Cress-Reference to Related Application

|O001J This application claims priority to U.S. Provisional Application Serial No. 61/712,913, filed 12 October 20.12, entitled "Thermally Stable Alginate Lyase", the entirety of which is hereby incorporated herein in its entirety.

Background

[00021 Polysaccharide lyases degrade anionic polysaccharides that are important in bacterial biofiim formation and host-pathogen interactions.

{0003} Stenotrophomonas m ltophili is a ubiquitous, gram-negative bacteria that has become an increasingly important nocosomial pathogen in immunocompromised patients. Approximately 1 % of all nosocoma! bacteremias are due to S. maltophilia infection, with the attributed mortality rate at nearly 28%, which places it among the highest attributed mortality rates observed for nosocomal bacteremias. A major reason for the high mortality associated with Λ ^' . maltophilia is its nearly universal resistance to broad-spectrum antibiotics such as imipenem and aminoglycosides, and increasing resistance to ' last-line' cephelasporin antibiotics such as ceftazidime and cefotaxime.

(0004| One unique mechanism that . maltophilia has evolved to become multi-drug resistant (MDR) is its production of highly branched, anionic exopoiysaccharides (EPS) to form biofilms. Biofilms are comprised of secreted, high molecular weight EPS such as alginic acid, which encapsulate a population of bacterial cells to create a 'niche' environment that is often impermeable to antimicrobials, detergents or other organic compounds.

[0Q05] While several pathogenic bacteria are capable of producing alginic acid biofilms., .V. maltophilia is unique in thai the EPS that comprise the biofiim are highly anionic due to the addition of branched poiy-D-galaeturonic acid (HexA)-ethyl-D-lactate (Lac) chains. Recent studies indicate the additional negative charge of S. maltophilia EPS due to the branched HexA- Lac group enables biofiim binding to metallic or plastic surfaces, as well as creating a heat-, acid- and detergent-resistant environment that, allows S. maltophilia to colonize water purification systems, ventilators and stents. As a result, S. maltophilia is the 2 ^ND leading cause of ventilator-associated pneumonia, with over 70% of ail 5. maliophilta-specifiiC, venti!ator-- associated MDR infections caused by hiofsim forming strains..

fOO06| In iofilm-positive MDR infections, surgery is the main option to either remove infected lung tissue or perforra a lung transplant, although both are often not feasible since the patient is immunocompromised and therefore ai greater risk of additional infections. This is especially a problem for cystic fibrosis (CF) patients, in which accumulation of mucin in the lungs creates a viscous, damp environment in which biofilra-producing S. maltophilia can. thrive. & maltophilia is the 2" ^d most common cause of chronic infection in CF patients after P. aeruginosa, with estimates as high as 20% of all MDR infections. Thus, biofilm formation plays a central role in the pathogenicity of S. maltophilia, and strategies targeting EPS degradation will enabie more effective treatments to prevent chronic S. maltophilia infection, particularly in CF and immunocompromised patients.

[0007J Embodiments of the present invention are directed to these and other ends.

Summary

Θ008| In some embodiments, the present invention provides a protein comprising an amino sequence selected from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7.

| 0 ⁽ >9| In some embodiments, the present invention provides a protei comprising an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; or SEQ ID NO: 7.

[OOlOj Some embodiments of the present invention provide a composition comprising: a protein comprising an amino sequence selected from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4;

SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7; and a carrier,

00I1| Other embodiments of the present invention provide a. protein having a signature motif that aligns with a reference sequence SEQ ID NO; 2, wherein the signature motif comprises 1 7, HI 68, R2I 5, Y222 residues.

|00 ^' I2| In some embodiments, the present invention provides method of preventing, treating or inhibiting a disease, disorder or condition selected from: a urinary tract infection, a catheter- originated infection, a midd!e-ear infection, dental plaque, gingivitis, an ey infection, endocarditis, cystic fibrosis, and nosocomial infections, comprising administering an effective

7 amount of a composition comprising an amino acid sequence selected from SEQ ID NO: 2; SEQ

ID NO; 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7, to a patient in need thereof.

|Θ0!3| Still further embodiments of the present invention provide methods of cleaning, disinfecting or sterilizing an inert surface. In some embodiments, the inert surface is selected from a prosthetic device, a ventilator, a catheter and an intrauterine device.

f 00141 Yet further embodiments, provide methods of producing a biotuel comprising contacting a bydrolysaie with a effective amount of a protein comprising an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7.

Brief Description of the Biological Sequences

{0015) The following sequences comply with 37 C.F.R. §§ 1.821 - 1.825 ("Requirements for

Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures - the Sequence Rules") and are consistent with World ntellectual Property Organization (WIPO) Standard ST.25 (2009 ) and the sequence listing requirements of the European Patent Convention (EPC) and the Patent Cooperation Treaty (PCT) Rules 5.2 and 49.5(a-his), and Section 208 and Annex C of the Administrative Instructions. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. § 1.822,

|Θ016| SEQ ID NO: 1 is the codon-opiimized nucleotide sequence of a polysaccharide lyase from Stenotrophomonm mahophUia (Smlt 1473).

0017| SEQ ID NO; 2 is the amino acid sequence of a polysaccharide lyase from Stenoirophomonm maltophiUa.

|6β18| SEQ ID NO: 3 is the amino acid sequence of a Yl 15F mutant of SEQ ID NO: 2.

{00191 SEQ I D NO: 4 is the ammo acid sequence of a W1.7 I A mutant of SEQ ID NO: 2.

