US20230203457A1 - Transglutaminase variants and applications of use thereof - Google Patents

Transglutaminase variants and applications of use thereof Download PDF

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US20230203457A1
US20230203457A1 US17/998,576 US202117998576A US2023203457A1 US 20230203457 A1 US20230203457 A1 US 20230203457A1 US 202117998576 A US202117998576 A US 202117998576A US 2023203457 A1 US2023203457 A1 US 2023203457A1
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enzyme
variant
tgase
product
transglutaminase
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William Shindel
Kamil S. GEDEON
Erika M. Milczek
Simone A. COSTA
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Curie Co Inc
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Curie Co Inc
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    • 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.)
    • C12N9/1022—Transferases (2.) transferring aldehyde or ketonic groups (2.2)
    • 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
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • 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
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • 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
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/35—Ketones, e.g. benzophenone
    • A—HUMAN NECESSITIES
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    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
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    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A—HUMAN NECESSITIES
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    • A61K8/40—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing nitrogen
    • A61K8/44—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof
    • A61K8/445—Aminocarboxylic acids or derivatives thereof, e.g. aminocarboxylic acids containing sulfur; Salts; Esters or N-acylated derivatives thereof aromatic, i.e. the carboxylic acid directly linked to the aromatic ring
    • 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
    • A61K8/46—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur
    • A61K8/466—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing sulfur containing sulfonic acid derivatives; Salts
    • A—HUMAN NECESSITIES
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    • 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
    • A—HUMAN NECESSITIES
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    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
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    • 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
    • A61K8/84—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds obtained by reactions otherwise than those involving only carbon-carbon unsaturated bonds
    • A61K8/89—Polysiloxanes
    • A61K8/891—Polysiloxanes saturated, e.g. dimethicone, phenyl trimethicone, C24-C28 methicone or stearyl dimethicone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the present invention relates to engineered variant microbial transglutaminase polypeptides comprising two or more amino acid substitutions, and the nucleic acids encoding them.
  • the variants may be used for conjugating proteins, peptides, or small molecules with increased specific activity compared to wild-type Streptomyces mobaraensis transglutaminase, such as in topical applications of use.
  • the variants may also be used as active biocidal enzymes and in formulations thereof for use as agents for broad spectrum microbial control.
  • Transglutaminases are enzymes capable of catalyzing an acyl transfer reaction in which a ⁇ -carboxy-amide group of a peptide bound glutamine residue is the acyl donor.
  • Primary amino groups in a variety of compounds may function as acyl acceptors with the subsequent formation of monosubstituted ⁇ -amides of peptide bound glutamine.
  • the Tgases form intramolecular or intermolecular ⁇ -glutamyl- ⁇ -lysyl crosslinks.
  • the catalytic reaction proceeds via glutamine deamination and formation of a protein-glutamyl-thioester at the active site of the enzyme.
  • Nucleophilic attack by a lysyl ⁇ -amino group of a second protein at the carbonyl moiety of the thioester intermediate generates isopeptide-crosslinked proteins that are largely resistant to proteolysis by common peptidases (Mariniello, et al. (2007) J Agr Food Chem. 55:4717-4721). Bonds formed by a Tgase exhibit high resistance to proteolytic degradation (proteolysis). Tgases from microbial origin are calcium-independent, which represents a major advantage for their practical use.
  • Tgase has found many applications in biotechnology and in the food processing industry, where it has earned the moniker “meat glue.”
  • the peptide crosslinking activity has shown useful for a variety of industrial purposes ranging from food processing, biotechnology, pharmaceuticals, medical devices, personal and household goods, and leather and textile treatment.
  • the most commonly used Tgase is microbial transglutaminase from Streptomyces mobaraensis , having the amino acid sequence depicted in SEQ ID NO:1 and referred to hereinafter as Tgase.
  • Lysyl oxidases (also known as protein-lysine 6-oxidase) are copper-dependent enzymes that catalyze formation of aldehydes from lysine residues in collagen and elastin precursors. These aldehydes are highly reactive, and undergo spontaneous chemical reactions with other lysyl oxidase-derived aldehyde residues, or with unmodified lysine residues. This results in cross-linking collagen and elastin. LOX proteins have been identified in animals, other eukaryotes, and in bacteria and archaea (reviewed in Grau-Bove, et al. (2015) Scientific Reports 5: Article number: 10568).
  • Powder-type formulations may be perceived as having a shorter useful life than the liquid-type formulations (i.e., do not provide the desired cosmetic benefit for as long) and/or are more susceptible to undesirable transfer from the skin to another surface. This is due to the fact that powder-type foundation products are essentially individual solid particles lying on the skin surface with little to prevent them from being rubbed or wiped away. Throughout the course of the day, the product is exposed to sebum (produced by the skin), moisture (sweat, tears, humidity, rain, washing) and oils (skin care products, other cosmetic products). As such, conventional liquid and semi-liquid products are not suited for staying on the skin for longer than one day.
  • One drawback shared by conventional concealers and foundations is that they may be unintentionally transferred to other surfaces (e.g., clothes, furniture, hair, and other areas of the body). Such transfers may result in clothing, furniture or other surfaces being soiled with makeup, and/or it may reduce the effective life of the makeup.
  • a variety of mechanisms may contribute to the undesired transfer of makeup from the skin. For example, sebum or other waxy/oily substances found on the skin can impede the ability of a foundation or concealer to adhere to the skin. In some instances, rubbing against a surface may abrade the concealer or foundation from the skin.
  • “long-wear” or “transfer-free” products are known. However, even conventional long-wear or transfer-free products may not provide a suitable level of coverage and/or beauty enhancement for more than 24 hours or provide suitable resistance to the abrasion encountered by skin during typical daily activities.
  • a new category of cosmetic and personal care products is desired: a semi-permanent technology that provides the same effect as when the product is initially applied, for multiple hours or days. Such a product does not require daily application or the frequent touch-ups of conventional technologies.
  • Such a method of dying has several disadvantages.
  • uniform dying can be obtained only with great difficulty.
  • the casing shows regions which are dyed more weakly or not at all, depending on how close together the sausages were arranged and how adequately they were wetted.
  • the dyestuff tends to both bleed out and also to migrate into the sausage material.
  • An improved method for dying sausage casings would be desirable.
  • Transglutaminase (Tgase) enzymes are provided herein.
  • the enzymes are variants of Streptomyces mobaraensis Tgase (SEQ ID NO:1). Some of the variants demonstrate improvement in transamidation activity that at least about 1.2-fold or at least about 2-fold, e.g., about 1.2-fold to about 10-fold greater than the wild-type Streptomyces mobaraensis enzyme (at least about or greater than any of about 20%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or 1000% improvement in enzymatic activity).
  • transglutaminase It is desirable to have high specific activity of transglutaminase to allow for lower quantities of enzyme for cross-linking glutamine-donor substrates with amine substrates in the transamidation reaction, to allow for lower cost of product development. Additionally, it is beneficial to identify mutational variants of transglutaminase that exhibit higher initial rates to a deliver shorter reaction times. Such variants may promote rapid cross-linking for applications of use such as microbial control.
  • microbial control One example is preservation, where rapid crosslinking of proteins, e.g., cellular surface proteins, leads to superior microbial control, such as, but not limited to, faster or more effective microbial kill rate.
  • the transglutaminase variants may be use in applications such as bonding of dye molecules or pigments to collagen, keratin, elastin, and/or other structural or accessory skin, hair, or nail proteins or peptides, which may be found in a product formulation or on the surface of skin, hair, or nails, such as bonding agents for semi-permanent or permanent application of functional ingredients, color, dye, or pigment in long-lasting topically applied products.
  • variants of the Streptomyces mobaraensis Tgase enzyme are provided.
  • the variant comprises or consists of: (i) S199G and S299V (SEQ ID NO:2); (ii) H289V and S299A (SEQ ID NO:3); (iii) N292M, H289T, and S299V (SEQ ID NO:4); (iv) N282E, H289V, and S299K (SEQ ID NO:5); (v) S284D, H289L, and S299K (SEQ ID NO:6); (vi) N282E, H289I, and S299K (SEQ ID NO:7); (vii) N282K, G283A, and S299V (SEQ ID NO:8); (viii) N282Q, S284P, H289E, and S299V (SEQ ID NO:9); (ix) N282K, G283
  • Variants of the Tgase sequence depicted in SEQ ID NO:1 are provided that comprise or consist of substitutions of amino acid residue A or G at position 199 and amino acid residue A, E, K, or V at position 299, or has the disclosed mutations and one or more conservative substitution within the remainder of the amino acid sequence, e.g., having the disclosed mutations and at least about 90%, at least about 95%, or at least about 98% sequence identity to the disclosed amino acid sequence, or is a circular permutant of a variant having the disclosed mutations.
  • Variants of the Tgase sequence depicted in SEQ ID NO:1 comprise or consist of substitutions of amino acid residue C, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, or Y at position 2, A or G at position 199 and amino acid residue A, E, K, or V at position 299, or has the disclosed mutations and one or more conservative substitution within the remainder of the amino acid sequence, e.g., having the disclosed mutations and at least about 90%, at least about 95%, or at least about 98% sequence identity to the disclosed amino acid sequence, or is a circular permutant of a variants having the disclosed mutations.
  • Variants of the Tgase sequence depicted in SEQ ID NO:1 are provided that comprise or consist of substitutions of amino acid residue E or M at position 282, amino acid residue I, T, or V at position 289, and amino acid residue K or V at position 299, or has the disclosed mutations and one or more conservative substitution within the remainder of the amino acid sequence, e.g., having the disclosed mutations and at least about 90%, at least about 95%, or at least about 98% sequence identity to the disclosed amino acid sequence, or is a circular permutant of a variant having the disclosed mutations.
  • a variant as described herein further comprises a C-terminal polyhistidine sequence. In some embodiments, a variant as described herein further comprises an N-terminal methionine residue. In some embodiments, the transglutaminase is a circular permutant of any of the amino acid sequences described herein, optionally further comprising an N-terminal methionine residue. In some embodiments, the transglutaminase enzyme further includes a pro-sequence.
  • a variant as disclosed herein, or a variant having disclosed mutations and at least about 90%, at least about 95%, or at least about 98% sequence identity to the disclosed amino acid sequence, or a variant that is a circular permutant of a variant having disclosed mutations has at least about 2-fold greater transglutaminase enzyme activity than the wild type enzyme having the amino acid sequence depicted in SEQ ID NO:1.
  • methods for increasing the shelf life and/or eliminating odor of a product.
  • the methods include incorporating a transglutaminase variant 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 include the composition.
  • products that include a transglutaminase variant as described herein in an effective amount to increase the shelf life of the product, in comparison to an identical product that does not include the enzyme.
  • the product may be a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy, plastic, packaging, or agricultural product.
  • the product 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, or sunscreen lotion.
  • the product is a wound care product selected from wound healing ointments, creams, and lotions, wound coverings, burn wound cream, bandages, tape, or steri-strips.
  • an enzyme composition in another aspect, includes: (i) a transglutaminase variant enzyme as described herein, a transglutaminase enzyme comprising or consisting of the amino acid sequence depicted in SEQ ID NO:1, a transglutaminase enzyme comprising or consisting of a; and (ii) a substrate for the transglutaminase enzyme, such as a sunscreen molecule, functional ingredient, a pigment, or a dye molecule.
  • the sunscreen molecule, functional ingredient, pigment or dye molecule is conjugated to a molecule that includes a free amino group ( FIG. 2 ).
  • the molecule that includes a free amino group may be derived from derivatization with a linker such as lysine, cadaverine, putrescine, hydrazine, adipic acid dihydrazide, sebacic dihydrazide, or hexamethylenediamine ( FIG. 4 ).
  • a linker such as lysine, cadaverine, putrescine, hydrazine, adipic acid dihydrazide, sebacic dihydrazide, or hexamethylenediamine ( FIG. 4 ).
