WO2001000800A1 - Low temperature enzymes derived from particular bacterial strain - Google Patents

Abstract

Stain removing compositions which can be granular detergents, liquid detergents, granular stain removers, liquid stain removers, household cleaners, food industrial cleaners, and the like, containing proteolytic enzymes derived from bacterium No. 177, are effective low temperature (0°C-50°C) stain removers.

Description

TITLE: LOW TEMPERATURE ENZYMES DERIVED FROM PARTICULAR BACTERIAL STRAIN

CROSS REFERENCE TO A RELATED APPLICATION

This application claims the benefit of the filing date of United States Provisional Application No. 60/141,187, filed June 25, 1999.

FIELD OF THE INVENTION

This invention relates to improved detergent compositions, both liquid and granular, household cleaners, and food industrial cleaners containing certain proteolytic enzymes having the benefit of enhanced cleaning at low temperatures. The invention also relates to the substantially pure enzymes and their isolation.

BACKGROUND OF THE INVENTION

Many applications exist for enzymes that have higher activities at temperatures lower than the currently available enzymes used in cleaning. For example, enzymes that remain active in cool and warm water would be useful for removing stains from clothes during lower temperature laundry conditions. This would decrease the energy consumption normally used to heat laundry water and would reduce the negative energy impact on the environment. Enzymes effective at lower temperatures would permit washing delicate or brightly colored fabrics in conditions that cause less shrinkage and dye bleeding. Under these low temperature conditions, stain removing enzymes which maintain high activity at low temperatures will remove more stain material from clothing than currently used higher water temperature active enzymes. Most currently available enzymes, including Savmase, have a higher temperature optima (45°C-55°C) and must be used at high water temperatures to be effective. The advantage of a low water temperature stain removing enzyme, if found, would be its ability to achieve the desired stain removal at lower temperatures, requiring less time and energy and offering less risk of substrate damage.

Enzymes effective at low temperatures could have additional utilities and applications not possible with currently available enzymes. For example, such enzymes could be used for other consumer applications such as household cleaners, with or without added surfactants. They could have utility m the baking and food processing industries where enzyme activity at lower temperatures allows faster processing or cleaning treatments at reduced temperatures which m turn lower the risk of growth of spoilage organisms. Other advantages of enzymes with higher activities at low temperatures include, but are not limited to, bioremediation m cool climates, industrial chemical conversions, scientific research, etc.

From the above description it can be seen that it would be desirable, or there is a need to develop low temperature activated proteolytic enzymes useful for a wide variety of effective stain removing compositions, with or without surfactants. Versatile proteolytic enzymes having low temperature effectiveness for stain removal could be used m liquid or granular detergents, liquid or granular stain removers, prewashes, household cleaners, and m food and industrial cleansing applications. This invention has as its primary ob ective the fulfillment of these needs. SUMMARY OF THE INVENTION

A novel bacterium strain 177, ATCC Deposit No.

(a deposit will be made upon indication of allowable subject matter) when isolated and purified has demonstrated high proteolytic enzyme activity m cleaning applications at low temperatures of from 0°C-50°C, preferably 15°C to 45°C. Tests of stain removal effectiveness with or without surfactants or builders have shown equal or exceeding stain removing capability from currently available proteolytic enzymes useful at higher temperatures, such as Savmase, which has an optimum temperature range of 45°C-55°C.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the effect of pH on stain removal of proteins with enzyme derived from strain No. 177.

Figures 2A, 2B, and 2C show gel analysis of stain removing enzymes produced by strain No. 177.

Figure 3 is a flow diagram showing growth strategy and separation technique for removing enzymes from strain No. 177.

Figure 4 shows thermal stability of proteolytic activities after incubation at various temperatures.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A description of the isolated novel bacterium, strain

177, shall first be provided. Then a description of specific industrial use compositions involving the stain remover enzyme (s) derived from bacterial strain No. 177 m combination with various cleansing compositions such as granular liquid detergents, pre- spot stain removers, household cleaners, food industrial cleaners, etc. will be given. Surprisingly, by careful observation and selection, a novel bacterium, strain 177, has been isolated, and by designing specific enrichments to target microorganisms that produce stain removing enzymes, and by screening for the maintenance of high enzymatic activity m cleaning applications at low temperatures (0°C-50°C), a successful result has been achieved.

The original source material was soil from a natural environmental enrichment . This material was used as the inoculum for further laboratory enrichments. From this work, an organism which produced several stain removing enzymes active m low temperature cleaning conditions was obtained and designated as strain 177. The microorganism was grown m media designed to induce the production of the stain removing activities. The spent media were assayed with and without surfactants and chelators to determine the efficacy of one or more of the stain removing enzymes to remove stains from cloth at various temperatures. A blend of active stain removing enzymes was detected on casein zymogram gels, and the stain removing activities have been partially separated. As earlier indicated, the stain removing enzymes from strain 177 will be deposited at the ATCC upon indication of allowable subject matter. The stain removing enzymes from strain 177 maintain higher stain removal activity than Savmase enzyme (commonly used m cleansers) within a temperature range of 0°C-37°C. In this comparison, strain 177 stain removing enzymes remove 50% more material from swatch patch stains. The addition of strain 177 stain removing enzymes to laundry detergent compositions will increase the stain removal activity within a broad low temperature range (0°C-50°C), increasing the effectiveness and the marketability of the product and decreasing energy requirements and costs. It therefore can be used effectively to fulfill the major objects of the present invention. Isolation and characterization of the strain is next discussed below.

