AU2016200189B2

AU2016200189B2 - Care enzyme system

Info

Publication number: AU2016200189B2
Application number: AU2016200189A
Authority: AU
Inventors: Richard Elliot Lilley; Neil James Parry
Original assignee: Unilever Global IP Ltd
Current assignee: Unilever Global IP Ltd
Priority date: 2012-05-10
Filing date: 2016-01-13
Publication date: 2017-07-27
Anticipated expiration: 2033-05-07
Also published as: IN2014MN02257A; AU2013258083A1; AU2016200189A1; WO2013167613A1; EP2847313A1

Abstract

Abstract A low temperature active enzymatic fabric care composition comprising the combination of one or more primary care enzymes and one or more auxiliary care enzymes. 7283959 1 (GHMatters) P98467.AU.1 JENNYP

Description

2016200189 20 Feb 2017 -1 -

CARE ENZYME SYSTEM

The present invention concerns combinations of fabric care enzymes, and in particular but not exclusively fabric care enzymes in a fabric treatment composition. 5 WO95/02675 discloses detergent compositions comprising cellulases capable of providing improved particulate soil removal and cellulases providing a colour clarification. Disclosed are compositions comprising a first cellulase component having retaining type activity and being capable of particulate soil removal a second 10 cellulase component having multiple domains comprising at least one non-catalytic domain attached to a catalytic domain and being capable of colour clarification.

Low temperature active enzyme formulations are needed for low temperature washing conditions. However, mesophilic and even more so thermophilic enzymes 15 are less effective at low temperatures so that levels added to washing formulations need to be increased which makes the formulation more expensive. In the case of care enzymes which act to hydrolyse the fabric material itself, high levels can lead to increased fabric damage which is counter productive. The present invention seeks to provide a low temperature enzymatic composition and process with improved care 20 but with no/minimal attendant fabric damage.

In a first aspect, the present invention provides a low temperature active enzymatic fabric care composition comprising the combination of one or more primary care enzymes and one or more auxiliary care enzymes wherein the one or more primary 25 care enzymes comprises one or more enzymes of the Glycoside Hydrolase Family 45.

In an aspect, the present invention provides a low temperature active enzymatic fabric treatment composition comprising the combination of: 30 1. one or more primary care enzymes; and 2. one or more auxiliary care enzymes. -2- 2016200189 20 Feb 2017 ln a second aspect the present invention provides a low temperature enzymatic fabric treatment process comprising the step of treating a fabric with the composition of the first aspect of the invention.

In a third aspect, the present invention provides use of an auxiliary care enzyme in 5 combination with a primary care enzyme in the treatment of fabrics for a care benefit.

In a fourth aspect, the present invention provides use of a low temperature active enzymatic fabric care composition comprising the combination of one or more primary care enzymes and one or more auxiliary care enzymes, in the treatment of fabrics for 10 a care benefit.

With the composition of the present invention, care of the fabric is improved using a low level of fabric care enzyme whose performance is synergistically improved at low temperature by the use of an auxiliary care enzyme. 15

Preferably the total amount of the enzymes of the invention ie. the total amount of the primary care enzyme and the auxiliary care enzyme is 0.01 -1.75 mg per Litre of the wash liquor. 20 Washing machines vary in wash liquor volume from 10 L to 48 L. Accordingly the total amount of enzymes of the invention per dose may be from 0.1 - 84 mg.

For hand washing, the wash liquor may be around 4 - 5 litres in which case the dose may be 0.04 - 8.75 mg of total enzymes of the invention. 25

The amount of detergent composition per dose will itself vary (e.g. 35 ml, 10 ml) depending on water levels, concentration of e.g. surfactants.

The primary care enzyme may be 0.003 mg/L of wash liquor. 30

Preferably the auxiliary care enzyme is therefore present either in equal weight (of enzyme protein) or, more preferably greater amounts than the primary care enzyme. -3-

Preferably the primary care enzyme and the auxiliary care enzyme are present in a weight ratio of enzyme protein in the range of from 1:1 to 1:29, more preferably 1:1 to 1:9 more preferably 1:5, more preferably 1:1.9. The preferred ranges of the invention exclude 1:30, 1:10 and 1:2.