  • the molecule that includes a free amino group is derived from an aliphatic amine of formula R(CH 2 ) n (NH 2 ), wherein n is an integer between 1 and 30, or n is an integer between 5 and 10, and R is a functional ingredient.
  • R(CH 2 ) n (NH 2 ) aliphatic amine of formula R(CH 2 ) n (NH 2 ), wherein n is an integer between 1 and 30, or n is an integer between 5 and 10, and R is a functional ingredient.
  • the sunscreen molecule, pigment, or dye molecule is conjugated to an amino acid, peptide, or protein with a free glutamine side chain ( FIG. 3 ).
  • Cosmetic compositions that include the enzyme composition are also provided.
  • methods for bonding color to a material or protein of interest.
  • the methods include contacting the material or protein of interest with a transglutaminase variant enzyme as described herein and a pigment or dye molecule, wherein the transglutaminase variant enzyme is present in an amount effective to covalently bind the pigment or dye molecule to the material or protein of interest ( FIG. 7 and FIG. 8 ).
  • the protein of interest is a protein that is present in skin.
  • the protein that is present in skin may be collagen, keratin, and/or elastin.
  • products that include a transglutaminase variant enzyme as described herein in an effective amount to add a color molecule onto a protein or a protein-, peptide-, or amino acid-containing material of interest when contacted with the product.
  • the product may be a personal care, cosmetic, leather, food, or agricultural product.
  • Methods for modifying the color of a protein or material of interest are also provided, which include contacting the protein or material of interest with the product.
  • compositions that include a transglutaminase variant enzyme as described herein in combination with one or more antimicrobial enzyme, peptide, or protein, wherein the composition possesses a preservative, biocidal, antimicrobial, or virucidal activity.
  • the antimicrobial enzyme, peptide, or protein is lysozyme, chitinase, lipase, lysin, lysostaphin, glucanase, DNase, RNase, lactoferrin, glucose oxidase, peroxidase, lactoperoxidase, lactonase, acylase, dispersin B, a-amylase, cellulase, nisin, bacteriocin, siderophore, polymyxin, or defensin.
  • a bacteriophage which includes a nucleic acid sequence that encodes a transglutaminase variant enzyme as described herein.
  • a composition that includes the bacteriophage provides antimicrobial activity.
  • the composition may further include a pharmaceutically acceptable excipient.
  • FIG. 1 schematically shows covalent addition of sunscreen conjugated to an aliphatic diamine (CH2) n (NH 2 ) 2 , where the sunscreen is oxybenzone or avobenzone, to an example protein, collagen, catalyzed by transglutaminase or lysyl oxidase to yield sunscreen-linker-collagen.
  • the enzyme catalyzes cross-linking between an amine group on the sunscreen molecule and either a Gln sidechain (transglutaminase) or Lys sidechain (lysyl oxidase) on collagen.
  • the enzyme can be encapsulated or unencapsulated.
  • FIG. 2 schematically shows covalent addition of sunscreen-linker-NH 2 to an example protein, collagen, catalyzed by transglutaminase to yield sunscreen-linker-collagen.
  • the enzyme catalyzes cross-linking between an amine group on the linker molecule and a Gln sidechain on collagen.
  • the enzyme can be encapsulated or unencapsulated.
  • FIG. 3 schematically shows covalent addition of sunscreen-linker-Gln to an example protein, collagen, catalyzed by transglutaminase to yield sunscreen-linker-collagen.
  • the enzyme catalyzes cross-linking between the Gln group on the linker molecule and an available amine on a Lys residue on collagen.
  • the enzyme can be encapsulated or unencapsulated.
  • FIGS. 4 A- 4 C illustrate formulas of sunscreen and linker molecules which may be combined to produce sunscreen-linker adducts.
  • FIG. 4 A shows a method for functionalizing oxybenzone via carbamate formation at the phenolic alcohol but not demonstrated to have reactivity with transglutaminase.
  • FIG. 4 B shows imine formation via the carbonyl of oxybenzone produces a transglutaminase compatible amine donor.
  • FIG. 4 C shows imine formation via the carbonyl of avobenzone produces a mixture of two transglutaminase compatible amine donors.
  • FIG. 5 illustrates the reaction of 1,6-hexane diamine (HMDA) with oxybenzone followed by covalent addition of the oxybenzone-linker adduct to an example protein, Cbz-Gln-Gly, catalyzed by transglutaminase to yield sunscreen-linker-protein.
  • the enzyme catalyzes cross-linking between a Gln sidechain on dipeptide and an available amine group on the sunscreen-linker.
  • the enzyme can be encapsulated or unencapsulated.
  • FIG. 6 shows characterization of the reaction product of Tgase catalyzed coupling of oxybenzone-linker to Cbz-Gln-Gly. This figure compares the amount of sunscreen-linker-protein product between wild-type Tgase, Tgase variant SEQ ID NO:28, and a negative control in the absence of Tgase.
  • Tgase SEQ ID NO:28 contains 11-fold more product than wild-type Tgase. No sunscreen-linker-protein product is observed in the absence of Tgase.
  • FIG. 7 schematically shows covalent addition of Dye-NH 2 (TAMRA-cadaverine) to Collagen catalyzed by transglutaminase to yield Dye-Collagen.
  • the enzyme catalyzes cross-linking between an amine group on the dye molecule and a Gln sidechain on collagen.
  • the enzyme can be encapsulated or unencapsulated.
  • FIG. 8 schematically shows covalent addition of Dye-Gln to Collagen catalyzed by transglutaminase to yield Dye-Collagen.
  • the enzyme catalyzes cross-linking between a Gln sidechain on the dye (ZQG-TAMRA) and a Lys, N-terminus, or other amine group on collagen.
  • ZQG-TAMRA ZQG-TAMRA
  • the enzyme can be encapsulated or unencapsulated.
  • FIG. 9 shows an enzyme dose response of TAMRA-cadaverine (1.7 g/L) in solution covalently bound to a collagen plate catalyzed by Tgase variant SEQ ID NO:28 (0 to 0.33% w/v).
  • Dye (TAMRA-cadaverine) was bound to the collagen plate by increasing amounts of Tgase variant SEQ ID NO:28 (left to right).
  • BSA bovine serum albumin
  • Bottom row shows the collagen-bound dye after removing residual, unbound dye through a washing process with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • FIG. 10 shows covalent addition of the short dye labeled peptide Cbz-Gln-Gly-TAMRA (2.5 g/L) in solution to a collagen plate catalyzed by Tgase variant SEQ ID NO:28.
  • Column 1 illustrates the Dye (Cbz-Gln-Gly-TAMRA) in buffer added to the collagen plate in the presence or absence of Tgase.
  • Column 2 illustrates removal of the excess reaction mixture after incubation.
  • Column 3 represents a washing step with PBS to remove unbound Dye.
  • Column 4 represents Dye bound to collagen after removal of excess unbound Dye. Dye was bound to the collagen in the presence of Tgase, and minimal or no binding was observed in the absence of Tgase.
  • Tgase enzymes are provided herein.
  • the enzymes are variants of Tgase (SEQ ID NO:1), and demonstrate improvements in transamidation activity that is at least about 1.2-fold, at least about 2-fold, at least about 10-fold, or at least about 11-fold greater than the wild-type Streptomyces mobaraensis enzyme.
  • lysyl oxidase (LOX) enzymes which may be utilized to form reactive aldehydes, for example, on cosmetically relevant proteins, such as collagen, to react with functional ingredients containing a nucleophile, such as a free R—NH 2 .
  • LOX lysyl oxidase
  • the cross-linking enzymes disclosed herein may be employed as novel biocidal agents for microbial control, with applications in healthcare products, personal care or cosmetic formulations, packaging (e.g., food, cosmetic, and pharmaceuticals), textile and leather production, paints and coatings, and marine applications including water treatment and purification.
  • Tgase enzymes disclosed herein may be employed for permanently modifying proteins of interest, such as, but not limited to, keratin and collagen, with dyes or proteins.
  • the Tgase enzymes may be used as preservatives.
  • Tgase enzymes that are mutant forms of the Streptomyces mobaraensis Tgase are disclosed herein.
  • the enzymes described herein are proteins obtained by mutating at least two amino acids in the polypeptide sequence of the wild-type Tgase, or circular permutants thereof, and observing transglutaminase transamidation activity between a glutamine amino acid residue and an amine (or hydroxylamine) acceptor.
  • Such methods can include, but are not limited to, using kits and commercially available reagents such as the Kunkel method, KLD method, or Gapped duplex method, and examples of the kit, for example, QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene), GeneArtTM Site-Directed Mutagenesis System (Invitrogen), Q5®, Site-Directed Mutagenesis System (New England Biolabs), TaKaRa Site-Directed Mutagenesis System (Prime STAR® Mutagenesis Basal kit, or Muta-DirectTM Site Directed Mutagenesis Kit (iNtRON), and the like.
  • kits and commercially available reagents such as the Kunkel method, KLD method, or Gapped duplex method, and examples of the kit, for example, QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene), GeneArtTM Site-Directed Mutagenesis System (Invitrogen), Q5®, Site-Directed Mutagenesis System (New England Biolabs), Ta
  • compositions and methods are provided herein for covalent bonding of functional ingredients such as UV-blocking molecules and/or color producing molecules (such as dye and pigment molecules) to proteins and peptides, in applications of use such as cosmetic and/or sunscreen products, for sunscreen and/or color application and binding to skin and skin-derived proteins and peptides, such as collagen, keratin, and/or elastin, and for color application and binding to food products such as edible food casings.
  • functional ingredients such as UV-blocking molecules and/or color producing molecules (such as dye and pigment molecules)
  • proteins and peptides in applications of use such as cosmetic and/or sunscreen products, for sunscreen and/or color application and binding to skin and skin-derived proteins and peptides, such as collagen, keratin, and/or elastin, and for color application and binding to food products such as edible food casings.
  • nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • 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.
  • 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 30 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.
  • antimicrobial refers to a substance that is intended to kill or inhibit the growth of bacteria, fungi, and viruses, for example, according to the Environmental Protection Agency (EPA).
  • EPA Environmental Protection Agency
  • base pair refers to a partnership (i.e., hydrogen bonded pairing) of adenine (A) with thymine (T), or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
  • a base pair may include A paired with Uracil (U), for example, in a DNA/RNA duplex.
  • a “bead” refers to a solid particle, comprising or consisting of a polymer as described herein.
  • biocide refers to a substance that kills microorganisms, for example, according to the Environmental Protection Agency (EPA).
  • EPA Environmental Protection Agency
  • Biodegradable refers to a substance that is capable of decomposition by microbes (e.g., bacteria) or other living organisms.”
  • catalyst refers to a chemical actor, such as a molecule or macromolecular structure, 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 After 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.
  • a “circular permutant” refers to a protein that has a changed order of amino acids in its amino acid sequence in comparison with a reference sequence. The result is a protein structure with different connectivity, but overall similar three-dimensional (3D) shape in comparison to the reference protein. For example, an N-terminal fragment may be moved to the C-terminal end of the protein amino acid sequence.
  • a “coloring agent” or “color producing molecule” refers to a molecule or compound that imparts a color to mammalian (e.g., human) skin, hair, or nails.
  • Coloring agents may include dyes and/or pigments. Nonlimiting examples of coloring agents include phenols, naphthols, and hydroxy azo derivatives.
  • a coloring agent may be modified to include an amino group (e.g., an alkylamino, alkylhydrazine, alkylhydrazide, or alkoxyamine moiety), attached either directly to the coloring agent or indirectly via a linker that is attached to the coloring agent.
  • composition refers to a combination of two or more substances, for example, a combination that includes one or more cross-linking enzyme as described herein and one or more chromophores, such as UV-blocking molecules, dyes, pigments, or other color-producing molecules.
