptomycin
sulfate (80 pg/ml), and chloramphenicoI ( 10 ~.g/ml) were filter sterilized
(0.22 pm) (alt 230) and added
to the stated final concentrations just prior to inoculation. Escherichia coli
XL-Blue, ~ZAPII,
pBluescript were products of Stratagene Cloning Systems (La Jolla, CA).
Example 2: Recovery of Extraceliular, Periplasmic and Cellular 1.3-1,4-li-D-
glucanase in
Recombinant E. coli
Expression of endo-1,3-1,4-/3-D-glucanase activity was detected according to
the procedures
of Neu and Heppel (1965) and Cornelis et al. (1982). E. coli was harvested by
centrifugation at 3,200
g for 10 min (Beckman CS-6R). Cell free culture media were used for
extracellular enzyme
preparation. The cell pellet was washed twice in half the volume of the
culture with 10 mM Tris-HCl
12
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pH 8.0 and suspended in the same volume of 25 % sucrose 5 mM EDTA. The
suspension was shaken
for 10 min at room temperature. After centrifugation, the cells were suspended
in the same volume
of ice-cold water, and the suspension was shaken for 10 min at 4~C. After
centrifugation, the
supernatant was used as the periplasmic fraction. The cell pellet was
sonicated to release intracellular
1,3-1,4-~3-D-glucanase activity.
Example 3: Construction and Screenine of an Orpinomyces cDNA Library
Extraction of RNA, purification of mRNA, and construction of a cDNA library
from
Orpinomyces PC-2 were described previously (Chen et a1.,1995).
Top agar of NZY plates containing S mM isopropyl-I-thio-(3-D-galactopyranoside
(IPTG) and
0.2% lichenan (Sigma Chemical Co., St. Louis, MO) was used to isolate I,3-1,4-
~3-D-glucanase-
producing clones. After growth at 37~C overnight, plates were stained by
flooding with 0.1 % Congo
red for 20 min: clear haloes around colonies on the background indicate ~3-
glucanase activity.
Improved clear zones were obtained by treatment of stained agar plates with 1
M NaCI for 20 min.
before observation. Pure positive clones were obtained after a secondary
screening. Positive ~ clones
were convened to pBluescript SK- clones by in vivo excision. Single colonies
were picked from the
LB plates and separately inoculated into 5 m1 LB +ampicillin ( 100 ~g/ml)
medium. The cultures were
shaken for 7-8 hours at 250 rpm) 37~C until the ODD of the cultures reached
about 1.5) and then 10
pl of 0.5 M IPTG was added to each culture and the tubes were incubated for
another 5 hours. The
cultures were then sonicated. After centrifugation, the clear supernatants
were used for testing
lichenase and CMCase activities. The pBluescript DNAs were purified from
overnight cultures in LB
medium containing 50 ~,g/ml ampicillin using the WizardT"' Maxipreps plasmid
purification system
(Promega, Madison, WI). Nucleotide sequences of insert DNA were determined
with an automatic
PCR sequencer (Applied Biosystems Foster City, CA). Both universal and
specific primers were used
to sequence both strands of the inserts. Sequence data were analyzed using the
Genetic Computer
Group (GCG) version 8 (University of Wisconsin Biotechnology Center, Madison,
WI) on the
VAX/VMS system of the BioScience Computing Resource at the University of
Georgia). The
nucleotide sequence of licA of Orpinomyces sp. PC-2 has been assigned
accession number U63813.
Example 4: Sodium Dodecyl Sulfate-Polyacrvlamide Gel Electrophoresis (SDS-
PAGE)
SDS-PAGE was carried out in Laemmli's buffer (Laemmli, U.K. (1970), Nature
227:680)
with Coomassie brilliant blue R-250 (Sigma Chemical Co., St. Louis, MO). To
visualize enzyme
activity, samples were pretreated by incubating for 1 h at 40~C in sample
buffer and proteins were size-
separated using SDS-PAGE at 4~C. To enhance removal of SDS and recovery of
enzymatic activity
following SDS-PAGE, gels were washed in SO mM sodium citrate buffer, pH 6.0
with 1 % (w/v)
bovine serum albumin (BSA) (McGrew and Green, 1983).
13
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Lichenase and CMCase activities were detected using the zymogram method of
Beguin (1983)
with a overlay containing 0.3 % (w/v) lichenan or carboxymethylcellulose and
agarose (2 % , w/v) in
50 mM sodium citrate buffer) pH 6Ø The bands of enzyme activity were
detected by staining the
agarose gel with Congo red and destraining with 1M NaCI.
