WO2002068575A1

WO2002068575A1 - Liquid detergent composition exhibiting enhanced α-amylase enzyme stability

Info

Publication number: WO2002068575A1
Application number: PCT/US2002/005512
Authority: WO
Inventors: Chandrika Kasturi; Mark Edward Wandsrat; Brian Xiaoqing Song
Original assignee: The Procter & Gamble Company
Priority date: 2001-02-28
Filing date: 2002-02-25
Publication date: 2002-09-06
Also published as: WO2002068575A8; DE60208777D1; US20110053824A1; MX254098B; US20060019856A1; US20020183226A1; JP2006257435A; JP2004525219A; US20100022434A1; DE60208777T2; ES2256453T3; ATE316132T1; CA2439379A1; MXPA03007787A; JP4267923B2; EP1373452B1; CA2439379C; EP1373452A1; US7579310B2

Abstract

An aqueous liquid or gel type detergent composition comprises boric acid or a boron compound, a polyhydroxy compound, and a relatively high level of calcium ion to stabilize a selected α-amylase enzyme.

Description

LIQUID DETERGENT COMPOSITION EXHIBITING ENHANCED α-AMYLASE

ENZYME STABILITY

TECHNICAL FIELD The present invention relates to aqueous liquid or gel type detergent compositions comprising a combination of boric acid or a boron compound capable of forming boric acid in the composition, a polyhydroxy compound, preferably propanediol, and a relatively high level of calcium ion to stabilize a selected α-amylase enzyme. The invention also relates to a process for enhancing stability of the α-amylase enzyme in a liquid or gel detergent composition.

BACKGROUND OF THE INVENTION

Aqueous liquid and gel detergent compositions containing enzymes, including amylases, are well known in the art. The major problem encountered with such compositions is that of ensuring a sufficient storage stability of the enzymes in the compositions. It is particularly difficult to stabilize amylases in the presence of proteases, which can readily degrade amylases in aqueous liquid or gel detergent compositions.

High-alkaline amylases such as alpha amylases are described in British Specification No. 1,296,839. The use of an enzyme stabilizing system comprising a mixture of boric acid or an alkali metal borate with calcium ion, and preferably with a polyol, is disclosed in U.S. Patent 4,537,706, Severson. Certain α-amylases that provide improved cleaning and stain removal are disclosed in WO97/32961, Baeck et al., and in ^09 12^13 and U.S. Patent 6,093,562. .

The present invention utilizes low levels of boric acid and polyhydroxy compound in combination with a relatively high level of calcium ion to provide surprisingly good stability of selected α-amylase enzymes. SUMMARY OF THE INVENTION The invention relates to an aqueous liquid or gel type detergent composition containing a selected α-amylase enzyme having improved stability, and a process for stabilizing the amylase enzyme in such a composition. The detergent compositions herein are useful for cleaning tableware (e.g., glassware, china, silverware, plastic, etc.), kitchenware, household surfaces such as floors, bathroom fixtures and countertops, and fabrics. The compositions may be fully formulated cleaning products or they may be additive or specialty products that can be used alone or with other cleaning products. Particularly preferred compositions herein are for use in automatic dishwashing machines.

In one aspect of the present invention, an aqueous liquid or gel type detergent composition comprises, by weight (1) from about 0.1% to about 15% of boric acid or a boron compound capable of forming boric acid in the composition; (2) from about 0.1 % to about 10% of a polyhydroxy compound selected from the group consisting of ethylene glycol, propylene glycol, 1,2-propanediol, butylene glycol, hexylene glycol, glycerol, mannitol, sorbitol, erythritol, glucose, fructose, lactose, erythritol-l,4-anhydride, and mixtures thereof; (3) from about 10 to about 100 millimoles of calcium ion per liter of composition; (4) from about 5% to about 90% water; and (5) an α-amylase enzyme, as defined hereinafter. In another aspect of the present invention, a process for stabilizing an amylase enzyme in an aqueous liquid or gel type detergent composition comprises mixing, with detergent ingredients (1) from about 0.1% to about 15% by weight, of boric acid or a boron compound capable of forming boric acid in the composition; (2) from about 0.1% to about 10% by weight, a polyhydroxy compound selected from the group consisting of ethylene glycol, propylene glycol, 1,2-propanediol, butylene glycol, hexylene glycol, glycerol, mannitol, sorbitol, erythritol, glucose, fructose, lactose, erythritol- 1,4-anhydride, and mixtures thereof; (3) from about 10 to about 100 millimoles of calcium ion per liter of composition; and (4) an α-amylase enzyme, as defined hereinafter.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an aqueous liquid or gel type detergent composition comprising boric acid or a boron compound capable of forming boric acid in the composition, a polyhydroxy compound, calcium ions, and selected α-amylase enzyme. The boric acid or boron compound capable of forming boric acid in the composition, is desirably present in an amount from about 0.5% to about 10% by weight, and preferably from about 1% to about 5%, and more preferably from about 2% to about 4% by weight (calculated on the basis of boric acid present). Boric acid is particularly preferred herein, although other compounds such as boric oxide, borax and other alkali metal borates (e.g., sodium ortho-, meta-, and pyroborate, and sodium pentaborate) are suitable. Substituted boric acids (e.g., phenylboronic acid, butane boronic acid, and p- bromo phenylboronic acid) can also be used in place of boric acid.