  • a “conservative modification” or “conservative substitution” means, in respect of a polypeptide, the replacement of an amino acid therein with another amino acid having a similar side chain.
  • Families of amino acids having similar side chains are known in the art. Such families include amino acids with basic side chains (lysine, arginine, histidine), acidic side chains (aspartic acid, glutamic acid), uncharged polar side chains (asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), branched side chains (threonine, valine, isoleucine), small side chains (glycine, alanine, serine), chain orientation changing side chains (glycine, proline) and aromatic side chains (tyrosine, phenylalanine, tryptophan).
  • derived from encompasses the terms “originated from,” “obtained from,” “obtainable from,” “isolated from,” “purified from,” and “created from,” and generally indicates that one specified material finds its origin in another specified material or has features that can be described with reference to another specified material.
  • duplex herein refers to a region of complementarity that exists between two polynucleotide sequences.
  • duplex region refers to the region of sequence complementarity that exists between two oligonucleotides or two portions of a single oligonucleotide.
  • a “dye” refers to a colored substance, e.g., a natural or synthetic substance used to add a color to or change the color of something, which is typically a water-soluble organic molecule.
  • 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.
  • MIC minimum inhibitory concentration
  • the preservative compositions described herein may be active against Gram positive bacteria, Gram negative bacteria, yeasts, and/or molds.
  • “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 Excipients”, 4 th _Ed., Pharmaceutical Press, 2003.
  • 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.
  • 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.
  • expression refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene.
  • the process includes both transcription and translation.
  • “Functional ingredient” refers to an ingredient which performs or fulfills a specific function within a product to deliver a benefit to the consumer.
  • sunscreen molecules block UV radiation and pigment or dye molecules modify skin, hair, or nail color.
  • a “gene” refers to a DNA segment that is involved in producing a polypeptide and includes regions preceding and following the coding regions as well as intervening sequences (introns) between individual coding segments (exons).
  • “Household products” are products, other than personal care products, that would be used by individual consumers.
  • Hybridization and “annealing” refer to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may include two nucleic acid strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of polymerase chain reaction (PCR), ligation reaction, sequencing reaction, or cleavage reaction, e.g., enzymatic cleavage of a polynucleotide by a ribozyme.
  • PCR polymerase chain reaction
  • ligation reaction ligation reaction
  • sequencing reaction or cleavage reaction, e.g., enzymatic cleavage of a polynucleotide by a ribozyme.
  • a first nucleic acid sequence that can be stabilized via hydrogen bonding with the bases of the nucleotide residues of a second sequence is said to be “hybridizable” to the second sequence.
  • the second sequence can also be said to be hybridizable to the first sequence.
  • hybridized refers to a polynucleotide in a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues
  • “Industrial products” refers to products that are used in industry.
  • isolated refers to a material (e.g., a protein, nucleic acid, or cell) that is removed from at least one component with which it is naturally associated, for example, at a concentration of at least 90% by weight, or at 15 least 95% by weight, or at least 98% by weight of the sample in which it is contained.
  • these terms may refer to a material which is substantially or essentially free from components which normally accompany it as found in its native state, such as, for example, an intact biological system.
  • An isolated nucleic acid molecule includes a nucleic acid molecule contained in cells that ordinarily express the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • a “mature” polypeptide, protein or enzyme refers to the activated form of a zymogen or proprotein following cleavage of its pro-sequence or in the absence of the pro-sequence.
  • the mature enzyme may be produced as a separate polypeptide from the pro-sequence in order to eliminate a post-translational processing (activation) step.
  • a “microbead” refers to a bead that is less than one millimeter in its largest dimension.
  • microorganism and “microbe” can include bacteria, protozoa, fungi, algae, amoebas, viruses, and molds life forms.
  • mutation refers to a change introduced into a parental sequence, including, but not limited to, substitutions, insertions, and deletions (including truncations), thereby producing a “mutant.”
  • the consequences of a mutation include, but are not limited to, the creation of a new character, property, function, phenotype or trait not found in the protein encoded by the parental sequence.
  • nucleotide herein refers to a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.
  • the base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of the pentose) and that combination of base and sugar is a nucleoside.
  • nucleoside contains a phosphate group bonded to the 3′ 30 or 5′ position of the pentose it is referred to as a nucleotide.
  • a sequence of polymeric operatively linked nucleotides is typically referred to herein as a “base sequence,” “nucleotide sequence,” “polynucleotide sequence,” “oligonucleotide sequence”, or nucleic acid or polynucleotide “strand,” and is represented herein by a formula whose left to right orientation is in the conventional direction of 5′-terminus to 3′-terminus, referring to the terminal 5′ phosphate group and the terminal 3′ hydroxyl group at the “5′” and “3′” ends of the polymeric sequence, respectively.
  • pathogen refers to microorganisms (e.g., bacteria, viruses, or parasites) that can cause disease in humans, animals, and/or plants.
  • “Peptide” refers to a compound consisting of two or more amino acids linked in a chain, the carboxyl group of each acid being joined to the amino group of the next by a bond of the type R—OC—NH—R′, for example, about 2 to about 50 amino acids.
  • “permanent” refers to the bonded application of either a sunscreen molecule or a color molecule, such as a dye or pigment molecule, on a surface or a protein molecule through a chemical covalent bond rather than a physical deposition or adsorption.
  • a “pigment” refers to a material that provides color, e.g., a material that changes the color of reflected or transmitted light as the result of wavelength-selective absorption, which is typically a water insoluble inorganic substance, such as, but not limited to, a mineral or a metal salt.
  • polymerase herein refers to an enzyme that catalyzes the polymerization of nucleotides (i.e., the polymerase activity).
  • the term polymerase encompasses DNA polymerases, RNA polymerases, and reverse transcriptases.
  • a “DNA polymerase” catalyzes the polymerization of deoxyribonucleotides.
  • An “RNA polymerase” catalyzes the polymerization of ribonucleotides.
  • a “reverse transcriptase” catalyzes the polymerization of deoxyribonucleotides that are complementary to an RNA template.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown, may be single- or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), and may contain deoxyribonucleotides, ribonucleotides, and/or analogs or modified forms of deoxyribonucleotides or ribonucleotides, including modified nucleotides or bases or their analogs. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present invention encompasses polynucleotides which encode a particular amino acid sequence.
  • modified nucleotide or nucleotide analog may be used, so long as the polynucleotide retains the desired functionality under conditions of use, including modifications that increase nuclease resistance (e.g., deoxy, 2′-O-Me,phosphorothioates, etc.).
  • Labels may also be incorporated for purposes of detection or capture, for example, radioactive or nonradioactive labels or anchors, e.g., biotin.
  • the term polynucleotide also includes peptide nucleic acids (PNA).
  • Polynucleotides may be naturally occurring or non-naturally occurring. Polynucleotides may contain RNA, DNA, or both, and/or modified forms and/or analogs thereof.
  • a sequence of nucleotides may be interrupted by non-nucleotide components.
  • One or more phosphodiester linkages may be replaced by alternative linking groups.
  • These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, adapters, and primers.
  • loci locus
  • a polynucleotide may include modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component, tag, reactive moiety, or binding partner. Polynucleotide sequences, when provided, are listed in the 5′ to 3′ direction, unless stated otherwise.
  • 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.
  • “preservative” is an agent added to a product as described herein to prevent (for some period of time) the growth of microorganisms, or the occurrence of undesirable chemical reactions (such as oxidation or odor generation), that spoil or deteriorate, including deterioration of one or more utility, of the product.
  • a product is intended to refer to a preparation or composition that has a specific utility, such as a consumer packaged good. Examples include, but are not limited to, personal care products, household products, cosmetics, over the counter therapeutics, pharmaceutical preparations, paints, coatings, adhesives, and formulations for purchase by a consumer.
  • a product includes a composition for bonding a sunscreen molecule (chromophore), dye, or pigment or modifying a protein with a sunscreen or color-producing molecule, such as a cosmetic or topically applied product.
  • a “promoter” refers to a regulatory sequence that is involved in initiating transcription of a gene by RNA polymerase.
  • a promoter may be an inducible promoter or a constitutive 5 promoter.
  • An “inducible promoter” is a promoter that is active under environmental or developmental regulatory conditions.
  • a “pro-sequence” refers to a polypeptide sequence within an expressed protein, e.g., a zymogen or proprotein, such as transglutaminase, which is typically cleaved from the protein to produce an active protein, such as an enzyme.
  • a pro-sequence may be essential for correct folding of the protein.
  • cleavage of the pro-sequence results in transition of an inactive enzyme to active enzyme.
  • the pro-sequence may be cleaved at multiple positions within the proprotein sequence, which may result in improved enzyme activity or stability relative to the native mature enzyme sequence.
  • the term “recombinant,” refers to genetic material (i.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides) that has been modified to alter its sequence or expression characteristics, such as by mutating the coding sequence to produce an altered polypeptide, fusing the coding sequence to that of another gene, placing a gene under the control of a different promoter, expressing a gene in a heterologous organism, expressing a gene at a decreased or elevated levels, expressing a gene conditionally or constitutively in manner different from its natural expression profile, and the like.
  • genetic material i.e., nucleic acids, the polypeptides they encode, and vectors and cells comprising such polynucleotides
  • nucleic acids, polypeptides, and cells based thereon have been manipulated such that they are not identical to related nucleic acids, polypeptides, and cells found in nature.
  • a recombinant cell may also be referred to as “engineered.”
  • 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, temperature, or ionic strength. This transition process can be repeatably cycled between phases.
  • 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.
  • phrases “substantially similar” and “substantially identical” in the context of at least two nucleic acids typically means that a polynucleotide includes a sequence that has at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% sequence identity, in comparison with a reference (e.g., wild-type) polynucleotide or polypeptide. Sequence identity may be determined using known programs such as BLAST, ALIGN, and CLUSTAL using standard parameters.
  • nucleic acid molecules hybridize to each other under stringent conditions (e.g., within a range of medium to high stringency).
  • Nucleic acid “synthesis” herein refers to any in vitro method for making a new strand of polynucleotide or elongating an existing polynucleotide (i.e., DNA or RNA) in a template dependent manner. Synthesis, according to the invention, can include amplification, which increases the number of copies of a polynucleotide template sequence with the use of a polymerase.
  • Polynucleotide synthesis results in the incorporation of nucleotides into a polynucleotide (e.g., extension from a primer), thereby forming a new polynucleotide molecule complementary to the polynucleotide template.
  • the formed polynucleotide molecule and its template can be used as templates to synthesize additional polynucleotide molecules.
  • DNA synthesis includes, but is not limited to, polymerase chain reaction (PCR), and may include the use of labeled nucleotides, e.g., for probes and oligonucleotide primers, or for polynucleotide sequencing.
  • “Under transcriptional control” is a term well understood in the art that indicates that transcription of a polynucleotide sequence depends on its being operably linked to an element which contributes to the initiation of, or promotes transcription.
  • “Surfactants” are surface-active agents that lower surface tension and thereby increase the emulsifying, foaming, dispersing, spreading and wetting properties of a product.
  • UV-absorbing sunscreen compounds include benzoates, oxybenzones, and cinnamic acid.
  • Exemplary organic sunscreens include, but are not limited to, para-aminobenzoic acid, trolamine salicylate, cinoxate, dioxybenzone, ensulizole, homosalate, meradimate, octinoxate, octisalate, octocrylene, padimate O, sulisobenzone, oxybenzone, and avobenzone.
  • Sunscreens may be approved by the Food and Drug Administration (FDA) or other international regulatory bodies for the purpose of blocking UVA and/or UVB radiation. Sunscreens may also be molecules which provide UVA and/or UVB protection, but which are not yet approved by the FDA or other international regulatory bodies for the purpose of blocking UVA and/or UVB radiation. Sunscreens may also be molecules with analogous structures to those approved by the FDA or other international regulatory bodies and which also provide UVA and/or UVB protection (sunscreen analogs). Sunscreens may also be modified by linkers and/or other small molecules and still provide UVA and/or UVB protection, either with the linker attached or after hydrolysis (sunscreen-linker adducts).
