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
  • C11D3/166—Organic compounds containing borium
• • 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
  • C11D1/00—Detergent compositions based essentially on surface-active compounds; Use of these compounds as a detergent
  • C11D1/66—Non-ionic compounds
  • C11D1/72—Ethers of polyoxyalkylene glycols
• • 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
  • C11D3/38663—Stabilised liquid enzyme compositions

Definitions

• the present invention relates to stabilization of a concentrated liquid enzyme additive in order to prevent precipitation.
• the invention further relates to a process for manufacturing of said liquid enzyme additive.
• Boronic acids containing only alkyl groups such as methyl, butyl or 2-cyclohexylethyl are poor inhibitors with methylbo- ronic acid as the poorest inhibitor, whereas boronic acids bearing aromatic groups such as phenyl, 4-methoxyphenyl or 3,5-dichiorophenyl are good inhibitors with 3,5- dichlorophenylboronic acid as a particularly effective one (see Keller et al, Biochem. Biophys. Res. Com. 176, 1991 , pp. 401-405).
• aryl boronic acids which have a substitution at the 3-position relative to boron are unexpectedly good reversible protease inhibitors.
• acetamidophenyl boronic acid is claimed to be a superior inhibitor of proteolytic enzymes (see WO 92/19707).
• An object of the present invention is therefore to provide a liquid enzyme additive, comprising an enzyme and a boronic acid or a derivative of boronic acid, which does not form precipitate during preparation or upon storage.
• a further object of the present invention is to provide a method for manufacturing said liquid enzyme additive with a minimum formation of precipitate. It has surprisingly been found that adding a surfactant to the liquid enzyme additive decreases the amount of precipitate being formed during storage significantly.
• the present invention provides thus in a first aspect of the present invention a concentrated liquid enzyme additive comprising enzyme, a phenyl boronic acid or a derivative thereof and a surfactant, wherein the enzyme is present in the amount of more than 1.5 g/L.
• the present invention provides in a second aspect a process for manufacturing a concentrated liquid enzyme additive comprising the following steps: i) providing a liquid enzyme preparation; ii) mixing the liquid of i) with a boronic acid or a derivative thereof; iii) adding a surfactant to the liquid enzyme additive before or after step ii) or together with the boronic acid or derivative thereof.
• the present invention further relates to the use of said concentrated liquid enzyme additive.
• the HLB (hydrophilic-lipophilic balance) value is a kind of index numbered from 1 to 20.
• the HLB system is a semi-empirical method to predict what type of surfactant properties a molecular structure will provide.
• the HLB system is based on the concept that some molecules have hydrophilic. groups, other molecules have lipophilic groups, and some have both. Weight percentage of each type of group on a molecule or in a mixture predicts what behavior the molecular structure will exhibit.
• Water-in-oil emulsifiers have low HLB numbers, typically around 4. Solubilizing agents have high HLB numbers. Oil-in-water emulsifiers have intermediate to high HLB numbers.
• HLB is the hydrophile-lipophile balance as described by W.C. Griffin, “Calculation of HLB Values of Non-Ionic Surfactants," Journal of the Society of Cosmetic Chemists 5 (1954): p249.
• a chemical that acts as a surface active agent encompasses a multitude of materials that function as emulsifiers, dispersants, oil-wetters, water-wetters, foamers and defoam- ers.
• the pH was measured using a pH meter PHM93 from Radiometer and a Ross® semi-micro combination pH electrode (Orion 8103SC). Before being used, the pH electrode was calibrated using standard buffers from Radiometer Analytical (pH 4.005 order no.: S11 M002; pH 7.000 order no.: S11 M004 and pH 10.012 order no.: S11 M007). The pH was measured at room temperature, 23°C.
• Enzyme concentrate An enzyme concentrate is an enzyme fermentation broth which have been exposed to removal of liquid and/or removal of unwanted material.
• a concentrated liquid enzyme additive is a product to be used as a raw material or premix in manufacturing of a finished product such as detergents.
• the concentrated liquid enzyme additive is in the following referred to as the liquid enzyme additive or the concentrated liquid enzyme additive.
• the liquid enzyme additive of the present invention comprises enzyme, boronic acid or a derivative thereof and a surfactant.
• the liquid enzyme additive has a pH of 5.5 to 10. In a more particular embodiment of the resent invention the pH is 7.5 to 10.
• the liquid enzyme additive further comprises an alkaline substance such as the liquid enzyme additive has a pH of 7.5 to 10 provided by the alkaline substance.
• the liquid enzyme additive may comprise additional materials.
• the liquid enzyme additive does not comprise chelants.
• the liquid enzyme additive does not comprise ethanolamines.
• the liquid enzyme additive does not comprise phosphonates.
• the liquid enzyme additive does not comprise perfume.
• the liquid enzyme additive is a concentrated product to be added to liquid detergents.
• the amount of enzyme used in the liquid enzyme additive is thus very high.
• the amount of enzyme present in the liquid enzyme additive is at least 1.5 g/L.
• the amount of enzyme is at least 5 g/L.
• the amount of enzyme present is at least 10 g/L.
• the amount of enzyme present is at least 20 g/L such as even above 25 g/L.
• the amount of enzyme does not exceed 200 g/L.
• the amount of enzyme does not exceed 150 g/L.
• the amount of enzyme present in the liquid enzyme additive is less than 100 g/L.
• the liquid enzyme additive comprises above 4 % by weight of enzyme protein. In a more particular embodiment of the present invention the liquid enzyme additive comprises at least 5% by weight of enzyme protein. In a most particular embodiment of the present invention the liquid enzyme additive comprises at least 7.5% by weight of enzyme protein.
• the enzyme is typically produced by fermentation of a bacterium or fungus, and subsequently recovered by methods known within the art.
• a typical recovery process may consist of the following steps: removal of cells and other solids by centrifugation or filtration and concentration by ultra-filtration and/or evaporation at reduced pressure.
• a polyol such as 1 ,2- propanediol, sorbitol, a simple carbohydrate or glycerol, may be added either immediately before, during or shortly after the concentration step to stabilize the enzyme.
• a combination of polyols may also be used. Further steps may be included in the recovery process, depending on the specifications of the final product. In order to increase the stability during the processes, e.g.
• the processes are typically run at a pH where the enzyme is stable and their activity is low, e.g. at pH 4.0-6.5.
• Another way to increase the stability, including reduction of proteolysis is to run the processes at low temperatures, e.g. below 10 0 C.
• the liquid composition contains at least one enzyme.
• the enzyme may be any commercially available enzyme, in particular an enzyme selected from the group consisting of proteases, amylases, lipases, cellulases, lyases, oxidoreductases and any mixture thereof. Mixtures of enzymes from the same class (e.g. proteases) are also included.
• a liquid composition comprising a protease is preferred.
