The Personal Care field includes Oral Care, Hair Care, Contact Lens Cleaning and Skin Care.
Within the Oral Care field we have developed compositions and products to remove and prevent the formation of dental plaque. The compositions and products include a dextranase and a mutanase and, optionally, other enzymes. In the Hair Care field we have developed low-toxic dye precursors and methods for dyeing. The low-toxic precursors can be used to dye natural and synthetic fibres, includ-ing textiles, thread and yarns, as well as keratinous fibres, in particular hair.
Our technologies within the Contact Lens Cleaning field relate, for example, to cleaning, disinfecting and preserving contact lenses using an enzyme (a protease) that functions as a cleaning agent, an enzyme inhibitor that inhibits further enzyme activity and a mild disinfecting agent.
A few of our patents in the Personal Care field are listed below. If you are interested in any of the patents, please do not hesitate to contact us for more details.
Title of Technology: Polypeptides Having Mutanase Activity
Abstract: This technology relates to polypeptides having mutanase activity and isolated nucleic acid sequences encoding the polypeptides. The technology also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid sequences as well as methods for producing the polypeptides. The present technology further relates to oral cavity compositions and methods for degrading mutan.
The formation of dental plaque leads to dental caries, gingival inflam-mation, periodontal disease, and eventually tooth loss. Dental plaque is a mixture of bacteria, epithelial cells, leukocytes, macrophages, and other oral exudate. The bacteria produce glucans and levans from sucrose found in the oral cavity. These glucans, levans, and microorganisms form an adhesive matrix for the continued proliferation of plaque.
Although mutanases have commercial potential for use as an anti-plaque agent in dental applications and personal care products, 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 commercially useful.
It is an object of the technology to provide new mutanases which can be produced in commercially useful quantities.
Detailed Description: The technology relates to isolated polypeptides having mutanase activity with the amino acid sequence set forth in SEQ ID NO:3 (see US 5,853,702) or a fragment or subsequence thereof which retains mutanase activity.
The polypeptides of the present technology are preferably obtained from species of Penicillium. The polypeptide may also be obtained from teleomorphs of Penicillium, e.g., Eupenicillium and Talaromyces.
The polypeptide may further be obtained from other fungi which are synonyms of Penicillium as defined by Samson and Pitt In Samson and Pitt (eds.), Advances in Penicillium and Aspergillus Systematics, Plenum Press, ASI Series, New York, 1985.
The technology relates also to polypeptides which are encoded by nucleic acid sequences w
hich are capable of hybridizing under high stringency conditions with an oligonucleotide probe which hybridizes under the same conditions with the nucleic acid sequence set forth in SEQ ID NO:2 (see US 5,853702) or its complementary strand.
Furthermore, the technology relates to polypeptides which have an amino acid sequence which has a degree of identity to the amino acid sequence set forth in SEQ ID NO:3 (see US 5,853,702) of at least about 60%, preferably at least about 70%, more preferably at least about 80%, even more preferably at least about 90%, most preferably at least 95%, and even most preferably at least about 97%, which qualitatively retain the mutanase activity of the polypeptides.
The technology also relates to polypeptides having immunochemical identity or partial immunochemical identity to the polypeptide native to Penicillium purpurogenum CBS 238.95. The technology also relates to hybrid or fusion polypeptides, comprising the catalytic domain in-cluded in the amino acid sequence set forth in SEQ ID NO:3 (see US 5,853,702). Moreover, the technology also relates to hybrid or fusion polypeptides, comprising the linker included in the amino acid se-quence set forth in SEQ ID NO:3. Furthermore, the technology also relates to hybrid or fusion polypeptides, comprising the mutan binding domain included in the amino acid sequence set forth in SEQ ID NO:3.
The technology also relates to isolated nucleic acid sequences which encode a polypeptide described in e.g. US 5,853,702. The nucleic acid sequence has a degree of identity to the nucleic acid sequence set forth in SEQ ID NO:2 (see US 5,853,702) of at least about 60.
The present invention also relates to nucleic acid sequences which are capable of hybridizing under hi
gh stringency conditions with an oligonucleotide probe which hybridizes under the same conditions with the nucleic acid sequence set forth in SEQ ID NO:2 (see US 5,853,702) or its complementary strand.
The technology also relates to nucleic acid constructs comprising a nucleic acid sequence of the present invention operably linked to one or more control sequences capable of directing the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The control sequence may be an appropriate promoter sequence, a nucleic acid sequence which is recognized by a host cell for expres-sion of the nucleic acid sequence. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encod-ing the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.
The control sequence may also be a suitable leader sequence, a non-translated region of a mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the polypeptide. Any leader se-quence which is functional in the host cell of choice may be used in the present invention.
The control sequence may also be a polyadenylation sequence, a sequence which is operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence which is functional in the host cell of choice may be used in the prese
nt invention.
The control sequence may also be a signal peptide coding region, which codes for an amino acid sequence linked to the amino terminus of the polypeptide which can direct the expressed polypeptide into the cell's secretory pathway.
