The present invention provides methods and compositions comprising at least one perhydrolase enzyme for cleaning and other applications. In some embodiments, the present invention provides methods and compositions for generation of long chain peracids. Certain embodiments of the present invention find particular use in applications involving cleaning, bleaching and disinfecting.
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1. An isolated perhydrolase enzyme, comprising:
an amino acid substitution at position 204; or
amino acid substitutions at positions 12 and 22, 12 and 154, 12 and 194, 154 and 194, or 154 and 196;
wherein said enzyme perhydrolyzes long chain acyl ester substrates of at least six carbon atoms,; the amino acid positions are positionally equivalent to those in M. smegmatis perhydrolase having the amino acid sequence of SEQ ID NO: 2; and wherein the amino acid sequence of said enzyme is at least 90% identical to the amino acid sequence of the wild type perhydrolase of M. smegmatis, as set forth in SEQ ID NO: 2.
2. The isolated perhydrolase enzyme of
3. The isolated perhydrolase enzyme of
0. 4. The isolated perhydrolase enzyme of
0. 5. The isolated perhydrolase enzyme of
0. 6. The isolated perhydrolase enzyme of
7. The isolated perhydrolase enzyme of
8. The isolated perhydrolase enzyme of
0. 9. An isolated perhydrolase enzyme, wherein said enzyme hydrolyzes long chain acyl ester substrates of at least six carbon atoms, and wherein the amino acid sequence of said enzyme is at least 90% identical to the amino acid sequence of the wild type perhydrolase of M. smegmatis, as set forth in SEQ ID NO: 2.
0. 10. The isolated perhydolase enzyme of
0. 11. The isolated perhydrolase enzyme of
13. The cleaning composition of
14. The cleaning composition of
15. The cleaning composition of
16. A cleaning method comprising contacting a substrate in the presence of the cleaning composition of
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The present application is a Continuation-in-Part of pending U.S. patent application Ser. No. 10/581,014, filed on Sep. 11, 2007, which application is a U.S. National phase filing of International Patent Application Serial No. PCT/US04/040438 under 35 U.S.C. §371, which application claims the benefit of U.S. Provisional Patent Application Ser. No. 60/526,764, filed Dec. 3, 2003, now abandoned.
wherein R1 is a moiety selected from the group consisting of H or a substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in some embodiments of the present invention, R1 comprises from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon atoms, or from 2 to 100 carbon atoms;
each R2 is an optionally substituted alkoxylate moiety, in some embodiments of the present invention, each R2 is independently an ethoxylate, propoxylate or butoxylate moiety;
R3 is an ester-forming moiety having the formula:
In some embodiments of the present invention, the molecule comprising an ester moiety is an alkyl ethoxylate or propoxylate having the formula R1Ox[(R2)m(R3)n]p wherein:
In some embodiments of the present invention, the molecule comprising the ester moiety has the formula:
R1Ox[(R2)m(R3)n]p
wherein R1 is H or a moiety that comprises a primary, secondary, tertiary or quaternary amine moiety, said R1 moiety that comprises an amine moiety being selected from substituted or unsubstituted alkyl, heteroalkyl, alkenyl, alkynyl, aryl, alkylaryl, alkylheteroaryl, and heteroaryl; in some embodiments, R1 comprises from 1 to 50,000 carbon atoms, from 1 to 10,000 carbon atoms, or from 2 to 100 carbon atoms;
each R2 is an alkoxylate moiety, in some embodiments of the present invention each R2 is independently an ethoxylate, propoxylate or butoxylate moiety;
In any of the aforementioned embodiments of the present invention, the molecule comprising an ester moiety may have a weight average molecular weight of less than 600,000 Daltons, less than 300,000 Daltons, less than 100,000 Daltons or even less than 60,000 Daltons.
Suitable molecules that comprise an ester moiety include, but are not limited to polycarbohydrates that comprise an ester moiety.
Adjunct Materials
While not essential for use of the present invention, the non-limiting list of adjuncts illustrated hereinafter are suitable for use in the cleaning compositions of the present invention. In some embodiments, these materials are incorporated to assist or enhance cleaning performance, for treatment of the substrate to be cleaned, or to modify the aesthetics of the cleaning composition as is the case with perfumes, colorants, dyes or the like. It is understood that such adjuncts are in addition to the enzymes of the present invention, hydrogen peroxide source and ester substrate. The precise nature of these additional components, and levels of incorporation thereof, will depend on the physical form of the composition and the nature of the cleaning operation for which it is to be used. Suitable adjunct materials include, but are not limited to, surfactants, builders, chelating agents, dye transfer inhibiting agents, deposition aids, dispersants, additional enzymes, and enzyme stabilizers, catalytic materials, bleach activators, bleach boosters, preformed peracids, polymeric dispersing agents, clay soil removal/anti-redeposition agents, brighteners, suds suppressors, dyes, perfumes, structure elasticizing agents, fabric softeners, carriers, hydrotropes, processing aids and/or pigments. In addition to the disclosure below, suitable examples of such other adjuncts and levels of use are found in U.S. Pat. Nos. 5,576,282, 6,306,812, and 6,326,348, herein incorporated by reference. In some embodiments, the aforementioned adjunct ingredients constitute the balance of the cleaning compositions of the present invention.
Surfactants—In some embodiments, the cleaning compositions provided by the present invention comprise at least one surfactant or surfactant system wherein the surfactant is selected from nonionic surfactants, anionic surfactants, cationic surfactants, ampholytic surfactants, zwitterionic surfactants, semi-polar nonionic surfactants and mixtures thereof.
In some preferred embodiments, the surfactant is typically present at a level of from about 0.1% to about 60%, from about 1% to about 50% or even from about 5% to about 40% by weight of the subject cleaning composition.
A number of known compounds are suitable surfactants useful in compositions comprising the perhydrolase enzymes of the present invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents (See e.g., U.S. Pat. Nos. 4,404,128 and 4,261,868). A suitable detergent formulation is that described in U.S. Pat. No. 5,204,015 (incorporated by reference). Those in the art are familiar with the different formulations which find use as cleaning compositions.
