Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/10—Animals; Substances produced thereby or obtained therefrom
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/10—Animals; Substances produced thereby or obtained therefrom
  • A01N63/14—Insects
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
  • A01N63/27—Pseudomonas
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
  • C09D5/16—Antifouling paints; Underwater paints
  • C09D5/1687—Use of special additives
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
  • C09D7/00—Features of coating compositions, not provided for in group C09D5/00; Processes for incorporating ingredients in coating compositions
  • C09D7/40—Additives
  • C09D7/60—Additives non-macromolecular
  • C09D7/63—Additives non-macromolecular organic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
  • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
  • C12N9/20—Triglyceride splitting, e.g. by means of lipase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
  • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)

Definitions

• the invention relates generally to an active enzyme such as an esterase (e.g., a lipolytic enzyme, a sulfuric ester hydrolase, an organophosphorus compound degradation enzyme); an antifungal or antimicrobial peptide; an enzyme (e.g., a lysozyme, a lytic transglycosylase), that may degrade a cell wall, a viral proteinaceous molecule, and/or a biologial membrane (e.g., a cell membrane, a virus envelope); and/or a peptidase, in a composition and methods for using the same.
• the composition may comprise a surface treatment such as a coating, an elastomer, an adhesive, a sealant, a textile finish or a wax; or a filler typically used in such a surface treatment.
• the surface of a material may be subject to addition of a surface treatment such as a coating, an adhesive, a sealant, a textile finish, and/or a wax, with a surface treatment typically used, for example, to protect, decorate, attach, and/or seal a surface and/or the underlying material.
• a filler typically comprises a particulate material that may be used as a component of a surface treatment.
• An example of use of such items includes a coating such as paint comprising a filler forming a solid protective, decorative, or functional adherent film on a surface.
• a biomolecule comprises a molecule often produced and isolated from an organism, such as an enzyme which catalyzes a chemical reaction.
• an enzyme comprises a lipolytic enzyme (e.g., a lipase) that catalyzes a reaction on a lipid substrate, such as a vegetable oil, a phospholipid, a sterol, and other hydrophobic molecule.
• a lipolytic enzyme catalyzed reaction may be used for an industrial or a commercial purpose, such as an alcohol or an acid esterification, an interesterification, a transesterification, an acidolysis, an alcoholysis, and/or resolution of a racemic alcohol and an organic acid mixture.
• organophosphate compound examples include an organophosphorus hydrolase (“OPH”), an organophosphorus acid anhydrolase (“OPAA”), and a DFPase.
• organophosphorus compounds and organosulfur (“OS”) compounds are used extensively as insecticides and are toxic to many organisms, including humans. OP compounds function as nerve agents. OP compounds have been used both as pesticides and chemical warfare agents.
• Alexander Fleming discovered lysozyme during a search for antibiotics when adding a drop of mucus to a growing bacterial culture and discovered it killed the bacteria. Lysozymes have widespread distribution in animals and plants.
• a lysozyme serves as a "natural antibiotic” protecting fluids and tissues that are rich in potential food for bacterial growth, such as an egg white.
• lysozyme may be found in many mammalian secretions and tissues, saliva, tears, milk, cervical mucus, leucocytes, kidneys, etc. Other enzymes possess antibiotic activity.
• a sulfuric ester hydrolase catalyzes a reaction at a sulfuric ester bond.
• a peptidase catalyzes a reaction at a peptide bond, such as a bond found in a peptide, a polypeptide or a protein, and may function as a digestive enzyme. Other enzymes catalyze various reactions.
• the invention features a composition, comprising an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; wherein the composition comprises an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• the active enzyme comprises a plurality of active enzymes.
• the enzyme comprises an esterase, a ceramidase, or a combination thereof, and wherein the esterase comprises a lipolytic enzyme, a phosphoric triester hydrolase, a sulfuric ester hydrolase, or a combination thereof.
• the lipolytic enzyme, the ceramidase, or a combination thereof comprises a carboxylesterase, a lipase, a lipoprotein lipase, an acylglycerol lipase, a hormone-sensitive lipase, a phospholipase A 1 , a phospholipases A 2 , a phosphatidylinositol deacylase, a phospholipase C, a phospholipase D, a phosphoinositide phospholipase C, a phosphatidate phosphatase, a lysophospholipase, a sterol esterase, a galactolipase, a sphingomyelin phosphodiesterase, a sphingomyelin phosphodiesterases D, a ceramidase, a wax-ester hydrolase, a fatty-acyl-ethyl-ester synthe, a
• the lipolytic enzyme, the ceramidase, or a combination thereof comprises: a carboxylesterase derived from Actinidia deliciosa, Aedes aegypti, Aeropyrum pernix, Alicyclobacillus acidocaldarius, Aphis gossypii, Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus clavatus, Athalia rosae, Bacillus acidocaldarius, Bombyx mandarina, Bombyx mori, Bos taurus, Burkholderia gladioli, Caenorhabditis elegans, Canis familiaris, Cavia porcellus, Chloroflexus aurantiacus, Felis catus, Fervidobacterium nodosum, Helicoverpa armigera, Homo sapiens, Macaca fascicularis, Malus pumila, Mesocricetus auratus, Mus musculus, Mus
• 5842-02103 2 musculus, Mus spretus, Mycobacterium tuberculosis, Mycoplasma hyopneumoniae, Myxococcus xanthus, Neosartorya fischeri, Oryctolagus cuniculus, Oryza saliva, Penicillium cyclopium, Phlebotomus papatasi, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas tragi, Pseudomonas sp, Rattus norvegicus, Rhizomucor miehei, Rhizopus oryzae, Rhizopus stolonifer, Ricinus communis, Samia cynthia ricini, Schizosaccharomyces pombe, Serratia marcescens, Spermophilus tridecemlineatus, Staphylococcus simulans, Sta
• the lipolytic enzyme comprises: a thermophilic carboxylesterase derived from Aeropyrum pernix, Alicyclobacillus acidocaldarius, Archaeoglobus fulgidus, Bacillus acidocaldarius, Pseudomonas aeruginosa, Sulfolobus shibatae, Sulfolobus solfataricus, Thermotoga maritime, or a combination thereof; a themophilic lipase derived from Acinetobacter calcoaceticus, Acinetobacter sp., Bacillus sphaericus, Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Candida rugosa, Candida thermophila, GeoBacillus thermoleovorans Toshki, Pseudomonas fragi, Staphylococcus xylosus, Sulfolobus solfataric
• the phosphoric triester hydrolase comprises an aryldialkylphosphatase, a diisopropyl-fluorophosphatase, or a combination thereof.
• the aryldialkylphosphatase comprises an organophosphorus hydrolase, a human paraoxonase, an animal carboxylase, or a combination thereof; wherein the diisopropyl-fluorophosphatase comprises an organophosphorus acid anhydrolase, a squid-type DFPase, a Mazur-type DFPase, or a combination thereof; or a combination thereof of the forgoing.
• the organophosphorus hydrolase comprises an Agrobacterium radiobacter P230 organophosphate hydrolase, a Flavobacterium balustinum parathion hydrolase, a Pseudomonas diminuta phosphotriesterase, a Flavobacterium sp opd gene product, a Flavobacterium sp. parathion hydrolase opd gene product, or a combination thereof; wherein the animal carboxylase comprises an insect carboxylase; or a combination thereof; wherein the organophosphorus acid anhydrolase comprises an Agrobacterium radiobacter P230 organophosphate hydrolase, a Flavobacterium balustinum parathion hydrolase, a Pseudomonas diminuta phosphotriesterase, a Flavobacterium sp opd gene product, a Flavobacterium sp. parathion hydrolase opd gene product, or a combination thereof; wherein the animal carboxylase comprises an insect carboxylase; or a combination thereof; wherein the
• 5842-02103 4 Altermonas organophosphorus acid anhydrolase, a prolidase, or a combination thereof; wherein the squid- type DFPase comprises a Loligo vulgaris DFPase, a Loligo pealei DFPase, a Loligo opalescens DFPase, or a combination thereof; wherein the Mazur-type DFPase comprises a mouse liver DFPase, a hog kidney DFPase, a Bacillus stearothermophilus strain OT DFPase, an Escherichia coli DFPase, or a combination thereof; or a combination thereof the forgoing.
• the insect carboxylase comprises a Plodia interpunctella carboxylase, Chrysomya putoria carboxylase, Lucilia cuprina carboxylase, Musca domestica carboxylase, or a combination thereof;
• the Altermonas organophosphorus acid anhydrolase comprises an Alteromonas sp JD6.5 organophosphorus acid anhydrolase, an Alteromonas haloplanktis organophosphorus acid anhydrolase, an Altermonas undina organophosphorus acid anhydrolase, or a combination thereof;
• the prolidase comprises a human prolidase, a Mus musculus prolidase, a Lactobacillus helveticus prolidase, an Escherichia coli prolidase, an Escherichia coli aminopeptidase P, or a combination thereof;
• the phosphoric triester hydrolase comprises a Plesiomona
• strain M6 mpcf gene product a Xanthomonas sp. phosphoric triester hydrolase, a Tetrahymena phosphoric triester hydrolase, or a combination thereof; or a combination thereof the forgoing.
• the sulfuric ester hydrolase comprises an arylsulfatase.
• the peptidase comprises a trypsin, a chymotrypsin, or a combination thereof.
• the antibiological enzyme comprises a lysozyme, a lysostaphin, a libiase, a lysyl endopeptidase, a mutanolysin, a cellulase, a chitinase, an ⁇ -agarase, an ⁇ -agarase, a ⁇ /-acetylmuramoyl-L- alanine amidase, a lytic transglycosylase, a glucan endo-1 ,3- ⁇ -D-glucosidase, an endo-1 ,3(4)- ⁇ -glucanase, a ⁇ -lytic metalloendopeptidase, a 3-deoxy
• the antibiological peptidic agent comprises SEQ ID No. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100,
• the active enzyme comprises a mesophilic enzyme, a psychrophilic enzyme, a thermophilic enzyme, a halophilic enzyme, or a combination thereof.
• the active enzyme, the antibiological peptidic agent, or a combination thereof comprises an immobilization carrier.
• the active enzyme, the antibiological peptidic agent, or a combination thereof comprises a purified active enzyme, a purified antibiological peptidic agent, or a combination thereof, [0014] In some embodiments, the active enzyme, the antibiological peptidic agent, or a combination thereof, comprises a particulate material. In some aspects, the active enzyme, the antibiological peptidic agent, or a combination thereof, comprises a cell-based particulate material. In other aspects, the cell-based particulate material comprises a whole cell particulate material or a cell fragment particulate material.
• the average wet molecular weight or dry molecular weight of a primary particle of the particulate material is about 50 kDa to about 1.5 x 10 14 kDa.
• an average active enzyme content, an average antibiological peptidic agent content, or a combination thereof, per primary particle of the particulate material is about 0.01 % to about 100%.
• the active enzyme, the antibiological peptidic agent, or a combination thereof is attenuated, sterilized, or a combination thereof.
• the active enzyme, the antibiological peptidic agent, or a combination thereof comprises about 0.01 % to about 80% of the composition by weight or volume.
• the active enzyme, the antibiological peptidic agent, or a combination thereof is microencapsulated.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof is about 5 um to about 5000 um thick upon a surface.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a paint.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a clear coating.
• the clear coating comprises a lacquer, a varnish, a shellac, a stain, a water repellent coating, or a combination thereof.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a multicoat system.
• the multicoat system comprises 2 to 10 layers.
• a plurality of layers of the multicoat system comprise the active enzyme.
• the multicoat system comprises a sealer, a water repellent, a primer, an undercoat, a topcoat, or a combination thereof.
• the topcoat comprises the active enzyme.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a coating that is capable of film formation.
• film formation occurs between about -10°C to about 4O 0 C. In other aspects, film formation occurs at baking conditions.
• the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a volatile component and a non-volatile component, and wherein film formation occurs by loss of part of the volatile component. In other facets, film formation occurs by cross-linking of a binder.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof produces a self-cleaning film.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof produces a temporary film.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a non-film forming coating.
• the non-film forming coating comprises a non-film formation binder.
• the non-film forming coating comprises a coating component in a concentration that is insufficient to produce a solid film.
• the architectural coating comprises an architectural wood coating, an architectural masonry coating, an architectural artist's coating, an architectural plastic coating, an architectural metal coating, or a combination thereof.
• the architectural coating has a pot life of at least 12 months at about -1 O 0 C to about 4O 0 C.
• the composition comprises an automotive coating, a can coating, a sealant coating, or a combination thereof.
• the composition comprises a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating, or a combination thereof.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a coating for a plastic surface.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a water-borne coating.
• the water-borne coating comprises a latex coating.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a solvent-borne coating.
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof has a low-
• the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof comprises a binder, a liquid component, a colorant, an additive, or a combination thereof.
• the binder comprises a thermoplastic binder, a thermosetting binder, or a combination thereof.
• the binder comprises an oil-based binder, a polyester resin, a modified cellulose, a polyamide, an amino resin, a urethane binder, a phenolic resin, an epoxy resin, a polyhydroxyether binder, an acrylic resin, a polyvinyl binder, a rubber resin, a bituminous binder, a polysulfide binder, a silicone binder, an organic binder, or a combination thereof.
• the oil-based binder comprises an oil, an alkyd, an oleoresinous binder, a fatty acid epoxide ester, or a combination thereof; wherein the polyester resin comprises a hydroxy- terminated polyester, a carboxylic acid-terminated polyester, or a combination thereof; wherein the modified cellulose comprises a cellulose ester, a nitrocellulose, or a combination thereof; wherein the epoxy resin comprises a cycloaliphatic epoxy binder; wherein the rubber resin comprises a chlorinated rubber resin, a synthetic rubber resin, or a combination thereof; or a combination thereof the forgoing.
• the liquid component comprises a solvent, a thinner, a diluent, a plasticizer, or a combination thereof.
• the liquid component comprises a liquid organic compound, an inorganic compound, water, or a combination thereof.
• the liquid organic compound comprises a hydrocarbon, an oxygenated compound, a chlorinated hydrocarbon, a nitrated hydrocarbon, a miscellaneous organic liquid, a plasticizer, or a combination thereof; wherein the inorganic compound comprises ammonia, hydrogen cyanide, hydrogen fluoride, hydrogen cyanide, sulfur dioxide, or a combination thereof; wherein the water comprises methanol, ethanol, propanol, isopropyl alcohol, tert- butanol, ethylene glycol, methyl glycol, ethyl glycol, propyl glycol, butyl glycol, ethyl diglycol, methoxypropanol, methyldipropylene glycol, dioxane, tetrahydorfuran, acetone, diacetone alcohol, dimethylformamide, dimethyl sulfoxide, ethylbenzene, tetrachloroethylene, p-xylene, toluene, diiso
• the hydrocarbon comprises an aliphatic hydrocarbon, a cycloaliphatic hydrocarbon, a terpene, an aromatic hydrocarbon, or a combination thereof; wherein the oxygenated compound comprises an alcohol, an ester, a glycol ether, a ketone, an ether, or a combination thereof; or a combination thereof the forgoing.
• the hydrocarbon comprises a petroleum ether, pentane, hexane, heptane, isododecane, a kerosene, a mineral spirit, a VMP naphtha, cyclohexane, methylcyclohexane, ethylcyclohexane, tetrahydronaphthalene, decahydronaphthalene, wood terpentine oil, pine oil, ⁇ -pinene, ⁇ -pinene, dipentene, D-limonene, benzene, toluene, ethylbenzene, xylene, cumene, a type I high flash aromatic naphtha, a type Il high flash aromatic
• oxygenated compound comprises methanol, ethanol, propanol, isopropanol, 1-butanol, isobutanol, 2-butanol, tert- butanol, amyl alcohol, isoamyl alcohol, hexanol, methylisobutylcarbinol, 2-ethylbutanol, isooctyl alcohol, 2- ethylhexanol, isodecanol, cylcohexanol, methylcyclohexanol, trimethylcyclohexanol, benzyl alcohol, methylbenzyl alcohol, furfuryl alcohol, tetrahydrofurfuryl alcohol, diacetone alcohol, trimethylcyclohexanol, methyl formate, ethyl formate, buty
• the colorant comprises a pigment, a dye, or a combination thereof.
• the active enzyme comprises a particulate material comprising about 0.000001 % to about 100% of the pigment.
• the pigment comprises a corrosion resistance pigment, a camouflage pigment, a color property pigment, an extender pigment, or a combination thereof.
• the corrosion resistance pigment comprises aluminum flake, aluminum triphosphate, aluminum zinc phosphate, ammonium chromate, barium borosilicate, barium chromate, barium metaborate, basic calcium zinc molybdate, basic carbonate white lead, basic lead silicate, basic lead silicochromate, basic lead silicosulfate, basic zinc molybdate, basic zinc molybdate-phosphate, basic zinc molybdenum phosphate, basic zinc phosphate hydrate, bronze flake, calcium barium phosphosilicate, calcium borosilicate, calcium chromate, calcium plumbate, calcium strontium phosphosilicate, calcium strontium zinc phosphosilicate, dibasic lead phosphite, lead chromosilicate
• the color property pigment comprises aniline black; anthraquinone black; carbon black; copper carbonate; graphite; iron oxide; micaceous iron oxide; manganese dioxide, azo condensation, metal complex brown; antimony oxide; basic lead carbonate; lithopone; titanium dioxide; white lead; zinc oxide; zinc sulphide; titanium dioxide and ferric oxide covered mica, bismuth oxychloride crystal, dioxazine violet, carbazole Blue; cobalt blue; indanthrone; phthalocyanine blue; Prussian blue; ultramarine; chrome green; hydrated chromium oxide; phthalocyanine green; anthrapyrimidine; arylamide yellow; barium chromate; benzimidazolone yellow; bismuth vanadate; cadmium sulfide yellow; complex inorganic color; diarylide yellow; disazo condensation; flavanthrone; isoindoline; isoindolinone; lead chromate; nickel azo yellow; organic metal complex
• the additive comprises 0.000001 % to 20.0% by weight, of the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating, or a combination thereof.
• the additive comprises an accelerator, an adhesion promoter, an antifoamer, anti-insect additive, an antioxidant, an antiskinning agent, a buffer, a catalyst, a coalescing agent, a corrosion inhibitor, a defoamer, a dehydrator, a dispersant, a drier, electrical additive, an emulsifier, a filler, a flame/fire retardant, a flatting agent, a flow control agent, a gloss aid, a leveling agent, a marproofing agent, a preservative, a silicone additive, a slip agent, a surfactant, a light stabilizer, a rheological control agent, a wetting additive, a cryopreservative, a xeroprotectant, a pH indicator, or a combination thereof.
• the preservative comprises an in-can preservative, an in-film preservative, or a combination thereof.
• the preservative comprises a biocide, a biostatic, or a combination thereof.
• the biocide, the biostatic, or a combination thereof comprises an algaecide, an algaestatic, a bactericide, a bacteristatic, a fungicide, a fungistatic, a germicide, a germistatic, a herbicide, a herbistatic, a microbiocide, a microbiostatic, a mildewcide, a mildewstatic, a molluskicide, a molluskistatic, a slimicide, a slimistatic, a viricide, a viristatic, or a combination thereof.
• the preservative comprises 1-(3-chloroallyl)-3,5,7-triaza-1-azoniaadamantane chloride; 1 ,2- benzisothiazoline-3-one; 1 ,2-dibromo-2,4-dicyanobutane; 1 ,3-bis(hydroxymethyl)-5,5-dimethylhydantoin; 1- methyl-3,5,7-triaza-1-azonia-adamantane chloride; 2-bromo-2-nitropropane-1 ,3-diol; 2-(4- thiazolyl)benzimidazole; 2-(hydroxymethyl)-amino-2-methyl-1-propanol; 2(hydroxymethyl)-aminoethanol; 2,2- dibromo-3-nitrilopropionamide; 2,4,5,6-tetrachloro-isophthalonitrile; 2-mercaptobenzo-thiazole; 2-methyl-4- isothiazolin-3-one; 2-n-oc
• 5842-02103 11 dicarboximide; p-chloro-m-cresol; phenoxyethanol; phenylmercuric acetate; poly(hexamethylene biguanide) hydrochloride; potassium dimethyldithiocarbamate; potassium N-hydroxy-methyl-N-methyl-dithiocarbamate; propyl parahydroxybenzoate; sodium 2-pyridinethiol-1 -oxide; tetra-hydro-3,5-di-methyl-2H-1 ,3,5-thiadiazine- 2-thione; tributyltin benzoate; tributyltin oxide; tributyltin salicylate; zinc pyrithione; sodium pyrithione; copper pyrithione; zinc oxide; a zinc soap; or a combination thereof.
• the elastomer comprises a thermoplastic elastomer, a melt processable rubber, a synthetic rubber, a natural rubber, a propylene oxide elastomer, an ethylene-isoprene elastomer, an ethylene-vinyl acetate elastomer, a non-polymeric elastomer, or a combination thereof.
• the thermoplastic elastomer comprises an elastomeric polyolefin, a thermoplastic vulcanizate, a styrenic thermoplastic elastomer, a styrene butadiene rubber, a polyurethane elastomer, a thermoplastic copolyester elastomer, a polyamide, or a combination thereof; wherein the synthetic rubber comprises a nitrile butadiene rubber, a butadiene rubber, a butyl rubber, a chlorinated/chlorosulfonated polyethylene, an epichlorohydrin, an ethylene propylene copolymer, a fluoroelastomer, a polyacrylate rubber, a poly(ethylene acrylic), a polychloroprene, a polyisoprene, a polysulfide rubber, a silicone rubber, or a combination thereof; wherein the non-polymeric elastomer comprises a vulcanized oil
• the composition comprises an adhesive, a sealant, or a combination thereof.
• the adhesive, the sealant, or a combination thereof comprises an acrylic adhesive, an acrylic acid diester adhesive, a butyl rubber adhesive, a carbohydrate adhesive, a cellulosic adhesive, a cyanoacrylate adhesive, a cyanate ester adhesive, an epoxy adhesive, a melamine formaldehyde adhesive, a natural rubber adhesive, a neoprene rubber adhesive, a nitrile rubber adhesive, a phenolic adhesive, a phenoxy adhesive, a polyamide adhesive, a polybenzimidazole adhesive, a polyethylene adhesive, a polyester adhesive, a polyisobutylene adhesive, a polysulfide adhesive, a polyurethane adhesive, a polyvinyl acetal adhesive, a polyvinyl acetate adhesive, a polyvinyl alcohol adhesive, a protein adhesive, a reclaimed rubber adhesive, a
• the elastomer; the adhesive; the sealant, or a combination thereof comprises a polymeric material additive.
• the polymeric material additive comprises a curing agent, a cross-linking agent, an inhibitor, a nucleating agent, a plasticizer, a lubricant, a mold release agent, a slip agent, a diluent, a dispersant, a thickening agent, a thixotropic, a thinner, an anti-blocking agent, an antistatic agent, a flame retarder, a colorant, an antifogging agent, an odorant, a blowing agent, a surfactant, a defoamer, an anti-aging additive, a degrading agent, an anti-microbial agent, an adhesion promoter, an impact modifier, a low-profile additive, a filler, a pH indicator, or a combination thereof.
• the anti-microbial agent comprises a biocide
• the antibiological peptidic agent, the antibiological enzyme, or a combination thereof comprises a biocide, a biostatic, or a combination thereof.
• the composition is stored in a multi-pack container.
• a coating composition comprising an architectural coating comprising an active enzyme, an antibiological peptidic agent, or a combination thereof, wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• a multi-pack coating composition comprising a plurality of containers, wherein at least one container comprises an active enzyme, an antibiological peptidic agent, or a combination thereof; wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof; and wherein the coating comprises an architectural wood coating, an architectural masonry coating, an architectural artist coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating, or a combination thereof.
• an elastomer composition comprising an elastomer and an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• a filler composition comprising a filler and an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• an adhesive composition comprising an adhesive and an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• a sealant composition comprising a sealant and an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• a textile finish composition comprising a textile finish and an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• a wax composition comprising a wax and an active enzyme, an antibiological peptidic agent, or a combination thereof; and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• a method of preparing a bioactive surface treatment, a bioactive filler, or a combination thereof comprising the steps of: obtaining an active enzyme, an antibiological peptidic agent, or a combination thereof; wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof; and admixing at least one component of a surface treatment, a filler, or a combination thereof, with the active enzyme, the antibiological
• 5842-02103 13 peptidic agent, or a combination thereof a combination thereof; and then admixing any additional component of a surface treatment, a filler, or a combination thereof to complete the surface treatment, the filler, or a combination thereof.
• a method of preparing a bioactive surface treatment, a bioactive filler, or a combination thereof comprising the steps of: obtaining an active enzyme, an antibiological peptidic agent, or a combination thereof; wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof; and admixing a surface treatment, a filler, or a combination thereof, with the active enzyme, the antibiological peptidic agent, or a combination thereof.
• a method of reducing the concentration of a chemical on a surface comprising the steps of: applying a surface treatment to the surface, wherein the surface treatment comprises an active enzyme, and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof; and contacting the surface with a chemical, wherein the chemical comprises a substrate of the active enzyme; and wherein the substrate comprises an ester linkage, a peptide linkage, a lipid, a cell wall component, a cell membrane component, or a combination thereof.
• the surface treatment comprises an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, or a combination thereof.
• the substrate is a component of a living cell, a virus, or a combination thereof, and wherein the active enzyme produces a biocidal activity, a biostatic activity, or a combination thereof upon contact with the substrate.
• a method of cleaning a surface contaminated with a chemical comprising the steps of: contacting a surface contaminated with a chemical with a coating comprising an active enzyme, wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof, wherein the chemical comprises a substrate of the active enzyme; and wherein the substrate comprises an ester linkage, a peptide linkage, a lipid, a cell wall component, a cell membrane component, or a combination thereof.
• a method of reducing the concentration of a chemical on a surface comprising the steps of: applying a coating to the surface, wherein the coating comprises an architectural wood coating, an architectural masonry coating, an architectural artist coating, an automotive coating, a can coating, a sealant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating, or a combination thereof, and wherein the coating comprises an active enzyme, wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.and contacting the surface with a chemical, wherein the chemical comprises a substrate of the active enzyme; and wherein the chemical comprises an ester linkage, a peptide linkage, a lipid, a cell wall component, a cell membrane component, or a combination thereof.
• the step of applying to the surface a coating occurs prior to contacting the surface with the chemical.
• the surface is located on a stove, a sink, a drain pipe, a counter top, a floor, a wall, a cabinet, an appliance, or a combination thereof.
• the coating is formulated as
• the method further comprises the step of: applying a cleaning material to the surface, and removing the chemical, a product of the reaction of the chemical catalyzed by the active enzyme, or a combination thereof.
• the cleaning material comprises a cleaning solution, a cleaning devise, or a combination thereof.
• a method of cleaning a surface contaminated with a chemical comprising the steps of: obtaining a surface treatment comprising an active enzyme; and contacting a surface contaminated with a chemical with the surface treatment comprising an active enzyme, wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof, wherein the chemical comprises a substrate of the active enzyme; and wherein the chemical comprises an ester linkage, a peptide linkage, a lipid, a cell wall component, a cell membrane component, or a combination thereof.
• kit having component parts capable of being assembled comprising a container comprising an active enzyme, an antibiological peptidic agent, or a combination thereof, and a container comprising at least one component of an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• an article of manufacture comprising an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; wherein the article of manufacture comprises an active enzyme, an antibiological peptidic agent, or a combination thereof, and wherein the active enzyme comprises an esterase, a petroleum lipolytic enzyme, a ceramidase, a peptidase, an antibiological enzyme, or a combination thereof.
• Product is a composition.
• Product is a surface treatment.
• Product is a composition comprising a surface treatment.
• Product is a composition, comprising an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; comprising an active enzyme, an antibiological peptidic agent, or a combination thereof.
• composition obtainable by process of incorporation of an active enzyme, an antibiological peptidic agent, or a combination thereof; into an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof.
• composition comprising an architectural coating, an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; comprising an active enzyme, an antibiological peptidic agent,
• an architectural coating an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; characterized in that an active enzyme, an antibiological peptidic agent, or a combination thereof; is included as a component of the architectural coating, the automotive coating, the can coating, the sealant coating, the chemical agent resistant coating, the camouflage coating, the pipeline coating, the traffic marker coating, the aircraft coating, the nuclear power plant coating; the elastomer; the adhesive; the sealant, the wax, the textile finish, the filler, or the combination thereof.
• an architectural coating an automotive coating, a can coating, a sealant coating, a chemical agent resistant coating, a camouflage coating, a pipeline coating, a traffic marker coating, an aircraft coating, a nuclear power plant coating; an elastomer; an adhesive; a sealant, a wax, a textile finish, a filler, or a combination thereof; for the purpose of reducing the concentration of a chemical on a surface.
• the words “a,” “an,” “the,” and/or “said” may mean one or more than one.
• the terms “having,” “has,” “is,” “have,” “including,” “includes,” and/or “include” has the same meaning as “comprising,” “comprises,” and “comprise.”
• another may mean at least a second or more.
• compositions described as a coating suitable for use on a plastic surface described in different sections of the specification may be claimed individually and/or as a combination, as they are part of the same genera of plastic coatings.
• various monomers of a chemical type such as "amino acid” may be described in various parts of the specification, and such amino acid monomers may be claimed individually and/or in various combinations.
• Such related and/or like genera(s), sub-genera(s), specie(s), and/or embodiment(s) described herein are contemplated both in the form of an individual component that may be claimed, as well as a mixture and/or a combination that may be described in the claims as "at least one selected from,” "a mixture thereof” and/or "a combination thereof.”
• exemplary values are specified as a range, and all intermediate range(s), subrange(s), combination(s) of range(s) and individual value(s) within a cited range are contemplated and included herein.
• citation of a range "0.03% to 0.07%” provides specific values within the cited range, such as, for example, 0.03%, 0.04%, 0.05%, 0.06%, and 0.07%, as well as various combinations of such specific values, such as, for example, 0.03%, 0.06% and 0.07%, 0.04% and 0.06%, and/or 0.05% and 0.07%, as well as sub-ranges such as 0.03% to 0.05%, 0.04% to 0.07%, and/or 0.04% to 0.06%, etc.
• Example 15 provides additional descriptions of specific numeric values within any cited range that may be used for an integer, intermediate range(s), subrange(s), combinations of range(s) and individual value(s) within a cited range, including in the claims.
• the average weight per single particle (“primary particle”) of a biomolecular composition may be measured in "wet weight,” which refers to the weight of the particle prior to a drying and/or an extraction step that removes the liquid component of a
• the "wet weight" of a biomolecular composition e.g., a whole cell particulate material that has its liquid component replaced by some other liquid (e.g., an organic solvent) may also be measured in “wet weight.”
• the “dry weight” refers to the average per particle weight of a biomolecular composition after the majority of the liquid component has been removed.
• the term “majority” refers to about 50% to about 100%, with, for example, the greater values (e.g., about 85% to about 100%) contemplated in some aspects.
• the dry weight of a biomolecular composition may be about 5% to about 30% the wet weight, as a cell often may comprise about 70% to about 95% water.
• Any technique for measuring a biological cell's and/or a particle's size, volume, density, etc. used for various insoluble particulate materials (e.g., a pigment, an extender) that typically are comprised as a component of a material formulation may be applied to a biomolecular composition to determine a wet weight value, a dry weight value, a particle size, and/or a particle density, etc.
• Various examples of specific techniques are described herein. Further, such measurements of a cell's size, shape, density, numbers, etc.
• the average number of particles, size, shape, etc. of a biomolecular composition may be microscopically determined for a given volume and/or weight of a material, whether prepared as a "wet weight” and/or a "dry weight material," and the average particle weight, density, volume, etc. calculated.
• the average wet molecular weight or dry molecular weight of a primary particle of a biomolecular composition (e.g., a cell-based particulate material) comprises about 50 kDa to about 1.5 x 10 14 kDa.
• the average active enzyme content, average antibiological peptidic agent content, or a combination thereof, per primary particle and/or per the content of the material formulation may comprise about 0.00000001 % to about 100%.
• compositions and methods herein may produce materials ("material formulations”) (e.g., compositions, manufactured articles, etc) with a bioactivity.
• a biomolecule's activity e.g., an enzyme's catalytic reaction, a peptide's antimicrobial activity
• a material formulation include a surface treatment, a filler, a biomolecular composition, or a combination thereof.
• Examples of a property that may be altered include resistance to a microorganism; while examples of a property that may be conferred include enzymatic activity upon contact with a substrate (e.g., a lipid, an organophosphorus compound, etc.) of an enzyme, wherein the material comprises the enzyme.
• a substrate e.g., a lipid, an organophosphorus compound, etc.
• the material comprises the enzyme.
• Numberous examples of component(s), material formulation(s), composition(s), manufactured article(s), etc. are described herein, and inclusion of a biomolecular composition may alter
• 5842-02103 18 and/or confer a property that to modify such component(s), material formulation(s), composition(s), manufactured article(s), etc. to be useable for a different purpose and/or function.
• a lipolytic enzyme may confer a self-degreasing property to a material formulation.
• a proteinaceous composition e.g., a peptide composition, an enzyme
• possessing an antibiological activity may be incorporated into a material formulation to alter and/or confer a property (e.g., an antibiological activity, a sufficient antifungal activity) that may be exhibited in the material formulation.
• An example of a material formulation comprises a "surface treatment," which refers to a composition applied to a surface, and examples of such compositions specifically contemplated include a coating (e.g., a paint, a clear coat), a textile finish, a wax, an elastomer, an adhesive, a filler, and/or a sealant.
• a surface treatment may be prepared as an amorphous material (e.g., a liquid, a semisolid) and/or a simple geometric shape (e.g., a planar material) to allow ease of application to a surface.
• An adhesive refers to a composition capable of attachment to one or more surfaces ("substrates") of one or more objects ("adherents”), wherein the composition comprises a solid or is capable of converting into the solid, wherein the solid is capable of holding a plurality of objects (“adherents”) together by attachment to the surface of the objects while withstanding a normal operating stress load placed upon the objects and the solid.
• an adhesive e.g., a glue, a cement, an adhesive paste
• a sealant comprises a composition capable of attachment to a plurality of surfaces to fill a space and/or a gap between the plurality of surfaces and form a barrier to a gas, a liquid, a solid particle, an insect, or a combination thereof.
• An adhesive generally functions to prevent movement of the adherents, while a sealant typically functions to seal adherents that move.
• a sealant comprises a subtype of an adhesive based on purpose/function (i.e., a flexible adhesive), and a sealant typically possesses lower strength, greater flexibility, or a combination thereof, than many other types of adhesives (e.g., a structural adhesive).
• an abhesive comprises a material (e.g., a coating such as a clear coating or a paint; or a mold release agent such as a plastic release film) applied to a surface to inhibit adhesion/sticking of an additional material to the abhesive and/or a surface the abhesive covers.
• a material e.g., a coating such as a clear coating or a paint; or a mold release agent such as a plastic release film
• An elastomer (“elastomeric material”) comprises a "macromolecular material that returns rapidly to approximately the initial dimensions and shape after substantial deformation by a weak stress and release of the stress" while a rubber comprises a material "capable of recovering from a large deformation quickly and forcibly, and can be, and/or are already is, modified to a state in which it is essentially insoluble (but can swell) in a solvent.”
• a solvent commonly used to swell a rubber include benzene, methyl ethyl ketone, and/or ethanol toluene azeotrope (see, for example, definitions in ASTM D 1566).
• a rubber retracts within about one minute to less than about 1.5 times its original length after being held for about one minute at about twice its length at room temperature, while an elastomer retracts within about five minutes to within about 10% original length after being held for about five minutes at about twice its length at room temperature.
• cross-linking/vulcanization may be used to confer an elastomeric property, as the crosslinks promote maintenance of a material's dimensions.
• a plastic comprises a solid polymeric material solid at room temperature (i.e., about 23°C) in a finished state, and at some stage of the plastic's manufacture and/or processing was capable of being shaped by flow and/or molding into a finished article.
• a material such as an
• All plastics comprise a polymer, but not all polymers are a plastic, such as, for example, a cellulose that lacks a chemical modification to allow it to be processed as a plastic during manufacture, or a polymer that possesses an elastomeric property.
• All polymeric materials comprise a polymer, but not all polymers possess the physical/chemical properties to be classified as a specific material type, particularly when such a material type comprises another component in addition to the polymer.
• a "cell” in a biotechnology art described for production of a biomolecule refers to the smallest unit of living matter (viruses not withstanding), while a "cell” in a material art (e.g., an elastomer art) refers to a void in a material to produce a solid foam material (e.g., elastomer foam material).
• the word “mold” may be used in the context of a fungal cell, while in other context “mold” refers to a solid structure used to shape a material, such as a mold used to shape an elastomeric material into a geometric shape.
• mold refers to a solid structure used to shape a material, such as a mold used to shape an elastomeric material into a geometric shape.
• the appropriate definition and/or meaning for the term e.g., a biomolecular composition produced from a cell vs a void, a solid foamed material vs. a liquid or gas foam; a biological cell/organism vs. a device for material manufacture
• a biomolecular composition produced from a cell vs a void, a solid foamed material vs. a liquid or gas foam; a biological cell/organism vs. a device for material manufacture should be applied in accordance with the context of the term's use in light of the present disclosures
• a "biomolecular composition” or “biomolecule composition” refers to a composition comprising a biomolecule.
• a “biomolecule” refers to a molecule (e.g., a compound) comprising of one or more chemical moiety(s) ["specie(s),” “group(s),” “functionality(s),” “functional group(s)”] typically synthesized in living organisms, including but not limited to, an amino acid, a nucleotide, a polysaccharide, a simple sugar, a lipid, or a combination thereof.
• a biomolecule includes, a colorant (e.g., a chlorophyll), an enzyme, an antibody, a receptor, a transport protein, structural protein, a prion, an antibiological proteinaceous molecule (e.g., an antimicrobial proteinaceous molecule, an antifungal proteinaceous molecule), or a combination thereof.
• a biomolecule typically comprises a proteinaceous molecule.
• a “proteinaceous molecule,” proteinaceous composition,” and/or “peptidic agent” comprises a polymer formed from an amino acid, such as a peptide (i.e., about 3 to about 100 amino acids), a polypeptide (i.e., about 101 or more amino acids, such as about 50,000 or more amino acids), and/or a protein.
• a “protein” comprises a proteinaceous molecule comprising a contiguous molecular sequence three amino acids or greater in length, matching the length of a biologically produced proteinaceous molecule encoded by the genome of an organism. Examples of a proteinaceous molecule include an enzyme, an antibody, a receptor, a transport protein, a structural protein, or a combination thereof.
• a peptide e.g., an inhibitory peptide, an antifungal peptide
• a peptidic agent and/or proteinaceous molecule may comprise a mixture of such peptide(s) (e.g., an aliquot of a peptide library), polypeptide(s) and/or protein(s), and may also include materials such as any associated stabilizer(s), carrier(s), and/or inactive peptide(s), polypeptide(s), and/or protein(s).
• a proteinaceous molecule comprises an enzyme.
• a proteinaceous molecule that functions as an enzyme whether identical to the wild-type amino acid sequence encoded by an isolated gene, a functional equivalent of such a sequence, or a combination thereof, may be used.
• a wild-type enzyme refers to an amino acid sequence that functions as an enzyme and matches the sequence encoded by an isolated gene from a natural source.
• a "functional equivalent" to the wild-type enzyme generally comprises a proteinaceous molecule comprising a sequence and/or a structural analog of a wild-type enzyme's sequence and/or structure and functions as an enzyme.
• the functional equivalent enzyme may possess similar or the same enzymatic properties, such as catalyzing chemical reactions of the wild-type enzyme's EC classification; and/or may possess other enzymatic properties, such as catalyzing the chemical reactions of an enzyme related to the wild-type enzyme by sequence and/or structure.
• An enzyme encompasses its functional equivalents that catalyze the reaction catalyzed by the wild-type form of the enzyme (e.g., the reaction used for EC Classification).
• a functional equivalent of a wild-type enzyme examples include mutations to a wild-type enzyme sequence, such as a sequence truncation, an amino acid substitution, an amino acid modification, and/or a fusion protein, etc., wherein the altered sequence functions as an enzyme.
• the term "derived" refers to a biomolecule's (e.g., an enzyme) progenitor source, though the biomolecule may comprise a wild-type and/or a functional equivalent of the original source biomolecule, and thus the term "derived” encompasses both wild-type and functional equivalents.
• a coding sequence for a Homo sapiens enzyme may be mutated and recombinantly expressed in bacteria, and the bacteria comprising the enzyme processed into a biomolecular composition for use, but the enzyme, whether isolated and/or comprising other bacterial cellular material(s), comprises an enzyme "derived" from Homo sapiens.
• a wild-type enzyme isolated from an endogenous biological source such as, for example, a Pseudomonas putida lipase isolated from Pseudomonas putida, comprises an enzyme "derived" from Pseudomonas putida.
• a biomolecule may comprise a hybrid of various sequences, such as a fusion of a mammalian lipase and a non-mammalian lipase, and such a biomolecule may be considered derived from both sources.
• Other types of biomolecule(s) e.g., a ribozyme, a transport protein, etc.
• a biomolecule may be derived from a non-biological source, such as the case of a proteinaceous and/or a nucleotide sequence engineered by the hand of man.
• a nucleotide sequence encoding a synthetic peptide sequence from a peptide library may be recombinantly produced, and may thus "derived" from the originating peptide library.
• a biomolecular composition comprises a cell and/or cell debris (i.e.., a "cell- based” material), in contrast to a purified biomolecule (e.g., a purified enzyme).
• a cell used in a cell-based particulate material comprises a durable structure at the cell-external environment
• a cell may be obtained/isolated from a unicellular and/or an oligocellular organism, and a particulate material may be prepared from such an organism without a step to separate one or more cells from a multicellular tissue and/or a multicellular organism (e.g., a plant) into a smaller average particle size suitable for preparation of a material formulation (e.g., a biomolecular composition).
• a material formulation e.g., a biomolecular composition
• a biological material such as a virus (e.g., a bacteriophage), a biological cell (e.g., a microorganism), a virus, a tissue, and/or an organism (e.g., a plant) may be obtained from an environmental source using procedures of the art [see, for example, "Environmental Biotechnology Isolation of Biotechnological Organisms From Nature (Labeda, D. P., Ed.), 1990].