  • FDA Food and Drug Administration
  • Sunscreens may also be molecules which provide UVA and/or UVB protection, but which are not yet approved by the FDA or other international regulatory bodies for the purpose of blocking UVA and/or UVB radiation. Sunscreens may also be molecules with analogous structures to those approved by the FDA or other international regulatory bodies and which also
  • a “sunscreen” herein may refer to a sunscreen molecule, a sunscreen analog, and/or a sunscreen-linker adduct.
  • a sunscreen molecule may be modified to include an amino group (e.g., an alkylamino, alkylhydrazine, alkylhydrazide, or alkoxyamine moiety), attached either directly to the sunscreen molecule or indirectly via a linker (e.g., as part of a linker) that is attached to the sunscreen molecule.
  • TAMRA is an abbreviation for carboxytetramethylrhodamine.
  • variant proteins encompass “variant” proteins.
  • Variant proteins differ from another (i.e., parental) protein and/or from one another by a small number of amino acid residues.
  • a variant may include one or more amino acid mutations (e.g., amino acid deletion, insertion or substitution) as compared to the parental protein from which it is derived.
  • variants may have a specified degree of sequence identity with a reference protein or nucleic acid, e.g., as determined using a sequence alignment tool, such as BLAST, ALIGN, and CLUSTAL (see, infra).
  • variant proteins or nucleic acid may have at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with a reference sequence.
  • a “zymogen” or “proenzyme” refers to an inactive precursor of an enzyme, which may be converted into an active enzyme by catalytic action, such as via proteolytic cleavage of a pro-sequence.
  • Tgase enzymes are provided herein that are variants of the Ca 2+ -independent microbial transglutaminase (Tgase) Streptomyces mobaraensis .
  • Tgase variants with at least about 1.2-fold (20%) or 2-fold (100%) improvement in enzyme activity, versus the wild-type enzyme from Streptomyces mobaraensis (SEQ ID NO:1), are disclosed herein.
  • Tgase variants as described herein may be obtained by mutating at least two amino acids in the polypeptide sequence of the wild-type Tgase, and observing transglutaminase transamidation activity between a glutamine amino acid residue and an amine (or hydroxylamine) acceptor.
  • Methods for recombinant expression of proteins with mutational substitutions have been described previously and are well known in the art, for example, Molecular Cloning, A Laboratory Manual 4th ed., Cold Spring Harbor Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) and the like. Combinations of point mutations can be generated using a number of methods including error-prone PCR, gene shuffling, molecular breeding, and the like.
  • Tgase variants are disclosed in Table 3, along with enzymatic activity improvement, relative to the wild-type enzyme (SEQ ID NO:1).
  • Tgase variant of this invention has a double mutation (S199G and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase (SEQ ID NO: 1).
  • SEQ ID NO: 2 The amino acid sequence of variant M2 is shown in SEQ ID NO: 2.
  • Tgase variant of this invention has a double mutation (H289V and S299A), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M3 is shown in SEQ ID NO: 3.
  • Tgase variant of this invention has a triple mutation (N282M, H289T and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M4 is shown in SEQ ID NO: 4.
  • Tgase variant of this invention has a triple mutation (N282E, H289V, and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M5 is shown in SEQ ID NO: 5.
  • Tgase variant of this invention has a triple mutation (S284D, H289L, and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M6 is shown in SEQ ID NO: 6.
  • Tgase variant of this invention has a triple mutation (N282E, H289L, and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M7 is shown in SEQ ID NO: 7.
  • Tgase variant of this invention has a quadruple mutation (N282K, G283A, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M8 is shown in SEQ ID NO: 8.
  • Tgase variant of this invention has a quadruple mutation (N282Q, S284P, H289E, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M9 is shown in SEQ ID NO: 9.
  • Tgase variant of this invention has a quadruple mutation (N282K, G283A, S284P, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M10 is shown in SEQ ID NO: 10.
  • Tgase variant of this invention has a five-mutation (N282R, G283A, S284E, H289Q, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M11 is shown in SEQ ID NO: 11.
  • Tgase variant of this invention has a double mutation (S199A and S299A), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M12 is shown in SEQ ID NO: 12.
  • Tgase variant of this invention has a double mutation (S199A and S299E), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M13 is shown in SEQ ID NO: 13.
  • Tgase variant of this invention has a double mutation (S199A and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M14 is shown in SEQ ID NO: 14.
  • Tgase variant of this invention has a double mutation (S199A and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M15 is shown in SEQ ID NO: 15.
  • Tgase variant of this invention has a double mutation (S199G and S299A), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M16 is shown in SEQ ID NO: 16.
  • Tgase variant of this invention has a double mutation (S199G and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M17 is shown in SEQ ID NO: 17.
  • Tgase variant of this invention has a triple mutation (S2P, S199A, and S299A), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M18 is shown in SEQ ID NO: 18.
  • Tgase variant of this invention has a triple mutation (S2P, S199A, and S299E), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M19 is shown in SEQ ID NO: 19.
  • Tgase variant of this invention has a triple mutation (S2P, S199A, and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M20 is shown in SEQ ID NO: 20.
  • Tgase variant of this invention has a triple mutation (S2P, S199A, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M21 is shown in SEQ ID NO: 21.
  • Tgase variant of this invention has a triple mutation (S2P, S199G, and S299A), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M22 is shown in SEQ ID NO: 22.
  • Tgase variant of this invention has a triple mutation (S2P, S199G, and S299E), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M23 is shown in SEQ ID NO: 23.
  • Tgase variant of this invention has a triple mutation (S2P, S199G, and S299K), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M24 is shown in SEQ ID NO: 24.
  • Tgase variant of this invention has a triple mutation (S2P, S199G, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M25 is shown in SEQ ID NO: 25.
  • Tgase variant of this invention has a quadruple mutation (N282D, G283A, S284A, and S299V), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M26 is shown in SEQ ID NO: 26.
  • Tgase variant of this invention has a double mutation (S199G and S299E), relative to the sequence of the wild-type S. mobaraensis Tgase.
  • the amino acid sequence of variant M27 is shown in SEQ ID NO: 27.
  • Variants M2-M27 can have conservative substitutions thereto, provided their respective distinctive substitutions: (i) S199G/S299V, (ii) H289V/S299A, (iii) N292M/H289T/S299V, (iv) N282E/H289V/S299K, (v) S284D/H289L/S299K, (vi) N282E/H289I/S299K, (vii) N282K/G283A/S299V, (viii) N282Q/S284P/H289E/S299V, (ix) N282K/G283A/S284P/S299V, (x) N282R/G283A/S284E/H289Q/S299V, (xi) S199A/S299A, (xii) S199A/S299E, (xiii) S199A/S2
  • Such conservatively modified versions of variants M2-M27 are included in the scope of this invention. Plural conservative substitutions/modifications may be present. Conservatively modified versions of variants M2-M27 may be at least about 90%, 95%, or 98% identical to their respective unmodified sequences and may have at least about 2-fold greater transglutaminase enzymatic activity than the wild-type transglutaminase (SEQ ID NO:1).
  • Tgase variants disclosed herein may further comprise a polyhistidine peptide extension at their C-terminus, as exemplified with amino acid residues 334-339 of SEQ ID NO:29.
  • the polyhistidine peptide is a useful tag for purification purposes and does not affect enzymatic activity.
  • the polyhistidine peptide is 6-8 residues long.
  • Tgase variants disclosed herein may further comprise a methionine residue at their N-terminus.
  • the mature wild-type Streptomyces mobaraensis Tgase enzyme lacks the N-terminal methionine residue encoded by the gene sequence that encodes the enzyme.
  • the Tgase variant is expressed as a variant of the mature Streptomyces mobaraensis Tgase without an N-terminal methionine residue.
  • the Tgase is expressed as the mature Tgase with an additional N-terminal methionine residue, which may be provided by an expression vector from which the Tgase is expressed.
  • Tgase variants herein may further include a pro-sequence.
  • the variant is expressed with a pro-sequence, either as part of the variant polypeptide sequence (e.g., an additional amino acid sequence as an extension of an amino acid sequence described in Table 3) or as a separate polypeptide.
  • the mature variant polypeptide is expressed in the presence of a polypeptide Tgase pro-sequence.
  • a DNA sequence that encodes the pro-sequence and the DNA sequence that encodes the mature Tgase variant are expressed as discrete polypeptide sequences from the same DNA template.
  • the DNA sequence that encodes the mature polypeptide is expressed from a first DNA template, and the DNA sequence that encodes the pro-sequence is expressed from a separate second DNA template.
  • the pro-sequence is synthesized chemically and added to an expression system prior to, during, or after expression of the mature polypeptide.
  • the Tgase variant may be expressed in a cell free expression system, as disclosed in PCT Application No. US20/49226, which is incorporated by reference herein in its entirety.
  • the Tgase variant is expressed, e.g., expressed recombinantly, with a homologous pro-sequence, i.e., the native pro-sequence for the Tgase enzyme, i.e., the pro-sequence for the wild-type Tgase enzyme from the same organism.
  • the Tgase variant is expressed, e.g., expressed recombinantly, with a heterologous pro-sequence, i.e., a pro-sequence for the same enzyme but from a different organism or a pro-sequence for a different enzyme from the same or different organism.
  • a Tgase variant may be a circular permutant of a Tgase variant described herein (e.g., a variant described in Table 3).
  • the Tgase variant may be a circular permutant of a Tgase variant as described in Table 3, optionally further including an N-terminal methionine residue.
  • the circular permutants may provide novel substrate specificities, product profiles, and reaction kinetics versus the parent enzyme, i.e., the wild-type enzyme or a disclosed variant, e.g., as depicted in Table 3.
  • a circular permutant retains the same basic folding of the parent enzyme, but has the N-terminus in a different position, with the original N- and C-termini connected, optionally by a linking sequence.
  • the N-terminal residue of the wild-type or variant enzyme is positioned at a site in the protein other than the natural N-terminus.
  • compositions e.g., biocidal, preservative, antimicrobial, anti-bacterial, and anti-viral (virucidal) compositions that include one or more Tgase variant enzyme as described herein, such as any of the variants disclosed in Table 3, optionally with an N-terminal methionine residue, including circular permutants thereof, and optionally with a pro-sequence as described herein.
  • Tgase variant enzyme as described herein, such as any of the variants disclosed in Table 3, optionally with an N-terminal methionine residue, including circular permutants thereof, and optionally with a pro-sequence as described herein.
  • Such a composition may be included in or with (e.g., within or associated with) products to be preserved, e.g., for microbial control.
  • the Tgase variant 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.
  • the compositions include one or more Tgase variant enzyme, e.g., comprising or consisting of one or more Tgase variant as disclosed herein, 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
  • 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) (https://www.fda.gov/files/about %20fda/published/Pharmaceutical-Microbiology-Manual.pdf).
  • the effectiveness of the preservative system disclosed herein is determined based on the MIC (minimum inhibitory concentration) against a variety of microbes, including, but not limited to, Gram positive bacteria, Gram negative bacteria, yeast and/or mold (e.g. E. coli DH10B, E. coli ATCC 8739, B. subtilis BGSC 1A976, C. albicans ATCC 10231, and/or A. brasiliensis ATCC 16404).
  • Minimum inhibitory concentrations (MICs) are defined as the lowest concentration of an antimicrobial that will inhibit the growth of a microorganism. 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 one or more additional biocidal enzymes, such as a cross-linking enzyme, oxidase, nuclease, hydrolase, protease, and/or lytic enzyme.
  • the composition further includes one or more biocidal chemical, such as, but not limited to, chitosan, polylysine, and/or quaternary ammonium compounds. Exemplary, but nonlimiting examples of biocidal enzymes, compositions, and formulations, and methods of use thereof, are disclosed in PCT/US20/21211, which is incorporated by reference herein in its entirety.