• a liquid composition comprising two or more enzymes in which the first enzyme is a protease and the second enzyme is selected from the group consisting of amylases, lipases, cellulases, lyases and oxidoreductases is preferred.
• the second enzyme is a lipase.
• enzyme variants 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). Enzymes can be classified on the basis of the handbook Enzyme Nomenclature from NC- IUBMB, 1992), see also the ENZYME site at the internet: http://www.expasv.ch/enzyme/. ENZYME is a repository of information relative to the nomenclature of enzymes.
• IUB-MB International Union of Biochemistry and Molecular Biology
• glycoside hydrolase enzymes such as endoglucanase, xy- lanase, galactanase, mannanase, dextranase and alpha-galactosidase
• endoglucanase xy- lanase
• galactanase galactanase
• mannanase mannanase
• dextranase alpha-galactosidase
• alpha-galactosidase alpha-galactosidase
• the liquid enzyme additive preferably comprise a protease, such as a serine protease.
• protease such as a serine protease.
• proteases include those of animal, vegetable or microbial origin. Microbial origin is preferred. Chemically or genetically modified mutants are included.
• the protease may be a serine protease, preferably an alkaline microbial protease or a trypsin-like protease.
• al-kaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279).
• trypsin-like proteases are tryp-sin (e.g. of porcine or bovine origin) and the Fusarium pro-tease described in WO 89/06270.
• the protease is a serine protease.
• Serine proteases or serine endopeptidases are a class of peptidases which are characterised by the presence of a serine residue in the active center of the enzyme.
• a serine protease is an enzyme which catalyzes the hydrolysis of peptide bonds, and in which there is an essential serine residue at the active site (White, Handler and Smith, 1973 "Principles of Biochemistry,” Fifth Edition, McGraw-Hill Book Company, NY, pp. 271-272).
• the bacterial serine proteases have molecular weights in the 20,000 to 45,000 Daltons range. They are inhibited by diisopropylfluorophosphate. They hydrolyze simple terminal esters and are similar in activity to eukaryotic chymotrypsin, also a serine protease. A more narrow term, alkaline protease, covering a sub group, reflects the high pH optimum of some of the serine proteases, from pH 9.0 to 11.0 (for review, see Priest (1977) Bacteriological Rev. 41 711-753). Subtilases: A sub-group of the serine proteases tentatively designated subtilases has been proposed by Siezen et al. (1991), Protein Eng., 4 719-737.
• subtilisin-like proteases They are defined by homology analysis of more than 40 amino acid sequences of serine proteases previously referred to as subtilisin-like proteases.
• a subtilisin was previously defined as a serine protease produced by Gram-positive bacteria or fungi, and according to Siezen et al. now is a subgroup of the subtilases.
• a wide variety of subtilisins have been identified, and the amino acid sequence of a number of subtilisins have been determined. These include more than six subtilisins from Bacillus strains, namely, subtilisin 168, subtilisin BPN', subtilisin Carlsberg, subtilisin Y, subtilisin amylosacchariticus, and mesentericopeptidase (Kurihara et al.
• subtilisins are well-characterized physically and chemically. In addition to knowledge of the primary structure (amino acid sequence) of these enzymes, over 50 high resolution X-ray structures of subtilisins have been determined which delineate the binding of substrate, transition state, products, at least three different protease inhibitors, and define the structural consequences for natural variation (Kraut (1977) Ann. Rev. Biochem. 46 331-358).
• subtilases I-S1
• subtilisin 168 subtilisin 168
• subtilisin BPN 1 subtilisin Carlsberg
• subtilisin Carlsberg A further subgroup of the subtilases I-S2
• Siezen et al. is recognised by Siezen et al. (supra).
• Sub-group I- S2 proteases are described as highly alkaline subtilisins and comprise enzymes such as subtilisin PB92 (MAXACAL®, Gist-Brocades NV), subtilisin 309 (SAVINASE®, Novozymes A/S), subtilisin 147 (ESPERASE®, Novozymes A/S), and alkaline elastase YaB.
• subtilisin PB92 MAXACAL®, Gist-Brocades NV
• subtilisin 309 SAVINASE®, Novozymes A/S
• subtilisin 147 ESPERASE®, Novozymes A/S
• alkaline elastase YaB alkaline elastase YaB.
• proteases examples include KannaseTM, EverlaseTM, EsperaseTM, AlcalaseTM, NeutraseTM, DurazymTM, SavinaseTM, OvozymeTM, PyraseTM, Pancreatic Trypsin NOVO (PTN), Bio-FeedTM Pro and Clear-LensTM Pro (all available from Novozymes A/S, Bagsvaerd, Denmark).
• Other preferred proteases include those described in WO 01/58275 and WO 01/58276.
• proteases include RonozymeTM Pro, MaxataseTM, MaxacalTM, MaxapemTM, OpticleanTM, PropeaseTM, PurafectTM and Purafect OxTM (available from Genencor International Inc., Gist-Brocades, BASF, or DSM Nutritional Products).
• Suitable Upases include those of bacterial or fungal origin. Chemically or genetically modified mutants are included.
• useful lipases include a Humicola lanugi-nosa lipase, e.g., as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase, e.g., as described in EP 238 023, a Candida lipase, such as a C. antarctica lipase, e.g., the C. antarctica lipase A or B described in EP 214 761 , a Pseu-domonas lipase such as a P. pseudoalcaligenes and P. alcali-genes lipase, e.g., as described in EP 218 272, a P.
• a Humicola lanugi-nosa lipase e.g., as described in EP 258 068 and EP 305 216
• a Rhizomucor miehei lipase e.g., as described in EP 238 023
• cepacia lipase e.g., as described in EP 331 376, a P. stutzeri li-pase, e.g., as disclosed in BP 1 ,372,034, a P. fluorescens lipase, a Bacillus lipase, e.g., a B. subtilis lipase (Dar-tois et al., (1993), Biochemica et Biophysica acta 1131 , 253-260), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (WO 91/16422).
• cloned lipases may be useful, including the Penicillium camenbertii lipase described by Ya-maguchi et al., (1991), Gene 103, 61-67), the Geotricum can-didum lipase (Schimada, Y. et al., (1989), J. Biochem. 106, 383-388), and various Rhizopus lipases such as a R. delemar lipase (Hass, MJ et al., (1991 ), Gene 109, 117-113), a R. niveus lipase (Kugimiya et al., (1992), Biosci. Biotech. Bio-chem. 56, 716-719) and a R. oryzae lipase.
• R. delemar lipase Hass, MJ et al., (1991 ), Gene 109, 117-113
• R. niveus lipase Kugi
• lipolytic enzymes such as cutinases may also be useful, e.g., a cutinase derived from Pseudomonas mendocina as described in WO 88/09367, or a cutinase derived from Fusarium solani pisi (e.g. described in WO 90/09446).
