EP2744882A1 - Enzyme system - Google Patents

Enzyme system

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Publication number
EP2744882A1
EP2744882A1 EP12751483.4A EP12751483A EP2744882A1 EP 2744882 A1 EP2744882 A1 EP 2744882A1 EP 12751483 A EP12751483 A EP 12751483A EP 2744882 A1 EP2744882 A1 EP 2744882A1
Authority
EP
European Patent Office
Prior art keywords
enzymes
aha
psychrophilic
mesophilic
stainzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP12751483.4A
Other languages
German (de)
French (fr)
Inventor
Neil James Parry
Stephen Wilson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unilever PLC
Unilever NV
Original Assignee
Unilever PLC
Unilever NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Unilever PLC, Unilever NV filed Critical Unilever PLC
Priority to EP12751483.4A priority Critical patent/EP2744882A1/en
Publication of EP2744882A1 publication Critical patent/EP2744882A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
    • C11D2111/10—Objects to be cleaned
    • C11D2111/12—Soft surfaces, e.g. textile

Definitions

  • the present invention concerns the delivery of enzymes in a washing process.
  • the use of enzyme mixtures during a laundering process is known.
  • a first aspect of the present invention provides an enzymatic fabric treatment composition comprising the combination of:
  • a second aspect of the invention provides a method of treating a fabric using an enzymatic fabric treatment composition according to the first aspect wherein the temperature of the wash liquor varies between psychrophilic and mesophilic and optionally thermophilic temperatures throughout a single washing cycle.
  • the term "single washing cycle" as used herein means a single washing process, including one or more aqueous washing phases and one or more aqueous rinse phases. The cycle is completed after a final rinsing and the fabric is then ready for drying and re-use. The cycle may include a pre-treatment phase. Preferably the temperature varies between psychrophilic and mesophilic and thermophilic temperatures throughout a single washing cycle.
  • the invention is highly advantageous where the temperature of the wash liquor is uncontrolled in at least one part by the user.
  • the wash liquor temperature is uncontrolled in a substantial amount and more preferably is uncontrolled to the extent that temperature is entirely driven by ambient conditions.
  • the method may comprise a hand-washing treatment or be carried out in a washing machine, the washing machine preferably without any water heating.
  • wash liquor temperature is often both varying and uncontrolled. Varying of the temperature may happen within a single washing process or over successive washes.
  • the washing water may be drawn from a tap fed by underground pipes and the washing process may begin at low temperatures (5-15 °C) and then rise under warmer ambient conditions to 20°C, 30°C, 40°C or higher.
  • Varying climatic conditions mean ambient temperatures differ seasonally at the least.
  • pyschrophilic enzyme means enzymes that are effective at a temperature of 0°C - 25°C.
  • the term "mesophilic enzyme” means enzymes that are effective at a temperature in the range 25°C-50°C.
  • thermoophilic means enzymes that are effective at a temperature in the range 50°-90° C.
  • the term “effective” means that the enzyme has the ability to achieve stain removal or catalytic capability (in the given temperature zone).
  • the term “enzyme” includes enzyme variants (produced, for example, by recombinant techniques). 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).
  • stain removal is measured in terms of Remission units or a Remission index. Effective stain removal is preferably represented by remission equal to or greater than 2 Remission units.
  • treatment in the context of enzymatic fabric treatment composition preferably means cleaning and more preferably stain removal.
  • Enzymes may be from bacterial or fungal origin. Chemically modified or protein engineered mutants are included.
  • the one or more psychrophilic and/or one or more mesophilic and/or one or more thermophilic enzymes comprise a common class of enzymes. This has the advantage of addressing a particular type of stains at different
  • the common class is a protease or a lipase or a glycosyl hydrolase or a lyase or a oxidoreductase.
  • the invention may comprise a mixed class of enzymes.
  • this comprises a psychrophilic lipase and a mesophilic protease.
  • the advantage of this combination is with washing processes which begin with low temperature, in which the lipase is effective but where the protease is inhibited from attacking the lipase.
  • the one or more psychrophilic enzymes comprise esterases (ester hydrolases) and more preferably carboxylic ester hydrolases and more preferably e.g. lipases and/or phospholipases.
  • esterases ester hydrolases
  • carboxylic ester hydrolases preferably e.g. lipases and/or phospholipases.
  • lipases are highly advantageous psychrophilic enzymes because fats and oil based stains are more difficult to remove at psychrohilic temperatures.
  • the one or more psychrophilic enzymes comprise glycosyl hydrolases (glycosylases) for example cellulases, amylases (including alpha-amylases), xylanases, etc.
  • Psychrophilic lipases include lipases from Acinetobacter sp. Strain No. 6 (Suzuki et al. (2001 ) J. Biosci. Bioeng. 92: 144-148; Acinetobacter sp. Strain No.Oi 6
  • Psychrophilic esterases preferably include esterases EstATI and EstAT1 1 described by Jeon et al. Mar Biotechnol (2009) 1 1 :307-316.
  • Psychrophlic glycosyl hyrdolases preferably include glycosidases such as amylases, eg. a-amylases from Pseudoalteromonas haloplanktis strain TAC 125 and from Alteromonas haloplanktis A23 (Feller et al (1998) Journal Biological Chemistry Vol 273, No. 20 pp 12109-121 15) and from Nocardiopsis sp. 7326; cellulases and xylanase from e.g. Clostridium sp. PXYL1 (G. Akila, T.S.Chandra (2003) FEMS Microbiol. Letters 219, 63-67). Psychrophilic xylanases include E.coli phagemid (Lee et al. 2006b).
  • Preferred pyschrophilic proteases include those derived from Flavobacterium balustinum P104 (isolated from the internal organs of salmon and has been deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology as the deposit number of FERM BP-5006 on February 17, 1995 and described in WO/1996/025489) and from Arthrobacter globiformis S155 (Poitier et al, (1995) J. Gen. Microbiol. 133:2797-2806).
  • Pschrophilic lyases preferably include pectate lyases e.g. from
  • the one or more mesophilic enzymes comprise proteases and/or glycosidases and/or pectate lyases.
  • Preferred mesophilic proteases include serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease.
  • Alkaline proteases include subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168.
  • Trypsin-like i.e. capable of cleaving peptide bonds at the C-terminal side of lysine or arginine.
  • proteases may be of porcine or bovine origin. Fusarium derived trypsin proteases are also included.
  • protease enzymes include AlcalaseTM, SavinaseTM, PrimaseTM, DuralaseTM, DyrazymTM, EsperaseTM, EverlaseTM, PolarzymeTM, and KannaseTM, (Novozymes A/S), MaxataseTM, MaxacalTM, MaxapemTM,
  • Preferred mesophilic lipases include lipases from Humicola (synonym
  • Thermomyces e.g. from H. lanuginosa (7. lanuginosus) or from H. insolens, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes,
  • mesophilic lipase enzymes include LipolaseTM and CrelipolaseTM
  • Lipolase UltraTM LipexTM (Novozymes A/S).
  • Preferred mesophilic Phospholipases include enzymes which hydrolyse phospholipids.
  • Phospholipases Ai and A 2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in lysophospholipid are included as are Phospholipase C and phospholipase D (phosphodiesterases)which release diacyl glycerol or phosphatidic acid respectively.
  • phospholipase A used herein in connection with an enzyme of the invention is intended to cover an enzyme with Phospholipase Ai and/or
  • Phospholipase A 2 activity The phospholipase activity may be provided by enzymes having other activities as well, such as, e.g., a lipase with phospholipase activity.
  • the mesophilic phospholipase may be of any origin, e.g., of animal origin (such as, e.g., mammalian), e.g. from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom.
  • animal origin such as, e.g., mammalian
  • pancreas e.g., bovine or porcine pancreas
  • snake venom or bee venom e.g., from snake venom or bee venom.
  • the phospholipase may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g., A. niger, Dictyostelium, e.g., D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtil
  • Rhizomucor e.g., R. pusillus
  • Rhizopus e.g. R. arrhizus, R. japonicus, R.