Enrichments were designed to isolate microorganisms producing stain removing enzymes. For example, a soil sample, naturally enriched with protemaceous and other biological materials obtained from north central Pennsylvania was placed m sterile M9 medium (6 g Na₂HP0₄; 3 g KH₂P0₄; 0.5 g NaCl; 1.0 g NH₄C1 ; per liter) containing some or all of the following with allowable substitutions: 0.05% casammo acids, 0.05% sodium casemate, 0.02% glucose, trace elements, and autoclaved human hair. After incubation at 10°C-12°C for 20 days, samples were taken and inoculated onto different solid media and incubated at 18°C-19°C. The original colony of strain 177 was picked from a plate containing solid medium containing all or some of the following: (1.5 g lanolin, 1.0 g cetyl alcohol, 0.5 g Na Stearate, 2.5 g lard, 2.0 g Na Casemate, 0.5 g α-cellulose, 1.0 g potato starch, 1.0 g cornstarch, 0.2 g glucose, and 1.5 g Bacto-agar per liter) and purified by restreakmg many times. Strain 177 was selected as a potential stain removing enzyme producer because, among its traits, it grew on plates containing skim milk, indicating that it hydrolyzed casein, as shown by areas of clearing. Strain 177 forms raised, round, and translucent yellow to cream or tan colonies on Trypticase Soy Agar (TSA) depending upon growth temperature and age of the culture. On Brain Heart Infusion/Skim milk plates, the colonies are flatter with a raised center. They have a translucent/cream edge and cream to yellow center depending on growth temperature. Strain 177 cells are gram negative.

Strain 177 does not form isolated colonies on TSA at 37 °C, indicating that it would probably not grow well at normal body temperature. It formed isolated colonies at 5°C to 7°C. In addition, strain 177 does not exhibit β-hemolysis on blood agar plates at 31°C. Table I shows some physiological characteristics of strain 177.

TABLE I

Table 1. Comparison of some physiological characteristics between Strain 177, Xanthomonas sp, Stenotrophomas mal tophilia and S . africana

Chromosomal DNA was obtained from strain 177 and used as template DNA for polymerase chain reaction (PCR) amplification of the small subunit 16s rRNA gene. The sequence was aligned and compared to other sequences in the Ribosomal Database Project and GenBank. The most closely related genera to strain 177 were Xanthomonas and Stenotrophomas . The 16s rRNA gene sequence for strain 177:

(ID Sequence No. 1)

The term strain 177, or the deposit number for the ATCC,

Deposit No. , as used herein, is intended to refer to strain No. 177, and derived enzyme activities such as the proteolytic enzyme (s) derived from bacterial strain No. 177, or other enzymes derived from genetic equivalents of bacterial strain No. 177, or mutants or variants thereof, or cloned genes or derivatives thereof. The determining factor as to a genetic equivalent or a mutant or variant thereof is whether or not the isolated bacterial strain, such as No. 177, produces a proteolytic enzyme which is an effective low temperature cleaner useful in a wide variety of cleansing compositions such as those described herein.

The N-Terminal ammo acid sequence of one enzyme extracted from bacterial strain 177 is: L-T-P-N-D-T-R-F-S-E. Next, the description moves to specific examples of stam removing activities of the enzyme (s) derived from strain 177.

The term "conservatively modified variants" applies to both ammo acid and nucleic acid sequences . With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or conservatively modified variants of the ammo acid sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the ammo acid alanme. Thus, at every position where an alanme is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also, by reference to the genetic code, describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon m a nucleic acid (except AUG, which is ordinarily the only codon for methionine; and UGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide of the present invention is implicit m each described polypeptide sequence and is withm the scope of the present invention.

As to ammo acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single ammo acid or a small percentage of ammo acids m the encoded sequence is a "conservatively modified variant" where the alteration results m the substitution of an ammo acid with a chemically similar ammo acid. Thus, any number of ammo acid residues selected from the group of integers consisting of from 1 to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made. Conservatively modified variants typically provide similar biological activity as the unmodified polypeptide sequence from which they are derived. For example, substrate specificity, enzyme activity, or ligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the native protein for its native substrate. Conservative substitution tables providing functionally similar ammo acids are well known the art .

The following six groups each contain ammo acids that are conservative substitutions for one another:

1) Alanme (A) , Serine (S) , Threonme (T) ;

2) Aspartic acid (D) , Glutamic acid (E) ^•

3) Asparagme (N) , Glutamme (Q) ;

4) Argmine (R) , Lysme (K) ; 5) Isoleucme (I) , Leucme (L) , Methionine (M) , Valme (V) ; and 6) Phenylalanme (F) , Tyrosme (Y) , Tryptophan (W) . See also, Creighton (1984) Proteins W.H. Freeman and Company . Some of the stam removing enzymes were partially separated and characterized using protease substrates. Additional enzyme screening may be performed for other enzymes including, but not limited to, cellulases, lipases, and amylases . Strain 177 was grown m liquid media for various times, and spent media aliquots were assayed for activity using protease substrates m buffer with and without surfactants and chelators. This simulated cleaning buffer and enzyme mixture was incubated at 20°C to determine activity m presence of detergent formulation components. These initial assays indicated that there were stam removing enzymes produced by strain 177 that maintain high activity cleaning conditions at temperatures ranging from 0°C to 50°C. Figure 3 of the drawings represents a flow diagram which is but one example of growth strategy for strain 177, and the separation of the stam removing enzymes from strain 177. It can equally by applied to genetic equivalent mutant or variant strains of 177.