Example 5: Enzyme Assay
All enzyme assays were carried out in duplicate in 50 mM sodium citrate buffer
(pH 6.0) at
40~C unless otherwise stated.
~3-Glucanase activity was assayed by mixing a 0.2 ml aliquot of appropriately
diluted enzyme
with 0.4 m1 buffer containing 0.4% (w/v) lichenan or barley (~-glucan (Sigma
Chemical Co., St.
Louis, MO). The reaction was for 15 min and terminated by the addition 1.2 ml
of 31 mM
dinitrosalicylic acid (DNS) (Miller, 1959). The reaction tube was then placed
in boiling water for 5
min before determining the absorbance at 550 nm. Glucose was used as standard.
Activities on other
polysaccharides were assayed in assays similar to that using lichenan.
One unit {U) of enzyme activity was defined as the amount of enzyme releasing
one p,mol
glucose per min. Specific activity was expressed as units per mg of protein.
Protein concentration
was determined by the Bradford method and the Coomassie protein assay reagent
(Pierce Chemical
Co. , Rockford, IL) in duplicate sets using BSA as standard.
Example 6: Enzyme Purification
An overnight culture (10 ml) of E. coli XL1-blue (pBluescript-licA) was
inoculated into 500
ml LB-ampicillin (SO pg/ml) medium and grown to an ODD of 1.5 to 2Ø /3-
Glucanase expression
was induced by the addition of 1 mM IPTG, and each culture was aerated for an
additional 8 h at
37~C. A cell-free supernatant was obtained by centrifuging the culture at 4~C,
7,000 x g for 10 min.
The cell pellet was set aside, and the supernatant was concentrated to a
volume of about 50 ml by using
an ultrafiltration cell (Amicon Co., Beverly, Mass.) equipped with a PM 10
membrane. The
concentrated supernatant was dialyzed against 500 ml of 20 mM potassium
phosphate, pH 7Ø
Ammonium sulfate was added to a concentration of 0.8 M. The solution was
centrifuged at 4~C and
20,000 x g for 10 min to remove precipitated material. The clear solution was
loaded on a Phenyl
Superose 10/ 10 column (7.85 ml) equilibrated with 20 mM potassium phosphate,
pH 7.0) containing
0.8 M ammonium sulphate. ~3-Glucanase was eluted with a 200 ml linear gradient
of ammonium
sulphate, from 0.8 to 0 M, then further with 100 ml distilled water. Fractions
containing (3-glucanase
activity were pooled and concentrated, and the buffer was changed to 20 mM
piperazine-HCI, pH 5.5.
The solution was applied to a Mono Q 5/5 anion exchange column (I ml)
equilibrated with 20 mM
piperazine-HCI buffer, pH 5.5. The /3-glucanase fractions did not bind to the
column, and the enzyme
was eluted by applying 5 column volumes of the buffer. The /3-glucanase-
containing fractions were
pooled and concentrated, and the buffer was changed to 20 mM sodium acetate,
pH 5Ø The enzyme
14
CA 02267078 1999-03-29
WO 98I14595 PCT/US97/17811
sample applied to a canon exchange Resource S column (1 ml). It did not adsorb
to the column, and
it was eluted out by further passing through 5 column volumes of the buffer.
Final purification was
achieved by gel filtration over Superdex 75 10/30 column (composite of cross-
linked agarose and
dextran gel filtration resin, Pharmacia, Piscataway, NJ) equilibrated with 20
mM sodium phosphate,
100 mM NaCI, pH 6Ø Fractions exhibiting ~i-glucanase activity were combined
and stored at -20~C.
For partial purification of native (3-glucanase from the culture supernatant
of Orpinomyces
PC-2 culture, purification procedures as above were employed.
Example 7: N-Terminal Amino Acid Sequencine
Amino acid sequencing was done with protein bands isolated and purified after
SDS-PAGE.
The proteins were transferred onto a poly-vinylidene difluoride (PVDF)
membrane in a Mini Trans
Blot cell (Bio-Rad Laboratories) Hercules) CA). The transferred proteins were
visualized by Ponceau
S staining and then excised with a razor blade. N-terminal amino acid
sequencing was performed on
an Applied Biosystems model 477A gas-phase sequencer equipped with an
automatic on-line
phenyIthiohydantoin analyzer.