  • Sclerotinia e.g., S. libertiana
  • Trichophyton e.g. T. rubrum
  • Whetzelinia e.g., W. sclerotiorum
  • Bacillus e.g., B. megaterium, B. subtilis
  • Citrobacter e.g., C. freundii
  • Enterobacter e.g., E. aerogenes, E. cloacae
  • Edwardsiella E. tarda
  • Erwinia e.g., E. herbicola
  • Escherichia e.g., E. coli
  • Klebsiella e.g., K. pneumoniae
  • Proteus e.g., P. vulgaris
  • Providencia e.g., P. stuartii
  • Salmonella e.g. S. typhimurium
  • Serratia e.g., S. liquefasciens, S.
  • the phospholipase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum.
  • the class Pyrenomycetes such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum.
  • phospholipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus,
  • Preferred mesophilic phospholipases are derived from a strain of Humicola, especially Humicola lanuginosa or variant; and from strains of Fusarium, especially Fusarium oxysporum.
  • the phospholipase may be derived from
  • mesophilic phospholipases comprise a phospholipase Ai (EC.
  • mesophilic phospholipases examples include LECITASETM and LECITASETM ULTRA, YIELSMAX, or LIPOPAN F (available from Novozymes A/S, Denmark).
  • Preferred mesophilic cutinases are derived from a strain of
  • Aspergillus in particular Aspergillus oryzae, a strain of Alternaria, in particular Alternaria brassiciola, a strain of Fusarium, in particular Fusarium solani,
  • Fusarium solani pisi Fusarium roseum culmorum, or Fusarium roseum
  • sambucium a strain of Heiminthosporum, in particular Heiminthosporum sativum, a strain of Humicoia, in particular Humicoia insoiens, a strain of Pseudomonas, in particular Pseudomonas mendocina, or Pseudomonas putida, a strain of
  • Rhizoctonia in particular Rhizoctonia solani
  • a strain of Streptomyces in particular Streptomyces scabies
  • a strain of Ulocladium in particular Ulocladium
  • cutinase is derived from a strain of Humicoia insoiens, in particular the strain Humicoia insoiens DSM 1800.
  • cutinases include NOVOZYMTM 51032 (available from Novozymes A/S, Denmark).
  • Preferred mesophilic amylases are included for example, alpha-amylases obtained from Bacillus, e.g. from strains of B. licheniformis
  • mesophilic amylases are DuramylTM, TermamylTM,
  • Preferred mesophilic cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicoia, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum.
  • Especially preferred mesophilic cellulases are the alkaline or neutral cellulases having color care benefits.
  • Commercially available cellulases include
  • Preferred mesophilic pectate lyases include pectate lyases that are derived/cloned from bacterial genera such as Erwinia, Pseudomonas, Klebsiella and
  • alkaline mesophilic pectate lyases examples include BIOPREPTM and SCOURZYMETM L from Novozymes A/S, Denmark.
  • Preferred mesophilic mnanases include derived from a strain of the filamentous fungus genus Aspergillus, preferably Aspergillus niger or Aspergillus aculeatus or Trichoderma reseei or from the Bacillus microorganism FERM P- 8856 which produces beta-mannanase and beta-mannosidase or from alkalophilic Bacillus sp. AM-001 or from Bacillus amyloliquefaciens.
  • the mannanase may comprise alkaline family 5 and 26 mannanases derived from Bacillus
  • mannanases examples include MannawayTM available from Novozymes A/S Denmark.
  • Preferred mesophilic peroxidases/oxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof.
  • Commercially available peroxidases include GuardzymeTM and NovozymTM 51004 (Novozymes A/S).
  • Thermophilic proteases include proteases derived from Thermophilic Bacillus strain HS08 (African Journal of Biotechnology Vol. 5 (24), pp. 2433-2438, 18 December 2006) and B.Stearothermophilius 1503; Thermos caldophilus GK24; T. Aquaticus lZS ; T. aquaticus Yl Aq. l and Aq. II.
  • Thermophilic Lipases include those derived from Bacillus thermocatenulatus BTL1 and preferably BTL2 (Schimdt-Dannert et at., Biochim. Biophys. Acta (1994) 1214, pp. 43-5 and Biochim. Biophys. Acta (1996) 1301 , pp. 105-1 14).
  • Thermophilic glycosyl hydrolases include alpha-amylases from B.
  • Thermophilic lyases include the pectate lyases from Thermoanaerobacter italicus sp. nov. strain Ab9 (Kozianowski et al., (1997) Extremophiles Vol 1 , 4: 171 -182).
  • 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 direct application e.g. roll-on or spray-on compositions which may be used as a pre-treatment of e.g. localised portions of fabric prior to a 'main' wash.
  • the enzymes may be present at 0-5 wt%, preferably 2-4 wt%, and most preferably 2.5-3.5 wt% of the composition (where wt% means percentage of the total weight of the composition).
  • the total protein concentration (of the total range of enzymes according to the invention) in the wash liquor preferably ranges from 0.01 to 10.0 mg /L and more preferably 2 to 5 mg/L.
  • composition will comprise individual levels of psychrophilic/mesophilic and optionally thermophilic enzymes
  • the enzymes may be the sole fabric treatment agent or other stain removal agents may be incorporated.
  • 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), anti- corrosion 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.
  • fabric conditioners including clays, foam boosters, suds suppressors (anti-foams), anti- corrosion 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.
  • 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.
  • Figures 1 -3 correspond with Tables 1 -3a,b respectively, and show effectiveness data for enzymes AHA and Stainzyme separately and both together in various conditions.
  • an alpha amylase (hereafter termed AHA) was identified from peer-reviewed literature as having desirable psychrophilic properties (Feller, G. et al, 1992; J. Biol. Chem. 267 (8), 5217-5221 ).
  • the gene sequence (GenBank accession number: X58627.1 ) comprising the amylase was accessed from a publicly available database
  • FIG. 1 shows the modified sequence, for protein production in E. coli and figure 3 is the sequence of AHA once incorporated into the pUC19 vector.
  • the sequence shown in figure 2 was chemically synthesized and inserted into the pUC19 plasmid (as shown in figure 3) as a service by Eurogentec Ltd (Southampton, UK) using their own proprietary methodologies.
  • amylase The constitutive production of amylase was confirmed using reagents supplied by Roche Diagnostics in kit form (catalogue number 1 1876473 316) in 96-well microtitre plates as follows: 10 ⁇ of each sample to be assessed (or dilution thereof) was added to a well of the microtitre plate (in duplicate). 75 ⁇ reagent 1 was added followed by 15 ⁇ of reagent 2.
  • An assay blank comprised the additions described, replacing AHA-containing growth media with unused LB. Plates were incubate for 30 minutes at prescribed temperatures (normally 20°C) and then assessed for p-nitrophenol release (as a result of amylase activity) on a FluoStar Optima microplate reader set for absorbance detection at 405nm.
  • Enzyme combinations A, B, C, D, E are as follows.
  • the combination comprises the psychrophilic enzyme/s and the mesophilic enzyme and/or the thermophilic enzyme.
  • the a-amylases enzymatic activity of AHA, Stainzyme and of AHA and Stainzyme together between the temperatures of 20°C and 50°C was determined using reagents supplied by Roche Diagnostics in kit form (catalogue number 1 1876473 316).
  • the instrument was set up to run a temperature gradient between 20°C and 50°C.
  • the samples aliquoted into the 96-well PCR plate were incubated for exactly 30 minutes under the above conditions, at which point the PCR plate was rapidly cooled to 4°C.
  • 50 ⁇ of 1 M tris.base (unbuffered) was added to each well to stop the reaction (prevention of further p-nitrophenol reaction product release).
  • 10 ⁇ of each reaction cocktail was transferred to a clear flat-bottomed microtitre plate and assessed for absorbance at 405nm using a Fluostar Optima plate reader. Blank values at each tested temperature were subtracted from the results obtained for enzyme- containing cocktails. Results are shown in Figure 1 .
  • the instrument was set up to run a temperature gradient between 20 and 50°C.
  • the samples aliquoted into the 96-well PCR plate were incubated for exactly 30 minutes under the above conditions, at which point the PCR plate was rapidly cooled to 4°C.
