Note: Descriptions are shown in the official language in which they were submitted.
CA 022~8291 1998-12-1~
WO 98/00525~ 1 PCT/DK97/00283
Title: A recombinant enzyme with mutanase activity
FIELD OF THE lNvk..llON
The present invention relates to a method for constructing an
5 expression vector comprising a mutanase gene obtained from a
filamentous fungus suitable for heterologous production, a re-
combinant expression vector comprising said mutanase gene
sequence and a kex2 cleavage site between the DNA sequence
encoding the pro-peptide and the DN~ sequence encoding the mature
10 mutanase, a filamentous fungus host cell, a process of producing
recombinant mutanase, and said recombinant mutanase.
It is also the object of the invention to provide compositions
useful in oral care products for humans and animals.
15 B~CK~R~UND OF T~E INVENTION
Mutanases are a-1,3-glucanases (also known as a-1,3-
glucanohydrolases) which degrade the a-1,3-glycosidic linkages in
mutan. Mutanases have been described from two species of Tricho-
derma (Hasegawa et al., (1969), Journal of Biological Chemistry
20 244, p. 5460-5470; Guggenheim and Haller, (1972), Journal of
Dental Research 51, p. 394-402) and from a strain of Streptomyces
(Takehara et al., (1981), Journal of Bacteriology 145, p. 729-
735), Cladosporium resinae (Hare et al. (1978), Carbohydrate
Research 66, p. 245-264), Pseudomonas sp. (US patent no.
25 4,438,093), Flavobacterium sp. (JP 77038113), Bacillus circulanse
(JP 63301788) and Aspergillus sp.. A mutanase gene from Tricho-
derma harzianum has been cloned and sequenced (Japanese Patent
No. 4-58889-A from Nissin Shokuhin Kaisha LDT).
Although mutanases have commercial potential for use as an
30 antiplaque agent in dental applications and personal care pro-
ducts, e.g., toothpaste, chewing gum, or other oral and dental
care products, the art has been unable to produce mutanases in
significant quantities to be commercial useful.
US patent no. 4,353,891 (Guggenheim et al.) concerns plac~e
35 removal using mutanase produced by Trichoderma harzianum CBS
~ 243.71 to degrade mutan synthesized by cultivating Streptococcus
CA 022~8291 1998-12-l~
WO 98/00528 PCT/DK97100283
mutans strain CBS 350.71 identifiable as OMZ 176.
It is an object of the present invention to provide a recom-
binant mutanase from Trichoderma harzianum which can be produced
in commercially useful quantities.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows plasmid pMT1796
Figure 2 shows plasmid construction of plasmids pMT1796, pMT1802,
and pMT1815,
10 Figure 3 shows an outline of the construction of the A. oryzae
recombinant mutanase expression vector pMT1802,
Figure 4 shows the pH-profile of recombinant and wild-type T.
harzianum CBS 243.71 mutanase
Figure 5 shows the temperature profile of recombinant and wild-
15 type T. harzianum CBS 243.71 mutanase at pH 7,
Figure 6 shows the temperature stability of recombinant and wild-
type T. harzianum CBS 243.71 mutanase at pH 7,
Figure 7 shows the indirect Malthus standard curve for a mix
culture of S. mutans, A. viscosus and F. nucleatum grown in
20 BHI at 37~C.
SUMM~RY OF THE lNV~iNllON
The object of the invention is to provide a recombinant
mutanase derived from a filamentous fungus by heterologous
25 expression.
The present inventors have as the first been able to express
the mutanase gene of a filamentous fungus heterologously and
thus cleared the way for providing a single component, recom-
binant mutanase essentially free of any contaminants.
30 In the first aspect the invention relates to a method for
constructing an expression vector comprising a mutanase gene
obtained from a filamentous fungus suitable for heterologous
production comprising the steps of:
a) isolating a DNA sequence encoding a mutanase from a
35 filamentous fungus,
b) introducing a kex2 site or kex2-like site between the DNA
sequences encoding the pro-peptide and the mature region of the
CA 022~829l l998- l2- l~
W098/00528 PCT~K97/00283
mutanase, or replacing the mutanase (pre)pro-sequence with a
(pre)pro-sequence comprising a kex2 or kex2-like site of
another fungal enzyme,
c) cloning the DNA sequence obtained in step b) into a suitable
5 expression vector.
In a preferred embodiment the mutanase is obtained from a
strain within the genus Trichoderma.
In step b) the mutanase (pre)pro-sequence may for instance
be replaced with the Lipolase~ (pre)pro-sequence or the TAKA-
lo amylase (pre)pro-sequence.
It is also an object of the invention to provide an ex-
pression vector comprising a mutanase gene and a DNA sequence
encoding a (pre)pro-peptide with a kex2 site or kex2-like site
between the DNA sequences encoding said (pre)pro-peptide and
15 the mature region of the mutanase.
The invention also relates to a filamentous host cell for
production of recombinant mutanase derived from a filamentous
fungus. Preferred host cells include filamentous fungi of the
genera Trichoderma, Aspergillus, and Fusarium.
Further, the invention relates to a process for producing a
recombinant mutanase in a host cell, comprising the steps:
a) transforming an expression vector comprising a mutanase gene
with a kex2 site or kex2-like site between the DNA sequences
encoding the pro-peptide and the mature region of the mutanase
25 into a suitable filamentous fungus host cell,
b) cultivating the host cell in a suitable culture medium under
conditions permitting expression and secretion of an active
mutanase,
c) recovering and optionally purifying the secreted active re-
30 combinant mutanase from the culture medium.
The expression vector may be prepared according to the abovedescribed method of the invention.
A recombinant mutanase may according to the invention be
produced according to the process of the invention.
35 A substantially pure wild-type mutanase obtained from ~ri-
choderma harzianum CBS 243.71 essentially free of any
contaminants is also part of the invention.
CA 022~829l l998-l2-l~
W098/00528 PCT~K97/00283
The invention also relates to a composition comprising a
recombinant mutanase of the invention or a substantially pure
mutanase of the invention useful in oral care products and
-food, feed and/or pet food products.
s Finally the invention relates to the use of the recombinant
mutanase of the invention or the substantially purified mutanase
of the invention or composition or product of the invention
preventing the formation of human or animal dental plaque or
removing dental plaque and for the use in food, feed and/or pet
10 food products.
DET~TT~n DESCRIPTION OF THE lNv~lON
The object of the invention is to provide a recombinant muta-
nase derived from a filamentous fungus by heterologous
15 expression.
The present inventors have as the first been able to express
the mutanase gene of a filamentous fungus heterologously and
thus cleared the way for providing a single component
recombinant mutanase essentially free of any contaminants.
The principle of the invention can be used for all mutanases
derivable from filamentous fungi, such as from filamentous
fungi of the genus Trichoderma, such a strain of Trichoderma
harzianum, especially Trichoderma harzianum CBS 243.71, and the
genera Streptomyces, Cladosporium or Aspergillus.
2s Previously it has not been possible to produce mutanases of
filamentous fungi heterologously. Consequently, according to
prior art mutanases are produced homologously and comprise a
mixture of other enzyme activities besides the mutanase (i.e.
with undesired contaminants).
An example of this is Trichoderma harzianum CBS 243.71 which
are known to produce a mutanase as also described above. The
mutanase derived from Trichoderma harzianum CBS 243.71 has
before the successful findings of the present invention only
been produced homologously.
3s It is advantageous to be able to produce the mutanase hete-
rologously, as it is then possible to provide a single
component mutanase free of undesired contaminants. Further, it
CA 022~829l l998- l2- l~
W098/00528 PCT~K~7/00283
facilitates providing an isolated and purified enzyme of the
invention in industrial scale.
According to the invention it is possible to express
mutanases derived from filamentous fungi in a suitable host
5 cell by introducing a kex2 cleavage site or ~ex2-like site
between the DNA sequences encoding the pro-peptide and the
mature mutanase, or replacing the mutanase (pre)pro-sequence
with a (pre)pro-sequence comprising a kex2 site or kex2-like
site of another fungal enzyme.
lo The (pre)pro-sequence have for instance be the Lipolase~
(pre)pro-sequence or the TAKA-amylase (pre)pro-sequence.
Pro-peptides
A large number of mature proteins are initially synthesised
15 with a N-terminal extension, the pro-peptide, varying from very
small peptides (e.g. GLA 6 amino acids) to relatively long pep-
tides (e.g. PEPA 49 amino acids).
The pro-peptide can perform a number of different functions.
Firstly, pro-peptides might contribute to the efficiency of co-
20 translational translocation of the protein across the ER-mem-
brane. Secondly, pro-peptides might contribute to co-transla-
tional proteolytic processing of the polypeptide. Thirdly, they
might act as intracellular targeting signal for routing to
specific cellular compartments. Fourthly, in some pro-proteins
25 the pro-peptide keeps the protein inactive until it reaches its
site of action.
Removal of the pro-peptide from the mature protein occurs in
general by processing by a specific endopeptidase, usually
after the two positively charged amino acid residues Arg-Arg,
30 Arg-Lys or Lys-Arg. However, also other amino acid
combinations, containing at least one basic amino acid, have
been found to be processed.
The absence of these doublets in mature, endogenous secreted
proteins might protect them from proteolytic cleavage. As di-
35 basic cleavage is thought to occur in the Golgi, the internaldi-basic peptide sequences in cytoplasmic proteins will not be
attacked by this processing.
CA 022~829l l998-l2-l~
W098/00528 PCT~K97/00283
Rex2 sites
Kex2 sites (see e.g. Methods in Enzymology Vol 185, ed. D.
Goeddel, Academic Press Inc. (1990), San Diego, CA, "Gene
5 Expression Technology") and kex2-like sites are di-basic recog-
nition sites (i.e. cleavage sites) found between the pro-
peptide encoding region and the mature region of some proteins.
Insertion of a kex2 site or a kex2-like site have in certain
cases been shown to improve correct endopeptidase processing at
o the pro-peptide cleavage site resulting in increased protein
secretion levels.
However, in a number of other cases insertion of a Kex2
cleavage site did not increase the secretion level. For
instance, Cullen et al., (1987), Bio/Technology, vol. 5, p.
15 369-376, found that insertion of a kex2 site in the secretion
signal of chymosin (i . e . signal peptide and pro-peptide), which
encoded the glucoamylase signal peptide and pro-peptide fused
to prochymosin, did not increase the secretion level of
recombinant chymosin expressed in a Aspergillus nidulans host
20 cell.
Other examples of references showing that insertion of a
kex2 site or a kex2-like site do not always increase the
secretion level include Valverde et al., (1995), J. of Biolog.
Chem, p. 15821-15826)
2 5 In the context of the present invention the term
"heterologous" production means expression of a recombinant
enzyme in an host organism different from the original donor
organism or expression of a recombinant enzyme by the donor
organism.
The term "homologous" production means expression of the wild-
type enzyme by the original organism.
In the first aspect the invention relates to a method for
construction of an expression vector comprising a mutanase gene
suitable for heterologous production comprising the steps of:
35 a) isolating a DNA sequence encoding a mutanase from a
filamentous fungus known to produce a mutanase,
CA 022~8291 1998-12-1~
W098/OOS28 PCT~K97/00283
b) introducing a kex2 site or kex2-like site between the DNA
sequences encoding the pro-peptide and the mature region of the
mutanase, or replacing the mutanase (pre)pro-sequence with a
(pre)pro-sequence comprising a kex2 or kex2-like site of
5 another fungal enzyme,
c) cloning the mutanase gene with the kex2 site or kex2-like
site obtained in step b) into a suitable expression vector.