As indicated above, in some preferred embodiments, the detergent compositions of the present invention employ a surface active agent (i.e., surfactant) including anionic, nonionic and ampholytic surfactants well known for their use in detergent compositions. Some surfactants suitable for use in the present invention are described in British Patent Application No. 2 094 826 A, incorporated herein by reference. In some embodiments, mixtures of surfactants are used in the present invention.
Suitable anionic surfactants for use in the detergent composition of the present invention include, but are not limited to linear or branched alkylbenzene sulfonates; alkyl or alkenyl ether sulfates having linear or branched alkyl groups or alkenyl groups; alkyl or alkenyl sulfates; olefin sulfonates; alkane sulfonates and the like. Suitable counter ions for anionic surfactants include, but are not limited to alkali metal ions such as sodium and potassium; alkaline earth metal ions such as calcium and magnesium; ammonium ion; and alkanolamines having 1 to 3 alkanol groups of carbon number 2 or 3.
Ampholytic surfactants that find use in the present invention include, but are not limited to quaternary ammonium salt sulfonates, betaine-type ampholytic surfactants, and the like. Such ampholytic surfactants have both the positive and negative charged groups in the same molecule.
Nonionic surfactants that find use in the present invention generally comprise polyoxyalkylene ethers, as well as higher fatty acid alkanolamides or alkylene oxide adduct thereof, fatty acid glycerine monoesters, and the like.
In some preferred embodiments, the surfactant or surfactant mixture included in the detergent compositions of the present invention is provided in an amount from about 1 weight percent to about 95 weight percent of the total detergent composition and preferably from about 5 weight percent to about 45 weight percent of the total detergent composition. As indicated herein, in various embodiments of the present invention, numerous other components are included in the compositions of the present invention. However, it is not intended that the present invention be limited to these specific examples. Indeed, it is contemplated that additional compounds will find use in the present invention. The descriptions below merely illustrate some optional components.
Proteins, particularly the perhydrolase of the present invention can be formulated into known powdered and liquid detergents having pH between 3 and 12.0, at levels of about 0.001 to about 5% (preferably 0.1% to 0.5%) by weight. In some embodiments, these detergent cleaning compositions further include other enzymes such as proteases, amylases, mannanases, peroxidases, oxido reductases, cellulases, lipases, cutinases, pectinases, pectin lyases, xylanases, and/or endoglycosidases, as well as builders and stabilizers.
The addition of proteins to conventional cleaning compositions does not create any special use limitations. In other words, any temperature and pH suitable for the detergent are also suitable for the present compositions, as long as the pH is within the range in which the enzyme(s) is/are active, and the temperature is below the described protein's denaturing temperature. In addition, proteins of the invention find use in cleaning, bleaching, and disinfecting compositions without detergents, again either alone or in combination with a source of hydrogen peroxide, an ester substrate (e.g., either added to or inherent in the system utilized, such as with stains that contain esters, pulp that contains esters etc), other enzymes, surfactants, builders, stabilizers, etc. Indeed it is not intended that the present invention be limited to any particular formulation or application.
Builders—The cleaning compositions of the present invention may comprise one or more detergent builders or builder systems. When a builder is used, the cleaning composition typically comprises at least about 1%, from about 3% to about 60%, or from about 5% to about 40% builder by weight of the cleaning composition.
Builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates, alkali metal silicates, alkaline earth and alkali metal carbonates, aluminosilicate builders polycarboxylate compounds, ether hydroxypolycarboxylates, copolymers of maleic anhydride with ethylene or vinyl methyl ether, 1,3,5-trihydroxy benzene-2,4,6-trisulphonic acid, and carboxymethyloxysuccinic acid, the various alkali metal, ammonium and substituted ammonium salts of polyacetic acids such as ethylenediamine tetraacetic acid and nitrilotriacetic acid, as well as polycarboxylates such as mellitic acid, succinic acid, citric acid, oxydisuccinic acid, polymaleic acid, benzene 1,3,5-tricarboxylic acid, carboxymethyloxysuccinic acid, and soluble salts thereof.
Chelating Agents—In some embodiments, the cleaning compositions provided herein contain at least one chelating agent. Suitable chelating agents include but are not limited to copper, iron and/or manganese chelating agents and mixtures thereof.
When a chelating agent is used, the cleaning composition typically comprises from about 0.1% to about 15%, or from about 3.0% to about 10% chelating agent by weight of the subject cleaning composition.
Deposition Aid—In some embodiments, the cleaning compositions provided herein further comprise at lease one deposition aid. Suitable deposition aids include, but are not limited to polyethylene glycol, polypropylene glycol, polycarboxylate, soil release polymers such as polyterephthalic acid, clays such as kaolinite, montmorillonite, atapulgite, illite, bentonite, halloysite, and mixtures thereof.
Dye Transfer Inhibiting Agents—In yet some further embodiments, the cleaning compositions of the present invention also comprise one or more dye transfer inhibiting agents. Suitable polymeric dye transfer inhibiting agents include, but are not limited to, polyvinylpyrrolidone polymers, polyamine N-oxide polymers, copolymers of N-vinylpyrrolidone and N-vinylimidazole, polyvinyloxazolidones and polyvinylimidazoles or mixtures thereof.
When present in a subject cleaning composition, the dye transfer inhibiting agents are typically present at levels from about 0.0001% to about 10%, from about 0.01% to about 5%, or from about 0.1% to about 3% by weight of the cleaning composition.
Dispersants—In some additional embodiments, the cleaning compositions of the present invention comprise dispersants. Suitable water-soluble organic materials include, but are not limited to the homo- or co-polymeric acids or their salts, in which the polycarboxylic acid comprises at least two carboxyl radicals separated from each other by not more than two carbon atoms.
Enzymes—In some further embodiments, the cleaning compositions of the present invention comprise one or more detergent enzymes which provide cleaning performance and/or fabric care benefits. Examples of suitable enzymes include, but are not limited to, hemicellulases, peroxidases, proteases, cellulases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, β-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase, and amylases, or mixtures thereof. A typical combination is cocktail of conventional applicable enzymes like protease, lipase, cutinase and/or cellulase in conjunction with amylase.