  • 50 ⁇ of 1 M tris.base (unbuffered) was added to each well to stop the reaction (prevention of further p-nitrophenol reaction product release).
  • 10 ⁇ of each reaction cocktail was transferred to a clear flat-bottomed microtitre plate and assessed for absorbance at 405nm using a Fluostar Optima plate reader. Blank values at each tested temperature were subtracted from the results obtained for enzyme- containing cocktails. Results are shown in Figure 2.
  • the instrument was set up to run a temperature gradient between 20 and 50°C.
  • the samples aliquoted into the 96-well PCR plate were incubated for exactly 30 minutes under the above conditions, at which point the PCR plate was rapidly cooled to 4°C.
  • 50 ⁇ of 1 M tris.base (unbuffered) was added to each well to stop the reaction (prevention of further p-nitrophenol reaction product release).
  • 10 ⁇ of each reaction cocktail was transferred to a clear flat-bottomed microtitre plate and assessed for absorbance at 405nm using a Fluostar Optima plate reader. Blank values at each tested temperature were subtracted from the results obtained for enzyme- containing cocktails. Results are shown in Figure 3.
  • Table 1 a and 1 b Activity data for AHA, Stainzyme and both enzymes together, where stock solutions of each enzyme were diluted 1 in 125 for use in assay (data values are Absorbance readings using a Fluostar Optima microplate reader set to 405nm, after incubation with assay substrate for 30 minutes in a gradient of temperatures between 20°C and 50°C). Graphically represented in Figure 5.
  • n data Mean value 1.044 1.060 1.116 1.171 1.157 1.108 (0.5x Mean minus 0.913 0.931 0.988 1.039 1.021 0.971
  • AHA data Raw data value 1 1.674 1.709 1.770 1.742 1.653 1.444
  • Tables 2a, 2b Activity data for AHA, Stainzyme and both enzymes together, where stock solutions of each enzyme were diluted 1 in 250 for use in assay (data values are Absorbance readings using a Fluostar Optima microplate reader set to 405nm, after incubation with assay substrate for 30 minutes in a gradient of temperatures between 20°C and 50°C). Graphically represented in Figure 2. Table 2a Temperatures 22.5
  • Table 3 Activity data for AHA, Stainzyme and both enzymes together, where stock solutions of each enzyme were diluted 1 in 417 for use in assay (data values are Absorbance readings using a Fluostar Optima microplate reader set to 405nm, after incubation with assay substrate for 30 minutes in a gradient of temperatures between 20°C and 50°C). Graphically represented in Figure 3. Table 3a Temperatures 22.5
  • Stainzyme mean blank value 0.513 0.518 0.545 0.566 0.561 0.525 + 0.5x 1 Standard
  • AHA data Raw data value 1 0.906 0.925 0.947 0.924 0.871 0.752
  • AHA and Stainzyme a-amylase enzymes were diluted to appropriate concentrations (as indicated) using 100mM HEPES buffer pH7 plus 100mM sodium chloride and 1 mM calcium chloride.
  • Table 4 Data demonstrating stain removal of C-S-27 potato starch-stained fabric by AHA and Stainzyme, either alone or in combination. Wash temperatures were 10°C, 20°C and 60°C (top, middle and bottom panels respectively). Data is expressed as SRI's and replicates are presented along with the arithmetic mean values and the standard deviation (1 SD).
  • Non-Limiting examples of laundry enzymatic fabric treatment compositions are described below where the following example enzyme combinations are: A. Lipase Combination
  • Psychrophilic Lipase from Pseudoalteromonas sp W27.
  • Thermophilic Lipase from Bacillus thermocatenlatus BTL2 B. Protease Combination
  • Thermophilic Protease from Thermophilic Bacillus strain HS08
  • Psychrophilic amylase alpha-amylase from Pseudoalteromonas haloplanktis strain TAC 125 and/or Pseudoalteromonas haloplanktis strain TAB 23 (as above)
  • Mesophilic amylase StainzymeTM 2.0 T (Novozymes)
  • Thermophilic amylase alpha-amylase from B. stearothermophilus Donk BS-1 .
  • Psychrophilic pectate lyase from Pseudoalteromonas haloplanktis ANT/505 Mesophilic pectate lyase: BioprepTM Novozymes
  • Thermophilic pectate lyase from Thermoanaerobacter italicus sp. Nov. AB9.
  • Psychrophilic Lipase from Pseudoalteromonas sp W27 and
  • Thermophilic amylase alpha-amylase from B. stearothermophilus Donk BS-1 .
  • Liquid Enzymatic Fabric Treatment Composition comprising enzyme combinations above :
  • Sequestrant (Dequest 2066) 0.5 Fluorescor 0.3

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Abstract

An enzymatic fabric treatment composition comprising the combination of (i) one or more psychrophilic enzymes, and (ii) one or more mesophilic enzymes and/or one or more thermophilic enzymes.

Description

ENZYME SYSTEM
The present invention concerns the delivery of enzymes in a washing process. The use of enzyme mixtures during a laundering process is known.
An objective is to provide an improved washing/stain removal process involving enzymes for a wash liquor with variable uncontrolled temperature. A first aspect of the present invention provides an enzymatic fabric treatment composition comprising the combination of:
1 . one or more psychrophilic enzymes; and
2. one or more mesophilic enzymes and/or one or more thermophilic enzymes. A second aspect of the invention provides a method of treating a fabric using an enzymatic fabric treatment composition according to the first aspect wherein the temperature of the wash liquor varies between psychrophilic and mesophilic and optionally thermophilic temperatures throughout a single washing cycle. The term "single washing cycle" as used herein means a single washing process, including one or more aqueous washing phases and one or more aqueous rinse phases. The cycle is completed after a final rinsing and the fabric is then ready for drying and re-use. The cycle may include a pre-treatment phase. Preferably the temperature varies between psychrophilic and mesophilic and thermophilic temperatures throughout a single washing cycle.
The invention is highly advantageous where the temperature of the wash liquor is uncontrolled in at least one part by the user. Preferably the wash liquor temperature is uncontrolled in a substantial amount and more preferably is uncontrolled to the extent that temperature is entirely driven by ambient conditions. The method may comprise a hand-washing treatment or be carried out in a washing machine, the washing machine preferably without any water heating. With the invention, the combined problems of varying and uncontrolled (by the user) wash liquor temperature are both addressed in the context of enzyme performance. Wash liquor temperature is often both varying and uncontrolled. Varying of the temperature may happen within a single washing process or over successive washes. For example, in warm climates, for hand washing the washing water may be drawn from a tap fed by underground pipes and the washing process may begin at low temperatures (5-15 °C) and then rise under warmer ambient conditions to 20°C, 30°C, 40°C or higher. Varying climatic conditions mean ambient temperatures differ seasonally at the least. With the invention, these problems are addressed by providing a combination of enzymes which offer desirable functionality within different temperature zones. Even with automatic washing machines the temperature of the wash liquor may vary over the lifetime of one laundry product.
As used herein the term "pyschrophilic enzyme" means enzymes that are effective at a temperature of 0°C - 25°C.
As used herein the term "mesophilic enzyme" means enzymes that are effective at a temperature in the range 25°C-50°C. As used herein the term "thermophilic" means enzymes that are effective at a temperature in the range 50°-90° C.
As used herein the term "effective" means that the enzyme has the ability to achieve stain removal or catalytic capability (in the given temperature zone). As used herein the term "enzyme" includes enzyme variants (produced, for example, by recombinant techniques). 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). Preferably stain removal is measured in terms of Remission units or a Remission index. Effective stain removal is preferably represented by remission equal to or greater than 2 Remission units.
As used herein the term "treatment" in the context of enzymatic fabric treatment composition preferably means cleaning and more preferably stain removal.
Enzymes may be from bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Preferably, the one or more psychrophilic and/or one or more mesophilic and/or one or more thermophilic enzymes comprise a common class of enzymes. This has the advantage of addressing a particular type of stains at different
temperatures. In this case preferably the common class is a protease or a lipase or a glycosyl hydrolase or a lyase or a oxidoreductase.