In a preferred embodiment of the mutanase gene is obtained
from the genus Trichoderma, preferably a strain of the species
lO T. harzianum, especially the strain T. harzianum CBS 243.71.
The complete mutanase gene DNA sequence derived from
Trichoderma harzianum CBS 243.71 is shown in SEQ ID No. l
In step b) the mutanase (pre)pro-sequence may for instance
be replaced with the Lipolase~ (pre)pro-sequence or the TAKA-
15 amylase (pre)pro-sequence.
In the examples below illustrating the present invention a
kex2-site is inserted into the Tric~oderma harzianum mutanase
gene presented in SEQ ID No. l as the site specific mutation
E36 ~ K36.
Isolation of the mutanase gene
The DNA sequence encoding a mutanase may, in accordance with
well-known procedures, conveniently be isolated from DNA from a
suitable source, such as any of the above mentioned organisms
2s known to comprise a mutanase gene, by use of synthetic oligo-
nucleotide probes prepared on the basis of the DNA sequence
disclosed herein.
For instance, a suitable oligonucleotide probe may be pre-
pared on the basis of the nucleotide sequences shown in SEQ ID
30 no. l or the amino acid sequence shown in SEQ ID no. 2 or any
suitable sub-sequence thereof.
According to this method primers are designed from the
knowledge to at least a part of SEQ ID No. 2. Fragments of
mutanase gene are then PCR amplified by the use of these
35 primers. These fragments are used as probes for cloning the
complete gene.
CA 022~8291 1998-12-1~
WO 98/OQ5'~ PCTIDK97/00283
Alternatively, the DNA sequence encoding a mutanase may be
isolated by a general method involving
- cloning, in suitable vectors, a DNA or cDNA library from a
strain of genus Trichoderma,
5 - transforming suitable host cells with said vectors,
- culturing the host cells under suitable conditions to
express any enzyme of interest encoded by a clone in the DNA
library,
- screening for positive clones by determining any mutanase
o activity of the enzyme produced by such clones, and
- isolating the DNA coding an enzyme from such clones.
The general method is further disclosed in WO 93/11249 the
contents of which are hereby incorporated by reference.
15 Expression vector
In another aspect the invention relates to an expression
vector comprising a mutanase gene and a DNA sequence encoding a
pro-peptide with a kex2 site or kex2-like site inserted between
the DNA sequences encoding said pro-peptide and the mature
2 0 region of the mutanase.
In preferred embodiments of the invention the expression
vector comprises besides the kex2 site or kex2-like site an
operably linked DNA sequence encoding a prepro-peptide (i.e.
signal peptide and a pro-peptide). The prepro-sequence may
25 advantageously be the original mutanase signal-sequence or the
Lipolase~ signal-sequence or the TAKA signal-sequence and the
original mutanase pro-sequence or the Lipolase~ pro-sequence or
the TAKA pro-sequence.
The promoter may be the TAKA promoter or the TAKA:TPI
30 promoter.
In a specific embodiment of the invention the expression
vector is the pMT1796 used to illustrate the concept of the
invention in Example 3 below.
The choice of vector will often depend on the host cell into
35 which it is to be introduced.
Thus, the vector may be an autonomously replicating vector,
i.e. a vector which exists as an extrachromosomal entity, the
CA 022~8291 1998-12-l~
W098/00528 PCT~K97/00283
replication of which is independent of chromosomal replication,
e.g. a plasmid. Alternatively, the vector may be one which,
when introduced into a host cell, is integrated into the host
cell genome and replicated together with the chromosome(s) into
5 which it has been integrated.
In the vector, the DNA sequence encoding the mutanase should
also be operably connected to a suitable promoter and
terminator sequence. The promoter may be any DNA sequence which
shows transcriptional activity in the host cell of choice and
o may be derived from genes encoding proteins either homologous
or heterologous to the host cell.
The procedures used to ligate the DNA sequences coding for
the mutanase, a prepro-sequence including the kex2 site or
kex2-like site, the promoter and the terminator, respectively,
15 and to insert them into suitable vectors are well known to
persons skilled in the art (cf., for instance, Sambrook et al.,
(1989), Molecular Cloning. A Laboratory Manual, Cold Spring
Harbor, NY).
20 Host Cell
A third aspect of the invention relates to a filamentous
fungi host cell for production of recombinant mutanase derived
from a filamentous fungus of the genus Trichoderma, such as a
strain of T. harzianum, especially T. harzianum CBS 243.71, or
25 the genus Aspergillus, such as a strain of A. oryzae or A.
niger, or a strain of the genus Fusarium, such as a strain of
Fusarium oxysporium, ~usarium graminearum (in the perfect state
named Gribberella zeae, previously Sphaeria zeae, synonym with
Gibberella roseum and Gibberella roseum f. sp. cerealis)~ or
30 Fusarium sulphureum (in the prefect state named Gibberella
- puricaris, synonym with Fusarium trichothecioides, Fusarium
bactridioides, Fusarium sambucium, Fusarium roseum, and
Fusarium roseum var. graminearum), Fusarium cerealis (synonym
with Fusarium crokkwellnseJ or Fusarium venenatum.
35 The host cell may advantageously be a F. graminearum described
in WO 96/00787 (from Novo Nordisk A/S), e.g. the strain
deposited as Fusarium graminearum ATCC 20334. The strain ATCC
CA 022~829l l998-l2-l~
W098/00528 PCT~K97/00283
20334 was previously wrongly classified as Fusarium graminearum
(Yoder, W. and Christianson, L. 1997). RAPD-based and classical
taxonomic analyses have now revealed that the true identity of
the Quorn fungus, ATCC 20334, is Fusarium venenatum Nirenburg
5 Sp. nov.
In a preferred embodiment of the invention the host cell is
a protease deficient or protease minus strain.
This may for instance be the protease deficient strain
Aspergillus oryzae JaL125 having the alkaline protease gene
o named "alp" deleted. This strain is described in PCT/DK97/00135
(from Novo Nordisk A/S).
Filamentous fungi cells may be transformed by a process
involving protoplast formation and transformation of the
protoplasts followed by regeneration of the cell wall in a
15 manner known per se. The use of Aspergillus as a host
microorganism is described in EP 238 023 (Novo Nordisk A/S),
the contents of which are hereby incorporated by reference.
According to a further aspect the invention relates to a
process for producing a recombinant mutanase in a host cell. Said
20 process comprises the following steps:
a) transforming an expression vector encoding a mutanase gene
with a kex2 site or a kex2-like site between the DNA sequences
encoding the pro-peptide and the mature region of the mutanase
into a suitable filamentous fungus host cell,
25 b) cultivating the host cell in a suitable culture medium under
conditions permitting the expression of the expression vector,
c) recovering the secreted recombinant mutanase from the
culture medium,
d) and optionally purifying the recombinant mutanase.
The recombinant expression vector may advantageously be any
of the above described.
Further, the filamentous fungi host cells to be used for
production of the recombinant mutanase of the invention
according to the process of the invention may be any of the
35 above mentioned host cell, especially of the genera
Aspergillus, Fusarium or Trichoderma.
CA 022~829l l998-l2-l~
W098/00528 PCT~K~7/00283
11
The medium used to culture the transformed host cells may be
any conventional medium suitable for growing the host cells in
question. The expressed mutanase is secreted into the culture
medium and may be recovered from there by well-known procedures
5 including separating the cells from the medium by
centrifugation or filtration, precipitating proteinaceous com-
ponents of the medium by means of a salt such as ammonium
sulphate, followed by chromatographic procedures such as ion
exchange chromatography, affinity chromatography, or the like.
It is also an important object of the invention to provide a
recombinant mutanase produced according to the process of the
invention.
The isolated recombinant mutanase has essentially an amino
acid sequence as shown in SEQ ID no. 2. From SDS-PAGE a mole-
15 cular weight around 80 kDa was found.
The pH optimum of the recombinant mutanase was found to liein the range from 3.5 to 5.5 which equals the pH optimum of the
wild-type mutanase (see Figure 4). The temperature optimum of
both the recombinant and wild-type mutanase was found to be
20 around 45~C at pH 7 and around 55~C at pH 5.5 (see Figure 5).
Further, the residual activity starts to decline at 40~C at pH
7, while the enzyme is more stable at pH 5.5, where the
residual activity starts to decline at 55~C.
The inventors have also provided a substantially pure wild-
25 type mutanase obtained from Trichoderma harzianum CBS 243.71essentially free of any active contaminants, such as other
enzyme activities.
Composition
It is also an object of the invention to provide a
composition comprising the recombinant mutanase of the
invention or the purified wild-type mutanase essentially free
- of any active contaminants of the invention.
Oral care comPosition
....
CA 022~8291 1998-12-l~
W098/00528 PCT~K~7/00283
12
In a still further aspect, the present invention relates to
an oral care composition useful as an ingredient in oral care
products.
An oral care composition of the invention may suitably
5 comprise an amount of the recombinant Trichoderma harzianum
mutanase equivalent to an enzyme activity, calculated as enzyme
activity units in the final oral care product, in the range from
0.001 MU to 1000 MU/ml, preferably from 0.01 MU/ml to 500 MU/ml,
such as from 0.1 MU/ml to 100 MU/ml, especially 0.05 MU/ml to 100
10 MU/ml.
It is also contemplated according to the invention to
include other enzyme activities than mutanase activity in the
oral care composition. Contemplated enzyme activities include
activities from the group of enzymes comprising dextranases,
15 oxidases, such as glucose oxidase, L-amino acid oxidase,
peroxidases, such as e.g. the Coprinus sp . peroxidases described
in WO 95/10602 (from Novo Nordisk AtS) or lactoperoxidaseor,
haloperoxidases, laccases, proteases, such as papain, acidic
protease (e.g. the acidic proteases described in WO 95/02044
20 (Novo Nordisk A/S)), endoglucosidases, lipases, amylases,
including amyloglucosidases, such as AMG (from Novo Nordisk A/S),
and mixtures thereof.
Oral care Products
25 The oral care product may have any suitable physical form
(i.e. powder, paste, gel, liquid, ointment, tablet etc.). An
"oral care product" can be defined as a product which can be used
for maintaining or improving the oral hygiene in the mouth of
humans and animals, by preventing dental caries, preventing the
30 formation of dental plaque and tartar, removing dental plaque and
tartar, preventing and/or treating dental diseases etc.
At least in the context of the present invention oral care
products do also encompass products for cleaning dentures,
artificial teeth and the like.
35 Examples of such oral care products include toothpaste, dental
cream, gel or tooth powder, odontic, mouth washes, pre- or post
brushing rinse formulations, chewing gum, lozenges, and candy.
CA 022~829l l998- l2- l~
W098/~528 PCT~K~7/00283
13
Toothpastes and tooth gels typically include abrasive
polishing materials, foaming agents, flavouring agents,
humectants, binders, thickeners, sweetening agents,
whitening/bleaching/ stain removing agents, water, and optionally
5 enzymes.
