Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
  • A01N25/10—Macromolecular compounds
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
  • A01N25/28—Microcapsules or nanocapsules
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
  • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
  • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
  • A61K8/66—Enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
  • A61K8/73—Polysaccharides
  • A61K8/736—Chitin; Chitosan; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/52—Genes encoding for enzymes or proenzymes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/104—Aminoacyltransferases (2.3.2)
  • C12N9/1044—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/10—General cosmetic use
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/52—Stabilizers
  • A61K2800/524—Preservatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y203/00—Acyltransferases (2.3)
  • C12Y203/02—Aminoacyltransferases (2.3.2)
  • C12Y203/02013—Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII

Definitions

• the present invention relates to active enzymes derived from zymogen proteins and their use as biocidal (e.g ., cross-linking) agents.
• biocidal enzymes can be bound to a polymeric support to form an enzymatic device, for increased shelf life of a biocidal enzyme.
• the enzymatic device can be used to cross-link proteins or to modify proteins of interest, such as binding a molecule, protein, or peptide to another protein. More specifically, the enzyme(s) may be employed as biocidal agents for novel preservatives and as antimicrobials for healthcare products, personal care or cosmetic formulations, food, pharmaceuticals, packaging, and marine applications.
• Preservative compositions for protecting and preserving formulations against bacterial or fungal attack are known in the art, and have a wide variety of applications in fields such as personal care products, household and industrial products, health and hygiene products, and pharmaceuticals. There are many chemicals, small molecules and preservatives that are used as biocidal agents. Conventional preservative blends have included traditional active ingredients such as formaldehyde releasers and/or parabens, due to the good bacterial and fungicidal properties achieved by these types of compounds.
• One of the more common biocidal mechanisms is protein acylation and subsequent protein cross-linking using formaldehyde and glutaraldehyde.
• Gamma irradiation is another method that catalyzes cellular decomposition through cross-linking activity. Gamma irradiation is frequently employed for sterilization.
• biocidal enzymes and proteins have been used as biocompatible preservatives in the food (Malhotra, et al., Frontiers in Microbiology 2015, 6, 611), healthcare (Kaplan, et al., Journal of Dental Research 2010, 89, 205-218), and marine (Olsen, et al. (2007) Biofouling 23:369-383) industries.
• oxidases and peroxidases which generate oxidizing species for biocidal activity
• lytic enzymes like proteases and lyases (e.g., savmase, lysozyme, iysostaphin, subtilisin), which degrade the surface of microbes (e.g., fungi, viruses, bacteria); lactoferrin, which hydrolyzes nucleic acids, such as RNA; and antimicrobial peptides (e.g., nisin, periocin), which are believed to kill microbes by creating pores in the cell wall, resulting in cell rupture and leakage of cell contents.
• lytic enzymes like proteases and lyases (e.g., savmase, lysozyme, iysostaphin, subtilisin), which degrade the surface of microbes (e.g., fungi, viruses, bacteria); lactoferrin, which hydrolyzes nucleic acids, such as RNA
• biocidal agents often cause damage to the products that they are deployed to protect from microbial contamination.
• US Patent No. 5,326,561 discloses an enzyme fungicide cocktail using chitinolytic enzymes, glucanolytic enzymes and cellulases, which are lytic enzymes.
• lytic enzymes can destroy consumer product formulations that contain esters, which are used as conditioners and shine increasing agents, proteins (e.g., keratin and peptide hair/skin conditions), and/or carbohydrates (e.g., gums and other thickeners).
• proteins e.g., keratin and peptide hair/skin conditions
• carbohydrates e.g., gums and other thickeners
• biologic preservatives can deteriorate over time, reducing the shelf life of a product.
• agents with anti-microbial (e.g., bactericidal and fungicidal) activity which avoid these problems.
• compositions that can be incorporated into products to be preserved are disclosed herein.
• the disclosed compositions include immobilized or conjugated enzymes, such as enzymes which are initially secreted as zymogens, on a polymer support structure.
• zymogen classes of enzymes include, but are not limited to, hydrolases, proteases, lytic enzymes, and cross-linking enzymes.
• the compositions include a cross-linking enzyme, or another zymogen class of enzyme, immobilized or conjugated (e.g., covalently bound) to a polymer support, which may improve the shelf life, e.g., preserve catalytic activity, of the enzyme over a commercially relevant time frame, and/or protect the enzyme from auto-cross-linking and/or deterioration (e.g., decrease in catalytic activity) over extended storage periods.
• the enzyme is a cross-linking enzyme or an enzyme from another class of zymogen enzymes.
• the enzyme may react amino acid residues on a protein through cross-linking or binding a molecule of interest to a protein (e.g., conjugation of a small molecule, protein, or peptide to another protein).
• a cross-linking enzyme or other zymogen class enzyme is covalently bound to a polymeric support, e.g., chitosan, via a linker.
• a cross-linking enzyme or other zymogen class enzyme is immobilized on, e.g., covalently bound directly to, the polymeric support without use of a linker.
• the polymer is a biopolymer.
• the polymer is a biocidal polymer, such as, but not limited to, chitosan or carboxymethylchitosan.
• the compositions include one or more enzyme, such as a cross-linking enzyme, hydrolase, protease, and/or lytic enzyme,
• microbial e.g., bacterial
• a method of increasing the shelf-like of products including incorporating an effective amount of a composition as disclosed herein into the product or a formulation or composition that includes the product.
• the product does not include any other preservative substance or composition in addition to the enzyme/polymer composition disclosed herein.
• the product includes at least one other preservative substance or composition in addition to the enzyme/polymer composition disclosed herein.
• the product does not include
• the product includes at least one other preservative substance or composition, in addition to the enzyme/polymer composition disclosed herein, that is not formaldehyde or glutaraldehyde
• the shelf life of the product incorporating the composition is increased when compared to the same product not incorporating the composition.
• a polymer immobilized or conjugated enzyme as disclosed herein may bind a molecule of interest to a protein.
• Nonlimiting examples include: binding of a dye molecule to a protein, such as collagen, keratin, or elastin; binding of a protein or peptide to another protein, such as collagen, keratin, or elastin; and binding of a pharmaceutical (drug) molecule to a protein, for example, formation of an antibody-drug conjugate.
• preservative compositions include one or more zymogen-class enzyme immobilized on or encapsulated within a polymeric support structure.
• the enzyme may be selected from a hydrolase, a protease, a lytic enzyme, and a cross-linking enzyme, or combinations thereof.
• the enzyme is a cross- linking enzyme, such as a transglutaminase, a laccase, a peroxidase, a transferase, a lysyl oxidase, or a tyrosinase.
• the polymeric support structure includes a biocidal polymer. In an embodiment, the polymer is reversibly soluble.
• the enzyme may be covalently bound to the polymer, such as via a linker.
• the polymer is in the form of beads, such as microbeads.
• the polymer is in the form of biodegradable polymeric beads, such as biodegradable microbeads.
• the enzyme is encapsulated in the polymer.
• the polymer is selected from chitin, chitosan, carboxymethylchitosan, polylysine, cellulose, quaternary ammonium cellulose, alginate, pectin, and carboxy cellulose, or combinations thereof.
• methods for increasing the shelf-like of a product which include incorporating a zymogen-class enzyme into a product in an amount effective to prevent or decrease growth of one or more microbe, in comparison to an identical product that does not include the enzyme.
