BACKGROUND OF THE INVENTION There are various types of calcium binding type enzymes such as proteases and amylases. These calcium binding type enzymes are usually purified from cell culture medium or other materials.

A typical purification process is, for example, to add ammonium sulfate or organic solvents to the unpurified, or"crude"solution, to precipitate the desired enzymes. The precipitate is then put into an ion-exchange column and a gel filtration column, or an affinity chromatography column to separate the desired enzyme from other undesirable materials that were present in the unpurified solution. However, although the combination of the ion-exchange column and the gel-filtration column is very popular, it does not produce a sufficient amount of the purified enzyme because the total amount of enzymes which can be applied to these columns is limited. Also, although affinity chromatography has some advantages, it is not a popular method for purifying enzymes in an industrial use since it is very expensive.

Therefore, there is a need for a calcium binding type enzyme purification process that easily separates the desired enzymes from the other materials present in the crude solution, that has a high recovery rate, and is relatively quick.

An enzyme purification process for calcium binding type enzyme has now been found that is relatively fast and uncomplicated and generally is less expensive than prior processes.

SUMMARY OF THE INVENTION The present invention relates to an enzyme purification process. More particularly, the present invention relates to an enzyme purification process for calcium binding type enzyme.

Specifically, in one aspect of the present invention there is provided a process for purifying a calcium binding type enzyme comprising: (a) preparing unpurified solution such as a crude solution or a cell free culture medium solution which contains calcium binding type enzyme; (b) concentrating the solution; (c) diluting the solution by adding calcium free buffer; (d) adjusting buffer concentration and pH; (e) incubating the concentrated solution to precipitate the enzyme; and (f) collecting said precipitate. Further, the step (b) and (c) is repeated at least once.

Preferably, the calcium free buffer is selected from the group consisting of sodium phosphate buffer, sodium borate buffer, sodium acetate buffer, sodium carbonate buffer, potassium phosphate buffer, potassium borate buffer, potassium acetate buffer, potassium carbonate buffer, tris buffer, MES (2-N- Morphilino ethanesulfonic acid) buffer, MOBS (4-N-Morpholino butanesulfunic acid) buffer, HEPES (N-2-hydroxyethyl piperazine N-2-ethanesulfonic acid) buffer, BES (N, N-bis-2-hydroxyethyl 2-aminoethanesulfunic acid) buffer and mixtures thereof.

In another aspect of the present invention there is provided a detergent composition comprising (a) a detersive surfactant; (b) a calcium binding type enzyme wherein the calcium binding type enzyme is purified from the process of the present invention.

These and other features, aspects, and advantages of the present invention will become evident to those skilled in the art from a reading of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION While the specification conclues with claims particularly pointing out and distinctly claiming the invention, it is believed the present invention will be better understood from the following description.

All percentages are by weight of total composition unless specifically stated otherwise.

All ratios are weight ratios unless specifically stated otherwise.

Definitions As used herein,"comprising"means that other steps and other ingredients which do not affect the end result can be added. This term encompasses the terms"consisting of"and"consisting essentially of'.

All cited references are incorporated herein by reference in their entireties.

Citation of any reference is not an admission regarding any determination as to its availability as prior art to the claimed invention.

As used herein, the term"detergent composition"or"detergent"is intended to designate any of the agents conventionally used for removing soil, such as general household detergents or laundry detergents of the synthetic or soap type.

A calcium binding type enzyme purification process The present invention provides a calcium binding type enzyme purification process. As used herein,"A calcium binding type enzyme"is an enzyme which can bind calcium via a calcium-binding site in the enzyme molecule. These enzymes have at least 1 calcium binding site per molecule. Preferably, these enzymes have from about 2 to about 10 calcium binding sites per molecule.

The process of the present invention contains; (a) preparing a unpurified solution such as which contains calcium binding type enzyme, (b) concentrating, (c) diluting by adding calcium free buffer; (d) adjusting, (e) incubating the solution

to precipitate the protein; and (f) collecting said precipitate.

(a) Preparing step The first step of the present invention is preparing an unpurified solution which contains calcium binding type enzyme. An"unpurified solution"is any solution that contains at least one calcium binding type enzyme, such as cell free culture medium solutions, cell extracts, tissue extracts such as muscle homogenate, and Bacillus Subtilis free culture medium solutions. Commercially available enzyme solutions are, for example, Protease N TM (provided by Amano Seiyaku, Japan, Tokyo), Orientase 90N (provided by Hankyu Bioindustries Ltd.), and NC-25 (provided by Daiwa Kasei K. K). A preferable commercially available enzyme solution is Protease N. Commercially available products are preferably adjusted to have at least 1 mg/ml, preferably, from about 1 Omg/ml to 20mg/ml of total protein in a solution. Any calcium free diluted buffers in the pH range of from about 5.0 to about 9.0 can be used. The concentration of the buffer is to be from about 10 mM to about 25 mM, preferably.

(b) Concentrating step The second step of the present invention is concentrating the crude solution from step (a) The solution is concentrated to at least about 1/5, preferably about 1/10 of the volume. To concentrate the solution, Pellicon concentrator (Millipore) with Pellicon XL filter (Molecular Weight (MW) cut of 5000 (5K) or 10000 (10K)) is preferable concentrator.

(c) Diluting step The third step of the present invention is diluting the solution from step (b) by adding a calcium free buffer to remove calcium ions from the solution. Any kind of buffer can be used in step (c) as long as the buffer is calcium free.

Although not wanting to be limited by theory, the electrical charge of a calcium binding type enzyme depended on the number of calcium ion it binds. For example, BNAP, a metallo-protease from Bacillus Subtilis var.

Amylosacchariticus usually binds 3-4 calcium ions per molecule of enzyme. The iso-electric point of BNAP reported to be 4.5 with no calcium and 8.5 with 3-4 calcium binding (D. Tsuru, H. Kira, T. Yamamoto and J. Fukumoto, Agri. BioL Chem. Usually, the fermentation broths and the crude commercial samples contain lot of free calcium ions. If free calcium ions from the solution and partially from the protein molecule are removed and the enzyme is concentrated in a very diluted buffer (less than about 10 mM) of pH between 5.0- to 9.0, then, this calcium binding type enzyme selectively precipitates.

Thus, in this step, it is necessary to reduce the ionic strength of the solution specially calcium ions from the solution. In order to fulfill this purpose, step (b) and (c) may be repeated. Preferably step (b) and step (c) are repeated at least on time.

The buffers used in this step are preferably selected from the group consisting of sodium phosphate buffer, sodium borate buffer, sodium acetate buffer, sodium carbonate buffer, sodium citrate buffer, potassium phosphate buffer, potassium borate buffer, potassium acetate buffer, potassium carbonate buffer, potassium citrate buffer, tris buffer, MES (2-N-Morphilino ethanesulfonic acid) buffer, MOBS (4-N-Morpholino butanesulfunic acid) buffer, HEPES (N-2- hydroxyethyl piperazine N-2-ethanesulfonic acid) buffer, BES (N, N-bis-2- hydroxyethyl 2-aminoethanesulfunic acid) buffer and mixtures thereof, more preferably, selected from the group consisting of sodium phosphate, potassium phosphate, tris, sodium acetate, further preferably, sodium phosphate and potassium phosphate buffers. The concentration of the buffers for adding the solution above are preferably from about 5 mM to 50 mM, more preferably, from about 10 mM to about 25mM and the pH of buffers above is from about 5.0 to 9.0, preferably, from about 6.0 to about 8.0.

In Step (c), the concentration of the buffer is very important because if the concentration of the buffer is too low, the calcium binding type enzyme may partially precipitate during concentrating and these precipitates prevent from concentrating by a concentrator since the molecular-filter of the concentrator is blocked by the precipitates. Or if the concentration of the buffer is too high, a

large amount buffer/water is necessary to adjust to an appropriate concentration of buffer for final precipitation.

(d) Adjusting step The forth step of the present invention is to adjust a buffer concentration and pH. As explained above, step (b) and step (c) are preferably repeated at least once. After repeating step (b) and (c) enough, moving to step (b) and concentrate the solution according to step (b), described above. Then, the concentrated solution from step (b) is diluted from about 5 to about 10 times with a calcium free buffer or water, preferably the calcium free buffer is selected from the group consisting of sodium phosphate buffer, sodium borate buffer, sodium acetate buffer, sodium carbonate buffer, sodium citrate buffer, potassium phosphate buffer, potassium borate buffer, potassium acetate buffer, potassium carbonate buffer, potassium citrate buffer, tris buffer, MES (2-N-Morphilino ethanesulfonic acid) buffer, MOBS (4-N-Morpholino butanesulfunic acid) buffer, HEPES (N-2-hydroxyethyl piperazine N-2-ethanesulfonic acid) buffer, BES (N, N- bis-2-hydroxyethyl 2-aminoethanesulfunic acid) buffer and mixtures thereof and pH of the calcium free buffer is adjusted to from about 5.0 to about 9.0, preferably from about 6.0 to about 8.0 in order to reduce calcium ions.

Preferably, the concentration of the calcium ion is less than about 1000 micro- molar, more preferably, less than about 100 micro-molar. The concentration of the calcium free buffer is from about 0.1 mM to about 5 mM, preferably from about 2 mM to about 5 mM. This final dilution is to concentrate again to about 1/5 to about 1/25, preferably 1/10.

