HK1171339B - Enzymatic in situ preparation of peracid-based removable antimicrobial coating compositions and methods of use - Google Patents

Description

Enzymatic in situ preparation of peracid-based removable antimicrobial coating compositions and methods of use thereof

This patent application claims the benefit of four U.S. provisional applications 61/228732, 61/228735, 61/228737, and 61/228746, all filed on 7/27/2009.

Technical Field

The present invention relates to a method of controlling microorganisms comprising coating a surface with a removable antimicrobial film-forming composition comprising an in situ enzymatically generated peroxyacid, and a method of applying said composition.

Background

Chemical disinfectants are currently used in an increasing number of industries to ensure food safety and to comply with stricter health and safety regulations. Compositions for decontamination, disinfection and sterilization must have excellent germicidal efficacy, be fast acting, non-corrosive and be minimally effective. The ideal composition must have multiple mechanisms for killing microorganisms, thus providing efficacy against a broad spectrum of microorganisms and reducing the likelihood of microbial evolution leading to resistance to anti-bactericides.

Compositions comprising peroxides, especially Hydrogen Peroxide (HP) and peracetic acid (also known as peracetic acid, PAA) have proven to be very effective antimicrobial agents. Many of these formulations have passed the necessary tests and are registered products such as disinfectants, disinfectants and sporicides. Many of these peroxide/peroxyacid compositions are solutions that are useful for treating aqueous solutions, surfaces, and objects. Some compositions are approved for use in contacting food surfaces and for the disinfection of some food products. Some compositions are also registered as disinfectants or sterilants for vapor phase treatments.

Methods of cleaning, sterilizing, and/or disinfecting hard surfaces, meat products, living plant tissue, and medical equipment to eradicate the growth of harmful microorganisms have been described (U.S. Pat. No. 6,545,047; U.S. Pat. No. 6,183,807; U.S. Pat. No. 6,518,307; U.S. patent application publication No. 20030026846; and U.S. Pat. No. 5,683,724). Peracids have also been reported to be useful in the preparation of bleaching compositions for laundry detergent applications (U.S. Pat. No. 3,974,082; U.S. Pat. No. 5,296,161; and U.S. Pat. No. 5,364,554).

There are several methods for preparing peracids. For example, WO2006/076334 describes a microbicidal and decontamination composition comprising an aqueous solution of a peroxide and a peracid; U.S. Pat. No. 5,130,124 describes aqueous antimicrobial film-forming compositions with hydrogen peroxide; WO1996/022687 describes an oxidation to film composition comprising hydrogen peroxide. Enzyme catalysts may also be used to promote peracid generation. Co-pending U.S. patent publication No. 2008/0176299A1, which is incorporated herein by reference in its entirety, describes a process for the enzymatic production of peracids. Enzymes such as esterases (U.S. Pat. No. 3,974,082), proteases (U.S. Pat. No. 5,364,554), esterases and lipases (U.S. Pat. No. 5,296,161) have also been described for the enzymatic production of peracids.

Despite the peracid production process described above, there is a need for in situ enzymatic generation of peracid-based, easily removed, homogenous antimicrobial coating compositions that provide both short-term and long-term (or residual) antimicrobial efficacy after application to a surface.

Disclosure of Invention

The present invention solves the above problems by providing methods and compositions that are antimicrobial and also provide long-term antimicrobial effects by forming an antimicrobial coating on a target surface.

In one aspect, the present invention relates to a method of providing control of microorganisms at a locus, the method comprising the steps of:

a) forming a composition by combining components comprising:

i) a film-forming, water-soluble or water-dispersible agent, wherein the agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinylpyrrolidone;

ii) an inert solvent;

iii) a carboxylate substrate;

iv) a source of peroxygen;

v) an enzyme catalyst having perhydrolase activity to form at least one peroxyacid to provide at least a first antimicrobial agent; and

vi) a rheology modifier for providing shear thinning properties;

b) applying the composition to the site; and is

c) Allowing the composition to dry to form a coating on the site.

Wherein the composition further comprises a second antimicrobial agent, wherein the second antimicrobial agent comprises a quaternary ammonium compound.

Another aspect of the invention relates to a method of providing a control of a microorganism at a site, the method comprising the steps of:

(a) providing a first premix component comprising an inert solvent, a rheology modifier for providing shear thinning properties, and a film forming agent, wherein the film forming agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinyl pyrrolidone;

(b) providing a second premix component comprising an enzyme catalyst having perhydrolase activity, a carboxylic acid ester substrate, and a source of peroxygen;

(c) mixing the first pre-mix component and the second pre-mix component to obtain a liquid coating composition comprising a first antimicrobial agent having a peroxyacid;

(d) applying the coating composition to the site; and is

(e) Allowing the coating composition to dry to form a coating on the site.

Wherein at least one of the pre-mixed components further comprises a second antimicrobial agent, wherein the second antimicrobial agent comprises a quaternary ammonium compound.

Another aspect of the invention relates to an antimicrobial composition comprising components comprising:

a) a film-forming, water-soluble or water-dispersible agent, wherein the agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinylpyrrolidone;

b) an inert solvent;

c) a carboxylate substrate;

d) a source of peroxygen;

e) an enzyme catalyst having perhydrolase activity; and

f) a rheology modifier for providing shear thinning characteristics;

wherein a peroxyacid is formed upon combining the components; wherein the component further comprises a component that is an antimicrobial agent comprising a quaternary ammonium compound.

Another aspect of the present invention relates to an article comprising a coating having an antimicrobial composition on at least one surface of the article, wherein the antimicrobial composition comprises:

a) a film-forming, water-soluble or water-dispersible agent, wherein the agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinylpyrrolidone;

b) an inert solvent;

c) a carboxylate substrate;

d) a source of peroxygen;

e) an enzyme catalyst having perhydrolase activity;

f) a rheology modifier for providing shear thinning characteristics; and

g) a quaternary ammonium compound;

wherein peroxy acids are formed upon combining components (a) to (g).

Brief description of biological sequences

The following Sequences follow 37c.f.r. § 1.821-1.825 ("requirement-Sequence Rules for Patent applications Containing nucleotide and/or Amino Acid Sequence publications") (Requirements for nucleotide and/or Amino Acid Sequence disclosures), "and conform to the Sequence listing Requirements of the world intellectual Property Organization (intellectual Property Organization) (WIPO) st.25 standard (1998) and of the European Patent description (EPC) and Patent Cooperation Treatment (PCT) (Rules 5.2 and 49.5(a-bis) and of the administrative Instructions (appendix C) 208 and appendix C). The symbols and formats used for nucleotide and amino acid sequence data comply with the regulations set forth in 37c.f.r. § 1.822.

SEQ ID NO: 1 is the amino acid sequence of Bacillus subtilis ATCC31954 cephalosporin C deacetylase.

SEQ ID NO: 2 is the amino acid sequence of Bacillus subtilis subspecies Bacillus subtilis strain 168(GenBank Accession # NP-388200) cephalosporin c deacetylase.

SEQ ID NO: 3 is the amino acid sequence of Bacillus subtilis ATCC6633(GenBank Accession # YP-077621.1) cephalosporin c deacetylase.

SEQ ID NO: 4 is the amino acid sequence of Bacillus licheniformis (Bacillus licheniformis) ATCC14580(GenBank Accession # YP-077621.1) cephalosporin c deacetylase.

SEQ ID NO: 5 is the amino acid sequence of Bacillus pumilus (GenBank Accession # CAB76451.2) acetyl xylan esterase.

SEQ ID NO: 6 is the amino acid sequence of Clostridium thermocellum (Clostridium thermocellum) ATCC27405(GenBank Accession # ZP-00504991) acetylxylan esterase.

SEQ ID NO: 7 is the amino acid sequence of Thermotoga neapoliana (Thermotoga neocolitana) GenBank accession # AAB70869.1) acetylxylan esterase.

SEQ ID NO: 8 is the amino acid sequence of Thermotoga maritima (Thermotoga maritima) MSB8(GenBank accession # NP-227893.1) acetylxylan esterase.

SEQ ID NO: 9 is the amino acid sequence of Thermoanaerobacterium sp (GemBank Accession # AAB68821.1) acetylxylan esterase.

SEQ ID NO: 10 is the amino acid sequence of Bacillus (Bacillus sp.) NRRL B14911(GenBank accession # ZP-01168674) cephalosporin c deacetylase.

SEQ ID NO: 11 is the amino acid sequence of the acetyl xylan esterase of Bacillus halodurans (Bacillus halodurans) C-125(GenBank Accession # NP-244192).

SEQ ID NO: 12 is the amino acid sequence of Bacillus clausii (Bacillus clausii) KSM-K16 cephalosporin c deacetylase.

SEQ ID NO: 13 is the amino acid sequence of acetylxylan esterase from high temperature hydrogen producing bacteria (Thermoanaerobacterium saccharolyticum).

SEQ ID NO: 14 is the amino acid sequence of the acetylxylan esterase of Thermotoga rettingae (Thermotoga lettingeae).

SEQ ID NO: 15 is the amino acid sequence of Thermotoga petrophilia Thermotoga acetylxylan esterase.

SEQ ID NO: 16 is the amino acid sequence of Thermotoga (Thermotoga sp.) RQ2 "a" acetyl xylan esterase.

SEQ ID NO: 17 is the amino acid sequence of Thermotoga (Thermotoga sp.) RQ2 "b" acetyl xylan esterase.

SEQ ID NO: 18 is the nucleotide sequence of primer f27405 used in example 1.

SEQ ID NO: 19 is the nucleotide sequence of primer r27405 used in example 1.

SEQ ID NO: 20 is the plasmid encoded by seq id NO: 18 and 19, and a nucleic acid sequence of the amplified nucleic acid product.

SEQ ID NO: 21 is a contiguous amino acid sequence of 4 conserved motifs present in the CE-7 glycoesterase.

SEQ ID NO: 22 is the nucleotide sequence of the forward primer [ TAACTGCAGTAAGGAGGAAT AGGACATGGC CTTCTTCGAT TTACCCACTC ] used in example 8.

SEQ ID NO: 23 is the nucleotide sequence of the reverse primer [ TGATCTAGATTAGCCTTTCT CAAATAGTTT TTTCAAGA ] used in example 8.

SEQ ID NO: 24 is a plasmid consisting of SEQ ID NO: 22 and 23, and a nucleic acid sequence of the amplified nucleic acid product.

SEQ ID NO: 25 is the nucleotide sequence of the forward primer [ TAAGAATTCTAAGGAATAGG ACATGGCGTT TCTTCGACCT GCCTCTG ] used in example 8.

SEQ ID NO: 26 is the nucleotide sequence of the reverse primer [ AAACTGCAGTTAGCCCTTCT CAAACAGTTT CTTCAG ] used in example 8.

SEQ ID NO: 27 is the plasmid encoded by seq id NO: 25 and 26 nucleic acid sequences of the amplified nucleic acid products.

SEQ ID NO: 28 is the nucleotide sequence of forward primer Tma _ C277Sf [ GGACAACATCTCACCTCCTTCTA ] used in example 9.

SEQ ID NO: 29 is the nucleotide sequence of forward reverse primer Tma _ C227Sr [ TAGAAGGAGGTGAGATGTTGTCC ] used in example 9.

SEQ ID NO: 30 is the codon optimised sequence of t.maritime thermotoga acetylxylan esterase of plasmid pSW228 used in example 9.

Detailed Description

All percentages, parts, ratios, etc., are by weight unless otherwise indicated. Trademarks are shown in upper case. Furthermore, when an amount, concentration, or other value or parameter is given as either a range, preferred range, or a list of upper preferable values and lower preferable values, this disclosure will have the same effect if each individual value within the specified range, and any range derived from any combination of two individual values within the disclosed range, is specifically described, even if the individual values are not expressly disclosed herein either individually or separately. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. When a range is defined, it is not intended that the scope of the invention be limited to the specific values recited, unless specifically indicated.

For clarity, the terms used herein should be understood as described herein, or as understood by one of ordinary skill in the art of the present invention. Further, the interpretation of certain terms used herein is provided as follows:

"peracids" are carboxylic acids in which the acidic-OH groups have been substituted with-OOH groups. As used herein, "peracid" is synonymous with peroxyacid (peroxyacid), percarboxylic acid (percarboxylic acid), and peroxyacid (peroxyoic acid). Peracids include peracetic acid, which is generally known. As used herein, the term "peracetic acid" or "PAA" is synonymous with all other synonyms for peracetic acid, ethylene peroxy acid (etoposic acid), and CAS registry number 79-21-0.

"glyceryl triacetate" and glyceryl triacetate; glyceryl triacetate; triacetin, 1,2, 3-triacetoxypropane, 1,2, 3-glycerol triacetate, and all other synonyms for CAS registry number 102-76-1 are synonymous.

By "peroxygen source" is meant any peroxide compound or compound containing hydrogen peroxide that can be released into solution.

"shear rate" refers to the velocity gradient in a flowing substance and is expressed in the reciprocal of seconds(s)^-1) Is measured in SI units of (a).

"shear-thinning behavior" or "pseudoplastic behavior" refers to fluids that exhibit a decrease in viscosity as shear rate increases.

By "readily removable" is meant that the formed coating is readily removed after application of the liquid coating composition to a surface of interest, but not so removed by inadvertent physical contact.

"non-volatile" refers to compounds whose vapor pressure is less than 1000 pascals at 25 ℃.

By "inert solvent or aqueous solvent" is meant water or any other solvent that facilitates application of the coating composition to the site. Aqueous solvents may also be used to clean the coated surface to remove the coating as desired.

"film-forming agent" or "water-soluble or water-dispersible coating agent" are used interchangeably herein and refer to an agent that forms a film and is used to provide a protective coating to a surface of interest. These agents are either water soluble or water dispersible. These agents are described in further detail below.

"Metal chelating agent" or "sequestering agent" refers to agents that bind metals or metal-containing impurities and prevent their catalysis of the decomposition of hydrogen peroxide or peroxyacids.

"rheology modifier" or "rheology agent" refers to a compound that increases viscosity and/or provides shear thinning properties to the composition and causes the aqueous treating agent or coating composition to adhere to the surface of interest.

"wt%" refers to weight percent relative to the total weight of the solution or dispersion.

By "liquid coating composition" is meant a composition comprising at least one water-soluble film forming agent, an inert solvent, a carboxylic acid ester substrate, a peroxygen source, and an enzyme catalyst having perhydrolase activity, wherein at least one peroxyacid is produced.

As used herein, "antimicrobial agent" or "antimicrobial property" refers to the ability of an agent to kill microorganisms, prevent or prevent microbial contamination (e.g., form a barrier), or inhibit or prevent microbial growth, capture microorganisms to kill, prevent biofilm formation, or remove/remove biofilm.

As used herein, "antimicrobial agent" refers to a compound or substance having antimicrobial properties.

"microorganism" is intended to include any organism consisting of phylogenetically-ranging bacteria and archaea, as well as unicellular (e.g., yeast) and filamentous (e.g., mold) fungi, unicellular and filamentous algae, unicellular and multicellular parasites, viruses, prions, and viroids.

As used herein, "biocide" refers to a generally broad spectrum chemical agent that inactivates or destroys microorganisms. Chemical agents that exhibit the ability to inactivate or destroy microorganisms are said to have "biocidal" activity.

"biofilm" refers to a structured community of microorganisms encapsulated within a self-growing polymer matrix and attached to a living or inert surface.

"drying" refers to the process by which the inert solvent or any other liquid present in the formulation is removed by evaporation.

"disinfectant" refers to an agent that provides antimicrobial activity. According to the official disinfectant test, a disinfectant is a chemical that kills 99.9% of a particular test microorganism within 10 minutes under test conditions. (Germidal and reagent clearing Action of diagnostics, Official methods of Analysis of the Association of Official Analytical Chemists, paragraph 960.09 and applicable chapters, 15 th edition, 1990(EPA guidelines 91-2)).

"one unit of enzyme activity" or "one unit of activity" or "U" is defined as the amount of perhydrolase activity required to produce 1 micromole peracid product per minute at a given temperature.

"half-life" or "τ" is defined as the time at which the PAA concentration has dropped to half its maximum.

"enzyme catalyst" and "perhydrolase catalyst" are used interchangeably and refer to a catalyst comprising an enzyme having perhydrolytic activity, i.e., an enzyme that exhibits the ability to catalyze the reaction between a peroxide source and a substrate to form peracids, and which may be in the form of an intact microbial cell, one or more cells of a permeabilized microorganism, one or more cellular components of a microbial cell extract, a partially purified enzyme, or a purified enzyme.

"acetylxylan esterase" refers to an enzyme (E.C. 3.1.1.72; AXE) which catalyzes the deacetylation of acetylated xylan and other acetylated sugars, and also has significant perhydrolytic activity suitable for the preparation of percarboxylic acids from carboxylic acid esters.

"Gene" refers to a nucleic acid molecule capable of expressing a particular protein, which includes regulatory sequences preceding the coding sequence (5 'non-coding sequences) and regulatory sequences following the coding sequence (3' non-coding sequences).

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

"expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the invention. Expression may also refer to translation of mRNA into a polypeptide.