The compositions of the present invention also contain a polyhydroxy compound as described above. The polyhydroxy compound preferably contains from 2 to 6 carbon atoms and from 2 to 6 hydroxy groups, and is preferably selected from propylene glycol, ethylene glycol, glycerol, sorbitol, and glucose, and mixtures thereof. The polyhydroxy compound is preferably 1,2-propanediol. In the preferred embodiment, the polyhydroxy compound is desirably present in an amount from about 0.1 % to about 7% by weight, preferably from about 0.1% to about 5% by weight, and more preferably, from about 0.1% to about 3% by weight. Most preferably, the polyhydroxy compound is present at a level of from about 0.2% to about 1 % by weight.

The compositions herein also contain from about 10 to about 100, preferably from about 13 to about 50, more preferably from about 15 to about 30, and most preferably from about 18 to about 25, millimoles of calcium ion per liter of composition. The level of calcium ion should be selected so that there is always some minimum level available for the enzyme, after allowing for complexation with components such as builders, fatty acid, etc., in the composition. Any water-soluble calcium salt can be used as the source of calcium ion, including calcium chloride, calcium formate, and calcium acetate. A small amount of calcium ion, generally from about 0.05 to about 0.4 millimoles per liter, is often also present in the composition due to calcium in the enzyme slurry and formula water. The compositions herein contain from about 5% to about 90%, preferably from about 20% to about 80%, more preferably from about 40% to about 75% of water.

The compositions of the present invention also contain from about 0.01%o to about

5%, preferably from about 0.1% to about 2%, by weight of the α-amylase enzyme herein, which is typically available as a dilute (e.g., 2-4% active) slurry in water. On a pure, active enzyme basis, the compositions of the invention can contain from about 0.0001% to about 0.1%, preferably from about 0.001% to about 0.05%, by weight of the α-amylase.

The α-amylases herein are described in WO97/32961, incorporated herein by reference, as "specific amylase enzymes". These amylases include: (a) α-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 H value in the range of 8 to 10, measured by the Phadebas® α-amylase activity assay. Such

Phadebas® α-amylase activity assay is described at pages 9-10, WO95/26397.

(b) α-amylases according (a) comprising the amino sequence shown in SEQ ID No. 1 of WO97/32961 or an α-amylase being at least 80%) homologous with the amino acid sequence shown in SEQ ID No.l.

(c) α-amylases according (a) comprising the amino sequence shown in SEQ ID No.2 of WO97/32961 or an α-amylase being at least 80% homologous with the amino acid sequence shown in SEQ ID No.2. (d) α-amylases according (a) comprising the following amino sequence in the N-terminal : His-His-Asn-Gly-Thr-Asn-Gly-Thr-Met-Met-Gln-Tyr-Phe-Glu-Trp-Tyr-Leu-Pro-Asn- Asp (SEQ ID No.3) or an α-amylase being at least 80% homologous with the amino acid sequence shown (SEQ ID No.3) in the N-terminal. A polypeptide is considered to be X% homologous to the parent amylase if a comparison of the respective amino acid sequences, performed via algorithms, such as the one described by Lipman and Pearson in Science 227, 1985, p. 1435, reveals an identity of X%.

(e) α-amylases according (a-d) wherein the α-amylase is obtainable from an alkalophilic Bacillus species; and in particular, from any of the strains NCIB 12289, NCDB 12512, NOB 12513 and DSM 935. In the context of the present invention, the term "obtainable from" is intended not only to indicate an amylase produced by a Bacillus strain but also an amylase encoded by a DNA sequence isolated from such a Bacillus strain and produced in an host organism transformed with said DNA sequence. (f) α-amylase showing positive immunological cross-reactivity with antibodies raised against an α-amylase having an amino acid sequence corresponding respectively to SEQ ID No.1, ID No.2 or ID No.3.

(g) Variants of the following parent α-amylases which (i) have one of the amino acid sequences shown in SEQ ID No.l, ID No.2 or ID No.4 respectively, or (ii)displays at least 80% homology with one or more of said amino acid sequences, and/or displays immunological cross-reactivity with an antibody raised against an α-amylase having one of said amino acid sequences, and/or is encoded by a DNA sequence wich hybridizes with the same probe as a DNA sequence encoding an α-amylase having one of said amino acid sequence; in which variants : 1. at least one amino acid residue of said parent α-amylase has been deleted; and/or

2. at least one amino acid residue of said parent α-amylase has been replaced by a different amino acid residue; and/or

3. at least one amino acid residue has been inserted relative to said parent α-amylase; said variant having an α-amylase activity and exhibiting at least one of the following properties relative to said parent α-amylase : increased thermostability, increased stability towards oxidation, reduced Ca ion dependency, increased stability and/or α-amylolytic activity at neutral to relatively high pH values, increased α-amylolytic activity at relatively high temperature and increase or decrease of the isoelectric point (pi) so as to better match the pi value for α-amylase variant to the pH of the medium. . Said variants are described in WO96/23873 and U.S. Patent 6,093,562, issued July

25, 2000, both incorporated herein by reference.