(e) Incubating step The fifth step of the present invention is incubating the solution to precipitate the enzyme in the concentrated solution from step (d). The target enzyme sometimes starts to precipitate in step (d), but by this step, precipitates are yielded sufficiently. The target enzyme concentration in this solution should be higher than 1 mg/ml, preferably higher than 5 mg/ml. Incubating hours can be

flexibly changed depending on conditions from about 6 hours to about 48 hours, but preferably, from about 12 hours to about 24 hours. Also the Incubating temperature is from about 1 °C to about 10°C, preferably from about 2°C to about 5°C, more preferably, around 4°C. In this step, desired enzyme is obtained as a precipitate, usually white precipitate. In order to obtain proper precipitate, pH of the concentrated solution, enzyme concentration, incubating time, temperature described above are very important. Without this range, yield and purity of the enzyme would be very low.

(f) Collecting step The sixth step of the present invention is collecting the precipitate of step (e) by centrifuging or filtration, preferably by centrifuging. The speed of centrifuge is widely changed from about 1000 rpm to about 12000 rpm, preferably, from about 3000 rpm to about 4000 rpm. These precipitates washed with the buffer in step (d) or with water, and after mixing, centrifuge the solution and obtain precipitate again. Washing process is repeated more than 1 times, preferably twice. After washing, the precipitate is freeze dried to prepare powder sample or dissolved in some solution with calcium ions to prepare liquid sample.

The recovery and purity test method of the calcium binding type enzyme obtained by the process The calcium binding type enzyme obtained by the process of the present invention shows high recovery and purity. These are characterized by HPLC (High Performance Liquid Chromatography) analysis. A cationic ion exchange column (SP 8HR, Waters) is used. 10 mM sodium acetate buffer containing 10 mM of CaCI2 of pH 6.0 is used as mobil phase and a linear gradient of 0.5 M NaCI in same buffer is used. The purity and the total percent recovery of the calcium binding type enzyme is calculated based on the HPLC peak area under HPLC chromatograms. Total enzyme concentation of this precipitates is measured by BCA method. The recovery of the target enzyme is as high as 90%

is obtained by this method. About 70% of the precipitates is enzyme whereas about 50% of the total enzyme is a calcium binding type enzyme.

A preferable example of calcium binding type enzymes is a protease or an amylase. More preferably, the calcium-binding type enzyme is a metallo- protease. Since the enzymes which is obtained by the process of the present invention has high purity, these enzymes could be used in many ways such as an ingredient of the detergent composition.

The present invention also relates to a detergent composition having a detersive surfactant and an enzyme, wherein the enzyme is obtained by the process of described above. Preferably, the detergent composition of the present invention has from about 0.0001 % to about 5 % of the enzyme, more preferably, from about 0.001% to about 0.2%. Also the detergent composition optionally contains a detergent builder, and other ingredients.

Detersive surfactant The detergent compositions of the present invention includes surfactants wherein the surfactant can be selected from the group consisting of nonionic and/or anionic and/or cationic and/or ampholytic and/or zwitterionic and/or semi- polar surfactants.

The surfactant is typically present at a level of from 0.01% to 60% by weight. More preferred levels of incorporation are 1% to 35% by weight, most preferably from 1 % to 30% by weight of detergent compositions in accord with the invention.

The surfactant is preferably formulated to be compatible with enzyme components present in the composition. In liquid or gel compositions the surfactant is most preferably formulated such that it promotes, or at least does not degrade, the stability of any enzyme in these compositions.

Preferred surfactants to be used according to the present invention comprise as a surfactant one or more of the nonionic and/or anionic surfactants described herein.

Polyethylene, polypropylene, and polybutylene oxide condensates of alkyi phenols are suitable for use as the nonionic surfactant of the present invention, with the polyethylene oxide condensates being preferred. These compounds include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight-chain or branched-chain configuration with the alkylene oxide. In a preferred embodiment, the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol.

Commercially available nonionic surfactants of this type include IgepalTM CO- 630, marketed by the GAF Corporation; and TritonTM X-45, X-114, X-100 and X- 102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (e. g., alkyl phenol ethoxylates).

The condensation products of primary and secondary aliphatic alcools with from about 1 to about 25 moles of ethylene oxide are suitable for use as the nonionic surfactant of the nonionic surfactant s of the present invention. The alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms.

Preferred are the condensation products of alcohol having an alkyl group containing from about 8 to about 20 carbon atoms, more preferably from about 10 to about 18 carbon atoms, with from about 2 to about 10 moles of ethylene oxide per mole of alcool. About 2 to about 7 moles of ethylene oxide and most preferably from 2 to 5 moles of ethylene oxide per mole of alcohol are present in said condensation products. Examples of commercially available nonionic surfactants of this type include TergitolTM 15-S-9 (the condensation product of C11-C1s linear alcohol with 9 moles ethylene oxide), TergitolTM 24-L-6 NMW (the condensation product of C12-C14 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of C14-C1s linear alcohol with 9 moles of ethylene oxide), NeodolTM 23-3 (the condensation

product of C12-C13 linear alcohol with 3.0 moles of ethylene oxide), NeodolTM 45-7 (the condensation product of C14-C1s linear alcohol with 7 moles of ethylene oxide), NeodolTM 45-5 (the condensation product of C14-C1s linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C13-C1s alcohol with 9 moles ethylene oxide), marketed by The Procter & Gamble Company, and Genapol LA 030 or 050 (the condensation product of C12-C14 alcohol with 3 or 5 moles of ethylene oxide) marketed by Hoechst. Preferred range of HLB in these products is from 8-11 and most preferred from 8-10.

Also useful nonionic surfactants of the present invention are the alkylpolysaccharides disclosed in U. S. Patent 4,565,647, Llenado, issued January 21,1986, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e. g. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units. Any reducing saccharide containing 5 or 6 carbon atoms can be used, e. g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside). The intersaccharide bonds can be, e. g., between the one position of the additional saccharide units and the 2-, 3-, 4-, and/or 6-positions on the preceding saccharide units.

The preferred alkylpolyglycosides have the formula R20 n H2nO) t (glYcosyl) x wherein R2 is selected from the group consisting of alkyl, alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon

atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7. The glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the 1-position). The additional glycosyl units can then be attached between their 1-position and the preceding glycosyl units 2-, 3-, 4-and/or 6- position, preferably predominately the 2-position.

The condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable for use as the additional nonionic detersive surfactant of the present invention. The hydrophobic portion of these compounds will preferably have a molecular weight of from about 1500 to about 1800 and will exhibit water insolubility. The addition of polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide.

Examples of compounds of this type include certain of the commercially-available PlurafacTM LF404 and PluronicTM surfactants, marketed by BASF.

Also suitable for use as the nonionic surfactant of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamine. The hydrophobic moiety of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000. This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000. Examples of this type of nonionic surfactant include certain of the commercially available TetronicTM compounds, marketed by BASF.

Preferred for use as the nonionic surfactant of the present invention are polyethylene oxide condensates of alkyl phenols, condensation products of primary and secondary aliphatic alcools with from about 1 to about 25 moles of ethylene oxide, alkylpolysaccharides, and mixtures thereof. Most preferred are Cg-C14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and Cl-ci alcohol ethoxylates (preferably C10 avg.) having from 2 to 10 ethoxy groups, and mixtures thereof.

Highly preferred nonionic surfactants are polyhydroxy fatty acid amide surfactants of the formula. wherein R1 is H, or R1 is C- hydrocarby), 2-hydroxy ethyl, 2-hydroxy propyl or a mixture thereof, R2 is Cs 31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is a straight C11 15 alkyl or C16-18 alkyl or alkenyl chain such as coconut alkyl or mixtures thereof, and Z is derived from a reducing sugar such as glucose, fructose, maltose, lactose, in a reductive amination reaction.

Suitable anionic surfactants to be used are linear alkyl benzene sulfonate, alkyl ester sulfate, branched alkyl sulfate, methyl-branched alkyl sulfate surfactants including linear esters of Cg-C20 carboxylic acids (i. e., fatty acids) which are sulfonated with gaseous S03 according to"The Journal of the American Oil Chemists Society", 52 (1975), pp. 323-329. Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc.

The preferred alkyl ester sulfonate surfactant, especially for laundry applications, comprise alkyl ester sulfonate surfactants of the structural formula: wherein R3 is a Cg-C20 hydrocarbyl, preferably an alkyl, or combination thereof, R4 is a Cl-C6 hydrocarbyl, preferably an alkyl, or combination thereof, and M is a cation which forms a water soluble salt with the alkyl ester sulfonate. Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethanolamine, and triethanolamine. Preferably, R3 is C10-C16 alkyl, and R4 is methyl, ethyl or isopropyl. Especially preferred are the methyl ester sulfonates wherein R3 is C10-C16 alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO3M wherein R preferably is a C10-C24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl component, more preferably a C12-C1g alkyl or hydroxyalkyl, and M is H or a cation, e. g., an alkali metal cation (e. g. sodium, potassium, lithium), or ammonium or substituted ammonium (e. g. methyl-, dimethyl-, and trimethyl ammonium cations and quaternary ammonium cations such as tetramethyl- ammonium and dimethyl piperdinium cations and quaternary ammonium cations derived from alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like). Typically, alkyl chains of C12-C16 are preferred for lower wash temperatures (e. g. below about 50°C) and C16 18 alkyl chains are preferred for higher wash temperatures (e. g. above about 50°C).

Other anionic surfactants useful for detersive purposes can also be included in the detergent compositions of the present invention. These can

include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono-, di-and triethanolamine salts) of soap, Cg-C22 primary of secondary alkanesulfonates, Cg-C24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e. g., as described in British patent specification No. 1,082,179, Cg-C24 alkylpolyglycolethersulfates (containing up to 10 moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as the acyl isethionates, N-acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated C12-C1g monoesters) and diesters of sulfosuccinates (especially saturated and unsaturated C6-C12 diesters), acyl sarcosinates, sulfates of alkylpolysaccharides such as the sulfates of alkylpolyglucoside (the nonionic nonsulfated compounds being described below), branched primary alkyl sulfates, and alkyl polyethoxy carboxylates such as those of the formula RO (CH2CH20) k-CH2COO-M+ wherein R is a Cg-C22 alkyl, k is an integer from 1 to 10, and M is a soluble salt-forming cation. Resin acids and hydrogenated resin acids are also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated resin acids present in or derived from tall oil.