Mouth washes, including plaque removing liquids, typically
comprise a water/alcohol solution, flavour, humectant, sweetener,
foaming agent, colorant, and optionally enzymes.
lo Abrasives
Abrasive polishing material might also be incorporated into
the dentifrice product of the invention. According to the inven-
tion said abrasive polishing material includes alumina and
hydrates thereof, such as alpha alumina trihydrate, magnesium
15 trisilicate, magnesium carbonate, kaolin, aluminosilicates, such
as calcined aluminum silicate and aluminum silicate, calcium
carbonate, zirconium silicate, and also powdered plastics, such
as polyvinyl chloride, polyamides, polymethyl methacrylate,
polystyrene, phenol-formaldehyde resins, melamine-formaldehyde
20 resins, urea-formaldehyde resins, epoxy resins, powdered
polyethylene, silica xerogels, hydrogels and aerogels and the
like. Also suitable as abrasive agents are calcium
pyrophosphate, water-insoluble alkali metaphosphates, dicalcium
phosphate and/or its dihydrate, dicalcium orthophosphate,
25 tricalcium phosphate, particulate hydroxyapatite and the like. It
is also possible to employ mixtures of these substances.
Dependent on the oral care product the abrasive product may be
present in from 0 to 70% by weight, preferably from 1% to 70%.
For toothpastes the abrasive material content typically lies in
30 the range of from 10% to 70% by weight of the final toothpaste
product.
Humectants are employed to prevent loss of water from e.g.
toothpastes. Suitable humectants for use in oral care products
according to the invention include the following compounds and
35 mixtures thereof: glycerol, polyol, sorbitol, polyethylene
glycols (PEG), propylene glycol, 1,3-propanediol, 1,4-butanediol,
hydrogenated partially hydrolysed polysaccharides and the like.
CA 022~8291 1998-12-l~
W098/00528 PCT~K97/00283
14
Humectants are in general present in from 0% to 80%, preferably 5
to 70% by weight in toothpaste.
Silica, starch, tragacanth gum, xanthan gum, extracts of Irish
moss, alginates, pectin, cellulose derivatives, such as
5 hydroxyethyl cellulose, sodium carboxymethyl cellulose and
hydroxypropyl cellulose, polyacrylic acid and its salts,
polyvinylpyrrolidone, can be mentioned as examples of suitable
thickeners and binders, which helps stabilizing the dentifrice
product. Thickeners may be present in toothpaste creams and gels
o in an amount of from 0.1 to 20% by weight, and binders to the
extent of from 0.01 to 10% by weight of the final product.
Foaminq aqents
As foaming agent soap, an-ionic, cat-ionic, non-ionic, ampho-
15 teric and/or zwitterionic surfactants can be used. These may bepresent at levels of from o% to 15%, preferably from 0.1 to 13%,
more preferably from 0.25 to 10% by weight of the final product.
Surfactants
Surfactants are only suitable to the extent that they do not
exert an inactivation effect on the present enzymes. Surfactants
include fatty alcohol sulphates, salts of sulphonated mono-
glycerides or fatty acids having 10 to 20 carbon atoms, fatty
acid-albumen condensation products, salts of fatty acids amides
25 and taurines and/or salts of fatty acid esters of isethionic
acid.
Sweeteninq aqents
Suitable sweeteners include saccharin.
Flavourinq aqents
Flavours, such as spearmint, are usually present in low
amounts, such as from 0.01% to about 5% by weight, especially
from 0.1% to 5%.
CA 022~829l l998-l2-l~
W098/00528 PCT~K~7/00283
Whiteninq/bleaching aqents
Whitening/bleaching agents include H2~2 and may be added in
amounts less that 5%, preferably from 0.25 to 4%, calculated on
the basis of the weight of the final product.
The whitening/bleaching agents may be an enzyme, such as an
oxidoreductase. Examples of suitable teeth bleaching enzymes are
described in WO 97/06775 (from Novo Nordisk A/S).
Water
10 Water is usually added in an amount giving e.g. toothpaste a
flowable form.
Additional aqents
Further water-soluble anti-bacterial agents, such as
15 chlorhexidine digluconate, hexetidine, alexidine, quaternary
ammonium anti-bacterial compounds and water-soluble sources of
certain metal ions such as zinc, copper, silver and stannous
(e.g. zinc, copper and stannous chloride, and silver nitrate) may
also be included.
20 Also contemplated according to the invention is the addition
of compounds which can be used as fluoride source,
dyes/colorants, preservatives, vitamins, pH-adjusting agents,
anti-caries agents, desensitizing agents etc.
2 5 EnzYmes
Other essential components used in oral care products and in
oral care products of the invention are enzymes. Enzymes are
biological catalysts of chemical reactions in living systems.
Enzymes combine with the substrates on which they act forming an
30 intermediate enzyme-substrate complex. This complex is then
converted to a reaction product and a liberated enzyme which
continue its specific enzymatic function.
Enzymes provide several benefits when used for cleansing of
the oral cavity. Proteases break down salivary proteins, which
35 are adsorbed onto the tooth surface and form the pellicle, the
first layer of resulting plaque. Proteases along with lipases
destroy bacteria by lysing proteins and lipids which form the
CA 022~8291 1998-12-l~
W098/00528 PCT~K97100283
16
structural components of bacterial cell walls and membranes.
Dextranase breaks down the organic skeletal structure produced by
bacteria that forms a matrix for bacterial adhesion. Proteases
and amylases, not only prevents pla~ue formation, but also
5 prevents the development of calculus by breaking-up the
carbohydrate-protein complex that binds calcium, preventing
mineralization.
Toothpaste
A toothpaste produced from an oral care composition of the
invention (in weight % of the final toothpaste composition) may
typically comprise the following ingredients:
Abrasive material lO to 70%
Humectant 0 to 80%
15 Thickener O.l to 20%
Binder O.Ol to 10%
Sweetener 0.1% to 5%
Foaming agent 0 to l5%
Whitener 0 to 5%
20 Enzymes 0.0001% to 20%
In a specific embodiment of the invention the oral care
product is toothpaste having a pH in the range from 6.0 to about
8.0 comprising
a) 10% to 70% Abrasive material
25 b) 0 to 80% Humectant
c) O.l to 20% Thickener
d) O.Ol to 10% Binder
e) 0.1% to 5% Sweetener
f) 0 to 15% Foaming agent
30 g) 0 to 5% Whitener
i) 0.0001% to 20% Enzymes.
Said enzymes referred to under i) include the recombinant
mutanase of the invention, and optionally other types of enzymes
mentioned above known to be used in toothpastes and the like.
CA 022~829l l998-l2-l~
W098/00528 PCT~K97/00283
17
Mouth wash
A mouth wash produced from an oral care composition of the
invention (in weight % of the final mouth wash composition) may
5 typically comprise the following ingredients:
0-20% Humectant
0-2% Surfactant
o-5% Enzymes
0-20% Ethanol
0 0-2% Other ingredients (e.g. flavour, sweetener
active ingredients such as fluorides).
0-70% Water
The mouth wash composition may be buffered with an appropriate
buffer e.g. sodium citrate or phosphate in the pH-range 6-7.5.
15 The mouth wash may be in none-diluted form (i. e. must be
diluted before use).
Method of Manufacture
The oral care composition and products of the present
20 invention can be made using methods which are common in the oral
product area.
According to the present invention the recombinant mutanase
and/or the substantially purified mutanase free of active
contaminants can be use in food, feed and/or pet food products.
MAT~T~TR AND N~,n~D~
Materials
Micro-orqanisms
30 Trichoderma harzianum CBS 243.71
A. oryzae JaL 125: Aspergillus oryzae IFO 4177 available from
Institute for Fermentation, Osaka; 17-25 Juso Hammachi 2-Chome
Yodogawa-ku, Osaka, Japan, having the alkaline protease gene
named "alp" (described by Murakami K et al., (1991), Agric. Biol.
35 Chem. 55, p. 2807-2811) deleted by a one step gene replacement
method (described by G. May in "Applied Molecular Genetics of
Filamentous Fungi" (1992), p. 1-25. Eds. J. R. Kinghorn and G.
CA 022~8291 1998-12-1~
W098/00528 PCT~K97/00283
18
Turner; Blackie Academic and Professional), using the A. oryzae
pyrG gene as marker.
E. col i DH5a
5 Plasmids and Vectors:
pMT1796 (Figure 1 and Figure 2)
pMT1802 (Figure 2)
pMT1815 (Figure 2)
pHD414: Aspergillus expression vector is a derivative of the
o plasmid p775 (described in EP 238.023). The construction of the
pHD414 is further described in W0 93/11249. pHD414 contains the
A. niger glucoamylase terminator and the A. oryzae TAKA amylase
promoter.
pHD414+mut (Figure 3)
15 pHan37 containing the TAKA:TPI promoter
Linkers:
Linker #1:
GATCCTCACA ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GG
GAGTGT TAC AAC CCG CAA CAG GCT GCA GAT CCG GAT CCG C
Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly
Linker #2:
C CAA TAC TGT TAG T
GT ACG GTT ATG ACA ATC AGATC
Ala Cys Gln Tyr Cys ***
Primers:
Primer 1: 5' GGGGGGATCCACCATGAG 3' (SEQ ID No. 3)
30 Primer 2: 5' ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC 3' (SEQ ID
No. 4)
Primer 3: 5' GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT 3' (SEQ ID
No. 5)
Primer 4: 5' CCACGGTCACCAACAATAC 3' (SEQ ID No. 6)
35 Primer 5: GGGGGGATCCACCATGAG (SEQ ID No. 7),
Primer 6: ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC (SEQ ID No. 8)
Primer 7: GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT (SEQ ID N0. 9),
Primer 8: CCACGGTCACCAACAATAC (SEQ ID No. 10).
CA 022~8291 1998-12-1~
W098/~S28 PCT~K97/~283
19
~ Enzymes:
lysyl-specific protease from Achromobacter
Trichoderma harzianum CBS 243.71 fermentation broth (Batch no.
5 PPM 3897)
Media, Substrates and Solutions:
YPM: 2~ maltose, 1% bactopeptone and 0.5% yeast extract)
DAPI: 4',6-diamidino-2-phenylindole (Sigma D-9542)
lo Britton-Robinson Buffer
BHI: Brain Heart Infusion broth
Equipment:
10 kDa cut-off ultra-filtration cassette (Alpha Minisette from
15 Filtron).
Phenyl-sepharose FF (high sub) column (Pharmacia)
Seitz EK1 filter plate
Q-sepharose FF column (Pharmacia)
Applied Biosystems 473A protein sequencer
20 2 litre Kieler fermenter
Olympus model BX50 microscope
Malthus Flexi M2060 (Malthus Instrument Limited)
Methods:
25 Molecular biology procedures
All molecular biology procedures including restriction
digests, DNA ligations, E. coli transformations, DNA
isolations, Southern hybridizations, PCR amplifications, and
library constructions and screenings were completed using stan-
30 dard techniques (Sambrook, J., Fritsch, E. F., and Maniatis, T.
1989. Molecular cloning: A laboratory manual /E.F. Cold Spring
Harbor Laboratory Press, Plainview, NY).
Preparation of Mutan
Mutan is prepared by growing streptococcus mutans CBS 350.71
at pH 6.5, 37~C (kept constant), and with an aeration rate of 75
rpm in a medium comprised of the following components:
CA 022~829l l998-l2-l~
W098/00528 PCT~K~7/00283
NZ-Case 6.5 g/litre
Yeast Extract 6 g/litre
(NH4)2SO4 20 g/litre
K2PO4 3 g/litre
Glucose 50 g/litre
Pluronic PE6100 0.1%
After 35 hours, sucrose is added to a final concentration of
60 g/litre to induce glucosyltransferase. The total fermentation
time is 75 hours. The supernatant from the fermentation is
10 centrifuged and filtered (sterile). Sucrose is then added to the
supernatant to a final concentration of 5% (pH is adjusted to pH
7.0 with acetic acid) and the solution is stirred overnight at
37~C. The solution is filtered and the insoluble mutan is
harvested on propex and washed extensively with deionized water
15 containing 1% sodium benzoate, pH 5 (adjusted with acetic acid).