A particularly preferred α-amylase herein is Natalase®, available from Novo, which has amino acid sequence shown in Seq. ID No. 2 in WO 97/32961 with the Aspartic Acid (Asp or D) at position 183 and the Glycine (Gly or G) at position 184 deleted.

In the present invention, it has surprisingly been found that the combination of boric acid or boron compound, polyhydroxy compound, and calcium ion at the levels herein unexpectedly stabilizes the selected α-amylase enzyme compared to other α- amylase enzymes such as Termamyl^®.

Other detergent ingredients

The compositions of the invention may also contain additional components generally found in detergent compositions. The compositions may contain surfactants, especially anionic and/or nonionic surfactants, solvents, clay, polycarboxylate thickeners, baking soda, brighteners, carbonates, phosphates, dicarboxylic acid, siloxanes, perfumes, bleach and bleach catalysts, and mixtures thereof. Preferred components are discussed in more detail hereafter.

(a) Thickeners The physical stability of the liquid product may be improved and the thickness of the liquid product may be altered by the addition of a cross-linking polyacrylate thickener to the liquid detergent product as a thixotropic thickener.

Thickeners for use herein include those selected from clay, polycarboxylates, such as Polygel^®, gums, carboxymethyl cellulose, polyacrylates, and mixtures thereof. Clay thickeners herein preferably have a double-layer structure. The clay may be naturally occurring, e.g., Bentonites, or artificially made, e.g., Laponite^®. Laponite^® is supplied by Southern Clay Products, Inc. See The Chemistry and Physics of Clays, Grimshaw, 4^th ed., 1971, pages 138-155, Wiley-Interscience.

(b) pH adjusting components The above liquid detergent product is preferably low foaming, readily soluble in the washing medium and most effective at pH values best conducive to improved cleaning performance, such as in a range of desirably from about pH 6.5 to about pH 12.5, and preferably from about pH 7.0 to about pH 12.0, more preferably from about pH 8.0 to about pH 11.0, when measured at a concentration of 1% by weight in water. Preferably the pH is from about 8.5 to about 10.5, most preferably from about 8.5 to about 10.0. The pH adjusting components are desirably selected from sodium or potassium hydroxide, sodium or potassium carbonate or sesquicarbonate, sodium or potassium silicate, boric acid, sodium or potassium bicarbonate, sodium or potassium borate, and mixtures thereof. NaOH or KOH are the preferred ingredients for increasing the pH to within the above ranges. Other preferred pH adjusting ingredients are sodium carbonate, potassium carbonate, and mixtures thereof, (c) Surfactant

Compositions of the present invention preferably contain a low foaming nonionic surfactant, preferably an alkyl ethoxylate surfactant. A preferred surfactant is SLF18® manufactured by BASF Corporation. Surfactants herein are generally present in a range of from about 0.1% to about 10% by weight of the composition. Surfactants useful herein are described in more detail in WO 98/03622, published January 29, 1998, and in U.S. Patent 4,537,707, both incorporated herein by reference. (d) Builder

The compositions of the present invention also preferably contain one or more detergent builders to assist in controlling mineral hardness and in the removal of particulate soils. Inorganic as well as organic builders can be used.

The level of builder can vary widely depending upon the end use of the composition and its desired physical form. When present, the compositions will typically comprise at least about 1% builder. Preferred compositions comprise from about 5% to about 50%, more preferably about 10% to about 30%>, by weight, of detergent builder. Lower or higher levels of builder, however, are not meant to be excluded.

Inorganic or P-containing detergent builders include, but are not limited to, the alkali metal, ammonium and alkanolammonium salts of polyphosphates (exemplified by the tripolyphosphates, and glassy polymeric meta-phosphates), phosphonates, phytic acid, silicates, carbonates (including bicarbonates and sesquicarbonates), and aluminosilicates.

Examples of silicate builders are the alkali metal silicates, particularly those having a SiO₂:Na₂O ratio in the range 1.6:1 to 3.2:1 and layered silicates, such as the layered sodium silicates described in U.S. Patent 4,664,839, issued May 12, 1987 to H. P. Rieck. NaSKS-6 is the trademark for a crystalline layered silicate marketed by Hoechst (commonly abbreviated herein as "SKS-6"). NaSKS-6 can be prepared by methods such as those described in German DE-A-3,417,649 and DE-A-3,742,043. Other layered silicates, such as those having the general formula NaMSi_xO_2x+ι -yH₂O wherein M is sodium or hydrogen, x is a number from 1.9 to 4, preferably 2, and y is a number from 0 to 20 can be used herein. Various other layered silicates from Hoechst include NaSKS-5, NaSKS-7 and NaSKS-11, as the alpha, beta and gamma forms.

Examples of carbonate builders are the alkaline earth and alkali metal carbonates as disclosed in German Patent Application No. 2,321,001 published on November 15, 1973.

Aluminosilicate builders may be useful in the present invention. Aluminosilicate builders include those having the empirical formula:

M_z(zAlO₂)y]xH₂O . wherein z and y are integers of at least 6, the molar ratio of z to y is in the range from 1.0 to about 0.5, and x is an integer from about 15 to about 264.