Preferably, the one or more primary care enzymes comprises one or more enzymes of the Glycoside Hydrolase Family 45.

Preferably the one or more primary care enzymes comprises one or cellulolytic enzymes (commonly known as cellulases but not restricted to the class EC 3.1.2.4) active in restoring colour to cotton or cotton-based fabrics by removal of fuzz and pills from the surface of the fabric.

The one or more auxiliary care enzymes preferably comprise one or more glycosyl hydrolases of Family 5 and/or Family 7.

DEFINITIONS

In this patent specification: “Primary care enzymes” means enzymes active at restoring colour to fabrics by removing fuzz and pills from the surface of the fabric. “care benefit” means restoration of colour to or improvement of feel of fabrics by removing fuzz and pills from the surface of the fabric. “Auxiliary care enzymes” means any enzyme which is active to hydrolyse cel lool igosaccha rides. “Low temperature active” means active at temperatures of 25 degrees Celcius or lower. 2016200189 13 Jan 2016 -4- “Cellooligosaccharides” means any subchain of cellulose which is a reaction product of the hydrolysis cellulose by the primary care enzyme. Preferably the sub chain is soluble and preferably it contain 2 - 8 glucose units: cellobiose (DP=2), cellotriose (DP=3), cellotetraose (DP=4), cellopentaose (DP=5), 5 cellohexaose (DP=6), celloseptose (DP=7), cellooctanose (DP=8); more preferably 4-8 such units. (DP means degrees of polymerisation). “Glycoside Hydrolase Family” means any Glycoside Hydrolase Family (designated by number) of the Glycoside Hydrolase Family Classification system, ίο based on amino acid similarities, being part of the Carbohydrate-Active Enzymes database (CAZy) developed by the Glycogenomics group at Architecture et Fonction des Macromolecules Biologiques, Unite Mixte de Recherches UMR6098, CNRS,Universite de Provence Universite de la Mediterranee.

is “Glycoside Hydrolase Family 5” includes the retaining enzymes of chitosanase (EC 3.2.1.132): · -mannosidase (EC 3.2.1.25): cellulase (EC 3.2.1.4): glucan · -1,3-glucosidase (EC 3.2.1.58): licheninase (EC 3.2.1.73): glucan endo-1,6-· -glucosidase (EC 3.2.1.75): mannan endo-· -1,4-mannosidase (EC 3.2.1.78): endo-· -1,4-xylanase (EC 3.2.1.8): cellulose · -1,4-cellobiosidase (EC 3.2.1.91): · -20 1,3-mannanase (EC 3.2.1.-): xyloglucan-specific endo-· -1,4-glucanase (EC 3.2.1.151): mannan transglycosylase (EC 2.4.1.-): endo-· -1,6-galactanase (EC 3.2.1.164): endoglycoceramidase (EC 3.2.1.123): · -primeverosidase (EC 3.2.1.149) 25 “Glycoside Hydrolase Family 7” includes endo-· -1,4-glucanase (EC 3.2.1.4): reducing end-acting cellobiohydrolase (EC 3.2.1.176): chitosanase (EC 3.2.1.132): endo-· -1,3-1,4-glucanase (EC 3.2.1.73) “Glycoside Hydrolase Family 45” includes the inverting enzymes of 30 endoglucanase (EC 3.2.1.4) -5-

The enzymes of the invention may be from bacterial or fungal origin. Chemically modified or protein engineered mutants are included.

Preferably the primary care and/or the auxiliary care enzymes are cellulases.

Preferred cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum.