In the assay experiments summarized m Figure 4, spent media isolated from bacterium 177 and containing the enzyme were tested for enzymatic activity after incubation at various temperatures from low to high. The tests revealed substantial stability within the low temperature range.

The flow diagram and separation techniques represented by Figure 3 are self-explanatory, and their application is known to those of ordinary skill m the art with enzyme isolation and purification for use with strain 177. Filtered and concentrated spent media from strain 177 was assayed at different pH values to determine an optimal pH for activity. This was performed on all of the stam removing activities the spent media without separation or purification. Figure 1 summarizes the results, indicating a broad pH range of activity for the stam removing enzymes, with preferred pH of 7-10.

The purification of any or all of the stam removing enzymes produced by strain 177 can include any of the many currently used techniques practiced m the art which may include, but are not limited to size exclusion, ion exchange, and affinity chromatography procedures. The first attempts to separate the different sta removing enzymes were done on a hydrophobic interaction column. The preliminary results indicated that there was separation of the different stam removing enzymes as seen on a gelatin zymogram activity gel . Another type of separation, anion exchange, was performed. Subsequent batch purifications separated upper and lower protein bands as illustrated by SDS-polyacrylamide gel electrophoresis (Figure 2A lanes 2, 3, and 4) . This separation also distinguished separate activities observed with zymogram gels. The zymogram gels show that the unbound fraction contains a lower molecular weight protein (between 15-22 kDa) (Figure 2B and C) . The fraction binding to tne anion exchange resm and eluted with 500 mM salt separated a higher molecular weight subunit protein (between 20-30 kDa) . Another purification method which could be used is HPLC using a size exclusion column. Specific cleansing carrier compositions useful with enzymes derived from the novel bacterium strain 177 can be formulated, and are exemplified below.

Granular detergents are one significant industrial application. The detergent compositions of the present invention contain an organic surfactant, a water-soluble phosphorus or non-phosphorus detergent builder (non- phosphorus preferred) .

The compositions of the present invention can be prepared by drying an aqueous slurry comprising the components, or by agglomeration, or by mixing the ingredients to an aqueous solution or suspension. The effect is obtained regardless of the method of preparation.

The detergent compositions herein contain from about 5% to about 50% by weight of an organic surfactant selected from the group consisting of anionic, noniomc, zwitterionic, ampholytic and cationic surfactants, and mixtures thereof. The surfactant preferably represents from about 10% to about 30% by weight of the detergent composition. Surfactants useful herein are listed m U.S. Patent No. 3,664,961, Noms, issued May 23, 1972, and m U.S. Patent No.

3,919,678, Laughlm, et al . , issued December 30, 1975, both incorporated herein by reference. Useful cationic surfactants also include those described m U.S. Patent No. 4,222,905, Cockrell, issued September 16, 1980, and m U.S. Patent No. 4,239,659, Murphy, issued December 16, 1980, both incorporated herein by reference.

Water soluble salts of the higher fatty acids, i.e., "soaps", are useful anionic surfactants m the compositions herein. This includes alkali metal soaps such as the sodium, potassium, ammonium, and substituted ammonium salts of higher fatty acids containing from about 8 to about 24 carbon atoms, and preferably from about 12 to about 18 carbon atoms. Soaps can be made by direct saponification of fats and oils or by the neutralization of free fatty acids. Particularly useful are the sodium and potassium salts of the mixtures of fatty acids derived from coconut oil and tallow, i.e., sodium or potassium tallow and coconut soap. Useful anionic surfactants also include the water soluble salts, preferably the alkali metal, ammonium and substituted ammonium salts, of organic sulfuπc reaction products having m their molecular structure an alkyl group containing from about 10 to about 20 carbon atoms and a sulfonic acid or sulfuπc acid ester group. (Included m the term "alkyl" is the alkyl portion of acyl groups.) Examples of this group of synthetic surfactants are the sodium and potassium alkyl sulfates, especially those obtained by sulfatmg the higher alcohols (C₈-C_ιa carbon atoms) such as those produced by reducing the glyceπdes of tallow or coconut oil; and the sodium and potassium alkylbenzene sulfonates m which the alkyl group contains from about 9 to about 15 carbon atoms, m straight chain or branched chain configuration, e.g., those of the type described U.S. Patent Nos. 2,220,099 and 2,477,383, both of which are incorporated herein by reference. Especially valuable are linear straight chain alkylbenzene sulfonates m which the average number of carbon atoms m the alkyl group is from about 11 to 13, abbreviated as C^-C^LAS.

Other anionic surfactants suitable for use herein are the sodium alkyl glyceryl ether sulfonates, especially those ethers of higher alcohols derived from tallow and coconut oil; sodium coconut oil fatty acid monoglyceride sulfonates and sulfates; sodium or potassium salts of alkyl phenol ethylene oxide ether sulfates containing from about 1 to about 10 units of ethylene oxide per molecule and from about 8 to about 12 carbon atoms the alkyl group; and sodium or potassium salts of alkyl ethylene oxide ether sulfates containing from about 1 to about 10 units of ethylene oxide per molecule, and from about 10 to about 20 carbon atoms m the alkyl group. Other useful anionic surfactants include the water soluble salts of esters of alpha-sulfonated fatty acids containing from about 6 to 20 carbon atoms m the fatty acid group and from about 1 to 10 carbon atoms m the ester group; water soluble salts of 2 -acyloxy-alkane-1-sulfonic acids containing from about 2 to 9 carbon atoms m the acyl group and from about 9 to about 23 carbon atoms the alkane moiety; alkyl ether sulfates containing from about 10 to 20 carbon atoms m the alkyl group and from about 1 to 30 moles of ethylene oxide; water soluble salts of olefin sulfonates containing from about 12 to 24 carbon atoms; and beta- alkyloxy alkane sulfonates containing from about 1 to 3 carbon atoms m the alkyl group and from about 8 to 20 carbon atoms m the alkane moiety.