Enzyme Stabilizers—Enzymes for use in detergents can be stabilized by various techniques. The enzymes employed herein can be stabilized by the presence of water-soluble sources of calcium and/or magnesium ions in the finished compositions that provide such ions to the enzymes. It is contemplated that enzyme stabilizers will find use in some embodiments of the cleaning compositions provided herein.
Catalytic Metal Complexes—In some embodiments, the cleaning compositions of the present invention comprise catalytic metal complexes. One type of metal-containing bleach catalyst is a catalyst system comprising a transition metal cation of defined bleach catalytic activity, such as copper, iron, titanium, ruthenium, tungsten, molybdenum, or manganese cations, an auxiliary metal cation having little or no bleach catalytic activity, such as zinc or aluminum cations, and a sequestrate having defined stability constants for the catalytic and auxiliary metal cations, particularly ethylenediaminetetraacetic acid, ethylenediaminetetra (methylenephosphonic acid) and water-soluble salts thereof. Examples of these catalysts are described in U.S. Pat. No. 4,430,243, herein incorporated by reference.
In some embodiments, the compositions provided herein are catalyzed by means of a manganese compound. Such compounds and levels of use are well known in the art and include, for example, the manganese-based catalysts disclosed in U.S. Pat. No. 5,576,282, which is herein incorporated by reference.
Cobalt bleach catalysts useful herein are known, and are described, for example, in U.S. Pat. No. 5,597,936; and U.S. Pat. No. 5,595,967, both of which are incorporated herein by reference. Such cobalt catalysts are readily prepared by known procedures, such as taught for example in U.S. Pat. No. 5,597,936, and U.S. Pat. No. 5,595,967.
In some embodiments, the compositions provided herein also comprise at least one transition metal complex of a macropolycyclic rigid ligand (“MRL”). As a practical matter, and not by way of limitation, in some embodiments of the compositions and cleaning processes provided herein are adjusted to provide on the order of at least one part per hundred million of the active MRL species in the aqueous washing medium, and will preferably provide from about 0.005 ppm to about 25 ppm, more preferably from about 0.05 ppm to about 10 ppm, and most preferably from about 0.1 ppm to about 5 ppm, of the MRL in the wash liquor.
Preferred transition-metals in the instant transition-metal bleach catalyst include manganese, iron and chromium. Preferred MRLs herein are a special type of ultra-rigid ligand that is cross-bridged such as 5,12-diethyl-1,5,8,12-tetraazabicyclo[6.6.2]hexadecane.
Suitable transition metal MRLs are readily prepared by known procedures, such as taught for example in WO 00/332601, and U.S. Pat. No. 6,225,464, both of which are incorporated by reference herein.
Cleaning and Detergent Formulations
The detergent compositions of the present invention are provided in any suitable form, including but not limited to liquids, granules, emulsions, gels, and pastes. When a solid detergent composition is employed, the detergent is preferably formulated in the form of granules. Preferably, the granules are formulated to additionally contain a protecting agent (See e.g., U.S. application Ser. No. 07/642,669 filed Jan. 17, 1991, incorporated herein by reference). Likewise, in some embodiments, the granules are formulated so as to contain materials to reduce the rate of dissolution of the granule into the wash medium (See e.g., U.S. Pat. No. 5,254,283, incorporated herein by reference). In addition, the perhydrolase enzymes of the present invention find use in formulations in which substrate and enzyme are present in the same granule. Thus, in some embodiments, the efficacy of the enzyme is increased by the provision of high local concentrations of enzyme and substrate (See e.g., U.S. Patent Appln. Publ. No. US 2003/0191033, herein incorporated by reference).
The cleaning compositions of the present invention are formulated into any suitable form and prepared by any process chosen by the formulator, non-limiting examples of which are described in U.S. Pat. No. 5,879,584, U.S. Pat. No. 5,691,297, U.S. Pat. No. 5,574,005, U.S. Pat. No. 5,569,645, U.S. Pat. No. 5,565,422, U.S. Pat. No. 5,516,448, U.S. Pat. No. 5,489,392, and U.S. Pat. No. 5,486,303; all of which are incorporated herein by reference.
The cleaning compositions provided herein are typically be formulated such that, during use in aqueous cleaning operations, the wash water will have a pH of from about 5.0 to about 11.5, or from about 7.5 to about 10.5. Liquid product formulations are typically formulated to have a pH from 15 about 3.0 and about 9.0. Granular laundry products are typically formulated to have a pH from about 9 to about 11. Techniques for controlling pH at recommended usage levels include the use of buffers, alkalis, acids, etc., and are well known to those skilled in the art.
When the enzyme(s) of the present invention is/are employed in a granular composition or liquid, it is sometimes desirable for the enzyme(s) to be in the form of an encapsulated particle to protect such enzyme from other components of the granular composition during storage. In addition, encapsulation is also a means of controlling the availability of the enzyme(s) during the cleaning process and may enhance performance of the enzyme(s). In this regard, the enzyme(s) are encapsulated with any suitable encapsulating material known in the art.
The encapsulating material typically encapsulates at least part of the enzyme(s). Typically, the encapsulating material is water-soluble and/or water-dispersible. The encapsulating material may have a glass transition temperature (Tg) of 0° C. or higher (See e.g., WO 97/11151, incorporated herein by reference).
In some embodiments, the encapsulating is selected from carbohydrates, natural or synthetic gums, chitin and chitosan, cellulose and cellulose derivatives, silicates, phosphates, borates, polyvinyl alcohol, polyethylene glycol, paraffin waxes and combinations thereof. When the encapsulating material is a carbohydrate, it is typically selected from monosaccharides, oligosaccharides, polysaccharides, and combinations thereof. Typically, the encapsulating material is a starch. Suitable starches are described in EP 0 922 499, U.S. Pat. No. 4,977,252, U.S. Pat. No. 5,354,559, and U.S. Pat. No. 5,935,826, each of which is herein incorporated by reference.