• a virus e.g., a bacteriophage
• a biological cell e.g., a microorganism
• a virus e.g., a tissue, and/or an organism
• an organism e.g., a plant
• the identification of a biological material usually comprises characterization of suitable growth conditions for the cell and/or a virus, such as energy source (e.g., a digestible organic molecule), vitamin requirements, mineral requirements, pH conditions, light conditions, temperature, etc.
• energy source e.g., a digestible organic molecule
• CCAP Culture Collection of Algae and Protozoa
• CIP Collection de I'lnstitut Pasteur
• DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen
• IHEM IHEM Biomedical Fungi and Yeasts Collection
• unicellular refers to 1 cell that generally does not live in contact with a second cell.
• oligocellular refers to about 2 to about 100 cells, which generally live in contiguous contact with the other cells. Common specific types of oligocellular biological material includes 2 contacting cells (“dicellular”), three contacting cells (“tricellular”) and four contacting cells (“tetracellular”).
• multicellular refers to 101 or more cells (e.g., hundreds, thousands, millions, billions, trillions), which generally live in contiguous contact with the other cells.
• the composition may be referred to herein as a "unicellular-based particulate material.”
• the composition may be known herein as an "oligocellular-based particulate material,” as well as a "dicellular-based particulate material,” tricellular-based particulate material,” or "tetracellular-based particulate material,” as appropriate.
• the composition may be known herein as a "multicellular-based particulate material.”
• a cell-based particulate material may be referred to herein based upon the type of biological material from which it was derived, including taxonomic/phylogenetic classification and/or biochemical composition, as well as one or more processing steps used in its preparation.
• Examples of such lexicography for a cell-based particulate material include an "eurkaryotic-based particulate material,” a “prokaryotic-based particulate material,” a “plant-based particulate material,” a “microorganism-based particulate material,” a “ Eubacteria-based particulate material,” an “/Arcftaea-based particulate material,” a “fungi-based particulate material,” a “yeast- based particulate material,” a “Prof/sfa-based particulate material,” an "algae-based particulate material,” a “Chrysophyta-based particulate material,” a “Methanolacinia-based particulate material,” a “Microscilla aggregans-based particulate material,” a “bacteriophage HER-6 [44Lindberg]-based particulate material,” a “bacteria and algae-based particulate material,” a “peptidoglycan-
• Certain cell(s) and/or virus(s) are capable of growth in environmental conditions typically harmful to many other types of cells ("extremophiles"), such as conditions of extreme temperature, salt and/or pH.
• a biomolecule derived from such a cell and/or a virus may be useful in certain embodiments for durability, activity, or other property of a biomolecular composition (e.g., a material formulation comprising a biomolecular composition) that undergoes conditions similar to (e.g., the same or overlapping ranges) as those found in the cell's and/or the virus's growth environment.
• a hyperthermophile-based biomolecular composition may find usefulness in a material formulation where high temperature thermal extremes may occur, including extremes of temperature that may occur during coating based film formation and/or use of a coating produced film near a heat source.
• a "hyperthermophile" or “thermophile” typically grows in temperatures considered herein to comprise a baking temperature for a
• compositions may comprise a biomolecule derived from a thermophile.
• a biomolecular composition with prolonged stability, enzymatic activity, or a combination thereof, at other temperature ranges may be used depending upon the application.
• a "psychrophile” typically grows at about -1O 0 C to about 2O 0 C
• a “mesophile” typically grows at about 2O 0 C to about 4O 0 C
• an "extreme halophile” may be capable of living in salt-water conditions of about 1.5 M (8.77% w/v) sodium chloride to about 2.7 M (15.78% w/v) or more sodium chloride.
• An extreme halophile's biomolecule component(s) may be relatively resistant to an ionic-salt component of a material formulation.
• an "extreme acidophile” may be capable of growing in about pH 1 to about pH 6, while an “extreme alkaliphile” may be capable of growing in about pH 8 to about pH 14.
• One or more biomolecules such as an enzyme derived from such a cell and/or a virus may be selected on the basis the cell's and/or a virus's growth conditions for incorporation into the compositions, articles, etc. described herein.
• a material and/or a chemical formula thereof may be obtained from convenient source such as a public database, a biological depository, and/or a commercial vendor.
• convenient source such as a public database, a biological depository, and/or a commercial vendor.
• nucleotide sequences including those that encode amino acid sequences, may be obtained at a public database, such as the Entrez Nucleotides database, which includes sequences from other databases including GenBank (e.g., CoreNucleotide), RefSeq, and PDB.
• nucleotide and amino acid sequences includes the Kyoto Encyclopedia of Genes and Genomes (“KEEG") (Kanehisa, M.et al., 2008; Kanehisa, M. et al., 2006; Kanehisa, M. and Goto, S., 2000).
• KEEG Kyoto Encyclopedia of Genes and Genomes
• various amino acid sequences may be obtained at a public database, such as the Entrez databank, which includes sequences from other databases including SwissProt, PIR, PRF, PDB, Gene, GenBank, and RefSeq. Numerous nucleic acid sequences and/or encoded amino acid sequences can be obtained from such sources.
• a biological material comprising, or are capable of comprising such a biomolecule (e.g., a living cell, a virus), may be obtained from a depository such as the American Type Culture Collection ("ATCC"), P.O. Box 1549 Manassas, VA 20108, USA.
• ATCC American Type Culture Collection
• a biomolecule, a chemical reagent, a biological material, and/or an equipment may be obtained from a commercial vendor such as Amersham Biosciences ® , 800 Centennial Avenue, P.O.
• a biomolecule, a chemical reagent, a biological material, and/or an equipment may be obtained from commercial vendors such as Amersham Biosciences ® , 800 Centennial Avenue, P.O.
• a cell, nucleic acid sequence, amino acid sequence, and the like may be manipulated in light of the present disclosures, using standard techniques [see, for example, In “Molecular Cloning” (Sambrook, J., and Russell, D. W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 2001”; In “Current Protocols in Molecular Biology” (Chanda, V. B. Ed.) John Wiley & Sons, 2002”; In “Current Protocols in Nucleic Acid
• a biomolecule for use depends on the property to be conferred to a composition, an article, etc.
• a biomolecule comprises an enzyme, to confer a property such as as enzymatic activity to a material formulation (e.g., a surface treatment, a filler, a biomolecular composition).
• a material formulation e.g., a surface treatment, a filler, a biomolecular composition.
• the term "enzyme” refers to a molecule that possesses the ability to accelerate a chemical reaction, and comprises one or more chemical moiety(s) typically synthesized in living organisms, including but not limited to, an amino acid, a nucleotide, a polysaccharide, a simple sugar, a lipid, or a combination thereof.
• Enzymes are identified by a numeric classification system [See, for example, IUBM B (1992) Enzyme Nomenclature: Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. (NC-ICBMB and Edwin C. Webb Eds.) Academic Press, San Diego, CA.; Enzyme nomenclature. Recommendations 1992, 1994; Enzyme nomenclature. Recommendations 1992, 1995; Enzyme nomenclature. Recommendations 1992, 1996; Enzyme nomenclature. Recommendations 1992, 1997; Enzyme nomenclature. Recommendations 1992, 1999].
• An enzyme may function in synthesis and/or degradation, a catabolic reaction and/or an anabolic reaction, and other types of reversible reactions.
• an enzyme normally described as an esterase may function as an ester synthetase depending upon the concentration of the substrate(s) and/or the product(s), such as an excess of hydrolyzed esters, typically considered the product of an esterase reaction, relative to unhydrolyzed esters, typically considered the substrate of the esterase reaction.
• a lipase may function as a lipid synthetase due to a relative abundance of free fatty acid(s) and alcohol moiety(s) to catalyze the synthesis of a fatty acid ester.
• any reaction that an enzyme may be capable of is contemplated, such as, for example, a transesterification, an interesterification, and/or an intraesterification, and the like, being conducted by an esterase.
• an esterase may alter the odor and/or fragrance of a composition by degrading an odor causing chemical, such as those produced by a microorganism, as well as synthesize a fragrant compound, as odor or fragrant compounds often comprises an ester linkage.
• active or “bioactive” refers to the effect of biomolecule, such as conferring and/or altering a property of a material formulation.
• a material formulation comprising an "active" or “bioactive” antibiological peptide refers to the material formulation possessing altered and/or conferred antibiological effect (e.g., a biocidal effect, a biostatic effect) on a living cell (e.g., a living organism, a fungal cell) and/or a virus relative to a like material formulation lacking a similar content of the antibiological peptide, when the context allows.
• the term "bioactive" or “active” refers to the ability of an enzyme, in the context of an enzyme, to accelerate a chemical reaction differentiating such activity from a like ability of a composition, an article, a method, etc. that does not comprise an enzyme to accelerate a chemical reaction.
• a surface treatment comprising lysozyme that displays lysozyme activity comprises an active enzyme (e.g., a lysozyme EC 3.2.1.17).
• a surface treatment comprising a lipolytic enzyme and a non-enzyme catalyst of a lipolytic reaction that demonstrates an improved lipolytic activity (e.g., a statistically difference in activity; an improvement in a property as scored, such as from "good” to "excellent", by an assay; etc.) relative to a similar surface treatment lacking an active lipolytic enzyme.
• An "effective amount” refers to a concentration of component of a material formulation and/or the material formulation itself (e.g., an antifungal peptide, a biomolecular composition) capable of exerting a desired effect (e.g., an antifungal effect).
• an enzyme may comprise a simple enzyme, a complex enzyme, or a combination thereof.
• a "simple enzyme” comprises an enzyme wherein a chemical property of one or more moiety(s) found in its amino acid sequence produces enzymatic activity.
• a “complex enzyme” comprises an enzyme whose catalytic activity functions when an apo-enzyme combines with a prosthetic group, a co-factor, or a combination thereof.
• An "apo-enzyme” comprises a proteinaceous molecule and may be relatively catalytically inactive without a prosthetic group and/or a co- factor.
• a "prosthetic group” or “co-enzyme” comprises a non-proteinaceous molecule that may be attached to the apo-enzyme to produce a catalytically active complex enzyme.
• a "holo-enzyme” comprises a complex enzyme comprising an apo-enzyme and a co-enzyme.
• a "co-factor” comprises a molecule that acts in combination with the apo-enzyme to produce a catalytically active complex enzyme.
• a prosthetic group comprises one or more bound metal atoms, a vitamin derivative, or a combination thereof.
• Examples of a metal atom that may be used in a prosthetic group and/or a co-factor include Ca, Cd, Co, Cu, Fe, Mg, Mn, Ni, Zn, or a combination thereof.
• the metal atom comprises an ion, such as Ca 2+ , Cd 2+ , Co 2+ , Cu 2+ , Fe +2 , Mg 2+ , Mn 2+ , Ni 2+ , Zn 2+ , or a combination thereof.
• a "metalloenzyme” comprises a complex enzyme comprising an apo- enzyme and a prosthetic group, wherein the prosthetic group comprises a metal atom.
• a "metal activated enzyme” comprises a complex enzyme comprising an apo-enzyme and a co-factor, wherein the co-factor comprises a metal atom.
• a chemical that is capable of binding and/or is bound by a biomolecule may be known herein as a "ligand.”
• ligand e.g., a proteinaceous molecule
• binding refers to a physical contact between the biomolecule (e.g., a proteinaceous molecule) at a specific region of the biomolecule (e.g., a proteinaceous molecule) and the ligand in a reversible fashion.
• Examples of a binding interaction include such interactions as a ligand known as an "antigen" binding an antibody, a ligand binding a receptor, a ligand binding an enzyme, a ligand binding a peptide and/or a polypeptide, and the like.
• a portion of the biomolecule (e.g., a proteinaceous molecule) wherein ligand binding occurs may be known herein as a "binding site.”
• a ligand acted upon by an enzyme in an accelerated chemical reaction may be known herein as a "substrate.”
• a contact between the enzyme and a substrate in a fashion suitable for the accelerated chemical reaction to proceed may be known herein as "substrate binding.”
• a portion of the enzyme involved in the chemical interactions that contributed to the accelerated chemical reaction may be known herein as an "active site.”
• a chemical that slows and/or prevents the enzyme from conducting the accelerated chemical reaction may be known herein as an "inhibitor.”
• inhibitor binding occurs at a binding site, an active site, or a combination thereof.
• an inhibitor's binding occurs without the inhibitor undergoing the chemical reaction.
• the inhibitor may also comprise a substrate such as in the case of an inhibitor that precludes and/or reduces the ability of the enzyme in catalyzing the chemical reaction of a target substrate for the period of time inhibitor binding occurs at an active site and/or a binding site.
• an inhibitor undergoes the chemical reaction at a slower rate relative to a target substrate.
• enzymes may be described by the classification system of The International Union of Biochemistry and Molecular Biology (“IUBMB").
• IUBMB The International Union of Biochemistry and Molecular Biology
• the IUBMB classifies enzymes by the type of reaction catalyzed and enumerates a sub-class by a designated enzyme commission number ("EC").
• an enzyme may comprise an oxidoreductase (EC 1 ), a transferase (EC 2), a hydrolase (EC 3), a lyase (EC 4), an isomerase (EC 5), a ligase (EC 6), or a combination thereof.
• An enzyme may be able to catalyze multiple reactions, and thus have activities of multiple EC classifications.
• a "moiety,” “group,” and/or “species” in the context of the field of chemistry refers to a chemical substructure that may be a part of a larger molecule.
• Examples of a moiety include an acid halide, an acid anhydride, an alcohol, an aldehyde, an alkane, an alkene, an alkyl halide, an alkyne, an amide, an amine, an arene, an aryl halide, a carboxylic acid, an ester, an ether, a ketone, a nitrile, a phenol, a sulfide, a sulfonic acid, a thiol, etc.
• An oxidoreductase catalyzes an oxido-reduction of a substrate, wherein the substrate comprises either a hydrogen donor and/or an electron donor.
• An oxidoreductase may be classified by the substrate moiety of the donor and/or the acceptor.
• Examples of an oxidoreductase include an oxidoreductase that acts on a donor CH-OH moiety, (EC 1.1 ); a donor aldehyde or a donor oxo moiety, (EC 1.2); a donor CH-CH moiety, (EC 1.3); a donor CH-NH 2 moiety, (EC 1.4); a donor CH-NH moiety, (EC 1.5); a donor nicotinamide adenine dinucleotide ("NADH") or a donor nicotinamide adenine dinucleotide phosphate ("NADPH”), (EC 1.6); a donor nitrogenous compound, (EC 1.7); a donor sulfur moiety, (EC 1.8); a donor heme moiety, (EC 1.9); a donor diphenol and/or a related moiety as donor, (EC 1.10); a peroxid
• a transferase catalyzes the transfer of a moiety from a donor compound to an acceptor compound.
• a transferase may be classified based on the chemical moiety transferred. Examples of a transferase include a transferase that catalyzes the transfer of an one-carbon moiety, (EC 2.1 ); an aldehyde and/or a ketonic moiety, (EC 2.2); an acyl moiety, (EC 2.3); a glycosyl moiety, (EC 2.4); an alkyl and/or an aryl moiety other than a methyl moiety, (EC 2.5); a nitrogenous moiety, (EC 2.6); a phosphorus-containing moiety, (EC 2.7); a sulfur-containing moiety, (EC 2.8); a selenium-containing moiety, (EC 2.9); or a combination thereof.
• a hydrolase catalyzes the hydrolysis of a chemical bond.
• a hydrolase may be classified based on the chemical bond cleaved or the moiety released or transferred by the hydrolysis reaction.
• Examples of a hydrolase include a hydrolase that catalyzes the hydrolysis of an ester bond, (EC 3.1 ); a glycosyl released/transferred moiety, (EC 3.2); an ether bond, (EC 3.3); a peptide bond, (EC 3.4); a carbon-nitrogen bond, other than a peptide bond, (EC 3.5); an acid anhydride, (EC 3.6); a carbon-carbon bond, (EC 3.7); a halide bond, (EC 3.8); a phosphorus-nitrogen bond, (EC 3.9); a sulfur-nitrogen bond, (EC 3.10); a carbon- phosphorus bond, (EC 3.11 ); a sulfur-sulfur bond, (EC 3.12); a carbon-sulfur bond, (
• Examples of an esterase include a carboxylic ester hydrolase (EC 3.1.1 ); a thioester hydrolase (EC 3.1.2); a phosphoric monoester hydrolase (EC 3.1.3); a phosphoric diester hydrolase (EC 3.1.1 ); a carboxylic ester hydrolase (EC 3.1.1 ); a thioester hydrolase (EC 3.1.2); a phosphoric monoester hydrolase (EC 3.1.3); a phosphoric diester hydrolase (EC
• Examples of a carboxylic ester hydrolase include a carboxylesterase (EC 3.1.1.1 ); an arylesterase (EC 3.1.1.2); a triacylglycerol ipase (EC 3.1.1.3); a phospholipase A2 (EC 3.1.1.4); a lysophospholipase (EC 3.1.1.5); an acetylesterase (EC 3.1.1.6); an acetylcholinesterase (EC 3.1.1.7); a cholinesterase (EC 3.1.1.8); a tropinesterase (EC 3.1.1.10); a pectinesterase (EC 3.1.1.1 1 ); a sterol esterase (EC 3.1.1.13); a chlorophyllase (EC 3.1.1.14); a L-arabinonolactonase (EC 3.1.1.15); a gluconolactonase (EC 3.1.1.17); an uronolact
• Examples of an enzyme that acts on a carbon-nitrogen bond, other than a peptide bond include an enzyme acting on a linear amide (EC 3.5.1 ); a cyclic amide (EC 3.5.2); a linear amidine (EC 3.5.3); a cyclic amidine (EC 3.5.4); a nitrile (EC 3.5.5); an other compound (EC 3.5.99); or a combination thereof.
• Examples of an enzyme that catalyzes a reaction on a carbon-nitrogen bond of a non-peptide linear amide include an asparaginase (EC 3.5.1.1 ); a glutaminase (EC 3.5.1.2); a ⁇ -amidase (EC 3.5.1.3); an amidase (EC 3.5.1.4); a urease (EC 3.5.1.5); a ⁇ -ureidopropionase (EC 3.5.1.6); a ureidosuccinase (EC 3.5.1.7); a formylaspartate deformylase (EC 3.5.1.8); an arylformamidase (EC 3.5.1.9); a formyltetrahydrofolate deformylase (EC 3.5.1.10); a penicillin amidase (EC 3.5.1.11 ); a biotinidase (EC 3.5.1.12); an aryl-acylamidase (EC 3.5.1.13); an aminoacylase (EC 3.5.1.10)
• 5842-02103 30 deacetylase (EC 3.5.1.48); a formamidase (EC 3.5.1.49); a pentanamidase (EC 3.5.1.50); a A- acetamidobutyryl-CoA deacetylase (EC 3.5.1.51 ); a peptide-N4-(N-acetyl- ⁇ -glucosaminyl)asparagines amidase (EC 3.5.1.52); a N-carbamoylputrescine amidase (EC 3.5.1.53); an allophanate hydrolase (EC 3.5.1.54); a long-chain-fatty-acyl-glutamate deacylase (EC 3.5.1.55); a N,N-dimethylformamidase (EC 3.5.1.56); a tryptophanamidase (EC 3.5.1.57); a N-benzyloxycarbonylglycine hydrolase (EC 3.5.1.58); a N- carbamoylsarco
• Examples of an enzyme that catalyzes a reaction on a carbon-nitrogen bond of a non-peptide cyclic amide include a barbiturase (EC 3.5.2.1 ); a dihydropyrimidinase (EC 3.5.2.2); a dihydroorotase (EC 3.5.2.3); a carboxymethylhydantoinase (EC 3.5.2.4); an allantoinase (EC 3.5.2.5); a ⁇ -lactamase (EC 3.5.2.6); an imidazolonepropionase (EC 3.5.2.7); a 5-oxoprolinase (ATP-hydrolysing) (EC 3.5.2.9); a creatininase (EC 3.5.2.10); a L-lysine-lactamase (EC 3.5.2.1 1 ); a 6-aminohexanoate-cyclic-dimer hydrolase (EC 3.5.2.12); a 2,5-di
• Examples of an enzyme that acts on an acid anhydride(EC 3.6) include an enzyme acting on: a phosphorus-containing anhydride (EC 3.6.1 ); a sulfonyl-containing anhydride (EC 3.6.2); an acid anhydride catalyzing transmembrane movement of a substance (EC 3.6.3); an acid anhydride involved in cellular and/or subcellular movement (EC 3.6.4); a GTP involved in cellular and/or subcellular movement (EC 3.6.5); or a combination thereof.
• a lyase catalyzes the cleavage of a chemical bond by reactions other than hydrolysis and/or oxidation.
• a lyase may be classified based on the chemical bond cleaved. Examples of a lyase include a
• 5842-02103 31 lyase that catalyzes the cleavage of a carbon-carbon bond, (EC 4.1 ); a carbon-oxygen bond, (EC 4.2); a carbon-nitrogen bond, (EC 4.3); a carbon-sulfur bond, (EC 4.4); a carbon-halide bond, (EC 4.5); a phosphorus-oxygen bond, (EC 4.6); an other lyase, (EC 4.99); or a combination thereof.
• An isomerase catalyzes a change within one molecule.
• an isomerase examples include a racemase and/or an epimerase, (EC 5.1 ); a c/s-frans-isomerase, (EC 5.2); an intramolecular isomerase, (EC 5.3); an intramolecular transferase, (EC 5.4); an intramolecular lyase, (EC 5.5); an other isomerases, (EC 5.99); or a combination thereof.
• a ligase catalyzes the formation of a chemical bond between two substrates with the hydrolysis of a diphosphate bond of a triphosphate such as ATP.
• a ligase may be classified based on the chemical bond created. Examples of a lyase include a ligase that form a carbon — oxygen bond, (EC 6.1 ); a carbon — sulfur bond, (EC 6.2); a carbon — nitrogen bond, (EC 6.3); a carbon — carbon bond, (EC 6.4); a phosphoric ester bond, (EC 6.5); or a combination thereof.
• An enzyme in various embodiments comprises a lipolytic enzyme, which as used herein comprises an enzyme that catalyzes a reaction or series of reactions on a lipid substrate.
• a lipolytic enzyme produces one or more products that are more soluble in a liquid component such as a polar liquid component (e.g., water); absorb easier into a material formulation than the lipid substrate.
• the enzyme catalyzes hydrolysis of a fatty acid bond (e.g., an ester bond).
• the products produced comprise a carboxylic acid moiety (e.g., a free fatty acid), an alcohol moiety (e.g., a glycerol), or a combination thereof.
• at least one product may be relatively more soluble in an aqueous media (e.g., a water comprising detergent) than the substrate.
• a "lipid" comprises a hydrophobic and/or an amphipathic organic molecule extractable with a non-aqueous solvent.
• lipid incude a triglyceride; a diglyceride; a monoglyceride; a phospholipid; a glycolipid (e.g., galactolipid); a steroid (e.g., cholesterol); a wax; a fat- soluble vitamin (e.g., vitamin A, D, E, K); a petroleum based material, such as, for example, a hydrocarbon composition such as gasoline, a crude petroleum oil, a petroleum grease, etc.; or a combination thereof.
• a lipid may comprise a combination (mixture) of lipids, such as a grease comprising both a fatty acid based lipid and a petroleum based lipid.
• a lipid may comprise an apolar ("nonpolar") lipid (e.g., a hydrocarbons, a carotene), a polar lipid (e.g., triacylglycerol, a retinol, a wax, a sterol), or a combination thereof.
• a polar lipid may possess partial solubility in water (e.g., a lysophospholipid). Because of the prevalence of these types of lipids in activities such as, for example, a restaurant food preparation and a counterpart use in a household application, a material formulation may be formulated to comprise one or more lipolytic enzymes to promote lipid removal from a material formulation contaminated with a lipid in these and/or other environments.
• Lipolytic enzymes have been identified in cells across the phylogenetic categories, and purified for analysis and/or use in commercial applications (Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes," 1974). Further, numerous nucleotide sequences for lipolytic enzymes have been isolated, the encoded protein sequence determined, and in many cases the nucleotide sequences recombinantly
• 5842-02103 32 expressed for high level production of a lipolytic enzyme (e.g., a lipase), particularly for isolation, purification and subsequent use in an industrial/commercial application ["Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.) 1994].
• a lipolytic enzyme e.g., a lipase
• alpha/beta hydrolase Many lipolytic enzymes are classified as an alpha/beta fold hydrolase (“alpha/beta hydrolase"), due to a structural configuration generally comprising an 8 member beta pleated sheet, where many sheets are parallel, with several alpha helices on both sides of the sheet.
• a lipolytic enzyme's amino acid sequence commonly comprises Ser, Glu/Asp, His active site residues (e.g., Ser152, Asp176, and His263 by human pancreatic numbering).
• the Ser may be comprised in a GXSXG substrate binding consensus sequence for many types of lipolytic enzymes, with a GGYSQGXA sequence being present in a cutinase.
• the active site serine may be at a turn between a beta-strand and an alpha helix, and these lipolytic enzymes are classified as serine esterases.
• a substitution at the 1 st position GIy e.g., Thr
• GIy e.g., Thr
• a Pro residue may be found at the residues 1 and 4 down from the Asp, and the His may be typically within a CXHXR sequence.
• a lipolytic enzyme generally comprises an alpha helix flap (a.k.a.
• lid region (around amino acid residues 240-260 by human pancreatic lipase numbering) covering the active site, with a conserved tryptophan in this region in proximity of the active site serine in many lipolytic enzymes [In “Advances in Protein Chemistry, Volume 45 Lipoproteins, Apolipoproteins, and Lipases.” (Anfinsen, C. B., Edsall, JT. , Richards, Frederic, R. M., Eisenberg, D. S., and Schumaker, V.N. Eds.) Academic Press, Inc., San Diego, California, pp. 1-152, 1994; “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 1-243-270, 337-354, 1994.]. Any such alpha/beta hydrolase, particularly one possessing a lipolytic activity, may be used.
• a lipolytic alpha/beta hydrolase's catalysis usually depends upon and/or becomes stimulated by interfacial activation, which refers to the contact of such an enzyme with an interface where two layers of materials with differing hydrophobic/hydrophilic character meet, such as a water/oil interface of a micelle and/or an emulsion, an air/water interface, and/or a solid carrier/organic solvent interface of an immobilized enzyme.
• Interfacial activation may result from lipid substrate forming an ordered confirmation in a localized hydrophobic environment, so that the substrate more easily binds a lipolytic enzyme than a lipid substrate's conformation in a hydrophilic environment.
• Cutinase comprises a lipolytic alpha/beta hydrolase that may be not substantially enhanced by interfacial activation.
• a cutinase generally lacks a Nd, and may possess the ability to bury an aliphatic fatty acid chain in the active site cleft without the charge effects of an interface prompting a conformational change in the enzyme [In "Engineering of/with Lipases” (F. Xavier Malcata., Ed.), pp. 125-142, 1996].
• a lipolytic enzyme contemplated for use hydrolyzes an ester of a glycerol based lipid (e.g., a triglyceride, a phospholipid).
• Glycerol typically comprises a naturally produced alcohol having a 3 carbon backbone with 3 alcohol moieties (positions 1 , 2, and 3). One or more of these positions are often esterified with a fatty acid in many naturally produced and/or synthetic lipids.
• Common examples of a triglyceride include a fat, which comprises a solid at room temperature; or an oil, which comprises a liquid at room temperature.
• a "fatty acid” (“FA”) refers to saturated, monounsaturated, or polyunsaturated aliphatic acid.
• a short chain fatty acid comprises about 2 to about 6 carbons (“C2 to C6”) in
• a medium chain fatty acid comprises about 8 to about 10 carbons in the acid and main chain; and a long chain fatty acid comprises about 12 or more carbons (e.g., 12 to about 60 carbons).
• a main chain carbons substituted by another element (e.g., oxygen).
• a short chain fatty acid generally possesses solubility in water and other polar solvents, but solubility tends to decrease with increased carbon chain length in polar solvents, though solubility in non-polar solvents tends to increase.
• a common solvent for a medium and/or a long chain fatty acid includes an acetone, an acetic acid, an acetonitrile, a benzene, a chloroform, a chyclohexane, an alcohol (e.g., ethanol, methanol), or a combination thereof.
• a lipolytic enzyme hydrolyzes an ester at one or more of glycerol's alcohol position(s) (e.g., a 1 , 3 lipase), though a lipolytic enzyme often hydrolyzes a non-glycerol ester of an alcohol other than glycerol.
• a naturally produced wax comprises a fatty acid ester of ethylene glycol, which has a 2 carbon backbone and 2 alcohol moieties, where one or both of the alcohol moiety(s) are esterified with a fatty acid.
• a fatty acid forms an ester with an alcohol group of a non-glycerol and/or an ethylene glycol molecule, such as sterol lipid (e.g., cholesterol), and an enzyme that catalyzes the formation and/or cleavage of that linkage may be considered to comprise a lipolytic enzyme (e.g., a sterol hydrolase).
• one or more hydroxyl moiety(s) of an alcohol comprise a fatty acid and one or more hydroxyl moiety(s) comprise an ester of a chemical structure other than a fatty acid
• an enzyme that catalyzes hydrolysis and/or cleavage of the non-FA linkage comprises a lipolytic enzyme (e.g., a phospholipase).
• a phospholipid (“phosphoglyceride”) comprises a diglyceride with the 3 rd remaining position esterified to a phosphate group.
• the phosphate moiety may be esterified to a hydrophilic moiety such as a polyhydroxyl alcohol (e.g., a glycerol, an inositol) and/or an amino alcohol (e.g., a choline, a serine, an ethanolamine).
• a hydrophilic moiety such as a polyhydroxyl alcohol (e.g., a glycerol, an inositol) and/or an amino alcohol (e.g., a choline, a serine, an ethanolamine).
• Examples of a phospholipid includes a phosphatidic acid (“PA”), a phosphatidylcholine (“PC,” “lecithin”), a phosphotidyl ethanolamine (“PE,” “cephalin”), a phosphotidylglycerol (“PG”), a phosphotidylinositol (“Pl,” “monophosphoinositide”), a phosphotidylserine (“PE,” “serine”), a phosphotidylinositol 4,5-diphosphate (“PIP 2 ,” “triphosphoinositide”), a diphosphotidylglycerol (“DPG,” “cardiolipin”), or a combination thereof.
• PA phosphatidic acid
• PC phosphatidylcholine
• PE phosphotidyl ethanolamine
• PG phosphotidylglycerol
• Pl phosphotidylinositol
• an alcohol e.g., a glycerol, an ethylene glycol
• a lipolytic enzyme may act on that substrate to hydrolyze that linkage.
• sphingomyelin comprises a glycerol having a fatty acid amide bond and 2 phosphate ester bonds
• a lipolytic enzyme may cleave the amide linkage.
• An enzyme may be identified and referred to by the primary catalytic function (E. C. classification), but often catalyze another reaction, and examples of such an enzyme may be referred to herein (e.g., a carboxylesterase/lipase) based on the multiple activities.
• a material formulation comprising one or more enzymes lipolytic enzyme(s) may possess the ability to cleave (e.g., hydrolyze) all positions of an alcohol for ease of removal of the product(s) of the reaction.
• a multifunction enzyme may be used instead a plurality of enzymes to expand the range of different substrates that are acted upon, though a plurality of single and/or multifunctional enzymes may be used as well.
• degrading enzymes derived from a mesophile and an extremophile may be incorporated into a material formulation to expand the catalytic effectiveness against various substrates in differing temperature conditions experienced in an outdoor application and/or near a heat source.
• a lipolytic enzyme often produces a product that may be more aqueous soluble and/or removable after a single chemical reaction
• a series of enzyme reactions releases a fatty acid and/or degrades a lipid, such as in the case of a combination of a sphingomyelin phosphodiesterase that produces a ⁇ /-acylsphingosine from a sphingomyelin phospholipid, followed by a ceramidase hydrolyzing an amide bond in a ⁇ /-acylsphingosine to produce a free fatty acid and a sphingosine.
• a lipolytic enzyme comprises a hydrolase.
• a hydrolase generally comprises an esterase, a ceramidase (EC 3.5.1.23), or a combination thereof.
• Examples of an esterase comprise those identified by enzyme commission number (EC 3.1 ): a carboxylic ester hydrolase, (EC 3.1.3), a phosphoric monoester hydrolase (EC 3.1.3), a phosphoric diester hydrolase (EC 3.1.4), or a combination thereof.
• a carboxylic ester hydrolase catalyzes the hydrolytic cleavage of an ester to produce an alcohol and a carboxylic acid product.
• a phosphoric monoester hydrolase catalyzes the hydrolytic cleavage of an O-P ester bond.
• a "phosphoric diester hydrolase” catalyzes the hydrolytic cleavage of a phosphate group's phosphorus atom and two other moieties over two ester bonds.
• a “ceramidase” hydrolyzes the N-acyl bond of ceramide to release a fatty acid and sphingosine.
• a lipolytic esterase and a ceramidase include a carboxylesterase (EC 3.1.1.1 ), a lipase (EC 3.1.1.3), a lipoprotein lipase (EC 3.1.1.34), an acylglycerol lipase (EC 3.1.1.23), a hormone-sensitive lipase (EC 3.1.1.79), a phospholipase A 1 (EC 3.1.1.32), a phospholipase A 2 (EC 3.1.1.4), a phosphatidylinositol deacylase (EC 3.1.1.52), a phospholipase C (EC 3.1.4.3), a phospholipase D (EC 3.1.4.4), a phosphoinositide phospholipase C (EC 3.1.4.11 ), a phosphatidate phosphata
• Carboxylesterases [0100] Carboxylesterase (EC 3.1.1.1 ) has been also referred to in that art as “carboxylic-ester hydrolase,” “ali-esterase,” “B-esterase,” “monobutyrase,” “cocaine esterase,” “procaine esterase,” “methylbutyrase,” “vitamin A esterase,” “butyryl esterase,” “carboxyesterase,” “carboxylate esterase,” “carboxylic esterase,” “methylbutyrate esterase,” “triacetin esterase,” “carboxyl ester hydrolase,” “butyrate esterase,” “methylbutyrase,” “ ⁇ -carboxylesterase,” “propionyl esterase,” “nonspecific carboxylesterase,” “esterase D,”
• carboxylic ester + H 2 O an alcohol + a carboxylate.
• the carboxylate comprises a fatty acid.
• the fatty acid comprises about 10 or less carbons, to differentiate its preferred substrate and classification from a lipase, though a carboxylesterase (e.g., a microsome carboxylesterase) may possess the catalytic activity of an arylesterase, a lysophospholipase, an acetylesterase, an acylglycerol lipase, an acylcarnitine hydrolase, a palmitoyl-CoA hydrolase, an amidase, an aryl-acylamidase, a vitamin A esterase, or a combination thereof.
• a carboxylesterase e.g., a microsome carboxylesterase
• a carboxylesterase may possess the catalytic activity of an arylesterase, a lysophospholipase, an acetylesterase, an acylglycerol lipase, an acylcarnitine hydrolase, a palmitoyl-
• Carboxylesterase producing cells and methods for isolating a carboxylesterase from a cellular material and/or a biological source have been described [see, for example, Augusteyn, R. C. et al., 1969; Horgan, D. J., et al., 1969; In “Upases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type carboxylesterase and/or a functional equivalent amino acid sequence for producing a carboxylesterase and/or a functional equivalent include Protein database bank entries: 1AUO, 1AUR, 1CI8, 1CI9, 1 EVQ, UJI, 1 K4Y, 1 L7Q, 1 L7R, 1 MX1 , 1 MX5, 1 MX9, 1QZ3, 1 R1 D, 1TQH, 1 U4N, 1YA4, 1YA8, 1YAH, 1YAJ, 2C7B, 2DQY, 2DQZ, 2DR0, 2FJ0, 2H11, 2H7C, 2HM7, 2HRQ, 2HRR, 2JEY, 2JEZ, 2JF0, 2O7R, 207V, 2OGS, 2OGT, and/or 2R11.
• Protein database bank entries 1AUO, 1AUR, 1CI8, 1CI9, 1 EVQ, UJI, 1 K4Y, 1 L7
• Lipase (EC 3.1.1.3) has been also referred to in that art as “triacylglycerol acylhydrolase,” “triacylglycerol lipase,” “triglyceride lipase,” “tributyrase,” “butyrinase,” “glycerol ester hydrolase,” “tributyrinase,” “Tween hydrolase,” “steapsin,” “triacetinase,” “tributyrin esterase,” “Tweenase,” “amno N-AP,” “Takedo 1969-4-9,” “Meito MY 30,” “Tweenesterase,” “GA 56,” “capalase L,” “triglyceride hydrolase,” “triolein hydrolase,” “tween-hydrolyzing esterase,” “amano CE,” “cacordase,” “triglyceridase,” “triacylglycerol ester hydrolase,” “amano P,”
• the carboxylate comprises a fatty acid.
• Lipase and/or co-lipase producing cells and methods for isolating a lipase and/or a co-lipase from a cellular material and/or a biological source have been described, [see, for example, Korn, E. D. and Quigley., 1957; Lynn, W. S. and Perryman, N. C,. 1960; Tani, T. and Tominaga, Y. J., 1991 ; Sugihara, A. et al., 1992; in "Methods and Molecular Biology, Volume 109 Lipase and Phospholipase Protocols.” (Mark Doolittle and Karen Reue, Eds.), pp.
• a lipase may often catalyze the hydrolysis of short and/or medium chain fatty acid(s) less than about 12 carbons ("12C"), but has a preference and/or specificity for about 12C or greater fatty acid(s).
• a lipolytic enzyme classified as a carboxylesterase prefers short and/or medium chain fatty acid(s), though some carboxylesterases may also hydrolyze esters of longer fatty acids.
• the chain length preference for a lipase may be applicable to the other lipolytic fatty acid esterase(s) and/or a ceramidase, other than a carboxylesterase unless otherwise noted.
• a lipase may be obtained from a commercial vendor, such as a type VII lipase from Candida rugosa (Sigma-Aldrich product no. L1754; >700 unit/mg solid; CAS No. 9001-62-1 ) comprising lactose; a Lipoase
• Novozymes Lipolase 100 L, Type EX
• An enzyme stabilizing compound such as a propylene glycol and/or a sucrose may promote a property such as enzyme activity/stability in a material formulation (e.g., a water-borne paint, a 2k epoxy system).
• a mammalian lipase may be classified into one of four groups: gastric, hepatic, lingual, and pancreatic, and has homology to lipoprotein lipase.
• a pancreatic lipase generally are inactivated by a bile salt, which comprise an amphiphilic molecule found in an animal intestine that may bind a lipid and confer a negative charge that inhibits a pancreatic lipase.
• a colipase comprises a protein that binds a pancreatic lipase and reactivates it in the presence of a bile salt [In "Engineering of/with Lipases" (F. Xavier Malcata., Ed.) p. 168, 1996].
• a co-lipase may be combined with a pancreatic lipase in a composition to promote a lipase's (e.g., a pancreatic lipase) activity.
• Structural information for a wild-type lipase and/or a functional equivalent amino acid sequence for producing a lipase and/or a functional equivalent include Protein database bank entries: 1AKN, 1 BU8, 1CRL, 1CUA, 1CUB, 1CUC, 1CUD, 1CUE, 1 CUF, 1CUG, 1CUH, 1CUI, 1CUJ, 1CUU, 1CUV, 1CUW, 1CUX, 1CUY, 1 CUZ, 1CVL, 1 DT3, 1 DT5, 1 DTE, 1 DU4, 1 EIN, 1 ETH, 1 EX9, 1 F6W, 1 FFA, 1 FFB, 1 FFC, 1 FFD, 1 FFE, 1GPL, 1GT6, 1GZ7, 1 HLG, 1 HPL, 1 HQD, 116W 1 1 ISP, 1JI3, UMY, 1 K8Q, 1 KU0, 1 LBS, 1 LBT, 1 LGY, 1 LLF, 1 LPA
• Lipoprotein lipase (EC 3.1.1.34) has been also referred to in that art as "triacylglycero-protein acylhydrolase,” “clearing factor lipase,” “diglyceride lipase,” “diacylglycerol lipase,” “postheparin esterase,” “diglyceride lipase,” “postheparin lipase,” “diacylglycerol hydrolase,” and/or “lipemia-clearing factor.”
• a lipoprotein lipase's biological function comprises hydrolyzing a triglyceride found in an animal lipoprotein.
• a protein such as apolipoprotein may be combined with a lipoprotein lipase.
• Lipoprotein lipase producing cells and methods for isolating a lipoprotein lipase from a cellular material and/or a biological source have been described, [see, for example, Egelrud, T. and Olivecrona, T., 1973; Greten, H.
• Acylglycerol lipase (EC 3.1.1.23) has been also referred to in that art as “glycerol-ester acylhydrolase,” “monoacylglycerol lipase,” “monoacylglycerolipase,” “monoglyceride lipase,” “monoglyceride hydrolase,” “fatty acyl monoester lipase,” “monoacylglycerol hydrolase,” “monoglyceridyllipase,” and/or “monoglyceridase.”
• Acylglycerol lipase catalyzes a glycerol monoester's hydrolysis, particularly a fatty acid ester's hydrolysis.
• a hormone-sensitive lipase generally may be also active against a steroid fatty acid ester and/or a retinyl ester, and/or has a preference for a 1- or a 3-ester bond of an acylglycerol substrate.
• Hormone-sensitive lipase producing cells and methods for isolating a hormone-sensitive lipase from a cellular material and/or a biological source have been described, [see, for example, Tsujita, T.
• Phospholipase A 1 (EC 3.1.1.32) has been also referred to in that art as "phosphatidylcholine 1- acylhydrolase.”
• a phospholipases A 1 substrate's specificity may be broader
• Phospholipase A 1 producing cells and methods for isolating a phospholipase A 1 from a cellular material and/or a biological source have been described [see, for example, Gatt, S., 1968; van den Bosch, H., et al., 1974; In “Upases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp.243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type phospholipase A 1 and/or a functional equivalent amino acid sequence for producing a phospholipase A 1 and/or a functional equivalent include Protein database bank entries: 1FW2, 1FW3, 1ILD, 1ILZ, 1IM0, 1QD5, and/or 1QD6.