100201 SEQ ID NO; 5 is the amino acid se uence of a H223 F mutant of SEQ ID NO: 2.

002i) SEQ ID NO 6 is the amino acid sequence of a Y225F mutant of SEQ ID NO: 2.

[0022 | SEQ ID NO: 7 is the amino acid sequence of a 167L mutant of SEQ ID NO; 2,

Brief Description of the Drawing

(0023 j Figure 1 (A-D) depicts the pH dependent activity of an exemplary protein of the present invention, against various substrates.

Detailed Description

|00241 used herein, "contacting" refers to placing a composition in contact with the target body surface for a period of time sufficient to achieve the desired result. In some embodiments, "contacting" may refer to placing a composition comprising (or capable of producing) an efficacious concentration of a lyase, as described herein, in contact with a target body surface for a period of time sufficient to achieve the desired result. Contacting includes spraying, treating, immersing, flushing, pouring on or in, mixing, combining, painting, coating, applying, affixing to and otherwise communicating a lyase solution or a composition comprising an efficacious concentration of lyase, a solutio or composition that forms an efficacious concentration of lyase or a component of the composition that forms an efficacious concentration of lyase with the body surface.

|0025| As used herein, an "isolated nucleic acid molecule", "isolated polynucleotide", and "isolated nucleic acid fragment" may be used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid molecule in the form of a. polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

f0026| As used herein, the term "amino acid" refers to the basic chemical structural unit of a protein or polypeptide. Table .1 (below) provides a list of abbreviations used herein to identify specific amino acids;

Table 1

|ft027| It is well known in the art that alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not affect the functional properties of the encoded protein are common. For the purposes of the present invention substitutions are defined as exchanges within one of the following five groups:

L Small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly);

2. Polar, negatively charged residues and their amides; Asp, Asn, Glu, Gin;

3. Polar, positively charged residues: His, Arg, Lys;

4. Large aliphatic, nonpolar residues; Met.. Leu, lie, Val (Cys); and

5. Large aromatic residues; Phe, Tyr, and Trp.

|0028| Thus, a eodon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a eodon encoding another less hydrophobic residue (such as glycine) or a more hydrophobic residue (such as valine, leucine, or isoleucine). Similarly, changes which result in substitution of one negatively charged residue for another (such as aspartic acid for glutamic acid) or one positively charged residue for another {such as lysine for arginine) can also be expected to produce a functionally equivalent product. In many cases, nucleotide changes which result in alteration of the -terminal and C-terminal portions of the protein raoiecuie would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products.

j0029| As used herein, the terms "signature motif and "diagnostic motif refer to conserved structures shared among a family of enzymes having a defined activity. The signature motif can be used to define and/or identify the family of struciurally-rekted enzymes havins similar enzvmatic activity for certain substrates. The signature motif can be a sinele contiguous amin acid sequence or a collection of discontiguous, conserved motifs or residues that together form the signature motif.

[6036} As used herein, the term "eodon optimized", as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflec t the typic al eodon usage of the host organism without altering the polypeptide for which the DNA codes. As used herein, "synthetic genes" can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art These building blocks are ligated and annealed to form gene segments that are then enzymatically assembled to construct the entire gene.

(0031| As used herein, the term "chemically synthesized", as pertaining to a DNA sequence, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well-established procedures, or automated chemical synthesis can be performed using one of a number of commercially a vailable machines. Accordingly, the genes can be tailored tor optimal gene expression based on optimization of nucleotide sequences to reflect the codon bias of the host ceil. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

(0032) As used herein, "pharmaceiitically-acceptable" means thai drags, medicaments and/or inert ingredients which the term describes are suitable for use in contact with the tissues of humans and other animals without undue toxicity, incompatibility, instability, irritation, allergic response, and the like, commensurate with a reasonable benefit risk ratio.

(0033) As used herein, the terms "pharmaceutically acceptable carrier", "orally acceptable carrier" and "cosmetically acceptable carrier" may have overlapping scope. However, one skilled in the art would appreciate that some ingredients suitable for use in a "pharmaceutically acceptable carrier' ⁵ or a "cosmetically acceptable carrier" may not be suitable for use in an "orally acceptable carrier".

(0034) Compositions envisioned by the present application include oral care compositions, persona! care compositions (e.g., skin care, hair care and ocular compositions), and pharmaceutical compositions (topical and systemic).

(0035) As used herein, the term "oral care composition" refers to a composition for which the intended use can include oral care, oral hygiene, or oral appearance, or for which the intended method of use can comprise administration to the oral cavity.

(0036) As used herein, an "orally acceptable carrier' and "orally acceptable vehicle" are used interchangeably, and refer to a material or combination of materials that is safe for use in the oral cavity, commensurate with a reasonable benefit/risk ratio, with which the other ingredients may be associated while retaining efficacy. Such carrier materials should be selected for compatibility with the other ingredients of the compositions, and preferably do not substantially reduce the efficac of the other ingredients. Selection of specific carrier components is dependent on the desired product form, including dentifrices, rinses, gels, and paints.

{O037J Materials useful in an "orally acceptable carrier" include, bat are not limited to: adhesion agents, viscosity modifiers, diluents, surfactants, foam modulators, peroxide activators, peroxide stability agents, abrasives, pH modifying agents, humectants, mouth feel agents, sweeteners, fiavorants, colorants, and combinations thereof

(O038J As used herein, an "adhesion agent" is a material or combination of materials that enhances the retention of an ingredient to a oral cavity surface onto which it is applied. Such adhesion agents include without limitation: adhesives, film forming materials and viscosity enhancers; for example, hydrophi ic organic polymers, hydrophobic organic polymers, silicone gums, silicone adhesives, silicas, and combinations thereof.

f003 | As used herein, the term "personal care composition" refers to a composition for which the intended use can include promotion or improvement of health, cleanliness, odor, appearance, or attractiveness of skin.