In some embodiments, the encapsulating material is a microsphere made from plastic (e.g., thermoplastics, acrylonitrile, methacrylonitrile, polyacrylonitrile, polymethacrylonitrile and mixtures thereof). Commercially available microspheres that find use include but are not limited to those supplied by Expancel (Stockviksverken, Sweden) under the trademark EXPANCEL®, and those supplied by PQ Corp. (Valley Forge, Pa.) under the tradenames PM 6545, PM 6550, PM 7220, PM 7228, EXTENDOSPHERES®, LUXSIL®, Q-CEL® and SPHERICEL®.
In addition to the ingredients described above, perfumes, buffers, preservatives, dyes and the like also find use with the present invention. These components are provided in concentrations and forms known to those in the art.
In some embodiments, the powdered detergent bases of the present invention are prepared by any known preparation methods including spray-drying methods and granulation methods. The detergent base obtained particularly by the spray-drying method and/or spray-drying granulation method are preferred. The detergent base obtained by the spray-drying method is not restricted with respect to preparation conditions. The detergent base obtained by the spray-drying method is the form of hollow granules which are obtained by spraying an aqueous slurry of heat-resistant ingredients, such as surface active agents and builders, into a hot space. After the spray-drying, perfumes, enzymes, bleaching agents, inorganic alkaline builders are added, as desired. With a highly dense, granular detergent base obtained such as by the spray-drying-granulation method, various ingredients may also be added after the preparation of the base.
In some embodiments comprising liquid detergent bases the base is a homogenous solution, while in other embodiments, it is a non-homogenous dispersion.
In some embodiments, the detergent compositions of the present invention are incubated with fabric (e.g., soiled fabrics), in industrial and household uses at temperatures, reaction times and liquor ratios conventionally employed in these environments. The incubation conditions (i.e., the conditions effective for treating materials with detergent compositions according to the present invention), are readily ascertainable by those of skill in the art. Accordingly, the appropriate conditions effective for treatment with the present detergents correspond to those using similar detergent compositions which include wild-type perhydrolase.
As indicated above, in some embodiments of the detergents provided by the present invention are formulated as a pre-wash in the appropriate solution at an intermediate pH, where sufficient activity exists to provide desired improvements in softening, depilling, pilling prevention, surface fiber removal or cleaning. When the detergent composition is a pre-soak (e.g., pre-wash or pre-treatment) composition, either as a liquid, spray, gel or paste composition, the perhydrolase enzyme is generally employed from about 0.00001% to about 5% weight percent based on the total weight of the pre-soak or pre-treatment composition. In such compositions, surfactant(s) may optionally be employed and when employed, is/are generally present at a concentration of from about 0.0005 to about 1 weight percent based on the total weight of the pre-soak. The remainder of the composition comprises conventional components used in the pre-soak (e.g., diluent, buffers, other enzymes (proteases), etc.) at their conventional concentrations.
In some embodiments, the cleaning compositions provided by the present invention find use in cleaning a situs (e.g., a surface or fabric). Typically at least a portion of the situs is contacted with at least one cleaning composition provided herein, in neat form or diluted in a wash liquor, and then the situs is optionally washed and/or rinsed. For purposes of the present invention, washing includes but is not limited to, scrubbing, and mechanical agitation. The fabric comprises most any fabric capable of being laundered in normal consumer use conditions. The cleaning compositions provided herein are typically employed at concentrations of from about 500 ppm to about 15,000 ppm in solution. When the wash solvent is water, the water temperature typically ranges from about 5° C. to about 90° C. and, when the situs comprises a fabric, the water to fabric mass ratio is typically from about 1:1 to about 30:1.
The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
PCT publication WO05/056782 provides methods for the identification and use of perhydrolase enzymes. Each of the Examples in this publication is individually incorporated by reference herein for disclosure of all methods and disclosed therein including but not limited to disclosure of: methods of making perhydrolases, methods of identifying perhydrolases, methods of testing perhydrolases, perhydrolase polynucleotide and polypeptide sequences, methods of using perhydrolases and compositions in which perhydrolases may be employed.
In the experimental disclosure which follows, the following abbreviations apply: ° C. (degrees Centigrade); rpm (revolutions per minute); H2O (water); HCl (hydrochloric acid); aa (amino acid); by (base pair); kb (kilobase pair); kD (kilodaltons); gm (grams); μg and ug (micrograms); mg (milligrams); ng (nanograms); μl and ul (microliters); ml (milliliters); mm (millimeters); nm (nanometers); μm and um (micrometer); M (molar); mM (millimolar); μM and uM (micromolar); U (units); V (volts); MW (molecular weight); sec (seconds); min(s) (minute/minutes); hr(s) (hour/hours); MgCl2 (magnesium chloride); NaCl (sodium chloride); OD280 (optical density at 280 nm); OD600 (optical density at 600 nm); PAGE (polyacrylamide gel electrophoresis); EtOH (ethanol); PBS (phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2]); SDS (sodium dodecyl sulfate); Tris (tris(hydroxymethyl)aminomethane); TAED (N,N,N′N′-tetraacetylethylenediamine); w/v (weight to volume); v/v (volume to volume); Per (perhydrolase); per (perhydrolase gene); Ms (M. smegmatis); MS (mass spectroscopy); AATCC (American Association of Textile and Coloring Chemists); WFK (wfk Testgewebe GmbH, Bruggen-Bracht, Germany); Amersham (Amersham Life Science, Inc. Arlington Heights, Ill.); Pierce (Pierce Biotechnology, Rockford, Ill.); Amicon (Amicon, Inc., Beverly, Mass.); ATCC (American Type Culture Collection, Manassas, Va.); Amersham (Amersham Biosciences, Inc., Piscataway, N.J.); Becton Dickinson (Becton Dickinson Labware, Lincoln Park, N.J.); BioRad (BioRad, Richmond, Calif.); Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Difco (Difco Laboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL (Life Technologies, Inc., Gaithersburg, Md.); Novagen (Novagen, Inc., Madison, Wis.); Qiagen (Qiagen, Inc., Valencia, Calif.); Invitrogen (Invitrogen Corp., Carlsbad, Calif.); Dionex (Dionex Corp., Sunnyvale, Calif.); Sigma-Aldrich (Sigma-Aldrich Chemical Co., St. Louis, Mo.); Sorvall (Sorvall Instruments, a subsidiary of DuPont Co., Biotechnology Systems, Wilmington, Del.); Stratagene (Stratagene Cloning Systems, La Jolla, Calif.); Roche (Hoffmann La Roche, Inc., Nutley, N.J.); Molecular Devices (Molecular Devices, Corp, Sunnyvale, Calif.); and Agilent (Agilent Technologies, Palo Alto, Calif.).