Especially preferred primary care cellulases are the alkaline or neutral cellulases having color care benefits. Such preferred enzymes include those having a molecular weight of from 17kDa to 30 kDa, for example the endoglucanases sold under the tradename Biotouch(R) NCD, DCC and DCL (AB Enzymes, Darmstadt, Germany). Other preferred commercially available cellulases include Celluzyme™, Carezyme™,, Renozyme™ (Novozymes A/S), Clazinase™ and Puradax HA™, Puradax(R) EG-L and Puradax(R) HA (Genencor International Inc.), and KAC-500(B)™, KAC(R)-500(B) (Kao Corporation).

Especially preferred auxiliary care cellulases include those active to active to hydrolyse cellooligosaccharides and may include Endolase™ Celluclean™. Whitezyme(R) (Novozymes A/S, Bagsvaerd.

Other enzymes could be included in the fabric treatment composition such as proteases, lipases, phospholipases, amylases, pectate lyases, mannases, peroxidases/oxidases.

Whilst mesophilic enzymes are preferred, psychrophilic enzymes may be used and are included in the scope of the invention. Such enzymes include the cellulases and xylanase from e.g. Clostridium sp. PXYL1 (G. Akila, T.S.Chandra -6- (2003) FEMS Microbiol. Letters 219, 63-67). Psychrophilic xylanases include E.coli phagemid (Lee et al. 2006b).

Once a suitable enzyme has been selected, it is relatively easy for the skilled man to isolate a suitable micro-organism capable of producing the enzyme under washing conditions. To that end, micro-organisms are screened for their capability of producing the desired enzyme under washing conditions, in an assay that resembles the washing conditions as closely as possible.

For all enzymes of the invention, enzyme variants (produced, for example, by recombinant techniques) are included within the meaning of the term "enzyme". Examples of such enzyme variants are disclosed, e.g., in EP 251,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV).

The fabric treatment composition may comprise a laundry/fabric cleaning/care composition and may comprise one or more surfactants and/or optionally other ingredients.

Such compositions of the invention may be in dry solid form e.g. powdered, granules or tableted powders or liquid or gel form. It may also be in the form of a solid detergent bar. The composition may be a concentrate to be diluted, rehydrated and/or dissolved in a solvent, including water, before use. The composition may also be a ready-to-use (in-use) composition.

The present invention is suitable for use in industrial or domestic fabric wash compositions, fabric conditioning compositions and compositions for both washing and conditioning fabrics (so-called through the wash conditioner compositions). The present invention can also be applied to industrial or domestic non-detergent based fabric care compositions, for example spray-on compositions. -7-

Other detergent ingredients may be included including surfactants, builders, sequestring agents, hydrotropes, preservatives, complexing agents, polymers, stabilizers, perfumes, optical brighteners, or other ingredients such as e.g. fabric conditioners including clays, foam boosters, suds suppressors (anti-foams), anticorrosion agents, soil-suspending agents, anti-soil redeposition agents, antimicrobials, tarnish inhibitors, or combinations of one or more thereof, provided that these ingredients are compatible with the enzymes.

The fabric wash compositions may comprise a fabric wash detergent material selected from non-soap anionic surfactant, nonionic surfactants, soap, amphoteric surfactants, zwitterionic surfactants and mixtures thereof. The surfactants may be present in the composition at a level of from 0.1% to 60% by weight.

Any enzyme present in a composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative, e.g., an aromatic borate ester, or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid.

Examples Of Non Limiting Embodiments Of The Invention 1. Assay to determine the level of reducing sugars released from cotton I inters by cellulases. This shows the

Part I - Enzyme hydrolysis:

Enzymes concentrations/ratios A,B and C were prepared as follows. A total concentration of 4mg/L: 1:1 ratio of primary : auxiliary B. total concentration of 1.74 mg/L: 1:1.9 ratio of primary : auxiliary C. total concentration of 1.5 mg/L: 1:5 ratio of primary : auxilary -8-

The enzymes A,B and C were incubated in 200· I volume in microplate wells with 2% (w/v) cotton linters in 15mM MOPS buffer (pH 7) for 1 hour at room temperature/25°C. Rapid agitation was provided by a Heidolph microtitre plate shaker platform at 1,050rpm. No enzyme and no substrate controls, as well as a reducing sugar standard (glucose) range from 0 - 200· M, were included in the assays. At the end of the incubation, cotton linters were allowed to settle for 10 minutes before 100· I aliquots of supernatant solution were transferred to clean microplates.