(00401 Materials useful in a "cosmetically acceptable carrier" include, but are not limited to an ingredient selected from a surfactant, a conditioning agent, a moisturizer, an enzyme or other protein, a vitamin, a colorant, a denaCuram, a film f rming polymer, an antidandruff agent, a fragrance, an alcohol, an anticholinergic, an antipe spirant salt (such as aluminum and zirconium salts).

(00411 The compositions described herein may be adapted for oral, parenteral, sublingual, nasal or transdermal administration. The compositions may be in the form of tablets, capsules, powders, granules, lozenges, suppositories, reconstitutable powders, or liquid preparations, such as oral or sterile parenteral solutions or suspensions.

(0042| Unit dose presentation forms for oral administration may be tablets and capsules and may contain conventional exeipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesia stearaie; disintegTants, for example starch, polyvinylpyrrolidone, sodium starch giycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulphate. |ft043| In some embodiment suitable lyases may include enzymes comprising an amino acid sequence having at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the amino acid sequences reported herein.

[0044] In some embodiments, suitable isolated nucleic acid molecules encode a protein having an amino acid sequence that is at least about 20%, preferably at least 30%, 33%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences reported herein. Suitable nucleic acid molecules not only have the above homologies, but also typically encode a protein having about 210 to 380 amino acids in length, about 300 to about 360 amino acids, preferably about 310 to about 350 amino acids, and most preferably about 320 to about 335 amino acids in length wherein each protein is characterized as having lyase activity.

[0045] As used herein, the term "body surface" refers to any surface of the human body that may serve as the target tor a lyase benefit agent In some embodiments, the proteins described herein have an affinity for a particula ^' body surface.

|Θ046| As used herein, the term "effective amount" refers to the quantity of an amino acid sequence or composition necessary to achieve the enzymatic activity required i the specific application. Such effective amounts are readily ascertained by one of ordinary skill in the art and are based on many factors, such as the particular enzyme variant used, the intended use or application, the specific composition for the intended use or application, and the dosage form in which the enzyme is prepared (e.g. solid, liquid, etc.).

[0047] As used herein, the term "mutant" or "variant" refers to an enzyme having a genetic modification that results in at least one amino acid addition, deletion, and/or substitution when compared to the corresponding enzyme (typically the wild type enzyme) from which, the variant was derived; so long as the signature motif and the associated lyase activity are maintained. Examples of variants are provided as SEQ ID NOs: 3, 4, 5, 6, and 7.

[0048] As the skilled artisan will recognize, substantially similar sequences (retaining the signature motifs) to those described herein may also be used i the present compositions and methods. In some embodiments, the substantially similar sequences are defined by their ability to hybridize, under highly stringent conditions with the nucleic acid molecules associated with sequences exemplified herein, in some embodiments, sequence alignment algorithms ma be used to define substantially similar enzymes based on the percent ideality to the nucleotide or amino acid sequences provided herein.

f0049| As used herein, the term "percent identity" refers to the relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences, in the art, "identity" a!so means the degree of sequence relateduess between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences, ' dentity" can be readily calculated by known methods, included but not limited to those described in: Computational Molecular Biology (Lesk, A.M., ed.) Oxford University Press, NY (1 88); Biocomptsting: Informatics and Genome Projects (Smith, D.W,, ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data. Part I (Griffin, A.M., and Griffin, H.G., eds.) Humana Presss, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1 87); and Sequence Anal.ysi s P imer (Gribskov, M. and Devereax, Eds.) Stockton Press, NY f 1991 ).

j0O5O| Hyaluronic acid (HA) is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid. Hyaluronic acid is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with hyaluronic acid creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. Hyaluronic acid plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis. erabryological development, wound healing, angiogenesis, and tumorigenesis (Toole 1991 Cell Biol, ExtraceH. Matrix, Hay (ed). Plenum Press, New York, 1384-1386; Berirand et al. 1992 int. I. Cancer 52: 1-6; Knndaon et al, 1993 FASEB J. 7: 1233-1241 ). in addition, hyaluronic acid levels correlate with tumor aggressiveness (Ozello et al. I960 Cancer Res. 20:600-604; Takeuchi et al. 1976, Cancer Res. 36:2133-2139; Kimata et al. 1983 Cancer Res. 43:1347-1354).

(00511 Hyaluronic acid is found in the extracellular matri of many cells, especially in soft connective tissues. Hyaluronic acid has been assigned various physiological functions, such as in water and plasma, protein homeostasis (Laurent TC et al (1992) FASEB J 6: 2397-2404). Hyaluronic acid production increases in proliferating cells and may play a role in mitosis. It has also been implicated in locomotion and ceil migration. Hyaluronic acid seems to play important roles in ceil regulation, development, and differentiation (Laurent et al, supra). |ft052| Hyaluronic acid has been used in clinical medicine. Its tissue protective and Theological properties have proved useful in ophthalmic surgery to protect the corneal endothelium during cataract surgery. Serum hyaluronic acid is diagnostic of liver disease and various inflammatory conditions, such as rheumatoid arthritis. Interstitial edema caused by accumulation of hyaluronic acid may cause dysfunction in various organs {Laurent et al, supra).

f O053| Some embodiments of the present invention provide a protein comprising an amino sequence selected from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7.

[0054) Some embodiments of the present invention provide a protein having lyase activity comprising an amino sequence selected from SEQ ID NO; 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7.