Alternatively the invention may comprise a mixed class of enzymes. Preferably this comprises a psychrophilic lipase and a mesophilic protease. The advantage of this combination is with washing processes which begin with low temperature, in which the lipase is effective but where the protease is inhibited from attacking the lipase.
Psychrophilic Enzymes
Preferably the one or more psychrophilic enzymes comprise esterases (ester hydrolases) and more preferably carboxylic ester hydrolases and more preferably e.g. lipases and/or phospholipases. Lipases are highly advantageous psychrophilic enzymes because fats and oil based stains are more difficult to remove at psychrohilic temperatures.
Phospholipases are advantageous for pyschrophilic for much the same reason. Advantageously, alternatively or additionaly, the one or more psychrophilic enzymes comprise glycosyl hydrolases (glycosylases) for example cellulases, amylases (including alpha-amylases), xylanases, etc.
Psychrophilic lipases include lipases from Acinetobacter sp. Strain No. 6 (Suzuki et al. (2001 ) J. Biosci. Bioeng. 92: 144-148; Acinetobacter sp. Strain No.Oi6
(Brueuil and Kushner, (1975) Can. J. Microbiol. 21 :423-433; Achromobacter lipolyticum (Khan et al. , (1967), Biochem. Biophys. Acta. 132:68-77 1967), Aeromonas sp. Strain No. LPB 4 (Lee et al. (2003), J. Microbiol. 41 :22-27, Aeromonas hydrophiia (Pemberton et al. (1997) FEMS Microbiol. Lett. 152: 1 -10 ); Bacillus sphaericus MTCC 7526 (Joseph. PhD Thesis(2006) Allahabad
Agricultural Institute, Allahabd, IN); Microbacterium phyllosphaerae MTCC 7530, Moraxell sp. (Feller et al. (1990) FEMS Microbiol. Lett. 66:239-244; Moraxella sp TA144 (Feller et al. (1991 ) Gene.102: 1 1 1 -1 15; Photobacterium lipolyticum M37 (Ryu et al. (2006) Appl. Microbiol. Biotechnol. 70: 321 -326); Pseudoalteromonas sp. Wp27 (Zeng et al. (2004) J. Microbiol. Biotechnol. 14: 952-958 );
Pseudoalteromonas sp. (Giudice et al. (2006) J. Applied Microbiology 101 : 1039- 1048, Pscychrobacter sp. and Vibrio sp. ; Psychrobacter sp. Wp37 (Zeng et al. (2004) J. Microbiol. Biotechnol. 14: 952-958 ); Psychrobacter okhotskensis sp. (Yumoto et al. (2003) Int. J. Syst. Evol. Microbiol. 53: 1985-1989 ); Psychrobacter sp. Ant300 (Kulakovaa et al. (2004) Biochemica. Biophysica. Acta. 1696:59-65); Psychrobacter immobilis strain B 10 (Arpigny et al. (1997) J. Mol. Catal. B Enzy. 3: 29-35.), Serratia marcescens (Abdou, (2003) J. Dairy Sci. 86: 127-132,
Staphylococcus aureus (Alford and Pierce, (1961 ) J. Food Sci. 26:518-524), Staphylococcus epidermidis (Joseph et al. (2006) J. Gen. Appl. Microbiol. 52: 315- 320). Psychrophilic esterases preferably include esterases EstATI and EstAT1 1 described by Jeon et al. Mar Biotechnol (2009) 1 1 :307-316.
Psychrophlic glycosyl hyrdolases preferably include glycosidases such as amylases, eg. a-amylases from Pseudoalteromonas haloplanktis strain TAC 125 and from Alteromonas haloplanktis A23 (Feller et al (1998) Journal Biological Chemistry Vol 273, No. 20 pp 12109-121 15) and from Nocardiopsis sp. 7326; cellulases and xylanase from e.g. Clostridium sp. PXYL1 (G. Akila, T.S.Chandra (2003) FEMS Microbiol. Letters 219, 63-67). Psychrophilic xylanases include E.coli phagemid (Lee et al. 2006b).
Preferred pyschrophilic proteases include those derived from Flavobacterium balustinum P104 (isolated from the internal organs of salmon and has been deposited in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology as the deposit number of FERM BP-5006 on February 17, 1995 and described in WO/1996/025489) and from Arthrobacter globiformis S155 (Poitier et al, (1995) J. Gen. Microbiol. 133:2797-2806).
Pschrophilic lyases preferably include pectate lyases e.g. from
Pseudoalteromonas haloplanktis strain ANT/505 (Truong et al (2001 )
Extremophiles 5: 35-44).
Mesophilic enzymes
Preferably, the one or more mesophilic enzymes comprise proteases and/or glycosidases and/or pectate lyases.
Preferred mesophilic proteases include serine protease or a metallo protease, preferably an alkaline microbial protease or a trypsin-like protease. Alkaline proteases include subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168.
Trypsin-like (i.e. capable of cleaving peptide bonds at the C-terminal side of lysine or arginine.) Such proteases may be of porcine or bovine origin. Fusarium derived trypsin proteases are also included.
Commercially available protease enzymes include Alcalase™, Savinase™, Primase™, Duralase™, Dyrazym™, Esperase™, Everlase™, Polarzyme™, and Kannase™, (Novozymes A/S), Maxatase™, Maxacal™, Maxapem™,
Properase™, Purafect™, Purafect OxP™, FN2™, and FN3™ (Genencor
International Inc.). Preferred mesophilic lipases include lipases from Humicola (synonym
Thermomyces), e.g. from H. lanuginosa (7. lanuginosus) or from H. insolens, a Pseudomonas lipase, e.g. from P. alcaligenes or P. pseudoalcaligenes,
P. cepacia, P. stutzeri, P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P. wisconsinensis, a Bacillus lipase, e.g. from B. subtiiis (Dartois et al. (1993), Biochemica et Biophysica Acta, 1 131 , 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).
Commercially available mesophilic lipase enzymes include Lipolase™ and
Lipolase Ultra™, Lipex™ (Novozymes A/S).
Preferred mesophilic Phospholipases (EC 3.1 .1 .4 and/or EC 3.1 .1 .32) include enzymes which hydrolyse phospholipids. Phospholipases Ai and A2 which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B) which can hydrolyze the remaining fatty acyl group in lysophospholipid are included as are Phospholipase C and phospholipase D (phosphodiesterases)which release diacyl glycerol or phosphatidic acid respectively.
The term "phospholipase A" used herein in connection with an enzyme of the invention is intended to cover an enzyme with Phospholipase Ai and/or
Phospholipase A2 activity. The phospholipase activity may be provided by enzymes having other activities as well, such as, e.g., a lipase with phospholipase activity.
The mesophilic phospholipase may be of any origin, e.g., of animal origin (such as, e.g., mammalian), e.g. from pancreas (e.g., bovine or porcine pancreas), or snake venom or bee venom. Preferably the phospholipase may be of microbial origin, e.g., from filamentous fungi, yeast or bacteria, such as the genus or species Aspergillus, e.g., A. niger, Dictyostelium, e.g., D. discoideum; Mucor, e.g. M. javanicus, M. mucedo, M. subtilissimus; Neurospora, e.g. N. crassa;
Rhizomucor, e.g., R. pusillus; Rhizopus, e.g. R. arrhizus, R. japonicus, R.
stolonifer, Sclerotinia, e.g., S. libertiana; Trichophyton, e.g. T. rubrum;
Whetzelinia, e.g., W. sclerotiorum; Bacillus, e.g., B. megaterium, B. subtilis;
Citrobacter, e.g., C. freundii; Enterobacter, e.g., E. aerogenes, E. cloacae
Edwardsiella, E. tarda; Erwinia, e.g., E. herbicola; Escherichia, e.g., E. coli;
Klebsiella, e.g., K. pneumoniae; Proteus, e.g., P. vulgaris; Providencia, e.g., P. stuartii; Salmonella, e.g. S. typhimurium; Serratia, e.g., S. liquefasciens, S.
marcescens; Shigella, e.g., S. flexneri; Streptomyces, e.g., S. violeceoruber, Yersinia, e.g., V. enterocolitica. Thus, the phospholipase may be fungal, e.g., from the class Pyrenomycetes, such as the genus Fusarium, such as a strain of F. culmorum, F. heterosporum, F. solani, or a strain of F. oxysporum. The
phospholipase may also be from a filamentous fungus strain within the genus Aspergillus, such as a strain of Aspergillus awamori, Aspergillus foetidus,
Aspergillus japonicus, Aspergillus niger or Aspergillus oryzae. Preferred mesophilic phospholipases are derived from a strain of Humicola, especially Humicola lanuginosa or variant; and from strains of Fusarium, especially Fusarium oxysporum. The phospholipase may be derived from
Fusarium oxysporum DSM 2672. Preferably mesophilic phospholipases comprise a phospholipase Ai (EC.