"transformation" refers to the transfer of a nucleic acid molecule into the genome of a host organism such that the gene is stably inherited. In the present invention, the genome of a host cell includes chromosomal and extra-chromosomal (e.g., plasmid) genes. Host organisms containing the transformed nucleic acid molecules are referred to as "transgenic" or "recombinant" or "transformed" organisms.

"ATCC" means the American type culture Collection. The microorganism collection is located in Virginia, USA.

By "first antimicrobial agent" is meant an antimicrobial agent that is generated in situ in the coating composition according to the method of the present invention. In a preferred embodiment, the first antimicrobial agent is a peroxyacid.

By "second antimicrobial agent" is meant an additional antimicrobial agent in addition to the peroxyacid present in the coating composition. According to the present method, such preparations are not generated in situ. In a preferred embodiment, the second antimicrobial agent is a quaternary ammonium compound.

"RGQ" refers to the amino acid sequence set forth in SEQ ID NO: a highly conserved motif of amino acid residues 118-120 at position 1, which sequence is characteristic of the CE-7 enzyme family.

"GXSQG" refers to the amino acid sequence set forth in SEQ ID NO: a highly conserved motif of amino acid residues 179-183 at position 1, which sequence is characteristic of the CE-7 enzyme family.

"HE" refers to the amino acid sequence set forth in SEQ ID NO: a highly conserved motif of amino acid residue 298-299 at position 1, which sequence is characteristic of the CE-7 enzyme family.

"LXD" refers to the amino acid sequence set forth in SEQ ID NO: a highly conserved motif of amino acid residue 267-269 at position 1, which sequence is characteristic of the CE-7 enzyme family.

By "substantially similar" is meant a nucleic acid molecule in which a change in one or more nucleotide bases results in the addition, substitution, or deletion of one or more amino acids, but does not affect the functional properties (i.e., perhydrolytic activity) of the protein encoded by the DNA sequence.

"signature motif", "CE-7 signature motif" and "signature motif" refer to conserved structures shared by enzyme families having defined activities such as perhydrolase activity.

In one aspect, the invention is a method comprising using a perhydrolase enzyme in combination with a peroxygen source and a carboxylic acid ester to form a peroxyacid. Perhydrolases are enzymes that catalyze the reaction of a peroxygen source (e.g., hydrogen peroxide) with a selected substrate to form a peracid. As noted above, enzymes having perhydrolase activity include, but are not limited to, lipases, esterases, proteases, non-heme haloperoxidases, and carbohydrate esterases of the CE-7 family, such as acetylxylan esterase or cephalosporine C deacetylase.

The use of these enzymes for their perhydrolytic activity is described in commonly owned U.S. patent publication 2009/0005590A1, U.S. patent publication 2008/0176299A1, and U.S. patent publication 2007/042924A1 and U.S. patent publication 61/102,520, all of which are incorporated herein by reference in their entirety.

Certain enzymes that structurally belong to the family of CE-7 esterases, such as cephalosporin C deacetylase (CAH) and acetylxylan esterase (AXE), exhibit significant perhydrolysis activity for converting carboxylic acid esters (in the presence of inorganic sources of peroxygen, such as hydrogen peroxide, sodium percarbonate, sodium perborate) to peracids at concentrations sufficient for use as disinfectants and/or bleaches.

The perhydrolase catalyst may be used as one or more cellular components of an intact microbial cell, a permeabilized microbial cell, a microbial cell extract, a partially purified enzyme, or a purified enzyme. The enzymatic catalyst may also be chemically modified (e.g., by pegylation or by reaction with a crosslinking agent). The perhydrolase catalyst may also be immobilized to a soluble or insoluble support using methods well known to those skilled in the art.

Carbohydrate esterases of the CE-7 family

Members of the CE-7 family, cephalomycin C deacetylase (E.C. 3.1.1.41; systematic name cephalomycin C acetylhydrolase; CAH) and acetylxylan esterase (E.C.3.1.1.72) are present in plants, fungi (such as Cephalosporium acremonium), yeasts (such as Rhodosporidium toruloides, Rhodotorula glutinis) and bacteria (such as Thermoanaerobacter sp, Norcardia rhodobacter, and many members of the genus Bacillus) (Politino et al, appl. environ. Microbiol, 63: 4807-4811, 1997); sakai et al, j.ferent.bioeng.85: 53-57, 1998); lorenz, w. and Wiegel, j., j.bacteriol., 179: 5436-5441, 1997); cardza et al, appl.microbiol.biotechnol, 54: 406, 412, 2000); mitshushima et al, supra, Abbott, b. and Fukuda, d., appl.microbiol.30: 413-419, 1975); vincent et al, j.mol.biol., 330: 593-606, 2003, Takami et al, NAR, 28: 4317-4331, 2000); rey et al, Genome biol., 5: article 77, 2004); degrassi et al, microbiology, 146: 1585 1591, 2000); U.S. patent publication 6,645,233; U.S. patent publication 5,281,525; U.S. patent publication 5,338,676; and WO 99/03984. And SEQ ID NO: 1 an incomplete list of CE-7 family members with significant homology is provided in SEQ ID NOs 1-18.

As carbohydrate esterases of CE-7 family members (Vincent et al, supra), cephalosporin C deacetylase (E.C.3.1.1.41) and acetylxylan esterase (E.C.3.1.1.72) hydrolyze the acetyl groups on acetylated polymeric xylans, acetylated xylose, acetylated glucose and acetylated cellulose. Thus, acetylated saccharides may be suitable substrates for the production of percarboxylic acids using the process of the present invention (i.e. in the presence of a peroxygen source). Examples of acetylated saccharides include, but are not limited to, acetylated glucose (e.g., glucose pentaacetate), acetylated mannose, acetylated xylose (e.g., xylose tetraacetate), and acetylated cellulose.

Some enzymes, such as those suitable for use in the methods of the invention, have marker motifs that can be used to define and/or identify structurally related enzyme families that have similar enzymatic activity to a defined substrate family. The signature motif can be a single contiguous amino acid sequence or a discontinuous collection of conserved motifs that together form the signature motif. Generally, conserved motifs are represented by amino acid sequences. As described herein, the perhydrolases of the present invention belong to the CE-7 family of carbohydrate esterases. This enzyme family can be defined by the presence of a marker motif as described below.

The CE-7 family members are unusual in that they typically exhibit esterolytic activity against acetylated xylo-oligosaccharides and cephalosporin C, suggesting that the CE-7 family provides a single class of proteins with multifunctional deacetylase activity on a small range of substrates (Vincent et al, supra). Vincent et al describe the structural similarity between members of this family and define the signature sequence motif characteristic of the CE-7 family (SEQ ID NO: 21). Using CLUSTALW in combination with reference sequence SEQ ID NO: 1 has several highly conserved motifs that are characteristic of this family, including the sequence of SEQ ID NO: 1, residue 118-120(RGQ), SEQ ID NO: residue 179-183(GXSQG) of 1, SEQ ID NO: 1, residue 298-299(HE) and SEQ ID NO: residue 267-269(LXD) of 1. Multiple alignments of amino acid sequences can be performed using Higgins and Sharp, cabaos, 5: the Clustal alignment method of 151-. The default parameters for pairwise alignments using the Clustal method are typically KTUPLE 1, gap penalty 3, window 5, DIAGONALS SAVED 5.

The amino acid sequence of the perhydrolase has at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, even more preferably at least 95%, most preferably at least 99% similarity to the sequences reported herein, wherein the resulting enzymes retain the functional properties of the present invention (i.e., perhydrolytic activity), which are suitable for use in the methods of the present invention. The term "substantially similar to" may also refer to an enzyme with perhydrolytic activity which is encoded by a nucleic acid molecule which hybridizes under stringent conditions to a nucleic acid molecule of the amino acid sequence reported herein (e.g., SEQ ID NOS: 1-17 and 30). It is therefore to be understood that the invention encompasses more than the specific exemplary sequences.

Comparison of overall percent identity between the perhydrolases exemplified herein indicates that the identity of the perhydrolase with seq id NO: 1 (but retaining the characteristic motif) exhibit significant perhydrolase activity and are structurally classified as CE-7 carbohydrase.

In one embodiment, one of the perhydrolase catalysts used in the method comprises an enzyme having a CE-7 signature motif that hybridizes to the sequence of reference SEQ ID NO: 1 aligning the marker motif to comprise:

i) in SEQ ID NO: 1, amino acid position 118-120;

ii) in SEQ ID NO: 1, the GXSQG motif at amino acid position 179-183; and

iii) in SEQ ID NO: 1 amino acid position 298-299 of the HE motif;

wherein the enzyme is also homologous to SEQ ID NO: 1 have at least 30% amino acid identity.

In another embodiment, the signature motif further comprises a fourth conserved motif defined when CLUSTALW is used to align reference sequences SEQ ID NO: the LXD motif at amino acid residue position 267-269 at position 1.

In another embodiment, the perhydrolase catalyst comprises a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 1-17 and 30, or a substantially similar enzyme having perhydrolase activity, obtained by substitution, deletion or addition of one or more amino acids to said amino acid sequence.

In another embodiment, the substantially similar enzyme having perhydrolase activity is identical to an enzyme selected from the group consisting of SEQ ID NOs: 1-17 and 30 has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

In another embodiment, the perhydrolase catalyst comprises a substitution with a substitution as set forth in SEQ ID NO: 1, wherein the above-described marker motifs (e.g., RGQ, GXSQG, and HE, and optionally LXD) are retained.

In another embodiment, the perhydrolase system of the present methods is used in combination with additional enzymes, including, but not limited to, proteases, amylases, and the like. Indeed, it is contemplated that a variety of enzymes will be used in the present invention, including, but not limited to, microbial cell wall degrading and glycoprotein degrading enzymes, lysozyme, hemicellulase, peroxidase, protease, cellulase, xylanase, lipase, phospholipase, esterase, cutinase, pectinase, keratinase, isomeroreductase, oxidase, phenoloxidase, lipoxygenase, ligninase, pullulanase, tannase, pentosanase, mailanase, 13-glucanase, arabinosidase, hyaluronidase, chondroitinase, laccase, endoglucanase, PNGase, amylase, and the like, and mixtures thereof. In some embodiments, enzyme stabilizers are used in the methods of the invention. It is expected that by using a combination of enzymes, the amount of chemicals required in the process will be reduced at the same time.

While in a preferred embodiment, the perhydrolytic activity of a member of the family of structurally related carbohydrate esterase enzymes, CE-7, has been used in the present methods, any perhydrolase enzyme obtained from any source that converts primarily an ester substrate to a peracid in the presence of hydrogen peroxide may also be used in the present methods.

In addition, some hydrolases may be used in existing processes. For this purpose, suitable hydrolases include (1) proteases belonging to the peptidyl-peptide hydrolase class (e.g., pepsin B, chymosin, trypsin, chymotrypsin a, chymotrypsin B, elastase, enterokinase, cathepsin C, papain, chymopapain, ficin, thrombin, plasmin, renin, subtilisin, aspergillopeptidase a, collagenase, clostripase B, kallikrein, pepsin, cathepsin D, bromelain, keratinase, chymotrypsin C, pepsin C, aspergillopeptidase B, urokinase, carboxypeptidases a and B, and aminopeptidases); (2) formate hydrolases including carboxyl esterase, lipase, pectinesterase and chlorophyllase; and (3) enzymes with high perhydrolysis to hydrolysis ratios. Especially effective among them are lipases characterized by primary, secondary, tertiary, and/or quaternary structure using the perhydrolase enzymes of the present methods, as well as esterases exhibiting high perhydrolysis to hydrolysis ratios, as well as protein engineered esterases, cutinases, and lipases.

Suitable enzymatic reaction mixtures

"suitable enzymatic reaction mixture", "components suitable for the in situ production of peracid", "suitable reaction components", and "suitable aqueous reaction mixture" are used interchangeably herein and refer to materials and solutions in which the reactants and enzyme catalyst come into contact. The components of suitable aqueous reaction mixtures are provided herein, and one skilled in the art will appreciate the range of component variations suitable for the process of the present invention.

In one embodiment, a suitable enzymatic reaction mixture produces peracids in situ after the reaction components are combined. As such, the reaction components can be provided as a multi-component system in which one or more of the reaction components are kept separate until used. The design of systems for combining multiple active components is known in the art and will generally depend on the physical form of the individual reaction components. For example, multiple active fluid (liquid-liquid) systems typically use multi-chamber dispensing bottles or two-phase systems (U.S. patent application publication 2005/0139608; U.S. patent 5,398,846; U.S. patent 5,624,634; U.S. patent 6,391,840; EP patent 0807156B 1; U.S. patent application publication 2005/0008526; and PCT publication WO 00/11713A1), such as found in certain bleaching applications in which the desired bleaching agent is produced upon mixing of reactive fluids. Other forms of multi-component systems for generating peracids can include, but are not limited to, those designed for one or more solid components or solid-liquid component combinations, such as powders (e.g., various commercially available bleaching compositions, U.S. patent publication 5,116,575), multi-layer tablets (U.S. patent 6,210,639), water-soluble packets with multiple compartments (U.S. patent 6,995,125), and solid coacervates that react upon the addition of water (U.S. patent 6,319,888).

In another aspect, a suitable system for combining the reaction components uses a dual nozzle bottle, as disclosed in U.S. patent publication 2005/014427. An alternative device suitable for use in the method of the present invention is a dual compartment trigger-activated fluid dispenser as disclosed in EP patent 0715899B 1.

In another aspect, a suitable system for mixing the appropriate reaction components may be a container having a membrane separating the reaction components, wherein the reaction components are mixed upon rupture of the membrane by mechanical force prior to use. In another aspect, a suitable device may be an inner bag.

In another aspect, devices for combining or mixing suitable enzymatic reaction mixtures according to the methods of the present invention include systems, devices, containers, kits, multi-packs, bags, dispensers and applicators known to those skilled in the art for keeping the reaction components separate prior to use.

In another aspect, the enzymatic perhydrolysis product composition may comprise additional components that provide the desired functionality. Such additional components include, but are not limited to, buffers, detergent builders, thickeners, emulsifiers, surfactants, wetting agents, corrosion inhibitors (e.g., benzotriazole), enzyme stabilizers, and peroxide stabilizers (e.g., metal ion chelating agents). A plurality of attachmentsThe components are well known in the detergent industry (see, e.g., U.S. patent publication 5,932,532; incorporated herein by reference). Examples of emulsifiers include, but are not limited to, polyvinyl alcohol or polyvinyl pyrrolidone. Examples of thickeners include, but are not limited toRD, corn starch, PVP,Polysorbate 20, polyvinyl alcohol (PVA), and lecithin. Examples of buffer systems include, but are not limited to, sodium dihydrogen phosphate/disodium hydrogen phosphate; sulfamic acid/triethanolamine; citric acid/triethanolamine; tartaric acid/triethanolamine; succinic acid/triethanolamine; acetic acid/triethanolamine; and sodium bicarbonate/carbonate. Examples of surfactants include, but are not limited to: a) nonionic surfactants such as block copolymers of ethylene oxide or propylene oxide, ethoxylated or propoxylated linear and branched primary and secondary alcohols, and aliphatic phosphine oxides, b) cationic surfactants such as quaternary ammonium compounds, especially quaternary ammonium compounds having a C8-C20 alkyl group in combination with a nitrogen atom, additionally in combination with three C1-C2 alkyl groups, C) anionic surfactants such as alkane carboxylic acids (e.g., C8-C20 fatty acids), alkyl phosphonates, alkyl sulfonates (e.g., sodium dodecyl sulfate "SDS"), or linear or branched alkyl benzene sulfonates, alkenyl sulfonates, and d) amphoteric and zwitterionic surfactants such as aminocarboxylic acids, aminodicarboxylic acids, alkylbetaines, and mixtures thereof. Additional components may include fragrances, dyes, hydrogen peroxide stabilizers (e.g. metal chelating agents such as 1-hydroxyethylidene-1, 1-diphosphonic acid)2010, Solutia inc, st.louis, mo, and ethylenediaminetetraacetic acid (EDTA)),SL、0520、0531. enzyme activity stabilizers (e.g., polyethylene glycol (PEG)), and detergent builders.

In another aspect, the enzymatic perhydrolyzate may be premixed prior to contacting the surface or inanimate object to be disinfected to produce the desired concentration of peroxycarboxylic acid.

In another aspect, the enzymatic perhydrolysis product may be premixed to produce peroxycarboxylic acid at a desired concentration and optionally diluted with water or a solution consisting essentially of water to produce a mixture having a desired lower concentration of peracid.

In another aspect, the enzymatic perhydrolyzate is not premixed prior to contacting the surface to be disinfected or the inanimate object to produce the desired concentration of peroxycarboxylic acid, but rather, the reaction mixture components that produce the desired concentration of peroxycarboxylic acid are contacted with the surface to be disinfected or the inanimate object to produce the desired concentration of peroxycarboxylic acid. In some embodiments, the components of the reaction mixture are combined or mixed at the site. In some embodiments, the reactive components are delivered or applied to the site and then mixed or combined to generate the desired peroxyacid.