  • a biocidal enzyme enhances the antimicrobial properties of a biocidal chemical 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 Tgase 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.
  • compositions include one or more antimicrobial peptides.
  • antimicrobial peptides include, but are not limited to, nisin and pediocin.
  • compositions include one or more antimicrobial proteins.
  • antimicrobial proteins include, but are not limited to, casein.
  • a Tgase variant enzyme as described herein may be utilized in a biocidal, preservative, anti-bacterial, or anti-viral (virucidal) composition in combination with one or more of the antimicrobial enzymes, peptides, or proteins described in Table 1.
  • insects and fungi 10 3032 Lipase Hydrolyzes extracellular lipids and Prabhawathi et al. polymers. (2014) PLoS One 9(5) Lysin Utilized by bacteriophages to Hoops et al. (2008) Appl. hydrolyze the glycan component of Environ. Microbiol. bacterial cell wall 75: 5, 1388-1394 Lysostaphin Metalloendopeptidase which Kokai-Kun et al. (2003) cleaves the pentaglycine bridges Antimicrob Agents found in cell wall peptidoglycan. Chermother 47(5): 1589-1597 Glucanase Secreted by soil bacteria including Shafi et al.
  • a Tgase variant as described herein may be formulated with one or more biocidal chemical, including, but not limited to chitosan, polylysine, or quaternary ammonium compounds, for example, for use as a biocidal, preservative, anti-bacterial, or anti-viral (virucidal) composition.
  • biocidal chemicals are shown in Table 2.
  • Quaternary ammonium compounds 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).
  • the antimicrobial activity of chitin, chitosan and their derivatives against different groups of microorganisms, such as bacteria, yeast, and fungi, is known.
  • Quaternary ammonium compounds (non-limiting examples include, cetyl pyridinium chloride, benzethonium chloride, benzalkonium chloride, polyaminopropyl biguanide), have limited use for personal care industry due to specific incompatibilities with other cosmetic ingredients.
  • Geogard 233S Geogard 233S, Geogard 233S, Geogard 361
  • these antimicrobial compositions are based on cationic benzethonium chloride which gets deactivated by many anionic ingredients that form important part of topical personal care formulations.
  • Formaldehyde is classified as Category 3 CMR (carcinogenic, mutagenic and reproductive toxicity). However, it is interesting to note that a few antimicrobials that slowly release formaldehyde are still being used and being commercially manufactured. Due to the paucity of effective and well-accepted antimicrobials, the industry is forced to continue with the use of formaldehyde donors like DMDM hydantoin (CAS 6440-58-0), imidazolidinyl urea, and diazolidinyl urea (CAS 39236-46-9). The formaldehyde released by these substances is capable of reacting with several cosmetic ingredients via its very reactive aldehydic carbonyl functionality.
  • UV-A absorber Avobenzone
  • avobenzone reacts with formaldehyde that is released by formaldehyde derivatives.
  • This is a significant disadvantage for sunscreen formulations.
  • Preservative blends Clariant's Niapaguard PDU and Cognis's Elestab 305
  • ISP's Germaben II, Germaben H-E exploit combinations of parabens with diazolidinyl urea.
  • ISP's Germall Plus and Lonza's Glydant Plus utilize diazolidinyl urea along with iodopropynyl butyl carbamate (IPBC).
  • McIntyre's Paragon series has DMDM hydantoin and other antimicrobials like paraben, phenoxy ethanol and IPBC. Symrise's Neo-Dragocide and Thor's Microcare IMP exploit synergy between parabens and imidazolidinyl urea.
  • Parabens are esters of p-hydroxy benzoic acid.
  • Paraben compounds include in particular Methyl-paraben (CAS 99-76-3), Ethyl-paraben (CAS 120-47-8), Propyl-paraben (CAS 94-13-3), Butyl-paraben (CAS 94-26-8), Isopropyl-paraben (CAS 4191-73-5), and Benzyl-paraben (CAS 94-18-8).
  • Clariant's ‘Phenonip’ is a blend of six antimicrobials out of which the five are parabens.
  • the same company offers blends of only parabens as ‘Nipastat’ and ‘Nipasept’, Cognis's Elestab FL 15, Elestab 48, Elestab 50J, Elestab 305, Elestab 388, Elestab 3344, Elestab 4112, Elestab 4121, Elestab 4150 Lipo are all blends of antimicrobials with at least one paraben in them.
  • Induchem's Uniphen P23, ISP's Germaben and LiquaPar series of blends contain several parabens.
  • Galaxy Surfactants offers Galguard NK1 and Galguard NK2 blends that are based on four and five paraben blends respectively with phenoxy ethanol.
  • Neolone MXP of Rohm and Haas has parabens with methyl isothiazolinone.
  • Neo-Dragocide series of blends from Symrise has parabens.
  • Euxyl K 300 of Schulke and Mayr has five parabens.
  • Thor's Microcare PM4 and Microcare PM5 have four and five parabens respectively.
  • Parabens are phenol derivatives; all phenolic antimicrobials have phenolic ‘hydroxyl’ group and that is a very reactive organic functionality with very acidic hydrogen with pKa of 10.
  • Nalco's Merguard series (four blends) relies on halogenated molecules, methyl dirbromo glutaronitrile and 2-bromo-2-nitro-1,3-diol.
  • Several blends of Euxyl series from Schulke and Mayr are based on chlorothiazolinones, methyl dibromo glutaronitrile, 2-bromo-2-nitro-1,3-diol and diazolidinyl urea.
  • Microcare series from Thor employs parabens, 2-bromo-2-nitro-1,3-diol, iodopropynyl butylcarbamate (IPBC), imidazolidynyl urea, and diazolidinyl urea.
  • halogenated antimicrobials are chlorphenesin, and chlorhexidine. It is common knowledge that like phenolic compounds, the halogenated organic molecules exhibit significant levels of toxic effects. For example, IPBC has risk of thyroid hormonal disturbances due to its iodine content. It has not been allowed in Japan and in the EU is allowed only up to 0.02% in leave-on products. Similarly, the EU permits usage of methyl dirbromo glutaronitrile only up to 0.1% in rinse-off products only. Bronopol, 2-bromo-2-nitropropane-1,3-diol, is implicated in generation of carcinogenic nitrosoamines on interacting with some of the nitrogen containing cosmetic ingredients.
  • the antimicrobial efficacy of methyl chloro isothiazolinone is so powerful that it is allowed only in rinse-off products at 15 ppm concentration.
  • Chloromethyl isothiazolinone does have a very broad spectrum of anti-microbial activity, but the toxicity of such powerful anti-microbials is extremely high and hence cosmetic formulators do not prefer to use this kind of powerful antimicrobial in the cosmetics that remain on human skin for a long time. It is reasonable to expect that any strong bactericide at a low concentration (ppm level) is likely to be equally lethal to any other cells of a living organism, including human cells. This is the precise reason why in Japan chloromethyl isothiazolinone is not allowed for preservation if the product is going to come in contact with the mucous membrane.
  • Halogenated compounds include 2,4-dichlorobenzyl-alcohol, Chloroxylenol (also known as 4-chloro-3,5-dimethyl-phenol, Bronopol (also known as 2-bromo-2-nitropropane-1,3-diol, iodopropynyl butyl carbamate.
  • compositions described herein may include vectors (e.g., bacteriophage), for the delivery of genetic material encoding one or more biocidal enzyme(s) (e.g., Tgase variant(s)) as described herein.
  • vectors e.g., bacteriophage
  • biocidal enzyme(s) e.g., Tgase variant(s)
  • bacteriophage and “phage” are used interchangeably to refer to a bacteriophage isolate in which members of the isolate have substantially the same genetic makeup, such as sharing at least about any of 90%, 95%, 99%, 99.9% or more sequence identity in the genome.
  • Bacteriophage or “phage” refers to the parent bacteriophage as well as the progeny or derivatives (such as genetically engineered versions) thereof.
  • the bacteriophage can be a naturally occurring phage isolate, or an engineered phage, including vectors, or nucleic acids that encode at least all essential genes, or the full genome of a phage to carry out the life cycle of the phage inside a host bacterium.
  • compositions that can be included in products to be used for long-lasting application of functional ingredients including UV-blocking sunscreens, and/or coloring agents, such as pigments or dyes.
  • the disclosed compositions include one or more cross-linking enzyme, such as a Tgase enzyme (e.g., a Tgase variant as disclosed herein) and/or a lysyl oxidase enzyme, for the purpose of reacting amino acid residues on skin protein or other protein-, peptide-, or amino acid-containing materials with a molecule of interest, such as a sunscreen, or color producing molecule, e.g., a pigment or dye.
  • a cross-linking enzyme such as a Tgase enzyme (e.g., a Tgase variant as disclosed herein) and/or a lysyl oxidase enzyme, for the purpose of reacting amino acid residues on skin protein or other protein-, peptide-, or amino acid-containing materials with a molecule of interest, such as a sunscreen,
  • the composition includes any of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme.
  • the Tgase and/or lysyl oxidase enzyme(s) acts as a catalyst to crosslink the active or functional ingredient (e.g., sunscreen molecule or coloring agent) to a protein or peptide, such as a protein or peptide of mammalian (e.g., human) skin, hair, or nails.
  • the transglutaminase enzyme e.g., Tgase variant enzyme
  • lysyl oxidase enzyme(s) acts as a catalyst to crosslink an amino group (e.g., an alkylamino, alkylhydrazine, alkylhydrazide, or alkoxyamine moiety) of the active or functional ingredient or of a linker with an amino acid (e.g., side chain of glutamine or lysine residues) in skin, hair, or nail proteins or peptides.
  • an amino group e.g., an alkylamino, alkylhydrazine, alkylhydrazide, or alkoxyamine moiety
  • an amino acid e.g., side chain of glutamine or lysine residues
  • Examples of skin, hair, or nail proteins include but are not limited to collagen, keratin, elastin, and/or cornified cell envelope proteins including involucrin, loricrin, small proline-rich proteins, periplakin, envoplakin, and filaggrin.
  • Examples of peptides include but are not limited to hydrolyzed collagen, hydrolyzed keratin, and/or hydrolyzed elastin.
  • the compositions include UV-blocking molecules such as sunscreens.
  • the compositions include coloring agents (color producing molecules), such as dye or pigment molecules.
  • the enzyme is immobilized on or encapsulated in a polymeric support. In some embodiments, the enzyme is a transglutaminase enzyme.
  • a composition described herein includes a pharmaceutically or cosmetically acceptable vehicle or carrier, e.g., to act as a diluent or dispersant for the active or functional ingredient and the Tgase and/or lysyl oxidase enzyme(s), or cross-linked active or functional ingredient with proteins or peptides, in the composition, for example, to promote or facilitate distribution of the active or functional ingredient and the cross-linking enzyme, or cross-linked active or functional ingredient with proteins or peptides, when the composition is applied to the skin, hair, or nails of a subject.
  • a pharmaceutically or cosmetically acceptable vehicle or carrier e.g., to act as a diluent or dispersant for the active or functional ingredient and the Tgase and/or lysyl oxidase enzyme(s), or cross-linked active or functional ingredient with proteins or peptides, in the composition, for example, to promote or facilitate distribution of the active or functional ingredient and the cross-linking enzyme, or cross-linked active or functional ingredient
  • the pharmaceutically or cosmetically acceptable vehicle or carrier may comprise or consist of water, and may include liquid or solid emollients, solvents, humectants, thickeners, and/or powders, and in some embodiments, may form about 10% to about 99.9%, or about 50% to about 99%, by weight of the composition.
  • the composition is in the form of an emulsion, which may contain an oil or oily material in an amount up to about 90%, or about 10% to about 80% by volume of the composition.
  • the composition includes one or more emulsifier and/or one or more surfactant.