[0055] hi some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO; 3. in some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 4. in some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the protein comprises the amino acid sequence of SEQ ID NO: 7.

[0056) Some embodiments of the present invention prov ide a protein comprising a amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2,

[0057] Some embodiments of the present invention provide a protein having lyase activity comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 2.

[0058) Some embodiments of the present invention provide a protei comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 3.

[0059) Some embodiments of the present invention provide a protein having lyase activity comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 3. f §060| Some embodiments of the present invention provide a protein comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4.

10061) Some embodiments of the present invention provide a protein having lyase activity comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 4.

{0062) Some embodiments of the present invention provide a protein comprising an amino acid sequence having at least 50%. 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 5.

100631 Some embodiments of the present invention provide a protein having lyase activity comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence .identity to SEQ ID NO: 5.

[0064) Some embodiments of the present invention provide a protein comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 6.

0065) Some embodiments of the present invention provide a protein having lyase activity comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 6.

(0066| Some embodiments of the present invention provide a protein comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 7.

[0067J Some embodiments of the present invention provide a protein having lyase activity comprising an amino acid sequence having at least 50%, 75%, 80%, 85%, 90%, 95% or 99% sequence identity to SEQ ID NO: 7.

{0068} Further embodiments of the present invention provide a protein comprising a. signature motif comprising N1 7, HI 68, R215, Y222 residues.

[0069J Still further embodiments of the present invention provide a protein comprising a signature motif consisting essentially of 67, H.I 68, R2.15, Y222 residues.

|OO70| Yet further embodiments of the present invention provide a protein comprising a signature motif consisting of N.167, H I 68, R215, Y222 residues. [0071] Other embodiments of the present invention provide a protein having lyase activity comprising a signature motif comprising N 167, HI 68, R2I5, Y222 residues.

|0072| Still other embodiments of the present invention provide a protein having lyase activity comprising a signature motif consisting essentially of N 1 7, HI 68, R2I5, Y222 residues.

[00731 While other embodiments of the present invention provide a protein having lyase activity comprising a signature motif consisting of N I67, HI 68, 215, Y222 residues.

[0074 f Some embodiments of the present invention provide a protein having a signature motif that aligns with a reference sequence SEQ ID NO; 2, wherein the signature motif comprises

N1 7, H168, R235, Y222 residues.

[00751 Yet other embodiments of the present invention provide a protein having lyase activity having a signature motif that aligns with a reference sequence SEQ ID NO: 2, wherein the signature motif comprises N.167, H 68, R21 S, Y222 residues.

[0076J In some embodiments, the present invention provides a composition comprising: a. protein comprising an amino sequence selected f om SEQ ID NO: 2; SEQ ID NO: 3; SEQ I D NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7; and a carrier.

[0077] 1» some embodiments, the present invention provides a composition comprising: a protein having lyase activity comprising an amino sequence selected from SEQ ID NO: 2; SEQ ID NO; 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7; and a carrier.

[0078] In some embodiments, the carrier is selected from a pharmaceutically acceptable carrier; an orally acceptable carrier; and a cosmetically acceptable carrier. In some embodiments, the carrier comprises a poiyol. in some embodiments, the polyol is selected from selected from a dioi; a triol; and other alcohols containing multiple hydroxy! groups, and a combination of two or more thereof. In some embodiments, the polyol is selected from glycerin; glycerol; polyethylene glycol; polypropylene glycol; isomalt; lactitoS; maltitoi; mannitol; sorbitol; xylitol; and combination of two or more thereof, in some embodiments, the polyol comprises glycerin.

|0079| h* some embodiments, the carrier further comprises a chelating agent, in some embodiments, the chelating agent is selected from ethyienediaminetetraacetic acid (EDTA); citric acid, phosphono-acetic acid (PAA), and nitroiotriacetic acid (NTA). In some embodiments, the chelating agent comprieses a calcium chelating agent. In some embodiments, the chelating agent comprises EDTA. |ft08 | In some embodiments, the carrier may comprise a detergent, In some embodiments, the detergent is selected from: octyl glycoside; laarytdimethylamme N-oxide (LDAO) n-Decyl-β- D~thiomaltoside (Anapoe C ¾); and a polysorbate (e.g. Tsveen 20).

|0081| In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at, temperatures in excess of 20 deg. C. In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at temperatures in excess of 25 deg. C. In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at temperatures in excess of 30 deg. C. In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at temperatures in excess of 35 deg. C. In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at temperatures in excess of 40 deg. C. In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at temperatures in excess of 45 deg. C. In some embodiments of the present invention, the proteins described herein are capable of maintaining their activity at temperatures in excess of 50 deg. C.

|Θ082| In some embodiments, the compositions described herein are formulated to maintain enzyme activity at temperatures of from about 0 deg. C to about 70 deg. C.

[0-983J In some embodiments, the compositions described herein are formulated to have pH~ selective activity. In some embodiments, the pH-selective activity allows the composition to target specific sites on a body surface, in some embodiments, the pH-selective activity allows the composition to target specific sites within the body.

f0084J In some embodiments, the present invention provides a nucleic acid molecule comprising SEQ ID NO: 1.

{0Ο85| Some embodiments of the present invention provide a method of producing a bio uei comprising contacting a hydrolysate with an effective amount of a protein having lyase activity comprising an amino acid sequence selected from SEQ ID NO: 2; SEQ ID ^' NO: 3; SEQ ID NO; 4; SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO; 7.