The concentration of the catalyst in the aqueous reaction mixture depends on the specific catalytic activity of the catalyst, and is selected to achieve the desired reaction rate. The concentration of the catalyst in the perhydrolysis reaction typically has a range of 0.0001mg to 10mg, preferably 0.0005mg to 2.0mg, per ml of total reaction volume. The catalyst may also be immobilized on a soluble or insoluble support using methods well known to those skilled in the art; see, e.g., Immobilization of Enzymes and Cells; gordon f. bickerstaff, Editor; humana Press, Totowa, N.J., USA, 1997. The use of an immobilized catalyst allows for recovery and reuse in subsequent reactions. The enzyme catalyst may be in the form of whole microbial cells, permeabilized microbial cells, microbial cell extracts, partially purified enzymes, or purified enzymes, and mixtures thereof.

In one aspect, the concentration of peracid generated by combining chemical perhydrolysis and enzymatic perhydrolysis of a carboxylic acid ester is sufficient to provide an effective concentration of peracid for a desired application at a desired pH. In another aspect, the methods of the present invention provide a combination of an enzyme and an enzyme substrate to produce a desired effective concentration of peracid, wherein the concentration of peracid produced is significantly lower without the addition of enzyme. While basic chemical perhydrolysis of the enzyme substrate may exist in some cases by direct chemical reaction of the inorganic peroxide with the enzyme substrate, the generated peracid concentration may be insufficient to provide an effective peracid concentration in the desired application, and the addition of a suitable perhydrolase enzyme catalyst to the reaction mixture significantly increases the total peracid concentration.

The concentration of peracid (e.g., peracetic acid) generated by perhydrolysis of at least one carboxylic acid ester is at least about 2ppm, preferably at least 20ppm, preferably at least 100ppm, more preferably at least 200ppm peracid, more preferably at least 300ppm, more preferably at least 500ppm, more preferably at least 700ppm, more preferably at least about 1000ppm peracid, most preferably at least about 2000ppm peracid within 10 minutes, preferably within 5 minutes, and most preferably within 1 minute of the start of the perhydrolysis reaction. In another aspect, the concentration of peracid generated by the perhydrolysis catalyst within one hour, more preferably within 30 minutes, most preferably within 5 minutes after initiation of the perhydrolysis reaction is at least about 3000ppm, preferably at least 5000ppm, more preferably 8000ppm, most preferably at least 10000 ppm. In one aspect, the reaction time required to produce the desired concentration of peracid is no more than about two hours, preferably no more than about 30 minutes, more preferably no more than about 10 minutes, even more preferably no more than about 5 minutes, and most preferably in about 1 minute or less. In other aspects, a hard surface or an inanimate object contaminated with a concentration of a collection of microorganisms is contacted with a peracid formed according to the methods described herein within about 1 minute to about 168 hours, or within about 1 minute to about 48 hours, or within about 1 minute to 8 hours, or within 1 minute to 2 hours, or any such time interval therein, after combining the reaction components.

The reaction temperature is selected to control the reaction rate and stability of the enzyme catalyst activity. The reaction temperature may be in the range of just above the freezing point of the reaction mixture (about 0 ℃) to about 75 ℃, preferably in the range of about 5 ℃ to about 55 ℃.

The pH of the final reaction mixture comprising the peracid is from about 5 to about 9.5, preferably from about 6 to about 8, more preferably from about 6.5 to about 7.5. In another embodiment, the pH of the reaction mixture is acidic (pH < 7). The pH of the reaction mixture during or after completion of the reaction may optionally be controlled by the addition of suitable buffers, including but not limited to phosphates, pyrophosphates, bicarbonates, acetates, or citrates. When a buffer is used, its concentration is generally 0.1mM to 1.0M, preferably 1mM to 300mM, most preferably 10mM to 100 mM.

In one embodiment of the invention, at least some of the components are premixed. By pre-mixing is meant mixing one or more individual components of the coating mixture, but not all of the desired components of the film-forming composition. Additional components of the coating composition must be added to the pre-mix components prior to final application or use at a particular site. In another embodiment, two pre-mixed components must be mixed according to the method of the present invention to form the desired coating composition.

The components of the coating composition of the present invention may be contained in a multi-compartment containment system, also referred to herein as a multi-compartment system. A multi-compartment system refers to a device that keeps two or more reaction components of a multi-compartment system coating system separate prior to use. In one aspect, the multi-compartment system comprises at least two compartments and may comprise a multi-compartment dispense vial or a dual phase system for combining reactive compounds in liquid form. In another aspect, a powder, a multilayer tablet, or a water-insoluble packet with multiple compartments may be used for the compound in solid form or a combination of solid and liquid forms. In another aspect, any kind of system, device, container, package, bag, kit, multi-pack, dispenser, or applicator for keeping reaction components separate prior to use may be used in accordance with the methods of the present invention.

The coating of the invention can be removed with an aqueous solution after application. An effective aqueous solution for removing the coating is any solution comprising from 60 to 100% by weight of water, the remaining components of the dissolved or dispersed components. Dissolved or dispersed components include, but are not limited to, solvents such as alcohols, solubilizers, surfactants, salts, chelating agents, acids and bases.

The antimicrobial coating of the present invention is a durable coating. Durability in the context of the present invention is the characteristic or feature of an applied antimicrobial coating that, after drying, remains on the surface under typical use conditions until it is intentionally started to be removed or allowed to be removed. The conditions of use are the prevailing environmental conditions during which the coating remains on the target surface at the site of application of the invention, and may include situations where there is inadvertent contact with water.

The antimicrobial coating applied to the target surface preferably may be continuous or substantially continuous. Continuous or substantially continuous in the context of the present invention means that the coating covering the target surface is free of voids, interruptions, uncovered areas, or coating defects that inadvertently leave exposed surface areas.

The coating of the present invention is preferably uniform or substantially uniform. Uniform or substantially uniform in the context of the present invention means that the coating has only small thickness variations across the coated surface, the standard deviation of the coating thickness across the coated surface being in the range of 0-40% of the coating thickness. A non-uniform or substantially non-uniform coating will not provide uniform antimicrobial and removal properties across the surface to which the coating is applied, and generally a non-uniform coating appearance is considered aesthetically undesirable for many applications.

The pseudoplasticity index or Shear Thinning Index (STI) provides an indication of the resistance to sagging and dripping that the composition may exhibit. The value recorded at the lower shear rate is divided by the value at the higher shear rate to give the STI. Generally, the higher the STI of the coating material, the higher its resistance to sagging and dripping will be. In this publication the shear thinning index is defined as the ratio of the viscosities measured at a first shear rate and a second shear rate, wherein the value of the second shear rate is 10 times the value of the first shear rate. Alternatively, if s^-1The shear rate in (a) is known and the shear thinning index can also be calculated by measuring the viscosity with a rotary viscometer at a first rpm and a second rpm, where the second rpm is 10 times the value of the first rpm. The shear thinning index is calculated as the ratio of the viscosities measured at 1rpm and 10rpm, independent of the particular first and second shear rates used or the particular first and second rpm values used to calculate the STI in the examples.

The antimicrobial coating of the present invention is useful as a disinfectant. As defined herein, a sanitizing agent is a chemical or mixture of chemicals that can be either (i) a sanitizing agent that contacts food if its purpose is to control microorganisms that actually or potentially contact food on a surface, or (ii) a sanitizing agent that does not contact food if the surface is not intended to contact food. As defined herein, a disinfectant that contacts food kills at least 99.999% of a particular test microorganism within 30 seconds under test method conditions, such as EPA policy DIS/TSS-4: "effective data requirements-verifying information for previous clean food-contact surfaces", United States environmental protection Agency, described in 30/1/1979. As defined herein, a disinfectant that does not contact food kills at least 99.9% of a particular test microorganism within 5 minutes under test method conditions, such as ASTM standard E1153-03, published in month 4 and 10, 2003, and at month 7, 2003: "Standard Test Method for effectiveness of Sanitizers Recommended for improving Non-Food Contact Surfaces".

The coating compositions of the present invention are capable of exhibiting residual antimicrobial efficacy and exhibit self-disinfecting properties. "residual antimicrobial efficacy" or "self-disinfecting properties" refers to the properties of a coating formed as described herein that retains antimicrobial activity after drying. Antimicrobial activity of the dried coating can be measured using the residual self-disinfection (RSS) test.

There is a long felt need for antimicrobial agents with improved antimicrobial efficacy and improved speed of action. The specific requirements for such formulations vary depending on the intended application (e.g., germicide, disinfectant, sterilant, sterile packaging process, etc.) and the applicable public health needs. For example, as suggested by Germidic and Detergent disinfecting Action of disinfecting, Official Methods of analysis of the Association of Official Analytical Chemists, paragraph 960.09 and the applicable chapters, 15 th edition, 1990(EPA Guideline 91-2), the disinfectant should provide a 99.999% reduction (5-log reduction) in 30 seconds for several test organisms at room temperature (23-27 ℃).

The in situ enzymatically generated peracid-based, removable antimicrobial coating compositions used in the present invention can be used as a replacement for or in addition to standard disinfecting products (e.g., dilute quaternary ammonium compound solutions, peracid foams, etc.) and as a protective coating for routine disinfection of equipment, whether in use or not, and for long term protection (weeks or months).

The in situ enzymatically generated peracid-based, removable antimicrobial coating compositions of the present disclosure provide several advantages, including, but not limited to, killing stand-alone or planktonic microorganisms and microorganisms living in biofilms, reducing or preventing microbial growth by preventing biofilm formation and trapping microorganisms in, under, or otherwise in contact with the coating.

The coating compositions disclosed herein can be modified by formulating the compositions with rheology modifiers to coat vertical, sloped, complex geometry, or difficult to reach surfaces. This enables the antimicrobial agent to be applied to surfaces on or within equipment that are difficult to apply to conventional antimicrobial solutions having conventional shear-viscosity characteristics and viscosities of less than about 0.01 pascal-second at 25 ℃. Horizontal and vertical surfaces may be covered with a thin protective coating free of biocide waste, since the coating prevents or greatly reduces the occurrence of dripping by the rheology modifier. By formulating the composition with suitable rheology modifiers and a degree of crosslinking, coating compositions can be prepared having a variety of coating characteristics, which will vary in surface finish and protective properties as well as ease of removal.

The coating composition of the present invention provides several mechanisms to prevent contamination from microbial or non-microbial sources, such as soil. For example, when a liquid coating composition is applied, cells that are planktonic or loosely attached to a surface are killed, or their growth reduced or prevented, by an antimicrobial agent in the coating formulation.

Furthermore, by spreading the antimicrobial agent into the hydrated biofilm after application of the liquid antimicrobial coating composition of the present invention, before the applied film-forming composition is completely dried to provide an antimicrobial film, cells on the surface protected by the biofilm will be killed, or their growth may be reduced or prevented. For sustained antimicrobial activity, it is desirable that the antimicrobial films of the present invention be semi-permeable. The antimicrobial film thus formed constitutes a reservoir for the antimicrobial agent which provides a much longer contact time than conventional disinfecting rinses which typically drip in seconds or minutes.

When a coating is present on a site, activity that is retained for a long period of time is particularly beneficial in a variety of applications. The film-forming antimicrobial compositions of the present invention do not drip rapidly from the target surface and are not easily removed, for example, by incidental contact. The flexibility, viscosity, strength and adhesion of the films of the coatings of the present invention are varied such that they are tailored to specific uses, thus providing a consistently useful antimicrobial protection in a variety of situations where such sustained activity (residual benefit) was previously unavailable.

The film-forming, water-soluble or water-dispersible agent used in the practice of the present invention can be at least one of any formulation that is durable and removable. The films of the present invention are designed to be removable under relatively mild conditions. For example, the film of the invention can be removed when treated with an aqueous solution having a temperature above 15 ℃, preferably above 30 ℃. Suitable film forming agents are selected from, but are not limited to, polyvinyl alcohol copolymers, polyvinyl pyrrolidone, polyacrylic acid, acrylate copolymers, ionic hydrocarbon polymers, polyurethanes, polysaccharides, functionalized polysaccharides, rice bran extracts, glucomannan, guar gum, gum arabic, johannis gum, cellulose, methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose starch, hydroxyethyl starch, xanthan gum, carrageenan, curdlan, pullulan, pectin, dextran, chitosan, glycerol, sodium alginate cross-linked with calcium salts, carrageenan, ethylene oxide/propylene oxide/ethylene oxide block copolymers, and combinations thereof. One skilled in the art can readily select an appropriate molecular weight range to provide a range of water solubilities to provide an easily removable coating according to the methods of the invention.

Polyvinyl alcohols, sometimes referred to as poly (vinyl alcohol), are made from polyethylene ethyl esters by hydrolysis and are suitable for use in the present invention. The physical properties of polyvinyl alcohol are controlled by molecular weight and degree of hydrolysis. The most commonly available grades of polyvinyl alcohol are classified by degree of hydrolysis as: an 87-89% grade (containing 11-13 mole% residual vinyl acetate units), a 96% hydrolyzed grade (containing 4 mole% residual vinyl acetate units), and "fully hydrolyzed" and "super hydrolyzed" grades, which are about 98% and greater than 99% hydrolyzed, respectively. Lower degrees of hydrolysis (e.g., 74% and 79%) are also commercially available. Some preferred degrees of hydrolysis are greater than 85 mole%, or greater than 92 mole%. The polyvinyl alcohol component of the invention may also be a copolymer of vinyl alcohol, for example obtained by hydrolysis of a copolymer of vinyl acetate with a small amount (up to about 15 mole%) of other monomers. Suitable comonomers are, for example, esters of acrylic acid, methacrylic acid, maleic or fumaric acid, itaconic acid, etc. In addition, copolymerization of vinyl acetate with hydrocarbons such as alpha-olefins (e.g., ethylene, propylene or octadecene, etc.), with higher vinyl esters such as vinyl butyrate, ethyl 2-hexanoate, stearate, trimethyl acetate, or their homologs ("VV-10" type vinyl esters sold by shellchem. Other suitable comonomers are N-substituted acrylamides, vinyl fluoride, allyl acetate, allyl alcohol, etc. Furthermore, free unsaturated acids such as acrylic acid, methacrylic acid, monomethylmaleic acid and the like can also be used as comonomers.

Due to the variety of grades known in the literature or commercially available, one skilled in the art can formulate polyvinyl alcohol solutions having an average degree of hydrolysis ranging from 74% to greater than 99% by simply mixing known or commercial grades in any desired ratio.

The flexibility, water sensitivity, ease of dissolution, viscosity, film strength and adhesion of the polyvinyl alcohol film of the film can be varied by adjusting the molecular weight and the degree of hydrolysis.

In one embodiment, the polyvinyl alcohol used in the process of the present invention has a degree of hydrolysis of from about 85% to greater than 99%. In another embodiment, the polyvinyl alcohol has a degree of hydrolysis of from about 87% to greater than 89%.

In one embodiment, the polyvinyl alcohol has a number average molecular weight (Mn) in a range between about 4,000 to about 200,000, or about 4,000 to about 150,000, or 10,000 to about 100,000.

In one embodiment, the molecular weight of the polyvinyl alcohol is in a range between about 10,000 and 130,000. In another embodiment, polyvinyl alcohols of different molecular weights may be mixed to provide desired characteristics.

In one embodiment, the polyvinyl alcohol is used at about 2% to about 30% by weight of the solution weight. In a more specific embodiment, the polyvinyl alcohol is used at about 2% to about 15% by weight of the solution weight. In a more specific embodiment, the polyvinyl alcohol is used at about 5% to about 12% by weight of the solution weight.

The film-forming compositions of the present invention may comprise PVP at a concentration of about 0.25% to about 50% by weight. Suitable grades of PVP are available from International Specialty Products (Wayne, NJ, USA). Such grades include: k-15 having an average molecular weight (Mw) in the range of about 6,000 to about 15,000; k-30 having a molecular weight in the range of about 40,000 to about 80,000; k-60 having a molecular weight in the range of about 240,000 to about 450,000; k-90 having a molecular weight in the range of about 900,000 to about 1,500,000; and K-120 having a molecular weight in the range of about 2,000,000 to about 3,000,000. Mixtures of PVP, as well as combinations of PVP and other film-forming compounds, may be used. Suitable grades are described in co-owned and co-pending U.S. patent applications: described in U.S. patent 2007/0275101a1 and U.S. patent 2008/0026026a1, both of which are incorporated herein by reference in their entirety.

Generally, a lower molecular weight PVP will produce a less viscous product than a higher molecular weight PVP at the same concentration. For a given concentration of PVP, as the molecular weight range increases, the viscosity will also increase. PVP having any of a variety of molecular weight ranges may be used in the present invention. However, it is preferred to use PVP having a molecular weight distribution of from about 15,000 to about 3,000,000 g/mol. Having such a molecular weight distribution generally results in a film-forming composition having a viscosity that can be easily adjusted and that can be easily washed away without significant signs of interaction with the coated surface. In a preferred embodiment, PVP having a molecular weight distribution of between about 15,000 and about 3,000,000g/mol is present at a concentration of between about 0.25% and about 40% by weight. In another preferred embodiment, PVP having a molecular weight distribution of between about 30,000 and about 1,200,000g/mol is present at a concentration of between about 0.25% and about 10% by weight.

