Cellulose binding domains (CBDs) for oral care products

Details Cellulose binding domains (CBDs) for oral care products Cellulose binding domains (CBDs) for oral care products

1999-04-08

2001-07-24

Rose, Shep K. (Department: 1614)

Drug, bio-affecting and body treating compositions

Dentifrices

Ferment containing

C424S048000, C424S440000, C424S441000, C424S464000, C424S465000, C424S489000

Reexamination Certificate

active

06264925

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to an oral care composition comprising a
C
ellulose
B
inding
D
omain, an oral care product comprising an oral care composition of the invention, and further to the use of a Cellulose 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 micro-organisms 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 micro-organisms, 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 a-1,6 glucosidic linkage and as the major component a water-insoluble extra-cellular polysaccharide called “mutan” comprised of a backbone with a-1,3-glycosidic linkages and branches with a-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. Said material may be an enzyme, such as galactose oxidase (see Example 6 of said PCT 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 described 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 found that
C
ellulose
B
inding
D
omains (CBDs) have an dispersing effect on oral polysaccharides. Consequently, Cellulose Binding Domains are suitable for removing and/or preventing dental plaque.
In the following the abbreviation “CBD” will be used for “
C
ellulose
B
inding
D
omain”.
Cellulose Binding Domain (CBD)
A CBD is a polypeptide which has high affinity for or binds to water-insoluble forms of cellulose and chitin, including crystalline forms.
CBDs are found as integral parts of large protein complexes consisting of two or more different polypeptides, for example in hydrolytic enzymes (hydrolases) which typically are composed of a catalytic domain containing the active site for substrate hydrolysis, and a Carbohydrate Binding Domain or Cellulose Binding Domain (CBD) for binding to the insoluble matrix. Such enzymes can comprise more than one catalytic domain and one, two or three CBDs and optionally one or more polypeptide regions linking the CBD(s) with the catalytic domain(s), the latter regions usually being denoted a “linker”. Examples of hydrolytic enzymes comprising a CBD are cellulases, xylanases, mannanases, arabinofuranosidases, acetyl esterases and chitinases. CBDs have also been found in algae, e.g. the red alga
Porphyra purpurea
as a non-hydrolytic polysaccharide binding protein, see Peter Tomme et al. “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, 1996. However, most of the known CBDs are from cellulases and xylanases.
In this context, the term “Cellulose Binding Domain” is intended to be understood as defined by Tomme et al., op. cit. This definition classifies more than 120 Cellulose Binding Domains into 10 families (I-X) which may have different functions or roles in connection with the mechanism of substrate binding. However, it is anticipated that new family representatives and additional CBD families will appear in the future.
In the protein complex, typically a hydrolytic enzyme, a CBD is located at the N or C termini or is internal.
A monomeric CBD typically consists of more than about 30 and less than about 250 amino acid residues. For example, a CBD classified in Family I consists of 33-37 amino acid residues; a CBD classified in Family IIa consists of 95-108 amino acid residues; and a CBD classified in Family VI consists of 85-92 amino acid residues. Accordingly, the molecular weight of a monomeric CBD will typically be in the range of from about 4 kD to about 40 kD, and usually below about 35 kD.
CBDs may be useful as a single domain polypeptide (single unit CBD) or as a dimer, a trimer, or a polymer; or as a part of a protein hybrid.
Single Unit Cellulose Binding Domain (single unit CBD)
The term “Single Unit CBD” may also be referred to as “Isolated CBD” or “Separate CBD”.
In the context of the present invention a “Single Unit CBD” includes up to the entire part of the amino acid sequence of a CBD-containing enzyme, e.g. a polysaccharide hydrolysing enzyme, being essentially free of the catalytic domain, but retaining the CBD(s).
Thus, in the context of the invention, the entire catalytic amino acid sequence of a cellulolytic enzyme (e.g. a cellulase) or other enzymes comprising one or more CBDs is not to be regarded as a Single Unit CBD.
Typically a Single Unit CBD constitutes one or more CBDs of a polysaccharide hydrolysing enzyme, one or more CBDs of a cellulose binding protein or a protein designed and/or engineered to be capable of binding to cellulosic carbohydrates.
The Single Unit CBD is at least as large as the minimum number of amino acids in the amino acid sequence required to bind to cellulosic carbohydrates.
A Single Unit CBD may also be an amino acid sequence in which the binding and catalytic domain are one and the same.
Cellulases useful for preparation of Cellulose Binding Domains
The techniques used in isolating a cellulase gene are well-known in the art.
In the present context, the term “cellulase” refers to an enzyme which catalyses the degradation of cellulose to glucose, cellobiose, triose and other cello-oligosaccharides.
Preferably, the cellulase is a microbial cellulase, more preferably a bacterial or fungal cellulase.
Examples of bacterial cellulases are cellulases derived from or producible by bacteria from the group consisting of Pseudomonas, Bacillus, Cellulomonas, Clostridium, Microspora, Thermotoga, Caldocellum and Actinomycets such as Streptomyces, Termomonospora and Acidothemus, in particular from the group consisting of
Pseudomonas cellulolyticus, Bacillus lautus, Cellulomonas fimi, Microspora bispora, Termomonospora fusca, Termomonospora cellulolyticum
and
Acidothemus celluloly

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