Water soluble nonionic surfactants are also useful the compositions of the invention. Such nonionic materials include compounds produced by the condensation of alkylene oxide groups (hydrophilic m nature) with an organic hydrophobic compound, which may be aliphatic or alkyl aromatic m nature. The length of the polyoxyalkylene group which is condensed with any particular hydrophobic group can be readily adjusted to yield a water soluble compound having the desired degree of balance between hydrophilic and hydrophobic elements.

Suitable nonionic surfactants include the polyethylene oxide condensates of alkyl phenols, e.g., the condensation products of alkyl phenols having an alkyl group containing from about 6 to 15 carbon atoms, m either a straight chain or branched chain configuration, with from about 3 to 12 moles of ethylene oxide per mole of alkyl phenol .

Preferred nonionics are the water soluble condensation products of aliphatic alcohols containing from 8 to 22 carbon atoms, either straight chain or branched configuration, with from 3 to 12 moles of ethylene oxide per mole of alcohol . Particularly preferred are the condensation products of alcohols having an alkyl group containing from about 9 to 15 carbon atoms with from about 4 to 8 moles of ethylene oxide per mole of alcohol . Semi-polar nonionic surfactants useful herein include water-soluble am e oxides containing one alkyl moiety of from about 10 to 18 carbon atoms and two moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from 1 to 3 carbon atoms; water soluble phosphme oxides containing one alkyl moiety of about 10 to 18 carbon atoms and two moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from 1 to about 3 carbon atoms; and water soluble sulfoxides containing one alkyl moiety of from about 10 to 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from 1 to about 3 carbon atoms . Ampholytic surfactants include derivatives of aliphatic or aliphatic derivatives of heterocyclic secondary and tertiary ammes m which the aliphatic moiety can be straight chain or branched, and wherein one of the aliphatic substituents contains from about 8 to 18 carbon atoms and at least one aliphatic substituent contains an antiomc water solubiliz g group.

Zwitteπonic surfactants include derivatives of aliphatic quaternary ammonium, phosphonium, and sulfonium compounds m which one of the aliphatic substituents contains from about 8 to about 18 carbon atoms.

Particularly preferred surfactants herein are anionic surfactants selected from the group consisting of the alkali metal salts of C_{lλ 13} alkylbenzene sulfonates, C_{14 18} alkyl sulfates, C_{14 18} alkyl linear polyethoxy sulfates containing from about 1 to about 4 moles of ethylene oxide, and mixtures thereof .

The preferred compositions of the present invention also contain from about 5% to about 80%, preferably from about 10% to about 70%, and most preferably from about 15% to about 60% by weight of a non-phosphorous detergent builder. The non- phosphorous detergent builder can be either organic or inorganic m nature.

Non-phosphorous detergent builders are generally selected from the various water soluble, alkali metal, ammonium or substituted ammonium carbonates, and silicates. Preferred are the alkali metal, especially sodium, salts of the above. However, the present compositions preferably contain less than about 6%, more preferably less than about 4% by weight of silicate materials for optimum granule solubility.

Specific examples of non-phosphorus, inorganic builders are sodium and potassium carbonate, bicarbonate, sesquicarbonate, tetraborate decahydrate, and silicate having a molar ratio of Sι0₂ to alkali metal oxide of from about 0.5 to about 4.0, preferably from about 1.0 to about 2.4. An especially preferred detergency builder is crystalline alummosilicate ion exchange material of the formula

Na_z[ (Al0₂)_z(Sι0₂)_y]xH₂0 wherein z and y are at least about 6, the molar ratio of z to y is from about 1.0 to about 0.5 and x is from about 10 to about 264. Amorphous hydrated alummosilicate materials useful herein have the empirical formula

M_z(zAl0₂ ^SiO;,) wherein M is sodium, potassium, ammonium or substituted ammonium, z is from about 0.5 to about 2 and y is 1, said material having a magnesium ion exchange capacity of at least about 50 milligram equivalents of CaC0₃ hardness per gram of anhydrous alummosilicate.

The alummosilicate ion exchange builder materials herein are m hydrated form and contain from about 10% to about 28% of water by weight if crystalline, and potentially even higher amounts of water if amorphous. Highly preferred crystalline alummosilicate ion exchange materials contain from about 18% to about 22% water m their crystal matrix. The crystalline alummosilicate ion exchange materials are further characterized by a particle size diameter of from about 0.1 micron to about 10 microns. Amorphous materials are often smaller, e.g., down to less than about 0.01 micron. Preferred ion exchange materials have a particle size diameter of from about 0.2 micron to about 4 microns. The term "particle size diameter" herein represents the average particle size diameter of a given ion exchange material as determined by conventional analytical techniques such as, for example, microscopic determination utilizing a scanning electron microscope. The crystalline alummosilicate ion exchange materials herein are usually further characterized by their calcium ion exchange capacity, which is at least about 200 mg equivalent of CaC0₃ water hardness/g of alummosilicate, calculated on an anhydrous basis, and which generally is the range of from about 300 mg . eq./g. to about 352 mg . eq./g. The alummosilicate ion exchange materials herein are still further characterized by their calcium ion exchange rate which is at least about 2 grains Ca⁺7gallon/mιnute/gram/gallon of alummosilicate (anhydrous basis) , and generally lies within the range of from about 2 grams/gallon/mmute/gram/gallon to about 6 grams/gallon/mmute/gram/gallon, based on calcium ion hardness. Optimum alummosilicate for builder purposes exhibit a calcium ion exchange rate of at least about 4 grams/gallon/mmute/gram/gallon.