One class of molecules that is widely recognized as including high level disinfectants are organic peroxyacid molecules. In recent years research has been carried out on these compounds, with the aim at least of providing them with stable and safe delivery devices. Of the peroxyacids, peroxyacetic acid (often referred to as peracetic acid or PAA) has received the most intense attention. This particular peroxyacid rarely separates into pure compounds, but is usually a solution in an aqueous and non-aqueous medium. Peracetic acid is often supplied as an equilibrium mixture, mainly comprising water, acetic acid and hydrogen peroxide, as well as many other minor components such as stabilizers (WO 2006/076334; U.S. patent 5,508,046). However, even equilibrium systems of peracetic acid are thermodynamically unstable in nature due to decomposition of peracetic acid into acetic acid and oxygen. Such decomposition can be delayed, but not completely prevented, if special measures are taken. Other characteristics which make the peroxy acids difficult to use or store are their rapid and violent decomposition, especially of low molecular weight and high purity. To address this problem, peroxy acids are typically generated in situ to achieve specific desired functions. Such in situ generation has advantages in that the amount of peroxyacid produced can be stoichiometrically controlled by selection of the relative compositions of the starting materials. Furthermore, due to the non-equilibrium nature of the in situ system, higher concentrations of peroxy acids may be obtained than peroxy acids obtained from an equilibrium system.

It is also desirable to generate peroxyacids at a desired rate: too slow a rate may result in poor performance. The methods of the present invention provide a combination of an enzyme and an enzyme substrate to produce a desired effective concentration of peracid, wherein the concentration of peracid produced is significantly lower without the addition of enzyme. While perhydrolase substrates may be substantially chemically synthesized in some cases by direct chemical reaction of inorganic peroxides with enzyme substrates, the generated peracid concentration may not be sufficient to provide an effective peracid concentration in the desired application, and the addition of a suitable perhydrolase catalyst to the reaction mixture significantly increases the total peracid concentration.

One solution to the intrinsic lack of stability of peracids is to provide a system wherein the reaction components that generate or react to form the peracid are kept separate until needed (WO 2006/016145). Such a solution to the problem is contemplated in the methods described herein.

The film-forming component is combined in the present invention with other components including a carboxylic acid ester substrate, an enzyme catalyst having perhydrolase activity, and a source of peroxygen. The film-forming component may be combined with one or more of the following components: the carboxylic acid ester substrate, enzyme catalyst, and peroxygen source are stored together in one compartment, or in separate compartments, prior to combining to form the antimicrobial coating composition and applying to a target surface. For example, the film-forming components from the carboxylic ester substrate, the enzyme catalyst, and the peroxygen source may be stored in separate compartments and combined just prior to application to the target surface, or the film-forming components may be stored with the carboxylic ester substrate but separated from the enzyme catalyst or peroxygen source until they are combined and applied to the target surface.

The process of the present invention enzymatically generates effective concentrations of peracids in situ in the form of a durable, continuous and easily removable film-forming composition using an enzyme having perhydrolase activity from the CE-7 family of carbohydrate esterases. As described herein, genes encoding the enzymes (e.g., enzymes having the amino acid sequence SEQ ID NO: 7 isolated from Thermotoga neapoliana (Thermotoga nerolitana), the amino acid sequence SEQ ID NO: 8 isolated from Thermotoga maritima (Thermotoga maritima), or a substantially similar amino acid sequence) are suitable for use in the methods of the invention. As disclosed above, other enzymes having perhydrolase activity from different sources may also be used in the methods disclosed herein.

In a preferred embodiment, the inorganic peroxide source is hydrogen peroxide. The present invention is not intended to be limited by inorganic hydrogen peroxide. Hydrogen peroxide can be generated using an enzyme catalyst and a suitable substrate. Any enzyme that generates hydrogen peroxide and an acid is used in the process of the invention, as well as suitable substrates. For example, lactoperoxidase from Lactobacillus (Lactobacillus) species, which is known to produce hydrogen peroxide from lactic acid and oxygen, may be used in the process of the invention.

Other enzymes capable of producing hydrogen peroxide (e.g., glucose oxidase, alcohol oxidase, ethylene glycol oxidase, glycerol oxidase, amino acid oxidase, etc.) also produce peracids in combination with the ester substrate and perhydrolase enzymes of the present methods. The present invention also uses enzymes that generate acid from a substrate without generating hydrogen peroxide. Examples of such enzymes include, but are not limited to, proteases.

In another aspect, the peroxygen source may be any peroxide compound and may be selected from, but is not limited to, the following: hydrogen peroxide, sodium perborate monohydrate, sodium perborate tetrahydrate, sodium perpyrophosphate, urea peroxide, sodium percarbonate, sodium peroxide, and mixtures thereof.

In one embodiment, suitable substrates include esters represented by the formula:

[X]_mR₅

wherein X is R₆C (O) ester group of O

R₆(iii) a linear, branched or cyclic hydrocarbyl moiety from C1 to C7, optionally substituted with hydroxy or C1 to C4 alkoxy, wherein when R6 is from C2 to C7, R₆Optionally containing one or more ether linkages;

R₅a ═ C1 to C6 linear, branched, or cyclic hydrocarbyl moiety, optionally substituted with hydroxyl; wherein R is₅Each carbon atom in (a) independently contains no more than one hydroxyl group or no more than one ester group; wherein R is₅Optionally containing one or more ether linkages;

m is 1 to R₅The number of carbon atoms; and wherein the ester has a solubility in water of at least 5ppm at 25 ℃.

In another embodiment, suitable substrates also include esters represented by the formula: r₁-COO-R₂Wherein R is₁Linear or branched alkyl of ═ C1 to C7, optionally substituted with hydroxy or C1 to C4 alkoxy, and R₂Linear or branched alkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, - (CH) of ═ C1 to C10₂CH₂-O)_nH or- (CH)₂CH(CH₃)-O)_nH and n is 1 to 10.

In another embodiment, suitable substrates also include glycerides represented by the formula: r₁-COO-CH₂-CH(OR₃)-CH₂-OR₄Wherein R is₁Linear or branched alkyl of ═ C1 to C7, optionally substituted with hydroxy or C1 to C4 alkoxy, and R₃And R₄Is H or R alone₁C(O)。

In another embodiment, R₆Is a C1 to C7 branched chain hydrocarbyl moiety optionally substituted with hydroxyl or C1 to C4 alkoxy, optionally containing one or more ether linkages. In a more preferred embodiment, R₆Is a C2 to C7 straight chain hydrocarbyl moiety optionally substituted with hydroxyl groups, and/or optionally contains one or more ether linkages.

In another embodiment, suitable substrates also include acetylated sugars selected from the group consisting of acetylated monosaccharides, disaccharides and polysaccharides. In a preferred embodiment, the acetylated saccharides include acetylated monosaccharides, disaccharides and polysaccharides. In another embodiment, the acetylated saccharide is selected from the group consisting of acetylated xylan, acetylated xylan fragments, acetylated xylose (e.g., xylose tetraacetate), acetylated glucose (e.g., glucose pentaacetate), β -D-ribofuranose-1, 2, 3, 5-tetraacetate, tri-o-acetyl-D-galactal and tri-o-acetyl-D-glucal, and acetylated cellulose. In a preferred embodiment, the acetylated saccharide is selected from the group consisting of β -D-ribofuranose-1, 2, 3, 5-tetraacetic acid ester, tri-o-acetyl-D-galactal and tri-o-acetyl-D-glucal, and acetylated cellulose. Thus, acetylated saccharides may be suitable substrates for the production of percarboxylic acids using the process of the present invention (i.e. in the presence of a peroxygen source).

In one embodiment, examples of the substrate are selected from: glycerol monoacetate; a glycerol diacetate; triacetin; glycerol monopropionate; glycerol dipropionate; glyceryl tripropionate; monobutyric acid ester of glycerol; dibutylglycerol; glycerol tributyrate; glucose pentaacetate; xylose tetraacetate; acetylated xylan; acetylated xylan fragments; beta-D-ribofuranose-1, 2, 3, 5-tetraacetate; tri-o-acetyl-D-galactal; tri-o-acetyl-glucal; mono-or diesters of 1, 2-ethanediol, 1, 2-propanediol, 1, 3-propanediol, 1, 2-butanediol, 1, 3-butanediol, 2, 3-butanediol, 1, 4-butanediol, 1, 2-pentanediol, 2, 5-pentanediol, 1, 6-pentanediol, 1, 2-hexanediol, 2, 5-hexanediol, 1, 6-hexanediol; and mixtures thereof.

In a preferred embodiment, the substrate is selected from the group consisting of ethyl acetate, methyl lactate, ethyl lactate, methyl glycolate, ethyl glycolate, methyl methoxyacetate, ethyl methoxyacetate, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, triethyl 2-acetylcitrate, glucose pentaacetate, gluconolactone, glycerides (mono-, di-and triglycerides) such as monoacetin, diacetin, triacetin, monopropionate, dipropionate, tripropionate (1, 2, 3-tripropionylglycerol), monobutyrate, dibutyrate (dibutyrylglycerol), tributyrin (1, 2, 3-tributyrin), acetylated sugars, and mixtures thereof.

In another preferred aspect, the carboxylate substrate is selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, ethyl acetate, and ethyl lactate. In yet another aspect, the carboxylate substrate is selected from the group consisting of diacetin, triacetin, ethyl acetate, and ethyl lactate. In a preferred aspect, the carboxylic acid ester is a glycerol ester selected from the group consisting of monoacetin, diacetin, triacetin, and mixtures thereof.

The inert solvent used in the present invention includes water or a solution comprising at least 50% by weight of water. Additional solvents include monohydric, monofunctional, and polyfunctional alcohols, preferably containing from about 1 to about 6 carbon atoms and from 1 to about 6 hydroxyl groups. Examples include ethanol, isopropanol, n-propanol, 1, 2-propanediol, 1, 2-butanediol, 2-methyl-2, 4-pentanediol, mannitol, and glucose. Higher glycols, polyglycols, polyoxides, glycol ethers and propylene glycol ethers are also useful. Additional solvents include the free acids and the following alkali metal salts: sulfonated alkylaryl groups such as toluene, xylene, cumene and phenol or phenol ethers or diphenyl ether sulfonates; alkyl and dialkyl naphthalene sulfonates and alkoxylated derivatives.

Compositions useful in the present invention may also comprise one or more surfactants. Without being bound by theory, it is believed that the surfactant will help wet the surface to be coated and will help even coverage by the film. It is believed that the surfactant also helps to bubble through the film when removed, thereby assisting in removing the film and washing the protected surface. Suitable surfactants have a preferred hydrophilic-lipophilic balance (HLB) of about 9 to about 17. Suitable surfactants include, but are not limited to: amphoteric surfactants such as Ampoteric N from Tomah Products; silicone surfactants such as BYK 348 from BYK Chemie (BYK-Chemie GmbH, Wesel, Germany); fluorinated surfactants such as those available from DuPont (Wilmington, DE, USA)FS 300; and nonylphenol polyoxyethylene ether based surfactants such as Triton N-101 from Dow (Midland, MI, USA). Other suitable surfactants include ethoxylated decynediols such as those available from Air Products&Surfynol 465 from Chemicals (Allentown, Pa., USA); alkylaryl polyethers such as Triton CF-10 from Dow; octylphenoxy polyethoxyethanol such as Triton X-100 from Dow; ethoxylated alcohols such as Neodol 23-5 or Neodol 91-8 from Shell (The Hague, The Netherlands); tergitol 15-S-7 from Dow; from Stepan Company (North)field, IL, USA), Steol-4N, 28% sodium laureth sulfate; aminating oxygen, e.g. from StepanLO; EO/PO block copolymers such as those from BASF (Parsippany, NJ, USA)17R 4; sorbitan derivatives such as Tween 20 or Tween 60 available from Uniqema (New Castle, DE, USA); and quaternary ammonium compounds such as benzalkonium chloride. Other suitable surfactants include organo-siloxane surfactants, such as those available from Setre Chemical Company (Memphis, TN, USA)From Dow Corning Silicones (Midland, MI, USA)Q2-5211; or from Siltech Corporation (Toronto, ON, Canada)A008。

It can be important to the flexibility and integrity of the protective film, which is plasticized. For the purposes of the present invention, the plasticization of the film has been accomplished by incorporating suitable plasticizers, such as polyethylene glycol or glycerol. Other plasticizers suitable for use in the present invention include, but are not limited to, solvents, polyols, polyethylene glycols having an average molecular weight of 200 to 800g/mol, and sorbitol. PEG is preferred over glycerol because glycerol is readily metabolized by microorganisms, potentially leading to microbial growth.

The addition of a plasticizer also generally allows the film to retain a slightly tacky surface feel. As the plasticizer content increases, the resulting film will also exhibit increased tack. Such tackiness may be desirable at low levels in order to trap airborne particulates and scale or other substances. However, if the plasticizer content is too high, the coating becomes too tacky and will show a low resistance to accidental mechanical removal, such as wiping.

Preferred amounts of plasticizer are from about 1% to about 20% by weight, more preferably from about 5% to about 10% by weight, based on the weight of the film forming agent.

Compositions useful in the present invention may also contain one or more rheology modifiers for increasing the viscosity, or thickening and causing the aqueous treating agent or coating composition to adhere to a surface. Adhesion enables the composition to remain in contact with temporary and resident microorganisms for a longer period of time, enhances germicidal efficacy and prevents litter from excessive dripping. The rheology modifier may be a film forming agent or act synergistically with a film forming agent to form a barrier that provides additional protection. Useful water-soluble or water-dispersible rheology modifiers can be classified as inorganic or organic. Organic thickeners can be further classified into natural and synthetic polymers, the latter being further classified into synthetic natural-based and synthetic petroleum-based.

Inorganic thickeners are generally compounds which have been fumigated or precipitated to produce particles having a large surface to size ratio, for example colloidal magnesium aluminium silicatesColloidal clay (bentonite), or silicaUseful natural hydrogel thickeners are primarily vegetable-derived exudates. For example, gum tragacanth, karaya and gum acacia; and extracts such as carrageenan, locust bean gum, guar gum and pectin; or pure culture fermentation products such as xanthan gum are potentially useful in the present invention. Chemically, all of these materials are salts of complex anionic polysaccharides. Useful synthetic natural-based thickeners are cellulosic derivatives in which the free hydroxyl groups on the linear anhydroglucose polymer have been etherified or esterified to produce a class of materials that dissolve in water and form viscous solutions. Such materials include alkylcelluloses and hydroxylsAlkyl-alkylcelluloses, including in particular methylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, hydroxybutylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, hydroxypropylcellulose and carboxymethylcellulose. Another class of preferred thickeners includes polyacrylates such as proprietary Acusol thickeners, (e.g., Acusol 823, Rohmand Haas, Philadelphia, PA, USA), and Carbopol thickeners such as Carbopol 934 or Carbopol Aqua-30 polymers (B F Goodrich, Cleveland, OH, USA). Additional preferred acrylate-based rheology modifiers include proprietary cationsThickeners (Ciba, Basel, Switzerland). The polyacrylate thickener may be used in a concentration of up to 3% by weight based on the weight of the film forming agent. Mixtures of thickeners may also be used, wherein the total amount of the mixture may be up to 3% by weight depending on the thickener used and the desired viscosity of the final product.

For purposes of this patent application, other possible thickeners include dextrins, corn starch and hydrous magnesium silicates, such as sodium magnesium silicate sold under the trade name Laponite XLG (Southern Clay Products, inc., Gonzales, TX, USA).

In addition to the in situ generation of the peroxyacid, one or more additional antimicrobial agents may be present in the composition. Antimicrobial agents useful in the present invention may be inorganic or organic agents, or mixtures thereof.

The term "inorganic antimicrobial agent" as used herein is a generic term for inorganic compounds that contain metals or metal ions such as silver, zinc, copper, and the like and have antimicrobial properties.

The present invention is not limited by the selection of any particular antimicrobial agent, and any known water-soluble or water-dispersible antimicrobial agent can be included in the compositions of the present invention, such as antimicrobial agents, mildewcides, antiseptics, sanitizers, disinfectants, sanitizers, algaecides, antifouling agents, preservatives, combinations of the foregoing, and the like, provided that the antimicrobial agent is chemically compatible with the other components of the composition. Suitable classes of antimicrobial agents are described below.