• the enzyme may be selected from a hydrolase, a protease, a lytic enzyme, and a cross-linking enzyme, or combinations thereof.
• the enzyme is a cross-linking enzyme, such as a transglutaminase, a laccase, a peroxidase, a transferase, a lysyl oxidase, or a tyrosinase.
• methods for increasing the shelf-like of a product which include incorporating a preservative composition that includes one or more zymogen- class enzyme immobilized on or encapsulated within a polymeric support structure, as described herein, into the product in an amount effective to prevent or decrease growth of one or more microbe in comparison to an identical product that does not comprise the composition.
• the enzyme may be selected from a hydrolase, a protease, a lytic enzyme, and a cross-linking enzyme, or combinations thereof.
• the enzyme is a cross- linking enzyme, such as a transglutaminase, a laccase, a peroxidase, a transferase, a lysyl oxidase, or a tyrosinase.
• the polymeric support structure includes a biocidal polymer.
• the polymer is reversibly soluble.
• the enzyme may be covalently bound to the polymer, such as via a linker.
• the polymer is in the form of beads, such as microbeads.
• the polymer is in the form of biodegradable polymeric beads, such as biodegradable microbeads.
• the enzyme is encapsulated in the polymer.
• the polymer is selected from chitin, chitosan, carboxymethylchitosan, polylysine, cellulose, quaternary ammonium cellulose, alginate, pectin, and carboxy cellulose, or combinations thereof.
• products that include a zymogen-class enzyme in an effective amount to increase the shelf life of the product, in comparison to an identical product that does not comprise the enzyme.
• the enzyme is included in an amount that is effective to prevent or decrease growth of one or more microbe, in comparison to an identical product that does not comprise the composition.
• the enzyme is selected from a hydrolase, a protease, a lytic enzyme, and a cross-linking enzyme, or combinations thereof.
• the enzyme is a cross-linking enzyme, such as a transglutaminase, a laccase, a peroxidase, a transferase, a lysyl oxidase, or a tyrosinase.
• the polymeric support structure includes a biocidal polymer.
• the polymer is reversibly soluble.
• the enzyme may be covalently bound to the polymer, such as via a linker.
• the polymer is in the form of beads, such as microbeads.
• the polymer is in the form of biodegradable polymeric beads, such as biodegradable microbeads.
• the enzyme is encapsulated in the polymer.
• the polymer is selected from chitin, chitosan, carboxymethylchitosan, polylysine, cellulose, quaternary ammonium cellulose, alginate, pectin, and carboxy cellulose, or combinations thereof.
• the product is a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy, plastic, packaging, or agricultural product.
• the product may be a personal care product selected from bar soap, liquid soap, hand sanitizer, preoperative skin disinfectant, cleansing wipes, disinfecting wipes, body wash, acne treatment products, antifungal diaper rash cream, antifungal skin cream, shampoo, conditioner, cosmetics deodorant, antimicrobial creams, body lotion, hand cream, topical cream, aftershave lotion, skin toner, mouth wash, toothpaste, and sunscreen lotion.
• the product may be a wound care product selected from wound healing ointments, creams, and lotions, wound coverings, bum wound cream, bandages, tape, and steri-strips.
• At least about 90% of enzyme activity is retained over a period of 28 days.
• the enzyme activity is retained at pH of about 4 to about 9 and temperature of about 4°C to about 40°C.
• microbial growth is inhibited by at least about 80% over a period of 28 days at about pH 4 to about pH 9, and temperature of about 4°C to about 40°C.
• the product does not include any other preservative substance.
• the product includes at least one other preservative substance, such as, but not limited to, at least one petrochemical derived preservative substance.
• Figure 1 shows a cross-linking (or acylating) enzyme bound to a polymer support.
• the polymer support shown is chitosan (the fully deacetylated form).
• Figure 2 shows the use of the immobilized enzymes for cross-linking proteins via lysine, tyrosine, and glutamine amino acid residues.
• Figure 3 shows the use of the immobilized enzymes for modifying proteins with cargo (e.g., covalently attaching cargo, such as small molecules or polymers) via lysine, tyrosine, and glutamine amino acid residues.
• cargo e.g., covalently attaching cargo, such as small molecules or polymers
• Figure 4 shows stability of transglutaminase-polymer bead conjugates as described in Example 3.
• Figure 5 shows a method for encapsulating enzyme with free monomers as described in Example 4.
• Figures 6A-6B show stability of carboxymethylchitosan encapsulated transglutaminase as described in Example 5.
• Figure 7 shows transglutaminase activity after encapsulation with chitosan monomers as described in Example 6.
• Figures 8A-8B show stability of chitosan encapsulated transglutaminase as described in Example 6.
• Figure 9A shows growth inhibition of B. subtilis in the presence of 312 mg L -1 (or 0.03% w/v) Curie Co mTG (solid black line). Dashed grey line shows growth in media only.
• Figure 9B shows growth inhibition of E. coli (DHlOb, cloning strain) in the presence of 880 mg L -1 (0.088% w/v) Curie Co mTG (solid black line). Dashed grey line shows growth in media only.
• Figure 9C shows Growth inhibition of C. albicans in the presence of 880 mg L -1 (or 0.088% w/v) Curie Co mTG (solid black line). Dashed grey line shows growth in media only.
• Figure 9D shows final cell viability of E. coli ATCC 8739 cultures after 16 h growth as measured by the BacTiter Glow cell viability assay (Promega).
• Luminescent signal is an indicator of population viability as it is proportional to the amount of ATP produced by viable cells.
• Enzyme is 440 mg L -1 (or 0.044% w/v) Curie Co mTG.
• Chitosan is 0.025% w/v.
• Chitosan + enzyme is the combination of the two at the same concentrations.
• Biocidal enzymes, biocidal enzyme-polymer conjugates, and polymer encapsulated biocidal enzymes are described herein. Enzyme-polymer conjugates and encapsulated enzymes demonstrate superior stability relative to unconjugated or unencapsulated enzymes over several months at pH 4.5, which is a relevant pH range for cosmetic skincare applications. A biocidal enzyme and mutant form of the enzyme have been found to exhibit antimicrobial properties. When mixing the enzyme with known antimicrobial polymers the antimicrobial activity of both the enzyme and polymer have potentiated effect on antimicrobial activity. The polymer enhances the stability of the enzyme, allowing for longer shelf life of the enzyme, and mutant forms of the enzyme, and consequently a product into which the enzyme is
• a reference to“A and/or B,” when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
• composition refers to a combination of two or more substances, including an immobilized enzyme (e.g., preservative) composition as described herein.
• the term“product” is intended to refer to a preparation, composition, or article of manufacture that has a specific utility that is to be preserved with an immobilized enzyme composition as described herein.
• “preservative” is an agent added to a product as described to prevent (for some period of time) the growth of microorganisms, or the occurrence of undesirable chemical reactions (such as oxidation), that spoil or deteriorate, including deterioration of one or more utility, of the product.
• Encapsulate or“encapsulation” as used herein refers to the entrapment or enclosure of an enzyme in a matrix.
• the matrix can be polymer alone or polymer with a cross-linking agent to covalently bind the enzyme to the polymer or to a porous polymeric network structure of the matrix or to a semi-permeable membrane coating containing the enzyme.