As described in PCT publication WO05/056782, the perhydrolase gene of M. smegmatis perhydrolase gene was cloned. The nucleotide sequence of the perhydrolase gene of M. smegmatis perhydrolase gene is:
(SEQ ID NO: 1)
ATGGCCAAGCGAATTCTGTGTTTCGGTGATTCCCTGACCTGGGGCTGGGT
CCCCGTCGAAGACGGGGCACCCACCGAGCGGTTCGCCCCCGACGTGCGCT
GGACCGGTGTGCTGGCCCAGCAGCTCGGAGCGGACTTCGAGGTGATCGAG
GAGGGACTGAGCGCGCGCACCACCAACATCGACGACCCCACCGATCCGCG
GCTCAACGGCGCGAGCTACCTGCCGTCGTGCCTCGCGACGCACCTGCCGC
TCGACCTGGTGATCATCATGCTGGGCACCAACGACACCAAGGCCTACTTC
CGGCGCACCCCGCTCGACATCGCGCTGGGCATGTCGGTGCTCGTCACGCA
GGTGCTCACCAGCGCGGGCGGCGTCGGCACCACGTACCCGGCACCCAAGG
TGCTGGTGGTCTCGCCGCCACCGCTGGCGCCCATGCCGCACCCCTGGTTC
CAGTTGATCTTCGAGGGCGGCGAGCAGAAGACCACTGAGCTCGCCCGCGT
GTACAGCGCGCTCGCGTCGTTCATGAAGGTGCCGTTCTTCGACGCGGGTT
CGGTGATCAGCACCGACGGCGTCGACGGAATCCACTTCACCGAGGCCAAC
AATCGCGATCTCGGGGTGGCCCTCGCGGAACAGGTGCGGAGCCTGCTGTA
A
The amino acid sequence of the M. smegmatis perhydrolase enzyme is:
(SEQ ID NO: 2)
MAKRILCFGDSLTWGWVPVEDGAPTERFAPDVRWTGVLAQQLGADFEVIE
EGLSARTTNIDDPTDPRLNGASYLPSCLATHLPLDLVIIMLGTNDTKAYF
RRTPLDIALGMSVLVTQVLTSAGGVGTTYPAPKVLVVSPPPLAPMPHPWF
QLIFEGGEQKTTELARVYSALASFMKVPFFDAGSVISTDGVDGIHFTEAN
NRDLGVALAEQVRSLL.
Also, as described in PCT publication WO05/056782 each and every amino acid position of the M. smegmatis perhydrolase enzyme was mutated to each of the remaining 19 amino acids produce a site saturation library. Using the methods described in Example 2 of PCT publication WO05/056782, the wild-type perhydrolase each and of the perhydrolase variants in the site saturation library was tested for its ability to hydrolyze p-nitrophenylcaproate, a C6 acyl ester substrate.
Wild type perhydrolase was not able to hydrolyze pNC6. The following perhydrolase variants were identified as having an ability to hydrolyze pNC6:
TABLE 1
Perhydrolase variants able to hydrolyze pNC6
Wild-Type
Residue/Position
Amino Acid Variant(s)
L12
G, P, Q
G22
W
N59
P
I153
P
F154
Q, S, T, V
I194
G
F196
S, Q, V, G, P, I, H
L204
Y, W
The mutations identified in Table 1 were combined together to produce four different libraries, NSAL1, NSAL2, NSAL3 and NSAL4 using wild-type perhydrolase (SEQ ID NO:2) and the L12G variant as parent molecules. The primers used to make the combinatorial libraries are as follows, where to the “NNS” sequence represents a degenerate codon NNG/C (N=G, A, T or C) that encodes all 20 amino acids and one stop codon:
TABLE 2
Mutations and Primers Used for
Combinatorial Libraries
Mutations
Primer Sequence
L12G
GTGTTTCGGTGATTCCGGCACCTG
(SEQ ID NO: 3)
GGGCTGGGTCC
L12P
GTGTTTCGGTGATTCCCCGACCTG
(SEQ ID NO: 4)
GGGCTGGGTCCC
L12Q
GTGTTTCGGTGATTCCCAGACCTG
(SEQ ID NO: 5)
GGGCTGGGTCCC
L12NNS
GTGTTTCGGTGATTCCNNSACCTG
(SEQ ID NO: 6)
GGGCTGGGTCC
I194G
GACGGCGTCGACGGAGGCCACTTC
(SEQ ID NO: 7)
ACCGAGGCCAAC
I194NNS
GACGGCGTCGACGGANNSCACTTC
(SEQ ID NO: 8)
ACCGAGGCCAAC
F154T
TGGTTCCAGTTGATCACCGAGGGC
(SEQ ID NO: 9)
GGCGAGCAGAAG
F154S
TGGTTCCAGTTGATCAGCGAGGGC
(SEQ ID NO: 10)
GGCGAGCAGAAG
F154NNS
TGGTTCCAGTTGATCNNSGAGGGC
(SEQ ID NO: 11)
GGCGAGCAGAAG
F196S
GCGTCGACGGAATCCACAGCACCG
(SEQ ID NO: 12)
AGGCCAACAATCG
F196Q
GCGTCGACGGAATCCACCAGACCG
(SEQ ID NO: 13)
AGGCCAACAATCG
F196V
GCGTCGACGGAATCCACGTTACCG
(SEQ ID NO: 14)
AGGCCAACAATCG
F196G
GCGTCGACGGAATCCACGGTACCG
(SEQ ID NO: 15)
AGGCCAACAATCG
F196P
GCGTCGACGGAATCCACCCGACCG
(SEQ ID NO: 16)
AGGCCAACAATCG
F1961
GCGTCGACGGAATCCACATCACCG
(SEQ ID NO: 17)
AGGCCAACAATCG
F196NNS
GCGTCGACGGAATCCACNNSACCG
(SEQ ID NO: 18)
AGGCCAACAATCG
F154V
TGGTTCCAGTTGATCGTTGAGGGC
(SEQ ID NO: 19)
GGCGAGCAGAAG
F196H
GCGTCGACGGAATCCACCATACCG
(SEQ ID NO: 20)
AGGCCAACAATCG
F154Q
TGGTTCCAGTTGATCCAGGAGGGC
(SEQ ID NO: 21)
GGCGAGCAGAAG
N59P
AGCGCGCGCACCACCCCGATCGAC
(SEQ ID NO: 22)
GACCCCACCGATC
L204Y