Examples of useful antimicrobial agents include chlorhexidine, chlorhexidine gluconate, glutaraldehyde, halazone, hexachlorophene, nitrofurazone, labflufen, thimerosal, C1-C5-paraben, hypochlorite, halocarban, chlorobenzophenol, phenolic resins, mafenide acetate, amsacrine hydrochloride, quaternary ammonium salts, chlorine and bromine releasing compounds (e.g., alkali and alkaline earth hypochlorites and hypobromites, isocyanurates, chlorinated derivatives of hydantoin, sulfonamides, amines, and the like), peroxide and peroxyacid compounds (e.g., peracetic acid, peroctanoic acid), protonated short chain carboxylic acids, oxychlorobenzenesulfonic acid, meflon, merbromin, dibromsalan, glyceryl laurate, sodium and/or zinc 1-oxo-2-mercaptopyridine, trisodium phosphate, (dodecyl) (tetramethyldiamine) glycine and/or (dodecyl) (aminopropyl) glycine, and the like. Useful quaternary ammonium salts include N-C₁₀-C₂₄-alkyl-N-benzyl-quaternary ammonium salts comprising water-solubilizing anions such as halides, e.g. chloride, bromide and iodide; sulfates, dimethyl sulfate, and the like, and heterocyclic imines such as imidazolinium salts. The quaternary ammonium salts may also include other non-halogenated anions such as propionate and saccharin. Useful phenolic resin fungicides include phenol, m-cresol, o-cresol, p-cresol, o-phenyl-phenol, 4-chloro-m-cresol, chloroxylenol, 6-n-pentyl-m-cresol, resorcinol-acetate, p-tert-butylphenol, and o-benzyl-p-chlorophenol. Useful antimicrobial agents known to be effective in preventing visible growth of mold colonies include, for example, butyl 3-iodo-2-propargyl carbamate, 2- (4-thiazolyl) benzimidazole, p-tolyl diiodomethyl sulfone, tetrachloroisophthalonitrile, zinc complexes of 2-pyridinethiol-1-oxide (including salts thereof), and combinations of the foregoing. Coating compositions comprising antimicrobial agents provide protection against a variety of different microorganisms.

In one embodiment, the coating composition is protective against gram positive or gram negative bacteria. Gram-positive bacteria that are inhibited or killed by the coating include, but are not limited to, Clostridium tetani (Clostridium tetani), Clostridium perfringens (c.perfringens), Clostridium botulinum (c.botulinum), other Clostridium species (Clostridium), mycobacterium tuberculosis (mycobacterium tuberculosis), mycobacterium bovis (m.bovis), mycobacterium typhimurium (m.typhimurium), mycobacterium bovis (m.bovis) strain, BCG sublaterial, mycobacterium avium (m.avium), mycobacterium intracellulare (m.intracellularis), mycobacterium africanum (m.africanum), mycobacterium kawasabi (m.kansasanii), mycobacterium marinum (m.marinum), mycobacterium ulcerosa (m.ulcerans), mycobacterium avium subspecies (m.avium paratuberis), staphylococcus aureus (staphylococcus aureus), Streptococcus pyogenes (Streptococcus epidermidis), Streptococcus pyogenes (Streptococcus pyogenes) Bacillus anthracis (Bacillus antrhricus), Bacillus subtilis (B. subtilis), Nocardia asteroides and other Nocardia species, Streptococcus viridans (Streptococcus viridans group), Peptococcus species (Peptococcus species), Peptostreptococcus species (Peptostreptococcus species), Actinomyces chlamydomonas (Actinomyces israelii) and other Actinomyces species (Actinomyces species), Propionibacterium acnes (Propionibacterium acnes) and Enterococcus species (Enterococcus species). Gram-negative bacteria that are inhibited or killed by the coating include, but are not limited to, Pseudomonas aeruginosa (Pseudomonas spec), other Pseudomonas species (Pseudomonas specs), Campylobacter species (Campylobacter speces), Vibrio cholerae (Vibrio cholerae), Escherichia species (Ehrlichia specs), Actinobacillus pleuropneumoniae (Actinobacillus pleuropneumoniae), Pasteurella haemolytica (Pasteurella haemolytica), Pasteurella multocida (P.multocida), other Pasteurella species (Pasteurella specs), Legionella retrograded (Leginella pneumophila), other Legionella species (Legionella vulgaris), Salmonella typhi (Salmonella typhimurium), other Salmonella typhimurium (Salmonella specula), Shigenia Chlamydia (Schizoea), Salmonella typhimurium Chlamydia (Brevibacterium), Salmonella choleraesuis (Brevibacterium), and Salmonella choleraesuis Meningococcus (Neiserria meningitidis), gonococci (N.gonorrhoea), Haemophilus influenzae (Haemophilus influenzae), Haemophilus ducreyi (H.ducreyi), other Haemophilus species (Haemophilus species), Yersinia pestis (Yersinia pestis), Yersinia enterocolitica (Y.enterolitica), other Yersinia species (Yersinia speces), Escherichia coli (Escherichia coli), enterococcus hirae (E.hirae), and other Escherichia species (Escherichia specs), and other enterobacteria (Enterobacteriaceae), Burkholderia cepacia (Burkholderia cepacia), Burkholderia pseudomallei (B. pseudomonas), Francisella tularensis (Francisella tularensis), Bacteroides fragilis (Bacteroides fragilis), Clostridium nucleatum (Fusobacterium subclautus), Prevotella species (Provetella species), ruminant Cowderia ruminants (Cowdria ruminants), Klebsiella species (Klebsiella species), and Proteus species (Proteus species).

In another embodiment, the coating provides protection against fungi including, but not limited to, Alternaria (Alternaria alternata), Aspergillus niger (Aspergillus niger), Aureobasidium pullulans (Aureobasidium pullulans), Mycobacterium sporotrichum (Cladosporium cladosporioides), Deuteromyces australis (Drechslera australis), Mucor graminis (Gliomastix cerealis), Pediobolus oryzae (Monilia grisea), Penicillium globosum (Penicillium commune), Phoma annulata (Phoma fimeti), Cissus dermatum (Pihromyces chartarum), and human Basidiomycetes lineare (Scolebasidium humicola).

Trace impurities, especially metals, can react with hydrogen peroxide and peroxy acids and cause decomposition. Many peroxide/peroxyacid compositions therefore include stabilizing ingredients, such as metal-chelating compounds and metal-containing hybridsA material. Examples of preferred chelating agents include, but are not limited to, alkylene phosphoramidates or salts thereof, some of which are under the trade name(Thermphos, Valissingen, the Netherlands), 1-hydroxyethylidene-1, 1-diphosphonic acid and salts thereof, some of which are sold under the trade name(Thermphos) and 2-phosphono-1, 2, 4-butanetricarboxylic acid or salts thereof, in the form of their saltsIs available under the trade name LANXESS Corporation (Pittsburgh, PA, USA). Another class of suitable compounds are aminocarboxylic acids or salts thereof. One example is ethylenediaminetetraacetic acid.

In addition, other suitable chelating agents include dipicolinic acid, ethane-1, 1, 2-triphosphonic acid, and ethylene-1, 1-diphosphonic acid. In addition, chelating agents also well known in the art are phosphates, polyphosphates, pyrophosphates, and carboxylic acids such as citric acid or salicylic acid. A sufficient amount of stabilizer or stabilizers will be present to inhibit the decomposition of the peroxide/peroxyacid.

The present invention may optionally include a colorant or dye. Colorants useful in the present invention include dyes and pigments such as food grade pigments.

Dyes useful in the present invention include both water soluble and water insoluble dyes. Water soluble dyes can be readily formulated into the aqueous systems of the present invention. Water insoluble dyes can be included in the oil phase that can be dispersed or suspended in the antimicrobial coating compositions useful in the present invention. For the purposes of the present invention, useful dyes are generally organic compounds which absorb visible light and thus lead to the development of a detectable color. Fluorescent dyes may also be used, for example, in order to develop the film by ultraviolet light.

Dyes typically useful in the present invention are colorants approved for use in food, pharmaceutical, cosmetic and medical devices. The colorants currently used and their state are as follows: the colorants allowed for use in the food product are (1) certified: FD & C blue No.1, FD & C blue No.2, FD & C green No. 3, FD & C red No. 40, FD & C yellow No. 5, FD & C yellow No. 6, Citrus red No.2, and orange (B) (2) certificate-exempt: annatto extract, theta-derived-8' -carotenal, canthaxanthin, caramel, theta-carotene, carrot oil, cochineal extract (carmine), corn germ cream, dehydrated beets (beet powder), dried algal meal, ferrous gluconate, fruit juice, grape color extract, grape skin extract, sweet pepper, chili oil, riboflavin, saffron, synthetic iron oxide, tagetes meal and extract, titanium dioxide, partially roasted defatted cooked cottonseed flour, turmeric butter, ultramarine blue, and vegetable juice. The colorants allowed for use in pharmaceuticals (including colorants allowed for use in food) are (1) certified: FD & C red No. 4, D & C blue No. 9, D & C green No. 5, D & C green No. 6, D & C green No. 8, D & C orange yellow No. 4, D & C orange yellow No. 5, D & C orange yellow No. 10, D & C orange yellow No. 11, D & C red No. 6, D & C red No. 7, D & C red No. 17, D & C red 21, D & C red No. 22, D & C red No. 27, D & C red No.28, D & C red No. 30, D & C red No. 31, D & C red No. 33, D & C red No. 34, D & C red No. 36, D & C red No. 39, D & C violet No.2, D & C yellow No. 7, D & C yellow No. 8, D & C yellow No. 10, D & C yellow No. 11, and ext. Furthermore, canthaxanthin, beta-carotene, chlorophyllin and other pigments are known. For a more detailed listing and/or discussion of approved pigments, see d.m. marmion, Handbook of u.s.colors, Foods, Drugs, cosmetics and Medical Devices, John Wiley & Sons inc, New York (1991) and U.S. code of Federal Regulations, Title 21, parts 70-82.

The present invention may optionally include a crosslinking agent. Advantages of using a crosslinker with a film-forming composition include affecting the mechanical properties of the film, such as tack and mechanical strength, and the solubility of the coating. In addition, crosslinking reduces adhesion and prevents the physical attachment of scale and microorganisms to the polymer film, which may be desirable for certain applications. The degree of crosslinking is adjusted to achieve the desired combination of properties.

Suitable crosslinking agents for polyvinyl alcohol and copolymers thereof include, but are not limited to: aldehydes (e.g., formaldehyde, glyoxal, glutaraldehyde), boric acid, sodium tetraborate, metal ions (e.g., zinc, iron, aluminum, nickel, vanadium, cobalt, copper, zirconium, titanium, manganese ions), organometallic compounds (e.g., organotitanates such as DuPont)Organic Cr (III) complexes such as DuPontSiloxanes (e.g., tetraethoxysilane, polydimethylsiloxane), isocyanates (e.g., blocked, water-soluble or dispersible isocyanates), epoxides (e.g., diglycidyl ether), dicarboxylic acids (e.g., oxalic acid, maleic acid, fumaric acid, phthalic acid), urea-based crosslinkers (e.g., Sunrez 700). Bismuth and trivalent metal cations (e.g., fe (ii), fe (iii), al (iii)) are preferred because they form covalent bonds between PVOH polymer chains when the film is dried. This allows the crosslinker to be added to the film-forming liquid as a 'one pot' mixture. In order to efficiently crosslink the polymer without precipitating other ingredients such as particulate rheology control agents, the appropriate concentration must be carefully selected.

In most cases the cross-linking agent will be mixed with the other ingredients using standard mixing techniques. The crosslinking reaction may optionally be carried out in the presence of a catalyst, as is well known to those skilled in the art. In the case of aldehydes, isocyanates, siloxanes, diglycidyl ethers and dicarboxylic acids, heat and acid catalysts or metal catalysts may additionally be used.

The crosslinker concentration in the formulation can be zero to an upper limit, either determined by the formulation stability limit at which precipitation begins to form, or by instability at which the resulting film is efficiently removed.

The preferred crosslinker concentration may depend largely on the type of crosslinker and is generally less than 25% by weight of the polymer content, more preferably less than 10% by weight of the polymer content.

In addition to the aforementioned components, the compositions of the present invention may also contain one or more performance enhancing additives, also referred to as "performance enhancers". These enhancers include flash rust inhibitors, which include any number of organic or inorganic materials used in water-based systems to prevent rusting of metals that contact the material and are unmasked. Two examples are sodium benzoate or benzotriazole.

Another optional performance enhancing additive includes a combination of one or more defoamers recommended for use in water-based systems, which are used to prevent undesirable foaming of the product during application. Too much foam can disrupt the desired continuous film formation of the product and lead to failure. The addition of a foam control product, such as Drewplus L475 from Ashland Chemical, Inc. of Drew Industrial Division (Covington, KY, USA), may also be advantageous for mixing and processing of the masking composition. An additional optional performance enhancing additive is an antioxidant for improving the shelf life of the coating formulation. One example is butylated hydroxytoluene. Additional additives include fragrances.

A foaming agent may additionally be added to generate bubbles in the applied coating. The air bubbles may act as an opacifier to facilitate application and/or allow contact with the surface for longer periods of time; for example by preventing dripping from the bevel and/or reducing the amount of coating formulation required to treat a certain surface area or volume.

Application indicators may also be added. Some of these indicators are as described above, but include pigments, dyes, fluorescent dyes, pH indicators, or generate bubbles during application.

Small amounts (typically less than 1% by weight) of these additional materials may be added with appropriate adjustment of water or other components. It will be appreciated that mixtures of any one or more of the above optional components may also be used.

An optional performance enhancing ingredient is an agent that provides a surface effect with respect to the area comprised of the fibrous matrix. Such surface effects include easy-care, shrink-control, anti-wrinkle, durable-setting, moisture control, softness, strength, anti-slip, anti-static, anti-snag, anti-pilling, stain-resist, stain-release, soil-repellency, soil release, water repellency, oil repellency, odor control, antimicrobial, or sun-protection.

The film or coating may be applied to the target surface or area by any means, including pouring. The film or coating is used to achieve a continuous and/or uniform layer on the target surface. Coating systems conventionally used for paints and coatings may be used for coatings such as, but not limited to, brushes, roller brushes, peck brushes, pads, sponges, combs, hand pump dispensers, compressed air driven spray guns, airless spray guns, electronic or electrostatic atomizers, backpack spray equipment, aerosol spray cans, clothing, paper, feathers, styluses, knives, and other application tools. If dipping is used as the method of applying the coating, no special equipment is required. If an aerosol spray is used for application, the coating composition may be mixed with an aerosol propellant (e.g., compressed gas) or the coating composition may be physically separated from the propellant by a barrier material such as a polymeric bag within the can; if the coating composition and propellant are mixed, the mixture may constitute one or more liquid phases.

For fibrous substrates such as textiles and carpets, the coating may be applied by blotting, foaming, elastic nipping, padding, kissing rolls, rolling, skeining, capstan, liquid injection, flooding, rollers, brushes, rollers, spraying, dipping, and the like. The coating may also be applied by using a conventional vat dyeing process, a continuous dyeing process, or a spin line application.

In one embodiment of the present disclosure, an electrostatic sprayer may be used to coat the surface. Electrostatic sprayers impart energy to the aqueous coating composition through a high electrical potential. This energy acts to atomize and charge the aqueous coating composition to produce a fine charged particle spray. Electrostatic sprayers are readily available from suppliers such as Tae In Tech co, South Korea and Spectrum, Houston, TX, USA.

In another embodiment of the invention, an airless spray gun may be used to coat a target surface. Airless spray guns utilize high fluid pressure and special nozzles to deliver and atomize liquids rather than compressed air. The liquid is supplied to the airless spray gun by a fluid pump at a pressure typically in the range of 3.5 to 45 MPa. When paint leaves the fluid nozzle at such a pressure, it will spread slightly and atomize into droplets without the influence of atomizing air. The high velocity of the sprayed paint pushes the droplets toward the target surface. The fluid nozzles on airless spray guns are substantially different from the fluid nozzles on air atomizing spray guns. The selection of a suitable nozzle determines how much paint is delivered and the air blast pattern of application. The size of the airless nozzle orifice determines the amount of paint sprayed out. Airless fluid delivery is high speed, in the range of 700 and 2000 mL/min. The recommended lance distance is about 30cm from the target, and depending on the nozzle type, a 10 to 45cm blast pattern is possible. Thus, the nozzle may be selected for each application based on the size and shape of the target surface and the thickness of the coating to be applied. Airless spray guns produce minor air flow disturbances that can drain liquid from "hard to reach areas" that would be present in food processing equipment, hatcheries, and the like. The high flow rate makes the absence of gas advantageous in cleaning and disinfecting where the antimicrobial coating will be applied over a large surface area and multiple surfaces.

The thickness of the applied and dried film will depend on a number of factors. These factors include the concentration of the film forming agent, the concentration of the rheology control additives and/or other additives, and the temperature and humidity of application. The thickness of the film and the uniformity of the film also depend, at least in part, on the parameters of the application equipment, such as the delivery of the fluid, the diameter of the sprayer orifice, the air pressure or piston pump pressure for airless application and the distance of the spray applicator from the target surface. Thus, the liquid formulation can be adjusted to produce the desired film thickness. The atomization of the coating solution is selected so that a uniform thin film is applied over the target area.