• conjugate refers to a covalent, ionic, or electrostatic bond, such as a bond between an enzyme and a polymer.
• A“reversibly soluble polymer” refers to a polymer which can phase transition from a soluble to insoluble material in solution in response to controllable stimuli in the environment, such as, but not limited to, pH or ionic strength. This transition process can be repeatably cycled between phases.
• Effective amount refers to an amount (e.g., minimum inhibitory concentration (MIC)) of a preservative composition as disclosed herein that is sufficient to prevent or inhibit microbial growth.
• the preservative compositions of this patent are active against Gram positive bacteria, Gram negative bacteria, yeast, and/or mold.
• shelf life refers to the length of time for which an item (e.g., a product as described herein) remains usable, fit for consumption, or saleable.
• “Household products” are products, other than personal care products, that would be used by individual consumers.
• “Industrial products” refers to products that are used in industry.
• “Emollients” are externally applied agents that soften or soothe skin, and are generally known in the art and listed in compendia, such as the“Handbook of Pharmaceutical
• Emmulsifiers are surface active substances which promote the suspension of one liquid in another and promote the formation of a stable mixture, or emulsion, of hydrophobic and hydrophilic substances, such as oil and water.
• “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading, and/or wetting properties of a composition or product.
• A“bead” refers to a solid particle, comprising or consisting of a polymer as described herein.
• A“microbead” refers to a bead that is less than one millimeter in its largest dimension.
• Biodegradable refers to a substance that is capable of decomposition by microbes (e.g., bacteria) or other living organisms.
• amino acid refers to a molecule containing both an amine group and a carboxyl group that are bound to a carbon, which is designated the alpha-carbon.
• Suitable amino acids include, without limitation, both the D- and L-isomers of the naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.
• a single“amino acid” might have multiple sidechain moieties, as available per an extended aliphatic or aromatic backbone scaffold. Unless the context specifically indicates otherwise, the term amino acid, as used herein, is intended to include amino acid analogs.
• catalyst refers to a chemical actor, such as a molecule or
• a catalyst which accelerates the speed at which a chemical reaction occurs where a reactant or reactants is converted into a product or products, while the catalyst is not turned into a product itself, or otherwise changed or consumed at the completion of the chemical reaction.
• a catalyst participates in one chemical reaction, because it is unchanged, it may participate in further chemical reactions, acting on additional reactants to create additional products.
• a catalyst decreases the activation energy barrier across the reaction path allowing it to occur at a colder temperature, or faster at a given temperature. In this way, a more rapid approach of the system to chemical equilibrium may be achieved.
• Catalysts subsume enzymes, which are protein catalysts.
• lysate refers to the liquid containing a mixture and/or a solution of cell contents that result from cell lysis.
• the methods described herein comprise a purification of chemicals or mixture of chemicals in a cellular lysate. In some embodiments, the methods comprise a purification of amino acids and/or protein in a cellular lysate.
• lysis refers to the rupture of the plasma membrane and if present, the cell wall of a cell such that a significant amount of intracellular material escapes to the extracellular space. Lysis can be performed using electrochemical, mechanical, osmotic, thermal, or viral means.
• the methods described herein comprise performing a lysis of cells or microorganisms as described herein in order to separate a chemical or mixture of chemicals from the contents of a bioreactor. In some embodiments, the methods comprise performing a lysis of cells or microorganisms described herein in order to separate an amino acid or mixture of amino acids and/or proteins from the contents of a bioreactor or cellular growth medium.
• polypeptide refers to a composition comprised of amino acids and recognized as a protein by those of skill in the art.
• the conventional one-letter or three-letter code for amino acid residues is used herein.
• polypeptide and“protein” are used interchangeably herein to refer to polymers of amino acids of any length.
• the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non- amino acids.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
• polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids, etc.
• other modifications known in the art include, for example, amino acids, etc.
• Ranges may be expressed herein as from“about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
• compositions e.g., preservative compositions, that can be included in or with (e.g., within or associated with) products to be preserved.
• the disclosed compositions include immobilized enzymes, e.g., cross-linking enzymes (see, e.g., Fig. 1).
• the immobilized enzyme may catalyze a reaction of amino acid residues on a protein, thereby effecting, for example, protein cross-linking or binding a molecule of interest to a protein (see, e.g., Figs. 2 and 3).
• the compositions include one or more enzyme, e.g., comprising or consisting of one or more cross-linking enzyme, in an amount effective to inhibit microbial (e.g., bacterial) growth, e.g., inhibition of 80% to 100%, or any of at least about 80%, 85%, 90%, 95%, 98%, or 99% of microbial growth, in a product to be preserved.
• microbial e.g., bacterial
• the enzyme(s) are immobilized on a support that comprises or consists of one or more polymer, optionally a biocidal polymer and/or biopolymer, optionally via a linker, as described herein.
• the enzyme(s) are encapsulated in a polymer, e.g., a biocidal polymer and/or biopolymer, as described herein.
• the polymer comprises or consists of chitosan or carboxymethylchitosan.
• the polymeric support is in the form of a bead, e.g., a biodegradable bead, e.g., a microbead.
• the bead, e.g., biodegradable bead, e.g., microbead comprises or consists of chitosan or carboxymethylchitosan.
• Preservatives are antimicrobial ingredients added to product formulations to maintain the microbiological safety of the products by inhibiting the growth of and reducing the amount of microbial contaminants. US Pharmacopeia has published protocols for acceptable microbial survival for preservatives in cosmetics and personal care products. These tests include USP 51 (Antimicrobial Effectiveness Test) and USP 61 (Microbial Limits Test)
• the effectiveness of the preservative system disclosed herein is determined based on the MIC (minimum inhibitory concentration) against a variety of microbes (e.g. E. coli DH 10b. E. coli ATCC 8739, B. subtilis BGSC 1A976, C. albicans ATCC 10231, and/or A.
• MIC minimum inhibitory concentration
• MICs Minimum inhibitory concentrations
• Microbial growth may be determined, for example, by spectrophotometric methods (the optical density at 600 nm) or with a cell viability assay (BacTiter Glo, Promega).
• the compositions include an active enzyme, such as a zymogen- class enzyme, for example, but not limited to, a cross-linking enzyme, immobilized on, e.g., covalently bound to or encapsulated in, a polymer support, such as a biocidal polymer, such as, but not limited to, chitosan or carboxymethylchitosan, which may improve the shelf life of the enzyme, and/or protect the enzyme from auto-cross-linking and/or deterioration over extended storage periods.
• the enzyme may be covalently bound to the support via a linker.
• a zymogen class enzyme such as a cross-linking enzyme
• a polymer support e.g., a biopolymer, such as chitosan or carboxymethylchitosan
• a zymogen class enzyme such as a cross-linking enzyme
• a polymer support e.g., a biopolymer, such as chitosan or
• the enzyme is a transglutaminase (e.g., Streptomyces mobaraensis transglutaminase mutant S2P (e.g., as described in Javitt, et al. (2017) BMC Biotechnol.
• a transglutaminase e.g., Streptomyces mobaraensis transglutaminase mutant S2P (e.g., as described in Javitt, et al. (2017) BMC Biotechnol.