Further examples are described in"Surface Active Agents and Detergents" (Vol. I and 11 by Schwartz, Perry and Berch). A variety of such surfactants are also generally disclosed in U. S. Patent 3,929,678, issued December 30,1975 to Laughlin, et al. at Column 23, line 58 through Column 29, line 23 (herein incorporated by reference).

When included therein, the detergent compositions of the present invention typically comprise from about 1% to about 40%, preferably from about 3% to about 20% by weight of such anionic surfactants.

Highly preferred anionic surfactants include alkyl alkoxylated sulfate surfactants hereof are water soluble salts or acids of the formula RO (A) mSO3M wherein R is an unsubstituted C10-C24 alkyl or hydroxyalkyl group having a Calo-

C24 alkyl component, preferably a C12-C20 alkyl or hydroxyalkyl, more preferably C12-C1g alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e. g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein. Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl-ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like. Exemplary surfactants are C12-C1g alkyl polyethoxylate (1.0) sulfate (C12-C1gE (1.0) M), C12-C1g alkyl polyethoxylate (2.25) sulfate (C12-C1gE (2.25) M), C12-C1g alkyl polyethoxylate (3.0) sulfate (C12-C1gE (3.0) M), and C12-C1g alkyl polyethoxylate (4.0) sulfate (C12-C1gE (4.0) M), wherein M is conveniently selected from sodium and potassium.

The detergent compositions of the present invention may also contain cationic, ampholytic, zwitterionic, and semi-polar surfactants, as well as the nonionic and/or anionic surfactants other than those already described herein.

Cationic detersive surfactants suitable for use in the detergent compositions of the present invention are those having one long-chain hydrocarbyl group. Examples of such cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula: [R2 (OR3) y] [R4 (OR3) y] 2R5N+X- wherein R2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain, each R3 is selected from the group consisting of -CH2CH2-,-CH2CH (CH3)-,-CH2CH (CH20H)-,-CH2CH2CH2-, and mixtures

thereof; each R4 is selected from the group consisting of Cl-C4 alkyl, C-C4 hydroxyalkyl, benzyl ring structures formed by joining the two R4 groups,- CH2CHOH-CHOHCOR6CHOHCH20H wherein R6 is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not 0; R5 is the same as R4 or is an alkyl chain wherein the total number of carbon atoms of R2 plus R5 is not more than about 18; each y is from 0 to about 10 and the sum of the y values is from 0 to about 15; and X is any compatible anion.

Quaternary ammonium surfactant suitable for the present invention has the formula (I):

Formula I whereby R1 is a short chainlength alkyl (C6-C10) or alkylamidoalkyl of the formula(II):

Formula II y is 2-4, preferably 3. whereby R2 is H or a C1-C3 alkyl, whereby x is 0-4, preferably 0-2, most preferably 0, whereby R3, R4 and R5 are either the same or different and can be either a short chain alkyl (C1-C3) or alkoxylated alkyl of the formula III, whereby X-is a counterion, preferably a halide, e. g. chloride or methylsulfate.

Formula III R6 is Cl-C4 and z is 1 or 2.

Preferred quaternary ammonium surfactants are those as defined in formula I whereby Rl is Cg, Cio or mixtures thereof, x=o, R3, R4 = CH3 and R5 = CH2CH20H.

Highly preferred cationic surfactants are the water-soluble quaternary ammonium compounds useful in the present composition having the formula : R1R2R3R4N+X-(i) wherein Rl is Cl-ci alkyl, each of R2, R3 and R4 is independently Cl-C4 alkyl, Cl-C4 hydroxy alkyl, benzyl, and- (C2H40) xhi where x has a value from 2 to 5, and X is an anion. Not more than one of R2, R3 or R4 should be benzyl.

The preferred alkyl chain length for R1 is C12-C1s particularly where the alkyl group is a mixture of chain lengths derived from coconut or palm kernel fat or is derived synthetically by olefin build up or OXO alcools synthesis. Preferred groups for R2R3 and R4 are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions.

Examples of suitable quaternary ammonium compounds of formulae (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl dihydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide;

C12-15 dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide; choline esters (compounds of formula (i) wherein Rl is alkyl and R2R3R4 are methyl). di-alkyl imidazolines [compounds of formula (i)].

Other cationic surfactants useful herein are also described in U. S. Patent 4,228,044, Cambre, issued October 14,1980 and in European Patent Application EP 000,224.

Typical cationic fabric softening components include the water-insoluble quaternary-ammonium fabric softening actives or thei corresponding amine precursor, the most commonly used having been di-long alkyl chain ammonium chloride or methyl sulfate.

Preferred cationic softeners among these include the following: 1) ditallow dimethylammonium chloride (DTDMAC); 2) dihydrogenated tallow dimethylammonium chloride; 3) dihydrogenated tallow dimethylammonium methylsulfate; 4) distearyl dimethylammonium chloride; 5) dioleyl dimethylammonium chloride; 6) dipalmityl hydroxyethyl methylammonium chloride; 7) stearyl benzyl dimethylammonium chloride; 8) tallow trimethylammonium chloride; 9) hydrogenated tallow trimethylammonium chloride; 10) C12-14 alkyl hydroxyethyl dimethylammonium chloride; 11) C12 18 alkyl dihydroxyethyl methylammonium chloride;

12) di (stearoyloxyethyl) dimethylammonium chloride (DSOEDMAC); 13) di (tallow-oxy-ethyl) dimethylammonium chloride; 14) ditallow imidazolinium methylsulfate; 15) 1- (2-tallowylamidoethyl)-2-tallowyl imidazolinium methylsulfate.

Biodegradable quaternary ammonium compounds have been presented as alternatives to the traditionally used di-long alkyl chain ammonium chlorides and methyl sulfates. Such quaternary ammonium compounds contain long chain alk (en) yl groups interrupted by functional groups such as carboxy groups. Said materials and fabric softening compositions containing them are disclosed in numerous publications such as EP-A-0,040,562, and EP-A-0,239,910.

The quaternary ammonium compounds and amine precursors herein have the formula (I) or (II), below:

wherein Q is selected from-O-C (O)-,-C (O)-O-,-O-C (O)-O-,-NR4-C (O)-,-C (O)- NR4-; Rl is (CH2) n-Q-T2 or T3; R2 is (CH2) m-Q-T4 or T5 or R3; R3 is Cl-C4 alkyl or Cl-C4 hydroxyalkyl or H; R4 is H or C1-C4 alkyl or C1-C4 hydroxyalkyl; T1, T2, T3, T4, T5 are independently C-C22 alkyl or alkenyl;

n and m are integers from 1 to 4; and X-is a softener-compatible anion. Non-limiting examples of softener-compatible anions include chloride or methyl sulfate.

The alkyl, or alkenyl, chain T1, T2, T3, T4, T5 must contain at least 11 carbon atoms, preferably at least 16 carbon atoms. The chain may be straight or branched. Tallow is a convenient and inexpensive source of long chain alkyl and alkenyl material. The compounds wherein T1, T2, T3, T4, T5 represents the mixture of long chain materials typical for tallow are particularly preferred.

Specific examples of quaternary ammonium compounds suitable for use in the aqueous fabric softening compositions herein include: 1) N, N-di (tallowyl-oxy-ethyl)-N, N-dimethyl ammonium chloride; 2) N, N-di (tallowyl-oxy-ethyl)-N-methyl, N- (2-hydroxyethyl) ammonium methyl sulfate; 3) N, N-di (2-tallowyl-oxy-2-oxo-ethyl)-N, N-dimethyl ammonium chloride; 4) N, N-di (2-tallowyl-oxy-ethylcarbonyl-oxy-ethyl)-N, N-dimethyl ammonium chloride; 5) N- (2-tallowyl-oxy-2-ethyl)-N- (2-tallowyl-oxy-2-oxo-ethyl)-N, N-dimethyl ammonium chloride; 6) N, N, N-tri (tallowyl-oxy-ethyl)-N-methyl ammonium chloride; 7) N- (2-tallowyl-oxy-2-oxo-ethyl)-N- (tallowyl-N, N-dimethyl-ammonium chloride; and 8) 1,2-ditallowyl-oxy-3-trimethylammoniopropane chloride; and mixtures of any of the above materials.

When included therein, the detergent compositions of the present invention typically comprise from 0.2% to about 25%, preferably from about 1% to about 8% by weight of such cationic surfactants.

Ampholytic surfactants are also suitable for use in the detergent compositions of the present invention. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic

derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight-or branched-chain. One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e. g. carboxy, sulfonate, sulfate. See U. S. Patent No. 3,929,678 to Laughlin et al., issued December 30,1975 at column 19, lines 18-35, for examples of ampholytic surfactants.

When included therein, the detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in detergent compositions.

These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See U. S. Patent No. 3,929,678 to Laughlin et al., issued December 30,1975 at column 19, line 38 through column 22, line 48, for examples of zwitterionic surfactants.

When included therein, the detergent compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such zwitterionic surfactants.