Target surfaces (areas) include all surfaces that potentially can be contaminated with microorganisms, including surfaces that are often difficult to apply disinfectants or sanitizers (e.g., hard to reach surfaces). Examples of target surfaces include equipment surfaces found in the food or beverage industry (e.g., tanks, conveyors, floors, drains, coolers, freezers, refrigerators, equipment surfaces, ceilings, walls, valves, belts, pipes, drains, piping systems, fittings, cracks, combinations thereof, etc.); architectural surfaces, including surfaces in or on buildings under construction, new-dwelling buildings, and seasonal buildings such as vacation homes (e.g., ceilings, walls, wooden frames, floors, windows, plumbing), kitchens (sewers, drains, countertops, refrigerators, cutting boards), bathrooms (showers, lavatories, drains, pipes, plumbing, bathtubs), (especially for mold removal), decks, wooden surfaces, siding and other home surfaces, asphalt shingle roofing, platforms, or stone areas (especially algae treatment); ship and ship equipment surfaces; garbage disposal, garbage bins and large garbage bins or other garbage removal equipment and surfaces; associated piping and discharge pipes for the non-food industry; present on hospital surfaces; or surfaces in which surgical, outpatient, or veterinary services are provided (e.g., ceilings, walls, floors, piping, beds, equipment, clothing in a hospital/veterinary facility or other healthcare facility, including wipes, shoes, and other hospital or veterinary surfaces), first reactors or other emergency services equipment and clothing; saw yard equipment, surfaces and wood products; a restaurant surface; supermarket, grocery store, retail and convenience store equipment and surfaces; deli equipment and surfaces and food preparation surfaces; brewery and bakery surfaces; bathroom surfaces such as waterways, showers, countertops, and toilets; clothing and shoes; a toy; school and gym equipment, ceilings, walls, floors, windows, plumbing, and other surfaces; the kitchen surface is like sewer, table top and facilities; wood or composite decks, pools, hot tubs and spa surfaces; a carpet; paper materials; leather; animal carcasses, fur and hides; barn or stable surfaces, such as poultry, cattle, cows, goats, horses and pigs; and hatcheries of poultry or shrimp. Surfaces within buildings in which animals reside, such as birdcages and stalls, may be coated with the antimicrobial coatings described herein. Additional surfaces also include food products such as beef, poultry, pork, vegetables, fruits, seafood, combinations thereof, and the like.

Additional areas suitable for use in the present invention include fibrous substrate surfaces and include fibers, yarns, fabrics, textiles, nonwovens, carpets, leather, or paper. The fibrous substrate is made of natural fibers such as wool, cotton, jute, sisal, seaweed, paper, coir and cellulose, or mixtures thereof; or from synthetic fibers such as polyamides, polyesters, polyolefins, aramids, polyacrylics, and blends thereof; or from a blend of at least one natural fiber and at least one synthetic fiber. "Fabric" means a natural or synthetic fabric comprised of fibers, such as cotton, rayon, silk, wool, polyester, polypropylene, polyolefin, nylon, and aramid (such asAnd). "fabric blend" refers to a fabric made from two or more types of fibers. Typically these blends are a combination of at least one natural fiber and at least one synthetic fiber, but can also be a blend of two or more natural fibers or a blend of two or more synthetic fibers. Nonwoven substrates include, for example, spunlaced nonwovens such as those available from e.i. du Pont DE Nemours and Company (Wilmington, DE, USA), and laminated nonwovens such as spunbond-meltblown-spunbond nonwovens.

Examples of surface materials are metals (e.g., steel, stainless steel, chromium, titanium, iron, copper, brass, aluminum, and alloys thereof), minerals (e.g., concrete), natural or synthetic polymers and plastics (e.g., polyolefins such as polyethylene, polypropylene, polystyrene, poly (meth) acrylate, polyacrylonitrile, polybutadiene, polyacrylonitrile butadiene styrene, poly (acrylonitrile, butadiene), acrylonitrile butadiene; polyesters such as polyethylene terephthalate; and polyamides such as nylon). Additional surfaces include bricks, tiles, ceramics, porcelain, glass, wood (flooring), polyethylene (flooring) and linoleum.

The device or surface protected with the temporary coating may or may not be used in a protected state. The target surface may be hydrophobic or hydrophilic.

The coating system may also be one or more components and may include a catalyst. Generally, the coating is allowed to set or dry for greater than about 5 minutes to form a film. However, the coating may be antimicrobially effective in a relatively short time, for example after 30 seconds. The coating may be removed at any time before or after it is dried, depending on the desired use. The drying time will depend in part on several factors, including environmental conditions such as humidity and temperature. The drying time will also depend on the thickness of the applied coating.

The thickness of the film or coating applied to the target surface affects the time required for removal and the amount of biocide applied per unit area to the surface. Thicker films increase the time interval over which the film needs to be reapplied to maintain the desired antimicrobial properties. The thinner the film, the easier and faster it will be removed by rinsing. It is therefore important to administer the formulation by: the application results in a film thickness that both facilitates removal of the coating and has long-term antimicrobial properties. As noted above, the film or coating has a thickness of about 0.3 to about 300 microns. In a more specific embodiment, the film or coating has a thickness of from about 0.5 to about 100 microns. In a more specific embodiment, the film or coating has a thickness of from about 1.0 to about 30 microns.

The present invention relates to a film that can be removed when the user deems it appropriate. The coating is easily removable and can be removed by washing the surface with an aqueous solvent or solution. The removal time may be determined based on the following factors: or (i) a desired minimum contact time to produce a desired antimicrobial activity, typically expressed as killing or inactivating the amount of microorganisms in the initial microbial population, or (ii) a need or desire to remove the coating from the surface prior to beginning a subsequent operation or processing step. Although the coating can be removed at any time, such as after drying, the film thickness, biocide concentration and specific application determine the appropriate removal time. For example, a user may desire that the treated device resume normal operation after a period of inactivity. For example, washing is required before eating the fruit. When the biocide in the film is exhausted, the film can be removed and a new coating applied. For example, the drain may be treated periodically, such as daily, weekly, or biweekly. Antimicrobial activity can be measured as early as after 30 seconds, hours, days, weeks, months, or even years after application of the film. Thus, the time to remove the coating is a function of the application of the coating.

Film removal can be accomplished by dissolving or dispersing the resulting coating. This can be achieved by applying an aqueous solution onto the coating. In one embodiment, the solution temperature is in the range of about 15 ℃ to about 100 ℃. In another embodiment, the solution temperature is from about 30 ℃ to about 80 ℃. The application of the solution or water can be achieved by simple rinsing or spraying onto the surface. Coating removal can also be accomplished by using a pressurized scrubber, which facilitates removal through additional mechanical stress. Coating removal can also be accomplished by washing with a cloth or sponge that is wetted with water. In addition, mild additives including commonly used acids or bases, chelating agents, or detergents may be utilized or mixed with the aqueous solution to aid in the dissolution or dispersion of the film forming agent or aqueous dispersion. Alternatively, the membrane, for example, in the drain may be degraded by repeatedly washing the drain with water and/or other components. The film may also be removed by peeling the film off the surface, wiping or brushing off the surface, or other mechanical removal mechanism.

In addition to intentional removal by an operator, removal also includes removal with automated or robotic systems as well as unintentional removal, such as removal after a period of continuous or periodic contact of the coating with a liquid within a pipe or drain, which continuously or periodically applies mechanical stress such as wear-induced removal.

Contact time refers to the time that a coating or coating composition provides antimicrobial properties to microorganisms that are in contact with or near the coating or coating composition. Depending on the particular need for the antimicrobial agent, the contact time will vary, as willGermicidal and Detergent Sanitizing Action of Disinfectants， Official Methods of Analysis of the Association of Official Analytical ChemistsSection 960.09 and applicable chapter, 15 th edition, 1990(EPA Guideline 91-2). For example, if the intended application of the present disclosure is to be used as a disinfectant to contact food surfaces, the composition should provide a 99.999% reduction (5-log reduction) to several test microorganisms within 30 seconds at room temperature. If the intended application is to be used as a disinfectant that does not contact food surfaces, the composition should provide a 99.9% reduction (3-log reduction) of several tested microorganisms within 5 minutes at room temperature. If it is intended to use the disclosure as a disinfectant, the composition will provide a 99.9% reduction (3-log reduction) within 10 minutes. If the intended application is to provide residual antimicrobial activity, the present method will allow greater than 10 minutes of time to contact the microorganisms.

The coating of the present invention provides a physical barrier. Physical barrier is defined herein as a film formed from the film-forming composition of the present invention. The resulting film seals the treated surface against environmental contaminants such as dirt, fat, dust, microorganisms, and the like. These contaminants will remain on the coating surface and will be washed away when the coating is removed.

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods and compositions of the present disclosure have been described in terms of various aspects and preferred embodiments of the invention, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

General procedure

Recombinant techniques and microbial growth

Standard recombinant DNA and molecular cloning techniques used in the examples are well known in the art and are described in Sambrook, j., Fritsch, e.f. and manitis, t.,Molecular Cloning：A Laboratory Manualcold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, T.J.Silhavy, M.L.Bennan and L.W.Enquist,Experiments with Gene Fusionscold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and Ausubel, F.M. et al,Current Protocols in Molecular Biologygreene Publishing Assoc. and Wiley-Interscience, N.Y., 1987.

Materials and methods suitable for the maintenance and growth of bacterial cultures are also well known in the art. Techniques suitable for use in the following examples may be found in the following documents:Manual of Methods for General Bacteriologyphillipp Gerhardt, R.G.E.Murray, Ralph N.Costilow, Eugene W.Nester, Willis A.Wood, Noel R.Krieg and G.Brigg Schillips editors, American Society for Microbiology, Washington, DC, USA, 1994, or Thomas D.Brock inBiotechnology：A Textbook of Industrial MicrobiologySecond edition, Sinauer Associates, inc., Sunderland, MA, USA, 1989.

Unless otherwise indicated, all reagents, restriction enzymes and materials used for bacterial cell growth and maintenance were obtained from Aldrich Chemicals (Milwaukee, Wis., USA), BDdiagnostic Systems (Sparks, MD, USA), Life Technologies (Rockville, Md., USA) or Sigma Chemical Company (St. Louis, Mo., USA).

SDS gel electrophoresis

SDS gel electrophoresis is performed using methods well known in the art.

Method for testing antimicrobial activity on hard surfaces

The peracid can have biocidal activity. Typical alternative biocides known in the art to be suitable for use in the present invention include, for example, chlorine dioxide, chloroisocyanurates, hypochlorites, ozone, amines, chlorophenols, copper salts, organic sulfur compounds and quaternary ammonium salts. The biocidal or antimicrobial efficacy of the coating compositions was measured using the two test methods described below.

Disinfectant testing without food contact: to evaluate the antimicrobial activity of the coating composition under the following conditions: wherein microbial contamination is already present on the target surface at the time of application of the antimicrobial coating composition, using a "Standard Test Method for effectiveness of Sanitizers Recommanded for Inanimate Non-Food contact surfaces" as described in ASTM Standard E1153-03. This test method is referred to as the disinfectant test or "NFC test" without contact with food.

Residual self-sterilization test: to evaluate the antimicrobial activity of the coating composition under the following conditions: where microbial contamination contacts the dried coating, the following residual self-disinfection test method is used. This test method is referred to as the residual self-disinfection test or "RSS test". Using non-holes of 25.4X 25.4mm in sizePre-cleaned, stainless steel (type SS 316) test blocks. One colony was selected from an initial plate of organisms (three transfers in succession but no more than 30 transfers) and placed in 10mL AOAC nutrient broth (2.5 g sodium chloride; 2.5g beef extract; Anatone 5 g; 500mL deionized water). The inoculated culture was cultured statically at 35 ℃ for 24 hours. In preparing the test inoculum, the static culture was vigorously stirred with a vortex stirrer, allowed to stand for 15 minutes, and the upper two-thirds of the culture was transferred to a sterile tube (approximately 6 mL). Organic scale loading with Fetal Bovine Serum (FBS) was added to a final concentration of 5 wt% organic scale loading of this test strain. The final inoculum density tested was about 1X 10⁸CFU/mL。

The test block was placed in 70 wt% ethanol overnight, soaked in mild detergent for at least 30 minutes, rinsed thoroughly with tap water and air dried. Once the surface is cleaned, all grasping actions are accomplished with sterile forceps. The test block was sprayed with 70 wt% ethanol and thoroughly dried. An aliquot (50 μ L) of the coating composition to be tested was applied to each stainless steel test block, spread evenly with a sterile plastic spreader, placed in a sterile plastic petri dish and air dried overnight in a biosafety cabinet. The ambient air temperature and relative humidity were recorded. An uncoated coupon was used as a control surface, which was treated under the same conditions as the coupon treated with the coating composition.

The test block was inoculated by spot staining 0.01mL of the inoculum on the surface of the coated test block, using at least 30 plaques. At least two replicate test blocks were inoculated per coating composition. After 5 minutes, the test coupons were aseptically transferred to 20mL Bacto^TMD/E neutralizes the broth. The tube was shaken vigorously by hand, treated in an ultrasonic water bath at maximum power for 10s, and finally shaken on a rotary shaker at 250rpm for 4 minutes. Cell density of cultures Using Serial dilution plating technique with Butterfield PhosphoteBuffer dilution tube and Trypticase^TMSoy Agar (TSA) dish calculation. The amount of all samples transferred to the D/E neutralization broth in approximately 30 minutes was calculated.

The mean cell density (CFU/mL) of the treated and untreated coupons in parallel was calculated as the geometric mean of individual CFU measurements. All logarithms are logarithms based on 10.

Examples

To demonstrate preferred aspects of the present invention, the following examples are provided herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Abbreviations used in the examples

The following abbreviations in the specification correspond to the following measurement, technique, property, or compound units: "sec" or "s" refers to seconds, "min" refers to minutes, "h" or "hr" refers to hours, "mm" refers to millimeters; "μ L" means microliter, "mL" means milliliter, "L" means liter, "mM" means millimole per liter, "M" means mole per liter, "mmol" means millimole, "DEG C" means degree Celsius; "ppm" means parts per million and refers to mg/L (milligrams per liter), in the examples below, "wt" means weight, "wt%" means percent by weight, "g" means grams, "μ g" means micrograms, "g" means gravitational constant, "HPLC" means high performance liquid chromatography, "dd H₂O "means distilled and deionized water," DI "means deionized," dcw "means dry cell weight," ATCC "orRefers to the American type culture Collection (Manassas, VA, USA), "U" refers to the unit of perhydrolase activity, and "U/mg protein" refers to each milliThe perhydrolase activity units for the gram-protein, "rpm" means revolutions per minute, "EDTA" means ethylenediaminetetraacetic acid, "PAA" means peracetic acid, "PEG" means polyethylene glycol, "Pa · s" means pascal seconds; "IPTG" refers to isopropyl-beta-D-thiogalactoside; "MPa" means megapascals; "NMWCO" indicates nominal molecular weight cut-off, "LB" refers to Luria broth, "OD" refers to optical density, "g feed/min" refers to grams fed per minute, "SDS" refers to sodium dodecyl sulfate, "o-DA" refers to o-dianisidine, "pNPA" refers to p-nitrophenylacetate, "L.cm^-1·mol^-1"means liter per mole per centimeter," mg/mL "means milligrams per milliliter," kg "means kilograms," U/mg "means units per milligram," CFU "means colony forming units; "FBS" refers to fetal bovine serum; "log CFU" refers to the common logarithm of the number of CFUs of 10; "CFU/mL" means CFU per milliliter; "NFC" refers to a disinfectant test without contact with food; "RSS" refers to residual self-disinfecting activity; "SS 316" refers to stainless steel type 316 (ASTM standard); "TAED" refers to tetraacetylethylenediamine.

Chemical product

All chemicals were obtained from Sigma-Aldrich (st. louis, MO, USA) unless otherwise indicated.51-04 (partially hydrolyzed grade polyvinyl alcohol, 88% grade) from DuPont (Wilmington, DE, USA). Average molecular weight 300g/mol (Carbowax)^TMPEG-300) from Dow (Midland, MI, USA).885、3300 andn25-7 was obtained from Stepan (Northfield, IL, USA).885 contains 50% by weight of a quaternary ammonium compound as an antimicrobial agent and 50% by weight of an inert material.FRC is obtained from Ciba (Basel, Switzerland).215UP is available from Cognis Corporation (Cincinnati, OH). PC 5450NF was obtained from Performance Chemicals, LLC (Concord, NH). Tetraacetylethylenediamine (TAED B675) was obtained from Warwick International Ltd (Flintshire, UK). Sodium percarbonateC) Obtained from OCI Chemical Corp (Decatur, AL, USA).SL is obtained from Thermphos (Vlissingen, the Netherlands).L-77 was obtained from Setre Chemical Company (Memphis, TN, USA). Microbial media were obtained from DIFCO Laboratories (Detroit, MI, USA), GIBCO/BRL (Gaithersburg, MD, USA), TCIAmerica (Portland, OR, USA), Roche Diagnostics Corporation (Indianapolis, IN, USA) OR Sigma-Aldrich Chemical Company St. Bacto^TMD/E neutralization Broth was obtained from Difco (Cat. No.281910, Difco)^TMLaboratories，Detroit，MI，USA)。

pNPA microplate assay

To every four wells of a 96-well microplate, 170. mu.L of potassium phosphate buffer (50mM, pH7.2) and or 10. mu.L of 0.1mg total protein/mL cell lysate, or 10. mu.L of 0.02mg total protein/mL spray-dried enzyme solution was added. Cover the microplate and place it in a microplate reader (molecular devices)384Plus), then mixed for several seconds and equilibrated at 30 ℃ for 10 minutes. Then 20. mu.L of 30mM pNPA solution prepared by mixing 0.6mL of 100mM pNPA with 1.4mL of 50mM potassium phosphate buffer (pH 7.2) in acetonitrile (immediately before use) was added simultaneously to each well of the microplate. The change in absorbance at 400nm was measured over a reaction time of 1 minute. The background rate of pNPA hydrolysis (subtracted from the rate of hydrolysis measured in the presence of enzyme) was measured in four wells, respectively, by replacing the cell lysate or enzyme-containing solution with 10. mu.L/well of potassium phosphate buffer (50mM, pH 7.2). The extinction coefficient of p-nitrophenol measured in a 1-cm path length cell at pH7.2 was 10,909L cm^-1·mol^-1. The enzymatic activity of one pNPA unit is equivalent to the amount of protein that catalyzes the hydrolysis of one micromole of pNPA in one minute.