GCCAACAATCGCGATTATGGGGTG
(SEQ ID NO: 23)
GCCCTCGCGGAAC
L204W
GCCAACAATCGCGATTGGGGGGTG
(SEQ ID NO: 24)
GCCCTCGCGGAAC
L204NNS
GCCAACAATCGCGATNNSGGGGTG
(SEQ ID NO: 25)
GCCCTCGCGGAAC
I153P
CCCTGGTTCCAGTTGCCGTTCGAG
(SEQ ID NO: 26)
GGCGGCGAGCAG
G22W
GTCCCCGTCGAAGACTGGGCACCC
(SEQ ID NO: 27)
ACCGAGCGGTTC
QuikChange multi site-directed mutagenesis (QCMS) was used to create combinatorial libraries NSAL1-NSAL4 using method described in WO 05/056782. The QCMS reaction consisted of 16.5 uL of sterile distilled H2O, 2.5 uL of 10×buffer from the kit, 1 uL dNTPs from the kit, 3 uL of the 20 primers mix (10 uL of each 100 ng/uL primer was mixed together ahead of time), 1 uL of pMSAT-NcoI miniprep DNA as template (˜50 ng), and 1 uL of the enzyme blend from the kit for a total of 25 uL. The cycling conditions were 95° C. for 1 mM once, 95° C. for 1 min, 55° C. for 1 mM, 65° C. for 10 min for 30 cycles. Next, DpnI digestion was carried out twice sequentially with 1 or 0.5 uL of enzyme (QCMS kit) at 37° C. for 4 hours. 2 uL of the reaction was transformed into BL21 (DE3) pLysS competent cells (Novagen) as per the manufacturer's instructions. The transformation was plated on LB plates containing 100 ppm carbenicillin, 0.1 mM IPTG and 0.25% of tricaproin (a C6 acyl chain substrate that was mixed in the media by sonication).
After incubation of the plates at 37° C. for 24 hours followed by room temperature for 2 days, a majority of the halo-forming colonies were grown overnight at 37° C. in 96-well plates containing LB with 100 ppm of carbenicillin. To re-assess the halo-formers, the cultures were replica-stamped onto a large agar plate containing LB, 100 ppm carbenicillin, 0.1 mM IPTG and 0.25% of tricaproin.
Table 3 describes further details of the combinatorial libraries and their screening.
TABLE 3
Description of Libraries and Colonies Screened
COLONIES
LI-
PARENT
COLONIES
WITH
BRARY
PRIMERS USED
MOLECULE
SCREENED
HALOS**
NSAL1
L12NNS, F154NNS,
WILD
182
21
F196NNS, I194NNS,
TYPE
L204NNS
NSAL2
L12NNS, F154NNS,
L12G
169
40
F196NNS, I194NNS,
L204NNS
NSAL3
All primers in Table 1
WILD
~1200*
4
except NNS codon primers
TYPE
NSAL4
All primers in Table 1
L12G
~1000*
~100-200*
except NNS codon primers
*This number is approximate. The exact number of colonies was not determined.
**Some of the halo forming colonies did not form halos upon re-testing. The number of halo-formers in NSAL2 and NSAL4 is higher than in NSAL1 and NSAL3 due to the L12G parent that was the present in 25% of the NSAL2 and NSAL4 libraries.
The polynucleotides encoding the perhydrolase enzyme of the halo-forming colonies were sequenced to determine which mutations contribute to halo formation (Table 4).
TABLE 4
Sequence of Halo-Forming Clones*
LIBRARY
SEQUENCE
NSAL1
I194G (3 clones)
NSAL3
I194G
NSAL4
L12G G22W
NSAL2
L12G I194M
NSAL1
F154A I194M (2 clones)
NSAL1
F154A
NSAL1
F154G I194V
NSAL1
F154E I194S (3 clones)
NSAL1
F154E
NSAL3
F154T F196I (2 clones)
NSAL3
F154V
NSAL3
L12Q F154V
NSAL3
L12M F154E
NSAL3
L12G F154G I194V
*Halo-producing L12G clones are not listed in the table, since this mutation was known to produce halos on tricaproin plates.
Variants that formed halos on tricaproin plates that had an amino acid sequence different from a parent sequence were tested for their ability to hydrolyze p-nitrophenylcaproate (pNC6) and p-nitrophenyloctanoate (pNC8) in 100 mM Tris/HCl pH 8, 0.1% Triton-X100 and 1 mM of the pNC6 or pNC8 using methods described in Example 2 of PCT publication WO 05/056782.
The rate of p-nitrophenol appearance was recorded for each of the halo-forming variants. The wild type enzyme showed no hydrolysis of pNC6 or pNC8. Ratios of hydrolysis of pNC6/pNC8 are shown in Table 5 below.