Useful aluminosilicate ion exchange materials are commercially available. A method for producting aluminosilicate ion exchange materials is disclosed in U.S. Patent 3,985,669, Krummel, et al, issued October 12, 1976. Preferred synthetic crystalline aluminosilicate ion exchange materials useful herein are available under the designations Zeolite A, Zeolite P (B), Zeolite MAP and Zeolite X. In an especially preferred embodiment, the crystalline aluminosilicate ion exchange material has the formula:

Na₁₂[AlO₂)₁₂(SiO₂)₁₂] xH₂O wherein x is from about 20 to about 30, especially about 27. This material is know as Zeolite A. Dehydrated zeolites (x = 0 - 10) may also be used herein. Preferably, the aluminosilicate has a particle size of about 0.1-10 microns in diameter.

Organic detergent builders suitable for the purposes of the present invention include, but are not restricted to, a wide variety of polycarboxylate compounds. As used herein, "polycarboxylate" refers to compounds having a plurality of carboxylate groups, preferably at least 3 carboxylates. Polycarboxylate builder can generally be added to the composition in acid form, but can also be added in the form of a neutralized salt. When utilized in salt form, alkali metals, such as sodium, potassium, and lithium, or alkanolammonium salts are preferred.

Included among the polycarboxylate builders are a variety of categories of useful materials. One important category of polycarboxylate builders encompasses the ether polycarboxylates, including oxydisuccinate, as disclosed in Berg, U.S. Patent 3,128,287, issued April 1, 1964, and Lamberti et al, U.S. Patent 3,635,830, issued January 18, 1972. See also "TMS/TDS" builders of U.S. Patent 4,663,071, issued to Bush et al, on May 5, 1987. Suitable ether polycarboxylates also include cyclic compounds, particularly alicyclic compounds, such as those described in U.S. Patents 3,923,679; 3,835,163; 4,158,635; 4,120,874 and 4,102,903.

Citrate builders, e.g., citric acid and soluble salts thereof (particularly sodium salt), are polycarboxylate builders of importance for liquid detergent formulations due to their availability from renewable resources and their biodegradability. Oxydisuccinates are also especially useful in such compositions and combinations. Also suitable in the compositions of the present invention are the 3,3-dicarboxy-4- oxa-l,6-hexanedioates and the related compounds disclosed in U.S. Patent 4,566,984, Bush, issued January 28, 1986. Laurylsuccinates are the preferred builders of this group, and are described in European Patent Application 86200690.5/0,200,263, published November 5, 1986. Other suitable polycarboxylates are disclosed in U.S. Patent 4,144,226,

Crutchfield et al, issued March 13, 1979 and in U.S. Patent 3,308,067, Diehl, issued March 7, 1967. See also Diehl U.S. Patent 3,723,322.

Fatty acids, e.g., C₁₂-C₁₈ monocarboxylic acids, can also be incorporated into the compositions alone, or in combination with the aforesaid builders, especially citrate and/or the succinate builders, to provide additional builder activity.

Preferred builders herein include the various alkali metal phosphates such as the well-known sodium tripolyphosphates, sodium pyrophosphate and sodium orthophosphate. Phosphonate builders such as ethane- l-hydroxy-l,l-diphosphonate and other known phosphonates (see, for example, U.S. Patents 3,159,581; 3,213,030; 3,422,021; 3,400,148; and 3,422,137) can also be used though such materials are more commonly used in a low-level mode as chelants or stabilizers. Sodium and/or potassium tripolyphosphate is a particularly preferred builder herein, and preferably is used at a level of from about 15% to 35%, more preferably from about 20% to about 30%, by weight of the composition. (e) Other adjunct detergent ingredients

The liquid or gel detergent composition may optionally contain up to about 20%> of a dispersant polymer selected from the group consisting of polyacrylates and polyacrylate copolymers.

The compositions of the present invention may also contain other enzymes and enzyme stabilizing agents such as short chain carboxylic acids as disclosed in WO 98/03622, published January 29, 1998, U.S. Patent 4,537,707, Severson, and U.S. Patent 4,318,818, Letton, et. al., all incorporated herein by reference.

The compositions herein may also contain bleaching agents and activators, material care agents, and chelating agents such as disclosed in WO 98/03622, incorporated herein by reference.

To exemplify the present invention and demonstrate its benefits, the following gel detergent formulas are prepared containing α-amylase, boric acid, 1-2-propanediol and calcium ion at the levels indicated.

Table 1

* As indicated in Table 2.

The above compositions are prepared by mixing the ingredients in the following order. A solution premix is made by mixing water, potassium hydroxide, sulfuric acid, propanediol, boric acid and sodium tripolyphosphate (STP) in a stainless steel tank. The premix is recirculated through a high shear mixer to grind the STP to a particle size range of about 10-70 microns. A heat exchanger is used to remove heat from the batch. A polymer premix is prepared by dissolving the polyacrylate thickener in a weakly acidified water - nitric acid solution. The polymer solution is then neutralized with the first premix to make a gel base. Continuous mixing with the first premix causes the polymer to swell and provide a gel-like texture. The product is then cooled prior to the addition of the nonionic surfactant, enzymes, perfume and minors. The finished product is a stable gel detergent particularly useful as an automatic dishwashing detergent composition. The stability of the α-amylase in the above formulas, as determined by % amylase remaining after storage at 90°F (32.2°C) for 1, 2, 3 and 4 weeks, is shown in Table 2.