  • the active or functional ingredient includes at least one alkyl-amino (—RNH 2 ), hydrazine, hydrazide, or hydroxylamine moiety, either directly on the active or functional ingredient, or indirectly on a linker attached (e.g., covalently bound) thereto.
  • the alkyl (R) group may be an aliphatic hydrocarbon chain including 1 to 8 carbon atoms.
  • a composition for application to mammalian e.g., human skin, hair, or nails
  • an effective amount of at least one active or functional ingredient such as, for example, a sunscreen molecule or coloring agent
  • one or more transglutaminase enzyme e.g., a Tgase variant as described herein or any of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a
  • the composition is formulated for topical application to skin, hair, or nails of a mammalian (e.g., human) individual, and in certain embodiments may contain: (c) a pharmaceutically or acceptable carrier in an amount effective to deliver the Tgase and/or lysyl oxidase enzyme(s) and the active or functional ingredient to the skin, hair, or nails of the individual.
  • a pharmaceutically or acceptable carrier in an amount effective to deliver the Tgase and/or lysyl oxidase enzyme(s) and the active or functional ingredient to the skin, hair, or nails of the individual.
  • the active or functional ingredient may include at least one alkylamino (—RNH 2 ), hydrazine, hydrazide, or hydroxylamine moiety, either directly on the active or functional ingredient, or indirectly on a linker attached (e.g., covalently bound) thereto.
  • compositions described herein may contain one or more color producing molecule, such as a dye or pigment molecule, for application and binding to a surface, such as binding to one or more protein on the surface of skin, such as collagen, keratin, and/or elastin, or binding to an edible casing for a food product, such as a sausage casing.
  • a color producing molecule such as a dye or pigment molecule
  • Nonlimiting examples of color producing molecules are described in “Summary of Color Additives for Use in the United States in Foods, Drugs, Cosmetics, and Medical Devices,” US Food and Drug Administration, https://www.fda.gov/industry/color-additive-inventories/summary-color-additives-use-united-states-foods-drugs-cosmetics-and-medical-devices.
  • compositions described herein may contain one or more UV-blocking molecule(s), such as a sunscreen molecule, for application and binding to a protein or peptide within the product formulation or on the surface of skin, such as collagen, keratin, elastin, hydrolyzed collagen, hydrolyzed keratin, and/or hydrolyzed elastin.
  • a sunscreen molecule for application and binding to a protein or peptide within the product formulation or on the surface of skin, such as collagen, keratin, elastin, hydrolyzed collagen, hydrolyzed keratin, and/or hydrolyzed elastin.
  • Nonlimiting examples of sunscreen and/or sunscreen analog molecules include but are not limited to, para-aminobenzoic acid, trolamine salicylate, cinoxate, dioxybenzone, ensulizole, homosalate, meradimate, octinoxate, octisalate, octocrylene, padimate O, sulisobenzone, oxybenzone, avobenzone, and benzophenone hydrazone.
  • the sunscreen is functionalized with a linker molecule to provide a substrate handle for enzymatic binding to a protein or peptide.
  • a non-limiting example of this functionalization may be accomplished through formation of a Schiff base between the sunscreen molecule and linker.
  • a non-limiting example of this functionalization may be accomplished through formation of a carbamate linkage between the sunscreen molecule and linker.
  • the linker may include an available amine for enzyme recognition in the form of a primary amine, hydrazine, hydrazide, or alkoxyamine moiety.
  • the linker may also include a glutamine residue for enzyme recognition.
  • the linker may consist of two functional chemical end groups linked by an aliphatic carbon chain of varying length for in situ formation of the sunscreen-linker molecule.
  • Nonlimiting examples of linkers include cadaverine, putrescine, hydrazine, adipic acid dihydrazide, sebacic dihydrazide, and hexamethylenediamine.
  • the sunscreen-linker adduct is bound to a protein or peptide of interest and the sunscreen can be subsequently released by hydrolysis.
  • the sunscreen molecule is hydrolysable or otherwise releasable from the linker.
  • the sunscreen-linker adduct remains bound to a protein or peptide, e.g., a protein or peptide present on skin, to provide UV-blocking protection.
  • compositions described herein may contain one or more proteins or peptides of interest for sunscreen, skin care, and/or cosmetic products or applications of use.
  • Nonlimiting examples of proteins and peptides of interest for skin care products and cosmetics are: collagen, hydrolyzed collagen, keratin, hydrolyzed keratin, elastin, hydrolyzed elastin, silk, hydrolyzed silk, silk fibroin peptide, acetyl hexapeptide-3, acetyl hexapeptide-8, acetyl tetrapeptide-5, acetyl tetrapeptide-9, acetylarginyltryptophyl diphenylglycine, copper tripeptide-1, CT-2, dipeptide-2, heptapeptide-7, hexanoyl dipeptide-3 norleucine acetate, hexapeptide-9, hexapeptide-11, manganese tripeptide-1, myristoyl hexapeptide-16, myristoyl hexapeptide-16, myristoyl pentapeptide-17, nonapeptide-1, palm
  • compositions described herein may contain one or more model peptides of interest.
  • a model peptide of interest includes Cbz-Gln-Gly.
  • compositions described herein include a polymeric support.
  • Tgase enzyme e.g., Tgase variant enzyme
  • lysyl oxidase enzyme 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.
  • polymeric supports include: chitin, chitosan, carboxymethylchitosan, oxidized cellulose, quaternary ammonium cellulose, alginates, pectin, and carboxycellulose. Examples of polymeric supports and immobilization or encapsulation of cross-linking enzymes therein are described in PCT/US20/21211, which is incorporated herein by reference in its entirety.
  • the Tgase (e.g., Tgase variant enzyme as disclosed herein) and/or lysyl oxidase enzyme(s) are immobilized on particles, e.g., chitosan particles, such as beads, e.g., chitosan beads (e.g., microbeads), or nanoparticles.
  • the beads (e.g., microbeads) may be biodegradable.
  • 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 (1 ⁇ 4)-linked 2-acetamido-2-deoxy- ⁇ -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 (1 ⁇ 4)-linked A-units and 2-amino-2-deoxy- ⁇ -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).
  • the antimicrobial activity of chitin, chitosan and their derivatives against different groups of microorganisms, such as bacteria, yeast, and fungi, is known.
  • 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 (Dinçer, et al. (2012) Int. 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.
  • glutaraldehyde as a linker to immobilize the enzyme on chitosan.
  • tyrosinase-chitosan biocatalysts have been explored for the production of L-DOPA (Carvalho, et al., Appl . Biochem. Biotechnol. (2000) 84-86:791-800).
  • Microbial transglutaminase has been immobilized on chitosan using glutaraldehyde as a linker for the purpose of deamidation of food proteins (Nonaka, et al.
  • one or more Tgase enzyme e.g., Tgase variant enzyme as disclosed herein
  • a lysyl oxidase enzyme is immobilized on a polymer, 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), dicarbonyl (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.
  • 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.
  • the Tgase e.g., Tgase variant enzyme
  • lysyl oxidase enzyme(s) are 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, paints, coatings, adhesives, energy, plastic, packaging, or agricultural products, optionally immobilized on or encapsulated in a polymeric support, which include an effective amount, for example, about 0.0001% w/v to about 5% w/v, of one or more Tgase variant enzyme as described herein, or a composition thereof as described herein, to act as an antimicrobial agent, e.g., preservative, in the product.
  • an antimicrobial agent e.g., preservative
  • products disclosed herein include cosmetics and personal care products which include compositions described herein, compositions that include one or more Tgase enzyme (e.g., Tgase variant enzyme as described herein) and/or lysyl oxidase enzyme, optionally immobilized on or encapsulated in a polymeric support, and one or more active or functional ingredient which may include a sunscreen and/or color producing molecule, in an amount effective to bond a sunscreen molecule or color to a surface, such as covalently binding to one or more skin-derived protein or peptide either on the surface of skin or within the product formulation, e.g., collagen, keratin, and/or elastin.
  • Tgase enzyme e.g., Tgase variant enzyme as described herein
  • lysyl oxidase enzyme optionally immobilized on or encapsulated in a polymeric support
  • active or functional ingredient which may include a sunscreen and/or color producing molecule, in an amount effective to bond a sunscreen molecule or color
  • the product composition includes any of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme.
  • an effective amount of the Tgase (e.g., Tgase variant(s)) and/or lysyl oxidase is in a range of about 0.0001% to about 5% w/v, such as about 0.001% to about 1% or about 0.01% to 0.1%, by weight of the composition.
  • the active or functional ingredient e.g., sunscreen molecule or coloring agent
  • the active or functional ingredient is present in the composition in an amount effective to provide a benefit, such as a UV protecting benefit or a cosmetic or aesthetic benefit, e.g., to hair, skin, or nail proteins or peptides or to the hair, skin, or nails of an individual to whom the composition is topically applied.
  • the effective amount of the active or functional ingredient e.g., sunscreen molecule or coloring agent is in a range of about 0.1% to about 70%, such as about 1% to about 35%, by weight of the composition.
  • one or more Tgase variant as described herein is included in a personal care product, such as, but 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.
  • a personal care product such as, but not limited to, bar soap, liquid soap (e.g., hand soap
  • one or more Tgase variant is included in a wound care item, such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, burn wound cream, bandages, tape, and steri-strips, and medical articles such as medical gowns, caps, face masks, and shoe-covers, surgical drops, etc.
  • a wound care item such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, burn wound cream, bandages, tape, and steri-strips, and medical articles such as medical gowns, caps, face masks, and shoe-covers, surgical drops, etc.
  • one or more Tgase variant is included in an oral care product, such as mouth rinse, toothpaste, or dental floss coating, a veterinary or pet care product, a preservative composition, or a surface disinfectant, such as a disinfectant solution, spray or wipe.
  • one or more Tgase variant as described herein is incorporated into a household or industrial product, for example, as a preservative substance.
  • the Tgase variant(s) may be included in a household cleaner, such as concentrated a liquid cleaner or spray cleaner, 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.
  • one or more Tgase variant may be included in a food wash product, e.g., designed to clean fruits and vegetables prior to consumption, packaging, and food coatings.
  • Tgase variants as described herein include, but are not limited to, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, energy (e.g., fracking fluid), plastic, packaging, and agricultural products.
  • the Tgase variant 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.
  • Products disclosed herein include cosmetics and personal care products which include a Tgase enzyme (e.g., Tgase variant as described herein) and/or lysyl oxidase enzyme, or composition thereof as described herein, and one or more color producing molecule, in an amount effective to bond color to a surface, such as covalently binding to one or more protein of skin, e.g., collagen, keratin, and/or elastin, or to a protein of a food product, such as an edible casing for a processed food product, e.g., a sausage casing.
  • a Tgase enzyme e.g., Tgase variant as described herein
  • lysyl oxidase enzyme or composition thereof as described herein
  • color producing molecule in an amount effective to bond color to a surface, such as covalently binding to one or more protein of skin, e.g., collagen, keratin, and/or elastin, or to a protein of a food product
  • the product composition includes any of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme.
  • an effective amount of the Tgase enzyme (e.g., Tgase variant enzyme) and/or lysyl oxidase enzyme is up to about 1% w/v.
  • Products disclosed herein may include one or more Tgase variant enzyme(s), optionally immobilized on or encapsulated in a polymeric support, and one or more functional ingredients including a sunscreen and/or color producing molecule, in an amount effective to bond a sunscreen and/or color molecule to a surface.
  • the product covalently binds sunscreen molecules to skin-derived proteins, e.g. collagen, keratin, and/or elastin, found within the product formulation.
  • the product covalently binds sunscreen molecules to skin proteins, e.g. collagen, keratin, and/or elastin.
  • the product covalently binds color to skin proteins, e.g.
  • the product contains the functional ingredient with a linker which is sufficient to react with the native enzymes on the skin's surface to crosslink to skin's proteins.