• a biocidal enzyme utilized in a composition described herein is an enzyme that is initially secreted as a zymogen.
• Zymogens are inactive enzyme precursors (proenzymes) that are expressed with a pro-sequence that must be cleaved to afford active enzyme. Cleavage of a pro-sequence affords active enzyme that is often highly toxic to cells.
• a proenzyme is expressed with a cleavable leader sequence to suppress activity of the enzyme, due to related enzyme toxicity to the cell. Therefore, zymogens present a useful class of enzymes for use as antimicrobial agents.
• the mature active enzyme form (i.e., without the pro- sequence) is used in the disclosed compositions immobilized on a polymeric support, such as chitosan or carboxymethylchitosan, for preparation of a biocidal preservative composition.
• a polymeric support such as chitosan or carboxymethylchitosan
• useful enzymes within this category include, but are not limited to, hydrolases, proteases, nucleic acid lytic-enzymes, and cross-linking enzymes.
• cross-linking enzymes include, but are not limited to: transglutaminases, laccases, peroxidases, transferases, lysyl oxidases, and tyrosinases. These enzymes have been used to covalently bind small organic molecules, peptides, proteins, cells, and other molecules of interest to polymer or other biopolymer (such as protein or saccharide) scaffolds.
• Preferred cross-linking enzymes include transglutaminases, lysyl oxidases, and tyrosinases, which exhibit for cellular toxicity in the active enzyme forms. Due to their auto- cross-linking activity, these cross-linking enzymes are bound to polymer supports, such as chitosan or carboxymethylchitosan, to prevent self-destruction and improve shelf-life. In some embodiments, the polymer possesses biocidal activity. Chitosan and carboxymethyl chitosan have known antimicrobial (biocidal) properties.
• a chitosan-enzyme composition e.g., biocidal enzyme immobilized on chitosan
• a biocidal enzyme immobilized on any of the biocidal polymers disclosed herein has enhanced antimicrobial action, in comparison to the biocidal action of the enzyme or the polymer alone.
• the biocidal enzyme may be any of the enzymes disclosed herein, such as a zymogen class enzyme, for example, a cross-linking enzyme.
• a cross-linking enzyme enhances the antimicrobial properties of chitosan or other biocidal support by providing an additional mechanism of antimicrobial action.
• Chitosan for example, ruptures the cell membrane and leads to spillage of the cell contents.
• the cross-linking enzyme can cross-link proteins vital for cell function both on the surface of the cell and within the cell. This combination of both materials together reduce the quantity of the materials needed and provide additional stability to the enzyme allowing for greater activity over time (less chitosan and less enzyme) and reduce the undesirable effects that may accompany the use of biocidal chitosan.
• Using higher weight percent chitosan in formulations results in acidic product formulations because only protonated chitosan has antimicrobial properties.
• higher weight compositions of chitosan correlates with the addition of multiple protonated amines (on the chitosan backbone) into the product in which chitosan is added, for example, personal care products.
• Highly acidic product formulations may not be desirable for sensory feel or compatible with existing product formulation.
• higher weight percent of chitosan in a formulation leads to increases in viscosity, which also may be undesirable for product formulation.
• a second biocidal agent e g., a cross-linking enzyme, will allow the amount of chitosan to be reduced while still maintaining biocidal properties.
• the enzyme alone may be sufficient for preservative/antimicrobial properties, however, immobilization of the enzyme on or encapsulation in a polymeric support, such as chitosan or carboxymethylchitosan, increases the enzymes shelf-life allowing the product formulator to use less enzyme to achieve desirable preservative properties.
• a transglutaminase is an enzyme that catalyzes the formation of an isopeptide bond between a free amine group of a protein, for example, and the acyl group at the end of the side chain of protein- or peptide-bound glutamine. Such enzymes are classified as EC 2.3.2.13.
• Transglutaminases catalyze a transamidation reaction between glutamyl and lysyl side chains of target proteins. The catalytic reaction proceeds via glutamine deamination and formation of a protein-glutamyl-thioester at the active site of the enzyme.
• the transglutaminase is a microbial transglutaminase, for example Ca2 + -independent microbial transglutaminase (MTGase) of a variant of Streptomyces mobaraensis.
• the Tgase is a microbial Tgase and preferably is the Ca2 + -independent microbial transglutaminase (MTGase) of a variant of Streptomyces mobaraensis.
• the Tgase is a more stable mutational variant of Streptomyces mobaraensis Tgase, such as S2P-Tgase (Javitt, et al. (2017) BMC Biotechnol. 17:23).
• S2P-Tgase Well defined prokaryotic Tgases are shown in Table 1, reproduced from Zhang, et al. (2010) Biotechnol. Genet. Eng. Rev. 26:205-222, with additions from Steffen, et al. ( 2017) J. Biol. Chem. 292(38): 15622-15635.
• GRAS Generally Recognized as Safe
• FDA/CFSAN agency response letters GRAS notice numbers 000004 (1998), 000029 (1999), 000055 (2001), and 000095 (2002).
• Commercially available microbial transglutaminase is produced on large scale and distributed under the trade name ACTIVA® by Ajinomoto US, Inc.
• Lysyl oxidases (also known as protein-lysine 6-oxidase) are copper- dependent enzymes that oxidize primary amine substrates to reactive aldehydes.
• LOX Lysyl oxidases
• Five different LOX enzymes have been identified in mammals, LOX and LOX-bke (LOXL) 1 to 4, showing a highly conserved catalytic carboxy terminal domain and more divergence in the rest of the sequence.
• LOX proteins have been identified not only in animals, but also in many other eukaryotes, as well as in bacteria and archaea, reviewed in Grau-Bove, et al. (2015) Scientific Reports 5: Article number: 10568.
• Tyrosinase (EC 1.14.18.1), usually known as the enzyme responsible for the enzymatic browning of fruits and vegetables, has been demonstrated to induce cross-linking of the whey proteins a-lactalbumin and b-lactoglobubn. Tyrosinases have been isolated and studied from a wide variety of plant, animal, and fungal species.
• tyrosinases are of mammalian origin.
• a few bacterial tyrosinases have been reported, of which Streptomyces tyrosinases are the most thoroughly characterized (U.S. Pat. Nos. 5,801,047 and 5,814,495).
• tyrosinases have been disclosed, e.g.,. from Bacillus and Myrothecimi (EP 919 628), Mucor (JP
• Laccases are multi-copper oxidases found in plants, fungi, and bacteria, which oxidize phenolic substrates, performing one-electron oxidations, resulting in crosslinking.
• Methods for cross-linking proteins by laccases have been disclosed, e.g., in US2002/009770. Plant proteins derived from beans, cereals, and animal proteins, including milk, egg, meat, blood, and tendon are listed as suitable substrates.
• Fungal laccases are disclosed in US2002/019038.
• compositions described herein include a polymeric support.
• a biocidal enzyme, as described herein is immobilized on the support, with or without a linker, or encapsulated within a polymeric support, such as a reversibly soluble polymer, including, but not limited to, chitosan, carboxymethylchitosan, or polylysine.
• the polymer is a biocidal polymer.
• Nonlimiting examples of polymeric supports include: chitin, chitosan, carboxymethylchitosan, oxidized cellulose, quaternary ammonium cellulose, alginates, pectin, and carboxy cellulose.