3.1 .1 .32). or a phospholipase A2 (EC.3.1 .1.4.). Examples of commercial mesophilic phospholipases include LECITASE™ and LECITASE™ ULTRA, YIELSMAX, or LIPOPAN F (available from Novozymes A/S, Denmark). Preferred mesophilic cutinases (EC 3.1 .1 .74.) are derived from a strain of
Aspergillus, in particular Aspergillus oryzae, a strain of Alternaria, in particular Alternaria brassiciola, a strain of Fusarium, in particular Fusarium solani,
Fusarium solani pisi, Fusarium roseum culmorum, or Fusarium roseum
sambucium, a strain of Heiminthosporum, in particular Heiminthosporum sativum, a strain of Humicoia, in particular Humicoia insoiens, a strain of Pseudomonas, in particular Pseudomonas mendocina, or Pseudomonas putida, a strain of
Rhizoctonia, in particular Rhizoctonia solani, a strain of Streptomyces, in particular Streptomyces scabies, or a strain of Ulocladium, in particular Ulocladium
consortiale. Most preferably cutinase is derived from a strain of Humicoia insoiens, in particular the strain Humicoia insoiens DSM 1800.
Commercial cutinases include NOVOZYM™ 51032 (available from Novozymes A/S, Denmark). Preferred mesophilic amylases (alpha and/or beta) are included for example, alpha-amylases obtained from Bacillus, e.g. from strains of B. licheniformis
NCIB8059, ATCC6634, ATCC6598, ATCC11945, ATCC 8480, ATCC9945a, or the Bacillus sp. strains DSM 12649 (AA560 alpha-amylase) or Bacillus sp. DSM 12648 (AA349 alpha-amylase).
Commercially available mesophilic amylases are Duramyl™, Termamyl™,
Termamyl Ultra™, Natalase™, Stainzyme™, Fungamyl™ and BAN™
(Novozymes A/S), Rapidase™ and Purastar™ (from Genencor International Inc.).
Preferred mesophilic cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicoia, Fusarium, Thielavia, Acremonium, e.g. the fungal cellulases produced from Humicola insolens, Thielavia terrestris, Myceliophthora thermophila, and Fusarium oxysporum.
Especially preferred mesophilic cellulases are the alkaline or neutral cellulases having color care benefits. Commercially available cellulases include
Celluzyme™, Carezyme™, Endolase™, Renozyme™ (Novozymes A/S),
Clazinase™ and Puradax HA™ (Genencor International Inc.), and KAC-500(B)™ (Kao Corporation). Preferred mesophilic pectate lyases include pectate lyases that are derived/cloned from bacterial genera such as Erwinia, Pseudomonas, Klebsiella and
Xanthomonas, as well as from Bacillus subtilis (Nasser et al. (1993) FEBS Letts. 335:319-326) and Bacillus sp. YA-14 (Kim et al. (1994) Biosci. Biotech. Biochem. 58:947-949); Bacillus pumilus (Dave and Vaughn (1971 ) J. Bacteriol. 108: 166- 174), B. polymyxa (Nagel and Vaughn (1961 ) Arch. Biochem. Biophys. 93:344- 352), B. stearothermophilus (Karbassi and Vaughn (1980) Can. J. Microbiol.
26:377-384), Bacillus sp. (Hasegawa and Nagel (1966) J. Food Sci. 31 :838-845) and Bacillus sp. RK9 (Kelly and Fogarty (1978) Can. J. Microbiol. 24: 1 164-1 172. Divalent cation-independent and/or thermostable pectate lyases may be used.
Examples of commercially available alkaline mesophilic pectate lyases include BIOPREP™ and SCOURZYME™ L from Novozymes A/S, Denmark.
Preferred mesophilic mnanases (EC 3.2.1 .78) include derived from a strain of the filamentous fungus genus Aspergillus, preferably Aspergillus niger or Aspergillus aculeatus or Trichoderma reseei or from the Bacillus microorganism FERM P- 8856 which produces beta-mannanase and beta-mannosidase or from alkalophilic Bacillus sp. AM-001 or from Bacillus amyloliquefaciens. The mannanase may comprise alkaline family 5 and 26 mannanases derived from Bacillus
agaradhaerens, Bacillus licheniformis, Bacillus halodurans, Bacillus clausii, Bacillus sp., and Humicola insolens. Examples of commercially available mannanases include Mannaway™ available from Novozymes A/S Denmark. Preferred mesophilic peroxidases/oxidases include peroxidases from Coprinus, e.g. from C. cinereus, and variants thereof. Commercially available peroxidases include Guardzyme™ and Novozym™ 51004 (Novozymes A/S).
Thermophilic Enzymes
Thermophilic proteases include proteases derived from Thermophilic Bacillus strain HS08 (African Journal of Biotechnology Vol. 5 (24), pp. 2433-2438, 18 December 2006) and B.Stearothermophilius 1503; Thermos caldophilus GK24; T. Aquaticus lZS ; T. aquaticus Yl Aq. l and Aq. II.
Thermophilic Lipases include those derived from Bacillus thermocatenulatus BTL1 and preferably BTL2 (Schimdt-Dannert et at., Biochim. Biophys. Acta (1994) 1214, pp. 43-5 and Biochim. Biophys. Acta (1996) 1301 , pp. 105-1 14). Thermophilic glycosyl hydrolases include alpha-amylases from B.
stearothermophilus Donk, strain BS-1 (Journal Biochemistry, Vol 67, 1 :65-75) and from Bacillus sp. ANT-6 (Process Biochemistry (May 2003) Vol 38, 10: 1397-1403) Thermophilic lyases include the pectate lyases from Thermoanaerobacter italicus sp. nov. strain Ab9 (Kozianowski et al., (1997) Extremophiles Vol 1 , 4: 171 -182).
Once each suitable enzyme is chosen according to the invention, it is relatively easy for the skilled man to isolate a suitable micro-organism capable of producing the enzyme and to carry out optimization procedures known in the art for making enzymes which have the required stability/performance in e.g. powder or liquid compositions and/or in certain washing conditions etc. 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 direct application e.g. roll-on or spray-on compositions which may be used as a pre-treatment of e.g. localised portions of fabric prior to a 'main' wash.
The enzymes may be present at 0-5 wt%, preferably 2-4 wt%, and most preferably 2.5-3.5 wt% of the composition (where wt% means percentage of the total weight of the composition).
The total protein concentration (of the total range of enzymes according to the invention) in the wash liquor preferably ranges from 0.01 to 10.0 mg /L and more preferably 2 to 5 mg/L.
The total protein concentration and wt % of composition will comprise individual levels of psychrophilic/mesophilic and optionally thermophilic enzymes
respectively; and within that combination the levels may be lower then e.g. half that used when individual enzymes are used alone. This is possible because of the realisation that whilst a high peak in a one temperature may seem to offer great effectiveness in that temperature range, there is little or no activity outside of this range in a wash process where temperature is uncontrolled. With the invention it is the combination of enzymes with effectiveness across an
uncontrolled temperature profile of the actual wash which achieves effectiveness, and so lower than (e.g. half of the) individual levels which means temperature sensitive enzymatic performance compositions can be provided whilst maintaining the costs of the composition to a reasonable level as total enzyme content is not higher and may even be lower. The enzymes may be the sole fabric treatment agent or other stain removal agents may be incorporated.