• Phospholipase A 2 (EC 3.1.1.4) has been also referred to in that art as "phosphatidylcholine 2- acylhydrolase,” “lecithinase A,” “phosphatidase,” and/or “phosphatidolipase,” ad “phospholipase A.”
• a phospholipases A 2 also catalyzes reactions on a phosphatidylethanolamine, a choline plasmalogen and/or a phosphatide, and/or acts on a 2-position ester bond.
• Ca 2+ generally improves enzyme function.
• Phospholipase A 2 producing cells and methods for isolating a phospholipase A 2 from a cellular material and/or a biological source have been described, [see, for example, Saito, K. and Hanahan, D.J., 1962; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp.243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type phospholipase A 2 and/or a functional equivalent amino acid sequence for producing a phospholipase A 2 and/or a functional equivalent include Protein database bank entries: 1A2A, 1A3D, 1A3F, 1AE7, 1AOK, 1AYP, 1B4W, 1BBC, 1BCI, 1BJJ 1BK9, 1BP2, 1BPQ, 1BUN, 1BVM, 1C1J, 1C74, 1CEH, 1CJY, 1CL51CLP, 1DB4, 1DB5, 1DCY, 1DPY, 1FAZ, 1FDK, 1FE5, 1FX9, 1FXF 1G0Z, 1G2X, 1G4I, 1GH4, 1GMZ, 1GOD, 1GP7, 1HN4, 1IJL, 1IRB 1IT4, 1IT5, 1J1A, UIA, ULT, 1JQ8, 1JQ9, 1KP4, 1KPM, 1
• Phosphatidylinositol deacylase (EC 3.1.1.52) has been also referred to in that art as "1- phosphatidyl-D-myo-inositol 2-acy I hydrolase,” “phosphatidylinositol phospholipase A 2 ,” and/or
• Phosphatidylinositol deacylase producing cells and methods for isolating a phosphatidylinositol deacylase from a cellular material and/or a biological source have been described, [see, for example, Gray, N. C. C. and Strickland, K.P., 1982; Gray, N. C. C. and Strickland, K.
• Phospholipases C [0112] Phospholipase C (EC 3.1.4.3) has been also referred to in that art as "phosphatidylcholine cholinephosphohydrolase,” “lipophosphodiesterase I,” “lecithinase C,” “Clostridium welchii ⁇ -toxin,” “Clostridium oedematiens ⁇ - and ⁇ -toxins,” “lipophosphodiesterase C,” “ phosphatidase C,” “heat-labile hemolysin,” and/or “ ⁇ -toxin.”
• a bacterial phospholipase C may have activity against sphingomyelin and phosphatidylinositol.
• Phospholipase C producing cells and methods for isolating a phospholipase C from a cellular material and/or a biological source have been described [see, for example, Sheiknejad, R. G. and Srivastava, P. N., 1986; Takahashi, T., et al., 1974; In “Upases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G.
• Structural information for a wild-type phospholipase C and/or a functional equivalent amino acid sequence for producing a phospholipase C and/or a functional equivalent include Protein database bank entries: 1AH7, 1 CA1 , 1 GYG, 1 IHJ, 1OLP, 1 P5X, 1 P6D, 1 P6E, 1QM6, 1 QMD, 2FFZ, 2FGN, and/or 2HUC.
• Phospholipase D producing cells and methods for isolating a phospholipase D from a cellular material and/or a biological source have been described, [see, for example, Astrachan, L. 1973; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type phospholipase D and/or a functional equivalent amino acid sequence for producing a phospholipase D and/or a functional equivalent include Protein database bank entries: 1 FOI, IVOR, 1V0S, 1V0T, 1V0U, 1V0V, 1V0W, 1V0Y, 2ZE4, and/or 2ZE9.
• Phosphoinositide phospholipase C (EC 3.1.4.11 ) has been also referred to in that art as "1- phosphatidyl-1 D-myo-inositol-4,5-bisphosphate inositoltrisphosphohydrolase," 'triphosphoinositide
• phosphodiesterase "phosphoinositidase C,” “1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase,” “monophosphatidylinositol phosphodiesterase,” “phosphatidylinositol phospholipase C,” “PI-PLC,” and/or "1- phosphatidyl-D-myo-inositol-4,5-bisphosphate inositoltrisphosphohydrolase.”
• a phosphoinositide phospholipase C may have activity against other phosphatidyl esters.
• a phosphoinositide phospholipase C producing cells and methods for isolating a phosphoinositide phospholipase C from a cellular material and/or a biological source have been described, [see, for example, Downes, CP. and Michell, R. H. 1981 ; Rhee, S. G. and Bae, Y. S. 1997; In “Upases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G.
• Structural information for a wild-type phosphoinositide phospholipase C and/or a functional equivalent amino acid sequence for producing a phosphoinositide phospholipase C and/or a functional equivalent include Protein database bank entries: 1 DJG, 1 DJH, 1 DJI, 1 DJW, 1 DJX, 1 DJY, 1 DJZ, 1 HSQ, UAD, 1 MAI, 1QAS, 1QAT, 1Y0M, 1YW0, 1YWP, 2C5L, 2EOB, 2FCI, 2FJL, 2FJU, 2HSP, 2ISD, 2K2J, 2PLD, 2PLE, and/or 2ZKM.
• Protein database bank entries 1 DJG, 1 DJH, 1 DJI, 1 DJW, 1 DJX, 1 DJY, 1 DJZ, 1 HSQ, UAD, 1 MAI, 1QAS, 1QAT, 1Y0M, 1YW0, 1YWP, 2C5L, 2EOB, 2
• Phosphatidate phosphatase (EC 3.1.3.4) has been also referred to in that art as "3-sn- phosphatidate phosphohydrolase,” “phosphatic acid phosphatase,” “acid phosphatidyl phosphatase,” and “phosphatic acid phosphohydrolase.”
• a phosphatidate phosphatase may have activity against other phosphatidyl esters.
• a phosphatidate phosphatase producing cells and methods for isolating a phosphatidate phosphatase from a cellular material and/or a biological source have been described, [see, for example, Smith, S. W., et al., 1957; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and
• Lysophospholipase (EC 3.1.1.5) has been also referred to in that art as "2-lysophosphatidylcholine acylhydrolase,” “lecithinase B,” “lysolecithinase,” “phospholipase B,” “lysophosphatidase,” “lecitholipase,” “phosphatidase B,” “lysophosphatidylcholine hydrolase,” “lysophospholipase A1 ,” “lysophopholipase L2,” “lysophospholipaseOtransacylase,” "neuropathy target esterase,” “NTE,” “NTE-LysoPLA,” and “NTE- lysophospholipase.”
• Lysophospholipase producing cells and methods for isolating a lysophospholipase from a cellular material and/or a biological source have been described, [see, for example, van den Bosch, H., et al., 1981 ; van den Bosch, H., et al., 1973; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein. Structural information for a wild-type lysophospholipase and/or a functional equivalent
• Protein database bank entries 1G86, 1 HDK, 1 IVN, UOO, URL, 1 LCL, 1QKQ, 1 U8U, 1V2G, 2G07, 2G08, 2G09, and/or 2G0A.
• Sterol esterase (EC 3.1.1.13) has been also referred to in that art as “lysosomal acid lipase,” “sterol esterase,” “cholesterol esterase,” “cholesteryl ester synthase,” “triterpenol esterase,” “cholesteryl esterase,” “cholesteryl ester hydrolase,” “sterol ester hydrolase,” “cholesterol ester hydrolase,” “cholesterase,” and/or “acylcholesterol lipase.”
• a sterol esterase may be active against a triglyceride as well.
• Cholesterol may comprise the substrate used to characterize a sterol esterase, though the enzyme also hydrolyzes a lipid vitamin ester (e.g., vitamin E acetate, vitamin E palmate, vitamin D 3 acetate).
• a bile salt often activates the enzyme.
• Sterol esterase producing cells and methods for isolating a sterol esterase from a cellular material and/or a biological source have been described [see, for example, Okawa, Y. and Yamaguchi, T., 1977; via recombinant expression in a baculoviral system in "Methods and Molecular Biology, Volume 109 Lipase and Phospholipase Protocols.” (Mark Doolittle and Karen Reue, Eds.), pp.
• Structural information for a wild- type sterol esterase and/or a functional equivalent amino acid sequence for producing a sterol esterase and/or a functional equivalent include Protein database bank entries: 1AQL and/or 2BCE.
• Galactolipase (EC 3.1.1.26) has been also referred to in that art as "1 ,2-diacyl-3- ⁇ -D-galactosyl-sn- glycerol acylhydrolase," "galactolipid lipase,” “polygalactolipase,” and/or “galactolipid acylhydrolase.”
• a galactolipase also may have activity against a phospholipid.
• the substrate for galactolipase comprises a galactolipid abundantly found in plant cells, and organisms that digest plant material (e.g., an animal) also produce this enzyme.
• Galactolipase producing cells and methods for isolating a galactolipase from a cellular material and/or a biological source have been described, [see, for example, Helmsing, 1969; Hirayama, O., et al., 1975 In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Sphingomyelin phosphodiesterase (EC 3.1.4.12) has been also referred to in that art as “sphingomyelinase,” “neutral sphingomyelinase,” “sphingomyelin cholinephosphohydrolase,” and/or “sphingomyelin N-acylsphingoosine-hydrolase.”
• a sphingomyelin phosphodiesterase also
• 5842-02103 42 may have activity against a phospholipid.
• Sphingomyelin phosphodiesterase producing cells and methods for isolating a sphingomyelin phosphodiesterase from a cellular material and/or a biological source have been described, [see, for example, Chatterjee, S. and Ghosh, N. 1989; Kanfer, J.N., et al., 1966; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Sphingomyelin phosphodiesterase D (EC 3.1.4.41 ) has been also referred to in that art as "sphingomyelin ceramide-phosphohydrolase" and/or "sphingomyelinase D.”
• a sphingomyelin phosphodiesterase D also may catalyze the reaction: hydrolyses 2-lysophosphatidylcholine to choline and 2-lysophosphatidate.
• Sphingomyelin phosphodiesterase D producing cells and methods for isolating a sphingomyelin phosphodiesterase D from a cellular material and/or a biological source have been described, [see, for example, Soucek, A. et al., 1971 ; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Ceramidase (EC 3.5.1.23) has been also referred to in that art as " ⁇ /-acylsphingosine amidohydrolase,” “acylsphingosine deacylase,” andor “glycosphingolipid ceramide deacylase sphingomyelin.”
• Ceramidase producing cells and methods for isolating a ceramidase from a cellular material and/or a biological source have been described [see, for example, E.
• Wax-ester hydrolase (EC 3.1.1.50) has been also referred to in that art as “wax-ester acylhydrolase,” and “jojoba wax esterase,” and/or "WEH.”
• a wax-ester hydrolase may also hydrolyze a long-chain acylglycerol.
• Wax-ester hydrolase producing cells and methods for isolating a wax- ester hydrolase from a cellular material and/or a biological source have been described, [see, for example, Huang, A.H.C.
• Fatty-acyl-ethyl-ester synthase (EC 3.1.1.67) has been also referred to in that art as "long-chain- fatty-acyl-ethyl-ester acylhydrolase," and/or "FAEES.”
• Fatty-acyl-ethyl-ester synthase producing cells and methods for isolating a fatty-acyl-ethyl-ester synthase from a cellular material and/or a biological source have been described [see, for example, Mogelson, S. and Lange, L. G. 1984; In “Upases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• Retinyl-palmitate esterase (EC 3.1.1.21 ) has been also referred to in that art as “retinyl-palmitate palmitohydrolase,” “retinyl palmitate hydrolase,” “retinyl palmitate hydrolyase,” and/or “retinyl ester hydrolase.”
• a retinyl-palmitate esterase may also hydrolyze a long-chain acylglycerol.
• Retinyl-palmitate esterase producing cells and methods for isolating a retinyl-palmitate esterase from a cellular material and/or a biological source have been described, [see, for example, T. et al., 2005; Gao, J. and Simon, 2005; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes," 1974], and may be used in conjunction with the disclosures herein.
• 11-c/s-retinyl-palmitate hydrolase (EC 3.1.1.63) has been also referred to in that art as "11-c/s- retinyl-palmitate acylhydrolase,” “1 1-cis-retinol palmitate esterase,” and/or "RPH.”
• 11-c/s-retinyl- palmitate hydrolase producing cells and methods for isolating a 1 1-c/s-retinyl-palmitate hydrolase from a cellular material and/or a biological source have been described, [see, for example, Blaner, W.S., et al., 1987; Blaner, W.S., et al., 1984; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• / ⁇ //-frans-retinyl-palmitate hydrolase (EC 3.1.1.64) has been also referred to in that art as "all-trans- retinyl-palmitate acylhydrolase.”
• a detergent generally promotes this enzyme's activity.
• AII- frans-retinyl-palmitate hydrolase producing cells and methods for isolating an / ⁇ //-frans-retinyl-palmitate hydrolase from a cellular material and/or a biological source have been described, [see, for example, Blaner, W.S., Das, et al., 1987; In “Lipases their Structure, Biochemistry and Application” (Paul Woolley and Steffen B. Peterson, Eds.), pp. 243-270, 1994; Brockerhoff, Hans and Jensen, Robert G. "Lipolytic Enzymes,” 1974], and may be used in conjunction with the disclosures herein.
• An acyloxyacyl hydrolase generally prefers a lipopolysaccharide from a Salmonella typhimurium and related organisms.
• an acyloxyacyl hydrolase may also possess a phospholipase, an acyltransferase, a phospholipase A 2 , a lysophospholipase, a phospholipase A 1 , a phosphatidylinositol deacylase, a diacylglycerol lipase, and/or a phosphatidyl lipase activity.
• An acyloxyacyl hydrolase generally prefers saturated Ci 2 -Ci 6 fatty acid esters.
• a petroleum hydrocarbon generally comprises a mixture of an alkane, a cycloalkane, an aromatic hydrocarbons, and/or a polycyclic aromatic hydrocarbon.
• This type of lipid differ from a lipid typically catalyzed by an alpha/beta hydrolase, in that a petroleum hydrocarbon lacks a chemical moiety such as an alcohol, an ester bond, and/or a carboxylic acid.
• Some microorganisms are capable of digesting one or more petroleum lipids, generally by adding one or more oxygen moiety(s) prior to integration of the lipid into cellular metabolic pathways. Often petroleum degradation occurs via a metabolic pathway comprising numerous enzymes and proteins, in some cases bound to various cellular membranes.
• a biomolecular composition may be prepared from a cell and/or a virus that produces such a petroleum lipolytic enzyme.
• a type of petroleum lipolytic enzyme comprises one that first adds, rather than modifies, a polar solvent solubility enhancing moiety (e.g., an alcohol, an acid), as that initial modification in a degradation pathway may be sufficient to improve solubility and/or an absorptive property of a target petroleum lipid.
• a polar solvent solubility enhancing moiety e.g., an alcohol, an acid
• a petroleum alkane substrate undergoes catalysis by a plurality of enzymes and/or proteins (e.g., an alkane hydroxylase, a rubredoxins, an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-CoA synthetase) and proteins (e.g., an outer membrane protein, a methyl-accepting transducer protein), that convert the alkane into an aldehyde and an acid with the participation of additional enzymes and proteins not encoded by the operon.
• enzymes and/or proteins e.g., an alkane hydroxylase, a rubredoxins, an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-CoA synthetase
• proteins e.g., an outer membrane protein, a methyl-accepting transducer protein
• a membrane bound monooxygenase, a rubredioxin, and a soluble rubredioxin add an alcohol moiety to the petroleum alkane by shunting electrons through a NADH compound to a hydroxylase.
• These initial enzymatic activities that result in improvement of solubility by addition of an alcohol may be used to select an enzyme.
• the alcohol may be further catalyzed into an aldehyde, then an acid, before entering regular cellular metabolic pathways (e.g., energy production).
• Another example of petroleum degradation comprises a polycyclic aromatic hydrocarbon having oxygenated moiety(s) added by the enzymes and proteins expressed from the nahAaAbAcAdBFCED operon for naphthalene degradation.
• These enzymes and proteins include: a reductase (nahAa), a ferredoxin (nahAb), an iron sulfur protein large subunit (nahAc), an iron sulfur protein small subunit (nahAd), a cis- naphthalene dihydrodiol dehydrogenase (nahB), a salicyaldehyde dehydrogenase (nahF), a 1 ,2- dihydroxynaphthalene oxygenase (nahC), a 2-hydroxybenzalpyruvate aldolase (nahE), a 2- hydroxychromene-2-carboxylate isomerase (nahD).
• a reductase nahAa
• nahAb ferredoxin
• nahAc iron sulfur protein large subunit
• the nahAa to nahAd genes encode a naphthalene dioxygenase.
• Pseudomonas putida strains may also have the salicylate degradation pathway, which includes the following enzymes: a salicylate hydroxylase (nahG), a chloroplast-type ferredoxin (nahf), a catechol oxygenase (nahH), a 2-hydroxymuconic semialdehyde dehydrogenase (nahl), a 2-hydroxymuconic semialdehyde dehydrogenase (nahN), a 2-oxo-4-pentenoate hydratase (nahL), a 4-hydroxy-2-oxovalerate aldolase (nahO), an acetaldehyde dehydrogenase (nahM), a 4-oxalocrotonate decarboxylase (nahK), and/or a 2-hydroxymuconate tautomerase (nahJ). Both operons are regulated by salicy
• a plurality of petroleum lipolytic enzymes in a biomolecular composition e.g., a plurality of cells that act one or more petroleum substrates, a plurality of semipurified or purified petroleum lipolytic enzymes, etc.
• conversion of the petroleum may occur through a plurality of the steps of a petroleum degradation pathway (e.g., via a cell-based composition comprising the degradation pathway's enzymes).
• a material formulation may comprise a lipolytic, a petroleum lipolytic enzyme, another enzyme, or a combination thereof.
• a lipolytic enzyme may be combined with another enzyme that either does not possess lipolytic activity or has such activity as an additional function, for the purpose to confer an additional catalytic and/or binding property to a material
• the additional enzyme comprises a hydrolase.
• An additional hydrolase may comprise an esterase.
• a type of an additional esterase comprises an esterase that catalyzes the hydrolysis of an organophosphorus compound. Examples of such an additional esterase include those identified by enzyme commission number EC 3.1.8, the phosphoric triester hydrolases.
• a phosphoric triester hydrolase catalyzes the hydrolytic cleavage of an ester from a phosphorus moiety. Examples of a phosphoric triester hydrolase include an aryldialkylphosphatase (EC 3.1.8.1 ), a diisopropyl- fluorophosphatase (EC 3.1.8.2), or a combination thereof.
• a material formulation with multiple biomolecule activities such as a dual enzymatic function (e.g., ease of lipid and organophosphorus compound removal/detoxification), may be of benefit depending upon the type of compounds that contact and/or are comprised as part of such an item.
• An "organophosphorus compound” comprises a phosphoryl center, and further comprises two or three ester linkages.
• the type of phosphoester bond and/or additional covalent bond at the phosphoryl center classifies an organophosphorus compound.
• the OP compound may be known as an "oxon OP compound” and/or “oxon organophosphorus compound.”
• the OP compound may be known as a "thion OP compound” and/or "thion organophosphorus compound.”
• Additional examples of bond-type classified OP compounds include a phosphonocyanate, which comprises a P-CN bond; a phosphoroamidate, which comprises a P-N bond; a phosphotriester, which comprises a P-O bond; a phosphodiester, which comprises a P-O bond; a phosphonofluoridate, which comprises a P-F bond; and a phosphonothiolate, which comprises a P-S bond.
• a "dimethyl OP compound” comprises two methyl moieties covalently bonded to the
• an OP compound comprises two ethoxy moieties covalently bonded to the phosphorus atom, such as, for example, a diazinon.
• an OP compound comprises an organophosphorus nerve agent and/or an organophosphorus pesticide.
• a "nerve agent” functions as an inhibitor of a cholinesterase, including but not limited to, an acetyl cholinesterase, a butyl cholinesterase, or a combination thereof.
• a nerve agent comprises an inhibitor of a cholinesterase (e.g., acetyl cholinesterase) whose catalytic activity may be used for health and survival in an animal, including a human.
• a cholinesterase e.g., acetyl cholinesterase
• Certain OP compounds are so toxic to humans that they have been adapted for use as chemical warfare agents, such as a tabun, a soman, a sarin, a cyclosarin, a GX, and/or a VX (e.g., a R-VX).
• a CWA may comprise an airborne form and such a formulation may be known herein as an "OP-nerve gas.”
• Examples of an airborne form include a gas, a vapor, an aerosol, a dust, or a combination thereof.
• Examples of an OP compound that may be formulated as an OP nerve gas include a tabun, a sarin, a soman, a VX, a cyclosarin, a GX, or a combination thereof.
• a CWA such as a persistent agent (e.g., a VX, a thickened soman)
• a persistent agent e.g., a VX, a thickened soman
• pose a threat through dermal absorption [In “Chemical Warfare Agents: Toxicity at Low Levels,” (Satu M. Somani and James A. Romano, Jr., Eds.) p. 414, 2001].
• a “persistent agent” comprises a CWA formulated [e.g., comprising a thickener such as one or more carbon based polymer(s)] to be less volatile (e.g., non-volatile) and thus remain as a solid and/or liquid (e.g., remain upon a contaminated surface) while exposed to the open air for more than about three hours.
• a persistent agent may convert from an airborne dispersal form to a solid and/or liquid residue on a surface, thus providing the opportunity to contact the skin of a human and/or other target.
• the toxicities for common OP chemical warfare agents after contact with skin are shown at Table 2.
• an OP compound may comprise a particularly poisonous organophosphorus nerve agent.
• a "particularly poisonous” agent possesses a LD 50 of 35 mg/kg or less for an organism after percutaneous ("skin") administration of the agent.
• a particularly poisonous OP nerve agent include a tabun, a sarin, a cyclosarin, a soman, a VX, a R-VX, or a combination thereof.
• a terms such as “detoxification,” “detoxify,” “detoxified,” “degradation,” “degrade,” and/or “degraded” refers to a chemical reaction of a compound that produces a chemical product less harmful to the health and/or survival of a target organism contacted with the chemical product relative to contact with the parent compound.
• OP compounds may be detoxified using chemical hydrolysis and/or through enzymatic hydrolysis (Yang, Y.-C. et al., 1992; Yang, Y.-C. et al., 1996; Yang, Y.-C. et al., 1990; LeJeune, K. E. et al., 1998a).
• the enzymatic hydrolysis comprises a specifically targeted reaction wherein the OP compound may be cleaved at the phosphoryl center's chemical bond resulting in predictable products that are acidic in nature but benign from a neurotoxicity perspective (Kolakowski, J. E. et al., 1997; Rastogi, V. K. et al., 1997; Dumas, D. P. et al., 1990; Raveh, L. et al., 1992).
• chemical hydrolysis may be much less specific, and in the case of VX may produce some quantity of byproducts that approach the toxicity of the intact agent (Yang, Y.-C. et al., 1996; Yang, Y.-C.
• an enzyme composition degrades a CWA, a particularly poisonous organophosphorus nerve agent, or a combination thereof, into product that may be not particularly poisonous.
• Many OP compounds are pesticides that are not particularly poisonous to a human, though they do possess varying degrees of toxicity to a human and/or another animal.
• Examples of an OP pesticide include a bromophos-ethyl, a chlorpyrifos, a chlorfenvinphos, a chlorothiophos, a chlorpyrifos-methyl, a coumaphos, a crotoxyphos, a crufomate, a cyanophos, a diazinon, a dichlofenthion, a dichlorvos, a dursban, an EPN, an ethoprop, an ethyl-parathion, an etrimifos, a famphur, a fensulfothion, a fenthion, a fenthrothion, an isofenphos, a jodfenphos, a leptophos-oxon, a malathion, a methyl-parathion, a mevinphos, a paraoxon, a parathion, a parathion-
• An aryldialkylphosphatase (EC 3.1.8.1 ) may be also known by its systemic name “aryltriphosphate dialkylphosphohydrolase” and various enzymes in this category have been known in the art by names such as “organophosphate hydrolase”; “paraoxonase”; “A-esterase”; “aryltriphosphatase”; “organophosphate esterase”; “esterase B1”; “esterase E4"; "paraoxon esterase”; “pirimiphos-methyloxon esterase”; "OPA anhydrase”; “organophosphorus hydrolase”; “phosphotriesterase”; “PTE”; “paraoxon hydrolase”; “OPH”; and/or “organophosphorus acid anhydrase.”
• Examples of an aryl dialkyl phosphate include an organophosphorus compound comprising a phosphonic acid ester, a phosphinic acid ester, or a combination thereof.
• Aryldialkylphosphatase producing cells and methods for isolating an aryldialkylphosphatase from a cellular material and/or a biological source have been described, [see, for example, Bosmann, H. B., 1972; and Mackness, M.I. et al., 1987.], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type aryldialkylphosphatase and/or a functional equivalent amino acid sequence for producing an aryldialkylphosphatase and/or a functional equivalent include Protein database bank entries: 1 EYW, 1 EZ2, 1 HZY, 1 I0B, 1 I0D, UGM, 1 P6B, 1 P6C, 1 P9E, 1 QW7,
• 5842-02103 49 1V04, 2D2G, 2D2H, 2D2J, 2O4M, 2O4Q, 2OB3, 2OQL, 2R1 K, 2R1 L, 2R1 M, 2R1 N, 2R1 P, 2VC5, 2VC7, 2ZC1 , 3C86, 3CAK, and/or 3E3H.
• Examples of an aryldialkylphosphatase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA - 5444(PON 1 ), 5445(PON2), 5446(PON3); PTR - 463547(PON 1 ), 463548(PON3), 463549(PON2); MCC - 699107, 699236, 699355(PON1 ); MMU - 18979(Pon1 ), 269823(Pon3),
• Organophosphorus hydrolase (E. C.3.1.8.1 ) has been also referred to in that art as "organophosphate-hydrolyzing enzyme,” “phosphotriesterase,” “PTE,” “organophosphate-degrading enzyme,” “OP anhydrolase,” “OP hydrolase,” “OP thiolesterase,” “organophosphorus triesterase,” “parathion hydrolase,” “paraoxonase,” “DFPase,” “somanase,” “VXase,” and/or “sarinase.” As used herein, this type of enzyme may be referred to herein as “organophosphorus hydrolase” and/or "OPH.” [0144] The initial discovery of OPH was from two bacterial strains from the closely related genera: Pseudomonas diminuta and Flavobacterium spp.
• Examples of an opd gene and a gene product that may be used include an Agrobacterium radiobacter P230 organophosphate hydrolase gene, opdA (Genbank accession no. AY043245; Entrez databank no. AAK85308); a Flavobacterium balustinum opd gene for parathion hydrolase (Genbank accession no. AJ426431 ; Entrez databank no. CAD19996); a Pseudomonas diminuta phosphodiesterase opd gene (Genbank accession no. M20392; Entrez databank no.
• AAA98299 Protein Data Bank entries UGM, 1 DPM, 1 EYW, 1 EZ2, 1 HZY, 1 IOB, 1 IOD, 1 PSC and 1 PTA); a Flavobacterium sp opd gene (Genbank accession no. M22863; Entrez databank no. AAA24931 ; ATCC 27551 ); a Flavobacterium sp. parathion hydrolase opd gene (Genbank accession no. M29593; Entrez databank no. AAA24930; ATCC 27551 ); or a combination thereof (Home, I. et al., 2002; Somara, S. et al., 2002; McDaniel, C. S.
• OPH OP detoxifying enzyme that has been often studied and characterized, with the enzyme obtained from Pseudomonas being the target of focus for many studies.
• This OPH was initially purified following expression from a recombinant baculoviral vector in insect tissue culture of the Fall Armyworm, Spodoptera frugiperda (Dumas, D. P. et al., 1989b). Purified enzyme preparations have been shown to be able to detoxify via hydrolysis a wide spectrum of structurally related insect and mammalian neurotoxins that
• this detoxification ability included a number of organophosphorofluoridate nerve agents such as a sarin and a soman. This was the first recombinant DNA construction encoding an enzyme capable of degrading these nerve gases. This enzyme was capable of degrading the common organophosphorus insecticide analog (paraoxon) at rates exceeding 2 x 10 7 M “1 (mole enzyme) "1 , which may be equivalent to the catalytically efficient enzymes observed in nature.
• the purified enzyme preparations are capable of detoxifying a sarin and the less toxic model mammalian neurotoxin 0,0-diisopropyl phosphorofluoridate ("DFP") at the equivalent rates of 50-60 molecules per molecule of enzyme-dimer per second.
• DFP mammalian neurotoxin 0,0-diisopropyl phosphorofluoridate
• the enzyme may hydrolyze a soman and a VX at approximately 10% and 1 % of the rate of a sarin, respectively.
• the breadth of substrate utility e.g., a V agent, a sarin, a soman, a tabun, a cycosarin, an OP pesticide
• the efficiency for the hydrolysis exceeds the known abilities of other prokaryotic and eukaryotic organophosphorus acid anhydrases, and this detoxification may be due to a single enzyme rather than a family of related, substrate-limited proteins.
• the X-ray crystal structure of Pseudomonas OPH has been determined (Benning, M. M. et al., 1994; Benning, M. M. et al., 1995; Vanhooke, J. L. et al., 1996).
• OPH monomer's active site binds two atoms of Zn 2+ ; however, OPH may be prepared wherein Co 2+ replaces Zn 2+ , which enhances catalytic rates.
• Examples of the catalytic rates (k cat ) and specificities (k cat /K m ) for Co 2+ substituted OPH against various OP compounds are shown at Table 3 below.
• Wild-type Zn OPH was used in obtaining these kinetic parameters; diSioudi, B. et al., 1999a; Kolakoski, J. E. et al., 1997; c Rastogi, V. K. et al., 1997; d Raveh, L. et al., 1992.
• the phosphoryl center of OP compounds is chiral, and Pseudomonas OPH preferentially binds and/or cleaves S p enantiomers over R 9 enantiomers of the chiral phosphorus in various substrates by a ratio of about 10:1 to about 90:1 (Chen-Goodspeed, M. et al., 2001a; Hong, S.-B. and Raushel, F. M., 1999a; Hong, S.-B. and Raushel, F. M., 1999b).
• a CWA such as a VX, a sarin, and/or a soman are usually prepared and used as a mixture of sterioisomers of varying toxicity, with VX and sarin having two enantiomers each, with the chiral center around the phosphorus of the cleavable bond.
• Soman possesses four enantiomers, with one chiral center based on the phosphorus and an additional chiral center based on a pinacolyl moiety [In "Chemical Warfare Agents: Toxicity at Low Levels" (Satu M. Somani and James A. Romano, Jr., Eds.) pp 26-29, 2001 ; Li, W.-S. et al., 2001 ; Yang, Y.-C. et al., 1992; Benshop, H. P. et al.,
• the S P enantiomer of sarin may be about 10 4 times faster in inactivating acetylcholinesterase than the Rp enantiomer (Benschop, H. P. and De Jong, L. P. A. 1988), while the two S p enantiomers of soman may be about 10 5 times faster in inactivating acetylcholinesterase than the R P enantiomers (Li, W.-S. et al., 2001 ; Benschop, H. P. et al., 1984). Wild-type organophosphorus hydrolase seems to have greater specificity for the less toxic enantiomers of sarin and soman.
• OPH may be about 9-fold faster cleaving an analog of the R P enantiomer of sarin relative to an analog of the S p enantiomer, and about 10-fold faster in cleaving analogs of the R c enantiomers of soman relative to analogs of the S c enantiomers (Li, W.-S. et al., 2001 ).
• a peraoxonase such as a human paraoxonase comprises a calcium dependent protein, and may be also known as an "arylesterase” and/or “aryl-ester hydrolase” (Josse, D. et al., 1999; Vitarius, J. A. and Sultanos, L. G., 1995).
• Examples of the human paraoxonase (“HPON1 ") gene and gene products may be accessed at (Genbank accession no. M63012; Entrez databank no. AAB59538) (Hassett, C. et al., 1991 ).
• a diisopropyl-fluorophosphatase (EC 3.1.8.2) may be also known by its systemic name “diisopropyl- fluorophosphate fluorohydrolase,” and various enzymes in this category have been known in the art by names such as "DFPase”; “tabunase”; “somanase”; “organophosphorus acid anhydrolase”; “organophosphate acid anhydrase”; “OPA anhydrase”; "diisopropylphosphofluoridase”; “dialkylfluorophosphatase”; “diisopropyl phosphorofluoridate hydrolase”; “isopropylphosphorofluoridase”; and/or “diisopropylfluorophosphonate dehalogenase.”
• a diisopropyl-fluorophosphatase catalyzes the following reaction: diisopropyl fluorophosphate + H
• Examples of a diisopropyl fluorophosphate include an organophosphorus compound comprising a phosphorus-halide, a phosphorus-cyanide, or a combination thereof.
• Diisopropyl-fluorophosphatase producing cells and methods for isolating a diisopropyl-fluorophosphatase from a cellular material and/or a biological source have been described, [see, for example, Cohen, J.A. and Warring, M. G., 1957], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type diisopropyl-fluorophosphatase and/or a functional equivalent amino acid sequence for producing a diisopropyl-fluorophosphatase and/or a functional equivalent include Protein database bank entries: 1 E1A, 1 PJX, 2GVU, 2GVV, 2GVW, 2GVX, 2IAO, 2IAP, 2IAQ, 2IAR, 2IAS, 2IAT, 2IAU, 2IAV, 2IAW, 2IAX, 2W43, and/or 3BYC.
• OPAAs Organophosphorus acid anhydrolases (E. C.3.1.8.2), known as "OPAAs,” have been isolated from microorganisms and identified as enzymes that detoxify OP compounds (Serdar, C. M. and Gibson, D. T., 1985; Mulbry, W. W. et al., 1986; DeFrank, J. J. and Cheng, T. -C, 1991 ).
• the better-characterized OPAAs have been isolated from an Altermonas species, such as an Alteromonas sp JD6.5, an Alteromonas haloplanktis, and an Altermonas undina (ATCC 29660) (Cheng, T.-C. et al., 1996; Cheng, T.-C. et al., 1997;
• an OPAA gene and a gene product that may be used include an Alteromonas sp JD6.5 opaA gene, (GeneBank accession no. U29240; Entrez databank no. AAB05590); an Alteromonas haloplanktis prolidase gene (GeneBank accession no. U56398; Entrez databank AAA99824; ATCC 23821 ); or a combination thereof (Cheng, T. C. et al., 1996; Cheng, T.-C. et al., 1997).
• the wild-type encoded OPAA from an Alteromonas sp JD6.5 comprises 517 amino acids, while the wild-type encoded OPAA from an Alteromonas haloplanktis comprises 440 amino acids (Cheng, T. C. et al., 1996; Cheng, T.-C. et al., 1997).
• the Alteromonas OPAAs accelerates the hydrolysis of a phosphotriester and/or a phosphofluoridate, including a cyclosarin, a sarin and/or a soman (Table 4).
• OPAA from an Alteromonas sp JD6.5 (“OPAA-2”) possesses a general binding and cleavage preference up to 112:1 for the S p enantiomers of various p-nitrophenyl phosphotriesters (Hill, C. M. et al., 2000). Additionally, an OPAA from an Alteromonas sp JD6.5 may be over 2 fold faster at cleaving a S p enantiomer of a sarin analog, and over 15-fold faster in cleaving analogs of the R 0 enantiomers of soman relative to analogs of the S c enantiomers (Hill, C. M. et al., 2001 ).
• a "squid-type DFPase” (EC 3.1.8.2) refers to an enzyme that catalyzes the cleavage of both a DFP and a soman, and may be isolated from organisms of the Loligo genus. Generally, a squid-type DFPase cleaves a DFP at a faster rate than a soman.
• Squid-type DFPases include, for example, a DFPase obtained from a Loligo vulgaris, a Loligo pealei, a Loligo opalescens, or a combination thereof (Hoskin, F. C. G. et al., 1984; Hoskin, F. C. G. et al., 1993; Garden, J. M. et al., 1975).
• a well-characterized example of a squid-type DFPase includes the DFPase that has been isolated from the optical ganglion of a Loligo vulgaris (Hoskin, F. C. G. et al., 1984).
• This squid-type DFPase cleaves a variety of OP compounds, including a DFP, a sarin, a cyclosarin, a soman, and a tabun (Hartleib, J. and Ruterjans, H., 2001a).
• the gene encoding this squid-type DFP has been isolated, and may be accessed at GeneBank accession no. AX018860 (International patent publication: WO 9943791-A).
• This enzyme's X-ray crystal structure has been determined (Protein Data Bank entry 1 E1A) (Koepke, J. et al., 2002; Scharff, E. I. et al., 2001 ).
• This squid-type DFPase binds two Ca 2+ ions, which function in catalytic activity and enzyme stability (Hartleib, J. et al., 2001 ). Both the DFPase from a Loligo vulgaris and a Loligo
• 5842-02103 53 pea/e are susceptible to proteolytic cleavage into a 26-kDa and 16 kDa fragments, and the fragments from a Loligo vulgaris are capable of forming active enzyme when associated together (Hartleib, J. and Ruterjans, H., 2001a).
• a "Mazur-type DFPase” (EC 3.1.8.2) refers to an enzyme that catalyzes the cleavage of both DFP and soman.
• a Mazur-type DFPase cleaves a soman at a faster rate than a DFP.
• Examples of a Mazur-type DFPase include the DFPase isolated from a mouse liver (Billecke, S. S. et al., 1999), which may be the same as the DFPase known as a SMP-30 (Fujita,T. et al., 1996; Billecke, S. S. et al., 1999; Genebank accession no.
• Any phosphoric triester hydrolase known in the art may be used.
• An example of an additional phosphoric triester hydrolase includes a product of the gene, mpd, (GenBank accession number AF338729; Entrez databank AAK14390) isolated from a Plesiomonas sp. strain M6 (Zhongli, C. et al., 2001 ).
• Other examples include a phosphoric triester hydrolase identified in a Xanthomonas sp. (Tchelet, R. et al., 1993); a Tetrahymena (Landis, W. G.
• a sulfuric ester hydrolase catalyzes the hydrolysis of a sulfuric ester bond.
• a sulfuric ester hydrolase include an arylsulfatase (EC 3.1.6.1 ), a steryl-sulfatase (EC 3.1.6.2), a glycosulfatase (EC 3.1.6.3), a N-acetylgalactosamine-6-sulfatase (EC 3.1.6.4), a choline-sulfatase (EC 3.1.6.6), a cellulose-polysulfatase (EC 3.1.6.7), a cerebroside-sulfatase (EC 3.1.6.8), a chondro-4-sulfatase (EC 3.1.6.9), a chondro-6-sulfatase (EC 3.1.6.10), a disulfoglucosamine-6-sulfatase (EC 3.1.6).
• An example of a sulfuric ester hydrolase includes an arylsulfatase (EC 3.1.6.1 ), which has been also referred to as “sulfatase,” “nitrocatechol sulfatase,” “phenolsulfatase,” “phenylsulfatase,” “p-nitrophenyl sulfatase,” “arylsulfohydrolase,” “4-methylumbelliferyl sulfatase,” “estrogen sulfatase,” “arylsulfatase C,” “arylsulfatase B,” “arylsulfatase A,” and/or “aryl-sulfate sulfohydrolase.”
• An arylsulfatase catalyzes the
• arylsulfatase producing cells and methods for isolating an arylsulfatase from a cellular material and/or a biological source have been described, [see, for example, Dodgson, K.S. et al., 1956; Roy, A.B. 1960; Roy, A.B., 1976; Webb, E. C. and Morrow, P. F. W., 1959), and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type arylsulfatase and/or a functional equivalent amino acid sequence for producing an arylsulfatase and/or a functional equivalent include Protein database bank entries: 1 HDH.
• Examples of an arylsulfatase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA - 414(ARSD), 415(ARSE); MCC - 704070, 720575(ARSE); CFA - 491718(ARSD), 491719(ARSE); BTA - 505899(ARSE); MDO - 100010082, 100010127; GGA - 418658(ARSD); KLA - KLLA0F03146g; DHA - DEHA0F17710g; YLI - YALI0D26488g; SPO - SPBPB10D8.02c; MGR - MGG_10308;
• a peptidase catalyzes a reaction on a peptide bond, though other secondary reactions (e.g., an esterase activity) may also be catalyzed in some cases.
• a peptidase generally may be categorized as either an exopeptidase (EC 3.4.11-19) or an endopeptidase (EC 3.4.21-24 and EC 3.4.99).
• Examples of a peptidase include an alpha-amino-acyl-peptide hydrolase (EC 3.4.11 ), a peptidyl-amino-acid hydrolase (EC 3.4.17), a dipeptide hydrolase (EC 3.4.13), a peptidyl peptide hydrolase (EC 3.4), a peptidylamino-acid hydrolase (EC 3.4), an acylamino-acid hydrolase (EC 3.4), an aminopeptidase (EC 3.4.11 ), a dipeptidase (EC 3.4.13), a dipeptidyl-peptidase (EC 3.4.14), a tripeptidyl-peptidase (EC 3.4.14), a peptidyl-dipeptidase (EC 3.4.15), a serine-type carboxypeptidase (EC 3.4.16), a metallocarboxypeptidase (EC 3.4.17), a cysteine- type carboxypeptidase (
• Examples of a serine endopeptidase includes a chymotrypsin (EC 3.4.21.1 ); a chymotrypsin C (EC 3.4.21.2); a metridin (EC 3.4.21.3); a trypsin (EC 3.4.21.4); a thrombin (EC 3.4.21.5); a coagulation factor Xa (EC 3.4.21.6); a plasmin (EC 3.4.21.7); an enteropeptidase (EC 3.4.21.9); an acrosin (EC 3.4.21.10); an ⁇ -Lytic endopeptidase (EC 3.4.21.12); a glutamyl endopeptidase (EC 3.4.21.19); a cathepsin G (EC 3.4.21.20); a coagulation factor Vila (EC 3.4.21.21 ); a coagulation factor IXa (EC 3.4.21.22); a cucumisin (EC 3.4.21.25); a prolyl
• venombin AB EC 3.4.21.55
• a leucyl endopeptidase EC 3.4.21.57
• a tryptase EC 3.4.21.59
• a scutelarin EC 3.4.21.60
• a kexin EC 3.4.21.61
• a subtilisin EC 3.4.21.62
• an oryzin EC 3.4.21.63
• a peptidase K EC 3.4.21.64
• a thermomycolin EC 3.4.21.65)
• a thermitase EC 3.4.21.66
• an endopeptidase So EC 3.4.21.67
• a t-plasminogen activator EC 3.4.21.68
• a protein C activated
• EC 3.4.21.69 a pancreatic endopeptidase E
• a pancreatic elastase Il EC 3.4.21.