Determination of the rheological Properties

The rheological properties of the liquid antimicrobial formulations were evaluated using a Brookfield Digital Viscometer Model DV-II (Brookfield engineering laboratories, Middleboro, MA, USA) with RV series spindle #6 and a high glass beaker. The sample was poured into a glass beaker to load the sample. The viscosity was measured at different rpm.

High Performance Liquid Chromatography (HPLC)

The PAA concentration in the reaction mixture was determined using HPLC. A Superco Discovery C8 column (15 cm. times.4.0 mm, 5 μm) and a pre-column Superguard Discovery C8 were used. The injection volume was 10. mu.L. The following table shows the gradient method with acetonitrile and deionized water at 1.0mL/min and ambient temperature.

The retention times of the compounds of interest using this HPLC method were: MTSO (methyl p-toluene sulfoxide) ═ 2.34 minutes; DEET (N, N-diethyl-m-toluamide) ═ 4.40 minutes; TPPO ═ triphenylphosphine oxide ═ 5.02 minutes; MTS ═ 5.99 minutes (p-methylthiotoluene), and TPP ═ triphenylphosphine ═ 6.48 minutes.

Example 1

Cloning and expression of acetyl from Thermotoga neapoliana (Thermomotoga neapolitana) Xylan esterase

Synthesis of the coding region encoding an acetylxylan esterase from Thermotoga neapoliana (THERMOTOGA neapolitana) Using codons optimized for expression in E.coliAccession # AAB70869, SEQ ID NO: 7) (DNA 2.0, Menlo Park, CA, USA). The PCR was then carried out (94 ℃ for 0.5 min, 55 ℃ for 0.5 min, 70 ℃ for 1 min, 30 cycles) using the primers SEQ ID NO: 18 and SEQ ID NO: 19 amplifying the coding region. The resulting nucleic acid product (SEQ ID NO: 20) was subcloned into(Invitrogen, Carlsbad, Calif., USA) to generate plasmid pSW 196. Plasmid pSW196 was used to transform E.coli KLP18 to produce strain KLP18/pSW196 (see published U.S. patent application 2008/0176299, which is incorporated herein by reference in its entirety). KLP18/pSW196 was shake-cultured in LB medium at 37 ℃ to OD_600nm0.4-0.5, at which time isopropyl-beta-D-thiogalactoside (IPTG) was added to a final concentration of 1mM and incubation was continued for 2-3 hours. Cells were harvested by centrifugation and stored in 20 wt% glycerol at-80 ℃; perhydrolase expression in 20-40% of total soluble protein as determined by SDS-PAGE。

Example 2

Escherichia coli expressing Thermotoga naresicola (T.neapolitana) perhydrolase Fermentation of KLP18/pSW196

The fermentation is carried out in three stages: preparation of pre-seeds in flasks, seed preparation in 14-L fermentors, and fermentation scale-up in 200-L fermentors. Seed cultures were prepared by feeding a 2L shake flask with 0.5L of seed medium containing yeast extract (Difco, 1.0g/L), K₂HPO₄(10.0g/L)，KH₂PO₄(7.0g/L), sodium citrate dihydrate (1.0g/L), (NH)₄)₂SO₄(4.0g/L)，MgSO₄Heptahydrate (1.0g/L) and ammonium ferric citrate (0.10 g/L). The pH of the medium was adjusted to 6.8, the medium was packed in a flask, and then sterilized. After sterilization, glucose (50 wt%, 10.0mL) and 1mL stock of ampicillin (25mg/mL) were added. 1-mL of a culture of E.coli KLP18/pSW196 in 20% glycerol was inoculated into seed medium and cultured at 35 ℃ and 300 rpm.

The seed medium was transferred to a 14-L seed fermentor (Braun, B.Braun Biotech Inc., Allentown, Pa., USA) having an optical density of about 1-2 at 550nm with 8L of medium in the fermentor, which contained KH₂PO₄(5.0g/L)，FeSO₄Heptahydrate (0.05g/L), MgSO₄Heptahydrate (1.0g/L), sodium citrate dihydrate (1.90g/L), Biospumex153K antifoam agent (0.5mL/L, Cognis Corporation), sodium chloride (1.0g/L), CaCl₂Dihydrate (0.1g/L), and a trace element solution (10mL/L) comprising citric acid monohydrate (10g/L), MnSO₄Hydrate (2g/L), sodium chloride (2g/L), FeSO₄Heptahydrate (0.5g/L), ZnSO₄Heptahydrate (0.2g/L), CuSO₄Pentahydrate (0.02g/L) and NaMoO₄Dihydrate (0.02 g/L). The sterilized additive comprises glucose solution (5)0 wt%, 80.0g), ampicillin (25mg/mL) stock (16.00mL), and thiamine (3.3g/L) stock (6.00 mL). Dissolved Oxygen (DO) concentration was controlled at 25% air saturation. DO is controlled first by impeller agitation (at a rate of 400 to 1400rpm) followed by aeration (at a rate of 2 to 10L/min under standard conditions). The temperature was controlled at 35 ℃. The pH was controlled at 6.8. NH (NH)₄OH (29% by weight) and H₂SO₄(200g/L) was used for pH control. The overhead pressure was maintained at 0.50 bar. When the culture density reached OD₅₅₀When nm was greater than 5 or when the glucose concentration in the broth dropped to less than 1g/L, 8L of the culture was transferred to a 200L Sartorius Biostet-DCU-D production fermentor.

The initial volume of culture added to a 200-L Sartorius Biostet-DCU-D production fermentor (Sartorius StediumNorth America Inc., Bohemia, NY, USA) was 150L, where the medium was prepared using the same ingredients as in the 14-L seed fermentor. The sterilized additive comprises glucose-MgSO₄Solution (glucose 50 wt%, MgSO)₄1% by weight of heptahydrate; 1600g) Ampicillin (25g/L) stock solution (3200mL) and thiamine (3.3g/L) stock solution (120 mL). Dissolved Oxygen (DO) concentration was controlled at 25% air saturation. DO is controlled first by impeller agitation (at a rate of 200 to 450rpm) followed by aeration (at a rate of 35 to 80L/min under standard conditions). The temperature was controlled at 35 ℃. The pH was controlled at 6.8. NH (NH)₄OH (29% by weight) and H₂SO₄(200g/L) was used for pH control. The overhead pressure was maintained at 0.50 bar. Mixing glucose-MgSO₄Solutions (glucose (50 wt%) and MgSO4 heptahydrate (1 wt%) were used for fed-batch growth glucose was fed when the glucose concentration dropped to 0.5g/L, starting at a rate of 6.30 g/min, gradually increasing to 7.30, 8.50, 9.8, 11.40, 13.30, 15.40, 17.2 g/min, respectively, per hour, then the rate remained constant.the glucose concentration in the medium was monitored, and if the concentration exceeded 0.1g/L, the feed rate was decreased or the feed was temporarily stopped₅₅₀75 nm (at 26.7 h) was started by adding 180mL of IPTG (0.5M). Cells were harvested by centrifugation 16 hours after IPTG addition. Fermenting for 42.7 hoursTerminated, and approximately 21kg of wet cell slurry was harvested, having a perhydrolase specific activity of 3.4pNPA U/mg dry cell weight.

Example 3

Preparation of Thermotoga neapoliana (T.neapolitana) perhydrolase

The cell slurry (13 kg; E.coli KLP18/pSW196, prepared as described in example 2) was suspended in 40L 50mM sodium phosphate buffer, pH 7.4, the cell suspension was homogenized using an APV 16.56 homogenizer (APVFluid Handling and Homogenizers, Delavan, Wis., USA) at 83MPa and room temperature, and the resulting homogenized product was heated to 65 ℃, held at this temperature for 30 minutes, and then cooled to room temperature. A 0.1 micron hollow fiber membrane cartridge (GEHealthcare, Little Chalfont, United Kingdom) was used to separate the solids from the protein solution. The clear protein solution was then concentrated with a 30,000NMWCO hollow fiber membrane cartridge (GE Healthcare) to obtain 14.7L of the final concentrate, which was frozen at-80 ℃. This material was then thawed and further concentrated (30,000NMWCO) to provide 7.5 liters of solution containing approximately 256 grams of protein (34.15g protein/L; determined using the Micro-BCA protein assay (Sigma Life sciences, Sigma-Aldrich Corp., St. Louis, Mo, USA.) to this solution was added 500g Maltrin M100 maltodextrin, 110g Maltrin M250 corn syrup solid, and 110g Maltrin M040 maltodextrin (all available from Gain Processing Corporation (GPC), Muscatine, IA, USA).

The resulting mixture (ca.7.9kg) was spray dried (inlet temperature 225 ℃, outlet temperature 76 ℃, feed rate 60g/min) to produce a total of 786 grams of solid (68% yield) enzyme powder: 93.2% dry weight, 20.3% protein by weight, 34.5pNPA U/mg protein.

Example 4

In situ generation of peracetic acid using a proenzyme having perhydrolase activity in the presence of suspected vinyl alcohol

This example shows the in situ generation of peracetic acid by a zymogen having perhydrolase activity in the presence of polyvinyl alcohol. The reaction is carried out with a perhydrolase isolated from Thermotoga neapoliana (T.neapolitana). In a 75mL glass vial equipped with a magnetic stir bar were placed 12.3mL of 0.2M sodium bicarbonate buffer (pH 7.2; 44mM), 36.2mL of deionized water, 41.7mgSL (Thermphos, Vlissingen, the Netherlands), 0.95mL of triacetin (90mL) and 6.0g51-04 (20% aqueous solution, DuPont, Wilmington, DE, USA) and stirred at room temperature until the sample is homogeneous. At this time, 0.51mL of 30 wt% hydrogen peroxide (89mM) was added, followed by 125. mu.L of 20mg/mL Thermotoga neapoliana (T.neapolitana) perhydrolase in 50mM sodium bicarbonate buffer (pH 7.2). The hydrogen peroxide and peracetic acid concentrations were monitored over time using a cerium sulfate/sodium thiosulfate titration assay (Greenspan, f.p.; MacKeller, d.g. anal. chem., 20: 1061-1063, 1948). Control reactions (12.3mL of 0.2M sodium bicarbonate buffer, pH7.2, 50 mM; 0.51mL of 30% hydrogen peroxide, 100 mM; 36.2mL of deionized water; 41.7mgSL; 0.95mL of triacetin, 0.1M; 125 μ L of Thermotoga neapolitana (T.neapolitana) perhydrolase (based on the weight of protein in the enzyme powder)) in 50mM sodium bicarbonate buffer, pH7.2, and no enzyme was added (same as the enzyme-free control conditions described above). After 30 minutes, a control reaction was carried out with triacetin and hydrogen peroxide without the addition of enzyme, resulting in 114ppm peracetic acid from the chemical reaction of triacetin and hydrogen peroxide. With triacetin and hydrogen peroxide, with addition of enzymesThe control reaction was run with 380ppm peracetic acid in 1 minute and increased to 2280ppm over 30 minutes. In situ enzymatic generation of PAA in the presence of polyvinyl alcohol generates 456ppm within 1 minute and increases to 1976ppm over 30 minutes. Table 1 shows PAA generated by in situ enzymatic generation of PAA in the presence of a film forming agent.

TABLE 1

Enzymatic production of PAA in the Presence of polyvinyl alcohol

Example 5

In situ generation of peracetic acid in a film-forming composition using a proenzyme having perhydrolase activity

This example illustrates the in situ generation of peracetic acid with a zymogen having perhydrolase activity in the presence of a film-forming composition and an additional performance-enhancing additive. The reaction was performed with a perhydrolase derived from Thermotoga neapoliana (T. neapolitana). In a 75mL glass vial equipped with a magnetic stir bar were placed 12.3mL of 0.2M sodium bicarbonate buffer (pH 7.2; 44mM), 36.2mL of deionized water, 41.7mgSL (Thermphos, Vlissingen, the Netherlands), 0.95mL of triacetin (89mM), 6.0g51-04 (20% by weight aqueous solution, DuPont, Wilmington, DE, USA), 0.5g PEG-300(Dow, Midland, MI, USA) andL-77(Setre Chemical Ccompany, Memphis, TN, USA) and stirred at room temperature until the sample was homogeneous. At this point 0.51mL of 30% hydrogen peroxide (88mM) was added, followed by 125. mu.L of 20mg/mL Thermotoga neapoliana (T.neapoliana) perhydrolase (based on the weight% of protein in the enzyme powder) in 50mM sodium bicarbonate buffer (pH 7.2). The hydrogen peroxide and peracetic acid concentrations were monitored over time using a cerium sulfate/sodium thiosulfate titration test (Greenspan, f.p.; MacKeller, d.g. anal.chem., 20: 1061-1063, 1948). Control reactions (12.3mL of 0.2M sodium bicarbonate buffer, pH7.2, 50 mM; 0.51mL of 30% hydrogen peroxide, 100 mM; 36.2mL DI (deionized water); 41.7mgSL; 0.95mL of triacetin, 0.1M; 125 μ L of Thermotoga neapolitana (T.neapolitana) perhydrolase (based on the weight of protein in the enzyme powder)) in 50mM sodium bicarbonate buffer, pH7.2, and no enzyme was added (same as the enzyme-free control conditions described above). After 30 minutes, a control reaction was carried out with triacetin and hydrogen peroxide without the addition of enzyme, resulting in 114ppm peracetic acid from the chemical reaction of triacetin and hydrogen peroxide. The control reaction was carried out with triacetin and hydrogen peroxide with the addition of enzyme, producing 380ppm peracetic acid in 1 minute and increasing to 2280ppm over 30 minutes. In situ enzymatic generation of PAA in a film-forming composition generates 342ppm within 1 minute and increases to 1900ppm over 30 minutes. Table 2 shows the concentration of PAA enzymatically generated in situ in the presence of a film-forming composition and additional performance-enhancing additives.

TABLE 2

Enzymatic production of PAA in film-forming compositions

Example 6

Enzymatically generated in situ in a film-forming composition comprising a second biocide and a rheology modifier Acetic acid

This example illustrates the in situ generation of peracetic acid with a zymogen having perhydrolase activity in the presence of a film-forming composition comprising a second biocide and a rheology modifier. In a 100mL glass bottle equipped with a stirring rod was placed 25g of 20 wt%51-04 aqueous solution, 14g deionized water, 0.8gFRC, 0.5g PEG 300 and 0.15g885 and stirred at room temperature until the sample is homogeneous. At this point 1.09g of triacetin (100mM) and 0.67g of 30% by weight hydrogen peroxide (100mM) were added and mixed with stirring. The pH was adjusted to above 7.0 by the addition of 8g of 0.2M sodium bicarbonate buffer (32 mM). After pH adjustment, 125. mu.L of Thermotoga neapoliana (T.neapolitana) perhydrolase (20 mg/mL solution in 50mM sodium bicarbonate buffer (% based on the weight of protein in the enzyme powder)) was added and the timer was started. The control reaction was carried out using the same conditions as above except that no enzyme was used. Peracetic acid concentration was monitored at 5 minutes, 30 minutes and 1 hour using a cerium sulfate/sodium thiosulfate titration test. After a period of 1 hour, a control reaction without added enzyme produced 487ppm peracetic acid from the chemical reaction of triacetin and hydrogen peroxide. In situ enzymatic production of PAA in a film-forming composition produced 2112ppm in 5 minutes and increased to 2593ppm over 1 hour. Table 3 showsConcentration of enzymatically produced PAA in a film-forming composition.

TABLE 3

Enzymatically produced PAA in a film-forming composition comprising a second biocide and a rheology modifier

Example 7

Shear thinning index of removable antimicrobial coating compositions

The "pseudoplasticity index" or "shear thinning index" (STI) provides an indication of the resistance to sagging and dripping of the composition. Common measurements determine viscosity at two different shear rates. The value recorded at the lower shear rate divided by the value at the higher shear rate gives the STI. Generally, the higher the STI of the coating material, the higher its resistance to sagging and dripping will be. The STI value used herein was determined by measuring the viscosity 30 minutes after the addition of the enzyme. The viscosity was measured at room temperature using a Brookfield Digital Viscometer Model DV-II (Brookfield engineering laboratories, Middleboro, MA, USA) with RV series spindle # 6. The Shear Thinning Index (STI) was calculated by dividing the viscosity measured at 1rpm by the viscosity measured at 10 rpm. The STI for coating composition a of the present invention (see composition of table 4) is given in table 5. The film-forming composition A had a viscosity of 12.8 pas at 1rpm and dropped to 4.86 pas at 10 rpm. The coating composition had a shear thinning index of 2.74.