Semi-polar nonionic surfactants are a special category of nonionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; water-soluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms.

detergentsurfactantsincludetheSemi-polarnonionic amine oxide surfactants having the formula wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures therof containing from about 8 to about 22 carbon atoms; R4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof; x is from 0 to about 3; and each R5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups. The R5 groups can be attached to each other, e. g., through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C10-C1g alkyl dimethyl amine oxides and Cg-C12 alkoxy ethyl dihydroxy ethyl amine oxides.

When included therein, the cleaning compositions of the present invention typically comprise from 0.2% to about 15%, preferably from about 1% to about 10% by weight of such semi-polar nonionic surfactants.

The detergent composition of the present invention may further comprise a cosurfactant selected from the group of primary or tertiary amines.

Suitable primary amines for use herein include amines according to the formula R1NH2 wherein Rl is a C6-C12 preferably C6-C10 alkyl chain or R4X (CH2) n, X is-O-,-C (O) NH- or-NH-, R4 is a C6-C12 alkyl chain n is between 1 to 5, preferably 3. Ri alkyl chains may be straight or branched and may be interrupted with up to 12, preferably less than 5 ethylene oxide moieties.

Preferred amines according to the formula herein above are n-alkyl amines.

Suitable amines for use herein may be selected from 1-hexylamine, 1- octylamine, 1-decylamine and laurylamine. Other preferred primary amines include C8-C10 oxypropylamine, octyloxypropylamine, 2-ethylhexyl- oxypropylamine, lauryl amido propylamine and amido propylamine.

Suitable tertiary amines for use herein include tertiary amines having the formula Ri R2R3N wherein R1 and R2 are Cl-C8 alkylchains or R3 is either a C6-C12, preferably C6-C10 alkyl chain, or R3 is R4X (CH2) n, whereby X is-O-,-C (O) NH- or-NH-, R4 is a C4-C12 n is between 1 to 5, preferably 2-3. R5 is H or Cl-C2 alkyl and x is between 1 to 6.

R3 and R4 may be linear or branched; R3 alkyl chains may be interrupted with up to 12, preferably less than 5, ethylene oxide moieties.

Preferred tertiary amines are R1R2R3N where R1 is a C6-C12 alkyl chain, R2 and R3 are C1-C3 alkyl or where R5 is H or CH3 and x = 1-2.

Also preferred are the amidoamines of the formula: wherein R1 is C6-C12 alkyl; n is 2-4, preferably n is 3; R2 and R3 is Cl-C4 Most preferred amines of the present invention include 1-octylamine, 1- hexylamine, 1-decylamine, 1-dodecylamine, C8-10oxypropylamine, N coco 1- 3diaminopropane, coconutalkyldimethylamine, lauryldimethylamine, lauryl bis (hydroxyethyl) amine, coco bis (hydroxyehtyl) amine, lauryl amine 2 moles propoxylated, octyl amine 2 moles propoxylated, lauryl amidopropyidimethylamine, C8-10 amidopropyldimethylamine and C10 amidopropyldimethylamine.

The most preferred amines for use in the compositions herein are 1-hexylamine, 1-octylamine, 1-decylamine, 1-dodecylamine. Especially desirable are n- dodecyldimethylamine and bishydroxyethylcoconutalkylamine and oleylamine 7 times ethoxylated, lauryl amido propylamine and cocoamido propylamine.

ADDITIONAL INGREDIENTS Builder The compositions according to the present invention may further comprise a builder. Any conventional builder is suitable for use herein including aluminosilicate materials, silicates, polycarboxylates, alkyl-or alkenyl-succinic acid and fatty acids, materials such as ethylenediamine tetraacetate, diethylene triamine pentamethyleneacetate, metal ion sequestrants such as aminopolyphosphonates, particularly ethylenediamine tetramethylene phosphonic acid and diethylene triamine pentamethylenephosphonic acid.

Phosphate builders can also be used herein.

Suitable builders can be an inorganic ion exchange material, commonly an inorganic hydrated aluminosilicate material, more particularly a hydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic builder material is layered silicate, e. g. SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting of sodium silicate (Na2Si205).

Suitable polycarboxylates containing one carboxy group include lactic acid, glycolic acid and ether derivatives thereof as disclosed in Belgian Patent Nos. 831,368,821,369 and 821,370. Polycarboxylates containing two carboxy groups include the water-soluble salts of succinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid, diglycollic acid, tartaric acid, tartronic acid and fumaric acid, as well as the ether carboxylates described in German Offenlegenschrift 2,446,686, and 2,446,687 and U. S. Patent No. 3,935,257 and the sulfinyl carboxylates described in Belgian Patent No. 840,623.

Polycarboxylates containing three carboxy groups include, in particular, water-

soluble citrates, aconitrates and citraconates as well as succinate derivatives such as the carboxymethyloxysuccinates described in British Patent No.

1,379,241, lactoxysuccinates described in Netherlands Application 7205873, and the oxypolycarboxylate materials such as 2-oxa-1,1,3-propane tricarboxylates described in British Patent No. 1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinates disclosed in British Patent No. 1,261,829,1,1,2,2-ethane tetracarboxylates, 1,1,3,3-propane tetracarboxylates and 1,1,2,3-propane tetracarboxylates.

Polycarboxylates containing sulfo substituents include the sulfosuccinate derivatives disclosed in British Patent Nos. 1,398,421 and 1,398,422 and in U. S.

Patent No. 3,936,448, and the sulfonated pyrolyse citrates described in British Patent No. 1,082,179, while polycarboxylates containing phosphone substituents are disclosed in British Patent No. 1,439,000.

Alicyclic and heterocyclic polycarboxylates include cyclopentane- cis, cis, cis-tetracarboxylates, cyclopentadienide pentacarboxylates, 2,3,4,5- tetrahydro-furan-cis, cis, cis-tetracarboxylates, 2,5-tetrahydro-furan-cis- dicarboxylates, 2,2,5,5-tetrahydrofuran-tetracarboxylates, 1,2,3,4,5,6-hexane- hexacar-boxylates and and carboxymethyl derivatives of polyhydric alcools such as sorbitol, mannitol and xylitol. Aromatic poly-carboxylates include mellitic acid, pyromellitic acid and the phthalic acid derivatives disclosed in British Patent No. 1,425,343.

Of the above, the preferred polycarboxylates are hydroxycarboxylates containing up to three carboxy groups per molecule, more particularly citrates.

Preferred builder s for use in the present compositions include a mixture of a water-insoluble aluminosilicate builder such as zeolite A or of a layered silicate (SKS-6), and a water-soluble carboxylate chelating agent such as citric acid.

Other preferred builders include a mixture of a water-insoluble aluminosilicate builder such as zeolite A, and a watersoluble carboxylate chelating agent such as citric acid. Preferred builders for use in liquid detergent compositions of the present invention are soaps and polycarboxylates.

Other builder materials that can form part of the builder for use in granular compositions include inorganic materials such as alkali metal carbonates, bicarbonates, silicates, and organic materials such as the organic phosphonates, amino polyalkylene phosphonates and amino polycarboxylates.

Other suitable water-soluble organic salts are 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.- Polymers of this type are disclosed in GB-A-1,596,756. Examples of such salts are polyacrylates of MW 2000-5000 and their copolymers with maleic anhydride, such copolymers having a molecular weight of from 20,000 to 70,000, especially about 40,000.

Detergency builder salts are normally included in amounts of from 1% to 80% by weight of the composition preferably from 10% to 70% and most usually from 30% to 60% by weight. detergent enzymes The detergent compositions can further comprise one or more enzymes which provide cleaning performance, fabric care and/or sanitisation benefits.

Said enzymes include enzymes selected from cellulases, hemicellulases, peroxidases, serine-proteases, gluco-amylases, amylases, xylanases, lipases, phospholipases, esterases, cutinases, pectinases, keratanases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, ß-glucanases, arabinosidases, hyaluronidase, chondroitinase, laccase or mixtures thereof.

A preferred combination is a detergent composition having cocktail of conventional applicable enzymes like serine-protease, amylase, lipase, cutinase and/or cellulase in conjunction with one or more plant cell wall degrading enzymes.

The cellulases usable in the present invention include both bacterial or fungal cellulases. Preferably, they will have a pH optimum of between 5 and 12 and an activity above 50 CEVU (Cellulose Viscosity Unit). Suitable cellulases are

disclosed in U. S. Patent 4,435,307, Barbesgoard et al, J61078384 and W096/02653 which discloses fungal cellulase produced respectively from Humicola insolens, Trichoderma, Thielavia and Sporotrichum. EP 739 982 describes cellulases isolated from novel Bacillus species. Suitable cellulases are also disclosed in GB-A-2.075.028; GB-A-2.095.275; DE-OS-2.247.832 and W095/26398.

Examples of such cellulases are cellulases produced by a strain of Humicola insolens (Humicola grisea var. thermoidea), particularly the Humicola strain DSM 1800.

Other suitable cellulases are cellulases originated from Humicola insolens having a molecular weight of about 50KDa, an isoelectric point of 5.5 and containing 415 amino acids; and a"43kD endoglucanase derived from Humicola insolens, DSM 1800, exhibiting cellulase activity; a preferred endoglucanase component has the amino acid sequence disclosed in PCT Patent Application No. WO 91/17243.

Also suitable cellulases are the EGIII cellulases from Trichoderma longibrachiatum described in W094/21801, Genencor, published September 29, 1994. Especially suitable cellulases are the cellulases having color care benefits.

Examples of such cellulases are cellulases described in European patent application No. 91202879.2, filed November 6,1991 (Novo). Carezyme and Celluzyme (Novo Nordisk A/S) are especially useful. See also W091/17244 and W091/21801. Other suitable cellulases for fabric care and/or cleaning properties are described in W096/34092, W096/17994 and W095/24471.