  • the product covalently binds color to a protein of a food product, such as an edible casing for a processed food product, e.g., a sausage casing.
  • an effective amount of the cross-linking enzyme e.g., a transglutaminase enzyme
  • a product or composition which includes Tgase variant as described herein further includes one or more additional enzymes selected from acyl transferases, alpha-amylases, beta-amylases, alpha-galactosidases, arabinosidases, aryl esterases, beta-galactosidases, carrageenases, catalases, cellobiohydrolases, cellulases, chondroitinases, cutinases, endo-beta-1,4-glucanases, endo-beta-mannases, esterases, exo-mannanases, galactanases, glucoamylases, hemicellulases, hyaluronidases, keratinases, laccases, lactases, ligninases, lipases, lipoxygenases, mannanases, oxidases, pectate lyases, pectin ace
  • a Tgase variant enzyme such as any of the variants disclosed in Table 3, optionally with an N-terminal methionine residue, including circular permutants thereof, and optionally with a pro-sequence as described herein, or a composition thereof as described 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 Tgase variant enzyme of composition thereof 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 a Tgase variant enzyme or composition thereof as 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, phenoxyethanol, and/or organic acids (such as sodium benzoate).
  • a Tgase variant enzyme as described herein, alone or in combination with a biocidal chemical, e.g., chitosan is the only antimicrobial, e.g., antibacterial or preservative, agent in the product.
  • a Tgase variant 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). In some embodiments, a Tgase variant 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).
  • preservative blends are compatible with products, stable towards oxidizing or reducing agents and to normal range of pH (4.5 to 8.0) of various products.
  • Non-limiting examples of products in which the Tgase variants described herein may be incorporated are described in PCT Application No. PCT/US20/21211, and in U.S. Provisional Application No. 63/075,763, which are incorporated herein by reference in their entireties.
  • a Tgase variant enzyme as described herein or composition thereof, e.g., preservative composition, as described herein can be incorporated into any personal care product.
  • Personal care products into which the disclosed Tgase variant enzymes 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 wipe
  • wound care items such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, burn 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.
  • a Tgase variant enzyme as disclosed herein can be incorporated into any suitable personal care product intended for use in modifying the appearance of skin, such as a cosmetic product (e.g., lipstick, foundation, blush, or eye makeup).
  • Cosmetic products into which the disclosed compositions may be incorporated include, but are not limited to, liquid or powder foundation, liquid or solid eyeliner, blush, eye shadow, tinted powder, “pancake” type powder to be used dry or moistened, lip color, or makeup setting sprays, etc.
  • the disclosed compositions may also be incorporated into a bronzer or artificial tanning product.
  • compositions may be incorporated into a sunscreen product, such as a chemical sunscreen, e.g., to bind a sunscreen chromophore (such as, but not limited to, oxybenzone avobenzone, octisalate, octocrylene, homosalate, or octinoxate, or a derivative thereof) to skin protein.
  • a sunscreen chromophore such as, but not limited to, oxybenzone avobenzone, octisalate, octocrylene, homosalate, or octinoxate, or a derivative thereof
  • the personal care products that are protected from the microbial contamination by the disclosed enzymes and compositions can be of any type of such as emulsions, gels, serums, solutions, toners, lotions, creams, spray, gel, powder, stick and cleansers.
  • the personal care product formulation typically includes a base formulation to which the enzyme 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. In some embodiments, active ingredients suited for topical applications are included.
  • 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, Hexachlorophene, Hexylresorcinol, Iodine complex (ammonium ether sulfate and polyoxyethylene sorbitan monolaurate), Iodine complex (phosphate ester of alkylaryloxy polyethylene glycol), Nonylphenoxypoly (ethyleneoxy) ethanoliodine, Poloxamer-iodine complex, Povidone, Undecoylium chloride iodine complex, Methylbenzethonium chloride, Phenol, Phenol 16, Secondary amyltricresols, Sodium oxychlorosene, Tribromsalan, Triclocarban, Triclosan, and 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 Tgase variant enzyme as described herein, alone or in combination with (e.g., blend) a biocidal chemical, including but not limited to, chitosan is the only antibacterial, antifungal, antimicrobial, or preservative agent in the product.
  • the Tgase variant enzyme, alone or in combination (e.g., blend) a biocidal chemical, 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., Tgase variant enzyme or composition thereof as disclosed herein
  • one or more synthetic preservative e.g., petrochemical derived substance
  • the preservative e.g., antimicrobial
  • one or more biobased preservative i.e., Tgase variant enzyme or composition thereof 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, monoethanolamine, nonionic emulsifying wax, oleic acid, poloxamer, poloxamers, polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, propylene glycol alginate, self-e
  • 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, hydroxyethylcellulose, 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 quaternium-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.
  • Suitable moisturizers include, e.g., butylene glycol, cetyl alcohol, dimethicone, dimy
  • an enzyme composition as disclosed herein e.g., a composition including one or more Tgase variant enzyme(s), optionally immobilized on or encapsulated in a polymeric support, and sunscreen or color producing molecule(s)
  • a cosmetic product e.g., lipstick, foundation, blush, or eye makeup
  • Cosmetic products into which the disclosed compositions may be incorporated include, but are not limited to, liquid or powder foundation, liquid or solid eyeliner, blush, eye shadow, tinted powder, “pancake” type powder to be used dry or moistened, lip color, or makeup setting sprays, etc.
  • compositions may also be incorporated into a bronzer or artificial tanning product. Additionally, the disclosed compositions may be incorporated into a sunscreen product, such as a chemical sunscreen, e.g., to bind a sunscreen chromophore (such as, but not limited to, oxybenzone, avobenzone, octisalate, octocrylene, homosalate, or octinoxate, or a derivative thereof) to skin protein.
  • a sunscreen chromophore such as, but not limited to, oxybenzone, avobenzone, octisalate, octocrylene, homosalate, or octinoxate, or a derivative thereof
  • Non-limiting embodiments of household/industrial products which may incorporate the disclosed Tgase variant enzymes or compositions thereof as disclosed herein 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.
  • one or more biobased preservative i.e., Tgase variant enzyme or composition thereof 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.
  • a Tgase enzyme e.g., Tgase variant enzyme as described herein
  • lysyl oxidase enzyme can be incorporated into any natural collagen containing product or used during leather processing to modify the leather such that color is covalently bound to one or more protein in leather, such as animal or non-animal derived collagen, keratin, silk, and/or elastin proteins.
  • a Tgase enzyme e.g., Tgase variant enzyme as described herein
  • lysyl oxidase enzyme can be incorporated into any food protein or used during food processing, to modify the color of food protein.
  • Food products into which the Tgase and/or lysyl oxidase enzyme(s) may be incorporated include, but are not limited to, animal-derived products containing collagen or gelatin (hydrolyzed collagen). These include, but are not limited to, gelatin products, meat products or meat analogue products such as sausage casings, pork rinds, or any meat or marine product including the skin layer of the animal and/or collagen.
  • the enzyme composition may be incorporated into non-animal derived collagen-containing products or any collagen-containing product.
  • Tgase variant enzymes or compositions thereof as disclosed herein include, but are not limited to, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, adhesive, 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 i.e., Tgase variant enzyme as disclosed herein
  • one or more additional preservative substance such as one or more petrochemically derived preservative substance.
  • one or more biobased preservative i.e., Tgase variant enzyme or composition thereof 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.
  • Tgase variants disclosed herein including any of the variants disclosed in Table 3
  • an N-terminal methionine residue including circular permutants thereof, and optionally with a pro-sequence as described herein
  • Methods are provided for use of the Tgase variants disclosed herein (including any of the variants disclosed in Table 3), optionally with an N-terminal methionine residue, including circular permutants thereof, and optionally with a pro-sequence as described herein) in various applications of use in which crosslinking of proteins or peptides is desired or beneficial.
  • Tgase variants as described herein may be used in applications of use such as, but not limited to, preservative, antimicrobial, odor control, pharmaceutical, cosmetic, topical, industrial, energy, healthcare, or marine applications.
  • the disclosed variants may be used as alternatives or in addition to conventional preservatives, such as, but not limited to, parabens, formaldehyde, and glutaraldehyde and conventional biocidal agents, 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.
  • conventional preservatives such as, but not limited to, parabens, formaldehyde, and glutaraldehyde and conventional biocidal agents, including silver (used in wound care products)
  • 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 variants may be 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.
  • anti-microbial e.g., preservative
  • Nonlimiting examples of such applications of use are described, for example, in PCT/US20/21211, which is incorporated by reference herein in its entirety.
  • the Tgase variants may be employed as antimicrobial agents with applications in healthcare products, personal care or cosmetic formulations, packaging (food, cosmetic, and pharmaceuticals), textile and leather production, paints and coatings, and marine applications including water treatment and purification.
  • the Tgase variants may be employed for permanently modifying proteins of interest, such as, but not limited to, keratin and collagen, with functional ingredients, dyes, or proteins.
  • Tgase enzymes such as any of the disclosed variants
  • lysyl oxidase enzymes may be incorporated into products to facilitate covalent bonding of color, dye, or pigment molecules to proteins or peptides.
  • the methods include contacting a protein or material of interest with one or more Tgase enzyme (e.g., Tgase variant as described herein) and/or lysyl oxidase enzyme and one or more color-producing molecule, e.g., a dye or pigment molecule.
  • the Tgase and/or lysyl oxidase enzyme(s) are present in an amount that is sufficient (i.e., effective) to covalently bind the color molecule(s) to the protein or material of interest.
  • the protein of interest is one or more protein present in skin, and the Tgase and/or lysyl oxidase enzyme(s) and/or color molecule(s) may be in the form of a cosmetic or personal care product.
  • the protein present in skin may be collagen, keratin, and/or elastin.
  • the protein or material of interest is leather, a food product, or an agricultural product, or a protein of interest therein
  • the Tgase enzyme(s) e.g., Tgase variant(s)
  • lysyl oxidase e.g., lysyl oxidase
  • color molecule(s) are in the form of a composition that is suitable for modifying or adding color to the leather, a food product, or an agricultural product, or a protein of interest therein.
  • a Tgase variant as described herein may be used as an alternative to or in addition to conventional preservatives, such as, but not limited to, parabens, formaldehyde, and glutaraldehyde and conventional biocidal agents, 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, adhesive, energy, plastic, packaging, and agricultural products.
  • a Tgase variant may be used as an antimicrobial (e.g., preservative) ingredient that inhibits the growth of potentially harmful bacteria, fungi, and/or other microbes, and accordingly, is added to a product to be preserved in an effective amount to inhibit bacterial, fungal, and/or microbial growth in such a products.
  • antimicrobial e.g., preservative
  • Nonlimiting examples of such applications of use are described, for example, in PCT/US20/21211, which is incorporated by reference herein in its entirety.
  • 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 characterized by MIC (minimum inhibitory concentration) against gram-positive and gram-negative bacteria as well as fungi, which results in reduction of microbial growth by approximately 80-100%, or any of at least about 80%, 85%, 90%, 95%, 98%, or 99% of microbial growth.
  • the composition When combined with a product as described herein, e.g., a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, adhesive, 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, adhesive, 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 Tgase variant or a composition thereof as described herein) to a product or system, such as a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, marine, paint, coating, adhesive, 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.
  • 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-life, 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 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.
  • the Tgase variant enzyme may be included at a concentration of about 0.0001% w/v to about 1% w/v, 0.0001% w/v to about 0.01% w/v, about 0.0001% w/v to about 2.5% w/v, about 0.0001% w/v to about 5% w/v, about 0.0001% w/v to about 0.001% w/v, about 0.001% w/v to about 0.01% w/v, about 0.01% w/v to about 0.1% w/v, 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,
  • Non-limiting examples of personal care products to which the preservative methods may be applied 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
  • wound care items such as, but not limited to, wound healing ointments, creams, and lotions, wound coverings, burn 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 to which the preservative methods may be applied 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.