The amorphous alummosilicate ion exchange materials usually have a Mg⁺⁺ exchange capacity of at least about 50 mg . eq. CaC0₃/g. (12 mg . Mg⁺⁺/g.) and a Mg⁺⁺ exchange rate of at least about 1 gram/gallon/mmute/gram/gallon. Amorphous materials do not exhibit an observable diffraction pattern when examined by Cu radiation (1.54 Angstrom Units) .

Alummosilicate ion exchange materials useful m the practice of this invention are commercially available. The alummosilicates useful m this invention can be crystalline or amorphous structure and can be naturally-occurring alummosilicates or synthetically derived. A method for producing alummosilicate ion exchange materials is discussed m U.S. Patent No. 3,985,669, Krummel, et al . , issued October 12, 1976, incorporated herein by reference. Preferred synthetic crystalline alummosilicate ion exchange materials useful herein are available under the designations Zeolite A, Zeolite B, and Zeolite X. In an especially preferred embodiment, the crystalline alummosilicate ion exchange material m Zeolite A and has the formula

Na₁₂ [A10₂) ₁₂. (Sι0₂) ₁₂] .χH₂0 wherein x is from about 20 to about 30, especially about 27. Water soluble, non-phosphorus organic builders useful herein include the various alkali metal, ammonium and substituted ammonium, carboxylates, non-polymeric polycarboxylates and polyhydroxysulfonates . Examples of non- polymeric polycarboxylate builders are the sodium, potassium, lithium, ammonium and substituted ammonium salts of ethylenediammetetraacetic acid, nitπlotriacetic acid, oxydisuccmic acid, mellitic acid, benzene polycarboxylic acids, and citric acid. The compositions of this invention only contain the limited amount of polyacrylate defined hereinafter.

Other useful builders herein are sodium and potassium carboxymethyloxymalonate, carboxymethyloxysucc ate, cis- cyclohexanehexacarboxylate , ciscyclopentanetetracarboxylate , and phloroglucmol tπsulfonate . Other suitable non-polymeric polycarboxylates are the polyacetal carboxylates described m U.S. Patent No. 4,144,226, issued March 13, 1979 to Crutchfield, et al . , and U.S. Patent No. 4,246,495, issued March 27, 1979 to Crutchfield, et al . , both incorporated herein by reference. These polyacetal carboxylates can be prepared by bringing together under polymerization conditions an ester of glyoxylic acid and a polymerization initiator. The resulting polyacetal carboxylate ester is then attached to chemically stable end groups to stabilize the polyacetal carboxylate against rapid depolymerization m alkaline solution, converted to the corresponding salt, and added to a surfactant . Other detergency builder materials useful herein are the "seeded builder" compositions disclosed m Belgian Patent No. 798,856, issued October 29, 1973, incorporated herein by reference. Specific examples of such seeded builder mixtures are: 3.1 wt . mixtures of sodium carbonate and calcium carbonate having 5 micron particle diameter; 2.7:1 wt . mixtures of sodium sesquicarbonate and calcium carbonate having a particle diameter of 0.5 microns; 20:1 wt . mixtures of sodium sesquicarbonate and calcium hydroxide having a particle diameter of 0.01 micron; and a 3:3:1 wt . mixture of sodium carbonate, sodium alummate and calcium oxide having a particle diameter of 5 microns.

Preferably the builder is selected from the group consisting of zeolites, especially Zeolite A; carbonates, especially sodium carbonate; and citrates, especially sodium citrate .

Soaps, as described hereinbefore, can also act as builders depending upon the pH of the wash solution, the insolubility of the calcium and/or magnesium soaps, and the presence of other builders and soap dispersants.

The compositions herein preferably contain as part of the non-phosphorous builder from about 0% to about 6%, preferably from about 0.5% to about 5%, and most preferably from about 1% to about 4% by weight of an alkali metal silicate having a molar ratio of Sι0₂ to alkali metal oxide of from about 1.0 to about 3.2, 2.4. Sodium silicate, particularly one having a molar ratio of from about 1.8 to about 2.2, is preferred.

The alkali metal silicates can be purchased m either liquid or granular form. Silicate slurries can conveniently be used to avoid having to dissolve the dried form m the aqueous slurry (e.g., crutcher mix) of the components herein. The amount of the enzyme derived from bacterial strain No. 177 useful m the granular detergent compositions of the present invention will be apparent to skilled detergent formulary petitioners of ordinary skill. However, some general guidelines are given.

The pure enzyme component is incorporated herein m an amount of from about 0.005% to about 0.2%, preferably from about 0.02% to about 0.09%. The preferred proteolytic enzyme component should give to the composition a proteolytic activity of at least about 0.003 Anson Units per liter, preferably from about 0.003 to about 1.125 Anson Units per liter of wash solution. Most preferably, from about 0.016 to about 0.063 Anson Units per liter of wash solution. Above about 0.1 Anson Units per liter of wash solution additional pure enzyme provides only minimal increase m performance . Other enzymes including amylolytic enzymes can also be included.