• examples of commercially available lipases include LipexTM, LipoprimeTM, LipopanTM, LipolaseTM, LipolaseTM Ultra, LipozymeTM, PalataseTM, ResinaseTM, NovozymTM 435 and LecitaseTM (all available from Novozymes A/S).
• lipases include LumafastTM (Pseudomonas mendocina lipase from Genencor International Inc.); LipomaxTM (Ps. pseudoalcaligenes lipase from Gist- Brocades/Genencor Int. Inc.; and Bacillus sp. lipase from Solvay enzymes. Further lipases are available from other suppliers such as Lipase P "Amano” (Amano Pharmaceutical Co. Ltd.).
• Amylases include those of bacterial or fungal origin.
• Amylases include, for example, a- amylases obtained from a special strain of B. licheniformis, described in more detail in British Patent Specification No. 1 ,296,839.
• Commercially available amylases are DuramylTM, TermamylTM, FungamylTM and BANTM (available from Novozymes A/S) and RapidaseTM and Maxamyl PTM (available from Gist-Brocades).
• Suitable cellulases include those of bacterial or fungal origin. Chemically or genetically modified mu-tants are included. Suitable cellulases are disclosed in US 4,435,307, which discloses fungal cellulases produced from Humicola insolens. Especially suitable cellulases are the cellulases having color care benefits. Examples of such cellulases are cellulases described in European patent application No. 0 495 257.
• Oxidoreductases Any oxidoreductase suitable for use in a liquid composition, e.g., peroxidases or oxidases such as laccases, can be used herein.
• Suitable peroxidases herein include those of plant, bacterial or fungal origin. Chemically or genetically modified mutants are included. Examples of suitable peroxidases are those derived from a strain of Coprinus, e.g., C. cinerius or C. macrorhizus, or from a strain of Bacillus, e.g., B. pumilus, particularly peroxidase according to WO 91/05858.
• Suitable laccases herein include those of bacterial or fungal origin.
• laccases are those obtainable from a strain of Trametes, e.g., T. villosa or T. versicolor, or from a strain of Coprinus, e.g., C. cinereus, or from a strain of Myceliophthora, e.g., M. thermophila.
• the types of enzymes which may be present in the liquid of the invention include oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.- .-), isomerases (EC 5.-.-.-) and ligases (EC 6.-.-.-).
• Preferred oxidoreductases in the context of the invention are peroxidases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4)].
• An Example of a commercially available oxidoreductase (EC 1.-.-.-) is GluzymeTM (enzyme available from Novozymes A/S). Further oxidoreductases are available from other suppliers.
• Preferred transferases are transferases in any of the following sub-classes: a Transferases transferring one-carbon groups (EC 2.1 ); b transferases transferring aldehyde or ketone residues (EC 2.2); acyltransferases (EC 1.11.1), laccases (EC 1.10.3.2) and glucose oxidases (EC 1.1.3.4)].
• An Example of a commercially available oxidoreductase (EC 1.-.-.-) is GluzymeTM (enzyme available from Novozymes A/S). Further
• a most preferred type of transferase in the context of the invention is a transglutaminase (protein-glutamine ⁇ -glutamyltransferase; EC 2.3.2.13). Further examples of suitable transglutaminases are described in WO 96/06931 (Novo Nordisk A/S).
• Preferred hydrolases in the context of the invention are: carboxylic ester hydrolases (EC 3.1.1.-) such as lipases (EC 3.1.1.3); phytases (EC 3.1.3.-), e.g. 3-phytases (EC 3.1.3.8) and 6-phytases (EC 3.1.3.26); glycosidases (EC 3.2, which fall within a group denoted herein as "carbohydrases”), such as ⁇ -amylases (EC 3.2.1.1); peptidases (EC 3.4, also known as proteases); and other carbonyl hydrolases.
• carboxylic ester hydrolases EC 3.1.1.-
• phytases EC 3.1.3.-
• 3-phytases EC 3.1.3.8
• 6-phytases EC 3.1.3.26
• glycosidases EC 3.2, which fall within a group denoted herein as "carbohydrases”
• ⁇ -amylases EC
• phytases examples include Bio-FeedTM Phytase (Novozymes), RonozymeTM P (DSM Nutritional Products), NatuphosTM (BASF), FinaseTM (AB Enzymes), and the PhyzymeTM product series (Danisco).
• Other preferred phytases include those described in WO 98/28408, WO 00/43503, and WO 03/066847.
• carbohydrase is used to denote not only enzymes capable of breaking down carbohydrate chains (e.g. starches or cellulose) of especially five- and six- membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six-membered ring structures such as D-glucose to five-membered ring structures such as D-fructose.
• Carbohydrases of relevance include the following (EC numbers in parentheses): ⁇ -amylases (EC 3.2.1.1), ⁇ -amylases (EC 3.2.1.2), glucan 1 ,4- ⁇ -glucosidases (EC 3.2.1.3), endo-1 ,4-beta-glucanase (cellulases, EC 3.2.1.4), endo-1 ,3(4)- ⁇ -glucanases (EC 3.2.1.6), endo-1 ,4- ⁇ -xylanases (EC 3.2.1.8), dextranases (EC 3.2.1.11 ), chitinases (EC 3.2.1.14), polygalacturonases (EC 3.2.1.15), lysozymes (EC 3.2.1.17), ⁇ -glucosidases (EC 3.2.1.21), ⁇ - galactosidases (EC 3.2.1.22), ⁇ -galactosidases (EC 3.2.1.23), amylo-1
• carbohydrases examples include Alpha-GalTM, Bio-FeedTM Alpha, Bio-FeedTM Beta, Bio-FeedTM Plus, Bio-FeedTM Wheat, Bio-FeedTM Z, NovozymeTM 188, CarezymeTM, CelluclastTM, CellusoftTM, CelluzymeTM, CeremylTM, CitrozymTM, DenimaxTM, DezymeTM, DextrozymeTM, DuramylTM, EnergexTM, FinizymTM, FungamylTM, GamanaseTM, GlucanexTM, LactozymTM, LiquezymeTM, MaltogenaseTM, NatalaseTM, PentopanTM, PectinexTM, PromozymeTM, PulpzymeTM, NovamylTM, TermamylTM, AMGTM (Amyloglucosidase Novo), MaltogenaseTM, SweetzymeTM and AquazymTM (all available from Novozymes NS).
• CarezymeTM Celluc
• carbohydrases are available from other suppliers, such as the RoxazymeTM and RonozymeTM product series (DSM Nutritional Products), the AvizymeTM, PorzymeTM and GrindazymeTM product series (Danisco, Finnfeeds), and NatugrainTM (BASF) , PurastarTM and PurastarTM
• OxAm (Genencor).
• Other commercially available enzymes include MannawayTM, PectawayTM, StainzymeTM and RenozymeTM.
• Suitable surfactants to avoid precipitation in the liquid enzyme additive may be any surfactant.