Said cellulases are normally incorporated in the detergent composition at levels from 0.0001 % to 2% of pure enzyme by weight of the detergent composition.

Peroxidase enzymes are used in combination with oxygen sources, e. g. percarbonate, perborate, persulfate, hydrogen peroxide, etc and with a phenolic substrate as bleach enhancing molecule. They are used for"solution bleaching", i. e. to prevent transfer of dyes or pigments removed from substrates during wash operations to other substrates in the wash solution. Peroxidase enzymes are known in the art, and include, for example, horseradish peroxidase, ligninase and

haloperoxidase such as chloro-and bromo-peroxidase. Peroxidase-containing detergent compositions are disclosed, for example, in PCT International Application WO 89/099813, W089/09813 and in European Patent application EP No. 91202882.6, filed on November 6,1991 and EP No. 96870013.8, filed February 20,1996. Also suitable is the laccase enzyme.

Enhancers are generally comprised at a level of from 0.1% to 5% by weight of total composition. Preferred enhancers are substitued phenthiazine and phenoxasine 10-Phenothiazinepropionicacid (PPT), 10-ethylphenothiazine-4- carboxylic acid (EPC), 10-phenoxazinepropionic acid (POP) and 10- methylphenoxazine (described in WO 94/12621) and substitued syringates (C3- C5 substitued alkyl syringates) and phenols. Sodium percarbonate or perborate are preferred sources of hydrogen peroxide.

Said peroxidases are normally incorporated in the detergent composition at levels from 0.0001% to 2% of pure enzyme by weight of the detergent composition.

Other preferred enzymes that can be included in the detergent compositions of the present invention include lipases. Suitable lipase enzymes for detergent usage include those produced by microorganisms of the Pseudomonas group, such as Pseudomonas stutzeri ATCC 19.154, as disclosed in British Patent 1,372,034. Suitable lipases include those which show a positive immunological cross-reaction with the antibody of the lipase, produced by the microorganism Pseudomonas fluorescent IAM 1057. This lipase is available from Amano Pharmaceutical Co. Ltd., Nagoya, Japan, under the trade name Lipase P "Amano,"hereinafter referred to as"Amano-P". Other suitable commercial lipases include Amano-CES, lipases ex Chromobacter viscosum, e. g.

Chromobacter viscosum var. lipolyticum NRRLB 3673 from Toyo Jozo Co., Tagata, Japan; Chromobacter viscosum lipases from U. S. Biochemical Corp., U. S. A. and Disoynth Co., The Netherlands, and lipases ex Pseudomonas gladioli. Especially suitable lipases are lipases such as M1 LipaseR and LipomaxR (Gist-Brocades) and LipolaseR and Lipolase UltraR (Novo) which have found to be very effective when used in combination with the compositions of the

present invention. Also suitables are the lipolytic enzymes described in EP 258 068, WO 92/05249 and WO 95/22615 by Novo Nordisk and in WO 94/03578, WO 95/35381 and WO 96/00292 by Unilever.

Also suitable are cutinases [EC 3.1.1.50] which can be considered as a special kind of lipase, namely lipases which do not require interfacial activation. Addition of cutinases to detergent compositions have been described in e. g. WO-A- 88/09367 (Genencor); WO 90/09446 (Plant Genetic) and WO 94/14963 and WO 94/14964 (Unilever).

The lipases and/or cutinases are normally incorporated in the detergent composition at levels from 0.0001% to 2% of pure enzyme by weight of the detergent composition.

Suitable serine-proteases are the subtilisins which are obtained from particular strains of B. subtilis and B. licheniformis (subtilisin BPN and BPN').

One suitable serine-protease is obtained from a strain of Bacillus, having maximum activity throughout the pH range of 8-12, developed and sold as ESPERASE by Novo Industries A/S of Denmark, hereinafter"Novo". The preparation of this enzyme and analogous enzymes is described in GB 1,243,784 to Novo. Other suitable proteases include ALCALASE DURAZYM and SAVINASE from Novo and MAXATASE MAXACALO, PROPERASE (E and MAXAPEMO (protein engineered Maxacal) from Gist-Brocades. Proteolytic enzymes also encompass modified bacterial serine proteases, such as those described in European Patent Application Serial Number 87 303761.8, filed April 28,1987 (particularly pages 17,24 and 98), and which is called herein"Protease B", and in European Patent Application 199,404, Venegas, published October 29,1986, which refers to a modified bacterial serine protealytic enzyme which is called"Protease A"herein. Suitable is the protease called herein"Protease C", which is a variant of an alkaline serine protease from Bacillus in which lysine replaced arginine at position 27, tyrosine replaced valine at position 104, serine replaced asparagine at position 123, and alanine replaced threonine at position 274. Protease C is described in EP 90915958: 4, corresponding to WO 91/06637,

Published May 16,1991. Genetically modified variants, particularly of Protease C, are also included herein.

A preferred serine-protease referred to as"Protease D"is a carbonyl hydrolase variant having an amino acid sequence not found in nature, which is derived from a precursor carbonyl hydrolase by substituting a different amino acid for a plurality of amino acid residues at a position in said carbonyl hydrolase equivalent to position +76, preferably also in combination with one or more amino acid residue positions equivalent to those selected from the group consisting of +99, +101, +103, +104, +107, +123, +27, +105, +109, +126, +128, +135, +156, +166, +195, +197, +204, +206, +210, +216, +217, +218, +222, +260, +265, and/or +274 according to the numbering of Bacillus amyloliquefaciens subtilisin, as described in W095/10591 and in the patent application of C. Ghosh, et al, "Bleaching Compositions Comprising Protease Enzymes"having US Serial No.

08/322,677, filed October 13,1994. Also suitable is a carbonyl hydrolase variant of the protease described in W095/10591, having an amino acid sequence derived by replacement of a plurality of amino acid residues replaced in the precursor enzyme corresponding to position +210 in combination with one or more of the following residues: +33, +62, +67, +76, +100, +101, +103, +104, +107, +128, +129, +130, +132, +135, +156, +158, +164, +166, +167, +170, +209, +215, +217, +218, and +222, where the numbered position corresponds to naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other carbonyl hydrolases or subtilisins, such as Bacillus lentus subtilisin (co-pending patent application US Serial No. 60/048,550, filed June 04,1997).

Also suitable for the present invention are serine-protease described in patent applications EP 251 446 and WO 91/06637, protease BLAP described in W091/02792 and their variants described in WO 95/23221.

See also a high pH serine-protease from Bacillus sp. NCIMB 40338 described in WO 93/18140 A to Novo. Enzymatic detergents comprising serine-protease, one or more other enzymes, and a reversible protease inhibitor are described in WO 92/03529 A to Novo. When desired, a serine-protease having decreased

adsorption and increased hydrolysis is available as described in WO 95/07791 to Procter & Gamble. A recombinant trypsin-like protease for detergents suitable herein is described in WO 94/25583 to Novo. Other suitable serine-proteases are described in EP 516 200 by Unilever.

The proteolytic enzymes are incorporated in the detergent compositions of the present invention a level of from 0.0001% to 2%, preferably from 0.001% to 0.2%, more preferably from 0.005% to 0.1% pure enzyme by weight of the composition.

Amylases (a and/or ß) can be included for removal of carbohydrate-based stains. W094/02597, Novo Nordisk A/S published February 03,1994, describes detergent compositions which incorporate mutant amylases. See also W095/10603, Novo Nordisk A/S, published April 20,1995. Other amylases known for use in detergent compositions include both a-and p-amylases. a- Amylases are known in the art and include those disclosed in US Pat. no.

5,003,257; EP 252,666; WO/91/00353; FR 2,676,456; EP 285,123; EP 525,610; EP 368,341; and British Patent specification no. 1,296,839 (Novo). Other suitable amylases are stability-enhanced amylases described in W094/18314, published August 18,1994 and W096/05295, Genencor, published February 22,1996 and amylase variants having additional modification in the immediate parent available from Novo Nordisk A/S, disclosed in WO 95/10603, published April 95. Also suitable are amylases described in EP 277 216, W095/26397 and W096/23873 (all by Novo Nordisk).

Examples of commercial a-amylases products are Purafect Ox Am from Genencor and Termamyl, Ban@ Fungamyl and Duramyl), all available from Novo Nordisk A/S Denmark. W095/26397 describes other suitable amylases: a- amylases characterised by having a specific activity at least 25% higher than the specific activity of Termamyl@ at a temperature range of 25°C to 55°C and at a pH value in the range of 8 to 10, measured by the Phadebas a-amylase activity assay. Suitable are variants of the above enzymes, described in W096/23873 (Novo Nordisk). Other amylolytic enzymes with improved properties with respect

to the activity level and the combination of thermostability and a higher activity level are described in W095/35382.

The amylolytic enzymes are incorporated in the detergent compositions of the present invention a level of from 0.0001% to 2%, preferably from 0.00018% to 0.06%, more preferably from 0.00024% to 0.048% pure enzyme by weight of the composition.

The above-mentioned enzymes may be of any suitable origin, such as vegetable, animal, bacterial, fungal and yeast origin. Origin can further be mesophilic or extremophilic (psychrophilic, psychrotrophic, thermophilic, barophilic, alkalophilic, acidophilic, halophilic, etc.). Purified or non-purified forms of these enzymes may be used. Nowadays, it is common practice to modify wild- type enzymes via protein/genetic engineering techniques in order to optimise their performance efficiency in the detergent compositions of the invention. For example, the variants may be designed such that the compatibility of the enzyme to commonly encountered ingredients of such compositions is increased.

Alternatively, the variant may be designed such that the optimal pH, bleach or chelant stability, catalytic activity and the like, of the enzyme variant is tailored to suit the particular cleaning application.