TABLE 5
Variants having pNC6/pNC8 Hydrolytic Activity
Ratio pNC6 Hydrolysis:pNC8
Sequences
Hydrolysis
F154A I194M
1.13
F154G I194V
0.34
L12G
0.79
L12G I194M
0.65
Variants F154T F1961,L12Q F154V, L12M F154E, L12G F154G, F154E I194S, and L12G G22W had the ability to hydrolyze tricaproin but did not hydrolyze pNC6 or pNC8.
The data show that specific variants of the M. smegmatis perhydrolase are capable of using medium and long chain acyl esters as a substrate.
In this Example, methods that find use in assessing enzyme purity and activity are described. However, it is not intended that the present invention be limited to these specific methods, as other suitable methods find use.
Enzyme Activity Assay (pNB Assay)
This activity is measured by hydrolysis of p-nitrophenylbutyrate or other long chain p-nitrophenyl compounds. The reaction mixture was prepared by adding 10 ul of 100 mM p-nitrophenylbutyrate in dimethylsulfoxide to 990 ml of 100 mM Tris-HCl buffer, pH 8.0 containing 0.1% Triton X-100. The background rate of hydrolysis was measured before the addition of enzyme at 410 nm. The reaction was initiated by the addition of 10 ul of enzyme to 990 ml of the reaction and the change of absorbance at 410 nm was measured at room temperate (˜23° C.). The background corrected results are reported as δA410/min/ml or δA410/min/mg protein.
Transesterification
Transesterification is measured by GC separation of products in buffered aqueous reactions. Reactions to measure ethyl acetate transesterification with propanol contained in 1 ml of 50 mM KPO4, pH 7.0; 200 mM ethyl acetate, 200 mM 1-propanol, and enzyme. Reactions to measure ethyl acetate transesterification with neopentyl glycol (NPG) contained in 1 ml of 50 mM KPO4, pH 7.0; 303 mM ethyl acetate, 100 mM NPG, and enzyme. The reactions were incubated at the indicated temperatures and for the indicated times. Separations are performed using a 30M FFAP column (Phenomenex). The inlet split ratio was approximately 1:25, the injector is 250° C., head pressure of 10 psi He, and detection was by FID at 250° C. The chromatography program was set at 40° C. initial for 4 min, followed by a gradient of 15° C./min to 180° C. Components eluted in the following order and were not quantified; ethyl acetate, ethyl alcohol, propyl acetate, propyl alcohol, acetic acid, NPG diacetate, NPG monoacetate, and NPG.
Preparation of Substrate
The substrates were prepared as described herein. Ethyl acetate (EtOAc) or other water soluble esters were diluted in a desired buffer to a concentration of 10 mM of ester. tributyrin and other water insoluble substrates are prepared by making substrate swatches. Polyester swatches were cut from non-dyed polyester fabric (Polycotton, PCW 22) using a ⅝ inch punch and placed in a 24-well microtiter plate (Costar, Cell Culture Plate). The insoluble ester was diluted to 1.03 M in hexane. Then, 10 μL of the insoluble ester solution were then adsorbed onto the polyester swatch.
Determination of Hydrolysis (GC Assay)
The hydrolytic assay described below finds use in determining the amount of substrate hydrolysis. In this assay, the assay solution was comprised of 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, and 20 mM potassium chloride in a total volume of 0.99 ml and an amount of enzyme that would generate 20 nmoles of acetic acid per minute at 25° C.
For measuring water insoluble ester hydrolysis, the reaction mixture was added to the insoluble ester fabric swatch. The swatch was prepared as described above (“Preparation of Substrate”). All the other conditions for the assay were the same except for exclusion of other ester substrates.
Hydrolytic activity was measured by monitoring the increase of acids generated by the enzyme from acyl donor substrates using gas chromatography coupled with flame ionization detection. The assay was conducted by first pipetting 50 μL of assay solution containing all the components except the enzyme into 200 μL of methanol (HPLC grade) to determine the amount of acid in the assay solution at time 0. Then, 10 μL of enzyme was added to the assay solution to a desired final concentration which produced approximately 20 nanomoles of acid per minute. A timer was started and 50 μL aliquots were taken from the assay solution and added to 200 μL of methanol at various times, typically 2, 5, 10, 15, 25, 40, and 60 minutes, after addition of the enzyme.
These methanol-quenched samples were then injected into a gas chromatograph coupled with a flame ionization detector (Agilent 6890N) and analyzed for hydrolytic components, acetic, and butyric acids, etc. Gas chromatography was conducted using a nitroterephthalic acid modified polyethylene glycol column (Zebron FFAP; with dimensions: 30 m long, 250 um diameter, 250 nm film thickness). A 3 μL aliquot of sample was applied to the column by a splitless injection under constant a helium flow of 1.0 mL/minute. The inlet was maintained at a temperature of 250° C., and was purged of any remaining sample components after 2 minutes. When analyzing acetic acid, the temperature of the column was maintained at 75° C. for 1 minute after injection, increased 25° C./minute to 100° C., then increased 15° C./minute to 200° C.
When analyzing butyric acid, the temperature of the column was controlled as described above, except the temperature was additionally increased 25° C./minute to 225° C. and held at 225° C. for 1 minute. The flame ionization detector was maintained throughout the chromatography at 250° C. and under constant hydrogen flow of 25 mL/minute, air flow of 200 mL/minute, and a combined column and makeup helium flow of 30 mL/minute. The amount of hydrolyzed acid in the sample was then determined by integrating the acid peak in the chromatogram for total ion counts and calculating the acid from the ion count using a standard curve generated under the above conditions for acetic and butyric acids at varying concentrations in the assay solution (without enzyme).
Determination of Perhydrolysis (OPD Assay)
The perhydrolytic activity assay described below finds use in determining the amount of peracid formed in the reaction. In these assays, the solution comprised 50 mM potassium phosphate pH 7.5, 10 mM ester substrate, 29 mM hydrogen peroxide, 20 mM potassium chloride, and 10 mM O-phenylenediamine
When using water insoluble ester as the acyl donor, an ester-adsorbed fabric swatch was used as the substrate, prepared as described above (“Preparation of Substrate”).