• the surfactant of the present invention may be anionic, nonionic, cationic, or amphoteric (zwit- terionic).
• the HLB value of the surfactant is at least 9 such as at least 10. In a more particular embodiment the HLB value is between 10 and 20. In a more particular embodiment the HLB value of the surfactant is between 11 and 15. In a particular embodiment of the present invention the surfactant is soluble in the enzyme liquid additive in the temperature range of 0 to 40 0 C and do not phase separate. In a more par- ticular embodiment the surfactant can be added as a mixture of two or more surfactants.
• the amount of surfactant added is in particular 0.1 to 10 % w/w of the total liquid additive more particular 0.25 to 8 % w/w such as even more particular 0.5 to 5% w/w. In a particular embodiment of the present invention the amount of surfactant is less than 1 % w/w of the total liquid enzyme additive. In a particular embodiment of the present invention the amount of surfactant is less than 0.7 % w/w of the total liquid enzyme additive.
• the amount of surfactant added to the enzyme liquid additive is at least 0.1% w/w. In a more particular embodiment of the present invention the surfactant is added to the liquid enzyme additive is at least 0.25% w/w. In an even more particular embodiment the surfactant is added to the liquid enzyme additive is at least 0.5% w/w. In a most particular embodiment of the present invention the surfactant is added to the liquid enzyme additive is at least 1% w/w.
• the amount of surfactant added to the enzyme liquid additive is less than 20% w/w. In a more particular embodiment of the present invention the amount of surfactant added to the enzyme liquid additive is less than 15% w/w. In an even more particular embodiment of the present invention the amount of surfactant added to the enzyme liquid additive is less than 10% w/w. In a most particular embodiment of the present invention the amount of surfactant added to the enzyme liquid additive is less than 5%.
• the surfactant is a non-ionic surfactant.
• the nonionic surfactants are alcohol ethoxylate (AEO or AE), alcohol propoxylate, carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamine oxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (e.g. as described in WO 92/06154).
• Polyethylene, polypropylene, and polybutylene oxide condensates of alkyl phenols include the condensation products of alkyl phenols having an alkyl group containing from about 6 to about 14 carbon atoms, preferably from about 8 to about 14 carbon atoms, in either a straight chain or branched-chain configuration with the alkylene oxide.
• the ethylene oxide is present in an amount equal to from about 2 to about 25 moles, more preferably from about 3 to about 15 moles, of ethylene oxide per mole of alkyl phenol.
• nonionic surfactants of this type include IgepalTM CO-630, marketed by the GAF Corporation; and TritonTM X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company. These surfactants are commonly referred to as alkylphenol alkoxylates (e.g., alkyl phenol ethoxylates).
• the condensation products of primary and secondary aliphatic alcohols with about 1 to about 25 moles of ethylene oxide are preferred as the nonionic surfactant.
• the alkyl chain of the aliphatic alcohol can either be straight or branched, primary or secondary, and generally contains from about 8 to about 22 carbon atoms.
• nonionic surfactants of this type include TergitolTM 15-S-9 (The condensation product of C 11 -Ci 5 linear alcohol with 9 moles ethylene oxide), TergitolTM 24-L-6 NMW (the condensation product of C 12 -Ci 4 primary alcohol with 6 moles ethylene oxide with a narrow molecular weight distribution), both marketed by Union Carbide Corporation; NeodolTM 45-9 (the condensation product of Ci 4 -Ci 5 linear alcohol with 9 moles of ethylene oxide), NeodolTM 23-3 (the condensation product of Ci 2 -Ci 3 linear alcohol with 3.0 moles of ethylene oxide), NeodolTM 45-7 (the condensation product of Ci 4 -Ci 5 linear alcohol with 7 moles of ethylene oxide), NeodolTM 45-5 (the condensation product of C 14 - C 15 linear alcohol with 5 moles of ethylene oxide) marketed by Shell Chemical Company, KyroTM EOB (the condensation product of C 13 -Ci 5 alcohol with 9 moles ethylene oxide), marketed by The
• nonionic surfactants of this type include Softanol® from lneos Oxide, Belgium.
• alkylpolysaccharides disclosed in US 4,565,647, having a hydrophobic group containing from about 6 to about 30 carbon atoms, preferably from about 10 to about 16 carbon atoms and a polysaccharide, e.g. a polyglycoside, hydrophilic group containing from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7 saccharide units.
• Any reducing saccharide containing 5 or 6 carbon atoms can be used, e.g., glucose, galactose and galactosyl moieties can be substituted for the glucosyl moieties (optionally the hydrophobic group is attached at the 2-, 3-, A-, etc. positions thus giving a glucose or galactose as opposed to a glucoside or galactoside).
• the intersaccharide bonds can be, e.g., between the one position of the additional saccharide units and the 2-, 3-, A-, and/or 6- positions on the preceding saccharide units.
• the preferred alkylpolyglycosides have the formula:
• R 2 is selected from the group consisting of alky], alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which the alkyl groups contain from about 10 to about 18, preferably from about 12 to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 to about 10, preferably 0; and x is from about 1.3 to about 10, preferably from about 1.3 to about 3, most preferably from about 1.3 to about 2.7.
• the glycosyl is preferably derived from glucose. To prepare these compounds, the alcohol or alkylpolyethoxy alcohol is formed first and then reacted with glucose, or a source of glucose, to form the glucoside (attachment at the
• the additional glycosyl units can then be attached between their 1 -position and the preceding glycosyl units 2-, 3-, A-, and/or 6-position, preferably predominantly the 2-position.
• the condensation products of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol are also suitable as surfactant.
• the hydrophobic portion of these compounds will preferably have a molecular weight from about 1500 to about 1800 and will exhibit water insolubility.
• polyoxyethylene moieties to this hydrophobic portion tends to increase the water solubility of the molecule as a whole, and the liquid character of the product is retained up to the point where the polyoxyethylene content is about 50% of the total weight of the condensation product, which corresponds to condensation with up to about 40 moles of ethylene oxide.
• examples of compounds of this type include certain of the commercially available PluronicTM surfactants, marketed by BASF.
• PluronicTM surfactants also suitable for use as the nonionic surfactant of the nonionic surfactant system of the present invention, are the condensation products of ethylene oxide with the product resulting from the reaction of propylene oxide and ethylenediamine.
• the hydrophobic moiety of these products consists of the reaction product of ethylenediamine and excess propylene oxide, and generally has a molecular weight of from about 2500 to about 3000.
• This hydrophobic moiety is condensed with ethylene oxide to the extent that the condensation product contains from about 40% to about 80% by weight of polyoxyethylene and has a molecular weight of from about 5,000 to about 11,000.
• Examples of this type of nonionic surfactant include certain of the commercially available TetronicTM compounds, marketed by BASF.