The control sequence may also be a propeptide coding region, which codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the pro-polypeptide. The propeptide coding region may be obtained from the Bacillus subtilis alkaline protease gene (aprE), the Bacillus subtilis neutral protease gene (nprT), the Saccharomyces cerevisiae alpha-factor gene, or the Myceliophthora thermophilum laccase gene (WO 95/33836).
The nucleic acid constructs of the present invention may also com-prise one or more nucleic acid sequences which encode one or more factors that are advantageous in the expression of the polypeptide, e.g., an activator (e.g., a transacting factor), a chaperone, and a processing protease.
The technology also relates to recombinant expression vectors com-prising a nucleic acid sequence of the present invention, a promoter, and transcriptional and translational stop signals.
The technology also relates to recombinant host cells, comprising a nucleic acid sequence of the invention, which are advantageously used in the recombinant production of the polypeptides.
The cell is preferably transformed with a vector compri
sing a nucleic acid sequence of the invention followed by integration of the vector into the host chromosome.
The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source. The host cell may be a uni-cellular microorganism, e.g., a prokaryote, or a non-unicellular micro-organism, e.g., a eukaryote. Useful unicellular cells are bacterial cells such as gram positive bacteria.
The host cell may be a eukaryote, such as a mammalian cell, an in-sect cell, a plant cell or a fungal cell. Useful mammalian cells include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or any number of other immortalized cell lines available, e.g., from the American Type Culture Collection.
In a preferred embodiment, the host cell is a fungal cell. In a preferred embodiment, the fungal host cell is a yeast cell.
The technology also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a Penicillium strain to produce a supernatant comprising the polypeptide; and (b) recovering the polypeptide.
The technology also relates to methods for producing a polypeptide of the present invention comprising (a) cultivating a host cell under con-ditions conducive to expression of the polypeptide; and (b) recovering the polypeptide.
The mutanase of the present technology can be used as an anti-plaque agent to degrade mutan produced by Streptococcus mutans in the oral cavity (Guggenheim, 1970, Helv. Odont. Acta 14:89-108). Mutan plays an important role for adhesion and proliferation of bacteria on the surface of teeth and, hence,
may be important in the etiology of dental caries (Kelstrup, 1978, Danish Dental Journal 82:431-437).
The present technology is also directed to oral cavity compositions, particularly dentifrices, comprising the mutanase in an effective amount and a suitable oral carrier for use as an antiplaque agent in dental applications and personal care products. The compositions of the present technology can be made using methods which are common in the oral product area. Dentifrices are compositions used in conjunction with a toothbrush to remove stains from teeth and to leave the mouth feeling clean and refreshed after brushing. Dentifrices are also used to deliver agents with specific therapeutic and cosmetic functions. Examples of personal care products include, but are not limited to, toothpaste, toothgel, mouthwash, chewing gum, and denture cleaners.
The composition ingredients will vary depending on the particular product (Kirk-Othmer, John Wiley & Son, New York). Examples of ingredients include, but are not limited to, an abrasive, a humectant, a surfactant, an emulsifier, a colloid, a chelating agent, an adhesive, one or more gums or resins for cohesiveness and structure, one or more flavor agents, color, a preservative and active agents for specific effects (e.g., fluoride and whiteners). Mouthwashes can deliver active agents that cannot be provided by toothpaste because of chemical incompatibilty between the agent and the toothpaste ingredients. For example, sodium fluoride, calcium-containing abrasives, sodium lauryl sulfate, and chlorhexidine are incompatible.
The technology is also directed to a method for degrading mutan in an oral cavity comprising applying to the oral cavity an effective amount of the compositions of the present invention. The compositions of the present technology can
be applied in a dry, paste, gum, or liquid form. The composition may be a concentrate which requires dilution with a suitable quantity of water or other diluent before application. The con-centrations of each component in the composition will vary depending on the use and method of application. The mutanase concentration will vary depending upon the nature of the particular composition, specifically, whether it is a concentrate or to be used directly. After application, the composition is then allowed to remain in contact with the tissues of the oral cavity for a period of time ranging from about 15 seconds to about 12 hours until removed by rinsing or brushing. Al-ternatively, the composition may be left indefinitely until the composi-tion is removed by a mechanical process, e.g., drinking liquid or chewing food.