• 5842-02103 56 3.4.21.100); a xanthomonalisin (EC 3.4.21.101 ); a C-terminal processing peptidase (EC 3.4.21.102); a physarolisin (EC 3.4.21.103); a mannan-binding lectin-associated serine protease-2 (EC 3.4.21.104); a rhomboid protease (EC 3.4.21.105); a hepsin (EC 3.4.21.106); a peptidase Do (EC 3.4.21.107); a HtrA2 peptidase (EC 3.4.21.108); a matriptase (EC 3.4.21.109); a C5a peptidase (EC 3.4.21.110); an aqualysin 1 (EC 3.4.21.111 ); a site-1 protease (EC 3.4.21.1 12); a pestivirus NS3 polyprotein peptid
• Trypsin (EC 3.4.21.4; CAS registry number: 9002-07-7) has been also referred to in that art as " ⁇ - trypsin,” “ ⁇ -trypsin,” “cocoonase,” “parenzyme,” “parenzymol,” “tryptar,” “trypure,” “pseudotrypsin,” “tryptase,” “tripcellim,” and/or “sperm receptor hydrolase.”
• a trypsin catalyzes the reaction: a preferential cleavage at an Arg and/or a Lys residue.
• Trypsin producing cells and methods for isolating a trypsin from a cellular material and/or a biological source have been described [see, for example, Huber, R. and Bode, W., 1978; Walsh, K.A., 1970; Read, R.J. et al., 1984; Fiedler, F. 1987; Fletcher, T.S. et al., 1987; Polgar, L. Structure and function of serine proteases. In New Comprehensive Biochemistry Vol. 16, Hydrolytic Enzymes (Neuberger, A. and Brocklehurst, K. eds), pp. 159-200, 1987; Tani, T., et al. 1990), and may be used in conjunction with the disclosures herein.
• Examples of a trypsin and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA - 5644(PRSSI ), 5645(PRSS2), 5646(PRSS3); PTR - 747006(PRSS3); MCC - 698352(PRSS2), 698729(PRSSI ), 699238(PRSS2); MMU - 22072(Prss2), 435889(1810049H19Rik), 436522(Try10); RNO - 24691(Prss1 ), 25052(Prss2), 286960, 362347; CFA - 475521 (PRSS3); BTA - 282603(PRSS2), 780933; MDO - 100010059, 100010109, 100010619, 100010951 ; GGA - 396344(PRSS2), 396345(PRSS3),
• Dmel_CG2056 (spirit), Dmel_CG30002, Dmel_CG30025; , Dmel_CG30031 , Dmel_CG30371 , Dmel_CG30414, Dmel_CG3066(Sp7); , Dmel_CG31219, Dmel_CG31265, Dmel_CG31269, Dmel_CG31681 ; , Dmel_CG31728, Dmel_CG31822, Dmel_CG31824, Dmel_CG31954; , Dmel_CG32269, Dmel_CG32271 , Dmel_CG32277, Dmel_CG32374; , Dmel_CG32383(sphinx1 ), Dmel_CG32755,
• Structural information for a wild-type trypsin and/or a functional equivalent amino acid sequence for producing a trypsin and/or a functional equivalent include Protein database bank entries: 1A0J, 1AKS, 1AMH, 1AN1, 1ANB, 1ANC, 1AND, 1ANE, 1AQ7, 1AUJ, 1AVW, 1AVX, 1AZ8, 1BJU, 1BJV, 1BRA, 1BRB, 1BRC, 1BTP, 1BTW, 1BTX, 1BTY, 1BTZ, 1BZX, 1C1N, 1C1O, 1C1P, 1C1Q, 1C1R, 1C1S, 1C1T, 1C2D, 1C2E, 1C2F, 1C2G, 1C2H, 1C2I, 1C2J, 1C2K, 1C2L, 1C2M, 1C5P, 1C5Q, 1C5R, 1C5S, 1C5T,
• Chymotrypsin (EC 3.4.21.1) has been also referred to as “chymotrypsins A and B," “ ⁇ -chymar ophth,” “avazyme,” “chymar,” “chymotest,” “enzeon,” “quimar,” “quimotrase,” “ ⁇ -chymar,” “ ⁇ -chymotrypsin
• a chymotrypsin generally cleaves peptide bonds at the carboxyl side of amino acids, with a preference for a substrate comprising a Tyr, a Trp, a Phe, and/or a Leu.
• chymotrypsin producing cells and methods for isolating a chymotrypsin from a cellular material and/or a biological source have been described, [see, for example, Dodgson, K. S. et al., 1956; Roy, A.B. 1960; Roy, A.B., 1976; Webb, E. C. and Morrow, P. F. W., 1959), and may be used in conjunction with the disclosures herein.
• Examples of a chymotrypsin and/or a functional equivalent KEEG sequences for production of wild- type and/or a functional equivalent nucleotide and protein sequence include: HSA - 1504(CTRBI ), 440387(CTRB2); PTR - 736467(CTRBI ); MCC - 711100, 713851(CTRBI ); MMU - 66473(Ctrb1 ); RNO - 24291 (Ctrbi ); CFA - 479649(CTRB2), 479650(CTRBI ), 610373; BTA - 504241(CTRBI ); XLA - 379495, 379607(MGC64417), 444360; XTR - 496968(ctrl), 548358(ctrb1 ); DRE - 322451 (ctrbi ), 562139; NVE - NEMVE_v1g 140545; DME -
• Structural information for a wild-type chymotrypsin and/or a functional equivalent amino acid sequence for producing a chymotrypsin and/or a functional equivalent include Protein database bank entries: 1AB9, 1ACB, 1AFQ, 1CA0, 1CBW, 1CHO, 1 DLK, 1 EQ9, 1 EX3, 1GCD, 1GCT, 1GG6, 1 GGD, 1GHA, 1GHB, 1GL0, 1GL1 , 1GMC, 1GMD, 1GMH, 1 HJA, 1 K2I, 1 KDQ, 1 MTN, 1 N8O, 1 OXG, 1 P2M, 1 P2N, 1 P2O, 1 P2Q, 1T7C, 1T8L, 1T8M, 1T8N, 1T8O, 1VGC, 1YPH, 2CHA, 2GCH, 2GCT, 2GMT, 2JET, 2P8O, 2VGC, 3BG4, 3GCH, 3G
• Chymotrypsin C (EC 3.4.21.2; CAS no. 9036-09-3) hydrolyzes a peptide bond, particularly those comprising a Leu, a Tyr, a Phe, a Met, a Trp, a GIn, and/or an Asn.
• Chymotrypsin C producing cells and methods for isolating a chymotrypsin C from a cellular material and/or a biological source have been described, [see, for example, Peanasky, R.J. et al., 1969; Folk, J. E., 1970; and Wilcox, P. E., 1970], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type chymotrypsin C and/or a functional equivalent amino acid sequence for producing a chymotrypsin C and/or a functional equivalent include Protein database bank entries: HSA * - * 1 1330(CTRC); PTR * - * 739685(CTRC); MCC * - * 700270, 700762(CTRC); MMU * - * 76701 (Ctrc); RNO * - * 362653(Ctrc); CFA * - * 478220(CTRC); and/or BTA * - * 514047(CTRC).
• Subtilisin (EC 3.4.21.62; CAS No. 9014-01-1 ) has been also referred to as "alcalase 0.6L,” “alcalase 2.5L,” “alcalase,” “alcalase,” “ALK-enzyme,” “bacillopeptidase A,” “bacillopeptidase B,” “Bacillus subtilis alkaline proteinase bioprase,” “Bacillus subtilis alkaline proteinase,” “bioprase AL 15,” “bioprase APL
• subtilisin comprises a serine endopeptidase
• subtilisin producing cells and methods for isolating a subtilisin from a cellular material and/or a biological source have been described, [see, for example, Nedkov, P., et al., 1985; Ikemura, H., et al., 1987), and may be used in conjunction with the disclosures herein.
• a subtilisin has esterase activity.
• Structural information for a wild-type subtilisin and/or a functional equivalent amino acid sequence for producing a subtilisin and/or a functional equivalent include Protein database bank entries: 1A2Q, 1 AF4, 1AK9, 1AQN, 1AU9, 1AV7, 1AVT, 1 BE6, 1 BE8, 1 BFK, 1 BFU, 1 BH6, 1C3L, 1C9J, 1C9M, 1C9N, 1CSE, 1 DUI, 1GCI, 1GNS, 1GNV, 1 IAV, UEA, 1 LW6, 1 MPT, 1 NDQ, 1 NDU, 1OYV, 1Q5P, 1 R0R, 1SBC, 1SBH, 1SBI, 1 SBN, 1SCA, 1SCB, 1 SCD, 1SCJ, 1SCN, 1SIB, 1SPB, 1ST3, 1SUA, 1SUB, 1SUC, 1SUD, 1SUE, 1SUP, 1SVN, 1TK2, 1
• a peroxidase may be categorized by the donor. Examples of a peroxidase includes a NADH peroxidase (EC 1.11.1.1 ;
• CAS registry number: 9032-24-0 which uses a NADH as a donor; a NADPH peroxidase (EC 1.1 1.1.2; CAS registry number: 9029-51-0), which uses a NADPH as a donor; a fatty-acid peroxidase (EC 1.11.1.3; CAS registry number: 9029-52-1 ), which uses a palmitate as a donor; a cytochrome-c peroxidase (EC 1.11.1.5; CAS registry number: 9029-53-2), which uses a ferrocytochrome c as a donor; a catalase (EC 1.11.1.6; CAS registry number: 9001-05-2), which uses a H 2 O 2 as a donor; a peroxidase (EC 1.11.1.7; CAS registry number: 9003-99-0), which uses various substrates as a donor; an iodide peroxidase (EC 1.1 1.1.8; CAS registry number: 9031-28-1 ), which uses an iodide as a
• 5842-02103 60 which uses a L-ascorbate as a donor; a phospholipid-hydroperoxide glutathione peroxidase (EC 1.11.1.12; CAS registry number: 97089-70-8), which uses a glutathione and a lipid hydroperoxide as a donor; a manganese peroxidase (EC 1.11.1.13; CAS registry number: 114995-15-2), which uses a Mn(II) and a H + as a donor; a lignin peroxidase (EC 1.11.1.14; CAS registry number: 93792-13-3), which uses a 1 ,2-bis(3,4- dimethoxyphenyl)propane-1 ,3-diol as a donor; a peroxiredoxin (EC 1.11.1.15; CAS registry number: 207137- 51-7); a versatile peroxidase (EC 1.11.1.16; CAS registry number: 42613-30
• Peroxidases (EC 1.11.1.7) [0170] Peroxidase (EC 1.11.1.7; CAS registry number: 9003-99-0) has been also referred to as
• a peroxidase (EC 1.11.1.7) may be referred herein by its EC classification number (EC 1.11.1.7) to distinguish from the subgenus of "peroxidases,” which are referred to herein by the EC classification number (EC 1.11.1 ).
• a peroxidase generally comprises a hemoprotein.
• Peroxidase (EC 1.1 1.1.7) producing cells and methods for isolating a peroxidase from a cellular material and/or a biological source have been described [see, for example, Kenten, R. H. and Mann, P.J. G., 1954; Morrison, M. et al., 1957; Paul, K. G. Peroxidases. In: Boyer, P. D., Lardy, H. and Myrback, K.
• Examples of a peroxidase (EC 1.11.1.7) and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA - 4025(LPO), 4353(MPO), 8288(EPX), 9588(PRDX6); PTR - 468420(EPX), 469589(PRDX6), 738041 (PRDX6), 748680(MPO); MCC - 706486(PRDX6), 707299, 709655(EPX), 709848(LPO), 714246(MPO); MMU - 1 1758(Prdx6), 13861(Epx), 17523(Mpo), 320769(Prdx6-rs1 ), 76113(Lpo); RNO - 303413(Mpo), 303414(Epx), 94167(Prdx6); CFA - 480069(PRDX6)
• AT2G38380 AT2G38390, AT2G39040, AT2G41480, AT2G43480, AT3G01190; AT3G03670, AT3G17070, AT3G21770, AT3G28200; AT3G491 ⁇ (ATPCA/ATPRXSS/PRXSS/PRXCA);
• AT3G49120 AT3G49120(ATPCB/ATPERX34/PERX34/PRXCB), AT3G49960, AT4G08770; AT4G08780, AT4G1 1290, AT4G16270, AT4G17690, AT4G21960(PRXRI ); AT4G26010, AT4G30170, AT4G31760, AT4G33420, AT4G36430, AT4G37520; AT4G37530, AT5G05340, AT5G06720, AT5G06730, AT5G14130, AT5G15180; AT5G17820, AT5G19880, AT5G19890, AT5G22410, AT5G24070, AT5G40150; AT5G42180, AT5G47000, AT5G51890, AT5G58390, AT5G58400, AT5G64100; AT5G64110, AT5G64120, AT5G66390, AT5G67400; OSA -
• Structural information for a wild-type peroxidase (EC 1.11.1.7) and/or a functional equivalent amino acid sequence for producing a peroxidase and/or a functional equivalent include Protein database bank entries: 1ARP; 1ARU; 1ARV; 1ARW; 1ARX; 1ARY; 1ATJ; 1 BGP; 1 C8I; 1CK6; 1CXP; 1 D2V; 1 D5L; 1 D7W; 1 DNU; 1 DNW; 1 FHF; 1GW2; 1GW0; 1GWT; 1GWU; 1GX2; 1GZA; 1GZB; 1 H3J; 1 H55; 1 H57; 1 H58; 1 H5A; 1 H5C; 1 H5D; 1 H5E; 1 H5F; 1 H5G; 1 H5H; 1 H5I; 1 H5J; 1 H5K; 1 H5L; 1 H5M; 1 HCH; 1 HSR; 1 KZM; 1 LY8
• a material formulation (e.g., a surface treatment, a filler, a biomolecular composition, a textile finish, etc.) comprises an antibiological agent.
• An antibiological agent may comprise a biomolecular composition such as a proteinaceous molecule ("antibiological proteinaceous molecule”) such
• a material formulation may comprise an antibiological agent by being formulated, prepared, processed, post-cured processed, manufactured, and/or applied (e.g., applied to a surface), in a fashion to be suitable to possess an antibiological activity and/or function (e.g., an antimicrobial activity, an antifouling activity).
• antibiological agent e.g., an antimicrobial agent, an antifouling agent
• may act against a biological entity e.g., a cell, a virus
• contacts e.g., a surface contact, an internal incorporation, an infiltration, an infestation
• An antibiological agent may act by treating an infestation, preventing infestation, inhibiting infestation (e.g., preventing cell attachment), inhibiting growth, preventing growth, lysing, and/or killing; a biological entity such as a cell and/or a virus (e.g., one or more genera and/or species of a cell and/or a virus).
• a biological entity such as a cell and/or a virus (e.g., one or more genera and/or species of a cell and/or a virus).
• some embodiments comprise a process for treating an infestation, preventing infestation, inhibiting infestation (e.g., preventing cell attachment), inhibiting growth, preventing growth, lysing, and/or killing a cell and/or a virus (e.g., a fungal cell) comprising contacting the cell and/or the virus with a material formulation (e.g., a paint, a coating composition, a biomolecular composition) comprising at least one proteinaceous molecule (e.g., an effective amount of an antibiological peptide, antibiological polypeptide, an antibiological enzyme, and/or an antibiological protein).
• a material formulation e.g., a paint, a coating composition, a biomolecular composition
• proteinaceous molecule e.g., an effective amount of an antibiological peptide, antibiological polypeptide, an antibiological enzyme, and/or an antibiological protein
• an antibiological agent e.g., an antibiological proteinaceous molecule
• an antibiological proteinaceous molecule may possess a biocidal and/or a biostatic activity.
• an antimicrobial and/or an antifouling enzyme may act as a biocide and/or a biostatic.
• an antibiological proteinaceous molecule e.g., a biostatic
• a coating comprising an antimicrobial agent may act against a microbial cell and/or a virus adapted for growth in a non-marine environment and/or does not produces fouling; while a coating comprising an antifouling agent may act against a marine cell that produces fouling.
• a virus may be a target of such an antibiological agent, as the virus (e.g., a membrane enveloped virus) may comprise a biomolecule target of an antibiological agent (e.g., an enzyme, an antibiological proteinaceous molecule such as a peptide).
• a target cell and/or a target virus may be capable of infesting an inanimate object (e.g., a building material, an indoor structure, an outdoor structure).
• an "inanimate object” refers to structures and objects other than a living cell (e.g., a living organism).
• Examples of an inanimate object include an architectural structure that may comprise a painted and/or an unpainted surface such as the exterior wall of a building; the interior wall of a building; an industrial equipment; an outdoor sculpture; an outdoor furniture; a construction material for indoor and/or outdoor use such as a wood, a stone, a brick, a wall board (e.g., a sheetrock), a ceiling tile, a concrete, an unglazed tile, a stucco, a grout, a roofing tile, a shingle, a painted and/or a treated wood, a synthetic composite material, a leather, a textile, or a combination thereof.
• a painted and/or an unpainted surface such as the exterior wall of a building; the interior wall of a building; an industrial equipment; an outdoor sculpture; an outdoor furniture; a construction material for indoor and/or outdoor use such as a wood, a stone, a brick, a wall board (e.g., a sheetrock), a ceiling tile, a concrete, an unglazed tile
• Such an inanimate object may comprise (e.g., a plastic building material, a wood coated with a surface treatment) a material formulation.
• a building material includes a conventional and/or a non-conventional indoor and/or an outdoor construction and/or a decorative material, such as a wood; a sheet-rock (e.g., a wallboard); a paper and/or vinyl coated wallboard; a fabric (e.g., a
• 5842-02103 63 textile a carpet; a leather; a ceiling tile; a cellulose resin wall board (e.g., a fiberboard); a stone; a brick; a concrete; an unglazed tile; a stucco; a grout; a painted surface; a roofing tile; a shingle; a cellulose-rich material; a material capable of providing nutrient(s) to a cell (e.g., fungi) and/or a virus, capable of harboring nutrient material(s) and/or supporting a biological (e.g., a fungal) infestation; or a combination thereof.
• a cell e.g., fungi
• a virus capable of harboring nutrient material(s) and/or supporting a biological (e.g., a fungal) infestation
• a biological e.g., a fungal
• One or more cells may, for example, infest, survive upon, survive within, grow on the surface, and/or grow within, an inanimate object.
• a target cell and/or a target virus include those that can infest and/or survive upon and/or within: an inanimate object such as an indoor structure, an outdoor structure, a building material, or a combination thereof, and may cause defacement (e.g., deterioration or discoloration), odor, environment hazards, and other undesirable effects.
• a material e.g., an object
• a material may be susceptible (“prone") to infestation by a cell and/or a virus when it is capable of serving as a food source for a cell (e.g., the material comprises a substance that serves as a food source).
• the material comprises a substance that serves as a food source.
• any described formulation of a cell and/or a virus (e.g., a fungus) prone material formulation may be modified to incorporate an antibiological agent (e.g., an antifungal peptidic agent).
• a fungal-prone material may comprise a binder comprising a carbon-based polymer that serves as a nutrient for a fungus, and a coating comprising the binder as a component may also comprise an antibiological proteinaceous composition.
• a susceptible material formulation such as a grout and/or a caulk that may be in frequent contact with or constantly exposed to fungal nutrients and moisture may comprise a proteinaceous molecule effective against a fungus on and/or within the susceptible material formulation (e.g., a surface).
• Antibiological activity can provide and/or facilitate disinfection, decontamination and/or sanitization of an material and/or an object (e.g., an inanimate object, a building material), which refer to the process of reducing the number of cell(s) (e.g., a fungus microorganism) and/or viruses to levels that no longer pose a threat (e.g., a threat to property, a threat to the health of a desired organism such as human).
• a bioactive antifungal agent can be accompanied by removal (e.g., manual removal, machine aided removal) of the cell(s) and/or the virus(s).
• a material formulation comprising an antimicrobial proteinaceous composition
• an application such as a hospital and/or a health care application, such as reducing and/or preventing a hospital-acquired infection (e.g., a so-called "super bugs" infection); and/or reducing (e.g., reducing the spread) and/or preventing infection(s) (e.g., a viral infection such as SARS); as well as a hygienic surface application (e.g., an antimicrobial cleaner, an antimicrobial utensil, an antimicrobial food preparation surface, an antimicrobial coating system); reducing and/or preventing food poisoning; or a combination thereof.
• a hospital-acquired infection e.g., a so-called "super bugs" infection
• reducing e.g., reducing the spread
• infection(s) e.g., a viral infection such as SARS
• a hygienic surface application e.g., an antim
• a strain of bacteria that may be resistant to a conventional antibiotic, such as a Staphalococcus [e.g., a Methicillin-resistant Staphylococcus aureus (“MRSA”)], a Streptococcus bacteria, and/or a Vero-cytotoxin producing variants of Escherichia coli.
• MRSA Methicillin-resistant Staphylococcus aureus
• Vero-cytotoxin producing variants of Escherichia coli e.g., a Vero-cytotoxin producing variants of Escherichia coli.
• a material formulation e.g., a coating
• a proteinaceous molecule e.g., a peptide
• a biological cell e.g., a fungal cell
• a virus e.g., a virus
• 64 fungal cell may be used in assaying and/or screening for an antifungal composition (e.g., a peptide library), may comprise a fungal organism known to, or suspected of, infesting a vulnerable material(s) and/or surface(s) (e.g., a construction material).
• an antifungal composition e.g., a peptide library
• Such methods may be used to assay and/or screen, for example, antifungal activity against a wide variety of fungus genera and species, such as in the case of selecting a composition comprising a broad-spectrum antifungal activity.
• Similar methods may be used to identify particular proteinaceous composition(s) (e.g., a peptide, a plurality peptides) that target specific fungus genera or species.
• Examples of such a fungal cell often used in such an assay include members of the genera Stachybotrys (especially Stachybotrys chartarum), Aspergillus species (sp.), Penicillium sp., Fusarium sp., Alternaria dianthicola, Aureobasidium pullulans (aka Pullularia pullulans), Phoma pigmentivora and Cladosporium sp, though an assay may be adapted for other cell(s).
• a proteinaceous molecule may be effective (e.g., inhibit growth, treat infestation, etc.) against a cell (e.g., a fungal cell, a bacterial cell) and/or a virus from a genera and/or a species of, for example, an Alternaria (e.g., an Alternaria dianthicola), an Aspergillus [(e.g., an Aspergillus species (sp.), an Aspergillus fumigatus, an Aspergillus Parasiticus], an Aureobasidium (e.g., an Aureobasidium pullulans a.k.a.
• a cell e.g., a fungal cell, a bacterial cell
• viruses e.g., a virus from a genera and/or a species of, for example, an Alternaria (e.g., an Alternaria dianthicola), an Aspergillus [(e.g., an Aspergillus species (
• a Pullularia pullulans a Candida
• a Ceratocystis e.g., a Ceratocystis Fagacearum
• a Cladosporium e.g., a Cladosporium sp.
• a Fusarium e.g., a Fusarium sp., a Fusarium oxysporum, a Fusariam Sambucinum
• a Magaporthe e.g., a Magaporthe Aspergillus nidulans
• Mycosphaerella e.g., a Penicillium (e.g., a Penicillium sp.), a Phoma (e.g., a Phoma pigmentivora), a Pphiostoma (e.g., a Pphiostoma ulmi), a Pythium (e.g,.
• Cell and/or viral culture conditions may be modified appropriately to provide favorable growth and proliferation conditions, using the techniques of the art, and to assay and/or screen for activity against a target cell (e.g., a bacteria, an algae, etc.) and/or a virus.
• a target cell e.g., a bacteria, an algae, etc.
• any suitable peptide/polypeptide/protein screening method in the art may be used to identify an antibiological proteinaceous molecule (e.g., an antifungal peptide) for an assay as active antibiological agent (e.g., an antifungal agent) in a material formulation (e.g., a paint, a coating material, a biomolecular composition).
• a material formulation e.g., a paint, a coating material, a biomolecular composition
• an in vitro method to determine bioactivity of a peptide such as a peptide from a synthetic peptide combinational library, may be used (Furka, A., et al., 1991 ; Houghten, R. A., et al., 1991 ; Houghten, R. A., et al., 1992).
• An antibiological biomolecular composition may be combined with any other antibiological agent described herein and/or known in the art, such as a preservative (e.g., a chemical biocide, a chemical biostatic) typically used in a surface treatment (e.g., a coating, a paint) and/or an antimicrobial agent (e.g., a chemical biocide, a chemical biostatic) typically used in a polymeric material (e.g., a plastic, an elastomer, etc).
• a preservative e.g., a chemical biocide, a chemical biostatic
• a surface treatment e.g., a coating, a paint
• an antimicrobial agent e.g., a chemical biocide, a chemical biostatic typically used in a polymeric material (e.g., a plastic, an elastomer, etc).
• one or more antibiological proteinaceous molecule(s) may be used in combination with and/or as a substitute for one or more existing antibiological agents (e.g., a preservative, an antimicrobial agent, a fungicide, a fungistatic, a bactericide, an algaecide, etc.) identified herein and/or in the art.
• an antifungal peptidic agent e.g., an enzyme
• one or more existing antibiological agents e.g., a preservative, an antimicrobial agent, a fungicide, a fungistatic, a bactericide, an algaecide, etc.
• an antibiological agent e.g., a preservative
• an antibiological proteinaceous molecule e.g., an antimicrobial proteinaceous molecule, an antifungal peptidic agent, an antimicrobial enzyme
• examples of an antibiological proteinaceous molecule include, but are not limited to those non-peptidic antimicrobial compounds (i.e., biocides, fungicides, algaecides, mildewcides, etc.) which have
• antibiological proteinaceous molecule(s) and/or combinations with another antibiological agent may provide an advantage such as a broader range of activity against various organisms (e.g., a bacteria, an algae, a fungi, etc.), a synergistic antibiological and/or preservative effect, a longer duration of effect, or a combination thereof.
• an antimicrobial and/or an antifouling agent comprising an enzyme (e.g., an antimicrobial enzyme, an antifouling enzyme) and/or a peptide (e.g., an antifouling peptide, an antimicrobial peptide, an antifungal peptide, an antialgae peptide, an antibacterial peptide, an antimildew peptide, etc) may be used alone or in combination with one or more additional antibiological agent(s) (e.g., an antimicrobial agent, an antifouling agent, a preservative, a biocide, a biostatic agent) and/or technique (see for example, Baldridge, G.
• an antimicrobial agent e.g., an antimicrobial agent, an antifouling agent, a preservative, a biocide, a biostatic agent
• technique see for example, Baldridge, G.
• an antimicrobial peptide comprises ProteCoat ® (Reactive Surfaces, Ltd.; also described in U.S. Patent Nos. U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097, and Patent Application Nos. 10/884,355 and 11/368,086).
• certain peptides contemplated for use have been shown to involve synergy between the peptides (e.g., antifungal peptides) and non-peptide antifungal agents that may be useful in controlling growth of a Fusarium, a Rhizoctonia, a Ceratocystis, a Pythium, a Mycosphaerella, an Aspergillus and/or a Candida genera of fungi.
• synergistic combinations have been described and successfully used to inhibit the growth of an Aspergillus fumigatus and an A.
• peptide and non-peptide agent(s) may be useful as, for example, a component (e.g., an additive) in a material formulation (e.g., a paint, a coating) such as for deterring, preventing, and/or treating a fungal infestation.
• a component e.g., an additive
• a material formulation e.g., a paint, a coating
• an antibiological agent e.g., an antimicrobial agent, an antifouling agent
• a detergent e.g., a nonionic detergent, a zwitterionic detergent, an ionic detergent
• CHAPS zwitterionic
• Triton X series detergent nonionic
• SDS ionic
• a basic protein such as a protamine
• a cationic polysaccharide such as chitosan
• a metal ion chelator such as EDTA; or a combination thereof, all of which have may have effectiveness against a lipid cellular membrane, and may be incorporated into a material formulation and/or used in a washing composition (e.g., a washing solution, a washing suspension, a washing emulsion) applied to a material formulation.
• a washing composition e.g., a washing solution, a washing suspension, a washing emulsion
• a material formulation comprising an antimicrobial peptide and an antimicrobial enzyme may be washed with a commercial washing solution that may also comprise an antimicrobial peptide.
• an additional preservative, an biocide, an biostatic agent, or a combination thereof comprises a non-peptidic antimicrobial agent, a non-amino based antimicrobial agent, a compounded peptide antimicrobial agent, an enzyme-based antimicrobial agent, or a combination thereof, such as those described in U.S. Patent Application 11/865,514 filed 10/1/07, incorporated by reference.
• an antibiological agent e.g., an antimicrobial agent, an antifouling agent
• 5842-02103 66 combined with a non-peptidic antimicrobial agent, a non-amino based antimicrobial agent, a compounded peptide antimicrobial agent, an enzyme-based antimicrobial agent, or a combination thereof, and an improved (e.g., additive, synergistic) effect may occur, so that the concentration of one or more components of the antibiological agent may be reduced relative to the component's use alone or in a combination comprising fewer components.
• the concentration of any individual antibiological agent component comprises about 0.000000001 % to about 20% (e.g., about 0.000000001 % to about 4%) or more, of a material formulation, an antibiological agent (e.g., an antimicrobial agent, an antifouling agent), a washing composition, or a combination thereof.
• an antibiological agent e.g., an antimicrobial agent, an antifouling agent, an enzyme, a peptide, a preservative
• another biomolecular composition e.g., an enzyme, a cell based particulate material
• an additional property e.g., a catalytic activity, a binding property
• biomolecular compositions examples include an enzyme such as a lipolytic enzyme, though some lipolytic enzymes may have antimicrobial and/or antifouling activity; a phosphoric triester hydrolase; a sulfuric ester hydrolase; a peptidase, some of which may have an antimicrobial and/or antifouling activity; a peroxidase, or a combination thereof.
• a biomolecular composition may be used with little or no antimicrobial and/or antifouling function.
• a material formation may comprise a combination of active enzymes with little or no active antimarine, antifouling, and/or antimicrobial enzyme present.
• an antibiological agent comprises an enzyme (e.g., an antimicrobial enzyme, an antifungal enzyme, an antialgae enzyme, an antibacterial enzyme, antimildew enzyme, an antifouling enzyme, etc.) that may catalyze a reaction.
• an enzyme may promote cleavage of a chemical bond in a biological cell wall, a viral proteinaceous molecule, and/or a cellular membrane component (e.g., a viral envelope component).
• an antimicrobial proteinaceous molecule e.g., a peptide
• a biostatic and/or a biocidal activity e.g., activity via cell membrane permeablization.
• An antibiological proteinaceous molecule may compromise a cellular membrane (e.g., the cell membrane enclosing the cytoplasm, a viral envelope) to allow for cell wall and/or viral proteinaceous molecule disruption.
• a cellular membrane e.g., the cell membrane enclosing the cytoplasm, a viral envelope
• antibiological activities e.g., an antimicrobial activity, an antifouling activity
• an enzymatic antibiological agent e.g., an antimicrobial agent
• an enzymatic antibiological agent may comprise a hydrolytic enzyme, such as a lysozyme that may cleave a peptidoglycan cell wall component.
• a lysozyme active in a coating may confer a catalytic, antimicrobial
• a lysozyme may be used in a material formulation such as a cream, an ointment, and/or a pharmaceutical, partly due to its size (14.4 kDa).
• an antimicrobial peptide ProteCoatTM
• ProteCoatTM may be efficacious against a Gram positive organism, and a combination of an antimicrobial and/or an antifouling enzyme (e.g., a lysozyme) demonstrates activity against cell(s).
• a material formulation comprising a lipolytic enzyme such as a phospholipase and/or a cholesterol esterase that acts to compromise the integrity of a cell membrane, may allow ease of access for one or more enzyme(s) that degrade cell wall and/or viral proteinaceous coat component(s), and/or a preservative to act in a biocidal and/or a biostatic function as well (e.g., acts against a cell component).
• an enzyme that possesses an antiobiological activity e.g., an antimicrobial activity, an antifouling activity
• the enzyme comprises a glycosylase (EC 3.2).
• the enzyme comprises a glycosidase (EC 3.2.1 ), which comprises an enzyme that hydrolyses an O- glycosyl compound, a S-glycosyl compound, or a combination thereof.
• a glycosidase EC 3.2.1
• the glycosidase acts on an O-glycosyl compound, and examples of such an enzyme include a lysozyme, an agarase, a cellulose, a chitinase, or a combination thereof.
• an antibiological enzyme acts on a cell wall, a viral proteinaceous molecule, and/or a cellular membrane component
• examples of such enzymes include a lysozyme, a lysostaphin, a libiase, a lysyl endopeptidase, a mutanolysin, a cellulase, a chitinase, an ⁇ -agarase, an ⁇ -agarase, a ⁇ /-acetylmuramoyl-L-alanine amidase, a lytic transglycosylase, a glucan endo-1 ,3- ⁇ -D-glucosidase, an endo-1 ,3(4)- ⁇ -glucanase, a ⁇ -lytic metalloendopeptidase, a 3-deoxy-2- octulosonidase,
• Lysozyme (EC 3.2.1.17; CAS registry number: 9001-63-2) has been also referred to in that art as "peptidoglycan N-acetylmuramoylhydrolase,” “1 ,4-N-acetylmuramidase,” “globulin G,” “globulin G1 ,” “L- 7001 ,” “lysozyme g,” “mucopeptide glucohydrolase,” “mucopeptide N-acetylmuramoylhydrolase,” “muramidase,” “N,O-diacetylmuramidase,” and "PR1-lysozyme.”
• a lysozyme catalyzes the reaction: in a peptidoglycan, hydrolyzes a (1 ,4)- ⁇ -linkage between N-acetylmuramic acid and a N-acetyl-D-glucosamine; in a chitodextrin (a polymer of (1
• a lysozyme demonstrates endo-N-acetylmuramidase activity, and may cleave a glycan comprising linked peptides, but has little or no activity toward a glycan that lack linked peptide.
• a lysozyme comprises a single chain protein with a MW of 14.3 kD. Lysozyme producing cells and methods for isolating a lysozyme from a cellular material and/or a biological source have been described [see, for example, Blade, CCF. et al., 1967a; Blake, CCF.
• a common example of a lysozyme comprises a chicken egg white lysozyme ("CEWL").
• the general activity range of a CEWL lysozyme may comprise about pH 6.0 to about 9.0, with maximal activity of the lysozyme at about pH 6.2 may be at an ionic strength of about 0.02 M to about 0.100 M, while at about pH 9.2 the maximal activity may be between an ionic strength of about 0.01 M to about 0.06 M.
• Another example of a lysozyme comprises a commercially available lysozyme (e.g., Sigma Aldrich).
• Lysozymes comprise proteins with similar folding structures, generally divided into 9 classes.
• a bacteriophage T4 lysozyme a goose egg-white lysozyme, a hen egg-white lysozyme, and a Chaloropsis lysozyme.
• Two domains connected by an alpha helix form the active site, with a glutamic acid located in the N-terminal half of the protein, in the C-terminal end of an alpha-helix.
• Another active site residue typically comprises an aspartic acid.
• Chalaropsis lysozyme comprises a cellosyl, which differs in having an active site comprising a single, flattened ellipsoid domain with a beta/alpha fold with a long groove comprising an electronegative hole on the C-terminal face.
• a cellosyl may be produced from Streptomyces coelicolor.
• An additional Chalaropsis lysozyme comprises LytC produced from Streptomyces pneumonia.
• an autolytic lysozyme examples include a SF muramidase from an Enterococus faecium ("Enterococcus hirae"; ATCC 9790); and/or a pesticin, encoded by the pst gene on the pPCP1 plasmid from Yersinia pestis.
• a lysozyme has been recombinantly expressed in Aspergillus niger (Gheshlaghi et al, 2005; Archer et al. 1990; Gyamerah et al. 2002; Mainwaring et al. 1999).
• lysozyme examples include denaturation of the lysozyme, an attachment of a polysaccharide and/or a hydrophobic polypeptide to enhance effectiveness against a Gram negative bacterial, or a combination thereof (Touch et al., 2003; Aminlari et al., 2005; Web et al., 1994).
• a lysozyme damages and/or destroys a bacterial cell wall, and exemplifies an action many antimicrobial and/or antifouling enzymes.
• a lysozyme catalyzes cleavage of a peptidoglycan's glycosidic bond between a N-acetylmuramic acid (“NAM”) and a N-acetylglucosamine (“NAG”) that often comprise part of a cell wall.
• NAM N-acetylmuramic acid
• NAG N-acetylglucosamine
• a lysozyme acts, the structural integrity of the cell wall may be reduced (e.g., destroyed), and the bacteria cell bursts ("lysis") under internal osmotic pressure.
• a lysozyme may act by an additional antimicrobial and/or antifouling mechanisms of action, other than enzymatic action, triggered by contact with a cell such as cell membrane damage, induction of an autolysin's activity, or a combination thereof (Masschalck and Michiels, 2003).
• a lysozyme may be effective against a Gram positive bacteria since the peptidoglycan layer may be relatively accessible to the enzyme, although a lysozyme may be also effective against Gram negative bacteria that possess relatively less peptidoglycan in a cell wall, particularly after the outer membrane has been compromised, such as by contact with an anti-cellular membrane agent such as an antimicrobial and/or antifouling peptide, a detergent, a metal chelator (e.g., a metal ion chelator, EDTA), or a combination thereof.
• an anti-cellular membrane agent such as an antimicrobial and/or antifouling peptide, a detergent, a metal chelator (e.g., a metal ion chelator, EDTA), or a combination thereof.
• Structural information for a wild-type lysozyme and/or a functional equivalent amino acid sequence for producing a lysozyme and/or a functional equivalent include Protein database bank entries: 1021, 1031, 1041, 1071, 1081, 1091, 1 101, 1111, 1121, 1131, 1141, 1 151, 1161, 1181, 1191, 1201, 1221, 1231, 1251, 1261, 1271, 1281, 1291, 1301, 1311, 1321, 1331, 1341, 1351, 1371, 1381, 1391, 1401, 1411, 1421, 1431, 1441, 1451, 1461, 1471, 1481, 1491, 1501, 1511, 1521, 1531, 1541, 1551, 1561, 1571, 1581, 1591, 1601, 1611, 1621, 1631, 1641, 1651, 1661,
• lysozyme examples include: a bacteriophage T4 lysozyme a from Escherichia coli expression; a mutant T4 lysozyme (e.g., a lysozyme comprising an engineered metal-binding site; an engineered thermostable lysozyme; a 199a; 199a and/or m102q mutant; a cavity producing mutants; an engineered salt bridge stability mutant; an engineered disulfide bond mutant; a g28a/i29a/g30a/c54t/c97a mutant; a I32a/l33a/t34a/c54t/c97a/e1 ⁇ 8v; r14a/k16a/i17a/k19a/t21a/e22a/c54t/c97a mutant; a y24a/y25a/t26a/i27a/c54t/c97a mutant; a lys
• Nucleotide and protein sequences for a lysozyme from various organisms are available via database such as, for example, KEGG.
• Examples of lysozyme and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA - 4069(LYZ); PTR - 450190(LYZ); MCC - 718361 (LYZ); MMU - 17105(Lyz2) 17110(Lyz1 ); RNO - 25211(Lyz2); DPO - Dpse_GA11118 Dpse_GA20595; AGA - AgaP_AGAP005717
• Lysostaphin (EC 3.4.24.75; CAS registry number: 9011-93-2) has been also referred to in that art as "glycyl-glycine endopeptidase.” Lysostaphin catalyzes the reaction: in a staphylococcal (e.g., S. aureus) peptidoglycan, hydrolyzes a -GIyGIy- bond in a pentaglycine inter-peptide link (e.g., cleaves the polyglycine cross-links in the peptidoglycan layer of the cell wall of a Staphylococcus sp.).
• staphylococcal e.g., S. aureus
• pentaglycine inter-peptide link e.g., cleaves the polyglycine cross-links in the peptidoglycan layer of the cell wall of a Staphylococcus sp.
• a lysostaphin typically comprises a zinc-dependent, 25-kDa endopeptidase with an activity optimum of about pH 7.5.
• Lysostaphin producing cells e.g., Staphylococcus simulans, ATCC 67080, 69764, 67079, 67076, and 67078
• methods for isolating a lysostaphin from a cellular material and/or a biological source have been described [see, for example, Recsei, P.A., et al., 1987; Thumm, G. and Gotz, F. 1997; Trayer, H. R., and Buckley, C. E., 1970; Browder, H.
• a lysostaphin comprises a commercially available lysostaphin (e.g., Sigma Aldrich).
• Structural information for a wild-type lysostaphin and/or a functional equivalent amino acid sequence for producing a lysostaphin and/or a functional equivalent include Protein database bank entries: 1QWY, 2B0P, 2B13, and/or 2B44.
• Examples of a lysostaphin and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HAR: HEAR2799; SAU: SA0265(lytM); SAV: SAV0276(lytM); SAW: SAHV_0274(lytM); SAM: MW0252(lytM); SAR: SAR0273(lytM); SAS: SAS0252; SAC: SACOL0263(lytM); SAB: SAB0215(lytM); SAA: SAUSA300_0270(lytM); SAX: USA300HOU_0289(lytM); SAO: SAOUHSC_00248; SAJ: SaurJH9_0260;
• SAH SaurJH1_0267; SAE: NWMN_0210(lytM); NPU: Npun_F1058 Npun_F4149 Npun_F4637 Npun_F5024 Npun_F6078; AVA: Ava_0183 Ava_2410 Ava_3195 Ava_4756 Ava_4929 Ava_C0210; AMR: AM1_4073 AM1 5374 and/or AM1 B0175.