ExamQle 8: Enzvme Characterization
The pH optimum was determined at 40~C using the following buffers: 0.1 M
sodium acetate
(pH 4.2 to 5.4), sodium phosphate (pH 5.8 to 7.8), and Hepes-NaOH (pH 8.2 and
8.6) with
increments of 0.4. Enzyme stability at different pH values was determined by
measuring the residual
activity after incubating the enzyme for 24 h at 4~C at pH 3.0 to 10.2
(glycine-HCI buffer for pH 3.0
to 3.4; Hepes-NaOH for pH 9.0; piperazine-HCl for pH 9.4 to 10.2). For other
pH ranges, buffers
were the same as those used for optimum pH determinations).
The effect of temperature on (3-glucanase activity was determined by assaying
the enzyme at
temperatures from 30 to 65~C with increments of 5~C. Thermostability was
measured by incubating
the enzyme in 50 mM sodium citrate buffer, pH 6.0 for 5 min to 24 h at
temperatures from 40 to 60~C
with increments of 5~C. The enzyme solution was chilled in an ice bath for 5
min and then analyzed
by running the standard assay at 40~C. In all these assays, lichenan was used
as substrate.
For determination of Km and V",~, suitably diluted (3-glucanase was incubated
with lichenan
and barley-a-glucan at concentrations ranging from 0.02 to 1.0% (w/v) under
the assay conditions
given. Km and V",~ values were obtained from Lineweaver-Burk plots.
Analysis of lichenan and barley-(3-glucan degradation products was carried out
with 5 U of
the purified recombinant a-glucanase with 5 mg individual substrate in 1 m1 of
a 50 mM sodium
citrate buffer) pH 6.0, at 40~C. Samples were periodically withdrawn and
hydrolysis was stopped by
placing the reaction in boiling water for 5 min. A 10 ~1 portion of each
sample was spotted onto thin-
layer chromatography (TLC) silica gel plate (Analtech) Inc.) Newark, DE) and
chromatographed in
a solvent system containing chloroform, glacial acetic acid, and water (6:7:1,
vol/vol) (Lake and
CA 02267078 1999-03-29
WO 98I14595 PCT/LTS97/17811
Goodwin, 1976). Plates were sprayed with a reagent consisting of aniline (2
ml), diphenylamine (2
g), acetone (100 ml)) and 85% H~P04 (15 ml). Then sugars were visualized by
heating the plate for
15 min at 105~C (Hansen, 1975). Glucose, cellobiose, cellotriose,
cellotetraose and cellopentaose were
used as standards.
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WO 98/14595 PCTILTS97/178i i
Table 1. Iiomoiogy comparison between Orpinomyces PC-2 LICA and other
~-glucanases
Strain hio. of AA overlap Identity (%)
Bacillus polymyxa 207 58
Bacillus subtilis 199 56.8
Clostridium thermocellum 243 SZ.7
Bacillus macerans 204 58.3
Bacillus licheniformis 200 57
to Bacillus amyloliquefaciens200 55.5
Clostridium thermocellum 194 55.2
Ruminococcus 201 55.7
flavefaciens
Fibrobacter succinogenes 170 30.6
20
Table 2. Summary of the purification of the recombinant LICA
Step Enzyme Specific activity
U U mg 1
Supernatant 4,388 156.7
Phenyl Superose3,149 1,850
Mono Q 2,648 2,400
Resource S 2,421 2,950
Superdex 75 1,909 3,786
17
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WO 98l14595 PCT/US97/17811
Table 3. Substrate specificity of purified recombinant LICA of
Orpinomyces PC-2'
Substrate Linkage Specific %
activity
U mg''
Lichenan ,B-1,3; ~-1,4 3,786 100
Barley ~-glucan~-1,3; S-1,4 5,317 140
Laminarin ~-1,3; S-1,6 0 0
Pachvman S-1,3 0 0
CMC ~-1,4 0 0
Acid swollen cellulose ~-1,4 0 0
Pustulan ~-1,6 0 0
The following substrates were not hydrolyzed: Avicel, arabinogalactan,
mannan, araban, starch, xylan, pullulan, galactan, and Gum arabic
(0.35% wt vol-1), PNP-S-D-xyloside, PNP-~S-D-glucoside, and PNP-~-D-
cellobiose { 1 1nM) .
