Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing
Reexamination Certificate
1999-03-25
2001-03-27
Prouty, Rebecca E. (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
C424S094200, C435S183000, C435S200000, C435S202000, C435S211000, C435S219000
Reexamination Certificate
active
06207149
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an oral care composition comprising a Starch Binding Domain, an oral care product comprising an oral care composition of the invention, and further to the use of a Starch Binding Domain for oral care purposes, including prevention of the formation of dental plaque and/or removal of dental plaque.
BACKGROUND OF THE INVENTION
The formation of dental plaque leads to dental caries, gingival inflammation, periodontal disease, and eventually tooth loss. Dental plaque is a mixture of bacteria, epithelial cells, leukocytes, macrophages, and other oral exudate. Said bacteria produce highly branched polysaccharides which together with microorganisms from the oral cavity form an adhesive matrix for the continued proliferation of dental plaque.
As dental plaque continues to accumulate rock hard white or yellowish deposits arise. These deposits are called calcified plaque, calculus or tartar, and are formed in the saliva from plaque and minerals, such as in particular calcium.
Oral Polysaccharides
Oral polysaccharides mainly consist of the adhesive polysaccharides termed “fructans” and “glucans”.
Glucans are produced from carbohydrates, such as sucrose introduced into the mouth, e.g. as a food or beverage constituent, by the action of cariogenic microorganisms, such as
Streptococcus mutans
or
Streptococcus sanguis
, growing in the oral cavity.
The term “glucan” is a general common term covering a number of polysaccharides and includes cellulose, starch, dextran, mutan, pullulan etc.
Oral glucans comprise water-soluble dextran, having large portions of &agr;-1,6 glucosidic linkage and as the major component a water-insoluble extra-cellular polysaccharide called “mutan” comprised of a backbone with &agr;-1,3-glycosidic linkages and branches with &agr;-1,6-glycosidic linkages.
Mutan binds to almost any surface such as the surface of teeth, (i.e. hydroxyapatite constituting the hard outer porous layer of the teeth), pellicle, the cell surface of oral micro-organisms as well as to acceptor proteins on the cell of said cariogenic bacteria adhering to the teeth surface.
WO 95/31556 (Unilever) discloses an oral composition comprising the Glucan Binding Domain of glycosyltransferase having specific binding affinity for dextran (being a polysaccharide with mainly &agr;-1,6-glucosidic linkages).
According to WO 95/31556 the Glucan Binding Domain is covalently chemically bound to material having an activity, such as inhibitory effect against the formation of dental plaque. The material may be an enzyme, such as galactose oxidase (see Example 6 of said WO application).
A number of Cellulose Binding Domains are known in the art. Peter Tomme et al., (1996), “Cellulose-Binding Domains: Classification and Properties” in “Enzymatic Degradation of Insoluble Carbohydrates”, John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618; Ong et al. (1989), TIBTech 7, p. 239-243; and WO 93/21331 describe a vast number of Cellulose Binding Domains.
SUMMARY OF THE INVENTION
It is the object of the present invention to provide oral care products which can be used for improving the oral hygiene of humans and animals, by effectively preventing the formation of dental plaque and/or removing already deposited dental plaque.
The present inventors have seen indications that Starch Binding Domains (SBDs) have a dispersing effect on oral polysaccharides. Consequently, Starch Binding Domains are suitable for removing and/or preventing dental plaque.
Starch Binding Domains (SBD)
In the following “
S
tarch
B
inding
D
omain” will be abbreviated as “SBD” and is meant to define all polypeptide sequences or peptide sequences having affinity for binding to Starch.
Most known SBDs today are found in CGTases, i.e. cyclodextrin glucanotransferases (E.C. 2.4.1.19), and glucoamylases (E.C. 3.2.1.3). See also Chen et al. (1991), Gene 991, p. 121-126, describing Starch Binding Domain hybrids.
