Transglutaminase variants and applications of use thereof

Abstract

Variants of the transglutaminase enzyme of Streptomyces mobaraensis are provided. The disclosed variants exhibit at least about 2-fold increased enzymatic activity versus the wild-type enzyme. Methods and compositions are provided for biocidal applications of use and for covalently binding small organic molecules to a protein or material of interest. Methods are provided for increasing the self-life of products, such as personal care, household and industrial products, by incorporating an effective amount of the disclosed variant enzymes into the product. The transglutaminase variants may also be used to covalently bind functional ingredients, such as UV-blocking molecules, dyes, or pigments to proteins. The transglutaminase enzymes and functional ingredients may be incorporated into a cosmetic formulation for modifying skin, hair, or nail proteins or skin-derived proteins, such as collagen, keratin, and/or elastin.

We claim: 
     
         1 . A variant of the transglutaminase enzyme amino acid sequence depicted in SEQ ID NO:1, comprising substitutions of amino acid residue A or G at position 199 and amino acid residue A, E, K, or V at position 299. 
     
     
         2 . The transglutaminase variant of  claim 1 , further comprising an N-terminal methionine residue, a C-terminal histidine polyhistidine sequence, and/or a pro-sequence. 
     
     
         3 . The transglutaminase variant of  claim 1 , further comprising substitution of amino acid residue C, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W, Y at position 2. 
     
     
         4 . The transglutaminase variant of  claim 3 , further comprising an N-terminal methionine residue, a C-terminal histidine polyhistidine sequence, and/or a pro-sequence. 
     
     
         5 . A variant of the transglutaminase enzyme amino acid sequence depicted in SEQ ID NO:1, comprising substitutions of amino acid residue D, E, K, M, R, or Q at position 282, amino acid residue E, I, L, T, Q, or V at position 289, and amino acid residue A, E, K, or V at position 299. 
     
     
         6 . The transglutaminase variant of  claim 5 , further comprising an N-terminal methionine residue, a C-terminal histidine polyhistidine sequence, and/or a pro-sequence. 
     
     
         7 . A variant of the transglutaminase enzyme amino acid sequence depicted in SEQ ID NO:1, comprising the amino acid sequence depicted in any of SEQ ID NOs: 2-27. 
     
     
         8 . The transglutaminase variant of  claim 7 , further comprising an N-terminal methionine residue, a C-terminal histidine polyhistidine sequence, and/or a pro-sequence. 
     
     
         9 . A transglutaminase enzyme, comprising a sequence having at least about 90% sequence identity to any of the amino acid sequences depicted in SEQ ID NOs:2-27 or a circular permutant of any of the amino acid sequences depicted in SEQ ID NOs: 2-27, wherein the variant comprises at least about 2-fold greater transglutaminase enzymatic activity than the transglutaminase enzyme comprising the amino acid sequence depicted in SEQ ID NO:1. 
     
     
         10 . The transglutaminase variant of  claim 9 , further comprising an N-terminal methionine residue, a C-terminal histidine polyhistidine sequence, and/or a pro-sequence. 
     
     
         11 . A method for increasing the shelf life of a product, comprising incorporating the transglutaminase variant of any of  claims 1  to  10  into the product in an amount effective to prevent or decrease growth of one or more microbe in comparison to an identical product that does not comprise the variant. 
     
     
         12 . A product comprising the transglutaminase variant of any of  claims 1  to  10  in an effective amount to increase the shelf life of the product, in comparison to an identical product that does not comprise the variant. 
     
     
         13 . The product of  claim 12 , wherein the product is a personal care, household, industrial, food, pharmaceutical, cosmetic, healthcare, wound care, marine, paint, coating, energy, plastic, packaging, textile, leather, or agricultural product. 
     
     
         14 . The product of  13 , wherein the product is a personal care product selected from bar soap, liquid soap, hand sanitizer, preoperative skin disinfectant, cleansing wipes, disinfecting wipes, body wash, acne treatment products, antifungal diaper rash cream, antifungal skin cream, shampoo, conditioner, cosmetic, deodorant, antimicrobial cream, body lotion, hand cream, topical cream, aftershave lotion, skin toner, mouth wash, toothpaste, and sunscreen lotion. 
     