Glossary of Terms
| Term: |
Definition: |
| Nucleic acid construct |
A nucleic acid molecule, either single- or double-stranded. The term nucleic acid construct may be synonymous with the term expression cassette when the nucleic acid construct contains all the control sequences required for expression of a coding sequence of the present invention. |
| Coding sequence |
The term coding sequence as defined herein is a sequence which is transcribed into mRNA and translated into a polypeptide of the present invention when placed under the control of the above mentioned control sequences. nucleic acid sequences. |
| Isolated |
The term isolated nucleic acid sequence as used herein refer
s to a nucleic acid sequence which is essentially free of other nucleic acid sequences, e.g., at least about 20% pure preferably about 90% pure, and even most preferably about 95% pure, as determined by agarose gel electrophoresis |
| Control sequences |
The term control sequences is defined herein to include all components which are necessary or advantageous for expression of the coding sequence of the nucleic acid sequence. |
| Activator |
An activator is a protein which activates transcription of a nucleic acid sequence encoding a polypeptide |
| Chaperone |
A chaperone is a protein which assists another polypeptide in folding properly . |
| Processing protease |
A processing protease is a protease that cleaves a propeptide to generate a mature biochemically active polypeptide endoprotease. |
| Host cell |
The term host cell encompasses any progeny of a parent cell which is not identical to the parent cell due to mutations that occur during replication. |
| Transformation |
Transformation means introducing a vector comprising a nucleic acid sequence of the present invention into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extrachromosomal vector. |
| Fungi |
Fungi as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota |
| Yeast |
Yeast
as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The ascosporogenous yeasts are divided into the families Spermophthoraceae and Saccharomycetaceae. |
| Effective amount |
Effective amount is defined herein as a sufficient amount of the mutanase to reduce plaque. |
| Suitable oral carrier |
Suitable oral carrier is defined herein as a suitable vehicle which can be used to apply the compositions of the present invention to the oral cavity in a safe and effective manner. |
In general hair dyeing compositions on the market today can be divided into three main groups:
The temporary hair dyes are only intended to change the natural hair colour for a short period of time and usually functions by depositing dyes on the surface of the hair. Such hair dyes are easy to remove with normal shampooing.
When using semi-permanent hair dyes the colour of the dyed hair can survive for five or more shampooings. This is achieved by using dyes having a high affinity for hair keratin and which is able penetrate into the interior of the hair
Permanent hair dyes are very durable to sunlight, shampooing and other hair treatments and need only to be refreshed once a month as new hair grows out. With these dyeing systems the dyes are created directly in and on the hair. Small aromatic co
lourless dye precursors (e.g. p-phenylene-diamine, o-aminophenol, o-phenylendiamine (OPD)) penetrate deep into the hair where said dye precursors are oxidised by an oxidising agent into coloured polymeric compounds. These coloured compounds are larger than the dye precursors and can not be washed out of the hair.
By including compounds referred to as modifiers (or couplers) in the hair dyeing composition a number of hair color tints can be obtained. Cathecol and Resorcinol are examples of such modifiers.
Some of the today most widely used dye precursors such as OPD are known to be both mutagenic and carcinogenic.
The use of H2O2 in dyeing compositions have some disadvantages as H2O2 damages the hair. Further, oxidative dyeing often demands high pH (normally around pH 9-10), which also inflicts damage on the hair and on the skin. Consequently, if using dye compositions comprising H2O2 it is not recommendable to dye the hair often.
To overcome the disadvantages of using H2O2 it has been suggested to use oxidation enzymes to replace H2O2.
The aim of the technology is to use the present findings to make available a substrate which to all intents and purposes is non-toxic, non - mutagenic and/or non-carcinogenic and which may be used in immuno - chemical assays, for the dying of keratinous fibers, in particular hair and for dying both natural and synthetic fibers, e.g.
textiles.
In another aspect the technology relates to a method for quantitative and/or qualitative analysis of a material of biological interest. In this case a peroxidase enzyme together with a marker is bound to the compound in question. Hydrogen peroxide is then converted with a cromogenetic substrate (i.e. color forming compound) in the presence of the peroxidase, the substrate includes a bond with the general formulae.
The colored product produced by the method of the invention is especially suited to the dying of textiles, thread, yarn, wool, hides and skins and human hair. Other natural fibres such as cotton and silk may also be dyed with the product, as may synthetic fibres such as polyamides, polyurethane and polyester.
The colored product may either be made immediately before it is to be used for dying or it may be synthesized in the immediate vicinity of the substance to be dyed. For example this may be done by mixing the substrate and the oxidation system in a person's hair.
The dying process may be carried out rinsing the person's hair with a mixture of the substrate of the invention and hydrogen peroxide or an oxidation enzyme. A peroxidase is then added and distributed in the hair. When the desired degree of coloring has been obtained the hair is rinsed with water.
The substrate may be mixed with the oxidation system before it is applied to the hair. As stated above the substrate may be oxidised with hydrogen peroxide or an oxidation enzyme generating hydrogen peroxidase in the presence of a peroxidase.
A preferred use of the composition is as a permanent dye for the dyeing of human hair.&nb
sp;Preferred dye precursors (i.e. substrates) are benzoic acid esters, especially diamino benzoic acid esters, in particular 3,4-diamino benzoic acid methyl ester (DABA-Me), 3,4-diamino benzoic acid ethyl ester and 3,4-diamino benzoic acid isopropyl ester.
In a further aspect the technology relates to a method for dyeing keratinous fibers, in particular hair, fur, hide and wool, using a composition as described above. The dyeing method can be conducted with one or more dye precursors (i.e. substrates of the invention) and optionally in combination with one or more modifiers.