Finally, the insoluble mutan is lyophilized and ground.
Determination of mutanase activity (MU)
One Mutanase _nit (MU) is the amount of enzyme which under
20 standard conditions liberates 1 ~mol reducing sugar (calculated
as glucose) per minute. Reducing sugars were measured with
alkaline K3Fe(CN)6.
Standard Conditions
Substrate............ 1.5% mutan
25 Reaction time........ 15 minutes
Temperature.......... 40~C
pH................... .5.5
A detailed description of Novo Nordisk's analytical method (AF
180/1-GB) is available from Novo Nordisk A/S on request.
Mutanase Plate Assay
A 5% mutan suspension is made in 50 mM sodium acetate, pH 5.5
and the suspension is homogenised for 15 minutes in an Ultra
Turrax T25 homogenizer at 4~C. 1% agarose in 50 mM sodium
35 acetate, pH 5.5 is made 0.2% with respect to mutan and 12.5 ml
- agarose is casted in each petri dish (d=lo cm). The sample to be
CA 022~829l l998- l2- l~
W098/00528 PCT~K97/00283
21
analyzed for mutanase activity is applied in sample wells punched
in the agarose, and the plate is incubated overnight at 37~C,
whereafter clearing zones are formed around mutanase containing
samples.
Western hybridization
Western hybridizations are performed using the ECL western
blotting system (Amersham International, plc, Buckinghamshire,
England) and a primary antibody solution containing polyclonal
o rabbit-anti-mutanase. The limit of detection is O.OO1 MU/ml.
Mass spectrometry
Mass spectrometry of purified wild-type mutanase is done using
matrix assisted laser desorption ionization time-of-flight mass
15 spectrometry in a VG Analytical TofSpec. For mass spectrometry 2
ml of sample is mixed with 2 ml saturated matrix solution (a-
cyano-4-hydroxycinnamic acid in 0.1% TFA:acetonitrile (70:30))
and 2 ml of the mixture spotted onto the target plate. Before
introduction into the mass spectrometer the solvent is removed by
20 evaporation. Samples are desorbed and ionized by 4 ns laser
pulses (337 nm) at threshold laser power and accelerated into the
field-free flight tube by an accelerating voltage of 25 kV. Ions
are detected by a microchannel plate set at 1850 V.
2 5 Preparation of Hydroxyapatite disks IHA)
Hydroxyapatite tablets are prepared by compressing 250 mg of
hydroxyapatite in a tablet die at about 5,900 kg (13,000 lbs) of
pressure for 5 minutes. The tablets are then sintered at 600~C
for 4 hours and finally hydrated with sterile deionized water.
Plaque coating of Hydroxyapatite disks (HA)
Hydroxyapatite disks (HA) were dry sterilised (121~C, 2 bar,
20 minutes) and coated with filter sterilised saliva for 18 hours
at 37~C. The HA disks were placed in a sterile rack in a beaker,
35 Brain Heart Infusion broth (BHI) containing 0.2% sucrose was
poured into the beaker covering the disks. Sterile Na2S (pH 7.0)
was added immediately before inoculation given the final concen-
~, .
CA 022~829l l998-l2-l~
W098/00528 PCT~Kg7/00283
22
tration of 5 g/litre. A mixture 1:1:1 of Streptococcus mutans,
Actinomyces viscosus and Fusobacterium nucleatum grown anaero-
bically (BHI, 37~C, 24 h) was used as inoculum in the concen-
tration of approximately 106 cfu/ml. The disks were incubated
5 anaerobic at 37~C for 4 days with slight stirring.
Malthus-method for plaque
The Malthus-method is based on the methods described in
Johnston et al., (1995), Journal of Microbiological Methods 21,
o p. 15-26 and Johansem et al. (1995), Journal of Applied
Bacteriology 78, p. 297-303.
EXAMPLES
15 Example 1
Purification of wild-type Mutanase
lOO g fermentation broth of Trlchoderma harzianum CBS 243.71
(Batch no. PPM 3897) were dissolved in 1 litre 10 mM sodium
acetate, pH 5.2 overnight at 4~C.
65 g DEAE-Sephadex A-50 were swelled in 3 litre lO mM sodium
acetate, pH 5.2. Excess buffer was removed after swelling. DEAE-
Sephadex was mixed with the crude mutanase preparation for 1 hour
and unbound material was collected by filtration through Propex
cloth. The gel was further washed with 2.5 l of 10 mM sodium ace-
25 tate, pH 5.2. A pool containing the unbound material was made;
volume 4 litre. Remaining DEAE-Sephadex particles were removed by
filtration through a Whatman GF/F filter.
350 ml S-Sepharose was equilibrated in 10 mM sodium acetate,
pH 5.2 and mixed with 600 ml of the pool from the DEAE-Sephadex
30 for 10 minutes. Unbound material was collected by filtration
through Propex cloth and the gel was washed with 500 ml 10 mM
sodium acetate buffer, pH 5.2. Bound material was eluted with the
same buffer containing 1 M NaCl. The procedure was repeated 7
times. The combined pool containing the unbound material (7
35 litre) was concentrated on a Filtron concentrator equipped with a
10 kDa cut-off membrane and followed by a buffer change to 10 mM
sodium acetate, pH 4.7. The concentrate was filtrated through a
CA 022~829l l998- l2- l~
W098/00528 PCT~K97/00283
23
Whatmann GF/F filter. The final volume of the concentrate was 600
ml.
An S-Sepharose column (180 ml, 2.6 x 33 cm) was equilibrated
with 10 mM sodium acetate, pH 4.7. The pH adjusted concentrate
5 from the S-Sepharose batch ion exchange was applied onto the
column in 50 ml portions with a flow of 10 ml/min. The mutanase
was eluted with a linear gradient from 0 to 20 mM NaCl in 3
column volumes. The residual protein was eluted with the same
buffer containing 1 M NaCl. Fractions were analyzed for mutanase
o activity (plate assay) and fractions with high activity were
pooled. The procedure was repeated 12 times. The combined
mutanase pool was concentrated in a Filtron concentrator equipped
with a lo kDa cut-off membrane and followed by a buffer change to
10 mM Tris-HCl, pH 8Ø The final volume of the concentrate was
15 870 ml.
The concentrated pool from the S-Sepharose column was further
purified on a HiLoad Q-Sepharose column (50 ml, 2.6 x 10 cm)
equilibrated with 10 mM Tris-HCl, pH 8Ø Portions of 130 ml was
applied with a flow of 8 ml/min. Elution of the mutanase was per-
20 formed with a linear gradient from 0 to 50 mM NaCl in 12 columnvolumes. Fractions with high mutanase activity (plate assay) were
pooled, concentrated in an Amicon cell equipped with a 10 kDa
cut-off membrane. Finally, the mutanase preparation was dialyzed
extensively against 10 mM sodium phosphate, pH 7.0 and filtrated
25 through a 0.45 mm filter.
The yield of the mutanase in the purification described above
was 300 mg. The purity of the HiLoad-Q preparation was analyzed
by SDS-PAGE and N-terminal sequencing and judged by both methods
the purity was around 95%.
Example 2
N-terminal sequencing of wild-type Mutanase
N-terminal amino acid sequencing was carried out in an Applied
Biosystems 473A protein sequencer.
35 To generate peptides reduced and S-carboxymethylated mutanase
(" 450 mg) was digested with the lysyl-specific protease from
Achromobacter (lO mg) in 20 mM NH4HC03 for 16 hours at 37~C. The
. ~ .. _ .... . .
CA 022~829l lsss-l2-l~
W098/00528 PCT~K~7/00283
24
resulting peptides were separated by reversed phase HPLC using a
Vydac C18 column eluted with a linear gradient of 80% 2-propanol
containing 0.08% TFA in 0.1% aqueous TFA. Peptides were
repurified by reversed-phase-HPLC using a Vydac C1g column eluted
5 with linear gradients of 80% acetonitrile containing 0.08% TFA in
0.1% aqueous TFA before being subjected to N-terminal amino acid
sequencing.
The amino acid sequences determined are given below.
N-terminal:
lO Ala-Ser-Ser-Ala-Asp-Arg-Leu-Val-Phe-Cys-His-Phe-Met-Ile-Gly-Ile-
Val-Gly-Asp-Arg-Gly-Ser-Ser-Ala-Asp-Tyr-Asp-Asp-Asp-
Peptide 1:
Val Phe-Ile-Ser-Phe-Asp-Phe-Asn-Trp-Trp-Ser-Pro-~ly-Asn-Ala-Val-
Gly-Val-Gly-Gln-Lys
15 Peptide 2:
Pro-Tyr-Leu-Ala-Pro-Val-Ser-Pro-Trp-Phe-Phe-Thr-His-Phe-Gly-Pro-
Glu-Val-Ser-Tyr-Ser-
Peptide 3:
Trp-Val-Asn-Asp-Met-Pro-His-Asp-Gly-Phe-Leu-Asp-Leu-Ser-Lys
Example 3
Construction of the mutanase expression vectors, pMT1796,
pMT1802 and pMT1815
A cDNA clone encoding mutanase was identified in a
25 Trichoderma harzianum CBS 243.71 library by hybridization with
a fragment of the gene amplified by PCR using primers based on
the mutanase sequence shown in SEQ ID NO. 1.
DNA sequence analysis of the isolated clone, pHD414+mut,
showed that it indeed encoded the mutanase gene, and that the
30 5 ' end of the construct contained a long leader sequence. To
remove this leader, pHD414+mut was restricted with the enzymes
EcoRI, NarI and XhoI. From this digestion a 3499 nt
(nucleotide) vector fragment and a 610 nt NarI/XhoI fragment
were isolated. These two fragments were then ligated with
35 linker #1 (see above) and a 618 nt EcoRI/BamHI fragment from
pHan37 containing the TAKA:TPI promoter, giving plasmid pJW99.
HD414+mut was next digested with XhoI and SphI, and a 1790 nt
CA 022~829l lsss-l2-l~
W098/00528 PCT~K97/00283
fragment encoding amino acids 35-598 of the mutanase gene was
isolated.
This fragment was ligated with linker #2 (see above) and
pJW99 that had been linearized with the restriction enzymes
5 XbaI and XhoI. The resulting plasmid, pMT1802, contains the ~.
harzianum mutanase gene under the control of the TAKA:TPI pro-
moter. Plasmid pMT1796 is identical to pMT1802 except that E36
of the mutanase protein has been changed to K36 by replacing
the XhoI/RpnI fragment of pMT1802 with a PC~ amplified fragment
lo containing the desired mutation.
This PCR fragment was created in a two step procedure as
reported in Ho, et al. (198g), Gene, 77, p. 51-59, using the
following primers:
Primer 1 (nt 2751 5'CAGCGTCCACATCACGAGC nt 2769) and
~5 Primer 2 (nt 3306 5'GAAGAAGCACGTTTCTCGAGAGACCG nt 3281);
Primer 3 (nt 3281 5' CGGTCTCTGAGAAACGT~CTTCTTC nt 3306) and
Primer 4 (nt 4266 5 GCCACTTCCGTTATTAGCC nt 4248); nucleotide
numbers refer to the pMT1802 plasmid (See SEQ ID No. 11).