In particular, attention should be focused on amino acids sensitive to oxidation in the case of bleach stability and on surface charges for the surfactant compatibility. The isoelectric point of such enzymes may be modified by the substitution of some charged amino acids, e. g. an increase in isoelectric point may help to improve compatibility with anionic surfactants. The stability of the enzymes may be further enhanced by the creation of e. g. additional salt bridges and enforcing calcium binding sites to increase chelant stability. Special attention must be paid to the cellulases as most of the cellulases have separate binding domains (CBD). Properties of such enzymes can be altered by modifications in these domains.

Said enzymes are normally incorporated in the detergent composition at levels from 0.0001 % to 2% of pure enzyme by weight of the detergent composition. The enzymes can be added as separate single ingredients (prills,

granulates, stabilized liquids, etc... containing one enzyme) or as mixtures of two or more enzymes (e. g. cogranulates).

Other suitable detergent ingredients that can be added are enzyme oxidation scavengers which are described in Copending European Patent application 92870018.6 filed on January 31,1992. Examples of such enzyme oxidation scavengers are ethoxylated tetraethylene polyamines.

A range of enzyme materials and means for their incorporation into synthetic detergent compositions is also disclosed in WO 9307263 A and WO 9307260 A to Genencor International, WO 8908694 A to Novo, and U. S.

3,553,139, January 5,1971 to McCarty et al. Enzymes are further disclosed in U. S. 4,101,457, Place et al, July 18,1978, and in U. S. 4,507,219, Hughes, March 26,1985. Enzyme materials useful for liquid detergent formulations, and their incorporation into such formulations, are disclosed in U. S. 4,261,868, Hora et al, April 14,1981. Enzymes for use in detergents can be stabilised by various techniques. Enzyme stabilisation techniques are disclosed and exemplified in U. S. 3,600,319, August 17,1971, Gedge et al, EP 199,405 and EP 200,586, October 29,1986, Venegas. Enzyme stabilisation s are also described, for example, in U. S. 3,519,570. A useful Bacillus, sp. AC13 giving proteases, xylanases and cellulases, is described in WO 9401532 A to Novo.

Color care and fabric care benefits Technologies which provide a type of color care benefit can also be included. Examples of these technologies are metallo catalysts for color maintenance. Such metallo catalysts are described in copending European Patent Application No. 92870181.2. Dye fixing agents, polyolefin dispersion for anti-wrinkles and improved water absorbancy, perfume and amino-functional polymer for color care treatment and perfume substantivity are further examples of color care/fabric care technologies and are described in the co-pending Patent Application No. 96870140.9, filed November 07,1996.

Fabric softening agents can also be incorporated into detergent compositions in accordance with the present invention. These agents may be

inorganic or organic in type. Inorganic softening agents are exemplified by the smectite clays disclosed in GB-A-1 400 898 and in USP 5,019,292. Organic fabric softening agents include the water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP-BO 011 340 and their combination with mono C12-C14 quaternary ammonium salts are disclosed in EP-B-0 026 527 and EP-B-0 026 528 and di-long-chain amides as disclosed in EP-B-0 242 919. Other useful organic ingredients of fabric softening s include high molecular weight polyethylene oxide materials as disclosed in EP-A-0 299 575 and 0 313 146.

Levels of smectite clay are normally in the range from 2% to 20%, more preferably from 5% to 15% by weight, with the material being added as a dry mixed component to the remainder of the formulation. Organic fabric softening agents such as the water-insoluble tertiary amines or dilong chain amide materials are incorporated at levels of from 0.5% to 5% by weight, normally from 1 % to 3% by weight whilst the high molecular weight polyethylene oxide materials and the water soluble cationic materials are added at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by weight. These materials are normally added to the spray dried portion of the composition, although in some instances it may be more convenient to add them as a dry mixed particulate, or spray them as molten liquid on to other solid components of the composition.

Bleaching agent Additional optional detergent ingredients that can be included in the detergent compositions of the present invention include bleaching agents.

Preferred peroxygen bleaching agents include those peroxygen bleaching compounds which are capable of yielding hydrogen peroxide in an aqueous solution. These compounds are well known in the art and include hydrogen peroxide and the alkali metal peroxides, organic peroxide bleaching compounds such as urea peroxide, and inorganic persalt bleaching compounds, such as the alkali metal perborates, percarbonates, perphosphates, and the like.

Preferred peroxygen bleaching agents include peroxygen bleach selected from the group consisting of perborates, percarbonates, peroxyhydrates,

peroxides, persulfates, and mixtures thereof. Specific preferred examples include: sodium perborate, commercially available in the form of mono-and tetra-hydrates, sodium carbonate peroxyhydrate, sodium pyrophosphate peroxyhydrate, urea peroxyhydrate, and sodium peroxide. Particular preferred are sodium perborate tetrahydrate, and especially, sodium perborate monohydrate. Sodium perborate monohydrate is especially preferred because it is very stable during storage and yet still dissolves very quickly in the bleaching solution.

These bleaching agent components can include one or more oxygen bleaching agents and, depending upon the bleaching agent chosen, one or more bleach activators. When present oxygen bleaching compounds will typically be present at levels of from about 1 % to about 25%.

The bleaching agent component for use herein can be any of the bleaching agents useful for detergent compositions including oxygen bleaches as well as others known in the art. The bleaching agent suitable for the present invention can be an activated or non-activated bleaching agent.

One category of oxygen bleaching agent that can be used encompasses percarboxylic acid bleaching agents and salts thereof. Suitable examples of this class of agents include magnesium monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro perbenzoic acid, 4-nonylamino-4- oxoperoxybutyric acid and diperoxydodecanedioic acid. Such bleaching agents are disclosed in U. S. Patent 4,483,781, U. S. Patent Application 740,446, European Patent Application 0,133,354 and U. S. Patent 4,412,934. Highly preferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproic acid as described in U. S. Patent 4, 634,551.

Another category of bleaching agents that can be used encompasses the halogen bleaching agents. Examples of hypohalite bleaching agents, for example, include trichloro isocyanuric acid and the sodium and potassium dichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides. Such materials are normally added at 0.5-10% by weight of the finished product, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination with bleach activators such as tetraacetylethylenediamine (TAED), nonanoyloxybenzene-sulfonate (NOBS, described in US 4,412,934), 3,5,- trimethylhexanoloxybenzenesulfonate (ISONOBS, described in EP 120,591) or pentaacetylglucose (PAG) or Phenolsulfonate ester of N-nonanoyl-6- aminocaproic acid (NACA-OBS, described in W094/28106), which are perhydrolyzed to form a peracid as the active bleaching species, leading to improved bleaching effect. Also suitable activators are acylated citrate esters such as disclosed in Copending European Patent Application No. 91870207.7 and unsymetrical acyclic imide bleach activator of the following formula as disclosed in the Procter & Gamble co-pending patent applications US serial No.

60/022,786 (filed July 30,1996) and No. 60/028,122 (filed October 15,1996): wherein R is a C7-C13 linear or branched chain saturated or unsaturated alkyl group, R2 is a Cl-C8, linear or branched chain saturated or unsaturated alkyl group and R3 is a Cl-C4 linear or branched chain saturated or unsaturated alkyl group.

Useful bleaching agents, including peroxyacids and bleaching s comprising bleach activators and peroxygen bleaching compounds for use in detergent compositions according to the invention are described in our co- pending applications USSN 08/136,626, PCT/US95/07823, W095/27772, W095/27773, W095/27774 and W095/27775.

The hydrogen peroxide may also be present by adding an enzymatic (i. e. an enzyme and a substrate therefore) which is capable of generating hydrogen peroxide at the beginning or during the washing and/or rinsing process. Such enzymatic s are disclosed in EP Patent Application 91202655.6 filed October 9, 1991.

Metal-containing catalysts for use in bleach compositions, include cobalt- containing catalysts such as Pentaamine acetate cobalt (iil) salts and manganese-containing catalysts such as those described in EPA 549 271; EPA 549 272; EPA 458 397; US 5,246,621; EPA 458 398; US 5,194,416 and US 5,114,611. Bleaching composition comprising a peroxy compound, a manganese-containing bleach catalyst and a chelating agent is described in the patent application No 94870206.3.

Bleaching agents other than oxygen bleaching agents are also known in the art and can be utilized herein. One type of non-oxygen bleaching agent of particular interest includes photoactivated bleaching agents such as the sulfonated zinc and/or aluminum phthalocyanines. These materials can be deposited upon the substrate during the washing process. Upon irradiation with light, in the presence of oxygen, such as by hanging clothes out to dry in the daylight, the sulfonated zinc phthalocyanine is activated and, consequently, the substrate is bleached. Preferred zinc phthalocyanine and a photoactivated bleaching process are described in U. S. Patent 4,033,718. Typically, detergent compositions will contain about 0.025% to about 1.25%, by weight, of sulfonated zinc phthalocyanine.

Chelating Agents The detergent compositions herein may also optionally contain one or more iron and/or manganese chelating agents. Such chelating agents can be selected from the group consisting of amino carboxylates, amino phosphonates, polyfunctionally-substituted aromatic chelating agents and mixtures therein, all as hereinafter defined. Without intending to be bound by theory, it is believed that the benefit of these materials is due in part to their exceptional ability to remove iron and manganese ions from washing solutions by formation of soluble chelates.

Amino carboxylates useful as optional chelating agents include ethylenediaminetetracetates, N-hydroxyethylethylenediaminetriacetates, nitrilo-

triacetates, ethylenediamine tetraproprionates, triethylenetetraaminehexacetates, diethylenetriaminepentaacetates, and ethanoldiglycines, alkali metal, ammonium, and substituted ammonium salts therein and mixtures therein.