• Suitable surfactants may be polyethylene oxide condensates of alkyl phenols, condensa- tion products of primary and secondary aliphatic alcohols with from about 1 to about 25 moles of ethyleneoxide, alkylpolysaccharides, and mixtures hereof. Most preferred are C 8 -C 14 alkyl phenol ethoxylates having from 3 to 15 ethoxy groups.
• nonionic surfactants may be polyhydroxy fatty acid amide surfactants of the formula
• R 1 is H, or R 1 is C 1-4 hydrocarbyl, 2-hydroxyethyl, 2-hydroxypropyl or a mixture thereof, R 2 is C 5-31 hydrocarbyl, and Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least 3 hydroxyls directly connected to the chain, or an alkoxylated derivative thereof.
• R 1 is methyl
• R 2 is straight C 1 - I-15 alkyl or C 16-18 alkyl or alkenyl chain such as coconut alkyl or mixtures thereof
• Z is derived from a reducing sugar such as glucose, fructose, maltose or lactose, in a reductive amination reaction.
• the surfactants are selected from the group consisting of:
• R-O-(CH 2 CH 2 O) n H wherein R is a branched or linear alkane with 8 to 22 carbon atoms and n is equal to or higher than 3. In a preferred embodiment n is equal to or higher than 4, in a more preferred embodiment n is equal to or higher than 5. n may be but is not limited to 3, 7, 8, 15 and 80.
• the average chain length is C12 to C18, in a more preferred embodiment the average chain length is C13 to C16, in a more particular embodiment the aver- age chain length is C13 to C15,
• CAS. No. 68131-40-8 which covers a group of compounds with following synonyms C11-15-sec- alkyl-omega-hydroxypoly(oxy-1 ,2-ethanediyl); C11-15-secondary alcohols, ethoxylated; Eth- oxylated Secondary Alcohols; SM-9; Tergitol 15-S-9 and CAS. No. 69227-20-9 Which covers ethoxylated alcohol-(C16-C22),
• the surfactant is selected from the group consisting of:
• the surfactant may also be ethoxylates of amines, amides and acids.
• the surfactants may be a co-polymer of ethoxylate and propoxylate, including but not limited to ethoxy- propoxy block co-polymers of alcohol, amines, amides and acids.
• Suitable anionic surfactants include alkyl alkoxylated sulfate surfactants.
• Examples hereof are water soluble salts or acids of the formula RO(A) m SO3M wherein R is an unsubstituted C 10 -C- 24 alkyl or hydroxyalkyl group having a Ci 0 -C 24 alkyl component, preferably a C 12 -C 20 alkyl or hydro-xyalkyl, more preferably Ci 2 -C 18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero, typically between about 0.5 and about 6, more preferably between about 0.5 and about 3, and M is H or a cation which can be, for example, a metal cation (e.g., sodium, potassium, lithium, calcium, magnesium, etc.), ammonium or substituted-ammonium cation.
• R is an unsubstituted C 10 -C- 24 alkyl or hydroxyalkyl group having a Ci 0 -C 24 alkyl component, preferably a C 12 -C 20 al
• Alkyl ethoxylated sulfates as well as alkyl propoxylated sulfates are contemplated herein.
• Specific examples of substituted ammonium cations include methyl-, dimethyl, trimethyl-ammonium cations and quaternary ammonium cations such as tetramethyl- ammonium and dimethyl piperdinium cations and those derived from alkylamines such as ethylamine, diethylamine, triethylamine, mixtures thereof, and the like.
• Exemplary surfactants are C 12 -Ci 8 alkyl polyethoxylate (1.0) sulfate (Ci 2 -Ci 8 E(1.0)M), C 12 -C t8 alkyl polyethoxylate (2.25) sulfate (C 12 -Ci 8 (2.25)M, and C 12 -C 18 alkyl polyethoxylate (3.0) sulfate (C 12 -C 18 E(3.0)M), and Ci 2 -C 18 alkyl polyethoxylate (4.0) sulfate (Ci 2 -C 18 E(4.0)M), wherein M is conveniently selected from sodium and potassium.
• Suitable anionic surfactants to be used may be alkylbenzenesulfonate (LAS), alpha- olefinsulfonate (AOS), alkyl sulfate (fatty alcohol sulfate) (AS), alcohol ethoxysulfate (AEOS or AES), secondary alkanesulfonat.es (SAS), alpha-sulfo fatty acid methyl esters, alkyl- or alkenyl- succinic acid, xylene sulfonate or soap, alkyl ester sulfonate surfactants including linear esters of C 8 -C 20 carboxylic acids (i.e., fatty acids) which are sulfonated with gaseous SO 3 according to "The Journal of the American Oil Chemists Society", 52 (1975), pp.
• LAS alkylbenzenesulfonate
• AOS alpha- olefinsulfonate
• AS fatty alcohol sulfate
• Suitable starting materials would include natural fatty substances as derived from tallow, palm oil, etc.
• a pre- ferred anionic surfactant is a sodium, potassium or ammonium salt of xylenesulfonic acid such as (CH3)2C6H3SO3Na.
• Suitable alkyl ester sulfonate surfactant comprise alkyl ester sulfonate surfactants of the structural formula:
• R 3 is a C 8 -C 2O hydrocarbyl, preferably an alkyl, or combination thereof
• R 4 is a C 1 -C 6 hydrocarbyl, preferably an alkyl, or combination thereof
• M is a cation which forms a water soluble salt with the alkyl ester sulfonate.
• Suitable salt-forming cations include metals such as sodium, potassium, and lithium, and substituted or unsubstituted ammonium cations, such as monoethanolamine, diethonolamine, and triethanolamine.
• R 3 is C 10 -C 16 alkyl
• R 4 is methyl, ethyl or isopropyl.
• the methyl ester sulfonates wherein R 3 is Ci O -C 16 alkyl.
• alkyl sulfate surfactants which are water soluble salts or acids of the formula ROSO 3 M wherein R preferably is a C 10 -C 24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a Ci 0 -C 2O alkyl component, more preferably a C 12 -Ci 8 alkyl or hydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or ammonium or substituted ammonium (e.g.
• R preferably is a C 10 -C 24 hydrocarbyl, preferably an alkyl or hydroxyalkyl having a Ci 0 -C 2O alkyl component, more preferably a C 12 -Ci 8 alkyl or hydroxyalkyl
• M is H or a cation, e.g., an alkali metal cation (e.g. sodium, potassium, lithium), or am
• alkylamines such as ethylamine, diethylamine, triethylamine, and mixtures thereof, and the like.
• alkyl chains of C 12 -C 16 are preferred for lower wash temperatures (e.g. below about 50 0 C) and C 16 -C 18 alkyl chains are preferred for higher wash temperatures (e.g. above about 50 0 C).