18
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WO 98/14595 PCT/US97/17811
Table 4. Nucleotide and deduced amino acid sequences of a S-1, 3-1, 4-
glucanase (lichenase) cDNA (licA) of Orpinomyces PC-2
AATAAAAGAAAAAA
AAAATATATATTAAATAATAATATATATTAAAGTAAATJ?~~AAAAAAATTTAAGAAAATAT
TTTCATTATATAATTAATATTTTTTGATAAAATAAAGATTATAATAAAATGAAAAGTATA
M K S I
ATTTCTATTGCTGCTTTATCTGTTTTAGGATTGATTTCTAAAACTATGGCTGCTCCTGCT
I S I A A L S V L G L I S K T M A A P A
1
CCCGCTCCTGTTCCTGGTACTGCTTGGAATGGTAGTCATGATGTCATGGATTTCAACTAT
P A P V P G T A W N G 8 H D V M D F N Y
1
CATGAAAGTAACCGTTTTGAAATGTCAAACTGGCCAAATGGTGAAATGTTTAATTGTAGA
H E S N R F E M S N W P N G E M F N C R
TGGACTCCAAATAATGACAAATTTGAAAATGGTAAATTAAAGCTTACTATTGATAGAGAT
W T P N N D K F E N G K L K L T I D R D
GGTTCCGGATATACTTGTGGTGAATATCGTACTAAAAACTATTATGGATATGGTATGTTC
G S G Y T C G E Y R T K N Y Y G Y G M F
CAAGTTAATATGAAACCAATTAAGAATCCAGGAGTTGTTTCTTCCTTCTTTACTTACACA
Q V N M K P I K N P G V V S S F F T Y T
GGACCAAGTGATGGAACTAAGTGGGATGAAATTGATATAGAATTCCTTGGTTATGATACA
G P S . D G T K W D E I D I E F L G Y D T
ACCAAAGTTCAATTTAACTACTACACTAATGGACAAGGTCATCATGAACATATTCATTAT
T K V Q F N Y Y T N G Q G H H E H I H Y
CTTGGATTTGATGCCTCTCAAGGATTCCATACCTATGGTTTCTTCTGGGCGAGAAATTCT
L G F D A S Q G F H T Y G F F W A R N S
ATTACATGGTATGTAGATGGTACAGCCGTTTACACTGCTTACGACAATATTCCAGATACA
I T W Y V D G T A V Y T A Y D N I P D T
CCAGGTAAGATTATGATGAATGCTTGGAATGGTATTGGAGTTGATGACTGGCTTAGACCA
P G K I M M N A W N G I G V D D W L R P
TTTAATGGAAGAACTAATATTAGTGCCTACTATGATTGGGTATCTTATGATGCACCAAGA
F N G R T N I S A Y Y D W V S Y D A P R
AACTAAATTATTTAAATAAATATATAATTTTTGTTTTAAAATTTAAAAAAATATATATAT
N
ATATATTATAAATTAATATGAAAAATAAAAATAAGATGT
1. t: Cleavage site for the recombinant protein
2. Underline: n-terminal sequence of the recombinant protein
3. t: Cleavage site for the native protein from the fungus
4. Bold and underline: n-terminal sequence of native protein from
the fungus
5. Double underline: poly-A tail
19
Table 5. Alignment of some homologous sequences with Orpinomyces LICA sequence
0
Li c a_Orp in 1 . . . . ~~- AL~G~~~R ~ . . . PAP ~ P~P G~AWN$SHD~MDF~H~ NRF .
(~P
Gub_Bacpo 1 . MMRR FAT ITG 1351l~~lFF . tV8 FA. . . . N!1l~~FWEPL . . . . . .