Part II - BCA assay:

For concentration as in A Bicinchoninic Acid (BCA) method was used to determine the release of reducing sugars into solution. BCA reagent solution was prepared by mixing BCA reagents A and B in a 50:1 (v/v) ratio. BCA reagent solution was then mixed in a 1:1 (v/v) ratio with 100μΙ aliquots of sample supernatant solutions in sealed microplate wells and incubated at 65-70°C for 40 minutes. Microplates were then allowed to cool to room temperature in a refrigerator, before well seals were carefully removed. Absorbance of reaction products was measured at 540nm in a spectrophotometer. Interference by protein was accounted for by subtracting protein only (no cotton linters) control readings. Total reducing sugar released was calculated using a standard curve of glucose from 0 - 200μΜ.

Results

Tables 1,2 and 3 show the results from A, B and C (respectively) as described above : synergistic release of reducing sugars from cotton linters by a 1:1 mix of three different Family 45 EG-V type endoglucanases (Carezyme, Renozyme and Biotouch) and a Family 7 EG-I type endoglucanase (Endolase). The assay was carried out at 25°C. No enzyme control values have been subtracted from all data. 2016200189 13 Jan 2016 -9-

Table 1 Enzyme A (4mg/L total; 1:1 primary : care ratio) Reducing sugars released (· M) 50% Endolase 11.4 100% Endolase 16.4 50% Carezyme 53.5 100% Carezyme 72.6 Carezyme + Endolase 108.5 50% Renozyme 67.0 100% Renozyme 83.9 Renozyme + Endolase 130.1 50% Biotouch 21.7 100% Biotouch 51.1 Biotouch + Endolase 113.6 5

Table 2 Reducing sugars released (· M) 1.14mg/L Endolase 19.5 1.74mg/L Endolase 20.9 0.6mg/L Renozyme 30.8 1.74mg/L Renozyme 57.6 0.6:1.14mg/L Renozyme:Endolase 90.0 0.6mg/L Biotouch 8.3 1.74mg/L Biotouch 26.1 0.6:1.14mg/L Biotouch:Endolase 56.9 2016200189 13 Jan 2016 - 10- Table 3

Reducing sugars released (· M) 1.25mg/L Endolase 15.3 1.5mg/L Endolase 16.1 0.25mg/L Renozyme 9.3 1.5mg/L Renozyme 40.8 0.25:1.25mg/L Renozyme:Endolase 51.8 0.25mg/L Biotouch 0.0 1,5mg/L Biotouch 8.4 0.25:1.25mg/L Biotouch:Endolase 30.3 5 Table 4- (corresponds to Figure 4) showing synergistic release of reducing sugars from cotton linters by a 1:1 mix of two different Family 45 EG-V type endoglucanases (Carezyme and Biotouch) and a Family 5 EG-I type endoglucanase (Celluclean). The assay was carried out at 25°C. No enzyme control values have been subtracted from all data.

Reducing sugars released (· M) 50% Celluclean 7.7 100% Celluclean 15.5 50% Carezyme 35.1 100% Carezyme 47.7 Carezyme + Celluclean 60.8 50% Biotouch 7.7 100% Biotouch 34.4 Biotouch + Celluclean 71.3 - 11 -

Conclusions • A 1:1 mix of the two types of cellulase according to the invention was found to act synergistically in the release of reducing sugars from cotton linters at 25°C. The first cellulase component in this mix is a primary care enzyme, being a Family 45 EG-V type endoglucanase. • The secondary cellulase component is an auxiliary care enzyme with low activity towards cotton linters. Family 45 endoglucanase. The action of the secondary cellulase is thought to reduce the pool of soluble oligo- / polysaccharides which effectively inhibit the activity of the Family 45 endoglucanase towards the insoluble cotton linters.