To create pMT1815, a 127 nt DNA fragment was PCR amplified
20 using again a two step procedure and the primers:
Primer 5: GGGGGGATCCACCATGAG;
Primer 6: ACGGTCAGCAGAAGAAGCTCGACGAATAGGACTGGC;
Primer 7: GCCAGTCCTATTCGTCGAGCTTCTTCTGCTGACCGT;
Primer 8: CCACGGTCACCAACAATAC,
25 and the plasmids pHan37 and pMT1802 as templates in the first
round of amplification.
This fragment contains a BamHI restriction enzyme site
followed by the Lipolase~ prepro-sequence in frame with
residues 38-54 of the mutanase protein and ending with a BstEII
30 site.
The fragment was digested with the restriction enzymes
BstEII and BamHI and inserted into pMTl802 that had been
linearized with the same pair of enzymes. Changes in constructs
were confirmed and the integrity of the resulting coding
35 regions were checked by nucleotide sequencing.
CA 022~829l l998-l2-l~
W O g8/OQ~2~ PCT~DK97/00283
26
Example 4
Expression of recombinant Mutanase in Aspergillus oryzae
The strain A. oryzae JaL125 was transformed using a PEG-
mediated protocol (see EP 238 023) and a DNA mixture containing
O. 5 ~g of a plasmid encoding the gene that confers resistance
to the herbicide Basta and 8.0 ~g of one of the three mutanase
expression plasmids. Transformants were selected on minimal
plates containing 0.5~ basta and 50 mM urea as a nitrogen
source.
Shake flask cultures
Transformed colonies were spore purified twice on selection
media and spores were harvested. A 20 ml universal container
(Nunc, cat #364211) containing 10 ml YPM (2~ maltose, 1%
15 bactopeptone and 0.5% yeast extract) was inoculated with spores
and grown for 5 days with shaking at 30~C. The supernatant was
harvested after 5 days growth.
highest mutanase number of
Construct level detected transformants tested
pMT1802, mutanase <0.001 10
prepro + mutanase
pMT1796, mutanase 3.8 4
prepro + KEX2 +
mutanase
pMT1815, Lipolase~ 0.16 22
prepro + mutanase
Table 1 Compari~on of mutanase expression from the three different
20 expression constructs. The limit of detection was O.OO1 MU/ml
The presence of mutanase in culture supernatants was
examined by western hybridizations. SDS-PAGE and protein
transfers were performed using standard protocols.
Example 5
Purification of recombinant mutanase
700 ml fermentation broth was filtered and concentrated. The
pH was adjusted to 4.7 (conductivity around 300 ~S/cm) and the
30 broth was loaded onto an S-Sepharose column (XK 50/22)
- (Pharmacia) equilibrated in 10 mM sodium acetate pH 4.7. The
CA 022~829l l998-l2-l~
W098/00528 PCT~K97/00283
27
mutanase was eluted in a linear NaCl gradient. The major part
of the mutanase appeared in the unbound fractions. These
fractions were pooled and concentrated. Then the concentrate
was loaded onto a HiLoad Q-Sepharose column (Pharmacia)
5 equilibrated in 10 mM Tris-HCl, pH 8.0 (around 600 ~S/cm). The
mutanase was eluted in a linear gradient of NaCl and the
mutanase containing-fractions were pooled according to purity
and activity. The pooled fractions were concentrated and a
fraction was further purified by gelfiltration on a Superdex 75
10 (16/60) column (Pharmacia) in sodium acetate pH 6Ø
The purified mutanase has a specific activity around l9 MU
pr. absorption unit at 280 nm. From SDS-PAGE (Novex 4-20 %; run
according to the manufacturer's instructions) a molecular
weight around 80 kDa is found.
15 The N-terminal amino acid sequence was confirmed to be
identical to the N-terminal amino acid sequence of the wt
mutanase (Ala-Ser-Ser-Ala-) (see Example 2)
Example 6
20 pH-profile of mutana~e
500 ml 5 % mutan in 50 mM Britton-Robinson buffer at varying
pH was added 2 ml enzyme sample (diluted in MilliQ-filtered
water) in large vials (to ensure sufficient agitation) and
incubated for 15 minutes at 40~C while shaking vigorously. The
25 reaction was terminated by adding 0.5 ml 0.4 M NaOH and the
samples were filtered on Munktell filters. lO0 ~l filtrate in
Eppendorf vials were added 750 ~l ferricyanide reagent (0.4 g/l
K3Fe(CN)6, 20 g/l Na2CO3) and incubated 15 minutes at 85~C.
After allowing the samples to cool, the decrease in absorption
30 at 420 nm was measured. A dilution series of glucose was
included as a standard. Substrate and enzyme blanks were always
included. Samples were run in duplicate. The pH-optimum for
both wild-type and recombinant enzyme is around pH 3.5-5.5 (see
Figure 4).
Example 7
. .
CA 022~829l l998-l2-l~
W098/00528 PCT~K97/00283
28
Temperature profile of mutanase:
500 ml 5 % mutan in 100 mM sodium acetate, pH 5.5 or in 100
mM sodium phosphate, pH 7 was added 2 ml enzyme sample (diluted
in MilliQ-filtered water) in large vials (to ensure sufficient
5 agitation) and incubated for 15 minutes at various temperatures
while shaking vigorously. The reaction was terminated by adding
0.5 ml 0.4 M NaOH and the samples were filtered on Munktell
filters. 100 ~l filtrate in Eppendorf vials were added 750 ~l
ferricyanide reagent (0.4 g/l K3Fe(CN)6, 20 g/l Na2CO3) and in-
10 cubated 15 minutes at 85~C. After allowing the samples to cool,the drop in absorption at 420 nm was measured. A dilution
series of glucose was included as a standard. Substrate and
enzyme blanks were always included. Samples were run in
duplicate. The temperature profiles for the recombinant and wt
15 mutanase were identical. The temperature optimum at pH 7 was
around 45 ~C. The temperature optimum at pH 5.5 was above 55~
(See Figure 5).
Example 8
20 Temperature stability of mutanase:
The temperature stability was investigated by pre-incubating
enzyme samples for 30 minutes at various temperatures in 0.1 M
sodium acetate, pH 5.5 or in 0.1 M sodium phosphate, pH 7
before assaying the residual activity. Both recombinant and wt
25 mutanase have similar temperature stability profiles. The
residual activity starts to decline at 40 oc at pH 7, while the
enzyme is more stable at pH 5.5, where the residual activity
starts to decline at 55~C (See Figure 6).
30 Example 9
Molecular weight of purified wild-type MutAn~e
The mass spectrometry, performed as described above, of the
mutanase revealed an average mass around 75 kDa. In addition, it
was clear from the spectra that the glycosylation of the mutanase
35 is heterogeneous. The peptide mass of the mutanase is more than
64 kDa meaning that more than 11 kDa of carbohydrate is attached
CA 022~8291 1998-12-1~
W098/00528 PCT~K97/00283
29
to the enzyme.
Example 10
Activity of mutanase against Dental Plaque
5 A plaque biofilm was grown anaerobic on saliva coated hydro-
xyapatite disks as described in the Material and Methods Section
above. The plaque was a mixed culture of Streptococcus mutans
(SFAG, CBS 350.71), Actinomyces viscosus (DSM 43329) and F~so-
bacterium nucleatum subsp. polymorphum (DSM 20482).
o HA disks with plaque were transferred to acetate buffer (pH
5.5) containing recombinant Trichoderma mutanase 1 MU/ml and
whirled for 2 minutes (sterile buffer was used as control).
After enzyme treatment, the disks were either DAPI stained or
transferred to Malthus cells, as indirect impedance measurements
15 were used when enumerating living adherent cells (Malthus Flexi
M2060, Malthus Instrument Limited).
For the impedance measurements 3 ml of BHI were transferred to
the outer chamber of the indirect Malthus cells, and 0.5 ml of
sterile KOH (0.1 M) was transferred to the inner chamber. After
20 mutanase treatment the disks with plaque were slightly rinsed
with phosphate buffer and transferred to the outer chamber. The
detection times (dt) in Malthus were converted to colony counts
by use of a calibration curve relating cfu/ml to dt (Figure 7).
The calibration curve was constructed by a series of 10-fold
25 dilution rate prepared from the mixed culture. Conductance dt of
each dilution step was determined in BHI and a calibration curve
relating cfu/ml of the 10 fold dilutions to dt in BHI was
constructed for the mixed culture (Figure 7).
The removal of plaque from the disks was also determined by
30 fluorescent microscopy, after mutanase treatment disks were
stained with DAPI (3 mM) and incubated in the dark for 5 minutes
(20~C). The DAPI stained cells were examined with the x 100 oil
immersion fluorescence objective on an Olympus model BX50
microscope equipped with a 200 W mercury lamp and an W - filter.
35 The result was compared with the quantitative data obtained by
the impedance measurements.
,
CA 022~829l l998-l2-l~
W O 98/00~28 PCTADK97/00283
The number of living cells on the saliva treated HA-surface
after enzyme treatment was determined by the Malthus method and
shown in Table 1. However, by the Malthus method it is not
possible to distinguish between a bactericidal activity of
5 mutanase or an enzymatic removal of the plaque. Therefore a
decrease in living bacteria on the surface has to be compared
with the simultaneously removal of plaque from the surface which
is estimated by the DA~I staining.
Mutanase Log10 reduction Removal ofNo. of
(MU/ml) (cfu/cm )plaque (%) observations
~ ~ ~ 10
1 1.4 96 6
10 Table 2: Enzymatic plaque removal (pH 5.5, 2 minutes) from saliva trea-ed
hydroxyapatite det~rminAd by ~ nAe measurements.
A significant removal of plaque was determined by fluorescent
microscopy after treatment with mutanase. Thus mutanase reduced
15 the amount of adhering cells. However, the activity was observed
as a removal of plaque and not as a bactericidal activity against
cells in plaque.