• Preferred supports are biocidal polymers, nonlimiting examples of which are shown in Table 2. Table 2. Examples of Biocidal Polymers for Antimicrobial Applications
• the polymeric support is a biocidal biopolymer, such as chitosan or carboxymethylchitosan.
• the enzyme is immobilized on particles, e.g., chitosan particles, such as beads, e.g., chitosan beads (e.g., microbeads), or nanoparticles.
• the beads e.g., microbeads
• the enzyme may be immobilized by encapsulation with free monomers (e.g., chitosan or carboxymethylchitosan monomers), for example, utilizing a linker.
• Chitosan is a linear aminopolysaccharide of glucosamine and N-acetylglucosamine units and is obtained by alkaline deacetylation of chitin extracted from the exoskeleton of crustaceans such as shrimps and crabs, as well from the cell walls of some fungi.
• Chitin is a linear polymer of (l 4)-linked 2-acetamido-2-deoxy-b-D-glucopyranose (GlcNAc; A-unit), which is insoluble in aqueous solvents. It also has many structural similarities with cellulose, such as conformation of the monomers and diequatorial glycosidic linkages.
• Chitosan may be considered as a family of linear binary copolymers of (l 4)-linked A-units and 2-amino-2- deoxy-b-D-glucopyranose (GlcN; D-unit).
• Carboxymethylchitosan e.g., of fungal origin
• N,O-carboxymethylchitosan is > 80% substituted with carboxyl groups.
• Quaternary ammonium containing biopolymers like chitosan and its more acetylated form chitin, are well known for their antimicrobial activity (Kong, et al. (2010) Int. J. of Food Microbiol. 144: 51-63).
• Chitin, chitosan, and other related polymers are excellent scaffolds to immobilize enzymes (Muzzarelli (1980) Enzyme Microb. Technol. 2: 177-184).
• Tyrosinase has been immobilized on chitosan for dephenolization of industrial waste (Dinner, et al. (2012) Int. J. Biol. Macromol. 50:815-820) and for optical detection of phenol compounds (Abdullah, et al. (2006) Sensors and Actuators B: Chemical 114:604-609).
• the tyrosinase is either directly ligated to the chitosan support without a linker or using glutaraldehyde as a linker to immobilize the enzyme on chitosan. Additionally, tyrosinase-chitosan biocatalysts have been explored for the production of L-DOPA (Carvalho, et al. , Appl. Biochem.
• Microbial transglutaminase has been immobilized on chitosan using glutaraldehyde as a linker for the purpose of deamidation of food proteins (Nonaka, et al. (1996) Biosci, Biotechnol, and Biochem. 60:532-533), using Chitopearl 3007, a microbead form of chitosan, for the polymer support, with glutaraldehyde as a linker.
• polymeric supports are provided in, for example, Nonaka, et al. (1996) Biosci, Biotechnol, and Biochem. 60:532-533 and Hayashi, T et al. (1991 ) J Appl Polymer Sci 42: 85-92, which is incorporated by reference herein in its entirety.
• an enzyme as disclosed herein is immobilized on a polymer, e.g., a biocidal polymer, such as a biocidal biopolymer, e.g., chitosan, via a chemical linker, which covalently links the enzyme to the polymer.
• the linker is an alkylene (e.g. methylene), a diimine (1,5-diimine), a diamine (1,5-diamine), di carbonyl (e.g. 1,4-dicarbonyl), an amide bond, a polypeptide, an alkyl linker, or contains a phenyl group, a fused heterocycle, or an aromatic group, such as:
• reagents which can be used to provide linkers include, but are not limited to: formaldehyde, glutaraldehyde, succinate anhydride, phenolic compounds, genipin, carbodiimide reagents, proteins or peptides (e.g., zein, gelatin, collagen).
• the linking reagent is genepin, epichlorohydrin, formaldehyde, or glutaraldehyde. (see Fig. 1). In a preferred embodiment, the linking reagent is glutaraldehyde.
• the enzyme is covalently linked to a carrier (polymeric support), without the use of a linker.
• Products disclosed herein include personal care products, household products, industrial food, pharmaceutical, cosmetic, healthcare, marine, painting, coating, or energy products, which include an effective amount, for example, about 0.0001% w/v to about 5% w/v, of the disclosed enzymes (e.g., zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme) or compositions (e.g., zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme, immobilized on or encapsulated in a polymer, such as a biopolymer and/or a biocidal polymer) to act as an antimicrobial agent, e.g., preservative, in the product.
• the disclosed enzymes e.g., zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g
• an enzyme or enzyme/polymer composition as disclosed herein is included as an antimicrobial agent in any of the products disclosed herein at a concentration of any of at least about 0.0001% w/v, 0.0005% w/v, 0.001% w/v, 0.005% w/v, 0.01% w/v, 0.05% w/v, 0.1% w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, or 5% w/v.
• the enzyme or enzyme/polymer composition is included at a concentration of any of about 0.0001% w/v to about 0.0005% w/v, about 0.001% w/v to about 0.005% w/v, about 0.005% w/v to about 0.01% w/v, about 0.01% w/v to about 0.05% w/v, about 0.05% w/v to about 0.1% w/v, about 0.1% w/v to about 0.5% w/v, about 0.5% w/v to about 1% w/v, about 1% w/v to about 1.5% w/v, about 1.5% w/v to about 2% w/v, about 2% w/v to about 2.5% w/v, about 2.5% w/v to about 3% w/v, about 3% w/v to about 3.5% w/v, about 3.5% w/v to about 4% w/v, about 4% w/v, about 4% w/v to about 4.5%
• products in which an enzyme or enzyme/polymer composition described herein is included as an antimicrobial agent do not include a petrochemically derived preservative substance, such as, but not limited to, parabens, formaldehyde and formaldehyde releasers, isothiazolinones, phenoxy ethanol, and/or organic acids (such as sodium benzoate).
• a biocidal enzyme e.g., cross-linking or lytic enzyme, alone or in combination with a biocidal polymer, e.g., chitosan, is the only amtimicrobial, e.g., antibacterial or preservative, agent in the product.
• an enzyme as described herein e.g., zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme
• an enzyme as described herein is included as an antimicrobial agent in combination with one or more additional antimicrobial agent(s), such as, but not limited to, one or more petrochemically derived preservative substance(s).
• a composition as described herein e.g., zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme, immobilized on or encapsulated in a polymer, such as a biopolymer and/or a biocidal polymer, for example, but not limited to, chitosan
• zymogen-class enzyme such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme, immobilized on or encapsulated in a polymer, such as a biopolymer and/or a biocidal polymer, for example, but not limited to, chitosan
• additional antimicrobial agent(s) such as, but not limited to, one or more petrochemically derived preservative substance(s).
• Products disclosed herein also include industrial biocatalysis products, which include an amount of the disclosed compositions effective to remove or inactivate a biocatalyst, or biocatalytic activity, from a reaction mixture such as an industrial process (e.g., act as a purification handle for removing enzymes from industrial chemical reactions).
• a reaction mixture such as an industrial process (e.g., act as a purification handle for removing enzymes from industrial chemical reactions).
• an industrial process e.g., act as a purification handle for removing enzymes from industrial chemical reactions.
• “smart” biocatalytic enzyme-immobilization technologies are of interest to industries that employ biocatalysts for chemical or biologic manufacture.