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), anti- corrosion 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
Various non-limiting embodiments of the invention will now be more particularly described with reference to the following figures in which:
Figures 1 -3 correspond with Tables 1 -3a,b respectively, and show effectiveness data for enzymes AHA and Stainzyme separately and both together in various conditions. An exemplary method of Cloning, production and purification of a
psychrophilic amylase (from Pseudoalteromonas haloplanktis strain
TAB23):
Method to clone a psychrophilic enzyme from a publically-available database of gene sequences
In an embodiment of the invention an alpha amylase (hereafter termed AHA) was identified from peer-reviewed literature as having desirable psychrophilic properties (Feller, G. et al, 1992; J. Biol. Chem. 267 (8), 5217-5221 ). The gene sequence (GenBank accession number: X58627.1 ) comprising the amylase was accessed from a publicly available database
(http://www.ncbi.nlm.nih.gov/nuccore/2467084) (figure 1 ), and was modified in silico to enable its incorporation into the E. coli compatible plasmid vector pUC19, in a manner that would facilitate expression of the recombinant protein. Figure 2 shows the modified sequence, for protein production in E. coli and figure 3 is the sequence of AHA once incorporated into the pUC19 vector. The sequence shown in figure 2 was chemically synthesized and inserted into the pUC19 plasmid (as shown in figure 3) as a service by Eurogentec Ltd (Southampton, UK) using their own proprietary methodologies. Method to produce the psychrophilic alpha-amylase AHA
10ng of pUC19 plasmid containing AHA-encoding DNA was transformed into chemically competent E. coli strain HB101 , plated out onto LB agar containing 100 g/ml carbenicillin and incubated overnight at 37°C. Single colonies were then used to inoculate 0.5L volumes of LB media containing 100 g/ml carbenicillin (in 2L shake-flasks). Flasks were incubated on an orbital shaker at 120rpm at 18°C for 24-36 hours, at which point the cells were separated from the media
(centrifugation at 10,000g, 15 minutes, 4°C). Media containing the recombinant AHA was stored at 4°C until needed.
The constitutive production of amylase was confirmed using reagents supplied by Roche Diagnostics in kit form (catalogue number 1 1876473 316) in 96-well microtitre plates as follows: 10μΙ of each sample to be assessed (or dilution thereof) was added to a well of the microtitre plate (in duplicate). 75μΙ reagent 1 was added followed by 15μΙ of reagent 2. An assay blank comprised the additions described, replacing AHA-containing growth media with unused LB. Plates were incubate for 30 minutes at prescribed temperatures (normally 20°C) and then assessed for p-nitrophenol release (as a result of amylase activity) on a FluoStar Optima microplate reader set for absorbance detection at 405nm.
Method to purify recombinant AHA
1 -2L at a time of LB growth media containing AHA was first diluted 1 in 2 with sterile distilled water and was then concentrated to between 300 and 500ml using tangential cross-flow filtration using a regenerated cellulose filter device with a nominal molecular weight cut-off of 30,000 daltons. The concentrated LB
(containing the AHA) was then purified by ion-exchange chromatography (using 100ml of Macro-Prep High Q ion-exchange support - BioRad) followed by gel filtration using a 1 .5cm x 60cm S200 chromatography column (GE Healthcare). Fractions containing AHA were identified using the amylase assay described above and presence of a protein at the expected 49000 daltons (along with its purity) was assessed by SDS-PAGE analysis, using a coommassie-based protein stain (Imperial stain - ThermoPierce).
Enzyme combinations A, B, C, D, E are as follows.
In each combination A-D, the combination comprises the psychrophilic enzyme/s and the mesophilic enzyme and/or the thermophilic enzyme.
Enzyme effectiveness is tested in the following compostions Method to demonstrate enhanced catalytic activity of a-amylase
combination solutions across wide temperature ranges including
psychrophilic, mesophilic and thermophilic (terms as defined herein).
The a-amylases enzymatic activity of AHA, Stainzyme and of AHA and Stainzyme together between the temperatures of 20°C and 50°C was determined using reagents supplied by Roche Diagnostics in kit form (catalogue number 1 1876473 316).
Stock solutions of each enzyme (1 mg/ml of AHA, 0.5mg/ml of Stainzyme) was prepared in buffer A (100mM Hepes, 100mM NaCI, 1 mM CaCI, pH7.0). The enzyme stock solutions were further diluted 1 in 125 in buffer A and mixed in reaction cocktails, on ice, as follows:
1 ) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of diluted Stainzyme
2) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of diluted AHA
3) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of buffer A (negative control)
4) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.15ml of diluted Stainzyme, 0.15ml of diluted AHA 10ΟμΙ aliquots were immediately transferred to a 0.2ml thin-walled 96-well PCR plate (2 complete rows per reaction cocktail, giving duplicate readings for each temperature assessed) and incubated on a PCR instrument capable of running a thermal gradient (Techne TC-500) thus:
The instrument was set up to run a temperature gradient between 20°C and 50°C. The samples, aliquoted into the 96-well PCR plate were incubated for exactly 30 minutes under the above conditions, at which point the PCR plate was rapidly cooled to 4°C. During the cooling step, 50μΙ of 1 M tris.base (unbuffered) was added to each well to stop the reaction (prevention of further p-nitrophenol reaction product release). After thorough mixing, 10ΟμΙ of each reaction cocktail was transferred to a clear flat-bottomed microtitre plate and assessed for absorbance at 405nm using a Fluostar Optima plate reader. Blank values at each tested temperature were subtracted from the results obtained for enzyme- containing cocktails. Results are shown in Figure 1 .
In a variation of the described experiment, stock solutions of each enzyme
(1 mg/ml of AHA, 0.5mg/ml Stainzyme) was prepared in buffer A (100mM Hepes, 100mM NaCI, 1 mM CaCI, pH7.0). The enzyme stock solutions were further diluted 1 in 250 in buffer A and mixed in reaction cocktails, on ice, as follows:
1 ) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.15ml of buffer A, 0.15ml of diluted Stainzyme
2) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.15ml of buffer A, 0.15ml of diluted AHA
3) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of buffer A (negative control)
4) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.15ml of diluted Stainzyme, 0.15ml of diluted AHA
10ΟμΙ aliquots were immediately transferred to a 0.2ml thin-walled 96-well PCR plate (2 complete rows per reaction cocktail, giving duplicate readings for each temperature assessed) and incubated on a PCR instrument capable of running a thermal gradient (Techne TC-500) thus:
The instrument was set up to run a temperature gradient between 20 and 50°C. The samples, aliquoted into the 96-well PCR plate were incubated for exactly 30 minutes under the above conditions, at which point the PCR plate was rapidly cooled to 4°C. During the cooling step, 50μΙ of 1 M tris.base (unbuffered) was added to each well to stop the reaction (prevention of further p-nitrophenol reaction product release). After thorough mixing, 10ΟμΙ of each reaction cocktail was transferred to a clear flat-bottomed microtitre plate and assessed for absorbance at 405nm using a Fluostar Optima plate reader. Blank values at each tested temperature were subtracted from the results obtained for enzyme- containing cocktails. Results are shown in Figure 2. In yet another variation of the described experiment, stock solutions of each enzyme (1 mg/ml of AHA, 0.5mg/ml Stainzyme) was prepared in buffer A (100mM Hepes, 100mM NaCI, 1 mM CaCI, pH7.0). The enzyme stock solutions were further diluted 1 in 415 in buffer A and mixed in reaction cocktails, on ice, as follows:
1 ) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of diluted Stainzyme
2) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of diluted AHA
3) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.3ml of buffer A (negative control)
4) 2.25ml amylase detection reagent 1 , 0.45ml detection reagent 2, 0.15ml of diluted Stainzyme, 0.15ml of diluted AHA
10ΟμΙ aliquots were immediately transferred to a 0.2ml thin-walled 96-well PCR plate (2 complete rows per reaction cocktail, giving duplicate readings for each temperature assessed) and incubated on a PCR instrument capable of running a thermal gradient (Techne TC-500) thus:
The instrument was set up to run a temperature gradient between 20 and 50°C. The samples, aliquoted into the 96-well PCR plate were incubated for exactly 30 minutes under the above conditions, at which point the PCR plate was rapidly cooled to 4°C. During the cooling step, 50μΙ of 1 M tris.base (unbuffered) was added to each well to stop the reaction (prevention of further p-nitrophenol reaction product release). After thorough mixing, 10ΟμΙ of each reaction cocktail was transferred to a clear flat-bottomed microtitre plate and assessed for absorbance at 405nm using a Fluostar Optima plate reader. Blank values at each tested temperature were subtracted from the results obtained for enzyme- containing cocktails. Results are shown in Figure 3. Table 1 a and 1 b: Activity data for AHA, Stainzyme and both enzymes together, where stock solutions of each enzyme were diluted 1 in 125 for use in assay (data values are Absorbance readings using a Fluostar Optima microplate reader set to 405nm, after incubation with assay substrate for 30 minutes in a gradient of temperatures between 20°C and 50°C). Graphically represented in Figure 5.