• anionic surfactants may include salts (including, for example, sodium, potassium, ammonium, and substituted ammonium salts such as mono- di- and triethanolamine salts) of soap, C 8 -C 22 primary or secondary alkanesulfonat.es, C 8 -C 24 olefinsulfonates, sulfonated polycarboxylic acids prepared by sulfonation of the pyrolyzed product of alkaline earth metal citrates, e.g., as described in British patent specification No.
• alkylpo- lyglycolethersulfates (containing up to 10 moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether sulfates, paraffin sulfonates, alkyl phosphates, isethionates such as the acyl isethionates, N- acyl taurates, alkyl succinamates and sulfosuccinates, monoesters of sulfosuccinates (especially saturated and unsaturated Ci 2 -Ci 8 monoesters) and diesters of sulfosuccinates (especially saturated and unsaturated C 6 -C 12 diesters), acyl sarcosinates, sulfates of alkylpolysaccharide
• Resin acids and hydrogenated resin acids are also suitable, such as rosin, hydrogenated rosin, and resin acids and hydrogenated resin acids present in or derived from tall oil.
• Alkylbenzene sulfonates may be suitable. Especially linear straight-chain) alkyl benzene sulfonates (LAS) wherein the alkyl group preferably contains from 10 to 18 carbon atoms. Further examples are described in "Surface Active Agents and Detergents" (Vol. I and Il by Schwartz, Perrry and Berch). A variety of such surfactants are also generally disclosed in US 3,929,678, (Column 23, line 58 through Column 29, line 23, herein incorporated by reference).
• Cationic surfactants suitable are those having one long-chain hydrocarbyl group.
• cationic surfactants include the ammonium surfactants such as alkyltrimethylammonium halogenides, and those surfactants having the formula:
• R 2 is an alkyl or alkyl benzyl group having from about 8 to about 18 carbon atoms in the alkyl chain
• each R 3 is selected form the group consisting of -CH 2 CH 2 -, -CH 2 CH(CH 3 )-, - CH 2 CH(CH 2 OH)-, -CH 2 CH 2 CH 2 -, and mixtures thereof
• each R 4 is selected from the group consisting of C 1 -C 4 alkyl, C 1 -C 4 hydroxyalkyl, benzyl ring structures formed by joining the two R 4 groups, -CH 2 CHOHCHOHCOR 6 CHOHCH 2 OH, wherein R 6 is any hexose or hexose polymer having a molecular weight less than about 1000, and hydrogen when y is not 0
• R 5 is the same as R 4 or is an alkyl chain, wherein the total number of carbon atoms or R 2 plus R 5 is not more than about 18; each y is from 0 to
• R 1 is C 8 -C 16 alkyl
• each of R 2 , R 3 and R 4 is independently C 1 -C 4 alkyl, C 1 -C 4 hydroxy alkyl, benzyl, and -(C 2 H 40 ) x H where x has a value from 2 to 5, and X is an anion.
• R 2 , R 3 or R 4 should be benzyl.
• the preferred alkyl chain length for R 1 is C 12 -C 15 , particularly where the alkyl group is a mixture of chain lengths derived from coconut or palm kernel fat or is derived synthetically by olefin build up or OXO alcohols synthesis.
• R 2 R 3 and R 4 are methyl and hydroxyethyl groups and the anion X may be selected from halide, methosulphate, acetate and phosphate ions.
• suitable quaternary ammonium compounds of formulae (i) for use herein are: coconut trimethyl ammonium chloride or bromide; coconut methyl di hydroxyethyl ammonium chloride or bromide; decyl triethyl ammonium chloride; decyl dimethyl hydroxyethyl ammonium chloride or bromide; C 12- - I5 dimethyl hydroxyethyl ammonium chloride or bromide; coconut dimethyl hydroxyethyl ammonium chloride or bromide; myristyl trimethyl ammonium methyl sulphate; lauryl dimethyl benzyl ammonium chloride or bromide; lauryl dimethyl (ethenoxy) 4 ammonium chloride or bromide; choline esters (compounds),
• Ampholytic surfactants may also be suitable. These surfactants can be broadly described as aliphatic derivatives of secondary or tertiary amines, or aliphatic derivatives of heterocyclic secondary and tertiary amines in which the aliphatic radical can be straight- or branched-chain.
• One of the aliphatic substituents contains at least about 8 carbon atoms, typically from about 8 to about 18 carbon atoms, and at least one contains an anionic water-solubilizing group, e.g. carboxy, sulfonate, sulfate. See US 3,929,678 (column 19, lines 18-35) for examples of ampholytic surfactants.
• Zwitterionic surfactants may also be suitable. These surfactants can be broadly described as derivatives of secondary and tertiary amines, derivatives of heterocyclic secondary and tertiary amines, or derivatives of quaternary ammonium, quaternary phosphonium or tertiary sulfonium compounds. See US 3,929,678 (column 19, line 38 through column 22, line 48) for examples of zwitterionic surfactants.
• Semi-polar nonionic surfactants are a special category of nonionic surfactants which include water-soluble amine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; watersoluble phosphine oxides containing one alkyl moiety of from about 10 to about 18 carbon atoms and 2 moieties selected from the group consisting of alkyl groups and hydroxyalkyl groups containing from about 1 to about 3 carbon atoms; and water-soluble sulfoxides containing one alkyl moiety from about 10 to about 18 carbon atoms and a moiety selected from the group consisting of alkyl and hydroxyalkyl moieties of from about 1 to about 3 carbon atoms.
• Semi-polar nonionic surfactants include the amine oxide surfactants having the formula: O
• R 3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixtures thereof containing from about 8 to about 22 carbon atoms
• R 4 is an alkylene or hydroxyalkylene group containing from about 2 to about 3 carbon atoms or mixtures thereof
• x is from 0 to about 3
• each R 5 is an alkyl or hydroxyalkyl group containing from about 1 to about 3 carbon atoms or a polyethylene oxide group containing from about 1 to about 3 ethylene oxide groups.
• the R 5 groups can be attached to each other, e.g., through an oxygen or nitrogen atom, to form a ring structure.
• amine oxide surfactants in particular include C 10 -Ci 8 alkyl dimethyl amine oxides and C 8 -C 12 alkoxy ethyl dihydroxy ethyl amine oxides.
• Enzyme stabilizer The enzyme stabilizer of the present invention is either boronic acid and/or a derivative thereof.
• the stabilizer is a phenyl boronic acid and/or a derivative thereof.
• the present invention covers liquid enzyme additives comprising boronic acid or derivatives thereof. In a particular embodiment the invention covers liquid enzyme additives comprising phenyl boronic acid or derivatives thereof.
• the stabilizer is a naphthalene boronic acid derivative.
• the amount of stabilizer present in the liquid enzyme additive is dependent of the amount of enzyme present in the liquid enzyme additive.
• the amount of stabilizer added is in particular 0.1 to 20 % (w/w) of the total liquid additive, more particular 0.5 to 8 % (w/w) such as even more particular 1 to 5% (w/w).