• the enzyme immobilization platform features reversible solubility that allows for the catalyst to be employed for homogeneous catalysis at low pH conditions or heterogeneous catalysis
• immobilization platform described herein will reduce the cost and time associated with purifying protein products and facilitate catalyst recycling.
• compositions described herein provide the ability to immobilize enzymes on a reversibly soluble polymer, such as a reversibly soluble biopolymer, e.g., chitosan, to streamline production to purification protocols in biomanufacturing.
• Chitosan demonstrates reversible solubility in water upon pH shift (soluble below pH 6.5 and insoluble above pH 6.5).
• Immobilized enzymes are typically employed under heterogeneous conditions (with the enzyme remaining in the solid phase, insoluble under the reaction conditions) because most carriers are insoluble organic resins derived from petrochemicals. These reactions are plagued by poor mass transfer and result in slower reaction rates and reduced product yields.
• Described herein is an immobilization platform that can be utilized under homogeneous conditions (in which the enzyme may exist in the liquid phase, soluble under the reaction conditions) and quickly precipitated to remove and recycle the biocatalyst.
• the enzyme is“tagged” with a polymer to control solubility upon a mild shift in pH. This allows for rapid quenching of the reaction without employing excess heat or anti-solvent for removal of the enzyme, which compromises the integrity of the desired reaction product (e.g., protein product) and increases waste volume, resulting in higher manufacturing expenses.
• An enzyme or composition, e.g., preservative composition, as described herein can be incorporated into any personal care product.
• Personal care products into which the disclosed compositions may be incorporated include, but are not limited to, bar soap, liquid soap (e.g., hand soap), hand sanitizer (including rinse off and leave-on alcohol based and aqueous-based hand disinfectants), preoperative skin disinfectant, cleansing wipes, disinfecting wipes, body wash, acne treatment products, antifungal diaper rash cream, antifungal skin cream, shampoo, conditioner, cosmetics (including but not limited to liquid or powder foundation, liquid or solid eyeliner, mascara, cream eye shadow, tinted powder, "pancake” type powder to be used dry or moistened, make up removal products, etc.), deodorant, antimicrobial creams, body lotion, hand cream, topical cream, aftershave lotion, skin toner, mouth wash, toothpaste, sunscreen lotion, and baby products such as, but not limited to, cleansing wipes, baby shampoo, baby soap, and diaper cream.
• wound care items such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, bum wound cream, bandages, tape, and steri-strips, and medical articles such as medical gowns, caps, face masks, and shoe-covers, surgical drops, etc.
• Additional personal care products include, but are not limited to, oral products such as mouth rinse, toothpaste, dental floss coatings, veterinary and pet care products, preservative compositions, and surface disinfectants, including solutions, sprays or wipes.
• the personal care product formulation typically includes a base formulation to which the preservative composition of the present disclosure is added.
• the base formulation may contain numerous and different ingredients depending upon the end use application.
• the personal care product formulation may contain solvents, surfactants, emulsifiers, consistency factors, conditioners, emollients, skin care ingredients, moisturizers, thickeners, lubricants, fillers, antioxidants, other preservatives, active ingredients, in particular dermatologically active ingredients, fragrances and the like, as well as mixtures thereof.
• Active ingredients as mentioned herein include, for example, anti-inflammatories, and optionally, anti- bacterials, antifungals and the like agents. Active ingredients suited for topical applications are particularly preferred.
• the personal care product does not contain any additional preservatives, such as a petrochemical derived preservative substance.
• the personal care product includes one or more additional preservative substance, such as a petrochemical derived preservative, in addition to the enzyme or enzyme/polymer composition described herein.
• the personal care product does not include conventional anti- bacterial and/or antifungal“active agents” that are typically included in personal care products.
• Conventional anti-bacterials used in hand soap include: Cloflucarban, Fluorosalan,
• polyoxyethylene sorbitan monolaurate polyoxyethylene sorbitan monolaurate
• Iodine complex phosphate ester of alkylaryloxy polyethylene glycol
• Nonylphenoxypoly (ethyleneoxy) ethanoliodine Poloxamer-iodine complex
• Poloxamer-iodine complex Poloxamer-iodine complex
• Povidone Undecoylium chloride iodine complex, Methylbenzethonium chloride, Phenol, Phenol 16, Secondary amyltricresols, Sodium oxychlorosene, Tribromsalan,
• Triclocarban Triclosan
• Triple dye Conventional antimicrobials used as preservatives in consumer product formulations include: parabens, formaldehyde and formaldehyde releasers, isothiazolinones, phenoxyethanol, and organic acids (such as sodium benzoate).
• a biocidal enzyme e.g., zymogen-class enzyme, such as a cross- linking or lytic enzyme, alone or in combination with (e.g., immobilized on or encapsulated in) a polymer, e.g., a biocidal polymer, such as but not limited to, chitosan, is the only
• a biocidal enzyme e.g., zymogen-class enzyme, such as a cross-linking or lytic enzyme, alone or in combination with (e.g., immobilized on or encapsulated in) a polymer, e.g., a biocidal polymer, such as but not limited to, chitosan, is combined with one or more additional preservative substance, such as one or more petrochemically derived preservative substance.
• one or more biobased preservative i.e., enzyme or enzyme/polymer composition as disclosed herein
• one or more synthetic preservative e.g., petrochemical derived substance
• the preservative e.g., antimicrobial effect achieved between the biobased and synthetic preservatives is additive or synergistic.
• one or more biobased preservative i.e., enzyme or enzyme/polymer composition as disclosed herein
• one or more additional preservative substance for example, a biocidal substance selected from polylysine, chitosan, benzoate, nisin, lysozyme, and chitosan, or any combination thereof, and the preservative (e.g., antimicrobial) effect achieved between the biobased preservative and the additional preservative substance(s) is additive or synergistic.
• the personal care product may include emollients.
• Emollients include, without limitation, almond oil, castor oil, ceratonia extract, cetostearoyl alcohol, cetyl alcohol, cetyl esters wax, cholesterol, cottonseed oil, cyclomethicone, ethylene glycol palmitostearate, glycerin, glycerin monostearate, glyceryl monooleate, isopropyl myristate, isopropyl palmitate, lanolin, lecithin, light mineral oil, medium-chain triglycerides, mineral oil and lanolin alcohols, petrolatum, petrolatum and lanolin alcohols, soybean oil, starch, stearyl alcohol, sunflower oil, xylitol and combinations thereof.
• the emollients are ethylhexylstearate and ethylhexyl palmitate.
• Common emulsifiers are: metallic soaps, certain animal and vegetable oils, and various polar compounds. Suitable emulsifiers include acacia, anionic emulsifying wax, calcium stearate, carbomers, cetostearyl alcohol, cetyl alcohol, cholesterol, diethanolamine, ethylene glycol palmitostearate, glycerin monostearate, glyceryl monooleate, hydroxpropyl cellulose, hypromellose, lanolin, hydrous, lanolin alcohols, lecithin, medium-chain triglycerides, methylcellulose, mineral oil and lanolin alcohols, monobasic sodium phosphate,
• the emulsifier is glycerol stearate.