. . . S FN STW R GYS Q
Gub_Bacau 1 MPY.L_LVTG FMS F._~TASAQT GSFFDPF......... GYN GFW R GYS N
Gub_Clotm 1 ...MRNR,~ LMA~~~L~ I ~PFYRAEA~T~TN~PFV~...OP.S .~ SQW R~ S
Gub_Bacli 1 MSYR~R IjM LYTGI~FLSF .~F ~ ~ SASAQT GSFYEPF. . . . . . . . . YN
GLW~R ' G
Lami_Clotm 1 . . .MRNR ~ ~ LMA~L'~L ~ I ~ PFYRAEA~TQVN~PFV~. . .FRS
.~'tTQWKIC.R.~AR. .FVS
Gub_Fibsu 1
....................................................................M~
Lica_Orpin 63 ~ DR~E~. ~G~..~fi ~I P
n.~ ~ ~ ~ ~ - ,r
Gub_Hacpo 56 T RA N T D 8 TS..P NR D T N ~~ $ S T
Gub_Bncau 60 T RA SMTSL EldR TS..P NR D N- T IJ L R T I
Gub_Clotm 62 R-S, VT~S~. ~ I 'm~7CGSYP RS ~~~F Y : R ~A I n
~~'. L
Gub_Bacli 61 T~RA SMTSL E A~~TS..PSYNR D N- ~T _ 8
l~:~ili v -
Lami_Clotm 60 TVLEAFTGDIB~. I ~ YG SYP RS - S~-~ Y R ~A ~ ~ . ~
Gub_Fibeu 3 I~FC~AVRSALA.V~AAAA~TT ~.RD G LY LEEVQ RAR AAAS T L QNG E
Lica_Orpin 128 . ~
o Gub_Bacpo 123 .
dub Bacau 127 .
Gub'_Clotm 130 . ~o
Gub_Bacli 128 .
Lami_Clotm 128 . ~
Gub Fibau 71 IA
N
Lica_Orpin 193 A ...Y ~D ~ RP .N y ~ S D~PRN*...........
Gub_Bacpo 188 LRH ...TT ~S L T ~S G9 N.P Y~E ~..R T N..............
dub_Baceu 192 LRH ...T~Q -T L T ~E G8~ N.P Y~H ~ R TRR..............
Gub_Clotm 195 R~R, ...TR L P ~H GR ~ .pvQ~E R!Y R YPNGVPQDNPTPTPTIA
Gub_Bacli 193 LKH ...TT ~Q L ~E G8 .p' RSLB R TRR..............
Lami_Clotm 193 R~R ....TR ~ - L P ~E GR~.P Q~E~GICRIL~...............
Gub_Fibsu 141 E RR EGGQVB I,~TG Q AF L SSES.AA GQ ESRLP
FQBI~RVYRYTPGQGEGGSDFTLD
Lica_Orpin .................... "d
Gub_Bacpo .................... H
Gub_Baceu .. ..... .... ......
Gub Clotm 260 PSTPTNPNLPLRGDVNGDGH
GubT$acli _ s
~ ~ Y~ ~ Q H X ~ ~_ F R T ~ T
H Q ~ ~ R ~ ~ G~. . 1C IN ~ T8 QPGY R
~ P ~ ~ R ~ 1 N R D ~ _ D QP ' R.. ~ ~ Q
n
~ NP ~ ~ R ~ ~ GN . . . Y ' N ~ ~ ~~ D E RP Y DF ~ R
p ~ ~ R~ ~ N~.. R N ~~ S D QP R ~ Q
~NNP ~ ~ R~ ~ R GN... Y~~N ~ ~D E Rp Y DF ~ R
~ RP ~ R~PGSF 8 II GRA QRTS H SPA D~ LE P Y R ~ Q
,.
Lami_Clotm ....................
Gub_Fibau 209 WTDNFDTFDGSRWGRGDWTF
CA 02267078 1999-03-29
WO 98/1A595 PCT/US97/17811
REFERENCES CITED
Guliga, G.S., and Brant, D.A. (1986) Carbohydr. Res. 157, 139-156.
Fleming, M., and Kawami, K. (1977) Carbohydr. Res. 57, 15-23.
Anderson, M.A., and Stone, B.A. (1975) FEBS Lett. 52) 202-207.
Godfrey, T. (1983) Industrial Enzymology (Godfrey) T. & Reichelt, J.) eds) p.
466, MacMillan,
London.
White et al. (1983) Poult. Sci. 62, 853-862.
Murphy et al. (1984) Nucleic Acids Res. 12, 5355-5367.
Hofemeister et al. (1986) Gene (Amst.) 49, 177-187.
Borriss et al. (1990) Mol. Gen. Genet. 222, 278-283.
Lloberas et al. (1991) Eur. J. Biochem. 197, 337-343.
Louw et al. (1993) Appl. Microbiol. Biotechnol. 38, 507-513.
Gosalbes et al. (l991) J. Bacteriol. 173, 7705-7710.
Schimming et al. (1992) Eur. J. Biochem. 204, 13-19.
Teather, R., and Airflow, 3.D. (1990) J. Bacteriol. 172, 3837-3841.
Flint et al. (1993) J. Bacteriol. I75) 2943-2951.