• Libiase comprises an enzyme obtained from Streptomyces fulvissimus (e.g., Streptomyces fulvissimus TU-6) that it typically used to promote the lysis of Gram-positive bacteria (e.g., a Lactobacillus, an Aerococcus, a Listeria, a Pneumococcus, a Streptococcus).
• Gram-positive bacteria e.g., a Lactobacillus, an Aerococcus, a Listeria, a Pneumococcus, a Streptococcus.
• a libiase possesses a lysozyme and a ⁇ -N- acetyl-D-glucosaminidase activity, with activity optimum of about pH 4, and a stability optimum of about pH 4 to about pH 8.
• Commercial preparations of a libiase are available (Sigma-Aldrich).
• Libiase producing cells and methods for isolating a libiase from a cellular material and/or a biological source have been described (see, for example, Niwa et al. 2005; Ohbuchi, K. et al., 2001 ), and may be used in conjunction with the disclosures herein.
• Lysyl endopeptidase (EC 3.4.21.50; CAS registry number: 123175-82-6) has been also referred to in that art as "Achromobacter lyticus alkaline proteinase I"; “Achromobacter proteinase I”; “achromopeptidase”; “lysyl bond specific proteinase”; and/or “protease I,”
• a lysyl endopeptidase catalyzes the peptide cleavage reaction: at a Lys, including -LysPro-.
• the lysyl endopeptidase comprises a (trypsin family) family S1 peptidase.
• Lysyl endopeptidase producing cells and methods for isolating a lysyl endopeptidase from a cellular material and/or a biological source have been described (see, for example, Ahmed et al, 2003; Chohnan et al. 2002; Elliott, B.W. and Cohen, C. 1986; Ezaki, T and Suzuki, S., 1982; Jekel, P.A., et al., 1983; Li et al. 1998;
• Masaki, T. et al. 1981 Masaki, T. et al., 1981 ; Masaki, T. et al., 1981 ; Ohara, T. et al., 1989; Tsunasawa, S. et al., 1989), and may be used in conjunction with the disclosures herein.
• An example of a lysyl endopeptidase comprises a 27kDa "achromopeptidase" obtained from Achromobacter lyticus M497-1 that may be used to promote lysis of a Gram positive bacterium typically resistant to a lysozyme.
• the achromopeptidase has an activity optimum of about pH 8.5 to about pH 9, and an example of an achromopeptidase comprises a commercially available achromopeptidase (e.g., Sigma Aldrich; Wako Pure Chemical Industries, Ltd.).
• Structural information for a wild-type lysyl endopeptidase and/or a functional equivalent amino acid sequence for producing a lysyl endopeptidase and/or a functional equivalent include Protein database bank entries: 1arb and/or 1arc.
• Examples of a lysyl endopeptidase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: SRU: SRlM 622.
• Mutanolysin (EC 3.4.99.-) comprises a 23kD N-acetyl muramidase obtained from Streptomyces globisporus (e.g., ATCC 21553).
• a mutanolysin catalyzes the reaction: in a cell wall peptidoglycan- polysaccharide, cleavage of a N-acetylmuramyl- ⁇ (1-4)-N-acetylglucosamine bond.
• Examples of cells that mutanolysin acts on include Gram positive bacteria (e.g., a Listeria, a Lactobacillus, a Lactococcus).
• Mutanolysin producing cells and methods for isolating a mutanolysin from a cellular material and/or a biological source have been described (see, for example, Assaf, N. A., and Dick, W. A., 1993; Calandra, G.
• a mutanolysin's binding of a cell wall polymer uses carboxy terminal moiety(s) of the enzyme, so mutagenesis and/or truncation of those amino acids may effect binding and enzyme activity.
• An example of a mutanolysin comprises a commercially available mutanolysin (e.g., Sigma Aldrich).
• Cellulase (EC 3.2.1.4; CAS registry number: 9012-54-8) has been also referred to in that art as "4- (1 ,3;1 ,4)- ⁇ -D-glucan 4-glucanohydrolase,” “1 ,4-(1 ,3;1 ,4)- ⁇ -D-glucan 4-glucanohydrolase,” "9.5 cellulase,” “alkali cellulase,” “avicelase,” “celluase A; cellulosin AP,” “celludextrinase,” “cellulase A 3,” “endo-1 ,4- ⁇ -D- glucanase,” “endoglucanase D,” “pancellase SS,” “ ⁇ -1 ,4-endoglucan hydrolase,” and/or “ ⁇ -1 ,4-glucanase.”
• Cellulase catalyzes the reaction: in a cellulose, endohydrolysis of a (1 ,4)- ⁇ -glu
• a cellulase may possess the catalytic activity of: hydrolyse of a 1 ,4-linkage in a ⁇ -D-glucan also comprising a 1 ,3-linkage.
• hydrolyse of a 1 ,4-linkage in a ⁇ -D-glucan also comprising a 1 ,3-linkage.
• Cellulase producing cells and methods for isolating a cellulase from a cellular material and/or a biological source have been described [see, for example, Datta, P. K., et al., 1963; Myers, F. L. and Northcote, D. H., 1959; Whitaker, D. R. et al., 1963; Hatfield, R. and Nevins, D.J., 1986; Inohue, M.
• a commercially available cellulase preparation e.g., Sigma-Aldrich
• an additional enzyme retained and/or added during preparation such as a hemicellulase, to aid digestion of cellulose comprising substrates.
• Structural information for a wild-type cellulase and/or a functional equivalent amino acid sequence for producing a cellulase and/or a functional equivalent include Protein database bank entries: 1A39; 1A3H; 1AIW; 1CEC; 1CEM; 1CEN; 1CEO; 1CLC; 1CX1 ; 1 DAQ; 1 DAV; 1 DYM; 1 DYS; 1 E5J; 1 ECE; 1 EDG; 1 EG1 ; 1 EGZ; 1 F9D; 1 F9O; 1 FAE; 1 FBO; 1 FBW; 1 FCE; 1 G01 ; 1G0C; 1 G87; 1G9G; 1G9J; 1 GA2; 1GU3; 1GZJ; 1 H0B; 1 H11 ; 1 H1 N; 1 H2J; 1 H5V; 1 H8V; 1 HD5; 1 HF6; 1 IA6; 1 IA7; 1 IS9; 1J83; 1
• Examples of a cellulase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: DFRU: 144551(NEWSINFRUG00000162829) 157531(NEWSINFRUG00000148215)
• ECI UTI89_C4063(yhjM); ECP: ECP_3631 ; ECV: APECO1_2917(bcsZ); ECW: EcE24377A_4019(bcsZ); ECM: EcSMS35_3840(bcsZ); ECL: EcolC_0186; STY: STY4183(yhjM); STT: t3900(yhjM); SPT: SPA3473(yhjM); SEK: SSPA3243; SPQ: SPAB_04494; SEC: SC3551 ; SEH: SeHA_C3933(bcsZ); SEE: SNSL254_A3889(bcsZ); SEW: SeSA_A3812(bcsZ); SEA: SeAg_B3825(bcsZ); SED: SeD_A3993(bcsZ); SEG: SG3819(bcsZ); BCN: Bc
• Chitinase (EC 3.2.1.14; CAS registry number: 9001-06-3) has been also referred to in that art as "(1 ⁇ 4)-2-acetamido-2-deoxy- ⁇ -D-glucan glycanohydrolase," “1 ,4- ⁇ -poly-N-acetylglucosaminidase,” “chitodextrinase,” “poly[1 ,4-(N-acetyl- ⁇ -D-glucosaminide)] glycanohydrolase,” “poly- ⁇ -glucosaminidase,” and/or " ⁇ -1 ,4-poly-N-acetyl glucosamidinase.”
• a chitinase catalyzes the reaction: random hydrolysis of a N- acetyl- ⁇ -D-glucosaminide (1 ⁇ 4)- ⁇ -linkage in a chitin; and random hydrolysis of a N-acetyl
• a chitinase may possess the catalytic activity of a lysozyme.
• Chitinase producing cells and methods for isolating a chitinase from a cellular material and/or a biological source have been described [see, for example, Fischer, E. H. and Stein, E.A. Cleavage of O- and S-glycosidic bonds (survey), in Boyer, P. D., Lardy, H. and Myrback, K. (Eds.), The Enzymes, 2nd end., vol. 4, pp. 301-312, 1960; Tracey, M.V., 1955], and may be used in conjunction with the disclosures herein.
• a chitinase comprises a commercially available chitinase (e.g., Sigma Aldrich).
• Structural information for a wild-type chitinase and/or a functional equivalent amino acid sequence for producing a chitinase and/or a functional equivalent include Protein database bank entries: 1CNS; 1CTN; 1 D2K; 1 DXJ; 1 E6Z; 1 ED7; 1 EDQ; 1 EHN; 1 EIB; 1 FFQ; 1 FFR; 1GOI; 1 GPF; 1 H0G; 1 H0I; 1 HKI; 1 HKJ; 1 HKK; 1 HKM; 1 HVQ; 1 ITX; 1 K85; 1 K9T; 1 KFW; 1 KQY; 1 KQZ; 1 KR0; 1 KR1 ; 1 LL4; 1 LL6; 1 LL7; 1 LLO; 1 NH6; 1 O6I; 1OGB;
• Examples of a chitinase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA: 1 118(CHITI ) 27159(CHIA); PTR: 457641(CHITI ); MCC: 703284(CHIA) 703286(CHITI ); MMU: 71884(Chit1 ) 81600(Chia); CFA: 479904(CHIA); BTA: 282645(CHIA); DECB: 100065255(LOC100065255); MDO: 100015954(LOC100015954) 100030396(LOC100030396) 100030417(LOC100030417) 100033109(LOC100033109) 100033117(LOC1000331 17) 100033119(LOC100033119); OAA: 100089089(LOC100089089); GGA: 395072(CHIA); XLA: 444170(MGC80644); XTR: 44
• ⁇ -agarase (EC 3.2.1.158; CAS no. 63952-00-1 ) has been also referred to in that art as "agarose 3- glycanohydrolase,” “agarase,” and/or “agaraseA33.”
• ⁇ -agarase catalyzes the reaction: in an agarose, endohydrolysis of a 1 ,3- ⁇ -L-galactosidic linkage, producing an agarotetraose.
• Porphyran, a sulfated agarose may also be cleaved.
• an ⁇ -agarase obtained from a Thalassomonas sp.
• ⁇ -agarase activity may be enhanced by Ca 2+ .
• ⁇ -agarase producing cells and methods for isolating an ⁇ -agarase from a cellular material and/or a biological
• 5842-02103 75 source have been described (see, for example, Ohta, Y., et al., 2005; Potin, P., et al., 1993), and may be used in conjunction with the disclosures herein.
• ⁇ -agarase (EC 3.2.1.81 ; CAS registry number: 37288-57-6) has been also referred to in that art as "agarose 4-glycanohydrolase,” “AgaA,” “AgaB,” “agarase,” “agarose 3-glycanohydrolase,” and/or "endo- ⁇ - agarase.”
• a ⁇ -agarase catalyzes the reaction: in agarose, hydrolysis of a 1 ,4- ⁇ -D-galactosidic linkage, producing a tetramer.
• An AgaA derived from Zobellia galactanivorans produces a neoagarohexaose and a neoagarotetraose
• an AgaB produces a neoagarobiose and a neoagarotetraose.
• a ⁇ -agarase also cleaves a porphyran. ⁇ -agarase producing cells and methods for isolating a ⁇ -agarase from a cellular material and/or a biological source have been described (see, for example, Allouch, J., et al., 2003; Duckworth, M. and Turvey, J. R. 1969; Jam, M. et al., 2005; Ohta, Y.
• Structural information for a wild-type ⁇ -agarase and/or a functional equivalent amino acid sequence for producing a ⁇ -agarase and/or a functional equivalent include Protein database bank entries: 1 O4Y, 1O4Z, and/or 1 URX.
• Examples of a ⁇ -agarase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: PPF: Pput_1162; PAT: Patl_1904 Patl_1971 Patl_2341 Patl_2640 Patl_2642; SDE: Sde_1 175 Sde_1176 Sde_2644 Sde_2650 Sde_2655; RPB: RPB_3029; RPD: RPD_2419; RPE: RPE_4620; SCO: SCO3471(dagA); and/or RBA: RB3421(agrA).
• N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28; CAS registry number: 9013-25-6) has been also referred to in that art as "peptidoglycan amidohydrolase," “acetylmuramoyl-alanine amidase,” “acetylmuramyl-alanine amidase,” “acetylmuramyl-L-alanine amidase,” “murein hydrolase,” "N- acetylmuramic acid L-alanine amidase,” “N-acetylmuramoyl-L-alanine amidase type I,” “N-acetylmuramoyl-L- alanine amidase type II,” “N-acetylmuramylalanine amidase,” “N-acetylmuramyl-L-alanine amidase,” and/or "N-acylmuramyl-L-
• ⁇ /-acetylmuramoyl-L-alanine amidase producing cells and methods for isolating a N- acetylmuramoyl-L-alanine amidase from a cellular material and/or a biological source have been described [see, for example, Ghuysen, J.-M. et al. 1969; Herbold, D. R. and Glaser, L. 1975; Ward, J.B.et al., 1982), and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type N- acetylmuramoyl-L-alanine amidase and/or a functional equivalent amino acid sequence for producing a N- acetylmuramoyl-L-alanine amidase and/or a functional equivalent include Protein database bank entries: 1ARO, 1GVM, 1 H8G, 1 HCX, 1J3G, UWQ, 1 LBA, 1X60, 1XOV, 2AR3, 2BGX, 2BH7, and/or 2BML.
• acetylmuramoyl-L-alanine amidase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA: 114770(PGLYRP2) 1 14771(PGLYRP3) 571 15(PGLYRP4) 8993(PGLYRPI ); PTR: 455797(PGLYRP2) 737434(PGLYRP3) 737562(PGLYRP4); MCC: 714583(LOC714583) 718287(PGLYRP2)
• a lytic transglycosylase (“lytic murein transglycosylase,” EC 3.2.1.-) demonstrates exo-N- acetylmuramidase activity, and can cleave a glycan strand comprising linked a peptide and/or a glycan strand that lack linked peptides with similar efficiency.
• a lysozyme and a lytic transglycosylase cleaves the ⁇ 1 ,4-glycosidic bond between a N-Acetyl-D-Glucosamine ("GIcNAc”) and a N-Acetylmuramic acid (“MurNAc”), but a lytic transglycosylase has a transglycosylation reaction producing a 1 ,6-anhydro ring at the MurNAc.
• a lytic transglycosylase may be inhibited by a ⁇ /-acetylglucosamine thiazoline.
• An example of a lytic transglycosylase includes a MItB produced from Psudomonas aeruginosa.
• a lytic transglycosylase generally may be classified as a family 1 , a family 2 (e.g., MItA), a family 3 (e.g., MItB) or a family 4 lytic transglycosylase (i.e., generally bacteriophage), based on a similar amino acid sequence, particularly comprising a conserved amino acid.
• a family 1 lytic transglycosylase may be classified as a 1 A type (e.g., Slt70), a 1 B type (e.g., MItC), a 1 C type (e.g., EmtA), a 1 D type (e.g., MItD), or a 1 E type (e.g., YfhD).
• Lytic transglycosylase producing cells and methods for isolating a lytic transglycosylase from a cellular material and/or a biological source have been described [see, for example, Holtje et al, 1975; Thunnissen et al. 1994; Scheurwater et al, 2007; Reid et al., 2004; Blackburn and Clark, 2001 ), and may be used in conjunction with the disclosures herein.
• Crystal structures for various lytic transglycosylases include those for a Neisseria gonorrhoeae MItA and an E. coli MItA; an E. coli Slt70; a phage ⁇ lytic transglycosylase; and an E. coli Slt35 (Powell et al., 2006; van Straaten et al., 2005; van Straaten et al., 2007; van Asselt et al., 1999a; Thunnissen et al., 1994; Leung et al., 2001 ; van Asselt et al., 1999b).
• a lytic transglycosylase active site generally comprises a glutamic acid (e.g., a Glu162 of Slt35; a Glu478 of Slt70), with a relatively more hydrophobic active site than a goose egg white lysozyme.
• Another active site residue may comprise an aspartic acid (e.g., an Asp308 of MItA). Structural information for a wild-type lytic transglycosylase and/or a functional equivalent amino acid
• sequence for producing a lytic transglycosylase and/or a functional equivalent include Protein database bank entries: 1Q2R, 1Q2S, 2PJJ, 2PIC, 1QSA, 2PNW, 1QTE, 1QUS, 1QUT, 1QDR, 1SLY, 1 D0K, 1 D0L, 1 D0M, 3BKH, 3BKV, and/or 2AE0.
• Examples of lytic transglycosylase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: ECO: b2701(mltB); ECJ: JW2671(mltB); ECE: Z4004(mltB); ECS: ECs3558; ECC: c3255(mltB); YPY: YPK_1464; YEN: YE1242(mltB); SFL: SF2724(mltB); SFX: S2915(mltB); SFV: SFV_2804(mltB); SSN: SSON_2845(mltB); SBO: SBO_2817(mltB); SBC: SbBS512_E3176(mltB); SDY: SDY_2897(mltB); ECA: ECA1083(mltB); ENT: Ent638_31
• Bcen2424_0538 BAM: Bamb_0443; BMU: Bmul_2856; BPS: BPSL3046; BPM: BURPS1710b_3570(mltA); BPL: BURPS1106A_3578(mltA); BPD: BURPS668_3551(mltA); BTE: BTHJ2905; PNU: Pnuc_0151 ; PNE: Pnec_0165; BPE: BP3268; BPA: BPP4152; BJA: blrO643; BRA: BRADO0205; MAG: amb4542; MGM: Mmc1_0484; and/or SYP: SYNPCC7002_A2370(mltA).
• Glucan endo-1 ,3- ⁇ -D-glucosidase (EC 3.2.1.39; CAS registry number: 9025-37-0) has been also referred to in that art as "3- ⁇ -D-glucan glucanohydrolase,” “(1 ⁇ 3)- ⁇ -glucan 3-glucanohydrolase,” “1 ,3- ⁇ -D- glucan 3-glucanohydrolase,” “1 ,3- ⁇ -D-glucan glucanohydrolase,” “callase,” "endo-(1 ,3)- ⁇ -D-glucanase,” “endo-1 ,3- ⁇ -D-glucanase,” “endo-1 ,3- ⁇ -glucanase,” “endo-1 ,3- ⁇ -glucosidase,” “kitalase,” “laminaranase,” “laminarinase,” “oligo-1 ,3-glucosidase,” and/or " ⁇ -1 ,3-glucana
• a glucan endo-1 ,3- ⁇ -D-glucosidase may possess the catalytic activity of hydrolyzing a laminarin, a pachyman, a paramylon, or a combination thereof, and also have a limited hydrolysis activity against a mixed-link (1 ,3-1 ,4-)- ⁇ -D-glucan.
• a glucan endo-1 ,3- ⁇ -D-glucosidase may be useful against fungal cell walls.
• Glucan endo-1 , 3- ⁇ -D-glucosidase producing cells and methods for isolating a glucan endo-1 , 3- ⁇ -D- glucosidase from a cellular material and/or a biological source have been described [see, for example, Chesters, C. G. C. and Bull, AT., 1963; Reese, ET. and Mandels, M., 1959; Tsuchiya, D., and Taga, M., 2001 ; Petit, J., et al., 10:4-5, 1994], and may be used in conjunction with the disclosures herein.
• An enzyme preparation comprising a glucan endo-1 ,3- ⁇ -D-glucosidase prepared from a Rhizoctonia solani ("Kitalase"), or a Trichoderma harzianum (Glucanex®) (Sigma-Aldrich).
• Structural information for a wild-type glucan endo-1 ,3- ⁇ -D-glucosidase and/or a functional equivalent amino acid sequence for producing a glucan endo- 1 ,3- ⁇ -D-glucosidase and/or a functional equivalent include Protein database bank entries: 1GHS, 2CYG, 2HYK, and/or 3DGT.
• Examples of an endo-1 ,3- ⁇ -D-glucosidase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: DBMO: Bmb007310; ATH: AT3G57260(BGL2); DPOP: 769807(fgenesh4_pg.C_LG_X001297); MGR: MGG_09733; TET: TTHERM_00243770 TTHERM_00637420 TTHERM_00956460 TTHERM_00956480; SFR: Sfri_1319; SAZ: Sama_1396; SDE: Sde_3121 ; PIN: Ping_0554; RLE: RL3815; MMR: Mmar10_0247; NAR: Saro_1608; SAL: Sala_0919; RHA: RHA1_ro05769 RHA1_ro05771 ; and/or FJO
• Endo-1 , 3(4)- ⁇ -glucanase (EC 3.2.1.6; CAS registry number: 62213-14-3) has been also referred to in that art as "3-(1 ⁇ 3;1 ⁇ 4)- ⁇ -D-glucan 3(4)-glucanohydrolase,” “1 ,3-(1 ,3;1 ,4)- ⁇ -D-glucan 3(4)- glucanohydrolase,” "endo-1 ,3-1 ,4- ⁇ -D-glucanase,” “endo-1 ,3- ⁇ -D-glucanase,” “endo-1 ,3- ⁇ -D-glucanase,” “endo-1 ,3- ⁇ -glucanase,” “endo-1 ,3- ⁇ -glucanase,” “endo- ⁇ -(1 ⁇ 3)-D-glucanase,” “endo- ⁇ -(1-3)-D-glucanase,” “endo- ⁇ -1 , 3(4)- glucanase,” “end
• Endo-1 ,3(4)- ⁇ -glucanase producing cells and methods for isolating an endo-1 ,3(4)- ⁇ - glucanase from a cellular material and/or a biological source have been described [see, for example, Barras, D. R. and Stone, B.A., 1969a; Barras, D. R. and Stone, B.A., 1969b; Cunningham, L. W. and Manners, D.J., 1961 ; Reese, ET.
• Structural information for a wild-type endo-1 , 3(4 )- ⁇ - glucanase and/or a functional equivalent amino acid sequence for producing an endo-1 ,3(4)- ⁇ -glucanase and/or a functional equivalent include Protein database bank entries: 1 UP4, 1 UP6, 1 UP7, and/or 2CL2.
• Examples of an endo-1 ,3(4)- ⁇ -glucanase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: NCR: NCU04431 NCU07076; PAN: PODANSg699 PODANSg9033; FGR: FG04768.1 FG06119.1 FG08757.1 ; AFM: AFUA_1G04260 AFUA_1G05290 AFUA_3G03080 AFUA_4G 13360; AFUA_5G02280 AFUA_5G 13990 AFUA_5G14030 AFUA_6G14540; ANG: An01g03090; DPCH: 10833(fgenesh1_pm.C_scaffold_14000004) 123909(e_gwh2.6.417.1 ); LBC: LACBIDRAFT_174636 LACBIDRAFT_191735 LACBIDRAF
• ⁇ -lytic metalloendopeptidase (EC 3.4.24.32; CAS no. 37288-92-9) has been also referred to in that art as "achromopeptidase component," “Myxobacter ⁇ -lytic proteinase,” “Myxobacter495 ⁇ -lytic proteinase,” “Myxobacterium sorangium ⁇ -lytic proteinase,” “ ⁇ -lytic metalloproteinase,” and/or " ⁇ -lytic protease.”
• a ⁇ -lytic metalloendopeptidase catalyzes the reaction: a N-acetylmuramoyl Ala cleavage, as well as an insulin B chain cleavage.
• a ⁇ -lytic metalloendopeptidase may be used, for example, against a bacterial cell wall, ⁇ - lytic metalloendopeptidase producing cells and methods for isolating a ⁇ -lytic metalloendopeptidase from a cellular material and/or a biological source (e.g., an Achromobacter lyticus; a Lysobacter enzymogenes) have been described [see, for example, Whitaker, D. R. et al.,1965; Whitaker, D. R. and Roy, C, 1967; Li, S. L. et al., 1990; Altmann, F. et al., 1986; Plummer, T.H., Jr.
• a biological source e.g., an Achromobacter lyticus; a Lysobacter enzymogenes
• 3-deoxy-2-octulosonidase (EC 3.2.1.124; CAS no. 103171-48-8) has been also referred to in that art as "capsular-polysaccharide 3-deoxy-D-manno-2-octulosonohydrolase," “2-keto-3-deoxyoctonate hydrolase,” “octulofuranosylono hydrolase,” “octulopyranosylonohydrolase,” and/or “octulosylono hydrolase.”
• a 3-deoxy-2-octulosonidase catalyzes the reaction: endohydrolysis of the ⁇ -ketopyranosidic linkage of a 3- deoxy-D-manno-2-octulosonate in a capsular polysaccharide.
• a 3-deoxy-2-octulosonidase acts on a polysaccharide of a bacterial (e.g., an Escherichia coli) cell wall.
• 3-deoxy-2-octulosonidase producing cells and methods for isolating a 3-deoxy-2-octulosonidase from a cellular material and/or a biological source
• the reaction may promote the glycosylation of the glyglucosamine residue, and produce a peptide comprising an aspartate and a substituted ⁇ /-acetyl- ⁇ -D-glucosaminylamine.
• Peptide- ⁇ / ⁇ /V-acetyl- ⁇ -glucosaminyOasparagine amidase does not substantively act on (GlcNAc)Asn, as 3 or more amino acids in the substrate promotes the reaction.
• Peptide- ⁇ / 4 -( ⁇ /-acetyl- ⁇ - glucosaminyl)asparagine amidase producing cells and methods for isolating an eptide- ⁇ /4-( ⁇ /-acetyl- ⁇ - glucosaminyl)asparagine amidase from a cellular material and/or a biological source have been described [see, for example, Plummer, T. H., Jr. and Tarentino, A.L., 1981 ; Takahashi, N. and Nishibe, H., 1978; Takahashi, N., 1977; Tarentino, A.L. et al., 1985], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type peptide-/V*-( ⁇ /-acetyl- ⁇ -glucosaminyl) asparagine amidase and/or a functional equivalent amino acid sequence for producing a peptide- ⁇ /4-( ⁇ /-acetyl- ⁇ - glucosaminyl)asparagine amidase and/or a functional equivalent include Protein database bank entries:
• peptide-N 4 -(N-acetyl- ⁇ -glucosaminyl)asparagine amidase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA: 55768(NGLYI ); PTR: 460233(NGLYI ); MCC: 700842(LOC700842); DECB: 100059456(LOC100059456); OAA: 100075786(LOC100075786); GGA: 420655(NGLYI ); DRE: 553627(zgc: 110561 ); DFRU:
• Mannosyl-glycoprotein endo- ⁇ - ⁇ /-acetylglucosaminidase (EC 3.2.1.g6; CAS no. 37278-88-g) has been also referred to in that art as "glycopeptide-D-mannosyl- ⁇ /4-( ⁇ /-acetyl-D-glucosaminyl)2-asparagine 1 ,4- ⁇ /-acetyl- ⁇ -glucosaminohydrolase," "di- ⁇ /-acetylchitobiosyl ⁇ - ⁇ /-acetylglucosaminidase,” “endoglycosidase S,”
• Mannosyl-glycoprotein endo- ⁇ - ⁇ /- acetylglucosaminidase producing cells and methods for isolating a mannosyl-glycoprotein endo- ⁇ - ⁇ /- acetylglucosaminidase from a cellular material and/or a biological source have been described [see, for example, Chien, S., et al., 1977; Koide, N. and Muramatsu, T. ,1974; Pierce, R.J. et al.,1979; Pierce, R.J. et al., 1980; Tai, T.
• Structural information for a wild-type mannosyl-glycoprotein endo- ⁇ - ⁇ /- acetylglucosaminidase and/or a functional equivalent amino acid sequence for producing a mannosyl- glycoprotein endo- ⁇ - ⁇ /-acetylglucosaminidase and/or a functional equivalent include Protein database bank entries: 1C3F, 1C8X, 1C8Y, 1C90, 1C91 , 1C92, 1C93, 1 EDT, 1 EOK, 1 EOM, and/or 2EBN.
• Examples of mannosyl-glycoprotein endo- ⁇ - ⁇ /-acetylglucosaminidase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: HSA: 64772(FLJ21865); OAA: 100089364(LOC100089364); DCIN: 254322(gw1.55.22.1 ); DAME: 24424(ENSAPMG00000015707) 33583(ENSAPMG00000015707); DBMO: BmbO29819; TCA: 658146(LOC658146); BMY: Bm1_17595; DHA: DEHA0F20174g; PIC: PICST_32069(HEX1 ); MBR: MONBRDRAFT_34057; TBR: TbO9.160.2050; BCL: ABC3097; LSP: Bsph_1040; SAU: SA0905(atl); SAV: SAV
• i-carrageenase (EC 3.2.1.157) has been also referred to in that art as "i-carrageenan 4- ⁇ -D- glycanohydrolase (configuration-inverting)."
• An ⁇ -carrageenase catalyzes the reaction: in an ⁇ -carrageenan, endohydrolysis of a 1 ,4- ⁇ -D-linkage between a 3,6-anhydro-D-galactose-2-sulfate and a D-galactose A- sulfate, i-carrageenase producing cells and methods for isolating an ⁇ -carrageenase from a cellular material and/or a biological source have been described [see, for example, Barbeyron, T.
• Structural information for a wild-type i-carrageenase and/or a functional equivalent amino acid sequence for producing a i-carrageenase and/or a functional equivalent include Protein database bank entries: 1 H80 and/or 1 KTW.
• ⁇ -carrageenase (EC 3.2.1.83; CAS no. 37288-59-8) has been also referred to in that art as "K- carrageenan 4- ⁇ -D-glycanohydrolase," “ ⁇ -carrageenan 4- ⁇ -D-glycanohydrolase (configuration-retaining).” K- carrageenase catalyzes the reaction: in a ⁇ -carrageenans, endohydrolysis of a 1 ,4- ⁇ -D-linkage between a 3,6-anhydro-D-galactose and a D-galactose 4-sulfate. ⁇ -carrageenase often acts against an algae (e.g., red algae).
• algae e.g., red algae
• ⁇ -carrageenase producing cells and methods for isolating a ⁇ -carrageenase from a cellular material and/or a biological source have been described [see, for example, Weigl, J. and Yashe, W., 1966; Potin, P. et al., 1991 ; Potin, P. et al., 1995; Michel, G. et al., 1999; Michel, G., et al., 2001.], and may be used in conjunction with the disclosures herein.
• Structural information for a wild-type ⁇ -carrageenase and/or a functional equivalent amino acid sequence for producing a ⁇ -carrageenase and/or a functional equivalent include Protein database bank entries: 1 DYP.
• Examples of ⁇ -carrageenase and/or a functional equivalent KEEG sequences for production of wild-type and/or a functional equivalent nucleotide and protein sequence include: RBA: RB2702.
• ⁇ -carrageenase (EC 3.2.1.162) has been also referred to in that art as "endo-(1 ⁇ 4)- ⁇ -carrageenose 2,6,2'-trisulfate-hydrolase," and/or "endo- ⁇ -1 ,4-carrageenose 2,6,2'-trisulfate-hydrolase.”
• a ⁇ -carrageenase catalyzes the reaction: in a ⁇ -carrageenan, endohydrolysis of a (1 ,4)- ⁇ -linkage, producing a ⁇ -D-Galp2,6S2- (1 ,3)- ⁇ -D-Galp2S-(1 ,4)- ⁇ -D-Galp2,6S2-(1 ,3)-D-Galp2S tetrasaccharide.
• ⁇ -carrageenase producing cells and methods for isolating a ⁇ -carrageenase from cellular materials e.g., Pseudoalteromonas sp
• biological sources e.g., Ohta, Y. and Hatada, 2006
• ⁇ -neoagaro-oligosaccharide hydrolase (EC 3.2.1.159) has been also referred to in that art as " ⁇ - neoagaro-oligosaccharide 3-glycohydrolase,” " ⁇ -neoagarooligosaccharide hydrolase,” and/or " ⁇ -NAOS hydrolase.”
• An ⁇ -neoagaro-oligosaccharide hydrolase catalyzes the reaction: hydrolysis of a 1 ,3- ⁇ -L- galactosidic linkage in a neoagaro-oligosaccharide, wherein the substrate is a pentamer or smaller, producing a D-galactose and a 3,6-anhydro-L-galactose.
• ⁇ -neoagaro-oligosaccharide hydrolase producing cells and methods for isolating a NAME from a cellular material and/or a biological source have been described [see, for example, Sugano, Y., et al. 1994], and may be used in conjunction with the disclosures herein.
• An endolysin may be used for a Gram positive bacteria, such as one that may be resistant to a lysozyme.
• An endolysin comprises a phage encoded enzyme that fosters release of a new phage by destruction of a cell wall.
• An endolysin may comprise a N-acetylmuramidase, a N-acetylglucosaminidae, an emdopeptidase, and/or an amidase.
• An endolysin may be translocated by phage encoded holin protein in disrupting a cytosolic membrane (Wang et al., 2000).
• 5842-02103 83 monocytogenes bacteriophage-lysin have been recombinantly expressed in a Lactoccus lactus and/or an E. coli (Loessner et al. 1995; Gaeng et al. 2000; O'Flaherty et al. 2005).
• An autolysin such as, for example, from Staphylococcus aureus, Bacillus subtilis, or Streptococcus pneumonia, may also be used as an antimicrobial and/or an antifouling enzyme (Smith et al, 2000; Lopez et al. 2000; Foster et al. 1995).
• a protease may be used to cleave the mannoprotein outer cell wall layer, such as for a fungi such as a yeast.
• a glucanase such as, for example, a beta(1->6) glucanase, a glucan endo-1 ,3- ⁇ -D-glucosidase, and/or an endo-1 , 3(4)- ⁇ -glucanase can then more easily cleave glucan from the inner cell wall layer(s). Combinations of a protease and a glucanase may be used to produce an improved lytic activity.
• a reducing agent such as a dithiothreitol of beta-mercaptoethanol, may aid in allowing enzyme contact with the inner cell wall by breaking a disulfide linkage, such as between a cell wall protein and a mannose.
• a mannose, a chitinase, a proteinase, a pectinase, an amylase, or a combination thereof may also be used, such as for aiding cell wall component cleavage.
• Examples of enzymes that degrade fungal cell walls include those produced by an Arthrobacter sp., a Celluloseimicrobium cellulans ("Oerskovia xanthineolytica LL G109") (DSM 10297), a Cellulosimicrobium cellulans ⁇ "Arthobacter lueus 73/14") (ATCC 21606), a Cellulosimicrobium cellulans TK-1 , a Rarobacter faecitabidus, a Rhizoctonia sp., or a combination thereof.
• An Arthrobacter sp. a Celluloseimicrobium cellulans ("Oerskovia xanthineolytica LL G109") (DSM 10297), a Cellulosimicrobium cellulans ⁇ "Arthobacter lueus 73/14”) (ATCC 21606), a Cellulosimicrobium cellulans TK-1 , a
• a Celluloseimicrobium cellulans (ATCC 21606) produces a protease and a glucanase ("lyticase") with a functional optimum of about pH 10 and about pH 8.0, respectively (Scott and Schekman, 1980; Shen et al., 1991 ).
• a Celluloseimicrobium cellulans (DSM 10297) produces a protease with functional optimums of about pH 9.5 to about pH 10, and a glucanase with a functional optimum of about pH 8.0 and about 4O 0 C (Salazar et al.
• a Rarobacter faecitabidus produces a protease effective against cell wall a component (Shimoi et al, 1992).
• a Rarobacter sp. produces a glucanase with a functional optimum of about pH 6 to about pH 7, and about 4O 0 C (Kobayashi et al.1981 ).
• commercially available enzyme preparations such as a zymolase and/or a lyticase (Sigma-Aldrich), generally comprising a ⁇ -1 ,3-glucanase and another enzyme, may be used.
• antibiological proteinaceous molecule examples include the peptide sequences described in U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097, and Patent Application Nos. 10/884,355 and 11/368,086, and these antibiological peptides (e.g., antifungal peptides) include those of SEQ ID No.
• 5842-02103 84 library may be used individually (e.g., SEQ ID No. 14, SEQ ID No. 41 ), or in a combination (e.g., a mixture of SEQ ID Nos. 25-47).
• These sequences establish a number of precise chemical compositions which possess antibiological (e.g., antifungal) activity.
• antibiological e.g., antifungal
• one or more of these proteinaceous sequences may be used against a spectrum of fungi.
• One or more of these sequences may be useful, for example, in a material formulation and/or an application for an antibiological proteinaceous composition (e.g., for treating and/or protecting building materials and other non-living objects from infestation by a cell such as a fungi).
• a proteinaceous molecule e.g., a peptide
• a sequence may be produced and used in the forward and/or reverse pattern (e.g., synthesized C-terminal to N-terminal manner, or the reverse N-terminal to C-terminal).
• a relatively variable composition e.g., "XXXXRF"; SEQ ID No.
• a proteinaceous composition (e.g., a peptide composition) may exhibit variable abilities to, for example, prevent and/or inhibit growth (e.g., fungal growth) as adjudged by the minimal inhibitory concentrations (MIC mg/ml) and/or the concentrations necessary to inhibit growth of fifty percent of a population of cells (e.g., a fungal spore, a cell, a mycelia) (IC50 mg/ml).
• MIC mg/ml minimal inhibitory concentrations
• the MICs may range depending upon the proteinaceous additive (e.g., a peptide additive comprising one or more SEQ ID Nos. 1 to 199) and target organism from about 3 to about 1700 mg/ml (e.g., about 3 to about 300 mg/ml), while the IC50's may range depending upon the proteinaceous additive (e.g., a peptide additive) and target organisms from about 2 to about 1700 mg/ml (e.g., about 2 to about 100 mg/ml).
• the proteinaceous additive e.g., a peptide additive comprising one or more SEQ ID Nos. 1 to 199
• target organism e.g., about 3 to about 300 mg/ml
• the IC50's may range depending upon the proteinaceous additive (e.g., a peptide additive) and target organisms from about 2 to about 1700 mg/ml (e.g., about 2 to about 100 mg/ml).
• Target organisms susceptible to these amounts include, for example, a Fusarium oxysporum, a Fusariam Sambucinum, a Rhizoctonia Solani, a Ceratocystis Fagacearum, a Pphiostoma ulmi, a Pythium ultimum, a Magaporthe Aspergillus nidulans, an Aspergillus fumigatus, and/or an Aspergillus Parasiticus.
• a peptide e.g., an antifungal peptide
• a peptide sequence such as SEQ ID Nos. 6, 7, 8, 9, and/or 10, may act on a cell such as a bacteria and a fungi.
• a peptide sequence such as SEQ ID Nos.
• 41 , 197, 198, and 199 can inhibit growth of an Erwinia amylovora, an Erwinia carotovora, an Escherichia coli, an Ralstonia solanocerum, an Staphylococcus aureus, and/or an Streptococcus faecalis in standard media at IC50's of between about 10 to about 1100 mg/ml and MICs of between about 20 to about 1700 mg/ml.
• an active antibiological agent e.g., an antifungal agent
• an antibiological agent used in a material formulation e.g., a paint, a coating composition
• possible modes of action of a peptide, a polypeptide, and/or a protein, by which they exert their effect(s) may include, for example, destabilizing a cellular (e.g., a fungal cell) membrane (e.g., perturb membrane functions responsible for osmotic balance); a disruption of macromolecular
• 5842-02103 85 synthesis (e.g., cell wall biosynthesis) and/or metabolism; disruption of appressorium formation; or a combination thereof, (see, for example, Fiedler, H. P., et al. 1982; Isono, K. and S. Suzuki. 1979; Zasloff, M. 1987; US Patent Application 10/601 ,207).
• a proteinaceous composition may comprise one or more peptide(s), polypeptide(s), and/or protein(s) (e.g., an enzyme, an antimicrobial enzyme, an anti-cell wall enzyme, an anti-cell membrane enzyme).
• one or more peptide(s) and enzyme(s) may be selected for a mixture due to related activity(s) (e.g., antibiological activity).
• a proteinaceous composition e.g., a peptide composition
• a homogeneous peptide composition may comprise a single active peptide specie of a well-defined sequence, though a minor amount (e.g., less than about 20% by moles) of impurity(s) may coexist with the peptide in the peptide composition so long as the impurity does not interfere with a desired property(s) of the active peptide (e.g., a growth inhibitory property).
• a peptide may have a completely defined sequence.
• an antifungal peptidic agent may comprise a single peptide of a precise sequence (e.g., the hexapeptide of SEQ ID No. 198, SEQ ID No. 41 , SEQ ID No.
• a proteinaceous composition e.g., a peptide
• a demonstrable activity e.g., antibiotic activity, antifungal activity
• the peptide composition may instead comprise a mixture of peptides (e.g., an aliquot of a peptide library, a mixture of isolated peptides).
• the peptide composition comprising a mixture of peptides may comprise at least one active peptide (e.g., a peptide having antifungal activity).
• a peptide composition may comprise an active (e.g., an antifungal) peptide, wherein the peptide composition may be impure to the extent that the peptide composition may comprise one or more peptides of unknown exact sequence which may or may not have activity (e.g., an antifungal activity).
• a mixed proteinaceous composition may be used treat a target (e.g., a biological target, a fungal target, a viral target) with lower concentrations of numerous active additives (e.g., a plurality of active peptides, a plurality of antifungal peptides) rather than a higher concentration of a single chemical composition (e.g., a single peptide sequence); a mixed proteinaceous composition may be used to treat an array of targets (e.g., a plurality of target organisms, a plurality of fungal organisms) each with a different causative agent; or combination thereof.
• a target e.g., a biological target, a fungal target, a viral target
• numerous active additives e.g., a plurality of active peptides, a plurality of antifungal peptides
• a single chemical composition e.g., a single peptide sequence
• a mixed proteinaceous composition may be used to treat an array of
• a proteinaceous (e.g., a peptide mixture, a synthetic peptide combinatorial library) comprises an equimolar mixture of proteinaceous molecules (e.g., an equimolar mixture of peptides).
• at least one (e.g., 1 , 2, 3, 4, 5, 6, or more such as to about 10,000 amino acids) of the amino acid residue(s) e.g., an N-terminal amino acid residue, a C-terminal amino acid residue
• proteinaceous molecule e.g., a peptide
• a proteinaceous molecule mixture e.g., a peptide mixture such as a peptide library.