[0086f In some embodiments, the hydrolysate is derived from an. extract of a seaweed selected from the group consisting of red. algae, brown algae, green alga and a combination thereof. In some embodiments, the red algae comprise laver, agar-agar, sea string, and Grateioupiaceae. In some embodiments, the brown algae comprise sea mustard, laminaria, seaweed fitsiforme, gulfweed, Ecklonia sfo mfer , rhubarb, and Poiamogekm ax phyllus. hi some embodiments, the green algae comprise green laver, sea lattuce, Monastroma niiidum, and sea staghorn.

f008?| hi some embodiments, the hydrolysate is derived trom an extract of a seaweed selected from the group consisting of red algae, brown algae, green algae and combination thereof the red algae comprises laver, agar-agar, sea string, and Grateloupiaceae, the brown algae comprises sea mustard, laminaria, seaweed fusiforme, gulfweed, Ecklonia stolonifera, rhubarb, and Potamogeton oxyphyllm, and the green algae comprises green laver, sea lattuce, Monaslroma niihiwn., and sea staghorn.

|0088| In some embodiments, the extract comprises a red algae extract selected trom among agar, cellulose, carrageenan, xylan, and raannan, a green algae extract selected trom among cellulose, xylan, mannan, starch, fractan, and pararnylon, or a brown algae extract selected from among cellulose, alginate, fucoidan and lammaran.

[0089J In some embodiments, the hydrolysate is a monosaccharide, a furan compound or an organic acid. In some embodiments, the hydrolysate comprises a compound selected from galactose, glucose, xylose, mamiose and a combination thereof when the extract from red algae is hydtoiyzed. In some embodiments, the hydrolysate comprises a compound selected from, glucose, xylose, mannose, fructose and a combination thereof when the extract from green algae is hydrolyzed. In some embodiments, the hydrolysate comprises a compound selected from glucose, glueronie acid, fueose, galactose, xylose, manoitol and a combination thereof when the extract from brown algae is hydro!yzed.

{009β| hi some embodiments, the biofuel comprises an oxygen-containing compound or a biohydrocarbon. In some embodiments, the oxygen-containing compound is selected from ethanoi, propano!, butanof, peotanol, hexanol and a combination thereof. In some embodiments, the biohydrocarbon is selected from biogaso ^' lme, biodiesel, a jet fuel, an additive and a combination thereof

[0O9IJ Still further embodiments provide a method of cleaning, disinfecting or sterilizing an inert surface, comprising contacting the inert surface with an effective amount of a protein having lyase activity comprising an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID MO: 6; and SEQ ID NO; 7.

|0G92J in some embodiments, the inert surface is selected from a prosthetic device; a ventilator, a catheter and an intrauterine device. {§093| Yet other embodiments provide a method of treating, preventing or inhibiting a disease, disorder or condition marked by the presence of biofilm, comprising administering an effective amount of a protein having lyase activity comprising an amino acid sequence selected from SEQ ID NO: 2; SEQ ID HO: 3; SEQ ID NO: 4: SEQ ID NO: 5; SEQ ID NO: 6: and SEQ ID NO: 7, to a body surface of a patient in need thereo

[0094 j In some embodiments, the disease, disorder or condition marked by the presence of biofilm is selected from a urinary tract infection, a catheter-originated infection, a middle-ear infection, dental plaque, gingivitis, an eye infection, endocarditis, cystic fibrosis, and a nosocomial infection.

[00951 In some embodiments, the present invention provides a protein comprising an amino acid sequence selected from SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4: SEQ ID NO; 5; SEQ ID NO: 6; and SEQ ID NO: 7, wherein the protei is derived from a bacterial genus selected from Pseudomonas, Ach omobacter, Bordeteila, Ulfginosibacferhtm and Duganella.

{6096) In some embodiments, the present invention provides a protein comprising N1 7, HI 68, R215, Y222 residues, wherein the protein is derived from a bacterial genus selected from Pseudomonas, Achromobaeter, Bordeteila, Uliginosibaclerium and Duganella. in some embodiments, the nucleic acid sequence of SEQ ID NO; 1 is derived from a bacterial genus selected, from Pxeudotmmas, Achromobaeter, Bordeteila, (lliginosibacieriwn and Duganella. (0697) In some embodiments, the present invention provides peptides which have an affinity for a specific target site. For example, skin, oral cavity (soft tissue and hard surfaces, e.g. teeth), a binding protein to a particular extracellular matrix protein or ligand for a cell-surface receptor, and the like. In some embodiments, a peptides which has an affinity for a specific target site is referred to as a "binding peptide''. In some embodiments, a binding peptide is fused to any one of the amino acid sequences described herein.

[0098j in some embodiments, multiple binding peptides are fused to any one of the amino acid sequences described herein. Multiple binding elements can be linked directly together or they can be linked together using peptide spaces. Certain peptide spacers/liners are from i to 100 or 1 to 50 amino acids in. length, in some embodiments, the peptide spaces are about 1 to about 25, 3 to about 40, or 3 to 30 amino acids in length. In other embodiments, the spacers are from about .1 to about 20 or from 5 to about 20 amino acids in ienat .

[0099) In some embodiments, the amino acid sequences described herein, themselves contain domains which have an affinity for particular body surfaces.

[00100] The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any manner. Those skilled i the art will readily recognize a variety of noncritica! parameters, which can be changed or modified to yield essentially the same results.

Examples

[00101 j Unless otherwise stated, standard molecular biology techniques were used for subcloiimg and site-directed mutagenesis. An Escherichia coll t >doH -optimized nucleotide sequence of S li/473 was subcloned into pET28a(-r) (invitrogen) as a BamHl-X ^' hol insert. Mutagenic primers were designed via FrimerX (http://www.bioinfonnatics.org^nmerX) and point mutations generated via the QuikChange IS Site Directed Mutagenesis kit (Agilent Technologies). Nucleotide sequences containing point moiations were confirmed by DNA sequencing (GeneWiz).