Becker et al. (1993) Mol. Gen. Genet. 238, 145-154.
Sakellaris et al. (1993) FEMS Microbiol. Lett. 109, 269-272.
Fincher et al. (1986) Proc. Natl. Acad. Sci. USA. 83, 2081-2085.
Zverlov, V . V , and V elikodvorskaya, G. A. ( 1990) Biotechnol. Lett. 12, 811-
816.
Orpin, C.G., and Joblin, K.N. (1988) in The Rumen Microbial Ecosystem, ed.
Hobson, P.N.
(Elsevier, London), pp. 129-151.
Akin et al. (l983) Appl. Environ. Microbiol. 46,738-748.
Barichievich, E.M., and R.E. Calza. (1990) Appl. Environ. Microbiol. 56:43-48.
Barr et al. (1989) Can. J. Bot. 67:2815-2824.
Borneman et al. (1989) Appl. Environ. Microbiol. 55:1066-1073.
Breton et al. (1990) FEMS Microbiol. Lett. 70:177-182.
Chen et al. (1994) Appl. Environ. Microbiol. 60:64-70.
Chen et al. (1995) Proc. Acad. Sci. USA. 92,2587-2591.
Chen et al. (1995) in S.K. Ballal (ed.) SAAS Bulletin, Biochemistry and
Biotechnolog 8:1-6.
Heath et al. (1983) Can. J. Bot. 61:295-307.
Ho, Y.W. and T. Bauchop (1990). Mycotaxon 38:397-A05.
Miller,G.L. (1959) Anal. Chem. 31:426-428.
Orpin, G.C. (1975) J. Gen. Microbiol. 91:249-262.
Wubah, D.A. and M.S. Fuller (1991) Mycologia 83,303-310.
Laemmli, U.K. (1970) Nature (London) 227, 680-683.
McGrew, B.R. and Green, M. (1990) Ailal. Biochem. 189, 68-74.
21
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Beguin, P. (1983) Anal. Biochem. 131, 333-336.
Lake, B.D. and Goodwin, H.J. (1976) Chromatographic and electrophoretic
techniques, vol.l, p.
345-366. In LSmith and J.M.T. Seakins (ed.), Lipids) 4th ed. Pitman Press,
Bath, England.
Hansen, S.A. (1975) J. Chromatogr. 105:388-390.
Andersson, H. and von Heijne, G. (1991) Proc. Natl. Acad. Sci. USA 88:9751-
9754.
Li et al. (1988) Proc. Natl. Acad. Sci. USA 85:7685-7689.
Kohara et al. (1991) J. Biol. Chem. 266, 20363-20368.
Von Heijne, G. (1986) Nucleic Acids Research 14, 1i:4683-4690.
Airflow et al. (1988) Biochem. J. 255, 833-841.
Huber, D.J. and Nevin, D.J. (1977) Plant Physiol. 60, 300-304.
Buliga et al. (1986) Carbohydr. Res. 157,139-156.
Kemp et al. (1984) J. Gen. Microbiol. 130,27-37.
Neu, H.C. and Heppel, L.A. (196S) J. Biol. Chem. 240, 3685-3692.
Cornelis et al. (1982) Mol. Gen. Genet. 186, 507-511.
Juncosa et al. (1994) J. Biol. Chem. 269, 14530-14535.
Henrissat, B. and Bairoch, A. (1993) Biochem. J. 293, 781-788.
Gilbert et al. (1992) Mol. Microbiol. 6, 2065-2072.
Denman et al. (1996) Appl. Environ. Microbiol. 62, 1889-1896.
Black et al. (1994) Biochem. J. 299, 381-387.
Zhou et al. (1994) Biochem. J. 297) 359-364.
22
CA 02267078 1999-03-29
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: University of Georgia Research Foundation, Inc.
Li) Xin-Liang
Ljungdahl, Lars G.
Chen, Huizhong
(ii) TITLE OF INVENTION: Lichenases and Coding Sequences
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Greenlee, Winner and Sullivan, P.C.
(B) STREET: 5370 Manhattan Circle, Suite 201
(C) CITY: Boulder
(D) STATE: Colorado
(E) COUNTRY: US
(F) ZIP: 80303
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
{D) SOFTWARE: PatentIn Release #1.0, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: WO
(B) FILING DATE: 03-OCT-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/027,882
(B) FILING DATE: 04-OCT-l996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Ferber, Donna M.