Certain minors can be added to the granular detergent compositions commonly found m laundry and detergent cleaning compositions of a dry nature. For example, such compositions can contain thickeners and cell-suspending agents such as carboxymethylcellulose and the like. It can contain, as well as the proteolytic enzyme, combinations with lipolitic enzymes. Also as minors, various perfumes, optical bleaches, fillers, anti-cakmg agents, fabric softeners and the like can be present m the compositions to provide the usual benefits occasioned by the use of such materials m granular detergent compositions. It can be recognized that all such adjuvant materials are useful herein inasmuch as they are compatible and stable m the presence of bacterial strain derived proteolytic enzyme described herein. Likewise, peroxy bleaching components can be commonly added. In certain instances, liquid detergents are desirable alternatives to dry granular detergents. They have, for example, a large degree of consumer acceptance, since they can be applied directly to stains and dirty spots on fabrics without being pre-dissolved m water or other fluid media. Further, they have the advantage that a stream of liquid detergent can be more easily directed to a targeted location m the wash water or clothing than a dry granular product . The enzymes of the present invention can be used usefully m liquid detergent compositions.

In the present invention, a liquid, aqueous detergent is specially formulated to contain nonionic and anionic surfactants, enzymes and an enzyme stabilizer comprising relatively high amounts of calcium ion, but m which phase separation is prevented, for example, by the use of an alkyl ether carboxylate. Separation into various layers is disadvantageous to liquid detergents, since various cleaning actives will then be separated from one another, and complete cleaning may not result. Further, phase instability results an aesthetically unattractive product.

The nonionic surfactants present m the invention will preferably have a pour point of less than 40°C, more preferably less than 35°C, and most preferably below about 30°C. They will have an HLB (hydrophile-lipophile balance) of between 2 and 16, more preferably between 4 and 15, and most preferably between 10 and 14. However, mixtures of lower HLB surfactants with higher HLB surfactants can be present, the resulting HLB usually being an average of the two or more surfactants. Additionally, the pour points of the mixtures can be, but are not necessarily, weighted averages of the surfactants used.

The nonionic surfactants are preferably selected from the group consisting of C_{6 18} alcohols with 1-15 moles of ethylene oxide per mole of alcohol, C_{6 18} alcohols with 1-10 moles of propylene oxide per mole of alcohol, C_{6 18} alcohols with 1-15 moles of ethylene oxide and 1-10 moles of propylene oxide per mole of alcohol, C_{6 18} alkylphenols with 1-15 moles of ethylene oxide or propylene oxide or both, and mixtures of any of the foregoing. Certain suitable surfactants are available form Shell Chemical Company under the trademark Neodol . Suitable surfactants include Neodol 25-9 (C_{12 15} alcohol with an average 9 moles of ethylene oxide per mole of alcohol) . Another suitable surfactant may be Alfonic 1218- 70, which is a C_{12 18} alcohol, which is ethoxylated with about 10.7 moles of ethylene oxide per mole of alcohol, from Vista Chemical, Inc. These and other nonionic surfactants used m the invention can be either linear or branched, or primary or secondary alcohols. If surfactants used are partially unsaturated, they can vary from C₁₀₂₂ alkoxylated alcohols, with a minimum iodine value of at least 40, such as exemplified by Drozd et al . , U.S. Patent No. 4,668,423, incorporated herein by reference. An example of an ethoxylated, propoxylated alcohol is Surfonic JL-80X (C_{9 X1} alcohol with about 9 moles of ethylene oxide and 1.5 moles of propylene oxide per mole of alcohol) , available from Texaco Chemical Company.

Other suitable nonionic surfactants may include polyoxyethylene carboxylic acid esters, fatty acid glycerol esters, fatty acid and ethoxylated fatty acid alkanolamides , certain block copolymers of propylene oxide and ethylene oxide and block polymers of propylene oxide and ethylene oxide with a propoxylated ethylene diamme (or some other suitable initiator) . Still further, such semi-polar nonionic surfactants as amme oxides, phosphme oxides, sulfoxides and their ethoxylated derivatives, may be suitable for use herein. Nonionic surfactants are especially preferred for use m this invention since they are generally found m liquid form, usually contain 100% active content, and are particularly effective at removing oily soils, such as sebum and glycerides . The nonionic surfactant should be present m the liquid detergent at about 5-65%, more preferably 15-45%, and most preferably 25-40%, by weight of the composition. It is actually most preferred to have the surfactant system include about at least 50% nonionic surfactant. The ratio of the surfactants should be, preferably, about 10:1 to 1:1 nonionic to anionic surfactants, more preferably 4:1 to 1:1. The resulting liquid composition should preferably have a viscosity of about 1-5,000 centipoises (CPS), more preferably 5-3,000 CPS, and most preferably about 10-1,500 CPS. One of the anionic surfactants used herein may be an alkyl ether sulfate. The other two can be an alkyl ether carboxylate phase stabilizer and an unsaturated fatty acid. However, the latter two materials are utilized their roles as, respectively, phase stabilizers and foam controllers. The alkyl ether sulfates are also known as alcohol alkoxysulfate anionic surfactants. These types of surfactants have the following structure:

R—(—0—CH₂CH₂—) nS0₄M wherein R is a C_{10 16} alkyl, and n is an integer from about 1- 5, and M is H or an alkali metal cation (sodium, potassium or lithium) .