Table 2

% Amylase remaining at 90°F 32.2°C after # weeks

As can be seen above, the Natalase® in Formula 2 of the present invention has better stability with 20 millimoles of calcium ion per liter than with the lower level of calcium in Formula 1.

In contrast, increasing the calcium level from 3.3 to 20 millimoles of calcium ion per liter does not significantly improve Termamyl® stability in a similar base Formula B (compare results for Formula 4 versus Formula 3).

The Natalase® in Formula 6 of the present invention containing 13.3 millimoles of calcium ion per liter also has better stability than in Formula 5 containing only 6.7 millimoles of calcium ion per liter. Even at the higher level of 20 millimoles of calcium ion per liter, both boric acid and diol are necessary for good Natalase® stability, as can be seen by comparing the results for Formula 9 of the invention versus Formula 7 with no diol and Formula 8 with no boric acid.

Other compositions of the present invention are as follows:

Table 3

Other compositions of the invention are obtained when, in the above Formulas A- D, the boric acid is replaced with sodium borate, and/or the 1 ,2-propanediol is replaced with ethylene glycol, propylene glycol, glycerol and sorbitol. Accordingly, having thus described the invention in detail, it will be obvious to those skilled in the art that various changes may be made without departing from the scope of the invention, and the invention is not to be considered limited to what is described in the specification.

Claims

What is claimed is:

1. An aqueous liquid or gel type detergent composition comprising, by weight:

(1) from about 0.1 % to about 15% of boric acid or a boron compound capable of forming boric acid in the composition;

(2) from about 0.1 %> to about 10%) of a polyhydroxy compound selected from the group consisting of ethylene glycol/ propylene glycol, 1,2-propanediol, butylene glycol, hexylene glycol, glycerol, mannitol, sorbitol, erythritol, glucose, fructose, lactose, erythritol- 1 ,4-anhydride, and mixtures thereof; (3) from about 10 to about 100 millimoles of calcium ion per liter of composition;

(4) from about 5% to about 90% of water; and

(5) an α-amylase enzyme selected from the group consisting of:

(a) α-amylase 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® α-amylase activity assay and/or;

(b) α-amylase according (a) comprising the amino sequence shown in SEQ ID No. l or an α-amylase being at least 80% homologous with the amino acid sequence shown in SEQ ID No.l and/or;

(c) α-amylase according (a) comprising the amino sequence shown in SEQ ID No.2 or an α-amylase being at least 80% homologous with the amino acid sequence shown in SEQ

ID No.2 and/or;

(d) α-amylase according (a) comprising the following amino sequence in the N-terminal : His-His-Asn-Gly-Thr-Asn-Gly-Thr-Met-Met-Gln-Tyr-Phe-Glu-Trp-Tyr-Leu-Pro-Asn- Asp (SEQ ID No.3) or an α-amylase being at least 80% homologous with the amino acid sequence shown (SEQ ID No.3) in the N-terminal and/or;

(e) α-amylase according (a-d) wherein the α-amylase is obtainable from an alkalophilic Bacillus species and/or (f) α-amylase according to (e) wherein the amylase is obtainable from any of the strains NCIB 12289, NCIB 12512, NCU3 12513 and DSM 935 and/or;

(g)α-amylase showing positive immunological cross-reactivity with antibodies raised against an α-amylase having an amino acid sequence corresponding respectively to SEQ ID No.l, ID No.2 or ID No.3 and/or;

(h) Variant of a parent α-amylase, which parent α-amylase (i) has one of the amino acid sequences shown in SEQ ID No.l, ID No.2 or ID No.4 respectively, or (ii)displays at least 80% homology with one or more of said amino acid sequences, and/or displays immunological cross-reactivity with an antibody raised against an α-amylase having one of said amino acid sequences, and/or is encoded by a DNA sequence wich hybridizes with the same probe as a DNA sequence encoding an α-amylase having one of said amino acid sequence; in which variants :

(i) at least one amino acid residue of said parent α-amylase has been deleted; and/or (ii) at least one amino acid residue of said parent α-amylase has been replaced by a different amino acid residue; and/or

(iii) at least one amino acid residue has been inserted relative to said parent α-amylase; said variant having an α-amylase activity and exhibiting at least one of the following properties relative to said parent α-amylase : increased thermostability, increased stability towards oxidation, reduced Ca ion dependency, increased stability and/or α-amylolytic activity at neutral to relatively high pH values, increased α-amylolytic activity at relatively high temperature and increase or decrease of the isoelectric point (pi) so as to better match the pi value for α-amylase variant to the pH of the medium.

2. The detergent composition according to claim 1, comprising from about 0.5% to about 10% by weight boric acid.

3. The detergent composition according to claim 2, comprising from about 1% to about 5% by weight boric acid.

4. The detergent composition according to claim 1 , wherein said polyhydroxy compound is 1,2-propanediol.

5. The detergent composition according to claim 1 , comprising from about 0.1 % to about 7% by weight said polyhydroxy compound.

6. The detergent composition according to claim 5, comprising from about 0.1 % to about 3% by weight said polyhydroxy compound.

7. The detergent composition according to claim 1, comprising from about 13 to about 50 millimoles of calcium ion per liter of composition.