Amino phosphonates are also suitable for use as chelating agents in the compositions of the invention when at lease low levels of total phosphorus are permitted in detergent compositions, and include ethylenediaminetetrakis (methylenephosphonates) as DEQUEST. Preferred, these amino phosphonates to not contain alkyl or alkenyl groups with more than about 6 carbon atoms.

Polyfunctionally-substituted aromatic chelating agents are also useful in the compositions herein. See U. S. Patent 3,812,044, issued May 21,1974, to Connor et al. Preferred compounds of this type in acid form are dihydroxydisulfobenzenes such as 1,2-dihydroxy-3,5-disulfobenzene.

A preferred biodegradable chelator for use herein is ethylenediamine disuccinate ("EDDS"), especially the [S, S] isomer as described in U. S. Patent 4,704,233, November 3,1987, to Hartman and Perkins.

The compositions herein may also contain water-soluble methyl glycine diacetic acid (MGDA) salts (or acid form) as a chelant or co-builder useful with, for example, insoluble builders such as zeolites, layered silicates and the like.

If utilized, these chelating agents will generally comprise from about 0.1% to about 15% by weight of the detergent compositions herein. More preferably, if utilized, the chelating agents will comprise from about 0.1% to about 3.0% by weight of such compositions.

Suds suppressor Another optional ingredient is a suds suppressor, exemplified by silicones, and silica-silicone mixtures. Silicones can be generally represented by alkylated polysiloxane materials while silica is normally used in finely divided forms exemplified by silica aerogels and xerogels and hydrophobic silicas of various types. These materials can be incorporated as particulates in which the suds suppressor is advantageously releasably incorporated in a water-soluble or water-dispersible, substantially non-surface-active detergent impermeable

carrier. Alternatively the suds suppressor can be dissolve or dispersed in a liquid carrier and applied by spraying on to one or more of the other components.

A preferred silicone suds controlling agent is disclosed in Bartollota et al.

U. S. Patent 3 933 672. Other particularly useful suds suppressors are the self- emulsifying silicone suds suppressors, described in German Patent Application DTOS 2 646 126 published April 28,1977. An example of such a compound is DC-544, commercially available from Dow Corning, which is a siloxane-glycol copolymer. Especially preferred suds controlling agent are the suds suppressor comprising a mixture of silicone oils and 2-alkyl-alcanols. Suitable 2-alkyl- alkanols are 2-butyl-octanol which are commercially available under the trade name Isofol 12 R.

Such suds suppressor are described in Copending European Patent application N 92870174.7 filed 10 November, 1992.

Especially preferred silicone suds controlling agents are described in Copending European Patent application N°92201649.8. Said compositions can comprise a silicone/silica mixture in combination with fumed nonporous silica such as AerosilR.

The suds suppressors described above are normally employed at levels of from 0.001 % to 2% by weight of the composition, preferably from 0.01 % to 1 % by weight.

Others Other components used in detergent compositions may be employed, such as soil-suspending agents, soil-release agents, optical brighteners, abrasives, bactericides, tarnish inhibitors, coloring agents, and/or encapsulated or non-encapsulated perfumes.

Especially suitable encapsulating materials are water soluble capsules which consist of a matrix of polysaccharide and polyhydroxy compounds such as described in GB 1,464,616. Other suitable water soluble encapsulating materials comprise dextrins derived from ungelatinized starch acid-esters of substituted

dicarboxylic acids such as described in US 3,455,838. These acid-ester dextrins are, preferably, prepared from such starches as waxy maize, waxy sorghum, sago, tapioca and potato. Suitable examples of said encapsulating materials include N-Lok manufactured by National Starch. The N-Lok encapsulating material consists of a modified maize starch and glucose. The starch is modified by adding monofunctional substituted groups such as octenyl succinic acid anhydride.

Preferred optical brighteners are anionic in character, examples of which are disodium 4,4'-bis- (2-diethanolamino-4-anilino-s-triazin-6-ylamino) stilbene- 2: 2'disulphonate, disodium 4,-4'-bis- (2-morpholino-4-anilino-s-triazin-6-ylamino- stilbene-2: 2'-disulphonate, disodium 4,4'-bis- (2, 4-dianilino-s-triazin-6- ylamino) stilbene-2: 2'-disulphonate, monosodium 4', 4"-bis- (2, 4-dianilino-s-tri- azin-6 ylamino) stilbene-2-sulphonate, disodium 4,4'-bis- (2-anilino-4- (N-methyl-N- <BR> <BR> <BR> 2-hydroxyethylamino)-s-triazin-6-ylamino) stilbene-2, 2'-disulphonate, di-sodium 3-triazol-2-yl)-stilbene-2, 2'disulphonate, di-so-dium 4,4'bis (2-anilino-4- (1-methyl-2-hydroxyethylamino)-s-triazin-6- ylami-no) stilbene- 2,2'disulphonate, sodium 2 (stilbyl-4"-(naphtho-1', 2': 4,5)-1,2,3-triazole-2"- sulphonate and 4,4'-bis (2-sulphostyryl) biphenyl. Highly preferred brighteners are the specific brighteners of copending European Patent application No.

95201943.8.

Other useful polymeric materials are the polyethylene glycols, particularly those of molecular weight 1000-10000, more particularly 2000 to 8000 and most preferably about 4000. These are used at levels of from 0.20% to 5% more preferably from 0.25% to 2.5% by weight. These polymers and the previously mentioned homo-or co-polymeric polycarboxylate salts are valable for improving whiteness maintenance, fabric ash deposition, and cleaning performance on clay, proteinaceous and oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in compositions of the present invention are conventionally copolymers or terpolymers of terephthalic acid with ethylene glycol and/or propylene glycol units in various arrangements. Examples of such

polymers are disclosed in the commonly assigned US Patent Nos. 4116885 and 4711730 and European Published Patent Application No. 0 272 033. A particular preferred polymer in accordance with EP-A-0 272 033 has the formula (CH3 (PEG) 43) 0.75 (POH) 0. 25 [T-PO) 2.8 (T-PEG) p. 4] T (PO- H) o. 25 ( (PEG) 43CH3) 0.75 where PEG is- (OC2H4) 0-, PO is (OC3H60) and T is (pcOC6H4CO).

Also very useful are modified polyesters as random copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and 1-2 propane diol, the end groups consisting primarily of sulphobenzoate and secondarily of mono esters of ethylene glycol and/or propane-diol. The target is to obtain a polymer capped at both end by sulphobenzoate groups,"primarily", in the present context most of said copolymers herein will be end-capped by sulphobenzoate groups. However, some copolymers will be less than fully capped, and therefore their end groups may consist of monoester of ethylene glycol and/or propane 1-2 diol, thereof consist"secondarily"of such species.

The selected polyesters herein contain about 46% by weight of dimethyl terephthalic acid, about 16% by weight of propane-1.2 diol, about 10% by weight ethylene glycol about 13% by weight of dimethyl sulfobenzoic acid and about 15% by weight of sulfoisophthalic acid, and have a molecular weight of about 3.000. The polyesters and their method of preparation are described in detail in EPA 311 342.

The enzyme stabilizer useful herein depends upon characteristics such as the enzyme used, and the active ingredient. However, preferred examples of an enzyme stabilizer useful herein includes calcium ion, borates, borate-diols, propylene glycol, short chain carboxylic acids, boronic acids, and mixtures thereof.

It is well known in the art that free chlorine in tap water rapidly deactivates the enzymes comprised in detergent compositions. Therefore, using chlorine

scavenger such as perborate, ammonium sulfate, sodium sulphite or polyethyleneimine at a level above 0.1% by weight of total composition, in the formulas will provide improved through the wash stability of the detergent enzymes. Compositions comprising chlorine scavenger are described in the European patent application 92870018.6 filed January 31,1992.

Alkoxylated polycarboxylates such as those prepared from polyacrylates are useful herein to provide additional grease removal performance. Such materials are described in WO 91/08281 and PCT 90/01815 at p. 4 et seq., incorporated herein by reference. Chemically, these materials comprise polyacrylates having one ethoxy side-chain per every 7-8 acrylate units. The side-chains are of the formula- (CH2CH2O) m (CH2) nCH3 wherein m is 2-3 and n is 6-12. The side-chains are ester-linked to the polyacrylate"backbone"to provide a"comb"polymer type structure. The molecular weight can vary, but is typically in the range of about 2000 to about 50,000. Such alkoxylated polycarboxylates can comprise from about 0.05% to about 10%, by weight, of the compositions herein.

The following examples are meant to exemplify compositions of the present invention, but are not necessarily meant to limit or otherwise define the scope of the invention.

In the detergent compositions, the enzymes levels are expressed by pure enzyme by weight of the total composition and unless otherwise specified, the detergent ingredients are expressed by weight of the total compositions.

EXAMPLES The following examples further describe and demonstrate the preferred embodiments within the scope of the present invention. The examples are given solely for the purpose of illustration, and are not to be constructed as limitations of the present invention since many variations thereof are possible without departing from its spirit and scope.

An example of calcium binding type enzyme purification

The following example shows an enzyme purification process for a metallo-protease.

(a) Made about 400 ml of crude samples (Protease N TM (provided by Amano Seiyaku, Japan, Tokyo), in 25 mM sodium phosphate buffer of pH 6 at XC.

The concentration was adjusted to 10 mg/ml.

(b) Then, concentrated the solution from step (a) by Pellicon concentrator (Millipore) with Pellicon XL filter (Molecular Weight cut of 5k) and obtained 40 mi concentrated solution.