CA 022~8291 1998-12-1~
WO 98/OOS28 PCT/DK97/00283
31
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
'A' NAME: Novo Nordisk A/S
B STREET: Novo Alle
C CITY: Bagsvaerd
E COUN~ : Denmark
F POSTAL CODE (ZIP): DK-2880
G TELEPHONE: +45 4444 8888
H TELEFAX: +45 4449 3256
~ T-TLE OF lNV~ lON: A recombinant enzyme with mutanase activity
(iii) NUMBER OF ~Q~N~S: 11
~iv) COMPUTER ~An~Rn~ FORN:
'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 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
~'A) LENGTH: 1905 base pairs
B) TYPE: nucleic acid
'C) STRANDEDNESS: single
D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
~vi) ORIGINAL SOURCE:
(B) STRAIN: Trich~enma harzianum CBS 243.71
(ix) FEATURE:
'A) NAME/KEY: CDS
B) LOCATION:1..1905
'A) NAME/KEY: sig peptide
IB) LOCATION:1..120
(xi) S':QUENCE DESCRIPTION: SEQ ID NO: 1:
ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GGC GCC CTT GCT GCC GCA 48
Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala Leu Ala Ala Ala
1 5 10 15
GCT CTG TCT TCT CTC GGC AGT GCC GCT CCC GCC AAT GTT GCT ATT CGG 96
Ala Leu Ser Ser Leu Gly Ser Ala Ala Pro Ala Asn Val Ala Ile Arg
20 25 30
TCT CTC GAG GAA CGT GCT TCT TCT GCT GAC CGT CTC GTA TTC TGT CAC 144
Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His
35 40 45
TTC ATG ATT GGT ATT GTT GGT GAC CGT GGC AGC TCA GCA GAC TAT GAT 192
Phe Met Ile Gly Ile Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp
50 55 60
GAT GAC ATG CAA CGT GCC AAA GCC GCT GGC ATT GAC GCA TTC GCT CTG 240
Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu
65 70 75 80
AAC ATC GGC GTT GAC GGC TAT ACC GAC CAG CAA CTC GGG TAT GCC TAT 288
Asn Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Gly Tyr Ala Tyr
85 90 95
GAC TCT GCC GAC CGT AAT GGC ATG AAA GTC TTC ATT TCA TTC GAT TTC 336
Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe Ile Ser Phe Asp Phe
100 105 110
AAC TGG TGG AGC CCC GGT AAT GCA GTT GGT GTT GGC CAG AAG ATT GCG 384
Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gln Lys Ile Ala
115 120 125
CA 022~829l l998-l2-l~
W O 98/00528 PCT~DK~7/00283
32
CAG TAT GCC AGC CGT CCC GCC CAG CTG TAT GTT GAC AAC CGG CCA TTC 432
Gln Tyr Ala Ser Arg Pro Ala Gln Leu Tyr Val Asp Asn Arg Pro Phe
130 135 140
GCC TCT TCC TTC GCT GGT GAC GGT TTG GAT GTA AAT GCG TTG CGC TCT 480
Ala Ser Ser Phe Ala Gly Asp Gly Leu A5p Val Asn Ala Leu Arg Ser
145 150 155 160
GCT GCA GGC TCC AAC GTT TAC TTT GTG CCC AAC TTC CAC CCT GGT CAA 528
Ala Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly Gln
165 170 175
TCT TCC CCC TCC AAC ATT GAT GGC GCC CTC AAC TGG ATG GCC TGG GAT 576
Ser Ser Pro Ser Asn Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp
180 185 190
AAT GAT GGA AAC AAC AAG GCA CCC AAG CCG GGC CAG ACT GTC ACG GTG 624
Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gln Thr Val Thr Val
195 200 205
GCA GAC GGT GAC AAC GCT TAC AAG AAT TGG TTG GGT GGC AAG CCT TAC 672
Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys Pro Tyr
210 215 220
CTA GCG CCT GTC TCC CCT TGG TTT TTC ACC CAT TTT GGC CCT GAA GTT 720
Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val
225 230 235 240
TCA TAT TCC AAG AAC TGG GTC TTC CCA GGT GGT CCT CTG ATC TAT AAC 768
Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Pro Leu Ile Tyr Asn
245 250 255
CGG TGG CAA CAG GTC TTG CAG CAG GGC TTC CCC ATG GTT GAG ATT GTT 816
Arg Trp Gln Gln Val Leu Gln Gln Gly Phe Pro Met Val Glu Ile Val
260 265 270
ACC TGG AAT GAC TAC GGC GAG TCT CAC TAC GTC GGT CCT CTG AAG TCT 864
Thr Trp Asn Asp Tyr Gly Glu Ser Hi~ Tyr Val Gly Pro Leu Lys Ser
275 280 285
AAG CAT TTC GAT GAT GGC AAC TCC A~A TGG GTC AAT GAT ATG CCC CAT 912
Lys His Phe Asp Asp Gly Asn Ser Lys Trp Val Asn Asp Met Pro His
290 295 300
GAT GGA TTC TTG GAT CTT TCA A~G CCG TTT ATT GCT GCA TAT AAG AAC 960
Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe Ile Ala Ala Tyr Lys Asn
305 310 315 320
AGG GAT ACT GAT ATA TCT AAG TAT GTT CAA AAT GAG CAG CTT GTT TAC 1008
Arg Asp Thr Asp Ile Ser Lys Tyr Val Gln Asn Glu Gln Leu Val Tyr
325 330 335
TGG TAC CGC CGC AAC TTG AAG GCA TTG GAC TGC GAC GCC ACC GAC ACC 1056
Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr Asp Thr
340 345 350
ACC TCT AAC CGC CCG GCT AAT AAC GGA AGT GGC AAT TAC TTT ATG GGA 1104
Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met Gly
355 360 365
CGC CCT GAT GGT TGG CAA ACT ATG GAT GAT ACC GTT TAT GTT GCC GCA 1152
Arg Pro Asp Gly Trp Gln Thr Met Asp Asp Thr Val Tyr Val Ala Ala
370 375 380
CTT CTC AAG ACC GCC GGT AGC GTC ACG GTC ACG TCT GGC GGC ACC ACT 1200
Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly Thr Thr
CA 022~X291 1998-12-1~
WO 98/00528 PCT~DK97/00283
33
385 390 395 400
CAA ACG TTC CAG GCC AAC GCC GGA GCC AAC CTC TTC CAA ATC CCT GCC 1248
Gln Thr Phe Gln Ala Asn Ala Gly Ala Asn Leu Phe Gln Ile Pro Ala
405 410 415
AGC ATC GGC CAG CAA AAG TTT GCT CTA ACT CGC AAC GGT CAG ACC GTC 1296
Ser Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gly Gln Thr Val
420 425 430
TTT AGC GGA ACC TCA TTG ATG GAT ATC ACC AAC GTT TGC TCT TGC GGT 1344
Phe Ser Gly Thr Ser Leu Met Asp Ile Thr Asn Val Cys Ser Cys Gly
435 440 445
ATC TAC AAT TTC AAC CCA TAT GTT GGC ACC ATT CCT GCC GGC TTT GAC 1392
Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile Pro Ala Gly Phe Asp
450 455 460
GAC CCT CTT CAG GCT GAC GGT CTT TTC TCT TTG ACC ATC GGA TTG CAT 1440
Asp Pro Leu Gln Ala Asp Gly Leu Phe Ser Leu Thr Ile Gly Leu His
465 470 475 480
GTC ACG ACT TGT CAG GCC AAG CCA TCT CTT GGA ACC AAC CCT CCT GTC 1488
Val Thr Thr Cys Gln Ala Lys Pro Ser Leu Gly Thr Asn Pro Pro Val
485 490 495
ACT TCT GGC CCT GTG TCC TCG CTG CCA GCT TCC TCC ACC ACC CGC GCA 1536
Thr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser Thr Thr Arg Ala
500 505 510
TCC TCG CCT CCT GTT TCT TCA ACT CGT GTC TCT TCT CCC CCT GTC TCT 1584
Ser Ser Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro Val Ser
515 520 525
TCC CCT CCA GTT TCT CGC ACC TCT TCT CCC CCT CCC CCT CCG GCC AGC 1632
Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro Ala Ser
530 535 540
AGC ACG CCG CCA TCG GGT CAG GTT TGC GTT GCC GGC ACC GTT GCT GAC 1680
Ser Thr Pro Pro Ser Gly Gln Val Cys Val Ala Gly Thr Val Ala Asp
545 550 555 560
GGC GAG TCC GGC AAC TAC ATC GGC CTG TGC CAA TTC AGC TGC AAC TAC 1728
Gly Glu Ser Gly Asn Tyr Ile Gly Leu Cys Gln Phe Ser Cys Asn Tyr
565 570 575
GGT TAC TGT CCA CCG GGA CCG TGT AAG TGC ACC GCC TTT GGT GCT CCC 1776
Gly Tyr Cy5 Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala Pro
580 585 590
ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC GGC TGC CCT CTA CCG GGA 1824
Ile Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu Pro Gly
595 600 605
GAA GGC GAT GGT TAT CTG GGC CTG TGC AGT TTC AGT TGT AAC CAT AAT 1872
Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn His Asn
610 615 620
TAC TGC CCG CCA ACG GCA TGC CAA TAC TGT TAG 1905
Tyr Cys Pro Pro Thr Ala Cys Gln Tyr Cys *
625 630 635
~2) INFORMATION FOR SEQ ID NO: 2:
yU~N~ CHARACTERISTICS:
(A) LENGTH: 635 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
CA 022~829l l998-l2-l~
W O ~8/005~8 PCT~DK~7100283
34
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala Leu Ala Ala Ala
1 5 10 15
~la Leu Ser Ser Leu Gly Ser Ala Ala Pro Ala Asn Val Ala Ile Arg
Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe Cys His
Phe Met Ile Gly Ile Val Gly Asp Arg Gly Ser Ser Ala Asp Tyr Asp
Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe Ala Leu
~sn Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Gly Tyr Ala Tyr
~sp Ser Ala Asp Arg Asn Gly Met Lys Val Phe Ile Ser Phe Asp Phe
100 105 110
Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gln Lys Ile Ala
115 120 125
Gln Tyr Ala Ser Arg Pro Ala Gln Leu Tyr Val Asp Asn Arg Pro Phe
130 135 140
Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp Val Asn Ala Leu Arg Ser
145 150 155 160
~la Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro Gly Gln