Table 1a Temperatures 22.5
Temperature (°C) 22.5 23.9 26.4 29.3 31.3 33.8
Blank Raw data value 1 0.130 0.129 0.131 0.133 0.137 0.137 values Raw data value 2 0.132 0.129 0.127 0.130 0.136 0.137
Average value 0.131 0.129 0.129 0.131 0.136 0.137
Stainzyme Raw data value 1 0.507 0.553 0.632 0.738 0.866 0.975 data Raw data value 2 0.536 0.565 0.643 0.733 0.842 0.957
Mean value 0.521 0.559 0.637 0.735 0.854 0.966
Mean minus 0.391 0.430 0.509 0.604 0.717 0.829 mean blank value
1 Standard 0.015 0.006 0.005 0.002 0.012 0.009 deviation from the
mean value
Stainzyme Raw data value 1 1.053 1.069 1.124 1.178 1.158 1.112 and AHA Raw data value 2 1.034 1.052 1.109 1.164 1.157 1.104 combinatio
n data Mean value 1.044 1.060 1.116 1.171 1.157 1.108 (0.5x Mean minus 0.913 0.931 0.988 1.039 1.021 0.971
Stainzyme mean blank value
+ 0.5x 1 Standard 0.010 0.008 0.007 0.007 0.000 0.004 AHA) deviation from the
mean value
AHA data Raw data value 1 1.674 1.709 1.770 1.742 1.653 1.444
Raw data value 2 1.659 1.748 1.787 1.776 1.641 1.436
Mean value 1.666 1.728 1.778 1.759 1.647 1.440
Mean minus 1.536 1.599 1.650 1.628 1.511 1.303 mean blank value
1 Standard 0.008 0.019 0.008 0.017 0.006 0.004 deviation from the
mean value
Table 1 b Temperatures 35.7
Tables 2a, 2b: Activity data for AHA, Stainzyme and both enzymes together, where stock solutions of each enzyme were diluted 1 in 250 for use in assay (data values are Absorbance readings using a Fluostar Optima microplate reader set to 405nm, after incubation with assay substrate for 30 minutes in a gradient of temperatures between 20°C and 50°C). Graphically represented in Figure 2. Table 2a Temperatures 22.5
Temperature (°C) 22.5 23.9 26.4 29.3 31 .3 33.8
Blank Raw data value 1 0.1 16 0.1 16 0.1 19 0.1 18 0.121 0.124 values Raw data value 2 0.1 18 0.1 17 0.1 14 0.1 19 0.122 0.123
Average value 0.1 17 0.1 17 0.1 17 0.1 18 0.122 0.124
Stainzyme Raw data value 1 0.256 0.272 0.300 0.344 0.385 0.418 data Raw data value 2 0.268 0.273 0.299 0.332 0.371 0.414
Mean value 0.262 0.272 0.299 0.338 0.378 0.416
Mean minus
mean blank value 0.145 0.156 0.182 0.220 0.256 0.292
1 Standard
deviation from the
mean value 0.009 0.000 0.001 0.008 0.010 0.002
Stainzyme Raw data value 1 0.858 0.870 0.907 0.940 0.922 0.878 and AHA Raw data value 2 0.838 0.849 0.895 0.924 0.921 0.874 combinatio
n data (1x Mean value 0.848 0.859 0.901 0.932 0.921 0.876 Stainzyme Mean minus
+ 1x AHA) mean blank value 0.731 0.743 0.784 0.814 0.800 0.752
1 Standard
deviation from the
mean value 0.014 0.015 0.008 0.01 1 0.001 0.003
AHA data Raw data value 1 0.677 0.686 0.700 0.684 0.639 0.546
Raw data value 2 0.673 0.694 0.703 0.692 0.633 0.547
Mean value 0.675 0.690 0.702 0.688 0.636 0.547
Mean minus
mean blank value 0.558 0.573 0.585 0.570 0.514 0.423
1 Standard
deviation from the
mean value 0.003 0.006 0.002 0.005 0.005 0.001
Table 2b Temperatures 35.7
Table 3: Activity data for AHA, Stainzyme and both enzymes together, where stock solutions of each enzyme were diluted 1 in 417 for use in assay (data values are Absorbance readings using a Fluostar Optima microplate reader set to 405nm, after incubation with assay substrate for 30 minutes in a gradient of temperatures between 20°C and 50°C). Graphically represented in Figure 3. Table 3a Temperatures 22.5
Temperature (°C) 22.5 23.9 26.4 29.3 31 .3 33.8
Blank Raw data value 1 0.125 0.124 0.126 0.126 0.129 0.134 values Raw data value 2 0.124 0.123 0.122 0.123 0.126 0.131
Average value 0.125 0.123 0.124 0.124 0.127 0.133
Stainzyme Raw data value 1 0.326 0.349 0.391 0.452 0.520 0.576 data Raw data value 2 0.341 0.352 0.395 0.448 0.505 0.568
Mean value 0.334 0.351 0.393 0.450 0.513 0.572
Mean minus
mean blank value 0.209 0.227 0.269 0.326 0.385 0.439
1 Standard
deviation from the
mean value 0.010 0.002 0.002 0.003 0.010 0.006
Stainzyme Raw data value 1 0.640 0.650 0.679 0.697 0.694 0.658 and AHA Raw data value 2 0.636 0.633 0.659 0.684 0.682 0.657 combinatio
n data Mean value 0.638 0.642 0.669 0.691 0.688 0.658 (0.5x Mean minus
Stainzyme mean blank value 0.513 0.518 0.545 0.566 0.561 0.525 + 0.5x 1 Standard
AHA) deviation from the
mean value 0.003 0.012 0.014 0.009 0.009 0.001
AHA data Raw data value 1 0.906 0.925 0.947 0.924 0.871 0.752
Raw data value 2 0.903 0.935 0.956 0.941 0.865 0.742
Mean value 0.904 0.930 0.951 0.932 0.868 0.747
Mean minus
mean blank value 0.780 0.806 0.827 0.808 0.741 0.614
1 Standard
deviation from the
mean value 0.003 0.007 0.006 0.013 0.004 0.007
Table 3b Temperatures 35.7
Concentrations for Table 1 a, 1 b are : AHA alone = 0.8mg/L, Stainzyme alone = 0.4mg/L, together = 0.4mg/L AHA and 0.2mg/L Stainzyme (total protein = 0.6mg/L)
Concentrations for Table 2a, 2b are AHA alone = 0.2mg/L, Stainzyme alone
together = 0.2mg/L AHA and 0.1 mg/L Stainzyme (total protein = 0.3mg/L)
Concentrations for Table 3a, 3b are AHA alone = 0.24mg/L, Stainzyme alone = 0.12mg/L, together = 0.12mg/L AHA and 0.06mg/L Stainzyme (total protein = 0.18mg/L). The results from Tables 1 a,b & 3a, b show also that using both enzymes together at lower concentration than the single enzyme controls gives a more balanced profile of activity across a broad temperature range. The results from Table 2a, b shows also that adding the two enzymes together at the same concentration as used in the single enzyme controls gives the highest activity across the temperature range.