Example I

(00102] For expression, constructs are elecrroporatec! into coli BL21 (DE3) cells and plated on LB agar plates containing 50 ug mL kanamycin. An individual colony is cultured in 5 niL LB medium supplemented with 50 pg rnL kanamycin for 16 hours at 37"C, 200 rpm. Then 2 mL of saturated culture is added to 200 mL LB and incubated for 1.6 hours at 18°C, 200 rpm. The culture is then diluted to an OD«¾o of 0.8 in 800 mL LB and grown for 1 hour at 38 C, 200 rpm. After 1 hour, protein expression is induced by the addition of 1 mM IPTG and the culture is incubated at 18"C, 200 rpm for another 16-20 hours.

Exa mple 2

[00103] For purification, cells are harvested at 8,000 g for 15 minutes at 4°C, washed once in 20 mL of ice-cold PBS, resuspended in .15 mL lysis buffer (100 mM HEPES, 500 mM NaCI, 10% w/v glycerol, 10 mM imidazole) and sonicated at .15 W, 50% duty, .15 minutes total processing time. The soluble cell lysate is clarified by cenfrirugation at 17,000 g for 20 minutes at 4°C and bexahistidme tagged Sm.lil.473 is purified from the sample by immobilized metal ion affinity chromatography (IMAC). Cell lysate is passed over a colum containing 15 mL of Ni ^{2 *} -bound Chelating Sepharose Fast Flow resin (GE Healthcare) pre-equilibrated in IMAC Buffer A (20 mM HEPES. 500 mM NaCI., 10% w/v glycerol, 10 raM imidazole) at a flow rate of 1.8 niL/min via a BioLogic LP chromatography system (Bio-Rad) with fraction collector. The column, is washed for 70 min with IM C Buffer A to remove any unbound proteins, before applying a gradient from 0% to 100% IMAC Buffer B (20 m HEPES, 500 mM NaCl, 10% w/v glycerol 500 mM imidazole) over the course of 200 minutes. Fractions are assayed for protein content via Bradford reagent ( Bio-Rad) and samples containing purified Sm.lt .1473 are pooled together and dialyzed against 4 L of 20 mM sodium phosphate buffer pH 8 for 40 hours at 4"C with one buffer exchange. Purit is assessed using SDS-PA.GE, western blotting and. MALDTTOF. For MALDI-TOF, 1 uL of protein sample is mixed with 1 pL of sroapinic acid matrix, spotted onto a MSP 96 target ground steel plate (Broker), and allowed to air dry. Samples are then analyzed via a Microflex mass spectrometer (Broker Daitomcs).

Example 3

[00104] Two 1 niL samples of cultures induced for 12 hours are harvested by centrifugation at 8,000 g for 10 minutes at 4°C. On pellet is resuspended in 200 pL of 4 M urea and designated the whole cell lysate sample. The second pellet is resuspended in 200 pL of ice-cold sucrose buffer (20 mM HEPES, 1 mM EDTA, 20% w/v sucrose), incubated on ice for 15 minutes, centrifiiged, resuspended in 200 pL ice-cold 5 mM MgS0 ₄ , and incubated on ice tor 15 minutes. The resulting supernatant containing the periplasmic proteins is collected by centrifugation at 17,000 g for 10 minutes at 4 ^" C.

Example 4

[00105] Forty (40) uL of protein sample mixed with 10 pL of 5x Lammeli sample buffer is heated for 5 minutes at 90°C before loading 10 p.L onto a 4% stacking/ 12% separating acrylamide gel with MES running buffer. Five (5) pL of Precision Plus Protein All Blue Standard (Bio-Rad) is used as a molecular weight standard. Samples are run at 100 V and transferred to a nitrocellulose membrane (Amersham Hybond ECL) for 90 minutes at 17 V, 4°C. Transfer is confirmed by staining the membrane with 0.1 % Ponceau S stain. The membrane is then blocked overnight at 4 ^t! C with 5% fat-free milk in TBST, washed once with TBST incubated for 1 hour at room temperature with mouse monoclonal, anti-hexahistidine tag primary antibody (Cell Signaling) diluted 3000 fold in 5% at-free milk, washed five times with. TBST, incubated for 1 hour at room temperature with horseradish peroxidase-conjugated. anti-mouse IgG secondary antibody (Cell Signaling) diluted 3000 fold in 5% fat-free milk, washed five times with TBST, and then twice with TBS. The membrane is developed with a c emil miaescent horseradish peroxidase substrate (GE Healthcare) and imaged with a Storm 860 scanner.

Example 5

90106) Five (5) uL of induced culture is spotted onto LB plates solidified with S % agarose and supplemented with 50 pg/mL kanamycin and 1 mg mL of polyManA or polyGlcA. Plates are incubated at room temperature for 12 hours, gently flooded with 10% cetyl pyridmrum chloride, and incubated at room temperature for 30 minutes. A clearing zone surrounding the area treated with culture is indicative of extracellular lyase activity.