165 170 175
~er Ser Pro Ser Asn Ile Asp Gly Ala Leu Asn Trp Met Ala Trp Asp
1~0 185 190
Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gln Thr Val Thr Val
195 200 205
Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys Pro Tyr
210 215 220
Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro Glu Val
225 230 235 240
~er Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Pro Leu Ile Tyr Asn
245 250 255
~rg Trp Gln Gln Val Leu Gln Gln Gly Phe Pro Met Val Glu Ile Val
260 265 270
Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr Val Gly Pro Leu Lys Ser
275 280 285
Lys His Phe Asp Asp Gly Asn Ser Lys Trp Val Asn Asp Met Pro His
290 295 300
Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe Ile Ala Ala Tyr Lys Asn
305 310 315 320
~rg Asp Thr Asp Ile Ser Lys Tyr Val Gln Asn Glu Gln Leu Val Tyr
325 330 335
~rp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr Asp Thr
CA 022~8291 1998- 12- 1~
WO ~8t0052X PCT/DK97/00283
340 345 350
Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe Met Gly
355 360 365
Arg Pro Asp Gly Trp Gln Thr Met Asp Asp Thr Val Tyr Val Ala Ala
370 375 380
Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly Thr Thr
385 390 395 400
~ln Thr Phe Gln Ala Asn Ala Gly Ala Aan Leu Phe Gln Ile Pro Ala
405 410 415
~er Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gly Gln Thr Val
420 425 430
Phe Ser Gly Thr Ser Leu Met Asp Ile Thr Asn Val Cys Ser Cys Gly
435 440 445
Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile Pro Ala Gly Phe Asp
450 455 460
Asp Pro Leu Gln Ala Asp Gly Leu Phe Ser Leu Thr Ile Gly Leu His
465 470 475 480
~al Thr Thr Cys Gln Ala Lys Pro Ser Leu Gly Thr Asn Pro Pro Val
485 490 495
~hr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser Thr Thr Arg Ala
500 505 510
Ser Ser Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro Val Ser
515 520 525
Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro Ala Ser
530 535 540
Ser Thr Pro Pro Ser Gly Gln Val Cys Val Ala Gly Thr Val Ala Asp
545 550 555 560
~ly Glu Ser Gly Asn Tyr Ile Gly Leu Cys Gln Phe Ser Cys Asn l'yr
565 570 575
~ly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly Ala Pro
580 585 590
Ile Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu Pro Gly
595 600 605
Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn His Asn
610 615 620
Tyr Cys Pro Pro Thr Ala Cys Gln Tyr Cys *
625 630 635
~2) INFORMATION FOR SEQ ID NO: 3:
;yu~;N~: CEIARACTERISTICS:
A LEN-GTH: 19 baqe pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
tii) MOL 'CULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 1"
CA 022~829l l998-l2-l~
W O 98/00528 PCT~DK97/00283
36
CAGCGTCCAC ATCACGAGC 19
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
I~A) LENGTH: 26 base pairs
.B) TYPE: nucleic acid
,C) STRANDEDNESS: single
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 2"
GAAGAAGCAC GTTTCTGCAG AGACCG 26
(2) INFORMATION FOR SEQ ID NO: 5:
Qu~N~ CHARACTERISTICS:
'A' LENGTH: 26 base pairs
Bl TYPE: nucleic acid
,C STR~NDEDNESS: single
D TOPOLOGY: linear
(ii) MOL,CULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 3"
CGGTCTCTCG AGAAACGTGC TTCTTC 26
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(Al LENGTH: 19 base pairs
(B TYPE: nucleic acid
(C STRANDEDNESS: single
~D TOPOLOGY: linear
(ii) MOL-CULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 4"
GCCACTTCCG TTATTAGCC 19
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
'A LENGTH: 18 base pairs
B TYPE: nucleic acid
C STRANDEDNESS: single
D TOPOLOGY: linear
(ii) MOL.'CULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 5"
GGGGGGATCC ACCATGAG 18
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
'A) LENGTH: 36 base pairs
B) TYPE: nucleic acid
C) STR~NDEDNESS: single
,D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = ~Primer 6"
ACGGTCAGCA GAAGAAGCTC GAC~.AATAGG ACTGGC 36
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
~ (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 022~8291 1998-12-1~
WO 98/00528 PCT/DK97/00283
37
~D) TOPOLOGY: linear
(ii) MOLECULE TYPE: other nucleic acid
(A) DESCRIPTION: /de6c = "Primer 7"
GCCAGTCCTA TTCGTCGAGC ll~Ll~lGCT GACCGT 36
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
~A' LENGTH: l9 base pairs
B TYPE: nucleic acid
,C STRANDEDNESS: single
~D TOPOLOGY: linear
(ii) MOL''CULE TYPE: other nucleic acid
(A) DESCRIPTION: /desc = "Primer 8"
CCACGGTCAC CAACAATAC 19
(2) INFORMATION FOR SEQ ID NO: ll:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6032 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(vi) ORIGINAL SOURCE:
(B) STRAIN: Trichoderma harzianum CBS 243.71
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION:3188..5092
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: ll:
GACGAAAGGG CCTCGTGATA CGCCTATTTT TATAGGTTAA TGTCATGATA ATAATGGTTT 60
CTTAGACGTC AGGTGGCACT TTTCGGGGAA ATGTGCGCGG AACCCCTATT TGTTTATTTT 120
TCTAAATACA TTCAAATATG TATCCGCTCA T~-AGA~AATA ACCCTGATAA ATGCTTCAAT 180
AATATTGAAA AAGGAAGAGT ATGAGTATTC AACATTTCCG TGTCGCCCTT ATTCCCTTTT 240
TTGCGGCATT TTGCCTTCCT Gllll~GCTC ACCCAGAAAC GCTGGTGAAA GTAAAAGATG 300
CTGAAGATCA GTTGGGTGCA CGAGTGGGTT ACATCGAACT GGATCTCAAC AGCGGTAAGA 360
TCCTTGAGAG TTTTCGCCCC GAAGAACGTT TTCCAATGAT GAGCACTTTT AAAGTTCTGC 420
TATGTGGCGC GGTATTATCC CGTATTGACG CCGGGCAAGA GCAACTCGGT CGCCGCATAC 480
ACTATTCTCA GAATGACTTG GTTGAGTACT CACCAGTCAC AGAAAAGCAT CTTACGGATG 540
GCATGACAGT AAGAGAATTA TGCAGTGCTG CCATAACCAT GAGTGATAAC ACTGCGGCCA 600
ACTTACTTCT GACAACGATC GGAGGACCGA AGGAGCTAAC CG~l L 111 ~G ~ACAA~ATGG 660
GGGATCATGT AACTCGCCTT GATCGTTGGG AACCGGAGCT GAATGAAGCC ATACCAAACG 720
ACGAGCGTGA CACCACGATG CCTGTAGCAA TGGCAACAAC GTTGCGCAAA CTATTAACTG 780
GCGAACTACT TACTCTAGCT TCCCGGCAAC AATTAATA~A CTGGATGGAG GCGGATAAAG 840
TTGCAGGACC ACTTCTGCGC TCGGCCCTTC CGGCTGGCTG GTTTATTGCT GATAAATCTG 900
GAGCCGGTGA GCGTGGGTCT CGCGGTATCA TTGCAGCACT GGGGCCAGAT GGTAAGCCCT 960
CCCGTATCGT AGTTATCTAC ACGACGGGGA GTCAGGCAAC TATGGATGAA CGAAATAGAC 1020
AGATCGCTGA GATAGGTGCC TCACTGATTA AGCATTGGTA ACTGTCAGAC CAAGTTTACT 1080
CATATATACT TTAGATTGAT TTAAAACTTC ATTTTTAATT TAAAAGGATC TAGGTGAAGA 1140
TC~lllllGA TAATCTCATG ACCAAAATCC CTTAACGTGA GTTTTCGTTC CACTGAGCGT 1200
CAGACCCCGT AGAAAAGATC AAAGGATCTT CTTGAGATCC 'l--.LllCTG CGCGTAATCT 1260
GCTGCTTGCA AA~AAAAAAA CCACCGCTAC CAGCGGTGGT llGlllGCCG GATCAAGAGC 1320
TACCAACTCT TTTTCCGAAG GTAACTGGCT TCAGCAGAGC GCAGATACCA AATACTGTCC 1380
TTCTAGTGTA GCCGTAGTTA GGCCACCACT TCAAGAACTC TGTAGCACCG CCTACATACC 1440
TCGCTCTGCT AATCCTGTTA CCAGTGGCTG CTGCCAGTGG CGATAAGTCG TGTCTTACCG 1500
GGTTGGACTC AAGACGATAG TTACCGGATA AGGCGCAGCG GTCGGGCTGA ACGGGGGGTT 1560
CGTGCACACA GCCCAGCTTG GAGCGAACGA CCTACACCGA ACTGAGATAC CTACAGCGTG 1620
AGCATTGAGA AAGCGCCACG CTTCCCGAAG GGAGAAAGGC GGACAGGTAT CCGGTAAGCG 1680
.. . . . .
CA 022~829l l998-l2-l~
W O 98/00528 PCT~DK97/00283
38
GCAGGGTCGG AACAGGAGAG CGCACGAGGG AGCTTCCAGG GGGAAACGCC TGGTATCTTT 1740
ATAGTCCTGT CGGGTTTCGC CACCTCTGAC TTGAGCGTCG Alllll~lGA TGCTCGTCAG 1800
GGGGGCGGAG CCTATGGAAA AACGCCAGCA ACGCGGCCTT TTTACGGTTC CTGGCCTTTT 1860
GCTGGCCTTT TGCTCACATG TTCTTTCCTG CGTTATCCCC TGATTCTGTG GATAACCGTA 1920
TTACCGCCTT TGAGTGAGCT GATACCGCTC GCCGCAGCCG AACGACCGAG CGCAGCGAGT 1980
CAGTGAGCGA GGAAGCGGAA GAGCGCCCAA TACGCAAACC GCCTCTCCCC GCGCGTTGGC 2040
CGATTCATTA ATGCAGCCTG ATTAATGATT ACATACGCCT CCGGGTAGTA GACCGAGCAG 2100
CCGAGCCAGT TCAGCGCCTA AAACGCCTTA TACAATTAAG CAGTTAAAGA AGTTAGAATC 2160
TACGCTTAAA AAGCTACTTA AAAATCGATC TCGCAGTCCC GATTCGCCTA TCAAAACCAG 2220
TTTAAATCAA CTGATTAAAG GTGCCGAACG AGCTATAAAT GATATAACAA TATTAAAGCA 2280
TTAATTAGAG CAATATCAGG CCGCGCACGA AAGGCAACTT AAAAAGCGAA AGCGCTCTAC 2340
TAAACAGATT ACTTTTGAAA AAGGCACATC AGTATTTAAA GCCCGAATCC TTATTAAGCG 2400
CCGAAATCAG GCAGATAAAG CCATACAGGC AGATAGACCT CTACCTATTA AATCGGCTTC 2460
TAGGCGCGCT CCATCTAAAT GTTCTGGCTG TGGTGTACAG GGGCATAAAA TTACGCACTA 2520