(B) REGISTRATION NUMBER: 33,878
{C) REFERENCE/D6~KET NUMBER: 55-96 WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (303) 499-B080
(B) TELEFAX: (303) 499-8089
(2) INFORMATION FOR SEQ ID N0:1:
ii) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 971 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: not relevant
(ii) MOLECUIJE TYPE: CDNA to mRNA
(iii) HYPOTHETTCAL: NO
{iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Orpinomyces
(B) STRAIN: PC2
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: l23..860
23
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WO 98/14595 PCT/US97/17811
(ix) FEATURE:
(A) NAME/KEY: mat peptide
(B) LOCATION: 210..857
(D) OTHER INFORMATION: /EC number= 3.2.1.73
/product= "mature lichenase produced by
Orpinomyces PC2"
{xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AATAAAAGAA AAAAAAAATA TATATTAAAT AATAATATAT ATTAAAGTAA ATAAAAAAAA
ATTTAAGAAA ATATTTTCAT TATATAATTA ATATTTTTTG ATAAAATAAA GATTATAATA
120
AA ATG AAA AGT ATA ATT TCT ATT GCT GCT TTA TCT GTT TTA GGA TTG
167
Met Lys Ser Ile Ile Ser Ile Ala Ala Leu Ser Val Leu Gly Leu
-29 -25 -20 -15
ATT TCT AAA ACT ATG GCT GCT CCT GCT CCC GCT CCT GTT CCT GGT ACT
215 .
Ile Ser Lys Thr Met Ala Ala Pro Ala Pro Ala Pro Val Pro Gly Thr
-10 -5 1
GCT TGG AAT GGT AGT CAT GAT GTC ATG GAT TTC AAC TAT CAT GAA AGT
263
Ala Trp Asn Gly Ser His Asp Val Met Asp Phe Asn Tyr His Glu Ser
- 5 10 15
AAC CGT TTT GAA ATG TCA AAC TGG CCA AAT GGT GAA ATG TTT AAT TGT
311
Asn Arg Phe Glu Met Ser Asn Trp Pro Asn Gly Glu Met Phe Asn Cys
20 25 30
AGA TGG ACT CCA AAT AAT GAC AAA TTT GAA AAT GGT AAA TTA AAG CTT
359
Arg Trp Thr Pro Asn Asn Asp Lys Phe Glu Asn Gly Lys Leu Lys Leu
35 40 45 50
ACT ATT GAT AGA GAT GGT TCC GGA TAT ACT TGT GGT GAA TAT CGT ACT
407
Thr Ile Asp Arg Asp Gly Ser Gly Tyr Thr Cys Gly Glu Tyr Arg Thr
55 60 65
AAA AAC TAT TAT GGA TAT GGT ATG TTC CAA GTT AAT ATG AAA CCA ATT
455
Lys Asn Tyr Tyr Gly Tyr Gly Met Phe Gln Val Asn Met Lys Pro Ile
75 80
AAG AAT CCA GGA GTT GTT TCT TCC TTC TTT ACT TAC ACA GGA CCA AGT
503
Lys Asn Pro Gly Val Val Ser Ser Phe Phe Thr Tyr Thr Gly Pro Ser
85 90 95
GAT GGA ACT AAG TGG GAT GAA ATT GAT ATA GAA TTC CTT GGT TAT GAT
551
Asp Gly Thr Lys Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly Tyr Asp
100 105 110
ACA ACC AAA GTT CAA TTT AAC TAC TAC ACT AAT GGA CAA GGT CAT CAT
599
Thr Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Gln Gly His His
115 120 l25 130
24
CA 02267078 1999-03-29
WO 98I14595 - PCTiUS97/17811
GAA CAT ATT CAT TAT CTT GGA TTT GAT GCC TCT CAA GGA TTC CAT ACC
697
Glu His Ile His Tyr Leu Gly Phe Asp Ala Ser Gln Gly Phe His Thr
l35 140 145
TAT GGT TTC TTC TGG GCG AGA AAT TCT ATT ACA TGG TAT GTA GAT GGT
695
Tyr Gly Phe Phe Trp Ala Arg Asn Ser Ile Thr Trp Tyr Val Asp Gly
150 l55 160
ACA GCC GTT TAC ACT GCT TAC GAC AAT ATT CCA GAT ACA CCA GGT AAG
743
-Thr Ala Val Tyr Thr Ala Tyr Asp Asn Ile Pro Asp Thr Pro Gly Lys
l65 l70 ~ 175