These alkyl ether sulfates are manufactured by condensing a fatty alcohol with ethylene oxide and sulfonat g the resulting product. This is then neutralized with an appropriate base. Normally, it is typical to calculate the amount of surfactant on a non-neutralized or acid basis. Some ethanol or other solvent may be present m the commercial surfactant as a carrier. In the present invention, it is preferred to have about 0-30% of the alkoxylated, sulfated fatty alcohol, more preferably 2-25%, and most preferably 5-20% thereof.

The alkyl ether carboxylate stabilizer (also known as an alcohol alkoxycarboxylate) is preferably a C_{8 18}, more preferably C_{10 16}, and most preferably C_{12 14} fatty alcohol, which has been ethoxylated with an average of about 1-20, more preferably 2-15, and most preferably 3-10 moles of ethylene oxide per mole of alcohol, and subsequently carboxylated. They are also known as carboxylated fatty alcohol ethoxylates . It is preferred that if a mixture of fatty alcohols is used, the higher molecular weight portions (i.e., C₁₄ and greater) are present m lesser amounts, although higher alkyl ether carboxylates may be utilized by having higher amounts of ethylene oxide to aid m dispersing the compound m aqueous solution. The use of the carboxylated, fatty alcohol ethoxylate phase stabilizer is preferred since, unlike other anionic surfactants, e.g., alkyl benzene sulfonate (LAS) , there are less deleterious effects on enzymes. More importantly, unlike regular fatty acid soaps or LAS< phase instability because of co- precipitation with the calcium salts is avoided.

Although m typical liquid and dry detergent applications, alkylpolysiloxanes, such as dimethylpolysiloxane, have been used as anti- foaming agents, such agents may not be optimal for use m the present invention since they provide little, if any, cleaning performance. It has been found that unsaturated fatty acids m relatively low amounts are effective as foam-controllmg agents. Additionally, these materials are relatively soluble and thus dispersed very well m the inventive liquid detergent. In the present application, it is preferred that less than 5% of this unsaturated C₆₂₀ fatty acid be present, more preferably less than 4%, and most preferably less than 3%. Even as little as 1% saturated fatty acid can cause a precipitate to form, and so they should be avoided. An especially preferred fatty acid is oleic acid. A lower alkanol, i.e., a C_{λ 4} alcohol, is used m the present invention to enhance the dispersibility of the composition and possibly to thin a relatively viscous formulation. Ethanol and propanol are preferred, with ethanol being most preferred. 0-25% of the alkanol is present, more preferably 1-20%, and most preferably 1-15%. A further solvent may also be substituted for the alkanol, or combined with the alkanol, and added to the present invention. These are selected from C_{2 6} glycols and glycol ethers. Examples of such glycols include ethylene glycol and propylene glycol, and an exemplary glycol ether is 2 -butoxyethanol (also called butyl Cellosolve, available from Union Carbide) . If both solvents, i.e., alkanol and either glycol or glycol ether are present, it is preferred that they be in a ratio of about 10:1 to 1:10, more preferably about 3:1 to 1:3, and most preferably about 1:1. Propylene glycol is especially preferred because of the added phase stability it produces, as well as enhanced rinsability of the liquid detergent .

The enzyme should be present m liquid detergents an amount of about 0.01-5%, more preferably about 0.01-3%, and most preferably about 0.1-2% by weight of the detergent. Mixtures of enzymes are desirable, and can be used.

The present invention may require that an enzyme stabilizer be present to prevent substantial deactivation or denaturation of the enzymes m the aqueous phase of the liquid detergent. Thus, water soluble calcium salts which can provide calcium ions are suitable for use herein. Thus, any water soluble calcium salt able to provide available calcium ions m aqueous solution is suitable. Examples of such sources of calcium ions include, but are not limited to, calcium chloride, calcium acetate, calcium propionate and calcium formate. It is not exactly understood why calcium ions help to stabilize enzymes against deactivation.

However, unlike the prior art, surprisingly much higher amounts of calcium salt can be present and still maintain good phase stability. It is preferred that about 0.01-1%, more preferably 0.01-0.5%, and most preferably about 0.05- 0.5% calcium ion be present m the liquid detergent.

The present invention m liquid detergents is preferably near neutral. Thus, m contrast to most dry, granular detergents, the pH is somewhat more acidic. Thus, the pH of the invention varies from about 6-10, more preferably between 6-8, and most preferably, no more than about 8. In order to attain the pH, the pH can be adjusted by the use of various buffers. A large number of the materials added to these aqueous detergents are acidic nature, such as the alkyl ether sulfate, the alkyl ether carboxylate, and the unsaturated fatty acids. Additionally, discussed m 9 below, additional stabilizers are selected from short chain carboxylic acids. Therefore, buffers and pH-adjustmg agents such as sodium hydroxide and sodium bicarbonate can be used to modify the pH. In the event that more acidity is desired, hydrochloric acid, sulfuπc acid, and citric acid would be suitable for maintaining or adjusting to a more acidic pH.

Additionally, desirable phase stabilizers are water soluble, short chain carboxylic acids, and the salts thereof. These include acetic acid, formic acid and propionic acid, and their alkali metal and ammonium salts. Sodium chloride and other water soluble chlorides can also be used. It is preferred that these particular types of salts vary from about 1-15%, more preferably about 1-10%, and most preferably about 1-7.5% by weight of the composition. Sodium acetate is especially preferred for use here. When these short chain carboxylates are added, the minimum phase stabilizing amount of the fatty alcohol carboxylate is actually lowered. These salts differ from the calcium salts 7. (above) used as enzyme stabilizers.