• Suitable non-ionic surfactants include emulsifying wax, glyceryl monooleate, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polysorbate, sorbitan esters, benzyl alcohol, benzyl benzoate, cyclodextrins, glycerin monostearate, poloxamer, povidone and combinations thereof.
• the non-ionic surfactant is stearyl alcohol.
• Suitable antioxidants include, e.g., sulfites (e.g., sodium sulfite), tocopherol or derivates thereof, ascorbic acid or derivates thereof, citric acid, propyl gallate, chitosan glycolate, cysteine, N-acetyl cysteine plus zinc sulfate, thiosulfates (e.g. sodium thiosulfate), polyphenols glutathione, dithiothreitol (DTT), superoxide dismutase, catalase and the like.
• sulfites e.g., sodium sulfite
• tocopherol or derivates thereof ascorbic acid or derivates thereof
• citric acid propyl gallate
• chitosan glycolate cysteine
• cysteine N-acetyl cysteine plus zinc sulfate
• thiosulfates e.g. sodium thiosulfate
• Chelators such as ethylene diamine tetraacetic acid (EDTA), may also be included.
• EDTA ethylene diamine tetraacetic acid
• Suitable thickeners include, e.g., acrylates/steareth-20 methacrylate copolymer, carbomer, carboxymethyl starch, cera alba, dimethicone/vinyl dimethicone crosspolymer, propylene glycol alginate, hydroxy ethylcellulose, hydroxypropyl methylcellulose, silica, silica dimethyl silylate, xanthan gum, and hydrogenated butylenes/ethylene/styrene copolymer.
• Suitable moisturizers include, e.g., butylene glycol, cetyl alcohol, dimethicone, dimyristyl tartrate, glucose glycereth-26, glycerin, glyceryl stearate, hydrolyzed milk protein, lactic acid, lactose and other sugars, laureth-8, lecithin, octoxyglycerin, PEG- 12, PEG 135, PEG-150, PEG-20, PEG-8, pentylene glycol, hexylene glycol, phytantriol, poly quatemium-39 PPG-20 methyl glucose ether, propylene glycol, sodium hyaluronate, sodium lactate, sodium PCA, sorbitol, succinoglycan, synthetic beeswax, tri-C14-15 alkyl citrate, and starch.
• Non-limiting embodiments of household/industrial products which may incorporate the disclosed enzymes or enzyme/polymer compositions as a preservative substance, either alone or in combination with one or more additional preservative substance, such as one or more petrochemically derived preservative substance, include, but are not limited to, householder cleaners, such as concentrated liquid cleaners and spray cleaners, cleaning wipes, dish washing liquid, dish washer detergent, spray-mop liquid, furniture polish, indoor paint, outdoor paint, dusting spray, laundry detergent, fabric softener, rug/fabric cleaner, window and glass cleaner, toilet bowl cleaner, liquid/cream cleanser, etc.
• householder cleaners such as concentrated liquid cleaners and spray cleaners, cleaning wipes, dish washing liquid, dish washer detergent, spray-mop liquid, furniture polish, indoor paint, outdoor paint, dusting spray, laundry detergent, fabric softener, rug/fabric cleaner, window and glass cleaner, toilet bowl cleaner, liquid/cream cleanser, etc.
• compositions described herein may be used in a food wash product, e.g., designed to clean fruits and vegetables prior to consumption.
• “Household products” are products, other than personal care products, that would be used by individual consumers.
• “Industrial products” refers to products that are used in industry.
• a biocidal enzyme e.g., zymogen-class enzyme, such as a cross- linking or lytic enzyme, alone or in combination with (e.g., immobilized on or encapsulated in) a polymer, e.g., a biocidal polymer, such as but not limited to, chitosan, is combined with one or more additional preservative substance, such as one or more petrochemical derived preservative substance.
• one or more biobased preservative i.e., enzyme or enzyme/polymer composition as disclosed herein
• one or more synthetic preservative e.g., petrochemical derived substance
• the preservative e.g., antimicrobial effect achieved between the biobased and synthetic preservatives is additive or synergistic.
• one or more biobased preservative i.e., enzyme or enzyme/polymer composition as disclosed herein
• one or more additional preservative substance for example, a biocidal substance selected from polylysine, chitosan, benzoate, nisin, lysozyme, and chitosan, or any combination thereof, and the preservative (e.g., antimicrobial) effect achieved between the biobased preservative and the additional preservative substance(s) is additive or synergistic.
• Other products into which the disclosed enzymes or enzyme-polymer compositions as disclosed herein may be incorporated include, but are not limited to, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy (e.g., fracking fluid), plastic, packaging, and agricultural products.
• the disclosed enzymes or enzyme-polymer compositions disclosed herein may be incorporated into HVAC systems, cooling ponds, water purification systems, or may be used in an industrial application, such as, but not limited to, pulp and paper processing.
• a biocidal enzyme e.g., zymogen-class enzyme, such as a cross- linking or lytic enzyme, alone or in combination with (e.g., immobilized on or encapsulated in) a polymer, e.g., a biocidal polymer, such as but not limited to, chitosan, is combined with one or more additional preservative substance, such as one or more petrochemically derived preservative substance.
• one or more biobased preservative i.e., enzyme or enzyme/polymer composition as disclosed herein
• one or more synthetic preservative e.g., petrochemically derived substance
• the preservative e.g., antimicrobial effect achieved between the biobased and synthetic preservatives is additive or synergistic.
• the disclosed enzymes and enzyme/polymer compositions may be used as alternatives or in addition to conventional preservatives, such as, but not limited to, parabens,
• biocidal agents such as those disclosed herein, including silver (used in wound care products), in various applications that require preservatives for example, personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy, plastic, packaging, and agricultural products, or in any of the products or systems disclosed herein.
• the disclosed enzymes and compositions are used as anti-microbial (e.g., preservative) ingredients that inhibit the growth of potentially harmful bacteria, fungi, and/or other microbes, and accordingly, are added to the product to be preserved in an effective amount to inhibit bacterial, fungal, and/or microbial growth in these products.
• USP ⁇ 51> passing criteria are achieved, i.e., for Category 2 Products: Bacteria: No less than 2.0 log reduction from the initial calculated count at 14 days, and no increase from the 14 days' count at 28 days; for Yeast and Molds: No increase from the initial calculated count at 14 and 28 days.
• the antimicrobial behavior of the enzymes and enzyme-biopolymer coformulations are
• MIC minimum inhibitory concentration
• the composition When combined with a product as described herein, e.g., a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy, plastic, packaging, or agricultural product, or in any of the products or systems disclosed herein, e.g., in a formulation or incorporated into a product or system as a preservative, the composition may have effective broad spectrum preservation activity over a broad pH range.
• a product as described herein e.g., a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy, plastic, packaging, or agricultural product, or in any of the products or systems disclosed herein, e.g., in a formulation or incorporated into a product or system as a preservative, the composition may have effective broad spectrum preservation activity over a broad pH range.
• the method includes adding a preservative composition as described herein (e.g., a zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme) or compositions (e.g., zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme, immobilized on or encapsulated in a polymer, such as a biopolymer and/or a biocidal polymer to a product or system, such as a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy, plastic, packaging, or agricultural product, or in any of the products or systems disclosed herein, e.g., in a formulation or incorporated into a product or system, wherein microbial growth is decreased and/or shelf life of the product is increased in comparison to an identical product that does not contain the preservative composition as described herein (
• the enzyme is a zymogen-class enzymes, such as an enzyme selected from a hydrolase, a protease, a lytic enzyme, a cross- linking enzyme, and combinations thereof.