• the amount of stabilizer is above 1 % (w/w) of the total liquid additive.
• the amount of stabilizer is above 1.5 % (w/w) of the total liquid additive.
• the amount of stabilizer is above 2 % (w/w) of the total liquid additive.
• the amount of stabilizer added to the enzyme liquid additive is at least 0.1% (w/w).
• the stabilizer is added to the liquid enzyme additive is at least 0.5% (w/w). In an even more particular embodiment the stabilizer is added to the liquid enzyme additive is at least 1% (w/w). In a most particular embodiment of the present invention the stabilizer is added to the liquid enzyme additive is at least 1.5 % (w/w).
• the amount of stabilizer added to the enzyme liquid additive is less than 20% (w/w). In a more particular embodiment of the present invention the amount of stabilizer added to the enzyme liquid additive is less than 15% (w/w). In an even more particular embodiment of the present invention the amount of stabilizer added to the enzyme liquid additive is less than 10% (w/w). In a most particular embodiment of the present invention the amount of stabilizer added to the enzyme liquid additive is less than 5% (w/w).
• R is selected from the group consisting of hydrogen, hydroxy, C 1 -C 6 alkyl, substituted C 1 - C 6 alkyl, C 1 -C 6 alkenyl and substituted C 1 -C 6 alkenyl.
• a preferred embodiment of the present invention provides a liquid composition comprising an enzyme and a phenyl boronic acid derivative enzyme stabilizer of the formula disclosed above, wherein R is a C 1 -C 6 alkyl, in particular wherein R is CH 3 , CH 3 CH 2 or CH 3 CH 2 CH 2 , or wherein R is hydrogen.
• the stabilizer of the enzyme is A- formyl-phenyl-boronic acid (4-FPBA).
• the stabilizer is selected from the group consisting of: thiophene-2 boronic acid, thiophene-3 boronic acid, acetamidophenyl boronic acid, benzofu- ran-2 boronic acid, naphtalene-1 boronic acid, naphtalene-2 boronic acid, 2-FPBA, 3-FBPA, 4- FPBA, 1-thianthrene boronic acid, 4-dibenzofuran boronic acid, 5-methylthiophene-2 boronic, acid, thionaphtrene boronic acid, furan-2 boronic acid, furan-3 boronic acid, 4,4 biphenyl- diborinic acid, 6-hydroxy-2-naphtalene, 4-(methylthio) phenyl boronic acid, 4 (trimethyl- silyOphenyl boronic acid, 3-bromothiophene boronic acid, 4-methylthiophene boronic acid, 2- naphtyl
• boronic acid derivatives suitable as stabilizers are described in US 4,963,655, US 5,159,060, WO 95/12655, WO 95/29223, WO 92/19707, WO 94/04653, WO 94/04654, US 5442100, US 5488157 and US 5472628.
• Any compound providing a pH above 7 when added to the liquid enzyme additive may be used to adjust the pH of the enzyme comprising mixture.
• Suitable compounds may be bases e.g. sodium hydroxide, potassium hydroxide or alkaline buffer salts.
• Suitable buffer salts may be potassium bicarbonate, potassium carbonate, tetra potassium pyrophosphate, potassium tripolyphosphate, sodium bicarbonate and sodium carbonate.
• Other suitable salts or compounds able of providing an alkaline pH may be used.
• the present invention further relates to the preparation of the liquid enzyme additive.
• the liquid enzyme additive comprises the enzyme, the boronic acid or derivative thereof and a surfactant. These compounds may be mixed in random order or all at the same time.
• the process of the present invention com- prises the steps of: i) providing a liquid enzyme preparation; ii) mixing the liquid enzyme preparation of i) with a surfactant and a boronic acid or derivative thereof;
• the process of the present invention comprises the steps of: i) providing a liquid enzyme preparation; ii) mixing the liquid enzyme preparation of i) with a boronic acid or derivative thereof; and iii) adding a surfactant to the liquid enzyme additive together, before or after adding boronic acid or a derivative thereof.
• the surfactant is mixed with the liquid enzyme preparation before boronic acid or a derivative thereof is added.
• the process of manufacturing a concentrated liquid enzyme additive comprising 1.5 g/L enzyme comprises the steps of: i) providing a liquid enzyme preparation; ii) mixing the liquid enzyme preparation of i) with a boronic acid or derivative thereof; and iii) adding a surfactant to the liquid enzyme additive either before or after adding boronic acid or a derivative thereof
• the surfactant may be added to the fermentation broth, to a cell free fermentation broth, or to a concentrate containing the one or more enzymes before the boronic acid or a derivative thereof is added.
• the surfactant is added to a fermentation broth. In a more particular embodiment of the present invention the surfactant is added to a cell free fermentation broth. In a most preferred embodiment of the present invention the surfactant is added to an enzyme concentrate.
• the preparation method of the liquid enzyme additive in some cases may have an effect on the stability of the additive and thus on the forming of precipitates therein.
• the pH of the enzyme additive is from 4.5 to 11. In a more particular embodiment of the present invention the pH of the enzyme additive is 5.5 to 10. We have found that adjusting pH and the time of which the adjustment takes place have an effect on the forming of precipitate in the liquid enzyme additive.
• pH of the enzyme containing liquid could be adjusted to pH 7.5 to 10 to avoid precipitation, more particularly to 8 to 9.
• the pH is preferably adjusted before mixing of the enzyme containing liquid and boronic acid or derivative thereof whereby the formation of precipitate is avoided.
• the adjustment can also take place after mixing of the enzyme and boronic acid or derivative thereof whereby the formed precipitate is dissolved.
• the process of the present invention comprises the steps of: i) providing a liquid enzyme preparation; ii) mixing the liquid enzyme preparation of i) with a boronic acid or derivative thereof; adjusting the pH of the liquid to 7.5 to 10 either before or after adding the boronic acid or derivative thereof.
• the process of the present invention comprises the steps of: i) providing a liquid enzyme preparation; ii) mixing the liquid enzyme preparation of i) with a boronic acid or derivative thereof; adjusting the pH of the liquid to 7.5 to 10 either before or after adding the boronic acid or derivative thereof and adding a surfactant to the liquid before or after adding the boronic acid or derivative thereof.
• the pH may be adjusted with any suitable alkaline substance.
• the pH is adjusted with NaOH.
• the process for manufacturing the liquid enzyme additive comprises the following steps: i) providing a liquid enzyme preparation comprising one or more enzymes; ii) mixing the liquid enzyme preparation of i) with a surfactant; iii) adjusting the pH of the liquid enzyme preparation of ii) to 7.5 to 10; iv) mixing the liquid composition of ii) with boronic acid or a derivative thereof.
• a liquid enzyme preparation comprising one or more enzymes; ii) adjusting the pH of the liquid enzyme preparation of i) to 7.5 to 10; iii) mixing the liquid enzyme preparation of ii) with a surfactant; iv) mixing the liquid composition of ii) with boronic acid or a derivative thereof.