• the peptidic agent may comprise a peptide library aliquot comprising a mixture of peptides in which at least two, three and/or four or more of the N-terminal amino acid residues are known.
• the amino acid residue(s) may be in common for a plurality of proteinaceous molecules (e.g., for
• a mixed proteinaceous composition (e.g., a mixed peptide composition) comprises one or more variable amino acid residue(s), and such a proteinaceous molecule mixture (e.g., a peptide mixture, a peptide library) may be selected for use due to the increased cost of testing and/or the cost of producing a completely defined proteinaceous molecule (e.g., an defined antibiotic peptide).
• the sequence of a peptide may be defined for only certain of the C-terminal amino acid residues leaving the remaining amino acid residues defined as equimolar ratios.
• certain of the peptides of SEQ ID Nos. 1 to 199 have somewhat variable amino acid compositions.
• the variable residue(s) in each aliquot of the SPCL comprising a given SEQ ID Nos. having a variable residue, may each be uniformly represented in equimolar amounts by one of nineteen different naturally-occurring amino acids in one or the other stereoisomeric form.
• the variable residue(s) may be rapidly defined using the method described in one or more of U.S.
• activity e.g., controlling fungal growth
• peptides encompassed by the C-terminal sequence "XXXXRF" SEQ ID No. 1
• SEQ ID No. 1 SEQ ID No. 1
• peptides may have a mixed equimolar array of peptides representing the same nineteen amino acid residues, some of which may have antibiological (e.g., antifungal activity) and some of which may not have such activity.
• the "XXXLRF" (SEQ ID No. 9) peptide composition comprises an antibiological (e.g., an antifungal agent). This process may be carried out to the point where completely defined peptide(s) are produced and assayed for antibiological (e.g., antifungal) activity.
• FHLRF SEQ ID No. 31
• all amino acid residues in a six residue peptide may be known.
• a proteinaceous composition may also be non-homogenous, comprising, for example, both D-, L- and/or cyclic amino acids.
• a proteinaceous composition comprises a plurality (e.g., a mixture) of different proteinaceous molecules, including proteinacous molecule(s) that comprise an L-amino acid, a D amino acid, a cyclic amino acid, or a combination thereof.
• a mixture of different proteinaceous molecules may comprises one or more peptides comprising L amino acids; one or more peptides comprising D amino acids; and/or one or more peptides comprising both an L amino acid and an D- amino acid.
• a retroinversopeptidomimetic of SEQ ID No. (41 ) demonstrated inhibitory function, albeit less so than either the D- or L- configurations, against certain household fungi such as a Fusarium and an Aspergillus (Guichard, 1994).
• a peptide composition may comprise or be modified to comprises fewer cysteines and/or exclude cysteine(s) to reduce and/or prevent disulfide linkage problem that may occur in certain
• one or more peptides may be prepared as a peptide library, which typically comprises a plurality (e.g., about 2 to about 10 10 peptides).
• a peptide library may comprise a D- amino acid, an L-amino acid, a cyclic amino acid, a common amino acid, an uncommon amino acid (e.g., a non-naturally occurring amino acid), a stereoisomer (e.g., a D-amino acid stereoisomer, an L-amino acid stereoisomer), or a combination thereof.
• a peptide library may comprise a synthetically produced peptide and/or a biologically produced peptide (e.g., a recombinantly produced peptide, see for example U.S. Patent No. 4,935,351 ).
• a synthetic peptide combinational library typically comprises a mixture (e.g., an equimolar mixture) of free peptide(s).
• a SPCL peptide may possess activity (e.g., an antifungal activity, antipathogen activity), such as, for example, a SPCL comprising 52,128,400 six-residue peptides, wherein each peptide comprised D-amino acids and having non-acetylated N-termini and amidated C-termini.
• activity e.g., an antifungal activity, antipathogen activity
• a SPCL comprising 52,128,400 six-residue peptides, wherein each peptide comprised D-amino acids and having non-acetylated N-termini and amidated C-termini.
• a hexapeptide library comprised peptides with the first two amino acids in each peptide chain individually and specifically defined and with the last four amino acids comprising an equimolar mixtures of 20 amino acids.
• the final concentration for each peptide was about 9.38 ng/ml in a mixture comprising about 1.5 mg (peptide mix)/ml solution.
• This mixture profile assumed that an average peptide has a molecular weight of about 785. This concentration was sufficient to permit testing for antifungal activity.
• an antibiotic composition(s) comprising equimolar mixture of peptides produced in a synthetic peptide combinatorial library (see U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097, and Patent Application Nos. 10/884,355 and 11/368,086,) have been derived and shown to have desirable antibiotic activity.
• these relatively variable compositions are based upon the sequences of one or more of the peptides disclosed in any of the U.S. Patent Nos. 6,020,312; 5,885,782; and 5,602,097, and Patent Application Nos. 10/884,355 and 11/368,086.
• a peptide composition comprises a peptide derived from amino acids of a length readily accomplished using standard peptide synthesis procedures, such as, for example, between about 3 to about 100 amino acids in length (e.g., about 3 to about 25 residues in length, about 6 residues in length, etc.).
• a proteinaceous molecule e.g., an antifungal peptide sequence identified as described herein
• suitable cell(s) e.g., a bacterial cell, an insect cell
• DNA encoding the proteinaceous molecule's sequence e.g., encoding an antifungal peptide's sequence described herein
• an expression vector may comprise a DNA sequence encoding SEQ ID No. 1 in the correct orientation and reading frame with respect to the promoter sequence to allow translation of the DNA encoding the SEQ ID No. 1.
• such a proteinaceous sequence may comprise one or more other sequences (e.g., extracellular and/or intracellular signal sequence(s) to target a proteinaceous molecule, restriction enzyme site(s), ion and/or metal binding sites such as a His-Tag), for ease of processing, preparation, and/or to alter and/or confer an additional sequences (e.g., extracellular and/or intracellular signal sequence(s) to target a proteinaceous molecule, restriction enzyme site(s), ion and/or metal binding sites such as a His-Tag), for ease of processing, preparation, and/or to alter and/or confer an additional
• a plurality of peptide sequence(s), which may comprise multiple copies of the same and/or different sequences, may be produced.
• One or more restriction enzyme site(s) may expressed between selected sequence(s), to allow cleavage into smaller proteinaceous molecules (e.g., cleavage into smaller peptide sequences).
• a metal binding site such as a His-tag may be added for ease of purification and/or to confer a metal binding property.
• a peptide sequence may be included as part of a polypeptide by incorporation of one or more copies of peptide sequence(s), additional sequences (e.g., His- tags, restriction enzyme sites).
• one or more peptide sequence(s) and/or one or more such additional sequences may be added to the C-terminus and/or the N-terminus of another proteinacous sequence (e.g., an enzyme).
• an enzyme e.g., an antibiological enzyme, an esterase
• an enzyme may be modified to comprise an antimicrobial peptide sequence, a restriction enzyme site, and/or a metal binding domain (e.g., a His-Tag), with the additional proteinaceous sequence(s) added at the N-terminus, the C- terminus, or a combination thereof.
• a proteinaceous composition may comprise a carrier (e.g., a microsphere, a liposome, a saline solution, a buffer, a solvent, a soluble carrier, an insoluble carrier).
• the carrier may be one suitable for a permanent, a semi-permanent, and/or a temporary material formulation (e.g., a permanent surface coating application, a semi-permanent coating, a non-film forming coating, a temporary coating).
• a carrier may be selected to comprise a chemical and/or a physical characteristic which does not significantly interfere with the antibiotic activity of a proteinaceous (e.g., a peptide) composition.
• a microsphere carrier may be effectively utilized with a proteinaceous composition in order to deliver the composition to a selected site of activity (e.g., onto a surface).
• a liposome may be similarly utilized to deliver an antibiotic (e.g., a labile antibiotic).
• a saline solution a material formulation (e.g., a coating) acceptable buffer, a solvent, and/or the like may also be utilized as a carrier for a proteinaceous (e.g., a peptide) composition.
• a material formulation e.g., a coating
• a solvent e.g., a solvent
• a proteinaceous e.g., a peptide
• An antibiological agent may act on a biological entity such as a biological cell and/or a biological virus.
• a biological entity such as a biological cell and/or a biological virus.
• Examples of a cell include a prokaryotic cell and/or an eukaryotic cell.
• An antibiological agent generally binds a biomolecule ligand to act on the biological entity, such as, for example an enzyme cleaving a cellular biomolecule and/or a peptide associating with and disrupting a cellular membrane.
• Prokaryotic organisms are generally classified in the Kingdom Monera as an Archaea ("Archaebacteria”) or an Eubacteria ("bacteria”). Eukaryotic organisms are generally classified in the
• a virus does not possess a cell wall, but comprises a proteinaceous outer coat, that may be surrounded by a phospholipid membrane ("envelope").
• a cell and/or a virus that may be a target of an antibiological agent comprises an Animalia cell (e.g., a mollusk cell), a Plantae cell, an
• a cell and/or a virus that may be a target of an antibiological agent may comprise a microorganism, a marine fouling organism, or a combination thereof.
• An antibiological proteinaceous composition may be referred to by the target cell it effects, such as an "antifungal peptidic agent.”
• such a cell may comprise a pathogen (e.g., a fungal pathogen, a plant pathogen, an animal pathogen such as a human pathogen, etc.).
• An Archaea typically comprises a cell wall comprising a pseudopeptidoglycan, a peptide, a polypeptide, a protein (e.g., a glycoprotein), or a combination thereof.
• Examples of an Archaea genus includes an Acidianus, an Acidilobus, an Aeropyrum, an Archaeoglobus, a Caldivirga, a Desulfurococcus, a Ferroglobus, a Ferroplasma, a Haloarcula, a Halobacterium, a Halobaculum, a Halococcus, a Haloferax, a Halogeometricum, a Halomicrobium, a Halorhabdus, a Halorubrum, a Haloterrigena, a Hyperthermus, an Ignicoccus, a Metallosphaera, a Methanobacterium, a Methanobrevibacter, a Methane-calculus, a Methanocal
• An Eubacteria typically comprises a cell wall comprising a peptidoglycan, a peptide, a polypeptide, a protein (e.g., a glycoprotein), a lipid, or a combination thereof.
• the members of the Eubacteria phyla are divided into Gram-positive Eubacteria or Gram-negative Eubacteria (e.g., Cyanobacteria, Proteobacteria, Spirochetes) based on biochemical and structural differences between the cell wall and/or an associated a phospholipid bilayer ("cell membrane") of the organism(s).
• a “Gram-positive Eubacteria” (“Gram-positive bacteria”) refers to an Eubacteria comprising a cell wall that typically stains positive with Gram stain reaction (see, for example, Scherrer, R., 1984) and may not be surrounded by an outer cell membrane.
• a Gram positive bacteria generally have a cell wall composed of a thick layer of peptidoglycan overlaid by a thinner layer of techoic acid.
• a "Gram-negative Eubacteria” (“Gram negative bacteria”) refer to Eubacteria comprising a cell wall that typically stains negative with Gram stain reaction and may be surrounded by a second lipid bilayer ("outer cell membrane”). Gram negative bacteria have a thinner layer of peptidoglycan.
• Gram-negative Eubacteria do not stain well using a standard Gram stain procedure. However, these bacteria may be classified as a Gram-negative Eubacteria by the presence of an outer cell membrane, a morphological feature typically not present in a Gram-positive Eubacteria.
• Examples of a Gram-positive Eubacteria comprise an Acetobacterium, an Actinokineospora, an Actinomadura, an Actinomyces, an Actinoplanes, an Actinopolyspora, an Actinosynnema, an Aerococcus, an Aeromicrobium, an Agromyces, an Amphibacillus, an Amycolatopsis, an Arcanobacterium, an Arthrobacter, an Aureobacterium, a Bacillus, a Bifidobacterium, a Brachy bacterium, a Brevibacterium, a Brochothrix, a Carnobacterium, a Caryophanon, a Catellatospora, a Cellulomonas, a Clavibacter, a Clostridium, a Coprococcus, a Coriobacterium, a Corynebacterium, a Curtobacterium, a Dactylosporangium
• Marinococcus a Melissococcus, a Microbacterium, a Microbispora, a Micrococcus, a Micromonospora, a Microtetraspora, a Mobiluncus, a Mycobacterium, a Nocardia, a Nocardioides, a Nocardiopsis, an Oerskovia, a Pediococcus, a Peptococcus, a Peptostreptococcus, a Pilimelia, a Planobispora, a Planococcus, a Planomonospora, a Promicromonospora, a Propionibacterium, a Pseudonocardia, a Rarobacter, a Renibacterium, a Rhodococcus, a Rothia, a Rubrobacter, a Ruminococcus, a Saccharococcus, a
• Saccharomonospora a Saccharopolyspora, a Saccharothrix, a Salinicoccus, a Sarcina, a Sphaerobacter, a Spirillospora, a Sporichthya, a Sporohalobacter, a Sporolactobacillus, a Sporosarcina, a Staphylococcus, a Streptoalloteichus, a Streptococcus, a Streptomyces, a Streptosporangium, a Syntrophospora, a Terrabacter, a Thermacetogenium, a Thermoactinomyces, a Thermoanaerobacter, a Thermoanaerobium, a Thermomonospora, a Trichococcus, a Tsukamurella, a Vagococcus, or a combination thereof.
• Examples of a Gram-negative Eubacteria comprises an Acetivibrio, an Acetoanaerobium, an Acetobacter, an Acetomicrobium, an Acidaminobacter, an Acidaminococcus, an Acidiphilium, an Acidomonas, an Acidovorax, an Acinetobacter, an Aeromonas, an Agitococcus, an Agrobacterium, an Agromonas, an Alcaligenes, an Allochromatium, an Alteromonas, an Alysiella, an Aminobacter, an /Anabaena, an Anaerobiospirillum, an Anaerorhabdus, an Anaerovibrio, an Ancalomicrobium, an
• Ancylobacter an Angulomicrobium, an Aquaspirillum, an Archangium, an Arsenophonus, an Arthrospira, an Asticcacaulis, an /Azomonas, an Azorhizobium, an Azospirillum, an Azotobacter, a Bacteroides, a Bdellovibrio, a Beggiatoa, a Beijerinckia, a Blastobacter, a Blastochloris, a Bordetella, a Borrelia, a Brachyspira, a Bradyrhizobium, a Brevundimonas, a Brucella, a Budvicia, a Buttiauxella, a Butyrivibrio, a Calothrix, a Campylobacter, a Capnocytophaga, a Cardiobacterium, a Caulobacter, a Cedecea, a
• an Eubacteria comprises an Abiotrophia, an Acetitomaculum, an Acetohalobium, an Acetonema, an Achromobacter, an Acidimicrobium, an Acidithiobacillus, an Acidobacterium, an Acidocella, an Acrocarpospora, an Actinoalloteichus, an Actinobacillus, an Actinobaculum, an Actinocorallia, an Aequorivita, an Afipia, an Agreia, an Agrococcus, an Ahrensia, an Albibacter, an Albidovulum, an Alcanivorax, an Alicycliphilus, an Alicyclobacillus, an Alkalibacterium, an
• Halothermothrix a Halothiobacillus, a Helcococcus, a Hellophllum, a Heliorestis, a Herbidospora, a Hippea, a Holdemania, a Holophaga, a Hydrogenobacter, a Hydrogenobaculum, a Hydrogenophilus, a Hydrogenothermus, a Hydrogenovibrio, a Hymenobacter, an Ignavigranum, an Iodobacter, an Isobaculum, a Janibacter, a Kineococcus, a Kineosphaera, a Kitasatosporia, a Knoellia, a Kocuria, a Kozakia, a Kribbella, a Kutzneria, a Kytococcus, a Lachnobacterium, a Laribacter, a Lautropia, a Lechevalieria, a Leifsonia, a Leisingera, a Lentze
• Pseudobutyrivibrio a Pseudoramibacter, a Pseudorhodobacter, a Pseudospirillum, a Pseudoxanthomonas, a Psychroflexus, a Psychromonas, a Psychroserpens, a Ralstonia, a Ramlibacter, a Raoultella, a Rathayibacter, a Rhodothermus, a Roseateles, a Roseburia, a Roseiflexus, a Roseinatronobacter, a Roseospirillum, a Roseovarius, a Rubritepida, a Ruegeria, a Sagittula, a Salana, a Salegentibacter, a Salinibacter, a Salinivibrio, a Sanguibacter, a Scardovia, a Schineria, a Schwartzia, a Sedimentibacter, a
• Thermosyntropha a Thermoterrabacterium, a Thermovenabulum, a Thermovibrio, a Thialkalimicrobium, a Thialkalivibrio, a Thioalkalivibrio, a Thiobaca, a Thiomonas, a Tindallia, a Tolumonas, a Turicella, a Turicibacter, an Ureibacillus, a Verrucosispora, a Victivallis, a Virgibacillus, a Vogesella, a Weissella, a Williamsia, a Xenophilus, a Zavarzinia, a Zooshikella, a Zymobacter, or a combination thereof.
• Organisms of the eukaryotic Fung/ Kingdom include organisms commonly referred to as a molds, morels, mildews, mushrooms, puffballs, rusts, smuts, truffles, and yeasts.
• a fungal organism typically comprises multicellular filaments that grow into a food supply (e.g., a carbon based polymer), but may become unicellular spore(s) in nutrient poor conditions.
• “Mold” may be used herein as a synonym for fungi, where the context permits, especially when referring to indoor contaminants. However, the term “mold” also, and more specifically, denotes certain types of fungi.
• the plasmodial slime molds the cellular slime molds, water molds, and the everyday common mold.
• True molds refer to filamentous fungi comprising the mycelium, specialized, spore-bearing structures called conidiophores, and conidia ("spores").
• "Mildew” is another common name for certain fungi, including a powdery mildew and a downy mildew.
• "Yeasts" are unicellular members of the fungus family.
• fungus for the purposes of the present disclosure, where any of the terms fungus, a mold, a morel, a mildew, a mushroom, a puffball, a rust, a smut, a truffle, and/or a yeast is used, the others are implied where the context permits.
• a fungi cell wall typically comprises a beta-1 ,4-linked homopolymers of N-acetylglucosamine ("chitin") and a glucan.
• the glucan is usually an alpha-glucan, such as a polymer comprising an alpha-1 ,3- and alpha-1 , 6- linkage (Griffin, 1993).
• Some Ascomycota species e.g., Ophiostomataceae
• Certain species of Chytridiomycota e.g., Coelomomycetales
• do not possess a cell wall Alexopoulos et al., 1996. Examples of a fungi genus includes an Aciculoconidium, an fungi genus, and others.
• Agaricostilbum an Ambrosiozyma, an Arxiozyma, an Arxula, an Ascoidea, a Babjevia, a Bensingtonia, a Blastobotrys, a Botryozyma, a Bullera, a Bulleromyces, a Candida, a Cephaloascus, a Chionosphaera, a Citeromyces, a Clavispora, a Cryptococcus, a Cystofilobasidium, a Debaryomyces, a Dekkera, a Dipodascopsis, a Dipodascus, an Endomyces, an Eremothecium, an Erythrobasidium, a Fellomyces, a Filobasidiella, a Filobasidium, a Galactomyces, a Geotrichum, a Hanseniaspora, a Hyalodendron, an Issatchenkia
• Sporobolomyces a Sporopachydermia, a Stephanoascus, a Sterigmatomyces, a Sterigmatosporidium, a Sympodiomyces, a Sympodiomycopsis, a Taphrina, a Tilletiaria, a Tilletiopsis, a Torulaspora, a Trichosporon, a Trichosporonoides, a Trigonopsis, a Tsuchiyaea, a Wickerhamia, a Wickerhamiella, a Williopsis, a Xanthophyllomyces, a Yarrowia, a Zygoascus, a Zygosaccharomyces, a Zygozyma, or a combination thereof.
• Examples of a fungal genus sometimes found in a building having excess indoor moisture comprises a Stachybotrys (e.g., a Stachybotrys chartarum), which is commonly found in nature growing on a cellulose-rich plant material and/or a water-damaged building material, such as ceiling tiles, wallpaper, sheet- rock and cellulose resin wallboard (e.g., a fiberboard).
• a Stachybotrys may produce mycotoxins, compounds that have toxic properties.
• a proteinaceous composition e.g., a peptide composition
• Organisms of the Kingdom Protista refer to a heterogenous set of eukaryotic unicellular, oligocellular and/or multicellular organisms that may not have been classified as belonging to the other eukaryotic Kingdoms, though they typically have features related to the Plant Kingdom (e.g., an algae, which generally are photosynthetic), the Fungi Kingdom (e.g., an Oomycota) and/or the Animal Kingdom (e.g., a protozoa).
• Organisms of certain Protista Phyla particularly those organisms commonly known as "algae,"
• 5842-02103 95 comprise a cell wall, silica based shell and/or exoskeleton (e.g., a test, a frustule), or other durable material at the cell-external environment interface.
• Examples of a Protista comprises an Acetabularia, an Achnanthes, an Amphidinium, an Ankistrodesmus, an Anophryoides, an Aphanomyces, an Astasia, an Asterionella, a Blepharisma, a Botrydiopsis, a Botrydium, a Botryococcus, a Bracteacoccus, a Brevilegnia, a Bulbochaete, a Caenomorpha, a Cephaleuros, a Ceratium, a Chaetoceros, a Chaetophora, a Characiosiphon, a Chlamydomonas, a Chlorella, a Chloridella, a Chlorobotrys, a Chlorococcum, a Chromulina, a Chroodactylon, a Chrysamoeba, a Chrysocapsa, a Cladophora,
• Draparnaldia a Dunaliella, a Dysmorphococcus, an Enteromorpha, an Entosiphon, an Eudorina, an Euglena, an Euplotes, an Eustigmatos, a Flintiella, a Fragilaria, a Fritschiella, a Glaucoma, a Gonium, a Gonyaulax, a Gymnodinium, a Gyropaigne, a Haematococcus, a Halophytophthora, a Heterosigma, a Hyalotheca, a Hydrodictyon, a Khawkinea, a Lagenidium, a Leptolegnia, a Mallomonas, a Mantoniella, a Melosira, a Menoidium, a Mesanophrys, a Mesotaenium, a Metopus, a Micrasterias, a Microspore,
• a diatom refers to a unicellular algae that possess a cell wall comprising silicon. Examples of a diatom include organisms of the phyla Chrysophyta and/or Bacillariphyta.
• a Chrysophyta (“golden algae,” “golden-brown algae”) typically comprises a freshwater diatom.
• Examples of a Chrysophyta includes a Chlorobotrys, a Chromulina, a Chrysamoeba, a Chrysocapsa, a Dinobryon, an Eustigmatos, a Heterosigma, a Mallomonas, a Monodopsis, a Nannochloropsis, an Ochromonas, a Paraphysomonas, a Pleurochloris, a Polyedriella, a Pseudocharaciopsis, a Rhizochromulina, a Synura, a Thaumatomastix, a Vischeria, or a combination thereof.
• a Bacillariphyta typically comprises a marine diatom.
• Bacillariphyta examples include an Achnanthes, an Asterionella, a Chaetoceros, a Cocconeis, a Cyclotella, a Fragilaria, a Melosira, a Navicula, a Nitzschia, a Skeletonema, a Stauroneis, a Stephanodiscus, a Synedra, a Thalassiosira, or a combination thereof.
• a Xanthophyta (“yellow-green algae”) is typically yellowish-green in color, with examples including a Botrydiopsis, a Botrydium, a Botryococcus, a Chloridella, a Mischococcus, an Ophiocytium, a Tribonema, a Vaucheria, or a combination thereof.
• An Euglenophyta (“euglenoids”) generally is unicellular, aquatic algae and comprises a pellicle, which comprises an outer membrane reinforced by proteins, rather than a cell wall.
• Euglenophyta examples include an Astasia, a Colacium, a Cryptoglena, a Distigma, an Entosiphon, an Euglena, a Gyropaigne, a Khawkinea, a Menoidium, a Personaldium, a Peranema, a Petalomonas, a Phacus, a Ploeotia, a Rhabdomonas, a Rhynchopus, a Scytomonas, a Trachelomonas, or a combination thereof.
• a Chlorophyta (“green algae”) typically forms unicellular to oligocellular cluster(s), and comprises a cell wall comprising a cellulose.
• Examples of a Chlorophyta include a Vo/vox, a Chlorella, a Pleurococcus, a Spirogyra, a Chlamydomonas, a Gonium, a Mantoniella, a Nephroselmis, a Pyramimonas, a Tetraselmis, an Ulothrix, an Enteromorpha, a Cephaleuros, a Cladophora, a Pithophora, a Rhizoclonium, a Derbesia, an Acetabularia, a Chlorella, a Microthamnion, a Prototheca, a Stichococcus, a Trebouxia, an Ankistrodesmus, a Bracteacoccus, a Bulbochaete, a Cha
• Fritschiella a Gonium, a Haematococcus, a Hydrodictyon, an Oedogonium, a Microspore, a Pandorina, a Pediastrum, a Pleodorina, a Scenedesmus, a Selenastrum, a Sphaerocystis, a Stephanosphaera, a Stigeoclonium, a Tetracystis, a Tetraedron, a Trentepohlia, an Uronema, a Vo/vox, a Closterium, a Cosmarium, a Cylindrocystis, a Hyalotheca, a Mesotaenium, a Micrasterias, a Mougeotia, a Pleurotaenium, a Spirogyra, a Spondylosium, a Staurastrum, a Xanthidium, a Zygnema, or a combination therof.
• a Rhodophyta (“red algae”) generally is multicellular and comprises a cell wall comprising a sulfated polysaccharide, such as, for example, an agar, a carrageenan, a cellulose, or a combination thereof.
• a Rhodophyta genera that are typically unicellular include a Chroodactylon, a Flintiella, a Porphyridium, a Rhodella, a Rhodosorus, or a combination thereof.
• a Pyrrophyta (“fire algae,” “dinoflagellate”) generally is a unicellular marine organism possessing a cell wall comprising cellulose.
• a Pyrrophyta typically is red, and examples include a dinoflagellate genera such as an Amphidinium, a Ceratium, a Gonyaulax, a Gymnodinium, an Oxyrrhis, a Peridinium, a Prorocentrum, or a combination thereof.
• a Ciliophora ("ciliate") generally is unicellular and comprises a pellicle. Examples of a Ciliophora includes an Anophryoides, a Blepharisma, a Caenomorpha, a Cohnilembus, a Coleps, a Colpidium, a
• Colpoda a Cyclidium, a Dexiostoma, a Didinium, an Euplotes, a Glaucoma, a Mesanophrys, a Metopus, an Opisthonecta, a Paramecium, a Paranophrys, a Plagiopyla, a Platyophrya, a Pseudocohnilembus, a Spathidium, a Spirostomum, a Stentor, a Tetrahymena, a Trimyema, an Uronema, a Vorticella, or a combination thereof.
• An Oomycota (“oomycete,” “water mold”) is a fungi-like organism, and is often listed in the fungal sections of biological culture collections.
• An Oomycota is typically unicellular but differ from a fungi by possessing a cell wall that comprises a cellulose and/or a glycan.
• Examples of an Oomycota an Aphanomyces, a Brevilegnia, a Dictyuchus, a Halophytophthora, a Lagenidium, a Leptolegnia, a
• 5842-02103 97 Peronophythora, a Plasmopara, a Plectospira, a Pythiopsis, a Pythium, a Saprolegnia, a Thraustotheca, or a combination thereof.
• a virus e.g., an enveloped virus
• a DNA virus such as a Herpesviridae (“herpesviruses”), a Poxviridae (“poxviruse”), and/or a Baculoviridae (“baculooviruses”); an RNA virus such as a Flaviviridae (“flavivirus”), a Togaviridae (“togavirus”), a Coronaviridae ("coronavirus”; e.g., Severe Acute Respiratory Syndrome - “SARS”), a Deltaviridae (“deltavirus”; e.g., Hepatitis D), an Orthomyxoviridae (“orthomyxovirus”), a Paramyxoviridae (“paramyxovirus”), a Rhabdoviridae (“rhabdovirus”), a Bunyavirid
• a component of a cell wall, a viral proteinaceous molecule, and/or a cellular membrane may comprise a target of an antibiological agent; may comprise a component of a cell-based particulate material, or a combination thereof.
• Examples of such a cell wall, a viral proteinaceous molecule, and/or a cellular membrane component includes a peptidoglycan, a pseudopeptidoglycan, a teichoic acid, a teichuronic acid, a cellulose, a neutral polysaccharide, a chitin, a mannin, a glucan, a proteinaceous molecule, a lipid (e.g., a phospholipid), or a combination thereof.
• cell and/or viral component(s) may function as an antibiological agent's target such as an antibiological enzyme substrate and/or a ligand for a proteinaceous molecule's binding interaction (e.g., an antibiological peptide binding); as well as possibly function as a component(s) of a cell-based particulate material.
• an antibiological agent's target such as an antibiological enzyme substrate and/or a ligand for a proteinaceous molecule's binding interaction (e.g., an antibiological peptide binding); as well as possibly function as a component(s) of a cell-based particulate material.
• An Eubacteria cell wall typicallys comprise a peptidoglycan ("mucopeptide,” "murein"), as well as a glycoprotein, a protein, a polysaccharide, a lipid, or a combination thereof.
• Peptidoglycan generally comprises alternating monomers of the amino-sugars ⁇ /-acetylglucosamine and ⁇ /-acetylmuramic acid.
• the ⁇ /-acetylmuramic acid monomers often further comprise a tetra-peptide of the sequence L-alanine-D- glutamic acid-L-diamino acid-D-alanine covalently bonded to the muramic acid.
• the attached tetrapeptides of peptidoglycan participate in cross-linking a plurality of polymers to contribute to the cell wall structure.
• the tetrapeptides may form the cross-linkages by direct covalent bonds, and/or one or more amino acids may form the cross-linking bonds between the tetrapeptides.
• a biomolecule used in many embodiments may comprise a peptidoglycan for conferring particulate nature and durability to various cell-based particulate materials, given the general ease of growth of Eubacteria.
• Archaea do not possess peptidoglycan, but many Archaea may comprise a pseudopeptidoglycan, which comprises N- acetyltalosaminuronic acid, instead of N-acetylmuramic in peptidoglycan.
• a cell wall particularly of Gram-positive Eubacteria, may comprise up to 50% teichoic acid.
• Teichoic acid comprises an acidic polymer comprising monomers of a phosphate and a glycerol; a phosphate and a ribitol; and/or a ⁇ /-acetylglucosamine and a glycerol.
• a sugar e.g., glucose
• an amino acid e.g., D-alanine
• a teichoic acid may be associated with a phospholipid bilayer adjacent to a cell wall. Often, a teichoic acid may be covalently bonded to a glycolipid of a cell membrane, and may be known as a "lipoteichoic acid.” Teichic acids are common in a Staphylococcus, a Micrococcus, a Bacillus, and/or a Lactobacillus genera. [0255] A cell wall of certain species of Gram-positive Eubacteria may comprise teichuronic acid.
• Teichuronic acid comprises a polymer comprising a ⁇ /-acetylglucosamine and a glucuronic acid; and/or a glucose and an amino-mannuronic acid.
• acidic conditions may damage this cell wall component, as an uronic acid such as a glucuronic acid, and particularly an amino-mannuronic acid, may be hydrolyzed in an acid. Exposure to acid during processing and/or in a material formulation may reduce this component from a cell based particulate matter.
• a cell wall particularly of a Gram-positive Eubacteria, may comprise a neutral polysaccharide, other than those described for a peptidoglycan, a teichoic acid, a cellulose, and/or a lipopolysacharide.
• a neutral polysaccharide comprises a polymer comprising a majority of neutral sugars, wherein the neutral sugar typically comprises a hexose, a pentose, and/or an amino sugar thereof.
• Examples of a neutral sugar found in a neutral polysaccharide include an arabinose, a galactose, a 3-O-methyl-D- galactose, a mannose, a xylose, a rhamnose, a glucose, a fructose, or a combination thereof.
• Examples of an amino sugar found in a neutral polysaccharide include a glucosamine, a galactosamine, or a combination thereof.
• a cell wall and/or a virus may comprise a proteinaceous molecule, such as, for example, a polypeptide, a peptide, a protein.
• a proteinaceous material may dominate the structural integrity that confers particulate material durability to a virus and/or a cell comprising a pellicle.
• peptide linkage(s) are common throughout a peptidoglycan and/or a pseudopeptidoglycan.
• a cell wall may comprise a lipid, other than those described for a peptidoglycan, a teichoic acid, and/or a lipopolysacharide.
• a cell comprises various lipid biomolecules, which generally comprise fatty acids.
• lipids may be removed from a cell and/or a cell wall.
• the lipid components of a cell and/or a cell wall remaining in the particulate matter may affect a material formulation's reactions wherein lipid (e.g., a fatty acid double bond) cross-linking
• Lipids of particular relevance for such a potential cross-linking reaction include those of the outer membrane, which comprise a fatty acid, the cell wall, or a combination thereof.
• Gram-negative cells comprise a phospholipid bilayer often refered to as the "outer cell membrane” that surrounds the cell wall.
• a "phospholipid bilayer” comprises two layers of phospholipid molecules, wherein the fatty acids components of each layer's phospholipids contact each other, thereby creating a hydrophobic inner region, and the head groups of each layer's phospholipids, which are generally hydrophilic, contact the external environment.
• Examples of a phospholipid include a glycerophospholipid, which comprises two fatty acids and one hydrophilic moiety called a "head group" covalently connected to a trihydroxyl alcohol glycerol.
• Non-limiting examples of a head group include a choline, an ethanolamine, a serine, an inositol, an additional glycerol, or a combination thereof.
• a phospholipid bilayer generally comprises a plurality of peptides and/or polypeptides with hydrophobic regions that are retained in the phospholipid bilayer's hydrophobic inner region.
• the cell wall peptidoglycan may be linked to the phospholipid membrane by a periplasmic space lipoprotein.
• Gram-positive Eubacteria cell walls generally comprise about 0% to about 2% lipid. However, certain categories of Gram-positive Eubacteria may comprise up to about 50% or more lipid content as part of the cell wall.
• Such Eubacteria include different species of Gordonia, Mycobacterium, Nocardia, and Rhodococcus. Additionally, the lipids of such Eubacteria generally comprise a branched chain fatty acid, particularly mycolic acids (Barry, C. E. et al., Prog Lipid Res 37:143, 1998). A mycolic acid may be covalently bound and/or loosely associated with a cell wall sugar. The type of Eubacteria may be sometimes used to identify the carbon-backbone length of a mycolic acid. For example, an eumycolic acid may be isolated from a Mycobacterium, and generally comprises about 60 to about 90 carbon atoms.
• a corynomycolic acid isolated from a Corynobacterium generally comprises 22 to 36 carbons.
• a nocardomycoic acid isolated from a Nocardia generally comprises 44 to 60 carbons.
• a mycolic acid generally comprises a fatty acid branch ("alpha branch") and an aldehyde (“meromycolate branch”).
• a mycolic acid may further comprise a carbon double bond, an epoxy ester moiety, a cyclopropane ring moiety, a keto moiety, a methoxy moiety, or a combination thereof, generally located on a meromycolate branch.
• a mycolic acid may comprise an ⁇ -mycolic acid, a methoxymycolic acid, a ketomycolic acid, an epoxymycolic acid, a wax ester, or a combination thereof.
• a ⁇ -mycolic acid comprises a cis or trans carbon double bond and/or a cyclopropane, and may further comprise a methyl branch adjacent to such a moiety.
• a methoxymycolic acid comprises a methoxy moiety and a double bond or a cyclopropane.
• a ketomycolic acid comprises a-methyl-branched ketone.
• An epoxymycolic acid comprises an ⁇ -methyl-branch epoxide.
• a wax ester comprises an internal ester group and a carbon double bond or a cyclopropane ring.
• a cell lipid may comprise a glycolipid, which refers to a glycan covalently attached to a lipid.
• Non-limiting examples of a glycolipid include a dolichyl phosphoryl glycan, a pyrophosphoryl glycan, an undecaprenyl phosphoryl glycan, a pryophosphoryl glycan, a retinyl phosphoryl glycan, a glycosphingolipid (e.g., a ceramide, a galactosphingolipid, a glucosphingolipid including a ganlioside), a glycoglycerolipid (e.g., a monogalactosyldiacylglycerol), a steroidal glycoside (e.g., ouabain, digoxin,
• a glycosphingolipid e.g., a ceramide, a galactosphingolipid, a glucosphingolipid including a ganlioside
• a glycoglycerolipid e.g., a monogalactosyldi
• glycosylated phosphoinositide e.g., a GPI anchor, a lipophosphoglycan, a lipopeptidophosphoglycan, a glycoinositol phospholipid
• a glycosylated phosphoinositide e.g., a GPI anchor, a lipophosphoglycan, a lipopeptidophosphoglycan, a glycoinositol phospholipid
• the phospholipid bilayers of Archaea are biochemically distinct from the lipids described above, as they comprise branched hydrocarbon chains attached to glycerol by ether linkages instead of fatty acids attached to glycerol by ester linkages.
• a cell wall of organisms primarily of the Kingdom Planta, comprises cellulose.
• Cellulose comprises a polysaccharide polymer (e.g., a linear polymer) typically hundreds to thousands of glucose monomer units long, and commonly functions as a structural component of the primary cell wall of green plants and many forms of algae.
• some bacteria form a biofilm by secreting cellulose
• some Ascomycota fungal species e.g., an Ophiostomataceae
• Ascomycota fungal species e.g., an Ophiostomataceae
• Fungi cells and spore wall components typically include beta-1 ,4-linked homopolymers of a N- acetylglucosamine ("chitin") and a glucan.
• a chitin is similar to a cellulose, though an acetylamine moiety (N- acetylglucosamine) substitutes for a hydroxyl moiety on the glucose monomer(s).
• the relative increase in hydrogen bonding between chitin polymer chains enhances the strength of a chitin-polymer matrix.
• the glucan usually comprises an alpha-glucan, such as a polymer comprising an alpha-1 ,3- and an alpha-1 ,6- linkage (Griffin, 1993).
• Agarose and porphyran comprise polysaccharide polymers, and are components of some algae (e.g., red algae).
• a fungal cell wall (e.g., a yeast cell wall) may comprise an oligo-mannan, a helical ⁇ (1-6)-D-glucan, and/or a ⁇ (1-3)-D-glucan, well as a chitin, lipid(s) and/or protein(s).
• a linkage (e.g., a ⁇ (1-4)-linkage) may occur, for example between the nonreducing ends of a glucan and a glycoprotein; and the reducing ends of chitin (Kollar, R., et al., 1995; Kapteyn, J. C, et al., 1996).
• a biomolecule such as an enzyme may possess one or more secondary characteristics, functions and/or activities (e.g., a binding activity, a catalytic activity) in addition to the characteristic, the function and/or the activity of its classification (e.g., EC classification) and/or characterization.
• secondary characteristics, functions and/or activities e.g., a binding activity, a catalytic activity
• a multifunctional enzyme may be selected for use based on the secondary activity over the primary activity of its classification.
• an enzyme may be selected for both its primary activity and a secondary activity.
• carboxylesterases EC 3.1.1.1
• a diazinon and/or a malathion e.g., Rattus norvegicus ES4 and ES10; enzymes from a Plodia interpunctella, a Chrysomya putoria, a Lucilia cuprina, a Musca domestica, a Myzus persicae, and/or a Homo sapiens liver cell.
• an organophosphorus compound acts as an inhibitor of the carboxylesterase, though hydrolysis occurs in some instances [In “Esterases, Lipases, and Phospholipases from Structure to Clinical Significance.” (Mackness, M.I. and Clerc, M., Eds.), pp. 91-98, 1994].
• Many genes in an organism e.g., an eukaryatic organism
• an allele of a carboxylesterase gene possessing an organophosphate hydrolase (EC 3.1.8.1 ) activity may be responsible for OP compound resistance.
• Examples of such a carboxylesterase gene include an allele isolated from Lucilia cuprina
• Such a multifunctional carboxylesterase may be selected for a lipolytic activity in one application, and selected for an organophosphorus compound binding and/or hydrolytic activity in a different application.
• Such a multifunctional carboxylesterase may be differentiated herein by the use of "carboxylesterase” when referring to an enzyme as a lipolytic enzyme, and a “carboxylase” when referring to an enzyme used for function as an organophosphorus compound binding/degrading enzyme.
• a carboxylesterase and/or a carbamoyl lyase may be useful against a carbamate nerve agent, and are specifically contemplated for use in a biomolecular composition and/or a material formulation for use against such a carbamate nerve agent.
• a prolidase (“imidodipeptidase,” “proline dipeptidase,” “peptidase D,” “g- peptidase”), a PepQ and/or an aminopeptidase P gene and/or a gene product may possess, for example, an OPAA activity.
• OPAAs possess sequence and structural similarity to a human prolidase, an Escherichia coli aminopeptidase P and/or an Escherichia coli PepQ (Cheng, T.-C. et al., 1997; Cheng, T.-C. et al., 1996).
• a prolidase and/or a PepQ protein (E. C.
• prolidase genes and gene products include a Mus musculus prolidase gene (GeneBank accession no.
• cholinesterases e.g., an acetyl cholinesterase
• OP degrading activity have been identified in insects resistant OP pesticides (see, for example, Baxter, G. D. et al., 1998; Baxter, G. D. et al., 2002; Rodrigo, L., et al., 1997, Vontas, J. G., et al., 2002; Walsh, S. B., et al., 2001 ; Zhu, K. Y., et al., 1995), and are contemplate for use.
• a proteinaceous molecule e.g., an enzyme, an antibody, a receptor, a peptide, a polypeptide
• An alteration in a property is possible because such molecules may be manipulated, for example, by chemical modification, including but not limited to, modifications described herein.
• alter or “alteration” may result in an increase or a decrease in the measured value for a particular property.
• a property in the context of a proteinaceous molecule, includes, but is not limited to, a ligand binding property, a catalytic property, a stability property, a property related to environmental safety, a charge property, or a combination thereof.