(c) Then, added 360 ml of 25 mM sodium phosphate buffer of pH 6 to the solution from step (b) and obtained 400 ml of a solution. The solution was applied to step (b) again and repeated step (b) and (c) twice. Finally obtained about 40 ml of solution.

(d) Added about 360 mi of Milli-Q water to the solution from step (c) and obtained 400 mi of a solution and pH was adjusted to 6.7 with few drops of 0.1 M NaOH. Then, solution was further concentrated by Pellicon concentrator to 80-100 ml.

(e) Incubated the solution from step (d) for 24 hours at 4 C in the refrigerator (Mitsubishi) and obtained white precipitate.

(f) Centrifuged the mixer from step (e) by a centrifuger ( (CR 5B, Hitachi) with 4000 rpm of a speed of centrifuge for 15 minutes. Washed the precipitates with Milli-Q water twice. Then freeze dried the precipitate and finally obtained about 1g of precipitate.

The precipitate contained 32% of active metallo-protease and showed 58 AU/mg of caseinolytic activity.

Examples of detergent compositions comprising a metallo-protease purified by the present invention

Examples 1 Liquid Hard Surface Cleaning Compositions Example No.

Component A B C D E F G Metallo-protease 0.01 0.02 0.03 0.01 0.02 Serine-Protease-----0.02 0.01 EDA*---2.90 2.90-- Citrate-----2.90 2.90 C13 linear alky-1.95-1.95-1.95 benzene sulfonate (LAS) Alkyl sulfate (AS) 2.00-2.20-2.20-2.20 Alkylpolyoxyethlene 2.00-2.20-2.20-2.20 sulfate (AES) Amine Oxide 0.40-0.50-0.50-0.50 Hydrotrope-1.30-1.30-1.30 Solvent**-6.30 6.30 6.30 6.30 6.30 6.30 Ca++ (as CaCi2)-~ 0 40 0.40 0.40 Water and Minors balance to 100% *Na4 ethylenediamine diacetic acid **Diethyleneglycol monohexyl ether In Examples 1-F and 1-G, any combination of the protease enzymes useful in the present invention recited herein, among others, are substituted for metallo-protease and serine-protease, with substantially similar results.

2. Dishwashing Compositions Examples 2 Dishwashing Composition Example No.

Component A B C D E F Metallo-protease 0.001 0.005 0.01 0.05 0.01 0.003 Serine-protease----0.04 0.01 TFAA* 0.90 0.90 0.90 0.90 0.90 0.90 AES 12.00 12.00 12.00 12.00 12.00 12.00 2-methyl undecanoic acid 4.50 4.50--4.50 4.50-- C 12 alcohol ethoxylate (4) 3.00 3.00 3.00 3.00 3.00 3.00 Amine oxide 3.00 3.00 3.00 3.00 3.00 3.00 Hydrotrope 2.00 2.00 2.00 2.00 2.00 2.00 Ethanol 4.00 4.00 4.00 4.00 4.00 4.00 Mg++ (as MgCl2) 0. 20 0.20 0.20 0.20 0.20 0.20 Ca++ (as CaCl2) 0. 40 0.40 0.40 0.40 0.40 0.40 Water and Minors balance to 100% *C16-C1g alkyl N-methyl glucamide In Examples 2-E and 2-F, any combination of the protease enzymes useful in the present invention recited herein, among others, are substituted for metallo-protease and serine-protease, with substantially similar results.

3. Fabric cleaning compositions Granular Fabric Cleaning Composition The granular fabric cleaning compositions of the present invention contain an effective amount of one or more protease enzymes, preferably from about 0.001% to about 10%, more preferably, from about 0.005% to about 5%, more preferably from 0.01 % to about 1 % by weight of active protease enzyme of the composition. (See U. S. Patent No. 5,679,630 Examples).

Example 3 Granular Fabric Composition Example No.

Component A B C D Metallo-protease 0.01 0.02 0.02 0.02 Serine-protease--0.01 0.02 C13 linear alkyl benzene sulfonate 22.00 22.00 22.00 22.00 Phosphate (as sodium 23.00 23.00 23.00 23.00 tripolyphosphates) Sodium carbonate 23.00 23.00 23.00 23.00 Sodium silicate 14.00 14.00 14.00 14.00 Zeolite 8.20 8.20 8.20 8.20 DPTA* 0.40-0.40- Ca++ (as CaCl2) 0.40 0.40 0.40 0.40 Sodium sulfate 5.50 5.50 5.50 5.50 Water balance to 100% *Diethylene triamine pentaacetic acid In Examples 3-C and 3-D, any combination of the protease enzymes useful in the present invention recited herein, among others, are substituted for metallo-protease and serine-protease, with substantially similar results.

Example 4 Granular Fabric Cleaning Composition Example No.

Component A B C D Metallo-protease'0.01 0.02 0.007 0.005 Serine-protease2--0.003 0.005 C12 alkyl benzene sulfonate 12.00 12.00 12.00 12.00 Zeolite A (1-10 micrometer) * 26.00 26.00 26.00 26.00 C12-Cl4 secondary (2,3) alkyl sulfate, 5.00 5.00 5.00 5.00 sodium salt Sodium citrate 5.00 5.00 5.00 5.00 Optical brightener 0.10 0.10 0.10 0.10 Sodium sulfate 17.00 17.00 17.00 17.00 Ca++ (as CaCl2) 0.40 0.40 0.40 0.40 Fillers, water, minors balance to 100% *A Hydrated sodium aluminosilicate of formula Nal2 (AlO2SiO2) 12. 27H20 having a primary particle size in the range from 0.1 to 10 micrometers (weight expressed on an anhydrous basis) In Examples 4-C and 4-D, any combination of the protease enzymes useful in the present invention recited herein, among others, are substituted for metallo-protease and serine-protease, with substantially similar results.

Example 5 Liquid Fabric Cleaninq Compositions Example No.

Component A B Di H20 38. 63 MEA (monoethanolamine) 0.48 9.00 NaOH 4. 40 1.00 Pdiol 4.00 10.0 Citric acid 2.50 2.00 Sodiumsulfate 1. 75- DTPA 0.50 1.00 FWA Premix (Br 15/MEA/NI 23-9) 0.15 0.15 Na C25AE1. 80S 23. 50- AE3S(H)-4.00 C11. 8HLAS* 3.00 14.00 Neodol 2.00 6.00 EtOH 0. 50 2.00 Ca++ (as CaCl2) 0. 10 0.10 Borax premix (Borax/MEA/Pdiol/CitricAcid) 2.50 Boricacid-1.00 C10 amido propyl dimethyl amine 1. 50- 1.20-TEPA105** Dye 0. 0040 0.0015 Cellulase 0.053 0.20 Amylase 0. 15 0.20 Metallo-protease 0. 05 0 05 Waters and minors up to 100%

*HLAS: Acid form of linear alkyl benzene sulfonate (a synthetic anionic surfactant) **TEPA: tetraethylene pentamine Example 6 Bar Fabric Cleaning Compositions Example No.

Component A B C D Metallo-protease 0.01 0.03 0.01 0.02 Serine-protease--0.01 0.01 C12-C16 alkyl sulfate, Na 20.0 20.0 20.0 20.00 C12-C14 N-methyl glucamide 5.0 5.0 5.0 5.00 C11-C13 alkyl benzene sulfonate, Na 10.0 10.0 10.0 10.00 Sodium pyrophosphate 7.0 7.0 7.0 7.00 Sodium tripolyphosphate 7.0 7.0 7.0 7.00 Zeolite A (0.1-. 10µl) 5.0 5.0 5.0 5.00 Carboxymethylcellulose 0.2 0.2 0.2 0.20 Polyacrylate (MW 1400) 0.2 0.2 0.2 0.20 Coconut monethanolamide 5.0 5.0 5.0 5.00 Brightener, perfume 0.2 0.2 0.2 0.20 CaS04 1.0 1.0 1.0 1.00 MgS04 1.0 1.0 1.0 1.00 Water 4.0 4.0 4.0 4.00 Filler* balance to 100% *Can be selected from convenient materials such as CaCO3, talc, clay, silicates, and the like.

In Examples 6-C and 6-D any combination of the protease enzymes useful in the present invention recited herein, among others, are substituted for metallo- protease and serine-protease, with substantially similar results.

The compositions of the present invention can be suitably prepared by any process chosen by the formulator, non-limiting examples of which are described in U. S. 5,691,297 Nassano et al., issued November 11,1997; U. S. 5,574,005 Welch et al., issued November 12,1996; U. S. 5,569,645 Dinniwell et al., issued October 29,1996; U. S. 5,565,422 Del Greco et al., issued October 15,1996; U. S. 5,516,448 Capeci et al., issued May 14,1996; U. S. 5,489,392 Capeci et al. i issued February 6,1996; U. S. 5,486,303 Capeci et al., issued January 23,1996 all of which are incorporated herein by reference.

In addition to the above examples, the cleaning compositions of the present invention can be formulated into any suitable laundry detergent composition, non-limiting examples of which are described in U. S. 5,679,630 Baeck et al., issued October 21,1997; U. S. 5,565,145 Watson et al., issued October 15,1996; U. S. 5,478,489 Fredj et al., issued December 26,1995; U. S.

5,470,507 Fredj et al., issued November 28,1995; U. S. 5,466,802 Panandiker et al., issued November 14,1995; U. S. 5,460,752 Fredj et al., issued October 24, 1995; U. S. 5,458,810 Fredj et al., issued October 17,1995; U. S. 5,458,809 Fredj et al., issued October 17,1995; U. S. 5,288,431 Huber et al., issued February 22, 1994 all of which are incorporated herein by reference.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to one skilled in the art without departing from the scope of the present invention.