The standard detergent adjuncts can be included m the present invention. These include dyes such as Monastral blue and anthraqumone dyes (such as those described m Zielske, U.S. Patent No. 4,661,293, and U.S. Patent No. 4,746,461).

Pigments, which are also suitable colorants, can be selected, without limitation, from titanium dioxide, ultramarine blue (see also, Chang, et al . , U.S. Patent No. 4,708,816), and colored alummosilicates. Fluorescent whitening agents are still other desirable adjuncts. These include the stilbene, styrene, and naphthalene derivatives which, upon being impinged by visible light, emit or fluoresce light at a different wavelength. These FWA's or bπghteners are useful for improving the appearance of fabrics which have become dingy through repeated soilings and washings. Preferred

FWA's are Tmopal CBS-X and Tmopal RBS, both from Ciba Geigy A.G., and Phorwhite BBH, from Mobay Chemicals. Examples of suitable FWA's can be found m U.S. Patent Nos. 1,298,577, 2,076,011, 2,026,054, 2,026,566, 1,393,042; and U.S. Patent Nos. 3,951,960, 4,298,290, 3,993,659, 3,980,713 and 3,627,758, incorporated herein by reference. Anti- redeposition agents such as carboxymethylcellulose are potentially desirable. Next, foam boosters, such as appropriate anionic surfactants, may be appropriate for inclusion herein. Also, m the case of excess foaming resulting from the use of certain nonionic surfactants, further anti-foammg agents, such as alkylated polysiloxanes , e.g., dimethylpolysiloxane, would be desirable. Next, bleach activators could well be very desirable for inclusion herein and a liquid oxidant, specifically hydrogen peroxide. Suitable examples of appropriate bleach activators may be found m Mitchell, et al . , U.S. patent No. 4,772,290. Mitchell may be especially appropriate since it describes stable activators m an aqueous liquid hydrogen peroxide composition, and it is incorporated herein by reference. In this detergent matrix it may also be desirable to stabilize the liquid hydrogen peroxide against decomposition. Thus, stabilizers therefor may be appropriate, such as those disclosed m Baker, et al . , U.S. Patent No. 4,764,302, and m Mitchell, et al . , published European Patent Application EP 209,228, both of which are incorporated herein by reference. Lastly, m case the composition is too thin, some thickeners such as gums (xanthan gum and guar gum) , and various resms (e.g., polyvmyl alcohol and polyvmyl pyrrolidone) , may be suitable for use. Fragrances are also desirable adjuncts m these compositions.

The additives may be present m amounts ranging from 0- 30%, more preferably 0-20%, and most preferably 0-10%. In certain cases, some of the individual adjuncts may overlap m other categories. For example, some buffers, such as silicates, may be also builders. However, builders are to be avoided m this invention, since even small amounts of either organic or inorganic builders will cause phase instability by reacting with one or more of the ingredients m the inventive liquid detergents. Also, some surface active esters may actually function to a limited extent as surfactants. However, the present invention contemplates each of the adjuncts as providing discrete performance benefits m their various categories.

High water liquid enzyme prewashes can also be used with the compositions of the present invention. For examples of the details of such high water liquid enzyme prewashes, see U.S. Patent No. 5,589,448 issued December 31, 1996, the disclosure of which, with respect to the liquid enzyme prewash composition, is incorporated herein. It can be suitably used with proteolytic enzyme derived from bacterial strain No. 177. An additional patent describing suitable high water liquid enzyme prewash compositions is U.S. Patent No. 5,789,364, again the disclosure of which is incorporated herein by reference. Additional industrial cleaners used m the baking industry and other food processing industries can also be used effectively with the enzymes of the present invention to effectively clean food-soiled surfaces m the food manufacturing and preparation areas of, for example, bakeries. Such compositions typically will contain a detergent composition, enzymes that degrade food compositions, surfactants, low alkaline builders, water conditioning agents, and optionally a variety of formulary adjuvants, depending on the form. For details of such proteolytic enzyme cleaners, see U.S. Patent No. 5,858,117 issued January 12, 1999, the disclosure of which is incorporated herein by reference. The enzymes of the present invention can be used m lieu of the enzymes m such type cleaners . It can be seen from the above that a description and examples of sta removing effectiveness of a highly useful proteolytic enzyme effective at low temperatures has been both discovered and successfully isolated from bacterial strain No. 177 and demonstrated useful. It has therefore been demonstrated to accomplish all of the objectives of the invention.

It goes without saying that certain modifications to the preferred description presented herein can be made. Such modifications are contemplated as being within the scope of the present invention, and therefore the examples of the preferred embodiment (s) are to be taken as illustrative, and not limiting.

Claims

What is claimed is:

1. A substantially pure stam removal effective and low temperature effective proteolytic enzyme derived from bacterium strain No. 177 or a mutant or variant thereof, or cloned genes thereof.

2. The substantially pure stam removal effective and low temperature effective enzyme of claim 1 derived from a bacterium which has the 16s rRNA (sequence I.D. No. 1) .

3. The substantially pure sta removal low temperature effective enzyme of claim 2 which has a terminal ammo acid sequence of L-T-P-N-D-T-R-F-S-E .

4. A proteolytic enzyme, said enzyme comprising: a N- termmal ammo acid sequence of L-T-P-N-D-T-R-F-S-E and/or conservatively modified variants thereof and which is effective as a stam remover at low temperature, (0°C-50°C)

5. A purified and isolated bacterium comprising: a rRNA sequence of SEQ ID No .1 and which produces the enzyme of 21

6. The bacterium of claim 5 wherein said bacterium is strain no . 177.