8. The detergent composition according to claim 3 comprising by weight from about 0.1% to about 7% of 1,2-propanediol, from about 15 to about 30 millimoles of calcium ion per liter of composition, from about 0.1 % to about 2% of the α-amylase enzyme, and from about 40%> to about 70% of water.

9. The detergent composition according to claim 1 further comprising a protease enzyme.

10. The detergent composition according to claim 8 further comprising from about 0.1 % to about 2% of a protease enzyme.

11. The detergent composition according to claim 10 wherein the α-amylase enzyme is Natalase^®.

12. A process for stabilizing an amylase enzyme in an aqueous liquid or gel type detergent composition, comprising mixing, with detergent ingredients:

(1) from about 0.1% to about 15% by weight, of boric acid or a boron compound capable of forming boric acid in the composition; (2) from about 0.1% to about 10% by weight, of a polyhydroxy compound selected from the group consisting of ethylene glycol, propylene glycol, 1,2- propanediol, butylene glycol, hexylene glycol, glycerol, mannitol, sorbitol, erythritol, glucose, fructose, lactose, erythritol- 1 ,4-anhydride, and mixtures thereof;

(3) from about 10 to about 100 millimoles of calcium ion per liter of composition; and

(4) an α-amylase enzyme selected from the group consisting of:

(a) α-amylase 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® α-amylase activity assay and/or;

(b) α-amylase according (a) comprising the amino sequence shown in SEQ ID No. 1 or an α-amylase being at least 80% homologous with the amino acid sequence shown in SEQ ID No.l and/or; (c) α-amylase according (a) comprising the amino sequence shown in SEQ ID No.2 or an α-amylase being at least 80% homologous with the amino acid sequence shown in SEQ ID No.2 and/or;

(d) α-amylase according (a) comprising the following amino sequence in the N-terminal : His-His-Asn-Gly-Thr-Asn-Gly-Thr-Met-Met-Gln-Tyr-Phe-Glu-Trp-Tyr-Leu-Pro-Asn- Asp (SEQ ID No.3) or an α-amylase being at least 80%) homologous with the amino acid sequence shown (SEQ ID No.3) in the N-terminal and/or;

(e) α-amylase according (a-d) wherein the α-amylase is obtainable from an alkalophilic Bacillus species and/or

(f) α-amylase according to (e) wherein the amylase is obtainable from any of the strains NCIB 12289, NCIB 12512, NCIB 12513 and DSM 935 and/or;

(g)α-amylase showing positive immunological cross-reactivity with antibodies raised against an α-amylase having an amino acid sequence corresponding respectively to SEQ ID No.l, ID No.2 or ID No.3 and/or; (h) Variant of a parent α-amylase, which parent α-amylase (i) has one of the amino acid sequences shown in SEQ ID No.l, ID No.2 or ID No.4 respectively, or (ii)displays at least 80% homology with one or more of said amino acid sequences, and/or displays immunological cross-reactivity with an antibody raised against an α-amylase having one of said amino acid sequences, and/or is encoded by a DNA sequence wich hybridizes with the same probe as a DNA sequence encoding an α-amylase having one of said amino acid sequence; in which variants :

(iii) at least one amino acid residue has been inserted relative to said parent α-amylase; said variant having an α-amylase activity and exhibiting at least one of the following properties relative to said parent α-amylase : increased thermostability, increased stability towards oxidation, reduced Ca ion dependency, increased stability and/or α-amylolytic activity at neutral to relatively high pH values, increased α-amylolytic activity at relatively high temperature and increase or decrease of the isoelectrie point (pi) so as to better match the pi value for α-amylase variant to the pH of the medium.

13. A process according to claim 12 comprising, by weight of the composition, from about 1% to about 5% of boric acid, from about 0.1% to about 7% of the polyhydroxy compound, from about 13 to about 50 millimoles of calcium ion per liter of composition, and from about 0.1 %> to about 2% of the α-amylase enzyme.

14. A process according to claim 13 comprising from about 40%) to. about 10% water, and wherein the polyhydroxy compound is 1,2-propanediol.

15. A process according to claim 13 further comprising from about 0.1% to about 2% of a protease enzyme.

16. A process according to claim 15 comprising from about 15 to about 30 millimoles of calcium ion per liter of composition.