CCCGAATCGA TAGAACTACT CATTTTTATA TAGAAGTCAG AATTCATAGT GTTTTGATCA 2580
TTTTAAATTT TTATATGGCG GGTGGTGGGC AACTCGCTTG CGCGGGCAAC TCGCTTACCG 2640
ATTACGTTAG GGCTGATATT TACGTGAAAA TCGTCAAGGG ATGCAAGACC AAAGTAGTAA 2700
AACCCCGGAA GTCAACAGCA TCCAAGCCCA AGTCCTTCAC GGAGAAACCC CAGCGTCCAC 2760
ATCACGAGCG AAGGACCACC TCTAGGCATC GGACGCACCA TCCAATTAGA AGCAGCAAAG 2820
CGAAACAGCC CAAGAAAAAG GTCGGCCCGT CGGCCTTTTC TGCAACGCTG ATCACGGGCA 2880
GCGATCCAAC CAACACCCTC CAGAGTGACT AGGGGCGGAA ATTTAAAGGG ATTAATTTCC 2940
ACTCAACCAC AAATCACAGT CGTCCCCGGT ATTGTCCTGC AGAATGCAAT TTAAACTCTT 3000
CTGCGAATCG CTTGGATTCC CCGCCCCTAG TCGTAGAGCT TAAAGTATGT CC~llGlCGA 3060
TGCGATGTAT CACAACATAT AAATACTAGC AAGGGATGCC ATGCTTGGAG TTTCCAACTC 3120
AATTTACCTC TATCCACACT TCT~llC~Ll CCTCAATCCT CTATATACAC AACTGGGGAT 3180
CCTCACA ATG TTG GGC GTT GTC CGC CGT CTA GGC CTA GGC GCC CTT GCT 3229
Met Leu Gly Val Val Arg Arg Leu Gly Leu Gly Ala Leu Ala
1 5 10
GCC GCA GCT CTG TCT TCT CTC GGC AGT GCC GCT CCC GCC AAT GTT GCT 3277
Ala Ala Ala Leu Ser Ser Leu Gly Ser Ala Ala Pro Ala Asn Val Ala
15 20 25 30
ATT CGG TCT CTC GAG GAA CGT GCT TCT TCT GCT GAC CGT CTC GTA TTC 3325
Ile Arg Ser Leu Glu Glu Arg Ala Ser Ser Ala Asp Arg Leu Val Phe
35 40 45
TGT CAC TTC ATG ATT GGT ATT GTT GGT GAC CGT GGC AGC TCA GCA GAC 3373
Cys His Phe Met Ile Gly Ile Val Gly Asp Arg Gly Ser Ser Ala Asp
50 55 60
TAT GAT GAT GAC ATG CAA CGT GCC AAA GCC GCT GGC ATT GAC GCA TTC 3421
Tyr Asp Asp Asp Met Gln Arg Ala Lys Ala Ala Gly Ile Asp Ala Phe
65 70 75
GCT CTG AAC ATC GGC GTT GAC GGC TAT ACC GAC CAG CAA CTC GGG TAT 3469
Ala Leu Asn Ile Gly Val Asp Gly Tyr Thr Asp Gln Gln Leu Gly Tyr
80 85 90
GCC TAT GAC TCT GCC GAC CGT AAT GGC ATG AAA GTC TTC ATT TCA TTC 3517
Ala Tyr Asp Ser Ala Asp Arg Asn Gly Met Lys Val Phe Ile Ser Phe
95 100 105 110
GAT TTC AAC TGG TGG AGC CCC GGT AAT GCA GTT GGT GTT GGC CAG AAG 3565
Asp Phe Asn Trp Trp Ser Pro Gly Asn Ala Val Gly Val Gly Gln Lys
115 120 125
ATT GCG CAG TAT GCC AGC CGT CCC GCC CAG CTG TAT GTT GAC AAC CGG 3613
Ile Ala Gln Tyr Ala Ser Arg Pro Ala Gln Leu Tyr Val Asp Asn Arg
130 135 140
CCA TTC GCC TCT TCC TTC GCT GGT GAC GGT TTG GAT GTA AAT GCG TTG 3661
Pro Phe Ala Ser Ser Phe Ala Gly Asp Gly Leu Asp Val Asn Ala Leu
145 150 155
CGC TCT GCT GCA GGC TCC AAC GTT TAC TTT GTG CCC AAC TTC CAC CCT 3709
Arg Ser Ala Ala Gly Ser Asn Val Tyr Phe Val Pro Asn Phe His Pro
160 165 170
GGT CAA TCT TCC CCC TCC AAC ATT GAT GGC GCC CTC AAC TGG ATG GCC 3757
Gly Gln Ser Ser Pro Ser Asn Ile Asp Gly Ala Leu Asn Trp Met Ala
175 180 185 190
TGG GAT AAT GAT GGA AAC AAC AAG GCA CCC AAG CCG GGC CAG ACT GTC 3805
Trp Asp Asn Asp Gly Asn Asn Lys Ala Pro Lys Pro Gly Gln Thr Val
195 200 205
ACG GTG GCA GAC GGT GAC AAC GCT TAC AAG AAT TGG TTG GGT GGC AAG 3853
Thr Val Ala Asp Gly Asp Asn Ala Tyr Lys Asn Trp Leu Gly Gly Lys
210 215 220
CA 022~829l l998-l2-l~
WO 98/00528 PCT/DK97/00283
39
CCT TAC CTA GCG CCT GTC TCC CCT TGG TTT TTC ACC CAT TTT GGC CCT 3901
Pro Tyr Leu Ala Pro Val Ser Pro Trp Phe Phe Thr His Phe Gly Pro
225 230 235
GAA GTT TCA TAT TCC AAG AAC TGG GTC TTC CCA GGT GGT CCT CTG ATC 3949
Glu Val Ser Tyr Ser Lys Asn Trp Val Phe Pro Gly Gly Pro Leu Ile
240 245 250
TAT AAC CGG TGG CAA CAG GTC TTG CAG CAG GGC TTC CCC ATG GTT GAG 3997
Tyr Asn Arg Trp Gln Gln Val Leu Gln Gln Gly Phe Pro Met Val Glu
255 260 265 270
ATT GTT ACC TGG AAT GAC TAC GGC GAG TCT CAC TAC GTC GGT CCT CTG 4045
Ile Val Thr Trp Asn Asp Tyr Gly Glu Ser His Tyr Val Gly Pro Leu
AAG TCT AAG CAT TTC GAT GAT GGC AAC TCC AAA TGG GTC AAT GAT ATG 4093
Lys Ser Lys His Phe Asp Asp Gly Agn Ser Lys Trp Val Asn A p Met
290 295 300
CCC CAT GAT GGA TTC TTG GAT CTT TCA AAG CCG TTT ATT GCT GCA TAT 4141
Pro His Asp Gly Phe Leu Asp Leu Ser Lys Pro Phe Ile Ala Ala Tyr
305 310 315
AAG AAC AGG GAT ACT GAT ATA TCT AAG TAT GTT CAA AAT GAG CAG CTT 4189
Lys Asn Arg Asp Thr Asp Ile Ser Lys Tyr Val Gln Asn Glu Gln Leu
320 325 330
GTT TAC TGG TAC CGC CGC AAC TTG AAG GCA TTG GAC TGC GAC GCC ACC 4237
Val Tyr Trp Tyr Arg Arg Asn Leu Lys Ala Leu Asp Cys Asp Ala Thr
335 340 345 350
GAC ACC ACC TCT AAC CGC CCG GCT AAT AAC GGA AGT GGC AAT TAC TTT 4285
Asp Thr Thr Ser Asn Arg Pro Ala Asn Asn Gly Ser Gly Asn Tyr Phe
355 360 365
ATG GGA CGC CCT GAT GGT TGG CAA ACT ATG GAT GAT ACC GTT TAT GTT 4333
Met Gly Arg Pro Asp Gly Trp Gln Thr Met Asp Asp Thr Val Tyr Val
370 375 380
GCC GCA CTT CTC AAG ACC GCC GGT AGC GTC ACG GTC ACG TCT GGC GGC 4381
Ala Ala Leu Leu Lys Thr Ala Gly Ser Val Thr Val Thr Ser Gly Gly
385 390 395
ACC ACT CAA ACG TTC CAG GCC AAC GCC GGA GCC AAC CTC TTC CAA ATC 4429
Thr Thr Gln Thr Phe Gln Ala Asn Ala Gly Ala Asn Leu Phe Gln Ile
400 405 410
CCT GCC AGC ATC GGC CAG CAA AAG TTT GCT CTA ACT CGC AAC GGT CAG 4477
Pro Ala Ser Ile Gly Gln Gln Lys Phe Ala Leu Thr Arg Asn Gl.y Gln
415 420 425 430
ACC GTC TTT AGC GGA ACC TCA TTG ATG GAT ATC ACC AAC GTT TGC TCT 4525
Thr Val Phe Ser Gly Thr Ser Leu Met Asp Ile Thr Asn Val Cys Ser
435 440 445
TGC GGT ATC TAC AAT TTC AAC CCA TAT GTT GGC ACC ATT CCT GCC GGC 4573
Cys Gly Ile Tyr Asn Phe Asn Pro Tyr Val Gly Thr Ile Pro Ala Gly
450 455 460
TTT GAC GAC CCT CTT CAG GCT GAC GGT CTT TTC TCT TTG ACC ATC GGA 4621
Phe Asp Asp Pro Leu Gln Ala A~p Gly Leu Phe Ser Leu Thr Ile Gly
465 470 475
TTG CAT GTC ACG ACT TGT CAG GCC AAG CCA TCT CTT GGA ACC AAC CCT 4669
Leu His Val Thr Thr Cys Gln Ala Lys Pro Ser Leu Gly Thr Asn Pro
480 485 490
CCT GTC ACT TCT GGC CCT GTG TCC TCG CTG CCA GCT TCC TCC ACC ACC 4717
Pro Val Thr Ser Gly Pro Val Ser Ser Leu Pro Ala Ser Ser Thr Thr
495 500 505 510
CGC GCA TCC TCG CCT CCT GTT TCT TCA ACT CGT GTC TCT TCT CCC CCT 4765
Arg Ala Ser Ser Pro Pro Val Ser Ser Thr Arg Val Ser Ser Pro Pro
515 520 525
GTC TCT TCC CCT CCA GTT TCT CGC ACC TCT TCT CCC CCT CCC CCT CCG 4813
Val Ser Ser Pro Pro Val Ser Arg Thr Ser Ser Pro Pro Pro Pro Pro
530 535 540
GCC AGC AGC ACG CCG CCA TCG GGT CAG GTT TGC GTT GCC GGC ACC GTT 4861
Ala Ser Ser Thr Pro Pro Ser Gly Gln Val Cys Val Ala Gly Thr Val
545 550 555
GCT GAC GGC GAG TCC GGC AAC TAC ATC GGC CTG TGC CAA TTC AGC TGC 4909
Ala Asp Gly Glu Ser Gly Asn Tyr Ile Gly Leu Cys Gln Phe Ser Cys
~ 560 565 570
AAC TAC GGT TAC TGT CCA CCG GGA CCG TGT AAG TGC ACC GCC TTT GGT 4957
, . . . . . . .
CA 022~829l l998-l2-l~
W O 98/00528 PCT~DK97/00283
Asn Tyr Gly Tyr Cys Pro Pro Gly Pro Cys Lys Cys Thr Ala Phe Gly
575 580 585 590
GCT CCC ATC TCG CCA CCG GCA AGC AAT GGG CGC AAC GGC TGC CCT CTA 5005
Ala Pro Ile Ser Pro Pro Ala Ser Asn Gly Arg Asn Gly Cys Pro Leu
595 600 605
CCG GGA GAA GGC GAT GGT TAT CTG GGC CTG TGC AGT TTC AGT TGT AAC 5053
Pro Gly Glu Gly Asp Gly Tyr Leu Gly Leu Cys Ser Phe Ser Cys Asn
610 615 620
CAT AAT TAC TGC CCG CCA ACG GCA TGC CAA TAC TGT TAG TCTAGAGGGT 5102
His Asn Tyr Cys Pro Pro Thr Ala Cys Gln Tyr Cys *
625 630 635
GACTGACACC TGGCGGTAGA CAATCAATCC ATTTCGCTAT AGTTAAAGGA TGGGGATGAG 5162
GGCAATTGGT TATATGATCA TGTATGTAGT GGGTGTGCAT AATAGTAGTG A~ATGGAAGC 5222
CAAGTCATGT GATTGTAATC GACCGACGGA ATTGAGGATA TCCGGAAATA CAGACACCGT 5282
GAAAGCCATG Gl~lllC~ll CGTGTAGAAG ACCAGACAGA CAGTCCCTGA TTTACCCTGC 5342
ACAAAGCACT A~AAAATTAG CATTCCATCC ll~l~iGCTT GCTCTGCTGA TATCACTGTC 5402
ATTCAATGCA TAGCCATGAG CTCATCTTAG ATCCAAGCAC GTAATTCCAT AGCCGAGGTC 5462
CACAGTGGAG CAGCAACATT CCCCATCATT GCTTTCCCCA GGGGCCTCCC AACGACTAAA 5522
TCAAGAGTAT ATCTCTACCG TCCAATAGAT CG1~llCGCT TCAAAATCTT TGACAATTCC 5582
AAGAGGGTCC CCATCCATCA AACCCAGTTC AATAATAGCC GAGATGCATG GTGGAGTCAA 5642
TTAGGCAGTA TTGCTGGAAT GTCGGGGCCA GTTGGCCGGG TGGTCATTGG CCGCCTGTGA 5702
TGCCATCTGC CACTAAATCC GATCATTGAT CCACCGCCCA CGAGGGCGTC TTTGCTTTTT 5762
GCGCGGCGTC CAGGTTCAAC TCTCTCCTCT AGCGCCTGAT GCGGTATTTT CTCCTTACGC 5822
ATCTGTGCGG TATTTCACAC CGCATATGGT GCACTCTCAG TACAATCTGC TCTGATGCCG 5882
CATAGTTAAG CCAGCCCCGA CACCCGCCAA CACCCGCTGA CGCGCCCTGA CGGGCTTGTC 5942
TGCTCCCGGC ATCCGCTTAC AGACAAGCTG TGACCGTCTC CGGGAGCTGC Ai~l~l~AGA 6002
GGTTTTCACC GTCATCACCG AAACGCGCGA 6032