Example 6

100107] Sodium alginate, medium viscosity, is obtained from MP Biomedicals. Hyaluronic acid potassium salt from human umbilical cord is obtained from The Wistar Institute. PolyGlcA is prepared from Avicel PH I 05 (FMC Biopolymer) by first converting the macrocrystalline cellulose to regenerated amorphous cellulose by dissolution in 86,2% H PO4 and then converting the regenerated amorphous cellulose to polyGlcA via 2,2,6 ,6-tetrameihylpipelidine-l -oxyi radical (TEM PO)-NaBr-NaC10 mediated oxidation. PolyGlcA samples are precipitated with ethanol, dried, resuspended in dd¾Q and dialyzed against 4 L of ddHjO for 40 hours at 4°C with one buffer exchange. polyManA, polyGulA, and polyMG block structures are prepared from sodium alginate by partial acid hydrolysis as described by previously. Each fraction is resuspended in ddl¾0 and dialyzed against 4 L of d l ) for 40 hours at 4"C with one buffer exchange. Sugar concentration is determined via the phenol-su!foric acid method. The composition of each alginate fraction is determined via circular dichroism. Briefly, 300 pL of a 5 mg/mL solution of each fraction is added to a 1 mm pathlength quartz cuvette (Starna) and ellipticity measured from 190 to 260 nm in a 3-815 circular dichroism spectrometer (JASCO). The scan speed is 200 nm min; with three accu ulations per sample. The polyManA fraction is calculated to contain S5.4 ± 0.6% MaoA, the poly G fraction 54.1 ± 0.4% ManA, and the polyGulA fraction 17.3 ± 1.6% ManA, Circular dichroism spectra and associated calculations are used to determine polysaccharide composition.

Example 7

[00198) The β-eHmination mechanism of PLs results in the formation of a double bond whose accumulation is monitored by measuring the change in absorbance at 235 run, Absorbance measurements are made in 1 second intervals using an Ultrospec 3300 pro UV-Vis spectrophotometer with a detection limit of 0.001. absorbaoce units at 235 nm per minute. One unit (U) of enzyme activity is defined as an increase in absorbance at 235 nm of 1.0 per minute at 25°C (1 U - 1 ΑΑ ₂ 35 _ηί¾ miir ¹ ) (28-30). 17.5 μ of purified Sink 1473 is added to a final volume of 350 uL for reactions containing alginate, poiyManA, polyGulA, pol MG, or HA. Due to the higher activity against po!yGlcA versu ail other substrates, 1 ,75 pg of protein is added to reactions containing polyGlcA. The pH of ail reactions is maintained by using a specific buffer for a given pH (Acetate for pH 4-6, Phosphate for pH 6-8, Tris for pH 6.5-9, Glycine for pH 9- .10) at a total ionic strength of 30 ttiM. For the determination of optimal pH and buffer conditions, the enzyme is incubated in a given buffer without substrate for 10 minutes before being added to a solution containing a final polysaccharide concentration of I mg/mL in the same buffer. For the determination of Michaelis constant (KM) and maximum velocity ( V x) for each substrate, polysaccharide concentration (S) is varied from 0.0625 mg mL to 2 mg/mL and initial rates (ι·ν) are fitted to the ichaelts-Menten equation, v ~ Vmjc iKM+S _* with generalized reduced gradient (GRG2) nonlinear optimization program. For all substrates, R-squared and correlation values are greater than 0,965 and 0.985, respecti vely . Lyase activity is also confirmed by measuring the concentration of the resulting deoxy sugar via the thioharhituric acid (TBA) method as described previously.

Example 8

£00109) HPLC experiments are run on an Agilent .1 100 series HPLC value system equipped with a 96-wel! autosampler and UV-Vis detector. Smltl 473 at a final concentration of 50 pg/mL (5 pg/mL for polyGlcA samples) is added to either poiyManA (3 mg/mL, 30 ra Tris pH 9), poIyGicA (3 mg/mL, 30 niM Tris pH 7), or HA (1 mg/mL, 30 ni Acetate pH 5) at final concentrations and buffers indicated in parenthesis, 15 pL of the reaction mixture is injected at 30-minute intervals over the course of 24 hours onto a TS gel SuperOligoPW column (Tosoh) equipped with corresponding guard column. The mobile phase is 20 mM sodium phosphate buffer pH 8 plus 250 mM NaCI and the flo rate is 0.275 m ^' L'rnin. Unsaturated uronic acid products are detected by absorbaoce at 235 nm. A series of PEG standards are used to generate a calibration curve.

Example 9

j Olltlj Recombinant Smltl47 is purified to homogeneity from E. coli cell lysates via !MAC and purit is confirmed by immunoblotting and ALDI-TOF. The enzymatic activity of purified Sm1tl473 (SEQ ID NO: 2) is tested against the following polyuronides: alginate, polyManA, poiyGulA, polyMG, polyGicA, and hyaluronic acid. The results of this evaluation are illustrated in Table 2 (below), in which initial, rates for each substrate (1 rag/mL) are given at their respective optimal pH values.

Table 2

{001.11 Of particular note, specific activities for each of the three most active substrates (polyManA, poSyGIcA, HA) are found to be strongly dependent on pH, with optimal activity for HA at pH 5, polyGicA at pH 7 and polyManA at pH 9 (Figure I). The highest overall specific activity (848.3 ± 6.3 U/mg at pH 7) was for polyGicA (Figure 1 A), which is among the highest reported for polyGlcA-specific lyases. For polyManA the specific activity (68.5 ± 2.9 U/mg at pH 9) was also significant (Figure IB), being within an order of magnitude to reported values for polyMaoA-specific lyases. Likewise, while the specific activity for HA (42.3 ± 1.3 U/mg at pH 5) was the lowest for the three major substrates (Figure 1C), it is within an order of magnitude of reported values for bacterial hyalurorsidases. Optimal pH values for each substrate were independently confirmed by the TBA method (Figore 1 D). Kinetic parameters for each of the three substrates were also determined according to the Miehaelis-Menten equation described in Table 3 (below), with VMAX values similar in magnitude and trend to the specific activities for each of the three main substrates (polyManA, polyGicA,, HA); V _MAX for polyGicA is approximately 10-fold greater versus polyManA or HA.