17. A process according to claim 16 wherein the α-amylase enzyme is Natalase^®.

SEQUENCE LISTING

<110> The Procter & Gamble Company

<120> LIQUID DETERGENT COMPOSITION EXHIBITING ENCHANCED a-AMYL ASE ENZYME STABILITY

<130> Detergent Composition

<140> P&G Case 8437/VB, Priority Claim 09/795,211

<141> Filing PCT Case 2002-02-25, Priority Filing 2001-02-28

<160> 4

<170> Patentln version 3.1

<210> 1

<211> 485

<212> PRT

<213> alkaliphilicbacillus

<400> 1

His His Asn Gly Thr Asn Gly Thr Met Met Gin Tyr Phe Glu Trp Tyr

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala 20 25 30

Asn Leu Lys Ser Lys Gly lie Thr Ala Val Trp lie Pro Pro Ala Trp 35 40 45

Lys Gly Thr Ser Gin Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60

sp Leu Gly Glu Phe Asn Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80

Thr Arg Asn Gin Leu Gin Ala Ala Val Thr Ser Leu Lys Asn Asn Gly

85 90 95

He Gin Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110

Gly Thr Glu He Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn 115 120 125

Gin Glu Thr Ser Gly Glu Tyr Ala He Glu Ala Trp Thr Lys Phe Asp 130 135 140

Phe Pro Gly Arg Gly Asn Asn His Ser Ser Phe Lys Trp Arg Trp Tyr 145 150 155 160

His Phe Asp Gly Thr Asp Trp Asp Gin Ser Arg Gin Leu Gin Asn Lys

165 170 175

He Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190

Thr Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205

sp His Pro Glu Val He His Glu Leu Arg Asn Trp Gly Val Trp Tyr 210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg He Asp Ala Val Lys His

225 230 235 240

lie Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr

245 250 255

Thr Gly Lys Pro Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270

Gly Ala He Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val 275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 ' 300

Gly Tyr Tyr Asp Met Arg Asn He Leu Asn Gly Ser Val Val Gin Lys

305 310 315 320

His Pro Thr His Ala Val Thr Phe Val Asp Asn His Asp Ser Gin Pro

325 330 ^' 335

Gly Glu Ala Leu Glu Ser Phe Val Gin Gin Trp Phe Lys Pro Leu Ala 340 345 350 Tyr Ala Leu Val Leu Thr Arg Glu Gin Gly Tyr Pro Ser Val Phe Tyr 355 360 365

Gly Asp Tyr Tyr Gly He Pro Thr His Gly Val Pro Ala Met Lys Ser 370 375 380

Lys He Asp Pro Leu Leu Gin Ala Arg Gin Thr Phe Ala Tyr Gly Thr 385 390 395 400

Gin His Asp Tyr Phe Asp His His Asp He He Gly Trp Thr Arg Glu

405 410 415

Gly Asn Ser Ser His Pro Asn Ser Gly Leu Ala Thr He Met Ser Asp 420 425 430

Gly Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys Asn Lys Ala Gly 435 440 445

Gin Val Trp Arg Asp He Thr Gly Asn Arg Thr Gly Thr Val Thr He 450 455 460

Asn Ala Asp Gly Trp Gly Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480

Val Trp Val Lys Gin

485

<210> 2<211> 485<212> PRT<213> alakaliphilicbacillus<400> 2

His His Asn Gly Thr Asn Gly Thr Met Met Gin Tyr Phe Glu Trp His

1 5 10 15

Leu Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ser 20 25 30

Asn Leu Arg Asn Arg Gly He Thr Ala He Trp He Pro Pro Ala -Trp 35 40 45

Lys Gly Thr Ser Gin Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr 50 55 60

Asp Leu Gly Glu Phe Asn Gin Lys Gly Thr Val Arg Thr Lys Tyr Gly 65 70 75 80

Thr Arg Ser Gin Leu Glu Ser Ala He His Ala Leu Lys Asn Asn Gly

85 90 95

Val Gin Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp 100 105 110

Ala Thr Glu Asn Val Leu Ala Val Glu Val Asn Pro Asn Asn Arg Asn 115 120 125

Gin Glu He Ser Gly Asp Tyr Thr He Glu Ala Trp Thr Lys Phe Asp 130 135 140 Phe Pro Gly Arg Gly Asn Thr Tyr Ser Asp Phe Lys Trp Arg Trp Tyr 145 150 155 160