TABLE 4

Coating composition A for enzymatic peracid production

TABLE 5

Viscosity and shear thinning index

Example 8

Cloning and expression of acetyl xylan esterase from Thermotoga maritima (Thermotoga maritima)

A gene encoding an acetyl xylan esterase from Thermotoga maritima (T.maritima) MSB8 (e.g., as inAccession No. NP _227893.1, SEQ ID NO: 8) synthesis was performed (DNA 2.0, Menlo Park, CA, USA). The PCR was then carried out (94 ℃ for 0.5 min, 55 ℃ for 0.5 min, 70 ℃ for 1 min, 30 cycles) using the primers SEQ ID NO: 22 and SEQ ID NO: 23 amplifying the gene. The resulting nucleic acid product (SEQ ID NO: 24) was digested with restriction enzymes PstI and XbaI and subcloned into pUC19 between the PstI and XbaI cleavage sites to give plasmid pSW 207. A codon-optimized version of thermotoga maritima (t. maritima) MSB8 acetyl xylan esterase was synthesized for expression in e.coli and subsequently amplified by PCR (94 ℃ for 0.5 min, 55 ℃ for 0.5 min, 70 ℃ for 1 min, 30 cycles) using primers SEQ ID NO: 25 and 26 for amplification. The resulting nucleic acid product (SEQ ID NO: 27) was digested with restriction enzymes EcoRI and PstI and subcloned into pTrc99ALogin toNo. M22744) to generate plasmid pSW 228. Coli KLP18 (U.S. patent application publication 2008/0176299, incorporated herein by reference) was transformed with plasmids pSW207 and pSW228 to generate strains KLP18/pSW207 and KLP18/pSW228, respectively. KLP18/pSW207 and KLP18/pSW228 were shake-cultured at 37 ℃ in LB medium to OD_600nm0.4-0.5, at which time IPTG is added to a final concentration of 1mM and incubation is continued for 2-3 hours. Cells were harvested by centrifugation and subjected to SDS-PAGE to determine the expression of perhydrolase in amounts of 20-40% of total soluble protein.

Example 9

Thermotoga maritima (Thermotoga maritima) acetyl xylan built on residue C277 Esterase mutants

The C277(Cys277) site of Thermotoga maritima (T. maritima) acetylxylan esterase was changed to Ser using an oligonucleotide primer pair (SEQ ID N0: 28 and 29) designed based on the codon-optimized sequence of Thermotoga maritima (T. maritima) acetylxylan esterase (SEQ ID NO: 30) in plasmid pSW 228. Mutations were made using Quikchange (Stratagene, CedarCreek, TX, USA) according to the manufacturer's instructions. The amplified plasmid was treated with 1U of Dpnl at 37 ℃ for 1 hour. The treated plasmid was used to transform chemically competent E.coli XL1-blue (Stratagene). Transformants were streaked onto LB agar plates supplemented with 0.1mg ampicillin/mL and grown overnight at 37 ℃. A maximum of five individual colonies were picked and the plasmid DNA was sequenced to determine the expected mutation.

Example 10

Expression of Thermotoga maritima (Thermotoga maritima) acetyl wood in Escherichia coli KLP18 Glycan esterase C277S mutant

With confirmed acetyl xylan estersThe plasmid of the mutant enzyme C277S was used to transform E.coli KLP18 (example 1) to produce strain KLP18/pSW 228/C277S. The transformant was cultured at 37 ℃ in LB medium with shaking to OD_600nm0.4-0.5, at which time IPTG is added to a final concentration of 1mM and incubation is continued for 2-3 hours. Cells were harvested by centrifugation and subjected to SDS-PAGE to determine the expression of the acetylxylan esterase C277S mutant in amounts of 20-40% of total soluble protein.

Example 11

Preparation of spray-dried Thermotoga maritima (T. maritima) acetyl xylan esterase wild-type mutations Body C277S

The fermentation protocol for the cultivation of E.coli KLP18/pSW228 (for the preparation of Thermotoga thermosiphon (T. maritima) acetylxylan esterase) or E.coli KLP18/pSW228/C277S (for the preparation of Thermotoga maritima (T. maritima) acetylxylan esterase C277S mutant) was similar to the protocol described in example 2, except that it was carried out on a 10-L scale. Cell slurry was harvested by centrifugation at 5,000 Xg for 15 minutes and frozen at-80 ℃ for later use.

The cell slurry sample was resuspended (200g/L) in 50mM phosphate buffer, pH 7.0, which was supplemented with 1.0mM Dithiothreitol (DTT). The resuspended cells were passed twice through a French cell press to ensure > 95% cell lysis. The lysed cells were centrifuged at 12,000 Xg for 30 minutes and the supernatant was heated at 75 ℃ for 20 minutes and then quenched in an ice bath for 2 minutes. Precipitated proteins were removed by centrifugation at 11,000 Xg for 10 minutes. SDS-PAGE indicates that the CE-7 enzyme contains approximately 85-90% total protein in the formulation.

A method similar to that described in example 3 was repeated using the cell slurry prepared as described above. The spray-dried enzyme powder of the Thermotoga maritima (T.maritima) perhydrolase obtained was: 95.5% dry weight, 27.8% protein by weight, 86.1pNPA U/mg protein. The spray-dried enzyme powder of the Thermotoga maritima (T.maritima) C277S perhydrolase obtained was: 95.5% dry weight, 25.5% protein by weight, 129.5pNPA U/mg protein.

Example 12

In situ enzymatic generation of peracetic acid in film-forming compositions using various perhydrolases

The ability to enzymatically generate PAA in situ in a film-forming composition using different applicable perhydrolases and to use a second biocidal active and rheology modifier in the formulation is shown in this example.

A coating composition similar to that described in example 6 was prepared using the same method as described above, except that the ingredients and compositions were used in the weight% amounts listed in table 6. Coating composition B PAA was produced in a film-forming composition using TAED and sodium percarbonate without enzymes and was used for comparison only. Coating composition C lacks enzymes and serves as a control with enzyme-containing compositions D, E and F. The initial pH of each enzyme reaction mixture was about 7.0. The initial pH of the TAED reaction mixture was about 8.0.

The concentration of PAA in the reaction mixture was determined according to the method described by Karst et al (anal. chem., 69: 3623-3627, 1997). At the desired time point (1 min, 5 min, etc.), an aliquot of the reaction mixture (approximately 25-50mg) was removed and diluted with 5mM phosphoric acid by weight to terminate the reaction. Typical dilutions are 1: 35 for samples with PAA concentrations greater than 2000ppm, 1: 25 for samples with PAA concentrations of about 1000ppm, and 1: 10 for samples with PAA concentrations below 200 ppm. Approximately 200. mu.L (or enough to completely fill the filter cup) of the diluted aliquot was transferred to non-sterile 10,000NMWL (nominal molecular weight range) filtration units (Nanosep 10K Omega, PALL Life Sciences, East Hills, NY, USA) and filtered by centrifugation at 12,000rpm for 7 minutes. An aliquot (100 μ L) of the resulting filtrate was transferred to a 1.8-mL screw cap HPLC Vial (1.8mL ROBO Autosampler visual, VWRInterationary, West Chester, PA, USA) containing 300 μ L of deionized water, then 100 μ L of 20mM MTS (p-methylthiotoluene) in acetonitrile was added, the Vial capped, the contents mixed slightly, and then incubated at about 25 ℃ for 10 minutes in the absence of light. Then 400. mu.L of acetonitrile and 100. mu.L of triphenylphosphine solution (120mM) in acetonitrile were added to each vial, the vials were capped, the resulting solutions were mixed and incubated at 25 ℃ for 30 minutes in the absence of light. Then 100. mu.L of 2.5mM N, N-diethyl-m-toluamide (DEET; HPLC external standard) and the resulting solution analyzed by HPLC as described above were added to each vial.

The PAA concentrations over time are given in table 7. The maximum PAA concentration for all enzyme-containing as well as TAED-containing compositions was observed between 0.5 and 2 hours after the start of the respective chemical reaction. After the maximum concentration has been reached, the concentration decreases with time, which can be expressed mathematically approximately by an exponential decay function, of the form c (t) k · exp [ -t/(ln (2) · τ) ], where c (t) describes the PAA concentration as a function of the reaction time t. The parameters k and τ are constants that may be different for each composition, and τ represents the approximate half-life, i.e., the time that the PAA concentration has dropped to half its maximum. The approximate half-life was calculated by least squares fitting the PAA concentration data over time to times between 2 and 120 hours by the exponential decay function described above. The half-life τ is also given in table 7. It can be seen that the half-lives of enzymatically produced PAA are similar and between about 27 and 29 hours. As a control, the non-enzymatically produced PAA half-life of coating composition B was shortened by more than 7 hours. Thus, the enzymatically generated composition results in a slower decrease in PAA concentration over time in the liquid coating composition. This may be beneficial in cases where a particular minimum PAA concentration is expected to be present for a longer period of time.

TABLE 7

PAA enzymatically prepared in a film-forming composition using a plurality of perhydrolases

n.d. denotes "indeterminate"

Example 13

Method for preparing film-forming antimicrobial composition G for Staphylococcus aureus (Staphylococcus aureus) Efficacy of

Antimicrobial properties of film-forming composition G (table 8) was tested using the NFC test method with Staphylococcus aureus (ATCC 6538) as the test microorganism. Table 8 gives composition G in weight%. The combined reagents after mixing were 100 wt%. Each test piece was uniformly applied with a volume of 0.05mL of the coating composition. After five minutes of exposure, a reduction in Colony Forming Units (CFU) of either 5.6 logs or more was observed, which equates to at least a 99.999% reduction in the number of CFUs.

TABLE 8

Antimicrobial NFC testing of composition G against staphylococcus aureus (s

Example 14

Method for preparing film-forming antimicrobial composition H for Staphylococcus aureus (Staphylococcus aureus) Efficacy of

Antimicrobial properties of film-forming composition H (table 9) was tested using the RSS test method and Staphylococcus aureus (ATCC 6538) as the test microorganism. Table 9 gives composition H in weight%. All combined reagents equal 100 wt%. Each test piece was uniformly applied with a volume of 0.05mL of the coating composition. As shown in table 9, composition H achieved a 3.1 order of magnitude reduction in CFU, which equates to at least a 99.9% reduction in CFU.

TABLE 9

Antimicrobial RSS testing of composition H against Staphylococcus aureus (S.aureus)

Example 15

Comparison of PAA generated in situ in commercially available PAA solutions and films

This example compares the PAA concentration in the film over time between a commercially available equilibrium PAA solution added to the film-forming composition Pack a and PAA generated in situ in the film-forming composition.

Table 10 shows film-forming compositions H and #141B in weight%. All ingredients were mixed to produce 100 wt% antimicrobial coating composition. For composition #141B, commercially available PAA (90 μ Ι _ of a 32 wt% solution) was added to Pack a instead of being generated in situ.

Watch 10

Film Forming compositions H and #141B

After all the components of the composition were mixed together, the film was applied to a plastic panel with an 8-channel wet film applicator (5 mil film thickness, model No.15, Paul n. gardner co. inc., Pompano Beach, FL, USA). The concentration of PAA in the membrane was monitored at intervals when the membrane was dry. In a typical experiment, the film was tacky to the touch within 30 minutes and completely dried over an hour. The concentration of PAA in the reaction mixture was determined according to the method described by Karst et al (anal. chem., 69: 3623-3627, 1997). See example 12 for details.

The PAA concentration in coating mixture H was observed to increase from 1925ppm PAA at 1 minute to a maximum of 2599ppm PAA at 10 minutes, decreasing to 732ppm PAA within one hour, while the PAA concentration in composition #141B steadily decreased from a maximum of 2849ppm PAA at one minute to 22ppm PAA at one hour over one hour. This example highlights the benefits of the film-forming composition of the present invention, where PAA is generated in situ rather than being prepared in advance.

TABLE 11

PAA concentration composition H and #141B in coating film over time

Example 16

Application and removal of coating composition H

Coating composition H (table 9) was applied to an aluminum panel using an 8-channel wet film applicator (5 mil thick, type No.15, Paul n. gardner co. inc., Pompano Beach, FL, USA). The resulting coating after drying has an excellent appearance characterized by the absence of coating defects such as sagging, foam or bubbles, pits, or uncovered areas. The dried film was easily removed by rinsing the plate with tap water at 30-32 ℃. No visible residue remained on the aluminum plate, indicating good removal characteristics of the film.

Claims (12)

1. A method of providing microbial control at vertical, oblique, complex geometric or hard to reach sites, the method comprising the steps of:

a) forming a composition by combining components comprising:

i) a film-forming, water-soluble or water-dispersible agent, wherein the agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinylpyrrolidone;

ii) an inert solvent;

iii) a carboxylate substrate;

iv) a source of peroxygen;

v) an enzyme catalyst having perhydrolase activity to form at least one in situ peroxyacid to provide at least a first antimicrobial agent; and

vi) a rheology modifier for providing shear thinning behavior wherein the rheology modifier is a polyacrylate, a cationA thickener or Carbopol thickener;

b) applying the composition to the site; and is

c) Allowing the composition to dry to form a coating on the site;

wherein the composition further comprises a second antimicrobial agent, wherein the second antimicrobial agent comprises a quaternary ammonium compound.

2. The process of claim 1 wherein the enzyme catalyst is selected from the group consisting of carbohydrate esterases of the CE-7 family.

3. The method of claim 1, wherein said peroxyacid is generated at a concentration of at least 20ppm within 5 minutes to 2 hours of combining said components.

4. A method of providing microbial control at vertical, oblique, complex geometric or hard to reach sites, the method comprising the steps of:

(a) providing a first premix component comprising an inert solvent, a rheology modifier for providing shear thinning properties, and a film forming agent, wherein the rheology modifier is a polyacrylate, a cationA thickener or Carbopol thickener, wherein the film forming agent is polyvinyl alcohol, a polyvinyl alcohol copolymer or polyvinylpyrrolidone;

(b) providing a second premix component comprising an enzyme catalyst having perhydrolase activity, a carboxylic acid ester substrate, and a source of peroxygen;

(c) mixing the first pre-mix component and the second pre-mix component to obtain a liquid coating composition comprising a first antimicrobial agent comprising a peroxy acid, wherein the peroxy acid is formed in situ;

(d) applying the coating composition to the site; and is

(e) Allowing the coating composition to dry to form a coating on the site;

wherein at least one of the pre-mixed components further comprises a second antimicrobial agent, wherein the second antimicrobial agent comprises a quaternary ammonium compound.

5. The method of claim 4, wherein the first premix component and the second premix component are provided in a multi-compartment system; wherein the first premix component and the second premix component are kept separate prior to step (c).

6. The method of claim 4, wherein one of the premixed components is in liquid form and one of the premixed components is in solid form.

7. The process of claim 4 wherein the enzyme catalyst comprises a carbohydrate esterase of the CE-7 family.

8. An antimicrobial composition comprising components comprising:

a) a film-forming, water-soluble or water-dispersible agent, wherein the agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinylpyrrolidone;

b) an inert solvent;

c) a carboxylate substrate;

d) a source of peroxygen;

e) an enzyme catalyst having perhydrolase activity, thereby forming at least one peroxyacid in situ to provide at least a first antimicrobial agent; and

f) rheology modifier for providing shear thinning behavior, wherein said streamThe modifier is polyacrylate and cationA thickener or Carbopol thickener;

wherein the peroxyacid is formed in situ upon combining the components;

wherein the component further comprises a component that is an antimicrobial agent comprising a quaternary ammonium compound.

9. The composition of claim 8 wherein at least one or more of said components are packaged separately from said other components in a multi-compartment system prior to their combination to form said peroxyacid.

10. The composition of claim 8, wherein said peroxyacid is generated at a concentration of at least 20ppm within 5 minutes to 2 hours of combining said components.

11. An article comprising a coating having an antimicrobial composition on at least one vertical, sloped, complex geometry, or hard to reach surface of the article, wherein the antimicrobial composition comprises:

a) a film-forming, water-soluble or water-dispersible agent, wherein the agent is polyvinyl alcohol, a polyvinyl alcohol copolymer, or polyvinylpyrrolidone;

b) an inert solvent;

c) a carboxylate substrate;

d) a source of peroxygen;

e) an enzyme catalyst having perhydrolase activity, thereby forming at least one peroxyacid in situ to provide at least a first antimicrobial agent;

f) rheology modifier for providing shear thinning behavior wherein said rheology modifier is polyacrylate, cationicA thickener or Carbopol thickener; and

g) a quaternary ammonium compound;

wherein peroxy acids are formed upon combining components (a) to (g).

12. The article of claim 11, wherein the article is equipment used in the food or beverage industry.