• the enzyme is a cross- linking enzyme, such as a transglutaminase, laccase, peroxidase, transferase, lysyl oxidase, or tyrosinase, or a combination thereof.
• polymeric support is a biocidal polymer, such as chitosan or carboxymethylchitosan.
• the enzyme is immobilized on the support via a linker. In some embodiments, no other preservative is included in the product composition, such as, but not limited to formaldehyde and/or glutaraldehyde.
• a method for increasing the shelf-like, integrity, or microbial free (e.g., bacterial and/or fungal free) status of a product composition such as a personal care, household or industrial product
• the method includes incorporating an effective amount of a preservative composition as described herein (e.g., a biocidal enzyme immobilized on a polymeric support (e.g., a biocidal polymer)) into the product (e.g., personal care, household or industrial product).
• the effective amount may be an amount, referred to as the MIC (minimum inhibitory concentration), which results in reduction of microbial growth by approximately 80 - 100%, or any of at least about 80%, 85%, 90%, 95%, 98%, or 99% reduction of microbial growth as described herein.
• MIC minimum inhibitory concentration
• an enzyme e.g., a zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme
• a concentration of about 0.01% w/v to about 5% w/v or any of at least about 0.01% w/v, 0.05% w/v, 0.1% w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, or 5% w/v, or any of about 0.01% w/v to about 0.05% w/v, about 0.1% w/v to about 0.5% w/v, about 1% w/v to about 1.5% w/v, about 1.5% w/v to about 2%
• enzyme/polymer composition such as a zymogen-class enzyme, such as a crosslinking or lytic enzyme or other enzyme disclosed herein, e.g., a biocidal enzyme, immobilized on or encapsulated in a polymer, such as a biopolymer and/or a biocidal polymer, may be included at a concentration of about 0.04% w/v enzyme and about 0.025% w/v polymer (such as, but not limited to, chitosan).
• Examples of personal care products which may utilize the disclosed compositions of the include bar soap, liquid soap (e.g., hand soap), hand sanitizer (including rinse off and leave-on alcohol based and aqueous-based hand disinfectants), preoperative skin disinfectant, cleansing wipes, disinfecting wipes, body wash, acne treatment products, antifungal diaper rash cream, antifungal skin cream, shampoo, conditioner, cosmetics (including but not limited to liquid or powder foundation, liquid or solid eyeliner, mascara, cream eye shadow, tinted powder, "pancake” type powder to be used dry or moistened, make up removal products etc.) deodorant, antimicrobial creams, body lotion, hand cream, topical cream, aftershave lotion, skin toner, mouth wash, toothpaste, sunscreen lotion, and baby products such as, but not limited to, cleansing wipes, baby shampoo, baby soap, and diaper cream.
• bar soap liquid soap
• hand soap hand sanitizer
• preoperative skin disinfectant including rinse off and leave-on alcohol based and aqueous-based hand disinfect
• wound care items such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, bum wound cream, bandages, tape, and steri- strips, and medical articles such as medical gowns, caps, face masks, and shoe-covers, surgical drops, etc.
• Additional products include but are not limited to oral products such as mouth rinse, toothpaste, and dental floss coatings, veterinary and pet care products, preservative
• compositions, and surface disinfectants including solutions, sprays or wipes.
• Non-limiting examples of household/industrial products which may incorporate the disclosed compositions include householder cleaners such as concentrated liquid cleaners and spray cleaners, cleaning wipes, dish washing liquid, dish washer detergent, spray-mop liquid, furniture polish, indoor paint, outdoor paint, dusting spray, laundry detergent, fabric softener, rug/fabric cleaner, window and glass cleaner, toilet bowl cleaner, liquid/cream cleanser, etc.
• the compositions of the present subject matter may be used in a food wash product, designed to clean fruits and vegetables prior to consumption, packaging, and food coatings.
• TI formulation Commercially available wild-type Streptomyces mobaraensis transglutaminase (TI formulation) was secured from Ajinimoto. TGase is available from Ajinomoto USA under the trade name Activa-TI. This product is sold as a solid preparation of 99% maltodextrin and 1% microbial enzyme. Ajinimoto reports the enzyme activity is 81-135 U/g. The Activa-TI was used as received as well as purified from the maltodextrin by dialysis to concentrate the enzyme.
• the cells were grown in shake flasks, lysed by homogenization, and the S2P-TG was isolated from the cell debris by centrifugation.
• the resulting semi-purified enzyme (clarified lysate) were compared on an SDS-PAGE gel, by spectroscopy, and activity for concentration of active enzyme.
• the S2P-TG was further purified by affinity column on a Ni-IMAC resin prior to MIC assay.
• the clarified lysate containing S2P-TG was compared to His-tag purified S2P-TG for polymer conjugation studies and found to perform similarly.
• the His-tag purified S2P-TG was used for MIC studies.
• Microbeads were collected via filtration and washed with hexane, ethanol, and water.
• Enzyme immobilization The chitosan beads were pre-activated 0.1% - 5% v/v glutaraldehyde at room temperature for 5 hr. Excess glutaraldehyde was removed by washing twice with water. Semi-purified enzyme (clarified lysate) was added at total protein concentration 0.2 - 0.4 mg/mL and incubated at 4 °C for 16 hr. Unbound enzyme was removed by washing twice with water. Negative controls containing chitosan beads and glutaraldehyde alone or chitosan beads and clarified lysate alone were also included. The samples were assayed for activity using the colorimetric hydroxamate assay and HPLC based assay.
• the mixture was then centrifuged to collect the precipitated chitosan, effectively washing this phase of free, unbound enzyme, which remained in the supernatant.
• the supernatant was removed and the resulting pellet then resolubilized with pH adjustment, typically into acidic solution or buffer (either 1% acetic acid or 10 mM Tris, pH 4.3).
• the three samples resulting from this process labeled“R” (Reaction),“S” (Supernatant), and“P” (Pellet), were assayed for activity using the hydroxamate assay.
• Transglutaminase was immobilized by encapsulation with free carboxymethylchitosan monomers, as described in Example 4.
• the linker was l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) linker.
• EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride
• Transglutaminase was immobilized by encapsulation with free chitosan monomers, as described in Example 4.
• the linker was EDC.
• Control reactions were also performed for samples prepared in the absence of enzyme and linker. The results are shown in Fig. 7.
• excess unbound enzyme was removed by centrifugation and removal of the supernatant.
• the resolubilized pellet (P) containing EDC, S2P-TG, and chitosan together is the sole reaction that demonstrates enzymatic activity (roughly 82% of the total enzyme activity is retained).
• the plate was then incubated with shaking at the appropriate temperature for the strain being tested in a plate reader (Biotek Synergy HI) and the optical density at 600 nm recorded every 10 min over the course of the assay (9 - 18 hours).
• the BacTiter Glo assay was performed according to the manufacturer’s instructions with 25 mL of each culture and 25 mL of BacTiter Glo Assay Reagent in a black-walled 96 well half area plate (Promega). The assay was incubated with shaking for 5 minutes at room temperature and then the luminescent signal intensity quantified.

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