Specifically, an SBD has been found in the commercially available enzyme AMG (a glucoamylase) from
Aspergillus niger.
SBDs may be useful as a single domain polypeptide or as a dimer, a trimer, or a polymer; or as a part of a protein hybrid.
Single Unit Starch Binding Domain (Single Unit SBD)
The term “Single Unit SBD” may also be referred to as “Isolated SBD” or “Separate SBD”.
In the context of the present invention a “Single Unit SBD” includes up to the entire part of the amino acid sequence of an SBD-containing enzyme, e.g. a polysaccharide hydrolyzing enzyme, being essentially free of the catalytic domain, but retaining the SBD(s).
Thus, in the context of the invention, the entire catalytic amino acid sequence of a starch degrading enzyme (e.g. a glucoamylase) or other enzymes comprising one or more SBDs is not to be regarded as a Single Unit SBD.
Typically a Single Unit SBD constitutes one or more SBDs of a polysaccharide hydrolyzing enzyme, one or more SBDs of a starch binding protein or a protein designed and/or engineered to be capable of binding to starch.
The Single Unit SBD is at least as large as the minimum number of amino acids in the amino acid sequence required to bind to starch.
A Single Unit SBD may also be an amino acid sequence in which the binding and catalytic domain are one and the same.
Isolation of a Starch Binding Domain
In order to isolate the Starch Binding Domain of e.g. a glucoamylase, several genetic approaches may be used. One method uses restriction enzymes to remove a portion of the gene and then to fuse the remaining gene-vector fragment in frame to obtain a mutated gene that encodes a protein truncated for a particular gene fragment. Another method involves the use of exonucleases such as Ba131 to systematically delete nucleotides either externally from the 5′ and the 3′ ends of the DNA or internally from a restricted gap within the gene. These gene deletion methods result in a mutated gene encoding a shortened gene molecule which may then be evaluated for substrate binding ability. Appropriate substrates for evaluating the binding activity include compounds such as starch.
Once a nucleotide sequence encoding the substrate binding region has been identified, either as cDNA or chromosomal DNA, it may then be manipulated in a variety of ways to fuse it to a DNA sequence encoding the enzyme of interest. The starch binding encoding fragment and the DNA encoding the enzyme of interest are then ligated with or without a linker. The resulting ligated DNA may then be manipulated in a variety of ways to provide for expression. Microbial hosts such as Aspergillus, e.g.,
A. niger
and
A. oryzae
, Bacillus,
E. coli
or
S. cerevisiae
are preferred.
In the first aspect, the invention relates to an oral care composition comprising an SBD and ingredients conventionally used in oral care compositions.
The SBD may be any SBD, such as a Single Unit SBD of any kind. SBDs specifically contemplated are SBDs isolated from microorganisms, such as bacteria, filamentous fungi or yeasts, such as SBDs derived from e.g. a strain of Aspergillus sp. especially
A. niger.
Example 1 below describes the cloning and expression of an SBD as being a region in the maltogenic amylase enzyme product produced by
Bacillus stearothermophilus
C599 disclosed in EP patent no. 120,693 (Novo Industri A/S).
In an embodiment of the invention the oral care composition further comprises an enzyme capable of degrading polysaccharides.
In the second aspect, the invention relates to an oral care product comprising an oral care composition of the invention.
In the third aspect, the invention relates to the use of an SBD for oral care purposes, including preventing the formation of dental plaque and/or removing dental plaque.
DETAILED DESCRIPTION OF THE INVENTION
It is the object of the present invention to provide oral care products which can be used for improving the oral hygiene of humans and animals, by effectively preventing the formation of dental plaque and/or removing already deposited dental plaque.
The present inventors have seen indications that Starch Binding Do
Fuglsang Claus Crone
Tsuchiya Rie
Green, Esq. Reza
Lambiris Esq. Elias J.
Novo Nordisk A S
Prouty Rebecca E.
Rao Manjunath
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