Table 3

(00112 j The KM values for the three substrates also follow a similar trend to VM ^' M and specific activity, with a 5-fold larger ¾ for HA versus polyG!cA. Thus, based on the highest specific activity for polyGlcA (Figure LA), one could tentatively conclude that Smlt 1473 is a polyGIeA- specific lyase. However, the multiple pH optima exhibited by Smitl.473 for maximal HA, polyGlcA and polyManA cleavage (Figure I), as well as comparable kinetic parameters and specific acti vities to published values for lyases specific to each of these substrates, indicate that Smlt 1473 is a multifunctional PL with potent activity against -polyManA, polyGlcA and HA. Example 10

1001 13] As stated previously, the β-elimination mechanism of PLs requires a neutralizing group, proton acceptor, and protein donor. Despite diverse substrate specificity, secondary structure content, and tertiary folds, the residues responsible for each of the aforementioned roles appears to be highly conserved across many polysaccharide lyase families. Based on sequence alignments with related polysaccharide lyases using COBALT, four conserved, putative active- site residues in S«i.Stl 473 were identified (N1.67, H 168, 2 I5, and Y222) and predicted inactivating mutants (N S.67L, HI 68 A, R215L, and Y222F) were expressed, purified and characterized. Enzymatic activity of each mutant was observed directly from a plate assay with polyGlcA and poly ManA as the substrates, and quantified in terms of the change in absorbance at 235 nm and TBA method. All four mutations ( 167L, HI 68 A, R215L, and Y222F) were found to abolish activit against all three substrates (polyGlcA, polyManA, HA) with the exception of HI 68 A, which retained low activity (1.0% of wild-type) against polyGlcA at. pH 7. Interestingly, while the H I68A -mutant retained approximately 10% of wild-type activity against polyGlcA, its specific activity (59.9 ± 1.8 U/mg at pH 7) was comparable to optimal, wild-type Smlfi.473 activity against polyManA (68.5 ± 2.9 U/mg at pH 9} and HA (42.3 ± 1.3 U/mg at pH 5). To confirm that the .mutant lyases retained secondary structure equivalent to wild-type, CD spectra were collected for wild-type and each mutant (N1 7L, H168A, R215L, and Y222F), For wild-type and mutant Smlt 1473, a predominantl a-helical secondary structure was observed in terms of specific minima at 208 nm and 222 nm. The calculated percent he!icity for wild-type was 48,3%, with less than 5% change in percent helicity for each mutant, indicating the overall secondary structure was not perturbed significantly by the mutations. Therefore, the catalytic mechanism of Sm!tl473 is dependent on four highly conserved residues (NI67, HI 68, R215, Y222) that are consistent with an A.sn/Arg-Tyr/His neutralization-acid/base mechanism.

Example 11

{0011 1 Various concentrations of alginic acid are added to 0, 1 mg/mL samples of SEQ ID NO: 2. Reaction velocities are calculated from the absorbance at 235 nm generated from unsaturated products of alginic acid. The data described in Table 4 (below) demonstrates that the reaction velocit increases with increases in alginic acid concentration.

Tab!e 4

Example 12

|00115] Thermal stability of an exemplary protein of the present invention is evaluated by adding a sample of alginic acid to a fixed concentration of an exemplary protein of the present invention. The mixture is then heated to a temperature of about 50 deg. C for approximately 10 minutes, and then cooled rapidly to room temperature (20 deg. C). Initial reaction velocities are caicuiated from the absorbance at 235 nm generated from unsaturated products of alginic acid. The data described in Table 5 (below) demonstrates the thermal stability of an exemplary protein of the present invention, as it is able to maintain activity at temperatures in excess of 50 deg. C.

Table 5

Example 13

(00116] Reaction velocities of wild-type and mutant enzymes of the present invention are evaluated. 1 mg/mL samples of alginic acid are added to a 0.1 mg/mL samples of wild-type (WT) and mutant enzymes (Y 115F, W 17 I A, H221 F and Y225F). Reaction velocities are calculated from the absorbance at 235 nra generated from unsaturated products of atginic acid. The data provided in Table 6 (below) describes the resiilts of this evaluation.

Table 6

Example 14

(001171 The degradation of ceHuronic acid by a fixed concentration of an exerapiary protem of the present invention (SEQ ID NO: 2) in the presence of various detergents (1%) and acids is evaluated. The data described in Table 7 (below) demonstrates that stability (msensMvity) of an exemplary protem of the present invention to various detergents. Activity is measured in terms of the percentage of activity of the SEQ ID NO: 2 in the absence of a detergent.

Table 7

Example 15

[00118] initial reaction velocities are measured for exemplary proteins of the present invention

(SEQ ID NO: 2 [WT], YI 15F, W171A, H221F and Y225F). Samples of cellurotiic acid are added to samples of exemplary proteins of the present, in vention. The data provided in Table 8

(below) describes the results of this evaluation.

Table 8

{Θ0Ϊ19] It is imeoded thai any patents, patent applications or printed publications, including books, mentioned in this patent document be hereby incorporated by reference in their entirety. {00120 j As those skilled in the art will appreciate, numerous changes and modifications may be made to the embodiments described herein, without departing from the spirit of the invention, it is intended that all such variations fail within the scope of the invention.