• Examples of a catalytic property that may be altered include a kinetic parameter, such as K m , a catalytic rate (k cat ) for a substrate, an enzyme's specificity for a substrate (k cat /K m ), or a combination thereof.
• Examples of a stability property that may be altered include thermal stability, half-life of activity, stability after exposure to a weathering condition, or a combination thereof.
• Examples of a property related to environmental safety include an alteration in toxicity, antigenicity, bio-degradability, or a combination thereof.
• an alteration to increase an enzyme's catalytic rate for a substrate, an proteinaceous molecule's specificity and/or binding property(s) for a ligand, a proteinaceous molecule's thermal stability, a proteinaceous molecule's half-life of activity, and/or a proteinaceous molecule's stability after exposure to a weathering condition may be selected for some applications, while a decrease in toxicity and/or antigenicity for a proteinaceous molecule may be selected in additional applications.
• a proteinaceous molecule e.g., an enzyme, an antibody, a receptor, a peptide, a polypeptide
• a proteinaceous molecule comprising a chemical modification and/or a sequence modification that functions the same or similar (e.g., a modified enzyme of the same EC classification as the unmodified enzyme) comprises a "functional equivalent" to, and "in accordance" with, an un-modified proteinaceous molecule.
• assays for determining whether a composition possesses one or more properties including, for example, an enzymatic activity, a stability property, a binding property, etc.
• a given chemical modification to a proteinaceous molecule e.g., an enzyme, an antibody, a receptor, a peptide, a polypeptide
• a functional equivalent enzyme comprising a plurality of different chemical modifications may be produced.
• a functional equivalent proteinaceous molecule comprising a structural analog and/or a sequence analog may possess an altered, an enhanced property and/or a reduced property, in comparison to the proteinaceous molecule upon which it is based.
• a "structural analog" refers to one or more
• 5842-02103 103 chemical modifications to the peptide backbone and/or non-side chain chemical moiety(s) of a proteinaceous molecule.
• a subcomponent of an proteinaceous molecule such as an apo-enzyme, a prosthetic group, a co-factor, or a combination thereof, may be modified to produce a functional equivalent structural analog.
• such an proteinaceous molecule sub-component that does not comprise a proteinaceous molecule may be altered to produce a functional equivalent structural analog of an proteinaceous molecule when combined with the other sub-components.
• sequence analog refers to one or more chemical modifications to the side chain chemical moiety(s), also known herein as a “residue” of one or more amino acids that define a proteinaceous molecule's sequence. Often such a “sequence analog” comprises an amino acid substitution, which may be produced by recombinant expression of a nucleic acid comprising a genetic mutation to produce a mutation in the expressed amino acid sequence.
• an "amino acid' may comprise a common and/or an uncommon amino acid.
• the common amino acids include: alanine (Ala, A); arginine (Arg, R); aspartic acid (a.k.a. aspartate; Asp, D); asparagine (Asn, N); cysteine (Cys, C); glutamic acid (a.k.a.
• An uncommon amino acid refers to an analog of a common amino acid (e.g., a D isomer of an L-amino acid), as well as a synthetic amino acid whose side chain may be chemically unrelated to the side chains of the common amino acids (e.g., a norleucine),.
• An amino acid may comprise a D-amino acid, an L-amino acid, and/or a cyclic (non-racemic) amino acid.
• a proteinaceous sequence e.g., a peptide
• a proteinaceous sequence may be constructed as retroinversopeptidomimetic of a proteinaceous sequence (e.g., a D- config u ration, an L- configuration.
• a proteinaceous molecule may comprise an amino acid such as a common amino acid, an uncommon amino acid, an L-amino acid, a D-amino acid, a cyclic (non-racemic) amino, or a combination thereof.
• an amino acid such as a common amino acid, an uncommon amino acid, an L-amino acid, a D-amino acid, a cyclic (non-racemic) amino, or a combination thereof.
• such a proteinaceous molecule may act rapidly and/or have reduced stability.
• a D-amino acid may increase the stability of a proteinaceous molecule, such as making the proteinaceous molecule insensitive and/or less susceptible to an L-amino acid biodegradation pathway.
• an L-amino acid peptide may be stabilized by addition of a D-amino acid at one or both of the peptide termini.
• biochemical pathways are available which may degrade a proteinaceous molecule comprising a D-amino acid, and may reduce long-term environmental persistence of such a proteinaceous molecule.
• the side chains of amino acids comprise one or more moiety(s) with specific chemical and physical properties. Certain side chains contribute to a ligand binding property, a catalytic property, a stability
• cysteines may form covalent bonds between different parts of a contiguous amino acid sequence, and/or between noncontiguous amino acid sequences to confer enhanced stability to a secondary, tertiary and/or quaternary structure.
• the presence of hydrophobic or hydrophilic side chains exposed to the outer environment may alter the hydrophobicity or hydrophilicity of part of a proteinaceous sequence, such as in the case of a transmembrane domain embedded in a lipid layer of a membrane.
• hydrophilic side chains may be exposed to the environment surrounding a proteinaceous molecule, which may enhance the overall solubility of a proteinaceous molecule in a polar liquid, such as water and/or a liquid component of a material formulation.
• various acidic, basic, hydrophobic, hydrophilic, and/or aromatic side chains present at or near a binding site of a proteinaceous structure may affect the affinity for a proteinaceous sequence for binding a ligand and/or a substrate, based on the covalent, ionic, Van der Waal forces, hydrogen bond, hydrophilic, hydrophobic, and/or aromatic interactions at a binding site.
• a residue may be "at or near" a residue and/or a group of residues when it is within about 15A, about 14A, about 13A, about 12A, about 11A, about 1 ⁇ A, about 9A, about 8A, about 7A, about 6A, about 5A, about 4A, about 3A, about 2k, and/or about 1 A the residue or group of residues such as residues identified as contributing to the active site and/or the binding site of a proteinaceous molecule.
• Identification of an amino acid whose chemical modification may likely change a property of a proteinaceous molecule may be accomplished using such methods as a chemical reaction, mutation, X-ray crystallography, nuclear magnetic resonance ("NMR"), computer based modeling, or a combination thereof. Selection of an amino acid on the basis of such information may then be used in the rational design of a mutant proteinaceous sequence that may possess an altered property. Alterations include those that alter a proteinaceous molecule's activity and/or function (e.g., binding activity, enzymatic activity, antimicrobial activity) to produce a functional equivalent of a proteinaceous moleculee.
• NMR nuclear magnetic resonance
• residues of a proteinaceous molecule that contribute to the properties of a proteinaceous molecule comprise chemically reactive moiety(s). These residues are often susceptible to chemical reactions that may inhibit their ability to contribute to a property of the proteinaceous molecule.
• a chemical reaction may be used to identify one or more amino acids comprised within the proteinaceous molecule that may contribute to a property. The identified amino acids then may be subject to modifications such as amino acid substitutions to produce a functional equivalent.
• 5842-02103 105 bis(dimethylamino)phenothiazin-5-ium chloride ("methylene blue”); Lys, which may be reacted with dimethylsuberimidate; Lys and/or Arg, which may be reacted with 2,4-dinitrofluorobenzene; Lys and/or Arg, which may be reacted with trinitrobenzene sulfonic acid ("TNBS”); Trp, which may be reacted with 2-hydroxy- 5-nitrobenzyl bromide 1-ethyl-3(3-dimethylaminopropyl); Trp, which may be reacted with 2-acetoxy-5- nitrobenzyl chloride; Trp, which may be reacted with ⁇ /-bromosucinimide; Tyr, which may be reacted with N- acetylimidazole (“NAI”); or a combination thereof (Hartleib, J. and Ruterjans, H., 2001 b; Josse, D. et al
• a variety of modifications of the art can be made to a proteinaceous molecule (e.g., a peptide), particularly a modification that may confer, retain, and/or alter a property (e.g., an antibiological activity).
• a modification may be used to increase the intrinsic antifungal potency of a peptide.
• a modification may reduce an antibiological activity of a proteinaceous molecule, such a reduction may still produce a proteinaceous molecule with suitable antibiological activlity.
• Other modifications may facilitate handling of a peptide.
• Other modifications may alter a binding property.
• a proteinaceous molecule's (e.g., a peptide) functional moiety that may typically be modified include a hydroxyl, an amino, a guanidinium, a carboxyl, an amide, a phenol, an imidazol ring(s), and/or a sulfhydryl.
• Typical reactions of these moieties include, for example, acetylation of a hydroxyl group by an alkyl halide; esterification, amidation (e.g., carbodiimides or other catalyst mediated amidation), and/or reduction to an alcohol of a carboxyl moiety; acidic or basic condition deamidation of an asparagine and/or a glutamine; an acylation, an alkylation, an arylation, and/or an amidation reaction of an amino group such as the primary amino group of a proteinaceous molecule (e.g., a peptide) and/or the amino group of a lysine residue; halogenation and/or nitration of the phenolic moiety of a tyrosine; or a combination thereof.
• esterification amidation (e.g., carbodiimides or other catalyst mediated amidation), and/or reduction to an alcohol of a carboxyl moiety
• solubility of a proteinaceous molecule examples include acylating a charged lysine residue and/or acetylating a carboxyl moiety of an aspartic acid and/or a glutamic acid.
• a cysteine may be eliminated from a proteinaceous molecule's (e.g., a peptide, an antibiological peptide) sequence, which may reduce cross linking via the cysteine's amino acid's free sulfhydryl moiety.
• a proteinaceous molecule e.g., an antifungal peptide, an antibiological peptide
• an activity e.g., an antibiological activity
• a proteinaceous composition (e.g., a peptide composition, an antibiotic peptide composition) comprises proteinaceous molecule (e.g., a peptide, a peptide library) has not been purified (e.g., impure by comprising one or more peptides of unknown exact sequence), comprises a side chain that has not been de-blocked (i.e., comprises a blocked side chain), comprises a covalent attachment to the synthetic resin (e.g., has not been cleared from a synthetic resin) used to anchor the growing amino acid chain of a peptide, or a combination thereof (e.g., both blocked at a side chain and attached to a resin).
• proteinaceous molecule e.g., a peptide, a peptide library
• has not been purified e.g., impure by comprising one or more peptides of unknown exact sequence
• comprises a side chain that has not been de-blocked i.e., comprises a blocked side chain
• the secondary, tertiary and/or quaternary structure of a proteinaceous molecule may be modeled using techniques known in the art, including X-ray crystallography, nuclear magnetic resonance, computer based modeling, or a combination thereof to aid in the identification of active- site, binding site, and other residues for the design and production of a mutant form of a proteinaceous molecule (e.g., an enzyme) (Bugg, C. E. et al., 1993; Cohen, A. A. and Shatzmiller, S. E., 1993; Hruby, V. J.,
• the secondary, tertiary and/or quaternary structures of a proteinaceous molecule may be directly determined by techniques such as X-ray crystallography and/or nuclear magnetic resonance to identify amino acids likely to effect one or more properties. Additionally, many primary, secondary, tertiary, and/or quaternary structures of proteinaceous molecules may be obtained using a public computerized database.
• An example of such a databank that may be used for this purpose comprises the Protein Data Bank (PDB), an international repository of the 3- dimensional structures of many biological macromolecules.
• PDB Protein Data Bank
• Computer modeling may be used to identify amino acids likely to affect one or more properties.
• a structurally related proteinaceous molecule comprises primary, secondary, tertiary and/or quaternary structures that are evolutionarily conserved in the wild-type protein sequences of various organisms.
• the secondary, tertiary and/or quaternary structure of a proteinaceous molecule may be modeled using a computer to overlay the proteinaceous molecule's amino acid sequence, which may be also known as the "primary structure," onto the computer model of a described primary, secondary, tertiary, and/or quaternary structure of another, structurally related proteinaceous molecule.
• amino acids that may participate in an active site, a binding site, a transmembrane domain, the general hydrophobicity and/or hydrophilicity of a proteinaceous molecule, the general positive and/or negative charge of a proteinaceous molecule, etc, may be identified by such comparative computer modeling.
• a selected proteinaceous molecule e.g., an active peptide
• functional equivalents may be created using mutations that substitute a different amino acid for the identified amino acid of interest.
• substitutions of an amino acid side chain to produce a "functional equivalent" proteinaceous molecule are also known in the art, and may involve a conservative side chain substitution a non-conservative side chain substitution, or a combination thereof, to rationally alter a property of a proteinaceous molecule.
• conservative side chain substitutions include, when applicable, replacing an amino acid side chain with one similar in charge (e.g., an arginine, a histidine, a lysine); similar in hydropathic index; similar in hydrophilicity; similar in hydrophobicity; similar in shape (e.g., a phenylalanine, a tryptophan, a tyrosine); similar in size (e.g., an alanine, a glycine, a serine); similar in chemical type (e.g., acidic side chains, aromatic side chains, basic side chains); or a combination thereof.
• an amino acid side chain with one similar in charge e.g., an arginine, a histidine, a lysine
• similar in hydropathic index similar in hydrophilicity
• similar in hydrophobicity similar in shape
• similar in shape e.g., a phenylalanine, a tryptophan, a tyrosine
• the relative hydropathic character of the amino acid may determine the secondary structure of the resultant protein, which in turn defines the interaction of the protein with a ligand (e.g., a substrate) molecule.
• a ligand e.g., a substrate
• a proteinaceous molecule e.g., a peptide, a polypeptide
• a principal aspect of the interaction of the proteinaceous molecule e.g., a peptide
• position within the proteinaceous molecule e.g., a peptide
• a characteristic of the amino acid residue may determine the interaction the proteinaceous molecule (e.g., a peptide) has in a biological system.
• An amino acid sequence may be varied in some embodiments. For example, certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain similar if not identical biological activity.
• the hydropathic index of the common amino acids are: Arg (-4.5); Lys (-3.9); Asn (-3.5); Asp (-3.5); GIn (-3.5); GIu (-3.5); His (-3.2); Pro (-1.6); Tyr (-1.3); Trp (-0.9); Ser (-0.8); Thr (-0.7); GIy (-0.4); Ala (+1.8); Met (+1.9); Cys (+2.5); Phe (+2.8); Leu (+3.8); VaI (+4.2); and Ne (+4.5). Additionally, a value has also been given to various amino acids based on hydrophilicity, which may also be used as a criterion for substitution (U.S. Pat. No. 4,554,101 ).
• the hydrophilicity values for the common amino acids are: Trp (-3.4); Phe (-2.5); Tyr (-2.3); Ne (-1.8); Leu (-1.8); VaI (-1.5); Met (-1.3); Cys (-1.0); Ala (-0.5); His (-0.5); Pro (-0.5+/-0.1 ); Thr (-0.4); GIy (0); Asn (+0.2); GIn (+0.2); Ser (+0.3); Asp (+3.0+/-0.1 ); GIu (+3.0+/-0.1 ); Arg (+3.0); and/or Lys (+3.0).
• an amino acid may be conservatively substituted (i.e., exchanged) for an amino acid comprising a similar or same hydropathic index and/or hydrophilic value
• the difference between the respective index and/or value may be generally within +/- 2, within +/- 1 , and/or within +/-0.5.
• a biological functional equivalence may typically be maintained wherein an amino acid substituted (e.g., conservatively substituted).
• isoleucine for example, which has a hydropathic index of +4.5, can be substituted for valine (+4.2) or leucine (+3.8), and still obtain a proteinaceous molecule (e.g., a protein) having similar activity (e.g., a biologic activity).
• a lysine (-3.9) can be substituted for arginine (-4.5), and so on.
• These amino acid substitutions are generally based on the relative similarity of R-group substituents, for example, in terms of size, electrophilic character, charge, and the like.
• substitutions which take the foregoing characteristics into consideration, for example for a hydropathic index include An alanine substituted with a GIy and/or a Ser; an arginine substituted with a Lys; an asparagine substituted with a GIn and/or a His; an aspartate substituted with a GIu; a cysteine substituted with a Ser; a glutamate substituted with an Asp; a glutamine substituted with an Asn; a glycine substituted with an Ala; a histidine substituted with an Asn and/or a GIn; an isoleucine substituted with a Leu and/or VaI; a leucine substituted with an Ne and/or a VaI; a lysine substituted with an Arg, a GIn, and/or a GIu; a methionine substituted with a Met, a Leu, a Tyr; a serotinine substituted with a Ser, an
• a functional equivalent may be produced by a non-mutation based chemical modification to an amino acid, a peptide, and/or a polypeptide.
• chemical modifications include, when applicable, a hydroxylation of a proline and/or a lysine; a phosphorylation of a hydroxyl group of a serine and/or a threonine; a methylation of an alpha-amino group of a lysine, an arginine and/or a histidine (Creighton, T.
• FITC fluorescein isothiocyanate compound
• Such modifications may produce an alteration in a property of a proteinaceous molecule.
• a N-terminal glycosylation may enhance a proteinaceous molecule's stability (Powell, M. F. et al., 1993).
• substitution of a beta-amino acid isoserine for a serine may enhance the aminopeptidase resistance a proteinaceous molecule (Coller, B. S. et al., 1993).
• a proteinaceous molecule may comprise a proteinaceous molecule longer or shorter than the wild- type amino acid sequence(s).
• an enzyme comprising longer or shorter sequence(s) may be encompassed, insofar as it retains enzymatic activity.
• a proteinaceous molecule may comprise one or more peptide and/or polypeptide sequence(s).
• a modification to a proteinaceous molecule may add and/or subtract one or two amino acids from a peptide and/or polypeptide sequence.
• a change to a proteinaceous molecule may add and/or remove one or more peptide and/or polypeptide sequence(s). Often a peptide or a polypeptide sequence may be added or removed to confer or remove a specific property from the proteinaceous molecule, and numerous examples of such modifications to a proteinaceous molecule are described herein, particularly in reference to fusion proteins.
• the native OPH of Pseudomonas diminuta may be produced with a short amino acide sequence at its N-terminas that promotes the exportation of the protein through the cell membrane and later cleaved.
• this signal sequence's amino acid sequence may be deleted by genetic modification in the DNA construction placed into Escherichia coli host cells to enhance its production.
• a "peptide" comprises a contiguous molecular sequence from about 3 to about 100 amino acids in length.
• a sequence of a peptide may comprise about 3 to about 100 amino acids in length.
• a "polypeptide" comprises a contiguous molecular sequence about 101 amino acids or greater.
• a sequence length of a polypeptide examples include about 101 to about 10,000 amino acids.
• a "protein” may comprise a proteinaceous molecule comprising a contiguous molecular sequence three amino acids or greater in length, matching the length of a biologically produced proteinaceous molecule encoded by the genome of an organism.
• Removal of one or more amino acids from a proteinaceous moleculee's sequence may reduce or eliminate a detectable property such as enzymatic activity, binding activity, etc.
• a longer sequence, particularly a proteinaceous molecule may consecutively and/or non-consecutively comprises and/or even repeats one or more sequences of a proteinaceous molecule (e.g., a repeated enzymatic
• fusion proteins may be bioengineered to comprise a wild-type sequence and/or a functional equivalent of a proteinaceous molecule's sequence and an additional peptide and/or polypeptide sequence that confers a property and/or function.
• An example of a functional equivalent includes a lipolytic enzyme functional equivalent.
• a lipolytic enzyme functional equivalent Using recombinant DNA technology, wild-type and mutant forms of numerous lipolytic genes have been expressed in various cell types and expression systems, for further characterization and analysis, as well as large scale production of lipolytic enzymes for industrial and/or commercial use.
• signaling sequences are added, deleted and/or modified to redirect an expressed enzyme's targeting to extracellular secretion to allow rapid purification from cellular material, and additional sequences, particularly tags (e.g., a poly His tag) are added to aid in purification.
• tags e.g., a poly His tag
• an enzyme may be targeted to the cell surface and/or to intercellular expression. Codon optimization may be used to enhance yield of enzyme produced in a host cell.
• mutations converting one or more residues of a protease cleavage site may enhance resistance to protease digestion.
• chymotrypsin cleavage site residues 149-156 identified in Pseudomonas glumae lipase may be converted into a proline, an arginine, and/or other residue(s) for enhance enzyme stability against protease inactivation.
• a mutation may be made that mimic the differences between a thermophilic lipolytic enzyme and a psychrophilic and/or a mesophilic lipolytic enzyme.
• thermostability comprises ones that improve the hydrophobic core packaging (i.e., enhance the ratio of the residues' volume within the van der Waals distances to total residues' volume; reduce the total enzyme surface-to-volume ratio); increases the percentage of arginine as charged residues, as arginine forms stabilizing ion-pairs; mutating a peptide bond that are liable to spontaneous and/or chemical (i.e., asn-gln, asp-pro) breakage; replaces a residue susceptible to oxidation, such as a methionine (e.g., a met with a leu) and aromatic residues, particularly those on the surface; and make such changes isomorphic (e.g., by use of a residue of similar size during substitution mutation) to prevent voids from being created [In "Engineering of/with Lipases" (F.
• 5842-02103 110 hydrophobic surface area of the enzyme and formation of an oxyanion transition state binding site ("oxyanion hole") that promotes catalysis.
• oxyanion hole oxyanion transition state binding site
• a cutinase lacks a lid structure and has a preformed oxyanion hole, so it typically does not use interfacial activation for lipolytic activity (Martinez, C. et al., 1994; Nicolas, A. et al., 1996).
• Ligand preference may be changed by alterations to binding site residue(s) and/or residue(s) of domains near the binding site.
• the preference for a cutinase for esters of about 4 to about 5 carbon fatty acids was shifted to esters of about 7 to about 8 carbon fatty acids by a binding site A85F mutation.
• a Phe139Trp mutation of the lid domain of a Candida antartica lipase improved activity against tributyrine substrate about 4-fold after comparison to the crystal structures of the more active lipases from a Rhizomucor miehei and a Humicola lanuginosa.
• enantioselectivity for a Humicola lanuginosa lipase was increased for 1-heptyl 2-methyldcanoate and decreased for phenyl 2- methyldecanoate by mutation to alter the open-lid conformation's electrostatic stability (In “Engineering of/with Upases” (F. Xavier Malcata., Ed.) pp. 197-202, 1996).
• a LipolaseTM and a Lipolase UltraTM are industrial lipases produced by multiple mutations to improve enzyme properties of temperature stability, proteolytic cleavage resistance, oxidation resistance, detergent resistance, and pH optimization. These lipases are mutated forms of the lipase isolated from a Humicola lanuginsa, where negatively charged residue(s) on the lid domain were replaced with positive and/or hydrophobic residue(s) (e.g., D96L) to reduce repulsion of negatively charged FAs and/or surfactant(s) associated with lipid(s), resulting in about 4 to about 5 fold or greater improvement in multicycle activity tests.
• positive and/or hydrophobic residue(s) e.g., D96L
• Mutations at a SavinaseTM cleavage sites also improved resistance to a proteolytic digestion.
• bulk mutations via random mutation libraries may be used directed domain sequences implicated with stability and/or activity (e.g., lid domain in a lipolytic enzyme, an active site region) to generate large numbers of mutants under selective screening protocols to mimic evolution and identify a modified enzyme (In "Engineering of/with Lipases” (F. Xavier Malcata., Ed.) pp. 203-217, 1996).
• lipolytic enzymes particularly enzymes having one or more mutations from the wild-type sequence (e.g., tags, signal sequences, mutations altering activity, etc.), are shown on the Table below.
• a glutaraldehyde e.g., cross-linking to produce a cross-linked enzyme crystal know as a "CLEC”
• a polystyrene e.g., cross-linking to produce a cross-linked enzyme crystal know as a "CLEC”
• a polystyrene e.g., cross-linking to produce a cross-linked enzyme crystal know as a "CLEC”
• a polystyrene e.g., cross-linking to produce a cross-linked enzyme crystal know as a "CLEC”
• a polystyrene e.g., cross-linking to produce a cross-linked enzyme crystal know as a "CLEC”
• a polystyrene e.g., cross-linking to produce a cross-linked enzyme crystal know as a "CLEC”
• a polystyrene e.g., cross-linking to produce a cross-linked enzyme crystal know as a
• lipase with didodecyl /V-D-glucono-L-glutamate enhanced activity 100-fold and improved organic solubility, presumably because the surfactant acted as an interface to alter the lid conformation.
• Production of a Psuedomonas cepacia and Candida rugosa lipase CLECs enhanced stability, and the C. rugosa CLEC has enhanced enantioselectivity for ketoprofen (Lalonde, J.J. et al., 1995; Persichetti, R.A., 1996).
• the presence of some chemicals may also enhance stability, such as hexanol, which has been described as improving cutinase's stability (In
• OPH normally binds two atoms of Zn 2+ per monomer when endogenously expressed. While binding a Zn 2+ , this enzyme may comprise a stable dimeric enzyme, with a thermal temperature of melting (“T m ”) of approximately 75 0 C and a conformational stability of approximately 40 killocalorie per mole (“kcal/mol”)
• a structural analog of an OPH sequence may be prepared comprising a Zn 2+ , a Co 2+ , a Fe 2+ , a Cu 2+ , a Mn 2+ , a Cd 2+ , a Ni 2+ , or a combination thereof.
• changes in the bound metal may be achieved by using cell growth media during cell expression of the enzyme wherein the concentration of a metal present may be defined, and/or removing the bound metal with a chelator (e.g., 1 ,10-phenanthroline; 8-hydroxyquinoline-5-sulfphonic acid; ethylenediaminetetraacetic acid) to produce an apo-enzyme, followed by reconstitution of a catalytically active enzyme by contact with a selected metal (Omburo, G. A. et al., 1992; Watkins, L. M. et al., 1997a; Watkins, L. M. et al., 1997b).
• a chelator e.g., 1 ,10-phenanthroline; 8-hydroxyquinoline-5-sulfphonic acid; ethylenediaminetetraacetic acid
• a structural analog of an OPH sequence may be prepared to comprise one metal atom per monomer.
• OPH structure analysis has been conducted using NMR (Omburo, G. A. et al., 1993).
• the X-ray crystal structure for OPH has been determined (Benning, M. M. et al., 1994; Benning, M. M. et al., 1995; Vanhooke, J. L. et al., 1996), including the structure of the enzyme while binding a substrate, further identifying residues involved in substrate binding and catalytic activity (Benning, M. M. et al., 2000). From these structure evaluations, the amino acids His55, His57, His201 ,
• the amino acid Asp301 may aid a nucleophilic attack by a bound hydroxide upon the phosphorus to promote cleavage of an OP compound, while the amino acid His354 may aid the transfer of a proton from the active site to the surrounding liquid in the latter stages of the reaction (Raushel, F. M., 2002).
• the amino acids His254 and His257 are not thought to comprise direct metal binding amino acids, but may comprise residues that interact (e.g., a hydrogen bond, a Van der Waal interaction) with each other and other active site residue(s), such as a residue that directly contact a substrate and/or bind a metal atom.
• amino acid His254 may interact with the amino acids His230, Asp232, Asp233, and Asp301.
• Amino acid His257 may comprise a participant in a hydrophobic substrate-binding pocket.
• the active site pocket comprises various hydrophobic amino acids, Trp131 , Phe132, Leu271 , Phe306, and Tyr309. These amino acids may aid the binding of a hydrophobic OP compound (Benning, M. M. et al., 1994; Benning, M. M. et al., 1995; Vanhooke, J. L. et al., 1996). Electrostatic interactions may occur between phosphoryl oxygen, when present, and the side chains of Trp131 and His201.
• the amino acids Gly60, Ne106, Leu303, and Ser308 are thought to comprise the small subsite.
• the amino acids Cys59 and Ser61 are near the small subsite, but with the side chains thought to be orientated away from the subsite.
• the amino acids His254, His257, Leu271 , and Met317 are thought to comprise the large subsite.
• the amino acids Trp131 , Phe132, Phe306, and Tyr309 are thought to comprise the leaving group subsite, though Leu271 may be considered part of this subsite as well (Watkins, L. M. et al., 1997a).
• OPH sequence analog mutants include H55C, H57C, C59A, G60A, S61A, I106A, I106G, W131A, W131 F, W131 K, F132A, F132H, F132Y, L136Y, L140Y, H201 C, H230C, H254A, H254R, H254S, H257A, H257L, H257Y, L271A, L271Y, L303A, F306A, F306E, F306H, F306K, F306Y, S308A, S308G, Y309A, M317A, M317H, M317K, M317R, H55C/H57C,
• the sequence and structural information has been used in production of mutants of OPH possessing cysteine substitutions at the metal binding histidines His55, His57, His201 , and His230.
• the H57C mutant had between 50% ⁇ i.e., binding a Cd 2+ , a Zn 2+ ) and 200% ⁇ i.e., binding a Co 2+ ) wild- type OPH activity for paraoxon cleavage.
• the H201C mutant had about 10% activity, the H230C mutant had less than 1 % activity, and the H55C mutant bound one atom of a Co 2+ and possessed little detectable activity, but may still be useful if possessing an useful property (e.g., enhanced stability) (Watkins, L. M., 1997b).
• the sequence and structural information has been used in production of mutants of OPH possessing altered metal binding and/or bond-type cleavage properties.
• OPH mutants H254R, H257L, and H254R/H257L have been made to alter amino acids that are thought to interact with nearby metal-binding amino acids. These mutants also reduced the number of metal ions ⁇ i.e., Co 2+ , Zn 2+ ) binding the enzyme dimer from four to two, while still retaining 5% to greater than 100% catalytic rates for the various substrates. These reduced metal mutants possess enhanced specificity for larger substrates such as NPPMP and demeton-S, and reduced specificity for the smaller substrate diisopropyl fluorophosphonate (diSioudi, B. et al., 1999).
• the H254R mutant and the H257L mutant each demonstrated a greater than four-fold increase in catalytic activity and specificity against VX and its analog demeton S.
• the H257L mutant also demonstrated a five-fold enhanced specificity against soman and its analog NPPMP (diSioudi, B. D. et al., 1999).
• mutants of OPH ⁇ i.e., a phosphotriesterase
• These substrates either comprised a negative charge and/or a large amide moiety.
• a M317A mutant was created to enlarge the size of the large subsite, and M317H, M317K, and M317R mutants were created to incorporate a cationic group in the active site.
• the M317A mutant demonstrated a 200-fold cleavage rate enhancement in the presence of alkylamines, which were added to reduce the substrate's negative charge.
• M317H, M317K, and M317R mutants demonstrated modest improvements in rate and/or specificity, including a 7-fold k cat /K m improvement for the M317K mutant (Shim, H. et al., 1998).
• the W131 K, F132Y, F132H, F306Y, F306H, F306K, F306E, F132H/F306H, F132Y/F306Y, F132Y/F306H, and F132H/F306Y mutants were made to add and/or change the side chain of active site residues to form a hydrogen bond and/or donate a hydrogen to a cleaved substrate's leaving group, to enhance the rate of cleavage for certain substrates, such as phosphofluoridates.
• F132Y, F132H, F306Y, F306H, F132H/F306H, F132Y/F306Y, F132Y/F306H, and F132H/F306Y mutants all demonstrated enhanced enzymatic cleavage rates, of about three- to ten-fold improvement, against the phosphonofluoridate, diisopropyl fluorophosphonate (Watkins, L. M. et al., 1997a).
• OPH mutants W131 F, F132Y, L136Y, L140Y, L271Y and H257L were designed to modify the active site size and placement of amino acid side chains to refine the structure of binding subsites to specifically fit the binding of a VX substrate.
• the refinement of the active site structure produced a 33% increase in cleavage activity against VX in the L136Y mutant (Gopal, S. et al., 2000).
• Various mutants of OPH have been made to alter the steriospecificity, and in some cases, the rate of reaction, by substitutions in substrate binding subsites.
• the C59A, G60A, S61A, I106A, W131A, F132A, H254A, H257A, L271A, L303A, F306A, S308A, Y309A, and M317A mutants of OPH have been produced to alter the size of various amino acids associated with the small subsite, the large subsite and the leaving group subsite, to alter enzyme activity and selectivity, including sterioselectivity, for various OP compounds.
• the G60A mutant reduced the size of the small subsite, and decreased both rate (k cat ) and specificity (k cat /K a ) for R p -enantiomers, thereby enhancing the overall specificity for some S p -enantiomers to over 11 ,000: 1.
• Mutants H254Y, H254F, H257Y, H257F, H257W, H257L, L271Y, L271 F, L271W, M317Y, M317F, and M317W were produced to shrink the large subsite, with the H257Y mutant, for example, demonstrating a reduced selectivity for S p -enantiomers (Chen-Goodspeed, M. et al., 2001 b).
• I106A/H257Y, F132A/H257Y, I106A/F132A/H257Y, 1106A/H257Y/S308A, I106A/F132A/H257W, F132A/H257Y/S308A, I106G/H257Y, F132G/H257Y, I106G/F132G/H257Y, I106G/H257Y/S308G, and I106G/F132G/H257Y/S308G were made to simultaneously enlarge the small subsite and shrink the large subsite.
• Mutants such as H257Y, I106A/H257Y, I106G, I106A/F132A, and I106G/F132G/S308G were effective in altering steriospecificity for S P :R P enantiomer ratios of some substrates to less than 3:1 ratios.
• Mutants including F132A/H257Y, I106A/F132A/H257W, I106G/F132G/H257Y, and I106G/F132G/H257Y/S308G demonstrated a reversal of selectivity for S P :R P enantiomer ratios of some substrates to ratios from 3.6:1 to 460:1.
• Such alterations in sterioselectivity may enhance OPH performance against a specific OP compound that may comprise a target of detoxification, including a CWA. Enlargement of the small subsite
• a mutant of OPH designated G60A has also been created with enhanced steriospecificity relative to specific analogs of enantiomers of sarin and soman (Li, W.-S. et al., 2001 ; Raushel, F. M., 2002). Of greater interest, these mutant forms of OPH have been directly assayed against sarin and soman nerve agents, and demonstrated enhanced detoxification rates for racemic mixtures of sarin or soman enantiomers.
• Wild-type OPH has a k cat for sarin of 56 s ⁇ 1
• the I106A/F132A/H257Y mutant has k cat for sarin of 1000 s ⁇ ⁇
• wild-type OPH has a k cat for soman of 5 s "1
• the G60A Mutant has k cat for soman of 10 s "1 (Kolakoski, Jan E. et al. 1997; Li, W.-S. et al., 2001 ).
• the mutants identified may possess substitutions at amino acids that have not been identified as directly comprising the active site, or its binding subsites, using techniques such as NMR, X-ray crystallography and computer structure analysis, but still contribute to activity for one or more substrates.
• selection of OPH mutants based upon enhanced cleavage of methyl parathion identified the A80V/S365P, L182S/V310A, I274N, H257Y, H257Y/I274N/S365P, L130M/H257Y/I274N, and A14T/A80V/L185R/H257Y/I274N mutants as having enhanced activity.
• Amino acids Ne274 and Val310 are within 1 ⁇ A of the active site, though not originally identified as part of the active site from X-ray and computer structure analysis. However, mutants with substitutions at these amino acids demonstrated improved activity, with mutants comprising the I274N and H257Y substitutions particularly active against methyl parathion. Additionally, the mutant, A14T/A80V/L185R/H257Y/I274N, further comprising a L185R substitution, was active having a 25-fold improvement against methyl parathion (Cho, C. M. -H. et al., 2002). [0312] In an example, a functional equivalent of OPH may be prepared that lacks the first 29-31 amino acids of the wild-type enzyme.
• the wild-type form of OPH endogenously or recombinantly expressed in Pseudomonas or Flavobacterium removes the first N-terminal 29 amino acids from the precursor protein to produce the mature, enzymatically active protein (Mulbry, W. and Karns, J., 1989; Serdar, C. M. et al., 1989).
• Recombinant expressed OPH in Gliocladium virens apparently removes part or all of this sequence (Dave, K. I. et al., 1994b).
• Recombinant expressed OPH in Streptomyces lividans primarily has the first 29 or 30 amino acids removed during processing, with a few percent of the functional equivalents having the first 31 amino acids removed (Rowland, S. S. et al., 1992).
• Recombinant expressed OPH in Spodoptera frugiperda cells has the first 30 amino acids removed during processing (Dave, K. I. et al., 1994a).
• the 29 amino acid leader peptide sequence targets OPH enzyme to the cell membrane in Escherichia coli, and this sequence may be partly or fully removed during cellular processing (Dave, K. I. et al., 1994a; Miller, C. E., 1992; Serdar, C. M. et al., 1989; Mulbry, W. and Karns, J., 1989).
• the association of OPH comprising the leader peptide sequence with the cell membrane in Escherichia coli expression systems seems to be relatively weak, as brief 15 second sonication releases most of the activity into the extracellular environment (Dave, K. I. et al., 1994a).
• recombinant OPH may be expressed without this leader peptide sequence to enhance enzyme stability and expression efficiency in Escherichia coli (Serdar, C. M., et al. 1989).
• recombinant expression efficiency in Pseudomonas putida for OPH was improved by retaining this sequence, indicating that different species of bacteria may have varying preferences for a signal sequence (Walker, A. W. and Keasling, J. D., 2002).
• the length of an enzymatic sequence may be readily modified to improve expression or other properties in a particular organism, or select a cell with a relatively good ability to express a biomolecule, in light of the present disclosures and methods in the art (see U.S. Patent Nos.
• mutants of OPH comprising one or more amino acid substitutions such as the C59A, G60A, S61A, I106A, W131A, F132A, H254A, H257A, L271A, L303A, F306A, S308A, Y309A, M317A, I106A/F132A, I106A/S308A, F132A/S308A, I106G, F132G, S308G, I106G/F132G, I106G/S308G, F132G/S308G, I106G/F132G/S308G, H254Y, H254F, H257Y, H257F, H257W, H257L, L271Y, L271W, M317Y, M317F, M317W, I106A/H257Y, F132A/H257Y, I106A/F132A/
• LacZ-OPH fusion protein mutants lacking the 29 amino acid leader peptide sequence and comprising an amino acid substitution mutant such as W131 F, F132Y, L136Y, L140Y, H257L, L271 L, L271Y, F306A, or F306Y have been recombinantly expressed (Gopal, S. et al., 2000).
• OPH mutants that comprise additional amino acid sequences are also known in the art.
• An OPH fusion protein lacking the 29 amino acid leader sequence and possessing an additional C-terminal flag octapeptide sequence was expressed and localized in the cytoplasm of Escherichia coli (Wang, J. et al., 2001 ).
• nucleic acids encoding truncated versions of the ice nucleation protein ("InaV") from Pseudomonas syringae have been used to construct vectors that express OPH-lnaV fusion proteins in Escherichia coli.
• the InaV sequences targeted and anchored the OPH- InaV fusion proteins to the cells' outer membrane (Shimazu, M. et al., 2001a; Wang, A. A. et al., 2002).
• a vector encoding a similar fusion protein was expressed in Moraxella sp., and demonstrated a 70-fold improved OPH activity on the cell surface compared to Escherichia coli expression (Shimazu, M. et al., 2001 b).
• fusion proteins comprising the signal sequence and first nine amino acids of lipoprotein, a transmembrane domain of outer membrane protein A ("Lpp-OmpA"), and either a wild-type OPH sequence or an OPH truncation mutant lacking the first 29 amino acids has been expressed in Escherichia coli.
• Lpp-OmpA transmembrane domain of outer membrane protein A
• a fusion protein comprising N-terminus to C-terminus, a (His)6 polyhistidine tag, a green fluorescent protein ("GFP"), an enterokinase recognition site, and an OPH sequence lacking the 29 amino acid leader sequence has been expressed within Escherichia coli cells (Wu, C-F. et al., 2000b, Wu, C-F. et al., 2002).
• a similar fusion protein a (His)6 polyhistidine tag, an enterokinase recognition site, and an OPH sequence lacking the 29 amino acid leader sequence has also been expressed within Escherichia coli cells (Wu, C-F. et al., 2002). Additionally, variations of these GFP-OPH fusion proteins have been expressed within Escherichia coli cells where a second enterokinase recognition site was placed at the C-terminus of the OPH gene fragment sequence, followed by a second OPH gene fragment sequence (Wu, C-F. et al., 2001 b).
• the GFP sequence produced fluorescence that was proportional to both the quantity of the fusion protein, and the activity of the OPH sequence, providing a fluorescent assay of enzyme activity and stability in GFP-OPH fusion proteins (Wu, C-F. et al., 2000b, Wu, C-F. et al., 2002).
• a fusion protein comprising an elastin-like polypeptide ("ELP") sequence, a polyglycine linker sequence, and an OPH sequence was expressed in Escherichia coli (Shimazu, M. et al., 2002).
• a cellulose-binding domain at the N-terminus of an OPH fusion protein lacking the 29 amino acid leader sequence and a similar fusion protein wherein OPH possessed the leader sequence, where both predominantly excreted into the external medium as soluble proteins by recombinant expression in Escherichia coli (Richins, R. D. et al., 2000).
• Site-directed mutagenesis was used to alter the enzymatic activity of human paraoxonase through conservative and non-conservative substitutions, and thus clarify the specific amino acids functioning in enzymatic activity.
• Specific paraoxonase mutants include the sequence analogs E32A, E48A, E52A, D53A, D88A, D107A, H114N, D121A, H133N, H154N, H160N, W193A, W193F, W201A, W201 F, H242N, H245N, H250N, W253A, W253F, D273A, W280A, W280F, H284N, and/or H347N.
• the various paraoxonase mutants generally had different enzymatic properties.
• W253A had a 2-fold greater k cat ; and W201 F, W253A and W253F each had a 2 to 4 fold increase in k cat , though W201 F also had a lower substrate affinity.
• a non-conservative substitution mutant W280A had 1 % wild-type paraoxonase activity, but the conservative substitution mutant W280F had similar activity as the wild-type paraoxonase (Josse, D. et al., 1999; Josse, D. et al., 2001 ).
• Specific squid-type DFPase mutants include the sequence analogs H181 N, H224N, H274N, H219N, H248N, and/or H287N.
• the H287N mutant lost about 96% activity, and may act as a hydrogen acceptor in active site reactions.
• the H181 N and H274N mutants lost between 15% and 19% activity, and are thought to help stabilize the enzyme.
• the H224N mutant gained about 14% activity, indicating that alterations to this residue may also affect activity (Hartleib, J. and Ruterjans, H., 2001 b).
• squid-type DFPase functional equivalents recombinant squid-type DFPase sequence-length mutants have been expressed wherein a (His)6 tag sequence and a thrombin cleavage site has been added to the squid-type DFPase (Hartleib, J. and Ruterjans, H., 2001a).