Perhydrolytic activity was measured by monitoring the absorbance increase at 458 nm of oxidized o-phenylenediamine (OPD) by peracid generated with the enzyme. The perhydrolytic activity assay solution was prepared in the same manner as the hydrolytic activity assay solution, except that OPD was added to the assay solution to a final concentration of 10 mM. The OPD solution was prepared immediately before conducting the assay by dissolving 72 mg OPD (Sigma-Aldrich, dihydrochloride) in 19.94 mL of the same buffer and the pH was adjusted by slowly adding 60 μL of 13.5 M potassium hydroxide. The pH was measured and if needed, small quantities of potassium hydroxide were added to return the pH to the original pH of the buffer. Then, 495 μL of this OPD solution were added with the other assay components to a final assay volume of 0.990 mL. An assay quenching solution was also prepared by dissolving 36 mg OPD in 20 mL 100 mM citric acid and 70% ethanol.
The assay was typically conducted at 25° C. The assay was started by pipetting 100 μL of assay solution before the addition of the enzyme into 200 μL of quenching solution to determine the amount of perhydrolytic components and background absorbance in the assay solution at time 0. Then, 10 μL of enzyme were added to the assay solution to a desired final concentration which produced approximately 10 nanomoles of peracid per minute. A timer was started and 100 μL aliquots were taken from the assay solution and added to 200 μL of quenching solution at various times, typically 2, 5, 10, 15, 25, 40, and 60 minutes, after adding the enzyme. The quenched assay solutions were incubated for 30 minutes to allow any remaining peracid to oxidize the OPD. Then, 100 μL of each quenched assay solution was transferred to a 96-well microtiter plate (Costar) and the absorbance of the solution was measured at 458 nm by a spectrophotometric plate reader (Molecular Devices, SpectraMAX 250). The amount of peracid in each quenched sample was calculated using a standard curve generated under the above conditions with peracetic acid at varying concentrations in the assay solution (without enzyme).
Perhydrolysis/Hydrolysis Ratio:
Perhydrolysis/Hydrolysis ratio=Perhydrolysis measured in the Perhydrolysis assay/(Total acid detected in the hydrolysis assay−Perhydrolysis measured in the perhydrolysis assay)
Perhydrolase Peracid Generation Assay
For perhydrolysis measurements, the enzyme is incubated in the buffer of choice at a specified temperature with a substrate ester in the presence of hydrogen peroxide. Typical substrates to measure perhydrolysis of medium or long chain esters include methyl or ethyl esters of hexanoate, heptanoate, octanoate, nonanoate or C10-C22 or longer fatty acid esters, and others. In addition, the wild type enzyme was found able to hydrolyze nitrophenylesters of short chain acids. The latter are convenient substrates to measure enzyme concentration. In some embodiments, peracid acid and acetic acid are measured by the ABTS or HPLC assays. Nitrophenylester hydrolysis is also described below.
ARTS Assay (One Milliliter):
This assay provides a determination of peracetic acid produced by perhydrolase. This protocol was adapted from Karst et al. (Karst et al., Analyst, 122:567-571 [1997]). Briefly, a 100 μL aliquot of solution to be analyzed was added to 1 mL 125 mM K+ citrate pH 5, 1 mM ABTS, 50 μM KI. Absorbance was measured at 420 nm for highest sensitivity. However, multiple additional wavelengths were sometimes used over the broad absorption spectrum of ABTS. Calibration curves were constructed based on known peracid concentration series.
HPLC (Model—Agilent 1100) Determination of Perhydrolase Reaction Products:
For determination of the ratio of perhydrolysis to hydrolysis of the perhydrolase reaction, perhydrolase reaction samples were quenched by acidification to a final concentration of 0.24% methanesulfonic acid, and the products were separated by reverse phase HPLC on a Dionex OA column (cat #062903; Dionex). The mobile phase was 100 mM NaPO4, pH 3.9 (buffer was prepared by titrating 100 mM Na2PO4 with methanesulfonic acid to pH 3.9) run under isocratic conditions at 30° C. Detection was at 210 nm. Concentrations of products were calculated by comparison of the integrated peak areas against calibration standards.
Nitrophenylester Hydrolysis Kinetic Assay
Enzyme and substrate were incubated in 100 mM Tris/HCl pH 8.0 (or 50 mM B(OH)3 pH 9.5 or another buffer). Absorbance at 402 nm was monitored. In some experiments, the assay was carried out in standard 1 mL cuvettes, while in other experiments, microtiter plate wells were used. The latter method was used for the screening of mutant libraries. Enzyme concentration was determined by comparison to standard curves obtained under the same reaction conditions.
Para-Nitrophenylcaproate Hydrolysis Assay
The pNC6 substrate solution was prepared by mixing 1 mM pNC6 (100 mM stock solution), 1 ml DMSO, 19 ml 100 mM Phosphate (pH8), and glycerol to a final concentration of 10%. To assay samples, 10 μl of the cell lysate were added to 190 μl of the substrate solution, and assayed at 405 nm for 15 minutes in a spectrophotometer. The results were presented as the average of two experiments.
Para-Pitrophenyl Acetate (pNA) Hydrolysis Assay
Aliquots of the lysed cell supernatant were diluted 1-100 in 100 mM phosphate buffer (pH 8). To assay the samples, 5 μl of the 1-100 diluted cell supernatant were placed into each well of a microtiter plate. Then, 195 μl of reaction buffer/substrate mix (1 mM pNA, 100 mM phosphate, pH 8, 10% glycerol) were added, and the absorbance rate at 405 nm measured over 3 minutes (kinetics program, microtiter plate reader). The results were presented as the average of two experiments.
All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.
Having described the preferred embodiments of the present invention, it will appear to those ordinarily skilled in the art that various modifications may be made to the disclosed embodiments, and that such modifications are intended to be within the scope of the present invention.
Those of skill in the art readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The compositions and methods described herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It is readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
Cervin, Marguerite A., Bott, Richard R., Poulose, Ayrookaran J., Amin, Neelam S., Weyler, Walter
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