Exemplary Fabric Treatment Compositons (% wt)

Alkybenzenesulfonic (LAS) acid 12.8

NaOH 2.58 C12-14 alcohol 7-ethoxolate 8.6 C12-13 alcohol 3-ethoxolate Sulphate Na salt 8.6

Citric acid 2.9 C12-18 fatty acid 5

Total Enzymes according to the invention 1

Other enzymes (e.g. amylase and/or protease) 1

Triethanolamine 3.1

Sequestrant (Dequest 2066) 0.5

Fluorescor 0.3

Propylene glycol 8.6

Glycerol 4.78

Perfume 1.6

Water 22.1

Perfume, dyes, miscellaneous minors balance.

In further embodiments, the enzymes of the invention are included at 0.02 wt % . 2016200189 20 Feb 2017 - 12-

The viscosity of the above exemplary composition is 2700 cps at 20 s'1. However, lower viscosity formulations are possible.

Unless stated otherwise, all proportions are given in weight percent by weight of the 5 total composition.

It is of course to be understood that the invention is not intended to be restricted to the details of the above embodiment which are described by way of example only. 10 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. 15

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general 20 knowledge in the field of endeavour to which this specification relates.

Claims

The claims defining the invention are as follows:

1. A low temperature active enzymatic fabric care composition comprising the combination of one or more primary care enzymes and one or more auxiliary care enzymes wherein the one or more primary care enzymes comprises one or more enzymes of the Glycoside Hydrolase Family 45.
2. A low temperature active enzymatic fabric care composition according to claim 1, wherein the total amount of enzymes of the invention ie. the total amount of the primary care enzyme and the auxiliary care enzyme is 0.01 -1.75 mg per Litre of the wash liquor.
3. A low temperature active enzymatic fabric care composition according to any one of the preceding claims, wherein the total amount of enzymes of the invention ie. the total amount of the primary care enzyme and the auxiliary care enzyme is 0.1 - 84 mg per dose of total composition.
4. A low temperature active enzymatic fabric care composition according to any one of the preceding claims wherein the auxiliary care enzyme is present in equal amounts with the primary care enzyme.
5. A low temperature active enzymatic fabric care composition according to any one of claims 1 to 3 wherein the auxiliary care enzyme is present in an amount greater than the primary care enzyme.
6. A low temperature active enzymatic fabric care composition according to any one of the preceding claims wherein the primary care enzyme and the auxiliary care enzyme are present in a weight ration of enzyme protein in the range selected from 1:1 to 1:29, or 1:1 to 1:9, or 1:1 to 1:5, or 1:1 to 1:1.9.
7. A low temperature active enzymatic fabric care composition according to any one of the preceding claims wherein the primary care enzyme and the auxiliary care enzyme are present in a weight ratio of enzyme protein excluding 1:30, or 1:10, or 1:2.
8. A low temperature active enzymatic fabric care composition according to any one of the preceding claims wherein one or more primary care enzymes comprises one or cellulolytic enzymes active in restoring colour to cotton or cotton-based fabrics by removal of fuzz and pills from the surface of the fabric.
9. A low temperature active enzymatic fabric care composition according to any one of the preceding claims wherein one or more auxiliary care enzymes comprise one or more glycosyl hydrolases of Family 5 and/or Family 7.
10. A low temperature enzymatic fabric care process comprising the step of treating a fabric with the composition of any one of the preceding claims.
11. Use of an auxiliary care enzyme in combination with a primary care enzyme in the treatment of fabrics for a care benefit wherein the primary care enzyme comprises an enzyme of the Glycoside Hydrolase Family 45.
12. Use of a low temperature active enzymatic fabric care composition comprising the combination of one or more primary care enzymes and one or more auxiliary care enzymes, in the treatment of fabrics for a care benefit, wherein the one or more primary care enzymes comprises one or more enzymes of the Glycoside Hydrolase Family 45.