ATT ATG ATG AAT GCT TGG AAT GGT ATT GGA GTT GAT GAC TGG CTT AGA
791
Ile Met Met Asn Ala Trp Asn Gly Ile Gly Va1 Asp Asp Trp Leu Arg
180 185 190
CCA TTT AAT GGA AGA ACT AAT ATT AGT GCC TAC TAT GAT TGG GTA TCT
839
Pro Phe Asn Gly Arg Thr Asn Ile Ser Ala Tyr Tyr Asp Trp Val Ser
l95 200 205 210
TAT GAT GCA CCA AGA AAC TAA ATTATTTAAA TAAATATATA ATTTTTGTTT
890
Tyr Asp Ala Pro Arg Asn
215
TAAAATTTAA AAAAATATAT ATATATATAT TATAAATTAA TATGAAAAAT AAAAATAAGA
950
TGTAAAAAAA Ai~AAAAe~AAA A
971
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 246 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Lys Ser Ile Ile Ser Tle A1a Ala Leu Ser Val Leu Gly Leu Ile
-29 -25 -20 -15
Ser Lys Thr Met Ala Ala Pro Ala Pro Ala Pro Val Pro Gly Thr Ala
-10 -5 1
Trp Asn Gly Ser His Asp Val Met Asp Phe Asn Tyr His Glu Ser Asn
10 15
Arg Phe Glu Met Ser Asn Trp Pro Asn Gly Glu Met Phe Asn Cys Arg
20 25 30 35
Trp Thr Pro Asn Asn Asp Lys Phe Glu Asn Gly Lys Leu Lys Leu Thr
40 45 50
CA 02267078 1999-03-29
WO 98114595 PCT/US97/17811
Ile Asp Arg Asp Gly Ser Gly Tyr Thr Cys Gly Glu Tyr Arg Thr Lys
55 60 65
Asn Tyr Tyr Gly Tyr Gly Met Phe Gln Val Asn Met Lys Pro Ile Lys
70 75 80
Asn Pro Gly Val Val Ser Ser Phe Phe Thr Tyr Thr Gly Pro Ser Asp
85 90 95
Gly Thr Lys Trp Asp Glu Ile Asp Ile Glu Phe Leu Gly Tyr Asp Thr
100 105 110 115
Thr Lys Val Gln Phe Asn Tyr Tyr Thr Asn Gly Gln Gly His His Glu
120 l25 I30
His Ile His Tyr Leu Gly Phe Asp Ala Ser Gln Gly Phe His Thr Tyr
135 l40 145
Gly Phe Phe Trp Ala Arg Asn Ser Ile Thr Trp Tyr Val Asp Gly Thr
150 155 l60
Ala Val Tyr Thr Ala Tyr Asp Asn Ile Pro Asp Thr Pro Gly Lys Ile
l65 170 175
Met Met Asn Ala Trp Asn Gly Ile Gly Val Asp Asp Trp Leu Arg Pro
180 1B5 190 195
Phe Asn Gly Arg Thr Asn Ile Ser Ala Tyr Tyr Asp Trp Val Ser Tyr
200 205 210
Asp Ala Pro Arg Asn
2l5
(2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii} MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: N-terminal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
Gly Thr Ala Trp Asn Gly Leu His Asp Val Met Asp
1 5 10
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 102 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: cDNA to mRNA
(iii} HYPOTHETICAL: NO
26
CA 02267078 1999-03-29
WO 98I14595 PCT/US97/17811
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..l02
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
ATG AAG TTC TTC GCC ACC ATT GCT GCT CTC GTT GTG GGA GCT GTT GCT
48
Met Lys Phe Phe Ala Thr Ile Ala Ala Leu Val Val Gly Ala Val Ala
1 5 10 15
GCC CCA GTC GCA GAG GCT GAG GCT GAG GCC AGC AGC CCC ATG CTG ATC
96
Ala Pro Val Ala Glu Ala Glu Ala Glu Ala Ser Ser Pro Met Leu Ile
20 25 30
GAA CGT
l02
G1u Arg
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Met Lys Phe Phe Ala Thr Ile Ala Ala Leu Val Val Gly Ala Val Ala
1 5 10 15
Ala Pro Val Ala Glu Ala Glu Ala Glu Ala Ser Ser Pro Met Leu Ile
20 25 30
Glu Arg
(2) INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ TD N0:6:
Asp Glu Ile Asp Ile
1 5
27