• the process for manufacturing the liquid enzyme additive comprises the following steps: i) providing a liquid enzyme preparation comprising one or more enzymes; ii) mixing the liquid enzyme preparation of i) with a surfactant; iii) adjusting the pH of the liquid enzyme preparation of ii) to 7.5 to 10; iv) mixing the liquid composition of ii) with phenyl boronic acid or a derivative thereof.
• a liquid enzyme preparation comprising one or more enzymes; ii) adjusting the pH of the liquid enzyme preparation of i) to 7.5 to 10; iii) mixing the liquid enzyme preparation of ii) with a surfactant; iv) mixing the liquid composition of ii) with phenyl boronic acid or a derivative thereof.
• the boronic acid or derivative thereof may be added to the liquid enzyme preparation as a solid or as a liquid containing the boronic acid or derivative thereof on a partly or fully dissolved form.
• the boronic acid or its derivative thereof is dissolved in glycerol or 1 ,2-propanediol which is adjusted to pH 8.5-10 with sodium hydroxide or potasium hydroxide prior to the addition.
• compositions comprising the liquid enzyme additive of the invention
• the invention also relates to compositions comprising the liquid enzyme additive of the invention.
• the composition may be any composition, but particularly suitable compositions are cleaning compositions, personal care compositions, textile processing compositions e.g. bleaching, pharmaceutical compositions, leather processing compositions, pulp or paper processing compositions, food and beverage compositions and animal feed compositions.
• the liquid enzyme additive is used as a raw material in liquid detergents, e.g. laundry detergents.
• the invention is further directed to the use of the liquid enzyme additive in liquid detergent composition.
• a liquid composition comprising the liquid enzyme additive
• the liquid enzyme additive may be present in a concentration of 0.01 - 20 % w/w, preferably the composition may contain 0.05 - 10% w/w of the liquid enzyme additive, more preferably the liquid composition may contain 0.1 - 5% w/w of the liquid enzyme additive, most preferably the liquid composition may contain 0.1 - 3% w/w of the liquid enzyme additive.
• the liquid enzyme additive of the present invention is used in a liquid composition such as a detergent the amount of each enzyme will typically be 1-1000 mg/L, in particular 5-750 mg/L, especially 10-500 mg/L calculated as pure enzyme protein.
• a liquid composition comprising the liquid enzyme additive
• the liquid composition may contain 0.001 - 7.5 % w/w of the boronic acid or its derivative, preferably the composition may contain 0.005 - 4% w/w of the boronic acid or its derivative, more preferably the composition may contain 0.005 - 1.2% w/w of boronic acid or its derivative, most preferably the liquid composition may contain 0.01 - 0.15% w/w of boronic acid or its derivative.
• the boronic acid or its derivative may be an acid or the alkali metal salt of said acid.
• a protease was formulated with 4-FPBA and surfactant.
• the protease was a concentrated Savinase solution containing about 40 g enzyme protein/L and 55% 1 ,2-propane diol, pH 5.5
• the surfactant was added to the protease (2% w/w on final composition).
• the pH was measured using a pH meter PHM93 from Radiometer and a Ross® semi-micro combination pH electrode (Orion 8103SC). Before being used, the pH electrode was calibrated using standard buffers from Radiometer analytical (pH 4.005 order no.: S11 M002; pH 7.000 order no.: S11 M004 and pH 10.012 order no.: S11 M007). The p H was measured at room temperature. A solution of 30 % 4-FPBA in 1 ,2-propanediol adjusted to pH 9.6 with 10 M NaOH was added to the enzyme/surfactant mixture to a final concentration of 1.6% w/w 4-FPBA. The final enzyme concentration was 36 mg/ml The samples were then transferred to two vials, which after sealing were incubated for 4 weeks at 5° and 4O 0 C, respectively.
• a protease was formulated with 4-FPBA and surfactant. The same three surfactants used in example 1 were tried out.
• the protease was a concentrated Alcalase® solution containing 44 g enzyme/L and 30% 1 ,2- propane diol, pH 5.2
• the final enzyme concentration was 40 mg/ml.
• the samples were then transferred to two vials, which after sealing were incubated for 4 weeks at 5° and 40 0 C, respectively. After storage, the physical stability of the samples was determined by visual inspection.
• Example 2 The experiment was carried out as outlined in Example 1 and 2, only the concentration of the surfactant was varied. The results are shown in table 5.
• a protease (same as in example 1 ) was formulated with 4-FPBA using the following procedure: 1 ) Surfactant was added to the liquid protease sample to a final concentration of 2% w/w (see table 1 ). 2) After mixing, the pH was adjusted to 8.7 with 10 M NaOH
• 4-FPBA solution 30 % 4-FPBA in 1 ,2-propanediol adjusted to pH 9.6 with 10 M NaOH
• the enzyme protein concentration of an enzyme solution can be determined by a number of methods. If the specific activity of the enzyme is known, the enzyme protein concentration can be determined by first determining the enzyme activity (in units/g material) at a selected set of conditions and divide by the specific activity (in units/mg enzyme protein). The specific activity of an enzyme is determined by first purifying the enzyme to homogeneity by means known in the art. The enzyme activity is then determined in the purified sample at the same set of conditions as used for measuring the enzyme activity in the enzyme protein concentrate. The total protein concentration is also determined in the purified sample and the specific activity is then obtained by dividing the enzyme activity with the protein concentration of the purified sample.
• the total protein concentration can be determined by one of the many total protein assays well known in the art (A review of different colorimetric protein assays is given by Christine V. Sa- pan, Roger L. Lundblad and Nicholas C. Price in Biotechnol. Appl. Biochem. (1999) 29, p99- 108). If the enzyme containing solution only contains the protein of interest on active form, the enzyme protein concentration can be determined directly by measuring the total protein concentration.
• the used method in the present invention is an assay based on the hydrolysis of N 1 N- dimethylcasein (DMC). Briefly, the protease activity is followed spectrophotometrically at 420 nm for 10 minutes after a pre-incubation period of 8 minutes. The assay is run at pH 8.3 and at 37 0 C. The following solutions are used for the assay:
• DMC-substrate 0.4% N,N-dimethylcasein in 90 mM sodium tetraborate, 120 mM sodium phosphate, 0.2% Brij 35, adjusted to pH 8.0.
• TNBS solution 1.73 mM 2,4,6-trinitrobenzenesulfonic acid in water.
• Dilution buffer 0.15 M KCI, 0.05 M boric acid, 0.16 M sodium sulfite, 0.2% Brij 35 adjusted to pH 9.0.
• 80 ⁇ l_ TNBS solution is mixed with 45 ⁇ l_ sample or standard (diluted in dilution buffer) and the reaction is started by the addition of 160 ⁇ l_ DMC-substrate.

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