His Phe Asp Gly Val Asp Trp Asp Gin Ser Arg Gin Phe Gin Asn Arg

165 170 175

He Tyr Lys Phe Arg Gly Asp Gly Lys Ala Trp Asp Trp Glu Val Asp 180 185 190

Ser Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Val Asp Met 195 200 205

Asp His Pro Glu Val Val Asn Glu Leu Arg Arg Trp Gly Glu Trp Tyr 210 215 220

Thr Asn Thr Leu Asn Leu Asp Gly Phe Arg He Asp Ala Val Lys His 225 230 235 240

He Lys Tyr Ser Phe Thr Arg Asp Trp Leu Thr His Val Arg Asn Ala

245 250 255

Thr Gly Lys Glu Met Phe Ala Val Ala Glu Phe Trp Lys Asn Asp Leu 260 265 270

Gly Ala Leu Glu Asn T'yr Leu Asn Lys Thr Asn Trp Asn His Ser Val 275 280 285

Phe Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Gly 290 295 300

Gly Asn Tyr Asp Met Ala Lys Leu Leu Asn Gly Thr Val Val Gin Lys 305 310 315 320

His Pro Met His Ala Val Thr Phe Val Asp Asn His Asp Ser Gin Pro

325 330 335

Gly Glu Ser Leu Glu Ser Phe Val Gin Glu Trp Phe Lys Pro Leu Ala 340 345 350

Tyr Ala Leu He Leu Thr Arg Glu Gin Gly Tyr Pro Ser Val Phe Tyr 355 360 365

Gly Asp Tyr Tyr Gly He Pro Thr His Ser Val Pro Ala Met Lys Ala 370 375 380

Lys He Asp Pro He Leu Glu Ala Arg Gin Asn Phe Ala Tyr Gly Thr 385 390 395 400

Gin His Asp Tyr Phe Asp His His Asn He He Gly Trp Thr Arg Glu

405 410 415

Gly Asn Thr Thr His Pro Asn Ser Gly Leu Ala Thr He Met Ser Asp 420 425 430

Gly Pro Gly Gly Glu Lys Trp Met Tyr Val Gly Gin Asn Lys Ala Gly 435 440 445

Gin Val Trp His Asp He Thr Gly Asn Lys Pro Gly Thr Val Thr He 450 455 . 460

Asn Ala Asp Gly Trp Ala Asn Phe Ser Val Asn Gly Gly Ser Val Ser 465 470 475 480

He Trp Val Lys Arg

485

<210> 3<211> 20<212> PRT<213> akaliphilicbacillus<400> 3 His His Asn Gly Thr Asn Gly Thr Met Met Gin Tyr Phe Glu Trp Tyr 1 5 10 15

Leu Pro Asn Asp 20

<210> 4<211> 515<212> PRT<213> alkaliphilicbacillus<400> 4 Ala Ala Pro Phe Asn Gly Thr Met Met Gin Tyr Phe Glu Trp Tyr Leu 1 5 10 15

Pro Asp Asp Gly Thr Leu Trp Thr Lys Val Ala Asn Glu Ala Asn Asn 20 25 30 Leu Ser Ser Leu Gly He Thr Ala Leu Trp Leu Pro Pro Ala Tyr Lys 35 40 45

Gly Thr Ser Arg Ser Asp Val Gly Tyr Gly Val Tyr Asp Leu Tyr Asp 50 55 60

Leu Gly Glu Phe Asn Gin Lys Gly Ala Val Arg Thr Lys Tyr Gly Thr 65 70 75 80

Lys Ala Gin Tyr Leu Gin Ala He Gin Ala Ala His Ala Ala Gly Met

85 90 95

Gin Val Tyr Ala Asp Val Val Phe Asp His Lys Gly Gly Ala Asp Gly 100 105 110

Thr Glu Trp Val Asp Ala Val Glu Val Asn Pro Ser Asp Arg Asn Gin 115 120 125

Glu He Ser Gly Thr Tyr Gin He Gin Ala Trp Thr Lys Phe Asp Phe 130 135 140

Pro Gly Arg Gly Asn Thr Tyr Ser Ser Phe Lys Trp Arg Trp Tyr His 145 150 155 160

Phe Asp Gly Val Asp Trp Asp Glu Ser Arg Lys Leu Ser Arg He Tyr

165 170 175 Lys Phe Arg Gly He Gly Lys Ala Trp Asp Trp Glu Val Asp Thr Glu 180 185 190

Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Leu Asp Met Asp His 195 200 205

Pro Glu Val Val Thr Glu Leu Lys Ser Trp Gly Lys Trp Tyr Val Asn 210 215 220

Thr Thr Asn He Asp Gly Phe Arg Leu Asp Ala Val Lys His He Lys 225 230 235 240

Phe Ser Phe Phe Pro Asp Trp Leu Ser Asp Val Arg Ser Gin Thr Gly

245 250 255

Lys Pro Leu Phe Thr Val Gly Glu Tyr Trp Ser Tyr Asp He Asn Lys 260 265 270

Leu His Asn Tyr -He Met Lys Thr Asn Gly Thr Met Ser Leu Phe Asp 275 280 285

Ala Pro Leu His Asn Lys Phe Tyr Thr Ala Ser Lys Ser Gly Gly Thr 290 295 300

Phe Asp Met Arg Thr Leu Met Thr Asn Thr Leu Met Lys Asp Gin Pro 305 310 315 320

10 Thr Leu Ala Val Thr Phe Val Asp Asn His Asp Thr Glu Pro Gly Gin

325 330 335

Ala Leu Gin Ser Trp Val Asp Pro Trp Phe Lys Pro Leu Ala Tyr Ala 340 345 350

Phe He Leu Thr Arg Gin Glu Gly Tyr Pro Cys Val Phe Tyr Gly Asp 355 ^• 360 365

Tyr Tyr Gly He Pro Gin Tyr Asn He Pro Ser Leu Lys Ser Lys He 370 375 380

Asp Pro Leu Leu He Ala Arg Arg Asp Tyr Ala Tyr Gly Thr Gin His 385 390 395 400

Asp Tyr Leu Asp His Ser Asp He He Gly Trp Thr Arg Glu Gly Val

405 410 415

Thr Glu Lys Pro Gly Ser Gly Leu Ala Ala Leu He Thr Asp Gly Pro 420 425 430

Gly Gly Ser Lys Trp Met Tyr Val Gly Lys Gin His Ala Gly Lys Val 435 440 445

Phe Tyr Asp Leu Thr Gly Asn Arg Ser Asp Thr Val Thr He Asn Ser 450 455 460

11 Asp Gly Trp Gly Glu Phe Lys Val Asn Gly Gly Ser Val Ser Val Trp 465 470 475 480

Val Pro Arg Lys Thr Thr Val Ser Thr He Ala Trp Ser He Thr Thr

485 490 495

Arg Pro Trp Thr Asp Glu Phe Val Arg Trp Thr Glu Pro Arg Leu Val 500 505 510

Ala Trp Pro 515

12