Method to demonstrate starch-based stain removal from fabric using psychrophilic and mesophilic a-amylase combinations
4mm C-S-27 potato starch stained fabric micro-swatches in wells of 96-well microtitre plates (supplied by CFT, Vlaardingen, NL) were pre-washed for 5 minutes (200μΙ per well, 1000rpm on an orbital shaker and at the temperature at which the stain removal assay was conducted) using 100mM HEPES buffer pH7 plus 100mM sodium chloride and 1 mM calcium chloride. Pre-wash solution was discarded.
AHA and Stainzyme a-amylase enzymes were diluted to appropriate concentrations (as indicated) using 100mM HEPES buffer pH7 plus 100mM sodium chloride and 1 mM calcium chloride.
200μΙ diluted enzyme was added to swatches in triplicate. Microtitre plates containing swatches and enzyme solution were incubated for 1 hour at either 10°C, 20°C or 60°C, shaken at 1000rpm.
After incubation, enzyme-containing solutions were removed from the microtitre plate wells and the treated swatches were rinsed with 5 changes of water (5 minute incubation each, at the same temperature as was conducted for the wash step, shaking at
1000rpm). Treated swatches were then allowed to dry overnight at room temperature. Once dry, fabric micro-swatches were analysed at 410nm using a flatbed remission spectrophotometer to measure removal of the stain. The results are expressed as SRI units, which were generated using the delta remission values generated. SRI values obtained for C-S-27 fabric microswatches after stain removal using AHA and Stainzyme either alone or in combination are shown in table 8. The tabulated data clearly demonstrates that at high temperatures Stainzyme delivers the majority of the cleaning benefit. At low temperatures, Stainzyme performance is reduced and AHA enhances the stain removal. Table 4 Data demonstrating stain removal of C-S-27 potato starch-stained fabric by AHA and Stainzyme, either alone or in combination. Wash temperatures were 10°C, 20°C and 60°C (top, middle and bottom panels respectively). Data is expressed as SRI's and replicates are presented along with the arithmetic mean values and the standard deviation (1 SD).
Assayed at 10UC AHA mg/L
1 1 1 mean 1 SD
Stainzyme 0.125 85.77 84.33 84.53 84.88 0.78
mg/L 0.062 84.46 84.14 84.41 84.34 0.17
0.031 83.60 83.75 82.82 83.39 0.50
0 81.87 81.29 81.44 81.53 0.30
Assayed al 10UC AHA mg/L
0.5 0.5 0.5 mean 1 SD
Stainzyme 0.125 83.81 82.58 83.35 83.25 0.62
mg/L 0.062 80.63 80.87 81.03 80.84 0.20
0.031 79.53 79.79 80.34 79.88 0.41
0 77.03 76.49 75.80 76.44 0.61
Assayed al 10UC AHA mg/L
0 0 0 mean 1 SD
Stainzyme 0.125 79.77 79.60 79.12 79.50 0.34
mg/L 0.062 76.88 76.66 75.93 76.49 0.50
0.031 72.45 72.33 72.61 72.46 0.14
0 64.02 63.34 65.22 64.19 0.95
Assayed al 20UC AHA mg/L
1 1 1 mean 1 SD
Stainzyme 0.125 89.46 89.39 89.13 89.32 0.17
mg/L 0.062 87.89 87.56 87.47 87.64 0.22
0.031 86.41 87.35 86.84 86.87 0.47
0 83.67 84.33 84.36 84.12 0.39
Assayed al 20UC AHA mg/L
0.5 0.5 0.5 mean 1 SD Stainzyme 0.125 88.39 87.75 88.33 88.16 0.35
mg/L 0.062 85.73 85.82 86.24 85.93 0.27
0.031 84.25 83.73 85.27 84.42 0.78
0 80.15 77.92 79.58 79.22 1.16
Assayed al 20UC AHA mg/L
0 0 0 mean 1 SD
Stainzyme 0.125 85.95 86.32 85.93 86.07 0.22
mg/L 0.062 82.70 82.77 82.81 82.76 0.06
0.031 80.86 79.33 79.1 1 79.77 0.95
0 65.50 65.25 66.08 65.61 0.43
Assayed al 60UC AHA mg/L
1 1 1 mean 1 SD
Stainzyme 0.125 92.55 92.54 92.33 92.47 0.12
mg/L 0.062 91.60 91.86 92.07 91.84 0.24
0.031 90.10 90.56 89.89 90.18 0.34
0 71.31 71.02 71.72 71.35 0.36
Assayed al 60UC AHA mg/L
0.5 0.5 0.5 mean 1 SD
Stainzyme 0.125 93.38 92.75 93.24 93.12 0.33
mg/L 0.062 91.76 91.91 92.40 92.02 0.34
0.031 90.52 90.39 91.18 90.70 0.43
0 67.44 67.1 1 67.32 67.29 0.16
Assayed al 60UC AHA mg/L
0 0 0 mean 1 SD
Stainzyme 0.125 93.40 92.59 92.34 92.78 0.55
mg/L 0.062 91.71 92.12 91.62 91.82 0.27
0.031 90.82 90.62 90.28 90.57 0.27
0 65.57 64.59 66.27 65.47 0.84
Non-Limiting examples of laundry enzymatic fabric treatment compositions are described below where the following example enzyme combinations are: A. Lipase Combination
Psychrophilic Lipase: from Pseudoalteromonas sp W27.
Mesophilic Lipase: Lipex 100 T (Novozymes A/A)
Thermophilic Lipase: from Bacillus thermocatenlatus BTL2 B. Protease Combination
Psychrophilic Protease: from Flavobacterium balustinum P104
Mesophilic Protease: Savinase™24.0 GTT (12.0 or 24.0 T) Novozymes
Thermophilic Protease: from Thermophilic Bacillus strain HS08
C. Amylase combination
Psychrophilic amylase: alpha-amylase from Pseudoalteromonas haloplanktis strain TAC 125 and/or Pseudoalteromonas haloplanktis strain TAB 23 (as above) Mesophilic amylase: Stainzyme™ 2.0 T (Novozymes)
Thermophilic amylase: alpha-amylase from B. stearothermophilus Donk BS-1 .
D. Lyase Combination
Psychrophilic pectate lyase: from Pseudoalteromonas haloplanktis ANT/505 Mesophilic pectate lyase: Bioprep™ Novozymes
Thermophilic pectate lyase: from Thermoanaerobacter italicus sp. Nov. AB9.
E. Mixed Combination
Psychrophilic Lipase: from Pseudoalteromonas sp W27 and
Mesophilic Protease: 24.0 GTT (12.0 or 24.0 T) Novozymes and/or
Thermophilic amylase: alpha-amylase from B. stearothermophilus Donk BS-1 .
Examples of Liquid Enzymatic Fabric Treatment Composition comprising enzyme combinations above :
Alkyl polyglucosides (APG) 12.8
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
Enzymes A or B or C or D or E (as above) 3
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.
Unless stated otherwise, all proportions are given in weight percent by weight of any 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

Claims

An enzymatic fabric treatment composition comprising the combination of: i. one or more psychrophilic enzymes, and
ii. one or more mesophilic enzymes/and or one or more thermophilic enzymes.
An enzymatic fabric treatment composition according to any preceding claim wherein the psychrophilic, mesophilic and preferably thermophilic enzymes all comprise a common class of enzymes.
An enzymatic fabric treatment composition according to claim 2 wherein the common class is a protease or a lipase or glycosyl hydrolase or a lyase.
An enzymatic fabric treatment composition according to any preceding claim wherein the one or more psychrophilic enzymes comprises an esterase.
An enzymatic fabric treatment composition according to claim 4 wherein the one or more psychrophilic enzymes comprises a lipase.
A method of treating a fabric using an enzymatic fabric treatment composition according to any preceding claim wherein the temperature of the wash liquor varies between psychrophilic and mesophilic temperatures throughout a single washing cycle.
A method according to claim 6 wherein the temperature varies between psychrophilic and mesophilic and thermophilic temperatures throughout a single washing cycle. A method of treating a fabric according to any of claims 6 or 7 wherein the method comprises a hand-washing treatment and/or treatment using a washing machine.
A method of treating a fabric according to any of claims 4-8 wherein the temperature of the wash liquor is uncontrolled in at least one part by the user.
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