     
         15 . The product of  claim 13 , wherein the product is a wound care product selected from wound healing ointment, cream, and lotion, wound covering, burn wound cream, bandages, tape, and steri-strips. 
     
     
         16 . An enzyme composition comprising: (i) the transglutaminase variant of any of  claims 1  to  10 , a transglutaminase enzyme comprising the amino acid sequence depicted in SEQ ID NO:1, 28, or 29, and/or a lysyl oxidase enzyme; and (ii) a substrate for the transglutaminase or lysyl oxidase enzyme, comprising a sunscreen molecule, a pigment or dye molecule, or a functional ingredient molecule. 
     
     
         17 . The enzyme composition of  claim 16 , wherein the sunscreen molecule, pigment or dye molecule, or functional ingredient molecule is conjugated to a molecule that comprises a free amino group. 
     
     
         18 . The enzyme composition of  claim 17 , wherein the molecule that comprises 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 and R is a functional ingredient. 
     
     
         19 . The enzyme composition of  claim 18 , wherein n is an integer between 5 and 10. 
     
     
         20 . The enzyme composition of  claim 17 , wherein the molecule that comprises a free amino group is derived from lysine, cadaverine, putrescine, hydrazine, adipic acid dihydrazide, sebacic dihydrazide, and hexamethylenediamine. 
     
     
         21 . The enzyme composition of  claim 16 , wherein the sunscreen molecule, pigment or dye molecule, or functional ingredient molecule is conjugated to an amino acid, peptide, or protein with a free glutamine side chain. 
     
     
         22 . A cosmetic composition comprising the enzyme composition of  claim 16 . 
     
     
         23 . A method for bonding color to a material or protein of interest, comprising contacting the material or protein of interest with the transglutaminase variant of any of  claims 1  to  10 , a transglutaminase enzyme comprising the amino acid sequence depicted in SEQ ID NO:1, 28, or 29, and/or a lysyl oxidase enzyme and a pigment or dye molecule, wherein the transglutaminase or lysyl oxidase enzyme is present in an amount effective to covalently bind the pigment or dye molecule to the material or protein of interest. 
     
     
         24 . The method of  claim 23 , wherein the protein of interest is a protein that is present in skin. 
     
     
         25 . The method of  claim 24 , wherein the protein that is present in skin comprises collagen, keratin, and/or elastin. 
     
     
         26 . A product comprising the transglutaminase variant of any of  claims 1  to  10 , a transglutaminase enzyme comprising the amino acid sequence depicted in SEQ ID NO:1, 28, or 29, and/or a lysyl oxidase enzyme in an effective amount to add a functional ingredient or color molecule onto a protein or a protein-, peptide-, or amino acid-containing material of interest when contacted with the product. 
     
     
         27 . The product of  claim 26 , wherein the product is a personal care, cosmetic, textile, leather, food, or agricultural product. 
     
     
         28 . A method of modifying the color of a protein or material of interest, comprising contacting the protein or material of interest with the product according to  claim 26 . 
     
     
         29 . A composition comprising the transglutaminase variant of any of  claims 1  to  10  in combination with one or more antimicrobial enzyme, peptide, or protein, wherein the composition comprises preservative, biocidal, antimicrobial, or virucidal activity. 
     
     
         30 . The composition of  claim 29 , wherein the antimicrobial enzyme, peptide, or protein is selected from lysozyme, chitinase, lipase, lysin, lysostaphin, glucanase, DNase, RNase, lactoferrin, glucose oxidase, peroxidase, lactoperoxidase, lactonase, acylase, dispersin B, a-amylase, cellulase, nisin, bacteriocin, siderophore, polymyxin, and defensin. 
     
     
         31 . A bacteriophage, comprising a nucleic acid sequence that encodes the transglutaminase variant of any of  claims 1  to  10 . 
     
     
         32 . A composition comprising the bacteriophage of  claim 31 , wherein the composition comprises antimicrobial activity. 
     
     
         33 . The composition of  claim 32 , wherein the composition further comprises a pharmaceutically acceptable excipient.