• a polypeptide comprising amino acids 1-148 of squid-type DFPase has been admixed with a polypeptide comprising amino acids 149-314 of squid-type DFPase to produce an active enzyme (Hartleib, J. and Ruterjans, H., 2001a).
• a composition, an article, a method, etc. may comprise one or more selected biomolecules, in various combinations thereof, with a proteinaceous molecule (e.g., an enzyme, a peptide that binds a ligand, a polypeptide that binds a ligand, an antimicrobial peptide, an antifouling peptide) being a type of biomolecule in certain facets.
• a proteinaceous molecule e.g., an enzyme, a peptide that binds a ligand, a polypeptide that binds a ligand, an antimicrobial peptide, an antifouling peptide
• any combination of biomolecules such as an enzyme (e.g., an antimicrobial enzyme, organophosphorous compound degrading enzyme, an esterase, a peptidase, a lipolytic enzyme, an antifouling enzyme, etc) and/or a peptide (e.g., an antimicrobial peptide, an antifouling enzyme) described herein are contemplated for incorporation into a material formulation (e.g., a surface treatment, a filler, a biomolecular composition), and may be used to confer one or more properties (e.g., one or more enzyme activities, one or more binding activities, one or more antimicrobial activities, etc) to such compositions.
• an enzyme e.g., an antimicrobial enzyme, organophosphorous compound degrading enzyme, an esterase, a peptidase, a lipolytic enzyme, an antifouling enzyme, etc
• a peptide e.g., an antimicrobial peptide, an antifouling enzyme
• a composition may comprise an endogenous, recombinant, biologically manufactured, chemically synthesized, and/or chemically modified, biomolecule.
• a composition may comprises a wild-type enzyme, a recombinant enzyme, a biologically manufactured peptide and/or polypeptide (e.g., a biologically produced enzyme that may be subsequently chemically modified), a chemically synthesized peptide and/or polypeptide, or a combination thereof.
• a recombinant proteinaceous molecule comprises a wild-type proteinaceous molecule, a functional equivalent proteinaceous molecule, or a combination thereof.
• a biomolecule e.g., a proteinaceous molecule
• any such biomolecule in the art is contemplated for inclusion in a composition, an article, a method, etc.
• a combination of biomolecules may be selected for inclusion in a material formulation, to improve one or more properties of such a composition.
• a composition may comprise 1 to 1000 or more
• 5842-02103 146 different selected biomolecules of interest.
• various enzymes have differing binding properties, catalytic properties, stability properties, properties related to environmental safety, etc, one may select a combination of enzymes to confer an expanded range of properties to a composition.
• a plurality of lipolytic enzymes with differing abilities to cleave the lipid substrates, may be admixed to confer a larger range of catalytic properties to a composition than achievable by the selection of a single lipolytic enzyme.
• a material formulation may comprise a plurality of biomolecular compositions.
• one or more layers of a multicoat system comprise one or more different biomolecular compositions to confer differing properties between one layer and at least a second layer of the multicoat system.
• a multifunctional surface treatment e.g., a paint, a coating
• Such a surface treatment may provide functions upon application to a surface such as, for example, lend antifungal and antibacterial properties to the surface; avoid the problem human toxicity that may be associated with a conventional biocidal compound in a coating (e.g., a paint); usefulness in hospital environments and other health care settings (e.g., deter food poisoning, hospital acquired infections by antibiotic-resistant "super bugs," deter SARS-like outbreaks); reduce the contamination of a public facility and/or a surface by a toxic chemical (e.g., an OP compound) due to an accidental spill, an improper application of certain insecticide, and/or as a result of deliberate criminal and/or terroristic act; or a combination thereof.
• a toxic chemical e.g., an OP compound
• the concentration of any individual selected biomolecule (e.g., an enzyme, a peptide, a polypeptide) of a material formulation comprises about
• a cell-based particulate material may function as a filler, and may comprise up to about 80% of the volume of material formulation (e.g., a coating, a surface treatment), in some embodiments.
• an antibiological peptide may comprise about 0.000000001 % to about 20%, 10%, or 5% of a material formulation.
• a proteinaceous molecule may be biologically produced in a cell, a tissue and/or an organism transformed with a genetic expression vector.
• an "expression vector” refers to a carrier nucleic acid molecule, into which a nucleic acid sequence may be inserted, wherein the nucleic acid sequence may be capable of being transcribed into a ribonucleic acid (“RNA") molecule after introduction into a cell.
• RNA ribonucleic acid
• an expression vector comprises deoxyribonucleic acid ("DNA”).
• an “expression system” refers to an expression vector, and may further comprise additional reagents to promote insertion of a nucleic acid sequence, introduction into a cell, transcription and/or translation.
• a "vector” refers to a carrier nucleic acid molecule into which a nucleic acid sequence may be inserted for
• a viral vector may be used in conjunction with either an eukaryotic and/or a prokaryotic host cell, particularly one permissive for replication and/or expression of the vector.
• a cell capable of being transformed with a vector may be known herein as a "host cell.”
• the inserted nucleic acid sequence encodes for at least part of a gene product.
• the nucleic acid sequence may be transcribed into a RNA molecule, the RNA molecule may be then translated into a proteinaceous molecule.
• a “gene” refers to a nucleic acid sequence isolated from an organism, and/or man-made copies or mutants thereof, comprising a nucleic acid sequence capable of being transcribed and/or translated in an organism.
• a “gene product” comprises the transcribed RNA and/or translated proteinaceous molecule from a gene.
• partial nucleic acid sequences of a gene known herein as a “gene fragment,” are used to produce a part of the gene product.
• Many gene and gene fragment sequences are known in the art, and are both commercially available and/or publicly disclosed at a database such as Genbank.
• a gene and/or a gene fragment may be used to recombinantly produce a proteinaceous molecule and/or in construction of a fusion protein comprising a proteinaceous molecule.
• a nucleic acid sequence such as a nucleic acid sequence encoding an enzyme, and/or any other desired RNA and/or proteinaceous molecule (as well as a nucleic acid sequence comprising a promoter, a ribosome binding site, an enhancer, a transcription terminator, an origin of replication, and/or other nucleic acid sequences, including but not limited to those described herein may be recombinantly produced and/or synthesized using any method or technique in the art in various combinations. [In "Molecular Cloning” (Sambrook, J., and Russell, D.
• a gene and/or a gene fragment encoding an enzyme of interest may be isolated and/or amplified through polymerase chain reaction ("PCRTM") technology. Often such nucleic acid sequence may be readily available from a public database and/or a commercial vendor, as previously described.
• PCRTM polymerase chain reaction
• Nucleic acid sequences called codons, encoding for each amino acid are used to copy and/or mutate a nucleic acid sequence to produce a desired mutant in an expressed amino acid sequence.
• Codons comprise nucleotides such as adenine ("A”), cytosine ("C”), guanine ("G”), thymine (“T”) and uracil ("U”).
• the common amino acids are generally encoded by the following codons: alanine by GCU, GCC, GCA, or GCG; arginine by CGU, CGC, CGA, CGG, AGA, or AGG; aspartic acid by GAU or GAC; asparagine by AAU or AAC; cysteine by UGU or UGC; glutamic acid by GAA or GAG; glutamine by CAA or CAG; glycine by GGU, GGC, GGA, or GGG; histidine by CAU or CAC; isoleucine by AUU, AUC, or AUA; leucine by UUA, UUG, CUU, CUC, CUA ,or CUG; lysine by AAA or AAG; methionine by AUG; phenylalanine by UUU or UUC;
• a mutation in a nucleic acid encoding a proteinaceous molecule may be introduced into the nucleic acid sequence through any technique in the art.
• Such a mutation may be bioengineered to a specific region of a nucleic acid comprising one or more codons using a technique such as site-directed mutagenesis and/or cassette mutagenesis.
• a technique such as site-directed mutagenesis and/or cassette mutagenesis.
• Numerous examples of phosphoric triester hydrolase mutants have been produced using site-directed mutagenesis or cassette mutagenesis, and are described herein, as well as other enzymes.
• the choice of codons may be made to mimic the host cell's molecular biological activity, to improve the efficiency of expression from an expression vector.
• codons may be selected to match the preferred codons used by a host cell in expressing endogenous proteins.
• the codons selected may be chosen to approximate the G-C content of an expressed gene and/or a gene fragment in a host cell's genome, or the G-C content of the genome itself.
• a host cell may be genetically altered to recognize more efficiently use a variety of codons, such as Escherichia coli host cells that are cfnaYgene positive (Brinkmann, U. et al., 1989).
• An expression vector may comprise specific nucleic acid sequences such as a promoter, a ribosome binding site, an enhancer, a transcription terminator, an origin of replication, and/or other nucleic acid sequence, including but not limited to those described herein, in various combinations.
• a nucleic acid sequence may be "exogenous" when foreign to the cell into which the vector is being introduced and/or that the sequence is homologous to a sequence in the cell, but in a position within the host cell nucleic acid in which the sequence is ordinarily not found.
• An expression vector may have one or more nucleic acid sequences removed by restriction enzyme digestion, modified by mutagenesis, and/or replaced with another more appropriate nucleic acid sequence, for transcription and/or translation in a host cell suitable for the expression vector selected.
• a vector may be constructed by recombinant techniques in the art. Further, a vector may be expressed and/or transcribe a nucleic acid sequence and/or translate its cognate proteinaceous molecule.
• the conditions under which to incubate any of the above described host cells to maintain them and to permit replication of a vector, and techniques and conditions allowing large-scale production of a vector, as well as production of a nucleic acid sequence encoded by a vector into a RNA molecule and/or translation of the RNA molecule into a cognate proteinaceous molecule, may be used.
• a cell may express multiple gene and/or gene fragment products from the same vector, and/or express more than one vector. Often this occurs simply as part of the normal function of a multi-vector expression system.
• one gene or gene fragment may be used to produce a repressor that suppresses the activity of a promoter that controls the expression of a gene or a gene fragment of interest.
• the repressor gene and the desired gene may be on different vectors.
• multiple gene, gene fragment and/or expression systems may be used to express an enzymatic sequence of interest and another gene or gene fragment that may be desired for a particular function.
• a particular function may be desired for a particular function.
• recombinant Pseudomonas putida has co-expressed OPH from one vector, and the multigenes encoding the enzymes for converting p-nitrophenol to ⁇ -ketoadipate from a different vector.
• the expressed OPH catalyzed the cleavage of parathion to p-nitrophenol.
• the additionally expressed recombinant enzymes converted the p-nitrophenol, a moderately toxic compound, to ⁇ -ketoadipate, thereby detoxifying both an OP compound and the byproducts of its hydrolysis (Walker, A. W. and Keasling, J. D., 2002).
• Escherichia coli cells expressed a cell surface targeted INPNC-OPH fusion protein from one vector to detoxify OP compounds, and co-expressed from a different vector a cell surface targeted Lpp-OmpA- cellulose binding domain fusion protein to immobilize the cell to a cellulose support (Wang, A. A. et al., 2002).
• a vector co-expressed an antisense RNA sequence to the transcribed stress response gene ⁇ 32 and OPH in Escherichia coli. The antisense ⁇ 32 RNA was used to reduce the cell's stress response, including proteolytic damage, to an expressed recombinant proteinaceous molecule.
• OPH enzyme A sixfold enhanced specific activity of expressed OPH enzyme was seen (Srivastava, R. et al., 2000).
• multiple OPH fusion proteins were expressed from the same vector using the same promoter but separate ribosome binding sites (Wu, C-F. et al., 2001 b).
• An expression vector generally comprises a plurality of functional nucleic acid sequences that either comprise a nucleic acid sequence with a molecular biological function in a host cell, such as a promoter, an enhancer, a ribosome binding site, a transcription terminator, etc, and/or encode a proteinaceous sequence, such as a leader peptide, a polypeptide sequence with enzymatic activity, a peptide and/or a polypeptide with a binding property, etc.
• a nucleic acid sequence may comprise a "control sequence,” which refers to a nucleic acid sequence that functions in the transcription and possibly translation of an operatively linked coding sequence in a particular host cell.
• an "operatively linked” or “operatively positioned” nucleic acid sequence refers to the placement of one nucleic acid sequence into a functional relationship with another nucleic acid sequence.
• Vectors and expression vectors may further comprise one or more nucleic acid sequences that serve other functions as well and are described herein. [0336]
• the various functional nucleic acid sequences that comprise an expression vector are operatively linked so to position the different nucleic acid sequences for function in a host cell.
• the functional nucleic acid sequences may be contiguous such as placement of a nucleic acid sequence encoding a leader peptide sequence in correct amino acid frame with a nucleic acid sequence encoding a polypeptide comprising a polypeptide sequence with enzymatic activity.
• the functional nucleic acid sequences may be non-contiguous such as placing a nucleic acid sequence comprising an enhancer distal to a nucleic acid sequence comprising such sequences as a promoter, an encoded proteinaceous molecule, a transcription termination sequence, etc.
• a "promoter” comprises a control sequence comprising a region of a nucleic acid sequence at which initiation and rate of transcription are controlled.
• a nucleic acid sequence comprising a promoter and an additional nucleic acid sequence, particularly one encoding a gene and/or a gene fragment's product
• the phrases "operatively linked,” “operatively positioned,” “under control,” and “under transcriptional control” mean that a promoter is in a functional location and/or an orientation in relation to the
• a promoter may comprise genetic element(s) at which regulatory protein(s) and molecule(s) may bind such as an RNA polymerase and other transcription factor(s).
• a promoter employed may be constitutive, tissue-specific, inducible, and/or useful under the appropriate conditions to direct high level expression of the introduced nucleic acid sequence, such as the large-scale production of a recombinant proteinaceous molecule. Examples of a promoter include a lac, a tac, an amp, a heat shock promoter of a P- element of Drosophila, a baculovirus polyhedron gene promoter, or a combination thereof.
• the nucleic acids encoding OPH have been expressed using the polyhedron promoter of a baculoviral expression vector (Dumas, D. P. et al., 1990).
• a Cochliobolus heterostrophus promoter, prom ⁇ has been used to express a nucleic acid encoding OPH (Dave, K. I. et al., 1994b).
• the promoter may be endogenous or heterologous.
• An "endogenous promoter” comprises one naturally associated with a gene and/or a sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or an exon.
• the coding nucleic acid sequence may be positioned under the control of a “heterologous promoter” or “recombinant promoter,” which refers to a promoter that may be not normally associated with a nucleic acid sequence in its natural environment.
• a specific initiation signal also may be required for efficient translation of a coding sequence by the host cell.
• a signal may include an ATG initiation codon ("start codon") and/or an adjacent sequence.
• Exogenous translational control signals including the ATG initiation codon, may be provided. Techniques of the art may be used for determining this and providing the signals.
• the initiation codon may be "in-frame" with the reading frame of the desired coding sequence to ensure translation of the entire insert.
• the exogenous translational control signal and/or an initiation codon may be either natural or synthetic. The efficiency of expression may be enhanced by the inclusion of an appropriate transcription enhancer.
• a promoter may or may not be used in conjunction with an "enhancer,” which refers to a c/s-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.
• An enhancer may comprise one naturally associated with a nucleic acid sequence, located either downstream and/or upstream of that sequence.
• a recombinant or heterologous enhancer refers also to an enhancer not normally associated with a nucleic acid sequence in its natural environment.
• Such a promoter and/or enhancer may include a promoter and/or enhancer of another gene, a promoter and/or enhancer isolated from any other prokaryotic, viral, or eukaryotic cell, a promoter and/or enhancer not "naturally occurring," i.e., a promoter and/or enhancer comprising different elements of different transcriptional regulatory regions, and/or mutations that alter expression.
• a sequence may be produced using recombinant cloning and/or nucleic acid amplification technology, including PCRTM, in connection with the compositions disclosed herein (U.S. Patent 4,683,202, U.S. Patent 5,928,906).
• a promoter and/or an enhancer that effectively directs the expression of the nucleic acid sequence in the cell type may be chosen for expression.
• the art of molecular biology generally knows the use of promoters, enhancers, and cell type combinations for expression. Furthermore, the control sequences that
• Vectors may comprise a multiple cloning site ("MCS"), which comprises a nucleic acid region that comprises multiple restriction enzyme sites, any of which may be used in conjunction with standard recombinant technology to digest the vector.
• MCS multiple cloning site
• Restriction enzyme digestion refers to catalytic cleavage of a nucleic acid molecule with an enzyme which functions at specific locations in a nucleic acid molecule. Many of these restriction enzymes are commercially available. Use of such enzymes may be done in accordance with the art.
• a vector may be linearized and/or fragmented using a restriction enzyme that cuts within the MCS to enable an exogenous nucleic acid sequence to be ligated to the vector.
• "Ligation” refers to the process of forming phosphodiester bonds between two nucleic acid fragments, which may or may not be contiguous with each other. Techniques involving restriction enzymes and ligation reactions in the art of recombinant technology may be applied. .
• a "fusion protein,” as used herein, comprises an expressed contiguous amino acid sequence comprising a proteinaceous molecule of interest and one or more additional peptide and/or polypeptide sequences.
• the additional peptide and/or polypeptide sequence generally provides an useful additional property to the fusion protein, including but not limited to, targeting the fusion protein to a particular location within and/or external to the host cell (e.g., a signal peptide); promoting the ease of purification and/or detection of the fusion protein (e.g., a tag, a fusion partner); promoting the ease of removal of one or more additional sequences from the peptide and/or the polypeptide of interest (e.g., a protease cleavage site); and separating one or more sequences of the fusion protein to allow improved activity and/or function of the sequence(s) (e.g., a linker sequence).
• a "tag” comprises a peptide sequence operatively associated to the sequence of another peptide and/or polypeptide sequence.
• a tag include a His-tag, a strep-tag, a flag-tag, a T7-tag, a S-tag, a HSV-tag, a polyarginine-tag, a polycysteine-tag, a polyaspartic acid-tag, a polyphenylalanine-tag, or a combination thereof.
• a His-tag may comprise about 6 to about 10 amino acids in length, and can be incorporated at the N-terminus, C-terminus, and/or within an amino acid sequence for use in detection and purification.
• a His tag binds affinity columns comprising nickel, and may be eluted using low pH conditions or with imidazole as a competitor (Unger, T. F., 1997).
• a strep-tag may comprise about 10 amino acids in length, and may be incorporated at the C-terminus.
• a strep-tag binds streptavidin or affinity resins that comprise streptavidin.
• a flag-tag may comprise about 8 amino acids in length, and may be incorporated at the N-terminus and/or the C-terminus of an amino acid sequence for use in purification.
• a T7-tag may comprise about 1 1 to about 16 amino acids in length, and may be incorporated at the N-terminus and/or within an amino acid sequence for use in purification.
• a S-tag may comprise about 15 amino acids in length, and may be incorporated at the N-terminus, C-terminus and/or within an amino acid sequence for use in detection and purification.
• a HSV-tag may comprise about 11 amino acids in length, and may be incorporated at the C-terminus of an amino acid sequence for use in purification. The HSV tag binds an anti-HSV antibody in purification procedures (Unger, T. F., 1997).
• a polyarginine-tag may comprise about 5 to about 15 amino acids in length, and may be incorporated at the C-terminus of an amino acid sequence for use in purification.
• a polycysteine-tag may comprise about 4 amino acids in length, and may be incorporated
• a polyaspartic acid-tag may comprise about 5 to about 16 amino acids in length, and may be incorporated at the C-terminus of an amino acid sequence for use in purification.
• a polyphenylalanine-tag may comprise about 11 amino acids in length, and may be incorporated at the N-terminus of an amino acid sequence for use in purification.
• a (His)6 tag sequence has been used to purify fusion proteins comprising GFP- OPH or OPH using immobilized metal affinity chromatography ("IMAC") (Wu, C-F. et al., 2000b; Wu, C-F. et al., 2002).
• a (His)6 tag sequence followed by a thrombin cleavage site has been used to purify fusion proteins comprising squid-type DFPase using IMAC (Hartleib, J. and Ruterjans, H., 2001a).
• an OPH fusion protein comprising a C-terminal flag has been expressed (Wang, J. et al., 2001 ).
• a "fusion partner” comprises a polypeptide operatively associated to the sequence of another peptide and/or polypeptide of interest.
• Properties that a fusion partner may confer to a fusion protein include, but are not limited to, enhanced expression, enhanced solubility, ease of detection, and/or ease of purification of a fusion protein.
• Examples of a fusion partner include a thioredoxin, a cellulose-binding domain, a calmodulin binding domain, an avidin, a protein A, a protein G, a glutathione-S-transferase, a chitin-binding domain, an ELP, a maltose-binding domain, or a combination thereof.
• Thioredoxin may be incorporated at the N-terminus and/or the C-terminus of an amino acid sequence for use in purification.
• a cellulose-binding domain binds a variety of resins comprising cellulose or chitin (Unger, T. F., 1997).
• a calmodulin-binding domain binds affinity resins comprising calmodulin in the presence of calcium, and allows elution of the fusion protein in the presence of ethylene glycol tetra acetic acid ("EGTA”) (Unger, T. F., 1997).
• EGTA ethylene glycol tetra acetic acid
• Avidin may be useful in purification and/or detection.
• a protein A and/or a protein G binds a variety of anti-bodies for ease of purification.
• Protein A may be bound to an IgG sepharose resin (Unger, T. F., 1997). Streptavidin may be useful in purification and/or detection. Glutathione-S-transferase may be incorporated at the N-terminus of an amino acid sequence for use in detection and/or purification. Glutathione-S-transferase binds affinity resins comprising glutathione (Unger, T. F., 1997).
• An elastin-like polypeptide comprises repeating sequences (e.g., 78 repeats) which reversibly converts itself, and thus the fusion protein, from an aqueous soluble polypeptide to an insoluble polypeptide above an empirically determined transition temperature.
• the transition temperature may be affected by the number of repeats, and may be determined spectrographically using techniques known in the art, including measurements at 655 nano meters ("nm") over a 4 0 C to 8O 0 C range (Urry, D. W. 1992; Shimazu, M. et al., 2002).
• a chitin-binding domain comprises an intein cleavage site sequence, and may be incorporated at the C-terminus for purification.
• the chitin-binding domain binds affinity resins comprising chitin, and an intein cleavage site sequence allows the self-cleavage in the presence of thiols at reduced temperature to release the peptide and/or the polypeptide sequence of interest (Unger, T.
• a maltose-binding domain may be incorporated at the N-terminus and/or the C-terminus of an amino acid sequence for use in detection and/or purification.
• a maltose-binding domain sequence usually further comprises a ten amino acid poly asparagine sequence between the maltose binding domain and the sequence of interest to aid the maltose-binding domain in binding affinity resins comprising amylose (Unger, T. F., 1997).
• a fusion protein comprising an elastin-like polypeptide sequence and an OPH sequence has been expressed (Shimazu, M. et al., 2002).
• a cellulose-binding domain- OPH fusion protein has also been recombinantly expressed (Richins, R. D. et al., 2000).
• a maltose binding protein-E3 carboxylesterase fusion protein has been recombinantly expressed (Claudianos, C. et al., 1999)
• a protease cleavage site promotes proteolytic removal of the fusion partner from the peptide and/or the polypeptide of interest.
• a fusion protein may be bound to an affinity resin, and cleavage at the cleavage site promotes the ease of purification of a peptide and/or a polypeptide of interest with much (e.g., most) to about all of the tag and/or the fusion partner sequence removed (Unger, T. F., 1997).
• protease cleavage sites used in the art include the factor Xa cleavage site, which comprises about four amino acids in length; the enterokinase cleavage site, which comprises about five amino acids in length; the thrombin cleavage site, which comprises about six amino acids in length; the rTEV protease cleavage site, which comprises about seven amino acids in length; the 3C human rhino virus protease, which comprises about eight amino acids in length; and the PreScissionTM cleavage site, which comprises about eight amino acids in length.
• an enterokinase recognition site was used to separate an OPH sequence from a fusion partner (Wu, C-F. et al., 2000b; Wu, C-F. et al., 2001 b).
• the "terminator region” or “terminator” may also comprise a specific DNA sequence that permits site-specific cleavage of the new transcript so as to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of adenosine nucleotides ("polyA") of about 200 in number to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to more stable and are translated more efficiently.
• polyA adenosine nucleotides
• a terminator comprises a signal for the cleavage of the RNA, and in some aspects the terminator signal promote polyadenylation of the message.
• the terminator and/or polyadenylation site elements may serve to enhance message levels and/or to reduce read through from the cassette into other sequences.
• a terminator contemplated includes any known terminator of transcription, including but not limited to those described herein.
• a termination sequence of a gene such as for example, a bovine growth hormone terminator and/or a viral termination sequence, such as for example a SV40 terminator.
• the termination signal may lack of transcribable and/or translatable sequence, such as due to a sequence truncation.
• a trpC terminator from Aspergillus nidulans has been used in the expression of recombinant OPH (Dave, K. I. et al., 1994b).
• a polyadenylation signal may be included to effect proper polyadenylation of the transcript. Any such sequence may be employed. Some embodiments include the SV40 polyadenylation signal and/or the bovine growth hormone polyadenylation signal, convenient and/or known to function well in various target cells. Polyadenylation may increase the stability of the transcript and/or may facilitate cytoplasmic transport.
• a vector in a host cell may comprise one or more origins of replication sites (“ori"), which comprises a nucleic acid sequence at which replication initiates.
• ori origins of replication sites
• ARS autonomously replicating sequence
• prokaryotic and/or eukaryotic expression vectors are known in the art.
• types of expression vectors include a bacterial artificial chromosome ("BAC"), a cosmid, a plasmid [e.g., a pMB1/colE1 derived plasmid such as pBR322, pUC18; a Ti plasmid of Agrobacterium tumefaciens derived vector (Rogers, S. G.
• shuttle vectors may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells [e.g., a wheat dwarf virus ("WDV”) pW1-11 and/or pW1-GUS shuttle vector (Ugaki, M. et al., 1991 )].
• WDV wheat dwarf virus
• An expression vector operatively linked to a nucleic acid sequence encoding an enzymatic sequence may be constructed using techniques in the art in light of the present disclosures [In "Molecular Cloning” (Sambrook, J., and Russell, D.W., Eds.) 3rd Edition, Cold Spring Harbor, New York: Cold Spring
• Prokaryote- and/or eukaryote-based systems may be employed to produce nucleic acid sequences, and/or their cognate polypeptides, proteins and peptides. Many such systems are widely available, including those provide by commercial vendors.
• an insect cell/baculovirus system may produce a high level of protein expression of a heterologous nucleic acid sequence, such as described in U.S. Patent No. 5,871 ,986, 4,879,236, both incorporated herein by reference, and which may be bought, for example, under the name MAXBAC ® 2.0 from INVITROGEN ® and BACPACKTM BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH ® .
• an expression system include STRATAGENE ® 'S COMPLETE CONTROLTM Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an Escherichia coli expression system.
• Another example comprises an inducible expression system available from INVITROGEN ® , which carries the T-REXTM (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter.
• INVITROGEN ® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica.
• E3 carboxylesterase enzymatic sequences and phosphoric triester hydrolase functional equivalents have been recombinantly expressed in a BACPACKTM Baculovirus Expression System From CLONTECH ® (Newcomb, R. D. et al., 1997; Campbell, P. M. et a., 1998).
• a biomolecule may be expressed in a plant cell (e.g., a corn cell), using techniques such as those described in U.S. Patent Nos. 6,504,085, 6,136,320, 6,087,558, 6034,298, 5,914,123, and 5,804,694.
• the bacterium host cell comprises a Gram-negative bacterium cell.
• Various prokaryotic host cells have been
• a prokaryotic host cell known in the art may be used to express a peptide and/or a polypeptide (e.g., a polypeptide comprising an enzyme sequence).
• An expression vector for use in prokaryotic cells generally comprises nucleic acid sequences such as, a promoter, a ribosome binding site (e.g., a Shine-Delgarno sequence), a start codon, a multiple cloning site, a fusion partner, a protease cleavage site, a stop codon, a transcription terminator, an origin of replication, a repressor, and/or any other additional nucleic acid sequence that may be used in such an expression vector in the art [see, for example, Makrides, S. C, 1996; Hannig, G. and Makrides, S. C, 1998; Stevens, R.
• nucleic acid sequences such as, a promoter, a ribosome binding site (e.g., a Shine-Delgarno sequence), a start codon, a multiple cloning site, a fusion partner, a protease cleavage site, a stop codon, a transcription
• a promoter may be positioned about 10 to about 100 nucleotides 5' to a nucleic acid sequence comprising a ribosome binding site.
• Examples of promoters that have been used in a prokaryotic cell includes a T5 promoter, a lac promoter, a tac promoter, a trc promoter, an araBAD promoter, a P L promoter, a T7 promoter, a T7-lac operator promoter, and variations thereof.
• the lactose operator regulates the T5 promoter.
• a lac promoter e.g., a lac promoter, a /acUV5 promoter
• a tac promoter e.g., a tacl promoter, a tacll promoter
• a T7-lac operator promoter or a trc promoter are each suppressed by a lacl repressor, a more effective lacP repressor and/or an even stronger lacP ⁇ repressor (Glascock, C. B. and Weickert, M. J., 1998).
• Isopropyl- ⁇ -D-thiogalactoside IPTG
• IPTG Isopropyl- ⁇ -D-thiogalactoside
• An araBAD promoter may be suppressed by an araC repressor, and may be induced by 1-arabinose.
• a P L promoter or a T7 promoter are each suppressed by a ⁇ clts857 repressor, and induced by a temperature of 42 0 C.
• Nalidixic acid may be used to induce a P L promoter.
• recombinant amino acid substitution mutants of OPH have been expressed in Escherichia coli using a lac promoter induced by IPTG (Watkins, L. M. et al., 1997b).
• recombinant wild type and a signal sequence truncation mutant of OPH was expressed in Pseudomonas putida under control of a lactac and tac promoters (Walker, A. W. and Keasling, J. D., 2002).
• an OPH-Lpp-OmpA fusion protein has been expressed in Escherichia coli strains JM105 and XL1- Blue using a constitutive Ipp-lac promoter and/or a tac promoter induced by IPTG and controlled by a lacP repressor (Richins, R. D. et al., 1997; Kaneva, I. et al., 1998; Mulchandani, A.
• a cellulose-binding domain-OPH fusion protein has also been recombinantly expressed under the control of a T7 promoter (Richins, R. D. et al., 2000).
• recombinant Altermonas sp. JD6.5 OPAA has been expressed under the control of a trc promoter in Escherichia coli (Cheng, T.-C. et al., 1999).
• a (His)6 tag sequence-thrombin cleavage site-squid-type DFPase has been expressed using a Ptac promoter in Escherichia coli (Hartleib, J. and Ruterjans, H., 2001a).
• a ribosome binding site functions in transcription initiation, and may be positioned about 4 to about 14 nucleotides 5' from the start codon.
• a start codon signals initiation of transcription.
• a multiple cloning site comprises restriction sites for incorporation of a nucleic acid sequence encoding a peptide and/or a polypeptide of interest.
• a stop codon signals translation termination.
• the vectors and/or the constructs may comprise at least one termination signal.
• a “termination signal” or “terminator” comprises DNA sequences involved in specific termination of a RNA transcript by a RNA polymerase. Thus, in certain embodiments a termination signal ends the production of a RNA transcript.
• a terminator may be used in vivo to achieve a desired message level.
• a transcription terminator signals the end of transcription and often enhances mRNA stability. Examples of a transcription terminator include a rrnB T1 and/or a rrnB T2 transcription terminator (Unger, T. F., 1997).
• An origin of replication regulates the number of expression vector copies maintained in a transformed host cell.
• a selectable marker usually provides a transformed cell resistance to an antibiotic.
• a selectable marker used in a prokaryotic expression vector include a ⁇ -lactamase, which provides resistance to antibiotic such as an ampicillin and/or a carbenicillin; a tet gene product, which provides resistance to a tetracycline, and/or a Km gene product, which provides resistance to a kanamycin.
• a repressor regulatory gene suppresses transcription from the promoter. Examples of repressor regulatory genes include the lacl, the iacf, and/or the lacP ⁇ repressors (Glascock, C. B. and Weickert, M. J., 1998).
• the host cell's genome, and/or additional nucleic acid vector co-transfected into the host cell may comprise one or more of these nucleic acid sequences, such as, for example, a repressor.
• An expression vector for a prokaryotic host cell may comprise a nucleic acid sequence that encodes a periplasmic space signal peptide.
• this nucleic acid sequence may be operatively linked to a nucleic acid sequence comprising an enzymatic peptide and/or polypeptide, wherein the periplasmic space signal peptide directs the expressed fusion protein to be translocated into a prokaryotic host cell's periplasmic space. Fusion proteins secreted in the periplasmic space may be obtained through simplified purification protocols compared to non-secreted fusion proteins.
• a periplasmic space signal peptide may be operatively linked at or near the N-terminus of an expressed fusion protein.
• periplasmic space signal peptide examples include the Escherichia coli ompA, ompT, and malel leader peptide sequences and the T7 caspid protein leader peptide sequence (Unger, T. F., 1997).
• Mutated and/or recombinantly altered bacterium that release a peptide and/or a polypeptide (e.g., an enzyme sequence) into the environment may be used for purification and/or contact of a proteinaceous molecule with a target chemical ligand.
• a strain of bacteria such as, for example, a bacteriocin-release protein mutant strain of Escherichia coli, may be used to promote release of expressed proteins targeted to the periplasm into the extracellular environment (Van der WaI, F. J. et al., 1998).
• a bacterium may be transfected with an expression vector that produces a gene and/or a gene fragment product that promotes the release of a protenaceous molecule of interest from the periplasm into the extracellular environment.
• an expression vector that produces a gene and/or a gene fragment product that promotes the release of a protenaceous molecule of interest from the periplasm into the extracellular environment.
• a plasmid encoding the third topological domain of ToIA has been described as promoting the release of endogenous and recombinantly expressed proteins from the periplasm (Wan, E. W. and Baneyx, F., 1998).
• 5842-02103 157 include their progeny, which includes any and all subsequent generations. All progeny may not be identical due to deliberate and/or inadvertent mutations.
• "host cell” refers to a prokaryotic and/or an eukaryotic cell, and it includes any transformable organism capable of replicating a vector and/or expressing a heterologous gene and/or gene fragment encoded by a vector.
• a host cell can, and has been, used as a recipient for vectors.
• a host cell may be
• transfected or transformed, which refers to a process by which exogenous nucleic acid sequence may be transferred or introduced into the host cell.
• a transformed cell includes the primary subject cell and its progeny. Techniques for transforming a cell include, for example calcium phosphate precipitation, cell sonication, diethylaminoethanol (“DEAE”)-dextran, direct microinjection, DNA-loaded liposomes, electroporation, gene bombardment using high velocity microprojectiles, receptor-mediated transfection, viral-mediated transfection, or a combination thereof [In “Molecular Cloning” (Sambrook, J., and Russell, D.
• a suitable expression vector may be transformed into a cell, the cell may be grown in an appropriate environment, and in some cases, used to produce a tissue and/or whole multicellular organism.
• the terms "engineered” and “recombinant” cells and/or host cells are intended to refer to a cell comprising an introduced exogenous nucleic acid sequence. Therefore, engineered cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced exogenous nucleic acid sequence.
• Engineered cells are thus cells having a nucleic acid sequence introduced through the hand of man.
• Recombinant cells include those having an introduced cDNA and/or genomic gene and/or a gene fragment positioned adjacent to a promoter not naturally associated with the particular introduced nucleic acid sequence, a gene, and/or a gene fragment.
• An enzyme or a proteinaceous molecule produced from the introduced gene and/or gene fragment may be referred to, for example, as a recombinant enzyme or recombinant proteinaceous molecule, respectively. All tissues, offspring, progeny and/or descendants of such a cell, tissue, and/or organism comprising the transformed nucleic acid sequence thereof may be used.
• an expressed proteinaceous molecule may be purified from cellular material, some embodiments disclosed herein use the properties of a proteinaceous molecule composition comprising, a proteinaceous molecule expressed and retained within a cell, whether naturally and/or through recombinant expression.
• a proteinaceous molecule may be produced using recombinant nucleic acid expression systems in the cell.
• Cells are known herein based on the type of proteinaceous molecule expressed within the cell, whether endogenous and/or recombinant, so that, for example, a cell expressing an enzyme of interest may be known as an "enzyme cell," a cell expressing a lipase may be known herein as a "lipase cell,” etc.
• Additional examples of such nomenclature include a carboxylesterase cell, an OPAA cell, a human phospholipase A 1 cell, a carboxylase cell, a cutinase cell, an aminopeptideases cell, efc., respectively denoting cells that comprise, a carboxylesterase, an OPAA, a human phospholipase A 1 , a carboxylase, a cutinase, an aminopeptideases, efc.
• a cell comprises a bacterial cell, a fungal cell (e.g., a yeast cell), an animal cell (e.g., an insect cell), a plant cell, an algae cell, a mildew cell, or a combination thereof.
• the cell comprises a cell wall.
• Contemplated proteinaceous molecule comprising cell walls include, but are
• a microorganism comprises the proteinaceous molecule.
• contemplated microorganisms include a bacterium, a fungus, or a combination thereof.
• a bacterial host cell that have been used with expression vectors include an Aspergillus niger, a Bacillus (e.g., B. amyloliquefaciens, B. brevis, B. licheniformis, B.
• subtilis an Escherichia coli, a Kluyveromyces lactis, a Moraxella sp., a Pseudomonas (e.g., fluorescens, putida), Flavobacterium cell, a Plesiomonas cell, an Alteromonas cell, or a combination thereof.
• yeast cell include a Streptomyces lividans cell, a Gliocladium virens cell, a Saccharomyces cell, or a combination thereof.
• Host cells may be derived from prokaryotes and/or eukaryotes, which may be used for the desired result comprises replication of the vector and/or expression of part or all of the vector-encoded nucleic acid sequences.
• Numerous cell lines and cultures are available for use as a host cell, and they may be obtained through the American Type Culture Collection, an organization which serves as an archive for living cultures and genetic materials.
• An appropriate host may be determined based on the vector backbone and the desired result.
• a plasmid and/or cosmid for example, may be introduced into a prokaryote host cell for replication of many vectors.
• Examples of a bacterial cell used as a host cell for vector replication and/or expression include DH5a, JM109, and KC8, as well as a number of commercially available bacterial hosts such as NovablueTM Escherichia coli cells (NOVAGENE ® ), SURE ® Competent Cells and SOLOPACKTM Gold Cells (STRATAGENE ® ).
• NovablueTM Escherichia coli cells NOVAGENE ®
• SURE ® Competent Cells SOLOPACKTM Gold Cells
• Escherichia coli cells have been the common cell types used to express both wild type and mutant forms of OPH (Dumas, D. P. et al., 1989a; Dave, K. I. et al., 1993; Lai, K. et al., 1994; Wu, C-F. et al., 2001a).
• the OPH I106A/F132A/H257Y and G60A mutants have been expressed in Escherichia coli BL-21 host cells (Kuo, J. M. and Raushel, F. M., 1994; Li, W.-S. et al., 2001 ).
• maltose-binding domain-E3 carboxylesterase and phosphoric triester hydrolase functional equivalents have been expressed in Escherichia coli TB1 cells (Claudianos, C. et al., 1999).
• the OPH mutants designated W131 F, F132Y, L136Y, L140Y, H257L, L271Y, F306A, and F306Y each have been expressed in NovablueTM Escherichia coli cells (Gopal, S. et al., 2000).
• OPAA from Alteromonas sp JD6.5 has been recombinantly expressed in Escherichia coli cells (Hill, C. M., 2000).
• recombinant Altermonas sp. JD6.5 OPAA has been expressed in Escherichia coli (Cheng, T.-C. et al., 1999).
• the mpd gene has been recombinantly expressed in Escherichia coli, and the encoded enzyme demonstrated methyl parathion degradation activity (Zhongli, C. et al., 2001 ).
• a recombinant squid-type DFPase fusion protein has been expressed Escherichia coli BL-21 cells (Hartleib, J. and Ruterjans, H., 2001a).
• bacterial cells such as Escherichia coli LE392 may be used as host cells for phage viruses.
• a bacterium species may be selected to express a proteinaceous molecule due to a particular property.
• Examples of eukaryotic host cells for replication and/or expression of a vector include yeast cells HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, and PC12.
• OPH has been expressed in the host yeast cells of Streptomyces lividans (Steiert, J. G. et al., 1989).
• OPH has been
• OPH has been expressed in the cells of Drosophila melanogaster (Phillips, J. P. et al., 1990).
• OPH has been expressed in the fungus Gliocladium virens (Dave, K. I. et al., 1994b).
• the gene for human paraoxonase, PON1 has been recombinantly expressed in human embryonic kidney cells (Josse, D.
• an eukaryotic cell that may be selected for expression comprises a plant cell, such as, for example, a corn cell.
• Any size flask and/or fermentor may be used to grow a cell, a tissue and/or an organism that may express a recombinant proteinaceous molecule.
• bulk production of a composition, an article, etc. comprising an enzymatic sequence is contemplated.
• a fusion protein comprising, N-terminus to C-terminus, a (His)6 polyhistidine tag, a green fluorescent protein ("GFP"), an enterokinase recognition site, and an OPH lacking the 29 amino acid leader sequence, has been expressed in Escherichia coli.
• the GFP sequence produced fluorescence that was proportional both the quantity of the fusion protein, and the activity of the OPH sequence.
• the fusion protein was more soluble than an OPH expressed without the added sequences, and was expressed within the cells (Wu, C-F. et al., 2000b; Wu, C-F. et al., 2001a).
• the temperature selected may influence the rate and/or quality of recombinant proteinaceous molecule production.
• expression of a proteinaceous molecule may be conducted at about 4 0 C to about 5O 0 C
• Such combinations may include a shift from one temperature (e.g., about 37 0 C) to another temperature (e.g., about 3O 0 C) during the induction of the expression of proteinaceous molecule.
• both eukaryotic and prokaryotic expression of an OPH may be conducted at temperatures about 3O 0 C, which has increased the production of an enzymatically active OPH by reducing protein misfolding and/or inclusion body formation in some instances (Chen-Goodspeed, M.

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