  • 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 preservative methods 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.
  • one or more Tgase enzyme may be included in a product to be used for long-lasting application of functional ingredients including UV-blocking sunscreens, and/or coloring agents, such as pigments or dyes.
  • any of the Tgase enzymes disclosed in SEQ ID NOs:1-29, and/or a lysyl oxidase enzyme is included in the product composition.
  • the Tgase (e.g., Tgase variant) and/or lysyl oxidase enzyme(s) may be used in a composition for delivery of an active or functional ingredient to mammalian (e.g., human) skin, hair, or nails, such as, but not limited to, permanent (covalent) color modification of the surface of hair fibers.
  • Tgase (e.g., Tgase variant) and/or lysyl oxidase enzyme(s) may be incorporated in a product to be applied topically and which bonds to the skin of an individual, such as a UV-blocking (sunscreen) product, or a cosmetic product.
  • the Tgase (e.g., Tgase variant) and/or lysyl oxidase enzyme(s) may be used to provide permanent application of color to the skin of an animal such as in leather processing. In some embodiments, the Tgase (e.g., Tgase variant) and/or lysyl oxidase enzyme(s) may be used to provide a permanent application of color in food processing.
  • Methods are provided herein for modifying or adding color to a protein or material of interest.
  • the methods include contacting a protein, peptide, or material of interest with one or more Tgase (e.g., Tgase variant as described herein) and/or lysyl oxidase enzyme(s) and one or more functional ingredient including a sunscreen and/or color-producing molecule, e.g., a dye or pigment molecule.
  • Tgase e.g., Tgase variant
  • lysyl oxidase enzyme(s) are present in an amount that is sufficient (i.e., effective) to covalently bind the sunscreen and/or color molecule(s) to the protein, peptide, or material of interest.
  • the protein of interest is one or more protein present in skin
  • the Tgase e.g., Tgase variant
  • lysyl oxidase enzyme(s) and sunscreen(s) and/or color molecule(s) may be in the form of a cosmetic or personal care product.
  • the protein present in skin may be collagen, keratin, and/or elastin.
  • the material of interest is one or more protein or peptide derived from skin
  • the Tgase e.g., Tgase variant
  • lysyl oxidase enzyme(s) and/or sunscreen(s) and/or color molecule(s) may be in the form of a cosmetic or personal care product.
  • the protein present in the product formulation may be collagen, keratin, and/or elastin.
  • the peptide present in the product formulation may be hydrolyzed collagen, hydrolyzed keratin, and/or hydrolyzed elastin.
  • the protein or material of interest is leather, a food product, or an agricultural product, or a protein of interest therein
  • the Tgase e.g., Tgase variant
  • lysyl oxidase enzyme(s) and/or color molecule(s) are in the form of a composition that is suitable for modifying or adding color to the leather, a food product, or an agricultural product, or a protein of interest therein.
  • a method for delivering an active or functional ingredient (such as a sunscreen molecule or coloring agent) to proteins or peptides of mammalian (e.g., human) skin, hair, or nails.
  • an active or functional ingredient such as a sunscreen molecule or coloring agent
  • the method may include application of a composition as described herein to proteins or peptides of mammalian (e.g., human) skin, hair, or nails or topical application of the composition to skin, hair, or nails of a mammalian (e.g., human) individual.
  • the method includes contacting proteins and/or peptides of mammalian (e.g., human) skin, hair, or nails, with a composition that includes: (a) an effective amount of at least one active or functional ingredient (such as, for example, a sunscreen molecule or coloring agent); and (b) one or more Tgase (e.g., Tgase variant) and/or lysyl oxidase enzyme(s) in an amount effective to catalyze the crosslinking of the active or functional ingredient to a protein or peptide of mammalian (e.g., human) skin, hair, or nails.
  • a composition that includes: (a) an effective amount of at least one active or functional ingredient (such as, for example, a sunscreen molecule or coloring agent); and (b) one or more Tgase (e.g., Tgase variant) and/or lysyl oxidase enzyme(s) in an amount effective to catalyze the crosslinking of the active or functional ingredient to a protein
  • the method includes topical application of the composition to the skin, hair, or nails of a mammalian (e.g., human) individual, and in certain embodiments the composition may contain: (c) a pharmaceutically or acceptable carrier in an amount effective to deliver the Tgase variant enzyme and the active or functional ingredient to the skin, hair, or nails of the individual.
  • a mammalian e.g., human
  • the composition may contain: (c) a pharmaceutically or acceptable carrier in an amount effective to deliver the Tgase variant enzyme and the active or functional ingredient to the skin, hair, or nails of the individual.
  • the active or functional ingredient may include at least one alkylamino (—RNH 2 ), hydrazine, hydrazide, or hydroxylamine moiety, either directly on the active or functional ingredient, or indirectly on a linker attached (e.g., covalently bound) thereto, and the method includes catalysis by the transglutaminase enzyme of crosslinking (e.g., formation of covalent bonds) between the amino groups of the active or functional ingredient and amino groups (e.g., amino groups on glutamine and/or lysine amino acid residue side chains) in proteins or peptides of skin, hair, or nails.
  • crosslinking e.g., formation of covalent bonds
  • the genes coding for the pro-sequence and mature Tgase were codon optimized for expression in E. coli based on the published amino acid sequence (Kanaji, et al. (1993) J. Biol. Chem. 268(16):11565-11572), synthesized, and cloned onto pUC19-derived expression vectors as described in PCT/US20/49226.
  • the genes coding for the pro-sequence and mature Tgase were codon optimized for expression in E. coli based on the published amino acid sequence (Kanaji, et al. (1993) J. Biol. Chem. 268(16):11565-11572) and synthesized as described in PCT/US20/49226.
  • the DNA was cloned onto the pET9a vector under control of the T7 promoter for expression in E. coli.
  • Mutations were introduced into the mature Tgase gene using site directed mutagenesis methods known in the art.
  • Pro-sequence and mature Tgase variants were expressed simultaneously in a commercially available cell-free protein synthesis kit following the manufacturer's instructions as described in PCT/US20/49226.
  • Tgase specific activity was measured in the examples herein using a colorimetric hydroxamate activity assay (Folk and Cole (1965) J Biol Chemistry 240(7):2951-2960). Briefly, the hydroxamate assay uses N-carbobenzoxy-L-glutaminylglycine (Z-Gln-Gly or CBZ-Gln-Gly) as the amine acceptor substrate and hydroxylamine as an amine donor. In the presence of transglutaminase, the hydroxylamine is incorporated to form Z-glutamylhydroxamate-glycine, which develops a colored complex with iron (III), detectable at 525 nm after incubation at 37° C. for 5-60 minutes.
  • Z-Gln-Gly or CBZ-Gln-Gly N-carbobenzoxy-L-glutaminylglycine
  • Z-Gln-Gly or CBZ-Gln-Gly N-carbobenz
  • Tgase L-glutamic acid ⁇ -monohydroxamate (Millipore Sigma) as standard.
  • One unit of Tgase is defined as the amount of enzyme, determined using a commercially available ELISA kit (Zedira E021) following the manufacturer's protocol, that catalyzes formation of 1 ⁇ mol of the peptide derivative of ⁇ -glutamylhydroxylamine per minute.
  • Protein functionalization by Tgase variants was determined by one of two methods: (1) the fluorogenic labeling of casein using monodansylcadaverine; and (2) covalently locking a fluorescent substrate onto a collagen plate. All results were normalized to Tgase concentration using ELISA.
  • Initial rates of the active mutants for protein substrates were determined by measuring the increase in fluorescence over time associated with Tgase-catalyzed labeling of casein with dansylcadaverine (e.g., a commercially available kit, Fluorogenic Activity Assay Kit, Zedira T036).
  • TAMRA-cadaverine (1.7 g/L) was covalently bound to a collagen plate (Corning BioCoat Collagen I Multiwell Plates) in the presence of increasing amounts of Tgase, SEQ ID NO:28.
  • Tgase variant (0-0.33% w/v) in phosphate buffered saline (PBS) at pH 7.4, total volume of 60 ⁇ L.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • FIG. 7 A scheme of the reaction is illustrated in FIG. 7 .
  • the results are shown in FIG. 9 .
  • the wells are shown pre-color removal (top row) and post-wash with PBS (bottom row).
  • Glutamine donor dye Cbz-Gln-Gly-TAMRA (2.5 g/L) was bonded to a collagen plate (Corning BioCoat Collagen I Multiwell Plates) in the presence of Tgase with the amino acid sequence depicted in SEQ ID NO:28 (0.05-0.3% w/v) in PBS at pH 7.4.
  • a negative control containing BSA (0.33% w/v) and a negative control in the absence of BSA or Tgase were run in parallel.
  • the plate was incubated for 16 h at 37° C. Following incubation, the plates were washed with PBS to remove any residual, unbound dye.
  • FIG. 8 A scheme of the reaction is illustrated in FIG. 8 .
  • the results are shown in FIG. 10 .
  • the wells are shown pre-color removal and post-wash with PBS.
  • Yeast or bacterial starter cultures were grown at 30° C.-37° C. overnight. The following day, the cell density of the saturated cultures was calculated using OD600 and cultures were diluted to 10 5 -10 8 cells per mL. Cultures (100 ⁇ L) were made from the dilute starters in 96 well plates. Mutant or wild-type Tgase was added to each culture at 0.0001-1 weight percent. The cultures were grown overnight at 30° C.-37° C. and growth curves were measured by a BioTek Synergy Plate Reader. The following day, a cell viability assay such as BacTiter Glo (Promega following manufacturer's protocols) was used to assess cell survival rate following challenge with Tgase. A decrease in luminescence indicates a decrease in cell viability. Results are shown in Table 3.
  • E. coli was cultured in the presence of wild-type Tgase or Tgase variants at concentrations ranging from 1 mg/L-250 mg/L. Lower optical density (OD) measurements correlated to improvements in antibacterial efficacy relative to wild-type Tgase.
  • mobaraensis Tgase Numbering of amino acid positions is in reference to the mature S. mobaraensis Tgase amino acid sequence depicted in SEQ ID NO: 1.
  • a “ ⁇ ” indicates a reduction in activity.
  • a “+” indicates an improvement between 1.2- and 2-fold.
  • a “++” indicates an improvement between 2- and 5-fold.
  • a “+++” indicates an improvement greater than 5-fold.
  • Oxybenzone (0.25 mol), toluene (100 mL), and glacial acetic acid (1 drop) were charged in a 250 mL three neck flask fitted with a mechanical agitator, thermocouple, and dean stark trap.
  • Molten 1,6-hexanediamine (0.25 mol) was added to the flask and the reaction was refluxed overnight to remove water. The reaction was monitored by HPLC.
  • the resulting oxybenzone-imine was isolated by rotary evaporation under reduced pressure and further dried under vacuum to deliver the imine product, 2-(((6-aminohexyl)imino)(phenyl)methyl)-5-methoxyphenol, as a yellow solid in nearly quantitative yield (m/z 327.3).
  • the scheme is shown in FIG. 5 .
  • Example 7 The imine product in Example 7 (1.6 g/L) and Cbz-Gln-Gly dipeptide (1.65 g/L) were dissolved in 0.1M Tris-HCl pH 8.0. To aid in dissolution of the imine, 10% 1:1 dichloromethane in dimethyl sulfoxide was employed. Tgase (0.01 wt %) was added to the suspension and the reaction was incubated at 37° C. overnight with constant agitation. Tgase catalyzed, covalent addition of the dipeptide to the imine was confirmed by LCMS (m/z 647.25). In the absence of Tgase, no covalent addition was observed. The scheme is shown in FIG. 5 and results are shown in FIG. 6 . Tgase variant, SEQ ID NO:28 demonstrates 11-fold improvement in activity relative to wild-type Tgase. No product was observed in the absence of Tgase (negative control).

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